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Sample records for abundantly expressed transcripts

  1. Transcript abundance supercedes editing efficiency as a factor in developmental variation of chloroplast gene expression.

    PubMed Central

    Peeters, Nemo M; Hanson, Maureen R

    2002-01-01

    In maize plastids, transcripts are known to be modified at 27 C-to-U RNA editing sites, affecting the expression-of 15 different genes. The relative contribution of editing efficiency versus transcript abundance in regulation of chloroplast gene expression has previously been analyzed for only a few genes. We undertook a comprehensive analysis of the editing efficiency of each of the 27 maize editing sites in 10 different maize tissues, which contain a range of plastid types including chloroplasts, etioplasts, and amyloplasts. Using a reproducible poisoned primer extension assay, we detected variation between RNA editing extent of different sites in the same transcript in the same tissue, and between the same site in different tissues. The most striking editing deficiency is in an editing site in ndhB that is edited at only 8% and 1% in roots and callus plastids respectively, whereas green leaf chloroplasts edit this site at 100%. Editing efficiencies of some sites are not affected by the developmental stages we examined and are always edited close to 80-100%. The relative amounts of transcripts of each of the 10 genes that exhibited variable editing extents were determined by real-time PCR. Seven genes exhibited over 100 times lower transcript abundance in either roots or tissue-cultured cells relative to green leaf tissue. The quantitative analysis indicates that a particular editing site can be efficiently edited over a large range of transcript abundance, resulting in no general correlation of transcript abundance and editing extent. The independent variation of editing efficiency of different sites within the same transcript fits with a model that postulates individual trans-acting factors specific to each editing site. Because tissues where editing efficiency at certain sites is low invariably also exhibited greatly decreased abundance of the transcripts carrying those sites, decrease in the amounts of particular RNAs rather than a lack of editing is

  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. Steroidogenic acute regulatory protein in white sturgeon (Acipenser transmontanus): cDNA cloning, sites of expression and transcript abundance in corticosteroidogenic tissue after an acute stressor.

    PubMed

    Kusakabe, Makoto; Zuccarelli, Micah D; Nakamura, Ikumi; Young, Graham

    2009-06-01

    The white sturgeon, Acipenser transmontanus, is a primitive bony fish that is recognized as an important emerging species for aquaculture. However, many aspects of its stress and reproductive physiology remain unclear. These processes are controlled by various steroid hormones. In order to investigate the regulation of steroidogenesis associated with acute stress in sturgeon, a cDNA-encoding steroidogenic acute regulatory protein (StAR) was isolated from white sturgeon. The putative amino acid sequence of sturgeon StAR shares high homology (over 60%) with other vertebrates. Phylogenetic analysis grouped sturgeon StAR within Actinopterygii, but it was clearly segregated from teleost StARs. RT-PCR analysis revealed that transcripts were most abundant in yellow corpuscles found throughout the kidney and weaker signals were detected in gonad and kidney. Very weak signals were also detected in brain and spleen by quantitative real-time PCR. In situ hybridization revealed that StAR is expressed in the cells of yellow corpuscles. No significant changes in StAR gene expression were detected in response to an acute handling stress. These results suggest that StAR is highly conserved throughout vertebrates, but the expression of the functional protein during the stress response may be partially regulated post-transcriptionally.

  4. 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.

  5. Soy Glyceollins Regulate Transcript Abundance in the Female Mouse Brain

    PubMed Central

    Bamji, Sanaya F.; Page, Robert B.; Patel, Dharti; Sanders, Alexia; Alvarez, Alejandro R.; Gambrell, Caitlin; Naik, Kuntesh; Raghavan, Ashwin M.; Burow, Matthew E.; Boue, Stephen M.; Klinge, Carolyn M.; Ivanova, Margarita; Corbitt, Cynthia

    2015-01-01

    Glyceollins (Gly), produced by soy plants in response to stress, have anti-estrogenic activity in breast and ovarian cancer cell lines in vitro and in vivo. In addition to known anti-estrogenic effects, Gly exhibits mechanisms of action not involving estrogen receptor (ER) signaling. To date, effects of Gly on gene expression in the brain are unknown. For this study, we implanted 17-β estradiol (E2) or placebo slow-release pellets into ovariectomized CFW mice followed by 11 days of exposure to Gly or vehicle i.p. injections. We then performed a microarray on total RNA extracted from whole brain hemispheres and identified differentially expressed genes (DEGs) by a 2 × 2 factorial ANOVA with an FDR = 0.20. In total, we identified 33 DEGs with a significant E2 main effect, 5 DEGs with a significant Gly main effect, 74 DEGs with significant Gly and E2 main effects (but no significant interaction term), and 167 DEGs with significant interaction terms. Clustering across all DEGs revealed that transcript abundances were similar between the E2+Gly and E2-only treatments. However, gene expression after Gly-only treatment was distinct from both of these treatments and was generally characterized by higher transcript abundance. Collectively, our results suggest that whether Gly acts in the brain through ER-dependent or ER-independent mechanisms depends on the target gene. PMID:25953511

  6. Mapping quantitative trait loci for expression abundance.

    PubMed

    Jia, Zhenyu; Xu, Shizhong

    2007-05-01

    Mendelian loci that control the expression levels of transcripts are called expression quantitative trait loci (eQTL). When mapping eQTL, we often deal with thousands of expression traits simultaneously, which complicates the statistical model and data analysis. Two simple approaches may be taken in eQTL analysis: (1) individual transcript analysis in which a single expression trait is mapped at a time and the entire eQTL mapping involves separate analysis of thousands of traits and (2) individual marker analysis where differentially expressed transcripts are detected on the basis of their association with the segregation pattern of an individual marker and the entire analysis requires scanning markers of the entire genome. Neither approach is optimal because data are not analyzed jointly. We develop a Bayesian clustering method that analyzes all expressed transcripts and markers jointly in a single model. A transcript may be simultaneously associated with multiple markers. Additionally, a marker may simultaneously alter the expression of multiple transcripts. This is a model-based method that combines a Gaussian mixture of expression data with segregation of multiple linked marker loci. Parameter estimation for each variable is obtained via the posterior mean drawn from a Markov chain Monte Carlo sample. The method allows a regular quantitative trait to be included as an expression trait and subject to the same clustering assignment. If an expression trait links to a locus where a quantitative trait also links, the expressed transcript is considered to be associated with the quantitative trait. The method is applied to a microarray experiment with 60 F(2) mice measured for 25 different obesity-related quantitative traits. In the experiment, approximately 40,000 transcripts and 145 codominant markers are investigated for their associations. A program written in SAS/IML is available from the authors on request.

  7. 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

  8. 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.

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

    PubMed Central

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

    2010-01-01

    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 (∼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. PMID:20002133

  10. 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.

  11. The flavonoid pathway in tomato seedlings: transcript abundance and the modeling of metabolite dynamics.

    PubMed

    Groenenboom, Marian; Gomez-Roldan, Victoria; Stigter, Hans; Astola, Laura; van Daelen, Raymond; Beekwilder, Jules; Bovy, Arnaud; Hall, Robert; Molenaar, Jaap

    2013-01-01

    Flavonoids are secondary metabolites present in all terrestrial plants. The flavonoid pathway has been extensively studied, and many of the involved genes and metabolites have been described in the literature. Despite this extensive knowledge, the functioning of the pathway in vivo is still poorly understood. Here, we study the flavonoid pathway using both experiments and mathematical models. We measured flavonoid metabolite dynamics in two tissues, hypocotyls and cotyledons, during tomato seedling development. Interestingly, the same backbone of interactions leads to very different accumulation patterns in the different tissues. Initially, we developed a mathematical model with constant enzyme concentrations that described the metabolic networks separately in both tissues. This model was unable to fit the measured flavonoid dynamics in the hypocotyls, even if we allowed unrealistic parameter values. This suggested us to investigate the effect of transcript abundance on flavonoid accumulation. We found that the expression of candidate flavonoid genes varies considerably with time. Variation in transcript abundance results in enzymatic variation, which could have a large effect on metabolite accumulation. Candidate transcript abundance was included in the mathematical model as representative for enzyme concentration. We fitted the resulting model to the flavonoid dynamics in the cotyledons, and tested it by applying it to the data from hypocotyls. When transcript abundance is included, we are indeed able to explain flavonoid dynamics in both tissues. Importantly, this is possible under the biologically relevant restriction that the enzymatic properties estimated by the model are conserved between the tissues.

  12. System wide analyses have underestimated protein abundances and the importance of transcription in mammals.

    PubMed

    Li, Jingyi Jessica; Bickel, Peter J; Biggin, Mark D

    2014-01-01

    Large scale surveys in mammalian tissue culture cells suggest that the protein expressed at the median abundance is present at 8,000-16,000 molecules per cell and that differences in mRNA expression between genes explain only 10-40% of the differences in protein levels. We find, however, that these surveys have significantly underestimated protein abundances and the relative importance of transcription. Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al. (2011), we find that the median protein detected is expressed at 170,000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than do the uncorrected protein data. In addition, we estimated the impact of further errors in mRNA and protein abundances using direct experimental measurements of these errors. The resulting analysis suggests that mRNA levels explain at least 56% of the differences in protein abundance for the 4,212 genes detected by Schwanhausser et al. (2011), though because one major source of error could not be estimated the true percent contribution should be higher. We also employed a second, independent strategy to determine the contribution of mRNA levels to protein expression. We show that the variance in translation rates directly measured by ribosome profiling is only 12% of that inferred by Schwanhausser et al. (2011), and that the measured and inferred translation rates correlate poorly (R(2) = 0.13). Based on this, our second strategy suggests that mRNA levels explain ∼81% of the variance in protein levels. We also determined the percent contributions of transcription, RNA degradation, translation and protein degradation to the variance in protein abundances using both of our strategies. While the magnitudes of the two estimates vary, they both suggest that transcription plays a more important role than the earlier studies implied and translation a much smaller

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

    PubMed

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

    2016-09-29

    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.

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

    PubMed Central

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

    2015-01-01

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

  15. 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

  16. The use of Molecular Beacons to Directly Measure Bacterial mRNA Abundances and Transcript Degradation

    PubMed Central

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

    2009-01-01

    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. PMID:18992285

  17. Effect of oocyte quality on the relative abundance of specific gene transcripts in bovine mature oocytes and 16-cell embryos

    PubMed Central

    Bilodeau-Goeseels, Sylvie

    2003-01-01

    Although the developmental potential of oocytes is related to oocyte quality, whether the expression of specific genes is altered in oocytes of different quality and in resulting embryos is not known. Semi-quantitative reverse transcription-polymerase chain reaction was used to compare the relative abundance of 2 transcripts for housekeeping proteins (β-actin and ribosomal protein L30) and 3 transcripts for growth factor ligand or receptors (platelet derived growth factor receptor α (PDGFRα), basic fibroblast growth factor (bFGF)), in mature bovine oocytes of high versus low developmental potential. The transcripts for L30, PDGFRα, and bFGF in 16-cell embryos originating from these oocytes were also examined. No significant effect of oocyte quality was detected for any of the transcripts examined from oocytes or 16-cell embryos. In conclusion, a lower developmental potential of oocytes with advanced signs of atresia, was not associated with a lower level of abundance of the transcripts examined. PMID:12760483

  18. Gene Transcript Abundance Profiles Distinguish Kawasaki Disease from Adenovirus Infection

    PubMed Central

    Popper, Stephen J.; Watson, Virginia E.; Shimizu, Chisato; Kanegaye, John T.; Burns, Jane C.; Relman, David A.

    2010-01-01

    Background Acute Kawasaki disease (KD) is difficult to distinguish from other illnesses that involve acute rash or fever, in part because the etiologic agent(s) and pathophysiology remain poorly characterized. As a result, diagnosis and critical therapies may be delayed. Methods We used DNA microarrays to identify possible diagnostic features of KD. We compared gene expression patterns in the blood of 23 children with acute KD and 18 age-matched febrile children with 3 illnesses that resemble KD. Results Genes associated with platelet and neutrophil activation were expressed at higher levels in patients with KD than in patients with acute adenovirus infections or systemic adverse drug reactions, but levels in patients with KD were not higher than those in patients with scarlet fever. Genes associated with B cell activation were also expressed at higher levels in patients with KD than in control subjects. A striking absence of interferon-stimulated gene expression in patients with KD was confirmed in an independent cohort of patients with KD. Using a set of 38 gene transcripts, we successfully predicted the diagnosis for 21 of 23 patients with KD and 7 of 8 patients with adenovirus infection. Conclusions These findings provide insight into the molecular features that distinguish KD from other febrile illnesses and support the feasibility of developing novel diagnostic reagents for KD based on the host response. PMID:19583510

  19. 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

  20. Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism.

    PubMed

    Miller, Rachel; Wu, Guangxi; Deshpande, Rahul R; Vieler, Astrid; Gärtner, Katrin; Li, Xiaobo; Moellering, Eric R; Zäuner, Simone; Cornish, Adam J; Liu, Bensheng; Bullard, Blair; Sears, Barbara B; Kuo, Min-Hao; Hegg, Eric L; Shachar-Hill, Yair; Shiu, Shin-Han; Benning, Christoph

    2010-12-01

    Like many microalgae, Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient deprived. To begin studying the mechanisms underlying this process, nitrogen (N) deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and noninduced conditions was applied as a first approach to studying molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of eight biologically independent libraries, four for each condition, N replete and N deprived, allowing a statistically sound comparison of expression levels under the two tested conditions. As expected, N deprivation activated a subset of control genes involved in gametogenesis while down-regulating protein biosynthesis. Genes for components of photosynthesis were also down-regulated, with the exception of the PSBS gene. N deprivation led to a marked redirection of metabolism: the primary carbon source, acetate, was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogenesis but funneled directly into fatty acid biosynthesis. Additional fatty acids may be produced by membrane remodeling, a process that is suggested by the changes observed in transcript abundance of putative lipase genes. Inferences on metabolism based on transcriptional analysis are indirect, but biochemical experiments supported some of these deductions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in microalgae.

  1. 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.

  2. Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

    PubMed Central

    Gall, BJ; Wilson, A; Schroer, AB; Gross, JD; Stoilov, P; Setola, V; Watkins, CM; Siderovski, DP

    2015-01-01

    G protein signaling modulator 3 (GPSM3) is a regulator of G protein-coupled receptor signaling, with expression restricted to leukocytes and lymphoid organs. Previous genome-wide association studies have highlighted single-nucleotide polymorphisms (SNPs rs204989, rs204991) in a region upstream of the GPSM3 transcription start site as being inversely correlated to the prevalence of rheumatoid arthritis (RA) -- this association is supported by the protection afforded to Gpsm3-deficient mice in models of inflammatory arthritis. Here, we assessed the functional consequences of these polymorphisms. We collected biospecimens from 50 volunteers with RA diagnoses, 50 RA-free volunteers matched to the aforementioned group, and 100 unmatched healthy young volunteers. We genotyped these individuals for GPSM3 (rs204989, rs204991), CCL21 (rs2812378), and HLA gene region (rs6457620) polymorphisms, and found no significant differences in minor allele frequencies between the RA and disease-free cohorts. However, we identified that individuals homozygous for SNPs rs204989 and rs204991 had decreased GPSM3 transcript abundance relative to individuals homozygous for the major allele. In vitro promoter activity studies suggest that SNP rs204989 is the primary cause of this decrease in transcript levels. Knockdown of GPSM3 in THP-1 cells, a human monocytic cell line, was found to disrupt ex vivo migration to the chemokine MCP-1. PMID:26821282

  3. Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance.

    PubMed

    Gall, B J; Wilson, A; Schroer, A B; Gross, J D; Stoilov, P; Setola, V; Watkins, C M; Siderovski, D P

    2016-03-01

    G protein signaling modulator 3 (GPSM3) is a regulator of G protein-coupled receptor signaling, with expression restricted to leukocytes and lymphoid organs. Previous genome-wide association studies have highlighted single-nucleotide polymorphisms (SNPs; rs204989 and rs204991) in a region upstream of the GPSM3 transcription start site as being inversely correlated to the prevalence of rheumatoid arthritis (RA)-this association is supported by the protection afforded to Gpsm3-deficient mice in models of inflammatory arthritis. Here, we assessed the functional consequences of these polymorphisms. We collected biospecimens from 50 volunteers with RA diagnoses, 50 RA-free volunteers matched to the aforementioned group and 100 unmatched healthy young volunteers. We genotyped these individuals for GPSM3 (rs204989, rs204991), CCL21 (rs2812378) and HLA gene region (rs6457620) polymorphisms, and found no significant differences in minor allele frequencies between the RA and disease-free cohorts. However, we identified that individuals homozygous for SNPs rs204989 and rs204991 had decreased GPSM3 transcript abundance relative to individuals homozygous for the major allele. In vitro promoter activity studies suggest that SNP rs204989 is the primary cause of this decrease in transcript levels. Knockdown of GPSM3 in THP-1 cells, a human monocytic cell line, was found to disrupt ex vivo migration to the chemokine MCP-1.

  4. 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.

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

    DOE PAGES

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

    2014-10-23

    Lithium (Li) toxicity in plants is, at a minimum, a function of Li+ concentration, exposure time, species and growth conditions. Most plant studies with Li+ focus on short-term acute exposures. This study examines short- and long-term effects of Li+ exposure in Arabidopsis with Li+ uptake studies and measured shoot mRNA transcript abundance levels in treated and control plants. Stress, pathogen-response and arabinogalactan protein genes were typically more up-regulated in older (chronic, low level) Li+-treatment plants and in the much younger plants from acute high-level exposures. The gene regulation behavior of high-level Li+ resembled 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

  6. 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

  7. 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

  8. Human lung expresses unique gamma-glutamyl transpeptidase transcripts.

    PubMed Central

    Wetmore, L A; Gerard, C; Drazen, J M

    1993-01-01

    gamma-Glutamyl transpeptidase (EC 2.3.2.2, gamma GT) is a membrane-bound ectoenzyme that plays an important role in the metabolism of glutathione. It is composed of two subunits, both of which are encoded by a common mRNA. We examined the expression of gamma GT in human lung tissue by Northern blot analysis and screening a cDNA library made from human lung poly(A)+ RNA. Our results show that there are two gamma GT mRNA populations in human lung tissue. We define these as group I (2.4 kb) and group II (approximately 1.2 kb) transcripts. In the present communication, we characterize the unique lung transcript. Sequence analysis of representative clones shows that group I transcripts are virtually identical to those previously isolated from liver and placenta but possess a unique 5' untranslated region. In marked contrast, group II transcripts appear to be human-lung-specific. Group II transcripts appear on Northern blots probed with full-length or 3'-biased gamma GT cDNA. Sequence analysis of group II clones shows them to be homologous with group I clones in the region that encodes the reading frame for the light chain; however, they possess a series of unique 5' untranslated regions, which suggests that they arise from lung-specific message processing. Additionally, approximately 50% of the isolated group II clones contain 34 nt substitutions compared with the "wild-type" gamma GT transcripts. These data indicate that human lung expresses unique gamma GT transcripts of unknown function as well as the classical form. The abundant group II transcripts may encode part of a heterodimer related to gamma GT or represent processed lung-specific pseudogenes. Images Fig. 1 PMID:7689219

  9. A preliminary study on genotypic differences in transcript abundance of drought-responsive genes in sugar beet.

    PubMed

    Rajabi, Abazar; Ranji, Zabihallah; Griffiths, Zhoward; Ober, Eric S

    2007-10-15

    In this study, four sugar beet genotypes of differing responses to drought were selected from a field experiment conducted under well-watered and water-limited conditions in 2004. In addition, two candidate genes: 2-cysteine peroxiredoxin (2-cys prx) and Nucleoside Diphosphate Kinase (NDPK), thought to be associated with drought tolerance, were chosen from a previous proteomics study in sugar beet. An expression analysis of the two drought-regulated genes using semi-quantitative reverse transcription Polymerase Chain Reaction (RT-PCR) indicated that there were genotypic differences in the transcript abundance of the candidate genes with the differences in the expression level of 2-cys prx being likely associated with the drought responses of the genotypes in a two-year field study. However, the expression analysis of the genes has to be investigated at different stages of the stress period on more genotypes.

  10. DBATE: database of alternative transcripts expression.

    PubMed

    Bianchi, Valerio; Colantoni, Alessio; Calderone, Alberto; Ausiello, Gabriele; Ferrè, Fabrizio; Helmer-Citterich, Manuela

    2013-01-01

    The use of high-throughput RNA sequencing technology (RNA-seq) allows whole transcriptome analysis, providing an unbiased and unabridged view of alternative transcript expression. Coupling splicing variant-specific expression with its functional inference is still an open and difficult issue for which we created the DataBase of Alternative Transcripts Expression (DBATE), a web-based repository storing expression values and functional annotation of alternative splicing variants. We processed 13 large RNA-seq panels from human healthy tissues and in disease conditions, reporting expression levels and functional annotations gathered and integrated from different sources for each splicing variant, using a variant-specific annotation transfer pipeline. The possibility to perform complex queries by cross-referencing different functional annotations permits the retrieval of desired subsets of splicing variant expression values that can be visualized in several ways, from simple to more informative. DBATE is intended as a novel tool to help appreciate how, and possibly why, the transcriptome expression is shaped. DATABASE URL: http://bioinformatica.uniroma2.it/DBATE/.

  11. 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

  12. Transcript Abundance Explains mRNA Mobility Data in Arabidopsis thaliana.

    PubMed

    Calderwood, Alexander; Kopriva, Stanislav; Morris, Richard J

    2016-03-01

    Recently, a large population of mRNA was shown to be able to travel between plant organs via sieve elements as a putative long-distance signaling molecule. However, a mechanistic basis by which transcripts are selected for transport has not yet been identified. Here, we show that experimental mRNA mobility data in Arabidopsis can be explained by transcript abundance and half-life. This suggests that the majority of identified mobile transcripts can be accounted for by non-sequence-specific movement of mRNA from companion cells into sieve elements.

  13. Selenium regulation of transcript abundance and translational efficiency of glutathione peroxidase-1 and -4 in rat liver.

    PubMed Central

    Weiss Sachdev, S; Sunde, R A

    2001-01-01

    Glutathione peroxidase (GPX)1 mRNA in rat liver falls dramatically during Se deficiency to levels that are approx. 10% of Se-adequate levels. This regulation is mediated by mRNA stability, and is hypothesized to involve nonsense-mediated mRNA decay. mRNA levels for GPX4 and other selenoproteins are much less regulated by Se status. To evaluate the relative contribution of mRNA abundance versus translational efficiency to overall regulation of GPX1 expression, we quantified GPX1, GPX4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts per cell in rat liver. Surprisingly, we found that GPX1 transcripts in Se deficiency are moderately abundant and similar in abundance to GAPDH and other selenoprotein mRNAs; Se supplementation increases GPX1 mRNA so that it is 30-fold higher than GAPDH mRNA. Translational efficiency of GPX1 mRNA is half of that of GPX4. Translational efficiency of GPX1 mRNA increases approx. 20-fold with Se supplementation and appears to switch GPX1 mRNA from nonsense-mediated degradation to translation. This regulatory switch can explain why GPX1 expression is an excellent parameter for assessment of Se status. PMID:11463357

  14. Seasonal variations in developmental competence and relative abundance of gene transcripts in buffalo (Bubalus bubalis) oocytes.

    PubMed

    Abdoon, Ahmed S; Gabler, Christoph; Holder, Christoph; Kandil, Omaima M; Einspanier, Ralf

    2014-11-01

    Hot season is a major constraint to production and reproduction in buffaloes. The present work aimed to investigate the effect of season on ovarian function, developmental competence, and the relative abundance of gene expression in buffalo oocytes. Three experiments were conducted. In experiment 1, pairs of buffalo ovaries were collected during cold season (CS, autumn and winter) and hot season (HS, spring and summer), and the number of antral follicles was recorded. Cumulus oocyte complexes (COCs) were aspirated and evaluated according to their morphology into four Grades. In experiment 2, Grade A and B COCs collected during CS and HS were in vitro matured (IVM) for 24 hours under standard conditions at 38.5 °C in a humidified air of 5% CO2. After IVM, cumulus cells were removed and oocytes were fixed, stained with 1% aceto-orcein, and evaluated for nuclear configuration. In vitro matured buffalo oocytes harvested during CS or HS were in vitro fertilized (IVF) using frozen-thawed buffalo semen and cultured in vitro to the blastocyst stage. In experiment 3, buffalo COCs and in vitro matured oocytes were collected during CS and HS, and then snap frozen in liquid nitrogen for gene expression analysis. Total RNA was extracted from COCs and in vitro matured oocytes, and complementary DNA was synthesized; quantitative Reverse Transcription-Polymerase Chain Reaction was performed for eight candidate genes including GAPDH, ACTB, B2M, GDF9, BMP15, HSP70, and SOD2. The results indicated that HS significantly (P < 0.01) decreased the number of antral follicles and the number of COCs recovered per ovary. The number of Grade A, B, and C COCs was lower (P < 0.05) during HS than CS. In vitro maturation of buffalo oocytes during HS significantly (P < 0.01) reduced the number of oocytes reaching the metaphase II stage and increased the percentage of degenerated oocytes compared with CS. Oocytes collected during HS also showed signs of cytoplasmic degeneration. After IVF

  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. Transcriptional regulation of mammalian selenoprotein expression

    PubMed Central

    Stoytcheva, Zoia R.; Berry, Marla J.

    2009-01-01

    Background Selenoproteins contain the twenty-first amino acid, selenocysteine, and are involved in cellular defenses against oxidative damage, important metabolic and developmental pathways, and responses to environmental challenges. Elucidating the mechanisms regulating selenoprotein expression at the transcriptional level is key to understanding how these mechanisms are called into play to respond to the changing environment. Methods This review summarizes published studies on transcriptional regulation of selenoprotein genes, focused primarily on genes whose encoded protein functions are at least partially understood. This is followed by in silico analysis of predicted regulatory elements in selenoprotein genes, including those in the aforementioned category as well as the genes whose functions are not known. Results Our findings reveal regulatory pathways common to many selenoprotein genes, including several involved in stress-responses. In addition, tissue-specific regulatory factors are implicated in regulating many selenoprotein genes. Conclusions These studies provide new insights into how selenoprotein genes respond to environmental and other challenges, and the roles these proteins play in allowing cells to adapt to these changes. General Significance Elucidating the regulatory mechanisms affecting selenoprotein expression is essential for understanding their roles in human diseases, and for developing diagnostic and potential therapeutic approaches to address dysregulation of members of this gene family. PMID:19465084

  17. 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.

  18. In-Depth Global Analysis of Transcript Abundance Levels in Porcine Alveolar Macrophages Following Infection with Porcine Reproductive and Respiratory Syndrome Virus

    PubMed Central

    Miller, Laura C.; Neill, John D.; Harhay, Gregory P.; Lager, Kelly M.; Laegreid, William W.; Kehrli, Marcus E.

    2010-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide and causes considerable economic loss. Identifying specific cell signaling or activation pathways that associate with variation in PRRSV replication and macrophage function may lead to identification of novel gene targets for the control of PRRSV infection. Serial Analysis of Gene Expression (SAGE) was used to create and survey the transcriptome of in vitro mock-infected and PRRSV strain VR-2332-infected porcine alveolar macrophages (PAM) at 0, 6, 12, 16, and 24 hours after infection. The transcriptome data indicated changes in transcript abundance occurring in PRRSV-infected PAMs over time after infection with more than 590 unique tags with significantly altered transcript abundance levels identified (P < .01). Strikingly, innate immune genes (whose transcript abundances are typically altered in response to other pathogens or insults including IL-8, CCL4, and IL-1β) showed no or very little change at any time point following infection. PMID:22331987

  19. 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

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

    DOE PAGES

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

    2014-11-28

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

  1. 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.

  2. Trans-splicing Into Highly Abundant Albumin Transcripts for Production of Therapeutic Proteins In Vivo

    PubMed Central

    Wang, Jun; Mansfield, S Gary; Cote, Colette A; Jiang, Ping Du; Weng, Ke; Amar, Marcelo JA; Brewer, Bryan H; Remaley, Alan T; McGarrity, Gerard J; Garcia-Blanco, Mariano A; Puttaraju, M

    2008-01-01

    Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo. PMID:19066600

  3. Nested transcripts of gap junction gene have distinct expression patterns.

    PubMed

    Zhang, Z; Curtin, K D; Sun, Y A; Wyman, R J

    1999-09-05

    The shaking B locus (shakB, or Passover) codes for structural molecules of gap junctions in Drosophila. This report describes the complex set of transcripts from the shakB locus. A nested set of five transcripts is described. The transcripts share 3' exons, but each has its own 5' exon. The transcripts are arrayed as a series in the genomic DNA stretching over 60 kb. The 5' end of each successive transcript lies further proximal on the chromosome. Each new transcript shares all the 3' exons with the one preceding it, but adds one or two more 5' exons. The different transcripts are expressed in a wide variety of locations in the nervous system and in non-neural tissues. Some tissues express more than one transcript, and the expression pattern of each is developmentally regulated. Within the adult central nervous system (CNS), these transcripts have an expression pattern that is restricted to the giant fiber system (GFS). The GFS is a small set of neurons which mediates the visually induced escape jump. shakB is required for function of the GFS electrical synapses. The transcript previously defined as active in the giant fiber is not, in fact, expressed in that cell. Instead, we find that another transcript, shakB(N3), and perhaps shakB(N4) as well, is expressed in the GFS; this transcript is not expressed elsewhere in the adult CNS. Two other transcripts, shakB(N1) and shakB(N2), are expressed in the optic lamina but not elsewhere in the CNS. This expression pattern explains the neurophysiological and behavioral defects in escape exhibited in mutants of shakB.

  4. Gene expression in plant mitochondria: transcriptional and post-transcriptional control.

    PubMed Central

    Binder, Stefan; Brennicke, Axel

    2003-01-01

    The informational content of the mitochondrial genome in plants is, although small, essential for each cell. Gene expression in these organelles involves a number of distinct transcriptional and post-transcriptional steps. The complex post-transcriptional processes of plant mitochondria such as 5' and 3' RNA processing, intron splicing, RNA editing and controlled RNA stability extensively modify individual steady-state RNA levels and influence the mRNA quantities available for translation. In this overview of the processes in mitochondrial gene expression, we focus on confirmed and potential sites of regulatory interference and discuss the evolutionary origins of the transcriptional and post-transcriptional processes. PMID:12594926

  5. 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

  6. 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

  7. 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.

  8. Hepatic cytochrome P450 activity, abundance, and expression throughout human development

    SciTech Connect

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

    2016-07-01

    Cytochrome P450s are Phase I metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes can vary considerably throughout human development, especially when comparing fetal development to neonates, children, and adults. In an effort to develop a more comprehensive understanding of the ontogeny of P450 expression and activity we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. To quantify the functional activity of individual P450s we employ activity-based protein profiling, which uses modified mechanism-based inhibitors of P450s as chemical probes, in tandem with proteomic analyses to quantify activity. Our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. The results were used to distribute P450s into three general classes based upon developmental stage of expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that our ontogeny 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.

  9. Transcriptional and post-transcriptional regulation of pst2 operon expression in Vibrio cholerae O1.

    PubMed

    da C Leite, Daniel M; Barbosa, Livia C; Mantuano, Nathalia; Goulart, Carolina L; Veríssimo da Costa, Giovani C; Bisch, Paulo M; von Krüger, Wanda M A

    2017-02-27

    One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions

  10. Transcriptional and epigenetic regulation of PPARγ expression during adipogenesis

    PubMed Central

    2014-01-01

    The nuclear receptor PPARγ is a master regulator of adipogenesis. PPARγ is highly expressed in adipose tissues and its expression is markedly induced during adipogenesis. In this review, we describe the current knowledge, as well as future directions, on transcriptional and epigenetic regulation of PPARγ expression during adipogenesis. Investigating the molecular mechanisms that control PPARγ expression during adipogenesis is critical for understanding the development of white and brown adipose tissues, as well as pathological conditions such as obesity and diabetes. The robust induction of PPARγ expression during adipogenesis also serves as an excellent model system for studying transcriptional and epigenetic regulation of cell-type-specific gene expression. PMID:24904744

  11. Changes in abundance of an abscisic acid-responsive, early cysteine-labeled metallothionein transcript during pollen embryogenesis in bread wheat (Triticum aestivum).

    PubMed

    Reynolds, T L; Crawford, R L

    1996-12-01

    A clone for an embryoid-abundant, early cysteine-labeled metallothionein (EcMt) gene has been isolated from a wheat pollen embryoid cDNA library. The transcript of this gene was only expressed in embryogenic microspores, pollen embryoids, and developing zygotic embryos of wheat. Accumulation of the EcMt mRNA showed a direct and positive correlation with an increase of the plant hormone, abscisic acid (ABA) in developing pollen embryoids. Treating cultures with an inhibitor of ABA biosynthesis, fluridone, suppressed not only ABA accumulation but also the appearance of the EcMt gene transcript and the ability of microspores to form embryoids. These results suggest that the EcMt gene may act as a molecular marker for pollen embryogenesis because ABA biosynthesis is accompanied by the increased expression of the EcMt transcript that coincides with the differentiation of pollen embryoids in wheat anther cultures.

  12. Identification of differentially expressed sense and antisense transcript pairs in breast epithelial tissues

    PubMed Central

    Grigoriadis, Anita; Oliver, Gavin R; Tanney, Austin; Kendrick, Howard; Smalley, Matt J; Jat, Parmjit; Neville, A Munro

    2009-01-01

    Background More than 20% of human transcripts have naturally occurring antisense products (or natural antisense transcripts – NATs), some of which may play a key role in a range of human diseases. To date, several databases of in silico defined human sense-antisense (SAS) pairs have appeared, however no study has focused on differential expression of SAS pairs in breast tissue. We therefore investigated the expression levels of sense and antisense transcripts in normal and malignant human breast epithelia using the Affymetrix HG-U133 Plus 2.0 and Almac Diagnostics Breast Cancer DSA microarray technologies as well as massively parallel signature sequencing (MPSS) data. Results The expression of more than 2500 antisense transcripts were detected in normal breast duct luminal cells and in primary breast tumors substantially enriched for their epithelial cell content by DSA microarray. Expression of 431 NATs were confirmed by either of the other two technologies. A corresponding sense transcript could be identified on DSA for 257 antisense transcripts. Of these SAS pairs, 163 have not been previously reported. A positive correlation of differential expression between normal and malignant breast samples was observed for most SAS pairs. Orientation specific RT-QPCR of selected SAS pairs validated their expression in several breast cancer cell lines and solid breast tumours. Conclusion Disease-focused and antisense enriched microarray platforms (such as Breast Cancer DSA) confirm the assumption that antisense transcription in the human breast is more prevalent than previously anticipated. Expression of a proportion of these NATs has already been confirmed by other technologies while the true existence of the remaining ones has to be validated. Nevertheless, future studies will reveal whether the relative abundances of antisense and sense transcripts have regulatory influences on the translation of these mRNAs. PMID:19615061

  13. The Raccoon Polyomavirus Genome and Tumor Antigen Transcription Are Stable and Abundant in Neuroglial Tumors

    PubMed Central

    Brostoff, Terza; Dela Cruz, Florante N.; Church, Molly E.; Woolard, Kevin D.

    2014-01-01

    ABSTRACT Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including simian virus 40 (SV40), murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (nonintegrated) in neuroglial tumors. Here, we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further colocalized TAg DNA and mRNA by in situ hybridization and found that the majority of tumor cells showed positive staining. Lastly, we examined the stability of the viral genome and TAg transcription by quantitative reverse transcriptase PCR in cultured tumor cells in vitro and in a mouse xenograft model. When tumor cells were cultured in vitro, TAg transcription increased nearly 2 log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplantation of cultured cells in mice. This study represents a minimal criterion for RacPyV's association with neuroglial tumors and a novel mechanism of stability for a polyomavirus in cancer. IMPORTANCE The natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but they can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of free-ranging raccoons. Viral copy number was consistently high in these tumors but was low or undetectable in nontumor

  14. GRLD-1 regulates cell-wide abundance of glutamate receptor through post-transcriptional regulation

    PubMed Central

    Wang, George J.; Kang, Lijun; Kim, Julie E.; Maro, Géraldine S.; Xu, X. Z. Shawn; Shen, Kang

    2011-01-01

    AMPA receptors mediate most of the fast postsynaptic response at glutamatergic synapses. The abundance of AMPA receptors in neurons and at postsynaptic membranes is tightly regulated. Changes in synaptic AMPA receptor levels have been proposed to be a key regulatory event in synaptic plasticity and learning and memory. While the local, synapse-specific regulation of AMPA receptors has been intensely studied, the global, cell-wide control is less well understood. Using a forward genetic approach, we identified Glutamate Receptor Level Decreased-1 (GRLD-1), a putative RNA-binding protein that is required for efficient production of GLR-1 in the AVE interneurons in the nematode Caenorhabditis elegans. In grld-1 mutants, GLR-1 levels were drastically reduced. Consistently, both glutamate-induced currents in AVE and glr-1-dependent nose-touch avoidance behavior were defective in grld-1 mutants. We propose that this evolutionarily conserved family of proteins controls the abundance of GLR-1 by regulating glr-1 transcript splicing. PMID:21037582

  15. Abundant Gene-by-Environment Interactions in Gene Expression Reaction Norms to Copper within Saccharomyces cerevisiae

    PubMed Central

    Hodgins-Davis, Andrea; Adomas, Aleksandra B.; Warringer, Jonas; Townsend, Jeffrey P.

    2012-01-01

    Genetic variation for plastic phenotypes potentially contributes phenotypic variation to populations that can be selected during adaptation to novel ecological contexts. However, the basis and extent of plastic variation that manifests in diverse environments remains elusive. Here, we characterize copper reaction norms for mRNA abundance among five Saccharomyces cerevisiae strains to 1) describe population variation across the full range of ecologically relevant copper concentrations, from starvation to toxicity, and 2) to test the hypothesis that plastic networks exhibit increased population variation for gene expression. We find that although the vast majority of the variation is small in magnitude (considerably <2-fold), not just some, but most genes demonstrate variable expression across environments, across genetic backgrounds, or both. Plastically expressed genes included both genes regulated directly by copper-binding transcription factors Mac1 and Ace1 and genes indirectly responding to the downstream metabolic consequences of the copper gradient, particularly genes involved in copper, iron, and sulfur homeostasis. Copper-regulated gene networks exhibited more similar behavior within the population in environments where those networks have a large impact on fitness. Nevertheless, expression variation in genes like Cup1, important to surviving copper stress, was linked with variation in mitotic fitness and in the breadth of differential expression across the genome. By revealing a broader and deeper range of population variation, our results provide further evidence for the interconnectedness of genome-wide mRNA levels, their dependence on environmental context and genetic background, and the abundance of variation in gene expression that can contribute to future evolution. PMID:23019066

  16. Transcriptional Regulation of Gene Expression in C. elegans

    PubMed Central

    Reinke, Valerie; Krause, Michael; Okkema, Peter

    2013-01-01

    Protein coding gene sequences are converted to mRNA by the highly regulated process of transcription. The precise temporal and spatial control of transcription for many genes is an essential part of development in metazoans. Thus, understanding the molecular mechanisms underlying transcriptional control is essential to understanding cell fate determination during embryogenesis, post-embryonic development, many environmental interactions, and disease-related processes. Studies of transcriptional regulation in C. elegans exploit its genomic simplicity and physical characteristics to define regulatory events with single cell and minute time scale resolution. When combined with the genetics of the system, C. elegans offers a unique and powerful vantage point from which to study how chromatin-associated protein and their modifications interact with transcription factors and their binding sites to yield precise control of gene expression through transcriptional regulation. PMID:23801596

  17. 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.

  18. 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

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

    PubMed Central

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

    2016-01-01

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

  20. Identification of differentially expressed transcripts from maturing stem of sugarcane by in silico analysis of stem expressed sequence tags and gene expression profiling.

    PubMed

    Casu, Rosanne E; Dimmock, Christine M; Chapman, Scott C; Grof, Christopher P L; McIntyre, C Lynne; Bonnett, Graham D; Manners, John M

    2004-03-01

    Sugarcane accumulates high concentrations of sucrose in the mature stem and a number of physiological processes on-going in maturing stem tissue both directly and indirectly allow this process. To identify transcripts that are associated with stem maturation, we compared patterns of gene expression in maturing and immature stem tissue by expression profiling and bioinformatic analysis of sets of stem ESTs. This study complements a previous study of gene expression associated directly with sugar metabolism in sugarcane. A survey of sequences derived from stem tissue identified an abundance of several classes of sequence that are associated with fibre biosynthesis in the maturing stem. A combination of EST analyses and microarray hybridization revealed that genes encoding homologues of the dirigent protein, a protein that assists in the stereospecificity of lignin assembly, were the most abundant and most strongly differentially expressed transcripts in maturing stem tissue. There was also evidence of coordinated expression of other categories of fibre biosynthesis and putative defence- and stress-related transcripts in the maturing stem. This study has demonstrated the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques to highlight the very significant transcriptional investment the maturing stem of sugarcane has placed in fibre biosynthesis and stress tolerance, in addition to its already well-documented role in sugar accumulation.

  1. 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.

  2. 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 ...

  3. 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

  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 and posttranscriptional regulation of transcription factor expression in Arabidopsis roots

    PubMed Central

    Lee, Ji-Young; Colinas, Juliette; Wang, Jean Y.; Mace, Daniel; Ohler, Uwe; Benfey, Philip N.

    2006-01-01

    Understanding how the expression of transcription factor (TF) genes is modulated is essential for reconstructing gene regulatory networks. There is increasing evidence that sequences other than upstream noncoding can contribute to modulating gene expression, but how frequently they do so remains unclear. Here, we investigated the regulation of TFs expressed in a tissue-enriched manner in Arabidopsis roots. For 61 TFs, we created GFP reporter constructs driven by each TF’s upstream noncoding sequence (including the 5′UTR) fused to the GFP reporter gene alone or together with the TF’s coding sequence. We compared the visually detectable GFP patterns with endogenous mRNA expression patterns, as defined by a genome-wide microarray root expression map. An automated image analysis method for quantifying GFP signals in different tissues was developed and used to validate our visual comparison method. From these combined analyses, we found that (i) the upstream noncoding sequence was sufficient to recapitulate the mRNA expression pattern for 80% (35/44) of the TFs, and (ii) 25% of the TFs undergo posttranscriptional regulation via microRNA-mediated mRNA degradation (2/24) or via intercellular protein movement (6/24). The results suggest that, for Arabidopsis TFs, upstream noncoding sequences are major contributors to mRNA expression pattern establishment, but modulation of transcription factor protein expression pattern after transcription is relatively frequent. This study provides a systematic overview of regulation of TF expression at a cellular level. PMID:16581911

  6. 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.

  7. Sequential Logic Model Deciphers Dynamic Transcriptional Control of Gene Expressions

    PubMed Central

    Yeo, Zhen Xuan; Wong, Sum Thai; Arjunan, Satya Nanda Vel; Piras, Vincent; Tomita, Masaru; Selvarajoo, Kumar; Giuliani, Alessandro; Tsuchiya, Masa

    2007-01-01

    Background Cellular signaling involves a sequence of events from ligand binding to membrane receptors through transcription factors activation and the induction of mRNA expression. The transcriptional-regulatory system plays a pivotal role in the control of gene expression. A novel computational approach to the study of gene regulation circuits is presented here. Methodology Based on the concept of finite state machine, which provides a discrete view of gene regulation, a novel sequential logic model (SLM) is developed to decipher control mechanisms of dynamic transcriptional regulation of gene expressions. The SLM technique is also used to systematically analyze the dynamic function of transcriptional inputs, the dependency and cooperativity, such as synergy effect, among the binding sites with respect to when, how much and how fast the gene of interest is expressed. Principal Findings SLM is verified by a set of well studied expression data on endo16 of Strongylocentrotus purpuratus (sea urchin) during the embryonic midgut development. A dynamic regulatory mechanism for endo16 expression controlled by three binding sites, UI, R and Otx is identified and demonstrated to be consistent with experimental findings. Furthermore, we show that during transition from specification to differentiation in wild type endo16 expression profile, SLM reveals three binary activities are not sufficient to explain the transcriptional regulation of endo16 expression and additional activities of binding sites are required. Further analyses suggest detailed mechanism of R switch activity where indirect dependency occurs in between UI activity and R switch during specification to differentiation stage. Conclusions/Significance The sequential logic formalism allows for a simplification of regulation network dynamics going from a continuous to a discrete representation of gene activation in time. In effect our SLM is non-parametric and model-independent, yet providing rich biological

  8. Transcriptome discovery in non-model wild fish species for the development of quantitative transcript abundance assays

    USGS Publications Warehouse

    Hahn, Cassidy M.; Iwanowicz, Luke R.; Cornman, Robert S.; Mazik, Patricia M.; Blazer, Vicki S.

    2016-01-01

    Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. High throughput methodologies were employed to establish partial transcriptomes for three wild-caught, non-model fish species; smallmouth bass (Micropterus dolomieu), white sucker (Catostomus commersonii) and brown bullhead (Ameiurus nebulosus). Sequences from these transcriptome databases were utilized in the development of a custom nCounter CodeSet that allowed for direct multiplexed measurement of 50 transcript abundance endpoints in liver tissue. Sequence information was also utilized in the development of quantitative real-time PCR (qPCR) primers. Cross-species hybridization allowed the smallmouth bass nCounter CodeSet to be used for quantitative transcript abundance analysis of an additional non-model species, largemouth bass (Micropterus salmoides). We validated the nCounter analysis data system with qPCR for a subset of genes and confirmed concordant results. Changes in transcript abundance biomarkers between sexes and seasons were evaluated to provide baseline data on transcript modulation for each species of interest.

  9. Expression Profiling of Cell Lines Expressing Regulated NP2 Transcripts

    DTIC Science & Technology

    2004-09-01

    EGF in the presence or absence of exogenous HRS . The results will provide a framework fo r the interpretation of future gene expression studies in...e studies require further verification. Small sam- ple size, tissue heterogeneity, and inter-indivi- dual variations among human patients may result ... studies we proposed using gene expression profiling to determine change s in gene expression as a function of expression of the neurofibromatosis-2 (NF2

  10. 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.

  11. Rapid transcription fosters coordinate snail expression in the Drosophila embryo.

    PubMed

    Boettiger, Alistair Nicol; Levine, Michael

    2013-01-31

    Transcription is commonly held to be a highly stochastic process, resulting in considerable heterogeneity of gene expression among the different cells in a population. Here, we employ quantitative in situ hybridization methods coupled with high-resolution imaging assays to measure the expression of snail, a developmental patterning gene necessary for coordinating the invagination of the mesoderm during gastrulation of the Drosophila embryo. Our measurements of steady-state mRNAs suggest that there is very little variation in snail expression across the different cells that make up the mesoderm and that synthesis approaches the kinetic limits of Pol II processivity. We propose that rapid transcription kinetics and negative autoregulation are responsible for the remarkable homogeneity of snail expression and the coordination of mesoderm invagination.

  12. Dynamic Post-Transcriptional Regulation of HIV-1 Gene Expression

    PubMed Central

    Kula, Anna; Marcello, Alessandro

    2012-01-01

    Gene expression of the human immunodeficiency virus type 1 (HIV-1) is a highly regulated process. Basal transcription of the integrated provirus generates early transcripts that encode for the viral products Tat and Rev. Tat promotes the elongation of RNA polymerase while Rev mediates the nuclear export of viral RNAs that contain the Rev-responsive RNA element (RRE). These RNAs are exported from the nucleus to allow expression of Gag-Pol and Env proteins and for the production of full-length genomic RNAs. A balance exists between completely processed mRNAs and RRE-containing RNAs. Rev functions as an adaptor that recruits cellular factors to re-direct singly spliced and unspliced viral RNAs to nuclear export. The aim of this review is to address the dynamic regulation of this post-transcriptional pathway in light of recent findings that implicate several novel cellular cofactors of Rev function. PMID:24832221

  13. 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

  14. The expression and post-transcriptional regulation of FSTL1 transcripts in placental trophoblasts

    PubMed Central

    Mouillet, Jean-Francois; Mishima, Takuya; Paffaro, Andrea Mollica do Amarante; Parks, Tony W.; Ziegler, Judy A.; Chu, Tianjiao; Sadovsky, Yoel

    2015-01-01

    Introduction Follistatin-like-1 (FSTL1) is a widely expressed secreted protein with diverse but poorly understood functions. Originally described as a pro-inflammatory molecule, it has recently been reported to play a role in signaling pathways that regulate development and homeostasis. Distinctively, FSTL1 harbors within its 3′-UTR the sequence encoding microRNA-198 (miR-198), shown to be inversely regulated relative to FSTL1 expression and to exhibit opposite actions on cellular processes such as cell migration. We sought to investigate the expression of FSTL1 and to assess its interplay with miR-198 in human trophoblasts. Methods We used a combination of northern blot analyses, quantitative PCR, small RNA sequencing, western blot and immunohistochemistry to characterize FSTL1 and miR-198 expression in placental trophoblasts. We also used reporter assays to examine the post-transcriptional regulation of FSTL1 and assess its putative regulation by miR-198. Results We detected the expression of FSTL1 transcript in both the human extravillous trophoblast line HTR-8/SVneo and in primary term human villous trophoblasts. We also found that the expression of FSTL1 was largely restricted to extravillous trophoblasts. Hypoxia enhanced the expression of FSTL1 protein in cultured primary villous trophoblasts. Interestingly, we did not detect any evidence for expression or function of mature miR-198 in human trophoblasts. Discussion Our data indicate that placental FSTL1 is expressed particularly in extravillous trophoblasts. We also found no evidence for placental expression of miR-198, or for its regulation of FSTL1, implying that the post-transcriptional regulation of FSTL1 by miR-198 is tissue specific. PMID:26386648

  15. The influence of soil pH on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria.

    PubMed

    Nicol, Graeme W; Leininger, Sven; Schleper, Christa; Prosser, James I

    2008-11-01

    Autotrophic ammonia oxidation occurs in acid soils, even though laboratory cultures of isolated ammonia oxidizing bacteria fail to grow below neutral pH. To investigate whether archaea possessing ammonia monooxygenase genes were responsible for autotrophic nitrification in acid soils, the community structure and phylogeny of ammonia oxidizing bacteria and archaea were determined across a soil pH gradient (4.9-7.5) by amplifying 16S rRNA and amoA genes followed by denaturing gradient gel electrophoresis (DGGE) and sequence analysis. The structure of both communities changed with soil pH, with distinct populations in acid and neutral soils. Phylogenetic reconstructions of crenarchaeal 16S rRNA and amoA genes confirmed selection of distinct lineages within the pH gradient and high similarity in phylogenies indicated a high level of congruence between 16S rRNA and amoA genes. The abundance of archaeal and bacterial amoA gene copies and mRNA transcripts contrasted across the pH gradient. Archaeal amoA gene and transcript abundance decreased with increasing soil pH, while bacterial amoA gene abundance was generally lower and transcripts increased with increasing pH. Short-term activity was investigated by DGGE analysis of gene transcripts in microcosms containing acidic or neutral soil or mixed soil with pH readjusted to that of native soils. Although mixed soil microcosms contained identical archaeal ammonia oxidizer communities, those adapted to acidic or neutral pH ranges showed greater relative activity at their native soil pH. Findings indicate that different bacterial and archaeal ammonia oxidizer phylotypes are selected in soils of different pH and that these differences in community structure and abundances are reflected in different contributions to ammonia oxidizer activity. They also suggest that both groups of ammonia oxidizers have distinct physiological characteristics and ecological niches, with consequences for nitrification in acid soils.

  16. 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

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

    PubMed Central

    Tsai, Alex Yi-Lin; Chan, Kin; Ho, Chi-Yip; Canam, Thomas; Capron, Resmi; Master, Emma R.; Bräutigam, Katharina

    2017-01-01

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

  18. Development competence and relative transcript abundance of oocytes derived from small and medium follicles of prepubertal gilts.

    PubMed

    Kohata, Chiyuki; Izquierdo-Rico, María José; Romar, Raquel; Funahashi, Hiroaki

    2013-12-01

    The objective of this study was to examine the competence of mature oocytes aspirated from small follicles (SF, <2 mm in diameter) and medium follicles (MF, 3-6 mm) of abattoir-derived prepubertal gilt ovaries. Oocytes were selected by the presence of the first polar body (1pb) after IVM in a chemically defined medium, for sperm penetration, pronuclear formation, cleavage rate, and development to the blastocyst stage. Relative transcript abundance of genes associated with regulation of oocyte maturation (AURKA, AURKB, and MOS), fertilization (ZP3 and ZP4), maternal effect (NALP9 and HSF1), and anti-apoptosis (BCL2) were also examined in oocytes at germinal vesicle (GV) and metaphase-II (MII) stages. In SF, compared with MF, the maturation rate post-IVM was lower (P < 0.05), but there were no differences in sperm penetration rate (78.2% and 68.5% at 6 hours after insemination and 90.8% and 91.9% at 9 hours after insemination, P = 0.51 and P = 0.67, respectively), the percentage of oocytes that formed both female and male pronuclei (27.9% and 25.8% at 6 hours after insemination and 79.4% and 76.1% at 9 hours after insemination), or cleavage rate at 48 hours after insemination (85.9% and 89.7%, respectively, P = 0.46), whereas blastocyst formation rate was lower (P < 0.05) in oocytes from SF versus MF (14.7% and 31.0%). Transcript abundances decreased (P < 0.05) in all genes examined between the GV and MII stages, although only transcript abundance for MOS was lower (P < 0.05) in GV oocytes from SF versus MF. In conclusion, mature oocytes from SF and MF of prepubertal gilts with a visible 1pb had similar fertilizability in vitro and relative transcript abundance of nine genes. However, follicle size affected meiotic competence, early embryonic development to the blastocyst stage, and transcript abundance of the MOS gene.

  19. Age-related changes in transcriptional abundance and circulating levels of anti-Mullerian hormone and Sertoli cell count in crossbred and Zebu bovine males.

    PubMed

    Rajak, S K; Kumaresan, A; Attupuram, N M; Chhillar, S; Baithalu, R K; Nayak, S; Sreela, L; Singh, Raushan K; Tripathi, U K; Mohanty, T K; Yadav, Savita

    2017-02-01

    Age-related changes in peripheral anti-Mullerian hormone (AMH) concentrations and transcriptional abundance of AMH gene in testicular tissue were studied in crossbred (Holstein Friesian × Tharparkar) and Zebu (Tharparkar) males. In both the breeds, basal AMH concentrations were estimated using ELISA method in blood plasma obtained from six males each at 1, 6, 12, 18, and 24 months age. After blood collection at respective ages, all the males were castrated and expression and immunolocalization of AMH was performed in the testicular tissue. The concentration of AMH in blood plasma was found to be highest at 1 month of age in both crossbred and Zebu males, which subsequently decreased with advancing age. Significantly (P < 0.05) lower concentration of AMH was observed in crossbred as compared with Zebu males at 24 months of age. In line with peripheral AMH concentrations, the expression of AMH gene was also higher (P < 0.05) at 1 month of age, which thereafter declined significantly with advancement of age in crossbred males. Furthermore, the expression of AMH gene differed significantly between Zebu and crossbred males at all the age groups studied. Immunolocalization of AMH in testicular tissue also revealed a stronger expression at 1 month age, which gradually decreased till 24 months of age. The true Sertoli cell count was significantly higher in Zebu compared with crossbred males at all age groups studied except at 6 months age. The relationship between Sertoli cell count and circulating AMH concentrations was negative and significant (r = -0.81; P = 0.004). In conclusion, expression of AMH gene in testicular tissue and peripheral blood concentrations of AMH were higher in young compared with adults in both crossbred and Zebu males; however, the transcriptional abundance and circulating levels of AMH were higher in Zebu compared with crossbred males.

  20. 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

  1. Specificity and transcriptional activity of microbiota associated with low and high microbial abundance sponges from the Red Sea.

    PubMed

    Moitinho-Silva, Lucas; Bayer, Kristina; Cannistraci, Carlo V; Giles, Emily C; Ryu, Taewoo; Seridi, Loqmane; Ravasi, Timothy; Hentschel, Ute

    2014-03-01

    Marine sponges are generally classified as high microbial abundance (HMA) and low microbial abundance (LMA) species. Here, 16S rRNA amplicon sequencing was applied to investigate the diversity, specificity and transcriptional activity of microbes associated with an LMA sponge (Stylissa carteri), an HMA sponge (Xestospongia testudinaria) and sea water collected from the central Saudi Arabia coast of the Red Sea. Altogether, 887 068 denoised sequences were obtained, of which 806 661 sequences remained after quality control. This resulted in 1477 operational taxonomic units (OTUs) that were assigned to 27 microbial phyla. The microbial composition of S. carteri was more similar to that of sea water than to that of X. testudinaria, which is consistent with the observation that the sequence data set of S. carteri contained many more possibly sea water sequences (~24%) than the X. testudinaria data set (~6%). The most abundant OTUs were shared between all three sources (S. carteri, X. testudinaria, sea water), while rare OTUs were unique to any given source. Despite this high degree of overlap, each sponge species contained its own specific microbiota. The X. testudinaria-specific bacterial taxa were similar to those already described for this species. A set of S. carteri-specific bacterial taxa related to Proteobacteria and Nitrospira was identified, which are likely permanently associated with S. carteri. The transcriptional activity of sponge-associated microorganisms correlated well with their abundance. Quantitative PCR revealed the presence of Poribacteria, representing typical sponge symbionts, in both sponge species and in sea water; however, low transcriptional activity in sea water suggested that Poribacteria are not active outside the host context.

  2. 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.

  3. Transient Phenomena in Gene Expression after Induction of Transcription

    PubMed Central

    Deneke, Carlus; Rudorf, Sophia; Valleriani, Angelo

    2012-01-01

    When transcription of a gene is induced by a stimulus, the number of its mRNA molecules changes with time. Here we discuss how this time evolution depends on the shape of the mRNA lifetime distribution. Analysis of the statistical properties of this change reveals transient effects on polysomes, ribosomal profiles, and rate of protein synthesis. Our studies reveal that transient phenomena in gene expression strongly depend on the specific form of the mRNA lifetime distribution. PMID:22558114

  4. Transcriptional and Post-Transcriptional Regulation of Thrombospondin-1 Expression: A Computational Model

    PubMed Central

    Isenberg, Jeffrey S.; Popel, Aleksander S.

    2017-01-01

    Hypoxia is an important physiological stress signal that drives angiogenesis, the formation of new blood vessels. Besides an increase in the production of pro-angiogenic signals such as vascular endothelial growth factor (VEGF), hypoxia also stimulates the production of anti-angiogenic signals. Thrombospondin-1 (TSP-1) is one of the anti-angiogenic factors whose synthesis is driven by hypoxia. Cellular synthesis of TSP-1 is tightly regulated by different intermediate biomolecules including proteins that interact with hypoxia-inducible factors (HIFs), transcription factors that are activated by receptor and intracellular signaling, and microRNAs which are small non-coding RNA molecules that function in post-transcriptional modification of gene expression. Here we present a computational model that describes the mechanistic interactions between intracellular biomolecules and cooperation between signaling pathways that together make up the complex network of TSP-1 regulation both at the transcriptional and post-transcriptional level. Assisted by the model, we conduct in silico experiments to compare the efficacy of different therapeutic strategies designed to modulate TSP-1 synthesis in conditions that simulate tumor and peripheral arterial disease microenvironment. We conclude that TSP-1 production in endothelial cells depends on not only the availability of certain growth factors but also the fine-tuned signaling cascades that are initiated by hypoxia. PMID:28045898

  5. 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.

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

    PubMed Central

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

    2016-01-01

    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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. The Role of the Ubiquitously Expressed Transcription Factor Sp1 in Tissue-specific Transcriptional Regulation and in Disease

    PubMed Central

    O’Connor, Leigh; Gilmour, Jane; Bonifer, Constanze

    2016-01-01

    Sp1 belongs to the 26 member strong Sp/KLF family of transcription factors. It is a paradigm for a ubiquitously expressed transcription factor and is involved in regulating the expression of genes associated with a wide range of cellular processes in mammalian cells. Sp1 can interact with a range of proteins, including other transcription factors, members of the transcription initiation complex and epigenetic regulators, enabling tight regulation of its target genes. In this review, we discuss the mechanisms involved in Sp1-mediated transcriptional regulation, as well as how a ubiquitous transcription factor can be involved in establishing a tissue-specific pattern of gene expression and mechanisms by which its activity may be regulated. We also consider the role of Sp1 in human diseases, such as cancer. PMID:28018142

  14. Interaction between the yeast mitochondrial and nuclear genomes influences the abundance of novel transcripts derived from the spacer region of the nuclear ribosomal DNA repeat.

    PubMed Central

    Parikh, V S; Conrad-Webb, H; Docherty, R; Butow, R A

    1989-01-01

    We have identified stable transcripts from the so-called nontranscribed spacer region (NTS) of the nuclear ribosomal DNA repeat in certain respiration-deficient strains of Saccharomyces cerevisiae. These RNAs, which are transcribed from the same strand as is the 37S rRNA precursor, are 500 to 800 nucleotides long and extend from the 5' end of the 5S rRNA gene to three major termination sites about 1,780, 1,830, and 1,870 nucleotides from the 3' end of the 26S rRNA gene. A survey of various wild-type and respiration-deficient strains showed that NTS transcript abundance depended on the mitochondrial genotype and a single codominant nuclear locus. In strains with that nuclear determinant, NTS transcripts were barely detected in [rho+] cells, were slightly more abundant in various mit- derivatives, and were most abundant in petites. However, in one petite that was hypersuppressive and contained a putative origin of replication (ori5) within its 757-base-pair mitochondrial genome, NTS transcripts were no more abundant than in [rho+] cells. The property of low NTS transcript abundance in the hypersuppressive petite was unstable, and spontaneous segregants that contained NTS transcripts as abundant as in the other petites examined could be obtained. Thus, respiration deficiency per se is not the major factor contributing to the accumulation of these unusual RNAs. Unlike RNA polymerase I transcripts, the abundant NTS RNAs were glucose repressible, fractionated as poly(A)+ RNAs, and were sensitive to inhibition by 10 micrograms of alpha-amanitin per ml, a concentration that had no effect on rRNA synthesis. Abundant NTS RNAs are therefore most likely derived by polymerase II transcription. Images PMID:2473390

  15. 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.

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

    PubMed Central

    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

    2015-01-01

    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

  17. Association of triacylglyceride content and transcript abundance of genes involving in lipid synthesis of nitrogen deficient Phaeodactylum tricornutum

    NASA Astrophysics Data System (ADS)

    Zhang, Lin; Han, Jichang; Yang, Guanpin; Zhu, Baohua; Pan, Kehou

    2014-03-01

    Phaeodactylum tricornutum is a diatom that is rich in lipids. Recently, it has received much attention as a feedstock for biodiesel production. Nitrogen deficiency is widely known to increase the content of neutral lipids (mainly triacylglycerides, or TAGs) of microalgae, including P. tricornutum, but the mechanism is unclear. In this study, we deciphered the correlations between TAG content and nine key enzymatic genes involved in lipid synthesis in P. tricornutum. After being cultured under nitrogen-free conditions for 0, 4, 24, 48, 72, 120, and 168 h, the TAG contents of P. tricornutum cells were assayed and the transcript abundances of the target genes were monitored by quantitative real-time PCR. The results show that the abundances of four target gene transcripts ( LACS3, G3PDH2, G3PDH3, and G3PDH5) were positively correlated with TAG content, indicating that these genes may be involved in TAG synthesis in P. tricornutum. The findings improve our understanding of the metabolic network and regulation of lipid synthesis and will guide the future genetic improvement of the TAG content of P. tricornutum.

  18. 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.

  19. Transcriptional regulation of 15-lipoxygenase expression by promoter methylation.

    PubMed

    Liu, Cheng; Xu, Dawei; Sjöberg, Jan; Forsell, Pontus; Björkholm, Magnus; Claesson, Hans-Erik

    2004-07-01

    15-Lipoxygenase type 1 (15-LO), a lipid-peroxidating enzyme implicated in physiological membrane remodeling and the pathogenesis of atherosclerosis, inflammation, and carcinogenesis, is highly regulated and expressed in a tissue- and cell-type-specific fashion. It is known that interleukins (IL) 4 and 13 play important roles in transactivating the 15-LO gene. However, the fact that they only exert such effects on a few types of cells suggests additional mechanism(s) for the profile control of 15-LO expression. In the present study, we demonstrate that hyper- and hypomethylation of CpG islands in the 15-LO promoter region is intimately associated with the transcriptional repression and activation of the 15-LO gene, respectively. The 15-LO promoter was exclusively methylated in all examined cells incapable of expressing 15-LO (certain solid tumor and human lymphoma cell lines and human T lymphocytes) while unmethylated in 15-LO-competent cells (the human airway epithelial cell line A549 and human monocytes) where 15-LO expression is IL4-inducible. Inhibition of DNA methylation in L428 lymphoma cells restores IL4 inducibility to 15-LO expression. Consistent with this, the unmethylated 15-LO promoter reporter construct exhibited threefold higher activity in A549 cells compared to its methylated counterpart. Taken together, demethylation of the 15-LO promoter is a prerequisite for the gene transactivation, which contributes to tissue- and cell-type-specific regulation of 15-LO expression.

  20. Comparative analysis of transcript abundance in Pinus sylvestris after challenge with a saprotrophic, pathogenic or mutualistic fungus.

    PubMed

    Adomas, Aleksandra; Heller, Gregory; Olson, Ake; Osborne, Jason; Karlsson, Magnus; Nahalkova, Jarmila; Van Zyl, Len; Sederoff, Ron; Stenlid, Jan; Finlay, Roger; Asiegbu, Frederick O

    2008-06-01

    To investigate functional differences in the recognition and response mechanisms of conifer roots to fungi with different trophic strategies, Pinus sylvestris L. was challenged with a saprotrophic fungus Trichoderma aureoviride Rifai. The results were compared with separate studies investigating pine interactions with a pathogen, Heterobasidion annosum (Fr.) Bref. sensu stricto and an ectomycorrhizal symbiont, Laccaria bicolor Maire (Orton). Global changes in the expression of 2109 conifer genes were assayed 1, 5 and 15 days after inoculation. Gene expression data from a cDNA microarray were analyzed by the 2-interconnected mixed linear model statistical approach. The total number of genes differentially expressed compared with the uninfected control was similar after challenge with the pathogen and the ectomycorrhizal symbiont, but the number of differentially expressed genes increased over time for H. annosum, and decreased for L. bicolor. Inoculation of pine roots with T. aureoviride resulted overall in a much lower number of genes with changed transcript levels compared with inoculation with H. annosum or L. bicolor. Functional classification of the differentially expressed genes revealed that the ectomycorrhizal fungus triggered transient induction of defence-related genes. The response and induction of defence against the pathogen was delayed and the magnitude increased over time. Thus, there were specific transcriptional responses depending on whether the conifer roots were challenged with mutualistic, saprotrophic or pathogenic fungi. This suggests that pine trees are able to recognize diverse fungal species and specifically distinguish whether they are pathogenic, neutral or beneficial microbial agents.

  1. A Weakened Transcriptional Enhancer Yields Variegated Gene Expression

    PubMed Central

    Collins, Cathy; Azmi, Peter; Berru, Maribel; Zhu, Xiaofu; Shulman, Marc J.

    2006-01-01

    Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, ∼1.4 kb 3′ of the promoter, but inactive ∼6 kb 3′, indicating that the activity of the core enhancer was distance-dependent. A longer segment – the core enhancer plus ∼1 kb of 3′ flanking material, including the 3′ matrix attachment region – was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3′ flank includes binding sites for at least two activators. PMID:17183661

  2. 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

    2016-08-24

    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.

  3. 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

  4. Agouti revisited: transcript quantification of the ASIP gene in bovine tissues related to protein expression and localization.

    PubMed

    Albrecht, Elke; Komolka, Katrin; Kuzinski, Judith; Maak, Steffen

    2012-01-01

    Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species.

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

    DOE PAGES

    Poovaiah, Charleson R.; Bewg, William P.; Lan, Wu; ...

    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

  6. NikR mediates nickel-responsive transcriptional induction of urease expression in Helicobacter pylori.

    PubMed

    van Vliet, Arnoud H M; Poppelaars, Sophie W; Davies, Beverly J; Stoof, Jeroen; Bereswill, Stefan; Kist, Manfred; Penn, Charles W; Kuipers, Ernst J; Kusters, Johannes G

    2002-06-01

    The important human pathogen Helicobacter pylori requires the abundant expression and activity of its urease enzyme for colonization of the gastric mucosa. The transcription, expression, and activity of H. pylori urease were previously demonstrated to be induced by nickel supplementation of growth media. Here it is demonstrated that the HP1338 protein, an ortholog of the Escherichia coli nickel regulatory protein NikR, mediates nickel-responsive induction of urease expression in H. pylori. Mutation of the HP1338 gene (nikR) of H. pylori strain 26695 resulted in significant growth inhibition of the nikR mutant in the presence of supplementation with NiCl(2) at > or =100 microM, whereas the wild-type strain tolerated more than 10-fold-higher levels of NiCl(2). Mutation of nikR did not affect urease subunit expression or urease enzyme activity in unsupplemented growth media. However, the nickel-induced increase in urease subunit expression and urease enzyme activity observed in wild-type H. pylori was absent in the H. pylori nikR mutant. A similar lack of nickel responsiveness was observed upon removal of a 19-bp palindromic sequence in the ureA promoter, as demonstrated by using a genomic ureA::lacZ reporter gene fusion. In conclusion, the H. pylori NikR protein and a 19-bp operator sequence in the ureA promoter are both essential for nickel-responsive induction of urease expression in H. pylori.

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

    PubMed

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

    2017-03-01

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

  8. Progressive lung cancer determined by expression profiling and transcriptional regulation.

    PubMed

    Han, Namshik; Dol, Zulkifli; Vasieva, Olga; Hyde, Russell; Liloglou, Triantafillos; Raji, Olaide; Brambilla, Elisabeth; Brambilla, Christian; Martinet, Yves; Sozzi, Gabriella; Risch, Angela; Montuenga, Luis M; Brass, Andy; Field, John K

    2012-07-01

    Clinically, our ability to predict disease outcome for patients with early stage lung cancer is currently poor. To address this issue, tumour specimens were collected at surgery from non-small cell lung cancer (NSCLC) patients as part of the European Early Lung Cancer (EUELC) consortium. The patients were followed-up for three years post-surgery and patients who suffered progressive disease (PD, tumour recurrence, metastasis or a second primary) or remained disease-free (DF) during follow-up were identified. RNA from both tumour and adjacent-normal lung tissue was extracted from patients and subjected to microarray expression profiling. These samples included 36 adenocarcinomas and 23 squamous cell carcinomas from both PD and DF patients. The microarray data was subject to a series of systematic bioinformatics analyses at gene, network and transcription factor levels. The focus of these analyses was 2-fold: firstly to determine whether there were specific biomarkers capable of differentiating between PD and DF patients, and secondly, to identify molecular networks which may contribute to the progressive tumour phenotype. The experimental design and analyses performed permitted the clear differentiation between PD and DF patients using a set of biomarkers implicated in neuroendocrine signalling and allowed the inference of a set of transcription factors whose activity may differ according to disease outcome. Potential links between the biomarkers, the transcription factors and the genes p21/CDKN1A and Myc, which have previously been implicated in NSCLC development, were revealed by a combination of pathway analysis and microarray meta-analysis. These findings suggest that neuroendocrine-related genes, potentially driven through p21/CDKN1A and Myc, are closely linked to whether or not a NSCLC patient will have poor clinical outcome.

  9. 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

  10. Transcriptional and post-transcriptional control of PHO8 expression by PHO regulatory genes in Saccharomyces cerevisiae.

    PubMed Central

    Kaneko, Y; Tamai, Y; Toh-e, A; Oshima, Y

    1985-01-01

    A DNA fragment bearing the PHO8 gene, which encodes repressible alkaline phosphatase of Saccharomyces cerevisiae, was cloned. Northern hybridizations with the PHO8 DNA as probe indicated that the PHO8 transcript is 1.8 kilobases in length and is more abundant in cells grown in low-phosphate medium than in high-phosphate medium. The pho9 mutant, whose phenotype is defective in the activity of repressible alkaline phosphatase, produced as much of the PHO8 transcript as did the PHO9+ cells. Hence, the PHO9 product should act at the post-transcriptional level. The pho4 mutant could not derepress the PHO8 transcript, whereas the pho80 mutant could, irrespective of the amount of Pi in the medium, as has been suggested by genetic study. Images PMID:2984552

  11. Expression of a Mutant kcnj2 Gene Transcript in Zebrafish

    PubMed Central

    Leong, Ivone U. S.; Skinner, Jonathan R.; Shelling, Andrew N.; Love, Donald R.

    2013-01-01

    Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the human KCNJ2 gene, which encodes for the subunit of the potassium inwardly-rectifying channel (IK1), have been associated with the disorder. The majority of mutations are considered to be dominant-negative as mutant proteins interact to limit the function of wild type KCNJ2 proteins. Several LQT7 syndrome mouse models have been created that vary in the physiological similarity to the human disease. To complement the LQT7 mouse models, we investigated the usefulness of the zebrafish as an alternative model via a transient approach. Initial bioinformatic analysis identified the zebrafish orthologue of the human KCNJ2 gene, together with a spatial expression profile that was similar to that of human. The expression of a kcnj2-12 transcript carrying an in-frame deletion of critical amino acids identified in human studies resulted in embryos that exhibited defects in muscle development, thereby affecting movement, a decrease in jaw size, pupil-pupil distance, and signs of scoliosis. These defects correspond to some phenotypes expressed by human LQT7 patients. PMID:27335675

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

    PubMed

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

    2017-04-01

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

  13. Expression of SRY transcripts in preimplantation human embryos

    SciTech Connect

    Fiddler, M.; Abdel-Rahman, B.; Rappolee, D.A.

    1995-01-02

    We have examined the expression of SRY mRNA in individual in vitro fertilized preimplantation human embryos; because of ethical constraints, these studies were confined to embryos with one and three pronuclei. Using a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we observed SRY mRNA at the one-cell through the blastula stages but not in spermatoza. These results indicate that the de novo transcription of this sex-specific gene occurs at a developmental time considerably earlier than that of gonadal differentiation. Our results also indicate that in vitro fertilized embryos with one pronucleus are likely to be diploid. 39 refs., 1 fig., 2 tabs.

  14. Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose

    PubMed Central

    Meng, Jian; Feng, Ming; Dong, Weibing; Zhu, Yemin; Li, Yakui; Zhang, Ping; Wu, Lifang; Li, Minle; Lu, Ying; Chen, Hanbei; Liu, Xing; Lu, Yan; Sun, Haipeng; Tong, Xuemei

    2016-01-01

    Transcription factor carbohydrate responsive element binding protein (ChREBP) promotes glycolysis and lipogenesis in metabolic tissues and cancer cells. ChREBP-α and ChREBP-β, two isoforms of ChREBP transcribed from different promoters, are both transcriptionally induced by glucose. However, the mechanism by which glucose increases ChREBP mRNA levels remains unclear. Here we report that hepatocyte nuclear factor 4 alpha (HNF-4α) is a key transcription factor for glucose-induced ChREBP-α and ChREBP-β expression. Ectopic HNF-4α expression increased ChREBP transcription while knockdown of HNF-4α greatly reduced ChREBP mRNA levels in liver cancer cells and mouse primary hepatocytes. HNF-4α not only directly bound to an E-box-containing region in intron 12 of the ChREBP gene, but also promoted ChREBP-β transcription by directly binding to two DR1 sites and one E-box-containing site of the ChREBP-β promoter. Moreover, HNF-4α interacted with ChREBP-α and synergistically promoted ChREBP-β transcription. Functionally, HNF-4α suppression reduced glucose-dependent ChREBP induction. Increased nuclear abundance of HNF-4α and its binding to cis-elements of ChREBP gene in response to glucose contributed to glucose-responsive ChREBP transcription. Taken together, our results not only revealed the novel mechanism by which HNF-4α promoted ChREBP transcription in response to glucose, but also demonstrated that ChREBP-α and HNF-4α synergistically increased ChREBP-β transcription. PMID:27029511

  15. Expression-based discovery of candidate ovule development regulators through transcriptional profiling of ovule mutants

    PubMed Central

    Skinner, Debra J; Gasser, Charles S

    2009-01-01

    Background Arabidopsis ovules comprise four morphologically distinct parts: the nucellus, which contains the embryo sac, two integuments that become the seed coat, and the funiculus that anchors the ovule within the carpel. Analysis of developmental mutants has shown that ovule morphogenesis relies on tightly regulated genetic interactions that can serve as a model for developmental regulation. Redundancy, pleiotropic effects and subtle phenotypes may preclude identification of mutants affecting some processes in screens for phenotypic changes. Expression-based gene discovery can be used access such obscured genes. Results Affymetrix microarrays were used for expression-based gene discovery to identify sets of genes expressed in either or both integuments. The genes were identified by comparison of pistil mRNA from wild type with mRNA from two mutants; inner no outer (ino, which lacks the outer integument), and aintegumenta (ant, which lacks both integuments). Pools of pistils representing early and late stages of ovule development were evaluated and data from the three genotypes were used to designate genes that were predominantly expressed in the integuments using pair-wise and cluster analyses. Approximately two hundred genes were found to have a high probability of preferential expression in these structures, and the predictive nature of the expression classes was confirmed with reverse transcriptase polymerase chain reaction and in situ hybridization. Conclusion The results showed that it was possible to use a mutant, ant, with broad effects on plant phenotype to identify genes expressed specifically in ovules, when coupled with predictions from known gene expression patterns, or in combination with a more specific mutant, ino. Robust microarray averaging (RMA) analysis of array data provided the most reliable comparisons, especially for weakly expressed genes. The studies yielded an over-abundance of transcriptional regulators in the identified genes, and

  16. Correlation between transcript abundance of the RB gene and the level of the RB-mediated late blight resistance in potato.

    PubMed

    Kramer, Lara C; Choudoir, Mallory J; Wielgus, Susan M; Bhaskar, Pudota B; Jiang, Jiming

    2009-04-01

    Numerous disease-resistance genes have been cloned and characterized in various plant species. Only a few of these reported genes were transcriptionally induced or had enhanced transcription upon pathogen infection. Here, we report that transcription of the RB gene, which was cloned from the wild potato species Solanum bulbocastanum and confers resistance to potato late blight, was significantly increased after inoculation with the late blight pathogen Phytophthora infestans. Different RB transgenic lines showed different levels of resistance, which were correlated with the amounts of RB transcript in the transgenic plants. Different transgenic lines also showed different patterns of RB transcription 1, 3, and 5 days after P. infestans inoculation. Interestingly, the RB gene showed a higher basal level of transcription and a more dramatic transcriptional increase upon inoculation in S. bulbocastanum than in all potato transgenic lines. Our results revealed a predictive correlation between transcript abundance of the RB gene and the level of the RB-mediated late blight resistance. High level of resistance was associated with a combination of rapid RB transcript induction immediately after pathogen infection followed by the steady production of RB transcript. Thus, the transcription level of the RB gene provides a valuable marker for selecting and deploying RB-containing potato lines for late blight control.

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

    SciTech Connect

    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 plant 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.

  18. Linking chloroplast antioxidant defense to carbohydrate availability: the transcript abundance of stromal ascorbate peroxidase is sugar-controlled via ascorbate biosynthesis.

    PubMed

    Heiber, Isabelle; Cai, Wenguo; Baier, Margarete

    2014-01-01

    All genes encoding chloroplast antioxidant enzymes are nuclear-encoded and posttranscriptionally targeted to chloroplasts. The transcript levels of most of them decreased upon sucrose feeding like the transcript levels of many genes encoding components of the photosynthetic electron transport chain. However, the transcript abundance of stromal ascorbate peroxidase (s-APX; At4g08390) increased. Due to mild sugar application conditions, the plants kept the phosphorylation status of the ADP+ATP pool and the redox states of the NADPH+NADP+ and the ascorbate pools under control, which excludes them as signals in s-APX regulation. Correlation with ascorbate pool size regulation and comparison of transcript abundance regulation in the starch-biosynthetic mutant adg1, the ascorbate biosynthesis mutant vtc1, and the abscisic acid (ABA) biosynthetic mutant aba2 showed a link between sugar induction of s-APX and ascorbate biosynthesis.

  19. 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...

  20. Relationships between inflammation- and immunity-related transcript abundance in the rumen and jejunum of beef steers with divergent average daily gain.

    PubMed

    Reynolds, J G; Foote, A P; Freetly, H C; Oliver, W T; Lindholm-Perry, A K

    2017-02-21

    The bovine rumen papillae are in contact with a wide array of microorganisms and the metabolites they produce, which may activate an inflammatory and/or immune response. Cytokines, chemokines and their receptor genes were tested for differential expression in the rumen and jejunum of beef steers with greater and lesser average daily body weight gain (ADG) near the average daily dry matter intake (DMI) for the population. Angus-sired steers (n = 16) were used to represent the greater (ADG = 2.2 ± 0.07 kg/day; DMI = 10.1 ± 0.05 kg/day) and lesser (ADG = 1.7 ± 0.05 kg/day; DMI = 10.1 ± 0.05 kg/day) ADG groups with eight steers each. Rumen epithelium and jejunum mucosal samples were collected at slaughter, and gene expression was evaluated using a commercially available qRT-PCR array containing 84 genes representing chemokines, cytokines and their receptors. None of the genes on the array were differentially expressed in the jejunum of the steers with greater vs. lesser ADG. However, in the rumen, two chemokine genes (CCL11, CXCL5) and one receptor gene (IL10RA) were detected as differentially expressed (P < 0.05). The genes IL1A, BMP2, CXCL12 and TNFSF13 also displayed trends for differential expression (P < 0.10). All of the genes identified were lower in transcript abundance in the greater ADG animals. Thus, greater ADG steers have a lesser inflammatory response in the rumen papillae, which may lead to a more efficient use of nutrients.

  1. Stochastic model of transcription factor-regulated gene expression

    NASA Astrophysics Data System (ADS)

    Karmakar, Rajesh; Bose, Indrani

    2006-09-01

    We consider a stochastic model of transcription factor (TF)-regulated gene expression. The model describes two genes, gene A and gene B, which synthesize the TFs and the target gene proteins, respectively. We show through analytic calculations that the TF fluctuations have a significant effect on the distribution of the target gene protein levels when the mean TF level falls in the highest sensitive region of the dose-response curve. We further study the effect of reducing the copy number of gene A from two to one. The enhanced TF fluctuations yield results different from those in the deterministic case. The probability that the target gene protein level exceeds a threshold value is calculated with the knowledge of the probability density functions associated with the TF and target gene protein levels. Numerical simulation results for a more detailed stochastic model are shown to be in agreement with those obtained through analytic calculations. The relevance of these results in the context of the genetic disorder haploinsufficiency is pointed out. Some experimental observations on the haploinsufficiency of the tumour suppressor gene, Nkx 3.1, are explained with the help of the stochastic model of TF-regulated gene expression.

  2. Cell type-specific regulation of von Willebrand factor expression by the E4BP4 transcriptional repressor.

    PubMed

    Hough, Christine; Cuthbert, Carla D; Notley, Colleen; Brown, Christine; Hegadorn, Carol; Berber, Ergul; Lillicrap, David

    2005-02-15

    Mechanisms of tissue-restricted patterns of von Willebrand factor (VWF) expression involve activators and repressors that limit expression to endothelial cells and megakaryocytes. The relative transcriptional activity of the proximal VWF promoter was assessed in VWF-producing and -nonproducing cells, and promoter activity was highest in endothelial cells followed by megakaryocytes. Only basal VWF promoter activity was seen in nonendothelial cells. Here we identify a negative response element located at nucleotides (nts) +96/+105 and demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that in vivo this sequence interacts with the E4BP4 transcriptional repressor. Differences in size and relative abundance of nuclear E4BP4 were observed. In HepG2 cells, low levels of larger forms of E4BP4 are present that directly interact with the negative response element. In VWF-expressing cells, high levels of smaller forms predominate with no evidence of direct DNA binding. However, in endothelial cells, mutation of the VWF E4BP4 binding motif not only restores but also further elevates VWF promoter activity, suggesting that E4BP4 may be part of a coordinated binding complex. These observations implicate this binding motif in repressing both activated and basal levels of VWF transcription by different cell type-specific mechanisms, and support the hypothesis that E4BP4 sequesters negative regulators of transcription, thereby enhancing activated gene expression.

  3. 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...

  4. Analysis of transcripts expressed by Eimeria tenella oocysts using subtractive hybridization methods.

    PubMed

    Miska, K B; Fetterer, R H; Barfield, R C

    2004-12-01

    To characterize the genes expressed by Eimeria tenella oocysts, the sequence of 499 expressed sequence tags (ESTs) was obtained from complementary DNA (cDNAs) enriched for transcripts expressed by unsporulated or sporulated oocysts. Of these, 225 clones were isolated from cDNA of sporulated oocysts and 274 from unsporulated oocysts. A total of 163 unique sequences were found, and the majority of these (64%) represent novel genes with no significant homology to the proteins in GenBank. Approximately half of the unique transcripts generated from sporulated oocysts are also expressed by sporozoites and merozoites, whereas the expression of most (79%) of the transcripts from unsporulated oocysts has not yet been detected at other stages of development. The expression of 4 transcripts obtained from the subtracted cDNAs was confirmed by quantitative reverse transcriptase-polymerase chain reaction. The results confirmed that these transcripts are in fact differentially expressed between sporulated and unsporulated oocysts.

  5. Transcriptional control of transglutaminase 2 expression in mouse apoptotic thymocytes.

    PubMed

    Sándor, Katalin; Daniel, Bence; Kiss, Bea; Kovács, Fruzsina; Szondy, Zsuzsa

    2016-08-01

    Transglutaminase 2 (TGM2) is a ubiquitously expressed multifunctional protein, which participates in various biological processes including thymocyte apoptosis. As a result, the transcriptional regulation of the gene is complex and must depend on the cell type. Previous studies from our laboratory have shown that in dying thymocytes the expression of Tgm2 is induced by external signals derived from engulfing macrophages, such as retinoids, transforming growth factor (TGF)-β and adenosine, the latter triggering the adenylate cyclase signaling pathway. The existence of TGF-β and retinoid responsive elements in the promoter region of Tgm2 has already been reported, but the intergenic regulatory elements participating in the regulation of Tgm2 have not yet been identified. Here we used publicly available results from DNase I hypersensitivity analysis followed by deep sequencing and chromatin immunoprecipitation followed by deep sequencing against CCCTC-binding factor (CTCF), H3K4me3, H3K4me1 and H3K27ac to map a putative regulatory element set for Tgm2 in thymocytes. By measuring eRNA expressions of these putative enhancers in retinoid, rTGF-β or dibutiryl cAMP-exposed thymocytes we determined which of them are functional. By applying ChIP-qPCR against SMAD4, retinoic acid receptor, retinoid X receptor, cAMP response element binding protein, P300 and H3K27ac under the same conditions, we identified two enhancers of Tgm2, which seem to act as integrators of the TGF-β, retinoid and adenylate cyclase signaling pathways in dying thymocytes. Our study describes a novel strategy to identify and characterize the signal-specific functional enhancer set of a gene by integrating genome-wide datasets and measuring the production of enhancer specific RNA molecules.

  6. 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.

  7. Hierarchical Post-transcriptional Regulation of Colicin E2 Expression in Escherichia coli

    PubMed Central

    Opitz, Madeleine; Frey, Erwin

    2016-01-01

    Post-transcriptional regulation of gene expression plays a crucial role in many bacterial pathways. In particular, the translation of mRNA can be regulated by trans-acting, small, non-coding RNAs (sRNAs) or mRNA-binding proteins, each of which has been successfully treated theoretically using two-component models. An important system that includes a combination of these modes of post-transcriptional regulation is the Colicin E2 system. DNA damage, by triggering the SOS response, leads to the heterogeneous expression of the Colicin E2 operon including the cea gene encoding the toxin colicin E2, and the cel gene that codes for the induction of cell lysis and release of colicin. Although previous studies have uncovered the system’s basic regulatory interactions, its dynamical behavior is still unknown. Here, we develop a simple, yet comprehensive, mathematical model of the colicin E2 regulatory network, and study its dynamics. Its post-transcriptional regulation can be reduced to three hierarchically ordered components: the mRNA including the cel gene, the mRNA-binding protein CsrA, and an effective sRNA that regulates CsrA. We demonstrate that the stationary state of this system exhibits a pronounced threshold in the abundance of free mRNA. As post-transcriptional regulation is known to be noisy, we performed a detailed stochastic analysis, and found fluctuations to be largest at production rates close to the threshold. The magnitude of fluctuations can be tuned by the rate of production of the sRNA. To study the dynamics in response to an SOS signal, we incorporated the LexA-RecA SOS response network into our model. We found that CsrA regulation filtered out short-lived activation peaks and caused a delay in lysis gene expression for prolonged SOS signals, which is also seen in experiments. Moreover, we showed that a stochastic SOS signal creates a broad lysis time distribution. Our model thus theoretically describes Colicin E2 expression dynamics in detail and

  8. Subtractive transcriptome analysis of leaf and rhizome reveals differentially expressed transcripts in Panax sokpayensis.

    PubMed

    Gurung, Bhusan; Bhardwaj, Pardeep K; Talukdar, Narayan C

    2016-11-01

    In the present study, suppression subtractive hybridization (SSH) strategy was used to identify rare and differentially expressed transcripts in leaf and rhizome tissues of Panax sokpayensis. Out of 1102 randomly picked clones, 513 and 374 high quality expressed sequenced tags (ESTs) were generated from leaf and rhizome subtractive libraries, respectively. Out of them, 64.92 % ESTs from leaf and 69.26 % ESTs from rhizome SSH libraries were assembled into different functional categories, while others were of unknown function. In particular, ESTs encoding galactinol synthase 2, ribosomal RNA processing Brix domain protein, and cell division cycle protein 20.1, which are involved in plant growth and development, were most abundant in the leaf SSH library. Other ESTs encoding protein KIAA0664 homologue, ubiquitin-activating enzyme e11, and major latex protein, which are involved in plant immunity and defense response, were most abundant in the rhizome SSH library. Subtractive ESTs also showed similarity with genes involved in ginsenoside biosynthetic pathway, namely farnesyl pyrophosphate synthase, squalene synthase, and dammarenediol synthase. Expression profiles of selected ESTs validated the quality of libraries and confirmed their differential expression in the leaf, stem, and rhizome tissues. In silico comparative analyses revealed that around 13.75 % of unigenes from the leaf SSH library were not represented in the available leaf transcriptome of Panax ginseng. Similarly, around 18.12, 23.75, 25, and 6.25 % of unigenes from the rhizome SSH library were not represented in available root/rhizome transcriptomes of P. ginseng, Panax notoginseng, Panax quinquefolius, and Panax vietnamensis, respectively, indicating a major fraction of novel ESTs. Therefore, these subtractive transcriptomes provide valuable resources for gene discovery in P. sokpayensis and would complement the available transcriptomes from other Panax species.

  9. Implication of a multisubunit Ets-related transcription factor in synaptic expression of the nicotinic acetylcholine receptor.

    PubMed Central

    Schaeffer, L; Duclert, N; Huchet-Dymanus, M; Changeux, J P

    1998-01-01

    In adult muscle, transcription of the nicotinic acetylcholine receptor (AChR) is restricted to the nuclei located at the neuromuscular junction. The N-box, a new promoter element, was identified recently and shown to contribute to this compartmentalized synaptic expression of the AChR delta- and epsilon-subunits. We demonstrate that the N-box mediates transcriptional activation in cultured myotubes and identify the transcription factor that binds to the N-box as a heterooligomer in myotubes and adult muscle. The GABP (GA-binding protein) alpha-subunit belongs to the Ets family of transcription factors, whereas the beta-subunit shares homology with IkappaB and Drosophila Notch protein. GABP binding specificity to mutated N-box in vitro strictly parallels the sequence requirement for beta-galactosidase targeting to the endplate in vivo. In situ hybridization studies reveal that the mRNAs of both GABP subunits are abundant in mouse diaphragm, with preferential expression of the alpha-subunit at motor endplates. In addition, heregulin increases GABPalpha protein levels and regulates phosphorylation of both subunits in cultured chick myotubes. Finally, dominant-negative mutants of either GABPalpha or GABPbeta block heregulin-elicited transcriptional activation of the AChR delta and epsilon genes. These findings establish the expected connection with a presynaptic trophic factor whose release contributes to the accumulation of AChR subunit mRNAs at the motor endplate. PMID:9606190

  10. Transcriptional regulation of 15-lipoxygenase expression by histone h3 lysine 4 methylation/demethylation.

    PubMed

    Liu, Cheng; Xu, Dawei; Han, Hongya; Fan, Yidong; Schain, Frida; Xu, Zhonghua; Claesson, Hans-Erik; Björkholm, Magnus; Sjöberg, Jan

    2012-01-01

    15-Lipoxygenase-1 (15-LOX-1) oxidizes polyunsaturated fatty acids to a rich spectrum of biologically active metabolites and is implicated in physiological membrane remodelling, inflammation and apoptosis. Its deregulation is involved in the pathogenesis of diverse cancer and immune diseases. Recent experimental evidence reveals that dynamic histone methylation/demethylation mediated by histone methyltransferases and demethylases plays a critical role in regulation of chromatin remodelling and gene expression. In the present study, we compared the histone 3 lysine 4 (H3-K4) methylation status of the 15-LOX-1 promoter region of the two Hodgkin lymphoma (HL) cell lines L1236 and L428 with abundant and undetectable 15-LOX-1 expression, respectively. We identified a potential role of H3-K4 methylation in positive regulation of 15-LOX-1 transcription. Furthermore, we found that histone methyltransferase SMYD3 inhibition reduced 15-LOX-1 expression by decreasing promoter activity in L1236 cells. SMYD3 knock down in these cells abolished di-/trimethylation of H3-K4, attenuated the occupancy by the transactivator STAT6, and led to diminished histone H3 acetylation at the 15-LOX-1 promoter. In contrast, inhibition of SMCX, a JmjC-domain-containing H3-K4 tri-demethylase, upregulated 15-LOX-1 expression through induction of H3-K4 trimethylation, histone acetylation and STAT6 recruitment at the 15-LOX-1 promoter in L428 cells. In addition, we observed strong SMYD3 expression in the prostate cancer cell line LNCaP and its inhibition led to decreased 15-LOX-1 expression. Taken together, our data suggest that regulation of histone methylation/demethylation at the 15-LOX-1 promoter is important in 15-LOX-1 expression.

  11. Alternatively spliced transcripts of the thymus-specific protease PRSS16 are differentially expressed in human thymus.

    PubMed

    Luther, C; Wienhold, W; Oehlmann, R; Heinemann, M K; Melms, A; Tolosa, E

    2005-02-01

    The putative serine protease PRSS16 is abundantly expressed in the thymic cortex and the gene is encoded within the HLA I complex. Although its function is not yet defined, the very restricted expression points to a role in T-cell development in the thymus. In this study, we show that the PRSS16 mRNA is alternatively spliced to generate at least five transcripts. Apart from the full-length sequence, we found two other isoforms with all putative active site residues of the serine protease, suggesting that those variants may also be functional. Semi-quantitative analysis of the splice variants in different tissue samples revealed a strong correlation between the specific formation of alternatively spliced PRSS16 transcripts and the age and thymus pathology status of the donor. Newborn thymi express mostly the PRSS16-4 and -5 isoforms and lack the PRSS16-1 transcript, which appears around 2 years of age and stays until adulthood. Incidentally, thymi from myasthenia gravis (MG) patients with thymoma showed a marked decrease in the expression of the full-length PRSS16-1 and increased expression of the smaller isoforms. The data suggest a potential role of the PRSS16 isoforms in the postnatal morphogenesis of the thymus and in the thymus pathology related to MG.

  12. Congenital human thyroglobulin defect due to low expression of the thyroid-specific transcription factor TTF-1.

    PubMed Central

    Acebrón, A; Aza-Blanc, P; Rossi, D L; Lamas, L; Santisteban, P

    1995-01-01

    TTF-1 and Pax-8 are thyroid-specific transcription factors, from homeo and paired box genes, respectively, that are responsible for thyroid development and for thyroglobulin and thyroperoxidase gene expression. However, TTF-1 and Pax-8 preferentially bind to the thyroglobulin and thyroperoxidase promoters, respectively. Here, we have studied a patient with defective thyroglobulin synthesis. Thyroglobulin mRNA was found at very low levels while the mRNA for thyroperoxidase was found to be more abundant compared with control tissue. The low levels of thyroglobulin mRNA are caused by a transcriptional defect due to the virtual absence of TTF-1 expression as determined by Northern blot analysis, reverse transcriptase-PCR, and electrophoretic mobility shift assays. The level of Pax-8 mRNA was the same in the goiter and in the control thyroid. These results are the first reported evidence of a congenital goiter with a thyroglobulin synthesis defect due to the low expression of the thyroid-specific transcription factor TTF-1. Moreover, these data suggest that TTF-1 and Pax-8 would be differentially regulating thyroglobulin and thyroperoxidase gene transcription. Images PMID:7635972

  13. 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.

  14. Water-deficit-induced changes in transcription factor expression in maize seedlings.

    PubMed

    Seeve, Candace M; Cho, In-Jeong; Hearne, Leonard B; Srivastava, Gyan Prakash; Joshi, Trupti; Smith, Dante O; Sharp, Robert E; Oliver, Melvin J

    2016-12-31

    Plants tolerate water deficits by regulating gene networks controlling cellular and physiological traits to modify growth and development. Transcription factor (TF)-directed regulation of transcription within these gene networks is key to eliciting appropriate responses. In this study, reverse transcription quantitative PCR (RT-qPCR) was used to examine the abundance of 618 transcripts from 536 TF genes in individual root and shoot tissues of maize seedlings grown in vermiculite under well-watered (water potential of -0.02 MPa) and water-deficit conditions (water potentials of -0.3 and -1.6 MPa). A linear mixed model identified 433 TF transcripts representing 392 genes that differed significantly in abundance in at least one treatment, including TFs that intersect growth and development and environmental stress responses. TFs were extensively differentially regulated across stressed maize seedling tissues. Hierarchical clustering revealed TFs with stress-induced increased abundance in primary root tips that likely regulate root growth responses to water deficits, possibly as part of abscisic acid and/or auxin-dependent signaling pathways. Ten of these TFs were selected for validation in nodal root tips of drought-stressed field-grown plants (late V1 to early V2 stage). Changes in abundance of these TF transcripts under a field drought were similar to those observed in the seedling system.

  15. Changes in endogenous gene transcript and protein levels in maize plants expressing the soybean ferritin transgene

    PubMed Central

    Kanobe, Milly N.; Rodermel, Steven R.; Bailey, Theodore; Scott, M. Paul

    2013-01-01

    Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337) on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium, and iron. We have shown that the soybean ferritin transgene affected the expression of native iron homeostasis genes in the maize plant. These results underscore the importance of taking a holistic approach to the evaluation of transgenic events in target plants, comparing the transgenic plant to the untransformed controls. PMID:23785377

  16. Changes in endogenous gene transcript and protein levels in maize plants expressing the soybean ferritin transgene.

    PubMed

    Kanobe, Milly N; Rodermel, Steven R; Bailey, Theodore; Scott, M Paul

    2013-01-01

    Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337) on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium, and iron. We have shown that the soybean ferritin transgene affected the expression of native iron homeostasis genes in the maize plant. These results underscore the importance of taking a holistic approach to the evaluation of transgenic events in target plants, comparing the transgenic plant to the untransformed controls.

  17. A gene-expression-based neural code for food abundance that modulates lifespan

    PubMed Central

    Entchev, Eugeni V; Patel, Dhaval S; Zhan, Mei; Steele, Andrew J; Lu, Hang; Ch'ng, QueeLim

    2015-01-01

    How the nervous system internally represents environmental food availability is poorly understood. Here, we show that quantitative information about food abundance is encoded by combinatorial neuron-specific gene-expression of conserved TGFβ and serotonin pathway components in Caenorhabditis elegans. Crosstalk and auto-regulation between these pathways alters the shape, dynamic range, and population variance of the gene-expression responses of daf-7 (TGFβ) and tph-1 (tryptophan hydroxylase) to food availability. These intricate regulatory features provide distinct mechanisms for TGFβ and serotonin signaling to tune the accuracy of this multi-neuron code: daf-7 primarily regulates gene-expression variability, while tph-1 primarily regulates the dynamic range of gene-expression responses. This code is functional because daf-7 and tph-1 mutations bidirectionally attenuate food level-dependent changes in lifespan. Our results reveal a neural code for food abundance and demonstrate that gene expression serves as an additional layer of information processing in the nervous system to control long-term physiology. DOI: http://dx.doi.org/10.7554/eLife.06259.001 PMID:25962853

  18. A gene-expression-based neural code for food abundance that modulates lifespan.

    PubMed

    Entchev, Eugeni V; Patel, Dhaval S; Zhan, Mei; Steele, Andrew J; Lu, Hang; Ch'ng, QueeLim

    2015-05-12

    How the nervous system internally represents environmental food availability is poorly understood. Here, we show that quantitative information about food abundance is encoded by combinatorial neuron-specific gene-expression of conserved TGFβ and serotonin pathway components in Caenorhabditis elegans. Crosstalk and auto-regulation between these pathways alters the shape, dynamic range, and population variance of the gene-expression responses of daf-7 (TGFβ) and tph-1 (tryptophan hydroxylase) to food availability. These intricate regulatory features provide distinct mechanisms for TGFβ and serotonin signaling to tune the accuracy of this multi-neuron code: daf-7 primarily regulates gene-expression variability, while tph-1 primarily regulates the dynamic range of gene-expression responses. This code is functional because daf-7 and tph-1 mutations bidirectionally attenuate food level-dependent changes in lifespan. Our results reveal a neural code for food abundance and demonstrate that gene expression serves as an additional layer of information processing in the nervous system to control long-term physiology.

  19. Transcript profiling of expressed sequence tags from semimembranosus muscle of commercial and naturalized pig breeds.

    PubMed

    Nascimento, C S; Peixoto, J O; Verardo, L L; Campos, C F; Weller, M M C; Faria, V R; Botelho, M E; Martins, M F; Machado, M A; Silva, F F; Lopes, P S; Guimarães, S E F

    2012-09-17

    In general, genetic differences across different breeds of pig lead to variation in mature body size and slaughter age. The Commercial breeds Duroc and Large White and the local Brazilian breed Piau are ostensibly distinct in terms of growth and muscularity, commercial breeds are much leaner while local breeds grow much slower and are fat type pigs. However, the genetic factors that underlie such distinctions remain unclear. We used expressed sequence tags (ESTs) to characterize and compare transcript profiles in the semimembranosus muscle of these pig breeds. Our aim was to identify differences in breed-related gene expression that might influence growth performance and meat quality. We constructed three non-normalized cDNA libraries from semimembranosus muscle, using two samples from each one, of these three breeds; 6902 high-quality ESTs were obtained. Cluster analysis was performed and these sequences were clustered into 3670 unique sequences; 24.7% of the sequences were categorized as contigs and 75.3% of the sequences were singletons. Based on homology searches against the SwissProt protein database, we were able to assign a putative protein identity to only 1050 unique sequences. Among these, 58.5% were full-length protein sequences and 17.2% were pig-specific sequences. Muscle structural and cytoskeletal proteins, such as actin, and myosin, were the most abundant transcripts (16.7%) followed by those related to mitochondrial function (12.9%), and ribosomal proteins (12.4%). Furthermore, ESTs generated in this study provide a rich source for identification of novel genes and for the comparative analysis of gene expression patterns in divergent pig breeds.

  20. Abundantly and rarely expressed Lhc protein genes exhibit distinct regulation patterns in plants.

    PubMed

    Klimmek, Frank; Sjödin, Andreas; Noutsos, Christos; Leister, Dario; Jansson, Stefan

    2006-03-01

    We have analyzed gene regulation of the Lhc supergene family in poplar (Populus spp.) and Arabidopsis (Arabidopsis thaliana) using digital expression profiling. Multivariate analysis of the tissue-specific, environmental, and developmental Lhc expression patterns in Arabidopsis and poplar was employed to characterize four rarely expressed Lhc genes, Lhca5, Lhca6, Lhcb7, and Lhcb4.3. Those genes have high expression levels under different conditions and in different tissues than the abundantly expressed Lhca1 to 4 and Lhcb1 to 6 genes that code for the 10 major types of higher plant light-harvesting proteins. However, in some of the datasets analyzed, the Lhcb4 and Lhcb6 genes as well as an Arabidopsis gene not present in poplar (Lhcb2.3) exhibited minor differences to the main cooperative Lhc gene expression pattern. The pattern of the rarely expressed Lhc genes was always found to be more similar to that of PsbS and the various light-harvesting-like genes, which might indicate distinct physiological functions for the rarely and abundantly expressed Lhc proteins. The previously undetected Lhcb7 gene encodes a novel plant Lhcb-type protein that possibly contains an additional, fourth, transmembrane N-terminal helix with a highly conserved motif. As the Lhcb4.3 gene seems to be present only in Eurosid species and as its regulation pattern varies significantly from that of Lhcb4.1 and Lhcb4.2, we conclude it to encode a distinct Lhc protein type, Lhcb8.

  1. 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

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

    PubMed Central

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

    2012-01-01

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

  3. 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.

  4. 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.

  5. Abundance, diversity and functional gene expression of denitrifier communities in adjacent riparian and agricultural zones.

    PubMed

    Dandie, Catherine E; Wertz, Sophie; Leclair, Caissie L; Goyer, Claudia; Burton, David L; Patten, Cheryl L; Zebarth, Bernie J; Trevors, Jack T

    2011-07-01

    Lands under riparian and agricultural management differ in soil properties, water content, plant species and nutrient content and are therefore expected to influence denitrifier communities, denitrification and nitrous oxide (N(2) O) emissions. Denitrifier community abundance, denitrifier community structure, denitrification gene expression and activity were quantified on three dates in a maize field and adjacent riparian zone. N(2) O emissions were greater in the agricultural zone, whereas complete denitrification to N(2) was greater in the riparian zone. In general, the targeted denitrifier community abundance did not change between agricultural and riparian zones. However, nosZ gene expression was greater in the riparian zone than the agricultural zone. The community structure of nirS-gene-bearing denitrifiers differed in June only, whereas the nirK-gene-bearing community structure differed significantly between the riparian and the agricultural zones at all dates. The nirK-gene-bearing community structure was correlated with soil pH, while no significant correlations were found between nirS-gene-bearing community structure and soil environmental variables or N(2) O emissions, denitrification or denitrifier enzyme activity. The results suggested for the nirK and nirS-gene-bearing communities different factors control abundance vs. community structure. The nirK-gene-bearing community structure was also more responsive than the nirS-gene-bearing community structure to change between the two ecosystems.

  6. Dietary protein-related changes in hepatic transcription correspond to modifications in hepatic protein expression in growing pigs.

    PubMed

    Junghans, Peter; Kaehne, Thilo; Beyer, Manfred; Metges, Cornelia C; Schwerin, Manfred

    2004-01-01

    In a previous investigation we showed by expression profiling based on transcription analysis using differential display RT-PCR (DDRT-PCR) and real-time RT-PCR that a soy protein diet (SPI) significantly changes the hepatic transcription pattern compared with a casein diet (CAS). The present study was conducted to determine whether the transcriptional modulation is translated into protein expression. The hepatic mRNA abundance of four genes (EP24.16, LC3, NPAP60L, RFC2) that showed diet-related expression in previous DDRT-PCR experiments was analyzed by real-time RT-PCR. Two pigs that showed the most prominent SPI-related changes of transcription and two casein-fed pigs were selected and their hepatic protein pattern was studied comparatively by two-dimensional gel electrophoresis and peptide mass fingerprinting. The two-dimensional protein gel electrophoresis revealed a predominant SPI-associated upregulation of protein expression that corresponded to the results of the mRNA study. Of 380 diet-related protein spots displayed, 215 appeared exclusively or enlarged in the two SPI pigs; 10 of 39 diet-related expressed protein spots extracted could be identified by peptide mass fingerprinting and database search. Compared with the transcriptomics approach, the proteomics approach led in part to the identification of the same diet-associated expressed molecules (plasminogen, trypsin, phospholipase A2, glutathione-S-transferase alpha, retinal binding protein) or at least molecules belonging to the same metabolic pathways (protein and amino acid metabolism, oxidative stress response, lipid metabolism). The present results at the proteome level confirm SPI-related increased oxidative stress response and significant effects on protein biosynthesis already observed at the transcriptome level.

  7. 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

  8. The abundance of processed pseudogenes derived from glycolytic genes is correlated with their expression level.

    PubMed

    McDonell, Laura; Drouin, Guy

    2012-02-01

    The abundance of processed pseudogenes in different vertebrate species is known to be proportional to the length of their oogenesis. However, this hypothesis cannot explain why, in a given species, certain genes produce more processed pseudogenes than others. In particular, one would expect that all genes of the glycolytic pathway would generate roughly the same number of processed pseudogenes. However, some glycolitic genes generate more processed pseudogenes than others. Here, we show that there is a positive correlation between the abundance of processed pseudogene generated from glycolytic genes and their level of expression. The variation in expression level of different glycolytic genes likely reflects the fact that some of them, such a GAPDH, have functions other than those they play in glycolysis. Furthermore, the age distribution of GAPDH-processed pseudogenes corresponds to the age distribution of LINE1 elements, which are the source of the reverse transcriptase that generates processed pseudogenes. These results support the hypothesis that gene expression levels affect the level of processed pseudogene production.

  9. 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.

  10. 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

  11. 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

  12. 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

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

    2016-09-01

    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.

  13. Agouti Revisited: Transcript Quantification of the ASIP Gene in Bovine Tissues Related to Protein Expression and Localization

    PubMed Central

    Albrecht, Elke; Komolka, Katrin; Kuzinski, Judith; Maak, Steffen

    2012-01-01

    Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species. PMID:22530003

  14. 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.

  15. 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.

  16. Mining SAGE data allows large-scale, sensitive screening of antisense transcript expression.

    PubMed

    Quéré, Ronan; Manchon, Laurent; Lejeune, Mireille; Clément, Oliver; Pierrat, Fabien; Bonafoux, Béatrice; Commes, Thérèse; Piquemal, David; Marti, Jacques

    2004-11-23

    As a growing number of complementary transcripts, susceptible to exert various regulatory functions, are being found in eukaryotes, high throughput analytical methods are needed to investigate their expression in multiple biological samples. Serial Analysis of Gene Expression (SAGE), based on the enumeration of directionally reliable short cDNA sequences (tags), is capable of revealing antisense transcripts. We initially detected them by observing tags that mapped on to the reverse complement of known mRNAs. The presence of such tags in individual SAGE libraries suggested that SAGE datasets contain latent information on antisense transcripts. We raised a collection of virtual tags for mining these data. Tag pairs were assembled by searching for complementarities between 24-nt long sequences centered on the potential SAGE-anchoring sites of well-annotated human expressed sequences. An analysis of their presence in a large collection of published SAGE libraries revealed transcripts expressed at high levels from both strands of two adjacent, oppositely oriented, transcription units. In other cases, the respective transcripts of such cis-oriented genes displayed a mutually exclusive expression pattern or were co-expressed in a small number of libraries. Other tag pairs revealed overlapping transcripts of trans-encoded unique genes. Finally, we isolated a group of tags shared by multiple transcripts. Most of them mapped on to retroelements, essentially represented in humans by Alu sequences inserted in opposite orientations in the 3'UTR of otherwise different mRNAs. Registering these tags in separate files makes possible computational searches focused on unique sense-antisense pairs. The method developed in the present work shows that SAGE datasets constitute a major resource of rapidly investigating with high sensitivity the expression of antisense transcripts, so that a single tag may be detected in one library when screening a large number of biological samples.

  17. Cloning and expression of a transcription factor activator protein-1 (AP-1) member identified from manila clam Venerupis philippinarum.

    PubMed

    Wu, Luning; Zhang, Lei; Zhao, Jianmin; Ning, Xuanxuan; Mu, Changkao; Wang, Chunlin

    2015-02-15

    The transcription factor activator protein-1 (AP-1) proteins are implicated to play a major role in the regulation of numerous genes involved in the function and development of the immune system, cell differentiation, proliferation, apoptosis, etc. It can bind to promoter of its target genes in a sequence-specific manner to transactivate or repress them. In this study, the full-length cDNA of an AP-1 was identified from Venerupis philippinarum (denoted as VpAP-1) by EST analysis and RACE approaches. Phylogenetic analysis indicated that VpAP-1 had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the AP-1 protein family. Spatial expression analysis found that VpAP-1 transcript was most abundantly expressed in the hemocytes and hepatopancreas, weakly expressed in the tissues of the gills, mantle and muscle. After Vibrio anguillarum challenge, the expression of VpAP-1 transcript in overall hemocyte population was up-regulated in the first 6h, and then decreased to 1.5-fold of the control group at 12h. As time progressed, a second peak of VpAP-1 expression was detected at 24h post-infection, which was 5-fold compared with that of the control group (P<0.01). After that, the expression level was sharply decreased and dropped to 0.5-fold of the control at 96h. The above results indicated that VpAP-1 was perhaps involved in the immune responses against microbe infection and might be contributed to the clearance of bacterial pathogens.

  18. SOLiD-SAGE of Endophyte-Infected Red Fescue Reveals Numerous Effects on Host Transcriptome and an Abundance of Highly Expressed Fungal Secreted Proteins

    PubMed Central

    Ambrose, Karen V.; Belanger, Faith C.

    2012-01-01

    One of the most important plant-fungal symbiotic relationships is that of cool season grasses with endophytic fungi of the genera Epichloë and Neotyphodium. These associations often confer benefits, such as resistance to herbivores and improved drought tolerance, to the hosts. One benefit that appears to be unique to fine fescue grasses is disease resistance. As a first step towards understanding the basis of the endophyte-mediated disease resistance in Festuca rubra we carried out a SOLiD-SAGE quantitative transcriptome comparison of endophyte-free and Epichloë festucae-infected F. rubra. Over 200 plant genes involved in a wide variety of physiological processes were statistically significantly differentially expressed between the two samples. Many of the endophyte expressed genes were surprisingly abundant, with the most abundant fungal tag representing over 10% of the fungal mapped tags. Many of the abundant fungal tags were for secreted proteins. The second most abundantly expressed fungal gene was for a secreted antifungal protein and is of particular interest regarding the endophyte-mediated disease resistance. Similar genes in Penicillium and Aspergillus spp. have been demonstrated to have antifungal activity. Of the 10 epichloae whole genome sequences available, only one isolate of E. festucae and Neotyphodium gansuense var inebrians have an antifungal protein gene. The uniqueness of this gene in E. festucae from F. rubra, its transcript abundance, and the secreted nature of the protein, all suggest it may be involved in the disease resistance conferred to the host, which is a unique feature of the fine fescue–endophyte symbiosis. PMID:23285269

  19. SOLiD-SAGE of endophyte-infected red fescue reveals numerous effects on host transcriptome and an abundance of highly expressed fungal secreted proteins.

    PubMed

    Ambrose, Karen V; Belanger, Faith C

    2012-01-01

    One of the most important plant-fungal symbiotic relationships is that of cool season grasses with endophytic fungi of the genera Epichloë and Neotyphodium. These associations often confer benefits, such as resistance to herbivores and improved drought tolerance, to the hosts. One benefit that appears to be unique to fine fescue grasses is disease resistance. As a first step towards understanding the basis of the endophyte-mediated disease resistance in Festuca rubra we carried out a SOLiD-SAGE quantitative transcriptome comparison of endophyte-free and Epichloë festucae-infected F. rubra. Over 200 plant genes involved in a wide variety of physiological processes were statistically significantly differentially expressed between the two samples. Many of the endophyte expressed genes were surprisingly abundant, with the most abundant fungal tag representing over 10% of the fungal mapped tags. Many of the abundant fungal tags were for secreted proteins. The second most abundantly expressed fungal gene was for a secreted antifungal protein and is of particular interest regarding the endophyte-mediated disease resistance. Similar genes in Penicillium and Aspergillus spp. have been demonstrated to have antifungal activity. Of the 10 epichloae whole genome sequences available, only one isolate of E. festucae and Neotyphodium gansuense var inebrians have an antifungal protein gene. The uniqueness of this gene in E. festucae from F. rubra, its transcript abundance, and the secreted nature of the protein, all suggest it may be involved in the disease resistance conferred to the host, which is a unique feature of the fine fescue-endophyte symbiosis.

  20. Transcriptional regulation of fucosyltransferase 1 gene expression in colon cancer cells.

    PubMed

    Taniuchi, Fumiko; Higai, Koji; Tanaka, Tomomi; Azuma, Yutaro; Matsumoto, Kojiro

    2013-01-01

    The α 1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses the FUT1 gene, and shows α 1,2-fucosyltransferase (FUT) activity. 5'-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site -10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay, FUT1 gene expression was shown to be regulated at the region -91 to -81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation of FUT1 gene expression in DLD-1 cells. These results suggest that a defined region in the 5'-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression of FUT1 is regulated by Elk-1 in DLD-1 cells.

  1. 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

  2. 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...

  3. 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...

  4. 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.

  5. Foxp transcription factors suppress a non-pulmonary gene expression program to permit proper lung development.

    PubMed

    Li, Shanru; Morley, Michael; Lu, MinMin; Zhou, Su; Stewart, Kathleen; French, Catherine A; Tucker, Haley O; Fisher, Simon E; Morrisey, Edward E

    2016-08-15

    The inhibitory mechanisms that prevent gene expression programs from one tissue to be expressed in another are poorly understood. Foxp1/2/4 are forkhead transcription factors that repress gene expression and are individually important for endoderm development. We show that combined loss of all three Foxp1/2/4 family members in the developing anterior foregut endoderm leads to a loss of lung endoderm lineage commitment and subsequent development. Foxp1/2/4 deficient lungs express high levels of transcriptional regulators not normally expressed in the developing lung, including Pax2, Pax8, Pax9 and the Hoxa9-13 cluster. Ectopic expression of these transcriptional regulators is accompanied by decreased expression of lung restricted transcription factors including Nkx2-1, Sox2, and Sox9. Foxp1 binds to conserved forkhead DNA binding sites within the Hoxa9-13 cluster, indicating a direct repression mechanism. Thus, Foxp1/2/4 are essential for promoting lung endoderm development by repressing expression of non-pulmonary transcription factors.

  6. Effects of porcine oocytes on the expression levels of transcripts encoding glycolytic enzymes in granulosa cells.

    PubMed

    Matsuno, Yuta; Onuma, Asuka; Fujioka, Yoshie A; Emori, Chihiro; Fujii, Wataru; Naito, Kunihiko; Sugiura, Koji

    2016-09-01

    Oocytes play critical roles in regulating the expression of transcripts encoding the glycolytic enzymes phosphofructokinase, platelet (PFKP) and lactate dehydrogenase A (LDHA) in granulosa cells in mice, but whether this is the case in pigs or other mammals has not been adequately investigated. Therefore, the aim of this study was to determine whether porcine oocytes regulate the expression levels of these transcripts in granulosa cells in vitro. Porcine cumulus cells expressed higher levels of PFKP and LDHA transcripts than mural granulosa cells (MGCs). However, co-culturing with oocytes had no significant effect on the isolated cumulus cells. While murine oocytes promoted the expression of both Pfkp and Ldha transcripts by murine MGCs, porcine oocytes promoted the expression of only Pfkp, but not Ldha transcripts by murine MGCs. Neither murine nor porcine oocytes affected PFKP and LDHA expression by porcine MGCs. Moreover, in the presence of porcine follicular fluid, porcine oocytes maintained the expression of PFKP, but not LDHA by porcine cumulus cells. Therefore, porcine oocytes are capable of regulating the expression of PFKP but not LDHA in granulosa cells in coordination with unknown factor(s) present in the follicular fluid.

  7. 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.

  8. Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene

    PubMed Central

    Yeganeh, Meghdad; Praz, Viviane; Cousin, Pascal; Hernandez, Nouria

    2017-01-01

    Overlapping gene arrangements can potentially contribute to gene expression regulation. A mammalian interspersed repeat (MIR) nested in antisense orientation within the first intron of the Polr3e gene, encoding an RNA polymerase III (Pol III) subunit, is conserved in mammals and highly occupied by Pol III. Using a fluorescence assay, CRISPR/Cas9-mediated deletion of the MIR in mouse embryonic stem cells, and chromatin immunoprecipitation assays, we show that the MIR affects Polr3e expression through transcriptional interference. Our study reveals a mechanism by which a Pol II gene can be regulated at the transcription elongation level by transcription of an embedded antisense Pol III gene. PMID:28289142

  9. Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene.

    PubMed

    Yeganeh, Meghdad; Praz, Viviane; Cousin, Pascal; Hernandez, Nouria

    2017-02-15

    Overlapping gene arrangements can potentially contribute to gene expression regulation. A mammalian interspersed repeat (MIR) nested in antisense orientation within the first intron of the Polr3e gene, encoding an RNA polymerase III (Pol III) subunit, is conserved in mammals and highly occupied by Pol III. Using a fluorescence assay, CRISPR/Cas9-mediated deletion of the MIR in mouse embryonic stem cells, and chromatin immunoprecipitation assays, we show that the MIR affects Polr3e expression through transcriptional interference. Our study reveals a mechanism by which a Pol II gene can be regulated at the transcription elongation level by transcription of an embedded antisense Pol III gene.

  10. 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.

  11. Expression of mRNA encoding the macrophage colony-stimulating factor receptor (c-fms) is controlled by a constitutive promoter and tissue-specific transcription elongation.

    PubMed Central

    Yue, X; Favot, P; Dunn, T L; Cassady, A I; Hume, D A

    1993-01-01

    The gene encoding the receptor for macrophage colony-stimulating factor 1 (CSF-1), the c-fms protooncogene, is selectively expressed in immature and mature mononuclear phagocytes and trophoblasts. Exon 1 is expressed only in trophoblasts. Isolation and sequencing of genomic DNA flanking exon 2 of the murine c-fms gene revealed a TATA-less promoter with significant homology to human c-fms. Reverse transcriptase primer extension analysis using exon 2 primers identified multiple clustered transcription initiation sites. Their position was confirmed by RNase protection. The same primer extension products were detected in equal abundance from macrophage or nonmacrophage sources of RNA. c-fms mRNA is acutely down-regulated in primary macrophages by CSF-1, bacterial lipopolysaccharide (LPS), and phorbol myristate acetate (PMA). Each of these agents reduced the abundance of c-fms RNA detectable by primer extension using an exon 3 primer without altering the abundance of presumptive short c-fms transcripts detected with exon 2 primers. Primer extension analysis with an intron 2 primer detected products at greater abundance in nonmacrophages. Templates detected with the intronic primer were induced in macrophages by LPS, PMA, and CSF-1, suggesting that each of the agents caused a shift from full-length c-fms mRNA production to production of unspliced, truncated transcripts. The c-fms promoter functioned constitutively in the RAW264 macrophage cell line, the B-cell line MOPC.31C, and several nonhematopoietic cell lines. Macrophage-specific expression and responsiveness to selective repression by LPS and PMA was achieved by the incorporation of intron 2 into the c-fms promoter-reporter construct. The results suggest that expression of the c-fms gene in macrophages is controlled by sequences in intron 2 that act by regulating transcription elongation. Images PMID:8497248

  12. Ovarian dysgerminomas are characterised by frequent KIT mutations and abundant expression of pluripotency markers

    PubMed Central

    Hoei-Hansen, Christina E; Kraggerud, Sigrid M; Abeler, Vera M; Kærn, Janne; Rajpert-De Meyts, Ewa; Lothe, Ragnhild A

    2007-01-01

    Background Ovarian germ cell tumours (OGCTs) typically arise in young females and their pathogenesis remains poorly understood. We investigated the origin of malignant OGCTs and underlying molecular events in the development of the various histological subtypes of this neoplasia. Results We examined in situ expression of stem cell-related (NANOG, OCT-3/4, KIT, AP-2γ) and germ cell-specific proteins (MAGE-A4, NY-ESO-1, TSPY) using a tissue microarray consisting of 60 OGCT tissue samples and eight ovarian small cell carcinoma samples. Developmental pattern of expression of NANOG, TSPY, NY-ESO-1 and MAGE-A4 was determined in foetal ovaries (gestational weeks 13–40). The molecular genetic part of our study included search for the presence of Y-chromosome material by fluorescence in situ hybridisation (FISH), and mutational analysis of the KIT oncogene (exon 17, codon 816), which is often mutated in testicular GCTs, in a subset of tumour DNA samples. We detected a high expression of transcription factors related to the embryonic stem cell-like pluripotency and undifferentiated state in OGCTs, but not in small cell carcinomas, supporting the view that the latter do not arise from a germ cell progenitor. Bilateral OGCTs expressed more stem cell markers than unilateral cases. However, KIT was mutated in 5/13 unilateral dysgerminomas, whereas all bilateral dysgerminomas (n = 4) and all other histological types (n = 22) showed a wild type sequence. Furthermore, tissue from five phenotypic female patients harbouring combined dysgerminoma/gonadoblastoma expressed TSPY and contained Y-chromosome material as confirmed by FISH. Conclusion This study provides new data supporting two distinct but overlapping pathways in OGCT development; one involving spontaneous KIT mutation(s) leading to increased survival and proliferation of undifferentiated oogonia, the other related to presence of Y chromosome material and ensuing gonadal dysgenesis in phenotypic females. PMID:17274819

  13. 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

  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. Integrated pathway-based transcription regulation network mining and visualization based on gene expression profiles.

    PubMed

    Kibinge, Nelson; Ono, Naoaki; Horie, Masafumi; Sato, Tetsuo; Sugiura, Tadao; Altaf-Ul-Amin, Md; Saito, Akira; Kanaya, Shigehiko

    2016-06-01

    Conventionally, workflows examining transcription regulation networks from gene expression data involve distinct analytical steps. There is a need for pipelines that unify data mining and inference deduction into a singular framework to enhance interpretation and hypotheses generation. We propose a workflow that merges network construction with gene expression data mining focusing on regulation processes in the context of transcription factor driven gene regulation. The pipeline implements pathway-based modularization of expression profiles into functional units to improve biological interpretation. The integrated workflow was implemented as a web application software (TransReguloNet) with functions that enable pathway visualization and comparison of transcription factor activity between sample conditions defined in the experimental design. The pipeline merges differential expression, network construction, pathway-based abstraction, clustering and visualization. The framework was applied in analysis of actual expression datasets related to lung, breast and prostrate cancer.

  16. Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes

    PubMed Central

    van der Does, H. Charlotte; Schmidt, Sarah M.; Langereis, Léon; Hughes, Timothy R.

    2016-01-01

    Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called ‘effectors’. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the ‘pathogenicity’ chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol

  17. Introns and gene expression: Cellular constraints, transcriptional regulation, and evolutionary consequences

    PubMed Central

    Heyn, Patricia; Kalinka, Alex T; Tomancak, Pavel; Neugebauer, Karla M

    2015-01-01

    A gene's “expression profile” denotes the number of transcripts present relative to all other transcripts. The overall rate of transcript production is determined by transcription and RNA processing rates. While the speed of elongating RNA polymerase II has been characterized for many different genes and organisms, gene-architectural features – primarily the number and length of exons and introns – have recently emerged as important regulatory players. Several new studies indicate that rapidly cycling cells constrain gene-architecture toward short genes with a few introns, allowing efficient expression during short cell cycles. In contrast, longer genes with long introns exhibit delayed expression, which can serve as timing mechanisms for patterning processes. These findings indicate that cell cycle constraints drive the evolution of gene-architecture and shape the transcriptome of a given cell type. Furthermore, a tendency for short genes to be evolutionarily young hints at links between cellular constraints and the evolution of animal ontogeny. PMID:25400101

  18. Trypanosoma brucei: Enrichment by UV of intergenic transcripts from the variable surface glycoprotein gene expression site

    SciTech Connect

    Coquelet, H.; Tebabi, P.; Pays, A.; Steinert, M.; Pays, E. )

    1989-09-01

    The expression site for the variable surface glycoprotein (VSG) gene AnTat 1.3A of Trypanosoma brucei is 45 kilobases long and encompasses seven expression site-associated genes (ESAGs). After UV irradiation, several large transcripts from the putative promoter region were strongly enriched. We report that one such major transcript starts near the poly(A) addition site of the first gene (ESAG 7), spans the intergenic region, and extends to the poly(A) addition site of the second gene (ESAG 6), thus bypassing the normal 3' splice site of the ESAG 6 mRNA. Since this transcript is spliced, we conclude that UV irradiation does not inhibit splicing but stabilizes unstable processing products. This demonstrates that at least some intergenic regions of the VSG gene expression site are continuously transcribed in accordance with a polycistronic transcription model.

  19. Identification of sequences regulating the transcription of a Dictyostelium gene selectively expressed in prespore cells.

    PubMed Central

    Early, A E; Williams, J G

    1989-01-01

    There has been considerable debate about the relative contributions of transcriptional and post-transcriptional mechanisms to the regulation of prespore gene expression in Dictyostelium. We have determined the DNA sequence upstream of D19, the Dictyostelium gene encoding PsA, a prespore-specific, cell surface protein of unknown function. Our analysis of gene fusions, in which D19 upstream sequences are placed adjacent to a heterologous reporter gene, indicates that transcriptional signals alone are sufficient for the correct temporal and cell-type specific expression of this gene. We also show that the 5' and 3' boundaries of the minimal sequences necessary for correct developmental regulation lie within the region 338 to 122 nucleotides upstream of the start site of transcription but that flanking sequences seem to be necessary for optimal expression. Images PMID:2550894

  20. Expression of E2F transcription factor family genes during chick wing development.

    PubMed

    Towers, Matthew; Fisunov, Gleb; Tickle, Cheryll

    2009-10-01

    The E2F family of transcriptional regulators activate or repress gene expression during specific phases of the cell cycle and control various processes including proliferation, apoptosis and differentiation. However, little is known about the developmental roles of E2F transcription factors in higher vertebrates. The chick wing is an excellent system for studying these processes because, in addition to having a rich classical embryology, it is increasingly amenable to molecular and genomic approaches. We show that the human and mouse complement of eight E2F transcription factors is conserved in the chicken and that chicken E2F genes are expressed in different spatial and temporal patterns during wing development. We discuss how the expression patterns of the eight chicken E2F transcription factors might be related to important morphogenetic events.

  1. Ikaros family transcription factors expression in rat thymus: detection of impaired development.

    PubMed

    Paradzik, M; Novak, S; Mokrovic, G; Bordukalo Niksic, T; Heckel, D; Stipic, J; Pavicic Baldani, D; Cicin-Sain, L; Antica, M

    2012-01-01

    The expression of Ikaros family transcription factors and consequently their signalling pathway is limiting for hematopoietic and lymphocyte development in mice and human. Due to their importance, these transcription factors are highly homologous between species. As an initial approach to examining the possible involvement of Ikaros transcription factors in pathogenesis of rat lymphoid development, we analyzed the expression of all known Ikaros family members, Ikaros, Aiolos, Helios, Eos and Pegasus in the rat thymus. We established a semi-quantitative RT-PCR to detect mRNA of each transcription factor. For the first time we give evidence of the expression of Ikaros family transcription factors in the rat thymus. Further, we evaluated whether their mRNA expression was succumbed to changes when the rats were exposed to ethanol, as a known debilitating agent during development. Therefore we analyzed the thymus of adult rats whose mothers were forced to drink ethanol during gestation, to detect possible changes in thymus mRNA expression levels of Ikaros, Aiolos, Helios, Eos and Pegasus. We found that rats prenatally exposed to ethanol show a slightly higher expression of Ikaros family transcription factors in the adult thymus when compared to control rats, but these differences were not statistically significant. We further studied the distribution of the major lymphocyte subpopulations in the rat thymus according to CD3, CD4 and CD8 expression by four color flow cytometry. We found a higher incidence of CD3 positive cells in the double positive, CD4+CD8+ thymic subpopulation of rats prenatally exposed to ethanol when compared to non-exposed animals. Our findings indicate that ethanol exposure of pregnant rats might influence the development of CD3 positive cells in the thymus of the offspring but this result should be further tackled at the level of transcription factor expression.

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

    PubMed Central

    Barker, Brandon E.; Smallbone, Kieran; Myers, Christopher R.; Xi, Hongwei; Locasale, Jason W.; Gu, Zhenglong

    2015-01-01

    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

  3. Transcription factor co-localization patterns affect human cell type-specific gene expression

    PubMed Central

    2012-01-01

    Background Cellular development requires the precise control of gene expression states. Transcription factors are involved in this regulatory process through their combinatorial binding with DNA. Information about transcription factor binding sites can help determine which combinations of factors work together to regulate a gene, but it is unclear how far the binding data from one cell type can inform about regulation in other cell types. Results By integrating data on co-localized transcription factor binding sites in the K562 cell line with expression data across 38 distinct hematopoietic cell types, we developed regression models to describe the relationship between the expression of target genes and the transcription factors that co-localize nearby. With K562 binding sites identifying the predictors, the proportion of expression explained by the models is statistically significant only for monocytic cells (p-value< 0.001), which are closely related to K562. That is, cell type specific binding patterns are crucial for choosing the correct transcription factors for the model. Comparison of predictors obtained from binding sites in the GM12878 cell line with those from K562 shows that the amount of difference between binding patterns is directly related to the quality of the prediction. By identifying individual genes whose expression is predicted accurately by the binding sites, we are able to link transcription factors FOS, TAF1 and YY1 to a sparsely studied gene LRIG2. We also find that the activity of a transcription factor may be different depending on the cell type and the identity of other co-localized factors. Conclusion Our approach shows that gene expression can be explained by a modest number of co-localized transcription factors, however, information on cell-type specific binding is crucial for understanding combinatorial gene regulation. PMID:22721266

  4. A transcription map in the CATCH22 critical region: identification, mapping, and ordering of four novel transcripts expressed in heart.

    PubMed

    Lindsay, E A; Rizzu, P; Antonacci, R; Jurecic, V; Delmas-Mata, J; Lee, C C; Kim, U J; Scambler, P J; Baldini, A

    1996-02-15

    The acronym CATCH22 is used to indicate collectively a group of related phenotypes, namely velocardiofacial syndrome (VCFS), DiGeorge anomaly (DGA), and conotruncal anomaly face, which are associated with deletions within 22q11.2 in the great majority of patients. A deletion map has allowed us to delimit a smallest region of deletion overlap, considerably smaller than the commonly deleted region. We have mapped within this region the chromosomal breakpoint of a balanced translocation patient presenting with a DGA/VCFS phenotype, making this region the strongest candidate for the location of the gene(s) responsible for the disease phenotype. We report a systematic gene search in this region and show the presence of at least six distinct transcripts, two of which have been previously described. The region searched was approximately 270 kb; therefore, an average of one transcript every 45 kb was found. We generated eight new ESTs and mapped two ESTs present in public databases. All six transcripts are expressed in heart, an organ involved in 70%-80% of CATCH22 patients. We show that the multimethod approach to search for expressed sequences is effective and indeed necessary for a comprehensive search and provides molecular tools for further characterization of the potential genes identified.

  5. 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.

  6. Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise.

    PubMed

    Mendell, Joshua T; Sharifi, Neda A; Meyers, Jennifer L; Martinez-Murillo, Francisco; Dietz, Harry C

    2004-10-01

    Premature termination codons induce rapid transcript degradation in eukaryotic cells through nonsense-mediated mRNA decay (NMD). This pathway can modulate phenotypes arising from nonsense or frameshift mutations, but little is known about the physiologic role of NMD in higher eukaryotes. To address this issue, we examined expression profiles in mammalian cells depleted of Rent1 (also called hUpf1), a factor essential for NMD. Upregulated transcripts included those with upstream open reading frames in the 5' untranslated region, alternative splicing that introduces nonsense codons or frameshifts, introns in the 3' untranslated region or selenocysteine codons. Transcripts derived from ancient transposons and endogenous retroviruses were also upregulated. These RNAs are unified by the presence of a spliced intron at least 50 nucleotides downstream of a termination codon, a context sufficient to initiate NMD. Consistent with direct regulation by NMD, representative upregulated transcripts decayed more slowly in cells deficient in NMD. In addition, inhibition of NMD induced by amino acid starvation upregulated transcripts that promote amino acid homeostasis. These results document that nonsense surveillance is a crucial post-transcriptional regulatory event that influences the expression of broad classes of physiologic transcripts, has been functionally incorporated into essential homeostatic mechanisms and suppresses expression of evolutionary remnants.

  7. The transcription factor ultraspiracle influences honey bee social behavior and behavior-related gene expression.

    PubMed

    Ament, Seth A; Wang, Ying; 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.

  8. 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

  9. 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.

  10. A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei.

    PubMed

    Navarro, M; Gull, K

    2001-12-13

    In the mammalian host, African trypanosomes generate consecutive waves of parasitaemia by changing their antigenic coat. Because this coat consists of a single type of variant surface glycoprotein (VSG), the question arises of how a trypanosome accomplishes the transcription of only one of a multi-allelic family of VSG expression site loci to display a single VSG type on the surface at any one time. No major differences have been detected between the single active expression site and the cohort of inactive expression sites. Here we identify an extranucleolar body containing RNA polymerase I (pol I) that is transcriptionally active and present only in the bloodstream form of the parasite. Visualization of the active expression site locus by tagging with green fluorescent protein shows that it is specifically located at this unique pol I transcriptional factory. The presence of this transcriptional body in postmitotic nuclei and its stability in the nucleus after DNA digestion provide evidence for a coherent structure. We propose that the recruitment of a single expression site and the concomitant exclusion of inactive loci from a discrete transcriptional body define the mechanism responsible for VSG mono-allelic expression.

  11. 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

    2015-05-11

    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.

  12. TRANSLUCENT GREEN, an ERF family transcription factor, controls water balance in Arabidopsis by activating the expression of aquaporin genes.

    PubMed

    Zhu, Danling; Wu, Zhe; Cao, Guangyu; Li, Jigang; Wei, Jia; Tsuge, Tomohiko; Gu, Hongya; Aoyama, Takashi; Qu, Li-Jia

    2014-04-01

    Water is the most abundant molecule in almost all living organisms. Aquaporins are channel proteins that play critical roles in controlling the water content of cells. Here, we report the identification of an AP2/EREBP family transcription factor in Arabidopsis thaliana, TRANSLUCENT GREEN (TG), whose overexpression in transgenic plants gave enhanced drought tolerance and vitrified leaves. TG protein is localized in the nucleus, binds DRE and GCC elements in vitro, and acts as a transcriptional activator in yeast cells. Microarray analysis revealed a total of 330 genes regulated by TG, among which five genes encode aquaporins. A transient expression assay showed that TG directly binds to the promoters of three aquaporin genes, such as AtTIP1;1, AtTIP2;3, and AtPIP2;2, indicating that TG directly regulates the expression of these genes. Moreover, overexpression of AtTIP1;1 resulted in vitrified phenotypes in transgenic Arabidopsis plants, similar to those observed in TG overexpression lines. Water injection into wild-type leaves recapitulated the vitrified leaf phenotypes, which was reversed by cutting off the water supply from vascular bundles. Taken together, our data support that TG controls water balance in Arabidopsis through directly activating the expression of aquaporin genes.

  13. 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

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

    PubMed

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

    2016-06-27

    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.

  15. 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

  16. Control of transferrin expression by β-amyloid through the CP2 transcription factor.

    PubMed

    Jang, Sang-Min; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Kang, Eun-Jin; Kim, Chul Geun; Kim, Hyun-Jung; Choi, Kyung-Hee

    2010-10-01

    Accumulation of β-amyloid protein (Aβ) is one of the most important pathological features of Alzheimer's disease. Although Aβ induces neurodegeneration in the cortex and hippocampus through several molecular mechanisms, few studies have evaluated the modulation of transcription factors during Aβ-induced neurotoxicity. Therefore, in this study, we investigated the transcriptional activity of transcription factor CP2 in neuronal damage mediated by Aβ (Aβ(1-42) and Aβ(25-35) ). An unbiased motif search of the transferrin promoter region showed that CP2 binds to the transferrin promoter, an iron-regulating protein, and regulates transferrin transcription. Ectopic expression of CP2 led to increased transferrin expression at both the mRNA and protein levels, whereas knockdown of CP2 down-regulated transferrin mRNA and protein expression. Moreover, CP2 trans-activated transcription of a transferrin reporter gene. An electrophoretic mobility shift assay and a chromatin immunoprecipitation assay showed that CP2 binds to the transferrin promoter region. Furthermore, the binding affinity of CP2 to the transferrin promoter was regulated by Aβ, as Aβ (Aβ(1-42) and Aβ(25-35) ) markedly increased the binding affinity of CP2 for the transferrin promoter. Taken together, these results suggest that CP2 contributes to the pathogenesis of Alzheimer's disease by inducing transferrin expression via up-regulating its transcription.

  17. GLUT4, GLUT1, and GLUT8 are the dominant GLUT transcripts expressed in the murine left ventricle

    PubMed Central

    2012-01-01

    Background The heart derives energy from a wide variety of substrates including fatty acids, carbohydrates, ketones, and amino acids. The healthy heart generates up to 30% of its ATP from glucose. Under conditions of cardiac injury or stress, the heart relies even more heavily on glucose as a source of fuel. Glucose is transported into the heart by members of the family of facilitative glucose transporters (GLUTs). While research examining the transport of glucose into the heart has primarily focused on the roles of the classical glucose transporters GLUT1 and GLUT4, little is known about the functions of more newly identified GLUT isoforms in the myocardium. Methods In this study the presence and relative RNA message abundance of each of the known GLUT isoforms was determined in left ventricular tissue from two commonly used inbred laboratory mouse strains (C57BL/6J and FVB/NJ) by quantitative real time PCR. Relative message abundance was also determined in GLUT4 null mice and in murine models of dilated and hypertrophic cardiomyopathy. Results GLUT4, GLUT1, and GLUT8 were found to be the most abundant GLUT transcripts in the normal heart, while GLUT3, GLUT10, and GLUT12 are present at relatively lower levels. Assessment of relative GLUT expression in left ventricular myocardium from mice with dilated cardiomyopathy revealed increased expression of GLUT1 with reduced levels of GLUT4, GLUT8, and GLUT12. Compensatory increase in the expression of GLUT12 was observed in genetically altered mice lacking GLUT4. Conclusions Glucose transporter expression varies significantly among murine models of cardiac dysfunction and involves several of the class III GLUT isoforms. Understanding how these more newly identified GLUT isoforms contribute to regulating myocardial glucose transport will enhance our comprehension of the normal physiology and pathophysiology of the heart. PMID:22681646

  18. Polyester: simulating RNA-seq datasets with differential transcript expression

    PubMed Central

    Frazee, Alyssa C.; Jaffe, Andrew E.; Langmead, Ben; Leek, Jeffrey T.

    2015-01-01

    Motivation: Statistical methods development for differential expression analysis of RNA sequencing (RNA-seq) requires software tools to assess accuracy and error rate control. Since true differential expression status is often unknown in experimental datasets, artificially constructed datasets must be utilized, either by generating costly spike-in experiments or by simulating RNA-seq data. Results: Polyester is an R package designed to simulate RNA-seq data, beginning with an experimental design and ending with collections of RNA-seq reads. Its main advantage is the ability to simulate reads indicating isoform-level differential expression across biological replicates for a variety of experimental designs. Data generated by Polyester is a reasonable approximation to real RNA-seq data and standard differential expression workflows can recover differential expression set in the simulation by the user. Availability and implementation: Polyester is freely available from Bioconductor (http://bioconductor.org/). Contact: jtleek@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25926345

  19. 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

  20. Scaling of gene expression with transcription-factor fugacity.

    PubMed

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

    2014-12-19

    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.

  1. Studying Gene Expression: Database Searches and Promoter Fusions to Investigate Transcriptional Regulation in Bacteria†

    PubMed Central

    Martinez-Vaz, Betsy M.; Makarevitch, Irina; Stensland, Shane

    2010-01-01

    A laboratory project was designed to illustrate how to search biological databases and utilize the information provided by these resources to investigate transcriptional regulation in Escherichia coli. The students searched several databases (NCBI Genomes, RegulonDB and EcoCyc) to learn about gene function, regulation, and the organization of transcriptional units. A fluorometer and GFP promoter fusions were used to obtain fluorescence data and measure changes in transcriptional activity. The class designed and performed experiments to investigate the regulation of genes necessary for biosynthesis of amino acids and how expression is affected by environmental signals and transcriptional regulators. Assessment data showed that this activity enhanced students’ knowledge of databases, reporter genes and transcriptional regulation. PMID:23653697

  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. 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.

  4. Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens.

    PubMed

    Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Lam, Siew H; Ip, Yuen K

    2017-01-01

    The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance

  5. Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

    PubMed Central

    Chng, You R.; Ong, Jasmine L. Y.; Ching, Biyun; Chen, Xiu L.; Hiong, Kum C.; Wong, Wai P.; Chew, Shit F.; Lam, Siew H.; Ip, Yuen K.

    2017-01-01

    The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance

  6. Improved transgene expression fine-tuning in mammalian cells using a novel transcription-translation network.

    PubMed

    Malphettes, Laetitia; Fussenegger, Martin

    2006-08-05

    Following the discovery of RNA interference (RNAi) and related phenomena, novel regulatory processes, attributable to small non-protein-coding RNAs, continue to emerge. Capitalizing on the ability of artificial short interfering RNAs (siRNAs) to trigger degradation of specific target transcripts, and thereby silence desired gene expression, we designed and characterized a generic transcription-translation network in which it is possible to fine-tune heterologous protein production by coordinated transcription and translation interventions using macrolide and tetracycline antibiotics. Integration of siRNA-specific target sequences (TAGs) into the 5' or 3' untranslated regions (5'UTR, 3'UTR) of a desired constitutive transcription unit rendered transgene-encoded protein (erythropoietin, EPO; human placental alkaline phosphatase, SEAP; human vascular endothelial growth factor 121, VEGF(121)) production in mammalian cells responsive to siRNA levels that can be fine-tuned by macrolide-adjustable RNA polymerase II- or III-dependent promoters. Coupling of such macrolide-responsive siRNA-triggered translation control with tetracycline-responsive transcription of tagged transgene mRNAs created an antibiotic-adjustable two-input transcription-translation network characterized by elimination of detectable leaky expression with no reduction in maximum protein production levels. This transcription-translation network revealed transgene mRNA depletion to be dependent on siRNA and mRNA levels and that translation control was able to eliminate basal expression inherent to current transcription control modalities. Coupled transcription-translation circuitries have the potential to lead the way towards composite artificial regulatory networks, to enable complex therapeutic interventions in future biopharmaceutical manufacturing, gene therapy and tissue engineering initiatives.

  7. 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.

  8. Embryonic Expression of the Chicken Krüppel-like (KLF) Transcription Factor Gene Family

    PubMed Central

    Antin, Parker B.; Pier, Maricela; Sesepasara, Terry; Yatskievych, Tatiana A; Darnell, Diana K.

    2010-01-01

    The Krüppel-like transcription factors are zinc finger proteins that activate and suppress target gene transcription. Although KLF factors have been implicated in regulating many developmental processes, a comprehensive gene expression analysis has not been reported. Here we present the chicken KLF gene family and expression during the first five days of embryonic development. Fourteen chicken KLF genes or expressed sequences have been previously identified. Through synteny analysis and cDNA mapping we have identified the KLF9 gene and determined that the gene presently named KLF1 is the true ortholog of KLF17 in other species. In situ hybridization expression analyses show that in general KLFs are broadly expressed in multiple cell and tissue types. Expression of KLFs 3, 7, 8, and 9, is widespread at all stages examined. KLFs 2, 4, 5, 6, 10, 11, 15 and 17 show more restricted patterns that suggest multiple functions during early stages of embryonic development. PMID:20503383

  9. The E2F transcription factor family regulates CENH3 expression in Arabidopsis thaliana.

    PubMed

    Heckmann, Stefan; Lermontova, Inna; Berckmans, Barbara; De Veylder, Lieven; Bäumlein, Helmut; Schubert, Ingo

    2011-11-01

    To elucidate the epigenetic maintenance mechanism for functional plant centromeres, we studied transcriptional regulation of the centromere-specific histone H3 variant CENH3 in Arabidopsis thaliana. We focused on the structure and activity of the CENH3 promoter (CENH3pro) and its regulation by E2F transcription factors. Use of CENH3pro::GUS reporter gene constructs showed that CENH3pro is active in dividing tissues, and that full expression in root meristems depends on intragenic regulatory elements within the second intron. Chromatin immunoprecipitation identified CENH3 as an E2F target gene. Transient co-expression of a CENH3pro::GUS reporter gene construct with various E2F transcription factors in A. thaliana protoplasts showed that E2Fa and E2Fb (preferentially with dimerization protein DPb) activate CENH3pro. Stable over-expression of E2Fa and E2Fb increased the CENH3 transcript level in planta, whereas over-expression of E2Fc decreased the CENH3 transcript level. Surprisingly, mutation of the two E2F binding sites of CENH3pro, in particular the more upstream one (E2F2), caused an increase in CENH3pro activity, indicating E2F-dependent transcriptional repression. CENH3pro repression may be triggered by the interplay of typical and atypical E2Fs in a cell cycle-dependent manner, and/or by interaction of typical E2Fs with retinoblastoma-related (RBR) protein. We speculate that E2Fs are involved in differential transcriptional regulation of CENH3 versus H3, as H3 promoters lack E2F binding motifs. E2F binding motifs are also present in human and Drosophila CENH3pro regions, thus cell cycle-dependent transcriptional regulation of CENH3 may be highly conserved.

  10. Distinct Expression Patterns of Natural Antisense Transcripts in Arabidopsis1[C][W

    PubMed Central

    Henz, Stefan R.; Cumbie, Jason S.; Kasschau, Kristin D.; Lohmann, Jan U.; Carrington, James C.; Weigel, Detlef; Schmid, Markus

    2007-01-01

    It has been shown that overlapping cis-natural antisense transcripts (cis-NATs) can form a regulatory circuit in which small RNAs derived from one transcript regulate stability of the other transcript, which manifests itself as anticorrelated expression. However, little is known about how widespread antagonistic expression of cis-NATs is. We have determined how frequently cis-NAT pairs, which make up 7.4% of annotated transcription units in the Arabidopsis (Arabidopsis thaliana) genome, show anticorrelated expression patterns. Indeed, global expression profiles of pairs of cis-NATs on average have significantly lower pairwise Pearson correlation coefficients than other pairs of neighboring genes whose transcripts do not overlap. However, anticorrelated expression that is greater than expected by chance is found in only a small number of cis-NAT pairs. The degree of anticorrelation does not depend on the length of the overlap or on the distance of the 5′ ends of the transcripts. Consistent with earlier findings, cis-NATs do not exhibit an increased likelihood to give rise to small RNAs, as determined from available small RNA sequences and massively parallel signature sequencing tags. However, the overlapping regions of cis-NATs appeared to be enriched for small RNA loci compared to nonoverlapping regions. Furthermore, expression of cis-NATs was not disproportionately affected in various RNA-silencing mutants. Our results demonstrate that there is a trend toward anticorrelated expression of cis-NAT pairs in Arabidopsis, but currently available data do not produce a strong signature of small RNA-mediated silencing for this process. PMID:17496106

  11. 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.

  12. 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

  13. Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites

    PubMed Central

    Foth, Bernardo J; Zhang, Neng; Mok, Sachel; Preiser, Peter R; Bozdech, Zbynek

    2008-01-01

    Background Malaria is a one of the most important infectious diseases and is caused by parasitic protozoa of the genus Plasmodium. Previously, quantitative characterization of the P. falciparum transcriptome demonstrated that the strictly controlled progression of these parasites through their intra-erythrocytic developmental cycle is accompanied by a continuous cascade of gene expression. Although such analyses have proven immensely useful, the correlations between abundance of transcripts and their cognate proteins remain poorly characterized. Results Here, we present a quantitative time-course analysis of relative protein abundance for schizont-stage parasites (34 to 46 hours after invasion) based on two-dimensional differential gel electrophoresis of protein samples labeled with fluorescent dyes. For this purpose we analyzed parasite samples taken at 4-hour intervals from a tightly synchronized culture and established more than 500 individual protein abundance profiles with high temporal resolution and quantitative reproducibility. Approximately half of all profiles exhibit a significant change in abundance and 12% display an expression peak during the observed 12-hour time interval. Intriguingly, identification of 54 protein spots by mass spectrometry revealed that 58% of the corresponding proteins - including actin-I, enolase, eukaryotic initiation factor (eIF)4A, eIF5A, and several heat shock proteins - are represented by more than one isoform, presumably caused by post-translational modifications, with the various isoforms of a given protein frequently showing different expression patterns. Furthermore, comparisons with transcriptome data generated from the same parasite samples reveal evidence of significant post-transcriptional gene expression regulation. Conclusions Together, our data indicate that both post-transcriptional and post-translational events are widespread and of presumably great biological significance during the intra

  14. 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.

  15. Identification of Embryoid-Abundant Genes That Are Temporally Expressed during Pollen Embryogenesis in Wheat Anther Cultures.

    PubMed

    Reynolds, T L; Kitto, S L

    1992-12-01

    Uninucleate microspores in anther cultures of bread wheat (Triticum aestivum cv Pavon) are capable of producing haploid pollen embryoids and plants. To gain an understanding of this alternate pathway of pollen development, we constructed a cDNA library to young pollen embryoids, isolated embryoid-specific genes, and analyzed their expression patterns during morphogenesis. Two embryoid-abundant clones, pEMB4 and 94, were expressed very early during culture, suggesting that these genes are associated with development and are not simply expressed as a consequence of differentiation. The accumulation patterns of five cloned mRNAs may indicate the activation of specific genes associated with the major morphological and physiological activities connected with the differentiation of embryoids in vitro. These results suggest that embryoid-abundant gene expression is causally related to this pathway because gene expression is spatially and temporally specific and is not observed when microspores are cultured under noninductive conditions.

  16. Inferring biological functions and associated transcriptional regulators using gene set expression coherence analysis

    PubMed Central

    Kim, Tae-Min; Chung, Yeun-Jun; Rhyu, Mun-Gan; Ho Jung, Myeong

    2007-01-01

    Background Gene clustering has been widely used to group genes with similar expression pattern in microarray data analysis. Subsequent enrichment analysis using predefined gene sets can provide clues on which functional themes or regulatory sequence motifs are associated with individual gene clusters. In spite of the potential utility, gene clustering and enrichment analysis have been used in separate platforms, thus, the development of integrative algorithm linking both methods is highly challenging. Results In this study, we propose an algorithm for discovery of molecular functions and elucidation of transcriptional logics using two kinds of gene information, functional and regulatory motif gene sets. The algorithm, termed gene set expression coherence analysis first selects functional gene sets with significantly high expression coherences. Those candidate gene sets are further processed into a number of functionally related themes or functional clusters according to the expression similarities. Each functional cluster is then, investigated for the enrichment of transcriptional regulatory motifs using modified gene set enrichment analysis and regulatory motif gene sets. The method was tested for two publicly available expression profiles representing murine myogenesis and erythropoiesis. For respective profiles, our algorithm identified myocyte- and erythrocyte-related molecular functions, along with the putative transcriptional regulators for the corresponding molecular functions. Conclusion As an integrative and comprehensive method for the analysis of large-scaled gene expression profiles, our method is able to generate a set of testable hypotheses: the transcriptional regulator X regulates function Y under cellular condition Z. GSECA algorithm is implemented into freely available software package. PMID:18021416

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

    PubMed Central

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

    2015-01-01

    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 (DRG). In the present study we investigated 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, X, as well as other brainstem nuclei during different stages of development into adult stage. We find that genetically identified Brn3bKO 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. PMID:26356988

  18. 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.

  19. Transcription factor AP-2α regulates acute myeloid leukemia cell proliferation by influencing Hoxa gene expression.

    PubMed

    Ding, Xiaofeng; Yang, Zijian; Zhou, Fangliang; Wang, Fangmei; Li, Xinxin; Chen, Cheng; Li, Xiaofeng; Hu, Xiang; Xiang, Shuanglin; Zhang, Jian

    2013-08-01

    Transcription factor AP-2α mediates transcription of a number of genes implicated in mammalian development, cell proliferation and carcinogenesis. In the current study, we identified Hoxa7, Hoxa9 and Hox cofactor Meis1 as AP-2α target genes, which are involved in myeloid leukemogenesis. Luciferase reporter assays revealed that overexpression of AP-2α activated transcription activities of Hoxa7, Hoxa9 and Meis1, whereas siRNA of AP-2α inhibited their transcription activities. We found that AP-2 binding sites in regulatory regions of three genes activated their transcription by mutant analysis and AP-2α could interact with AP-2 binding sites in vivo by chromatin immunoprecipitation (ChIP). Further results showed that the AP-2α shRNA efficiently inhibited mRNA and protein levels of Hoxa7, Hoxa9 and Meis1 in AML cell lines U937 and HL60. Moreover, decreased expression of AP-2α resulted in a significant reduction in the growth and proliferation of AML cells in vitro. Remarkably, AP-2α knockdown leukemia cells exhibit decreased tumorigenicity in vivo compared with controls. Finally, AP-2α and target genes in clinical acute myeloid leukemia samples of M5b subtype revealed variable expression levels and broadly paralleled expression. These data support a role of AP-2α in mediating the expression of Hoxa genes in acute myeloid leukemia to influence the proliferation and cell survival.

  20. Expressed transcripts associated with high rates of egg production in chicken ovarian follicles.

    PubMed

    Yang, K T; Lin, C Y; Huang, H L; Liou, J S; Chien, C Y; Wu, C P; Huang, C W; Ou, B R; Chen, C F; Lee, Y P; Lin, E C; Tang, P C; Lee, W C; Ding, S T; Cheng, W T K; Huang, M C

    2008-02-01

    The purpose of this study was to characterize differentially expressed transcripts associated with varying rates of egg production in Taiwan country chickens. Ovarian follicles were isolated from two strains of chicken which showed low (B) or high (L2) rates of egg production, then processed for RNA extraction and cDNA library construction. Three thousand and eight forty clones were randomly selected from the cDNA library and amplified by PCR, then used in microarray analysis. Differentially expressed transcripts (P<0.05, log(2)> or = 1.75) were sequenced, and aligned using GenBank. This analysis revealed 20 non-redundant sequences which corresponded to known transcripts. Eight transcripts were expressed at a higher level in ovarian tissue prepared from chicken strain B, and 12 transcripts were expressed at a higher level in L2 birds. These differential patterns of expression were confirmed by semi-quantitative RT-PCR. We show that transcripts of cyclin B2 (cycB2), ferritin heavy polypeptide 1 (FTH1), Gag-Pol polyprotein, thymosin beta4 (TB4) and elongation factor 1 alpha1 (EEF1A1) were enriched in B strain ovarian follicles. In contrast, thioredoxin (TXN), acetyl-CoA dehydrogenase long chain (ACADL), inhibitor of growth family member 4 (ING4) and annexin II (ANXA2) were expressed in at higher levels in the L2 strain. We suggest that our approach may lead to the isolation of effective molecular markers that can be used in selection programs in Taiwan country chickens.

  1. Regulation of microcin C51 operon expression: the role of global regulators of transcription.

    PubMed

    Fomenko, D; Veselovskii, A; Khmel, I

    2001-06-01

    Expression of the microcin C51 operon in Escherichia coli cells is regulated as a function of the phase of growth; it is stimulated during the decelerating phase of growth. Using single-copy P(mcc)-lac transcriptional fusion (the promoter region of the microcin C51 operon fused to a promoterless lac operon in lambda phage), we showed that transcription from the microcin operon promoter is dependent on sigma(s) (RpoS) factor. However, some level of P(mcc)-lac expression is possible in rpoS null mutants, indicating that another sigma factor might be involved in transcription of the microcin C51 operon. Overproduction of sigma70 decreased Pmcc-directed transcription, presumably as a result of competition of sigma factors for the limited amount of core RNA polymerase. The cyclic AMP-CRP complex was shown to stimulate transcription from Pmcc: the absence of CRP or cAMP in crp or cya mutant cells strongly decreased the level of P(mcc)-lac expression. The production of C51 microcin decreased or was absent in rpoS, crp and cya mutant cells. Leucine-responsive protein Lrp and histone-like protein H-NS repressed P(mcc)-lac expression in the exponential and decelerating phases of growth. In studies of P(mcc)-lac expression in double mutant cells, we showed that proteins CRP, Lrp and H-NS acted in rpoS-dependent and rpoS-independent ways in transcription of the microcin C51 operon. Mutation hns(-) resulted in an increase in P(mcc)-lac expression in crp, rpoS and lrp mutant cells, as in wild-type cells.

  2. 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.

  3. Expression of peroxisome proliferator-activated receptor and CCAAT/enhancer binding protein transcription factors in cultured human sebocytes.

    PubMed

    Chen, WenChieh; Yang, Chao-Chun; Sheu, Hamm-Ming; Seltmann, Holger; Zouboulis, Christos C

    2003-09-01

    Lipid synthesis and accumulation represent a major step in sebocyte differentiation and it may be of importance for sebocytes to express two families of transcription factors, CCAAT/enhancer binding proteins (c/EBPs) and peroxisome proliferator-activated receptors (PPARs), which were found to play a crucial role in the differentiation of adipocytes. Using the immortalized human sebaceous gland cell line SZ95 we examined the expression of the molecules before and after treatment with testosterone, 5alpha-dihydrotestosterone, dexamethasone, 17beta-estradiol and genistein, at 6, 12, 24, and 48 h, respectively. Reverse transcription-PCR analysis showed expression of peroxisome proliferator-activated receptors -alpha, -delta, -gamma1, -gamma2 and CCAAT/enhancer binding proteins-alpha, -beta, -gamma-delta in native SZ95 sebocytes. In western blot studies, high levels of CCAAT/enhancer binding proteins-alpha and -beta, and peroxisome proliferator-activated receptors-gamma were expressed at 6, 24, and 12 h, respectively. Immunostaining of the cultured sebocytes showed the CCAAT/enhancer binding proteins-alpha and -beta mainly localized within nuclei, whereas peroxisome proliferator-activated receptors-gamma in the cytoplasm. Strong staining of sebocytes was immunohistochemically revealed in the basal layer of sebaceous glands in human scalp and sebaceous nevus. Genistein down-regulated the expression of CCAAT/enhancer binding proteins-alpha and -beta, and peroxisome proliferator-activated receptors-gamma on the protein level. Treatment with linoleic acid for 48 h induced further differentiation of sebocytes leading to abundant lipid synthesis.

  4. Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response.

    PubMed

    Fornara, Fabio; Panigrahi, Kishore C S; Gissot, Lionel; Sauerbrunn, Nicolas; Rühl, Mark; Jarillo, José A; Coupland, George

    2009-07-01

    Flowering of Arabidopsis is induced by long summer days (LDs). The transcriptional regulator CONSTANS (CO) promotes flowering, and its transcription is increased under LDs. We systematically misexpressed transcription factors in companion cells and identified several DOF proteins that delay flowering by repressing CO transcription. Combining mutations in four of these, including CYCLING DOF FACTOR 2 (CDF2), caused photoperiod-insensitive early flowering by increasing CO mRNA levels. CO transcription is promoted to differing extents by GIGANTEA (GI) and the F-box protein FKF1. We show that GI stabilizes FKF1, thereby reducing CDF2 abundance and allowing transcription of CO. Despite the crucial function of GI in wild-type plants, introducing mutations in the four DOF-encoding genes into gi mutants restored the diurnal rhythm and light inducibility of CO. Thus, antagonism between GI and DOF transcription factors contributes to photoperiodic flowering by modulating an underlying diurnal rhythm in CO transcript levels.

  5. A primer on molecular biology for imagers: II. Transcription and gene expression.

    PubMed

    Pandit, Sunil D; Li, King C P

    2004-03-01

    The process of gene expression is complex and highly regulated to ensure that the right gene is expressed at the right place, at the right time, and in regulated amounts. The cell has multiple levels at which it controls the expression of a transcript including gene expression, alternate splicing, and stability of the transcript. Alternate splicing to generate different RNA species from a given gene and DNA rearrangements where genes are rearranged during cellular differentiation (eg, immunoglobulin genes) are additional mechanisms used to generate diversity in complex organisms. Epigenetic mechanisms such as methylation where CpG-rich islands in the promoter region depending on their methylation status can also modulate gene expression. The reader is requested to refer to the books, review articles, and web sites for additional information.

  6. Insight into transcription factor gene duplication from Caenorhabditis elegans Promoterome-driven expression patterns

    PubMed Central

    Reece-Hoyes, John S; Shingles, Jane; Dupuy, Denis; Grove, Christian A; Walhout, Albertha JM; Vidal, Marc; Hope, Ian A

    2007-01-01

    Background The C. elegans Promoterome is a powerful resource for revealing the regulatory mechanisms by which transcription is controlled pan-genomically. Transcription factors will form the core of any systems biology model of genome control and therefore the promoter activity of Promoterome inserts for C. elegans transcription factor genes was examined, in vivo, with a reporter gene approach. Results Transgenic C. elegans strains were generated for 366 transcription factor promoter/gfp reporter gene fusions. GFP distributions were determined, and then summarized with reference to developmental stage and cell type. Reliability of these data was demonstrated by comparison to previously described gene product distributions. A detailed consideration of the results for one C. elegans transcription factor gene family, the Six family, comprising ceh-32, ceh-33, ceh-34 and unc-39 illustrates the value of these analyses. The high proportion of Promoterome reporter fusions that drove GFP expression, compared to previous studies, led to the hypothesis that transcription factor genes might be involved in local gene duplication events less frequently than other genes. Comparison of transcription factor genes of C. elegans and Caenorhabditis briggsae was therefore carried out and revealed very few examples of functional gene duplication since the divergence of these species for most, but not all, transcription factor gene families. Conclusion Examining reporter expression patterns for hundreds of promoters informs, and thereby improves, interpretation of this data type. Genes encoding transcription factors involved in intrinsic developmental control processes appear acutely sensitive to changes in gene dosage through local gene duplication, on an evolutionary time scale. PMID:17244357

  7. 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.

  8. HSF1 functions as a transcription regulator for Dp71 expression.

    PubMed

    Tan, Jin; Tan, Sichuang; Zheng, Hexin; Liu, Meidong; Chen, Guangwen; Zhang, Huali; Wang, Kangkai; Tan, Sipin; Zhou, Jiang; Xiao, Xian-zhong

    2015-03-01

    Heat shock factor 1 (HSF1) is one of the most important transcriptional molecules in the heat shock process; however, HSF1 can also regulate the expression of other proteins. Dystrophin Dp71 is one of the most widely expressed isoforms of the dystrophin gene family. In our experiments, we showed for the first time that HSF1 can function as a transcriptional factor for endogenous Dp71 expression in vivo and in vitro. We demonstrated that the messenger RNA (mRNA) and protein expression of Dp71 were significantly reduced in HSF1-knockout mice compared with wild-type mice in brain, lung, liver, spleen, and kidney. Overexpression of HSF1 significantly enhanced the mRNA and protein expression of Dp71 in HeLa cells. Inhibiting the expression of HSF1 in HeLa cells significantly reduced the expression of Dp71. By use of the EMSA technique, the chromatin immunoprecipitation assay, and the luciferase reporter system, we demonstrated that HSF1 can directly bind the HSE in the Dp71 promoter region. We concluded from our data that HSF1 functions as a transcriptional regulator of Dp71 expression.

  9. Regulation of photoreceptor gene expression by Crx-associated transcription factor network

    PubMed Central

    Hennig, Anne K.; Peng, Guang-Hua; Chen, Shiming

    2008-01-01

    Rod and cone photoreceptors in the mammalian retina are special types of neurons that are responsible for phototransduction, the first step of vision. Development and maintenance of photoreceptors require precisely regulated gene expression. This regulation is mediated by a network of photoreceptor transcription factors centered on Crx, an Otx-like homeodomain transcription factor. The cell type (subtype) specificity of this network is governed by factors that are preferentially expressed by rods or cones or both, including the rod-determining factors neural retina leucine zipper protein (Nrl) and the orphan nuclear receptor Nr2e3; and cone-determining factors, mostly nuclear receptor family members. The best-documented of these include thyroid hormone receptor β2 (Trβ2), retinoid related orphan receptor Rorβ, and retinoid X receptor Rxrγ. The appropriate function of this network also depends on general transcription factors and co-factors that are ubiquitously expressed, such as the Sp zinc finger transcription factors and STAGA coactivator complexes. These cell type-specific and general transcription regulators form complex interactomes; mutations that interfere with any of the interactions can cause photoreceptor development defects or degeneration. In this manuscript, we review recent progress on the roles of various photoreceptor transcription factors and interactions in photoreceptor subtype development. We also provide evidence of auto-, para-, and feedback regulation among these factors at the transcriptional level. These protein-protein and protein-promoter interactions provide precision and specificity in controlling photoreceptor subtype-specific gene expression, development and survival. Understanding these interactions may provide insights to more effective therapeutic interventions for photoreceptor diseases. PMID:17662965

  10. 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

  11. Effects of transcription induction homogeneity and transcript stability on expression of two genes in a constructed operon.

    PubMed

    Smolke, C D; Khlebnikov, A; Keasling, J D

    2001-12-01

    A synthetic operon was constructed using the reporter genes gfp and lacZ and the arabinose-inducible araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 3' and 5' ends of the genes and a putative RNase E site was placed between the genes. These mRNA control elements have been shown to affect transcript processing and decay, resulting in altered protein levels. These constructs were transformed into cells harboring the native arabinose-inducible araE gene encoding the arabinose transport protein and engineered cells harboring a constitutively expressed araE. In the strains with arabinose-dependent transport the linear response in the production of both reporter proteins to inducer concentration occurred over a narrow range of arabinose concentrations. In the strains with constitutive transport the linear range of gene expression occurred over a much larger arabinose concentration range than in strains with the arabinose-inducible transport. Strains with the arabinose-inducible transport harboring different operon constructs produced the two reporter proteins at very different levels at low arabinose concentrations; as inducer concentrations increased, differences in relative expression levels decreased. In contrast, strains with constitutive transport harboring different operon constructs produced the reporter proteins at very different levels across the entire range of inducer concentrations, pointing to the importance of optimizing gene expression control at various levels to control the production of heterologous proteins.

  12. 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

  13. Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression.

    PubMed

    Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

    2014-05-02

    To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

  14. No association between HPV positive breast cancer and expression of human papilloma viral transcripts.

    PubMed

    Gannon, Orla M; Antonsson, Annika; Milevskiy, Michael; Brown, Melissa A; Saunders, Nicholas A; Bennett, Ian C

    2015-12-14

    Infectious agents are thought to be responsible for approximately 16% of cancers worldwide, however there are mixed reports in the literature as to the prevalence and potential pathogenicity of viruses in breast cancer. Furthermore, most studies to date have focused primarily on viral DNA rather than the expression of viral transcripts. We screened a large cohort of fresh frozen breast cancer and normal breast tissue specimens collected from patients in Australia for the presence of human papilloma virus (HPV) DNA, with an overall prevalence of HPV of 16% and 10% in malignant and non-malignant tissue respectively. Samples that were positive for HPV DNA by nested PCR were screened by RNA-sequencing for the presence of transcripts of viral origin, using three different bioinformatic pipelines. We did not find any evidence for HPV or other viral transcripts in HPV DNA positive samples. In addition, we also screened publicly available breast RNA-seq data sets for the presence of viral transcripts and did not find any evidence for the expression of viral transcripts (HPV or otherwise) in other data sets. This data suggests that transcription of viral genomes is unlikely to be a significant factor in breast cancer pathogenesis.

  15. Abundant 5S rRNA-Like Transcripts Encoded by the Mitochondrial Genome in Amoebozoa ▿ †

    PubMed Central

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

    2010-01-01

    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. PMID:20304999

  16. Intracompartmental and intercompartmental transcriptional networks coordinate the expression of genes for organellar functions.

    PubMed

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

    2011-09-01

    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.

  17. Selective up-regulation of JunD transcript and protein expression in vasopressinergic supraoptic nucleus neurones in water-deprived rats.

    PubMed

    Yao, S T; Gouraud, S S; Qiu, J; Cunningham, J T; Paton, J F R; Murphy, D

    2012-12-01

    The magnocellular neurones (MCN) of the supraoptic nucleus (SON) undergo reversible changes during dehydration. We hypothesise that alterations in steady-state transcript levels might be partially responsible for this plasticity. In turn, regulation of transcript abundance might be mediated by transcription factors. We have previously used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation. We observed down-regulation of 11 and up-regulation of 31 transcription factor transcripts, including members of the activator protein-1 gene family, namely c-fos, c-jun, fosl1 and junD. Because JunD expression and regulation within the SON has not been previously described, we have used in situ hybridisation and the quantitative reverse transcriptase-polymerase chain reaction to confirm the array results, demonstrating a significant increase in JunD mRNA levels following 24 and 72 h of water deprivation. Western blot and immunohistochemistry revealed a significant increase in JunD protein expression following dehydration. Double-staining fluorescence immunohistochemistry with a neurone-specific marker (NeuN) demonstrated that JunD staining is predominantly neuronal. Additionally, JunD immunoreactivity is observed primarily in vasopressin-containing neurones with markedly less staining seen in oxytocin-containing MCNs. Furthermore, JunD is highly co-expressed with c-Fos in MCNs of the SON following dehydration. These results suggest that JunD plays a role in the regulation of gene expression within MCNs of the SON in association with other Fos and Jun family members.

  18. 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.

  19. 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.

  20. Transcript expression patterns illuminate the mechanistic background of hormesis in caenorhabditis elegans maupas.

    PubMed

    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.

  1. 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

  2. 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.

  3. 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.

  4. Transcriptional network inference from functional similarity and expression data: a global supervised approach.

    PubMed

    Ambroise, Jérôme; Robert, Annie; Macq, Benoit; Gala, Jean-Luc

    2012-01-06

    An important challenge in system biology is the inference of biological networks from postgenomic data. Among these biological networks, a gene transcriptional regulatory network focuses on interactions existing between transcription factors (TFs) and and their corresponding target genes. A large number of reverse engineering algorithms were proposed to infer such networks from gene expression profiles, but most current methods have relatively low predictive performances. In this paper, we introduce the novel TNIFSED method (Transcriptional Network Inference from Functional Similarity and Expression Data), that infers a transcriptional network from the integration of correlations and partial correlations of gene expression profiles and gene functional similarities through a supervised classifier. In the current work, TNIFSED was applied to predict the transcriptional network in Escherichia coli and in Saccharomyces cerevisiae, using datasets of 445 and 170 affymetrix arrays, respectively. Using the area under the curve of the receiver operating characteristics and the F-measure as indicators, we showed the predictive performance of TNIFSED to be better than unsupervised state-of-the-art methods. TNIFSED performed slightly worse than the supervised SIRENE algorithm for the target genes identification of the TF having a wide range of yet identified target genes but better for TF having only few identified target genes. Our results indicate that TNIFSED is complementary to the SIRENE algorithm, and particularly suitable to discover target genes of "orphan" TFs.

  5. Genome-Wide Association between Transcription Factor Expression and Chromatin Accessibility Reveals Regulators of Chromatin Accessibility

    PubMed Central

    Rueedi, Rico

    2017-01-01

    To better understand genome regulation, it is important to uncover the role of transcription factors in the process of chromatin structure establishment and maintenance. Here we present a data-driven approach to systematically characterise transcription factors that are relevant for this process. Our method uses a linear mixed modelling approach to combine datasets of transcription factor binding motif enrichments in open chromatin and gene expression across the same set of cell lines. Applying this approach to the ENCODE dataset, we confirm already known and imply numerous novel transcription factors that play a role in the establishment or maintenance of open chromatin. In particular, our approach rediscovers many factors that have been annotated as pioneer factors. PMID:28118358

  6. Genome-Wide Association between Transcription Factor Expression and Chromatin Accessibility Reveals Regulators of Chromatin Accessibility.

    PubMed

    Lamparter, David; Marbach, Daniel; Rueedi, Rico; Bergmann, Sven; Kutalik, Zoltán

    2017-01-01

    To better understand genome regulation, it is important to uncover the role of transcription factors in the process of chromatin structure establishment and maintenance. Here we present a data-driven approach to systematically characterise transcription factors that are relevant for this process. Our method uses a linear mixed modelling approach to combine datasets of transcription factor binding motif enrichments in open chromatin and gene expression across the same set of cell lines. Applying this approach to the ENCODE dataset, we confirm already known and imply numerous novel transcription factors that play a role in the establishment or maintenance of open chromatin. In particular, our approach rediscovers many factors that have been annotated as pioneer factors.

  7. Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar): Increased Abundance and Expression of a Calreticulin-Like Protein.

    PubMed

    Micallef, Giulia; Cash, Phillip; Fernandes, Jorge M O; Rajan, Binoy; Tinsley, John W; Bickerdike, Ralph; Martin, Samuel A M; Bowman, Alan S

    2017-01-01

    In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts.

  8. Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar): Increased Abundance and Expression of a Calreticulin-Like Protein

    PubMed Central

    Micallef, Giulia; Cash, Phillip; Fernandes, Jorge M. O.; Rajan, Binoy; Tinsley, John W.; Bickerdike, Ralph

    2017-01-01

    In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts. PMID:28046109

  9. 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

  10. CITA/NLRC5: A critical transcriptional regulator of MHC class I gene expression.

    PubMed

    Downs, Isaac; Vijayan, Saptha; Sidiq, Tabasum; Kobayashi, Koichi S

    2016-07-08

    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.

  11. G =  MAT: linking transcription factor expression and DNA binding data.

    PubMed

    Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

    2011-01-31

    Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/.

  12. G = MAT: Linking Transcription Factor Expression and DNA Binding Data

    PubMed Central

    Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

    2011-01-01

    Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/. PMID:21297945

  13. Epigenetic and Transcriptional Regulation of IRAK-M Expression in Macrophages.

    PubMed

    Lyroni, Konstantina; Patsalos, Andreas; Daskalaki, Maria G; Doxaki, Christina; Soennichsen, Birte; Helms, Mike; Liapis, Ioannis; Zacharioudaki, Vassiliki; Kampranis, Sotirios C; Tsatsanis, Christos

    2017-02-01

    During macrophage activation, expression of IL-1R-associated kinase (IRAK)-M is induced to suppress TLR-mediated responses and is a hallmark of endotoxin tolerance. Endotoxin tolerance requires tight regulation of genes occurring at the transcriptional and epigenetic levels. To identify novel regulators of IRAK-M, we used RAW 264.7 macrophages and performed a targeted RNA interference screen of genes encoding chromatin-modifying enzymes, signaling molecules, and transcription factors involved in macrophage activation. Among these, the transcription factor CCAAT/enhancer binding protein (C/EBP)β, known to be involved in macrophage inactivation, was necessary for the induction of IRAK-M expression. Chromatin immunoprecipitation showed that C/EBPβ was recruited to the IRAK-M promoter following LPS stimulation and was indispensable for IRAK-M transcriptional activation. Among histone 3-modifying enzymes, our screen showed that knockdown of the histone 3 lysine 27 (H3K27) methyltransferase and part of the polycomb recessive complex 2, enhancer of Zeste 2, resulted in IRAK-M overexpression. In contrast, knockdown of the H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat X chromosome suppressed the induction of IRAK-M in response to LPS stimulation. Accordingly, we demonstrated that H3K27 on the IRAK-M promoter is trimethylated in unstimulated cells and that this silencing epigenetic mark is removed upon LPS stimulation. Our data propose a mechanism for IRAK-M transcriptional regulation according to which, in the naive state, polycomb recessive complex 2 repressed the IRAK-M promoter, allowing low levels of expression; following LPS stimulation, the IRAK-M promoter is derepressed, and transcription is induced to allow its expression.

  14. Breast Cancer Resistance Protein Abundance, but Not mRNA Expression, Correlates With Estrone-3-Sulfate Transport in Caco-2.

    PubMed

    Harwood, Matthew D; Neuhoff, Sibylle; Rostami-Hodjegan, Amin; Warhurst, Geoffrey

    2016-04-01

    Transporter mRNA and protein expression data are used to extrapolate in vitro transporter kinetics to in vivo drug disposition predictions. Breast cancer resistance protein (BCRP) possesses broad substrate specificity; therefore, understanding BCRP expression-activity relationships are necessary for the translation to in vivo. Bidirectional transport of estrone-3-sulfate (E-3-S), a BCRP probe, was evaluated with respect to relative BCRP mRNA expression and absolute protein abundance in 10- and 29-day cultured Caco-2 cells. BCRP mRNA expression was quantified by real-time PCR against a housekeeper gene, Cyclophilin A. The BCRP protein abundance in total membrane fractions was quantified by targeted proteomics, and [(3)H]-E-3-S bidirectional transport was determined in the presence or absence of Ko143, a potent BCRP inhibitor. BCRP mRNA expression was 1.5-fold higher in 29- versus 10-day cultured cells (n = 3), whereas a 2.4-fold lower (p < 0.001) BCRP protein abundance was observed in 29- versus 10-day cultured cells (1.28 ± 0.33 and 3.06 ± 0.22 fmol/μg protein, n = 6, respectively). This correlated to a 2.45-fold lower (p < 0.01) efflux ratio for E-3-S in 29- versus 10-day cultured cells (8.97 ± 2.51 and 3.32 ± 0.66, n = 6, respectively). Caco-2 cell BCRP protein abundance, but not mRNA levels, correlates with BCRP activity, suggesting that extrapolation strategies incorporating BCRP protein abundance-activity relationships may be more successful.

  15. 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

  16. 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

  17. Mitotic retention of gene expression patterns by the cell fate-determining transcription factor Runx2

    PubMed Central

    Young, Daniel W.; Hassan, Mohammad Q.; Yang, Xiao-Qing; Galindo, Mario; Javed, Amjad; Zaidi, Sayyed K.; Furcinitti, Paul; Lapointe, David; Montecino, Martin; Lian, Jane B.; Stein, Janet L.; van Wijnen, Andre J.; Stein, Gary S.

    2007-01-01

    During cell division, cessation of transcription is coupled with mitotic chromosome condensation. A fundamental biological question is how gene expression patterns are retained during mitosis to ensure the phenotype of progeny cells. We suggest that cell fate-determining transcription factors provide an epigenetic mechanism for the retention of gene expression patterns during cell division. Runx proteins are lineage-specific transcription factors that are essential for hematopoietic, neuronal, gastrointestinal, and osteogenic cell fates. Here we show that Runx2 protein is stable during cell division and remains associated with chromosomes during mitosis through sequence-specific DNA binding. Using siRNA-mediated silencing, mitotic cell synchronization, and expression profiling, we identify Runx2-regulated genes that are modulated postmitotically. Novel target genes involved in cell growth and differentiation were validated by chromatin immunoprecipitation. Importantly, we find that during mitosis, when transcription is shut down, Runx2 selectively occupies target gene promoters, and Runx2 deficiency alters mitotic histone modifications. We conclude that Runx proteins have an active role in retaining phenotype during cell division to support lineage-specific control of gene expression in progeny cells. PMID:17360627

  18. Digital expression profiling of novel diatom transcripts provides insight into their biological functions

    PubMed Central

    2010-01-01

    Background Diatoms represent the predominant group of eukaryotic phytoplankton in the oceans and are responsible for around 20% of global photosynthesis. Two whole genome sequences are now available. Notwithstanding, our knowledge of diatom biology remains limited because only around half of their genes can be ascribed a function based onhomology-based methods. High throughput tools are needed, therefore, to associate functions with diatom-specific genes. Results We have performed a systematic analysis of 130,000 ESTs derived from Phaeodactylum tricornutum cells grown in 16 different conditions. These include different sources of nitrogen, different concentrations of carbon dioxide, silicate and iron, and abiotic stresses such as low temperature and low salinity. Based on unbiased statistical methods, we have catalogued transcripts with similar expression profiles and identified transcripts differentially expressed in response to specific treatments. Functional annotation of these transcripts provides insights into expression patterns of genes involved in various metabolic and regulatory pathways and into the roles of novel genes with unknown functions. Specific growth conditions could be associated with enhanced gene diversity, known gene product functions, and over-representation of novel transcripts. Comparative analysis of data from the other sequenced diatom, Thalassiosira pseudonana, helped identify several unique diatom genes that are specifically regulated under particular conditions, thus facilitating studies of gene function, genome annotation and the molecular basis of species diversity. Conclusions The digital gene expression database represents a new resource for identifying candidate diatom-specific genes involved in processes of major ecological relevance. PMID:20738856

  19. 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

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

    PubMed

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

    2014-02-21

    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.

  1. Gene Expression Under the Influence: Transcriptional Profiling of Ethanol in the Brain

    PubMed Central

    Contet, Candice

    2013-01-01

    Sensitivity to ethanol intoxication, propensity to drink ethanol and vulnerability to develop alcoholism are all influenced by genetic factors. Conversely, exposure to ethanol or subsequent withdrawal produce gene expression changes, which, in combination with environmental variables, may participate in the emergence of compulsive drinking and relapse. The present review offers an integrated perspective on brain gene expression profiling in rodent models of predisposition to differential ethanol sensitivity or consumption, in rats and mice subjected to acute or chronic ethanol exposure, as well as in human alcoholics. The functional categories over-represented among differentially expressed genes suggest that the transcriptional effects of chronic ethanol consumption contribute to the neuroplasticity and neurotoxicity characteristic of alcoholism. Importantly, ethanol produces distinct transcriptional changes within the different brain regions involved in intoxication, reinforcement and addiction. Special emphasis is put on recent profiling studies that have provided some insights into the molecular mechanisms potentially mediating genome-wide regulation of gene expression by ethanol. In particular, current evidence for a role of transcription factors, chromatin remodeling and microRNAs in coordinating the expression of large sets of genes in animals predisposed to excessive ethanol drinking or exposed to protracted abstinence, as well as in human alcoholics, is presented. Finally, studies that have compared ethanol with other drugs of abuse have highlighted common gene expression patterns that may play a central role in drug addiction. The availability of novel technologies and a focus on mechanistic approaches are shaping the future of ethanol transcriptomics. PMID:24078902

  2. Gene expression profiling of M. truncatula transcription factors identifies putative regulators of grain legume seed filling.

    PubMed

    Verdier, Jérôme; Kakar, Klementina; Gallardo, Karine; Le Signor, Christine; Aubert, Grégoire; Schlereth, Armin; Town, Christopher D; Udvardi, Michael K; Thompson, Richard D

    2008-08-01

    Legume seeds represent a major source of proteins for human and livestock diets. The model legume Medicago truncatula is characterized by a process of seed development very similar to that of other legumes, involving the interplay of sets of transcription factors (TFs). Here, we report the first expression profiling of over 700 M. truncatula genes encoding putative TFs throughout seven stages of seed development, obtained using real-time quantitative RT-PCR. A total of 169 TFs were selected which were expressed at late embryogenesis, seed filling or desiccation. The site of expression within the seed was examined for 41 highly expressed transcription factors out of the 169. To identify possible target genes for these TFs, the data were combined with a microarray-derived transcriptome dataset. This study identified 17 TFs preferentially expressed in individual seed tissues and 135 corresponding co-expressed genes, including possible targets. Certain of the TFs co-expressed with storage protein mRNAs correspond to those already known to regulate seed storage protein synthesis in Arabidopsis, whereas the timing of expression of others may be more specifically related to the delayed expression of the legumin-class storage proteins observed in legumes.

  3. Two novel transcripts encoding two Ankyrin repeat containing proteins have preponderant expression during the mouse spermatogenesis.

    PubMed

    Wang, Fei; Hu, Jiarui; Song, Ping; Gong, Wuming

    2007-12-01

    The clone 4921537P18 expressed preponderantly in mouse testis was identified by screening the Riken cDNA database, and two new full-length isoforms of this clone, which were named gsarp1 (Gonad Specific Ankyrin Repeat (ANK) Protein 1) and gsarp2, were found and isolated from mouse testis in the course of the research. Both of the GSARP1 and GSARP2 contain an ANK region circular composed by seven ANKs, and their structural feature is very similar to that of the IkappaB family proteins, while IkappaB proteins associate with the transcription factor NF-kappaB via their ANKs in the NF-kappaB pathway. We investigated the expression pattern at the mRNA level by Reverse transcription PCR. The gsarp1 has high expression level in mouse testis, while has low expression level in the ovary, and the gsarp2 is only expressed in mouse testis. The gsarp1 and gsarp2 begin to be detected at the early and later pachytene stage of meiosis separately, while both have high-expression level at the stage of MI and MII. The result of in situ hybridization reveals that the gsarp1 is primarily expressed in spermatocytes, while gsarp2 is expressed in spermatocytes and spermatids. In view of the structural feature and expression pattern of the GSARP1 and GSARP2, we speculate that they may play a certain role in a signal pathway of meiosis.

  4. Comprehensive Expression Map of Transcription Regulators in the Adult Zebrafish Telencephalon Reveals Distinct Neurogenic Niches

    PubMed Central

    Diotel, Nicolas; Rodriguez Viales, Rebecca; Armant, Olivier; März, Martin; Ferg, Marco; Rastegar, Sepand; Strähle, Uwe

    2015-01-01

    The zebrafish has become a model to study adult vertebrate neurogenesis. In particular, the adult telencephalon has been an intensely studied structure in the zebrafish brain. Differential expression of transcriptional regulators (TRs) is a key feature of development and tissue homeostasis. Here we report an expression map of 1,202 TR genes in the telencephalon of adult zebrafish. Our results are summarized in a database with search and clustering functions to identify genes expressed in particular regions of the telencephalon. We classified 562 genes into 13 distinct patterns, including genes expressed in the proliferative zone. The remaining 640 genes displayed unique and complex patterns of expression and could thus not be grouped into distinct classes. The neurogenic ventricular regions express overlapping but distinct sets of TR genes, suggesting regional differences in the neurogenic niches in the telencephalon. In summary, the small telencephalon of the zebrafish shows a remarkable complexity in TR gene expression. The adult zebrafish telencephalon has become a model to study neurogenesis. We established the expression pattern of more than 1200 transcription regulators (TR) in the adult telencephalon. The neurogenic regions express overlapping but distinct sets of TR genes suggesting regional differences in the neurogenic potential. J. Comp. Neurol. 523:1202–1221, 2015. © 2015 Wiley Periodicals, Inc. PMID:25556858

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

    PubMed Central

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

    2009-01-01

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

  6. Robust stratification of breast cancer subtypes using differential patterns of transcript isoform expression.

    PubMed

    Stricker, Thomas P; Brown, Christopher D; Bandlamudi, Chaitanya; McNerney, Megan; Kittler, Ralf; Montoya, Vanessa; Peterson, April; Grossman, Robert; White, Kevin P

    2017-03-01

    Breast cancer, the second leading cause of cancer death of women worldwide, is a heterogenous disease with multiple different subtypes. These subtypes carry important implications for prognosis and therapy. Interestingly, it is known that these different subtypes not only have different biological behaviors, but also have distinct gene expression profiles. However, it has not been rigorously explored whether particular transcriptional isoforms are also differentially expressed among breast cancer subtypes, or whether transcript isoforms from the same sets of genes can be used to differentiate subtypes. To address these questions, we analyzed the patterns of transcript isoform expression using a small set of RNA-sequencing data for eleven Estrogen Receptor positive (ER+) subtype and fourteen triple negative (TN) subtype tumors. We identified specific sets of isoforms that distinguish these tumor subtypes with higher fidelity than standard mRNA expression profiles. We found that alternate promoter usage, alternative splicing, and alternate 3'UTR usage are differentially regulated in breast cancer subtypes. Profiling of isoform expression in a second, independent cohort of 68 tumors confirmed that expression of splice isoforms differentiates breast cancer subtypes. Furthermore, analysis of RNAseq data from 594 cases from the TCGA cohort confirmed the ability of isoform usage to distinguish breast cancer subtypes. Also using our expression data, we identified several RNA processing factors that were differentially expressed between tumor subtypes and/or regulated by estrogen receptor, including YBX1, YBX2, MAGOH, MAGOHB, and PCBP2. RNAi knock-down of these RNA processing factors in MCF7 cells altered isoform expression. These results indicate that global dysregulation of splicing in breast cancer occurs in a subtype-specific and reproducible manner and is driven by specific differentially expressed RNA processing factors.

  7. Robust stratification of breast cancer subtypes using differential patterns of transcript isoform expression

    PubMed Central

    Stricker, Thomas P.; Bandlamudi, Chaitanya; Kittler, Ralf; Montoya, Vanessa; Peterson, April; Grossman, Robert

    2017-01-01

    Breast cancer, the second leading cause of cancer death of women worldwide, is a heterogenous disease with multiple different subtypes. These subtypes carry important implications for prognosis and therapy. Interestingly, it is known that these different subtypes not only have different biological behaviors, but also have distinct gene expression profiles. However, it has not been rigorously explored whether particular transcriptional isoforms are also differentially expressed among breast cancer subtypes, or whether transcript isoforms from the same sets of genes can be used to differentiate subtypes. To address these questions, we analyzed the patterns of transcript isoform expression using a small set of RNA-sequencing data for eleven Estrogen Receptor positive (ER+) subtype and fourteen triple negative (TN) subtype tumors. We identified specific sets of isoforms that distinguish these tumor subtypes with higher fidelity than standard mRNA expression profiles. We found that alternate promoter usage, alternative splicing, and alternate 3’UTR usage are differentially regulated in breast cancer subtypes. Profiling of isoform expression in a second, independent cohort of 68 tumors confirmed that expression of splice isoforms differentiates breast cancer subtypes. Furthermore, analysis of RNAseq data from 594 cases from the TCGA cohort confirmed the ability of isoform usage to distinguish breast cancer subtypes. Also using our expression data, we identified several RNA processing factors that were differentially expressed between tumor subtypes and/or regulated by estrogen receptor, including YBX1, YBX2, MAGOH, MAGOHB, and PCBP2. RNAi knock-down of these RNA processing factors in MCF7 cells altered isoform expression. These results indicate that global dysregulation of splicing in breast cancer occurs in a subtype-specific and reproducible manner and is driven by specific differentially expressed RNA processing factors. PMID:28263985

  8. 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.

  9. 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.

  10. Differential expression of KRT83 regulated by the transcript factor CAP1 in Chinese Tan sheep.

    PubMed

    Liu, Yufang; Kang, Xiaolong; Yang, Wanjie; Xie, Minggui; Zhang, Jibin; Fang, Meiying

    2017-03-08

    Keratin 83 (KRT83) is an important keratin protein in hair development. In this study, expression of KRT83 was compared among different tissues and between 1-month-old lambs and 48-month adult of Chinese Tan sheep, which showed different fleece phenotypes. The results showed that KRT83 was only expressed in skin, and KRT83 mRNA level in skin was significantly higher in Tan lambs than in adult sheep. To further understand the expression regulation of KRT83 by transcription factors in Tan sheep, amplified sequences coving different ranges of KRT83 promoter region were inserted into a pGL3-basic vector and then transfected into sheep primary fibroblast cells. Luciferase assay indicated that the sequence from -218bp to -10bp in the KRT83 promoter induced the highest transcription activity of the vector in the fibroblast cells. Transcription factor adenylate cyclase-associated protein 1 (CAP1) was predicted by online tools within this region. Electrophoretic mobility shift assay (EMSA) confirmed binding of the purified CAP1 protein to the target core region from -88bp to -10bp, because mutation in the target core sequence resulted in failure of CAP1 binding to the target region. Moreover, overexpression of CAP1 protein led to repression of the KRT83 promoter activity in sheep primary fibroblast cells, and expression of CAP1 was lower in lambs than in adult sheep. Therefore, we concluded that CAP1 is a key transcription factor involved in negative regulation of KRT83 expression in Tan sheep skin. Our study provides new insights into the transcriptional regulation of KRT83 and further hints of its critical role in curly hair phenotype in sheep.

  11. 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

  12. TIGER: Toolbox for integrating genome-scale metabolic models, expression data, and transcriptional regulatory networks

    PubMed Central

    2011-01-01

    Background Several methods have been developed for analyzing genome-scale models of metabolism and transcriptional regulation. Many of these methods, such as Flux Balance Analysis, use constrained optimization to predict relationships between metabolic flux and the genes that encode and regulate enzyme activity. Recently, mixed integer programming has been used to encode these gene-protein-reaction (GPR) relationships into a single optimization problem, but these techniques are often of limited generality and lack a tool for automating the conversion of rules to a coupled regulatory/metabolic model. Results We present TIGER, a Toolbox for Integrating Genome-scale Metabolism, Expression, and Regulation. TIGER converts a series of generalized, Boolean or multilevel rules into a set of mixed integer inequalities. The package also includes implementations of existing algorithms to integrate high-throughput expression data with genome-scale models of metabolism and transcriptional regulation. We demonstrate how TIGER automates the coupling of a genome-scale metabolic model with GPR logic and models of transcriptional regulation, thereby serving as a platform for algorithm development and large-scale metabolic analysis. Additionally, we demonstrate how TIGER's algorithms can be used to identify inconsistencies and improve existing models of transcriptional regulation with examples from the reconstructed transcriptional regulatory network of Saccharomyces cerevisiae. Conclusion The TIGER package provides a consistent platform for algorithm development and extending existing genome-scale metabolic models with regulatory networks and high-throughput data. PMID:21943338

  13. Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development

    PubMed Central

    Anderson, Kelly M.; Anderson, Douglas M.; McAnally, John R.; Shelton, John M.; Bassel-Duby, Rhonda; Olson, Eric N.

    2017-01-01

    HAND2 is an ancestral regulator of heart development and one of four transcription factors that control the reprogramming of fibroblasts into cardiomyocytes1–4. Deletion of Hand2 in mice results in right ventricle hypoplasia and embryonic lethality1,5. Hand2 expression is tightly regulated by upstream enhancers6,7 that reside within a super-enhancer delineated by histone H3 acetyl Lys27 (H3K27ac) modifications8. Here we show that transcription of a Hand2-associated long non-coding RNA, which we named upperhand (Uph), is required to maintain the super-enhancer signature and elongation of RNA polymerase II through the Hand2 enhancer locus. Blockade of Uph transcription, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated long non-coding RNAs encoded by the mammalian genome9, the Uph–Hand2 regulatory partnership offers a mechanism by which divergent non-coding transcription can establish a permissive chromatin environment. PMID:27783597

  14. Gastrointestinal Fibroblasts Have Specialized, Diverse Transcriptional Phenotypes: A Comprehensive Gene Expression Analysis of Human Fibroblasts

    PubMed Central

    Ishii, Genichiro; Aoyagi, Kazuhiko; Sasaki, Hiroki; Ochiai, Atsushi

    2015-01-01

    Background Fibroblasts are the principal stromal cells that exist in whole organs and play vital roles in many biological processes. Although the functional diversity of fibroblasts has been estimated, a comprehensive analysis of fibroblasts from the whole body has not been performed and their transcriptional diversity has not been sufficiently explored. The aim of this study was to elucidate the transcriptional diversity of human fibroblasts within the whole body. Methods Global gene expression analysis was performed on 63 human primary fibroblasts from 13 organs. Of these, 32 fibroblasts from gastrointestinal organs (gastrointestinal fibroblasts: GIFs) were obtained from a pair of 2 anatomical sites: the submucosal layer (submucosal fibroblasts: SMFs) and the subperitoneal layer (subperitoneal fibroblasts: SPFs). Using hierarchical clustering analysis, we elucidated identifiable subgroups of fibroblasts and analyzed the transcriptional character of each subgroup. Results In unsupervised clustering, 2 major clusters that separate GIFs and non-GIFs were observed. Organ- and anatomical site-dependent clusters within GIFs were also observed. The signature genes that discriminated GIFs from non-GIFs, SMFs from SPFs, and the fibroblasts of one organ from another organ consisted of genes associated with transcriptional regulation, signaling ligands, and extracellular matrix remodeling. Conclusions GIFs are characteristic fibroblasts with specific gene expressions from transcriptional regulation, signaling ligands, and extracellular matrix remodeling related genes. In addition, the anatomical site- and organ-dependent diversity of GIFs was also discovered. These features of GIFs contribute to their specific physiological function and homeostatic maintenance, and create a functional diversity of the gastrointestinal tract. PMID:26046848

  15. 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.

  16. Impact of improving dietary amino acid balance for lactating sows on efficiency of dietary amino acid utilization and transcript abundance of genes encoding lysine transporters in mammary tissue.

    PubMed

    Huber, L; de Lange, C F M; Ernst, C W; Krogh, U; Trottier, N L

    2016-11-01

    Lactating multiparous Yorkshire sows ( = 64) were used in 2 experiments to test the hypothesis that reducing dietary CP intake and improving AA balance through crystalline AA (CAA) supplementation improves apparent dietary AA utilization efficiency for milk production and increases transcript abundance of genes encoding Lys transporter proteins in mammary tissue. In Exp. 1, 40 sows were assigned to 1 of 4 diets: 1) high CP (HCP; 16.0% CP, as-fed basis; analyzed concentration), 2) medium-high CP (MHCP; 15.7% CP), 3) medium-low CP (MLCP; 14.3% CP), and 4) low CP (LCP; 13.2% CP). The HCP diet was formulated using soybean meal and corn as the only Lys sources. The reduced-CP diets contained CAA to meet estimated requirements for essential AA that became progressively limiting with reduction in CP concentration, that is, Lys, Ile, Met + Cys, Thr, Trp, and Val. Dietary standardized ileal digestible (SID) Lys concentration was 80% of the estimated requirement. In Exp. 2, 24 sows were assigned to the HCP or LCP diets. In Exp. 1, blood samples were postprandially collected 15 h on d 3, 7, 14, and 18 of lactation and utilization efficiency of dietary AA for milk production was calculated during early (d 3 to 7) and peak (d 14 to 18) lactation. Efficiency values were estimated from daily SID AA intakes and milk AA yield, with corrections for maternal AA requirement for maintenance and AA contribution from body protein losses. In Exp. 2, mammary tissue was biopsied on d 4 and 14 of lactation to determine the mRNA abundance of genes encoding Lys transporter proteins. In peak lactation, Lys, Thr, Trp, and Val utilization efficiency increased with decreasing dietary CP (linear for Trp and Val, < 0.05; in sows fed the MHCP diet vs. sows fed the HCP diet for Lys and Thr, < 0.05). Total essential and nonessential 15-h postprandial serum AA concentrations increased with decreasing dietary CP (linear, = 0.09 and < 0.05, respectively), suggesting increased maternal body protein

  17. 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

  18. 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.

  19. Transcriptional abundance of antioxidant enzymes in endometrium and their circulating levels in Zebu cows with and without uterine infection.

    PubMed

    Baithalu, R K; Singh, S K; Kumaresan, A; Mohanty, A K; Mohanty, T K; Kumar, S; Kerketta, S; Maharana, B R; Patbandha, T K; Attupuram, N; Agarwal, S K

    2017-02-01

    Oxidative stress during peripartum period may compromise the uterine immunity. In the present study, we assessed the oxidative stress and antioxidant status during peripartum period and studied their relationship with postpartum uterine infection in dairy cows. Peripheral blood concentrations of total antioxidant capacity (TAC), malondialdehyde (MDA) and nitric oxide (NO) were determined (day -21, -7, on the day of calving and day +7, +21, +35) in normal (n=11), puerperal metritic (n=7) and clinical endometritic (n=6) cows. Endometrial biopsy was performed on the day of calving and expression of CAT, GPx4 and SOD2 genes was studied using qRT-PCR. Puerperal metritic cows had significantly (P<0.05) lower TAC (on day -7, day 0, day +7, +21 & +35), higher MDA (on day -21, -7 & on the day of calving) and NO (on day 0, +7 & day +35) concentrations compared to normal cows. Similarly, clinical endometritic cows had significantly (P<0.05) lower TAC (on day -7, 0, +7 & +21), higher MDA (on day -21, -7, +7 and +35) and NO (on day +7, +21 & +35) concentrations compared to normal cows. The expression of CAT and GPx4 genes was lower (P<0.05) and SOD2 gene was higher (P<0.05) in endometrial tissue of cows that developed uterine infection compared to normal cows. The relationship of peripheral levels of MDA and NO with antioxidant enzymes expression in endometrial tissue was found significant. Receiver operator characteristic analysis revealed that the concentrations of TAC on day -7 to day +35, MDA on day -21 to day +7 and NO on the day of calving to day +35 were highly correlated to the development of postpartum uterine infection in cows. It may be inferred that the low serum TAC level and high level of lipid peroxidation and NO during peripartum period influenced the endometrial expression of anitioxidative genes that compromised the uterine health during postpartum period.

  20. The Transcription Factor FoxK Participates with Nup98 To Regulate Antiviral Gene Expression

    PubMed Central

    Panda, Debasis; Gold, Beth; Tartell, Michael A.; Rausch, Keiko; Casas-Tinto, Sergio

    2015-01-01

    ABSTRACT Upon infection, pathogen recognition leads to a rapidly activated gene expression program that induces antimicrobial effectors to clear the invader. We recently found that Nup98 regulates the expression of a subset of rapidly activated antiviral genes to restrict disparate RNA virus infections in Drosophila by promoting RNA polymerase occupancy at the promoters of these antiviral genes. How Nup98 specifically targets these loci was unclear; however, it is known that Nup98 participates with transcription factors to regulate developmental-gene activation. We reasoned that additional transcription factors may facilitate the Nup98-dependent expression of antiviral genes. In a genome-wide RNA interference (RNAi) screen, we identified a relatively understudied forkhead transcription factor, FoxK, as active against Sindbis virus (SINV) in Drosophila. Here we find that FoxK is active against the panel of viruses that are restricted by Nup98, including SINV and vesicular stomatitis virus (VSV). Mechanistically, we show that FoxK coordinately regulates the Nup98-dependent expression of antiviral genes. Depletion of FoxK significantly reduces Nup98-dependent induction of antiviral genes and reduces the expression of a forkhead response element-containing luciferase reporter. Together, these data show that FoxK-mediated activation of gene expression is Nup98 dependent. We extended our studies to mammalian cells and found that the mammalian ortholog FOXK1 is antiviral against two disparate RNA viruses, SINV and VSV, in human cells. Interestingly, FOXK1 also plays a role in the expression of antiviral genes in mammals: depletion of FOXK1 attenuates virus-inducible interferon-stimulated response element (ISRE) reporter expression. Overall, our results demonstrate a novel role for FOXK1 in regulating the expression of antiviral genes, from insects to humans. PMID:25852164

  1. Expression of growth hormone and its transcription factor, Pit-1, in early bovine development.

    PubMed

    Joudrey, E M; Lechniak, D; Petrik, J; King, W A

    2003-03-01

    During bovine embryogenesis, bovine growth hormone (bGH) contributes to proliferation, differentiation, and modulation of embryo metabolism. Pituitary-specific transcription factor-1 (Pit-1) is a transcription factor that binds to promoters of GH, prolactin (PRL), and thyroid-stimulating hormone-beta (TSHbeta) encoding genes. A polymorphism in the fifth exon of the bGH gene resulting in a leucine (Leu) to valine (Val) substitution provides an Alu I restriction site when the Leu allele is present. To determine the onset of embryonic expression of the bGH gene, oocytes derived from ovaries homozygous for Leu alleles were fertilized in vitro with spermatozoa obtained from a Val homozygote. For each developmental stage examined, three separate pools of embryos composed of approximately 100 cell samples underwent RNA isolation, reverse transcription to cDNA, and amplification by nested PCR (nPCR). Bovine GH gene transcripts were identified at 2- to 4-cell (n = 162), 8- to 16-cell (n = 73), morulae (n = 51), and blastocyst (n = 15) stages. Likewise, transcripts for Pit-1 were detected at 2-cell (n = 125), 4-cell (n = 114), 8-cell (n = 56), 12-to-32-cell (n = 32), morulae (n = 68), and blastocyst (n = 14) stages. After digestion with Alu1, bGH cDNA was genotyped by restriction fragment length polymorphism (RFLP) analysis. Bovine GH mRNA was present in all pools of stages examined. Both Leu and Val alleles (maternal and paternal) were only detected in pools of embryos that had reached 8- to 16-cell stage. Results suggest that transcription of the bGH gene begins at the 8- to 16-cell stage in bovine embryos, possibly under control of the transcription factor, Pit-1, and that RFLP analysis of the bGH gene can be used to determine parental origin of transcripts in early embryonic development.

  2. The differentially DNA-methylated region responsible for expression of runt-related transcription factor 2

    PubMed Central

    WAKITANI, Shoichi; YOKOI, Daigo; HIDAKA, Yuichi; NISHINO, Koichiro

    2016-01-01

    Runt-related transcription factor 2 (Runx2) is essential for osteogenesis. This study aimes at identification of the genomic region differentially methylated in DNA for regulation of Runx2 expression. In the proximal promoter of mouse Runx2, DNA methylation was frequent at the region further than 3 kb relative to the transcription start site, in contrast to lower methylation status of the closer locus within 2 kb from the transcription start site. At the intermediate part, we identified a novel differentially methylated region in the Runx2 promoter region (Runx2-DMR): from −2.7 to −2.2 kb relative to the start site of Runx2 transcription in mice. In this region, the DNA methylation rate correlated negatively with Runx2 expression among mouse organs as well as among primary cultures of bone marrow from different dogs. Induction of mouse and dog mesenchymal-like cells into osteoblastic differentiation decreased the methylation rate of Runx2-DMR. Thus, in this study, we identified a novel genomic region in which DNA methylation status is related to Runx2 expression and detected demethylation of Runx2-DMR during osteoblastic differentiation in mouse and dog. PMID:27916785

  3. Expression of transcripts for cysteine-rich secretory proteins (CRISPs) in the murine lacrimal gland.

    PubMed

    Haendler, B; Toda, I; Sullivan, D A; Schleuning, W D

    1999-03-01

    Cysteine-rich secretory proteins (CRISPs) represent a family of evolutionarily conserved proteins which may play a role in the innate immune system and are transcriptionally regulated by androgens in several tissues. Transcripts for all three members of the CRISP family have now been identified in the murine lacrimal gland. RT-PCR using primers able to discriminate between the related CRISP forms allowed the amplification of fragments with the expected length. DNA sequencing revealed a complete identity with the hitherto characterized epididymal CRISP-1, testicular CRISP-2, and salivary gland CRISP-3. An analysis of several mouse strains indicated that all expressed the three CRISP forms, but in differing amounts. RT-PCR analysis of RNA isolated from acinar cells of lacrimal glands revealed that they expressed CRISP-1 and CRISP-2. Semiquantitative and quantitative analyses furthermore showed higher CRISP-1 and CRISP-3 mRNA levels in the lacrimal glands of male BALB/c and NOD mice when compared to females. Testosterone treatment of C3H/HeJ female mice was followed by an upregulation of the steady-state CRISP-1 but not CRISP-2 transcript levels. A comparable stimulation was observed for the mRNAs coding for parotid secretory protein (PSP), a factor previously shown to exhibit sexual dimorphism in the murine lacrimal gland. The expression of CRISP transcripts in the lacrimal gland is consistent with a function in the innate immune system.

  4. Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes.

    PubMed

    Cestari, Igor; Stuart, Ken

    2015-05-26

    African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.

  5. Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

    PubMed Central

    Cestari, Igor; Stuart, Ken

    2015-01-01

    African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing. PMID:25964327

  6. 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.

  7. Proposed Method for Estimating Health-Promoting Glucosinolates and Hydrolysis Products in Broccoli (Brassica oleracea var. italica) Using Relative Transcript Abundance.

    PubMed

    Becker, Talon M; Jeffery, Elizabeth H; Juvik, John A

    2017-01-18

    Due to the importance of glucosinolates and their hydrolysis products in human nutrition and plant defense, optimizing the content of these compounds is a frequent breeding objective for Brassica crops. Toward this goal, we investigated the feasibility of using models built from relative transcript abundance data for the prediction of glucosinolate and hydrolysis product concentrations in broccoli. We report that predictive models explaining at least 50% of the variation for a number of glucosinolates and their hydrolysis products can be built for prediction within the same season, but prediction accuracy decreased when using models built from one season's data for prediction of an opposing season. This method of phytochemical profile prediction could potentially allow for lower phytochemical phenotyping costs and larger breeding populations. This, in turn, could improve selection efficiency for phase II induction potential, a type of chemopreventive bioactivity, by allowing for the quick and relatively cheap content estimation of phytochemicals known to influence the trait.

  8. 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...

  9. 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…

  10. Expression of SDF-1 and CXCR4 transcript variants and CXCR7 in epithelial ovarian cancer.

    PubMed

    Jaszczynska-Nowinka, Karolina; Rucinski, Marcin; Ziolkowska, Agnieszka; Markowska, Anna; Malendowicz, Ludwik K

    2014-05-01

    Chemokine stromal cell-derived factor-1 (SDF-1) and its receptors, CXCR4 and CXCR7, have been implicated in epithelial ovarian cancer progression and metastasis. However, limited data are available on the expression levels of SDF-1 and CXCR4 variants and CXCR7 in human epithelial ovarian cancer. The present study aimed to characterize the expression pattern and levels of SDF-1, CXCR4 and CXCR7 in normal human ovaries and epithelial ovarian cancer. The expression of SDF-1 and CXCR4 transcript variants and CXCR7 was determined by quantitative polymerase chain reaction (qPCR). Plasma SDF-1α levels were determined by commercially available EIA kits and cancer antigen 125 (CA 125) levels were quantified by automated microparticle enzyme immunosorbent assay. High expression levels of SDF-1 transcript variant 1 were identified in ovarian cancer and control ovaries. By contrast, in both groups the expression levels of SDF-1 transcript variants 3 and 4 were extremely low. Furthermore, SDF-1 variant 1 levels were notably higher in epithelial ovarian cancer than in control ovaries, while data for the remaining transcripts were similar in both groups. CXCR4 transcript variant 2 and CXCR7 expression levels in normal and neoplastic ovaries were similar. In both groups, CXCR4 transcript variant 2 was not detected. Plasma SDF-1α levels were notably higher in females with epithelial ovarian cancer than in the control ovaries. Elevated levels of blood SDF-1α were found prior to surgery, 6 days after surgery and following completion of the first chemotherapy course. These increases were independent of the type of epithelial ovarian cancer. Our results suggest that the expression of SDF-1 and the genes controlling alternative splicing are elevated in epithelial ovarian cancer, leading to an increased formation of SDF-1 variant 1. Elevated plasma SDF-1α levels in epithelial ovarian cancer patients are not associated with the presence of tumors and/or metastases, however reflect a

  11. 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.

  12. Transcription factors that directly regulate the expression of CSLA9 encoding mannan synthase in Arabidopsis thaliana.

    PubMed

    Kim, Won-Chan; Reca, Ida-Barbara; Kim, Yongsig; Park, Sunchung; Thomashow, Michael F; Keegstra, Kenneth; Han, Kyung-Hwan

    2014-03-01

    Mannans are hemicellulosic polysaccharides that have a structural role and serve as storage reserves during plant growth and development. Previous studies led to the conclusion that mannan synthase enzymes in several plant species are encoded by members of the cellulose synthase-like A (CSLA) gene family. Arabidopsis has nine members of the CSLA gene family. Earlier work has shown that CSLA9 is responsible for the majority of glucomannan synthesis in both primary and secondary cell walls of Arabidopsis inflorescence stems. Little is known about how expression of the CLSA9 gene is regulated. Sequence analysis of the CSLA9 promoter region revealed the presence of multiple copies of a cis-regulatory motif (M46RE) recognized by transcription factor MYB46, leading to the hypothesis that MYB46 (At5g12870) is a direct regulator of the mannan synthase CLSA9. We obtained several lines of experimental evidence in support of this hypothesis. First, the expression of CSLA9 was substantially upregulated by MYB46 overexpression. Second, electrophoretic mobility shift assay (EMSA) was used to demonstrate the direct binding of MYB46 to the promoter of CSLA9 in vitro. This interaction was further confirmed in vivo by a chromatin immunoprecipitation assay. Finally, over-expression of MYB46 resulted in a significant increase in mannan content. Considering the multifaceted nature of MYB46-mediated transcriptional regulation of secondary wall biosynthesis, we reasoned that additional transcription factors are involved in the CSLA9 regulation. This hypothesis was tested by carrying out yeast-one hybrid screening, which identified ANAC041 and bZIP1 as direct regulators of CSLA9. Transcriptional activation assays and EMSA were used to confirm the yeast-one hybrid results. Taken together, we report that transcription factors ANAC041, bZIP1 and MYB46 directly regulate the expression of CSLA9.

  13. The Fli-1 transcription factor regulates the expression of CCL5/RANTES1

    PubMed Central

    Lennard Richard, Mara L.; Sato, Shuzo; Suzuki, Eiji; Williams, Sarah; Nowling, Tamara K.; Zhang, Xian K.

    2014-01-01

    The Fli-1 transcription factor, an Ets family member, is implicated in the pathogenesis of systemic lupus erythematosus (SLE) in human patients and murine models of lupus. Lupus-prone mice with reduced Fli-1 expression have significantly less nephritis, prolonged survival and decreased infiltrating inflammatory cells into the kidney. Inflammatory chemokines, including CCL5, are critical for attracting inflammatory cells. In this study, decreased CCL5 mRNA expression was observed in kidneys of lupus prone NZM2410 mice, with reduced Fli-1 expression. CCL5 protein expression was significantly decreased in endothelial cells transfected with Fli-1 specific siRNA compared to controls. Fli-1 binds to endogenous Ets binding sites in the distal region of the CCL5 promoter. Transient transfection assays demonstrate that Fli-1 drives transcription from the CCL5 promoter in a dose-dependent manner. Both Ets1, another Ets family member, and Fli-1 drive transcription from the CCL5 promoter, although Fli-1 transactivation was significantly stronger. Ets1 acts as a dominant negative transcription factor to Fli-1, indicating that they may have at least one DNA binding site in common. Systematic deletion of DNA binding sites demonstrates the importance of the sites located within a 225bp region of the promoter. Mutation of the Fli-1 DNA binding domain significantly reduces transactivation of the CCL5 promoter by Fli-1. We have identified a novel regulator of transcription for CCL5. These results suggest that Fli-1 is a novel and critical regulator of proinflammatory chemokines and affects the pathogenesis of disease through the regulation of factors that recruit inflammatory cells to sites of inflammation. PMID:25098295

  14. The Fli-1 transcription factor regulates the expression of CCL5/RANTES.

    PubMed

    Lennard Richard, Mara L; Sato, Shuzo; Suzuki, Eiji; Williams, Sarah; Nowling, Tamara K; Zhang, Xian K

    2014-09-15

    The friend leukemia insertion site 1 (Fli-1) transcription factor, an Ets family member, is implicated in the pathogenesis of systemic lupus erythematosus in human patients and murine models of lupus. Lupus-prone mice with reduced Fli-1 expression have significantly less nephritis, prolonged survival, and decreased infiltrating inflammatory cells into the kidney. Inflammatory chemokines, including CCL5, are critical for attracting inflammatory cells. In this study, decreased CCL5 mRNA expression was observed in kidneys of lupus-prone NZM2410 mice with reduced Fli-1 expression. CCL5 protein expression was significantly decreased in endothelial cells transfected with Fli-1-specific small interfering RNA compared with controls. Fli-1 binds to endogenous Ets binding sites in the distal region of the CCL5 promoter. Transient transfection assays demonstrate that Fli-1 drives transcription from the CCL5 promoter in a dose-dependent manner. Both Ets1, another Ets family member, and Fli-1 drive transcription from the CCL5 promoter, although Fli-1 transactivation was significantly stronger. Ets1 acts as a dominant-negative transcription factor for Fli-1, indicating that they may have at least one DNA binding site in common. Systematic deletion of DNA binding sites demonstrates the importance of the sites located within a 225-bp region of the promoter. Mutation of the Fli-1 DNA binding domain significantly reduces transactivation of the CCL5 promoter by Fli-1. We identified a novel regulator of transcription for CCL5. These results suggest that Fli-1 is a novel and critical regulator of proinflammatory chemokines and affects the pathogenesis of disease through the regulation of factors that recruit inflammatory cells to sites of inflammation.

  15. 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.

  16. 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

  17. Phosphorus acquisition efficiency in arbuscular mycorrhizal maize is correlated with the abundance of root-external hyphae and the accumulation of transcripts encoding PHT1 phosphate transporters.

    PubMed

    Sawers, Ruairidh J H; Svane, Simon F; Quan, Clement; Grønlund, Mette; Wozniak, Barbara; Gebreselassie, Mesfin-Nigussie; González-Muñoz, Eliécer; Chávez Montes, Ricardo A; Baxter, Ivan; Goudet, Jerome; Jakobsen, Iver; Paszkowski, Uta

    2017-04-01

    Plant interactions with arbuscular mycorrhizal fungi have long attracted interest for their potential to promote more efficient use of mineral resources in agriculture. Their use, however, remains limited by a lack of understanding of the processes that determine the outcome of the symbiosis. In this study, the impact of host genotype on growth response to mycorrhizal inoculation was investigated in a panel of diverse maize lines. A panel of 30 maize lines was evaluated with and without inoculation with arbuscular mycorrhizal fungi. The line Oh43 was identified to show superior response and, along with five other reference lines, was characterized in greater detail in a split-compartment system, using (33) P to quantify mycorrhizal phosphorus uptake. Changes in relative growth indicated variation in host capacity to profit from the symbiosis. Shoot phosphate content, abundance of root-internal and -external fungal structures, mycorrhizal phosphorus uptake, and accumulation of transcripts encoding plant PHT1 family phosphate transporters varied among lines. Superior response in Oh43 is correlated with extensive development of root-external hyphae, accumulation of specific Pht1 transcripts and high phosphorus uptake by mycorrhizal plants. The data indicate that host genetic factors influence fungal growth strategy with an impact on plant performance.

  18. PJA1, encoding a RING-H2 finger ubiquitin ligase, is a novel human X chromosome gene abundantly expressed in brain.

    PubMed

    Yu, Ping; Chen, Yiwang; Tagle, Danilo A; Cai, Tao

    2002-06-01

    RING-finger proteins contain cysteine-rich, zinc-binding domains and are involved in the formation of macromolecular scaffolds important for transcriptional repression and ubiquitination. In this study, we have identified a RING-H2 finger gene, PJA1 (for praja-1), from a human brain cDNA library and mapped it to human chromosome Xq12 between markers DXS983 and DXS1216, a region implicated in X-linked mental retardation (MRX). Northern blot analysis indicated a 2.7-kb transcript that was abundantly expressed in the brain, including regions of the cerebellum, cerebral cortex, medulla, occipital pole, frontal lobe, temporal lobe, and putamen. Amino acid sequence analysis of the 71-kDa protein PJA1 showed 52.3% identity to human PJA2 (for praja-2, also known as NEURODAP1/KIAA0438) and also a significant identity to its homologs in rat, mouse, and zebrafish. In vitro binding and immunoprecipitation assays demonstrated that both PJA1 and PJA2 are able to bind the ubiquitin-conjugating enzyme UbcH5B. Moreover, the ubiquitination assay indicated that PJA1 and PJA2 have an E2-dependent E3 ubiquitin ligase activity. Thus our findings demonstrate that PJA1 can be involved in protein ubiquitination in the brain and is a suitable candidate gene for MRX.

  19. Overexpression of Transcription Factor Sp1 Leads to Gene Expression Perturbations and Cell Cycle Inhibition

    PubMed Central

    Deniaud, Emmanuelle; Baguet, Joël; Chalard, Roxane; Blanquier, Bariza; Brinza, Lilia; Meunier, Julien; Michallet, Marie-Cécile; Laugraud, Aurélie; Ah-Soon, Claudette; Wierinckx, Anne; Castellazzi, Marc; Lachuer, Joël; Gautier, Christian

    2009-01-01

    Background The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression. Methodology and Principal Findings We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis. Conclusion This study shows that the binding to DNA of overexpressed Sp1

  20. Variability of Gene Expression Identifies Transcriptional Regulators of Early Human Embryonic Development

    PubMed Central

    Hasegawa, Yu; Taylor, Deanne; Ovchinnikov, Dmitry A.; Wolvetang, Ernst J.; de Torrenté, Laurence; Mar, Jessica C.

    2015-01-01

    An analysis of gene expression variability can provide an insightful window into how regulatory control is distributed across the transcriptome. In a single cell analysis, the inter-cellular variability of gene expression measures the consistency of transcript copy numbers observed between cells in the same population. Application of these ideas to the study of early human embryonic development may reveal important insights into the transcriptional programs controlling this process, based on which components are most tightly regulated. Using a published single cell RNA-seq data set of human embryos collected at four-cell, eight-cell, morula and blastocyst stages, we identified genes with the most stable, invariant expression across all four developmental stages. Stably-expressed genes were found to be enriched for those sharing indispensable features, including essentiality, haploinsufficiency, and ubiquitous expression. The stable genes were less likely to be associated with loss-of-function variant genes or human recessive disease genes affected by a DNA copy number variant deletion, suggesting that stable genes have a functional impact on the regulation of some of the basic cellular processes. Genes with low expression variability at early stages of development are involved in regulation of DNA methylation, responses to hypoxia and telomerase activity, whereas by the blastocyst stage, low-variability genes are enriched for metabolic processes as well as telomerase signaling. Based on changes in expression variability, we identified a putative set of gene expression markers of morulae and blastocyst stages. Experimental validation of a blastocyst-expressed variability marker demonstrated that HDDC2 plays a role in the maintenance of pluripotency in human ES and iPS cells. Collectively our analyses identified new regulators involved in human embryonic development that would have otherwise been missed using methods that focus on assessment of the average expression

  1. PML/RARa inhibits PTEN expression in hematopoietic cells by competing with PU.1 transcriptional activity

    PubMed Central

    Noguera, Nélida Inés; Piredda, Maria Liliana; Taulli, Riccardo; Catalano, Gianfranco; Angelini, Giulia; Gaur, Girish; Nervi, Clara; Voso, Maria Teresa; Lunardi, Andrea; Pandolfi, Pier Paolo; Lo-Coco, Francesco

    2016-01-01

    Acute promyelocitic leukemia (APL) is characterized by the pathognomonic presence in leukemic blasts of the hybrid protein PML/RARA, that acts as a transcriptional repressor impairing the expression of genes that are critical to myeloid differentiation. Here, we show that primary blasts from APL patients express lower levels of the oncosuppressor protein PTEN, as compared to blast cells from other AML subtypes or normal bone marrow, and demonstrate that PML-RARA directly inhibits PTEN expression. We show that All-Trans Retinoic Acid (ATRA) triggers in APL cells an active chromatin status at the core regulatory region of the PTEN promoter, that allows the binding of the myeloid-regulating transcription factor PU.1, and, in turn, the transcriptional induction of PTEN. ATRA, via PML/RARA degradation, also promotes PTEN nuclear re-localization and decreases expression of the PTEN target Aurora A kinase. In conclusion, our findings support the notion that PTEN is one of the primary targets of PML/RARA in APL PMID:27626703

  2. Continued Discovery of Transcriptional Units Expressed in Cells of the Mouse Mononuclear Phagocyte Lineage

    PubMed Central

    Wells, Christine A.; Ravasi, Timothy; Sultana, Razvan; Yagi, Ken; Carninci, Piero; Bono, Hidemasa; Faulkner, Geoffrey; Okazaki, Yasushi; Quackenbush, John; Hume, David A.; Lyons, Paul A.

    2003-01-01

    The current RIKEN transcript set represents a significant proportion of the mouse transcriptome but transcripts expressed in the innate and acquired immune systems are poorly represented. In the present study we have assessed the complexity of the transcriptome expressed in mouse macrophages before and after treatment with lipopolysaccharide, a global regulator of macrophage gene expression, using existing RIKEN 19K arrays. By comparison to array profiles of other cells and tissues, we identify a large set of macrophage-enriched genes, many of which have obvious functions in endocytosis and phagocytosis. In addition, a significant number of LPS-inducible genes were identified. The data suggest that macrophages are a complex source of mRNA for transcriptome studies. To assess complexity and identify additional macrophage expressed genes, cDNA libraries were created from purified populations of macrophage and dendritic cells, a functionally related cell type. Sequence analysis revealed a high incidence of novel mRNAs within these cDNA libraries. These studies provide insights into the depths of transcriptional complexity still untapped amongst products of inducible genes, and identify macrophage and dendritic cell populations as a starting point for sampling the inducible mammalian transcriptome. PMID:12819134

  3. Gene expression analysis of WRKY transcription factors in Arabidopsis thaliana cell cultures during a parabolic flight

    NASA Astrophysics Data System (ADS)

    Babbick, Maren; Barjaktarović, Žarko; Hampp, Ruediger

    Plants sense gravity by specialized cells (statocytes) and adjust growth and development accordingly. It has, however, also been shown that plant cells which are not part of specialized tissues are also able to sense gravitational forces. Therefore we used undifferentiated, homogeneous cell cultures of Arabidopsis thaliana (cv. Columbia) in order to identify early alterations in gene expression as a response to altered gravitational field strengths. In this contribution we report on cell cultures exposed to parabolic flights (approximately 20 sec of microgravity). For this short-term exposure study, we specifically checked for genes at the beginning of signal transduction chains, such as those coding for transcription factors (TFs). TFs are small proteins that regulate expression of their target genes by binding to specific promoter sequences. Our main focus were members of the so-called WRKY TF family. WRKY TFs are known to be involved in various physiological processes like senescence and pathogen defense. By quantifying transcriptional changes of these genes by real-time RT-PCR, we wanted to find out, how gene expression is affected by both hyperand microgravity conditions during a parabolic flight. For this purpose Arabidopsis thaliana callus cultures were metabolically quenched by the injection of RNAlater at the end of the microgravity-phase of each parabola. The data we present will show how fast changes in amounts of transcripts will occur, and to what degree the expression profiles are comparable with data obtained from exposures to hypergravity and simulated microgravity.

  4. The hematopoietic transcription factor PU.1 regulates RANK gene expression in myeloid progenitors

    SciTech Connect

    Kwon, Oh Hyung; Lee, Chong-Kil; Lee, Young Ik; Paik, Sang-Gi; Lee, Hyun-Jun . E-mail: hjlee7@kribb.re.kr

    2005-09-23

    Osteoclasts are bone resorbing cells of hematopoietic origin. The hematopoietic transcription factor PU.1 is critical for osteoclastogenesis; however, the molecular mechanisms of PU.1-regulated osteoclastogenesis have not been explored. Here, we present evidence that the receptor activator of nuclear factor {kappa}B (RANK) gene that has been shown to be crucial for osteoclastogenesis is a transcriptional target of PU.1. The PU.1 {sup -/-} progenitor cells failed to express the RANK gene and reconstitution of PU.1 in these cells induced RANK expression. Treatment of the PU.1 reconstituted cells with M-CSF and RANKL further augmented the RANK gene expression. To explore the regulatory mechanism of the RANK gene expression by PU.1, we have cloned the human RANK promoter. Transient transfection assays have revealed that the 2.2-kb RANK promoter was functional in a monocyte line RAW264.7, whereas co-transfection of PU.1 transactivated the RANK promoter in HeLa cells. Taken together, these results suggest that PU.1 regulates the RANK gene transcription and this may represent one of the key roles of PU.1 in osteoclast differentiation.

  5. Expression of HSF2 decreases in mitosis to enable stress-inducible transcription and cell survival.

    PubMed

    Elsing, Alexandra N; Aspelin, Camilla; Björk, Johanna K; Bergman, Heidi A; Himanen, Samu V; Kallio, Marko J; Roos-Mattjus, Pia; Sistonen, Lea

    2014-09-15

    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.

  6. DDX6 post-transcriptionally down-regulates miR-143/145 expression through host gene NCR143/145 in cancer cells.

    PubMed

    Iio, Akio; Takagi, Takeshi; Miki, Kohei; Naoe, Tomoki; Nakayama, Atsuo; Akao, Yukihiro

    2013-10-01

    In various human malignancies, widespread dysregulation of microRNA (miRNA) expression is reported to occur and affects various cell growth programs. Recent studies suggest that the expression levels of miRNAs that act as tumor suppressors are frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription, and disturbances in miRNA processing. MiR-143 and -145 are well-recognized miRNAs that are highly expressed in several tissues, but down-regulated in most types of cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that DEAD-box RNA helicase 6, DDX6 (p54/RCK), post-transcriptionally down-regulated miR-143/145 expression by prompting the degradation of its host gene product, NCR143/145 RNA. In human gastric cancer cell line MKN45, DDX6 protein was abundantly expressed and accumulated in processing bodies (P-bodies). DDX6 preferentially increased the instability of non-coding RNA, NCR143/145, which encompasses the miR-143/145 cluster, and down-regulated the expression of mature miR-143/145. In human monocytic cell line THP-1, lipopolysaccharide treatment promoted the assembly of P-bodies and down-regulated the expression of NCR143/145 and its miR-143/145 rapidly. In these cells, cycloheximide treatment led to a loss of P-bodies and to an increase in NCR143/145 RNA stability, thus resulting in up-regulation of miR-143/145 expression. These data demonstrate that DDX6 contributed to the control of NCR143/145 RNA stability in P-bodies and post-transcriptionally regulated miR-143/145 expression in cancer cells.

  7. Activity-Based Anorexia Alters the Expression of BDNF Transcripts in the Mesocorticolimbic Reward Circuit

    PubMed Central

    Ho, Emily V.; Klenotich, Stephanie J.; McMurray, Matthew S.; Dulawa, Stephanie C

    2016-01-01

    Anorexia nervosa (AN) is a complex eating disorder with severe dysregulation of appetitive behavior. The activity-based anorexia (ABA) paradigm is an animal model in which rodents exposed to both running wheels and scheduled feeding develop aspects of AN including paradoxical hypophagia, dramatic weight loss, and hyperactivity, while animals exposed to only one condition maintain normal body weight. Brain-derived neurotrophic factor (BDNF), an activity-dependent modulator of neuronal plasticity, is reduced in the serum of AN patients, and is a known regulator of feeding and weight maintenance. We assessed the effects of scheduled feeding, running wheel access, or both on the expression of BDNF transcripts within the mesocorticolimbic pathway. We also assessed the expression of neuronal cell adhesion molecule 1 (NCAM1) to explore the specificity of effects on BDNF within the mesocorticolimbic pathway. Scheduled feeding increased the levels of both transcripts in the hippocampus (HPC), increased NCAM1 mRNA expression in the ventral tegmental area (VTA), and decreased BDNF mRNA levels in the medial prefrontal cortex (mPFC). In addition, wheel running increased BDNF mRNA expression in the VTA. No changes in either transcript were observed in the nucleus accumbens (NAc). Furthermore, no changes in either transcript were induced by the combined scheduled feeding and wheel access condition. These data indicate that scheduled feeding or wheel running alter BDNF and NCAM1 expression levels in specific regions of the mesocorticolimbic pathway. These findings contribute to our current knowledge of the molecular alterations induced by ABA and may help elucidate possible mechanisms of AN pathology. PMID:27861553

  8. 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

  9. Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei.

    PubMed

    Nguyen, Tu N; Müller, Laura S M; Park, Sung Hee; Siegel, T Nicolai; Günzl, Arthur

    2014-03-01

    Monoallelic expression within a gene family is found in pathogens exhibiting antigenic variation and in mammalian olfactory neurons. Trypanosoma brucei, a lethal parasite living in the human bloodstream, expresses variant surface glycoprotein (VSG) from 1 of 15 bloodstream expression sites (BESs) by virtue of a multifunctional RNA polymerase I. The active BES is transcribed in an extranucleolar compartment termed the expression site body (ESB), whereas silent BESs, located elsewhere within the nucleus, are repressed epigenetically. The regulatory mechanisms, however, are poorly understood. Here we show that two essential subunits of the basal class I transcription factor A (CITFA) predominantly occupied the promoter of the active BES relative to that of a silent BES, a phenotype that was maintained after switching BESs in situ. In these experiments, high promoter occupancy of CITFA was coupled to high levels of both promoter-proximal RNA abundance and RNA polymerase I occupancy. Accordingly, fluorescently tagged CITFA-7 was concentrated in the nucleolus and the ESB. Because a ChIP-seq analysis found that along the entire BES, CITFA-7 is specifically enriched only at the promoter, our data strongly indicate that monoallelic BES transcription is activated by a mechanism that functions at the level of transcription initiation.

  10. Differential transcript expression between the microfilariae of the filarial nematodes, Brugia malayi and B. pahangi

    PubMed Central

    2010-01-01

    Background Brugia malayi and B. pahangi are two closely related nematodes that cause filariasis in humans and animals. However, B. pahangi microfilariae are able to develop in and be transmitted by the mosquito, Armigeres subalbatus, whereas most B. malayi are rapidly melanized and destroyed within the mosquito hemocoel. A cross-species microarray analysis employing the B. malayi V2 array was carried out to determine the transcriptional differences between B. malayi and B. pahangi microfilariae with similar age distribution. Results Following microarray data analysis, a list of preferentially expressed genes in both microfilariae species was generated with a false discovery rate estimate of 5% and a signal intensity ratio of 2 or higher in either species. A total of 308 probes were preferentially expressed in both species with 149 probes, representing 123 genes, in B. pahangi microfilariae and 159 probes, representing 107 genes, in B. malayi microfilariae. In B. pahangi, there were 76 (62%) up-regulated transcripts that coded for known proteins that mapped into the KEGG pathway compared to 61 (57%) transcripts in B. malayi microfilariae. The remaining 47 (38%) transcripts in B. pahangi and 46 (43%) transcripts in B. malayi microfilariae were comprised almost entirely of hypothetical genes of unknown function. Twenty-seven of the transcripts in B. pahangi microfilariae coded for proteins that associate with the secretory pathway compared to thirty-nine in B. malayi microfilariae. The data obtained from real-time PCR analysis of ten genes selected from the microarray list of preferentially expressed genes showed good concordance with the microarray data, indicating that the microarray data were reproducible. Conclusion In this study, we identified gene transcripts that were preferentially expressed in the microfilariae of B. pahangi and B. malayi, some of which coded for known immunomodulatory proteins. These comparative transcriptome data will be of interest to

  11. Serine 574 phosphorylation alters transcriptional programming of FOXO3 by selectively enhancing apoptotic gene expression.

    PubMed

    Li, Z; Zhao, J; Tikhanovich, I; Kuravi, S; Helzberg, J; Dorko, K; Roberts, B; Kumer, S; Weinman, S A

    2016-04-01

    Forkhead box O3 (FOXO3) is a multispecific transcription factor that is responsible for multiple and conflicting transcriptional programs such as cell survival and apoptosis. The protein is heavily post-translationally modified and there is considerable evidence that post-transcriptional modifications (PTMs) regulate protein stability and nuclear-cytosolic translocation. Much less is known about how FOXO3 PTMs determine the specificity of its transcriptional program. In this study we demonstrate that exposure of hepatocytes to ethanol or exposure of macrophages to lipopolysaccharide (LPS) induces the c-Jun N-terminal kinase (JNK)-dependent phosphorylation of FOXO3 at serine-574. Chromatin immunoprecipitation (ChIP), mRNA and protein measurements demonstrate that p-574-FOXO3 selectively binds to promoters of pro-apoptotic genes but not to other well-described FOXO3 targets. Both unphosphorylated and p-574-FOXO3 bound to the B-cell lymphoma 2 (Bcl-2) promoter, but the unphosphorylated form was a transcriptional activator, whereas p-574-FOXO3 was a transcriptional repressor. The combination of increased TRAIL (TNF-related apoptosis-inducing ligand) and decreased Bcl-2 was both necessary and sufficient to induce apoptosis. LPS treatment of a human monocyte cell line (THP-1) induced FOXO3 S-574 phosphorylation and apoptosis. LPS-induced apoptosis was prevented by knockdown of FOXO3. It was restored by overexpressing wild-type FOXO3 but not by overexpressing a nonphosphorylatable S-574A FOXO3. Expression of an S-574D phosphomimetic form of FOXO3 induced apoptosis even in the absence of LPS. A similar result was obtained with mouse peritoneal macrophages where LPS treatment increased TRAIL, decreased Bcl-2 and induced apoptosis in wild-type but not FOXO3(-/-) cells. This work thus demonstrates that S-574 phosphorylation generates a specifically apoptotic form of FOXO3 with decreased transcriptional activity for other well-described FOXO3 functions.

  12. Expression profiles of key transcription factors involved in lipid metabolism in Beijing-You chickens.

    PubMed

    Fu, R Q; Liu, R R; Zhao, G P; Zheng, M Q; Chen, J L; Wen, J

    2014-03-01

    Intramuscular fat (IMF) is a crucial factor for the meat quality of chickens. With the aim of studying the molecular mechanisms underlying IMF deposition in chickens, the expression profiles of five candidate transcription factors involved in lipid metabolism in several tissues were examined in Beijing-You (BJY) chickens at five ages (0, 4, 8, 14 and 20 wk). Results showed that accumulation of IMF in breast (IMFbr), thigh (IMFth) and abdominal fat weight increased significantly (P<0.01) after 8 wk. Accumulation of both IMFbr and IMFth from 8 to 14 wk exceeded that from 14 to 20 wk; IMFth was 4-7 times of IMFbr. As for the expression profiles of key transcription factors: 1) expression of C/EBPα and PPARγ in abdominal fat was significantly higher than that in breast and thigh muscles at all ages. The expression of C/EBPα was positively correlated with PPARγ in both breast and thigh muscles, which indicated that both C/EBPα and PPARγ promoted fat deposition and might act through a unified pathway; 2) the expression of SREBP-1 in 0, 4, and 8 wk in thigh muscle was significantly higher than that in breast; 3) expression of C/EBPβ at 4 and 8 wk was significantly higher than that at 14 and 20 wk; and it was positively correlated with IMFth and IMFbr from 0 to 8 wk; 4) expression of PPARα in breast and thigh muscles was significantly higher than that in abdominal fat. Taken together, all five transcription factors studied play roles in lipid metabolism in chickens with C/EBPα and PPARγ being important effectors.

  13. Validation of an algorithm for delay stochastic simulation of transcription and translation in prokaryotic gene expression

    NASA Astrophysics Data System (ADS)

    Roussel, Marc R.; Zhu, Rui

    2006-12-01

    The quantitative modeling of gene transcription and translation requires a treatment of two key features: stochastic fluctuations due to the limited copy numbers of key molecules (genes, RNA polymerases, ribosomes), and delayed output due to the time required for biopolymer synthesis. Recently proposed algorithms allow for efficient simulations of such systems. However, it is critical to know whether the results of delay stochastic simulations agree with those from more detailed models of the transcription and translation processes. We present a generalization of previous delay stochastic simulation algorithms which allows both for multiple delays and for distributions of delay times. We show that delay stochastic simulations closely approximate simulations of a detailed transcription model except when two-body effects (e.g. collisions between polymerases on a template strand) are important. Finally, we study a delay stochastic model of prokaryotic transcription and translation which reproduces observations from a recent experimental study in which a single gene was expressed under the control of a repressed lac promoter in E. coli cells. This demonstrates our ability to quantitatively model gene expression using these new methods.

  14. Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator.

    PubMed

    Westbrook, Alexandra M; Lucks, Julius B

    2017-04-06

    RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression leak, which impedes the construction of larger synthetic regulatory networks as leak propagation can interfere with desired network function. To address this, we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and ribosome binding site sequestration increases repression from 85% to 98%, or activation from 10-fold to over 900-fold, in response to cognate antisense RNAs. We also show that orthogonal repressive versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this mechanism, we use it to reduce network leak in an RNA-only cascade. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of more sophisticated regulatory networks.

  15. Transcription without XPB Establishes a Unified Helicase-Independent Mechanism of Promoter Opening in Eukaryotic Gene Expression.

    PubMed

    Alekseev, Sergey; Nagy, Zita; Sandoz, Jérémy; Weiss, Amélie; Egly, Jean-Marc; Le May, Nicolas; Coin, Frederic

    2017-02-02

    Transcription starts with the assembly of pre-initiation complexes on promoters followed by their opening. Current models suggest that class II gene transcription requires ATP and the TFIIH XPB subunit to open a promoter. Here, we observe that XPB depletion surprisingly leaves transcription virtually intact. In contrast, inhibition of XPB ATPase activity affects transcription, revealing that mRNA expression paradoxically accommodates the absence of XPB while being sensitive to the inhibition of its ATPase activity. The XPB-depleted TFIIH complex is recruited to active promoters and contributes to transcription. We finally demonstrate that the XPB ATPase activity is only used to relieve a transcription initiation block imposed by XPB itself. In the absence of this block, transcription initiation can take place without XPB ATPase activity. These results suggest that a helicase is dispensable for mRNA transcription, thereby unifying the mechanism of promoter DNA opening for the three eukaryotic RNA polymerases.

  16. 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

  17. Effects of dietary roughage levels on the expression of adipogenic transcription factors in Wagyu steers.

    PubMed

    Yamada, T; Kawakami, S-I; Nakanishi, N

    2009-12-01

    We hypothesized that dietary roughage level would alter the expression levels of adipogenic transcription factors in adipose tissue of Japanese black (Wagyu) steers. Steers were fed whole crop rice silage at three levels: (1) high-roughage feeding group, fed 8kg silage and 5kg concentrate (HR); (2) middle roughage feeding group, fed 5kg silage and 6kg concentrate (MR); and (3) low roughage feeding group, fed 2kg silage and 7kg concentrate (LR) from 22 to 30months of age. In subcutaneous adipose tissue, there were no significant differences in the expression of the adipogenic transcription factors and adipocyte size among feeding groups. In mesenteric adipose tissue, the expression of C/EBPα in the LR and MR groups was significantly higher than that in the HR group. Adipocyte size in the mesenteric adipose tissue of the LR group was significantly larger than that of the HR group. In intermuscular adipose tissue, the expression of C/EBPβ-LAP in the LR group was significantly higher than that in the HR group, and the expression of C/EBPβ-LIP in the LR and MR groups was significantly higher than that in the HR group. Adipocyte size in the intermuscular adipose tissue of the LR and MR groups was significantly smaller than that of the HR group. These results suggest that dietary roughage revel affects the adipose tissue depot-specific differences in C/EBP family expression pattern and adipocyte cellularity in Wagyu steers.

  18. Human population-specific gene expression and transcriptional network modification with polymorphic transposable elements.

    PubMed

    Wang, Lu; Rishishwar, Lavanya; Mariño-Ramírez, Leonardo; Jordan, I King

    2016-12-19

    Transposable element (TE) derived sequences are known to contribute to the regulation of the human genome. The majority of known TE-derived regulatory sequences correspond to relatively ancient insertions, which are fixed across human populations. The extent to which human genetic variation caused by recent TE activity leads to regulatory polymorphisms among populations has yet to be thoroughly explored. In this study, we searched for associations between polymorphic TE (polyTE) loci and human gene expression levels using an expression quantitative trait loci (eQTL) approach. We compared locus-specific polyTE insertion genotypes to B cell gene expression levels among 445 individuals from 5 human populations. Numerous human polyTE loci correspond to both cis and trans eQTL, and their regulatory effects are directly related to cell type-specific function in the immune system. PolyTE loci are associated with differences in expression between European and African population groups, and a single polyTE loci is indirectly associated with the expression of numerous genes via the regulation of the B cell-specific transcription factor PAX5 The polyTE-gene expression associations we found indicate that human TE genetic variation can have important phenotypic consequences. Our results reveal that TE-eQTL are involved in population-specific gene regulation as well as transcriptional network modification.

  19. Expression of transcription factors during sodium phenylacetate induced erythroid differentiation in K562 cells.

    PubMed

    Rath, A V; Schmahl, G E; Niemeyer, C M

    1997-01-01

    During 15 days of treatment of K562 cells with sodium phenylacetate, we observed an increase in the cellular hemoglobin concentration with a similar increase in the expression of gamma-globin mRNA. Morphological studies demonstrated characteristic features of erythroid differentiation and maturation. At the same time there was no change in the level of expression of the cell surface antigenes CD33, CD34, CD45, CD71 and glycophorin A. Likewise, the level of expression of the erythroid transcription factors GATA-1, GATA-2, NF-E2, SCL and RBTN2, all expressed in untreated K562 cells, did not increase during sodium phenylacetate induced erythroid differentiation. The expression of the nuclear factors Evi-1 and c-myb, known to inhibit erythroid differentiation, did not decrease. We conclude that sodium phenylacetate treatment of K562 cells increases gamma-globin mRNA and induces cell maturation as judged by morphology without affecting the expression of the erythroid transcription factors, some of which are known to be involved in the regulation of beta-like globin genes.

  20. Leveraging skewed transcript abundance by RNA-Seq to increase the genomic depth of the tree of life.

    PubMed

    Hittinger, Chris Todd; Johnston, Mark; Tossberg, John T; Rokas, Antonis

    2010-01-26

    Assembling the tree of life is a major goal of biology, but progress has been hindered by the difficulty and expense of obtaining the orthologous DNA required for accurate and fully resolved phylogenies. Next-generation DNA sequencing technologies promise to accelerate progress, but sequencing the genomes of hundreds of thousands of eukaryotic species remains impractical. Eukaryotic transcriptomes, which are smaller than genomes and biased toward highly expressed genes that tend to be conserved, could potentially provide a rich set of phylogenetic characters. We sampled the transcriptomes of 10 mosquito species by assembling 36-bp sequence reads into phylogenomic data matrices containing hundreds of thousands of orthologous nucleotides from hundreds of genes. Analysis of these data matrices yielded robust phylogenetic inferences, even with data matrices constructed from surprisingly few sequence reads. This approach is more efficient, data-rich, and economical than traditional PCR-based and EST-based methods and provides a scalable strategy for generating phylogenomic data matrices to infer the branches and twigs of the tree of life.

  1. Comprehensive analysis of TCP transcription factors and their expression during cotton (Gossypium arboreum) fiber early development.

    PubMed

    Ma, Jun; Liu, Fang; Wang, Qinglian; Wang, Kunbo; Jones, Don C; Zhang, Baohong

    2016-02-09

    TCP proteins are plant-specific transcription factors implicated to perform a variety of physiological functions during plant growth and development. In the current study, we performed for the first time the comprehensive analysis of TCP gene family in a diploid cotton species, Gossypium arboreum, including phylogenetic analysis, chromosome location, gene duplication status, gene structure and conserved motif analysis, as well as expression profiles in fiber at different developmental stages. Our results showed that G. arboreum contains 36 TCP genes, distributing across all of the thirteen chromosomes. GaTCPs within the same subclade of the phylogenetic tree shared similar exon/intron organization and motif composition. In addition, both segmental duplication and whole-genome duplication contributed significantly to the expansion of GaTCPs. Many these TCP transcription factor genes are specifically expressed in cotton fiber during different developmental stages, including cotton fiber initiation and early development. This suggests that TCP genes may play important roles in cotton fiber development.

  2. Identification of Embryoid-Abundant Genes That Are Temporally Expressed during Pollen Embryogenesis in Wheat Anther Cultures 1

    PubMed Central

    Reynolds, Thomas L.; Kitto, Sherry L.

    1992-01-01

    Uninucleate microspores in anther cultures of bread wheat (Triticum aestivum cv Pavon) are capable of producing haploid pollen embryoids and plants. To gain an understanding of this alternate pathway of pollen development, we constructed a cDNA library to young pollen embryoids, isolated embryoid-specific genes, and analyzed their expression patterns during morphogenesis. Two embryoid-abundant clones, pEMB4 and 94, were expressed very early during culture, suggesting that these genes are associated with development and are not simply expressed as a consequence of differentiation. The accumulation patterns of five cloned mRNAs may indicate the activation of specific genes associated with the major morphological and physiological activities connected with the differentiation of embryoids in vitro. These results suggest that embryoid-abundant gene expression is causally related to this pathway because gene expression is spatially and temporally specific and is not observed when microspores are cultured under noninductive conditions. Images Figure 1 Figure 2 Figure 3 PMID:16653192

  3. 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

  4. 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

  5. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells

    PubMed Central

    2011-01-01

    Background Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. Results We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. Conclusion These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP. PMID:21929758

  6. Transcriptional control of two ribosome-inactivating protein genes expressed in spinach (Spinacia oleracea) embryos.

    PubMed

    Kawade, Kensuke; Masuda, Kiyoshi

    2009-05-01

    SoRIP1 and SoRIP2 are ribosome-inactivating protein (RIP: EC 3.2.2.22) genes identified in spinach (Spinacia oleracea). They are differentially expressed in a development-dependent manner during spinach somatic embryogenesis. Here, we isolated genomic clones of SoRIP1 and SoRIP2. These two RIP genes have different genomic organization. Phylogenetic analysis of predicted amino acid sequences of RIPs in Caryophyllales plants revealed that they are divided into two major subfamilies, corresponding to SoRIP1 and SoRIP2. To gain further insight into the transcriptional control of SoRIP1 and SoRIP2, we obtained their 5'-flanking sequences by inverse PCR. Comparison of two 5'-flanking sequences revealed the characteristic cis elements in each region that confer differential transcriptional control. In the 5'-flanking region of SoRIP1, we found several motifs with functions related to embryonic development. The 5'-flanking region of SoRIP2 contains some defense-responsive motifs. Expression of SoRIP1 was detected in various tissues. In particular, SoRIP1 was highly expressed in the early immature fruits, and immunohistochemistry showed that SoRIP1 accumulated in the peripheral region of the immature embryo, with weaker expression in internal cells. During fruit development, the expression of SoRIP2 was low. However, the accumulation of SoRIP2 was conspicuous in the epidermis of the immature embryo. The expression of SoRIP2, but not SoRIP1, in leaves was induced by salicylic acid treatment. This differential transcriptional regulation of SoRIP1 and SoRIP2 suggests that the corresponding proteins may have different functions, one being related to embryonic development and the other to embryo defense.

  7. 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

  8. Transcript profiling reveals rewiring of iron assimilation gene expression in Candida albicans and C. dubliniensis.

    PubMed

    Moran, Gary P

    2012-12-01

    Hyphal growth is repressed in Candida albicans and Candida dubliniensis by the transcription factor Nrg1. Transcript profiling of a C. dubliniensis NRG1 mutant identified a common group of 28 NRG1-repressed genes in both species, including the hypha-specific genes HWP1, ECE1 and the regulator of cell elongation UME6. Unexpectedly, C. dubliniensis NRG1 was required for wild-type levels of expression of 10 genes required for iron uptake including seven ferric reductases, SIT1, FTR1 and RBT5. However, at alkaline pH and during filamentous growth in 10% serum, most of these genes were highly induced in C. dubliniensis. Conversely, RBT5, PGA10, FRE10 and FRP1 did not exhibit induction during hyphal growth when NRG1 is downregulated, indicating that in C. dubliniensis NRG1 is also required for optimal expression of these genes in alkaline environments. In iron-depleted medium at pH 4.5, reduced growth of the NRG1 mutant relative to wild type was observed; however, growth was restored to wild-type levels or greater at pH 6.5, indicating that alkaline induction of iron assimilation gene expression could rescue this phenotype. These data indicate that transcriptional control of iron assimilation and pseudohypha formation has been separated in C. albicans, perhaps promoting growth in a wider range of niches.

  9. The POU Transcription Factor Drifter/Ventral veinless Regulates Expression of Drosophila Immune Defense Genes▿

    PubMed Central

    Junell, Anna; Uvell, Hanna; Davis, Monica M.; Edlundh-Rose, Esther; Antonsson, Åsa; Pick, Leslie; Engström, Ylva

    2010-01-01

    Innate immunity operates as a first line of defense in multicellular organisms against infections caused by different classes of microorganisms. Antimicrobial peptides (AMPs) are synthesized constitutively in barrier epithelia to protect against microbial attack and are also upregulated in response to infection. Here, we implicate Drifter/Ventral veinless (Dfr/Vvl), a class III POU domain transcription factor, in tissue-specific regulation of the innate immune defense of Drosophila. We show that Dfr/Vvl is highly expressed in a range of immunocompetent tissues, including the male ejaculatory duct, where its presence overlaps with and drives the expression of cecropin, a potent broad-spectrum AMP. Dfr/Vvl overexpression activates transcription of several AMP genes in uninfected flies in a Toll pathway- and Imd pathway-independent manner. Dfr/Vvl activates a CecA1 reporter gene both in vitro and in vivo by binding to an upstream enhancer specific for the male ejaculatory duct. Further, Dfr/Vvl and the homeodomain protein Caudal (Cad) activate transcription synergistically via this enhancer. We propose that the POU protein Dfr/Vvl acts together with other regulators in a combinatorial manner to control constitutive AMP gene expression in a gene-, tissue-, and sex-specific manner, thus promoting a first-line defense against infection in tissues that are readily exposed to pathogens. PMID:20457811

  10. Pituitary transcription factor Prop-1 stimulates porcine follicle-stimulating hormone beta subunit gene expression.

    PubMed

    Aikawa, Satoko; Kato, Takako; Susa, Takao; Tomizawa, Kyoko; Ogawa, Satoshi; Kato, Yukio

    2004-11-12

    Molecular cloning of the transcription factor that modulates the expression of porcine follicle-stimulating hormone beta subunit (FSHbeta) gene was performed by the yeast one-hybrid cloning system using the -852/-746 upstream region (Fd2) as a bait sequence. We eventually cloned a pituitary transcription factor, Prop-1, which has been identified as an upstream transcription factor of Pit-1 gene. Binding ability of Prop-1 to the bait sequence was confirmed using recombinant Prop-1, and the binding property was investigated by DNase I footprinting, revealing that Prop-1 certainly bound to the large AT-rich region throughout the Fd2. Co-transfection of Prop-1 expression vector together with a reporter gene fused with Fd2 in CHO cells demonstrated an attractive stimulation of reporter gene expression. Immunohistochemistry of adult porcine pituitary confirmed the colocalization of the Prop-1 and FSHbeta subunit. This study is the first to report that Prop-1 participates in the regulation of FSHbeta gene. The present finding will provide new insights into the development of pituitary cell lineage and combined pituitary hormone deficiency (CPHD), since why the defect of Prop-1 causes CPHD including gonadotropins (FSH and LH) has yet to be clarified.

  11. Expression and epigenetic dynamics of transcription regulator Lhx8 during mouse oogenesis.

    PubMed

    Zhang, Lian-Jun; Pan, Bo; Chen, Bo; Zhang, Xi-Feng; Liang, Gui-Jin; Feng, Yan-Ni; Wang, Lin-Qing; Ma, Jin-Mei; Li, Lan; Shen, Wei

    2012-09-10

    The spatial and temporal specific activation and inhibition of numerous genes are required for successful oogenesis which is precisely regulated by germ cell-related transcription factors, and appropriate epigenetic modifications, including DNA methylation, histone modification and other mechanisms that closely regulate the functional exertion of these transcription factors. In this study, we characterized the correlation between the expression and epigenetic dynamics of Lhx8, a germ cell specific transcription factor during mouse oogenesis. Immunohistochemistry, quantitative PCR and western blots were performed to localize and quantify the expressional characteristics of Lhx8 in oocytes of 13.5 dpc (day post coitum), 17.5 dpc, 0 dpp (day post partum), 3 dpp, 7 dpp and 14 dpp. The results showed that LHX8 protein was located in the nucleus of oocytes, and increasingly expressed during primordial follicle activation. Sequencing of bisulfite-converted genomic DNAs revealed that the methylation dynamics of Lhx8-3' was highly changeable but almost no change occurred in Lhx8-5'. ChIP-QPCR analysis showed that histone H3 acetylation of Lhx8 was also increased during primordial follicle assembly and activation. In conclusion, Lhx8 expression is related with the activation of primordial follicles, which is highly correlated with the demethylation of Lhx8-3' untranslated region and the high acetylation of histone H3.

  12. 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.

  13. Expression of PITX2 homeodomain transcription factor during rat gonadal development in a sexually dimorphic manner.

    PubMed

    Nandi, Shyam Sundar; Ghosh, Pamela; Roy, Sib Sankar

    2011-01-01

    PITX2, a multifunctional Paired-like homeodomain transcription factor, plays obligatory role during development of organs like heart, brain and pituitary. It regulates differentiation of vascular smooth muscle cells and hematopoietic stem cells. Although we earlier reported the Pitx2/PITX2 expression in gonad, but the expression pattern of its different isoforms in mammalian gonads especially during development is still not known. As PITX2 participates in the development of multiple organs and different homeobox genes have been shown to control gonadal functions, we wanted to investigate the role of PITX2 in gonadal development and its function. The objective of our study was to know the expression profile of different Pitx2/PITX2 isoforms and its localization throughout the development of gonads. Here we show the temporal and spatial expression pattern of Pitx2/PITX2 and its localization throughout the embryonic and postnatal stages of rat gonads. Pitx2/PITX2 expression profile reveals the differential and dimorphic expression pattern of its two isoforms PITX2B2 and-Cβ throughout the embryonic development stages and also in the postnatal stages, where it becomes more prominent. This is the first report where PITX2 homeodomain transcription factor shows isoform-specific sexually dimorphic expression. In addition, PITX2 localization was found in the embryonic ovarian primordial germ cell clusters and germ cells inside the testicular cords and also in somatic cells. In adults, ovarian granulosa and theca cells as well as germ cells inside the seminiferous tubules in testis express PITX2. All the evidences suggest that the differential expression of PITX2 might be associated with sex-specific embryonic and postnatal gonadal development and the physiological processes.

  14. Expression profile and transcription factor binding site exploration of imprinted genes in human and mouse

    PubMed Central

    Steinhoff, Christine; Paulsen, Martina; Kielbasa, Szymon; Walter, Jörn; Vingron, Martin

    2009-01-01

    Background In mammals, imprinted genes are regulated by an epigenetic mechanism that results in parental origin-specific expression. Though allele-specific regulation of imprinted genes has been studied for several individual genes in detail, little is known about their overall tissue-specific expression patterns and interspecies conservation of expression. Results We performed a computational analysis of microarray expression data of imprinted genes in human and mouse placentae and in a variety of adult tissues. For mouse, early embryonic stages were also included. The analysis reveals that imprinted genes are expressed in a broad spectrum of tissues for both species. Overall, the relative tissue-specific expression levels of orthologous imprinted genes in human and mouse are not highly correlated. However, in both species distinctive expression profiles are found in tissues of the endocrine pathways such as adrenal gland, pituitary, pancreas as well as placenta. In mouse, the placental and embryonic expression patterns of imprinted genes are highly similar. Transcription factor binding site (TFBS) prediction reveals correlation of tissue-specific expression patterns and the presence of distinct TFBS signatures in the upstream region of human imprinted genes. Conclusion Imprinted genes are broadly expressed pre- and postnatally and do not exhibit a distinct overall expression pattern when compared to non-imprinted genes. The relative expression of most orthologous gene pairs varies significantly between human and mouse suggesting rapid species-specific changes in gene regulation. Distinct expression profiles of imprinted genes are confined to certain human and mouse hormone producing tissues, and placentae. In contrast to the overall variability, distinct expression profiles and enriched TFBS signatures are found in human and mouse endocrine tissues and placentae. This points towards an important role played by imprinted gene regulation in these tissues. PMID

  15. [An immunohistochemical study of the expression of transcription factor Oct3/4 in mouse spermatogenesis].

    PubMed

    Tomilin, A N; Kostyleva, E I; Drosdovskiĭ, M A; Seralini, J E; Vorob'ev, V I

    1996-01-01

    The expression of POU-domain transcription factor Oct3/4 in the testis of adult mice has been studied using indirect immunofluorescence with highly specific antibodies. The protein is shown to be expressed in germ cells of seminiferous epithelium in a stage specific manner. The protein synthesis is initiated in mid-pachytene spermatocytes, increases to reach its peak during meiotic division. The Oct3/4 level remains augmented in early spermatids, but gradually declines during their further developmental advancement. These findings imply that Oct3/4 may have a regulatory function providing for the control of meiosis and/or terminal differentiation of spermatogenic cells.

  16. Expression of adipogenic transcription factors in adipose tissue of fattening Wagyu and Holstein steers.

    PubMed

    Yamada, T; Kawakami, S-I; Nakanishi, N

    2009-01-01

    In this experiment, we studied the effects of breed differences on the protein expression of adipogenic transcription factors, the C/EBP family (C/EBPα, C/EBPβ-LAP, C/EBPβ-LIP and C/EBPδ) and PPARγ, in the adipose tissues of Japanese Black (Wagyu) and Holstein steers from various anatomical sites (subcutaneous, intermuscular, and mesenteric) at different fattening periods (19 and 24 months of age). The expression of C/EBPβ-LAP and C/EBPα in the mesenteric fat tissue of Wagyu at 19 months of age was significantly higher than that of Holstein. The expression of C/EBPδ in the subcutaneous, intermuscular and mesenteric fat tissue of Wagyu at 19 months of age was significantly higher than that of Holstein. The plasma insulin concentrations of Wagyu steers at 19 months of age tended to be higher than those of Holstein. No significant differences in the expression of the adipogenic transcription factors and plasma insulin concentration were observed between the breeds at 24 months of age. These results suggest the existence of breed difference on the expression of the C/EBP family between fattening Wagyu and Holstein steers at 19 months of age, whereas breed difference might have disappeared before 24 months of age.

  17. Chicken ovalbumin upstream promoter transcription factor (COUP-TF): expression during mouse embryogenesis.

    PubMed

    Pereira, F A; Qiu, Y; Tsai, M J; Tsai, S Y

    1995-06-01

    Members of the steroid/thyroid hormone receptor superfamily such as TR, RAR, RXR and VDR are known to play important roles in regulation of gene expression during development, differentiation and homeostasis. COUP-TFs are orphan members of this superfamily of nuclear receptors and have been shown to negatively regulate the ability of these nuclear receptors to transactivate target genes. Two different mechanisms are implicated in this repression. First, COUP-TFs bind to AGGTCA direct repeats and palindromes with various spacings, which include response elements for TR, RAR, RXR and VDR, allowing for direct competition of COUP-TFs for the response elements. Second, COUP-TFs can heterodimerize with RXRs, the essential cofactor for effective binding of VDR, TRs and RARs to their cognate response elements. The physiological significance of this negative effect of COUP-TF on the activity of these receptors has been analyzed. Detection of COUP-TF transcripts during mouse development reveal discrete spatial and temporal expression domains consistent with COUP-TFs being involved in regulation of gene expression during embryogenesis. Transcripts are localized within discrete regions of the central and peripheral nervous system including the inner ear. In addition, COUP-TFs are found in many tissues including testes, ovary, prostate, skin, kidney, lung, stomach, intestine, pancreas and salivary gland. Some of these expression domains colocalize with those of TR, RAR, and RXR. The simultaneous expression of these genes raise the possibility that COUP-TFs can act as negative regulatory factors during development and differentiation.

  18. 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.

  19. 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

  20. Application of anti-listerial bacteriocins: monitoring enterocin expression by multiplex relative reverse transcription-PCR.

    PubMed

    Williams, D Ross; Chanos, Panagiotis

    2012-12-01

    Listeriosis is a deadly food-borne disease, and its incidence may be limited through the biotechnological exploitation of a number of anti-listerial biocontrol agents. The most widely used of these agents are bacteriocins and the Class II enterocins are characterized by their activity against Listeria. Enterocins are primarily produced by enterococci, particularly Enterococcus faecium and many strains have been described, often encoding multiple bacteriocins. The use of these strains in food will require that they are free of virulence functions and that they exhibit a high level expression of anti-listerial enterocins in fermentation conditions. Multiplex relative RT (reverse transcription)-PCR is a technique that is useful in the discovery of advantageous expression characteristics among enterocin-producing strains. It allows the levels of individual enterocin gene expression to be monitored and determination of how expression is altered under different growth conditions.

  1. Measuring the expressed abundance of the three phases of water with an imaging spectrometer over melting snow

    NASA Astrophysics Data System (ADS)

    Green, Robert O.; Painter, Thomas H.; Roberts, Dar A.; Dozier, Jeff

    2006-10-01

    From imaging spectrometer data, we simultaneously estimate the abundance of the three phases of water in an environment that includes melting snow, basing the analysis on the spectral shift in the absorption coefficient between water vapor, liquid water, and ice at 940, 980, and 1030 nm respectively. We apply a spectral fitting algorithm that measures the expressed abundance of the three phases of water to a data set acquired by the Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) over Mount Rainier, Washington, on 14 June 1996. Precipitable water vapor varies from 1 mm over the summit of Mount Rainier to 10 mm over the lower valleys to the northwest. Equivalent path absorption of liquid water varies from 0 to 13 mm, with the zero values over rocky areas and high-elevation snow and the high values associated with liquid water held in vegetation canopies and in melting snow. Ice abundance varies from 0 to 30 mm equivalent path absorption in the snow- and glacier-covered portions of Mount Rainier. The water and ice abundances are related to the amount of liquid water and the sizes of the ice grains in the near-surface layer. Precision of the estimates, calculated over locally homogeneous areas, indicates an uncertainty of better than 1.5% for all three phases, except for liquid water in vegetation, where an optimally homogeneous site was not found. The analysis supports new strategies for hydrological research and applications as imaging spectrometers become more available.

  2. 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

  3. Regulation and expression of multidrug resistance (MDR) transcripts in the intestinal epithelium

    PubMed Central

    Li, M; Hurren, R; Zastawny, R L; Ling, V; Buick, R N

    1999-01-01

    A paucity of information exists on the regulation of gene expression in the undifferentiated intestine. The intestinal epithelium is one of the few normal tissues expressing the multidrug resistance (MDR) genes that confer the multidrug resistant phenotype to a variety of tumours. Expression of mdr1a has been observed in the primitive rat intestinal epithelial cell line, IEC-18. It is hypothesized that characterization of MDR gene expression in IEC-18 cells will provide insight into gene regulation in undifferentiated intestinal cells. A series of hamster mdr1a promoter deletion constructs was studied in IEC-18 and a region with 12–13-fold enhancer activity was identified. This region was shown to function in an orientation- and promoter context-independent manner, specifically in IEC-18 cells. Unexpectedly, Northern probing revealed a greater expression of mdr1b than mdr1a in IEC-18 cells. A quantitative reverse transcription polymerase chain reaction assay was used to compare the relative expression of MDR genes in IEC cells, fetal intestine, and in the undifferentiated and differentiated components of adult intestinal epithelium. MDR transcript levels in IEC cells were found to resemble those of fetal intestine and small intestinal crypts, where a conversion from mixed mdr1a/mdr1b to predominantly mdr1a expression occurs as cells mature. This work describes two contributions to the field of gene regulation in the undifferentiated intestine – first, the initial characterization of a putative mdr1a enhancer region with specificity for primitive intestinal cells and secondly, the first report of mdr1b detection in the intestine and its expression in primitive cell types. © 1999 Cancer Research Campaign PMID:10376961

  4. c-Maf Transcription Factor Regulates ADAMTS-12 Expression in Human Chondrogenic Cells

    PubMed Central

    Hong, Eunmee; Yik, Jasper; Amanatullah, Derek F.; Di Cesare, Paul E.

    2013-01-01

    Objective: ADAMTS (a disintegrin and metalloproteinase with thrombospondin type-1 motif) zinc metalloproteinases are important during the synthesis and breakdown of cartilage extracellular matrix. ADAMTS-12 is up-regulated during in vitro chondrogenesis and embryonic limb development; however, the regulation of ADAMTS-12 expression in cartilage remains unknown. The transcription factor c-Maf is a member of Maf family of basic ZIP (bZIP) transcription factors. Expression of c-Maf is highest in hypertrophic chondrocytes during embryonic development and postnatal growth. We hypothesize that c-Maf and ADAMTS-12 are co-expressed during chondrocyte differentiation and that c-Maf regulates ADAMTS-12 expression during chondrogenesis. Design: Promoter analysis and species alignments identified potential c-Maf binding sites in the ADAMTS-12 promoter. c-Maf and ADAMTS-12 co-expression was monitored during chondrogenesis of stem cell pellet cultures. Luciferase expression driven by ADAMTS-12 promoter segments was measured in the presence and absence of c-Maf, and synthetic oligonucleotides were used to confirm specific binding of c-Maf to ADAMTS-12 promoter sequences. Results: In vitro chondrogenesis from human mesenchymal stem cells revealed co-expression of ADAMTS-12 and c-Maf during differentiation. Truncation and point mutations of the ADAMTS-12 promoter evaluated in reporter assays localized the response to the proximal 315 bp of the ADAMTS-12 promoter, which contained a predicted c-Maf recognition element (MARE) at position -61. Electorphoretic mobility shift assay confirmed that c-Maf directly interacted with the MARE at position -61. Conclusions: These data suggest that c-Maf is involved in chondrocyte differentiation and hypertrophy, at least in part, through the regulation of ADAMTS-12 expression at a newly identified MARE in its proximal promoter. PMID:26069660

  5. Regulation of Notch-mediated transcription by a bovine herpesvirus 1 encoded protein (ORF2) that is expressed in latently infected sensory neurons.

    PubMed

    Liu, Yilin; Jones, Clinton

    2016-08-01

    Bovine herpesvirus 1 (BoHV-1) is an Alphaherpesvirinae subfamily member that establishes life-long latency in sensory neurons. The latency-related RNA (LR-RNA) is abundantly expressed during latency. An LR mutant virus containing stop codons at the amino-terminus of open reading frame (ORF)2 does not reactivate from latency and replicates less efficiently in tonsils and trigeminal ganglia. ORF2 inhibits apoptosis, interacts with Notch family members, and interferes with Notch-dependent transcription suggesting ORF2 expression enhances survival of infected neurons. The Notch signaling pathway is crucial for neuronal differentiation and survival suggesting that interactions between ORF2 and Notch family members regulate certain aspects of latency. Consequently, for this study, we compared whether ORF2 interfered with the four mammalian Notch family members. ORF2 consistently interfered with Notch1-3-mediated transactivation of three cellular promoters. Conversely, Notch4-mediated transcription was not consistently inhibited by ORF2. Electrophoretic shift mobility assays using four copies of a consensus-DNA binding site for Notch/CSL (core binding factor (CBF)-1, Suppressor of Hairless, Lag-2) as a probe revealed ORF2 interfered with Notch1 and 3 interactions with a CSL family member bound to DNA. Additional studies demonstrated ORF2 enhances neurite sprouting in mouse neuroblastoma cells that express Notch1-3, but not Notch4. Collectively, these studies indicate that ORF2 inhibits Notch-mediated transcription and signaling by interfering with Notch interacting with CSL bound to DNA.

  6. 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

  7. 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.

  8. Structure, expression, and biological function of INSM1 transcription factor in neuroendocrine differentiation

    PubMed Central

    Lan, Michael S.; Breslin, Mary B.

    2009-01-01

    Zinc-finger transcription factors are DNA-binding proteins that are implicated in many diverse biological functions. INSM1 (formerly IA-1) contains five zinc-finger motifs and functions as a transcription factor. INSM1 protein structure is highly conserved in homologues of different species. It is predominantly expressed in developing neuroendocrine tissues and the nervous system in mammals. INSM1 represents an important player in early embryonic neurogenesis. In pancreatic endocrine cell differentiation, Ngn3 first activates INSM1 and subsequently NeuroD/β2. Conversely, INSM1 exerts a feedback mechanism to suppress NeuroD/β2 and its own gene expression. INSM1 gene ablation in the mouse results in the impairment of pancreatic endocrine cell maturation. Further, deletion of INSM1 severely impairs catecholamine biosynthesis and secretion from the adrenal gland that results in early embryonic lethality. Genetically, INSM1 acts as a downstream factor of Mash 1 and Phox2b in the differentiation of the sympatho-adrenal lineage. In the developing neocortex, mouse embryos lacking INSM1 expression contain half the number of basal progenitors and show a reduction in cortical plate radial thickness. Cell signaling studies reveal that INSM1 contributes to the induction of cell cycle arrest/exit necessary to facilitate cellular differentiation. INSM1 is highly expressed in tumors of neuroendocrine origin. Hence, its promoter could serve as a tumor-specific promoter that drives a specific targeted cancer gene therapy for the treatment of neuroendocrine tumors. Taken together, all of these features of INSM1 strongly support its role as an important regulator during neuroendocrine differentiation.—Lan, M. S., Breslin, M. B. Structure, expression, and biological function of INSM1 transcription factor in neuroendocrine differentiation. PMID:19246490

  9. 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.

  10. Tissue- and environmental response-specific expression of 10 PP2C transcripts in Mesembryanthemum crystallinum.

    PubMed

    Miyazaki, S; Koga, R; Bohnert, H J; Fukuhara, T

    1999-03-01

    Ten transcripts (Mpc1-10) homologous to protein phosphatases of the 2C family have been isolated from the halophyte Mesembryanthemum crystallinum (common ice plant). Transcripts range in size from 1.6 to 2.6 kb, and encode proteins whose catalytic domains are between 24% and 62% identical to that of the Arabidopsis PP2C, ABI1. Transcript expression is tissue specific. Two isoforms are present only in roots (Mpc1 and Mpc5), three in young leaves (Mpc6, 8 and 9), two in old leaves (Mpc6 and Mpc8), and two in post-flowering leaves (Mpc8 and Mpc9). Mpc2 is strongly expressed in roots and also in seeds, meristematic tissues and mature flowers. Mpc3 is specific for leaf meristems, and Mpc4 is found in root and leaf meristems. Mpc7 is restricted to meristematic tissues. Mpc10 is only present in mature flowers. Mpc2 (in roots and leaves), Mpc5 (in roots) and Mpc8 (weakly in leaves) are induced by salinity stress and drought conditions with different kinetics in different tissues, but other Mpcs are downregulated by stress. Cold stress (4 degrees C) leads to a decline in Mpc5 and Mp6, but low temperature provoked a long-term (days) increase in Mpc2 levels in leaves and a transient increase (less than 24 h) in roots. Four full-length transcripts have been obtained. In each case, after over-expression in E. coli, the isolated proteins exhibited (Mg2+-dependent, okadeic acid-insensitive) protein phosphatase activity, although activity against 32P-phosphocasein varied among different PP2Cs. Determination of tissue developmental and stress response specificity of PP2C will facilitate functional studies of signal-transducing enzymes in this halophytic organism.

  11. Over-expression of Dof-type transcription factor increases lipid production in Chlamydomonas reinhardtii.

    PubMed

    Ibáñez-Salazar, Alejandro; Rosales-Mendoza, Sergio; Rocha-Uribe, Alejandro; Ramírez-Alonso, Jocelín Itzel; Lara-Hernández, Ignacio; Hernández-Torres, Araceli; Paz-Maldonado, Luz María Teresita; Silva-Ramírez, Ana Sonia; Bañuelos-Hernández, Bernardo; Martínez-Salgado, José Luis; Soria-Guerra, Ruth Elena

    2014-08-20

    The high demand for less polluting, newer, and cheaper fuel resources has increased the search of the most innovative options for the production of the so-called biofuels. Chlamydomonas reinhardtii is a photosynthetic unicellular algae with multiple biotechnological advantages such as easy handling in the laboratory, a simple scale-up to industrial levels, as well as a feasible genetic modification at nuclear and chloroplast levels. Besides, its fatty acids can be used to produce biofuels. Previous studies in plants have found that the over expression of DOF-type transcription factor genes increases the synthesis and the accumulation of total lipids in seeds. In this context, the over-expression of a DOF-type transcription factor in C. reinhardtii was applied as approach to increase the amount of lipids. The results indicate higher amounts (around 2-fold) of total lipids, which are mainly fatty acids, in the genetically C. reinhardtii modified strains when compared with the non-genetically modified strain. In order to elucidate the possible function of the introduced Dof-type transcription factor, we performed a transcription profile of 8 genes involved in fatty acid biosynthesis and 6 genes involved in glycerolipid biosynthesis, by quantitative real time (qRT-PCR). Differential expression profile was observed, which can explain the increase in lipid accumulation. However, these strains did not show notable changes in the fatty acid profile. This work represents an early effort in generating a strategy to increase fatty acids production in C. reinhardtii and their use in biofuel synthesis.

  12. 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.

  13. 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.

  14. In tobacco BY-2 cells xyloglucan oligosaccharides alter the expression of genes involved in cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

    PubMed

    González-Pérez, Lien; Perrotta, Lara; Acosta, Alexis; Orellana, Esteban; Spadafora, Natasha; Bruno, Leonardo; Bitonti, Beatrice M; Albani, Diego; Cabrera, Juan Carlos; Francis, Dennis; Rogers, Hilary J

    2014-10-01

    Xyloglucan oligosaccharides (XGOs) are breakdown products of XGs, the most abundant hemicelluloses of the primary cell walls of non-Poalean species. Treatment of cell cultures or whole plants with XGOs results in accelerated cell elongation and cell division, changes in primary root growth, and a stimulation of defence responses. They may therefore act as signalling molecules regulating plant growth and development. Previous work suggests an interaction with auxins and effects on cell wall loosening, however their mode of action is not fully understood. The effect of an XGO extract from tamarind (Tamarindus indica) on global gene expression was therefore investigated in tobacco BY-2 cells using microarrays. Over 500 genes were differentially regulated with similar numbers and functional classes of genes up- and down-regulated, indicating a complex interaction with the cellular machinery. Up-regulation of a putative XG endotransglycosylase/hydrolase-related (XTH) gene supports the mechanism of XGO action through cell wall loosening. Differential expression of defence-related genes supports a role for XGOs as elicitors. Changes in the expression of genes related to mitotic control and differentiation also support previous work showing that XGOs are mitotic inducers. XGOs also affected expression of several receptor-like kinase genes and transcription factors. Hence, XGOs have significant effects on expression of genes related to cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

  15. Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages.

    PubMed

    Gautier, Emmanuel L; Shay, Tal; Miller, Jennifer; Greter, Melanie; Jakubzick, Claudia; Ivanov, Stoyan; Helft, Julie; Chow, Andrew; Elpek, Kutlu G; Gordonov, Simon; Mazloom, Amin R; Ma'ayan, Avi; Chua, Wei-Jen; Hansen, Ted H; Turley, Shannon J; Merad, Miriam; Randolph, Gwendalyn J

    2012-11-01

    We assessed gene expression in tissue macrophages from various mouse organs. The diversity in gene expression among different populations of macrophages was considerable. Only a few hundred mRNA transcripts were selectively expressed by macrophages rather than dendritic cells, and many of these were not present in all macrophages. Nonetheless, well-characterized surface markers, including MerTK and FcγR1 (CD64), along with a cluster of previously unidentified transcripts, were distinctly and universally associated with mature tissue macrophages. TCEF3, C/EBP-α, Bach1 and CREG-1 were among the transcriptional regulators predicted to regulate these core macrophage-associated genes. The mRNA encoding other transcription factors, such as Gata6, was associated with single macrophage populations. We further identified how these transcripts and the proteins they encode facilitated distinguishing macrophages from dendritic cells.

  16. E2A is a transcriptional regulator of CD38 expression in chronic lymphocytic leukemia.

    PubMed

    Saborit-Villarroya, I; Vaisitti, T; Rossi, D; D'Arena, G; Gaidano, G; Malavasi, F; Deaglio, S

    2011-03-01

    CD38, a nucleotide-metabolizing ectoenzyme and a receptor, is a negative prognostic marker for chronic lymphocytic leukemia (CLL) patients. CD38 has a genetic polymorphism, with a C → G variation in a putative E-box located in a regulatory region. E2A, the predominant E-box factor in B lymphocytes, was found to be highly expressed by CD38(+) CLL patients. The highest CD38 levels scored by E2A(+)/G carrier patients suggested that E2A is (i) directly associated with CD38 expression, and that (ii) the binding of the transcription factor is influenced by the CD38 genotype. Chromatin immunoprecipitation indicated that E2A directly interacts with the CD38 regulatory region. Furthermore, E2A binding was stronger in the presence of the G allele. Experiments of E2A silencing led to a significant reduction of surface levels of CD38, confirming the working hypothesis. A direct functional interplay between E2A and CD38 was shown by exposing CLL cells to interleukin-2 and TLR-9 ligands, both inducers of CD38 expression. Under these conditions, CD38 upregulation was primarily conditioned by the presence of E2A and then by the G allele. The results of this study link E2A and CD38 expression within a common pathway, in which E-protein activity is required for the efficient induction of CD38 transcription.

  17. 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

    2015-02-17

    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.

  18. Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates.

    PubMed

    Hiruta, Jin; Mazet, Francoise; Yasui, Kinya; Zhang, Peijun; Ogasawara, Michio

    2005-07-01

    The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/FoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage.

  19. 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.

  20. 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.

  1. Regulation of myostatin expression and myoblast differentiation by FoxO and SMAD transcription factors.

    PubMed

    Allen, David L; Unterman, Terry G

    2007-01-01

    Myostatin, a member of the transforming growth factor (TGF)-beta family, plays an important role in regulating skeletal muscle growth and differentiation. Here we examined the role of FoxO1 and SMAD transcription factors in regulating myostatin gene expression and myoblast differentiation in C(2)C(12) myotubes in vitro. Both myostatin and FoxO1 mRNA expression were greater in fast- vs. slow-twitch skeletal muscles in vivo. Moreover, expression of a constitutively active form of FoxO1 increased myostatin mRNA and increased activity of a myostatin promoter reporter construct in differentiated C(2)C(12) myotubes. Mutagenesis of highly conserved FoxO or SMAD binding sites significantly decreased myostatin promoter activity, and binding assays showed that both FoxO1 and SMADs bind to their respective sites in the myostatin promoter. Treatment with TGF-beta and/or overexpression of SMAD2, -3, or -4 also resulted in a significant increase in myostatin promoter activity. Treatment with TGF-beta along with overexpression of SMAD2 and FoxO1 resulted in the largest increase in myostatin promoter activity. Finally, TGF-beta treatment and SMAD2 overexpression greatly potentiated FoxO1-mediated suppression of myoblast differentiation. Together these data demonstrate that FoxO1 and SMAD transcription factors regulate the expression of myostatin and contribute to the control of muscle cell growth and differentiation.

  2. Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha.

    PubMed

    Davison, James M; Lickwar, Colin R; Song, Lingyun; Breton, Ghislain; Crawford, Gregory E; Rawls, John F

    2017-04-06

    Microbiota influence diverse aspects of intestinal physiology and disease in part by controlling tissue-specific transcription of host genes. However, host genomic mechanisms mediating microbial control of intestinal gene expression are poorly understood. Hepatocyte nuclear factor 4 (HNF4) is the most ancient family of nuclear receptor transcription factors with important roles in human metabolic and inflammatory bowel diseases, but a role in host response to microbes is unknown. Using an unbiased screening strategy, we found that zebrafish Hnf4a specifically binds and activates a microbiota-suppressed intestinal epithelial transcriptional enhancer. Genetic analysis revealed that zebrafish hnf4a activates nearly half of the genes that are suppressed by microbiota, suggesting microbiota negatively regulate Hnf4a. In support, analysis of genomic architecture in mouse intestinal epithelial cells disclosed that microbiota colonization leads to activation or inactivation of hundreds of enhancers along with drastic genome-wide reduction of HNF4A and HNF4G occupancy. Interspecies meta-analysis suggested interactions between HNF4A and microbiota promote gene expression patterns associated with human inflammatory bowel diseases. These results indicate a critical and conserved role for HNF4A in maintaining intestinal homeostasis in response to microbiota.

  3. Genome-wide identification and expression analysis of TCP transcription factors in Gossypium raimondii

    PubMed Central

    Ma, Jun; Wang, Qinglian; Sun, Runrun; Xie, Fuliang; Jones, Don C.; Zhang, Baohong

    2014-01-01

    Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play versatile functions in multiple aspects of plant growth and development. However, no systematical study has been performed in cotton. In this study, we performed for the first time the genome-wide identification and expression analysis of the TCP transcription factor family in Gossypium raimondii. A total of 38 non-redundant cotton TCP encoding genes were identified. The TCP transcription factors were divided into eleven subgroups based on phylogenetic analysis. Most TCP genes within the same subfamily demonstrated similar exon and intron organization and the motif structures were highly conserved among the subfamilies. Additionally, the chromosomal distribution pattern revealed that TCP genes were unevenly distributed across 11 out of the 13 chromosomes; segmental duplication is a predominant duplication event for TCP genes and the major contributor to the expansion of TCP gene family in G. raimondii. Moreover, the expression profiles of TCP genes shed light on their functional divergence. PMID:25322260

  4. Genome-wide identification and expression analysis of TCP transcription factors in Gossypium raimondii.

    PubMed

    Ma, Jun; Wang, Qinglian; Sun, Runrun; Xie, Fuliang; Jones, Don C; Zhang, Baohong

    2014-10-16

    Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play versatile functions in multiple aspects of plant growth and development. However, no systematical study has been performed in cotton. In this study, we performed for the first time the genome-wide identification and expression analysis of the TCP transcription factor family in Gossypium raimondii. A total of 38 non-redundant cotton TCP encoding genes were identified. The TCP transcription factors were divided into eleven subgroups based on phylogenetic analysis. Most TCP genes within the same subfamily demonstrated similar exon and intron organization and the motif structures were highly conserved among the subfamilies. Additionally, the chromosomal distribution pattern revealed that TCP genes were unevenly distributed across 11 out of the 13 chromosomes; segmental duplication is a predominant duplication event for TCP genes and the major contributor to the expansion of TCP gene family in G. raimondii. Moreover, the expression profiles of TCP genes shed light on their functional divergence.

  5. 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.

  6. Indian Hedgehog Signaling Regulates Transcription and Expression of Collagen Type X via Runx2/Smads Interactions*

    PubMed Central

    Amano, Katsuhiko; Densmore, Michael; Nishimura, Riko; Lanske, Beate

    2014-01-01

    Indian hedgehog (Ihh) is essential for chondrocyte differentiation and endochondral ossification and acts with parathyroid hormone-related peptide in a negative feedback loop to regulate early chondrocyte differentiation and entry to hypertrophic differentiation. Independent of this function, we and others recently reported independent Ihh functions to promote chondrocyte hypertrophy and matrix mineralization in vivo and in vitro. However, the molecular mechanisms for these actions and their functional significance are still unknown. We recently discovered that Ihh overexpression in chondrocytes stimulated the expression of late chondrocyte differentiation markers and induced matrix mineralization. Focusing on collagen type X (Col10α1) expression and transcription, we observed that hedgehog downstream transcription factors GLI-Krüppel family members (Gli) 1/2 increased COL10A1 promoter activity and identified a novel Gli1/2 response element in the 250-bp basic promoter. In addition, we found that Ihh induced Runx2 expression in chondrocytes without up-regulating other modulators of chondrocyte maturation such as Mef2c, Foxa2, and Foxa3. Runx2 promoted Col10α1 expression in cooperation with Ihh. Further analyses using promoter assays, immunofluorescence, and binding assays showed the interaction of Gli1/2 in a complex with Runx2/Smads induces chondrocyte differentiation. Finally, we could demonstrate that Ihh promotes in vitro matrix mineralization using similar molecular mechanisms. Our data provide an in vitro mechanism for Ihh signaling to positively regulate Col10α1 transcription. Thus, Ihh signaling could be an important player for not only early chondrocyte differentiation but maturation and calcification of chondrocytes. PMID:25028519

  7. cDNA cloning, tissue distribution, and chromosomal localization of Ocp2, a gene encoding a putative transcription-associated factor predominantly expressed in the auditory organs

    SciTech Connect

    Chen, Hong; Thalmann, I.; Thalmann, R.

    1995-06-10

    We report the cloning of the Ocp2 gene encoding OCP-II from a guinea pig organ-of-Corti cDNA library. The predicted open reading frame encodes a protein of 163 amino acids with an estimated molecular mass of 18.6 kDa. A homology search revealed that Ocp2 shares significant sequence similarity with p15, a sub-unit of transcription factor SIII that regulates the activity of the RNA polymerase II elongation complex. The Ocp2 messenger RNA is expressed abundantly in the cochlea while not significantly in any other tissues examined, including brain, eye, heart, intestine, kidney, liver, lung, thigh muscle, and testis, demonstrating that the expression of this gene may be restricted to auditory organs. A polyclonal antiserum was raised against the N-terminal region of OCP-II. Immunohistochemical staining of paraffin-embedded sections of the cochlea showed that OCP-II is localized abundantly in nonsensory cells in the organ of Corti; in addition, it was also detected, at a lower concentration, in vestibular sensory organs, as well as auditory and vestibular brain stem nuclei. The Ocp2 gene was mapped to mouse chromosome 4 as well as 11. Our results suggest that OCP-II may be involved in transcription regulation for the development or maintenance of specialized functions of the inner ear. 40 refs., 5 figs.

  8. Transcriptional regulatory network refinement and quantification through kinetic modeling, gene expression microarray data and information theory

    PubMed Central

    Sayyed-Ahmad, Abdallah; Tuncay, Kagan; Ortoleva, Peter J

    2007-01-01

    Background Gene expression microarray and other multiplex data hold promise for addressing the challenges of cellular complexity, refined diagnoses and the discovery of well-targeted treatments. A new approach to the construction and quantification of transcriptional regulatory networks (TRNs) is presented that integrates gene expression microarray data and cell modeling through information theory. Given a partial TRN and time series data, a probability density is constructed that is a functional of the time course of transcription factor (TF) thermodynamic activities at the site of gene control, and is a function of mRNA degradation and transcription rate coefficients, and equilibrium constants for TF/gene binding. Results Our approach yields more physicochemical information that compliments the results of network structure delineation methods, and thereby can serve as an element of a comprehensive TRN discovery/quantification system. The most probable TF time courses and values of the aforementioned parameters are obtained by maximizing the probability obtained through entropy maximization. Observed time delays between mRNA expression and activity are accounted for implicitly since the time course of the activity of a TF is coupled by probability functional maximization, and is not assumed to be proportional to expression level of the mRNA type that translates into the TF. This allows one to investigate post-translational and TF activation mechanisms of gene regulation. Accuracy and robustness of the method are evaluated. A kinetic formulation is used to facilitate the analysis of phenomena with a strongly dynamical character while a physically-motivated regularization of the TF time course is found to overcome difficulties due to omnipresent noise and data sparsity that plague other methods of gene expression data analysis. An application to Escherichia coli is presented. Conclusion Multiplex time series data can be used for the construction of the network of

  9. 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

  10. Post-Transcriptional Control of Cytokine Gene Expression in Health and Disease

    PubMed Central

    2014-01-01

    Post-transcriptional control of cytokine gene expression is essential for rapid and transient response to stimuli and external stress. In health, post-transcriptional control is exerted by a number of trans-acting RNA-binding proteins and cis-acting sequence elements. These elements exist largely in the 3′ untranslated region and comprise microRNA targets and notably AU-rich elements, and exert regulated mRNA decay and translation repression. Defects in this control can lead to increased and sustained production of pro-inflammatory mediators contributing to several chronic inflammatory disease and cancer states. This introduction to the Journal's special issue on the topic summarizes, in a non-comprehensive list, the types of RNA-binding protein and their target cytokines, and potential contributions to disease, and presents the highlights of the individual reviews. PMID:24552151

  11. Specific alternative HOX11 transcripts are expressed in paediatric neural tumours and T-cell acute lymphoblastic leukaemia.

    PubMed

    Watt, Paul M; Hoffmann, Katrin; Greene, Wayne K; Brake, Rachael L; Ford, Jette; Kees, Ursula R

    2003-12-24

    HOX11 is a proto-oncogene, which is silent in normal mature T-cells, while being aberrantly activated in T-cell acute lymphoblastic leukaemia (T-ALL) by translocations t(10;14)(q24;q11) or t(7;10)(q35;q24). Although many oncogenes are expressed in alternative forms in cancer, thus far, only one form of the human HOX11 transcript has been reported. We describe here the identification of three alternative transcripts of the HOX11 proto-oncogene, expressed in primary T-ALL specimens. Using rapid amplification of cDNA ends (RACE) and targeted RT-PCR, we have sequenced 23 individual cDNA clones characterising these novel transcripts. Northern hybridisation identified particular novel exons expressed in T-ALL, which are not expressed in normal T-cells. To date, aberrant expression of HOX11 has only been associated with leukaemia. Our survey of a range of neuroblastoma and primitive neuroectodermal tumour (PNET) cell lines demonstrated the expression of these novel HOX11 transcripts in tumours of neural origin, while their expression was not detected in normal brain tissues. Strikingly, the dominant transcript in these neural tumour cell lines is more than 1 kb larger than the dominant transcript in T-ALL. These observations, combined with sequence data from several EST clones derived from medulloblastoma cDNA libraries, support a new hypothesis that HOX11 may also function as a neural oncogene or brain tumour marker.

  12. Transcriptomic analysis of the mussel Elliptio complanata identifies candidate stress-response genes and an abundance of novel or noncoding transcripts.

    PubMed

    Cornman, Robert S; Robertson, Laura S; Galbraith, Heather; Blakeslee, Carrie

    2014-01-01

    Mussels are useful indicator species of environmental stress and degradation, and the global decline in freshwater mussel diversity and abundance is of conservation concern. Elliptio complanata is a common freshwater mussel of eastern North America that can serve both as an indicator and as an experimental model for understanding mussel physiology and genetics. To support genetic components of these research goals, we assembled transcriptome contigs from Illumina paired-end reads. Despite efforts to collapse similar contigs, the final assembly was in excess of 136,000 contigs with an N50 of 982 bp. Even so, comparisons to the CEGMA database of conserved eukaryotic genes indicated that ∼ 20% of genes remain unrepresented. However, numerous candidate stress-response genes were present, and we identified lineage-specific patterns of diversification among molluscs for cytochrome P450 detoxification genes and two saccharide-modifying enzymes: 1,3 beta-galactosyltransferase and fucosyltransferase. Less than a quarter of contigs had protein-level similarity based on modest BLAST and Hmmer3 statistical thresholds. These results add comparative genomic resources for molluscs and suggest a wealth of novel proteins and noncoding transcripts.

  13. Transcriptomic analysis of the mussel Elliptio complanata identifies candidate stress-response genes and an abundance of novel or noncoding transcripts

    USGS Publications Warehouse

    Cornman, Robert S.; Robertson, Laura S.; Galbraith, Heather S.; Blakeslee, Carrie J.

    2014-01-01

    Mussels are useful indicator species of environmental stress and degradation, and the global decline in freshwater mussel diversity and abundance is of conservation concern. Elliptio complanata is a common freshwater mussel of eastern North America that can serve both as an indicator and as an experimental model for understanding mussel physiology and genetics. To support genetic components of these research goals, we assembled transcriptome contigs from Illumina paired-end reads. Despite efforts to collapse similar contigs, the final assembly was in excess of 136,000 contigs with an N50 of 982 bp. Even so, comparisons to the CEGMA database of conserved eukaryotic genes indicated that ∼20% of genes remain unrepresented. However, numerous candidate stress-response genes were present, and we identified lineage-specific patterns of diversification among molluscs for cytochrome P450 detoxification genes and two saccharide-modifying enzymes: 1,3 beta-galactosyltransferase and fucosyltransferase. Less than a quarter of contigs had protein-level similarity based on modest BLAST and Hmmer3 statistical thresholds. These results add comparative genomic resources for molluscs and suggest a wealth of novel proteins and noncoding transcripts.

  14. 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.

  15. Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits.

    PubMed

    Zermiani, Monica; Zonin, Elisabetta; Nonis, Alberto; Begheldo, Maura; Ceccato, Luca; Vezzaro, Alice; Baldan, Barbara; Trentin, Annarita; Masi, Antonio; Pegoraro, Marco; Fadanelli, Livio; Teale, William; Palme, Klaus; Quintieri, Luigi; Ruperti, Benedetto

    2015-12-01

    Apple (Malus×domestica Borkh) fruits are stored for long periods of time at low temperatures (1 °C) leading to the occurrence of physiological disorders. 'Superficial scald' of Granny Smith apples, an economically important ethylene-dependent disorder, was used as a model to study relationships among ethylene action, the regulation of the ROP-GAP rheostat, and maintenance of H2O2 homeostasis in fruits during prolonged cold exposure. The ROP-GAP rheostat is a key module for adaptation to low oxygen in Arabidopsis through Respiratory Burst NADPH Oxidase Homologs (RBOH)-mediated and ROP GTPase-dependent regulation of reactive oxygen species (ROS) homeostasis. Here, it was shown that the transcriptional expression of several components of the apple ROP-GAP machinery, including genes encoding RBOHs, ROPs, and their ancillary proteins ROP-GEFs and ROP-GAPs, is coordinately and negatively regulated by ethylene in conjunction with the progressive impairment of apoplastic H2O2 homeostatic levels. RNA sequencing analyses showed that several components of the known ROP- and ROS-associated transcriptional networks are regulated along with the ROP-GAP rheostat in response to ethylene perception. These findings may extend the role of the ROP-GAP rheostat beyond hypoxic responses and suggest that it may be a functional regulatory node involved in the integration of ethylene and ROS signalling pathways in abiotic stress.

  16. 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.

  17. Growth Arrest Specific 1 (GAS1) Is Abundantly Expressed in the Adult Mouse Central Nervous System

    PubMed Central

    Zarco, Natanael; Bautista, Elizabeth; Cuéllar, Manola; Vergara, Paula; Flores-Rodriguez, Paola; Aguilar-Roblero, Raúl

    2013-01-01

    Growth arrest specific 1 (GAS1) is a pleiotropic protein that induces apoptosis and cell arrest in different tumors, but it is also involved in the development of the nervous system and other tissues and organs. This dual ability is likely caused by its capacity to interact both by inhibiting the intracellular signaling cascade induced by glial cell-line derived neurotrophic factor and by facilitating the activity of the sonic hedgehog pathway. The presence of GAS1 mRNA has been described in adult mouse brain, and here we corroborated this observation. We then proceeded to determine the distribution of the protein in the adult central nervous system (CNS). We detected, by western blot analysis, expression of GAS1 in olfactory bulb, caudate-putamen, cerebral cortex, hippocampus, mesencephalon, medulla oblongata, cerebellum, and cervical spinal cord. To more carefully map the expression of GAS1, we performed double-label immunohistochemistry and noticed expression of GAS1 in neurons in all brain areas examined. We also observed expression of GAS1 in astroglial cells, albeit the pattern of expression was more restricted than that seen in neurons. Briefly, in the present article, we report the widespread distribution and cellular localization of the GAS1 native protein in adult mammalian CNS. PMID:23813868

  18. Transcriptional and computational study of expansins differentially expressed in response to inclination in radiata pine.

    PubMed

    Mateluna, Patricio; Valenzuela-Riffo, Felipe; Morales-Quintana, Luis; Herrera, Raúl; Ramos, Patricio

    2017-03-09

    Plants have the ability to reorient their vertical growth when exposed to inclination. This response can be as quick as 2 h in inclined young pine (Pinus radiata D. Don) seedlings, with over accumulation of lignin observed after 9 days s. Several studies have identified expansins involved in cell expansion among other developmental processes in plants. Six putative expansin genes were identified in cDNA libraries isolated from inclined pine stems. A differential transcript abundance was observed by qPCR analysis over a time course of inclination. Five genes changed their transcript accumulation in both stem sides in a spatial and temporal manner compared with non-inclined stem. To compare these expansin genes, and to suggest a possible mechanism of action at molecular level, the structures of the predicted proteins were built by comparative modeling methodology. An open groove on the surface of the proteins composed of conserved zresidues was observed. Using a cellulose polymer as ligand the protein-ligand interaction was evaluated, with the results showing differences in the protein-ligand interaction mode. Differences in the binding energy interaction can be explained by changes in some residues that generate differences in electrostatic surface in the open groove region, supporting the participation of six members of multifamily proteins in this specific process. The data suggests participation of different expansin proteins in the dissembling and remodeling of the complex cell wall matrix during the reorientation response to inclination.

  19. Mouse Mesenchymal Progenitor Cells Expressing Adipogenic and Osteogenic Transcription Factors Suppress the Macrophage Inflammatory Response.

    PubMed

    Fernandez, Natalie; Renna, Heather; McHugh, Lauren; Mazolkova, Katie; Crugnola, William; Evans, Jodi F

    2017-01-01

    Mesenchymal progenitor cell characteristics that can identify progenitor populations with specific functions in immunity are actively being investigated. Progenitors from bone marrow and adipose tissue regulate the macrophage (MΦ) inflammatory response by promoting the switch from an inflammatory to an anti-inflammatory phenotype. Conversely, mesenchymal progenitors from the mouse aorta (mAo) support and contribute to the MΦ response under inflammatory conditions. We used cell lines with purported opposing immune-regulatory function, a bone marrow derived mesenchymal progenitor cell line (D1) and a mouse aorta derived mesenchymal progenitor cell line (mAo). Their interaction and regulation of the MΦ cell response to the inflammatory mediator, lipopolysaccharide (LPS), was examined by coculture. As expected, D1 cells suppressed NO, TNF-α, and IL-12p70 production but MΦ phagocytic activity remained unchanged. The mAo cells enhanced NO and TNF-α production in coculture and enhanced MΦ phagocytic activity. Using flow cytometry and PCR array, we then sought to identify sets of MSC-associated genes and markers that are expressed by these progenitor populations. We have determined that immune-supportive mesenchymal progenitors highly express chondrogenic and tenogenic transcription factors while immunosuppressive mesenchymal progenitors highly express adipogenic and osteogenic transcription factors. These data will be useful for the isolation, purification, and modification of mesenchymal progenitors to be used in the treatment of inflammatory diseases.

  20. Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide

    PubMed Central

    Green, Jeffrey; Rolfe, Matthew D; Smith, Laura J

    2014-01-01

    Molecular oxygen (O2) and nitric oxide (NO) are diatomic gases that play major roles in infection. The host innate immune system generates reactive oxygen species and NO as bacteriocidal agents and both require O2 for their production. Furthermore, the ability to adapt to changes in O2 availability is crucial for many bacterial pathogens, as many niches within a host are hypoxic. Pathogenic bacteria have evolved transcriptional regulatory systems that perceive these gases and respond by reprogramming gene expression. Direct sensors possess iron-containing co-factors (iron–sulfur clusters, mononuclear iron, heme) or reactive cysteine thiols that react with O2 and/or NO. Indirect sensors perceive the physiological effects of O2 starvation. Thus, O2 and NO act as environmental cues that trigger the coordinated expression of virulence genes and metabolic adaptations necessary for survival within a host. Here, the mechanisms of signal perception by key O2- and NO-responsive bacterial transcription factors and the effects on virulence gene expression are reviewed, followed by consideration of these aspects of gene regulation in two major pathogens, Staphylococcus aureus and Mycobacterium tuberculosis. PMID:25603427

  1. A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity

    PubMed Central

    Xu, Yuejuan; Fang, Shaohai; Zhang, Erge; Pu, Tian; Cao, Ruixue; Fu, Qihua; Li, Fen; Chen, Sun; Sun, Kun; Xu, Rang

    2017-01-01

    Transcription factor TBX1 plays a pivotal role in heart development and has been implicated in 22q11.2 deletion syndrome. The structure of this protein has been elucidated, and several mutations have been identified that disrupt TBX1 localization, DNA/protein-binding, or mRNA expression. This study reports a mutation in the TBX1 gene that leads to significantly reduced expression of the mutant protein. A total of 773 conotruncal heart defect patients and 516 unrelated healthy control individuals were enrolled, none of which harbored a 22q11.2 deletion or duplication. We identified a mutation, c.303-305delGAA, located in the third exon of TBX1 that does not disrupt TBX1 mRNA expression or DNA binding activity, but results in decreased TBX1 protein levels and transcriptional activity. Through protein degradation studies we demonstrated that TBX1 is degraded primarily in proteasomes. Although the c.303-305delGAA mutation leads to low levels of the mutant protein, we found that increased protein degradation was not the cause, and we hypothesize that an alternate mechanism, such as translational inhibition, may be the cause. PMID:28272434

  2. 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

  3. RNA Sequencing Reveals Xyr1 as a Transcription Factor Regulating Gene Expression beyond Carbohydrate Metabolism

    PubMed Central

    Ma, Liang; Chen, Ling; Zhang, Lei; Zou, Gen; Liu, Rui; Jiang, Yanping

    2016-01-01

    Xyr1 has been demonstrated to be the main transcription activator of (hemi)cellulases in the well-known cellulase producer Trichoderma reesei. This study comprehensively investigates the genes regulated by Xyr1 through RNA sequencing to produce the transcription profiles of T. reesei Rut-C30 and its xyr1 deletion mutant (Δxyr1), cultured on lignocellulose or glucose. xyr1 deletion resulted in 467 differentially expressed genes on inducing medium. Almost all functional genes involved in (hemi)cellulose degradation and many transporters belonging to the sugar porter family in the major facilitator superfamily (MFS) were downregulated in Δxyr1. By contrast, all differentially expressed protease, lipase, chitinase, some ATP-binding cassette transporters, and heat shock protein-encoding genes were upregulated in Δxyr1. When cultured on glucose, a total of 281 genes were expressed differentially in Δxyr1, most of which were involved in energy, solute transport, lipid, amino acid, and monosaccharide as well as secondary metabolism. Electrophoretic mobility shift assays confirmed that the intracellular β-glucosidase bgl2, the putative nonenzymatic cellulose-attacking gene cip1, the MFS lactose transporter lp, the nmrA-like gene, endo T, the acid protease pepA, and the small heat shock protein hsp23 were probable Xyr1-targets. These results might help elucidate the regulation system for synthesis and secretion of (hemi)cellulases in T. reesei Rut-C30. PMID:28116297

  4. Using Blood Informative Transcripts in Geographical Genomics: Impact of Lifestyle on Gene Expression in Fijians

    PubMed Central

    Nath, Artika Praveeta; Arafat, Dalia; Gibson, Greg

    2012-01-01

    In previous geographical genomics studies of the impact of lifestyle on gene expression inferred from microarray analysis of peripheral blood samples, we described the complex influences of culture, ethnicity, and gender in Morocco, and of pregnancy in Brisbane. Here we describe the use of nanofluidic Fluidigm quantitative RT-PCR arrays targeted at a set of 96 transcripts that are broadly informative of the major axes of immune gene expression, to explore the population structure of transcription in Fiji. As in Morocco, major differences are seen between the peripheral blood transcriptomes of rural villagers and residents of the capital city, Suva. The effect is much greater in Indian villages than in Melanesian highlanders and appears to be similar with respect to the nature of at least two axes of variation. Gender differences are much smaller than ethnicity or lifestyle effects. Body mass index is shown to associate with one of the axes as it does in Atlanta and Brisbane, establishing a link between the epidemiological transition of human metabolic disease, and gene expression profiles. PMID:23162571

  5. Aryl hydrocarbon receptor modulates NADPH oxidase activity via direct transcriptional regulation of p40phox expression.

    PubMed

    Wada, Taira; Sunaga, Hiroshi; Ohkawara, Reiko; Shimba, Shigeki

    2013-05-01

    A member of the NADPH oxidase subunits, p40(phox) plays an important role in the regulation of NADPH oxidase activity and the subsequent production of reactive oxygen species (ROS). In this study, we show that mouse p40(phox) is a novel transcriptional target of the aryl hydrocarbon receptor (AhR), known as a dioxin receptor or xenobiotic receptor, in the liver. Treatment of mice with 3-methylcholanthrene (3MC) increased p40(phox) gene expression in the liver, but this induction of p40(phox) gene expression was diminished by the deletion of the AhR gene in the liver. Consistent with the in vivo results, the expression of the p40(phox) gene was increased in 3MC-treated Hepa1c1c7 cells in an AhR-dependent manner. In addition, promoter analysis established p40(phox) as a transcriptional target of AhR. Studies using the RNA-interference technique revealed that p40(phox) is involved in the increase of NADPH oxidase activity and the subsequent ROS production in AhR-activated Hepa1c1c7 cells. Consequently, the results obtained here may provide a novel molecular mechanism for ROS production after exposure to dioxins.

  6. Mouse Mesenchymal Progenitor Cells Expressing Adipogenic and Osteogenic Transcription Factors Suppress the Macrophage Inflammatory Response

    PubMed Central

    Fernandez, Natalie; Renna, Heather; McHugh, Lauren; Mazolkova, Katie; Crugnola, William

    2017-01-01

    Mesenchymal progenitor cell characteristics that can identify progenitor populations with specific functions in immunity are actively being investigated. Progenitors from bone marrow and adipose tissue regulate the macrophage (MΦ) inflammatory response by promoting the switch from an inflammatory to an anti-inflammatory phenotype. Conversely, mesenchymal progenitors from the mouse aorta (mAo) support and contribute to the MΦ response under inflammatory conditions. We used cell lines with purported opposing immune-regulatory function, a bone marrow derived mesenchymal progenitor cell line (D1) and a mouse aorta derived mesenchymal progenitor cell line (mAo). Their interaction and regulation of the MΦ cell response to the inflammatory mediator, lipopolysaccharide (LPS), was examined by coculture. As expected, D1 cells suppressed NO, TNF-α, and IL-12p70 production but MΦ phagocytic activity remained unchanged. The mAo cells enhanced NO and TNF-α production in coculture and enhanced MΦ phagocytic activity. Using flow cytometry and PCR array, we then sought to identify sets of MSC-associated genes and markers that are expressed by these progenitor populations. We have determined that immune-supportive mesenchymal progenitors highly express chondrogenic and tenogenic transcription factors while immunosuppressive mesenchymal progenitors highly express adipogenic and osteogenic transcription factors. These data will be useful for the isolation, purification, and modification of mesenchymal progenitors to be used in the treatment of inflammatory diseases. PMID:28191017

  7. Regulated expression of the neurofibromin type I transcript in the developing chicken brain.

    PubMed

    Baizer, L; Ciment, G; Hendrickson, S K; Schafer, G L

    1993-12-01

    Neurofibromatosis type 1 (NF-1) is among the most common inherited diseases affecting cells of the central and peripheral nervous systems. A region of the NF-1 gene is similar in sequence to the ras-GTPase activator protein (ras-GAP), and investigations have confirmed that the NF-1 gene product (now known as neurofibromin) stimulates ras-GTPase activity in vitro and in vivo. Neurofibromin modulates the ability of ras proteins to regulate cellular proliferation and/or differentiation, suggesting a possible role in normal development. An alternative form of the neurofibromin transcript with an additional 63-bp exon inserted in the GAP-related domain (GRD) has been described recently. To determine whether differential expression of the two forms of neurofibromin GRD mRNA plays a role in embryonic development, we have isolated and characterized the corresponding chicken cDNA. The predicted amino acid sequence for the inserted exon is identical between chick and human, as are the exon-intron boundaries. RNase protection and RNA-polymerase chain reaction analyses demonstrate that most tissues express predominantly type II mRNA (which contains the insert) throughout embryonic development. In contrast, whereas type II is the major form in the brain early in development, expression of the type I transcript (without the insert) in this tissue increases dramatically at later times. Analysis of primary cultures derived from chick embryo brain indicates that the type I mRNA is enriched in neurons.

  8. 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).

  9. 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

  10. 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

  11. 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

  12. 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.

  13. Characterization of expression dynamics of WOX homeodomain transcription factors during somatic embryogenesis in Vitis vinifera.

    PubMed

    Gambino, Giorgio; Minuto, Martina; Boccacci, Paolo; Perrone, Irene; Vallania, Rosalina; Gribaudo, Ivana

    2011-01-01

    Different cultivars of Vitis vinifera vary in their potential to form embryogenic tissues. The WUSCHEL (WUS)-related homeobox (WOX) genes have been shown to play an important role in coordinating the gene transcription involved in the early phases of embryogenesis. The expression dynamics of 12 VvWOX genes present in the V. vinifera genome in embryogenic and other tissues of 'Chardonnay' were analysed. In order to understand the influence of WOX genes on the somatic embryogenic process, their expression profiles were compared in two cultivars of V. vinifera ('Chardonnay' and 'Cabernet Sauvignon') that show different aptitudes for embryogenesis. The expression of all VvWOX genes was influenced by culture conditions. VvWOX2 and VvWOX9 were the principal WOX genes expressed during the somatic embryogenesis process, and the low aptitude for embryogenesis of 'Cabernet Sauvignon' was generally correlated with the low expression levels of these VvWOX genes. VvWOX3 and VvWOX11 were strongly activated in correspondence to torpedo and cotyledonary stages of somatic embryos, with low expression in the earlier developmental stages (pre-embryogenic masses and globular embryos) and during embryo germination. VvWOX genes appeared to be key regulators of somatic embryogenesis in grapevine, and the regulation of these genes during early phases of somatic embryogenesis differed between the two cultivars of the same species.

  14. 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

  15. 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.

  16. 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.

  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. 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.

  19. Triptolide Upregulates Myocardial Forkhead Helix Transcription Factor p3 Expression and Attenuates Cardiac Hypertrophy

    PubMed Central

    Ding, Yuan-Yuan; Li, Jing-Mei; Guo, Feng-Jie; Liu, Ya; Tong, Yang-Fei; Pan, Xi-Chun; Lu, Xiao-Lan; Ye, Wen; Chen, Xiao-Hong; Zhang, Hai-Gang

    2016-01-01

    The forkhead/winged helix transcription factor (Fox) p3 can regulate the expression of various genes, and it has been reported that the transfer of Foxp3-positive T cells could ameliorate cardiac hypertrophy and fibrosis. Triptolide (TP) can elevate the expression of Foxp3, but its effects on cardiac hypertrophy remain unclear. In the present study, neonatal rat ventricular myocytes (NRVM) were isolated and stimulated with angiotensin II (1 μmol/L) to induce hypertrophic response. The expression of Foxp3 in NRVM was observed by using immunofluorescence assay. Fifty mice were randomly divided into five groups and received vehicle (control), isoproterenol (Iso, 5 mg/kg, s.c.), one of three doses of TP (10, 30, or 90 μg/kg, i.p.) for 14 days, respectively. The pathological morphology changes were observed after Hematoxylin and eosin, lectin and Masson’s trichrome staining. The levels of serum brain natriuretic peptide (BNP) and troponin I were determined by enzyme-linked immunosorbent assay and chemiluminescence, respectively. The mRNA and protein expressions of α- myosin heavy chain (MHC), β-MHC and Foxp3 were determined using real-time PCR and immunohistochemistry, respectively. It was shown that TP (1, 3, 10 μg/L) treatment significantly decreased cell size, mRNA and protein expression of β-MHC, and upregulated Foxp3 expression in NRVM. TP also decreased heart weight index, left ventricular weight index and, improved myocardial injury and fibrosis; and decreased the cross-scetional area of the myocardium, serum cardiac troponin and BNP. Additionally, TP markedly reduced the mRNA and protein expression of myocardial β-MHC and elevated the mRNA and protein expression of α-MHC and Foxp3 in a dose-dependent manner. In conclusion, TP can effectively ameliorate myocardial damage and inhibit cardiac hypertrophy, which is at least partly related to the elevation of Foxp3 expression in cardiomyocytes. PMID:27965581

  20. The transcription factor regulatory factor X1 increases the expression of neuronal glutamate transporter type 3.

    PubMed

    Ma, Kaiwen; Zheng, Shuqiu; Zuo, Zhiyi

    2006-07-28

    Glutamate transporters (excitatory amino acid transporters, EAAT) play an important role in maintaining extracellular glutamate homeostasis and regulating glutamate neurotransmission. However, very few studies have investigated the regulation of EAAT expression. A binding sequence for the regulatory factor X1 (RFX1) exists in the promoter region of the gene encoding for EAAT3, a neuronal EAAT, but not in the promoter regions of the genes encoding for EAAT1 and EAAT2, two glial EAATs. RFX proteins are transcription factors binding to X-boxes of DNA sequences. Although RFX proteins are necessary for the normal function of sensory neurons in Caenorhabditis elegans, their roles in the mammalian brain are not known. We showed that RFX1 increased EAAT3 expression and activity in C6 glioma cells. RFX1 binding complexes were found in the nuclear extracts of C6 cells. The activity of EAAT3 promoter as measured by luciferase reporter activity was increased by RFX1 in C6 cells and the neuron-like SH-SY5Y cells. However, RFX1 did not change the expression of EAAT2 proteins in the NRK52E cells. RFX1 proteins were expressed in the neurons of rat brain. A high expression level of RFX1 proteins was found in the neurons of cerebral cortex and Purkinje cells. Knockdown of the RFX1 expression by RFX1 antisense oligonucleotides decreased EAAT3 expression in rat cortical neurons in culture. These results suggest that RFX1 enhances the activity of EAAT3 promoter to increase the expression of EAAT3 proteins. This study provides initial evidence for the regulation of gene expression in the nervous cells by RFX1.

  1. Novel expression and transcriptional regulation of FoxJ1 during oro-facial morphogenesis

    PubMed Central

    Venugopalan, Shankar R.; Amen, Melanie A.; Wang, Jianbo; Wong, Leeyean; Cavender, Adriana C.; D'Souza, Rena N.; Akerlund, Mikael; Brody, Steve L.; Hjalt, Tord A.; Amendt, Brad A.

    2008-01-01

    Axenfeld–Rieger syndrome (ARS) patients with PITX2 point mutations exhibit a wide range of clinical features including mild craniofacial dysmorphism and dental anomalies. Identifying new PITX2 targets and transcriptional mechanisms are important to understand the molecular basis of these anomalies. Chromatin immunoprecipitation assays demonstrate PITX2 binding to the FoxJ1 promoter and PITX2C transgenic mouse fibroblasts and PITX2-transfected cells have increased endogenous FoxJ1 expression. FoxJ1 is expressed at embryonic day 14.5 (E14.5) in early tooth germs, then down-regulated from E15.5–E17.5 and re-expressed in the inner enamel epithelium, oral epithelium, tongue epithelium, sub-mandibular salivary gland and hair follicles during E18.5 and neonate day 1. FoxJ1 and Pitx2 exhibit overlapping expression patterns in the dental and oral epithelium. PITX2 activates the FoxJ1 promoter and, Lef-1 and β-catenin interact with PITX2 to synergistically regulate the FoxJ1 promoter. FoxJ1 physically interacts with the PITX2 homeodomain to synergistically regulate FoxJ1, providing a positive feedback mechanism for FoxJ1 expression. Furthermore, FoxJ1, PITX2, Lef-1 and β-catenin act in concert to activate the FoxJ1 promoter. The PITX2 T68P ARS mutant protein physically interacts with FoxJ1; however, it cannot activate the FoxJ1 promoter. These data indicate a mechanism for the activity of the ARS mutant proteins in specific cell types and provides a basis for craniofacial/ tooth anomalies observed in these patients. These data reveal novel transcriptional mechanisms of FoxJ1 and demonstrate a new role of FoxJ1 in oro-facial morphogenesis. PMID:18723525

  2. FOXL2 transcriptionally represses Sf1 expression by antagonizing WT1 during ovarian development in mice

    PubMed Central

    Takasawa, Kei; Kashimada, Kenichi; Pelosi, Emanuele; Takagi, Masatoshi; Morio, Tomohiro; Asahara, Hiroshi; Schlessinger, David; Mizutani, Shuki; Koopman, Peter

    2014-01-01

    Steroidogenic factor 1 (SF1; Ad4BP/NR5A1) plays key roles in gonadal development. Initially, the Sf1 gene is expressed in mouse fetal gonads of both sexes, but later is up-regulated in testes and down-regulated in ovaries. While Sf1 expression is activated and maintained by Wilms tumor 1 (WT1) and LIM homeobox 9 (LHX9), the mechanism of sex-specific regulation remains unclear. We hypothesized that Sf1 is repressed by the transcription factor Forkhead box L2 (FOXL2) during ovarian development. In an in vitro system (TM3 cells), up-regulation of Sf1 by the WT1 splice variant WT1-KTS was antagonized by FOXL2, as determined by quantitative RT-PCR. Using reporter assays, we localized the Sf1 proximal promoter region involved in this antagonism to a 674-bp interval. A conserved FOXL2 binding site was identified in this interval by in vitro chromatin immunoprecipitation. Introducing mutations into this site abolished negative regulation by FOXL2 in reporter assays. Finally, in Foxl2-null mice, Sf1 expression was increased 2-fold relative to wild-type XX fetal gonads. Our results support the hypothesis that FOXL2 negatively regulates Sf1 expression by antagonizing WT1-KTS during early ovarian development in mice.—Takasawa, K., Kashimada, K., Pelosi, E., Takagi, M., Morio, T., Asahara, H., Schlessinger, D., Mizutani, S., Koopman, P. FOXL2 transcriptionally represses Sf1 expression by antagonizing WT1 during ovarian development in mice. PMID:24451388

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. Developmental Expression and Hypoxic Induction of Hypoxia Inducible Transcription Factors in the Zebrafish

    PubMed Central

    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

  8. Isolation of All CD44 Transcripts in Human Epidermis and Regulation of Their Expression by Various Agents

    PubMed Central

    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

  9. Ethanol induced astaxanthin accumulation and transcriptional expression of carotenogenic genes in Haematococcus pluvialis.

    PubMed

    Wen, Zewen; Liu, Zhiyong; Hou, Yuyong; Liu, Chenfeng; Gao, Feng; Zheng, Yubin; Chen, Fangjian

    2015-10-01

    Haematococcus pluvialis is one of the most promising natural sources of astaxanthin. However, inducing the accumulation process has become one of the primary obstacles in astaxanthin production. In this study, the effect of ethanol on astaxanthin accumulation was investigated. The results demonstrated that astaxanthin accumulation occurred with ethanol addition even under low-light conditions. The astaxanthin productivity could reach 11.26 mg L(-1) d(-1) at 3% (v/v) ethanol, which was 2.03 times of that of the control. The transcriptional expression patterns of eight carotenogenic genes were evaluated using real-time PCR. The results showed that ethanol greatly enhanced transcription of the isopentenyl diphosphate (IPP) isomerase genes (ipi-1 and ipi-2), which were responsible for isomerization reaction of IPP and dimethylallyl diphosphate (DMAPP). This finding suggests that ethanol induced astaxanthin biosynthesis was up-regulated mainly by ipi-1 and ipi-2 at transcriptional level, promoting isoprenoid synthesis and substrate supply to carotenoid formation. Thus ethanol has the potential to be used as an effective reagent to induce astaxanthin accumulation in H. pluvialis.

  10. Role of transcription factor-mediated nucleosome disassembly in PHO5 gene expression.

    PubMed

    Kharerin, Hungyo; Bhat, Paike J; Marko, John F; Padinhateeri, Ranjith

    2016-02-04

    Studying nucleosome dynamics in promoter regions is crucial for understanding gene regulation. Nucleosomes regulate gene expression by sterically occluding transcription factors (TFs) and other non-histone proteins accessing genomic DNA. How the binding competition between nucleosomes and TFs leads to transcriptionally compatible promoter states is an open question. Here, we present a computational study of the nucleosome dynamics and organization in the promoter region of PHO5 gene in Saccharomyces cerevisiae. Introducing a model for nucleosome kinetics that takes into account ATP-dependent remodeling activity, DNA sequence effects, and kinetics of TFs (Pho4p), we compute the probability of obtaining different "promoter states" having different nucleosome configurations. Comparing our results with experimental data, we argue that the presence of local remodeling activity (LRA) as opposed to basal remodeling activity (BRA) is crucial in determining transcriptionally active promoter states. By modulating the LRA and Pho4p binding rate, we obtain different mRNA distributions-Poisson, bimodal, and long-tail. Through this work we explain many features of the PHO5 promoter such as sequence-dependent TF accessibility and the role of correlated dynamics between nucleosomes and TFs in opening/coverage of the TATA box. We also obtain possible ranges for TF binding rates and the magnitude of LRA.

  11. NusA-dependent transcription termination prevents misregulation of global gene expression

    PubMed Central

    Mondal, Smarajit; Yakhnin, Alexander V.; Sebastian, Aswathy; Albert, Istvan; Babitzke, Paul

    2017-01-01

    Intrinsic transcription terminators consist of an RNA hairpin followed by a U-rich tract, and these signals can trigger termination without the involvement of additional factors. Although NusA is known to stimulate intrinsic termination in vitro, the in vivo targets and global impact of NusA are not known because it is essential for viability. Using genome-wide 3′ end-mapping on an engineered Bacillus subtilis NusA depletion strain, we show that weak suboptimal terminators are the principle NusA substrates. Moreover, a subclass of weak non-canonical terminators was identified that completely depend on NusA for effective termination. NusA-dependent terminators tend to have weak hairpins and/or distal U-tract interruptions, supporting a model in which NusA is directly involved in the termination mechanism. Depletion of NusA altered global gene expression directly and indirectly via readthrough of suboptimal terminators. Readthrough of NusA-dependent terminators caused misregulation of genes involved in essential cellular functions, especially DNA replication and metabolism. We further show that nusA is autoregulated by a transcription attenuation mechanism that does not rely on antiterminator structures. Instead, NusA-stimulated termination in its 5′ UTR dictates the extent of transcription into the operon, thereby ensuring tight control of cellular NusA levels. PMID:27571753

  12. The GATA transcription factor GtaC regulates early developmental gene expression dynamics in Dictyostelium.

    PubMed

    Santhanam, Balaji; Cai, Huaqing; Devreotes, Peter N; Shaulsky, Gad; Katoh-Kurasawa, Mariko

    2015-07-06

    In many systems, including the social amoeba Dictyostelium discoideum, development is often marked by dynamic morphological and transcriptional changes orchestrated by key transcription factors. However, efforts to examine sequential genome-wide changes of gene regulation in developmental processes have been fairly limited. Here we report the developmental regulatory dynamics of GtaC, a GATA-type zinc-finger transcription factor, through the analyses of serial ChIP- and RNA-sequencing data. GtaC is essential for developmental progression, decoding extracellular cAMP pulses during early development and may play a role in mediating cell-type differentiation at later stages. We find that GtaC exhibits temporally distinctive DNA-binding patterns concordant with each developmental stage. We identify direct GtaC targets and observe cotemporaneous GtaC-binding and developmental expression regulation. Our results suggest that GtaC regulates multiple physiological processes as Dictyostelium transitions from a group of unicellular amoebae to an integrated multicellular organism.

  13. Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin.

    PubMed

    Ortells, M Carmen; Morancho, Beatriz; Drews-Elger, Katherine; Viollet, Benoit; Laderoute, Keith R; López-Rodríguez, Cristina; Aramburu, Jose

    2012-05-01

    Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses.

  14. Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin

    PubMed Central

    Ortells, M. Carmen; Morancho, Beatriz; Drews-Elger, Katherine; Viollet, Benoit; Laderoute, Keith R.; López-Rodríguez, Cristina; Aramburu, Jose

    2012-01-01

    Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses. PMID:22287635

  15. Role of transcription factor-mediated nucleosome disassembly in PHO5 gene expression

    NASA Astrophysics Data System (ADS)

    Kharerin, Hungyo; Bhat, Paike J.; Marko, John F.; Padinhateeri, Ranjith

    2016-02-01

    Studying nucleosome dynamics in promoter regions is crucial for understanding gene regulation. Nucleosomes regulate gene expression by sterically occluding transcrip