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Sample records for abundantly expressed transcripts

  1. Abundant and broad expression of transcription-induced chimeras and protein products in mammalian genomes.

    PubMed

    Lu, Guanting; Wu, Jin; Zhao, Gangbin; Wang, Zhiqiang; Chen, Weihua; Mu, Shijie

    2016-02-12

    The expression of transcription-induced chimeras (TICs) was underestimated due to strategic and logical reasons. In order to thoroughly examine TICs, systematic survey of TIC events was conducted in mammalian genomes using ESTs, followed by experimental validation using RT-PCR and real-time quantitative PCR (qPCR). The expression of ∼98% TIC events in at least one tissue or cell line from both mouse and human was verified. Besides, ∼40% TICs were broadly expressed, and ∼33% of TICs showed expression levels comparable to or higher than their upstream parental genes. We further identified putative chimeric proteins in public databases and validated two using Western blotting. GO analysis revealed that proteins resided in one multi-protein complex or functioning in metabolic or signaling pathway tended to produce fused products. Taken together, we have shown substantial evidence for the underestimated TIC events; and TICs could be a novel regulated way to further increases the proteome complexity in mammalian genomes. Possible regulation mechanisms and evolution of TICs were also discussed. PMID:26718406

  2. Leaf aquaporin transcript abundance in peanut genotypes diverging in expression of the limited-transpiration trait when subjected to differing vapor pressure deficits and aquaporin inhibitors.

    PubMed

    Devi, M Jyostna; Sinclair, Thomas R; Jain, Mukesh; Gallo, Maria

    2016-04-01

    A plant trait currently being exploited to decrease crop yield loss under water-deficit conditions is limited-transpiration rate (TRlim ) under high atmospheric vapor pressure deficit (VPD) conditions. Although limited genotype comparisons for the TRlim trait have been performed in peanut (Arachis hypogaea), no detailed study to describe the basis for this trait in peanut has been reported. Since it has been hypothesized that the TRlim trait may be a result of low leaf hydraulic conductance associated with aquaporins (AQPs), the first objective of this study was to examine a possible correlation of TRlim to leaf AQP transcriptional profiles in six peanut cultivars. Five of the studied cultivars were selected because they expressed TRlim while the cultivar York did not. Transcripts of six AQPs were measured. Under exposure to high vapor pressure deficit, cultivar C 76-16 had decreased AQP transcript abundance for four of the six AQPs but in York only one AQP had decreased abundance. The second objective was to explore the influence of AQP inhibitors mercury and silver on expression of TRlim and AQP transcription profiles. Quantitative RT-PCR data were compared in cultivars York and C 76-16, which had the extreme response in TR to VPD. Inhibitor treatment resulted in increased abundance of AQP transcripts in both. The results of these experiments indicate that AQP transcript abundance itself may not be useful in identifying genotypes expressing the TRlim trait under high VPD conditions.

  3. Microdissected double-minute DNA detects variable patterns of chromosomal localizations and multiple abundantly expressed transcripts in normal and leukemic cells

    SciTech Connect

    Sen, S.; Zhou, Hongyi; Stass, S.A.; Sen, P. ); Mulac-Jericevic, B.; Pirrotta, V. )

    1994-02-01

    Double-minute (dm) chromosomes are cytogenetically resolvable DNA amplification-mediating acentric extrachromosomal structures that are commonly seen in primary tumors, tumor cell lines, and drug-resistant cells grown in vitro. Selective isolation of dm DNAs with standard molecular biological techniques is difficult, and thus, detailed studies to elucidate their structure, site of chromosomal origin, and chromosomal reintegration patterns have been limited. In those instances in which a gene has been localized on dms, characterization of the remainder of the DNA, which far exceeds the size of the gene identified, has remained inconclusive. dms seen in the acute myeloid leukemia cell line HL-60 have been shown to harbor the c-myc protooncogene. In this paper, the authors report the successful isolation of the dm-specific DNAs from these cells by the microdissection/polymerase chain reaction technique and demonstrate that the dm DNAs derived from a single discrete normal chromosome segment 8q24.1-q24.2 reintegrate at various specific locations in the leukemic cells. The microdissected dm DNA detects multiple abundantly expressed transcripts distinct from c-myc mRNA on Northern blots. By devising a [open quotes]transcript selection[close quotes] strategy, they cloned the partial genomic sequence of a gene from the microdissected DNA that encodes two of these RNAs. This strategy will be generally applicable for rapid cloning of unknown amplified genes harbored on dms. With DNA from 20 microdissected dms, they constructed a genomic library of about 20,000 recombinant microclones with an average insert size of about 450 bp. The microclones should help in isolating corresponding yeast artificial chromosome clones for high-resolution physical mapping of dms in HL-60 cells. Furthermore, application of the microdissection technique appears to be an extremely feasible approach to characterization of dms in other cell types. 42 refs., 6 figs., 1 tab.

  4. Ultrasensitive Detection of Bacteria by Targeting Abundant Transcripts.

    PubMed

    Wang, Xinhui; Li, Xinran; Liu, Shiwei; Ren, Hang; Yang, Mingjuan; Ke, Yuehua; Huang, Liuyu; Liu, Chao; Liu, Bo; Chen, Zeliang

    2016-01-01

    Molecular detection assays are increasingly becoming routine diagnostic techniques for bacterial infection; however, their sensitivities are restricted by the low concentrations of bacteria in clinical samples. Here, we report a new paradigm for ultrasensitive detection of bacteria. The principle of this approach is that by choosing highly transcribed genes as signature sequences and detecting both DNA and its RNA transcripts, assay sensitivity can be greatly improved. First, signature genes with abundant transcripts were screened by RNA-Seq. We confirmed that RT-PCR efficiently amplifies both DNA and RNA, while PCR amplifies only DNA. Unexpectedly, we found that the RNA extraction efficiency is relatively low, while simplified denaturation was more appropriate for transcript detection. For highly transcribed genes, RT-PCR consistently generated lower cycle threshold (Ct) values than those of PCR. The sensitivity of RT-PCR targeting abundant transcripts could detect quantities as low as one bacterium, which was not possible using PCR. Amplification of different genes among several other common bacteria also confirmed that transcript detection by RT-PCR is more sensitive than is DNA detection by PCR. Therefore, abundant transcript detection represents a universal strategy for ultrasensitive detection of bacteria.

  5. RNA-Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation

    PubMed Central

    Reyes, Juan M; Chitwood, James L; Ross, Pablo J

    2014-01-01

    Molecular changes occurring during mammalian oocyte maturation are partly regulated by cytoplasmic polyadenylation (CP) and affect oocyte quality, yet the extent of CP activity during oocyte maturation remains unknown. Single bovine oocyte RNA sequencing (RNA-Seq) was performed to examine changes in transcript abundance during in vitro oocyte maturation in cattle. Polyadenylated RNA from individual germinal-vesicle and metaphase-II oocytes was amplified and processed for Illumina sequencing, producing approximately 30 million reads per replicate for each sample type. A total of 10,494 genes were found to be expressed, of which 2,455 were differentially expressed (adjusted P<0.05 and fold change >2) between stages, with 503 and 1,952 genes respectively increasing and decreasing in abundance. Differentially expressed genes with complete 3’-untranslated-region sequence (279 increasing and 918 decreasing in polyadenylated transcript abundance) were examined for the presence, position, and distribution of motifs mediating CP, revealing enrichment (85%) and lack there of (18%) in up- and down-regulated genes, respectively. Examination of total and polyadenylated RNA abundance by quantitative PCR validated these RNA-Seq findings. The observed increases in polyadenylated transcript abundance within the RNA-Seq data are likely due to CP, providing novel insight into targeted transcripts and resultant differential gene expression profiles that contribute to oocyte maturation. PMID:25560149

  6. RNA-Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation.

    PubMed

    Reyes, Juan M; Chitwood, James L; Ross, Pablo J

    2015-02-01

    Molecular changes occurring during mammalian oocyte maturation are partly regulated by cytoplasmic polyadenylation (CP) and affect oocyte quality, yet the extent of CP activity during oocyte maturation remains unknown. Single bovine oocyte RNA sequencing (RNA-Seq) was performed to examine changes in transcript abundance during in vitro oocyte maturation in cattle. Polyadenylated RNA from individual germinal-vesicle and metaphase-II oocytes was amplified and processed for Illumina sequencing, producing approximately 30 million reads per replicate for each sample type. A total of 10,494 genes were found to be expressed, of which 2,455 were differentially expressed (adjusted P < 0.05 and fold change >2) between stages, with 503 and 1,952 genes respectively increasing and decreasing in abundance. Differentially expressed genes with complete 3'-untranslated-region sequence (279 increasing and 918 decreasing in polyadenylated transcript abundance) were examined for the presence, position, and distribution of motifs mediating CP, revealing enrichment (85%) and lack thereof (18%) in up- and down-regulated genes, respectively. Examination of total and polyadenylated RNA abundance by quantitative PCR validated these RNA-Seq findings. The observed increases in polyadenylated transcript abundance within the RNA-Seq data are likely due to CP, providing novel insight into targeted transcripts and resultant differential gene expression profiles that contribute to oocyte maturation.

  7. Accumulation of Transcripts Abundance after Barley Inoculation with Cochliobolus sativus

    PubMed Central

    Arabi, Mohammad Imad Eddin; AL-Daoude, Antonious; Shoaib, Amina; Jawhar, Mohammad

    2015-01-01

    Spot blotch caused by the hemibiotrophic pathogen Cochliobolus sativus has been the major yield-reducing factor for barley production during the last decade. Monitoring transcriptional reorganization triggered in response to this fungus is an essential first step for the functional analysis of genes involved in the process. To characterize the defense responses initiated by barley resistant and susceptible cultivars, a survey of transcript abundance at early time points of C. sativus inoculation was conducted. A notable number of transcripts exhibiting significant differential accumulations in the resistant and susceptible cultivars were detected compared to the non-inoculated controls. At the p-value of 0.0001, transcripts were divided into three general categories; defense, regulatory and unknown function, and the resistant cultivar had the greatest number of common transcripts at different time points. Quantities of differentially accumulated gene transcripts in both cultivars were identified at 24 h post infection, the approximate time when the pathogen changes trophic lifestyles. The unique and common accumulated transcripts might be of considerable interest for enhancing effective resistance to C. sativus. PMID:25774113

  8. Metaproteomics reveals abundant transposase expression in mutualistic endosymbionts

    SciTech Connect

    Kleiner, Manuel; Young, Jacque C; Shah, Manesh B; Verberkmoes, Nathan C; Dubilier, Nicole

    2013-01-01

    Transposases, enzymes that catalyze the movement of mobile genetic elements, are the most abundant genes in nature. While many bacteria encode an abundance of transposases in their genomes, the current paradigm is that transposase gene expression is tightly regulated and generally low due to its severe mutagenic effects. In the current study, we detected the highest number of transposase proteins ever reported in bacteria, in symbionts of the gutless marine worm Olavius algarvensis using metaproteomics. At least 26 different transposases from 12 different families were detected and genomic and proteomic analyses suggest many of these are active. This high expression of transposases indicates that the mechanisms for their tight regulation have been disabled or destroyed. Based on recent studies on other symbionts and pathogens that showed high transposase transcription, we speculate that abundant transposase expression might be common in symbionts and pathogens.

  9. Transcriptional abundance is not the single force driving the evolution of bacterial proteins

    PubMed Central

    2013-01-01

    Background Despite rapid progress in understanding the mechanisms that shape the evolution of proteins, the relative importance of various factors remain to be elucidated. In this study, we have assessed the effects of 16 different biological features on the evolutionary rates (ERs) of protein-coding sequences in bacterial genomes. Results Our analysis of 18 bacterial species revealed new correlations between ERs and constraining factors. Previous studies have suggested that transcriptional abundance overwhelmingly constrains the evolution of yeast protein sequences. This transcriptional abundance leads to selection against misfolding or misinteractions. In this study we found that there was no single factor in determining the evolution of bacterial proteins. Not only transcriptional abundance (codon adaptation index and expression level), but also protein-protein associations (PPAs), essentiality (ESS), subcellular localization of cytoplasmic membrane (SLM), transmembrane helices (TMH) and hydropathicity score (HS) independently and significantly affected the ERs of bacterial proteins. In some species, PPA and ESS demonstrate higher correlations with ER than transcriptional abundance. Conclusions Different forces drive the evolution of protein sequences in yeast and bacteria. In bacteria, the constraints are involved in avoiding a build-up of toxic molecules caused by misfolding/misinteraction (transcriptional abundance), while retaining important functions (ESS, PPA) and maintaining the cell membrane (SLM, TMH and HS). Each of these independently contributes to the variation in protein evolution. PMID:23914835

  10. Transcription factor abundance controlled by an auto-regulatory mechanism involving a transcription start site switch

    PubMed Central

    Ngondo, Richard Patryk; Carbon, Philippe

    2014-01-01

    A transcriptional feedback loop is the simplest and most direct means for a transcription factor to provide an increased stability of gene expression. In this work performed in human cells, we reveal a new negative auto-regulatory mechanism involving an alternative transcription start site (TSS) usage. Using the activating transcription factor ZNF143 as a model, we show that the ZNF143 low-affinity binding sites, located downstream of its canonical TSS, play the role of protein sensors to induce the up- or down-regulation of ZNF143 gene expression. We uncovered that the TSS switch that mediates this regulation implies the differential expression of two transcripts with an opposite protein production ability due to their different 5′ untranslated regions. Moreover, our analysis of the ENCODE data suggests that this mechanism could be used by other transcription factors to rapidly respond to their own aberrant expression level. PMID:24234445

  11. Abundances of crenarchaeal amoA genes and transcripts in the Pacific Ocean.

    PubMed

    Church, Matthew J; Wai, Brenner; Karl, David M; DeLong, Edward F

    2010-03-01

    Planktonic Crenarchaea are thought to play a key role in chemolithotrophic ammonia oxidation, a critical step of the marine nitrogen (N) cycle. In this study, we examined the spatial distributions of ammonia-oxidizing Crenarchaea across a large (approximately 5200 km) region of the central Pacific Ocean. Examination of crenarchaeal 16S rRNA, ammonia monooxygenase subunit A (amoA) genes, and amoA transcript abundances provided insight into their spatial distributions and activities. Crenarchaeal gene abundances increased three to four orders of magnitude with depth between the upper ocean waters and dimly lit waters of the mesopelagic zone. The resulting median value of the crenarchaeal amoA: 16S rRNA gene ratio was 1.3, suggesting the majority of Crenarchaea in the epi- and mesopelagic regions of the Pacific Ocean have the metabolic machinery for ammonia oxidation. Crenarchaeal amoA transcript abundances typically increased one to two orders of magnitude in the transitional zone separating the epipelagic waters from the mesopelagic (100-200 m), before decreasing into the interior of the mesopelagic zone. The resulting gene copy normalized transcript abundances revealed elevated amoA expression in the upper ocean waters (0-100 m) where crenarchaeal abundances were low, with transcripts decreasing into the mesopelagic zone as crenarchaeal gene abundances increased. These results suggest ammonia-oxidizing Crenarchaea are active contributors to the N cycle throughout the epi- and mesopelagic waters of the Pacific Ocean.

  12. The human protein p19ARF is not detected in hemopoietic human cell lines that abundantly express the alternative beta transcript of the p16INK4a/MTS1 gene.

    PubMed

    Della Valle, V; Duro, D; Bernard, O; Larsen, C J

    1997-11-13

    The p16/MTS1/CDKN2 gene on human chromosome band 9p21 encodes two unrelated proteins: p16INK4a, a specific inhibitor of the cyclin D-dependent kinases CKD4 and CDK6, and the structurally unrelated p19ARF protein that arrests cell growth in G1/S and also in G2/M. By use of polyclonal antibodies, the human p19ARF (hp19ARF) protein has been identified in the nucleus of various cells including normal cultured fibroblasts. The level of this protein did not fluctuate throughout the cell cycle and was more elevated in fibroblasts with limited or arrested growth, suggesting that p19ARF accumulated in presenescent or senescent cells. Interestingly, hp19ARF was not detected in several hemopoietic tumor cell lines (mainly of B-type lymphoid origin) that expressed abundant amounts of the p16beta transcript. This finding indicates that in certain tumors, the expression of hp19ARF RNA and protein may be uncoupled. Furthermore, it suggests that disruption of a translational mechanism may be involved in the inactivation of hp19ARF. PMID:9395243

  13. Ethylene and pollination decrease transcript abundance of an ethylene receptor gene in Dendrobium petals.

    PubMed

    Thongkum, Monthathip; Burns, Parichart; Bhunchoth, Anjana; Warin, Nuchnard; Chatchawankanphanich, Orawan; van Doorn, Wouter G

    2015-03-15

    We studied the expression of a gene encoding an ethylene receptor, called Ethylene Response Sensor 1 (Den-ERS1), in the petals of Dendrobium orchid flowers. Transcripts accumulated during the young floral bud stage and declined by the time the flowers had been open for several days. Pollination or exposure to exogenous ethylene resulted in earlier flower senescence, an increase in ethylene production and a lower Den-ERS1 transcript abundance. Treatment with 1-methylcyclopropene (1-MCP), an inhibitor of the ethylene receptor, decreased ethylene production and resulted in high transcript abundance. The literature indicates two kinds of ethylene receptor genes with regard to the effects of ethylene. One group shows ethylene-induced down-regulated transcription, while the other has ethylene-induced up-regulation. The present gene is an example of the first group. The 5' flanking region showed binding sites for Myb and myb-like, homeodomain, MADS domain, NAC, TCP, bHLH and EIN3-like transcription factors. The binding site for the EIN3-like factor might explain the ethylene effect on transcription. A few other transcription factors (RAV1 and NAC) seem also related to ethylene effects.

  14. Transcript Abundance of Photorhabdus Insect-Related (Pir) Toxin in Manduca sexta and Galleria mellonella Infections

    PubMed Central

    Castagnola, Anaïs; Mulley, Geraldine; Davis, Nathaniel; Waterfield, Nicholas; Stock, S. Patricia

    2016-01-01

    In this study, we assessed pirAB toxin transcription in Photorhabdus luminescens laumondii (strain TT01) (Enterobacteriaceae) by comparing mRNA abundance under in vivo and in vitro conditions. In vivo assays considered both natural and forced infections with two lepidopteran hosts: Galleria mellonella and Manduca sexta. Three portals of entry were utilized for the forced infection assays: (a) integument; (b) the digestive route (via mouth and anus); and (c) the tracheal route (via spiracles). We also assessed plu4093-2 transcription during the course of a natural infection; this is when the bacteria are delivered by Heterorhabditis bacteriophora nematodes. Transcript abundance in G. mellonella was higher than in M. sexta at two of the observed time points: 15 and 18 h. Expression of pirAB plu4093-2 reached above endogenous control levels at 22 h in G. mellonella but not in M. sexta. Overall, pirAB plu4093-2 transcripts were not as highly expressed in M. sexta as in G. mellonella, from 15 to 22 h. This is the first study to directly compare pirAB plu4093-2 toxin transcript production considering different portals of entry. PMID:27690103

  15. The use of molecular beacons to directly measure bacterial mRNA abundances and transcript degradation.

    PubMed

    Kuechenmeister, Lisa J; Anderson, Kelsi L; Morrison, John M; Dunman, Paul M

    2009-02-01

    The regulation of mRNA turnover is a dynamic means by which bacteria regulate gene expression. Although current methodologies allow characterization of the stability of individual transcripts, procedures designed to measure alterations in transcript abundance/turnover on a high throughput scale are lacking. In the current report, we describe the development of a rapid and simplified molecular beacon-based procedure to directly measure the mRNA abundances and mRNA degradation properties of well-characterized Staphylococcus aureus pathogenicity factors. This method does not require any PCR-based amplification, can monitor the abundances of multiple transcripts within a single RNA sample, and was successfully implemented into a high throughput screen of transposon mutant library members to detect isolates with altered mRNA turnover properties. It is expected that the described methodology will provide great utility in characterizing components of bacterial RNA degradation processes and can be used to directly measure the mRNA levels of virtually any bacterial transcript.

  16. Transcript Abundance of Putative Lipid Phosphate Phosphatases During Development of Trypanosoma brucei in the Tsetse Fly.

    PubMed

    Alves e Silva, Thiago Luiz; Savage, Amy F; Aksoy, Serap

    2016-04-01

    African trypanosomes (Trypanosoma brucei spp.) cause devastating diseases in sub-Saharan Africa. Trypanosomes differentiate repeatedly during development in tsetse flies before gaining mammalian infectivity in fly salivary glands. Lipid phosphate phosphatases (LPPs) are involved in diverse biological processes, such as cell differentiation and cell migration. Gene sequences encoding two putative T. brucei LPP proteins were used to search the T. brucei genome, revealing two additional putative family members. Putative structural features and transcript abundance during parasite development in tsetse fly were characterized. Three of the four LPP proteins are predicted to have six transmembrane domains, while the fourth shows only one. Semiquantitative gene expression revealed differential regulation of LPPs during parasite development. Transcript abundance for three of the four putative LPP genes was elevated in parasites infecting salivary glands, but not mammalian-infective metacyclic cells in fly saliva, indicating a potential role of this family in parasite establishment in tsetse salivary glands.

  17. cAMP post-transcriptionally diminishes the abundance of adrenodoxin reductase mRNA.

    PubMed Central

    Brentano, S T; Black, S M; Lin, D; Miller, W L

    1992-01-01

    Adrenodoxin reductase (AR; ferridoxin: NADP+ oxidoreductase, EC 1.18.1.2) is a flavoprotein that mediates electron transport from NADPH to all known mitochondrial forms of cytochrome P450. AR mRNA was found in all human adult and fetal tissues examined; however, it was vastly more abundant in tissues that synthesize steroid hormones. The ratio of the 18- form of mRNA lacking 18 alternately spliced bases to the 18+ form was approximately 100:1 and remained constant irrespective of the tissue or hormonal manipulation, indicating that the alternate splicing is a passive nonregulated event. AR protein was unchanged by forskolin treatment of human JEG-3 cytotrophoblast cells for 24 h, but the mRNA diminished. Phorbol 12-myristate 13-acetate and cycloheximide had no effect, even though these agents had the expected effects on P450scc and adrenodoxin mRNAs. cAMP decreased the abundance of AR mRNA expressed from both transfected plasmids and the endogenous gene, indicating the effect was post-transcriptional. AR gene transcription in JEG-3 cells and promoter-chloramphenicol acetyltransferase constructs transfected into JEG-3 cells were unresponsive to forskolin. Powerful basal transcription elements were identified between -46 and -214 bases from the principal transcriptional initiation site, a region containing six elements closely resembling the binding site for transcription factor SP1. Images PMID:1315050

  18. 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

  19. Stochastic variation of transcript abundance in C57BL/6J mice

    PubMed Central

    2011-01-01

    Background Transcripts can exhibit significant variation in tissue samples from inbred laboratory mice. We have designed and carried out a microarray experiment to examine transcript variation across samples from adipose, heart, kidney, and liver tissues of C57BL/6J mice and to partition variation into within-mouse and between-mouse components. Within-mouse variance captures variation due to heterogeneity of gene expression within tissues, RNA-extraction, and array processing. Between-mouse variance reflects differences in transcript abundance between genetically identical mice. Results The nature and extent of transcript variation differs across tissues. Adipose has the largest total variance and the largest within-mouse variance. Liver has the smallest total variance, but it has the most between-mouse variance. Genes with high variability can be classified into groups with correlated patterns of expression that are enriched for specific biological functions. Variation between mice is associated with circadian rhythm, growth hormone signaling, immune response, androgen regulation, lipid metabolism, and the extracellular matrix. Genes showing correlated patterns of within-mouse variation are also associated with biological functions that largely reflect heterogeneity of cell types within tissues. Conclusions Genetically identical mice can experience different individual outcomes for medically important traits. Variation in gene expression observed between genetically identical mice can identify functional classes of genes that are likely to vary in the absence of experimental perturbations, can inform experimental design decisions, and provides a baseline for the interpretation of gene expression data in interventional studies. The extent of transcript variation among genetically identical mice underscores the importance of stochastic and micro-environmental factors and their phenotypic consequences. PMID:21450099

  20. Characterization of differential transcript abundance through time during Nematostella vectensis development

    PubMed Central

    2013-01-01

    Background Nematostella vectensis, a burrowing sea anemone, has become a popular species for the study of cnidarian development. In previous studies, the expression of a variety of genes has been characterized during N. vectensis development with in situ mRNA hybridization. This has provided detailed spatial resolution and a qualitative perspective on changes in expression. However, little is known about broad transcriptome-level patterns of gene expression through time. Here we examine the expression of N. vectensis genes through the course of development with quantitative RNA-seq. We provide an overview of changes in the transcriptome through development, and examine the maternal to zygotic transition, which has been difficult to investigate with other tools. Results We measured transcript abundance in N. vectensis with RNA-seq at six time points in development: zygote (2 hours post fertilization (HPF)), early blastula (7 HPF), mid-blastula (12 HPF), gastrula (24 HPF), planula (5 days post fertilization (DPF)) and young polyp (10 DPF). The major wave of zygotic expression appears between 7–12 HPF, though some changes occur between 2–7 HPF. The most dynamic changes in transcript abundance occur between the late blastula and early gastrula stages. More transcripts are upregulated between the gastrula and planula than downregulated, and a comparatively lower number of transcripts significantly change between planula and polyp. Within the maternal to zygotic transition, we identified a subset of maternal factors that decrease early in development, and likely play a role in suppressing zygotic gene expression. Among the first genes to be expressed zygotically are genes whose proteins may be involved in the degradation of maternal RNA. Conclusions The approach presented here is highly complementary to prior studies on spatial patterns of gene expression, as it provides a quantitative perspective on a broad set of genes through time but lacks spatial resolution. In

  1. 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

  2. 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.

  3. Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants

    PubMed Central

    Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

    2015-01-01

    Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5′ transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5′UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5′end can modulate protein levels up to 160%–300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple

  4. Identifying Novel Transcriptional Regulators with Circadian Expression

    PubMed Central

    Schick, Sandra; Thakurela, Sudhir; Fournier, David; Hampel, Mareike Hildegard

    2015-01-01

    Organisms adapt their physiology and behavior to the 24-h day-night cycle to which they are exposed. On a cellular level, this is regulated by intrinsic transcriptional-translational feedback loops that are important for maintaining the circadian rhythm. These loops are organized by members of the core clock network, which further regulate transcription of downstream genes, resulting in their circadian expression. Despite progress in understanding circadian gene expression, only a few players involved in circadian transcriptional regulation, including transcription factors, epigenetic regulators, and long noncoding RNAs, are known. Aiming to discover such genes, we performed a high-coverage transcriptome analysis of a circadian time course in murine fibroblast cells. In combination with a newly developed algorithm, we identified many transcription factors, epigenetic regulators, and long intergenic noncoding RNAs that are cyclically expressed. In addition, a number of these genes also showed circadian expression in mouse tissues. Furthermore, the knockdown of one such factor, Zfp28, influenced the core clock network. Mathematical modeling was able to predict putative regulator-effector interactions between the identified circadian genes and may help for investigations into the gene regulatory networks underlying circadian rhythms. PMID:26644408

  5. Autoregulatory loop of Msx1 expression involving its antisense transcripts.

    PubMed

    Petit, Stéphane; Meary, Fleur; Pibouin, Laurence; Jeanny, Jean-Claude; Fernandes, Isabelle; Poliard, Anne; Hotton, Dominique; Berdal, Ariane; Babajko, Sylvie

    2009-08-01

    The Msx1 homeogene plays an important role in epithelial-mesenchymal interactions leading organogenesis. Msx1 gene is submitted to bidirectional transcription generating a long non-coding antisense (AS) RNA potentially involved in Msx1 expression regulation. RT-Q-PCR and RNA-FISH studies indicated that transient overexpression of the Msx1 AS transcript in 705IC5 mouse odontoblasts decreased the abundance of endogenous Msx1 S mRNA at the post-transcriptional level. Conversely, Msx1 overexpression increased the AS RNA level probably by activating AS transcription. In vivo mapping by RT-PCR evidenced both Msx1 RNAs in all adult mouse tissues tested raising the issue of Msx1 function during adulthood. The expression patterns of the two RNAs were similar, confirming the tight S/AS relationship. In particular, both Msx1 mRNAs and Msx1 protein were similarly distributed in eyes, and were found in regions with a common ectodermic origin and in cells potentially involved in regeneration. In conclusion, we report that Msx1 S RNA is negatively controlled by its AS RNA at a post-transcriptional level, and that the AS RNA is retrocontrolled positively by Msx1. The tight link between Msx1 S and AS RNAs constitutes a regulatory loop resulting in a fine-tuned expression of Msx1 which appears to be significant for adult homeostasis.

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

    DOE PAGES

    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.; et al

    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 prior studies due to its influence on:more » 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

  7. 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.

  8. Impacts of Different Exposure Scenarios on Transcript Abundances in Danio rerio Embryos when Investigating the Toxicological Burden of Riverine Sediments

    PubMed Central

    Bluhm, Kerstin; Otte, Jens C.; Yang, Lixin; Zinsmeister, Christian; Legradi, Jessica; Keiter, Steffen; Kosmehl, Thomas; Braunbeck, Thomas; Strähle, Uwe; Hollert, Henner

    2014-01-01

    Purpose Recently, a proof-of-concept study revealed the suitability of transcriptome analyses to obtain and assess changes in the abundance of transcripts in zebrafish (Danio rerio) embryos after exposure to organic sediment extracts. The present study investigated changes in the transcript abundance in zebrafish embryos exposed to whole sediment samples and corresponding organic extracts in order to identify the impact of different exposure pathways on sediment toxicity. Materials and Methods Danio rerio embryos were exposed to sublethal concentrations of three sediment samples from the Danube River, Germany. The sediment samples were investigated both as freeze-dried samples and as organic extracts. Silica dust and a process control of the extraction procedure were used as references. After exposure, mRNA was isolated and changes in profiles of gene expression levels were examined by an oligonucleotide microarray. The microarray results were compared with bioassays, chemical analysis of the sediments and profiles of gene expression levels induced by several single substances. Results and Discussion The microarray approach elucidated significant changes in the abundance of transcripts in exposed zebrafish embryos compared to the references. Generally, results could be related to Ah-receptor-mediated effects as confirmed by bioassays and chemical analysis of dioxin-like contaminants, as well as to exposure to stress-inducing compounds. Furthermore, the results indicated that mixtures of chemicals, as present in sediment and extract samples, result in complex changes of gene expression level profiles difficult to compare with profiles induced by single chemical substances. Specifically, patterns of transcript abundances were less influenced by the chemical composition at the sampling site compared t the method of exposure (sediment/extract). This effect might be related to different bioavailability of chemicals. Conclusions The apparent difference between the

  9. A novel method to prioritize RNAseq data for post-hoc analysis based on absolute changes in transcript abundance.

    PubMed

    McNutt, Patrick; Gut, Ian; Hubbard, Kyle; Beske, Phil

    2015-06-01

    The use of fold-change (FC) to prioritize differentially expressed genes (DEGs) for post-hoc characterization is a common technique in the analysis of RNA sequencing datasets. However, the use of FC can overlook certain population of DEGs, such as high copy number transcripts which undergo metabolically expensive changes in expression yet fail to exceed the ratiometric FC cut-off, thereby missing potential important biological information. Here we evaluate an alternative approach to prioritizing RNAseq data based on absolute changes in normalized transcript counts (ΔT) between control and treatment conditions. In five pairwise comparisons with a wide range of effect sizes, rank-ordering of DEGs based on the magnitude of ΔT produced a power curve-like distribution, in which 4.7-5.0% of transcripts were responsible for 36-50% of the cumulative change. Thus, differential gene expression is characterized by the high production-cost expression of a small number of genes (large ΔT genes), while the differential expression of the majority of genes involves a much smaller metabolic investment by the cell. To determine whether the large ΔT datasets are representative of coordinated changes in the transcriptional program, we evaluated large ΔT genes for enrichment of gene ontologies (GOs) and predicted protein interactions. In comparison to randomly selected DEGs, the large ΔT transcripts were significantly enriched for both GOs and predicted protein interactions. Furthermore, enrichments were were consistent with the biological context of each comparison yet distinct from those produced using equal-sized populations of large FC genes, indicating that the large ΔT genes represent an orthagonal transcriptional response. Finally, the composition of the large ΔT gene sets were unique to each pairwise comparison, indicating that they represent coherent and context-specific responses to biological conditions rather than the non-specific upregulation of a family of genes

  10. Cell-Specific Expression of Mitochondrial Transcripts in Maize Seedlings.

    PubMed Central

    Li, X. Q.; Zhang, M.; Brown, G. G.

    1996-01-01

    Although mitochondria are thought to assume crucial and possibly novel physiological functions during male gametogenesis, it is not known to what extent mitochondrial function is necessary for other aspects of plant development or to what degree the expression of plant mitochondrial genes is subject to cell-specific regulation, particularly during vegetative growth. We have used in situ hybridization to show that extensive differences exist in the levels of mitochondrial RNAs (mtRNAs) among different tissues and among different individual cell types within the same organ of maize seedlings. The expression of all examined mtRNAs is enhanced in vascular bundles, particularly in procambium- and xylem-forming cells. Mitochondrial transcript levels correlated highly with cell division activity. For example, in roots, the transcripts are abundant in the dividing cells of the meristem but drop to very low levels in the nondividing cells of the root cap and the meristem quiescent center. By comparison, levels of functional mitochondria, as assessed by rhodamine-123 fluorescence, did not vary greatly among the same group of cells. In shoots, in situ hybridization and blot hybridization revealed differences in the patterns of localization among different mtRNAs. The results indicate that during vegetative growth, mitochondrial gene expression at the transcript level is subject to an unexpected degree of cell-specific regulation and that different controls may operate on different trancripts. PMID:12239371

  11. Patterns of Transcript Abundance of Eukaryotic Biogeochemically-Relevant Genes in the Amazon River Plume.

    PubMed

    Zielinski, Brian L; Allen, Andrew E; Carpenter, Edward J; Coles, Victoria J; Crump, Byron C; Doherty, Mary; Foster, Rachel A; Goes, Joaquim I; Gomes, Helga R; Hood, Raleigh R; McCrow, John P; Montoya, Joseph P; Moustafa, Ahmed; Satinsky, Brandon M; Sharma, Shalabh; Smith, Christa B; Yager, Patricia L; Paul, John H

    2016-01-01

    silicon became limiting. Expression of these genes, including carbonic anhydrase and transporters for nitrate and phosphate, were found to reflect the physiological status and biogeochemistry of river plume environments. These relatively stable patterns of eukaryotic transcript abundance occurred over modest spatiotemporal scales, with similarity observed in sample duplicates collected up to 2.45 km in space and 120 minutes in time. These results confirm the use of metatranscriptomics as a valuable tool to understand and predict microbial community function. PMID:27598790

  12. Patterns of Transcript Abundance of Eukaryotic Biogeochemically-Relevant Genes in the Amazon River Plume.

    PubMed

    Zielinski, Brian L; Allen, Andrew E; Carpenter, Edward J; Coles, Victoria J; Crump, Byron C; Doherty, Mary; Foster, Rachel A; Goes, Joaquim I; Gomes, Helga R; Hood, Raleigh R; McCrow, John P; Montoya, Joseph P; Moustafa, Ahmed; Satinsky, Brandon M; Sharma, Shalabh; Smith, Christa B; Yager, Patricia L; Paul, John H

    2016-01-01

    silicon became limiting. Expression of these genes, including carbonic anhydrase and transporters for nitrate and phosphate, were found to reflect the physiological status and biogeochemistry of river plume environments. These relatively stable patterns of eukaryotic transcript abundance occurred over modest spatiotemporal scales, with similarity observed in sample duplicates collected up to 2.45 km in space and 120 minutes in time. These results confirm the use of metatranscriptomics as a valuable tool to understand and predict microbial community function.

  13. Patterns of Transcript Abundance of Eukaryotic Biogeochemically-Relevant Genes in the Amazon River Plume

    PubMed Central

    Allen, Andrew E.; Carpenter, Edward J.; Coles, Victoria J.; Crump, Byron C.; Doherty, Mary; Foster, Rachel A.; Goes, Joaquim I.; Gomes, Helga R.; Hood, Raleigh R.; McCrow, John P.; Montoya, Joseph P.; Moustafa, Ahmed; Satinsky, Brandon M.; Sharma, Shalabh; Smith, Christa B.; Yager, Patricia L.; Paul, John H.

    2016-01-01

    silicon became limiting. Expression of these genes, including carbonic anhydrase and transporters for nitrate and phosphate, were found to reflect the physiological status and biogeochemistry of river plume environments. These relatively stable patterns of eukaryotic transcript abundance occurred over modest spatiotemporal scales, with similarity observed in sample duplicates collected up to 2.45 km in space and 120 minutes in time. These results confirm the use of metatranscriptomics as a valuable tool to understand and predict microbial community function. PMID:27598790

  14. Controlling for gene expression changes in transcription factor protein networks.

    PubMed

    Banks, Charles A S; Lee, Zachary T; Boanca, Gina; Lakshminarasimhan, Mahadevan; Groppe, Brad D; Wen, Zhihui; Hattem, Gaye L; Seidel, Chris W; Florens, Laurence; Washburn, Michael P

    2014-06-01

    The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein-protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions.

  15. AtRTD - a comprehensive reference transcript dataset resource for accurate quantification of transcript-specific expression in Arabidopsis thaliana.

    PubMed

    Zhang, Runxuan; Calixto, Cristiane P G; Tzioutziou, Nikoleta A; James, Allan B; Simpson, Craig G; Guo, Wenbin; Marquez, Yamile; Kalyna, Maria; Patro, Rob; Eyras, Eduardo; Barta, Andrea; Nimmo, Hugh G; Brown, John W S

    2015-10-01

    RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression.

  16. Environmental stresses modulate abundance and timing of alternatively spliced circadian transcripts in Arabidopsis.

    PubMed

    Filichkin, Sergei A; Cumbie, Jason S; Dharmawardhana, Palitha; Jaiswal, Pankaj; Chang, Jeff H; Palusa, Saiprasad G; Reddy, A S N; Megraw, Molly; Mockler, Todd C

    2015-02-01

    Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron-retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad-range thermal cycles triggered a sharp increase in the long IR CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or Pseudomonas syringae infection induced a similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts, whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out-of-phase oscillations of CCA1 IR transcripts. Taken together, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of nonsense-mediated mRNA decay (NMD)-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs. PMID:25680774

  17. Environmental Stresses Modulate Abundance and Timing of Alternatively Spliced Circadian Transcripts in Arabidopsis.

    PubMed

    Filichkin, Sergei A; Cumbie, Jason S; Dharmawadhana, J Palitha; Jaiswal, Pankaj; Chang, Jeff H; Palusa, Saiprasad G; Reddy, A S N; Megraw, Molly; Mockler, Todd C

    2014-11-01

    Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad range thermal cycles triggered a sharp increase in the long intron retaining CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or P. syringae infection induced similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out of phase oscillations of CCA1 IR transcripts. Altogether, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of NMD-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs. PMID:25366180

  18. Brugia malayi abundant larval transcript 2 protein treatment attenuates experimentally-induced colitis in mice.

    PubMed

    Khatri, Vishal; Amdare, Nitin; Yadav, Ravi Shankar; Tarnekar, Aaditya; Goswami, Kalyan; Reddy, Maryada Venkata Rami

    2015-11-01

    Helminths are known to modulate host's immunity by suppressing host protective pro-inflammatory responses. Such immunomodulatory effects have been experimentally shown to have therapeutic implications in immune mediated disorders. In the present study, we have explored a filarial protein i.e. Brugia malayi recombinant abundant larval transcript 2 (rBmALT2) for its therapeutic effect in dextran sodium sulfate (DSS) induced colitis in mouse model. The immunomodulatory activity of rBmALT-2 was initially confirmed by demonstrating that it suppressed the lipopolysaccharide (LPS) induced nitric oxide synthesis and down-regulated the expression of pro-inflammatory cytokines in vitro by peritoneal exudate cells of mice. Treatment with rBmALT2 reduced severity of colitis associated with significant reduction in weight loss, disease activity, colon damage, mucosal edema and histopathological score including myeloperoxidase activity in colon tissues. rBmALT2 was comparatively more effective in attenuation of colitis when used in the preventive mode than when used for curative purpose. The therapeutic effect of rBmALT2 was found to be associated with downregulation of IFN-γ, IL-6, IL-17 and upregulation of IL-10 cytokines. These results provide strong experimental evidence that BmALT2 could be a potential alternative therapeutic agent in colitis. PMID:26669016

  19. Abundance of specific mRNA transcripts impacts hatching success in European eel, Anguilla anguilla L.

    PubMed

    Rozenfeld, Christoffer; Butts, Ian A E; Tomkiewicz, Jonna; Zambonino-Infante, Jose-Luis; Mazurais, David

    2016-01-01

    Maternal mRNA governs early embryonic development in fish and variation in abundance of maternal transcripts may contribute to variation in embryonic survival and hatch success in European eel, Anguilla anguilla. Previous studies have shown that quantities of the maternal gene products β-tubulin, insulin-like growth factor 2 (igf2), nucleoplasmin (npm2), prohibitin 2 (phb2), phosphatidylinositol glycan biosynthesis class F protein 5 (pigf5), and carnitine O-palmitoyltransferase liver isoform-like 1 (cpt1) are associated with embryonic developmental competence in other teleosts. Here, the relations between relative mRNA abundance of these genes in eggs and/or embryos and egg quality, was studied and analyzed. We compared egg quality of the two groups: i) batches with hatching and ii) batches with no hatching. Results showed no significant differences in relative mRNA abundance between the hatch and no hatching groups for any of the selected genes at 0, 2.5, and 5HPF. However, at 30HPF the hatch group showed significantly higher abundance of cpt1a, cpt1b, β-tubulin, phb2, and pigf5 transcripts than the no hatch group. Therefore, these results indicate that up-regulation of the transcription of these genes in European eel after the mid-blastula transition, may be needed to sustain embryonic development and hatching success.

  20. Post-transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans

    PubMed Central

    Wells, Melissa L.; Washington, Onica L.; Hicks, Stephanie N.; Nobile, Clarissa J.; Hartooni, Nairi; Wilson, Gerald M.; Zucconi, Beth E.; Huang, Weichun; Li, Leping; Fargo, David C.; Blackshear, Perry J.

    2015-01-01

    Summary Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins bind to AU-rich regions in target mRNAs, leading to their deadenylation and decay. Family members in Saccharomyces cerevisiae influence iron metabolism, whereas the single protein expressed in Schizosaccharomyces pombe, Zfs1, regulates cell–cell interactions. In the human pathogen Candida albicans, deep sequencing of mutants lacking the orthologous protein, Zfs1, revealed significant increases (> 1.5-fold) in 156 transcripts. Of these, 113 (72%) contained at least one predicted TTP family member binding site in their 3′UTR, compared with only 3 of 56 (5%) down-regulated transcripts. The zfs1Δ/Δ mutant was resistant to 3-amino-1,2,4-triazole, perhaps because of increased expression of the potential target transcript encoded by HIS3. Sequences of the proteins encoded by the putative Zfs1 targets were highly conserved among other species within the fungal CTG clade, while the predicted Zfs1 binding sites in these mRNAs often ‘disappeared’ with increasing evolutionary distance from the parental species. C. albicans Zfs1 bound to the ideal mammalian TTP binding site with high affinity, and Zfs1 was associated with target transcripts after co-immunoprecipitation. Thus, the biochemical activities of these proteins in fungi are highly conserved, but Zfs1-like proteins may target different transcripts in each species. PMID:25524641

  1. Differential regulation of abundance and deadenylation of maternal transcripts during bovine oocyte maturation in vitro and in vivo

    PubMed Central

    Thélie, Aurore; Papillier, Pascal; Pennetier, Sophie; Perreau, Christine; Traverso, Juan Martin; Uzbekova, Svetlana; Mermillod, Pascal; Joly, Catherine; Humblot, Patrice; Dalbiès-Tran, Rozenn

    2007-01-01

    Background In bovine maturing oocytes and cleavage stage embryos, gene expression is mostly controlled at the post-transcriptional level, through degradation and deadenylation/polyadenylation. We have investigated how post transcriptional control of maternal transcripts was affected during in vitro and in vivo maturation, as a model of differential developmental competence. Results Using real time PCR, we have analyzed variation of maternal transcripts, in terms of abundance and polyadenylation, during in vitro or in vivo oocyte maturation and in vitro embryo development. Four genes are characterized here for the first time in bovine: ring finger protein 18 (RNF18) and breast cancer anti-estrogen resistance 4 (BCAR4), whose oocyte preferential expression was not previously reported in any species, as well as Maternal embryonic leucine zipper kinase (MELK) and STELLA. We included three known oocyte marker genes (Maternal antigen that embryos require (MATER), Zygote arrest 1 (ZAR1), NACHT, leucine rich repeat and PYD containing 9 (NALP9)). In addition, we selected transcripts previously identified as differentially regulated during maturation, peroxiredoxin 1 and 2 (PRDX1, PRDX2), inhibitor of DNA binding 2 and 3 (ID2, ID3), cyclin B1 (CCNB1), cell division cycle 2 (CDC2), as well as Aurora A (AURKA). Most transcripts underwent a moderate degradation during maturation. But they displayed sharply contrasted deadenylation patterns that account for variations observed previously by DNA array and correlated with the presence of a putative cytoplasmic polyadenylation element in their 3' untranslated region. Similar variations in abundance and polyadenylation status were observed during in vitro maturation or in vivo maturation, except for PRDX1, that appears as a marker of in vivo maturation. Throughout in vitro development, oocyte restricted transcripts were progressively degraded until the morula stage, except for MELK ; and the corresponding genes remained silent after

  2. Seasonal variation in transcript abundance in cork tissue analyzed by real time RT-PCR.

    PubMed

    Soler, Marçal; Serra, Olga; Molinas, Marisa; García-Berthou, Emili; Caritat, Antònia; Figueras, Mercè

    2008-05-01

    The molecular processes underlying cork biosynthesis and differentiation are mostly unknown. Recently, a list of candidate genes for cork biosynthesis and regulation was made available opening new possibilities for molecular studies in cork oak (Quercus suber L.). Based on this list, we analyzed the seasonal variation in mRNA abundance in cork tissue of selected genes by real time reverse-transcriptase polymerase chain reaction (RT-PCR). Relative transcript abundance was evaluated by principal component analysis and genes were clustered in several functional subgroups. Structural genes of suberin pathways such as CYP86A1, GPAT and HCBT, and regulatory genes of the NAM and WRKY families showed highest transcript accumulation in June, a crucial month for cork development. Other cork structural genes, such as FAT and F5H, were significantly correlated with temperature and relative humidity. The stress genes HSP17.4 and ANN were strongly positively correlated to temperature, in accord with their protective role.

  3. Genome-wide identification and characterization of reference genes with different transcript abundances for Streptomyces coelicolor

    PubMed Central

    Li, Shanshan; Wang, Weishan; Li, Xiao; Fan, Keqiang; Yang, Keqian

    2015-01-01

    The lack of reliable reference genes (RGs) in the genus Streptomyces hampers effort to obtain the precise data of transcript levels. To address this issue, we aimed to identify reliable RGs in the model organism Streptomyces coelicolor. A pool of potential RGs containing 1,471 genes was first identified by determining the intersection of genes with stable transcript levels from four time-series transcriptome microarray datasets of S. coelicolor M145 cultivated in different conditions. Then, following a strict rational selection scheme including homology analysis, disturbance analysis, function analysis and transcript abundance analysis, 13 candidates were selected from the 1,471 genes. Based on real-time quantitative reverse transcription PCR assays, SCO0710, SCO6185, SCO1544, SCO3183 and SCO4758 were identified as the top five genes with the most stable transcript levels among the 13 candidates. Further analyses showed these five genes also maintained stable transcript levels in different S. coelicolor strains, as well as in Streptomyces avermitilis MA-4680 and Streptomyces clavuligerus NRRL 3585, suggesting they could fulfill the requirements of accurate data normalization in streptomycetes. Moreover, the systematic strategy employed in this work could be used for reference in other microorganism to select reliable RGs. PMID:26527303

  4. Transcript length mediates developmental timing of gene expression across Drosophila.

    PubMed

    Artieri, Carlo G; Fraser, Hunter B

    2014-11-01

    The time required to transcribe genes with long primary transcripts may limit their ability to be expressed in cells with short mitotic cycles, a phenomenon termed intron delay. As such short cycles are a hallmark of the earliest stages of insect development, we tested the impact of intron delay on the Drosophila developmental transcriptome. We find that long zygotically expressed genes show substantial delay in expression relative to their shorter counterparts, which is not observed for maternally deposited transcripts. Patterns of RNA-seq coverage along transcripts show that this delay is consistent with their inability to completely transcribe long transcripts, but not with transcriptional initiation-based regulatory control. We further show that highly expressed zygotic genes maintain compact transcribed regions across the Drosophila phylogeny, allowing conservation of embryonic expression patterns. We propose that the physical constraints of intron delay affect patterns of expression and the evolution of gene structure of a substantial portion of the Drosophila transcriptome.

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

    SciTech Connect

    Schwender, Jorg; Konig, Christina; Klapperstuck, Matthias; Heinzel, Nicolas; Munz, Eberhard; Hebbelmann, Inga; Hay, Jordan O.; Denolf, Peter; De Bodt, Stefanie; Redestig, Henning; Caestecker, Evelyne; Jakob, Peter M.; Borisjuk, Ljudmilla; Rolletschek, Hardy

    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, 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.

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

    DOE PAGES

    Schwender, Jorg; Konig, Christina; Klapperstuck, Matthias; Heinzel, Nicolas; Munz, Eberhard; Hebbelmann, Inga; Hay, Jordan O.; Denolf, Peter; De Bodt, Stefanie; Redestig, Henning; et al

    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

  7. A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance

    PubMed Central

    Spandidos, Athanasia; Wang, Xiaowei; Wang, Huajun; Dragnev, Stefan; Thurber, Tara; Seed, Brian

    2008-01-01

    Background Quantitative polymerase chain reaction (QPCR) is a widely applied analytical method for the accurate determination of transcript abundance. Primers for QPCR have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. Although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not designed for use under a common PCR thermal profile. Results We previously reported the implementation of an algorithm to predict PCR primers for most known human and mouse genes. We now report the use of that resource to identify 17483 pairs of primers that have been experimentally verified to amplify unique sequences corresponding to distinct murine transcripts. The primer pairs have been validated by gel electrophoresis, DNA sequence analysis and thermal denaturation profile. In addition to the validation studies, we have determined the uniformity of amplification using the primers and the technical reproducibility of the QPCR reaction using the popular and inexpensive SYBR Green I detection method. Conclusion We have identified an experimentally validated collection of murine primer pairs for PCR and QPCR which can be used under a common PCR thermal profile, allowing the evaluation of transcript abundance of a large number of genes in parallel. This feature is increasingly attractive for confirming and/or making more precise data trends observed from experiments performed with DNA microarrays. PMID:19108745

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

    PubMed Central

    Schwender, Jörg; König, Christina; Klapperstück, Matthias; Heinzel, Nicolas; Munz, Eberhard; Hebbelmann, Inga; Hay, Jordan O.; Denolf, Peter; De Bodt, Stefanie; Redestig, Henning; Caestecker, Evelyne; Jakob, Peter M.; Borisjuk, Ljudmilla; Rolletschek, Hardy

    2014-01-01

    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, 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. This limitation needs to be borne in mind in evaluating transcriptome data and designing metabolic engineering experiments. PMID:25506350

  9. Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development

    PubMed Central

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo; Ansong, Charles; Anderson, Lindsey N.; Smith, Jordan N.; Corley, Richard A.

    2016-01-01

    Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography–mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage–dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics. PMID:27084891

  10. Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development.

    PubMed

    Sadler, Natalie C; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo; Ansong, Charles; Anderson, Lindsey N; Smith, Jordan N; Corley, Richard A; Wright, Aaron T

    2016-07-01

    Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography-mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics. PMID:27084891

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

    PubMed

    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

    2016-05-01

    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.

  12. 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

  13. Resistance locus pyramids alter transcript abundance in soybean roots inoculated with Fusarium solani f.sp. glycines.

    PubMed

    Iqbal, M J; Yaegashi, S; Njiti, V N; Ahsan, R; Cryder, K L; Lightfoot, D A

    2002-11-01

    Soybean Sudden Death Syndrome (SDS) is caused by Fusarium solani f.sp. glycines (Fsg). Six quantitative trait loci (QTLs), each conferring partial resistance to SDS, have been discovered in an Essex x Forrest recombinant inbred line (RIL) population, but their mode of action is not clear. This study aimed to identify genes (ESTs) whose mRNA transcripts were altered in abundance in soybean roots following inoculation of Fsg. Roots of the soybean variety Forrest (four resistance alleles) were inoculated with Fsg, and 14 days later RNA sequences that were differentially expressed relative to uninoculated roots were enriched using suppression subtraction and differential display. The abundance of these RNAs was quantified in inoculated and non-inoculated roots by macroarray hybridizations. A unigene set of 135 ESTs was identified and used in a further macroarray analysis. The abundance of 28 cDNA fragments was increased more than two-fold in inoculated compared to uninoculated roots of RIL 23 (six resistance alleles). In Forrest and Essex (two resistance alleles), the level of only one mRNA was increased two-fold in inoculated roots compared to the uninoculated roots. In Essex most of the mRNAs analyzed decreased in abundance (61/135 showed a two-fold decrease), while in Forrest most mRNA abundances did not change. Among the 28 cDNAs that revealed a two-fold or higher increase in mRNA abundance in RIL 23, 14% code for proteins known to be involved in plant defense, 21% in metabolism, 14% in cell structure and 4% in transport. Unannotated ESTs accounted for 43% of the genes, and 4% of the sequences were previously unknown. The plant defense-related genes that showed a differential response to Fsg inoculation suggested a role for the phenylproponoid pathway in soybean defense against Fsg. In Essex, genes involved in plant defense, cell wall synthesis, ethylene synthesis and metabolism were expressed at lower levels in inoculated roots. The difference in response between

  14. Transcriptional profiling by cDNA-AFLP analysis showed differential transcript abundance in response to water stress in Populus hopeiensis

    PubMed Central

    2012-01-01

    Background Drought is one of the main environmental factors limiting tree growth and productivity of plantation forests worldwide. Populus hopeiensis Hu et Chow is one of the most important commercial plantation tree species in China. However, the genes controlling drought tolerance in this species have not been identified or characterized. Here, we conducted differential expression analyses and identified a number of genes that were up- or downregulated in P. hopeiensis during water stress. To the best of our knowledge, this is the first comprehensive study of differentially expressed genes in water-stressed P. hopeiensis. Results Using the cDNA-AFLP detection technique, we used 256 primer combinations to identify differentially expressed genes in P. hopeiensis during water stress. In total, 415 transcript derived-fragments (TDFs) were obtained from 10× deep sequencing of 473 selected TDFs. Of the 415 TDFs, 412 were annotated by BLAST searches against various databases. The majority of these genes encoded products involved in ion transport and compartmentalization, cell division, metabolism, and protein synthesis. The TDFs were clustered into 12 groups on the basis of their expression patterns. Of the 415 reliable TDFs, the sequences of 35 were homologous to genes that play roles in short or long-term resistance to drought stress. Some genes were further selected for validation of cDNA-AFLP expression patterns using real-time PCR analyses. The results confirmed the expression patterns that were detected using the cDNA-AFLP technique. Conclusion The cDNA-AFLP technique is an effective and powerful tool for identifying candidate genes that are differentially expressed under water stress. We demonstrated that 415 TDFs were differentially expressed in water-stressed poplar. The products of these genes are involved in various biological processes in the drought response of poplar. The results of this study will aid in the identification of candidate genes of future

  15. [Function and expression of transcription factors implicated in gonadal differentiation].

    PubMed

    Morohashi, K

    1998-07-01

    Several transcription factors such as SRY, DAX-1, Ad4BP/SF-1, WT-1 and SOX -9, have been revealed to be implicated in gonadal development by analyzing the genetic disorders of human and the gene disrupted mice. All of these transcription factors are expressed in the early stage of the gonadal development and the expression profiles during the gonadal development are clearly different between the two sexes. Functions of the transcription factors are discussed by referring genes under the control of these factors. Effects of endocrine disruptants on the expression of Ad4BP/SF-1 in the fetal gonads was also discussed. PMID:9702047

  16. Stochastic models of gene expression and post-transcriptional regulation

    NASA Astrophysics Data System (ADS)

    Pendar, Hodjat; Kulkarni, Rahul; Jia, Tao

    2011-10-01

    The intrinsic stochasticity of gene expression can give rise to phenotypic heterogeneity in a population of genetically identical cells. Correspondingly, there is considerable interest in understanding how different molecular mechanisms impact the 'noise' in gene expression. Of particular interest are post-transcriptional regulatory mechanisms involving genes called small RNAs, which control important processes such as development and cancer. We propose and analyze general stochastic models of gene expression and derive exact analytical expressions quantifying the noise in protein distributions [1]. Focusing on specific regulatory mechanisms, we analyze a general model for post-transcriptional regulation of stochastic gene expression [2]. The results obtained provide new insights into the role of post-transcriptional regulation in controlling the noise in gene expression. [4pt] [1] T. Jia and R. V. Kulkarni, Phys. Rev. Lett.,106, 058102 (2011) [0pt] [2] T. Jia and R. V. Kulkarni, Phys. Rev. Lett., 105, 018101 (2010)

  17. Laccase Gene Family in Cerrena sp. HYB07: Sequences, Heterologous Expression and Transcriptional Analysis.

    PubMed

    Yang, Jie; Xu, Xinqi; Ng, Tzi Bun; Lin, Juan; Ye, Xiuyun

    2016-01-01

    Laccases are a class of multi-copper oxidases with industrial potential. In this study, eight laccases (Lac1-8) from Cerrena sp. strain HYB07, a white-rot fungus with high laccase yields, were analyzed. The laccases showed moderate identities to each other as well as with other fungal laccases and were predicted to have high redox potentials except for Lac6. Selected laccase isozymes were heterologously expressed in the yeast Pichia pastoris, and different enzymatic properties were observed. Transcription of the eight laccase genes was differentially regulated during submerged and solid state fermentation, as shown by quantitative real-time polymerase chain reaction and validated reference genes. During 6-day submerged fermentation, Lac7 and 2 were successively the predominantly expressed laccase gene, accounting for over 95% of all laccase transcripts. Interestingly, accompanying Lac7 downregulation, Lac2 transcription was drastically upregulated on days 3 and 5 to 9958-fold of the level on day 1. Consistent with high mRNA abundance, Lac2 and 7, but not other laccases, were identified in the fermentation broth by LC-MS/MS. In solid state fermentation, less dramatic differences in transcript abundance were observed, and Lac3, 7 and 8 were more highly expressed than other laccase genes. Elucidating the properties and expression profiles of the laccase gene family will facilitate understanding, production and commercialization of the fungal strain and its laccases. PMID:27527131

  18. Transcriptional and post-transcriptional regulation of chloroplast gene expression in Petunia hybrida.

    PubMed

    van Grinsven, M Q; Gielen, J J; Zethof, J L; Nijkamp, H J; Kool, A J

    1986-11-01

    To study the control of differential gene expression during plastid biogenesis in Petunia hybrida, we have investigated the in vivo translation and transcription of the rbc L gene, coding for the large subunit of ribulose bisphosphate carboxylase (LSU), and the psa A gene, coding for P700 chlorophyll-a apoprotein (AP700). Differential expression of these plastid-encoded genes was studied in two developmentally different plastid systems, proplastid-like organelles from the green cell suspension AK2401 and mature chloroplasts from green leaves. In vivo translation of rbc L and psa A transcripts was analysed using specific antibodies. Specific transcript levels were analysed using internal fragments of the rbc L and psa A genes. A standardization procedure was used so that a direct correlation could be made between the amount of products and gene copy number. In Petunia hybrida the amount of LSU polypeptides present in both plastid types does not correspond to the amount of specific mRNA for the gene. Although the rbc L transcripts are present in both plastid types, the LSU protein is only present in green leaf plastids and not in cell culture plastids. In vitro translation of isolated rbc L transcripts give similar results, thereby suggesting that differences in the primary structure of the transcripts are responsible for the observed discrepancy. In contrast to this, the amount of AP700 polypeptides does correspond to the amount of the psa A transcripts. Therefore, our results indicate that the expression of chloroplast genes during plastid biogenesis takes place on at least two different levels: expression of the rbc L gene is regulated post-transcriptionally while expression of the psa A gene is regulated at the transcriptional level.

  19. A combined expression-interaction model for inferring the temporal activity of transcription factors.

    PubMed

    Shi, Yanxin; Klutstein, Michael; Simon, Itamar; Mitchell, Tom; Bar-Joseph, Ziv

    2009-08-01

    Methods suggested for reconstructing regulatory networks can be divided into two sets based on how the activity level of transcription factors (TFs) is inferred. The first group of methods relies on the expression levels of TFs, assuming that the activity of a TF is highly correlated with its mRNA abundance. The second treats the activity level as unobserved and infers it from the expression of the genes that the TF regulates. While both types of methods were successfully applied, each suffers from drawbacks that limit their accuracy. For the first set, the assumption that mRNA levels are correlated with activity is violated for many TFs due to post-transcriptional modifications. For the second, the expression level of a TF which might be informative is completely ignored. Here we present the post-transcriptional modification model (PTMM) that, unlike previous methods, utilizes both sources of data concurrently. Our method uses a switching model to determine whether a TF is transcriptionally or post-transcriptionally regulated. This model is combined with a factorial HMM to reconstruct the interactions in a dynamic regulatory network. Using simulated and real data, we show that PTMM outperforms the other two approaches discussed above. Using real data, we also show that PTMM can recover meaningful TF activity levels and identify post-transcriptionally modified TFs, many of which are supported by other sources. Supporting website: www.sb.cs.cmu.edu/PTMM/PTMM.html.

  20. Regulation of nitrogenase gene expression by transcript stability in the cyanobacterium Anabaena variabilis.

    PubMed

    Pratte, Brenda S; Thiel, Teresa

    2014-10-01

    The nitrogenase gene cluster in cyanobacteria has been thought to comprise multiple operons; however, in Anabaena variabilis, the promoter for the first gene in the cluster, nifB1, appeared to be the primary promoter for the entire nif cluster. The structural genes nifHDK1 were the most abundant transcripts; however, their abundance was not controlled by an independent nifH1 promoter, but rather, by RNA processing, which produced a very stable nifH1 transcript and a moderately stable nifD1 transcript. There was also no separate promoter for nifEN1. In addition to the nifB1 promoter, there were weak promoters inside the nifU1 gene and inside the nifE1 gene, and both promoters were heterocyst specific. In an xisA mutant, which effectively separated promoters upstream of an 11-kb excision element in nifD1 from the downstream genes, the internal nifE1 promoter was functional. Transcription of the nif1 genes downstream of the 11-kb element, including the most distant genes, hesAB1 and fdxH1, was reduced in the xisA mutant, indicating that the nifB1 promoter contributed to their expression. However, with the exception of nifK1 and nifE1, which had no expression, the downstream genes showed low to moderate levels of transcription in the xisA mutant. The hesA1 gene also had a promoter, but the fdxH gene had a processing site just upstream of the gene. The processing of transcripts at sites upstream of nifH1 and fdxH1 correlated with increased stability of these transcripts, resulting in greater amounts than transcripts that were not close to processing sites. PMID:25092030

  1. Transcription mediated insulation and interference direct gene cluster expression switches.

    PubMed

    Nguyen, Tania; Fischl, Harry; Howe, Françoise S; Woloszczuk, Ronja; Serra Barros, Ana; Xu, Zhenyu; Brown, David; Murray, Struan C; Haenni, Simon; Halstead, James M; O'Connor, Leigh; Shipkovenska, Gergana; Steinmetz, Lars M; Mellor, Jane

    2014-11-19

    In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change.

  2. Transcription mediated insulation and interference direct gene cluster expression switches

    PubMed Central

    Nguyen, Tania; Brown, David; Murray, Struan C; Haenni, Simon; Halstead, James M; O'Connor, Leigh; Shipkovenska, Gergana; Steinmetz, Lars M; Mellor, Jane

    2014-01-01

    In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change. DOI: http://dx.doi.org/10.7554/eLife.03635.001 PMID:25407679

  3. Decoupling of evolutionary changes in transcription factor binding and gene expression in mammals.

    PubMed

    Wong, Emily S; Thybert, David; Schmitt, Bianca M; Stefflova, Klara; Odom, Duncan T; Flicek, Paul

    2015-02-01

    To understand the evolutionary dynamics between transcription factor (TF) binding and gene expression in mammals, we compared transcriptional output and the binding intensities for three tissue-specific TFs in livers from four closely related mouse species. For each transcription factor, TF-dependent genes and the TF binding sites most likely to influence mRNA expression were identified by comparing mRNA expression levels between wild-type and TF knockout mice. Independent evolution was observed genome-wide between the rate of change in TF binding and the rate of change in mRNA expression across taxa, with the exception of a small number of TF-dependent genes. We also found that binding intensities are preferentially conserved near genes whose expression is dependent on the TF, and the conservation is shared among binding peaks in close proximity to each other near the TSS. Expression of TF-dependent genes typically showed an increased sensitivity to changes in binding levels as measured by mRNA abundance. Taken together, these results highlight a significant tolerance to evolutionary changes in TF binding intensity in mammalian transcriptional networks and suggest that some TF-dependent genes may be largely regulated by a single TF across evolution.

  4. Antisense transcription as a tool to tune gene expression.

    PubMed

    Brophy, Jennifer A N; Voigt, Christopher A

    2016-01-14

    A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip-based oligo synthesis was applied to build a large library of 5,668 terminator-promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering.

  5. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

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

    SciTech Connect

    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.

    2014-10-23

    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 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.

  7. 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

  8. Transcriptional regulation of chemokine expression in ovarian cancer.

    PubMed

    Singha, Bipradeb; Gatla, Himavanth R; Vancurova, Ivana

    2015-01-01

    The increased expression of pro-inflammatory and pro-angiogenic chemokines contributes to ovarian cancer progression through the induction of tumor cell proliferation, survival, angiogenesis, and metastasis. The substantial potential of these chemokines to facilitate the progression and metastasis of ovarian cancer underscores the need for their stringent transcriptional regulation. In this Review, we highlight the key mechanisms that regulate the transcription of pro-inflammatory chemokines in ovarian cancer cells, and that have important roles in controlling ovarian cancer progression. We further discuss the potential mechanisms underlying the increased chemokine expression in drug resistance, along with our perspective for future studies. PMID:25790431

  9. Transcriptional regulation of chemokine expression in ovarian cancer.

    PubMed

    Singha, Bipradeb; Gatla, Himavanth R; Vancurova, Ivana

    2015-01-01

    The increased expression of pro-inflammatory and pro-angiogenic chemokines contributes to ovarian cancer progression through the induction of tumor cell proliferation, survival, angiogenesis, and metastasis. The substantial potential of these chemokines to facilitate the progression and metastasis of ovarian cancer underscores the need for their stringent transcriptional regulation. In this Review, we highlight the key mechanisms that regulate the transcription of pro-inflammatory chemokines in ovarian cancer cells, and that have important roles in controlling ovarian cancer progression. We further discuss the potential mechanisms underlying the increased chemokine expression in drug resistance, along with our perspective for future studies.

  10. Identification and characterization of jute LTR retrotransposons:: Their abundance, heterogeneity and transcriptional activity.

    PubMed

    Ahmed, Salim; Shafiuddin, Md; Azam, Muhammad Shafiul; Islam, Md Shahidul; Ghosh, Ajit; Khan, Haseena

    2011-05-01

    Long Terminal Repeat (LTR) retrotransposons constitute a significant part of eukaryotic genomes and play an important role in genome evolution especially in plants. Jute is an important fiber crop with a large genome of 1,250 Mbps. This genome is still mostly unexplored. In this study we aimed at identifying and characterizing the LTR retrotransposons of jute with a view to understanding the jute genome better. In this study, the Reverse Transcriptase domain of Ty1-copia and Ty3-gypsy LTR retrotransposons of jute were amplified by degenerate primers and their expressions were examined by reverse transcription PCR. Copy numbers of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy elements were determined by dot blot analysis. Sequence analysis revealed higher heterogeneity among Ty1-copia retrotransposons than Ty3-gypsy and clustered each of them in three groups. Copy number of RT genes in Ty1-copia was found to be higher than that of Ty3-gypsy elements from dot blot hybridization. Cumulatively Ty1-copia and Ty3-gypsy may constitute around 19% of the jute genome where two groups of Ty1-copia were found to be transcriptionally active. Since the LTR retrotransposons constitute a large portion of jute genome, these findings imply the importance of these elements in the evolution of jute genome. PMID:22016842

  11. Identification and characterization of jute LTR retrotransposons:: Their abundance, heterogeneity and transcriptional activity.

    PubMed

    Ahmed, Salim; Shafiuddin, Md; Azam, Muhammad Shafiul; Islam, Md Shahidul; Ghosh, Ajit; Khan, Haseena

    2011-05-01

    Long Terminal Repeat (LTR) retrotransposons constitute a significant part of eukaryotic genomes and play an important role in genome evolution especially in plants. Jute is an important fiber crop with a large genome of 1,250 Mbps. This genome is still mostly unexplored. In this study we aimed at identifying and characterizing the LTR retrotransposons of jute with a view to understanding the jute genome better. In this study, the Reverse Transcriptase domain of Ty1-copia and Ty3-gypsy LTR retrotransposons of jute were amplified by degenerate primers and their expressions were examined by reverse transcription PCR. Copy numbers of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy elements were determined by dot blot analysis. Sequence analysis revealed higher heterogeneity among Ty1-copia retrotransposons than Ty3-gypsy and clustered each of them in three groups. Copy number of RT genes in Ty1-copia was found to be higher than that of Ty3-gypsy elements from dot blot hybridization. Cumulatively Ty1-copia and Ty3-gypsy may constitute around 19% of the jute genome where two groups of Ty1-copia were found to be transcriptionally active. Since the LTR retrotransposons constitute a large portion of jute genome, these findings imply the importance of these elements in the evolution of jute genome.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  13. Transcript abundance profiles reveal larger and more complex responses of grapevine to chilling compared to osmotic and salinity stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, iso-osmotic salinity (120 mM NaCl, 12mM CaCl2) and osmotic (PEG) stresses, along with chilling (5oC) stress, were applied to the cold-sensitive grapevine species V. vinifera cv. Cabernet Sauvignon. Microarray analysis of transcript abundance in shoot tips revealed that 43% of gene exp...

  14. The Role of Multiple Transcription Factors In Archaeal Gene Expression

    SciTech Connect

    Charles J. Daniels

    2008-09-23

    Since the inception of this research program, the project has focused on two central questions: What is the relationship between the 'eukaryal-like' transcription machinery of archaeal cells and its counterparts in eukaryal cells? And, how does the archaeal cell control gene expression using its mosaic of eukaryal core transcription machinery and its bacterial-like transcription regulatory proteins? During the grant period we have addressed these questions using a variety of in vivo approaches and have sought to specifically define the roles of the multiple TATA binding protein (TBP) and TFIIB-like (TFB) proteins in controlling gene expression in Haloferax volcanii. H. volcanii was initially chosen as a model for the Archaea based on the availability of suitable genetic tools; however, later studies showed that all haloarchaea possessed multiple tbp and tfb genes, which led to the proposal that multiple TBP and TFB proteins may function in a manner similar to alternative sigma factors in bacterial cells. In vivo transcription and promoter analysis established a clear relationship between the promoter requirements of haloarchaeal genes and those of the eukaryal RNA polymerase II promoter. Studies on heat shock gene promoters, and the demonstration that specific tfb genes were induced by heat shock, provided the first indication that TFB proteins may direct expression of specific gene families. The construction of strains lacking tbp or tfb genes, coupled with the finding that many of these genes are differentially expressed under varying growth conditions, provided further support for this model. Genetic tools were also developed that led to the construction of insertion and deletion mutants, and a novel gene expression scheme was designed that allowed the controlled expression of these genes in vivo. More recent studies have used a whole genome array to examine the expression of these genes and we have established a linkage between the expression of specific tfb

  15. Chromatin features, RNA polymerase II and the comparative expression of lens genes encoding crystallins, transcription factors, and autophagy mediators

    PubMed Central

    Sun, Jian; Rockowitz, Shira; Chauss, Daniel; Wang, Ping; Kantorow, Marc; Zheng, Deyou

    2015-01-01

    Purpose Gene expression correlates with local chromatin structure. Our studies have mapped histone post-translational modifications, RNA polymerase II (pol II), and transcription factor Pax6 in lens chromatin. These data represent the first genome-wide insights into the relationship between lens chromatin structure and lens transcriptomes and serve as an excellent source for additional data analysis and refinement. The principal lens proteins, the crystallins, are encoded by predominantly expressed mRNAs; however, the regulatory mechanisms underlying their high expression in the lens remain poorly understood. Methods The formaldehyde-assisted identification of regulatory regions (FAIRE-Seq) was employed to analyze newborn lens chromatin. ChIP-seq and RNA-seq data published earlier (GSE66961) have been used to assist in FAIRE-seq data interpretation. RNA transcriptomes from murine lens epithelium, lens fibers, erythrocytes, forebrain, liver, neurons, and pancreas were compared to establish the gene expression levels of the most abundant mRNAs versus median gene expression across other differentiated cells. Results Normalized RNA expression data from multiple tissues show that crystallins rank among the most highly expressed genes in mammalian cells. These findings correlate with the extremely high abundance of pol II all across the crystallin loci, including crystallin genes clustered on chromosomes 1 and 5, as well as within regions of “open” chromatin, as identified by FAIRE-seq. The expression levels of mRNAs encoding DNA-binding transcription factors (e.g., Foxe3, Hsf4, Maf, Pax6, Prox1, Sox1, and Tfap2a) revealed that their transcripts form “clusters” of abundant mRNAs in either lens fibers or lens epithelium. The expression of three autophagy regulatory mRNAs, encoding Tfeb, FoxO1, and Hif1α, was found within a group of lens preferentially expressed transcription factors compared to the E12.5 forebrain. Conclusions This study reveals novel features of

  16. SENSITIVE TO PROTON RHIZOTOXICITY1, CALMODULIN BINDING TRANSCRIPTION ACTIVATOR2, and other transcription factors are involved in ALUMINUM-ACTIVATED MALATE TRANSPORTER1 expression.

    PubMed

    Tokizawa, Mutsutomo; Kobayashi, Yuriko; Saito, Tatsunori; Kobayashi, Masatomo; Iuchi, Satoshi; Nomoto, Mika; Tada, Yasuomi; Yamamoto, Yoshiharu Y; Koyama, Hiroyuki

    2015-03-01

    In Arabidopsis (Arabidopsis thaliana) the root apex is protected from aluminum (Al) rhizotoxicity by excretion of malate, an Al chelator, by ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1). AtALMT1 expression is fundamentally regulated by the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) zinc finger protein, but other transcription factors have roles that enable Al-inducible expression with a broad dynamic range. In this study, we characterized multiple cis-elements in the AtALMT1 promoter that interact with transcription factors. In planta complementation assays of AtALMT1 driven by 5' truncated promoters of different lengths showed that the promoter region between -540 and 0 (the first ATG) restored the Al-sensitive phenotype of atalm1 and thus contains cis-elements essential for AtALMT1 expression for Al tolerance. Computation of overrepresented octamers showed that eight regions in this promoter region contained potential cis-elements involved in Al induction and STOP1 regulation. Mutation in a position around -297 from the first ATG completely inactivated AtALMT1 expression and Al response. In vitro binding assays showed that this region contained the STOP1 binding site, which accounted for the recognition by four zinc finger domains of the protein. Other positions were characterized as cis-elements that regulated expression by repressors and activators and a transcription factor that determines root tip expression of AtALMT1. From the consensus of known cis-elements, we identified CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR2 to be an activator of AtALMT1 expression. Al-inducible expression of AtALMT1 changed transcription starting sites, which increased the abundance of transcripts with a shortened 5' untranslated region. The present analyses identified multiple mechanisms that regulate AtALMT1 expression.

  17. 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.

  18. Abundance, transcription levels and phylogeny of bacteria capable of nitrous oxide reduction in a municipal wastewater treatment plant.

    PubMed

    Song, Kang; Suenaga, Toshikazu; Hamamoto, Aki; Satou, Kouichi; Riya, Shohei; Hosomi, Masaaki; Terada, Akihiko

    2014-09-01

    Nitrous oxide (N2O) production and expression of genes capable of its reduction were investigated in two full-scale parallel plug-flow activated sludge systems. These two systems continuously received wastewater with the same constituents, but operated under distinct nitrification efficiencies due to mixed liquor suspended solid (MLSS) concentration and the different hydraulic retention times (HRTs). A shorter HRT in system 2 resulted in a lower nitrification efficiency (40-60%) in conjunction with a high N2O emission (50.6 mg-N/L/day), whereas there was a higher nitrification efficiency (>99%) in system 1 with low N2O emission (22.6 mg-N/L/day). The DNA abundance of functional genes responsible for nitrification and denitrification were comparable in both systems, but transcription of nosZ mRNA in the lower N2O emission system (system 1) was one order of magnitude higher than that in the higher N2O emission system (system 2). The diversity and evenness of the nosZ gene were nearly identical; however, the predominant N2O reducing bacteria were phylogenetically distinct. Phylogenetic analysis indicated that N2O-reducing strains only retrieved in system 1 were close to the genera Rhodobacter, Oligotropha and Shinella, whereas they were close to the genera Mesorhizobium only in system 2. The distinct predominant N2O reducers may directly or indirectly influence N2O emissions.

  19. Abundance, transcription levels and phylogeny of bacteria capable of nitrous oxide reduction in a municipal wastewater treatment plant.

    PubMed

    Song, Kang; Suenaga, Toshikazu; Hamamoto, Aki; Satou, Kouichi; Riya, Shohei; Hosomi, Masaaki; Terada, Akihiko

    2014-09-01

    Nitrous oxide (N2O) production and expression of genes capable of its reduction were investigated in two full-scale parallel plug-flow activated sludge systems. These two systems continuously received wastewater with the same constituents, but operated under distinct nitrification efficiencies due to mixed liquor suspended solid (MLSS) concentration and the different hydraulic retention times (HRTs). A shorter HRT in system 2 resulted in a lower nitrification efficiency (40-60%) in conjunction with a high N2O emission (50.6 mg-N/L/day), whereas there was a higher nitrification efficiency (>99%) in system 1 with low N2O emission (22.6 mg-N/L/day). The DNA abundance of functional genes responsible for nitrification and denitrification were comparable in both systems, but transcription of nosZ mRNA in the lower N2O emission system (system 1) was one order of magnitude higher than that in the higher N2O emission system (system 2). The diversity and evenness of the nosZ gene were nearly identical; however, the predominant N2O reducing bacteria were phylogenetically distinct. Phylogenetic analysis indicated that N2O-reducing strains only retrieved in system 1 were close to the genera Rhodobacter, Oligotropha and Shinella, whereas they were close to the genera Mesorhizobium only in system 2. The distinct predominant N2O reducers may directly or indirectly influence N2O emissions. PMID:24725963

  20. Molecular cloning and expression of hardening-induced genes in Chlorella vulgaris C-27: the most abundant clone encodes a late embryogenesis abundant protein.

    PubMed

    Joh, T; Honjoh, K; Yoshimoto, M; Funabashi, J; Miyamoto, T; Hatano, S

    1995-01-01

    To investigate the effects of hardening on gene expression in Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27), a frost-hardy strain, 17 cDNA clones corresponding to hardening-induced Chlorella (hiC) genes were isolated by differential screening of a cDNA library from 6-h hardened cells. Northern blot analysis of transcripts of hiC genes showed that these genes are specifically induced by hardening and that their patterns of induction vary. Southern blots of genomic DNAs from two strains (Chlorella ellipsoidea Gerneck IAM C-102, chilling-sensitive; and C. vulgaris C-27, frost-hardy) of Chlorella indicated that ten hiC clones out of 17 hybridized only with DNA of strain C-27 and the other seven clones hybridized with DNA of both strains. However, of these seven clones, transcripts corresponding to six clones did not accumulate in strain C-102 at low temperatures. The sequence of a deduced protein encoded by the most abundant clone, hiC6, exhibited homology to sequences of Group III LEA (late embryogenesis abundant) proteins and had an amino-terminal amino acid sequence that was similar to the sequences of chloroplast transit peptides. PMID:7719632

  1. Developmental-stage-dependent transcriptional response to leukaemic oncogene expression

    PubMed Central

    Regha, Kakkad; Assi, Salam A.; Tsoulaki, Olga; Gilmour, Jane; Lacaud, Georges; Bonifer, Constanze

    2015-01-01

    Acute myeloid leukaemia (AML) is characterized by a block in myeloid differentiation the stage of which is dependent on the nature of the transforming oncogene and the developmental stage of the oncogenic hit. This is also true for the t(8;21) translocation that gives rise to the RUNX1-ETO fusion protein and initiates the most common form of human AML. Here we study the differentiation of mouse embryonic stem cells expressing an inducible RUNX1-ETO gene into blood cells as a model, combined with genome-wide analyses of transcription factor binding and gene expression. RUNX1-ETO interferes with both the activating and repressive function of its normal counterpart, RUNX1, at early and late stages of blood cell development. However, the response of the transcriptional network to RUNX1-ETO expression is developmental stage specific, highlighting the molecular mechanisms determining specific target cell expansion after an oncogenic hit. PMID:26018585

  2. Expression analysis of TALE family transcription factors during avian development.

    PubMed

    Coy, Sarah E; Borycki, Anne-Gaëlle

    2010-04-01

    The TALE family of homeodomain containing transcription factors consists of the Meis, Prep and Tgif, and the Pbx subfamily of proteins. Several TALE orthologues have been identified in amniotes, but no comprehensive analysis of their expression pattern during embryogenesis has been performed. Here, we report on TALE gene expression in the avian embryo. During embryonic development, Pbx genes are predominantly expressed in the neural ectoderm and paraxial mesoderm, although Pbx3 is restricted to the intermediate and lateral mesoderm, and anterior central nervous system. Members of the Meis, Prep, and Tgif subfamilies are expressed at high levels in the paraxial mesoderm, and display differential expression along the anterior-posterior and dorsoventral axes of the developing neural tube. Overall the expression patterns reported in this study are consistent with the known function of the TALE gene family in controlling early patterning of limb, neural tube and paraxial mesoderm tissues during embryogenesis.

  3. Variable expressions of Staphylococcus aureus bicomponent leucotoxins semiquantified by competitive reverse transcription-PCR.

    PubMed

    Bronner, S; Stoessel, P; Gravet, A; Monteil, H; Prévost, G

    2000-09-01

    A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/10(4) CFU to 10(2) mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system in S. aureus, except that expression of hlgA was not affected in the agr mutant.

  4. Changes in transcript abundance for cuticular proteins and other genes three hours after a blood meal in Anopheles gambiae

    PubMed Central

    Vannini, Laura; Dunn, W. Augustine; Reed, Tyler W.; Willis, Judith H.

    2014-01-01

    Numerous studies have examined changes in transcript levels after Anopheles gambiae takes a blood meal. Marinotti et al. (2006) used microarrays and reported massive changes in transcript levels 3 h after feeding (BF3h) compared to non-blood fed (NBF). We were intrigued by the number of transcripts for structural cuticular proteins (CPs) that showed such major differences in levels and employed paired-end (50 bp) RNA-seq technology to compare whole body transcriptomes from 5-day-old females NBF and BF3h. We detected transcripts for the majority of CPs (164/243) but levels of only 12 were significantly altered by the blood meal. While relative transcript levels of NBF females were somewhat similar to the microarray data, there were major differences in BF3h animals, resulting in levels of many transcripts, both for CPs and other genes changing in the opposite direction. We compared our data also to other studies done with both microarrays and RNA-seq. Findings were consistent that a small number of CP genes have transcripts that persist even in 5-day-old adults. Some of these transcripts showed diurnal rhythms (Rund et al., 2013; Rinker et al., 2013). In situ hybridization revealed that transcripts for several of these CP genes were found exclusively or predominantly in the eye. Transcripts other than for CPs that changed in response to blood-feeding were predominantly expressed in midgut and Malpighian tubules. Even in these tissues, genes responsible for proteins with similar functions, such as immunity or digestion, responded differently, with transcript levels for some rising and others falling. These data demonstrate that genes coding for some CPs are dynamic in expression even in adults and that the response to a blood meal is rapid and precisely orchestrated. PMID:24269292

  5. Changes in transcript abundance for cuticular proteins and other genes three hours after a blood meal in Anopheles gambiae.

    PubMed

    Vannini, Laura; Augustine Dunn, W; Reed, Tyler W; Willis, Judith H

    2014-01-01

    Numerous studies have examined changes in transcript levels after Anopheles gambiae takes a blood meal. Marinotti et al. (2006) used microarrays and reported massive changes in transcript levels 3 h after feeding (BF3h) compared to non-blood fed (NBF). We were intrigued by the number of transcripts for structural cuticular proteins (CPs) that showed such major differences in levels and employed paired-end (50 bp) RNA-seq technology to compare whole body transcriptomes from 5-day-old females NBF and BF3h. We detected transcripts for the majority of CPs (164/243) but levels of only 12 were significantly altered by the blood meal. While relative transcript levels of NBF females were somewhat similar to the microarray data, there were major differences in BF3h animals, resulting in levels of many transcripts, both for CPs and other genes changing in the opposite direction. We compared our data also to other studies done with both microarrays and RNA-seq. Findings were consistent that a small number of CP genes have transcripts that persist even in 5-day-old adults. Some of these transcripts showed diurnal rhythms (Rund et al., 2013; Rinker et al., 2013). In situ hybridization revealed that transcripts for several of these CP genes were found exclusively or predominantly in the eye. Transcripts other than for CPs that changed in response to blood-feeding were predominantly expressed in midgut and Malpighian tubules. Even in these tissues, genes responsible for proteins with similar functions, such as immunity or digestion, responded differently, with transcript levels for some rising and others falling. These data demonstrate that genes coding for some CPs are dynamic in expression even in adults and that the response to a blood meal is rapid and precisely orchestrated.

  6. Post-Transcriptional Control of Chloroplast Gene Expression

    PubMed Central

    del Campo, Eva M.

    2009-01-01

    Chloroplasts contain their own genome, organized as operons, which are generally transcribed as polycistronic transcriptional units. These primary transcripts are processed into smaller RNAs, which are further modified to produce functional RNAs. The RNA processing mechanisms remain largely unknown and represent an important step in the control of chloroplast gene expression. Such mechanisms include RNA cleavage of pre-existing RNAs, RNA stabilization, intron splicing, and RNA editing. Recently, several nuclear-encoded proteins that participate in diverse plastid RNA processing events have been characterised. Many of them seem to belong to the pentatricopeptide repeat (PPR) protein family that is implicated in many crucial functions including organelle biogenesis and plant development. This review will provide an overview of current knowledge of the post-transcriptional processing in chloroplasts. PMID:19838333

  7. Spatial expression of transcription factors in Drosophila embryonic organ development

    PubMed Central

    2013-01-01

    Background Site-specific transcription factors (TFs) bind DNA regulatory elements to control expression of target genes, forming the core of gene regulatory networks. Despite decades of research, most studies focus on only a small number of TFs and the roles of many remain unknown. Results We present a systematic characterization of spatiotemporal gene expression patterns for all known or predicted Drosophila TFs throughout embryogenesis, the first such comprehensive study for any metazoan animal. We generated RNA expression patterns for all 708 TFs by in situ hybridization, annotated the patterns using an anatomical controlled vocabulary, and analyzed TF expression in the context of organ system development. Nearly all TFs are expressed during embryogenesis and more than half are specifically expressed in the central nervous system. Compared to other genes, TFs are enriched early in the development of most organ systems, and throughout the development of the nervous system. Of the 535 TFs with spatially restricted expression, 79% are dynamically expressed in multiple organ systems while 21% show single-organ specificity. Of those expressed in multiple organ systems, 77 TFs are restricted to a single organ system either early or late in development. Expression patterns for 354 TFs are characterized for the first time in this study. Conclusions We produced a reference TF dataset for the investigation of gene regulatory networks in embryogenesis, and gained insight into the expression dynamics of the full complement of TFs controlling the development of each organ system. PMID:24359758

  8. Transcript abundance of amino acid transporters, β-casein, and α-lactalbumin in mammary tissue of periparturient, lactating, and postweaned sows.

    PubMed

    Manjarin, R; Steibel, J P; Zamora, V; Am-In, N; Kirkwood, R N; Ernst, C W; Weber, P S; Taylor, N P; Trottier, N L

    2011-07-01

    The objective of these experiments was to test the hypothesis that transcript abundance of cationic AA transporter- and milk protein-encoding genes increase in the porcine mammary gland in response to higher lactation demand. Genes of interest included those encoding for the milk proteins α-lactalbumin (α-LA) and β-casein (β-CN; LALBA and CSN2, respectively), and AA transporter b(0,+)AT, y(+)LAT1, y(+)LAT2, ATB(0,+), CAT-1, and CAT-2b (SLC7A9, SLC7A7, SLC7A6, SLC6A14, SLC7A1, and SLC7A2, respectively). Mammary tissue was biopsied from 4 sows on d 110 of gestation (prepartum), on d 2 (early postpartum), on d 5 (early), and d 17 (peak) of lactation, and on d 5 after weaning (postweaning), and mRNA of target genes quantified by reverse transcription quantitative PCR. Compared with prepartum, CAT-1, ATB(0,+), y(+)LAT2, β-CN, and α-LA mRNA abundance was higher at early lactation, whereas compared with early lactation, only CAT-1 and α-LA mRNA abundance was higher at peak lactation. The CAT-2b, y(+)LAT1, and b(0,+)AT mRNA abundance did not differ when comparing either prepartum or peak lactation to early lactation. Compared with peak lactation, postweaning mRNA abundance of CAT-1, ATB(0,+), α-LA, and β-CN decreased, y(+)LAT2, CAT-2b, and b(0,+)AT remained unchanged, and y(+)LAT1 increased. The mRNA abundance of y(+)LAT2 increased from early postpartum to early lactation, and remained unchanged for CAT-1, ATB(0,+), α-LA, and β-CN. From prepartum to peak lactation, the mRNA abundance of CAT-1, y(+)LAT2, and ATB(0,+) was positively correlated with that of β-CN and α-LA. In conclusion, the expression of genes encoding for y(+)LAT1, CAT-2b, and b(0,+)AT remained unchanged in porcine mammary glands over prepartum to peak lactation period, whereas expression of genes encoding for CAT-1, ATB(0,+), and y(+)LAT2 was upregulated and positively correlated to expression of genes encoding for the mammary synthesized milk proteins β-CN and α-LA.

  9. Transcription factors expressed in soybean roots under drought stress.

    PubMed

    Pereira, S S; Guimarães, F C M; Carvalho, J F C; Stolf-Moreira, R; Oliveira, M C N; Rolla, A A P; Farias, J R B; Neumaier, N; Nepomuceno, A L

    2011-10-21

    To gain insight into stress-responsive gene regulation in soybean plants, we identified consensus sequences that could categorize the transcription factors MYBJ7, BZIP50, C2H2, and NAC2 as members of the gene families myb, bzip, c2h2, and nac, respectively. We also investigated the evolutionary relationship of these transcription factors and analyzed their expression levels under drought stress. The NCBI software was used to find the predicted amino acid sequences of the transcription factors, and the Clustal X software was used to align soybean and other plant species sequences. Phylogenetic trees were built using the Mega 4.1 software by neighbor joining and the degree of confidence test by Bootstrap. Expression level studies were carried out using hydroponic culture; the experiments were designed in completely randomized blocks with three repetitions. The blocks consisted of two genotypes, MG/BR46 Conquista (drought-tolerant) and BR16 (drought-sensitive) and the treatments consisted of increasingly long dehydration periods (0, 25, 50, 75, and 100 min). The transcription factors presented domains and/or conserved regions that characterized them as belonging to the bzip, c2h2, myb, and nac families. Based on the phylogenetic trees, it was found that the myb, bzip and nac genes are closely related to myb78, bzip48 and nac2 of soybean and that c2h2 is closely related to c2h2 of Brassica napus. Expression of all genes was in general increased under drought stress in both genotypes. Major differences between genotypes were due to the lowering of the expression of the mybj7 and c2h2 genes in the drought-tolerant variety at some times. Over-expression or silencing of some of these genes has the potential to increase stress tolerance.

  10. Towards resolving the transcription factor network controlling myelin gene expression

    PubMed Central

    Fulton, Debra L.; Denarier, Eric; Friedman, Hana C.; Wasserman, Wyeth W.; Peterson, Alan C.

    2011-01-01

    In the central nervous system (CNS), myelin is produced from spirally-wrapped oligodendrocyte plasma membrane and, as exemplified by the debilitating effects of inherited or acquired myelin abnormalities in diseases such as multiple sclerosis, it plays a critical role in nervous system function. Myelin sheath production coincides with rapid up-regulation of numerous genes. The complexity of their subsequent expression patterns, along with recently recognized heterogeneity within the oligodendrocyte lineage, suggest that the regulatory networks controlling such genes drive multiple context-specific transcriptional programs. Conferring this nuanced level of control likely involves a large repertoire of interacting transcription factors (TFs). Here, we combined novel strategies of computational sequence analyses with in vivo functional analysis to establish a TF network model of coordinate myelin-associated gene transcription. Notably, the network model captures regulatory DNA elements and TFs known to regulate oligodendrocyte myelin gene transcription and/or oligodendrocyte development, thereby validating our approach. Further, it links to numerous TFs with previously unsuspected roles in CNS myelination and suggests collaborative relationships amongst both known and novel TFs, thus providing deeper insight into the myelin gene transcriptional network. PMID:21729871

  11. Post-Transcriptional Regulation of Gene Expression in Yersinia Species

    PubMed Central

    Schiano, Chelsea A.; Lathem, Wyndham W.

    2012-01-01

    Proper regulation of gene expression is required by bacterial pathogens to respond to continually changing environmental conditions and the host response during the infectious process. While transcriptional regulation is perhaps the most well understood form of controlling gene expression, recent studies have demonstrated the importance of post-transcriptional mechanisms of gene regulation that allow for more refined management of the bacterial response to host conditions. Yersinia species of bacteria are known to use various forms of post-transcriptional regulation for control of many virulence-associated genes. These include regulation by cis- and trans-acting small non-coding RNAs, RNA-binding proteins, RNases, and thermoswitches. The effects of these and other regulatory mechanisms on Yersinia physiology can be profound and have been shown to influence type III secretion, motility, biofilm formation, host cell invasion, intracellular survival and replication, and more. In this review, we discuss these and other post-transcriptional mechanisms and their influence on virulence gene regulation, with a particular emphasis on how these processes influence the virulence of Yersinia in the host. PMID:23162797

  12. Characterization of GPR101 transcript structure and expression patterns.

    PubMed

    Trivellin, Giampaolo; Bjelobaba, Ivana; Daly, Adrian F; Larco, Darwin O; Palmeira, Leonor; Faucz, Fabio R; Thiry, Albert; Leal, Letícia F; Rostomyan, Liliya; Quezado, Martha; Schernthaner-Reiter, Marie Helene; Janjic, Marija M; Villa, Chiara; Wu, T John; Stojilkovic, Stanko S; Beckers, Albert; Feldman, Benjamin; Stratakis, Constantine A

    2016-08-01

    We recently showed that Xq26.3 microduplications cause X-linked acrogigantism (X-LAG). X-LAG patients mainly present with growth hormone and prolactin-secreting adenomas and share a minimal duplicated region containing at least four genes. GPR101 was the only gene highly expressed in their pituitary lesions, but little is known about its expression patterns. In this work, GPR101 transcripts were characterized in human tissues by 5'-Rapid Amplification of cDNA Ends (RACE) and RNAseq, while the putative promoter was bioinformatically predicted. We investigated GPR101 mRNA and protein expression by RT-quantitative PCR (qPCR), whole-mount in situ hybridization, and immunostaining, in human, rhesus monkey, rat and zebrafish. We identified four GPR101 isoforms characterized by different 5'-untranslated regions (UTRs) and a common 6.1kb long 3'UTR. GPR101 expression was very low or absent in almost all adult human tissues examined, except for specific brain regions. Strong GPR101 staining was observed in human fetal pituitary and during adolescence, whereas very weak/absent expression was detected during childhood and adult life. In contrast to humans, adult monkey and rat pituitaries expressed GPR101, but in different cell types. Gpr101 is expressed in the brain and pituitary during rat and zebrafish development; in rat pituitary, Gpr101 is expressed only after birth and shows sexual dimorphism. This study shows that different GPR101 transcripts exist and that the brain is the major site of GPR101 expression across different species, although divergent species- and temporal-specific expression patterns are evident. These findings suggest an important role for GPR101 in brain and pituitary development and likely reflect the very different growth, development and maturation patterns among species. PMID:27282544

  13. Transcriptional regulation of IGF-I expression in skeletal muscle

    NASA Technical Reports Server (NTRS)

    McCall, G. E.; Allen, D. L.; Haddad, F.; Baldwin, K. M.

    2003-01-01

    The present study investigated the role of transcription in the regulation of insulin-like growth factor (IGF)-I expression in skeletal muscle. RT-PCR was used to determine endogenous expression of IGF-I pre-mRNA and mRNA in control (Con) and functionally overloaded (FO) rat plantaris. The transcriptional activities of five different-length IGF-I promoter fragments controlling transcription of a firefly luciferase (FLuc) reporter gene were tested in vitro by transfection of myoblasts or in vivo during FO by direct gene transfer into the plantaris. Increased endogenous IGF-I gene transcription during 7 days of plantaris FO was evidenced by an approximately 140-160% increase (P < 0.0001) in IGF-I pre-mRNA (a transcriptional marker). IGF-I mRNA expression also increased by approximately 90% (P < 0.0001), and it was correlated (R = 0.93; P < 0.0001) with the pre-mRNA increases. The three longest IGF-I exon 1 promoters induced reporter gene expression in proliferating C2C12 and L6E9 myoblasts. In differentiated L6E9 myotubes, promoter activity increased approximately two- to threefold over myoblasts. Overexpression of calcineurin and MyoD increased the activity of the -852/+192 promoter in C2C12 myotubes by approximately 5- and approximately 18-fold, respectively. However, FO did not induce these exogenous promoter fragments. Nevertheless, the present findings are consistent with the hypothesis that the IGF-I gene is transcriptionally regulated during muscle hypertrophy in vivo as evidenced by the induction of the endogenous IGF-I pre-mRNA during plantaris FO. The exon 1 promoter region of the IGF-I gene is sufficient to direct inducible expression in vitro; however, an in vivo response to FO may require elements outside the -852/+346 region of the exon 1 IGF-I promoter or features inherent to the endogenous IGF-I gene.

  14. Autoregulation of topoisomerase I expression by supercoiling sensitive transcription

    PubMed Central

    Ahmed, Wareed; Menon, Shruti; D. N. B. Karthik, Pullela V.; Nagaraja, Valakunja

    2016-01-01

    The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Escherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobacterium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of sub-optimal spacing between the −35 and −10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the −35 and −10 elements to facilitate the optimal transcription of topoI. PMID:26496944

  15. Transcriptional regulation of human thromboxane synthase gene expression

    SciTech Connect

    Lee, K.D.; Baek, S.J.; Fleischer, T

    1994-09-01

    The human thromboxane synthase (TS) gene encodes a microsomal enzyme catalyzing the conversion of prostaglandin endoperoxide into thromboxane A{sub 2}(TxA{sub 2}), a potent inducer of vasoconstriction and platelet aggregation. A deficiency in platelet TS activity results in bleeding disorders, but the underlying molecular mechanism remains to be elucidated. Increased TxA{sub 2} has been associated with many pathophysiological conditions such as cardiovascular disease, pulmonary hypertension, pre-eclampsia, and thrombosis in sickle cell patients. Since the formation of TxA{sub 2} is dependent upon TS, the regulation of TS gene expression may presumably play a crucial role in vivo. Abrogation of the regulatory mechanism in TS gene expression might contribute, in part, to the above clinical manifestations. To gain insight into TS gene regulation, a 1.7 kb promoter of the human TS gene was cloned and sequenced. RNase protection assay and 5{prime} RACE protocols were used to map the transcription initiation site to nucleotide A, 30 bp downstream from a canonical TATA box. Several transcription factor binding sites, including AP-1, PU.1, and PEA3, were identified within this sequence. Transient expression studies in HL-60 cells transfected with constructs containing various lengths (0.2 to 5.5 kb) of the TS promoter/luciferase fusion gene indicated the presence of multiple repressor elements within the 5.5 kb TS promoter. However, a lineage-specific up-regulation of TS gene expression was observed in HL-60 cells induced by TPA to differentiate along the macrophage lineage. The increase in TS transcription was not detectable until 36 hr after addition of the inducer. These results suggest that expression of the human TS gene may be regulated by a mechanism involving repression and derepression of the TS promoter.

  16. Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung.

    PubMed

    Gifford, Alex H; Willger, Sven D; Dolben, Emily L; Moulton, Lisa A; Dorman, Dana B; Bean, Heather; Hill, Jane E; Hampton, Thomas H; Ashare, Alix; Hogan, Deborah A

    2016-10-01

    The discovery of therapies that modulate Pseudomonas aeruginosa virulence or that can eradicate chronic P. aeruginosa lung infections associated with cystic fibrosis (CF) will be advanced by an improved understanding of P. aeruginosa behavior in vivo We demonstrate the use of multiplexed Nanostring technology to monitor relative abundances of P. aeruginosa transcripts across clinical isolates, in serial samples, and for the purposes of comparing microbial physiology in vitro and in vivo The expression of 75 transcripts encoded by genes implicated in CF lung disease was measured in a variety of P. aeruginosa strains as well as RNA serial sputum samples from four P. aeruginosa-colonized subjects with CF collected over 6 months. We present data on reproducibility, the results from different methods of normalization, and demonstrate high concordance between transcript relative abundance data obtained by Nanostring or transcriptome sequencing (RNA-Seq) analysis. Furthermore, we address considerations regarding sequence variation between strains during probe design. Analysis of P. aeruginosa grown in vitro identified transcripts that correlated with the different phenotypes commonly observed in CF clinical isolates. P. aeruginosa transcript profiles in RNA from CF sputum indicated alginate production in vivo, and transcripts involved in quorum-sensing regulation were less abundant in sputum than strains grown in the laboratory. P. aeruginosa gene expression patterns from sputum clustered closely together relative to patterns for laboratory-grown cultures; in contrast, laboratory-grown P. aeruginosa showed much greater transcriptional variation with only loose clustering of strains with different phenotypes. The clustering within and between subjects was surprising in light of differences in inhaled antibiotic and respiratory symptoms, suggesting that the pathways represented by these 75 transcripts are stable in chronic CF P. aeruginosa lung infections. PMID:27481238

  17. Expression and Functional Analysis of WRKY Transcription Factors in Chinese Wild Hazel, Corylus heterophylla Fisch.

    PubMed

    Zhao, Tian-Tian; Zhang, Jin; Liang, Li-Song; Ma, Qing-Hua; Chen, Xin; Zong, Jian-Wei; Wang, Gui-Xi

    2015-01-01

    Plant WRKY transcription factors are known to regulate various biotic and abiotic stress responses. In this study we identified a total of 30 putative WRKY unigenes in a transcriptome dataset of the Chinese wild Hazel, Corylus heterophylla, a species that is noted for its cold tolerance. Thirteen full-length of these ChWRKY genes were cloned and found to encode complete protein sequences, and they were divided into three groups, based on the number of WRKY domains and the pattern of zinc finger structures. Representatives of each of the groups, Unigene25835 (group I), Unigene37641 (group II) and Unigene20441 (group III), were transiently expressed as fusion proteins with yellow fluorescent fusion protein in Nicotiana benthamiana, where they were observed to accumulate in the nucleus, in accordance with their predicted roles as transcriptional activators. An analysis of the expression patterns of all 30 WRKY genes revealed differences in transcript abundance profiles following exposure to cold, drought and high salinity conditions. Among the stress-inducible genes, 23 were up-regulated by all three abiotic stresses and the WRKY genes collectively exhibited four different patterns of expression in flower buds during the overwintering period from November to April. The organ/tissue related expression analysis showed that 18 WRKY genes were highly expressed in stem but only 2 (Unigene9262 and Unigene43101) were greatest in male anthotaxies. The expression of Unigene37641, a member of the group II WRKY genes, was substantially up-regulated by cold, drought and salinity treatments, and its overexpression in Arabidopsis thaliana resulted in better seedling growth, compared with wild type plants, under cold treatment conditions. The transgenic lines also had exhibited higher soluble protein content, superoxide dismutase and peroxidase activiety and lower levels of malondialdehyde, which collectively suggets that Unigene37641 expression promotes cold tolerance.

  18. Expression and Functional Analysis of WRKY Transcription Factors in Chinese Wild Hazel, Corylus heterophylla Fisch.

    PubMed

    Zhao, Tian-Tian; Zhang, Jin; Liang, Li-Song; Ma, Qing-Hua; Chen, Xin; Zong, Jian-Wei; Wang, Gui-Xi

    2015-01-01

    Plant WRKY transcription factors are known to regulate various biotic and abiotic stress responses. In this study we identified a total of 30 putative WRKY unigenes in a transcriptome dataset of the Chinese wild Hazel, Corylus heterophylla, a species that is noted for its cold tolerance. Thirteen full-length of these ChWRKY genes were cloned and found to encode complete protein sequences, and they were divided into three groups, based on the number of WRKY domains and the pattern of zinc finger structures. Representatives of each of the groups, Unigene25835 (group I), Unigene37641 (group II) and Unigene20441 (group III), were transiently expressed as fusion proteins with yellow fluorescent fusion protein in Nicotiana benthamiana, where they were observed to accumulate in the nucleus, in accordance with their predicted roles as transcriptional activators. An analysis of the expression patterns of all 30 WRKY genes revealed differences in transcript abundance profiles following exposure to cold, drought and high salinity conditions. Among the stress-inducible genes, 23 were up-regulated by all three abiotic stresses and the WRKY genes collectively exhibited four different patterns of expression in flower buds during the overwintering period from November to April. The organ/tissue related expression analysis showed that 18 WRKY genes were highly expressed in stem but only 2 (Unigene9262 and Unigene43101) were greatest in male anthotaxies. The expression of Unigene37641, a member of the group II WRKY genes, was substantially up-regulated by cold, drought and salinity treatments, and its overexpression in Arabidopsis thaliana resulted in better seedling growth, compared with wild type plants, under cold treatment conditions. The transgenic lines also had exhibited higher soluble protein content, superoxide dismutase and peroxidase activiety and lower levels of malondialdehyde, which collectively suggets that Unigene37641 expression promotes cold tolerance. PMID

  19. Expression and Functional Analysis of WRKY Transcription Factors in Chinese Wild Hazel, Corylus heterophylla Fisch

    PubMed Central

    Liang, Li-Song; Ma, Qing-Hua; Chen, Xin; Zong, Jian-Wei; Wang, Gui-Xi

    2015-01-01

    Plant WRKY transcription factors are known to regulate various biotic and abiotic stress responses. In this study we identified a total of 30 putative WRKY unigenes in a transcriptome dataset of the Chinese wild Hazel, Corylus heterophylla, a species that is noted for its cold tolerance. Thirteen full-length of these ChWRKY genes were cloned and found to encode complete protein sequences, and they were divided into three groups, based on the number of WRKY domains and the pattern of zinc finger structures. Representatives of each of the groups, Unigene25835 (group I), Unigene37641 (group II) and Unigene20441 (group III), were transiently expressed as fusion proteins with yellow fluorescent fusion protein in Nicotiana benthamiana, where they were observed to accumulate in the nucleus, in accordance with their predicted roles as transcriptional activators. An analysis of the expression patterns of all 30 WRKY genes revealed differences in transcript abundance profiles following exposure to cold, drought and high salinity conditions. Among the stress-inducible genes, 23 were up-regulated by all three abiotic stresses and the WRKY genes collectively exhibited four different patterns of expression in flower buds during the overwintering period from November to April. The organ/tissue related expression analysis showed that 18 WRKY genes were highly expressed in stem but only 2 (Unigene9262 and Unigene43101) were greatest in male anthotaxies. The expression of Unigene37641, a member of the group II WRKY genes, was substantially up-regulated by cold, drought and salinity treatments, and its overexpression in Arabidopsis thaliana resulted in better seedling growth, compared with wild type plants, under cold treatment conditions. The transgenic lines also had exhibited higher soluble protein content, superoxide dismutase and peroxidase activiety and lower levels of malondialdehyde, which collectively suggets that Unigene37641 expression promotes cold tolerance. PMID

  20. Fast and accurate approximate inference of transcript expression from RNA-seq data

    PubMed Central

    Hensman, James; Papastamoulis, Panagiotis; Glaus, Peter; Honkela, Antti; Rattray, Magnus

    2015-01-01

    Motivation: Assigning RNA-seq reads to their transcript of origin is a fundamental task in transcript expression estimation. Where ambiguities in assignments exist due to transcripts sharing sequence, e.g. alternative isoforms or alleles, the problem can be solved through probabilistic inference. Bayesian methods have been shown to provide accurate transcript abundance estimates compared with competing methods. However, exact Bayesian inference is intractable and approximate methods such as Markov chain Monte Carlo and Variational Bayes (VB) are typically used. While providing a high degree of accuracy and modelling flexibility, standard implementations can be prohibitively slow for large datasets and complex transcriptome annotations. Results: We propose a novel approximate inference scheme based on VB and apply it to an existing model of transcript expression inference from RNA-seq data. Recent advances in VB algorithmics are used to improve the convergence of the algorithm beyond the standard Variational Bayes Expectation Maximization algorithm. We apply our algorithm to simulated and biological datasets, demonstrating a significant increase in speed with only very small loss in accuracy of expression level estimation. We carry out a comparative study against seven popular alternative methods and demonstrate that our new algorithm provides excellent accuracy and inter-replicate consistency while remaining competitive in computation time. Availability and implementation: The methods were implemented in R and C++, and are available as part of the BitSeq project at github.com/BitSeq. The method is also available through the BitSeq Bioconductor package. The source code to reproduce all simulation results can be accessed via github.com/BitSeq/BitSeqVB_benchmarking. Contact: james.hensman@sheffield.ac.uk or panagiotis.papastamoulis@manchester.ac.uk or Magnus.Rattray@manchester.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online

  1. Transcriptional and posttranscriptional control of hepatitis B virus gene expression

    PubMed Central

    Uprichard, Susan L.; Wieland, Stefan F.; Althage, Alana; Chisari, Francis V.

    2003-01-01

    Hepatitis B virus (HBV) infects humans and certain nonhuman primates. Viral clearance and acute disease are associated with a strong, polyclonal, multispecific cytotoxic T lymphocyte response. Infiltrating T cells, as well as other activated inflammatory cells, produce cytokines that can regulate hepatocellular gene expression. Using an HBV transgenic mouse model, our laboratory has previously demonstrated that adoptive transfer of HBV-specific cytotoxic T lymphocytes or injection of IL-2 can noncytopathically inhibit HBV gene expression by a posttranscriptional IFN-γ- and/or tumor necrosis factor α-dependent mechanism. Here, we report that HBV gene expression can also be controlled at the posttranscriptional level during persistent lymphocytic choriomeningitis virus infection. In contrast, it is controlled at the transcriptional level during acute murine cytomegalovirus infection or after repetitive polyinosinic-polycytidylic acid injection. Finally, we show that transcriptional inhibition of HBV is associated with changes in liver-specific gene expression. These results elucidate pathways that regulate the viral life cycle and suggest additional approaches for the treatment of chronic HBV infection. PMID:12552098

  2. The transcriptional repressor DREAM is involved in thyroid gene expression

    SciTech Connect

    D'Andrea, Barbara; Di Palma, Tina; Mascia, Anna; Motti, Maria Letizia; Viglietto, Giuseppe; Nitsch, Lucio; Zannini, Mariastella . E-mail: stella@szn.it

    2005-04-15

    Downstream regulatory element antagonistic modulator (DREAM) was originally identified in neuroendocrine cells as a calcium-binding protein that specifically binds to downstream regulatory elements (DRE) on DNA, and represses transcription of its target genes. To explore the possibility that DREAM may regulate the endocrine activity of the thyroid gland, we analyzed its mRNA expression in undifferentiated and differentiated thyroid cells. We demonstrated that DREAM is expressed in the normal thyroid tissue as well as in differentiated thyroid cells in culture while it is absent in FRT poorly differentiated cells. In the present work, we also show that DREAM specifically binds to DRE sites identified in the 5' untranslated region (UTR) of the thyroid-specific transcription factors Pax8 and TTF-2/FoxE1 in a calcium-dependent manner. By gel retardation assays we demonstrated that thapsigargin treatment increases the binding of DREAM to the DRE sequences present in Pax8 and TTF-2/Foxe1 5' UTRs, and this correlates with a significant reduction of the expression of these genes. Interestingly, in poorly differentiated thyroid cells overexpression of exogenous DREAM strongly inhibits Pax8 expression. Moreover, we provide evidence that a mutated form of DREAM unable to bind Ca{sup 2+} interferes with thyroid cell proliferation. Therefore, we propose that in thyroid cells DREAM is a mediator of the calcium-signaling pathway and it is involved in the regulation of thyroid cell function.

  3. Calcium regulates caveolin-1 expression at the transcriptional level

    SciTech Connect

    Yang, Xiao-Yan; Huang, Cheng-Cheng; Kan, Qi-Ming; Li, Yan; Liu, Dan; Zhang, Xue-Cheng; Sato, Toshinori; Yamagata, Sadako; Yamagata, Tatsuya

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Caveolin-1 expression is regulated by calcium signaling at the transcriptional level. Black-Right-Pointing-Pointer An inhibitor of or siRNA to L-type calcium channel suppressed caveolin-1 expression. Black-Right-Pointing-Pointer Cyclosporine A or an NFAT inhibitor markedly reduced caveolin-1 expression. Black-Right-Pointing-Pointer Caveolin-1 regulation by calcium signaling is observed in several mouse cell lines. -- Abstract: Caveolin-1, an indispensable component of caveolae serving as a transformation suppressor protein, is highly expressed in poorly metastatic mouse osteosarcoma FBJ-S1 cells while highly metastatic FBJ-LL cells express low levels of caveolin-1. Calcium concentration is higher in FBJ-S1 cells than in FBJ-LL cells; therefore, we investigated the possibility that calcium signaling positively regulates caveolin-1 in mouse FBJ-S1 cells. When cells were treated with the calcium channel blocker nifedipine, cyclosporin A (a calcineurin inhibitor), or INCA-6 (a nuclear factor of activated T-cells [NFAT] inhibitor), caveolin-1 expression at the mRNA and protein levels decreased. RNA silencing of voltage-dependent L-type calcium channel subunit alpha-1C resulted in suppression of caveolin-1 expression. This novel caveolin-1 regulation pathway was also identified in mouse NIH 3T3 cells and Lewis lung carcinoma cells. These results indicate that caveolin-1 is positively regulated at the transcriptional level through a novel calcium signaling pathway mediated by L-type calcium channel/Ca{sup 2+}/calcineurin/NFAT.

  4. Transcript expression profiling for adventitious roots of Panax ginseng Meyer.

    PubMed

    Subramaniyam, Sathiyamoorthy; Mathiyalagan, Ramya; Natarajan, Sathishkumar; Kim, Yu-Jin; Jang, Moon-Gi; Park, Jun-Hyung; Yang, Deok Chun

    2014-08-01

    Panax ginseng Meyer is one of the major medicinal plants in oriental countries belonging to the Araliaceae family which are the primary source for ginsenosides. However, very few genes were characterized for ginsenoside pathway, due to the limited genome information. Through this study, we obtained a comprehensive transcriptome from adventitious roots, which were treated with methyl jasmonic acids for different time points (control, 2h, 6h, 12h, and 24h) and sequenced by RNA 454 pyrosequencing technology. Reference transcriptome 39,304,529 (0.04GB) was obtained from 5,724,987,880 bases (5.7GB) of 22 libraries by de novo assembly and 35,266 (58.5%) transcripts were annotated with biological schemas (GO and KEGG). The digital gene expression patterns were obtained from in vitro grown adventitious root sequences which mapped to reference, from that, 3813 (6.3%) unique transcripts were involved in ≥2 fold up and downregulations. Finally, candidates for ginsenoside pathway genes were predicted from observed expression patterns. Among them, 30 transcription factors, 20 cytochromes, and 11 glycosyl transferases were predicted as ginsenoside candidates. These data can remarkably expand the existing transcriptome resources of Panax, especially to predict existence of gene networks in P. ginseng. The entity of the data provides a valuable platform to reveal more on secondary metabolism and abiotic stresses from P. ginseng in vitro grown adventitious roots.

  5. Neurotoxocarosis alters myelin protein gene transcription and expression.

    PubMed

    Heuer, Lea; Beyerbach, Martin; Lühder, Fred; Beineke, Andreas; Strube, Christina

    2015-06-01

    Neurotoxocarosis is an infection of the central nervous system caused by migrating larvae of the common dog and cat roundworms (Toxocara canis and Toxocara cati), which are zoonotic agents. As these parasites are prevalent worldwide and neuropathological and molecular investigations on neurotoxocarosis are scare, this study aims to characterise nerve fibre demyelination associated with neurotoxocarosis on a molecular level. Transcription of eight myelin-associated genes (Cnp, Mag, Mbp, Mog, Mrf-1, Nogo-A, Plp1, Olig2) was determined in the mouse model during six time points of the chronic phase of infection using qRT-PCR. Expression of selected proteins was analysed by Western blotting or immunohistochemistry. Additionally, demyelination and neuronal damage were investigated histologically. Significant differences (p ≤ 0.05) between transcription rates of T. canis-infected and uninfected control mice were detected for all analysed genes while T. cati affected five of eight investigated genes. Interestingly, 2', 3 ´-cyclic nucleotide 3'-phosphodiesterase (Cnp) and myelin oligodendrocyte glycoprotein (Mog) were upregulated in both T. canis- and T. cati-infected mice preceding demyelination. Later, CNPase expression was additionally enhanced. As expected, myelin basic protein (Mbp) was downregulated in cerebra and cerebella of T. canis-infected mice when severe demyelination was present 120 days post infectionem (dpi). The transcriptional pattern observed in the present study appears to reflect direct traumatic and hypoxic effects of larval migration as well as secondary processes including host immune reactions, demyelination and attempts to remyelinate damaged areas.

  6. Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation

    PubMed Central

    Tsuchiya, Megumi; Isogai, Shin; Taniguchi, Hiroaki; Tochio, Hidehito; Shirakawa, Masahiro; Morohashi, Ken-ichirou; Hiraoka, Yasushi; Haraguchi, Tokuko; Ogawa, Hidesato

    2015-01-01

    Transcriptional coregulators contribute to several processes involving nuclear receptor transcriptional regulation. The transcriptional coregulator androgen receptor-interacting protein 4 (ARIP4) interacts with nuclear receptors and regulates their transcriptional activity. In this study, we identified p62 as a major interacting protein partner for ARIP4 in the nucleus. Nuclear magnetic resonance analysis demonstrated that ARIP4 interacts directly with the ubiquitin-associated (UBA) domain of p62. ARIP4 and ubiquitin both bind to similar amino acid residues within UBA domains; therefore, these proteins may possess a similar surface structure at their UBA-binding interfaces. We also found that p62 is required for the regulation of ARIP4 protein levels under nutrient starvation conditions. We propose that p62 is a novel binding partner for ARIP4, and that its binding regulates the cellular protein level of ARIP4 under conditions of metabolic stress. PMID:26412716

  7. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown.

    PubMed

    Pertea, Mihaela; Kim, Daehwan; Pertea, Geo M; Leek, Jeffrey T; Salzberg, Steven L

    2016-09-01

    High-throughput sequencing of mRNA (RNA-seq) has become the standard method for measuring and comparing the levels of gene expression in a wide variety of species and conditions. RNA-seq experiments generate very large, complex data sets that demand fast, accurate and flexible software to reduce the raw read data to comprehensible results. HISAT (hierarchical indexing for spliced alignment of transcripts), StringTie and Ballgown are free, open-source software tools for comprehensive analysis of RNA-seq experiments. Together, they allow scientists to align reads to a genome, assemble transcripts including novel splice variants, compute the abundance of these transcripts in each sample and compare experiments to identify differentially expressed genes and transcripts. This protocol describes all the steps necessary to process a large set of raw sequencing reads and create lists of gene transcripts, expression levels, and differentially expressed genes and transcripts. The protocol's execution time depends on the computing resources, but it typically takes under 45 min of computer time. HISAT, StringTie and Ballgown are available from http://ccb.jhu.edu/software.shtml. PMID:27560171

  8. Molecular characterization of BZR transcription factor family and abiotic stress induced expression profiling in Brassica rapa.

    PubMed

    Saha, Gopal; Park, Jong-In; Jung, Hee-Jeong; Ahmed, Nasar Uddin; Kayum, Md Abdul; Kang, Jong-Goo; Nou, Ill-Sup

    2015-07-01

    BRASSINAZOLE-RESISTANT (BZR) transcription factors (TFs) are primarily well known as positive regulators of Brassinosteroid (BR) signal transduction in different plants. BR is a plant specific steroid hormone, which has multiple stress resistance functions besides various growth regulatory roles. Being an important regulator of the BR synthesis, BZR TFs might have stress resistance related activities. However, no stress resistance related functional study of BZR TFs has been reported in any crop plants so far. Therefore, this study identified 15 BZR TFs of Brassica rapa (BrBZR) from a genome-wide survey and characterized them through sequence analysis and expression profiling against several abiotic stresses. Various systematic in silico analysis of these TFs validated the fundamental properties of BZRs, where a high degree of similarity also observed with recognized BZRs of other plant species from the comparison studies. In the organ specific expression analyses, 6 BrBZR TFs constitutively expressed in flower developmental stages indicating their flower specific functions. Subsequently, from the stress resistance related expression profiles differential transcript abundance levels were observed by 6 and 11 BrBZRs against salt and drought stresses, respectively. All BrBZRs showed several folds up-regulation against exogenous ABA treatment. All BrBZRs also showed differential expression against low temperature stress treatments and these TFs were proposed as transcriptional activators of CBF cold response pathway of B. rapa. Notably, three BrBZRs gave co-responsive expression against all the stresses tested here, suggesting their multiple stress resistance related functions. Thus, the findings would be helpful in resolving the complex regulatory mechanism of BZRs in stress resistance and further functional genomics study of these potential TFs in different Brassica crops. PMID:25931321

  9. Molecular characterization of BZR transcription factor family and abiotic stress induced expression profiling in Brassica rapa.

    PubMed

    Saha, Gopal; Park, Jong-In; Jung, Hee-Jeong; Ahmed, Nasar Uddin; Kayum, Md Abdul; Kang, Jong-Goo; Nou, Ill-Sup

    2015-07-01

    BRASSINAZOLE-RESISTANT (BZR) transcription factors (TFs) are primarily well known as positive regulators of Brassinosteroid (BR) signal transduction in different plants. BR is a plant specific steroid hormone, which has multiple stress resistance functions besides various growth regulatory roles. Being an important regulator of the BR synthesis, BZR TFs might have stress resistance related activities. However, no stress resistance related functional study of BZR TFs has been reported in any crop plants so far. Therefore, this study identified 15 BZR TFs of Brassica rapa (BrBZR) from a genome-wide survey and characterized them through sequence analysis and expression profiling against several abiotic stresses. Various systematic in silico analysis of these TFs validated the fundamental properties of BZRs, where a high degree of similarity also observed with recognized BZRs of other plant species from the comparison studies. In the organ specific expression analyses, 6 BrBZR TFs constitutively expressed in flower developmental stages indicating their flower specific functions. Subsequently, from the stress resistance related expression profiles differential transcript abundance levels were observed by 6 and 11 BrBZRs against salt and drought stresses, respectively. All BrBZRs showed several folds up-regulation against exogenous ABA treatment. All BrBZRs also showed differential expression against low temperature stress treatments and these TFs were proposed as transcriptional activators of CBF cold response pathway of B. rapa. Notably, three BrBZRs gave co-responsive expression against all the stresses tested here, suggesting their multiple stress resistance related functions. Thus, the findings would be helpful in resolving the complex regulatory mechanism of BZRs in stress resistance and further functional genomics study of these potential TFs in different Brassica crops.

  10. Loss of the Yeast SR Protein Npl3 Alters Gene Expression Due to Transcription Readthrough.

    PubMed

    Holmes, Rebecca K; Tuck, Alex C; Zhu, Chenchen; Dunn-Davies, Hywel R; Kudla, Grzegorz; Clauder-Munster, Sandra; Granneman, Sander; Steinmetz, Lars M; Guthrie, Christine; Tollervey, David

    2015-12-01

    Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3' end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3' end formation. Tiling arrays and RNAPII mapping data revealed 3' extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression. PMID:26694144

  11. 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.

  12. Neurotoxocarosis alters myelin protein gene transcription and expression.

    PubMed

    Heuer, Lea; Beyerbach, Martin; Lühder, Fred; Beineke, Andreas; Strube, Christina

    2015-06-01

    Neurotoxocarosis is an infection of the central nervous system caused by migrating larvae of the common dog and cat roundworms (Toxocara canis and Toxocara cati), which are zoonotic agents. As these parasites are prevalent worldwide and neuropathological and molecular investigations on neurotoxocarosis are scare, this study aims to characterise nerve fibre demyelination associated with neurotoxocarosis on a molecular level. Transcription of eight myelin-associated genes (Cnp, Mag, Mbp, Mog, Mrf-1, Nogo-A, Plp1, Olig2) was determined in the mouse model during six time points of the chronic phase of infection using qRT-PCR. Expression of selected proteins was analysed by Western blotting or immunohistochemistry. Additionally, demyelination and neuronal damage were investigated histologically. Significant differences (p ≤ 0.05) between transcription rates of T. canis-infected and uninfected control mice were detected for all analysed genes while T. cati affected five of eight investigated genes. Interestingly, 2', 3 ´-cyclic nucleotide 3'-phosphodiesterase (Cnp) and myelin oligodendrocyte glycoprotein (Mog) were upregulated in both T. canis- and T. cati-infected mice preceding demyelination. Later, CNPase expression was additionally enhanced. As expected, myelin basic protein (Mbp) was downregulated in cerebra and cerebella of T. canis-infected mice when severe demyelination was present 120 days post infectionem (dpi). The transcriptional pattern observed in the present study appears to reflect direct traumatic and hypoxic effects of larval migration as well as secondary processes including host immune reactions, demyelination and attempts to remyelinate damaged areas. PMID:25773181

  13. Sugarcane transgenics expressing MYB transcription factors show improved glucose release

    DOE PAGES

    Poovaiah, Charleson R.; Bewg, William P.; Lan, Wu; Ralph, John; Coleman, Heather D.

    2016-07-15

    In this study, sugarcane, a tropical C4 perennial crop, is capable of producing 30-100 tons or more of biomass per hectare annually. The lignocellulosic residue remaining after sugar extraction is currently underutilized and can provide a significant source of biomass for the production of second-generation bioethanol. As a result, MYB31 and MYB42 were cloned from maize and expressed in sugarcane with and without the UTR sequences. The cloned sequences were 98 and 99 % identical to the published nucleotide sequences. The inclusion of the UTR sequences did not affect any of the parameters tested. There was little difference in plantmore » height and the number of internodes of the MYB-overexpressing sugarcane plants when compared with controls. MYB transgene expression determined by qPCR exhibited continued expression in young and maturing internodes. MYB31 downregulated more genes within the lignin biosynthetic pathway than MYB42. MYB31 and MYB42 expression resulted in decreased lignin content in some lines. All MYB42 plants further analyzed showed significant increases in glucose release by enzymatic hydrolysis in 72 h, whereas only two MYB31 plants released more glucose than control plants. This correlated directly with a significant decrease in acid-insoluble lignin. Soluble sucrose content of the MYB42 transgenic plants did not vary compared to control plants. In conclusion, this study demonstrates the use of MYB transcription factors to improve the production of bioethanol from sugarcane bagasse remaining after sugar extraction.« less

  14. Myelin inhibits oligodendroglial maturation and regulates oligodendrocytic transcription factor expression.

    PubMed

    Plemel, Jason R; Manesh, Sohrab B; Sparling, Joseph S; Tetzlaff, Wolfram

    2013-09-01

    Myelin loss is a hallmark of multiple sclerosis (MS) and promoting central nervous system myelin repair has become a major therapeutic target. Despite the presence of oligodendrocytes precursors cells (OPCs) in chronic lesions of MS, remyelination often fails. The mechanism underlying this failure of remyelination remains unknown, but it is hypothesized that environmental cues act to inhibit the maturation/differentiation of oligodendroglia, preventing remyelination. The rate of CNS remyelination is correlated to the speed of phagocytosis of myelin debris, which is present following demyelination and trauma. Thus, myelin debris could inhibit CNS remyelination. Here, we demonstrate that OPCs cultured on myelin were robustly inhibited in their maturation, as characterized by the decreased expression of immature and mature oligodendrocytes markers, the impaired production of myelin gene products, as well as their stalled morphological complexity relative to OPCs cultured on a control substrate. OPCs in contact with myelin stopped proliferating and decreased the expression of OPC markers to a comparable degree as cells grown on a control substrate. The expression of two transcription factors known to prevent OPC differentiation and maturation were increased in cells that were in contact with myelin: inhibitor of differentiation family (ID) members 2 and 4. Overexpression of ID2 and ID4 in OPCs was previously reported to decrease the percentage of cells expressing mature oligodendrocyte markers. However, knockdown of ID2 and/or ID4 in OPCs did not increase oligodendroglial maturation on or off of myelin, suggesting that contact with myelin regulates additional regulatory elements.

  15. PPP1, a plant-specific regulator of transcription controls Arabidopsis development and PIN expression

    PubMed Central

    Benjamins, René; Barbez, Elke; Ortbauer, Martina; Terpstra, Inez; Lucyshyn, Doris; Moulinier-Anzola, Jeanette; Khan, Muhammad Asaf; Leitner, Johannes; Malenica, Nenad; Butt, Haroon; Korbei, Barbara; Scheres, Ben; Kleine-Vehn, Jürgen; Luschnig, Christian

    2016-01-01

    Directional transport of auxin is essential for plant development, with PIN auxin transport proteins representing an integral part of the machinery that controls hormone distribution. However, unlike the rapidly emerging framework of molecular determinants regulating PIN protein abundance and subcellular localization, insights into mechanisms controlling PIN transcription are still limited. Here we describe PIN2 PROMOTER BINDING PROTEIN 1 (PPP1), an evolutionary conserved plant-specific DNA binding protein that acts on transcription of PIN genes. Consistent with PPP1 DNA-binding activity, PPP1 reporter proteins are nuclear localized and analysis of PPP1 null alleles and knockdown lines indicated a function as a positive regulator of PIN expression. Furthermore, we show that ppp1 pleiotropic mutant phenotypes are partially reverted by PIN overexpression, and results are presented that underline a role of PPP1-PIN promoter interaction in PIN expression control. Collectively, our findings identify an elementary, thus far unknown, plant-specific DNA-binding protein required for post-embryonic plant development, in general, and correct expression of PIN genes, in particular. PMID:27553690

  16. PPP1, a plant-specific regulator of transcription controls Arabidopsis development and PIN expression.

    PubMed

    Benjamins, René; Barbez, Elke; Ortbauer, Martina; Terpstra, Inez; Lucyshyn, Doris; Moulinier-Anzola, Jeanette; Khan, Muhammad Asaf; Leitner, Johannes; Malenica, Nenad; Butt, Haroon; Korbei, Barbara; Scheres, Ben; Kleine-Vehn, Jürgen; Luschnig, Christian

    2016-01-01

    Directional transport of auxin is essential for plant development, with PIN auxin transport proteins representing an integral part of the machinery that controls hormone distribution. However, unlike the rapidly emerging framework of molecular determinants regulating PIN protein abundance and subcellular localization, insights into mechanisms controlling PIN transcription are still limited. Here we describe PIN2 PROMOTER BINDING PROTEIN 1 (PPP1), an evolutionary conserved plant-specific DNA binding protein that acts on transcription of PIN genes. Consistent with PPP1 DNA-binding activity, PPP1 reporter proteins are nuclear localized and analysis of PPP1 null alleles and knockdown lines indicated a function as a positive regulator of PIN expression. Furthermore, we show that ppp1 pleiotropic mutant phenotypes are partially reverted by PIN overexpression, and results are presented that underline a role of PPP1-PIN promoter interaction in PIN expression control. Collectively, our findings identify an elementary, thus far unknown, plant-specific DNA-binding protein required for post-embryonic plant development, in general, and correct expression of PIN genes, in particular. PMID:27553690

  17. The Expression of BAFF Is Controlled by IRF Transcription Factors.

    PubMed

    Sjöstrand, Maria; Johansson, Alina; Aqrawi, Lara; Olsson, Tomas; Wahren-Herlenius, Marie; Espinosa, Alexander

    2016-01-01

    Patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) are typically characterized by the presence of autoantibodies and an IFN-signature. The strength of the IFN-signature positively correlates with disease severity, suggesting that type I IFNs are active players in these diseases. BAFF is a cytokine critical for development and proper selection of B cells, and the targeting of BAFF has emerged as a successful treatment strategy of SLE. Previous reports have suggested that BAFF expression is directly induced by type I IFNs, but the precise mechanism for this remains unknown. In this article, we demonstrate that BAFF is a bona fide ISG and that IFN regulatory factors (IRFs) control the expression of BAFF. We identify IRF1 and IRF2 as positive regulators of BAFF transcription and IRF4 and IRF8 as potent repressors; in addition, we have mapped the precise binding site for these factors in the BAFF promoter. IFN-β injections induced BAFF expression mainly in neutrophils and monocytes, and BAFF expression in neutrophils from pSS patients strongly correlated with the strength of the IFN-signature. In summary, we show that BAFF expression is directly induced by type I IFNs via IRF1 and IRF2, whereas IRF4 and IRF8 are negative regulators of BAFF expression. These data suggest that type I IFN blockade in SLE and pSS patients will lead to downregulation of BAFF and a consequential reduction of autoreactive B cell clones and autoantibodies. PMID:26590315

  18. The Expression of BAFF Is Controlled by IRF Transcription Factors

    PubMed Central

    Sjöstrand, Maria; Johansson, Alina; Aqrawi, Lara; Olsson, Tomas; Wahren-Herlenius, Marie

    2016-01-01

    Patients with systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome (pSS) are typically characterized by the presence of autoantibodies and an IFN-signature. The strength of the IFN-signature positively correlates with disease severity, suggesting that type I IFNs are active players in these diseases. BAFF is a cytokine critical for development and proper selection of B cells, and the targeting of BAFF has emerged as a successful treatment strategy of SLE. Previous reports have suggested that BAFF expression is directly induced by type I IFNs, but the precise mechanism for this remains unknown. In this article, we demonstrate that BAFF is a bona fide ISG and that IFN regulatory factors (IRFs) control the expression of BAFF. We identify IRF1 and IRF2 as positive regulators of BAFF transcription and IRF4 and IRF8 as potent repressors; in addition, we have mapped the precise binding site for these factors in the BAFF promoter. IFN-β injections induced BAFF expression mainly in neutrophils and monocytes, and BAFF expression in neutrophils from pSS patients strongly correlated with the strength of the IFN-signature. In summary, we show that BAFF expression is directly induced by type I IFNs via IRF1 and IRF2, whereas IRF4 and IRF8 are negative regulators of BAFF expression. These data suggest that type I IFN blockade in SLE and pSS patients will lead to downregulation of BAFF and a consequential reduction of autoreactive B cell clones and autoantibodies. PMID:26590315

  19. FMR1 transcript isoforms: association with polyribosomes; regional and developmental expression in mouse brain.

    PubMed

    Brackett, David M; Qing, Feng; Amieux, Paul S; Sellers, Drew L; Horner, Philip J; Morris, David R

    2013-01-01

    The primary transcript of the mammalian Fragile X Mental Retardation-1 gene (Fmr1), like many transcripts in the central nervous system, is alternatively spliced to yield mRNAs encoding multiple proteins, which can possess quite different biochemical properties. Despite the fact that the relative levels of the 12 Fmr1 transcript isoforms examined here vary by as much as two orders of magnitude amongst themselves in both adult and embryonic mouse brain, all are associated with polyribosomes, consistent with translation into the corresponding isoforms of the protein product, FMRP (Fragile X Mental Retardation Protein). Employing the RiboTag methodology developed in our laboratory, the relative proportions of the 7 most abundant transcript isoforms were measured specifically in neurons and found to be similar to those identified in whole brain. Measurements of isoform profiles across 11 regions of adult brain yielded similar distributions, with the exceptions of the hippocampus and the olfactory bulb. These two regions differ from most of the brain in relative amounts of transcripts encoding an alternate form of one of the KH RNA binding domains. A possible relationship between patterns of expression in the hippocampus and olfactory bulb and the presence of neuroblasts in these two regions is suggested by the isoform patterns in early embryonic brain and in cultured neural progenitor cells. These results demonstrate that the relative levels of the Fmr1 isoforms are modulated according to developmental stage, highlighting the complex ramifications of losing all the protein isoforms in individuals with Fragile X Syndrome. It should also be noted that, of the eight most prominent FMRP isoforms (1-3, 6-9 and 12) in mouse, only two have the major site of phosphorylation at Ser-499, which is thought to be involved in some of the regulatory interactions of this protein.

  20. Abundant 5S rRNA-like transcripts encoded by the mitochondrial genome in amoebozoa.

    PubMed

    Bullerwell, Charles E; Burger, Gertraud; Gott, Jonatha M; Kourennaia, Olga; Schnare, Murray N; Gray, Michael W

    2010-05-01

    5S rRNAs are ubiquitous components of prokaryotic, chloroplast, and eukaryotic cytosolic ribosomes but are apparently absent from mitochondrial ribosomes (mitoribosomes) of many eukaryotic groups including animals and fungi. Nevertheless, a clearly identifiable, mitochondrion-encoded 5S rRNA is present in Acanthamoeba castellanii, a member of Amoebozoa. During a search for additional mitochondrial 5S rRNAs, we detected small abundant RNAs in other members of Amoebozoa, namely, in the lobose amoeba Hartmannella vermiformis and in the myxomycete slime mold Physarum polycephalum. These RNAs are encoded by mitochondrial DNA (mtDNA), cosediment with mitoribosomes in glycerol gradients, and can be folded into a secondary structure similar to that of bona fide 5S rRNAs. Further, in the mtDNA of another slime mold, Didymium nigripes, we identified a region that in sequence, potential secondary structure, and genomic location is similar to the corresponding region encoding the Physarum small RNA. A mtDNA-encoded small RNA previously identified in Dictyostelium discoideum is here shown to share several characteristics with known 5S rRNAs. Again, we detected genes encoding potential homologs of this RNA in the mtDNA of three other species of the genus Dictyostelium as well as in a related genus, Polysphondylium. Taken together, our results indicate a widespread occurrence of small, abundant, mtDNA-encoded RNAs with 5S rRNA-like structures that are associated with the mitoribosome in various amoebozoan taxa. Our working hypothesis is that these novel small abundant RNAs represent radically divergent mitochondrial 5S rRNA homologs. We posit that currently unrecognized 5S-like RNAs may exist in other mitochondrial systems in which a conventional 5S rRNA cannot be identified.

  1. Expression of transcripts for two interleukin 8 receptors in human phagocytes, lymphocytes and melanoma cells.

    PubMed Central

    Moser, B; Barella, L; Mattei, S; Schumacher, C; Boulay, F; Colombo, M P; Baggiolini, M

    1993-01-01

    Two cDNAs coding for distinct interleukin 8 (IL-8) receptors, IL-8R1 [Murphy and Tiffany (1991) Science 253, 1280-1283] and IL-8R2 [Holmes, Lee, Kuang, Rice and Wood (1991) Science 253, 1278-1280] have been reported, and biochemical studies on human neutrophils have revealed two proteins (p70 and p44) that bind IL-8 with high affinity [Moser, Schumacher, von Tscharner, Clark-Lewis and Baggiolini (1991), J. Biol. Chem. 266, 10666-10671]. We have cloned the cDNA coding for IL-8R1 from a library of differentiated HL-60 cells. Transfection of this cDNA into Jurkat cells resulted in the expression of high-affinity binding for IL-8 and two related cytokines, GRO alpha and neutrophil-activating peptide 2 (Kd 0.5-1.0 nM). Northern-blot analysis with the IL-8R1 cDNA as probe revealed abundant expression of transcripts of different size in human neutrophils and low-level expression of a single RNA species in HL-60 cells differentiated with dimethyl sulphoxide and retinoic acid. Because of the extensive nucleotide sequence similarity of the cDNAs for IL-8R1 and IL-8R2, the reverse-transcription PCR method was used for analysis of RNA expression in myeloid and lymphoid cells, 19 cell lines established from human primary melanomas or metastases, two melanocyte and one fibroblast cell lines. IL-8R1 mRNA transcripts were expressed at high levels in neutrophils, and to a lesser extent in blood monocytes and the myeloid cell lines, HL-60 and AML 193, but were not found in THP-1 cells, lymphocytes and Jurkat cells. IL-8R2 mRNA transcripts, by contrast, were found in all blood cells and related cell lines, as well as in all melanoma, melanocyte and fibroblast cell lines tested. As for IL-8R1, IL-8R2 mRNA expression was highest in neutrophils. These results suggest that IL-8R1 and IL-8R2 may both be involved in neutrophil activation by IL-8 and related cytokines, and presumably correspond to p70 and p44, the receptors that were identified biochemically. Possible IL-8 functions on

  2. A family of transcripts encoding water channel proteins: tissue-specific expression in the common ice plant.

    PubMed Central

    Yamada, S; Katsuhara, M; Kelly, W B; Michalowski, C B; Bohnert, H J

    1995-01-01

    Seawater-strength salt stress of the ice plant (Mesembryanthemum crystallinum) initially results in wilting, but full turgor is restored within approximately 2 days. We are interested in a mechanistic explanation for this behavior and, as a requisite for in-depth biochemical studies, have begun to analyze gene expression changes in roots coincident with the onset of stress. cDNAs that suggested changes in mRNA amount under stress were found; their deduced amino acid sequences share homologies with proteins of the Mip (major intrinsic protein) gene family and potentially encode aquaporins. One transcript, MipB, was found only in root RNA, whereas two other transcripts, MipA and MipC, were detected in roots and leaves. Transcript levels of MipB were of low abundance. All transcripts declined initially during salt stress but later recovered to at least prestress level. The most drastic decline was in MipA and MipC transcripts. MipA mRNA distribution in roots detected by in situ hybridization indicated that the transcript was present in all cells in the root tip. In the expansion zone of the root where vascular bundles differentiate, MipA transcript amounts were most abundant in the endodermis. In older roots, which had undergone secondary growth, MipA was highly expressed in cell layers surrounding individual xylem strands. MipA was also localized in leaf vascular tissue and, in lower amounts, in mesophyll cells. Transcripts for MipB seemed to be present exclusively in the tip of the root, in a zone before and possibly coincident with the development of a vascular system. MipA- and MipB-encoded proteins expressed in Xenopus oocytes led to increased water permeability. mRNA fluctuations of the most highly expressed MipA and MipC coincided with turgor changes in leaves under stress. As the leaves regained turgor, transcript levels of these water channel proteins increased. PMID:7549476

  3. Transcriptional profiling of pea ABR17 mediated changes in gene expression in Arabidopsis thaliana

    PubMed Central

    Krishnaswamy, Sowmya S; Srivastava, Sanjeeva; Mohammadi, Mohsen; Rahman, Muhammad H; Deyholos, Michael K; Kav, Nat NV

    2008-01-01

    Background Pathogenesis-related proteins belonging to group 10 (PR10) are elevated in response to biotic and abiotic stresses in plants. Previously, we have shown a drastic salinity-induced increase in the levels of ABR17, a member of the PR10 family, in pea. Furthermore, we have also demonstrated that the constitutive expression of pea ABR17 cDNA in Arabidopsis thaliana and Brassica napus enhances their germination and early seedling growth under stress. Although it has been reported that several members of the PR10 family including ABR17 possess RNase activity, the exact mechanism by which the aforementioned characteristics are conferred by ABR17 is unknown at this time. We hypothesized that a study of differences in transcriptome between wild type (WT) and ABR17 transgenic A. thaliana may shed light on this process. Results The molecular changes brought about by the expression of pea ABR17 cDNA in A. thaliana in the presence or absence of salt stress were investigated using microarrays consisting of 70-mer oligonucleotide probes representing 23,686 Arabidopsis genes. Statistical analysis identified number of genes which were over represented among up- or down-regulated transcripts in the transgenic line. Our results highlight the important roles of many abscisic acid (ABA) and cytokinin (CK) responsive genes in ABR17 transgenic lines. Although the transcriptional changes followed a general salt response theme in both WT and transgenic seedlings under salt stress, many genes exhibited differential expression patterns when the transgenic and WT lines were compared. These genes include plant defensins, heat shock proteins, other defense related genes, and several transcriptional factors. Our microarray results for selected genes were validated using quantitative real-time PCR. Conclusion Transcriptional analysis in ABR17 transgenic Arabidopsis plants, both under normal and saline conditions, revealed significant changes in abundance of transcripts for many stress

  4. Building gene expression signatures indicative of transcription factor activation to predict AOP modulation

    EPA Science Inventory

    Building gene expression signatures indicative of transcription factor activation to predict AOP modulation Adverse outcome pathways (AOPs) are a framework for predicting quantitative relationships between molecular initiatin...

  5. Identification of a novel herpes simplex virus type 1 transcript and protein (AL3) expressed during latency

    PubMed Central

    Jaber, Tareq; Henderson, Gail; Li, Sumin; Perng, Guey-Chuen; Carpenter, Dale; Wechsler, Steven L.; Jones, Clinton

    2009-01-01

    The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected sensory neurons. In small animal models of infection, expression of the first 1.5 kb of LAT coding sequences is necessary and sufficient for wild-type reactivation from latency. The ability of LAT to inhibit apoptosis is important for reactivation from latency. Within the first 1.5 kb of LAT coding sequences and LAT promoter sequences, additional transcripts have been identified. For example, the anti-sense to LAT transcript (AL) is expressed in the opposite direction to LAT from the 5′ end of LAT and LAT promoter sequences. In addition, the upstream of LAT (UOL) transcript is expressed in the LAT direction from sequences in the LAT promoter. Further examination of the first 1.5 kb of LAT coding sequences revealed two small ORFs that are anti-sense with respect to LAT (AL2 and AL3). A transcript spanning AL3 was detected in productively infected cells, mouse neuroblastoma cells stably expressing LAT and trigeminal ganglia (TG) of latently infected mice. Peptide-specific IgG directed against AL3 specifically recognized a protein migrating near 15 kDa in cells stably transfected with LAT, mouse neuroblastoma cells transfected with a plasmid containing the AL3 ORF and TG of latently infected mice. The inability to detect the AL3 protein during productive infection may have been because the 5′ terminus of the AL3 transcript was downstream of the first in-frame methionine of the AL3 ORF during productive infection. PMID:19570955

  6. ß-catenin, a transcription factor activated by canonical Wnt signaling, is expressed in sensory neurons of calves latently infected with bovine herpesvirus 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Detection and transcript expression of S-RNase gene associated with self-incompatibility in apricot (Prunus armeniaca L.).

    PubMed

    Feng, Jianrong; Chen, Xuesen; Wu, Yan; Liu, Wen; Liang, Qing; Zhang, Lijie

    2006-09-01

    The identity and expression of S-RNase genotypes in the self-compatible (SC) apricot cultivar 'Katy' and the self-incompatible (SI) cultivar 'Xinshiji' were examined. We used allele specific polymerase chain reaction (AS-PCR) and designated the alleles in 'Katy' and 'Xinshiji' as S(8)Sc and S(9)S(10), respectively. The S-RNase gene was expressed in style at the balloon stage in both genotypes. Using real-time fluorescence quantification RT-PCR technology (FQRT-PCR), spatio-temporal expression patterns of S-RNase gene between 'Katy' and 'Xinshiji' were compared. The results revealed that the expression of the S-RNase gene in 'Katy' and 'Xinshiji' were different. The transcript abundance was distinctly diverse at the key stage (i.e., at 24 h after self-pollination) in both genotypes, and was greater in 'Xinshiji' (SI) than 'Katy' (SC). In addition, the abundance of the S-RNase transcript was higher in upper-half of style than in the lower-half of style or in the ovary. In the SI cultivar 'Xinshiji', the expression of S-RNase reminded a relatively high level after cross-pollination, but it dropped continuously after self-pollination and un-pollination.

  8. Express yourself: Transcriptional regulation of plant innate immunity.

    PubMed

    Garner, Christopher M; Kim, Sang Hee; Spears, Benjamin J; Gassmann, Walter

    2016-08-01

    The plant immune system is a complex network of components that function together to sense the presence and activity of potential biotic threats, and integrate these signals into an appropriate output, namely the transcription of genes that activate an immune response that is commensurate with the perceived threat. Given the variety of biotic threats a plant must face the immune response must be plastic, but because an immune response is costly to the plant in terms of energy expenditure and development it must also be under tight control. To meet these needs transcriptional control is exercised at multiple levels. In this article we will review some of the latest developments in understanding how the plant immune response is regulated at the level of transcription. New roles are being discovered for the long-studied WRKY and TGA transcription factor families, while additional critical defense functions are being attributed to TCPs and other transcription factors. Dynamically controlling access to DNA through post-translational modification of histones is emerging as an essential component of priming, maintaining, attenuating, and repressing transcription in response to biotic stress. Unsurprisingly, the plant's transcriptional response is targeted by pathogen effectors, and in turn resistance proteins stand guard over and participate in transcriptional regulation. Together, these multiple layers lead to the observed complexity of the plant transcriptional immune response, with different transcription factors or chromatin components playing a prominent role depending on the plant-pathogen interaction being studied. PMID:27174437

  9. Express yourself: Transcriptional regulation of plant innate immunity.

    PubMed

    Garner, Christopher M; Kim, Sang Hee; Spears, Benjamin J; Gassmann, Walter

    2016-08-01

    The plant immune system is a complex network of components that function together to sense the presence and activity of potential biotic threats, and integrate these signals into an appropriate output, namely the transcription of genes that activate an immune response that is commensurate with the perceived threat. Given the variety of biotic threats a plant must face the immune response must be plastic, but because an immune response is costly to the plant in terms of energy expenditure and development it must also be under tight control. To meet these needs transcriptional control is exercised at multiple levels. In this article we will review some of the latest developments in understanding how the plant immune response is regulated at the level of transcription. New roles are being discovered for the long-studied WRKY and TGA transcription factor families, while additional critical defense functions are being attributed to TCPs and other transcription factors. Dynamically controlling access to DNA through post-translational modification of histones is emerging as an essential component of priming, maintaining, attenuating, and repressing transcription in response to biotic stress. Unsurprisingly, the plant's transcriptional response is targeted by pathogen effectors, and in turn resistance proteins stand guard over and participate in transcriptional regulation. Together, these multiple layers lead to the observed complexity of the plant transcriptional immune response, with different transcription factors or chromatin components playing a prominent role depending on the plant-pathogen interaction being studied.

  10. Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos.

    PubMed

    Bothma, Jacques P; Garcia, Hernan G; Esposito, Emilia; Schlissel, Gavin; Gregor, Thomas; Levine, Michael

    2014-07-22

    We present the use of recently developed live imaging methods to examine the dynamic regulation of even-skipped (eve) stripe 2 expression in the precellular Drosophila embryo. Nascent transcripts were visualized via MS2 RNA stem loops. The eve stripe 2 transgene exhibits a highly dynamic pattern of de novo transcription, beginning with a broad domain of expression during nuclear cycle 12 (nc12), and progressive refinement during nc13 and nc14. The mature stripe 2 pattern is surprisingly transient, constituting just ∼15 min of the ∼90-min period of expression. Nonetheless, this dynamic transcription profile faithfully predicts the limits of the mature stripe visualized by conventional in situ detection methods. Analysis of individual transcription foci reveals intermittent bursts of de novo transcription, with duration cycles of 4-10 min. We discuss a multistate model of transcription regulation and speculate on its role in the dynamic repression of the eve stripe 2 expression pattern during development.

  11. Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

    PubMed Central

    Aragon, Anthony D; Quiñones, Gabriel A; Thomas, Edward V; Roy, Sushmita; Werner-Washburne, Margaret

    2006-01-01

    Background As carbon sources are exhausted, Saccharomyces cerevisiae cells exhibit reduced metabolic activity and cultures enter the stationary phase. We asked whether cells in stationary phase cultures respond to additional stress at the level of transcript abundance. Results Microarrays were used to quantify changes in transcript abundance in cells from stationary phase cultures in response to stress. More than 800 mRNAs increased in abundance by one minute after oxidative stress. A significant number of these mRNAs encode proteins involved in stress responses. We tested whether mRNA increases were due to new transcription, rapid poly-adenylation of message (which would not be detected by microarrays), or potential release of mature mRNA present in the cell but resistant to extraction during RNA isolation. Examination of the response to oxidative stress in an RNA polymerase II mutant, rpb1-1, suggested that new transcription was not required. Quantitative RT-PCR analysis of a subset of these transcripts further suggested that the transcripts present in isolated total RNA from stationary phase cultures were polyadenylated. In contrast, over 2,000 transcripts increased after protease treatment of cell-free lysates from stationary phase but not exponentially growing cultures. Different subsets of transcripts were released by oxidative stress and temperature upshift, suggesting that mRNA release is stress-specific. Conclusions Cells in stationary phase cultures contain a large number of extraction-resistant mRNAs in a protease-labile, rapidly releasable form. The transcript release appears to be stress-specific. We hypothesize that these transcripts are associated with P-bodies. PMID:16507144

  12. Nonspecific transcription factor binding can reduce noise in the expression of downstream proteins

    NASA Astrophysics Data System (ADS)

    Soltani, M.; Bokes, P.; Fox, Z.; Singh, A.

    2015-10-01

    Transcription factors (TFs) interact with a multitude of binding sites on DNA and partner proteins inside cells. We investigate how nonspecific binding/unbinding to such decoy binding sites affects the magnitude and time-scale of random fluctuations in TF copy numbers arising from stochastic gene expression. A stochastic model of TF gene expression, together with decoy site interactions is formulated. Distributions for the total (bound and unbound) and free (unbound) TF levels are derived by analytically solving the chemical master equation under physiologically relevant assumptions. Our results show that increasing the number of decoy binding sides considerably reduces stochasticity in free TF copy numbers. The TF autocorrelation function reveals that decoy sites can either enhance or shorten the time-scale of TF fluctuations depending on model parameters. To understand how noise in TF abundances propagates downstream, a TF target gene is included in the model. Intriguingly, we find that noise in the expression of the target gene decreases with increasing decoy sites for linear TF-target protein dose-responses, even in regimes where decoy sites enhance TF autocorrelation times. Moreover, counterintuitive noise transmissions arise for nonlinear dose-responses. In summary, our study highlights the critical role of molecular sequestration by decoy binding sites in regulating the stochastic dynamics of TFs and target proteins at the single-cell level.

  13. Transcripts involved in steroid biosynthesis and steroid receptor signaling are expressed early in development in the fathead minnow (Pimephales promelas).

    PubMed

    Wood, Richard K; Seidel, Jason S; Martyniuk, Christopher J

    2015-04-01

    Sex differentiation in organisms is correlated to sex steroid production and receptor signaling pathways involving androgens and estrogens. Timing of expression is critical, and characterization of sensitive windows is needed to determine how environmental stressors may perturb sex differentiation. The objectives of this study were to determine whether genes related to steroid biosynthesis, steroid receptor signaling, and those related to sex differentiation were expressed in pre-differentiated fathead minnow (FHM) embryos, an ecotoxicological model. Transcripts were measured over two weeks (1 day post fertilization (dpf) to 14 days), prior to sex differentiation. The first three time points investigated (1, 3, and 5 dpf) corresponded to the neurula stage, dorsal swim bladder pigmentation, and pre-hatch. The fourth time point (6 dpf) was collected immediately post-hatch and the fifth time point investigated was after 8 days of larval growth (14 dpf). The majority of transcripts investigated, for example estrogen, androgen, and thyroid receptors as well as steroid biosynthesis transcripts, were expressed within the first 72 hours of development; exceptions were star (steroidogenic acute regulatory protein) and cyp19a, which did not have detectable expression until 5 dpf (pre-hatch). Transcripts that increased in relative mRNA abundance over the first two weeks of development included ar, dax1, hsd11b2, hsd17b, cyp19a and thra. This study demonstrates that there is early expression of transcripts related to steroid biosynthesis, steroid receptor signaling, and sex differentiation in pre-hatch FHM embryos. Additional studies are required to determine their relative roles in male and female differentiation during these early developmental periods.

  14. Expression of the bmpB Gene of Borrelia burgdorferi Is Modulated by Two Distinct Transcription Termination Events

    PubMed Central

    Ramamoorthy, Ramesh; McClain, Natalie A.; Gautam, Aarti; Scholl-Meeker, Dorothy

    2005-01-01

    bmp gene family 36 of Borrelia burgdorferi, the agent of Lyme disease, comprises four paralogs: bmpA, bmpB, bmpC, and bmpD. The bmpA and bmpB genes constitute an operon. All four genes have been found to be transcribed in cultured spirochetes. Expression from the bmpAB operon results in three distinct transcripts of 1.1, 1.6, and 2.4 kb, and the relative expression of bmpA mRNA is three- to fourfold greater than that of bmpB mRNA. However, thus far only expression of the BmpA protein has been demonstrated. Therefore, in this study we characterized the origins of the three transcripts and compared the relative expression of the BmpA and BmpB proteins. Northern blotting revealed that the three distinct transcripts originated from a single promoter located upstream of bmpA but terminated either 3′ to the bmpA (1.1-kb RNA) or bmpB (2.4-kb RNA) gene or, most unusually, within the bmpB gene (1.6-kb RNA). Termination within the bmpB gene was associated with a functional Rho-independent transcription terminator. At the protein level, we also observed a 4.3-fold greater abundance of BmpA compared to that of BmpB. These studies identify a transcription termination mechanism in B. burgdorferi resulting in the disparate expression of the two genes of the bmpAB operon. PMID:15805505

  15. Analysis of LPS-induced, NFκB-dependent interleukin-8 transcription in kidney embryonic cell line expressing TLR4 using luciferase assay.

    PubMed

    Yunusova, Tamara; Akhtar, Mumtaz; Poltoratsky, Vladimir

    2014-01-01

    Gene expression is orchestrated by a complex network of signal transduction pathways that typically originate on cell surface receptors and culminate in DNA-binding transcription factors, which translocate to the nucleus and bind cis-regulatory elements in promoter regions of genes, thereby inducing de novo synthesis of the nascent RNA transcripts and their splicing. Gene expression arrays monitor abundance of the matured, spliced cDNA, which undergoes additional posttranscriptional modifications that greatly affect the half-life of the cDNA. Thus, the relative abundance of cDNA is not necessarily commensurable with the activity of promoters of the corresponding genes. In contrast, reporter gene assays provide valuable insight into the regulation of gene expression at the level of transcription and allow for discerning the contribution of individual transcription factors into changes in gene expression. Here, we describe a robust reporter gene assay method that is useful for exploration of transcription regulatory network, which regulates gene expression in response to inflammation. The method is exemplified by using the promoter region of the prototypic pro-inflammatory chemokine interleukin-8 (IL-8, CXCL8), which plays an important role in immune response as well as carcinogenesis. Using the luciferase reporter gene assay, we analyze the activation status of the IL-8 promoter in lipopolysaccharide (LPS)-stimulated human embryonic kidney cells. PMID:24908317

  16. Analysis of LPS-induced, NFκB-dependent interleukin-8 transcription in kidney embryonic cell line expressing TLR4 using luciferase assay.

    PubMed

    Yunusova, Tamara; Akhtar, Mumtaz; Poltoratsky, Vladimir

    2014-01-01

    Gene expression is orchestrated by a complex network of signal transduction pathways that typically originate on cell surface receptors and culminate in DNA-binding transcription factors, which translocate to the nucleus and bind cis-regulatory elements in promoter regions of genes, thereby inducing de novo synthesis of the nascent RNA transcripts and their splicing. Gene expression arrays monitor abundance of the matured, spliced cDNA, which undergoes additional posttranscriptional modifications that greatly affect the half-life of the cDNA. Thus, the relative abundance of cDNA is not necessarily commensurable with the activity of promoters of the corresponding genes. In contrast, reporter gene assays provide valuable insight into the regulation of gene expression at the level of transcription and allow for discerning the contribution of individual transcription factors into changes in gene expression. Here, we describe a robust reporter gene assay method that is useful for exploration of transcription regulatory network, which regulates gene expression in response to inflammation. The method is exemplified by using the promoter region of the prototypic pro-inflammatory chemokine interleukin-8 (IL-8, CXCL8), which plays an important role in immune response as well as carcinogenesis. Using the luciferase reporter gene assay, we analyze the activation status of the IL-8 promoter in lipopolysaccharide (LPS)-stimulated human embryonic kidney cells.

  17. Stochastic expression dynamics of a transcription factor revealed by single-molecule noise analysis.

    PubMed

    Hensel, Zach; Feng, Haidong; Han, Bo; Hatem, Christine; Wang, Jin; Xiao, Jie

    2012-08-01

    Gene expression is inherently stochastic; precise gene regulation by transcription factors is important for cell-fate determination. Many transcription factors regulate their own expression, suggesting that autoregulation counters intrinsic stochasticity in gene expression. Using a new strategy, cotranslational activation by cleavage (CoTrAC), we probed the stochastic expression dynamics of cI, which encodes the bacteriophage λ repressor CI, a fate-determining transcription factor. CI concentration fluctuations influence both lysogenic stability and induction of bacteriophage λ. We found that the intrinsic stochasticity in cI expression was largely determined by CI expression level irrespective of autoregulation. Furthermore, extrinsic, cell-to-cell variation was primarily responsible for CI concentration fluctuations, and negative autoregulation minimized CI concentration heterogeneity by counteracting extrinsic noise and introducing memory. This quantitative study of transcription factor expression dynamics sheds light on the mechanisms cells use to control noise in gene regulatory networks. PMID:22751020

  18. An inducible promoter mediates abundant expression from the immediate-early 2 gene region of human cytomegalovirus at late times after infection.

    PubMed Central

    Puchtler, E; Stamminger, T

    1991-01-01

    An abundant late transcript of 1.5 kb originates from the immediate-early 2 (IE-2) gene region of human cytomegalovirus (HCMV) at late times after infection. The transcriptional start of this RNA was precisely mapped, and the putative promoter region was cloned in front of the CAT gene as reporter. This region, which comprises 78 nucleotides of IE-2 sequence upstream of the determined cap site, was strongly activated by viral superinfection at late times in the replicative cycle. As shown by RNase protection analyses, the authentic transcription start is used. No activation of this late promoter was observed after cotransfection with an expression plasmid containing the HCMV IE-1 and -2 gene region. This result suggests that, compared with early and early late promoters of HCMV, different or additional viral functions are required for the activation of true late promoters. Images PMID:1656096

  19. Measles virus nucleocapsid transcript expression is not restricted to the osteoclast lineage in patients with Paget's disease of bone.

    PubMed

    Reddy, S V; Menaa, C; Singer, F R; Cundy, T; Cornish, J; Whyte, M P; Roodman, G D

    1999-10-01

    Abundant evidence supports a viral etiology for Paget's disease of bone (PD), however, an infectious virus has not been isolated from PD patients. Thus, it is unclear how the virus is maintained for the many years that the disease persists in patients. We considered if a primitive multipotential hematopoietic stem cell (HSC), which is self-renewing, passes the virus to its differentiated progeny and serves as a reservoir for the pathogen. If a primitive stem cell harbored measles virus (MV), then other hematopoietic lineages derived from this stem cell in PD patients should also express MV transcripts. Therefore, because the human hematopoietic stem cell has not been clearly identified or isolated in large numbers, we isolated RNA from highly purified erythroid and multipotential hematopoietic progenitors that are the precursors for erythroid, granulocyte, megakaryocyte and macrophages (CFU-GEMM), and used RT-PCR to determine if MV nucleocapsid transcripts were present. MV transcripts were detected in PD patients in early erythroid (BFU-E) and more primitive multipotential myeloid progenitors (CFU-GEMM). Nonhematopoietic stromal cells from PD patients did not express MV transcripts. The expression of MV transcripts in erythroid progenitors was further confirmed by in situ hybridization using antisense riboprobes to MV nucleocapsid transcripts. Thus, our findings suggest that the pluripotent HSCs may be a potential reservoir for the virus. We propose that when HSCs, which contain MV, divide they produce a second HSC that serves as a reservoir for the virus and also transmit the virus to their more differentiated progeny in the erythroid and myeloid lineages. This mechanism would permit a defective virus to persist in HSCs of PD patients for many years, since HSCs are usually in G0 phase, and then be transmitted to more differentiated cells. This model further suggests that a mature complete virus that affects cell function could only act pathogenetically in the

  20. Differences in expression, actions and cocaine regulation of two isoforms for the brain transcriptional regulator NAC1.

    PubMed

    Korutla, L; Wang, P J; Lewis, D M; Neustadter, J H; Stromberg, M F; Mackler, S A

    2002-01-01

    BTB/POZ proteins can influence the cell cycle and contribute to oncogenesis. Many family members are present in the mammalian CNS. Previous work demonstrated elevated NAC1 mRNA levels in the rat nucleus accumbens in response to cocaine. NAC1 acts like other BTB/POZ proteins that regulate transcription but is unusual because of the absence of identifiable DNA binding domains. cDNAs were isolated encoding two NAC1 isoforms differing by only 27 amino acids (the longer isoform contains 514 amino acids). The mRNAs for both isoforms were simultaneously expressed throughout the rat brain and peripheral tissues. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that the mRNA of the longer isoform was more abundant than the mRNA of the shorter isoform. Western blot analysis demonstrated a similar unequal distribution between the isoforms in the CNS. The longer isoform was the more abundant of the two NAC1 proteins and the ratio between them differed throughout the rat brain. The shorter isoform was not detected in most of the examined peripheral tissues, suggesting differences from the CNS in post-transcriptional processing. Both isoforms repressed transcription in H293T cells using a Gal4-luciferase reporter system. However, the shorter isoform did not repress transcription as effectively as the longer isoform. Transfection of different ratios for both isoforms, in order to replicate the relative amounts observed throughout the CNS, supported an interaction between the isoforms. The net effect on transcriptional repression was determined by the ratio of the two NAC1 isoforms. Each isoform exhibited the subnuclear localization that is characteristic of many BTB/POZ proteins. A rapid and transient increase in the level of the shorter isoform occurred in the nucleus accumbens 2 h following a single i.p. cocaine injection. We conclude that the two isoforms of NAC1 may differentially affect neuronal functions, including the regulation of

  1. Cloning, expression, and transcription analysis of L-arabinose isomerase gene from Mycobacterium smegmatis SMDU.

    PubMed

    Takata, Goro; Poonperm, Wayoon; Rao, Devendar; Souda, Akane; Nishizaki, Tomoe; Morimoto, Kenji; Izumori, Ken

    2007-12-01

    The L-arabinose metabolic gene cluster, araA, araB, araD, araG, araH and araR, encoding L-arabinose isomerase (L-AI) and its accessory proteins was cloned from Mycobacterium smegmatis SMDU and sequenced. The deduced amino acid sequence of araA displayed highest identity with that of Bacillus subtilis (52%). These six genes comprised the L-arabinose operon, and its genetic arrangement was similar to that of B. subtilis. The L-AI gene (araA), encoding a 501 amino acid protein with a calculated molecular mass of 54,888 Da, was expressed in Escherichia coli. The productivity and overall enzymatic properties of the recombinant L-AI were almost same as the authentic L-AI from M. smegmatis. Although the recombinant L-AI showed high substrate specificity, as did L-AI from other organisms, this enzyme catalyzed not only isomerization of L-arabinose-L-ribulose and D-galactose-D-tagatose but also isomerization of L-altrose-L-psicose and L-erythrulose-L-threose. In combination with L-AI from M. smegmatis, L-threose and L-altrose can be produced from cheap and abundant erythritol and D-fructose respectively, indicating that this enzyme has great potential for biological application in rare sugar production. Transcription analysis using various sugars revealed that this enzyme was significantly induced not only by L-arabinose and D-galactose but also by L-ribose, galactitol, L-ribulose, and L-talitol. This different result of transcription mediated by sugars from that of E. coli suggests that the transcriptional regulation of araA from M. smegmatis against sugar is loose compared with that from E. coli, and that it depends on the hydroxyl configuration at C2, C3 and C4 positions of sugars.

  2. A Molecular Profile of Cocaine Abuse Includes the Differential Expression of Genes that Regulate Transcription, Chromatin, and Dopamine Cell Phenotype

    PubMed Central

    Bannon, Michael J; Johnson, Magen M; Michelhaugh, Sharon K; Hartley, Zachary J; Halter, Steven D; David, James A; Kapatos, Gregory; Schmidt, Carl J

    2014-01-01

    Chronic drug abuse, craving, and relapse are thought to be linked to long-lasting changes in neural gene expression arising through transcriptional and chromatin-related mechanisms. The key contributions of midbrain dopamine (DA)-synthesizing neurons throughout the addiction process provide a compelling rationale for determining the drug-induced molecular changes that occur in these cells. Yet our understanding of these processes remains rudimentary. The postmortem human brain constitutes a unique resource that can be exploited to gain insights into the pathophysiology of complex disorders such as drug addiction. In this study, we analyzed the profiles of midbrain gene expression in chronic cocaine abusers and well-matched drug-free control subjects using microarray and quantitative PCR. A small number of genes exhibited robust differential expression; many of these are involved in the regulation of transcription, chromatin, or DA cell phenotype. Transcript abundances for approximately half of these differentially expressed genes were diagnostic for assigning subjects to the cocaine-abusing vs control cohort. Identification of a molecular signature associated with pathophysiological changes occurring in cocaine abusers' midbrains should contribute to the development of biomarkers and novel therapeutic targets for drug addiction. PMID:24642598

  3. 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

  4. A post-transcriptional mechanism regulates calpastatin expression in bovine skeletal muscle.

    PubMed

    Nattrass, G S; Cafe, L M; McIntyre, B L; Gardner, G E; McGilchrist, P; Robinson, D L; Wang, Y H; Pethick, D W; Greenwood, P L

    2014-02-01

    the abundance of an alternative polyadenylated variant of the CAST transcript, terminated at the proximal polyadenylation site, provides a unique insight into the potential involvement of a post-transcriptional regulatory mechanism which may influence protein expression levels in bovine skeletal muscle. PMID:24664555

  5. IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene.

    PubMed Central

    Miller, C P; McGehee, R E; Habener, J F

    1994-01-01

    We describe the cloning from a rat islet somatostatin-producing cell line of a 1.4 kb cDNA encoding a new homeoprotein, IDX-1 (islet/duodenum homeobox-1), with close sequence similarity to the Drosophila melanogaster homeobox protein Antennapedia (Antp) and the Xenopus laevis endoderm-specific homeoprotein XlHbox8. Analyses of IDX-1 mRNA and protein in rat tissues show that IDX-1 is expressed in pancreatic islets and ducts and in the duodenum. In electrophoretic mobility shift assays IDX-1 binds to three sites in the 5' flanking region of the rat somatostatin gene. In co-transfection experiments IDX-1 transactivates reporter constructs containing somatostatin promoter sequences, and mutation of the IDX-1 binding sites attenuates transactivation. Reverse transcription-polymerase chain reaction of islet RNA using degenerate amplimers for mRNAs encoding homeoproteins indicates that IDX-1 is the most abundant of 12 different Antp-like homeodomain mRNAs expressed in adult rat islets. The pattern of expression, relative abundance and transcriptional regulatory activity suggests that IDX-1 may be involved in the regulation of islet hormone genes and in cellular differentiation in the endocrine pancreas and the duodenum. Images PMID:7907546

  6. Wuchereria bancrofti 20/22 a homologue of abundant larval transcript L3 stage filarial antigen: molecular and immunological characterization.

    PubMed

    Aparnaa, Ramanathan; Mahalakshmi, Natarajan; Harini, Asai; Jeyaprita, Parasurama Jawaharlal; Anugraha, Gandhirajan; Amdare, N P; Khatri, V K; Reddy, M V R; Kaliraj, P

    2014-10-01

    The chromadorea abundant larval transcript (ALT) family of proteins contains ALT one of the most studied putative vaccine candidate in experimental filariasis. This study reports the characterization of Wuchereria bancrofti 20/22 (Wb20/22) as a member of chromadorea, the ALT family of proteins from the L3 stage of W. bancrofti. The high reactivity with serum from the endemic normal (EN) population suggests that Wb20/22 could be a target of elicit protective immunity. The glutamic acid-rich region of Wb20/22 was predicted to harbour the longest linear B-cell epitope by insilico prediction tools. The significance of this region was revealed by studying the mutant form of Wb20/22, without acidic domain (WOAD) which was cloned, and the immune response was compared with Wb20/22. The signal sequence of Wb20/22 was also an immunodominant region, and mutant construct without signal sequence (WOSS) was cloned and characterized. The peak antibody titre elicited by WOAD was higher than Wb20/22 or WOSS, which pointed to the immunomodulatory role of glutamic acid-rich region. Wb20/22 elicited very high levels of IL-10 and diminished levels of IL-4 and IL-5 which could be the reason for low antibody titre. The prophylactic efficacy of WOAD conferred protection (62·26%) which was higher than Wb20/22 (49·82%) and WOSS (54·78%). PMID:24888320

  7. Expression and regulation of the Msx1 natural antisense transcript during development.

    PubMed

    Coudert, Amélie E; Pibouin, Laurence; Vi-Fane, Brigitte; Thomas, Bethan L; Macdougall, Mary; Choudhury, Anuradha; Robert, Benoît; Sharpe, Paul T; Berdal, Ariane; Lezot, Frédéric

    2005-01-01

    Bidirectional transcription, leading to the expression of an antisense (AS) RNA partially complementary to the protein coding sense (S) RNA, is an emerging subject in mammals and has been associated with various processes such as RNA interference, imprinting and transcription inhibition. Homeobox genes do not escape this bidirectional transcription, raising the possibility that such AS transcription occurs during embryonic development and may be involved in the complexity of regulation of homeobox gene expression. According to the importance of the Msx1 homeobox gene function in craniofacial development, especially in tooth development, the expression and regulation of its recently identified AS transcripts were investigated in vivo in mouse from E9.5 embryo to newborn, and compared with the S transcript and the encoded protein expression pattern and regulation. The spatial and temporal expression patterns of S, AS transcripts and protein are consistent with a role of AS RNA in the regulation of Msx1 expression in timely controlled developmental sites. Epithelial-mesenchymal interactions were shown to control the spatial organization of S and also AS RNA expression during early patterning of incisors and molars in the odontogenic mesenchyme. To conclude, this study clearly identifies the Msx1 AS RNA involvement during tooth development and evidences a new degree of complexity in craniofacial developmental biology: the implication of endogenous AS RNAs.

  8. Members of a large retroposon family are determinants of post-transcriptional gene expression in Leishmania.

    PubMed

    Bringaud, Frédéric; Müller, Michaela; Cerqueira, Gustavo Coutinho; Smith, Martin; Rochette, Annie; El-Sayed, Najib M A; Papadopoulou, Barbara; Ghedin, Elodie

    2007-09-01

    Trypanosomatids are unicellular protists that include the human pathogens Leishmania spp. (leishmaniasis), Trypanosoma brucei (sleeping sickness), and Trypanosoma cruzi (Chagas disease). Analysis of their recently completed genomes confirmed the presence of non-long-terminal repeat retrotransposons, also called retroposons. Using the 79-bp signature sequence common to all trypanosomatid retroposons as bait, we identified in the Leishmania major genome two new large families of small elements--LmSIDER1 (785 copies) and LmSIDER2 (1,073 copies)--that fulfill all the characteristics of extinct trypanosomatid retroposons. LmSIDERs are approximately 70 times more abundant in L. major compared to T. brucei and are found almost exclusively within the 3'-untranslated regions (3'UTRs) of L. major mRNAs. We provide experimental evidence that LmSIDER2 act as mRNA instability elements and that LmSIDER2-containing mRNAs are generally expressed at lower levels compared to the non-LmSIDER2 mRNAs. The considerable expansion of LmSIDERs within 3'UTRs in an organism lacking transcriptional control and their role in regulating mRNA stability indicate that Leishmania have probably recycled these short retroposons to globally modulate the expression of a number of genes. To our knowledge, this is the first example in eukaryotes of the domestication and expansion of a family of mobile elements that have evolved to fulfill a critical cellular function.

  9. Adipocyte expression of PU.1 transcription factor causes insulin resistance through upregulation of inflammatory cytokine gene expression and ROS production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have reported previously that ETS family transcription factor PU.1 is expressed in mature adipocytes of white adipose tissue. PU.1 expression is increased greatly in mouse models of genetic or diet-induced obesity. Here, we show that PU.1 expression is increased only in visceral but not subcutane...

  10. The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion

    PubMed Central

    Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W.; Kulozik, Andreas E.

    2016-01-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. PMID:27407180

  11. Construction of co-expression network based on natural expression variation of xylogenesis-related transcripts in Eucalyptus tereticornis.

    PubMed

    Dharanishanthi, Veeramuthu; Dasgupta, Modhumita Ghosh

    2016-10-01

    Natural genetic variation is randomly distributed and gene expression patterns vary widely in natural populations. These variations are an effect of multifactorial genetic perturbations resulting in different phenotypes. Genome-wide analysis can be used to comprehend the genetic basis governing this naturally occurring developmental variation. Secondary growth is a highly complex trait and systems genetics models are presently being applied to understand the molecular architecture of wood formation. In the present study, the natural variation in expression patterns of 18,987 transcripts expressed in the developing xylem tissues were documented across four phenotypes of Eucalyptus tereticornis with distinct holocellulose/klason lignin content. The differentially expressed genes across all the phenotypes were used to construct co-expression networks and sub-network 2 with 380 nodes and 17,711 edges was determined as the network of relevance, including 30 major cell wall biogenesis related transcripts with 2394 interactions and 10 families of transcription factors with 3360 interactions. EYE [EMBRYO YELLOW] was identified as major hub transcript with 173 degrees which interacted with known cell wall biogenesis genes. K-mean clustering was also performed for differentially expressed transcripts and two clusters discriminated the phenotypes based on their holocellulose/klason lignin content. The cluster based networks were enriched with GOs related to cell wall biogenesis and sugar metabolism. The networks developed in the present study enabled identification of critical regulators and novel transcripts whose expression variation could presumably govern the phenotypic variation in wood properties across E. tereticornis. PMID:27465117

  12. Identification of new SOX2OT transcript variants highly expressed in human cancer cell lines and down regulated in stem cell differentiation.

    PubMed

    Saghaeian Jazi, Marie; Samaei, Nader Mansour; Ghanei, Mostafa; Shadmehr, Mohammad Behgam; Mowla, Seyed Javad

    2016-02-01

    Long non-coding RNAs are manifested as a new paradigm of molecular effectors in a wide range of human diseases. Human SOX2 overlapping transcript (SOX2OT) gene can generate six lncRNA transcript variants which are functionally assumed to be correlated with cellular differentiation and carcinogenesis. However, the circumstances determining expressional and functional differences between SOX2OT transcript variants remain to be explored. Here, we studied the expression of all SOX2OT transcript variants specifically in five human cancer cell lines by real-time RT-PCR. Changes of the new SOX2OT transcript variants expression were measured during the NT2 teratocarcinoma cell line neuronal-like differentiation and were compared to pluripotency regulators, SOX2 and OCT4A gene expressions. Surprisingly, we identified two new SOX2OT transcripts, named SOX2OT-7, SOX2OT-8 which lack exon 8. We discovered that beside active proximal and distal SOX2OT promoters, different cancer cell lines express high levels of some SOX2OT transcript variants differentially by alternative splicing. Significantly, both SOX2OT-7 and SOX2OT-8 are highly expressed in human cancer cell lines coinciding with SOX2, one of the pluripotency regulators. Our results revealed that SOX2OT-7 is almost the most abundant form of SOX2OT transcript variants in the examined cancer cell lines particularly in NT2 teratocarcinoma cell line where its expression falls upon neuronal-like differentiation similar to SOX2 and OCT4A. We suggest that at least some of SOX2OT transcripts are significantly associated with cancer and stem cell related pathways.

  13. Exogenous GDF9 but not Activin A, BMP15 or TGFβ alters tight junction protein transcript abundance in zebrafish ovarian follicles.

    PubMed

    Clelland, Eric S; Kelly, Scott P

    2011-04-01

    The tight junction (TJ) complex plays an important role in regulating paracellular permeability and provides mechanical stability in vertebrate epithelia and endothelia. In zebrafish ovarian follicles, TJ complexes in the follicular envelope degenerate as the follicles develop towards maturation. In the current study, transcript abundance of claudins (cldn d, g, h, 1, and 12) and occludins (ocln, and ocln b) were assessed in mid-vitellogenic follicles in response to treatment with exogenous growth factors that are reported to be involved in zebrafish follicle development (i.e. Activin A, BMP15, GDF9 and TGFβ). Exogenous GDF9 reduced the transcript abundance of cldn g, ocln and ocln b in mid-vitellogenic follicles, whereas Activin A, BMP15, and TGFβ had no effect. Subsequent studies with GDF9 revealed that this factor did not alter TJ protein transcript abundance in pre-vitellogenic follicles but did increase the abundance of ocln b in fully grown (maturing) follicles. GDF9 was also seen to increase the abundance of StAR mRNA in all but primary stage follicles. These data suggest a role for GDF9 in the regulation of TJ integrity in zebrafish ovarian follicles, perhaps in the facilitation of ovulation, and support a previously postulated role for GDF9 in zebrafish ovarian follicle development. In addition, data also support the idea that endocrine factors play an important role in the regulation of TJ proteins during ovarian follicle development.

  14. Increased expression of the Hutchinson-Gilford progeria syndrome truncated lamin A transcript during cell aging.

    PubMed

    Rodriguez, Sofia; Coppedè, Fabio; Sagelius, Hanna; Eriksson, Maria

    2009-07-01

    Most cases of the segmental progeroid syndrome, Hutchinson-Gilford progeria syndrome (HGPS), are caused by a de novo dominant mutation within a single codon of the LMNA gene. This mutation leads to the increased usage of an internal splice site that generates an alternative lamin A transcript with an internal deletion of 150 nucleotides, called lamin A Delta 150. The LMNA gene encodes two major proteins of the inner nuclear lamina, lamins A and C, but not much is known about their expression levels. Determination of the overall expression levels of the LMNA gene transcripts is an important step to further the understanding of the HGPS. In this study, we have performed absolute quantification of the lamins A, C and A Delta 150 transcripts in primary dermal fibroblasts from HGPS patients and unaffected age-matched and parent controls. We show that the lamin A Delta 150 transcript is present in unaffected controls but its expression is >160-fold lower than that in samples from HGPS patients. Analysis of transcript expression during in vitro aging shows that although the levels of lamin A and lamin C transcripts remain unchanged, the lamin A Delta 150 transcript increases in late passage cells from HGPS patients and parental controls. This study provides a new method for LMNA transcript analysis and insights into the expression of the LMNA gene in HGPS and normal cells.

  15. Transcript-Specific Expression Profiles Derived from Sequence-Based Analysis of Standard Microarrays

    PubMed Central

    Moll, Anton G.; Lindenmeyer, Maja T.; Kretzler, Matthias; Nelson, Peter J.; Zimmer, Ralf; Cohen, Clemens D.

    2009-01-01

    Background Alternative mRNA processing mechanisms lead to multiple transcripts (i.e. splice isoforms) of a given gene which may have distinct biological functions. Microarrays like Affymetrix GeneChips measure mRNA expression of genes using sets of nucleotide probes. Until recently probe sets were not designed for transcript specificity. Nevertheless, the re-analysis of established microarray data using newly defined transcript-specific probe sets may provide information about expression levels of specific transcripts. Methodology/Principal Findings In the present study alignment of probe sequences of the Affymetrix microarray HG-U133A with Ensembl transcript sequences was performed to define transcript-specific probe sets. Out of a total of 247,965 perfect match probes, 95,008 were designated “transcript-specific”, i.e. showing complete sequence alignment, no cross-hybridization, and transcript-, not only gene-specificity. These probes were grouped into 7,941 transcript-specific probe sets and 15,619 gene-specific probe sets, respectively. The former were used to differentiate 445 alternative transcripts of 215 genes. For selected transcripts, predicted by this analysis to be differentially expressed in the human kidney, confirmatory real-time RT-PCR experiments were performed. First, the expression of two specific transcripts of the genes PPM1A (PP2CA_HUMAN and P35813) and PLG (PLMN_HUMAN and Q5TEH5) in human kidneys was determined by the transcript-specific array analysis and confirmed by real-time RT-PCR. Secondly, disease-specific differential expression of single transcripts of PLG and ABCA1 (ABCA1_HUMAN and Q5VYS0_HUMAN) was computed from the available array data sets and confirmed by transcript-specific real-time RT-PCR. Conclusions Transcript-specific analysis of microarray experiments can be employed to study gene-regulation on the transcript level using conventional microarray data. In this study, predictions based on sufficient probe set size and

  16. The transcription factor titration effect dictates level of gene expression.

    PubMed

    Brewster, Robert C; Weinert, Franz M; Garcia, Hernan G; Song, Dan; Rydenfelt, Mattias; Phillips, Rob

    2014-03-13

    Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number-in multiple identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally, we use these experiments to dynamically measure plasmid copy number through the cell cycle.

  17. The Transcription Factor Titration Effect Dictates Level of Gene Expression

    PubMed Central

    Brewster, Robert C.; Weinert, Franz M.; Garcia, Hernan G.; Song, Dan; Rydenfelt, Mattias; Phillips, Rob

    2014-01-01

    Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number; in multiple, identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally we use these experiments to dynamically measure plasmid copy number through the cell cycle. PMID:24612990

  18. Green tea polyphenols added to IVM and IVC media affect transcript abundance, apoptosis, and pregnancy rates in bovine embryos.

    PubMed

    Wang, Zhengguang; Fu, Chunquan; Yu, Songdong

    2013-01-01

    Three experiments were conducted to examine the effects of green tea polyphenols (GTP) during IVM and IVC on apoptosis and relative transcript abundance (RA) of three genes controlling antioxidant enzymes, as well as subsequent pregnancy rates. In experiment 1, oocytes were matured in the presence of 0, 10, 15, or 25 μM GTP for 24 hours. The GTP dose applied to IVM medium was followed by the same dose supplemented to IVC medium, so oocytes and embryos of a given group were cultured in similar conditions. This resulted in a total of four groups (three experimental groups and the control). After IVF, presumptive zygotes were cultured in medium containing 0 to 25 μM GTP for 8 days. The addition of 15 μM GTP during IVM and IVC increased RA of SOD1, CAT, and GPX genes in blastocysts compared with the control (P < 0.05). Increase in GTP doses from 15 to 25 μM did not further increase the transcript level. In experiment 2, effects of GTP doses on apoptosis were investigated in bovine blastocysts. Two of the applied GTP doses (10 and 15 μM) decreased the apoptotic index (AI) in blastocysts (7.4% and 6.2% respectively) compared with the control (9.3%; P < 0.05). However, the highest GTP dose used (25 μM) caused an increase in AI compared with a dose of 15 μM (P < 0.05). Considering the results of experiment 1 and 2, the effects of 15 μM GTP treatment during IVM and IVC on pregnancy rate was evaluated after embryo transfer in experiment 3. Cows receiving embryos treated with 15 μM GTP had higher pregnancy rates on Day 30 (34.8% vs. 28.6%) and Day 60 (34.8% vs. 23.9%) than those receiving control embryos (P < 0.05). In conclusion, addition of 15 μM GTP during IVM and IVC improved pregnancy rates; this improvement seemed to be associated with the increase of RA of antioxidant enzyme genes and the decrease in AI in bovine blastocysts.

  19. Identification of brain transcriptional variation reproduced in peripheral blood: an approach for mapping brain expression traits

    PubMed Central

    Jasinska, Anna J.; Service, Susan; Choi, Oi-wa; DeYoung, Joseph; Grujic, Olivera; Kong, Sit-yee; Jorgensen, Matthew J.; Bailey, Julia; Breidenthal, Sherry; Fairbanks, Lynn A.; Woods, Roger P.; Jentsch, J. David; Freimer, Nelson B.

    2009-01-01

    Genome-wide gene expression studies may provide substantial insight into gene activities and biological pathways differing between tissues and individuals. We investigated such gene expression variation by analyzing expression profiles in brain tissues derived from eight different brain regions and from blood in 12 monkeys from a biomedically important non-human primate model, the vervet (Chlorocebus aethiops sabaeus). We characterized brain regional differences in gene expression, focusing on transcripts for which inter-individual variation of expression in brain correlates well with variation in blood from the same individuals. Using stringent criteria, we identified 29 transcripts whose expression is measurable, stable, replicable, variable between individuals, relevant to brain function and heritable. Polymorphisms identified in probe regions could, in a minority of transcripts, confound the interpretation of the observed inter-individual variation. The high heritability of levels of these transcripts in a large vervet pedigree validated our approach of focusing on transcripts that showed higher inter-individual compared with intra-individual variation. These selected transcripts are candidate expression Quantitative Trait Loci, differentially regulating transcript levels in the brain among individuals. Given the high degree of conservation of tissue expression profiles between vervets and humans, our findings may facilitate the understanding of regional and individual transcriptional variation and its genetic mechanisms in humans. The approach employed here—utilizing higher quality tissue and more precise dissection of brain regions than is usually possible in humans—may therefore provide a powerful means to investigate variation in gene expression relevant to complex brain related traits, including human neuropsychiatric diseases. PMID:19692348

  20. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    SciTech Connect

    Rumi, Mohammad; Ishihara, Shunji . E-mail: si360405@med.shimane-u.ac.jp; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-13

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor {alpha}-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.

  1. Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression.

    EPA Science Inventory

    Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression Exposure to many drugs and environmentally-relevant chemicals can cause adverse outcomes. These adverse outcomes, such as cancer, have been linked to mol...

  2. EXPRESSION PROFILING OF FIVE RAT STRAINS REVEAL TRANSCRIPTIONAL MODES IN THE ANTIGEN PROCESSING PATHWAY

    EPA Science Inventory

    Comparative gene expression profiling of rat strains with genetic predisposition to diverse cardiovascular diseases can help decode the transcriptional program that governs cellular behavior. We hypothesized that co-transcribed, intra-pathway, functionally coherent genes can be r...

  3. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae.

    PubMed

    Rantasalo, Anssi; Czeizler, Elena; Virtanen, Riitta; Rousu, Juho; Lähdesmäki, Harri; Penttilä, Merja; Jäntti, Jussi; Mojzita, Dominik

    2016-01-01

    This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications. PMID:26901642

  4. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae

    PubMed Central

    Rantasalo, Anssi; Czeizler, Elena; Virtanen, Riitta; Rousu, Juho; Lähdesmäki, Harri; Penttilä, Merja

    2016-01-01

    This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications. PMID:26901642

  5. Ectopic expression of MYB46 identifies transcriptional regulatory genes involved in secondary wall biosynthesis in Arabidopsis.

    PubMed

    Ko, Jae-Heung; Kim, Won-Chan; Han, Kyung-Hwan

    2009-11-01

    MYB46 functions as a transcriptional switch that turns on the genes necessary for secondary wall biosynthesis. Elucidating the transcriptional regulatory network immediately downstream of MYB46 is crucial to our understanding of the molecular and biochemical processes involved in the biosynthesis and deposition of secondary walls in plants. To gain insights into MYB46-mediated transcriptional regulation, we first established an inducible secondary wall thickening system in Arabidopsis by expressing MYB46 under the control of dexamethasone-inducible promoter. Then, we used an ATH1 GeneChip microarray and Illumina digital gene expression system to obtain a series of transcriptome profiles with regard to the induction of secondary wall development. These analyses allowed us to identify a group of transcription factors whose expression coincided with or preceded the induction of secondary wall biosynthetic genes. A transient transcriptional activation assay was used to confirm the hierarchical relationships among the transcription factors in the network. The in vivo assay showed that MYB46 transcriptionally activates downstream target transcription factors, three of which (AtC3H14, MYB52 and MYB63) were shown to be able to activate secondary wall biosynthesis genes. AtC3H14 activated the transcription of all of the secondary wall biosynthesis genes tested, suggesting that AtC3H14 may be another master regulator of secondary wall biosynthesis. The transcription factors identified here may include direct activators of secondary wall biosynthesis genes. The present study discovered novel hierarchical relationships among the transcription factors involved in the transcriptional regulation of secondary wall biosynthesis, and generated several testable hypotheses.

  6. Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p

    PubMed Central

    Nguyen, Ha T.; Jia, Wei; Beedle, Aaron M.; Kennedy, Eileen J.; Murph, Mandi M.

    2015-01-01

    Although microRNAs (miRNAs) are small, non-protein-coding entities, they have important roles in post-transcriptional regulation of most of the human genome. These small entities generate fine-tuning adjustments in the expression of mRNA, which can mildly or massively affect the abundance of proteins. Previously, we found that the expression of miR-30c-2-3p is induced by lysophosphatidic acid and has an important role in the regulation of cell proliferation in ovarian cancer cells. The goal here is to confirm that ATF3 mRNA is a target of miR-30c-2-3p silencing, thereby further establishing the functional role of miR-30c-2-3p. Using a combination of bioinformatics, qRT-PCR, immunoblotting and luciferase assays, we uncovered a regulatory pathway between miR-30c-2-3p and the expression of the transcription factor, ATF3. Lysophosphatidic acids triggers the expression of both miR-30c-2-3p and ATF3, which peak at 1 h and are absent 8 h post stimulation in SKOV-3 and OVCAR-3 serous ovarian cancer cells. The 3´-untranslated region (3´-UTR) of ATF3 was a predicted, putative target for miR-30c-2-3p, which we confirmed as a bona-fide interaction using a luciferase reporter assay. Specific mutations introduced into the predicted site of interaction between miR-30c-2-3p and the 3´-UTR of ATF3 alleviated the suppression of the luciferase signal. Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after lysophosphatidic acid stimulation. Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by lysophosphatidic acid, subsequently miR-30c-2-3p negatively regulates the expression of ATF3 through post-transcriptional silencing, which prevents further ATF3-related outcomes as a consequence of lysophosphatidic acid signaling. PMID:26418018

  7. Dietary phosphorus transcriptionally regulates 25-hydroxyvitamin D-1alpha-hydroxylase gene expression in the proximal renal tubule.

    PubMed

    Zhang, Martin Y H; Wang, Xuemei; Wang, Jonathan T; Compagnone, Nathalie A; Mellon, Synthia H; Olson, Jean L; Tenenhouse, Harriet S; Miller, Walter L; Portale, Anthony A

    2002-02-01

    Synthesis of the hormone 1,25-dihydroxyvitamin D, the biologically active form of vitamin D, occurs in the kidney and is catalyzed by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). We sought to characterize the effects of changes in dietary phosphorus on the kinetics of renal mitochondrial 1alpha-hydroxylase activity and the renal expression of P450c1alpha and P450c24 mRNA, to localize the nephron segments involved in such regulation, and to determine whether transcriptional mechanisms are involved. In intact mice, restriction of dietary phosphorus induced rapid, sustained, approximately 6- to 8-fold increases in renal mitochondrial 1alpha-hydroxylase activity and renal P450c1alpha mRNA abundance. Immunohistochemical analysis of renal sections from mice fed the control diet revealed the expression of 1alpha-hydroxylase protein in the proximal convoluted and straight tubules, epithelial cells of Bowman's capsule, thick ascending limb of Henle's loop, distal tubule, and collecting duct. In mice fed a phosphorus-restricted diet, immunoreactivity was significantly increased in the proximal convoluted and proximal straight tubules and epithelial cells of Bowman's capsule, but not in the distal nephron. Dietary phosphorus restriction induced a 2-fold increase in P450c1alpha gene transcription, as shown by nuclear run-on assays. Thus, the increase in renal synthesis of 1,25-dihydroxyvitamin D induced in normal mice by restricting dietary phosphorus can be attributed to an increase in the renal abundance of P450c1alpha mRNA and protein. The increase in P450c1alpha gene expression, which occurs exclusively in the proximal renal tubule, is due at least in part to increased transcription of the P450c1alpha gene.

  8. Quercetin represses apolipoprotein B expression by inhibiting the transcriptional activity of C/EBPβ.

    PubMed

    Shimizu, Makoto; Li, Juan; Inoue, Jun; Sato, Ryuichiro

    2015-01-01

    Quercetin is one of the most abundant polyphenolic flavonoids found in fruits and vegetables and has anti-oxidative and anti-obesity effects. Because the small intestine is a major absorptive organ of dietary nutrients, it is likely that highly concentrated food constituents, including polyphenols, are present in the small intestinal epithelial cells, suggesting that food factors may have a profound effect in this tissue. To identify novel targets of quercetin in the intestinal enterocytes, mRNA profiling using human intestinal epithelial Caco-2 cells was performed. We found that mRNA levels of some apolipoproteins, particularly apolipoprotein B (apoB), are downregulated in the presence of quercetin. On the exposure of Caco-2 cells to quercetin, both mRNA and protein levels of apoB were decreased. Promoter analysis of the human apoB revealed that quercetin response element is localized at the 5'-proximal promoter region, which contains a conserved CCAAT enhancer-binding protein (C/EBP)-response element. We found that quercetin reduces the promoter activity of apoB, driven by the enforced expression of C/EBPβ. Quercetin had no effect on either mRNA or protein levels of C/EBPβ. In contrast, we found that quercetin inhibits the transcriptional activity of C/EBPβ but not its recruitment to the apoB promoter. On the exposure of Caco-2 cells to quercetin 3-O-glucuronide, which is in a cell-impermeable form, no notable change in apoB mRNA was observed, suggesting an intracellular action of quercetin. In vitro interaction experiments using quercetin-conjugated beads revealed that quercetin binds to C/EBPβ. Our results describe a novel regulatory mechanism of transcription of apolipoprotein genes by quercetin in the intestinal enterocytes. PMID:25875015

  9. SNAP-25 is abundantly expressed in enteric neuronal networks and upregulated by the neurotrophic factor GDNF.

    PubMed

    Barrenschee, M; Böttner, M; Harde, J; Lange, C; Cossais, F; Ebsen, M; Vogel, I; Wedel, T

    2015-06-01

    Control of intestinal motility requires an intact enteric neurotransmission. Synaptosomal-associated protein 25 (SNAP-25) is an essential component of the synaptic vesicle fusion machinery. The aim of the study was to investigate the localization and expression of SNAP-25 in the human intestine and cultured enteric neurons and to assess its regulation by the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF). SNAP-25 expression and distribution were analyzed in GDNF-stimulated enteric nerve cell cultures, and synaptic vesicles were evaluated by scanning and transmission electron microscopy. Human colonic specimens were processed for site-specific SNAP-25 gene expression analysis and SNAP-25 immunohistochemistry including dual-labeling with the pan-neuronal marker PGP 9.5. Additionally, gene expression levels and distributional patterns of SNAP-25 were analyzed in colonic specimens of patients with diverticular disease (DD). GDNF-treated enteric nerve cell cultures showed abundant expression of SNAP-25 and exhibited granular staining corresponding to synaptic vesicles. SNAP-25 gene expression was detected in all colonic layers and isolated myenteric ganglia. SNAP-25 co-localized with PGP 9.5 in submucosal and myenteric ganglia and intramuscular nerve fibers. In patients with DD, both SNAP-25 mRNA expression and immunoreactive profiles were decreased compared to controls. GDNF-induced growth and differentiation of cultured enteric neurons is paralleled by increased expression of SNAP-25 and formation of synaptic vesicles reflecting enhanced synaptogenesis. The expression of SNAP-25 within the human enteric nervous system and its downregulation in DD suggest an essential role in enteric neurotransmission and render SNAP-25 as a marker for impaired synaptic plasticity in enteric neuropathies underlying intestinal motility disorders.

  10. Microbial Gene Abundance and Expression Patterns across a River to Ocean Salinity Gradient.

    PubMed

    Fortunato, Caroline S; Crump, Byron C

    2015-01-01

    Microbial communities mediate the biogeochemical cycles that drive ecosystems, and it is important to understand how these communities are affected by changing environmental conditions, especially in complex coastal zones. As fresh and marine waters mix in estuaries and river plumes, the salinity, temperature, and nutrient gradients that are generated strongly influence bacterioplankton community structure, yet, a parallel change in functional diversity has not been described. Metagenomic and metatranscriptomic analyses were conducted on five water samples spanning the salinity gradient of the Columbia River coastal margin, including river, estuary, plume, and ocean, in August 2010. Samples were pre-filtered through 3 μm filters and collected on 0.2 μm filters, thus results were focused on changes among free-living microbial communities. Results from metagenomic 16S rRNA sequences showed taxonomically distinct bacterial communities in river, estuary, and coastal ocean. Despite the strong salinity gradient observed over sampling locations (0 to 33), the functional gene profiles in the metagenomes were very similar from river to ocean with an average similarity of 82%. The metatranscriptomes, however, had an average similarity of 31%. Although differences were few among the metagenomes, we observed a change from river to ocean in the abundance of genes encoding for catabolic pathways, osmoregulators, and metal transporters. Additionally, genes specifying both bacterial oxygenic and anoxygenic photosynthesis were abundant and expressed in the estuary and plume. Denitrification genes were found throughout the Columbia River coastal margin, and most highly expressed in the estuary. Across a river to ocean gradient, the free-living microbial community followed three different patterns of diversity: 1) the taxonomy of the community changed strongly with salinity, 2) metabolic potential was highly similar across samples, with few differences in functional gene abundance

  11. Microbial Gene Abundance and Expression Patterns across a River to Ocean Salinity Gradient

    PubMed Central

    Fortunato, Caroline S.; Crump, Byron C.

    2015-01-01

    Microbial communities mediate the biogeochemical cycles that drive ecosystems, and it is important to understand how these communities are affected by changing environmental conditions, especially in complex coastal zones. As fresh and marine waters mix in estuaries and river plumes, the salinity, temperature, and nutrient gradients that are generated strongly influence bacterioplankton community structure, yet, a parallel change in functional diversity has not been described. Metagenomic and metatranscriptomic analyses were conducted on five water samples spanning the salinity gradient of the Columbia River coastal margin, including river, estuary, plume, and ocean, in August 2010. Samples were pre-filtered through 3 μm filters and collected on 0.2 μm filters, thus results were focused on changes among free-living microbial communities. Results from metagenomic 16S rRNA sequences showed taxonomically distinct bacterial communities in river, estuary, and coastal ocean. Despite the strong salinity gradient observed over sampling locations (0 to 33), the functional gene profiles in the metagenomes were very similar from river to ocean with an average similarity of 82%. The metatranscriptomes, however, had an average similarity of 31%. Although differences were few among the metagenomes, we observed a change from river to ocean in the abundance of genes encoding for catabolic pathways, osmoregulators, and metal transporters. Additionally, genes specifying both bacterial oxygenic and anoxygenic photosynthesis were abundant and expressed in the estuary and plume. Denitrification genes were found throughout the Columbia River coastal margin, and most highly expressed in the estuary. Across a river to ocean gradient, the free-living microbial community followed three different patterns of diversity: 1) the taxonomy of the community changed strongly with salinity, 2) metabolic potential was highly similar across samples, with few differences in functional gene abundance

  12. Microbial Gene Abundance and Expression Patterns across a River to Ocean Salinity Gradient.

    PubMed

    Fortunato, Caroline S; Crump, Byron C

    2015-01-01

    Microbial communities mediate the biogeochemical cycles that drive ecosystems, and it is important to understand how these communities are affected by changing environmental conditions, especially in complex coastal zones. As fresh and marine waters mix in estuaries and river plumes, the salinity, temperature, and nutrient gradients that are generated strongly influence bacterioplankton community structure, yet, a parallel change in functional diversity has not been described. Metagenomic and metatranscriptomic analyses were conducted on five water samples spanning the salinity gradient of the Columbia River coastal margin, including river, estuary, plume, and ocean, in August 2010. Samples were pre-filtered through 3 μm filters and collected on 0.2 μm filters, thus results were focused on changes among free-living microbial communities. Results from metagenomic 16S rRNA sequences showed taxonomically distinct bacterial communities in river, estuary, and coastal ocean. Despite the strong salinity gradient observed over sampling locations (0 to 33), the functional gene profiles in the metagenomes were very similar from river to ocean with an average similarity of 82%. The metatranscriptomes, however, had an average similarity of 31%. Although differences were few among the metagenomes, we observed a change from river to ocean in the abundance of genes encoding for catabolic pathways, osmoregulators, and metal transporters. Additionally, genes specifying both bacterial oxygenic and anoxygenic photosynthesis were abundant and expressed in the estuary and plume. Denitrification genes were found throughout the Columbia River coastal margin, and most highly expressed in the estuary. Across a river to ocean gradient, the free-living microbial community followed three different patterns of diversity: 1) the taxonomy of the community changed strongly with salinity, 2) metabolic potential was highly similar across samples, with few differences in functional gene abundance

  13. Diversity, abundance, and consistency of microbial oxygenase expression and biodegradation in a shallow contaminated aquifer

    SciTech Connect

    Yagi, J.M.; Madsen, E.L.

    2009-10-15

    The diversity of Rieske dioxygenase genes and short-term temporal variability in the abundance of two selected dioxygenase gene sequences were examined in a naphthalene-rich, coal tar waste-contaminated subsurface study site. Using a previously published PCR-based approach (S. M. Ni Chadhain, R. S. Norman, K. V. Pesce, J. J. Kukor, and G. J. Zylstra, Appl. Environ. Microbiol. 72: 4078-4087, 2006) a broad suite of genes was detected, ranging from dioxygenase sequences associated with Rhodococcus and Sphingomonas to 32 previously uncharacterized Rieske gene sequence clone groups. The nag genes appeared frequently (20% of the total) in two groundwater monitoring wells characterized by low (similar to 10{sup 2} ppb; similar to 1 {mu} M) ambient concentrations of naphthalene. A quantitative competitive PCR assay was used to show that abundances of nag genes (and archetypal nah genes) fluctuated substantially over a 9-month period. To contrast short-term variation with long-term community stability, in situ community gene expression (dioxygenase mRNA) and biodegradation potential (community metabolism of naphthalene in microcosms) were compared to measurements from 6 years earlier. cDNA sequences amplified from total RNA extracts revealed that nah- and nag-type genes were expressed in situ, corresponding well with structural gene abundances. Despite evidence for short-term (9-month) shifts in dioxygenase gene copy number, agreement in field gene expression (dioxygenase mRNA) and biodegradation potential was observed in comparisons to equivalent assays performed 6 years earlier. Thus, stability in community biodegradation characteristics at the hemidecadal time frame has been documented for these subsurface microbial communities.

  14. Evaluation of data-dependent versus targeted shotgun proteomic approaches for monitoring transcription factor expression in breast cancer.

    PubMed

    Sandhu, Charanjit; Hewel, Johannes A; Badis, Gwenael; Talukder, Shaheynoor; Liu, Jian; Hughes, Timothy R; Emili, Andrew

    2008-04-01

    In breast cancer, there is a significant degree of molecular diversity among tumors. Multiple perturbations in signal transduction pathways impinge on transcriptional networks that in turn dictate malignant transformation and metastatic progression. Detailed knowledge of the sequence-specific transcription factors that become activated or repressed within a tumor and comparison of their relative levels of expression in cancer versus normal tissue should therefore provide insight into disease mechanisms, improving patient stratification and facilitating personalized treatment. While high-throughput tandem mass spectrometry methods for global proteome profiling have been developed, existing approaches have limited sensitivity and are often unable to detect low-abundance transcription factors in a complex biological specimen like a biopsy or tumor cell extract. To this end, we have undertaken a systematic comparative evaluation of three MS/MS methods for the ability to detect reference transcription factors spiked in known amounts into a cell-free breast cancer nuclear extract: Data-Dependent Acquisition (DDA), wherein precursor ion intensity dictates selection for fragmentation; Targeted Peptide Monitoring (TPM), a directed approach using successive isolation and fragmentation of predefined m/ z ratios; and Multiple Reaction Monitoring (MRM), in which specific precursor ion to product ion transitions are selectively monitored. Through a series of controlled, parallel benchmarking experiments, we have determined the relative figures-of-merit of each approach, and have established that prior knowledge of signature proteotypic peptides markedly improves overall detection sensitivity, reliability, and quantification.

  15. Chronic high fat feeding increases anxiety-like behaviour and reduces transcript abundance of glucocorticoid signalling genes in the hippocampus of female rats.

    PubMed

    Sivanathan, Shathveekan; Thavartnam, Kabriya; Arif, Shahneen; Elegino, Trisha; McGowan, Patrick O

    2015-06-01

    The consumption of diets high in saturated fats and obesity have been associated with impaired physical and mental health. Previous studies indicate that chronic high fat diet consumption leads to systemic inflammation in humans and non-human animal models. Studies in non-human animals suggest that altered physiological responses to stress are also a consequence of high fat diet consumption. Glucocorticoid signalling mechanisms may link immune and stress-related pathways in the brain, and were shown to be significantly altered in the brains of female rat offspring of mothers exposed to chronic high fat diet during pregnancy and lactation. For adult females, the consequence of chronic high fat diet consumption on these signalling pathways and their relationship to stress-related behaviour is not known. In this study, we examined the effects of chronic consumption of a high fat diet compared to a low fat control diet among adult female Long Evans rats. We found significant differences in weight gain, caloric intake, anxiety-related behaviours, and glucocorticoid-related gene expression over a 10-week exposure period. As expected, rats in the high fat diet group gained the most weight and consumed the greatest number of calories. Rats in the high fat diet group showed significantly greater levels of anxiety-related behaviour in the Light Dark and Open Field tasks compared to rats in the low fat diet group. Rats consuming high fat diet also exhibited reduced transcript abundance in the hippocampus of stress-related mineralocorticoid receptor and glucocorticoid receptor genes, as well as nuclear factor kappa beta gene expression, implicated in inflammatory processes. Together, these data indicate that chronic high fat diet consumption may increase anxiety-like behaviour at least in part via alterations in glucocorticoid signalling mechanisms in limbic brain regions.

  16. The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression

    PubMed Central

    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. PMID:25983739

  17. 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. PMID:25983739

  18. Operator Sequence Alters Gene Expression Independently of Transcription Factor Occupancy in Bacteria

    PubMed Central

    Garcia, Hernan G.; Sanchez, Alvaro; Boedicker, James Q.; Osborne, Melisa; Gelles, Jeff; Kondev, Jane; Phillips, Rob

    2012-01-01

    SUMMARY A canonical quantitative view of transcriptional regulation holds that the only role of operator sequence is to set the probability of transcription factor binding, with operator occupancy determining the level of gene expression. In this work, we test this idea by characterizing repression in vivo and the binding of RNA polymerase in vitro in experiments where operators of various sequences were placed either upstream or downstream from the promoter in Escherichia coli. Surprisingly, we find that operators with a weaker binding affinity can yield higher repression levels than stronger operators. Repressor bound to upstream operators modulates promoter escape, and the magnitude of this modulation is not correlated with the repressor-operator binding affinity. This suggests that operator sequences may modulate transcription by altering the nature of the interaction of the bound transcription factor with the transcriptional machinery, implying a new layer of sequence dependence that must be confronted in the quantitative understanding of gene expression. PMID:22840405

  19. AnimalTFDB 2.0: a resource for expression, prediction and functional study of animal transcription factors.

    PubMed

    Zhang, Hong-Mei; Liu, Teng; Liu, Chun-Jie; Song, Shuangyang; Zhang, Xiantong; Liu, Wei; Jia, Haibo; Xue, Yu; Guo, An-Yuan

    2015-01-01

    Transcription factors (TFs) are key regulators for gene expression. Here we updated the animal TF database AnimalTFDB to version 2.0 (http://bioinfo.life.hust.edu.cn/AnimalTFDB/). Using the improved prediction pipeline, we identified 72 336 TF genes, 21 053 transcription co-factor genes and 6502 chromatin remodeling factor genes from 65 species covering main animal lineages. Besides the abundant annotations (basic information, gene model, protein functional domain, gene ontology, pathway, protein interaction, ortholog and paralog, etc.) in the previous version, we made several new features and functions in the updated version. These new features are: (i) gene expression from RNA-Seq for nine model species, (ii) gene phenotype information, (iii) multiple sequence alignment of TF DNA-binding domains, and the weblogo and phylogenetic tree based on the alignment, (iv) a TF prediction server to identify new TFs from input sequences and (v) a BLAST server to search against TFs in AnimalTFDB. A new nice web interface was designed for AnimalTFDB 2.0 allowing users to browse and search all data in the database. We aim to maintain the AnimalTFDB as a solid resource for TF identification and studies of transcription regulation and comparative genomics.

  20. Aire mediates thymic expression and tolerance of pancreatic antigens via an unconventional transcriptional mechanism.

    PubMed

    Danso-Abeam, Dina; Staats, Kim A; Franckaert, Dean; Van Den Bosch, Ludo; Liston, Adrian; Gray, Daniel H D; Dooley, James

    2013-01-01

    The autoimmune regulator (Aire), mediates central tolerance of peripheral self. Its activity in thymic epithelial cells (TECs) directs the ectopic expression of thousands of tissue-restricted antigens (TRAs), causing the deletion of autoreactive thymocytes. The molecular mechanisms orchestrating the breadth of transcriptional regulation by Aire remain unknown. One prominent model capable of explaining both the uniquely high number of Aire-dependent targets and their specificity posits that tissue-specific transcription factors induced by Aire directly activate their canonical targets, exponentially adding to the total number of Aire-dependent TRAs. To test this "Hierarchical Transcription" model, we analysed mice deficient in the pancreatic master transcription factor pancreatic and duodenal homeobox 1 (Pdx1), specifically in TECs (Pdx1(ΔFoxn1) ), for the expression and tolerance of pancreatic TRAs. Surprisingly, we found that lack of Pdx1 in TECs did not reduce the transcription of insulin or somatostatin, or alter glucagon expression. Moreover, in a model of thymic deletion driven by a neo-TRA under the control of the insulin promoter, Pdx1 in TECs was not required to affect thymocyte deletion or the generation of regulatory T (Treg) cells. These findings suggest that the capacity of Aire to regulate expression of a huge array of TRAs relies solely on an unconventional transcriptional mechanism, without intermediary transcription factors.

  1. The gene for transcription factor GATA-6 resides on mouse chromosome 18 and is expressed in myocardium and vascular smooth muscle

    SciTech Connect

    Narita, Naoko; Wilson, D.B.; Bielinska, M.

    1996-09-01

    We report the mapping and developmental expression pattern of the gene encoding mouse GATA-6, a member of a family of transcription factors involved in tissue-specific gene expression. Using backcross analysis, the Gata6 gene was localized to mouse chromosome 18, linked to the gene encoding transthyretin. RNase protection analysis showed that Gata6 is abundantly expressed in the heart, stomach, intestine, and ovaries of the adult mouse. The developmental expression patterns of Gata6 and the closely related gene Gata4 were directly compared using in situ hybridization. Both genes were found to be highly expressed in the myocardium, stomach epithelium, and small intestinal epithelium throughout mouse development. Of the two genes, however, only Gata6 was expressed in vascular smooth muscle. The overlapping distributions of GATA-4 and GATA-6 transcripts in the heart support the possibility of functional redundancy or interplay between these two transcription factors in this tissue. The presence of GATA-6 mRNA in vascular smooth muscle suggests that this transcription factor may play a distinctive role in gene expression in this cell type. 5 refs., 2 figs.

  2. Transcriptional repression of Caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.

    PubMed

    Boopathi, Ettickan; Gomes, Cristiano Mendes; Goldfarb, Robert; John, Mary; Srinivasan, Vittala Gopal; Alanzi, Jaber; Malkowicz, S Bruce; Kathuria, Hasmeena; Zderic, Stephen A; Wein, Alan J; Chacko, Samuel

    2011-05-01

    Hypertrophy occurs in urinary bladder wall smooth muscle (BSM) in men with partial bladder outlet obstruction (PBOO) caused by benign prostatic hyperplasia (BPH) and in animal models of PBOO. Hypertrophied BSM from the rabbit model exhibits down-regulation of caveolin-1, a structural and functional protein of caveolae that function as signaling platforms to mediate interaction between receptor proteins and adaptor and effector molecules to regulate signal generation, amplification, and diversification. Caveolin-1 expression is diminished in PBOO-induced BSM hypertrophy in mice and in men with BPH. The proximal promoter of the human and mouse caveolin-1 (CAV1) gene was characterized, and it was observed that the transcription factor GATA-6 binds this promoter, causing reduced expression of caveolin-1. Furthermore, caveolin-1 expression levels inversely correlate with the abundance of GATA-6 in BSM hypertrophy in mice and human beings. Silencing of GATA6 gene expression up-regulates caveolin-1 expression, whereas overexpression of GATA-6 protein sustains the transcriptional repression of caveolin-1 in bladder smooth muscle cells. Together, these data suggest that GATA-6 acts as a transcriptional repressor of CAV1 gene expression in PBOO-induced BSM hypertrophy in men and mice. GATA-6-induced transcriptional repression represents a new regulatory mechanism of CAV1 gene expression in pathologic BSM, and may serve as a target for new therapy for BPH-induced bladder dysfunction in aging men.

  3. The Transcriptional Cascade in the Heat Stress Response of Arabidopsis Is Strictly Regulated at the Level of Transcription Factor Expression.

    PubMed

    Ohama, Naohiko; Kusakabe, Kazuya; Mizoi, Junya; Zhao, Huimei; Kidokoro, Satoshi; Koizumi, Shinya; Takahashi, Fuminori; Ishida, Tetsuya; Yanagisawa, Shuichi; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2016-01-01

    Group A1 heat shock transcription factors (HsfA1s) are the master regulators of the heat stress response (HSR) in plants. Upon heat shock, HsfA1s trigger a transcriptional cascade that is composed of many transcription factors. Despite the importance of HsfA1s and their downstream transcriptional cascade in the acquisition of thermotolerance in plants, the molecular basis of their activation remains poorly understood. Here, domain analysis of HsfA1d, one of several HsfA1s in Arabidopsis thaliana, demonstrated that the central region of HsfA1d is a key regulatory domain that represses HsfA1d transactivation activity through interaction with HEAT SHOCK PROTEIN70 (HSP70) and HSP90. We designated this region as the temperature-dependent repression (TDR) domain. We found that HSP70 dissociates from HsfA1d in response to heat shock and that the dissociation is likely regulated by an as yet unknown activation mechanism, such as HsfA1d phosphorylation. Overexpression of constitutively active HsfA1d that lacked the TDR domain induced expression of heat shock proteins in the absence of heat stress, thereby conferring potent thermotolerance on the overexpressors. However, transcriptome analysis of the overexpressors demonstrated that the constitutively active HsfA1d could not trigger the complete transcriptional cascade under normal conditions, thereby indicating that other factors are necessary to fully induce the HSR. These complex regulatory mechanisms related to the transcriptional cascade may enable plants to respond resiliently to various heat stress conditions. PMID:26715648

  4. Functional expression and analysis of the pancreatic transcription factor PDX-1 in yeast.

    PubMed

    Ozcan, Sabire; Mosley, Amber L; Aryal, Bishwa K

    2002-07-19

    The pancreas-specific transcription factor Pdx-1 is important for pancreas development and beta-cell specific gene expression in insulin-producing cells. We have expressed the mouse PDX-1 gene in the yeast Saccharomyces cerevisiae and characterized its functional domains. Pdx-1 functions as a strong activator in yeast and stimulates gene expression by more than 80-fold. The transcriptional activation domain of Pdx-1 is located within the first 144 amino-terminal amino acids. Pdx-1 is also able to bind and activate transcription from the A3 element of the human insulin gene promoter in yeast. Analysis of the effects of two-point mutations (Q59L and R197H) in the PDX-1 gene found in type II diabetes patients showed that both point mutations interfere with the ability of Pdx-1 to bind to DNA and to activate transcription in yeast.

  5. Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors.

    PubMed

    Perdigão, Pedro; Gaj, Thomas; Santa-Marta, Mariana; Barbas, Carlos F; Goncalves, Joao

    2016-01-01

    The presence of replication-competent HIV-1 -which resides mainly in resting CD4+ T cells--is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection.

  6. A robust and efficient method for estimating enzyme complex abundance and metabolic flux from expression data.

    PubMed

    Barker, Brandon E; Sadagopan, Narayanan; Wang, Yiping; Smallbone, Kieran; Myers, Christopher R; Xi, Hongwei; Locasale, Jason W; Gu, Zhenglong

    2015-12-01

    A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with high-throughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability and improve our understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the creation of tissue-specific models, which reduces the available reactions in an organism model, and does not provide an objective function for the estimation of fluxes. We develop a method, flux assignment with LAD (least absolute deviation) convex objectives and normalization (FALCON), that employs metabolic network reconstructions along with expression data to estimate fluxes. In order to use such a method, accurate measures of enzyme complex abundance are needed, so we first present an algorithm that addresses quantification of complex abundance. Our extensions to prior techniques include the capability to work with large models and significantly improved run-time performance even for smaller models, an improved analysis of enzyme complex formation, the ability to handle large enzyme complex rules that may incorporate multiple isoforms, and either maintained or significantly improved correlation with experimentally measured fluxes. FALCON has been implemented in MATLAB and ATS, and can be downloaded from: https://github.com/bbarker/FALCON. ATS is not required to compile the software, as intermediate C source code is available. FALCON requires use of the COBRA Toolbox, also implemented in MATLAB.

  7. Conserved Units of Co-Expression in Bacterial Genomes: An Evolutionary Insight into Transcriptional Regulation

    PubMed Central

    Junier, Ivan; Rivoire, Olivier

    2016-01-01

    Genome-wide measurements of transcriptional activity in bacteria indicate that the transcription of successive genes is strongly correlated beyond the scale of operons. Here, we analyze hundreds of bacterial genomes to identify supra-operonic segments of genes that are proximal in a large number of genomes. We show that these synteny segments correspond to genomic units of strong transcriptional co-expression. Structurally, the segments contain operons with specific relative orientations (co-directional or divergent) and nucleoid-associated proteins are found to bind at their boundaries. Functionally, operons inside a same segment are highly co-expressed even in the apparent absence of regulatory factors at their promoter regions. Remote operons along DNA can also be co-expressed if their corresponding segments share a transcriptional or sigma factor, without requiring these factors to bind directly to the promoters of the operons. As evidence that these results apply across the bacterial kingdom, we demonstrate them both in the Gram-negative bacterium Escherichia coli and in the Gram-positive bacterium Bacillus subtilis. The underlying process that we propose involves only RNA-polymerases and DNA: it implies that the transcription of an operon mechanically enhances the transcription of adjacent operons. In support of a primary role of this regulation by facilitated co-transcription, we show that the transcription en bloc of successive operons as a result of transcriptional read-through is strongly and specifically enhanced in synteny segments. Finally, our analysis indicates that facilitated co-transcription may be evolutionary primitive and may apply beyond bacteria. PMID:27195891

  8. Rapid Transcriptional Pulsing Dynamics of High Expressing Retroviral Transgenes in Embryonic Stem Cells

    PubMed Central

    Lo, Mandy Y. M.; Rival-Gervier, Sylvie; Pasceri, Peter; Ellis, James

    2012-01-01

    Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES) cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells. PMID:22606340

  9. The Transcription Factor Ultraspiracle Influences Honey Bee Social Behavior and Behavior-Related Gene Expression

    PubMed Central

    Chen, Chieh-Chun; Blatti, Charles A.; Hong, Feng; Liang, Zhengzheng S.; Negre, Nicolas; White, Kevin P.; Rodriguez-Zas, Sandra L.; Mizzen, Craig A.; Sinha, Saurabh; Zhong, Sheng; Robinson, Gene E.

    2012-01-01

    Behavior is among the most dynamic animal phenotypes, modulated by a variety of internal and external stimuli. Behavioral differences are associated with large-scale changes in gene expression, but little is known about how these changes are regulated. Here we show how a transcription factor (TF), ultraspiracle (usp; the insect homolog of the Retinoid X Receptor), working in complex transcriptional networks, can regulate behavioral plasticity and associated changes in gene expression. We first show that RNAi knockdown of USP in honey bee abdominal fat bodies delayed the transition from working in the hive (primarily “nursing” brood) to foraging outside. We then demonstrate through transcriptomics experiments that USP induced many maturation-related transcriptional changes in the fat bodies by mediating transcriptional responses to juvenile hormone. These maturation-related transcriptional responses to USP occurred without changes in USP's genomic binding sites, as revealed by ChIP–chip. Instead, behaviorally related gene expression is likely determined by combinatorial interactions between USP and other TFs whose cis-regulatory motifs were enriched at USP's binding sites. Many modules of JH– and maturation-related genes were co-regulated in both the fat body and brain, predicting that usp and cofactors influence shared transcriptional networks in both of these maturation-related tissues. Our findings demonstrate how “single gene effects” on behavioral plasticity can involve complex transcriptional networks, in both brain and peripheral tissues. PMID:22479195

  10. Inferring the Transcriptional Landscape of Bovine Skeletal Muscle by Integrating Co-Expression Networks

    PubMed Central

    Hudson, Nicholas J.; Reverter, Antonio; Wang, YongHong; Greenwood, Paul L.; Dalrymple, Brian P.

    2009-01-01

    Background Despite modern technologies and novel computational approaches, decoding causal transcriptional regulation remains challenging. This is particularly true for less well studied organisms and when only gene expression data is available. In muscle a small number of well characterised transcription factors are proposed to regulate development. Therefore, muscle appears to be a tractable system for proposing new computational approaches. Methodology/Principal Findings Here we report a simple algorithm that asks “which transcriptional regulator has the highest average absolute co-expression correlation to the genes in a co-expression module?” It correctly infers a number of known causal regulators of fundamental biological processes, including cell cycle activity (E2F1), glycolysis (HLF), mitochondrial transcription (TFB2M), adipogenesis (PIAS1), neuronal development (TLX3), immune function (IRF1) and vasculogenesis (SOX17), within a skeletal muscle context. However, none of the canonical pro-myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6 and MEF2C) were linked to muscle structural gene expression modules. Co-expression values were computed using developing bovine muscle from 60 days post conception (early foetal) to 30 months post natal (adulthood) for two breeds of cattle, in addition to a nutritional comparison with a third breed. A number of transcriptional landscapes were constructed and integrated into an always correlated landscape. One notable feature was a ‘metabolic axis’ formed from glycolysis genes at one end, nuclear-encoded mitochondrial protein genes at the other, and centrally tethered by mitochondrially-encoded mitochondrial protein genes. Conclusions/Significance The new module-to-regulator algorithm complements our recently described Regulatory Impact Factor analysis. Together with a simple examination of a co-expression module's contents, these three gene expression approaches are starting to illuminate the in vivo

  11. Inducible transcript expressed by reactive epithelial cells at sites of olfactory sensory neuron proliferation.

    PubMed

    Stoss, Thomas D; Nickell, Melissa D; Hardin, Debra; Derby, Charles D; McClintock, Timothy S

    2004-02-15

    The continuous replacement of cells in the spiny lobster olfactory organ depends on proliferation of new cells at a specific site, the proximal proliferation zone (PPZ). Using representational difference analysis of cDNA, we identified transcripts enriched in the PPZ compared to the mature zone (MZ) of the organ. The 12 clones identified included four novel sequences, three exoskeletal proteins, a serine protease, two protease inhibitors, a putative growth factor, and a sequence named PET-15 that has similarity to antimicrobial proteins of the crustin type. PET-15 mRNA was only detected in epithelial cells. It was abundant in all epithelial cells of the PPZ, but was only detected in the MZ at sites of damage to the olfactory organ. PET-15 mRNA was increased by types of damage that are known to induce proliferation of new olfactory sensory neurons in the olfactory organ. It increased in the PPZ after partial ablation of the olfactory organ and in the MZ after shaving of aesthetasc sensilla. These ipsilateral effects were mirrored by smaller increases in the undamaged contralateral olfactory organ. These contralateral effects are most parsimoniously explained by the action of a diffusible signal. Because epithelial cells are the source of proliferating progenitors in the olfactory organ, the same diffusible signal may stimulate increases in both cellular proliferation and PET-15 mRNA. The uniformity of expression of PET-15 in the PPZ epithelium suggests that the epithelial cells that give rise to new olfactory sensory neurons are a subset of cells that express PET-15.

  12. Transcription factor expression in lipopolysaccharide-activated peripheral-blood-derived mononuclear cells

    PubMed Central

    Roach, Jared C.; Smith, Kelly D.; Strobe, Katie L.; Nissen, Stephanie M.; Haudenschild, Christian D.; Zhou, Daixing; Vasicek, Thomas J.; Held, G. A.; Stolovitzky, Gustavo A.; Hood, Leroy E.; Aderem, Alan

    2007-01-01

    Transcription factors play a key role in integrating and modulating biological information. In this study, we comprehensively measured the changing abundances of mRNAs over a time course of activation of human peripheral-blood-derived mononuclear cells (“macrophages”) with lipopolysaccharide. Global and dynamic analysis of transcription factors in response to a physiological stimulus has yet to be achieved in a human system, and our efforts significantly advanced this goal. We used multiple global high-throughput technologies for measuring mRNA levels, including massively parallel signature sequencing and GeneChip microarrays. We identified 92 of 1,288 known human transcription factors as having significantly measurable changes during our 24-h time course. At least 42 of these changes were previously unidentified in this system. Our data demonstrate that some transcription factors operate in a functional range below 10 transcripts per cell, whereas others operate in a range three orders of magnitude greater. The highly reproducible response of many mRNAs indicates feedback control. A broad range of activation kinetics was observed; thus, combinatorial regulation by small subsets of transcription factors would permit almost any timing input to cis-regulatory elements controlling gene transcription. PMID:17913878

  13. Expression of Drosophila forkhead transcription factors during kidney development.

    PubMed

    Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F; Zuo, Xiaofeng; Lipschutz, Joshua H

    2014-03-28

    The Drosophila forkhead (Dfkh) family of transcription factors has over 40 family members. One Dfkh family member, BF2 (aka FoxD1), has been shown, by targeted disruption, to be essential for kidney development. In order to determine if other Dfkh family members were involved in kidney development and to search for new members of this family, reverse transcriptase polymerase chain reaction (RT-PCR) was performed using degenerate primers of the consensus sequence of the DNA binding domain of this family and developing rat kidney RNA. The RT-PCR product was used to probe RNA from a developing rat kidney (neonatal), from a 20-day old kidney, and from an adult kidney. The RT-PCR product hybridized only to a developing kidney RNA transcript of ∼2.3 kb (the size of BF2). A lambda gt10 mouse neonatal kidney library was then screened, using the above-described RT-PCR product as a probe. Three lambda phage clones were isolated that strongly hybridized to the RT-PCR probe. Sequencing of the RT-PCR product and the lambda phage clones isolated from the developing kidney library revealed Dfkh BF2. In summary, only Dfkh family member BF2, which has already been shown to be essential for nephrogenesis, was identified in our screen and no other candidate Dfkh family members were identified.

  14. Expression of Drosophila Forkhead Transcription Factors During Kidney Development

    PubMed Central

    Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F.; Zuo, Xiaofeng; Lipschutz, Joshua H.

    2014-01-01

    The Drosophila forkhead (Dfkh) family of transcription factors has over 40 family members. One Dfkh family member, BF2 (aka FoxD1), has been shown, by targeted disruption, to be essential for kidney development. In order to determine if other Dfkh family members were involved in kidney development and to search for new members of this family, reverse transcriptase polymerase chain reaction (RT-PCR) was performed using degenerate primers of the consensus sequence of the DNA binding domain of this family and developing rat kidney RNA. The RT-PCR product was used to probe RNA from a developing rat kidney (neonatal), from a 20-day old kidney, and from an adult kidney. The RT-PCR product hybridized only to a developing kidney RNA transcript of ~2.3 kb (the size of BF2). A lambda gt10 mouse neonatal kidney library was then screened, using the above-described RT-PCR product as a probe. Three lambda phage clones were isolated that strongly hybridized to the RT-PCR probe. Sequencing of the RT-PCR product and the lambda phage clones isolated from the developing kidney library revealed Dfkh BF2. In summary, only Dfkh family member BF2, which has already been shown to be essential for nephrogenesis, was identified in our screen and no other candidate Dfkh family members were identified. PMID:24491558

  15. RNA-seq Transcriptional Profiling of an Arbuscular Mycorrhiza Provides Insights into Regulated and Coordinated Gene Expression in Lotus japonicus and Rhizophagus irregularis.

    PubMed

    Handa, Yoshihiro; Nishide, Hiroyo; Takeda, Naoya; Suzuki, Yutaka; Kawaguchi, Masayoshi; Saito, Katsuharu

    2015-08-01

    Gene expression during arbuscular mycorrhizal development is highly orchestrated in both plants and arbuscular mycorrhizal fungi. To elucidate the gene expression profiles of the symbiotic association, we performed a digital gene expression analysis of Lotus japonicus and Rhizophagus irregularis using a HiSeq 2000 next-generation sequencer with a Cufflinks assembly and de novo transcriptome assembly. There were 3,641 genes differentially expressed during arbuscular mycorrhizal development in L. japonicus, approximately 80% of which were up-regulated. The up-regulated genes included secreted proteins, transporters, proteins involved in lipid and amino acid metabolism, ribosomes and histones. We also detected many genes that were differentially expressed in small-secreted peptides and transcription factors, which may be involved in signal transduction or transcription regulation during symbiosis. Co-regulated genes between arbuscular mycorrhizal and root nodule symbiosis were not particularly abundant, but transcripts encoding for membrane traffic-related proteins, transporters and iron transport-related proteins were found to be highly co-up-regulated. In transcripts of arbuscular mycorrhizal fungi, expansion of cytochrome P450 was observed, which may contribute to various metabolic pathways required to accommodate roots and soil. The comprehensive gene expression data of both plants and arbuscular mycorrhizal fungi provide a powerful platform for investigating the functional and molecular mechanisms underlying arbuscular mycorrhizal symbiosis.

  16. Effects of inefficient transcription termination of rbcL on the expression of accD in plastids of Arabidopsis thaliana.

    PubMed

    He, Baoye; Mu, Ying; Chi, Wei

    2015-12-01

    The plastid accD gene encodes one subunit of a multimeric acetyl-CoA carboxylase that is required for fatty acid biosynthesis. In Arabidopsis thaliana, the accD gene is transcribed by the nuclear-encoded phage-type RNA polymerase, and the accumulation of accD transcripts is subjected to a dynamic pattern during chloroplast development. However, the mechanisms underlying the regulation of accD expression remain unknown. Here, we showed that the inefficient transcription termination of rbcL due to the absence of RHON1 impaired the developmental profile of accD, resulting in the constitutive expression of accD during chloroplast development. Moreover, the accumulation of accD transcripts accordingly resulted in an increase in accD protein levels, suggesting that transcript abundance is critical for accD gene production. Our study demonstrates that the interplay between accD and upstream rbcL regulates the expression of accD and highlights the significance of transcriptional regulation in plastid gene expression in higher plants.

  17. The Role of Transcription Factors at Antisense-Expressing Gene Pairs in Yeast

    PubMed Central

    Mostovoy, Yulia; Thiemicke, Alexander; Hsu, Tiffany Y.; Brem, Rachel B.

    2016-01-01

    Genes encoded close to one another on the chromosome are often coexpressed, by a mechanism and regulatory logic that remain poorly understood. We surveyed the yeast genome for tandem gene pairs oriented tail-to-head at which expression antisense to the upstream gene was conserved across species. The intergenic region at most such tandem pairs is a bidirectional promoter, shared by the downstream gene mRNA and the upstream antisense transcript. Genomic analyses of these intergenic loci revealed distinctive patterns of transcription factor regulation. Mutation of a given transcription factor verified its role as a regulator in trans of tandem gene pair loci, including the proximally initiating upstream antisense transcript and downstream mRNA and the distally initiating upstream mRNA. To investigate cis-regulatory activity at such a locus, we focused on the stress-induced NAD(P)H dehydratase YKL151C and its downstream neighbor, the metabolic enzyme GPM1. Previous work has implicated the region between these genes in regulation of GPM1 expression; our mutation experiments established its function in rich medium as a repressor in cis of the distally initiating YKL151C sense RNA, and an activator of the proximally initiating YKL151C antisense RNA. Wild-type expression of all three transcripts required the transcription factor Gcr2. Thus, at this locus, the intergenic region serves as a focal point of regulatory input, driving antisense expression and mediating the coordinated regulation of YKL151C and GPM1. Together, our findings implicate transcription factors in the joint control of neighboring genes specialized to opposing conditions and the antisense transcripts expressed between them. PMID:27190003

  18. Alternative transcription initiation leads to expression of a novel ALK isoform in cancer

    PubMed Central

    Wiesner, Thomas; Lee, William; Obenauf, Anna C.; Ran, Leili; Murali, Rajmohan; Zhang, Qi Fan; Wong, Elissa W. P.; Hu, Wenhuo; Scott, Sasinya N.; Shah, Ronak H.; Landa, Iñigo; Button, Julia; Lailler, Nathalie; Sboner, Andrea; Gao, Dong; Murphy, Devan A.; Cao, Zhen; Shukla, Shipra; Hollmann, Travis J.; Wang, Lu; Borsu, Laetitia; Merghoub, Taha; Schwartz, Gary K.; Postow, Michael A.; Ariyan, Charlotte E.; Fagin, James A.; Zheng, Deyou; Ladanyi, Marc; Busam, Klaus J.; Berger, Michael F.; Chen, Yu; Chi, Ping

    2016-01-01

    Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ~ 11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALKATI. In ALKATI-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites1. ALKATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALKATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALKATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALKATI, suggesting that patients with ALKATI-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation. PMID:26444240

  19. Encoding four gene expression programs in the activation dynamics of a single transcription factor.

    PubMed

    Hansen, Anders S; O'Shea, Erin K

    2016-04-01

    Cellular signaling response pathways often exhibit a bow-tie topology [1,2]: multiple upstream stress signals converge on a single shared transcription factor, which is thought to induce different downstream gene expression programs (Figure 1A). However, if several different signals activate the same transcription factor, can each signal then induce a specific gene expression response? A growing body of literature supports a temporal coding theory where information about environmental signals can be encoded, at least partially, in the temporal dynamics of the shared transcription factor [1,2]. For example, in the case of the budding yeast transcription factor Msn2, different stresses induce distinct Msn2 activation dynamics: Msn2 shows pulsatile nuclear activation with dose-dependent frequency under glucose limitation, but sustained nuclear activation with dose-dependent amplitude under oxidative stress [3]. These dynamic patterns can then lead to differential gene expression responses [3-5], but it is not known how much specificity can be obtained. Thus, a major question of this temporal coding theory is how many gene response programs or cellular functions can be robustly encoded by dynamic control of a single transcription factor. Here we provide the first direct evidence that, simply by regulating the activation dynamics of a single transcription factor, it is possible to preferentially induce four distinct gene expression programs. PMID:27046808

  20. A genome-scale study of transcription factor expression in the branching mouse lung

    PubMed Central

    Herriges, John C.; Yi, Lan; Hines, Elizabeth A.; Harvey, Julie F.; Xu, Guoliang; Gray, Paul; Ma, Qiufu; Sun, Xin

    2012-01-01

    Background Mammalian lung development consists of a series of precisely choreographed events that drive the progression from simple lung buds to the elaborately branched organ that fulfills the vital function of gas exchange. Strict transcriptional control is essential for lung development. Among the large number of transcription factors encoded in the mouse genome, only a small portion of them are known to be expressed and function in the developing lung. Thus a systematic investigation of transcription factors expressed in the lung is warranted. Results To enrich for genes that may be responsible for regional growth and patterning, we performed a screen using RNA in situ hybridization to identify genes that show restricted expression patterns in the embryonic lung. We focused on the pseudoglandular stage during which the lung undergoes branching morphogenesis, a cardinal event of lung development. Using a genome-scale probe set that represents over 90% of the transcription factors encoded in the mouse genome, we identified sixty-two transcription factor genes with localized expression in the epithelium, mesenchyme or both. Many of these genes have not been previously implicated in lung development. Conclusions Our findings provide new starting points for the elucidation of the transcriptional circuitry that controls lung development. PMID:22711520

  1. Identification of differentially expressed genes associated with flower color in peach using genome-wide transcriptional analysis.

    PubMed

    Zhou, Y; Wu, X X; Zhang, Z; Gao, Z H

    2015-01-01

    Flower color is an important trait of the ornamental peach (Prunus persica L.). However, the mechanism responsible for the different colors that appear in the same genotype remains unclear. In this study, red samples showed higher anthocyanins content (0.122 ± 0.009 mg/g), which was significantly different from that in white samples (0.066 ± 0.010 mg/g). Similarly to carotenoids content, red extract (0.058 ± 0.004 mg/L) was significantly higher in white extract (0.015 ± 0.004 mg/L). We estimated gene expression using Illumina sequencing technology in libraries from white and red flower buds. A total of 3,599,960 and 3,464,141 tags were sequenced from the 2 libraries, respectively. Moreover, we identified 106 significantly differentially expressed genes between the 2 libraries. Among these, 78 and 28 represented transcripts with a higher or lower abundance of more than 2-fold than in the white flower library, respectively. GO annotation indicated that highly ranked genes were involved in the pigment biosynthetic process. Expression patterns of 11 genes were verified using quantitative reverse transcription-polymerase chain reaction assays. The results suggest that hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase, 2-oxoglutarate-dependent dioxygenase, isoflavone reductase, riboflavin kinase, zeta-carotene desaturase, and ATP binding cassette transporter may be associated with the flower color formation. Our results may be useful for scientists focusing on Prunus persica floral development and biotechnology.

  2. Estrogen induced concentration dependent differential gene expression in human breast cancer (MCF7) cells: Role of transcription factors

    SciTech Connect

    Chandrasekharan, Sabarinath; Kandasamy, Krishna Kumar; Dayalan, Pavithra; Ramamurthy, Viraragavan

    2013-08-02

    Highlights: •Estradiol (E2) at low dose induced cell proliferation in breast cancer cells. •E2 at high concentration induced cell stress in breast cancer cells. •Estrogen receptor physically interacts only with a few transcription factors. •Differential expression of genes with Oct-1 binding sites increased under stress. •Transcription factor binding sites showed distinct spatial distribution on genes. -- Abstract: Background: Breast cancer cells respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. The mechanism of this concentration dependent differential outcome is not well understood yet. Methodology: Meta-analysis of the expression data of MCF7 cells treated with low (1 nM) or high (100 nM) dose of estradiol (E2) was performed. We identified genes differentially expressed at the low or the high dose, and examined the nature of regulatory elements in the vicinity of these genes. Specifically, we looked for the difference in the presence, abundance and spatial distribution of binding sites for estrogen receptor (ER) and selected transcription factors (TFs) in the genomic region up to 25 kb upstream and downstream from the transcription start site (TSS) of these genes. Results: It was observed that at high dose E2 induced the expression of stress responsive genes, while at low dose, genes involved in cell cycle were induced. We found that the occurrence of transcription factor binding regions (TFBRs) for certain factors such as Sp1 and SREBP1 were higher on regulatory regions of genes expressed at low dose. At high concentration of E2, genes with a higher frequency of Oct-1 binding regions were predominantly involved. In addition, there were differences in the spatial distribution pattern of the TFBRs in the genomic regions among the two sets of genes. Discussion: E2 induced predominantly proliferative/metabolic response at low concentrations; but at high concentration, stress–rescue responses were induced

  3. Expression profiling of muscle reveals transcripts differentially expressed in muscle that affect water-holding capacity of pork.

    PubMed

    Ponsuksili, Siriluck; Murani, Eduard; Phatsara, Chirawath; Jonas, Elisabeth; Walz, Christina; Schwerin, Manfred; Schellander, Karl; Wimmers, Klaus

    2008-11-12

    To identify biological processes as well as molecular markers for drip loss, a parameter for water holding capacity of meat, the M. longissimus dorsi transcriptomes of six divergent sib pairs were analyzed using Affymetrix Porcine Genome Array. Functional categories of differentially regulated transcripts were determined by single-gene analysis and gene set analysis. The transcripts being up-regulated at high drip loss belong to groups of genes functionally categorized as genes of membrane proteins, signal transduction, cell communication, response to stimulus, and cytoskeleton. Among genes down-regulated with high drip loss, functional groups of oxidoreductase activity, lipid metabolism, and electron transport were identified. Differential regulation of the abundance of transcripts of these biological networks in live muscle affect mortem biochemical processes of meat maturation. Knowledge of this functional link is indicative for the identification of candidate genes for improvement of meat quality. PMID:18922009

  4. 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

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

    PubMed

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

    2016-02-18

    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.

  6. Cloning, Sequencing, and Expression of Selenoprotein Transcripts in the Turkey (Meleagris gallopavo).

    PubMed

    Sunde, Roger A; Sunde, Gavin R; Sunde, Colin M; Sunde, Milton L; Evenson, Jacqueline K

    2015-01-01

    The minimum Se requirement for male turkey poults is 0.3 μg Se/g--three times higher than requirements found in rodents--based on liver and gizzard glutathione peroxidase-4 (GPX4) and GPX1 activities. In addition, turkey liver GPX4 activity is 10-fold higher and GPX1 activity is 10-fold lower than in rats, and both GPX1 and GPX4 mRNA levels are dramatically down-regulated by Se deficiency. Currently, the sequences of all annotated turkey selenoprotein transcripts and proteins in the NCBI database are only "predicted." Thus we initiated cloning and sequencing of the full turkey selenoprotein transcriptome to demonstrate expression of selenoprotein transcripts in the turkey, and to develop tools to investigate Se regulation of the full selenoproteome. Total RNA was isolated from six tissues of Se-adequate adult tom turkeys, and used to prepare reverse-transcription cDNA libraries. PCR primers were designed, based initially on chicken, rodent, porcine, bovine and human sequences and later on turkey shotgun cloning sequences. We report here the cloning of full transcript sequences for 9 selenoproteins, and 3'UTR portions for 15 additional selenoproteins, which include SECIS elements in 22 3'UTRs, and in-frame Sec (UGA) codons within coding regions of 19 selenoproteins, including 12 Sec codons in SEPP1. In addition, we sequenced the gap between two contigs from the shotgun cloning of the turkey genome, and found the missing sequence for the turkey Sec-tRNA. RTPCR was used to determine the relative transcript expression in 6 tissues. GPX3 expression was high in all tissues except kidney, GPX1 expression was high in kidney, SEPW1 expression was high in heart, gizzard and muscle, and SELU expression was high in liver. SEPP2, a selenoprotein not found in mammals, was highly expressed in liver but not in other tissues. In summary, transcripts for 24 selenoproteins are expressed in the turkey, not just predicted.

  7. Differential display of abundantly expressed genes of Trichoderma harzianum during colonization of tomato-germinating seeds and roots.

    PubMed

    Mehrabi-Koushki, Mehdi; Rouhani, Hamid; Mahdikhani-Moghaddam, Esmat

    2012-11-01

    The identification of Trichoderma genes whose expression is altered during early stages of interaction with developing roots of germinated seeds is an important step toward understanding the rhizosphere competency of Trichoderma spp. The potential of 13 Trichoderma strains to colonize tomato root and promote plant growth has been evaluated. All used strains successfully propagated in spermosphere and continued their growth in rhizoplane simultaneously root enlargement while the strains T6 and T7 were the most abundant in the apical segment of roots. Root colonization in most strains associated with promoting the roots and shoots growth while they significantly increased up to 43 and 40 % roots and shoots dry weights, respectively. Differential display reverse transcriptase-PCR (DDRT-PCR) has been developed to detect differentially expressed genes in the previously selected strain, Trichoderma harzianum T7, during colonization stages of tomato-germinating seeds and roots. Amplified DDRT-PCR products were analyzed on gel agarose and 62 differential bands excised, purified, cloned, and sequenced. Obtained ESTs were submit-queried to NCBI database by BLASTx search and gene ontology hierarchy. Most of transcripts (29 EST) corresponds to known and hypothetical proteins such as secretion-related small GTPase, 40S ribosomal protein S3a, 3-hydroxybutyryl-CoA dehydrogenase, DNA repair protein rad50, lipid phosphate phosphatase-related protein type 3, nuclear essential protein, phospholipase A2, fatty acid desaturase, nuclear pore complex subunit Nup133, ubiquitin-activating enzyme, and 60S ribosomal protein L40. Also, 13 of these sequences showed no homology (E > 0.05) with public databases and considered as novel genes. Some of these ESTs corresponded to genes encodes enzymes potentially involved in nutritional support of microorganisms which have obvious importance in the establishment of Trichoderma in spermosphere and rhizosphere, via potentially functioning in

  8. Scaling of Gene Expression with Transcription-Factor Fugacity

    PubMed Central

    Weinert, Franz M.; Brewster, Robert C.; Rydenfelt, Mattias; Phillips, Rob; Kegel, Willem K.

    2015-01-01

    The proteins associated with gene regulation are often shared between multiple pathways simultaneously. By way of contrast, models in regulatory biology often assume these pathways act independently. We demonstrate a framework for calculating the change in gene expression for the interacting case by decoupling repressor occupancy across the cell from the gene of interest by way of a chemical potential. The details of the interacting regulatory architecture are encompassed in an effective concentration, and thus, a single scaling function describes a collection of gene expression data from diverse regulatory situations and collapses it onto a single master curve. PMID:25554908

  9. Splicing of a non-coding antisense transcript controls LEF1 gene expression

    PubMed Central

    Beltran, Manuel; Aparicio-Prat, Estel; Mazzolini, Rocco; Millanes-Romero, Alba; Massó, Pere; Jenner, Richard G.; Díaz, Víctor M.; Peiró, Sandra; de Herreros, Antonio García

    2015-01-01

    In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms. PMID:25990740

  10. Matrix attachment regions and regulated transcription increase and stabilize transgene expression.

    PubMed

    Abranches, Rita; Shultz, Randall W; Thompson, William F; Allen, George C

    2005-09-01

    Transgene silencing has been shown to be associated with strong promoters, but it is not known whether the propensity for silencing is caused by the level of transcription, or some other property of the promoter. If transcriptional activity fosters silencing, then transgenes with inducible promoters may be less susceptible to silencing. To test this idea, a doxycycline-inducible luciferase transgene was transformed into an NT1 tobacco suspension culture cell line that constitutively expressed the tetracycline repressor. The inducible luciferase gene was flanked by tobacco Rb7 matrix attachment regions (MAR) or spacer control sequences in order to test the effects of MARs in conjunction with regulated transcription. Transformed lines were grown under continuous doxycycline (CI), or delayed doxycycline induction (DI) conditions. Delayed induction resulted in higher luciferase expression initially, but continued growth in the presence of doxycycline resulted in a reduction of expression to levels similar to those found in continuously induced lines. In both DI and CI treatments, the Rb7 MAR significantly reduced the percentage of silenced lines and increased transgene expression levels. These data demonstrate that active transcription increases silencing, especially in the absence of the Rb7 MAR. Importantly, the Rb7 MAR lines showed higher expression levels under both CI and DI conditions and avoided silencing that may occur in the absence of active transcription such as what would be expected as a result of condensed chromatin spreading. PMID:17173639

  11. Characterization and Improvement of RNA-Seq Precision in Quantitative Transcript Expression Profiling

    SciTech Connect

    Labaj, Pawel P.; Leparc, German G.; Linggi, Bryan E.; Markillie, Lye Meng; Wiley, H. S.; Kreil, David P.

    2011-07-01

    Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large scale RNA-Seq data sets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. Results: We report on a comprehensive study of target coverage and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive target coverage of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, less than 30% of all transcripts could be quantified reliably with a relative error < 20%. Based on established tools, we then introduce a new approach for mapping and analyzing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision.

  12. Tillage Management and Seasonal Effects on Denitrifier Community Abundance, Gene Expression and Structure over Winter.

    PubMed

    Tatti, Enrico; Goyer, Claudia; Burton, David L; Wertz, Sophie; Zebarth, Bernie J; Chantigny, Martin; Filion, Martin

    2015-10-01

    Tillage effects on denitrifier communities and nitrous oxide (N2O) emissions were mainly studied during the growing season. There is limited information for the non-growing season, especially in northern countries where winter has prolonged periods with sub-zero temperatures. The abundance and structure of the denitrifier community, denitrification gene expression and N2O emissions in fields under long-term tillage regimes [no-tillage (NT) vs conventional tillage (CT)] were assessed during two consecutive winters. NT exerted a positive effect on nirK and nosZ denitrifier abundance in both winters compared to CT. Moreover, the two contrasting managements had an opposite influence on nirK and nirS RNA/DNA ratios. Tillage management resulted in different denitrifier community structures during both winters. Seasonal changes were observed in the abundance and the structure of denitrifiers. Interestingly, the RNA/DNA ratios were greater in the coldest months for nirK, nirS and nosZ. N2O emissions were not influenced by management but changed over time with two orders of magnitude increase in the coldest month of both winters. In winter of 2009-2010, emissions were mainly as N2O, whereas in 2010-2011, when soil temperatures were milder due to persistent snow cover, most emissions were as dinitrogen. Results indicated that tillage management during the growing season induced differences in denitrifier community structure that persisted during winter. However, management did not affect the active cold-adapted community structure.

  13. Position dependent expression of a homeobox gene transcript in relation to amphibian limb regeneration.

    PubMed Central

    Savard, P; Gates, P B; Brockes, J P

    1988-01-01

    Adult urodele amphibians such as the newt Notophthalmus viridescens are capable of regenerating their limbs and tail by formation of a blastema, a growth zone of mesenchymal progenitor cells. In an attempt to identify genes implicated in specification of the regenerate, we screened a newt forelimb blastema cDNA library with homeobox probes, and isolated and sequenced clones that identify a 1.8 kb polyadenylated transcript containing a homeobox. The transcript is derived from a single gene called NvHbox 1, the newt homologue of XIHbox 1 (Xenopus), HHO.c8 (human) and Hox-6.1 (mouse). The cDNA for the 1.8 kb transcript has two exons as determined by isolation and partial sequencing of a genomic clone. The expression of the transcript shows several interesting features in relation to limb regeneration: (i) Hybridization of Northern blots of poly(A)+ RNA from limb and tail and their respective blastemas shows that the transcript in limb tissues has exons 1 and 2, whereas a 1.8 kb transcript in tail tissues has only exon 2. (ii) The transcript is expressed in limbs of adult newt but not of adult Xenopus, raising the possibility that this contributes to an explanation of the loss of regenerative ability with maturation in adult anurans. (iii) The transcript is expressed at a higher level in a proximal (mid-humerus) blastema than in a distal one (mid-radius). When distal blastemas were proximalized by treatment with retinoic acid, no change in the level of the transcript was detected by Northern analysis at a single time point after amputation.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2907476

  14. Abundant contribution of short tandem repeats to gene expression variation in humans

    PubMed Central

    Gymrek, Melissa; Willems, Thomas; Guilmatre, Audrey; Zeng, Haoyang; Markus, Barak; Georgiev, Stoyan; Daly, Mark J.; Price, Alkes L.; Pritchard, Jonathan; Sharp, Andrew

    2016-01-01

    The contribution of repetitive elements to quantitative human traits is largely unknown. Here, we report a genome-wide survey of the contribution of Short Tandem Repeats (STRs), one of the most polymorphic and abundant repeat classes, to gene expression in humans. Our survey identified 2,060 significant expression STRs (eSTRs). These eSTRs were replicable in orthogonal populations and expression assays. We used variance partitioning to disentangle the contribution of eSTRs from linked SNPs and indels and found that eSTRs contribute 10%–15% of the cis-heritability mediated by all common variants. Further functional genomic analyses showed that eSTRs are enriched in conserved regions, co-localize with regulatory elements, and can modulate certain histone modifications. By analyzing known GWAS hits and searching for new associations in 1,685 deeply-phenotyped whole-genomes, we found that eSTRs are enriched in various clinically-relevant conditions. These results highlight the contribution of short tandem repeats to the genetic architecture of quantitative human traits. PMID:26642241

  15. Aminopeptidase N gene expression and abundance in caprine mammary gland is influenced by circulating plasma peptide.

    PubMed

    Mabjeesh, S J; Gal-Garber, O; Milgram, J; Feuermann, Y; Cohen-Zinder, M; Shamay, A

    2005-06-01

    This study examined the localization and the effect of circulating peptides on the expression of aminopeptidase N (EC 3.4.11.2) in caprine mammary gland. Four lactating goats in mid to late lactation were used in a crossover design and were subjected to 2 dietary treatments. Abomasal infusion of casein hydrolysate was used to increase the concentration of peptide-bound amino acid in the circulation. Samples of mammary gland tissue from each goat were taken by biopsy at the end of each treatment period to measure gene and protein expression of aminopeptidase N in the tissue. There were no measurable effects on feed intake and milk production for any of the treatments. Western blot analysis showed that aminopeptidase N is located on the basolateral side of parenchymal cells and not on the apical membranes. Abomasal infusion of casein hydrolysate caused a marked change in the profile of arterial blood free amino acids and peptide-bound amino acids smaller than 1500 Da. Abundance of aminopeptidase N mRNA and protein increased by 51 and 58%, respectively, in casein hydrolysate-infused goats compared with the control treatment. It was concluded that aminopeptidase N is one candidate actively involved in the mammary gland to support protein synthesis and milk production. In accordance with the nutritional conditions in the current experiment, it is suggested that aminopeptidase N expression is partly controlled by the metabolic requirements of the gland and postabsorptive forms of amino acids in the circulation. PMID:15905436

  16. The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation.

    PubMed

    Collu-Marchese, Melania; Shuen, Michael; Pauly, Marion; Saleem, Ayesha; Hood, David A

    2015-01-01

    The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2-3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2-3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes.

  17. A distance difference matrix approach to identifying transcription factors that regulate differential gene expression

    PubMed Central

    De Bleser, Pieter; Hooghe, Bart; Vlieghe, Dominique; van Roy, Frans

    2007-01-01

    We introduce a method that considers target genes of a transcription factor, and searches for transcription factor binding sites (TFBSs) of secondary factors responsible for differential responses among these targets. Based on the distance difference matrix concept, the method simultaneously integrates statistical overrepresentation and co-occurrence of TFBSs. Our approach is validated on datasets of differentially regulated human genes and is shown to be highly effective in detecting TFBSs responsible for the observed differential gene expression. PMID:17504544

  18. The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation

    PubMed Central

    Collu-Marchese, Melania; Shuen, Michael; Pauly, Marion; Saleem, Ayesha; Hood, David A.

    2015-01-01

    The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2–3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2–3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes. PMID:26182383

  19. Parathyroid hormone inhibition of Na{sup +}/H{sup +} exchanger 3 transcription: Intracellular signaling pathways and transcription factor expression

    SciTech Connect

    Neri, Elida Adalgisa; Bezerra, Camila Nogueira Alves Queiroz-Leite, Gabriella Duarte; Polidoro, Juliano Zequini; Rebouças, Nancy Amaral

    2015-06-12

    The main transport mechanism of reabsorption of sodium bicarbonate and fluid in the renal proximal tubules involves Na{sup +}/H{sup +} exchanger 3 (NHE3), which is acutely and chronically downregulated by parathyroid hormone (PTH). Although PTH is known to exert an inhibitory effect on NHE3 expression and transcription, the molecular mechanisms involved remain unclear. Here, we demonstrated that, in opossum kidney proximal tubule (OKP) cells, PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter that controls expression of the reporter gene. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways transformed PTH from an inhibitor of promoter activity into an activator of that same activity, as did point mutations in the EGR1, Sp1, and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1 mRNA and of some Sp3 isoforms. Electrophoretic mobility shift assay showed a supershift of the −61 to −42-bp probe with an anti-EGR1 antibody in PTH-treated cells, suggesting that EGR1 binding is relevant for the inhibitory activity of PTH. We conclude that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression; to EGR1 binding to the proximal and core promoters; and to PKA and JAK/STAT pathway activation. This mechanism might be responsible, at least in part, for lower NHE3 expression and sodium reabsorption in renal proximal tubules in the presence of high PTH levels. - Highlights: • PTH regulation of Nhe3 promoter depends on EGR1 binding. • EGR1, PKA and JAK/STAT are involved in PTH inhibition of the Nhe3 promoter. • PTH alters expression of EGR1 and Sp3. • PTH inhibits the Nhe3 promoter by regulating PKA and JAK/STAT signaling.

  20. Evidence for the expression of abundant microRNAs in the locust genome.

    PubMed

    Wang, Yanli; Jiang, Feng; Wang, Huimin; Song, Tianqi; Wei, Yuanyuan; Yang, Meiling; Zhang, Jianzhen; Kang, Le

    2015-01-01

    Substantial accumulation of neutral sequences accounts for genome size expansion in animal genomes. Numerous novel microRNAs (miRNAs), which evolve in a birth and death manner, are considered evolutionary neutral sequences. The migratory locust is an ideal model to determine whether large genomes contain abundant neutral miRNAs because of its large genome size. A total of 833 miRNAs were discovered, and several miRNAs were randomly chosen for validation by Northern blot and RIP-qPCR. Three additional verification methods, namely, processing-dependent methods of miRNA biogenesis using RNAi, evolutionary comparison with closely related species, and evidence supported by tissue-specific expression, were applied to provide compelling results that support the authenticity of locust miRNAs. We observed that abundant local duplication events of miRNAs, which were unique in locusts compared with those in other insects with small genome sizes, may be responsible for the substantial acquisition of miRNAs in locusts. Together, multiple evidence showed that the locust genome experienced a burst of miRNA acquisition, suggesting that genome size expansion may have considerable influences of miRNA innovation. These results provide new insight into the genomic dynamics of miRNA repertoires under genome size evolution.

  1. Merkel cell carcinoma subgroups by Merkel cell polyomavirus DNA relative abundance and oncogene expression

    PubMed Central

    Bhatia, Kishor; Goedert, James J.; Modali, Rama; Preiss, Liliana; Ayers, Leona W.

    2010-01-01

    Merkel cell polyomavirus (MCPyV) was recently discovered in Merkel cell carcinoma (MCC), a clinically and pathologically heterogeneous malignancy of dermal neuroendocrine cells. To investigate this heterogeneity, we developed a tissue microarray (TMA) to characterize immunohistochemical staining of candidate tumor cell proteins and a quantitative PCR assay to detect MCPyV and measure viral loads. MCPyV was detected in 19 of 23 (74%) primary MCC tumors, but 8 of these had less than 1 viral copy per 300 cells. Viral abundance of 0.06–1.2viral copies/cell was directly related to presence of retinoblastoma gene product (pRb) and terminal deoxyribonucleotidyl transferase (TdT) by immunohistochemical staining (P≤0.003). Higher viral abundance tumors tended to be associated with less p53 expression, younger age at diagnosis, and longer survival (P≤0.08). These data suggest that MCC may arise through different oncogenic pathways, including ones independent of pRb and MCPyV. PMID:19551862

  2. Evidence for the expression of abundant microRNAs in the locust genome.

    PubMed

    Wang, Yanli; Jiang, Feng; Wang, Huimin; Song, Tianqi; Wei, Yuanyuan; Yang, Meiling; Zhang, Jianzhen; Kang, Le

    2015-01-01

    Substantial accumulation of neutral sequences accounts for genome size expansion in animal genomes. Numerous novel microRNAs (miRNAs), which evolve in a birth and death manner, are considered evolutionary neutral sequences. The migratory locust is an ideal model to determine whether large genomes contain abundant neutral miRNAs because of its large genome size. A total of 833 miRNAs were discovered, and several miRNAs were randomly chosen for validation by Northern blot and RIP-qPCR. Three additional verification methods, namely, processing-dependent methods of miRNA biogenesis using RNAi, evolutionary comparison with closely related species, and evidence supported by tissue-specific expression, were applied to provide compelling results that support the authenticity of locust miRNAs. We observed that abundant local duplication events of miRNAs, which were unique in locusts compared with those in other insects with small genome sizes, may be responsible for the substantial acquisition of miRNAs in locusts. Together, multiple evidence showed that the locust genome experienced a burst of miRNA acquisition, suggesting that genome size expansion may have considerable influences of miRNA innovation. These results provide new insight into the genomic dynamics of miRNA repertoires under genome size evolution. PMID:26329925

  3. Expression of cytokine mRNA transcripts in renal cell carcinoma.

    PubMed

    Olive, C; Cheung, C; Nicol, D; Falk, M C

    1998-08-01

    Renal cell carcinoma (RCC) is a solid tumour of the kidney and is the most common renal neoplasm. Despite the presence of tumour infiltrating lymphocytes (TIL) in RCC, these tumours continue to progress in vivo suggesting a poor host immune response to the tumour, and the suppression of TIL effector function. Cytokines are key molecules that modulate the function of T cells. The possibility is investigated that the local production of cytokines in RCC contributes to immunosuppression of TIL. The expression of pro-inflammatory (IFN-gamma/IL-2) and immunosuppressive (IL-10/TGF-beta) cytokine mRNA transcripts was determined in RCC, normal kidney and peripheral blood of RCC patients using a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with cytokine-specific primers. Following Southern blot hybridization of the PCR products with internal radiolabelled oligonucleotide probes, cytokine transcript levels were measured by densitometry and expressed relative to the glyceraldehyde-3-phosphate dehydrogenase densitometry score. With the exception of IL-10, there were no differences in expression of cytokine mRNA transcripts between the peripheral blood of patients and normal healthy individuals. It was found that TGF-beta transcripts were well represented in normal kidney and RCC. In contrast, the expression of IFN-gamma transcripts, while low in the majority of samples, was significantly increased in RCC when compared to normal kidney (P=0.05). The IL-2 and IL-10 transcripts showed a more variable expression in normal kidney and RCC, with no significant differences in expression between the sample groups. The data demonstrating pro-inflammatory and immunosuppressive cytokine expression in RCC do not support a prominent immunosuppressive cytokine profile in these tumours. PMID:9723777

  4. Pleiohomeotic Interacts with the Core Transcription Elongation Factor Spt5 to Regulate Gene Expression in Drosophila

    PubMed Central

    Jennings, Barbara H.

    2013-01-01

    The early elongation checkpoint regulated by Positive Transcription Elongation Factor b (P-TEFb) is a critical control point for the expression of many genes. Spt5 interacts directly with RNA polymerase II and has an essential role in establishing this checkpoint, and also for further transcript elongation. Here we demonstrate that Drosophila Spt5 interacts both physically and genetically with the Polycomb Group (PcG) protein Pleiohomeotic (Pho), and the majority of Pho binding sites overlap with Spt5 binding sites across the genome in S2 cells. Our results indicate that Pho can interact with Spt5 to regulate transcription elongation in a gene specific manner. PMID:23894613

  5. Regulation of RE1 protein silencing transcription factor (REST) expression by HIP1 protein interactor (HIPPI).

    PubMed

    Datta, Moumita; Bhattacharyya, Nitai P

    2011-09-30

    Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease. PMID:21832040

  6. The 5th Symposium on Post-Transcriptional Regulation of Plant Gene Expression (PTRoPGE)

    SciTech Connect

    Karen S. Browning; Marie Petrocek; Bonnie Bartel

    2006-06-01

    The 5th Symposium on Post-Transcriptional Regulation of Plant Gene Expression (PTRoPGE) will be held June 8-12, 2005 at the University of Texas at Austin. Exciting new and ongoing discoveries show significant regulation of gene expression occurs after transcription. These post-transcriptional control events in plants range from subtle regulation of transcribed genes and phosphorylation, to the processes of gene regulation through small RNAs. This meeting will focus on the regulatory role of RNA, from transcription, through translation and finally degradation. The cross-disciplinary design of this meeting is necessary to encourage interactions between researchers that have a common interest in post-transcriptional gene expression in plants. By bringing together a diverse group of plant molecular biologist and biochemists at all careers stages from across the world, this meeting will bring about more rapid progress in understanding how plant genomes work and how genes are finely regulated by post-transcriptional processes to ultimately regulate cells.

  7. Pervasive transcription read-through promotes aberrant expression of oncogenes and RNA chimeras in renal carcinoma

    PubMed Central

    Grosso, Ana R; Leite, Ana P; Carvalho, Sílvia; Matos, Mafalda R; Martins, Filipa B; Vítor, Alexandra C; Desterro, Joana MP; Carmo-Fonseca, Maria; de Almeida, Sérgio F

    2015-01-01

    Aberrant expression of cancer genes and non-canonical RNA species is a hallmark of cancer. However, the mechanisms driving such atypical gene expression programs are incompletely understood. Here, our transcriptional profiling of a cohort of 50 primary clear cell renal cell carcinoma (ccRCC) samples from The Cancer Genome Atlas (TCGA) reveals that transcription read-through beyond the termination site is a source of transcriptome diversity in cancer cells. Amongst the genes most frequently mutated in ccRCC, we identified SETD2 inactivation as a potent enhancer of transcription read-through. We further show that invasion of neighbouring genes and generation of RNA chimeras are functional outcomes of transcription read-through. We identified the BCL2 oncogene as one of such invaded genes and detected a novel chimera, the CTSC-RAB38, in 20% of ccRCC samples. Collectively, our data highlight a novel link between transcription read-through and aberrant expression of oncogenes and chimeric transcripts that is prevalent in cancer. DOI: http://dx.doi.org/10.7554/eLife.09214.001 PMID:26575290

  8. Uncovering a Macrophage Transcriptional Program by Integrating Evidence from Motif Scanning and Expression Dynamics

    PubMed Central

    Ramsey, Stephen A.; Klemm, Sandy L.; Zak, Daniel E.; Kennedy, Kathleen A.; Thorsson, Vesteinn; Li, Bin; Gilchrist, Mark; Gold, Elizabeth S.; Johnson, Carrie D.; Litvak, Vladimir; Navarro, Garnet; Roach, Jared C.; Rosenberger, Carrie M.; Rust, Alistair G.; Yudkovsky, Natalya; Aderem, Alan; Shmulevich, Ilya

    2008-01-01

    Macrophages are versatile immune cells that can detect a variety of pathogen-associated molecular patterns through their Toll-like receptors (TLRs). In response to microbial challenge, the TLR-stimulated macrophage undergoes an activation program controlled by a dynamically inducible transcriptional regulatory network. Mapping a complex mammalian transcriptional network poses significant challenges and requires the integration of multiple experimental data types. In this work, we inferred a transcriptional network underlying TLR-stimulated murine macrophage activation. Microarray-based expression profiling and transcription factor binding site motif scanning were used to infer a network of associations between transcription factor genes and clusters of co-expressed target genes. The time-lagged correlation was used to analyze temporal expression data in order to identify potential causal influences in the network. A novel statistical test was developed to assess the significance of the time-lagged correlation. Several associations in the resulting inferred network were validated using targeted ChIP-on-chip experiments. The network incorporates known regulators and gives insight into the transcriptional control of macrophage activation. Our analysis identified a novel regulator (TGIF1) that may have a role in macrophage activation. PMID:18369420

  9. A robust and efficient method for estimating enzyme complex abundance and metabolic flux from expression data.

    PubMed

    Barker, Brandon E; Sadagopan, Narayanan; Wang, Yiping; Smallbone, Kieran; Myers, Christopher R; Xi, Hongwei; Locasale, Jason W; Gu, Zhenglong

    2015-12-01

    A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with high-throughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability and improve our understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the creation of tissue-specific models, which reduces the available reactions in an organism model, and does not provide an objective function for the estimation of fluxes. We develop a method, flux assignment with LAD (least absolute deviation) convex objectives and normalization (FALCON), that employs metabolic network reconstructions along with expression data to estimate fluxes. In order to use such a method, accurate measures of enzyme complex abundance are needed, so we first present an algorithm that addresses quantification of complex abundance. Our extensions to prior techniques include the capability to work with large models and significantly improved run-time performance even for smaller models, an improved analysis of enzyme complex formation, the ability to handle large enzyme complex rules that may incorporate multiple isoforms, and either maintained or significantly improved correlation with experimentally measured fluxes. FALCON has been implemented in MATLAB and ATS, and can be downloaded from: https://github.com/bbarker/FALCON. ATS is not required to compile the software, as intermediate C source code is available. FALCON requires use of the COBRA Toolbox, also implemented in MATLAB. PMID:26381164

  10. RNA pol II transcript abundance controls condensin accumulation at mitotically up-regulated and heat-shock-inducible genes in fission yeast.

    PubMed

    Nakazawa, Norihiko; Sajiki, Kenichi; Xu, Xingya; Villar-Briones, Alejandro; Arakawa, Orie; Yanagida, Mitsuhiro

    2015-06-01

    Condensin plays fundamental roles in chromosome dynamics. In this study, we determined the binding sites of condensin on fission yeast (Schizosaccharomyces pombe) chromosomes at the level of nucleotide sequences using chromatin immunoprecipitation (ChIP) and ChIP sequencing (ChIP-seq). We found that condensin binds to RNA polymerase I-, II- and III-transcribed genes during both mitosis and interphase, and we focused on pol II constitutive and inducible genes. Accumulation sites for condensin are distinct from those of cohesin and DNA topoisomerase II. Using cell cycle stage and heat-shock-inducible genes, we show that pol II-mediated transcripts cause condensin accumulation. First, condensin's enrichment on mitotically activated genes was abolished by deleting the sep1(+) gene that encodes an M-phase-specific forkhead transcription factor. Second, by raising the temperature, condensin accumulation was rapidly induced at heat-shock protein genes in interphase and even during mid-mitosis. In interphase, condensin accumulates preferentially during the postreplicative phase. Pol II-mediated transcription was neither repressed nor activated by condensin, as levels of transcripts per se did not change when mutant condensin failed to associate with chromosomal DNA. However, massive chromosome missegregation occurred, suggesting that abundant pol II transcription may require active condensin before proper chromosome segregation. PMID:25847133

  11. Daily injection of tumor necrosis factor-{alpha} increases hepatic triglycerides and alters transcript abundance of metabolic genes in lactating dairy cattle.

    PubMed

    Bradford, Barry J; Mamedova, Laman K; Minton, J Ernest; Drouillard, James S; Johnson, Bradley J

    2009-08-01

    To determine whether inflammation can induce bovine fatty liver, we administered recombinant bovine tumor necrosis factor-alpha (rbTNF) to late-lactation Holstein cows. Cows (n = 5/treatment) were blocked by feed intake and parity and randomly assigned within block to control (CON; saline), rbTNF at 2 microg/(kg.d), or pair-fed control (saline, intake matched) treatments. Treatments were administered once daily by subcutaneous injection for 7 d. Plasma samples were collected daily for analysis of glucose and FFA and a liver biopsy was collected on d 7 for triglyceride (TG) and quantitative RT-PCR analyses. Data were analyzed using treatment contrasts to assess effects of tumor necrosis factor-alpha (TNFalpha) and decreased feed intake. By d 7, feed intake of both rbTNF and pair-fed cows was approximately 15% less than CON (P < 0.01). Administration of rbTNF resulted in greater hepatic TNFalpha mRNA and protein abundance and 103% higher liver TG content (P < 0.05) without affecting the plasma FFA concentration. Hepatic carnitine palmitoyltransferase 1 transcript abundance tended to be lower (P = 0.09) and transcript abundance of fatty acid translocase and 1-acyl-glycerol-3-phosphate acyltransferase was higher (both P < 0.05) after rbTNF treatment, consistent with increased FFA uptake and storage as TG. Transcript abundance of glucose-6-phosphatase (P < 0.05) and phosphoenolpyruvate carboxykinase 1 (P = 0.09), genes important for gluconeogenesis, was lower for rbTNF-treated cows. These findings indicate that TNFalpha promotes liver TG accumulation and suggest that inflammatory pathways may also be responsible for decreased glucose production in cows with fatty liver.

  12. O2 abundances in the Martian atmosphere determined using Mars Express SPICAM UV stellar occultation data

    NASA Astrophysics Data System (ADS)

    Lewis, N. K.; Sandel, B. R.; Yelle, R. V.; Bertaux, J.-L.; Montmessin, F.; Quémerais, E.

    2008-09-01

    The distribution of O2 with altitude, latitude, and season is an important factor in the evolution and current stability of Mars' CO2 rich atmosphere. CO2 is photolyzed in the Martian atmosphere to form CO and O according to the following process: CO2 + hn→ CO + O. The atomic oxygen then preferentially recombines to form O2. If this simple reaction is indeed the dominant process in the Martian atmosphere then O2 should be more abundant than the currently accepted value of 0.12 percent [1]. Nair et al. (1994) present a detailed photochemical model of the Martian atmosphere, which shows that the abundance of O2 is largely controlled by reactions with odd hydrogen radicals from photolyzed water in the lower atmosphere. While the Nair et al. (1994) model certainly helps to explain the major photochemical processes at work in theMartian atmosphere, it assumes the abundance of O2 does not vary with latitude and season and is roughly constant with altitude. Our study probes the abundance of O2 in theMartian atmosphere during winter in the southern hemisphere (Ls=90-180) when CO2 condenses out of the atmosphere to form a polar cap. This enrichment of O2 with respect to CO2 during southern Martian winter allows for a more robust detection of O2 in addition to probing the effect of seasonal variations on the photochemistry of the Martian atmosphere. The European Space Agency's Mars Express spacecraft was placed in orbit around Mars on 25 December 2003. The SPICAM (Spectroscopy for the Investigation of the Characteristics of the Atmosphere of Mars) instrument aboard Mars Express measures stellar occultations in the 118-320 nm wavelength region [2]. The stellar occultation technique determines the abundance of chemical species by comparing a reference stellar spectrum (I0) to the same stellar spectrum attenuated by the planetary atmosphere (I). The slant densities, Ni(z), are related to the transmission, Tz(l), through (1) where z is the minimum altitude along the line of

  13. Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

    PubMed

    Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

    2014-10-17

    The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

  14. Expression and Purification of Mitochondrial RNA Polymerase and Transcription Factor A from Drosophila melanogaster.

    PubMed

    Gajewski, John P; Arnold, Jamie J; Salminen, Tiina S; Kaguni, Laurie S; Cameron, Craig E

    2016-01-01

    Mitochondrial gene expression is essential in all organisms. Our understanding of mitochondrial transcription on a biochemical level has been limited by the inability to purify the individual protein components involved in mitochondrial gene expression. Recently, new systems have been identified that permit purification of these proteins from bacteria. However, the generalizability of these systems is not clear. Here, we have applied the technology from the Cameron lab to express and purify mitochondrial RNA polymerase and transcription factor A from Drosophila melanogaster. We show that the use of SUMO system to produce SUMO fusion proteins in bacteria is effective not only for the human and mouse proteins, but also for the fly proteins. The application of this system to produce the mitochondrial proteins from other organisms should permit detailed understanding of mitochondrial transcription from any organism.

  15. 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.

  16. Regulation of His4 Expression by the Saccharomyces Cerevisiae Sin4 Transcriptional Regulator

    PubMed Central

    Jiang, Y. W.; Stillman, D. J.

    1995-01-01

    The yeast SIN4 gene functions in the transcriptional activation and repression of diverse yeast genes. Previous experiments suggest a sin4 mutation affects chromatin structure and thus alters transcriptional regulation. In this report we show that SIN4 is required for full expression of the HIS4, Ty1, and MATα genes, in addition to the previously described SIN4-dependence of CTS1 expression. All of these genes contain within their promoters a binding site for the Rap1p transcriptional regulator. However, SIN4 does not play a direct role either in transcriptional activation or repression by Rap1p. The HIS4 gene can be activated by either of two pathways, the basal or the inducible pathway, and experiments are described that show that a sin4 mutation affects both pathways. It was shown previously that mutation of the Rap1p binding site in the HIS4 promoter causes a similar effect on HIS4 expression and that this promoter mutation also causes a change in chromatin structure. The SNF2/SWI2 gene is also required for full HIS4 expression, and we show that a sin4 snf2 double mutant is not synergistic compared to either single mutant. We show that nucleosomes are positioned at the HIS4 promoter and that this positioning is disrupted in a snf2 mutant but not in a sin4 mutant. These findings suggest that SIN4 plays a distinct role in transcriptional regulation. PMID:7635278

  17. Dynamic expression of transcription factor Brn3b during mouse cranial nerve development.

    PubMed

    Sajgo, Szilard; Ali, Seid; Popescu, Octavian; Badea, Tudor Constantin

    2016-04-01

    During development, transcription factor combinatorial codes define a large variety of morphologically and physiologically distinct neurons. Such a combinatorial code has been proposed for the differentiation of projection neurons of the somatic and visceral components of cranial nerves. It is possible that individual neuronal cell types are not specified by unique transcription factors but rather emerge through the intersection of their expression domains. Brn3a, Brn3b, and Brn3c, in combination with each other and/or transcription factors of other families, can define subgroups of retinal ganglion cells (RGC), spiral and vestibular ganglia, inner ear and vestibular hair cell neurons in the vestibuloacoustic system, and groups of somatosensory neurons in the dorsal root ganglia. The present study investigates the expression and potential role of the Brn3b transcription factor in cranial nerves and associated nuclei of the brainstem. We report the dynamic expression of Brn3b in the somatosensory component of cranial nerves II, V, VII, and VIII and visceromotor nuclei of nerves VII, IX, and X as well as other brainstem nuclei during different stages of development into adult stage. We find that genetically identified Brn3b(KO) RGC axons show correct but delayed pathfinding during the early stages of embryonic development. However, loss of Brn3b does not affect the anatomy of the other cranial nerves normally expressing this transcription factor.

  18. DNA methylation-mediated transcription factors regulate Piwil1 expression during chicken spermatogenesis

    PubMed Central

    QIU, Lingling; XU, Lu; CHANG, Guobin; GUO, Qixin; LIU, Xiangping; BI, Yulin; ZHANG, Yu; WANG, Hongzhi; WANG, Kehua; LU, Wei; REN, Lichen; ZHU, Pengfei; WU, Yun; ZHANG, Yang; XU, Qi; CHEN, Guohong

    2016-01-01

    The P-element induced wimpy testis (Piwi) protein family is responsible for initiating spermatogenesis and maintaining the integrity of germ cells and stem cells, but little is known regarding its transcriptional regulation in poultry. Here, we characterized the methylation status of the Piwil1 promoter in five different spermatogenic cell lines using direct bisulfite pyrosequencing and determined that methylation correlates negatively with germ cell type-specific expression patterns of piwil1. We demonstrated that methylation of the −148 CpG site, which is the predicted binding site for the transcription factors TCF3 and NRF1, was differentially methylated in different spermatogenic cells. This site was completely methylated in PGCs (primordial germ cells), but was unmethylated in round spermatids. A similar result was obtained in the region from +121 to +139 CpG sites of the Piwil1 promoter CpG island, which was predicted to contain SOX2 binding sites. In addition, demethylation assays further demonstrated that DNA methylation indeed regulates Piwil1 expression during chicken spermatogenesis. Combined with transcription factor binding site prediction, we speculate that methylation influences the recruitment of corresponding transcription factors. Collectively, we show the negative correlation between promoter methylation and piwil1 expression and that the spatiotemporal expression of chicken Piwil1 from the PGC stage to the round spermatid stage is influenced by methylation-mediated transcription factor regulation. PMID:27108736

  19. Comparative characteristics of mu chain and alpha chain transcripts expressed by individual tonsil plasma cells.

    PubMed

    Yavuz, S; Grammer, A C; Yavuz, A S; Nanki, T; Lipsky, P E

    2001-01-01

    Plasma cells (PCs) are one of the two major cell types generated during germinal center reactions. To test the hypothesis that PCs express a unique repertoire of immunoglobulin (Ig) genes resulting from intensive antigenic stimulation and selection, the mutational pattern and distribution of V(H) gene segments within 178 transcripts amplified from individual IgM and IgA secreting tonsil PCs were analyzed. The results demonstrated that both mu and alpha transcripts expressed repertoires with limited diversity. Moreover, both mu and alpha transcripts were heavily mutated, with a significantly increased mutational frequency noted for alpha compared to mu transcripts (5.0 x 10(-2) vs 1.8 x 10(-2), P<0.001). In addition, both mu and alpha transcripts showed significantly greater targeting of mutations to RGYW motifs (purine/guanine/pyrimidine/A or T) compared to memory B cells. Finally, clonally expanded cells were detected in alpha but not mu PC compartments. These results indicate that antigen driven stimulation and selection shape the entire expressed PC repertoire, but the impact is greater in alpha expressing PCs.

  20. Differential expression analysis of transcripts related to oil metabolism in maturing seeds of Jatropha curcas L.

    PubMed

    Chandran, Divya; Sankararamasubramanian, H M; Kumar, M Ashok; Parida, Ajay

    2014-04-01

    Jatropha curcas has been widely studied at the molecular level due to its potential as an alternative source of fuel. Many of the reports till date on this plant have focussed mainly on genes contributing to the accumulation of oil in its seeds. A suppression subtractive hybridization strategy was employed to identify genes which are differentially expressed in the mid maturation stage of J. curcas seeds. Random expressed sequence tag sequencing of the cDNA subtraction library resulted in 385 contigs and 1,428 singletons, with 591 expressed sequence tags mapping for enzymes having catalytic roles in various metabolic pathways. Differences in transcript levels in early and mid-to-late maturation stages of seeds were also investigated using sequence information obtained from the cDNA subtraction library. Seven out of 12 transcripts having putative roles in central carbon metabolism were up regulated in early seed maturation stage while lipid metabolism related transcripts were detected at higher levels in the later stage of seed maturation. Interestingly, 4 of the transcripts revealed putative alternative splice variants that were specifically present or up regulated in the early or late maturation stage of the seeds. Transcript expression patterns from the current study using maturing seeds of J. curcas reveal a subtle balancing of oil accumulation and utilization, which may be influenced by their energy requirements.

  1. Expression of Transcription Factor GATA-6 in Alveolar Epithelial Cells Is Linked to Neonatal Lung Disease

    PubMed Central

    Vähätalo, Riika; Asikainen, Tiina M.; Karikoski, Riitta; Kinnula, Vuokko L.; White, Carl W.; Andersson, Sture; Heikinheimo, Markku; Myllärniemi, Marjukka

    2011-01-01

    Background Premature birth and respiratory distress syndrome (RDS) are risk factors for disturbed lung development and bronchopulmonary dysplasia (BPD). The molecular mechanisms related to prematurity and BPD remain largely unknown. Epithelial expression of the transcription factor GATA-6 has been implicated in normal and abnormal murine lung development. Objectives The possible involvement of GATA-6 in the normal development and in RDS and BPD was investigated in the human and baboon lung. Methods Immunohistochemistry was used to study the expression of GATA-6 and thyroid transcription factor 1 in lung specimens from different age groups of human and baboon fetuses and newborns with lung disease. Furthermore, the regulatory role of TGF-β1 in GATA-6 expression was investigated in human pulmonary epithelial cell lines using RT-PCR. Results GATA-6 expression increased in the developing human airway epithelium along with advancing gestation, but diminished to negligible at birth. In RDS, GATA-6 expression was enhanced at 5–7 days after birth, and decreased thereafter. In BPD, the expression of GATA-6 in alveolar epithelial cells was low. These results were confirmed and extended using an established baboon model of prematurity. The in vitro experiments revealed that TGF-β1 induces GATA-6 and thyroid transcription factor 1 expression in lung epithelial cells. Conclusions Our results suggest that the expression of GATA-6 at the early stages of the preterm lung may be related to impaired postnatal alveolar development. PMID:21071980

  2. Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland.

    PubMed

    Stehle, J H; Foulkes, N S; Molina, C A; Simonneaux, V; Pévet, P; Sassone-Corsi, P

    1993-09-23

    Transcription factor CREM appears to play a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. This axis is modulated by the pineal hormone melatonin, whose production is in turn driven by the endogenous clock. There is striking circadian fluctuation of a novel CREM isoform, ICER, which is expressed at high levels during the night. ICER is generated from an alternative, intronic promoter and functions as a powerful repressor of cyclic AMP-induced transcription. Rhythmic adrenergic signals originated by the clock direct ICER expression by stimulation of the cAMP signal transduction pathway.

  3. The influence of salinity on the abundance, transcriptional activity, and diversity of AOA and AOB in an estuarine sediment: a microcosm study.

    PubMed

    Zhang, Yan; Chen, Lujun; Dai, Tianjiao; Tian, Jinping; Wen, Donghui

    2015-11-01

    Estuarine sediment-seawater microcosms were established to evaluate the influence of salinity on the population, transcriptional activity, and diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB). AOA was found to show the most abundant and the highest transcriptional activity under moderate salinity; on the other hand, AOB abundance was not sensitive to salinity variation but showed the highest transcriptional activity in the low-salinity microcosms. AOA exhibited more advantages than AOB on growth and ammonia-oxidizing activity under moderate- and high-salinity environments. The highest richness and diversity of active AOA were found under salinity of 15 psu. All the active AOA detected under the salinities studied were clustered into Nitrosopumilus maritimus linage, with the composition shifted from N. maritimus C12 cluster, N. maritimus like 1.1 cluster, N. maritimus SCM1 cluster, and N. maritimus like 1.2 cluster to N. maritimus C12 and N. maritimus A10 clusters when salinity was increased from 5 to 30 psu.

  4. Differential expression of the transcripts of Spartina alterniflora Loisel (smooth cordgrass) induced in response to petroleum hydrocarbon.

    PubMed

    RamanaRao, Mangu Venkata; Weindorf, David; Breitenbeck, Gary; Baisakh, Niranjan

    2012-05-01

    Petroleum hydrocarbons (PHC) in soil are potentially toxic to plants and exert negative effect on the environment and human health. To understand the effect of PHC on the gene expression profile of a wetland plant Spartina alterniflora in the coastal Louisiana, plants were subject up to 40% PHC under greenhouse conditions. The plants exposed to PHC showed 21% reduction of leaf total chlorophyll after 2 weeks of stress. Using 20 annealing control primers, 28 differentially expressing genes (DEGs) were identified in leaf and root tissues of S. alterniflora in response to PHC stress. Eleven of these 28 DEGs had role in either molecular function (chlorophyll a-b binding protein, HSP70, NADH, RAN1-binding protein, and RNA-binding protein), biological processes (cell wall protein, nucelosome/chromatin assembly factor) or cellular function (30 S ribosomal protein). This indicated that genes in different regulatory pathways of S. alterniflora were involved in response to PHC. All DEGs showed reduced transcript accumulation in root under oil stress, whereas they showed up- or down-regulation in their transcript abundance in leaf depending on the concentration of the PHC. The genes identified through this study could be used in the genetic screen of S. alterniflora for resistance to PHC.

  5. Dorsal transcription factor is involved in regulating expression of crustin genes during white spot syndrome virus infection.

    PubMed

    Huang, Xin; Wang, Wen; Ren, Qian

    2016-10-01

    Nuclear factor-kappa B (NF-κB) pathways play important roles in innate immune responses. In this study, we identified a dorsal homolog (MrDorsal) from freshwater prawn Macrobrachium rosenbergii. The full-length cDNA of MrDorsal comprised 2533 bp with an open reading frame of 1986 bp, which encoded a peptide of 661 amino acid residues. Amino acid sequence analysis showed that MrDorsal contains a Rel homolog domain and an IPT/TIG (i.e., Ig-like, plexin, and transcription factors) domain. The signature sequence of dorsal protein FRYMCEG existed in the deduced amino acid sequence. Sequence analysis showed that MrDorsal shared high similarities with Dorsal from invertebrate species. MrDorsal was abundant in the hemocytes and gills of healthy prawns but minute levels were detected in other tissues. The expression of MrDorsal was significantly upregulated 48 h after the white spot syndrome virus (WSSV-) challenge. Knockdown of MrDorsal using double-stranded RNA could suppress the transcription of crustin genes (MrCrustin2 and MrCrustin4) in gills of prawns after 48 h of the WSSV challenge. Results indicated that MrDorsal was involved to regulate the expression of crustin genes and it might play potential important roles during WSSV infection. PMID:27181712

  6. Cloning of nitric oxide associated 1 (NOA1) transcript from oil palm (Elaeis guineensis) and its expression during Ganoderma infection.

    PubMed

    Kwan, Yee-Min; Meon, Sariah; Ho, Chai-Ling; Wong, Mui-Yun

    2015-02-01

    Nitric oxide associated 1 (NOA1) protein is implicated in plant disease resistance and nitric oxide (NO) biosynthesis. A full-length cDNA encoding of NOA1 protein from oil palm (Elaeis guineensis) was isolated and designated as EgNOA1. Sequence analysis suggested that EgNOA1 was a circular permutated GTPase with high similarity to the bacterial YqeH protein of the YawG/YlqF family. The gene expression of EgNOA1 and NO production in oil palm root tissues treated with Ganoderma boninense, the causal agent of basal stem rot (BSR) disease were profiled to investigate the involvement of EgNOA1 during fungal infection and association with NO biosynthesis. Real-time PCR (qPCR) analysis revealed that the transcript abundance of EgNOA1 in root tissues was increased by G. boninense treatment. NO burst in Ganoderma-treated root tissue was detected using Griess reagent, in advance of the up-regulation of the EgNOA1 transcript. This indicates that NO production was independent of EgNOA1. However, the induced expression of EgNOA1 in Ganoderma-treated root tissues implies that it might be involved in plant defense responses against pathogen infection.

  7. Transcriptional coactivator p300 regulates glucose-induced gene expression in endothelial cells.

    PubMed

    Chen, Shali; Feng, Biao; George, Biju; Chakrabarti, Rana; Chen, Megan; Chakrabarti, Subrata

    2010-01-01

    Sustained hyperglycemia in diabetes causes alteration of a large number of transcription factors and mRNA transcripts, leading to tissue damage. We investigated whether p300, a transcriptional coactivator with histone acetyl transferase activity, regulates glucose-induced activation of transcription factors and subsequent upregulation of vasoactive factors and extracellular matrix (ECM) proteins in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated in varied glucose concentrations and were studied after p300 small interfering RNA (siRNA) transfection, p300 overexpression, or incubation with the p300 inhibitor curcumin. Histone H2AX phosphorylation and lysine acetylation were examined for oxidative DNA damage and p300 activation. Screening for transcription factors was performed with the Luminex system. Alterations of selected transcription factors were validated. mRNA expression of p300, endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), and fibronectin (FN) and its splice variant EDB(+)FN and FN protein production were analyzed. HUVECs in 25 mmol/l glucose showed increased p300 production accompanied by increased binding of p300 to ET-1 and FN promoters, augmented histone acetylation, H2AX phosphorylation, activation of multiple transcription factors, and increased mRNA expression of vasoactive factors and ECM proteins. p300 overexpression showed a glucose-like effect on the mRNA expression of ET-1, VEGF, and FN. Furthermore, siRNA-mediated p300 blockade or chemical inhibitor of p300 prevented such glucose-induced changes. Similar mRNA upregulation was also seen in the organ culture of vascular tissues, which was prevented by p300 siRNA transfection. Data from these studies suggest that glucose-induced p300 upregulation is an important upstream epigenetic mechanism regulating gene expression of vasoactive factors and ECM proteins in endothelial cells and is a potential therapeutic target for diabetic complications.

  8. PAT4 is abundantly expressed in excitatory and inhibitory neurons as well as epithelial cells.

    PubMed

    Roshanbin, Sahar; Hellsten, Sofie V; Tafreshiha, Atieh; Zhu, Yinan; Raine, Amanda; Fredriksson, Robert

    2014-04-01

    PAT4, the fourth member of the SLC36/proton dependent amino acid transporter (PAT) family, is a high-affinity, low capacity electroneutral transporter of neutral amino acids like proline and tryptophan. It has also been associated with the function of mTORC1, a complex in the mammalian target of rapamycin (mTOR) pathway. We performed in situ hybridization and immunohistological analysis to determine the expression profile of PAT4, as well as an RT-PCR study on tissue from mice exposed to leucine. We performed a phylogenetic analysis to determine the evolutionary origin of PAT4. The in situ hybridization and the immunohistochemistry on mouse brain sections and hypothalamic cells showed abundant PAT4 expression in the mouse brain intracellularly in both inhibitory and excitatory neurons, partially co-localizing with lysosomal markers and epithelial cells lining the ventricles. Its location in epithelial cells around the ventricles indicates a transport of substrates across the blood brain barrier. Phylogenetic analysis showed that PAT4 belongs to an evolutionary old family most likely predating animals, and PAT4 is the oldest member of that family. PMID:24530433

  9. Expression of transcription factor AP-2α predicts survival in epithelial ovarian cancer

    PubMed Central

    Anttila, M A; Kellokoski, J K; Moisio, K I; Mitchell, P J; Saarikoski, S; Syrjänen, K; Kosma, V-M

    2000-01-01

    The 52-kDa activator protein (AP)-2 is a DNA-binding transcription factor which has been reported to have growth inhibitory effects in cancer cell lines and in human tumours. In this study the expression of AP-2α was analysed in 303 epithelial ovarian carcinomas by immunohistochemistry (IHC) with a polyclonal AP-2α antibody and its mRNA status was determined by in situ hybridization (ISH) and reverse transcriptase-polymerase chain reaction (RT-PCR). The immunohistochemical expression of AP-2α was correlated with clinicopathological variables, p21/WAF1 protein expression and survival. In normal ovaries, epithelial cells expressed AP-2α protein only in the cytoplasm. In carcinomas nuclear AP-2α expression was observed in 28% of the cases although cytoplasmic expression was more common (51%). The expression of AP-2α varied according to the histological subtype and differentiation. AP-2α and p21/WAF1 expressions did not correlate with each other. Both in univariate (P = 0.002) and multivariate analyses (relative risks (RR) 1.6, 95% confidence interval (CI) 1.13–2.18, P = 0.007) the high cytoplasmic AP-2α expression favoured the overall survival. In contrast, the nuclear AP-2α expression combined with low cytoplasmic expression increased the risk of dying of ovarian cancer (RR = 2.10, 95% CI 1.13–3.83, P = 0.018). The shift in the expression pattern of AP-2α (nuclear vs cytoplasmic) in carcinomas points out to the possibility that this transcription factor may be used by oncogenes in certain histological subtypes. Based on the mRNA analyses, the incomplete expression and translation of AP-2α in ovarian cancer may be due to post-transcriptional regulation. © 2000 Cancer Research Campaign PMID:10864206

  10. Identification of 30 MYB transcription factor genes and analysis of their expression during abiotic stress in peanut (Arachis hypogaea L.).

    PubMed

    Chen, Na; Yang, Qingli; Pan, Lijuan; Chi, Xiaoyuan; Chen, Mingna; Hu, Dongqing; Yang, Zhen; Wang, Tong; Wang, Mian; Yu, Shanlin

    2014-01-01

    The MYB superfamily constitutes one of the most abundant groups of transcription factors and plays central roles in developmental processes and defense responses in plants. In the work described in this article, 30 unique peanut MYB genes that contained full-length cDNA sequences were isolated. The 30 genes were grouped into three categories: one R1R2R3-MYB, nine R2R3-MYBs and 20 MYB-related members. The sequence composition of the R2 and R3 repeats was conserved among the nine peanut R2R3-MYB proteins. Phylogenetic comparison of the members of this superfamily between peanut and Arabidopsis revealed that the putative functions of some peanut MYB proteins were clustered into the Arabidopsis functional groups. Expression analysis during abiotic stress identified a group of MYB genes that responded to at least one stress treatment. This is the first comprehensive study of the MYB gene family in peanut.

  11. MicroRNA-218 inhibits melanogenesis by directly suppressing microphthalmia-associated transcription factor expression

    PubMed Central

    Guo, Jia; Zhang, Jin-Fang; Wang, Wei-Mao; Cheung, Florence Wing-ki; Lu, Ying-fei; Ng, Chi-fai; Kung, Hsiang-fu; Liu, Wing-keung

    2014-01-01

    The microphthalmia-associated transcription factor (MITF) is a pivotal regulator of melanogenic enzymes for melanogenesis, and its expression is modulated by many transcriptional factors at the transcriptional level or post-transcriptional level through microRNAs (miRNAs). Although several miRNAs modulate melanogenic activities, there is no evidence of their direct action on MITF expression. Out of eight miRNAs targeting the 3′-UTR of Mitf predicted by bioinformatic programs, our results show miR-218 to be a novel candidate for direct action on MITF expression. Ectopic miR-218 dramatically reduced MITF expression, suppressed tyrosinase activity, and induced depigmentation in murine immortalized melan-a melanocytes. MiR-218 also suppressed melanogenesis in human pigmented skin organotypic culture (OTC) through the repression of MITF. An inverse correlation between MITF and miR-218 expression was found in human primary skin melanocytes and melanoma cell lines. Taken together, our findings demonstrate a novel mechanism involving miR-218 in the regulation of the MITF pigmentary process and its potential application for skin whitening therapy. PMID:24824743

  12. Transcriptional control of MHC class II gene expression during differentiation from B cells to plasma cells.

    PubMed

    Dellabona, P; Latron, F; Maffei, A; Scarpellino, L; Accolla, R S

    1989-04-15

    In this study we investigated the molecular mechanisms responsible for the extinction of the constitutive MHC class II gene expression of human B cells on somatic cell hybridization with murine plasmocytoma cells. We found that this event is due to trans-acting suppressor functions of mouse origin pre-existing in the plasmocytoma cells and acting at transcriptional level. Transcription of the entire family of human class II genes is suppressed, including genes as DO beta for which a distinct regulation of expression in B cells had been previously demonstrated. Suppression appears specific for class II genes because in the hybrids expression of MHC class I genes of mouse is unaffected and of human only partially reduced. Interestingly, also murine invariant chain gene is expressed in both parental plasmocytoma and hybrid cells although at reduced amounts as compared to a murine class II positive B cell line. The class II negative phenotype of hybrid cells and parental plasmocytoma cells is highly stable and unaffected by treatment with protein synthesis inhibitors, suggesting that the transcriptional suppressor function is not mediated by rapid, labile turning-over proteins. Possible mechanisms responsible for transcriptional regulation of MHC class II gene expression during terminal differentiation of B cells to plasma cells are discussed. PMID:2495328

  13. Intracompartmental and Intercompartmental Transcriptional Networks Coordinate the Expression of Genes for Organellar Functions1[W

    PubMed Central

    Leister, Dario; Wang, Xi; Haberer, Georg; Mayer, Klaus F.X.; Kleine, Tatjana

    2011-01-01

    Genes for mitochondrial and chloroplast proteins are distributed between the nuclear and organellar genomes. Organelle biogenesis and metabolism, therefore, require appropriate coordination of gene expression in the different compartments to ensure efficient synthesis of essential multiprotein complexes of mixed genetic origin. Whereas organelle-to-nucleus signaling influences nuclear gene expression at the transcriptional level, organellar gene expression (OGE) is thought to be primarily regulated posttranscriptionally. Here, we show that intracompartmental and intercompartmental transcriptional networks coordinate the expression of genes for organellar functions. Nearly 1,300 ATH1 microarray-based transcriptional profiles of nuclear and organellar genes for mitochondrial and chloroplast proteins in the model plant Arabidopsis (Arabidopsis thaliana) were analyzed. The activity of genes involved in organellar energy production (OEP) or OGE in each of the organelles and in the nucleus is highly coordinated. Intracompartmental networks that link the OEP and OGE gene sets serve to synchronize the expression of nucleus- and organelle-encoded proteins. At a higher regulatory level, coexpression of organellar and nuclear OEP/OGE genes typically modulates chloroplast functions but affects mitochondria only when chloroplast functions are perturbed. Under conditions that induce energy shortage, the intercompartmental coregulation of photosynthesis genes can even override intracompartmental networks. We conclude that dynamic intracompartmental and intercompartmental transcriptional networks for OEP and OGE genes adjust the activity of organelles in response to the cellular energy state and environmental stresses, and we identify candidate cis-elements involved in the transcriptional coregulation of nuclear genes. Regarding the transcriptional regulation of chloroplast genes, novel tentative target genes of σ factors are identified. PMID:21775496

  14. Transcriptome Profiling Reveals Differentially Expressed Transcripts Between the Human Adrenal Zona Fasciculata and Zona Reticularis

    PubMed Central

    Rege, Juilee; Nakamura, Yasuhiro; Wang, Tao; Merchen, Todd D.; Sasano, Hironobu

    2014-01-01

    Context: The human adrenal zona fasciculata (ZF) and zona reticularis (ZR) are responsible for the production of cortisol and 19-carbon steroids (often called adrenal androgens), respectively. However, the gene profiles and exact molecular mechanisms leading to the functional phenotype of the ZF and ZR are still not clearly defined. In the present study, we identified the transcripts that are differentially expressed in the ZF and ZR. Objective: The objective of the study was to compare the transcriptome profiles of ZF and ZR. Design and Methods: ZF and ZR were microdissected from 10 human adrenals. Total RNA was extracted from 10 ZF/ZR pairs and hybridized to Illumina microarray chips. The 10 most differentially expressed transcripts were studied with quantitative RT-PCR (qPCR). Immunohistochemistry was also performed on four zone-specific genes. Results: Microarray results demonstrated that only 347 transcripts of the 47 231 were significantly different by 2-fold or greater in the ZF and ZR. ZF had 195 transcripts with 2-fold or greater increase compared with its paired ZR, whereas ZR was found to have 152 transcripts with 2-fold or greater higher expression than in ZF. Microarray and qPCR analysis of transcripts encoding steroidogenic enzymes (n = 10) demonstrated that only 3β-hydroxysteroid dehydrogenase, steroid sulfotransferase, type 5 17β-hydroxysteroid dehydrogenase, and cytochrome b5 were significantly different. Immunohistochemistry and qPCR studies confirmed that the ZF had an increased expression of lymphoid enhancer-binding factor 1 and nephroblastoma overexpressed, whereas ZR showed an increased expression of solute carrier family 27 (fatty acid transporter) (SLC27A2), member 2 and TSPAN12 (tetraspanin 12) Conclusion: Microarray revealed several novel candidate genes for elucidating the molecular mechanisms governing the ZF and ZR, thereby increasing our understanding of the functional zonation of these two adrenocortical zones. PMID:24423296

  15. Modulation of gap junction transcript and protein expression during pregnancy in the rat

    PubMed Central

    1990-01-01

    The expression of three different gap junction transcripts, alpha 1 (Cx43), beta 1 (Cx32), and beta 2 (Cx26) was examined in several organs during pregnancy in the rat. In all of the organs that were examined-- uterus, ovary, heart, and liver--there was a strong correlation between levels of gap junction mRNA and gap junction antigens that were detected at different stages of pregnancy. A striking change in alpha 1 transcript levels (a 5.5-fold increase) was detected in the uterine myometrium on the day before parturition. This elevation of the alpha 1 transcript is thought to be associated with the formation of gap junctions that are required for synchronizing the contractility of the myometrial cells during parturition. 2 d before parturition, there was a detectable elevation of beta 2 transcripts and protein in the endometrial epithelium, which was then followed by a dramatic decrease in beta 2 gap junctional protein on the day before parturition. There was also a substantial elevation of alpha 1 transcripts (a 6.7-fold increase) in the stromal regions of the ovary on the day before parturition that was identical to the temporal pattern of alpha 1 expression in the myometrium. In all three instances--the alpha 1 transcripts in the myometrium, beta 2 transcripts in the endometrium, and alpha 1 transcripts in the ovary--the transcript modulation appeared to be cell specific, because the changes in transcript levels of these three gene products occurred independently of the poly(A) + RNA concentrations at the same pregnancy stages in the respective organs. There were no specific changes detected in gap junction transcript levels in the heart and liver during pregnancy. These observations indicate that a cell-specific modulation of gap junction expression occurs in two regions of the uterus and the ovary during pregnancy. Further, it appears that the same gap junction gene in different organs, such as the alpha 1 gene in the uterine myometrium and the heart, can be

  16. Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

    SciTech Connect

    Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

    2006-05-25

    Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

  17. Transcript Expression Patterns Illuminate the Mechanistic Background of Hormesis in Caenorhabditis Elegans Maupas

    PubMed Central

    Steinberg, Christian E.W.; Pietsch, Kerstin; Saul, Nadine; Menzel, Stefanie; Swain, Suresh C.; Stürzenbaum, Stephen R.; Menzel, Ralph

    2013-01-01

    The animal model Caenorhabditis elegans was employed to study polyphenol- and humic substances-induced hormetic changes in lifespan. A detailed insight into the underlying mechanism of hormesis was uncovered by applying whole genome DNA microarray experimentation over a range of quercetin (Q), tannic acid (TA), and humic substances (HuminFeed®, HF) concentrations. The transcriptional response to all exposures followed a non-linear mode which highlighted differential signaling and metabolic pathways. While low Q concentrations regulated processes improving the health of the nematodes, higher concentrations extended lifespan and modulated substantially the global transcriptional response. Over-represented transcripts were notably part of the biotransformation process: enhanced catabolism of toxic intermediates possibly contributes to the lifespan extension. The regulation of transcription, Dauer entry, and nucleosome suggests the presence of distinct exposure dependent differences in transcription and signaling pathways. TA- and HF-mediated transcript expression patterns were overall similar to each other, but changed across the concentration range indicating that their transcriptional dynamics are complex and cannot be attributed to a simple adaptive response. In contrast, Q-mediated hormesis was well aligned to fit the definition of an adaptive response. Simple molecules are more likely to induce an adaptive response than more complex molecules.

  18. NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei.

    PubMed

    Narayanan, Mani Shankar; Kushwaha, Manish; Ersfeld, Klaus; Fullbrook, Alexander; Stanne, Tara M; Rudenko, Gloria

    2011-03-01

    Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

  19. STAT5 Outcompetes STAT3 To Regulate the Expression of the Oncogenic Transcriptional Modulator BCL6

    PubMed Central

    Walker, Sarah R.; Nelson, Erik A.; Yeh, Jennifer E.; Pinello, Luca; Yuan, Guo-Cheng

    2013-01-01

    Inappropriate activation of the transcription factors STAT3 and STAT5 has been shown to drive cancer pathogenesis through dysregulation of genes involved in cell survival, growth, and differentiation. Although STAT3 and STAT5 are structurally related, they can have opposite effects on key genes, including BCL6. BCL6, a transcriptional repressor, has been shown to be oncogenic in diffuse large B cell lymphoma. BCL6 also plays an important role in breast cancer pathogenesis, a disease in which STAT3 and STAT5 can be activated individually or concomitantly. To determine the mechanism by which these oncogenic transcription factors regulate BCL6 transcription, we analyzed their effects at the levels of chromatin and gene expression. We found that STAT3 increases expression of BCL6 and enhances recruitment of RNA polymerase II phosphorylated at a site associated with transcriptional initiation. STAT5, in contrast, represses BCL6 expression below basal levels and decreases the association of RNA polymerase II at the gene. Furthermore, the repression mediated by STAT5 is dominant over STAT3-mediated induction. STAT5 exerts this effect by displacing STAT3 from one of the two regulatory regions to which it binds. These findings may underlie the divergent biology of breast cancers containing activated STAT3 alone or in conjunction with activated STAT5. PMID:23716595

  20. Regulating expression of cell and tissue-specific genes by modifying transcription

    SciTech Connect

    Beachy, Roger N; Dai, Shunhong

    2010-06-14

    Transcriptional regulation is the primary step to control gene expression, therefore function. Such regulation is achieved primarily via a combination of the activities of the promoter cis regulatory DNA elements and trans regulatory proteins that function through binding to these DNA elements. Rice bZIP transcription factors RF2a, RF2b and RLP1 play key roles in regulating the activity of a vascular tissue specific promoter isolated from Rice Tungro Bacilliform Virus (RTBV), through their interactions with the Box II essential cis element located in the promoter (Dai et al., 2006., Dai et al., 2004., Yin et al., 1997). RF2a, RF2b and RLP1 possess multiple regulatory domains. Functional characterization reveals that those domains can activate or repress the activity of the RTBV promoter. It is equally as important to recognize that these proteins control plant development by regulating differentiation and/or function of the vascular tissues. Studies of transcriptional regulation of the RTBV promoter by this group of bZIP proteins will not only provide insights about gene expression in the vascular tissue, but also insights about general mechanisms of transcription activation and repression. The knowledge gained from this research will also enable us to develop a well-described set of tools that can be used to control expression of multiple genes in transgenic plants. We have proposed characterize the function domains of RF2a, RF2b and RLP1 and explore the biological function of the transcription repressor RLP1.

  1. Links between Transcription, Environmental Adaptation and Gene Variability in Escherichia coli: Correlations between Gene Expression and Gene Variability Reflect Growth Efficiencies.

    PubMed

    Feugeas, Jean-Paul; Tourret, Jerome; Launay, Adrien; Bouvet, Odile; Hoede, Claire; Denamur, Erick; Tenaillon, Olivier

    2016-10-01

    Gene expression is known to be the principle factor explaining how fast genes evolve. Highly transcribed genes evolve slowly because any negative impact caused by a particular mutation is magnified by protein abundance. However, gene expression is a phenotype that depends both on the environment and on the strains or species. We studied this phenotypic plasticity by analyzing the transcriptome profiles of four Escherichia coli strains grown in three different culture media, and explored how expression variability was linked to gene allelic diversity. Genes whose expression changed according to the media and not to the strains were less polymorphic than other genes. Genes for which transcription depended predominantly on the strain were more polymorphic than other genes and were involved in sensing and responding to environmental changes, with an overrepresentation of two-component system genes. Surprisingly, we found that the correlation between transcription and gene diversity was highly variable among growth conditions and could be used to quantify growth efficiency of a strain in a medium. Genetic variability was found to increase with gene expression in poor growth conditions. As such conditions are also characterized by down-regulation of all DNA repair systems, including transcription-coupled repair, we suggest that gene expression under stressful conditions may be mutagenic and thus leads to a variability in mutation rate among genes in the genome which contributes to the pattern of protein evolution.

  2. 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.

  3. ORTI: An Open-Access Repository of Transcriptional Interactions for Interrogating Mammalian Gene Expression Data

    PubMed Central

    Ma, Xiuquan; Burykin, Timur; James, David E.; Kuncic, Zdenka

    2016-01-01

    Transcription factors (TFs) play a fundamental role in coordinating biological processes in response to stimuli. Consequently, we often seek to determine the key TFs and their regulated target genes (TGs) amidst gene expression data. This requires a knowledge-base of TF-TG interactions, which would enable us to determine the topology of the transcriptional network and predict novel regulatory interactions. To address this, we generated an Open-access Repository of Transcriptional Interactions, ORTI, by integrating available TF-TG interaction databases. These databases rely on different types of experimental evidence, including low-throughput assays, high-throughput screens, and bioinformatics predictions. We have subsequently categorised TF-TG interactions in ORTI according to the quality of this evidence. To demonstrate its capabilities, we applied ORTI to gene expression data and identified modulated TFs using an enrichment analysis. Combining this with pairwise TF-TG interactions enabled us to visualise temporal regulation of a transcriptional network. Additionally, ORTI enables the prediction of novel TF-TG interactions, based on how well candidate genes co-express with known TGs of the target TF. By filtering out known TF-TG interactions that are unlikely to occur within the experimental context, this analysis predicts context-specific TF-TG interactions. We show that this can be applied to experimental designs of varying complexities. In conclusion, ORTI is a rich and publicly available database of experimentally validated mammalian transcriptional interactions which is accompanied with tools that can identify and predict transcriptional interactions, serving as a useful resource for unravelling the topology of transcriptional networks. PMID:27723773

  4. Transcriptional regulation of hydroxypyruvate reductase gene expression by cytokinin in etiolated pumpkin cotyledons.

    PubMed

    Andersen, B R; Jin, G; Chen, R; Ertl, J R; Chen, C M

    1996-01-01

    To understand the mechanisms by which the expression of a specific gene is modulated by cytokinin, the regulation of hydroxypyruvate reductase (HPR) transcript levels by N6-benzyladenine (BA) in etiolated pumpkin (Cucurbita pepo L. cv. Halloween) cotyledons was investigated. A pumpkin HPR cDNA was generated by reverse transcriptase-polymerase chain reaction and its nucleotide sequence was determined. An antisense HPR RNA was prepared for RNase protection analysis of HPR-mRNA expression patterns in the cotyledons of dark-grown pumpkin seedlings. Treatment of the cotyledons with BA was shown to modulate HPR mRNA levels in a dose- and time-dependent manner. Similarly, nuclear run-on studies showed that the rate of transcription was also enhanced by BA treatment of the cotyledons. These results suggest that the enhancement of HPR mRNA by cytokinin is, at least in part, at the level of transcription. PMID:8580766

  5. Real-time reverse-transcription polymerase chain reaction: technical considerations for gene expression analysis.

    PubMed

    Doak, Shareen H; Zaïr, Zoulikha M

    2012-01-01

    The reverse transcription - polymerase chain reaction (RT-PCR) is a sensitive technique for the quantification of steady-state mRNA levels, particularly in samples with limited quantities of extracted RNA, or for analysis of low level transcripts. The procedure amplifies defined mRNA transcripts by taking advantage of retroviral enzymes with reverse transcriptase (RT) activity, coupled to PCR. The resultant PCR product concentration is directly proportional to the initial starting quantity of mRNA, therefore allowing quantification of gene expression by incorporation of a fluorescence detector for the appropriate amplicons. In this chapter, we describe a number of the most popular techniques for performing RT-PCR and detail the subsequent analysis methodologies required to interpret the resultant data in either a relative manner or through absolute quantification of gene expression levels.

  6. Genetic variability of transcript abundance in pig peri-mortem skeletal muscle: eQTL localized genes involved in stress response, cell death, muscle disorders and metabolism

    PubMed Central

    2011-01-01

    Background The genetics of transcript-level variation is an exciting field that has recently given rise to many studies. Genetical genomics studies have mainly focused on cell lines, blood cells or adipose tissues, from human clinical samples or mice inbred lines. Few eQTL studies have focused on animal tissues sampled from outbred populations to reflect natural genetic variation of gene expression levels in animals. In this work, we analyzed gene expression in a whole tissue, pig skeletal muscle sampled from individuals from a half sib F2 family shortly after slaughtering. Results QTL detection on transcriptome measurements was performed on a family structured population. The analysis identified 335 eQTLs affecting the expression of 272 transcripts. The ontologic annotation of these eQTLs revealed an over-representation of genes encoding proteins involved in processes that are expected to be induced during muscle development and metabolism, cell morphology, assembly and organization and also in stress response and apoptosis. A gene functional network approach was used to evidence existing biological relationships between all the genes whose expression levels are influenced by eQTLs. eQTLs localization revealed a significant clustered organization of about half the genes located on segments of chromosome 1, 2, 10, 13, 16, and 18. Finally, the combined expression and genetic approaches pointed to putative cis-drivers of gene expression programs in skeletal muscle as COQ4 (SSC1), LOC100513192 (SSC18) where both the gene transcription unit and the eQTL affecting its expression level were shown to be localized in the same genomic region. This suggests cis-causing genetic polymorphims affecting gene expression levels, with (e.g. COQ4) or without (e.g. LOC100513192) potential pleiotropic effects that affect the expression of other genes (cluster of trans-eQTLs). Conclusion Genetic analysis of transcription levels revealed dependence among molecular phenotypes as being

  7. Transcriptional Factor PU.1 Regulates Decidual C1q Expression in Early Pregnancy in Human.

    PubMed

    Madhukaran, Shanmuga Priyaa; Kishore, Uday; Jamil, Kaiser; Teo, Boon Heng Dennis; Choolani, Mahesh; Lu, Jinhua

    2015-01-01

    C1q is the first recognition subcomponent of the complement classical pathway, which in addition to being synthesized in the liver, is also expressed by macrophages and dendritic cells (DCs). Trophoblast invasion during early placentation results in accumulation of debris that triggers the complement system. Hence, both early and late components of the classical pathway are widely distributed in the placenta and decidua. In addition, C1q has recently been shown to significantly contribute to feto-maternal tolerance, trophoblast migration, and spiral artery remodeling, although the exact mechanism remains unknown. Pregnancy in mice, genetically deficient in C1q, mirrors symptoms similar to that of human preeclampsia. Thus, regulated complement activation has been proposed as an essential requirement for normal successful pregnancy. Little is known about the molecular pathways that regulate C1q expression in pregnancy. PU.1, an Ets-family transcription factor, is required for the development of hematopoietic myeloid lineage immune cells, and its expression is tissue-specific. Recently, PU.1 has been shown to regulate C1q gene expression in DCs and macrophages. Here, we have examined if PU.1 transcription factor regulates decidual C1q expression. We used immune-histochemical analysis, PCR, and immunostaining to localize and study the gene expression of PU.1 transcription factor in early human decidua. PU.1 was highly expressed at gene and protein level in early human decidual cells including trophoblast and stromal cells. Surprisingly, nuclear as well as cytoplasmic PU.1 expression was observed. Decidual cells with predominantly nuclear PU.1 expression had higher C1q expression. It is likely that nuclear and cytoplasmic PU.1 localization has a role to play in early pregnancy via regulating C1q expression in the decidua during implantation.

  8. Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae

    PubMed Central

    Yuzenkova, Yulia; Gamba, Pamela; Herber, Martijn; Attaiech, Laetitia; Shafeeq, Sulman; Kuipers, Oscar P.; Klumpp, Stefan; Zenkin, Nikolay; Veening, Jan-Willem

    2014-01-01

    Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in ‘transcription traffic jams’ on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes. PMID:25190458

  9. Transcriptional modulation of transin gene expression by epidermal growth factor and transforming growth factor beta

    SciTech Connect

    Machida, C.M.; Muldoon, L.L.; Rodland, K.D.; Magun, B.E.

    1988-06-01

    Transin is a transformation-associated gene which is expressed constitutively in rat fibroblasts transformed by a variety of oncogenes and in malignant mouse skin carcinomas but not benign papillomas or normal skin. It has been demonstrated that, in nontransformed Rat-1 cells, transin RNA expression is modulated positively by epidermal growth factor (EGF) and negatively by transforming growth factor beta (TGF-BETA); other peptide growth factors were found to have no effect on transin expression. Results presented here indicate that both protein synthesis and continuous occupancy of the EGF receptor by EGF were required for sustained induction of transin RNA. Treatment with TGF-BETA inhibited the ability of EGF to induce transin, whether assayed at the transcriptional level by nuclear run-on analysis or at the level of transin RNA accumulation by Northern (RNA) blot analysis of cellular RNA. TGF-BETA both blocked initial production of transin transcription by EGF and halted established production of transin transcripts during prolonged treatment. These results suggest that TGF-BETA acts at the transcriptional level to antagonize EGF-mediated induction of transin gene expression.

  10. Transcription factors expressed in olfactory bulb local progenitor cells revealed by genome-wide transcriptome profiling

    PubMed Central

    Campbell, Gordon R. O.; Baudhuin, Ariane; Vranizan, Karen; Ngai, John

    2011-01-01

    The local progenitor population in the olfactory bulb (OB) gives rise to mitral and tufted projection neurons during embryonic development. In contrast, OB interneurons are derived from sources outside the bulb where neurogenesis continues throughout life. While many of the genes involved in OB interneuron development have been characterized, the genetic pathways driving local progenitor cell differentiation in this tissue are largely unknown. To better understand this process, we used transcriptional profiling to monitor gene expression of whole OB at daily intervals from embryonic day 11 through birth, generating a compendium of gene expression encompassing the major developmental events of this tissue. Through hierarchical clustering, bioinformatics analysis, and validation by RNA in situ hybridizations, we identified a large number of transcription factors, DNA binding proteins, and cell cycle-related genes expressed by the local neural progenitor cells (NPCs) of the embryonic OB. Further in silico analysis of transcription factor binding sites identified an enrichment of genes regulated by the E2F-Rb pathway among those expressed in the local NPC population. Together these results provide initial insights into the molecular identity of the OB local NPC population and the transcription factor networks that may regulate their function. PMID:21194568

  11. SATB1 Packages Densely Looped, Transcriptionally Active Chromatin for Coordinated Expression of Cytokine Genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    SATB1 (special AT-rich sequence binding protein 1) organizes cell type–specific nuclear architecture by anchoring specialized DNA sequences and recruiting chromatin remodeling factors to control gene transcription. We studied the role of SATB1 in regulating the coordinated expression of Il5, Il4 and...

  12. cDNA-AFLP transcriptional profiling reveals genes expressed during flower development in Oncidium Milliongolds.

    PubMed

    Qian, X; Gong, M J; Wang, C X; Tian, M

    2014-01-01

    The flower developmental process, which is crucial to the whole lifecycle of higher plants, is influenced by both environmental and endogenous factors. The genus Oncidium is commercially important for cut flower and houseplant industry and is ideal for flower development studies. Using cDNA-amplified restriction fragment length polymorphism analysis, we profiled transcripts that are differentially expressed during flower development of Oncidium Milliongolds. A total of 15,960 transcript-derived fragments were generated, with 114 primer sets. Of these, 1248 were sequenced, producing 993 readable sequences. BLASTX/N analysis showed that 833 of the 993 transcripts showed homology to genes in the NCBI databases, exhibiting functions involved in various processes, such as signal transduction, energy conversion, metabolism, and gene expression regulation. The full-length mRNAs of SUCROSE SYNTHASE 1 (SUS1) and LEAFY (LFY) were cloned, and their expression patterns were characterized. The results showed that the expression levels of SUS1 and LFY were similar during flower development. To confirm the function of SUS1 in flower buds, carbohydrate content and sucrose synthase activity were determined. The results showed that changes in sucrose content and sucrose synthase activity reflected SUS1 expression levels. Collectively, these results indicate that SUS1 influences flower development by regulating LFY expression levels through changing the sucrose content of flower buds.

  13. Transcriptional coactivator undifferentiated embryonic cell transcription factor 1 expressed in spermatogonial stem cells: a putative marker of boar spermatogonia.

    PubMed

    Lee, Won-Young; Lee, Kyung-Hoon; Heo, Young-Tae; Kim, Nam-Hyung; Kim, Jin-Hoi; Kim, Jae-Hwan; Moon, Sung-Hwan; Chung, Hak-Jae; Yoon, Min-Jung; Song, Hyuk

    2014-11-30

    Spermatogenesis is initiated from spermatogonial stem cells (SSCs), which are derived from gonocytes. Although some rodent SSC markers have been investigated, other species- and developmental stage-specific markers of spermatogonia have not been identified. The objective of this study was to characterize the expression of undifferentiated embryonic cell transcription factor 1 (UTF1) gene as a potential marker for spermatogonia and SSCs in the boar testis. In boar testis tissue at pre-pubertal stages (tissues collected at 5, 30, and 60 days of age), UTF1 gene expression was detected in almost all spermatogonia cells that expressed a protein gene product 9.5 (PGP9.5), and immunocytochemical analysis of isolated total testicular cells showed that 91.14% of cells staining for PGP9.5 also stained for UTF1. However, in boar testis tissue at pubertal and post-pubertal stages (tissues collected at 90, 120, 150, and 180 days of age), UTF1 was not detected in all PGP9.5-positive cells in the basement membrane. While some PGP9.5-positive cells stained for UTF1, other cells stained only for PGP9.5 or UTF1. PGP9.5, UTF1, and NANOG was assessed in in vitro cultures of pig SSCs (pSSCs) from testes collected at 5 days of age. The relative amounts of PGP9.5, NANOG, and UTF1 mRNA were greater in pSSC colonies than in testis and muscle tissue. Thus, the UTF1 gene is expressed in PGP9.5-positive spermatogonia cells of pigs at 5 days of age, and its expression is maintained in cultured pSSC colonies, suggesting that UTF1 is a putative marker for early-stage spermatogonia in the pre-pubertal pig testis. These findings will facilitate the study of spermatogenesis and applications in germ cell research.

  14. Transcripts encoding HAND genes are differentially expressed and regulated by BMP4 and GDNF in developing avian gut.

    PubMed

    Wu, Xiaodong; Howard, Marthe J

    2002-01-01

    Growth and transcription factors provide important developmental cues to neural crest-derived precursors of enteric neurons. The basic helix-loop-helix transcription factors, HAND2 and HAND1, are expressed in the gastrointestinal tract, but neither the growth factors that induce their expression nor the cell types that express them in the gut are known. We show that transcripts encoding HAND2 are expressed in all segments of the developing gut while those encoding HAND1 are confined to the small intestine and colon. Using in situ hybridization combined with immunostaining using cell type-specific antigens, we demonstrate that transcripts encoding HAND2 are expressed in neurons of both the myenteric and submucosal ganglia. Transcripts encoding HAND1 are expressed by cells in the epithelial lining of the small intestine and colon. The differential localization of HAND2 and HAND1 is reflected in nonoverlapping patterns of regulation by gut-derived factors. The expression of transcripts encoding HAND2 is increased in neural crest-derived cells when cocultured with E4 gut, suggesting a gut-derived factor regulates expression of HAND genes. Exposure of gut-derived neural crest-derived cells to BMP4 significantly increased the expression of HAND2 in all gut segments. In the esophagus and gizzard, where HAND1 is not normally expressed, treatment with BMP4 induced the expression of transcripts encoding HAND1 in nonneural crest-derived cells. GDNF failed to induce consistent expression of transcripts encoding HAND2 in neural crest cells but did support a modest increase in HAND2 expression in gut-derived crest cells obtained from the esophagus and colon. GDNF had no detectable effect on the expression of transcripts encoding HAND1. These results suggest; 1) that HAND2 has a function in the development of enteric neurons, and 2) that BMP and GDNF differentially regulate HAND2 and HAND1 gene expression in the developing gastrointestinal tract.

  15. RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

    PubMed

    Stefanovic, Branko

    2013-01-01

    Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments.

  16. Reciprocal regulation of transcription factors and PLC isozyme gene expression in adult cardiomyocytes.

    PubMed

    Singal, Tushi; Dhalla, Naranjan S; Tappia, Paramjit S

    2010-06-01

    By employing a pharmacological approach, we have shown that phospholipase C (PLC) activity is involved in the regulation of gene expression of transcription factors such as c-Fos and c-Jun in cardiomyocytes in response to norepinephrine (NE). However, there is no information available regarding the identity of specific PLC isozymes involved in the regulation of c-Fos and c-Jun or on the involvement of these transcription factors in PLC isozyme gene expression in adult cardiomyocytes. In this study, transfection of cardiomyocytes with PLC isozyme specific siRNA was found to prevent the NE-mediated increases in the corresponding PLC isozyme gene expression, protein content and activity. Unlike PLC gamma(1) gene, silencing of PLC beta(1), beta(3) and delta(1) genes with si RNA prevented the increases in c-Fos and c-Jun gene expression in response to NE. On the other hand, transfection with c-Jun si RNA suppressed the NE-induced increase in c-Jun as well as PLC beta(1), beta(3) and delta(1) gene expression, but had no effect on PLC gamma(1) gene expression. Although transfection of cardiomyocytes with c-Fos si RNA prevented NE-induced expression of c-Fos, PLC beta(1) and PLC beta(3) genes, it did not affect the increases in PLC delta(1) and PLC gamma(1) gene expression. Silencing of either c-Fos or c-Jun also depressed the NE-mediated increases in PLC beta(1), beta(3) and gamma(1) protein content and activity in an isozyme specific manner. Furthermore, silencing of all PLC isozymes as well as of c-Fos and c-Jun resulted in prevention of the NE-mediated increase in atrial natriuretic factor gene expression. These findings, by employing gene silencing techniques, demonstrate that there occurs a reciprocal regulation of transcription factors and specific PLC isozyme gene expression in cardiomyocytes.

  17. Advanced Glycation End-Products affect transcription factors regulating insulin gene expression

    SciTech Connect

    Puddu, A.; Storace, D.; Odetti, P.; Viviani, G.L.

    2010-04-23

    Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic {beta}-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation prevents FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.

  18. The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts

    PubMed Central

    Lindholm, Maléne E; Giacomello, Stefania; Werne Solnestam, Beata; Kjellqvist, Sanela

    2016-01-01

    Regularly performed endurance training has many beneficial effects on health and skeletal muscle function, and can be used to prevent and treat common diseases e.g. cardiovascular disease, type II diabetes and obesity. The molecular adaptation mechanisms regulating these effects are incompletely understood. To date, global transcriptome changes in skeletal muscles have been studied at the gene level only. Therefore, global isoform expression changes following exercise training in humans are unknown. Also, the effects of repeated interventions on transcriptional memory or training response have not been studied before. In this study, 23 individuals trained one leg for three months. Nine months later, 12 of the same subjects trained both legs in a second training period. Skeletal muscle biopsies were obtained from both legs before and after both training periods. RNA sequencing analysis of all 119 skeletal muscle biopsies showed that training altered the expression of 3,404 gene isoforms, mainly associated with oxidative ATP production. Fifty-four genes had isoforms that changed in opposite directions. Training altered expression of 34 novel transcripts, all with protein-coding potential. After nine months of detraining, no training-induced transcriptome differences were detected between the previously trained and untrained legs. Although there were several differences in the physiological and transcriptional responses to repeated training, no coherent evidence of an endurance training induced transcriptional skeletal muscle memory was found. This human lifestyle intervention induced differential expression of thousands of isoforms and several transcripts from unannotated regions of the genome. It is likely that the observed isoform expression changes reflect adaptational mechanisms and processes that provide the functional and health benefits of regular physical activity. PMID:27657503

  19. Synergistic interactions between transcription factors control expression of the apolipoprotein AI gene in liver cells.

    PubMed Central

    Widom, R L; Ladias, J A; Kouidou, S; Karathanasis, S K

    1991-01-01

    The gene coding for apolipoprotein AI (apoAI), a plasma protein involved in the transport of cholesterol and other lipids in the plasma, is expressed predominantly in liver and intestine. Previous work in our laboratory has shown that different cis-acting elements in the 5'-flanking region of the human apoAI gene control its expression in human hepatoma (HepG2) and colon carcinoma (Caco-2) cells. Hepatocyte-specific expression is mediated by elements within the -256 to -41 DNA region relative to the apoAI gene transcription start site (+1). In this study it was found that the -222 to -110 apoAI gene region is necessary and sufficient for expression in HepG2 cells. It was also found that this DNA region functions as a powerful hepatocyte-specific transcriptional enhancer. Gel retardation and DNase I protection experiments showed that HepG2 cells contain proteins that bind to specific sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within this enhancer. Site-directed mutagenesis that prevents binding of these proteins to individual or different combinations of these sites followed by functional analysis of these mutants in HepG2 cells revealed that protein binding to any one of these sites in the absence of binding to the others was not sufficient for expression. Binding to any two of these sites in any combination was sufficient for only low levels of expression. Binding to all three sites was essential for maximal expression. These results indicate that the transcriptional activity of the apoAI gene in liver cells is dependent on synergistic interactions between transcription factors bound to its enhancer. Images PMID:1846669

  20. Transcriptional regulation of human RANK ligand gene expression by E2F1

    SciTech Connect

    Hu Yan; Sun Meng; Nadiminty, Nagalakshmi; Lou Wei; Pinder, Elaine; Gao, Allen C.

    2008-06-06

    Receptor activator of nuclear factor kappa B ligand (RANKL) is a critical osteoclastogenic factor involved in the regulation of bone resorption, immune function, the development of mammary gland and cardiovascular system. To understand the transcriptional regulation of RANKL, we amplified and characterized a 1890 bp 5'-flanking sequence of human RANKL gene (-1782 bp to +108 bp relative to the transcription start site). Using a series of deletion mutations of the 1890 bp RANKL promoter, we identified a 72 bp region (-172 to -100 bp) mediating RANKL basal transcriptional activity. Sequence analysis revealed a putative E2F binding site within this 72 bp region in the human RANKL promoter. Overexpression of E2F1 increased RANKL promoter activity, while down-regulation of E2F1 expression by small interfering RNA decreased RANKL promoter activity. RT-PCR and enzyme linked immunosorbent assays (ELISA) further demonstrated that E2F1 induced the expression of RANKL. Electrophoretic gel mobility shift assays (EMSA) and antibody competition assays confirmed that E2F1 proteins bind to the consensus E2F binding site in the RANKL promoter. Mutation of the E2F consensus binding site in the RANKL promoter profoundly reduced the basal promoter activity and abolished the transcriptional modulation of RANKL by E2F1. These results suggest that E2F1 plays an important role in regulating RANKL transcription through binding to the E2F consensus binding site.

  1. Alternative promoter usage and differential expression of multiple transcripts of mouse Prkar1a gene.

    PubMed

    Banday, Abdul Rouf; Azim, Shafquat; Tabish, Mohammad

    2011-11-01

    Prkar1a gene encodes regulatory type 1 alpha subunit (RIα) of cAMP-dependent protein kinase (PKA) in mouse. The role of this gene has been implicated in Carney complex and many cancer types that suggest its involvement in physiological processes like cell cycle regulation, growth and/or proliferation. We have identified and sequenced partial cDNA clones encoding four alternatively spliced transcripts of mouse Prkar1a gene. These transcripts have alternate 5' UTR structure which results from splicing of three exons (designated as E1a, E1b, and E1c) to canonical exon 2. The designated transcripts T1, T2, T3, and T4 contain 5' UTR exons as E1c, E1a + E1b, E1a, and E1b, respectively. The transcript T1 corresponded to earlier reported transcript in GenBank. In silico study of genomic DNA sequence revealed three distinct promoter regions namely, P1, P2, and P3 upstream of the exons E1a, E1b, and E1c, respectively. P1 is non-CpG-related promoter but P2 and P3 are CpG-related promoters; however, all three are TATA less. RT-PCR analysis demonstrated the expression of all four transcripts in late postnatal stages; however, these were differentially regulated in early postnatal stages of 0.5 day, 3 day, and 15 day mice in different tissue types. Variations in expression of Prkar1a gene transcripts suggest their regulation from multiple promoters that respond to a variety of signals arising in or out of the cell in tissue and developmental stage-specific manner. PMID:21638026

  2. Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors.

    PubMed

    Norton, Nadine; Sun, Zhifu; Asmann, Yan W; Serie, Daniel J; Necela, Brian M; Bhagwate, Aditya; Jen, Jin; Eckloff, Bruce W; Kalari, Krishna R; Thompson, Kevin J; Carr, Jennifer M; Kachergus, Jennifer M; Geiger, Xochiquetzal J; Perez, Edith A; Thompson, E Aubrey

    2013-01-01

    Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs) and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter (226 gene panel) and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlations of >0.94 and >0.80 with NanoString and ScriptSeq protocols, respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes) and ScriptSeq whole transcriptome protocols respectively, p<2x10(-16). Specifically for lincRNAs, we observed superb Pearson correlation (0.988) between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads). Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transcriptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol performed

  3. The impaired intestinal mucosal immune system by valine deficiency for young grass carp (Ctenopharyngodon idella) is associated with decreasing immune status and regulating tight junction proteins transcript abundance in the intestine.

    PubMed

    Luo, Jian-Bo; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Zhang, Yong-An; Zhou, Xiao-Qiu

    2014-09-01

    This study investigated the effects of dietary valine on the growth, intestinal immune response, tight junction proteins transcript abundance and gene expression of immune-related signaling molecules in the intestine of young grass carp (Ctenopharyngodon idella). Six iso-nitrogenous diets containing graded levels of valine (4.3-19.1 g kg(-)(1) diet) were fed to the fish for 8 weeks. The results showed that percentage weight gain (PWG), feed intake and feed efficiency of fish were the lowest in fish fed the valine-deficient diet (P < 0.05). In addition, valine deficiency decreased lysozyme, acid phosphatase activities and complement 3 content in the intestine (P < 0.05), down-regulated mRNA levels of interleukin 10, transforming growth factor β1, IκBα and target of rapamycin (TOR) (P < 0.05), and up-regulated tumor necrosis factor α, interleukin 8 and nuclear factor κB P65 (NF-κB P65) gene expression (P < 0.05). Additionally, valine deficiency significantly decreased transcript of Occludin, Claudin b, Claudin c, Claudin 3, and ZO-1 (P < 0.05), and improved Claudin 15 expression in the fish intestine (P < 0.05). However, valine did not have a significant effect on expression of Claudin 12 in the intestine of grass carp (P > 0.05). In conclusion, valine deficiency decreased fish growth and intestinal immune status, as well as regulated gene expression of tight junction proteins, NF-κB P65, IκBα and TOR in the fish intestine. Based on the quadratic regression analysis of lysozyme activity or PWG, the dietary valine requirement of young grass carp (268-679 g) were established to be 14.47 g kg(-1) diet (4.82 g 100 g(-1) CP) or 14.00 g kg(-1) diet (4.77 g 100 g(-1) CP), respectively.

  4. Dual transcriptional-translational cascade permits cellular level tuneable expression control

    PubMed Central

    Morra, Rosa; Shankar, Jayendra; Robinson, Christopher J.; Halliwell, Samantha; Butler, Lisa; Upton, Mathew; Hay, Sam; Micklefield, Jason; Dixon, Neil

    2016-01-01

    The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems. PMID:26405200

  5. Dual transcriptional-translational cascade permits cellular level tuneable expression control.

    PubMed

    Morra, Rosa; Shankar, Jayendra; Robinson, Christopher J; Halliwell, Samantha; Butler, Lisa; Upton, Mathew; Hay, Sam; Micklefield, Jason; Dixon, Neil

    2016-02-18

    The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems. PMID:26405200

  6. Molecular mining of GGAA tagged transcripts and their expression in water buffalo Bubalus bubalis.

    PubMed

    Rawal, Leena; Ali, Safdar; Ali, Sher

    2012-01-15

    Repeat sequences are involved in regulation of gene expression both at the transcriptional and translational level. In the mammalian genomes, tri- and tetranucleotide repeats like ATA, AATA, GGAA and GAAA have been associated with diseases. In silico analysis of (GGAA)5 distribution across the species showed maximum number of this repeat in the mouse transcriptome compared to that in other species. Following this, we conducted minisatellite associated sequence amplification (MASA) to explore the buffalo's transcriptome using cDNA from different tissues and an oligo based on (GGAA)5 repeats. MASA uncovered twenty six mRNA transcripts showing homology to known genes in the database. qPCR studies showed the highest expression of twelve transcripts in the spleen. A transcript, pLRC107 with its partial sequence of 203 nucleotides showed sequence variation at several positions in spleen as compared to other four tissues examined. Transcript pLRC100 was found to represent the partial coding sequence of Bos taurus HECT {(homologous to E6-associated protein (UBE3A) carboxyl-terminus domain) and RCC1 (CHC1)-like domain (RLD) 1}, mRNA. We ascertained full length coding sequence of HECT gene and localized the same on buffalo chromosome 10 employing FISH. This gene was found to be conserved across the species. Another gene LRP8 uncovered in the process showed copy number variation between buffalo males (4-9) and females (34-54). The MASA approach enabled us to identify several genes in Bubalus bubalis without screening an entire cDNA library. The highest expression of 12 mRNA transcripts in spleen suggests their likely involvement with immuno transaction. A comprehensive knowledge of the repeat tagged transcriptomes is envisaged to help in understanding their significance in genome organization and evolution forming rich basis of functional and comparative genomics.

  7. Hepcidin expression in liver cells: evaluation of mRNA levels and transcriptional regulation.

    PubMed

    Kanamori, Yohei; Murakami, Masaru; Matsui, Tohru; Funaba, Masayuki

    2014-08-01

    Hepcidin produced in the liver negatively regulates intestinal iron absorption, and the bone morphogenetic protein (BMP) pathway is well-known to stimulate hepcidin expression. However, the regulation of hepcidin expression has not been fully elucidated. In this study, we evaluate different systems that can be used to determine how hepcidin expression is regulated. The basal expression of hepcidin in liver cell lines, such as HepG2 cells and Hepa1-6 cells, was lower than that in the liver and primary hepatocytes; the expression levels of hepcidin in the cell lines were near the limit of detection for RT-PCR and RT-qPCR analyses. Treatment with trichostatin A, RNAlater, or MG-132 enhanced the expression of hepcidin in HepG2 cells, suggesting that histone deacetylation, instability of mRNA, or proteosomal degradation of the protein(s) that positively regulate hepcidin expression may be responsible for the decreased expression of hepcidin in HepG2 cells. In luciferase-based reporter assays, BMP induced the transcription of a reporter, hepcidin(-2018)-luc, that contains nt -2018 through nt -35 of the hepcidin promoter in HepG2 cells and Hepa1-6 cells. However, BRE-luc, a representative reporter used to evaluate BMP signaling, was unresponsive to BMP in HepG2 cells. These results suggest that hepcidin transcription can be best evaluated in liver cell lines and that the hepcidin promoter senses BMP signaling with high sensitivity. The present study demonstrates that studies regarding the regulation of hepcidin expression at the mRNA level should be evaluated in primary hepatocytes, and liver cell lines are well-suited for studies examining the transcriptional regulation of hepcidin.

  8. Pregnancy complicated by obesity induces global transcript expression alterations in visceral and subcutaneous fat.

    PubMed

    Bashiri, Asher; Heo, Hye J; Ben-Avraham, Danny; Mazor, Moshe; Budagov, Temuri; Einstein, Francine H; Atzmon, Gil

    2014-08-01

    Maternal obesity is a significant risk factor for development of both maternal and fetal metabolic complications. Increase in visceral fat and insulin resistance is a metabolic hallmark of pregnancy, yet not much is known how obesity alters adipose cellular function and how this may contribute to pregnancy morbidities. We sought to identify alterations in genome-wide transcription expression in both visceral (omental) and abdominal subcutaneous fat deposits in pregnancy complicated by obesity. Visceral and abdominal subcutaneous fat deposits were collected from normal weight and obese pregnant women (n = 4/group) at the time of scheduled uncomplicated cesarean section. A genome-wide expression array (Affymetrix Human Exon 1.0 st platform), validated by quantitative real-time PCR, was utilized to establish the gene transcript expression profile in both visceral and abdominal subcutaneous fat in normal weight and obese pregnant women. Global alteration in gene expression was identified in pregnancy complicated by obesity. These regions of variations led to identification of indolethylamine N-methyltransferase, tissue factor pathway inhibitor-2, and ephrin type-B receptor 6, not previously associated with fat metabolism during pregnancy. In addition, subcutaneous fat of obese pregnant women demonstrated increased coding protein transcripts associated with apoptosis as compared to lean counterparts. Global alteration of gene expression in adipose tissue may contribute to adverse pregnancy outcomes associated with obesity.

  9. NFAT1 transcription factor regulates cell cycle progression and cyclin E expression in B lymphocytes.

    PubMed

    Teixeira, Leonardo K; Carrossini, Nina; Sécca, Cristiane; Kroll, José E; DaCunha, Déborah C; Faget, Douglas V; Carvalho, Lilian D S; de Souza, Sandro J; Viola, João P B

    2016-09-01

    The NFAT family of transcription factors has been primarily related to T cell development, activation, and differentiation. Further studies have shown that these ubiquitous proteins are observed in many cell types inside and outside the immune system, and are involved in several biological processes, including tumor growth, angiogenesis, and invasiveness. However, the specific role of the NFAT1 family member in naive B cell proliferation remains elusive. Here, we demonstrate that NFAT1 transcription factor controls Cyclin E expression, cell proliferation, and tumor growth in vivo. Specifically, we show that inducible expression of NFAT1 inhibits cell cycle progression, reduces colony formation, and controls tumor growth in nude mice. We also demonstrate that NFAT1-deficient naive B lymphocytes show a hyperproliferative phenotype and high levels of Cyclin E1 and E2 upon BCR stimulation when compared to wild-type B lymphocytes. NFAT1 transcription factor directly regulates Cyclin E expression in B cells, inhibiting the G1/S cell cycle phase transition. Bioinformatics analysis indicates that low levels of NFAT1 correlate with high expression of Cyclin E1 in different human cancers, including Diffuse Large B-cell Lymphomas (DLBCL). Together, our results demonstrate a repressor role for NFAT1 in cell cycle progression and Cyclin E expression in B lymphocytes, and suggest a potential function for NFAT1 protein in B cell malignancies.

  10. Pregnancy Complicated by Obesity Induces Global Transcript Expression Alterations in Visceral and Subcutaneous Fat

    PubMed Central

    Bashiri, Asher; Heo, Hye J.; Ben-Avraham, Danny; Mazor, Moshe; Budagov, Temuri; Einstein, Francine H.; Atzmon, Gil

    2014-01-01

    Maternal obesity is a significant risk factor for development of both maternal and fetal metabolic complications. Increase in visceral fat and insulin resistance is a metabolic hallmark of pregnancy, yet little is known how obesity alters adipose cellular function and how this may contribute to pregnancy morbidities. We sought to identify alterations in genome-wide transcription expression in both visceral (omental) and abdominal subcutaneous fat deposits in pregnancy complicated by obesity. Visceral and abdominal subcutaneous fat deposits were collected from normal weight and obese pregnant women (n=4/group) at time of scheduled uncomplicated cesarean section. A genome-wide expression array (Affymetrix Human Exon 1.0 st platform), validated by quantitative real-time PCR, was utilized to establish the gene transcript expression profile in both visceral and abdominal subcutaneous fat in normal weight and obese pregnant women. Global alteration in gene expression was identified in pregnancy complicated by obesity. These regions of variations lead to identification of indolethylamine N-methyltransferase (INMT), tissue factor pathway inhibitor-2 (TFPI-2), and ephrin type-B receptor 6 (EPHB6), not previously associated with fat metabolism during pregnancy. In addition, subcutaneous fat of obese pregnant women demonstrated increased coding protein transcripts associated with apoptosis compared to lean counterparts. Global alteration of gene expression in adipose tissue may contribute to adverse pregnancy outcomes associated with obesity. PMID:24696292

  11. Transcription Factors Are Targeted by Differentially Expressed miRNAs in Primates

    PubMed Central

    Dannemann, Michael; Prüfer, Kay; Lizano, Esther; Nickel, Birgit; Burbano, Hernán A.; Kelso, Janet

    2012-01-01

    MicroRNAs (miRNAs) are small RNA molecules involved in the regulation of mammalian gene expression. Together with other transcription regulators, miRNAs modulate the expression of genes and thereby potentially contribute to tissue and species diversity. To identify miRNAs that are differentially expressed between tissues and/or species, and the genes regulated by these, we have quantified expression of miRNAs and messenger RNAs in five tissues from multiple human, chimpanzee, and rhesus macaque individuals using high-throughput sequencing. The breadth of this tissue and species data allows us to show that downregulation of target genes by miRNAs is more pronounced between tissues than between species and that downregulation is more pronounced for genes with fewer binding sites for expressed miRNAs. Intriguingly, we find that tissue- and species-specific miRNAs target transcription factor genes (TFs) significantly more often than expected. Through their regulatory effect on transcription factors, miRNAs may therefore exert an indirect influence on a larger proportion of genes than previously thought. PMID:22454130

  12. Suppressing the expression of a forkhead transcription factor disrupts the chitin biosynthesis pathway in Spodoptera exigua.

    PubMed

    Zhao, Lina; Wei, Ping; Guo, Hongshuang; Wang, Shigui; Tang, Bin

    2014-05-01

    Forkhead (Fox) transcription factors display functional diversity and are involved in various metabolic and developmental processes. The Spodoptera exigua Fox (SeFox) encodes a protein of 353 amino acids with a theoretical molecular mass of approximately 38.99 kDa and an isoelectric point of 8.86. qPCR results revealed that SeFox was expressed mainly in the brain, fat body, epidermis, midgut, Malpighian tubules, and testis. SeFox was expressed, with some changes, throughout development in the fat body and whole body. Injection of dsSeFox (SeFox dsRNA) into larvae resulted in incidences of albino plus molting deformity (4.8%), molting deformity (26.2%), and albino phenotypes (69.1%). dsSeFox injection resulted in approximately 50% knockdown of transcript levels at 36 h. Compared with control groups, hexokinase (HK) expression was reduced to approximately 40% at 48 h postinjection. Chitin synthase A (CHSA) expression was reduced to two-thirds at 24 h, but increased at 72 h. Compared with untreated control and green fluorescent protein-treated groups, Chitin synthase B (CHSB) expression decreased to 33% following dsSeFox injection by 36 h. We infer from our results that forkhead transcription factors act in chitin synthesis in S. exigua. PMID:24464395

  13. Discovering Transcriptional Modules by Combined Analysis of Expression Profiles and Regulatory Sequences

    NASA Astrophysics Data System (ADS)

    Halperin, Yonit; Linhart, Chaim; Ulitsky, Igor; Shamir, Ron

    A key goal of gene expression analysis is the characterization of transcription factors (TFs) and micro-RNAs (miRNAs) regulating specific transcriptional programs. The most common approach to address this task is a two-step methodology: In the first step, a clustering procedure is executed to partition the genes into groups that are believed to be co-regulated, based on expression profile similarity. In the second step, a motif discovery tool is applied to search for over-represented cis-regulatory motifs within each group. In an effort to obtain better results by simultaneously utilizing all available information, several studies have suggested computational schemes for a single-step combined analysis of expression and sequence data. Despite extensive research, reverse engineering complex regulatory networks from microarray measurements remains a difficult challenge with limited success, especially in metazoans.

  14. Exploring the transcription factor activity in high-throughput gene expression data using RLQ analysis

    PubMed Central

    2013-01-01

    Background Interpretation of gene expression microarray data in the light of external information on both columns and rows (experimental variables and gene annotations) facilitates the extraction of pertinent information hidden in these complex data. Biologists classically interpret genes of interest after retrieving functional information from a subset of genes of interest. Transcription factors play an important role in orchestrating the regulation of gene expression. Their activity can be deduced by examining the presence of putative transcription factors binding sites in the gene promoter regions. Results In this paper we present the multivariate statistical method RLQ which aims to analyze microarray data where additional information is available on both genes and samples. As an illustrative example, we applied RLQ methodology to analyze transcription factor activity associated with the time-course effect of steroids on the growth of primary human lung fibroblasts. RLQ could successfully predict transcription factor activity, and could integrate various other sources of external information in the main frame of the analysis. The approach was validated by means of alternative statistical methods and biological validation. Conclusions RLQ provides an efficient way of extracting and visualizing structures present in a gene expression dataset by directly modeling the link between experimental variables and gene annotations. PMID:23742070

  15. Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression.

    PubMed

    Kabadi, Ami M; Gersbach, Charles A

    2014-09-01

    Engineered DNA-binding proteins that can be targeted to specific sites in the genome to manipulate gene expression have enabled many advances in biomedical research. This includes generating tools to study fundamental aspects of gene regulation and the development of a new class of gene therapies that alter the expression of endogenous genes. Designed transcription factors have entered clinical trials for the treatment of human diseases and others are in preclinical development. High-throughput and user-friendly platforms for designing synthetic DNA-binding proteins present innovative methods for deciphering cell biology and designing custom synthetic gene circuits. We review two platforms for designing synthetic transcription factors for manipulating gene expression: Transcription activator-like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. We present an overview of each technology and a guide for designing and assembling custom TALE- and CRISPR/Cas9-based transcription factors. We also discuss characteristics of each platform that are best suited for different applications.

  16. Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Altering expression of transcription factors can be an effective means to coordinately modulate entire metabolic pathways in plants. It can also provide useful information concerning the identities of genes that constitute metabolic networks. Here, we used ectopic expression of a MYB transcription f...

  17. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    ERIC Educational Resources Information Center

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  18. Transcription factor expression dynamics of early T-lymphocyte specification and commitment

    PubMed Central

    David-Fung, Elizabeth-Sharon; Butler, Robert; Buzi, Gentian; Yui, Mary A.; Diamond, Rochelle A.; Anderson, Michele K.; Rowen, Lee; Rothenberg, Ellen V.

    2009-01-01

    Summary Mammalian T lymphocytes are a prototype for development from adult pluripotent stem cells. While T-cell specification is driven by Notch signaling, T-lineage commitment is only finalized after prolonged Notch activation. However, no T-lineage specific regulatory factor has been reported that mediates commitment. We used a gene-discovery approach to identify additional candidate T-lineage transcription factors and characterized expression of >100 regulatory genes in early T-cell precursors using realtime RT-PCR. These regulatory genes were also monitored in multilineage precursors as they entered T-cell or non-T-cell pathways in vitro; in non-T cells ex vivo; and in later T-cell developmental stages after lineage commitment. At least three major expression patterns were observed. Transcription factors in the largest group are expressed at relatively stable levels throughout T-lineage specification as a legacy from prethymic precursors, with some continuing while others are downregulated after commitment. Another group is highly expressed in the earliest stages only, and is downregulated before or during commitment. Genes in a third group undergo upregulation at one of three distinct transitions, suggesting a positive regulatory cascade. However, the transcription factors induced during commitment are not T-lineage specific. Different members of the same transcription factor family can follow opposite trajectories during specification and commitment, while factors co-expressed early can be expressed in divergent patterns in later T-cell development. Some factors reveal new regulatory distinctions between αβ and γδ T-lineage differentiation. These results show that T-cell identity has an essentially complex regulatory basis and provide a detailed framework for regulatory network modeling of T-cell specification. PMID:19013443

  19. Molecular cloning and expression profile of an abiotic stress and hormone responsive MYB transcription factor gene from Panax ginseng.

    PubMed

    Afrin, Sadia; Zhu, Jie; Cao, Hongzhe; Huang, Jingjia; Xiu, Hao; Luo, Tiao; Luo, Zhiyong

    2015-04-01

    The v-myb avian myeloblastosis viral oncogene homolog (MYB) family constitutes one of the most abundant groups of transcription factors and plays vital roles in developmental processes and defense responses in plants. A ginseng (Panax ginseng C.A. Meyer) MYB gene was cloned and designated as PgMYB1. The cDNA of PgMYB1 is 762 base pairs long and encodes the R2R3-type protein consisting 238 amino acids. Subcellular localization showed that PgMYB1-mGFP5 fusion protein was specifically localized in the nucleus. To understand the functional roles of PgMYB1, we investigated the expression patterns of PgMYB1 in different tissues and under various conditions. Quantitative real-time polymerase chain reaction and western blot analysis showed that PgMYB1 was expressed at higher level in roots, leaves, and lateral roots than in stems and seeds. The expression of PgMYB1 was up-regulated by abscisic acid, salicylic acid, NaCl, and cold (chilling), and down-regulated by methyl jasmonate. These results suggest that PgMYB1 might be involved in responding to environmental stresses and hormones. PMID:25791525

  20. Ubiquitin-conjugating enzyme E2-like gene associated to pathogen response in Concholepas concholepas: SNP identification and transcription expression.

    PubMed

    Núñez-Acuña, Gustavo; Aguilar-Espinoza, Andrea; Chávez-Mardones, Jacqueline; Gallardo-Escárate, Cristian

    2012-10-01

    Ubiquitin-conjugated E2 enzyme (UBE2) is one of the main components of the proteasome degradation cascade. Previous studies have shown an increase of expression levels in individuals challenged to some pathogen organism such as virus and bacteria. The study was to characterize the immune response of UBE2 gene in the gastropod Concholepas concholepas through expression analysis and single nucleotide polymorphisms (SNP) discovery. Hence, UBE2 was identified from a cDNA library by 454 pyrosequencing, while SNP identification and validation were performed using De novo assembly and high resolution melting analysis. Challenge trials with Vibrio anguillarum was carried out to evaluate the relative transcript abundance of UBE2 gene from two to thirty-three hours post-treatment. The results showed a partial UBE2 sequence of 889 base pair (bp) with a partial coding region of 291 bp. SNP variation (A/C) was observed at the 546th position. Individuals challenged by V. anguillarum showed an overexpression of the UBE2 gene, the expression being significantly higher in homozygous individuals (AA) than (CC) or heterozygous individuals (A/C). This study contributes useful information relating to the UBE2 gene and its association with innate immune response in marine invertebrates. PMID:22971731

  1. Molecular cloning and expression profile of an abiotic stress and hormone responsive MYB transcription factor gene from Panax ginseng.

    PubMed

    Afrin, Sadia; Zhu, Jie; Cao, Hongzhe; Huang, Jingjia; Xiu, Hao; Luo, Tiao; Luo, Zhiyong

    2015-04-01

    The v-myb avian myeloblastosis viral oncogene homolog (MYB) family constitutes one of the most abundant groups of transcription factors and plays vital roles in developmental processes and defense responses in plants. A ginseng (Panax ginseng C.A. Meyer) MYB gene was cloned and designated as PgMYB1. The cDNA of PgMYB1 is 762 base pairs long and encodes the R2R3-type protein consisting 238 amino acids. Subcellular localization showed that PgMYB1-mGFP5 fusion protein was specifically localized in the nucleus. To understand the functional roles of PgMYB1, we investigated the expression patterns of PgMYB1 in different tissues and under various conditions. Quantitative real-time polymerase chain reaction and western blot analysis showed that PgMYB1 was expressed at higher level in roots, leaves, and lateral roots than in stems and seeds. The expression of PgMYB1 was up-regulated by abscisic acid, salicylic acid, NaCl, and cold (chilling), and down-regulated by methyl jasmonate. These results suggest that PgMYB1 might be involved in responding to environmental stresses and hormones.

  2. SOX9 modulates the expression of key transcription factors required for heart valve development.

    PubMed

    Garside, Victoria C; Cullum, Rebecca; Alder, Olivia; Lu, Daphne Y; Vander Werff, Ryan; Bilenky, Mikhail; Zhao, Yongjun; Jones, Steven J M; Marra, Marco A; Underhill, T Michael; Hoodless, Pamela A

    2015-12-15

    Heart valve formation initiates when endothelial cells of the heart transform into mesenchyme and populate the cardiac cushions. The transcription factor SOX9 is highly expressed in the cardiac cushion mesenchyme, and is essential for heart valve development. Loss of Sox9 in mouse cardiac cushion mesenchyme alters cell proliferation, embryonic survival, and valve formation. Despite this important role, little is known about how SOX9 regulates heart valve formation or its transcriptional targets. Therefore, we mapped putative SOX9 binding sites by ChIP-Seq in E12.5 heart valves, a stage at which the valve mesenchyme is actively proliferating and initiating differentiation. Embryonic heart valves have been shown to express a high number of genes that are associated with chondrogenesis, including several extracellular matrix proteins and transcription factors that regulate chondrogenesis. Therefore, we compared regions of putative SOX9 DNA binding between E12.5 heart valves and E12.5 limb buds. We identified context-dependent and context-independent SOX9-interacting regions throughout the genome. Analysis of context-independent SOX9 binding suggests an extensive role for SOX9 across tissues in regulating proliferation-associated genes including key components of the AP-1 complex. Integrative analysis of tissue-specific SOX9-interacting regions and gene expression profiles on Sox9-deficient heart valves demonstrated that SOX9 controls the expression of several transcription factors with previously identified roles in heart valve development, including Twist1, Sox4, Mecom and Pitx2. Together, our data identify SOX9-coordinated transcriptional hierarchies that control cell proliferation and differentiation during valve formation.

  3. Ribavirin-induced intracellular GTP depletion activates transcription elongation in coagulation factor VII gene expression.

    PubMed

    Suzuki, Atsuo; Miyawaki, Yuhri; Okuyama, Eriko; Murata, Moe; Ando, Yumi; Kato, Io; Takagi, Yuki; Takagi, Akira; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

    2013-01-01

    Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.

  4. E2F Transcription Factors Control the Roller Coaster Ride of Cell Cycle Gene Expression.

    PubMed

    Thurlings, Ingrid; de Bruin, Alain

    2016-01-01

    Initially, the E2F transcription factor was discovered as a factor able to bind the adenovirus E2 promoter and activate viral genes. Afterwards it was shown that E2F also binds to promoters of nonviral genes such as C-MYC and DHFR, which were already known at that time to be important for cell growth and DNA metabolism, respectively. These findings provided the first clues that the E2F transcription factor might be an important regulator of the cell cycle. Since this initial discovery in 1987, several additional E2F family members have been identified, and more than 100 targets genes have been shown to be directly regulated by E2Fs, the majority of these are important for controlling the cell cycle. The progression of a cell through the cell cycle is accompanied with the increased expression of a specific set of genes during one phase of the cell cycle and the decrease of the same set of genes during a later phase of the cell cycle. This roller coaster ride, or oscillation, of gene expression is essential for the proper progression through the cell cycle to allow accurate DNA replication and cell division. The E2F transcription factors have been shown to be critical for the temporal expression of the oscillating cell cycle genes. This review will focus on how the oscillation of E2Fs and their targets is regulated by transcriptional, post-transcriptional and post-translational mechanism in mammals, yeast, flies, and worms. Furthermore, we will discuss the functional impact of E2Fs on the cell cycle progression and outline the consequences when E2F expression is disturbed. PMID:26254918

  5. Transcriptional Regulation of Zein Gene Expression in Maize through the Additive and Synergistic Action of opaque2, Prolamine-Box Binding Factor, and O2 Heterodimerizing Proteins

    PubMed Central

    Zhang, Zhiyong; Yang, Jun; Wu, Yongrui

    2015-01-01

    Maize (Zea mays) zeins are some of the most abundant cereal seed storage proteins (SSPs). Their abundance influences kernel hardness but compromises its nutritional quality. Transcription factors regulating the expression of zein and other SSP genes in cereals are endosperm-specific and homologs of maize opaque2 (O2) and prolamine-box binding factor (PBF). This study demonstrates that the ubiquitously expressed transcription factors, O2 heterodimerizing proteins (OHPs), specifically regulate 27-kD γ-zein gene expression (through binding to an O2-like box in its promoter) and interact with PBF. The zein content of double mutants OhpRNAi;o2 and PbfRNAi;o2 and the triple mutant PbfRNAi;OhpRNAi;o2 is reduced by 83, 89, and 90%, respectively, compared with the wild type. The triple mutant developed the smallest zein protein bodies, which were merely one-tenth the wild type’s size. Total protein levels in these mutants were maintained in a relatively constant range through proteome rebalancing. These data show that OHPs, O2, and PBF are master regulators of zein storage protein synthesis, acting in an additive and synergistic mode. The differential expression patterns of OHP and O2 genes may cause the slight differences in the timing of 27-kD γ-zein and 22-kD α-zein accumulation during protein body formation. PMID:25901087

  6. Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

    PubMed

    Ferreira, Savio Siqueira; Hotta, Carlos Takeshi; Poelking, Viviane Guzzo de Carli; Leite, Debora Chaves Coelho; Buckeridge, Marcos Silveira; Loureiro, Marcelo Ehlers; Barbosa, Marcio Henrique Pereira; Carneiro, Monalisa Sampaio; Souza, Glaucia Mendes

    2016-05-01

    Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production. PMID:26820137

  7. Expression Profiles of the Nuclear Receptors and Their Transcriptional Coregulators During Differentiation of Neural Stem Cells

    PubMed Central

    Androutsellis-Theotokis, A.; Chrousos, G. P.; McKay, R. D.; DeCherney, A. H.; Kino, T.

    2013-01-01

    Neural stem cells (NSCs) are pluripotent precursors with the ability to proliferate and differentiate into 3 neural cell lineages, neurons, astrocytes and oligodendrocytes. Elucidation of the mechanisms underlying these biologic processes is essential for understanding both physiologic and pathologic neural development and regeneration after injury. Nuclear hormone receptors (NRs) and their transcriptional coregulators also play crucial roles in neural development, functions and fate. To identify key NRs and their transcriptional regulators in NSC differentiation, we examined mRNA expression of 49 NRs and many of their coregulators during differentiation (0–5 days) of mouse embryonic NSCs induced by withdrawal of fibroblast growth factor-2 (FGF2). 37 out of 49 NRs were expressed in NSCs before induction of differentiation, while receptors known to play major roles in neural development, such as THRα, RXRs, RORs, TRs, and COUPTFs, were highly expressed. CAR, which plays important roles in xenobiotic metabolism, was also highly expressed. FGF2 withdrawal induced mRNA expression of RORγ, RXRγ, and MR by over 20-fold. Most of the transcriptional coregulators examined were expressed basally and throughout differentiation without major changes, while FGF2 withdrawal strongly induced mRNA expression of several histone deacetylases (HDACs), including HDAC11. Dexamethasone and aldosterone, respectively a synthetic glucocorticoid and natural mineralocorticoid, increased NSC numbers and induced differentiation into neurons and astrocytes. These results indicate that the NRs and their coregulators are present and/or change their expression during NSC differentiation, suggesting that they may influence development of the central nervous system in the absence or presence of their ligands. PMID:22990992

  8. Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

    PubMed

    Ferreira, Savio Siqueira; Hotta, Carlos Takeshi; Poelking, Viviane Guzzo de Carli; Leite, Debora Chaves Coelho; Buckeridge, Marcos Silveira; Loureiro, Marcelo Ehlers; Barbosa, Marcio Henrique Pereira; Carneiro, Monalisa Sampaio; Souza, Glaucia Mendes

    2016-05-01

    Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production.

  9. Gene Expression in Mouse Thyrotrope Adenoma: Transcription Elongation Factor Stimulates Proliferation.

    PubMed

    Gergics, Peter; Christian, Helen C; Choo, Monica S; Ajmal, Adnan; Camper, Sally A

    2016-09-01

    Thyrotrope hyperplasia and hypertrophy are common responses to primary hypothyroidism. To understand the genetic regulation of these processes, we studied gene expression changes in the pituitaries of Cga(-/-) mice, which are deficient in the common α-subunit of TSH, LH, and FSH. These mice have thyrotrope hypertrophy and hyperplasia and develop thyrotrope adenoma. We report that cell proliferation is increased, but the expression of most stem cell markers is unchanged. The α-subunit is required for secretion of the glycoprotein hormone β-subunits, and mutants exhibit elevated expression of many genes involved in the unfolded protein response, consistent with dilation and stress of the endoplasmic reticulum. Mutants have elevated expression of transcription factors that are important in thyrotrope function, such as Gata2 and Islet 1, and those that stimulate proliferation, including Nupr1, E2f1, and Etv5. We characterized the expression and function of a novel, overexpressed gene, transcription elongation factor A (SII)-like 5 (Tceal5). Stable expression of Tceal5 in a pituitary progenitor cell line is sufficient to increase cell proliferation. Thus, Tceal5 may act as a proto-oncogene. This study provides a rich resource for comparing pituitary transcriptomes and an analysis of gene expression networks. PMID:27580811

  10. Transcription Factors Expressed in Mouse Cochlear Inner and Outer Hair Cells

    PubMed Central

    Li, Yi; Liu, Huizhan; Barta, Cody L.; Judge, Paul D.; Zhao, Lidong; Zhang, Weiping J.; Gong, Shusheng; Beisel, Kirk W.; He, David Z. Z.

    2016-01-01

    Regulation of gene expression is essential to determining the functional complexity and morphological diversity seen among different cells. Transcriptional regulation is a crucial step in gene expression regulation because the genetic information is directly read from DNA by sequence-specific transcription factors (TFs). Although several mouse TF databases created from genome sequences and transcriptomes are available, a cell type-specific TF database from any normal cell populations is still lacking. We identify cell type-specific TF genes expressed in cochlear inner hair cells (IHCs) and outer hair cells (OHCs) using hair cell-specific transcriptomes from adult mice. IHCs and OHCs are the two types of sensory receptor cells in the mammalian cochlea. We show that 1,563 and 1,616 TF genes are respectively expressed in IHCs and OHCs among 2,230 putative mouse TF genes. While 1,536 are commonly expressed in both populations, 73 genes are differentially expressed (with at least a twofold difference) in IHCs and 13 are differentially expressed in OHCs. Our datasets represent the first cell type-specific TF databases for two populations of sensory receptor cells and are key informational resources for understanding the molecular mechanism underlying the biological properties and phenotypical differences of these cells. PMID:26974322

  11. Transcription Factors Expressed in Mouse Cochlear Inner and Outer Hair Cells.

    PubMed

    Li, Yi; Liu, Huizhan; Barta, Cody L; Judge, Paul D; Zhao, Lidong; Zhang, Weiping J; Gong, Shusheng; Beisel, Kirk W; He, David Z Z

    2016-01-01

    Regulation of gene expression is essential to determining the functional complexity and morphological diversity seen among different cells. Transcriptional regulation is a crucial step in gene expression regulation because the genetic information is directly read from DNA by sequence-specific transcription factors (TFs). Although several mouse TF databases created from genome sequences and transcriptomes are available, a cell type-specific TF database from any normal cell populations is still lacking. We identify cell type-specific TF genes expressed in cochlear inner hair cells (IHCs) and outer hair cells (OHCs) using hair cell-specific transcriptomes from adult mice. IHCs and OHCs are the two types of sensory receptor cells in the mammalian cochlea. We show that 1,563 and 1,616 TF genes are respectively expressed in IHCs and OHCs among 2,230 putative mouse TF genes. While 1,536 are commonly expressed in both populations, 73 genes are differentially expressed (with at least a twofold difference) in IHCs and 13 are differentially expressed in OHCs. Our datasets represent the first cell type-specific TF databases for two populations of sensory receptor cells and are key informational resources for understanding the molecular mechanism underlying the biological properties and phenotypical differences of these cells. PMID:26974322

  12. [Expression and clinical significance of caudal type homeobox transcription factor-2 in adult acute lymphocytic leukemia].

    PubMed

    Lu, Hai-Yan; Xia, Hai-Long; Chen, Xiao-Wen; Zhu, Li-Xin; Wang, Qing-Yi; Cheng, Xin

    2011-04-01

    This study was aimed to investigate the expression and clinical significance of CDX1, CDX2 and CDX4 genes in acute lymphocytic leukemia (ALL). Expressions of CDX1, CDX2, and CDX4 in 51 adult acute lymphocytic leukemia patients and 14 healthy subjects were detected by reverse transcription polymerase chain reaction (RT-PCR). The results indicated that CDX1, CDX2 and CDX4 were not expressed in 14 healthy persons and 15 CR ALL patients, the positive expression rate of CDX2 gene in de novo ALL patients was 60.8%, while it obviously decreased in patients with complete remission (CR) (p < 0.05); the expression of CDX2 was increased again in relapsed patients (81.8%). When the expression of CDX2 was analyzed in different risk groups of ALL patients, the CDX2 expression rate in high risk (HR) patients was 91.7%, and that in the standard risk (SR) group was 45.7%. Furthermore, analyses of CDX1 and CDX4 expression in series of ALL samples did not show the expression of these genes. In patients with adult ALL at diagnosis and relapse, the CR rate of patients with CDX2 positive expression was lower than that of patients with CDX2 negative expression (p < 0.05). The median survival time in CDX2 positive expression patients was shorter than that in negative expression patient. It is concluded that expression of CDX2 may correlated with pathogenesis and relapse of adult ALL, but the expression of CDX1 and CDX4 don' t associated with pathogenesis and relapse of adult ALL; the CR rate and prognosis of patients with CDX2 positive expression is lower and poor. The expression of CDX2 may be used as a marker for occurrence, relapse and poor prognosis of adult ALL patients.

  13. The Epstein-Barr virus (EBV) early protein EB2 is a posttranscriptional activator expressed under the control of EBV transcription factors EB1 and R.

    PubMed Central

    Buisson, M; Manet, E; Trescol-Biemont, M C; Gruffat, H; Durand, B; Sergeant, A

    1989-01-01

    From the cloning and characterization of cDNAs, we found that the Epstein-Barr virus (EBV) open reading frame (ORF) BMLF1-BSLF2 coding for the early protein EB2 is present in several mRNAs generated by alternative splicing and expressed in the leftward direction from two promoters PM and PM1. The PM promoter controls the expression of two abundant mRNA species of 1.9 and 2 kilobases (kb), whereas the PM1 promoter controls the expression of at least three mRNAs 3.6, 4.0, and 4.4 kb long. The PM promoter probably overlaps with the PS promoter which controls the transcription of a 3.6-kb mRNA expressed in the rightward direction and containing the ORF BSRF1. Although it increases the amount of chloramphenicol acetyltransferase enzyme expressed from the chimeric pMCAT gene, EB2 is not a promiscuous trans-activator of gene expression and does not positively regulate its own expression from promoter PM. The EB2 activation is not promoter dependent but could possibly act by stabilizing mRNAs and increasing their translation. The PM promoter is, however, activated by the two EBV transcription trans-acting factors, EB1 and R, encoded by the EBV ORFs BZLF1 and BRLF1, respectively. EB1 activates the PM promoter from a consensus AP-1 binding site, and R activates the PM promoter from an enhancer. Images PMID:2555554

  14. Seasonal variation in nifH abundance and expression of cyanobacterial communities associated with boreal feather mosses.

    PubMed

    Warshan, Denis; Bay, Guillaume; Nahar, Nurun; Wardle, David A; Nilsson, Marie-Charlotte; Rasmussen, Ulla

    2016-09-01

    Dinitrogen (N2)-fixation by cyanobacteria living in symbiosis with pleurocarpous feather mosses (for example, Pleurozium schreberi and Hylocomium splendens) represents the main pathway of biological N input into N-depleted boreal forests. Little is known about the role of the cyanobacterial community in contributing to the observed temporal variability of N2-fixation. Using specific nifH primers targeting four major cyanobacterial clusters and quantitative PCR, we investigated how community composition, abundance and nifH expression varied by moss species and over the growing seasons. We evaluated N2-fixation rates across nine forest sites in June and September and explored the abundance and nifH expression of individual cyanobacterial clusters when N2-fixation is highest. Our results showed temporal and host-dependent variations of cyanobacterial community composition, nifH gene abundance and expression. N2-fixation was higher in September than June for both moss species, explained by higher nifH gene expression of individual clusters rather than higher nifH gene abundance or differences in cyanobacterial community composition. In most cases, 'Stigonema cluster' made up less than 29% of the total cyanobacterial community, but accounted for the majority of nifH gene expression (82-94% of total nifH expression), irrespective of sampling date or moss species. Stepwise multiple regressions showed temporal variations in N2-fixation being greatly explained by variations in nifH expression of the 'Stigonema cluster'. These results suggest that Stigonema is potentially the most influential N2-fixer in symbiosis with boreal forest feather mosses. PMID:26918665

  15. Seasonal variation in nifH abundance and expression of cyanobacterial communities associated with boreal feather mosses

    PubMed Central

    Warshan, Denis; Bay, Guillaume; Nahar, Nurun; Wardle, David A; Nilsson, Marie-Charlotte; Rasmussen, Ulla

    2016-01-01

    Dinitrogen (N2)-fixation by cyanobacteria living in symbiosis with pleurocarpous feather mosses (for example, Pleurozium schreberi and Hylocomium splendens) represents the main pathway of biological N input into N-depleted boreal forests. Little is known about the role of the cyanobacterial community in contributing to the observed temporal variability of N2-fixation. Using specific nifH primers targeting four major cyanobacterial clusters and quantitative PCR, we investigated how community composition, abundance and nifH expression varied by moss species and over the growing seasons. We evaluated N2-fixation rates across nine forest sites in June and September and explored the abundance and nifH expression of individual cyanobacterial clusters when N2-fixation is highest. Our results showed temporal and host-dependent variations of cyanobacterial community composition, nifH gene abundance and expression. N2-fixation was higher in September than June for both moss species, explained by higher nifH gene expression of individual clusters rather than higher nifH gene abundance or differences in cyanobacterial community composition. In most cases, ‘Stigonema cluster' made up less than 29% of the total cyanobacterial community, but accounted for the majority of nifH gene expression (82–94% of total nifH expression), irrespective of sampling date or moss species. Stepwise multiple regressions showed temporal variations in N2-fixation being greatly explained by variations in nifH expression of the ‘Stigonema cluster'. These results suggest that Stigonema is potentially the most influential N2-fixer in symbiosis with boreal forest feather mosses. PMID:26918665

  16. SIRT1 Suppresses Activator Protein-1 Transcriptional Activity and Cyclooxygenase-2 Expression in Macrophages*

    PubMed Central

    Zhang, Ran; Chen, Hou-Zao; Liu, Jin-Jing; Jia, Yu-Yan; Zhang, Zhu-Qin; Yang, Rui-Feng; Zhang, Yuan; Xu, Jing; Wei, Yu-Sheng; Liu, De-Pei; Liang, Chih-Chuan

    2010-01-01

    SIRT1 (Sirtuin type 1), a mammalian orthologue of yeast SIR2 (silent information regulator 2), has been shown to mediate a variety of calorie restriction (CR)-induced physiological events, such as cell fate regulation via deacetylation of the substrate proteins. However, whether SIRT1 deacetylates activator protein-1 (AP-1) to influence its transcriptional activity and target gene expression is still unknown. Here we demonstrate that SIRT1 directly interacts with the basic leucine zipper domains of c-Fos and c-Jun, the major components of AP-1, by which SIRT1 suppressed the transcriptional activity of AP-1. This process requires the deacetylase activity of SIRT1. Notably, SIRT1 reduced the expression of COX-2, a typical AP-1 target gene, and decreased prostaglandin E2 (PGE2) production of peritoneal macrophages (pMΦs). pMΦs with SIRT1 overexpression displayed improved phagocytosis and tumoricidal functions, which are associated with depressed PGE2. Furthermore, SIRT1 protein level was up-regulated in CR mouse pMΦs, whereas elevated SIRT1 decreased COX-2 expression and improved PGE2-related macrophage functions that were reversed following inhibition of SIRT1 deacetylase activity. Thus, our results indicate that SIRT1 may be a mediator of CR-induced macrophage regulation, and its deacetylase activity contributes to the inhibition of AP-1 transcriptional activity and COX-2 expression leading to amelioration of macrophage function. PMID:20042607

  17. Expression of HSF2 decreases in mitosis to enable stress-inducible transcription and cell survival

    PubMed Central

    Elsing, Alexandra N.; Aspelin, Camilla; Björk, Johanna K.; Bergman, Heidi A.; Himanen, Samu V.; Kallio, Marko J.; Roos-Mattjus, Pia

    2014-01-01

    Unless mitigated, external and physiological stresses are detrimental for cells, especially in mitosis, resulting in chromosomal missegregation, aneuploidy, or apoptosis. Heat shock proteins (Hsps) maintain protein homeostasis and promote cell survival. Hsps are transcriptionally regulated by heat shock factors (HSFs). Of these, HSF1 is the master regulator and HSF2 modulates Hsp expression by interacting with HSF1. Due to global inhibition of transcription in mitosis, including HSF1-mediated expression of Hsps, mitotic cells are highly vulnerable to stress. Here, we show that cells can counteract transcriptional silencing and protect themselves against proteotoxicity in mitosis. We found that the condensed chromatin of HSF2-deficient cells is accessible for HSF1 and RNA polymerase II, allowing stress-inducible Hsp expression. Consequently, HSF2-deficient cells exposed to acute stress display diminished mitotic errors and have a survival advantage. We also show that HSF2 expression declines during mitosis in several but not all human cell lines, which corresponds to the Hsp70 induction and protection against stress-induced mitotic abnormalities and apoptosis. PMID:25202032

  18. Ectopic expression of single transcription factors directs differentiation of a medaka spermatogonial cell line.

    PubMed

    Thoma, Eva C; Wagner, Toni U; Weber, Isabell P; Herpin, Amaury; Fischer, Andreas; Schartl, Manfred

    2011-08-01

    The capability to form all cell types of the body is a unique feature of stem cells. However, many questions remain concerning the mechanisms regulating differentiation potential. The derivation of spermatogonial cell lines (SGs) from mouse and human, which can differentiate across germ-layer borders, suggested male germ cells as a potential stem cell source in addition to embryonic stem cells. Here, we present a differentiation system using an SG of the vertebrate model organism Oryzias latipes (medaka). We report differentiation of this cell line into 4 different ectodermal and mesodermal somatic cell types. In addition to differentiation into adipocytes by retinoic acid treatment, we demonstrate for the first time that directed differentiation of an SG can be induced by ectopic expression of single transcription factors, completely independent of culture conditions. Transient transfection with mitf-m, a transcription factor that has been shown to induce differentiation into melanocytes in medaka embryonic stem cells, resulted in the formation of the same cell type in spermatogonia. Similarly, the formation of neuron-like cells and matrix-depositing osteoblasts was induced by ectopic expression of mash1 and cbfa1, respectively. Interestingly, we found that the expression of all mentioned fate-inducing transcription factors leads to recapitulation of the temporal pattern of marker gene expression known from in vivo studies. PMID:21090990

  19. Identification and Expression Profiles of Six Transcripts Encoding Carboxylesterase Protein in Vitis flexuosa Infected with Pathogens.

    PubMed

    Islam, Md Zaherul; Yun, Hae Keun

    2016-08-01

    Plants protect themselves from pathogen attacks via several mechanisms, including hypersensitive cell death. Recognition of pathogen attack by the plant resistance gene triggers expression of carboxylesterase genes associated with hypersensitive response. We identified six transcripts of carboxylesterase genes, Vitis flexuosa carboxylesterase 5585 (VfCXE5585), VfCXE12827, VfCXE13132, VfCXE17159, VfCXE18231, and VfCXE47674, which showed different expression patterns upon transcriptome analysis of V. flexuosa inoculated with Elsinoe ampelina. The lengths of genes ranged from 1,098 to 1,629 bp, and their encoded proteins consisted of 309 to 335 amino acids. The predicted amino acid sequences showed hydrolase like domains in all six transcripts and contained two conserved motifs, GXSXG of serine hydrolase characteristics and HGGGF related to the carboxylesterase family. The deduced amino acid sequence also contained a potential catalytic triad consisted of serine, aspartic acid and histidine. Of the six transcripts, VfCXE12827 showed upregulated expression against E. ampelina at all time points. Three genes (VfCXE5585, VfCXE12827, and VfCXE13132) showed upregulation, while others (VfCXE17159, VfCXE18231, and VfCXE47674) were down regulated in grapevines infected with Botrytis cinerea. All transcripts showed upregulated expression against Rhizobium vitis at early and later time points except VfCXE12827, and were downregulated for up to 48 hours post inoculation (hpi) after upregulation at 1 hpi in response to R. vitis infection. All tested genes showed high and differential expression in response to pathogens, indicating that they all may play a role in defense pathways during pathogen infection in grapevines. PMID:27493610

  20. Identification and Expression Profiles of Six Transcripts Encoding Carboxylesterase Protein in Vitis flexuosa Infected with Pathogens

    PubMed Central

    Islam, Md. Zaherul; Yun, Hae Keun

    2016-01-01

    Plants protect themselves from pathogen attacks via several mechanisms, including hypersensitive cell death. Recognition of pathogen attack by the plant resistance gene triggers expression of carboxylesterase genes associated with hypersensitive response. We identified six transcripts of carboxylesterase genes, Vitis flexuosa carboxylesterase 5585 (VfCXE5585), VfCXE12827, VfCXE13132, VfCXE17159, VfCXE18231, and VfCXE47674, which showed different expression patterns upon transcriptome analysis of V. flexuosa inoculated with Elsinoe ampelina. The lengths of genes ranged from 1,098 to 1,629 bp, and their encoded proteins consisted of 309 to 335 amino acids. The predicted amino acid sequences showed hydrolase like domains in all six transcripts and contained two conserved motifs, GXSXG of serine hydrolase characteristics and HGGGF related to the carboxylesterase family. The deduced amino acid sequence also contained a potential catalytic triad consisted of serine, aspartic acid and histidine. Of the six transcripts, VfCXE12827 showed upregulated expression against E. ampelina at all time points. Three genes (VfCXE5585, VfCXE12827, and VfCXE13132) showed upregulation, while others (VfCXE17159, VfCXE18231, and VfCXE47674) were down regulated in grapevines infected with Botrytis cinerea. All transcripts showed upregulated expression against Rhizobium vitis at early and later time points except VfCXE12827, and were downregulated for up to 48 hours post inoculation (hpi) after upregulation at 1 hpi in response to R. vitis infection. All tested genes showed high and differential expression in response to pathogens, indicating that they all may play a role in defense pathways during pathogen infection in grapevines. PMID:27493610

  1. Two modified RNA extraction methods compatible with transcript profiling and gene expression analysis for cotton roots.

    PubMed

    Xie, Chengjian; Wang, Chunyan; Wang, Xiaokun; Yang, Xingyong

    2013-01-01

    Efficient isolation of high-quality RNA is of prime importance for optimal transcript profiling results and further gene expression analysis. However, it is difficult for cotton roots because of lower-than-average RNA content and high content of polysaccharides, polyphenols, and other secondary metabolites. To develop simple and reliable protocols for high-quality RNA extraction from cotton roots for transcript profiling and gene expression analysis, some modifications were introduced to a reported plant RNA isolation protocol and a reagent kit method. Using method A, we successfully extracted high-quality RNA for transcript profiling from cotton roots. Gel electrophoresis analysis and polymerase chain reaction (PCR) assay indicated that RNA had good integrity without protein and genomic DNA contamination. Furthermore, the A260/280 (1.9) and A260/230 (1.6) ratios indicated that the isolated RNA was of high purity. Using method B, about 7 µg total RNA of high quality could be obtained from 0.1 g samples from cotton roots, which can be used for reverse-transcription (RT)-PCR and quantitative real-time RT-PCR. The two RNA extraction methods were used to investigate different gene expression of cotton roots (Gossypium hirsutum) infected by weak pathogenic Verticillium dahliae and the results showed they can satisfy the transcript profiling and quantitative real-time RT-PCR requirements for RNA. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

  2. Differential expression of two activating transcription factor 5 isoforms in papillary thyroid carcinoma

    PubMed Central

    Vicari, Luisa; La Rosa, Cristina; Forte, Stefano; Calabrese, Giovanna; Colarossi, Cristina; Aiello, Eleonora; Salluzzo, Salvatore; Memeo, Lorenzo

    2016-01-01

    Background Activating transcription factor 5 (ATF5) is a member of the activating transcription/cAMP response element-binding protein family of basic leucine zipper proteins that plays an important role in cell survival, differentiation, proliferation, and apoptosis. The ATF5 gene generates two transcripts: ATF5 isoform 1 and ATF5 isoform 2. A number of studies indicate that ATF5 could be an attractive target for therapeutic intervention in several tumor types; however, so far, the role of ATF5 has not been investigated in papillary thyroid carcinoma (PTC). Methods Quantitative real-time reverse transcription polymerase chain reaction and immuno-histochemical staining were used to study ATF5 mRNA and protein expression in PTC. Results We report here that ATF5 is expressed more in PTC tissue than in normal thyroid tissue. Furthermore, this is the first study that describes the presence of both ATF5 isoforms in PTC. Conclusion These findings could provide potential applications in PTC cancer treatment. PMID:27785070

  3. Post-transcriptional regulation of gene expression in bacterial pathogens by toxin-antitoxin systems

    PubMed Central

    Bertram, Ralph; Schuster, Christopher F.

    2014-01-01

    Toxin-antitoxin (TA) systems are small genetic elements ubiquitous in prokaryotic genomes that encode toxic proteins targeting various vital cellular functions. Typically, toxin activity is controlled by adjacently encoded protein or RNA antitoxins and unleashed as a consequence of genetic fluctuations or stressful conditions. Whereas some TA systems interfere with replication or cell wall synthesis, most of them influence transcriptional and post-transcriptional gene regulation. Antitoxin proteins often act as DNA binding transcriptional regulators and many TA toxins exhibit endoribonuclease activity to selectively degrade different RNA species and thus alter gene expression patterns. Some TA RNases cleave tRNA, tmRNAs or rRNAs, whereas most commonly mRNAs either in association with the ribosome or as free transcripts, are targeted. Examples are provided on how TA toxins differentially shape gene expression in bacterial pathogens by creating specialized ribosomes or by altering the transcriptome and how this may be tied in the control of pathogenicity factors. PMID:24524029

  4. Engineering secondary metabolism in maize cells by ectopic expression of transcription factors

    PubMed Central

    Grotewold, E; Chamberlin, M; Snook, M; Siame, B; Butler, L; Swenson, J; Maddock, S; Clair, GS; Bowen, B

    1998-01-01

    Manipulation of plant natural product biosynthesis through genetic engineering is an attractive but technically challenging goal. Here, we demonstrate that different secondary metabolites can be produced in cultured maize cells by ectopic expression of the appropriate regulatory genes. Cell lines engineered to express the maize transcriptional activators C1 and R accumulate two cyanidin derivatives, which are similar to the predominant anthocyanin found in differentiated plant tissues. In contrast, cell lines that express P accumulate various 3-deoxy flavonoids. Unexpectedly, P-expressing cells in culture also accumulate phenylpropanoids and green fluorescent compounds that are targeted to different subcellular compartments. Two endogenous biosynthetic genes (c2 and a1, encoding chalcone synthase and flavanone/dihydroflavonol reductase, respectively) are independently activated by ectopic expression of either P or C1/R, and there is a dose-response relationship between the transcript level of P and the degree to which c2 or a1 is expressed. Our results support a simple model showing how the gene encoding P may act as a quantitative trait locus controlling insecticidal C-glycosyl flavone level in maize silks, and they suggest how p1 might confer a selective advantage against insect predation in maize. PMID:9596632

  5. Endothelial Aquaporin-1 (AQP1) Expression Is Regulated by Transcription Factor Mef2c

    PubMed Central

    Jiang, Yong; Liu, He; Liu, Wen-jing; Tong, Hai-bin; Chen, Chang-jun; Lin, Fu-gui; Zhuo, Yan-hang; Qian, Xiao-zhen; Wang, Zeng-bin; Wang, Yu; Zhang, Peng; Jia, Hong-liang

    2016-01-01

    Aquaporin 1 (AQP1) is expressed in most microvasculature endothelial cells and forms water channels that play major roles in a variety of physiologic processes. This study aimed to delineate the transcriptional regulation of AQP1 by Mef2c in endothelial cells. Mef2c cooperated with Sp1 to activate human AQP1 transcription by binding to its proximal promoter in human umbilical cord vein endothelial cells (HUVEC). Over-expression of Mef2c, Sp1, or Mef2c/Sp1 increased HUVEC migration and tube-forming ability, which can be abolished AQP1 knockdown. These data indicate that AQP1 is a direct target of Mef2c in regulating angiogenesis and vasculogenesis of endothelial cells. PMID:26923194

  6. Multiple breast cancer risk variants are associated with differential transcript isoform expression in tumors

    PubMed Central

    Caswell, Jennifer L.; Camarda, Roman; Zhou, Alicia Y.; Huntsman, Scott; Hu, Donglei; Brenner, Steven E.; Zaitlen, Noah; Goga, Andrei; Ziv, Elad

    2015-01-01

    Genome-wide association studies have identified over 70 single-nucleotide polymorphisms (SNPs) associated with breast cancer. A subset of these SNPs are associated with quantitative expression of nearby genes, but the functional effects of the majority remain unknown. We hypothesized that some risk SNPs may regulate alternative splicing. Using RNA-sequencing data from breast tumors and germline genotypes from The Cancer Genome Atlas, we tested the association between each risk SNP genotype and exon-, exon–exon junction- or transcript-specific expression of nearby genes. Six SNPs were associated with differential transcript expression of seven nearby genes at FDR < 0.05 (BABAM1, DCLRE1B/PHTF1, PEX14, RAD51L1, SRGAP2D and STXBP4). We next developed a Bayesian approach to evaluate, for each SNP, the overlap between the signal of association with breast cancer and the signal of association with alternative splicing. At one locus (SRGAP2D), this method eliminated the possibility that the breast cancer risk and the alternate splicing event were due to the same causal SNP. Lastly, at two loci, we identified the likely causal SNP for the alternative splicing event, and at one, functionally validated the effect of that SNP on alternative splicing using a minigene reporter assay. Our results suggest that the regulation of differential transcript isoform expression is the functional mechanism of some breast cancer risk SNPs and that we can use these associations to identify causal SNPs, target genes and the specific transcripts that may mediate breast cancer risk. PMID:26472073

  7. A new transcription factor for mitosis: in Schizosaccharomyces pombe, the RFX transcription factor Sak1 works with forkhead factors to regulate mitotic expression.

    PubMed

    Garg, Angad; Futcher, Bruce; Leatherwood, Janet

    2015-08-18

    Mitotic genes are one of the most strongly oscillating groups of genes in the eukaryotic cell cycle. Understanding the regulation of mitotic gene expression is a key issue in cell cycle control but is poorly understood in most organisms. Here, we find a new mitotic transcription factor, Sak1, in the fission yeast Schizosaccharomyces pombe. Sak1 belongs to the RFX family of transcription factors, which have not previously been connected to cell cycle control. Sak1 binds upstream of mitotic genes in close proximity to Fkh2, a forkhead transcription factor previously implicated in regulation of mitotic genes. We show that Sak1 is the major activator of mitotic gene expression and also confirm the role of Fkh2 as the opposing repressor. Sep1, another forkhead transcription factor, is an activator for a small subset of mitotic genes involved in septation. From yeasts to humans, forkhead transcription factors are involved in mitotic gene expression and it will be interesting to see whether RFX transcription factors may also be involved in other organisms.

  8. Expression Atlas update—a database of gene and transcript expression from microarray- and sequencing-based functional genomics experiments

    PubMed Central

    Petryszak, Robert; Burdett, Tony; Fiorelli, Benedetto; Fonseca, Nuno A.; Gonzalez-Porta, Mar; Hastings, Emma; Huber, Wolfgang; Jupp, Simon; Keays, Maria; Kryvych, Nataliya; McMurry, Julie; Marioni, John C.; Malone, James; Megy, Karine; Rustici, Gabriella; Tang, Amy Y.; Taubert, Jan; Williams, Eleanor; Mannion, Oliver; Parkinson, Helen E.; Brazma, Alvis

    2014-01-01

    Expression Atlas (http://www.ebi.ac.uk/gxa) is a value-added database providing information about gene, protein and splice variant expression in different cell types, organism parts, developmental stages, diseases and other biological and experimental conditions. The database consists of selected high-quality microarray and RNA-sequencing experiments from ArrayExpress that have been manually curated, annotated with Experimental Factor Ontology terms and processed using standardized microarray and RNA-sequencing analysis methods. The new version of Expression Atlas introduces the concept of ‘baseline’ expression, i.e. gene and splice variant abundance levels in healthy or untreated conditions, such as tissues or cell types. Differential gene expression data benefit from an in-depth curation of experimental intent, resulting in biologically meaningful ‘contrasts’, i.e. instances of differential pairwise comparisons between two sets of biological replicates. Other novel aspects of Expression Atlas are its strict quality control of raw experimental data, up-to-date RNA-sequencing analysis methods, expression data at the level of gene sets, as well as genes and a more powerful search interface designed to maximize the biological value provided to the user. PMID:24304889

  9. The over-expression of a chrysanthemum WRKY transcription factor enhances aphid resistance.

    PubMed

    Li, Peiling; Song, Aiping; Gao, Chunyan; Jiang, Jiafu; Chen, Sumei; Fang, Weimin; Zhang, Fei; Chen, Fadi

    2015-10-01

    Members of the large WRKY transcription factor family are responsible for the regulation of plant growth, development and the stress response. Here, five WRKY members were isolated from chrysanthemum. They each contained a single WRKY domain and a C2H2 zinc finger motif, so were classified into group II. Transient expression experiments demonstrated that all five were expressed in the nucleus, although CmWRKY42 was also expressed in the cytoplasm. When expressed heterologously in yeast, the products of CmWRKY22 and CmWRKY48 exhibited transactivation activity, while those of CmWRKY21, CmWRKY40 and CmWRKY42 did not. The transcription of the five CmWRKY genes was profiled when the plants were challenged with a variety of abiotic and biotic stress agents, as well as being treated with various phytohormones. CmWRKY21 proved to be markedly induced by salinity stress, and suppressed by high temperature exposure; CmWRKY22 was induced by high temperature exposure; CmWRKY40 was highly induced by salinity stress, and treatment with either abscisic acid (ABA) or methyl jasmonate (MeJA); CmWRKY42 was up-regulated by salinity stress, low temperature, ABA and MeJA treatment and aphid infestation; CmWRKY48 was induced by drought stress, ABA and MeJA treatment and aphid infestation. The function of CmWRKY48 was further investigated by over-expressing it transgenically. The constitutive expression of this transcription factor inhibited the aphids' population growth capacity, suggesting that it may represent an important component of the plant's defense machinery against aphids. PMID:26184088

  10. A Comparative Analysis of Transcription Factor Expression during Metazoan Embryonic Development

    PubMed Central

    Schep, Alicia N.; Adryan, Boris

    2013-01-01

    During embryonic development, a complex organism is formed from a single starting cell. These processes of growth and differentiation are driven by large transcriptional changes, which are following the expression and activity of transcription factors (TFs). This study sought to compare TF expression during embryonic development in a diverse group of metazoan animals: representatives of vertebrates (Danio rerio, Xenopus tropicalis), a chordate (Ciona intestinalis) and invertebrate phyla such as insects (Drosophila melanogaster, Anopheles gambiae) and nematodes (Caenorhabditis elegans) were sampled, The different species showed overall very similar TF expression patterns, with TF expression increasing during the initial stages of development. C2H2 zinc finger TFs were over-represented and Homeobox TFs were under-represented in the early stages in all species. We further clustered TFs for each species based on their quantitative temporal expression profiles. This showed very similar TF expression trends in development in vertebrate and insect species. However, analysis of the expression of orthologous pairs between more closely related species showed that expression of most individual TFs is not conserved, following the general model of duplication and diversification. The degree of similarity between TF expression between Xenopus tropicalis and Danio rerio followed the hourglass model, with the greatest similarity occuring during the early tailbud stage in Xenopus tropicalis and the late segmentation stage in Danio rerio. However, for Drosophila melanogaster and Anopheles gambiae there were two periods of high TF transcriptome similarity, one during the Arthropod phylotypic stage at 8–10 hours into Drosophila development and the other later at 16–18 hours into Drosophila development. PMID:23799133

  11. Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor.

    PubMed

    Núñez-López, Lizeth; Aguirre-Cruz, Andrés; Barrera-Figueroa, Blanca Estela; Peña-Castro, Julián Mario

    2015-01-01

    Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene) can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0), mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200-300% more glucose, adult vegetative plants yielded 40-90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1), juvenile transition (SPL3-5) and meristematic identity (FUL, SOC1) but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production.

  12. Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor

    PubMed Central

    Núñez-López, Lizeth; Aguirre-Cruz, Andrés

    2015-01-01

    Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene) can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0), mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200–300% more glucose, adult vegetative plants yielded 40–90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1), juvenile transition (SPL3-5) and meristematic identity (FUL, SOC1) but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production. PMID:25780769

  13. Hog1 Targets Whi5 and Msa1 Transcription Factors To Downregulate Cyclin Expression upon Stress

    PubMed Central

    González-Novo, Alberto; Jiménez, Javier; Clotet, Josep; Nadal-Ribelles, Mariona; Cavero, Santiago

    2015-01-01

    Yeast cells have developed complex mechanisms to cope with extracellular insults. An increase in external osmolarity leads to activation of the stress-activated protein kinase Hog1, which is the main regulator of adaptive responses, such as gene expression and cell cycle progression, that are essential for cellular survival. Upon osmostress, the G1-to-S transition is regulated by Hog1 through stabilization of the cyclin-dependent kinase inhibitor Sic1 and the downregulation of G1 cyclin expression by an unclear mechanism. Here, we show that Hog1 interacts with and phosphorylates components of the core cell cycle transcriptional machinery such as Whi5 and the coregulator Msa1. Phosphorylation of these two transcriptional regulators by Hog1 is essential for inhibition of G1 cyclin expression, for control of cell morphogenesis, and for maximal cell survival upon stress. The control of both Whi5 and Msa1 by Hog1 also revealed the necessity for proper coordination of budding and DNA replication. Thus, Hog1 regulates G1 cyclin transcription upon osmostress to ensure coherent passage through Start. PMID:25733686

  14. CaMKII inhibition promotes neuronal apoptosis by transcriptionally upregulating Bim expression.

    PubMed

    Zhao, Yiwei; Zhu, Lin; Yu, Shaojun; Zhu, Jing; Wang, Chong

    2016-09-28

    The effects of Ca/calmodulin-dependent protein kinase II (CaMKII) on neuronal apoptosis are complex and contradictory, and the underlying mechanisms remain unclear. Bcl-2-interacting mediator of cell death (Bim) is an important proapoptotic protein under many physiological and pathophysiological conditions. However, there is no evidence that CaMKII and Bim are mechanistically linked in neuronal apoptosis. In this study, we showed that CaMKII inhibition by the inhibitors KN-62 and myristoylated autocamtide-2-related inhibitory peptide promoted apoptosis in cerebellar granule neurons in a dose-dependent manner. CaMKII inhibition increased Bim protein and messenger RNA levels. The expression of early growth response factor-1, a transcription factor of Bim, was also induced by CaMKII inhibitors. These data suggested that CaMKII repressed the transcriptional expression of Bim. Moreover, knockdown of Bim using small interfering RNAs attenuated the proapoptotic effects of CaMKII inhibition. Taken together, this is the first report to show that CaMKII inhibition transcriptionally upregulates Bim expression to promote neuronal apoptosis, providing new insights into the proapoptotic mechanism of CaMKII inhibition.

  15. CaMKII inhibition promotes neuronal apoptosis by transcriptionally upregulating Bim expression.

    PubMed

    Zhao, Yiwei; Zhu, Lin; Yu, Shaojun; Zhu, Jing; Wang, Chong

    2016-09-28

    The effects of Ca/calmodulin-dependent protein kinase II (CaMKII) on neuronal apoptosis are complex and contradictory, and the underlying mechanisms remain unclear. Bcl-2-interacting mediator of cell death (Bim) is an important proapoptotic protein under many physiological and pathophysiological conditions. However, there is no evidence that CaMKII and Bim are mechanistically linked in neuronal apoptosis. In this study, we showed that CaMKII inhibition by the inhibitors KN-62 and myristoylated autocamtide-2-related inhibitory peptide promoted apoptosis in cerebellar granule neurons in a dose-dependent manner. CaMKII inhibition increased Bim protein and messenger RNA levels. The expression of early growth response factor-1, a transcription factor of Bim, was also induced by CaMKII inhibitors. These data suggested that CaMKII repressed the transcriptional expression of Bim. Moreover, knockdown of Bim using small interfering RNAs attenuated the proapoptotic effects of CaMKII inhibition. Taken together, this is the first report to show that CaMKII inhibition transcriptionally upregulates Bim expression to promote neuronal apoptosis, providing new insights into the proapoptotic mechanism of CaMKII inhibition. PMID:27483389

  16. Heterologous expression of Translocated promoter region protein, Tpr, identified as a transcription factor from Rattus norvegicus.

    PubMed

    Agarwal, Shivani; Yadav, Sunita Kumari; Dixit, Aparna

    2011-05-01

    Our earlier studies have demonstrated that the 35 kDa isoform of Translocated promoter region protein (Tpr) of Rattus norvegicus was able to augment c-jun transcription efficiently. Identification of direct targets that may in part downregulate c-jun transcription might prove to be an ideal target to curtail the proliferation of normal cells under pathophysiological conditions. In order to evaluate its potential as a pharmaceutical target, the protein must be produced and purified in sufficiently high yields. In the present study, we report the high level expression of Tpr protein of R. norvegicus employing heterologous host, Escherichia coli, to permit its structural characterization in great detail. We here demonstrate that the Tpr protein was expressed in soluble form and approximately 90 mg/L of the purified protein at the shake flask level could be achieved to near homogeneity using single step-metal chelate affinity chromatography. The amino acid sequence of the protein was confirmed by mass spectroscopic analysis. The highly unstable and disordered Tpr protein was imparted structural and functional stability by the addition of glycerol and it has been shown that the natively unfolded Tpr protein retains DNA binding ability under these conditions only. Thus, the present study emphasizes the significance of an efficient prokaryotic system, which results in a high level soluble expression of a DNA binding protein of eukaryotic origin. Thus, the present strategy employed for purification of the R. norvegicus Tpr protein bypasses the need for the tedious expression strategies associated with the eukaryotic expression systems.

  17. Identification of a transcriptional regulator that controls intracellular gene expression in Salmonella Typhi.

    PubMed

    Haghjoo, Erik; Galán, Jorge E

    2007-06-01

    Salmonella enterica serovar Typhi (S. Typhi), the aetiological agent of typhoid fever, is an exclusively human pathogen. Little is known about specific factors that may confer to this bacterium its unique pathogenic features. One of these determinants is CdtB, a homologue of the active subunit of the cytolethal distending toxin, which causes DNA damage leading to cell cycle arrest and distension of intoxicated cells. A unique property of S. Typhi CdtB is that it is only synthesized when this bacterium is within an intracellular compartment. Through a genetic screen, we have identified a transcriptional regulatory protein that controls the intracellular expression of cdtB. This regulator, which we have named IgeR, is a member of the DeoR family of transcriptional regulatory proteins and is highly conserved in all S. enterica serovars. IgeR directly binds the cdtB promoter and represses its expression in the extracellular environment. Microarray analysis identified additional IgeR-regulated genes that are involved in virulence. Constitutive expression of igeR resulted in the reduction of intracellular expression of cdtB by S. Typhi and in significant impairment of the virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium) in mice. We propose that IgeR may co-ordinate gene expression during Salmonella's transition from an extracellular to an intracellular environment. PMID:17555437

  18. Myosin heavy chain-2b transcripts and isoform are expressed in human laryngeal muscles.

    PubMed

    Smerdu, Vika; Cvetko, Erika

    2013-01-01

    Three fast myosin heavy chain (MyHC) isoforms, i.e. MyHC-2a, -2x and -2b, are expressed in skeletal muscles of smaller mammals. In contrast, only MyHC-2a and -2x have been revealed in humans so far. The expression of MyHC isoforms is known to be wider in the functionally more specialized laryngeal muscles. Though mRNA transcripts of the MyHC-2b gene were found to be expressed in certain human skeletal and laryngeal muscles, the corresponding isoform has not been demonstrated in these muscles. To our knowledge, we are the first to demonstrate not only the expression of MyHC-2b transcripts using an in situ hybridization technique but also the corresponding protein, i.e. the MyHC-2b isoform, in some human laryngeal muscles by immunohistochemistry but not by polyacrylamide gel electrophoresis. Using a set of antibodies specific to MyHC isoforms, we demonstrated that MyHC-2b was always co-expressed with the major MyHC isoforms, not only with the fast ones (MyHC-2a and -2x) but with the slow isoform (MyHC-1) as well.

  19. Reactive oxygen species in signalling the transcriptional activation of WIPK expression in tobacco.

    PubMed

    Xu, Juan; Yang, Kwang-Yeol; Yoo, Seung Jin; Liu, Yidong; Ren, Dongtao; Zhang, Shuqun

    2014-07-01

    Plant mitogen-activated protein kinases represented by tobacco WIPK (wounding-induced protein kinase) and its orthologs in other species are unique in their regulation at transcriptional level in response to stress and pathogen infection. We previously demonstrated that transcriptional activation of WIPK is essential for induced WIPK activity, and activation of salicylic acid-induced protein kinase (SIPK) by the constitutively active NtMEK2(DD) is sufficient to induce WIPK gene expression. Here, we report that the effect of SIPK on WIPK gene expression is mediated by reactive oxygen species (ROS). Using a combination of pharmacological and gain-of-function transgenic approaches, we studied the relationship among SIPK activation, WIPK gene activation in response to fungal cryptogein, light-dependent ROS generation in chloroplasts, and ROS generated via NADPH oxidase. In the conditional gain-of-function GVG-NtMEK2(DD) transgenic tobacco, induction of WIPK expression is dependent on the ROS generation in chloroplasts. Consistently, methyl viologen, an inducer of ROS generation in chloroplasts, highly activated WIPK expression. In addition to chloroplast-originated ROS, H(2)O(2) generated from the cell-surface NADPH oxidase could also activate WIPK gene expression, and inhibition of cryptogein-induced ROS generation also abolished WIPK gene activation. Our data demonstrate that WIPK gene activation is mediated by ROS, which provides a mechanism by which ROS influence cellular signalling processes in plant stress/defence response.

  20. Tissue-specific mtDNA abundance from exome data and its correlation with mitochondrial transcription, mass and respiratory activity.

    PubMed

    D'Erchia, Anna Maria; Atlante, Anna; Gadaleta, Gemma; Pavesi, Giulio; Chiara, Matteo; De Virgilio, Caterina; Manzari, Caterina; Mastropasqua, Francesca; Prazzoli, Gian Marco; Picardi, Ernesto; Gissi, Carmela; Horner, David; Reyes, Aurelio; Sbisà, Elisabetta; Tullo, Apollonia; Pesole, Graziano

    2015-01-01

    Eukaryotic cells contain a population of mitochondria, variable in number and shape, which in turn contain multiple copies of a tiny compact genome (mtDNA) whose expression and function is strictly coordinated with the nuclear one. mtDNA copy number varies between different cell or tissues types, both in response to overall metabolic and bioenergetics demands and as a consequence or cause of specific pathological conditions. Here we present a novel and reliable methodology to assess the effective mtDNA copy number per diploid genome by investigating off-target reads obtained by whole-exome sequencing (WES) experiments. We also investigate whether and how mtDNA copy number correlates with mitochondrial mass, respiratory activity and expression levels. Analyzing six different tissues from three age- and sex-matched human individuals, we found a highly significant linear correlation between mtDNA copy number estimated by qPCR and the frequency of mtDNA off target WES reads. Furthermore, mtDNA copy number showed highly significant correlation with mitochondrial gene expression levels as measured by RNA-Seq as well as with mitochondrial mass and respiratory activity. Our methodology makes thus feasible, at a large scale, the investigation of mtDNA copy number in diverse cell-types, tissues and pathological conditions or in response to specific treatments. PMID:25446395

  1. Transcriptome-wide profiling and expression analysis of transcription factor families in a liverwort, Marchantia polymorpha

    PubMed Central

    2013-01-01

    Background Transcription factors (TFs) are vital elements that regulate transcription and the spatio-temporal expression of genes, thereby ensuring the accurate development and functioning of an organism. The identification of TF-encoding genes in a liverwort, Marchantia polymorpha, offers insights into TF organization in the members of the most basal lineages of land plants (embryophytes). Therefore, a comparison of Marchantia TF genes with other land plants (monocots, dicots, bryophytes) and algae (chlorophytes, rhodophytes) provides the most comprehensive view of the rates of expansion or contraction of TF genes in plant evolution. Results In this study, we report the identification of TF-encoding transcripts in M. polymorpha for the first time, as evidenced by deep RNA sequencing data. In total, 3,471 putative TF encoding transcripts, distributed in 80 families, were identified, representing 7.4% of the generated Marchantia gametophytic transcriptome dataset. Overall, TF basic functions and distribution across families appear to be conserved when compared to other plant species. However, it is of interest to observe the genesis of novel sequences in 24 TF families and the apparent termination of 2 TF families with the emergence of Marchantia. Out of 24 TF families, 6 are known to be associated with plant reproductive development processes. We also examined the expression pattern of these TF-encoding transcripts in six male and female developmental stages in vegetative and reproductive gametophytic tissues of Marchantia. Conclusions The analysis highlighted the importance of Marchantia, a model plant system, in an evolutionary context. The dataset generated here provides a scientific resource for TF gene discovery and other comparative evolutionary studies of land plants. PMID:24365221

  2. Dietary carbohydrates enhance lactase/phlorizin hydrolase gene expression at a transcription level in rat jejunum.

    PubMed

    Tanaka, T; Kishi, K; Igawa, M; Takase, S; Goda, T

    1998-04-01

    We have previously shown that dietary sucrose stimulates the lactase/phlorizin hydrolase (LPH) mRNA accumulation along with a rise in lactase activity in rat jejunum [Goda, Yasutake, Suzuki, Takase and Koldovský (1995) Am. J. Physiol. 268, G1066-G1073]. To elucidate the mechanisms whereby dietary carbohydrates enhance the LPH mRNA expression, 7-week-old rats that had been fed a low-carbohydrate diet (5.5% of energy as starch) were given diets containing various monosaccharides or sucrose for 12h. Among carbohydrates examined, fructose, sucrose, galactose and glycerol elicited an increase in LPH mRNA accumulation along with a rise in lactase activity in the jejunum. By contrast, glucose and alpha-methylglucoside were unable to elicit a significant increase in LPH mRNA levels. To explore a transcriptional mechanism for the carbohydrate-induced increases in LPH mRNA levels, we employed two techniques currently available to estimate transcriptional rate, i.e. RNA protection assays of pre-mRNA using an intron probe, and nuclear run-on assays. Both assays revealed that fructose elicited an increase in transcription of the LPH gene, and that the transcription of LPH was influenced only slightly, if at all, by glucose intake. These results suggest that certain monosaccharides such as fructose or their metabolite(s) are capable of enhancing LPH mRNA levels in the small intestine, and that transcriptional control might play a major role in the carbohydrate-induced increase of LPH mRNA expression.

  3. Resistance of Biomphalaria glabrata 13–16-R1 snails to Schistosoma mansoni PR1 is a function of haemocyte abundance and constitutive levels of specific transcripts in haemocytes☆

    PubMed Central

    Larson, Maureen K.; Bender, Randal C.; Bayne, Christopher J.

    2014-01-01

    Continuing transmission of human intestinal schistosomiasis depends on the parasite’s access to susceptible snail intermediate hosts (often Biomphalaria glabrata). Transmission fails when parasite larvae enter resistant individuals in wild snail populations. The genetic basis for differences in snail susceptibility/resistance is being intensively investigated as a means to devise novel control strategies based on resistance genes. Reactive oxygen species produced by the snail’s defence cells (haemocytes) are effectors of resistance. We hypothesised that genes relevant to production and consumption of reactive oxygen species would be expressed differentially in the haemocytes of snail hosts with different susceptibility/resistance phenotypes. By restricting the genetic diversity of snails, we sought to facilitate identification of resistance genes. By inbreeding, we procured from a 13–16-R1 snail population with both susceptible and resistant individuals 52 lines of B. glabrata (expected homozygosity ~87.5%), and determined the phenotype of each in regard to susceptibility/resistance to Schistosoma mansoni. The inbred lines were found to have line-specific differences in numbers of spreading haemocytes; these were enumerated in both juvenile and adult snails. Lines with high cell numbers were invariably resistant to S. mansoni, whereas lines with lower cell numbers could be resistant or susceptible. Transcript levels in haemocytes were quantified for 18 potentially defence-related genes. Among snails with low cell numbers, the different susceptibility/resistance phenotypes correlated with differences in transcript levels for two redox-relevant genes: an inferred phagocyte oxidase component and a peroxiredoxin. Allograft inflammatory factor (potentially a regulator of leucocyte activation) was expressed at higher levels in resistant snails regardless of spread cell number. Having abundant spreading haemocytes is inferred to enable a snail to kill parasite

  4. Expression of root-related transcription factors associated with flooding tolerance of soybean (Glycine max).

    PubMed

    Valliyodan, Babu; Van Toai, Tara T; Alves, Jose Donizeti; de Fátima P Goulart, Patricia; Lee, Jeong Dong; Fritschi, Felix B; Rahman, Mohammed Atiqur; Islam, Rafiq; Shannon, J Grover; Nguyen, Henry T

    2014-01-01

    Much research has been conducted on the changes in gene expression of the model plant Arabidopsis to low-oxygen stress. Flooding results in a low oxygen environment in the root zone. However, there is ample evidence that tolerance to soil flooding is more than tolerance to low oxygen alone. In this study, we investigated the physiological response and differential expression of root-related transcription factors (TFs) associated with the tolerance of soybean plants to soil flooding. Differential responses of PI408105A and S99-2281 plants to ten days of soil flooding were evaluated at physiological, morphological and anatomical levels. Gene expression underlying the tolerance response was investigated using qRT-PCR of root-related TFs, known anaerobic genes, and housekeeping genes. Biomass of flood-sensitive S99-2281 roots remained unchanged during the entire 10 days of flooding. Flood-tolerant PI408105A plants exhibited recovery of root growth after 3 days of flooding. Flooding induced the development of aerenchyma and adventitious roots more rapidly in the flood-tolerant than the flood-sensitive genotype. Roots of tolerant plants also contained more ATP than roots of sensitive plants at the 7th and 10th days of flooding. Quantitative transcript analysis identified 132 genes differentially expressed between the two genotypes at one or more time points of flooding. Expression of genes related to the ethylene biosynthesis pathway and formation of adventitious roots was induced earlier and to higher levels in roots of the flood-tolerant genotype. Three potential flood-tolerance TFs which were differentially expressed between the two genotypes during the entire 10-day flooding duration were identified. This study confirmed the expression of anaerobic genes in response to soil flooding. Additionally, the differential expression of TFs associated with soil flooding tolerance was not qualitative but quantitative and temporal. Functional analyses of these genes will be

  5. Expression of forkhead box transcription factor genes Foxp1 and Foxp2 during jaw development.

    PubMed

    Cesario, Jeffry M; Almaidhan, Asma A; Jeong, Juhee

    2016-03-01

    Development of the face is regulated by a large number of genes that are expressed in temporally and spatially specific patterns. While significant progress has been made on characterizing the genes that operate in the oral region of the face, those regulating development of the aboral (lateral) region remain largely unknown. Recently, we discovered that transcription factors LIM homeobox (LHX) 6 and LHX8, which are key regulators of oral development, repressed the expression of the genes encoding forkhead box transcription factors, Foxp1 and Foxp2, in the oral region. To gain insights into the potential role of the Foxp genes in region-specific development of the face, we examined their expression patterns in the first pharyngeal arch (primordium for the jaw) of mouse embryos at a high spatial and temporal resolution. Foxp1 and Foxp2 were preferentially expressed in the aboral and posterior parts of the first pharyngeal arch, including the developing temporomandibular joint. Through double immunofluorescence and double fluorescent RNA in situ hybridization, we found that Foxp1 was expressed in the progenitor cells for the muscle, bone, and connective tissue. Foxp2 was expressed in subsets of bone and connective tissue progenitors but not in the myoblasts. Neither gene was expressed in the dental mesenchyme nor in the oral half of the palatal shelf undergoing extensive growth and morphogenesis. Together, we demonstrated for the first time that Foxp1 and Foxp2 are expressed during craniofacial development. Our data suggest that the Foxp genes may regulate development of the aboral and posterior regions of the jaw.

  6. Expression of root-related transcription factors associated with flooding tolerance of soybean (Glycine max).

    PubMed

    Valliyodan, Babu; Van Toai, Tara T; Alves, Jose Donizeti; de Fátima P Goulart, Patricia; Lee, Jeong Dong; Fritschi, Felix B; Rahman, Mohammed Atiqur; Islam, Rafiq; Shannon, J Grover; Nguyen, Henry T

    2014-01-01

    Much research has been conducted on the changes in gene expression of the model plant Arabidopsis to low-oxygen stress. Flooding results in a low oxygen environment in the root zone. However, there is ample evidence that tolerance to soil flooding is more than tolerance to low oxygen alone. In this study, we investigated the physiological response and differential expression of root-related transcription factors (TFs) associated with the tolerance of soybean plants to soil flooding. Differential responses of PI408105A and S99-2281 plants to ten days of soil flooding were evaluated at physiological, morphological and anatomical levels. Gene expression underlying the tolerance response was investigated using qRT-PCR of root-related TFs, known anaerobic genes, and housekeeping genes. Biomass of flood-sensitive S99-2281 roots remained unchanged during the entire 10 days of flooding. Flood-tolerant PI408105A plants exhibited recovery of root growth after 3 days of flooding. Flooding induced the development of aerenchyma and adventitious roots more rapidly in the flood-tolerant than the flood-sensitive genotype. Roots of tolerant plants also contained more ATP than roots of sensitive plants at the 7th and 10th days of flooding. Quantitative transcript analysis identified 132 genes differentially expressed between the two genotypes at one or more time points of flooding. Expression of genes related to the ethylene biosynthesis pathway and formation of adventitious roots was induced earlier and to higher levels in roots of the flood-tolerant genotype. Three potential flood-tolerance TFs which were differentially expressed between the two genotypes during the entire 10-day flooding duration were identified. This study confirmed the expression of anaerobic genes in response to soil flooding. Additionally, the differential expression of TFs associated with soil flooding tolerance was not qualitative but quantitative and temporal. Functional analyses of these genes will be

  7. Expression of Root-Related Transcription Factors Associated with Flooding Tolerance of Soybean (Glycine max)

    PubMed Central

    Valliyodan, Babu; Van Toai, Tara T.; Alves, Jose Donizeti; de Fátima P. Goulart, Patricia; Lee, Jeong Dong; Fritschi, Felix B.; Rahman, Mohammed Atiqur; Islam, Rafiq; Shannon, J. Grover; Nguyen, Henry T.

    2014-01-01

    Much research has been conducted on the changes in gene expression of the model plant Arabidopsis to low-oxygen stress. Flooding results in a low oxygen environment in the root zone. However, there is ample evidence that tolerance to soil flooding is more than tolerance to low oxygen alone. In this study, we investigated the physiological response and differential expression of root-related transcription factors (TFs) associated with the tolerance of soybean plants to soil flooding. Differential responses of PI408105A and S99-2281 plants to ten days of soil flooding were evaluated at physiological, morphological and anatomical levels. Gene expression underlying the tolerance response was investigated using qRT-PCR of root-related TFs, known anaerobic genes, and housekeeping genes. Biomass of flood-sensitive S99-2281 roots remained unchanged during the entire 10 days of flooding. Flood-tolerant PI408105A plants exhibited recovery of root growth after 3 days of flooding. Flooding induced the development of aerenchyma and adventitious roots more rapidly in the flood-tolerant than the flood-sensitive genotype. Roots of tolerant plants also contained more ATP than roots of sensitive plants at the 7th and 10th days of flooding. Quantitative transcript analysis identified 132 genes differentially expressed between the two genotypes at one or more time points of flooding. Expression of genes related to the ethylene biosynthesis pathway and formation of adventitious roots was induced earlier and to higher levels in roots of the flood-tolerant genotype. Three potential flood-tolerance TFs which were differentially expressed between the two genotypes during the entire 10-day flooding duration were identified. This study confirmed the expression of anaerobic genes in response to soil flooding. Additionally, the differential expression of TFs associated with soil flooding tolerance was not qualitative but quantitative and temporal. Functional analyses of these genes will be

  8. ZBTB2 increases PDK4 expression by transcriptional repression of RelA/p65

    PubMed Central

    Kim, Min-Young; Koh, Dong-In; Choi, Won-Il; Jeon, Bu-Nam; Jeong, Deok-yoon; Kim, Kyung-Sup; Kim, Kunhong; Kim, Se-Hoon; Hur, Man-Wook

    2015-01-01

    The NF-κB is found in almost all animal cell types and is involved in a myriad of cellular responses. Aberrant expression of NF-κB has been linked to cancer, inflammatory diseases and improper development. Little is known about transcriptional regulation of the NF-κB family member gene RelA/p65. Sp1 plays a key role in the expression of the RelA/p65 gene. ZBTB2 represses transcription of the gene by inhibiting Sp1 binding to a Sp1-binding GC-box in the RelA/p65 proximal promoter (bp, −31 to −21). Moreover, recent studies revealed that RelA/p65 directly binds to the peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α) to decrease transcriptional activation of the PGC1α target gene PDK4, whose gene product inhibits pyruvate dehydrogenase (PDH), a key regulator of TCA cycle flux. Accordingly, we observed that RelA/p65 repression by ZBTB2 indirectly results in increased PDK4 expression, which inhibits PDH. Consequently, in cells with ectopic ZBTB2, the concentrations of pyruvate and lactate were higher than those in normal cells, indicating changes in glucose metabolism flux favoring glycolysis over the TCA cycle. Knockdown of ZBTB2 in mouse xenografts decreased tumor growth. ZBTB2 may increase cell proliferation by reprogramming glucose metabolic pathways to favor glycolysis by upregulating PDK4 expression via repression of RelA/p65 expression. PMID:25609694

  9. Conserved Regional Patterns of GABA-Related Transcript Expression in the Neocortex of Subjects With Schizophrenia

    PubMed Central

    Hashimoto, Takanori; Bazmi, H. Holly; Mirnics, Karoly; Wu, Qiang; Sampson, Allan R.; Lewis, David A.

    2010-01-01

    Objective Individuals with schizophrenia exhibit disturbances in a number of cognitive, affective, sensory, and motor functions that depend on the circuitry of different cortical areas. The cognitive deficits associated with dysfunction of the dorsolateral prefrontal cortex result, at least in part, from abnormalities in GABA neurotransmission, as reflected in a specific pattern of altered expression of GABA-related genes. Consequently, the authors sought to determine whether this pattern of altered gene expression is restricted to the dorsolateral prefrontal cortex or could also contribute to the dysfunction of other cortical areas in subjects with schizophrenia. Method Real-time quantitative polymerase chain reaction was used to assess the levels of eight GABA-related transcripts in four cortical areas (dorsolateral prefrontal cortex, anterior cingulate cortex, and primary motor and primary visual cortices) of subjects (N=12) with schizophrenia and matched normal comparison subjects. Results Expression levels of seven transcripts were lower in subjects with schizophrenia, with the magnitude of reduction for each transcript comparable across the four areas. The largest reductions were detected for mRNA encoding somatostatin and parvalbumin, followed by moderate decreases in mRNA expression for the 67-kilodalton isoform of glutamic acid decarboxylase, the GABA membrane transporter GAT-1, and the α1 and δ subunits of GABAA receptors. In contrast, the expression of calretinin mRNA did not differ between the subject groups in any of the four areas. Conclusions Because the areas examined represent the major functional domains (e.g., association, limbic, motor, and sensory) of the cerebral cortex, our findings suggest that a conserved set of molecular alterations affecting GABA neurotransmission contribute to the pathophysiology of different clinical features of schizophrenia. PMID:18281411

  10. Neoplasms with schwannian differentiation express transcription factors known to regulate normal schwann cell development.

    PubMed

    Pytel, Peter; Karrison, Theodore; Can Gong; Tonsgard, James H; Krausz, Thomas; Montag, Anthony G

    2010-12-01

    A number of transcription factors have been identified as important in guiding normal Schwann cell development. This study used immunohistochemistry on tissue arrays to assess the expression of some of these transcription factors (Sox5, Sox9, Sox10, AP-2α, Pax7, and FoxD3) on 76 schwannomas, 105 neurofibromas, and 34 malignant peripheral nerve sheath tumors (MPNSTs). Sox9 and Sox10 were found to be widely expressed in all tumor types. FoxD3 reactivity was stronger and more frequently found in schwannomas and MPNSTs than neurofibromas. AP-2α was positive in 31% to 49% of all tumors, but strong reactivity was limited to MPNSTs and schwannomas. Pax7 and Sox5 expression was restricted to subsets of MPNSTs. Statistical analysis showed significant differences between the 3 tumor types in the expression of these markers. No differences were found in the analyzed tumor subgroups, including schwannomas of different sites, schwannomas with or without NF2 association, neurofibromas of different types, or sporadic versus NF1-associated MPNSTs. These results suggest that the transcription factors that guide normal Schwann cell development also play a role in the biology of neoplastic cells with Schwannian differentiation. FoxD3, AP-2α, Pax7, and Sox5 are upregulated in MPNSTs compared with neurofibromas and may be markers of malignant transformation. Screening the expression of FoxD3, Sox9, and Sox10 on 23 cases of other spindle-cell proliferations that may be considered in the differential diagnosis of MPNST, including synovial sarcoma and spindle cell melanoma, suggests that these 3 are helpful markers of Schwannian differentiation in the context of diagnosing MPNSTs.

  11. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat.

    PubMed

    Goswami, Suneha; Kumar, Ranjeet R; Dubey, Kavita; Singh, Jyoti P; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C; Kala, Yugal K; Singh, Gyanendra P; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D

    2016-01-01

    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat-a novel step toward the development of "climate-smart" wheat.

  12. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat

    PubMed Central

    Goswami, Suneha; Kumar, Ranjeet R.; Dubey, Kavita; Singh, Jyoti P.; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C.; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C.; Kala, Yugal K.; Singh, Gyanendra P.; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D.

    2016-01-01

    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat—a novel step toward the development of

  13. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat.

    PubMed

    Goswami, Suneha; Kumar, Ranjeet R; Dubey, Kavita; Singh, Jyoti P; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C; Kala, Yugal K; Singh, Gyanendra P; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D

    2016-01-01

    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat-a novel step toward the development of "climate-smart" wheat

  14. Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts.

    PubMed

    Oppert, Brenda; Martynov, Alexander G; Elpidina, Elena N

    2012-09-01

    The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa protoxin diet for 24 h (RNA-Seq), or in larvae exposed to the protoxin for 6, 12, or 24 h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases.

  15. Posttranscriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA, and genomic RNA

    PubMed Central

    LeBlanc, Jason; Weil, Jason; Beemon, Karen

    2013-01-01

    After reverse transcription of the retroviral RNA genome and integration of the DNA provirus into the host genome, host machinery is used for viral gene expression along with viral proteins and RNA regulatory elements. Here, we discuss co-transcriptional and posttranscriptional regulation of retroviral gene expression, comparing simple and complex retroviruses. Cellular RNA polymerase II synthesizes full-length viral primary RNA transcripts that are capped and polyadenylated. All retroviruses generate a singly spliced env mRNA from this primary transcript, which encodes the viral glycoproteins. In addition, complex viral RNAs are alternatively spliced to generate accessory proteins, such as Rev, which is involved in posttranscriptional regulation of HIV-1 RNA. Importantly, the splicing of all retroviruses is incomplete; they must maintain and export a fraction of their primary RNA transcripts. This unspliced RNA functions both as the major mRNA for Gag and Pol proteins and as the packaged genomic RNA. Different retroviruses export their unspliced viral RNA from the nucleus to the cytoplasm by either Tap-dependent or Rev/CRM1-dependent routes. Translation of the unspliced mRNA involves frame-shifting or termination codon suppression so that the Gag proteins, which make up the capsid, are expressed more abundantly than the Pol proteins, which are the viral enzymes. After the viral polyproteins assemble into viral particles and bud from the cell membrane, a viral encoded protease cleaves them. Some retroviruses have evolved mechanisms to protect their unspliced RNA from decay by nonsense-mediated RNA decay and to prevent genome editing by the cellular APOBEC deaminases. PMID:23754689

  16. Poly(C)-binding proteins as transcriptional regulators of gene expression

    SciTech Connect

    Choi, Hack Sun Hwang, Cheol Kyu; Song, Kyu Young; Law, P.-Y.; Wei, L.-N.; Loh, Horace H.

    2009-03-13

    Poly(C)-binding proteins (PCBPs) are generally known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). They can be divided into two groups: hnRNP K and PCBP1-4. These proteins are involved mainly in various posttranscriptional regulations (e.g., mRNA stabilization or translational activation/silencing). In this review, we summarize and discuss how PCBPs act as transcriptional regulators by binding to specific elements in gene promoters that interact with the RNA polymerase II transcription machinery. Transcriptional regulation of PCBPs might itself be regulated by their localization within the cell. For example, activation by p21-activated kinase 1 induces increased nuclear retention of PCBP1, as well as increased promoter activity. PCBPs can function as a signal-dependent and coordinated regulator of transcription in eukaryotic cells. We address the molecular mechanisms by which PCBPs binding to single- and double-stranded DNA mediates gene expression.

  17. PU.1 Suppresses Th2 Cytokine Expression via Silencing of GATA3 Transcription in Dendritic Cells.

    PubMed

    Yashiro, Takuya; Kubo, Masato; Ogawa, Hideoki; Okumura, Ko; Nishiyama, Chiharu

    2015-01-01

    The transcription factor PU.1 is predominantly expressed in dendritic cells (DCs) and is essential for DC differentiation. Although there are several reports that PU.1 positively regulates the expression of DC-specific genes, whether PU.1 also has a suppressive effect on DCs is largely unknown. Here we demonstrate that PU.1 suppresses the expression of Th2 cytokines including IL-13 and IL-5 in bone marrow-derived DCs (BMDCs), through repression of the expression of GATA3, which is a master regulator of Th2 differentiations. When PU.1 siRNA was introduced into BMDCs, LPS-induced expression of IL-13 and IL-5 was increased along with upregulation of the constitutive expression of GATA2 and GATA3. The additional introduction of GATA3 siRNA but not of GATA2 siRNA abrogated PU.1 siRNA-mediated upregulation of IL-13 and IL-5. A chromatin immunoprecipitation assay showed that PU.1 bound to Gata3 proximal promoter region, which is more dominant than the distal promoter in driving GATA3 transcription in DCs. The degree of histone acetylation at the Gata3 promoter was decreased in PU.1 siRNA-introduced DCs, suggesting the involvement of PU.1 in chromatin modification of the Gata3 promoter. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, increased the degree of histone H3 acetylation at the Gata3 promoter and induced the subsequent expression of GATA3. Experiments using HDAC inhibitors and siRNAs showed that HDAC3 suppressed GATA3 expression. The recruitment of HDAC3 to the Gata3 promoter was decreased by PU.1 knockdown. LPS-induced IL-13 expression was dramatically reduced in BMDCs generated from mice lacking the conserved GATA3 response element, termed CGRE, which is an essential site for the binding of GATA3 on the Il-13 promoter. The degree of H3K4me3 at CGRE was significantly increased in PU.1 siRNA-transfected stimulated DCs. Our results indicate that PU.1 plays pivotal roles in DC development and function, serving not only as a transcriptional

  18. Homeobox transcription factor Six7 governs expression of green opsin genes in zebrafish.

    PubMed

    Ogawa, Yohey; Shiraki, Tomoya; Kojima, Daisuke; Fukada, Yoshitaka

    2015-08-01

    Colour discrimination in vertebrates requires cone photoreceptor cells in the retina, and high-acuity colour vision is endowed by a set of four cone subtypes expressing UV-, blue-, green- and red-sensitive opsins. Previous studies identified transcription factors governing cone photoreceptor development in mice, although loss of blue and green opsin genes in the evolution of mammals make it difficult to understand how high-acuity colour vision was organized during evolution and development. Zebrafish (Danio rerio) represents a valuable vertebrate model for studying colour vision as it retains all the four ancestral vertebrate cone subtypes. Here, by RT-qPCR and in situ hybridization analysis, we found that sine oculis homeobox homolog 7 (six7), a transcription factor widely conserved in ray-finned fish, is expressed predominantly in the cone photoreceptors in zebrafish at both the larval and the adult stages. TAL effector nuclease-based six7 knock-out revealed its roles in expression of green, red and blue cone opsin genes. Most prominently, the six7 deficiency caused a loss of expression of all the green opsins at both the larval and adult stages. six7 is indispensable for the development and/or maintenance of the green cones. PMID:26180064

  19. Comprehensive analysis suggests overlapping expression of rice ONAC transcription factors in abiotic and biotic stress responses.

    PubMed

    Sun, Lijun; Huang, Lei; Hong, Yongbo; Zhang, Huijuan; Song, Fengming; Li, Dayong

    2015-01-01

    NAC (NAM/ATAF/CUC) transcription factors comprise a large plant-specific gene family that contains more than 149 members in rice. Extensive studies have revealed that NAC transcription factors not only play important roles in plant growth and development, but also have functions in regulation of responses to biotic and abiotic stresses. However, biological functions for most of the members in the NAC family remain unknown. In this study, microarray data analyses revealed that a total of 63 ONAC genes exhibited overlapping expression patterns in rice under various abiotic (salt, drought, and cold) and biotic (infection by fungal, bacterial, viral pathogens, and parasitic plants) stresses. Thirty-eight ONAC genes exhibited overlapping expression in response to any two abiotic stresses, among which 16 of 30 selected ONAC genes were upregulated in response to exogenous ABA. Sixty-five ONAC genes showed overlapping expression patterns in response to any two biotic stresses. Results from the present study suggested that members of the ONAC genes with overlapping expression pattern may have pleiotropic biological functions in regulation of defense response against different abiotic and biotic stresses, which provide clues for further functional analysis of the ONAC genes in stress tolerance and pathogen resistance. PMID:25690040

  20. Characterization and expression analysis of WOX2 homeodomain transcription factor in Aegilops tauschii.

    PubMed

    Zhao, Shan; Jiang, Qian-Tao; Ma, Jian; Wang, Ji-Rui; Liu, Ya-Xi; Chen, Guo-Yue; Qi, Peng-Fei; Pu, Zhi-En; Lu, Zhen-Xiang; Zheng, You-Liang; Wei, Yu-Ming

    2015-03-01

    The WUSCHEL (WUS)-related homeobox (WOX) gene family coordinates transcription during the early phases of embryogenesis. In this study, a putative WOX2 homolog was isolated and characterized from Aegilops tauschii, the donor of D genome of Triticum aestivum. The sequence consisted of 2045 bp, and contained an open reading frame (ORF), encoded 322 amino acids. The predicted protein sequence contained a highly conserved homeodomain and the WUS-box domain, which is present in some members of the WOX protein family. The full-length ORF was subcloned into prokaryotic expression vector pET-30a, and an approximately 34-kDa protein was expressed in Escherichia coli BL21 (DE3) cells with IPTG induction. The molecular mass of the expressed protein was identical to that predicted by the cDNA sequence. Phylogenetic analysis suggested that Ae. tauschii WOX2 is closely related to the rice and maize orthologs. Quantitative PCR analysis showed that WOX2 from Ae. tauschii was primarily expressed in the seeds; transcription increased during seed development and declined after the embryos matured, suggesting that WOX2 is associated with embryo development in Ae. tauschii.

  1. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus.

    PubMed

    Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus; Nielsen, Henrik Bjørn; Botanga, Christopher J; Thorgrimsen, Stephan; Palma, Kristoffer; Suarez-Rodriguez, Maria Cristina; Sandbech-Clausen, Signe; Lichota, Jacek; Brodersen, Peter; Grasser, Klaus D; Mattsson, Ole; Glazebrook, Jane; Mundy, John; Petersen, Morten

    2008-08-20

    Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

  2. PPAR{gamma} transcriptionally regulates the expression of insulin-degrading enzyme in primary neurons

    SciTech Connect

    Du, Jing; Zhang, Lang; Liu, Shubo; Zhang, Chi; Huang, Xiuqing; Li, Jian; Zhao, Nanming; Wang, Zhao

    2009-06-12

    Insulin-degrading enzyme (IDE) is a protease that has been demonstrated to play a key role in degrading both A{beta} and insulin and deficient in IDE function is associated with Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2) pathology. However, little is known about the cellular and molecular regulation of IDE expression. Here we show IDE levels are markedly decreased in DM2 patients and positively correlated with the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) levels. Further studies show that PPAR{gamma} plays an important role in regulating IDE expression in rat primary neurons through binding to a functional peroxisome proliferator-response element (PPRE) in IDE promoter and promoting IDE gene transcription. Finally, we demonstrate that PPAR{gamma} participates in the insulin-induced IDE expression in neurons. These results suggest that PPAR{gamma} transcriptionally induces IDE expression which provides a novel mechanism for the use of PPAR{gamma} agonists in both DM2 and AD therapies.

  3. Differential expression of forkhead box transcription factors following butylated hydroxytoluene lung injury.

    PubMed

    Kalinichenko, V V; Lim, L; Shin, B; Costa, R H

    2001-04-01

    The forkhead box (Fox) proteins are a growing family of transcription factors that have important roles in cellular proliferation and differentiation and in organ morphogenesis. The Fox family members hepatocyte nuclear factor (HNF)-3beta (Foxa2) and HNF-3/forkhead homolog (HFH)-8 (FREAC-1, Foxf1) are expressed in adult pulmonary epithelial and mesenchymal cells, respectively, but these cells display only low expression levels of the proliferation-specific HFH-11B gene (Trident, Foxm1b). The regulation of these Fox transcription factors in response to acute lung injury, however, has yet to be determined. We report here on the use of butylated hydroxytoluene (BHT)-mediated lung injury to demonstrate that HFH-11 protein and RNA levels were markedly increased throughout the period of lung repair. The maximum levels of HFH-11 were observed by day 2 following BHT injury when both bronchiolar and alveolar epithelial cells were undergoing extensive proliferation. Although BHT lung injury did not alter epithelial cell expression of HNF-3beta, a 65% reduction in HFH-8 mRNA levels was observed during the period of mesenchymal cell proliferation. HFH-8-expressing cells were colocalized with platelet endothelial cell adhesion molecule-1-positive alveolar endothelial cells and with alpha-smooth muscle actin-positive peribronchiolar smooth muscle cells.

  4. Post-transcriptional regulation of gene expression in neural stem cells.

    PubMed

    Kim, Do-Yeon

    2016-06-01

    Expression of each gene can be controlled at several steps during the flow of genetic information from DNA to protein. Tight regulation of gene expression is especially important for stem cells because of their greater ripple effects, compared with terminally differentiated cells. Dysregulation of gene expression arising in stem cells can be perpetuated within the stem cell pool via self-renewal throughout life. In addition, transcript profiles within stem cells can determine the selective advantage or disadvantage of each cell, leading to changes in cell fate, such as a tendency for proliferation, death, and differentiation. The identification of neural stem/progenitor cells (NSPCs) and greater understanding of their cellular physiology have raised the possibility of using NSPCs to replace damaged or injured neurons. However, an accurate grasp of gene expression control must take precedence in order to use NSPCs in therapies for neurological diseases. Recently, accumulating evidence has demonstrated the importance of post-transcriptional regulation in NSPC fate decisions. In this review, we will summarize and discuss the recent findings on key mRNA modulators and their vital roles in NSPC homeostasis. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Stress-impaired transcription factor expression and insulin secretion in transplanted human islets

    PubMed Central

    Dai, Chunhua; Kayton, Nora S.; Shostak, Alena; Poffenberger, Greg; Cyphert, Holly A.; Aramandla, Radhika; Thompson, Courtney; Papagiannis, Ioannis G.; Shiota, Masakazu; Stafford, John M.; Greiner, Dale L.; Herrera, Pedro L.; Shultz, Leonard D.; Stein, Roland; Powers, Alvin C.

    2016-01-01

    Type 2 diabetes is characterized by insulin resistance, hyperglycemia, and progressive β cell dysfunction. Excess glucose and lipid impair β cell function in islet cell lines, cultured rodent and human islets, and in vivo rodent models. Here, we examined the mechanistic consequences of glucotoxic and lipotoxic conditions on human islets in vivo and developed and/or used 3 complementary models that allowed comparison of the effects of hyperglycemic and/or insulin-resistant metabolic stress conditions on human and mouse islets, which responded quite differently to these challenges. Hyperglycemia and/or insulin resistance impaired insulin secretion only from human islets in vivo. In human grafts, chronic insulin resistance decreased antioxidant enzyme expression and increased superoxide and amyloid formation. In human islet grafts, expression of transcription factors NKX6.1 and MAFB was decreased by chronic insulin resistance, but only MAFB decreased under chronic hyperglycemia. Knockdown of NKX6.1 or MAFB expression in a human β cell line recapitulated the insulin secretion defect seen in vivo. Contrary to rodent islet studies, neither insulin resistance nor hyperglycemia led to human β cell proliferation or apoptosis. These results demonstrate profound differences in how excess glucose or lipid influence mouse and human insulin secretion and β cell activity and show that reduced expression of key islet-enriched transcription factors is an important mediator of glucotoxicity and lipotoxicity. PMID:27064285

  6. Transcription factor GATA-6 activates expression of gastroprotective trefoil genes TFF1 and TFF2.

    PubMed

    Al-azzeh, E D; Fegert, P; Blin, N; Gött, P

    2000-02-29

    One of the early events in inflammation and epithelial restitution of the gastrointestinal tract is the up-regulation of secretory peptides belonging to the trefoil factor family (TFF) that promote cell migration, protect and heal the mucosa. Their major expression site is stomach (TFF1, TFF2) and intestine (TFF3). Located in the 5'-flanking region of the genes are several consensus sites for members of the GATA transcription factors known to control gut-specific gene expression. By reverse transcription-PCR (RT-PCR), GATA-6 was shown to be expressed in a variety of tumor cell lines of gastric, intestinal and pancreatic origin. In MKN45, KATOIII and LS174T, cotransfection with TFF reporter genes and GATA-6 expression vectors revealed that GATA-6 activates TFF1 and TFF2 4-6-fold, without an effect on TFF3. The functional contribution of GATA binding sequences in the reverse orientation was further characterized by reporter gene assays using TFF2 deletion constructs and by gel shift experiments. PMID:10684977

  7. Homeobox transcription factor Six7 governs expression of green opsin genes in zebrafish.

    PubMed

    Ogawa, Yohey; Shiraki, Tomoya; Kojima, Daisuke; Fukada, Yoshitaka

    2015-08-01

    Colour discrimination in vertebrates requires cone photoreceptor cells in the retina, and high-acuity colour vision is endowed by a set of four cone subtypes expressing UV-, blue-, green- and red-sensitive opsins. Previous studies identified transcription factors governing cone photoreceptor development in mice, although loss of blue and green opsin genes in the evolution of mammals make it difficult to understand how high-acuity colour vision was organized during evolution and development. Zebrafish (Danio rerio) represents a valuable vertebrate model for studying colour vision as it retains all the four ancestral vertebrate cone subtypes. Here, by RT-qPCR and in situ hybridization analysis, we found that sine oculis homeobox homolog 7 (six7), a transcription factor widely conserved in ray-finned fish, is expressed predominantly in the cone photoreceptors in zebrafish at both the larval and the adult stages. TAL effector nuclease-based six7 knock-out revealed its roles in expression of green, red and blue cone opsin genes. Most prominently, the six7 deficiency caused a loss of expression of all the green opsins at both the larval and adult stages. six7 is indispensable for the development and/or maintenance of the green cones.

  8. Homeobox transcription factor Six7 governs expression of green opsin genes in zebrafish

    PubMed Central

    Ogawa, Yohey; Shiraki, Tomoya; Kojima, Daisuke; Fukada, Yoshitaka

    2015-01-01

    Colour discrimination in vertebrates requires cone photoreceptor cells in the retina, and high-acuity colour vision is endowed by a set of four cone subtypes expressing UV-, blue-, green- and red-sensitive opsins. Previous studies identified transcription factors governing cone photoreceptor development in mice, although loss of blue and green opsin genes in the evolution of mammals make it difficult to understand how high-acuity colour vision was organized during evolution and development. Zebrafish (Danio rerio) represents a valuable vertebrate model for studying colour vision as it retains all the four ancestral vertebrate cone subtypes. Here, by RT-qPCR and in situ hybridization analysis, we found that sine oculis homeobox homolog 7 (six7), a transcription factor widely conserved in ray-finned fish, is expressed predominantly in the cone photoreceptors in zebrafish at both the larval and the adult stages. TAL effector nuclease-based six7 knock-out revealed its roles in expression of green, red and blue cone opsin genes. Most prominently, the six7 deficiency caused a loss of expression of all the green opsins at both the larval and adult stages. six7 is indispensable for the development and/or maintenance of the green cones. PMID:26180064

  9. CITA/NLRC5: A critical transcriptional regulator of MHC class I gene expression.

    PubMed

    Downs, Isaac; Vijayan, Saptha; Sidiq, Tabasum; Kobayashi, Koichi S

    2016-07-01

    Major histocompatibility complex (MHC) class I and class II molecules play essential roles in the development and activation of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator) has been recognized as a master regulator of MHC class II gene expression, albeit knowledge about the regulatory mechanism of MHC class I gene expression had been limited. Recently identified MHC class I transactivator (CITA), or NLRC5, also belongs to the NLR protein family and constitutes a critical regulator for the transcriptional activation of MHC class I genes. In addition to MHC class I genes, CITA/NLRC5 induces the expression of β2 -microglobulin, TAP1 and LMP2, essential components of the MHC class I antigen presentation pathway. Therefore, CITA/NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and class II pathways, respectively. © 2016 BioFactors, 42(4):349-357, 2016. PMID:27087581

  10. Chicken ovalbumin upstream promoter transcription factor II regulates renin gene expression.

    PubMed

    Mayer, Sandra; Roeser, Marc; Lachmann, Peter; Ishii, Sumiyashi; Suh, Jae Mi; Harlander, Sabine; Desch, Michael; Brunssen, Coy; Morawietz, Henning; Tsai, Sophia Y; Tsai, Ming-Jer; Hohenstein, Bernd; Hugo, Christian; Todorov, Vladimir T

    2012-07-13

    This study aimed to investigate the possible involvement of the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in the regulation of renin gene expression. COUP-TFII colocalized with renin in the juxtaglomerular cells of the kidney, which are the main source of renin in vivo. Protein-DNA binding studies demonstrated that COUP-TFII binds to an imperfect direct repeat COUP-TFII recognition sequence (termed hereafter proxDR) in the proximal renin promoter. Because cAMP signaling plays a central role in the control of the renin gene expression, we suggested that COUP-TFII may modulate this cAMP effect. Accordingly, knockdown of COUP-TFII in the clonal renin-producing cell lines As4.1 and Calu-6 diminished the stimulation of the renin mRNA expression by cAMP agonists. In addition, the mutation of the proxDR element in renin promoter reporter gene constructs abrogated the inducibility by cAMP. The proxDR sequence was found to be necessary for the function of a proximal renin promoter cAMP-response element (CRE). Knockdown of COUP-TFII or cAMP-binding protein (CREB), which is the archetypal transcription factor binding to CRE, decreased the basal renin gene expression. However, the deficiency of COUP-TFII did not further diminish the renin expression when CREB was knocked down. In agreement with the cell culture studies, mutant mice deficient in COUP-TFII have lower renin expression than their control strain. Altogether our data show that COUP-TFII is involved in the control of renin gene expression.

  11. Chicken Ovalbumin Upstream Promoter Transcription Factor II Regulates Renin Gene Expression*

    PubMed Central

    Mayer, Sandra; Roeser, Marc; Lachmann, Peter; Ishii, Sumiyashi; Suh, Jae Mi; Harlander, Sabine; Desch, Michael; Brunssen, Coy; Morawietz, Henning; Tsai, Sophia Y.; Tsai, Ming-Jer; Hohenstein, Bernd; Hugo, Christian; Todorov, Vladimir T.

    2012-01-01

    This study aimed to investigate the possible involvement of the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in the regulation of renin gene expression. COUP-TFII colocalized with renin in the juxtaglomerular cells of the kidney, which are the main source of renin in vivo. Protein-DNA binding studies demonstrated that COUP-TFII binds to an imperfect direct repeat COUP-TFII recognition sequence (termed hereafter proxDR) in the proximal renin promoter. Because cAMP signaling plays a central role in the control of the renin gene expression, we suggested that COUP-TFII may modulate this cAMP effect. Accordingly, knockdown of COUP-TFII in the clonal renin-producing cell lines As4.1 and Calu-6 diminished the stimulation of the renin mRNA expression by cAMP agonists. In addition, the mutation of the proxDR element in renin promoter reporter gene constructs abrogated the inducibility by cAMP. The proxDR sequence was found to be necessary for the function of a proximal renin promoter cAMP-response element (CRE). Knockdown of COUP-TFII or cAMP-binding protein (CREB), which is the archetypal transcription factor binding to CRE, decreased the basal renin gene expression. However, the deficiency of COUP-TFII did not further diminish the renin expression when CREB was knocked down. In agreement with the cell culture studies, mutant mice deficient in COUP-TFII have lower renin expression than their control strain. Altogether our data show that COUP-TFII is involved in the control of renin gene expression. PMID:22645148

  12. SR proteins in vertical integration of gene expression from transcription to RNA processing to translation.

    PubMed

    Zhong, Xiang-Yang; Wang, Pingping; Han, Joonhee; Rosenfeld, Michael G; Fu, Xiang-Dong

    2009-07-10

    SR proteins have been studied extensively as a family of RNA-binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and colocalize with genes that are engaged in specific intra- and interchromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings, therefore, highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell-cycle progression in higher eukaryotic cells.

  13. Transcription Factors Expressed in Lateral Organ Boundaries: Identification of Downstream Targets

    SciTech Connect

    Springer, Patricia S

    2010-07-12

    The processes of lateral organ initiation and patterning are central to the generation of mature plant form. Characterization of the molecular mechanisms underlying these processes is essential to our understanding of plant development. Communication between the shoot apical meristem and initiating organ primordia is important both for functioning of the meristem and for proper organ patterning, and very little is known about this process. In particular, the boundary between meristem and leaf is emerging as a critical region that is important for SAM maintenance and regulation of organogenesis. The goal of this project was to characterize three boundary-expressed genes that encode predicted transcription factors. Specifically, we have studied LATERAL ORGAN BOUNDARIES (LOB), LATERAL ORGAN FUSION1 (LOF1), and LATERAL ORGAN FUSION2 (LOF2). LOB encodes the founding member of the LOB-DOMAIN (LBD) plant-specific DNA binding transcription factor family and LOF1 and LOF2 encode paralogous MYB-domain transcription factors. We characterized the genetic relationship between these three genes and other boundary and meristem genes. We also used an ectopic inducible expression system to identify direct targets of LOB.

  14. Differential sensitivities of transcription factor target genes underlie cell type-specific gene expression profiles

    PubMed Central

    Johnson, Kirby D.; Kim, Shin-Il; Bresnick, Emery H.

    2006-01-01

    Changes in transcription factor levels and activities dictate developmental fate. Such a change might affect the full ensemble of target genes for a factor or only uniquely sensitive targets. We investigated the relationship among activity of the hematopoietic transcription factor GATA-1, chromatin occupancy, and target gene sensitivity. Graded activation of GATA-1 in GATA-1-null cells revealed high-, intermediate-, and low-sensitivity targets. GATA-1 activity requirements for occupancy and transcription often correlated. A GATA-1 amino-terminal deletion mutant severely deregulated the low-sensitivity gene Tac-2. Thus, cells expressing different levels of a cell type-specific activator can have qualitatively distinct target gene expression patterns, and factor mutations preferentially deregulate low-sensitivity genes. Unlike other target genes, GATA-1-mediated Tac-2 regulation was bimodal, with activation followed by repression, and the coregulator Friend of GATA-1 (FOG-1) selectively mediated repression. A GATA-1 mutant defective in FOG-1 binding occupied a Tac-2 regulatory region at levels higher than wild-type GATA-1, whereas FOG-1 facilitated chromatin occupancy at a distinct target site. These results indicate that FOG-1 is a determinant of GATA factor target gene sensitivity by either facilitating or opposing chromatin occupancy. PMID:17043224

  15. rRNA Gene Expression of Abundant and Rare Activated-Sludge Microorganisms and Growth Rate Induced Micropollutant Removal.

    PubMed

    Vuono, David C; Regnery, Julia; Li, Dong; Jones, Zackary L; Holloway, Ryan W; Drewes, Jörg E

    2016-06-21

    The role of abundant and rare taxa in modulating the performance of wastewater-treatment systems is a critical component of making better predictions for enhanced functions such as micropollutant biotransformation. In this study, we compared 16S rRNA genes (rDNA) and rRNA gene expression of taxa in an activated-sludge-treatment plant (sequencing batch membrane bioreactor) at two solids retention times (SRTs): 20 and 5 days. These two SRTs were used to influence the rates of micropollutant biotransformation and nutrient removal. Our results show that rare taxa (<1%) have disproportionally high ratios of rRNA to rDNA, an indication of higher protein synthesis, compared to abundant taxa (≥1%) and suggests that rare taxa likely play an unrecognized role in bioreactor performance. There were also significant differences in community-wide rRNA expression signatures at 20-day SRT: anaerobic-oxic-anoxic periods were the primary driver of rRNA similarity. These results indicate differential expression of rRNA at high SRTs, which may further explain why high SRTs promote higher rates of micropollutant biotransformation. An analysis of micropollutant-associated degradation genes via metagenomics and direct measurements of a suite of micropollutants and nutrients further corroborates the loss of enhanced functions at 5-day SRT operation. This work advances our knowledge of the underlying ecosystem properties and dynamics of abundant and rare organisms associated with enhanced functions in engineered systems. PMID:27196630

  16. Understanding Transcription Factors in Sugar Beets: Genetic and Physical Mapping, Differential Expression, and Conservation Between Related Germplasm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcription factors control all biological processes at the cellular level, but their role in sugar beets is still widely unknown. In order to develop a greater understanding, 47 primer pairs were designed around expressed tag sequences (ESTs) whose putative functions are various transcription fac...

  17. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis

    PubMed Central

    Donaldson, Michael E; Saville, Barry J

    2013-01-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense–antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  18. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    PubMed

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis.

  19. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    PubMed

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  20. Human androgen receptor expressed in HeLa cells activates transcription in vitro.

    PubMed Central

    De Vos, P; Schmitt, J; Verhoeven, G; Stunnenberg, H G

    1994-01-01

    The androgen receptor (AR) is a ligand-responsive transcription factor, belonging to the class of steroid receptors. AR mutations have been associated with various X-linked diseases, characterized by complete or partial resistance to androgens. To further analyse the molecular mechanism of action of the AR, we have produced the human AR in HeLa cells with a Vaccinia virus expression system. Binding studies on infected HeLa cells demonstrate that the recombinant AR interacts specifically and with high affinity with natural and synthetic androgens. In a gel retardation assay the partially purified AR specifically recognizes an androgen response element of the rat prostatic binding protein gene. Moreover, the recombinant AR activates transcription in vitro from a synthetic promoter construct containing glucocorticoid response elements (GRE). Images PMID:8165128

  1. PTRcombiner: mining combinatorial regulation of gene expression from post-transcriptional interaction maps

    PubMed Central

    2014-01-01

    Background The progress in mapping RNA-protein and RNA-RNA interactions at the transcriptome-wide level paves the way to decipher possible combinatorial patterns embedded in post-transcriptional regulation of gene expression. Results Here we propose an innovative computational tool to extract clusters of mRNA trans-acting co-regulators (RNA binding proteins and non-coding RNAs) from pairwise interaction annotations. In addition the tool allows to analyze the binding site similarity of co-regulators belonging to the same cluster, given their positional binding information. The tool has been tested on experimental collections of human and yeast interactions, identifying modules that coordinate functionally related messages. Conclusions This tool is an original attempt to uncover combinatorial patterns using all the post-transcriptional interaction data available so far. PTRcombiner is available at http://disi.unitn.it/~passerini/software/PTRcombiner/. PMID:24758252

  2. A Progenitor Cell Expressing Transcription Factor RORγt Generates All Human Innate Lymphoid Cell Subsets.

    PubMed

    Scoville, Steven D; Mundy-Bosse, Bethany L; Zhang, Michael H; Chen, Li; Zhang, Xiaoli; Keller, Karen A; Hughes, Tiffany; Chen, Luxi; Cheng, Stephanie; Bergin, Stephen M; Mao, Hsiaoyin C; McClory, Susan; Yu, Jianhua; Carson, William E; Caligiuri, Michael A; Freud, Aharon G

    2016-05-17

    The current model of murine innate lymphoid cell (ILC) development holds that mouse ILCs are derived downstream of the common lymphoid progenitor through lineage-restricted progenitors. However, corresponding lineage-restricted progenitors in humans have yet to be discovered. Here we identified a progenitor population in human secondary lymphoid tissues (SLTs) that expressed the transcription factor RORγt and was unique in its ability to generate all known ILC subsets, including natural killer (NK) cells, but not other leukocyte populations. In contrast to murine fate-mapping data, which indicate that only ILC3s express Rorγt, these human progenitor cells as well as human peripheral blood NK cells and all mature ILC populations expressed RORγt. Thus, all human ILCs can be generated through an RORγt(+) developmental pathway from a common progenitor in SLTs. These findings help establish the developmental signals and pathways involved in human ILC development.

  3. Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Longissimus Muscle in Growing Pigs

    PubMed Central

    Men, X. M.; Deng, B.; Tao, X.; Qi, K. K.; Xu, Z. W.

    2016-01-01

    The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM) during postnatal growth of pigs. Five DLY (Duroc×Landrace×Yorkshire) crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA), to quantity myosin heavy-chains (MyHCs) mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (%) of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01), and MyHC I mRNA proportion was positively correlated with creatine kinase (CK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activities (p<0.05). These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics. PMID:26949945

  4. Accurate Gene Expression-Based Biodosimetry Using a Minimal Set of Human Gene Transcripts

    SciTech Connect

    Tucker, James D.; Joiner, Michael C.; Thomas, Robert A.; Grever, William E.; Bakhmutsky, Marina V.; Chinkhota, Chantelle N.; Smolinski, Joseph M.; Divine, George W.; Auner, Gregory W.

    2014-03-15

    Purpose: Rapid and reliable methods for conducting biological dosimetry are a necessity in the event of a large-scale nuclear event. Conventional biodosimetry methods lack the speed, portability, ease of use, and low cost required for triaging numerous victims. Here we address this need by showing that polymerase chain reaction (PCR) on a small number of gene transcripts can provide accurate and rapid dosimetry. The low cost and relative ease of PCR compared with existing dosimetry methods suggest that this approach may be useful in mass-casualty triage situations. Methods and Materials: Human peripheral blood from 60 adult donors was acutely exposed to cobalt-60 gamma rays at doses of 0 (control) to 10 Gy. mRNA expression levels of 121 selected genes were obtained 0.5, 1, and 2 days after exposure by reverse-transcriptase real-time PCR. Optimal dosimetry at each time point was obtained by stepwise regression of dose received against individual gene transcript expression levels. Results: Only 3 to 4 different gene transcripts, ASTN2, CDKN1A, GDF15, and ATM, are needed to explain ≥0.87 of the variance (R{sup 2}). Receiver-operator characteristics, a measure of sensitivity and specificity, of 0.98 for these statistical models were achieved at each time point. Conclusions: The actual and predicted radiation doses agree very closely up to 6 Gy. Dosimetry at 8 and 10 Gy shows some effect of saturation, thereby slightly diminishing the ability to quantify higher exposures. Analyses of these gene transcripts may be advantageous for use in a field-portable device designed to assess exposures in mass casualty situations or in clinical radiation emergencies.

  5. Differentially Expressed Transcripts and Dysregulated Signaling Pathways and Networks in African American Breast Cancer

    PubMed Central

    Stewart, Paul A.; Luks, Jennifer; Roycik, Mark D.; Sang, Qing-Xiang Amy; Zhang, Jinfeng

    2013-01-01

    African Americans (AAs) have higher mortality rate from breast cancer than that of Caucasian Americans (CAs) even when socioeconomic factors are accounted for. To better understand the driving biological factors of this health disparity, we performed a comprehensive differential gene expression analysis, including subtype- and stage-specific analysis, using the breast cancer data in the Cancer Genome Atlas (TCGA). In total, 674 unique genes and other transcripts were found differentially expressed between these two populations. The numbers of differentially expressed genes between AA and CA patients increased in each stage of tumor progression: there were 26 in stage I, 161 in stage II, and 223 in stage III. Resistin, a gene that is linked to obesity, insulin resistance, and breast cancer, was expressed more than four times higher in AA tumors. An uncharacterized, long, non-coding RNA, LOC90784, was down-regulated in AA tumors, and its expression was inversely related to cancer stage and was the lowest in triple negative AA breast tumors. Network analysis showed increased expression of a majority of components in p53 and BRCA1 subnetworks in AA breast tumor samples, and members of the aurora B and polo-like kinase signaling pathways were also highly expressed. Higher gene expression diversity was observed in more advanced stage breast tumors suggesting increased genomic instability during tumor progression. Amplified resistin expression may indicate insulin-resistant type II diabetes and obesity are associated with AA breast cancer. Expression of LOC90784 may have a protective effect on breast cancer patients, and its loss, particularly in triple negative breast cancer, could be having detrimental effects. This work helps elucidate molecular mechanisms of breast cancer health disparity and identifies putative biomarkers and therapeutic targets such as resistin, and the aurora B and polo-like kinase signaling pathways for treating AA breast cancer patients. PMID

  6. In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

    PubMed Central

    2011-01-01

    Background Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1) in vitro (derived from LB cell cultures) and in vivo (derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Results Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of in silico predicted SD1 membrane proteome) contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while β-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria

  7. hebp3, a novel member of the heme-binding protein gene family, is expressed in the medaka meninges with higher abundance in females due to a direct stimulating action of ovarian estrogens.

    PubMed

    Nakasone, Kiyoshi; Nagahama, Yoshitaka; Okubo, Kataaki

    2013-02-01

    The brains of teleost fish exhibit remarkable sexual plasticity throughout their life span. To dissect the molecular basis for the development and reversal of sex differences in the teleost brain, we screened for genes differentially expressed between sexes in the brain of medaka (Oryzias latipes). One of the genes identified in the screen as being preferentially expressed in females was found to be a new member of the heme-binding protein gene family that includes hebp1 and hebp2 and was designated here as hebp3. The medaka hebp3 is expressed in the meninges with higher abundance in females, whereas there is no expression within the brain parenchyma. This female-biased expression of hebp3 is not attributable to the direct action of sex chromosome genes but results from the transient and reversible action of estrogens derived from the ovary. Moreover, estrogens directly activate the transcription of hebp3 via a palindromic estrogen-responsive element in the hebp3 promoter. Taken together, our findings demonstrate that hebp3 is a novel transcriptional target of estrogens, with female-biased expression in the meninges. The definite but reversible sexual dimorphism of the meningeal hebp3 expression may contribute to the development and reversal of sex differences in the teleost brain.

  8. Phosphorylated STAT3 physically interacts with NPM and transcriptionally enhances its expression in cancer.

    PubMed

    Ren, Z; Aerts, J L; Pen, J J; Heirman, C; Breckpot, K; De Grève, J

    2015-03-26

    The signal transducer and activator of transcription 3 (STAT3) can be activated by the tyrosine kinase domain of the chimeric protein nucleophosmin/anaplastic lymphoma kinase (NPM/ALK), and has a pivotal role in mediating NPM/ALK-related malignant cell transformation. Although the role of STAT3 and wild-type NPM in oncogenesis has been extensively investigated, the relationship between both molecules in cancer remains poorly understood. In the present study, we first demonstrate that STAT3 phosphorylation at tyrosine 705 is accompanied by a concomitant increase in the expression level of NPM. Nuclear co-translocation of phosphorylated STAT3 with NPM can be triggered by interferon-alpha (IFN-α) stimulation of Jurkat cells and phosphorylated STAT3 co-localizes with NPM in cancer cells showing constitutive STAT3 activation. We further demonstrate that STAT3 phosphorylation can transcriptionally mediate NPM upregulation in IFN-α-stimulated Jurkat cells and is responsible for maintaining its expression in cancer cells showing constitutive STAT3 activation. Inhibition of STAT3 phosphorylation or knockdown of NPM expression abrogates their simultaneous transnuclear movements. Finally, we found evidence for a physical interaction between NPM and STAT3 in conditions of STAT3 activation. In conclusion, NPM is a downstream effector of the STAT3 signaling, and can facilitate the nuclear entry of phosphorylated STAT3. These observations might open novel opportunities for targeting the STAT3 pathway in cancer.

  9. Identifying Stress Transcription Factors Using Gene Expression and TF-Gene Association Data.

    PubMed

    Wu, Wei-Sheng; Chen, Bor-Sen

    2009-11-24

    Unicellular organisms such as yeasts have evolved to survive environmental stresses by rapidly reorganizing the genomic expression program to meet the challenges of harsh environments. The complex adaptation mechanisms to stress remain to be elucidated. In this study, we developed Stress Transcription Factor Identification Algorithm (STFIA), which integrates gene expression and TF-gene association data to identify the stress transcription factors (TFs) of six kinds of stresses. We identified some general stress TFs that are in response to various stresses, and some specific stress TFs that are in response to one specific stress. The biological significance of our findings is validated by the literature. We found that a small number of TFs may be sufficient to control a wide variety of expression patterns in yeast under different stresses. Two implications can be inferred from this observation. First, the adaptation mechanisms to different stresses may have a bow-tie structure. Second, there may exist extensive regulatory cross-talk among different stress responses. In conclusion, this study proposes a network of the regulators of stress responses and their mechanism of action.

  10. An inducible transcription factor activates expression of human immunodeficiency virus in T cells

    NASA Astrophysics Data System (ADS)

    Nabel, Gary; Baltimore, David

    1987-04-01

    Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines1,2. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III (refs 3-6) and art genes7. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-κB (ref. 8), with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-κB acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).

  11. Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene

    PubMed Central

    Zhou, Jiawei

    2016-01-01

    Klotho (KL), originally discovered as an aging suppressor, is a membrane protein that shares sequence similarity with the β-glucosidase enzymes. Recent reports showed Klotho might play a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcine KL gene. Deletion fragment analysis identified KL-D2 (−418 bp to −3 bp) as the porcine KL core promoter. MARC0022311SNP (A or G) in KL intron 1 was detected in Landrace × DIV pigs using the Porcine SNP60 BeadChip. The pGL-D2-A and pGL-D2-G were constructed with KL-D2 and the intron fragment of different alleles and relative luciferase activity of pGL3-D2-G was significantly higher than that of pGL3-D2-A in the PK cells and ST cells. This was possibly the result of a change in KL binding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immune-precipitation (ChIP). Moreover, OCT-1 regulated endogenous KL expression by RNA interference experiments. Our study indicates SNP MARC0022311 affects porcine KL expression by regulating its promoter activity via OCT-1. PMID:27478698

  12. Transcriptional activity and expression of liver X receptor in the ascidian Halocynthia roretzi.

    PubMed

    Raslan, Ahmed Ahmed; Lee, Jung Hwan; Shin, Jihye; Shin, Yun Kyung; Sohn, Young Chang

    2013-09-01

    Liver X receptors, LXRs, are ligand-activated transcription factors that belong to the group H nuclear receptor (NR) superfamily. In this study, an LXR (HrLXR) cDNA was cloned from the ascidian Halocynthia roretzi hepatopancreas and characterized to examine the functional conservation of ancestral LXRs in chordates. A phylogenetic analysis of HrLXR showed that it belongs to the tunicate (urochordate) LXR subgroup, which is distinct from vertebrate LXRs. Quantitative real-time PCR analysis revealed that HrLXR mRNA was expressed predominantly in the gills, and highly expressed in unfertilized eggs followed by decrease at later embryonic and larval stages. Unexpectedly, HrLXR was not activated by GW3965, whereas a synthetic ligand for a farnesoid X receptor, GW4064, activated HrLXR. This activation was abolished by the deletion of 51 amino acids from the N-terminus. In a mammalian two-hybrid system, HrLXR interacted with HrRXR in the presence of GW4064 or 9-cis retinoic acid. The injection of GW3965 and GW4064 in vivo increased the ATPbinding cassette sub-family G member 4 and HrLXR mRNA levels in the hepatopancreas and gills. These results suggest that the mRNA expression and transcriptional properties of HrLXR are different from those of vertebrate LXRs, although HrLXR is likely responsive to the related NR ligand, GW4064.

  13. Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene.

    PubMed

    Li, Yan; Wang, Lei; Zhou, Jiawei; Li, Fenge

    2016-01-01

    Klotho (KL), originally discovered as an aging suppressor, is a membrane protein that shares sequence similarity with the β-glucosidase enzymes. Recent reports showed Klotho might play a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcine KL gene. Deletion fragment analysis identified KL-D2 (-418 bp to -3 bp) as the porcine KL core promoter. MARC0022311SNP (A or G) in KL intron 1 was detected in Landrace × DIV pigs using the Porcine SNP60 BeadChip. The pGL-D2-A and pGL-D2-G were constructed with KL-D2 and the intron fragment of different alleles and relative luciferase activity of pGL3-D2-G was significantly higher than that of pGL3-D2-A in the PK cells and ST cells. This was possibly the result of a change in KL binding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immune-precipitation (ChIP). Moreover, OCT-1 regulated endogenous KL expression by RNA interference experiments. Our study indicates SNP MARC0022311 affects porcine KL expression by regulating its promoter activity via OCT-1. PMID:27478698

  14. Differentially expressed transcripts in stomach of Penaeus monodon in response to AHPND infection.

    PubMed

    Soonthornchai, Wipasiri; Chaiyapechara, Sage; Klinbunga, Sirawut; Thongda, Wilawan; Tangphatsornruang, Sithichoke; Yoocha, Thippawan; Jarayabhand, Padermsak; Jiravanichpaisal, Pikul

    2016-12-01

    Acute Hepatopancreatic Necrosis Disease (AHPND) is an emerging disease in aquacultured shrimp caused by a pathogenic strain of Vibrio parahaemolyticus. As with several pathogenic bacteria, colonization of the stomach appeared to be the initial step of the infection for AHPND-causing Vibrio. To understand the immune responses in the stomach of black tiger shrimp (Penaeus monodon), differentially expressed transcripts (DETs) in the stomach during V. parahaemolyticus strain 3HP (VP3HP) infection was examined using Ion Torrent sequencing. From the total 42,998 contigs obtained, 1585 contigs representing 1513 unigenes were significantly differentially expressed with 1122 and 391 unigenes up- and down-regulated, respectively. Among the DETs, there were 141 immune-related unigenes in 10 functional categories: antimicrobial peptide, signal transduction pathway, proPO system, oxidative stress, proteinases/proteinase inhibitors, apoptotic tumor-related protein, pathogen recognition immune regulator, blood clotting system, adhesive protein and heat shock protein. Expression profiles of 20 of 22 genes inferred from RNA sequencing were confirmed with the results from qRT-PCR. Additionally, a novel isoform of anti-lipopolysaccharide factor, PmALF7 whose transcript was induced in the stomach after challenge with VP3HP was discovered. This study provided a fundamental information on the molecular response in the shrimp stomach during the AHPND infection that would be beneficial for future research. PMID:27339467

  15. Correlation of mRNA expression and protein abundance affected by multiple sequence features related to translational efficiency in Desulfovibrio vulgaris: A quantitative analysis

    SciTech Connect

    Nie, Lei; Wu, Gang; Zhang, Weiwen

    2006-12-01

    The modest correlation between mRNA expression and protein abundance in large scale datasets is explained in part by experimental challenges, such as technological limitations, and in part by fundamental biological factors in the transcription and translation processes. Among various factors affecting the mRNA-protein correlation, the roles of biological factors related to translation are poorly understood. In this study, using experimental mRNA expression and protein abundance data collected from Desulfovibrio vulgaris by DNA microarray and LC-MS/MS proteomic analysis, we quantitatively examined the effects of several translational-efficiency-related sequence features on mRNA-protein correlation. Three classes of sequence features were investigated according to different translational stages: (1) initiation: Shine-Dalgarno sequences, start codon identity and start codon context; (2) elongation: codon usage and amino acid usage; and (3) termination: stop codon identity and stop codon context. Surprisingly, although it is widely accepted that translation initiation is a rate-limiting step for translation, our results showed that the mRNA-protein correlation was affected the most by the features at elongation stages, codon usage and amino acid composition (7.4-12.6% and 5.3-9.3% of the total variation of mRNA-protein correlation, respectively), followed by stop codon context and the Shine-Dalgarno sequence (2.5-4.2% and 2.3%, respectively). Taken together, all sequence features contributed to 18.4-21.8% of the total variation of mRNA-protein correlation. As the first comprehensive quantitative analysis of the mRNA-protein correlation in bacterial D. vulgaris, our results suggest that the traditional view of the relative importance of various sequence features in prokaryotic protein translation might be questionable.

  16. Alterations in ZENK and glucagon RNA transcript expression during increased ocular growth in chickens

    PubMed Central

    Kozulin, Peter; Megaw, Pam L.; Morgan, Ian G.

    2010-01-01

    Purpose To examine in detail the time-course of changes in Zif268, Egr-1, NGFI-A, and Krox-24 (ZENK) and pre-proglucagon (PPG) RNA transcript levels in the chick retina during periods of increased ocular growth induced by form-deprivation and negative-lens wear. To further elucidate the role of ZENK in the modulation of ocular growth, we investigated the effect of intravitreal injections of the muscarinic antagonist atropine and the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (ADTN), both of which block the development of experimental myopia, on the expression of ZENK in eyes fitted with negative-lenses. Methods Myopia was induced by fitting translucent diffusers or −10D polymethyl methacrylate (PMMA) lenses over one eye of the chicken. At times from 1 h to 10 days after fitting of the diffusers or negative lenses, retinal RNA transcript levels of the selected genes were determined by semi-quantitative real-time reverse transcriptase polymerase chain reaction (RT–PCR). For the pharmacology experiments, −10D lenses were fitted over the left eye of chicks for a period of 1h. Intravitreal injections of atropine (10 μl–25 mM), ADTN (10 μl–10 mM), or a vehicle solution were made immediately before fitting of the lenses. Results ZENK RNA transcript levels were rapidly and persistently down-regulated following the attachment of the optical devices over the eye. With a delay relative to ZENK, PPG transcript levels were also down-regulated. Induced changes in gene expression were similar for both form-deprivation and negative-lens wear. When atropine or ADTN were administered immediately before lens attachment, the rapid down-regulation in ZENK RNA transcript levels normally seen following 1 h of negative-lens wear was not seen, and ZENK transcript levels rose above those values seen in control eyes. However, injection of atropine or ADTN into untreated eyes had no effect on ZENK transcript levels. Conclusions Both form

  17. Expression of the rat protamine 2 gene is suppressed at the level of transcription and translation

    SciTech Connect

    Bunick, D.; Hecht, N.B. ); Balhorn, R.; Stanker, L.H. )

    1990-05-01

    The authors have compared the rat protamine 2 gene sequence (rP2) to that of the mouse protamine 2 (mP2) gene. The sequence encompasses 435 nucleotides of the coding region which includes an intron of 120 nucleotides, 461 nucleotides 5{prime} to the coding sequence and 181 bases 3{prime} to it. In the mouse the protamine 2 gene is abundantly transcribed and translated. The mP2 protein is initially synthesized as a precursor and then proteolytically processed to yield the mature protein. In contrast, in the rat, protamine 2 transcripts are present at 2-5% that found in the mouse and the mature protein has never been detected in spermatozoa. Analyses of total sperm basic nuclear proteins extracted from epididymal sperm using a monoclonal antibody specific for protamine 2 suggest that the rat P2 mRNA is translated in vivo but is not properly processed. These results suggest that the lowered transcription rate and altered processing sites of the rat protamine 2 gene are likely to contribute to the lack of protamine 2 in rat spermatozoa.

  18. Two Novel Heat-Soluble Protein Families Abundantly Expressed in an Anhydrobiotic Tardigrade

    PubMed Central

    Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D.; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

    2012-01-01

    Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals. PMID:22937162

  19. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

    PubMed

    Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

    2012-01-01

    Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

  20. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

    PubMed

    Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

    2012-01-01

    Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals. PMID:22937162

  1. Transcriptional identification and characterization of differentially expressed genes associated with embryogenesis in radish (Raphanus sativus L.).

    PubMed

    Zhai, Lulu; Xu, Liang; Wang, Yan; Zhu, Xianwen; Feng, Haiyang; Li, Chao; Luo, Xiaobo; Everlyne, Muleke M; Liu, Liwang

    2016-01-01

    Embryogenesis is an important component in the life cycle of most plant species. Due to the difficulty in embryo isolation, the global gene expression involved in plant embryogenesis, especially the early events following fertilization are largely unknown in radish. In this study, three cDNA libraries from ovules of radish before and after fertilization were sequenced using the Digital Gene Expression (DGE) tag profiling strategy. A total of 5,777 differentially expressed transcripts were detected based on pairwise comparison in the three libraries (0_DAP, 7_DAP and 15_DAP). Results from Gene Ontology (GO) and pathway enrichment analysis revealed that these differentially expressed genes (DEGs) were implicated in numerous life processes including embryo development and phytohormones biosynthesis. Notably, some genes encoding auxin response factor (ARF ), Leafy cotyledon1 (LEC1) and somatic embryogenesis receptor-like kinase (SERK ) known to be involved in radish embryogenesis were differentially expressed. The expression patterns of 30 genes including LEC1-2, AGL9, LRR, PKL and ARF8-1 were validated by qRT-PCR. Furthermore, the cooperation between miRNA and mRNA may play a pivotal role in the radish embryogenesis process. This is the first report on identification of DEGs profiles related to radish embryogenesis and seed development. These results could facilitate further dissection of the molecular mechanisms underlying embryogenesis and seed development in radish. PMID:26902837

  2. Transcriptional identification and characterization of differentially expressed genes associated with embryogenesis in radish (Raphanus sativus L.)

    PubMed Central

    Zhai, Lulu; Xu, Liang; Wang, Yan; Zhu, Xianwen; Feng, Haiyang; Li, Chao; Luo, Xiaobo; Everlyne, Muleke M.; Liu, Liwang

    2016-01-01

    Embryogenesis is an important component in the life cycle of most plant species. Due to the difficulty in embryo isolation, the global gene expression involved in plant embryogenesis, especially the early events following fertilization are largely unknown in radish. In this study, three cDNA libraries from ovules of radish before and after fertilization were sequenced using the Digital Gene Expression (DGE) tag profiling strategy. A total of 5,777 differentially expressed transcripts were detected based on pairwise comparison in the three libraries (0_DAP, 7_DAP and 15_DAP). Results from Gene Ontology (GO) and pathway enrichment analysis revealed that these differentially expressed genes (DEGs) were implicated in numerous life processes including embryo development and phytohormones biosynthesis. Notably, some genes encoding auxin response factor (ARF ), Leafy cotyledon1 (LEC1) and somatic embryogenesis receptor-like kinase (SERK ) known to be involved in radish embryogenesis were differentially expressed. The expression patterns of 30 genes including LEC1-2, AGL9, LRR, PKL and ARF8-1 were validated by qRT-PCR. Furthermore, the cooperation between miRNA and mRNA may play a pivotal role in the radish embryogenesis process. This is the first report on identification of DEGs profiles related to radish embryogenesis and seed development. These results could facilitate further dissection of the molecular mechanisms underlying embryogenesis and seed development in radish. PMID:26902837

  3. Transcriptional identification and characterization of differentially expressed genes associated with embryogenesis in radish (Raphanus sativus L.).

    PubMed

    Zhai, Lulu; Xu, Liang; Wang, Yan; Zhu, Xianwen; Feng, Haiyang; Li, Chao; Luo, Xiaobo; Everlyne, Muleke M; Liu, Liwang

    2016-02-23

    Embryogenesis is an important component in the life cycle of most plant species. Due to the difficulty in embryo isolation, the global gene expression involved in plant embryogenesis, especially the early events following fertilization are largely unknown in radish. In this study, three cDNA libraries from ovules of radish before and after fertilization were sequenced using the Digital Gene Expression (DGE) tag profiling strategy. A total of 5,777 differentially expressed transcripts were detected based on pairwise comparison in the three libraries (0_DAP, 7_DAP and 15_DAP). Results from Gene Ontology (GO) and pathway enrichment analysis revealed that these differentially expressed genes (DEGs) were implicated in numerous life processes including embryo development and phytohormones biosynthesis. Notably, some genes encoding auxin response factor (ARF ), Leafy cotyledon1 (LEC1) and somatic embryogenesis receptor-like kinase (SERK ) known to be involved in radish embryogenesis were differentially expressed. The expression patterns of 30 genes including LEC1-2, AGL9, LRR, PKL and ARF8-1 were validated by qRT-PCR. Furthermore, the cooperation between miRNA and mRNA may play a pivotal role in the radish embryogenesis process. This is the first report on identification of DEGs profiles related to radish embryogenesis and seed development. These results could facilitate further dissection of the molecular mechanisms underlying embryogenesis and seed development in radish.

  4. Expression of transcription factor grainyhead-like 2 is diminished in cervical cancer

    PubMed Central

    Torres-Reyes, Luis A; Alvarado-Ruiz, Liliana; Piña-Sánchez, Patricia; Martínez-Silva, María G; Ramos-Solano, Moisés; Olimón-Andalón, Vicente; Ortiz-Lazareno, Pablo C; Hernández-Flores, Georgina; Bravo-Cuellar, Alejandro; Aguilar-Lemarroy, Adriana; Jave-Suarez, Luis F

    2014-01-01

    The transcription factor grainyhead-like 2 (GRHL2) is evolutionarily conserved in many different species, and is involved in morphogenesis, epithelial differentiation, and the control of the epithelial-mesenchymal transition. It has also recently been implicated in carcinogenesis, but its role in this remains controversial. Expression of GRHL2 has not previously been reported in cervical cancer, so the present study aimed to characterize GRHL2 expression in cervical cancer-derived cell lines (CCCLs) and cervical tissues with different grades of lesions. Microarray analysis found that the expression of 58 genes was down-regulated in CCCLs compared to HaCaT cells (non-tumorigenic human epithelial cell line). The expression of eight of these genes was validated by quantitative real-time PCR (qPCR), and GRHL2 was found to be the most down-regulated. Western blot assays corroborated that GRHL2 protein levels were strongly down-regulated in CCCLs. Cervical cells from women without cervical lesions were shown to express GRHL2, while immunohistochemistry found that positivity to GRHL2 decreased in cervical cancer tissues. In conclusion, a loss or strong reduction in GRHL2 expression appears to be a characteristic of cervical cancer, suggesting that GRHL2 down-regulation is a necessary step during cervical carcinogenesis. However, further studies are needed to delineate the role of GRHL2 in cervical cancer and during malignant progression. PMID:25550776

  5. The forkhead transcription factor, Foxd1, is necessary for pituitary luteinizing hormone expression in mice.

    PubMed

    Gumbel, Jason H; Patterson, Elizabeth M; Owusu, Sarah A; Kabat, Brock E; Jung, Deborah O; Simmons, Jasmine; Hopkins, Torin; Ellsworth, Buffy S

    2012-01-01

    The pituitary gland regulates numerous physiological functions including growth, reproduction, temperature and metabolic homeostasis, lactation, and response to stress. Pituitary organogenesis is dependent on signaling factors that are produced in and around the developing pituitary. The studies described in this report reveal that the forkhead transcription factor, Foxd1, is not expressed in the developing mouse pituitary gland, but rather in the mesenchyme surrounding the pituitary gland, which is an essential source of signaling factors that regulate pituitary organogenesis. Loss of Foxd1 causes a morphological defect in which the anterior lobe of the pituitary gland protrudes through the cartilage plate that is developing ventral to the pituitary at embryonic days (e)14.5, e16.5, and e18.5. The number of proliferating pituitary cells is increased at e14.5 and e16.5. Loss of Foxd1 also results in significantly decreased levels of Lhb expression at e18.5. This decrease in Lhb expression does not appear to be due to a change in the number of gonadotrope cells in the pituitary gland. Previous studies have shown that loss of the LIM homeodomain factor, Lhx3, which is activated by the FGF signaling pathway, results in loss of LH production. Although there is a difference in Lhb expression in Foxd1 null mice, the expression pattern of LHX3 is not altered in Foxd1 null mice. These studies suggest that Foxd1 is indirectly required for normal Lhb expression and cartilage formation. PMID:23284914

  6. Transcriptional Profiling of mRNA Expression in the Mouse Distal Colon

    PubMed Central

    HOOGERWERF, WILLEMIJNTJE A.; SINHA, MALA; CONESA, ANA; LUXON, BRUCE A.; SHAHINIAN, VAHAKN B.; CORNÉLISSEN, GERMAINE; HALBERG, FRANZ; BOSTWICK, JONATHON; TIMM, JOHN; CASSONE, VINCENT M.

    2009-01-01

    Background & Aims Intestinal epithelial cells and the myenteric plexus of the mouse gastrointestinal tract contain a circadian clock–based intrinsic timekeeping system. Because disruption of the biological clock has been associated with increased susceptibility to colon cancer and gastrointestinal symptoms, we aimed to identify rhythmically expressed genes in the mouse distal colon. Methods Microarray analysis was used to identify genes that were rhythmically expressed over a 24-hour light/dark cycle. The transcripts were then classified according to expression pattern, function, and association with physiologic and pathophysiologic processes of the colon. Results A circadian gene expression pattern was detected in approximately 3.7% of distal colonic genes. A large percentage of these genes were involved in cell signaling, differentiation, and proliferation and cell death. Of all the rhythmically expressed genes in the mouse colon, approximately 7% (64/906) have been associated with colorectal cancer formation (eg, B-cell leukemia/lymphoma-2 [Bcl2]) and 1.8% (18/906) with various colonic functions such as motility and secretion (eg, vasoactive intestinal polypeptide, cystic fibrosis transmembrane conductance regulator). Conclusions A subset of genes in the murine colon follows a rhythmic expression pattern. These findings may have significant implications for colonic physiology and pathophysiology. PMID:18848557

  7. Expression and transcriptional regulation of the GnRH receptor gene in human neuronal cells.

    PubMed

    Yeung, Chung-Man; An, Beum-Soo; Cheng, Chi Keung; Chow, Billy K C; Leung, Peter C K

    2005-11-01

    GnRH, acts via the GnRH receptor (GnRHR), plays a pivotal role in human reproduction by stimulating the synthesis and secretion of gonadotropins from pituitary gonadotropes. Studies have also suggested that it has other extra-pituitary functions. To date, the transcriptional regulation of human GnRHR gene in the brain remains largely unknown. Recently, the human cerebellar medulloblastoma cell line TE-671 is found to express GnRH. We report here for the first time that GnRHR is also expressed in this neuronal cell line. Treatment with GnRHR agonist stimulated the phosphorylation of both ERK1/2 and JNK in the cells. Moreover, transient transfection of various human GnRHR promoter-luciferase constructs into the cells identified an upstream promoter region located between -2197 and -1018. Important cis-acting regulatory elements were found at -1300/-1018 and -2197/- 1900, as deletion of either region caused a dramatic decrease in the promoter activity. An upstream GnRHR promoter element was identified to be important for basal transcription in the human neuronal TE-671 cells, in contrast to the previous finding that a downstream promoter is responsible for the gonadotrope-specific expression. Furthermore, we showed that antide (GnRHR antagonist) significantly stimulated the GnRHR promoter activity and inhibition of protein kinase C (PKC) pathway by staurosporine could also up-regulate the promoter activity in dose- and time-dependent manners. Taken together, these data suggest that activation of the GnRHR by interacting with GnRH may transcriptionally down-regulate itself via the PKC pathway in human neuronal cells.

  8. Green tea extracts reduce adipogenesis by decreasing expression of transcription factors C/EBPα and PPARγ

    PubMed Central

    Yang, Xiuling; Yin, Lei; Li, Tang; Chen, Zhihong

    2014-01-01

    Objectives: This study is to determine if green tea (Camellia sinensis) extracts (GTE) affects adipogenesis and further investigate the related molecular mechanisms. Methods: Patients with metabolic syndrome were recruited in this study. Of them, 70 patients received GTE and 64 received water to serve as the control group. The human serum adiponectin, visfatin, and leptin concentrations were determined by enzyme-linked immunosorbent assay. Adipogenesis of 3T3-L1 preadipocytes was induced with reagents and then the cells were treated with GTE. The lipids were stained with Oil Red O for analysis of adipogenesis of 3T3-L1 preadipocytes. The 3T3-L1 preadipocytes were treated with increasing concentrations (0.2-0.5%, w/v) of GTE for 2 days and the cell viability was determined by MTT assay. Reverse transcription real-time PCR and immunoblotting assays were performed to determine RNA and protein levels of relative molecules. Results: GTE increases the serum concentrations of adiponectin but decreases visfatin levels in patients received GTE. The leptin concentrations in serum were not significantly affected. The GTE reduces the adipogenesis-induced lipid accumulation in 3T3-L1 preadipocytes. GTE decreases the mRNA and protein expression of adipogenic transcription factors C/EBPα and PPARγ in 3T3-L1 cells. Expression levels of the adipocyte-specific genes encoding adipocyte protein 2, lipoprotein lipase, and glucose transporter 4 were also decreased by GTE. Furthermore, it was found that GTE reduces phosphorylation of Akt during adipocyte differentiation. Conclusions: GTE reduces adipogenesis by decreasing expression of transcription factors C/EBPα and PPARγ by reduction of phosphorylation of Akt during adipocyte differentiation. PMID:25663987

  9. Quantitative Protein and mRNA Profiling Shows Selective Post-Transcriptional Control of Protein Expression by Vasopressin in Kidney Cells*

    PubMed Central

    Khositseth, Sookkasem; Pisitkun, Trairak; Slentz, Dane H.; Wang, Guanghui; Hoffert, Jason D.; Knepper, Mark A.; Yu, Ming-Jiun

    2011-01-01

    Previous studies in yeast have supported the view that post-transcriptional regulation of protein abundances may be more important than previously believed. Here we ask the question: “In a physiological regulatory process (the response of mammalian kidney cells to the hormone vasopressin), what fraction of the expressed proteome undergoes a change in abundance and what fraction of the regulated proteins have corresponding changes in mRNA levels?” In humans and other mammals, vasopressin fulfills a vital homeostatic role (viz. regulation of renal water excretion) by regulating the water channel aquaporin-2 in collecting duct cells. To address the question posed, we utilized large-scale quantitative protein mass spectrometry (LC-MS/MS) employing stable isotopic labeling in cultured mpkCCD cells (‘SILAC’) coupled with transcriptomic profiling using oligonucleotide expression arrays (Affymetrix). Preliminary studies analyzing two nominally identical control samples by SILAC LC-MS/MS yielded a relative S.D. of 13% (for ratios), establishing the precision of the SILAC approach in our hands. We quantified nearly 3000 proteins with nontargeted SILAC LC-MS/MS, comparing vasopressin- versus vehicle-treated samples. Of these proteins 786 of them were quantified in each of 3 experiments, allowing statistical analysis and 188 of these showed significant vasopressin-induced changes in abundance, including aquaporin-2 (20-fold increase). Among the proteins with statistically significant abundance changes, a large fraction (at least one-third) was found to lack changes in the corresponding mRNA species (despite sufficient statistical power), indicating that post-transcriptional regulation of protein abundance plays an important role in the vasopressin response. Bioinformatic analysis of the regulated proteins (versus all transcripts) shows enrichment of glutathione S-transferase isoforms as well as proteins involved in organization of the actin cytoskeleton. The latter

  10. Hepatic Transporter Expression in Metabolic Syndrome: Phenotype, Serum Metabolic Hormones, and Transcription Factor Expression.

    PubMed

    Donepudi, Ajay C; Cheng, Qiuqiong; Lu, Zhenqiang James; Cherrington, Nathan J; Slitt, Angela L

    2016-04-01

    Metabolic syndrome is a multifactorial disease associated with obesity, insulin resistance, diabetes, and the alteration of multiple metabolic hormones. Obesity rates have been rising worldwide, which increases our need to understand how this population will respond to drugs and exposure to other chemicals. The purpose of this study was to determine in lean and obese mice the ontogeny of clinical biomarkers such as serum hormone and blood glucose levels as well as the physiologic markers that correlate with nuclear receptor- and transporter-related pathways. Livers from male and female wild-type (WT) (C57BL/6) and ob/ob mice littermates were collected before, during, and after the onset of obesity. Serum hormone and mRNA levels were analyzed. Physiologic changes and gene expression during maturation and progression to obesity were performed and correlation analysis was performed using canonical correlations. Significant ontogenic changes in both WT and ob/ob mice were observed and these ontogenic changes differ in ob/ob mice with the development of obesity. In males and females, the ontogenic pattern of the expression of genes such as Abcc3, 4, Abcg2, Cyp2b10, and 4a14 started to differ from week 3, and became significant at weeks 4 and 8 in ob/ob mice compared with WT mice. In obese males, serum resistin, glucagon, and glucose levels correlated with the expression of most hepatic ATP-binding cassette (Abc) transporters, whereas in obese females, serum glucagon-like peptide 1 levels were correlated with most hepatic uptake transporters and P450 enzymes. Overall, the correlation between physiologic changes and gene expression indicate that metabolism-related hormones may play a role in regulating the genes involved in drug metabolism and transport. PMID:26847773

  11. Contributions of transcription and mRNA decay to gene expression dynamics of fission yeast in response to oxidative stress

    PubMed Central

    Marguerat, Samuel; Lawler, Katherine; Brazma, Alvis; Bähler, Jürg

    2014-01-01

    The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, affords insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putative cis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress. PMID:25007214

  12. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    NASA Astrophysics Data System (ADS)

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-05-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

  13. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    PubMed

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-04-26

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

  14. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

    PubMed Central

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J.; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  15. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    PubMed Central

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-01-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks. PMID:27161996

  16. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks.

    PubMed

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-01-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks. PMID:27161996

  17. Analysis of liver connexin expression using reverse transcription quantitative real-time polymerase chain reaction

    PubMed Central

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin mRNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction and data analysis. PMID:27207283

  18. Genome-Wide Identification and Expression Analysis of the NAC Transcription Factor Family in Cassava.

    PubMed

    Hu, Wei; Wei, Yunxie; Xia, Zhiqiang; Yan, Yan; Hou, Xiaowan; Zou, Meiling; Lu, Cheng; Wang, Wenquan; Peng, Ming

    2015-01-01

    NAC [no apical meristem (NAM), Arabidopsis transcription activation factor [ATAF1/2] and cup-shaped cotyledon (CUC2)] proteins is one of the largest groups of plant specific transcription factors and plays a crucial role in plant growth, development, and adaption to the environment. Currently, no information is known about the NAC family in cassava. In this study, 96 NAC genes (MeNACs) were identified from the cassava genome. Phylogenetic analysis of the NACs from cassava and Arabidopsis showed that MeNAC proteins can be clustered into 16 subgroups. Gene structure analysis found that the number of introns of MeNAC genes varied from 0 to 5, with the majority of MeNAC genes containing two introns, indicating a small gene structure diversity of cassava NAC genes. Conserved motif analysis revealed that all of the identified MeNACs had the conserved NAC domain and/or NAM domain. Global expression analysis suggested that MeNAC genes exhibited different expression profiles in different tissues between wild subspecies and cultivated varieties, indicating their involvement in the functional diversity of different accessions. Transcriptome analysis demonstrated that MeNACs had a widely transcriptional response to drought stress and that they had differential expression profiles in different accessions, implying their contribution to drought stress resistance in cassava. Finally, the expression of twelve MeNAC genes was analyzed under osmotic, salt, cold, ABA, and H2O2 treatments, indicating that cassava NACs may represent convergence points of different signaling pathways. Taken together, this work found some excellent tissue-specific and abiotic stress-responsive candidate MeNAC genes, which would provide a solid foundation for functional investigation of the NAC family, crop improvement and improved understanding of signal transduction in plants. These data bring new insight on the complexity of the transcriptional control of MeNAC genes and support the hypothesis that

  19. Analysis of Liver Connexin Expression Using Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction.

    PubMed

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin RNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction, and data analysis. PMID:27207283

  20. Mining regulatory network connections by ranking transcription factor target genes using time series expression data.

    PubMed

    Honkela, Antti; Rattray, Magnus; Lawrence, Neil D

    2013-01-01

    Reverse engineering the gene regulatory network is challenging because the amount of available data is very limited compared to the complexity of the underlying network. We present a technique addressing this problem through focussing on a more limited problem: inferring direct targets of a transcription factor from short expression time series. The method is based on combining Gaussian process priors and ordinary differential equation models allowing inference on limited potentially unevenly sampled data. The method is implemented as an R/Bioconductor package, and it is demonstrated by ranking candidate targets of the p53 tumour suppressor.

  1. Role of PU.1 in MHC Class II Expression via CIITA Transcription in Plasmacytoid Dendritic Cells.

    PubMed

    Miura, Ryosuke; Kasakura, Kazumi; Nakano, Nobuhiro; Hara, Mutsuko; Maeda, Keiko; Okumura, Ko; Ogawa, Hideoki; Yashiro, Takuya; Nishiyama, Chiharu

    2016-01-01

    The cofactor CIITA is a master regulator of MHC class II expression and several transcription factors regulating the cell type-specific expression of CIITA have been identified. Although the MHC class II expression in plasmacytoid dendritic cells (pDCs) is also mediated by CIITA, the transcription factors involved in the CIITA expression in pDCs are largely unknown. In the present study, we analyzed the role of a hematopoietic lineage-specific transcription factor, PU.1, in CIITA transcription in pDCs. The introduction of PU.1 siRNA into mouse pDCs and a human pDC cell line, CAL-1, reduced the mRNA levels of MHC class II and CIITA. When the binding of PU.1 to the 3rd promoter of CIITA (pIII) in CAL-1 and mouse pDCs was analyzed by a chromatin immunoprecipitation assay, a significant amount of PU.1 binding to the pIII was detected, which was definitely decreased in PU.1 siRNA-transfected cells. Reporter assays showed that PU.1 knockdown reduced the pIII promoter activity and that three Ets-motifs in the human pIII promoter were candidates of cis-enhancing elements. By electrophoretic mobility shift assays, it was confirmed that two Ets-motifs, GGAA (-181/-178) and AGAA (-114/-111), among three candidates, were directly bound with PU.1. When mouse pDCs and CAL-1 cells were stimulated by GM-CSF, mRNA levels of PU.1, pIII-driven CIITA, total CIITA, MHC class II, and the amount of PU.1 binding to pIII were significantly increased. The GM-CSF-mediated up-regulation of these mRNAs was canceled in PU.1 siRNA-introduced cells. Taking these results together, we conclude that PU.1 transactivates the pIII through direct binding to Ets-motifs in the promoter in pDCs.

  2. Differential expression pattern of rubber elongation factor (REF) mRNA transcripts from high and low yielding clones of rubber tree (Hevea brasiliensis Muell. Arg.).

    PubMed

    Priya, P; Venkatachalam, P; Thulaseedharan, A

    2007-10-01

    In Hevea tree, rubber elongation factor (REF) is a key gene involved in rubber biosynthesis. Since the immaturity period for Hevea is 6 years, identification of molecular marker for latex yield potential will be beneficial for early selection of high yielding clones. The main objective of this research is to study the expression pattern of the REF gene in contrasting latex yield rubber clones (high and low yielding) by Northern blot as well as RT-PCR analysis. Accumulation of REF mRNA transcripts was significantly higher in latex cells compared to other cells of seedlings and mature Hevea trees. Northern results revealed that the level of REF gene expression in latex cells of high yielding rubber clones was significantly higher than in low yielders. According to RT-PCR results, the abundance of REF mRNA transcripts in latex cells was fivefold higher in the RRII105 clone, one of the most high yielding rubber clones. It is evident from the results that both tapping and ethephon treatment had a direct effect on induction of REF gene expression. Results demonstrate a positive correlation between REF gene expression pattern and latex yield.

  3. Molecular Characterization and Expression Profiling of NAC Transcription Factors in Brachypodium distachyon L.

    PubMed

    Zhu, Gengrui; Chen, Guanxing; Zhu, Jiantang; Zhu, Yan; Lu, Xiaobing; Li, Xiaohui; Hu, Yingkao; Yan, Yueming

    2015-01-01

    NAC (NAM, ATAF1/2, CUC2) transcription factors are involved in regulating plant developmental processes and response to environmental stresses. Brachypodium distachyon is an emerging model system for cereals, temperate grasses and biofuel crops. In this study, a comprehensive investigation of the molecular characterizations, phylogenetics and expression profiles under various abiotic stresses of the NAC gene family in Brachypodium distachyon was performed. In total, 118 BNAC genes in B. distachyon were identified, of which 22 (18.64%) were tandemly duplicated and segmentally duplicated, respectively. The Bayesian phylogenetic inference using Markov Chain Monte Carlo (MCMC) algorithms showed that they were divided into two clades and fourteen subfamilies, supported by similar motif compositions within one subfamily. Some critical amino acids detected using DIVERGE v3.0 might contribute to functional divergence among subfamilies. The different exon-intron organizations among subfamilies revealed structural differentiation. Promoter sequence predictions showed that the BNAC genes were involved in various developmental processes and diverse stress responses. Three NAC domain-encoding genes (BNAC012, BNAC078 and BNAC108), orthologous of NAC1, were targeted by five miRNA164 (Bdi-miR164a-c, e, f), suggesting that they might function in lateral organ enlargement, floral development and the responses to abiotic stress. Eleven (~9.32%) BNAC proteins containing α-helical transmembrane motifs were identified. 23 representative BNAC genes were analyzed by quantitative real-time PCR, showing different expression patterns under various abiotic stresses, of which 18, 17 and 11 genes were up-regulated significantly under drought, H2O2 and salt stresses, respectively. Only four and two genes were up-regulated under cold and cadmium stresses, respectively. Dynamic transcriptional expression analysis revealed that six genes showed constitutive expression and period

  4. Aberrant expression of the neuronal transcription factor FOXP2 in neoplastic plasma cells.

    PubMed

    Campbell, Andrew J; Lyne, Linden; Brown, Philip J; Launchbury, Rosalind J; Bignone, Paola; Chi, Jianxiang; Roncador, Giovanna; Lawrie, Charles H; Gatter, Kevin C; Kusec, Rajko; Banham, Alison H

    2010-04-01

    FOXP2 mutation causes a severe inherited speech and language defect, while the related transcription factors FOXP1, FOXP3 and FOXP4 are implicated in cancer. FOXP2 mRNA and protein expression were characterised in normal human tissues, haematological cell lines and multiple myeloma (MM) patients' samples. FOXP2 mRNA and protein were absent in mononuclear cells from different anatomical sites, lineages and stages of differentiation. However, FOXP2 mRNA and protein was detected in several lymphoma (8/20) and all MM-derived cell lines (n = 4). FOXP2 mRNA was expressed in bone marrow samples from 96% of MM patients (24/25), 66.7% of patients with the pre-neoplastic plasma cell proliferation monoclonal gammopathy of undetermined significance (MGUS) (6/9), but not in reactive plasma cells. The frequency of FOXP2 protein expression in CD138(+) plasma cells was significantly higher in MGUS (P = 0.0005; mean 46.4%) and MM patients (P < or = 0.0001; mean 57.3%) than in reactive marrows (mean 2.5%). FOXP2 (>10% nuclear positivity) was detectable in 90.2% of MM (55/61) and 90.9% of MGUS (10/11) patients, showing more frequent expression than CD56 and labelling 75% of CD56-negative MM (9/12). FOXP2 represents the first transcription factor whose expression consistently differentiates normal and abnormal plasma cells and FOXP2 target genes are implicated in MM pathogenesis.

  5. Molecular Characterization and Expression Profiling of NAC Transcription Factors in Brachypodium distachyon L.

    PubMed

    Zhu, Gengrui; Chen, Guanxing; Zhu, Jiantang; Zhu, Yan; Lu, Xiaobing; Li, Xiaohui; Hu, Yingkao; Yan, Yueming

    2015-01-01

    NAC (NAM, ATAF1/2, CUC2) transcription factors are involved in regulating plant developmental processes and response to environmental stresses. Brachypodium distachyon is an emerging model system for cereals, temperate grasses and biofuel crops. In this study, a comprehensive investigation of the molecular characterizations, phylogenetics and expression profiles under various abiotic stresses of the NAC gene family in Brachypodium distachyon was performed. In total, 118 BNAC genes in B. distachyon were identified, of which 22 (18.64%) were tandemly duplicated and segmentally duplicated, respectively. The Bayesian phylogenetic inference using Markov Chain Monte Carlo (MCMC) algorithms showed that they were divided into two clades and fourteen subfamilies, supported by similar motif compositions within one subfamily. Some critical amino acids detected using DIVERGE v3.0 might contribute to functional divergence among subfamilies. The different exon-intron organizations among subfamilies revealed structural differentiation. Promoter sequence predictions showed that the BNAC genes were involved in various developmental processes and diverse stress responses. Three NAC domain-encoding genes (BNAC012, BNAC078 and BNAC108), orthologous of NAC1, were targeted by five miRNA164 (Bdi-miR164a-c, e, f), suggesting that they might function in lateral organ enlargement, floral development and the responses to abiotic stress. Eleven (~9.32%) BNAC proteins containing α-helical transmembrane motifs were identified. 23 representative BNAC genes were analyzed by quantitative real-time PCR, showing different expression patterns under various abiotic stresses, of which 18, 17 and 11 genes were up-regulated significantly under drought, H2O2 and salt stresses, respectively. Only four and two genes were up-regulated under cold and cadmium stresses, respectively. Dynamic transcriptional expression analysis revealed that six genes showed constitutive expression and period

  6. Molecular Characterization and Expression Profiling of NAC Transcription Factors in Brachypodium distachyon L

    PubMed Central

    Zhu, Yan; Lu, Xiaobing; Li, Xiaohui; Hu, Yingkao; Yan, Yueming

    2015-01-01

    NAC (NAM, ATAF1/2, CUC2) transcription factors are involved in regulating plant developmental processes and response to environmental stresses. Brachypodium distachyon is an emerging model system for cereals, temperate grasses and biofuel crops. In this study, a comprehensive investigation of the molecular characterizations, phylogenetics and expression profiles under various abiotic stresses of the NAC gene family in Brachypodium distachyon was performed. In total, 118 BNAC genes in B. distachyon were identified, of which 22 (18.64%) were tandemly duplicated and segmentally duplicated, respectively. The Bayesian phylogenetic inference using Markov Chain Monte Carlo (MCMC) algorithms showed that they were divided into two clades and fourteen subfamilies, supported by similar motif compositions within one subfamily. Some critical amino acids detected using DIVERGE v3.0 might contribute to functional divergence among subfamilies. The different exon-intron organizations among subfamilies revealed structural differentiation. Promoter sequence predictions showed that the BNAC genes were involved in various developmental processes and diverse stress responses. Three NAC domain-encoding genes (BNAC012, BNAC078 and BNAC108), orthologous of NAC1, were targeted by five miRNA164 (Bdi-miR164a-c, e, f), suggesting that they might function in lateral organ enlargement, floral development and the responses to abiotic stress. Eleven (~9.32%) BNAC proteins containing α-helical transmembrane motifs were identified. 23 representative BNAC genes were analyzed by quantitative real-time PCR, showing different expression patterns under various abiotic stresses, of which 18, 17 and 11 genes were up-regulated significantly under drought, H2O2 and salt stresses, respectively. Only four and two genes were up-regulated under cold and cadmium stresses, respectively. Dynamic transcriptional expression analysis revealed that six genes showed constitutive expression and period

  7. Runx3-regulated expression of two Ntrk3 transcript variants in dorsal root ganglion neurons.

    PubMed

    Ogihara, Yuuki; Masuda, Tomoyuki; Ozaki, Shigeru; Yoshikawa, Masaaki; Shiga, Takashi

    2016-03-01

    Somatosensation is divided into proprioception and cutaneous sensation. Dorsal root ganglion (DRG) neurons project their fibers toward peripheral targets including muscles and skin, and centrally to the spinal cord. Proprioceptive DRG neurons transmit information from muscle spindles and Golgi tendon organs to the spinal cord. We previously showed that Runt-related transcription factor 3 (Runx3) is expressed in these neurons and their projections to the ventral spinal cord and muscle spindles are lost in Runx3-deficient (Runx3(-/-) ) mouse embryos. Although Runx3 is likely to contribute to the fate decision and projection of proprioceptive DRG neurons, the precise roles for Runx3 in these phenomena are unknown. To identify genes regulated by Runx3 in embryonic DRGs, we performed microarray analyses using cDNAs isolated from wild-type and Runx3(-/-) DRGs of embryonic day (E) 12.5 and selected two transcript variants of the tyrosine kinase receptor C (TrkC) gene. These variants, Ntrk3 variant 1 (Ntrk3-v1) and variant 2 (Ntrk3-v2), encode full-length and truncated receptors of neurotrophin-3, respectively. Using double in situ hybridization, we found that most of Ntrk3-v1 mRNA expression in E14.5 DRGs depended on Runx3 but that more than half of Ntrk3-v2 mRNA one were expressed in a Runx3-independent manner. Furthermore, our data revealed that the rate of Ntrk3-v1 and Ntrk3-v2 colocalization in DRGs changed from E14.5 to E18.5. Together, our data suggest that Runx3 may play a crucial role in the development of DRGs by regulating the expression of Ntrk3 variants and that DRG neurons expressing Ntrk3-v1 but not Ntrk3-v2 may differentiate into proprioceptive ones. PMID:26061886

  8. A Snapshot of the Expression Signature of Androgen Receptor Splicing Variants and Their Distinctive Transcriptional Activities

    PubMed Central

    Hu, Rong; Isaacs, William B.; Luo, Jun

    2012-01-01

    BACKGROUND The diversity and complexity of the human androgen receptor (AR) splicing variants are well appreciated but not fully understood. The goal of this study is to generate a comprehensive expression signature of AR variants in castration-resistant prostate cancer (CRPC), and to address the relative importance of the individual variants in conferring the castration-resistant phenotype. METHODS A modified RNA amplification method, termed selective linear amplification of sense RNA, was developed to amplify all AR transcripts containing AR exon 3 in CRPC specimens, which were profiled using tiling expression microarrays. Coding sequences for the AR variants were cloned into expression vectors and assessed for their transcriptional activities. Quantitative RT-PCR was used to determine their in vivo expression patterns in an expanded set of clinical specimens. RESULTS In addition to expression peaks in AR intron 3, a novel AR exon, termed exon 9, was discovered. Exon 9 was spliced into multiple novel AR variants. Different AR splicing variants were functionally distinctive, with some demonstrating constitutive activity while others were conditionally active. Conditionally active AR-Vs may activate AR signaling depending on the cellular context. Importantly, AR variant functions did not appear to depend on the full-length AR. CONCLUSIONS This study provided the first unbiased snapshot of the AR variant signature consisting of multiple AR variants with distinctive functional properties, directly in CRPC specimens. Study findings suggest that the aggregate function of multiple AR variants may confer a castration-resistant phenotype independent of the full-length AR. PMID:21446008

  9. Developmental Expression and Hypoxic Induction of Hypoxia Inducible Transcription Factors in the Zebrafish.

    PubMed

    Köblitz, Louise; Fiechtner, Birgit; Baus, Katharina; Lussnig, Rebecca; Pelster, Bernd

    2015-01-01

    The hypoxia inducible transcription factor (HIF) has been shown to coordinate the hypoxic response of vertebrates and is expressed in three different isoforms, HIF-1α, HIF-2α and HIF-3α. Knock down of either Hif-1α or Hif-2α in mice results in lethality in embryonic or perinatal stages, suggesting that this transcription factor is not only controlling the hypoxic response, but is also involved in developmental phenomena. In the translucent zebrafish embryo the performance of the cardiovascular system is not essential for early development, therefore this study was designed to analyze the expression of the three Hif-isoforms during zebrafish development and to test the hypoxic inducibility of these transcription factors. To complement the existing zfHif-1α antibody we expressed the whole zfHif-2α protein and used it for immunization and antibody generation. Similarly, fragments of the zfHif-3α protein were used for immunization and generation of a zfHif-3α specific antibody. To demonstrate presence of the Hif-isoforms during development [between 1 day post fertilization (1 dpf) and 9 dpf] affinity-purified antibodies were used. Hif-1α protein was present under normoxic conditions in all developmental stages, but no significant differences between the different developmental stages could be detected. Hif-2α was also present from 1 dpf onwards, but in post hatching stages (between 5 and 9 dpf) the expression level was significantly higher than prior to hatching. Similarly, Hif-3α was expressed from 1 dpf onwards, and the expression level significantly increased until 5 dpf, suggesting that Hif-2α and Hif-3α play a particular role in early development. Hypoxic exposure (oxygen partial pressure = 5 kPa) in turn caused a significant increase in the level of Hif-1α protein even at 1 dpf and in later stages, while neither Hif-2α nor Hif-3α protein level were affected. In these early developmental stages Hif-1α therefore appears to be more important for

  10. Isolation of All CD44 Transcripts in Human Epidermis and Regulation of Their Expression by Various Agents.

    PubMed

    Teye, Kwesi; Numata, Sanae; Ishii, Norito; Krol, Rafal P; Tsuchisaka, Atsunari; Hamada, Takahiro; Koga, Hiroshi; Karashima, Tadashi; Ohata, Chika; Tsuruta, Daisuke; Saya, Hideyuki; Haftek, Marek; Hashimoto, Takashi

    2016-01-01

    CD44, a cell surface proteoglycan, is involved in many biological events. CD44 transcripts undergo complex alternative splicing, resulting in many functionally distinct isoforms. To date, however, the nature of these isoforms in human epidermis has not been adequately determined. In this study, we isolated all CD44 transcripts from normal human epidermis, and studied how their expressions are regulated. By RT-PCR, we found that a number of different CD44 transcripts were expressed in human epidermis, and we obtained all these transcripts from DNA bands in agarose and acrylamide gels by cloning. Detailed sequence analysis revealed 18 CD44 transcripts, 3 of which were novel. Next, we examined effects of 10 different agents on the expression of CD44 transcripts in cultured human keratinocytes, and found that several agents, particularly epidermal growth factor, hydrogen peroxide, phorbol 12-myristate 13-acetate, retinoic acid, calcium and fetal calf serum differently regulated their expressions in various patterns. Furthermore, normal and malignant keratinocytes were found to produce different CD44 transcripts upon serum stimulation and subsequent starvation, suggesting that specific CD44 isoforms are involved in tumorigenesis via different CD44-mediated biological pathways. PMID:27505250