Alteration of respiration capacity and transcript accumulation level of alternative oxidase genes in necrosis lines of common wheat.

PubMed

Sugie, Atsushi; Murai, Koji; Takumi, Shigeo

2007-06-01

Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in plants. AOX is a key enzyme of the alternative respiration pathway. To study the effects of necrotic cell death on the mitochondrial function, production of reactive oxygen species (ROS), respiration capacities and accumulation patterns of mitochondria-targeted protein-encoding gene transcripts were compared between wild-type, lesion-mimic mutant and hybrid necrosis wheat plants. Around cells with the necrosis symptom, ROS accumulated abundantly in the intercellular spaces. The ratio of the alternative pathway to the cytochrome pathway was markedly enhanced in the necrotic leaves. Transcripts of a wheat AOX gene, Waox1a, were more abundant in a novel lesion-mimic mutant of common wheat than in the wild-type plants. An increased level of the Waox1a transcripts was also observed in hybrid plants containing Ne1 and Ne2 genes. These results indicated that an increase of the wheat AOX transcript level resulted in enhancement of respiration capacity of the alternative pathway in the necrotic cells.

Defining the microbial transcriptional response to colitis through integrated host and microbiome profiling

PubMed Central

Ilott, Nicholas Edward; Bollrath, Julia; Danne, Camille; Schiering, Chris; Shale, Matthew; Adelmann, Krista; Krausgruber, Thomas; Heger, Andreas; Sims, David; Powrie, Fiona

2016-01-01

The gut microbiome is significantly altered in inflammatory bowel diseases, but the basis of these changes is not well understood. We have combined metagenomic and metatranscriptomic profiling of the gut microbiome to assess modifications to both bacterial community structure and transcriptional activity in a mouse model of colitis. By using transcriptomic analysis of colonic tissue and luminal RNA derived from the host, we have also characterised how host transcription relates to the microbial transcriptional response in inflammation. In colitis, increased abundance and transcription of diverse microbial gene families involved in responses to nutrient deprivation, antimicrobial peptide production and oxidative stress support an adaptation of multiple commensal genera to withstand a diverse set of environmental stressors in the inflammatory environment. These data are supported by a transcriptional signature of activated macrophages and granulocytes in the gut lumen during colitis, a signature that includes the transcription of the key antimicrobial genes S100a8 and S100a9 (calprotectin). Genes involved in microbial resistance to oxidative stress, including Dps/ferritin, Fe-dependent peroxidase and glutathione S-transferase were identified as changing to a greater extent at the level of transcription than would be predicted by DNA abundance changes, implicating a role for increased oxygen tension and/or host-derived reactive oxygen species in driving transcriptional changes in commensal microbes. PMID:27003245

Transgenic expression of fungal accessory hemicellulases in Arabidopsis thaliana triggers transcriptional patterns related to biotic stress and defense response

DOE PAGES

Tsai, Alex Yi-Lin; Chan, Kin; Ho, Chi-Yip; ...

2017-03-02

The plant cell wall is an abundant and renewable resource for lignocellulosic applications such as the production of biofuel. Due to structural and compositional complexities, the plant cell wall is, however, recalcitrant to hydrolysis and extraction of platform sugars. A cell wall engineering strategy to reduce this recalcitrance makes use of microbial cell wall modifying enzymes that are expressed directly in plants themselves. Previously, we constructed transgenic Arabidopsis thaliana constitutively expressing the fungal hemicellulases: Phanerochaete carnosa glucurnoyl esterase (PcGCE) and Aspergillus nidulans α-arabinofuranosidase (AnAF54). While the PcGCE lines demonstrated improved xylan extractability, they also displayed chlorotic leaves leading to themore » hypothesis that expression of such enzymes in planta resulted in plant stress. The objective of this study is to investigate the impact of transgenic expression of the aforementioned microbial hemicellulases in planta on the host arabidopsis. More specifically, we investigated transcriptome profiles by short read high throughput sequencing (RNAseq) from developmentally distinct parts of the plant stem. When compared to non-transformed wild-type plants, a subset of genes was identified that showed differential transcript abundance in all transgenic lines and tissues investigated. Intriguingly, this core set of genes was significantly enriched for those involved in plant defense and biotic stress responses. While stress and defense-related genes showed increased transcript abundance in the transgenic plants regardless of tissue or genotype, genes involved in photosynthesis (light harvesting) were decreased in their transcript abundance potentially reflecting wide-spread effects of heterologous microbial transgene expression and the maintenance of plant homeostasis. Additionally, an increase in transcript abundance for genes involved in salicylic acid signaling further substantiates our finding that transgenic expression of microbial cell wall modifying enzymes induces transcriptome responses similar to those observed in defense responses.« less

Transgenic expression of fungal accessory hemicellulases in Arabidopsis thaliana triggers transcriptional patterns related to biotic stress and defense response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Alex Yi-Lin; Chan, Kin; Ho, Chi-Yip

The plant cell wall is an abundant and renewable resource for lignocellulosic applications such as the production of biofuel. Due to structural and compositional complexities, the plant cell wall is, however, recalcitrant to hydrolysis and extraction of platform sugars. A cell wall engineering strategy to reduce this recalcitrance makes use of microbial cell wall modifying enzymes that are expressed directly in plants themselves. Previously, we constructed transgenic Arabidopsis thaliana constitutively expressing the fungal hemicellulases: Phanerochaete carnosa glucurnoyl esterase (PcGCE) and Aspergillus nidulans α-arabinofuranosidase (AnAF54). While the PcGCE lines demonstrated improved xylan extractability, they also displayed chlorotic leaves leading to themore » hypothesis that expression of such enzymes in planta resulted in plant stress. The objective of this study is to investigate the impact of transgenic expression of the aforementioned microbial hemicellulases in planta on the host arabidopsis. More specifically, we investigated transcriptome profiles by short read high throughput sequencing (RNAseq) from developmentally distinct parts of the plant stem. When compared to non-transformed wild-type plants, a subset of genes was identified that showed differential transcript abundance in all transgenic lines and tissues investigated. Intriguingly, this core set of genes was significantly enriched for those involved in plant defense and biotic stress responses. While stress and defense-related genes showed increased transcript abundance in the transgenic plants regardless of tissue or genotype, genes involved in photosynthesis (light harvesting) were decreased in their transcript abundance potentially reflecting wide-spread effects of heterologous microbial transgene expression and the maintenance of plant homeostasis. Additionally, an increase in transcript abundance for genes involved in salicylic acid signaling further substantiates our finding that transgenic expression of microbial cell wall modifying enzymes induces transcriptome responses similar to those observed in defense responses.« less

ChloroSeq, an optimized chloroplast RNA-Seq bioinformatic pipeline, reveals remodeling of the organellar transcriptome under heat stress

DOE PAGES

Castandet, Benoît; Hotto, Amber M.; Strickler, Susan R.; ...

2016-07-06

Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsismore » thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. In conclusion, ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators.« less

ChloroSeq, an optimized chloroplast RNA-Seq bioinformatic pipeline, reveals remodeling of the organellar transcriptome under heat stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castandet, Benoît; Hotto, Amber M.; Strickler, Susan R.

Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsismore » thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. In conclusion, ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators.« less

A monoallelic-to-biallelic T-cell transcriptional switch regulates GATA3 abundance

PubMed Central

Ku, Chia-Jui; Lim, Kim-Chew; Kalantry, Sundeep; Maillard, Ivan; Engel, James Douglas; Hosoya, Tomonori

2015-01-01

Protein abundance must be precisely regulated throughout life, and nowhere is the stringency of this requirement more evident than during T-cell development: A twofold increase in the abundance of transcription factor GATA3 results in thymic lymphoma, while reduced GATA3 leads to diminished T-cell production. GATA3 haploinsufficiency also causes human HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome, often accompanied by immunodeficiency. Here we show that loss of one Gata3 allele leads to diminished expansion (and compromised development) of immature T cells as well as aberrant induction of myeloid transcription factor PU.1. This effect is at least in part mediated transcriptionally: We discovered that Gata3 is monoallelically expressed in a parent of origin-independent manner in hematopoietic stem cells and early T-cell progenitors. Curiously, half of the developing cells switch to biallelic Gata3 transcription abruptly at midthymopoiesis. We show that the monoallelic-to-biallelic transcriptional switch is stably maintained and therefore is not a stochastic phenomenon. This unique mechanism, if adopted by other regulatory genes, may provide new biological insights into the rather prevalent phenomenon of monoallelic expression of autosomal genes as well as into the variably penetrant pathophysiological spectrum of phenotypes observed in many human syndromes that are due to haploinsufficiency of the affected gene. PMID:26385963

Gene expression patterns of two dominant tallgrass prairie species differ in response to warming and altered precipitation

NASA Astrophysics Data System (ADS)

Smith, Melinda D.; Hoffman, Ava M.; Avolio, Meghan L.

2016-05-01

To better understand the mechanisms underlying plant species responses to climate change, we compared transcriptional profiles of the co-dominant C4 grasses, Andropogon gerardii Vitman and Sorghastrum nutans (L.) Nash, in response to increased temperatures and more variable precipitation regimes in a long-term field experiment in native tallgrass prairie. We used microarray probing of a closely related model species (Zea mays) to assess correlations in leaf temperature (Tleaf) and leaf water potential (LWP) and abundance changes of ~10,000 transcripts in leaf tissue collected from individuals of both species. A greater number of transcripts were found to significantly change in abundance levels with Tleaf and LWP in S. nutans than in A. gerardii. S. nutans also was more responsive to short-term drought recovery than A. gerardii. Water flow regulating transcripts associated with stress avoidance (e.g., aquaporins), as well as those involved in the prevention and repair of damage (e.g., antioxidant enzymes, HSPs), were uniquely more abundant in response to increasing Tleaf in S. nutans. The differential transcriptomic responses of the co-dominant C4 grasses suggest that these species may cope with and respond to temperature and water stress at the molecular level in distinct ways, with implications for tallgrass prairie ecosystem function.

Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog

PubMed Central

Wolff, Stephanie E.; Veldhoen, Nik; Helbing, Caren C.; Ramirez, Claire A.; Malpas, Janae M.; Propper, Catherine R.

2015-01-01

Wildlife and human populations are exposed to anthropogenic mixtures of chemicals in the environment that may adversely influence normal reproductive function and development. We determined the effects of exposure to estrogenic chemicals and wastewater effluent (WWE) on developing gonads of the American bullfrog, Rana (Lithobates) catesbeiana, a species whose widespread distribution make it an ideal model for environmental monitoring for endocrine effects of chemical contaminants. Premetamorphic bullfrog tadpoles were exposed to treatment vehicle, 17β-estradiol (E2; 10−9 M) or 4-tert-octylphenol (OP; 10−9 M, 10−8 M, and 10−7 M). Additionally, gonadal differentiation was evaluated in bullfrog tadpoles from a WWE-containing site versus those from a reference location receiving no WWE. In both studies, phenotypic sex, steroidogenic factor-1 (nr5a1), and aromatase (cyp19a1) mRNA levels using quantitative real-time PCR were determined. Exposure to E2 or OP did not alter sex ratios. In controls, both nr5a1 and cyp19a1 transcript levels exhibited sexual dimorphism, with males demonstrating higher levels of nr5a1 and females greater abundance of cyp19a1. However, E2 exposure increased cyp19a1 mRNA abundance in testes and decreased levels in ovaries, eliminating the sexual dimorphism observed in controls. E2-exposed males exhibited increased nr5a1 transcript levels in the testes compared to controls, while females demonstrated no E2 effect. OP treatment had no effect on female cyp19a1 mRNA abundance, but exposure to 10−7 M OP increased testicular transcript levels. Treatment with 10−9 and 10−8 M OP, but not 10−7 M, resulted in decreased abundance of nr5a1 transcript in both ovaries and testes. Animals from the field had sexually dimorphic gonadal levels of cyp19a1, but both sexes from the WWE site exhibited elevated cyp19a1 transcript abundance compared to the reference location. Individual chemical compounds and anthropogenic wastewater effluent dispersed within the environment influence the levels of gonadal mRNA encoding key proteins involved in gonadal differentiation. PMID:25863316

Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root.

PubMed

Carvalho, Luiz Jcb; Agustini, Marco Av; Anderson, James V; Vieira, Eduardo A; de Souza, Claudia Rb; Chen, Songbi; Schaal, Barbara A; Silva, Joseane P

2016-06-10

Cassava (Manihot esculenta Crantz) storage root provides a staple food source for millions of people worldwide. Increasing the carotenoid content in storage root of cassava could provide improved nutritional and health benefits. Because carotenoid accumulation has been associated with storage root color, this study characterized carotenoid profiles, and abundance of key transcripts associated with carotenoid biosynthesis, from 23 landraces of cassava storage root ranging in color from white-to-yellow-to-pink. This study provides important information to plant breeding programs aimed at improving cassava storage root nutritional quality. Among the 23 landraces, five carotenoid types were detected in storage root with white color, while carotenoid types ranged from 1 to 21 in storage root with pink and yellow color. The majority of storage root in these landraces ranged in color from pale-to-intense yellow. In this color group, total β-carotene, containing all-E-, 9-Z-, and 13-Z-β-carotene isomers, was the major carotenoid type detected, varying from 26.13 to 76.72 %. Although no α-carotene was observed, variable amounts of a α-ring derived xanthophyll, lutein, was detected; with greater accumulation of α-ring xanthophylls than of β-ring xanthophyll. Lycopene was detected in a landrace (Cas51) with pink color storage root, but it was not detected in storage root with yellow color. Based on microarray and qRT-PCR analyses, abundance of transcripts coding for enzymes involved in carotenoid biosynthesis were consistent with carotenoid composition determined by contrasting HPLC-Diode Array profiles from storage root of landraces IAC12, Cas64, and Cas51. Abundance of transcripts encoding for proteins regulating plastid division were also consistent with the observed differences in total β-carotene accumulation. Among the 23 cassava landraces with varying storage root color and diverse carotenoid types and profiles, landrace Cas51 (pink color storage root) had low LYCb transcript abundance, whereas landrace Cas64 (intense yellow storage root) had decreased HYb transcript abundance. These results may explain the increased amounts of lycopene and total β-carotene observed in landraces Cas51 and Cas64, respectively. Overall, total carotenoid content in cassava storage root of color class representatives were associated with spatial patterns of secondary growth, color, and abundance of transcripts linked to plastid division. Finally, a partial carotenoid biosynthesis pathway is proposed.

RNA-Seq Alignment to Individualized Genomes Improves Transcript Abundance Estimates in Multiparent Populations

PubMed Central

Munger, Steven C.; Raghupathy, Narayanan; Choi, Kwangbom; Simons, Allen K.; Gatti, Daniel M.; Hinerfeld, Douglas A.; Svenson, Karen L.; Keller, Mark P.; Attie, Alan D.; Hibbs, Matthew A.; Graber, Joel H.; Chesler, Elissa J.; Churchill, Gary A.

2014-01-01

Massively parallel RNA sequencing (RNA-seq) has yielded a wealth of new insights into transcriptional regulation. A first step in the analysis of RNA-seq data is the alignment of short sequence reads to a common reference genome or transcriptome. Genetic variants that distinguish individual genomes from the reference sequence can cause reads to be misaligned, resulting in biased estimates of transcript abundance. Fine-tuning of read alignment algorithms does not correct this problem. We have developed Seqnature software to construct individualized diploid genomes and transcriptomes for multiparent populations and have implemented a complete analysis pipeline that incorporates other existing software tools. We demonstrate in simulated and real data sets that alignment to individualized transcriptomes increases read mapping accuracy, improves estimation of transcript abundance, and enables the direct estimation of allele-specific expression. Moreover, when applied to expression QTL mapping we find that our individualized alignment strategy corrects false-positive linkage signals and unmasks hidden associations. We recommend the use of individualized diploid genomes over reference sequence alignment for all applications of high-throughput sequencing technology in genetically diverse populations. PMID:25236449

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

NASA Astrophysics Data System (ADS)

Fagerquist, Clifton K.; Zaragoza, William J.

2015-05-01

We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H- strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) with post source decay (PSD) and real time-quantitative polymerase chain reaction (RT-qPCR). Stx2a and Stx2c in STEC strain E32511 were quantified from the integrated peak area of their singly charged disulfide-intact B-subunit ions at m/z ~7819 and m/z ~7774, respectively. We found that the Stx2a subtype was 21-fold more abundant than the Stx2c subtype. The two amino acid substitutions (16D ↔ 16 N and 24D ↔ 24A) that distinguish Stx2a from Stx2c not only result in a mass difference of 45 Da between their respective B-subunits but also result in distinctly different fragmentation channels by MS/MS-PSD because both substitutions involve an aspartic acid (D) residue. Importantly, these two substitutions have also been linked to differences in subtype toxicity. We measured the relative abundances of mRNA transcripts using RT-qPCR and determined that the stx2a transcript is 13-fold more abundant than stx2c transcript. In silico secondary structure analysis of the full mRNA operons of stx2a and stx2c suggest that transcript structural differences may also contribute to a relative increase of Stx2a over Stx2c. In consequence, toxin expression may be under both transcriptional and post-transcriptional control.

Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed

USGS Publications Warehouse

Rocca, Jennifer D.; Hall, Edward K.; Lennon, Jay T.; Evans, Sarah E.; Waldrop, Mark P.; Cotner, James B.; Nemergut, Diana R.; Graham, Emily B.; Wallenstein, Matthew D.

2015-01-01

For any enzyme-catalyzed reaction to occur, the corresponding protein-encoding genes and transcripts are necessary prerequisites. Thus, a positive relationship between the abundance of gene or transcripts and corresponding process rates is often assumed. To test this assumption, we conducted a meta-analysis of the relationships between gene and/or transcript abundances and corresponding process rates. We identified 415 studies that quantified the abundance of genes or transcripts for enzymes involved in carbon or nitrogen cycling. However, in only 59 of these manuscripts did the authors report both gene or transcript abundance and rates of the appropriate process. We found that within studies there was a significant but weak positive relationship between gene abundance and the corresponding process. Correlations were not strengthened by accounting for habitat type, differences among genes or reaction products versus reactants, suggesting that other ecological and methodological factors may affect the strength of this relationship. Our findings highlight the need for fundamental research on the factors that control transcription, translation and enzyme function in natural systems to better link genomic and transcriptomic data to ecosystem processes.

Sodium selenite increases the transcript levels of iodothyronine deiodinases I and II in ovine and bovine fetal thyrocytes in vitro.

PubMed

Foroughi, Mohammad Ali; Dehghani, Hesam; Mahdavi-Shahri, Naser; Bassami, Mohammad Reza

2013-07-01

Selenium is essential for thyroid hormone homeostasis. Selenium is co-translationally incorporated into the protein backbone of 5' deiodinase enzymes, which are responsible for the intra- and extra-thyroidal activation of thyroid hormones. The objective of this study was to evaluate the effects of sodium selenite on the transcript levels of type I (DIO1) and II (DIO2) deiodinases in the primary culture of ovine and bovine fetal thyroid. By culture of fetal thyrocytes in the presence or absence of sodium selenite, and quantification of DIO1 and DIO2 transcripts using real-time reverse transcription polymerase chain reaction (RT-qPCR), we found that sodium selenite is able to increase the abundance of transcripts for DIO1 and DIO2 genes. We also found that cultured thyrocytes in the presence of sodium selenite compared to control cultured thyrocytes release more T3 into the culture medium. This indicates that in the presence of sodium selenite higher levels of DIO1 and DIO2 enzymes are produced, which are able to convert T4 to T3. In conclusion, we have shown that sodium selenite is increasing the abundance of DIO1 and DIO2 transcripts and increasing the production and release of T3 from cultured fetal thyrocytes. This finding emphasizes the role of selenium in transcriptional and expression processes during development and suggests that selenium deficiency during pregnancy in sheep and cattle may lead to the lower levels of DIO1 and DIO2 transcription in fetal thyroid, and thus, lower level of thyroidal T3 release into the fetal serum. Copyright © 2013 Elsevier GmbH. All rights reserved.

SMAD3 augments FoxO3-induced MuRF-1 promoter activity in a DNA-binding-dependent manner

PubMed Central

Bollinger, Lance M.; Witczak, Carol A.; Houmard, Joseph A.

2014-01-01

Muscle-specific RING finger-1 (MuRF-1), a ubiquitin ligase and key regulator of proteasome-dependent protein degradation, is highly expressed during skeletal muscle atrophy. The transcription factor forkhead box O3 (FoxO3) induces MuRF-1 expression, but the direct role of other major atrophy-related transcription factors, such as SMAD3, is largely unknown. The goal of this study was to determine whether SMAD3 individually regulates, or with FoxO3 coordinately regulates, MuRF-1 expression. In cultured myotubes or human embryonic kidney cells, MuRF-1 mRNA content and promoter activity were increased by FoxO3 but not by SMAD3 overexpression. However, FoxO3 and SMAD3 coexpression synergistically increased MuRF-1 mRNA and promoter activity. Mutation of the SMAD-binding element (SBE) in the proximal MuRF-1 promoter or overexpression of a SMAD3 DNA-binding mutant attenuated FoxO3-dependent MuRF-1 promoter activation, showing that SMAD binding to DNA is required for optimal activation of FoxO3-induced transcription of MuRF-1. Using chromatin immunoprecipitation, SMAD3 DNA binding increased FoxO3 abundance and SBE mutation reduced FoxO3 abundance on the MuRF-1 promoter. Furthermore, SMAD3 overexpression dose-dependently increased FoxO3 protein content, and coexpression of FoxO3 and SMAD3 synergistically increased FoxO-dependent gene transcription [assessed with a FoxO response element (FRE)-driven reporter]. Collectively, these results show that SMAD3 regulates transcription of MuRF-1 by increasing FoxO3 binding at a conserved FRE-SBE motif within the proximal promoter region, and by increasing FoxO3 protein content and transcriptional activity. These data are the first to indicate that two major transcription factors regulating protein degradation, FoxO3 and SMAD3, converge to coordinately and directly regulate transcription of MuRF-1. PMID:24920680

Unbalanced Activation of Glutathione Metabolic Pathways Suggests Potential Involvement in Plant Defense against the Gall Midge Mayetiola destructor in Wheat

PubMed Central

Liu, Xuming; Zhang, Shize; Whitworth, R. Jeff; Stuart, Jeffrey J.; Chen, Ming-Shun

2015-01-01

Glutathione, γ-glutamylcysteinylglycine, exists abundantly in nearly all organisms. Glutathione participates in various physiological processes involved in redox reactions by serving as an electron donor/acceptor. We found that the abundance of total glutathione increased up to 60% in resistant wheat plants within 72 hours following attack by the gall midge Mayetiola destructor, the Hessian fly. The increase in total glutathione abundance, however, is coupled with an unbalanced activation of glutathione metabolic pathways. The activity and transcript abundance of glutathione peroxidases, which convert reduced glutathione (GSH) to oxidized glutathione (GSSG), increased in infested resistant plants. However, the enzymatic activity and transcript abundance of glutathione reductases, which convert GSSG back to GSH, did not change. This unbalanced regulation of the glutathione oxidation/reduction cycle indicates the existence of an alternative pathway to regenerate GSH from GSSG to maintain a stable GSSG/GSH ratio. Our data suggest the possibility that GSSG is transported from cytosol to apoplast to serve as an oxidant for class III peroxidases to generate reactive oxygen species for plant defense against Hessian fly larvae. Our results provide a foundation for elucidating the molecular processes involved in glutathione-mediated plant resistance to Hessian fly and potentially other pests as well. PMID:25627558

Prostanoid-induced expression of matrix metalloproteinase-1 messenger ribonucleic acid in rat osteosarcoma cells

NASA Technical Reports Server (NTRS)

Clohisy, J. C.; Connolly, T. J.; Bergman, K. D.; Quinn, C. O.; Partridge, N. C.

1994-01-01

Individual prostanoids have distinct potencies in activating intracellular signaling pathways and regulating gene expression in osteoblastic cells. The E-series prostaglandins (PGs) are known to stimulate matrix metalloproteinase-1 (MMP-1) synthesis and secretion in certain rodent and human osteoblastic cells, yet the intracellular events involved remain unclear. To further characterize this response and its signal transduction pathway(s), we examined prostanoid-induced expression of the MMP-1 gene in the rat osteoblastic osteosarcoma cell line UMR 106-01. Northern blot analysis demonstrated that prostaglandin E2 (PGE2) and PGE1 were very potent stimulators (40-fold) of MMP-1 transcript abundance, PGF2 alpha and prostacyclin were weak stimulators (4-fold), and thromboxane-B2 had no effect. The marked increase in MMP-1 transcript abundance after PGE2 treatment was first detected at 2 h, became maximal at 4 h, and persisted beyond 24 h. This response was dose dependent and elicited maximal and half-maximal effects with concentrations of 10(-6) and 0.6 x 10(-7) M, respectively. Cycloheximide, a protein synthesis inhibitor, completely blocked this effect of PGE2, suggesting that the expression of other genes is required. Nuclear run-on experiments demonstrated that PGE2 rapidly activates MMP-1 gene transcription, with a maximal increase at 2-4 h. The second messenger analog, 8-bromo-cAMP, mimicked the effects of PGE2 by stimulating a dose-dependent increase in MMP-1 messenger RNA (mRNA) levels, with a maximal effect quantitatively similar to that observed with PGE2. Thus, in UMR 106-01 cells, different prostanoids have distinct potencies in stimulating MMP-1 mRNA abundance. Our data suggest that PGE2 stimulation of MMP-1 synthesis is due to activation of MMP-1 gene transcription and a subsequent marked increase in MMP-1 mRNA abundance. This effect is dependent on de novo protein synthesis and is mimicked by protein kinase-A activation.

Gene expression patterns of two dominant tallgrass prairie species differ in response to warming and altered precipitation

DOE PAGES

Smith, Melinda D.; Hoffman, Ava M.; Avolio, Meghan L.

2016-05-13

To better understand the mechanisms underlying plant species responses to climate change, we compared transcriptional profiles of the co-dominant C 4 grasses, Andropogon gerardii Vitman and Sorghastrum nutans (L.) Nash, in response to increased temperatures and more variable precipitation regimes in a long-term field experiment in native tallgrass prairie. We used microarray probing of a closely related model species ( Zea mays) to assess correlations in leaf temperature (T leaf) and leaf water potential (LWP) and abundance changes of ~10,000 transcripts in leaf tissue collected from individuals of both species. A greater number of transcripts were found to significantly changemore » in abundance levels with T leaf and LWP in S. nutans than in A. gerardii. S. nutans also was more responsive to short-term drought recovery than A. gerardii. Water flow regulating transcripts associated with stress avoidance (e.g., aquaporins), as well as those involved in the prevention and repair of damage (e.g., antioxidant enzymes, HSPs), were uniquely more abundant in response to increasing T leaf in S. nutans. Furthermore, the differential transcriptomic responses of the co-dominant C 4 grasses suggest that these species may cope with and respond to temperature and water stress at the molecular level in distinct ways, with implications for tallgrass prairie ecosystem function.« less

Land use type significantly affects microbial gene transcription in soil.

PubMed

Nacke, Heiko; Fischer, Christiane; Thürmer, Andrea; Meinicke, Peter; Daniel, Rolf

2014-05-01

Soil microorganisms play an essential role in sustaining biogeochemical processes and cycling of nutrients across different land use types. To gain insights into microbial gene transcription in forest and grassland soil, we isolated mRNA from 32 sampling sites. After sequencing of generated complementary DNA (cDNA), a total of 5,824,229 sequences could be further analyzed. We were able to assign nonribosomal cDNA sequences to all three domains of life. A dominance of bacterial sequences, which were affiliated to 25 different phyla, was found. Bacterial groups capable of aromatic compound degradation such as Phenylobacterium and Burkholderia were detected in significantly higher relative abundance in forest soil than in grassland soil. Accordingly, KEGG pathway categories related to degradation of aromatic ring-containing molecules (e.g., benzoate degradation) were identified in high abundance within forest soil-derived metatranscriptomic datasets. The impact of land use type forest on community composition and activity is evidently to a high degree caused by the presence of wood breakdown products. Correspondingly, bacterial groups known to be involved in lignin degradation and containing ligninolytic genes such as Burkholderia, Bradyrhizobium, and Azospirillum exhibited increased transcriptional activity in forest soil. Higher solar radiation in grassland presumably induced increased transcription of photosynthesis-related genes within this land use type. This is in accordance with high abundance of photosynthetic organisms and plant-infecting viruses in grassland.

Microarray analysis identifies keratin loci as sensitive biomarkers for thyroid hormone disruption in the salamander Ambystoma mexicanum.

PubMed

Page, Robert B; Monaghan, James R; Samuels, Amy K; Smith, Jeramiah J; Beachy, Christopher K; Voss, S Randal

2007-02-01

Ambystomatid salamanders offer several advantages for endocrine disruption research, including genomic and bioinformatics resources, an accessible laboratory model (Ambystoma mexicanum), and natural lineages that are broadly distributed among North American habitats. We used microarray analysis to measure the relative abundance of transcripts isolated from A. mexicanum epidermis (skin) after exogenous application of thyroid hormone (TH). Only one gene had a >2-fold change in transcript abundance after 2 days of TH treatment. However, hundreds of genes showed significantly different transcript levels at days 12 and 28 in comparison to day 0. A list of 123 TH-responsive genes was identified using statistical, BLAST, and fold level criteria. Cluster analysis identified two groups of genes with similar transcription patterns: up-regulated versus down-regulated. Most notably, several keratins exhibited dramatic (1000 fold) increases or decreases in transcript abundance. Keratin gene expression changes coincided with morphological remodeling of epithelial tissues. This suggests that keratin loci can be developed as sensitive biomarkers to assay temporal disruptions of larval-to-adult gene expression programs. Our study has identified the first collection of loci that are regulated during TH-induced metamorphosis in a salamander, thus setting the stage for future investigations of TH disruption in the Mexican axolotl and other salamanders of the genus Ambystoma.

Induction of lcc2 expression and activity by Agaricus bisporus provides defence against Trichoderma aggressivum toxic extracts

PubMed Central

Sjaarda, Calvin P; Abubaker, Kamal S; Castle, Alan J

2015-01-01

Laccases are used by fungi for several functions including defence responses to stresses associated with attack by other fungi. Laccase activity changes and the induction of two laccase genes, lcc1 and lcc2, in Agaricus bisporus were measured in response to toxic extracts of medium in which Trichoderma aggressivum, the cause of green mould disease, was grown. A strain of A. bisporus that shows resistance to the extracts showed higher basal levels and greater enzymatic activity after extract exposure than did a sensitive strain. Furthermore, pre-incubation of T. aggressivum extract with laccases reduced toxicity. Faster induction and greater numbers of lcc2 transcripts in response to the extract were noted in the resistant strain than in the sensitive strain. The timing and increase in lcc2 transcript abundance mirrored changes in total laccase activity. No correlation between resistance and lcc1 transcription was apparent. Transcript abundance in transformants with a siRNA construct homologous to both genes varied widely. A strong negative correlation between transcript abundance and sensitivity of the transformant to toxic extract was observed in plate assays. These results indicated that laccase activity and in particular that encoded by lcc2 contributes to toxin metabolism and by extension green mould disease resistance. PMID:25824278

Integration of multi-omics data of a genome-reduced bacterium: Prevalence of post-transcriptional regulation and its correlation with protein abundances

PubMed Central

Chen, Wei-Hua; van Noort, Vera; Lluch-Senar, Maria; Hennrich, Marco L.; H. Wodke, Judith A.; Yus, Eva; Alibés, Andreu; Roma, Guglielmo; Mende, Daniel R.; Pesavento, Christina; Typas, Athanasios; Gavin, Anne-Claude; Serrano, Luis; Bork, Peer

2016-01-01

We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated ‘-omics’ data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. PMID:26773059

Signatures from Tissue-specific MPSS Libraries Identify Transcripts Preferentially Expressed in the Mouse Inner Ear

PubMed Central

Peters, Linda M.; Belyantseva, Inna A.; Lagziel, Ayala; Battey, James F.; Friedman, Thomas B.; Morell, Robert J.

2007-01-01

Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well-suited for identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome (MRT) online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome. PMID:17049805

The Argonaute protein TbAGO1 contributes to large and mini-chromosome segregation and is required for control of RIME retroposons and RHS pseudogene-associated transcripts.

PubMed

Durand-Dubief, Mickaël; Absalon, Sabrina; Menzer, Linda; Ngwabyt, Sandra; Ersfeld, Klaus; Bastin, Philippe

2007-12-01

The protist Trypanosoma brucei possesses a single Argonaute gene called TbAGO1 that is necessary for RNAi silencing. We previously showed that in strain 427, TbAGO1 knock-out leads to a slow growth phenotype and to chromosome segregation defects. Here we report that the slow growth phenotype is linked to defects in segregation of both large and mini-chromosome populations, with large chromosomes being the most affected. These phenotypes are completely reversed upon inducible re-expression of TbAGO1 fused to GFP, demonstrating their link with TbAGO1. Trypanosomes that do not express TbAGO1 show a general increase in the abundance of transcripts derived from the short retroposon RIME (Ribosomal Interspersed Mobile Element). Supplementary large RIME transcripts emerge in the absence of RNAi, a phenomenon coupled to the disappearance of short transcripts. These fluctuations are reversed by inducible expression of GFP::TbAGO1. Furthermore, we use a combination of Northern blots, RT-PCR and sequencing to reveal that RNAi controls expression of transcripts derived from RHS (Retrotransposon Hot Spot) pseudogenes (RHS genes with retro-element(s) integrated within their coding sequence). Absence of RNAi also leads to an increase of steady-state transcripts from regular RHS genes (those without retro-element), indicating a role for pseudogene in control of gene expression. However, analysis of retroposon abundance and arrangement in the genome of multiple clonal cell lines of TbAGO1-/- failed to reveal movement of mobile elements despite the increased amounts of retroposon transcripts.

Transcriptional blood signatures distinguish pulmonary tuberculosis, pulmonary sarcoidosis, pneumonias and lung cancers.

PubMed

Bloom, Chloe I; Graham, Christine M; Berry, Matthew P R; Rozakeas, Fotini; Redford, Paul S; Wang, Yuanyuan; Xu, Zhaohui; Wilkinson, Katalin A; Wilkinson, Robert J; Kendrick, Yvonne; Devouassoux, Gilles; Ferry, Tristan; Miyara, Makoto; Bouvry, Diane; Valeyre, Dominique; Dominique, Valeyre; Gorochov, Guy; Blankenship, Derek; Saadatian, Mitra; Vanhems, Phillip; Beynon, Huw; Vancheeswaran, Rama; Wickremasinghe, Melissa; Chaussabel, Damien; Banchereau, Jacques; Pascual, Virginia; Ho, Ling-Pei; Lipman, Marc; O'Garra, Anne

2013-01-01

New approaches to define factors underlying the immunopathogenesis of pulmonary diseases including sarcoidosis and tuberculosis are needed to develop new treatments and biomarkers. Comparing the blood transcriptional response of tuberculosis to other similar pulmonary diseases will advance knowledge of disease pathways and help distinguish diseases with similar clinical presentations. To determine the factors underlying the immunopathogenesis of the granulomatous diseases, sarcoidosis and tuberculosis, by comparing the blood transcriptional responses in these and other pulmonary diseases. We compared whole blood genome-wide transcriptional profiles in pulmonary sarcoidosis, pulmonary tuberculosis, to community acquired pneumonia and primary lung cancer and healthy controls, before and after treatment, and in purified leucocyte populations. An Interferon-inducible neutrophil-driven blood transcriptional signature was present in both sarcoidosis and tuberculosis, with a higher abundance and expression in tuberculosis. Heterogeneity of the sarcoidosis signature correlated significantly with disease activity. Transcriptional profiles in pneumonia and lung cancer revealed an over-abundance of inflammatory transcripts. After successful treatment the transcriptional activity in tuberculosis and pneumonia patients was significantly reduced. However the glucocorticoid-responsive sarcoidosis patients showed a significant increase in transcriptional activity. 144-blood transcripts were able to distinguish tuberculosis from other lung diseases and controls. Tuberculosis and sarcoidosis revealed similar blood transcriptional profiles, dominated by interferon-inducible transcripts, while pneumonia and lung cancer showed distinct signatures, dominated by inflammatory genes. There were also significant differences between tuberculosis and sarcoidosis in the degree of their transcriptional activity, the heterogeneity of their profiles and their transcriptional response to treatment.

Transcriptomic analysis reveals numerous diverse protein kinases and transcription factors involved in desiccation tolerance in the resurrection plant Myrothamnus flabellifolia

PubMed Central

Ma, Chao; Wang, Hong; Macnish, Andrew J; Estrada-Melo, Alejandro C; Lin, Jing; Chang, Youhong; Reid, Michael S; Jiang, Cai-Zhong

2015-01-01

The woody resurrection plant Myrothamnus flabellifolia has remarkable tolerance to desiccation. Pyro-sequencing technology permitted us to analyze the transcriptome of M. flabellifolia during both dehydration and rehydration. We identified a total of 8287 and 8542 differentially transcribed genes during dehydration and rehydration treatments respectively. Approximately 295 transcription factors (TFs) and 484 protein kinases (PKs) were up- or down-regulated in response to desiccation stress. Among these, the transcript levels of 53 TFs and 91 PKs increased rapidly and peaked early during dehydration. These regulators transduce signal cascades of molecular pathways, including the up-regulation of ABA-dependent and independent drought stress pathways and the activation of protective mechanisms for coping with oxidative damage. Antioxidant systems are up-regulated, and the photosynthetic system is modified to reduce ROS generation. Secondary metabolism may participate in the desiccation tolerance of M. flabellifolia as indicated by increases in transcript abundance of genes involved in isopentenyl diphosphate biosynthesis. Up-regulation of genes encoding late embryogenesis abundant proteins and sucrose phosphate synthase is also associated with increased tolerance to desiccation. During rehydration, the transcriptome is also enriched in transcripts of genes encoding TFs and PKs, as well as genes involved in photosynthesis, and protein synthesis. The data reported here contribute comprehensive insights into the molecular mechanisms of desiccation tolerance in M. flabellifolia. PMID:26504577

Effect of nitrogen fertilizer and/or rice straw amendment on methanogenic archaeal communities and methane production from a rice paddy soil.

PubMed

Bao, Qiongli; Huang, Yizong; Wang, Fenghua; Nie, Sanan; Nicol, Graeme W; Yao, Huaiying; Ding, Longjun

2016-07-01

Nitrogen fertilization and returning straw to paddy soil are important factors that regulate CH4 production. To evaluate the effect of rice straw and/or nitrate amendment on methanogens, a paddy soil was anaerobically incubated for 40 days. The results indicated that while straw addition increased CH4 production and the abundances of mcrA genes and their transcripts, nitrate amendment showed inhibitory effects on them. The terminal restriction fragment length polymorphism (T-RFLP) analysis based on mcrA gene revealed that straw addition obviously changed methanogenic community structure. Based on mcrA gene level, straw-alone addition stimulated Methanosarcinaceaes at the early stage of incubation (first 11 days), but nitrate showed inhibitory effect. The relative abundance of Methanobacteriaceae was also stimulated by straw addition during the first 11 days. Furthermore, Methanosaetaceae were enriched by nitrate-alone addition after 11 days, while Methanocellaceae were enriched by nitrate addition especially within the first 5 days. The transcriptional methanogenic community indicated more dynamic and complicated responses to straw and/or nitrate addition. Based on mcrA transcript level, nitrate addition alone resulted in the increase of Methanocellaceae and the shift from Methanosarcinaceae to Methanosaetaceae during the first 5 days of incubation. Straw treatments increased the relative abundance of Methanobacteriaceae after 11 days. These results demonstrate that nitrate addition influences methanogens which are transcriptionally and functionally active and can alleviate CH4 production associated with straw amendment in paddy soil incubations, presumably through competition for common substrates between nitrate-utilizing organisms and methanogens.

The transcription factor C/EBPβ in the dorsal root ganglion contributes to peripheral nerve trauma–induced nociceptive hypersensitivity

PubMed Central

Li, Zhisong; Mao, Yuanyuan; Liang, Lingli; Wu, Shaogen; Yuan, Jingjing; Mo, Kai; Cai, Weihua; Mao, Qingxiang; Cao, Jing; Bekker, Alex; Zhang, Wei; Tao, Yuan-Xiang

2017-01-01

Changes in gene transcription in the dorsal root ganglion (DRG) after nerve trauma contribute to the genesis of neuropathic pain. We report that peripheral nerve trauma caused by chronic constriction injury (CCI) increased the abundance of the transcription factor C/EBPβ (CCAAT/enhancer binding protein β) in the DRG. Blocking this increase mitigated the development and maintenance of CCI-induced mechanical, thermal, and cold pain hypersensitivities without affecting basal responses to acute pain and locomotor activity. Conversely, mimicking this increase produced hypersensitivity to mechanical, thermal, or cold pain. In the ipsilateral DRG, C/EBPβ promoted a decrease in the abundance of the voltage-gated potassium channel subunit Kv1.2 and µ opioid receptor (MOR) at the mRNA and protein levels, which would be predicted to increase excitability in the ipsilateral DRG neurons and reduce the efficacy of morphine analgesia. These effects required C/EPBβ-mediated transcriptional activation of Ehmt2 (euchromatic histonelysine N-methyltransferase 2), which encodes G9a, an epigenetic silencer of the genes encoding Kv1.2 and MOR. Blocking the increase in C/EBPβ in the DRG improved morphine analgesia after CCI. These results suggest that C/EBPβ is an endogenous initiator of neuropathic pain and could be a potential target for the prevention and treatment of this disorder. PMID:28698219

Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone

PubMed Central

Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

2016-01-01

Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

Regulation of Rat Hepatic L-Pyruvate Kinase Promoter Composition and Activity by Glucose, n-3 Polyunsaturated Fatty Acids, and Peroxisome Proliferator-activated Receptor-α Agonist*S

PubMed Central

Xu, Jinghua; Christian, Barbara; Jump, Donald B.

2009-01-01

Carbohydrate regulatory element-binding protein (ChREBP), MAX-like factor X(MLX), and hepatic nuclear factor-4α (HNF-4α)are key transcription factors involved in the glucose-mediated induction of hepatic L-type pyruvate kinase (L-PK) gene transcription. n-3 polyunsaturated fatty acids (PUFA) and WY14643 (peroxisome proliferator-activated receptor α (PPARα) agonist) interfere with glucose-stimulated L-PK gene transcription in vivo and in rat primary hepatocytes. Feeding rats a diet containing n-3 PUFA or WY14643 suppressed hepatic mRNAL-PK but did not suppress hepatic ChREBP or HNF-4α nuclear abundance. Hepatic MLX nuclear abundance, however, was suppressed by n-3 PUFA but not WY14643. In rat primary hepatocytes, glucose-stimulated accumulation of mRNALPK and L-PK promoter activity correlated with increased ChREBP nuclear abundance. This treatment also increased L-PK promoter occupancy by RNA polymerase II (RNA pol II), acetylated histone H3 (Ac-H3), and acetylated histone H4 (Ac-H4) but did not significantly impact L-PK promoter occupancy by ChREBP or HNF-4α. Inhibition of L-PK promoter activity by n-3 PUFA correlated with suppressed RNA pol II, Ac-H3, and Ac-H4 occupancy on the L-PK promoter. Although n-3 PUFA transiently suppressed ChREBP and MLX nuclear abundance, this treatment did not impact ChREBP-LPK promoter interaction. HNF4α-LPK promoter interaction was transiently suppressed by n-3 PUFA. Inhibition of L-PK promoter activity by WY14643 correlated with a transient decline in ChREBP nuclear abundance and decreased Ac-H4 interaction with the L-PK promoter. WY14643, however, had no impact on MLX nuclear abundance or HNF4α-LPK promoter interaction. Although overexpressed ChREBP or HNF-4α did not relieve n-3 PUFA suppression of L-PK gene expression, overexpressed MLX fully abrogated n-3 PUFA suppression of L-PK promoter activity and mRNAL-PK abundance. Overexpressed ChREBP, but not MLX, relieved the WY14643 inhibition of L-PK. In conclusion, n-3 PUFA and WY14643/PPARα target different transcription factors to control L-PK gene transcription. MLX, the heterodimer partner for ChREBP, has emerged as a novel target for n-3 PUFA regulation. PMID:16644726

Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includens.

PubMed

Lavine, M D; Chen, G; Strand, M R

2005-12-01

Inducible expression of antimicrobial peptides and other humoral immune factors by the insect fat body is well documented. Hemocytes comprise the second essential arm of the insect immune system but it is unclear whether antimicrobial peptide genes are expressed by all or only some types of hemocytes. Here we report the cloning of cecropin A (Pi-cecA), lebocin (Pi-leb) and lysozyme (Pi-lys) homologs from the moth Pseudoplusia includens. Relative-quantitative real-time PCR (rq-rtPCR) indicated that transcript abundance for each antimicrobial gene increased in fat body and hemocytes following immune challenge with the Gram-negative bacterium Escherichia coli. Relative transcript abundance of Pi-cecA was much higher in fat body than hemocytes. In contrast, transcript levels of Pi-leb were three-fold lower in hemocytes than fat body while transcript levels of Pi-lys were three-fold higher. Estimates for the overall contribution of the fat body and hemocytes to antimicrobial peptide expression suggested that hemocytes contribute significantly to Pi-lys transcript levels in larvae but produce much smaller amounts of Pi-cecA and Pi-leb compared to the fat body. Each antimicrobial peptide was also inducibly expressed in hemocytes following challenge with the Gram-positive bacterium Micrococcus luteus or when hemocytes formed capsules around chromatography beads. Analysis of hemocyte types indicated that granulocytes and plasmatocytes expressed all three antimicrobial peptides, whereas spherule cells and oenocytoids expressed only lysozyme. Transcriptional profiles of these antimicrobial genes were similar in granulocytes and plasmatocytes in vivo but were very different in vitro.

Occupancy of RNA Polymerase II Phosphorylated on Serine 5 (RNAP S5P) and RNAP S2P on Varicella-Zoster Virus Genes 9, 51, and 66 Is Independent of Transcript Abundance and Polymerase Location within the Gene.

PubMed

Henderson, Heather H; Timberlake, Kensey B; Austin, Zoe A; Badani, Hussain; Sanford, Bridget; Tremblay, Keriann; Baird, Nicholas L; Jones, Kenneth; Rovnak, Joel; Frietze, Seth; Gilden, Don; Cohrs, Randall J

2016-02-01

Regulation of gene transcription in varicella-zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and the occupancy of phosphorylated RNA polymerase II (RNAP) on the gene promoter, body, and terminus of VZV genes 9, 51, and 66. The abundance of VZV gene 9, 51, and 66 transcripts in VZV-infected human fetal lung fibroblasts was determined by reverse transcription-linked quantitative PCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5(P)) on VZV genes 9, 51, and 66 independently of transcript abundance and the location within the virus gene at both 1 and 3 days postinfection (dpi). In contrast, phosphorylated serine 2 (S2(P))-modified RNAP was not detected at any virus gene location at 3 dpi and was detected at levels only slightly above background levels at 1 dpi. Regulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromatin immunoprecipitation (ChIP). We used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that hyperphosphorylation at serine 5 of the C-terminal domain of RNAP along the lengths of VZV genes (the promoter, body, and transcription termination site) was independent of mRNA abundance. In contrast, little to no enrichment of serine 3 phosphorylation of RNAP was detected at these virus gene regions. This is distinct from the findings for RNAP at highly regulated host genes, where RNAP S5(P) occupancy decreased and S2(P) levels increased as the polymerase transited through the gene. Overall, these results suggest that RNAP associates with human and virus transcriptional units through different mechanisms. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Selenium treatment differentially affects sulfur metabolism in high and low glucosinolate producing cultivars of broccoli (Brassica oleracea L.).

PubMed

McKenzie, Marian J; Chen, Ronan K Y; Leung, Susanna; Joshi, Srishti; Rippon, Paula E; Joyce, Nigel I; McManus, Michael T

2017-12-01

The effect of selenium (Se) application on the sulfur (S)-rich glucosinolate (GSL)-containing plant, broccoli (Brassica oleracea L. var. italica) was examined with a view to producing germplasm with increased Se and GSL content for human health, and to understanding the influence of Se on the regulation of GSL production. Two cultivars differing in GSL content were compared. Increased Se application resulted in an increase in Se uptake in planta, but no significant change in total S or total GSL content in either cultivar. Also no significant change was observed in the activity of ATP sulfurylase (ATPS, EC 2.7.7.4) or O-acetylserine(thiol) lyase (OASTL, EC 2.5.1.47) with increased Se application. However, in the first investigation of APS kinase (APSK, EC 2.7.1.25) expression in response to Se fertilisation, an increase in transcript abundance of one variant of APS kinase 1 (BoAPSK1A) was observed in both cultivars, and an increase in BoAPSK2 transcript abundance was observed in the low GSL producing cultivar. A mechanism by which increased APSK transcription may provide a means of controlling the content of S-containing compounds, including GSLs, following Se uptake is proposed. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Juvenile Hormone Biosynthesis Gene Expression in the corpora allata of Honey Bee (Apis mellifera L.) Female Castes

PubMed Central

Rosa, Gustavo Conrado Couto; Moda, Livia Maria; Martins, Juliana Ramos; Bitondi, Márcia Maria Gentile; Hartfelder, Klaus; Simões, Zilá Luz Paulino

2014-01-01

Juvenile hormone (JH) controls key events in the honey bee life cycle, viz. caste development and age polyethism. We quantified transcript abundance of 24 genes involved in the JH biosynthetic pathway in the corpora allata-corpora cardiaca (CA-CC) complex. The expression of six of these genes showing relatively high transcript abundance was contrasted with CA size, hemolymph JH titer, as well as JH degradation rates and JH esterase (jhe) transcript levels. Gene expression did not match the contrasting JH titers in queen and worker fourth instar larvae, but jhe transcript abundance and JH degradation rates were significantly lower in queen larvae. Consequently, transcriptional control of JHE is of importance in regulating larval JH titers and caste development. In contrast, the same analyses applied to adult worker bees allowed us inferring that the high JH levels in foragers are due to increased JH synthesis. Upon RNAi-mediated silencing of the methyl farnesoate epoxidase gene (mfe) encoding the enzyme that catalyzes methyl farnesoate-to-JH conversion, the JH titer was decreased, thus corroborating that JH titer regulation in adult honey bees depends on this final JH biosynthesis step. The molecular pathway differences underlying JH titer regulation in larval caste development versus adult age polyethism lead us to propose that mfe and jhe genes be assayed when addressing questions on the role(s) of JH in social evolution. PMID:24489805

Spatial and temporal transcriptome changes occurring during flower opening and senescence of the ephemeral hibiscus flower, Hibiscus rosa-sinensis

PubMed Central

Trivellini, Alice; Cocetta, Giacomo; Hunter, Donald A.; Vernieri, Paolo; Ferrante, Antonio

2016-01-01

Flowers are complex systems whose vegetative and sexual structures initiate and die in a synchronous manner. The rapidity of this process varies widely in flowers, with some lasting for months while others such as Hibiscus rosa-sinensis survive for only a day. The genetic regulation underlying these differences is unclear. To identify key genes and pathways that coordinate floral organ senescence of ephemeral flowers, we identified transcripts in H. rosa-sinensis floral organs by 454 sequencing. During development, 2053 transcripts increased and 2135 decreased significantly in abundance. The senescence of the flower was associated with increased abundance of many hydrolytic genes, including aspartic and cysteine proteases, vacuolar processing enzymes, and nucleases. Pathway analysis suggested that transcripts altering significantly in abundance were enriched in functions related to cell wall-, aquaporin-, light/circadian clock-, autophagy-, and calcium-related genes. Finding enrichment in light/circadian clock-related genes fits well with the observation that hibiscus floral development is highly synchronized with light and the hypothesis that ageing/senescence of the flower is orchestrated by a molecular clock. Further study of these genes will provide novel insight into how the molecular clock is able to regulate the timing of programmed cell death in tissues. PMID:27591432

Staying alive in adversity: transcriptome dynamics in the stress-resistant dauer larva.

PubMed

Holt, Suzan J

2006-10-01

In response to food depletion and overcrowding, the soil nematode Caenorhabditis elegans can arrest development and form an alternate third larval stage called the dauer. Though nonfeeding, the dauer larva is long lived and stress resistant. Metabolic and transcription rates are lowered but the transcriptome of the dauer is complex. In this study, distribution analysis of transcript profiles generated by Serial Analysis of Gene Expression (SAGE) in dauer larvae and in mixed developmental stages is presented. An inverse relationship was observed between frequency and abundance/copy number of SAGE tag types (transcripts) in both profiles. In the dauer profile, a relatively greater proportion of highly abundant transcripts was counterbalanced by a smaller fraction of low to moderately abundant transcripts. Comparisons of abundant tag counts between the two profiles revealed relative enrichment in the dauer profile of transcripts with predicted or known involvement in ribosome biogenesis and protein synthesis, membrane transport, and immune responses. Translation-coupled mRNA decay is proposed as part of an immune-like stress response in the dauer larva. An influence of genomic region on transcript level may reflect the coordination of transcription and mRNA turnover.

Transcriptional Control in Marine Copiotrophic and Oligotrophic Bacteria with Streamlined Genomes.

PubMed

Cottrell, Matthew T; Kirchman, David L

2016-10-01

Bacteria often respond to environmental stimuli using transcriptional control, but this may not be the case for marine bacteria such as "Candidatus Pelagibacter ubique," a cultivated representative of the SAR11 clade, the most abundant organism in the ocean. This bacterium has a small, streamlined genome and an unusually low number of transcriptional regulators, suggesting that transcriptional control is low in Pelagibacter and limits its response to environmental conditions. Transcriptome sequencing during batch culture growth revealed that only 0.1% of protein-encoding genes appear to be under transcriptional control in Pelagibacter and in another oligotroph (SAR92) whereas >10% of genes were under transcriptional control in the copiotrophs Polaribacter sp. strain MED152 and Ruegeria pomeroyi When growth levels changed, transcript levels remained steady in Pelagibacter and SAR92 but shifted in MED152 and R. pomeroyi Transcript abundances per cell, determined using an internal RNA sequencing standard, were low (<1 transcript per cell) for all but a few of the most highly transcribed genes in all four taxa, and there was no correlation between transcript abundances per cell and shifts in the levels of transcription. These results suggest that low transcriptional control contributes to the success of Pelagibacter and possibly other oligotrophic microbes that dominate microbial communities in the oceans. Diverse heterotrophic bacteria drive biogeochemical cycling in the ocean. The most abundant types of marine bacteria are oligotrophs with small, streamlined genomes. The metabolic controls that regulate the response of oligotrophic bacteria to environmental conditions remain unclear. Our results reveal that transcriptional control is lower in marine oligotrophic bacteria than in marine copiotrophic bacteria. Although responses of bacteria to environmental conditions are commonly regulated at the level of transcription, metabolism in the most abundant bacteria in the ocean appears to be regulated by other mechanisms. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Claudin-31 contributes to corticosteroid-induced alterations in the barrier properties of the gill epithelium.

PubMed

Kolosov, Dennis; Donini, Andrew; Kelly, Scott P

2017-01-05

The contribution of Claudin-31 (Cldn-31) to corticosteroid-induced tightening of the trout gill epithelium was examined using a primary cultured model preparation. Cldn-31 is a ∼23 kDa protein that localizes to the periphery of gill epithelial cells and diffusely in select gill cells that are Na + -K + -ATPase-immunoreactive. Transcriptional knockdown (KD) of cldn-31 reduced Cldn-31 abundance and increased epithelium permeability. Under simulated in vivo conditions (apical freshwater), cldn-31 KD increased net ion flux rates (≡ efflux). Cortisol treatment increased Cldn-31 abundance and decreased epithelium permeability. This tightening effect was diminished, but not eliminated, by cldn-31 KD, most likely due to other cortisol-sensitive TJ proteins that were transcriptionally unperturbed or enhanced in cortisol-treated cldn-31 KD preparations. However, cldn-31 KD abolished a cortisol-induced increase in Cldn-8d abundance, which may contribute to compromised cldn-31 KD epithelium permeability. Data suggest an important barrier function for Cldn-31 and an integral role for Cldn-31 in corticosteroid-induced gill epithelium tightening. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Transcriptional Blood Signatures Distinguish Pulmonary Tuberculosis, Pulmonary Sarcoidosis, Pneumonias and Lung Cancers

PubMed Central

Bloom, Chloe I.; Graham, Christine M.; Berry, Matthew P. R.; Rozakeas, Fotini; Redford, Paul S.; Wang, Yuanyuan; Xu, Zhaohui; Wilkinson, Katalin A.; Wilkinson, Robert J.; Kendrick, Yvonne; Devouassoux, Gilles; Ferry, Tristan; Miyara, Makoto; Bouvry, Diane; Dominique, Valeyre; Gorochov, Guy; Blankenship, Derek; Saadatian, Mitra; Vanhems, Phillip; Beynon, Huw; Vancheeswaran, Rama; Wickremasinghe, Melissa; Chaussabel, Damien; Banchereau, Jacques; Pascual, Virginia; Ho, Ling-pei; Lipman, Marc; O’Garra, Anne

2013-01-01

Rationale New approaches to define factors underlying the immunopathogenesis of pulmonary diseases including sarcoidosis and tuberculosis are needed to develop new treatments and biomarkers. Comparing the blood transcriptional response of tuberculosis to other similar pulmonary diseases will advance knowledge of disease pathways and help distinguish diseases with similar clinical presentations. Objectives To determine the factors underlying the immunopathogenesis of the granulomatous diseases, sarcoidosis and tuberculosis, by comparing the blood transcriptional responses in these and other pulmonary diseases. Methods We compared whole blood genome-wide transcriptional profiles in pulmonary sarcoidosis, pulmonary tuberculosis, to community acquired pneumonia and primary lung cancer and healthy controls, before and after treatment, and in purified leucocyte populations. Measurements and Main Results An Interferon-inducible neutrophil-driven blood transcriptional signature was present in both sarcoidosis and tuberculosis, with a higher abundance and expression in tuberculosis. Heterogeneity of the sarcoidosis signature correlated significantly with disease activity. Transcriptional profiles in pneumonia and lung cancer revealed an over-abundance of inflammatory transcripts. After successful treatment the transcriptional activity in tuberculosis and pneumonia patients was significantly reduced. However the glucocorticoid-responsive sarcoidosis patients showed a significant increase in transcriptional activity. 144-blood transcripts were able to distinguish tuberculosis from other lung diseases and controls. Conclusions Tuberculosis and sarcoidosis revealed similar blood transcriptional profiles, dominated by interferon-inducible transcripts, while pneumonia and lung cancer showed distinct signatures, dominated by inflammatory genes. There were also significant differences between tuberculosis and sarcoidosis in the degree of their transcriptional activity, the heterogeneity of their profiles and their transcriptional response to treatment. PMID:23940611

Drosophila mitochondrial transcription factor B1 modulates mitochondrial translation but not transcription or DNA copy number in Schneider cells.

PubMed

Matsushima, Yuichi; Adán, Cristina; Garesse, Rafael; Kaguni, Laurie S

2005-04-29

We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor (d-mtTF) B1. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB1 to 5% of its normal level in Schneider cells. In striking contrast with our previous study on d-mtTFB2, we found that RNAi knock-down of d-mtTFB1 does not change the abundance of specific mitochondrial RNA transcripts, nor does it affect the copy number of mitochondrial DNA. In a corollary manner, overexpression of d-mtTFB1 did not increase either the abundance of mitochondrial RNA transcripts or mitochondrial DNA copy number. Our data suggest that, unlike d-mtTFB2, d-mtTFB1 does not have a critical role in either transcription or regulation of the copy number of mitochondrial DNA. Instead, because we found that RNAi knockdown of d-mtTFB1 reduces mitochondrial protein synthesis, we propose that it serves its primary role in modulating translation. Our work represents the first study to document the role of mtTFB1 in vivo and establishes clearly functional differences between mtTFB1 and mtTFB2.

DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis

PubMed Central

Zhong, Xuehua; Hale, Christopher J.; Nguyen, Minh; Ausin, Israel; Groth, Martin; Hetzel, Jonathan; Vashisht, Ajay A.; Henderson, Ian R.; Wohlschlegel, James A.; Jacobsen, Steven E.

2015-01-01

DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation. PMID:25561521

Integrative analysis with ChIP-seq advances the limits of transcript quantification from RNA-seq

PubMed Central

Liu, Peng; Sanalkumar, Rajendran; Bresnick, Emery H.; Keleş, Sündüz; Dewey, Colin N.

2016-01-01

RNA-seq is currently the technology of choice for global measurement of transcript abundances in cells. Despite its successes, isoform-level quantification remains difficult because short RNA-seq reads are often compatible with multiple alternatively spliced isoforms. Existing methods rely heavily on uniquely mapping reads, which are not available for numerous isoforms that lack regions of unique sequence. To improve quantification accuracy in such difficult cases, we developed a novel computational method, prior-enhanced RSEM (pRSEM), which uses a complementary data type in addition to RNA-seq data. We found that ChIP-seq data of RNA polymerase II and histone modifications were particularly informative in this approach. In qRT-PCR validations, pRSEM was shown to be superior than competing methods in estimating relative isoform abundances within or across conditions. Data-driven simulations suggested that pRSEM has a greatly decreased false-positive rate at the expense of a small increase in false-negative rate. In aggregate, our study demonstrates that pRSEM transforms existing capacity to precisely estimate transcript abundances, especially at the isoform level. PMID:27405803

Community structure of the metabolically active rumen bacterial and archaeal communities of dairy cows over the transition period

PubMed Central

Zhu, Zhigang; Noel, Samantha Joan; Difford, Gareth Frank; Al-Soud, Waleed Abu; Brejnrod, Asker; Sørensen, Søren Johannes; Lassen, Jan; Løvendahl, Peter; Højberg, Ole

2017-01-01

Dairy cows experience dramatic changes in host physiology from gestation to lactation period and dietary switch from high-forage prepartum diet to high-concentrate postpartum diet over the transition period (parturition +/- three weeks). Understanding the community structure and activity of the rumen microbiota and its associative patterns over the transition period may provide insight for e.g. improving animal health and production. In the present study, rumen samples from ten primiparous Holstein dairy cows were collected over seven weeks spanning the transition period. Total RNA was extracted from the rumen samples and cDNA thereof was subsequently used for characterizing the metabolically active bacterial (16S rRNA transcript amplicon sequencing) and archaeal (qPCR, T-RFLP and mcrA and 16S rRNA transcript amplicon sequencing) communities. The metabolically active bacterial community was dominated by three phyla, showing significant changes in relative abundance range over the transition period: Firmicutes (from prepartum 57% to postpartum 35%), Bacteroidetes (from prepartum 22% to postpartum 18%) and Proteobacteria (from prepartum 7% to postpartum 32%). For the archaea, qPCR analysis of 16S rRNA transcript number, revealed a significant prepartum to postpartum increase in Methanobacteriales, in accordance with an observed increase (from prepartum 80% to postpartum 89%) in relative abundance of 16S rRNA transcript amplicons allocated to this order. On the other hand, a significant prepartum to postpartum decrease (from 15% to 2%) was observed in relative abundance of Methanomassiliicoccales 16S rRNA transcripts. In contrast to qPCR analysis of the 16S rRNA transcripts, quantification of mcrA transcripts revealed no change in total abundance of metabolically active methanogens over the transition period. According to T-RFLP analysis of the mcrA transcripts, two Methanobacteriales genera, Methanobrevibacter and Methanosphaera (represented by the T-RFs 39 and 267 bp), represented more than 70% of the metabolically active methanogens, showing no significant changes over the transition period; minor T-RFs, likely to represent members of the order Methanomassiliicoccales and with a relative abundance below 5% in total, decreased significantly over the transition period. In accordance with the T-RFLP analysis, the mcrA transcript amplicon sequencing revealed Methanobacteriales to cover 99% of the total reads, dominated by the genera Methanobrevibacter (75%) and Methanosphaera (24%), whereas the Methanomassiliicoccales order covered only 0.2% of the total reads. In conclusion, the present study showed that the structure of the metabolically active bacterial and archaeal rumen communities changed over the transition period, likely in response to the dramatic changes in physiology and nutritional factors like dry matter intake and feed composition. It should be noted however that for the methanogens, the observed community changes were influenced by the analyzed gene (mcrA or 16S rRNA). PMID:29117259

Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular.

PubMed

Izuogu, Osagie G; Alhasan, Abd A; Mellough, Carla; Collin, Joseph; Gallon, Richard; Hyslop, Jonathon; Mastrorosa, Francesco K; Ehrmann, Ingrid; Lako, Majlinda; Elliott, David J; Santibanez-Koref, Mauro; Jackson, Michael S

2018-04-20

Circular RNAs (circRNAs) are predominantly derived from protein coding genes, and some can act as microRNA sponges or transcriptional regulators. Changes in circRNA levels have been identified during human development which may be functionally important, but lineage-specific analyses are currently lacking. To address this, we performed RNAseq analysis of human embryonic stem (ES) cells differentiated for 90 days towards 3D laminated retina. A transcriptome-wide increase in circRNA expression, size, and exon count was observed, with circRNA levels reaching a plateau by day 45. Parallel statistical analyses, controlling for sample and locus specific effects, identified 239 circRNAs with expression changes distinct from the transcriptome-wide pattern, but these all also increased in abundance over time. Surprisingly, circRNAs derived from long non-coding RNAs (lncRNAs) were found to account for a significantly larger proportion of transcripts from their loci of origin than circRNAs from coding genes. The most abundant, circRMST:E12-E6, showed a > 100X increase during differentiation accompanied by an isoform switch, and accounts for > 99% of RMST transcripts in many adult tissues. The second most abundant, circFIRRE:E10-E5, accounts for > 98% of FIRRE transcripts in differentiating human ES cells, and is one of 39 FIRRE circRNAs, many of which include multiple unannotated exons. Our results suggest that during human ES cell differentiation, changes in circRNA levels are primarily globally controlled. They also suggest that RMST and FIRRE, genes with established roles in neurogenesis and topological organisation of chromosomal domains respectively, are processed as circular lncRNAs with only minor linear species.

Post-transcriptional Mechanisms Contribute Little to Phenotypic Variation in Snake Venoms.

PubMed

Rokyta, Darin R; Margres, Mark J; Calvin, Kate

2015-09-09

Protein expression is a major link in the genotype-phenotype relationship, and processes affecting protein abundances, such as rates of transcription and translation, could contribute to phenotypic evolution if they generate heritable variation. Recent work has suggested that mRNA abundances do not accurately predict final protein abundances, which would imply that post-transcriptional regulatory processes contribute significantly to phenotypes. Post-transcriptional processes also appear to buffer changes in transcriptional patterns as species diverge, suggesting that the transcriptional changes have little or no effect on the phenotypes undergoing study. We tested for concordance between mRNA and protein expression levels in snake venoms by means of mRNA-seq and quantitative mass spectrometry for 11 snakes representing 10 species, six genera, and three families. In contrast to most previous work, we found high correlations between venom gland transcriptomes and venom proteomes for 10 of our 11 comparisons. We tested for protein-level buffering of transcriptional changes during species divergence by comparing the difference between transcript abundance and protein abundance for three pairs of species and one intraspecific pair. We found no evidence for buffering during divergence of our three species pairs but did find evidence for protein-level buffering for our single intraspecific comparison, suggesting that buffering, if present, was a transient phenomenon in venom divergence. Our results demonstrated that post-transcriptional mechanisms did not contribute significantly to phenotypic evolution in venoms and suggest a more prominent and direct role for cis-regulatory evolution in phenotypic variation, particularly for snake venoms. Copyright © 2015 Rokyta et al.

A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia.

PubMed

Yin, Jing; Chang, Xiaoxiao; Kasuga, Takao; Bui, Mai; Reid, Michael S; Jiang, Cai-Zhong

2015-01-01

The basic helix-loop-helix (bHLH) transcription factors (TFs) play important roles in regulating multiple biological processes in plants. However, there are few reports about the function of bHLHs in flower senescence. In this study, a bHLH TF, PhFBH4, was found to be dramatically upregulated during flower senescence. Transcription of PhFBH4 is induced by plant hormones and abiotic stress treatments. Silencing of PhFBH4 using virus-induced gene silencing or an antisense approach extended flower longevity, while transgenic petunia flowers with an overexpression construct showed a reduction in flower lifespan. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was significantly changed in petunia PhFBH4 transgenic flowers. Furthermore, silencing or overexpression of PhFBH4 reduced or increased, respectively, transcript abundances of important ethylene biosynthesis-related genes, ACS1 and ACO1, thereby influencing ethylene production. An electrophoretic mobility shift assay showed that the PhFBH4 protein physically interacted with the G-box cis-element in the promoter of ACS1, suggesting that ACS1 was a direct target of the PhFBH4 protein. In addition, ectopic expression of this gene altered plant development including plant height, internode length, and size of leaves and flowers, accompanied by alteration of transcript abundance of the gibberellin biosynthesis-related gene GA2OX3. Our results indicate that PhFBH4 plays an important role in regulating plant growth and development through modulating the ethylene biosynthesis pathway.

Effect of postharvest ethylene treatment on sugar content, glycosidase activity and its gene expression in mango fruit.

PubMed

Chidley, Hemangi G; Deshpande, Ashish B; Oak, Pranjali S; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

2017-03-01

Ripening-associated softening is one of the important attributes that largely determines the shelf-life of mango (Mangifera indica Linn.) fruits. To reveal the effect of pre-climacteric ethylene treatment on ripening-related softening of Alphonso mango, ethylene treatment was given to mature, raw Alphonso fruits. Changes in the pool of reducing and non-reducing sugars, enzymatic activity of three glycosidases: β-d-galactosidase, α-d-mannosidase and β-d-glucosidase and their relative transcript abundance were analysed for control and ethylene treated fruits during ripening. Early activity of all the three glycosidases and accelerated accumulation of reducing and non-reducing sugars on ethylene treatment was evident. β-d-Galactosidase showed the highest activity among three glycosidases in control fruits and marked increase in activity upon ethylene treatment. This was confirmed by the histochemical assay of its activity in control and ethylene treated ripe fruits. Relative transcript abundance revealed high transcript levels of β-d-galactosidase in control fruits. Ethylene-treated fruits showed early and remarkable increase in the β-d-galactosidase transcripts while α-d-mannosidase transcript variants displayed early accumulation. The findings suggest reduction in the shelf-life of Alphonso mango upon pre-climacteric ethylene treatment, a significant role of β-d-galactosidase and α-d-mannosidase in the ripening related softening of Alphonso fruits and transcriptional regulation of their expression by ethylene. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Inflammatory Stimuli Increase Progesterone Receptor-A Stability and Transrepressive Activity in Myometrial Cells

PubMed Central

Peters, Gregory A.; Yi, Lijuan; Skomorovska-Prokvolit, Yelenna; Patel, Bansari; Amini, Peyvand; Tan, Huiqing

2017-01-01

The steroid hormone progesterone acting via the nuclear progesterone receptor (PR) isoforms, progesterone receptor A (PR-A) and progesterone receptor B (PR-B), is essential for the maintenance of uterine quiescence during pregnancy. Inhibition of PR signaling augments uterine contractility and induces labor. Human parturition is thought to be triggered by modulation of PR signaling in myometrial cells to induce a functional progesterone withdrawal. One mechanism for functional progesterone withdrawal is increased abundance of PR-A, which decreases progesterone responsiveness by inhibiting the transcriptional activity of PR-B. Human parturition also involves tissue-level inflammation within the myometrium. This study examined the control of PR-A abundance and transrepressive activity in myometrial cells and the role of the inflammatory stimuli in the form of interleukin-1β (IL-1β) and lipopolysaccharide (LPS) in these processes. We found that abundance of PR-A was markedly increased by progesterone and by exposure to IL-1β and LPS via posttranslational mechanisms involving increased PR-A protein stability. In contrast, progesterone decreased abundance of PR-B by increasing its rate of degradation. Together, progesterone and proinflammatory stimuli induced a PR-A–dominant state in myometrial cells similar to that observed in term laboring myometrium. IL-1β and LPS also increased the capacity for PR-A to inhibit the transcriptional activity of PR-B. Taken together, our data suggest that proinflammatory stimuli increase the steady-state levels of PR-A and its transrepressive activity in myometrial cells and support the hypothesis that tissue-level inflammation triggers parturition by inducing PR-A–mediated functional progesterone withdrawal. PMID:27886516

Aquaporin-2 Regulation in Health and Disease

PubMed Central

Radin, M. Judith; Yu, Ming-Jiun; Stoedkilde, Lene; Miller, R. Lance; Hoffert, Jason D.; Frokiaer, Jorgen; Pisitkun, Trairak; Knepper, Mark A.

2012-01-01

Aquaporin-2 (AQP2), the vasopressin-regulated water channel of the renal collecting duct, is dysregulated in numerous water balance disorders in humans and animals including those associated with polyuria (e.g. urinary tract obstruction, hypokalemia, inflammation, and lithium toxicity) and dilutional hyponatremia (e.g. SIADH). Normal regulation of AQP2 by vasopressin involves two independent regulatory mechanisms: 1) short-term regulation of AQP2 trafficking to and from the apical plasma membrane, and 2) long term regulation of the total abundance of the AQP2 protein in the cells. Most water balance disorders are the result of dysregulation of processes that regulate the total abundance of AQP2 in collecting duct cells. In general, the level of AQP2 in a collecting duct cell is determined by a balance between production via translation of AQP2 mRNA and removal via degradation and/or secretion into the urine in exosomes. AQP2 abundance increases in response to vasopressin chiefly due to increased translation subsequent to increases in AQP2 mRNA. Vasopressin-mediated regulation AQP2 gene transcription is poorly understood, although several transcription factor binding elements in the 5’ flanking region of the AQP2 gene have been identified and candidate transcription factors corresponding to these element have been discovered in proteomics studies. Here we review progress in this area and discuss elements of vasopressin signaling in the collecting duct that may impinge on regulation of AQP2 in health and in the context of examples of polyuric diseases. PMID:23130944

Molecular Analysis of the In Situ Growth Rates of Subsurface Geobacter Species

PubMed Central

Giloteaux, Ludovic; Barlett, Melissa; Chavan, Milind A.; Smith, Jessica A.; Williams, Kenneth H.; Wilkins, Michael; Long, Philip; Lovley, Derek R.

2013-01-01

Molecular tools that can provide an estimate of the in situ growth rate of Geobacter species could improve understanding of dissimilatory metal reduction in a diversity of environments. Whole-genome microarray analyses of a subsurface isolate of Geobacter uraniireducens, grown under a variety of conditions, identified a number of genes that are differentially expressed at different specific growth rates. Expression of two genes encoding ribosomal proteins, rpsC and rplL, was further evaluated with quantitative reverse transcription-PCR (qRT-PCR) in cells with doubling times ranging from 6.56 h to 89.28 h. Transcript abundance of rpsC correlated best (r2 = 0.90) with specific growth rates. Therefore, expression patterns of rpsC were used to estimate specific growth rates of Geobacter species during an in situ uranium bioremediation field experiment in which acetate was added to the groundwater to promote dissimilatory metal reduction. Initially, increased availability of acetate in the groundwater resulted in higher expression of Geobacter rpsC, and the increase in the number of Geobacter cells estimated with fluorescent in situ hybridization compared well with specific growth rates estimated from levels of in situ rpsC expression. However, in later phases, cell number increases were substantially lower than predicted from rpsC transcript abundance. This change coincided with a bloom of protozoa and increased attachment of Geobacter species to solid phases. These results suggest that monitoring rpsC expression may better reflect the actual rate that Geobacter species are metabolizing and growing during in situ uranium bioremediation than changes in cell abundance. PMID:23275510

Epigenetics regulates transcription and pathogenesis in the parasite Trichomonas vaginalis.

PubMed

Pachano, Tomas; Nievas, Yesica R; Lizarraga, Ayelen; Johnson, Patricia J; Strobl-Mazzulla, Pablo H; de Miguel, Natalia

2017-06-01

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Different T. vaginalis strains vary greatly in their adherence and cytolytic capacities. These phenotypic differences might be attributed to differentially expressed genes as a consequence of extra-genetic variation, such as epigenetic modifications. In this study, we explored the role of histone acetylation in regulating gene transcription and pathogenesis in T. vaginalis. Here, we show that histone 3 lysine acetylation (H3KAc) is enriched in nucleosomes positioned around the transcription start site of active genes (BAP1 and BAP2) in a highly adherent parasite strain; compared with the low acetylation abundance in contrast to that observed in a less-adherent strain that expresses these genes at low levels. Additionally, exposition of less-adherent strain with a specific histone deacetylases inhibitor, trichostatin A, upregulated the transcription of BAP1 and BAP2 genes in concomitance with an increase in H3KAc abundance and chromatin accessibility around their transcription start sites. Moreover, we demonstrated that the binding of initiator binding protein, the transcription factor responsible for the initiation of transcription of ~75% of known T. vaginalis genes, depends on the histone acetylation state around the metazoan-like initiator to which initiator binding protein binds. Finally, we found that trichostatin A treatment increased parasite aggregation and adherence to host cells. Our data demonstrated for the first time that H3KAc is a permissive histone modification that functions to mediate both transcription and pathogenesis of the parasite T. vaginalis. © 2017 John Wiley & Sons Ltd.

Temperature gradient affects differentiation of gene expression and SNP allele frequencies in the dominant Lake Baikal zooplankton species.

PubMed

Bowman, Larry L; Kondrateva, Elizaveta S; Timofeyev, Maxim A; Yampolsky, Lev Y

2018-06-01

Local adaptation and phenotypic plasticity are main mechanisms of organisms' resilience in changing environments. Both are affected by gene flow and are expected to be weak in zooplankton populations inhabiting large continuous water bodies and strongly affected by currents. Lake Baikal, the deepest and one of the coldest lakes on Earth, experienced epilimnion temperature increase during the last 100 years, exposing Baikal's zooplankton to novel selective pressures. We obtained a partial transcriptome of Epischura baikalensis (Copepoda: Calanoida), the dominant component of Baikal's zooplankton, and estimated SNP allele frequencies and transcript abundances in samples from regions of Baikal that differ in multiyear average surface temperatures. The strongest signal in both SNP and transcript abundance differentiation is the SW-NE gradient along the 600+ km long axis of the lake, suggesting isolation by distance. SNP differentiation is stronger for nonsynonymous than synonymous SNPs and is paralleled by differential survival during a laboratory exposure to increased temperature, indicating directional selection operating on the temperature gradient. Transcript abundance, generally collinear with the SNP differentiation, shows samples from the warmest, less deep location clustering together with the southernmost samples. Differential expression is more frequent among transcripts orthologous to candidate thermal response genes previously identified in model arthropods, including genes encoding cytoskeleton proteins, heat-shock proteins, proteases, enzymes of central energy metabolism, lipid and antioxidant pathways. We conclude that the pivotal endemic zooplankton species in Lake Baikal exists under temperature-mediated selection and possesses both genetic variation and plasticity to respond to novel temperature-related environmental pressures. © 2018 John Wiley & Sons Ltd.

The metatranscriptome of a deep-sea hydrothermal plume is dominated by water column methanotrophs and lithotrophs

PubMed Central

Lesniewski, Ryan A; Jain, Sunit; Anantharaman, Karthik; Schloss, Patrick D; Dick, Gregory J

2012-01-01

Microorganisms mediate geochemical processes in deep-sea hydrothermal vent plumes, which are a conduit for transfer of elements and energy from the subsurface to the oceans. Despite this important microbial influence on marine geochemistry, the ecology and activity of microbial communities in hydrothermal plumes is largely unexplored. Here, we use a coordinated metagenomic and metatranscriptomic approach to compare microbial communities in Guaymas Basin hydrothermal plumes to background waters above the plume and in the adjacent Carmen Basin. Despite marked increases in plume total RNA concentrations (3–4 times) and microbially mediated manganese oxidation rates (15–125 times), plume and background metatranscriptomes were dominated by the same groups of methanotrophs and chemolithoautotrophs. Abundant community members of Guaymas Basin seafloor environments (hydrothermal sediments and chimneys) were not prevalent in the plume metatranscriptome. De novo metagenomic assembly was used to reconstruct genomes of abundant populations, including Marine Group I archaea, Methylococcaceae, SAR324 Deltaproteobacteria and SUP05 Gammaproteobacteria. Mapping transcripts to these genomes revealed abundant expression of genes involved in the chemolithotrophic oxidation of ammonia (amo), methane (pmo) and sulfur (sox). Whereas amo and pmo gene transcripts were abundant in both plume and background, transcripts of sox genes for sulfur oxidation from SUP05 groups displayed a 10–20-fold increase in plumes. We conclude that the biogeochemistry of Guaymas Basin hydrothermal plumes is mediated by microorganisms that are derived from seawater rather than from seafloor hydrothermal environments such as chimneys or sediments, and that hydrothermal inputs serve as important electron donors for primary production in the deep Gulf of California. PMID:22695860

The metatranscriptome of a deep-sea hydrothermal plume is dominated by water column methanotrophs and lithotrophs.

PubMed

Lesniewski, Ryan A; Jain, Sunit; Anantharaman, Karthik; Schloss, Patrick D; Dick, Gregory J

2012-12-01

Microorganisms mediate geochemical processes in deep-sea hydrothermal vent plumes, which are a conduit for transfer of elements and energy from the subsurface to the oceans. Despite this important microbial influence on marine geochemistry, the ecology and activity of microbial communities in hydrothermal plumes is largely unexplored. Here, we use a coordinated metagenomic and metatranscriptomic approach to compare microbial communities in Guaymas Basin hydrothermal plumes to background waters above the plume and in the adjacent Carmen Basin. Despite marked increases in plume total RNA concentrations (3-4 times) and microbially mediated manganese oxidation rates (15-125 times), plume and background metatranscriptomes were dominated by the same groups of methanotrophs and chemolithoautotrophs. Abundant community members of Guaymas Basin seafloor environments (hydrothermal sediments and chimneys) were not prevalent in the plume metatranscriptome. De novo metagenomic assembly was used to reconstruct genomes of abundant populations, including Marine Group I archaea, Methylococcaceae, SAR324 Deltaproteobacteria and SUP05 Gammaproteobacteria. Mapping transcripts to these genomes revealed abundant expression of genes involved in the chemolithotrophic oxidation of ammonia (amo), methane (pmo) and sulfur (sox). Whereas amo and pmo gene transcripts were abundant in both plume and background, transcripts of sox genes for sulfur oxidation from SUP05 groups displayed a 10-20-fold increase in plumes. We conclude that the biogeochemistry of Guaymas Basin hydrothermal plumes is mediated by microorganisms that are derived from seawater rather than from seafloor hydrothermal environments such as chimneys or sediments, and that hydrothermal inputs serve as important electron donors for primary production in the deep Gulf of California.

A microRNA feedback loop regulates global microRNA abundance during aging.

PubMed

Inukai, Sachi; Pincus, Zachary; de Lencastre, Alexandre; Slack, Frank J

2018-02-01

Expression levels of many microRNAs (miRNAs) change during aging, notably declining globally in a number of organisms and tissues across taxa. However, little is known about the mechanisms or the biological relevance for this change. We investigated the network of genes that controls miRNA transcription and processing during C. elegans aging. We found that miRNA biogenesis genes are highly networked with transcription factors and aging-associated miRNAs. In particular, miR-71, known to influence life span and itself up-regulated during aging, represses alg-1 /Argonaute expression post-transcriptionally during aging. Increased ALG-1 abundance in mir-71 loss-of-function mutants led to globally increased miRNA expression. Interestingly, these mutants demonstrated widespread mRNA expression dysregulation and diminished levels of variability both in gene expression and in overall life span. Thus, the progressive molecular decline often thought to be the result of accumulated damage over an organism's life may be partially explained by a miRNA-directed mechanism of age-associated decline. © 2018 Inukai et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Genomic Regulation of the Response of an Agroecosystem to Elements of Global Change

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeLucia, Evan, H.

This document outlines some of the major accomplishments from this project: (1) New tools for analyzing and visualizing microarray data from soybean gene expression experiments; (2) Physiological, biochemical, and gene array evidence that acclimation of carbon metabolism to elevated CO{sub 2} is governed in significant part by changes in gene expression associated with respiratory metabolism; (3) Increased carbon assimilation in soybeans grown at elevated CO{sub 2} altered pools of carbohydrates and transcripts that control growth and expansion of young leaves; (4) Growth at elevated CO{sub 2} increases the abundance of transcripts controlling cell wall polysaccharide synthesis but not transcripts controllingmore » lignin synthesis; (5) The total antioxidant capacity of soybeans varies among cultivars and in response to atmospheric change; (6) Accelerated leaf senescence at elevated O{sub 3} coincides with reduced abundance of transcripts controlling protein synthesis; (7) Growth under elevated CO{sub 2} increases the susceptibility of soybean to insect herbivores by increasing insect lifespan and fecundity through altered leaf chemistry and by defeating molecular induction of plant defenses; (8) Exposure to elevated CO{sub 2} and O{sub 3} alters flavonoid metabolism in soybean; (9) Exposure to elevated CO{sub 2} or O{sub 3} conferred resistance to soybean mosaic virus by cross inducing defense- and stress-related signaling pathways; and (10) Exposure to elevated CO{sub 2} accelerates decomposition by changing chemical and biotic properties of the soil.« less

A genome-wide survey of maternal and embryonic transcripts during Xenopus tropicalis development.

PubMed

Paranjpe, Sarita S; Jacobi, Ulrike G; van Heeringen, Simon J; Veenstra, Gert Jan C

2013-11-06

Dynamics of polyadenylation vs. deadenylation determine the fate of several developmentally regulated genes. Decay of a subset of maternal mRNAs and new transcription define the maternal-to-zygotic transition, but the full complement of polyadenylated and deadenylated coding and non-coding transcripts has not yet been assessed in Xenopus embryos. To analyze the dynamics and diversity of coding and non-coding transcripts during development, both polyadenylated mRNA and ribosomal RNA-depleted total RNA were harvested across six developmental stages and subjected to high throughput sequencing. The maternally loaded transcriptome is highly diverse and consists of both polyadenylated and deadenylated transcripts. Many maternal genes show peak expression in the oocyte and include genes which are known to be the key regulators of events like oocyte maturation and fertilization. Of all the transcripts that increase in abundance between early blastula and larval stages, about 30% of the embryonic genes are induced by fourfold or more by the late blastula stage and another 35% by late gastrulation. Using a gene model validation and discovery pipeline, we identified novel transcripts and putative long non-coding RNAs (lncRNA). These lncRNA transcripts were stringently selected as spliced transcripts generated from independent promoters, with limited coding potential and a codon bias characteristic of noncoding sequences. Many lncRNAs are conserved and expressed in a developmental stage-specific fashion. These data reveal dynamics of transcriptome polyadenylation and abundance and provides a high-confidence catalogue of novel and long non-coding RNAs.

Spatial and temporal transcriptome changes occurring during flower opening and senescence of the ephemeral hibiscus flower, Hibiscus rosa-sinensis.

PubMed

Trivellini, Alice; Cocetta, Giacomo; Hunter, Donald A; Vernieri, Paolo; Ferrante, Antonio

2016-10-01

Flowers are complex systems whose vegetative and sexual structures initiate and die in a synchronous manner. The rapidity of this process varies widely in flowers, with some lasting for months while others such as Hibiscus rosa-sinensis survive for only a day. The genetic regulation underlying these differences is unclear. To identify key genes and pathways that coordinate floral organ senescence of ephemeral flowers, we identified transcripts in H. rosa-sinensis floral organs by 454 sequencing. During development, 2053 transcripts increased and 2135 decreased significantly in abundance. The senescence of the flower was associated with increased abundance of many hydrolytic genes, including aspartic and cysteine proteases, vacuolar processing enzymes, and nucleases. Pathway analysis suggested that transcripts altering significantly in abundance were enriched in functions related to cell wall-, aquaporin-, light/circadian clock-, autophagy-, and calcium-related genes. Finding enrichment in light/circadian clock-related genes fits well with the observation that hibiscus floral development is highly synchronized with light and the hypothesis that ageing/senescence of the flower is orchestrated by a molecular clock. Further study of these genes will provide novel insight into how the molecular clock is able to regulate the timing of programmed cell death in tissues. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Development of intestinal ion-transporting mechanisms during smoltification and seawater acclimation in Atlantic salmon Salmo salar

USGS Publications Warehouse

Sundh, Henrik; Nilsen, Tom O.; Lindström, Jenny; Hasselberg-Frank, Linda; Stefansson, Sigurd O.; McCormick, Stephen D.; Sundell, K.

2014-01-01

This study investigated the expression of ion transporters involved in intestinal fluid absorption and presents evidence for developmental changes in abundance and tissue distribution of these transporters during smoltification and seawater (SW) acclimation of Atlantic salmonSalmo salar. Emphasis was placed on Na+, K+-ATPase (NKA) and Na+, K+, Cl− co-transporter (NKCC) isoforms, at both transcriptional and protein levels, together with transcription of chloride channel genes. The nka α1c was the dominant isoform at the transcript level in both proximal and distal intestines; also, it was the most abundant isoform expressed in the basolateral membrane of enterocytes in the proximal intestine. This isoform was also abundantly expressed in the distal intestine in the lower part of the mucosal folds. The protein expression of intestinal Nkaα1c increased during smoltification. Immunostaining was localized to the basal membrane of the enterocytes in freshwater (FW) fish, and re-distributed to a lateral position after SW entry. Two other Nka isoforms, α1a and α1b, were expressed in the intestine but were not regulated to the same extent during smoltification and subsequent SW transfer. Their localization in the intestinal wall indicates a house-keeping function in excitatory tissues. The absorptive form of the NKCC-like isoform (sub-apically located NKCC2 and/or Na+, Cl−co-transporter) increased during smoltification and further after SW transfer. The cellular distribution changed from a diffuse expression in the sub-apical regions during smoltification to clustering of the transporters closer to the apical membrane after entry to SW. Furthermore, transcript abundance indicates that the mechanisms necessary for exit of chloride ions across the basolateral membrane and into the lateral intercellular space are present in the form of one or more of three different chloride channels: cystic fibrosis transmembrane conductance regulator I and II and chloride channel 3.

Microarray Analyses and Comparisons of Upper or Lower Flanks of Rice Shoot Base Preceding Gravitropic Bending

PubMed Central

Zang, Aiping; Chen, Haiying; Dou, Xianying; Jin, Jing; Cai, Weiming

2013-01-01

Gravitropism is a complex process involving a series of physiological pathways. Despite ongoing research, gravitropism sensing and response mechanisms are not well understood. To identify the key transcripts and corresponding pathways in gravitropism, a whole-genome microarray approach was used to analyze transcript abundance in the shoot base of rice (Oryza sativa sp. japonica) at 0.5 h and 6 h after gravistimulation by horizontal reorientation. Between upper and lower flanks of the shoot base, 167 transcripts at 0.5 h and 1202 transcripts at 6 h were discovered to be significantly different in abundance by 2-fold. Among these transcripts, 48 were found to be changed both at 0.5 h and 6 h, while 119 transcripts were only changed at 0.5 h and 1154 transcripts were changed at 6 h in association with gravitropism. MapMan and PageMan analyses were used to identify transcripts significantly changed in abundance. The asymmetric regulation of transcripts related to phytohormones, signaling, RNA transcription, metabolism and cell wall-related categories between upper and lower flanks were demonstrated. Potential roles of the identified transcripts in gravitropism are discussed. Our results suggest that the induction of asymmetrical transcription, likely as a consequence of gravitropic reorientation, precedes gravitropic bending in the rice shoot base. PMID:24040303

Water deficit alters differentially metabolic pathways affecting important flavor and quality traits in grape berries of Cabernet Sauvignon and Chardonnay

PubMed Central

Deluc, Laurent G; Quilici, David R; Decendit, Alain; Grimplet, Jérôme; Wheatley, Matthew D; Schlauch, Karen A; Mérillon, Jean-Michel; Cushman, John C; Cramer, Grant R

2009-01-01

Background Water deficit has significant effects on grape berry composition resulting in improved wine quality by the enhancement of color, flavors, or aromas. While some pathways or enzymes affected by water deficit have been identified, little is known about the global effects of water deficit on grape berry metabolism. Results The effects of long-term, seasonal water deficit on berries of Cabernet Sauvignon, a red-wine grape, and Chardonnay, a white-wine grape were analyzed by integrated transcript and metabolite profiling. Over the course of berry development, the steady-state transcript abundance of approximately 6,000 Unigenes differed significantly between the cultivars and the irrigation treatments. Water deficit most affected the phenylpropanoid, ABA, isoprenoid, carotenoid, amino acid and fatty acid metabolic pathways. Targeted metabolites were profiled to confirm putative changes in specific metabolic pathways. Water deficit activated the expression of numerous transcripts associated with glutamate and proline biosynthesis and some committed steps of the phenylpropanoid pathway that increased anthocyanin concentrations in Cabernet Sauvignon. In Chardonnay, water deficit activated parts of the phenylpropanoid, energy, carotenoid and isoprenoid metabolic pathways that contribute to increased concentrations of antheraxanthin, flavonols and aroma volatiles. Water deficit affected the ABA metabolic pathway in both cultivars. Berry ABA concentrations were highly correlated with 9-cis-epoxycarotenoid dioxygenase (NCED1) transcript abundance, whereas the mRNA expression of other NCED genes and ABA catabolic and glycosylation processes were largely unaffected. Water deficit nearly doubled ABA concentrations within berries of Cabernet Sauvignon, whereas it decreased ABA in Chardonnay at véraison and shortly thereafter. Conclusion The metabolic responses of grapes to water deficit varied with the cultivar and fruit pigmentation. Chardonnay berries, which lack any significant anthocyanin content, exhibited increased photoprotection mechanisms under water deficit conditions. Water deficit increased ABA, proline, sugar and anthocyanin concentrations in Cabernet Sauvignon, but not Chardonnay berries, consistent with the hypothesis that ABA enhanced accumulation of these compounds. Water deficit increased the transcript abundance of lipoxygenase and hydroperoxide lyase in fatty metabolism, a pathway known to affect berry and wine aromas. These changes in metabolism have important impacts on berry flavor and quality characteristics. Several of these metabolites are known to contribute to increased human-health benefits. PMID:19426499

Integrative analysis with ChIP-seq advances the limits of transcript quantification from RNA-seq.

PubMed

Liu, Peng; Sanalkumar, Rajendran; Bresnick, Emery H; Keleş, Sündüz; Dewey, Colin N

2016-08-01

RNA-seq is currently the technology of choice for global measurement of transcript abundances in cells. Despite its successes, isoform-level quantification remains difficult because short RNA-seq reads are often compatible with multiple alternatively spliced isoforms. Existing methods rely heavily on uniquely mapping reads, which are not available for numerous isoforms that lack regions of unique sequence. To improve quantification accuracy in such difficult cases, we developed a novel computational method, prior-enhanced RSEM (pRSEM), which uses a complementary data type in addition to RNA-seq data. We found that ChIP-seq data of RNA polymerase II and histone modifications were particularly informative in this approach. In qRT-PCR validations, pRSEM was shown to be superior than competing methods in estimating relative isoform abundances within or across conditions. Data-driven simulations suggested that pRSEM has a greatly decreased false-positive rate at the expense of a small increase in false-negative rate. In aggregate, our study demonstrates that pRSEM transforms existing capacity to precisely estimate transcript abundances, especially at the isoform level. © 2016 Liu et al.; Published by Cold Spring Harbor Laboratory Press.

Differential response of ammonia-oxidizing archaea and bacteria to the wetting of salty arid soil.

PubMed

Sher, Yonatan; Ronen, Zeev; Nejidat, Ali

2016-08-01

Ammonia-oxidizing archaea and bacteria (AOA, AOB) catalyze the first and rate-limiting step of nitrification. To examine their differential responses to the wetting of dry and salty arid soil, AOA and AOB amoA genes (encoding subunit A of the ammonia monooxygenase) and transcripts were enumerated in dry (summer) and wet (after the first rainfall) soil under the canopy of halophytic shrubs and between the shrubs. AOA and AOB were more abundant under shrub canopies than between shrubs in both the dry and wetted soil. Soil wetting caused a significant decrease in AOB abundance under the canopy and an increase of AOA between the shrubs. The abundance of the archaeal amoA gene transcript was similar for both the wet and dry soil, and the transcript-to-gene ratios were < 1 independent of niche or water content. In contrast, the bacterial amoA transcript-to-gene ratios were between 78 and 514. The lowest ratio was in dry soil under the canopy and the highest in the soil between the shrubs. The results suggest that the AOA are more resilient to stress conditions and maintain a basic activity in arid ecosystems, while the AOB are more responsive to changes in the biotic and abiotic conditions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome-wide analysis on Chlamydomonas reinhardtii reveals the impact of hydrogen peroxide on protein stress responses and overlap with other stress transcriptomes

DOE PAGES

Blaby, Ian K.; Blaby-Haas, Crysten E.; Pérez-Pérez, María Esther; ...

2015-12-07

Reactive oxygen species (ROS) are produced by and have the potential to be damaging to all aerobic organisms. In photosynthetic organisms, they are an unavoidable byproduct of electron transfer in both the chloroplast and mitochondrion. Here, in this paper, we employ the reference unicellular green alga Chlamydomonas reinhardtii to identify the effect of H 2O 2 on gene expression by monitoring the changes in the transcriptome in a time-course experiment. Comparison of transcriptomes from cells sampled immediately prior to the addition of H 2O 2 and 0.5 and 1 h subsequently revealed 1278 differentially abundant transcripts. Of those transcripts thatmore » increase in abundance, many encode proteins involved in ROS detoxification, protein degradation and stress responses, whereas among those that decrease are transcripts encoding proteins involved in photosynthesis and central carbon metabolism. In addition to these transcriptomic adjustments, we observe that addition of H 2O 2 is followed by an accumulation and oxidation of the total intracellular glutathione pool, and a decrease in photosynthetic O 2 output. Additionally, we analyze our transcriptomes in the context of changes in transcript abundance in response to singlet O 2 (O 2 *), and relate our H 2O 2-induced transcripts to a diurnal transcriptome, where we demonstrate enrichments of H 2O 2-induced transcripts early in the light phase, late in the light phase and 2 h prior to light. In conclusion, on this basis several genes that are highlighted in this work may be involved in previously undiscovered stress remediation pathways or acclimation responses.« less

Rhizosphere heterogeneity shapes abundance and activity of sulfur-oxidizing bacteria in vegetated salt marsh sediments

PubMed Central

Thomas, François; Giblin, Anne E.; Cardon, Zoe G.; Sievert, Stefan M.

2014-01-01

Salt marshes are highly productive ecosystems hosting an intense sulfur (S) cycle, yet little is known about S-oxidizing microorganisms in these ecosystems. Here, we studied the diversity and transcriptional activity of S-oxidizers in salt marsh sediments colonized by the plant Spartina alterniflora, and assessed variations with sediment depth and small-scale compartments within the rhizosphere. We combined next-generation amplicon sequencing of 16S rDNA and rRNA libraries with phylogenetic analyses of marker genes for two S-oxidation pathways (soxB and rdsrAB). Gene and transcript numbers of soxB and rdsrAB phylotypes were quantified simultaneously, using newly designed (RT)-qPCR assays. We identified a diverse assemblage of S-oxidizers, with Chromatiales and Thiotrichales being dominant. The detection of transcripts from S-oxidizers was mostly confined to the upper 5 cm sediments, following the expected distribution of root biomass. A common pool of species dominated by Gammaproteobacteria transcribed S-oxidation genes across roots, rhizosphere, and surrounding sediment compartments, with rdsrAB transcripts prevailing over soxB. However, the root environment fine-tuned the abundance and transcriptional activity of the S-oxidizing community. In particular, the global transcription of soxB was higher on the roots compared to mix and rhizosphere samples. Furthermore, the contribution of Epsilonproteobacteria-related S-oxidizers tended to increase on Spartina roots compared to surrounding sediments. These data shed light on the under-studied oxidative part of the sulfur cycle in salt marsh sediments and indicate small-scale heterogeneities are important factors shaping abundance and potential activity of S-oxidizers in the rhizosphere. PMID:25009538

Altered Expression of Porcine Piwi Genes and piRNA during Development

PubMed Central

Kowalczykiewicz, Dorota; Pawlak, Piotr; Lechniak, Dorota; Wrzesinski, Jan

2012-01-01

Three Sus scrofa Piwi genes (Piwil1, Piwil2 and Piwil4) encoding proteins of 861, 985 and 853 aminoacids, respectively, were cloned and sequenced. Alignment of the Piwi proteins showed the high identity between Sus scrofa and Homo sapiens. Relative transcript abundance of porcine Piwil1, Piwil2 and Piwil4 genes in testes, ovaries and oocytes derived from sexually immature and mature animals was examined using Real-Time PCR. Expression of the three Piwi mRNAs was proved to be tissue specific and restricted exclusively to the gonads. In testes of adult pigs the highest relative transcript abundance was observed for the Sus scrofa Piwil1 gene. On the other hand, in testes of neonatal pigs the Piwil1 transcript level was over 2–fold reduced while the level of Piwil2 transcript was higher. As regards the expression of the Piwil4 transcript, its level was 34-fold elevated in testes of neonatal piglet when compared to adult male. In ovaries of prepubertal and pubertal female pigs transcript abundance of the three Piwi genes was significantly reduced in comparison with testes. However, similarly to testes, in ovaries of neonatal pigs the Piwil2 gene was characterized by the highest relative transcript abundance among the three Piwi genes analysed. In prepubertal and pubertal oocytes Piwil1 transcript was the most abundant whereas the expression of Piwil4 was undetectable. We also demonstrated that expression of piRNA occurs preferentially in the gonads of adult male and female pigs. Moreover, a piRNA subset isolated from ovaries was 2–3 nucleotides longer than the piRNA from testes. PMID:22952772

Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments.

PubMed

Yang, Bo; Jiang, Yuanqing; Rahman, Muhammad H; Deyholos, Michael K; Kav, Nat N V

2009-06-03

Members of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napus L.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship to Arabidopsis thaliana, we sought to characterize a subset of canola WRKY genes in the context of pathogen and hormone responses. In this study, we identified 46 WRKY genes from canola by mining the expressed sequence tag (EST) database and cloned cDNA sequences of 38 BnWRKYs. A phylogenetic tree was constructed using the conserved WRKY domain amino acid sequences, which demonstrated that BnWRKYs can be divided into three major groups. We further compared BnWRKYs to the 72 WRKY genes from Arabidopsis and 91 WRKY from rice, and we identified 46 presumptive orthologs of AtWRKY genes. We examined the subcellular localization of four BnWRKY proteins using green fluorescent protein (GFP) and we observed the fluorescent green signals in the nucleus only.The responses of 16 selected BnWRKY genes to two fungal pathogens, Sclerotinia sclerotiorum and Alternaria brassicae, were analyzed by quantitative real time-PCR (qRT-PCR). Transcript abundance of 13 BnWRKY genes changed significantly following pathogen challenge: transcripts of 10 WRKYs increased in abundance, two WRKY transcripts decreased after infection, and one decreased at 12 h post-infection but increased later on (72 h). We also observed that transcript abundance of 13/16 BnWRKY genes was responsive to one or more hormones, including abscisic acid (ABA), and cytokinin (6-benzylaminopurine, BAP) and the defense signaling molecules jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes in Arabidopsis and rice, and observed both similarities and differences in expression patterns. We identified a set of 13 BnWRKY genes from among 16 BnWRKY genes assayed, that are responsive to both fungal pathogens and hormone treatments, suggesting shared signaling mechanisms for these responses. This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.

Regulation of Glycan Structures in Animal Tissues

PubMed Central

Nairn, Alison V.; York, William S.; Harris, Kyle; Hall, Erica M.; Pierce, J. Michael; Moremen, Kelley W.

2008-01-01

Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of >700 genes, including enzymes involved in sugar-nucleotide biosynthesis, transporters, glycan extension, modification, recognition, catabolism, and numerous glycosylated core proteins. Comparison with parallel microarray analyses indicates a significantly greater sensitivity and dynamic range for our quantitative real time reverse transcriptase-PCR approach, particularly for the numerous low abundance glycan-related enzymes. Mapping of the genes and transcript levels to their respective biosynthetic pathway steps allowed a comparison with glycan structural data and provides support for a model where many, but not all, changes in glycan abundance result from alterations in transcript expression of corresponding biosynthetic enzymes. PMID:18411279

Developmental reprogramming of rat GLUT-5 requires de novo mRNA and protein synthesis.

PubMed

Jiang, L; Ferraris, R P

2001-01-01

Fructose transporter (GLUT-5) expression is low in mid-weaning rat small intestine, increases normally after weaning is completed, and can be precociously induced by premature consumption of a high-fructose (HF) diet. In this study, an in vivo perfusion model was used to determine the mechanisms regulating this substrate-induced reprogramming of GLUT-5 development. HF (100 mM) but not high-glucose (HG) perfusion increased GLUT-5 activity and mRNA abundance. In contrast, HF and HG perfusion had no effect on Na(+)-dependent glucose transporter (SGLT-1) expression but increased c-fos and c-jun expression. Intraperitoneal injection of actinomycin D before intestinal perfusion blocked the HF-induced increase in fructose uptake rate and GLUT-5 mRNA abundance. Actinomycin D also prevented the perfusion-induced increase in c-fos and c-jun mRNA abundance but did not affect glucose uptake rate and SGLT-1 mRNA abundance. Cycloheximide blocked the HF-induced increase in fructose uptake rate but not the increase in GLUT-5 mRNA abundance and had no effect on glucose uptake rate and SGLT-1 mRNA abundance. In neonatal rats, the substrate-induced reprogramming of intestinal fructose transport is likely to involve transcription and translation of the GLUT-5 gene.

Source of metabolizable energy affects gene transcription in metabolic pathways in adipose and liver tissue of nonlactating, pregnant dairy cows.

PubMed

Crookenden, M A; Mandok, K S; Grala, T M; Phyn, C V C; Kay, J K; Greenwood, S L; Roche, J R

2015-02-01

The objective of this experiment was to determine if transcript abundance of genes involved in metabolic pathways in adipose and liver tissue could provide some explanation for the low efficiency with which ME in autumn pasture is used for BW gain. Nonlactating, pregnant (208 ± 19 d of gestation or approximately 75 d precalving) dairy cows (n = 90) were randomly allocated to either a control diet (i.e., offered fresh autumn pasture to maintenance requirements: 0.55 MJ ME/kg of measured metabolic BW [BW0.75] per day) or, in addition to the control diet, 1 of 2 supplement amounts (2.5 and 5.0 kg DM/d) of autumn pasture or 1 of 4 supplementary feeds (i.e., a control and 2 levels of feeding for each of 5 feeds: 11 groups of cows). Along with autumn pasture, evaluated feeds included spring pasture silage, maize silage, maize grain, and palm kernel expeller. Adipose and liver tissues were biopsied in wk 4 of the experiment and transcript abundance of genes involved in metabolic pathways associated with energy metabolism, lipolysis, and lipogenesis was determined. Additional feed, irrespective of type, increased BW gain (P < 0.01) and this effect was reflected in the expression of genes in adipose and liver tissue. However, autumn pasture had lower energy-use efficiency than the other feeds. Genes involved in both lipogenesis (ACACA, THRSP, GPAM, GPD1, and LPL) and lipolysis (PNPLA2) were upregulated (P < 0.05) in adipose tissue in response to increased ME intake/kilogram BW0.75. Hepatic expression of APOA1 decreased and that of APOB increased (P < 0.05) in cows offered maize grain and maize silage (i.e., starch-containing feeds). In comparison, pasture-fed cows demonstrated a degree of uncoupling of the somatotropic axis, with lower hepatic transcript abundance of both GHR1A and IGF-1 compared with cows offered any of the other 4 feeds. Changes to gene transcription indicate a possible molecular mechanism for the poor BW gain evident in ruminants consuming autumn pasture.

Insulin-Like Growth Factor-I Regulates LH Release by Modulation of Kisspeptin and NMDA-Mediated Neurotransmission in Young and Middle-Aged Female Rats

PubMed Central

Yao, Dachun; Shu, Jun; Sun, Yan; Etgen, Anne M.

2014-01-01

This study investigated potential mechanisms by which age and IGF-I receptor (IGF-Ir) signaling in the neuroendocrine hypothalamus affect estradiol-positive feedback effects on GnRH neuronal activation and on kisspeptin and N-methyl-D-aspartate (NMDA)-induced LH release and on the abundance of NMDA receptor subunits Nr1 and Nr2b and Kiss1r transcript and protein in the hypothalamus of young and middle-aged female rats. We infused vehicle, IGF-I, or JB-1, a selective antagonist of IGF-Ir, into the third ventricle of ovariectomized female rats primed with estradiol or vehicle and injected with vehicle, kisspeptin (3 or 30 nmol/kg), or NMDA (15 or 30 mg/kg). Regardless of dose, NMDA and kisspeptin resulted in significantly more LH release, GnRH/c-Fos colabeling, and c-Fos immunoreative cells in young than in middle-aged females. Estradiol priming significantly increased Kiss1r, Nr1, and Nr2b receptor transcript and protein abundance in young but not middle-aged female hypothalamus. JB-1 attenuated kisspeptin and NMDA-induced LH release, numbers of GnRH/c-Fos and c-Fos cells, and Kiss1r, Nr1, and Nr2b transcript and protein abundance in young females to levels observed in middle-aged females. IGF-I significantly enhanced NMDA and kisspeptin-induced LH release in middle-aged females without increasing numbers of GnRH/c-Fos or c-Fos immunoreactive cells. IGF-I infusion in middle-aged females also increased Kiss1r, Nr1, and Nr2b protein and transcript to levels that were equivalent to young estradiol-primed females. These findings indicate that age-related changes in estradiol-regulated responsiveness to excitatory input from glutamate and kisspeptin reflect reduced IGF-Ir signaling. PMID:24617524

Effects of Pb(Ⅱ) exposure on Chlorella protothecoides and Chlorella vulgaris growth, malondialdehyde, and photosynthesis-related gene transcription.

PubMed

Xiong, Bang; Zhang, Wei; Chen, Lin; Lin, Kuang-Fei; Guo, Mei-Jin; Wang, Wei-Liang; Cui, Xin-Hong; Bi, Hua-Song; Wang, Bin

2014-11-01

Greater exposure to Pb(Ⅱ) increases the likelihood of harmful effects in the environment. In this study, the aquatic unicellular alga Chlorella protothecoides (C. protothecoides) and Chlorella vulgaris (C. vulgaris) were chosen to assess the acute and chronic toxicity of Pb(Ⅱ) exposure. Results of the observations show dose-response relationships could be clearly observed between Pb(Ⅱ) concentration and percentage inhibition (PI). Exposure to Pb(Ⅱ) increased malondialdehyde (MDA) content by up to 4.22 times compared with the control, suggesting that there was some oxidative damage. ANOVA analysis shows that Pb(Ⅱ) decreased chlorophyll (chl) content, indicating marked concentration-dependent relationships, and the lowest levels of chl a, chl b, and total-chl were 14.53, 18.80, and 17.95% of the controls, respectively. A real-time PCR assay suggests the changes in transcript abundances of three photosynthetic-related genes. After 120 h exposure Pb(Ⅱ) reduced the transcript abundance of rbcL, psaB, and psbC, and the relative abundances of the three genes of C. protothecoides and C. vulgaris in response to Pb(Ⅱ) were 54.66-98.59, 51.68-95.59, 37.89-95.48, 36.04-94.94, 41.19-91.20, and 58.75-96.80% of those of the controls, respectively. As for 28 d treatments, the three genes displayed similar inhibitory trend. This research provides a basic understanding of Pb(Ⅱ) toxicity to aquatic organisms. Copyright © 2013 Wiley Periodicals, Inc.

Tyrosine kinase inhibitors and immunosuppressants perturb the myo-inositol but not the betaine cotransporter in isotonic and hypertonic MDCK cells

PubMed Central

Atta, Mohamed G.; Dahl, Stephen C.; Kwon, H. Moo; Handler, Joseph S.

2008-01-01

Background The sodium/myo-inositol cotransporter (SMIT) and the betaine cotransporter (BGT1) are essential for the accumulation of myo-inositol and betaine, and hence cell survival in a hypertonic environment. The underlying molecular mechanism involves an increase in transcription of the SMIT and BGT1 genes through binding of a trans-acting factor to enhancer elements in the 5′ flanking region of both genes, resulting in increased mRNA abundance and increased activity of the cotransporters. Current evidence regarding transcriptional and post-transcriptional regulation indicates that both cotransporters are regulated in parallel. Methods To investigate the signal transduction of hypertonic stress, we examined the effect of tyrosine kinase inhibitors and immunosuppressants on the hypertonicity-induced activity of the two cotransporters in Madin-Darby canine kidney (MDCK) cells. Results None of the agents studied affected BGT1 activity in isotonic or hypertonic conditions. Treatment of MDCK cells with genistein, a tyrosine kinase inhibitor, increased SMIT activity in hypertonic but not isotonic conditions. The stimulation of SMIT by genistein was accompanied by a parallel increase in mRNA abundance. In contrast, treating cells with tyrphostin A23, another tyrosine kinase inhibitor, or cyclosporine A, an immunosuppressant, inhibited SMIT activity in hypertonic cells. FK506, another immunosuppressant, increased SMIT activity, but only in isotonic conditions. Conclusions These results provide the first evidence of divergent regulatory pathways modulating SMIT and BGT activity. PMID:10027932

Expression of key lipid metabolism genes in adipose tissue is not altered by once-daily milking during a feed restriction of grazing dairy cows.

PubMed

Grala, T M; Roche, J R; Phyn, C V C; Rius, A G; Boyle, R H; Snell, R G; Kay, J K

2013-01-01

The objective of this study was to investigate the effect of reduced milking frequency, at 2 feeding levels, on gene expression in adipose tissue of grazing dairy cows during early lactation. Multiparous Holstein-Friesian and Holstein-Friesian × Jersey cows (n=120) were grazed on pasture and milked twice daily (2×) from calving to 34±6d in milk (mean ± standard deviation). Cows were then allocated to 1 of 4 treatments in a 2×2 factorial arrangement. Treatments consisted of 2 milking frequencies (2× or once daily; 1×) and 2 feeding levels for 3 wk: adequately fed (AF), consuming 14.3 kg of dry matter/cow per day, or underfed (UF), consuming 8.3 kg of dry matter/cow per day. After the treatment period, all cows were fed to target grazing residuals ≥1,600 kg of DM/cow per day and milked 2× for 20 wk. Adipose tissue was collected from 12 cows per treatment by subcutaneous biopsy at -1, 3, and 5 wk relative to treatment start, RNA was extracted, and transcript abundance of genes involved in lipid metabolism was quantified using a linear mixed model. At the end of the 3-wk treatment period, transcript abundance of genes involved in fatty acid (FA) uptake into adipose tissue (LPL), FA synthesis [FA synthase (FASN) and stearoyl-coenzyme A desaturase (SCD)], FA oxidation [acyl-coenzyme A synthetase long-chain family member 1 (ACSL1) and carnitine palmitoyltransferase 2 (CPT2)], glyceroneogenesis [glycerol-3-phosphate dehydrogenase 1 (GPD1) and pyruvate carboxylase (PC)], and triacylglyceride synthesis [diacylglycerol O-acyltransferase 2 (DGAT2)] were greater in AF1× cows compared with all other treatments. However, when cows were underfed, no effects of milking frequency were observed on transcript abundance of genes involved in adipose lipid metabolism. Despite increases in plasma NEFA concentrations in UF cows, no effects of underfeeding were observed on the transcription of lipolytic genes. At 5 wk, after cows were returned to 2× milking and standard feed allowance, transcript abundances of genes involved in FA synthesis [acetyl-coenzyme A carboxylase α (ACACA) and SCD)] were increased in cows previously UF. Expression of ACSL1 was decreased in UF1× cows relative to UF2× cows and CPT2 expression was greater in AF1× cows compared with AF2× cows. In conclusion, after 3 wk of reduced milking frequency during a feed restriction, transcription of genes involved in lipid metabolism in adipose tissue were not altered, possibly due to the reduced milk production in these animals. However, 3 wk of 1× milking in AF cows increased transcription of genes involved in FA synthesis, oxidation, and triacylglyceride synthesis. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Rapid transcriptome responses of maize (Zea mays) to UV-B in irradiated and shielded tissues

PubMed Central

Casati, Paula; Walbot, Virginia

2004-01-01

Background Depletion of stratospheric ozone has raised terrestrial levels of ultraviolet-B radiation (UV-B), an environmental change linked to an increased risk of skin cancer and with potentially deleterious consequences for plants. To better understand the processes of UV-B acclimation that result in altered plant morphology and physiology, we investigated gene expression in different organs of maize at several UV-B fluence rates and exposure times. Results Microarray hybridization was used to assess UV-B responses in directly exposed maize organs and organs shielded by a plastic that absorbs UV-B. After 8 hours of high UV-B, the abundance of 347 transcripts was altered: 285 were increased significantly in at least one organ and 80 were downregulated. More transcript changes occurred in directly exposed than in shielded organs, and the levels of more transcripts were changed in adult compared to seedling tissues. The time course of transcript abundance changes indicated that the response kinetics to UV-B is very rapid, as some transcript levels were altered within 1 hour of exposure. Conclusions Most of the UV-B regulated genes are organ-specific. Because shielded tissues, including roots, immature ears, and leaves, displayed altered transcriptome profiles after exposure of the plant to UV-B, some signal(s) must be transmitted from irradiated to shielded tissues. These results indicate that there are integrated responses to UV-B radiation above normal levels. As the same total UV-B irradiation dose applied at three intensities elicited different transcript profiles, the transcriptome changes exhibit threshold effects rather than a reciprocal dose-effect response. Transcriptome profiling highlights possible signaling pathways and molecules for future research. PMID:15003119

Knock-down of transcript abundance of a family of Kunitz proteinase inhibitor genes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function.

PubMed

Islam, Afsana; Leung, Susanna; Burgess, Elisabeth P J; Laing, William A; Richardson, Kim A; Hofmann, Rainer W; Dijkwel, Paul P; McManus, Michael T

2015-12-01

The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role. The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 over-expressing lines. RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes. These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Plane of nutrition affects the phylogenetic diversity and relative abundance of transcriptionally active methanogens in the bovine rumen.

PubMed

McGovern, Emily; McCabe, Matthew S; Cormican, Paul; Popova, Milka; Keogh, Kate; Kelly, Alan K; Kenny, David A; Waters, Sinead M

2017-10-12

Methane generated during enteric fermentation in ruminant livestock species is a major contributor to global anthropogenic greenhouse gas emissions. A period of moderate feed restriction followed by ad libitum access to feed is widely applied in cattle management to exploit the animal's compensatory growth potential and reduce feed costs. In the present study, we utilised microbial RNA from rumen digesta samples to assess the phylogenetic diversity of transcriptionally active methanogens from feed-restricted and non-restricted animals. To determine the contribution of different rumen methanogens to methanogenesis during dietary restriction of cattle, we conducted high-throughput mcrA cDNA amplicon sequencing on an Illumina MiSeq and analysed both the abundance and phylogenetic origin of different mcrA cDNA sequences. When compared to their unrestricted contemporaries, in feed-restricted animals, the methanogenic activity, based on mcrA transcript abundance, of Methanobrevibacter gottschalkii clade increased while the methanogenic activity of the Methanobrevibacter ruminantium clade and members of the Methanomassiliicoccaceae family decreased. This study shows that the quantity of feed consumed can evoke large effects on the composition of methanogenically active species in the rumen of cattle. These data potentially have major implications for targeted CH 4 mitigation approaches such as anti-methanogen vaccines and/or tailored dietary management.

Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

DOE PAGES

Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; ...

2016-07-28

Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

VARIATION IN THE ABUNDANCE OF SYNECHOCOCCUS SP. CC9311 NARB MRNA RELATIVE TO CHANGES IN LIGHT, NITROGEN GROWTH CONDITIONS AND NITRATE ASSIMILATION(1).

PubMed

Paerl, Ryan W; Tozzi, Sasha; Kolber, Zbigniew S; Zehr, Jonathan P

2012-08-01

Synechococcus- and Prochlorococcus-specific narB genes that encode for an assimilatory nitrate reductase are found in coastal to open-ocean waters. However, it remains uncertain if these picocyanobacteria assimilate nitrate in situ. This unknown can potentially be addressed by examining narB mRNA from the environment, but this requires a better understanding of the influence of environmental factors on narB gene transcription. In laboratory experiments with Synechococcus sp. CC9311 cultures exposed to diel light fluctuations and grown on nitrate or ammonium, there was periodic change in narB transcript abundance. This periodicity was broken in cultures subjected to a doubling of irradiance (40-80 μmol photons · m(-2)  · s(-1) ) during the mid-light period. Therefore, the irradiance level, not circadian rhythm, was the dominant factor controlling narB transcription. In nitrate-grown cultures, diel change in narB transcript abundance and nitrate assimilation rate did not correlate; suggesting narB mRNA levels better indicate nitrate assimilation activity than assimilation rate. Growth history also affected narB transcription, as changes in narB mRNA levels in nitrogen-deprived CC9311 cultures following nitrate amendment were distinct from cultures grown solely on nitrate. Environmental sampling for narB transcripts should consider time, irradiance, and the growth status of cells to ecologically interpret narB transcript abundances. © 2012 Phycological Society of America.

Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

PubMed

Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

2009-12-08

Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L. interrogans to adapt to conditions encountered in the host and to cause disease. Our results suggest down-regulation of protein expression in response to temperature, and decreased expression of outer membrane proteins may facilitate minimal interaction with host immune mechanisms.

Increased abundance of ADAM9 transcripts in the blood is associated with tissue damage

PubMed Central

Rinchai, Darawan; Kewcharoenwong, Chidchamai; Kessler, Bianca; Lertmemongkolchai, Ganjana; Chaussabel, Damien

2016-01-01

Background: Members of the ADAM (a disintegrin and metalloprotease domain) family have emerged as critical regulators of cell-cell signaling during development and homeostasis. ADAM9 is consistently overexpressed in various human cancers, and has been shown to play an important role in tumorigenesis. However, little is known about the involvement of ADAM9 during immune-mediated processes. Results: Mining of an extensive compendium of transcriptomic datasets identified important gaps in knowledge regarding the possible role of ADAM9 in immunological homeostasis and inflammation: 1) The abundance of ADAM9 transcripts in the blood was increased in patients with acute infection but, 2) changed very little after in vitro exposure to a wide range of pathogen-associated molecular patterns (PAMPs). 3) Furthermore it was found to increase significantly in subjects as a result of tissue injury or tissue remodeling, in absence of infectious processes. Conclusions: Our findings indicate that ADAM9 may constitute a valuable biomarker for the assessment of tissue damage, especially in clinical situations where other inflammatory markers are confounded by infectious processes. PMID:27990250

Genome-wide Determinants of Proviral Targeting, Clonal Abundance and Expression in Natural HTLV-1 Infection

PubMed Central

Melamed, Anat; Laydon, Daniel J.; Gillet, Nicolas A.; Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.

2013-01-01

The regulation of proviral latency is a central problem in retrovirology. We postulate that the genomic integration site of human T lymphotropic virus type 1 (HTLV-1) determines the pattern of expression of the provirus, which in turn determines the abundance and pathogenic potential of infected T cell clones in vivo. We recently developed a high-throughput method for the genome-wide amplification, identification and quantification of proviral integration sites. Here, we used this protocol to test two hypotheses. First, that binding sites for transcription factors and chromatin remodelling factors in the genome flanking the proviral integration site of HTLV-1 are associated with integration targeting, spontaneous proviral expression, and in vivo clonal abundance. Second, that the transcriptional orientation of the HTLV-1 provirus relative to that of the nearest host gene determines spontaneous proviral expression and in vivo clonal abundance. Integration targeting was strongly associated with the presence of a binding site for specific host transcription factors, especially STAT1 and p53. The presence of the chromatin remodelling factors BRG1 and INI1 and certain host transcription factors either upstream or downstream of the provirus was associated respectively with silencing or spontaneous expression of the provirus. Cells expressing HTLV-1 Tax protein were significantly more frequent in clones of low abundance in vivo. We conclude that transcriptional interference and chromatin remodelling are critical determinants of proviral latency in natural HTLV-1 infection. PMID:23555266

A tapeworm molecule manipulates vitellogenin expression in the beetle Tenebrio molitor

PubMed Central

Warr, E.; Meredith, J. M.; Nimmo, D. D.; Basu, S.; Hurd, H.; Eggleston, P.

2006-01-01

Metacestodes of Hymenolepis diminuta secrete a molecule that decreases vitellogenin (Vg) synthesis in the beetle host, Tenebrio molitor. The 5608 bp T. molitor Vg cDNA represents a single-copy gene encoding a single open reading frame of 1821 amino acids with a predicted molecular mass of 206 kDa. Northern blot analysis revealed detectable levels of transcripts only in adult females. In vivo, Vg mRNA abundance was significantly higher in fat bodies from infected females compared with control females at all but the earliest time point. In vitro, Vg mRNA abundance was significantly increased in fat bodies incubated with live stage I–II parasites. The apparent conflict between increased Vg mRNA abundance and decreased Vg protein in fat bodies from infected females is discussed. PMID:16907836

Coordinated regulation of growth, activity and transcription in natural populations of the unicellular nitrogen-fixing cyanobacterium Crocosphaera.

PubMed

Wilson, Samuel T; Aylward, Frank O; Ribalet, Francois; Barone, Benedetto; Casey, John R; Connell, Paige E; Eppley, John M; Ferrón, Sara; Fitzsimmons, Jessica N; Hayes, Christopher T; Romano, Anna E; Turk-Kubo, Kendra A; Vislova, Alice; Armbrust, E Virginia; Caron, David A; Church, Matthew J; Zehr, Jonathan P; Karl, David M; DeLong, Edward F

2017-07-31

The temporal dynamics of phytoplankton growth and activity have large impacts on fluxes of matter and energy, yet obtaining in situ metabolic measurements of sufficient resolution for even dominant microorganisms remains a considerable challenge. We performed Lagrangian diel sampling with synoptic measurements of population abundances, dinitrogen (N 2 ) fixation, mortality, productivity, export and transcription in a bloom of Crocosphaera over eight days in the North Pacific Subtropical Gyre (NPSG). Quantitative transcriptomic analyses revealed clear diel oscillations in transcript abundances for 34% of Crocosphaera genes identified, reflecting a systematic progression of gene expression in diverse metabolic pathways. Significant time-lagged correspondence was evident between nifH transcript abundance and maximal N 2 fixation, as well as sepF transcript abundance and cell division, demonstrating the utility of transcriptomics to predict the occurrence and timing of physiological and biogeochemical processes in natural populations. Indirect estimates of carbon fixation by Crocosphaera were equivalent to 11% of net community production, suggesting that under bloom conditions this diazotroph has a considerable impact on the wider carbon cycle. Our cross-scale synthesis of molecular, population and community-wide data underscores the tightly coordinated in situ metabolism of the keystone N 2 -fixing cyanobacterium Crocosphaera, as well as the broader ecosystem-wide implications of its activities.

Diversity and spatial distribution of hydrazine oxidoreductase (hzo) gene in the oxygen minimum zone off Costa Rica.

PubMed

Kong, Liangliang; Jing, Hongmei; Kataoka, Takafumi; Buchwald, Carolyn; Liu, Hongbin

2013-01-01

Anaerobic ammonia oxidation (anammox) as an important nitrogen loss pathway has been reported in marine oxygen minimum zones (OMZs), but the community composition and spatial distribution of anammox bacteria in the eastern tropical North Pacific (ETNP) OMZ are poorly determined. In this study, anammox bacterial communities in the OMZ off Costa Rica (CRD-OMZ) were analyzed based on both hydrazine oxidoreductase (hzo) genes and their transcripts assigned to cluster 1 and 2. The anammox communities revealed by hzo genes and proteins in CRD-OMZ showed a low diversity. Gene quantification results showed that hzo gene abundances peaked in the upper OMZs, associated with the peaks of nitrite concentration. Nitrite and oxygen concentrations may therefore colimit the distribution of anammox bacteria in this area. Furthermore, transcriptional activity of anammox bacteria was confirmed by obtaining abundant hzo mRNA transcripts through qRT-PCR. A novel hzo cluster 2x clade was identified by the phylogenetic analysis and these novel sequences were abundant and widely distributed in this environment. Our study demonstrated that both cluster 1 and 2 anammox bacteria play an active role in the CRD-OMZ, and the cluster 1 abundance and transcriptional activity were higher than cluster 2 in both free-living and particle-attached fractions at both gene and transcriptional levels.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai

Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

Phosphate Control of Oxytetracycline Production by Streptomyces rimosus Is at the Level of Transcription from Promoters Overlapped by Tandem Repeats Similar to Those of the DNA-Binding Sites of the OmpR Family

PubMed Central

McDowall, Kenneth J.; Thamchaipenet, Arinthip; Hunter, Iain S.

1999-01-01

Physiological studies have shown that Streptomyces rimosus produces the polyketide antibiotic oxytetracycline abundantly when its mycelial growth is limited by phosphate starvation. We show here that transcripts originating from the promoter for one of the biosynthetic genes, otcC (encoding anhydrotetracycline oxygenase), and from a promoter for the divergent otcX genes peak in abundance at the onset of antibiotic production induced by phosphate starvation, indicating that the synthesis of oxytetracycline is controlled, at least in part, at the level of transcription. Furthermore, analysis of the sequences of the promoters for otcC, otcX, and the polyketide synthase (otcY) genes revealed tandem repeats having significant similarity to the DNA-binding sites of ActII-Orf4 and DnrI, which are Streptomyces antibiotic regulatory proteins (SARPs) related to the OmpR family of transcription activators. Together, the above results suggest that oxytetracycline production by S. rimosus requires a SARP-like transcription factor that is either produced or activated or both under conditions of low phosphate concentrations. We also provide evidence consistent with the otrA resistance gene being cotranscribed with otcC as part of a polycistronic message, suggesting a simple mechanism of coordinate regulation which ensures that resistance to the antibiotic increases in proportion to production. PMID:10322002

Differential gene expression in ripening banana fruit.

PubMed Central

Clendennen, S K; May, G D

1997-01-01

During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants. PMID:9342866

Transcription of ethylene perception and biosynthesis genes is altered by putrescine, spermidine and aminoethoxyvinylglycine (AVG) during ripening in peach fruit (Prunus persica).

PubMed

Ziosi, Vanina; Bregoli, Anna Maria; Bonghi, Claudio; Fossati, Tiziana; Biondi, Stefania; Costa, Guglielmo; Torrigiani, Patrizia

2006-01-01

The time course of ethylene biosynthesis and perception was investigated in ripening peach fruit (Prunus persica) following treatments with the polyamines putrescine (Pu) and spermidine (Sd), and with aminoethoxyvinylglycine (AVG). Fruit treatments were performed in planta. Ethylene production was measured by gas chromatography, and polyamine content by high-performance liquid chromatography; expression analyses were performed by Northern blot or real-time polymerase chain reaction. Differential increases in the endogenous polyamine pool in the epicarp and mesocarp were induced by treatments; in both cases, ethylene production, fruit softening and abscission were greatly inhibited. The rise in 1-aminocyclopropane-1-carboxylate oxidase (PpACO1) mRNA was counteracted and delayed in polyamine-treated fruit, whereas transcript abundance of ethylene receptors PpETR1 (ethylene receptor 1) and PpERS1 (ethylene sensor 1) was enhanced at harvest. Transcript abundance of arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) was transiently reduced in both the epicarp and mesocarp. AVG, here taken as a positive control, exerted highly comparable effects to those of Pu and Sd. Thus, in peach fruit, increasing the endogenous polyamine pool in the epicarp or in the mesocarp strongly interfered, both at a biochemical and at a biomolecular level, with the temporal evolution of the ripening syndrome.

A mixture of the novel brominated flame retardants TBPH and TBB affects fecundity and transcript profiles of the HPGL-axis in Japanese medaka.

PubMed

Saunders, David M V; Podaima, Michelle; Codling, Garry; Giesy, John P; Wiseman, Steve

2015-01-01

The novel brominated flame retardants (NBFRs), bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH) and 2-ethylhexyl-2,3,4,5 tetrabromobenzoate (TBB) are components of the flame retardant mixture Firemaster 550 and both TBPH and TBB have recently been listed as high production volume chemicals by the US EPA. These NBFRs have been detected in several environmental matrices but very little is known about their toxic effects or potencies. Results of in vitro assays demonstrated potentials of these NBFRs to modulate endocrine function through interactions with estrogen (ER) and androgen receptors (AR) and via alterations to synthesis of 17-β-estradiol (E2) and testosterone (T), but in vivo effects of these chemicals on organisms are not known. Therefore a 21-day short term fish fecundity assay with Japanese medaka (Oryzias latipes) was conducted to investigate if these NBFRs affect endocrine function in vivo. Medaka were fed a diet containing either 1422 TBPH:1474 TBB or 138:144 μg/g food, wet weight (w/w). Cumulative production of eggs was used as a measure of fecundity and abundances of transcripts of 34 genes along the hypothalamus-pituitary-gonadal-liver (HPGL) axis were quantified to determine mechanisms of observed effects. Cumulative fecundity was impaired by 32% in medaka exposed to the greatest dose of the mixture of TBPH/TBB. A pattern of global down-regulation of gene transcription at all levels of the HPGL axis was observed, but effects were sex-specific. In female medaka the abundance of transcripts of ERβ was lesser in livers, while abundances of transcripts of VTG II and CHG H were greater. In male medaka, abundances of transcripts of ERα, ERβ, and ARα were lesser in gonads and abundances of transcripts of ERβ and ARα were lesser in brain. Abundances of transcripts of genes encoding proteins for synthesis of cholesterol (HMGR), transport of cholesterol (HDLR), and sex hormone steroidogenesis (CYP 17 and 3β-HSD) were significantly lesser in male medaka, which might have implications for concentrations of sex hormones. The results of this study demonstrate that exposure to components of the flame retardant mixture Firemaster(®) 550 has the potential to impair the reproductive axis of fishes. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression profiling of genes modulated by minocycline in a rat model of neuropathic pain

PubMed Central

2014-01-01

Background The molecular mechanisms underlying neuropathic pain are constantly being studied to create new opportunities to prevent or alleviate neuropathic pain. The aim of our study was to determine the gene expression changes induced by sciatic nerve chronic constriction injury (CCI) that are modulated by minocycline, which can effectively diminish neuropathic pain in animal studies. The genes associated with minocycline efficacy in neuropathic pain should provide insight into the etiology of neuropathic pain and identify novel therapeutic targets. Results We screened the ipsilateral dorsal part of the lumbar spinal cord of the rat CCI model for differentially expressed genes. Out of 22,500 studied transcripts, the abundance levels of 93 transcripts were altered following sciatic nerve ligation. Percentage analysis revealed that 54 transcripts were not affected by the repeated administration of minocycline (30 mg/kg, i.p.), but the levels of 39 transcripts were modulated following minocycline treatment. We then selected two gene expression patterns, B1 and B2. The first transcription pattern, B1, consisted of 10 mRNA transcripts that increased in abundance after injury, and minocycline treatment reversed or inhibited the effect of the injury; the B2 transcription pattern consisted of 7 mRNA transcripts whose abundance decreased following sciatic nerve ligation, and minocycline treatment reversed the effect of the injury. Based on the literature, we selected seven genes for further analysis: Cd40, Clec7a, Apobec3b, Slc7a7, and Fam22f from pattern B1 and Rwdd3 and Gimap5 from pattern B2. Additionally, these genes were analyzed using quantitative PCR to determine the transcriptional changes strongly related to the development of neuropathic pain; the ipsilateral DRGs (L4-L6) were also collected and analyzed in these rats using qPCR. Conclusion In this work, we confirmed gene expression alterations previously identified by microarray analysis in the spinal cord and analyzed the expression of selected genes in the DRG. Moreover, we reviewed the literature to illustrate the relevance of these findings for neuropathic pain development and therapy. Further studies are needed to elucidate the roles of the individual genes in neuropathic pain and to determine the therapeutic role of minocycline in the rat neuropathic pain model. PMID:25038616

Transcriptional regulators TRIM28, SETDB1, and TP53 are aberrantly expressed in porcine embryos produced by in vitro fertilization in comparison to in vivo- and somatic-cell nuclear transfer-derived embryos

PubMed Central

Hamm, Jennifer; Tessanne, Kim; Murphy, Clifton N; Prather, Randall S

2014-01-01

In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos. Mol. Reprod. Dev. 81: 552–556, 2014. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:24659575

Potential toxic effect of trifloxystrobin on cellular microstructure, mRNA expression and antioxidant enzymes in Chlorella vulgaris.

PubMed

Shen, Yu-Feng; Liu, Lei; Gong, Yu-Xin; Zhu, Bin; Liu, Guang-Lu; Wang, Gao-Xue

2014-05-01

This study investigated the effects of trifloxystrobin that one strobilurin used widely in the world as an effective fungicidal agent to control Asian soybean rust on aquatic unicellular algae Chlorella vulgaris. We determined the potential toxic effect of trifloxystrobin on C. vulgaris, and found median inhibition concentration (IC(50)) value 255.58 (95% confidence interval, 207.81-330.29)μgL(-1). In addition, the algal cells were obviously depressed or shrunk at different concentrations by electron microscopy. In the study, a real-time polymerase chain reaction (PCR) assay showed changes in transcript abundances of three photosynthetic genes, psaB, psbC, and rbcL, and one energy gene, ATPs. The results showed that trifloxystrobin reduced the transcript abundances of the three genes and enhanced expression of ATPs after 48 and 96 h. The lowest abundances of psaB, psbC and rbcL transcripts in response to trifloxystrobin exposure were 58%, 79% and 60% of those of the control, respectively. For the potential toxic influences, trifloxystrobin could decrease the soluble protein and total antioxidant contents (T-AOC), and increase superoxide dismutase (SOD) and peroxidase (POD) activity with a gradual concentration-response relationship. Overall, the present study demonstrated that trifloxystrobin could affect the activities of antioxidant enzymes, disrupts photosynthesis in C. vulgaris, and damage cellular structure. Copyright © 2014 Elsevier B.V. All rights reserved.

Irradiation influence on the phenoloxidase pathway and an anti-oxidant defense mechanism in Spodoptera litura (Lepidoptera: Noctuidae) and its implication in radio-genetic 'F 1 sterility' and biorational pest suppression tactics.

PubMed

Sachdev, B; Khan, Z; Zarin, M; Malhotra, P; Seth, R K; Bhatnagar, R K

2017-06-01

The present study was conducted to appraise the ontogenic radio-sensitivity of a serious tropical pest, Spodoptera litura (Fabr.). The molecular responses pertaining to the phenoloxidase (PO) pathway and an anti-oxidant defense mechanism were evaluated in order to understand its implication in pest control at pre-harvest and post-harvest intervals. Irradiation exhibited an inverse relationship with age with respect to impact on developmental and transcriptional responses. Transcript abundance of PO cascade enzymes, prophenoloxidase (slppo-2), its activating enzyme (slppae-1) and free-radical scavenging enzymes, superoxide dismutase (slsod) and catalase (slcat) was evaluated upon gamma irradiation alone and the dual-stress of radiation plus microbial challenge. The slppo-2, slppae-1, slsod and slcat transcripts were significantly up-regulated in F 1 L6 larvae (6th-instar) resulting from 100 Gy sub-sterilized male adults and unirradiated female moths. The extent of upregulation was relatively higher in comparison with L6 survivors (6th-instar larvae) developed from irradiated neonates (L1) treated with 100 Gy. Upon Photorhabdus challenge, the transcripts were down-regulated in irradiated L1 suggesting increased larval susceptibility to bacterial infections. Radioresistance increased with the age of the insect, and molecular responses (transcript abundance) of insect defense mechanism were less influenced when older age (F 1 progeny) were irradiated. These findings will help to optimize the gamma dose to be employed in inherited sterility technique for (pre-harvest) pest suppression and (post-harvest) phytosanitation and quarantine, and suggest compatible integration of biorational tactics including nuclear technology.

Inventory of high-abundance mRNAs in skeletal muscle of normal men.

PubMed

Welle, S; Bhatt, K; Thornton, C A

1999-05-01

G42875rial analysis of gene expression (SAGE) method was used to generate a catalog of 53,875 short (14 base) expressed sequence tags from polyadenylated RNA obtained from vastus lateralis muscle of healthy young men. Over 12,000 unique tags were detected. The frequency of occurrence of each tag reflects the relative abundance of the corresponding mRNA. The mRNA species that were detected 10 or more times, each comprising >/=0.02% of the mRNA population, accounted for 64% of the mRNA mass but <10% of the total number of mRNA species detected. Almost all of the abundant tags matched mRNA or EST sequences cataloged in GenBank. Mitochondrial transcripts accounted for approximately 20% of the polyadenylated RNA. Transcripts encoding proteins of the myofibrils were the most abundant nuclear-encoded mRNAs. Transcripts encoding ribosomal proteins, and those encoding proteins involved in energy metabolism, also were very abundant. The database can be used as a reference for investigations of alterations in gene expression associated with conditions that influence muscle function, such as muscular dystrophies, aging, and exercise.

The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling.

PubMed

Miao, Min; Niu, Xiangli; Kud, Joanna; Du, Xinran; Avila, Julian; Devarenne, Timothy P; Kuhl, Joseph C; Liu, Yongsheng; Xiao, Fangming

2016-07-01

We recently identified a defense-related tomato (Solanum lycopersicum) NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, that is subjected to ubiquitin-proteasome system-dependent degradation in plant cells. In this study, we identified a tomato ubiquitin ligase (termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1, promoting its degradation. We conducted coimmunoprecipitation and bimolecular fluorescence complementation to determine that SINA3 specifically interacts with the NAC1 transcription factor in the nucleus. Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and promotes NAC1 degradation via polyubiquitination in vivo, indicating that SINA3 is a ubiquitin ligase that ubiquitinates NAC1, promoting its degradation. Our real-time PCR analysis indicated that, in contrast to our previous finding that NAC1 mRNA abundance increases upon Pseudomonas infection, the SINA3 mRNA abundance decreases in response to Pseudomonas infection. Moreover, using Agrobacterium-mediated transient expression, we found that overexpression of SINA3 interferes with the hypersensitive response cell death triggered by multiple plant resistance proteins. These results suggest that SINA3 ubiquitinates a defense-related NAC transcription factor for degradation and plays a negative role in defense signaling. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Moderate nutrient restriction influences transcript abundance of genes impacting production efficiencies of beef cattle in fetal liver, muscle, and cerebrum by d 50 of gestation

USDA-ARS?s Scientific Manuscript database

We hypothesized that a moderate maternal nutrient restriction during the first 50 d of gestation in beef heifers would affect transcript abundance of genes impacting production efficiency phenotypes in fetal liver, muscle, and cerebrum. Fourteen Angus-cross heifers were estrus synchronized and assig...

Myo-inositol phosphate synthase expression in the European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus): effect of seawater acclimation.

PubMed

Kalujnaia, Svetlana; Hazon, Neil; Cramb, Gordon

2016-08-01

A single MIPS gene (Isyna1/Ino1) exists in eel and tilapia genomes with a single myo-d-inositol 3-phosphate synthase (MIPS) transcript identified in all eel tissues, although two MIPS spliced variants [termed MIPS(s) and MIPS(l)] are found in all tilapia tissues. The larger tilapia transcript [MIPS(l)] results from the inclusion of the 87-nucleotide intron between exons 5 and 6 in the genomic sequence. In most tilapia tissues, the MIPS(s) transcript exhibits much higher abundance (generally >10-fold) with the exception of white skeletal muscle and oocytes, in which the MIPS(l) transcript predominates. SW acclimation resulted in large (6- to 32-fold) increases in mRNA expression for both MIPS(s) and MIPS(l) in all tilapia tissues tested, whereas in the eel, changes in expression were limited to a more modest 2.5-fold increase and only in the kidney. Western blots identified a number of species- and tissue-specific immunoreactive MIPS proteins ranging from 40 to 67 kDa molecular weight. SW acclimation failed to affect the abundance of any immunoreactive protein in any tissue tested from the eel. However, a major 67-kDa immunoreactive protein (presumed to be MIPS) found in tilapia tissues exhibited 11- and 54-fold increases in expression in gill and fin samples from SW-acclimated fish. Immunohistochemical investigations revealed specific immunoreactivity in the gill, fin, skin, and intestine taken from only SW-acclimated tilapia. Immunofluorescence indicated that MIPS was expressed within gill chondrocytes and epithelial cells of the primary filaments, basal epithelial cell layers of the skin and fin, the cytosol of columnar intestinal epithelial and mucous cells, as well as unknown entero-endocrine-like cells. Copyright © 2016 the American Physiological Society.

Myo-inositol phosphate synthase expression in the European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus): effect of seawater acclimation

PubMed Central

Kalujnaia, Svetlana; Hazon, Neil

2016-01-01

A single MIPS gene (Isyna1/Ino1) exists in eel and tilapia genomes with a single myo-d-inositol 3-phosphate synthase (MIPS) transcript identified in all eel tissues, although two MIPS spliced variants [termed MIPS(s) and MIPS(l)] are found in all tilapia tissues. The larger tilapia transcript [MIPS(l)] results from the inclusion of the 87-nucleotide intron between exons 5 and 6 in the genomic sequence. In most tilapia tissues, the MIPS(s) transcript exhibits much higher abundance (generally >10-fold) with the exception of white skeletal muscle and oocytes, in which the MIPS(l) transcript predominates. SW acclimation resulted in large (6- to 32-fold) increases in mRNA expression for both MIPS(s) and MIPS(l) in all tilapia tissues tested, whereas in the eel, changes in expression were limited to a more modest 2.5-fold increase and only in the kidney. Western blots identified a number of species- and tissue-specific immunoreactive MIPS proteins ranging from 40 to 67 kDa molecular weight. SW acclimation failed to affect the abundance of any immunoreactive protein in any tissue tested from the eel. However, a major 67-kDa immunoreactive protein (presumed to be MIPS) found in tilapia tissues exhibited 11- and 54-fold increases in expression in gill and fin samples from SW-acclimated fish. Immunohistochemical investigations revealed specific immunoreactivity in the gill, fin, skin, and intestine taken from only SW-acclimated tilapia. Immunofluorescence indicated that MIPS was expressed within gill chondrocytes and epithelial cells of the primary filaments, basal epithelial cell layers of the skin and fin, the cytosol of columnar intestinal epithelial and mucous cells, as well as unknown entero-endocrine-like cells. PMID:27252471

HIV-1 Tat protein promotes formation of more-processive elongation complexes.

PubMed Central

Marciniak, R A; Sharp, P A

1991-01-01

The Tat protein of HIV-1 trans-activates transcription in vitro in a cell-free extract of HeLa nuclei. Quantitative analysis of the efficiency of elongation revealed that a majority of the elongation complexes generated by the HIV-1 promoter were not highly processive and terminated within the first 500 nucleotides. Tat trans-activation of transcription from the HIV-1 promoter resulted from an increase in processive character of the elongation complexes. More specifically, the analysis suggests that there exist two classes of elongation complexes initiating from the HIV promoter: a less-processive form and a more-processive form. Addition of purified Tat protein was found to increase the abundance of the more-processive class of elongation complex. The purine nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits transcription in this reaction by decreasing the efficiency of elongation. Surprisingly, stimulation of transcription elongation by Tat was preferentially inhibited by the addition of DRB. Images PMID:1756726

Expression of human placental lactogen and variant growth hormone genes in placentas.

PubMed

Martinez-Rodriguez, H G; Guerra-Rodriguez, N E; Iturbe-Cantu, M A; Martinez-Torres, A; Barrera-Saldaña, H A

1997-01-01

Previous studies comparing the expression levels of human placental lactogen (hPL) genes have shown varying results, due to, perhaps, the fact that in all of them only one placenta was being analyzed. Here, the expression of hPL and growth hormone variant (hGH-V) genes in fifteen term placentas was comparatively analyzed at the RNA level, using reverse transcription coupled to polymerase chain reaction (RT-PCR). The abundance of the combined RNA transcripts derived from these genes varied from one placenta to another. The authors found that hPL-4 transcripts were more abundant than those of hPL-3 in most samples (ratios from 1:1 to 6:1), transcripts from the putative hPL-1 pseudogene were more abundant at the unprocessed stage while those of the hGH-V gene were mostly processed. Again, the authors of this study observed wide variation from placenta to placenta in the abundance of both of these types of transcripts. The same was observed when a group of six placentas from abortuses and nine from pregnancies complicated by preclampsia, diabetes and hypertension was studied. The authors conclude that the disagreeing results reported in the literature which are not in agreement concerning the expression levels of hPL genes could be explained by normal variations of their expression levels among the different placentas analyzed.

Diversity and Spatial Distribution of Hydrazine Oxidoreductase (hzo) Gene in the Oxygen Minimum Zone Off Costa Rica

PubMed Central

Kong, Liangliang; Jing, Hongmei; Kataoka, Takafumi; Buchwald, Carolyn; Liu, Hongbin

2013-01-01

Anaerobic ammonia oxidation (anammox) as an important nitrogen loss pathway has been reported in marine oxygen minimum zones (OMZs), but the community composition and spatial distribution of anammox bacteria in the eastern tropical North Pacific (ETNP) OMZ are poorly determined. In this study, anammox bacterial communities in the OMZ off Costa Rica (CRD-OMZ) were analyzed based on both hydrazine oxidoreductase (hzo) genes and their transcripts assigned to cluster 1 and 2. The anammox communities revealed by hzo genes and proteins in CRD-OMZ showed a low diversity. Gene quantification results showed that hzo gene abundances peaked in the upper OMZs, associated with the peaks of nitrite concentration. Nitrite and oxygen concentrations may therefore colimit the distribution of anammox bacteria in this area. Furthermore, transcriptional activity of anammox bacteria was confirmed by obtaining abundant hzo mRNA transcripts through qRT-PCR. A novel hzo cluster 2x clade was identified by the phylogenetic analysis and these novel sequences were abundant and widely distributed in this environment. Our study demonstrated that both cluster 1 and 2 anammox bacteria play an active role in the CRD-OMZ, and the cluster 1 abundance and transcriptional activity were higher than cluster 2 in both free-living and particle-attached fractions at both gene and transcriptional levels. PMID:24205176

C3 exoenzyme impairs cell proliferation and apoptosis by altering the activity of transcription factors.

PubMed

von Elsner, Leonie; Hagemann, Sandra; Just, Ingo; Rohrbeck, Astrid

2016-09-01

C3 exoenzyme from C. botulinum is an ADP-ribosyltransferase that inactivates selectively RhoA, B, and C by coupling an ADP-ribose moiety. Rho-GTPases are involved in various cellular processes, such as regulation of actin cytoskeleton, cell proliferation, and apoptosis. Previous studies of our group with the murine hippocampal cell line HT22 revealed a C3-mediated inhibition of cell proliferation after 48 h and a prevention of serum-starved cells from apoptosis. For both effects, alterations of various signaling pathways are already known, including also changes on the transcriptional level. Investigations on the transcriptional activity in HT22 cells treated with C3 for 48 h identified five out of 48 transcription factors namely Sp1, ATF2, E2F-1, CBF, and Stat6 with a significantly regulated activity. For validation of identified transcription factors, studies on the protein level of certain target genes were performed. Western blot analyses exhibited an enhanced abundance of Sp1 target genes p21 and COX-2 as well as an increase in phosphorylation of c-Jun. In contrast, the level of p53 and apoptosis-inducing GADD153, a target gene of ATF2, was decreased. Our results reveal that C3 regulates the transcriptional activity of Sp1 and ATF2 resulting downstream in an altered protein abundance of various target genes. As the affected proteins are involved in the regulation of cell proliferation and apoptosis, thus the C3-mediated anti-proliferative and anti-apoptotic effects are consequences of the Rho-dependent alterations of the activity of certain transcriptional factors.

Energy status of ripening and postharvest senescent fruit of litchi (Litchi chinensis Sonn.)

PubMed Central

2013-01-01

Background Recent studies have demonstrated that cellular energy is a key factor switching on ripening and senescence of fruit. However, the factors that influence fruit energy status remain largely unknown. Results HPLC profiling showed that ATP abundance increased significantly in developing preharvest litchi fruit and was strongly correlated with fruit fresh weight. In contrast, ATP levels declined significantly during postharvest fruit senescence and were correlated with the decrease in the proportion of edible fruit. The five gene transcripts isolated from the litchi fruit pericarp were highly expressed in vegetative tissues and peaked at 70 days after flowering (DAF) consistent with fruit ADP concentrations, except for uncoupling mitochondrial protein 1 (UCP1), which was predominantly expressed in the root, and ATP synthase beta subunit (AtpB), which was up-regulated significantly before harvest and peaked 2 days after storage. These results indicated that the color-breaker stage at 70 DAF and 2 days after storage may be key turning points in fruit energy metabolism. Transcript abundance of alternative oxidase 1 (AOX1) increased after 2 days of storage to significantly higher levels than those of LcAtpB, and was down-regulated significantly by exogenous ATP. ATP supplementation had no significant effect on transcript abundance of ADP/ATP carrier 1 (AAC1) and slowed the changes in sucrose non-fermenting-1-related kinase 2 (SnRK2) expression, but maintained ATP and energy charge levels, which were correlated with delayed senescence. Conclusions Our results suggest that senescence of litchi fruit is closely related with energy. A surge of LcAtpB expression marked the beginning of fruit senescence. The findings may provide a new strategy to extend fruit shelf life by regulating its energy level. PMID:23547657

Bile acids modulate glucocorticoid metabolism and the hypothalamic–pituitary–adrenal axis in obstructive jaundice☆

PubMed Central

McNeilly, Alison D.; Macfarlane, David P.; O’Flaherty, Emmett; Livingstone, Dawn E.; Mitić, Tijana; McConnell, Kirsty M.; McKenzie, Scott M.; Davies, Eleanor; Reynolds, Rebecca M.; Thiesson, Helle C.; Skøtt, Ole; Walker, Brian R.; Andrew, Ruth

2010-01-01

Background & Aims Suppression of the hypothalamic–pituitary–adrenal axis occurs in cirrhosis and cholestasis and is associated with increased concentrations of bile acids. We investigated whether this was mediated through bile acids acting to impair steroid clearance by inhibiting glucocorticoid metabolism by 5β-reductase. Methods The effect of bile acids on glucocorticoid metabolism was studied in vitro in hepatic subcellular fractions and hepatoma cells, allowing quantitation of the kinetics and transcript abundance of 5β-reductase. Metabolism was subsequently examined in vivo in rats following dietary manipulation or bile duct ligation. Finally, glucocorticoid metabolism was assessed in humans with obstructive jaundice. Results In rat hepatic cytosol, chenodeoxycholic acid competitively inhibited 5β-reductase (Ki 9.19 ± 0.40 μM) and reduced its transcript abundance (in H4iiE cells) and promoter activity (reporter system, HepG2 cells). In Wistar rats, dietary chenodeoxycholic acid (1% w/w chow) inhibited hepatic 5β-reductase activity, reduced urinary excretion of 3α,5β-tetrahydrocorticosterone and reduced adrenal weight. Conversely, a fat-free diet suppressed bile acid levels and increased hepatic 5β-reductase activity, supplementation of the fat-free diet with CDCA reduced 5β-reductase activity, and urinary 3α,5β-reduced corticosterone. Cholestasis in rats suppressed hepatic 5β-reductase activity and transcript abundance. In eight women with obstructive jaundice, relative urinary excretion of 3α,5β-tetrahydrocortisol was significantly lower than in healthy controls. Conclusion These data suggest a novel role for bile acids in inhibiting hepatic glucocorticoid clearance, of sufficient magnitude to suppress hypothalamic–pituitary–adrenal axis activity. Elevated hepatic bile acids may account for adrenal insufficiency in liver disease. PMID:20347173

Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis

NASA Technical Reports Server (NTRS)

Scott, D. K.; Brakenhoff, K. D.; Clohisy, J. C.; Quinn, C. O.; Partridge, N. C.

1992-01-01

Collagenase is synthesized and secreted by rat osteoblastic cells in response to PTH. We have previously demonstrated that this effect involves a substantial increase in collagenase mRNA via transcription. Northern blots and nuclear run-on assays were performed to further investigate the induction of collagenase by PTH in the rat osteoblastic cell line UMR 106-01. Detectable amounts of collagenase mRNA were not apparent until 2 h of PTH treatment, showed the greatest abundance at 4 h, and declined to approximately 30% of maximum by 8 h. The changes in the rate of transcription of the collagenase gene in response to PTH paralleled and preceded the changes in the steady state mRNA levels. After an initial lag period of about 1 h, collagenase transcription rates increased from very low levels to a maximal response at 2 h, returning to about 50% of maximum by 10 h. The increased transcriptional rate of the collagenase gene was found to be dependent on the concentration of PTH, with a half-maximal response at approximately 7 x 10(-10) M rat PTH-(1-34) and a maximal effect with a dose of 10(-8) M. The PTH-mediated induction of collagenase transcriptional activity was completely abolished by cycloheximide, while transcription of the beta-actin gene was unaffected by the translation inhibitor. These data suggest that a protein factor(s) is required for PTH-mediated transcriptional induction of collagenase. Since PTH increases intracellular levels of several potential second messengers, agents that mimic these substances were employed to determine which signal transduction pathway is predominant in the PTH-mediated stimulation of collagenase transcription.(ABSTRACT TRUNCATED AT 250 WORDS).

Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

NASA Technical Reports Server (NTRS)

Murashov, A. K.; Wolgemuth, D. J.

1996-01-01

We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

Metatranscriptomics of N2-fixing cyanobacteria in the Amazon River plume

PubMed Central

Hilton, Jason A; Satinsky, Brandon M; Doherty, Mary; Zielinski, Brian; Zehr, Jonathan P

2015-01-01

Biological N2 fixation is an important nitrogen source for surface ocean microbial communities. However, nearly all information on the diversity and gene expression of organisms responsible for oceanic N2 fixation in the environment has come from targeted approaches that assay only a small number of genes and organisms. Using genomes of diazotrophic cyanobacteria to extract reads from extensive meta-genomic and -transcriptomic libraries, we examined diazotroph diversity and gene expression from the Amazon River plume, an area characterized by salinity and nutrient gradients. Diazotroph genome and transcript sequences were most abundant in the transitional waters compared with lower salinity or oceanic water masses. We were able to distinguish two genetically divergent phylotypes within the Hemiaulus-associated Richelia sequences, which were the most abundant diazotroph sequences in the data set. Photosystem (PS)-II transcripts in Richelia populations were much less abundant than those in Trichodesmium, and transcripts from several Richelia PS-II genes were absent, indicating a prominent role for cyclic electron transport in Richelia. In addition, there were several abundant regulatory transcripts, including one that targets a gene involved in PS-I cyclic electron transport in Richelia. High sequence coverage of the Richelia transcripts, as well as those from Trichodesmium populations, allowed us to identify expressed regions of the genomes that had been overlooked by genome annotations. High-coverage genomic and transcription analysis enabled the characterization of distinct phylotypes within diazotrophic populations, revealed a distinction in a core process between dominant populations and provided evidence for a prominent role for noncoding RNAs in microbial communities. PMID:25514535

Expression profiles of putative defence-related proteins in oil palm (Elaeis guineensis) colonized by Ganoderma boninense.

PubMed

Tan, Yung-Chie; Yeoh, Keat-Ai; Wong, Mui-Yun; Ho, Chai-Ling

2013-11-01

Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection. Copyright © 2013 Elsevier GmbH. All rights reserved.

Differences in transcript abundance of genes on BTA15 located within a region associated with gain in beef steers

USDA-ARS?s Scientific Manuscript database

Six markers on the Illumina Bovine 50k BeadChip within a 229 Kb region on bovine chromosome 15 were associated (P=0.002) with average daily gain (ADG) in beef cattle. We chose to evaluate seven genes located within this region for variation in RNA transcript abundance in a library of tissue samples ...

Molecular analysis of phosphate limitation in Geobacteraceae during the bioremediation of a uranium-contaminated aquifer

DOE Office of Scientific and Technical Information (OSTI.GOV)

N'Guessan, L.A.; Elifantz, H.; Nevin, K.P.

2009-09-01

Nutrient limitation is an environmental stress that may reduce the effectiveness of bioremediation strategies, especially when the contaminants are organic compounds or when organic compounds are added to promote microbial activities such as metal reduction. Genes indicative of phosphate-limitation were identified via microarray analysis of chemostat cultures of Geobacter sulfureducens. This analysis revealed that genes in the pst-pho operon, which is associated with a high affinity phosphate uptake system in other microorganisms, had significantly higher transcript abundance under phosphate-limiting conditions, with the genes pstB and phoU the most up-regulated. Quantitative PCR analysis of pstB and phoU transcript levels in G.more » sulfurreducens grown in chemostats demonstrated that the expression of these genes increased when phosphate was removed from the culture medium. Transcripts of pstB and phoU within the subsurface Geobacter species predominating during an in situ uranium bioremediation field experiment were more abundant than in chemostat cultures of G. sulfurreducens that were not limited for phosphate. Addition of phosphate to incubations of subsurface sediments did not stimulate dissimilatory metal reduction. The added phosphate was rapidly adsorbed onto the sediments. The results demonstrate that Geobacter species can effectively reduce U(VI) even when experiencing suboptimal phosphate concentrations and that increasing phosphate availability with phosphate additions is difficult to achieve due to the high reactivity of this compound. This transcript-based approach developed for diagnosing phosphate limitation should be applicable to assessing the potential need for additional phosphate in other bioremediation processes.« less

Molecular Analysis of Phosphate Limitation in Geobacteraceae During the Bioremediation of a Uranium-Contaminated Aquifer

DOE Office of Scientific and Technical Information (OSTI.GOV)

N'Guessan, A. Lucie; Elifantz, H.; Nevin, Kelly P.

2010-02-01

Nutrient limitation is an environmental stress that may reduce the effectiveness of bioremediation strategies, especially when the contaminants are organic compounds or when organic compounds are added to promote microbial activities such as metal reduction. Genes indicative of phosphate-limitation were identified via microarray analysis of chemostat cultures of Geobacter sulfureducens. This analysis revealed that genes in the pst-pho operon, which is associated with a high affinity phosphate uptake system in other microorganisms, had significantly higher transcript abundance under phosphate-limiting conditions, with the genes pstB and phoU the most up-regulated. Quantitative PCR analysis of pstB and phoU transcript levels in G.more » sulfurreducens grown in chemostats demonstrated that the expression of these genes increased when phosphate was removed from the culture medium. Transcripts of pstB and phoU within the subsurface Geobacter species predominating during an in situ uranium bioremediation field experiment were more abundant than in chemostat cultures of G. sulfurreducens that were not limited for phosphate. Addition of phosphate to incubations of subsurface sediments did not stimulate dissimilatory metal reduction. The added phosphate was rapidly adsorbed onto the sediments. The results demonstrate that Geobacter species can effectively reduce U(VI) even when experiencing suboptimal phosphate concentrations and that increasing phosphate availability with phosphate additions is difficult to achieve due to the high reactivity of this compound. This transcript-based approach developed for diagnosing phosphate limitation should be applicable to assessing the potential need for additional phosphate in other bioremediation processes.« less

Molecular analysis of phosphate limitation in Geobacteraceae during the bioremediation of a uranium-contaminated aquifer

DOE Office of Scientific and Technical Information (OSTI.GOV)

N'Guessan, A. Lucie; Elifantz, H.; Nevin, Kelly P.

2010-01-10

Nutrient limitation is an environmental stress that may reduce the effectiveness of bioremediation strategies, especially when the contaminants are organic compounds or when organic compounds are added to promote microbial activities such as metal reduction. Genes indicative of phosphatelimitation were identified by microarray analysis of chemostat cultures of Geobacter sulfureducens. This analysis revealed that genes in the pst-pho operon, which is associated with a high-affinity phosphate uptake system in other microorganisms, had significantly higher transcript abundance under phosphate-limiting conditions, with the genes pstB and phoU upregulated the most. Quantitative PCR analysis of pstB and phoU transcript levels in G. sulfurreducensmore » grown in chemostats demonstrated that the expression of these genes increased when phosphate was removed from the culture medium. Transcripts of pstB and phoU within the subsurface Geobacter species predominating during an in situ uranium-bioremediation field experiment were more abundant than in chemostat cultures of G. sulfurreducens that were not limited for phosphate. Addition of phosphate to incubations of subsurface sediments did not stimulate dissimilatory metal reduction. The added phosphate was rapidly adsorbed onto the sediments. The results demonstrate that Geobacter species can effectively reduce U(VI) even when experiencing suboptimal phosphate concentrations and that increasing phosphate availability with phosphate additions is difficult to achieve because of the high reactivity of this compound. This transcript-based approach developed for diagnosing phosphate limitation should be applicable to assessing the potential need for additional phosphate in other bioremediation processes.« less

Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism1[W][OA

PubMed Central

Zifkin, Michael; Jin, Alena; Ozga, Jocelyn A.; Zaharia, L. Irina; Schernthaner, Johann P.; Gesell, Andreas; Abrams, Suzanne R.; Kennedy, James A.; Constabel, C. Peter

2012-01-01

Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3′-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3′5′-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis. PMID:22086422

Gene expression and metabolite profiling of developing highbush blueberry fruit indicates transcriptional regulation of flavonoid metabolism and activation of abscisic acid metabolism.

PubMed

Zifkin, Michael; Jin, Alena; Ozga, Jocelyn A; Zaharia, L Irina; Schernthaner, Johann P; Gesell, Andreas; Abrams, Suzanne R; Kennedy, James A; Constabel, C Peter

2012-01-01

Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3'-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3'5'-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis.

Elevated ATF4 Expression, in the Absence of Other Signals, Is Sufficient for Transcriptional Induction via CCAAT Enhancer-binding Protein-activating Transcription Factor Response Elements*

PubMed Central

Shan, Jixiu; Örd, Daima; Örd, Tõnis; Kilberg, Michael S.

2009-01-01

Protein limitation in vivo or amino acid deprivation of cells in culture causes a signal transduction cascade consisting of activation of the kinase GCN2 (general control nonderepressible 2), phosphorylation of eukaryotic initiation factor 2, and increased synthesis of activating transcription factor (ATF) 4 by a translational control mechanism. In a self-limiting transcriptional program, ATF4 transiently activates a wide range of downstream target genes involved in transport, cellular metabolism, and other cell functions. Simultaneous activation of other signal transduction pathways by amino acid deprivation led to the question of whether or not the increased abundance of ATF4 alone was sufficient to trigger the transcriptional control mechanisms. Using 293 cells that ectopically express ATF4 in a tetracycline-inducible manner showed that ATF4 target genes were activated in the absence of amino acid deprivation. Ectopic expression of ATF4 alone resulted in effective recruitment of the general transcription machinery, but some reduction in histone modification was observed. These data document that ATF4 alone is sufficient to trigger the amino acid-responsive transcriptional control program. However, the absolute amount of ectopic ATF4 required to achieve the same degree of transcriptional activation observed after amino acid limitation was greater, suggesting that other factors may serve to enhance ATF4 function. PMID:19509279

Multiple Transcript Properties Related to Translation Affect mRNA Degradation Rates in Saccharomyces cerevisiae

PubMed Central

Neymotin, Benjamin; Ettorre, Victoria; Gresham, David

2016-01-01

Degradation of mRNA contributes to variation in transcript abundance. Studies of individual mRNAs have shown that both cis and trans factors affect mRNA degradation rates. However, the factors underlying transcriptome-wide variation in mRNA degradation rates are poorly understood. We investigated the contribution of different transcript properties to transcriptome-wide degradation rate variation in the budding yeast, Saccharomyces cerevisiae, using multiple regression analysis. We find that multiple transcript properties are significantly associated with variation in mRNA degradation rates, and that a model incorporating these properties explains ∼50% of the genome-wide variance. Predictors of mRNA degradation rates include transcript length, ribosome density, biased codon usage, and GC content of the third position in codons. To experimentally validate these factors, we studied individual transcripts expressed from identical promoters. We find that decreasing ribosome density by mutating the first translational start site of a transcript increases its degradation rate. Using coding sequence variants of green fluorescent protein (GFP) that differ only at synonymous sites, we show that increased GC content of the third position of codons results in decreased rates of mRNA degradation. Thus, in steady-state conditions, a large fraction of genome-wide variation in mRNA degradation rates is determined by inherent properties of transcripts, many of which are related to translation, rather than specific regulatory mechanisms. PMID:27633789

The Resilience of Microbial Community under Drying and Rewetting Cycles of Three Forest Soils.

PubMed

Zhou, Xue; Fornara, Dario; Ikenaga, Makoto; Akagi, Isao; Zhang, Ruifu; Jia, Zhongjun

2016-01-01

Forest soil ecosystems are associated with large pools and fluxes of carbon (C) and nitrogen (N), which could be strongly affected by variation in rainfall events under current climate change. Understanding how dry and wet cycle events might influence the metabolic state of indigenous soil microbes is crucial for predicting forest soil responses to environmental change. We used 454 pyrosequencing and quantitative PCR to address how present (DNA-based) and potentially active (RNA-based) soil bacterial communities might response to the changes in water availability across three different forest types located in two continents (Africa and Asia) under controlled drying and rewetting cycles. Sequencing of rRNA gene and transcript indicated that Proteobacteria, Actinobacteria, and Acidobacteria were the most responsive phyla to changes in water availability. We defined the ratio of rRNA transcript to rRNA gene abundance as a key indicator of potential microbial activity and we found that this ratio was increased following soil dry-down process whereas it decreased after soil rewetting. Following rewetting Crenarchaeota-like 16S rRNA gene transcript increased in some forest soils and this was linked to increases in soil nitrate levels suggesting greater nitrification rates under higher soil water availability. Changes in the relative abundance of (1) different microbial phyla and classes, and (2) 16S and amoA genes were found to be site- and taxa-specific and might have been driven by different life-strategies. Overall, we found that, after rewetting, the structure of the present and potentially active bacterial community structure as well as the abundance of bacterial (16S), archaeal (16S) and ammonia oxidizers (amoA), all returned to pre-dry-down levels. This suggests that microbial taxa have the ability to recover from desiccation, a critical response, which will contribute to maintaining microbial biodiversity in harsh ecosystems under environmental perturbations, such as significant changes in water availability.

The Resilience of Microbial Community under Drying and Rewetting Cycles of Three Forest Soils

PubMed Central

Zhou, Xue; Fornara, Dario; Ikenaga, Makoto; Akagi, Isao; Zhang, Ruifu; Jia, Zhongjun

2016-01-01

Forest soil ecosystems are associated with large pools and fluxes of carbon (C) and nitrogen (N), which could be strongly affected by variation in rainfall events under current climate change. Understanding how dry and wet cycle events might influence the metabolic state of indigenous soil microbes is crucial for predicting forest soil responses to environmental change. We used 454 pyrosequencing and quantitative PCR to address how present (DNA-based) and potentially active (RNA-based) soil bacterial communities might response to the changes in water availability across three different forest types located in two continents (Africa and Asia) under controlled drying and rewetting cycles. Sequencing of rRNA gene and transcript indicated that Proteobacteria, Actinobacteria, and Acidobacteria were the most responsive phyla to changes in water availability. We defined the ratio of rRNA transcript to rRNA gene abundance as a key indicator of potential microbial activity and we found that this ratio was increased following soil dry-down process whereas it decreased after soil rewetting. Following rewetting Crenarchaeota-like 16S rRNA gene transcript increased in some forest soils and this was linked to increases in soil nitrate levels suggesting greater nitrification rates under higher soil water availability. Changes in the relative abundance of (1) different microbial phyla and classes, and (2) 16S and amoA genes were found to be site- and taxa-specific and might have been driven by different life-strategies. Overall, we found that, after rewetting, the structure of the present and potentially active bacterial community structure as well as the abundance of bacterial (16S), archaeal (16S) and ammonia oxidizers (amoA), all returned to pre-dry-down levels. This suggests that microbial taxa have the ability to recover from desiccation, a critical response, which will contribute to maintaining microbial biodiversity in harsh ecosystems under environmental perturbations, such as significant changes in water availability. PMID:27486444

Heat stress during in vitro fertilization decreases fertilization success by disrupting anti-polyspermy systems of the oocytes.

PubMed

Sakatani, Miki; Yamanaka, Kenichi; Balboula, Ahmed Z; Takenouchi, Naoki; Takahashi, Masashi

2015-01-01

Low pregnancy rates during the summer are due, in part, to reduced fertilization. Given that elevated temperature is associated with this season, we investigated the effect of heat stress during fertilization using an in vitro model. Three experiments were performed to determine the mechanism by which exposure to elevated temperature disrupts fertilization. Oocytes were fertilized for 6 hr at 38.5°C or 41.0°C or 40.0°C with non-pre-incubated sperm, or for 6 hr at 38.5°C with sperm that had been pre-incubated at 38.5°C or 41.0°C for 4 hr. In each experiment, zygotes were cultured at 38.5°C in 5% CO(2) and 5% O(2). Rates of cleavage and blasocyst formation were reduced when fertilization occurs at elevated temperatures. The percent of sperm classified as alive, using fluorescein diacetate labeling, was decreased by pre-incubation and fertilization at 40.0°C. Although no difference was observed in sperm penetration rate, polyspermy tended to be increased by heat stress during fertilization. The zona pellucidae of zygotes formed following fertilization at 40.0°C for 6 hr were more sensitive to digestion with pronase. Furthermore, these zygotes exhibited higher hydrogen peroxide levels, measured by 2,7-dihydrodichlorofluorescein diacetate staining, and showed increased transcript abundance for HSPA1A, a gene involved in the heat-shock response, but decreased transcript abundance for UCHL1, a gene involved in preventing polyspermy. Results indicate that heat stress during fertilization is lethal to sperm, and causes oxidative stress, altered transcript abundance, and a defective block to polyspermy in the zygote. Thus, an increase in polyspermy is likely one cause of the reduced competency of zygotes fertilized under elevated temperatures to develop to the blastocyst stage. © 2014 Wiley Periodicals, Inc.

NR and High-Throughput Screening: Putting the Pieces Together Chemicals

EPA Science Inventory

Nuclear receptors (NR) are one of the most abundant classes of transcriptional regulators in animals and function as ligand-activated transcription factors. They provide a direct link between signaling molecules and transcriptional responses that impact diverse functions includin...

Ethylene-induced gene expression, enzyme activities, and water soaking in immature and ripe watermelon (Citrullus lanatus) fruit.

PubMed

Karakurt, Yasar; Huber, Donald J

2004-04-01

Watermelon fruit exhibit acute softening and placental-tissue water soaking following short exposure to exogenous ethylene. Experiments were performed to address transcript abundance and activities of cell wall and membrane hydrolases in placental tissue in response to treatment of watermelon fruit with ethylene. Watermelon fruit were harvested at immature and full-ripe stages and exposed to 50 microL L(-1) ethylene for 6 days at 20 degrees C. Ethylene affected the abundance of transcripts for PME (EC 3.2.1.11), and alpha-(EC 3.2.1.22) and beta-GAL (EC 3.2.1.23) but these effects were dependent on fruit maturity and appeared not to be associated with the water-soaking syndrome. PG (EC 3.2.1.15) and EXP mRNAs accumulated significantly in response to ethylene exposure. Additionally, the levels of mRNA and activities of LOX (EC 1.13.11.12), PLC (EC 3.1.4.3) and PLD (EC 3.1.4.4) were elevated in fruit of both maturity classes exposed to ethylene and were temporally associated with the visible symptoms of water soaking. The activity trends and transcript abundance in ethylene- compared with air-treated fruit indicate that PG, EXP, LOX, PLC and PLD levels increase with the onset and development of the water-soaking disorder and support the view that catabolic reactions targeting the membranes and cell-walls contribute to the disorder.

Transcriptomes That Confer to Plant Defense against Powdery Mildew Disease in Lagerstroemia indica

PubMed Central

Shi, Weibing; Rinehart, Timothy

2015-01-01

Transcriptome analysis was conducted in two popular Lagerstroemia cultivars: “Natchez” (NAT), a white flower and powdery mildew resistant interspecific hybrid and “Carolina Beauty” (CAB), a red flower and powdery mildew susceptible L. indica cultivar. RNA-seq reads were generated from Erysiphe australiana infected leaves and de novo assembled. A total of 37,035 unigenes from 224,443 assembled contigs in both genotypes were identified. Approximately 85% of these unigenes have known function. Of them, 475 KEGG genes were found significantly different between the two genotypes. Five of the top ten differentially expressed genes (DEGs) involved in the biosynthesis of secondary metabolites (plant defense) and four in flavonoid biosynthesis pathway (antioxidant activities or flower coloration). Furthermore, 5 of the 12 assembled unigenes in benzoxazinoid biosynthesis and 7 of 11 in flavonoid biosynthesis showed higher transcript abundance in NAT. The relative abundance of transcripts for 16 candidate DEGs (9 from CAB and 7 from NAT) detected by qRT-PCR showed general agreement with the abundances of the assembled transcripts in NAT. This study provided the first transcriptome analyses in L. indica. The differential transcript abundance between two genotypes indicates that it is possible to identify candidate genes that are associated with the plant defenses or flower coloration. PMID:26247009

Overexpression of antibiotic resistance genes in hospital effluents over time.

PubMed

Rowe, Will P M; Baker-Austin, Craig; Verner-Jeffreys, David W; Ryan, Jim J; Micallef, Christianne; Maskell, Duncan J; Pearce, Gareth P

2017-06-01

Effluents contain a diverse abundance of antibiotic resistance genes that augment the resistome of receiving aquatic environments. However, uncertainty remains regarding their temporal persistence, transcription and response to anthropogenic factors, such as antibiotic usage. We present a spatiotemporal study within a river catchment (River Cam, UK) that aims to determine the contribution of antibiotic resistance gene-containing effluents originating from sites of varying antibiotic usage to the receiving environment. Gene abundance in effluents (municipal hospital and dairy farm) was compared against background samples of the receiving aquatic environment (i.e. the catchment source) to determine the resistome contribution of effluents. We used metagenomics and metatranscriptomics to correlate DNA and RNA abundance and identified differentially regulated gene transcripts. We found that mean antibiotic resistance gene and transcript abundances were correlated for both hospital ( ρ  = 0.9, two-tailed P  <0.0001) and farm ( ρ  = 0.5, two-tailed P   <0.0001) effluents and that two β-lactam resistance genes ( bla GES and bla OXA ) were overexpressed in all hospital effluent samples. High β-lactam resistance gene transcript abundance was related to hospital antibiotic usage over time and hospital effluents contained antibiotic residues. We conclude that effluents contribute high levels of antibiotic resistance genes to the aquatic environment; these genes are expressed at significant levels and are possibly related to the level of antibiotic usage at the effluent source. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

Overexpression of antibiotic resistance genes in hospital effluents over time

PubMed Central

Baker-Austin, Craig; Verner-Jeffreys, David W.; Ryan, Jim J.; Micallef, Christianne; Maskell, Duncan J.; Pearce, Gareth P.

2017-01-01

Objectives: Effluents contain a diverse abundance of antibiotic resistance genes that augment the resistome of receiving aquatic environments. However, uncertainty remains regarding their temporal persistence, transcription and response to anthropogenic factors, such as antibiotic usage. We present a spatiotemporal study within a river catchment (River Cam, UK) that aims to determine the contribution of antibiotic resistance gene-containing effluents originating from sites of varying antibiotic usage to the receiving environment. Methods: Gene abundance in effluents (municipal hospital and dairy farm) was compared against background samples of the receiving aquatic environment (i.e. the catchment source) to determine the resistome contribution of effluents. We used metagenomics and metatranscriptomics to correlate DNA and RNA abundance and identified differentially regulated gene transcripts. Results: We found that mean antibiotic resistance gene and transcript abundances were correlated for both hospital (ρ = 0.9, two-tailed P <0.0001) and farm (ρ = 0.5, two-tailed P  <0.0001) effluents and that two β-lactam resistance genes (blaGES and blaOXA) were overexpressed in all hospital effluent samples. High β-lactam resistance gene transcript abundance was related to hospital antibiotic usage over time and hospital effluents contained antibiotic residues. Conclusions: We conclude that effluents contribute high levels of antibiotic resistance genes to the aquatic environment; these genes are expressed at significant levels and are possibly related to the level of antibiotic usage at the effluent source. PMID:28175320

Metabolic effects of dietary sugar beet pulp or wheat bran in growing female pigs.

PubMed

Weber, T E; Kerr, B J

2012-02-01

An experiment was conducted to determine the effects of feeding a moderate level of 2 different fiber sources on energy metabolites; mitochondrial biogenesis in the intestine, liver, and muscle; and the expression of some genes that regulate energy metabolism in intestine, liver, muscle, and adipose tissue. Female pigs (n = 36; BW = 15.0 ± 0.7 kg) were fed diets containing no added fiber, 12.5% sugar beet pulp (SBP), or 12.5% wheat bran (WB) for 24 d. Blood samples were collected on d 7 and 24 for cholesterol, glucose, NEFA, and triglyceride analyses. At completion of the experiment, ileum, colon, subcutaneous adipose, and LM samples were obtained from a subset (n = 6) of pigs fed each diet for analysis of tissue mitochondrial DNA (mtDNA) content and mRNA abundance by quantitative real-time reverse-transcription PCR. Glycogen and triglyceride content of liver and LM were determined, and colon content VFA was also determined. The addition of SBP or WB to the diet had no effect (P > 0.55) on ADG, ADFI, or G:F. Serum NEFA and triglycerides were increased (P < 0.05) in pigs fed SBP compared with pigs fed the control diet or WB on d 7, and NEFA remained increased (P < 0.05) on d 24 in pigs fed SBP. Dietary fiber had no effect (P > 0.24) on glycogen and triglyceride content of liver or LM, but colonic acetate concentrations were increased (P < 0.05) in pigs fed either SBP or WB. Pigs fed WB had an increased (P < 0.05) mtDNA content in ileum tissue and increased (P < 0.05) citrate synthase mRNA in colon tissue. In the liver, feeding either SBP or WB led to a decrease (P < 0.05) in mtDNA content, whereas feeding WB decreased (P < 0.05) mtDNA abundance in the LM, and feeding either SBP or WB decreased (P < 0.05) expression of citrate synthase mRNA. Quantitative reverse-transcription PCR revealed that feeding WB increased (P < 0.05) proliferating cell nuclear antigen mRNA abundance in the ileum and colon. Feeding WB increased (P < 0.05) mRNA abundance of a regulator of mitochondrial biogenesis, PPAR coactivator 1 α, in ileum tissue, and increased (P < 0.05) mRNA abundance of another mediator of mitochondrial biogensis, sirtuin 1, in colon tissue. Colonic mRNA expression of fasting-induced adipose factor was increased (P < 0.05) in pigs fed either SBP or WB, and adipose triglyceride lipase mRNA abundance was increased (P < 0.05) in adipose tissue of pigs fed SBP. These data indicate that increasing dietary fiber can increase the capacity of the intestine for oxidative metabolism and induce a repartitioning of energy metabolites depending on fiber source.

27-Hydroxycholesterol increases Myc protein stability via suppressing PP2A, SCP1 and FBW7 transcription in MCF-7 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Li-Ming; Liang, Zi-Rui; Zhou, Ke-Ren

27-hydroxycholesterol (27-HC), the most abundant metabolite of cholesterol, is a risk factor for breast cancer. It can increase the proliferation of breast cancer cells and promote the metastasis of breast tumours in mouse models. Myc is a critical oncoprotein overexpressed in breast cancer. However, whether 27-HC affects Myc expression has not been reported. In the current study, we aimed to investigate the effects of 27-HC on Myc and the underlying mechanisms in MCF-7 breast cancer cells. Our data demonstrated that 27-HC activated Myc via increasing its protein stability. Three key negative modulators of Myc protein stability, PP2A, SCP1 and FBW7,more » were suppressed by 27-HC at the transcriptional level. We performed a data-mining analysis of the chromatin immunoprecipitation with next-generation DNA sequencing (ChIP-Seq) data in the ChIPBase, and discovered that a number of putative transcription factors (TFs), including Myc itself, were involved in the transcriptional regulation of PP2A, SCP1 and FBW7. Our results provide a novel mechanistic insight into the activation of Myc by 27-HC via transcriptional repression of PP2A, SCP1 and FBW7 to increase Myc protein stability in breast cancer cells. - Highlights: • 27-Hydroxycholesterol (27-HC) activates Myc via increasing its protein stability. • 27-HC inhibits PP2A and SCP1 transcription to block pS62-Myc dephosphorylation. • 27-HC suppresses FBW7 transcription to prevent pT58-Myc degradation.« less

Regulation of alternative splicing by the circadian clock and food related cues

PubMed Central

2012-01-01

Background The circadian clock orchestrates daily rhythms in metabolism, physiology and behaviour that allow organisms to anticipate regular changes in their environment, increasing their adaptation. Such circadian phenotypes are underpinned by daily rhythms in gene expression. Little is known, however, about the contribution of post-transcriptional processes, particularly alternative splicing. Results Using Affymetrix mouse exon-arrays, we identified exons with circadian alternative splicing in the liver. Validated circadian exons were regulated in a tissue-dependent manner and were present in genes with circadian transcript abundance. Furthermore, an analysis of circadian mutant Vipr2-/- mice revealed the existence of distinct physiological pathways controlling circadian alternative splicing and RNA binding protein expression, with contrasting dependence on Vipr2-mediated physiological signals. This view was corroborated by the analysis of the effect of fasting on circadian alternative splicing. Feeding is an important circadian stimulus, and we found that fasting both modulates hepatic circadian alternative splicing in an exon-dependent manner and changes the temporal relationship with transcript-level expression. Conclusions The circadian clock regulates alternative splicing in a manner that is both tissue-dependent and concurrent with circadian transcript abundance. This adds a novel temporal dimension to the regulation of mammalian alternative splicing. Moreover, our results demonstrate that circadian alternative splicing is regulated by the interaction between distinct physiological cues, and illustrates the capability of single genes to integrate circadian signals at different levels of regulation. PMID:22721557

Systems-wide analysis of manganese deficiency-induced changes in gene activity of Arabidopsis roots

PubMed Central

Rodríguez-Celma, Jorge; Tsai, Yi-Hsiu; Wen, Tuan-Nan; Wu, Yu-Ching; Curie, Catherine; Schmidt, Wolfgang

2016-01-01

Manganese (Mn) is pivotal for plant growth and development, but little information is available regarding the strategies that evolved to improve Mn acquisition and cellular homeostasis of Mn. Using an integrated RNA-based transcriptomic and high-throughput shotgun proteomics approach, we generated a comprehensive inventory of transcripts and proteins that showed altered abundance in response to Mn deficiency in roots of the model plant Arabidopsis. A suite of 22,385 transcripts was consistently detected in three RNA-seq runs; LC-MS/MS-based iTRAQ proteomics allowed the unambiguous determination of 11,606 proteins. While high concordance between mRNA and protein expression (R = 0.87) was observed for transcript/protein pairs in which both gene products accumulated differentially upon Mn deficiency, only approximately 10% of the total alterations in the abundance of proteins could be attributed to transcription, indicating a large impact of protein-level regulation. Differentially expressed genes spanned a wide range of biological functions, including the maturation, translation, and transport of mRNAs, as well as primary and secondary metabolic processes. Metabolic analysis by UPLC-qTOF-MS revealed that the steady-state levels of several major glucosinolates were significantly altered upon Mn deficiency in both roots and leaves, possibly as a compensation for increased pathogen susceptibility under conditions of Mn deficiency. PMID:27804982

Maternal Choline Supplementation Modulates Placental Nutrient Transport and Metabolism in Late Gestation of Mouse Pregnancy.

PubMed

Kwan, Sze Ting Cecilia; King, Julia H; Yan, Jian; Wang, Zhen; Jiang, Xinyin; Hutzler, Jason S; Klein, Hallie R; Brenna, J Thomas; Roberson, Mark S; Caudill, Marie A

2017-11-01

Background: Fetal growth is dependent on placental nutrient supply, which is influenced by placental perfusion and transporter abundance. Previous research indicates that adequate choline nutrition during pregnancy improves placental vascular development, supporting the hypothesis that choline may affect placental nutrient transport. Objective: The present study sought to determine the impact of maternal choline supplementation (MCS) on placental nutrient transporter abundance and nutrient metabolism during late gestation. Methods: Female non-Swiss albino mice were randomly assigned to the 1×, 2×, or 4× choline diet (1.4, 2.8, and 5.6 g choline chloride/kg diet, respectively) 5 d before mating ( n = 16 dams/group). The placentas and fetuses were harvested on gestational day (E) 15.5 and E18.5. The placental abundance of macronutrient, choline, and acetylcholine transporters and glycogen metabolic enzymes, and the placental concentration of glycogen were quantified. Choline metabolites and docosahexaenoic acid (DHA) concentrations were measured in the placentas and/or fetal brains. Data were stratified by gestational day and fetal sex and were analyzed by using mixed linear models. Results: At E15.5, MCS downregulated the placental transcript and protein abundance of glucose transporter 1 (GLUT1) (-40% to -73%, P < 0.05) and the placental transcript abundance of glycogen-synthesizing enzymes (-24% to -50%, P ≤ 0.05). At E18.5, MCS upregulated GLUT3 protein abundance (+55%, P = 0.016) and the transcript abundance of glycogen-synthesizing enzymes only in the female placentas (+36% to +60%, P < 0.05), resulting in a doubling ( P = 0.01) of the glycogen concentration. A higher placental transcript abundance of the transporters for DHA, choline, and acetylcholine was also detected in response to MCS, consequently altering their concentrations in the placentas or fetal brains ( P ≤ 0.05). Conclusions: These data suggest that MCS modulates placental nutrient transporter abundance and nutrient metabolism in late gestation of mouse pregnancy, with subsequent effects on nutrient supply for the developing fetus. © 2017 American Society for Nutrition.

Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks.

PubMed

Han, L Q; Zhou, Z; Ma, Y; Batistel, F; Osorio, J S; Loor, J J

2018-04-18

Changes in the production of reactive oxygen species in the mammary gland of dairy cows during the periparturient period could lead to oxidative stress and potentially impair mammary function. Phosphorylation of the transcription factor nuclear factor erythroid 2-like 2 (NFE2L2), also known as nuclear factor-E2-related factor 2, controls mRNA abundance of genes encoding antioxidant proteins and enzymes. The hypothesis was that NFE2L2 phosphorylation status and target gene mRNA abundance in the mammary gland of dairy cows is altered around parturition. Total NFE2L2 protein, phosphorylated protein (p-NFE2L2), and ratio of p-NFE2L2 to NFE2L2 along with mRNA abundance of 24 genes related to the NFE2L2 signaling pathway, apoptosis, and cell proliferation were measured in mammary tissue samples from Holstein cows at -30, 1, 15, and 30 d relative to parturition. Although total NFE2L2 protein abundance did not differ, p-NFE2L2 and p-NFE2L2-to-NFE2L2 ratio were greater after parturition. The upregulation of DNA damage inducible transcript 3 (DDIT3) postpartum indicated a localized oxidative stress state. Among genes evaluated, thioredoxin (TXN), glutathione peroxidase 1 (GPX1), and glutathione S-transferase mu 1 (GSTM1) had the highest (37.1, 15.1, and 4.8% of total mRNA measured, respectively) abundance. The mRNA abundance of various target genes with detoxifying enzymatic functions and free radical scavenging activities [glutamate-cysteine ligase catalytic subunit (GCLC); glutathione reductase (GSR); ferrochelatase (FECH); TXN; thioredoxin reductase 1 (TXNRD1); and NAD(P)H quinone dehydrogenase 1 (NQO1)] were consistently upregulated (linear effect of time) as parturition approached and lactation began. Among the transcription regulators, NFE2L2 had the highest mRNA abundance (7.3% of total mRNA measured). Abundance of NFE2L2 and other transcription factors [nuclear factor kappa B subunit 1 (NFKB1), retinoid X receptor α (RXRA), and mitogen-activated protein kinase 14 (MAPK14)] were upregulated (linear effect of time) from -30 d to 30 d relative to parturition. Overall, NFE2L2 phosphorylation and downstream signaling leading to postpartal upregulation of genes associated with oxidative stress and inflammation in the mammary gland seem to be key components of normal cellular function to maintain proper redox homeostasis. However, if the longitudinal increases in mRNA and protein abundance of these antioxidant mechanisms are a reflection of cellular oxidative stress, then the likelihood of protein and DNA damage would be greater and might be one factor compromising cell viability and potentially lactation persistency. The actual cues coordinating these molecular responses remain to be determined. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity.

PubMed

Finley, Lydia W S; Vardhana, Santosha A; Carey, Bryce W; Alonso-Curbelo, Direna; Koche, Richard; Chen, Yanyang; Wen, Duancheng; King, Bryan; Radler, Megan R; Rafii, Shahin; Lowe, Scott W; Allis, C David; Thompson, Craig B

2018-05-01

A robust network of transcription factors and an open chromatin landscape are hallmarks of the naive pluripotent state. Recently, the acetyllysine reader Brd4 has been implicated in stem cell maintenance, but the relative contribution of Brd4 to pluripotency remains unclear. Here, we show that Brd4 is dispensable for self-renewal and pluripotency of embryonic stem cells (ESCs). When maintained in their ground state, ESCs retain transcription factor binding and chromatin accessibility independent of Brd4 function or expression. In metastable ESCs, Brd4 independence can be achieved by increased expression of pluripotency transcription factors, including STAT3, Nanog or Klf4, so long as the DNA methylcytosine oxidases Tet1 and Tet2 are present. These data reveal that Brd4 is not essential for ESC self-renewal. Rather, the levels of pluripotency transcription factor abundance and Tet1/2 function determine the extent to which bromodomain recognition of protein acetylation contributes to the maintenance of gene expression and cell identity.

Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.

PubMed

Borkowski, Olivier; Goelzer, Anne; Schaffer, Marc; Calabre, Magali; Mäder, Ulrike; Aymerich, Stéphane; Jules, Matthieu; Fromion, Vincent

2016-05-17

Complex regulatory programs control cell adaptation to environmental changes by setting condition-specific proteomes. In balanced growth, bacterial protein abundances depend on the dilution rate, transcript abundances and transcript-specific translation efficiencies. We revisited the current theory claiming the invariance of bacterial translation efficiency. By integrating genome-wide transcriptome datasets and datasets from a library of synthetic gfp-reporter fusions, we demonstrated that translation efficiencies in Bacillus subtilis decreased up to fourfold from slow to fast growth. The translation initiation regions elicited a growth rate-dependent, differential production of proteins without regulators, hence revealing a unique, hard-coded, growth rate-dependent mode of regulation. We combined model-based data analyses of transcript and protein abundances genome-wide and revealed that this global regulation is extensively used in B. subtilis We eventually developed a knowledge-based, three-step translation initiation model, experimentally challenged the model predictions and proposed that a growth rate-dependent drop in free ribosome abundance accounted for the differential protein production. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Transient dissociation of polyribosomes and concurrent recruitment of calreticulin and calmodulin transcripts in gravistimulated maize pulvini

NASA Technical Reports Server (NTRS)

Heilmann, I.; Shin, J.; Huang, J.; Perera, I. Y.; Davies, E.

2001-01-01

The dynamics of polyribosome abundance were studied in gravistimulated maize (Zea mays) stem pulvini. During the initial 15 min of gravistimulation, the amount of large polyribosomes transiently decreased. The transient decrease in polyribosome levels was accompanied by a transient decrease in polyribosome-associated mRNA. After 30 min of gravistimulation, the levels of polyribosomes and the amount of polyribosome-associated mRNA gradually increased over 24 h up to 3- to 4-fold of the initial value. Within 15 min of gravistimulation, total levels of transcripts coding for calreticulin and calmodulin were elevated 5-fold in maize pulvinus total RNA. Transcripts coding for calreticulin and calmodulin were recruited into polyribosomes within 15 min of gravistimulation. Over 4 h of gravistimulation, a gradual increase in the association of calreticulin and calmodulin transcripts with polyribosomes was seen predominantly in the lower one-half of the maize pulvinus; the association of transcripts for vacuolar invertase with polyribosomes did not change over this period. Our results suggest that within 15 min of gravistimulation, the translation of the majority of transcripts associated with polyribosomes decreased, resembling a general stress response. Recruitment of calreticulin and calmodulin transcripts into polyribosomes occurred predominantly in the lower pulvinus one-half during the first 4 h when the presentation time for gravistimulation in the maize pulvinus is not yet complete.

Gene expression in the twilight of death: The increase of thousands of transcripts has implications to transplantation, cancer, and forensic research.

PubMed

Pozhitkov, Alexander E; Noble, Peter A

2017-09-01

After a vertebrate dies, many of its organ systems, tissues, and cells remain functional while its body no longer works as a whole. We define this state as the "twilight of death" - the transition from a living body to a decomposed corpse. We claim that the study of the twilight of death is important to ethical, legal and medical science. We examined gene expression at the twilight of death in the zebrafish and mouse reaching the conclusion that apparently thousands of transcripts significantly increase in abundance from life to several hours/days postmortem relative to live controls. Transcript dynamics of different genes provided "proof-of-principle" that models accurately predict an individual's elapsed-time-of-death (i.e. postmortem interval). While many transcripts were associated with survival and stress compensation, others were associated with epigenetic factors, developmental control, and cancer. Future studies are needed to determine whether the high incidence of cancer in transplant recipients is due to the postmortem processes in donor organs. © 2017 WILEY Periodicals, Inc.

Quantitative Proteomic Profiling of Low Dose Ionizing Radiation Effects in a Human Skin Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hengel, Shawna; Aldrich, Joshua T.; Waters, Katrina M.

2014-07-29

To assess molecular responses to low doses of radiation that may be encountered during medical diagnostic procedures, nuclear accidents, or terrorist acts, a quantitative global proteomic approach was used to identify protein alterations in a reconstituted human skin tissue treated with 10 cGy of ionizing radiation. Subcellular fractionation was employed to remove highly abundant structural proteins and provide insight on radiation induced alterations in protein abundance and localization. In addition, peptides were post-fractionated using high resolution 2-dimensional liquid chromatography to increase the dynamic range of detection of protein abundance and translocation changes. Quantitative data was obtained by labeling peptides withmore » 8-plex isobaric iTRAQ tags. A total of 207 proteins were detected with statistically significant alterations in abundance and/or subcellular localization compared to sham irradiated tissues. Bioinformatics analysis of the data indicated that the top canonical pathways affected by low dose radiation are related to cellular metabolism. Among the proteins showing alterations in abundance, localization and proteolytic processing was the skin barrier protein filaggrin which is consistent with our previous observation that ionizing radiation alters profilaggrin processing with potential effects on skin barrier functions. In addition, a large number of proteases and protease regulators were affected by low dose radiation exposure indicating that altered proteolytic activity may be a hallmark of low dose radiation exposure. While several studies have demonstrated altered transcriptional regulation occurs following low dose radiation exposures, the data presented here indicates post-transcriptional regulation of protein abundance, localization, and proteolytic processing play an important role in regulating radiation responses in complex human tissues.« less

Poly A- transcripts expressed in HeLa cells.

PubMed

Wu, Qingfa; Kim, Yeong C; Lu, Jian; Xuan, Zhenyu; Chen, Jun; Zheng, Yonglan; Zhou, Tom; Zhang, Michael Q; Wu, Chung-I; Wang, San Ming

2008-07-30

Transcripts expressed in eukaryotes are classified as poly A+ transcripts or poly A- transcripts based on the presence or absence of the 3' poly A tail. Most transcripts identified so far are poly A+ transcripts, whereas the poly A- transcripts remain largely unknown. We developed the TRD (Total RNA Detection) system for transcript identification. The system detects the transcripts through the following steps: 1) depleting the abundant ribosomal and small-size transcripts; 2) synthesizing cDNA without regard to the status of the 3' poly A tail; 3) applying the 454 sequencing technology for massive 3' EST collection from the cDNA; and 4) determining the genome origins of the detected transcripts by mapping the sequences to the human genome reference sequences. Using this system, we characterized the cytoplasmic transcripts from HeLa cells. Of the 13,467 distinct 3' ESTs analyzed, 24% are poly A-, 36% are poly A+, and 40% are bimorphic with poly A+ features but without the 3' poly A tail. Most of the poly A- 3' ESTs do not match known transcript sequences; they have a similar distribution pattern in the genome as the poly A+ and bimorphic 3' ESTs, and their mapped intergenic regions are evolutionarily conserved. Experiments confirmed the authenticity of the detected poly A- transcripts. Our study provides the first large-scale sequence evidence for the presence of poly A- transcripts in eukaryotes. The abundance of the poly A- transcripts highlights the need for comprehensive identification of these transcripts for decoding the transcriptome, annotating the genome and studying biological relevance of the poly A- transcripts.

The genes encoding fructose bisphosphate aldolase in Trypanosoma brucei are interspersed with unrelated genes.

PubMed Central

Vijayasarathy, S; Ernest, I; Itzhaki, J E; Sherman, D; Mowatt, M R; Michels, P A; Clayton, C E

1990-01-01

The fructose bisphosphate aldolase genes of Trypanosoma brucei are interspersed with unrelated genes whose transcript levels show no developmental modulation. Transcription appears approximately constant across the entire locus, suggesting that aldolase mRNA abundance is regulated post-transcriptionally. Images PMID:2349093

The gene expression pattern induced by high plating density in cultured bovine and buffalo granulosa cells might be regulated by specific miRNA species

PubMed Central

YENUGANTI, Vengala Rao; BADDELA, Vijay Simha; BAUFELD, Anja; SINGH, Dheer; VANSELOW, Jens

2015-01-01

Precise regulation of cell type-specific gene expression profiles precedes the profound morphological reorganization of somatic cell layers during folliculogenesis, ovulation and luteinization. Cell culture models are essential to the study of corresponding molecular mechanisms of gene regulation. In a recent study, it was shown that an increased cell plating density can largely change gene expression profiles of cultured bovine granulosa cells. In our present study, we comparatively analyzed cell plating density effects on cultured bovine and buffalo granulosa cells. Cells were isolated from small- to medium-sized follicles (2–6 mm) and cultured under serum-free conditions at different plating densities. The abundance of selected marker transcripts and associated miRNA candidates was determined by quantitative real-time RT-PCR. We found in both species that the abundance of CYP19A1, CCNE1 and PCNA transcripts was remarkably lower at a high plating density, whereas VNN2 and RGS2 transcripts significantly increased. In contrast, putative regulators of CYP19A1, miR-378, miR-106a and let-7f were significantly higher in both species or only in buffalo, respectively. Also miR-15a, a regulator of CCNE1, was upregulated in both species. Thus, increased plating density induced similar changes of mRNA and miRNA expression in granulosa cells from buffalo and cattle. From these data, we conclude that specific miRNA species might be involved in the observed density-induced gene regulation. PMID:25740097

Proteomic analysis on mangrove plant Avicennia marina leaves reveals nitric oxide enhances the salt tolerance by up-regulating photosynthetic and energy metabolic protein expression.

PubMed

Shen, Zhi-Jun; Chen, Juan; Ghoto, Kabir; Hu, Wen-Jun; Gao, Gui-Feng; Luo, Mei-Rong; Li, Zan; Simon, Martin; Zhu, Xue-Yi; Zheng, Hai-Lei

2018-06-15

Avicennia marina (Forsk.) Vierh is one of the most salt-tolerant mangrove species. Our previous study revealed that nitric oxide (NO) enhanced the salt tolerance of A. marina by promoting salt secretion and Na+ sequestration under salt stress. However, little is known about the regulation of NO on proteomic profiling for this mangrove species. In this study, we used sodium nitroprusside (SNP), an NO donor, to investigate the regulatory mechanism of NO on salt tolerance of A. marina according to physiological and proteomic aspects. Photosynthesis data showed that the reduction in photosynthesis caused by high salinity treatment (400 mM NaCl) could be partially recovered by addition of SNP (100 μM). Further analysis revealed that the high salinity treatment could induce not only the stomatal limitation but also non-stomatal limitation on photosynthetic reduction, while SNP addition could restore the non-stomatal limitation, implying that the application of SNP was beneficial to the metabolic process in leaves. Proteomic analysis identified 49 differentially expressed proteins involved in various biological processes such as photosynthesis, energy metabolism, primary metabolism, RNA transcription, protein translation and stress response proteins. Under high salinity treatment, the abundances of proteins related to photosynthesis, such as ribulose-phosphate 3-epimerase (RPE, spot 3), RuBisCO large subunit (RBCL, spot 4, 5, 24), RuBisCO activase A (RCA, spot 17, 18) and quinine oxidoreductase-like protein isoform 1 (QOR1, spot 23), were significantly decreased. However, the abundance of proteins such as RBCL (spot 5, 9) and QOR1 (spot 23) were increased by SNP addition. In addition, exogenous NO supply alleviated salt tolerance by increasing the accumulation of some proteins involved in energy metabolism (spot 15), primary metabolism (spot 25, 45, 46), RNA transcription (spot 36) and stress response proteins (spot 12, 21, 26, 37, 43). The transcriptional levels of nine selected proteins were mostly consistent with their protein abundance except spot 46. Overall, the presented data demonstrated that NO has a positive effect on improving salt tolerance in A. marina by regulating the protein abundance involved in photosynthesis, energy metabolism, primary metabolism and stress response.

Secondary metabolite gene expression and interplay of bacterial functions in a tropical freshwater cyanobacterial bloom.

PubMed

Penn, Kevin; Wang, Jia; Fernando, Samodha C; Thompson, Janelle R

2014-09-01

Cyanobacterial harmful algal blooms (cyanoHABs) appear to be increasing in frequency on a global scale. The Cyanobacteria in blooms can produce toxic secondary metabolites that make freshwater dangerous for drinking and recreation. To characterize microbial activities in a cyanoHAB, transcripts from a eutrophic freshwater reservoir in Singapore were sequenced for six samples collected over one day-night period. Transcripts from the Cyanobacterium Microcystis dominated all samples and were accompanied by at least 533 genera primarily from the Cyanobacteria, Proteobacteria, Bacteroidetes and Actinobacteria. Within the Microcystis population, abundant transcripts were from genes for buoyancy, photosynthesis and synthesis of the toxin microviridin, suggesting that these are necessary for competitive dominance in the Reservoir. During the day, Microcystis transcripts were enriched in photosynthesis and energy metabolism while at night enriched pathways included DNA replication and repair and toxin biosynthesis. Microcystis was the dominant source of transcripts from polyketide and non-ribosomal peptide synthase (PKS and NRPS, respectively) gene clusters. Unexpectedly, expression of all PKS/NRPS gene clusters, including for the toxins microcystin and aeruginosin, occurred throughout the day-night cycle. The most highly expressed PKS/NRPS gene cluster from Microcystis is not associated with any known product. The four most abundant phyla in the reservoir were enriched in different functions, including photosynthesis (Cyanobacteria), breakdown of complex organic molecules (Proteobacteria), glycan metabolism (Bacteroidetes) and breakdown of plant carbohydrates, such as cellobiose (Actinobacteria). These results provide the first estimate of secondary metabolite gene expression, functional partitioning and functional interplay in a freshwater cyanoHAB.

Small-interfering RNAs from natural antisense transcripts derived from a cellulose synthase gene modulate cell wall biosynthesis in barley

PubMed Central

Held, Michael A.; Penning, Bryan; Brandt, Amanda S.; Kessans, Sarah A.; Yong, Weidong; Scofield, Steven R.; Carpita, Nicholas C.

2008-01-01

Small-interfering RNAs (siRNAs) from natural cis-antisense pairs derived from the 3′-coding region of the barley (Hordeum vulgare) CesA6 cellulose synthase gene substantially increase in abundance during leaf elongation. Strand-specific RT-PCR confirmed the presence of an antisense transcript of HvCesA6 that extends ≥1230 bp from the 3′ end of the CesA-coding sequence. The increases in abundance of the CesA6 antisense transcript and the 21-nt and 24-nt siRNAs derived from the transcript are coincident with the down-regulation of primary wall CesAs, several Csl genes, and GT8 glycosyl transferase genes, and are correlated with the reduction in rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Virus induced gene silencing using unique target sequences derived from HvCesA genes attenuated expression not only of the HvCesA6 gene, but also of numerous nontarget Csls and the distantly related GT8 genes and reduced the incorporation of D-14C-Glc into cellulose and into mixed-linkage (1 → 3),(1 → 4)-β-D-glucans of the developing leaves. Unique target sequences for CslF and CslH conversely silenced the same genes and lowered rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Our results indicate that the expression of individual members of the CesA/Csl superfamily and glycosyl transferases share common regulatory control points, and siRNAs from natural cis-antisense pairs derived from the CesA/Csl superfamily could function in this global regulation of cell-wall synthesis. PMID:19075248

Cigarette Smoking Decreases Global MicroRNA Expression in Human Alveolar Macrophages

PubMed Central

Graff, Joel W.; Powers, Linda S.; Dickson, Anne M.; Kim, Jongkwang; Reisetter, Anna C.; Hassan, Ihab H.; Kremens, Karol; Gross, Thomas J.

2012-01-01

Human alveolar macrophages are critical components of the innate immune system. Cigarette smoking-induced changes in alveolar macrophage gene expression are linked to reduced resistance to pulmonary infections and to the development of emphysema/COPD. We hypothesized that microRNAs (miRNAs) could control, in part, the unique messenger RNA (mRNA) expression profiles found in alveolar macrophages of cigarette smokers. Activation of macrophages with different stimuli in vitro leads to a diverse range of M1 (inflammatory) and M2 (anti-inflammatory) polarized phenotypes that are thought to mimic activated macrophages in distinct tissue environments. Microarray mRNA data indicated that smoking promoted an “inverse” M1 mRNA expression program, defined by decreased expression of M1-induced transcripts and increased expression of M1-repressed transcripts with few changes in M2-regulated transcripts. RT-PCR arrays identified altered expression of many miRNAs in alveolar macrophages of smokers and a decrease in global miRNA abundance. Stratification of human subjects suggested that the magnitude of the global decrease in miRNA abundance was associated with smoking history. We found that many of the miRNAs with reduced expression in alveolar macrophages of smokers were predicted to target mRNAs upregulated in alveolar macrophages of smokers. For example, miR-452 is predicted to target the transcript encoding MMP12, an important effector of smoking-related diseases. Experimental antagonism of miR-452 in differentiated monocytic cells resulted in increased expression of MMP12. The comprehensive mRNA and miRNA expression profiles described here provide insight into gene expression regulation that may underlie the adverse effects cigarette smoking has on alveolar macrophages. PMID:22952876

Identification of the septate junction protein gliotactin in the mosquito Aedes aegypti: evidence for a role in increased paracellular permeability in larvae.

PubMed

Jonusaite, Sima; Kelly, Scott P; Donini, Andrew

2017-07-01

Septate junctions (SJs) regulate paracellular permeability across invertebrate epithelia. However, little is known about the function of SJ proteins in aquatic invertebrates. In this study, a role for the transmembrane SJ protein gliotactin (Gli) in the osmoregulatory strategies of larval mosquito ( Aedes aegypti ) was examined. Differences in gli transcript abundance were observed between the midgut, Malpighian tubules, hindgut and anal papillae of A. aegypti , which are epithelia that participate in larval mosquito osmoregulation. Western blotting of Gli revealed its presence in monomer, putative dimer and alternatively processed protein forms in different larval mosquito organs. Gli localized to the entire SJ domain between midgut epithelial cells and showed a discontinuous localization along the plasma membranes of epithelial cells of the rectum as well as the syncytial anal papillae epithelium. In the Malpighian tubules, Gli immunolocalization was confined to SJs between the stellate and principal cells. Rearing larvae in 30% seawater caused an increase in Gli protein abundance in the anterior midgut, Malpighian tubules and hindgut. Transcriptional knockdown of gli using dsRNA reduced Gli protein abundance in the midgut and increased the flux rate of the paracellular permeability marker, polyethylene glycol (molecular weight 400 Da; PEG-400). Data suggest that in larval A. aegypti , Gli participates in the maintenance of salt and water balance and that one role for Gli is to participate in the regulation of paracellular permeability across the midgut of A. aegypti in response to changes in environmental salinity. © 2017. Published by The Company of Biologists Ltd.

Vemurafenib potently induces endoplasmic reticulum stress-mediated apoptosis in BRAFV600E melanoma cells

PubMed Central

Beck, Daniela; Niessner, Heike; Smalley, Keiran S.M.; Flaherty, Keith; Paraiso, Kim H.T.; Busch, Christian; Sinnberg, Tobias; Vasseur, Sophie; Iovanna, Juan Lucio; Drießen, Stefan; Stork, Björn; Wesselborg, Sebastian; Schaller, Martin; Biedermann, Tilo; Bauer, Jürgen; Lasithiotakis, Konstantinos; Weide, Benjamin; Eberle, Jürgen; Schittek, Birgit; Schadendorf, Dirk; Garbe, Claus; Kulms, Dagmar; Meier, Friedegund

2013-01-01

The V600E mutation in the kinase BRAF is frequently detected in melanomas and results in constitutive activation of BRAF, which then promotes cell proliferation by the mitogen-activated protein kinase (MAPK) signaling pathway. Although the BRAFV600E kinase inhibitor vemurafenib has remarkable antitumor activity in patients with BRAFV600E-mutated melanoma, its effects are limited by the onset of drug resistance. We found that exposure of melanoma cell lines with the BRAFV600E mutation to vemurafenib decreased the abundance of anti-apoptotic proteins and induced intrinsic mitochondrial apoptosis. Vemurafenib-treated melanoma cells showed increased cytosolic concentration of calcium, a potential trigger for endoplasmic reticulum (ER) stress, which can lead to apoptosis. Consistent with an ER stress-induced response, vemurafenib decreased the abundance of the ER chaperone protein GRP78, increased the abundance of the spliced isoform of the transcription factor X-box protein 1 (XBP1) (which transcriptionally activates genes involved in ER stress responses), increased the phosphorylation of the translation initiation factor eIF2α (which would be expected to inhibit protein synthesis), and induced the expression of ER stress-related genes. Knockdown of the ER stress response protein ATF4 significantly reduced vemurafenib-induced apoptosis. Moreover, the ER stress inducer thapsigargin prevented invasive growth of tumors formed from vemurafenib-sensitive melanoma cells in vivo. In melanoma cells with low sensitivity or resistance to vemurafenib, combination treatment with thapsigargin augmented or induced apoptosis. Thus, thapsigargin or other inducers of ER stress may be useful in combination therapies to overcome vemurafenib resistance. PMID:23362240

Identification of Abundantly Expressed Novel and Conserved Genes from the Infective Larval Stage of Toxocara canis by an Expressed Sequence Tag Strategy

PubMed Central

Tetteh, Kevin K. A.; Loukas, Alex; Tripp, Cindy; Maizels, Rick M.

1999-01-01

Larvae of Toxocara canis, a nematode parasite of dogs, infect humans, causing visceral and ocular larva migrans. In noncanid hosts, larvae neither grow nor differentiate but endure in a state of arrested development. Reasoning that parasite protein production is orientated to immune evasion, we undertook a random sequencing project from a larval cDNA library to characterize the most highly expressed transcripts. In all, 266 clones were sequenced, most from both 3′ and 5′ ends, and similarity searches against GenBank protein and dbEST nucleotide databases were conducted. Cluster analyses showed that 128 distinct gene products had been found, all but 3 of which represented newly identified genes. Ninety-five genes were represented by a single clone, but seven transcripts were present at high frequencies, each composing >2% of all clones sequenced. These high-abundance transcripts include a mucin and a C-type lectin, which are both major excretory-secretory antigens released by parasites. Four highly expressed novel gene transcripts, termed ant (abundant novel transcript) genes, were found. Together, these four genes comprised 18% of all cDNA clones isolated, but no similar sequences occur in the Caenorhabditis elegans genome. While the coding regions of the four genes are dissimilar, their 3′ untranslated tracts have significant homology in nucleotide sequence. The discovery of these abundant, parasite-specific genes of newly identified lectins and mucins, as well as a range of conserved and novel proteins, provides defined candidates for future analysis of the molecular basis of immune evasion by T. canis. PMID:10456930

The dynamics of cereal cyst nematode infection differ between susceptible and resistant barley cultivars and lead to changes in (1,3;1,4)-β-glucan levels and HvCslF gene transcript abundance.

PubMed

Aditya, Jessika; Lewis, John; Shirley, Neil J; Tan, Hwei-Ting; Henderson, Marilyn; Fincher, Geoffrey B; Burton, Rachel A; Mather, Diane E; Tucker, Matthew R

2015-07-01

Heterodera avenae (cereal cyst nematode, CCN) infects the roots of barley (Hordeum vulgare) forming syncytial feeding sites. In resistant host plants, relatively few females develop to maturity. Little is known about the physiological and biochemical changes induced during CCN infection. Responses to CCN infection were investigated in resistant (Rha2) and susceptible barley cultivars through histological, compositional and transcriptional analysis. Two phases were identified that influence CCN viability, including feeding site establishment and subsequent cyst maturation. Syncytial development progressed faster in the resistant cultivar Chebec than in the susceptible cultivar Skiff, and was accompanied by changes in cell wall polysaccharide abundance, particularly (1,3;1,4)-β-glucan. Transcriptional profiling identified several glycosyl transferase genes, including CELLULOSE SYNTHASE-LIKE F10 (HvCslF10), which may contribute to differences in polysaccharide abundance between resistant and susceptible cultivars. In barley, Rha2-mediated CCN resistance drives rapid deterioration of CCN feeding sites, specific changes in cell wall-related transcript abundance and changes in cell wall composition. During H. avenae infection, (1,3;1,4)-β-glucan may influence CCN feeding site development by limiting solute flow, similar to (1,3)-β-glucan during dicot cyst nematode infections. Dynamic transcriptional changes in uncharacterized HvCslF genes, possibly involved in (1,3;1,4)-β-glucan synthesis, suggest a role for these genes in the CCN infection process. © 2015 The University of Adelaide. New Phytologist © 2015 New Phytologist Trust.

Alternative Splicing Control of Abiotic Stress Responses.

PubMed

Laloum, Tom; Martín, Guiomar; Duque, Paula

2018-02-01

Alternative splicing, which generates multiple transcripts from the same gene, is an important modulator of gene expression that can increase proteome diversity and regulate mRNA levels. In plants, this post-transcriptional mechanism is markedly induced in response to environmental stress, and recent studies have identified alternative splicing events that allow rapid adjustment of the abundance and function of key stress-response components. In agreement, plant mutants defective in splicing factors are severely impaired in their response to abiotic stress. Notably, mounting evidence indicates that alternative splicing regulates stress responses largely by targeting the abscisic acid (ABA) pathway. We review here current understanding of post-transcriptional control of plant stress tolerance via alternative splicing and discuss research challenges for the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of EPSPS Copy Number Variation (CNV) and Glyphosate Application on the Aromatic and Branched Chain Amino Acid Synthesis Pathways in Amaranthus palmeri

PubMed Central

Fernández-Escalada, Manuel; Zulet-González, Ainhoa; Gil-Monreal, Miriam; Zabalza, Ana; Ravet, Karl; Gaines, Todd; Royuela, Mercedes

2017-01-01

A key enzyme of the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), is the known target of the widely used herbicide glyphosate. Glyphosate resistance in Amaranthus palmeri, one of the most troublesome weeds in agriculture, has evolved through increased EPSPS gene copy number. The aim of this work was to study the pleiotropic effects of (i) EPSPS increased transcript abundance due to gene copy number variation (CNV) and of (ii) glyphosate application on the aromatic amino acid (AAA) and branched chain amino acid (BCAA) synthesis pathways. Hydroponically grown glyphosate sensitive (GS) and glyphosate resistant (GR) plants were treated with glyphosate 3 days after treatment. In absence of glyphosate treatment, high EPSPS gene copy number had only a subtle effect on transcriptional regulation of AAA and BCAA pathway genes. In contrast, glyphosate treatment provoked a general accumulation of the transcripts corresponding to genes of the AAA pathway leading to synthesis of chorismate in both GS and GR. After chorismate, anthranilate synthase transcript abundance was higher while chorismate mutase transcription showed a small decrease in GR and remained stable in GS, suggesting a regulatory branch point in the pathway that favors synthesis toward tryptophan over phenylalanine and tyrosine after glyphosate treatment. This was confirmed by studying enzyme activities in vitro and amino acid analysis. Importantly, this upregulation was glyphosate dose dependent and was observed similarly in both GS and GR populations. Glyphosate treatment also had a slight effect on the expression of BCAA genes but no general effect on the pathway could be observed. Taken together, our observations suggest that the high CNV of EPSPS in A. palmeri GR populations has no major pleiotropic effect on the expression of AAA biosynthetic genes, even in response to glyphosate treatment. This finding supports the idea that the fitness cost associated with EPSPS CNV in A. palmeri may be limited. PMID:29201035

De-novo discovery of differentially abundant transcription factor binding sites including their positional preference.

PubMed

Keilwagen, Jens; Grau, Jan; Paponov, Ivan A; Posch, Stefan; Strickert, Marc; Grosse, Ivo

2011-02-10

Transcription factors are a main component of gene regulation as they activate or repress gene expression by binding to specific binding sites in promoters. The de-novo discovery of transcription factor binding sites in target regions obtained by wet-lab experiments is a challenging problem in computational biology, which has not been fully solved yet. Here, we present a de-novo motif discovery tool called Dispom for finding differentially abundant transcription factor binding sites that models existing positional preferences of binding sites and adjusts the length of the motif in the learning process. Evaluating Dispom, we find that its prediction performance is superior to existing tools for de-novo motif discovery for 18 benchmark data sets with planted binding sites, and for a metazoan compendium based on experimental data from micro-array, ChIP-chip, ChIP-DSL, and DamID as well as Gene Ontology data. Finally, we apply Dispom to find binding sites differentially abundant in promoters of auxin-responsive genes extracted from Arabidopsis thaliana microarray data, and we find a motif that can be interpreted as a refined auxin responsive element predominately positioned in the 250-bp region upstream of the transcription start site. Using an independent data set of auxin-responsive genes, we find in genome-wide predictions that the refined motif is more specific for auxin-responsive genes than the canonical auxin-responsive element. In general, Dispom can be used to find differentially abundant motifs in sequences of any origin. However, the positional distribution learned by Dispom is especially beneficial if all sequences are aligned to some anchor point like the transcription start site in case of promoter sequences. We demonstrate that the combination of searching for differentially abundant motifs and inferring a position distribution from the data is beneficial for de-novo motif discovery. Hence, we make the tool freely available as a component of the open-source Java framework Jstacs and as a stand-alone application at http://www.jstacs.de/index.php/Dispom.

Identifying the candidate genes involved in the calyx abscission process of 'Kuerlexiangli' (Pyrus sinkiangensis Yu) by digital transcript abundance measurements.

PubMed

Qi, Xiaoxiao; Wu, Jun; Wang, Lifen; Li, Leiting; Cao, Yufen; Tian, Luming; Dong, Xingguang; Zhang, Shaoling

2013-10-23

'Kuerlexiangli' (Pyrus sinkiangensis Yu), a native pear of Xinjiang, China, is an important agricultural fruit and primary export to the international market. However, fruit with persistent calyxes affect fruit shape and quality. Although several studies have looked into the physiological aspects of the calyx abscission process, the underlying molecular mechanisms remain unknown. In order to better understand the molecular basis of the process of calyx abscission, materials at three critical stages of regulation, with 6000 × Flusilazole plus 300 × PBO treatment (calyx abscising treatment) and 50 mg.L-1GA3 treatment (calyx persisting treatment), were collected and cDNA fragments were sequenced using digital transcript abundance measurements to identify candidate genes. Digital transcript abundance measurements was performed using high-throughput Illumina GAII sequencing on seven samples that were collected at three important stages of the calyx abscission process with chemical agent treatments promoting calyx abscission and persistence. Altogether more than 251,123,845 high quality reads were obtained with approximately 8.0 M raw data for each library. The values of 69.85%-71.90% of clean data in the digital transcript abundance measurements could be mapped to the pear genome database. There were 12,054 differentially expressed genes having Gene Ontology (GO) terms and associating with 251 Kyoto Encyclopedia of Genes and Genomes (KEGG) defined pathways. The differentially expressed genes correlated with calyx abscission were mainly involved in photosynthesis, plant hormone signal transduction, cell wall modification, transcriptional regulation, and carbohydrate metabolism. Furthermore, candidate calyx abscission-specific genes, e.g. Inflorescence deficient in abscission gene, were identified. Quantitative real-time PCR was used to confirm the digital transcript abundance measurements results. We identified candidate genes that showed highly dynamic changes in expression during the calyx abscission process. These genes are potential targets for future functional characterization and should be valuable for exploration of the mechanisms of calyx abscission, and eventually for developing methods based on small molecule application to induce calyx abscission in fruit production.

mRNA levels of imprinted genes in bovine in vivo oocytes, embryos and cross species comparisons in humans, mice and pigs

USDA-ARS?s Scientific Manuscript database

Twenty-six confirmed imprinted genes in the bovine were quantified in in vivo produced oocytes and embryos. Eighteen were detectable and their transcriptional abundance were categorized into five patterns: largely decreased (MEST and PLAGL1); first decreased and then increased (CDKN1C and IGF2R); p...

Micronuclei and other erythrocyte nuclear abnormalities in fishes from the Great Lakes Basin, USA

USGS Publications Warehouse

Braham, Ryan P.; Blazer, Vicki S.; Shaw, Cassidy H.; Mazik, Patricia M.

2017-01-01

Biological markers (biomarkers) sensitive to genotoxic and mutagenic contamination in fishes are widely used to identify exposure effects in aquatic environments. The micronucleus assay was incorporated into a suite of indicators to assess exposure to genotoxic and mutagenic contamination at five Great Lakes Areas of Concern (AOCs), as well as one non-AOC (reference) site. The assay allowed enumeration of micronuclei as well as other nuclear abnormalities for both site and species comparisons. Erythrocyte abnormality data was also compared to skin and liver tumor prevalence and hepatic transcript abundance. Erythrocyte abnormalities were observed at all sites with variable occurrence and severity among sites and species. Benthic-oriented brown bullhead (Ameiurus nebulosus) and white sucker (Catostomus commersonii) expressed lower rates of erythrocyte abnormalities, but higher rates of skin and liver neoplasms, when compared to pelagic-oriented largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) at the same site. The reduced erythrocyte abnormalities, increased transcript abundance associated with Phase I and II toxicant responsive pathways, and increased neoplastic lesions among benthic-oriented taxa may indicate the development of contaminant resistance of these species to more acute effects.

Towards an Understanding of the Function of the Phytochelatin Synthase of Schistosoma mansoni

PubMed Central

Rigouin, Coraline; Nylin, Elyse; Cogswell, Alexis A.; Schaumlöffel, Dirk; Dobritzsch, Dirk; Williams, David L.

2013-01-01

Phytochelatin synthase (PCS) is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain) work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis. PMID:23383357

Cell stress and translational inhibitors transiently increase the abundance of mammalian SINE transcripts.

PubMed Central

Liu, W M; Chu, W M; Choudary, P V; Schmid, C W

1995-01-01

The abundance of Alu RNA is transiently increased by heat shock in human cell lines. This effect is specific to Alu repeats among Pol III transcribed genes, since the abundance of 7SL, 7SK, 5S and U6 RNAs is essentially unaffected by heat shock. The rapid induction of Alu expression precedes the heat shock induction of mRNAs for the ubiquitin and HSP 70 heat shock genes. Heat shock mimetics also transiently induce Alu expression indicating that increased Alu expression is a general cell-stress response. Cycloheximide treatment rapidly and transiently increases the abundance of Alu RNA. Again, compared with other genes transcribed by Pol III, this increase is specific to Alu. However, as distinguished from the cell stress response, cycloheximide does not induce expression of HSP 70 and ubiquitin mRNAs. Puromycin also increases Alu expression, suggesting that this response is generally caused by translational inhibition. The response of mammalian SINEs to cell stress and translational inhibition is not limited to SINEs which are Alu homologues. Heat shock and cycloheximide each transiently induce Pol III directed expression of B1 and B2 RNAs in mouse cells and C-element RNA in rabbit cells. Together, these three species exemplify the known SINE composition of placental mammals, suggesting that mammalian SINEs are similarly regulated and may serve a common function. Images PMID:7784180

Characterization and transcription of arsenic respiration and resistance genes during in situ uranium bioremediation

PubMed Central

Giloteaux, Ludovic; Holmes, Dawn E; Williams, Kenneth H; Wrighton, Kelly C; Wilkins, Michael J; Montgomery, Alison P; Smith, Jessica A; Orellana, Roberto; Thompson, Courtney A; Roper, Thomas J; Long, Philip E; Lovley, Derek R

2013-01-01

The possibility of arsenic release and the potential role of Geobacter in arsenic biogeochemistry during in situ uranium bioremediation was investigated because increased availability of organic matter has been associated with substantial releases of arsenic in other subsurface environments. In a field experiment conducted at the Rifle, CO study site, groundwater arsenic concentrations increased when acetate was added. The number of transcripts from arrA, which codes for the α-subunit of dissimilatory As(V) reductase, and acr3, which codes for the arsenic pump protein Acr3, were determined with quantitative reverse transcription-PCR. Most of the arrA (>60%) and acr3-1 (>90%) sequences that were recovered were most similar to Geobacter species, while the majority of acr3-2 (>50%) sequences were most closely related to Rhodoferax ferrireducens. Analysis of transcript abundance demonstrated that transcription of acr3-1 by the subsurface Geobacter community was correlated with arsenic concentrations in the groundwater. In contrast, Geobacter arrA transcript numbers lagged behind the major arsenic release and remained high even after arsenic concentrations declined. This suggested that factors other than As(V) availability regulated the transcription of arrA in situ, even though the presence of As(V) increased the transcription of arrA in cultures of Geobacter lovleyi, which was capable of As(V) reduction. These results demonstrate that subsurface Geobacter species can tightly regulate their physiological response to changes in groundwater arsenic concentrations. The transcriptomic approach developed here should be useful for the study of a diversity of other environments in which Geobacter species are considered to have an important influence on arsenic biogeochemistry. PMID:23038171

Adipose and Muscle Tissue Gene Expression of Two Genes (NCAPG and LCORL) Located in a Chromosomal Region Associated with Cattle Feed Intake and Gain

PubMed Central

Lindholm-Perry, Amanda K.; Kuehn, Larry A.; Oliver, William T.; Sexten, Andrea K.; Miles, Jeremy R.; Rempel, Lea A.; Cushman, Robert A.; Freetly, Harvey C.

2013-01-01

A region on bovine chromosome 6 has been implicated in cattle birth weight, growth, and length. Non-SMC conodensin I complex subunit G (NCAPG) and ligand dependent nuclear receptor corepressor-like protein (LCORL) are positional candidate genes within this region. Previously identified genetic markers in both genes were associated with average daily gain (ADG) and average daily feed intake (ADFI) in a crossbred population of beef steers. These markers were also associated with hot carcass weight, ribeye area and adjusted fat thickness suggesting that they may have a role in lean muscle growth and/or fat deposition. The purpose of this study was to determine whether the transcript abundance of either of these genes in cattle adipose and muscle tissue was associated with variation in feed intake and average daily gain phenotypes. Transcript abundance for NCAPG and LCORL in adipose and muscle tissue was measured in heifers (adipose only), cows and steers using real-time polymerase chain reaction. In the adipose tissue from cows and heifers, a negative correlation between LCORL transcript abundance and ADFI were detected (P = 0.05). In the muscle tissue from cows, transcript abundance of NCAPG was associated with ADG (r = 0.26; P = 0.009). A positive correlation between LCORL transcript abundance from muscle tissue of steers and ADFI was detected (P = 0.04). LCORL protein levels in the muscle of steers were investigated and were associated with ADFI (P = 0.01). These data support our earlier genetic associations with ADFI and ADG within this region and represent the potential for biological activity of these genes in the muscle and adipose tissues of beef cattle; however, they also suggest that sex, age and/or nutrition-specific interactions may affect the expression of NCAPG and LCORL in these tissues. PMID:24278337

Transcriptomic profiles of Aspergillus flavus CA42, a strain that produces small sclerotia, by decanal treatment and after recovery.

PubMed

Chang, Perng-Kuang; Scharfenstein, Leslie L; Mack, Brian; Yu, Jiujiang; Ehrlich, Kenneth C

2014-07-01

Aspergillus flavus is a ubiquitous saprophyte and is capable of producing many secondary metabolites including the carcinogenic aflatoxins. The A. flavus population that produces small sclerotia (S strain) has been implicated as the culprit for persistent aflatoxin contamination in field crops. We investigated how the plant volatile decanal, a C10 fatty aldehyde, affected the growth and development of the S strain A. flavus. Decanal treatment yielded fluffy variants lacking sclerotia and conidia and exhibiting a dosage-dependent radial colony growth. We used RNA-Seq analysis to examine transcriptomic changes caused by decanal and after removal of decanal. Mature sclerotia contained only 80% of the total transcripts detected in all samples in comparison to 94% for the decanal treated culture. Gene ontology (GO) analysis showed that decanal treatment increased expression of genes involved in oxidoreductase activity, cellular carbohydrate metabolism, alcohol metabolism and aflatoxin biosynthesis. The treatment affected cellular components associated with cell wall, and gene expression of glucanases, α-amylases, pectinesterase and peptidase required for its biosynthesis was increased. After decanal was removed, the culture resumed sclerotial production. Moreover, its GO terms significantly overlapped with those of the untreated culture; five of the enriched molecular functions, oxidoreductase activity, monooxygenase activity, electron carrier activity, heme binding, and iron binding were found in the untreated culture. The GO term of cellular component enriched was mainly integral protein constituents of the membrane. The results suggested that decanal halted development at the vegetative state rendering the fungus unable to produce conidia and sclerotia. The induced fluffy phenotype could be related to lower transcript abundance of flbB, flbD, and flbE but not to veA expression. Increased abundance of the laeA transcript in the treated culture correlated with early transcriptional activation of aflatoxin and kojic acid biosynthesis gene clusters. Expression profiles revealed subtle differences in timing of activation of the respective 55 secondary metabolite gene clusters. Published by Elsevier Inc.

Correlations of carotenoid content and transcript abundances for fibrillin and carotenogenic enzymes in Capsicum annum fruit pericarp.

PubMed

Kilcrease, James; Rodriguez-Uribe, Laura; Richins, Richard D; Arcos, Juan Manuel Garcia; Victorino, Jesus; O'Connell, Mary A

2015-03-01

The fruits of Capsicum spp. are especially rich sites for carotenoid synthesis and accumulation, with cultivar-specific carotenoid accumulation profiles. Differences in chromoplast structure as well as carotenoid biosynthesis are correlated with distinct carotenoid accumulations and fruit color. In the present study, the inheritance of chromoplast shape, carotenoid accumulation profiles, and transcript levels of four genes were measured. Comparisons of these traits were conducted using fruit from contrasting variants, Costeño Amarillo versus Costeño Red, and from F1 hybrids; crosses between parental lines with novel versions of these traits. Intermediate chromoplast shapes were observed in the F1, but no association between specific carotenoid accumulation and chromoplast shape was detected. Increased total carotenoid content was associated with increased β-carotene and violaxanthin content. Transcript levels for phytoene synthase (Psy) and β-carotene hydroxylase (CrtZ-2) were positively correlated with increased levels of specific carotenoids. No correlation was detected between transcript levels of capsanthin/capsorubin synthase (Ccs) and carotenoid composition or chromoplast shape. Transcript levels of fibrillin, were differentially correlated with specific carotenoids, negatively correlated with accumulation of capsanthin, and positively correlated with violaxanthin. The regulation of carotenoid accumulation in chromoplasts in Capsicum fruit continues to be a complex process with multiple steps for control. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

CREB expression in the brains of two closely related parasitic wasp species that differ in long-term memory formation.

PubMed

van den Berg, M; Verbaarschot, P; Hontelez, S; Vet, L E M; Dicke, M; Smid, H M

2010-06-01

The cAMP/PKA signalling pathway and transcription factor cAMP response element-binding protein (CREB) play key roles in long-term memory (LTM) formation. We used two closely related parasitic wasp species, Cotesia glomerata and Cotesia rubecula, which were previously shown to be different in LTM formation, and sequenced at least nine different CREB transcripts in both wasp species. The splicing patterns, functional domains and amino acid sequences were similar to those found in the CREB genes of other organisms. The predicted amino acid sequences of the CREB isoforms were identical in both wasp species. Using real-time quantitative PCR we found that two low abundant CREB transcripts are differentially expressed in the two wasps, whereas the expression levels of high abundant transcripts are similar.

Isolation and expression analysis of FTZ-F1 encoding gene of black rock fish ( Sebastes schlegelii)

NASA Astrophysics Data System (ADS)

Shafi, Muhammad; Wang, Yanan; Zhou, Xiaosu; Ma, Liman; Muhammad, Faiz; Qi, Jie; Zhang, Quanqi

2013-03-01

Sex related FTZ-F1 is a transcriptional factor regulating the expression of fushi tarazu (a member of the orphan nuclear receptors) gene. In this study, FTZ-F1 gene ( FTZ-F1) was isolated from the testis of black rockfish ( Sebastes schlegeli) by homology cloning. The full-length cDNA of S. schlegeli FTZ-F1 ( ssFTZ-F1) contained a 232bp 5' UTR, a 1449bp ORF encoding FTZ-F1 (482 amino acid residules in length) with an estimated molecular weight of 5.4kD and a 105bp 3' UTR. Sequence, tissue distribution and phylogenic analysis showed that ssFTZ-F1 belonged to FTZ group, holding highly conserved regions including I, II and III FTZ-F1 boxes and an AF-2 hexamer. Relatively high expression was observed at different larva stages. In juveniles (105 days old), the transcript of ssFTZ-F1 can be detected in all tissues and the abuncance of the gene transcript in testis, ovary, spleen and brain was higher than that in other tissues. In mature fish, the abundance of gene transcript was higher in testis, ovary, spleen and brain than that in liver (trace amount), and the gene was not transcribed in other tissues. The highest abundance of gene transcript was always observed in gonads of both juvenile and mature fish. In addition, the abundance of gene transcript in male tissues were higher than that in female tissue counterparts ( P<0.05).

Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

NASA Technical Reports Server (NTRS)

Cook, T. F.; Burke, J. S.; Bergman, K. D.; Quinn, C. O.; Jeffrey, J. J.; Partridge, N. C.

1994-01-01

Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

Poly A- Transcripts Expressed in HeLa Cells

PubMed Central

Lu, Jian; Xuan, Zhenyu; Chen, Jun; Zheng, Yonglan; Zhou, Tom; Zhang, Michael Q.; Wu, Chung-I; Wang, San Ming

2008-01-01

Background Transcripts expressed in eukaryotes are classified as poly A+ transcripts or poly A- transcripts based on the presence or absence of the 3′ poly A tail. Most transcripts identified so far are poly A+ transcripts, whereas the poly A- transcripts remain largely unknown. Methodology/Principal Findings We developed the TRD (Total RNA Detection) system for transcript identification. The system detects the transcripts through the following steps: 1) depleting the abundant ribosomal and small-size transcripts; 2) synthesizing cDNA without regard to the status of the 3′ poly A tail; 3) applying the 454 sequencing technology for massive 3′ EST collection from the cDNA; and 4) determining the genome origins of the detected transcripts by mapping the sequences to the human genome reference sequences. Using this system, we characterized the cytoplasmic transcripts from HeLa cells. Of the 13,467 distinct 3′ ESTs analyzed, 24% are poly A-, 36% are poly A+, and 40% are bimorphic with poly A+ features but without the 3′ poly A tail. Most of the poly A- 3′ ESTs do not match known transcript sequences; they have a similar distribution pattern in the genome as the poly A+ and bimorphic 3′ ESTs, and their mapped intergenic regions are evolutionarily conserved. Experiments confirmed the authenticity of the detected poly A- transcripts. Conclusion/Significance Our study provides the first large-scale sequence evidence for the presence of poly A- transcripts in eukaryotes. The abundance of the poly A- transcripts highlights the need for comprehensive identification of these transcripts for decoding the transcriptome, annotating the genome and studying biological relevance of the poly A- transcripts. PMID:18665230

Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression.

PubMed

Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Matagne, Valerie; Toro, Carlos A; Ramaswamy, Suresh; Plant, Tony M; Ojeda, Sergio R

2015-12-16

In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty.

Metabolic potential and in situ activity of marine Marinimicrobia bacteria in an anoxic water column.

PubMed

Bertagnolli, Anthony D; Padilla, Cory C; Glass, Jennifer B; Thamdrup, Bo; Stewart, Frank J

2017-11-01

Marinimicrobia bacteria are widespread in subeuphotic areas of the oceans and particularly abundant in oxygen minimum zones (OMZs). Information on Marinimicrobia metabolism is sparse, making the biogeochemical influence of this group challenging to predict. Here, metagenome-assembled genomes representing Marinimicrobia subgroups PN262000N21 and ARCTIC96B-7 were retrieved to near completion (97% and 94%) from OMZ metagenomes, with contamination (14.1%) observed only in ARCTIC96B-7. Genes for aerobic carbon monoxide (CO) oxidation, polysulfide metabolism and hydrogen utilization were identified only in PN262000N21, while genes for partial denitrification occurred in both genomes. Transcripts mapping to these genomes increased from <0.3% of total mRNA from the oxic zone to a max of 22% under anoxia. ARCTIC96B-7 transcript representation decreased an order of magnitude from non-sulfidic to sulfidic depths. In contrast, PN262000N21 representation was relatively constant throughout the OMZ, although transcripts encoding sulfur-utilizing proteins, including sulfur transferases, were enriched at sulfidic depths. PN262000N21 transcripts encoding a protein with fibronectin domains similar to those in cellulosome-producing bacteria were also abundant, suggesting a potential for high molecular weight carbon cycling. These data provide omic-level descriptions of metabolic potential and activity in OMZ-associated Marinimicrobia, suggesting differentiation between subgroups with roles in carbon and dissimilatory inorganic nitrogen and sulfur cycling. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

EMSAR: estimation of transcript abundance from RNA-seq data by mappability-based segmentation and reclustering.

PubMed

Lee, Soohyun; Seo, Chae Hwa; Alver, Burak Han; Lee, Sanghyuk; Park, Peter J

2015-09-03

RNA-seq has been widely used for genome-wide expression profiling. RNA-seq data typically consists of tens of millions of short sequenced reads from different transcripts. However, due to sequence similarity among genes and among isoforms, the source of a given read is often ambiguous. Existing approaches for estimating expression levels from RNA-seq reads tend to compromise between accuracy and computational cost. We introduce a new approach for quantifying transcript abundance from RNA-seq data. EMSAR (Estimation by Mappability-based Segmentation And Reclustering) groups reads according to the set of transcripts to which they are mapped and finds maximum likelihood estimates using a joint Poisson model for each optimal set of segments of transcripts. The method uses nearly all mapped reads, including those mapped to multiple genes. With an efficient transcriptome indexing based on modified suffix arrays, EMSAR minimizes the use of CPU time and memory while achieving accuracy comparable to the best existing methods. EMSAR is a method for quantifying transcripts from RNA-seq data with high accuracy and low computational cost. EMSAR is available at https://github.com/parklab/emsar.

The abundance of homoeologue transcripts is disrupted by hybridization and is partially restored by genome doubling in synthetic hexaploid wheat.

PubMed

Hao, Ming; Li, Aili; Shi, Tongwei; Luo, Jiangtao; Zhang, Lianquan; Zhang, Xuechuan; Ning, Shunzong; Yuan, Zhongwei; Zeng, Deying; Kong, Xingchen; Li, Xiaolong; Zheng, Hongkun; Lan, Xiujin; Zhang, Huaigang; Zheng, Youliang; Mao, Long; Liu, Dengcai

2017-02-10

The formation of an allopolyploid is a two step process, comprising an initial wide hybridization event, which is later followed by a whole genome doubling. Both processes can affect the transcription of homoeologues. Here, RNA-Seq was used to obtain the genome-wide leaf transcriptome of two independent Triticum turgidum × Aegilops tauschii allotriploids (F1), along with their spontaneous allohexaploids (S1) and their parental lines. The resulting sequence data were then used to characterize variation in homoeologue transcript abundance. The hybridization event strongly down-regulated D-subgenome homoeologues, but this effect was in many cases reversed by whole genome doubling. The suppression of D-subgenome homoeologue transcription resulted in a marked frequency of parental transcription level dominance, especially with respect to genes encoding proteins involved in photosynthesis. Singletons (genes where no homoeologues were present) were frequently transcribed at both the allotriploid and allohexaploid plants. The implication is that whole genome doubling helps to overcome the phenotypic weakness of the allotriploid, restoring a more favourable gene dosage in genes experiencing transcription level dominance in hexaploid wheat.

Transcriptional and Posttranscriptional Control of Phaseolin and Phytohemagglutinin Gene Expression in Developing Cotyledons of Phaseolus vulgaris.

PubMed

Chappell, J; Chrispeels, M J

1986-05-01

The expression of phaseolin and phytohemagglutinin (PHA) in the developing cotyledons of a normal (Greensleeves) and a PHA-deficient (Pinto 111) cultivar of Phaseolus vulgaris was investigated. Phaseolin mRNA translational activity and abundance were present at similar levels in both cultivars. In contrast, PHA mRNA translational activity and abundance in Pinto 111 were less than 1% of the levels measured in Greensleeves. Using nuclear runoff assays, the transcription rate of phaseolin gene sequences was similar in both cultivars. The transcription rate of PHA gene sequences in Pinto 111 was only 20% of that measured in Greensleeves. Comparison of the transcription rates with the relative mRNA amounts measured in RNA blot hybridizations indicated that the normally expressed storage protein gene mRNAs were very stable with half-lives greater than several days. Because a low level of PHA gene transcription in Pinto 111 was measurable but no PHA mRNA accumulated, these results suggest that the PHA deficiency in Pinto 111 is due to a reduced transcription rate and possibly an instability of the mRNA.

Embryo Microinjection of Selenomethionine Reduces Hatchability and Modifies Oxidant Responsive Gene Expression in Zebrafish

NASA Astrophysics Data System (ADS)

Thomas, J. K.; Janz, D. M.

2016-05-01

In previous studies we demonstrated that exposure to selenomethionine (SeMet) causes developmental toxicities in zebrafish (Danio rerio). The objectives of this study were to establish a dose-response relationship for developmental toxicities in zebrafish after embryo microinjection of Se (8, 16 or 32 μg/g dry mass of eggs) in the form of SeMet, and to investigate potential underlying mechanism(s) of SeMet-induced developmental toxicities. A dose-dependent increase in frequencies of mortality and total deformities, and reduced hatchability were observed in zebrafish exposed to excess Se via embryo microinjection. The egg Se concentration causing 20% mortality was then used to investigate transcript abundance of proteins involved in antioxidant protection and methylation. Excess Se exposure modified gene expression of oxidant-responsive transcription factors (nuclear factor erythroid 2-related factor nrf2a and nrf2b), and enzymes involved in cellular methylation (methionine adenosyltransferase mat1a and mat2ab) in zebrafish larvae. Notably, excess Se exposure up-regulated transcript abundance of aryl hydrocarbon receptor 2 (ahr2), a signalling pathway involved in the toxicity of dioxin-related compounds. Our findings suggest that oxidative stress or modification of methylation, or a combination of these mechanisms, might be responsible for Se-induced developmental toxicities in fishes.

Hexokinase 2 drives glycogen accumulation in equine endometrium at day 12 of diestrus and pregnancy.

PubMed

Bramer, Sarah A; Macedo, Alysson; Klein, Claudia

2017-01-05

Secretion of histotroph during the prolonged pre-implantation phase in mares is crucial to pregnancy maintenance, manifested as increased embryonic loss in mares with age-related endometrial degeneration. Glycogen content of uterine histotroph is higher during the progesterone-dominated phase of the estrous cycle in mares, but regulatory mechanisms are not well understood. mRNA expression of glycogen-metabolizing enzymes (HK1, HK2, GSK3B, GYS1, PEPCK, PKM, PYGM) in endometrial samples were compared among mares in anestrus, estrus, and at Day 12 of diestrus and pregnancy. In addition, hexokinase 2 (HK2) activity was assessed using a colorimetric assay. HK2 was the key regulator of glycogen accumulation during diestrus and pregnancy; hexokinase transcript abundance and enzyme activity were significantly higher during diestrus and pregnancy than estrus and anestrus. In addition, despite similar relative transcript abundance, hexokinase activity was significantly greater in the pregnant versus diestrous endometrium. Therefore, we inferred there was regulation of hexokinase activity through phosphorylation, in addition to its regulation at the transcriptional level during early pregnancy. Based on immunohistochemistry, HK2 was localized primarily in luminal and glandular epithelial cells, with weaker staining in stromal cells. Among glycogen metabolizing enzymes identified, expression of HK2 was significantly greater during the progesterone-dominated phase of the cycle.

Intrinsic disorder in transcription factors†

PubMed Central

Liu, Jiangang; Perumal, Narayanan B.; Oldfield, Christopher J.; Su, Eric W.; Uversky, Vladimir N.; Dunker, A. Keith

2008-01-01

Intrinsic disorder (ID) is highly abundant in eukaryotes, which reflect the greater need for disorder-associated signaling and transcriptional regulation in nucleated cells. Although several well-characterized examples of intrinsically disordered proteins in transcriptional regulation have been reported, no systematic analysis has been reported so far. To test for a general prevalence of intrinsic disorder in transcriptional regulation, we used the Predictor Of Natural Disorder Regions (PONDR) to analyze the abundance of intrinsic disorder in three transcription factor datasets and two control sets. This analysis revealed that from 94.13% to 82.63% of transcription factors posses extended regions of intrinsic disorder, relative to 54.51% and 18.64% of the proteins in two control datasets, which indicates the significant prevalence of intrinsic disorder in transcription factors. This propensity of transcription factors for intrinsic disorder was confirmed by cumulative distribution function analysis and charge-hydropathy plots. The amino acid composition analysis showed that all three transcription factor datasets were substantially depleted in order-promoting residues, and significantly enriched in disorder-promoting residues. Our analysis of the distribution of disorder within the transcription factor datasets revealed that: (a) The AT-hooks and basic regions of transcription factor DNA-binding domains are highly disordered; (b) The degree of disorder in transcription factor activation regions is much higher than that in DNA-binding domains; (c) The degree of disorder is significantly higher in eukaryotic transcription factors than in prokaryotic transcription factors; (d) The level of α-MoRFs (molecular recognition feature) prediction is much higher in transcription factors. Overall, our data reflected the fact that the eukaryotes with well-developed gene transcription machinery require transcription factor flexibility to be more efficient. PMID:16734424

Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy.

PubMed

Potting, Christoph; Crochemore, Christophe; Moretti, Francesca; Nigsch, Florian; Schmidt, Isabel; Manneville, Carole; Carbone, Walter; Knehr, Judith; DeJesus, Rowena; Lindeman, Alicia; Maher, Rob; Russ, Carsten; McAllister, Gregory; Reece-Hoyes, John S; Hoffman, Gregory R; Roma, Guglielmo; Müller, Matthias; Sailer, Andreas W; Helliwell, Stephen B

2018-01-09

PARKIN, an E3 ligase mutated in familial Parkinson's disease, promotes mitophagy by ubiquitinating mitochondrial proteins for efficient engagement of the autophagy machinery. Specifically, PARKIN-synthesized ubiquitin chains represent targets for the PINK1 kinase generating phosphoS65-ubiquitin (pUb), which constitutes the mitophagy signal. Physiological regulation of PARKIN abundance, however, and the impact on pUb accumulation are poorly understood. Using cells designed to discover physiological regulators of PARKIN abundance, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. Testing identified genes individually resulted in a list of 53 positive and negative regulators. A transcriptional repressor network including THAP11 was identified and negatively regulates endogenous PARKIN abundance. RNAseq analysis revealed the PARKIN-encoding locus as a prime THAP11 target, and THAP11 CRISPR knockout in multiple cell types enhanced pUb accumulation. Thus, our work demonstrates the critical role of PARKIN abundance, identifies regulating genes, and reveals a link between transcriptional repression and mitophagy, which is also apparent in human induced pluripotent stem cell-derived neurons, a disease-relevant cell type. Copyright © 2018 the Author(s). Published by PNAS.

Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing.

PubMed

Vega-Arreguín, Julio C; Ibarra-Laclette, Enrique; Jiménez-Moraila, Beatriz; Martínez, Octavio; Vielle-Calzada, Jean Philippe; Herrera-Estrella, Luis; Herrera-Estrella, Alfredo

2009-07-06

In-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage. To explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20-454 pyrosequencing runs of a cDNA library obtained from 2 week-old Palomero Toluqueño maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20-454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20-454 sequences and corresponding levels of gene expression. A protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.

Transcriptional response of skeletal muscle to a low-protein gestation diet in porcine offspring accumulates in growth- and cell cycle-regulating pathways.

PubMed

Oster, Michael; Murani, Eduard; Metges, Cornelia C; Ponsuksili, Siriluck; Wimmers, Klaus

2012-08-17

Inadequate maternal protein supply during gestation represents an environmental factor that affects physiological signaling pathways with long-term consequences for growth, function, and structure of various tissues. Hypothesizing that the offspring's transcriptome is persistently altered by maternal diets, we used a porcine model to monitor the longitudinal expression changes in muscle to identify pathways relevant to fetal initiation of postnatal growth and development. German Landrace gilts were fed isoenergetic gestational diets containing 6.5% (LP) or 12.1% protein. The longissimus dorsi samples were collected from offspring at 94 days postconception (dpc) and 1, 28, and 188 days postnatum (dpn) for expression profiling. At 94 dpc, 1 dpn, and 28 dpn relatively few transcripts (<130) showed an altered abundance between the dietary groups. In fact, at 94 dpc genes of G2/M checkpoint regulation and mitotic roles of Polo-like kinases showed lowered transcript abundance in LP. At 188 dpn 677 transcripts were altered including those related to oxidative phosphorylation, citrate cycle, fatty acid metabolism (higher abundance in LP) and cell cycle regulation (lower abundance in LP). Correspondingly, transcriptional alterations during pre and postnatal development differed considerably among dietary groups, particularly for genes related to cell cycle regulation (G1/S and G2/M checkpoint regulation; cyclines), growth factor signaling (GH, IGF1, mTOR, RAN, VEGF, INSR), lipid metabolism, energy metabolism, and nucleic acid metabolism. In skeletal muscle, fetal programming related to maternal LP diets disturbed gene expression in growth-related pathways into adulthood. Diet-dependent gene expression may hamper proper development, thereby affecting signaling pathways related to energy utilization.

The CesA Gene Family of Barley. Quantitative Analysis of Transcripts Reveals Two Groups of Co-Expressed Genes1

PubMed Central

Burton, Rachel A.; Shirley, Neil J.; King, Brendon J.; Harvey, Andrew J.; Fincher, Geoffrey B.

2004-01-01

Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis. PMID:14701917

Galactinol synthase transcriptional profile in two genotypes of Coffea canephora with contrasting tolerance to drought

PubMed Central

Santos, Tiago Benedito Dos; de Lima, Rogério Barbosa; Nagashima, Getúlio Takashi; Petkowicz, Carmen Lucia de Oliveira; Carpentieri-Pípolo, Valéria; Pereira, Luiz Filipe Protasio; Domingues, Douglas Silva; Vieira, Luiz Gonzaga Esteves

2015-01-01

Increased synthesis of galactinol and raffinose family oligosaccharides (RFOs) has been reported in vegetative tissues in response to a range of abiotic stresses. In this work, we evaluated the transcriptional profile of a Coffea canephora galactinol synthase gene (CcGolS1) in two clones that differed in tolerance to water deficit in order to assess the contribution of this gene to drought tolerance. The expression of CcGolS1 in leaves was differentially regulated by water deficit, depending on the intensity of stress and the genotype. In clone 109A (drought-susceptible), the abundance of CcGolS1 transcripts decreased upon exposure to drought, reaching minimum values during recovery from severe water deficit and stress. In contrast, CcGolS1 gene expression in clone 14 (drought-tolerant) was stimulated by water deficit. Changes in galactinol and RFO content did not correlate with variation in the steady-state transcript level. However, the magnitude of increase in RFO accumulation was higher in the tolerant cultivar, mainly under severe water deficit. The finding that the drought-tolerant coffee clone showed enhanced accumulation of CcGolS1 transcripts and RFOs under water deficit suggests the possibility of using this gene to improve drought tolerance in this important crop. PMID:26273221

Galactinol synthase transcriptional profile in two genotypes of Coffea canephora with contrasting tolerance to drought.

PubMed

Santos, Tiago Benedito Dos; de Lima, Rogério Barbosa; Nagashima, Getúlio Takashi; Petkowicz, Carmen Lucia de Oliveira; Carpentieri-Pípolo, Valéria; Pereira, Luiz Filipe Protasio; Domingues, Douglas Silva; Vieira, Luiz Gonzaga Esteves

2015-05-01

Increased synthesis of galactinol and raffinose family oligosaccharides (RFOs) has been reported in vegetative tissues in response to a range of abiotic stresses. In this work, we evaluated the transcriptional profile of a Coffea canephora galactinol synthase gene (CcGolS1) in two clones that differed in tolerance to water deficit in order to assess the contribution of this gene to drought tolerance. The expression of CcGolS1 in leaves was differentially regulated by water deficit, depending on the intensity of stress and the genotype. In clone 109A (drought-susceptible), the abundance of CcGolS1 transcripts decreased upon exposure to drought, reaching minimum values during recovery from severe water deficit and stress. In contrast, CcGolS1 gene expression in clone 14 (drought-tolerant) was stimulated by water deficit. Changes in galactinol and RFO content did not correlate with variation in the steady-state transcript level. However, the magnitude of increase in RFO accumulation was higher in the tolerant cultivar, mainly under severe water deficit. The finding that the drought-tolerant coffee clone showed enhanced accumulation of CcGolS1 transcripts and RFOs under water deficit suggests the possibility of using this gene to improve drought tolerance in this important crop.

The CCAAT box in the proximal SERCA2 gene promoter regulates basal and stress-induced transcription in cardiomyocytes.

PubMed

Fragoso-Medina, Jorge; Rodriguez, Gabriela; Zarain-Herzberg, Angel

2018-05-01

The cardiac sarco/endoplasmic reticulum Ca 2+ -ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position -78 bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing -254 and -2579 bp of 5'-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPβ transcription factors to the SERCA2 gene proximal promoter.

A Gestational High Protein Diet Affects the Abundance of Muscle Transcripts Related to Cell Cycle Regulation throughout Development in Porcine Progeny

PubMed Central

Oster, Michael; Murani, Eduard; Metges, Cornelia C.; Ponsuksili, Siriluck; Wimmers, Klaus

2012-01-01

Background In various animal models pregnancy diets have been shown to affect offspring phenotype. Indeed, the underlying programming of development is associated with modulations in birth weight, body composition, and continual diet-dependent modifications of offspring metabolism until adulthood, producing the hypothesis that the offspring's transcriptome is permanently altered depending on maternal diet. Methodology/Principal Findings To assess alterations of the offspring's transcriptome due to gestational protein supply, German Landrace sows were fed isoenergetic diets containing protein levels of either 30% (high protein - HP) or 12% (adequate protein - AP) throughout their pregnancy. Offspring muscle tissue (M. longissimus dorsi) was collected at 94 days post conception (dpc), and 1, 28, and 188 days post natum (dpn) for use with Affymetrix GeneChip Porcine Genome Arrays and subsequent statistical and Ingenuity pathway analyses. Numerous transcripts were found to have altered abundance at 94 dpc and 1 dpn; at 28 dpn no transcripts were altered, and at 188 dpn only a few transcripts showed a different abundance between diet groups. However, when assessing transcriptional changes across developmental time points, marked differences were obvious among the dietary groups. Depending on the gestational dietary exposure, short- and long-term effects were observed for mRNA expression of genes related to cell cycle regulation, energy metabolism, growth factor signaling pathways, and nucleic acid metabolism. In particular, the abundance of transcripts related to cell cycle remained divergent among the groups during development. Conclusion Expression analysis indicates that maternal protein supply induced programming of the offspring's genome; early postnatal compensation of the slight growth retardation obvious at birth in HP piglets resulted, as did a permanently different developmental alteration and responsiveness to the common environment of the transcriptome. The transcriptome modulations are interpreted as the molecular equivalent of developmental plasticity of the offspring that necessitates adaptation and maintenance of the organismal phenotype. PMID:22496824

Seasonal variation in denitrification and dissimilatory nitrate reduction to ammonia process rates and corresponding key functional genes along an estuarine nitrate gradient

PubMed Central

Smith, Cindy J.; Dong, Liang F.; Wilson, John; Stott, Andrew; Osborn, A. Mark; Nedwell, David B.

2015-01-01

This research investigated spatial-temporal variation in benthic bacterial community structure, rates of denitrification and dissimilatory nitrate reduction to ammonium (DNRA) processes and abundances of corresponding genes and transcripts at three sites—the estuary-head, mid-estuary and the estuary mouth (EM) along the nitrate gradient of the Colne estuary over an annual cycle. Denitrification rates declined down the estuary, while DNRA rates were higher at the estuary head and middle than the EM. In four out of the six 2-monthly time-points, rates of DNRA were greater than denitrification at each site. Abundance of gene markers for nitrate-reduction (nitrate reductase narG and napA), denitrification (nitrite reductase nirS) and DNRA (DNRA nitrite reductase nrfA) declined along the estuary with significant relationships between denitrification and nirS abundance, and DNRA and nrfA abundance. Spatially, rates of denitrification, DNRA and corresponding functional gene abundances decreased along the estuary. However, temporal correlations between rate processes and functional gene and transcript abundances were not observed. PMID:26082763

Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

PubMed Central

Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

2016-01-01

Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage. PMID:26613339

Proteomic analysis of a high aluminum tolerant yeast Rhodotorula taiwanensis RS1 in response to aluminum stress.

PubMed

Wang, Chao; Wang, Chang Yi; Zhao, Xue Qiang; Chen, Rong Fu; Lan, Ping; Shen, Ren Fang

2013-10-01

Rhodotorula taiwanensis RS1 is a high-aluminum (Al)-tolerant yeast that can survive in Al concentrations up to 200mM. The mechanisms for the high Al tolerance of R. taiwanensis RS1 are not well understood. To investigate the molecular mechanisms underlying Al tolerance and toxicity in R. taiwanensis RS1, Al toxicity-induced changes in the total soluble protein profile were analyzed using two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry. A total of 33 differentially expressed proteins responding to Al stress were identified from approximately 850 reproducibly detected proteins. Among them, the abundance of 29 proteins decreased and 4 increased. In the presence of 100mM Al, the abundance of proteins involved in DNA transcription, protein translation, DNA defense, Golgi functions and glucose metabolism was decreased. By contrast, Al treatment led to increased abundance of malate dehydrogenase, which correlated with increased malate dehydrogenase activity and the accumulation of intracellular citrate, suggesting that Al-induced intracellular citrate could play an important role in detoxification of Al in R. taiwanensis RS1. © 2013.

Response of Methanogenic Microbial Communities to Desiccation Stress in Flooded and Rain-Fed Paddy Soil from Thailand

PubMed Central

Reim, Andreas; Hernández, Marcela; Klose, Melanie; Chidthaisong, Amnat; Yuttitham, Monthira; Conrad, Ralf

2017-01-01

Rice paddies in central Thailand are flooded either by irrigation (irrigated rice) or by rain (rain-fed rice). The paddy soils and their microbial communities thus experience permanent or arbitrary submergence, respectively. Since methane production depends on anaerobic conditions, we hypothesized that structure and function of the methanogenic microbial communities are different in irrigated and rain-fed paddies and react differently upon desiccation stress. We determined rates and relative proportions of hydrogenotrophic and aceticlastic methanogenesis before and after short-term drying of soil samples from replicate fields. The methanogenic pathway was determined by analyzing concentrations and δ13C of organic carbon and of CH4 and CO2 produced in the presence and absence of methyl fluoride, an inhibitor of aceticlastic methanogenesis. We also determined the abundance (qPCR) of genes and transcripts of bacterial 16S rRNA, archaeal 16S rRNA and methanogenic mcrA (coding for a subunit of the methyl coenzyme M reductase) and the composition of these microbial communities by T-RFLP fingerprinting and/or Illumina deep sequencing. The abundances of genes and transcripts were similar in irrigated and rain-fed paddy soil. They also did not change much upon desiccation and rewetting, except the transcripts of mcrA, which increased by more than two orders of magnitude. In parallel, rates of CH4 production also increased, in rain-fed soil more than in irrigated soil. The contribution of hydrogenotrophic methanogenesis increased in rain-fed soil and became similar to that in irrigated soil. However, the relative microbial community composition on higher taxonomic levels was similar between irrigated and rain-fed soil. On the other hand, desiccation and subsequent anaerobic reincubation resulted in systematic changes in the composition of microbial communities for both Archaea and Bacteria. It is noteworthy that differences in the community composition were mostly detected on the level of operational taxonomic units (OTUs; 97% sequence similarity). The treatments resulted in change of the relative abundance of several archaeal OTUs. Some OTUs of Methanobacterium, Methanosaeta, Methanosarcina, Methanocella and Methanomassiliicoccus increased, while some of Methanolinea and Methanosaeta decreased. Bacterial OTUs within Firmicutes, Cyanobacteria, Planctomycetes and Deltaproteobacteria increased, while OTUs within other proteobacterial classes decreased. PMID:28529503

Quantitative proteomics unravels that the post-transcriptional regulator Crc modulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosa.

PubMed

Reales-Calderón, Jose Antonio; Corona, Fernando; Monteoliva, Lucía; Gil, Concha; Martínez, Jose Luis

2015-09-08

Recent research indicates that the post-transcriptional regulator Crc modulates susceptibility to antibiotics and virulence in Pseudomonas aeruginosa. Several P. aeruginosa virulence factors are secreted or engulfed in vesicles. To decipher the Crc modulation of P. aeruginosa virulence, we constructed a crc deficient mutant and measure the proteome associated extracellular vesicles and the vesicle-free secretome using iTRAQ. Fifty vesicle-associated proteins were more abundant and 14 less abundant in the crc-defective strain, whereas 37 were more abundant and 17 less abundant in the vesicle-free secretome. Among them, virulence determinants, such as ToxA, protease IV, azurin, chitin-binding protein, PlcB and Hcp1, were less abundant in the crc-defective mutant. Transcriptomic analysis revealed that some of the observed changes were post-transcriptional and, thus, could be attributed to a direct Crc regulatory role; whereas, for other differentially secreted proteins, the regulatory role was likely indirect. We also observed that the crc mutant presented an impaired vesicle-associated secretion of quorum sensing signal molecules and less cytotoxicity than its wild-type strain. Our results offer new insights into the mechanisms by which Crc regulates P. aeruginosa virulence, through the modulation of vesicle formation and secretion of both virulence determinants and quorum sensing signals. This article is part of a Special Issue entitled: HUPO 2014. Published by Elsevier B.V.

Response of Spring Diatoms to CO2 Availability in the Western North Pacific as Determined by Next-Generation Sequencing.

PubMed

Endo, Hisashi; Sugie, Koji; Yoshimura, Takeshi; Suzuki, Koji

2016-01-01

Next-generation sequencing (NGS) technologies have enabled us to determine phytoplankton community compositions at high resolution. However, few studies have adopted this approach to assess the responses of natural phytoplankton communities to environmental change. Here, we report the impact of different CO2 levels on spring diatoms in the Oyashio region of the western North Pacific as estimated by NGS of the diatom-specific rbcL gene (DNA), which encodes the large subunit of RubisCO. We also examined the abundance and composition of rbcL transcripts (cDNA) in diatoms to assess their physiological responses to changing CO2 levels. A short-term (3-day) incubation experiment was carried out on-deck using surface Oyashio waters under different pCO2 levels (180, 350, 750, and 1000 μatm) in May 2011. During the incubation, the transcript abundance of the diatom-specific rbcL gene decreased with an increase in seawater pCO2 levels. These results suggest that CO2 fixation capacity of diatoms decreased rapidly under elevated CO2 levels. In the high CO2 treatments (750 and 1000 μatm), diversity of diatom-specific rbcL gene and its transcripts decreased relative to the control treatment (350 μatm), as well as contributions of Chaetocerataceae, Thalassiosiraceae, and Fragilariaceae to the total population, but the contributions of Bacillariaceae increased. In the low CO2 treatment, contributions of Bacillariaceae also increased together with other eukaryotes. These suggest that changes in CO2 levels can alter the community composition of spring diatoms in the Oyashio region. Overall, the NGS technology provided us a deeper understanding of the response of diatoms to changes in CO2 levels in terms of their community composition, diversity, and photosynthetic physiology.

Transcriptome analyses of the Giardia lamblia life cycle

PubMed Central

Birkeland, Shanda R.; Preheim, Sarah P.; Davids, Barbara J.; Cipriano, Michael J.; Palm, Daniel; Reiner, David S.; Svärd, Staffan G.; Gillin, Frances D.; McArthur, Andrew G.

2010-01-01

We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of Giardia lamblia. PMID:20570699

Using gene transcription to assess ecological and anthropological stressors in brown bears

USGS Publications Warehouse

Bowen, Lizabeth; Miles, A. Keith; Waters, Shannon C.; Gustine, Dave; Joly, Kyle; Hilderbrand, Grant V.

2018-01-01

Increasingly, population- and ecosystem-level health assessments are performed using sophisticated molecular tools. Advances in molecular technology enable the identification of synergistic effects of multiple stressors on the individual physiology of different species. Brown bears (Ursus arctos) are an apex predator; thus, they are ideal candidates for detecting potentially ecosystem-level systemic perturbations using molecular-based tools. We used gene transcription to analyze 130 brown bear samples from three National Parks and Preserves in Alaska. Although the populations we studied are apparently stable in abundance and exist within protected and intact environments, differences in transcript profiles were noted. The most prevalent differences were among locations. The transcript patterns among groups reflect the influence of environmental factors, such as nutritional status, disease, and xenobiotic exposure. However, these profiles also likely represent baselines for each unique environment by which future measures can be made to identify early indication of population-level changes due to, for example, increasing Arctic temperatures. Some of those environmental changes are predicted to be potentially positive for brown bears, but other effects such as the manifestation of disease or indirect effects of oceanic acidification may produce negative impacts.

Comparative tissue transcriptomics highlights dynamic differences among tissues but conserved metabolic transcript prioritization in preparation for arousal from torpor.

PubMed

Bogren, Lori K; Grabek, Katharine R; Barsh, Gregory S; Martin, Sandra L

2017-07-01

During the hibernation season, 13-lined ground squirrels spend days to weeks in torpor with body temperatures near freezing then spontaneously rewarm. The molecular drivers of the drastic physiological changes that orchestrate and permit torpor are not well understood. Although transcription effectively ceases at the low body temperatures of torpor, previous work has demonstrated that some transcripts are protected from bulk degradation in brown adipose tissue (BAT), consistent with the importance of their protein products for metabolic heat generation during arousal from torpor. We examined the transcriptome of skeletal muscle, heart, and liver to determine the patterns of differentially expressed genes in these tissues, and whether, like BAT, a subset of these were relatively increased during torpor. EDGE-tags were quantified from five distinct physiological states representing the seasonal and torpor-arousal cycles of 13-lined ground squirrels. Supervised clustering on relative transcript abundances with Random Forest separated the two states bracketing prolonged torpor, entrance into and aroused from torpor, in all three tissues. Independent analyses identified 3347, 6784, and 2433 differentially expressed transcripts among all sampling points in heart, skeletal muscle, and liver, respectively. There were few differentially expressed genes in common across all three tissues; these were enriched in mitochondrial and apoptotic pathway components. Divisive clustering of these data revealed unique cohorts of transcripts that increased across the torpor bout in each tissue with patterns reflecting various combinations of cycling within and between seasons as well as between torpor and arousal. Transcripts that increased across the torpor bout were likewise tissue specific. These data shed new light on the biochemical pathways that alter in concert with hibernation phenotype and provide a rich resource for further hypothesis-based studies.

Characterization and Transcription of Arsenic Respiration and Resistance Genes During In Situ Uranium Bioremediation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giloteaux, L.; Holmes, Dawn E.; Williams, Kenneth H.

2013-02-04

The possibility of arsenic release and the potential role of Geobacter in arsenic biogeochemistry during in situ uranium bioremediation was investigated because increased availability of organic matter has been associated with substantial releases of arsenic in other subsurface environments. In a field experiment conducted at the Rifle, CO study site, groundwater arsenic concentrations increased when acetate was added. The number of transcripts from arrA, which codes for the alpha subunit of dissimilatory As(V) reductase, and acr3, which codes for the arsenic pump protein Acr3, were determined with quantitative RT-PCR. Most of the arrA (> 60%) and acr3-1 (> 90%) sequencesmore » that were recovered were most similar to Geobacter species, while the majority of acr3-2 (>50%) sequences were most closely related to Rhodoferax ferrireducens. Analysis of transcript abundance demonstrated that transcription of acr3-1 by the subsurface Geobacter community was correlated with arsenic concentrations in the groundwater. In contrast, Geobacter arrA transcript numbers lagged behind the major arsenic release and remained high even after arsenic concentrations declined. This suggested that factors other than As(V) availability regulated transcription of arrA in situ even though the presence of As(V) increased transcription of arrA in cultures of G. lovleyi, which was capable of As(V) reduction. These results demonstrate that subsurface Geobacter species can tightly regulate their physiological response to changes in groundwater arsenic concentrations. The transcriptomic approach developed here should be useful for the study of a diversity of other environments in which Geobacter species are considered to have an important influence on arsenic biogeochemistry.« less

Spatial gradients in cell wall composition and transcriptional profiles along elongating maize internodes

PubMed Central

2014-01-01

Background The elongating maize internode represents a useful system for following development of cell walls in vegetative cells in the Poaceae family. Elongating internodes can be divided into four developmental zones, namely the basal intercalary meristem, above which are found the elongation, transition and maturation zones. Cells in the basal meristem and elongation zones contain mainly primary walls, while secondary cell wall deposition accelerates in the transition zone and predominates in the maturation zone. Results The major wall components cellulose, lignin and glucuronoarabinoxylan (GAX) increased without any abrupt changes across the elongation, transition and maturation zones, although GAX appeared to increase more between the elongation and transition zones. Microarray analyses show that transcript abundance of key glycosyl transferase genes known to be involved in wall synthesis or re-modelling did not match the increases in cellulose, GAX and lignin. Rather, transcript levels of many of these genes were low in the meristematic and elongation zones, quickly increased to maximal levels in the transition zone and lower sections of the maturation zone, and generally decreased in the upper maturation zone sections. Genes with transcript profiles showing this pattern included secondary cell wall CesA genes, GT43 genes, some β-expansins, UDP-Xylose synthase and UDP-Glucose pyrophosphorylase, some xyloglucan endotransglycosylases/hydrolases, genes involved in monolignol biosynthesis, and NAM and MYB transcription factor genes. Conclusions The data indicated that the enzymic products of genes involved in cell wall synthesis and modification remain active right along the maturation zone of elongating maize internodes, despite the fact that corresponding transcript levels peak earlier, near or in the transition zone. PMID:24423166

Differential transcriptional regulation of banana sucrose phosphate synthase gene in response to ethylene, auxin, wounding, low temperature and different photoperiods during fruit ripening and functional analysis of banana SPS gene promoter.

PubMed

Roy Choudhury, Swarup; Roy, Sujit; Das, Ranjan; Sengupta, Dibyendu N

2008-12-01

Sucrose phosphate synthase (SPS) (EC 2.3.1.14) is the key regulatory component in sucrose formation in banana (Musa acuminata subgroup Cavendish, cv Giant governor) fruit during ripening. This report illustrates differential transcriptional responses of banana SPS gene following ethylene, auxin, wounding, low temperature and different photoperiods during ripening in banana fruit. Whereas ethylene strongly stimulated SPS transcript accumulation, auxin and cold treatment only marginally increased the abundance of SPS mRNA level, while wounding negatively regulated SPS gene expression. Conversely, SPS transcript level was distinctly increased by constant exposure to white light. Protein level, enzymatic activity of SPS and sucrose synthesis were substantially increased by ethylene and increased exposure to white light conditions as compared to other treatments. To further study the transcriptional regulation of SPS in banana fruit, the promoter region of SPS gene was cloned and some cis-acting regulatory elements such as a reverse GCC-box ERE, two ARE motifs (TGTCTC), one LTRE (CCGAA), a GAGA-box (GAGA...) and a GATA-box LRE (GATAAG) were identified along with the TATA and CAAT-box. DNA-protein interaction studies using these cis-elements indicated a highly specific cis-trans interaction in the banana nuclear extract. Furthermore, we specifically studied the light responsive characteristics of GATA-box containing synthetic as well as native banana SPS promoter. Transient expression assays using banana SPS promoter have also indicated the functional importance of the SPS promoter in regulating gene expression. Together, these results provide insights into the transcriptional regulation of banana SPS gene in response to phytohormones and other environmental factors during fruit ripening.

Hypermutation in derepressed operons of Escherichia coli K12

PubMed Central

Wright, Barbara E.; Longacre, Angelika; Reimers, Jacqueline M.

1999-01-01

This article presents evidence that starvation for leucine in an Escherichia coli auxotroph triggers metabolic activities that specifically target the leu operon for derepression, increased rates of transcription, and mutation. Derepression of the leu operon was a prerequisite for its activation by the signal nucleotide, guanosine tetraphosphate, which accumulates in response to nutritional stress (the stringent response). A quantitative correlation was established between leuB mRNA abundance and leuB− reversion rates. To further demonstrate that derepression increased mutation rates, the chromosomal leu operon was placed under the control of the inducible tac promoter. When the leu operon was induced by isopropyl-d-thiogalactoside, both leuB mRNA abundance and leuB− reversion rates increased. These investigations suggest that guanosine tetraphosphate may contribute as much as attenuation in regulating leu operon expression and that higher rates of mutation are specifically associated with the derepressed leu operon. PMID:10220423

Progressive obesity alters the steroidogenic response to ovulatory stimulation and increases the abundance of mRNAs stored in the ovulated oocyte.

PubMed

Pohlmeier, William E; Xie, Fang; Kurz, Scott G; Lu, Ningxia; Wood, Jennifer R

2014-08-01

Obese women who are able to attain pregnancy are at increased risk for early-pregnancy loss due, in part, to reduced oocyte quality. We and others have demonstrated that female Lethal Yellow (LY) mice and female C57BL/6 mice fed a high fat diet (B6-HFD) exhibit phenotypes consistent with human obesity. These studies also showed that zygotes collected from LY and B6-HFD females have reduced developmental competence. The current hypothesis is that LY and B6-HFD females exhibit an abnormal response to gonadotropin stimulation compared to C57BL/6 controls fed normal rodent chow (B6-ND), resulting in the ovulation of oocytes with an altered molecular phenotype which may contribute to its reduced developmental competence. To test this hypothesis, age-matched B6-ND, B6-HFD, and LY females were stimulated with exogenous gonadotropins, then circulating hormone levels and the phenotypes of ovulated oocytes were analyzed. There was no difference in ovulation rate or in the percentage of morphologically abnormal oocytes collected from the oviduct of any females. Progesterone and progesterone/estradiol ratios, however, were increased in B6-HFD and LY compared to B6-ND females 16 hr post-human chorionic gonadotropin treatment. The transcript abundance of several candidate oocyte genes was also increased in B6-HFD- and LY-derived oocytes compared to B6-ND-derived oocytes. These data suggest that increased insulin and leptin levels of obese females elevated circulating progesterone concentrations, altered transcriptional activity during oocyte growth, and/or impaired mechanisms of RNA translation and degradation during oocyte maturation. These changes in mRNA abundance likely contribute to reduced oocyte quality and the subsequent poor embryogenesis associated with obesity. © 2014 Wiley Periodicals, Inc.

New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination.

PubMed

Ruiz-Ballesta, Isabel; Baena, Guillermo; Gandullo, Jacinto; Wang, Liqun; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

2016-05-01

Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Oxidative Damage to Rhesus Macaque Spermatozoa Results in Mitotic Arrest and Transcript Abundance Changes in Early Embryos1

PubMed Central

Burruel, Victoria; Klooster, Katie L.; Chitwood, James; Ross, Pablo J.; Meyers, Stuart A.

2013-01-01

ABSTRACT Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of metaphase II (MII) oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO2 in air for 2.25 h. Sperm were then assessed for motility, viability, and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the eight-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in two-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to four- and eight-cell stage was significantly lower for embryos generated with ROS-treated sperm than for controls. All embryos produced from ROS-treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of two-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS-induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the two-cell stage of development. PMID:23904511

Salinity alters snakeskin and mesh transcript abundance and permeability in midgut and Malpighian tubules of larval mosquito, Aedes aegypti.

PubMed

Jonusaite, Sima; Donini, Andrew; Kelly, Scott P

2017-03-01

This study examined the distribution and localization of the septate junction (SJ) proteins snakeskin (Ssk) and mesh in osmoregulatory organs of larval mosquito (Aedes aegypti), as well as their response to altered environmental salt levels. Ssk and mesh transcripts and immunoreactivity were detected in tissues of endodermal origin such as the midgut and Malpighian tubules of A. aegypti larvae, but not in ectodermally derived hindgut and anal papillae. Immunolocalization of Ssk and mesh in the midgut and Malpighian tubules indicated that both proteins are concentrated at regions of cell-cell contact between epithelial cells. Transcript abundance of ssk and mesh was higher in the midgut and Malpighian tubules of brackish water (BW, 30% SW) reared A. aegypti larvae when compared with freshwater (FW) reared animals. Therefore, [ 3 H]polyethylene glycol (MW 400Da, PEG-400) flux was examined across isolated midgut and Malpighian tubule preparations as a measure of their paracellular permeability. It was found that PEG-400 flux was greater across the midgut of BW versus FW larvae while the Malpighian tubules of BW-reared larvae had reduced PEG-400 permeability in conjunction with increased Cl - secretion compared to FW animals. Taken together, data suggest that Ssk and mesh are found in smooth SJs (sSJs) of larval A. aegypti and that their abundance alters in association with changes in epithelial permeability when larvae reside in water of differing salt content. This latter observation suggests that Ssk and mesh play a role in the homeostatic control of salt and water balance in larval A. aegypti. Copyright © 2016 Elsevier Inc. All rights reserved.

A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains

PubMed Central

Hutchinson, John N; Ensminger, Alexander W; Clemson, Christine M; Lynch, Christopher R; Lawrence, Jeanne B; Chess, Andrew

2007-01-01

Background Noncoding RNA species play a diverse set of roles in the eukaryotic cell. While much recent attention has focused on smaller RNA species, larger noncoding transcripts are also thought to be highly abundant in mammalian cells. To search for large noncoding RNAs that might control gene expression or mRNA metabolism, we used Affymetrix expression arrays to identify polyadenylated RNA transcripts displaying nuclear enrichment. Results This screen identified no more than three transcripts; XIST, and two unique noncoding nuclear enriched abundant transcripts (NEAT) RNAs strikingly located less than 70 kb apart on human chromosome 11: NEAT1, a noncoding RNA from the locus encoding for TncRNA, and NEAT2 (also known as MALAT-1). While the two NEAT transcripts share no significant homology with each other, each is conserved within the mammalian lineage, suggesting significant function for these noncoding RNAs. NEAT2 is extraordinarily well conserved for a noncoding RNA, more so than even XIST. Bioinformatic analyses of publicly available mouse transcriptome data support our findings from human cells as they confirm that the murine homologs of these noncoding RNAs are also nuclear enriched. RNA FISH analyses suggest that these noncoding RNAs function in mRNA metabolism as they demonstrate an intimate association of these RNA species with SC35 nuclear speckles in both human and mouse cells. These studies show that one of these transcripts, NEAT1 localizes to the periphery of such domains, whereas the neighboring transcript, NEAT2, is part of the long-sought polyadenylated component of nuclear speckles. Conclusion Our genome-wide screens in two mammalian species reveal no more than three abundant large non-coding polyadenylated RNAs in the nucleus; the canonical large noncoding RNA XIST and NEAT1 and NEAT2. The function of these noncoding RNAs in mRNA metabolism is suggested by their high levels of conservation and their intimate association with SC35 splicing domains in multiple mammalian species. PMID:17270048

HIF-1 and SKN-1 coordinate the transcriptional response to hydrogen sulfide in Caenorhabditis elegans.

PubMed

Miller, Dana L; Budde, Mark W; Roth, Mark B

2011-01-01

Hydrogen sulfide (H₂S) has dramatic physiological effects on animals that are associated with improved survival. C. elegans grown in H₂S are long-lived and thermotolerant. To identify mechanisms by which adaptation to H₂S effects physiological functions, we have measured transcriptional responses to H₂S exposure. Using microarray analysis we observe rapid changes in the abundance of specific mRNAs. The number and magnitude of transcriptional changes increased with the duration of H₂S exposure. Functional annotation suggests that genes associated with protein homeostasis are upregulated upon prolonged exposure to H₂S. Previous work has shown that the hypoxia-inducible transcription factor, HIF-1, is required for survival in H₂S. In fact, we show that hif-1 is required for most, if not all, early transcriptional changes in H₂S. Moreover, our data demonstrate that SKN-1, the C. elegans homologue of NRF2, also contributes to H₂S-dependent changes in transcription. We show that these results are functionally important, as skn-1 is essential to survive exposure to H₂S. Our results suggest a model in which HIF-1 and SKN-1 coordinate a broad transcriptional response to H₂S that culminates in a global reorganization of protein homeostasis networks.

Integration of oxygen signaling at the consensus HRE.

PubMed

Wenger, Roland H; Stiehl, Daniel P; Camenisch, Gieri

2005-10-18

The hypoxia-inducible factor 1 (HIF-1) was initially identified as a transcription factor that regulated erythropoietin gene expression in response to a decrease in oxygen availability in kidney tissue. Subsequently, a family of oxygen-dependent protein hydroxylases was found to regulate the abundance and activity of three oxygen-sensitive HIFalpha subunits, which, as part of the HIF heterodimer, regulated the transcription of at least 70 different effector genes. In addition to responding to a decrease in tissue oxygenation, HIF is proactively induced, even under normoxic conditions, in response to stimuli that lead to cell growth, ultimately leading to higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli-activated signaling pathways that influence the abundance and activity of HIFs include pathways in which kinases are activated and pathways in which reactive oxygen species are liberated. These pathways signal to the HIF protein hydroxylases, as well as to HIF itself, by means of covalent or redox modifications and protein-protein interactions. The final point of integration of all of these pathways is the hypoxia-response element (HRE) of effector genes. Here, we provide comprehensive compilations of the known growth stimuli that promote increases in HIF abundance, of protein-protein interactions involving HIF, and of the known HIF effector genes. The consensus HRE derived from a comparison of the HREs of these HIF effectors will be useful for identification of novel HIF target genes, design of oxygen-regulated gene therapy, and prediction of effects of future drugs targeting the HIF system.

RNAi Functions in Adaptive Reprogramming of the Genome | Center for Cancer Research

Cancer.gov

The regulation of transcribing DNA into RNA, including the production, processing, and degradation of RNA transcripts, affects the expression and the regulation of the genome in ways that are just beginning to be unraveled. A surprising discovery in recent years is that the vast majority of the genome is transcribed to yield an abundance of RNA transcripts. Many transcripts

Abscisic acid and ethephon regulation of cellulase in the endosperm cap and radicle during lettuce seed germination.

PubMed

Chen, Bingxian; Ma, Jun; Xu, Zhenjiang; Wang, Xiaofeng

2016-10-01

The purpose of this study was to investigate the role of cellulase in endosperm cap weakening and radicle elongation during lettuce (Lactuca sativa L.) seed germination. The application of abscisic acid (ABA) or ethephon inhibits or promotes germination, respectively, by affecting endosperm cap weakening and radicle elongation. Cellulase activities, and related protein and transcript abundances of two lettuce cellulase genes, LsCEL1 and LsCEL2, increase in the endosperm cap and radicle prior to radicle protrusion following imbibition in water. ABA or ethephon reduce or elevate, respectively, cellulase activity, and related protein and transcript abundances in the endosperm cap. Taken together, these observations suggest that cellulase plays a role in endosperm cap weakening and radicle elongation during lettuce seed germination, and that the regulation of cellulase in the endosperm cap by ABA and ethephon play a role in endosperm cap weakening. However, the influence of ABA and ethephon on radicle elongation may not be through their effects on cellulase. © 2016 Institute of Botany, Chinese Academy of Sciences.

A Cultivated Form of a Red Seaweed (Chondrus crispus), Suppresses β-Amyloid-Induced Paralysis in Caenorhabditis elegans.

PubMed

Sangha, Jatinder Singh; Wally, Owen; Banskota, Arjun H; Stefanova, Roumiana; Hafting, Jeff T; Critchley, Alan T; Prithiviraj, Balakrishnan

2015-10-20

We report here the protective effects of a methanol extract from a cultivated strain of the red seaweed, Chondrus crispus, against β-amyloid-induced toxicity, in a transgenic Caenorhabditis elegans, expressing human Aβ1-42 gene. The methanol extract of C. crispus (CCE), delayed β-amyloid-induced paralysis, whereas the water extract (CCW) was not effective. The CCE treatment did not affect the transcript abundance of amy1; however, Western blot analysis revealed a significant decrease of Aβ species, as compared to untreated worms. The transcript abundance of stress response genes; sod3, hsp16.2 and skn1 increased in CCE-treated worms. Bioassay guided fractionation of the CCE yielded a fraction enriched in monogalactosyl diacylglycerols (MGDG) that significantly delayed the onset of β-amyloid-induced paralysis. Taken together, these results suggested that the cultivated strain of C. crispus, whilst providing dietary nutritional value, may also have significant protective effects against β-amyloid-induced toxicity in C. elegans, partly through reduced β-amyloid species, up-regulation of stress induced genes and reduced accumulation of reactive oxygen species (ROS).

Coordinated Pulses of mRNA and of Protein Translation or Degradation Produce EGF-Induced Protein Bursts.

PubMed

Golan-Lavi, Roni; Giacomelli, Chiara; Fuks, Garold; Zeisel, Amit; Sonntag, Johanna; Sinha, Sanchari; Köstler, Wolfgang; Wiemann, Stefan; Korf, Ulrike; Yarden, Yosef; Domany, Eytan

2017-03-28

Protein responses to extracellular cues are governed by gene transcription, mRNA degradation and translation, and protein degradation. In order to understand how these time-dependent processes cooperate to generate dynamic responses, we analyzed the response of human mammary cells to the epidermal growth factor (EGF). Integrating time-dependent transcript and protein data into a mathematical model, we inferred for several proteins their pre-and post-stimulus translation and degradation coefficients and found that they exhibit complex, time-dependent variation. Specifically, we identified strategies of protein production and degradation acting in concert to generate rapid, transient protein bursts in response to EGF. Remarkably, for some proteins, for which the response necessitates rapidly decreased abundance, cells exhibit a transient increase in the corresponding degradation coefficient. Our model and analysis allow inference of the kinetics of mRNA translation and protein degradation, without perturbing cells, and open a way to understanding the fundamental processes governing time-dependent protein abundance profiles. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Environmental control of branching in petunia.

PubMed

Drummond, Revel S M; Janssen, Bart J; Luo, Zhiwei; Oplaat, Carla; Ledger, Susan E; Wohlers, Mark W; Snowden, Kimberley C

2015-06-01

Plants alter their development in response to changes in their environment. This responsiveness has proven to be a successful evolutionary trait. Here, we tested the hypothesis that two key environmental factors, light and nutrition, are integrated within the axillary bud to promote or suppress the growth of the bud into a branch. Using petunia (Petunia hybrida) as a model for vegetative branching, we manipulated both light quality (as crowding and the red-to-far-red light ratio) and phosphate availability, such that the axillary bud at node 7 varied from deeply dormant to rapidly growing. In conjunction with the phenotypic characterization, we also monitored the state of the strigolactone (SL) pathway by quantifying SL-related gene transcripts. Mutants in the SL pathway inhibit but do not abolish the branching response to these environmental signals, and neither signal is dominant over the other, suggesting that the regulation of branching in response to the environment is complex. We have isolated three new putatively SL-related TCP (for Teosinte branched1, Cycloidia, and Proliferating cell factor) genes from petunia, and have identified that these TCP-type transcription factors may have roles in the SL signaling pathway both before and after the reception of the SL signal at the bud. We show that the abundance of the receptor transcript is regulated by light quality, such that axillary buds growing in added far-red light have greatly increased receptor transcript abundance. This suggests a mechanism whereby the impact of any SL signal reaching an axillary bud is modulated by the responsiveness of these cells to the signal. © 2015 American Society of Plant Biologists. All Rights Reserved.

Environmental Control of Branching in Petunia1[OPEN

PubMed Central

Oplaat, Carla; Wohlers, Mark W.

2015-01-01

Plants alter their development in response to changes in their environment. This responsiveness has proven to be a successful evolutionary trait. Here, we tested the hypothesis that two key environmental factors, light and nutrition, are integrated within the axillary bud to promote or suppress the growth of the bud into a branch. Using petunia (Petunia hybrida) as a model for vegetative branching, we manipulated both light quality (as crowding and the red-to-far-red light ratio) and phosphate availability, such that the axillary bud at node 7 varied from deeply dormant to rapidly growing. In conjunction with the phenotypic characterization, we also monitored the state of the strigolactone (SL) pathway by quantifying SL-related gene transcripts. Mutants in the SL pathway inhibit but do not abolish the branching response to these environmental signals, and neither signal is dominant over the other, suggesting that the regulation of branching in response to the environment is complex. We have isolated three new putatively SL-related TCP (for Teosinte branched1, Cycloidia, and Proliferating cell factor) genes from petunia, and have identified that these TCP-type transcription factors may have roles in the SL signaling pathway both before and after the reception of the SL signal at the bud. We show that the abundance of the receptor transcript is regulated by light quality, such that axillary buds growing in added far-red light have greatly increased receptor transcript abundance. This suggests a mechanism whereby the impact of any SL signal reaching an axillary bud is modulated by the responsiveness of these cells to the signal. PMID:25911529

The Relative Abundance and Transcriptional Activity of Marine Sponge-Associated Microorganisms Emphasizing Groups Involved in Sulfur Cycle.

PubMed

Jensen, Sigmund; Fortunato, Sofia A V; Hoffmann, Friederike; Hoem, Solveig; Rapp, Hans Tore; Øvreås, Lise; Torsvik, Vigdis L

2017-04-01

During the last decades, our knowledge about the activity of sponge-associated microorganisms and their contribution to biogeochemical cycling has gradually increased. Functional groups involved in carbon and nitrogen metabolism are well documented, whereas knowledge about microorganisms involved in the sulfur cycle is still limited. Both sulfate reduction and sulfide oxidation has been detected in the cold water sponge Geodia barretti from Korsfjord in Norway, and with specimens from this site, the present study aims to identify extant versus active sponge-associated microbiota with focus on sulfur metabolism. Comparative analysis of small subunit ribosomal RNA (16S rRNA) gene (DNA) and transcript (complementary DNA (cDNA)) libraries revealed profound differences. The transcript library was predominated by Chloroflexi despite their low abundance in the gene library. An opposite result was found for Acidobacteria. Proteobacteria were detected in both libraries with representatives of the Alpha- and Gammaproteobacteria related to clades with presumably thiotrophic bacteria from sponges and other marine invertebrates. Sequences that clustered with sponge-associated Deltaproteobacteria were remotely related to cultivated sulfate-reducing bacteria. The microbes involved in sulfur cycling were identified by the functional gene aprA (adenosine-5'-phosphosulfate reductase) and its transcript. Of the aprA sequences (DNA and cDNA), 87 % affiliated with sulfur-oxidizing bacteria. They clustered with Alphaproteobacteria and with clades of deep-branching Gammaproteobacteria. The remaining sequences clustered with sulfate-reducing Archaea of the phylum Euryarchaeota. These results indicate an active role of yet uncharacterized Bacteria and Archaea in the sponge's sulfur cycle.

The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain

PubMed Central

Gehman, Lauren T.; Stoilov, Peter; Maguire, Jamie; Damianov, Andrey; Lin, Chia-Ho; Shiue, Lily; Ares, Manuel; Mody, Istvan; Black, Douglas L.

2011-01-01

The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts but their role in neuronal physiology is not clear1. We show here that central nervous system (CNS)-specific deletion of the Rbfox1 gene results in heightened susceptibility to spontaneous and kainic acid-induced seizures. Electrophysiological recording reveals a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole transcriptome analyses identify multiple splicing changes in the Rbfox1−/− brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation, some of which are implicated in human epilepsy. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function. PMID:21623373

Therapeutic Approaches Targeting MYC-Driven Prostate Cancer

PubMed Central

Rebello, Richard J.; Pearson, Richard B.; Hannan, Ross D.; Furic, Luc

2017-01-01

The transcript encoding the proto-oncogene MYC is commonly overexpressed in prostate cancer (PC). MYC protein abundance is also increased in the majority of cases of advanced and metastatic castrate-resistant PC (mCRPC). Accordingly, the MYC-directed transcriptional program directly contributes to PC by upregulating the expression of a number of pro-tumorigenic factors involved in cell growth and proliferation. A key cellular process downstream of MYC activity is the regulation of ribosome biogenesis which sustains tumor growth. MYC activity also cooperates with the dysregulation of the phosphoinositol-3-kinase (PI3K)/AKT/mTOR pathway to promote PC cell survival. Recent advances in the understanding of these interactions through the use of animal models have provided significant insight into the therapeutic efficacy of targeting MYC activity by interfering with its transcriptional program, and indirectly by targeting downstream cellular events linked to MYC transformation potential. PMID:28212321

Heritability and genetic basis of protein level variation in an outbred population

PubMed Central

Liu, Yi-Chun; Tekkedil, Manu M.; Steinmetz, Lars M.; Caudy, Amy A.; Fraser, Andrew G.

2014-01-01

The genetic basis of heritable traits has been studied for decades. Although recent mapping efforts have elucidated genetic determinants of transcript levels, mapping of protein abundance has lagged. Here, we analyze levels of 4084 GFP-tagged yeast proteins in the progeny of a cross between a laboratory and a wild strain using flow cytometry and high-content microscopy. The genotype of trans variants contributed little to protein level variation between individual cells but explained >50% of the variance in the population’s average protein abundance for half of the GFP fusions tested. To map trans-acting factors responsible, we performed flow sorting and bulk segregant analysis of 25 proteins, finding a median of five protein quantitative trait loci (pQTLs) per GFP fusion. Further, we find that cis-acting variants predominate; the genotype of a gene and its surrounding region had a large effect on protein level six times more frequently than the rest of the genome combined. We present evidence for both shared and independent genetic control of transcript and protein abundance: More than half of the expression QTLs (eQTLs) contribute to changes in protein levels of regulated genes, but several pQTLs do not affect their cognate transcript levels. Allele replacements of genes known to underlie trans eQTL hotspots confirmed the correlation of effects on mRNA and protein levels. This study represents the first genome-scale measurement of genetic contribution to protein levels in single cells and populations, identifies more than a hundred trans pQTLs, and validates the propagation of effects associated with transcript variation to protein abundance. PMID:24823668

Genome-enabled transcriptomics reveals archaeal populations that drive nitrification in a deep-sea hydrothermal plume.

PubMed

Baker, Brett J; Lesniewski, Ryan A; Dick, Gregory J

2012-12-01

Ammonia-oxidizing Archaea (AOA) are among the most abundant microorganisms in the oceans and have crucial roles in biogeochemical cycling of nitrogen and carbon. To better understand AOA inhabiting the deep sea, we obtained community genomic and transcriptomic data from ammonium-rich hydrothermal plumes in the Guaymas Basin (GB) and from surrounding deep waters of the Gulf of California. Among the most abundant and active lineages in the sequence data were marine group I (MGI) Archaea related to the cultured autotrophic ammonia-oxidizer, Nitrosopumilus maritimus. Assembly of MGI genomic fragments yielded 2.9 Mb of sequence containing seven 16S rRNA genes (95.4-98.4% similar to N. maritimus), including two near-complete genomes and several lower-abundance variants. Equal copy numbers of MGI 16S rRNA genes and ammonia monooxygenase genes and transcription of ammonia oxidation genes indicates that all of these genotypes actively oxidize ammonia. De novo genomic assembly revealed the functional potential of MGI populations and enhanced interpretation of metatranscriptomic data. Physiological distinction from N. maritimus is evident in the transcription of novel genes, including genes for urea utilization, suggesting an alternative source of ammonia. We were also able to determine which genotypes are most active in the plume. Transcripts involved in nitrification were more prominent in the plume and were among the most abundant transcripts in the community. These unique data sets reveal populations of deep-sea AOA thriving in the ammonium-rich GB that are related to surface types, but with key genomic and physiological differences.

c-myc, c-fos, and c-jun regulation in the regenerating livers of normal and H-2K/c-myc transgenic mice.

PubMed Central

Morello, D; Fitzgerald, M J; Babinet, C; Fausto, N

1990-01-01

We investigated the mechanisms of regulation of c-myc, c-fos, and c-jun at the early stages of liver regeneration in mice. We show that the transient increase in steady-state levels of c-myc mRNA at the start of liver regeneration is most probably regulated by posttranscriptional mechanisms. Although there was a marked increase in c-myc transcriptional initiation shortly after partial hepatectomy, a block in elongation prevented the completion of most transcripts. To gain further information on the mechanism of regulation of c-myc expression during liver regeneration, we used transgenic mice harboring the human c-myc gene driven by the H-2K promoter. In these animals, the murine c-myc responded to the growth stimulus generated by partial hepatectomy, whereas the expression of the transgene was constitutive and did not change in the regenerating liver. However, the mRNA from both genes increased markedly after cycloheximide injection, suggesting that the regulation of c-myc mRNA abundance in the regenerating liver differs from that occurring after protein synthesis inhibition. Furthermore, we show that in normal mice c-fos and c-jun mRNA levels and transcriptional rates increase within 30 min after partial hepatectomy. c-fos transcriptional elongation was restricted in nongrowing liver, but the block was partially relieved in the regenerating liver. Nevertheless, for both c-fos and c-jun, changes in steady-state mRNA detected after partial hepatectomy were much greater than the transcriptional increase. In the regenerating liver of H-2K/c-myc mice, c-fos and c-jun expression was diminished, whereas mouse c-myc expression was enhanced in comparison with that in nontransgenic animals. Images PMID:2111449

Mixture models reveal multiple positional bias types in RNA-Seq data and lead to accurate transcript concentration estimates.

PubMed

Tuerk, Andreas; Wiktorin, Gregor; Güler, Serhat

2017-05-01

Accuracy of transcript quantification with RNA-Seq is negatively affected by positional fragment bias. This article introduces Mix2 (rd. "mixquare"), a transcript quantification method which uses a mixture of probability distributions to model and thereby neutralize the effects of positional fragment bias. The parameters of Mix2 are trained by Expectation Maximization resulting in simultaneous transcript abundance and bias estimates. We compare Mix2 to Cufflinks, RSEM, eXpress and PennSeq; state-of-the-art quantification methods implementing some form of bias correction. On four synthetic biases we show that the accuracy of Mix2 overall exceeds the accuracy of the other methods and that its bias estimates converge to the correct solution. We further evaluate Mix2 on real RNA-Seq data from the Microarray and Sequencing Quality Control (MAQC, SEQC) Consortia. On MAQC data, Mix2 achieves improved correlation to qPCR measurements with a relative increase in R2 between 4% and 50%. Mix2 also yields repeatable concentration estimates across technical replicates with a relative increase in R2 between 8% and 47% and reduced standard deviation across the full concentration range. We further observe more accurate detection of differential expression with a relative increase in true positives between 74% and 378% for 5% false positives. In addition, Mix2 reveals 5 dominant biases in MAQC data deviating from the common assumption of a uniform fragment distribution. On SEQC data, Mix2 yields higher consistency between measured and predicted concentration ratios. A relative error of 20% or less is obtained for 51% of transcripts by Mix2, 40% of transcripts by Cufflinks and RSEM and 30% by eXpress. Titration order consistency is correct for 47% of transcripts for Mix2, 41% for Cufflinks and RSEM and 34% for eXpress. We, further, observe improved repeatability across laboratory sites with a relative increase in R2 between 8% and 44% and reduced standard deviation.

mRNA Transcript Abundance during Plant Growth and the Influence of Li + Exposure

DOE PAGES

Duff, M. C.; Kuhne, W. W.; Halverson, N. V.; ...

2014-10-23

Lithium (Li) toxicity in plants is, at a minimum, a function of Li + concentration, exposure time, species and growth conditions. Most plant studies with Li + focus on short-term acute exposures. This study examines short- and long-term effects of Li + exposure in Arabidopsis with Li + uptake studies and measured shoot mRNA transcript abundance levels in treated and control plants. Stress, pathogen-response and arabinogalactan protein genes were typically more up-regulated in older (chronic, low level) Li +-treatment plants and in the much younger plants from acute high-level exposures. The gene regulation behavior of high-level Li + resembled priormore » studies due to its influence on: inositol synthesis, 1-aminocyclopropane-1-carboxylate synthases and membrane ion transport. In contrast, chronically-exposed plants had gene regulation responses that were indicative of pathogen, cold, and heavy-metal stress, cell wall degradation, ethylene production, signal transduction, and calcium-release modulation. Acute Li + exposure phenocopies magnesium-deficiency symptoms and is associated with elevated expression of stress response genes that could lead to consumption of metabolic and transcriptional energy reserves and the dedication of more resources to cell development. In contrast, chronic Li + exposure increases expression signal transduction genes. The identification of new Li +-sensitive genes and a gene-based “response plan” for acute and chronic Li + exposure are delineated.« less

IsoPlot: a database for comparison of mRNA isoforms in fruit fly and mosquitoes

PubMed Central

Ng, I-Man; Tsai, Shang-Chi

2017-01-01

Abstract Alternative splicing (AS), a mechanism by which different forms of mature messenger RNAs (mRNAs) are generated from the same gene, widely occurs in the metazoan genomes. Knowledge about isoform variants and abundance is crucial for understanding the functional context in the molecular diversity of the species. With increasing transcriptome data of model and non-model species, a database for visualization and comparison of AS events with up-to-date information is needed for further research. IsoPlot is a publicly available database with visualization tools for exploration of AS events, including three major species of mosquitoes, Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus, and fruit fly Drosophila melanogaster, the model insect species. IsoPlot includes not only 88,663 annotated transcripts but also 17,037 newly predicted transcripts from massive transcriptome data at different developmental stages of mosquitoes. The web interface enables users to explore the patterns and abundance of isoforms in different experimental conditions as well as cross-species sequence comparison of orthologous transcripts. IsoPlot provides a platform for researchers to access comprehensive information about AS events in mosquitoes and fruit fly. Our database is available on the web via an interactive user interface with an intuitive graphical design, which is applicable for the comparison of complex isoforms within or between species. Database URL: http://isoplot.iis.sinica.edu.tw/ PMID:29220459

mRNA Transcript abundance during plant growth and the influence of Li(+) exposure.

PubMed

Duff, M C; Kuhne, W W; Halverson, N V; Chang, C-S; Kitamura, E; Hawthorn, L; Martinez, N E; Stafford, C; Milliken, C E; Caldwell, E F; Stieve-Caldwell, E

2014-12-01

Lithium (Li) toxicity in plants is, at a minimum, a function of Li(+) concentration, exposure time, species and growth conditions. Most plant studies with Li(+) focus on short-term acute exposures. This study examines short- and long-term effects of Li(+) exposure in Arabidopsis with Li(+) uptake studies and measured shoot mRNA transcript abundance levels in treated and control plants. Stress, pathogen-response and arabinogalactan protein genes were typically more up-regulated in older (chronic, low level) Li(+)-treatment plants and in the much younger plants from acute high-level exposures. The gene regulation behavior of high-level Li(+) resembled prior studies due to its influence on: inositol synthesis, 1-aminocyclopropane-1-carboxylate synthases and membrane ion transport. In contrast, chronically-exposed plants had gene regulation responses that were indicative of pathogen, cold, and heavy-metal stress, cell wall degradation, ethylene production, signal transduction, and calcium-release modulation. Acute Li(+) exposure phenocopies magnesium-deficiency symptoms and is associated with elevated expression of stress response genes that could lead to consumption of metabolic and transcriptional energy reserves and the dedication of more resources to cell development. In contrast, chronic Li(+) exposure increases expression signal transduction genes. The identification of new Li(+)-sensitive genes and a gene-based "response plan" for acute and chronic Li(+) exposure are delineated. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis.

PubMed

Crisp, Peter A; Ganguly, Diep R; Smith, Aaron B; Murray, Kevin D; Estavillo, Gonzalo M; Searle, Iain; Ford, Ethan; Bogdanović, Ozren; Lister, Ryan; Borevitz, Justin O; Eichten, Steven R; Pogson, Barry J

2017-08-01

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants ( k ) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5'-3' RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory. © 2017 American Society of Plant Biologists. All rights reserved.

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis[OPEN

PubMed Central

Estavillo, Gonzalo M.

2017-01-01

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5ʹ-3ʹ RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory. PMID:28705956

C/EBPβ contributes to transcriptional activation of long non-coding RNA NEAT1 during APL cell differentiation.

PubMed

Wang, Yewei; Fu, Lei; Sun, Ailian; Tang, Doudou; Xu, Yunxiao; Li, Zheyuan; Chen, Mingjie; Zhang, Guangsen

2018-05-05

Emerging evidences have shown that long non-coding RNAs (lncRNAs) play critical roles in cancer development and cancer therapy. LncRNA Nuclear Enriched Abundant Transcript 1 (NEAT1) is indispensable during acute promyelocytic leukemia (APL) cell differentiation induced by all-trans retinoic acid (ATRA). However, the precise mechanism of NEAT1 upregulation has not been fully understood. In this study, we performed chromatin immunoprecipitation and luciferase reporter assays to demonstrate that C/EBP family transcription factor C/EBPβ bind to and transactivate the promoter of lncRNA NEAT1 through the C/EBPβ binding sites both around -54 bp and -1453 bp upstream of the transcription start site. Moreover, the expression of C/EBPβ was increased after ATRA treatment, and the binding of C/EBPβ in the NEAT1 promoter was also dramatically increased. Finally, knockdown of C/EBPβ significantly reduced the ATRA-induced upregulation of NEAT1. In conclusion, C/EBPβ directly activates the expression of NEAT1 through binding to the promoter of NEAT1. Knockdown of C/EBPβ impairs ATRA-induced transcriptional activation of NEAT1. Our data indicate that C/EBPβ contributes to ATRA-induced activation of NEAT1 during APL cell differentiation. Our results enrich our knowledge on the regulation of lncRNAs and the regulatory role of C/EBPβ in APL cell differentiation. Copyright © 2017. Published by Elsevier Inc.

Light-Regulated Transcription of Genes Encoding Peridinin Chlorophyll a Proteins and the Major Intrinsic Light-Harvesting Complex Proteins in the Dinoflagellate Amphidinium carterae Hulburt (Dinophycae)1

PubMed Central

ten Lohuis, Michael R.; Miller, David J.

1998-01-01

In the dinoflagellate Amphidinium carterae, photoadaptation involves changes in the transcription of genes encoding both of the major classes of light-harvesting proteins, the peridinin chlorophyll a proteins (PCPs) and the major a/c-containing intrinsic light-harvesting proteins (LHCs). PCP and LHC transcript levels were increased up to 86- and 6-fold higher, respectively, under low-light conditions relative to cells grown at high illumination. These increases in transcript abundance were accompanied by decreases in the extent of methylation of CpG and CpNpG motifs within or near PCP- and LHC-coding regions. Cytosine methylation levels in A. carterae are therefore nonstatic and may vary with environmental conditions in a manner suggestive of involvement in the regulation of gene expression. However, chemically induced undermethylation was insufficient in activating transcription, because treatment with two methylation inhibitors had no effect on PCP mRNA or protein levels. Regulation of gene activity through changes in DNA methylation has traditionally been assumed to be restricted to higher eukaryotes (deuterostomes and green plants); however, the atypically large genomes of dinoflagellates may have generated the requirement for systems of this type in a relatively “primitive” organism. Dinoflagellates may therefore provide a unique perspective on the evolution of eukaryotic DNA-methylation systems. PMID:9576788

Diazotrophic bacterioplankton in a coral reef lagoon: phylogeny, diel nitrogenase expression and response to phosphate enrichment.

PubMed

Hewson, Ian; Moisander, Pia H; Morrison, Amanda E; Zehr, Jonathan P

2007-05-01

We investigated diazotrophic bacterioplankton assemblage composition in the Heron Reef lagoon (Great Barrier Reef, Australia) using culture-independent techniques targeting the nifH fragment of the nitrogenase gene. Seawater was collected at 3 h intervals over a period of 72 h (i.e. over diel as well as tidal cycles). An incubation experiment was also conducted to assess the impact of phosphate (PO(4)3*) availability on nifH expression patterns. DNA-based nifH libraries contained primarily sequences that were most similar to nifH from sediment, microbial mat and surface-associated microorganisms, with a few sequences that clustered with typical open ocean phylotypes. In contrast to genomic DNA sequences, libraries prepared from gene transcripts (mRNA amplified by reverse transcription-polymerase chain reaction) were entirely cyanobacterial and contained phylotypes similar to those observed in open ocean plankton. The abundance of Trichodesmium and two uncultured cyanobacterial phylotypes from previous studies (group A and group B) were studied by quantitative-polymerase chain reaction in the lagoon samples. These were detected as transcripts, but were not detected in genomic DNA. The gene transcript abundance of these phylotypes demonstrated variability over several diel cycles. The PO(4)3* enrichment experiment had a clearer pattern of gene expression over diel cycles than the lagoon sampling, however PO(4)3* additions did not result in enhanced transcript abundance relative to control incubations. The results suggest that a number of diazotrophs in bacterioplankton of the reef lagoon may originate from sediment, coral or beachrock surfaces, sloughing into plankton with the flooding tide. The presence of typical open ocean phylotype transcripts in lagoon bacterioplankton may indicate that they are an important component of the N cycle of the coral reef.

Circular RNA Expression: Its Potential Regulation and Function.

PubMed

Salzman, Julia

2016-05-01

In 2012, a new feature of eukaryotic gene expression emerged: ubiquitous expression of circular RNA (circRNA) from genes traditionally thought to express messenger or linear noncoding (nc)RNA only. CircRNAs are covalently closed, circular RNA molecules that typically comprise exonic sequences and are spliced at canonical splice sites. This feature of gene expression was first recognized in humans and mouse, but it quickly emerged that it was common across essentially all eukaryotes studied by molecular biologists. CircRNA abundance, and even which alternatively spliced circRNA isoforms are expressed, varies by cell type and can exceed the abundance of the traditional linear mRNA or ncRNA transcript. CircRNAs are enriched in the brain and increase in abundance during fetal development. Together, these features raise fundamental questions regarding the regulation of circRNA in cis and in trans, and its function. Copyright © 2016. Published by Elsevier Ltd.

Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression

PubMed Central

Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Matagne, Valerie; Toro, Carlos A.; Ramaswamy, Suresh; Plant, Tony M.; Ojeda, Sergio R.

2015-01-01

In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty. PMID:26671628

Extreme heterogeneity of influenza virus infection in single cells

PubMed Central

Russell, Alistair B; Trapnell, Cole

2018-01-01

Viral infection can dramatically alter a cell’s transcriptome. However, these changes have mostly been studied by bulk measurements on many cells. Here we use single-cell mRNA sequencing to examine the transcriptional consequences of influenza virus infection. We find extremely wide cell-to-cell variation in the productivity of viral transcription – viral transcripts comprise less than a percent of total mRNA in many infected cells, but a few cells derive over half their mRNA from virus. Some infected cells fail to express at least one viral gene, but this gene absence only partially explains variation in viral transcriptional load. Despite variation in viral load, the relative abundances of viral mRNAs are fairly consistent across infected cells. Activation of innate immune pathways is rare, but some cellular genes co-vary in abundance with the amount of viral mRNA. Overall, our results highlight the complexity of viral infection at the level of single cells. PMID:29451492

Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells

NASA Technical Reports Server (NTRS)

Clohisy, J. C.; Scott, D. K.; Brakenhoff, K. D.; Quinn, C. O.; Partridge, N. C.

1992-01-01

PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS).

Salt stress differentially affects growth-mediating β-expansins in resistant and sensitive maize (Zea mays L.).

PubMed

Geilfus, Christoph-Martin; Zörb, Christian; Mühling, Karl H

2010-12-01

Salinity mainly reduces shoot growth by the inhibition of cell division and elongation. Expansins loosen plant cell walls. Moreover, the expression of some isoforms is clearly correlated with growth. Effects of salinity on β-expansin transcripts protein abundance were recently reported for different crop species. This study provides a broad analysis of the impact of an 8-day 100mM NaCl stress treatment on the mRNA expression of different maize (Zea mays L.) β-Expansin isoforms using real-time quantitative RT-PCR. The composite β-expansin protein expression was analyzed by western blotting using an anti-peptide antibody raised against a conserved 15-amino-acid region shared by vegetatively expressed β-expansin isoforms. For the first time, changes in β-expansin transcript and protein abundance have been analyzed together with the salinity-induced inhibition of shoot growth. A salt-resistant and a salt-sensitive cultivar were compared in order to elucidate physiological changes. Genotypic differences in the relative concentration of six β-expansin transcripts together with differences in the abundance β-expansin protein are shown in response NaCl stress. In salt-sensitive Lector, reduced β-expansin protein expression was found to correlate positively with reduced shoot growth under stress. A down-regulation of ZmExpB2, ZmExpB6, and ZmExpB8 transcripts possibly contribute to this decrease in protein abundance. In contrast, the maintenance of shoot growth in salt-resistant SR03 might be related to an unaffected abundance of growth-mediating β-expansin proteins in the shoot. Our data suggest that the up-regulation of ZmExpB2, ZmExpB6, and ZmExpB8 may sustain the stable expression of β-expansin protein under conditions of salt stress. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Conserved Transcriptional Responses to Nutrient Stress in Bloom-Forming Algae

PubMed Central

Harke, Matthew J.; Juhl, Andrew R.; Haley, Sheean T.; Alexander, Harriet; Dyhrman, Sonya T.

2017-01-01

The concentration and composition of bioavailable nitrogen (N) and phosphorus (P) in the upper ocean shape eukaryotic phytoplankton communities and influence their physiological responses. Phytoplankton are known to exhibit similar physiological responses to limiting N and P conditions such as decreased growth rates, chlorosis, and increased assimilation of N and P. Are these responses similar at the molecular level across multiple species? To interrogate this question, five species from biogeochemically important, bloom-forming taxa (Bacillariophyta, Dinophyta, and Haptophyta) were grown under similar low N, low P, and replete nutrient conditions to identify transcriptional patterns and associated changes in biochemical pools related to N and P stress. Metabolic profiles, revealed through the transcriptomes of these taxa, clustered together based on species rather than nutrient stressor, suggesting that the global metabolic response to nutrient stresses was largely, but not exclusively, species-specific. Nutrient stress led to few transcriptional changes in the two dinoflagellates, consistent with other research. An orthologous group analysis examined functionally conserved (i.e., similarly changed) responses to nutrient stress and therefore focused on the diatom and haptophytes. Most conserved ortholog changes were specific to a single nutrient treatment, but a small number of orthologs were similarly changed under both N and P stress in 2 or more species. Many of these orthologs were related to photosynthesis and may represent generalized stress responses. A greater number of orthologs were conserved across more than one species under low P compared to low N. Screening the conserved orthologs for functions related to N and P metabolism revealed increased relative abundance of orthologs for nitrate, nitrite, ammonium, and amino acid transporters under N stress, and increased relative abundance of orthologs related to acquisition of inorganic and organic P substrates under P stress. Although the global transcriptional responses were dominated by species-specific changes, the analysis of conserved responses revealed functional similarities in resource acquisition pathways among different phytoplankton taxa. This overlap in nutrient stress responses observed among species may be useful for tracking the physiological ecology of phytoplankton field populations. PMID:28769884

Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism.

PubMed

Matsuda, Fumio; Kinoshita, Syohei; Nishino, Shunsuke; Tomita, Atsumi; Shimizu, Hiroshi

2017-01-01

Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis.

Illegitimate transcription: transcription of any gene in any cell type.

PubMed Central

Chelly, J; Concordet, J P; Kaplan, J C; Kahn, A

1989-01-01

Using in vitro amplification of cDNA by the polymerase chain reaction, we have detected spliced transcripts of various tissue-specific genes (genes for anti-Müllerian hormone, beta-globin, aldolase A, and factor VIIIc) in human nonspecific cells, such as fibroblasts, hepatoma cells, and lymphoblasts. In rats, erythroid- and liver-type pyruvate kinase transcripts were also detected in brain, lung, and muscle. The abundance of these "illegitimate" transcripts is very low; yet, their existence and the possibility of amplifying them by the cDNA polymerase chain reaction provide a powerful tool to analyze pathological transcripts of any tissue-specific gene by using any accessible cell. Images PMID:2495532

Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

NASA Technical Reports Server (NTRS)

Vian, A.; Henry-Vian, C.; Davies, E.

1999-01-01

It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

Chromatin-remodeling SWI/SNF complex regulates coenzyme Q6 synthesis and a metabolic shift to respiration in yeast.

PubMed

Awad, Agape M; Venkataramanan, Srivats; Nag, Anish; Galivanche, Anoop Raj; Bradley, Michelle C; Neves, Lauren T; Douglass, Stephen; Clarke, Catherine F; Johnson, Tracy L

2017-09-08

Despite its relatively streamlined genome, there are many important examples of regulated RNA splicing in Saccharomyces cerevisiae Here, we report a role for the chromatin remodeler SWI/SNF in respiration, partially via the regulation of splicing. We find that a nutrient-dependent decrease in Snf2 leads to an increase in splicing of the PTC7 transcript. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q 6 (ubiquinone or CoQ 6 ) and a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ 6 levels. The increase in PTC7 splicing occurs at least in part due to down-regulation of ribosomal protein gene expression, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs to otherwise poorly spliced transcripts. In contrast, a protein encoded by the nonspliced isoform of PTC7 represses CoQ 6 biosynthesis. Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Hypercholesterolemia potentiates aortic endothelial response to inhaled diesel exhaust

PubMed Central

Maresh, J. Gregory; Campen, Matthew J.; Reed, Matthew D.; Darrow, April L.; Shohet, Ralph V.

2012-01-01

Background Inhalation of diesel exhaust induces vascular effects including impaired endothelial function and increased atherosclerosis. Objective To examine the in vivo effects of subchronic diesel exhaust exposure on endothelial cell transcriptional responses in the presence of hypercholesterolemia. Methods ApoE (−/−) and ApoE (+/+) mice inhaled diesel exhaust diluted to particulate matter levels of 300 or 1000 μg/m3 vs. filtered air. After 30 days, endothelial cells were harvested from dispersed aortic cells by fluorescent-activated cell sorting (FACS). Relative mRNA abundance was evaluated by microarray analysis to measure strain-specific transcriptional responses in mice exposed to dilute diesel exhaust vs. filtered air. Results Forty-nine transcripts were significantly dysregulated by >2.8-fold in the endothelium of ApoE (−/−) mice receiving diesel exhaust at 300 or 1000 μg/m3. These included transcripts with roles in plasminogen activation, endothelial permeability, inflammation, genomic stability, and atherosclerosis; similar responses were not observed in ApoE (+/+) mice. Conclusions The potentiation of diesel exhaust-related endothelial gene regulation by hypercholesterolemia helps to explain air pollution-induced vascular effects in animals and humans. The observed regulated transcripts implicate pathways important in the acceleration of atherosclerosis by air pollution. PMID:21222557

Silicon enhances suberization and lignification in roots of rice (Oryza sativa).

PubMed

Fleck, Alexander T; Nye, Thandar; Repenning, Cornelia; Stahl, Frank; Zahn, Marc; Schenk, Manfred K

2011-03-01

The beneficial element silicon (Si) may affect radial oxygen loss (ROL) of rice roots depending on suberization of the exodermis and lignification of sclerenchyma. Thus, the effect of Si nutrition on the oxidation power of rice roots, suberization and lignification was examined. In addition, Si-induced alterations of the transcript levels of 265 genes related to suberin and lignin synthesis were studied by custom-made microarray and quantitative Real Time-PCR. Without Si supply, the oxidation zone of 12 cm long adventitious roots extended along the entire root length but with Si supply the oxidation zone was restricted to 5 cm behind the root tip. This pattern coincided with enhanced suberization of the exodermis and lignification of sclerenchyma by Si supply. Suberization of the exodermis started, with and without Si supply, at 4-5 cm and 8-9 cm distance from the root tip (drt), respectively. Si significantly increased transcript abundance of 12 genes, while two genes had a reduced transcript level. A gene coding for a leucine-rich repeat protein exhibited a 25-fold higher transcript level with Si nutrition. Physiological, histochemical, and molecular-biological data showing that Si has an active impact on rice root anatomy and gene transcription is presented here.

Up-regulated transcripts in a compatible powdery mildew-grapevine interaction.

PubMed

Fekete, Csaba; Fung, Raymond W M; Szabó, Zoltán; Qiu, Wenping; Chang, Le; Schachtman, Daniel P; Kovács, László G

2009-08-01

Powdery mildews (Erysiphales) are obligate biotrophic pathogens that invade susceptible plant cells without triggering cell death. This suggests a highly adept mechanism of parasitism which enables powdery mildews to avoid detection or evade defenses by their host. To better understand this plant-pathogen interaction, we employed suppression subtractive hybridization (SSH), differential hybridization and quantitative real-time (qRT) PCR for the identification of grapevine (Vitis vinifera L.) genes that were specifically up-regulated in response to the grape powdery mildew Erysiphe necator Schwein. We identified 25 grapevine transcripts that increased in abundance upon infection in leaves of the susceptible host V. vinifera Cabernet Sauvignon. Despite the compatible interaction between the pathogen and plant, several of the E. necator-induced transcripts represented typical defense response genes. Among the transcripts identified were those that encoded a leucine-rich repeat serine/threonine kinase-like receptor, an MYB transcription factor, and two ubiquitination-associated proteins, indicating the stimulation of intracellular signal transduction and regulatory functions. A number of genes characteristic of senescence processes, including metallothioneins, a deoxyribonuclease, an aspartyl protease and a subtilase-like serine protease, also were identified. These transcripts expanded the list of previously identified E. necator-responsive grapevine genes and facilitated a more comprehensive view of the molecular events that underlie this economically important plant-pathogen interaction.

LocExpress: a web server for efficiently estimating expression of novel transcripts.

PubMed

Hou, Mei; Tian, Feng; Jiang, Shuai; Kong, Lei; Yang, Dechang; Gao, Ge

2016-12-22

The temporal and spatial-specific expression pattern of a transcript in multiple tissues and cell types can indicate key clues about its function. While several gene atlas available online as pre-computed databases for known gene models, it's still challenging to get expression profile for previously uncharacterized (i.e. novel) transcripts efficiently. Here we developed LocExpress, a web server for efficiently estimating expression of novel transcripts across multiple tissues and cell types in human (20 normal tissues/cells types and 14 cell lines) as well as in mouse (24 normal tissues/cell types and nine cell lines). As a wrapper to RNA-Seq quantification algorithm, LocExpress efficiently reduces the time cost by making abundance estimation calls increasingly within the minimum spanning bundle region of input transcripts. For a given novel gene model, such local context-oriented strategy allows LocExpress to estimate its FPKMs in hundreds of samples within minutes on a standard Linux box, making an online web server possible. To the best of our knowledge, LocExpress is the only web server to provide nearly real-time expression estimation for novel transcripts in common tissues and cell types. The server is publicly available at http://loc-express.cbi.pku.edu.cn .

A single alteration 20 nt 5′ to an editing target inhibits chloroplast RNA editing in vivo

PubMed Central

Reed, Martha L.; Peeters, Nemo M.; Hanson, Maureen R.

2001-01-01

Transcripts of typical dicot plant plastid genes undergo C→U RNA editing at approximately 30 locations, but there is no consensus sequence surrounding the C targets of editing. The cis-acting elements required for editing of the C located at tobacco rpoB editing site II were investigated by introducing translatable chimeric minigenes containing sequence –20 to +6 surrounding the C target of editing. When the –20 to +6 sequence specified by the homologous region present in the black pine chloroplast genome was incorporated, virtually no editing of the transcripts occurred in transgenic tobacco plastids. Nucleotides that differ between the black pine and tobacco sequence were tested for their role in C→U editing by designing chimeric genes containing one or more of these divergent nucleotides. Surprisingly, the divergent nucleotide that had the strongest negative effect on editing of the minigene transcript was located –20 nt 5′ to the C target of editing. Expression of transgene transcripts carrying the 27 nt sequence did not affect the editing extent of the endogenous rpoB transcripts, even though the chimeric transcripts were much more abundant than those of the endogenous gene. In plants carrying a 93 nt rpoB editing site sequence, transgene transcripts accumulated to a level three times greater than transgene transcripts in the plants carrying the 27 nt rpoB editing sites and resulted in editing of the endogenous transcripts from 100 to 50%. Both a lower affinity of the 27 nt site for a trans-acting factor and lower abundance of the transcript could explain why expression of minigene transcripts containing the 27 nt sequence did not affect endogenous editing. PMID:11266552

Characterization of the c-type lysozyme gene family in Anopheles gambiae.

PubMed

Li, Bin; Calvo, Eric; Marinotti, Osvaldo; James, Anthony A; Paskewitz, Susan M

2005-11-07

Seven new c-type lysozyme genes were found using the Anopheles gambiae genome sequence, increasing to eight the total number of genes in this family identified in this species. The eight lysozymes in An. gambiae have considerable variation in gene structure and expression patterns. Lys c-6 has the most unusual primary amino acid structure as the predicted protein consists of five lysozyme-like domains. Transcript abundance of each c-type lysozyme was determined by semiquantitative RT-PCR. Lys c-1, c-6 and c-7 are expressed constitutively in all developmental stages from egg to adult. Lys c-2 and c-4 also are found in all stages, but with relatively much higher levels in adults. Conversely, Lys c-3 and c-8 transcripts are highest in larvae. Lys c-1, c-6 and c-7 transcripts are found in nearly all the adult tissue samples examined while Lys c-2 and Lys c-4 are more restricted in their expression. Lys c-1 and c-2 transcripts are clearly immune responsive and are increased significantly 6-12 h post challenge with bacteria. The functional adaptive changes that may have evolved during the expansion of this gene family are briefly discussed in terms of the expression patterns, gene and protein structures.

Transcriptional analysis of sulfate reducing and chemolithoautotrophic sulfur oxidizing bacteria in the deep subseafloor.

PubMed

Orsi, William D; Barker Jørgensen, Bo; Biddle, Jennifer F

2016-08-01

Sulfate reducing bacteria (SRB) oxidize a significant proportion of subseafloor organic carbon, but their metabolic activities and subsistence mechanisms are poorly understood. Here, we report in depth phylogenetic and metabolic analyses of SRB transcripts in the Peru Margin subseafloor and interpret these results in the context of sulfate reduction activity in the sediment. Relative abundance of overall SRB gene transcripts declines strongly whereas relative abundance of ribosomal protein transcripts from sulfate reducing δ-Proteobacteria peak at 90 m below seafloor (mbsf) within a deep sulfate methane transition zone. This coincides with isotopically heavy δ(34) S values of pore water sulfate (70‰), indicating active subseafloor microbial sulfate reduction. Within the shallow sulfate reduction zone (0-5 mbsf), a transcript encoding the beta subunit of dissimilatory sulfite reductase (dsrB) was related to Desulfitobacterium dehalogenans and environmental sequences from Aarhus Bay (Denmark). At 159 mbsf we discovered a transcript encoding the reversely operating dissimilatory sulfite reductase α-subunit (rdsrA), with basal phylogenetic relation to the chemolithoautotrophic SUP05 Group II clade. A diversity of SRB transcripts involved in cellular maintenance point toward potential subsistence mechanisms under low-energy over long time periods, and provide a detailed new picture of SRB activities underlying sulfur cycling in the deep subseafloor. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

The Dynamics of Transcript Abundance during Cellularization of Developing Barley Endosperm1[OPEN

PubMed Central

Zhang, Runxuan; Burton, Rachel A; Shirley, Neil J.; Little, Alan; Morris, Jenny; Milne, Linda

2016-01-01

Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would help predict end-use properties such as yield, quality, and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm 3 d to 8 d after pollination (DAP). Comparisons of transcript abundance over time revealed 47 gene expression modules that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the datasets could be mined to explore and define the processes of cell wall biosynthesis, remodeling, and degradation. Using a combination of spatial molecular network and gene ontology enrichment analyses, it is shown that genes involved in cell wall metabolism are found in multiple modules, but cluster into two main groups that exhibit peak expression at 3 DAP to 4 DAP and 5 DAP to 8 DAP. The presence of transcription factor genes in these modules allowed candidate genes for the control of wall metabolism during early barley grain development to be identified. The data are publicly available through a dedicated web interface (https://ics.hutton.ac.uk/barseed/), where they can be used to interrogate co- and differential expression for any other genes, groups of genes, or transcription factors expressed during early endosperm development. PMID:26754666

Overexpression of Transcription Factor Sp2 Inhibits Epidermal Differentiation and Increases Susceptibility to Wound and Carcinogen-Induced Tumorigenesis

PubMed Central

Kim, Tae-Hyung; Chiera, Shannon L.; Linder, Keith E.; Trempus, Carol S.; Smart, Robert C.; Horowitz, Jonathan M.

2010-01-01

Sp proteins are evolutionarily-conserved transcription factors required for the expression of a wide variety of genes that are critical for development and cell-cycle progression. De-regulated expression of certain Sp proteins is associated with the formation of a variety of human tumors, however direct evidence that any given Sp protein is oncogenic has been lacking. Here we report that Sp2 protein abundance in mice increases in concert with the progression of carcinogen-induced murine squamous cell carcinomas. Transgenic mice specifically overexpressing murine Sp2 in epidermal basal keratinocytes were highly susceptible to wound- and carcinogen-induced papillomagenesis. Transgenic animals that were homozygous rather than hemizygous for the Sp2 transgene exhibited a striking arrest in the epidermal differentiation program, perishing within two weeks of birth. Our results directly support the likelihood that Sp2 overexpression occurring in various human cancers has significant functional impact. PMID:20959487

Gene expression in the deep biosphere.

PubMed

Orsi, William D; Edgcomb, Virginia P; Christman, Glenn D; Biddle, Jennifer F

2013-07-11

Scientific ocean drilling has revealed a deep biosphere of widespread microbial life in sub-seafloor sediment. Microbial metabolism in the marine subsurface probably has an important role in global biogeochemical cycles, but deep biosphere activities are not well understood. Here we describe and analyse the first sub-seafloor metatranscriptomes from anaerobic Peru Margin sediment up to 159 metres below the sea floor, represented by over 1 billion complementary DNA (cDNA) sequence reads. Anaerobic metabolism of amino acids, carbohydrates and lipids seem to be the dominant metabolic processes, and profiles of dissimilatory sulfite reductase (dsr) transcripts are consistent with pore-water sulphate concentration profiles. Moreover, transcripts involved in cell division increase as a function of microbial cell concentration, indicating that increases in sub-seafloor microbial abundance are a function of cell division across all three domains of life. These data support calculations and models of sub-seafloor microbial metabolism and represent the first holistic picture of deep biosphere activities.

Integrative Analysis Reveals an Outcome-associated and Targetable Pattern of p53 and Cell Cycle Deregulation in Diffuse Large B-cell Lymphoma

PubMed Central

Monti, Stefano; Chapuy, Bjoern; Takeyama, Kunihiko; Rodig, Scott J; Hao, Yangsheng; Yeda, Kelly T.; Inguilizian, Haig; Mermel, Craig; Curie, Treeve; Dogan, Ahmed; Kutok, Jeffery L; Beroukim, Rameen; Neuberg, Donna; Habermann, Thomas; Getz, Gad; Kung, Andrew L; Golub, Todd R; Shipp, Margaret A

2013-01-01

Summary Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate. By integrating copy number data with transcriptional profiles and performing pathway analysis in primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary tumors either had multiple complementary alterations of p53 and cell cycle components or largely lacked these lesions. DLBCLs with p53 and cell cycle pathway CNAs had decreased abundance of p53 target transcripts and increased expression of E2F target genes and the Ki67 proliferation marker. CNAs of the CDKN2A-TP53-RB-E2F axis provide a structural basis for increased proliferation in DLBCL, predict outcome with current therapy and suggest targeted treatment approaches. PMID:22975378

Tubulin C-terminal Post-translational Modifications Do Not Occur in Wood Forming Tissue of Populus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Hao; Gu, Xi; Xue, Liang-Jiao

Cortical microtubules (MTs) are evolutionarily conserved cytoskeletal components with specialized roles in plants, including regulation of cell wall biogenesis. MT functions and dynamics are dictated by the composition of their monomeric subunits, α- (TUA) and β-tubulins (TUB), which in animals and protists are subject to both transcriptional regulation and post-translational modifications (PTM). While spatiotemporal regulation of tubulin gene expression has been reported in plants, whether and to what extent tubulin PTMs occur in these species remain poorly understood. We chose the woody perennial Populus for investigation of tubulin PTMs in this study, with a particular focus on developing xylem wheremore » high tubulin transcript levels support MT-dependent secondary cell wall deposition. Mass spectrometry and immunodetection concurred that detyrosination, non-tyrosination and glutamylation were essentially absent in tubulins isolated from wood-forming tissues of P. deltoides and P. tremula ×alba. Label-free quantification of tubulin isotypes and RNA-Seq estimation of tubulin transcript abundance were largely consistent with transcriptional regulation. However, two TUB isotypes were detected at noticeably lower levels than expected based on RNA-Seq transcript abundance in both Populus species. These findings led us to conclude that MT composition during wood formation depends exclusively on transcriptional and, to a lesser extent, translational regulation of tubulin isotypes.« less

Tubulin C-terminal Post-translational Modifications Do Not Occur in Wood Forming Tissue of Populus

DOE PAGES

Hu, Hao; Gu, Xi; Xue, Liang-Jiao; ...

2016-10-13

Cortical microtubules (MTs) are evolutionarily conserved cytoskeletal components with specialized roles in plants, including regulation of cell wall biogenesis. MT functions and dynamics are dictated by the composition of their monomeric subunits, α- (TUA) and β-tubulins (TUB), which in animals and protists are subject to both transcriptional regulation and post-translational modifications (PTM). While spatiotemporal regulation of tubulin gene expression has been reported in plants, whether and to what extent tubulin PTMs occur in these species remain poorly understood. We chose the woody perennial Populus for investigation of tubulin PTMs in this study, with a particular focus on developing xylem wheremore » high tubulin transcript levels support MT-dependent secondary cell wall deposition. Mass spectrometry and immunodetection concurred that detyrosination, non-tyrosination and glutamylation were essentially absent in tubulins isolated from wood-forming tissues of P. deltoides and P. tremula ×alba. Label-free quantification of tubulin isotypes and RNA-Seq estimation of tubulin transcript abundance were largely consistent with transcriptional regulation. However, two TUB isotypes were detected at noticeably lower levels than expected based on RNA-Seq transcript abundance in both Populus species. These findings led us to conclude that MT composition during wood formation depends exclusively on transcriptional and, to a lesser extent, translational regulation of tubulin isotypes.« less

Transcript abundance on its own cannot be used to infer fluxes in central metabolism

DOE PAGES

Schwender, Jorg; Konig, Christina; Klapperstuck, Matthias; ...

2014-11-28

An attempt has been made to define the extent to which metabolic flux in central plant metabolism is reflected by changes in the transcriptome and metabolome, based on an analysis of in vitro cultured immature embryos of two oilseed rape (Brassica napus) accessions which contrast for seed lipid accumulation. Metabolic flux analysis (MFA) was used to constrain a flux balance metabolic model which included 671 biochemical and transport reactions within the central metabolism. This highly confident flux information was eventually used for comparative analysis of flux vs. transcript (metabolite). Metabolite profiling succeeded in identifying 79 intermediates within the central metabolism,more » some of which differed quantitatively between the two accessions and displayed a significant shift corresponding to flux. An RNA-Seq based transcriptome analysis revealed a large number of genes which were differentially transcribed in the two accessions, including some enzymes/proteins active in major metabolic pathways. With a few exceptions, differential activity in the major pathways (glycolysis, TCA cycle, amino acid, and fatty acid synthesis) was not reflected in contrasting abundances of the relevant transcripts. The conclusion was that transcript abundance on its own cannot be used to infer metabolic activity/fluxes in central plant metabolism. Lastly, this limitation needs to be borne in mind in evaluating transcriptome data and designing metabolic engineering experiments.« less

Distant sequences determine 5′ end formation of cox3 transcripts in Arabidopsis thaliana ecotype C24

PubMed Central

Forner, Joachim; Weber, Bärbel; Wiethölter, Caterina; Meyer, Rhonda C.; Binder, Stefan

2005-01-01

The genomic environments and the transcripts of the mitochondrial cox3 gene are investigated in three Arabidopsis thaliana ecotypes. While the proximate 5′ sequences up to nucleotide position −584, the coding regions and the 3′ flanking regions are identical in Columbia (Col), C24 and Landsberg erecta (Ler), genomic variation is detected in regions further upstream. In the mitochondrial DNA of Col, a 1790 bp fragment flanked by a nonanucleotide direct repeat is present beyond position −584 with respect to the ATG. While in Ler only part of this insertion is conserved, this sequence is completely absent in C24, except for a single copy of the nonanucleotide direct repeat. Northern hybridization reveals identical major transcripts in the three ecotypes, but identifies an additional abundant 60 nt larger mRNA species in C24. The extremities of the most abundant mRNA species are identical in the three ecotypes. In C24, an extra major 5′ end is abundant. This terminus and the other major 5′ ends are located in identical sequence regions. Inspection of Atcox3 transcripts in C24/Col hybrids revealed a female inheritance of the mRNA species with the extra 5′ terminus. Thus, a mitochondrially encoded factor determines the generation of an extra 5′ mRNA end. PMID:16107557

Potassium Channel Interacting Protein 2 (KChIP2) is not a transcriptional regulator of cardiac electrical remodeling

PubMed Central

Winther, Sine V.; Tuomainen, Tomi; Borup, Rehannah; Tavi, Pasi; Antoons, Gudrun; Thomsen, Morten B.

2016-01-01

The heart-failure relevant Potassium Channel Interacting Protein 2 (KChIP2) augments CaV1.2 and KV4.3. KChIP3 represses CaV1.2 transcription in cardiomyocytes via interaction with regulatory DNA elements. Hence, we tested nuclear presence of KChIP2 and if KChIP2 translocates into the nucleus in a Ca2+ dependent manner. Cardiac biopsies from human heart-failure patients and healthy donor controls showed that nuclear KChIP2 abundance was significantly increased in heart failure; however, this was secondary to a large variation of total KChIP2 content. Administration of ouabain did not increase KChIP2 content in nuclear protein fractions in anesthetized mice. KChIP2 was expressed in cell lines, and Ca2+ ionophores were applied in a concentration- and time-dependent manner. The cell lines had KChIP2-immunoreactive protein in the nucleus in the absence of treatments to modulate intracellular Ca2+ concentration. Neither increasing nor decreasing intracellular Ca2+ concentrations caused translocation of KChIP2. Microarray analysis did not identify relief of transcriptional repression in murine KChIP2−/− heart samples. We conclude that although there is a baseline presence of KChIP2 in the nucleus both in vivo and in vitro, KChIP2 does not directly regulate transcriptional activity. Moreover, the nuclear transport of KChIP2 is not dependent on Ca2+. Thus, KChIP2 does not function as a conventional transcription factor in the heart. PMID:27349185

Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions

PubMed Central

Depke, Maren; Pané-Farré, Jan; Debarbouille, Michel; van der Kooi-Pol, Magdalena M.; Guérin, Cyprien; Dérozier, Sandra; Hiron, Aurelia; Jarmer, Hanne; Leduc, Aurélie; Michalik, Stephan; Reilman, Ewoud; Schaffer, Marc; Schmidt, Frank; Bessières, Philippe; Noirot, Philippe; Hecker, Michael; Msadek, Tarek; Völker, Uwe; van Dijl, Jan Maarten

2016-01-01

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria. PMID:27035918

Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought. Impaired Coordination of Selected Transcript and Proteomic Responses, and Regulation of Various Multifunctional Proteins

PubMed Central

Rurek, Michał; Czołpińska, Magdalena; Staszak, Aleksandra Maria; Nowak, Witold; Krzesiński, Włodzimierz; Spiżewski, Tomasz

2018-01-01

Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis) cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly) in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Respiratory (e.g., complex II, IV (CII, CIV) and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g., components of RNA editing machinery) were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots) immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX) regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation. PMID:29642585

Environmental Control of Phosphoenolpyruvate Carboxylase Induction in Mature Mesembryanthemum crystallinum L. 1

PubMed Central

Piepenbrock, Mechtild; Schmitt, Jürgen M.

1991-01-01

Mesembryanthemum crystallinum L. plants shift the mode of carbon assimilation from C3 to Crassulacean acid metabolism when stressed by high salinity. A prerequisite for Crassulacean acid metabolism induction is the synthesis of phosphoenolpyruvate carboxylase (PEPCase). A moderate increase in the abundance of PEPCase transcripts and activity is observed in 7-week-old, well-watered plants. This increase in PEPCase coincides in time with a decrease in the growth rate of the shoots. The steady-state level of PEPCase activity is uniform along the leaves of well-watered plants, as can be shown by comparing leaves of different age from individual 7-week-old plants. In contrast, the rate of induction in response to salt stress varies with the age of plants and to a lesser extent with the age of the leaves. Two-week-old seedlings induce PEPCase slowly under a moderate salt stress regimen, whereas older plants induce faster. When individual leaves from a seven-week-old plant are compared with respect to induction velocity, no clear-cut correlation with leaf age is apparent. The highest induction rate is observed in leaves from node five that are about 2 weeks old at the beginning of the experiment. PEPCase transcripts are readily down-regulated to minute levels when detached leaves are hydrated. The levels reached after 8 hours of rehydration are very similar, regardless of whether the leaves were cut from young or old plants or whether the plants were previously salt-stressed or well-watered. It is concluded that environmental rather than developmental factors are predominant in determining abundance of PEPCase activity and transcripts in leaves of mature M. crystallinum plants. ImagesFigure 1Figure 3Figure 5 PMID:16668542

Pre-hatching embryo-dependent and -independent programming of endometrial function in cattle

PubMed Central

Sponchiado, Mariana; Gomes, Nathália Souza; Fontes, Patrícia Kubo; Martins, Thiago; del Collado, Maite; Pastore, Athos de Assumpção; Pugliesi, Guilherme; Nogueira, Marcelo Fábio Gouveia

2017-01-01

The bovine pre-implantation embryo secretes bioactive molecules from early development stages, but effects on endometrial function are reported to start only after elongation. Here, we interrogated spatially defined regions of the endometrium transcriptome for responses to a day 7 embryo in vivo. We hypothesize that exposure to an embryo changes the abundance of specific transcripts in the cranial region of the pregnant uterine horn. Endometrium was collected from the uterotubal junction (UTJ), anterior (IA), medial (IM) and posterior (IP) regions of the uterine horn ipsilateral to the CL 7 days after estrus from sham-inseminated (Con) or artificially inseminated, confirmed pregnant (Preg) cows. Abundance of 86 transcripts was evaluated by qPCR using a microfluidic platform. Abundance of 12 transcripts was modulated in the Preg endometrium, including classical interferon-stimulated genes (ISG15, MX1, MX2 and OAS1Y), prostaglandin biosynthesis genes (PTGES, HPGD and AKR1C4), water channel (AQP4) and a solute transporter (SLC1A4) and this was in the UTJ and IA mainly. Additionally, for 71 transcripts, abundance varied according to region of the reproductive tract. Regulation included downregulation of genes associated with proliferation (IGF1, IGF2, IGF1R and IGF2R) and extracellular matrix remodeling (MMP14, MMP19 and MMP2) and upregulation of anti-adhesive genes (MUC1) in the cranial regions of uterine horn. Physical proximity to the embryo provides paracrine regulation of endometrial function. Embryo-independent regulation of the endometrial transcriptome may support subsequent stages of embryo development, such as elongation and implantation. We speculate that successful early embryo-dependent and -independent programming fine-tune endometrial functions that are important for maintenance of pregnancy in cattle. PMID:28423001

Use of transcriptome sequencing to understand the pistillate flowering in hickory (Carya cathayensis Sarg.).

PubMed

Huang, You-Jun; Liu, Li-Li; Huang, Jian-Qin; Wang, Zheng-Jia; Chen, Fang-Fang; Zhang, Qi-Xiang; Zheng, Bing-Song; Chen, Ming

2013-10-10

Different from herbaceous plants, the woody plants undergo a long-period vegetative stage to achieve floral transition. They then turn into seasonal plants, flowering annually. In this study, a preliminary model of gene regulations for seasonal pistillate flowering in hickory (Carya cathayensis) was proposed. The genome-wide dynamic transcriptome was characterized via the joint-approach of RNA sequencing and microarray analysis. Differential transcript abundance analysis uncovered the dynamic transcript abundance patterns of flowering correlated genes and their major functions based on Gene Ontology (GO) analysis. To explore pistillate flowering mechanism in hickory, a comprehensive flowering gene regulatory network based on Arabidopsis thaliana was constructed by additional literature mining. A total of 114 putative flowering or floral genes including 31 with differential transcript abundance were identified in hickory. The locations, functions and dynamic transcript abundances were analyzed in the gene regulatory networks. A genome-wide co-expression network for the putative flowering or floral genes shows three flowering regulatory modules corresponding to response to light abiotic stimulus, cold stress, and reproductive development process, respectively. Totally 27 potential flowering or floral genes were recruited which are meaningful to understand the hickory specific seasonal flowering mechanism better. Flowering event of pistillate flower bud in hickory is triggered by several pathways synchronously including the photoperiod, autonomous, vernalization, gibberellin, and sucrose pathway. Totally 27 potential flowering or floral genes were recruited from the genome-wide co-expression network function module analysis. Moreover, the analysis provides a potential FLC-like gene based vernalization pathway and an 'AC' model for pistillate flower development in hickory. This work provides an available framework for pistillate flower development in hickory, which is significant for insight into regulation of flowering and floral development of woody plants.

Use of transcriptome sequencing to understand the pistillate flowering in hickory (Carya cathayensis Sarg.)

PubMed Central

2013-01-01

Background Different from herbaceous plants, the woody plants undergo a long-period vegetative stage to achieve floral transition. They then turn into seasonal plants, flowering annually. In this study, a preliminary model of gene regulations for seasonal pistillate flowering in hickory (Carya cathayensis) was proposed. The genome-wide dynamic transcriptome was characterized via the joint-approach of RNA sequencing and microarray analysis. Results Differential transcript abundance analysis uncovered the dynamic transcript abundance patterns of flowering correlated genes and their major functions based on Gene Ontology (GO) analysis. To explore pistillate flowering mechanism in hickory, a comprehensive flowering gene regulatory network based on Arabidopsis thaliana was constructed by additional literature mining. A total of 114 putative flowering or floral genes including 31 with differential transcript abundance were identified in hickory. The locations, functions and dynamic transcript abundances were analyzed in the gene regulatory networks. A genome-wide co-expression network for the putative flowering or floral genes shows three flowering regulatory modules corresponding to response to light abiotic stimulus, cold stress, and reproductive development process, respectively. Totally 27 potential flowering or floral genes were recruited which are meaningful to understand the hickory specific seasonal flowering mechanism better. Conclusions Flowering event of pistillate flower bud in hickory is triggered by several pathways synchronously including the photoperiod, autonomous, vernalization, gibberellin, and sucrose pathway. Totally 27 potential flowering or floral genes were recruited from the genome-wide co-expression network function module analysis. Moreover, the analysis provides a potential FLC-like gene based vernalization pathway and an 'AC’ model for pistillate flower development in hickory. This work provides an available framework for pistillate flower development in hickory, which is significant for insight into regulation of flowering and floral development of woody plants. PMID:24106755

Large scale real-time PCR analysis of mRNA abundance in rainbow trout eggs in relationship with egg quality and post-ovulatory ageing.

PubMed

Aegerter, Sandrine; Jalabert, Bernard; Bobe, Julien

2005-11-01

The mRNA levels of 39 target genes were monitored in unfertilized eggs of 14 rainbow trout sampled the day of ovulation and again 5, 14, and 21 days later. For all 56 collected egg batches, an egg sample was fertilized to estimate egg quality by monitoring embryonic development. Remaining eggs were used for RNA extraction and subsequent real-time PCR analysis. A significant drop of egg quality was observed when eggs were held in the body cavity for 14 or 21 days post-ovulation (dpo). During the same period, eight transcripts (nucleoplasmin or Npm2, ferritin H, tubulin beta, JNK1, cyclin A1, cyclin A2, cathepsin Z, and IGF2) exhibited a differential abundance at one or several collection time(s). Interestingly, we observed higher levels of cyclins A1 and A2 mRNAs in eggs taken 5 days post-ovulation than in eggs taken, from the same females, at the time of ovulation. In addition, seven transcripts exhibited a differential abundance between low quality and high quality eggs. Low quality eggs were characterized by lower levels of Npm2, tubulin beta, and IGF1 transcripts. In contrast, keratins 8 and 18, cathepsin Z, and prostaglandin synthase 2 were more abundant in low quality eggs than in high quality eggs. In this study, we have demonstrated differences in mRNA levels in the rainbow trout egg that are reflective of developmental competence differences induced by post-ovulatory ageing. The putative role of these transcripts in post-ovulatory ageing-induced egg quality defects is discussed with special attention for corresponding cellular functions.

A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia

PubMed Central

Schwartz, Jason R.; Sarvaiya, Purvaba J.; Leiva, Lily E.; Velez, Maria C.; Singleton, Tammuella C.; Yu, Lolie C.; Vedeckis, Wayne V.

2012-01-01

Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells. However, not all leukemia cells are sensitive to GC, and no assay to stratify patients is available. In the GC-sensitive T-cell ALL cell line CEM-C7, auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response. This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations. The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay. There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples, but not for a probe set that detects a specific, low abundance GR transcript (exon 1A3). Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and, with further development, may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients. PMID:22739263

The role of proteosome-mediated proteolysis in modulating potentially harmful transcription factor activity in Saccharomyces cerevisiae

PubMed Central

Bonzanni, Nicola; Zhang, Nianshu; Oliver, Stephen G.; Fisher, Jasmin

2011-01-01

Motivation: The appropriate modulation of the stress response to variable environmental conditions is necessary to maintain sustained viability in Saccharomyces cerevisiae. Particularly, controlling the abundance of proteins that may have detrimental effects on cell growth is crucial for rapid recovery from stress-induced quiescence. Results: Prompted by qualitative modeling of the nutrient starvation response in yeast, we investigated in vivo the effect of proteolysis after nutrient starvation showing that, for the Gis1 transcription factor at least, proteasome-mediated control is crucial for a rapid return to growth. Additional bioinformatics analyses show that potentially toxic transcriptional regulators have a significantly lower protein half-life, a higher fraction of unstructured regions and more potential PEST motifs than the non-detrimental ones. Furthermore, inhibiting proteasome activity tends to increase the expression of genes induced during the Environmental Stress Response more than those in the rest of the genome. Our combined results suggest that proteasome-mediated proteolysis of potentially toxic transcription factors tightly modulates the stress response in yeast. Contact: jasmin.fisher@microsoft.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21685082

Regulatory consequences of neuronal ELAV-like protein binding to coding and non-coding RNAs in human brain

PubMed Central

Scheckel, Claudia; Drapeau, Elodie; Frias, Maria A; Park, Christopher Y; Fak, John; Zucker-Scharff, Ilana; Kou, Yan; Haroutunian, Vahram; Ma'ayan, Avi

2016-01-01

Neuronal ELAV-like (nELAVL) RNA binding proteins have been linked to numerous neurological disorders. We performed crosslinking-immunoprecipitation and RNAseq on human brain, and identified nELAVL binding sites on 8681 transcripts. Using knockout mice and RNAi in human neuroblastoma cells, we showed that nELAVL intronic and 3' UTR binding regulates human RNA splicing and abundance. We validated hundreds of nELAVL targets among which were important neuronal and disease-associated transcripts, including Alzheimer's disease (AD) transcripts. We therefore investigated RNA regulation in AD brain, and observed differential splicing of 150 transcripts, which in some cases correlated with differential nELAVL binding. Unexpectedly, the most significant change of nELAVL binding was evident on non-coding Y RNAs. nELAVL/Y RNA complexes were specifically remodeled in AD and after acute UV stress in neuroblastoma cells. We propose that the increased nELAVL/Y RNA association during stress may lead to nELAVL sequestration, redistribution of nELAVL target binding, and altered neuronal RNA splicing. DOI: http://dx.doi.org/10.7554/eLife.10421.001 PMID:26894958

Experimental assessment of diazotroph responses to elevated seawater pCO2 in the North Pacific Subtropical Gyre

NASA Astrophysics Data System (ADS)

Böttjer, Daniela; Karl, David M.; Letelier, Ricardo M.; Viviani, Donn A.; Church, Matthew J.

2014-06-01

We examined short-term (24-72 h) responses of naturally occurring marine N2 fixing microorganisms (termed diazotrophs) to abrupt increases in the partial pressure of carbon dioxide (pCO2) in seawater during nine incubation experiments conducted between May 2010 and September 2012 at Station ALOHA (A Long-term Oligotrophic Habitat Assessment) (22°45'N, 158°W) in the North Pacific Subtropical Gyre (NPSG). Rates of N2 fixation, nitrogenase (nifH) gene abundances and transcripts of six major groups of cyanobacterial diazotrophs (including both unicellular and filamentous phylotypes), and rates of primary productivity (as measured by 14C-bicarbonate assimilation into plankton biomass) were determined under contemporary (~390 ppm) and elevated pCO2 conditions (~1100 ppm). Quantitative polymerase chain reaction (QPCR) amplification of planktonic nifH genes revealed that unicellular cyanobacteria phylotypes dominated gene abundances during these experiments. In the majority of experiments (seven out of nine), elevated pCO2 did not significantly influence rates of dinitrogen (N2) fixation or primary productivity (two-way analysis of variance (ANOVA), P > 0.05). During two experiments, rates of N2 fixation and primary productivity were significantly lower (by 79 to 82% and 52 to 72%, respectively) in the elevated pCO2 treatments relative to the ambient controls (two-way ANOVA, P < 0.05). QPCR amplification of nifH genes and gene transcripts revealed that diazotroph abundances and nifH gene expression were largely unchanged by the perturbation of the seawater pCO2. Our results suggest that naturally occurring N2 fixing plankton assemblages in the NPSG are relatively resilient to large, short-term increases in pCO2.

Plastocyanin Controls the Stabilization of the Thylakoid Cu-transporting P-type ATPase PAA2/HMA8 in Response to Low Copper in Arabidopsis*

PubMed Central

Tapken, Wiebke; Ravet, Karl; Pilon, Marinus

2012-01-01

PAA2/HMA8 (P-type ATPase of Arabidopsis/Heavy-metal-associated 8) is a thylakoid located copper (Cu)-transporter in Arabidopsis thaliana. In tandem with PAA1/HMA6, which is located in the inner chloroplast envelope, it supplies Cu to plastocyanin (PC), an essential cuproenzyme of the photosynthetic machinery. We investigated whether the chloroplast Cu transporters are affected by Cu addition to the growth media. Immunoblots showed that PAA2 protein abundance decreased significantly and specifically when Cu in the media was increased, while PAA1 remained unaffected. The function of SPL7, the transcriptional regulator of Cu homeostasis, was not required for this regulation of PAA2 protein abundance and Cu addition did not affect PAA2 transcript levels, as determined by qRT-PCR. We used the translational inhibitor cycloheximide to analyze turnover and observed that the stability of the PAA2 protein was decreased in plants grown with elevated Cu. Interestingly, PAA2 protein abundance was significantly increased in paa1 mutants, in which the Cu content in the chloroplast is half of that of the wild-type, due to impaired Cu import into the organelle. In contrast in a pc2 insertion mutant, which has strongly reduced plastocyanin expression, the PAA2 protein levels were low regardless of Cu addition to the growth media. Together, these data indicate that plastid Cu levels control PAA2 stability and that plastocyanin, which is the target of PAA2 mediated Cu delivery in thylakoids, is a major determinant of this regulatory mechanism. PMID:22493454

Complex Interplay among DNA Modification, Noncoding RNA Expression and Protein-Coding RNA Expression in Salvia miltiorrhiza Chloroplast Genome

PubMed Central

Chen, Haimei; Zhang, Jianhui; Yuan, George; Liu, Chang

2014-01-01

Salvia miltiorrhiza is one of the most widely used medicinal plants. As a first step to develop a chloroplast-based genetic engineering method for the over-production of active components from S. miltiorrhiza, we have analyzed the genome, transcriptome, and base modifications of the S. miltiorrhiza chloroplast. Total genomic DNA and RNA were extracted from fresh leaves and then subjected to strand-specific RNA-Seq and Single-Molecule Real-Time (SMRT) sequencing analyses. Mapping the RNA-Seq reads to the genome assembly allowed us to determine the relative expression levels of 80 protein-coding genes. In addition, we identified 19 polycistronic transcription units and 136 putative antisense and intergenic noncoding RNA (ncRNA) genes. Comparison of the abundance of protein-coding transcripts (cRNA) with and without overlapping antisense ncRNAs (asRNA) suggest that the presence of asRNA is associated with increased cRNA abundance (p<0.05). Using the SMRT Portal software (v1.3.2), 2687 potential DNA modification sites and two potential DNA modification motifs were predicted. The two motifs include a TATA box–like motif (CPGDMM1, “TATANNNATNA”), and an unknown motif (CPGDMM2 “WNYANTGAW”). Specifically, 35 of the 97 CPGDMM1 motifs (36.1%) and 91 of the 369 CPGDMM2 motifs (24.7%) were found to be significantly modified (p<0.01). Analysis of genes downstream of the CPGDMM1 motif revealed the significantly increased abundance of ncRNA genes that are less than 400 bp away from the significantly modified CPGDMM1motif (p<0.01). Taking together, the present study revealed a complex interplay among DNA modifications, ncRNA and cRNA expression in chloroplast genome. PMID:24914614

Complex interplay among DNA modification, noncoding RNA expression and protein-coding RNA expression in Salvia miltiorrhiza chloroplast genome.

PubMed

Chen, Haimei; Zhang, Jianhui; Yuan, George; Liu, Chang

2014-01-01

Salvia miltiorrhiza is one of the most widely used medicinal plants. As a first step to develop a chloroplast-based genetic engineering method for the over-production of active components from S. miltiorrhiza, we have analyzed the genome, transcriptome, and base modifications of the S. miltiorrhiza chloroplast. Total genomic DNA and RNA were extracted from fresh leaves and then subjected to strand-specific RNA-Seq and Single-Molecule Real-Time (SMRT) sequencing analyses. Mapping the RNA-Seq reads to the genome assembly allowed us to determine the relative expression levels of 80 protein-coding genes. In addition, we identified 19 polycistronic transcription units and 136 putative antisense and intergenic noncoding RNA (ncRNA) genes. Comparison of the abundance of protein-coding transcripts (cRNA) with and without overlapping antisense ncRNAs (asRNA) suggest that the presence of asRNA is associated with increased cRNA abundance (p<0.05). Using the SMRT Portal software (v1.3.2), 2687 potential DNA modification sites and two potential DNA modification motifs were predicted. The two motifs include a TATA box-like motif (CPGDMM1, "TATANNNATNA"), and an unknown motif (CPGDMM2 "WNYANTGAW"). Specifically, 35 of the 97 CPGDMM1 motifs (36.1%) and 91 of the 369 CPGDMM2 motifs (24.7%) were found to be significantly modified (p<0.01). Analysis of genes downstream of the CPGDMM1 motif revealed the significantly increased abundance of ncRNA genes that are less than 400 bp away from the significantly modified CPGDMM1motif (p<0.01). Taking together, the present study revealed a complex interplay among DNA modifications, ncRNA and cRNA expression in chloroplast genome.

Lithium-induced neuroprotection in stroke involves increased miR-124 expression, reduced RE1-silencing transcription factor abundance and decreased protein deubiquitination by GSK3β inhibition-independent pathways.

PubMed

Doeppner, Thorsten R; Kaltwasser, Britta; Sanchez-Mendoza, Eduardo H; Caglayan, Ahmet B; Bähr, Mathias; Hermann, Dirk M

2017-03-01

Lithium promotes acute poststroke neuronal survival, which includes mechanisms that are not limited to GSK3β inhibition. However, whether lithium induces long-term neuroprotection and enhanced brain remodeling is unclear. Therefore, mice were exposed to transient middle cerebral artery occlusion and lithium (1 mg/kg bolus followed by 2 mg/kg/day over up to 7 days) was intraperitoneally administered starting 0-9 h after reperfusion onset. Delivery of lithium no later than 6 h reduced infarct volume on day 2 and decreased brain edema, leukocyte infiltration, and microglial activation, as shown by histochemistry and flow cytometry. Lithium-induced neuroprotection persisted throughout the observation period of 56 days and was associated with enhanced neurological recovery. Poststroke angioneurogenesis and axonal plasticity were also enhanced by lithium. On the molecular level, lithium increased miR-124 expression, reduced RE1-silencing transcription factor abundance, and decreased protein deubiquitination in cultivated cortical neurons exposed to oxygen-glucose deprivation and in brains of mice exposed to cerebral ischemia. Notably, this effect was not mimicked by pharmacological GSK3β inhibition. This study for the first time provides efficacy data for lithium in the postacute ischemic phase, reporting a novel mechanism of action, i.e. increased miR-124 expression facilitating REST degradation by which lithium promotes postischemic neuroplasticity and angiogenesis.

Lithium-induced neuroprotection in stroke involves increased miR-124 expression, reduced RE1-silencing transcription factor abundance and decreased protein deubiquitination by GSK3β inhibition-independent pathways

PubMed Central

Kaltwasser, Britta; Sanchez-Mendoza, Eduardo H; Caglayan, Ahmet B; Bähr, Mathias; Hermann, Dirk M

2016-01-01

Lithium promotes acute poststroke neuronal survival, which includes mechanisms that are not limited to GSK3β inhibition. However, whether lithium induces long-term neuroprotection and enhanced brain remodeling is unclear. Therefore, mice were exposed to transient middle cerebral artery occlusion and lithium (1 mg/kg bolus followed by 2 mg/kg/day over up to 7 days) was intraperitoneally administered starting 0–9 h after reperfusion onset. Delivery of lithium no later than 6 h reduced infarct volume on day 2 and decreased brain edema, leukocyte infiltration, and microglial activation, as shown by histochemistry and flow cytometry. Lithium-induced neuroprotection persisted throughout the observation period of 56 days and was associated with enhanced neurological recovery. Poststroke angioneurogenesis and axonal plasticity were also enhanced by lithium. On the molecular level, lithium increased miR-124 expression, reduced RE1-silencing transcription factor abundance, and decreased protein deubiquitination in cultivated cortical neurons exposed to oxygen–glucose deprivation and in brains of mice exposed to cerebral ischemia. Notably, this effect was not mimicked by pharmacological GSK3β inhibition. This study for the first time provides efficacy data for lithium in the postacute ischemic phase, reporting a novel mechanism of action, i.e. increased miR-124 expression facilitating REST degradation by which lithium promotes postischemic neuroplasticity and angiogenesis. PMID:27126323

Ascorbic acid deficiency leads to increased grain chalkiness in transgenic rice for suppressed of L-GalLDH.

PubMed

Yu, Le; Liu, Yonghai; Lu, Lina; Zhang, Qilei; Chen, Yezheng; Zhou, Liping; Chen, Hua; Peng, Changlian

2017-04-01

The grain chalkiness of rice (Oryza sativa L.), which determines the rice quality and price, is a major concern in rice breeding. Reactive oxygen species (ROS) plays a critical role in regulating rice endosperm chalkiness. Ascorbic acid (Asc) is a major plant antioxidant, which strictly regulates the levels of ROS. l-galactono-1, 4-lactone dehydrogenase (L-GalLDH, EC 1.3.2.3) is an enzyme that catalyzes the last step of Asc biosynthesis in higher plants. Here we show that the L-GalLDH-suppressed transgenic rice, GI-1 and GI-2, which have constitutively low (between 30% and 50%) leaf and grain Asc content compared with the wild-type (WT), exhibit significantly increased grain chalkiness. Further examination showed that the deficiency of Asc resulted in a higher lipid peroxidation and H 2 O 2 content, accompanied by a lower hydroxyl radical scavenging rate, total antioxidant capacity and photosynthetic ability. In addition, changes of the enzyme activities and gene transcript abundances related to starch synthesis were also observed in GI-1 and GI-2 grains. The results we presented here suggest a close correlation between Asc deficiency and grain chalkiness in the L-GalLDH-suppressed transgenics. Asc deficiency leads to the accumulation of H 2 O 2 , affecting antioxidant capacity and photosynthetic function, changing enzyme activities and gene transcript abundances related to starch synthesis, finally leading to the increased grain chalkiness. Copyright © 2017 Elsevier GmbH. All rights reserved.

Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting.

PubMed

Jackson, Angelyca A; Daniels, Emily F; Hammond, John H; Willger, Sven D; Hogan, Deborah A

2014-10-01

Haemolytic phospholipase C (PlcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PlcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the catabolism of PlcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed plcH transcription and we proposed that this regulation allowed for PlcH levels to be maintained in a way that promotes productive host-pathogen interactions. Evidence for Anr-mediated regulation of PlcH came from data showing that growth at low oxygen (1%) repressed PlcH abundance and plcH transcription in the WT, and that plcH transcription was enhanced in an Δanr mutant. The plcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased plcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress plcH transcription as GbdR, a positive regulator of plcH, was required for expression. Overexpression of Anr was sufficient to repress plcH transcription even at 21 % oxygen. Anr repressed plcH expression and phospholipase C activity in a cell culture model for P. aeruginosa-epithelial cell interactions. The Authors.

Whole-body transcriptome of selectively bred, resistant-, control-, and susceptible-line rainbow trout following experimental challenge with Flavobacterium psychrophilum

PubMed Central

Marancik, David; Gao, Guangtu; Paneru, Bam; Ma, Hao; Hernandez, Alvaro G.; Salem, Mohamed; Yao, Jianbo; Palti, Yniv; Wiens, Gregory D.

2014-01-01

Genetic improvement for enhanced disease resistance in fish is an increasingly utilized approach to mitigate endemic infectious disease in aquaculture. In domesticated salmonid populations, large phenotypic variation in disease resistance has been identified but the genetic basis for altered responsiveness remains unclear. We previously reported three generations of selection and phenotypic validation of a bacterial cold water disease (BCWD) resistant line of rainbow trout, designated ARS-Fp-R. This line has higher survival after infection by either standardized laboratory challenge or natural challenge as compared to two reference lines, designated ARS-Fp-C (control) and ARS-Fp-S (susceptible). In this study, we utilized 1.1 g fry from the three genetic lines and performed RNA-seq to measure transcript abundance from the whole body of naive and Flavobacterium psychrophilum infected fish at day 1 (early time-point) and at day 5 post-challenge (onset of mortality). Sequences from 24 libraries were mapped onto the rainbow trout genome reference transcriptome of 46,585 predicted protein coding mRNAs that included 2633 putative immune-relevant gene transcripts. A total of 1884 genes (4.0% genome) exhibited differential transcript abundance between infected and mock-challenged fish (FDR < 0.05) that included chemokines, complement components, tnf receptor superfamily members, interleukins, nod-like receptor family members, and genes involved in metabolism and wound healing. The largest number of differentially expressed genes occurred on day 5 post-infection between naive and challenged ARS-Fp-S line fish correlating with high bacterial load. After excluding the effect of infection, we identified 21 differentially expressed genes between the three genetic lines. In summary, these data indicate global transcriptome differences between genetic lines of naive animals as well as differentially regulated transcriptional responses to infection. PMID:25620978

Molecular characterization and transcriptional regulation of the renin-angiotensin system genes in Senegalese sole (Solea senegalensis Kaup, 1858): differential gene regulation by salinity.

PubMed

Armesto, Paula; Cousin, Xavier; Salas-Leiton, Emilio; Asensio, Esther; Manchado, Manuel; Infante, Carlos

2015-06-01

In this work, the complete cDNA sequence encoding angiotensinogen (agt) in the euryhaline flatfish Senegalese sole was obtained. Additionally, putative coding sequences belonging to other renin-angiotensin system (RAS) genes including renin (ren), angiotensin-converting enzyme (ace), angiotensin-converting enzyme 2 (ace2), as well as angiotensin II receptor type I (agtr1) and type II (agtr2), were also identified. In juvenile tissues, agt transcripts were mainly detected in liver, ren in kidney, ace and ace2 in intestine, agtr1 in kidney and brain, and agtr2 in liver and kidney. Expression analysis of the six RAS genes after a salinity shift revealed a clear increase of agt mRNA abundance in liver just after transferring soles to high salinity water (60 ppt) with a peak at 48 h. Moreover, gene expression analysis in gills showed transcriptional regulation of ace and agtr1 at 48 h and agtr2 at 96 h after transferring soles to 60 ppt. Incubation of larvae before mouth opening (until 3 days post hatch; dph) at low salinity (10 ppt) resulted in a coordinated transcriptional up-regulation of RAS genes. Nevertheless, no differences in mRNA abundance between salinities were observed when larvae were cultivated to low salinity after mouth opening. Whole-mount in situ hybridization (WISH) signal for agt and ace in 3 dph larvae incubated at 10 ppt and 35 ppt confirmed that the former gene was mainly expressed in liver whereas the later gene was mainly located in pharynx and posterior gut, without pronounced differences in intensity between salinities. Possible physiological significance of all these results is discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

A wheat salinity-induced WRKY transcription factor TaWRKY93 confers multiple abiotic stress tolerance in Arabidopsis thaliana.

PubMed

Qin, Yuxiang; Tian, Yanchen; Liu, Xiuzhi

2015-08-21

Wheat is an important crop in the world. But most of the cultivars are salt sensitive, and often adversely affected by salt stress. WRKY transcription factors play a major role in plant responses to salt stress, but the effective salinity regulatory WRKYs identified in bread wheat are limited and the mechanism of salt stress tolerance is also not well explored. Here, we identified a salt (NaCl) induced class II WRKY transcription factor TaWRKY93. Its transcript level was strongly induced by salt (NaCl) and exogenous abscisic acid (ABA). Over-expression of TaWRKY93 in Arabidopsis thaliana enhanced salt (NaCl), drought, low temperature and osmotic (mannitol) stress tolerance, mainly demonstrated by transgenic plants forming longer primary roots or more lateral roots on MS plates supplemented with NaCl and mannitol individually, higher survival rate under drought and low temperature stress. Further, transgenic plants maintained a more proline content, higher relative water content and less electrolyte leakage than the wild type plants. The transcript abundance of a series of abiotic stress-related genes was up-regulated in the TaWRKY93 transgenic plants. In summary, TaWRKY93 is a new positive regulator of abiotic stress, it may increase salinity, drought and low temperature stress tolerance through enhancing osmotic adjustment, maintaining membrane stability and increasing transcription of stress related genes, and contribute to the superior agricultural traits of SR3 through promoting root development. It can be used as a candidate gene for wheat transgenic engineering breeding against abiotic stress. Copyright © 2015 Elsevier Inc. All rights reserved.

Transcriptional and Physiological Responses to Nutrient Loading on Toxin Formation and Photosynthesis in Microcystis Aeruginosa FACHB-905

PubMed Central

Peng, Guotao; Lin, Sijie; Fan, Zhengqiu; Wang, Xiangrong

2017-01-01

An important goal of understanding harmful algae blooms is to determine how environmental factors affect the growth and toxin formation of toxin-producing species. In this study, we investigated the transcriptional responses of toxin formation gene (mcyB) and key photosynthesis genes (psaB, psbD and rbcL) of Microcystis aeruginosa FACHB-905 in different nutrient loading conditions using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Three physio-biochemical parameters (malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH)) were also evaluated to provide insight into the physiological responses of Microcystis cells. We observed an upregulation of mcyB gene in nutrient-deficient conditions, especially in nitrogen (N) limitation condition, and the transcript abundance declined after the nutrient were resupplied. Differently, high transcription levels were seen in phosphorus (P) deficient treatments for key photosynthesis genes throughout the culture period, while those in N-deficient cells varied with time, suggesting an adaptive regulation of Microsystis cells to nutrient stress. Increased contents of antioxidant enzymes (SOD and GSH) were seen in both N and P-deficient conditions, suggesting the presence of excess amount of free radical generation caused by nutrient stress. The amount of SOD and GSH continued to increase even after the nutrient was reintroduced and a strong correlation was seen between the MDA and enzyme activities, indicating the robust effort of rebalancing the redox system in Microcystis cells. Based on these transcriptional and physiological responses of M. aeruginosa to nutrient loading, these results could provide more insight into Microcystis blooms management and toxin formation regulation. PMID:28513574

Transcriptional and Physiological Responses to Nutrient Loading on Toxin Formation and Photosynthesis in Microcystis Aeruginosa FACHB-905.

PubMed

Peng, Guotao; Lin, Sijie; Fan, Zhengqiu; Wang, Xiangrong

2017-05-17

An important goal of understanding harmful algae blooms is to determine how environmental factors affect the growth and toxin formation of toxin-producing species. In this study, we investigated the transcriptional responses of toxin formation gene ( mcyB ) and key photosynthesis genes ( psaB , psbD and rbcL) of Microcystis aeruginosa FACHB-905 in different nutrient loading conditions using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Three physio-biochemical parameters (malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH)) were also evaluated to provide insight into the physiological responses of Microcystis cells. We observed an upregulation of mcyB gene in nutrient-deficient conditions, especially in nitrogen (N) limitation condition, and the transcript abundance declined after the nutrient were resupplied. Differently, high transcription levels were seen in phosphorus (P) deficient treatments for key photosynthesis genes throughout the culture period, while those in N-deficient cells varied with time, suggesting an adaptive regulation of Microsystis cells to nutrient stress. Increased contents of antioxidant enzymes (SOD and GSH) were seen in both N and P-deficient conditions, suggesting the presence of excess amount of free radical generation caused by nutrient stress. The amount of SOD and GSH continued to increase even after the nutrient was reintroduced and a strong correlation was seen between the MDA and enzyme activities, indicating the robust effort of rebalancing the redox system in Microcystis cells. Based on these transcriptional and physiological responses of M. aeruginosa to nutrient loading, these results could provide more insight into Microcystis blooms management and toxin formation regulation.

A role for tight junction-associated MARVEL proteins in larval sea lamprey (Petromyzon marinus) osmoregulation.

PubMed

Kolosov, Dennis; Bui, Phuong; Donini, Andrew; Wilkie, Mike P; Kelly, Scott P

2017-10-15

This study reports on tight junction-associated MARVEL proteins of larval sea lamprey ( Petromyzon marinus ) and their potential role in ammocoete osmoregulation. Two occludin isoforms (designated Ocln and Ocln-a) and a tricellulin (Tric) were identified. Transcripts encoding ocln , ocln-a and tric were broadly expressed in larval lamprey, with the greatest abundance of ocln in the gut, liver and kidney, ocln-a in the gill and skin, and tric in the kidney. Ocln and Ocln-a resolved as ∼63 kDa and ∼35 kDa MW proteins, respectively, while Tric resolved as a ∼50 kDa protein. Ocln immunolocalized to the gill vasculature and in gill mucous cells while Ocln-a localized to the gill pouch and gill epithelium. Both Ocln and Ocln-a localized in the nephron, the epidermis and the luminal side of the gut. In branchial tissue, Tric exhibited punctate localization, consistent with its presence at regions of tricellular contact. Following ion-poor water (IPW) acclimation of ammocoetes, serum [Na + ] and [Cl - ] decreased, but not [Ca 2+ ], and carcass moisture content increased. In association, Ocln abundance increased in the skin and kidney, but reduced in the gill of IPW-acclimated ammocoetes while Ocln-a abundance reduced in the kidney only. Tric abundance increased in the gill. Region-specific alterations in ocln , ocln-a and tric mRNA abundance were also observed in the gut. Data support a role for Ocln, Ocln-a and Tric in the osmoregulatory strategies of a basal vertebrate. © 2017. Published by The Company of Biologists Ltd.

Benzoate Mediates Repression of C4-Dicarboxylate Utilization in “Aromatoleum aromaticum” EbN1

PubMed Central

Trautwein, Kathleen; Grundmann, Olav; Wöhlbrand, Lars; Eberlein, Christian; Boll, Matthias

2012-01-01

Diauxic growth was observed in anaerobic C4-dicarboxylate-adapted cells of “Aromatoleum aromaticum” EbN1 due to preferred benzoate utilization from a substrate mixture of a C4-dicarboxylate (succinate, fumarate, or malate) and benzoate. Differential protein profiles (two-dimensional difference gel electrophoresis [2D DIGE]) revealed dynamic changes in abundance for proteins involved in anaerobic benzoate catabolism and C4-dicarboxylate uptake. In the first active growth phase, benzoate utilization was paralleled by maximal abundance of proteins involved in anaerobic benzoate degradation (e.g., benzoyl-coenzyme A [CoA] reductase) and minimal abundance of DctP (EbA4158), the periplasmic binding protein of a predicted C4-dicarboxylate tripartite ATP-independent periplasmic (TRAP) transporter (DctPQM). The opposite was observed during subsequent succinate utilization in the second active growth phase. The increased dctP (respectively, dctPQM) transcript and DctP protein abundance following benzoate depletion suggests that repression of C4-dicarboxylate uptake seems to be a main determinant for the observed diauxie. PMID:22081395

Quantitative proteomics unravels that the post-transcriptional regulator Crc modulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosa.

PubMed

Reales-Calderón, Jose Antonio; Corona, Fernando; Monteoliva, Lucía; Gil, Concha; Martínez, Jose Luis

2015-09-01

Crc is a post-transcriptional regulator in Pseudomonas aeruginosa that modulates its metabolism, but also its susceptibility to antibiotics and virulence. Most of P. aeruginosa virulence factors are secreted or engulfed in vesicles. A Crc deficient mutant was created and the extracellular vesicles associated exoproteome and the vesicle-free secretome was quantified using iTRAQ. Fifty vesicles-associated proteins were more abundant and 14 less abundant in the Crc-defective strain, whereas 37 were more abundant and 17 less abundant in the vesicle-free secretome. Different virulence determinants, such as ToxA, protease IV, azurin, chitin-binding protein, PlcB and Hcp1, were less abundant in the Crc-defective mutant. We also observed that the crc mutant presented an impaired vesicle-associated secretion of quorum sensing signal molecules and less cytotoxicity than its wild-type strain, in agreement with the low secretion of proteins related to virulence. Our results offer new insights into the mechanisms by which Crc regulates P. aeruginosa virulence, through the modulation of vesicle formation and secretion of both virulence determinants and quorum sensing signals.

Tissue-specific regulation of medium-chain acyl-CoA dehydrogenase gene by thyroid hormones in the developing rat.

PubMed

Djouadi, F; Riveau, B; Merlet-Benichou, C; Bastin, J

1997-05-15

During development, gene expression of medium-chain acyl-CoA dehydrogenase (MCAD), a nuclear-encoded mitochondrial enzyme that catalyses the first step of medium-chain fatty acid beta-oxidation, is highly regulated in tissues in accordance with fatty acid utilization, but the factors involved in this regulation are largely unknown. To investigate a possible role of thyroid hormones, rat pups were made hypothyroid by the administration of propylthiouracyl to the mother from day 12 of gestation, and their kidneys, heart and liver were removed on postnatal day 16 to determine MCAD mRNA abundance, protein level and enzyme activity. Similar experiments were run in 3,3',5-tri-iodothyronine (T3)-replaced hypothyroid (1 microg of T3/100 g body weight from postnatal day 5 to 15) and euthyroid pups. Hypothyroidism led to an increase in MCAD mRNA abundance in kidney and a decrease in abundance in heart, but had no effect in liver. The protein levels and enzyme activity were lowered in hypothyroid heart and kidney, suggesting that hypothyroidism affects post-transcriptional steps of gene expression in the kidney. All the effects of hypothyroidism were completely reversed in both heart and kidney by T3 replacement. Injection of a single T3 dose into 16-day-old euthyroid rats also led to tissue-specific changes in mRNA abundance. Nuclear run-on assays performed from hypothyroid and hypothyroid plus T3 rats showed that T3 stimulates MCAD gene transcription in heart and represses it in the kidney. These results indicate that the postnatal rise in circulating T3 is essential to the developmental regulation of the MCAD gene in vivo.

Light-Dependent Transcriptional Regulation of Genes of Biogeochemical Interest in the Diploid and Haploid Life Cycle Stages of Emiliania huxleyi▿ †

PubMed Central

Richier, Sophie; Kerros, Marie-Emmanuelle; de Vargas, Colomban; Haramaty, Liti; Falkowski, Paul G.; Gattuso, Jean-Pierre

2009-01-01

The expression of genes of biogeochemical interest in calcifying and noncalcifying life stages of the coccolithophore Emiliania huxleyi was investigated. Transcripts potentially involved in calcification were tested through a light-dark cycle. These transcripts were more abundant in calcifying cells and were upregulated in the light. Their application as potential candidates for in situ biogeochemical proxies is also suggested. PMID:19304825

Sperm Competitive Ability in Drosophila melanogaster Associated With Variation in Male Reproductive Proteins

PubMed Central

Fiumera, Anthony C.; Dumont, Bethany L.; Clark, Andrew G.

2005-01-01

Multiple mating by females establishes the opportunity for postcopulatory sexual selection favoring males whose sperm is preferentially employed in fertilizations. Here we use natural variation in a wild population of Drosophila melanogaster to investigate the genetic basis of sperm competitive ability. Approximately 101 chromosome 2 substitution lines were scored for components of sperm competitive ability (P1′, P2′, fecundity, remating rate, and refractoriness), genotyped at 70 polymorphic markers in 10 male reproductive genes, and measured for transcript abundance of those genes. Permutation tests were applied to quantify the statistical significance of associations between genotype and phenotype. Nine significant associations were identified between polymorphisms in the male reproductive genes and sperm competitive ability and 13 were identified between genotype and transcript abundance, but no significant associations were found between transcript abundance and sperm competitive ability. Pleiotropy was evident in two genes: a polymorphism in Acp33A associated with both P1′ and P2′ and a polymorphism in CG17331 associated with both elevated P2′ and reduced refractoriness. The latter case is consistent with antagonistic pleiotropy and may serve as a mechanism maintaining genetic variation. PMID:15466425

Detection and characterization of latent HSV RNA by in situ and northern blot hybridization in guinea pigs.

PubMed

Burke, R L; Hartog, K; Croen, K D; Ostrove, J M

1991-04-01

Following intravaginal infection of guinea pigs, herpes simplex virus establishes a latent infection in the sensory lumbosacral ganglia. Using the techniques of in situ and Northern blot hybridization, we have characterized this latent HSV-2 virus and compared it to latent HSV-1 at the same anatomical site. For HSV-2, a single 1.8-kb latency-associated transcript (LAT) was detected. In contrast, as described for latent HSV-1 in the trigeminal ganglia of rabbits and mice, two HSV-1 LAT species were detected in the lumbosacral ganglia, an abundant transcript of 1.8 kb and a less abundant transcript of 1.55 kb. Despite these differences in LAT expression, the clinical course of the acute and recurrent genital disease was similar for both viruses. LAT was detected in 0.3-6.0% of the sensory neurons of sacral but not in lumbar ganglia. The abundance of LAT correlated with the severity of the initial infection, but not with the frequency of recurrent disease. Thus, vaccination strategies that substantially reduced or eliminated symptomatic disease following challenge infection appeared to block the establishment of a latent infection.

Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

PubMed Central

Petrova, Olga E.; Garcia-Alcalde, Fernando; Zampaloni, Claudia; Sauer, Karin

2017-01-01

Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested. PMID:28117413

Micronuclei and other erythrocyte nuclear abnormalities in fishes from the Great Lakes Basin, USA

PubMed Central

Braham, Ryan P.; Shaw, Cassidy H.; Mazik, Patricia M.; Umbuzeiro, G.

2017-01-01

Biological markers (biomarkers) sensitive to genotoxic and mutagenic contamination in fishes are widely used to identify exposure effects in aquatic environments. The micronucleus assay was incorporated into a suite of indicators to assess exposure to genotoxic and mutagenic contamination at five Great Lakes Areas of Concern (AOCs), as well as one non‐AOC (reference) site. The assay allowed enumeration of micronuclei as well as other nuclear abnormalities for both site and species comparisons. Erythrocyte abnormality data was also compared to skin and liver tumor prevalence and hepatic transcript abundance. Erythrocyte abnormalities were observed at all sites with variable occurrence and severity among sites and species. Benthic‐oriented brown bullhead (Ameiurus nebulosus) and white sucker (Catostomus commersonii) expressed lower rates of erythrocyte abnormalities, but higher rates of skin and liver neoplasms, when compared to pelagic‐oriented largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) at the same site. The reduced erythrocyte abnormalities, increased transcript abundance associated with Phase I and II toxicant responsive pathways, and increased neoplastic lesions among benthic‐oriented taxa may indicate the development of contaminant resistance of these species to more acute effects. Environ. Mol. Mutagen. 58:570–581, 2017. © 2017 This article is a U.S. Government work and is in the public domain in the USA. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society PMID:28868735

In situ expression of eukaryotic ice-binding proteins in microbial communities of Arctic and Antarctic sea ice.

PubMed

Uhlig, Christiane; Kilpert, Fabian; Frickenhaus, Stephan; Kegel, Jessica U; Krell, Andreas; Mock, Thomas; Valentin, Klaus; Beszteri, Bánk

2015-11-01

Ice-binding proteins (IBPs) have been isolated from various sea-ice organisms. Their characterisation points to a crucial role in protecting the organisms in sub-zero environments. However, their in situ abundance and diversity in natural sea-ice microbial communities is largely unknown. In this study, we analysed the expression and phylogenetic diversity of eukaryotic IBP transcripts from microbial communities of Arctic and Antarctic sea ice. IBP transcripts were found in abundances similar to those of proteins involved in core cellular processes such as photosynthesis. Eighty-nine percent of the IBP transcripts grouped with known IBP sequences from diatoms, haptophytes and crustaceans, but the majority represented novel sequences not previously characterized in cultured organisms. The observed high eukaryotic IBP expression in natural eukaryotic sea ice communities underlines the essential role of IBPs for survival of many microorganisms in communities living under the extreme conditions of polar sea ice.

Metagenomic recovery of phage genomes of uncultured freshwater actinobacteria.

PubMed

Ghai, Rohit; Mehrshad, Maliheh; Mizuno, Carolina Megumi; Rodriguez-Valera, Francisco

2017-01-01

Low-GC Actinobacteria are among the most abundant and widespread microbes in freshwaters and have largely resisted all cultivation efforts. Consequently, their phages have remained totally unknown. In this work, we have used deep metagenomic sequencing to assemble eight complete genomes of the first tailed phages that infect freshwater Actinobacteria. Their genomes encode the actinobacterial-specific transcription factor whiB, frequently found in mycobacteriophages and also in phages infecting marine pelagic Actinobacteria. Its presence suggests a common and widespread strategy of modulation of host transcriptional machinery upon infection via this transcriptional switch. We present evidence that some whiB-carrying phages infect the acI lineage of Actinobacteria. At least one of them encodes the ADP-ribosylating component of the widespread bacterial AB toxins family (for example, clostridial toxin). We posit that the presence of this toxin reflects a 'trojan horse' strategy, providing protection at the population level to the abundant host microbes against eukaryotic predators.

Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™.

PubMed

Luck, Ashley N; Slatko, Barton E; Foster, Jeremy M

2017-01-01

Efficient transcriptomic sequencing of microbial mRNA derived from host-microbe associations is often compromised by the much lower relative abundance of microbial RNA in the mixed total RNA sample. One solution to this problem is to perform extensive sequencing until an acceptable level of transcriptome coverage is obtained. More cost-effective methods include use of prokaryotic and/or eukaryotic rRNA depletion strategies, sometimes in conjunction with depletion of polyadenylated eukaryotic mRNA. Here, we report use of Cappable-seq™ to specifically enrich, in a single step, Wolbachia endobacterial mRNA transcripts from total RNA prepared from the parasitic filarial nematode, Brugia malayi. The obligate Wolbachia endosymbiont is a proven drug target for many human filarial infections, yet the precise nature of its symbiosis with the nematode host is poorly understood. Insightful analysis of the expression levels of Wolbachia genes predicted to underpin the mutualistic association and of known drug target genes at different life cycle stages or in response to drug treatments is typically challenged by low transcriptomic coverage. Cappable-seq resulted in up to ~ 5-fold increase in the number of reads mapping to Wolbachia. On average, coverage of Wolbachia transcripts from B. malayi microfilariae was enriched ~40-fold by Cappable-seq. Additionally, this method has an additional benefit of selectively removing abundant prokaryotic ribosomal RNAs.The deeper microbial transcriptome sequencing afforded by Cappable-seq facilitates more detailed characterization of gene expression levels of pathogens and symbionts present in animal tissues.

FGF-23 Regulates CYP27B1 Transcription in the Kidney and in Extra-Renal Tissues

PubMed Central

Chanakul, Ankanee; Zhang, Martin Y. H.; Louw, Andrew; Armbrecht, Harvey J.; Miller, Walter L.; Portale, Anthony A.; Perwad, Farzana

2013-01-01

The mitochondrial enzyme 25-hydroxyvitamin D 1α-hydroxylase, which is encoded by the CYP27B1 gene, converts 25OHD to the biological active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D). Renal 1α-hydroxylase activity is the principal determinant of the circulating 1,25(OH)2D concentration and enzyme activity is tightly regulated by several factors. Fibroblast growth factor-23 (FGF-23) decreases serum 1,25(OH)2D concentrations by suppressing CYP27B1 mRNA abundance in mice. In extra-renal tissues, 1α-hydroxylase is responsible for local 1,25(OH)2D synthesis, which has important paracrine actions, but whether FGF-23 regulates CYP27B1 gene expression in extra-renal tissues is unknown. We sought to determine whether FGF-23 regulates CYP27B1 transcription in the kidney and whether extra-renal tissues are target sites for FGF-23-induced suppression of CYP27B1. In HEK293 cells transfected with the human CYP27B1 promoter, FGF-23 suppressed promoter activity by 70%, and the suppressive effect was blocked by CI-1040, a specific inhibitor of extracellular signal regulated kinase 1/2. To examine CYP27B1 transcriptional activity in vivo, we crossed fgf-23 null mice with mice bearing the CYP27B1 promoter-driven luciferase transgene (1α-Luc). In the kidney of FGF-23 null/1α-Luc mice, CYP27B1 promoter activity was increased by 3-fold compared to that in wild-type/1α-Luc mice. Intraperitoneal injection of FGF-23 suppressed renal CYP27B1 promoter activity and protein expression by 26% and 60% respectively, and the suppressive effect was blocked by PD0325901, an ERK1/2 inhibitor. These findings provide evidence that FGF-23 suppresses CYP27B1 transcription in the kidney. Furthermore, we demonstrate that in FGF-23 null/1α-Luc mice, CYP27B1 promoter activity and mRNA abundance are increased in several extra-renal sites. In the heart of FGF-23 null/1α-Luc mice, CYP27B1 promoter activity and mRNA were 2- and 5-fold higher, respectively, than in control mice. We also observed a 3- to 10-fold increase in CYP27B1 mRNA abundance in the lung, spleen, aorta and testis of FGF-23 null/1α-Luc mice. Thus, we have identified novel extra-renal target sites for FGF-23-mediated regulation of CYP27B1. PMID:24019880

Changes in transcript abundance relating to colony collapse disorder in honey bees (Apis mellifera).

PubMed

Johnson, Reed M; Evans, Jay D; Robinson, Gene E; Berenbaum, May R

2009-09-01

Colony collapse disorder (CCD) is a mysterious disappearance of honey bees that has beset beekeepers in the United States since late 2006. Pathogens and other environmental stresses, including pesticides, have been linked to CCD, but a causal relationship has not yet been demonstrated. Because the gut acts as a primary interface between the honey bee and its environment as a site of entry for pathogens and toxins, we used whole-genome microarrays to compare gene expression between guts of bees from CCD colonies originating on both the east and west coasts of the United States and guts of bees from healthy colonies sampled before the emergence of CCD. Considerable variation in gene expression was associated with the geographical origin of bees, but a consensus list of 65 transcripts was identified as potential markers for CCD status. Overall, elevated expression of pesticide response genes was not observed. Genes involved in immune response showed no clear trend in expression pattern despite the increased prevalence of viruses and other pathogens in CCD colonies. Microarray analysis revealed unusual ribosomal RNA fragments that were conspicuously more abundant in the guts of CCD bees. The presence of these fragments may be a possible consequence of picorna-like viral infection, including deformed wing virus and Israeli acute paralysis virus, and may be related to arrested translation. Ribosomal fragment abundance and presence of multiple viruses may prove to be useful diagnostic markers for colonies afflicted with CCD.

Changes in transcript abundance relating to colony collapse disorder in honey bees (Apis mellifera)

PubMed Central

Johnson, Reed M.; Evans, Jay D.; Robinson, Gene E.; Berenbaum, May R.

2009-01-01

Colony collapse disorder (CCD) is a mysterious disappearance of honey bees that has beset beekeepers in the United States since late 2006. Pathogens and other environmental stresses, including pesticides, have been linked to CCD, but a causal relationship has not yet been demonstrated. Because the gut acts as a primary interface between the honey bee and its environment as a site of entry for pathogens and toxins, we used whole-genome microarrays to compare gene expression between guts of bees from CCD colonies originating on both the east and west coasts of the United States and guts of bees from healthy colonies sampled before the emergence of CCD. Considerable variation in gene expression was associated with the geographical origin of bees, but a consensus list of 65 transcripts was identified as potential markers for CCD status. Overall, elevated expression of pesticide response genes was not observed. Genes involved in immune response showed no clear trend in expression pattern despite the increased prevalence of viruses and other pathogens in CCD colonies. Microarray analysis revealed unusual ribosomal RNA fragments that were conspicuously more abundant in the guts of CCD bees. The presence of these fragments may be a possible consequence of picorna-like viral infection, including deformed wing virus and Israeli acute paralysis virus, and may be related to arrested translation. Ribosomal fragment abundance and presence of multiple viruses may prove to be useful diagnostic markers for colonies afflicted with CCD. PMID:19706391

The Role of the EGF Receptor First Intron in Its Regulation in Breast Canceer.

DTIC Science & Technology

1997-08-01

of EGFR, to malignantly transform mammary glands in transgenic mice (46). Future work could determine if these putative oncogene factor binding sites...general transcription factors, and the use or overuse of one promoter could monopolize the abundance of these transcription factors within a cell. The ideal

ptr-MIR169 is a posttranscriptional repressor of PtrHAP2 during vegetative bud dormancy period of aspen (Populus tremuloides) trees

DOE Office of Scientific and Technical Information (OSTI.GOV)

Potkar, Rewati; Recla, Jill; Busov, Victor, E-mail: vbusov@mtu.edu

2013-02-15

Highlights: ► We show a novel microRNA-mediated mechanism for control of bud dormancy in trees. ► ptr-MIR169a and PtrHAP2–5 gene showed inverse expression during dormancy period. ► The PtrHAP2–5 decline in abundance correlated with high ptr-MIR169a levels. ► PtrHAP2–5 cleavage occurred at the miR169 site during PtrHAP2–5 transcript decline. ► Our results show that miR169 attenuates PtrHAP2–5 transcript during dormancy. -- Abstract: Dormancy is a mechanism evolved in woody perennial plants to survive the winter freezing and dehydration stress via temporary suspension of growth. We have identified two aspen microRNAs (ptr-MIR169a and ptr-MIR169h) which were highly and specifically expressed inmore » dormant floral and vegetative buds. ptr-MIR169a and its target gene PtrHAP2–5 showed inverse expression patterns during the dormancy period. ptr-MIR169a transcript steadily increased through the first half of the dormancy period and gradually declined with the approach of active growing season. PtrHAP2–5 abundance was higher in the beginning of the dormancy period but rapidly declined thereafter. The decline of PtrHAP2–5 correlated with the high levels of ptr-MIR169a accumulation, suggesting miR169-mediated attenuation of the target PtrHAP2–5 transcript. We experimentally verified the cleavage of PtrHAP2–5 at the predicted miR169a site at the time when PtrHAP2–5 transcript decline was observed. HAP2 is a subunit of a nuclear transcription factor Y (NF-Y) complex consisting of two other units, HAP3 and HAP5. Using digital expression profiling we show that poplar HAP2 and HAP5 are preferentially detected in dormant tissues. Our study shows that microRNAs play a significant and as of yet unknown and unstudied role in regulating the timing of bud dormancy in trees.« less

Characterisation of the transcriptome of male and female wild-type guppy brains with RNA-Seq and consequences of exposure to the pharmaceutical pollutant, 17α-ethinyl estradiol.

PubMed

Saaristo, Minna; Wong, Bob B M; Mincarelli, Laura; Craig, Allison; Johnstone, Christopher P; Allinson, Mayumi; Lindström, Kai; Craft, John A

2017-05-01

Waterways are increasingly being contaminated by chemical compounds that can disrupt the endocrinology of organisms. One such compound is 17α-ethinyl estradiol (EE2), a synthetic estrogen used in the contraceptive pill. Despite considerable research interest in the effects of EE2 on reproduction and gene expression, surprisingly, only a few studies have capitalised on technologies, such as next-generation sequencing (NGS), to uncover the molecular pathways related to EE2 exposure. Accordingly, using high-throughput sequencing technologies, the aim of our study was to explore the effects of EE2 on brain transcriptome in wild-type male and female guppy (Poecilia reticulata). We conducted two sets of experiments, where fish were exposed to EE2 (measured concentrations: 8ng/L and 38ng/L) in a flow-through system for 21days. The effects on the brain transcriptome on both males and females were assessed using Illumina sequencing (MiSeq and HiSeq) platform followed by bioinformatics analysis (edgeR, DESeq2). Here, we report that exposure to EE2 caused both up- and downregulation of specific transcript abundances, and affected transcript abundance in a sex-specific manner. Specifically, we found 773 transcripts, of which 60 were male-specific, 61 female-specific and 285 treatment-specific. EE2 affected expression of 165 transcripts in males, with 88 downregulated and 77 upregulated, while in females, 120 transcripts were affected with 62 downregulated and 58 upregulated. Finally, RT-qPCR validation demonstrated that expression of transcripts related to transposable elements, neuroserpin and heat shock protein were significantly affected by EE2-exposure. Our study is the first to report brain transcriptome libraries for guppies exposed to EE2. Not only does our study provide a valuable resource, it offers insights into the mechanisms underlying the feminizing effects on the brains of organisms exposed to environmentally realistic concentrations of EE2. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic Regulation of Hypothalamic Cocaine and Amphetamine-Regulated Transcript (CART) in BxD Inbred Mice

PubMed Central

Hawks, Brian W.; Li, Wei; Garlow, Steven J.

2009-01-01

Cocaine-Amphetamine Regulated Transcript (CART) peptides are implicated in a wide range of behaviors including in the reinforcing properties of psychostimulants, feeding and energy balance and stress and anxiety responses. We conducted a complex trait analysis to examine natural variation in the regulation of CART transcript abundance (CARTta) in the hypothalamus. CART transcript abundance was measured in total hypothalamic RNA from 26 BxD recombinant inbred (RI) mouse strains and in the C57BL/6 (B6) and DBA/2J (D2) progenitor strains. The strain distribution pattern for CARTta was continuous across the RI panel, which is consistent with this being a quantitative trait. Marker regression and interval mapping revealed significant quantitative trait loci (QTL) on mouse chromosome 4 (around 58.2cM) and chromosome 11 (between 20–36cM) that influence CARTta and account for 31% of the between strain variance in this phenotype. There are numerous candidate genes and QTL in these chromosomal regions that may indicate shared genetic regulation between CART expression and other neurobiological processes referable to known actions of this neuropeptide. PMID:18199428

Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl

PubMed Central

Paynel, Florence; Schaumann, Annick; Arkoun, Mustapha; Douchiche, Olfa; Morvan, Claudine

2009-01-01

Background and Aims In hypocotyls of flax (Linum usitatissimum) cadmium-induced reorientation of growth (i.e. an increase in expansion and a decrease in elongation) coincides with marked changes in the methylesterification and cross-linking of homogalacturonans within various cell-wall (CW) domains. The aim of the present study was to examine the involvement of pectin methylesterase (PME) and peroxidase (PER) in this cadmium-induced CW remodelling. Methods CW proteins were extracted from hypocotyls of 10- and 18-d-old flax that had been treated or not treated with 0·5 mm Cd(NO3)2. PME and PER expression within these extracts was detected by LC/MS, by isoelectric focusing and enzyme activity assays. Transcript expression by RT-PCR of known flax PME and PER genes was also measured in corresponding samples. Key Results In cadmium-treated seedlings, PME activity increased as compared with controls, particularly at day 10. The increased activity of PME was accompanied by increased abundance of both a basic protein isoform (B2) and a particular transcript (Lupme5). In contrast, induction of PER activity by cadmium was highest at day 18. Among the four reported PER genes, Flxper1 and 3 increased in abundance in the presence of cadmium at day 18. Conclusions The temporal regulation of Lupme and Flxper genes and of their respective enzyme activities fits the previously reported cadmium-induced structural changes of homogalacturonans within the CWs. After PME-catalysed de-esterification of homogalacturonans, their cross-linking would depend on the activity of PERs interacting with calcium-dimerized blocks and reinforce the cell cohesion during the cadmium-induced swelling. PMID:19815572

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

PubMed

Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela

2017-09-06

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.

Mitochondrial DNA Depletion in Respiratory Chain-Deficient Parkinson Disease Neurons.

PubMed

Grünewald, Anne; Rygiel, Karolina A; Hepplewhite, Philippa D; Morris, Christopher M; Picard, Martin; Turnbull, Doug M

2016-03-01

To determine the extent of respiratory chain abnormalities and investigate the contribution of mtDNA to the loss of respiratory chain complexes (CI-IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single-neuron level. Multiple-label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI-IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2, and COXI) and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser-capture microdissection, were assayed for mtDNA deletions, copy number, and presence of transcription/replication-associated 7S DNA employing a triplex real-time polymerase chain reaction (PCR) assay. Whereas mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in the abundances of CI and II subunits. At the single-cell level, CI and II deficiencies were correlated in patients. The CI deficiency concomitantly occurred with low abundances of the mtDNA transcription factors TFAM and TFB2M, which also initiate transcription-primed mtDNA replication. Consistent with this, real-time PCR analysis revealed fewer transcription/replication-associated mtDNA molecules and an overall reduction in mtDNA copy number in patients. This effect was more pronounced in single IPD neurons with severe CI deficiency. Respiratory chain dysfunction in IPD neurons not only involves CI, but also extends to CII. These deficiencies are possibly a consequence of the interplay between nDNA and mtDNA-encoded factors mechanistically connected via TFAM. © 2016 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

Quantitative high-throughput profiling of snake venom gland transcriptomes and proteomes (Ovophis okinavensis and Protobothrops flavoviridis)

PubMed Central

2013-01-01

Background Advances in DNA sequencing and proteomics have facilitated quantitative comparisons of snake venom composition. Most studies have employed one approach or the other. Here, both Illumina cDNA sequencing and LC/MS were used to compare the transcriptomes and proteomes of two pit vipers, Protobothrops flavoviridis and Ovophis okinavensis, which differ greatly in their biology. Results Sequencing of venom gland cDNA produced 104,830 transcripts. The Protobothrops transcriptome contained transcripts for 103 venom-related proteins, while the Ovophis transcriptome contained 95. In both, transcript abundances spanned six orders of magnitude. Mass spectrometry identified peptides from 100% of transcripts that occurred at higher than contaminant (e.g. human keratin) levels, including a number of proteins never before sequenced from snakes. These transcriptomes reveal fundamentally different envenomation strategies. Adult Protobothrops venom promotes hemorrhage, hypotension, incoagulable blood, and prey digestion, consistent with mammalian predation. Ovophis venom composition is less readily interpreted, owing to insufficient pharmacological data for venom serine and metalloproteases, which comprise more than 97.3% of Ovophis transcripts, but only 38.0% of Protobothrops transcripts. Ovophis venom apparently represents a hybrid strategy optimized for frogs and small mammals. Conclusions This study illustrates the power of cDNA sequencing combined with MS profiling. The former quantifies transcript composition, allowing detection of novel proteins, but cannot indicate which proteins are actually secreted, as does MS. We show, for the first time, that transcript and peptide abundances are correlated. This means that MS can be used for quantitative, non-invasive venom profiling, which will be beneficial for studies of endangered species. PMID:24224955

Quantitative response of nitrifying and denitrifying communities to environmental variables in a full-scale membrane bioreactor.

PubMed

Gómez-Silván, C; Vílchez-Vargas, R; Arévalo, J; Gómez, M A; González-López, J; Pieper, D H; Rodelas, B

2014-10-01

The abundance and transcription levels of specific gene markers of total bacteria, ammonia-oxidizing Betaproteobacteria, nitrite-oxidizing bacteria (Nitrospira-like) and denitrifiers (N2O-reducers) were analyzed using quantitative PCR (qPCR) and reverse-transcription qPCR during 9 months in a full-scale membrane bioreactor treating urban wastewater. A stable community of N-removal key players was developed; however, the abundance of active populations experienced sharper shifts, demonstrating their fast adaptation to changing conditions. Despite constituting a small percentage of the total bacterial community, the larger abundances of active populations of nitrifiers explained the high N-removal accomplished by the MBR. Multivariate analyses revealed that temperature, accumulation of volatile suspended solids in the sludge, BOD5, NH4(+) concentration and C/N ratio of the wastewater contributed significantly (23-38%) to explain changes in the abundance of nitrifiers and denitrifiers. However, each targeted group showed different responses to shifts in these parameters, evidencing the complexity of the balance among them for successful biological N-removal. Copyright © 2014 Elsevier Ltd. All rights reserved.

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR.

PubMed

Hanning, Jennifer E; Groves, Ian J; Pett, Mark R; Coleman, Nicholas

2013-05-21

Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR

PubMed Central

2013-01-01

Background Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. Findings We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. Conclusions These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects. PMID:23693071

A Guideline to Family-Wide Comparative State-of-the-Art Quantitative RT-PCR Analysis Exemplified with a Brassicaceae Cross-Species Seed Germination Case Study[W][OA

PubMed Central

Graeber, Kai; Linkies, Ada; Wood, Andrew T.A.; Leubner-Metzger, Gerhard

2011-01-01

Comparative biology includes the comparison of transcriptome and quantitative real-time RT-PCR (qRT-PCR) data sets in a range of species to detect evolutionarily conserved and divergent processes. Transcript abundance analysis of target genes by qRT-PCR requires a highly accurate and robust workflow. This includes reference genes with high expression stability (i.e., low intersample transcript abundance variation) for correct target gene normalization. Cross-species qRT-PCR for proper comparative transcript quantification requires reference genes suitable for different species. We addressed this issue using tissue-specific transcriptome data sets of germinating Lepidium sativum seeds to identify new candidate reference genes. We investigated their expression stability in germinating seeds of L. sativum and Arabidopsis thaliana by qRT-PCR, combined with in silico analysis of Arabidopsis and Brassica napus microarray data sets. This revealed that reference gene expression stability is higher for a given developmental process between distinct species than for distinct developmental processes within a given single species. The identified superior cross-species reference genes may be used for family-wide comparative qRT-PCR analysis of Brassicaceae seed germination. Furthermore, using germinating seeds, we exemplify optimization of the qRT-PCR workflow for challenging tissues regarding RNA quality, transcript stability, and tissue abundance. Our work therefore can serve as a guideline for moving beyond Arabidopsis by establishing high-quality cross-species qRT-PCR. PMID:21666000

Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus

PubMed Central

Obermeier, Christian; Hosseini, Bashir; Friedt, Wolfgang; Snowdon, Rod

2009-01-01

Background Serial analysis of gene expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana. Results Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development. Conclusion This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species. PMID:19575793

Identification of the thiamin pyrophosphokinase gene in rainbow trout: Characteristic structure and expression of seven splice variants in tissues and cell lines and during embryo development

USGS Publications Warehouse

Yuge, Shinya; Richter, Catherine A.; Wright-Osment, Maureen K.; Nicks, Diane; Saloka, Stephanie K.; Tillitt, Donald E.; Li, Weiming

2012-01-01

Thiamin pyrophosphokinase (TPK) converts thiamin to its active form, thiamin diphosphate. In humans, TPK expression is down-regulated in some thiamin deficiency related syndrome, and enhanced during pregnancy. Rainbow trout are also vulnerable to thiamin deficiency in wild life and are useful models for thiamin metabolism research. We identified the tpk gene transcript including seven splice variants in the rainbow trout. Almost all cell lines and tissues examined showed co-expression of several tpk splice variants including a potentially major one at both mRNA and protein levels. However, relative to other tissues, the longest variant mRNA expression was predominant in the ovary and abundant in embryos. During embryogenesis, total tpk transcripts increased abruptly in early development, and decreased to about half of the peak shortly after hatching. In rainbow trout, the tpk transcript complex is ubiquitously expressed for all tissues and cells examined, and its increase in expression could be important in the early-middle embryonic stages. Moreover, decimated tpk expression in a hepatoma cell line relative to hepatic and gonadal cell lines appears to be consistent with previously reported down-regulation of thiamin metabolism in cancer.

Carbohydrate metabolism changes in Prunus persica gummosis infected with Lasiodiplodia theobromae.

PubMed

Li, Z; Gao, L; Wang, Y T; Zhu, W; Ye, J L; Li, G H

2014-05-01

Peach gummosis represents a significant global disease of stone fruit trees and a major disease in the south peach production area of the Yangtze River of China. In this study, the carbohydrate composition of peach shoots during infection by Lasiodiplodia theobromae was examined. The expression of genes related to metabolic enzymes was also investigated. Control wounded and noninoculated tissue, lesion tissue, and wounded and inoculated surrounding lesion tissue of peach shoots were analyzed. Soluble sugars, glucose, mannose, arabinose, and xylose significantly increased in inoculated tissues of peach shoots compared with control tissues at different times after inoculation. Accumulation of polysaccharides was also observed by section observation and periodic acid Schiff's reagent staining during infection. Analysis using quantitative reverse-transcription polymerase chain reaction revealed that the abundance of key transcripts on the synthesis pathway of uridine diphosphate (UDP)-D-glucuronate, UDP-D-galactose, and UDP-D-arabinose increased but the synthesis of L-galactose and guanosine diphosphate-L-galactose were inhibited. After inoculation, the transcript levels of sugar transport-related genes (namely, SUT, SOT, GMT, and UGT) was induced. These changes in sugar content and gene expression were directly associated with peach gum polysaccharide formation and may be responsible for the symptoms of peach gummosis.

Identification of new members of the Escherichia coli K-12 MG1655 SlyA regulon

PubMed Central

Curran, Thomas D; Abacha, Fatima; Hibberd, Stephen P; Green, Jeffrey

2017-01-01

SlyA is a member of the MarR family of bacterial transcriptional regulators. Previously, SlyA has been shown to directly regulate only two operons in Escherichia coli K-12 MG1655, fimB and hlyE (clyA). In both cases, SlyA activates gene expression by antagonizing repression by the nucleoid-associated protein H-NS. Here, the transcript profiles of aerobic glucose-limited steady-state chemostat cultures of E. coli K-12 MG1655, slyA mutant and slyA over-expression strains are reported. The transcript profile of the slyA mutant was not significantly different from that of the parent; however, that of the slyA expression strain was significantly different from that of the vector control. Transcripts representing 27 operons were increased in abundance, whereas 3 were decreased. Of the 30 differentially regulated operons, 24 have previously been associated with sites of H-NS binding, suggesting that antagonism of H-NS repression is a common feature of SlyA-mediated transcription regulation. Direct binding of SlyA to DNA located upstream of a selection of these targets permitted the identification of new operons likely to be directly regulated by SlyA. Transcripts of four operons coding for cryptic adhesins exhibited enhanced expression, and this was consistent with enhanced biofilm formation associated with the SlyA over-producing strain. PMID:28073397

Identification of new members of the Escherichia coli K-12 MG1655 SlyA regulon.

PubMed

Curran, Thomas D; Abacha, Fatima; Hibberd, Stephen P; Rolfe, Matthew D; Lacey, Melissa M; Green, Jeffrey

2017-03-01

SlyA is a member of the MarR family of bacterial transcriptional regulators. Previously, SlyA has been shown to directly regulate only two operons in Escherichia coli K-12 MG1655, fimB and hlyE (clyA). In both cases, SlyA activates gene expression by antagonizing repression by the nucleoid-associated protein H-NS. Here, the transcript profiles of aerobic glucose-limited steady-state chemostat cultures of E. coli K-12 MG1655, slyA mutant and slyA over-expression strains are reported. The transcript profile of the slyA mutant was not significantly different from that of the parent; however, that of the slyA expression strain was significantly different from that of the vector control. Transcripts representing 27 operons were increased in abundance, whereas 3 were decreased. Of the 30 differentially regulated operons, 24 have previously been associated with sites of H-NS binding, suggesting that antagonism of H-NS repression is a common feature of SlyA-mediated transcription regulation. Direct binding of SlyA to DNA located upstream of a selection of these targets permitted the identification of new operons likely to be directly regulated by SlyA. Transcripts of four operons coding for cryptic adhesins exhibited enhanced expression, and this was consistent with enhanced biofilm formation associated with the SlyA over-producing strain.

Gene expression in honey bee (Apis mellifera) larvae exposed to pesticides and Varroa mites (Varroa destructor).

PubMed

Gregorc, Aleš; Evans, Jay D; Scharf, Mike; Ellis, James D

2012-08-01

Honey bee (Apis mellifera) larvae reared in vitro were exposed to one of nine pesticides and/or were challenged with the parasitic mite, Varroa destructor. Total RNA was extracted from individual larvae and first strand cDNAs were generated. Gene-expression changes in larvae were measured using quantitative PCR (qPCR) targeting transcripts for pathogens and genes involved in physiological processes, bee health, immunity, and/or xenobiotic detoxification. Transcript levels for Peptidoglycan Recognition Protein (PGRPSC), a pathogen recognition gene, increased in larvae exposed to Varroa mites (P<0.001) and were not changed in pesticide treated larvae. As expected, Varroa-parasitized brood had higher transcripts of Deformed Wing Virus than did control larvae (P<0.001). Varroa parasitism, arguably coupled with virus infection, resulted in significantly higher transcript abundances for the antimicrobial peptides abaecin, hymenoptaecin, and defensin1. Transcript levels for Prophenoloxidase-activating enzyme (PPOact), an immune end product, were elevated in larvae treated with myclobutanil and chlorothalonil (both are fungicides) (P<0.001). Transcript levels for Hexameric storage protein (Hsp70) were significantly upregulated in imidacloprid, fluvalinate, coumaphos, myclobutanil, and amitraz treated larvae. Definitive impacts of pesticides and Varroa parasitism on honey bee larval gene expression were demonstrated. Interactions between larval treatments and gene expression for the targeted genes are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Insulin-like growth factor-II regulates bone sialoprotein gene transcription.

PubMed

Choe, Jin; Sasaki, Yoko; Zhou, Liming; Takai, Hideki; Nakayama, Yohei; Ogata, Yorimasa

2016-09-01

Insulin-like growth factor-I and -II (IGF-I and IGF-II) have been found in bone extracts of several different species, and IGF-II is the most abundant growth factor stored in bone. Bone sialoprotein (BSP) is a noncollagenous extracellular matrix glycoprotein associated with mineralized connective tissues. In this study, we have investigated the regulation of BSP transcription by IGF-II in rat osteoblast-like ROS17/2.8 cells. IGF-II (50 ng/ml) increased BSP mRNA and protein levels after 6-h stimulation, and enhanced luciferase activities of the constructs pLUC3 (-116 to +60), pLUC4 (-425 to +60), pLUC5 (-801 to +60) and pLUC6 (-938 to +60). Effects of IGF-II were inhibited by tyrosine kinase, extracellular signal-regulated kinase1/2 and phosphatidylinositol 3-kinase inhibitors, and abrogated by 2-bp mutations in cAMP response element (CRE), FGF2 response element (FRE) and homeodomain protein-binding site (HOX). The results of gel shift assays showed that nuclear proteins binding to CRE, FRE and HOX sites were increased by IGF-II (50 ng/ml) at 3 and 6 h. CREB1, phospho-CREB1, c-Fos and c-Jun antibodies disrupted the formation of the CRE-protein complexes. Dlx5 and Runx2 antibodies disrupted the FRE- and HOX-protein complex formations. These studies therefore demonstrated that IGF-II increased BSP transcription by targeting CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB1, c-Fos, c-Jun, Dlx5 and Runx2 transcription factors appear to be key regulators of IGF-II effects on BSP transcription.

Cell division and turgor mediate enhanced plant growth in Arabidopsis plants treated with the bacterial signalling molecule lumichrome.

PubMed

Pholo, Motlalepula; Coetzee, Beatrix; Maree, Hans J; Young, Philip R; Lloyd, James R; Kossmann, Jens; Hills, Paul N

2018-05-17

Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis. Lumichrome (7,8 dimethylalloxazine), a novel multitrophic signal molecule produced by Sinorhizobium meliloti bacteria, has previously been shown to elicit growth promotion in different plant species (Phillips et al. in Proc Natl Acad Sci USA 96:12275-12280, https://doi.org/10.1073/pnas.96.22.12275 , 1999). However, the molecular mechanisms that underlie this plant growth promotion remain obscure. Global transcript profiling using RNA-seq suggests that lumichrome enhances growth by inducing genes impacting on turgor driven growth and mitotic cell cycle that ensures the integration of cell division and expansion of developing leaves. The abundance of XTH9 and XPA4 transcripts was attributed to improved mediation of cell-wall loosening to allow turgor-driven cell enlargement. Mitotic CYCD3.3, CYCA1.1, SP1L3, RSW7 and PDF1 transcripts were increased in lumichrome-treated Arabidopsis thaliana plants, suggesting enhanced growth was underpinned by increased cell differentiation and expansion with a consequential increase in biomass. Synergistic ethylene-auxin cross-talk was also observed through reciprocal over-expression of ACO1 and SAUR54, in which ethylene activates the auxin signalling pathway and regulates Arabidopsis growth by both stimulating auxin biosynthesis and modulating the auxin transport machinery to the leaves. Decreased transcription of jasmonate biosynthesis and responsive-related transcripts (LOX2; LOX3; LOX6; JAL34; JR1) might contribute towards suppression of the negative effects of methyl jasmonate (MeJa) such as chlorophyll loss and decreases in RuBisCO and photosynthesis. This work contributes towards a deeper understanding of how lumichrome enhances plant growth and development.

Elevated hepatic fatty acid elongase-5 activity corrects dietary fat-induced hyperglycemia in obese BL/6J mice[S

PubMed Central

Tripathy, Sasmita; Torres-Gonzalez, Moises; Jump, Donald B.

2010-01-01

Elevated hepatic fatty acid elongase-5 (Elovl5) activity lowers blood glucose in fasted chow-fed C57BL/6J mice. As high-fat diets induce hyperglycemia and suppress hepatic Elovl5 activity, we tested the hypothesis that elevated hepatic Elovl5 expression attenuates hyperglycemia in high-fat-diet-induced obese mice. Increasing hepatic Elovl5 activity by a recombinant adenoviral approach restored blood glucose and insulin, HOMA-IR, and glucose tolerance to normal values in obese mice. Elevated Elovl5 activity increased hepatic content of Elovl5 products (20:3,n-6, 22:4,n-6) and suppressed levels of enzymes (Pck1, G6Pc) and transcription factors (FoxO1 and PGC1α, but not CRTC2) involved in gluconeogenesis. Effects of Elovl5 on FoxO1 nuclear abundance correlated with increased phosphorylation of FoxO1, Akt, and the catalytic unit of PP2A, as well as a decline in cellular abundance of TRB3. Such changes are mechanistically linked to the regulation of FoxO1 nuclear abundance and gluconeogenesis. These results show that Elovl5 activity impacts the hepatic abundance and phosphorylation status of multiple proteins involved in gluconeogenesis. Our findings establish a link between fatty acid elongation and hepatic glucose metabolism and suggest a role for regulators of Elovl5 activity in the treatment of diet-induced hyperglycemia. PMID:20488798

Binary stress induces an increase in indole alkaloid biosynthesis in Catharanthus roseus

PubMed Central

Zhu, Wei; Yang, Bingxian; Komatsu, Setsuko; Lu, Xiaoping; Li, Ximin; Tian, Jingkui

2015-01-01

Catharanthus roseus is an important medicinal plant, which produces a variety of indole alkaloids of significant pharmaceutical relevance. In the present study, we aimed to investigate the potential stress-induced increase of indole alkaloid biosynthesis in C. roseus using proteomic technique. The contents of the detectable alkaloids ajmalicine, vindoline, catharanthine, and strictosidine in C. roseus were significantly increased under binary stress. Proteomic analysis revealed that the abundance of proteins related to tricarboxylic acid cycle and cell wall was largely increased; while, that of proteins related to tetrapyrrole synthesis and photosynthesis was decreased. Of note, 10-hydroxygeraniol oxidoreductase, which is involved in the biosynthesis of indole alkaloid was two-fold more abundant in treated group compared to the control. In addition, mRNA expression levels of genes involved in the indole alkaloid biosynthetic pathway indicated an up-regulation in their transcription in C. roseus under UV-B irradiation. These results suggest that binary stress might negatively affect the process of photosynthesis in C. roseus. In addition, the induction of alkaloid biosynthesis appears to be responsive to binary stress. PMID:26284098

Childhood tuberculosis is associated with decreased abundance of T cell gene transcripts and impaired T cell function.

PubMed

Hemingway, Cheryl; Berk, Maurice; Anderson, Suzanne T; Wright, Victoria J; Hamilton, Shea; Eleftherohorinou, Hariklia; Kaforou, Myrsini; Goldgof, Greg M; Hickman, Katy; Kampmann, Beate; Schoeman, Johan; Eley, Brian; Beatty, David; Pienaar, Sandra; Nicol, Mark P; Griffiths, Michael J; Waddell, Simon J; Newton, Sandra M; Coin, Lachlan J; Relman, David A; Montana, Giovanni; Levin, Michael

2017-01-01

The WHO estimates around a million children contract tuberculosis (TB) annually with over 80 000 deaths from dissemination of infection outside of the lungs. The insidious onset and association with skin test anergy suggests failure of the immune system to both recognise and respond to infection. To understand the immune mechanisms, we studied genome-wide whole blood RNA expression in children with TB meningitis (TBM). Findings were validated in a second cohort of children with TBM and pulmonary TB (PTB), and functional T-cell responses studied in a third cohort of children with TBM, other extrapulmonary TB (EPTB) and PTB. The predominant RNA transcriptional response in children with TBM was decreased abundance of multiple genes, with 140/204 (68%) of all differentially regulated genes showing reduced abundance compared to healthy controls. Findings were validated in a second cohort with concordance of the direction of differential expression in both TBM (r2 = 0.78 p = 2x10-16) and PTB patients (r2 = 0.71 p = 2x10-16) when compared to a second group of healthy controls. Although the direction of expression of these significant genes was similar in the PTB patients, the magnitude of differential transcript abundance was less in PTB than in TBM. The majority of genes were involved in activation of leucocytes (p = 2.67E-11) and T-cell receptor signalling (p = 6.56E-07). Less abundant gene expression in immune cells was associated with a functional defect in T-cell proliferation that recovered after full TB treatment (p<0.0003). Multiple genes involved in T-cell activation show decreased abundance in children with acute TB, who also have impaired functional T-cell responses. Our data suggest that childhood TB is associated with an acquired immune defect, potentially resulting in failure to contain the pathogen. Elucidation of the mechanism causing the immune paresis may identify new treatment and prevention strategies.

Integrated analysis of rice transcriptomic and metabolomic responses to elevated night temperatures identifies sensitivity- and tolerance-related profiles.

PubMed

Glaubitz, Ulrike; Li, Xia; Schaedel, Sandra; Erban, Alexander; Sulpice, Ronan; Kopka, Joachim; Hincha, Dirk K; Zuther, Ellen

2017-01-01

Transcript and metabolite profiling were performed on leaves from six rice cultivars under high night temperature (HNT) condition. Six genes were identified as central for HNT response encoding proteins involved in transcription regulation, signal transduction, protein-protein interactions, jasmonate response and the biosynthesis of secondary metabolites. Sensitive cultivars showed specific changes in transcript abundance including abiotic stress responses, changes of cell wall-related genes, of ABA signaling and secondary metabolism. Additionally, metabolite profiles revealed a highly activated TCA cycle under HNT and concomitantly increased levels in pathways branching off that could be corroborated by enzyme activity measurements. Integrated data analysis using clustering based on one-dimensional self-organizing maps identified two profiles highly correlated with HNT sensitivity. The sensitivity profile included genes of the functional bins abiotic stress, hormone metabolism, cell wall, signaling, redox state, transcription factors, secondary metabolites and defence genes. In the tolerance profile, similar bins were affected with slight differences in hormone metabolism and transcription factor responses. Metabolites of the two profiles revealed involvement of GABA signaling, thus providing a link to the TCA cycle status in sensitive cultivars and of myo-inositol as precursor for inositol phosphates linking jasmonate signaling to the HNT response specifically in tolerant cultivars. © 2016 John Wiley & Sons Ltd.

Cocaine alters Homer1 natural antisense transcript in the nucleus accumbens.

PubMed

Sartor, Gregory C; Powell, Samuel K; Velmeshev, Dmitry; Lin, David Y; Magistri, Marco; Wiedner, Hannah J; Malvezzi, Andrea M; Andrade, Nadja S; Faghihi, Mohammad A; Wahlestedt, Claes

2017-12-01

Natural antisense transcripts (NATs) are an abundant class of long noncoding RNAs that have recently been shown to be key regulators of chromatin dynamics and gene expression in nervous system development and neurological disorders. However, it is currently unclear if NAT-based mechanisms also play a role in drug-induced neuroadaptations. Aberrant regulation of gene expression is one critical factor underlying the long-lasting behavioral abnormalities that characterize substance use disorder, and it is possible that some drug-induced transcriptional responses are mediated, in part, by perturbations in NAT activity. To test this hypothesis, we used an automated algorithm that mines the NCBI AceView transcriptomics database to identify NAT overlapping genes linked to addiction. We found that 22% of the genes examined contain NATs and that expression of Homer1 natural antisense transcript (Homer1-AS) was altered in the nucleus accumbens (NAc) of mice 2h and 10days following repeated cocaine administration. In in vitro studies, depletion of Homer1-AS lead to an increase in the corresponding sense gene expression, indicating a potential regulatory mechanisms of Homer1 expression by its corresponding antisense transcript. Future in vivo studies are needed to definitely determine a role for Homer1-AS in cocaine-induced behavioral and molecular adaptations. Copyright © 2017 Elsevier Inc. All rights reserved.

A More Comprehensive Community of Ammonia-Oxidizing Archaea (AOA) Revealed by Genomic DNA and RNA Analyses of amoA Gene in Subtropical Acidic Forest Soils.

PubMed

Wu, Ruo-Nan; Meng, Han; Wang, Yong-Feng; Lan, Wensheng; Gu, Ji-Dong

2017-11-01

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are the main nitrifiers which are well studied in natural environments, and AOA frequently outnumber AOB by orders especially in acidic conditions, making AOA the most promising ammonia oxidizers. The phylogeny of AOA revealed in related studies, however, often varied and hardly reach a consensus on functional phylotypes. The objective of this study was to compare ammonia-oxidizing communities by amoA gene and transcript based on both genomic DNA and RNA in extremely acidic forest soils (pH <4.5). Our results support the numerical and functional dominance of AOA over AOB in acidic soils as bacterial amoA gene and transcript were both under detection limits and archaeal amoA, in contrast, were abundant and responded to the fluctuations of environmental factors. Organic matter from tree residues was proposed as the main source of microbial available nitrogen, and the potential co-precipitation of dissolved organic matter (DOM) with soluble Al 3+ species in acidic soil matrix may further restrict the amount of nitrogen sources required by AOB besides NH 3 /NH 4 + equilibrium. Although AOA were better adapted to oligotrophic environments, they were susceptible to the toxicity of exchangeable Al 3+ . Phylotypes affiliated to Nitrososphaera, Nitrososphaera sister group, and Nitrosotalea were detected by amoA gene and transcript. Nitrosotalea devantaerra and Nitrososphaera sister group were the major AOA. Compared to the genomic DNA data, higher relative abundances of Nitrososphaera and Nitrososphaera sister group were recognized in amoA transcript inferred AOA communities, where Nitrosotalea relative abundance was found lower, implying the functional activities of Nitrososphaera sister group and Nitrososphaera were easily underestimated and Nitrosotalea did not attribute proportionally to nitrification in extremely acidic soils. Further comparison of the different AOA community compositions and relative abundance of each phylotypes revealed by amoA genes and transcripts make it possible to identify the functional AOA species and assess their ecological role in extremely acidic soils.

A dynamic intron retention program enriched in RNA processing genes regulates gene expression during terminal erythropoiesis

DOE PAGES

Pimentel, Harold; Parra, Marilyn; Gee, Sherry L.; ...

2015-11-03

Differentiating erythroblasts execute a dynamic alternative splicing program shown here to include extensive and diverse intron retention (IR) events. Cluster analysis revealed hundreds of developmentallydynamic introns that exhibit increased IR in mature erythroblasts, and are enriched in functions related to RNA processing such as SF3B1 spliceosomal factor. Distinct, developmentally-stable IR clusters are enriched in metal-ion binding functions and include mitoferrin genes SLC25A37 and SLC25A28 that are critical for iron homeostasis. Some IR transcripts are abundant, e.g. comprising ~50% of highly-expressed SLC25A37 and SF3B1 transcripts in late erythroblasts, and thereby limiting functional mRNA levels. IR transcripts tested were predominantly nuclearlocalized. Splicemore » site strength correlated with IR among stable but not dynamic intron clusters, indicating distinct regulation of dynamically-increased IR in late erythroblasts. Retained introns were preferentially associated with alternative exons with premature termination codons (PTCs). High IR was observed in disease-causing genes including SF3B1 and the RNA binding protein FUS. Comparative studies demonstrated that the intron retention program in erythroblasts shares features with other tissues but ultimately is unique to erythropoiesis. Finally, we conclude that IR is a multi-dimensional set of processes that post-transcriptionally regulate diverse gene groups during normal erythropoiesis, misregulation of which could be responsible for human disease.« less

A dynamic intron retention program enriched in RNA processing genes regulates gene expression during terminal erythropoiesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pimentel, Harold; Parra, Marilyn; Gee, Sherry L.

Differentiating erythroblasts execute a dynamic alternative splicing program shown here to include extensive and diverse intron retention (IR) events. Cluster analysis revealed hundreds of developmentallydynamic introns that exhibit increased IR in mature erythroblasts, and are enriched in functions related to RNA processing such as SF3B1 spliceosomal factor. Distinct, developmentally-stable IR clusters are enriched in metal-ion binding functions and include mitoferrin genes SLC25A37 and SLC25A28 that are critical for iron homeostasis. Some IR transcripts are abundant, e.g. comprising ~50% of highly-expressed SLC25A37 and SF3B1 transcripts in late erythroblasts, and thereby limiting functional mRNA levels. IR transcripts tested were predominantly nuclearlocalized. Splicemore » site strength correlated with IR among stable but not dynamic intron clusters, indicating distinct regulation of dynamically-increased IR in late erythroblasts. Retained introns were preferentially associated with alternative exons with premature termination codons (PTCs). High IR was observed in disease-causing genes including SF3B1 and the RNA binding protein FUS. Comparative studies demonstrated that the intron retention program in erythroblasts shares features with other tissues but ultimately is unique to erythropoiesis. Finally, we conclude that IR is a multi-dimensional set of processes that post-transcriptionally regulate diverse gene groups during normal erythropoiesis, misregulation of which could be responsible for human disease.« less

Trees, fungi and bacteria: tripartite metatranscriptomics of a root microbiome responding to soil contamination.

PubMed

Gonzalez, E; Pitre, F E; Pagé, A P; Marleau, J; Guidi Nissim, W; St-Arnaud, M; Labrecque, M; Joly, S; Yergeau, E; Brereton, N J B

2018-03-21

One method for rejuvenating land polluted with anthropogenic contaminants is through phytoremediation, the reclamation of land through the cultivation of specific crops. The capacity for phytoremediation crops, such as Salix spp., to tolerate and even flourish in contaminated soils relies on a highly complex and predominantly cryptic interacting community of microbial life. Here, Illumina HiSeq 2500 sequencing and de novo transcriptome assembly were used to observe gene expression in washed Salix purpurea cv. 'Fish Creek' roots from trees pot grown in petroleum hydrocarbon-contaminated or non-contaminated soil. All 189,849 assembled contigs were annotated without a priori assumption as to sequence origin and differential expression was assessed. The 839 contigs differentially expressed (DE) and annotated from S. purpurea revealed substantial increases in transcripts encoding abiotic stress response equipment, such as glutathione S-transferases, in roots of contaminated trees as well as the hallmarks of fungal interaction, such as SWEET2 (Sugars Will Eventually Be Exported Transporter). A total of 8252 DE transcripts were fungal in origin, with contamination conditions resulting in a community shift from Ascomycota to Basidiomycota genera. In response to contamination, 1745 Basidiomycota transcripts increased in abundance (the majority uniquely expressed in contaminated soil) including major monosaccharide transporter MST1, primary cell wall and lamella CAZy enzymes, and an ectomycorrhiza-upregulated exo-β-1,3-glucanase (GH5). Additionally, 639 DE polycistronic transcripts from an uncharacterised Enterobacteriaceae species were uniformly in higher abundance in contamination conditions and comprised a wide spectrum of genes cryptic under laboratory conditions but considered putatively involved in eukaryotic interaction, biofilm formation and dioxygenase hydrocarbon degradation. Fungal gene expression, representing the majority of contigs assembled, suggests out-competition of white rot Ascomycota genera (dominated by Pyronema), a sometimes ectomycorrhizal (ECM) Ascomycota (Tuber) and ECM Basidiomycota (Hebeloma) by a poorly characterised putative ECM Basidiomycota due to contamination. Root and fungal expression involved transcripts encoding carbohydrate/amino acid (C/N) dialogue whereas bacterial gene expression included the apparatus necessary for biofilm interaction and direct reduction of contamination stress, a potential bacterial currency for a role in tripartite mutualism. Unmistakable within the metatranscriptome is the degree to which the landscape of rhizospheric biology, particularly the important but predominantly uncharacterised fungal genetics, is yet to be discovered.

Sequence and Expression Analyses of Ethylene Response Factors Highly Expressed in Latex Cells from Hevea brasiliensis

PubMed Central

Piyatrakul, Piyanuch; Yang, Meng; Putranto, Riza-Arief; Pirrello, Julien; Dessailly, Florence; Hu, Songnian; Summo, Marilyne; Theeravatanasuk, Kannikar; Leclercq, Julie; Kuswanhadi; Montoro, Pascal

2014-01-01

The AP2/ERF superfamily encodes transcription factors that play a key role in plant development and responses to abiotic and biotic stress. In Hevea brasiliensis, ERF genes have been identified by RNA sequencing. This study set out to validate the number of HbERF genes, and identify ERF genes involved in the regulation of latex cell metabolism. A comprehensive Hevea transcriptome was improved using additional RNA reads from reproductive tissues. Newly assembled contigs were annotated in the Gene Ontology database and were assigned to 3 main categories. The AP2/ERF superfamily is the third most represented compared with other transcription factor families. A comparison with genomic scaffolds led to an estimation of 114 AP2/ERF genes and 1 soloist in Hevea brasiliensis. Based on a phylogenetic analysis, functions were predicted for 26 HbERF genes. A relative transcript abundance analysis was performed by real-time RT-PCR in various tissues. Transcripts of ERFs from group I and VIII were very abundant in all tissues while those of group VII were highly accumulated in latex cells. Seven of the thirty-five ERF expression marker genes were highly expressed in latex. Subcellular localization and transactivation analyses suggested that HbERF-VII candidate genes encoded functional transcription factors. PMID:24971876

Expression and Stress-Dependent Induction of Potassium Channel Transcripts in the Common Ice Plant1

PubMed Central

Su, Hua; Golldack, Dortje; Katsuhara, Maki; Zhao, Chengsong; Bohnert, Hans J.

2001-01-01

We have characterized transcripts for three potassium channel homologs in the AKT/KAT subfamily (Shaker type) from the common ice plant (Mesembryanthemum crystallinum), with a focus on their expression during salt stress (up to 500 mm NaCl). Mkt1 and 2, Arabidopsis AKT homologs, and Kmt1, a KAT homolog, are members of small gene families with two to three isoforms each. Mkt1 is root specific; Mkt2 is found in leaves, flowers, and seed capsules; and Kmt1 is expressed in leaves and seed capsules. Mkt1 is present in all cells of the root, and in leaves a highly conserved isoform is detected present in all cells with highest abundance in the vasculature. MKT1 for which antibodies were made is localized to the plasma membrane. Following salt stress, MKT1 (transcripts and protein) is drastically down-regulated, Mkt2 transcripts do not change significantly, and Kmt1 is strongly and transiently (maximum at 6 h) up-regulated in leaves and stems. The detection and stress-dependent behavior of abundant transcripts representing subfamilies of potassium channels provides information about tissue specificity and the complex regulation of genes encoding potassium uptake systems in a halophytic plant. PMID:11161018

Sequence and expression analyses of ethylene response factors highly expressed in latex cells from Hevea brasiliensis.

PubMed

Piyatrakul, Piyanuch; Yang, Meng; Putranto, Riza-Arief; Pirrello, Julien; Dessailly, Florence; Hu, Songnian; Summo, Marilyne; Theeravatanasuk, Kannikar; Leclercq, Julie; Kuswanhadi; Montoro, Pascal

2014-01-01

The AP2/ERF superfamily encodes transcription factors that play a key role in plant development and responses to abiotic and biotic stress. In Hevea brasiliensis, ERF genes have been identified by RNA sequencing. This study set out to validate the number of HbERF genes, and identify ERF genes involved in the regulation of latex cell metabolism. A comprehensive Hevea transcriptome was improved using additional RNA reads from reproductive tissues. Newly assembled contigs were annotated in the Gene Ontology database and were assigned to 3 main categories. The AP2/ERF superfamily is the third most represented compared with other transcription factor families. A comparison with genomic scaffolds led to an estimation of 114 AP2/ERF genes and 1 soloist in Hevea brasiliensis. Based on a phylogenetic analysis, functions were predicted for 26 HbERF genes. A relative transcript abundance analysis was performed by real-time RT-PCR in various tissues. Transcripts of ERFs from group I and VIII were very abundant in all tissues while those of group VII were highly accumulated in latex cells. Seven of the thirty-five ERF expression marker genes were highly expressed in latex. Subcellular localization and transactivation analyses suggested that HbERF-VII candidate genes encoded functional transcription factors.

QsMYB1 expression is modulated in response to heat and drought stresses and during plant recovery in Quercus suber.

PubMed

Almeida, Tânia; Pinto, Glória; Correia, Barbara; Santos, Conceição; Gonçalves, Sónia

2013-12-01

Cork oak is an economically important forest species showing a great tolerance to high temperatures and shortage of water. However, the mechanisms underlying this plasticity are still poorly understood. Among the stress regulators, transcription factors (TFs) are especially important since they can control a wide range of stress-inducible genes, which make them powerful targets for genetic engineering of stress tolerance. Here we evaluated the influence of increasing temperatures (up to 55 °C) or drought (18% field capacity, FC) on the expression profile of an R2R3-MYB transcription factor of cork oak, the QsMYB1. QsMYB1 was previously identified as being preferentially expressed in cork tissues and as having an associated alternative splicing mechanism, which results in two different transcripts (QsMYB1.1 and QsMYB1.2). Expression analysis by reverse transcription quantitative PCR (RT-qPCR) revealed that increasing temperatures led to a gradual down-regulation of QsMYB1 transcripts with more effect on QsMYB1.1 abundance. On the other hand, under drought condition, expression of QsMYB1 variants, mainly the QsMYB1.2, was transiently up-regulated shortly after the stress imposition. Recovery from each stress has also resulted in a differential response by both QsMYB1 transcripts. Several physiological and biochemical parameters (plant water status, chlorophyll fluorescence, lipid peroxidation and proline content) were determined in order to monitor the plant performance under stress and recovery. In conclusion, this report provides the first evidence that QsMYB1 TF may have a putative function in the regulatory network of cork oak response to heat and drought stresses and during plant recovery. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Loss of epigenetic Kruppel-like factor 4 histone deacetylase (KLF-4-HDAC)-mediated transcriptional suppression is crucial in increasing vascular endothelial growth factor (VEGF) expression in breast cancer.

PubMed

Ray, Alpana; Alalem, Mohamed; Ray, Bimal K

2013-09-20

Vascular endothelial growth factor (VEGF) is recognized as an important angiogenic factor that promotes angiogenesis in a series of pathological conditions, including cancer, inflammation, and ischemic disorders. We have recently shown that the inflammatory transcription factor SAF-1 is, at least in part, responsible for the marked increase of VEGF levels in breast cancer. Here, we show that SAF-1-mediated induction of VEGF is repressed by KLF-4 transcription factor. KLF-4 is abundantly present in normal breast epithelial cells, but its level is considerably reduced in breast cancer cells and clinical cancer tissues. In the human VEGF promoter, SAF-1- and KLF-4-binding elements are overlapping, whereas SAF-1 induces and KLF-4 suppresses VEGF expression. Ectopic overexpression of KLF-4 and RNAi-mediated inhibition of endogenous KLF-4 supported the role of KLF-4 as a transcriptional repressor of VEGF and an inhibitor of angiogenesis in breast cancer cells. We show that KLF-4 recruits histone deacetylases (HDACs) -2 and -3 at the VEGF promoter. Chronological ChIP assays demonstrated the occupancy of KLF-4, HDAC2, and HDAC3 in the VEGF promoter in normal MCF-10A cells but not in MDA-MB-231 cancer cells. Co-transfection of KLF-4 and HDAC expression plasmids in breast cancer cells results in synergistic repression of VEGF expression and inhibition of angiogenic potential of these carcinoma cells. Together these results identify a new mechanism of VEGF up-regulation in cancer that involves concomitant loss of KLF-4-HDAC-mediated transcriptional repression and active recruitment of SAF-1-mediated transcriptional activation.

Evaluation and selection of internal reference genes from two- and six-row U.S. malting barley varieties throughout micromalting for use in RT-qPCR

USDA-ARS?s Scientific Manuscript database

Reverse Transcription quantitative Polymerase Chain Reaction (qRT-PCR) is a popular method for measuring transcript abundance. The most commonly used method of interpretation is relative quantification and thus necessitates the use of normalization controls (i.e. reference genes) to standardize tran...

Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

PubMed Central

Carter, Mark G; Sharov, Alexei A; VanBuren, Vincent; Dudekula, Dawood B; Carmack, Condie E; Nelson, Charlie; Ko, Minoru SH

2005-01-01

The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance. PMID:15998450

Obesity in mares promotes uterine inflammation and alters embryo lipid fingerprints and homeostasis.

PubMed

Sessions-Bresnahan, Dawn R; Heuberger, Adam L; Carnevale, Elaine M

2018-05-07

Maternal body composition can be an important determinant for development of obesity and metabolic syndrome in adult offspring. Obesity-related outcomes in offspring may include epigenetic alterations; however, mechanisms of fetal programming remain to be fully elucidated. This study was conducted to determine the impact of maternal obesity in the absence of a high fat diet on equine endometrium and preimplantation embryos. Embryos were collected from normal and obese mares at 8 and 16 d and a uterine biopsy at 16 d (0 d = ovulation). With the exception of 8 d embryos, each sample was divided into two pieces. One piece was analyzed for gene expression markers related to carbohydrate metabolism, lipid homeostasis, inflammation, endoplasmic reticulum stress, oxidative stress, mitochondrial stress, and components of the insulin-like growth factor (IGF) system. The second piece was analyzed for lipid content using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Obese mares had elevated concentrations of insulin, leptin and total cholesterol, and they tended to have increased triglycerides and decreased insulin sensitivity. Embryos from obese mares had altered transcript abundance in genes for inflammation and lipid homeostasis, as well as, endoplasmic reticulum, oxidative and mitochondrial stress and altered lipid fingerprints. Endometrium from obese mares had increased expression of inflammatory cytokines, lipid homeostasis regulation, mitochondrial stress, and the IGF2 system. This study demonstrates increased adiposity in mares alters the uterine environment, transcript abundance of genes for cellular functions, and lipid profiles of embryos. These alterations could affect prenatal programming, with potential long-term effects in offspring.

Chloroquine mediated modulation of Anopheles gambiae gene expression.

PubMed

Abrantes, Patrícia; Dimopoulos, George; Grosso, Ana Rita; do Rosário, Virgílio E; Silveira, Henrique

2008-07-02

Plasmodium development in the mosquito is crucial for malaria transmission and depends on the parasite's interaction with a variety of cell types and specific mosquito factors that have both positive and negative effects on infection. Whereas the defensive response of the mosquito contributes to a decrease in parasite numbers during these stages, some components of the blood meal are known to favor infection, potentiating the risk of increased transmission. The presence of the antimalarial drug chloroquine in the mosquito's blood meal has been associated with an increase in Plasmodium infectivity for the mosquito, which is possibly caused by chloroquine interfering with the capacity of the mosquito to defend against the infection. In this study, we report a detailed survey of the Anopheles gambiae genes that are differentially regulated by the presence of chloroquine in the blood meal, using an A. gambiae cDNA microarray. The effect of chloroquine on transcript abundance was evaluated separately for non-infected and Plasmodium berghei-infected mosquitoes. Chloroquine was found to affect the abundance of transcripts that encode proteins involved in a variety of processes, including immunity, apoptosis, cytoskeleton and the response to oxidative stress. This pattern of differential gene expression may explain the weakened mosquito defense response which accounts for the increased infectivity observed in chloroquine-treated mosquitoes. The results of the present study suggest that chloroquine can interfere with several putative mosquito mechanisms of defense against Plasmodium at the level of gene expression and highlight the need for a better understanding of the impacts of antimalarial agents on parasite transmission.

Downregulation of a UDP-Arabinomutase Gene in Switchgrass (Panicum virgatum L.) Results in Increased Cell Wall Lignin While Reducing Arabinose-Glycans

DOE PAGES

Willis, Jonathan D.; Smith, James A.; Mazarei, Mitra; ...

2016-10-26

Switchgrass (Panicum virgatum L.) is a C 4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall's natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf. The expression ofmore » a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control. Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.« less

Downregulation of a UDP-Arabinomutase Gene in Switchgrass (Panicum virgatum L.) Results in Increased Cell Wall Lignin While Reducing Arabinose-Glycans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willis, Jonathan D.; Smith, James A.; Mazarei, Mitra

Switchgrass (Panicum virgatum L.) is a C 4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall's natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf. The expression ofmore » a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control. Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.« less

Tide-related Changes in mRNA Abundance of Aromatases and Estrogen Receptors in the Ovary and Brain of the Threespot Wrasse Halichoeres trimaculatus

NASA Astrophysics Data System (ADS)

Oh, Dae-Ju; Hur, Sung-Pyo; Bouchekioua, Selma; Takeuchi, Yuki; Udagawa, Shingo; Aluru, Neelakanteswar; Park, Yong-Ju; Park, Ji-Gweon; Kim, Se-Jae; Moon, Thomas W.; Vijayan, Mathilakath M.; Takemura, Akihiro

2018-05-01

The threespot wrasse (Halichoeres trimaculatus; Family Labridae) is a common coral reef species of the Indo-Pacific Ocean. Given that this species spawns daily at high tide (HT), we hypothesized that endocrine changes in relation to gonadal development are synchronized with the tidal cycle. To test this, we examined the transcript abundance of two cytochrome P450 aromatases (cyp19a and cyp19b) and two estrogen receptors (erα and erβ) in the ovary and brain of this species in response to tidal change. When fish were collected around four tidal points [low tide (LT), flood tide (FT), high tide (HT), and ebb tide (ET)], gonadosomatic index and oocyte diameter increased around HT and FT, respectively. Ovulatory follicles were observed in ovaries around HT. Real-time quantitative polymerase-chain reaction revealed that mRNA abundance of cyp19a and erα, but not erβ, in the ovary increased around ET and HT, respectively. On the other hand, mRNA levels of cyp19b in the forebrain were significantly higher around FT. Increases of erα and erβ mRNA abundance around FT were observed in all areas of the brain and the midbrain, respectively. The changes in mRNA abundance of key genes involved in reproduction at specific tidal cycles, along with the development of the vitellogenic oocytes in the ovary, support our hypothesis that synchronization of endocrine changes to the tidal periodicity plays a role in the gonadal development of this species. We hypothesize that conversion of testosterone to E2 in the brain may be associated with the spawning behavior given that the wrasse exhibits group spawning with a territory-holding male around HT.

Glucocorticoids suppress tumor necrosis factor-alpha expression by human monocytic THP-1 cells by suppressing transactivation through adjacent NF-kappa B and c-Jun-activating transcription factor-2 binding sites in the promoter.

PubMed

Steer, J H; Kroeger, K M; Abraham, L J; Joyce, D A

2000-06-16

Glucocorticoid drugs suppress tumor necrosis factor-alpha (TNF-alpha) synthesis by activated monocyte/macrophages, contributing to an anti-inflammatory action in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells, glucocorticoids acted primarily on the TNF-alpha promoter to suppress a burst of transcriptional activity that occurred between 90 min and 3 h after LPS exposure. LPS increased nuclear c-Jun/ATF-2, NF-kappaB(1)/Rel-A, and Rel-A/C-Rel transcription factor complexes, which bound specifically to oligonucleotide sequences from the -106 to -88 base pair (bp) region of the promoter. The glucocorticoid, dexamethasone, suppressed nuclear binding activity of these complexes prior to and during the critical phase of TNF-alpha transcription. Site-directed mutagenesis in TNF-alpha promoter-luciferase reporter constructs showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-kappaB (-97 to -88 bp) binding sites each contributed to the LPS-stimulated expression. Mutating both sites largely prevented dexamethasone from suppressing TNF-alpha promoter-luciferase reporters. LPS exposure also increased nuclear Egr-1 and PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated expression but not to glucocorticoid response. Dexamethasone suppressed the abundance of the c-Fos/c-Jun complex in THP-1 cell nuclei, but there was no direct evidence for c-Fos/c-Jun transactivation through sites in the -172 to -52 bp region. Small contributions to glucocorticoid response were attributable to promoter sequences outside the -172 to -88 bp region and to sequences in the TNF-alpha 3'-untranslated region. We conclude that glucocorticoids suppress LPS-stimulated secretion of TNF-alpha from human monocytic cells largely through antagonizing transactivation by c-Jun/ATF-2 and NF-kappaB complexes at binding sites in the -106 to -88 bp region of the TNF-alpha promoter.

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lazarus, Kyren A.; Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122; Zhao, Zhe

2013-08-30

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels highermore » in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.« less

Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans

PubMed Central

Mitrovich, Quinn M.; Anderson, Philip

2000-01-01

Messenger RNA surveillance, the selective and rapid degradation of mRNAs containing premature stop codons, occurs in all eukaryotes tested. The biological role of this decay pathway, however, is not well understood. To identify natural substrates of mRNA surveillance, we used a cDNA-based representational difference analysis to identify mRNAs whose abundance increases in Caenorhabditis elegans smg(−) mutants, which are deficient for mRNA surveillance. Alternatively spliced mRNAs of genes encoding ribosomal proteins L3, L7a, L10a, and L12 are abundant natural targets of mRNA surveillance. Each of these genes expresses two distinct mRNAs. A productively spliced mRNA, whose abundance does not change in smg(−) mutants, encodes a normal, full-length, ribosomal protein. An unproductively spliced mRNA, whose abundance increases dramatically in smg(−) mutants, contains premature stop codons because of incomplete removal of an alternatively spliced intron. In transgenic animals expressing elevated quantities of RPL-12, a greater proportion of endogenous rpl-12 transcript is spliced unproductively. Thus, RPL-12 appears to autoregulate its own splicing, with unproductively spliced mRNAs being degraded by mRNA surveillance. We demonstrate further that alternative splicing of rpl introns is conserved among widely diverged nematodes. Our results suggest that one important role of mRNA surveillance is to eliminate unproductive by-products of gene regulation. PMID:10970881

Evidence of Geobacter-associated phage in a uranium-contaminated aquifer

PubMed Central

Holmes, Dawn E; Giloteaux, Ludovic; Chaurasia, Akhilesh K; Williams, Kenneth H; Luef, Birgit; Wilkins, Michael J; Wrighton, Kelly C; Thompson, Courtney A; Comolli, Luis R; Lovley, Derek R

2015-01-01

Geobacter species may be important agents in the bioremediation of organic and metal contaminants in the subsurface, but as yet unknown factors limit the in situ growth of subsurface Geobacter well below rates predicted by analysis of gene expression or in silico metabolic modeling. Analysis of the genomes of five different Geobacter species recovered from contaminated subsurface sites indicated that each of the isolates had been infected with phage. Geobacter-associated phage sequences were also detected by metagenomic and proteomic analysis of samples from a uranium-contaminated aquifer undergoing in situ bioremediation, and phage particles were detected by microscopic analysis in groundwater collected from sediment enrichment cultures. Transcript abundance for genes from the Geobacter-associated phage structural proteins, tail tube Gp19 and baseplate J, increased in the groundwater in response to the growth of Geobacter species when acetate was added, and then declined as the number of Geobacter decreased. Western blot analysis of a Geobacter-associated tail tube protein Gp19 in the groundwater demonstrated that its abundance tracked with the abundance of Geobacter species. These results suggest that the enhanced growth of Geobacter species in the subsurface associated with in situ uranium bioremediation increased the abundance and activity of Geobacter-associated phage and show that future studies should focus on how these phages might be influencing the ecology of this site. PMID:25083935

Probing the changes in gene expression due to α-crystallin mutations in mouse models of hereditary human cataract.

PubMed

Andley, Usha P; Tycksen, Eric; McGlasson-Naumann, Brittney N; Hamilton, Paul D

2018-01-01

The mammalian eye lens expresses a high concentration of crystallins (α, β and γ-crystallins) to maintain the refractive index essential for lens transparency. Crystallins are long-lived proteins that do not turnover throughout life. The structural destabilization of crystallins by UV exposure, glycation, oxidative stress and mutations in crystallin genes leads to protein aggregation and development of cataracts. Several destabilizing mutations in crystallin genes are linked with human autosomal dominant hereditary cataracts. To investigate the mechanism by which the α-crystallin mutations Cryaa-R49C and Cryab-R120G lead to cataract formation, we determined whether these mutations cause an altered expression of specific transcripts in the lens at an early postnatal age by RNA-seq analysis. Using knock-in mouse models previously generated in our laboratory, in the present work, we identified genes that exhibited altered abundance in the mutant lenses, including decreased transcripts for Clic5, an intracellular water channel in Cryaa-R49C heterozygous mutant lenses, and increased transcripts for Eno1b in Cryab-R120G heterozygous mutant lenses. In addition, RNA-seq analysis revealed increased histones H2B, H2A, and H4 gene expression in Cryaa-R49C mutant lenses, suggesting that the αA-crystallin mutation regulates histone expression via a transcriptional mechanism. Additionally, these studies confirmed the increased expression of histones H2B, H2A, and H4 by proteomic analysis of Cryaa-R49C knock-in and Cryaa;Cryab gene knockout lenses reported previously. Taken together, these findings offer additional insight into the early transcriptional changes caused by Cryaa and Cryab mutations associated with autosomal dominant human cataracts, and indicate that the transcript levels of certain genes are affected by the expression of mutant α-crystallin in vivo.

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury.

PubMed

Maia, Rafaela M; Valente, Valeria; Cunha, Marco A V; Sousa, Josane F; Araujo, Daniela D; Silva, Wilson A; Zago, Marco A; Dias-Neto, Emmanuel; Souza, Sandro J; Simpson, Andrew J G; Monesi, Nadia; Ramos, Ricardo G P; Espreafico, Enilza M; Paçó-Larson, Maria L

2007-07-24

The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury. Using the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

PubMed Central

Maia, Rafaela M; Valente, Valeria; Cunha, Marco AV; Sousa, Josane F; Araujo, Daniela D; Silva, Wilson A; Zago, Marco A; Dias-Neto, Emmanuel; Souza, Sandro J; Simpson, Andrew JG; Monesi, Nadia; Ramos, Ricardo GP; Espreafico, Enilza M; Paçó-Larson, Maria L

2007-01-01

Background The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. Results Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury. Conclusion Using the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data. PMID:17650329

Cadmium-induced ethylene production and responses in Arabidopsis thaliana rely on ACS2 and ACS6 gene expression

PubMed Central

2014-01-01

Background Anthropogenic activities cause metal pollution worldwide. Plants can absorb and accumulate these metals through their root system, inducing stress as a result of excess metal concentrations inside the plant. Ethylene is a regulator of multiple plant processes, and is affected by many biotic and abiotic stresses. Increased ethylene levels have been observed after exposure to excess metals but it remains unclear how the increased ethylene levels are achieved at the molecular level. In this study, the effects of cadmium (Cd) exposure on the production of ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and on the expression of the ACC Synthase (ACS) and ACC Oxidase (ACO) multigene families were investigated in Arabidopsis thaliana. Results Increased ethylene release after Cd exposure was directly measurable in a system using rockwool-cultivated plants; enhanced levels of the ethylene precursor ACC together with higher mRNA levels of ethylene responsive genes: ACO2, ETR2 and ERF1 also indicated increased ethylene production in hydroponic culture. Regarding underlying mechanisms, it was found that the transcript levels of ACO2 and ACO4, the most abundantly expressed members of the ACO multigene family, were increased upon Cd exposure. ACC synthesis is the rate-limiting step in ethylene biosynthesis, and transcript levels of both ACS2 and ACS6 showed the highest increase and became the most abundant isoforms after Cd exposure, suggesting their importance in the Cd-induced increase of ethylene production. Conclusions Cadmium induced the biosynthesis of ACC and ethylene in Arabidopsis thaliana plants mainly via the increased expression of ACS2 and ACS6. This was confirmed in the acs2-1acs6-1 double knockout mutants, which showed a decreased ethylene production, positively affecting leaf biomass and resulting in a delayed induction of ethylene responsive gene expressions without significant differences in Cd contents between wild-type and mutant plants. PMID:25082369

Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs

PubMed Central

2014-01-01

Background The genome is pervasively transcribed but most transcripts do not code for proteins, constituting non-protein-coding RNAs. Despite increasing numbers of functional reports of individual long non-coding RNAs (lncRNAs), assessing the extent of functionality among the non-coding transcriptional output of mammalian cells remains intricate. In the protein-coding world, transcripts differentially expressed in the context of processes essential for the survival of multicellular organisms have been instrumental in the discovery of functionally relevant proteins and their deregulation is frequently associated with diseases. We therefore systematically identified lncRNAs expressed differentially in response to oncologically relevant processes and cell-cycle, p53 and STAT3 pathways, using tiling arrays. Results We found that up to 80% of the pathway-triggered transcriptional responses are non-coding. Among these we identified very large macroRNAs with pathway-specific expression patterns and demonstrated that these are likely continuous transcripts. MacroRNAs contain elements conserved in mammals and sauropsids, which in part exhibit conserved RNA secondary structure. Comparing evolutionary rates of a macroRNA to adjacent protein-coding genes suggests a local action of the transcript. Finally, in different grades of astrocytoma, a tumor disease unrelated to the initially used cell lines, macroRNAs are differentially expressed. Conclusions It has been shown previously that the majority of expressed non-ribosomal transcripts are non-coding. We now conclude that differential expression triggered by signaling pathways gives rise to a similar abundance of non-coding content. It is thus unlikely that the prevalence of non-coding transcripts in the cell is a trivial consequence of leaky or random transcription events. PMID:24594072

Methanospirillum respiratory mRNA biomarkers correlate with hydrogenotrophic methanogenesis rate during growth and competition for hydrogen in an organochlorine-respiring mixed culture.

PubMed

Rowe, Annette R; Mansfeldt, Cresten B; Heavner, Gretchen L; Richardson, Ruth E

2013-01-02

Molecular biomarkers hold promise for inferring rates of key metabolic activities in complex microbial systems. However, few studies have assessed biomarker levels for simultaneously occurring (and potentially competing) respirations. In this study, methanogenesis biomarkers for Methanospirillum hungatei were developed, tested, and compared to Dehalococcoides mccartyi biomarkers in a well-characterized mixed culture. Proteomic analyses of mixed culture samples (n = 4) confirmed expression of many M. hungatei methanogenesis enzymes. The mRNAs for two oxidoreductases detected were explored as quantitative biomarkers of hydrogenotrophic methanogenesis: a coenzyme F(420)-reducing hydrogenase (FrcA) and an iron sulfur protein (MvrD). As shown previously in D. mccartyi, M. hungatei transcript levels correlated linearly with measured (R = 0.97 for FrcA, R = 0.91 for MvrD; n = 7) or calculated respiration rate (R = 0.81 for FrcA, R = 0.62 for MvrD; n = 35) across two orders of magnitude on a log-log scale. The average abundance of MvrD transcripts was consistently two orders of magnitude lower than FrcA, regardless of experimental condition. In experiments where M. hungatei was competing for hydrogen with D. mccartyi, transcripts for the key respiratory hydrogenase HupL were generally less abundant per mL than FrcA and more abundant than MvrD. With no chlorinated electron acceptor added, HupL transcripts fell below both targets. These biomarkers hold promise for the prediction of in situ rates of respiration for these microbes, even when growing in mixed culture and utilizing a shared substrate which has important implications for both engineered and environmental systems. However, the differences in overall biomarker abundances suggest that the strength of any particular mRNA biomarker relies upon empirically established quantitative trends under a range of pertinent conditions.

Finite-size effects in transcript sequencing count distribution: its power-law correction necessarily precedes downstream normalization and comparative analysis.

PubMed

Wong, Wing-Cheong; Ng, Hong-Kiat; Tantoso, Erwin; Soong, Richie; Eisenhaber, Frank

2018-02-12

Though earlier works on modelling transcript abundance from vertebrates to lower eukaroytes have specifically singled out the Zip's law, the observed distributions often deviate from a single power-law slope. In hindsight, while power-laws of critical phenomena are derived asymptotically under the conditions of infinite observations, real world observations are finite where the finite-size effects will set in to force a power-law distribution into an exponential decay and consequently, manifests as a curvature (i.e., varying exponent values) in a log-log plot. If transcript abundance is truly power-law distributed, the varying exponent signifies changing mathematical moments (e.g., mean, variance) and creates heteroskedasticity which compromises statistical rigor in analysis. The impact of this deviation from the asymptotic power-law on sequencing count data has never truly been examined and quantified. The anecdotal description of transcript abundance being almost Zipf's law-like distributed can be conceptualized as the imperfect mathematical rendition of the Pareto power-law distribution when subjected to the finite-size effects in the real world; This is regardless of the advancement in sequencing technology since sampling is finite in practice. Our conceptualization agrees well with our empirical analysis of two modern day NGS (Next-generation sequencing) datasets: an in-house generated dilution miRNA study of two gastric cancer cell lines (NUGC3 and AGS) and a publicly available spike-in miRNA data; Firstly, the finite-size effects causes the deviations of sequencing count data from Zipf's law and issues of reproducibility in sequencing experiments. Secondly, it manifests as heteroskedasticity among experimental replicates to bring about statistical woes. Surprisingly, a straightforward power-law correction that restores the distribution distortion to a single exponent value can dramatically reduce data heteroskedasticity to invoke an instant increase in signal-to-noise ratio by 50% and the statistical/detection sensitivity by as high as 30% regardless of the downstream mapping and normalization methods. Most importantly, the power-law correction improves concordance in significant calls among different normalization methods of a data series averagely by 22%. When presented with a higher sequence depth (4 times difference), the improvement in concordance is asymmetrical (32% for the higher sequencing depth instance versus 13% for the lower instance) and demonstrates that the simple power-law correction can increase significant detection with higher sequencing depths. Finally, the correction dramatically enhances the statistical conclusions and eludes the metastasis potential of the NUGC3 cell line against AGS of our dilution analysis. The finite-size effects due to undersampling generally plagues transcript count data with reproducibility issues but can be minimized through a simple power-law correction of the count distribution. This distribution correction has direct implication on the biological interpretation of the study and the rigor of the scientific findings. This article was reviewed by Oliviero Carugo, Thomas Dandekar and Sandor Pongor.

A transferrin gene associated with development and 2-tridecanone tolerance in Helicoverpa armigera

PubMed Central

Zhang, L; Shang, Q; Lu, Y; Zhao, Q; Gao, X

2015-01-01

The full-length cDNA (2320 bp) encoding a putative iron-binding transferrin protein from Helicoverpa armigera was cloned and named HaTrf. The putative HaTrf sequence included 670 amino acids with a molecular mass of approximately 76 kDa. Quantitative PCR results demonstrated that the transcriptional level of HaTrf was significantly higher in the sixth instar and pupa stages as compared with other developmental stages. HaTrf transcripts were more abundant in fat bodies and in the epidermis than in malpighian tubules. Compared with the control, the expression of HaTrf increased dramatically 24 h after treatment with 2-tridecanone. Apparent growth inhibition with a dramatic body weight decrease was observed in larvae fed with HaTrf double-stranded RNA (dsRNA), as compared with those fed with green fluorescent protein dsRNA. RNA interference of HaTrf also significantly increased the susceptibility of larvae to 2-tridecanone. These results indicate the possible involvement of HaTrf in tolerance to plant secondary chemicals. PMID:25430818

Response of the calcifying coccolithophore Emiliania huxleyi to low pH/high pCO2: from physiology to molecular level.

PubMed

Richier, Sophie; Fiorini, Sarah; Kerros, Marie-Emmanuelle; von Dassow, Peter; Gattuso, Jean-Pierre

2011-01-01

The emergence of ocean acidification as a significant threat to calcifying organisms in marine ecosystems creates a pressing need to understand the physiological and molecular mechanisms by which calcification is affected by environmental parameters. We report here, for the first time, changes in gene expression induced by variations in pH/pCO 2 in the widespread and abundant coccolithophore Emiliania huxleyi . Batch cultures were subjected to increased partial pressure of CO 2 (pCO 2 ; i.e. decreased pH), and the changes in expression of four functional gene classes directly or indirectly related to calcification were investigated. Increased pCO 2 did not affect the calcification rate and only carbonic anhydrase transcripts exhibited a significant down-regulation. Our observation that elevated pCO 2 induces only limited changes in the transcription of several transporters of calcium and bicarbonate gives new significant elements to understand cellular mechanisms underlying the early response of E. huxleyi to CO 2 -driven ocean acidification.

Programming of stress-related behavior and epigenetic neural gene regulation in mice offspring through maternal exposure to predator odor

PubMed Central

St-Cyr, Sophie; McGowan, Patrick O.

2015-01-01

Perinatal stress mediated through the mother can lead to long-term alterations in stress-related phenotypes in offspring. The capacity for adaptation to adversity in early life depends in part on the life history of the animal. This study was designed to examine the behavioral and neural response in adult offspring to prenatal exposure to predator odor: an ethologically-relevant psychological stressor. Pregnant mice were exposed daily to predator odors or distilled water control over the second half of the pregnancy. Predator odor exposure lead to a transient decrease in maternal care in the mothers. As adults, the offspring of predator odor-exposed mothers showed increased anti-predator behavior, a predator-odor induced decrease in activity and, in female offspring, an increased corticosterone (CORT) response to predator odor exposure. We found a highly specific response among stress-related genes within limbic brain regions. Transcript abundance of Corticotropin-releasing hormone receptor 1 (CRHR1) was elevated in the amygdala in adult female offspring of predator odor-exposed mothers. In the hippocampus of adult female offspring, decreased Brain-derived neurotrophic factor (BDNF) transcript abundance was correlated with a site-specific decrease in DNA methylation in Bdnf exon IV, indicating the potential contribution of this epigenetic mechanism to maternal programming by maternal predator odor exposure. These data indicate that maternal predator odor exposure alone is sufficient to induce an altered stress-related phenotype in adulthood, with implications for anti-predator behavior in offspring. PMID:26082698

Programming of stress-related behavior and epigenetic neural gene regulation in mice offspring through maternal exposure to predator odor.

PubMed

St-Cyr, Sophie; McGowan, Patrick O

2015-01-01

Perinatal stress mediated through the mother can lead to long-term alterations in stress-related phenotypes in offspring. The capacity for adaptation to adversity in early life depends in part on the life history of the animal. This study was designed to examine the behavioral and neural response in adult offspring to prenatal exposure to predator odor: an ethologically-relevant psychological stressor. Pregnant mice were exposed daily to predator odors or distilled water control over the second half of the pregnancy. Predator odor exposure lead to a transient decrease in maternal care in the mothers. As adults, the offspring of predator odor-exposed mothers showed increased anti-predator behavior, a predator-odor induced decrease in activity and, in female offspring, an increased corticosterone (CORT) response to predator odor exposure. We found a highly specific response among stress-related genes within limbic brain regions. Transcript abundance of Corticotropin-releasing hormone receptor 1 (CRHR1) was elevated in the amygdala in adult female offspring of predator odor-exposed mothers. In the hippocampus of adult female offspring, decreased Brain-derived neurotrophic factor (BDNF) transcript abundance was correlated with a site-specific decrease in DNA methylation in Bdnf exon IV, indicating the potential contribution of this epigenetic mechanism to maternal programming by maternal predator odor exposure. These data indicate that maternal predator odor exposure alone is sufficient to induce an altered stress-related phenotype in adulthood, with implications for anti-predator behavior in offspring.

Transcriptome analysis of root response to citrus blight based on the newly assembled Swingle citrumelo draft genome.

PubMed

Zhang, Yunzeng; Barthe, Gary; Grosser, Jude W; Wang, Nian

2016-07-08

Citrus blight is a citrus tree overall decline disease and causes serious losses in the citrus industry worldwide. Although it was described more than one hundred years ago, its causal agent remains unknown and its pathophysiology is not well determined, which hampers our understanding of the disease and design of suitable disease management. In this study, we sequenced and assembled the draft genome for Swingle citrumelo, one important citrus rootstock. The draft genome is approximately 280 Mb, which covers 74 % of the estimated Swingle citrumelo genome and the average coverage is around 15X. The draft genome of Swingle citrumelo enabled us to conduct transcriptome analysis of roots of blight and healthy Swingle citrumelo using RNA-seq. The RNA-seq was reliable as evidenced by the high consistence of RNA-seq analysis and quantitative reverse transcription PCR results (R(2) = 0.966). Comparison of the gene expression profiles between blight and healthy root samples revealed the molecular mechanism underneath the characteristic blight phenotypes including decline, starch accumulation, and drought stress. The JA and ET biosynthesis and signaling pathways showed decreased transcript abundance, whereas SA-mediated defense-related genes showed increased transcript abundance in blight trees, suggesting unclassified biotrophic pathogen was involved in this disease. Overall, the Swingle citrumelo draft genome generated in this study will advance our understanding of plant biology and contribute to the citrus breeding. Transcriptome analysis of blight and healthy trees deepened our understanding of the pathophysiology of citrus blight.

Digital gene expression for non-model organisms

PubMed Central

Hong, Lewis Z.; Li, Jun; Schmidt-Küntzel, Anne; Warren, Wesley C.; Barsh, Gregory S.

2011-01-01

Next-generation sequencing technologies offer new approaches for global measurements of gene expression but are mostly limited to organisms for which a high-quality assembled reference genome sequence is available. We present a method for gene expression profiling called EDGE, or EcoP15I-tagged Digital Gene Expression, based on ultra-high-throughput sequencing of 27-bp cDNA fragments that uniquely tag the corresponding gene, thereby allowing direct quantification of transcript abundance. We show that EDGE is capable of assaying for expression in >99% of genes in the genome and achieves saturation after 6–8 million reads. EDGE exhibits very little technical noise, reveals a large (106) dynamic range of gene expression, and is particularly suited for quantification of transcript abundance in non-model organisms where a high-quality annotated genome is not available. In a direct comparison with RNA-seq, both methods provide similar assessments of relative transcript abundance, but EDGE does better at detecting gene expression differences for poorly expressed genes and does not exhibit transcript length bias. Applying EDGE to laboratory mice, we show that a loss-of-function mutation in the melanocortin 1 receptor (Mc1r), recognized as a Mendelian determinant of yellow hair color in many different mammals, also causes reduced expression of genes involved in the interferon response. To illustrate the application of EDGE to a non-model organism, we examine skin biopsy samples from a cheetah (Acinonyx jubatus) and identify genes likely to control differences in the color of spotted versus non-spotted regions. PMID:21844123

Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

PubMed Central

Dupont, Chris L; McCrow, John P; Valas, Ruben; Moustafa, Ahmed; Walworth, Nathan; Goodenough, Ursula; Roth, Robyn; Hogle, Shane L; Bai, Jing; Johnson, Zackary I; Mann, Elizabeth; Palenik, Brian; Barbeau, Katherine A; Craig Venter, J; Allen, Andrew E

2015-01-01

Transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM. PMID:25333462

The Effects of Royal Jelly on Fitness Traits and Gene Expression in Drosophila melanogaster

PubMed Central

Shorter, John R.; Geisz, Matthew; Özsoy, Ergi; Magwire, Michael M.; Carbone, Mary Anna; Mackay, Trudy F. C.

2015-01-01

Royal Jelly (RJ) is a product made by honey bee workers and is required for queen differentiation and accompanying changes in queen body size, development time, lifespan and reproductive output relative to workers. Previous studies have reported similar changes in Drosophila melanogaster in response to RJ. Here, we quantified viability, development time, body size, productivity, lifespan and genome wide transcript abundance of D. melanogaster reared on standard culture medium supplemented with increasing concentrations of RJ. We found that lower concentrations of RJ do induce significant differences in body size in both sexes; higher concentrations reduce size, increase mortality, shorten lifespan and reduce productivity. Increased concentrations of RJ also consistently lengthened development time in both sexes. RJ is associated with changes in expression of 1,581 probe sets assessed using Affymetrix Drosophila 2.0 microarrays, which were enriched for genes associated with metabolism and amino acid degradation. The transcriptional changes are consistent with alterations in cellular processes to cope with excess nutrients provided by RJ, including biosynthesis and detoxification, which might contribute to accelerated senescence and reduced lifespan. PMID:26226016

A sunflower WRKY transcription factor stimulates the mobilization of seed-stored reserves during germination and post-germination growth.

PubMed

Raineri, Jesica; Hartman, Matías D; Chan, Raquel L; Iglesias, Alberto A; Ribichich, Karina F

2016-09-01

The sunflower transcription factor HaWRKY10 stimulates reserves mobilization in Arabidopsis. Gene expression and enzymes activity assays indicated that lipolysis and gluconeogenesis were increased. Microarray results suggested a parallelism in sunflower. Germinating oilseeds converts stored lipids into sugars, and thereafter in metabolic energy that is used in seedling growth and establishment. During germination, the induced lipolysis linked to the glyoxylate pathway and gluconeogenesis produces sucrose, which is then transported to the embryo and driven through catabolic routes. Herein, we report that the sunflower transcription factor HaWRKY10 regulates carbon partitioning by reducing carbohydrate catabolism and increasing lipolysis and gluconeogenesis. HaWRKY10 was regulated by abscisic acid and gibberellins in the embryo leaves 48 h after seed imbibition and highly expressed during sunflower seed germination and seedling growth, concomitantly with lipid mobilization. Sunflower leaf disks overexpressing HaWRKY10 showed repressed expression of genes related to sucrose cleavage and glycolysis compared with controls. Moreover, HaWRKY10 constitutive expression in Arabidopsis seeds produced higher decrease in lipid reserves, whereas starch and sucrose were more preserved compared with wild type. Gene transcripts abundance and enzyme activities involved in stored lipid mobilization and gluconeogenesis increased more in transgenic than in wild type seeds 36 h after imbibition, whereas the negative regulator of lipid mobilization, ABI4, was repressed. Altogether, the results point out a functional parallelism between tissues and plant species, and reveal HaWRKY10 as a positive regulator of storage reserve mobilization in sunflower.

Mesomere-derived glutamate decarboxylase-expressing blastocoelar mesenchyme cells of sea urchin larvae

PubMed Central

Katow, Hideki; Katow, Tomoko; Abe, Kouki; Ooka, Shioh; Kiyomoto, Masato; Hamanaka, Gen

2014-01-01

Summary The ontogenetic origin of blastocoelar glutamate decarboxylase (GAD)-expressing cells (GADCs) in larvae of the sea urchin Hemicentrotus pulcherrimus was elucidated. Whole-mount in situ hybridisation (WISH) detected transcription of the gene that encodes GAD in H. pulcherrimus (Hp-gad) in unfertilised eggs and all blastomeres in morulae. However, at and after the swimming blastula stage, the transcript accumulation was particularly prominent in clumps of ectodermal cells throughout the embryonic surface. During the gastrula stage, the transcripts also accumulated in the endomesoderm and certain blastocoelar cells. Consistent with the increasing number of Hp-gad transcribing cells, immunoblot analysis indicated that the relative abundance of Hp-Gad increased considerably from the early gastrula stage until the prism stage. The expression pattern of GADCs determined by immunohistochemistry was identical to the pattern of Hp-gad transcript accumulation determined using WISH. In early gastrulae, GADCs formed blastocoelar cell aggregates around the blastopore with primary mesenchyme cells. The increase in the number of blastocoelar GADCs was inversely proportional to the number of ectodermal GADCs ranging from a few percent of total GADCs in early gastrulae to 80% in late prism larvae; this depended on ingression of ectodermal GADCs into the blastocoel. Some of the blastocoelar GADCs were fluorescein-positive in the larvae that developed from the 16-cell stage chimeric embryos; these comprised fluorescein-labeled mesomeres and unlabelled macromeres and micromeres. Our finding indicates that some of the blastocoelar GADCs are derived from the mesomeres and thus they are the new group of mesenchyme cells, the tertiary mesenchyme cells. PMID:24357228

ß-catenin, a transcription factor activated by canonical Wnt signaling, is expressed in sensory neurons of calves latently infected with bovine herpesvirus 1

USDA-ARS?s Scientific Manuscript database

Like many a-herpesvirinae subfamily members, bovine herpes virus 1 (BoHV-1) expresses an abundant transcript in latently infected sensory neurons: the latency-related (LR) RNA. LR-RNA encodes a protein (ORF2) that inhibits apoptosis, interacts with Notch family members, interferes with Notch mediate...

A knotted1-like homeobox protein regulates abscission in tomato by modulating the auxin pathway

USDA-ARS?s Scientific Manuscript database

KD1, a gene encoding a KNOTTED1-LIKE HOMEOBOX transcription factor is known to be involved, in tomato, in ontogeny of the compound leaf. KD1 is also highly expressed in both leaf and flower abscission zones. Reducing abundance of transcripts of this gene in tomato, using both virus induced gene sile...

Transcriptional profiling of immune-related genes in Pacific white shrimp (Litopenaeus vannamei) during ontogenesis.

PubMed

Quispe, Ruth L; Justino, Emily B; Vieira, Felipe N; Jaramillo, Michael L; Rosa, Rafael D; Perazzolo, Luciane M

2016-11-01

We have performed here a gene expression analysis to determine the developmental stage at the main genes involved in crustacean immune response begin to be expressed and their changes in mRNA abundance during shrimp development. By using a quantitative PCR-based approach, we have measured the mRNA abundance of 24 immune-related genes from different functional categories in twelve developmental stages ranging from fertilized eggs to larval and postlarval stages and also in juveniles. We showed for the first time that the main genes from the RNAi-based post-transcriptional pathway involved in shrimp antiviral immunity are transcribed in all developmental stages, but exhibit a diverse pattern of gene expression during shrimp ontogenesis. On the other hand, hemocyte-expressed genes mainly involved in antimicrobial defenses appeared to be transcribed in larval stages, indicating that hematopoiesis initiates early in development. Moreover, transcript levels of some genes were early detected in fertilized eggs at 0-4 h post-spawning, suggesting a maternal contribution of immune-related transcripts to shrimp progeny. Altogether, our results provide important clues regarding the ontogenesis of hemocytes as well the establishment of antiviral and antimicrobial defenses in shrimp. Copyright © 2016 Elsevier Ltd. All rights reserved.

SAGE analysis of early oogenesis in the silkworm, Bombyx mori.

PubMed

Funaguma, Shunsuke; Hashimoto, Shin-ichi; Suzuki, Yutaka; Omuro, Naoko; Sugano, Sumio; Mita, Kazuei; Katsuma, Susumu; Shimada, Toru

2007-02-01

To identify genes involved in the differentiation of Bombyx cystoblast, we constructed two 3' long serial analysis of gene expression (Long SAGE) libraries from stage 1-3 or stage 2-3 egg chambers and compared their gene expression profiles. In both libraries, the most frequent tags were derived from the same novel transcript. The transcript does not have any open reading frame capable of encoding a protein with over 100 amino acids in length. RNA blot analysis revealed that this transcript is specifically and abundantly expressed in the Bombyx ovary, mainly the germ line cells in the ovarioles. These results suggest that Bombyx oogenesis may be regulated by a previously unidentified non-coding RNA. Comparison of the gene expression profiles between the stage 1-3 and stage 2-3 egg chamber libraries revealed that 272 tags were significantly more abundant in stage 1-3 egg chambers (p<0.05 and at least two-fold change) than in library 2. Among the differentially expressed transcripts were the sequences that correspond to ATP synthase subunit d (3.1-fold enriched) and ATP synthase coupling factor 6 (9.1-fold enriched), suggesting that they are involved in regulation of cell cycle of cystocytes.

Independent replication of mitochondrial genes supports the transcriptional program in developing fiber cells of cotton (Gossypium hirsutum L.).

PubMed

Thyssen, Gregory N; Song, Xianliang; Naoumkina, Marina; Kim, Hee-Jin; Fang, David D

2014-07-01

The mitochondrial genomes of flowering plants exist both as a "master circle" chromosome and as numerous subgenomic sublimons that are generated by intramolecular recombination. Differential stability or replication of these sublimons allows individual mitochondrial gene copy numbers to vary independently between different cell types and developmental stages. Our objective was to determine the relationship between mitochondrial gene copy number and transcript abundance in the elongating fiber cells of Upland cotton (Gossypium hirsutum L.). We compared RNA and DNA from cotton fiber cells at five developmental time points from early elongation through secondary cell wall thickening from the Ligon-lintless 2 (Li2) short fiber mutant and its wild type near isogenic line (NIL) DP5690. Mitochondrial gene copy number decreased from 3 to 8-DPA in the developing cotton fiber cells while transcript levels remained low. As secondary cell wall biosynthesis began in developing fibers, the expression levels and copy numbers of mitochondrial genes involved in energy production and respiration were up-regulated in wild type cotton DP5690. However, the short fiber mutant Li2, failed to increase expression of these genes, which include three subunits of ATP synthase, atp1, atp8 and atp9 and two cytochrome genes cox1 and cob. At the same time, Li2 failed to increase the copy numbers of these highly expressed genes. Surprisingly, we found that when mitochondrial genes were highly transcribed, they also had very high copy numbers. This observation suggests that in developing cotton fibers, increased mitochondrial sublimon replication may support increases in gene transcription. Published by Elsevier B.V.

Alterations in the neuropeptide galanin system in major depressive disorder involve levels of transcripts, methylation, and peptide

PubMed Central

Barde, Swapnali; Rüegg, Joelle; Prud’homme, Josée; Ekström, Tomas J.; Palkovits, Miklos; Turecki, Gustavo; Bagdy, Gyorgy; Ihnatko, Robert; Theodorsson, Elvar; Juhasz, Gabriella; Diaz-Heijtz, Rochellys; Mechawar, Naguib; Hökfelt, Tomas G. M.

2016-01-01

Major depressive disorder (MDD) is a substantial burden to patients, families, and society, but many patients cannot be treated adequately. Rodent experiments suggest that the neuropeptide galanin (GAL) and its three G protein-coupled receptors, GAL1–3, are involved in mood regulation. To explore the translational potential of these results, we assessed the transcript levels (by quantitative PCR), DNA methylation status (by bisulfite pyrosequencing), and GAL peptide by RIA of the GAL system in postmortem brains from depressed persons who had committed suicide and controls. Transcripts for all four members were detected and showed marked regional variations, GAL and galanin receptor 1 (GALR1) being most abundant. Striking increases in GAL and GALR3 mRNA levels, especially in the noradrenergic locus coeruleus and the dorsal raphe nucleus, in parallel with decreased DNA methylation, were found in both male and female suicide subjects as compared with controls. In contrast, GAL and GALR3 transcript levels were decreased, GALR1 was increased, and DNA methylation was increased in the dorsolateral prefrontal cortex of male suicide subjects, however, there were no changes in the anterior cingulate cortex. Thus, GAL and its receptor GALR3 are differentially methylated and expressed in brains of MDD subjects in a region- and sex-specific manner. Such an epigenetic modification in GALR3, a hyperpolarizing receptor, might contribute to the dysregulation of noradrenergic and serotonergic neurons implicated in the pathogenesis of MDD. Thus, one may speculate that a GAL3 antagonist could have antidepressant properties by disinhibiting the firing of these neurons, resulting in increased release of noradrenaline and serotonin in forebrain areas involved in mood regulation. PMID:27940914

The influence of postnatal nutrition on reproductive tract and endometrial gland development in dairy calves.

PubMed

Wilson, Meghan L; McCoski, Sarah R; Geiger, Adam J; Akers, R Michael; Johnson, Sally E; Ealy, Alan D

2017-04-01

Uterine gland development occurs after birth in cattle and other mammals. The timeline of gland development has been described in various species, but little is known about how postnatal diet influences uterine gland development. This is especially concerning in dairy heifers, where a variety of milk replacer and whole milk nutrition options exist. Little work also exists in cattle to describe how early exposure to steroids influences reproductive tract and uterine gland development. The objective of this work was to determine the effects of early postnatal plane of nutrition and estrogen supplementation on uterine gland development in calves. In both studies, Holstein heifer calves were assigned to restricted milk replacer (R-MR) or enhanced milk replacer (EH-MR) diets. In study 1, calves (R-MR, n = 6; EH-MR, n = 5) were euthanized at 8 wk. In study 2, calves were weaned at 8 wk and administered estradiol (R-MR, n = 6; EH-MR, n = 6) or placebo (R-MR, n = 6; EH-MR, n = 5) for an additional 14 d before euthanasia. Average daily gain and final body weight was greater in both studies in heifers fed the enhanced diet. At 8 wk, EH-MR calves had a greater number of glands and a smaller average gland size, but total gland area was not different from the R-MR group. At 10 wk, uterine gland number and size were not affected by diet or estrogen. Expression profiles of several paracrine mediators of gland development were examined. Increases in transcript abundance for IGF1 and IGFBP3 and a decrease in abundance of WNT7A were detected in calves fed the enhanced diet at 8 wk of age. Plane of nutrition did not affect transcript profiles at 10 wk of age, but estradiol supplementation decreased MET and WNT7A transcript abundance. To conclude, heifer calves on a restricted diet exhibited a uterine morphology and transcript profile suggestive of delayed uterine gland development. These changes appear to be corrected by wk 10 of life. Also, this work provides evidence supporting the contention that early estradiol exposure has detrimental effects on uterine gene expression. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Expression of the Arabidopsis thaliana BBX32 gene in soybean increases grain yield.

PubMed

Preuss, Sasha B; Meister, Robert; Xu, Qingzhang; Urwin, Carl P; Tripodi, Federico A; Screen, Steven E; Anil, Veena S; Zhu, Shuquan; Morrell, James A; Liu, Grace; Ratcliffe, Oliver J; Reuber, T Lynne; Khanna, Rajnish; Goldman, Barry S; Bell, Erin; Ziegler, Todd E; McClerren, Amanda L; Ruff, Thomas G; Petracek, Marie E

2012-01-01

Crop yield is a highly complex quantitative trait. Historically, successful breeding for improved grain yield has led to crop plants with improved source capacity, altered plant architecture, and increased resistance to abiotic and biotic stresses. To date, transgenic approaches towards improving crop grain yield have primarily focused on protecting plants from herbicide, insects, or disease. In contrast, we have focused on identifying genes that, when expressed in soybean, improve the intrinsic ability of the plant to yield more. Through the large scale screening of candidate genes in transgenic soybean, we identified an Arabidopsis thaliana B-box domain gene (AtBBX32) that significantly increases soybean grain yield year after year in multiple transgenic events in multi-location field trials. In order to understand the underlying physiological changes that are associated with increased yield in transgenic soybean, we examined phenotypic differences in two AtBBX32-expressing lines and found increases in plant height and node, flower, pod, and seed number. We propose that these phenotypic changes are likely the result of changes in the timing of reproductive development in transgenic soybean that lead to the increased duration of the pod and seed development period. Consistent with the role of BBX32 in A. thaliana in regulating light signaling, we show that the constitutive expression of AtBBX32 in soybean alters the abundance of a subset of gene transcripts in the early morning hours. In particular, AtBBX32 alters transcript levels of the soybean clock genes GmTOC1 and LHY-CCA1-like2 (GmLCL2). We propose that through the expression of AtBBX32 and modulation of the abundance of circadian clock genes during the transition from dark to light, the timing of critical phases of reproductive development are altered. These findings demonstrate a specific role for AtBBX32 in modulating soybean development, and demonstrate the validity of expressing single genes in crops to deliver increased agricultural productivity.

Identification of small RNAs abundant in Burkholderia cenocepacia biofilms reveal putative regulators with a potential role in carbon and iron metabolism.

PubMed

Sass, Andrea; Kiekens, Sanne; Coenye, Tom

2017-11-15

Small RNAs play a regulatory role in many central metabolic processes of bacteria, as well as in developmental processes such as biofilm formation. Small RNAs of Burkholderia cenocepacia, an opportunistic pathogenic beta-proteobacterium, are to date not well characterised. To address that, we performed genome-wide transcriptome structure analysis of biofilm grown B. cenocepacia J2315. 41 unannotated short transcripts were identified in intergenic regions of the B. cenocepacia genome. 15 of these short transcripts, highly abundant in biofilms, widely conserved in Burkholderia sp. and without known function, were selected for in-depth analysis. Expression profiling showed that most of these sRNAs are more abundant in biofilms than in planktonic cultures. Many are also highly abundant in cells grown in minimal media, suggesting they are involved in adaptation to nutrient limitation and growth arrest. Their computationally predicted targets include a high proportion of genes involved in carbon metabolism. Expression and target genes of one sRNA suggest a potential role in regulating iron homoeostasis. The strategy used for this study to detect sRNAs expressed in B. cenocepacia biofilms has successfully identified sRNAs with a regulatory function.

Unique cell-type-specific patterns of DNA methylation in the root meristem.

PubMed

Kawakatsu, Taiji; Stuart, Tim; Valdes, Manuel; Breakfield, Natalie; Schmitz, Robert J; Nery, Joseph R; Urich, Mark A; Han, Xinwei; Lister, Ryan; Benfey, Philip N; Ecker, Joseph R

2016-04-29

DNA methylation is an epigenetic modification that differs between plant organs and tissues, but the extent of variation between cell types is not known. Here, we report single-base-resolution whole-genome DNA methylomes, mRNA transcriptomes and small RNA transcriptomes for six cell populations covering the major cell types of the Arabidopsis root meristem. We identify widespread cell-type-specific patterns of DNA methylation, especially in the CHH sequence context, where H is A, C or T. The genome of the columella root cap is the most highly methylated Arabidopsis cell characterized so far. It is hypermethylated within transposable elements (TEs), accompanied by increased abundance of transcripts encoding RNA-directed DNA methylation (RdDM) pathway components and 24-nt small RNAs (smRNAs). The absence of the nucleosome remodeller DECREASED DNA METHYLATION 1 (DDM1), required for maintenance of DNA methylation, and low abundance of histone transcripts involved in heterochromatin formation suggests that a loss of heterochromatin may occur in the columella, thus allowing access of RdDM factors to the whole genome, and producing an excess of 24-nt smRNAs in this tissue. Together, these maps provide new insights into the epigenomic diversity that exists between distinct plant somatic cell types.

Determination of in vivo RNA kinetics using RATE-seq.

PubMed

Neymotin, Benjamin; Athanasiadou, Rodoniki; Gresham, David

2014-10-01

The abundance of a transcript is determined by its rate of synthesis and its rate of degradation; however, global methods for quantifying RNA abundance cannot distinguish variation in these two processes. Here, we introduce RNA approach to equilibrium sequencing (RATE-seq), which uses in vivo metabolic labeling of RNA and approach to equilibrium kinetics, to determine absolute RNA degradation and synthesis rates. RATE-seq does not disturb cellular physiology, uses straightforward normalization with exogenous spike-ins, and can be readily adapted for studies in most organisms. We demonstrate the use of RATE-seq to estimate genome-wide kinetic parameters for coding and noncoding transcripts in Saccharomyces cerevisiae. © 2014 Neymotin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Or mutation leads to photo-oxidative stress responses in cauliflower (Brassica oleracea) seedlings during de-etiolation.

PubMed

Men, Xiao; Dong, Kang

2013-11-01

The Orange (Or) gene is a gene mutation that can increase carotenoid content in plant tissues normally devoid of pigments. It affects plastid division and is involved in the differentiation of proplastids or non-colored plastids into chromoplasts. In this study, the de-etiolation process of the wild type (WT) cauliflower (Brassica oleracea L. var. botrytis) and Or mutant seedlings was investigated. We analyzed pigment content, plastid development, transcript abundance and protein levels of genes involved in the de-etiolation process. The results showed that Or can increase the carotenoid content in green tissues, although not as effectively as in non-green tissues, and this effect might be caused by the changes in biosynthetic pathway genes at both transcriptional and post-transcriptional levels. There was no significant difference in the plastid development process between the two lines. However, the increased content of antheraxanthin and anthocyanin, and higher expression levels of violaxanthin de-epoxidase gene (VDE) suggested a stress situation leading to photoinhibition and enhanced photoprotection in the Or mutant. The up-regulated expression levels of the reactive oxygen species (ROS)-induced genes, ZAT10 for salt tolerance zinc finger protein and ASCORBATE PEROXIDASE2 (APX2), suggested the existence of photo-oxidative stress in the Or mutant. In summary, abovementioned findings provide additional insight into the functions of the Or gene in different tissues and at different developmental stages.

Salt stress induces the formation of a novel type of 'pressure wood' in two Populus species.

PubMed

Janz, Dennis; Lautner, Silke; Wildhagen, Henning; Behnke, Katja; Schnitzler, Jörg-Peter; Rennenberg, Heinz; Fromm, Jörg; Polle, Andrea

2012-04-01

• Salinity causes osmotic stress and limits biomass production of plants. The goal of this study was to investigate mechanisms underlying hydraulic adaptation to salinity. • Anatomical, ecophysiological and transcriptional responses to salinity were investigated in the xylem of a salt-sensitive (Populus × canescens) and a salt-tolerant species (Populus euphratica). • Moderate salt stress, which suppressed but did not abolish photosynthesis and radial growth in P. × canescens, resulted in hydraulic adaptation by increased vessel frequencies and decreased vessel lumina. Transcript abundances of a suite of genes (FLA, COB-like, BAM, XET, etc.) previously shown to be activated during tension wood formation, were collectively suppressed in developing xylem, whereas those for stress and defense-related genes increased. A subset of cell wall-related genes was also suppressed in salt-exposed P. euphratica, although this species largely excluded sodium and showed no anatomical alterations. Salt exposure influenced cell wall composition involving increases in the lignin : carbohydrate ratio in both species. • In conclusion, hydraulic stress adaptation involves cell wall modifications reciprocal to tension wood formation that result in the formation of a novel type of reaction wood in upright stems named 'pressure wood'. Our data suggest that transcriptional co-regulation of a core set of genes determines reaction wood composition. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons.

PubMed

García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E

2016-05-05

We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma

PubMed Central

Gilmore, Sarah A.; Voorhies, Mark; Gebhart, Dana; Sil, Anita

2015-01-01

Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5’ leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature. PMID:26177267

Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma.

PubMed

Gilmore, Sarah A; Voorhies, Mark; Gebhart, Dana; Sil, Anita

2015-07-01

Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5' leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature.

SWATH-MS Quantitative Analysis of Proteins in the Rice Inferior and Superior Spikelets during Grain Filling

PubMed Central

Zhu, Fu-Yuan; Chen, Mo-Xian; Su, Yu-Wen; Xu, Xuezhong; Ye, Neng-Hui; Cao, Yun-Ying; Lin, Sheng; Liu, Tie-Yuan; Li, Hao-Xuan; Wang, Guan-Qun; Jin, Yu; Gu, Yong-Hai; Chan, Wai-Lung; Lo, Clive; Peng, Xinxiang; Zhu, Guohui; Zhang, Jianhua

2016-01-01

Modern rice cultivars have large panicle but their yield potential is often not fully achieved due to poor grain-filling of late-flowering inferior spikelets (IS). Our earlier work suggested a broad transcriptional reprogramming during grain filling and showed a difference in gene expression between IS and earlier-flowering superior spikelets (SS). However, the links between the abundances of transcripts and their corresponding proteins are unclear. In this study, a SWATH-MS (sequential window acquisition of all theoretical spectra-mass spectrometry) -based quantitative proteomic analysis has been applied to investigate SS and IS proteomes. A total of 304 proteins of widely differing functionality were observed to be differentially expressed between IS and SS. Detailed gene ontology analysis indicated that several biological processes including photosynthesis, protein metabolism, and energy metabolism are differentially regulated. Further correlation analysis revealed that abundances of most of the differentially expressed proteins are not correlated to the respective transcript levels, indicating that an extra layer of gene regulation which may exist during rice grain filling. Our findings raised an intriguing possibility that these candidate proteins may be crucial in determining the poor grain-filling of IS. Therefore, we hypothesize that the regulation of proteome changes not only occurs at the transcriptional, but also at the post-transcriptional level, during grain filling in rice. PMID:28066479

Expression analysis and identification of antimicrobial peptide transcripts from six North American frog species

USGS Publications Warehouse

Robertson, Laura S.; Fellers, Gary M.; Marranca, Jamie Marie; Kleeman, Patrick M.

2013-01-01

Frogs secrete antimicrobial peptides onto their skin. We describe an assay to preserve and analyze antimicrobial peptide transcripts from field-collected skin secretions that will complement existing methods for peptide analysis. We collected skin secretions from 4 North American species in the field in California and 2 species in the laboratory. Most frogs appeared healthy after release; however, Rana boylii in the Sierra Nevada foothills, but not the Coast Range, showed signs of morbidity and 2 died after handling. The amount of total RNA extracted from skin secretions was higher in R. boylii and R. sierrae compared to R. draytonii, and much higher compared to Pseudacris regilla. Interspecies variation in amount of RNA extracted was not explained by size, but for P. regilla it depended upon collection site and date. RNA extracted from skin secretions from frogs handled with bare hands had poor quality compared to frogs handled with gloves or plastic bags. Thirty-four putative antimicrobial peptide precursor transcripts were identified. This study demonstrates that RNA extracted from skin secretions collected in the field is of high quality suitable for use in sequencing or quantitative PCR (qPCR). However, some species do not secrete profusely, resulting in very little extracted RNA. The ability to measure transcript abundance of antimicrobial peptides in field-collected skin secretions complements proteomic analyses and may provide insight into transcriptional mechanisms that could affect peptide abundance.

Experimental incubations elicit profound changes in community transcription in OMZ bacterioplankton.

PubMed

Stewart, Frank J; Dalsgaard, Tage; Young, Curtis R; Thamdrup, Bo; Revsbech, Niels Peter; Ulloa, Osvaldo; Canfield, Don E; Delong, Edward F

2012-01-01

Sequencing of microbial community RNA (metatranscriptome) is a useful approach for assessing gene expression in microorganisms from the natural environment. This method has revealed transcriptional patterns in situ, but can also be used to detect transcriptional cascades in microcosms following experimental perturbation. Unambiguously identifying differential transcription between control and experimental treatments requires constraining effects that are simply due to sampling and bottle enclosure. These effects remain largely uncharacterized for "challenging" microbial samples, such as those from anoxic regions that require special handling to maintain in situ conditions. Here, we demonstrate substantial changes in microbial transcription induced by sample collection and incubation in experimental bioreactors. Microbial communities were sampled from the water column of a marine oxygen minimum zone by a pump system that introduced minimal oxygen contamination and subsequently incubated in bioreactors under near in situ oxygen and temperature conditions. Relative to the source water, experimental samples became dominated by transcripts suggestive of cell stress, including chaperone, protease, and RNA degradation genes from diverse taxa, with strong representation from SAR11-like alphaproteobacteria. In tandem, transcripts matching facultative anaerobic gammaproteobacteria of the Alteromonadales (e.g., Colwellia) increased 4-13 fold up to 43% of coding transcripts, and encoded a diverse gene set suggestive of protein synthesis and cell growth. We interpret these patterns as taxon-specific responses to combined environmental changes in the bioreactors, including shifts in substrate or oxygen availability, and minor temperature and pressure changes during sampling with the pump system. Whether such changes confound analysis of transcriptional patterns may vary based on the design of the experiment, the taxonomic composition of the source community, and on the metabolic linkages between community members. These data highlight the impressive capacity for transcriptional changes within complex microbial communities, underscoring the need for caution when inferring in situ metabolism based on transcript abundances in experimental incubations.

Experimental Incubations Elicit Profound Changes in Community Transcription in OMZ Bacterioplankton

PubMed Central

Stewart, Frank J.; Dalsgaard, Tage; Young, Curtis R.; Thamdrup, Bo; Revsbech, Niels Peter; Ulloa, Osvaldo; Canfield, Don E.; DeLong, Edward F.

2012-01-01

Sequencing of microbial community RNA (metatranscriptome) is a useful approach for assessing gene expression in microorganisms from the natural environment. This method has revealed transcriptional patterns in situ, but can also be used to detect transcriptional cascades in microcosms following experimental perturbation. Unambiguously identifying differential transcription between control and experimental treatments requires constraining effects that are simply due to sampling and bottle enclosure. These effects remain largely uncharacterized for “challenging” microbial samples, such as those from anoxic regions that require special handling to maintain in situ conditions. Here, we demonstrate substantial changes in microbial transcription induced by sample collection and incubation in experimental bioreactors. Microbial communities were sampled from the water column of a marine oxygen minimum zone by a pump system that introduced minimal oxygen contamination and subsequently incubated in bioreactors under near in situ oxygen and temperature conditions. Relative to the source water, experimental samples became dominated by transcripts suggestive of cell stress, including chaperone, protease, and RNA degradation genes from diverse taxa, with strong representation from SAR11-like alphaproteobacteria. In tandem, transcripts matching facultative anaerobic gammaproteobacteria of the Alteromonadales (e.g., Colwellia) increased 4–13 fold up to 43% of coding transcripts, and encoded a diverse gene set suggestive of protein synthesis and cell growth. We interpret these patterns as taxon-specific responses to combined environmental changes in the bioreactors, including shifts in substrate or oxygen availability, and minor temperature and pressure changes during sampling with the pump system. Whether such changes confound analysis of transcriptional patterns may vary based on the design of the experiment, the taxonomic composition of the source community, and on the metabolic linkages between community members. These data highlight the impressive capacity for transcriptional changes within complex microbial communities, underscoring the need for caution when inferring in situ metabolism based on transcript abundances in experimental incubations. PMID:22615914

Comparative expression profiling in grape (Vitis vinifera) berries derived from frequency analysis of ESTs and MPSS signatures.

PubMed

Iandolino, Alberto; Nobuta, Kan; da Silva, Francisco Goes; Cook, Douglas R; Meyers, Blake C

2008-05-12

Vitis vinifera (V. vinifera) is the primary grape species cultivated for wine production, with an industry valued annually in the billions of dollars worldwide. In order to sustain and increase grape production, it is necessary to understand the genetic makeup of grape species. Here we performed mRNA profiling using Massively Parallel Signature Sequencing (MPSS) and combined it with available Expressed Sequence Tag (EST) data. These tag-based technologies, which do not require a priori knowledge of genomic sequence, are well-suited for transcriptional profiling. The sequence depth of MPSS allowed us to capture and quantify almost all the transcripts at a specific stage in the development of the grape berry. The number and relative abundance of transcripts from stage II grape berries was defined using Massively Parallel Signature Sequencing (MPSS). A total of 2,635,293 17-base and 2,259,286 20-base signatures were obtained, representing at least 30,737 and 26,878 distinct sequences. The average normalized abundance per signature was approximately 49 TPM (Transcripts Per Million). Comparisons of the MPSS signatures with available Vitis species' ESTs and a unigene set demonstrated that 6,430 distinct contigs and 2,190 singletons have a perfect match to at least one MPSS signature. Among the matched sequences, ESTs were identified from tissues other than berries or from berries at different developmental stages. Additional MPSS signatures not matching to known grape ESTs can extend our knowledge of the V. vinifera transcriptome, particularly when these data are used to assist in annotation of whole genome sequences from Vitis vinifera. The MPSS data presented here not only achieved a higher level of saturation than previous EST based analyses, but in doing so, expand the known set of transcripts of grape berries during the unique stage in development that immediately precedes the onset of ripening. The MPSS dataset also revealed evidence of antisense expression not previously reported in grapes but comparable to that reported in other plant species. Finally, we developed a novel web-based, public resource for utilization of the grape MPSS data [1].

2-aminoimidazoles potentiate ß-lactam antimicrobial activity against Mycobacterium tuberculosis by reducing ß-lactamase secretion and increasing cell envelope permeability

PubMed Central

Obregón-Henao, Andrés; Ackart, David F.; Podell, Brendan K.; Belardinelli, Juan M.; Jackson, Mary; Nguyen, Tuan V.; Blackledge, Meghan S.; Melander, Roberta J.; Melander, Christian; Johnson, Benjamin K.; Abramovitch, Robert B.

2017-01-01

There is an urgent need to develop new drug treatment strategies to control the global spread of drug-sensitive and multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis). The ß-lactam class of antibiotics is among the safest and most widely prescribed antibiotics, but they are not effective against M. tuberculosis due to intrinsic resistance. This study shows that 2-aminoimidazole (2-AI)-based small molecules potentiate ß-lactam antibiotics against M. tuberculosis. Active 2-AI compounds significantly reduced the minimal inhibitory and bactericidal concentrations of ß-lactams by increasing M. tuberculosis cell envelope permeability and decreasing protein secretion including ß-lactamase. Metabolic labeling and transcriptional profiling experiments revealed that 2-AI compounds impair mycolic acid biosynthesis, export and linkage to the mycobacterial envelope, counteracting an important defense mechanism reducing permeability to external agents. Additionally, other important constituents of the M. tuberculosis outer membrane including sulfolipid-1 and polyacyltrehalose were also less abundant in 2-AI treated bacilli. As a consequence of 2-AI treatment, M. tuberculosis displayed increased sensitivity to SDS, increased permeability to nucleic acid staining dyes, and rapid binding of cell wall targeting antibiotics. Transcriptional profiling analysis further confirmed that 2-AI induces transcriptional regulators associated with cell envelope stress. 2-AI based small molecules potentiate the antimicrobial activity of ß-lactams by a mechanism that is distinct from specific inhibitors of ß-lactamase activity and therefore may have value as an adjunctive anti-TB treatment. PMID:28749949

Identification of a novel long noncoding RNA that promotes osteoblast differentiation.

PubMed

Nardocci, Gino; Carrasco, Margarita E; Acevedo, Elvis; Hodar, Christian; Meneses, Claudio; Montecino, Martín

2018-05-28

Long noncoding RNAs (lncRNAs) are a heterogeneous class of transcripts, longer than 200 nucleotides, 5'-capped, polyadenylated, and poorly conserved among mammalian species. Several studies have shown the contribution of lncRNAs to different cellular processes, including regulation of the chromatin structure, control of messenger RNA translation, regulation of gene transcription, regulation of embryonic pluripotency, and differentiation. Although limited numbers of functional lncRNAs have been identified so far, the immense regulatory potential of these RNAs is already evident, indicating that a functional characterization of lncRNAs is needed. In this study, mouse preosteoblastic cells were induced to differentiate into osteoblasts. At 3 sequential differentiation stages, total RNA was isolated and libraries were constructed for Illumina sequencing. The resulting sequences were aligned and transcript abundances were determined. New lncRNA candidates that displayed differential expression patterns during osteoblast differentiation were identified by combining bioinformatics and reverse transcription polymerase chain reaction analyses. Among these, lncRNA-1 that exhibited increased expression during osteogenesis and was downregulated during myogenesis. Importantly, knockdown of lncRNA-1 expression in primary mouse preosteoblasts was found to inhibit osteogenic differentiation, reflected by a reduced transcription of the Runx2/p57 and Sp7 bone master genes. Together, our results indicate that lncRNA-1 represents a new regulatory RNA that plays a relevant role during the early stages of osteogenesis. © 2018 Wiley Periodicals, Inc.

Decreasing Global Transcript Levels over Time Suggest that Phytoplasma Cells Enter Stationary Phase during Plant and Insect Colonization

PubMed Central

Pacifico, D.; Galetto, L.; Rashidi, M.; Abbà, S.; Palmano, S.; Firrao, G.; Bosco, D.

2015-01-01

To highlight different transcriptional behaviors of the phytoplasma in the plant and animal host, expression of 14 genes of “Candidatus Phytoplasma asteris,” chrysanthemum yellows strain, was investigated at different times following the infection of a plant host (Arabidopsis thaliana) and two insect vector species (Macrosteles quadripunctulatus and Euscelidius variegatus). Target genes were selected among those encoding antigenic membrane proteins, membrane transporters, secreted proteins, and general enzymes. Transcripts were detected for all analyzed genes in the three hosts; in particular, those encoding the antigenic membrane protein Amp, elements of the mechanosensitive channel, and two of the four secreted proteins (SAP54 and TENGU) were highly accumulated, suggesting that they play important roles in phytoplasma physiology during the infection cycle. Most transcripts were present at higher abundance in the plant host than in the insect hosts. Generally, transcript levels of the selected genes decreased significantly during infection of A. thaliana and M. quadripunctulatus but were more constant in E. variegatus. Such decreases may be explained by the fact that only a fraction of the phytoplasma population was transcribing, while the remaining part was aging to a stationary phase. This strategy might improve long-term survival, thereby increasing the likelihood that the pathogen may be acquired by a vector and/or inoculated to a healthy plant. PMID:25636844

Metabolic evolution of Corynebacterium glutamicum for increased production of L-ornithine

PubMed Central

2013-01-01

Background L-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. The commercial applications mean that efficient biotechnological production of L-ornithine has become increasingly necessary. Adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. The evolved strains can be further optimised by metabolic engineering. Thus, metabolic evolution strategy was used for engineering Corynebacterium glutamicum to enhance L-ornithine production. Results A C. glutamicum strain was engineered by using a combination of gene deletions and adaptive evolution with 70 passages of growth-based selection. The metabolically evolved C. glutamicum strain, named ΔAPE6937R42, produced 24.1 g/L of L-ornithine in a 5-L bioreactor. The mechanism used by C. glutamicum ΔAPE6937R42 to produce L-ornithine was investigated by analysing transcriptional levels of select genes and NADPH contents. The upregulation of the transcription levels of genes involved in the upstream pathway of glutamate biosynthesis and the elevated NADPH concentration caused by the upregulation of the transcriptional level of the ppnK gene promoted L-ornithine production in C. glutamicum ΔAPE6937R42. Conclusions The availability of NADPH plays an important role in L-ornithine production in C. glutamicum. Our results demonstrated that the combination of growth-coupled evolution with analysis of transcript abundances provides a strategy to engineer microbial strains for improving production of target compounds. PMID:23725060

GLUT4, GLUT1, and GLUT8 are the dominant GLUT transcripts expressed in the murine left ventricle

PubMed Central

2012-01-01

Background The heart derives energy from a wide variety of substrates including fatty acids, carbohydrates, ketones, and amino acids. The healthy heart generates up to 30% of its ATP from glucose. Under conditions of cardiac injury or stress, the heart relies even more heavily on glucose as a source of fuel. Glucose is transported into the heart by members of the family of facilitative glucose transporters (GLUTs). While research examining the transport of glucose into the heart has primarily focused on the roles of the classical glucose transporters GLUT1 and GLUT4, little is known about the functions of more newly identified GLUT isoforms in the myocardium. Methods In this study the presence and relative RNA message abundance of each of the known GLUT isoforms was determined in left ventricular tissue from two commonly used inbred laboratory mouse strains (C57BL/6J and FVB/NJ) by quantitative real time PCR. Relative message abundance was also determined in GLUT4 null mice and in murine models of dilated and hypertrophic cardiomyopathy. Results GLUT4, GLUT1, and GLUT8 were found to be the most abundant GLUT transcripts in the normal heart, while GLUT3, GLUT10, and GLUT12 are present at relatively lower levels. Assessment of relative GLUT expression in left ventricular myocardium from mice with dilated cardiomyopathy revealed increased expression of GLUT1 with reduced levels of GLUT4, GLUT8, and GLUT12. Compensatory increase in the expression of GLUT12 was observed in genetically altered mice lacking GLUT4. Conclusions Glucose transporter expression varies significantly among murine models of cardiac dysfunction and involves several of the class III GLUT isoforms. Understanding how these more newly identified GLUT isoforms contribute to regulating myocardial glucose transport will enhance our comprehension of the normal physiology and pathophysiology of the heart. PMID:22681646

GLUT4, GLUT1, and GLUT8 are the dominant GLUT transcripts expressed in the murine left ventricle.

PubMed

Aerni-Flessner, Lauren; Abi-Jaoude, Melissa; Koenig, Amanda; Payne, Maria; Hruz, Paul W

2012-06-08

The heart derives energy from a wide variety of substrates including fatty acids, carbohydrates, ketones, and amino acids. The healthy heart generates up to 30% of its ATP from glucose. Under conditions of cardiac injury or stress, the heart relies even more heavily on glucose as a source of fuel. Glucose is transported into the heart by members of the family of facilitative glucose transporters (GLUTs). While research examining the transport of glucose into the heart has primarily focused on the roles of the classical glucose transporters GLUT1 and GLUT4, little is known about the functions of more newly identified GLUT isoforms in the myocardium. In this study the presence and relative RNA message abundance of each of the known GLUT isoforms was determined in left ventricular tissue from two commonly used inbred laboratory mouse strains (C57BL/6J and FVB/NJ) by quantitative real time PCR. Relative message abundance was also determined in GLUT4 null mice and in murine models of dilated and hypertrophic cardiomyopathy. GLUT4, GLUT1, and GLUT8 were found to be the most abundant GLUT transcripts in the normal heart, while GLUT3, GLUT10, and GLUT12 are present at relatively lower levels. Assessment of relative GLUT expression in left ventricular myocardium from mice with dilated cardiomyopathy revealed increased expression of GLUT1 with reduced levels of GLUT4, GLUT8, and GLUT12. Compensatory increase in the expression of GLUT12 was observed in genetically altered mice lacking GLUT4. Glucose transporter expression varies significantly among murine models of cardiac dysfunction and involves several of the class III GLUT isoforms. Understanding how these more newly identified GLUT isoforms contribute to regulating myocardial glucose transport will enhance our comprehension of the normal physiology and pathophysiology of the heart.

Adrenal cortex expression quantitative trait loci in a German Holstein × Charolais cross.

PubMed

Brand, Bodo; Scheinhardt, Markus O; Friedrich, Juliane; Zimmer, Daisy; Reinsch, Norbert; Ponsuksili, Siriluck; Schwerin, Manfred; Ziegler, Andreas

2016-10-06

The importance of the adrenal gland in regard to lactation and reproduction in cattle has been recognized early. Caused by interest in animal welfare and the impact of stress on economically important traits in farm animals the adrenal gland and its function within the stress response is of increasing interest. However, the molecular mechanisms and pathways involved in stress-related effects on economically important traits in farm animals are not fully understood. Gene expression is an important mechanism underlying complex traits, and genetic variants affecting the transcript abundance are thought to influence the manifestation of an expressed phenotype. We therefore investigated the genetic background of adrenocortical gene expression by applying an adaptive linear rank test to identify genome-wide expression quantitative trait loci (eQTL) for adrenal cortex transcripts in cattle. A total of 10,986 adrenal cortex transcripts and 37,204 single nucleotide polymorphisms (SNPs) were analysed in 145 F2 cows of a Charolais × German Holstein cross. We identified 505 SNPs that were associated with the abundance of 129 transcripts, comprising 482 cis effects and 17 trans effects. These SNPs were located on all chromosomes but X, 16, 24 and 28. Associated genes are mainly involved in molecular and cellular functions comprising free radical scavenging, cellular compromise, cell morphology and lipid metabolism, including genes such as CYP27A1 and LHCGR that have been shown to affect economically important traits in cattle. In this study we showed that adrenocortical eQTL affect the expression of genes known to contribute to the phenotypic manifestation in cattle. Furthermore, some of the identified genes and related molecular pathways were previously shown to contribute to the phenotypic variation of behaviour, temperament and growth at the onset of puberty in the same population investigated here. We conclude that eQTL analysis appears to be a useful approach providing insight into the molecular and genetic background of complex traits in cattle and will help to understand molecular networks involved.

An intergenic non-coding rRNA correlated with expression of the rRNA and frequency of an rRNA single nucleotide polymorphism in lung cancer cells.

PubMed

Shiao, Yih-Horng; Lupascu, Sorin T; Gu, Yuhan D; Kasprzak, Wojciech; Hwang, Christopher J; Fields, Janet R; Leighty, Robert M; Quiñones, Octavio; Shapiro, Bruce A; Alvord, W Gregory; Anderson, Lucy M

2009-10-19

Ribosomal RNA (rRNA) is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA) upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics. Using quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately -1000 nucleotides upstream of the rRNA transcription start site (+1) and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014). During sequence analysis from -388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs) in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C) in the 5' leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014). Modelling of the secondary structure of the rRNA 5'-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences. The results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5' leader sequence of the primary rRNA transcript.

Environmental regulation of the long polar fimbriae 2 of enterohemorrhagic Escherichia coli O157:H7

PubMed Central

Arenas-Hernández, Margarita M.P.; Rojas-López, Maricarmen; Medrano-López, Abraham; Nuñez-Reza, Karen J.; Puente, José Luis; Martínez-Laguna, Ygnacio; Torres, Alfredo G.

2014-01-01

The molecular mechanisms controlling expression of the Long Polar Fimbriae 2 (Lpf2) of enterohemorrhagic E. coli (EHEC) O157:H7 were evaluated. Primer extension was used to locate the lpfA2 transcriptional start site in EHEC strain EDL933 at 171 bp upstream of the lpfA2 start codon. Semi-quantitative RT-PCR demonstrated that the highest lpfA2 expression occurs between an OD600 of 1.0 and 1.2 in DMEM at pH 6.5 and 37°C. The level of lpfA2 transcription at OD600 1.2 and pH 6.5 was 4-times greater than that at pH 7.2. Although lpfA2 expression was decreased under iron-depleted conditions, its expression was increased in a Ferric-uptake-regulator (Fur) mutant strain. The lpfA2 transcript was 0.7 and 2-times more abundant in wt EHEC grown in DMEM pH 6.5 plus iron and MacConkey broth at 25°C, respectively, than in DMEM at pH 6.5. The lpf2 expression in DMEM pH 6.5 plus iron and bile salts was 2.7-times more abundant and similar to MacConkey. Further, transcription in the EDL933Δfur was 0.6 and 0.8-times higher as compared to the wt strain grown in DMEM pH 6.5 plus iron and MacConkey broth, respectively. Electrophoretic mobility shift assays (EMSA) showed that purified Fur interacts with the lpf2 regulatory region, indicating that Fur-repression is exerted by direct binding to the promoter region. In summary, we demonstrated that the EHEC lpf2 operon is regulated in response to temperature, pH, bile salts and iron, during exponential phase of growth, and controlled by Fur. PMID:24966050

Differential expression of α-L-arabinofuranosidases during maize (Zea mays L.) root elongation.

PubMed

Kozlova, Liudmila V; Gorshkov, Oleg V; Mokshina, Natalia E; Gorshkova, Tatyana A

2015-05-01

Specific α- l -arabinofuranosidases are involved in the realisation of elongation growth process in cells with type II cell walls. Elongation growth in a plant cell is largely based on modification of the cell wall. In type II cell walls, the Ara/Xyl ratio is known to decrease during elongation due to the partial removal of Ara residues from glucuronoarabinoxylan. We searched within the maize genome for the genes of all predicted α-L-arabinofuranosidases that may be responsible for such a process and related their expression to the activity of the enzyme and the amount of free arabinose measured in six zones of a growing maize root. Eight genes of the GH51 family (ZmaABFs) and one gene of the GH3 family (ZmaARA-I) were identified. The abundance of ZmaABF1 and 3-6 transcripts was highly correlated with the measured enzymatic activity and free arabinose content that significantly increased during elongation. The transcript abundances also coincided with the pattern of changes in the Ara/Xyl ratio of the xylanase-extractable glucuronoarabinoxylan described in previous studies. The expression of ZmaABF3, 5 and 6 was especially up-regulated during elongation although corresponding proteins are devoid of the catalytic glutamate at the proper position. ZmaABF2 transcripts were specifically enriched in the root cap and meristem. A single ZmaARA-I gene was not expressed as a whole gene but instead as splice variants that encode the C-terminal end of the protein. Changes in the ZmaARA-I transcript level were rather moderate and had no significant correlation with free arabinose content. Thus, elongation growth of cells with type II cell walls is accompanied by the up-regulation of specific and predicted α-L-arabinofuranosidase genes, and the corresponding activity is indeed pronounced and is important for the modification of glucuronoarabinoxylan, which plays a key role in the modification of the cell wall supramolecular organisation.

Proteomic Analysis of Differentially Expressed Proteins Involved in Peel Senescence in Harvested Mandarin Fruit

PubMed Central

Li, Taotao; Zhang, Jingying; Zhu, Hong; Qu, Hongxia; You, Shulin; Duan, Xuewu; Jiang, Yueming

2016-01-01

Mandarin (Citrus reticulata), a non-climacteric fruit, is an economically important fruit worldwide. The mechanism underlying senescence of non-climacteric fruit is poorly understood. In this study, a gel-based proteomic study followed by LC-ESI-MS/MS analysis was carried out to investigate the proteomic changes involved in peel senescence in harvested mandarin “Shatangju” fruit stored for 18 days. Over the course of the storage period, the fruit gradually senesced, accompanied by a decreased respiration rate and increased chlorophyll degradation and disruption of membrane integrity. Sixty-three proteins spots that showed significant differences in abundance were identified. The up-regulated proteins were mainly associated with cell wall degradation, lipid degradation, protein degradation, senescence-related transcription factors, and transcription-related proteins. In contrast, most proteins associated with ATP synthesis and scavenging of reactive oxygen species were significantly down-regulated during peel senescence. Three thioredoxin proteins and three Ca2+ signaling-related proteins were significantly up-regulated during peel senescence. It is suggested that mandarin peel senescence is associated with energy supply efficiency, decreased antioxidant capability, and increased protein and lipid degradation. In addition, activation of Ca2+ signaling and transcription factors might be involved in cell wall degradation and primary or secondary metabolism. PMID:27303420

Role of Alternative Polyadenylation during Adipogenic Differentiation: An In Silico Approach

PubMed Central

Spangenberg, Lucía; Correa, Alejandro; Dallagiovanna, Bruno; Naya, Hugo

2013-01-01

Post-transcriptional regulation of stem cell differentiation is far from being completely understood. Changes in protein levels are not fully correlated with corresponding changes in mRNAs; the observed differences might be partially explained by post-transcriptional regulation mechanisms, such as alternative polyadenylation. This would involve changes in protein binding, transcript usage, miRNAs and other non-coding RNAs. In the present work we analyzed the distribution of alternative transcripts during adipogenic differentiation and the potential role of miRNAs in post-transcriptional regulation. Our in silico analysis suggests a modest, consistent, bias in 3′UTR lengths during differentiation enabling a fine-tuned transcript regulation via small non-coding RNAs. Including these effects in the analyses partially accounts for the observed discrepancies in relative abundance of protein and mRNA. PMID:24143171

Natural Genetic Variation Influences Protein Abundances in C. elegans Developmental Signalling Pathways

PubMed Central

Singh, Kapil Dev; Roschitzki, Bernd; Snoek, L. Basten; Grossmann, Jonas; Zheng, Xue; Elvin, Mark; Kamkina, Polina; Schrimpf, Sabine P.; Poulin, Gino B.; Kammenga, Jan E.; Hengartner, Michael O.

2016-01-01

Complex traits, including common disease-related traits, are affected by many different genes that function in multiple pathways and networks. The apoptosis, MAPK, Notch, and Wnt signalling pathways play important roles in development and disease progression. At the moment we have a poor understanding of how allelic variation affects gene expression in these pathways at the level of translation. Here we report the effect of natural genetic variation on transcript and protein abundance involved in developmental signalling pathways in Caenorhabditis elegans. We used selected reaction monitoring to analyse proteins from the abovementioned four pathways in a set of recombinant inbred lines (RILs) generated from the wild-type strains N2 (Bristol) and CB4856 (Hawaii) to enable quantitative trait locus (QTL) mapping. About half of the cases from the 44 genes tested showed a statistically significant change in protein abundance between various strains, most of these were however very weak (below 1.3-fold change). We detected a distant QTL on the left arm of chromosome II that affected protein abundance of the phosphatidylserine receptor protein PSR-1, and two separate QTLs that influenced embryonic and ionizing radiation-induced apoptosis on chromosome IV. Our results demonstrate that natural variation in C. elegans is sufficient to cause significant changes in signalling pathways both at the gene expression (transcript and protein abundance) and phenotypic levels. PMID:26985669

Microarray analysis of a salamander hopeful monster reveals transcriptional signatures of paedomorphic brain development

PubMed Central

2010-01-01

Background The Mexican axolotl (Ambystoma mexicanum) is considered a hopeful monster because it exhibits an adaptive and derived mode of development - paedomorphosis - that has evolved rapidly and independently among tiger salamanders. Unlike related tiger salamanders that undergo metamorphosis, axolotls retain larval morphological traits into adulthood and thus present an adult body plan that differs dramatically from the ancestral (metamorphic) form. The basis of paedomorphic development was investigated by comparing temporal patterns of gene transcription between axolotl and tiger salamander larvae (Ambystoma tigrinum tigrinum) that typically undergo a metamorphosis. Results Transcript abundances from whole brain and pituitary were estimated via microarray analysis on four different days post hatching (42, 56, 70, 84 dph) and regression modeling was used to independently identify genes that were differentially expressed as a function of time in both species. Collectively, more differentially expressed genes (DEGs) were identified as unique to the axolotl (n = 76) and tiger salamander (n = 292) than were identified as shared (n = 108). All but two of the shared DEGs exhibited the same temporal pattern of expression and the unique genes tended to show greater changes later in the larval period when tiger salamander larvae were undergoing anatomical metamorphosis. A second, complementary analysis that directly compared the expression of 1320 genes between the species identified 409 genes that differed as a function of species or the interaction between time and species. Of these 409 DEGs, 84% exhibited higher abundances in tiger salamander larvae at all sampling times. Conclusions Many of the unique tiger salamander transcriptional responses are probably associated with metamorphic biological processes. However, the axolotl also showed unique patterns of transcription early in development. In particular, the axolotl showed a genome-wide reduction in mRNA abundance across loci, including genes that regulate hypothalamic-pituitary activities. This suggests that an axolotls failure to undergo anatomical metamorphosis late in the larval period is indirectly associated with a mechanism(s) that acts earlier in development to broadly program transcription. The axolotl hopeful monster provides a model to identify mechanisms of early brain development that proximally and ultimately affect the expression of adult phenotypes. PMID:20584293

The venom gland transcriptome of the Desert Massasauga Rattlesnake (Sistrurus catenatus edwardsii): towards an understanding of venom composition among advanced snakes (Superfamily Colubroidea)

PubMed Central

Pahari, Susanta; Mackessy, Stephen P; Kini, R Manjunatha

2007-01-01

Background Snake venoms are complex mixtures of pharmacologically active proteins and peptides which belong to a small number of superfamilies. Global cataloguing of the venom transcriptome facilitates the identification of new families of toxins as well as helps in understanding the evolution of venom proteomes. Results We have constructed a cDNA library of the venom gland of a threatened rattlesnake (a pitviper), Sistrurus catenatus edwardsii (Desert Massasauga), and sequenced 576 ESTs. Our results demonstrate a high abundance of serine proteinase and metalloproteinase transcripts, indicating that the disruption of hemostasis is a principle mechanism of action of the venom. In addition to the transcripts encoding common venom proteins, we detected two varieties of low abundance unique transcripts in the library; these encode for three-finger toxins and a novel toxin possibly generated from the fusion of two genes. We also observed polyadenylated ribosomal RNAs in the venom gland library, an interesting preliminary obsevation of this unusual phenomenon in a reptilian system. Conclusion The three-finger toxins are characteristic of most elapid venoms but are rare in viperid venoms. We detected several ESTs encoding this group of toxins in this study. We also observed the presence of a transcript encoding a fused protein of two well-characterized toxins (Kunitz/BPTI and Waprins), and this is the first report of this kind of fusion in a snake toxin transcriptome. We propose that these new venom proteins may have ancillary functions for envenomation. The presence of a fused toxin indicates that in addition to gene duplication and accelerated evolution, exon shuffling or transcriptional splicing may also contribute to generating the diversity of toxins and toxin isoforms observed among snake venoms. The detection of low abundance toxins, as observed in this and other studies, indicates a greater compositional similarity of venoms (though potency will differ) among advanced snakes than has been previously recognized. PMID:18096037

Five omic technologies are concordant in differentiating the biochemical characteristics of the berries of five grapevine (Vitis vinifera L.) cultivars.

PubMed

Ghan, Ryan; Van Sluyter, Steven C; Hochberg, Uri; Degu, Asfaw; Hopper, Daniel W; Tillet, Richard L; Schlauch, Karen A; Haynes, Paul A; Fait, Aaron; Cramer, Grant R

2015-11-16

Grape cultivars and wines are distinguishable by their color, flavor and aroma profiles. Omic analyses (transcripts, proteins and metabolites) are powerful tools for assessing biochemical differences in biological systems. Berry skins of red- (Cabernet Sauvignon, Merlot, Pinot Noir) and white-skinned (Chardonnay, Semillon) wine grapes were harvested near optimum maturity (°Brix-to-titratable acidity ratio) from the same experimental vineyard. The cultivars were exposed to a mild, seasonal water-deficit treatment from fruit set until harvest in 2011. Identical sample aliquots were analyzed for transcripts by grapevine whole-genome oligonucleotide microarray and RNAseq technologies, proteins by nano-liquid chromatography-mass spectroscopy, and metabolites by gas chromatography-mass spectroscopy and liquid chromatography-mass spectroscopy. Principal components analysis of each of five Omic technologies showed similar results across cultivars in all Omic datasets. Comparison of the processed data of genes mapped in RNAseq and microarray data revealed a strong Pearson's correlation (0.80). The exclusion of probesets associated with genes with potential for cross-hybridization on the microarray improved the correlation to 0.93. The overall concordance of protein with transcript data was low with a Pearson's correlation of 0.27 and 0.24 for the RNAseq and microarray data, respectively. Integration of metabolite with protein and transcript data produced an expected model of phenylpropanoid biosynthesis, which distinguished red from white grapes, yet provided detail of individual cultivar differences. The mild water deficit treatment did not significantly alter the abundance of proteins or metabolites measured in the five cultivars, but did have a small effect on gene expression. The five Omic technologies were consistent in distinguishing cultivar variation. There was high concordance between transcriptomic technologies, but generally protein abundance did not correlate well with transcript abundance. The integration of multiple high-throughput Omic datasets revealed complex biochemical variation amongst five cultivars of an ancient and economically important crop species.

Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.

Heat shock proteins and heat shock factor 1 in carcinogenesis and tumor development: an update

PubMed Central

2013-01-01

Heat shock proteins (HSP) are a subset of the molecular chaperones, best known for their rapid and abundant induction by stress. HSP genes are activated at the transcriptional level by heat shock transcription factor 1 (HSF1). During the progression of many types of cancer, this heat shock transcriptional regulon becomes co-opted by mechanisms that are currently unclear, although evidently triggered in the emerging tumor cell. Concerted activation of HSF1 and the accumulation of HSPs then participates in many of the traits that permit the malignant phenotype. Thus cancers of many histologies exhibit activated HSF1 and increased HSP levels that may help to deter tumor suppression and evade therapy in the clinic. We review here the extensive work that has been carried out and is still in progress aimed at: (1) understanding the oncogenic mechanisms by which HSP genes are switched on, (2) determining the roles of HSF1 / HSP in malignant transformation and, (3) discovering approaches to therapy based on disrupting the influence of the HSF1 controlled transcriptome in cancer. PMID:22885793

Accumulation of transcription factors and cell signaling-related proteins in the nucleus during citrus-Xanthomonas interaction.

PubMed

Rani, T Swaroopa; Durgeshwar, P; Podile, Appa Rao

2015-07-20

The nucleus is the maestro of the cell and is involved in the modulation of cell signaling during stress. We performed a comprehensive nuclear proteome analysis of Citrus sinensis during interaction with host (Xanthomonas citri pv. citri-Xcc) and non-host (Xanthomonas oryzae pv. oryzae-Xoo) pathogens. The nuclear proteome was obtained using a sequential method of organelle enrichment and determined by nano-LC-MS/MS analysis. A total of 243 proteins accumulated differentially during citrus-Xanthomonas interaction, belonging to 11 functional groups, with signaling and transcription-related proteins dominating. MADS-box transcription factors, DEAD-box RNA helicase and leucine aminopeptidase, mainly involved in jasmonic acid (JA) responses, were in high abundance during non-host interaction (Xoo). Signaling-related proteins like serine/threonine kinase, histones (H3.2, H2A), phosphoglycerate kinase, dynamin, actin and aldolase showed increased accumulation early during Xoo interaction. Our results suggest that there is a possible involvement of JA-triggered defense responses during non-host resistance, with early recognition of the non-host pathogen. Copyright © 2015. Published by Elsevier GmbH.

Sugar is an endogenous cue for juvenile-to-adult phase transition in plants

PubMed Central

Yu, Sha; Cao, Li; Zhou, Chuan-Miao; Zhang, Tian-Qi; Lian, Heng; Sun, Yue; Wu, Jianqiang; Huang, Jirong; Wang, Guodong; Wang, Jia-Wei

2013-01-01

The transition from the juvenile to adult phase in plants is controlled by diverse exogenous and endogenous cues such as age, day length, light, nutrients, and temperature. Previous studies have shown that the gradual decline in microRNA156 (miR156) with age promotes the expression of adult traits. However, how age temporally regulates the abundance of miR156 is poorly understood. We show here that the expression of miR156 responds to sugar. Sugar represses miR156 expression at both the transcriptional level and post-transcriptional level through the degradation of miR156 primary transcripts. Defoliation and photosynthetic mutant assays further demonstrate that sugar from the pre-existing leaves acts as a mobile signal to repress miR156, and subsequently triggers the juvenile-to-adult phase transition in young leaf primordia. We propose that the gradual increase in sugar after seed germination serves as an endogenous cue for developmental timing in plants. DOI: http://dx.doi.org/10.7554/eLife.00269.001 PMID:23543845

Gravity-regulated gene expression in Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Sederoff, Heike; Brown, Christopher S.; Heber, Steffen; Kajla, Jyoti D.; Kumar, Sandeep; Lomax, Terri L.; Wheeler, Benjamin; Yalamanchili, Roopa

Plant growth and development is regulated by changes in environmental signals. Plants sense environmental changes and respond to them by modifying gene expression programs to ad-just cell growth, differentiation, and metabolism. Functional expression of genes comprises many different processes including transcription, translation, post-transcriptional and post-translational modifications, as well as the degradation of RNA and proteins. Recently, it was discovered that small RNAs (sRNA, 18-24 nucleotides long), which are heritable and systemic, are key elements in regulating gene expression in response to biotic and abiotic changes. Sev-eral different classes of sRNAs have been identified that are part of a non-cell autonomous and phloem-mobile network of regulators affecting transcript stability, translational kinetics, and DNA methylation patterns responsible for heritable transcriptional silencing (epigenetics). Our research has focused on gene expression changes in response to gravistimulation of Arabidopsis roots. Using high-throughput technologies including microarrays and 454 sequencing, we iden-tified rapid changes in transcript abundance of genes as well as differential expression of small RNA in Arabidopsis root apices after minutes of reorientation. Some of the differentially regu-lated transcripts are encoded by genes that are important for the bending response. Functional mutants of those genes respond faster to reorientation than the respective wild type plants, indicating that these proteins are repressors of differential cell elongation. We compared the gravity responsive sRNAs to the changes in transcript abundances of their putative targets and identified several potential miRNA: target pairs. Currently, we are using mutant and transgenic Arabidopsis plants to characterize the function of those miRNAs and their putative targets in gravitropic and phototropic responses in Arabidopsis.

Comparative day/night metatranscriptomic analysis of microbial communities in the North Pacific subtropical gyre.

PubMed

Poretsky, Rachel S; Hewson, Ian; Sun, Shulei; Allen, Andrew E; Zehr, Jonathan P; Moran, Mary Ann

2009-06-01

Metatranscriptomic analyses of microbial assemblages (< 5 microm) from surface water at the Hawaiian Ocean Time-Series (HOT) revealed community-wide metabolic activities and day/night patterns of differential gene expression. Pyrosequencing produced 75 558 putative mRNA reads from a day transcriptome and 75 946 from a night transcriptome. Taxonomic binning of annotated mRNAs indicated that Cyanobacteria contributed a greater percentage of the transcripts (54% of annotated sequences) than expected based on abundance (35% of cell counts and 21% 16S rRNA of libraries), and may represent the most actively transcribing cells in this surface ocean community in both the day and night. Major heterotrophic taxa contributing to the community transcriptome included alpha-Proteobacteria (19% of annotated sequences, most of which were SAR11-related) and gamma-Proteobacteria (4%). The composition of transcript pools was consistent with models of prokaryotic gene expression, including operon-based transcription patterns and an abundance of genes predicted to be highly expressed. Metabolic activities that are shared by many microbial taxa (e.g. glycolysis, citric acid cycle, amino acid biosynthesis and transcription and translation machinery) were well represented among the community transcripts. There was an overabundance of transcripts for photosynthesis, C1 metabolism and oxidative phosphorylation in the day compared with night, and evidence that energy acquisition is coordinated with solar radiation levels for both autotrophic and heterotrophic microbes. In contrast, housekeeping activities such as amino acid biosynthesis, membrane synthesis and repair, and vitamin biosynthesis were overrepresented in the night transcriptome. Direct sequencing of these environmental transcripts has provided detailed information on metabolic and biogeochemical responses of a microbial community to solar forcing.

Omic personality: implications of stable transcript and methylation profiles for personalized medicine.

PubMed

Tabassum, Rubina; Sivadas, Ambily; Agrawal, Vartika; Tian, Haozheng; Arafat, Dalia; Gibson, Greg

2015-08-13

Personalized medicine is predicated on the notion that individual biochemical and genomic profiles are relatively constant in times of good health and to some extent predictive of disease or therapeutic response. We report a pilot study quantifying gene expression and methylation profile consistency over time, addressing the reasons for individual uniqueness, and its relation to N = 1 phenotypes. Whole blood samples from four African American women, four Caucasian women, and four Caucasian men drawn from the Atlanta Center for Health Discovery and Well Being study at three successive 6-month intervals were profiled by RNA-Seq, miRNA-Seq, and Illumina Methylation 450 K arrays. Standard regression approaches were used to evaluate the proportion of variance for each type of omic measure among individuals, and to quantify correlations among measures and with clinical attributes related to wellness. Longitudinal omic profiles were in general highly consistent over time, with an average of 67 % variance in transcript abundance, 42 % in CpG methylation level (but 88 % for the most differentiated CpG per gene), and 50 % in miRNA abundance among individuals, which are all comparable to 74 % variance among individuals for 74 clinical traits. One third of the variance could be attributed to differential blood cell type abundance, which was also fairly stable over time, and a lesser amount to expression quantitative trait loci (eQTL) effects. Seven conserved axes of covariance that capture diverse aspects of immune function explained over half of the variance. These axes also explained a considerable proportion of individually extreme transcript abundance, namely approximately 100 genes that were significantly up-regulated or down-regulated in each person and were in some cases enriched for relevant gene activities that plausibly associate with clinical attributes. A similar fraction of genes had individually divergent methylation levels, but these did not overlap with the transcripts, and fewer than 20 % of genes had significantly correlated methylation and gene expression. People express an "omic personality" consisting of peripheral blood transcriptional and epigenetic profiles that are constant over the course of a year and reflect various types of immune activity. Baseline genomic profiles can provide a window into the molecular basis of traits that might be useful for explaining medical conditions or guiding personalized health decisions.

Pectin Methylesterase and Pectin Remodelling Differ in the Fibre Walls of Two Gossypium Species with Very Different Fibre Properties

PubMed Central

Liu, Qinxiang; Talbot, Mark; Llewellyn, Danny J.

2013-01-01

Pectin, a major component of the primary cell walls of dicot plants, is synthesized in Golgi, secreted into the wall as methylesters and subsequently de-esterified by pectin methylesterase (PME). Pectin remodelling by PMEs is known to be important in regulating cell expansion in plants, but has been poorly studied in cotton. In this study, genome-wide analysis showed that PMEs are a large multi-gene family (81 genes) in diploid cotton (Gossypium raimondii), an expansion over the 66 in Arabidopsis and suggests the evolution of new functions in cotton. Relatively few PME genes are expressed highly in fibres based on EST abundance and the five most abundant in fibres were cloned and sequenced from two cotton species. Their significant sequence differences and their stage-specific expression in fibres within a species suggest sub-specialisation during fibre development. We determined the transcript abundance of the five fibre PMEs, total PME enzyme activity, pectin content and extent of de-methylesterification of the pectin in fibre walls of the two cotton species over the first 25–30 days of fibre growth. There was a higher transcript abundance of fibre-PMEs and a higher total PME enzyme activity in G. barbadense (Gb) than in G. hirsutum (Gh) fibres, particularly during late fibre elongation. Total pectin was high, but de-esterified pectin was low during fibre elongation (5–12 dpa) in both Gh and Gb. De-esterified pectin levels rose thereafter when total PME activity increased and this occurred earlier in Gb fibres resulting in a lower degree of esterification in Gb fibres between 17 and 22 dpa. Gb fibres are finer and longer than those of Gh, so differences in pectin remodelling during the transition to wall thickening may be an important factor in influencing final fibre diameter and length, two key quality attributes of cotton fibres. PMID:23755181

The Abundance and Activity of Nitrate-Reducing Microbial Populations in Estuarine Sediments

NASA Astrophysics Data System (ADS)

Cardarelli, E.; Francis, C. A.

2014-12-01

Estuaries are productive ecosystems that ameliorate nutrient and metal contaminants from surficial water supplies. At the intersection of terrestrial and aquatic environments, estuarine sediments host major microbially-mediated geochemical transformations. These include denitrification (the conversion of nitrate to nitrous oxide and/or dinitrogen) and dissimilatory nitrate reduction to ammonium (DNRA). Denitrification has historically been seen as the predominant nitrate attenuation process and functions as an effective sink for nitrate. DNRA has previously been believed to be a minor nitrate reduction process and transforms nitrate within the ecosystem to ammonium, a more biologically available N species. Recent studies have compared the two processes in coastal environments and determined fluctuating environmental conditions may suppress denitrification, supporting an increased role for DNRA in the N cycle. Nitrate availability and salinity are factors thought to influence the membership of the microbial communities present, and the nitrate reduction process that predominates. The aim of this study is to investigate how nitrate concentration and salinity alter the transcript abundances of N cycling functional gene markers for denitrification (nirK, nirS) and DNRA (nrfA) in estuarine sediments at the mouth of the hypernutrified Old Salinas River, CA. Short-term whole core incubations amended with artificial freshwater/artificial seawater (2 psu, 35 psu) and with varying NO3- concentrations (200mM, 2000mM) were conducted to assess the activity as well as the abundance of the nitrate-reducing microbial populations present. Gene expression of nirK, nirS, and nrfA at the conclusion of the incubations was quantified using reverse transcription quantitative polymerase chain reaction (RT-qPCR). High abundances of nirK, nirS, and nrfA under particular conditions coupled with the resulting geochemical data ultimately provides insight onto how the aforementioned factors influence N cycling related gene expression and rates of nitrate reduction.

Molecular Characterization of Aquaporin 1 and Aquaporin 3 from the Gills of the African Lungfish, Protopterus annectens, and Changes in Their Branchial mRNA Expression Levels and Protein Abundance during Three Phases of Aestivation.

PubMed

Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Lam, Siew H; Ip, Yuen K

2016-01-01

African lungfishes can undergo long periods of aestivation on land during drought. During aestivation, lungfishes are confronted with desiccation and dehydration, and their gills become non-functional and covered with a thick layer of dried mucus. Aquaporins (Aqps) are a superfamily of integral membrane proteins which generally facilitate the permeation of water through plasma membranes. This study aimed to obtain the complete cDNA coding sequences of aqp1 and aqp3 from the gills of Protopterus annectens , and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Dendrogramic analyses of the deduced Aqp1 and Aqp3 amino acid sequences of P. annectens revealed their close relationships with those of Latimeria chalumnae and tetrapods. During the induction phase, there were significant decreases in the transcript levels of aqp1 and aqp3 in the gills of P. annectens , but the branchial Aqp1 and Aqp3 protein abundance remained unchanged. As changes in transcription might precede changes in translation, this could be regarded as an adaptive response to decrease the protein abundance of Aqp1 and Aqp3 in the subsequent maintenance phase of aestivation. As expected, the branchial transcript levels and protein abundance of aqp1 /Aqp1 and aqp3 /Aqp3 were significantly down-regulated during the maintenance phase, probably attributable to the shutdown of branchial functions and the cessation of volume regulation of branchial epithelial cells. Additionally, these changes could reduce the loss of water through branchial epithelial surfaces, supplementing the anti-desiccating property of the dried mucus. Upon arousal, it was essential for the lungfish to restore branchial functions. Indeed, the protein abundance of Aqp1 recovered partially, with complete recovery of mRNA expression level and protein abundance of Aqp3, in the gills of P. annectens after 3 days of arousal. These results provide insights into how P. annectens regulates branchial Aqp expression to cope with desiccation and rehydration during different phases of aestivation.

Dysregulation of autophagy in rat liver with mitochondrial DNA depletion induced by the nucleoside analogue zidovudine.

PubMed

Santos-Llamas, Ana; Monte, Maria J; Marin, Jose J G; Perez, Maria J

2018-03-28

The nucleoside reverse transcriptase inhibitor zidovudine (AZT), used in HIV infection treatment, induces mitochondrial DNA (mtDNA) depletion. A cause-effect relationship between mtDNA status alterations and autophagy has been reported. Both events are common in several liver diseases, including hepatocellular carcinoma. Here, we have studied autophagy activation in rat liver with mtDNA depletion induced by AZT administration in drinking water for 35 days. AZT at a concentration of 1 mg/ml, but not 0.5 mg/ml in the drinking water, decreased mtDNA levels in rat liver and extrahepatic tissues. In liver, mtDNA-encoded cytochrome c oxidase 1 protein levels were decreased. Although serum biomarkers of liver and kidney toxicity remained unaltered, β-hydroxybutyrate levels were increased in liver of AZT-treated rats. Moreover, autophagy was dysregulated at two levels: (i) decreased induction signalling of this process as indicated by increases in autophagy inhibitors activity (AKT/mTOR), and absence of changes (Beclin-1, Atg5, Atg7) or decreases (AMPK/ULK1) in the expression/activity of pro-autophagy proteins; and (ii) reduced autophagosome degradation as indicated by decreases in the lysosome abundance (LAMP2 marker) and the transcription factor TFEB controlling lysosome biogenesis. This resulted in increased autophagosome abundance (LC3-II marker) and accumulation of the protein selectively degraded by autophagy p62, and the transcription factor Nrf2 in liver of AZT-treated rats. Nrf2 was activated as indicated by the up-regulation of antioxidant target genes Nqo1 and Hmox-1. In conclusion, rat liver with AZT-induced mtDNA depletion presented dysregulations in autophagosome formation and degradation balance, which results in accumulation of these structures in parenchymal liver cells, favouring hepatocarcinogenesis.

Behavioural fever in zebrafish larvae.

PubMed

Rey, Sonia; Moiche, Visila; Boltaña, Sebastian; Teles, Mariana; MacKenzie, Simon

2017-02-01

Behavioural fever has been reported in different species of mobile ectotherms including the zebrafish, Danio rerio, in response to exogenous pyrogens. In this study we report, to our knowledge for the first time, upon the ontogenic onset of behavioural fever in zebrafish (Danio rerio) larvae. For this, zebrafish larvae (from first feeding to juveniles) were placed in a continuous thermal gradient providing the opportunity to select their preferred temperature. The novel thermal preference aquarium was based upon a continuous vertical column system and allows for non-invasive observation of larvae vertical distribution under isothermal (T R at 28 °C) and thermal gradient conditions (T CH : 28-32 °C). Larval thermal preference was assessed under both conditions with or without an immersion challenge, in order to detect the onset of the behavioural fever response. Our results defined the onset of the dsRNA induced behavioural fever at 18-20 days post fertilization (dpf). Significant differences were observed in dsRNA challenged larvae, which prefer higher temperatures (1-4 °C increase) throughout the experimental period as compared to non-challenged larvae. In parallel we measured the abundance of antiviral transcripts; viperin, gig2, irf7, trim25 and Mxb mRNAs in dsRNA challenged larvae under both thermal regimes: T R and T Ch . Significant increases in the abundance of all measured transcripts were recorded under thermal choice conditions signifying that thermo-coupling and the resultant enhancement of the immune response to dsRNA challenge occurs from 18 dpf onwards in the zebrafish. The results are of importance as they identify a key developmental stage where the neuro-immune interface matures in the zebrafish likely providing increased resistance to viral infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

CARTILAGE OLIGOMERIC MATRIX PROTEIN ENHANCES MATRIX ASSEMBLY DURING CHONDROGENESIS OF HUMAN MESENCHYMAL STEM CELLS

PubMed Central

Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S.; Chen, Faye H.

2011-01-01

Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate-hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention. PMID:22095699

Reduced Insulin/Insulin-like Growth Factor-1 Signaling and Dietary Restriction Inhibit Translation but Preserve Muscle Mass in Caenorhabditis elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Depuydt, Geert; Xie, Fang; Petyuk, Vladislav A.

Reduced signaling through the C. elegans insulin/IGF1 like tyrosine kinase receptor daf2 and dietary restriction via bacterial dilution are two well-characterized lifespan-extending interventions that operate in parallel or through (partially) independent mechanisms. Using accurate mass and time tag LCMS/MS quantitative proteomics we detected that the abundance of a large number of ribosomal subunits is decreased in response to dietary restriction as well as in the daf2(e1370) insulin/IGF1 receptor mutant. In addition, general protein synthesis levels in these long-lived worms are repressed. Surprisingly, ribosomal transcript levels were not correlated to actual protein abundance, suggesting that posttranscriptional regulation determines ribosome content. Proteomicsmore » also revealed increased presence of many structural muscle cell components in long-lived worms, which appears to result from prioritized preservation of muscle cell volume in nutrient-poor conditions or low insulin-like signaling. Activation of DAF16, but not diet-restriction, stimulates mRNA expression of muscle-related genes to prevent muscle atrophy. Important daf2 specific proteome changes include overexpression of aerobic metabolism enzymes and a general activation of stress responsive and immune defense systems, while increased abundance of many protein subunits of the proteasome core complex is a DR-specific characteristic.« less

Network analysis of transcriptomics expands regulatory landscapes in Synechococcus sp. PCC 7002

DOE Office of Scientific and Technical Information (OSTI.GOV)

McClure, Ryan S.; Overall, Christopher C.; McDermott, Jason E.

Cyanobacterial regulation of gene expression must contend with a genome organization that lacks apparent functional context, as the majority of cellular processes and metabolic pathways are encoded by genes found at disparate locations across the genome. In addition, the fact that coordinated regulation of cyanobacterial cellular machinery takes place with significantly fewer transcription factors, compared to other Eubacteria, suggests the involvement of post-transcriptional mechanisms and regulatory adaptations which are not fully understood. Global transcript abundance from model cyanobacterium Synechococcus sp. PCC 7002 grown under 42 different conditions was analyzed using context-likelihood of relatedness. The resulting 903-gene network, which was organizedmore » into 11 modules, not only allowed classification of cyanobacterial responses to specific environmental variables but provided insight into the transcriptional network topology and led to the expansion of predicted regulons. When used in conjunction with genome sequence, the global transcript abundance allowed identification of putative post-transcriptional changes in expression as well as novel potential targets of both DNA binding proteins and asRNA regulators. The results offer a new perspective into the multi-level regulation that governs cellular adaptations of fast-growing physiologically robust cyanobacterium Synechococcus sp. PCC 7002 to changing environmental variables. It also extends a methodological knowledge-based framework for studying multi-scale regulatory mechanisms that operate in cyanobacteria. Finally, it provides valuable context for integrating systems-level data to enhance evidence-driven genomic annotation, especially in organisms where traditional context analyses cannot be implemented due to lack of operon-based functional organization.« less

Metal contaminations impact archaeal community composition, abundance and function in remote alpine lakes.

PubMed

Compte-Port, Sergi; Borrego, Carles M; Moussard, Hélène; Jeanbille, Mathilde; Restrepo-Ortiz, Claudia Ximena; de Diego, Alberto; Rodriguez-Iruretagoiena, Azibar; Gredilla, Ainara; Fdez-Ortiz de Vallejuelo, Silvia; Galand, Pierre E; Kalenitchenko, Dimitri; Rols, Jean-Luc; Pokrovsky, Oleg S; Gonzalez, Aridane G; Camarero, Lluis; Muñiz, Selene; Navarro-Navarro, Enrique; Auguet, Jean-Christophe

2018-04-24

Using the 16S rRNA and mcrA genes, we investigated the composition, abundance and activity of sediment archaeal communities within 18 high-mountain lakes under contrasted metal levels from different origins (bedrock erosion, past-mining activities and atmospheric depositions). Bathyarchaeota, Euryarchaeota and Woesearchaeota were the major phyla found at the meta-community scale, representing 48%, 18.3% and 15.2% of the archaeal community respectively. Metals were equally important as physicochemical variables in explaining the assemblage of archaeal communities and their abundance. Methanogenesis appeared as a process of central importance in the carbon cycle within sediments of alpine lakes as indicated by the absolute abundance of methanogen 16S rRNA and mcrA gene transcripts (10 5 to 10 9 copies g -1 ). We showed that methanogen abundance and activity were significantly reduced with increasing concentrations of Pb and Cd, two indicators of airborne metal contaminations. Considering the ecological importance of methanogenesis in sediment habitats, these metal contaminations may have system wide implications even in remote area such as alpine lakes. Overall, this work was pioneer in integrating the effect of long-range atmospheric depositions on archaeal communities and indicated that metal contamination might significantly compromise the contribution of Archaea to the carbon cycling of the mountain lake sediments. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

DOE PAGES

Dupont, Chris L.; McCrow, John P.; Valas, Ruben; ...

2014-10-21

Here, transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO 2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteriamore » enriched in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM.« less

Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen

PubMed Central

Dai, Xin; Tian, Yan; Li, Jinting; Su, Xiaoyun; Wang, Xuewei; Zhao, Shengguo; Liu, Li; Luo, Yingfeng; Liu, Di; Zheng, Huajun; Wang, Jiaqi; Dong, Zhiyang

2014-01-01

The bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ∼1% and ∼0.1% of the total non-rRNAs, respectively. The majority (∼98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera Ruminococcus, Prevotella, and Fibrobacter. Most (∼82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen. PMID:25501482

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

PubMed

Antony, Edna; Taybi, Tahar; Courbot, Mikaël; Mugford, Sam T; Smith, J Andrew C; Borland, Anne M

2008-01-01

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

Mitochondrial DNA Depletion in Respiratory Chain–Deficient Parkinson Disease Neurons

PubMed Central

Rygiel, Karolina A.; Hepplewhite, Philippa D.; Morris, Christopher M.; Picard, Martin; Turnbull, Doug M.

2016-01-01

Objective To determine the extent of respiratory chain abnormalities and investigate the contribution of mtDNA to the loss of respiratory chain complexes (CI–IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single‐neuron level. Methods Multiple‐label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI–IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2, and COXI) and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser‐capture microdissection, were assayed for mtDNA deletions, copy number, and presence of transcription/replication‐associated 7S DNA employing a triplex real‐time polymerase chain reaction (PCR) assay. Results Whereas mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in the abundances of CI and II subunits. At the single‐cell level, CI and II deficiencies were correlated in patients. The CI deficiency concomitantly occurred with low abundances of the mtDNA transcription factors TFAM and TFB2M, which also initiate transcription‐primed mtDNA replication. Consistent with this, real‐time PCR analysis revealed fewer transcription/replication‐associated mtDNA molecules and an overall reduction in mtDNA copy number in patients. This effect was more pronounced in single IPD neurons with severe CI deficiency. Interpretation Respiratory chain dysfunction in IPD neurons not only involves CI, but also extends to CII. These deficiencies are possibly a consequence of the interplay between nDNA and mtDNA‐encoded factors mechanistically connected via TFAM. ANN NEUROL 2016;79:366–378 PMID:26605748

Effect of chloroquine on gene expression of Plasmodium yoelii nigeriensis during its sporogonic development in the mosquito vector

PubMed Central

Silveira, Henrique; Ramos, Susana; Abrantes, Patrícia; Lopes, Luís Filipe; do Rosario, Virgílio E; Abrahamsen, Mitchell S

2007-01-01

Background The anti-malarial chloroquine can modulate the outcome of infection during the Plasmodium sporogonic development, interfering with Plasmodium gene expression and subsequently, with transmission. The present study sets to identify Plasmodium genes that might be regulated by chloroquine in the mosquito vector. Methods Differential display RT-PCR (DDRT-PCR) was used to identify genes expressed during the sporogonic cycle that are regulated by exposure to chloroquine. Anopheles stephensi mosquitoes were fed on Plasmodium yoelii nigeriensis-infected mice. Three days post-infection, mosquitoes were fed a non-infectious blood meal from mice treated orally with 50 mg/kg chloroquine. Two differentially expressed Plasmodium transcripts (Pyn_chl091 and Pyn_chl055) were further characterized by DNA sequencing and real-time PCR analysis. Results Both transcripts were represented in Plasmodium EST databases, but displayed no homology with any known genes. Pyn_chl091 was upregulated by day 18 post infection when the mosquito had a second blood meal. However, when the effect of chloroquine on that transcript was investigated during the erythrocytic cycle, no significant differences were observed. Although slightly upregulated by chloroquine exposure the expression of Pyn_chl055 was more affected by development, increasing towards the end of the sporogonic cycle. Transcript abundance of Pyn_chl055 was reduced when erythrocytic stages were treated with chloroquine. Conclusion Chloroquine increased parasite load in mosquito salivary glands and interferes with the expression of at least two Plasmodium genes. The transcripts identified contain putative signal peptides and transmembrane domains suggesting that these proteins, due to their location, are targets of chloroquine (not as an antimalarial) probably through cell trafficking and recycling. PMID:17605769

Genome-Wide Transcriptional Profiling of the Purple Sulfur Bacterium Allochromatium vinosum DSM 180T during Growth on Different Reduced Sulfur Compounds

PubMed Central

Weissgerber, Thomas; Dobler, Nadine; Polen, Tino; Latus, Jeanette; Stockdreher, Yvonne

2013-01-01

The purple sulfur bacterium Allochromatium vinosum DSM 180T is one of the best-studied sulfur-oxidizing anoxygenic phototrophic bacteria, and it has been developed into a model organism for laboratory-based studies of oxidative sulfur metabolism. Here, we took advantage of the organism's high metabolic versatility and performed whole-genome transcriptional profiling to investigate the response of A. vinosum cells upon exposure to sulfide, thiosulfate, elemental sulfur, or sulfite compared to photoorganoheterotrophic growth on malate. Differential expression of 1,178 genes was observed, corresponding to 30% of the A. vinosum genome. Relative transcription of 551 genes increased significantly during growth on one of the different sulfur sources, while the relative transcript abundance of 627 genes decreased. A significant number of genes that revealed strongly enhanced relative transcription levels have documented sulfur metabolism-related functions. Among these are the dsr genes, including dsrAB for dissimilatory sulfite reductase, and the sgp genes for the proteins of the sulfur globule envelope, thus confirming former results. In addition, we identified new genes encoding proteins with appropriate subcellular localization and properties to participate in oxidative dissimilatory sulfur metabolism. Those four genes for hypothetical proteins that exhibited the strongest increases of mRNA levels on sulfide and elemental sulfur, respectively, were chosen for inactivation and phenotypic analyses of the respective mutant strains. This approach verified the importance of the encoded proteins for sulfur globule formation during the oxidation of sulfide and thiosulfate and thereby also documented the suitability of comparative transcriptomics for the identification of new sulfur-related genes in anoxygenic phototrophic sulfur bacteria. PMID:23873913

Efficient computation of co-transcriptional RNA-ligand interaction dynamics.

PubMed

Wolfinger, Michael T; Flamm, Christoph; Hofacker, Ivo L

2018-05-04

Riboswitches form an abundant class of cis-regulatory RNA elements that mediate gene expression by binding a small metabolite. For synthetic biology applications, they are becoming cheap and accessible systems for selectively triggering transcription or translation of downstream genes. Many riboswitches are kinetically controlled, hence knowledge of their co-transcriptional mechanisms is essential. We present here an efficient implementation for analyzing co-transcriptional RNA-ligand interaction dynamics. This approach allows for the first time to model concentration-dependent metabolite binding/unbinding kinetics. We exemplify this novel approach by means of the recently studied I-A 2 ' -deoxyguanosine (2 ' dG)-sensing riboswitch from Mesoplasma florum. Copyright © 2018 Elsevier Inc. All rights reserved.

Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus

DOE PAGES

Howard-Varona, Cristina; Roux, Simon; Dore, Hugo; ...

2016-05-17

Microbes impact human health and disease, industrial processes and natural ecosystems, but do so under the influence of viruses. Problematically, knowledge of viral infection efficiencies and outcomes (e.g. lysis, lysogeny) derives from few model systems that over-represent efficient, lytic infections and under-represent virus-host natural diversity. Here we sought to understand how infection efficiency is regulated in an environmental Bacteroidetes virus that represents a globally abundant viral group and has drastically different infection efficiencies when infecting two nearly identical bacterial strains. To this end, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout the infection of bothmore » bacterial hosts. While the phage transcriptome was similar during both infections, host transcriptional differences appeared to have altered infection efficiency. Specifically, host transcriptomes suggested that the phage failed to repress early host expression in the inefficient nfection, thereby allowing the host to respond against infection by delaying phage DNA replication and protein translation. Further measurements showed that phage DNA and particle production were delayed (by >30 minutes) and reduced (by >50%) in the inefficient versus efficient infection as the host over-expressed DNA degradation genes and under-expressed translation genes, respectively. Together these results suggest that multiple levels of regulation can impact infection efficiencies as failure to repress host transcription allowed the host to defend against both phage DNA and protein production. Given that this phage type is ubiquitous and abundant in the global oceans and that variably efficient viral infections are likely common in any ecosystem with varying phage-host abundances and physiological states, these data provide a critically needed foundation for understanding and modeling viral infection efficiency in nature.« less

Evidence for hydrogen oxidation and metabolic plasticity in widespread deep-sea sulfur-oxidizing bacteria.

PubMed

Anantharaman, Karthik; Breier, John A; Sheik, Cody S; Dick, Gregory J

2013-01-02

Hydrothermal vents are a well-known source of energy that powers chemosynthesis in the deep sea. Recent work suggests that microbial chemosynthesis is also surprisingly pervasive throughout the dark oceans, serving as a significant CO(2) sink even at sites far removed from vents. Ammonia and sulfur have been identified as potential electron donors for this chemosynthesis, but they do not fully account for measured rates of dark primary production in the pelagic water column. Here we use metagenomic and metatranscriptomic analyses to show that deep-sea populations of the SUP05 group of uncultured sulfur-oxidizing Gammaproteobacteria, which are abundant in widespread and diverse marine environments, contain and highly express genes encoding group 1 Ni, Fe hydrogenase enzymes for H(2) oxidation. Reconstruction of near-complete genomes of two cooccurring SUP05 populations in hydrothermal plumes and deep waters of the Gulf of California enabled detailed population-specific metatranscriptomic analyses, revealing dynamic patterns of gene content and transcript abundance. SUP05 transcripts for genes involved in H(2) and sulfur oxidation are most abundant in hydrothermal plumes where these electron donors are enriched. In contrast, a second hydrogenase has more abundant transcripts in background deep-sea samples. Coupled with results from a bioenergetic model that suggest that H(2) oxidation can contribute significantly to the SUP05 energy budget, these findings reveal the potential importance of H(2) as a key energy source in the deep ocean. This study also highlights the genomic plasticity of SUP05, which enables this widely distributed group to optimize its energy metabolism (electron donor and acceptor) to local geochemical conditions.

Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howard-Varona, Cristina; Roux, Simon; Dore, Hugo

Microbes impact human health and disease, industrial processes and natural ecosystems, but do so under the influence of viruses. Problematically, knowledge of viral infection efficiencies and outcomes (e.g. lysis, lysogeny) derives from few model systems that over-represent efficient, lytic infections and under-represent virus-host natural diversity. Here we sought to understand how infection efficiency is regulated in an environmental Bacteroidetes virus that represents a globally abundant viral group and has drastically different infection efficiencies when infecting two nearly identical bacterial strains. To this end, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout the infection of bothmore » bacterial hosts. While the phage transcriptome was similar during both infections, host transcriptional differences appeared to have altered infection efficiency. Specifically, host transcriptomes suggested that the phage failed to repress early host expression in the inefficient nfection, thereby allowing the host to respond against infection by delaying phage DNA replication and protein translation. Further measurements showed that phage DNA and particle production were delayed (by >30 minutes) and reduced (by >50%) in the inefficient versus efficient infection as the host over-expressed DNA degradation genes and under-expressed translation genes, respectively. Together these results suggest that multiple levels of regulation can impact infection efficiencies as failure to repress host transcription allowed the host to defend against both phage DNA and protein production. Given that this phage type is ubiquitous and abundant in the global oceans and that variably efficient viral infections are likely common in any ecosystem with varying phage-host abundances and physiological states, these data provide a critically needed foundation for understanding and modeling viral infection efficiency in nature.« less

Analysis of E2F factors during epidermal differentiation.

PubMed

Chang, Wing Y; Dagnino, Lina

2005-01-01

The multigene E2F family of transcription factors is central in the control of cell cycle progression. The expression and activity of E2F proteins is tightly regulated transcriptionally and posttranslationally as a function of the proliferation and differentiation status of the cell. In this chapter, we review protocols designed to determine E2F mRNA abundance in tissues by in situ hybridization techniques. The ability to culture primary epidermal keratinocytes and maintain them as either undifferentiated or terminally differentiated cells allows the biochemical and molecular characterization of changes in E2F expression and activity. Thus, we also discuss in detail methods to analyze E2F protein abundance by immunoblot and their ability to bind DNA in cultured cells using electrophoretic mobility shift assays.

The SAM-responsive SMK box is a reversible riboswitch

PubMed Central

Smith, Angela M.; Fuchs, Ryan T.; Grundy, Frank J.; Henkin, Tina M.

2010-01-01

The SMK (SAM-III) box is an S-adenosylmethionine (SAM)-responsive riboswitch found in the 5′ untranslated region of metK genes, encoding SAM synthetase, in many members of the Lactobacillales. SAM binding causes a structural rearrangement in the RNA that sequesters the Shine-Dalgarno (SD) sequence by pairing with a complementary anti-SD (ASD) sequence; sequestration of the SD sequence inhibits binding of the 30S ribosomal subunit and prevents translation initiation. We observed a slight increase in the half-life of the metK transcript in vivo when Enterococcus faecalis cells were depleted for SAM, but no significant change in overall transcript abundance, consistent with the model that this riboswitch regulates at the level of translation initiation. The half-life of the SAM-SMK box RNA complex in vitro is shorter than that of the metK transcript in vivo, raising the possibility of reversible binding of SAM. We used a fluorescence assay to directly visualize reversible switching between the SAM-free and SAM-bound conformations. We propose that the SMK box riboswitch can make multiple SAM-dependent regulatory decisions during the lifetime of the transcript in vivo, acting as a reversible switch that allows the cell to respond rapidly to fluctuations in SAM pools by modulating expression of the SAM synthetase gene. PMID:21143313

The post-transcriptional regulatory system CSR controls the balance of metabolic pools in upper glycolysis of Escherichia coli.

PubMed

Morin, Manon; Ropers, Delphine; Letisse, Fabien; Laguerre, Sandrine; Portais, Jean-Charles; Cocaign-Bousquet, Muriel; Enjalbert, Brice

2016-05-01

Metabolic control in Escherichia coli is a complex process involving multilevel regulatory systems but the involvement of post-transcriptional regulation is uncertain. The post-transcriptional factor CsrA is stated as being the only regulator essential for the use of glycolytic substrates. A dozen enzymes in the central carbon metabolism (CCM) have been reported as potentially controlled by CsrA, but its impact on the CCM functioning has not been demonstrated. Here, a multiscale analysis was performed in a wild-type strain and its isogenic mutant attenuated for CsrA (including growth parameters, gene expression levels, metabolite pools, abundance of enzymes and fluxes). Data integration and regulation analysis showed a coordinated control of the expression of glycolytic enzymes. This also revealed the imbalance of metabolite pools in the csrA mutant upper glycolysis, before the phosphofructokinase PfkA step. This imbalance is associated with a glucose-phosphate stress. Restoring PfkA activity in the csrA mutant strain suppressed this stress and increased the mutant growth rate on glucose. Thus, the carbon storage regulator system is essential for the effective functioning of the upper glycolysis mainly through its control of PfkA. This work demonstrates the pivotal role of post-transcriptional regulation to shape the carbon metabolism. © 2016 John Wiley & Sons Ltd.

The bromodomain protein BRD4 regulates splicing during heat shock

PubMed Central

Hussong, Michelle; Kaehler, Christian; Kerick, Martin; Grimm, Christina; Franz, Alexandra; Timmermann, Bernd; Welzel, Franziska; Isensee, Jörg; Hucho, Tim; Krobitsch, Sylvia; Schweiger, Michal R.

2017-01-01

The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies. PMID:27536004

Neuregulin 1 transcripts are differentially expressed in schizophrenia and regulated by 5′ SNPs associated with the disease

PubMed Central

Law, Amanda J.; Lipska, Barbara K.; Weickert, Cynthia Shannon; Hyde, Thomas M.; Straub, Richard E.; Hashimoto, Ryota; Harrison, Paul J.; Kleinman, Joel E.; Weinberger, Daniel R.

2006-01-01

Genetic variation in neuregulin 1 (NRG1) is associated with schizophrenia. The disease-associated SNPs are noncoding, and their functional implications remain unknown. We hypothesized that differential expression of the NRG1 gene explains its association to the disease. We examined four of the disease-associated SNPs that make up the original risk haplotype in the 5′ upstream region of the gene for their effects on mRNA abundance of NRG1 types I–IV in human postmortem hippocampus. Diagnostic comparisons revealed a 34% increase in type I mRNA in schizophrenia and an interaction of diagnosis and genotype (SNP8NRG221132) on this transcript. Of potentially greater interest, a single SNP within the risk haplotype (SNP8NRG243177) and a 22-kb block of this core haplotype are associated with mRNA expression for the novel type IV isoform in patients and controls. Bioinformatic promoter analyses indicate that both SNPs lead to a gain/loss of putative binding sites for three transcription factors, serum response factor, myelin transcription factor-1, and High Mobility Group Box Protein-1. These data implicate variation in isoform expression as a molecular mechanism for the genetic association of NRG1 with schizophrenia. PMID:16618933

Zinc in innate and adaptive tumor immunity

PubMed Central

2010-01-01

Zinc is important. It is the second most abundant trace metal with 2-4 grams in humans. It is an essential trace element, critical for cell growth, development and differentiation, DNA synthesis, RNA transcription, cell division, and cell activation. Zinc deficiency has adverse consequences during embryogenesis and early childhood development, particularly on immune functioning. It is essential in members of all enzyme classes, including over 300 signaling molecules and transcription factors. Free zinc in immune and tumor cells is regulated by 14 distinct zinc importers (ZIP) and transporters (ZNT1-8). Zinc depletion induces cell death via apoptosis (or necrosis if apoptotic pathways are blocked) while sufficient zinc levels allows maintenance of autophagy. Cancer cells have upregulated zinc importers, and frequently increased zinc levels, which allow them to survive. Based on this novel synthesis, approaches which locally regulate zinc levels to promote survival of immune cells and/or induce tumor apoptosis are in order. PMID:21087493

A Vitis vinifera basic helix-loop-helix transcription factor enhances plant cell size, vegetative biomass and reproductive yield

DOE PAGES

Lim, Sung Don; Yim, Won Choel; Liu, Degao; ...

2018-04-16

Strategies for improving plant size are critical targets for plant biotechnology to increase vegetative biomass or reproductive yield. To improve biomass production, a codon-optimized helix–loop–helix transcription factor (VvCEB1 opt) from wine grape was overexpressed in Arabidopsis thaliana resulting in significantly increased leaf number, leaf and rosette area, fresh weight and dry weight. Cell size, but typically not cell number, was increased in all tissues resulting in increased vegetative biomass and reproductive organ size, number and seed yield. Ionomic analysis of leaves revealed the VvCEB1 opt-overexpressing plants had significantly elevated, K, S and Mo contents relative to control lines. Increased Kmore » content likely drives increased osmotic potential within cells leading to greater cellular growth and expansion. To understand the mechanistic basis of VvCEB1 opt action, one transgenic line was genotyped using RNA-Seq mRNA expression profiling and revealed a novel transcriptional reprogramming network with significant changes in mRNA abundance for genes with functions in delayed flowering, pathogen–defence responses, iron homeostasis, vesicle-mediated cell wall formation and auxin-mediated signalling and responses. Direct testing of VvCEB1 opt-overexpressing plants showed that they had significantly elevated auxin content and a significantly increased number of lateral leaf primordia within meristems relative to controls, confirming that cell expansion and organ number proliferation were likely an auxin-mediated process. VvCEB1 opt overexpression in Nicotiana sylvestris also showed larger cells, organ size and biomass demonstrating the potential applicability of this innovative strategy for improving plant biomass and reproductive yield in crops.« less

A Vitis vinifera basic helix-loop-helix transcription factor enhances plant cell size, vegetative biomass and reproductive yield

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Sung Don; Yim, Won Choel; Liu, Degao

Strategies for improving plant size are critical targets for plant biotechnology to increase vegetative biomass or reproductive yield. To improve biomass production, a codon-optimized helix–loop–helix transcription factor (VvCEB1 opt) from wine grape was overexpressed in Arabidopsis thaliana resulting in significantly increased leaf number, leaf and rosette area, fresh weight and dry weight. Cell size, but typically not cell number, was increased in all tissues resulting in increased vegetative biomass and reproductive organ size, number and seed yield. Ionomic analysis of leaves revealed the VvCEB1 opt-overexpressing plants had significantly elevated, K, S and Mo contents relative to control lines. Increased Kmore » content likely drives increased osmotic potential within cells leading to greater cellular growth and expansion. To understand the mechanistic basis of VvCEB1 opt action, one transgenic line was genotyped using RNA-Seq mRNA expression profiling and revealed a novel transcriptional reprogramming network with significant changes in mRNA abundance for genes with functions in delayed flowering, pathogen–defence responses, iron homeostasis, vesicle-mediated cell wall formation and auxin-mediated signalling and responses. Direct testing of VvCEB1 opt-overexpressing plants showed that they had significantly elevated auxin content and a significantly increased number of lateral leaf primordia within meristems relative to controls, confirming that cell expansion and organ number proliferation were likely an auxin-mediated process. VvCEB1 opt overexpression in Nicotiana sylvestris also showed larger cells, organ size and biomass demonstrating the potential applicability of this innovative strategy for improving plant biomass and reproductive yield in crops.« less

The common transcriptional subnetworks of the grape berry skin in the late stages of ripening.

PubMed

Ghan, Ryan; Petereit, Juli; Tillett, Richard L; Schlauch, Karen A; Toubiana, David; Fait, Aaron; Cramer, Grant R

2017-05-30

Wine grapes are important economically in many countries around the world. Defining the optimum time for grape harvest is a major challenge to the grower and winemaker. Berry skins are an important source of flavor, color and other quality traits in the ripening stage. Senescent-like processes such as chloroplast disorganization and cell death characterize the late ripening stage. To better understand the molecular and physiological processes involved in the late stages of berry ripening, RNA-seq analysis of the skins of seven wine grape cultivars (Cabernet Franc, Cabernet Sauvignon, Merlot, Pinot Noir, Chardonnay, Sauvignon Blanc and Semillon) was performed. RNA-seq analysis identified approximately 2000 common differentially expressed genes for all seven cultivars across four different berry sugar levels (20 to 26 °Brix). Network analyses, both a posteriori (standard) and a priori (gene co-expression network analysis), were used to elucidate transcriptional subnetworks and hub genes associated with traits in the berry skins of the late stages of berry ripening. These independent approaches revealed genes involved in photosynthesis, catabolism, and nucleotide metabolism. The transcript abundance of most photosynthetic genes declined with increasing sugar levels in the berries. The transcript abundance of other processes increased such as nucleic acid metabolism, chromosome organization and lipid catabolism. Weighted gene co-expression network analysis (WGCNA) identified 64 gene modules that were organized into 12 subnetworks of three modules or more and six higher order gene subnetworks. Some gene subnetworks were highly correlated with sugar levels and some subnetworks were highly enriched in the chloroplast and nucleus. The petal R package was utilized independently to construct a true small-world and scale-free complex gene co-expression network model. A subnetwork of 216 genes with the highest connectivity was elucidated, consistent with the module results from WGCNA. Hub genes in these subnetworks were identified including numerous members of the core circadian clock, RNA splicing, proteolysis and chromosome organization. An integrated model was constructed linking light sensing with alternative splicing, chromosome remodeling and the circadian clock. A common set of differentially expressed genes and gene subnetworks from seven different cultivars were examined in the skin of the late stages of grapevine berry ripening. A densely connected gene subnetwork was elucidated involving a complex interaction of berry senescent processes (autophagy), catabolism, the circadian clock, RNA splicing, proteolysis and epigenetic regulation. Hypotheses were induced from these data sets involving sugar accumulation, light, autophagy, epigenetic regulation, and fruit development. This work provides a better understanding of berry development and the transcriptional processes involved in the late stages of ripening.

The acute salinity changes activate the dual pathways of endocrine responses in the brain and pituitary of tilapia.

PubMed

Aruna, Adimoolam; Nagarajan, Ganesan; Chang, Ching-Fong

2015-01-15

To analyze and compare the stress and osmoregulatory hormones and receptors in pituitary during acute salinity changes, the expression patterns of corticotropin releasing hormone (crh) in hypothalamus, prolactin (prl) releasing peptide (pRrp) in telencephalon and diencephalon, glucocorticoid receptors 2 (gr2), and mineralocorticoid receptor (mr), crh-r, pro-opiomelanocorticotropin (pomc), pRrp, prl, dopamine 2 receptor (d2-r), growth hormone (gh), gh-receptor (gh-r) and insulin-like growth hormone (igf-1) transcripts in pituitary were characterized in euryhaline tilapia. The results indicate that the crh transcripts increased in the hypothalamus and rostral pars distalis of the pituitary after the transfer of fish to SW. Similarly, the pRrp transcripts were more abundant in SW acclimated tilapia forebrain and hypothalamus. The crh-r, gr2 and mr transcripts were more expressed in rostral pars distalis and pars intermedia of pituitary at SW than FW tilapia. The data indicate that the SW acclimation stimulates these transcripts in the specific regions of the brain and pituitary which may be related to the activation of the hypothalamic-pituitary-interrenal (HPI)-axis. The results of dual in situ hybridization reveal that the transcripts of crh-r, gr2 and mr with pomc are highly co-localized in corticotrophs of pituitary. Furthermore, we demonstrate high expression of pRrp in the brain and low expression of pRrp and prl transcripts in the pituitary of SW fish. No crh-r and corticosteroid receptors were co-localized with prl transcripts in the pituitary. The gh-r and igf-1 mRNA levels were significantly increased in SW acclimated tilapia pituitary whereas there was no difference in the gh mRNA levels. The data suggest that the locally produced pRrp and d2-r may control and regulate the expression of prl mRNA in pituitary. Therefore, the dual roles of pRrp are involved in the stress (via brain-pituitary) and osmoregulatory (via pituitary) pathways in tilapia exposed to acute salinity changes. Copyright © 2014 Elsevier Inc. All rights reserved.

Growth at elevated ozone or elevated carbon dioxide concentration alters antioxidant capacity and response to acute oxidative stress in soybean (Glycine max)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gillespie, K.M.; Rogers, A.; Ainsworth, E. A.

2011-01-31

Soybeans (Glycine max Merr.) were grown at elevated carbon dioxide concentration ([CO{sub 2}]) or chronic elevated ozone concentration ([O{sub 3}]; 90 ppb), and then exposed to an acute O{sub 3} stress (200 ppb for 4 h) in order to test the hypothesis that the atmospheric environment alters the total antioxidant capacity of plants, and their capacity to respond to an acute oxidative stress. Total antioxidant metabolism, antioxidant enzyme activity, and antioxidant transcript abundance were characterized before, immediately after, and during recovery from the acute O{sub 3} treatment. Growth at chronic elevated [O{sub 3}] increased the total antioxidant capacity of plants,more » while growth at elevated [CO{sub 2}] decreased the total antioxidant capacity. Changes in total antioxidant capacity were matched by changes in ascorbate content, but not phenolic content. The growth environment significantly altered the pattern of antioxidant transcript and enzyme response to the acute O{sub 3} stress. Following the acute oxidative stress, there was an immediate transcriptional reprogramming that allowed for maintained or increased antioxidant enzyme activities in plants grown at elevated [O{sub 3}]. Growth at elevated [CO{sub 2}] appeared to increase the response of antioxidant enzymes to acute oxidative stress, but dampened and delayed the transcriptional response. These results provide evidence that the growth environment alters the antioxidant system, the immediate response to an acute oxidative stress, and the timing over which plants return to initial antioxidant levels. The results also indicate that future elevated [CO{sub 2}] and [O{sub 3}] will differentially affect the antioxidant system.« less

Stress-Induced Transcriptional Regulation in the Developing Rat Brain Involves Increased Cyclic Adenosine 3′,5′-Monophosphate-Regulatory Element Binding Activity

PubMed Central

Hatalski, Carolyn G.; Baram, Tallie Z.

2012-01-01

The cAMP-regulatory element (CRE) binding protein (CREB) functions as a trans-acting regulator of genes containing the CRE sequence in their promoter. These include a number of critical genes, such as CRF, involved in the hypothalamic response to stressful stimuli in the adult. The ability of the developing rat (during the first 2 postnatal weeks) to mount the full complement of this stress response has been questioned. We have previously demonstrated the stress-induced up-regulation of the transcription of hypothalamic CRF during the second postnatal week in the rat. The focus of the current study was to explore the mechanism of transcriptional regulation in response to stress through the physiological induction of transcriptional trans-activators that bind to the CRE in the developing rat brain. CRE-binding activity was detected via gel shift analysis in extracts from both the hypothalamus and the cerebral cortex of the developing rat. CREB was identified in these extracts by Western blot analysis and was shown to be the major contributor to the CRE-binding activity by gel shift analysis with two specific antibodies directed against CREB. After acute hypothermic stress, the abundance of CRE-binding activity (but not of total immunoreactive CREB), increased in hypothalamic extracts. This enhanced CRE-binding activity was blocked by an antiserum directed against CREB and was accompanied by an apparent increase in CREB phosphorylation. These results indicate that posttranslational enhancement of CRE-binding activity is likely to constitute an important mechanism for up-regulation of genes possessing the CRE sequence in the developing rat hypothalamus by adverse external signals. PMID:9415405

Roles of Protein Kinase A and Adenylate Cyclase in Light-Modulated Cellulase Regulation in Trichoderma reesei

PubMed Central

Schuster, André; Tisch, Doris; Seidl-Seiboth, Verena; Kubicek, Christian P.

2012-01-01

The cyclic AMP (cAMP) pathway represents a central signaling cascade with crucial functions in all organisms. Previous studies of Trichoderma reesei (anamorph of Hypocrea jecorina) suggested a function of cAMP signaling in regulation of cellulase gene expression. We were therefore interested in how the crucial components of this pathway, adenylate cyclase (ACY1) and cAMP-dependent protein kinase A (PKA), would affect cellulase gene expression. We found that both ACY1 and PKA catalytic subunit 1 (PKAC1) are involved in regulation of vegetative growth but are not essential for sexual development. Interestingly, our results showed considerably increased transcript abundance of cellulase genes in darkness compared to light (light responsiveness) upon growth on lactose. This effect is strongly enhanced in mutant strains lacking PKAC1 or ACY1. Comparison to the wild type showed that ACY1 has a consistently positive effect on cellulase gene expression in light and darkness, while PKAC1 influences transcript levels of cellulase genes positively in light but negatively in darkness. A function of PKAC1 in light-modulated cellulase gene regulation is also reflected by altered complex formation within the cel6a/cbh2 promoter in light and darkness and in the absence of pkac1. Analysis of transcript levels of cellulase regulator genes indicates that the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance. Consequently, both adenylate cyclase and protein kinase A are involved in light-modulated cellulase gene expression in T. reesei and have a dampening effect on the light responsiveness of this process. PMID:22286997

Functional Transcriptomics of Wild-Caught Lutzomyia intermedia Salivary Glands: Identification of a Protective Salivary Protein against Leishmania braziliensis Infection

PubMed Central

Carneiro, Marcia W.; Miranda, José Carlos; Clarêncio, Jorge; Barral-Netto, Manoel; Brodskyn, Cláudia; Barral, Aldina; Ribeiro, José M. C.; Valenzuela, Jesus G.; de Oliveira, Camila I.

2013-01-01

Background Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG) homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins. Methods and Findings A cDNA library from the Salivary Glands (SGs) of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11—coding for a 4.5-kDa protein—induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells. Conclusions We identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis. PMID:23717705

Functional transcriptomics of wild-caught Lutzomyia intermedia salivary glands: identification of a protective salivary protein against Leishmania braziliensis infection.

PubMed

de Moura, Tatiana R; Oliveira, Fabiano; Carneiro, Marcia W; Miranda, José Carlos; Clarêncio, Jorge; Barral-Netto, Manoel; Brodskyn, Cláudia; Barral, Aldina; Ribeiro, José M C; Valenzuela, Jesus G; de Oliveira, Camila I

2013-01-01

Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG) homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins. A cDNA library from the Salivary Glands (SGs) of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11--coding for a 4.5-kDa protein--induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells. We identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis.

Influence of nitrogen availability on the expression of genes involved in the biosynthesis of saxitoxin and analogs in Cylindrospermopsis raciborskii.

PubMed

Vico, Paula; Aubriot, Luis; Martigani, Fátima; Rigamonti, Natalia; Bonilla, Sylvia; Piccini, Claudia

2016-06-01

The development of cyanobacterial blooms in inland aquatic ecosystems is greatly promoted by nutrient availability, especially nitrogen and phosphorous. When blooms are dominated by toxigenic species the harmful effects of nutrient loading becomes particularly relevant. Among toxic species, Cylindrospermopsis raciborskii found in South American ecosystems is characterized by the production of saxitoxin and analogs (Paralytic Shellfish Poisoning, PSP), for which the factors that trigger their production have not been elucidated. In this study, the effect of nitrate availability on the relative transcript abundance of two genes (sxtU and sxtI), both involved in different steps of PSP biosynthetic pathway, was addressed in C. raciborskii MVCC19 by qPCR. The relative transcript abundance of both genes significantly increased from the beginning to the end of growth, independent of nitrate availability in the culture medium. Differences between the genes in terms of the levels of relative expression were also found, implying that during growth in nitrate-rich or nitrate-deprived conditions C. raciborskii MVCC19 has the ability to produce different kind of PSP molecules. The presence of nifH transcripts in the nitrogen-depleted treatment confirmed that in the absence of nitrate C. raciborskii fixed atmospheric N 2 . Moreover, after transferring filaments to nitrate-rich conditions the synthesis of nifH mRNA continued for few hours, suggesting that cell adjustments enabling the utilization of soluble nitrogen sources are not immediate. Our results show that biosynthesis of saxitoxin and analogs in C. raciborskii is not related to nitrate availability, but rather is linked to cyanobacteria growth rate. Copyright © 2016. Published by Elsevier B.V.

Defining the consequences of genetic variation on a proteome–wide scale

PubMed Central

Chick, Joel M.; Munger, Steven C.; Simecek, Petr; Huttlin, Edward L.; Choi, Kwangbom; Gatti, Daniel M.; Raghupathy, Narayanan; Svenson, Karen L.; Churchill, Gary A.; Gygi, Steven P.

2016-01-01

Genetic variation modulates protein expression through both transcriptional and post-transcriptional mechanisms. To characterize the consequences of natural genetic diversity on the proteome, here we combine a multiplexed, mass spectrometry-based method for protein quantification with an emerging outbred mouse model containing extensive genetic variation from eight inbred founder strains. By measuring genome-wide transcript and protein expression in livers from 192 Diversity outbred mice, we identify 2,866 protein quantitative trait loci (pQTL) with twice as many local as distant genetic variants. These data support distinct transcriptional and post-transcriptional models underlying the observed pQTL effects. Using a sensitive approach to mediation analysis, we often identified a second protein or transcript as the causal mediator of distant pQTL. Our analysis reveals an extensive network of direct protein–protein interactions. Finally, we show that local genotype can provide accurate predictions of protein abundance in an independent cohort of collaborative cross mice. PMID:27309819

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed Central

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-01-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis. PMID:8892842

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-11-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.

Transcriptional Responses to Sucrose Mimic the Plant-Associated Life Style of the Plant Growth Promoting Endophyte Enterobacter sp. 638

DOE PAGES

Taghavi, Safiyh; Wu, Xiao; Ouyang, Liming; ...

2015-01-21

Growth in sucrose medium was previously found to trigger the expression of functions involved in the plant associated life style of the endophytic bacterium Enterobacter sp. 638. Therefore, comparative transcriptome analysis between cultures grown in sucrose or lactate medium was used to gain insights in the expression levels of bacterial functions involved in the endophytic life style of strain 638. Growth on sucrose as a carbon source resulted in major changes in cell physiology, including a shift from a planktonic life style to the formation of bacterial aggregates. This shift was accompanied by a decrease in transcription of genes involvedmore » in motility (e.g. flagella biosynthesis) and an increase in the transcription of genes involved in colonization, adhesion and biofilm formation. The transcription levels of functions previously suggested as being involved in endophytic behavior and functions responsible for plant growth promoting properties, including the synthesis of indole-acetic acid, acetoin and 2,3-butanediol, also increased significantly for cultures grown in sucrose medium. Interestingly, despite an abundance of essential nutrients transcription levels of functions related to uptake and processing of nitrogen and iron became increased for cultures grown on sucrose as sole carbon source. Transcriptome data were also used to analyze putative regulatory relationships. In addition to the small RNA csrABCD regulon, which seems to play a role in the physiological adaptation and possibly the shift between free-living and plant-associated endophytic life style of Enterobacter sp. 638, our results also pointed to the involvement of rcsAB in controlling responses by Enterobacter sp. 638 to a plant-associated life style. Lastly, targeted mutagenesis was used to confirm this role and showed that compared to wild-type Enterobacter sp. 638 a ΔrcsB mutant was affected in its plant growth promoting ability.« less

Transcriptional Responses to Sucrose Mimic the Plant-Associated Life Style of the Plant Growth Promoting Endophyte Enterobacter sp. 638

PubMed Central

Taghavi, Safiyh; Wu, Xiao; Ouyang, Liming; Stadler, Andrea; McCorkle, Sean; Zhu, Wei; Maslov, Sergei; van der Lelie, Daniel

2015-01-01

Growth in sucrose medium was previously found to trigger the expression of functions involved in the plant associated life style of the endophytic bacterium Enterobacter sp. 638. Therefore, comparative transcriptome analysis between cultures grown in sucrose or lactate medium was used to gain insights in the expression levels of bacterial functions involved in the endophytic life style of strain 638. Growth on sucrose as a carbon source resulted in major changes in cell physiology, including a shift from a planktonic life style to the formation of bacterial aggregates. This shift was accompanied by a decrease in transcription of genes involved in motility (e.g. flagella biosynthesis) and an increase in the transcription of genes involved in colonization, adhesion and biofilm formation. The transcription levels of functions previously suggested as being involved in endophytic behavior and functions responsible for plant growth promoting properties, including the synthesis of indole-acetic acid, acetoin and 2,3-butanediol, also increased significantly for cultures grown in sucrose medium. Interestingly, despite an abundance of essential nutrients transcription levels of functions related to uptake and processing of nitrogen and iron became increased for cultures grown on sucrose as sole carbon source. Transcriptome data were also used to analyze putative regulatory relationships. In addition to the small RNA csrABCD regulon, which seems to play a role in the physiological adaptation and possibly the shift between free-living and plant-associated endophytic life style of Enterobacter sp. 638, our results also pointed to the involvement of rcsAB in controlling responses by Enterobacter sp. 638 to a plant-associated life style. Targeted mutagenesis was used to confirm this role and showed that compared to wild-type Enterobacter sp. 638 a ΔrcsB mutant was affected in its plant growth promoting ability. PMID:25607953

Transcriptional responses to sucrose mimic the plant-associated life style of the plant growth promoting endophyte Enterobacter sp. 638.

PubMed

Taghavi, Safiyh; Wu, Xiao; Ouyang, Liming; Zhang, Yian Biao; Stadler, Andrea; McCorkle, Sean; Zhu, Wei; Maslov, Sergei; van der Lelie, Daniel

2015-01-01

Growth in sucrose medium was previously found to trigger the expression of functions involved in the plant associated life style of the endophytic bacterium Enterobacter sp. 638. Therefore, comparative transcriptome analysis between cultures grown in sucrose or lactate medium was used to gain insights in the expression levels of bacterial functions involved in the endophytic life style of strain 638. Growth on sucrose as a carbon source resulted in major changes in cell physiology, including a shift from a planktonic life style to the formation of bacterial aggregates. This shift was accompanied by a decrease in transcription of genes involved in motility (e.g., flagella biosynthesis) and an increase in the transcription of genes involved in colonization, adhesion and biofilm formation. The transcription levels of functions previously suggested as being involved in endophytic behavior and functions responsible for plant growth promoting properties, including the synthesis of indole-acetic acid, acetoin and 2,3-butanediol, also increased significantly for cultures grown in sucrose medium. Interestingly, despite an abundance of essential nutrients transcription levels of functions related to uptake and processing of nitrogen and iron became increased for cultures grown on sucrose as sole carbon source. Transcriptome data were also used to analyze putative regulatory relationships. In addition to the small RNA csrABCD regulon, which seems to play a role in the physiological adaptation and possibly the shift between free-living and plant-associated endophytic life style of Enterobacter sp. 638, our results also pointed to the involvement of rcsAB in controlling responses by Enterobacter sp. 638 to a plant-associated life style. Targeted mutagenesis was used to confirm this role and showed that compared to wild-type Enterobacter sp. 638 a ΔrcsB mutant was affected in its plant growth promoting ability.

Transcriptional Responses to Sucrose Mimic the Plant-Associated Life Style of the Plant Growth Promoting Endophyte Enterobacter sp. 638

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taghavi, Safiyh; Wu, Xiao; Ouyang, Liming

Growth in sucrose medium was previously found to trigger the expression of functions involved in the plant associated life style of the endophytic bacterium Enterobacter sp. 638. Therefore, comparative transcriptome analysis between cultures grown in sucrose or lactate medium was used to gain insights in the expression levels of bacterial functions involved in the endophytic life style of strain 638. Growth on sucrose as a carbon source resulted in major changes in cell physiology, including a shift from a planktonic life style to the formation of bacterial aggregates. This shift was accompanied by a decrease in transcription of genes involvedmore » in motility (e.g. flagella biosynthesis) and an increase in the transcription of genes involved in colonization, adhesion and biofilm formation. The transcription levels of functions previously suggested as being involved in endophytic behavior and functions responsible for plant growth promoting properties, including the synthesis of indole-acetic acid, acetoin and 2,3-butanediol, also increased significantly for cultures grown in sucrose medium. Interestingly, despite an abundance of essential nutrients transcription levels of functions related to uptake and processing of nitrogen and iron became increased for cultures grown on sucrose as sole carbon source. Transcriptome data were also used to analyze putative regulatory relationships. In addition to the small RNA csrABCD regulon, which seems to play a role in the physiological adaptation and possibly the shift between free-living and plant-associated endophytic life style of Enterobacter sp. 638, our results also pointed to the involvement of rcsAB in controlling responses by Enterobacter sp. 638 to a plant-associated life style. Lastly, targeted mutagenesis was used to confirm this role and showed that compared to wild-type Enterobacter sp. 638 a ΔrcsB mutant was affected in its plant growth promoting ability.« less

Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo

PubMed Central

Rijal, Keshab; Maraia, Richard J.

2016-01-01

The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease. PMID:27518095

Transcriptomic and proteomic analyses of the Aspergillus fumigatus hypoxia response using an oxygen-controlled fermenter

PubMed Central

2012-01-01

Background Aspergillus fumigatus is a mold responsible for the majority of cases of aspergillosis in humans. To survive in the human body, A. fumigatus must adapt to microenvironments that are often characterized by low nutrient and oxygen availability. Recent research suggests that the ability of A. fumigatus and other pathogenic fungi to adapt to hypoxia contributes to their virulence. However, molecular mechanisms of A. fumigatus hypoxia adaptation are poorly understood. Thus, to better understand how A. fumigatus adapts to hypoxic microenvironments found in vivo during human fungal pathogenesis, the dynamic changes of the fungal transcriptome and proteome in hypoxia were investigated over a period of 24 hours utilizing an oxygen-controlled fermenter system. Results Significant increases in transcripts associated with iron and sterol metabolism, the cell wall, the GABA shunt, and transcriptional regulators were observed in response to hypoxia. A concomitant reduction in transcripts was observed with ribosome and terpenoid backbone biosynthesis, TCA cycle, amino acid metabolism and RNA degradation. Analysis of changes in transcription factor mRNA abundance shows that hypoxia induces significant positive and negative changes that may be important for regulating the hypoxia response in this pathogenic mold. Growth in hypoxia resulted in changes in the protein levels of several glycolytic enzymes, but these changes were not always reflected by the corresponding transcriptional profiling data. However, a good correlation overall (R2 = 0.2, p < 0.05) existed between the transcriptomic and proteomics datasets for all time points. The lack of correlation between some transcript levels and their subsequent protein levels suggests another regulatory layer of the hypoxia response in A. fumigatus. Conclusions Taken together, our data suggest a robust cellular response that is likely regulated both at the transcriptional and post-transcriptional level in response to hypoxia by the human pathogenic mold A. fumigatus. As with other pathogenic fungi, the induction of glycolysis and transcriptional down-regulation of the TCA cycle and oxidative phosphorylation appear to major components of the hypoxia response in this pathogenic mold. In addition, a significant induction of the transcripts involved in ergosterol biosynthesis is consistent with previous observations in the pathogenic yeasts Candida albicans and Cryptococcus neoformans indicating conservation of this response to hypoxia in pathogenic fungi. Because ergosterol biosynthesis enzymes also require iron as a co-factor, the increase in iron uptake transcripts is consistent with an increased need for iron under hypoxia. However, unlike C. albicans and C. neoformans, the GABA shunt appears to play an important role in reducing NADH levels in response to hypoxia in A. fumigatus and it will be intriguing to determine whether this is critical for fungal virulence. Overall, regulatory mechanisms of the A. fumigatus hypoxia response appear to involve both transcriptional and post-transcriptional control of transcript and protein levels and thus provide candidate genes for future analysis of their role in hypoxia adaptation and fungal virulence. PMID:22309491

Combined Training Enhances Skeletal Muscle Mitochondrial Oxidative Capacity Independent of Age

PubMed Central

Lanza, Ian R.; Henderson, Gregory C.; Rao, Rajesh R.; Spiegelman, Bruce M.

2015-01-01

Context: Skeletal muscle from sedentary older adults exhibits reduced mitochondrial abundance and oxidative capacity. Objective: The primary objective was to determine whether 8 weeks of combined training (CT) has a more robust effect than endurance training (ET) or resistance training (RT) on mitochondrial physiology in healthy young (18–30 years) and older (≥65 years) adults. Intervention: Thirty-four young and 31 older adults were randomly assigned to 8 weeks of ET, RT, and control/CT. Control subjects completed 8 weeks of no exercise (control) followed by 8 weeks of CT. Body composition, skeletal muscle strength, and peak oxygen uptake were measured before and after the intervention. Vastus lateralis muscle biopsy samples were obtained before and 48 hours after the intervention. Mitochondrial physiology was evaluated by high-resolution respirometry and expression of mitochondrial proteins and transcription factors by quantitative PCR and immunoblotting. Results: ET and CT significantly increased oxidative capacity and expression of mitochondrial proteins and transcription factors. All training modalities improved body composition, cardiorespiratory fitness, and skeletal muscle strength. CT induced the most robust improvements in mitochondria-related outcomes and physical characteristics despite lower training volumes for the ET and RT components. Importantly, most of the adaptations to training occurred independent of age. Conclusion: Collectively, these results demonstrate that both ET and CT increase muscle mitochondrial abundance and capacity although CT induced the most robust improvements in the outcomes measured. In conclusion, CT provides a robust exercise regimen to improve muscle mitochondrial outcomes and physical characteristics independent of age. PMID:25599385

Cortisol elevation post-hatch affects behavioural performance in zebrafish larvae.

PubMed

Best, Carol; Vijayan, Mathilakath M

2018-02-01

Maternal cortisol is essential for cortisol stress axis development and de novo production of this steroid commences only after hatch in zebrafish (Danio rerio). However, very little is known about the effect of elevated cortisol levels, during the critical period of stress axis activation, on larval performance. We tested the hypothesis that elevated cortisol levels post-hatch affect behavioural performance and this is mediated by glucocorticoid receptor (GR) activation in zebrafish larvae. The behavioural response included measuring larval activity in response to alternating light and dark cycles, as well as thigmotaxis. Zebrafish larvae at 3days post-fertilization were exposed to waterborne cortisol for 24h to mimic a steroid response to an early-life stressor exposure. Also, larvae were exposed to waterborne RU-486 (a GR antagonist) either in the presence or absence of cortisol to confirm GR activation. Co-treatment with RU-486 completely abolished the upregulation of cortisol-induced 11β-hydroxysteroid dehydrogenase type 2 transcript abundance, confirming GR signalling. Cortisol-exposed larvae displayed increased locomotor activity irrespective of light condition, but showed no changes in thigmotaxis. This cortisol-mediated behavioural response was not affected by co-treatment with RU-486. Cortisol exposure also did not modify the transcript abundances of GR and mineralocorticoid receptor (MR) in zebrafish larvae. Altogether, cortisol stress axis activation post-hatch increases locomotor activity in zebrafish larvae. Our results suggest that GR signalling may not be involved in this behavioural response, leading to the proposal that cortisol action via MR signalling may influence locomotor activity in zebrafish larvae. Copyright © 2017 Elsevier Inc. All rights reserved.

Stress axis regulation during social ascension in a group-living cichlid fish.

PubMed

Culbert, Brett M; Gilmour, Kathleen M; Balshine, Sigal

2018-06-19

Animals living in groups often form social hierarchies, with characteristic behaviours and physiologies associated with rank. However, when social opportunities arise and a subordinate ascends into a dominant position, quick adjustments are necessary to secure this position. Such periods of social transition are typically associated with elevated glucocorticoid production, but the precise regulation of the stress axis during these occasions is not well understood. Using the group-living cichlid, Neolamprologus pulcher, the effects of social ascension on the stress axis were assessed. Ascenders rapidly filled experimentally created vacancies, adopting a dominant behavioural phenotype within 72 h-elevating aggression, activity, and workload, while receiving high rates of affiliative behaviours from their group members. Despite assuming behavioural dominance within their groups, ascenders displayed higher cortisol levels than dominants three days post-ascension. Additionally, compared to subordinates, ascenders had increased transcript abundance of steroidogenic acute regulatory protein (star) and cytochrome p450 side-chain cleavage enzyme (p450scc) in the head kidney, indicating activation of the stress axis. Cortisol levels were lowest in ascenders that displayed low rates of aggression, potentially reflecting the reestablishment of social stability in these groups. Increased transcript abundance of both glucocorticoid receptors (gr1 and gr2) in the brain's preoptic area (POA) of ascenders compared to dominants suggested an enhanced capacity for cortisol regulation via negative feedback. Our results reveal a regulatory cascade of behavioural and physiological interactions and highlight the importance of investigating the underlying mechanisms regulating the stress axis. Copyright © 2018. Published by Elsevier Inc.

Micronuclei and other erythrocyte nuclear abnormalities in fishes from the Great Lakes Basin, USA.

PubMed

Braham, Ryan P; Blazer, Vicki S; Shaw, Cassidy H; Mazik, Patricia M

2017-10-01

Biological markers (biomarkers) sensitive to genotoxic and mutagenic contamination in fishes are widely used to identify exposure effects in aquatic environments. The micronucleus assay was incorporated into a suite of indicators to assess exposure to genotoxic and mutagenic contamination at five Great Lakes Areas of Concern (AOCs), as well as one non-AOC (reference) site. The assay allowed enumeration of micronuclei as well as other nuclear abnormalities for both site and species comparisons. Erythrocyte abnormality data was also compared to skin and liver tumor prevalence and hepatic transcript abundance. Erythrocyte abnormalities were observed at all sites with variable occurrence and severity among sites and species. Benthic-oriented brown bullhead (Ameiurus nebulosus) and white sucker (Catostomus commersonii) expressed lower rates of erythrocyte abnormalities, but higher rates of skin and liver neoplasms, when compared to pelagic-oriented largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) at the same site. The reduced erythrocyte abnormalities, increased transcript abundance associated with Phase I and II toxicant responsive pathways, and increased neoplastic lesions among benthic-oriented taxa may indicate the development of contaminant resistance of these species to more acute effects. Environ. Mol. Mutagen. 58:570-581, 2017. © 2017 This article is a U.S. Government work and is in the public domain in the USA. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society. © 2017 This article is a U.S. Government work and is in the public domain in the USA. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

S-phase duration is the main target of cell cycle regulation in neural progenitors of developing ferret neocortex.

PubMed

Turrero García, Miguel; Chang, YoonJeung; Arai, Yoko; Huttner, Wieland B

2016-02-15

The evolutionary expansion of the neocortex primarily reflects increases in abundance and proliferative capacity of cortical progenitors and in the length of the neurogenic period during development. Cell cycle parameters of neocortical progenitors are an important determinant of cortical development. The ferret (Mustela putorius furo), a gyrencephalic mammal, has gained increasing importance as a model for studying corticogenesis. Here, we have studied the abundance, proliferation, and cell cycle parameters of different neural progenitor types, defined by their differential expression of the transcription factors Pax6 and Tbr2, in the various germinal zones of developing ferret neocortex. We focused our analyses on postnatal day 1, a late stage of cortical neurogenesis when upper-layer neurons are produced. Based on cumulative 5-ethynyl-2'-deoxyuridine (EdU) labeling as well as Ki67 and proliferating cell nuclear antigen (PCNA) immunofluorescence, we determined the duration of the various cell cycle phases of the different neocortical progenitor subpopulations. Ferret neocortical progenitors were found to exhibit longer cell cycles than those of rodents and little variation in the duration of G1 among distinct progenitor types, also in contrast to rodents. Remarkably, the main difference in cell cycle parameters among the various progenitor types was the duration of S-phase, which became shorter as progenitors progressively changed transcription factor expression from patterns characteristic of self-renewal to those of neuron production. Hence, S-phase duration emerges as major target of cell cycle regulation in cortical progenitors of this gyrencephalic mammal. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

The expression of fibroblast growth factor receptors during early bovine conceptus development and pharmacological analysis of their actions on trophoblast growth in vitro.

PubMed

Ozawa, Manabu; Yang, Qi-En; Ealy, Alan D

2013-02-01

The overall aim of this work was to examine the expression profiles for fibroblast growth factor receptors (FGFRs) and describe their biological importance during bovine pre- and peri-implantation conceptus development. FGFR1 and FGFR2 mRNAs were detected at 1-, 2-, 8-cell, morula and blastocyst stages whereas FGFR3 and FGFR4 mRNAs were detected after the 8-cell stage but not earlier. The abundance of FGFR1, FGFR3, and FGFR4 mRNAs increased at the morula and blastocyst stages. Immunofluorescence microscopy detected FGFR2 and FGFR4 exclusively in trophoblast cells whereas FGFR1 and FGFR3 were detected in both trophoblast cells and inner cell mass in blastocysts. Neither transcripts for FGF10 nor its receptor (FGFR2b) were temporally related to interferon τ (IFNT) transcript profile during peri- and postimplantation bovine conceptus development. A series of studies used a chemical inhibitor of FGFR kinase function (PD173074) to examine FGFR activation requirements during bovine embryo development. Exposing embryos to the inhibitor (1 μM) beginning on day 5 post-fertilization did not alter the percentage of embryos that developed into blastocysts or blastocyst cell numbers. The inhibitor did not alter the abundance of CDX2 mRNA but decreased (P<0.05) the relative abundance of IFNT mRNA in blastocysts. Exposing blastocysts to the inhibitor from days 8 to 11 post-fertilization reduced (P<0.05) the percentage of blastocysts that formed outgrowths after transfer to Matrigel-coated plates. In conclusion, each FGFR was detected in bovine embryos, and FGFR activation is needed to maximize IFNT expression and permit outgrowth formation.

PSMA redirects cell survival signaling from the MAPK to the PI3K-AKT pathways to promote the progression of prostate cancer

PubMed Central

Caromile, Leslie Ann; Dortche, Kristina; Rahman, M. Mamunur; Grant, Christina L.; Stoddard, Christopher; Ferrer, Fernando A.; Shapiro, Linda H.

2017-01-01

Increased abundance of the prostate-specific membrane antigen (PSMA) on prostate epithelium is a hallmark of advanced metastatic prostate cancer (PCa) and correlates negatively with prognosis. However, direct evidence that PSMA functionally contributes to PCa progression remains elusive. We generated mice bearing PSMA-positive or PSMA-negative PCa by crossing PSMA-deficient mice with transgenic PCa (TRAMP) models, enabling direct assessment of PCa incidence and progression in the presence or absence of PSMA. Compared with PSMA-positive tumors, PSMA-negative tumors were smaller, lower-grade, and more apoptotic with fewer blood vessels, consistent with the recognized proangiogenic function of PSMA. Relative to PSMA-positive tumors, tumors lacking PSMA had less than half the abundance of type 1 insulin-like growth factor receptor (IGF-1R), less activity in the survival pathway mediated by PI3K-AKT signaling, and more activity in the proliferative pathway mediated by MAPK-ERK1/2 signaling. Biochemically, PSMA interacted with the scaffolding protein RACK1, disrupting signaling between the β1 integrin and IGF-1R complex to the MAPK pathway, enabling activation of the AKT pathway instead. Manipulation of PSMA abundance in PCa cell lines recapitulated this signaling pathway switch. Analysis of published databases indicated that IGF-1R abundance, cell proliferation, and expression of transcripts for antiapoptotic markers positively correlated with PSMA abundance in patients, suggesting that this switch may be relevant to human PCa. Our findings suggest that increase in PSMA in prostate tumors contributes to progression by altering normal signal transduction pathways to drive PCa progression and that enhanced signaling through the IGF-1R/β1 integrin axis may occur in other tumors. PMID:28292957

Coping with unpredictability: dopaminergic and neurotrophic responses to omission of expected reward in Atlantic salmon (Salmo salar L.).

PubMed

Vindas, Marco A; Sørensen, Christina; Johansen, Ida B; Folkedal, Ole; Höglund, Erik; Khan, Uniza W; Stien, Lars H; Kristiansen, Tore S; Braastad, Bjarne O; Øverli, Øyvind

2014-01-01

Comparative studies are imperative for understanding the evolution of adaptive neurobiological processes such as neural plasticity, cognition, and emotion. Previously we have reported that prolonged omission of expected rewards (OER, or 'frustrative nonreward') causes increased aggression in Atlantic salmon (Salmo salar). Here we report changes in brain monoaminergic activity and relative abundance of brain derived neurotrophic factor (BDNF) and dopamine receptor mRNA transcripts in the same paradigm. Groups of fish were initially conditioned to associate a flashing light with feeding. Subsequently, the expected food reward was delayed for 30 minutes during two out of three meals per day in the OER treatment, while the previously established routine was maintained in control groups. After 8 days there was no effect of OER on baseline brain stem serotonin (5-HT) or dopamine (DA) activity. Subsequent exposure to acute confinement stress led to increased plasma cortisol and elevated turnover of brain stem DA and 5-HT in all animals. The DA response was potentiated and DA receptor 1 (D1) mRNA abundance was reduced in the OER-exposed fish, indicating a sensitization of the DA system. In addition OER suppressed abundance of BDNF in the telencephalon of non-stressed fish. Regardless of OER treatment, a strong positive correlation between BDNF and D1 mRNA abundance was seen in non-stressed fish. This correlation was disrupted by acute stress, and replaced by a negative correlation between BDNF abundance and plasma cortisol concentration. These observations indicate a conserved link between DA, neurotrophin regulation, and corticosteroid-signaling pathways. The results also emphasize how fish models can be important tools in the study of neural plasticity and responsiveness to environmental unpredictability.

Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

PubMed Central

Calvanese, Vincenzo; Mallya, Meera; Campbell, R Duncan; Aguado, Begoña

2008-01-01

Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD). This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC). This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F) undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C) and not on their own. PMID:18817541

Annexin A7 deficiency potentiates cardiac NFAT activity promoting hypertrophic signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voelkl, Jakob; Alesutan, Ioana; Pakladok, Tatsiana

Highlights: • Cardiac Anxa7 expression was up-regulated following TAC. • The hypertrophic response following TAC was augmented in Anxa7-deficient mice. • Silencing of Anxa7 increased indicators of HL-1 cardiomyocytes hypertrophy. • Silencing of Anxa7 induced Nfatc1 nuclear translocation. • Silencing of Anxa7 enhanced NFAT-dependent transcriptional activity. - Abstract: Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca{sup 2+} signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7{sup −/−}) and wild-type mice (anxa7{supmore » +/+}) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7{sup −/−} mice than in anxa7{sup +/+} mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following β-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions.« less

Systematic analysis of phloem-feeding insect-induced transcriptional reprogramming in Arabidopsis highlights common features and reveals distinct responses to specialist and generalist insects.

PubMed

Foyer, Christine H; Verrall, Susan R; Hancock, Robert D

2015-02-01

Phloem-feeding insects (PFIs), of which aphids are the largest group, are major agricultural pests causing extensive damage to crop plants. In contrast to chewing insects, the nature of the plant response to PFIs remains poorly characterized. Scrutiny of the literature concerning transcriptional responses of model and crop plant species to PFIs reveals surprisingly little consensus with respect to the transcripts showing altered abundance following infestation. Nevertheless, core features of the transcriptional response to PFIs can be defined in Arabidopsis thaliana. This comparison of the PFI-associated transcriptional response observed in A. thaliana infested by the generalists Myzus persicae and Bemisia tabaci with the specialist Brevicoryne brassicae highlights the importance of calcium-dependent and receptor kinase-associated signalling. We discuss these findings within the context of the complex cross-talk between the different hormones regulating basal immune response mechanisms in plants. We identify PFI-responsive genes, highlighting the importance of cell wall-associated kinases in plant-PFI interactions, as well as the significant role of kinases containing the domain of unknown function 26. A common feature of plant-PFI interaction is enhanced abundance of transcripts encoding WRKY transcription factors. However, significant divergence was observed with respect to secondary metabolism dependent upon the insect attacker. Transcripts encoding enzymes and proteins associated with glucosinolate metabolism were decreased following attack by the generalist M. persicae but not by the specialist B. brassicae. This analysis provides a comprehensive overview of the molecular patterns associated with the plant response to PFIs and suggests that plants recognize and respond to perturbations in the cell wall occurring during PFI infestation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Systems-based analysis of Arabidopsis leaf growth reveals adaptation to water deficit

PubMed Central

Baerenfaller, Katja; Massonnet, Catherine; Walsh, Sean; Baginsky, Sacha; Bühlmann, Peter; Hennig, Lars; Hirsch-Hoffmann, Matthias; Howell, Katharine A; Kahlau, Sabine; Radziejwoski, Amandine; Russenberger, Doris; Rutishauser, Dorothea; Small, Ian; Stekhoven, Daniel; Sulpice, Ronan; Svozil, Julia; Wuyts, Nathalie; Stitt, Mark; Hilson, Pierre; Granier, Christine; Gruissem, Wilhelm

2012-01-01

Leaves have a central role in plant energy capture and carbon conversion and therefore must continuously adapt their development to prevailing environmental conditions. To reveal the dynamic systems behaviour of leaf development, we profiled Arabidopsis leaf number six in depth at four different growth stages, at both the end-of-day and end-of-night, in plants growing in two controlled experimental conditions: short-day conditions with optimal soil water content and constant reduced soil water conditions. We found that the lower soil water potential led to reduced, but prolonged, growth and an adaptation at the molecular level without a drought stress response. Clustering of the protein and transcript data using a decision tree revealed different patterns in abundance changes across the growth stages and between end-of-day and end-of-night that are linked to specific biological functions. Correlations between protein and transcript levels depend on the time-of-day and also on protein localisation and function. Surprisingly, only very few of >1700 quantified proteins showed diurnal abundance fluctuations, despite strong fluctuations at the transcript level. PMID:22929616

The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression.

PubMed

Zhou, Fei; Sun, Tian-Hu; Zhao, Lei; Pan, Xi-Wu; Lu, Shan

2015-01-01

The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, -421 bp from the translation initiation site) which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA), we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS) transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD) and constant dark (DD) conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC) driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant.

Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex.

PubMed

Chow, Keng-See; Wan, Kiew-Lian; Isa, Mohd Noor Mat; Bahari, Azlina; Tan, Siang-Hee; Harikrishna, K; Yeang, Hoong-Yeet

2007-01-01

Hevea brasiliensis is the most widely cultivated species for commercial production of natural rubber (cis-polyisoprene). In this study, 10,040 expressed sequence tags (ESTs) were generated from the latex of the rubber tree, which represents the cytoplasmic content of a single cell type, in order to analyse the latex transcription profile with emphasis on rubber biosynthesis-related genes. A total of 3,441 unique transcripts (UTs) were obtained after quality editing and assembly of EST sequences. Functional classification of UTs according to the Gene Ontology convention showed that 73.8% were related to genes of unknown function. Among highly expressed ESTs, a significant proportion encoded proteins related to rubber biosynthesis and stress or defence responses. Sequences encoding rubber particle membrane proteins (RPMPs) belonging to three protein families accounted for 12% of the ESTs. Characterization of these ESTs revealed nine RPMP variants (7.9-27 kDa) including the 14 kDa REF (rubber elongation factor) and 22 kDa SRPP (small rubber particle protein). The expression of multiple RPMP isoforms in latex was shown using antibodies against REF and SRPP. Both EST and quantitative reverse transcription-PCR (QRT-PCR) analyses demonstrated REF and SRPP to be the most abundant transcripts in latex. Besides rubber biosynthesis, comparative sequence analysis showed that the RPMPs are highly similar to sequences in the plant kingdom having stress-related functions. Implications of the RPMP function in cis-polyisoprene biosynthesis in the context of transcript abundance and differential gene expression are discussed.

Cleavage of rRNA ensures translational cessation in sperm at fertilization

PubMed Central

Johnson, G.D.; Sendler, E.; Lalancette, C.; Hauser, R.; Diamond, M.P.; Krawetz, S.A.

2011-01-01

Intact ribosomal RNAs (rRNAs) comprise the majority of somatic transcripts, yet appear conspicuously absent in spermatozoa, perhaps reflecting cytoplasmic expulsion during spermatogenesis. To discern their fate, total RNA retained in mature spermatozoa from three fertile donors was characterized by Next Generation Sequencing. In all samples, >75% of total sequence reads aligned to rRNAs. The distribution of reads along the length of these transcripts exhibited a high degree of non-uniformity that was reiterated between donors. The coverage of sequencing reads was inversely correlated with guanine-cytosine (GC)-richness such that sequences greater than ∼70% GC were virtually absent in all sperm RNA samples. To confirm the loss of sequence, the relative abundance of specific regions of the 28S transcripts in sperm was established by 7-Deaza-2′-deoxy-guanosine-5′-triphosphate RT–PCR. The inability to amplify specific regions of the 28S sequence from sperm despite the abundant representation of this transcript in the sequencing libraries demonstrates that approximately three-quarters of RNA retained in the mature male gamete are products of rRNA fragmentation. Hence, cleavage (not expulsion of the RNA component of the translational machinery) is responsible for preventing spurious translation following spermiogenesis. These results highlight the potential importance of those transcripts, including many mRNAs, which evade fragmentation and remain intact when sperm are delivered at fertilization. Sequencing data are deposited in GEO as: GSE29160. PMID:21831882

Plasticity of the Chemoreceptor Repertoire in Drosophila melanogaster

PubMed Central

Zhou, Shanshan; Stone, Eric A.; Mackay, Trudy F. C.; Anholt, Robert R. H.

2009-01-01

For most organisms, chemosensation is critical for survival and is mediated by large families of chemoreceptor proteins, whose expression must be tuned appropriately to changes in the chemical environment. We asked whether expression of chemoreceptor genes that are clustered in the genome would be regulated independently; whether expression of certain chemoreceptor genes would be especially sensitive to environmental changes; whether groups of chemoreceptor genes undergo coordinated rexpression; and how plastic the expression of chemoreceptor genes is with regard to sex, development, reproductive state, and social context. To answer these questions we used Drosophila melanogaster, because its chemosensory systems are well characterized and both the genotype and environment can be controlled precisely. Using customized cDNA microarrays, we showed that chemoreceptor genes that are clustered in the genome undergo independent transcriptional regulation at different developmental stages and between sexes. Expression of distinct subgroups of chemoreceptor genes is sensitive to reproductive state and social interactions. Furthermore, exposure of flies only to odor of the opposite sex results in altered transcript abundance of chemoreceptor genes. These genes are distinct from those that show transcriptional plasticity when flies are allowed physical contact with same or opposite sex members. We analyzed covariance in transcript abundance of chemosensory genes across all environmental conditions and found that they segregated into 20 relatively small, biologically relevant modules of highly correlated transcripts. This finely pixilated modular organization of the chemosensory subgenome enables fine tuning of the expression of the chemoreceptor repertoire in response to ecologically relevant environmental and physiological conditions. PMID:19816562

Bayesian Inference of Allele-Specific Gene Expression Indicates Abundant Cis-Regulatory Variation in Natural Flycatcher Populations

PubMed Central

Wang, Mi

2017-01-01

Abstract Polymorphism in cis-regulatory sequences can lead to different levels of expression for the two alleles of a gene, providing a starting point for the evolution of gene expression. Little is known about the genome-wide abundance of genetic variation in gene regulation in natural populations but analysis of allele-specific expression (ASE) provides a means for investigating such variation. We performed RNA-seq of multiple tissues from population samples of two closely related flycatcher species and developed a Bayesian algorithm that maximizes data usage by borrowing information from the whole data set and combines several SNPs per transcript to detect ASE. Of 2,576 transcripts analyzed in collared flycatcher, ASE was detected in 185 (7.2%) and a similar frequency was seen in the pied flycatcher. Transcripts with statistically significant ASE commonly showed the major allele in >90% of the reads, reflecting that power was highest when expression was heavily biased toward one of the alleles. This would suggest that the observed frequencies of ASE likely are underestimates. The proportion of ASE transcripts varied among tissues, being lowest in testis and highest in muscle. Individuals often showed ASE of particular transcripts in more than one tissue (73.4%), consistent with a genetic basis for regulation of gene expression. The results suggest that genetic variation in regulatory sequences commonly affects gene expression in natural populations and that it provides a seedbed for phenotypic evolution via divergence in gene expression. PMID:28453623

Nonhost Resistance of Barley to Different Fungal Pathogens Is Associated with Largely Distinct, Quantitative Transcriptional Responses1[W][OA

PubMed Central

Zellerhoff, Nina; Himmelbach, Axel; Dong, Wubei; Bieri, Stephane; Schaffrath, Ulrich; Schweizer, Patrick

2010-01-01

Nonhost resistance protects plants against attack by the vast majority of potential pathogens, including phytopathogenic fungi. Despite its high biological importance, the molecular architecture of nonhost resistance has remained largely unexplored. Here, we describe the transcriptional responses of one particular genotype of barley (Hordeum vulgare subsp. vulgare ‘Ingrid’) to three different pairs of adapted (host) and nonadapted (nonhost) isolates of fungal pathogens, which belong to the genera Blumeria (powdery mildew), Puccinia (rust), and Magnaporthe (blast). Nonhost resistance against each of these pathogens was associated with changes in transcript abundance of distinct sets of nonhost-specific genes, although general (not nonhost-associated) transcriptional responses to the different pathogens overlapped considerably. The powdery mildew- and blast-induced differences in transcript abundance between host and nonhost interactions were significantly correlated with differences between a near-isogenic pair of barley lines that carry either the Mlo wild-type allele or the mutated mlo5 allele, which mediates basal resistance to powdery mildew. Moreover, during the interactions of barley with the different host or nonhost pathogens, similar patterns of overrepresented and underrepresented functional categories of genes were found. The results suggest that nonhost resistance and basal host defense of barley are functionally related and that nonhost resistance to different fungal pathogens is associated with more robust regulation of complex but largely nonoverlapping sets of pathogen-responsive genes involved in similar metabolic or signaling pathways. PMID:20172964

Transcriptional Regulation of S Phase Kinase-associated Protein 2 by NR4A Orphan Nuclear Receptor NOR1 in Vascular Smooth Muscle Cells*

PubMed Central

Gizard, Florence; Zhao, Yue; Findeisen, Hannes M.; Qing, Hua; Cohn, Dianne; Heywood, Elizabeth B.; Jones, Karrie L.; Nomiyama, Takashi; Bruemmer, Dennis

2011-01-01

Members of the NR4A subgroup of the nuclear hormone receptor superfamily have emerged as key transcriptional regulators of proliferation and inflammation. NOR1 constitutes a ligand-independent transcription factor of this subgroup and induces cell proliferation; however, the transcriptional mechanisms underlying this mitogenic role remain to be defined. Here, we demonstrate that the F-box protein SKP2 (S phase kinase-associated protein 2), the substrate-specific receptor of the ubiquitin ligase responsible for the degradation of p27KIP1 through the proteasome pathway, constitutes a direct transcriptional target for NOR1. Mitogen-induced Skp2 expression is silenced in vascular smooth muscle cells (VSMC) isolated from Nor1-deficient mice or transfected with Nor1 siRNA. Conversely, adenovirus-mediated overexpression of NOR1 induces Skp2 expression in VSMC and decreases protein abundance of its target p27. Transient transfection experiments establish that NOR1 transactivates the Skp2 promoter through a nerve growth factor-induced clone B response element (NBRE). Electrophoretic mobility shift and chromatin immunoprecipitation assays further revealed that NOR1 is recruited to this NBRE site in the Skp2 promoter in response to mitogenic stimulation. In vivo Skp2 expression is increased during the proliferative response underlying neointima formation, and this transcriptional induction depends on the expression of NOR1. Finally, we demonstrate that overexpression of Skp2 rescues the proliferative arrest of Nor1-deficient VSMC. Collectively, these results characterize Skp2 as a novel NOR1-regulated target gene and detail a previously unrecognized transcriptional cascade regulating mitogen-induced VSMC proliferation. PMID:21868379

A nuclear gene for the iron-sulfur subunit of mitochondrial complex II is specifically expressed during Arabidopsis seed development and germination.

PubMed

Elorza, Alvaro; Roschzttardtz, Hannetz; Gómez, Isabel; Mouras, Armand; Holuigue, Loreto; Araya, Alejandro; Jordana, Xavier

2006-01-01

Three nuclear genes, SDH2-1, SDH2-2 and SDH2-3, encode the essential iron-sulfur subunit of mitochondrial complex II in Arabidopsis thaliana. SDH2-1 and SDH2-2 probably arose via a recent duplication event and we reported that both are expressed in all organs from adult plants. In contrast, transcripts from SDH2-3 were not detected. Here we present data demonstrating that SDH2-3 is specifically expressed during seed development. SDH2-3 transcripts appear during seed maturation, persist through desiccation, are abundant in dry seeds and markedly decline during germination. Analysis of transgenic Arabidopsis plants carrying the SDH2-3 promoter fused to the beta-glucuronidase reporter gene shows that the SDH2-3 promoter is activated in the embryo during maturation, from the bent-cotyledon stage. beta-Glucuronidase expression correlates with the appearance of endogenous SDH2-3 transcripts, suggesting that control of this nuclear gene is achieved through transcriptional regulation. Furthermore, progressive deletions of this promoter identified a 159 bp region (-223 to -65) important for SDH2-3 transcriptional activation in seeds. Interestingly, the SDH2-3 promoter remains active in embryonic tissues during germination and post-germinative growth, and is turned off in vegetative tissues (true leaves). In contrast to SDH2-3 transcripts, SDH2-1 and SDH2-2 transcripts are barely detected in dry seeds and increase during germination and post-germinative growth. The opposite expression patterns of SDH2 nuclear genes strongly suggest that during germination the embryo-specific SDH2-3 is replaced by SDH2-1 or SDH2-2 in mitochondrial complex II.

Circulating RNA transcripts identify therapeutic response in cystic fibrosis lung disease.

PubMed

Saavedra, Milene T; Hughes, Grant J; Sanders, Linda A; Carr, Michelle; Rodman, David M; Coldren, Christopher D; Geraci, Mark W; Sagel, Scott D; Accurso, Frank J; West, James; Nick, Jerry A

2008-11-01

Circulating leukocyte RNA transcripts are systemic markers of inflammation, which have not been studied in cystic fibrosis (CF) lung disease. Although the standard assessment of pulmonary treatment response is FEV(1), a measure of airflow limitation, the lack of systemic markers to reflect changes in lung inflammation critically limits the testing of proposed therapeutics. We sought to prospectively identify and validate peripheral blood leukocyte genes that could mark resolution of pulmonary infection and inflammation using a model by which RNA transcripts could increase the predictive value of spirometry. Peripheral blood mononuclear cells were isolated from 10 patients with CF and acute pulmonary exacerbations before and after therapy. RNA expression profiling revealed that 10 genes significantly changed with treatment when compared with matched non-CF and control subjects with stable CF to establish baseline transcript abundance. Peripheral blood mononuclear cell RNA transcripts were prospectively validated, using real-time polymerase chain reaction amplification, in an independent cohort of acutely ill patients with CF (n = 14). Patients who responded to therapy were analyzed using general estimating equations and multiple logistic regression, such that changes in FEV(1)% predicted were regressed with transcript changes. Three genes, CD64, ADAM9, and CD36, were significant and independent predictors of a therapeutic response beyond that of FEV(1) alone (P < 0.05). In both cohorts, receiver operating characteristic analysis revealed greater accuracy when genes were combined with FEV(1). Circulating mononuclear cell transcripts characterize a response to the treatment of pulmonary exacerbations. Even in small patient cohorts, changes in gene expression in conjunction with FEV(1) may enhance current outcomes measures for treatment response.

Genomic and Transcriptomic Analysis of Growth-Supporting Dehalogenation of Chlorinated Methanes in Methylobacterium

PubMed Central

Chaignaud, Pauline; Maucourt, Bruno; Weiman, Marion; Alberti, Adriana; Kolb, Steffen; Cruveiller, Stéphane; Vuilleumier, Stéphane; Bringel, Françoise

2017-01-01

Bacterial adaptation to growth with toxic halogenated chemicals was explored in the context of methylotrophic metabolism of Methylobacterium extorquens, by comparing strains CM4 and DM4, which show robust growth with chloromethane and dichloromethane, respectively. Dehalogenation of chlorinated methanes initiates growth-supporting degradation, with intracellular release of protons and chloride ions in both cases. The core, variable and strain-specific genomes of strains CM4 and DM4 were defined by comparison with genomes of non-dechlorinating strains. In terms of gene content, adaptation toward dehalogenation appears limited, strains CM4 and DM4 sharing between 75 and 85% of their genome with other strains of M. extorquens. Transcript abundance in cultures of strain CM4 grown with chloromethane and of strain DM4 grown with dichloromethane was compared to growth with methanol as a reference C1 growth substrate. Previously identified strain-specific dehalogenase-encoding genes were the most transcribed with chlorinated methanes, alongside other genes encoded by genomic islands (GEIs) and plasmids involved in growth with chlorinated compounds as carbon and energy source. None of the 163 genes shared by strains CM4 and DM4 but not by other strains of M. extorquens showed higher transcript abundance in cells grown with chlorinated methanes. Among the several thousand genes of the M. extorquens core genome, 12 genes were only differentially abundant in either strain CM4 or strain DM4. Of these, 2 genes of known function were detected, for the membrane-bound proton translocating pyrophosphatase HppA and the housekeeping molecular chaperone protein DegP. This indicates that the adaptive response common to chloromethane and dichloromethane is limited at the transcriptional level, and involves aspects of the general stress response as well as of a dehalogenation-specific response to intracellular hydrochloric acid production. Core genes only differentially abundant in either strain CM4 or strain DM4 total 13 and 58 CDS, respectively. Taken together, the obtained results suggest different transcriptional responses of chloromethane- and dichloromethane-degrading M. extorquens strains to dehalogenative metabolism, and substrate- and pathway-specific modes of growth optimization with chlorinated methanes. PMID:28919881

Genomic and Transcriptomic Analysis of Growth-Supporting Dehalogenation of Chlorinated Methanes in Methylobacterium.

PubMed

Chaignaud, Pauline; Maucourt, Bruno; Weiman, Marion; Alberti, Adriana; Kolb, Steffen; Cruveiller, Stéphane; Vuilleumier, Stéphane; Bringel, Françoise

2017-01-01

Bacterial adaptation to growth with toxic halogenated chemicals was explored in the context of methylotrophic metabolism of Methylobacterium extorquens , by comparing strains CM4 and DM4, which show robust growth with chloromethane and dichloromethane, respectively. Dehalogenation of chlorinated methanes initiates growth-supporting degradation, with intracellular release of protons and chloride ions in both cases. The core, variable and strain-specific genomes of strains CM4 and DM4 were defined by comparison with genomes of non-dechlorinating strains. In terms of gene content, adaptation toward dehalogenation appears limited, strains CM4 and DM4 sharing between 75 and 85% of their genome with other strains of M. extorquens . Transcript abundance in cultures of strain CM4 grown with chloromethane and of strain DM4 grown with dichloromethane was compared to growth with methanol as a reference C 1 growth substrate. Previously identified strain-specific dehalogenase-encoding genes were the most transcribed with chlorinated methanes, alongside other genes encoded by genomic islands (GEIs) and plasmids involved in growth with chlorinated compounds as carbon and energy source. None of the 163 genes shared by strains CM4 and DM4 but not by other strains of M. extorquens showed higher transcript abundance in cells grown with chlorinated methanes. Among the several thousand genes of the M. extorquens core genome, 12 genes were only differentially abundant in either strain CM4 or strain DM4. Of these, 2 genes of known function were detected, for the membrane-bound proton translocating pyrophosphatase HppA and the housekeeping molecular chaperone protein DegP. This indicates that the adaptive response common to chloromethane and dichloromethane is limited at the transcriptional level, and involves aspects of the general stress response as well as of a dehalogenation-specific response to intracellular hydrochloric acid production. Core genes only differentially abundant in either strain CM4 or strain DM4 total 13 and 58 CDS, respectively. Taken together, the obtained results suggest different transcriptional responses of chloromethane- and dichloromethane-degrading M. extorquens strains to dehalogenative metabolism, and substrate- and pathway-specific modes of growth optimization with chlorinated methanes.

The Severity of Plasmodium falciparum Infection Is Associated with Transcript Levels of var Genes Encoding Endothelial Protein C Receptor-Binding P. falciparum Erythrocyte Membrane Protein 1.

PubMed

Mkumbaye, Sixbert I; Wang, Christian W; Lyimo, Eric; Jespersen, Jakob S; Manjurano, Alphaxard; Mosha, Jacklin; Kavishe, Reginald A; Mwakalinga, Steven B; Minja, Daniel T R; Lusingu, John P; Theander, Thor G; Lavstsen, Thomas

2017-04-01

By attaching infected erythrocytes to the vascular lining, Plasmodium falciparum parasites leave blood circulation and avoid splenic clearance. This sequestration is central to pathogenesis. Severe malaria is associated with parasites expressing an antigenically distinct P. falciparum erythrocyte membrane protein 1 (PfEMP1) subset mediating binding to endothelial receptors. Previous studies indicate that PfEMP1 adhesins with so-called CIDRα1 domains capable of binding endothelial protein C receptor (EPCR) constitute the PfEMP1 subset associated with severe pediatric malaria. To analyze the relative importance of different subtypes of CIDRα1 domains, we compared Pf emp1 transcript levels in children with severe malaria (including 9 fatal and 114 surviving cases), children hospitalized with uncomplicated malaria ( n = 42), children with mild malaria not requiring hospitalization ( n = 10), and children with parasitemia and no ongoing fever ( n = 12). High levels of transcripts encoding EPCR-binding PfEMP1 were found in patients with symptomatic infections, and the abundance of these transcripts increased with disease severity. The compositions of CIDRα1 subtype transcripts varied markedly between patients, and none of the subtypes were dominant. Transcript-level analyses targeting other domain types indicated that subtypes of DBLβ or DBLζ domains might mediate binding phenomena that, in conjunction with EPCR binding, could contribute to pathogenesis. These observations strengthen the rationale for targeting the PfEMP1-EPCR interaction by vaccines and adjunctive therapies. Interventions should target EPCR binding of all CIDRα1 subtypes. Copyright © 2017 American Society for Microbiology.

Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation.

PubMed

Smith, Sarah R; Gillard, Jeroen T F; Kustka, Adam B; McCrow, John P; Badger, Jonathan H; Zheng, Hong; New, Ashley M; Dupont, Chris L; Obata, Toshihiro; Fernie, Alisdair R; Allen, Andrew E

2016-12-01

Environmental fluctuations affect distribution, growth and abundance of diatoms in nature, with iron (Fe) availability playing a central role. Studies on the response of diatoms to low Fe have either utilized continuous (24 hr) illumination or sampled a single time of day, missing any temporal dynamics. We profiled the physiology, metabolite composition, and global transcripts of the pennate diatom Phaeodactylum tricornutum during steady-state growth at low, intermediate, and high levels of dissolved Fe over light:dark cycles, to better understand fundamental aspects of genetic control of physiological acclimation to growth under Fe-limitation. We greatly expand the catalog of genes involved in the low Fe response, highlighting the importance of intracellular trafficking in Fe-limited diatoms. P. tricornutum exhibited transcriptomic hallmarks of slowed growth leading to prolonged periods of cell division/silica deposition, which could impact biogeochemical carbon sequestration in Fe-limited regions. Light harvesting and ribosome biogenesis transcripts were generally reduced under low Fe while transcript levels for genes putatively involved in the acquisition and recycling of Fe were increased. We also noted shifts in expression towards increased synthesis and catabolism of branched chain amino acids in P. tricornutum grown at low Fe whereas expression of genes involved in central core metabolism were relatively unaffected, indicating that essential cellular function is protected. Beyond the response of P. tricornutum to low Fe, we observed major coordinated shifts in transcript control of primary and intermediate metabolism over light:dark cycles which contribute to a new view of the significance of distinctive diatom pathways, such as mitochondrial glycolysis and the ornithine-urea cycle. This study provides new insight into transcriptional modulation of diatom physiology and metabolism across light:dark cycles in response to Fe availability, providing mechanistic understanding for the ability of diatoms to remain metabolically poised to respond quickly to Fe input and revealing strategies underlying their ecological success.

Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation

PubMed Central

McCrow, John P.; Badger, Jonathan H.; Zheng, Hong; New, Ashley M.; Dupont, Chris L.; Obata, Toshihiro; Fernie, Alisdair R.; Allen, Andrew E.

2016-01-01

Environmental fluctuations affect distribution, growth and abundance of diatoms in nature, with iron (Fe) availability playing a central role. Studies on the response of diatoms to low Fe have either utilized continuous (24 hr) illumination or sampled a single time of day, missing any temporal dynamics. We profiled the physiology, metabolite composition, and global transcripts of the pennate diatom Phaeodactylum tricornutum during steady-state growth at low, intermediate, and high levels of dissolved Fe over light:dark cycles, to better understand fundamental aspects of genetic control of physiological acclimation to growth under Fe-limitation. We greatly expand the catalog of genes involved in the low Fe response, highlighting the importance of intracellular trafficking in Fe-limited diatoms. P. tricornutum exhibited transcriptomic hallmarks of slowed growth leading to prolonged periods of cell division/silica deposition, which could impact biogeochemical carbon sequestration in Fe-limited regions. Light harvesting and ribosome biogenesis transcripts were generally reduced under low Fe while transcript levels for genes putatively involved in the acquisition and recycling of Fe were increased. We also noted shifts in expression towards increased synthesis and catabolism of branched chain amino acids in P. tricornutum grown at low Fe whereas expression of genes involved in central core metabolism were relatively unaffected, indicating that essential cellular function is protected. Beyond the response of P. tricornutum to low Fe, we observed major coordinated shifts in transcript control of primary and intermediate metabolism over light:dark cycles which contribute to a new view of the significance of distinctive diatom pathways, such as mitochondrial glycolysis and the ornithine-urea cycle. This study provides new insight into transcriptional modulation of diatom physiology and metabolism across light:dark cycles in response to Fe availability, providing mechanistic understanding for the ability of diatoms to remain metabolically poised to respond quickly to Fe input and revealing strategies underlying their ecological success. PMID:27973599

Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation

DOE PAGES

Smith, Sarah R.; Gillard, Jeroen T. F.; Kustka, Adam B.; ...

2016-12-14

Environmental fluctuations affect distribution, growth and abundance of diatoms in nature, with iron (Fe) availability playing a central role. Studies on the response of diatoms to low Fe have either utilized continuous (24 hr) illumination or sampled a single time of day, missing any temporal dynamics. We profiled the physiology, metabolite composition, and global transcripts of the pennate diatom Phaeodactylum tricornutum during steady-state growth at low, intermediate, and high levels of dissolved Fe over light:dark cycles, to better understand fundamental aspects of genetic control of physiological acclimation to growth under Fe-limitation. We greatly expand the catalog of genes involved inmore » the low Fe response, highlighting the importance of intracellular trafficking in Fe-limited diatoms. P. tricornutum exhibited transcriptomic hallmarks of slowed growth leading to prolonged periods of cell division/silica deposition, which could impact biogeochemical carbon sequestration in Fe-limited regions. Light harvesting and ribosome biogenesis transcripts were generally reduced under low Fe while transcript levels for genes putatively involved in the acquisition and recycling of Fe were increased. We also noted shifts in expression towards increased synthesis and catabolism of branched chain amino acids in P. tricornutum grown at low Fe whereas expression of genes involved in central core metabolism were relatively unaffected, indicating that essential cellular function is protected. Beyond the response of P. tricornutum to low Fe, we observed major coordinated shifts in transcript control of primary and intermediate metabolism over light:dark cycles which contribute to a new view of the significance of distinctive diatom pathways, such as mitochondrial glycolysis and the ornithine-urea cycle. This study provides new insight into transcriptional modulation of diatom physiology and metabolism across light:dark cycles in response to Fe availability, providing mechanistic understanding for the ability of diatoms to remain metabolically poised to respond quickly to Fe input and revealing strategies underlying their ecological success.« less

Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Sarah R.; Gillard, Jeroen T. F.; Kustka, Adam B.

Environmental fluctuations affect distribution, growth and abundance of diatoms in nature, with iron (Fe) availability playing a central role. Studies on the response of diatoms to low Fe have either utilized continuous (24 hr) illumination or sampled a single time of day, missing any temporal dynamics. We profiled the physiology, metabolite composition, and global transcripts of the pennate diatom Phaeodactylum tricornutum during steady-state growth at low, intermediate, and high levels of dissolved Fe over light:dark cycles, to better understand fundamental aspects of genetic control of physiological acclimation to growth under Fe-limitation. We greatly expand the catalog of genes involved inmore » the low Fe response, highlighting the importance of intracellular trafficking in Fe-limited diatoms. P. tricornutum exhibited transcriptomic hallmarks of slowed growth leading to prolonged periods of cell division/silica deposition, which could impact biogeochemical carbon sequestration in Fe-limited regions. Light harvesting and ribosome biogenesis transcripts were generally reduced under low Fe while transcript levels for genes putatively involved in the acquisition and recycling of Fe were increased. We also noted shifts in expression towards increased synthesis and catabolism of branched chain amino acids in P. tricornutum grown at low Fe whereas expression of genes involved in central core metabolism were relatively unaffected, indicating that essential cellular function is protected. Beyond the response of P. tricornutum to low Fe, we observed major coordinated shifts in transcript control of primary and intermediate metabolism over light:dark cycles which contribute to a new view of the significance of distinctive diatom pathways, such as mitochondrial glycolysis and the ornithine-urea cycle. This study provides new insight into transcriptional modulation of diatom physiology and metabolism across light:dark cycles in response to Fe availability, providing mechanistic understanding for the ability of diatoms to remain metabolically poised to respond quickly to Fe input and revealing strategies underlying their ecological success.« less

Microbial Mat Compositional and Functional Sensitivity to Environmental Disturbance

PubMed Central

Preisner, Eva C.; Fichot, Erin B.; Norman, Robert S.

2016-01-01

The ability of ecosystems to adapt to environmental perturbations depends on the duration and intensity of change and the overall biological diversity of the system. While studies have indicated that rare microbial taxa may provide a biological reservoir that supports long-term ecosystem stability, how this dynamic population is influenced by environmental parameters remains unclear. In this study, a microbial mat ecosystem located on San Salvador Island, The Bahamas was used as a model to examine how environmental disturbance affects the protein synthesis potential (PSP) of rare and abundant archaeal and bacterial communities and how these changes impact potential biogeochemical processes. This ecosystem experienced a large shift in salinity (230 to 65 g kg-1) during 2011–2012 following the landfall of Hurricane Irene on San Salvador Island. High throughput sequencing and analysis of 16S rRNA and rRNA genes from samples before and after the pulse disturbance showed significant changes in the diversity and PSP of abundant and rare taxa, suggesting overall compositional and functional sensitivity to environmental change. In both archaeal and bacterial communities, while the majority of taxa showed low PSP across conditions, the overall community PSP increased post-disturbance, with significant shifts occurring among abundant and rare taxa across and within phyla. Broadly, following the post-disturbance reduction in salinity, taxa within Halobacteria decreased while those within Crenarchaeota, Thaumarchaeota, Thermoplasmata, Cyanobacteria, and Proteobacteria, increased in abundance and PSP. Quantitative PCR of genes and transcripts involved in nitrogen and sulfur cycling showed concomitant shifts in biogeochemical cycling potential. Post-disturbance conditions increased the expression of genes involved in N-fixation, nitrification, denitrification, and sulfate reduction. Together, our findings show complex community adaptation to environmental change and help elucidate factors connecting disturbance, biodiversity, and ecosystem function that may enhance ecosystem models. PMID:27799927

Adipose triglyceride lipase protein abundance and translocation to the lipid droplet increase during leptin-induced lipolysis in bovine adipocytes.

PubMed

Koltes, D A; Spurlock, M E; Spurlock, D M

2017-10-01

Proper regulation of lipid metabolism is critical for preventing the development of metabolic diseases. It is clear that leptin plays a critical role in the regulation of energy homeostasis by regulating energy intake. However, leptin can also regulate energy homeostasis by inducing lipolysis in adipocytes, but it is unclear how the major lipases are involved in leptin-stimulated lipolysis. Therefore, the objectives of this study were to determine if (1) leptin acts directly to induce lipolysis in bovine adipocytes, (2) the potential lipases involved in leptin-induced lipolysis in bovine adipocytes, and (3) increases translocation of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) during leptin-stimulated lipolysis in bovine stromal vascular cell-derived adipocytes. As hypothesized, leptin induced a lipolytic response (P = 0.02) in isolated adipocytes which was accompanied by an increase in phosphorylation of signal transducer and activator of transcription (STAT)3 (P = 0.03), a well-documented secondary messenger of leptin, and ATGL protein abundance (P < 0.01). Protein abundance of STAT3, perilipin, HSL, and phosphorylation of HSL by PKA and AMPK were not altered during leptin-stimulated lipolysis (P > 0.05). Immunostaining techniques were employed to determine the location of HSL and ATGL. Both lipases translocated to the lipid droplet after 2 h of exposure to isoproterenol (P < 0.02). However, only ATGL was translocated to the lipid droplet during leptin-stimulated lipolysis (P = 0.04), indicating ATGL may be the active lipase in leptin-stimulated lipolysis. In summary, leptin stimulates lipolysis in bovine adipocytes. The lack of phosphorylated HSL and translocation of HSL to the lipid droplet during leptin-stimulated lipolysis suggest minimal activity by PKA. Interestingly, leptin-stimulated lipolysis is accompanied by an increase in ATGL protein abundance and translocation to the lipid droplet, indicating its involvement in leptin-stimulated lipolysis either due to an increase in protein abundance or through a novel lipolytic cascade. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: carbon and energy flow contribute to the distinct biofilm growth state.

PubMed

Clark, Melinda E; He, Zhili; Redding, Alyssa M; Joachimiak, Marcin P; Keasling, Jay D; Zhou, Jizhong Z; Arkin, Adam P; Mukhopadhyay, Aindrila; Fields, Matthew W

2012-04-16

Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations. The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells. Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion.

Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: Carbon and energy flow contribute to the distinct biofilm growth state

PubMed Central

2012-01-01

Background Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations. Results The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells. Conclusions Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion. PMID:22507456

WNK1 regulates skeletal muscle cell hypertrophy by modulating the nuclear localization and transcriptional activity of FOXO4.

PubMed

Mandai, Shintaro; Mori, Takayasu; Nomura, Naohiro; Furusho, Taisuke; Arai, Yohei; Kikuchi, Hiroaki; Sasaki, Emi; Sohara, Eisei; Rai, Tatemitsu; Uchida, Shinichi

2018-06-14

With-no-lysine (K) (WNK) kinases, which are mutated in the inherited form of hypertension pseudohypoaldosteronism type II, are essential regulators of membrane ion transporters. Here, we report that WNK1 positively regulates skeletal muscle cell hypertrophy via mediating the function of the pro-longevity transcription factor forkhead box protein O4 (FOXO4) independent of the conventional WNK signaling pathway linking SPS/STE20-related proline-alanine-rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1) to downstream effector ion transporters. Small interfering RNA (siRNA)-mediated silencing of WNK1, but not SPAK/OSR1 kinases, induced myotube atrophy and remarkable increases in the mRNA expression of the muscle atrophy ubiquitin ligases MAFbx and MuRF1 in C2C12 mouse skeletal muscle cells. WNK1 silencing also increased FOXO4 nuclear localization, and co-transfection of Foxo4-targeted siRNA completely reversed the myotube atrophy and upregulation of atrogene transcription induced by WNK1 silencing. We further illustrated that WNK1 protein abundance in skeletal muscle was increased by chronic voluntary wheel running exercise (hypertrophic stimulus) and markedly decreased by adenine-induced chronic kidney disease (atrophic stimulus) in mice. These findings suggest that WNK1 is involved in the physiological regulation of mammalian skeletal muscle hypertrophy and atrophy via interactions with FOXO4. The WNK1-FOXO4 axis may be a potential therapeutic target in human diseases causing sarcopenia.

Impaired Carbohydrate Digestion and Transport and Mucosal Dysbiosis in the Intestines of Children with Autism and Gastrointestinal Disturbances

PubMed Central

Williams, Brent L.; Hornig, Mady; Buie, Timothy; Bauman, Margaret L.; Cho Paik, Myunghee; Wick, Ivan; Bennett, Ashlee; Jabado, Omar; Hirschberg, David L.; Lipkin, W. Ian

2011-01-01

Gastrointestinal disturbances are commonly reported in children with autism, complicate clinical management, and may contribute to behavioral impairment. Reports of deficiencies in disaccharidase enzymatic activity and of beneficial responses to probiotic and dietary therapies led us to survey gene expression and the mucoepithelial microbiota in intestinal biopsies from children with autism and gastrointestinal disease and children with gastrointestinal disease alone. Ileal transcripts encoding disaccharidases and hexose transporters were deficient in children with autism, indicating impairment of the primary pathway for carbohydrate digestion and transport in enterocytes. Deficient expression of these enzymes and transporters was associated with expression of the intestinal transcription factor, CDX2. Metagenomic analysis of intestinal bacteria revealed compositional dysbiosis manifest as decreases in Bacteroidetes, increases in the ratio of Firmicutes to Bacteroidetes, and increases in Betaproteobacteria. Expression levels of disaccharidases and transporters were associated with the abundance of affected bacterial phylotypes. These results indicate a relationship between human intestinal gene expression and bacterial community structure and may provide insights into the pathophysiology of gastrointestinal disturbances in children with autism. PMID:21949732

The transcriptional repressor Sum1p counteracts Sir2p in regulation of the actin cytoskeleton, mitochondrial quality control and replicative lifespan in Saccharomyces cerevisiae.

PubMed

Higuchi-Sanabria, Ryo; Vevea, Jason D; Charalel, Joseph K; Sapar, Maria L; Pon, Liza A

2016-01-18

Increasing the stability or dynamics of the actin cytoskeleton can extend lifespan in C. elegans and S. cerevisiae . Actin cables of budding yeast, bundles of actin filaments that mediate cargo transport, affect lifespan control through effects on mitochondrial quality control. Sir2p, the founding member of the Sirtuin family of lifespan regulators, also affects actin cable dynamics, assembly, and function in mitochondrial quality control. Here, we obtained evidence for novel interactions between Sir2p and Sum1p, a transcriptional repressor that was originally identified through mutations that genetically suppress sir2 ∆ phenotypes unrelated to lifespan. We find that deletion of SUM1 in wild-type cells results in increased mitochondrial function and actin cable abundance. Furthermore, deletion of SUM1 suppresses defects in actin cables and mitochondria of sir2 ∆ yeast, and extends the replicative lifespan and cellular health span of sir2 ∆ cells. Thus, Sum1p suppresses Sir2p function in control of specific aging determinants and lifespan in budding yeast.

SIRT1 Activates MAO-A in the Brain to Mediate Anxiety and Exploratory Drive

PubMed Central

Libert, Sergiy; Pointer, Kelli; Bell, Eric L.; Das, Abhirup; Cohen, Dena E.; Asara, John M.; Kapur, Karen; Bergmann, Sven; Preisig, Martin; Otowa, Takeshi; Kendler, Kenneth S.; Chen, Xiangning; Hettema, John M.; van den Oord, Edwin J.; Rubio, Justin P.; Guarente, Leonard

2012-01-01

SUMMARY SIRT1 is a NAD+-dependent deacetylase that governs a number of genetic programs to cope with changes in the nutritional status of cells and organisms. Behavioral responses to food abundance are important for the survival of higher animals. Here we used mice with increased or decreased brain SIRT1 to show that this sirtuin regulates anxiety and exploratory drive by activating transcription of the gene encoding the monoamine oxidase A (MAO-A) to reduce serotonin levels in the brain. Indeed, treating animals with MAO-A inhibitors or selective serotonin reuptake inhibitors (SSRIs) normalized anxiety differences between wild-type and mutant animals. SIRT1 deacetylates the brain-specific helix-loop-helix transcription factor NHLH2 on lysine 49 to increase its activation of the MAO-A promoter. Both common and rare variations in the SIRT1 gene were shown to be associated with risk of anxiety in human population samples. Together these data indicate that SIRT1 mediates levels of anxiety, and this regulation may be adaptive in a changing environment of food availability. PMID:22169038

DNA-independent PARP-1 activation by phosphorylated ERK2 increases Elk1 activity: a link to histone acetylation.

PubMed

Cohen-Armon, Malka; Visochek, Leonid; Rozensal, Dana; Kalal, Adi; Geistrikh, Ilona; Klein, Rodika; Bendetz-Nezer, Sarit; Yao, Zhong; Seger, Rony

2007-01-26

PolyADP-ribose polymerases (PARPs) catalyze a posttranslational modification of nuclear proteins by polyADP-ribosylation. The catalytic activity of the abundant nuclear protein PARP-1 is stimulated by DNA strand breaks, and PARP-1 activation is required for initiation of DNA repair. Here we show that PARP-1 also acts within extracellular signal-regulated kinase (ERK) signaling cascade that mediates growth and differentiation. The findings reveal an alternative mode of PARP-1 activation, which does not involve binding to DNA or DNA damage. In a cell-free system, recombinant PARP-1 was intensively activated and thereby polyADP-ribosylated by a direct interaction with phosphorylated ERK2, and the activated PARP-1 dramatically increased ERK2-catalyzed phosphorylation of the transcription factor Elk1. In cortical neurons treated with nerve growth factors and in stimulated cardiomyocytes, PARP-1 activation enhanced ERK-induced Elk1-phosphorylation, core histone acetylation, and transcription of the Elk1-target gene c-fos. These findings constitute evidence for PARP-1 activity within the ERK signal-transduction pathway.

Impaired carbohydrate digestion and transport and mucosal dysbiosis in the intestines of children with autism and gastrointestinal disturbances.

PubMed

Williams, Brent L; Hornig, Mady; Buie, Timothy; Bauman, Margaret L; Cho Paik, Myunghee; Wick, Ivan; Bennett, Ashlee; Jabado, Omar; Hirschberg, David L; Lipkin, W Ian

2011-01-01

Gastrointestinal disturbances are commonly reported in children with autism, complicate clinical management, and may contribute to behavioral impairment. Reports of deficiencies in disaccharidase enzymatic activity and of beneficial responses to probiotic and dietary therapies led us to survey gene expression and the mucoepithelial microbiota in intestinal biopsies from children with autism and gastrointestinal disease and children with gastrointestinal disease alone. Ileal transcripts encoding disaccharidases and hexose transporters were deficient in children with autism, indicating impairment of the primary pathway for carbohydrate digestion and transport in enterocytes. Deficient expression of these enzymes and transporters was associated with expression of the intestinal transcription factor, CDX2. Metagenomic analysis of intestinal bacteria revealed compositional dysbiosis manifest as decreases in Bacteroidetes, increases in the ratio of Firmicutes to Bacteroidetes, and increases in Betaproteobacteria. Expression levels of disaccharidases and transporters were associated with the abundance of affected bacterial phylotypes. These results indicate a relationship between human intestinal gene expression and bacterial community structure and may provide insights into the pathophysiology of gastrointestinal disturbances in children with autism.

Wood reinforcement of poplar by rice NAC transcription factor

PubMed Central

Sakamoto, Shingo; Takata, Naoki; Oshima, Yoshimi; Yoshida, Kouki; Taniguchi, Toru; Mitsuda, Nobutaka

2016-01-01

Lignocellulose, composed of cellulose, hemicellulose, and lignin, in the secondary cell wall constitutes wood and is the most abundant form of biomass on Earth. Enhancement of wood accumulation may be an effective strategy to increase biomass as well as wood strength, but currently only limited research has been undertaken. Here, we demonstrated that OsSWN1, the orthologue of the rice NAC Secondary-wall Thickening factor (NST) transcription factor, effectively enhanced secondary cell wall formation in the Arabidopsis inflorescence stem and poplar (Populus tremula×Populus tremuloides) stem when expressed by the Arabidopsis NST3 promoter. Interestingly, in transgenic Arabidopsis and poplar, ectopic secondary cell wall deposition in the pith area was observed in addition to densification of the secondary cell wall in fiber cells. The cell wall content or density of the stem increased on average by up to 38% and 39% in Arabidopsis and poplar, respectively, without causing growth inhibition. As a result, physical strength of the stem increased by up to 57% in poplar. Collectively, these data suggest that the reinforcement of wood by NST3pro:OsSWN1 is a promising strategy to enhance wood-biomass production in dicotyledonous plant species. PMID:26812961

Ammonia oxidation kinetics and temperature sensitivity of a natural marine community dominated by Archaea

PubMed Central

Horak, Rachel E A; Qin, Wei; Schauer, Andy J; Armbrust, E Virginia; Ingalls, Anitra E; Moffett, James W; Stahl, David A; Devol, Allan H

2013-01-01

Archaeal ammonia oxidizers (AOAs) are increasingly recognized as prominent members of natural microbial assemblages. Evidence that links the presence of AOA with in situ ammonia oxidation activity is limited, and the abiotic factors that regulate the distribution of AOA natural assemblages are not well defined. We used quantitative PCR to enumerate amoA (encodes α-subunit of ammonia monooxygenase) abundances; AOA amoA gene copies greatly outnumbered ammonia-oxidizing bacteria and amoA transcripts were derived primarily from AOA throughout the water column of Hood Canal, Puget Sound, WA, USA. We generated a Michaelis–Menten kinetics curve for ammonia oxidation by the natural community and found that the measured Km of 98±14 nmol l−1 was close to that for cultivated AOA representative Nitrosopumilus maritimus SCM1. Temperature did not have a significant effect on ammonia oxidation rates for incubation temperatures ranging from 8 to 20 °C, which is within the temperature range for depths of measurable ammonia oxidation at the site. This study provides substantial evidence, through both amoA gene copies and transcript abundances and the kinetics response, that AOA are the dominant active ammonia oxidizers in this marine environment. We propose that future ammonia oxidation experiments use a Km for the natural community to better constrain ammonia oxidation rates determined with the commonly used 15NH4+ dilution technique. PMID:23657360

Ammonia oxidation kinetics and temperature sensitivity of a natural marine community dominated by Archaea.

PubMed

Horak, Rachel E A; Qin, Wei; Schauer, Andy J; Armbrust, E Virginia; Ingalls, Anitra E; Moffett, James W; Stahl, David A; Devol, Allan H

2013-10-01

Archaeal ammonia oxidizers (AOAs) are increasingly recognized as prominent members of natural microbial assemblages. Evidence that links the presence of AOA with in situ ammonia oxidation activity is limited, and the abiotic factors that regulate the distribution of AOA natural assemblages are not well defined. We used quantitative PCR to enumerate amoA (encodes α-subunit of ammonia monooxygenase) abundances; AOA amoA gene copies greatly outnumbered ammonia-oxidizing bacteria and amoA transcripts were derived primarily from AOA throughout the water column of Hood Canal, Puget Sound, WA, USA. We generated a Michaelis-Menten kinetics curve for ammonia oxidation by the natural community and found that the measured Km of 98±14 nmol l(-1) was close to that for cultivated AOA representative Nitrosopumilus maritimus SCM1. Temperature did not have a significant effect on ammonia oxidation rates for incubation temperatures ranging from 8 to 20 °C, which is within the temperature range for depths of measurable ammonia oxidation at the site. This study provides substantial evidence, through both amoA gene copies and transcript abundances and the kinetics response, that AOA are the dominant active ammonia oxidizers in this marine environment. We propose that future ammonia oxidation experiments use a Km for the natural community to better constrain ammonia oxidation rates determined with the commonly used (15)NH4(+) dilution technique.

Mitochondrial-nuclear crosstalk, haplotype and copy number variation distinct in muscle fiber type, mitochondrial respiratory and metabolic enzyme activities.

PubMed

Liu, Xuan; Trakooljul, Nares; Hadlich, Frieder; Murani, Eduard; Wimmers, Klaus; Ponsuksili, Siriluck

2017-10-25

Genes expressed in mitochondria work in concert with those expressed in the nucleus to mediate oxidative phosphorylation (OXPHOS), a process that is relevant for muscle metabolism and meat quality. Mitochondrial genome activity can be efficiently studied and compared in Duroc and Pietrain pigs, which harbor different mitochondrial haplotypes and distinct muscle fiber types, mitochondrial respiratory activities, and fat content. Pietrain pigs homozygous-positive for malignant hyperthermia susceptibility (PiPP) carried only haplotype 8 and showed the lowest absolute mtDNA copy number accompanied by a decrease transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6 and nuclear-encoded subunits NDUFA11 and NDUFB8. In contrast, we found that haplotype 4 of Duroc pigs had significantly higher mitochondrial DNA (mtDNA) copy numbers and an increase transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6. These results suggest that the variation in mitochondrial and nuclear genetic background among these animals has an effect on mitochondrial content and OXPHOS system subunit expression. We observed the co-expression pattern of mitochondrial and nuclear encoded OXPHOS subunits suggesting that the mitochondrial-nuclear crosstalk functionally involves in muscle metabolism. The findings provide valuable information for understanding muscle biology processes and energy metabolism, and may direct use for breeding strategies to improve meat quality and animal health.

Transcriptomic Changes in Coral Holobionts Provide Insights into Physiological Challenges of Future Climate and Ocean Change.

PubMed

Kaniewska, Paulina; Chan, Chon-Kit Kenneth; Kline, David; Ling, Edmund Yew Siang; Rosic, Nedeljka; Edwards, David; Hoegh-Guldberg, Ove; Dove, Sophie

2015-01-01

Tropical reef-building coral stress levels will intensify with the predicted rising atmospheric CO2 resulting in ocean temperature and acidification increase. Most studies to date have focused on the destabilization of coral-dinoflagellate symbioses due to warming oceans, or declining calcification due to ocean acidification. In our study, pH and temperature conditions consistent with the end-of-century scenarios of the Intergovernmental Panel on Climate Change (IPCC) caused major changes in photosynthesis and respiration, in addition to decreased calcification rates in the coral Acropora millepora. Population density of symbiotic dinoflagellates (Symbiodinium) under high levels of ocean acidification and temperature (Representative Concentration Pathway, RCP8.5) decreased to half of that found under present day conditions, with photosynthetic and respiratory rates also being reduced by 40%. These physiological changes were accompanied by evidence for gene regulation of calcium and bicarbonate transporters along with components of the organic matrix. Metatranscriptomic RNA-Seq data analyses showed an overall down regulation of metabolic transcripts, and an increased abundance of transcripts involved in circadian clock control, controlling the damage of oxidative stress, calcium signaling/homeostasis, cytoskeletal interactions, transcription regulation, DNA repair, Wnt signaling and apoptosis/immunity/ toxins. We suggest that increased maintenance costs under ocean acidification and warming, and diversion of cellular ATP to pH homeostasis, oxidative stress response, UPR and DNA repair, along with metabolic suppression, may underpin why Acroporid species tend not to thrive under future environmental stress. Our study highlights the potential increased energy demand when the coral holobiont is exposed to high levels of ocean warming and acidification.

Transcriptomic Changes in Coral Holobionts Provide Insights into Physiological Challenges of Future Climate and Ocean Change

PubMed Central

Kaniewska, Paulina; Chan, Chon-Kit Kenneth; Kline, David; Ling, Edmund Yew Siang; Rosic, Nedeljka; Edwards, David; Hoegh-Guldberg, Ove; Dove, Sophie

2015-01-01

Tropical reef-building coral stress levels will intensify with the predicted rising atmospheric CO2 resulting in ocean temperature and acidification increase. Most studies to date have focused on the destabilization of coral-dinoflagellate symbioses due to warming oceans, or declining calcification due to ocean acidification. In our study, pH and temperature conditions consistent with the end-of-century scenarios of the Intergovernmental Panel on Climate Change (IPCC) caused major changes in photosynthesis and respiration, in addition to decreased calcification rates in the coral Acropora millepora. Population density of symbiotic dinoflagellates (Symbiodinium) under high levels of ocean acidification and temperature (Representative Concentration Pathway, RCP8.5) decreased to half of that found under present day conditions, with photosynthetic and respiratory rates also being reduced by 40%. These physiological changes were accompanied by evidence for gene regulation of calcium and bicarbonate transporters along with components of the organic matrix. Metatranscriptomic RNA-Seq data analyses showed an overall down regulation of metabolic transcripts, and an increased abundance of transcripts involved in circadian clock control, controlling the damage of oxidative stress, calcium signaling/homeostasis, cytoskeletal interactions, transcription regulation, DNA repair, Wnt signaling and apoptosis/immunity/ toxins. We suggest that increased maintenance costs under ocean acidification and warming, and diversion of cellular ATP to pH homeostasis, oxidative stress response, UPR and DNA repair, along with metabolic suppression, may underpin why Acroporid species tend not to thrive under future environmental stress. Our study highlights the potential increased energy demand when the coral holobiont is exposed to high levels of ocean warming and acidification. PMID:26510159

Tumor Necrosis Factor Receptor-associated Factor 6 Is an Intranuclear Transcriptional Coactivator in Osteoclasts*

PubMed Central

Bai, Shuting; Zha, Jikun; Zhao, Haibo; Ross, F. Patrick; Teitelbaum, Steven L.

2008-01-01

Tumor necrosis factor receptor-associated factor 6 (TRAF6) associates with the cytoplasmic domain of receptor activator of NF-κB (RANK) and is an essential component of the signaling complex mediating osteoclastogenesis. However, the osteoclastic activity of TRAF6 is blunted by its association with four and half LIM domain 2 (FHL2), which functions as an adaptor protein in the cytoplasm and transcriptional regulator in the nucleus. We find that TRAF6 also localizes in the nuclei of osteoclasts but not their bone marrow macrophage precursors and that osteoclast intranuclear abundance is specifically increased by RANK ligand (RANKL). TRAF6 nuclear localization requires FHL2 and is diminished in fhl2-/- osteoclasts. Suggesting transcriptional activity, TRAF6 interacts with the transcription factor RUNX1 in the osteoclast nucleus. FHL2 also associates with RUNX1 but does so only in the presence of TRAF6. Importantly, TRAF6 recognizes FHL2 and RUNX1 in osteoclast nuclei, and the three molecules form a DNA-binding complex that recognizes and transactivates the RUNX1 response element in the fhl2 promoter. Finally, TRAF6 and its proximal activator, RANKL, polyubiquitinate FHL2, prompting its proteasomal degradation. These observations suggest a feedback mechanism whereby TRAF6 negatively regulates osteoclast formation by intracytoplasmic sequestration of FHL2 to blunt RANK activation and as a component of a transcription complex promoting FHL2 expression. PMID:18768464

Ethylene Response Factors Are Controlled by Multiple Harvesting Stresses in Hevea brasiliensis

PubMed Central

Putranto, Riza-Arief; Duan, Cuifang; Kuswanhadi; Chaidamsari, Tetty; Rio, Maryannick; Piyatrakul, Piyanuch; Herlinawati, Eva; Pirrello, Julien; Dessailly, Florence; Leclercq, Julie; Bonnot, François; Tang, Chaorong; Hu, Songnian; Montoro, Pascal

2015-01-01

Tolerance of recurrent mechanical wounding and exogenous ethylene is a feature of the rubber tree. Latex harvesting involves tapping of the tree bark and ethephon is applied to increase latex flow. Ethylene is an essential element in controlling latex production. The ethylene signalling pathway leads to the activation of Ethylene Response Factor (ERF) transcription factors. This family has been identified in Hevea brasiliensis. This study set out to understand the regulation of ERF genes during latex harvesting in relation to abiotic stress and hormonal treatments. Analyses of the relative transcript abundance were carried out for 35 HbERF genes in latex, in bark from mature trees and in leaves from juvenile plants under multiple abiotic stresses. Twenty-one HbERF genes were regulated by harvesting stress in laticifers, revealing an overrepresentation of genes in group IX. Transcripts of three HbERF-IX genes from HbERF-IXc4, HbERF-IXc5 and HbERF-IXc6 were dramatically accumulated by combining wounding, methyl jasmonate and ethylene treatments. When an ethylene inhibitor was used, the transcript accumulation for these three genes was halted, showing ethylene-dependent induction. Subcellular localization and transactivation experiments confirmed that several members of HbERF-IX are activator-type transcription factors. This study suggested that latex harvesting induces mechanisms developed for the response to abiotic stress. These mechanisms probably depend on various hormonal signalling pathways. Several members of HbERF-IX could be essential integrators of complex hormonal signalling pathways in Hevea. PMID:25906196