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Sample records for analysis reveals transcriptional

  1. Single cell transcriptional analysis reveals novel innate immune cell types

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    Linda E. Kippner

    2014-06-01

    Full Text Available Single-cell analysis has the potential to provide us with a host of new knowledge about biological systems, but it comes with the challenge of correctly interpreting the biological information. While emerging techniques have made it possible to measure inter-cellular variability at the transcriptome level, no consensus yet exists on the most appropriate method of data analysis of such single cell data. Methods for analysis of transcriptional data at the population level are well established but are not well suited to single cell analysis due to their dependence on population averages. In order to address this question, we have systematically tested combinations of methods for primary data analysis on single cell transcription data generated from two types of primary immune cells, neutrophils and T lymphocytes. Cells were obtained from healthy individuals, and single cell transcript expression data was obtained by a combination of single cell sorting and nanoscale quantitative real time PCR (qRT-PCR for markers of cell type, intracellular signaling, and immune functionality. Gene expression analysis was focused on hierarchical clustering to determine the existence of cellular subgroups within the populations. Nine combinations of criteria for data exclusion and normalization were tested and evaluated. Bimodality in gene expression indicated the presence of cellular subgroups which were also revealed by data clustering. We observed evidence for two clearly defined cellular subtypes in the neutrophil populations and at least two in the T lymphocyte populations. When normalizing the data by different methods, we observed varying outcomes with corresponding interpretations of the biological characteristics of the cell populations. Normalization of the data by linear standardization taking into account technical effects such as plate effects, resulted in interpretations that most closely matched biological expectations. Single cell transcription

  2. Controllability analysis of transcriptional regulatory networks reveals circular control patterns among transcription factors

    DEFF Research Database (Denmark)

    Österlund, Tobias; Bordel, Sergio; Nielsen, Jens

    2015-01-01

    we analyze the topology and organization of nine transcriptional regulatory networks for E. coli, yeast, mouse and human, and we evaluate how the structure of these networks influences two of their key properties, namely controllability and stability. We calculate the controllability for each network......Transcriptional regulation is the most committed type of regulation in living cells where transcription factors (TFs) control the expression of their target genes and TF expression is controlled by other TFs forming complex transcriptional regulatory networks that can be highly interconnected. Here...... as a measure of the organization and interconnectivity of the network. We find that the number of driver nodes n(D) needed to control the whole network is 64% of the TFs in the E. coli transcriptional regulatory network in contrast to only 17% for the yeast network, 4% for the mouse network and 8...

  3. Comparative transcriptional analysis reveals differential gene expression between asymmetric and symmetric zygotic divisions in tobacco.

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    Tian-Xiang Hu

    Full Text Available Asymmetric cell divisions occur widely during many developmental processes in plants. In most angiosperms, the first zygotic cell division is asymmetric resulting in two daughter cells of unequal size and with distinct fates. However, the critical molecular mechanisms regulating this division remain unknown. Previously we showed that treatment of tobacco zygotes with beta-glucosyl Yariv (βGlcY could dramatically alter the first zygotic asymmetric division to produce symmetric two-celled proembryos. In the present study, we isolated zygotes and two-celled asymmetric proembryos in vivo by micromanipulation, and obtained symmetric, two-celled proembryos by in vitro cell cultures. Using suppression-subtractive hybridization (SSH and macroarray analysis differential gene expression between the zygote and the asymmetric and symmetric two-celled proembryos was investigated. After sequencing of the differentially expressed clones, a total of 1610 EST clones representing 685 non-redundant transcripts were obtained. Gene ontology (GO term analysis revealed that these transcripts include those involved in physiological processes such as response to stimulus, regulation of gene expression, and localization and formation of anatomical structures. A homology search against known genes from Arabidopsis indicated that some of the above transcripts are involved in asymmetric cell division and embryogenesis. Quantitative real-time PCR confirmed the up- or down-regulation of the selected candidate transcripts during zygotic division. A few of these transcripts were expressed exclusively in the zygote, or in either type of the two-celled proembryos. Expression analyses of select genes in different tissues and organs also revealed potential roles of these transcripts in fertilization, seed maturation and organ development. The putative roles of few of the identified transcripts in the regulation of zygotic division are discussed. Further functional work on these

  4. Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells.

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    Roellig, Daniela; Tan-Cabugao, Johanna; Esaian, Sevan; Bronner, Marianne E

    2017-03-29

    The 'neural plate border' of vertebrate embryos contains precursors of neural crest and placode cells, both defining vertebrate characteristics. How these lineages segregate from neural and epidermal fates has been a matter of debate. We address this by performing a fine-scale quantitative temporal analysis of transcription factor expression in the neural plate border of chick embryos. The results reveal significant overlap of transcription factors characteristic of multiple lineages in individual border cells from gastrula through neurula stages. Cell fate analysis using a Sox2 (neural) enhancer reveals that cells that are initially Sox2+ cells can contribute not only to neural tube but also to neural crest and epidermis. Moreover, modulating levels of Sox2 or Pax7 alters the apportionment of neural tube versus neural crest fates. Our results resolve a long-standing question and suggest that many individual border cells maintain ability to contribute to multiple ectodermal lineages until or beyond neural tube closure.

  5. Meta-analysis of small RNA-sequencing errors reveals ubiquitous post-transcriptional RNA modifications.

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    Ebhardt, H Alexander; Tsang, Herbert H; Dai, Denny C; Liu, Yifeng; Bostan, Babak; Fahlman, Richard P

    2009-05-01

    Recent advances in DNA-sequencing technology have made it possible to obtain large datasets of small RNA sequences. Here we demonstrate that not all non-perfectly matched small RNA sequences are simple technological sequencing errors, but many hold valuable biological information. Analysis of three small RNA datasets originating from Oryza sativa and Arabidopsis thaliana small RNA-sequencing projects demonstrates that many single nucleotide substitution errors overlap when aligning homologous non-identical small RNA sequences. Investigating the sites and identities of substitution errors reveal that many potentially originate as a result of post-transcriptional modifications or RNA editing. Modifications include N1-methyl modified purine nucleotides in tRNA, potential deamination or base substitutions in micro RNAs, 3' micro RNA uridine extensions and 5' micro RNA deletions. Additionally, further analysis of large sequencing datasets reveal that the combined effects of 5' deletions and 3' uridine extensions can alter the specificity by which micro RNAs associate with different Argonaute proteins. Hence, we demonstrate that not all sequencing errors in small RNA datasets are technical artifacts, but that these actually often reveal valuable biological insights to the sites of post-transcriptional RNA modifications.

  6. Transcriptional regulation of rod photoreceptor homeostasis revealed by in vivo NRL targetome analysis.

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    Hong Hao

    Full Text Available A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP-Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP-Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis.

  7. Meta-analysis reveals conserved cell cycle transcriptional network across multiple human cell types.

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    Giotti, Bruno; Joshi, Anagha; Freeman, Tom C

    2017-01-05

    Cell division is central to the physiology and pathology of all eukaryotic organisms. The molecular machinery underpinning the cell cycle has been studied extensively in a number of species and core aspects of it have been found to be highly conserved. Similarly, the transcriptional changes associated with this pathway have been studied in different organisms and different cell types. In each case hundreds of genes have been reported to be regulated, however there seems to be little consensus in the genes identified across different studies. In a recent comparison of transcriptomic studies of the cell cycle in different human cell types, only 96 cell cycle genes were reported to be the same across all studies examined. Here we perform a systematic re-examination of published human cell cycle expression data by using a network-based approach to identify groups of genes with a similar expression profile and therefore function. Two clusters in particular, containing 298 transcripts, showed patterns of expression consistent with cell cycle occurrence across the four human cell types assessed. Our analysis shows that there is a far greater conservation of cell cycle-associated gene expression across human cell types than reported previously, which can be separated into two distinct transcriptional networks associated with the G 1 /S-S and G 2 -M phases of the cell cycle. This work also highlights the benefits of performing a re-analysis on combined datasets.

  8. Transcriptional Analysis of the Genetic Element pSSVx: Differential and Temporal Regulation of Gene Expression Reveals Correlation between Transcription and Replication

    DEFF Research Database (Denmark)

    Contursi, Patrizia; Cannio, Raffaele; Prato, Santina

    2007-01-01

    pSSVx from Sulfolobus islandicus strain REY15/4 is a hybrid between a plasmid and a fusellovirus. A systematic study performed by a combination of Northern blot analysis, primer extension, and reverse transcriptase PCR revealed the presence of nine major transcripts whose expression...... was differentially and temporally regulated over the growth cycle of S. islandicus. The map positions of the RNAs as well as the clockwise and the anticlockwise directions of their transcription were determined. Some genes were clustered and appeared to be transcribed as polycistronic messengers, among which one...... long transcriptional unit comprised the genes for the plasmid copy number control protein ORF60 (CopG), ORF91, and the replication protein ORF892 (RepA). We propose that a termination readthrough mechanism might be responsible for the formation of more than one RNA species from a single 5' end...

  9. Transcriptional Network Analysis Reveals Drought Resistance Mechanisms of AP2/ERF Transgenic Rice

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    Hongryul Ahn

    2017-06-01

    Full Text Available This study was designed to investigate at the molecular level how a transgenic version of rice “Nipponbare” obtained a drought-resistant phenotype. Using multi-omics sequencing data, we compared wild-type rice (WT and a transgenic version (erf71 that had obtained a drought-resistant phenotype by overexpressing OsERF71, a member of the AP2/ERF transcription factor (TF family. A comprehensive bioinformatics analysis pipeline, including TF networks and a cascade tree, was developed for the analysis of multi-omics data. The results of the analysis showed that the presence of OsERF71 at the source of the network controlled global gene expression levels in a specific manner to make erf71 survive longer than WT. Our analysis of the time-series transcriptome data suggests that erf71 diverted more energy to survival-critical mechanisms related to translation, oxidative response, and DNA replication, while further suppressing energy-consuming mechanisms, such as photosynthesis. To support this hypothesis further, we measured the net photosynthesis level under physiological conditions, which confirmed the further suppression of photosynthesis in erf71. In summary, our work presents a comprehensive snapshot of transcriptional modification in transgenic rice and shows how this induced the plants to acquire a drought-resistant phenotype.

  10. Microarray analysis of a salamander hopeful monster reveals transcriptional signatures of paedomorphic brain development

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    Putta Srikrishna

    2010-06-01

    Full Text Available Abstract Background The Mexican axolotl (Ambystoma mexicanum is considered a hopeful monster because it exhibits an adaptive and derived mode of development - paedomorphosis - that has evolved rapidly and independently among tiger salamanders. Unlike related tiger salamanders that undergo metamorphosis, axolotls retain larval morphological traits into adulthood and thus present an adult body plan that differs dramatically from the ancestral (metamorphic form. The basis of paedomorphic development was investigated by comparing temporal patterns of gene transcription between axolotl and tiger salamander larvae (Ambystoma tigrinum tigrinum that typically undergo a metamorphosis. Results Transcript abundances from whole brain and pituitary were estimated via microarray analysis on four different days post hatching (42, 56, 70, 84 dph and regression modeling was used to independently identify genes that were differentially expressed as a function of time in both species. Collectively, more differentially expressed genes (DEGs were identified as unique to the axolotl (n = 76 and tiger salamander (n = 292 than were identified as shared (n = 108. All but two of the shared DEGs exhibited the same temporal pattern of expression and the unique genes tended to show greater changes later in the larval period when tiger salamander larvae were undergoing anatomical metamorphosis. A second, complementary analysis that directly compared the expression of 1320 genes between the species identified 409 genes that differed as a function of species or the interaction between time and species. Of these 409 DEGs, 84% exhibited higher abundances in tiger salamander larvae at all sampling times. Conclusions Many of the unique tiger salamander transcriptional responses are probably associated with metamorphic biological processes. However, the axolotl also showed unique patterns of transcription early in development. In particular, the axolotl showed a genome

  11. New insights into molecular pathways associated with flatfish ovarian development and atresia revealed by transcriptional analysis

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    Agulleiro María J

    2009-09-01

    Full Text Available Abstract Background The Senegalese sole (Solea senegalensis is a marine flatfish of increasing commercial interest. However, the reproduction of this species in captivity is not yet controlled mainly because of the poor knowledge on its reproductive physiology, as it occurs for other non-salmonid marine teleosts that exhibit group-synchronous ovarian follicle development. In order to investigate intra-ovarian molecular mechanisms in Senegalese sole, the aim of the present study was to identify differentially expressed genes in the ovary during oocyte growth (vitellogenesis, maturation and ovarian follicle atresia using a recently developed oligonucleotide microarray. Results Microarray analysis led to the identification of 118 differentially expressed transcripts, of which 20 and 8 were monitored by real-time PCR and in situ hybridization, respectively. During vitellogenesis, many up-regulated ovarian transcripts had putative mitochondrial function/location suggesting high energy production (NADH dehydrogenase subunits, cytochromes and increased antioxidant protection (selenoprotein W2a, whereas other regulated transcripts were related to cytoskeleton and zona radiata organization (zona glycoprotein 3, alpha and beta actin, keratin 8, intracellular signalling pathways (heat shock protein 90, Ras homolog member G, cell-to-cell and cell-to-matrix interactions (beta 1 integrin, thrombospondin 4b, and the maternal RNA pool (transducer of ERBB2 1a, neurexin 1a. Transcripts up-regulated in the ovary during oocyte maturation included ion transporters (Na+-K+-ATPase subunits, probably required for oocyte hydration, as well as a proteinase inhibitor (alpha-2-macroglobulin and a vesicle calcium sensor protein (extended synaptotagmin-2-A. During follicular atresia, few transcripts were found to be up-regulated, but remarkably most of them were localized in follicular cells of atretic follicles, and they had inferred roles in lipid transport (apolipoprotein

  12. Transcript and metabolite analysis in Trincadeira cultivar reveals novel information regarding the dynamics of grape ripening

    Science.gov (United States)

    2011-01-01

    metabolism. These results were integrated with transcriptional profiling obtained using genome array to provide new information regarding the network of events leading to grape ripening. Conclusions Altogether the data obtained provides the most extensive survey obtained so far for gene expression and metabolites accumulated during grape ripening. Moreover, it highlighted information obtained in a poorly known variety exhibiting particular characteristics that may be cultivar specific or dependent upon climatic conditions. Several genes were identified that had not been previously reported in the context of grape ripening namely genes involved in carbohydrate and amino acid metabolisms as well as in growth regulators; metabolism, epigenetic factors and signaling pathways. Some of these genes were annotated as receptors, transcription factors, and kinases and constitute good candidates for functional analysis in order to establish a model for ripening control of a non-climacteric fruit. PMID:22047180

  13. Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria

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    Keeling Patrick J

    2007-09-01

    Full Text Available Abstract Background Dinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes. Results From two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression. Conclusion The dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements

  14. Spatially resolved metabolic analysis reveals a central role for transcriptional control in carbon allocation to wood.

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    Roach, Melissa; Arrivault, Stéphanie; Mahboubi, Amir; Krohn, Nicole; Sulpice, Ronan; Stitt, Mark; Niittylä, Totte

    2017-06-15

    The contribution of transcriptional and post-transcriptional regulation to modifying carbon allocation to developing wood of trees is not well defined. To clarify the role of transcriptional regulation, the enzyme activity patterns of eight central primary metabolism enzymes across phloem, cambium, and developing wood of aspen (Populus tremula L.) were compared with transcript levels obtained by RNA sequencing of sequential stem sections from the same trees. Enzymes were selected on the basis of their importance in sugar metabolism and in linking primary metabolism to lignin biosynthesis. Existing enzyme assays were adapted to allow measurements from ~1 mm3 sections of dissected stem tissue. These experiments provided high spatial resolution of enzyme activity changes across different stages of wood development, and identified the gene transcripts probably responsible for these changes. In most cases, there was a clear positive relationship between transcripts and enzyme activity. During secondary cell wall formation, the increases in transcript levels and enzyme activities also matched with increased levels of glucose, fructose, hexose phosphates, and UDP-glucose, emphasizing an important role for transcriptional regulation in carbon allocation to developing aspen wood. These observations corroborate the efforts to increase carbon allocation to wood by engineering gene regulatory networks. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  15. Proteomic analysis of FOXP proteins reveals interactions between cortical transcription factors associated with neurodevelopmental disorders.

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    Estruch, Sara B; Graham, Sarah A; Quevedo, Martí; Vino, Arianna; Dekkers, Dick H W; Deriziotis, Pelagia; Sollis, Elliot; Demmers, Jeroen; Poot, Raymond A; Fisher, Simon E

    2018-04-01

    FOXP transcription factors play important roles in neurodevelopment, but little is known about how their transcriptional activity is regulated. FOXP proteins cooperatively regulate gene expression by forming homo- and hetero-dimers with each other. Physical associations with other transcription factors might also modulate the functions of FOXP proteins. However, few FOXP-interacting transcription factors have been identified so far. Therefore, we sought to discover additional transcription factors that interact with the brain-expressed FOXP proteins, FOXP1, FOXP2 and FOXP4, through affinity-purifications of protein complexes followed by mass spectrometry. We identified seven novel FOXP-interacting transcription factors (NR2F1, NR2F2, SATB1, SATB2, SOX5, YY1 and ZMYM2), five of which have well-estabslished roles in cortical development. Accordingly, we found that these transcription factors are co-expressed with FoxP2 in the deep layers of the cerebral cortex and also in the Purkinje cells of the cerebellum, suggesting that they may cooperate with the FoxPs to regulate neural gene expression in vivo. Moreover, we demonstrated that etiological mutations of FOXP1 and FOXP2, known to cause neurodevelopmental disorders, severely disrupted the interactions with FOXP-interacting transcription factors. Additionally, we pinpointed specific regions within FOXP2 sequence involved in mediating these interactions. Thus, by expanding the FOXP interactome we have uncovered part of a broader neural transcription factor network involved in cortical development, providing novel molecular insights into the transcriptional architecture underlying brain development and neurodevelopmental disorders.

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

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    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.

  17. Microarray analysis reveals transcriptional plasticity in the reef building coral Acropora millepora

    DEFF Research Database (Denmark)

    Bay, L. K.; Ulstrup, K. E.; Nielsen, H. B.

    2009-01-01

    We investigated variation in transcript abundance in the scleractinian coral, Acropora millepora, within and between populations characteristically exposed to different turbidity regimes and hence different levels of light and suspended particulate matter. We examined phenotypic plasticity by com...

  18. Coxiella burnetii transcriptional analysis reveals serendipity clusters of regulation in intracellular bacteria.

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    Quentin Leroy

    Full Text Available Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is mainly transmitted to humans through an aerosol route. A spore-like form allows C. burnetii to resist different environmental conditions. Because of this, analysis of the survival strategies used by this bacterium to adapt to new environmental conditions is critical for our understanding of C. burnetii pathogenicity. Here, we report the early transcriptional response of C. burnetii under temperature stresses. Our data show that C. burnetii exhibited minor changes in gene regulation under short exposure to heat or cold shock. While small differences were observed, C. burnetii seemed to respond similarly to cold and heat shock. The expression profiles obtained using microarrays produced in-house were confirmed by quantitative RT-PCR. Under temperature stresses, 190 genes were differentially expressed in at least one condition, with a fold change of up to 4. Globally, the differentially expressed genes in C. burnetii were associated with bacterial division, (pppGpp synthesis, wall and membrane biogenesis and, especially, lipopolysaccharide and peptidoglycan synthesis. These findings could be associated with growth arrest and witnessed transformation of the bacteria to a spore-like form. Unexpectedly, clusters of neighboring genes were differentially expressed. These clusters do not belong to operons or genetic networks; they have no evident associated functions and are not under the control of the same promoters. We also found undescribed but comparable clusters of regulation in previously reported transcriptomic analyses of intracellular bacteria, including Rickettsia sp. and Listeria monocytogenes. The transcriptomic patterns of C. burnetii observed under temperature stresses permits the recognition of unpredicted clusters of regulation for which the trigger mechanism remains unidentified but which may be the result of a new mechanism of epigenetic regulation.

  19. Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest

    DEFF Research Database (Denmark)

    Sylvestersen, Kathrine B; Horn, Heiko; Jungmichel, Stephanie

    2014-01-01

    mono-methylation (MMA) sites. We thereby identify 1,027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization......, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers...... strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of transcriptional arrest. In contrast, the corresponding di-methylation or protein expression level is not altered in expression, confirming that MMA sites...

  20. Balanced transcription of cell division genes in Bacillus subtilis as revealed by single cell analysis

    NARCIS (Netherlands)

    Trip, Erik Nico; Veening, Jan-Willem; Stewart, Eric J.; Errington, Jeff; Scheffers, Dirk-Jan

    2013-01-01

    Cell division in bacteria is carried out by a set of conserved proteins that all have to function at the correct place and time. A cell cycle-dependent transcriptional programme drives cell division in bacteria such as Caulobacter crescentus. Whether such a programme exists in the Gram-positive

  1. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae

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    Ma Menggen

    2010-06-01

    Full Text Available Abstract Background Derived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain. Results A highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family and transcription factors. The parental strain showed repressed expressions for many genes and was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidate and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p/Pdr3p. Conclusion Enriched background of transcription abundance

  2. Genome-wide analysis of the Dof transcription factor gene family reveals soybean-specific duplicable and functional characteristics.

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    Yong Guo

    Full Text Available The Dof domain protein family is a classic plant-specific zinc-finger transcription factor family involved in a variety of biological processes. There is great diversity in the number of Dof genes in different plants. However, there are only very limited reports on the characterization of Dof transcription factors in soybean (Glycine max. In the present study, 78 putative Dof genes were identified from the whole-genome sequence of soybean. The predicted GmDof genes were non-randomly distributed within and across 19 out of 20 chromosomes and 97.4% (38 pairs were preferentially retained duplicate paralogous genes located in duplicated regions of the genome. Soybean-specific segmental duplications contributed significantly to the expansion of the soybean Dof gene family. These Dof proteins were phylogenetically clustered into nine distinct subgroups among which the gene structure and motif compositions were considerably conserved. Comparative phylogenetic analysis of these Dof proteins revealed four major groups, similar to those reported for Arabidopsis and rice. Most of the GmDofs showed specific expression patterns based on RNA-seq data analyses. The expression patterns of some duplicate genes were partially redundant while others showed functional diversity, suggesting the occurrence of sub-functionalization during subsequent evolution. Comprehensive expression profile analysis also provided insights into the soybean-specific functional divergence among members of the Dof gene family. Cis-regulatory element analysis of these GmDof genes suggested diverse functions associated with different processes. Taken together, our results provide useful information for the functional characterization of soybean Dof genes by combining phylogenetic analysis with global gene-expression profiling.

  3. Genome-wide analysis of the Dof transcription factor gene family reveals soybean-specific duplicable and functional characteristics.

    Science.gov (United States)

    Guo, Yong; Qiu, Li-Juan

    2013-01-01

    The Dof domain protein family is a classic plant-specific zinc-finger transcription factor family involved in a variety of biological processes. There is great diversity in the number of Dof genes in different plants. However, there are only very limited reports on the characterization of Dof transcription factors in soybean (Glycine max). In the present study, 78 putative Dof genes were identified from the whole-genome sequence of soybean. The predicted GmDof genes were non-randomly distributed within and across 19 out of 20 chromosomes and 97.4% (38 pairs) were preferentially retained duplicate paralogous genes located in duplicated regions of the genome. Soybean-specific segmental duplications contributed significantly to the expansion of the soybean Dof gene family. These Dof proteins were phylogenetically clustered into nine distinct subgroups among which the gene structure and motif compositions were considerably conserved. Comparative phylogenetic analysis of these Dof proteins revealed four major groups, similar to those reported for Arabidopsis and rice. Most of the GmDofs showed specific expression patterns based on RNA-seq data analyses. The expression patterns of some duplicate genes were partially redundant while others showed functional diversity, suggesting the occurrence of sub-functionalization during subsequent evolution. Comprehensive expression profile analysis also provided insights into the soybean-specific functional divergence among members of the Dof gene family. Cis-regulatory element analysis of these GmDof genes suggested diverse functions associated with different processes. Taken together, our results provide useful information for the functional characterization of soybean Dof genes by combining phylogenetic analysis with global gene-expression profiling.

  4. Meta-analysis of small RNA-sequencing errors reveals ubiquitous post-transcriptional RNA modifications

    OpenAIRE

    Ebhardt, H. Alexander; Tsang, Herbert H.; Dai, Denny C.; Liu, Yifeng; Bostan, Babak; Fahlman, Richard P.

    2009-01-01

    Recent advances in DNA-sequencing technology have made it possible to obtain large datasets of small RNA sequences. Here we demonstrate that not all non-perfectly matched small RNA sequences are simple technological sequencing errors, but many hold valuable biological information. Analysis of three small RNA datasets originating from Oryza sativa and Arabidopsis thaliana small RNA-sequencing projects demonstrates that many single nucleotide substitution errors overlap when aligning homologous...

  5. Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family.

    Science.gov (United States)

    Pearce, Stephen; Huttly, Alison K; Prosser, Ian M; Li, Yi-dan; Vaughan, Simon P; Gallova, Barbora; Patil, Archana; Coghill, Jane A; Dubcovsky, Jorge; Hedden, Peter; Phillips, Andrew L

    2015-06-05

    The gibberellin (GA) pathway plays a central role in the regulation of plant development, with the 2-oxoglutarate-dependent dioxygenases (2-ODDs: GA20ox, GA3ox, GA2ox) that catalyse the later steps in the biosynthetic pathway of particularly importance in regulating bioactive GA levels. Although GA has important impacts on crop yield and quality, our understanding of the regulation of GA biosynthesis during wheat and barley development remains limited. In this study we identified or assembled genes encoding the GA 2-ODDs of wheat, barley and Brachypodium distachyon and characterised the wheat genes by heterologous expression and transcript analysis. The wheat, barley and Brachypodium genomes each contain orthologous copies of the GA20ox, GA3ox and GA2ox genes identified in rice, with the exception of OsGA3ox1 and OsGA2ox5 which are absent in these species. Some additional paralogs of 2-ODD genes were identified: notably, a novel gene in the wheat B genome related to GA3ox2 was shown to encode a GA 1-oxidase, named as TaGA1ox-B1. This enzyme is likely to be responsible for the abundant 1β-hydroxylated GAs present in developing wheat grains. We also identified a related gene in barley, located in a syntenic position to TaGA1ox-B1, that encodes a GA 3,18-dihydroxylase which similarly accounts for the accumulation of unusual GAs in barley grains. Transcript analysis showed that some paralogs of the different classes of 2-ODD were expressed mainly in a single tissue or at specific developmental stages. In particular, TaGA20ox3, TaGA1ox1, TaGA3ox3 and TaGA2ox7 were predominantly expressed in developing grain. More detailed analysis of grain-specific gene expression showed that while the transcripts of biosynthetic genes were most abundant in the endosperm, genes encoding inactivation and signalling components were more highly expressed in the seed coat and pericarp. The comprehensive expression and functional characterisation of the multigene families encoding the 2-ODD

  6. Analysis of Transcriptional Signatures in Response to Listeria monocytogenes Infection Reveals Temporal Changes That Result from Type I Interferon Signaling

    Science.gov (United States)

    Potempa, Krzysztof; Graham, Christine M.; Moreira-Teixeira, Lucia; McNab, Finlay W.; Howes, Ashleigh; Stavropoulos, Evangelos; Pascual, Virginia; Banchereau, Jacques; Chaussabel, Damien; O’Garra, Anne

    2016-01-01

    Analysis of the mouse transcriptional response to Listeria monocytogenes infection reveals that a large set of genes are perturbed in both blood and tissue and that these transcriptional responses are enriched for pathways of the immune response. Further we identified enrichment for both type I and type II interferon (IFN) signaling molecules in the blood and tissues upon infection. Since type I IFN signaling has been reported widely to impair bacterial clearance we examined gene expression from blood and tissues of wild type (WT) and type I IFNαβ receptor-deficient (Ifnar1-/-) mice at the basal level and upon infection with L. monocytogenes. Measurement of the fold change response upon infection in the absence of type I IFN signaling demonstrated an upregulation of specific genes at day 1 post infection. A less marked reduction of the global gene expression signature in blood or tissues from infected Ifnar1-/- as compared to WT mice was observed at days 2 and 3 after infection, with marked reduction in key genes such as Oasg1 and Stat2. Moreover, on in depth analysis, changes in gene expression in uninfected mice of key IFN regulatory genes including Irf9, Irf7, Stat1 and others were identified, and although induced by an equivalent degree upon infection this resulted in significantly lower final gene expression levels upon infection of Ifnar1-/- mice. These data highlight how dysregulation of this network in the steady state and temporally upon infection may determine the outcome of this bacterial infection and how basal levels of type I IFN-inducible genes may perturb an optimal host immune response to control intracellular bacterial infections such as L. monocytogenes. PMID:26918359

  7. RNA-Seq analysis of rice roots reveals the involvement of post-transcriptional regulation in response to cadmium stress

    Directory of Open Access Journals (Sweden)

    Luqing eZheng

    2015-12-01

    Full Text Available Widely-spread cadmium (Cd pollution in the soil threatens both crop production and human health. How plants deal with the excess Cd are largely unknown. To evaluate the molecular mechanism by which plants respond to Cd stress, rice seedlings were treated with two concentrations of Cd and subjected to deep RNA sequencing. Comprehensive RNA-Seq analysis of rice roots under two gradients of Cd treatment revealed 1,169 Cd toxicity-responsive genes. These genes were involved in the reactive oxygen species scavenging system, stress response, cell wall formation, ion transport, and signal transduction. Nine out of 93 predicted long non coding RNAs (lncRNAs were detected as Cd-responsive lncRNAs due to their high correlation with the Cd stress response. In addition, we analyzed alternative splicing (AS events under different Cd concentrations. 476 differential alternatively spliced genes with 542 aberrant splicing events were identified. GO analysis indicated that these genes were highly enriched in oxidation reduction and cellular response to chemical stimulus. Real-time qRT-PCR validation analysis strengthened the reliability of our RNA-Seq results. The results suggest that post-transcriptional AS regulation may also be involved in plant responses to high Cd stress.

  8. Transcription analysis of recombinant industrial and laboratory Saccharomyces cerevisiae strains reveals the molecular basis for fermentation of glucose and xylose.

    Science.gov (United States)

    Matsushika, Akinori; Goshima, Tetsuya; Hoshino, Tamotsu

    2014-01-28

    There has been much research on the bioconversion of xylose found in lignocellulosic biomass to ethanol by genetically engineered Saccharomyces cerevisiae. However, the rate of ethanol production from xylose in these xylose-utilizing yeast strains is quite low compared to their glucose fermentation. In this study, two diploid xylose-utilizing S. cerevisiae strains, the industrial strain MA-R4 and the laboratory strain MA-B4, were employed to investigate the differences between anaerobic fermentation of xylose and glucose, and general differences between recombinant yeast strains, through genome-wide transcription analysis. In MA-R4, many genes related to ergosterol biosynthesis were expressed more highly with glucose than with xylose. Additionally, these ergosterol-related genes had higher transcript levels in MA-R4 than in MA-B4 during glucose fermentation. During xylose fermentation, several genes related to central metabolic pathways that typically increase during growth on non-fermentable carbon sources were expressed at higher levels in both strains. Xylose did not fully repress the genes encoding enzymes of the tricarboxylic acid and respiratory pathways, even under anaerobic conditions. In addition, several genes involved in spore wall metabolism and the uptake of ammonium, which are closely related to the starvation response, and many stress-responsive genes mediated by Msn2/4p, as well as trehalose synthase genes, increased in expression when fermenting with xylose, irrespective of the yeast strain. We further observed that transcript levels of genes involved in xylose metabolism, membrane transport functions, and ATP synthesis were higher in MA-R4 than in MA-B4 when strains were fermented with glucose or xylose. Our transcriptomic approach revealed the molecular events underlying the response to xylose or glucose and differences between MA-R4 and MA-B4. Xylose-utilizing S. cerevisiae strains may recognize xylose as a non-fermentable carbon source, which

  9. Integrated analysis of transcript-level regulation of metabolism reveals disease-relevant nodes of the human metabolic network.

    Science.gov (United States)

    Galhardo, Mafalda; Sinkkonen, Lasse; Berninger, Philipp; Lin, Jake; Sauter, Thomas; Heinäniemi, Merja

    2014-02-01

    Metabolic diseases and comorbidities represent an ever-growing epidemic where multiple cell types impact tissue homeostasis. Here, the link between the metabolic and gene regulatory networks was studied through experimental and computational analysis. Integrating gene regulation data with a human metabolic network prompted the establishment of an open-sourced web portal, IDARE (Integrated Data Nodes of Regulation), for visualizing various gene-related data in context of metabolic pathways. Motivated by increasing availability of deep sequencing studies, we obtained ChIP-seq data from widely studied human umbilical vein endothelial cells. Interestingly, we found that association of metabolic genes with multiple transcription factors (TFs) enriched disease-associated genes. To demonstrate further extensions enabled by examining these networks together, constraint-based modeling was applied to data from human preadipocyte differentiation. In parallel, data on gene expression, genome-wide ChIP-seq profiles for peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer binding protein (CEBP) α, liver X receptor (LXR) and H3K4me3 and microRNA target identification for miR-27a, miR-29a and miR-222 were collected. Disease-relevant key nodes, including mitochondrial glycerol-3-phosphate acyltransferase (GPAM), were exposed from metabolic pathways predicted to change activity by focusing on association with multiple regulators. In both cell types, our analysis reveals the convergence of microRNAs and TFs within the branched chain amino acid (BCAA) metabolic pathway, possibly providing an explanation for its downregulation in obese and diabetic conditions.

  10. Comparative transcriptional profiling analysis of the two daughter cells from tobacco zygote reveals the transcriptome differences in the apical and basal cells

    Directory of Open Access Journals (Sweden)

    Hu Tian-Xiang

    2010-08-01

    Full Text Available Abstract Background In angiosperm, after the first asymmetric zygotic cell division, the apical and basal daughter cells follow distinct development pathways. Global transcriptome analysis of these two cells is essential in understanding their developmental differences. However, because of the difficulty to isolate the in vivo apical and basal cells of two-celled proembryo from ovule and ovary in higher plants, the transcriptome analysis of them hasn't been reported. Results In this study, we developed a procedure for isolating the in vivo apical and basal cells of the two-celled proembryo from tobacco (Nicotiana tabacum, and then performed a comparative transcriptome analysis of the two cells by suppression subtractive hybridization (SSH combined with macroarray screening. After sequencing, we identified 797 differentially expressed ESTs corresponding to 299 unigenes. Library sequence analysis successfully identified tobacco homologies of genes involved in embryogenesis and seed development. By quantitative real-time PCR, we validated the differential expression of 40 genes, with 6 transcripts of them specifically expressed in the apical or basal cell. Expression analysis also revealed some transcripts displayed cell specific activation in one of the daughter cells after zygote division. These differential expressions were further validated by in situ hybridization (ISH. Tissue expression pattern analysis also revealed some potential roles of these candidate genes in development. Conclusions The results show that some differential or specific transcripts in the apical and basal cells of two-celled proembryo were successfully isolated, and the identification of these transcripts reveals that these two daughter cells possess distinct transcriptional profiles after zygote division. Further functional work on these differentially or specifically expressed genes will promote the elucidation of molecular mechanism controlling early embryogenesis.

  11. Systematic analysis of phloem-feeding insect-induced transcriptional reprogramming in Arabidopsis highlights common features and reveals distinct responses to specialist and generalist insects.

    Science.gov (United States)

    Foyer, Christine H; Verrall, Susan R; Hancock, Robert D

    2015-02-01

    Phloem-feeding insects (PFIs), of which aphids are the largest group, are major agricultural pests causing extensive damage to crop plants. In contrast to chewing insects, the nature of the plant response to PFIs remains poorly characterized. Scrutiny of the literature concerning transcriptional responses of model and crop plant species to PFIs reveals surprisingly little consensus with respect to the transcripts showing altered abundance following infestation. Nevertheless, core features of the transcriptional response to PFIs can be defined in Arabidopsis thaliana. This comparison of the PFI-associated transcriptional response observed in A. thaliana infested by the generalists Myzus persicae and Bemisia tabaci with the specialist Brevicoryne brassicae highlights the importance of calcium-dependent and receptor kinase-associated signalling. We discuss these findings within the context of the complex cross-talk between the different hormones regulating basal immune response mechanisms in plants. We identify PFI-responsive genes, highlighting the importance of cell wall-associated kinases in plant-PFI interactions, as well as the significant role of kinases containing the domain of unknown function 26. A common feature of plant-PFI interaction is enhanced abundance of transcripts encoding WRKY transcription factors. However, significant divergence was observed with respect to secondary metabolism dependent upon the insect attacker. Transcripts encoding enzymes and proteins associated with glucosinolate metabolism were decreased following attack by the generalist M. persicae but not by the specialist B. brassicae. This analysis provides a comprehensive overview of the molecular patterns associated with the plant response to PFIs and suggests that plants recognize and respond to perturbations in the cell wall occurring during PFI infestation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights

  12. Genome-wide location analysis reveals distinct transcriptional circuitry by paralogous regulators Foxa1 and Foxa2.

    Directory of Open Access Journals (Sweden)

    Irina M Bochkis

    Full Text Available Gene duplication is a powerful driver of evolution. Newly duplicated genes acquire new roles that are relevant to fitness, or they will be lost over time. A potential path to functional relevance is mutation of the coding sequence leading to the acquisition of novel biochemical properties, as analyzed here for the highly homologous paralogs Foxa1 and Foxa2 transcriptional regulators. We determine by genome-wide location analysis (ChIP-Seq that, although Foxa1 and Foxa2 share a large fraction of binding sites in the liver, each protein also occupies distinct regulatory elements in vivo. Foxa1-only sites are enriched for p53 binding sites and are frequently found near genes important to cell cycle regulation, while Foxa2-restricted sites show only a limited match to the forkhead consensus and are found in genes involved in steroid and lipid metabolism. Thus, Foxa1 and Foxa2, while redundant during development, have evolved divergent roles in the adult liver, ensuring the maintenance of both genes during evolution.

  13. Genome-Wide Location Analysis Reveals Distinct Transcriptional Circuitry by Paralogous Regulators Foxa1 and Foxa2

    Science.gov (United States)

    Bochkis, Irina M.; Schug, Jonathan; Ye, Diana Z.; Kurinna, Svitlana; Stratton, Sabrina A.; Barton, Michelle C.; Kaestner, Klaus H.

    2012-01-01

    Gene duplication is a powerful driver of evolution. Newly duplicated genes acquire new roles that are relevant to fitness, or they will be lost over time. A potential path to functional relevance is mutation of the coding sequence leading to the acquisition of novel biochemical properties, as analyzed here for the highly homologous paralogs Foxa1 and Foxa2 transcriptional regulators. We determine by genome-wide location analysis (ChIP-Seq) that, although Foxa1 and Foxa2 share a large fraction of binding sites in the liver, each protein also occupies distinct regulatory elements in vivo. Foxa1-only sites are enriched for p53 binding sites and are frequently found near genes important to cell cycle regulation, while Foxa2-restricted sites show only a limited match to the forkhead consensus and are found in genes involved in steroid and lipid metabolism. Thus, Foxa1 and Foxa2, while redundant during development, have evolved divergent roles in the adult liver, ensuring the maintenance of both genes during evolution. PMID:22737085

  14. Genome-wide analysis of Dof transcription factors reveals functional characteristics during development and response to biotic stresses in pepper

    Science.gov (United States)

    Kang, Won-Hee; Kim, Seungill; Lee, Hyun-Ah; Choi, Doil; Yeom, Seon-In

    2016-01-01

    The DNA-binding with one zinc finger proteins (Dofs) are a plant-specific family of transcription factors. The Dofs are involved in a variety of biological processes such as phytohormone production, seed development, and environmental adaptation. Dofs have been previously identified in several plants, but not in pepper. We identified 33 putative Dof genes in pepper (CaDofs). To gain an overview of the CaDofs, we analyzed phylogenetic relationships, protein motifs, and evolutionary history. We divided the 33 CaDofs, containing 25 motifs, into four major groups distributed on eight chromosomes. We discovered an expansion of the CaDofs dated to a recent duplication event. Segmental duplication that occurred before the speciation of the Solanaceae lineages was predominant among the CaDofs. The global gene-expression profiling of the CaDofs by RNA-seq analysis showed distinct temporal and pathogen-specific variation during development and response to biotic stresses (two TMV strains, PepMoV, and Phytophthora capsici), suggesting functional diversity among the CaDofs. These results will provide the useful clues into the responses of Dofs in biotic stresses and promote a better understanding of their multiple function in pepper and other species. PMID:27653666

  15. Comprehensive analysis of transcript start sites in ly49 genes reveals an unexpected relationship with gene function and a lack of upstream promoters.

    Science.gov (United States)

    Gays, Frances; Koh, Alan S C; Mickiewicz, Katarzyna M; Aust, Jonathan G; Brooks, Colin G

    2011-03-31

    Comprehensive analysis of the transcription start sites of the Ly49 genes of C57BL/6 mice using the oligo-capping 5'-RACE technique revealed that the genes encoding the "missing self" inhibitory receptors, Ly49A, C, G, and I, were transcribed from multiple broad regions in exon 1, in the intron1/exon2 region, and upstream of exon -1b. Ly49E was also transcribed in this manner, and uniquely showed a transcriptional shift from exon1 to exon 2 when NK cells were activated in vitro with IL2. Remarkably, a large proportion of Ly49E transcripts was then initiated from downstream of the translational start codon. By contrast, the genes encoding Ly49B and Q in myeloid cells, the activating Ly49D and H receptors in NK cells, and Ly49F in activated T cells, were predominantly transcribed from a conserved site in a pyrimidine-rich region upstream of exon 1. An ∼200 bp fragment from upstream of the Ly49B start site displayed tissue-specific promoter activity in dendritic cell lines, but the corresponding upstream fragments from all other Ly49 genes lacked detectable tissue-specific promoter activity. In particular, none displayed any significant activity in a newly developed adult NK cell line that expressed multiple Ly49 receptors. Similarly, no promoter activity could be found in fragments upstream of intron1/exon2. Collectively, these findings reveal a previously unrecognized relationship between the pattern of transcription and the expression/function of Ly49 receptors, and indicate that transcription of the Ly49 genes expressed in lymphoid cells is achieved in a manner that does not require classical upstream promoters.

  16. Integrated analysis of hematopoietic differentiation outcomes and molecular characterization reveals unbiased differentiation capacity and minor transcriptional memory in HPC/HSC-iPSCs.

    Science.gov (United States)

    Gao, Shuai; Hou, Xinfeng; Jiang, Yonghua; Xu, Zijian; Cai, Tao; Chen, Jiajie; Chang, Gang

    2017-01-23

    Transcription factor-mediated reprogramming can reset the epigenetics of somatic cells into a pluripotency compatible state. Recent studies show that induced pluripotent stem cells (iPSCs) always inherit starting cell-specific characteristics, called epigenetic memory, which may be advantageous, as directed differentiation into specific cell types is still challenging; however, it also may be unpredictable when uncontrollable differentiation occurs. In consideration of biosafety in disease modeling and personalized medicine, the availability of high-quality iPSCs which lack a biased differentiation capacity and somatic memory could be indispensable. Herein, we evaluate the hematopoietic differentiation capacity and somatic memory state of hematopoietic progenitor and stem cell (HPC/HSC)-derived-iPSCs (HPC/HSC-iPSCs) using a previously established sequential reprogramming system. We found that HPC/HSCs are amenable to being reprogrammed into iPSCs with unbiased differentiation capacity to hematopoietic progenitors and mature hematopoietic cells. Genome-wide analyses revealed that no global epigenetic memory was detectable in HPC/HSC-iPSCs, but only a minor transcriptional memory of HPC/HSCs existed in a specific tetraploid complementation (4 N)-incompetent HPC/HSC-iPSC line. However, the observed minor transcriptional memory had no influence on the hematopoietic differentiation capacity, indicating the reprogramming of the HPC/HSCs was nearly complete. Further analysis revealed the correlation of minor transcriptional memory with the aberrant distribution of H3K27me3. This work provides a comprehensive framework for obtaining high-quality iPSCs from HPC/HSCs with unbiased hematopoietic differentiation capacity and minor transcriptional memory.

  17. Comparative Analysis of mRNA Isoform Expression in Cardiac Hypertrophy and Development Reveals Multiple Post-Transcriptional Regulatory Modules

    Science.gov (United States)

    Park, Ji Yeon; Li, Wencheng; Zheng, Dinghai; Zhai, Peiyong; Zhao, Yun; Matsuda, Takahisa; Vatner, Stephen F.; Sadoshima, Junichi; Tian, Bin

    2011-01-01

    Cardiac hypertrophy is enlargement of the heart in response to physiological or pathological stimuli, chiefly involving growth of myocytes in size rather than in number. Previous studies have shown that the expression pattern of a group of genes in hypertrophied heart induced by pressure overload resembles that at the embryonic stage of heart development, a phenomenon known as activation of the “fetal gene program”. Here, using a genome-wide approach we systematically defined genes and pathways regulated in short- and long-term cardiac hypertrophy conditions using mice with transverse aortic constriction (TAC), and compared them with those regulated at different stages of embryonic and postnatal development. In addition, exon-level analysis revealed widespread mRNA isoform changes during cardiac hypertrophy resulting from alternative usage of terminal or internal exons, some of which are also developmentally regulated and may be attributable to decreased expression of Fox-1 protein in cardiac hypertrophy. Genes with functions in certain pathways, such as cell adhesion and cell morphology, are more likely to be regulated by alternative splicing. Moreover, we found 3′UTRs of mRNAs were generally shortened through alternative cleavage and polyadenylation in hypertrophy, and microRNA target genes were generally de-repressed, suggesting coordinated mechanisms to increase mRNA stability and protein production during hypertrophy. Taken together, our results comprehensively delineated gene and mRNA isoform regulation events in cardiac hypertrophy and revealed their relations to those in development, and suggested that modulation of mRNA isoform expression plays an importance role in heart remodeling under pressure overload. PMID:21799842

  18. Single-Cell Transcriptional Analysis Reveals Novel Neuronal Phenotypes and Interaction Networks Involved in the Central Circadian Clock.

    Science.gov (United States)

    Park, James; Zhu, Haisun; O'Sullivan, Sean; Ogunnaike, Babatunde A; Weaver, David R; Schwaber, James S; Vadigepalli, Rajanikanth

    2016-01-01

    Single-cell heterogeneity confounds efforts to understand how a population of cells organizes into cellular networks that underlie tissue-level function. This complexity is prominent in the mammalian suprachiasmatic nucleus (SCN). Here, individual neurons exhibit a remarkable amount of asynchronous behavior and transcriptional heterogeneity. However, SCN neurons are able to generate precisely coordinated synaptic and molecular outputs that synchronize the body to a common circadian cycle by organizing into cellular networks. To understand this emergent cellular network property, it is important to reconcile single-neuron heterogeneity with network organization. In light of recent studies suggesting that transcriptionally heterogeneous cells organize into distinct cellular phenotypes, we characterized the transcriptional, spatial, and functional organization of 352 SCN neurons from mice experiencing phase-shifts in their circadian cycle. Using the community structure detection method and multivariate analytical techniques, we identified previously undescribed neuronal phenotypes that are likely to participate in regulatory networks with known SCN cell types. Based on the newly discovered neuronal phenotypes, we developed a data-driven neuronal network structure in which multiple cell types interact through known synaptic and paracrine signaling mechanisms. These results provide a basis from which to interpret the functional variability of SCN neurons and describe methodologies toward understanding how a population of heterogeneous single cells organizes into cellular networks that underlie tissue-level function.

  19. Single-cell Transcriptional Analysis Reveals Novel Neuronal Phenotypes and Interaction Networks involved In the Central Circadian Clock

    Directory of Open Access Journals (Sweden)

    James Park

    2016-10-01

    Full Text Available Single-cell heterogeneity confounds efforts to understand how a population of cells organizes into cellular networks that underlie tissue-level function. This complexity is prominent in the mammalian suprachiasmatic nucleus (SCN. Here, individual neurons exhibit a remarkable amount of asynchronous behavior and transcriptional heterogeneity. However, SCN neurons are able to generate precisely coordinated synaptic and molecular outputs that synchronize the body to a common circadian cycle by organizing into cellular networks. To understand this emergent cellular network property, it is important to reconcile single-neuron heterogeneity with network organization. In light of recent studies suggesting that transcriptionally heterogeneous cells organize into distinct cellular phenotypes, we characterized the transcriptional, spatial, and functional organization of 352 SCN neurons from mice experiencing phase-shifts in their circadian cycle. Using the community structure detection method and multivariate analytical techniques, we identified previously undescribed neuronal phenotypes that are likely to participate in regulatory networks with known SCN cell types. Based on the newly discovered neuronal phenotypes, we developed a data-driven neuronal network structure in which multiple cell types interact through known synaptic and paracrine signaling mechanisms. These results provide a basis from which to interpret the functional variability of SCN neurons and describe methodologies towards understanding how a population of heterogeneous single cells organizes into cellular networks that underlie tissue-level function.

  20. Global MYCN transcription factor binding analysis in neuroblastoma reveals association with distinct E-box motifs and regions of DNA hypermethylation.

    LENUS (Irish Health Repository)

    Murphy, Derek M

    2009-01-01

    BACKGROUND: Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors. METHODOLOGY: We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified\\/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions. CONCLUSION: Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016), with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP). The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription factor affecting the

  1. Chat Transcript Analysis Reveals that Undergraduate Students are Open to Instruction, While Instructors and Librarians Care About Supporting Student Learning

    Directory of Open Access Journals (Sweden)

    Elaine Sullo

    2017-03-01

    Full Text Available A Review of: Jacoby, J., Ward, D., Avery, S., & Marcyk, E. (2016. The value of chat reference services: A pilot study. portal: Libraries and the Academy, 16(1, 109-129. https://doi.org/10.1353/pla.2016.0013 Objective – To investigate student, instructor, and librarian perspectives of chat reference service in the context of first-year undergraduate students conducting research for an introductory composition course. Design – Focus groups, individual interviews, and surveys. Setting – A large, public university in the United States of America. Subjects – 57 library reference providers, 36 instructors of an introductory composition course, and approximately 936 undergraduate students in certain sections of the introductory composition course who were assigned a specific research project. Methods – In spring of 2014, all participants were invited via email to respond to an anonymous chat transcript of a librarian interacting with a student working on his or her research project. Study participants could participate via a brief survey or by taking part in a focus group or individual interview. The invited instructors were asked to forward the invitation to the students in their sections, and reminder emails were sent two weeks after the initial email. Main Results – Nine instructors, 24 students, and 25 library reference providers participated in the study, representing a response rate of 25%, 3% (estimated, and 44%, respectively. The authors conducted a qualitative analysis of key themes that were derived from both the focus groups or individual interviews and the survey questions. The themes were: students as novice researchers, question negotiation, open and closed questions, instruction, speed and convenience, customer service, and referrals. The theme of “students as novice researchers” is based on student comments related to their frustrations of being inexperienced researchers, as well as librarian comments on strategies for

  2. A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors

    Directory of Open Access Journals (Sweden)

    Lior Izhar

    2015-06-01

    Full Text Available Localization to sites of DNA damage is a hallmark of DNA damage response (DDR proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose polymerase (PARP-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins.

  3. Analysis of temporal transcription expression profiles reveal links between protein function and developmental stages of Drosophila melanogaster.

    Science.gov (United States)

    Wan, Cen; Lees, Jonathan G; Minneci, Federico; Orengo, Christine A; Jones, David T

    2017-10-01

    Accurate gene or protein function prediction is a key challenge in the post-genome era. Most current methods perform well on molecular function prediction, but struggle to provide useful annotations relating to biological process functions due to the limited power of sequence-based features in that functional domain. In this work, we systematically evaluate the predictive power of temporal transcription expression profiles for protein function prediction in Drosophila melanogaster. Our results show significantly better performance on predicting protein function when transcription expression profile-based features are integrated with sequence-derived features, compared with the sequence-derived features alone. We also observe that the combination of expression-based and sequence-based features leads to further improvement of accuracy on predicting all three domains of gene function. Based on the optimal feature combinations, we then propose a novel multi-classifier-based function prediction method for Drosophila melanogaster proteins, FFPred-fly+. Interpreting our machine learning models also allows us to identify some of the underlying links between biological processes and developmental stages of Drosophila melanogaster.

  4. Analysis of temporal transcription expression profiles reveal links between protein function and developmental stages of Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Cen Wan

    2017-10-01

    Full Text Available Accurate gene or protein function prediction is a key challenge in the post-genome era. Most current methods perform well on molecular function prediction, but struggle to provide useful annotations relating to biological process functions due to the limited power of sequence-based features in that functional domain. In this work, we systematically evaluate the predictive power of temporal transcription expression profiles for protein function prediction in Drosophila melanogaster. Our results show significantly better performance on predicting protein function when transcription expression profile-based features are integrated with sequence-derived features, compared with the sequence-derived features alone. We also observe that the combination of expression-based and sequence-based features leads to further improvement of accuracy on predicting all three domains of gene function. Based on the optimal feature combinations, we then propose a novel multi-classifier-based function prediction method for Drosophila melanogaster proteins, FFPred-fly+. Interpreting our machine learning models also allows us to identify some of the underlying links between biological processes and developmental stages of Drosophila melanogaster.

  5. Transcriptional analysis and molecular dynamics simulations reveal the mechanism of toxic metals removal and efflux pumps in Lysinibacillus sphaericus OT4b.31

    KAUST Repository

    Shaw, Dario Rangel

    2017-11-23

    Lysinibacillus sphaericus strain OT4b.31 is a bacterium widely applied in bioremediation processes of hydrocarbon and metal polluted environments. In this study, we identified the molecular mechanism underlying the Pb2+ and Cr6+ resistance. Metal uptake and temporal transcription patterns of metal resistance operons were evaluated using reverse-transcribed quantitative PCR amplification. The function of the resistance determinants was studied applying docking and in silico mutagenesis methods. The results revealed that the adaptation of Lysinibacillus sphaericus OT4b.31 to elevated levels of lead and chromium involves the pbr and chr operons which comprise a transcriptional regulatory component (pbrR and chrB) and efflux ATPases (pbrA and chrA) to expel ions from the cytoplasm. Expression of metal resistance genes was constitutive and specifically inducible to the exposure of Pb2+ and Cr6+. The simultaneous presence of cations didn\\'t affect the bioaccumulation of metals, evidencing the multimetal resistance of L. sphaericus. Docking analysis revealed the key metal-protein interactions and the conformational changes after metal or ATP binding. Results showed that residues with aromatic rings or imidazole in the catalytic domain are crucial for metal binding and achievement of the function. To our knowledge, this is the first report of a specific mechanism for lead and chromium resistance in Lysinibacillus genus. From the findings of this study, it is possible to suggest the bacterium as a suitable candidate for rapid toxic metals bioremediation processes.

  6. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators

    DEFF Research Database (Denmark)

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone

    2017-01-01

    of which were assigned as regulators of gene expression. Among 288 identified transcriptional regulators, 49 were regulated within four hours of adipogenic stimulation including several known and many novel potential adipogenic regulators. A kinase-substrate database for 3T3-L1 preadipocytes established...... stages (4 hours) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation...... for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase (CDK) family and the mitogen activated protein kinase (MAPK) family including P...

  7. Transcript profile analysis reveals important roles of jasmonic acid signalling pathway in the response of sweet potato to salt stress.

    Science.gov (United States)

    Zhang, Huan; Zhang, Qian; Zhai, Hong; Li, Yan; Wang, Xiangfeng; Liu, Qingchang; He, Shaozhen

    2017-01-13

    Sweet potato is an important food and bio-energy crop, and investigating the mechanisms underlying salt tolerance will provide information for salt-tolerant breeding of this crop. Here, the root transcriptomes of the salt-sensitive variety Lizixiang and the salt-tolerant line ND98 were compared to identify the genes and pathways involved in salt stress responses. In total, 8,744 and 10,413 differentially expressed genes (DEGs) in Lizixiang and ND98, respectively, were involved in salt responses. A lower DNA methylation level was detected in ND98 than in Lizixiang. In both genotypes, the DEGs, which function in phytohormone synthesis and signalling and ion homeostasis, may underlie the different degrees of salt tolerance. Significant up-regulations of the genes involved in the jasmonic acid (JA) biosynthesis and signalling pathways and ion transport, more accumulation of JA, a higher degree of stomatal closure and a lower level of Na + were found in ND98 compared to Lizixiang. This is the first report on transcriptome responses to salt tolerance in sweet potato. These results reveal that the JA signalling pathway plays important roles in the response of sweet potato to salt stress. This study provides insights into the mechanisms and genes involved in the salt tolerance of sweet potato.

  8. Comparative transcriptional analysis of asexual and sexual morphs reveals possible mechanisms in reproductive polyphenism of the cotton aphid.

    Directory of Open Access Journals (Sweden)

    Li-Jun Liu

    Full Text Available Aphids, the destructive insect pests in the agriculture, horticulture and forestry, are capable of reproducing asexually and sexually upon environmental change. However, the molecular basis of aphid reproductive mode switch remains an enigma. Here we report a comparative analysis of differential gene expression profiling among parthenogenetic females, gynoparae and sexual females of the cotton aphid Aphis gossypii, using the RNA-seq approach with next-generation sequencing platforms, followed by RT-qPCR. At the cutoff criteria of fold change ≥2 and P<0.01, we identified 741 up- and 879 down-regulated genes in gynoparae versus parthenogenetic females, 2,101 up- and 2,210 down-regulated genes in sexual females compared to gynoparae, and 1,614 up- and 2,238 down-regulated genes in sexual females relative to parthenogenetic females. Gene ontology category and KEGG pathway analysis suggest the involvement of differentially expressed genes in multiple cellular signaling pathways into the reproductive mode transition, including phototransduction, cuticle composition, progesterone-mediated oocyte maturation and endocrine regulation. This study forms a basis for deciphering the molecular mechanisms underlying the shift from asexual to sexual reproduction in the cotton aphid. It also provides valuable resources for future studies on this host-alternating aphid species, and the insight into the understanding of reproductive mode plasticity in different aphid species.

  9. Transcriptional Analysis of Vitiligo Skin Reveals the Alteration of WNT Pathway: A Promising Target for Repigmenting Vitiligo Patients.

    Science.gov (United States)

    Regazzetti, Claire; Joly, Florence; Marty, Carine; Rivier, Michel; Mehul, Bruno; Reiniche, Pascale; Mounier, Carine; Rival, Yves; Piwnica, David; Cavalié, Marine; Chignon-Sicard, Bérengère; Ballotti, Robert; Voegel, Johannes; Passeron, Thierry

    2015-12-01

    Vitiligo affects 1% of the worldwide population. Halting disease progression and repigmenting the lesional skin represent the two faces of therapeutic challenge in vitiligo. We performed transcriptome analysis on lesional, perilesional, and non-depigmented skin from vitiligo patients and on matched skin from healthy subjects. We found a significant increase in CXCL10 in non-depigmented and perilesional vitiligo skin compared with levels in healthy control skin; however, neither CXCL10 nor other immune factors were deregulated in depigmented vitiligo skin. Interestingly, the WNT pathway, which is involved in melanocyte differentiation, was altered specifically in vitiligo skin. We demonstrated that oxidative stress decreases WNT expression/activation in keratinocytes and melanocytes. We developed an ex vivo skin model and confirmed the decrease activation of the WNT pathway in human skin subjected to oxidative stress. Finally, using pharmacological agents that activate the WNT pathway, we treated ex vivo depigmented skin from vitiligo patients and successfully induced differentiation of resident stem cells into pre-melanocytes. Our results shed light on the previously unrecognized role of decreased WNT activation in the prevention of melanocyte differentiation in depigmented vitiligo skin. Furthermore, these results support further clinical exploration of WNT agonists to repigment vitiligo lesions.

  10. RNA microarray analysis in prenatal mouse cochlea reveals novel IGF-I target genes: implication of MEF2 and FOXM1 transcription factors.

    Directory of Open Access Journals (Sweden)

    Hortensia Sanchez-Calderon

    2010-01-01

    Full Text Available Insulin-like growth factor-I (IGF-I provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness.A combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1(-/- null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1(-/- and Igf1(+/+ transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1(+/+ mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin

  11. Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells*

    Science.gov (United States)

    Zheng, Peng; Xiong, Qian; Wu, Ying; Chen, Ying; Chen, Zhuo; Fleming, Joy; Gao, Ding; Bi, Lijun; Ge, Feng

    2015-01-01

    Long noncoding RNAs (lncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective, we employed a quantitative proteomic strategy to systematically identify potential targets of HOTAIR. The expression of 170 proteins was significantly dys-regulated after inhibition of HOTAIR, implying that they could be potential targets of HOTAIR. Analysis of this data at the systems level revealed major changes in proteins involved in diverse cellular components, including the cytoskeleton and the respiratory chain. Further functional studies on vimentin (VIM), a key protein involved in the cytoskeleton, revealed that HOTAIR exerts its effects on migration and invasion of HeLa cells, at least in part, through the regulation of VIM expression. Inhibition of HOTAIR leads to mitochondrial dysfunction and ultrastructural alterations, suggesting a novel role of HOTAIR in maintaining mitochondrial function in cancer cells. Our results provide novel insights into the mechanisms underlying the function of HOTAIR in cancer cells. We expect that the methods used in this study will become an integral part of functional studies of lncRNAs. PMID:25762744

  12. Nascent RNA sequencing reveals distinct features in plant transcription

    OpenAIRE

    Hetzel, Jonathan; Duttke, Sascha H.; Benner, Christopher; Chory, Joanne

    2016-01-01

    Transcription is a fundamental and dynamic step in the regulation of gene expression, but the characteristics of plant transcription are poorly understood. We adapted the global nuclear run-on sequencing (GRO-seq) and 5′GRO-seq methods for plants and provide a plant version of the next-generation sequencing software HOMER (homer.ucsd.edu/homer/plants) to facilitate data analysis. Mapping nascent transcripts in Arabidopsis thaliana seedlings enabled identification of known and novel transcript...

  13. Analysis of transcription profile to reveal altered signaling pathways following the overexpression of human desumoylating isopeptidase 2 in pancreatic cancer cells

    Science.gov (United States)

    Fu, Yu-Yin; Kang, Yu-Huan; Shen, Cong-Cong; Wang, Rui-Xue; Yu, Lin; Li, Xin-Yue; Cui, Dan-Dan; Yang, Jin-Liang; Yao, Yu-Qin; Gou, Lan-Tu

    2016-01-01

    Human desumoylating isopeptidase 2 (DESI-2) is a member of the DESI family and contains a conserved PPPDE1 domain. Previous studies have demonstrated that DESI-2 overexpression may induce cell apoptosis. In the present study, differentially expressed genes were analyzed using a transcription microarray in DESI-2 overexpressing PANC-1 pancreatic cancer cells. A total of 45,033 genes were examined by microarray, which identified 1,766 upregulated and 1,643 downregulated genes. A series of altered signaling pathways were analyzed, in which certain essential signaling factors, including retinoid X receptor (RXR), BH3 interacting-domain death agonist, Ras homolog gene family member A (RhoA) and Rho-associated protein kinase, were further investigated at the protein level. The release of cytochrome c and the activation of caspase-3 were also detected by western blot analysis. Immunohistochemistry further revealed the expression features of RXR and RhoA in pancreatic ductal adenocarcinoma tissues with various DESI-2 expression levels. The results serve as a valuable reference for the further elucidation of the functions of DESI-2 in pancreatic cancer. PMID:28105175

  14. Global analysis of the transcriptional response of whitefly to tomato yellow leaf curl China virus reveals the relationship of coevolved adaptations.

    Science.gov (United States)

    Luan, Jun-Bo; Li, Jun-Min; Varela, Nélia; Wang, Yong-Liang; Li, Fang-Fang; Bao, Yan-Yuan; Zhang, Chuan-Xi; Liu, Shu-Sheng; Wang, Xiao-Wei

    2011-04-01

    The begomoviruses are the largest and most economically important group of plant viruses transmitted exclusively by the whitefly Bemisia tabaci in a circulative, persistent manner. The circulation of the viruses within the insect vectors involves complex interactions between virus and vector components; however, the molecular mechanisms of these interactions remain largely unknown. Here we investigated the transcriptional response of the invasive B. tabaci Middle East-Asia Minor 1 species to Tomato yellow leaf curl China virus (TYLCCNV) using Illumina sequencing technology. Results showed that 1,606 genes involved in 157 biochemical pathways were differentially expressed in the viruliferous whiteflies. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that TYLCCNV can perturb the cell cycle and primary metabolism in the whitefly, which explains the negative effect of this virus on the longevity and fecundity of B. tabaci. Our data also demonstrated that TYLCCNV can activate whitefly immune responses, such as autophagy and antimicrobial peptide production, which might lead to a gradual decrease of viral particles within the body of the viruliferous whitefly. Furthermore, PCR results showed that TYLCCNV can invade the ovary and fat body tissues of the whitefly, and Lysotracker and Western blot analyses revealed that the invasion of TYLCCNV induced autophagy in both the ovary and fat body tissues. Surprisingly, TYLCCNV also suppressed the whitefly immune responses by downregulating the expression of genes involved in Toll-like signaling and mitogen-activated protein kinase (MAPK) pathways. Taken together, these results reveal the relationship of coevolved adaptations between begomoviruses and whiteflies and will provide a road map for future investigations into the complex interactions between plant viruses and their insect vectors.

  15. Functional analysis of the conserved transcriptional regulator CfWor1 in Cladosporium fulvum reveals diverse roles in the virulence of plant pathogenic fungi

    NARCIS (Netherlands)

    Ökmen, B.; Collemare, J.; Griffiths, S.A.; Burgt, van der A.; Cox, R.; Wit, de P.J.G.M.

    2014-01-01

    Fungal Wor1-like proteins are conserved transcriptional regulators that are reported to regulate the virulence of several plant pathogenic fungi by affecting the expression of virulence genes. Here, we report the functional analysis of CfWor1, the homologue of Wor1 in Cladosporium fulvum. ¿cfwor1

  16. Genome-wide functional analysis of CREB/long-term memory-dependent transcription reveals distinct basal and memory gene expression programs.

    Science.gov (United States)

    Lakhina, Vanisha; Arey, Rachel N; Kaletsky, Rachel; Kauffman, Amanda; Stein, Geneva; Keyes, William; Xu, Daniel; Murphy, Coleen T

    2015-01-21

    Induced CREB activity is a hallmark of long-term memory, but the full repertoire of CREB transcriptional targets required specifically for memory is not known in any system. To obtain a more complete picture of the mechanisms involved in memory, we combined memory training with genome-wide transcriptional analysis of C. elegans CREB mutants. This approach identified 757 significant CREB/memory-induced targets and confirmed the involvement of known memory genes from other organisms, but also suggested new mechanisms and novel components that may be conserved through mammals. CREB mediates distinct basal and memory transcriptional programs at least partially through spatial restriction of CREB activity: basal targets are regulated primarily in nonneuronal tissues, while memory targets are enriched for neuronal expression, emanating from CREB activity in AIM neurons. This suite of novel memory-associated genes will provide a platform for the discovery of orthologous mammalian long-term memory components. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Transcriptional analysis of the sfa determinant revealing mmRNA processing events in the biogenesis of S fimbriae in pathogenic Escherichia coli.

    Science.gov (United States)

    Balsalobre, Carlos; Morschhäuser, Joachim; Jass, Jana; Hacker, Jörg; Uhlin, Bernt Eric

    2003-01-01

    Among the virulence factors present in pathogenic extraintestinal Escherichia coli strains, expression of fimbrial adhesins is necessary for attachment to the host tissues and subsequent colonization. Occurrence of the sfa determinant coding for the S fimbriae is widespread among the uropathogens and meningitis isolates. The sfa operon consists of nine genes. In the biogenesis of S fimbriae, the proteins encoded by the sfa genes are presumably required in a specific stoichiometry. In the present work we studied how differential expression of the sfa operon genes occurs. Our findings indicate that a number of endoribonucleolytic cleavages occur in the mRNA from the sfa operon, and we detected the presence of different distinct transcriptional products, including sfaBA, sfaA, sfaADE, and sfaGSH. The sfaGSH transcript represents the three distal genes of the sfa operon, which code for the minor subunits of the S fimbriae. Analysis of the proteins in S fimbriae suggested that expression of the sfaGSH transcript provides equimolar amounts of the minor subunits. Furthermore, we showed that in the generation of the major sfaA transcript, the processing included RNase E endoribonuceolytic cleavage of the precursor sfaBA transcript. We suggest that posttranscriptional mRNA processing events result in differential gene expression important to achieve the stoichiometry necessary for fimbrial adhesin biogenesis.

  18. Transcriptome analysis of paired primary colorectal carcinoma and liver metastases reveals fusion transcripts and similar gene expression profiles in primary carcinoma and liver metastases.

    Science.gov (United States)

    Lee, Ja-Rang; Kwon, Chae Hwa; Choi, Yuri; Park, Hye Ji; Kim, Hyun Sung; Jo, Hong-Jae; Oh, Nahmgun; Park, Do Youn

    2016-07-26

    Despite the clinical significance of liver metastases, the difference between molecular and cellular changes in primary colorectal cancers (CRC) and matched liver metastases is poorly understood. In order to compare gene expression patterns and identify fusion genes in these two types of tumors, we performed high-throughput transcriptome sequencing of five sets of quadruple-matched tissues (primary CRC, liver metastases, normal colon, and liver). The gene expression patterns in normal colon and liver were successfully distinguished from those in CRCs; however, RNA sequencing revealed that the gene expression between primary CRCs and their matched liver metastases is highly similar. We identified 1895 genes that were differentially expressed in the primary carcinoma and liver metastases, than that in the normal colon tissues. A major proportion of the transcripts, identified by gene expression profiling as significantly enriched in the primary carcinoma and metastases, belonged to gene ontology categories involved in the cell cycle, mitosis, and cell division. Furthermore, we identified gene fusion events in primary carcinoma and metastases, and the fusion transcripts were experimentally confirmed. Among these, a chimeric transcript resulting from the fusion of RNF43 and SUPT4H1 was found to occur frequently in primary colorectal carcinoma. In addition, knockdown of the expression of this RNF43-SUPT4H1 chimeric transcript was found to have a growth-inhibitory effect in colorectal cancer cells. The present study reports a high concordance of gene expression in the primary carcinoma and liver metastases, and reveals potential new targets, such as fusion genes, against primary and metastatic colorectal carcinoma.

  19. Transcriptome analysis of paired primary colorectal carcinoma and liver metastases reveals fusion transcripts and similar gene expression profiles in primary carcinoma and liver metastases

    International Nuclear Information System (INIS)

    Lee, Ja-Rang; Kwon, Chae Hwa; Choi, Yuri; Park, Hye Ji; Kim, Hyun Sung; Jo, Hong-Jae; Oh, Nahmgun; Park, Do Youn

    2016-01-01

    Despite the clinical significance of liver metastases, the difference between molecular and cellular changes in primary colorectal cancers (CRC) and matched liver metastases is poorly understood. In order to compare gene expression patterns and identify fusion genes in these two types of tumors, we performed high-throughput transcriptome sequencing of five sets of quadruple-matched tissues (primary CRC, liver metastases, normal colon, and liver). The gene expression patterns in normal colon and liver were successfully distinguished from those in CRCs; however, RNA sequencing revealed that the gene expression between primary CRCs and their matched liver metastases is highly similar. We identified 1895 genes that were differentially expressed in the primary carcinoma and liver metastases, than that in the normal colon tissues. A major proportion of the transcripts, identified by gene expression profiling as significantly enriched in the primary carcinoma and metastases, belonged to gene ontology categories involved in the cell cycle, mitosis, and cell division. Furthermore, we identified gene fusion events in primary carcinoma and metastases, and the fusion transcripts were experimentally confirmed. Among these, a chimeric transcript resulting from the fusion of RNF43 and SUPT4H1 was found to occur frequently in primary colorectal carcinoma. In addition, knockdown of the expression of this RNF43-SUPT4H1 chimeric transcript was found to have a growth-inhibitory effect in colorectal cancer cells. The present study reports a high concordance of gene expression in the primary carcinoma and liver metastases, and reveals potential new targets, such as fusion genes, against primary and metastatic colorectal carcinoma. The online version of this article (doi:10.1186/s12885-016-2596-3) contains supplementary material, which is available to authorized users

  20. A Bioinformatics Analysis Reveals a Group of MocR Bacterial Transcriptional Regulators Linked to a Family of Genes Coding for Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Teresa Milano

    2016-01-01

    Full Text Available The MocR bacterial transcriptional regulators are characterized by an N-terminal domain, 60 residues long on average, possessing the winged-helix-turn-helix (wHTH architecture responsible for DNA recognition and binding, linked to a large C-terminal domain (350 residues on average that is homologous to fold type-I pyridoxal 5′-phosphate (PLP dependent enzymes like aspartate aminotransferase (AAT. These regulators are involved in the expression of genes taking part in several metabolic pathways directly or indirectly connected to PLP chemistry, many of which are still uncharacterized. A bioinformatics analysis is here reported that studied the features of a distinct group of MocR regulators predicted to be functionally linked to a family of homologous genes coding for integral membrane proteins of unknown function. This group occurs mainly in the Actinobacteria and Gammaproteobacteria phyla. An analysis of the multiple sequence alignments of their wHTH and AAT domains suggested the presence of specificity-determining positions (SDPs. Mapping of SDPs onto a homology model of the AAT domain hinted at possible structural/functional roles in effector recognition. Likewise, SDPs in wHTH domain suggested the basis of specificity of Transcription Factor Binding Site recognition. The results reported represent a framework for rational design of experiments and for bioinformatics analysis of other MocR subgroups.

  1. Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare)WRKY transcription factor family reveals putatively retained functions betweenmonocots and dicots

    Energy Technology Data Exchange (ETDEWEB)

    Mangelsen, Elke; Kilian, Joachim; Berendzen, Kenneth W.; Kolukisaoglu, Uner; Harter, Klaus; Jansson, Christer; Wanke, Dierk

    2008-02-01

    WRKY proteins belong to the WRKY-GCM1 superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. For the cereal crop barley (Hordeum vulgare), three different WRKY proteins have been characterized so far, as regulators in sucrose signaling, in pathogen defense, and in response to cold and drought, respectively. However, their phylogenetic relationship remained unresolved. In this study, we used the available sequence information to identify a minimum number of 45 barley WRKY transcription factor (HvWRKY) genes. According to their structural features the HvWRKY factors were classified into the previously defined polyphyletic WRKY subgroups 1 to 3. Furthermore, we could assign putative orthologs of the HvWRKY proteins in Arabidopsis and rice. While in most cases clades of orthologous proteins were formed within each group or subgroup, other clades were composed of paralogous proteins for the grasses and Arabidopsis only, which is indicative of specific gene radiation events. To gain insight into their putative functions, we examined expression profiles of WRKY genes from publicly available microarray data resources and found group specific expression patterns. While putative orthologs of the HvWRKY transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of WRKY genes in Arabidopsis and barley. Indeed, highly correlative expression profiles were found between some of the putative orthologs. HvWRKY genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. HvWRKY proteins exhibited not only sequence similarities between orthologs with Arabidopsis, but also relatedness in their expression patterns. This correlative expression is indicative for a putative conserved function of related WRKY proteins in mono- and dicot species.

  2. Functional analysis of the conserved transcriptional regulator CfWor1 in Cladosporium fulvum reveals diverse roles in the virulence of plant pathogenic fungi.

    Science.gov (United States)

    Okmen, Bilal; Collemare, Jérôme; Griffiths, Scott; van der Burgt, Ate; Cox, Russell; de Wit, Pierre J G M

    2014-04-01

    Fungal Wor1-like proteins are conserved transcriptional regulators that are reported to regulate the virulence of several plant pathogenic fungi by affecting the expression of virulence genes. Here, we report the functional analysis of CfWor1, the homologue of Wor1 in Cladosporium fulvum. Δcfwor1 mutants produce sclerotium-like structures and rough hyphae, which are covered with a black extracellular matrix. These mutants do not sporulate and are no longer virulent on tomato. A CE.CfWor1 transformant that constitutively expresses CfWor1 produces fewer spores with altered morphology and is also reduced in virulence. RNA-seq and RT-qrtPCR analyses suggest that reduced virulence of Δcfwor1 mutants is due to global downregulation of transcription, translation and mitochondrial respiratory chain. The reduced virulence of the CE.CfWor1 transformant is likely due to downregulation of effector genes. Complementation of a non-virulent Δfosge1 (Wor1-homologue) mutant of Fusarium oxysporum f. sp. lycopersici with CfWor1 restored expression of the SIX effector genes in this fungus, but not its virulence. Chimeric proteins of CfWor1/FoSge1 also only partially restored defects of the Δfosge1 mutant, suggesting that these transcriptional regulators have functionally diverged. Altogether, our results suggest that CfWor1 primarily regulates development of C. fulvum, which indirectly affects the expression of a subset of virulence genes. © 2014 John Wiley & Sons Ltd.

  3. Transcriptional analysis of the HeT-A retrotransposon in mutant and wild type stocks reveals high sequence variability at Drosophila telomeres and other unusual features

    Directory of Open Access Journals (Sweden)

    Piñeyro David

    2011-11-01

    Full Text Available Abstract Background Telomere replication in Drosophila depends on the transposition of a domesticated retroelement, the HeT-A retrotransposon. The sequence of the HeT-A retrotransposon changes rapidly resulting in differentiated subfamilies. This pattern of sequence change contrasts with the essential function with which the HeT-A is entrusted and brings about questions concerning the extent of sequence variability, the telomere contribution of different subfamilies, and whether wild type and mutant Drosophila stocks show different HeT-A scenarios. Results A detailed study on the variability of HeT-A reveals that both the level of variability and the number of subfamilies are higher than previously reported. Comparisons between GIII, a strain with longer telomeres, and its parental strain Oregon-R indicate that both strains have the same set of HeT-A subfamilies. Finally, the presence of a highly conserved splicing pattern only in its antisense transcripts indicates a putative regulatory, functional or structural role for the HeT-A RNA. Interestingly, our results also suggest that most HeT-A copies are actively expressed regardless of which telomere and where in the telomere they are located. Conclusions Our study demonstrates how the HeT-A sequence changes much faster than previously reported resulting in at least nine different subfamilies most of which could actively contribute to telomere extension in Drosophila. Interestingly, the only significant difference observed between Oregon-R and GIII resides in the nature and proportion of the antisense transcripts, suggesting a possible mechanism that would in part explain the longer telomeres of the GIII stock.

  4. Genome-wide binding analysis of the transcription activator ideal plant architecture1 reveals a complex network regulating rice plant architecture.

    Science.gov (United States)

    Lu, Zefu; Yu, Hong; Xiong, Guosheng; Wang, Jing; Jiao, Yongqing; Liu, Guifu; Jing, Yanhui; Meng, Xiangbing; Hu, Xingming; Qian, Qian; Fu, Xiangdong; Wang, Yonghong; Li, Jiayang

    2013-10-01

    Ideal plant architecture1 (IPA1) is critical in regulating rice (Oryza sativa) plant architecture and substantially enhances grain yield. To elucidate its molecular basis, we first confirmed IPA1 as a functional transcription activator and then identified 1067 and 2185 genes associated with IPA1 binding sites in shoot apices and young panicles, respectively, through chromatin immunoprecipitation sequencing assays. The Squamosa promoter binding protein-box direct binding core motif GTAC was highly enriched in IPA1 binding peaks; interestingly, a previously uncharacterized indirect binding motif TGGGCC/T was found to be significantly enriched through the interaction of IPA1 with proliferating cell nuclear antigen promoter binding factor1 or promoter binding factor2. Genome-wide expression profiling by RNA sequencing revealed IPA1 roles in diverse pathways. Moreover, our results demonstrated that IPA1 could directly bind to the promoter of rice teosinte branched1, a negative regulator of tiller bud outgrowth, to suppress rice tillering, and directly and positively regulate dense and erect panicle1, an important gene regulating panicle architecture, to influence plant height and panicle length. The elucidation of target genes of IPA1 genome-wide will contribute to understanding the molecular mechanisms underlying plant architecture and to facilitating the breeding of elite varieties with ideal plant architecture.

  5. Genome-Wide Binding Analysis of the Transcription Activator IDEAL PLANT ARCHITECTURE1 Reveals a Complex Network Regulating Rice Plant Architecture[W

    Science.gov (United States)

    Lu, Zefu; Yu, Hong; Xiong, Guosheng; Wang, Jing; Jiao, Yongqing; Liu, Guifu; Jing, Yanhui; Meng, Xiangbing; Hu, Xingming; Qian, Qian; Fu, Xiangdong; Wang, Yonghong; Li, Jiayang

    2013-01-01

    IDEAL PLANT ARCHITECTURE1 (IPA1) is critical in regulating rice (Oryza sativa) plant architecture and substantially enhances grain yield. To elucidate its molecular basis, we first confirmed IPA1 as a functional transcription activator and then identified 1067 and 2185 genes associated with IPA1 binding sites in shoot apices and young panicles, respectively, through chromatin immunoprecipitation sequencing assays. The SQUAMOSA PROMOTER BINDING PROTEIN-box direct binding core motif GTAC was highly enriched in IPA1 binding peaks; interestingly, a previously uncharacterized indirect binding motif TGGGCC/T was found to be significantly enriched through the interaction of IPA1 with proliferating cell nuclear antigen PROMOTER BINDING FACTOR1 or PROMOTER BINDING FACTOR2. Genome-wide expression profiling by RNA sequencing revealed IPA1 roles in diverse pathways. Moreover, our results demonstrated that IPA1 could directly bind to the promoter of rice TEOSINTE BRANCHED1, a negative regulator of tiller bud outgrowth, to suppress rice tillering, and directly and positively regulate DENSE AND ERECT PANICLE1, an important gene regulating panicle architecture, to influence plant height and panicle length. The elucidation of target genes of IPA1 genome-wide will contribute to understanding the molecular mechanisms underlying plant architecture and to facilitating the breeding of elite varieties with ideal plant architecture. PMID:24170127

  6. Analysis of the transcriptome of the protozoan Theileria parva using MPSS reveals that the majority of genes are transcriptionally active in the schizont stage.

    Science.gov (United States)

    Bishop, Richard; Shah, Trushar; Pelle, Roger; Hoyle, David; Pearson, Terry; Haines, Lee; Brass, Andrew; Hulme, Helen; Graham, Simon P; Taracha, Evans L N; Kanga, Simon; Lu, Charles; Hass, Brian; Wortman, Jennifer; White, Owen; Gardner, Malcolm J; Nene, Vishvanath; de Villiers, Etienne P

    2005-01-01

    Massively parallel signature sequencing (MPSS) was used to analyze the transcriptome of the intracellular protozoan Theileria parva. In total 1,095,000, 20 bp sequences representing 4371 different signatures were generated from T.parva schizonts. Reproducible signatures were identified within 73% of potentially detectable predicted genes and 83% had signatures in at least one MPSS cycle. A predicted leader peptide was detected on 405 expressed genes. The quantitative range of signatures was 4-52,256 transcripts per million (t.p.m.). Rare transcripts (genes. Sequence signatures approximated a lognormal distribution, as in microarray. Transcripts were widely distributed throughout the genome, although only 47% of 138 telomere-associated open reading frames exhibited signatures. Antisense signatures comprised 13.8% of the total, comparable with Plasmodium. Eighty five predicted genes with antisense signatures lacked a sense signature. Antisense transcripts were independently amplified from schizont cDNA and verified by sequencing. The MPSS transcripts per million for seven genes encoding schizont antigens recognized by bovine CD8 T cells varied 1000-fold. There was concordance between transcription and protein expression for heat shock proteins that were very highly expressed according to MPSS and proteomics. The data suggests a low level of baseline transcription from the majority of protein-coding genes.

  7. The use of global transcriptional analysis to reveal the biological and cellular events involved in distinct development phases of Trichophyton rubrum conidial germination

    Directory of Open Access Journals (Sweden)

    Ding Guohui

    2007-04-01

    Full Text Available Abstract Background Conidia are considered to be the primary cause of infections by Trichophyton rubrum. Results We have developed a cDNA microarray containing 10250 ESTs to monitor the transcriptional strategy of conidial germination. A total of 1561 genes that had their expression levels specially altered in the process were obtained and hierarchically clustered with respect to their expression profiles. By functional analysis, we provided a global view of an important biological system related to conidial germination, including characterization of the pattern of gene expression at sequential developmental phases, and changes of gene expression profiles corresponding to morphological transitions. We matched the EST sequences to GO terms in the Saccharomyces Genome Database (SGD. A number of homologues of Saccharomyces cerevisiae genes related to signalling pathways and some important cellular processes were found to be involved in T. rubrum germination. These genes and signalling pathways may play roles in distinct steps, such as activating conidial germination, maintenance of isotropic growth, establishment of cell polarity and morphological transitions. Conclusion Our results may provide insights into molecular mechanisms of conidial germination at the cell level, and may enhance our understanding of regulation of gene expression related to the morphological construction of T. rubrum.

  8. Mixed-species genomic microarray analysis of fecal samples reveals differential transcriptional responses of bifidobacteria in breast- And formula-fed infants

    NARCIS (Netherlands)

    Klaassens, E.S.; Boesten, R.J.; Haarman, M.; Knol, J.; Schuren, F.H.; Vaughan, E.E.; Vos, W.M.de

    2009-01-01

    Although their exact function remains enigmatic, bifidobacteria are among the first colonizers of the newborn infant gut and further develop into abundant communities, notably in response to diet. Therefore, the transcriptional responses of bifidobacteria in rapidly processed fecal samples from

  9. Community structure analysis of transcriptional networks reveals distinct molecular pathways for early- and late-onset temporal lobe epilepsy with childhood febrile seizures.

    Directory of Open Access Journals (Sweden)

    Carlos Alberto Moreira-Filho

    Full Text Available Age at epilepsy onset has a broad impact on brain plasticity and epilepsy pathomechanisms. Prolonged febrile seizures in early childhood (FS constitute an initial precipitating insult (IPI commonly associated with mesial temporal lobe epilepsy (MTLE. FS-MTLE patients may have early disease onset, i.e. just after the IPI, in early childhood, or late-onset, ranging from mid-adolescence to early adult life. The mechanisms governing early (E or late (L disease onset are largely unknown. In order to unveil the molecular pathways underlying E and L subtypes of FS-MTLE we investigated global gene expression in hippocampal CA3 explants of FS-MTLE patients submitted to hippocampectomy. Gene coexpression networks (GCNs were obtained for the E and L patient groups. A network-based approach for GCN analysis was employed allowing: i the visualization and analysis of differentially expressed (DE and complete (CO - all valid GO annotated transcripts - GCNs for the E and L groups; ii the study of interactions between all the system's constituents based on community detection and coarse-grained community structure methods. We found that the E-DE communities with strongest connection weights harbor highly connected genes mainly related to neural excitability and febrile seizures, whereas in L-DE communities these genes are not only involved in network excitability but also playing roles in other epilepsy-related processes. Inversely, in E-CO the strongly connected communities are related to compensatory pathways (seizure inhibition, neuronal survival and responses to stress conditions while in L-CO these communities harbor several genes related to pro-epileptic effects, seizure-related mechanisms and vulnerability to epilepsy. These results fit the concept, based on fMRI and behavioral studies, that early onset epilepsies, although impacting more severely the hippocampus, are associated to compensatory mechanisms, while in late MTLE development the brain is less

  10. The hsp 16 Gene of the Probiotic Lactobacillus acidophilus Is Differently Regulated by Salt, High Temperature and Acidic Stresses, as Revealed by Reverse Transcription Quantitative PCR (qRT-PCR Analysis

    Directory of Open Access Journals (Sweden)

    Daniela Fiocco

    2011-08-01

    Full Text Available Small heat shock proteins (sHsps are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR procedure was developed and used to quantify the transcript level of a small heat shock gene (shs in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C, bile (0.3% w/v, hyperosmosis (1 M and 2.5 M NaCl, and low pH value (pH 4. The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5' noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR sequence (TTAGCACTC-N9-GAGTGCTAA homologue to the controlling IR of chaperone expression (CIRCE elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group.

  11. Genome-wide analysis of brain and gonad transcripts reveals changes of key sex reversal-related genes expression and signaling pathways in three stages of Monopterus albus.

    Directory of Open Access Journals (Sweden)

    Wei Chi

    Full Text Available The natural sex reversal severely affects the sex ratio and thus decreases the productivity of the rice field eel (Monopterus albus. How to understand and manipulate this process is one of the major issues for the rice field eel stocking. So far the genomics and transcriptomics data available for this species are still scarce. Here we provide a comprehensive study of transcriptomes of brain and gonad tissue in three sex stages (female, intersex and male from the rice field eel to investigate changes in transcriptional level during the sex reversal process.Approximately 195 thousand unigenes were generated and over 44.4 thousand were functionally annotated. Comparative study between stages provided multiple differentially expressed genes in brain and gonad tissue. Overall 4668 genes were found to be of unequal abundance between gonad tissues, far more than that of the brain tissues (59 genes. These genes were enriched in several different signaling pathways. A number of 231 genes were found with different levels in gonad in each stage, with several reproduction-related genes included. A total of 19 candidate genes that could be most related to sex reversal were screened out, part of these genes' expression patterns were validated by RT-qPCR. The expression of spef2, maats1, spag6 and dmc1 were abundant in testis, but was barely detected in females, while the 17β-hsd12, zpsbp3, gal3 and foxn5 were only expressed in ovary.This study investigated the complexity of brain and gonad transcriptomes in three sex stages of the rice field eel. Integrated analysis of different gene expression and changes in signaling pathways, such as PI3K-Akt pathway, provided crucial data for further study of sex transformation mechanisms.

  12. Genome-wide analysis of brain and gonad transcripts reveals changes of key sex reversal-related genes expression and signaling pathways in three stages of Monopterus albus.

    Science.gov (United States)

    Chi, Wei; Gao, Yu; Hu, Qing; Guo, Wei; Li, Dapeng

    2017-01-01

    The natural sex reversal severely affects the sex ratio and thus decreases the productivity of the rice field eel (Monopterus albus). How to understand and manipulate this process is one of the major issues for the rice field eel stocking. So far the genomics and transcriptomics data available for this species are still scarce. Here we provide a comprehensive study of transcriptomes of brain and gonad tissue in three sex stages (female, intersex and male) from the rice field eel to investigate changes in transcriptional level during the sex reversal process. Approximately 195 thousand unigenes were generated and over 44.4 thousand were functionally annotated. Comparative study between stages provided multiple differentially expressed genes in brain and gonad tissue. Overall 4668 genes were found to be of unequal abundance between gonad tissues, far more than that of the brain tissues (59 genes). These genes were enriched in several different signaling pathways. A number of 231 genes were found with different levels in gonad in each stage, with several reproduction-related genes included. A total of 19 candidate genes that could be most related to sex reversal were screened out, part of these genes' expression patterns were validated by RT-qPCR. The expression of spef2, maats1, spag6 and dmc1 were abundant in testis, but was barely detected in females, while the 17β-hsd12, zpsbp3, gal3 and foxn5 were only expressed in ovary. This study investigated the complexity of brain and gonad transcriptomes in three sex stages of the rice field eel. Integrated analysis of different gene expression and changes in signaling pathways, such as PI3K-Akt pathway, provided crucial data for further study of sex transformation mechanisms.

  13. Comparative Analysis of Muscle Hypertrophy Models Reveals Divergent Gene Transcription Profiles and Points to Translational Regulation of Muscle Growth through Increased mTOR Signaling

    Directory of Open Access Journals (Sweden)

    Marcelo G. Pereira

    2017-12-01

    Full Text Available Skeletal muscle mass is a result of the balance between protein breakdown and protein synthesis. It has been shown that multiple conditions of muscle atrophy are characterized by the common regulation of a specific set of genes, termed atrogenes. It is not known whether various models of muscle hypertrophy are similarly regulated by a common transcriptional program. Here, we characterized gene expression changes in three different conditions of muscle growth, examining each condition during acute and chronic phases. Specifically, we compared the transcriptome of Extensor Digitorum Longus (EDL muscles collected (1 during the rapid phase of postnatal growth at 2 and 4 weeks of age, (2 24 h or 3 weeks after constitutive activation of AKT, and (3 24 h or 3 weeks after overload hypertrophy caused by tenotomy of the Tibialis Anterior muscle. We observed an important overlap between significantly regulated genes when comparing each single condition at the two different timepoints. Furthermore, examining the transcriptional changes occurring 24 h after a hypertrophic stimulus, we identify an important role for genes linked to a stress response, despite the absence of muscle damage in the AKT model. However, when we compared all different growth conditions, we did not find a common transcriptional fingerprint. On the other hand, all conditions showed a marked increase in mTORC1 signaling and increased ribosome biogenesis, suggesting that muscle growth is characterized more by translational, than transcriptional regulation.

  14. A novel lineage transcription factor based analysis reveals differences in T helper cell subpopulation development in infected and intrauterine growth restricted (IUGR) piglets.

    Science.gov (United States)

    Ebner, F; Rausch, S; Scharek-Tedin, L; Pieper, R; Burwinkel, M; Zentek, J; Hartmann, S

    2014-10-01

    Research in mouse and human clearly identified subsets of T helper (Th) cells based on nuclear expression of specific lineage transcription factors. In swine, however, transcription factor based detection of functional subpopulations of porcine Th cells by flow cytometry is so far limited to regulatory T cells via Foxp3. T-bet and GATA-3 are the transcription factors that regulate commitment to Th1 or Th2 cells, respectively. In this study we prove GATA-3 and T-bet expression in porcine CD4(+) cells polarized in vitro. Importantly, GATA-3 and T-bet expressing cells were detectable in pigs infected with pathogens associated with Th2 and Th1 immune responses. Increased frequencies of GATA-3 positive CD4(+) cells are found in vivo in pigs experimentally infected with the nematode Trichuris suis, whereas porcine reproductive and respiratory syndrome virus (PRRSV) infection elicited T-bet positive CD4(+) T cells. Analysing the immune status of pre-weaning piglets with intrauterine growth restriction (IUGR) we found an increased expression of Foxp3, T-bet and GATA-3 in CD4(+) and CD4(+)CD8(+) double-positive T cells in systemic and intestinal compartments of IUGR piglets. Hence, we established the detection of porcine Th1 and Th2 cells via T-bet and GATA-3 and show that the porcine lineage transcription factors are differentially regulated very early in life depending on the developmental status. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Analysis of the human cytomegalovirus genomic region from UL146 through UL147A reveals sequence hypervariability, genotypic stability, and overlapping transcripts

    Directory of Open Access Journals (Sweden)

    Huang Diana D

    2006-01-01

    Full Text Available Abstract Background Although the sequence of the human cytomegalovirus (HCMV genome is generally conserved among unrelated clinical strains, some open reading frames (ORFs are highly variable. UL146 and UL147, which encode CXC chemokine homologues are among these variable ORFs. Results The region of the HCMV genome from UL146 through UL147A was analyzed in clinical strains for sequence variability, genotypic stability, and transcriptional expression. The UL146 sequences in clinical strains from two geographically distant sites were assigned to 12 sequence groups that differ by over 60% at the amino acid level. The same groups were generated by sequences from the UL146-UL147 intergenic region and the UL147 ORF. In contrast to the high level of sequence variability among unrelated clinical strains, the sequences of UL146 through UL147A from isolates of the same strain were highly stable after repeated passage both in vitro and in vivo. Riboprobes homologous to these ORFs detected multiple overlapping transcripts differing in temporal expression. UL146 sequences are present only on the largest transcript, which also contains all of the downstream ORFs including UL148 and UL132. The sizes and hybridization patterns of the transcripts are consistent with a common 3'-terminus downstream of the UL132 ORF. Early-late expression of the transcripts associated with UL146 and UL147 is compatible with the potential role of CXC chemokines in pathogenesis associated with viral replication. Conclusion Clinical isolates from two different geographic sites cluster in the same groups based on the hypervariability of the UL146, UL147, or the intergenic sequences, which provides strong evidence for linkage and no evidence for interstrain recombination within this region. The sequence of individual strains was absolutely stable in vitro and in vivo, which indicates that sequence drift is not a mechanism for the observed sequence hypervariability. There is also no

  16. Identification of an IMPDH1 mutation in autosomal dominant retinitis pigmentosa (RP10) revealed following comparative microarray analysis of transcripts derived from retinas of wild-type and Rho(-/-) mice.

    Science.gov (United States)

    Kennan, Avril; Aherne, Aileen; Palfi, Arpad; Humphries, Marian; McKee, Alex; Stitt, Alan; Simpson, David A C; Demtroder, Karin; Orntoft, Torben; Ayuso, Carmen; Kenna, Paul F; Farrar, G Jane; Humphries, Pete

    2002-03-01

    Comparative analysis of the transcriptional profiles of approximately 6000 genes in the retinas of wild-type mice with those carrying a targeted disruption of the rhodopsin gene was undertaken by microarray analysis. This revealed a series of transcripts, of which some were derived from genes known to map at retinopathy loci, levels of which were reduced or elevated in the retinas of Rho(-/-) mice lacking functional photoreceptors. The human homologue of one of these genes, encoding inosine monophosphate dehydrogenase type 1 (IMPDH1), maps to the region of 7q to which an adRP gene (RP10) had previously been localized. Mutational screening of DNA from the Spanish adRP family, originally used to localize the RP10 gene, revealed an Arg224Pro substitution co-segregating with the disease phenotype. The amino acid at position 224 of the IMPDH1 protein is conserved among species and the substitution is not present in healthy, unrelated individuals of European origin. These data provide strong evidence that mutations within the IMPDH1 gene cause adRP, and validate approaches to mutation detection involving comparative analysis of global transcription profiles in normal and degenerating retinal tissues. Other genes showing significant alterations in expression include some with anti-apoptotic functions and many encoding components of the extracellular matrix or cytoskeleton, a possible reflection of a response by Muller cells to preserve the remaining outer nuclear layer of the retina. We suggest that those genes identified are prime candidates for etiological involvement in degenerative retinal disease.

  17. Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways

    Directory of Open Access Journals (Sweden)

    Ying eZhao

    2015-06-01

    Full Text Available Metasequoiaglyptostroboidies is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as5-to-7years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia.

  18. What can digital transcript profiling reveal about human cancers?

    Directory of Open Access Journals (Sweden)

    J.M. Cerutti

    2003-08-01

    Full Text Available Important biological and clinical features of malignancy are reflected in its transcript pattern. Recent advances in gene expression technology and informatics have provided a powerful new means to obtain and interpret these expression patterns. A comprehensive approach to expression profiling is serial analysis of gene expression (SAGE, which provides digital information on transcript levels. SAGE works by counting transcripts and storing these digital values electronically, providing absolute gene expression levels that make historical comparisons possible. SAGE produces a comprehensive profile of gene expression and can be used to search for candidate tumor markers or antigens in a limited number of samples. The Cancer Genome Anatomy Project has created a SAGE database of human gene expression levels for many different tumors and normal reference tissues and provides online tools for viewing, comparing, and downloading expression profiles. Digital expression profiling using SAGE and informatics have been useful for identifying genes that have a role in tumor invasion and other aspects of tumor progression.

  19. Microarray analysis reveals increased transcriptional repression and reduced metabolic activity but not major changes in the core apoptotic machinery during maturation of sympathetic neurons

    Directory of Open Access Journals (Sweden)

    Mikk eRaba

    2016-03-01

    Full Text Available Postnatal maturation of the neurons whose main phenotype and basic synaptic contacts are already established includes neuronal growth, refinement of synaptic contacts, final steps of differentiation, programmed cell death period (PCD etc. In the sympathetic neurons, postnatal maturation includes permanent end of the PCD that occurs with the same time schedule in vivo and in vitro suggesting that the process could be genetically determined. Also many other changes in the neuronal maturation could be permanent and thus based on stable changes in the genome expression. However, postnatal maturation of the neurons is poorly studied. Here we compared the gene expression profiles of immature and mature sympathetic neurons using Affymetrix microarray assay. We found 1310 significantly up-regulated and 1151 significantly down-regulated genes in the mature neurons. Gene ontology analysis reveals up-regulation of genes related to neuronal differentiation, chromatin and epigenetic changes, extracellular factors and their receptors, and cell adhesion, whereas many down-regulated genes were related to metabolic and biosynthetic processes. We show that termination of PCD is not related to major changes in the expression of classical genes for apoptosis or cell survival. Our dataset is deposited to the ArrayExpress database and is a valuable source to select candidate genes in the studies of neuronal maturation. As an example, we studied the changes in the expression of selected genes Igf2bp3, Coro1A, Zfp57, Dcx, and Apaf1 in the young and mature sympathetic ganglia by quantitative PCR and show that these were strongly downregulated in the mature ganglia.

  20. Genomics analysis of genes expressed reveals differential ...

    African Journals Online (AJOL)

    Genomics analysis of genes expressed reveals differential responses to low chronic nitrogen stress in maize. ... Most induced clones were largely involved in various metabolism processes including physiological process, organelle regulation of biological process, nutrient reservoir activity, transcription regulator activity and ...

  1. Dual RNA-seq transcriptional analysis of wheat roots colonized by Azospirillum brasilense reveals up-regulation of nutrient acquisition and cell cycle genes.

    Science.gov (United States)

    Camilios-Neto, Doumit; Bonato, Paloma; Wassem, Roseli; Tadra-Sfeir, Michelle Z; Brusamarello-Santos, Liziane C C; Valdameri, Glaucio; Donatti, Lucélia; Faoro, Helisson; Weiss, Vinicius A; Chubatsu, Leda S; Pedrosa, Fábio O; Souza, Emanuel M

    2014-05-16

    The rapid growth of the world's population demands an increase in food production that no longer can be reached by increasing amounts of nitrogenous fertilizers. Plant growth promoting bacteria (PGPB) might be an alternative to increase nitrogenous use efficiency (NUE) in important crops such wheat. Azospirillum brasilense is one of the most promising PGPB and wheat roots colonized by A. brasilense is a good model to investigate the molecular basis of plant-PGPB interaction including improvement in plant-NUE promoted by PGPB. We performed a dual RNA-Seq transcriptional profiling of wheat roots colonized by A. brasilense strain FP2. cDNA libraries from biological replicates of colonized and non-inoculated wheat roots were sequenced and mapped to wheat and A. brasilense reference sequences. The unmapped reads were assembled de novo. Overall, we identified 23,215 wheat expressed ESTs and 702 A. brasilense expressed transcripts. Bacterial colonization caused changes in the expression of 776 wheat ESTs belonging to various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones and phytochemicals. In addition, genes encoding proteins related to bacterial chemotaxi, biofilm formation and nitrogen fixation were highly expressed in the sub-set of A. brasilense expressed genes. PGPB colonization enhanced the expression of plant genes related to nutrient up-take, nitrogen assimilation, DNA replication and regulation of cell division, which is consistent with a higher proportion of colonized root cells in the S-phase. Our data support the use of PGPB as an alternative to improve nutrient acquisition in important crops such as wheat, enhancing plant productivity and sustainability.

  2. Physiological, biochemical, and genome-wide transcriptional analysis reveals that elevated CO2 mitigates the impact of combined heat wave and drought stress in Arabidopsis thaliana at multiple organizational levels.

    Science.gov (United States)

    Zinta, Gaurav; AbdElgawad, Hamada; Domagalska, Malgorzata A; Vergauwen, Lucia; Knapen, Dries; Nijs, Ivan; Janssens, Ivan A; Beemster, Gerrit T S; Asard, Han

    2014-12-01

    Climate changes increasingly threaten plant growth and productivity. Such changes are complex and involve multiple environmental factors, including rising CO2 levels and climate extreme events. As the molecular and physiological mechanisms underlying plant responses to realistic future climate extreme conditions are still poorly understood, a multiple organizational level analysis (i.e. eco-physiological, biochemical, and transcriptional) was performed, using Arabidopsis exposed to incremental heat wave and water deficit under ambient and elevated CO2 . The climate extreme resulted in biomass reduction, photosynthesis inhibition, and considerable increases in stress parameters. Photosynthesis was a major target as demonstrated at the physiological and transcriptional levels. In contrast, the climate extreme treatment induced a protective effect on oxidative membrane damage, most likely as a result of strongly increased lipophilic antioxidants and membrane-protecting enzymes. Elevated CO2 significantly mitigated the negative impact of a combined heat and drought, as apparent in biomass reduction, photosynthesis inhibition, chlorophyll fluorescence decline, H2 O2 production, and protein oxidation. Analysis of enzymatic and molecular antioxidants revealed that the stress-mitigating CO2 effect operates through up-regulation of antioxidant defense metabolism, as well as by reduced photorespiration resulting in lowered oxidative pressure. Therefore, exposure to future climate extreme episodes will negatively impact plant growth and production, but elevated CO2 is likely to mitigate this effect. © 2014 John Wiley & Sons Ltd.

  3. Transcriptomic analysis reveals the roles of gibberellin-regulated genes and transcription factors in regulating bolting in lettuce (Lactuca sativa L.).

    Science.gov (United States)

    Liu, Xueying; Lv, Shanshan; Liu, Ran; Fan, Shuangxi; Liu, Chaojie; Liu, Renyi; Han, Yingyan

    2018-01-01

    A cool temperature is preferred for lettuce cultivation, as high temperatures cause premature bolting. Accordingly, exploring the mechanism of bolting and preventing premature bolting is important for agriculture. To explore this relationship in depth, morphological, physiological, and transcriptomic analyses of the bolting-sensitive line S39 at the five-leaf stage grown at 37°C were performed in the present study. Based on paraffin section results, we observed that S39 began bolting on the seventh day at 37°C. During bolting in the heat-treated plants, GA3 and GA4 levels in leaves and the indoleacetic acid (IAA) level in the stem reached a maximum on the sixth day, and these high contents were maintained. Additionally, bolting begins in the fifth day after GA3 treatment in S39 plants, GA3 and GA4 increased and then decreased, reaching a maximum on the fourth day in leaves. Similarly, IAA contents reached a maximum in the stem on the fifth day. No bolting was observed in the control group grown at 25°C, and significant changes were not observed in GA3 and GA4 levels in the controls during the observation period. RNA-sequencing data implicated transcription factors (TFs) in regulating bolting in lettuce, suggesting that the high GA contents in the leaves and IAA in the stem promote bolting. TFs possibly modulate the expression of related genes, such as those encoding hormones, potentially regulating bolting in lettuce. Compared to the control group, 258 TFs were identified in the stem of the treatment group, among which 98 and 156 were differentially up- and down-regulated, respectively; in leaves, 202 and 115 TFs were differentially up- and down-regulated, respectively. Significant changes in the treated group were observed for C2H2 zinc finger, AP2-EREBP, and WRKY families, indicating that these TFs may play important roles in regulating bolting.

  4. Nascent RNA sequencing reveals distinct features in plant transcription.

    Science.gov (United States)

    Hetzel, Jonathan; Duttke, Sascha H; Benner, Christopher; Chory, Joanne

    2016-10-25

    Transcriptional regulation of gene expression is a major mechanism used by plants to confer phenotypic plasticity, and yet compared with other eukaryotes or bacteria, little is known about the design principles. We generated an extensive catalog of nascent and steady-state transcripts in Arabidopsis thaliana seedlings using global nuclear run-on sequencing (GRO-seq), 5'GRO-seq, and RNA-seq and reanalyzed published maize data to capture characteristics of plant transcription. De novo annotation of nascent transcripts accurately mapped start sites and unstable transcripts. Examining the promoters of coding and noncoding transcripts identified comparable chromatin signatures, a conserved "TGT" core promoter motif and unreported transcription factor-binding sites. Mapping of engaged RNA polymerases showed a lack of enhancer RNAs, promoter-proximal pausing, and divergent transcription in Arabidopsis seedlings and maize, which are commonly present in yeast and humans. In contrast, Arabidopsis and maize genes accumulate RNA polymerases in proximity of the polyadenylation site, a trend that coincided with longer genes and CpG hypomethylation. Lack of promoter-proximal pausing and a higher correlation of nascent and steady-state transcripts indicate Arabidopsis may regulate transcription predominantly at the level of initiation. Our findings provide insight into plant transcription and eukaryotic gene expression as a whole.

  5. Revealing genome-scale transcriptional regulatory landscape of OmpR highlights its expanded regulatory roles under osmotic stress in Escherichia coli K-12 MG1655

    DEFF Research Database (Denmark)

    Seo, Sang Woo; Gao, Ye; Kim, Donghyuk

    2017-01-01

    A transcription factor (TF), OmpR, plays a critical role in transcriptional regulation of the osmotic stress response in bacteria. Here, we reveal a genome-scale OmpR regulon in Escherichia coli K-12 MG1655. Integrative data analysis reveals that a total of 37 genes in 24 transcription units (TUs...

  6. Whole-body single-cell sequencing reveals transcriptional domains in the annelid larval body.

    Science.gov (United States)

    Achim, Kaia; Eling, Nils; Vergara, Hernando Martinez; Bertucci, Paola Yanina; Musser, Jacob; Vopalensky, Pavel; Brunet, Thibaut; Collier, Paul; Benes, Vladimir; Marioni, John C; Arendt, Detlev

    2018-01-24

    Animal bodies comprise diverse arrays of cells. To characterise cellular identities across an entire body, we have compared the transcriptomes of single cells randomly picked from dissociated whole larvae of the marine annelid Platynereis dumerilii. We identify five transcriptionally distinct groups of differentiated cells, each expressing a unique set of transcription factors and effector genes that implement cellular phenotypes. Spatial mapping of cells into a cellular expression atlas, and wholemount in situ hybridisation of group-specific genes reveals spatially coherent transcriptional domains in the larval body, comprising e.g. apical sensory-neurosecretory cells vs. neural/epidermal surface cells. These domains represent new, basic subdivisions of the annelid body based entirely on differential gene expression, and are composed of multiple, transcriptionally similar cell types. They do not represent clonal domains, as revealed by developmental lineage analysis. We propose that the transcriptional domains that subdivide the annelid larval body represent families of related cell types that have arisen by evolutionary diversification. Their possible evolutionary conservation makes them a promising tool for evo-devo research. (167/250). © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  7. Global transcriptional analysis reveals the complex relationship between tea quality, leaf senescence and the responses to cold-drought combined stress in Camellia sinensis

    Directory of Open Access Journals (Sweden)

    Chao Zheng

    2016-12-01

    Full Text Available In field conditions, especially in arid and semi-arid areas, tea plants are often simultaneously exposed to various abiotic stresses such as cold and drought, which have profound effects on leaf senescence process and tea quality. However, most studies of gene expression in stress responses focus on a single inciting agent, and the confounding effect of multiple stresses on crop quality and leaf senescence remain unearthed. Here, global transcriptome profiles of tea leaves under separately cold and drought stress were compared with their combination using RNA-Seq technology. This revealed that tea plants shared a large overlap in unigenes displayed similar (26% expression pattern and avoid antagonistic responses (lowest level of prioritized mode: 0% to exhibit very congruent responses to co-occurring cold and drought stress; 31.5% differential expressed genes and 38% of the transcriptome changes in response to combined stresses were unpredictable from cold or drought single-case studies. We also identified 319 candidate genes for enhancing plant resistance to combined stress. We then investigated the combined effect of cold and drought on tea quality and leaf senescence. Our results showed that drought-induced leaf senescence were severely delayed by (i modulation of a number of senescence-associated genes and cold responsive genes, (ii enhancement of antioxidant capacity, (iii attenuation of lipid degradation, (iv maintenance of cell wall and photosynthetic system, (v alteration of senescence-induced sugar effect/sensitivity, as well as (vi regulation of secondary metabolism pathways that significantly influence the quality of tea during combined stress. Therefore, care should be taken when utilizing a set of stresses to try and maximize leaf longevity and tea quality.

  8. Phylogenetic analysis of the SAP30 family of transcriptional regulators reveals functional divergence in the domain that binds the nuclear matrix

    Directory of Open Access Journals (Sweden)

    Mäki Markku

    2009-06-01

    Full Text Available Abstract Background Deacetylation of histones plays a fundamental role in gene silencing, and this is mediated by a corepressor complex containing Sin3 as an essential scaffold protein. In this report we examine the evolution of two proteins in this complex, the Sin3-associated proteins SAP30L and SAP30, by using an archive of protein sequences from 62 species. Results Our analysis indicates that in tetrapods SAP30L is more similar than SAP30 to the ancestral protein, and the two copies in this group originated by gene duplication which occurred after the divergence of Actinopterygii and Sarcopterygii about 450 million years ago (Mya. The phylogenetic analysis and biochemical experiments suggest that SAP30 has diverged functionally from the ancestral SAP30L by accumulating mutations that have caused attenuation of one of the original functions, association with the nuclear matrix. This function is mediated by a nuclear matrix association sequence, which consists of a conserved motif in the C-terminus and the adjacent nucleolar localization signal (NoLS. Conclusion These results add further insight into the evolution and function of proteins of the SAP30 family, which share many characteristic with nuclear scaffolding proteins that are intimately involved in regulation of gene expression. Furthermore, SAP30L seems essential to eukaryotic biology, as it is found in animals, plants, fungi, as well as some taxa of unicellular eukaryotes.

  9. Transcriptional Analysis of Hair Follicle-Derived Keratinocytes from Donors with Atopic Dermatitis Reveals Enhanced Induction of IL32 Gene by IFN-γ

    Directory of Open Access Journals (Sweden)

    Yoshie Yoshikawa

    2013-02-01

    Full Text Available We cultured human hair follicle-derived keratinocytes (FDKs from plucked hairs. To gain insight into gene expression signatures that can distinguish atopic dermatitis from non-atopic controls without skin biopsies, we undertook a comparative study of gene expression in FDKs from adult donors with atopic dermatitis and non-atopic donors. FDK primary cultures (atopic dermatitis, n = 11; non-atopic controls, n = 7 before and after interferon gamma (IFN-γ treatment were used for microarray analysis and quantitative RT-PCR. Comparison of FDKs from atopic and non-atopic donors indicated that the former showed activated pathways with innate immunity and decreased pathways of cell growth, as indicated by increased NLRP2 expression and decreased DKK1 expression, respectively. Treatment with IFN-γ induced the enhanced expression of IL32, IL1B, IL8, and CXCL1 in the cells from atopic donors compared to that in cells from non-atopic donors at 24 h after treatment. IL1B expression in FDKs after IFN-γ treatment correlated with IL32 expression. We hypothesized that overexpression of IL32 in hair follicle keratinocytes of patients with atopic dermatitis would lead to the excessive production of pro-IL1β and that the activation of IL1β from pro-IL1β by inflammasome complex, in which NLRP2 protein might be involved, would be augmented. This is the first report to show enhanced induction of cytokine/chemokine genes by IFN-γ in atopic dermatitis using cultured FDKs.

  10. Metabolic network topology reveals transcriptional regulatory signatures of type 2 diabetes.

    Science.gov (United States)

    Zelezniak, Aleksej; Pers, Tune H; Soares, Simão; Patti, Mary Elizabeth; Patil, Kiran Raosaheb

    2010-04-01

    Type 2 diabetes mellitus (T2DM) is a disorder characterized by both insulin resistance and impaired insulin secretion. Recent transcriptomics studies related to T2DM have revealed changes in expression of a large number of metabolic genes in a variety of tissues. Identification of the molecular mechanisms underlying these transcriptional changes and their impact on the cellular metabolic phenotype is a challenging task due to the complexity of transcriptional regulation and the highly interconnected nature of the metabolic network. In this study we integrate skeletal muscle gene expression datasets with human metabolic network reconstructions to identify key metabolic regulatory features of T2DM. These features include reporter metabolites--metabolites with significant collective transcriptional response in the associated enzyme-coding genes, and transcription factors with significant enrichment of binding sites in the promoter regions of these genes. In addition to metabolites from TCA cycle, oxidative phosphorylation, and lipid metabolism (known to be associated with T2DM), we identified several reporter metabolites representing novel biomarker candidates. For example, the highly connected metabolites NAD+/NADH and ATP/ADP were also identified as reporter metabolites that are potentially contributing to the widespread gene expression changes observed in T2DM. An algorithm based on the analysis of the promoter regions of the genes associated with reporter metabolites revealed a transcription factor regulatory network connecting several parts of metabolism. The identified transcription factors include members of the CREB, NRF1 and PPAR family, among others, and represent regulatory targets for further experimental analysis. Overall, our results provide a holistic picture of key metabolic and regulatory nodes potentially involved in the pathogenesis of T2DM.

  11. Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing.

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    Sean P Gordon

    Full Text Available Genes in prokaryotic genomes are often arranged into clusters and co-transcribed into polycistronic RNAs. Isolated examples of polycistronic RNAs were also reported in some higher eukaryotes but their presence was generally considered rare. Here we developed a long-read sequencing strategy to identify polycistronic transcripts in several mushroom forming fungal species including Plicaturopsis crispa, Phanerochaete chrysosporium, Trametes versicolor, and Gloeophyllum trabeum. We found genome-wide prevalence of polycistronic transcription in these Agaricomycetes, involving up to 8% of the transcribed genes. Unlike polycistronic mRNAs in prokaryotes, these co-transcribed genes are also independently transcribed. We show that polycistronic transcription may interfere with expression of the downstream tandem gene. Further comparative genomic analysis indicates that polycistronic transcription is conserved among a wide range of mushroom forming fungi. In summary, our study revealed, for the first time, the genome prevalence of polycistronic transcription in a phylogenetic range of higher fungi. Furthermore, we systematically show that our long-read sequencing approach and combined bioinformatics pipeline is a generic powerful tool for precise characterization of complex transcriptomes that enables identification of mRNA isoforms not recovered via short-read assembly.

  12. Dynamic zebrafish interactome reveals transcriptional mechanisms of dioxin toxicity.

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    Andrey Alexeyenko

    2010-05-01

    Full Text Available In order to generate hypotheses regarding the mechanisms by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin causes toxicity, we analyzed global gene expression changes in developing zebrafish embryos exposed to this potent toxicant in the context of a dynamic gene network. For this purpose, we also computationally inferred a zebrafish (Danio rerio interactome based on orthologs and interaction data from other eukaryotes.Using novel computational tools to analyze this interactome, we distinguished between dioxin-dependent and dioxin-independent interactions between proteins, and tracked the temporal propagation of dioxin-dependent transcriptional changes from a few genes that were altered initially, to large groups of biologically coherent genes at later times. The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes. Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a as well as other genes that were dioxin-regulated one day after exposure. This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research.Thus, a powerful microarray experiment coupled with a flexible interactome and multi-pronged interactome tools (which are now made publicly available for microarray analysis and related work suggest the hypothesis that dioxin, best known in fish as a potent cardioteratogen, has many other targets. Many of these types of toxicity have been observed in mammalian species and are potentially caused by alterations to cyp1a.

  13. Global analysis of photosynthesis transcriptional regulatory networks.

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    Saheed Imam

    2014-12-01

    Full Text Available Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888, which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

  14. Global Analysis of Photosynthesis Transcriptional Regulatory Networks

    Science.gov (United States)

    Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.

    2014-01-01

    Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. PMID:25503406

  15. Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes

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    Deyholos Michael K

    2006-10-01

    Full Text Available Abstract Background Roots are an attractive system for genomic and post-genomic studies of NaCl responses, due to their primary importance to agriculture, and because of their relative structural and biochemical simplicity. Excellent genomic resources have been established for the study of Arabidopsis roots, however, a comprehensive microarray analysis of the root transcriptome following NaCl exposure is required to further understand plant responses to abiotic stress and facilitate future, systems-based analyses of the underlying regulatory networks. Results We used microarrays of 70-mer oligonucleotide probes representing 23,686 Arabidopsis genes to identify root transcripts that changed in relative abundance following 6 h, 24 h, or 48 h of hydroponic exposure to 150 mM NaCl. Enrichment analysis identified groups of structurally or functionally related genes whose members were statistically over-represented among up- or down-regulated transcripts. Our results are consistent with generally observed stress response themes, and highlight potentially important roles for underappreciated gene families, including: several groups of transporters (e.g. MATE, LeOPT1-like; signalling molecules (e.g. PERK kinases, MLO-like receptors, carbohydrate active enzymes (e.g. XTH18, transcription factors (e.g. members of ZIM, WRKY, NAC, and other proteins (e.g. 4CL-like, COMT-like, LOB-Class 1. We verified the NaCl-inducible expression of selected transcription factors and other genes by qRT-PCR. Conclusion Micorarray profiling of NaCl-treated Arabidopsis roots revealed dynamic changes in transcript abundance for at least 20% of the genome, including hundreds of transcription factors, kinases/phosphatases, hormone-related genes, and effectors of homeostasis, all of which highlight the complexity of this stress response. Our identification of these transcriptional responses, and groups of evolutionarily related genes with either similar or divergent

  16. Structural Model of RNA Polymerase II Elongation Complex with Complete Transcription Bubble Reveals NTP Entry Routes.

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    Lu Zhang

    2015-07-01

    Full Text Available The RNA polymerase II (Pol II is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the "secondary channel" is the only route for NTP to reach the active site of the enzyme or if the "main channel" could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription.

  17. Dynamic analysis of stochastic transcription cycles.

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    Claire V Harper

    2011-04-01

    Full Text Available In individual mammalian cells the expression of some genes such as prolactin is highly variable over time and has been suggested to occur in stochastic pulses. To investigate the origins of this behavior and to understand its functional relevance, we quantitatively analyzed this variability using new mathematical tools that allowed us to reconstruct dynamic transcription rates of different reporter genes controlled by identical promoters in the same living cell. Quantitative microscopic analysis of two reporter genes, firefly luciferase and destabilized EGFP, was used to analyze the dynamics of prolactin promoter-directed gene expression in living individual clonal and primary pituitary cells over periods of up to 25 h. We quantified the time-dependence and cyclicity of the transcription pulses and estimated the length and variation of active and inactive transcription phases. We showed an average cycle period of approximately 11 h and demonstrated that while the measured time distribution of active phases agreed with commonly accepted models of transcription, the inactive phases were differently distributed and showed strong memory, with a refractory period of transcriptional inactivation close to 3 h. Cycles in transcription occurred at two distinct prolactin-promoter controlled reporter genes in the same individual clonal or primary cells. However, the timing of the cycles was independent and out-of-phase. For the first time, we have analyzed transcription dynamics from two equivalent loci in real-time in single cells. In unstimulated conditions, cells showed independent transcription dynamics at each locus. A key result from these analyses was the evidence for a minimum refractory period in the inactive-phase of transcription. The response to acute signals and the result of manipulation of histone acetylation was consistent with the hypothesis that this refractory period corresponded to a phase of chromatin remodeling which significantly

  18. CYCLOPS reveals human transcriptional rhythms in health and disease.

    Science.gov (United States)

    Anafi, Ron C; Francey, Lauren J; Hogenesch, John B; Kim, Junhyong

    2017-05-16

    Circadian rhythms modulate many aspects of physiology. Knowledge of the molecular basis of these rhythms has exploded in the last 20 years. However, most of these data are from model organisms, and translation to clinical practice has been limited. Here, we present an approach to identify molecular rhythms in humans from thousands of unordered expression measurements. Our algorithm, cyclic ordering by periodic structure (CYCLOPS), uses evolutionary conservation and machine learning to identify elliptical structure in high-dimensional data. From this structure, CYCLOPS estimates the phase of each sample. We validated CYCLOPS using temporally ordered mouse and human data and demonstrated its consistency on human data from two independent research sites. We used this approach to identify rhythmic transcripts in human liver and lung, including hundreds of drug targets and disease genes. Importantly, for many genes, the circadian variation in expression exceeded variation from genetic and other environmental factors. We also analyzed hepatocellular carcinoma samples and show these solid tumors maintain circadian function but with aberrant output. Finally, to show how this method can catalyze medical translation, we show that dosage time can temporally segregate efficacy from dose-limiting toxicity of streptozocin, a chemotherapeutic drug. In sum, these data show the power of CYCLOPS and temporal reconstruction in bridging basic circadian research and clinical medicine.

  19. Transcriptional profiling reveals gland-specific differential expression in the three major salivary glands of the adult mouse.

    Science.gov (United States)

    Gao, Xin; Oei, Maria S; Ovitt, Catherine E; Sincan, Murat; Melvin, James E

    2018-04-01

    RNA-Seq was used to better understand the molecular nature of the biological differences among the three major exocrine salivary glands in mammals. Transcriptional profiling found that the adult murine parotid, submandibular, and sublingual salivary glands express greater than 14,300 protein-coding genes, and nearly 2,000 of these genes were differentially expressed. Principle component analysis of the differentially expressed genes revealed three distinct clusters according to gland type. The three salivary gland transcriptomes were dominated by a relatively few number of highly expressed genes (6.3%) that accounted for more than 90% of transcriptional output. Of the 912 transcription factors expressed in the major salivary glands, greater than 90% of them were detected in all three glands, while expression for ~2% of them was enriched in an individual gland. Expression of these unique transcription factors correlated with sublingual and parotid specific subsets of both highly expressed and differentially expressed genes. Gene ontology analyses revealed that the highly expressed genes common to all glands were associated with global functions, while many of the genes expressed in a single gland play a major role in the function of that gland. In summary, transcriptional profiling of the three murine major salivary glands identified a limited number of highly expressed genes, differentially expressed genes, and unique transcription factors that represent the transcriptional signatures underlying gland-specific biological properties.

  20. Discontent with content analysis of online transcripts

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    Judith Guevarra Enriquez

    2009-12-01

    Full Text Available Content analysis has dominated computer-mediated communication and educational technology studies for some time, and a review of its practices applied to online corpus of data or messages is overdue. We are confronted with complexity given the various foci, nuances and models for theorising learning and applying methods. One common suggestion to deal with the complexity in content analysis is a call for standardisation by replication or systematic research studies. This article presents its ‘discontent' with content analysis, discussing the issues and concerns that surround the analysis of online transcripts. It does not attempt to resolve nor provide a definitive answer. Instead, it is an open inquiry into another way of looking at online content. It presents an alternative or perhaps an extension of what we have come to know as content analysis. It argues for the notion of genres as another way of conceptualising online transcripts. It proposes two things: first that in performing transcript analysis, it is worthwhile to think how messages relate to a system of interactions that persists even beyond the online environment; secondly, there is an emergent and recurring metastructuring that is at work in online environments that is worth exploring, instead of imposing structures – models and frameworks that do not fit the emerging communicative practices of participants.

  1. Transcriptome sequencing reveals the roles of transcription factors in modulating genotype by nitrogen interaction in maize.

    Science.gov (United States)

    Chen, Qiuyue; Liu, Zhipeng; Wang, Baobao; Wang, Xufeng; Lai, Jinsheng; Tian, Feng

    2015-10-01

    Global transcriptome analysis in maize revealed differential nitrogen response between genotypes and implicate a crucial role of transcription factors in driving genotype by nitrogen interactions at gene expression level. Developing nitrogen-efficient cultivars are essential for sustainable and productive agriculture. Nitrogen use efficiency of plants is highly dependent on the interaction of environmental and genetic variation and results in adaptive phenotypes. This study used transcriptome sequencing to perform a comprehensive genotype by nitrogen (G × N) interaction analysis for two elite Chinese maize inbreds grown at normal and low nitrogen levels in field conditions. We demonstrated that the two maize inbreds showed contrasting agronomic and transcriptomic responses to changes in nitrogen availability. A total of 96 genes with a significant G × N interaction were detected. After characterizing the expression patterns of G × N interaction genes, we found that the G × N interaction genes tended to show condition-specific differential expression. The functional annotations of G × N interaction genes revealed that many different kinds of genes were involved in G × N interactions, but a significant enrichment for transcription factors was detected, particularly the AP2/EREBP and WRKY family, suggesting that transcription factors might play important roles in driving G × N interaction at gene expression level for nitrogen response in maize. Taken together, these results not only provide novel insights into the mechanism of nitrogen response in maize and set important basis for further characterization but also have important implications for other genotype by stress interaction.

  2. Quantitative analysis of tyrosinase transcripts in blood.

    Science.gov (United States)

    Johansson, M; Pisa, E K; Törmänen, V; Arstrand, K; Kågedal, B

    2000-07-01

    Tyrosinase is an enzyme unique to pigment-forming cells. Methods using this transcript for detection of melanoma cells in blood have given divergent results. Quantitative analytical procedures are therefore needed to study the analytical performance of the methods. Mononucleated cells were isolated by Percoll centrifugation. RNA was isolated by each of three methods: Ultraspec(TM)-II RNA isolation system, FastRNA(TM) GREEN Kit, and QIAamp RNA Blood Mini Kit. cDNA was synthesized using random hexamer primers. A tyrosinase-specific product of 207 bp was amplified by PCR. As an internal standard (and competitor) we used a 207-bp cDNA with a base sequence identical to the tyrosinase target except for a 20-bp probe-binding region. The PCR products were identified by 2, 4-dinitrophenol (DNP)-labeled probes specific for tyrosinase (5'DNP-GGGGAGCCTTGGGGTTCTGG-3') and internal standard (5'DNP-CGGAGCCCCGAAACCACATC-3') and quantified by ELISA. The calibration curves were linear and had a broad dynamic measuring range. A detection limit (2 SD above zero) of 48 transcripts/mL of blood was obtained from a low control. The analytical imprecision was 50% and 48% at concentrations of 1775 and 17 929 transcripts/mL (n = 12 and 14, respectively). With the cell line SK-Mel 28 added to blood and RNA extracted with the Ultraspec, Fast RNA, and QIAamp RNA methods, we found (mean +/- SD) 1716+/-1341, 2670+/-3174, and 24 320+/-5332 transcripts/mL of blood. Corresponding values were 527+/-497, 2497+/-1033, 14 930+/-1927 transcripts/mL of blood when the cell line JKM86-4 was added. One high-risk patient was followed by repeated analysis of tyrosinase transcripts in blood. The melanoma marker 5-S-cysteinyldopa in serum and urine was within reference values, but tyrosinase mRNA was slightly increased (120-168 transcripts/mL of blood). The tyrosinase mRNA increased to 1860 transcripts/mL concomitant with the increase in 5-S-cysteinyldopa; later a spleen metastasis was found. The results

  3. Identification and Transcript Analysis of the TCP Transcription Factors in the Diploid Woodland Strawberry Fragaria vesca

    Science.gov (United States)

    Wei, Wei; Hu, Yang; Cui, Meng-Yuan; Han, Yong-Tao; Gao, Kuan; Feng, Jia-Yue

    2016-01-01

    Plant-specific TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors play versatile functions in multiple processes of plant growth and development. However, no systematic study has been performed in strawberry. In this study, 19 FvTCP genes were identified in the diploid woodland strawberry (Fragaria vesca) accession Heilongjiang-3. Phylogenetic analysis suggested that the FvTCP genes were classified into two main classes, with the second class further divided into two subclasses, which was supported by the exon-intron organizations and the conserved motif structures. Promoter analysis revealed various cis-acting elements related to growth and development, hormone and/or stress responses. We analyzed FvTCP gene transcript accumulation patterns in different tissues and fruit developmental stages. Among them, 12 FvTCP genes exhibited distinct tissue-specific transcript accumulation patterns. Eleven FvTCP genes were down-regulated in different fruit developmental stages, while five FvTCP genes were up-regulated. Transcripts of FvTCP genes also varied with different subcultural propagation periods and were induced by hormone treatments and biotic and abiotic stresses. Subcellular localization analysis showed that six FvTCP-GFP fusion proteins showed distinct localizations in Arabidopsis mesophyll protoplasts. Notably, transient over-expression of FvTCP9 in strawberry fruits dramatically affected the expression of a series of genes implicated in fruit development and ripening. Taken together, the present study may provide the basis for functional studies to reveal the role of this gene family in strawberry growth and development. PMID:28066489

  4. Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity

    Science.gov (United States)

    Chiu, Isaac M; Barrett, Lee B; Williams, Erika K; Strochlic, David E; Lee, Seungkyu; Weyer, Andy D; Lou, Shan; Bryman, Gregory S; Roberson, David P; Ghasemlou, Nader; Piccoli, Cara; Ahat, Ezgi; Wang, Victor; Cobos, Enrique J; Stucky, Cheryl L; Ma, Qiufu; Liberles, Stephen D; Woolf, Clifford J

    2014-01-01

    The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4+SNS-Cre/TdTomato+, 2) IB4−SNS-Cre/TdTomato+, and 3) Parv-Cre/TdTomato+ cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation. DOI: http://dx.doi.org/10.7554/eLife.04660.001 PMID:25525749

  5. PPARα-independent transcriptional targets of perfluoroalkyl acids revealed by transcript profiling

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microarray datasets used in the analysis. This dataset is associated with the following publication: Rosen, M., K. Das, J. Rooney, B. Abbott, C. Lau, and C. Corton....

  6. The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation.

    Science.gov (United States)

    Cicchillitti, Lucia; Manni, Isabella; Mancone, Carmine; Regazzo, Giulia; Spagnuolo, Manuela; Alonzi, Tonino; Carlomosti, Fabrizio; Dell'Anna, Maria Lucia; Dell'Omo, Giulia; Picardo, Mauro; Ciana, Paolo; Capogrossi, Maurizio C; Tripodi, Marco; Magenta, Alessandra; Rizzo, Maria Giulia; Gurtner, Aymone; Piaggio, Giulia

    2017-01-10

    Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y-dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferation.

  7. Fibroblast growth factor receptor-1 (FGFR1) nuclear dynamics reveal a novel mechanism in transcription control.

    Science.gov (United States)

    Dunham-Ems, Star M; Lee, Yu-Wei; Stachowiak, Ewa K; Pudavar, Haridas; Claus, Peter; Prasad, Paras N; Stachowiak, Michal K

    2009-05-01

    Nuclear FGFR1 acts as a developmental gene regulator in cooperation with FGF-2, RSK1, and CREB-binding protein (CBP). FRAP analysis revealed three nuclear FGFR1 populations: i) a fast mobile, ii) a slower mobile population reflecting chromatin-bound FGFR1, and iii) an immobile FGFR1 population associated with the nuclear matrix. Factors (cAMP, CBP) that induce FGFR1-mediated gene activation shifted FGFR1 from the nuclear matrix (immobile) to chromatin (slow) and reduced the movement rate of the chromatin-bound population. Transcription inhibitors accelerated FGFR1 movement; the content of the chromatin-bound slow FGFR1 decreased, whereas the fast population increased. The transcriptional activation appears to involve conversion of the immobile matrix-bound and the fast nuclear FGFR1 into a slow chromatin-binding population through FGFR1's interaction with CBP, RSK1, and the high-molecular-weight form of FGF-2. Our findings support a general mechanism in which gene activation is governed by protein movement and collisions with other proteins and nuclear structures.

  8. Kinome-wide transcriptional profiling of uveal melanoma reveals new vulnerabilities to targeted therapeutics.

    Science.gov (United States)

    Bailey, Fiona P; Clarke, Kim; Kalirai, Helen; Kenyani, Jenna; Shahidipour, Haleh; Falciani, Francesco; Coulson, Judy M; Sacco, Joseph J; Coupland, Sarah E; Eyers, Patrick A

    2018-03-01

    Metastatic uveal melanoma (UM) is invariably fatal, usually within a year of diagnosis. There are currently no effective therapies, and clinical studies employing kinase inhibitors have so far demonstrated limited success. This is despite common activating mutations in GNAQ/11 genes, which trigger signalling pathways that might predispose tumours to a variety of targeted drugs. In this study, we have profiled kinome expression network dynamics in various human ocular melanomas. We uncovered a shared transcriptional profile in human primary UM samples and across a variety of experimental cell-based models. The poor overall response of UM cells to FDA-approved kinase inhibitors contrasted with much higher sensitivity to the bromodomain inhibitor JQ1, a broad transcriptional repressor. Mechanistically, we identified a repressed FOXM1-dependent kinase subnetwork in JQ1-exposed cells that contained multiple cell cycle-regulated protein kinases. Consistently, we demonstrated vulnerability of UM cells to inhibitors of mitotic protein kinases within this network, including the investigational PLK1 inhibitor BI6727. We conclude that analysis of kinome-wide signalling network dynamics has the potential to reveal actionable drug targets and inhibitors of potential therapeutic benefit for UM patients. © 2017 The Authors. Pigment Cell & Melanoma Research Published by John Wiley & Sons.

  9. Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation.

    Science.gov (United States)

    Bedard, Lynn Glowczewski; Dronamraju, Raghuvar; Kerschner, Jenny L; Hunter, Gerald O; Axley, Elizabeth DeVlieger; Boyd, Asha K; Strahl, Brian D; Mosley, Amber L

    2016-06-24

    Using affinity purification MS approaches, we have identified a novel role for casein kinase II (CKII) in the modification of the polymerase associated factor complex (PAF-C). Our data indicate that the facilitates chromatin transcription complex (FACT) interacts with CKII and may facilitate PAF complex phosphorylation. Posttranslational modification analysis of affinity-isolated PAF-C shows extensive CKII phosphorylation of all five subunits of PAF-C, although CKII subunits were not detected as interacting partners. Consistent with this, recombinant CKII or FACT-associated CKII isolated from cells can phosphorylate PAF-C in vitro, whereas no intrinsic kinase activity was detected in PAF-C samples. Significantly, PAF-C purifications combined with stable isotope labeling in cells (SILAC) quantitation for PAF-C phosphorylation from wild-type and CKII temperature-sensitive strains (cka1Δ cka2-8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1Δ cka2-8 strains. Consistent with a role of CKII in FACT and PAF-C function, we show that decreased CKII function in vivo results in decreased levels of histone H2B lysine 123 monoubiquitylation, a modification dependent on FACT and PAF-C. Taken together, our results define a coordinated role of CKII and FACT in the regulation of RNA polymerase II transcription through chromatin via phosphorylation of PAF-C. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Metabolic Network Topology Reveals Transcriptional Regulatory Signatures of Type 2 Diabetes

    DEFF Research Database (Denmark)

    Zelezniak, Aleksej; Pers, Tune Hannes; Pinho Soares, Simao Pedro

    2010-01-01

    mechanisms underlying these transcriptional changes and their impact on the cellular metabolic phenotype is a challenging task due to the complexity of transcriptional regulation and the highly interconnected nature of the metabolic network. In this study we integrate skeletal muscle gene expression datasets...... with human metabolic network reconstructions to identify key metabolic regulatory features of T2DM. These features include reporter metabolites—metabolites with significant collective transcriptional response in the associated enzyme-coding genes, and transcription factors with significant enrichment...... factor regulatory network connecting several parts of metabolism. The identified transcription factors include members of the CREB, NRF1 and PPAR family, among others, and represent regulatory targets for further experimental analysis. Overall, our results provide a holistic picture of key metabolic...

  11. Transcriptome Profiling of Tomato Fruit Development Reveals Transcription Factors Associated with Ascorbic Acid, Carotenoid and Flavonoid Biosynthesis

    Science.gov (United States)

    Ye, Jie; Hu, Tixu; Yang, Congmei; Li, Hanxia; Yang, Mingze; Ijaz, Raina; Ye, Zhibiao; Zhang, Yuyang

    2015-01-01

    Tomato (Solanum lycopersicum) serves as a research model for fruit development; however, while it is an important dietary source of antioxidant nutrients, the transcriptional regulation of genes that determine nutrient levels remains poorly understood. Here, the transcriptomes of fruit at seven developmental stages (7, 14, 21, 28, 35, 42 and 49 days after flowering) from two tomato cultivars (Ailsa Craig and HG6-61) were evaluated using the Illumina sequencing platform. A total of 26,397 genes, which were expressed in at least one developmental stage, were detected in the two cultivars, and the expression patterns of those genes could be divided into 20 groups using a K-mean cluster analysis. Gene Ontology term enrichment analysis indicated that genes involved in RNA regulation, secondary metabolism, hormone metabolism and cell wall metabolism were the most highly differentially expressed genes during fruit development and ripening. A co-expression analysis revealed several transcription factors whose expression patterns correlated with those of genes associated with ascorbic acid, carotenoid and flavonoid biosynthesis. This transcriptional correlation was confirmed by agroinfiltration mediated transient expression, which showed that most of the enzymatic genes in the ascorbic acid biosynthesis were regulated by the overexpression of each of the three transcription factors that were tested. The metabolic dynamics of ascorbic acid, carotenoid and flavonoid were investigated during fruit development and ripening, and some selected transcription factors showed transcriptional correlation with the accumulation of ascorbic acid, carotenoid and flavonoid. This transcriptome study provides insight into the regulatory mechanism of fruit development and presents candidate transcription factors involved in secondary metabolism. PMID:26133783

  12. Additional file 7: Figure S3. of Transcriptome analysis of paired primary colorectal carcinoma and liver metastases reveals fusion transcripts and similar gene expression profiles in primary carcinoma and liver metastases

    OpenAIRE

    Lee, Ja-Rang; Kwon, Chae; Choi, Yuri; Park, Hye; Kim, Hyun; Jo, Hong-Jae; Oh, Nahmgun; Park, Do

    2016-01-01

    Fusion transcripts in validation sets. (A) Gene fusion between ZMYND8 and SEPT9 gene by interchromosomal complex. (B) Gene fusion between ACE2 and PIR gene by intrachromosomal complex. Fusion junction was red arrow, and validation of fusion transcript by RT-PCR and Sanger sequencing in patient #3. Prediction of fusion protein was analyzed by conserved domain database.

  13. Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components.

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    Jeffrey A Pleiss

    2007-04-01

    Full Text Available Appropriate expression of most eukaryotic genes requires the removal of introns from their pre-messenger RNAs (pre-mRNAs, a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.

  14. Subcellular RNA sequencing reveals broad presence of cytoplasmic intron-sequence retaining transcripts in mouse and rat neurons.

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    Mugdha Khaladkar

    Full Text Available Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs, hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons. Intronic sub-sequence retention within IL1-β mRNA in anucleate platelets has been implicated in activity-dependent splicing and translation. In a recent study, we showed CIRTs harbor functional SINE ID elements which are hypothesized to mediate dendritic localization in neurons. Based on these studies and others, we hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation. We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions. We sequenced two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons. After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species (44-60% of the expressed transcripts. The sequence patterns, including stereotypical length, biased inclusion of specific introns, and intron-intron junctions, suggested CIRT-specific nuclear processing. Our analysis also suggested that these cytoplasmic intron-sequence retaining transcripts may serve as a primary transcript for ncRNAs. Our results show that retaining intronic sequences is not isolated to a few loci but may be a genome-wide phenomenon for embedding functional signals within certain mRNA. The results hypothesize a novel source of cis-sequences for post-transcriptional regulation. Our results hypothesize two potentially novel splicing pathways: one, within the nucleus for CIRT biogenesis; and another, within the

  15. Transcriptional analysis of apple fruit proanthocyanidin biosynthesis

    Science.gov (United States)

    Henry-Kirk, Rebecca A.

    2012-01-01

    Proanthocyanidins (PAs) are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. Many flavonoids have antioxidant properties and may have beneficial effects for human health. PAs are found in the seeds and fruits of many plants. In apple fruit (Malus × domestica Borkh.), the flavonoid biosynthetic pathway is most active in the skin, with the flavan-3-ols, catechin, and epicatechin acting as the initiating units for the synthesis of PA polymers. This study examined the genes involved in the production of PAs in three apple cultivars: two heritage apple cultivars, Hetlina and Devonshire Quarrenden, and a commercial cultivar, Royal Gala. HPLC analysis shows that tree-ripe fruit from Hetlina and Devonshire Quarrenden had a higher phenolic content than Royal Gala. Epicatechin and catechin biosynthesis is under the control of the biosynthetic enzymes anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR1), respectively. Counter-intuitively, real-time quantitative PCR analysis showed that the expression levels of Royal Gala LAR1 and ANR were significantly higher than those of both Devonshire Quarrenden and Hetlina. This suggests that a compensatory feedback mechanism may be active, whereby low concentrations of PAs may induce higher expression of gene transcripts. Further investigation is required into the regulation of these key enzymes in apple. Abbreviations:ANOVAanalysis of varianceANRanthocyanidin reductaseDADdiode array detectorDAFBdays after full bloomDFRdihydroflavonol reductaseLARleucoanthocyanidin reductaseLC-MSliquid chromatography/mass spectrometryPAproanthocyanidinqPCRreal-time quantitative PCR PMID:22859681

  16. Human disease modeling reveals integrated transcriptional and epigenetic mechanisms of NOTCH1 haploinsufficiency.

    Science.gov (United States)

    Theodoris, Christina V; Li, Molong; White, Mark P; Liu, Lei; He, Daniel; Pollard, Katherine S; Bruneau, Benoit G; Srivastava, Deepak

    2015-03-12

    The mechanisms by which transcription factor haploinsufficiency alters the epigenetic and transcriptional landscape in human cells to cause disease are unknown. Here, we utilized human induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) to show that heterozygous nonsense mutations in NOTCH1 that cause aortic valve calcification disrupt the epigenetic architecture, resulting in derepression of latent pro-osteogenic and -inflammatory gene networks. Hemodynamic shear stress, which protects valves from calcification in vivo, activated anti-osteogenic and anti-inflammatory networks in NOTCH1(+/+), but not NOTCH1(+/-), iPSC-derived ECs. NOTCH1 haploinsufficiency altered H3K27ac at NOTCH1-bound enhancers, dysregulating downstream transcription of more than 1,000 genes involved in osteogenesis, inflammation, and oxidative stress. Computational predictions of the disrupted NOTCH1-dependent gene network revealed regulatory nodes that, when modulated, restored the network toward the NOTCH1(+/+) state. Our results highlight how alterations in transcription factor dosage affect gene networks leading to human disease and reveal nodes for potential therapeutic intervention. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Genetic networks of liver metabolism revealed by integration of metabolic and transcriptional profiling.

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    Christine T Ferrara

    2008-03-01

    Full Text Available Although numerous quantitative trait loci (QTL influencing disease-related phenotypes have been detected through gene mapping and positional cloning, identification of the individual gene(s and molecular pathways leading to those phenotypes is often elusive. One way to improve understanding of genetic architecture is to classify phenotypes in greater depth by including transcriptional and metabolic profiling. In the current study, we have generated and analyzed mRNA expression and metabolic profiles in liver samples obtained in an F2 intercross between the diabetes-resistant C57BL/6 leptin(ob/ob and the diabetes-susceptible BTBR leptin(ob/ob mouse strains. This cross, which segregates for genotype and physiological traits, was previously used to identify several diabetes-related QTL. Our current investigation includes microarray analysis of over 40,000 probe sets, plus quantitative mass spectrometry-based measurements of sixty-seven intermediary metabolites in three different classes (amino acids, organic acids, and acyl-carnitines. We show that liver metabolites map to distinct genetic regions, thereby indicating that tissue metabolites are heritable. We also demonstrate that genomic analysis can be integrated with liver mRNA expression and metabolite profiling data to construct causal networks for control of specific metabolic processes in liver. As a proof of principle of the practical significance of this integrative approach, we illustrate the construction of a specific causal network that links gene expression and metabolic changes in the context of glutamate metabolism, and demonstrate its validity by showing that genes in the network respond to changes in glutamine and glutamate availability. Thus, the methods described here have the potential to reveal regulatory networks that contribute to chronic, complex, and highly prevalent diseases and conditions such as obesity and diabetes.

  18. Transcription regulatory networks analysis using CAGE

    KAUST Repository

    Tegnér, Jesper N.

    2009-10-01

    Mapping out cellular networks in general and transcriptional networks in particular has proved to be a bottle-neck hampering our understanding of biological processes. Integrative approaches fusing computational and experimental technologies for decoding transcriptional networks at a high level of resolution is therefore of uttermost importance. Yet, this is challenging since the control of gene expression in eukaryotes is a complex multi-level process influenced by several epigenetic factors and the fine interplay between regulatory proteins and the promoter structure governing the combinatorial regulation of gene expression. In this chapter we review how the CAGE data can be integrated with other measurements such as expression, physical interactions and computational prediction of regulatory motifs, which together can provide a genome-wide picture of eukaryotic transcriptional regulatory networks at a new level of resolution. © 2010 by Pan Stanford Publishing Pte. Ltd. All rights reserved.

  19. Multiple oxygen tension environments reveal diverse patterns of transcriptional regulation in primary astrocytes.

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    Wayne Chadwick

    Full Text Available The central nervous system normally functions at O(2 levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O(2-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O(2 tensions compared to the cell culture standard of 20% O(2, to investigate their ability to sense and translate this O(2 information to transcriptional activity. Variance of ambient O(2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O(2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O(2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O(2 tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional 'programs' may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity.

  20. Transcriptional profiling of developing mouse epidermis reveals novel patterns of coordinated gene expression.

    Science.gov (United States)

    Bazzi, Hisham; Fantauzzo, Katherine A; Richardson, Gavin D; Jahoda, Colin A B; Christiano, Angela M

    2007-04-01

    The mammalian epidermis is the first line of defense against external environmental challenges including dehydration. The epidermis undergoes a highly intricate developmental program in utero, transforming from a simple to a complex stratified epithelium. During this process of stratification and differentiation, epidermal keratinocytes express a defined set of structural proteins, mainly keratins, whose expression is controlled by largely unknown mechanisms. In order to identify novel factors contributing to epidermal morphogenesis, we performed a global transcriptional analysis of the developing mouse epidermis after separating it from the underlying dermis (E12.5-E15.5). Unexpectedly, the recently identified genes encoding secreted peptides dermokine (Dmkn), keratinocyte differentiation-associated protein (krtdap), and suprabasin (Sbsn) as well as a largely uncharacterized embryonic keratin (Krt77), were among the most highly differentially expressed genes. The three genes encoding the secreted proteins form a cluster in an approximately 40-Kb locus on human chromosome 19 and the syntenic region on mouse chromosome 7 known as the stratified epithelium secreted peptides complex (SSC). Using whole mount in situ hybridization, we show that these genes show a coordinated spatio-temporal expression pattern during epidermal morphogenesis. The expression of these genes initiates in the nasal epithelium and correlates with the initiation of other epidermal differentiation markers such as K1 and loricrin (Byrne et al. [1994] Development 120:2369-2383), as well as the initiation of barrier formation. Our observations reveal a coordinated mode of expression of the SSC genes as well as the correlation of their initiation in the nasal epithelium with the initiation of barrier formation at this site.

  1. Transcriptional Regulatory Network Analysis of MYB Transcription Factor Family Genes in Rice.

    Science.gov (United States)

    Smita, Shuchi; Katiyar, Amit; Chinnusamy, Viswanathan; Pandey, Dev M; Bansal, Kailash C

    2015-01-01

    MYB transcription factor (TF) is one of the largest TF families and regulates defense responses to various stresses, hormone signaling as well as many metabolic and developmental processes in plants. Understanding these regulatory hierarchies of gene expression networks in response to developmental and environmental cues is a major challenge due to the complex interactions between the genetic elements. Correlation analyses are useful to unravel co-regulated gene pairs governing biological process as well as identification of new candidate hub genes in response to these complex processes. High throughput expression profiling data are highly useful for construction of co-expression networks. In the present study, we utilized transcriptome data for comprehensive regulatory network studies of MYB TFs by "top-down" and "guide-gene" approaches. More than 50% of OsMYBs were strongly correlated under 50 experimental conditions with 51 hub genes via "top-down" approach. Further, clusters were identified using Markov Clustering (MCL). To maximize the clustering performance, parameter evaluation of the MCL inflation score (I) was performed in terms of enriched GO categories by measuring F-score. Comparison of co-expressed cluster and clads analyzed from phylogenetic analysis signifies their evolutionarily conserved co-regulatory role. We utilized compendium of known interaction and biological role with Gene Ontology enrichment analysis to hypothesize function of coexpressed OsMYBs. In the other part, the transcriptional regulatory network analysis by "guide-gene" approach revealed 40 putative targets of 26 OsMYB TF hubs with high correlation value utilizing 815 microarray data. The putative targets with MYB-binding cis-elements enrichment in their promoter region, functional co-occurrence as well as nuclear localization supports our finding. Specially, enrichment of MYB binding regions involved in drought-inducibility implying their regulatory role in drought response in rice

  2. Transcriptional Regulatory Network Analysis of MYB Transcription Factor Family Genes in Rice

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    Shuchi eSmita

    2015-12-01

    Full Text Available MYB transcription factor (TF is one of the largest TF families and regulates defense responses to various stresses, hormone signaling as well as many metabolic and developmental processes in plants. Understanding these regulatory hierarchies of gene expression networks in response to developmental and environmental cues is a major challenge due to the complex interactions between the genetic elements. Correlation analyses are useful to unravel co-regulated gene pairs governing biological process as well as identification of new candidate hub genes in response to these complex processes. High throughput expression profiling data are highly useful for construction of co-expression networks. In the present study, we utilized transcriptome data for comprehensive regulatory network studies of MYB TFs by top down and guide gene approaches. More than 50% of OsMYBs were strongly correlated under fifty experimental conditions with 51 hub genes via top down approach. Further, clusters were identified using Markov Clustering (MCL. To maximize the clustering performance, parameter evaluation of the MCL inflation score (I was performed in terms of enriched GO categories by measuring F-score. Comparison of co-expressed cluster and clads analyzed from phylogenetic analysis signifies their evolutionarily conserved co-regulatory role. We utilized compendium of known interaction and biological role with Gene Ontology enrichment analysis to hypothesize function of coexpressed OsMYBs. In the other part, the transcriptional regulatory network analysis by guide gene approach revealed 40 putative targets of 26 OsMYB TF hubs with high correlation value utilizing 815 microarray data. The putative targets with MYB-binding cis-elements enrichment in their promoter region, functional co-occurrence as well as nuclear localization supports our finding. Specially, enrichment of MYB binding regions involved in drought-inducibility implying their regulatory role in drought

  3. A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock.

    Science.gov (United States)

    Matsumoto, Akira; Ukai-Tadenuma, Maki; Yamada, Rikuhiro G; Houl, Jerry; Uno, Kenichiro D; Kasukawa, Takeya; Dauwalder, Brigitte; Itoh, Taichi Q; Takahashi, Kuniaki; Ueda, Ryu; Hardin, Paul E; Tanimura, Teiichi; Ueda, Hiroki R

    2007-07-01

    The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tiling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation.

  4. Transcription analysis of neonicotinoid resistance in Mediterranean (MED) populations of B. tabaci reveal novel cytochrome P450s, but no nAChR mutations associated with the phenotype.

    Science.gov (United States)

    Ilias, Aris; Lagnel, Jacques; Kapantaidaki, Despoina E; Roditakis, Emmanouil; Tsigenopoulos, Costas S; Vontas, John; Tsagkarakou, Anastasia

    2015-11-14

    Bemisia tabaci is one of the most damaging agricultural pests world-wide. Although its control is based on insecticides, B. tabaci has developed resistance against almost all classes of insecticides, including neonicotinoids. We employed an RNA-seq approach to generate genome wide expression data and identify genes associated with neonicotinoid resistance in Mediterranean (MED) B. tabaci (Q1 biotype). Twelve libraries from insecticide resistant and susceptible whitefly populations were sequenced on an Illumina Next-generation sequencing platform, and genomic sequence information of approximately 73 Gbp was generated. A reference transcriptome was built by de novo assembly and functionally annotated. A total of 146 P450s, 18 GSTs and 23 CCEs enzymes (unigenes) potentially involved in the detoxification of xenobiotics were identified, along with 78 contigs encoding putative target proteins of six different insecticide classes. Ten unigenes encoding nicotinic Acetylcholine Receptors (nAChR), the target of neoinicotinoids, were identified and phylogenetically classified. No nAChR polymorphism potentially related with the resistant phenotypes, was observed among the studied strains. DE analysis revealed that among the 550 differentially (logFC > 1) over-transcribed unigenes, 52 detoxification enzymes were over expressed including unigenes with orthologues in P450s, GSTs, CCE and UDP-glucuronosyltransferases. Eight P450 unigenes belonging to clades CYP2, CYP3 and CYP4 were highly up-regulated (logFC > 2) including CYP6CM1, a gene already known to confer imidacloprid resistance in B. tabaci. Using quantitative qPCRs, a larger screening of field MED B. tabaci from Crete with known neonicotinoid phenotype was performed to associate expression levels of P450s with resistance levels. Expression levels of five P450s, including CYP6CM1, were found associated with neonicotinoid resistance. However, a significant correlation was found only in CYP303 and CYP6CX3, with imidacloprid

  5. Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction

    Directory of Open Access Journals (Sweden)

    Valero Francisco

    2007-07-01

    Full Text Available Abstract Background The analysis of transcriptional levels of the genes involved in protein synthesis and secretion is a key factor to understand the host organism's responses to recombinant protein production, as well as their interaction with the cultivation conditions. Novel techniques such as the sandwich hybridization allow monitoring quantitatively the dynamic changes of specific RNAs. In this study, the transcriptional levels of some genes related to the unfolded protein response (UPR and central metabolism of Pichia pastoris were analysed during batch and fed-batch cultivations using an X-33-derived strain expressing a Rhizopus oryzae lipase under control of the formaldehyde dehydrogenase promoter (FLD1, namely the alcohol oxidase gene AOX1, the formaldehyde dehydrogenase FLD1, the protein disulfide isomerase PDI, the KAR2 gene coding for the BiP chaperone, the 26S rRNA and the R. oryzae lipase gene ROL. Results The transcriptional levels of the selected set of genes were first analysed in P. pastoris cells growing in shake flask cultures containing different carbon and nitrogen sources combinations, glycerol + ammonium, methanol + methylamine and sorbitol + methylamine. The transcriptional levels of the AOX1 and FLD1 genes were coherent with the known regulatory mechanism of C1 substrates in P. pastoris, whereas ROL induction lead to the up-regulation of KAR2 and PDI transcriptional levels, thus suggesting that ROL overexpression triggers the UPR. This was further confirmed in fed-batch cultivations performed at different growth rates. Transcriptional levels of the analysed set of genes were generally higher at higher growth rates. Nevertheless, when ROL was overexpressed in a strain having the UPR constitutively activated, significantly lower relative induction levels of these marker genes were detected. Conclusion The bead-based sandwich hybridization assay has shown its potential as a reliable instrument for quantification of

  6. Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum

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    Aquino-Ferreira Roseli

    2010-02-01

    Full Text Available Abstract Background Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST collection by constructing one cDNA library and nine suppression subtractive hybridization libraries. Results The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases. Conclusion In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.

  7. Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation.

    Science.gov (United States)

    Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard

    2016-05-26

    Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating - a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility.

  8. Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli.

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    Shan Goh

    Full Text Available BACKGROUND: Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development. METHODOLOGY/PRINCIPAL FINDINGS: Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50. When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50 values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required. CONCLUSIONS: RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.

  9. Transcriptional dynamics reveal critical roles for non-coding RNAs in the immediate-early response.

    Directory of Open Access Journals (Sweden)

    Stuart Aitken

    2015-04-01

    Full Text Available The immediate-early response mediates cell fate in response to a variety of extracellular stimuli and is dysregulated in many cancers. However, the specificity of the response across stimuli and cell types, and the roles of non-coding RNAs are not well understood. Using a large collection of densely-sampled time series expression data we have examined the induction of the immediate-early response in unparalleled detail, across cell types and stimuli. We exploit cap analysis of gene expression (CAGE time series datasets to directly measure promoter activities over time. Using a novel analysis method for time series data we identify transcripts with expression patterns that closely resemble the dynamics of known immediate-early genes (IEGs and this enables a comprehensive comparative study of these genes and their chromatin state. Surprisingly, these data suggest that the earliest transcriptional responses often involve promoters generating non-coding RNAs, many of which are produced in advance of canonical protein-coding IEGs. IEGs are known to be capable of induction without de novo protein synthesis. Consistent with this, we find that the response of both protein-coding and non-coding RNA IEGs can be explained by their transcriptionally poised, permissive chromatin state prior to stimulation. We also explore the function of non-coding RNAs in the attenuation of the immediate early response in a small RNA sequencing dataset matched to the CAGE data: We identify a novel set of microRNAs responsible for the attenuation of the IEG response in an estrogen receptor positive cancer cell line. Our computational statistical method is well suited to meta-analyses as there is no requirement for transcripts to pass thresholds for significant differential expression between time points, and it is agnostic to the number of time points per dataset.

  10. Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes.

    Science.gov (United States)

    Czechowski, Tomasz; Bari, Rajendra P; Stitt, Mark; Scheible, Wolf-Rüdiger; Udvardi, Michael K

    2004-04-01

    Summary To overcome the detection limits inherent to DNA array-based methods of transcriptome analysis, we developed a real-time reverse transcription (RT)-PCR-based resource for quantitative measurement of transcripts for 1465 Arabidopsis transcription factors (TFs). Using closely spaced gene-specific primer pairs and SYBR Green to monitor amplification of double-stranded DNA (dsDNA), transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4% of reactions produced non-specific PCR products. The amplification efficiency of each PCR was determined from the log slope of SYBR Green fluorescence versus cycle number in the exponential phase, and was used to correct the readout for each primer pair and run. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001 and 100 copies per cell. Only 13% of TF transcripts were undetectable in these organs. For comparison, 22K Arabidopsis Affymetrix chips detected less than 55% of TF transcripts in the same samples, the range of transcript levels was compressed by a factor more than 100, and the data were less accurate especially in the lower part of the response range. Real-time RT-PCR revealed 35 root-specific and 52 shoot-specific TF genes, most of which have not been identified as organ-specific previously. Finally, many of the TF transcripts detected by RT-PCR are not represented in Arabidopsis EST (expressed sequence tag) or Massively Parallel Signature Sequencing (MPSS) databases. These genes can now be annotated as expressed.

  11. RNA-seq transcriptional profiling of Leishmania amazonensis reveals an arginase-dependent gene expression regulation.

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    Juliana Ide Aoki

    2017-10-01

    Full Text Available Leishmania is a protozoan parasite that alternates its life cycle between the sand-fly vector and the mammalian host. This alternation involves environmental changes and leads the parasite to dynamic modifications in morphology, metabolism, cellular signaling and regulation of gene expression to allow for a rapid adaptation to new conditions. The L-arginine pathway in L. amazonensis is important during the parasite life cycle and interferes in the establishment and maintenance of the infection in mammalian macrophages. Host arginase is an immune-regulatory enzyme that can reduce the production of nitric oxide by activated macrophages, directing the availability of L-arginine to the polyamine pathway, resulting in parasite replication. In this work, we performed transcriptional profiling to identify differentially expressed genes in L. amazonensis wild-type (La-WT versus L. amazonensis arginase knockout (La-arg- promastigotes and axenic amastigotes.A total of 8253 transcripts were identified in La-WT and La-arg- promastigotes and axenic amastigotes, about 60% of them codifying hypothetical proteins and 443 novel transcripts, which did not match any previously annotated genes. Our RNA-seq data revealed that 85% of genes were constitutively expressed. The comparison of transcriptome and metabolome data showed lower levels of arginase and higher levels of glutamate-5-kinase in La-WT axenic amastigotes compared to promastigotes. The absence of arginase activity in promastigotes increased the levels of pyrroline 5-carboxylate reductase, but decreased the levels of arginosuccinate synthase, pyrroline 5-carboxylate dehydrogenase, acetylornithine deacetylase and spermidine synthase transcripts levels. These observations can explain previous metabolomic data pointing to the increase of L-arginine, citrulline and L-glutamate and reduction of aspartate, proline, ornithine and putrescine. Altogether, these results indicate that arginase activity is important

  12. Genome conformation capture reveals that the Escherichia coli chromosome is organized by replication and transcription.

    Science.gov (United States)

    Cagliero, Cedric; Grand, Ralph S; Jones, M Beatrix; Jin, Ding J; O'Sullivan, Justin M

    2013-07-01

    To fit within the confines of the cell, bacterial chromosomes are highly condensed into a structure called the nucleoid. Despite the high degree of compaction in the nucleoid, the genome remains accessible to essential biological processes, such as replication and transcription. Here, we present the first high-resolution chromosome conformation capture-based molecular analysis of the spatial organization of the Escherichia coli nucleoid during rapid growth in rich medium and following an induced amino acid starvation that promotes the stringent response. Our analyses identify the presence of origin and terminus domains in exponentially growing cells. Moreover, we observe an increased number of interactions within the origin domain and significant clustering of SeqA-binding sequences, suggesting a role for SeqA in clustering of newly replicated chromosomes. By contrast, 'histone-like' protein (i.e. Fis, IHF and H-NS) -binding sites did not cluster, and their role in global nucleoid organization does not manifest through the mediation of chromosomal contacts. Finally, genes that were downregulated after induction of the stringent response were spatially clustered, indicating that transcription in E. coli occurs at transcription foci.

  13. Deep sequencing reveals the complex and coordinated transcriptional regulation of genes related to grain quality in rice cultivars

    Directory of Open Access Journals (Sweden)

    An Gynheung

    2011-04-01

    Full Text Available Abstract Background Milling yield and eating quality are two important grain quality traits in rice. To identify the genes involved in these two traits, we performed a deep transcriptional analysis of developing seeds using both massively parallel signature sequencing (MPSS and sequencing-by-synthesis (SBS. Five MPSS and five SBS libraries were constructed from 6-day-old developing seeds of Cypress (high milling yield, LaGrue (low milling yield, Ilpumbyeo (high eating quality, YR15965 (low eating quality, and Nipponbare (control. Results The transcriptomes revealed by MPSS and SBS had a high correlation co-efficient (0.81 to 0.90, and about 70% of the transcripts were commonly identified in both types of the libraries. SBS, however, identified 30% more transcripts than MPSS. Among the highly expressed genes in Cypress and Ilpumbyeo, over 100 conserved cis regulatory elements were identified. Numerous specifically expressed transcription factor (TF genes were identified in Cypress (282, LaGrue (312, Ilpumbyeo (363, YR15965 (260, and Nipponbare (357. Many key grain quality-related genes (i.e., genes involved in starch metabolism, aspartate amino acid metabolism, storage and allergenic protein synthesis, and seed maturation that were expressed at high levels underwent alternative splicing and produced antisense transcripts either in Cypress or Ilpumbyeo. Further, a time course RT-PCR analysis confirmed a higher expression level of genes involved in starch metabolism such as those encoding ADP glucose pyrophosphorylase (AGPase and granule bound starch synthase I (GBSS I in Cypress than that in LaGrue during early seed development. Conclusion This study represents the most comprehensive analysis of the developing seed transcriptome of rice available to date. Using two high throughput sequencing methods, we identified many differentially expressed genes that may affect milling yield or eating quality in rice. Many of the identified genes are involved

  14. Deep sequencing reveals the complex and coordinated transcriptional regulation of genes related to grain quality in rice cultivars.

    Science.gov (United States)

    Venu, Rc; Sreerekha, Mv; Nobuta, Kan; Beló, André; Ning, Yuese; An, Gynheung; Meyers, Blake C; Wang, Guo-Liang

    2011-04-14

    Milling yield and eating quality are two important grain quality traits in rice. To identify the genes involved in these two traits, we performed a deep transcriptional analysis of developing seeds using both massively parallel signature sequencing (MPSS) and sequencing-by-synthesis (SBS). Five MPSS and five SBS libraries were constructed from 6-day-old developing seeds of Cypress (high milling yield), LaGrue (low milling yield), Ilpumbyeo (high eating quality), YR15965 (low eating quality), and Nipponbare (control). The transcriptomes revealed by MPSS and SBS had a high correlation co-efficient (0.81 to 0.90), and about 70% of the transcripts were commonly identified in both types of the libraries. SBS, however, identified 30% more transcripts than MPSS. Among the highly expressed genes in Cypress and Ilpumbyeo, over 100 conserved cis regulatory elements were identified. Numerous specifically expressed transcription factor (TF) genes were identified in Cypress (282), LaGrue (312), Ilpumbyeo (363), YR15965 (260), and Nipponbare (357). Many key grain quality-related genes (i.e., genes involved in starch metabolism, aspartate amino acid metabolism, storage and allergenic protein synthesis, and seed maturation) that were expressed at high levels underwent alternative splicing and produced antisense transcripts either in Cypress or Ilpumbyeo. Further, a time course RT-PCR analysis confirmed a higher expression level of genes involved in starch metabolism such as those encoding ADP glucose pyrophosphorylase (AGPase) and granule bound starch synthase I (GBSS I) in Cypress than that in LaGrue during early seed development. This study represents the most comprehensive analysis of the developing seed transcriptome of rice available to date. Using two high throughput sequencing methods, we identified many differentially expressed genes that may affect milling yield or eating quality in rice. Many of the identified genes are involved in the biosynthesis of starch, aspartate

  15. Enhancer transcription reveals subtype-specific gene expression programs controlling breast cancer pathogenesis.

    Science.gov (United States)

    Franco, Hector L; Nagari, Anusha; Malladi, Venkat S; Li, Wenqian; Xi, Yuanxin; Richardson, Dana; Allton, Kendra L; Tanaka, Kaori; Li, Jing; Murakami, Shino; Keyomarsi, Khandan; Bedford, Mark T; Shi, Xiaobing; Li, Wei; Barton, Michelle C; Dent, Sharon Y R; Kraus, W Lee

    2018-02-01

    Noncoding transcription is a defining feature of active enhancers, linking transcription factor (TF) binding to the molecular mechanisms controlling gene expression. To determine the relationship between enhancer activity and biological outcomes in breast cancers, we profiled the transcriptomes (using GRO-seq and RNA-seq) and epigenomes (using ChIP-seq) of 11 different human breast cancer cell lines representing five major molecular subtypes of breast cancer, as well as two immortalized ("normal") human breast cell lines. In addition, we developed a robust and unbiased computational pipeline that simultaneously identifies putative subtype-specific enhancers and their cognate TFs by integrating the magnitude of enhancer transcription, TF mRNA expression levels, TF motif P -values, and enrichment of H3K4me1 and H3K27ac. When applied across the 13 different cell lines noted above, the Total Functional Score of Enhancer Elements (TFSEE) identified key breast cancer subtype-specific TFs that act at transcribed enhancers to dictate gene expression patterns determining growth outcomes, including Forkhead TFs, FOSL1, and PLAG1. FOSL1, a Fos family TF, (1) is highly enriched at the enhancers of triple negative breast cancer (TNBC) cells, (2) acts as a key regulator of the proliferation and viability of TNBC cells, but not Luminal A cells, and (3) is associated with a poor prognosis in TNBC breast cancer patients. Taken together, our results validate our enhancer identification pipeline and reveal that enhancers transcribed in breast cancer cells direct critical gene regulatory networks that promote pathogenesis. © 2018 Franco et al.; Published by Cold Spring Harbor Laboratory Press.

  16. Detection of Pol IV/RDR2-dependent transcripts at the genomic scale in Arabidopsis reveals features and regulation of siRNA biogenesis

    Science.gov (United States)

    Li, Shaofang; Vandivier, Lee E.; Tu, Bin; Gao, Lei; Won, So Youn; Li, Shengben; Zheng, Binglian; Gregory, Brian D.

    2015-01-01

    Twenty-four-nucleotide small interfering (si)RNAs are central players in RNA-directed DNA methylation (RdDM), a process that establishes and maintains DNA methylation at transposable elements to ensure genome stability in plants. The plant-specific RNA polymerase IV (Pol IV) is required for siRNA biogenesis and is believed to transcribe RdDM loci to produce primary transcripts that are converted to double-stranded RNAs (dsRNAs) by RDR2 to serve as siRNA precursors. Yet, no such siRNA precursor transcripts have ever been reported. Here, through genome-wide profiling of RNAs in genotypes that compromise the processing of siRNA precursors, we were able to identify Pol IV/RDR2-dependent transcripts from tens of thousands of loci. We show that Pol IV/RDR2-dependent transcripts correspond to both DNA strands, whereas the RNA polymerase II (Pol II)-dependent transcripts produced upon derepression of the loci are derived primarily from one strand. We also show that Pol IV/RDR2-dependent transcripts have a 5′ monophosphate, lack a poly(A) tail at the 3′ end, and contain no introns; these features distinguish them from Pol II-dependent transcripts. Like Pol II-transcribed genic regions, Pol IV-transcribed regions are flanked by A/T-rich sequences depleted in nucleosomes, which highlights similarities in Pol II- and Pol IV-mediated transcription. Computational analysis of siRNA abundance from various mutants reveals differences in the regulation of siRNA biogenesis at two types of loci that undergo CHH methylation via two different DNA methyltransferases. These findings begin to reveal features of Pol IV/RDR2-mediated transcription at the heart of genome stability in plants. PMID:25414514

  17. Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia

    DEFF Research Database (Denmark)

    Bjerrum, Jacob T; Nielsen, Ole H; Riis, Lene B

    2014-01-01

    to identify potential biomarkers and transcripts of importance for the carcinogenic behavior of chronic inflammation. METHODS: The Affymetrix GeneChip Human Genome U133 Plus 2.0 was applied on colonic biopsies from UC patients with left-sided UC, pancolitis, dysplasia, and controls. Reverse transcription...... polymerase chain reaction and immunohistochemistry were performed for validating selected transcripts in the initial cohort and in 2 independent cohorts of patients with UC. Microarray data were analyzed by principal component analysis, and reverse transcription polymerase chain reaction...... and immunohistochemistry data by the Wilcoxon's rank-sum test. RESULTS: The principal component analysis results revealed separate clusters for left-sided UC, pancolitis, dysplasia, and controls. Close clustering of dysplastic and pancolitic samples indicated similarities in gene expression. Indeed, 101 and 656 parallel...

  18. Analysis of genomic sequence motifs for deciphering transcription factor binding and transcriptional regulation in eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Valentina eBoeva

    2016-02-01

    Full Text Available Eukaryotic genomes contain a variety of structured patterns: repetitive elements, binding sites of DNA and RNA associated proteins, splice sites and so on. Often, these structured patterns can be formalized as motifs and described using a proper mathematical model such as position weight matrix and IUPAC consensus. Two key tasks are typically carried out for motifs in the context of the analysis of genomic sequences. These are: identification in a set of DNA regions of over-represented motifs from a particular motif database, and de novo discovery of over-represented motifs. Here we describe existing methodology to perform these two tasks for motifs characterizing transcription factor binding. When applied to the output of ChIP-seq and ChIP-exo experiments, or to promoter regions of co-modulated genes, motif analysis techniques allow for the prediction of transcription factor binding events and enable identification of transcriptional regulators and co-regulators. The usefulness of motif analysis is further exemplified in this review by how motif discovery improves peak calling in ChIP-seq and ChIP-exo experiments and, when coupled with information on gene expression, allows insights into physical mechanisms of transcriptional modulation.

  19. Complexity of CNC transcription factors as revealed by gene targeting of the Nrf3 locus.

    Science.gov (United States)

    Derjuga, Anna; Gourley, Tania S; Holm, Teresa M; Heng, Henry H Q; Shivdasani, Ramesh A; Ahmed, Rafi; Andrews, Nancy C; Blank, Volker

    2004-04-01

    Cap'n'collar (CNC) family basic leucine zipper transcription factors play crucial roles in the regulation of mammalian gene expression and development. To determine the in vivo function of the CNC protein Nrf3 (NF-E2-related factor 3), we generated mice deficient in this transcription factor. We performed targeted disruption of two Nrf3 exons coding for CNC homology, basic DNA-binding, and leucine zipper dimerization domains. Nrf3 null mice developed normally and revealed no obvious phenotypic differences compared to wild-type animals. Nrf3(-/-) mice were fertile, and gross anatomy as well as behavior appeared normal. The mice showed normal age progression and did not show any apparent additional phenotype during their life span. We observed no differences in various blood parameters and chemistry values. We infected wild-type and Nrf3(-/-) mice with acute lymphocytic choriomeningitis virus and found no differences in these animals with respect to their number of virus-specific CD8 and CD4 T cells as well as their B-lymphocyte response. To determine whether the mild phenotype of Nrf3 null animals is due to functional redundancy, we generated mice deficient in multiple CNC factors. Contrary to our expectations, an absence of Nrf3 does not seem to cause additional lethality in compound Nrf3(-/-)/Nrf2(-/-) and Nrf3(-/-)/p45(-/-) mice. We hypothesize that the role of Nrf3 in vivo may become apparent only after appropriate challenge to the mice.

  20. Analysis artefacts of the INS-IGF2 fusion transcript

    DEFF Research Database (Denmark)

    Wernersson, Rasmus; Frogne, Thomas; Rescan, Claude

    2015-01-01

    Background: In gene expression analysis, overlapping genes, splice variants, and fusion transcripts are potential sources of data analysis artefacts, depending on how the observed intensity is assigned to one, or more genes. We here exemplify this by an in-depth analysis of the INS-IGF2 fusion...... proteomics analysis we could not demonstrate INS-IGF2 protein in samples of human islets nor in EndoC-βH1. Conclusions: Sequence features, such as fusion transcripts spanning multiple genes can lead to unexpected results in gene expression analysis, and care must be taken in generating and interpreting...... the results. For the specific case of INS-IGF2 we conclude that the abundance of the fusion transcript/protein is exceedingly lower than previously reported, and that current immuno-reagents available for detecting INS-IGF2 protein have a strong cross-reaction to native human proinsulin. Finally, we were...

  1. Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum.

    Directory of Open Access Journals (Sweden)

    Natalie J Garton

    2008-04-01

    Full Text Available Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis.Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body-positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body-positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R(2 = 0.64, p < 0.03 with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population.As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification

  2. Transcriptional responses of invasive and indigenous whiteflies to different host plants reveal their disparate capacity of adaptation

    Science.gov (United States)

    Xu, Hong-Xing; Hong, Yue; Zhang, Min-Zhu; Wang, Yong-Liang; Liu, Shu-Sheng; Wang, Xiao-Wei

    2015-01-01

    The whitefly Bemisia tabaci contains more than 35 cryptic species. The higher adaptability of Middle East-Asia Minor 1 (MEAM1) cryptic species has been recognized as one important factor for its invasion and displacement of other indigenous species worldwide. Here we compared the performance of the invasive MEAM1 and the indigenous Asia II 3 whitefly species following host plant transfer from a suitable host (cotton) to an unsuitable host (tobacco) and analyzed their transcriptional responses. After transfer to tobacco for 24 h, MEAM1 performed much better than Asia II 3. Transcriptional analysis showed that the patterns of gene regulation were very different with most of the genes up-regulated in MEAM1 but down-regulated in Asia II 3. Whereas carbohydrate and energy metabolisms were repressed in Asia II 3, the gene expression and protein metabolisms were activated in MEAM1. Compared to the constitutive high expression of detoxification genes in MEAM1, most of the detoxification genes were down-regulated in Asia II 3. Enzymatic activities of P450, GST and esterase further verified that the detoxification of MEAM1 was much higher than that of Asia II 3. These results reveal obvious differences in responses of MEAM1 and Asia II 3 to host transfer. PMID:26041313

  3. Transcriptional response of zebrafish embryos exposed to neurotoxic compounds reveals a muscle activity dependent hspb11 expression.

    Directory of Open Access Journals (Sweden)

    Nils Klüver

    Full Text Available Acetylcholinesterase (AChE inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM, in zebrafish embryos and compared the response with two non-AChE inhibiting unspecific control compounds, 1,4-dimethoxybenzene (DMB and 2,4-dinitrophenol (DNP. A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive induced gene in response to AChE inhibitors. Comparison of expression in wildtype, ache and sop(fixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds.

  4. RNA-SEQ reveals transcriptional level changes of poplar roots in different forms of nitrogen treatments

    Directory of Open Access Journals (Sweden)

    Chunpu eQu

    2016-02-01

    Full Text Available Poplar has emerged as a model plant for understanding molecular mechanisms of tree growth, development and response to environment. Long-term application of different forms of nitrogen (such as NO3--N and NH4+-N may cause morphological changes of poplar roots; however, the molecular level changes are still not well known. In this study, we analyzed the expression profiling of poplar roots treated by three forms of nitrogen: S1 (NH4+, S2 (NH4NO3 and S3 (NO3- by using RNA-SEQ technique. We found 463 genes significantly differentially expressed in roots by different N treatments, of which a total of 116 genes were found to differentially express between S1 and S2, 173 genes between S2 and S3, and 327 genes between S1 and S3. A cluster analysis shows significant difference in many transcription factor families and functional genes family under different N forms. Through an analysis of Mapman metabolic pathway, we found that the significantly differentially expressed genes are associated with fermentation, glycolysis and tricarboxylic acid cycle (TCA, secondary metabolism, hormone metabolism, and transport processing. Interestingly, we did not find significantly differentially expressed genes in N metabolism pathway, mitochondrial electron transport / ATP synthesis and mineral nutrition. We also found abundant candidate genes (20 transcription factors and 30 functional genes regulating morphology changes of poplar roots under the three N forms. The results obtained are beneficial to a better understanding of the potential molecular and cellular mechanisms regulating root morphology changes under different N treatments.

  5. Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia.

    Science.gov (United States)

    Bjerrum, Jacob T; Nielsen, Ole H; Riis, Lene B; Pittet, Valerie; Mueller, Christoph; Rogler, Gerhard; Olsen, Jørgen

    2014-12-01

    It is unknown why patients with extensive ulcerative colitis (UC) have a higher risk of colorectal cancer compared with patients with left-sided UC. This study characterizes the inflammatory processes in left-sided UC, pancolitis, and UC-associated dysplasia at the transcriptional level to identify potential biomarkers and transcripts of importance for the carcinogenic behavior of chronic inflammation. The Affymetrix GeneChip Human Genome U133 Plus 2.0 was applied on colonic biopsies from UC patients with left-sided UC, pancolitis, dysplasia, and controls. Reverse transcription polymerase chain reaction and immunohistochemistry were performed for validating selected transcripts in the initial cohort and in 2 independent cohorts of patients with UC. Microarray data were analyzed by principal component analysis, and reverse transcription polymerase chain reaction and immunohistochemistry data by the Wilcoxon's rank-sum test. The principal component analysis results revealed separate clusters for left-sided UC, pancolitis, dysplasia, and controls. Close clustering of dysplastic and pancolitic samples indicated similarities in gene expression. Indeed, 101 and 656 parallel upregulated and downregulated transcripts, respectively, were identified in specimens from dysplasia and pancolitis. Validation of selected transcripts hereof identified insulin receptor alpha (INSRA) and MAP kinase interacting serine/threonine kinase 2 (MKNK2) with an enhanced expression in dysplasia compared with left-sided UC and controls, whereas laminin γ2 (LAMC2) was found with a lower expression in dysplasia compared with the remaining 3 groups. This study demonstrates pancolitis and left-sided UC as distinct inflammatory processes at the transcriptional level, and identifies INSRA, MKNK2, and LAMC2 as potential critical transcripts in the inflammation-driven preneoplastic process of UC.

  6. Epigenetic landscapes reveal transcription factors that regulate CD8+ T cell differentiation.

    Science.gov (United States)

    Yu, Bingfei; Zhang, Kai; Milner, J Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P; Pereira, Renata M; Crotty, Shane; Chang, John T; Pipkin, Matthew E; Wang, Wei; Goldrath, Ananda W

    2017-05-01

    Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8 + T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8 + T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8 + T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8 + T cell differentiation.

  7. Epigenetic landscapes reveal transcription factors regulating CD8+ T cell differentiation

    Science.gov (United States)

    Yu, Bingfei; Zhang, Kai; Milner, J. Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P.; Pereira, Renata M.; Crotty, Shane; Chang, John T.; Pipkin, Matthew E.; Wang, Wei; Goldrath, Ananda W.

    2017-01-01

    Dynamic changes in the expression of transcription factors (TFs) can influence specification of distinct CD8+ T cell fates, but the observation of equivalent expression of TF among differentially-fated precursor cells suggests additional underlying mechanisms. Here, we profiled genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8+ T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that TF expression and binding contributed to establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal novel TFs influencing the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8+ T cell differentiation, regulated the formation of terminal-effector and memory-precursor cell-fates, respectively. Our data define the epigenetic landscape of differentiation intermediates, facilitating identification of TFs with previously unappreciated roles in CD8+ T cell differentiation. PMID:28288100

  8. A strong anti-inflammatory signature revealed by liver transcription profiling of Tmprss6-/- mice.

    Directory of Open Access Journals (Sweden)

    Michela Riba

    Full Text Available Control of systemic iron homeostasis is interconnected with the inflammatory response through the key iron regulator, the antimicrobial peptide hepcidin. We have previously shown that mice with iron deficiency anemia (IDA-low hepcidin show a pro-inflammatory response that is blunted in iron deficient-high hepcidin Tmprss6 KO mice. The transcriptional response associated with chronic hepcidin overexpression due to genetic inactivation of Tmprss6 is unknown. By using whole genome transcription profiling of the liver and analysis of spleen immune-related genes we identified several functional pathways differentially expressed in Tmprss6 KO mice, compared to IDA animals and thus irrespective of the iron status. In the effort of defining genes potentially targets of Tmprss6 we analyzed liver gene expression changes according to the genotype and independently of treatment. Tmprss6 inactivation causes down-regulation of liver pathways connected to immune and inflammatory response as well as spleen genes related to macrophage activation and inflammatory cytokines production. The anti-inflammatory status of Tmprss6 KO animals was confirmed by the down-regulation of pathways related to immunity, stress response and intracellular signaling in both liver and spleen after LPS treatment. Opposite to Tmprss6 KO mice, Hfe(-/- mice are characterized by iron overload with inappropriately low hepcidin levels. Liver expression profiling of Hfe(-/- deficient versus iron loaded mice show the opposite expression of some of the genes modulated by the loss of Tmprss6. Altogether our results confirm the anti-inflammatory status of Tmprss6 KO mice and identify new potential target pathways/genes of Tmprss6.

  9. Genomewide analysis of TCP transcription factor gene family in ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 93; Issue 3. Genomewide ... Teosinte branched1/cycloidea/proliferating cell factor1 (TCP) proteins are a large family of transcriptional regulators in angiosperms. They are ... To the best of our knowledge, this is the first study of a genomewide analysis of apple TCP gene family.

  10. In silico comparative genomic analysis of GABAA receptor transcriptional regulation

    Directory of Open Access Journals (Sweden)

    Joyce Christopher J

    2007-06-01

    Full Text Available Abstract Background Subtypes of the GABAA receptor subunit exhibit diverse temporal and spatial expression patterns. In silico comparative analysis was used to predict transcriptional regulatory features in individual mammalian GABAA receptor subunit genes, and to identify potential transcriptional regulatory components involved in the coordinate regulation of the GABAA receptor gene clusters. Results Previously unreported putative promoters were identified for the β2, γ1, γ3, ε, θ and π subunit genes. Putative core elements and proximal transcriptional factors were identified within these predicted promoters, and within the experimentally determined promoters of other subunit genes. Conserved intergenic regions of sequence in the mammalian GABAA receptor gene cluster comprising the α1, β2, γ2 and α6 subunits were identified as potential long range transcriptional regulatory components involved in the coordinate regulation of these genes. A region of predicted DNase I hypersensitive sites within the cluster may contain transcriptional regulatory features coordinating gene expression. A novel model is proposed for the coordinate control of the gene cluster and parallel expression of the α1 and β2 subunits, based upon the selective action of putative Scaffold/Matrix Attachment Regions (S/MARs. Conclusion The putative regulatory features identified by genomic analysis of GABAA receptor genes were substantiated by cross-species comparative analysis and now require experimental verification. The proposed model for the coordinate regulation of genes in the cluster accounts for the head-to-head orientation and parallel expression of the α1 and β2 subunit genes, and for the disruption of transcription caused by insertion of a neomycin gene in the close vicinity of the α6 gene, which is proximal to a putative critical S/MAR.

  11. Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA-seq

    Science.gov (United States)

    Kakaradov, Boyko; Arsenio, Janilyn; Widjaja, Christella E.; He, Zhaoren; Aigner, Stefan; Metz, Patrick J.; Yu, Bingfei; Wehrens, Ellen J.; Lopez, Justine; Kim, Stephanie H.; Zuniga, Elina I.; Goldrath, Ananda W.; Chang, John T.; Yeo, Gene W.

    2017-01-01

    SUMMARY During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants controlling CD8+ T lymphocyte fate specification. These findings suggest a model of terminal effector cell differentiation initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, highlighting the power and necessity of single-cell approaches. PMID:28218746

  12. Monosomic analysis reveals duplicated chromosomal segments in ...

    Indian Academy of Sciences (India)

    Monosomic analysis reveals duplicated chromosomal segments in maize genome. MAHESH C. YADAV1,2∗, J. K. S. ... cated chromosomal segments in maize genome. Materials and methods. Development and .... each in chromosomes 2 and 7, while 10 other pairs of du- plicate loci had one copy in chromosome 3 and the ...

  13. Analysis of Phonetic Transcriptions for Danish Automatic Speech Recognition

    DEFF Research Database (Denmark)

    Kirkedal, Andreas Søeborg

    2013-01-01

    Automatic speech recognition (ASR) relies on three resources: audio, orthographic transcriptions and a pronunciation dictionary. The dictionary or lexicon maps orthographic words to sequences of phones or phonemes that represent the pronunciation of the corresponding word. The quality of a speech...... recognition system depends heavily on the dictionary and the transcriptions therein. This paper presents an analysis of phonetic/phonemic features that are salient for current Danish ASR systems. This preliminary study consists of a series of experiments using an ASR system trained on the DK-PAROLE corpus...

  14. Large-scale transcriptome data reveals transcriptional activity of fission yeast LTR retrotransposons

    DEFF Research Database (Denmark)

    Mourier, Tobias; Willerslev, Eske

    2010-01-01

    makes it difficult to assess which elements are transcriptionally active, but data strongly indicates that only a subset of the LTR retrotransposons contribute significantly to the detected transcription. A considerable level of reverse strand transcription is also detected. Equal levels......BACKGROUND: Retrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. The numbers of retrotransposons within genomes and differences between closely related species may yield insight into the evolutionary history......-requisite for retrotransposition is transcription of the elements. Given their intrinsic sequence redundancy, transcriptome-level analyses of transposable elements are scarce. We have used recently published transcriptome data from the fission yeast Schizosaccharomyces pombe to assess the ability to detect and describe...

  15. Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene

    Directory of Open Access Journals (Sweden)

    Jeong-Woong Park

    2017-10-01

    Full Text Available Objective Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase (AXL gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form in equine skeletal muscle to gain insight(s into the role of each alternative transcript during exercise. Methods We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR, and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR. Results Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3 and immunoglobin (Ig domain was different between two alternative isoforms. Conclusion It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an NF-κB signaling pathway. Further study is necessary to uncover biological function(s and significance of the alternative splicing isoforms in race horse skeletal muscle.

  16. Pan-Cancer Mutational and Transcriptional Analysis of the Integrator Complex

    Directory of Open Access Journals (Sweden)

    Antonio Federico

    2017-04-01

    Full Text Available The integrator complex has been recently identified as a key regulator of RNA Polymerase II-mediated transcription, with many functions including the processing of small nuclear RNAs, the pause-release and elongation of polymerase during the transcription of protein coding genes, and the biogenesis of enhancer derived transcripts. Moreover, some of its components also play a role in genome maintenance. Thus, it is reasonable to hypothesize that their functional impairment or altered expression can contribute to malignancies. Indeed, several studies have described the mutations or transcriptional alteration of some Integrator genes in different cancers. Here, to draw a comprehensive pan-cancer picture of the genomic and transcriptomic alterations for the members of the complex, we reanalyzed public data from The Cancer Genome Atlas. Somatic mutations affecting Integrator subunit genes and their transcriptional profiles have been investigated in about 11,000 patients and 31 tumor types. A general heterogeneity in the mutation frequencies was observed, mostly depending on tumor type. Despite the fact that we could not establish them as cancer drivers, INTS7 and INTS8 genes were highly mutated in specific cancers. A transcriptome analysis of paired (normal and tumor samples revealed that the transcription of INTS7, INTS8, and INTS13 is significantly altered in several cancers. Experimental validation performed on primary tumors confirmed these findings.

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

    Science.gov (United States)

    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...

  18. Transcript profiling of crown rootless1 mutant stem base reveals new elements associated with crown root development in rice

    Directory of Open Access Journals (Sweden)

    Van Anh Le Thi

    2011-08-01

    Full Text Available Abstract Background In rice, the major part of the post-embryonic root system is made of stem-derived roots named crown roots (CR. Among the few characterized rice mutants affected in root development, crown rootless1 mutant is unable to initiate crown root primordia. CROWN ROOTLESS1 (CRL1 is induced by auxin and encodes an AS2/LOB-domain transcription factor that acts upstream of the gene regulatory network controlling CR development. Results To identify genes involved in CR development, we compared global gene expression profile in stem bases of crl1 mutant and wild-type (WT plants. Our analysis revealed that 250 and 236 genes are down- and up-regulated respectively in the crl1 mutant. Auxin induces CRL1 expression and consequently it is expected that auxin also alters the expression of genes that are early regulated by CRL1. To identify genes under the early control of CRL1, we monitored the expression kinetics of a selected subset of genes, mainly chosen among those exhibiting differential expression, in crl1 and WT following exogenous auxin treatment. This analysis revealed that most of these genes, mainly related to hormone, water and nutrient, development and homeostasis, were likely not regulated directly by CRL1. We hypothesized that the differential expression for these genes observed in the crl1 mutant is likely a consequence of the absence of CR formation. Otherwise, three CRL1-dependent auxin-responsive genes: FSM (FLATENNED SHOOT MERISTEM/FAS1 (FASCIATA1, GTE4 (GENERAL TRANSCRIPTION FACTOR GROUP E4 and MAP (MICROTUBULE-ASSOCIATED PROTEIN were identified. FSM/FAS1 and GTE4 are known in rice and Arabidopsis to be involved in the maintenance of root meristem through chromatin remodelling and cell cycle regulation respectively. Conclusion Our data showed that the differential regulation of most genes in crl1 versus WT may be an indirect consequence of CRL1 inactivation resulting from the absence of CR in the crl1 mutant. Nevertheless

  19. A compendium of antibiotic-induced transcription profiles reveals broad regulation of Pasteurella multocida virulence genes.

    Science.gov (United States)

    Melnikow, E; Schoenfeld, C; Spehr, V; Warrass, R; Gunkel, N; Duszenko, M; Selzer, P M; Ullrich, H J

    2008-10-15

    The transcriptional responses of Pasteurella multocida to eight antibiotics with known mode of actions (MoAs) and one novel antibiotic compound with an unknown MoA were collected to create a compendium of transcriptional profiles for MoA studies. At minimal inhibitory concentration the three bactericidal compounds enrofloxacin, cefquinome and the novel compound had a minor impact on gene regulation with approximately 1% of the P. multocida genome affected, whilst the bacteriostatic compounds florfenicol, tilmicosin, rifampin, trimethoprim and brodimoprim regulated 20% of the genome. Novobiocin was special in that it regulated 40% of all P. multocida genes. Regulation of target genes was observed for novobiocin, rifampin, florfenicol and tilmicosin and signature genes were identified for most antibiotics. The transcriptional profile induced by the novel compound was unrelated to the compendium profiles suggesting a new MoA. The transcription of many P. multocida virulence factors, particularly genes involved in capsule synthesis and export, LPS synthesis, competence, adherence and iron transport were altered in the presence of antibiotics. Virulence gene transcription was mainly negatively affected, however the opposite effect was also observed in the case of rifampin where the up-regulation of the tad locus involved in tight adherence was seen. Novobiocin and trimethoprim caused a marked reduction in the transcription of capsule genes, which correlated with a concomitant reduction of the capsular layer on the surface of P. multocida. The broad negative impact on virulence gene transcription supports the notion that the therapeutic effect of some antibiotics could be a combination of growth and virulence inhibition.

  20. Genetic and molecular analyses of PEG10 reveal new aspects of genomic organization, transcription and translation.

    Directory of Open Access Journals (Sweden)

    Heike Lux

    Full Text Available The paternally expressed gene PEG10 is a retrotransposon derived gene adapted through mammalian evolution located on human chromosome 7q21. PEG10 codes for at least two proteins, PEG10-RF1 and PEG10-RF1/2, by -1 frameshift translation. Overexpression or reinduced PEG10 expression was seen in malignancies, like hepatocellular carcinoma or B-cell acute and chronic lymphocytic leukemia. PEG10 was also shown to promote adipocyte differentiation. Experimental evidence suggests that the PEG10-RF1 protein is an inhibitor of apoptosis and mediates cell proliferation. Here we present new data on the genomic organization of PEG10 by identifying the major transcription start site, a new splice variant and report the cloning and analysis of 1.9 kb of the PEG10 promoter. Furthermore, we show for the first time that PEG10 translation is initiated at a non-AUG start codon upstream of the previously predicted AUG codon as well as at the AUG codon. The finding that PEG10 translation is initiated at different sides adds a new aspect to the already interesting feature of PEG10's -1 frameshift translation mechanism. It is now important to unravel the cellular functions of the PEG10 protein variants and how they are related to normal or pathological conditions. The generated promoter-reporter constructs can be used for future studies to investigate how PEG10 expression is regulated. In summary, our study provides new data on the genomic organization as well as expression and translation of PEG10, a prerequisite in order to study and understand the role of PEG10 in cancer, embryonic development and normal cell homeostasis.

  1. Evolutionary Analysis of DELLA-Associated Transcriptional Networks

    Directory of Open Access Journals (Sweden)

    Miguel A. Blázquez

    2017-04-01

    Full Text Available DELLA proteins are transcriptional regulators present in all land plants which have been shown to modulate the activity of over 100 transcription factors in Arabidopsis, involved in multiple physiological and developmental processes. It has been proposed that DELLAs transduce environmental information to pre-wired transcriptional circuits because their stability is regulated by gibberellins (GAs, whose homeostasis largely depends on environmental signals. The ability of GAs to promote DELLA degradation coincides with the origin of vascular plants, but the presence of DELLAs in other land plants poses at least two questions: what regulatory properties have DELLAs provided to the behavior of transcriptional networks in land plants, and how has the recruitment of DELLAs by GA signaling affected this regulation. To address these issues, we have constructed gene co-expression networks of four different organisms within the green lineage with different properties regarding DELLAs: Arabidopsis thaliana and Solanum lycopersicum (both with GA-regulated DELLA proteins, Physcomitrella patens (with GA-independent DELLA proteins and Chlamydomonas reinhardtii (a green alga without DELLA, and we have examined the relative evolution of the subnetworks containing the potential DELLA-dependent transcriptomes. Network analysis indicates a relative increase in parameters associated with the degree of interconnectivity in the DELLA-associated subnetworks of land plants, with a stronger effect in species with GA-regulated DELLA proteins. These results suggest that DELLAs may have played a role in the coordination of multiple transcriptional programs along evolution, and the function of DELLAs as regulatory ‘hubs’ became further consolidated after their recruitment by GA signaling in higher plants.

  2. Deep RNA sequencing reveals hidden features and dynamics of early gene transcription in Paramecium bursaria chlorella virus 1.

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    Guillaume Blanc

    Full Text Available Paramecium bursaria chlorella virus 1 (PBCV-1 is the prototype of the genus Chlorovirus (family Phycodnaviridae that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i., transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41% or the poly (A+-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i. was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most of these excision events have no functional role but are probably the result of the activity of misled splicesomes.

  3. RNA Sequencing of Murine Norovirus-Infected Cells Reveals Transcriptional Alteration of Genes Important to Viral Recognition and Antigen Presentation

    Directory of Open Access Journals (Sweden)

    Daniel Enosi Tuipulotu

    2017-08-01

    Full Text Available Viruses inherently exploit normal cellular functions to promote replication and survival. One mechanism involves transcriptional control of the host, and knowledge of the genes modified and their molecular function can aid in understanding viral-host interactions. Norovirus pathogenesis, despite the recent advances in cell cultivation, remains largely uncharacterized. Several studies have utilized the related murine norovirus (MNV to identify innate response, antigen presentation, and cellular recognition components that are activated during infection. In this study, we have used next-generation sequencing to probe the transcriptomic changes of MNV-infected mouse macrophages. Our in-depth analysis has revealed that MNV is a potent stimulator of the innate response including genes involved in interferon and cytokine production pathways. We observed that genes involved in viral recognition, namely IFIH1, DDX58, and DHX58 were significantly upregulated with infection, whereas we observed significant downregulation of cytokine receptors (Il17rc, Il1rl1, Cxcr3, and Cxcr5 and TLR7. Furthermore, we identified that pathways involved in protein degradation (including genes Psmb3, Psmb4, Psmb5, Psmb9, and Psme2, antigen presentation, and lymphocyte activation are downregulated by MNV infection. Thus, our findings illustrate that MNV induces perturbations in the innate immune transcriptome, particularly in MHC maturation and viral recognition that can contribute to disease pathogenesis.

  4. Host cell transcriptional profiling during malaria liver stage infection reveals a coordinated and sequential set of biological events.

    Science.gov (United States)

    Albuquerque, Sónia S; Carret, Céline; Grosso, Ana Rita; Tarun, Alice S; Peng, Xinxia; Kappe, Stefan H I; Prudêncio, Miguel; Mota, Maria M

    2009-06-17

    Plasmodium sporozoites migrate to the liver where they traverse several hepatocytes before invading the one inside which they will develop and multiply into thousands of merozoites. Although this constitutes an essential step of malaria infection, the requirements of Plasmodium parasites in liver cells and how they use the host cell for their own survival and development are poorly understood. To gain new insights into the molecular host-parasite interactions that take place during malaria liver infection, we have used high-throughput microarray technology to determine the transcriptional profile of P. berghei-infected hepatoma cells. The data analysis shows differential expression patterns for 1064 host genes starting at 6 h and up to 24 h post infection, with the largest proportion correlating specifically with the early stages of the infection process. A considerable proportion of those genes were also found to be modulated in liver cells collected from P. yoelii-infected mice 24 and 40 h after infection, strengthening the data obtained with the in vitro model and highlighting genes and pathways involved in the host response to rodent Plasmodium parasites. Our data reveal that host cell infection by Plasmodium sporozoites leads to a coordinated and sequential set of biological events, ranging from the initial stage of stress response up to the engagement of host metabolic processes and the maintenance of cell viability throughout infection.

  5. Systematic analysis of transcription start sites in avian development.

    Directory of Open Access Journals (Sweden)

    Marina Lizio

    2017-09-01

    Full Text Available Cap Analysis of Gene Expression (CAGE in combination with single-molecule sequencing technology allows precision mapping of transcription start sites (TSSs and genome-wide capture of promoter activities in differentiated and steady state cell populations. Much less is known about whether TSS profiling can characterize diverse and non-steady state cell populations, such as the approximately 400 transitory and heterogeneous cell types that arise during ontogeny of vertebrate animals. To gain such insight, we used the chick model and performed CAGE-based TSS analysis on embryonic samples covering the full 3-week developmental period. In total, 31,863 robust TSS peaks (>1 tag per million [TPM] were mapped to the latest chicken genome assembly, of which 34% to 46% were active in any given developmental stage. ZENBU, a web-based, open-source platform, was used for interactive data exploration. TSSs of genes critical for lineage differentiation could be precisely mapped and their activities tracked throughout development, suggesting that non-steady state and heterogeneous cell populations are amenable to CAGE-based transcriptional analysis. Our study also uncovered a large set of extremely stable housekeeping TSSs and many novel stage-specific ones. We furthermore demonstrated that TSS mapping could expedite motif-based promoter analysis for regulatory modules associated with stage-specific and housekeeping genes. Finally, using Brachyury as an example, we provide evidence that precise TSS mapping in combination with Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR-on technology enables us, for the first time, to efficiently target endogenous avian genes for transcriptional activation. Taken together, our results represent the first report of genome-wide TSS mapping in birds and the first systematic developmental TSS analysis in any amniote species (birds and mammals. By facilitating promoter-based molecular analysis and genetic

  6. A spatial point pattern analysis in Drosophila blastoderm embryos evaluating the potential inheritance of transcriptional states.

    Directory of Open Access Journals (Sweden)

    Feng He

    Full Text Available The Drosophila blastoderm embryo undergoes rapid cycles of nuclear division. This poses a challenge to genes that need to reliably sense the concentrations of morphogen molecules to form desired expression patterns. Here we investigate whether the transcriptional state of hunchback (hb, a target gene directly activated by the morphogenetic protein Bicoid (Bcd, exhibits properties indicative of inheritance between mitotic cycles. To achieve this, we build a dataset of hb transcriptional states at the resolution of individual nuclei in embryos at early cycle 14. We perform a spatial point pattern (SPP analysis to evaluate the spatial relationships among the nuclei that have distinct numbers of hb gene copies undergoing active transcription in snapshots of embryos. Our statistical tests and simulation studies reveal properties of dispersed clustering for nuclei with both or neither copies of hb undergoing active transcription. Modeling of nuclear lineages from cycle 11 to cycle 14 suggests that these two types of nuclei can achieve spatial clustering when, and only when, the transcriptional states are allowed to propagate between mitotic cycles. Our results are consistent with the possibility where the positional information encoded by the Bcd morphogen gradient may not need to be decoded de novo at all mitotic cycles in the Drosophila blastoderm embryo.

  7. Physiological, biochemical and transcriptional analysis of onion bulbs during storage

    Science.gov (United States)

    Chope, Gemma A.; Cools, Katherine; Hammond, John P.; Thompson, Andrew J.; Terry, Leon A.

    2012-01-01

    Background and Aims During the transition from endo-dormancy to eco-dormancy and subsequent growth, the onion bulb undergoes the transition from sink organ to source, to sustain cell division in the meristematic tissue. The mechanisms controlling these processes are not fully understood. Here, a detailed analysis of whole onion bulb physiological, biochemical and transcriptional changes in response to sprouting is reported, enabling a better knowledge of the mechanisms regulating post-harvest onion sprout development. Methods Biochemical and physiological analyses were conducted on different cultivars (‘Wellington’, ‘Sherpa’ and ‘Red Baron’) grown at different sites over 3 years, cured at different temperatures (20, 24 and 28 °C) and stored under different regimes (1, 3, 6 and 6 → 1 °C). In addition, the first onion oligonucleotide microarray was developed to determine differential gene expression in onion during curing and storage, so that transcriptional changes could support biochemical and physiological analyses. Key Results There were greater transcriptional differences between samples at harvest and before sprouting than between the samples taken before and after sprouting, with some significant changes occurring during the relatively short curing period. These changes are likely to represent the transition from endo-dormancy to sprout suppression, and suggest that endo-dormancy is a relatively short period ending just after curing. Principal component analysis of biochemical and physiological data identified the ratio of monosaccharides (fructose and glucose) to disaccharide (sucrose), along with the concentration of zeatin riboside, as important factors in discriminating between sprouting and pre-sprouting bulbs. Conclusions These detailed analyses provide novel insights into key regulatory triggers for sprout dormancy release in onion bulbs and provide the potential for the development of biochemical or transcriptional markers for sprout

  8. Genome-wide transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis reveals suppression of host immune genes

    Directory of Open Access Journals (Sweden)

    Killick Kate E

    2011-12-01

    Full Text Available Abstract Background Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB, a pathological infection with significant economic impact. Recent studies have highlighted the role of functional genomics to better understand the molecular mechanisms governing the host immune response to M. bovis infection. Furthermore, these studies may enable the identification of novel transcriptional markers of BTB that can augment current diagnostic tests and surveillance programmes. In the present study, we have analysed the transcriptome of peripheral blood leukocytes (PBL from eight M. bovis-infected and eight control non-infected age-matched and sex-matched Holstein-Friesian cattle using the Affymetrix® GeneChip® Bovine Genome Array with 24,072 gene probe sets representing more than 23,000 gene transcripts. Results Control and infected animals had similar mean white blood cell counts. However, the mean number of lymphocytes was significantly increased in the infected group relative to the control group (P = 0.001, while the mean number of monocytes was significantly decreased in the BTB group (P = 0.002. Hierarchical clustering analysis using gene expression data from all 5,388 detectable mRNA transcripts unambiguously partitioned the animals according to their disease status. In total, 2,960 gene transcripts were differentially expressed (DE between the infected and control animal groups (adjusted P-value threshold ≤ 0.05; with the number of gene transcripts showing decreased relative expression (1,563 exceeding those displaying increased relative expression (1,397. Systems analysis using the Ingenuity® Systems Pathway Analysis (IPA Knowledge Base revealed an over-representation of DE genes involved in the immune response functional category. More specifically, 64.5% of genes in the affects immune response subcategory displayed decreased relative expression levels in the infected animals compared to the control group. Conclusions This

  9. Transcription profile of boar spermatozoa as revealed by RNA-sequencing

    Science.gov (United States)

    High-throughput RNA sequencing (RNA-Seq) overcomes the limitations of the current hybridization-based techniques to detect the actual pool of RNA transcripts in spermatozoa. The application of this technology in livestock can speed the discovery of potential predictors of male fertility. As a first ...

  10. The volumes and transcript counts of single cells reveal concentration homeostasis and capture biological noise.

    NARCIS (Netherlands)

    Kempe, H.; Schwabe, A.; Crémazy, F.; Verschure, P.J.; Bruggeman, F.J.

    2015-01-01

    Transcriptional stochasticity can be measured by counting the number of mRNA molecules per cell. Cell-to-cell variability is best captured in terms of concentration rather than molecule counts, because reaction rates depend on concentrations. We combined single-molecule mRNA counting with

  11. CprK crystal structures reveal mechanism for transcriptional control of halorespiration

    NARCIS (Netherlands)

    Joyce, M.G.; Levy, C.; Gabor, K.; Pop, S.M.; Biehl, B.D.; Doukov, T.I.; Ryter, J.M.; Mazon, H.; Smidt, H.; Heuvel, van den R.H.H.; Ragsdale, S.W.; Oost, van der J.; Leys, D.

    2006-01-01

    Halorespiration is a bacterial respiratory process in which haloorganic compounds act as terminal electron acceptors. This process is controlled at transcriptional level by CprK, a member of the ubiquitous CRP-FNR family. Here we present the crystal structures of oxidized CprK in presence of the

  12. Development of a rapid and inexpensive method to reveal natural antisense transcripts

    Directory of Open Access Journals (Sweden)

    Collani Silvio

    2012-09-01

    Full Text Available Abstract Background Natural antisense transcripts (NATs are a group of RNAs encoded within a cell that have transcript complementarity to other RNA transcripts. NATs have been identified in multiple eukaryotes, including humans, mice, yeast and several plants, and are known to play crucial roles in gene regulation and modification via RNA interference, alternative splicing and genomic imprinting. NATs are also involved in several human diseases. Results We describe a novel method to detect the occurrence of target NATs in specific plant tissues. This method differs from the others currently used in molecular biology laboratories for a number of reasons, particularly the simplicity and versatility of application, low cost and lower material requirement. We demonstrate that NATs can be detected by using diluted cDNA, avoiding the need for a large amount of RNA, thus differing from basic techniques, such as Northern blot hybridisation and reverse-transcription PCR amplification. Furthermore, our method also allows the precise detection of long NATs and their cloning into plasmid vectors for downstream applications. We also reported the first case of a tissue-specific NAT occurring in Oleaceae family and, the antisense orientation of this transcript, allows the splicing of two introns otherwise impossible in the sense orientation. Conclusions This method is the first that combines the polymerisation and cleavage activity of DNA polymerase and exonuclease enzymes, respectively, to discover NATs in living organisms. It may simplify the discovery of NATs in plants providing a new strategy for an easy identification and characterization of this group of RNA molecules. Furthermore, since NATs are found in multiple eukaryotes, our method can be easily applied to a wide range of organisms, including human, mice and yeast.

  13. Untangling the Effect of Fatty Acid Addition at Species Level Revealed Different Transcriptional Responses of the Biogas Microbial Community Members

    DEFF Research Database (Denmark)

    Treu, Laura; Campanaro, Stefano; Kougias, Panagiotis

    2016-01-01

    In the present study, RNA-sequencing was used to elucidate the change of anaerobic digestion metatranscriptome after long chain fatty acids (oleate) exposure. To explore the general transcriptional behavior of the microbiome, the analysis was first performed on shotgun reads without considering a...

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

    Directory of Open Access Journals (Sweden)

    Andreas Tuerk

    2017-05-01

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

  15. Single-Cell RNA-Seq Reveals Transcriptional Heterogeneity in Latent and Reactivated HIV-Infected Cells.

    Science.gov (United States)

    Golumbeanu, Monica; Cristinelli, Sara; Rato, Sylvie; Munoz, Miguel; Cavassini, Matthias; Beerenwinkel, Niko; Ciuffi, Angela

    2018-04-24

    Despite effective treatment, HIV can persist in latent reservoirs, which represent a major obstacle toward HIV eradication. Targeting and reactivating latent cells is challenging due to the heterogeneous nature of HIV-infected cells. Here, we used a primary model of HIV latency and single-cell RNA sequencing to characterize transcriptional heterogeneity during HIV latency and reactivation. Our analysis identified transcriptional programs leading to successful reactivation of HIV expression. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Molecular insights of the first gastropod TLR counterpart from disk abalone (Haliotis discus discus), revealing its transcriptional modulation under pathogenic stress.

    Science.gov (United States)

    Elvitigala, Don Anushka Sandaruwan; Premachandra, H K A; Whang, Ilson; Nam, Bo-Hye; Lee, Jehee

    2013-08-01

    Toll-like receptors (TLRs) are well-characterized pattern recognition receptors of innate immunity, known to induce immune responses against the pathogens by interacting with evolutionarily conserved pathogen-associated molecular patterns (PAMPs). In this study, a novel TLR homolog from disk abalone (Haliotis discus discus) was identified and characterized at molecular level. The open reading frame (ORF) of AbTLR is 3804 bp in length and encodes a 1268 amino acid peptide with a calculated molecular mass of 143.5 kDa. The deduced protein shows typical TLR domain architecture, with leucine rich repeats (LRR) and the toll-interleukin receptor (TIR) domain. Phylogenetic analysis revealed a close evolutionary relationship for AbTLR to its invertebrate counterparts, with close clustering to the molluscan homologs. Quantitative real-time PCR detected ubiquitous transcription of AbTLR in healthy tissues, but with highest levels in hemocytes. Differential transcriptional modulation of AbTLR was observed in abalone hemocytes and gills upon immune challenge, whereby Vibrio parahaemolyticus and purified lipopolysaccharide (LPS) enhanced the transcript level prominently. In addition, the viral hemorrhagic septicemia virus induced AbTLR transcription in hemocytes and gills, representing the first evidence of viral-induced immune response in mollusks to date. Collectively, our findings support a putative role for AbTLR in abalone antiviral and antibacterial defense. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Pyrosequencing data reveals tissue-specific expression of lineage-specific transcripts in chickpea

    OpenAIRE

    Garg, Rohini; Jain, Mukesh

    2011-01-01

    Chickpea is a very important crop legume plant, which provides a protein-rich supplement to cereal-based diets and has the ability to fix atmospheric nitrogen. Despite its economic importance, the functional genomic resources for chickpea are very limited. Recently, we reported the complete transcriptome of chickpea using next generation sequencing technologies. We analyzed the tissue-specific expression of chickpea transcripts based on RNA-seq data. In addition, we identified two sets of lin...

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

    LENUS (Irish Health Repository)

    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.

  19. Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

    Science.gov (United States)

    Grunseich, Christopher; Wang, Isabel X; Watts, Jason A; Burdick, Joshua T; Guber, Robert D; Zhu, Zhengwei; Bruzel, Alan; Lanman, Tyler; Chen, Kelian; Schindler, Alice B; Edwards, Nancy; Ray-Chaudhury, Abhik; Yao, Jianhua; Lehky, Tanya; Piszczek, Grzegorz; Crain, Barbara; Fischbeck, Kenneth H; Cheung, Vivian G

    2018-02-01

    R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Determining physical constraints in transcriptional initiationcomplexes using DNA sequence analysis

    Energy Technology Data Exchange (ETDEWEB)

    Shultzaberger, Ryan K.; Chiang, Derek Y.; Moses, Alan M.; Eisen,Michael B.

    2007-07-01

    Eukaryotic gene expression is often under the control ofcooperatively acting transcription factors whose binding is limited bystructural constraints. By determining these structural constraints, wecan understand the "rules" that define functional cooperativity.Conversely, by understanding the rules of binding, we can inferstructural characteristics. We have developed an information theory basedmethod for approximating the physical limitations of cooperativeinteractions by comparing sequence analysis to microarray expressiondata. When applied to the coordinated binding of the sulfur amino acidregulatory protein Met4 by Cbf1 and Met31, we were able to create acombinatorial model that can correctly identify Met4 regulatedgenes.

  1. Theoretical analysis of transcription process with polymerase stalling

    Science.gov (United States)

    Li, Jingwei; Zhang, Yunxin

    2015-05-01

    Experimental evidence shows that in gene transcription RNA polymerase has the possibility to be stalled at a certain position of the transcription template. This may be due to the template damage or protein barriers. Once stalled, polymerase may backtrack along the template to the previous nucleotide to wait for the repair of the damaged site, simply bypass the barrier or damaged site and consequently synthesize an incorrect messenger RNA, or degrade and detach from the template. Thus, the effective transcription rate (the rate to synthesize correct product mRNA) and the transcription effectiveness (the ratio of the effective transcription rate to the effective transcription initiation rate) are both influenced by polymerase stalling events. So far, no theoretical model has been given to discuss the gene transcription process including polymerase stalling. In this study, based on the totally asymmetric simple exclusion process, the transcription process including polymerase stalling is analyzed theoretically. The dependence of the effective transcription rate, effective transcription initiation rate, and transcription effectiveness on the transcription initiation rate, termination rate, as well as the backtracking rate, bypass rate, and detachment (degradation) rate when stalling, are discussed in detail. The results showed that backtracking restart after polymerase stalling is an ideal mechanism to increase both the effective transcription rate and the transcription effectiveness. Without backtracking, detachment of stalled polymerase can also help to increase the effective transcription rate and transcription effectiveness. Generally, the increase of the bypass rate of the stalled polymerase will lead to the decrease of the effective transcription rate and transcription effectiveness. However, when both detachment rate and backtracking rate of the stalled polymerase vanish, the effective transcription rate may also be increased by the bypass mechanism.

  2. Transcription Profiles Reveal Sugar and Hormone Signaling Pathways Mediating Flower Induction in Apple (Malus domestica Borkh.).

    Science.gov (United States)

    Xing, Li-Bo; Zhang, Dong; Li, You-Mei; Shen, Ya-Wen; Zhao, Cai-Ping; Ma, Juan-Juan; An, Na; Han, Ming-Yu

    2015-10-01

    Flower induction in apple (Malus domestica Borkh.) is regulated by complex gene networks that involve multiple signal pathways to ensure flower bud formation in the next year, but the molecular determinants of apple flower induction are still unknown. In this research, transcriptomic profiles from differentiating buds allowed us to identify genes potentially involved in signaling pathways that mediate the regulatory mechanisms of flower induction. A hypothetical model for this regulatory mechanism was obtained by analysis of the available transcriptomic data, suggesting that sugar-, hormone- and flowering-related genes, as well as those involved in cell-cycle induction, participated in the apple flower induction process. Sugar levels and metabolism-related gene expression profiles revealed that sucrose is the initiation signal in flower induction. Complex hormone regulatory networks involved in cytokinin (CK), abscisic acid (ABA) and gibberellic acid pathways also induce apple flower formation. CK plays a key role in the regulation of cell formation and differentiation, and in affecting flowering-related gene expression levels during these processes. Meanwhile, ABA levels and ABA-related gene expression levels gradually increased, as did those of sugar metabolism-related genes, in developing buds, indicating that ABA signals regulate apple flower induction by participating in the sugar-mediated flowering pathway. Furthermore, changes in sugar and starch deposition levels in buds can be affected by ABA content and the expression of the genes involved in the ABA signaling pathway. Thus, multiple pathways, which are mainly mediated by crosstalk between sugar and hormone signals, regulate the molecular network involved in bud growth and flower induction in apple trees. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  3. Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

    Energy Technology Data Exchange (ETDEWEB)

    Newberry, K.J.; Huffman, J.L.; Miller, M.C.; Vazquez-Laslop, N.; Neyfakh, A.A.; Brennan, R.G.

    2009-05-22

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  4. Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics.

    Science.gov (United States)

    Brand, Marjorie; Ranish, Jeffrey A; Kummer, Nicolas T; Hamilton, Joan; Igarashi, Kazuhiko; Francastel, Claire; Chi, Tian H; Crabtree, Gerald R; Aebersold, Ruedi; Groudine, Mark

    2004-01-01

    During erythroid differentiation, beta-globin gene expression is regulated by the locus control region (LCR). The transcription factor NF-E2p18/MafK binds within this region and is essential for beta-globin expression in murine erythroleukemia (MEL) cells. Here we use the isotope-coded affinity tag (ICAT) technique of quantitative mass spectrometry to compare proteins interacting with NF-E2p18/MafK during differentiation. Our results define MafK as a 'dual-function' molecule that shifts from a repressive to an activating mode during erythroid differentiation. The exchange of MafK dimerization partner from Bach1 to NF-E2p45 is a key step in the switch from the repressed to the active state. This shift is associated with changes in the interaction of MafK with co-repressors and co-activators. Thus, our results suggest that in addition to its role as a cis-acting activator of beta-globin gene expression in differentiated erythroid cells, the LCR also promotes an active repression of beta-globin transcription in committed cells before terminal differentiation.

  5. Natural Guided Genome Engineering Reveals Transcriptional Regulators Controlling Quorum-Sensing Signal Degradation.

    Directory of Open Access Journals (Sweden)

    Abbas El Sahili

    Full Text Available Quorum-quenching (QQ are natural or engineered processes disrupting the quorum-sensing (QS signalling which controls virulence and persistence (e.g. biofilm in numerous bacteria. QQ involves different enzymes including lactonases, amidases, oxidases and reductases which degrade the QS molecules such as N-acylhomoserine lactones (NAHL. Rhodococcus erythropolis known to efficiently degrade NAHL is proposed as a biocontrol agent and a reservoir of QQ-enzymes for biotechnology. In R. erythropolis, regulation of QQ-enzymes remains unclear. In this work, we performed genome engineering on R. erythropolis, which is recalcitrant to reverse genetics, in order to investigate regulation of QQ-enzymes at a molecular and structural level with the aim to improve the QQ activity. Deep-sequencing of the R. erythropolis enhanced variants allowed identification of a punctual mutation in a key-transcriptional factor QsdR (Quorum sensing degradation Regulation which regulates the sole QQ-lactonase QsdA identified so far. Using biophysical and structural studies on QsdR, we demonstrate that QQ activity can be improved by modifying the regulation of QQ-enzymes degrading QS signal. This modification requiring the change of only one amino-acid in a transcriptional factor leads to an enhanced R. erythropolis in which the QS-signal degradation pathway is strongly activated.

  6. Comprehensive reanalysis of transcription factor knockout expression data in Saccharomyces cerevisiae reveals many new targets.

    Science.gov (United States)

    Reimand, Jüri; Vaquerizas, Juan M; Todd, Annabel E; Vilo, Jaak; Luscombe, Nicholas M

    2010-08-01

    Transcription factor (TF) perturbation experiments give valuable insights into gene regulation. Genome-scale evidence from microarray measurements may be used to identify regulatory interactions between TFs and targets. Recently, Hu and colleagues published a comprehensive study covering 269 TF knockout mutants for the yeast Saccharomyces cerevisiae. However, the information that can be extracted from this valuable dataset is limited by the method employed to process the microarray data. Here, we present a reanalysis of the original data using improved statistical techniques freely available from the BioConductor project. We identify over 100,000 differentially expressed genes-nine times the total reported by Hu et al. We validate the biological significance of these genes by assessing their functions, the occurrence of upstream TF-binding sites, and the prevalence of protein-protein interactions. The reanalysed dataset outperforms the original across all measures, indicating that we have uncovered a vastly expanded list of relevant targets. In summary, this work presents a high-quality reanalysis that maximizes the information contained in the Hu et al. compendium. The dataset is available from ArrayExpress (accession: E-MTAB-109) and it will be invaluable to any scientist interested in the yeast transcriptional regulatory system.

  7. Drosophila JAK/STAT pathway reveals distinct initiation and reinforcement steps in early transcription of Sxl.

    Science.gov (United States)

    Avila, Frank W; Erickson, James W

    2007-04-03

    X-linked signal elements (XSEs) communicate the dose of X chromosomes to the regulatory-switch gene Sex-lethal (Sxl) during Drosophila sex determination. Unequal XSE expression in precellular XX and XY nuclei ensures that only XX embryos will activate the establishment promoter, SxlPe, to produce a pulse of the RNA-binding protein, SXL [1]. Once XSE protein concentrations have been assessed, SxlPe is inactivated and the maintenance promoter, SxlPm, is turned on in both sexes; however, only in females is SXL present to direct the SxlPm-derived transcripts to be spliced into functional mRNA [2, 3]. Thereafter, Sxl is maintained in the on state by positive autoregulatory RNA splicing [2]. Once set in the stable on (female) or off (male) state, Sxl controls somatic sexual development through control of downstream effectors of sexual differentiation and dosage compensation [1, 4]. Most XSEs encode transcription factors that bind SxlPe, but the XSE unpaired (upd) encodes a secreted ligand for the JAK/STAT pathway [5-7]. We show that although STAT directly regulates SxlPe, it is dispensable for promoter activation. Instead, JAK/STAT is needed to maintain high-level SxlPe expression in order to ensure Sxl autoregulation in XX embryos. Thus, upd is a unique XSE that augments, rather than defines, the initial sex-determination signal.

  8. Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types.

    Science.gov (United States)

    Syring, Isabella; Klümper, Niklas; Offermann, Anne; Braun, Martin; Deng, Mario; Boehm, Diana; Queisser, Angela; von Mässenhausen, Anne; Brägelmann, Johannes; Vogel, Wenzel; Schmidt, Doris; Majores, Michael; Schindler, Anne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg; Shaikhibrahim, Zaki; Perner, Sven

    2016-04-26

    The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

  9. MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

    Directory of Open Access Journals (Sweden)

    Varun Kulkarni

    2016-01-01

    Full Text Available MicroRNAs are 18–22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type or resistant (mutant engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut, the reporter gene containing wild type (wt let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex. Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells.

  10. Genomic-wide transcriptional profiling in primary myoblasts reveals Runx1-regulated genes in muscle regeneration

    Directory of Open Access Journals (Sweden)

    Kfir Baruch Umansky

    2015-12-01

    Full Text Available In response to muscle damage the muscle adult stem cells are activated and differentiate into myoblasts that regenerate the damaged tissue. We have recently showed that following myopathic damage the level of the Runx1 transcription factor (TF is elevated and that during muscle regeneration this TF regulates the balance between myoblast proliferation and differentiation (Umansky et al.. We employed Runx1-dependent gene expression, Chromatin Immunoprecipitation sequencing (ChIP-seq, Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq and histone H3K4me1/H3K27ac modification analyses to identify a subset of Runx1-regulated genes that are co-occupied by the TFs MyoD and c-Jun and are involved in muscle regeneration (Umansky et al.. The data is available at the GEO database under the superseries accession number GSE56131.

  11. Comprehensive prediction in 78 human cell lines reveals rigidity and compactness of transcription factor dimers

    Science.gov (United States)

    Jankowski, Aleksander; Szczurek, Ewa; Jauch, Ralf; Tiuryn, Jerzy; Prabhakar, Shyam

    2013-01-01

    The binding of transcription factors (TFs) to their specific motifs in genomic regulatory regions is commonly studied in isolation. However, in order to elucidate the mechanisms of transcriptional regulation, it is essential to determine which TFs bind DNA cooperatively as dimers and to infer the precise nature of these interactions. So far, only a small number of such dimeric complexes are known. Here, we present an algorithm for predicting cell-type–specific TF–TF dimerization on DNA on a large scale, using DNase I hypersensitivity data from 78 human cell lines. We represented the universe of possible TF complexes by their corresponding motif complexes, and analyzed their occurrence at cell-type–specific DNase I hypersensitive sites. Based on ∼1.4 billion tests for motif complex enrichment, we predicted 603 highly significant cell-type–specific TF dimers, the vast majority of which are novel. Our predictions included 76% (19/25) of the known dimeric complexes and showed significant overlap with an experimental database of protein–protein interactions. They were also independently supported by evolutionary conservation, as well as quantitative variation in DNase I digestion patterns. Notably, the known and predicted TF dimers were almost always highly compact and rigidly spaced, suggesting that TFs dimerize in close proximity to their partners, which results in strict constraints on the structure of the DNA-bound complex. Overall, our results indicate that chromatin openness profiles are highly predictive of cell-type–specific TF–TF interactions. Moreover, cooperative TF dimerization seems to be a widespread phenomenon, with multiple TF complexes predicted in most cell types. PMID:23554463

  12. Preliminary crystallographic analysis of a possible transcription factor encoded by the mimivirus L544 gene

    International Nuclear Information System (INIS)

    Ciaccafava, Alexandre; Lartigue, Audrey; Mansuelle, Pascal; Jeudy, Sandra; Abergel, Chantal

    2011-01-01

    The mimivirus L544 gene product was expressed in E. coli and crystallized; preliminary phasing of a MAD data set was performed using the selenium signal present in a crystal of recombinant selenomethionine-substituted protein. Mimivirus is the prototype of a new family (the Mimiviridae) of nucleocytoplasmic large DNA viruses (NCLDVs), which already include the Poxviridae, Iridoviridae, Phycodnaviridae and Asfarviridae. Mimivirus specifically replicates in cells from the genus Acanthamoeba. Proteomic analysis of purified mimivirus particles revealed the presence of many subunits of the DNA-directed RNA polymerase II complex. A fully functional pre-transcriptional complex appears to be loaded in the virions, allowing mimivirus to initiate transcription within the host cytoplasm immediately upon infection independently of the host nuclear apparatus. To fully understand this process, a systematic study of mimivirus proteins that are predicted (by bioinformatics) or suspected (by proteomic analysis) to be involved in transcription was initiated by cloning and expressing them in Escherichia coli in order to determine their three-dimensional structures. Here, preliminary crystallographic analysis of the recombinant L544 protein is reported. The crystals belonged to the orthorhombic space group C222 1 with one monomer per asymmetric unit. A MAD data set was used for preliminary phasing using the selenium signal present in a selenomethionine-substituted protein crystal

  13. Changes in chromatin state reveal ARNT2 at a node of a tumorigenic transcription factor signature driving glioblastoma cell aggressiveness.

    Science.gov (United States)

    Bogeas, Alexandra; Morvan-Dubois, Ghislaine; El-Habr, Elias A; Lejeune, François-Xavier; Defrance, Matthieu; Narayanan, Ashwin; Kuranda, Klaudia; Burel-Vandenbos, Fanny; Sayd, Salwa; Delaunay, Virgile; Dubois, Luiz G; Parrinello, Hugues; Rialle, Stéphanie; Fabrega, Sylvie; Idbaih, Ahmed; Haiech, Jacques; Bièche, Ivan; Virolle, Thierry; Goodhardt, Michele; Chneiweiss, Hervé; Junier, Marie-Pierre

    2018-02-01

    Although a growing body of evidence indicates that phenotypic plasticity exhibited by glioblastoma cells plays a central role in tumor development and post-therapy recurrence, the master drivers of their aggressiveness remain elusive. Here we mapped the changes in active (H3K4me3) and repressive (H3K27me3) histone modifications accompanying the repression of glioblastoma stem-like cells tumorigenicity. Genes with changing histone marks delineated a network of transcription factors related to cancerous behavior, stem state, and neural development, highlighting a previously unsuspected association between repression of ARNT2 and loss of cell tumorigenicity. Immunohistochemistry confirmed ARNT2 expression in cell sub-populations within proliferative zones of patients' glioblastoma. Decreased ARNT2 expression was consistently observed in non-tumorigenic glioblastoma cells, compared to tumorigenic cells. Moreover, ARNT2 expression correlated with a tumorigenic molecular signature at both the tissue level within the tumor core and at the single cell level in the patients' tumors. We found that ARNT2 knockdown decreased the expression of SOX9, POU3F2 and OLIG2, transcription factors implicated in glioblastoma cell tumorigenicity, and repressed glioblastoma stem-like cell tumorigenic properties in vivo. Our results reveal ARNT2 as a pivotal component of the glioblastoma cell tumorigenic signature, located at a node of a transcription factor network controlling glioblastoma cell aggressiveness.

  14. Strand-specific RNA-seq reveals widespread occurrence of novel cis-natural antisense transcripts in rice

    Directory of Open Access Journals (Sweden)

    Lu Tingting

    2012-12-01

    Full Text Available Abstract Background Cis-natural antisense transcripts (cis-NATs are RNAs transcribed from the antisense strand of a gene locus, and are complementary to the RNA transcribed from the sense strand. Common techniques including microarray approach and analysis of transcriptome databases are the major ways to globally identify cis-NATs in various eukaryotic organisms. Genome-wide in silico analysis has identified a large number of cis-NATs that may generate endogenous short interfering RNAs (nat-siRNAs, which participate in important biogenesis mechanisms for transcriptional and post-transcriptional regulation in rice. However, the transcriptomes are yet to be deeply sequenced to comprehensively investigate cis-NATs. Results We applied high-throughput strand-specific complementary DNA sequencing technology (ssRNA-seq to deeply sequence mRNA for assessing sense and antisense transcripts that were derived under salt, drought and cold stresses, and normal conditions, in the model plant rice (Oryza sativa. Combined with RAP-DB genome annotation (the Rice Annotation Project Database build-5 data set, 76,013 transcripts corresponding to 45,844 unique gene loci were assembled, in which 4873 gene loci were newly identified. Of 3819 putative rice cis-NATs, 2292 were detected as expressed and giving rise to small RNAs from their overlapping regions through integrated analysis of ssRNA-seq data and small RNA data. Among them, 503 cis-NATs seemed to be associated with specific conditions. The deep sequence data from isolated epidermal cells of rice seedlings further showed that 54.0% of cis-NATs were expressed simultaneously in a population of homogenous cells. Nearly 9.7% of rice transcripts were involved in one-to-one or many-to-many cis-NATs formation. Furthermore, only 17.4-34.7% of 223 many-to-many cis-NAT groups were all expressed and generated nat-siRNAs, indicating that only some cis-NAT groups may be involved in complex regulatory networks. Conclusions

  15. Genomic organization, transcript variants and comparative analysis of the human nucleoporin 155 (NUP155) gene

    DEFF Research Database (Denmark)

    Zhang, Xiuqing; Yang, Huanming; Yu, Jun

    2002-01-01

    are approximately 80 and 8 kb in length, respectively. The human gene is ubiquitously expressed in many tissues analyzed and has two major transcript variants, resulted from an alternative usage of the 5' cryptic or consensus splice donor in intron 1 and two polyadenylation signals. We have also cloned DNA...... complementary to RNAs of the Nup155 orthologs from Fugu and mouse. Comparative analysis of the Nup155 orthologs in many species, including H. sapiens, Mus musculus, Rattus norvegicus, F. rubripes, Arabidopsis thaliana, Drosophila melanogaster, and Saccharomyces cerevisiae, has revealed two paralogs in S...

  16. R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters.

    Science.gov (United States)

    Chen, Liang; Chen, Jia-Yu; Zhang, Xuan; Gu, Ying; Xiao, Rui; Shao, Changwei; Tang, Peng; Qian, Hao; Luo, Daji; Li, Hairi; Zhou, Yu; Zhang, Dong-Er; Fu, Xiang-Dong

    2017-11-16

    R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms.

    Science.gov (United States)

    Allen, Mary Ann; Andrysik, Zdenek; Dengler, Veronica L; Mellert, Hestia S; Guarnieri, Anna; Freeman, Justin A; Sullivan, Kelly D; Galbraith, Matthew D; Luo, Xin; Kraus, W Lee; Dowell, Robin D; Espinosa, Joaquin M

    2014-05-27

    The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms.DOI: http://dx.doi.org/10.7554/eLife.02200.001. Copyright © 2014, Allen et al.

  18. GAM: a web-service for integrated transcriptional and metabolic network analysis.

    Science.gov (United States)

    Sergushichev, Alexey A; Loboda, Alexander A; Jha, Abhishek K; Vincent, Emma E; Driggers, Edward M; Jones, Russell G; Pearce, Edward J; Artyomov, Maxim N

    2016-07-08

    Novel techniques for high-throughput steady-state metabolomic profiling yield information about changes of nearly thousands of metabolites. Such metabolomic profiles, when analyzed together with transcriptional profiles, can reveal novel insights about underlying biological processes. While a number of conceptual approaches have been developed for data integration, easily accessible tools for integrated analysis of mammalian steady-state metabolomic and transcriptional data are lacking. Here we present GAM ('genes and metabolites'): a web-service for integrated network analysis of transcriptional and steady-state metabolomic data focused on identification of the most changing metabolic subnetworks between two conditions of interest. In the web-service, we have pre-assembled metabolic networks for humans, mice, Arabidopsis and yeast and adapted exact solvers for an optimal subgraph search to work in the context of these metabolic networks. The output is the most regulated metabolic subnetwork of size controlled by false discovery rate parameters. The subnetworks are then visualized online and also can be downloaded in Cytoscape format for subsequent processing. The web-service is available at: https://artyomovlab.wustl.edu/shiny/gam/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

    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.

  20. Transcriptional changes associated with resistance to inhibitors of epidermal growth factor receptor revealed using metaanalysis

    International Nuclear Information System (INIS)

    Younis, Sidra; Javed, Qamar; Blumenberg, Miroslav

    2015-01-01

    EGFR is important in maintaining metabolic homeostasis in healthy cells, but in tumors it activates downstream signaling pathways, causing proliferation, angiogenesis, invasion and metastasis. Consequently, EGFR is targeted in cancers using reversible, irreversible or antibody inhibitors. Unfortunately, tumors develop inhibitor resistance by mutations or overexpressing EGFR, or its ligand, or activating secondary, EGFR-independent pathways. Here we present a global metaanalysis comparing transcriptional profiles from matched pairs of EGFR inhibitor-sensitive vs. -resistant cell lines, using 15 datasets comprising 274 microarrays. We also analyzed separately pairs of cell lines derived using reversible, irreversible or antibody inhibitors. The metaanalysis identifies commonalities in cell lines resistant to EGFR inhibitors: in sensitive cell lines, the ontological categories involving the ErbB receptors pathways, cell adhesion and lipid metabolism are overexpressed; however, resistance to EGFR inhibitors is associated with overexpression of genes for ErbB receptors-independent oncogenic pathways, regulation of cell motility, energy metabolism, immunity especially inflammatory cytokines biosynthesis, cell cycle and responses to exogenous and endogenous stimuli. Specifically in Gefitinib-resistant cell lines, the immunity-associated genes are overexpressed, whereas in Erlotinib-resistant ones so are the mitochondrial genes and processes. Unexpectedly, lines selected using EGFR-targeting antibodies overexpress different gene ontologies from ones selected using kinase inhibitors. Specifically, they have reduced expression of genes for proliferation, chemotaxis, immunity and angiogenesis. This metaanalysis suggests that ‘combination therapies’ can improve cancer treatment outcomes. Potentially, use of mitochondrial blockers with Erlotinib, immunity blockers with Gefitinib, tyrosine kinase inhibitors with antibody inhibitors, may have better chance of avoiding

  1. Interactome analysis of transcriptional coactivator multiprotein bridging factor 1 unveils a yeast AP-1-like transcription factor involved in oxidation tolerance of mycopathogen Beauveria bassiana.

    Science.gov (United States)

    Chu, Xin-Ling; Dong, Wei-Xia; Ding, Jin-Li; Feng, Ming-Guang; Ying, Sheng-Hua

    2018-02-01

    Oxidation tolerance is an important determinant to predict the virulence and biocontrol potential of Beauveria bassiana, a well-known entomopathogenic fungus. As a transcriptional coactivator, multiprotein bridging factor 1 mediates the activity of transcription factor in diverse physiological processes, and its homolog in B. bassiana (BbMBF1) contributes to fungal oxidation tolerance. In this study, the BbMBF1-interactomes under oxidative stress and normal growth condition were deciphered by mass spectrometry integrated with the immunoprecipitation. BbMBF1p factor has a broad interaction with proteins that are involved in various cellular processes, and this interaction is dynamically regulated by oxidative stress. Importantly, a B. bassiana homolog of yeast AP-1-like transcription factor (BbAP-1) was specifically associated with the BbMBF1-interactome under oxidation and significantly contributed to fungal oxidation tolerance. In addition, qPCR analysis revealed that several antioxidant genes are jointly controlled by BbAP-1 and BbMBF1. Conclusively, it is proposed that BbMBF1p protein mediates BbAP-1p factor to transcribe the downstream antioxidant genes in B. bassiana under oxidative stress. This study demonstrates for the first time a proteomic view of the MBF1-interactome in fungi, and presents an initial framework to probe the transcriptional mechanism involved in fungal response to oxidation, which will provide a new strategy to improve the biocontrol efficacy of B. bassiana.

  2. Characterization of a novel radiation-inducible transcript, uscA, and analysis of its transcriptional regulation

    International Nuclear Information System (INIS)

    Lim, Sang Yong; Kim, Dong Ho; Joe, Min Ho

    2010-03-01

    The transcriptional expression of the uscA promote (P uscA ) only occurred under aerobic conditions and a dose of 2Gy maximally activated transcription of P uscA . However, various environmental stress including physical shocks (pH, temperature, osmotic shock), DNA damaging agents (UV and MMC) or oxidative stressagents (paraquat, menadione, and H 2 O 2 ) didn't cause the transcriptional activationof P uscA . The transcription of uscA was initiated at 170 bp upstream of the cyoA start codon, and ended around the ampG stop codon. The size of uscA was determined through reverse transcription assay, approximately 250 bp. The deletion analysis of uscA promoter demonstrates that radiation inducibility of P uscA is mediated by sequences present between -20 and +111 relativeto +1 of P uscA and radiation causes P uscA activation thorough permitting the expression that is repressed under non-irradiated conditions

  3. Characterization of a novel radiation-inducible transcript, uscA, and analysis of its transcriptional regulation

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Sang Yong; Kim, Dong Ho; Joe, Min Ho

    2010-03-15

    The transcriptional expression of the uscA promote (P{sub uscA}) only occurred under aerobic conditions and a dose of 2Gy maximally activated transcription of P{sub uscA}. However, various environmental stress including physical shocks (pH, temperature, osmotic shock), DNA damaging agents (UV and MMC) or oxidative stressagents (paraquat, menadione, and H{sub 2}O{sub 2}) didn't cause the transcriptional activationof P{sub uscA}. The transcription of uscA was initiated at 170 bp upstream of the cyoA start codon, and ended around the ampG stop codon. The size of uscA was determined through reverse transcription assay, approximately 250 bp. The deletion analysis of uscA promoter demonstrates that radiation inducibility of P{sub uscA} is mediated by sequences present between -20 and +111 relativeto +1 of P{sub uscA} and radiation causes P{sub uscA} activation thorough permitting the expression that is repressed under non-irradiated conditions

  4. Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling

    Directory of Open Access Journals (Sweden)

    Craigon Marie

    2009-08-01

    Full Text Available Abstract Background Interferons (IFNs are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs. Here we have used targeted RNA interference to suppress the expression of a number of key genes associated with IFN signalling in murine macrophages prior to stimulation with interferon-gamma. Genome-wide changes in transcript abundance caused by siRNA activity were measured using exon-level microarrays in the presence or absence of IFNγ. Results Transfection of murine bone-marrow derived macrophages (BMDMs with a non-targeting (control siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000 prior to stimulation with IFNγ. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. Network-based analysis of these data revealed six siRNAs to cause a marked shift in the macrophage transcriptome in the presence or absence IFNγ. These six siRNAs targeted the Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2 transcripts. The perturbation of the transcriptome by the six siRNAs was highly similar in each case and affected the expression of over 600 downstream transcripts. Regulated transcripts were clustered based on co-expression into five major groups corresponding to transcriptional networks associated with the type I and II IFN response, cell cycle regulation, and NF-KB signalling. In addition we have observed a significant non-specific immune stimulation of cells transfected with siRNA using Lipofectamine2000, suggesting use of this reagent in BMDMs, even at low concentrations, is enough to induce a type I IFN response. Conclusion Our results provide evidence that the type I IFN response in murine BMDMs is dependent on Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2, and that siRNAs targeted to these genes results in perturbation of key transcriptional networks associated

  5. The structure of the first representative of Pfam family PF09836 reveals a two-domain organization and suggests involvement in transcriptional regulation

    International Nuclear Information System (INIS)

    Das, Debanu; Grishin, Nick V.; Kumar, Abhinav; Carlton, Dennis; Bakolitsa, Constantina; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Burra, Prasad; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grzechnik, Anna; Grzechnik, Slawomir K.; Grant, Joanna C.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Johnson, Hope A.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2009-01-01

    The crystal structure of the NGO1945 gene product from N. gonorrhoeae (UniProt Q5F5IO) reveals that the N-terminal domain assigned as a domain of unknown function (DUF2063) is likely to bind DNA and that the protein may be involved in transcriptional regulation. Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (∼40–99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention

  6. Manual Transcription and Instrumental Analysis of Singing through Praat

    Directory of Open Access Journals (Sweden)

    Paolo Bravi

    2016-02-01

    Full Text Available In the field of ethnomusicology, the main tool-of-the-trade for music analysis has been musical transcription on the score, despite its acknowledged cultural bias and the limits of staff notation as a means of representing music conceived and performed outside of the Western music culture. In the digital era, a number of analytical tools have been designed which may serve to solve some of the problems related to the use of the score as a method for describing and visualizing music. These tools allow analyses to be performed, which were virtually impossible for most ethnomusicologists until a few decades ago. Praat, the well known software developed by Paul Boersma and David Weenink and designed for phonetic studies, may also be helpful for the annotation and analysis of the singing voice. This paper deals with some aspects of the use of this program for musicological aims, focussing on the relation between acoustic data and subjective musical interpretations and their relevance in analytic and perceptual investigations.

  7. Genome-wide analysis of the Mediator transcription complex

    NARCIS (Netherlands)

    Peppel, H.J. van de

    2006-01-01

    Transcription regulation is an essential process that enables living organisms to develop, to respond to extra-cellular signals and to environmental changes. In S. cerevisiae more than 300 proteins are required for accurate transcription regulation. This thesis focuses on one of the more central

  8. Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

    Energy Technology Data Exchange (ETDEWEB)

    Hubin, Elizabeth A.; Lilic, Mirjana; Darst, Seth A.; Campbell, Elizabeth A.

    2017-07-13

    The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the αCTD may play a role in Mtb transcription regulation. Our results reveal the structure of an Actinobacteria-unique insert of the RNAP β' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σA, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.

  9. Transcriptional Profiling Reveals a Common Metabolic Program in High-Risk Human Neuroblastoma and Mouse Neuroblastoma Sphere-Forming Cells

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    Mengling Liu

    2016-10-01

    Full Text Available High-risk neuroblastoma remains one of the deadliest childhood cancers. Identification of metabolic pathways that drive or maintain high-risk neuroblastoma may open new avenues of therapeutic interventions. Here, we report the isolation and propagation of neuroblastoma sphere-forming cells with self-renewal and differentiation potential from tumors of the TH-MYCN mouse, an animal model of high-risk neuroblastoma with MYCN amplification. Transcriptional profiling reveals that mouse neuroblastoma sphere-forming cells acquire a metabolic program characterized by transcriptional activation of the cholesterol and serine-glycine synthesis pathways, primarily as a result of increased expression of sterol regulatory element binding factors and Atf4, respectively. This metabolic reprogramming is recapitulated in high-risk human neuroblastomas and is prognostic for poor clinical outcome. Genetic and pharmacological inhibition of the metabolic program markedly decreases the growth and tumorigenicity of both mouse neuroblastoma sphere-forming cells and human neuroblastoma cell lines. These findings suggest a therapeutic strategy for targeting the metabolic program of high-risk neuroblastoma.

  10. Transcript profiling reveals auxin and cytokinin signaling pathways and transcription regulation during in vitro organogenesis of Ramie (Boehmeria nivea L. Gaud.

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    Xing Huang

    Full Text Available In vitro organogenesis, one of the most common pathways leading to in vitro plant regeneration, is widely used in biotechnology and the fundamental study of plant biology. Although previous studies have constructed a complex regulatory network model for Arabidopsis in vitro organogenesis, no related study has been reported in ramie. To generate more complete observations of transcriptome content and dynamics during ramie in vitro organogenesis, we constructed a reference transcriptome library and ten digital gene expression (DGE libraries for illumina sequencing. Approximately 111.34 million clean reads were obtained for transcriptome and the DGE libraries generated between 13.5 and 18.8 million clean reads. De novo assembly produced 43,222 unigenes and a total of 5,760 differentially expressed genes (DEGs were filtered. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database, 26 auxin related and 11 cytokinin related DEGs were selected for qRT-PCR validation of two ramie cultivars, which had high (Huazhu No. 5 or extremely low (Dazhuhuangbaima shoot regeneration abilities. The results revealed differing regulation patterns of auxin and cytokinin in different genotypes. Here we report the first genome-wide gene expression profiling of in vitro organogenesis in ramie and provide an overview of transcription and phytohormone regulation during the process. Furthermore, the auxin and cytokinin related genes have distinct expression patterns in two ramie cultivars with high or extremely low shoot regeneration ability, which has given us a better understanding of the in vitro organogenesis mechanism. This result will provide a foundation for future phytohormone research and lead to improvements of the ramie regeneration system.

  11. Virtual mutagenesis of the yeast cyclins genetic network reveals complex dynamics of transcriptional control networks.

    Directory of Open Access Journals (Sweden)

    Eliska Vohradska

    Full Text Available Study of genetic networks has moved from qualitative description of interactions between regulators and regulated genes to the analysis of the interaction dynamics. This paper focuses on the analysis of dynamics of one particular network--the yeast cyclins network. Using a dedicated mathematical model of gene expression and a procedure for computation of the parameters of the model from experimental data, a complete numerical model of the dynamics of the cyclins genetic network was attained. The model allowed for performing virtual experiments on the network and observing their influence on the expression dynamics of the genes downstream in the regulatory cascade. Results show that when the network structure is more complicated, and the regulatory interactions are indirect, results of gene deletion are highly unpredictable. As a consequence of quantitative behavior of the genes and their connections within the network, causal relationship between a regulator and target gene may not be discovered by gene deletion. Without including the dynamics of the system into the network, its functional properties cannot be studied and interpreted correctly.

  12. Dynamic transcriptional control of macrophage miRNA signature via inflammation responsive enhancers revealed using a combination of next generation sequencing-based approaches.

    Science.gov (United States)

    Czimmerer, Zsolt; Horvath, Attila; Daniel, Bence; Nagy, Gergely; Cuaranta-Monroy, Ixchelt; Kiss, Mate; Kolostyak, Zsuzsanna; Poliska, Szilard; Steiner, Laszlo; Giannakis, Nikolas; Varga, Tamas; Nagy, Laszlo

    2018-01-01

    MicroRNAs are important components of the post-transcriptional fine-tuning of macrophage gene expression in physiological and pathological conditions. However, the mechanistic underpinnings and the cis-acting genomic factors of how macrophage polarizing signals induce miRNA expression changes are not well characterized. Therefore, we systematically evaluated the transcriptional basis underlying the inflammation-mediated regulation of macrophage microRNome using the combination of different next generation sequencing datasets. We investigated the LPS-induced expression changes at mature miRNA and pri-miRNA levels in mouse macrophages utilizing a small RNA-seq method and publicly available GRO-seq dataset, respectively. Next, we identified an enhancer set associated with LPS-responsive pri-miRNAs based on publicly available H3K4 mono-methylation-specific ChIP-seq and GRO-seq datasets. This enhancer set was further characterized by the combination of publicly available ChIP and ATAC-seq datasets. Finally, direct interactions between the miR-155-coding genomic region and its distal regulatory elements were identified using a 3C-seq approach. Our analysis revealed 15 robustly LPS-regulated miRNAs at the transcriptional level. In addition, we found that these miRNA genes are associated with an inflammation-responsive enhancer network. Based on NFκB-p65 and JunB transcription factor binding, we showed two distinct enhancer subsets associated with LPS-activated miRNAs that possess distinct epigenetic characteristics and LPS-responsiveness. Finally, our 3C-seq analysis revealed the LPS-induced extensive reorganization of the pri-miR-155-associated functional chromatin domain as well as chromatin loop formation between LPS-responsive enhancers and the promoter region. Our genomic approach successfully combines various genome-wide datasets and allows the identification of the putative regulatory elements controlling miRNA expression in classically activated macrophages

  13. Transcriptional Profiling of Saccharomyces cerevisiae Reveals the Impact of Variation of a Single Transcription Factor on Differential Gene Expression in 4NQO, Fermentable, and Nonfermentable Carbon Sources

    Directory of Open Access Journals (Sweden)

    Xiaoqing Rong-Mullins

    2018-02-01

    . Hence, the complement to 4NQO resistance was poor growth on nonfermentable carbon sources, which in turn varied depending on the allele of Yrr1 expressed in the isogenic yeast. The oxidation state of the yeast affected the 4NQO toxicity by altering the reactive oxygen species (ROS generated by cellular metabolism. The integration of RNA-Seq and ChIP-Seq elucidated how Yrr1 regulates global gene transcription in response to 4NQO and how various Yrr1 alleles confer differential resistance to 4NQO. This study provides guidance for further investigation into how Yrr1 regulates cellular responses to 4NQO, as well as transcriptomic resources for further analysis of transcription factor variation on carbon source utilization.

  14. Transcriptional profiling reveals progeroid Ercc1-/Δ mice as a model system for glomerular aging

    Science.gov (United States)

    2013-01-01

    Background Aging-related kidney diseases are a major health concern. Currently, models to study renal aging are lacking. Due to a reduced life-span progeroid models hold the promise to facilitate aging studies and allow examination of tissue-specific changes. Defects in genome maintenance in the Ercc1-/Δ progeroid mouse model result in premature aging and typical age-related pathologies. Here, we compared the glomerular transcriptome of young and aged Ercc1-deficient mice to young and aged WT mice in order to establish a novel model for research of aging-related kidney disease. Results In a principal component analysis, age and genotype emerged as first and second principal components. Hierarchical clustering of all 521 genes differentially regulated between young and old WT and young and old Ercc1-/Δ mice showed cluster formation between young WT and Ercc1-/Δ as well as old WT and Ercc1-/Δ samples. An unexpectedly high number of 77 genes were differentially regulated in both WT and Ercc1-/Δ mice (p aging glomerulus. At the level of the transcriptome, the pattern of gene activities is similar in the progeroid Ercc1-/Δ mouse model constituting a valuable tool for future studies of aging-associated glomerular pathologies. PMID:23947592

  15. Suppression Subtractive Hybridization Reveals Transcript Profiling of Chlorella under Heterotrophy to Photoautotrophy Transition

    Science.gov (United States)

    Huang, Jianke; Wang, Weiliang; Yin, Weibo; Hu, Zanmin; Li, Yuanguang

    2012-01-01

    Background Microalgae have been extensively investigated and exploited because of their competitive nutritive bioproducts and biofuel production ability. Chlorella are green algae that can grow well heterotrophically and photoautotrophically. Previous studies proved that shifting from heterotrophy to photoautotrophy in light-induced environments causes photooxidative damage as well as distinct physiologic features that lead to dynamic changes in Chlorella intracellular components, which have great potential in algal health food and biofuel production. However, the molecular mechanisms underlying the trophic transition remain unclear. Methodology/Principal Findings In this study, suppression subtractive hybridization strategy was employed to screen and characterize genes that are differentially expressed in response to the light-induced shift from heterotrophy to photoautotrophy. Expressed sequence tags (ESTs) were obtained from 770 and 803 randomly selected clones among the forward and reverse libraries, respectively. Sequence analysis identified 544 unique genes in the two libraries. The functional annotation of the assembled unigenes demonstrated that 164 (63.1%) from the forward library and 62 (21.8%) from the reverse showed significant similarities with the sequences in the NCBI non-redundant database. The time-course expression patterns of 38 selected differentially expressed genes further confirmed their responsiveness to a diverse trophic status. The majority of the genes enriched in the subtracted libraries were associated with energy metabolism, amino acid metabolism, protein synthesis, carbohydrate metabolism, and stress defense. Conclusions/Significance The data presented here offer the first insights into the molecular foundation underlying the diverse microalgal trophic niche. In addition, the results can be used as a reference for unraveling candidate genes associated with the transition of Chlorella from heterotrophy to photoautotrophy, which holds

  16. Suppression subtractive hybridization reveals transcript profiling of Chlorella under heterotrophy to photoautotrophy transition.

    Directory of Open Access Journals (Sweden)

    Jianhua Fan

    Full Text Available Microalgae have been extensively investigated and exploited because of their competitive nutritive bioproducts and biofuel production ability. Chlorella are green algae that can grow well heterotrophically and photoautotrophically. Previous studies proved that shifting from heterotrophy to photoautotrophy in light-induced environments causes photooxidative damage as well as distinct physiologic features that lead to dynamic changes in Chlorella intracellular components, which have great potential in algal health food and biofuel production. However, the molecular mechanisms underlying the trophic transition remain unclear.In this study, suppression subtractive hybridization strategy was employed to screen and characterize genes that are differentially expressed in response to the light-induced shift from heterotrophy to photoautotrophy. Expressed sequence tags (ESTs were obtained from 770 and 803 randomly selected clones among the forward and reverse libraries, respectively. Sequence analysis identified 544 unique genes in the two libraries. The functional annotation of the assembled unigenes demonstrated that 164 (63.1% from the forward library and 62 (21.8% from the reverse showed significant similarities with the sequences in the NCBI non-redundant database. The time-course expression patterns of 38 selected differentially expressed genes further confirmed their responsiveness to a diverse trophic status. The majority of the genes enriched in the subtracted libraries were associated with energy metabolism, amino acid metabolism, protein synthesis, carbohydrate metabolism, and stress defense.The data presented here offer the first insights into the molecular foundation underlying the diverse microalgal trophic niche. In addition, the results can be used as a reference for unraveling candidate genes associated with the transition of Chlorella from heterotrophy to photoautotrophy, which holds great potential for further improving its lipid and

  17. Transcription, reverse transcription, and analysis of RNA containing artificial genetic components.

    Science.gov (United States)

    Leal, Nicole A; Kim, Hyo-Joong; Hoshika, Shuichi; Kim, Myong-Jung; Carrigan, Matthew A; Benner, Steven A

    2015-04-17

    Expanding the synthetic biology of artificially expanded genetic information systems (AEGIS) requires tools to make and analyze RNA molecules having added nucleotide "letters". We report here the development of T7 RNA polymerase and reverse transcriptase to catalyze transcription and reverse transcription of xNA (DNA or RNA) having two complementary AEGIS nucleobases, 6-amino-5-nitropyridin-2-one (trivially, Z) and 2-aminoimidazo[1,2a]-1,3,5-triazin-4(8H)-one (trivially, P). We also report MALDI mass spectrometry and HPLC-based analyses for oligomeric GACUZP six-letter RNA and the use of ribonuclease (RNase) A and T1 RNase as enzymatic tools for the sequence-specific degradation of GACUZP RNA. We then applied these tools to analyze the GACUZP and GACTZP products of polymerases and reverse transcriptases (respectively) made from DNA and RNA templates. In addition to advancing this 6-letter AEGIS toward the biosynthesis of proteins containing additional amino acids, these experiments provided new insights into the biophysics of DNA.

  18. Arabidopsis Pol II-Dependent in Vitro Transcription System Reveals Role of Chromatin for Light-Inducible rbcS Gene Transcription1

    Science.gov (United States)

    Ido, Ayaka; Iwata, Shinya; Iwata, Yuka; Igarashi, Hisako; Hamada, Takahiro; Sonobe, Seiji; Sugiura, Masahiro; Yukawa, Yasushi

    2016-01-01

    In vitro transcription is an essential tool to study the molecular mechanisms of transcription. For over a decade, we have developed an in vitro transcription system from tobacco (Nicotiana tabacum)-cultured cells (BY-2), and this system supported the basic activities of the three RNA polymerases (Pol I, Pol II, and Pol III). However, it was not suitable to study photosynthetic genes, because BY-2 cells have lost their photosynthetic activity. Therefore, Arabidopsis (Arabidopsis thaliana) in vitro transcription systems were developed from green and etiolated suspension cells. Sufficient in vitro Pol II activity was detected after the minor modification of the nuclear soluble extracts preparation method; removal of vacuoles from protoplasts and L-ascorbic acid supplementation in the extraction buffer were particularly effective. Surprisingly, all four Arabidopsis Rubisco small subunit (rbcS-1A, rbcS-1B, rbcS-2B, and rbcS-3B) gene members were in vitro transcribed from the naked DNA templates without any light-dependent manner. However, clear light-inducible transcriptions were observed using chromatin template of rbcS-1A gene, which was prepared with a human nucleosome assembly protein 1 (hNAP1) and HeLa histones. This suggested that a key determinant of light-dependency through the rbcS gene transcription was a higher order of DNA structure (i.e. chromatin). PMID:26662274

  19. cDNA cloning and transcriptional controlling of a novel low dose radiation-induced gene and its function analysis

    International Nuclear Information System (INIS)

    Zhou Pingkun; Sui Jianli

    2002-01-01

    Objective: To clone a novel low dose radiation-induced gene (LRIGx) and study its function as well as its transcriptional changes after irradiation. Methods: Its cDNA was obtained by DDRT-PCR and RACE techniques. Northern blot hybridization was used to investigate the gene transcription. Bioinformatics was employed to analysis structure and function of this gene. Results: LRIGx cDNA was cloned. The sequence of LRIGx was identical to a DNA clone located in human chromosome 20 q 11.2-12 Bioinformatics analysis predicted an encoded protein with a conserved helicase domain. Northern analysis revealed a ∼8.5 kb transcript which was induced after 0.2 Gy as well as 0.02 Gy irradiation, and the transcript level was increased 5 times at 4 h after 0.2 Gy irradiation. The induced level of LRIGx transcript by 2.0 Gy high dose was lower than by 0.2 Gy. Conclusion: A novel low dose radiation-induced gene has been cloned. It encodes a protein with a conserved helicase domain that could involve in DNA metabolism in the cellular process of radiation response

  20. Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

    DEFF Research Database (Denmark)

    Ronander, Elena; Bengtsson, Dominique C; Joergensen, Louise

    2012-01-01

    fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE(1). Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human...

  1. Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

    Science.gov (United States)

    Hardy, W Reef; Moldovan, Nicanor I; Moldovan, Leni; Livak, Kenneth J; Datta, Krishna; Goswami, Chirayu; Corselli, Mirko; Traktuev, Dmitry O; Murray, Iain R; Péault, Bruno; March, Keith

    2017-05-01

    Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31 - /CD45 - /CD34 + /CD146 - cells (adventitial stromal/stem cells [ASCs]) and CD31 - /CD45 - /CD34 - /CD146 + cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDH br ASC (most primitive); (b) ALDH dim ASC; (c) ALDH br PC; (d) ALDH dim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression

  2. Quantitative interactome analysis reveals a chemoresistant edgotype.

    Science.gov (United States)

    Chavez, Juan D; Schweppe, Devin K; Eng, Jimmy K; Zheng, Chunxiang; Taipale, Alex; Zhang, Yiyi; Takara, Kohji; Bruce, James E

    2015-08-03

    Chemoresistance is a common mode of therapy failure for many cancers. Tumours develop resistance to chemotherapeutics through a variety of mechanisms, with proteins serving pivotal roles. Changes in protein conformations and interactions affect the cellular response to environmental conditions contributing to the development of new phenotypes. The ability to understand how protein interaction networks adapt to yield new function or alter phenotype is limited by the inability to determine structural and protein interaction changes on a proteomic scale. Here, chemical crosslinking and mass spectrometry were employed to quantify changes in protein structures and interactions in multidrug-resistant human carcinoma cells. Quantitative analysis of the largest crosslinking-derived, protein interaction network comprising 1,391 crosslinked peptides allows for 'edgotype' analysis in a cell model of chemoresistance. We detect consistent changes to protein interactions and structures, including those involving cytokeratins, topoisomerase-2-alpha, and post-translationally modified histones, which correlate with a chemoresistant phenotype.

  3. Transient Shifts of Incubation Temperature Reveal Immediate and Long-Term Transcriptional Response in Chicken Breast Muscle Underpinning Resilience and Phenotypic Plasticity.

    Science.gov (United States)

    Naraballobh, Watcharapong; Trakooljul, Nares; Murani, Eduard; Brunner, Ronald; Krischek, Carsten; Janisch, Sabine; Wicke, Michael; Ponsuksili, Siriluck; Wimmers, Klaus

    2016-01-01

    Variations in egg incubation temperatures can have acute or long-term effects on gene transcription in avian species. Altered gene expression may, in turn, affect muscle traits in poultry and indirectly influence commercial production. To determine how changes in eggshell temperature affect gene expression, incubation temperatures were varied [36.8°C (low), 37.8°C (control), 38.8°C (high)] at specific time periods reflecting two stages of myogenesis [embryonic days (ED) 7-10 and 10-13]. Gene expression was compared between interventions and matching controls by microarrays in broiler breast muscle at ED10 or ED13 and post-hatch at day 35. Early (ED7-10) high incubation temperature (H10ΔC) resulted in 1370 differentially expressed genes (DEGs) in embryos. Ingenuity pathway analysis revealed temporary activation of cell maintenance, organismal development, and survival ability genes, but these effects were not maintained in adults. Late high incubation temperature (ED10-13) (H13ΔC) had slightly negative impacts on development of cellular components in embryos, but a cumulative effect was observed in adults, in which tissue development and nutrition metabolism were affected. Early low incubation temperature (L10ΔC) produced 368 DEGs, most of which were down-regulated and involved in differentiation and formation of muscle cells. In adults, this treatment down-regulated pathways of transcriptional processes, but up-regulated cell proliferation. Late low temperature incubation (L13ΔC) produced 795 DEGs in embryos, and activated organismal survival and post-transcriptional regulation pathways. In adults this treatment activated cellular and organ development, nutrition and small molecule activity, and survival rate, but deactivated size of body and muscle cells. Thermal interventions during incubation initiate immediate and delayed transcriptional responses that are specific for timing and direction of treatment. Interestingly, the transcriptional response to

  4. Transcriptome analysis reveals key differentially expressed genes involved in wheat grain development

    Directory of Open Access Journals (Sweden)

    Yonglong Yu

    2016-04-01

    Full Text Available Wheat seed development is an important physiological process of seed maturation and directly affects wheat yield and quality. In this study, we performed dynamic transcriptome microarray analysis of an elite Chinese bread wheat cultivar (Jimai 20 during grain development using the GeneChip Wheat Genome Array. Grain morphology and scanning electron microscope observations showed that the period of 11–15 days post-anthesis (DPA was a key stage for the synthesis and accumulation of seed starch. Genome-wide transcriptional profiling and significance analysis of microarrays revealed that the period from 11 to 15 DPA was more important than the 15–20 DPA stage for the synthesis and accumulation of nutritive reserves. Series test of cluster analysis of differential genes revealed five statistically significant gene expression profiles. Gene ontology annotation and enrichment analysis gave further information about differentially expressed genes, and MapMan analysis revealed expression changes within functional groups during seed development. Metabolic pathway network analysis showed that major and minor metabolic pathways regulate one another to ensure regular seed development and nutritive reserve accumulation. We performed gene co-expression network analysis to identify genes that play vital roles in seed development and identified several key genes involved in important metabolic pathways. The transcriptional expression of eight key genes involved in starch and protein synthesis and stress defense was further validated by qRT-PCR. Our results provide new insight into the molecular mechanisms of wheat seed development and the determinants of yield and quality.

  5. Comparing genomic expression patterns across plant species reveals highly diverged transcriptional dynamics in response to salt stress

    Directory of Open Access Journals (Sweden)

    Close Timothy J

    2009-08-01

    Full Text Available Abstract Background Rice and barley are both members of Poaceae (grass family but have a marked difference in salt tolerance. The molecular mechanism underlying this difference was previously unexplored. This study employs a comparative genomics approach to identify analogous and contrasting gene expression patterns between rice and barley. Results A hierarchical clustering approach identified several interesting expression trajectories among rice and barley genotypes. There were no major conserved expression patterns between the two species in response to salt stress. A wheat salt-stress dataset was queried for comparison with rice and barley. Roughly one-third of the salt-stress responses of barley were conserved with wheat while overlap between wheat and rice was minimal. These results demonstrate that, at transcriptome level, rice is strikingly different compared to the more closely related barley and wheat. This apparent lack of analogous transcriptional programs in response to salt stress is further highlighted through close examination of genes associated with root growth and development. Conclusion The analysis provides support for the hypothesis that conservation of transcriptional signatures in response to environmental cues depends on the genetic similarity among the genotypes within a species, and on the phylogenetic distance between the species.

  6. Global transcription profiling reveals differential responses to chronic nitrogen stress and putative nitrogen regulatory components in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Zhu Tong

    2007-08-01

    Full Text Available Abstract Background A large quantity of nitrogen (N fertilizer is used for crop production to achieve high yields at a significant economic and environmental cost. Efforts have been directed to understanding the molecular basis of plant responses to N and identifying N-responsive genes in order to manipulate their expression, thus enabling plants to use N more efficiently. No studies have yet delineated these responses at the transcriptional level when plants are grown under chronic N stress and the understanding of regulatory elements involved in N response is very limited. Results To further our understanding of the response of plants to varying N levels, a growth system was developed where N was the growth-limiting factor. An Arabidopsis whole genome microarray was used to evaluate global gene expression under different N conditions. Differentially expressed genes under mild or severe chronic N stress were identified. Mild N stress triggered only a small set of genes significantly different at the transcriptional level, which are largely involved in various stress responses. Plant responses were much more pronounced under severe N stress, involving a large number of genes in many different biological processes. Differentially expressed genes were also identified in response to short- and long-term N availability increases. Putative N regulatory elements were determined along with several previously known motifs involved in the responses to N and carbon availability as well as plant stress. Conclusion Differentially expressed genes identified provide additional insights into the coordination of the complex N responses of plants and the components of the N response mechanism. Putative N regulatory elements were identified to reveal possible new components of the regulatory network for plant N responses. A better understanding of the complex regulatory network for plant N responses will help lead to strategies to improve N use efficiency.

  7. Transcriptional regulation and signature patterns revealed by microarray analyses of Streptococcus pneumoniae R6 challenged with sublethal concentrations of translation inhibitors.

    Science.gov (United States)

    Ng, Wai-Leung; Kazmierczak, Krystyna M; Robertson, Gregory T; Gilmour, Raymond; Winkler, Malcolm E

    2003-01-01

    The effects of sublethal concentrations of four different classes of translation inhibitors (puromycin, tetracycline, chloramphenicol, and erythromycin) on global transcription patterns of Streptococcus pneumoniae R6 were determined by microarray analyses. Consistent with the general mode of action of these inhibitors, relative transcript levels of genes that encode ribosomal proteins and translation factors or that mediate tRNA charging and amino acid biosynthesis increased or decreased, respectively. Transcription of the heat shock regulon was induced only by puromycin or streptomycin treatment, which lead to truncation or mistranslation, respectively, but not by other antibiotics that block translation, transcription, or amino acid charging of tRNA. In contrast, relative transcript amounts of certain genes involved in transport, cellular processes, energy metabolism, and purine nucleotide (pur) biosynthesis were changed by different translation inhibitors. In particular, transcript amounts from a pur gene cluster and from purine uptake and salvage genes were significantly elevated by several translation inhibitors, but not by antibiotics that target other cellular processes. Northern blotting confirmed increased transcript amounts from part of the pur gene cluster in cells challenged by translation inhibitors and revealed the presence of a 10-kb transcript. Purine metabolism genes were negatively regulated by a homologue of the PurR regulatory protein, and full derepression in a DeltapurR mutant depended on optimal translation. Unexpectedly, hierarchical clustering of the microarray data distinguished among the global transcription patterns caused by antibiotics that inhibit different steps in the translation cycle. Together, these results show that there is extensive control of transcript amounts by translation in S. pneumoniae, especially for de novo purine nucleotide biosynthesis. In addition, these global transcription patterns form a signature that can be

  8. Analysis of microRNA transcription and post-transcriptional processing by Dicer in the context of CHO cell proliferation

    Science.gov (United States)

    Hackl, Matthias; Jadhav, Vaibhav; Klanert, Gerald; Karbiener, Michael; Scheideler, Marcel; Grillari, Johannes; Borth, Nicole

    2014-01-01

    CHO cells are the mammalian cell line of choice for recombinant production of therapeutic proteins. However, their low rate of proliferation limits obtainable space-time yields due to inefficient biomass accumulation. We set out to correlate microRNA transcription to cell-specific growth-rate by microarray analysis of 5 CHO suspension cell lines with low to high specific growth rates. Global microRNA expression analysis and Pearson correlation studies showed that mature microRNA transcript levels are predominately up-regulated in a state of fast proliferation (46 positively correlated, 17 negatively correlated). To further validate this observation, the expression of three genes that are central to microRNA biogenesis (Dicer, Drosha and Dgcr8) was analyzed. The expression of Dicer, which mediates the final step in microRNA maturation, was found to be strongly correlated to growth rate. Accordingly, knockdown of Dicer impaired cell growth by reducing growth-correlating microRNA transcripts. Moderate ectopic overexpression of Dicer positively affected cell growth, while strong overexpression impaired growth, presumably due to the concomitant increase of microRNAs that inhibit cell growth. Our data therefore suggest that Dicer dependent microRNAs regulate CHO cell proliferation and that Dicer could serve as a potential surrogate marker for cellular proliferation. PMID:24486028

  9. In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501.

    Science.gov (United States)

    Brantl, S; Nuez, B; Behnke, D

    1992-07-01

    Derivatives of the conjugative streptococcal plasmid pIP501 replicate stably in Bacillus subtilis. The region essential for replication of pIP501 has been narrowed down to a 2.2 kb DNA segment, the sequence of which has been determined. This region comprises two genes, copR and repR, proposed to be involved in copy control and replication. By in vitro and in vivo transcriptional analysis we characterized three active promoters, pI, pII and pIII within this region. A putative fourth promoter (pIV) was neither active in vitro nor in vivo. We showed that copR is transcribed from promoter pI while the repR gene is transcribed from promoter pII located just downstream of copR. The pII transcript encompasses a 329 nucleotide (nt) long leader sequence. A counter transcript that was complementary to a major part of this leader was found to originate from a third promoter pIII. The secondary structure of the counter transcript revealed several stem-loop regions. A regulatory function for this antisense RNA in the control of repR expression is proposed. Comparative analysis of the replication regions of pAM beta 1 and pSM19035 suggested a similar organization of transcriptional units, suggesting that an antisense RNA is produced by these plasmids also.

  10. Cloning, nucleotide sequence and transcriptional analysis of the uvrA gene from Neisseria gonorrhoeae

    International Nuclear Information System (INIS)

    Black, C.G.; Fyfe, J.A.M.; Davies, J.K.

    1997-01-01

    A recombinant plasmid capable of restoring UV resistance to an Escherichia coli uvrA mutant was isolated from a genomic library of Neisseria gonorrhoeae. Sequence analysis revealed an open reading frame whose deduced amino acid sequence displayed significant similarity to those of the UvrA proteins of other bacterial species. A second open reading frame (ORF259) was identified upstream from, and in the opposite orientation to the gonococcal uvrA gene. Transcriptional fusions between portions of the gonococcal uvrA upstream region and a reporter gene were used to localise promoter activity in both E. coli and N. gonorrhoeae. The transcriptional starting points of uvrA and ORF259 were mapped in E. coli by primer extension analysis, and corresponding σ 70 promoters were identified. The arrangement of the uvrA-ORF259 intergenic region is similar to that of the gonococcal recA-aroD intergenic region. Both contain inverted copies of the 10 bp neisserial DNA uptake sequence situated between divergently transcribed genes. However, there is no evidence that either the uptake sequence or the proximity of the promoters influences expression of these genes. (author)

  11. Functional analysis of limb transcriptional enhancers in the mouse.

    Science.gov (United States)

    Nolte, Mark J; Wang, Ying; Deng, Jian Min; Swinton, Paul G; Wei, Caimiao; Guindani, Michele; Schwartz, Robert J; Behringer, Richard R

    2014-01-01

    Transcriptional enhancers are genomic sequences bound by transcription factors that act together with basal transcriptional machinery to regulate gene transcription. Several high-throughput methods have generated large datasets of tissue-specific enhancer sequences with putative roles in developmental processes. However, few enhancers have been deleted from the genome to determine their roles in development. To understand the roles of two enhancers active in the mouse embryonic limb bud we deleted them from the genome. Although the genes regulated by these enhancers are unknown, they were selected because they were identified in a screen for putative limb bud-specific enhancers associated with p300, an acetyltransferase that participates in protein complexes that promote active transcription, and because the orthologous human enhancers (H1442 and H280) drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. We show that the orthologous mouse sequences, M1442 and M280, regulate dynamic expression in the developing limb. Although significant transcriptional differences in enhancer-proximal genes in embryonic limb buds accompany the deletion of M1442 and M280 no gross limb malformations during embryonic development were observed, demonstrating that M1442 and M280 are not required for mouse limb development. However, M280 is required for the development and/or maintenance of body size; M280 mice are significantly smaller than controls. M280 also harbors an "ultraconserved" sequence that is identical between human, rat, and mouse. This is the first report of a phenotype resulting from the deletion of an ultraconserved element. These studies highlight the importance of determining enhancer regulatory function by experiments that manipulate them in situ and suggest that some of an enhancer's regulatory capacities may be developmentally tolerated rather than developmentally required. © 2014 Wiley Periodicals, Inc.

  12. Single-cell profiling reveals that eRNA accumulation at enhancer-promoter loops is not required to sustain transcription.

    Science.gov (United States)

    Rahman, Samir; Zorca, Cornelia E; Traboulsi, Tatiana; Noutahi, Emmanuel; Krause, Matthew R; Mader, Sylvie; Zenklusen, Daniel

    2017-04-07

    Enhancers are intergenic DNA elements that regulate the transcription of target genes in response to signaling pathways by interacting with promoters over large genomic distances. Recent studies have revealed that enhancers are bi-directionally transcribed into enhancer RNAs (eRNAs). Using single-molecule fluorescence in situ hybridization (smFISH), we investigated the eRNA-mediated regulation of transcription during estrogen induction in MCF-7 cells. We demonstrate that eRNAs are localized exclusively in the nucleus and are induced with similar kinetics as target mRNAs. However, eRNAs are mostly nascent at enhancers and their steady-state levels remain lower than those of their cognate mRNAs. Surprisingly, at the single-allele level, eRNAs are rarely co-expressed with their target loci, demonstrating that active gene transcription does not require the continuous transcription of eRNAs or their accumulation at enhancers. When co-expressed, sub-diffraction distance measurements between nascent mRNA and eRNA signals reveal that co-transcription of eRNAs and mRNAs rarely occurs within closed enhancer-promoter loops. Lastly, basal eRNA transcription at enhancers, but not E2-induced transcription, is maintained upon depletion of MLL1 and ERα, suggesting some degree of chromatin accessibility prior to signal-dependent activation of transcription. Together, our findings suggest that eRNA accumulation at enhancer-promoter loops is not required to sustain target gene transcription. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. SVD identifies transcript length distribution functions from DNA microarray data and reveals evolutionary forces globally affecting GBM metabolism.

    Directory of Open Access Journals (Sweden)

    Nicolas M Bertagnolli

    Full Text Available To search for evolutionary forces that might act upon transcript length, we use the singular value decomposition (SVD to identify the length distribution functions of sets and subsets of human and yeast transcripts from profiles of mRNA abundance levels across gel electrophoresis migration distances that were previously measured by DNA microarrays. We show that the SVD identifies the transcript length distribution functions as "asymmetric generalized coherent states" from the DNA microarray data and with no a-priori assumptions. Comparing subsets of human and yeast transcripts of the same gene ontology annotations, we find that in both disparate eukaryotes, transcripts involved in protein synthesis or mitochondrial metabolism are significantly shorter than typical, and in particular, significantly shorter than those involved in glucose metabolism. Comparing the subsets of human transcripts that are overexpressed in glioblastoma multiforme (GBM or normal brain tissue samples from The Cancer Genome Atlas, we find that GBM maintains normal brain overexpression of significantly short transcripts, enriched in transcripts that are involved in protein synthesis or mitochondrial metabolism, but suppresses normal overexpression of significantly longer transcripts, enriched in transcripts that are involved in glucose metabolism and brain activity. These global relations among transcript length, cellular metabolism and tumor development suggest a previously unrecognized physical mode for tumor and normal cells to differentially regulate metabolism in a transcript length-dependent manner. The identified distribution functions support a previous hypothesis from mathematical modeling of evolutionary forces that act upon transcript length in the manner of the restoring force of the harmonic oscillator.

  14. The phylogeny of C/S1 bZIP transcription factors reveals a shared algal ancestry and the pre-angiosperm translational regulation of S1 transcripts

    NARCIS (Netherlands)

    Peviani, Alessia; Lastdrager, Jeroen; Hanson, Johannes; Snel, Berend

    2016-01-01

    Basic leucine zippers (bZIPs) form a large plant transcription factor family. C and S1 bZIP groups can heterodimerize, fulfilling crucial roles in seed development and stress response. S1 sequences also harbor a unique regulatory mechanism, termed Sucrose-Induced Repression of Translation (SIRT).

  15. Genomewide Analysis of Clp1 Function in Transcription in Budding Yeast

    OpenAIRE

    Al-Husini, Nadra; Sharifi, Ali; Mousavi, Seyed Ahmad; Chitsaz, Hamidreza; Ansari, Athar

    2017-01-01

    In budding yeast, the 3? end processing of mRNA and the coupled termination of transcription by RNAPII requires the CF IA complex. We have earlier demonstrated a role for the Clp1 subunit of this complex in termination and promoter-associated transcription of CHA1. To assess the generality of the observed function of Clp1 in transcription, we tested the effect of Clp1 on transcription on a genomewide scale using the Global Run-On-Seq (GRO-Seq) approach. GRO-Seq analysis showed the polymerase ...

  16. Analysis of carboxylesterase 2 transcript variants in cynomolgus macaque liver.

    Science.gov (United States)

    Uno, Yasuhiro; Igawa, Yoshiyuki; Tanaka, Maori; Ohura, Kayoko; Hosokawa, Masakiyo; Imai, Teruko

    2018-04-27

    Carboxylesterase (CES) is important for the detoxification of a wide range of drugs and xenobiotics. In this study, the hepatic level of CES2 mRNA was examined in cynomolgus macaques used widely in preclinical studies for drug metabolism. Three CES2 mRNAs were present in cynomolgus macaque liver. The mRNA level was highest for cynomolgus CES2A (formerly CES2v3), much lower for cynomolgus CES2B (formerly CES2v1) and extremely low for cynomolgus CES2C (formerly CES2v2). Most various transcript variants produced from cynomolgus CES2B gene did not contain a complete coding region. Thus, CES2A is the major CES2 enzyme in cynomolgus liver. A new transcript variant of CES2A, CES2Av2, was identified. CES2Av2 contained exon 3 region different from wild-type (CES2Av1). In cynomolgus macaques expressing only CES2Av2 transcript, CES2A contained the sequence of CES2B in exon 3 and vicinity, probably due to gene conversion. On genotyping, this CES2Av2 allele was prevalent in Indochinese cynomolgus macaques, but not in Indonesian cynomolgus or rhesus macaques. CES2Av2 recombinant protein showed similar activity to CES2Av1 protein for several substrates. It is concluded that CES2A is the major cynomolgus hepatic CES2, and new transcript variant, CES2Av2, has similar functions to CES2Av1.

  17. Gene transcription analysis during interaction between potato and Ralstonia solanacearum

    NARCIS (Netherlands)

    Li, G.C.; Jin, L.P.; Wang, X.W.; Xie, K.Y.; Yang, Y.; Vossen, van der E.A.G.; Huang, S.W.; Qu, D.Y.

    2010-01-01

    Bacterial wilt (BW) caused by Ralstonia solanacearum (Rs) is an important quarantine disease that spreads worldwide and infects hundreds of plant species. The BW defense response of potato is a complicated continuous process, which involves transcription of a battery of genes. The molecular

  18. DBTSS/DBKERO for integrated analysis of transcriptional regulation.

    Science.gov (United States)

    Suzuki, Ayako; Kawano, Shin; Mitsuyama, Toutai; Suyama, Mikita; Kanai, Yae; Shirahige, Katsuhiko; Sasaki, Hiroyuki; Tokunaga, Katsushi; Tsuchihara, Katsuya; Sugano, Sumio; Nakai, Kenta; Suzuki, Yutaka

    2018-01-04

    DBTSS (Database of Transcriptional Start Sites)/DBKERO (Database of Kashiwa Encyclopedia for human genome mutations in Regulatory regions and their Omics contexts) is the database originally initiated with the information of transcriptional start sites and their upstream transcriptional regulatory regions. In recent years, we updated the database to assist users to elucidate biological relevance of the human genome variations or somatic mutations in cancers which may affect the transcriptional regulation. In this update, we facilitate interpretations of disease associated genomic variation, using the Japanese population as a model case. We enriched the genomic variation dataset consisting of the 13,368 individuals collected for various genome-wide association studies and the reference epigenome information in the surrounding regions using a total of 455 epigenome datasets (four tissue types from 67 healthy individuals) collected for the International Human Epigenome Consortium (IHEC). The data directly obtained from the clinical samples was associated with that obtained from various model systems, such as the drug perturbation datasets using cultured cancer cells. Furthermore, we incorporated the results obtained using the newly developed analytical methods, Nanopore/10x Genomics long-read sequencing of the human genome and single cell analyses. The database is made publicly accessible at the URL (http://dbtss.hgc.jp/). © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine

    Directory of Open Access Journals (Sweden)

    Gosset Guillermo

    2007-09-01

    Full Text Available Abstract Background The rational design of L-phenylalanine (L-Phe overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase, reaching yields of 0.33 (g-Phe/g-Glc, which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed. Results MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB, gluconeogenic (ppsA, pckA and fermentative enzymes (ldhA were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt in the best PTS- L-Phe overproducing strain (PB12-ev2. Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to

  20. Functional analysis of Dof transcription factors controlling heading date and PPDK gene expression in rice

    NARCIS (Netherlands)

    Zhang, Yu

    2015-01-01

    Chapter 1 presents a general introduction to the factors involved in crop domestication and the specific role of transcription factors in this process. Chapter 2 describes the functional analysis of a Dof-type transcription factor, named OsDof24, from rice.chapter 3 describes the role of OsDof25 in

  1. Integrative analysis of histone ChIP-seq and transcription data using Bayesian mixture models

    DEFF Research Database (Denmark)

    Klein, Hans-Ulrich; Schäfer, Martin; Porse, Bo T

    2014-01-01

    Histone modifications are a key epigenetic mechanism to activate or repress the transcription of genes. Datasets of matched transcription data and histone modification data obtained by ChIP-seq exist, but methods for integrative analysis of both data types are still rare. Here, we present a novel...

  2. Network based transcription factor analysis of regenerating axolotl limbs

    Directory of Open Access Journals (Sweden)

    Cameron Jo Ann

    2011-03-01

    Full Text Available Abstract Background Studies on amphibian limb regeneration began in the early 1700's but we still do not completely understand the cellular and molecular events of this unique process. Understanding a complex biological process such as limb regeneration is more complicated than the knowledge of the individual genes or proteins involved. Here we followed a systems biology approach in an effort to construct the networks and pathways of protein interactions involved in formation of the accumulation blastema in regenerating axolotl limbs. Results We used the human orthologs of proteins previously identified by our research team as bait to identify the transcription factor (TF pathways and networks that regulate blastema formation in amputated axolotl limbs. The five most connected factors, c-Myc, SP1, HNF4A, ESR1 and p53 regulate ~50% of the proteins in our data. Among these, c-Myc and SP1 regulate 36.2% of the proteins. c-Myc was the most highly connected TF (71 targets. Network analysis showed that TGF-β1 and fibronectin (FN lead to the activation of these TFs. We found that other TFs known to be involved in epigenetic reprogramming, such as Klf4, Oct4, and Lin28 are also connected to c-Myc and SP1. Conclusions Our study provides a systems biology approach to how different molecular entities inter-connect with each other during the formation of an accumulation blastema in regenerating axolotl limbs. This approach provides an in silico methodology to identify proteins that are not detected by experimental methods such as proteomics but are potentially important to blastema formation. We found that the TFs, c-Myc and SP1 and their target genes could potentially play a central role in limb regeneration. Systems biology has the potential to map out numerous other pathways that are crucial to blastema formation in regeneration-competent limbs, to compare these to the pathways that characterize regeneration-deficient limbs and finally, to identify stem

  3. Deep sequencing of Lotus corniculatus L. reveals key enzymes and potential transcription factors related to the flavonoid biosynthesis pathway.

    Science.gov (United States)

    Wang, Ying; Hua, Wenping; Wang, Jian; Hannoufa, Abdelali; Xu, Ziqin; Wang, Zhezhi

    2013-04-01

    Lotus corniculatus L. is used worldwide as a forage crop due to its abundance of secondary metabolites and its ability to grow in severe environments. Although the entire genome of L. corniculatus var. japonicus R. is being sequenced, the differences in morphology and production of secondary metabolites between these two related species have led us to investigate this variability at the genetic level, in particular the differences in flavonoid biosynthesis. Our goal is to use the resulting information to develop more valuable forage crops and medicinal materials. Here, we conducted Illumina/Solexa sequencing to profile the transcriptome of L. corniculatus. We produced 26,492,952 short reads that corresponded to 2.38 gigabytes of total nucleotides. These reads were then assembled into 45,698 unigenes, of which a large number associated with secondary metabolism were annotated. In addition, we identified 2,998 unigenes based on homology with L. japonicus transcription factors (TFs) and grouped them into 55 families. Meanwhile, a comparison of four tag-based digital gene expression libraries, built from the flowers, pods, leaves, and roots, revealed distinct patterns of spatial expression of candidate unigenes in flavonoid biosynthesis. Based on these results, we identified many key enzymes from L. corniculatus which were different from reference genes of L. japonicus, and five TFs that are potential enhancers in flavonoid biosynthesis. Our results provide initial genetics resources that will be valuable in efforts to manipulate the flavonoid metabolic pathway in plants.

  4. Laser microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells.

    Science.gov (United States)

    Balestrini, Raffaella; Gómez-Ariza, Jorge; Lanfranco, Luisa; Bonfante, Paola

    2007-09-01

    The establishment of a symbiotic interaction between plant roots and arbuscular mycorrhizal (AM) fungi requires both partners to undergo significant morphological and physiological modifications which eventually lead to reciprocal beneficial effects. Extensive changes in gene expression profiles recently have been described in transcriptomic studies that have analyzed the whole mycorrhizal root. However, because root colonization by AM fungi involves different cell types, a cell-specific gene expression pattern is likely to occur. We have applied the laser microdissection (LMD) technology to investigate expression profiles of both plant and fungal genes in Lycopersicon esculentum roots colonized by Glomus mosseae. A protocol to harvest arbuscule-containing cells from paraffin sections of mycorrhizal roots has been developed using a Leica AS LMD system. RNA of satisfactory quantity and quality has been extracted for molecular analysis. Transcripts for plant phosphate transporters (LePTs), selected as molecular markers for a functional symbiosis, have been detected by reverse-transcriptase polymerase chain reaction assays and associated to distinct cell types, leading to novel insights into the distribution of LePT mRNAs. In fact, the transcripts of the five phosphate transporters (PTs) have been detected contemporaneously in the same arbusculated cell population, unlike from the neighboring noncolonized cells. In addition, fungal H(+)ATPase (GmHA5) and phosphate transporter (GmosPT) mRNAs were found exclusively in arbusculated cells. The discovery that five plant and one fungal PT genes are consistently expressed inside the arbusculated cells provides a new scenario for plant-fungus nutrient exchanges.

  5. Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis

    Science.gov (United States)

    2012-01-01

    Background The MYB gene family comprises one of the richest groups of transcription factors in plants. Plant MYB proteins are characterized by a highly conserved MYB DNA-binding domain. MYB proteins are classified into four major groups namely, 1R-MYB, 2R-MYB, 3R-MYB and 4R-MYB based on the number and position of MYB repeats. MYB transcription factors are involved in plant development, secondary metabolism, hormone signal transduction, disease resistance and abiotic stress tolerance. A comparative analysis of MYB family genes in rice and Arabidopsis will help reveal the evolution and function of MYB genes in plants. Results A genome-wide analysis identified at least 155 and 197 MYB genes in rice and Arabidopsis, respectively. Gene structure analysis revealed that MYB family genes possess relatively more number of introns in the middle as compared with C- and N-terminal regions of the predicted genes. Intronless MYB-genes are highly conserved both in rice and Arabidopsis. MYB genes encoding R2R3 repeat MYB proteins retained conserved gene structure with three exons and two introns, whereas genes encoding R1R2R3 repeat containing proteins consist of six exons and five introns. The splicing pattern is similar among R1R2R3 MYB genes in Arabidopsis. In contrast, variation in splicing pattern was observed among R1R2R3 MYB members of rice. Consensus motif analysis of 1kb upstream region (5′ to translation initiation codon) of MYB gene ORFs led to the identification of conserved and over-represented cis-motifs in both rice and Arabidopsis. Real-time quantitative RT-PCR analysis showed that several members of MYBs are up-regulated by various abiotic stresses both in rice and Arabidopsis. Conclusion A comprehensive genome-wide analysis of chromosomal distribution, tandem repeats and phylogenetic relationship of MYB family genes in rice and Arabidopsis suggested their evolution via duplication. Genome-wide comparative analysis of MYB genes and their expression analysis

  6. Transcriptional Profiles of Hybrid Eucalyptus Genotypes with Contrasting Lignin Content Reveal That Monolignol Biosynthesis-related Genes Regulate Wood Composition.

    Science.gov (United States)

    Shinya, Tomotaka; Iwata, Eiji; Nakahama, Katsuhiko; Fukuda, Yujiroh; Hayashi, Kazunori; Nanto, Kazuya; Rosa, Antonio C; Kawaoka, Akiyoshi

    2016-01-01

    Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, α-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants.

  7. Transcriptional profiles of hybrid Eucalyptus genotypes with contrasting lignin content reveal that monolignol biosynthesis-related genes regulate wood composition

    Directory of Open Access Journals (Sweden)

    Tomotaka eShinya

    2016-04-01

    Full Text Available Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected three-year old hybrid Eucalyptus (Eucalyptus urophylla x E. grandis genotypes (AM063 and AM380 that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0% and 48.2%, -cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA and sucrose synthase (SUSY were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase (UGP and xyloglucan endotransglucoxylase (XTH than those in AM380. Most monolignol biosynthesis- related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase (PAL, cinnamate-4-hydroxylase (C4H and 4-coumarate-CoA ligase (4CL. Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents

  8. Multiple independent analyses reveal only transcription factors as an enriched functional class associated with microRNAs

    Directory of Open Access Journals (Sweden)

    Croft Larry

    2012-07-01

    Full Text Available Abstract Background Transcription factors (TFs have long been known to be principally activators of transcription in eukaryotes and prokaryotes. The growing awareness of the ubiquity of microRNAs (miRNAs as suppressive regulators in eukaryotes, suggests the possibility of a mutual, preferential, self-regulatory connectivity between miRNAs and TFs. Here we investigate the connectivity from TFs and miRNAs to other genes and each other using text mining, TF promoter binding site and 6 different miRNA binding site prediction methods. Results In the first approach text mining of PubMed abstracts reveal statistically significant associations between miRNAs and both TFs and signal transduction gene classes. Secondly, prediction of miRNA targets in human and mouse 3’UTRs show enrichment only for TFs but not consistently across prediction methods for signal transduction or other gene classes. Furthermore, a random sample of 986 TarBase entries was scored for experimental evidence by manual inspection of the original papers, and enrichment for TFs was observed to increase with score. Low-scoring TarBase entries, where experimental evidence is anticorrelated miRNA:mRNA expression with predicted miRNA targets, appear not to select for real miRNA targets to any degree. Our manually validated text-mining results also suggests that miRNAs may be activated by more TFs than other classes of genes, as 7% of miRNA:TF co-occurrences in the literature were TFs activating miRNAs. This was confirmed when thirdly, we found enrichment for predicted, conserved TF binding sites in miRNA and TF genes compared to other gene classes. Conclusions We see enrichment of connections between miRNAs and TFs using several independent methods, suggestive of a network of mutual activating and suppressive regulation. We have also built regulatory networks (containing 2- and 3-loop motifs for mouse and human using predicted miRNA and TF binding sites and we have developed a web server

  9. Analysis of expression of transcription factors in early human retina.

    Science.gov (United States)

    Chen, Jing; Ma, Lixiang; Wang, Songtao; Wang, Xiaobing; Sun, Yan; Gao, Lu; Li, Jin; Zhou, Guomin

    2017-08-01

    The retina originates in the central nervous system. Due to its accessibility and simplicity, the retina has become an invaluable model for studying the basic mechanisms involved in development. To date, considerable knowledge regarding the interactions among genes that coordinate retinal development has been gained from extensive research in model animals. However, our understanding of retinal development in humans remains undeveloped. Here, we analyze the expression of transcription factors that are involved in the early development of the retina in human embryos at 6-12 weeks post-conception. Our work demonstrates that early developing neural retinas can be divided into two layers, the outer and inner neuroblast layers. Eye-field transcription factors and those related to the early development of the retina have distinct expression patterns in the two layers. Cell-type-specific transcription factors emerge at 8 weeks. These data provide clear and systemic structures for early retinal development in human. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

  10. Comparative transcriptional and genomic analysis of Plasmodium falciparum field isolates.

    Directory of Open Access Journals (Sweden)

    Margaret J Mackinnon

    2009-10-01

    Full Text Available Mechanisms for differential regulation of gene expression may underlie much of the phenotypic variation and adaptability of malaria parasites. Here we describe transcriptional variation among culture-adapted field isolates of Plasmodium falciparum, the species responsible for most malarial disease. It was found that genes coding for parasite protein export into the red cell cytosol and onto its surface, and genes coding for sexual stage proteins involved in parasite transmission are up-regulated in field isolates compared with long-term laboratory isolates. Much of this variability was associated with the loss of small or large chromosomal segments, or other forms of gene copy number variation that are prevalent in the P. falciparum genome (copy number variants, CNVs. Expression levels of genes inside these segments were correlated to that of genes outside and adjacent to the segment boundaries, and this association declined with distance from the CNV boundary. This observation could not be explained by copy number variation in these adjacent genes. This suggests a local-acting regulatory role for CNVs in transcription of neighboring genes and helps explain the chromosomal clustering that we observed here. Transcriptional co-regulation of physical clusters of adaptive genes may provide a way for the parasite to readily adapt to its highly heterogeneous and strongly selective environment.

  11. Comparative study between transcriptionally- and translationally-acting adenine riboswitches reveals key differences in riboswitch regulatory mechanisms.

    Directory of Open Access Journals (Sweden)

    Jean-François Lemay

    2011-01-01

    Full Text Available Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic. While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms.

  12. Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations

    Science.gov (United States)

    Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

    2017-01-01

    Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators. PMID:28301511

  13. Genome-wide identification, classification and analysis of heat shock transcription factor family in maize

    Directory of Open Access Journals (Sweden)

    Zhu Su-Wen

    2011-01-01

    Full Text Available Abstract Background Heat shock response in eukaryotes is transcriptionally regulated by conserved heat shock transcription factors (Hsfs. Hsf genes are represented by a large multigene family in plants and investigation of the Hsf gene family will serve to elucidate the mechanisms by which plants respond to stress. In recent years, reports of genome-wide structural and evolutionary analysis of the entire Hsf gene family have been generated in two model plant systems, Arabidopsis and rice. Maize, an important cereal crop, has represented a model plant for genetics and evolutionary research. Although some Hsf genes have been characterized in maize, analysis of the entire Hsf gene family were not completed following Maize (B73 Genome Sequencing Project. Results A genome-wide analysis was carried out in the present study to identify all Hsfs maize genes. Due to the availability of complete maize genome sequences, 25 nonredundant Hsf genes, named ZmHsfs were identified. Chromosomal location, protein domain and motif organization of ZmHsfs were analyzed in maize genome. The phylogenetic relationships, gene duplications and expression profiles of ZmHsf genes were also presented in this study. Twenty-five ZmHsfs were classified into three major classes (class A, B, and C according to their structural characteristics and phylogenetic comparisons, and class A was further subdivided into 10 subclasses. Moreover, phylogenetic analysis indicated that the orthologs from the three species (maize, Arabidopsis and rice were distributed in all three classes, it also revealed diverse Hsf gene family expression patterns in classes and subclasses. Chromosomal/segmental duplications played a key role in Hsf gene family expansion in maize by investigation of gene duplication events. Furthermore, the transcripts of 25 ZmHsf genes were detected in the leaves by heat shock using quantitative real-time PCR. The result demonstrated that ZmHsf genes exhibit different

  14. A Tutorial on Reliability Testing in AAC Language Sample Transcription and Analysis.

    Science.gov (United States)

    Kovacs, Thomas; Hill, Katya

    2015-06-01

    Establishing reliability is an essential step in language sample transcription and analysis. This tutorial provides an illustration of replicable procedures for reliability testing during transcription and analysis of language samples generated by people who use augmentative and alternative communication (AAC) systems. Statistical measures used for testing agreement between raters coding categorical data are summarized. Detailed procedures for reliability testing in AAC language sample transcription and analysis are provided, beginning with the collection of raw language sample data. Procedures include guidelines for (a) establishing inter-judge agreement during the transcription process, and (b) using Cohen's kappa to establish inter-rater reliability during deeper analysis of transcribed utterances. All procedures are demonstrated in a case example using language samples from children who use AAC.

  15. Transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans

    CSIR Research Space (South Africa)

    Crampton, Michael C

    2010-07-01

    Full Text Available This presentation focused on the transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans. It concludes to a successful implementation of a high throughput mRNA sandwich hybridisation...

  16. Transcriptome-Wide Identification and Expression Profiling Analysis of Chrysanthemum Trihelix Transcription Factors.

    Science.gov (United States)

    Song, Aiping; Wu, Dan; Fan, Qingqing; Tian, Chang; Chen, Sumei; Guan, Zhiyong; Xin, Jingjing; Zhao, Kunkun; Chen, Fadi

    2016-02-02

    Trihelix transcription factors are thought to feature a typical DNA-binding trihelix (helix-loop-helix-loop-helix) domain that binds specifically to the GT motif, a light-responsive DNA element. Members of the trihelix family are known to function in a number of processes in plants. Here, we characterize 20 trihelix family genes in the important ornamental plant chrysanthemum (Chrysanthemum morifolium). Based on transcriptomic data, 20 distinct sequences distributed across four of five groups revealed by a phylogenetic tree were isolated and amplified. The phylogenetic analysis also identified four pairs of orthologous proteins shared by Arabidopsis and chrysanthemum and five pairs of paralogous proteins in chrysanthemum. Conserved motifs in the trihelix proteins shared by Arabidopsis and chrysanthemum were analyzed using MEME, and further bioinformatic analysis revealed that 16 CmTHs can be targeted by 20 miRNA families and that miR414 can target 9 CmTHs. qPCR results displayed that most chrysanthemum trihelix genes were highly expressed in inflorescences, while 20 CmTH genes were in response to phytohormone treatments and abiotic stresses. This work improves our understanding of the various functions of trihelix gene family members in response to hormonal stimuli and stress.

  17. Mouse Obox and Crxos modulate preimplantation transcriptional profiles revealing similarity between paralogous mouse and human homeobox genes

    Directory of Open Access Journals (Sweden)

    Amy H. Royall

    2018-01-01

    Full Text Available Abstract Background ETCHbox genes are eutherian-specific homeobox genes expressed during preimplantation development at a time when the first cell lineage decisions are being made. The mouse has an unusual repertoire of ETCHbox genes with several gene families lost in evolution and the remaining two, Crxos and Obox, greatly divergent in sequence and number. Each has undergone duplication to give a double homeodomain Crxos locus and a large cluster of over 60 Obox loci. The gene content differences between species raise important questions about how evolution can tolerate loss of genes implicated in key developmental events. Results We find that Crxos internal duplication occurred in the mouse lineage, while Obox duplication was stepwise, generating subgroups with distinct sequence and expression. Ectopic expression of three Obox genes and a Crxos transcript in primary mouse embryonic cells followed by transcriptome sequencing allowed investigation into their functional roles. We find distinct transcriptomic influences for different Obox subgroups and Crxos, including modulation of genes related to zygotic genome activation and preparation for blastocyst formation. Comparison with similar experiments performed using human homeobox genes reveals striking overlap between genes downstream of mouse Crxos and genes downstream of human ARGFX. Conclusions Mouse Crxos and human ARGFX homeobox genes are paralogous rather than orthologous, yet they have evolved to regulate a common set of genes. This suggests there was compensation of function alongside gene loss through co-option of a different locus. Functional compensation by non-orthologous genes with dissimilar sequences is unusual but may indicate underlying distributed robustness. Compensation may be driven by the strong evolutionary pressure for successful early embryo development.

  18. A transcriptional analysis of carotenoid, chlorophyll and plastidial isoprenoid biosynthesis genes during development and osmotic stress responses in Arabidopsis thaliana

    KAUST Repository

    Meier, Stuart

    2011-05-19

    Background: The carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana.Results: A global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR) but was inhibited by abscisic acid (ABA). Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs) and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced and uncoupled from that of

  19. Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding.

    Science.gov (United States)

    de Castro, Minique Hilda; de Klerk, Daniel; Pienaar, Ronel; Rees, D Jasper G; Mans, Ben J

    2017-08-10

    Ticks secrete a diverse mixture of secretory proteins into the host to evade its immune response and facilitate blood-feeding, making secretory proteins attractive targets for the production of recombinant anti-tick vaccines. The largely neglected tick species, Rhipicephalus zambeziensis, is an efficient vector of Theileria parva in southern Africa but its available sequence information is limited. Next generation sequencing has advanced sequence availability for ticks in recent years and has assisted the characterisation of secretory proteins. This study focused on the de novo assembly and annotation of the salivary gland transcriptome of R. zambeziensis and the temporal expression of secretory protein transcripts in female and male ticks, before the onset of feeding and during early and late feeding. The sialotranscriptome of R. zambeziensis yielded 23,631 transcripts from which 13,584 non-redundant proteins were predicted. Eighty-six percent of these contained a predicted start and stop codon and were estimated to be putatively full-length proteins. A fifth (2569) of the predicted proteins were annotated as putative secretory proteins and explained 52% of the expression in the transcriptome. Expression analyses revealed that 2832 transcripts were differentially expressed among feeding time points and 1209 between the tick sexes. The expression analyses further indicated that 57% of the annotated secretory protein transcripts were differentially expressed. Dynamic expression profiles of secretory protein transcripts were observed during feeding of female ticks. Whereby a number of transcripts were upregulated during early feeding, presumably for feeding site establishment and then during late feeding, 52% of these were downregulated, indicating that transcripts were required at specific feeding stages. This suggested that secretory proteins are under stringent transcriptional regulation that fine-tunes their expression in salivary glands during feeding. No open

  20. Quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the JAK-STAT1 signaling pathway.

    Science.gov (United States)

    An, Kang; Fang, Liurong; Luo, Rui; Wang, Dang; Xie, Lilan; Yang, Jing; Chen, Huanchun; Xiao, Shaobo

    2014-12-05

    Transmissible gastroenteritis virus (TGEV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea and severe dehydration in piglets. In this study, liquid chromatography-tandem mass spectrometry coupled to isobaric tags for relative and absolute quantification labeling was used to quantitatively identify differentially expressed cellular proteins after TGEV infection in PK-15 cells. In total, 162 differentially expressed cellular proteins were identified, including 60 upregulated proteins and 102 downregulated proteins. These differentially expressed proteins were involved in the cell cycle, cellular growth and proliferation, the innate immune response, etc. Interestingly, many upregulated proteins were associated with interferon signaling, especially signal transducer and activator of transcription 1 (STAT1) and interferon-stimulated genes (ISGs). Immunoblotting and real-time quantitative reverse transcription polymerase chain reaction demonstrated that TGEV infection induces STAT1 phosphorylation and nuclear translocation, as well as ISG expression. This study for the first time reveals that TGEV induces interferon signaling from the point of proteomic analysis.

  1. Deubiquitylase Inhibition Reveals Liver X Receptor-independent Transcriptional Regulation of the E3 Ubiquitin Ligase IDOL and Lipoprotein Uptake

    NARCIS (Netherlands)

    Nelson, Jessica Kristine; Cook, Emma Clare Laura; Loregger, Anke; Hoeksema, Marten Anne; Scheij, Saskia; Kovacevic, Igor; Hordijk, Peter Lodewijk; Ovaa, Huib; Zelcer, Noam

    2016-01-01

    Cholesterol metabolism is subject to complex transcriptional and nontranscriptional regulation. Herein, the role of ubiquitylation is emerging as an important post-translational modification that regulates cholesterol synthesis and uptake. Similar to other post-translational modifications,

  2. DNA methylation-based chromatin compartments and ChIP-seq profiles reveal transcriptional drivers of prostate carcinogenesis.

    Science.gov (United States)

    Simmonds, Poppy; Loomis, Erick; Curry, Edward

    2017-06-07

    Profiles of DNA methylation of many tissues relevant in human disease have been obtained from microarrays and are publicly available. These can be used to generate maps of chromatin compartmentalization, demarcating open and closed chromatin across the genome. Additionally, large sets of genome-wide transcription factor binding profiles have been made available thanks to ChIP-seq technology. We have identified genomic regions with altered chromatin compartmentalization in prostate adenocarcinoma tissue relative to normal prostate tissue, using DNA methylation microarray data from The Cancer Genome Atlas. DNA binding profiles from the Encyclopedia of DNA Elements (ENCODE) ChIP-seq studies have been systematically screened to find transcription factors with inferred DNA binding sites located in discordantly open/closed chromatin in malignant tissue (compared with non-cancer control tissue). We have combined this with tests for corresponding up-/downregulation of the transcription factors' putative target genes to obtain an integrated measure of cancer-specific regulatory activity to identify likely transcriptional drivers of prostate cancer. Generally, we find that the degree to which transcription factors preferentially bind regions of chromatin that become more accessible during prostate carcinogenesis is significantly associated to the level of systematic upregulation of their targets, at the level of gene expression. Our approach has yielded 11 transcription factors that show strong cancer-specific transcriptional activation of targets, including the novel candidates KAT2A and TRIM28, alongside established drivers of prostate cancer MYC, ETS1, GABP and YY1. This approach to integrated epigenetic and transcriptional profiling using publicly available data represents a cheap and powerful technique for identifying potential drivers of human disease. In our application to prostate adenocarcinoma data, the fact that well-known drivers are amongst the top candidates

  3. Integration of transcriptomic and metabolic data reveals hub transcription factors involved in drought stress response in sunflower (Helianthus annuus L.).

    Science.gov (United States)

    Moschen, Sebastián; Di Rienzo, Julio A; Higgins, Janet; Tohge, Takayuki; Watanabe, Mutsumi; González, Sergio; Rivarola, Máximo; García-García, Francisco; Dopazo, Joaquin; Hopp, H Esteban; Hoefgen, Rainer; Fernie, Alisdair R; Paniego, Norma; Fernández, Paula; Heinz, Ruth A

    2017-07-01

    By integration of transcriptional and metabolic profiles we identified pathways and hubs transcription factors regulated during drought conditions in sunflower, useful for applications in molecular and/or biotechnological breeding. Drought is one of the most important environmental stresses that effects crop productivity in many agricultural regions. Sunflower is tolerant to drought conditions but the mechanisms involved in this tolerance remain unclear at the molecular level. The aim of this study was to characterize and integrate transcriptional and metabolic pathways related to drought stress in sunflower plants, by using a system biology approach. Our results showed a delay in plant senescence with an increase in the expression level of photosynthesis related genes as well as higher levels of sugars, osmoprotectant amino acids and ionic nutrients under drought conditions. In addition, we identified transcription factors that were upregulated during drought conditions and that may act as hubs in the transcriptional network. Many of these transcription factors belong to families implicated in the drought response in model species. The integration of transcriptomic and metabolomic data in this study, together with physiological measurements, has improved our understanding of the biological responses during droughts and contributes to elucidate the molecular mechanisms involved under this environmental condition. These findings will provide useful biotechnological tools to improve stress tolerance while maintaining crop yield under restricted water availability.

  4. Systematic mutagenesis of all predicted gntR genes in Xanthomonas campestris pv. campestris reveals a GntR family transcriptional regulator controlling hypersensitive response and virulence.

    Science.gov (United States)

    An, Shi-Qi; Lu, Guang-Tao; Su, Hui-Zhao; Li, Rui-Fang; He, Yong-Qiang; Jiang, Bo-Le; Tang, Dong-Jie; Tang, Ji-Liang

    2011-09-01

    The GntR family is one of the most abundant and widely distributed groups of helix-turn-helix transcriptional regulators in bacteria. Six open reading frames in the genome of the plant pathogen Xanthomonas campestris pv. campestris were predicted to encode GntR regulators. All six of the predicted GntR-encoding genes were individually mutagenized and mutants from five of them were successfully obtained. Plant disease response assays revealed that one, whose product belongs to the YtrA subfamily and has been named HpaR1, is involved in the hypersensitive response (HR) and virulence. Electrophoretic mobility shift assays and in vitro transcription assays revealed that HpaR1 could repress its own transcription level through binding to its promoter sequence, indicating an autoregulatory feedback inhibition mechanism for HpaR1 expression. Promoter-gusA reporter and reverse-transcription polymerase chain reaction analyses revealed that HpaR1 positively and negatively affects the expression of HR and pathogenicity (hrp) genes in host plant and standard media, respectively. Constitutive expression of the key hrp regulator, hrpG, in the hpaR1 mutant could bypass the requirement of HpaR1 for the induction of wild-type HR, suggesting that HpaR1 regulates the expression of hrp genes that encode the type III secretion system via hrpG.

  5. De Novo Transcriptional Analysis of Alfalfa in Response to Saline-Alkaline Stress.

    Science.gov (United States)

    An, Yi-Min; Song, Li-Li; Liu, Ying-Rui; Shu, Yong-Jun; Guo, Chang-Hong

    2016-01-01

    Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress that affects crop productivity. Alfalfa is a widely cultivated perennial forage legume with some tolerance to biotic and abiotic stresses, especially to saline-alkaline stress. To elucidate the mechanism underlying plant saline-alkaline tolerance, we conducted transcriptome analysis of whole alfalfa seedlings treated with saline-alkaline solutions for 0 day (control), 1 day (short-term treatment), and 7 days (long-term treatment) using ion torrent sequencing technology. A transcriptome database dataset of 53,853 unigenes was generated, and 2,286 and 2,233 genes were differentially expressed in the short-term and long-term treatment, respectively. Gene ontology analysis revealed 14 highly enriched pathways and demonstrated the differential response of metabolic pathways between the short-term and long-term treatment. The expression levels of 109 and 96 transcription factors were significantly altered significantly after 1 day and 7 days of treatment, respectively. Specific responses of peroxidase, flavonoids, and the light pathway component indicated that the antioxidant capacity was one of the central mechanisms of saline-alkaline stress tolerance response in alfalfa. Among the 18 differentially expressed genes examined by real time PCR, the expression levels of eight genes, including inositol transporter, DNA binding protein, raffinose synthase, ferritin, aldo/keto reductase, glutathione S-transferase, xyloglucan endotrans glucosylase, and a NAC transcription factor, exhibited different patterns in response to saline and alkaline stress. The expression levels of the NAC transcription factor and glutathione S-transferase were altered significantly under saline stress and saline-alkaline stress; they were upregulated under saline-alkaline stress and downregulated under salt stress. Physiology assays showed an increased concentration of reactive oxygen

  6. Transcriptional analysis of the mammalian heart with special reference to its endocrine function

    Directory of Open Access Journals (Sweden)

    de Bold Adolfo J

    2009-06-01

    Full Text Available Abstract Background Pharmacological and gene ablation studies have demonstrated the crucial role of the endocrine function of the heart as mediated by the polypeptide hormones ANF and BNP in the maintenance of cardiovascular homeostasis. The importance of these studies lies on the fact that hypertension and chronic congestive heart failure are clinical entities that may be regarded as states of relative deficiency of ANF and BNP. These hormones are produced by the atrial muscle cells (cardiocytes, which display a dual secretory/muscle phenotype. In contrast, ventricular cardiocytes display mainly a muscle phenotype. Comparatively little information is available regarding the genetic background for this important phenotypic difference with particular reference to the endocrine function of the heart. We postulated that comparison of gene expression profiles between atrial and ventricular muscles would help identify gene transcripts that underlie the phenotypic differences associated with the endocrine function of the heart. Results Comparison of gene expression profiles in the rat heart revealed a total of 1415 differentially expressed genes between the atria and ventricles based on a 1.8 fold cut-off. The identification of numerous chamber specific transcripts, such as ANF for the atria and Irx4 for the ventricles among several others, support the soundness of the GeneChip data and demonstrates that the differences in gene expression profiles observed between the atrial and ventricular tissues were not spurious in nature. Pathway analysis revealed unique expression profiles in the atria for G protein signaling that included Gαo1, Gγ2 and Gγ3, AGS1, RGS2, and RGS6 and the related K+ channels GIRK1 and GIRK4. Transcripts involved in vesicle trafficking, hormone secretion as well as mechanosensors (e.g. the potassium channel TREK-1 were identified in relationship to the synthesis, storage and secretion of hormones. Conclusion The data developed

  7. Transcriptional analysis of genes on human chromosome 21

    Energy Technology Data Exchange (ETDEWEB)

    Chiang, P.W.; Van Kueren, M.L.; Kurnit, D.M. [Univ. of Michigan, Ann Arbor, MI (United States)] [and others

    1994-09-01

    The polymerase chain reaction (PCR) was used to screen embryonic, fetal and adult human cDNA libraries for transcription on chromosome 21q22.1{yields}21q22.3. 73 pairs of oligonucleotide primers on chromosome 21 were applied to analyze 41 different cDNA libraries. Only phage eluate (and therefore no DNA isolation) was required for this sensitive screening. This technique was first used to demonstrate that huntingtin is expressed ubiquitiously throughout human life. 60 of the primers were positive with at least one library screening, indicating that the majority of primers derived from transcribed sequences. We examined embryonic (which we synthesized), fetal and adult human cDNA libraries. Even with our most complex human fetal brain library, we detected only ca. 50% of transcribed sequences, underscoring the need to screen multiple human cDNA libraries to determine if transcription occurred. Since only 2/73 clones were present in only one cDNA library, the vast majority of transcribed sequences are present in more than one tissue. Because this study involves random genes rather than genes selected because of their import, this demonstrates that only few anonymous genes are transcribed in a single tissue type. Expression of genes in different cDNA libraries aids in comparison between genotype and phenotype. Using combinations of gene-specific and vector-specific primers, we went on to develop a PCR-based technology that retrieves the largest sequences present in a cDNA library. This technology was used to isolate large genes on chromosome 21 that can be used in expression studies to examine the Down syndrome phenotype.

  8. Integrated Analysis of the Effects of Cold and Dehydration on Rice Metabolites, Phytohormones, and Gene Transcripts1[W][OPEN

    Science.gov (United States)

    Maruyama, Kyonoshin; Urano, Kaoru; Yoshiwara, Kyouko; Morishita, Yoshihiko; Sakurai, Nozomu; Suzuki, Hideyuki; Kojima, Mikiko; Sakakibara, Hitoshi; Shibata, Daisuke; Saito, Kazuki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2014-01-01

    Correlations between gene expression and metabolite/phytohormone levels under abiotic stress conditions have been reported for Arabidopsis (Arabidopsis thaliana). However, little is known about these correlations in rice (Oryza sativa ‘Nipponbare’), despite its importance as a model monocot. We performed an integrated analysis to clarify the relationships among cold- and dehydration-responsive metabolites, phytohormones, and gene transcription in rice. An integrated analysis of metabolites and gene expression indicated that several genes encoding enzymes involved in starch degradation, sucrose metabolism, and the glyoxylate cycle are up-regulated in rice plants exposed to cold or dehydration and that these changes are correlated with the accumulation of glucose (Glc), fructose, and sucrose. In particular, high expression levels of genes encoding isocitrate lyase and malate synthase in the glyoxylate cycle correlate with increased Glc levels in rice, but not in Arabidopsis, under dehydration conditions, indicating that the regulation of the glyoxylate cycle may be involved in Glc accumulation under dehydration conditions in rice but not Arabidopsis. An integrated analysis of phytohormones and gene transcripts revealed an inverse relationship between abscisic acid (ABA) signaling and cytokinin (CK) signaling under cold and dehydration stresses; these stresses increase ABA signaling and decrease CK signaling. High levels of Oryza sativa 9-cis-epoxycarotenoid dioxygenase transcripts correlate with ABA accumulation, and low levels of Cytochrome P450 (CYP) 735A transcripts correlate with decreased levels of a CK precursor in rice. This reduced expression of CYP735As occurs in rice but not Arabidopsis. Therefore, transcriptional regulation of CYP735As might be involved in regulating CK levels under cold and dehydration conditions in rice but not Arabidopsis. PMID:24515831

  9. Integration of transcript expression, copy number and LOH analysis of infiltrating ductal carcinoma of the breast

    Directory of Open Access Journals (Sweden)

    Hawthorn Lesleyann

    2010-08-01

    Full Text Available Abstract Background A major challenge in the interpretation of genomic profiling data generated from breast cancer samples is the identification of driver genes as distinct from bystander genes which do not impact tumorigenesis. One way to assess the relative importance of alterations in the transcriptome profile is to combine parallel analyses that assess changes in the copy number alterations (CNAs. This integrated analysis permits the identification of genes with altered expression that map within specific chromosomal regions which demonstrate copy number alterations, providing a mechanistic approach to identify the 'driver genes'. Methods We have performed whole genome analysis of CNAs using the Affymetrix 250K Mapping array on 22 infiltrating ductal carcinoma samples (IDCs. Analysis of transcript expression alterations was performed using the Affymetrix U133 Plus2.0 array on 16 IDC samples. Fourteen IDC samples were analyzed using both platforms and the data integrated. We also incorporated data from loss of heterozygosity (LOH analysis to identify genes showing altered expression in LOH regions. Results Common chromosome gains and amplifications were identified at 1q21.3, 6p21.3, 7p11.2-p12.1, 8q21.11 and 8q24.3. A novel amplicon was identified at 5p15.33. Frequent losses were found at 1p36.22, 8q23.3, 11p13, 11q23, and 22q13. Over 130 genes were identified with concurrent increases or decreases in expression that mapped to these regions of copy number alterations. LOH analysis revealed three tumors with whole chromosome or p arm allelic loss of chromosome 17. Genes were identified that mapped to copy neutral LOH regions. LOH with accompanying copy loss was detected on Xp24 and Xp25 and genes mapping to these regions with decreased expression were identified. Gene expression data highlighted the PPARα/RXRα Activation Pathway as down-regulated in the tumor samples. Conclusion We have demonstrated the utility of the application of

  10. Comparative transcript profiling of alloplasmic male-sterile lines revealed altered gene expression related to pollen development in rice (Oryza sativa L.).

    Science.gov (United States)

    Hu, Jihong; Chen, Guanglong; Zhang, Hongyuan; Qian, Qian; Ding, Yi

    2016-08-05

    Cytoplasmic male sterility (CMS) is an ideal model for investigating the mitochondrial-nuclear interaction and down-regulated genes in CMS lines which might be the candidate genes for pollen development in rice. In this study, a set of rice alloplasmic sporophytic CMS lines was obtained by successive backcrossing of Meixiang B, with three different cytoplasmic types: D62A (D type), ZS97A (WA type) and XQZ-A (DA type). Using microarray, the anther transcript profiles of the three indica rice CMS lines revealed 622 differentially expressed genes (DEGs) in each of the three CMS lines compared with the maintainer line Meixiang B. GO and MapMan analysis indicated that these DEGs were mainly involved in lipid metabolism and cell wall organization. Compared with the gene expression of sporophytic and gametophytic CMS lines, 303 DEGs were identified and 56 of them were down-regulated in all the CMS lines of rice. These down-regulated DEGs in the CMS lines were found to be involved in tapetum or cell wall formation and their suppressed expression might be related to male sterility. Weighted gene co-expression network analysis (WGCNA) revealed that two modules were significantly associated with male sterility and many hub genes that were differentially expressed in the CMS lines. A large set of putative genes involved in anther development was identified in the present study. The results will give some information for the nuclear gene regulation by different cytoplasmic genotypes and provide a rich resource for further functional research on the pollen development in rice.

  11. Comparative transcriptional profiling of 3 murine models of SLE nephritis reveals both unique and shared regulatory networks.

    Directory of Open Access Journals (Sweden)

    Ramalingam Bethunaickan

    Full Text Available To define shared and unique features of SLE nephritis in mouse models of proliferative and glomerulosclerotic renal disease.Perfused kidneys from NZB/W F1, NZW/BXSB and NZM2410 mice were harvested before and after nephritis onset. Affymetrix based gene expression profiles of kidney RNA were analyzed using Genomatix Pathway Systems and Ingenuity Pathway Analysis software. Gene expression patterns were confirmed using real-time PCR.955, 1168 and 755 genes were regulated in the kidneys of nephritic NZB/W F1, NZM2410 and NZW/BXSB mice respectively. 263 genes were regulated concordantly in all three strains reflecting immune cell infiltration, endothelial cell activation, complement activation, cytokine signaling, tissue remodeling and hypoxia. STAT3 was the top associated transcription factor, having a binding site in the gene promoter of 60/263 regulated genes. The two strains with proliferative nephritis shared a macrophage/DC infiltration and activation signature. NZB/W and NZM2410 mice shared a mitochondrial dysfunction signature. Dominant T cell and plasma cell signatures in NZB/W mice reflected lymphoid aggregates; this was the only strain with regulatory T cell infiltrates. NZW/BXSB mice manifested tubular regeneration and NZM2410 mice had the most metabolic stress and manifested loss of nephrin, indicating podocyte loss.These findings identify shared inflammatory mechanisms of SLE nephritis that can be therapeutically targeted. Nevertheless, the heterogeneity of effector mechanisms suggests that individualized therapy might need to be based on biopsy findings. Some common mechanisms are shared with non-immune-mediated renal diseases, suggesting that strategies to prevent tissue hypoxia and remodeling may be useful in SLE nephritis.

  12. The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms.

    LENUS (Irish Health Repository)

    Hamilton, Shea

    2009-12-11

    Abstract Background Biofilm formation enhances the capacity of pathogenic Salmonella bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir for the contamination of food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most gene expression studies of Salmonella have focused on the effect of infection-relevant stressors on virulence or the comparison of mutant and wild-type bacteria. However little is known about the physiological responses taking place inside a Salmonella biofilm. Results We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% of the S. Typhimurium genome (433 genes) showed a 2-fold or more change in the biofilm compared with planktonic cells. The genes that were significantly up-regulated implicated certain cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), and that a functional SPI2 secretion system regulator (ssrA) was required for S. Typhimurium biofilm formation. We identified STM0341 as a gene of unknown function that was needed for biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to decreased bacterial attachment and this biofilm defect was restored by

  13. RNA-Seq for enrichment and analysis of IRF5 transcript expression in SLE.

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    Rivka C Stone

    Full Text Available Polymorphisms in the interferon regulatory factor 5 (IRF5 gene have been consistently replicated and shown to confer risk for or protection from the development of systemic lupus erythematosus (SLE. IRF5 expression is significantly upregulated in SLE patients and upregulation associates with IRF5-SLE risk haplotypes. IRF5 alternative splicing has also been shown to be elevated in SLE patients. Given that human IRF5 exists as multiple alternatively spliced transcripts with distinct function(s, it is important to determine whether the IRF5 transcript profile expressed in healthy donor immune cells is different from that expressed in SLE patients. Moreover, it is not currently known whether an IRF5-SLE risk haplotype defines the profile of IRF5 transcripts expressed. Using standard molecular cloning techniques, we identified and isolated 14 new differentially spliced IRF5 transcript variants from purified monocytes of healthy donors and SLE patients to generate an IRF5 variant transcriptome. Next-generation sequencing was then used to perform in-depth and quantitative analysis of full-length IRF5 transcript expression in primary immune cells of SLE patients and healthy donors by next-generation sequencing. Evidence for additional alternatively spliced transcripts was obtained from de novo junction discovery. Data from these studies support the overall complexity of IRF5 alternative splicing in SLE. Results from next-generation sequencing correlated with cloning and gave similar abundance rankings in SLE patients thus supporting the use of this new technology for in-depth single gene transcript profiling. Results from this study provide the first proof that 1 SLE patients express an IRF5 transcript signature that is distinct from healthy donors, 2 an IRF5-SLE risk haplotype defines the top four most abundant IRF5 transcripts expressed in SLE patients, and 3 an IRF5 transcript signature enables clustering of SLE patients with the H2 risk haplotype.

  14. A novel approach for transcription factor analysis using SELEX with high-throughput sequencing (TFAST.

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    Daniel J Reiss

    Full Text Available BACKGROUND: In previous work, we designed a modified aptamer-free SELEX-seq protocol (afSELEX-seq for the discovery of transcription factor binding sites. Here, we present original software, TFAST, designed to analyze afSELEX-seq data, validated against our previously generated afSELEX-seq dataset and a model dataset. TFAST is designed with a simple graphical interface (Java so that it can be installed and executed without extensive expertise in bioinformatics. TFAST completes analysis within minutes on most personal computers. METHODOLOGY: Once afSELEX-seq data are aligned to a target genome, TFAST identifies peaks and, uniquely, compares peak characteristics between cycles. TFAST generates a hierarchical report of graded peaks, their associated genomic sequences, binding site length predictions, and dummy sequences. PRINCIPAL FINDINGS: Including additional cycles of afSELEX-seq improved TFAST's ability to selectively identify peaks, leading to 7,274, 4,255, and 2,628 peaks identified in two-, three-, and four-cycle afSELEX-seq. Inter-round analysis by TFAST identified 457 peaks as the strongest candidates for true binding sites. Separating peaks by TFAST into classes of worst, second-best and best candidate peaks revealed a trend of increasing significance (e-values 4.5 × 10(12, 2.9 × 10(-46, and 1.2 × 10(-73 and informational content (11.0, 11.9, and 12.5 bits over 15 bp of discovered motifs within each respective class. TFAST also predicted a binding site length (28 bp consistent with non-computational experimentally derived results for the transcription factor PapX (22 to 29 bp. CONCLUSIONS/SIGNIFICANCE: TFAST offers a novel and intuitive approach for determining DNA binding sites of proteins subjected to afSELEX-seq. Here, we demonstrate that TFAST, using afSELEX-seq data, rapidly and accurately predicted sequence length and motif for a putative transcription factor's binding site.

  15. Metabolite analysis of Mycobacterium species under aerobic and hypoxic conditions reveals common metabolic traits.

    Science.gov (United States)

    Drapal, Margit; Wheeler, Paul R; Fraser, Paul D

    2016-08-01

    A metabolite profiling approach has been implemented to elucidate metabolic adaptation at set culture conditions in five Mycobacterium species (two fast- and three slow-growing) with the potential to act as model organisms for Mycobacterium tuberculosis (Mtb). Analysis has been performed over designated growth phases and under representative environments (nutrient and oxygen depletion) experienced by Mtb during infection. The procedure was useful in determining a range of metabolites (60-120 compounds) covering nucleotides, amino acids, organic acids, saccharides, fatty acids, glycerols, -esters, -phosphates and isoprenoids. Among these classes of compounds, key biomarker metabolites, which can act as indicators of pathway/process activity, were identified. In numerous cases, common metabolite traits were observed for all five species across the experimental conditions (e.g. uracil indicating DNA repair). Amino acid content, especially glutamic acid, highlighted the different properties between the fast- and slow-growing mycobacteria studied (e.g. nitrogen assimilation). The greatest similarities in metabolite composition between fast- and slow-growing mycobacteria were apparent under hypoxic conditions. A comparison to previously reported transcriptomic data revealed a strong correlation between changes in transcription and metabolite content. Collectively, these data validate the changes in the transcription at the metabolite level, suggesting transcription exists as one of the predominant modes of cellular regulation in Mycobacterium. Sectors with restricted correlation between metabolites and transcription (e.g. hypoxic cultivation) warrant further study to elucidate and exploit post-transcriptional modes of regulation. The strong correlation between the laboratory conditions used and data derived from in vivo conditions, indicate that the approach applied is a valuable addition to our understanding of cell regulation in these Mycobacterium species.

  16. A Resource for the Transcriptional Signature of Bona Fide Trophoblast Stem Cells and Analysis of Their Embryonic Persistence

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    Georg Kuales

    2015-01-01

    Full Text Available Trophoblast stem cells (TSCs represent the multipotent progenitors that give rise to the different cells of the embryonic portion of the placenta. Here, we analysed the expression of key TSC transcription factors Cdx2, Eomes, and Elf5 in the early developing placenta of mouse embryos and in cultured TSCs and reveal surprising heterogeneity in protein levels. We analysed persistence of TSCs in the early placenta and find that TSCs remain in the chorionic hinge until E9.5 and are lost shortly afterwards. To define the transcriptional signature of bona fide TSCs, we used inducible gain- and loss-of-function alleles of Eomes or Cdx2, and EomesGFP, to manipulate and monitor the core maintenance factors of TSCs, followed by genome-wide expression profiling. Combinatorial analysis of resulting expression profiles allowed for defining novel TSC marker genes that might functionally contribute to the maintenance of the TSC state. Analyses by qRT-PCR and in situ hybridisation validated novel TSC- and chorion-specific marker genes, such as Bok/Mtd, Cldn26, Duox2, Duoxa2, Nr0b1, and Sox21. Thus, these expression data provide a valuable resource for the transcriptional signature of bona fide and early differentiating TSCs and may contribute to an increased understanding of the transcriptional circuitries that maintain and/or establish stemness of TSCs.

  17. Smith-Magenis syndrome results in disruption of CLOCK gene transcription and reveals an integral role for RAI1 in the maintenance of circadian rhythmicity.

    Science.gov (United States)

    Williams, Stephen R; Zies, Deborah; Mullegama, Sureni V; Grotewiel, Michael S; Elsea, Sarah H

    2012-06-08

    Haploinsufficiency of RAI1 results in Smith-Magenis syndrome (SMS), a disorder characterized by intellectual disability, multiple congenital anomalies, obesity, neurobehavioral abnormalities, and a disrupted circadian sleep-wake pattern. An inverted melatonin rhythm (i.e., melatonin peaks during the day instead of at night) and associated sleep-phase disturbances in individuals with SMS, as well as a short-period circadian rhythm in mice with a chromosomal deletion of Rai1, support SMS as a circadian-rhythm-dysfunction disorder. However, the molecular cause of the circadian defect in SMS has not been described. The circadian oscillator temporally orchestrates metabolism, physiology, and behavior largely through transcriptional modulation. Data support RAI1 as a transcriptional regulator, but the genes it might regulate are largely unknown. Investigation into the role that RAI1 plays in the regulation of gene transcription and circadian maintenance revealed that RAI1 regulates the transcription of circadian locomotor output cycles kaput (CLOCK), a key component of the mammalian circadian oscillator that transcriptionally regulates many critical circadian genes. Data further show that haploinsufficiency of RAI1 and Rai1 in SMS fibroblasts and the mouse hypothalamus, respectively, results in the transcriptional dysregulation of the circadian clock and causes altered expression and regulation of multiple circadian genes, including PER2, PER3, CRY1, BMAL1, and others. These data suggest that heterozygous mutation of RAI1 and Rai1 leads to a disrupted circadian rhythm and thus results in an abnormal sleep-wake cycle, which can contribute to an abnormal feeding pattern and dependent cognitive performance. Finally, we conclude that RAI1 is a positive transcriptional regulator of CLOCK, pinpointing a novel and important role for this gene in the circadian oscillator. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  18. Inferring a transcriptional regulatory network of the cytokinesis-related genes by network component analysis

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    Kao Cheng-Yan

    2009-11-01

    Full Text Available Abstract Background Network Component Analysis (NCA is a network structure-driven framework for deducing regulatory signal dynamics. In contrast to principal component analysis, which can be employed to select the high-variance genes, NCA makes use of the connectivity structure from transcriptional regulatory networks to infer dynamics of transcription factor activities. Using the budding yeast Saccharomyces cerevisiae as a model system, we aim to deduce regulatory actions of cytokinesis-related genes, using precise spatial proximity (midbody and/or temporal synchronicity (cytokinesis to avoid full-scale computation from genome-wide databases. Results NCA was applied to infer regulatory actions of transcription factor activity from microarray data and partial transcription factor-gene connectivity information for cytokinesis-related genes, which were a subset of genome-wide datasets. No literature has so far discussed the inferred results through NCA are independent of the scale of the gene expression dataset. To avoid full-scale computation from genome-wide databases, four cytokinesis-related gene cases were selected for NCA by running computational analysis over the transcription factor database to confirm the approach being scale-free. The inferred dynamics of transcription factor activity through NCA were independent of the scale of the data matrix selected from the four cytokinesis-related gene sets. Moreover, the inferred regulatory actions were nearly identical to published observations for the selected cytokinesis-related genes in the budding yeast; namely, Mcm1, Ndd1, and Fkh2, which form a transcription factor complex to control expression of the CLB2 cluster (i.e. BUD4, CHS2, IQG1, and CDC5. Conclusion In this study, using S. cerevisiae as a model system, NCA was successfully applied to infer similar regulatory actions of transcription factor activities from two various microarray databases and several partial transcription factor

  19. Inferring a transcriptional regulatory network of the cytokinesis-related genes by network component analysis.

    Science.gov (United States)

    Chen, Shun-Fu; Juang, Yue-Li; Chou, Wei-Kang; Lai, Jin-Mei; Huang, Chi-Ying F; Kao, Cheng-Yan; Wang, Feng-Sheng

    2009-11-27

    Network Component Analysis (NCA) is a network structure-driven framework for deducing regulatory signal dynamics. In contrast to principal component analysis, which can be employed to select the high-variance genes, NCA makes use of the connectivity structure from transcriptional regulatory networks to infer dynamics of transcription factor activities. Using the budding yeast Saccharomyces cerevisiae as a model system, we aim to deduce regulatory actions of cytokinesis-related genes, using precise spatial proximity (midbody) and/or temporal synchronicity (cytokinesis) to avoid full-scale computation from genome-wide databases. NCA was applied to infer regulatory actions of transcription factor activity from microarray data and partial transcription factor-gene connectivity information for cytokinesis-related genes, which were a subset of genome-wide datasets. No literature has so far discussed the inferred results through NCA are independent of the scale of the gene expression dataset. To avoid full-scale computation from genome-wide databases, four cytokinesis-related gene cases were selected for NCA by running computational analysis over the transcription factor database to confirm the approach being scale-free. The inferred dynamics of transcription factor activity through NCA were independent of the scale of the data matrix selected from the four cytokinesis-related gene sets. Moreover, the inferred regulatory actions were nearly identical to published observations for the selected cytokinesis-related genes in the budding yeast; namely, Mcm1, Ndd1, and Fkh2, which form a transcription factor complex to control expression of the CLB2 cluster (i.e. BUD4, CHS2, IQG1, and CDC5). In this study, using S. cerevisiae as a model system, NCA was successfully applied to infer similar regulatory actions of transcription factor activities from two various microarray databases and several partial transcription factor-gene connectivity datasets for selected cytokinesis

  20. Transcriptional profiling and in silico analysis of Dof transcription factor gene family for understanding their regulation during seed development of rice Oryza sativa L.

    Science.gov (United States)

    Gaur, Vikram Singh; Singh, U S; Kumar, Anil

    2011-04-01

    Seed development is a complex process controlled by temporal and spatial expression of many transcription factors (TF) inside the developing seed. In the present study, transcript profiles of all the 30 members of rice DofTFs from flowering to seed development stages were analyzed. It was found that 16 Dof genes besides a previously characterized Dof gene 'RPBF' are differentially expressed during the seed development and unlike RPBF are not seed specific. Based on the expression patterns, these rice DofTFs were categorized into four groups-6 genes were constitutive while 4 genes were up-regulated and 3 genes were down regulated and four genes were maximally expressed at specific stages of seed development viz. one gene at flowering, two genes at watery ripe and one gene at milky stage. The involvement of more than one gene at different stages of seed development is suggestive of combinatorial regulation of their downstream genes involved in seed development. In silico expression analysis of wheat and Arabidopsis Dof Tfs also revealed that more than 50% of the Dof genes are expressed during the seed development process. Further in silico study of regulatory elements present in the promoters of these genes revealed the presence of some unique and common motifs in the promoters of rice and wheat Dof genes which indicate that Dof genes are possibly involved in ethylene and jasmonate signaling pathways affecting grain filling and grain quality. These Dof genes containing ethylene responsive motifs in their promoter region could possibly be the targets of recently identified Sub1 gene which codes for a ethylene responsive factor.

  1. Deubiquitylase Inhibition Reveals Liver X Receptor-independent Transcriptional Regulation of the E3 Ubiquitin Ligase IDOL and Lipoprotein Uptake.

    Science.gov (United States)

    Nelson, Jessica Kristine; Cook, Emma Clare Laura; Loregger, Anke; Hoeksema, Marten Anne; Scheij, Saskia; Kovacevic, Igor; Hordijk, Peter Lodewijk; Ovaa, Huib; Zelcer, Noam

    2016-02-26

    Cholesterol metabolism is subject to complex transcriptional and nontranscriptional regulation. Herein, the role of ubiquitylation is emerging as an important post-translational modification that regulates cholesterol synthesis and uptake. Similar to other post-translational modifications, ubiquitylation is reversible in a process dependent on activity of deubiquitylating enzymes (DUBs). Yet whether these play a role in cholesterol metabolism is largely unknown. As a first step to test this possibility, we used pharmacological inhibition of cellular DUB activity. Short term (2 h) inhibition of DUBs resulted in accumulation of high molecular weight ubiquitylated proteins. This was accompanied by a dramatic decrease in abundance of the LDLR and attenuated LDL uptake into hepatic cells. Importantly, this occurred in the absence of changes in the mRNA levels of the LDLR or other SREBP2-regulated genes, in line with this phenotype being a post-transcriptional event. Mechanistically, we identify transcriptional induction of the E3 ubiquitin ligase IDOL in human and rodent cells as the underlying cause for ubiquitylation-dependent lysosomal degradation of the LDLR following DUB inhibition. In contrast to the established transcriptional regulation of IDOL by the sterol-responsive liver X receptor (LXR) transcription factors, induction of IDOL by DUB inhibition is LXR-independent and occurs in Lxrαβ(-/-) MEFs. Consistent with the role of DUBs in transcriptional regulation, we identified a 70-bp region in the proximal promoter of IDOL, distinct from that containing the LXR-responsive element, which mediates the response to DUB inhibition. In conclusion, we identify a sterol-independent mechanism to regulate IDOL expression and IDOL-mediated lipoprotein receptor degradation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Deubiquitylase Inhibition Reveals Liver X Receptor-independent Transcriptional Regulation of the E3 Ubiquitin Ligase IDOL and Lipoprotein Uptake*

    Science.gov (United States)

    Nelson, Jessica Kristine; Cook, Emma Clare Laura; Loregger, Anke; Hoeksema, Marten Anne; Scheij, Saskia; Kovacevic, Igor; Hordijk, Peter Lodewijk; Ovaa, Huib; Zelcer, Noam

    2016-01-01

    Cholesterol metabolism is subject to complex transcriptional and nontranscriptional regulation. Herein, the role of ubiquitylation is emerging as an important post-translational modification that regulates cholesterol synthesis and uptake. Similar to other post-translational modifications, ubiquitylation is reversible in a process dependent on activity of deubiquitylating enzymes (DUBs). Yet whether these play a role in cholesterol metabolism is largely unknown. As a first step to test this possibility, we used pharmacological inhibition of cellular DUB activity. Short term (2 h) inhibition of DUBs resulted in accumulation of high molecular weight ubiquitylated proteins. This was accompanied by a dramatic decrease in abundance of the LDLR and attenuated LDL uptake into hepatic cells. Importantly, this occurred in the absence of changes in the mRNA levels of the LDLR or other SREBP2-regulated genes, in line with this phenotype being a post-transcriptional event. Mechanistically, we identify transcriptional induction of the E3 ubiquitin ligase IDOL in human and rodent cells as the underlying cause for ubiquitylation-dependent lysosomal degradation of the LDLR following DUB inhibition. In contrast to the established transcriptional regulation of IDOL by the sterol-responsive liver X receptor (LXR) transcription factors, induction of IDOL by DUB inhibition is LXR-independent and occurs in Lxrαβ−/− MEFs. Consistent with the role of DUBs in transcriptional regulation, we identified a 70-bp region in the proximal promoter of IDOL, distinct from that containing the LXR-responsive element, which mediates the response to DUB inhibition. In conclusion, we identify a sterol-independent mechanism to regulate IDOL expression and IDOL-mediated lipoprotein receptor degradation. PMID:26719329

  3. A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq.

    Directory of Open Access Journals (Sweden)

    Sergey Y Druzhinin

    2015-07-01

    Full Text Available Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, "primer-dependent initiation," (PDI has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E. coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5' ends (5' RNA-seq that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T-1A+1 or G-1G+1 (where position +1 corresponds to the position of de novo initiation. Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´.

  4. A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq.

    Science.gov (United States)

    Druzhinin, Sergey Y; Tran, Ngat T; Skalenko, Kyle S; Goldman, Seth R; Knoblauch, Jared G; Dove, Simon L; Nickels, Bryce E

    2015-07-01

    Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, "primer-dependent initiation," (PDI) has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E. coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5' ends (5' RNA-seq) that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T-1A+1 or G-1G+1 (where position +1 corresponds to the position of de novo initiation). Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´.

  5. Genomewide Analysis of Clp1 Function in Transcription in Budding Yeast.

    Science.gov (United States)

    Al-Husini, Nadra; Sharifi, Ali; Mousavi, Seyed Ahmad; Chitsaz, Hamidreza; Ansari, Athar

    2017-07-31

    In budding yeast, the 3' end processing of mRNA and the coupled termination of transcription by RNAPII requires the CF IA complex. We have earlier demonstrated a role for the Clp1 subunit of this complex in termination and promoter-associated transcription of CHA1. To assess the generality of the observed function of Clp1 in transcription, we tested the effect of Clp1 on transcription on a genomewide scale using the Global Run-On-Seq (GRO-Seq) approach. GRO-Seq analysis showed the polymerase reading through the termination signal in the downstream region of highly transcribed genes in a temperature-sensitive mutant of Clp1 at elevated temperature. No such terminator readthrough was observed in the mutant at the permissive temperature. The poly(A)-independent termination of transcription of snoRNAs, however, remained unaffected in the absence of Clp1 activity. These results strongly suggest a role for Clp1 in poly(A)-coupled termination of transcription. Furthermore, the density of antisense transcribing polymerase upstream of the promoter region exhibited an increase in the absence of Clp1 activity, thus implicating Clp1 in promoter directionality. The overall conclusion of these results is that Clp1 plays a general role in poly(A)-coupled termination of RNAPII transcription and in enhancing promoter directionality in budding yeast.

  6. Tumor suppressor QM-like gene from disk abalone (Haliotis discus discus): molecular characterization and transcriptional analysis upon immune challenge.

    Science.gov (United States)

    Oh, Chulhong; De Zoysa, Mahanama; Nikapitiya, Chamilani; Whang, Ilson; Kim, Yu Cheol; Kang, Do-Hyung; Heo, Soo-jin; Choi, Young-Ung; Choi, Cheol Young; Lee, Jae-Seong; Lee, Jehee

    2010-09-01

    We describe molecular characterization and transcriptional analysis of the gene encoding tumor suppressor QM-like protein, AbQM, in the disk abalone Haliotis discus discus. The full-length cDNA (765-bp) of AbQM was found to consist of a 654-bp ORF coding for a 218 amino acid protein of a 25 kDa molecular mass with a 10.2 isoelectric point. Analysis of AbQM sequence revealed the presence of characteristic motifs, including the ribosomal protein L10 signature, SH3-binding motif and two antibiotic binding sites. Phylogenetic analysis confirmed that AbQM is closely related to other mollusk QM proteins, and altogether they form a mollusk QM protein sub-family which displays evolutionary conservation from yeast to human. Tissue-specific expression and transcriptional regulation of AbQM was analyzed by quantitative real-time PCR in response to bacterial (Vibrio alginolyticus and Vibrio parahemolyticus, Listeria monocytogenes) and viral (viral hemorrhagic septicemia virus, VHSV) challenge. AbQM transcripts were found to be expressed ubiquitously in all examined tissues in a constitutive manner, as similar expression levels were detected in hemocytes, mantle, digestive tract and muscle. Upon bacterial and VHSV challenge, AbQM showed significant up-regulation in gills, but not in hemocytes. Taken together, these findings suggest that AbQM in abalone-like mollusks can respond to and facilitate a defensive effect against pathogenic infection. Copyright 2010 Elsevier Ltd. All rights reserved.

  7. Transcriptional Analysis of Lactobacillus brevis to N-Butanol and Ferulic Acid Stress Responses

    Science.gov (United States)

    Winkler, James; Kao, Katy C.

    2011-01-01

    Background The presence of anti-microbial phenolic compounds, such as the model compound ferulic acid, in biomass hydrolysates pose significant challenges to the widespread use of biomass in conjunction with whole cell biocatalysis or fermentation. Currently, these inhibitory compounds must be removed through additional downstream processing or sufficiently diluted to create environments suitable for most industrially important microbial strains. Simultaneously, product toxicity must also be overcome to allow for efficient production of next generation biofuels such as n-butanol, isopropanol, and others from these low cost feedstocks. Methodology and Principal Findings This study explores the high ferulic acid and n-butanol tolerance in Lactobacillus brevis, a lactic acid bacterium often found in fermentation processes, by global transcriptional response analysis. The transcriptional profile of L. brevis reveals that the presence of ferulic acid triggers the expression of currently uncharacterized membrane proteins, possibly in an effort to counteract ferulic acid induced changes in membrane fluidity and ion leakage. In contrast to the ferulic acid stress response, n-butanol challenges to growing cultures primarily induce genes within the fatty acid synthesis pathway and reduced the proportion of 19∶1 cyclopropane fatty acid within the L. brevis membrane. Both inhibitors also triggered generalized stress responses. Separate attempts to alter flux through the Escherichia coli fatty acid synthesis by overexpressing acetyl-CoA carboxylase subunits and deleting cyclopropane fatty acid synthase (cfa) both failed to improve n-butanol tolerance in E. coli, indicating that additional components of the stress response are required to confer n-butanol resistance. Conclusions Several promising routes for understanding both ferulic acid and n-butanol tolerance have been identified from L. brevis gene expression data. These insights may be used to guide further engineering of

  8. Transcriptional responses of Italian ryegrass during interaction with Xanthomonas translucens pv. graminis reveal novel candidate genes for bacterial wilt resistance

    DEFF Research Database (Denmark)

    Wichmann, Fabienne; Asp, Torben; Widmer, Franko

    2011-01-01

    Xanthomonas translucens pv. graminis (Xtg) causes bacterial wilt, a severe disease of forage grasses such as Italian ryegrass (Lolium multiflorum Lam.). In order to gain a more detailed understanding of the genetic control of resistance mechanisms and to provide prerequisites for marker assisted...... selection, the partial transcriptomes of two Italian ryegrass genotypes, one resistant and one susceptible to bacterial wilt were compared at four time points after Xtg infection. A cDNA microarray developed from a perennial ryegrass (Lolium perenne) expressed sequence tag set consisting of 9,990 unique...... genes was used for transcriptome analysis in Italian ryegrass. An average of 4,487 (45%) of the perennial ryegrass sequences spotted on the cDNA microarray were detected by cross-hybridisation to Italian ryegrass. Transcriptome analyses of the resistant versus the susceptible genotype revealed...

  9. Transcriptome-wide identification and expression analysis of chrysanthemum SBP-like transcription factors.

    Science.gov (United States)

    Song, Aiping; Gao, Tianwei; Wu, Dan; Xin, Jingjing; Chen, Sumei; Guan, Zhiyong; Wang, Haibin; Jin, Lili; Chen, Fadi

    2016-05-01

    SQUAMOSA promoter-binding protein (SBP) transcription factors are known to function in a number of processes in plants. Here, we have characterized twelve SBP-like (SPL) genes in the important ornamental species chrysanthemum (Chrysanthemum morifolium). A total of twelve distinct sequences were isolated and amplified based on transcriptomic sequences. Phylogenetic analysis identified two pairs of orthologous proteins for Arabidopsis and chrysanthemum and two pairs of paralogous proteins in chrysanthemum. Conserved motifs in the SPL proteins shared by Arabidopsis and chrysanthemum were scanned using MEME. A bioinformatics analysis revealed that six of these genes contained a miR156 target site, while five CmSPLs were targeted by miR157. Moreover, we used 5' RLM-RACE to map the cleavage sites in CmSPL2 and CmSPL3. The expression of these twelve genes in response to a variety of phytohormone treatments and abiotic stresses was characterized. This work improves our understanding of the various functions of SPL gene family members in the stress response. Copyright © 2016. Published by Elsevier Masson SAS.

  10. Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene

    International Nuclear Information System (INIS)

    Kouzarides, T.; Bankier, A.T.; Satchwell, S.C.; Weston, K.; Tomlinson, P.; Barrell, B.G.

    1987-01-01

    DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. The authors present here a 5280 base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, they were able to analyze the extent of homology between the DNA polymerases of three distantly related herpes viruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpes viruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type I and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus

  11. Transcriptional profiling reveals developmental relationship and distinct biological functions of CD16+ and CD16- monocyte subsets

    Directory of Open Access Journals (Sweden)

    Zhou Xiaobo

    2009-08-01

    Full Text Available Abstract Background Human peripheral blood monocytes (Mo consist of subsets distinguished by expression of CD16 (FCγRIII and chemokine receptors. Classical CD16- Mo express CCR2 and migrate in response to CCL2, while a minor CD16+ Mo subset expresses CD16 and CX3CR1 and migrates into tissues expressing CX3CL1. CD16+ Mo produce pro-inflammatory cytokines and are expanded in certain inflammatory conditions including sepsis and HIV infection. Results To gain insight into the developmental relationship and functions of CD16+ and CD16- Mo, we examined transcriptional profiles of these Mo subsets in peripheral blood from healthy individuals. Of 16,328 expressed genes, 2,759 genes were differentially expressed and 228 and 250 were >2-fold upregulated and downregulated, respectively, in CD16+ compared to CD16- Mo. CD16+ Mo were distinguished by upregulation of transcripts for dendritic cell (DC (SIGLEC10, CD43, RARA and macrophage (MΦ (CSF1R/CD115, MafB, CD97, C3aR markers together with transcripts relevant for DC-T cell interaction (CXCL16, ICAM-2, LFA-1, cell activation (LTB, TNFRSF8, LST1, IFITM1-3, HMOX1, SOD-1, WARS, MGLL, and negative regulation of the cell cycle (CDKN1C, MTSS1, whereas CD16- Mo were distinguished by upregulation of transcripts for myeloid (CD14, MNDA, TREM1, CD1d, C1qR/CD93 and granulocyte markers (FPR1, GCSFR/CD114, S100A8-9/12. Differential expression of CSF1R, CSF3R, C1QR1, C3AR1, CD1d, CD43, CXCL16, and CX3CR1 was confirmed by flow cytometry. Furthermore, increased expression of RARA and KLF2 transcripts in CD16+ Mo coincided with absence of cell surface cutaneous lymphocyte associated antigen (CLA expression, indicating potential imprinting for non-skin homing. Conclusion These results suggest that CD16+ and CD16- Mo originate from a common myeloid precursor, with CD16+ Mo having a more MΦ – and DC-like transcription program suggesting a more advanced stage of differentiation. Distinct transcriptional programs, together

  12. Transcriptional analysis of aggressiveness and heterogeneity across grades of astrocytomas.

    Directory of Open Access Journals (Sweden)

    Chunjing Wang

    Full Text Available Astrocytoma is the most common glioma, accounting for half of all primary brain and spinal cord tumors. Late detection and the aggressive nature of high-grade astrocytomas contribute to high mortality rates. Though many studies identify candidate biomarkers using high-throughput transcriptomic profiling to stratify grades and subtypes, few have resulted in clinically actionable results. This shortcoming can be attributed, in part, to pronounced lab effects that reduce signature robustness and varied individual gene expression among patients with the same tumor. We addressed these issues by uniformly preprocessing publicly available transcriptomic data, comprising 306 tumor samples from three astrocytoma grades (Grade 2, 3, and 4 and 30 non-tumor samples (normal brain as control tissues. Utilizing Differential Rank Conservation (DIRAC, a network-based classification approach, we examined the global and individual patterns of network regulation across tumor grades. Additionally, we applied gene-based approaches to identify genes whose expression changed consistently with increasing tumor grade and evaluated their robustness across multiple studies using statistical sampling. Applying DIRAC, we observed a global trend of greater network dysregulation with increasing tumor aggressiveness. Individual networks displaying greater differences in regulation between adjacent grades play well-known roles in calcium/PKC, EGF, and transcription signaling. Interestingly, many of the 90 individual genes found to monotonically increase or decrease with astrocytoma grade are implicated in cancer-affected processes such as calcium signaling, mitochondrial metabolism, and apoptosis. The fact that specific genes monotonically increase or decrease with increasing astrocytoma grade may reflect shared oncogenic mechanisms among phenotypically similar tumors. This work presents statistically significant results that enable better characterization of different human

  13. An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription.

    Science.gov (United States)

    Giraud, Matthieu; Jmari, Nada; Du, Lina; Carallis, Floriane; Nieland, Thomas J F; Perez-Campo, Flor M; Bensaude, Olivier; Root, David E; Hacohen, Nir; Mathis, Diane; Benoist, Christophe

    2014-01-28

    Aire induces the expression of a large set of autoantigen genes in the thymus, driving immunological tolerance in maturing T cells. To determine the full spectrum of molecular mechanisms underlying the Aire transactivation function, we screened an AIRE-dependent gene-expression system with a genome-scale lentiviral shRNA library, targeting factors associated with chromatin architecture/function, transcription, and mRNA processing. Fifty-one functional allies were identified, with a preponderance of factors that impact transcriptional elongation compared with initiation, in particular members of the positive transcription elongation factor b (P-TEFb) involved in the release of "paused" RNA polymerases (CCNT2 and HEXIM1); mRNA processing and polyadenylation factors were also highlighted (HNRNPL/F, SFRS1, SFRS3, and CLP1). Aire's functional allies were validated on transfected and endogenous target genes, including the generation of lentigenic knockdown (KD) mice. We uncovered the effect of the splicing factor Hnrnpl on Aire-induced transcription. Transcripts sensitive to the P-TEFb inhibitor flavopiridol were reduced by Hnrnpl knockdown in thymic epithelial cells, independently of their dependence on Aire, therefore indicating a general effect of Hnrnpl on RNA elongation. This conclusion was substantiated by demonstration of HNRNPL interactions with P-TEFb components (CDK9, CCNT2, HEXIM1, and the small 7SK RNA). Aire-containing complexes include 7SK RNA, the latter interaction disrupted by HNRNPL knockdown, suggesting that HNRNPL may partake in delivering inactive P-TEFb to Aire. Thus, these results indicate that mRNA processing factors cooperate with Aire to release stalled polymerases and to activate ectopic expression of autoantigen genes in the thymus.

  14. Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair

    Directory of Open Access Journals (Sweden)

    Wang Yu

    2011-01-01

    , indicating that DNA-PKcs may also be involved in the TCR pathway of DNA damage repair. By means of immunoprecipitation and MALDI-TOF-Mass spectrometric analysis, we have revealed the interaction of DNA-PKcs and cyclin T2, which is a subunit of the human transcription elongation factor (P-TEFb. While the P-TEFb complex can phosphorylate the serine 2 of the carboxyl-terminal domain (CTD of RNA polymerase II and promote transcription elongation. Conclusion A new method of TCR assay was developed based the strand-specific-PCR (SS-PCR. Our data suggest that DNA-PKcs plays a role in the TCR pathway of UV-damaged DNA. One possible mechanistic hypothesis is that DNA-PKcs may function through associating with CyclinT2/CDK9 (P-TEFb to modulate the activity of RNA Pol II, which has already been identified as a key molecule recognizing and initializing TCR.

  15. Teacher Talk: Transcript Analysis as a Method of Improving Effectiveness during Comprehension Strategies Instruction

    Science.gov (United States)

    Robertson, Dana Andrew

    2012-01-01

    This mixed-methods study compared the outcomes of 5 general education teachers in a suburban, upper-middle-class elementary school engaged in transcript analysis during small-group reading strategies instruction to those obtained when engaged in audio analysis. Over a 5-month period, the teachers recorded one lesson per week and debriefed with the…

  16. Killer in our Midst: Part Two. An Analysis of Court Transcripts ...

    African Journals Online (AJOL)

    Killer in our Midst: Part Two. An Analysis of Court Transcripts Pertaining to the Defence of Stewart Wilken in \\"Die Staat Teen Stewart Wilken\\". Andrea Hurst. Abstract. This article forms the second part of an analysis of documents pertaining to the defence of serial killer Stewart Wilken in Die Staat Teen Stewart Wilken.

  17. Dynamic network of transcription and pathway crosstalk to reveal molecular mechanism of MGd-treated human lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Liyan Shao

    Full Text Available Recent research has revealed various molecular markers in lung cancer. However, the organizational principles underlying their genetic regulatory networks still await investigation. Here we performed Network Component Analysis (NCA and Pathway Crosstalk Analysis (PCA to construct a regulatory network in human lung cancer (A549 cells which were treated with 50 uM motexafin gadolinium (MGd, a metal cation-containing chemotherapeutic drug for 4, 12, and 24 hours. We identified a set of key TFs, known target genes for these TFs, and signaling pathways involved in regulatory networks. Our work showed that putative interactions between these TFs (such as ESR1/Sp1, E2F1/Sp1, c-MYC-ESR, Smad3/c-Myc, and NFKB1/RELA, between TFs and their target genes (such as BMP41/Est1, TSC2/Myc, APE1/Sp1/p53, RARA/HOXA1, and SP1/USF2, and between signaling pathways (such as PPAR signaling pathway and Adipocytokines signaling pathway. These results will provide insights into the regulatory mechanism of MGd-treated human lung cancer cells.

  18. Multiple independent analyses reveal only transcription factors as an enriched functional class associated with microRNAs

    DEFF Research Database (Denmark)

    Croft, Laurence J; Szklarczyk, Damian; Jensen, Lars J

    2012-01-01

    ABSTRACT: BACKGROUND: Transcription factors (TFs) have long been known to be principally activators of transcription in eukaryotes and prokaryotes. The growing awareness of the ubiquity of microRNAs (miRNAs) as suppressive regulators in eukaryotes, suggests the possibility of a mutual, preferential......RNA targets, appear not to select for real miRNA targets to any degree. Our manually validated text-mining results also suggests that miRNAs may be activated by more TFs than other classes of genes, as 7% of miRNA:TF co-occurrences in the literature were TFs activating miRNAs. This was confirmed when thirdly......, we found enrichment for predicted, conserved TF binding sites in miRNA and TF genes compared to other gene classes. CONCLUSIONS: We see enrichment of connections between miRNAs and TFs using several independent methods, suggestive of a network of mutual activating and suppressive regulation. We have...

  19. Transcriptional similarity in couples reveals the impact of shared environment and lifestyle on gene regulation through modified cytosines

    Directory of Open Access Journals (Sweden)

    Ke Tang

    2016-06-01

    Full Text Available Gene expression is a complex and quantitative trait that is influenced by both genetic and non-genetic regulators including environmental factors. Evaluating the contribution of environment to gene expression regulation and identifying which genes are more likely to be influenced by environmental factors are important for understanding human complex traits. We hypothesize that by living together as couples, there can be commonly co-regulated genes that may reflect the shared living environment (e.g., diet, indoor air pollutants, behavioral lifestyle. The lymphoblastoid cell lines (LCLs derived from unrelated couples of African ancestry (YRI, Yoruba people from Ibadan, Nigeria from the International HapMap Project provided a unique model for us to characterize gene expression pattern in couples by comparing gene expression levels between husbands and wives. Strikingly, 778 genes were found to show much smaller variances in couples than random pairs of individuals at a false discovery rate (FDR of 5%. Since genetic variation between unrelated family members in a general population is expected to be the same assuming a random-mating society, non-genetic factors (e.g., epigenetic systems are more likely to be the mediators for the observed transcriptional similarity in couples. We thus evaluated the contribution of modified cytosines to those genes showing transcriptional similarity in couples as well as the relationships these CpG sites with other gene regulatory elements, such as transcription factor binding sites (TFBS. Our findings suggested that transcriptional similarity in couples likely reflected shared common environment partially mediated through cytosine modifications.

  20. Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans

    Directory of Open Access Journals (Sweden)

    Stahl Stefanie

    2002-02-01

    Full Text Available Abstract Background Mucolipidosis type IV (MLIV is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. Results The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. Conclusions Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment.

  1. Boolean modelling reveals new regulatory connections between transcription factors orchestrating the development of the ventral spinal cord.

    KAUST Repository

    Lovrics, Anna

    2014-11-14

    We have assembled a network of cell-fate determining transcription factors that play a key role in the specification of the ventral neuronal subtypes of the spinal cord on the basis of published transcriptional interactions. Asynchronous Boolean modelling of the network was used to compare simulation results with reported experimental observations. Such comparison highlighted the need to include additional regulatory connections in order to obtain the fixed point attractors of the model associated with the five known progenitor cell types located in the ventral spinal cord. The revised gene regulatory network reproduced previously observed cell state switches between progenitor cells observed in knock-out animal models or in experiments where the transcription factors were overexpressed. Furthermore the network predicted the inhibition of Irx3 by Nkx2.2 and this prediction was tested experimentally. Our results provide evidence for the existence of an as yet undescribed inhibitory connection which could potentially have significance beyond the ventral spinal cord. The work presented in this paper demonstrates the strength of Boolean modelling for identifying gene regulatory networks.

  2. Transcriptional program of ciliated epithelial cells reveals new cilium and centrosome components and links to human disease.

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    Ramona A Hoh

    Full Text Available Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP. The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease.

  3. Innocence and Nostalgia in Conversation Analysis: The Dynamic Relations of Tape and Transcript

    Directory of Open Access Journals (Sweden)

    Malcolm Ashmore

    2000-12-01

    Full Text Available This paper attempts an analysis of some of the methodological practices of Conversation Analysis (CA; in particular, tape recording and transcription. The paper starts from the observation that, in the CA literature, these practices, and the analytic objects they create (the tape and the transcript, are accorded different treatment: simply put, for CA the tape is a "realist" object, while the transcript is a "constructivist" one. The significance of this difference is explored through an analysis of the dynamics of CA practice. We argue that the "constructivist transcript" is premised on an understanding of CA as predominantly concerned with maximising its "analytic utility": a concern of one distinct temporal stage of CA work: that of the "innocent" apprehension of objects in the "first time through". The "realist tape", in contrast, is based on a different aspect of the work of CA: its quest for greater "evidential utility", achieved by the "nostalgic" revisiting of previously produced objects for purposes of checking them against each other; work done in the "next time through". We further argue that both the ontology and the epistemology of CA's objects are changed in any next time encounter. We conclude with a cautionary speculation on the currently-projected, transcript-free, digital future of CA. URN: urn:nbn:de:0114-fqs000335

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

    Directory of Open Access Journals (Sweden)

    Youichi Higuchi

    Full Text Available 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.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.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.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.

  5. Validation of a reference gene for transcript analysis in cassava ...

    African Journals Online (AJOL)

    RT-qPCR) is a well-established method for the precise quantification of gene expression. For accurate relative real-time RT-qPCR analysis, validation of the expression of an appropriate reference gene is required. In this study, the expression of ...

  6. Transcriptional analysis of the bglP gene from Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Cote Christopher K

    2006-04-01

    Full Text Available Abstract Background An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II. Results To localize the promoter activity associated with the bglP locus, a series of transcriptional lacZ gene fusions was formed on a reporter shuttle vector using various DNA fragments from the bglP promoter region. Subsequent beta-galactosidase assays in S. mutans localized the bglP promoter region and identified putative -35 and -10 promoter elements. Primer extension analysis identified the bglP transcriptional start site. In addition, a terminated bglP transcript formed by transcriptional termination was identified via transcript mapping experiments. Conclusion The physical location of these genetic elements, the RAT site and the promoter regions, and the identification of a short terminated mRNA support the hypothesis that antitermination regulates the bglP transcript.

  7. Transcriptomic Profiling and H3K27me3 Distribution Reveal Both Demethylase-Dependent and Independent Regulation of Developmental Gene Transcription in Cell Differentiation.

    Science.gov (United States)

    Kang, Sung Chul; Kim, Se Kye; Chai, Jin Choul; Kim, Sun Hwa; Won, Kyoung-Jae; Lee, Young Seek; Jung, Kyoung Hwa; Chai, Young Gyu

    2015-01-01

    The removal of histone H3 trimethylation at lysine residue 27 (H3K27me3) plays a critical role in the transcriptional initiation of developmental genes. The H3K27me3-specific KDM6 demethylases JMJD3 and UTX are responsible for the transcriptional initiation of various developmental genes, but some genes are expressed in a KDM6 demethylase-independent manner. To address the role of H3K27me3 in the retinoic acid (RA)-induced differentiation of the human carcinoma NCCIT cell line, we inhibited JMJD3 and UTX using the H3K27me3 demethylase inhibitor GSK-J4. The commitment of JMJD3/UTX-inhibited cells to a specific fate was delayed, and transcriptome profiling also revealed the differential expression of genes related to cell fate specification in demethylase-inactivated cells; the expression levels of RA metabolism and HOX family genes significantly decreased. We observed a weak correlation between H3K27me3 enrichment and transcriptional repression in the control and JMJD/UTX-inhibited cells, except for a few sets of developmental genes that are indispensable for cell fate specification. Taken together, these results provide the H3K27me3 landscape of a differentiating cell line and suggest that both demethylase-dependent and demethylase-independent transcriptional regulation play a role in early differentiation and developmental gene expression activated by H3K27me3 demethylation.

  8. Functional analysis of a NF-κB transcription factor in the immune defense of Oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae).

    Science.gov (United States)

    Shi, Z; Liang, H; Hou, Y

    2017-04-01

    Although some novel antimicrobial peptides (AMP) have been successfully isolated from Bactrocera dorsalis Hendel, the mechanisms underlying the induction of these peptides are still elusive. The homolog of NF-κB transcription factor Relish, designated as BdRelish, was cloned from B. dorsalis. The full length cDNA of BdRelish is 3954 bp with an open reading frame that encodes 1013 amino acids. Similar to Drosophila Relish and the mammalian p100, it is a compound protein containing a conserved Rel homology domain, an IPT (Ig-like, plexins, transcription factors) domain and an IκB-like domain (four ankyrin repeats), the nuclear localization signal RKRRR is also detected at the residues 449-453, suggesting that it has homology to Relish and it is a member of the Rel family of transcription activator proteins. Reverse transcription quantitative polymerase chain reaction analysis reveals that BdRelish mRNAs are detected in different quantities from various tissues and the highest transcription level of BdRelish is determined in fat body. The injection challenge of Escherichia coli and Staphylococcus aureas significantly upregulated the expression of BdRelish. The injection of BdRelish dsRNA markedly reduced the expression of BdRelish and decreased the transcription magnitude of antimicrobial peptides. Individuals injected BdRelish dsRNA died at a significantly faster rate compared with the control groups. Therefore, BdRelish is vital for the transcription of AMPs to attack the invading bacteria.

  9. Phylogenetic, expression and functional characterizations of the maize NLP transcription factor family reveal a role in nitrate assimilation and signaling.

    Science.gov (United States)

    Wang, Zhangkui; Zhang, Lei; Sun, Ci; Gu, Riliang; Mi, Guohua; Yuan, Lixing

    2018-01-24

    Although nitrate represents an important nitrogen (N) source for maize, a major crop of dryland areas, the molecular mechanisms of nitrate uptake and assimilation remain poorly understood. Here, we identified nine maize NIN-like protein (ZmNLP) genes and analyzed the function of one member, ZmNLP3.1, in nitrate nutrition and signaling. The NLP family genes were clustered into three clades in a phylogenic tree. Comparative genomic analysis showed that most ZmNLP genes had collinear relationships to the corresponding NLPs in rice, and that the expansion of the ZmNLP family resulted from segmental duplications in the maize genome. Quantitative PCR analysis revealed the expression of ZmNLP2.1, ZmNLP2.2, ZmNLP3.1, ZmNLP3.2, ZmNLP3.3, and ZmNLP3.4 was induced by nitrate in maize roots. The function of ZmNLP3.1 was investigated by overexpressing it in the Arabidopsis nlp7-1 mutant, which is defective in the AtNLP7 gene for nitrate signaling and assimilation. Ectopic expression of ZmNLP3.1 restored the N-deficient phenotypes of nlp7-1 under nitrate-replete conditions in terms of shoot biomass, root morphology and nitrate assimilation. Furthermore, the nitrate induction of NRT2.1, NIA1, and NiR1 gene expression was recovered in the 35S::ZmNLP3.1/nlp7-1 transgenic lines, indicating that ZmNLP3.1 plays essential roles in nitrate signaling. Taken together, these results suggest that ZmNLP3.1 plays an essential role in regulating nitrate signaling and assimilation processes, and represents a valuable candidate for developing transgenic maize cultivars with high N-use efficiency. This article is protected by copyright. All rights reserved.

  10. Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes.

    Science.gov (United States)

    Hua, Qingzhu; Zhou, Qianjun; Gan, Susheng; Wu, Jingyu; Chen, Canbin; Li, Jiaqiang; Ye, Yaoxiong; Zhao, Jietang; Hu, Guibing; Qin, Yonghua

    2016-09-28

    Red dragon fruit or red pitaya ( Hylocereus polyrhizus ) is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus . RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to "phenylpropanoid biosynthesis", "tyrosine metabolism", "flavonoid biosynthesis", "ascorbate and aldarate metabolism", "betalains biosynthesis" and "anthocyanin biosynthesis". In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA) dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level.

  11. Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes

    Directory of Open Access Journals (Sweden)

    Qingzhu Hua

    2016-09-01

    Full Text Available Red dragon fruit or red pitaya (Hylocereus polyrhizus is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus. RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to “phenylpropanoid biosynthesis”, “tyrosine metabolism”, “flavonoid biosynthesis”, “ascorbate and aldarate metabolism”, “betalains biosynthesis” and “anthocyanin biosynthesis”. In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level.

  12. A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing

    KAUST Repository

    Zhang, Runxuan

    2017-04-05

    Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.

  13. A new in vivo model of pantothenate kinase-associated neurodegeneration reveals a surprising role for transcriptional regulation in pathogenesis.

    Directory of Open Access Journals (Sweden)

    Varun ePandey

    2013-09-01

    Full Text Available Pantothenate Kinase-Associated Neurodegeneration (PKAN is a neurodegenerative disorder with a poorly understood molecular mechanism. It is caused by mutations in Pantothenate Kinase, the first enzyme in the Coenzyme A (CoA biosynthetic pathway. Here, we developed a Drosophila model of PKAN (tim-fbl flies that allows us to continuously monitor the modeled disease in the brain. In tim-fbl flies, downregulation of fumble, the Drosophila PanK homologue in the cells containing a circadian clock results in characteristic features of PKAN such as developmental lethality, hypersensitivity to oxidative stress, and diminished life span. Despite quasi-normal circadian transcriptional rhythms, tim-fbl flies display brain-specific aberrant circadian locomotor rhythms, and a unique transcriptional signature. Comparison with expression data from flies exposed to paraquat demonstrates that, as previously suggested, pathways others than oxidative stress are affected by PANK downregulation. Surprisingly we found a significant decrease in the expression of key components of the photoreceptor recycling pathways, which could lead to retinal degeneration, a hallmark of PKAN. Importantly, these defects are not accompanied by changes in structural components in eye genes suggesting that changes in gene expression in the eye precede and may cause the retinal degeneration. Indeed tim-fbl flies have diminished response to light transitions, and their altered day/night patterns of activity demonstrates defects in light perception. This suggest that retinal lesions are not solely due to oxidative stress and demonstrates a role for the transcriptional response to CoA deficiency underlying the defects observed in dPanK deficient flies. Moreover, in the present study we developed a new fly model that can be applied to other diseases and that allows the assessment of neurodegeneration in the brains of living flies.

  14. Identification of genes specifically or preferentially expressed in maize silk reveals similarity and diversity in transcript abundance of different dry stigmas

    Directory of Open Access Journals (Sweden)

    Xu Xiao

    2012-07-01

    Full Text Available Abstract Background In plants, pollination is a critical step in reproduction. During pollination, constant communication between male pollen and the female stigma is required for pollen adhesion, germination, and tube growth. The detailed mechanisms of stigma-mediated reproductive processes, however, remain largely unknown. Maize (Zea mays L., one of the world’s most important crops, has been extensively used as a model species to study molecular mechanisms of pollen and stigma interaction. A comprehensive analysis of maize silk transcriptome may provide valuable information for investigating stigma functionality. A comparative analysis of expression profiles between maize silk and dry stigmas of other species might reveal conserved and diverse mechanisms that underlie stigma-mediated reproductive processes in various plant species. Results Transcript abundance profiles of mature silk, mature pollen, mature ovary, and seedling were investigated using RNA-seq. By comparing the transcriptomes of these tissues, we identified 1,427 genes specifically or preferentially expressed in maize silk. Bioinformatic analyses of these genes revealed many genes with known functions in plant reproduction as well as novel candidate genes that encode amino acid transporters, peptide and oligopeptide transporters, and cysteine-rich receptor-like kinases. In addition, comparison of gene sets specifically or preferentially expressed in stigmas of maize, rice (Oryza sativa L., and Arabidopsis (Arabidopsis thaliana [L.] Heynh. identified a number of homologous genes involved either in pollen adhesion, hydration, and germination or in initial growth and penetration of pollen tubes into the stigma surface. The comparison also indicated that maize shares a more similar profile and larger number of conserved genes with rice than with Arabidopsis, and that amino acid and lipid transport-related genes are distinctively overrepresented in maize. Conclusions Many of the

  15. Identification of genes specifically or preferentially expressed in maize silk reveals similarity and diversity in transcript abundance of different dry stigmas.

    Science.gov (United States)

    Xu, Xiao Hui; Chen, Hao; Sang, Ya Lin; Wang, Fang; Ma, Jun Ping; Gao, Xin-Qi; Zhang, Xian Sheng

    2012-07-02

    In plants, pollination is a critical step in reproduction. During pollination, constant communication between male pollen and the female stigma is required for pollen adhesion, germination, and tube growth. The detailed mechanisms of stigma-mediated reproductive processes, however, remain largely unknown. Maize (Zea mays L.), one of the world's most important crops, has been extensively used as a model species to study molecular mechanisms of pollen and stigma interaction. A comprehensive analysis of maize silk transcriptome may provide valuable information for investigating stigma functionality. A comparative analysis of expression profiles between maize silk and dry stigmas of other species might reveal conserved and diverse mechanisms that underlie stigma-mediated reproductive processes in various plant species. Transcript abundance profiles of mature silk, mature pollen, mature ovary, and seedling were investigated using RNA-seq. By comparing the transcriptomes of these tissues, we identified 1,427 genes specifically or preferentially expressed in maize silk. Bioinformatic analyses of these genes revealed many genes with known functions in plant reproduction as well as novel candidate genes that encode amino acid transporters, peptide and oligopeptide transporters, and cysteine-rich receptor-like kinases. In addition, comparison of gene sets specifically or preferentially expressed in stigmas of maize, rice (Oryza sativa L.), and Arabidopsis (Arabidopsis thaliana [L.] Heynh.) identified a number of homologous genes involved either in pollen adhesion, hydration, and germination or in initial growth and penetration of pollen tubes into the stigma surface. The comparison also indicated that maize shares a more similar profile and larger number of conserved genes with rice than with Arabidopsis, and that amino acid and lipid transport-related genes are distinctively overrepresented in maize. Many of the novel genes uncovered in this study are potentially involved

  16. Transfer entropy, symbolic transfer entropy and transcript mutual information indicators reveal a leading role of infragranular layers during slow oscillations

    Directory of Open Access Journals (Sweden)

    Núria Tort-Colet

    2015-04-01

    To determine the directionality of the information flow between different layers of the cortex and the connected thalamus during spontaneous activity we obtained multilayer local field potentials from the rat visual cortex and from its connected thalamus, the lateral geniculate nucleus, during deep anaesthesia. We analyzed directionality of information flow between thalamus, cortical infragranular layers (5 and 6 and supragranular layers (2/3 by means of three information theoretical indicators: transfer entropy, symbolic transfer entropy and transcript mutual information. These three indicators coincided in finding that infragranular layers lead the information flow during slow oscillations both towards supragranular layers and towards the thalamus.

  17. Genome-wide analysis of transcription factors during somatic embryogenesis in banana (Musa spp.) cv. Grand Naine.

    Science.gov (United States)

    Shivani; Awasthi, Praveen; Sharma, Vikrant; Kaur, Navjot; Kaur, Navneet; Pandey, Pankaj; Tiwari, Siddharth

    2017-01-01

    Transcription factors BABY BOOM (BBM), WUSCHEL (WUS), BSD, LEAFY COTYLEDON (LEC), LEAFY COTYLEDON LIKE (LIL), VIVIPAROUS1 (VP1), CUP SHAPED COTYLEDONS (CUC), BOLITA (BOL), and AGAMOUS LIKE (AGL) play a crucial role in somatic embryogenesis. In this study, we identified eighteen genes of these nine transcription factors families from the banana genome database. All genes were analyzed for their structural features, subcellular, and chromosomal localization. Protein sequence analysis indicated the presence of characteristic conserved domains in these transcription factors. Phylogenetic analysis revealed close evolutionary relationship among most transcription factors of various monocots. The expression patterns of eighteen genes in embryogenic callus containing somatic embryos (precisely isolated by Laser Capture Microdissection), non-embryogenic callus, and cell suspension cultures of banana cultivar Grand Naine were analyzed. The application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in the callus induction medium enhanced the expression of MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the embryogenic callus. It suggested 2, 4-D acts as an inducer for the expression of these genes. The higher expression of MaBBM2 and MaWUS2 in embryogenic cell suspension (ECS) as compared to non-embryogenic cells suspension (NECS), suggested that these genes may play a crucial role in banana somatic embryogenesis. MaVP1 showed higher expression in both ECS and NECS, whereas MaLEC2 expression was significantly higher in NECS. It suggests that MaLEC2 has a role in the development of non-embryogenic cells. We postulate that MaBBM2 and MaWUS2 can be served as promising molecular markers for the embryogencity in banana.

  18. Differential proteomic analysis reveals novel links between primary metabolism and antibiotic production in Amycolatopsis balhimycina

    DEFF Research Database (Denmark)

    Gallo, G.; Renzone, G.; Alduina, R.

    2010-01-01

    A differential proteomic analysis, based on 2-DE and MS procedures, was performed on Amycolatopsis balhimycina DSM5908, the actinomycete producing the vancomycin-like antibiotic balhimycin. A comparison of proteomic profiles before and during balhimycin production characterized differentially...... available over the World Wide Web as interactive web pages (http://www.unipa.it/ampuglia/Abal-proteome-maps). Functional clustering analysis revealed that differentially expressed proteins belong to functional groups involved in central carbon metabolism, amino acid metabolism and protein biosynthesis......, energetic and redox balance, sugar/amino sugar metabolism, balhimycin biosynthesis and transcriptional regulation or with hypothetical and/or unknown function. Interestingly, proteins involved in the biosynthesis of balhimycin precursors, such as amino acids, amino sugars and central carbon metabolism...

  19. Gene transcription analysis of carrot allergens by relative quantification with single and duplex reverse transcription real-time PCR.

    Science.gov (United States)

    Zagon, Jutta; Jansen, Bärbel; Knoppik, Meike; Ehlers, Anke; Kroh, Lothar W; Holzhauser, Thomas; Vieths, Stefan; Broll, Hermann

    2010-01-01

    Single and duplex real-time polymerase chain reaction (PCR) systems have been developed to quantify specific mRNA transcription of genes coding for the major Daucus carota allergen isoforms Dau c 1.01 and Dau c 1.02. Methods were tested with samples from the local market. Whereas the gene transcription levels for Dau c 1.01 were consistently high in all investigated samples, significant differences for the Dau c 1.02 transcription could be demonstrated in randomly collected market samples. The gene transcription level for the minor Dau c 1.02 variant is about one log below Dau c 1.01. Both formats, single or duplex real-time methods, exhibit ideal cycle threshold (CT) ranges and good reproducibility. In particular, the easily performed duplex real-time PCR system is potentially suited for the selection of hypoallergenic varieties and studying the impact of post-harvesting or environmental conditions.

  20. Genome-Wide Identification and Structural Analysis of bZIP Transcription Factor Genes in Brassica napus

    Directory of Open Access Journals (Sweden)

    Yan Zhou

    2017-10-01

    Full Text Available The basic region/leucine zipper motif (bZIP transcription factor family is one of the largest families of transcriptional regulators in plants. bZIP genes have been systematically characterized in some plants, but not in rapeseed (Brassica napus. In this study, we identified 247 BnbZIP genes in the rapeseed genome, which we classified into 10 subfamilies based on phylogenetic analysis of their deduced protein sequences. The BnbZIP genes were grouped into functional clades with Arabidopsis genes with similar putative functions, indicating functional conservation. Genome mapping analysis revealed that the BnbZIPs are distributed unevenly across all 19 chromosomes, and that some of these genes arose through whole-genome duplication and dispersed duplication events. All expression profiles of 247 bZIP genes were extracted from RNA-sequencing data obtained from 17 different B. napus ZS11 tissues with 42 various developmental stages. These genes exhibited different expression patterns in various tissues, revealing that these genes are differentially regulated. Our results provide a valuable foundation for functional dissection of the different BnbZIP homologs in B. napus and its parental lines and for molecular breeding studies of bZIP genes in B. napus.

  1. Genome-Wide Identification and Structural Analysis of bZIP Transcription Factor Genes in Brassica napus.

    Science.gov (United States)

    Zhou, Yan; Xu, Daixiang; Jia, Ledong; Huang, Xiaohu; Ma, Guoqiang; Wang, Shuxian; Zhu, Meichen; Zhang, Aoxiang; Guan, Mingwei; Lu, Kun; Xu, Xinfu; Wang, Rui; Li, Jiana; Qu, Cunmin

    2017-10-24

    The basic region/leucine zipper motif (bZIP) transcription factor family is one of the largest families of transcriptional regulators in plants. bZIP genes have been systematically characterized in some plants, but not in rapeseed ( Brassica napus ). In this study, we identified 247 BnbZIP genes in the rapeseed genome, which we classified into 10 subfamilies based on phylogenetic analysis of their deduced protein sequences. The BnbZIP genes were grouped into functional clades with Arabidopsis genes with similar putative functions, indicating functional conservation. Genome mapping analysis revealed that the BnbZIPs are distributed unevenly across all 19 chromosomes, and that some of these genes arose through whole-genome duplication and dispersed duplication events. All expression profiles of 247 bZIP genes were extracted from RNA-sequencing data obtained from 17 different B . napus ZS11 tissues with 42 various developmental stages. These genes exhibited different expression patterns in various tissues, revealing that these genes are differentially regulated. Our results provide a valuable foundation for functional dissection of the different BnbZIP homologs in B . napus and its parental lines and for molecular breeding studies of bZIP genes in B . napus .

  2. Transcriptional analysis of late ripening stages of grapevine berry

    Directory of Open Access Journals (Sweden)

    Guillaumie Sabine

    2011-11-01

    Full Text Available Abstract Background The composition of grapevine berry at harvest is a major determinant of wine quality. Optimal oenological maturity of berries is characterized by a high sugar/acidity ratio, high anthocyanin content in the skin, and low astringency. However, harvest time is still mostly determined empirically, based on crude biochemical composition and berry tasting. In this context, it is interesting to identify genes that are expressed/repressed specifically at the late stages of ripening and which may be used as indicators of maturity. Results Whole bunches and berries sorted by density were collected in vineyard on Chardonnay (white cultivar grapevines for two consecutive years at three stages of ripening (7-days before harvest (TH-7, harvest (TH, and 10-days after harvest (TH+10. Microvinification and sensory analysis indicate that the quality of the wines made from the whole bunches collected at TH-7, TH and TH+10 differed, TH providing the highest quality wines. In parallel, gene expression was studied with Qiagen/Operon microarrays using two types of samples, i.e. whole bunches and berries sorted by density. Only 12 genes were consistently up- or down-regulated in whole bunches and density sorted berries for the two years studied in Chardonnay. 52 genes were differentially expressed between the TH-7 and TH samples. In order to determine whether these genes followed a similar pattern of expression during the late stages of berry ripening in a red cultivar, nine genes were selected for RT-PCR analysis with Cabernet Sauvignon grown under two different temperature regimes affecting the precocity of ripening. The expression profiles and their relationship to ripening were confirmed in Cabernet Sauvignon for seven genes, encoding a carotenoid cleavage dioxygenase, a galactinol synthase, a late embryogenesis abundant protein, a dirigent-like protein, a histidine kinase receptor, a valencene synthase and a putative S

  3. De Novo Transcriptome Assembly (NGS) of Curcuma longa L. Rhizome Reveals Novel Transcripts Related to Anticancer and Antimalarial Terpenoids

    Science.gov (United States)

    Jayakumar, Vasanthan; Damodaran, Anand C.; Rao, Sudha Narayana; Katta, Mohan A. V. S. K.; Gopinathan, Sreeja; Sarma, Santosh Prasad; Senthilkumar, Vanitha; Niranjan, Vidya; Gopinath, Ashok; Mugasimangalam, Raja C.

    2013-01-01

    Herbal remedies are increasingly being recognised in recent years as alternative medicine for a number of diseases including cancer. Curcuma longa L., commonly known as turmeric is used as a culinary spice in India and in many Asian countries has been attributed to lower incidences of gastrointestinal cancers. Curcumin, a secondary metabolite isolated from the rhizomes of this plant has been shown to have significant anticancer properties, in addition to antimalarial and antioxidant effects. We sequenced the transcriptome of the rhizome of the 3 varieties of Curcuma longa L. using Illumina reversible dye terminator sequencing followed by de novo transcriptome assembly. Multiple databases were used to obtain a comprehensive annotation and the transcripts were functionally classified using GO, KOG and PlantCyc. Special emphasis was given for annotating the secondary metabolite pathways and terpenoid biosynthesis pathways. We report for the first time, the presence of transcripts related to biosynthetic pathways of several anti-cancer compounds like taxol, curcumin, and vinblastine in addition to anti-malarial compounds like artemisinin and acridone alkaloids, emphasizing turmeric's importance as a highly potent phytochemical. Our data not only provides molecular signatures for several terpenoids but also a comprehensive molecular resource for facilitating deeper insights into the transcriptome of C. longa. PMID:23468859

  4. De Novo transcriptome assembly (NGS) of Curcuma longa L. rhizome reveals novel transcripts related to anticancer and antimalarial terpenoids.

    Science.gov (United States)

    Annadurai, Ramasamy S; Neethiraj, Ramprasad; Jayakumar, Vasanthan; Damodaran, Anand C; Rao, Sudha Narayana; Katta, Mohan A V S K; Gopinathan, Sreeja; Sarma, Santosh Prasad; Senthilkumar, Vanitha; Niranjan, Vidya; Gopinath, Ashok; Mugasimangalam, Raja C

    2013-01-01

    Herbal remedies are increasingly being recognised in recent years as alternative medicine for a number of diseases including cancer. Curcuma longa L., commonly known as turmeric is used as a culinary spice in India and in many Asian countries has been attributed to lower incidences of gastrointestinal cancers. Curcumin, a secondary metabolite isolated from the rhizomes of this plant has been shown to have significant anticancer properties, in addition to antimalarial and antioxidant effects. We sequenced the transcriptome of the rhizome of the 3 varieties of Curcuma longa L. using Illumina reversible dye terminator sequencing followed by de novo transcriptome assembly. Multiple databases were used to obtain a comprehensive annotation and the transcripts were functionally classified using GO, KOG and PlantCyc. Special emphasis was given for annotating the secondary metabolite pathways and terpenoid biosynthesis pathways. We report for the first time, the presence of transcripts related to biosynthetic pathways of several anti-cancer compounds like taxol, curcumin, and vinblastine in addition to anti-malarial compounds like artemisinin and acridone alkaloids, emphasizing turmeric's importance as a highly potent phytochemical. Our data not only provides molecular signatures for several terpenoids but also a comprehensive molecular resource for facilitating deeper insights into the transcriptome of C. longa.

  5. De Novo transcriptome assembly (NGS of Curcuma longa L. rhizome reveals novel transcripts related to anticancer and antimalarial terpenoids.

    Directory of Open Access Journals (Sweden)

    Ramasamy S Annadurai

    Full Text Available Herbal remedies are increasingly being recognised in recent years as alternative medicine for a number of diseases including cancer. Curcuma longa L., commonly known as turmeric is used as a culinary spice in India and in many Asian countries has been attributed to lower incidences of gastrointestinal cancers. Curcumin, a secondary metabolite isolated from the rhizomes of this plant has been shown to have significant anticancer properties, in addition to antimalarial and antioxidant effects. We sequenced the transcriptome of the rhizome of the 3 varieties of Curcuma longa L. using Illumina reversible dye terminator sequencing followed by de novo transcriptome assembly. Multiple databases were used to obtain a comprehensive annotation and the transcripts were functionally classified using GO, KOG and PlantCyc. Special emphasis was given for annotating the secondary metabolite pathways and terpenoid biosynthesis pathways. We report for the first time, the presence of transcripts related to biosynthetic pathways of several anti-cancer compounds like taxol, curcumin, and vinblastine in addition to anti-malarial compounds like artemisinin and acridone alkaloids, emphasizing turmeric's importance as a highly potent phytochemical. Our data not only provides molecular signatures for several terpenoids but also a comprehensive molecular resource for facilitating deeper insights into the transcriptome of C. longa.

  6. A novel test for selection on cis-regulatory elements reveals positive and negative selection acting on mammalian transcriptional enhancers.

    Science.gov (United States)

    Smith, Justin D; McManus, Kimberly F; Fraser, Hunter B

    2013-11-01

    Measuring natural selection on genomic elements involved in the cis-regulation of gene expression--such as transcriptional enhancers and promoters--is critical for understanding the evolution of genomes, yet it remains a major challenge. Many studies have attempted to detect positive or negative selection in these noncoding elements by searching for those with the fastest or slowest rates of evolution, but this can be problematic. Here, we introduce a new approach to this issue, and demonstrate its utility on three mammalian transcriptional enhancers. Using results from saturation mutagenesis studies of these enhancers, we classified all possible point mutations as upregulating, downregulating, or silent, and determined which of these mutations have occurred on each branch of a phylogeny. Applying a framework analogous to Ka/Ks in protein-coding genes, we measured the strength of selection on upregulating and downregulating mutations, in specific branches as well as entire phylogenies. We discovered distinct modes of selection acting on different enhancers: although all three have experienced negative selection against downregulating mutations, the selection pressures on upregulating mutations vary. In one case, we detected positive selection for upregulation, whereas the other two had no detectable selection on upregulating mutations. Our methodology is applicable to the growing number of saturation mutagenesis data sets, and provides a detailed picture of the mode and strength of natural selection acting on cis-regulatory elements.

  7. The transcription analysis of duck enteritis virus UL49.5 gene using real-time quantitative reverse transcription PCR.

    Science.gov (United States)

    Lin, Meng; Jia, Renyong; Wang, Mingshu; Gao, Xinghong; Zhu, Dekang; Chen, Shun; Yin, Zhongqiong; Wang, Yin; Chen, Xiaoyue; Cheng, Anchun

    2013-10-01

    Duck enteritis virus (DEV) UL49.5 encoding glycoprotein N was a conserved gene. The transcription dynamic process of UL49.5 homologous genes in herpesviruses was reported. However, the transcription dynamic process of DEV UL49.5 gene has not yet been established. In this study, a real-time quantitative reverse transcription PCR (real-time qRT-PCR) assay was established to test the transcription dynamic process of DEV UL49.5 gene, and the recombinant plasmid pUCm-T/UL49.5 was constructed as the standard DNA. The samples prepared from DEV-infected (at different time points) and uninfected cell were detected and calculated. The results demonstrated that the real-time qRT-PCR assay was successfully established. The transcription product of DEV UL49.5 gene was first detected at 0.5 h post infection (p.i.), increased at 8 h p.i. and reached a peak at 60 h p.i. Our results illustrated that DEV UL49.5 gene could be regarded as a late gene. The transcription dynamic process of DEV UL49.5 gene may provide a significant clue for further studies of DEV UL49.5 gene.

  8. Killer in our Midst: Part One. An Analysis of Court Transcripts ...

    African Journals Online (AJOL)

    In the spirit of the work edited by Michel Foucault (1975) on Pierre Rivière, I propose to put philosophy to work by tackling a case study in which I shall analyse certain court transcripts that pertain to the defence of serial killer, Stewart Wilken, in Die Staat Teen Stewart Wilken. My analysis of these documents is intended to ...

  9. Functional analysis of jasmonate-responsive transcription factors in Arabidopsis thaliana

    NARCIS (Netherlands)

    Zarei, Adel

    2007-01-01

    The aim of the studies described in this thesis was the functional analysis of JA-responsive transcription factors in Arabidopsis with an emphasis on the interaction with the promoters of their target genes. In short, the following new results were obtained. The promoter of the PDF1.2 gene contains

  10. Microarray analysis of gender- and parasite-specific gene transcription in Strongyloides ratti

    NARCIS (Netherlands)

    Evans, Helen; Mello, Luciane V.; Fang, Yongxiang; Wit, Ernst; Thompson, Fiona J.; Viney, Mark E.; Paterson, Steve

    2008-01-01

    The molecular mechanisms by which parasitic nematodes reproduce and have adapted to life within a host are unclear. In the present study, microarray analysis was used to explore differential transcription among the different stages and sexes of Strongyloides ratti, a parasitic nematode of brown

  11. A Procedure for the Computerized Analysis of Cleft Palate Speech Transcription

    Science.gov (United States)

    Fitzsimons, David A.; Jones, David L.; Barton, Belinda; North, Kathryn N.

    2012-01-01

    The phonetic symbols used by speech-language pathologists to transcribe speech contain underlying hexadecimal values used by computers to correctly display and process transcription data. This study aimed to develop a procedure to utilise these values as the basis for subsequent computerized analysis of cleft palate speech. A computer keyboard…

  12. Invoking Thomas Kuhn: What Citation Analysis Reveals about Science Education

    Science.gov (United States)

    Loving, Cathleen C.; Cobern, William W.

    This paper analyzes how Thomas Kuhn's writings are used by others, especially science education researchers. Previous research in citation analysis is used to frame questions related to who cites Kuhn, in what manner and why. Research questions first focus on the variety of disciplines invoking Kuhn and to what extent Structure of Scientific Revolutions (SSR) is cited. The Web of Science database provides material from 1982 for this analysis. The science education literature is analyzed using back issues from 1985 of the Journal of Research in Science Teaching and Science Education. An article analysis reveals trends in terms of what Kuhnian ideas are most frequently invoked. Results indicate a wide array of disciplines from beekeeping to law cite Kuhn - especially generic citations to SSR. The science education journal analysis reveals pervasive use of the term paradigm, although use is quite varied. The two areas of research in science education most impacted by Kuhn appear to be conceptual change theory and constructivist epistemologies. Additional uses of Kuhn are discussed. The degree to which Kuhn is invoked in ways supporting the theoretical framework of citation analysis, whether his work is misappropriated, and the impact of Kuhn are discussed.

  13. Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq

    DEFF Research Database (Denmark)

    Sittka, A; Lucchini, S; Papenfort, K

    2008-01-01

    Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial...... transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co...... would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high...

  14. How to Identify Negative Attitudes towards Inclusive Education: Critical Discourse Analysis of Russian Transcripts Using Role and Reference Grammar

    Directory of Open Access Journals (Sweden)

    Mariia Rubtcova

    2016-09-01

    Full Text Available This paper presents the Role and Reference Grammar (RRG analysis that aims to reveal possibilities required for carrying out the interdisciplinary research development within Critical Discourse Analysis (CDA. It takes a closer look at conflicts, considering the example of a conflict situation occurred in reaction to the opening of the inclusive academic programme at one of St. Petersburg’s secondary schools. Role and Reference Grammar application demonstrates that the use of different verb types and macroroles has led to the various interpretations. These findings confirm that RRG could influence the increase of objectivity of the transcript analysis in qualitative social research. RRG provides new information which in combination with other methods can help us to understand the positions of participants involved into conflicts

  15. Network analysis of the transcriptional pattern of young and old cells of Escherichia coli during lag phase

    Directory of Open Access Journals (Sweden)

    Hinton Jay CD

    2009-11-01

    Full Text Available Abstract Background The aging process of bacteria in stationary phase is halted if cells are subcultured and enter lag phase and it is then followed by cellular division. Network science has been applied to analyse the transcriptional response, during lag phase, of bacterial cells starved previously in stationary phase for 1 day (young cells and 16 days (old cells. Results A genome scale network was constructed for E. coli K-12 by connecting genes with operons, transcription and sigma factors, metabolic pathways and cell functional categories. Most of the transcriptional changes were detected immediately upon entering lag phase and were maintained throughout this period. The lag period was longer for older cells and the analysis of the transcriptome revealed different intracellular activity in young and old cells. The number of genes differentially expressed was smaller in old cells (186 than in young cells (467. Relatively, few genes (62 were up- or down-regulated in both cultures. Transcription of genes related to osmotolerance, acid resistance, oxidative stress and adaptation to other stresses was down-regulated in both young and old cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were up-regulated in young cells while old cells up-regulated the Entner Doudoroff and gluconate pathways and down-regulated the pentose phosphate pathway. In both old and young cells, anaerobic respiration and fermentation pathways were down-regulated, but only young cells up-regulated aerobic respiration while there was no evidence of aerobic respiration in old cells. Numerous genes related to DNA maintenance and replication, translation, ribosomal biosynthesis and RNA processing as well as biosynthesis of the cell envelope and flagellum and several components of the chemotaxis signal transduction complex were up-regulated only in young cells. The genes for several transport proteins for iron compounds were up-regulated in both young

  16. Network analysis of the transcriptional pattern of young and old cells of Escherichia coli during lag phase

    LENUS (Irish Health Repository)

    Pin, Carmen

    2009-11-16

    Abstract Background The aging process of bacteria in stationary phase is halted if cells are subcultured and enter lag phase and it is then followed by cellular division. Network science has been applied to analyse the transcriptional response, during lag phase, of bacterial cells starved previously in stationary phase for 1 day (young cells) and 16 days (old cells). Results A genome scale network was constructed for E. coli K-12 by connecting genes with operons, transcription and sigma factors, metabolic pathways and cell functional categories. Most of the transcriptional changes were detected immediately upon entering lag phase and were maintained throughout this period. The lag period was longer for older cells and the analysis of the transcriptome revealed different intracellular activity in young and old cells. The number of genes differentially expressed was smaller in old cells (186) than in young cells (467). Relatively, few genes (62) were up- or down-regulated in both cultures. Transcription of genes related to osmotolerance, acid resistance, oxidative stress and adaptation to other stresses was down-regulated in both young and old cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were up-regulated in young cells while old cells up-regulated the Entner Doudoroff and gluconate pathways and down-regulated the pentose phosphate pathway. In both old and young cells, anaerobic respiration and fermentation pathways were down-regulated, but only young cells up-regulated aerobic respiration while there was no evidence of aerobic respiration in old cells. Numerous genes related to DNA maintenance and replication, translation, ribosomal biosynthesis and RNA processing as well as biosynthesis of the cell envelope and flagellum and several components of the chemotaxis signal transduction complex were up-regulated only in young cells. The genes for several transport proteins for iron compounds were up-regulated in both young and old cells

  17. Analysis of the transcriptional responses in inflorescence buds of Jatropha curcas exposed to cytokinin treatment.

    Science.gov (United States)

    Chen, Mao-Sheng; Pan, Bang-Zhen; Wang, Gui-Juan; Ni, Jun; Niu, Longjian; Xu, Zeng-Fu

    2014-11-30

    Jatropha curcas L. is a potential biofuel plant. Application of exogenous cytokinin (6-benzyladenine, BA) on its inflorescence buds can significantly increase the number of female flowers, thereby improving seed yield. To investigate which genes and signal pathways are involved in the response to cytokinin in J. curcas inflorescence buds, we monitored transcriptional activity in inflorescences at 0, 3, 12, 24, and 48 h after BA treatment using a microarray. We detected 5,555 differentially expressed transcripts over the course of the experiment, which could be grouped into 12 distinct temporal expression patterns. We also identified 31 and 131 transcripts in J. curcas whose homologs in model plants function in flowering and phytohormonal signaling pathways, respectively. According to the transcriptional analysis of genes involved in flower development, we hypothesized that BA treatment delays floral organ formation by inhibiting the transcription of the A, B and E classes of floral organ-identity genes, which would allow more time to generate more floral primordia in inflorescence meristems, thereby enhancing inflorescence branching and significantly increasing flower number per inflorescence. BA treatment might also play an important role in maintaining the flowering signals by activating the transcription of GIGANTEA (GI) and inactivating the transcription of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and TERMINAL FLOWER 1b (TFL1b). In addition, exogenous cytokinin treatment could regulate the expression of genes involved in the metabolism and signaling of other phytohormones, indicating that cytokinin and other phytohormones jointly regulate flower development in J. curcas inflorescence buds. Our study provides a framework to better understand the molecular mechanisms underlying changes in flowering traits in response to cytokinin treatment in J. curcas inflorescence buds. The results provide valuable information related to the mechanisms of cross-talk among

  18. Using network component analysis to dissect regulatory networks mediated by transcription factors in yeast.

    Directory of Open Access Journals (Sweden)

    Chun Ye

    2009-03-01

    Full Text Available Understanding the relationship between genetic variation and gene expression is a central question in genetics. With the availability of data from high-throughput technologies such as ChIP-Chip, expression, and genotyping arrays, we can begin to not only identify associations but to understand how genetic variations perturb the underlying transcription regulatory networks to induce differential gene expression. In this study, we describe a simple model of transcription regulation where the expression of a gene is completely characterized by two properties: the concentrations and promoter affinities of active transcription factors. We devise a method that extends Network Component Analysis (NCA to determine how genetic variations in the form of single nucleotide polymorphisms (SNPs perturb these two properties. Applying our method to a segregating population of Saccharomyces cerevisiae, we found statistically significant examples of trans-acting SNPs located in regulatory hotspots that perturb transcription factor concentrations and affinities for target promoters to cause global differential expression and cis-acting genetic variations that perturb the promoter affinities of transcription factors on a single gene to cause local differential expression. Although many genetic variations linked to gene expressions have been identified, it is not clear how they perturb the underlying regulatory networks that govern gene expression. Our work begins to fill this void by showing that many genetic variations affect the concentrations of active transcription factors in a cell and their affinities for target promoters. Understanding the effects of these perturbations can help us to paint a more complete picture of the complex landscape of transcription regulation. The software package implementing the algorithms discussed in this work is available as a MATLAB package upon request.

  19. Transcriptional Control of Dual Transporters Involved in α-Ketoglutarate Utilization Reveals Their Distinct Roles in UropathogenicEscherichia coli.

    Science.gov (United States)

    Cai, Wentong; Cai, Xuwang; Yang, Yongwu; Yan, Shigan; Zhang, Haibin

    2017-01-01

    Uropathogenic Escherichia coli (UPEC) are the primary causative agents of urinary tract infections. Some UPEC isolates are able to infect renal proximal tubule cells, and can potentially cause pyelonephritis. We have previously shown that to fulfill their physiological roles renal proximal tubule cells accumulate high concentrations of α-ketoglutarate (KG) and that gene cluster c5032 - c5039 contribute to anaerobic utilization of KG by UPEC str. CFT073, thereby promoting its in vivo fitness. Given the importance of utilizing KG for UPEC, this study is designed to investigate the roles of two transporters KgtP and C5038 in KG utilization, their transcriptional regulation, and their contributions to UPEC fitness in vivo . Our phylogenetic analyses support that kgtP is a widely conserved locus in commensal and pathogenic E. coli , while UPEC-associated c5038 was acquired through horizontal gene transfer. Global anaerobic transcriptional regulators Fumarate and nitrate reduction (FNR) and ArcA induced c5038 expression in anaerobiosis, and C5038 played a major role in anaerobic growth on KG. KgtP was required for aerobic growth on KG, and its expression was repressed by FNR and ArcA under anaerobic conditions. Analyses of FNR and ArcA binding sites and results of EMS assays suggest that FNR and ArcA likely inhibit kgtP expression through binding to the -35 region of kgtP promoter and occluding the occupancy of RNA polymerases. Gene c5038 can be specifically induced by KG, whereas the expression of kgtP does not respond to KG, yet can be stimulated during growth on glycerol. In addition, c5038 and kgtP expression were further shown to be controlled by different alternative sigma factors RpoN and RpoS, respectively. Furthermore, dual-strain competition assays in a murine model showed that c5038 mutant but not kgtP mutant was outcompeted by the wild-type strain during the colonization of murine bladders and kidneys, highlighting the importance of C5038 under in vivo

  20. Large-scale analysis by SAGE reveals new mechanisms of v-erbA oncogene action

    Directory of Open Access Journals (Sweden)

    Faure Claudine

    2007-10-01

    Full Text Available Abstract Background: The v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAα proto-oncogene that encodes the nuclear receptor for triiodothyronine (T3R. v-ErbA transforms erythroid progenitors in vitro by blocking their differentiation, supposedly by interference with T3R and RAR (Retinoic Acid Receptor. However, v-ErbA target genes involved in its transforming activity still remain to be identified. Results: By using Serial Analysis of Gene Expression (SAGE, we identified 110 genes deregulated by v-ErbA and potentially implicated in the transformation process. Bioinformatic analysis of promoter sequence and transcriptional assays point out a potential role of c-Myb in the v-ErbA effect. Furthermore, grouping of newly identified target genes by function revealed both expected (chromatin/transcription and unexpected (protein metabolism functions potentially deregulated by v-ErbA. We then focused our study on 15 of the new v-ErbA target genes and demonstrated by real time PCR that in majority their expression was activated neither by T3, nor RA, nor during differentiation. This was unexpected based upon the previously known role of v-ErbA. Conclusion: This paper suggests the involvement of a wealth of new unanticipated mechanisms of v-ErbA action.

  1. Comparative transcripts profiling reveals new insight into molecular processes regulating lycopene accumulation in a sweet orange (Citrus sinensis red-flesh mutant

    Directory of Open Access Journals (Sweden)

    Zhang Jianchen

    2009-11-01

    Full Text Available Abstract Background Interest in lycopene metabolism and regulation is growing rapidly because accumulative studies have suggested an important role for lycopene in human health promotion. However, little is known about the molecular processes regulating lycopene accumulation in fruits other than tomato so far. Results On a spontaneous sweet orange bud mutant with abnormal lycopene accumulation in fruits and its wild type, comparative transcripts profiling was performed using Massively Parallel Signature Sequencing (MPSS. A total of 6,877,027 and 6,275,309 reliable signatures were obtained for the wild type (WT and the mutant (MT, respectively. Interpretation of the MPSS signatures revealed that the total number of transcribed gene in MT is 18,106, larger than that in WT 17,670, suggesting that newly initiated transcription occurs in the MT. Further comparison of the transcripts abundance between MT and WT revealed that 3,738 genes show more than two fold expression difference, and 582 genes are up- or down-regulated at 0.05% significance level by more than three fold difference. Functional assignments of the differentially expressed genes indicated that 26 reliable metabolic pathways are altered in the mutant; the most noticeable ones are carotenoid biosynthesis, photosynthesis, and citrate cycle. These data suggest that enhanced photosynthesis and partial impairment of lycopene downstream flux are critical for the formation of lycopene accumulation trait in the mutant. Conclusion This study provided a global picture of the gene expression changes in a sweet orange red-flesh mutant as compared to the wild type. Interpretation of the differentially expressed genes revealed new insight into the molecular processes regulating lycopene accumulation in the sweet orange red-flesh mutant.

  2. Transcriptional response of Arabidopsis seedlings during spaceflight reveals peroxidase and cell wall remodeling genes associated with root hair development.

    Science.gov (United States)

    Kwon, Taegun; Sparks, J Alan; Nakashima, Jin; Allen, Stacy N; Tang, Yuhong; Blancaflor, Elison B

    2015-01-01

    • Plants will be an important component of advanced life support systems during space exploration missions. Therefore, understanding their biology in the spacecraft environment will be essential before they can be used for such systems.• Seedlings of Arabidopsis thaliana were grown for 2 wk in the Biological Research in Canisters (BRIC) hardware on board the second to the last mission of the space shuttle Discovery (STS-131). Transcript profiles between ground controls and space-grown seedlings were compared using stringent selection criteria.• Expression of transcripts associated with oxidative stress and cell wall remodeling was repressed in microgravity. These downregulated genes were previously shown to be enriched in root hairs consistent with seedling phenotypes observed in space. Mutations in genes that were downregulated in microgravity, including two uncharacterized root hair-expressed class III peroxidase genes (PRX44 and PRX57), led to defective polar root hair growth on Earth. PRX44 and PRX57 mutants had ruptured root hairs, which is a typical phenotype of tip-growing cells with defective cell walls and those subjected to stress.• Long-term exposure to microgravity negatively impacts tip growth by repressing expression of genes essential for normal root hair development. Whereas changes in peroxidase gene expression leading to reduced root hair growth in space are actin-independent, root hair development modulated by phosphoinositides could be dependent on the actin cytoskeleton. These results have profound implications for plant adaptation to microgravity given the importance of tip growing cells such as root hairs for efficient nutrient capture. © 2015 Botanical Society of America, Inc.

  3. Transcriptional and epigenetic analyses of the DMD locus reveal novel cis‑acting DNA elements that govern muscle dystrophin expression.

    Science.gov (United States)

    Gherardi, Samuele; Bovolenta, Matteo; Passarelli, Chiara; Falzarano, Maria Sofia; Pigini, Paolo; Scotton, Chiara; Neri, Marcella; Armaroli, Annarita; Osman, Hana; Selvatici, Rita; Gualandi, Francesca; Recchia, Alessandra; Mora, Marina; Bernasconi, Pia; Maggi, Lorenzo; Morandi, Lucia; Ferlini, Alessandra; Perini, Giovanni

    2017-11-01

    The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2Mb and accounts for approximately 0,1% of the entire human genome. Mutations in this gene cause Duchenne and Becker Muscular Dystrophy, X-linked Dilated Cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16h, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA polymerase II along the entire 2.2Mb of the DMD locus with just one site of pausing around intron 52. We also discovered epigenetic marks highlighting the existence of four novel cis‑DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies. Copyright © 2017. Published by Elsevier B.V.

  4. Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster.

    Science.gov (United States)

    de Vega-Bartol, José J; Simões, Marta; Lorenz, W Walter; Rodrigues, Andreia S; Alba, Rob; Dean, Jeffrey F D; Miguel, Célia M

    2013-08-30

    It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in

  5. Microbiota analysis to reveal temperature abuse of fresh pork

    DEFF Research Database (Denmark)

    Buschhardt, Tasja; Bahl, Martin Iain; Hansen, Tina Beck

    monitored during aerobic chill-storage (4 °C and 7 °C) and temperature abuse (12 °C and 16 °C) for 96 hours, by culture-based methods and 16S rRNA gene sequencing. Bacterial genera that dominated during prolonged temperature abuse were Acinetobacter, Serratia and Pseudomonas, whereas chill-stored meat...... was dominated by Pseudomonas only. We also showed that the initial community affects subsequent changes during storage. The results suggest that principal coordinate analysis of beta diversity could be a useful tool to reveal temperature abused meat. Sequence data and culturing data revealed a strong positive......Violations of temperature regulations in the meat chain may affect meat safety. Methods are lacking to estimate whether meat has been subjected to temperature abuse. Exposure to too high temperatures may lead to systematic changes in the diverse bacterial communities of fresh meat. We investigated...

  6. Microbiota analysis to reveal temperature abuse of fresh pork

    DEFF Research Database (Denmark)

    Buschhardt, Tasja; Bahl, Martin Iain; Hansen, Tina Beck

    2017-01-01

    Violations of temperature regulations in the meat chain may affect meat safety. Methods are lacking to estimate whether meat has been subjected to temperature abuse. Exposure to too high temperatures may lead to systematic changes in the diverse bacterial communities of fresh meat. We investigated...... monitored during aerobic chill-storage (4 °C and 7 °C) and temperature abuse (12 °C and 16 °C) for 96 hours, by culture-based methods and 16S rRNA gene sequencing. Bacterial genera that dominated during prolonged temperature abuse were Acinetobacter, Serratia and Pseudomonas, whereas chill-stored meat...... was dominated by Pseudomonas only. We also showed that the initial community affects subsequent changes during storage. The results suggest that principal coordinate analysis of beta diversity could be a useful tool to reveal temperature abused meat. Sequence data and culturing data revealed a strong positive...

  7. Global transcriptional analysis of nitrogen fixation and ammonium repression in root-associated Pseudomonas stutzeri A1501

    Directory of Open Access Journals (Sweden)

    Lu Wei

    2010-01-01

    Full Text Available Abstract Background Biological nitrogen fixation is highly controlled at the transcriptional level by regulatory networks that respond to the availability of fixed nitrogen. In many diazotrophs, addition of excess ammonium in the growth medium results in immediate repression of nif gene transcription. Although the regulatory cascades that control the transcription of the nif genes in proteobacteria have been well investigated, there are limited data on the kinetics of ammonium-dependent repression of nitrogen fixation. Results Here we report a global transcriptional profiling analysis of nitrogen fixation and ammonium repression in Pseudomonas stutzeri A1501, a root-associated and nitrogen-fixing bacterium. A total of 166 genes, including those coding for the global nitrogen regulation (Ntr and Nif-specific regulatory proteins, were upregulated under nitrogen fixation conditions but rapidly downregulated as early as 10 min after ammonium shock. Among these nitrogen fixation-inducible genes, 95 have orthologs in each of Azoarcus sp. BH72 and Azotobacter vinelandii AvoP. In particular, a 49-kb expression island containing nif and other associated genes was markedly downregulated by ammonium shock. Further functional characterization of pnfA, a new NifA-σ54-dependent gene chromosomally linked to nifHDK, is reported. This gene encodes a protein product with an amino acid sequence similar to that of five hypothetical proteins found only in diazotrophic strains. No noticeable differences in the transcription of nifHDK were detected between the wild type strain and pnfA mutant. However, the mutant strain exhibited a significant decrease in nitrogenase activity under microaerobic conditions and lost its ability to use nitrate as a terminal electron acceptor for the support of nitrogen fixation under anaerobic conditions. Conclusions Based on our results, we conclude that transcriptional regulation of nif gene expression in A1501 is mediated by the nif

  8. Analysis of intergenic spacer transcripts suggests ‘read-around’ transcription of the extrachromosomal circular rDNA in Euglena gracilis

    Science.gov (United States)

    Greenwood, Spencer J.; Schnare, Murray N.; Cook, James R.; Gray, Michael W.

    2001-01-01

    We report here the sequence of the 1743 bp intergenic spacer (IGS) that separates the 3′-end of the large subunit ribosomal RNA (rRNA) gene from the 5′-end of the small subunit (SSU) rRNA gene in the circular, extrachromosomal ribosomal DNA (rDNA) of Euglena gracilis. The IGS contains a 277 nt stretch of sequence that is related to a sequence found in ITS 1, an internal transcribed spacer between the SSU and 5.8S rRNA genes. Primer extension analysis of IGS transcripts identified three abundant reverse transcriptase stops that may be analogous to the transcription initiation site (TIS) and two processing sites (A′ and A0) that are found in this region in other eukaryotes. Features that could influence processing at these sites include an imperfect palindrome near site A0 and a sequence near site A′ that could potentially base pair with U3 small nucleolar RNA. Our identification of the TIS (verified by mung bean nuclease analysis) is considered tentative because we also detected low-abundance transcripts upstream of this site throughout the entire IGS. This result suggests the possibility of ‘read-around’ transcription, i.e. transcription that proceeds multiple times around the rDNA circle without termination. PMID:11353089

  9. Transcriptome analysis reveals dynamic changes in the gene ...

    Indian Academy of Sciences (India)

    significantly differentially expressed genes that were categorized as cation binding, transcription regulation, metabolic pro- ... are significantly altered in response to potassium deficiency, which can result in physiological and morphological changes in .... Tobacco seedling transcriptome response to low potassium stress.

  10. Analysis of geminivirus AL2 and L2 proteins reveals a novel AL2 silencing suppressor activity.

    Science.gov (United States)

    Jackel, Jamie N; Buchmann, R Cody; Singhal, Udit; Bisaro, David M

    2015-03-01

    L2 suppressor activities, we show that L2 is unable to suppress silencing in Nicotiana benthamiana plants that have undergone the vegetative-to-reproductive transition. As L2 was previously shown to be effective in mature Arabidopsis plants, these results illustrate that silencing mechanisms can change during development or in response to stress in ways that may be species specific. The AL2 and L2 proteins are known to share a suppression mechanism that correlates with the ability of both proteins to inhibit ADK, while AL2 in addition can inhibit silencing by transcriptionally activating cellular genes. Here, we also provide evidence for a third AL2 suppression mechanism that depends on neither transcription activation nor ADK inactivation. In addition to revealing the remarkable versatility of AL2, this work highlights the utility of viral suppressors as probes for the analysis of silencing pathways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Molecular genetic analysis of activation-tagged transcription factors thought to be involved in photomorphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Neff, Michael M.

    2011-06-23

    This is a final report for Department of Energy Grant No. DE-FG02-08ER15927 entitled “Molecular Genetic Analysis of Activation-Tagged Transcription Factors Thought to be Involved in Photomorphogenesis”. Based on our preliminary photobiological and genetic analysis of the sob1-D mutant, we hypothesized that OBP3 is a transcription factor involved in both phytochrome and cryptochrome-mediated signal transduction. In addition, we hypothesized that OBP3 is involved in auxin signaling and root development. Based on our preliminary photobiological and genetic analysis of the sob2-D mutant, we also hypothesized that a related gene, LEP, is involved in hormone signaling and seedling development.

  12. Two modified RNA extraction methods compatible with transcript profiling and gene expression analysis for cotton roots.

    Science.gov (United States)

    Xie, Chengjian; Wang, Chunyan; Wang, Xiaokun; Yang, Xingyong

    2013-01-01

    Efficient isolation of high-quality RNA is of prime importance for optimal transcript profiling results and further gene expression analysis. However, it is difficult for cotton roots because of lower-than-average RNA content and high content of polysaccharides, polyphenols, and other secondary metabolites. To develop simple and reliable protocols for high-quality RNA extraction from cotton roots for transcript profiling and gene expression analysis, some modifications were introduced to a reported plant RNA isolation protocol and a reagent kit method. Using method A, we successfully extracted high-quality RNA for transcript profiling from cotton roots. Gel electrophoresis analysis and polymerase chain reaction (PCR) assay indicated that RNA had good integrity without protein and genomic DNA contamination. Furthermore, the A260/280 (1.9) and A260/230 (1.6) ratios indicated that the isolated RNA was of high purity. Using method B, about 7 µg total RNA of high quality could be obtained from 0.1 g samples from cotton roots, which can be used for reverse-transcription (RT)-PCR and quantitative real-time RT-PCR. The two RNA extraction methods were used to investigate different gene expression of cotton roots (Gossypium hirsutum) infected by weak pathogenic Verticillium dahliae and the results showed they can satisfy the transcript profiling and quantitative real-time RT-PCR requirements for RNA. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

  13. Genome wide analysis of stress responsive WRKY transcription factors in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Shaiq Sultan

    2016-04-01

    Full Text Available WRKY transcription factors are a class of DNA-binding proteins that bind with a specific sequence C/TTGACT/C known as W-Box found in promoters of genes which are regulated by these WRKYs. From previous studies, 43 different stress responsive WRKY transcription factors in Arabidopsis thaliana, identified and then categorized in three groups viz., abiotic, biotic and both of these stresses. A comprehensive genome wide analysis including chromosomal localization, gene structure analysis, multiple sequence alignment, phylogenetic analysis and promoter analysis of these WRKY genes was carried out in this study to determine the functional homology in Arabidopsis. This analysis led to the classification of these WRKY family members into 3 major groups and subgroups and showed evolutionary relationship among these groups on the base of their functional WRKY domain, chromosomal localization and intron/exon structure. The proposed groups of these stress responsive WRKY genes and annotation based on their position on chromosomes can also be explored to determine their functional homology in other plant species in relation to different stresses. The result of the present study provides indispensable genomic information for the stress responsive WRKY transcription factors in Arabidopsis and will pave the way to explain the precise role of various AtWRKYs in plant growth and development under stressed conditions.

  14. A comprehensive study design reveals treatment- and transcript abundance–dependent concordance between RNA-seq and microarray data

    Science.gov (United States)

    Wang, Charles; Gong, Binsheng; Bushel, Pierre R.; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Xu, Joshua; Fang, Hong; Hong, Huixiao; Shen, Jie; Su, Zhenqiang; Meehan, Joe; Li, Xiaojin; Yang, Lu; Li, Haiqing; Łabaj, Paweł P.; Kreil, David P.; Megherbi, Dalila; Florian, Caiment; Gaj, Stan; van Delft, Joost; Kleinjans, Jos; Scherer, Andreas; Viswanath, Devanarayan; Wang, Jian; Yang, Yong; Qian, Hui-Rong; Lancashire, Lee J.; Bessarabova, Marina; Nikolsky, Yuri; Furlanello, Cesare; Chierici, Marco; Albanese, Davide; Jurman, Giuseppe; Riccadonna, Samantha; Filosi, Michele; Visintainer, Roberto; Zhang, Ke K.; Li, Jianying; Hsieh, Jui-Hua; Svoboda, Daniel L.; Fuscoe, James C.; Deng, Youping; Shi, Leming; Paules, Richard S.; Auerbach, Scott S.; Tong, Weida

    2014-01-01

    RNA-seq facilitates unbiased genome-wide gene-expression profiling. However, its concordance with the well-established microarray platform must be rigorously assessed for confident uses in clinical and regulatory application. Here we use a comprehensive study design to generate Illumina RNA-seq and Affymetrix microarray data from the same set of liver samples of rats under varying degrees of perturbation by 27 chemicals representing multiple modes of action (MOA). The cross-platform concordance in terms of differentially expressed genes (DEGs) or enriched pathways is highly correlated with treatment effect size, gene-expression abundance and the biological complexity of the MOA. RNA-seq outperforms microarray (90% versus 76%) in DEG verification by quantitative PCR and the main gain is its improved accuracy for low expressed genes. Nonetheless, predictive classifiers derived from both platforms performed similarly. Therefore, the endpoint studied and its biological complexity, transcript abundance, and intended application are important factors in transcriptomic research and for decision-making. PMID:25150839

  15. Transcript profiles of Blumeria graminis development during infection reveal a cluster of genes that are potential virulence determinants.

    Science.gov (United States)

    Both, Maike; Eckert, Sabine E; Csukai, Michael; Müller, Elisabeth; Dimopoulos, George; Spanu, Pietro D

    2005-02-01

    High-density cDNA microarrays (2,027 unigenes) were used to analyze transcript profiles of the plant-pathogenic fungus Blumeria graminis f. sp. hordei throughout its asexual life cycle and development of infection. RNA was obtained from four stages preceding penetration and four stages after penetration of the host cells. The microarray data was validated by comparing the expression of a plasma membrane H+-ATPase and fructose-1,6-bis phosphatase with the data obtained from a quantitative polymerase chain reaction (PCR) assay. The results showed that there was a global switch in expression between the pre- and postpenetrative stages. This was largely due to accumulation of RNA encoding protein biosynthesis genes in the late stages. Other functional clusters, such as virulence-related genes and sterol metabolism genes, are up-regulated in pre- and postpenetration stages, respectively. A group of RNAs whose abundance correlated with the expression of cap20, a gene known to be required for virulence in Colletotrichum gloeosporioides, identified genes that are strong candidates for pathogenicity factors in B. graminis.

  16. Global analysis of asymmetric RNA enrichment in oocytes reveals low conservation between closely related Xenopus species

    Science.gov (United States)

    Claußen, Maike; Lingner, Thomas; Pommerenke, Claudia; Opitz, Lennart; Salinas, Gabriela; Pieler, Tomas

    2015-01-01

    RNAs that localize to the vegetal cortex during Xenopus laevis oogenesis have been reported to function in germ layer patterning, axis determination, and development of the primordial germ cells. Here we report on the genome-wide, comparative analysis of differentially localizing RNAs in Xenopus laevis and Xenopus tropicalis oocytes, revealing a surprisingly weak degree of conservation in respect to the identity of animally as well as vegetally enriched transcripts in these closely related species. Heterologous RNA injections and protein binding studies indicate that the different RNA localization patterns in these two species are due to gain/loss of cis-acting localization signals rather than to differences in the RNA-localizing machinery. PMID:26337391

  17. Global analysis of cis-natural antisense transcripts and their heat-responsive nat-siRNAs in Brassica rapa.

    Science.gov (United States)

    Yu, Xiang; Yang, Jun; Li, Xiaorong; Liu, Xuxin; Sun, Chuanbao; Wu, Feijie; He, Yuke

    2013-12-10

    Brassica rapa includes several important leaf vegetable crops whose production is often damaged by high temperature. Cis-natural antisense transcripts (cis-NATs) and cis-NATs-derived small interfering RNAs (nat-siRNAs) play important roles in plant development and stress responses. However, genome-wide cis-NATs in B. rapa are not known. The NATs and nat-siRNAs that respond to heat stress have never been well studied in B. rapa. Here, we took advantage of RNA-seq and small RNA (sRNA) deep sequencing technology to identify cis-NATs and heat responsive nat-siRNAs in B. rapa. Analyses of four RNA sequencing datasets revealed 1031 cis-NATs B. rapa ssp. chinensis cv Wut and B. rapa ssp. pekinensis cv. Bre. Based on sequence homology between Arabidopsis thaliana and B. rapa, 303 conserved cis-NATs in B. rapa were found to correspond to 280 cis-NATs in Arabidopsis; the remaining 728 novel cis-NATs were identified as Brassica-specific ones. Using six sRNA libraries, 4846 nat-siRNAs derived from 150 cis-NATs were detected. Differential expression analysis revealed that nat-siRNAs derived from 12 cis-NATs were responsive to heat stress, and most of them showed strand bias. Real-time PCR indicated that most of the transcripts generating heat-responsive nat-siRNAs were upregulated under heat stress, while the transcripts from the opposite strands of the same loci were downregulated. Our results provide the first subsets of genome-wide cis-NATs and heat-responsive nat-siRNAs in B. rapa; these sRNAs are potentially useful for the genetic improvement of heat tolerance in B. rapa and other crops.

  18. Proteome and transcript analysis of Vitis vinifera cell cultures subjected to Botrytis cinerea infection.

    Science.gov (United States)

    Dadakova, K; Havelkova, M; Kurkova, B; Tlolkova, I; Kasparovsky, T; Zdrahal, Z; Lochman, J

    2015-04-24

    Gray mold caused by Botrytis cinerea is one of the most important diseases of grapevine resulting in significant reductions in yield and fruit quality. In order to examine the molecular mechanisms that characterize the interaction between B. cinerea and the host plant, the grapevine cytoplasmic proteome was analyzed by two-dimensional polyacrylamide gel electrophoresis. The interaction between Vitis vinifera cv. Gamay cells and B. cinerea was characterized by the increase in spot abundance of 30 proteins, of which 21 were successfully identified. The majority of these proteins were related to defence and stress responses and to cell wall modifications. Some of the modulated proteins have been previously found to be affected by other pathogens when they infect V. vinifera but interestingly, the proteins related to cell wall modification that were influenced by B. cinerea have not been shown to be modulated by any other pathogen studied to date. Transcript analysis using the quantitative real time polymerase chain reaction additionally revealed the up-regulation of several acidic, probably extracellular, chitinases. The results indicate that cell wall strengthening, accumulation of PR proteins and excretion of lytic enzymes are likely to be important mechanisms in the defence of grapevine against B. cinerea. Although gray mold caused by Botrytis cinerea is one of the most important diseases of grapevine, little information is available about proteomic changes in this pathosystem. These results suggest that cell wall strengthening, accumulation of PR proteins and excretion of lytic enzymes are important molecular mechanisms in the defence of grapevine against B. cinerea. Surprisingly, the proteins related to cell wall modification that were modulated by B. cinerea have not been shown to be affected by any other pathogen studied to date. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Analysis of transcription of plant 7SL RNA gene variants in HeLa in vitro transcription system

    Czech Academy of Sciences Publication Activity Database

    Vrba, Lukáš; Matoušek, Jaroslav

    2002-01-01

    Roč. 48, - (2002), s. 227-231 ISSN 0015-5500 R&D Projects: GA ČR GA503/95/1583; GA ČR GA521/99/1591 Institutional research plan: CEZ:AV0Z5051902 Keywords : plant genetic * gene transcription Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.615, year: 2002

  20. RNA-Sequencing Reveals Unique Transcriptional Signatures of Running and Running-Independent Environmental Enrichment in the Adult Mouse Dentate Gyrus

    Directory of Open Access Journals (Sweden)

    Catherine-Alexandra Grégoire

    2018-04-01

    Full Text Available Environmental enrichment (EE is a powerful stimulus of brain plasticity and is among the most accessible treatment options for brain disease. In rodents, EE is modeled using multi-factorial environments that include running, social interactions, and/or complex surroundings. Here, we show that running and running-independent EE differentially affect the hippocampal dentate gyrus (DG, a brain region critical for learning and memory. Outbred male CD1 mice housed individually with a voluntary running disk showed improved spatial memory in the radial arm maze compared to individually- or socially-housed mice with a locked disk. We therefore used RNA sequencing to perform an unbiased interrogation of DG gene expression in mice exposed to either a voluntary running disk (RUN, a locked disk (LD, or a locked disk plus social enrichment and tunnels [i.e., a running-independent complex environment (CE]. RNA sequencing revealed that RUN and CE mice showed distinct, non-overlapping patterns of transcriptomic changes versus the LD control. Bio-informatics uncovered that the RUN and CE environments modulate separate transcriptional networks, biological processes, cellular compartments and molecular pathways, with RUN preferentially regulating synaptic and growth-related pathways and CE altering extracellular matrix-related functions. Within the RUN group, high-distance runners also showed selective stress pathway alterations that correlated with a drastic decline in overall transcriptional changes, suggesting that excess running causes a stress-induced suppression of running’s genetic effects. Our findings reveal stimulus-dependent transcriptional signatures of EE on the DG, and provide a resource for generating unbiased, data-driven hypotheses for novel mediators of EE-induced cognitive changes.

  1. RNA-Sequencing Reveals Unique Transcriptional Signatures of Running and Running-Independent Environmental Enrichment in the Adult Mouse Dentate Gyrus.

    Science.gov (United States)

    Grégoire, Catherine-Alexandra; Tobin, Stephanie; Goldenstein, Brianna L; Samarut, Éric; Leclerc, Andréanne; Aumont, Anne; Drapeau, Pierre; Fulton, Stephanie; Fernandes, Karl J L

    2018-01-01

    Environmental enrichment (EE) is a powerful stimulus of brain plasticity and is among the most accessible treatment options for brain disease. In rodents, EE is modeled using multi-factorial environments that include running, social interactions, and/or complex surroundings. Here, we show that running and running-independent EE differentially affect the hippocampal dentate gyrus (DG), a brain region critical for learning and memory. Outbred male CD1 mice housed individually with a voluntary running disk showed improved spatial memory in the radial arm maze compared to individually- or socially-housed mice with a locked disk. We therefore used RNA sequencing to perform an unbiased interrogation of DG gene expression in mice exposed to either a voluntary running disk (RUN), a locked disk (LD), or a locked disk plus social enrichment and tunnels [i.e., a running-independent complex environment (CE)]. RNA sequencing revealed that RUN and CE mice showed distinct, non-overlapping patterns of transcriptomic changes versus the LD control. Bio-informatics uncovered that the RUN and CE environments modulate separate transcriptional networks, biological processes, cellular compartments and molecular pathways, with RUN preferentially regulating synaptic and growth-related pathways and CE altering extracellular matrix-related functions. Within the RUN group, high-distance runners also showed selective stress pathway alterations that correlated with a drastic decline in overall transcriptional changes, suggesting that excess running causes a stress-induced suppression of running's genetic effects. Our findings reveal stimulus-dependent transcriptional signatures of EE on the DG, and provide a resource for generating unbiased, data-driven hypotheses for novel mediators of EE-induced cognitive changes.

  2. Transcriptome Analysis of Polyhydroxybutyrate Cycle Mutants Reveals Discrete Loci Connecting Nitrogen Utilization and Carbon Storage in Sinorhizobium meliloti.

    Science.gov (United States)

    D'Alessio, Maya; Nordeste, Ricardo; Doxey, Andrew C; Charles, Trevor C

    2017-01-01

    Polyhydroxybutyrate (PHB) and glycogen polymers are produced by bacteria as carbon storage compounds under unbalanced growth conditions. To gain insights into the transcriptional mechanisms controlling carbon storage in Sinorhizobium meliloti , we investigated the global transcriptomic response to the genetic disruption of key genes in PHB synthesis and degradation and in glycogen synthesis. Under both nitrogen-limited and balanced growth conditions, transcriptomic analysis was performed with genetic mutants deficient in PHB synthesis ( phbA , phbB , phbAB , and phbC ), PHB degradation ( bdhA , phaZ , and acsA2 ), and glycogen synthesis ( glgA1 ). Three distinct genomic regions of the pSymA megaplasmid exhibited altered expression in the wild type and the PHB cycle mutants that was not seen in the glycogen synthesis mutant. An Fnr family transcriptional motif was identified in the upstream regions of a cluster of genes showing similar transcriptional patterns across the mutants. This motif was found at the highest density in the genomic regions with the strongest transcriptional effect, and the presence of this motif upstream of genes in these regions was significantly correlated with decreased transcript abundance. Analysis of the genes in the pSymA regions revealed that they contain a genomic overrepresentation of Fnr family transcription factor-encoding genes. We hypothesize that these loci, containing mostly nitrogen utilization, denitrification, and nitrogen fixation genes, are regulated in response to the intracellular carbon/nitrogen balance. These results indicate a transcriptional regulatory association between intracellular carbon levels (mediated through the functionality of the PHB cycle) and the expression of nitrogen metabolism genes. IMPORTANCE The ability of bacteria to store carbon and energy as intracellular polymers uncouples cell growth and replication from nutrient uptake and provides flexibility in the use of resources as they are available to

  3. Genome-wide identification of the regulatory targets of a transcription factor using biochemical characterization and computational genomic analysis

    Directory of Open Access Journals (Sweden)

    Jolly Emmitt R

    2005-11-01

    Full Text Available Abstract Background A major challenge in computational genomics is the development of methodologies that allow accurate genome-wide prediction of the regulatory targets of a transcription factor. We present a method for target identification that combines experimental characterization of binding requirements with computational genomic analysis. Results Our method identified potential target genes of the transcription factor Ndt80, a key transcriptional regulator involved in yeast sporulation, using the combined information of binding affinity, positional distribution, and conservation of the binding sites across multiple species. We have also developed a mathematical approach to compute the false positive rate and the total number of targets in the genome based on the multiple selection criteria. Conclusion We have shown that combining biochemical characterization and computational genomic analysis leads to accurate identification of the genome-wide targets of a transcription factor. The method can be extended to other transcription factors and can complement other genomic approaches to transcriptional regulation.

  4. Gene expression meta-analysis identifies metastatic pathways and transcription factors in breast cancer

    International Nuclear Information System (INIS)

    Thomassen, Mads; Tan, Qihua; Kruse, Torben A

    2008-01-01

    Metastasis is believed to progress in several steps including different pathways but the determination and understanding of these mechanisms is still fragmentary. Microarray analysis of gene expression patterns in breast tumors has been used to predict outcome in recent studies. Besides classification of outcome, these global expression patterns may reflect biological mechanisms involved in metastasis of breast cancer. Our purpose has been to investigate pathways and transcription factors involved in metastasis by use of gene expression data sets. We have analyzed 8 publicly available gene expression data sets. A global approach, 'gene set enrichment analysis' as well as an approach focusing on a subset of significantly differently regulated genes, GenMAPP, has been applied to rank pathway gene sets according to differential regulation in metastasizing tumors compared to non-metastasizing tumors. Meta-analysis has been used to determine overrepresentation of pathways and transcription factors targets, concordant deregulated in metastasizing breast tumors, in several data sets. The major findings are up-regulation of cell cycle pathways and a metabolic shift towards glucose metabolism reflected in several pathways in metastasizing tumors. Growth factor pathways seem to play dual roles; EGF and PDGF pathways are decreased, while VEGF and sex-hormone pathways are increased in tumors that metastasize. Furthermore, migration, proteasome, immune system, angiogenesis, DNA repair and several signal transduction pathways are associated to metastasis. Finally several transcription factors e.g. E2F, NFY, and YY1 are identified as being involved in metastasis. By pathway meta-analysis many biological mechanisms beyond major characteristics such as proliferation are identified. Transcription factor analysis identifies a number of key factors that support central pathways. Several previously proposed treatment targets are identified and several new pathways that may

  5. Metagenomic Analysis of Apple Orchard Soil Reveals Antibiotic Resistance Genes Encoding Predicted Bifunctional Proteins▿

    Science.gov (United States)

    Donato, Justin J.; Moe, Luke A.; Converse, Brandon J.; Smart, Keith D.; Berklein, Flora C.; McManus, Patricia S.; Handelsman, Jo

    2010-01-01

    To gain insight into the diversity and origins of antibiotic resistance genes, we identified resistance genes in the soil in an apple orchard using functional metagenomics, which involves inserting large fragments of foreign DNA into Escherichia coli and assaying the resulting clones for expressed functions. Among 13 antibiotic-resistant clones, we found two genes that encode bifunctional proteins. One predicted bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and a β-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional β-lactamase revealed a gene potentially involved in chloramphenicol resistance as well as a predicted transposase. A second clone that encodes a predicted bifunctional protein confers resistance to kanamycin and contains an aminoglycoside acetyltransferase domain fused to a second acetyltransferase domain that, based on nucleotide sequence, was predicted not to be involved in antibiotic resistance. This is the first report of a transcriptional regulator fused to a β-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance. PMID:20453147

  6. Active nuclear transcriptome analysis reveals inflammasome-dependent mechanism for early neutrophil response to Mycobacterium marinum.

    Science.gov (United States)

    Kenyon, Amy; Gavriouchkina, Daria; Zorman, Jernej; Napolitani, Giorgio; Cerundolo, Vincenzo; Sauka-Spengler, Tatjana

    2017-07-26

    The mechanisms governing neutrophil response to Mycobacterium tuberculosis remain poorly understood. In this study we utilise biotagging, a novel genome-wide profiling approach based on cell type-specific in vivo biotinylation in zebrafish to analyse the initial response of neutrophils to Mycobacterium marinum, a close genetic relative of M. tuberculosis used to model tuberculosis. Differential expression analysis following nuclear RNA-seq of neutrophil active transcriptomes reveals a significant upregulation in both damage-sensing and effector components of the inflammasome, including caspase b, NLRC3 ortholog (wu: fb15h11) and il1β. Crispr/Cas9-mediated knockout of caspase b, which acts by proteolytic processing of il1β, results in increased bacterial burden and less infiltration of macrophages to sites of mycobacterial infection, thus impairing granuloma development. We also show that a number of immediate early response genes (IEGs) are responsible for orchestrating the initial neutrophil response to mycobacterial infection. Further perturbation of the IEGs exposes egr3 as a key transcriptional regulator controlling il1β transcription.

  7. Molecular cloning, expression analysis and transcript localization of testicular orphan nuclear receptor 2 in the male catfish, Clarias batrachus.

    Science.gov (United States)

    Murugananthkumar, R; Akhila, M V; Rajakumar, A; Mamta, S K; Sudhakumari, C C; Senthilkumaran, B

    2016-12-01

    Testicular receptor 2 (TR2; also known as Nr2c1) is one of the first orphan nuclear receptors identified and known to regulate various physiological process with or without any ligand. In this study, we report the cloning of full length nr2c1 and its expression analysis during gonadal development, seasonal testicular cycle and after human chorionic gonadotropin (hCG) induction. In addition, in situ hybridization (ISH) was performed to localize nr2c1 transcripts in adult testis and whole catfish (1day post hatch). Tissue distribution and gonadal ontogeny studies revealed high expression of nr2c1 in developing and adult testis. Early embryonic stage-wise expression of nr2c1 seems to emphasize its importance in cellular differentiation and development. Substantial expression of nr2c1 during pre-spawning phase and localization of nr2c1 transcripts in sperm/spermatids were observed. Significant upregulation after hCG induction indicate that nr2c1 is under the regulation of gonadotropins. Whole mount ISH analysis displayed nr2c1 expression in notochord indicating its role in normal vertebrate development. Taken together, our findings suggest that nr2c1 may have a plausible role in the testicular and embryonic development of catfish. Copyright © 2015. Published by Elsevier Inc.

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

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    Miranda Lo

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

  9. Expression analysis and identification of antimicrobial peptide transcripts from six North American frog species

    Science.gov (United States)

    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.

  10. Genome-Wide Analysis of the Transcription Start Sites and Promoter Motifs of Phytoplasmas.

    Science.gov (United States)

    Nijo, Takamichi; Neriya, Yutaro; Koinuma, Hiroaki; Iwabuchi, Nozomu; Kitazawa, Yugo; Tanno, Kazuyuki; Okano, Yukari; Maejima, Kensaku; Yamaji, Yasuyuki; Oshima, Kenro; Namba, Shigetou

    2017-12-01

    Phytoplasmas are obligate intracellular parasitic bacteria that infect both plants and insects. We previously identified the sigma factor RpoD-dependent consensus promoter sequence of phytoplasma. However, the genome-wide landscape of RNA transcripts, including non-coding RNAs (ncRNAs) and RpoD-independent promoter elements, was still unknown. In this study, we performed an improved RNA sequencing analysis for genome-wide identification of the transcription start sites (TSSs) and the consensus promoter sequences. We constructed cDNA libraries using a random adenine/thymine hexamer primer, in addition to a conventional random hexamer primer, for efficient sequencing of 5'-termini of AT-rich phytoplasma RNAs. We identified 231 TSSs, which were classified into four categories: mRNA TSSs, internal sense TSSs, antisense TSSs (asTSSs), and orphan TSSs (oTSSs). The presence of asTSSs and oTSSs indicated the genome-wide transcription of ncRNAs, which might act as regulatory ncRNAs in phytoplasmas. This is the first description of genome-wide phytoplasma ncRNAs. Using a de novo motif discovery program, we identified two consensus motif sequences located upstream of the TSSs. While one was almost identical to the RpoD-dependent consensus promoter sequence, the other was an unidentified novel motif, which might be recognized by another transcription initiation factor. These findings are valuable for understanding the regulatory mechanism of phytoplasma gene expression.

  11. Real-time PCR analysis of carbon catabolite repression of cellobiose gene transcription in Trametes versicolor

    Energy Technology Data Exchange (ETDEWEB)

    Stapleton, P. C.; O' Mahoney, J.; Dobson, A. D. W. [National University of Ireland, Microbiology Department, Cork (Ireland)

    2004-02-01

    Previous reports indicate that in white rot fungi such as Trametes versicolor, the production of cellobiose dehydrogenase (CDH), an extracellular haemo-flavo-enzyme, is subject to carbon catabolite repression by both glucose and maltose, and that the repression is mediated at the transcriptional level. This paper describes the results of an investigation of CDH gene transcription in cellulolytic cultures of T. versicolor, in the presence of other additional carbon sources such as glucose, arabinose, and xylose. Using real time polymerase chain reaction (RT-PCR) assay methods in the presence of these other additional carbon sources, the levels of repression observed are quantitatively determined in an effort to obtain more accurate measurements of carbon catabolite repression of CDH production in this ligninolytic fungus. Ninety-six hours after addition, results of the analysis showed reduction in CDH transcript levels of 19-fold for galactose, 92-fold for arabinose and 114-fold for xylose. The greatest repressive effect was exhibited by glucose. In this case the reduction in CDH transcript levels was 3400-fold. CDH plays an important role in lignin degradation, and there is also substantial interest in the biotechnological applications of CDH, most particularly in the pulp and paper industry. 24 refs., 4 figs.

  12. Gene expression meta-analysis identifies metastatic pathways and transcription factors in breast cancer

    DEFF Research Database (Denmark)

    Thomassen, Mads; Tan, Qihua; Kruse, Torben

    2008-01-01

    studies. Besides classification of outcome, these global expression patterns may reflect biological mechanisms involved in metastasis of breast cancer. Our purpose has been to investigate pathways and transcription factors involved in metastasis by use of gene expression data sets. METHODS: We have......ABSTRACT: BACKGROUND: Metastasis is believed to progress in several steps including different pathways but the determination and understanding of these mechanisms is still fragmentary. Microarray analysis of gene expression patterns in breast tumors has been used to predict outcome in recent...... tumors compared to non-metastasizing tumors. Meta-analysis has been used to determine overrepresentation of pathways and transcription factors targets, concordant deregulated in metastasizing breast tumors, in several data sets. RESULTS: The major findings are upregulation of cell cycle pathways...

  13. Integrated mRNA and microRNA analysis identifies genes and small miRNA molecules associated with transcriptional and post-transcriptional-level responses to both drought stress and re-watering treatment in tobacco.

    Science.gov (United States)

    Chen, Qiansi; Li, Meng; Zhang, Zhongchun; Tie, Weiwei; Chen, Xia; Jin, Lifeng; Zhai, Niu; Zheng, Qingxia; Zhang, Jianfeng; Wang, Ran; Xu, Guoyun; Zhang, Hui; Liu, Pingping; Zhou, Huina

    2017-01-10

    Drought stress is one of the most severe problem limited agricultural productivity worldwide. It has been reported that plants response to drought-stress by sophisticated mechanisms at both transcriptional and post-transcriptional levels. However, the precise molecular mechanisms governing the responses of tobacco leaves to drought stress and water status are not well understood. To identify genes and miRNAs involved in drought-stress responses in tobacco, we performed both mRNA and small RNA sequencing on tobacco leaf samples from the following three treatments: untreated-control (CL), drought stress (DL), and re-watering (WL). In total, we identified 798 differentially expressed genes (DEGs) between the DL and CL (DL vs. CL) treatments and identified 571 DEGs between the WL and DL (WL vs. DL) treatments. Further analysis revealed 443 overlapping DEGs between the DL vs. CL and WL vs. DL comparisons, and, strikingly, all of these genes exhibited opposing expression trends between these two comparisons, strongly suggesting that these overlapping DEGs are somehow involved in the responses of tobacco leaves to drought stress. Functional annotation analysis showed significant up-regulation of genes annotated to be involved in responses to stimulus and stress, (e.g., late embryogenesis abundant proteins and heat-shock proteins) antioxidant defense (e.g., peroxidases and glutathione S-transferases), down regulation of genes related to the cell cycle pathway, and photosynthesis processes. We also found 69 and 56 transcription factors (TFs) among the DEGs in, respectively, the DL vs. CL and the WL vs. DL comparisons. In addition, small RNA sequencing revealed 63 known microRNAs (miRNA) from 32 families and 368 novel miRNA candidates in tobacco. We also found that five known miRNA families (miR398, miR390, miR162, miR166, and miR168) showed differential regulation under drought conditions. Analysis to identify negative correlations between the differentially expressed mi

  14. Genome wide identification of Dof transcription factor gene family in sorghum and its comparative phylogenetic analysis with rice and Arabidopsis.

    Science.gov (United States)

    Kushwaha, Hariom; Gupta, Shubhra; Singh, Vinay Kumar; Rastogi, Smita; Yadav, Dinesh

    2011-11-01

    The Dof (DNA binding with One Finger) family represents a classic zinc-finger transcription factors involved with multifarious roles exclusively in plants. There exists great diversity in terms of number of Dof genes observed in different crops. In current study, a total of 28 putative Dof genes have been predicted in silico from the recently available whole genome shotgun sequence of Sorghum bicolor (L.) Moench (with assigned accession numbers TPA:BK006983-BK007006 and TPA:BK007079-BK007082). The predicted SbDof genes are distributed on nine out of ten chromosomes of sorghum and most of these genes lack introns based on canonical intron/exon structure. Phylogenetic analysis of 28 SbDof proteins resulted in four subgroups constituting six clusters. The comparative phylogenetic analysis of these Dof proteins along with 30 rice and 36 Arabidopsis Dof proteins revealed six major groups similar to what has been observed earlier for rice and Arabidopsis. Motif analysis revealed the presence of conserved 50-52 amino acids Dof domain uniformly distributed across all the 28 Dof proteins of sorghum. The in silico cis-regulatory elements analysis of these SbDof genes suggested its diverse functions associated with light responsiveness, endosperm specific gene expression, hormone responsiveness, meristem specific expression and stress responsiveness.

  15. Genome-wide analysis of plant-specific Dof transcription factor family in tomato.

    Science.gov (United States)

    Cai, Xiaofeng; Zhang, Yuyang; Zhang, Chanjuan; Zhang, Tingyan; Hu, Tixu; Ye, Jie; Zhang, Junhong; Wang, Taotao; Li, Hanxia; Ye, Zhibiao

    2013-06-01

    The Dof (DNA binding with One Finger) family encoding single zinc finger proteins has been known as a family of plant-specific transcription factors. These transcription factors are involved in a variety of functions of importance for different biological processes in plants. In the current study, we identified 34 Dof family genes in tomato, distributed on 11 chromosomes. A complete overview of SlDof genes in tomato is presented, including the gene structures, chromosome locations, phylogeny, protein motifs and evolution pattern. Phylogenetic analysis of 34 SlDof proteins resulted in four classes constituting six clusters. In addition, a comparative analysis between these genes in tomato, Arabidopsis and rice was also performed. The tomato Dof family expansion has been dated to recent duplication events, and segmental duplication is predominant for the SlDof genes. Furthermore, the SlDof genes displayed differential expression either in their transcript abundance or in their expression patterns under normal growth conditions. This is the first step towards genome-wide analyses of the Dof genes in tomato. Our study provides a very useful reference for cloning and functional analysis of the members of this gene family in tomato and other species. © 2013 Institute of Botany, Chinese Academy of Sciences.

  16. A combination of improved differential and global RNA-seq reveals pervasive transcription initiation and events in all stages of the life-cycle of functional RNAs in Propionibacterium acnes, a major contributor to wide-spread human disease.

    Science.gov (United States)

    Lin, Yu-fei; A, David Romero; Guan, Shuang; Mamanova, Lira; McDowall, Kenneth J

    2013-09-14

    Sequencing of the genome of Propionibacterium acnes produced a catalogue of genes many of which enable this organism to colonise skin and survive exposure to the elements. Despite this platform, there was little understanding of the gene regulation that gives rise to an organism that has a major impact on human health and wellbeing and causes infections beyond the skin. To address this situation, we have undertaken a genome-wide study of gene regulation using a combination of improved differential and global RNA-sequencing and an analytical approach that takes into account the inherent noise within the data. We have produced nucleotide-resolution transcriptome maps that identify and differentiate sites of transcription initiation from sites of stable RNA processing and mRNA cleavage. Moreover, analysis of these maps provides strong evidence for 'pervasive' transcription and shows that contrary to initial indications it is not biased towards the production of antisense RNAs. In addition, the maps reveal an extensive array of riboswitches, leaderless mRNAs and small non-protein-coding RNAs alongside vegetative promoters and post-transcriptional events, which includes unusual tRNA processing. The identification of such features will inform models of complex gene regulation, as illustrated here for ribonucleotide reductases and a potential quorum-sensing, two-component system. The approach described here, which is transferable to any bacterial species, has produced a step increase in whole-cell knowledge of gene regulation in P. acnes. Continued expansion of our maps to include transcription associated with different growth conditions and genetic backgrounds will provide a new platform from which to computationally model the gene expression that determines the physiology of P. acnes and its role in human disease.

  17. Genome-wide integration on transcription factors, histone acetylation and gene expression reveals genes co-regulated by histone modification patterns.

    Directory of Open Access Journals (Sweden)

    Yayoi Natsume-Kitatani

    Full Text Available N-terminal tails of H2A, H2B, H3 and H4 histone families are subjected to posttranslational modifications that take part in transcriptional regulation mechanisms, such as transcription factor binding and gene expression. Regulation mechanisms under control of histone modification are important but remain largely unclear, despite of emerging datasets for comprehensive analysis of histone modification. In this paper, we focus on what we call genetic harmonious units (GHUs, which are co-occurring patterns among transcription factor binding, gene expression and histone modification. We present the first genome-wide approach that captures GHUs by combining ChIP-chip with microarray datasets from Saccharomyces cerevisiae. Our approach employs noise-robust soft clustering to select patterns which share the same preferences in transcription factor-binding, histone modification and gene expression, which are all currently implied to be closely correlated. The detected patterns are a well-studied acetylation of lysine 16 of H4 in glucose depletion as well as co-acetylation of five lysine residues of H3 with H4 Lys12 and H2A Lys7 responsible for ribosome biogenesis. Furthermore, our method further suggested the recognition of acetylated H4 Lys16 being crucial to histone acetyltransferase ESA1, whose essential role is still under controversy, from a microarray dataset on ESA1 and its bypass suppressor mutants. These results demonstrate that our approach allows us to provide clearer principles behind gene regulation mechanisms under histone modifications and detect GHUs further by applying to other microarray and ChIP-chip datasets. The source code of our method, which was implemented in MATLAB (http://www.mathworks.com/, is available from the supporting page for this paper: http://www.bic.kyoto-u.ac.jp/pathway/natsume/hm_detector.htm.

  18. Transcriptional analysis of the Arabidopsis ovule by massively parallel signature sequencing.

    Science.gov (United States)

    Sánchez-León, Nidia; Arteaga-Vázquez, Mario; Alvarez-Mejía, César; Mendiola-Soto, Javier; Durán-Figueroa, Noé; Rodríguez-Leal, Daniel; Rodríguez-Arévalo, Isaac; García-Campayo, Vicenta; García-Aguilar, Marcelina; Olmedo-Monfil, Vianey; Arteaga-Sánchez, Mario; de la Vega, Octavio Martínez; Nobuta, Kan; Vemaraju, Kalyan; Meyers, Blake C; Vielle-Calzada, Jean-Philippe

    2012-06-01

    The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization. The expression of 9775 genes was quantified in wild-type ovules, additionally detecting >2200 new transcripts mapping to antisense or intergenic regions. A quantitative comparison of global expression in wild-type and sporocyteless (spl) individuals resulted in 1301 genes showing 25-fold reduced or null activity in ovules lacking a female gametophyte, including those encoding 92 signalling proteins, 75 transcription factors, and 72 RNA-binding proteins not reported in previous studies based on microarray profiling. A combination of independent genetic and molecular strategies confirmed the differential expression of 28 of them, showing that they are either preferentially active in the female gametophyte, or dependent on the presence of a female gametophyte to be expressed in sporophytic cells of the ovule. Among 18 genes encoding pentatricopeptide-repeat proteins (PPRs) that show transcriptional activity in wild-type but not spl ovules, CIHUATEOTL (At4g38150) is specifically expressed in the female gametophyte and necessary for female gametogenesis. These results expand the nature of the transcriptional universe present in the ovule of Arabidopsis, and offer a large-scale quantitative reference of global expression for future genomic and developmental studies.

  19. The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

    Directory of Open Access Journals (Sweden)

    Maheswara Reddy Emani

    2015-03-01

    Full Text Available The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

  20. Whole genome transcript profiling of drug induced steatosis in rats reveals a gene signature predictive of outcome.

    Directory of Open Access Journals (Sweden)

    Nishika Sahini

    Full Text Available Drug induced steatosis (DIS is characterised by excess triglyceride accumulation in the form of lipid droplets (LD in liver cells. To explore mechanisms underlying DIS we interrogated the publically available microarray data from the Japanese Toxicogenomics Project (TGP to study comprehensively whole genome gene expression changes in the liver of treated rats. For this purpose a total of 17 and 12 drugs which are diverse in molecular structure and mode of action were considered based on their ability to cause either steatosis or phospholipidosis, respectively, while 7 drugs served as negative controls. In our efforts we focused on 200 genes which are considered to be mechanistically relevant in the process of lipid droplet biogenesis in hepatocytes as recently published (Sahini and Borlak, 2014. Based on mechanistic considerations we identified 19 genes which displayed dose dependent responses while 10 genes showed time dependency. Importantly, the present study defined 9 genes (ANGPTL4, FABP7, FADS1, FGF21, GOT1, LDLR, GK, STAT3, and PKLR as signature genes to predict DIS. Moreover, cross tabulation revealed 9 genes to be regulated ≥10 times amongst the various conditions and included genes linked to glucose metabolism, lipid transport and lipogenesis as well as signalling events. Additionally, a comparison between drugs causing phospholipidosis and/or steatosis revealed 26 genes to be regulated in common including 4 signature genes to predict DIS (PKLR, GK, FABP7 and FADS1. Furthermore, a comparison between in vivo single dose (3, 6, 9 and 24 h and findings from rat hepatocyte studies (2 h, 8 h, 24 h identified 10 genes which are regulated in common and contained 2 DIS signature genes (FABP7, FGF21. Altogether, our studies provide comprehensive information on mechanistically linked gene expression changes of a range of drugs causing steatosis and phospholipidosis and encourage the screening of DIS signature genes at the preclinical stage.

  1. Temporal analysis and spatial mapping of Lymantria dispar nuclear polyhedrosis virus transcripts and in vitro translation polypeptides

    Science.gov (United States)

    James M. Slavicek

    1991-01-01

    Genomic expression of the Lymantriu dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was studied. Viral specific transcripts expressed in cell culture at various times from 2 through 72 h postinfection were identified and their genomic origins mapped through Northern analysis. Sixty-five distinct transcripts were identified in this...

  2. Single-base methylome analysis reveals dynamic epigenomic differences associated with water deficit in apple.

    Science.gov (United States)

    Xu, Jidi; Zhou, Shasha; Gong, Xiaoqing; Song, Yi; van Nocker, Steve; Ma, Fengwang; Guan, Qingmei

    2018-02-01

    Cytosine methylation is an essential feature of epigenetic regulation and is involved in various biological processes. Although cytosine methylation has been analysed at the genomic scale for several plant species, there is a general lack of understanding of the dynamics of global and genic DNA methylation in plants growing in environments challenged with biotic and abiotic stresses. In this study, we mapped cytosine methylation at single-base resolution in the genome of commercial apple (Malus x domestica), and analysed changes in methylation patterns associated with water deficit in representative drought-sensitive and drought-tolerant cultivars. We found that the apple genome exhibits ~54%, ~38% and ~8.5% methylation at CG, CHG and CHH sequence contexts, respectively. We additionally documented changes in gene expression associated with water deficit in an attempt to link methylation and gene expression changes. Global methylation and transcription analysis revealed that promoter-unmethylated genes showed higher expression levels than promoter-methylated genes. Gene body methylation appears to be positively correlated with gene expression. Water deficit stress was associated with changes in methylation at a multitude of genes, including those encoding transcription factors (TFs) and transposable elements (TEs). These results present a methylome map of the apple genome and reveal widespread DNA methylation alterations in response to water deficit stress. These data will be helpful for understanding potential linkages between DNA methylation and gene expression in plants growing in natural environments and challenged with abiotic and biotic stresses. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  3. Genome-wide analysis of gene expression in primate taste buds reveals links to diverse processes.

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    Peter Hevezi

    Full Text Available Efforts to unravel the mechanisms underlying taste sensation (gustation have largely focused on rodents. Here we present the first comprehensive characterization of gene expression in primate taste buds. Our findings reveal unique new insights into the biology of taste buds. We generated a taste bud gene expression database using laser capture microdissection (LCM procured fungiform (FG and circumvallate (CV taste buds from primates. We also used LCM to collect the top and bottom portions of CV taste buds. Affymetrix genome wide arrays were used to analyze gene expression in all samples. Known taste receptors are preferentially expressed in the top portion of taste buds. Genes associated with the cell cycle and stem cells are preferentially expressed in the bottom portion of taste buds, suggesting that precursor cells are located there. Several chemokines including CXCL14 and CXCL8 are among the highest expressed genes in taste buds, indicating that immune system related processes are active in taste buds. Several genes expressed specifically in endocrine glands including growth hormone releasing hormone and its receptor are also strongly expressed in taste buds, suggesting a link between metabolism and taste. Cell type-specific expression of transcription factors and signaling molecules involved in cell fate, including KIT, reveals the taste bud as an active site of cell regeneration, differentiation, and development. IKBKAP, a gene mutated in familial dysautonomia, a disease that results in loss of taste buds, is expressed in taste cells that communicate with afferent nerve fibers via synaptic transmission. This database highlights the power of LCM coupled with transcriptional profiling to dissect the molecular composition of normal tissues, represents the most comprehensive molecular analysis of primate taste buds to date, and provides a foundation for further studies in diverse aspects of taste biology.

  4. The crystal structure of the AhRR-ARNT heterodimer reveals the structural basis of the repression of AhR-mediated transcription.

    Science.gov (United States)

    Sakurai, Shunya; Shimizu, Toshiyuki; Ohto, Umeharu

    2017-10-27

    2,3,7,8-Tetrachlorodibenzo- p -dioxin and related compounds are extraordinarily potent environmental toxic pollutants. Most of the 2,3,7,8-tetrachlorodibenzo- p -dioxin toxicities are mediated by aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor belonging to the basic helix-loop-helix (bHLH) Per-ARNT-Sim (PAS) family. Upon ligand binding, AhR forms a heterodimer with AhR nuclear translocator (ARNT) and induces the expression of genes involved in various biological responses. One of the genes induced by AhR encodes AhR repressor (AhRR), which also forms a heterodimer with ARNT and represses the activation of AhR-dependent transcription. The control of AhR activation is critical for managing AhR-mediated diseases, but the mechanisms by which AhRR represses AhR activation remain poorly understood, because of the lack of structural information. Here, we determined the structure of the AhRR-ARNT heterodimer by X-ray crystallography, which revealed an asymmetric intertwined domain organization presenting structural features that are both conserved and distinct among bHLH-PAS family members. The structures of AhRR-ARNT and AhR-ARNT were similar in the bHLH-PAS-A region, whereas the PAS-B of ARNT in the AhRR-ARNT complex exhibited a different domain arrangement in this family reported so far. The structure clearly disclosed that AhRR competitively represses AhR binding to ARNT and target DNA and further suggested the existence of an AhRR-ARNT-specific repression mechanism. This study provides a structural basis for understanding the mechanism by which AhRR represses AhR-mediated gene transcription. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Large-scale analysis of antisense transcription in wheat using the Affymetrix GeneChip Wheat Genome Array

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    Settles Matthew L

    2009-05-01

    -antisense transcript pairs, analysis of the gene ontology terms showed a significant over-representation of transcripts involved in energy production. These included several representations of ATP synthase, photosystem proteins and RUBISCO, which indicated that photosynthesis is likely to be regulated by antisense transcripts. Conclusion This study demonstrated the novel use of an adapted labeling protocol and a 3'IVT GeneChip array for large-scale identification of antisense transcription in wheat. The results show that antisense transcription is relatively abundant in wheat, and may affect the expression of valuable agronomic phenotypes. Future work should select potentially interesting transcript pairs for further functional characterization to determine biological activity.

  6. Sequencing and transcriptional analysis of the Streptococcus thermophilus histamine biosynthesis gene cluster: factors that affect differential hdcA expression

    DEFF Research Database (Denmark)

    Calles-Enríquez, Marina; Hjort, Benjamin Benn; Andersen, Pia Skov

    2010-01-01

    to produce histamine. The hdc clusters of S. thermophilus CHCC1524 and CHCC6483 were sequenced, and the factors that affect histamine biosynthesis and histidine-decarboxylating gene (hdcA) expression were studied. The hdc cluster began with the hdcA gene, was followed by a transporter (hdcP), and ended...... with the hdcB gene, which is of unknown function. The three genes were orientated in the same direction. The genetic organization of the hdc cluster showed a unique organization among the lactic acid bacterial group and resembled those of Staphylococcus and Clostridium species, thus indicating possible...... acquisition through a horizontal transfer mechanism. Transcriptional analysis of the hdc cluster revealed the existence of a polycistronic mRNA covering the three genes. The histidine-decarboxylating gene (hdcA) of S. thermophilus demonstrated maximum expression during the stationary growth phase, with high...

  7. Heat shock transcriptional factors in Malus domestica: identification, classification and expression analysis

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    Giorno Filomena

    2012-11-01

    Full Text Available Abstract Background Heat shock transcriptional factors (Hsfs play a crucial role in plant responses to biotic and abiotic stress conditions and in plant growth and development. Apple (Malus domestica Borkh is an economically important fruit tree whose genome has been fully sequenced. So far, no detailed characterization of the Hsf gene family is available for this crop plant. Results A genome-wide analysis was carried out in Malus domestica to identify heat shock transcriptional factor (Hsf genes, named MdHsfs. Twenty five MdHsfs were identified and classified in three main groups (class A, B and C according to the structural characteristics and to the phylogenetic comparison with Arabidopsis thaliana and Populus trichocarpa. Chromosomal duplications were analyzed and segmental duplications were shown to have occurred more frequently in the expansion of Hsf genes in the apple genome. Furthermore, MdHsfs transcripts were detected in several apple organs, and expression changes were observed by quantitative real-time PCR (qRT-PCR analysis in developing flowers and fruits as well as in leaves, harvested from trees grown in the field and exposed to the naturally increased temperatures. Conclusions The apple genome comprises 25 full length Hsf genes. The data obtained from this investigation contribute to a better understanding of the complexity of the Hsf gene family in apple, and provide the basis for further studies to dissect Hsf function during development as well as in response to environmental stimuli.

  8. Analysis of functional redundancies within the Arabidopsis TCP transcription factor family.

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    Danisman, Selahattin; van Dijk, Aalt D J; Bimbo, Andrea; van der Wal, Froukje; Hennig, Lars; de Folter, Stefan; Angenent, Gerco C; Immink, Richard G H

    2013-12-01

    Analyses of the functions of TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR1 (TCP) transcription factors have been hampered by functional redundancy between its individual members. In general, putative functionally redundant genes are predicted based on sequence similarity and confirmed by genetic analysis. In the TCP family, however, identification is impeded by relatively low overall sequence similarity. In a search for functionally redundant TCP pairs that control Arabidopsis leaf development, this work performed an integrative bioinformatics analysis, combining protein sequence similarities, gene expression data, and results of pair-wise protein-protein interaction studies for the 24 members of the Arabidopsis TCP transcription factor family. For this, the work completed any lacking gene expression and protein-protein interaction data experimentally and then performed a comprehensive prediction of potential functional redundant TCP pairs. Subsequently, redundant functions could be confirmed for selected predicted TCP pairs by genetic and molecular analyses. It is demonstrated that the previously uncharacterized class I TCP19 gene plays a role in the control of leaf senescence in a redundant fashion with TCP20. Altogether, this work shows the power of combining classical genetic and molecular approaches with bioinformatics predictions to unravel functional redundancies in the TCP transcription factor family.

  9. Characterization of Arabidopsis Transcriptional Responses to Different Aphid Species Reveals Genes that Contribute to Host Susceptibility and Non-host Resistance

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    Jaouannet, Maëlle; Morris, Jenny A.; Hedley, Peter E.; Bos, Jorunn I. B.

    2015-01-01

    Aphids are economically important pests that display exceptional variation in host range. The determinants of diverse aphid host ranges are not well understood, but it is likely that molecular interactions are involved. With significant progress being made towards understanding host responses upon aphid attack, the mechanisms underlying non-host resistance remain to be elucidated. Here, we investigated and compared Arabidopsis thaliana host and non-host responses to aphids at the transcriptional level using three different aphid species, Myzus persicae, Myzus cerasi and Rhopalosiphum pisum. Gene expression analyses revealed a high level of overlap in the overall gene expression changes during the host and non-host interactions with regards to the sets of genes differentially expressed and the direction of expression changes. Despite this overlap in transcriptional responses across interactions, there was a stronger repression of genes involved in metabolism and oxidative responses specifically during the host interaction with M. persicae. In addition, we identified a set of genes with opposite gene expression patterns during the host versus non-host interactions. Aphid performance assays on Arabidopsis mutants that were selected based on our transcriptome analyses identified novel genes contributing to host susceptibility, host defences during interactions with M. persicae as well to non-host resistance against R. padi. Understanding how plants respond to aphid species that differ in their ability to infest plant species, and identifying the genes and signaling pathways involved, is essential for the development of novel and durable aphid control in crop plants. PMID:25993686

  10. Characterization of Arabidopsis Transcriptional Responses to Different Aphid Species Reveals Genes that Contribute to Host Susceptibility and Non-host Resistance.

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    Maëlle Jaouannet

    2015-05-01

    Full Text Available Aphids are economically important pests that display exceptional variation in host range. The determinants of diverse aphid host ranges are not well understood, but it is likely that molecular interactions are involved. With significant progress being made towards understanding host responses upon aphid attack, the mechanisms underlying non-host resistance remain to be elucidated. Here, we investigated and compared Arabidopsis thaliana host and non-host responses to aphids at the transcriptional level using three different aphid species, Myzus persicae, Myzus cerasi and Rhopalosiphum pisum. Gene expression analyses revealed a high level of overlap in the overall gene expression changes during the host and non-host interactions with regards to the sets of genes differentially expressed and the direction of expression changes. Despite this overlap in transcriptional responses across interactions, there was a stronger repression of genes involved in metabolism and oxidative responses specifically during the host interaction with M. persicae. In addition, we identified a set of genes with opposite gene expression patterns during the host versus non-host interactions. Aphid performance assays on Arabidopsis mutants that were selected based on our transcriptome analyses identified novel genes contributing to host susceptibility, host defences during interactions with M. persicae as well to non-host resistance against R. padi. Understanding how plants respond to aphid species that differ in their ability to infest plant species, and identifying the genes and signaling pathways involved, is essential for the development of novel and durable aphid control in crop plants.

  11. Transcriptomic analysis of the late stages of grapevine (Vitis vinifera cv. Cabernet Sauvignon) berry ripening reveals significant induction of ethylene signaling and flavor pathways in the skin.

    Science.gov (United States)

    Cramer, Grant R; Ghan, Ryan; Schlauch, Karen A; Tillett, Richard L; Heymann, Hildegarde; Ferrarini, Alberto; Delledonne, Massimo; Zenoni, Sara; Fasoli, Marianna; Pezzotti, Mario

    2014-12-19

    Grapevine berry, a nonclimacteric fruit, has three developmental stages; the last one is when berry color and sugar increase. Flavors derived from terpenoid and fatty acid metabolism develop at the very end of this ripening stage. The transcriptomic response of pulp and skin of Cabernet Sauvignon berries in the late stages of ripening between 22 and 37 °Brix was assessed using whole-genome micorarrays. The transcript abundance of approximately 18,000 genes changed with °Brix and tissue type. There were a large number of changes in many gene ontology (GO) categories involving metabolism, signaling and abiotic stress. GO categories reflecting tissue differences were overrepresented in photosynthesis, isoprenoid metabolism and pigment biosynthesis. Detailed analysis of the interaction of the skin and pulp with °Brix revealed that there were statistically significantly higher abundances of transcripts changing with °Brix in the skin that were involved in ethylene signaling, isoprenoid and fatty acid metabolism. Many transcripts were peaking around known optimal fruit stages for flavor production. The transcript abundance of approximately two-thirds of the AP2/ERF superfamily of transcription factors changed during these developmental stages. The transcript abundance of a unique clade of ERF6-type transcription factors had the largest changes in the skin and clustered with genes involved in ethylene, senescence, and fruit flavor production including ACC oxidase, terpene synthases, and lipoxygenases. The transcript abundance of important transcription factors involved in fruit ripening was also higher in the skin. A detailed analysis of the transcriptome dynamics during late stages of ripening of grapevine berries revealed that these berries went through massive transcriptional changes in gene ontology categories involving chemical signaling and metabolism in both the pulp and skin, particularly in the skin. Changes in the transcript abundance of genes involved in

  12. Comparative analysis of regulatory elements between Escherichia coli and Klebsiella pneumoniae by genome-wide transcription start site profiling.

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    Donghyuk Kim

    Full Text Available Genome-wide transcription start site (TSS profiles of the enterobacteria Escherichia coli and Klebsiella pneumoniae were experimentally determined through modified 5' RACE followed by deep sequencing of intact primary mRNA. This identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Experimentally determined TSSs were then used to define promoter regions and 5' UTRs upstream of coding genes. Comparative analysis of these regulatory elements revealed the use of multiple TSSs, identical sequence motifs of promoter and Shine-Dalgarno sequence, reflecting conserved gene expression apparatuses between the two species. In both species, over 70% of primary transcripts were expressed from operons having orthologous genes during exponential growth. However, expressed orthologous genes in E. coli and K. pneumoniae showed a strikingly different organization of upstream regulatory regions with only 20% identical promoters with TSSs in both species. Over 40% of promoters had TSSs identified in only one species, despite conserved promoter sequences existing in the other species. 662 conserved promoters having TSSs in both species resulted in the same number of comparable 5' UTR pairs, and that regulatory element was found to be the most variant region in sequence among promoter, 5' UTR, and ORF. In K. pneumoniae, 48 sRNAs were predicted and 36 of them were expressed during exponential growth. Among them, 34 orthologous sRNAs between two species were analyzed in depth, and the analysis showed that many sRNAs of K. pneumoniae, including pleiotropic sRNAs such as rprA, arcZ, and sgrS, may work in the same way as in E. coli. These results reveal a new dimension of comparative genomics such that a comparison of two genomes needs to be comprehensive over all levels of genome organization.

  13. Comparative analysis reveals the underlying mechanism of vertebrate seasonal reproduction.

    Science.gov (United States)

    Ikegami, Keisuke; Yoshimura, Takashi

    2016-02-01

    Animals utilize photoperiodic changes as a calendar to regulate seasonal reproduction. Birds have highly sophisticated photoperiodic mechanisms and functional genomics analysis in quail uncovered the signal transduction pathway regulating avian seasonal reproduction. Birds detect light with deep brain photoreceptors. Long day (LD) stimulus induces secretion of thyroid-stimulating hormone (TSH) from the pars tuberalis (PT) of the pituitary gland. PT-derived TSH locally activates thyroid hormone (TH) in the hypothalamus, which induces gonadotropin-releasing hormone (GnRH) and hence gonadotropin secretion. However, during winter, low temperatures increase serum TH for adaptive thermogenesis, which accelerates germ cell apoptosis by activating the genes involved in metamorphosis. Therefore, TH has a dual role in the regulation of seasonal reproduction. Studies using TSH receptor knockout mice confirmed the involvement of PT-derived TSH in mammalian seasonal reproduction. In addition, studies in mice revealed that the tissue-specific glycosylation of TSH diversifies its function in the circulation to avoid crosstalk. In contrast to birds and mammals, one of the molecular machineries necessary for the seasonal reproduction of fish are localized in the saccus vasculosus from the photoreceptor to the neuroendocrine output. Thus, comparative analysis is a powerful tool to uncover the universality and diversity of fundamental properties in various organisms. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Gene expression in chicken reveals correlation with structural genomic features and conserved patterns of transcription in the terrestrial vertebrates.

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    Haisheng Nie

    Full Text Available BACKGROUND: The chicken is an important agricultural and avian-model species. A survey of gene expression in a range of different tissues will provide a benchmark for understanding expression levels under normal physiological conditions in birds. With expression data for birds being very scant, this benchmark is of particular interest for comparative expression analysis among various terrestrial vertebrates. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a gene expression survey in eight major chicken tissues using whole genome microarrays. A global picture of gene expression is presented for the eight tissues, and tissue specific as well as common gene expression were identified. A Gene Ontology (GO term enrichment analysis showed that tissue-specific genes are enriched with GO terms reflecting the physiological functions of the specific tissue, and housekeeping genes are enriched with GO terms related to essential biological functions. Comparisons of structural genomic features between tissue-specific genes and housekeeping genes show that housekeeping genes are more compact. Specifically, coding sequence and particularly introns are shorter than genes that display more variation in expression between tissues, and in addition intergenic space was also shorter. Meanwhile, housekeeping genes are more likely to co-localize with other abundantly or highly expressed genes on the same chromosomal regions. Furthermore, comparisons of gene expression in a panel of five common tissues between birds, mammals and amphibians showed that the expression patterns across tissues are highly similar for orthologous genes compared to random gene pairs within each pair-wise comparison, indicating a high degree of functional conservation in gene expression among terrestrial vertebrates. CONCLUSIONS: The housekeeping genes identified in this study have shorter gene length, shorter coding sequence length, shorter introns, and shorter intergenic regions, there seems

  15. A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.

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    Dariel Ashton-Beaucage

    2014-03-01

    Full Text Available The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing

  16. Genome-wide transcriptional profiling reveals two distinct outcomes in central Nervous system infections of rabies virus

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    Daiting eZhang

    2016-05-01

    Full Text Available Rabies remains a major public health concern in many developing countries. The precise neuropathogenesis of rabies is unknown, though it is hypothesized to be due to neuronal death or dysfunction. Mice that received intranasal inoculation of an attenuated rabies virus (RABV strain HEP-Flury exhibited subtle clinical signs, and eventually recovered, which is different from the fatal encephalitis caused by the virulent RABV strain CVS-11. To understand the neuropathogenesis of rabies and the mechanisms of viral clearance, we applied RNA sequencing (RNA-Seq to compare the brain transcriptomes of normal mice versus HEP-Flury or CVS-11 intranasally inoculated mice. Our results revealed that both RABV strains altered positively and negatively the expression levels of many host genes, including genes associated with innate and adaptive immunity, inflammation and cell death. It is found that HEP-Flury infection can activate the innate immunity earlier through the RIG-I/MDA-5 signaling, and the innate immunity pre-activated by HEP-Flury or Newcastle disease virus (NDV infection can effectively prevent the CVS-11 to invade central nervous system (CNS, but fails to clear the CVS-11 after its entry into the CNS. In addition, following CVS-11 infection, genes implicated in cell adhesion, blood vessel morphogenesis and coagulation were mainly up-regulated, while the genes involved in synaptic transmission and ion transport were significantly down-regulated. On the other hand, several genes involved in the MHC class II-mediated antigen presentation pathway were activated to a greater extent after the HEP-Flury infection as compared with the CVS-11 infection suggesting that the collaboration of CD4+ T cells and MHC class II-mediated antigen presentation is critical for the clearance of attenuated RABV from the CNS. The differentially regulated genes reported here are likely to include potential therapeutic targets for expanding the postexposure treatment window

  17. Understanding the role of the master regulator–XYR1–in Trichoderma reesei by global transcriptional analysis

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    Lilian Castro dos Santos

    2016-02-01

    Full Text Available We defined the role of the transcriptional factor–XYR1–in the filamentous fungus Trichoderma reesei during cellulosic material degradation. In this regard, we performed a global transcriptome analysis using RNA-Seq of the ∆xyr1 mutant strain of T. reesei compared with the parental strain QM9414 grown in the presence of cellulose, sophorose, and glucose as sole carbon sources. We found that 5885 genes were expressed differentially under the three tested carbon sources. Of these, 322 genes were upregulated in the presence of cellulose, while 367 and 188 were upregulated in sophorose and glucose, respectively. With respect to genes under the direct regulation of XYR1, 30 and 33 are exclusive to cellulose and sophorose, respectively. The most modulated genes in the ∆xyr1 belong to Carbohydrate-Active Enzymes (CAZymes, transcription factors, and transporters families. Moreover, we highlight the downregulation of transporters belonging to the MFS and ABC transporter families. Of these, MFS members were mostly downregulated in the presence of cellulose. In sophorose and glucose, the expression of these transporters was mainly upregulated. Our results revealed that MFS and ABC transporters could be new players in cellulose degradation and their role was shown to be carbon source-dependent. Our findings contribute to a better understanding of the regulatory mechanisms of XYR1 to control cellulase gene expression in T. reesei in the presence of cellulosic material, thereby potentially enhancing its application in several biotechnology fields.

  18. Identification, isolation, and expression analysis of heat shock transcription factors in the diploid woodland strawberry Fragaria vesca.

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    Hu, Yang; Han, Yong-Tao; Wei, Wei; Li, Ya-Juan; Zhang, Kai; Gao, Yu-Rong; Zhao, Feng-Li; Feng, Jia-Yue

    2015-01-01

    Heat shock transcription factors (Hsfs) are known to play dominant roles in plant responses to heat, as well as other abiotic or biotic stress stimuli. While the strawberry is an economically important fruit plant, little is known about the Hsf family in the strawberry. To explore the functions of strawberry Hsfs in abiotic and biotic stress responses, this study identified 17 Hsf genes (FvHsfs) in a wild diploid woodland strawberry (Fragaria vesca, 2n = 2x = 14) and isolated 14 of these genes. Phylogenetic analysis divided the strawberry FvHsfs genes into three main groups. The evolutionary and structural analyses revealed that the FvHsf family is conserved. The promoter sequences of the FvHsf genes contain upstream regulatory elements corresponding to different stress stimuli. In addition, 14 FvHsf-GFP fusion proteins showed differential subcellular localization in Arabidopsis mesophyll protoplasts. Furthermore, we examined the expression of the 17 FvHsf genes in wild diploid woodland strawberries under various conditions, including abiotic stresses (heat, cold, drought, and salt), biotic stress (powdery mildew infection), and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid). Fifteen of the seventeen FvHsf genes exhibited distinct changes on the transcriptional level during heat treatment. Of these 15 FvHsfs, 8 FvHsfs also exhibited distinct responses to other stimuli on the transcriptional level, indicating versatile roles in the response to abiotic and biotic stresses. Taken together, the present work may provide the basis for further studies to dissect FvHsf function in response to stress stimuli.

  19. Integrated analysis of whole genome and transcriptome sequencing reveals diverse transcriptomic aberrations driven by somatic genomic changes in liver cancers.

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    Yuichi Shiraishi

    Full Text Available Recent studies applying high-throughput sequencing technologies have identified several recurrently mutated genes and pathways in multiple cancer genomes. However, transcriptional consequences from these genomic alterations in cancer genome remain unclear. In this study, we performed integrated and comparative analyses of whole genomes and transcriptomes of 22 hepatitis B virus (HBV-related hepatocellular carcinomas (HCCs and their matched controls. Comparison of whole genome sequence (WGS and RNA-Seq revealed much evidence that various types of genomic mutations triggered diverse transcriptional changes. Not only splice-site mutations, but also silent mutations in coding regions, deep intronic mutations and structural changes caused splicing aberrations. HBV integrations generated diverse patterns of virus-human fusion transcripts depending on affected gene, such as TERT, CDK15, FN1 and MLL4. Structural variations could drive over-expression of genes such as WNT ligands, with/without creating gene fusions. Furthermore, by taking account of genomic mutations causing transcriptional aberrations, we could improve the sensitivity of deleterious mutation detection in known cancer driver genes (TP53, AXIN1, ARID2, RPS6KA3, and identified recurrent disruptions in putative cancer driver genes such as HNF4A, CPS1, TSC1 and THRAP3 in HCCs. These findings indicate genomic alterations in cancer genome have diverse transcriptomic effects, and integrated analysis of WGS and RNA-Seq can facilitate the interpretation of a large number of genomic alterations detected in cancer genome.

  20. Global analysis of WRKY transcription factor superfamily in Setaria identifies potential candidates involved in abiotic stress signalling

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    Mehanathan eMuthamilarasan

    2015-10-01

    Full Text Available Transcription factors (TFs are major players in stress signalling and constitute an integral part of signalling networks. Among the major TFs, WRKY proteins play pivotal roles in regulation of transcriptional reprogramming associated with stress responses. In view of this, genome- and transcriptome-wide identification of WRKY TF family was performed in the C4 model plants, Setaria italica (SiWRKY and S. viridis (SvWRKY, respectively. The study identified 105 SiWRKY and 44 SvWRKY proteins that were computationally analysed for their physicochemical properties. Sequence alignment and phylogenetic analysis classified these proteins into three major groups, namely I, II and III with majority of WRKY proteins belonging to group II (53 SiWRKY and 23 SvWRKY, followed by group III (39 SiWRKY and 11 SvWRKY and group I (10 SiWRKY and 6 SvWRKY. Group II proteins were further classified into 5 subgroups (IIa to IIe based on their phylogeny. Domain analysis showed the presence of WRKY motif and zinc finger-like structures in these proteins along with additional domains in a few proteins. All SiWRKY genes were physically mapped on the S. italica genome and their duplication analysis revealed that 10 and 8 gene pairs underwent tandem and segmental duplications, respectively. Comparative mapping of SiWRKY and SvWRKY genes in related C4 panicoid genomes demonstrated the orthologous relationships between these genomes. In silico expression analysis of SiWRKY and SvWRKY genes showed their differential expression patterns in different tissues and stress conditions. Expression profiling of candidate SiWRKY genes in response to stress (dehydration and salinity and hormone treatments (abscisic acid, salicylic acid and methyl jasmonate suggested the putative involvement of SiWRKY066 and SiWRKY082 in stress and hormone signalling. These genes could be potential candidates for further characterization to delineate their functional roles in abiotic stress signalling.

  1. Transcription profile of soybean-root-knot nematode interaction reveals a key role of phythormones in the resistance reaction.

    Science.gov (United States)

    Beneventi, Magda Aparecida; da Silva, Orzenil Bonfim; de Sá, Maria Eugênia Lisei; Firmino, Alexandre Augusto Pereira; de Amorim, Regina Maria Santos; Albuquerque, Erika Valéria Saliba; da Silva, Maria Cristina Mattar; da Silva, Joseane Padilha; Campos, Magnólia de Araújo; Lopes, Marcus José Conceição; Togawa, Roberto Coiti; Pappas, Georgios Joanis; Grossi-de-Sa, Maria Fatima

    2013-05-10

    Root-knot nematodes (RKN- Meloidogyne genus) present extensive challenges to soybean crop. The soybean line (PI 595099) is known to be resistant against specific strains and races of nematode species, thus its differential gene expression analysis can lead to a comprehensive gene expression profiling in the incompatible soybean-RKN interaction. Even though many disease resistance genes have been studied, little has been reported about phytohormone crosstalk on modulation of ROS signaling during soybean-RKN interaction. Using 454 technology to explore the common aspects of resistance reaction during both parasitism and resistance phases it was verified that hormone, carbohydrate metabolism and stress related genes were consistently expressed at high levels in infected roots as compared to mock control. Most noteworthy genes include those encoding glycosyltransferases, peroxidases, auxin-responsive proteins and gibberellin-regulated genes. Our data analysis suggests the key role of glycosyltransferases, auxins and components of gibberellin signal transduction, biosynthesis and deactivation pathways in the resistance reaction and their participation in jasmonate signaling and redox homeostasis in mediating aspects of plant growth and responses to biotic stress. Based on this study we suggest a reasonable model regarding to the complex mechanisms of crosstalk between plant hormones, mainly gibberellins and auxins, which can be crucial to modulate the levels of ROS in the resistance reaction to nematode invasion. The model also includes recent findings concerning to the participation of DELLA-like proteins and ROS signaling controlling plant immune or stress responses. Furthermore, this study provides a dataset of potential candidate genes involved in both nematode parasitism and resistance, which can be tested further for their role in this biological process using functional genomics approaches.

  2. Multi-Omics and Integrated Network Analyses Reveal New Insights into the Systems Relationships between Metabolites, Structural Genes, and Transcriptional Regulators in Developing Grape Berries (Vitis vinifera L. Exposed to Water Deficit

    Directory of Open Access Journals (Sweden)

    Stefania Savoi

    2017-07-01

    Full Text Available Grapes are one of the major fruit crops and they are cultivated in many dry environments. This study comprehensively characterizes the metabolic response of grape berries exposed to water deficit at different developmental stages. Increases of proline, branched-chain amino acids, phenylpropanoids, anthocyanins, and free volatile organic compounds have been previously observed in grape berries exposed to water deficit. Integrating RNA-sequencing analysis of the transcriptome with large-scale analysis of central and specialized metabolites, we reveal that these increases occur via a coordinated regulation of key structural pathway genes. Water deficit-induced up-regulation of flavonoid genes is also coordinated with the down-regulation of many stilbene synthases and a consistent decrease in stilbenoid concentration. Water deficit activated both ABA-dependent and ABA-independent signal transduction pathways by modulating the expression of several transcription factors. Gene-gene and gene-metabolite network analyses showed that water deficit-responsive transcription factors such as bZIPs, AP2/ERFs, MYBs, and NACs are implicated in the regulation of stress-responsive metabolites. Enrichment of known and novel cis-regulatory elements in the promoters of several ripening-specific/water deficit-induced modules further affirms the involvement of a transcription factor cross-talk in the berry response to water deficit. Together, our integrated approaches show that water deficit-regulated gene modules are strongly linked to key fruit-quality metabolites and multiple signal transduction pathways may be critical to achieve a balance between the regulation of the stress-response and the berry ripening program. This study constitutes an invaluable resource for future discoveries and comparative studies, in grapes and other fruits, centered on reproductive tissue metabolism under abiotic stress.

  3. Multi-Omics and Integrated Network Analyses Reveal New Insights into the Systems Relationships between Metabolites, Structural Genes, and Transcriptional Regulators in Developing Grape Berries (Vitis viniferaL.) Exposed to Water Deficit.

    Science.gov (United States)

    Savoi, Stefania; Wong, Darren C J; Degu, Asfaw; Herrera, Jose C; Bucchetti, Barbara; Peterlunger, Enrico; Fait, Aaron; Mattivi, Fulvio; Castellarin, Simone D

    2017-01-01

    Grapes are one of the major fruit crops and they are cultivated in many dry environments. This study comprehensively characterizes the metabolic response of grape berries exposed to water deficit at different developmental stages. Increases of proline, branched-chain amino acids, phenylpropanoids, anthocyanins, and free volatile organic compounds have been previously observed in grape berries exposed to water deficit. Integrating RNA-sequencing analysis of the transcriptome with large-scale analysis of central and specialized metabolites, we reveal that these increases occur via a coordinated regulation of key structural pathway genes. Water deficit-induced up-regulation of flavonoid genes is also coordinated with the down-regulation of many stilbene synthases and a consistent decrease in stilbenoid concentration. Water deficit activated both ABA-dependent and ABA-independent signal transduction pathways by modulating the expression of several transcription factors. Gene-gene and gene-metabolite network analyses showed that water deficit-responsive transcription factors such as bZIPs, AP2/ERFs, MYBs, and NACs are implicated in the regulation of stress-responsive metabolites. Enrichment of known and novel cis -regulatory elements in the promoters of several ripening-specific/water deficit-induced modules further affirms the involvement of a transcription factor cross-talk in the berry response to water deficit. Together, our integrated approaches show that water deficit-regulated gene modules are strongly linked to key fruit-quality metabolites and multiple signal transduction pathways may be critical to achieve a balance between the regulation of the stress-response and the berry ripening program. This study constitutes an invaluable resource for future discoveries and comparative studies, in grapes and other fruits, centered on reproductive tissue metabolism under abiotic stress.

  4. Distinct malaria parasite sporozoites reveal transcriptional changes that cause differential tissue infection competence in the mosquito vector and mammalian host.

    Science.gov (United States)

    Mikolajczak, Sebastian A; Silva-Rivera, Hilda; Peng, Xinxia; Tarun, Alice S; Camargo, Nelly; Jacobs-Lorena, Vanessa; Daly, Thomas M; Bergman, Lawrence W; de la Vega, Patricia; Williams, Jack; Aly, Ahmed S I; Kappe, Stefan H I

    2008-10-01

    The malaria parasite sporozoite transmission stage develops and differentiates within parasite oocysts on the Anopheles mosquito midgut. Successful inoculation of the parasite into a mammalian host is critically dependent on the sporozoite's ability to first infect the mosquito salivary glands. Remarkable changes in tissue infection competence are observed as the sporozoites transit from the midgut oocysts to the salivary glands. Our microarray analysis shows that compared to oocyst sporozoites, salivary gland sporozoites upregulate expression of at least 124 unique genes. Conversely, oocyst sporozoites show upregulation of at least 47 genes (upregulated in oocyst sporozoites [UOS genes]) before they infect the salivary glands. Targeted gene deletion of UOS3, encoding a putative transmembrane protein with a thrombospondin repeat that localizes to the sporozoite secretory organelles, rendered oocyst sporozoites unable to infect the mosquito salivary glands but maintained the parasites' liver infection competence. This phenotype demonstrates the significance of differential UOS expression. Thus, the UIS-UOS gene classification provides a framework to elucidate the infectivity and transmission success of Plasmodium sporozoites on a whole-genome scale. Genes identified herein might represent targets for vector-based transmission blocking strategies (UOS genes), as well as strategies that prevent mammalian host infection (UIS genes).

  5. Distinct Malaria Parasite Sporozoites Reveal Transcriptional Changes That Cause Differential Tissue Infection Competence in the Mosquito Vector and Mammalian Host▿

    Science.gov (United States)

    Mikolajczak, Sebastian A.; Silva-Rivera, Hilda; Peng, Xinxia; Tarun, Alice S.; Camargo, Nelly; Jacobs-Lorena, Vanessa; Daly, Thomas M.; Bergman, Lawrence W.; de la Vega, Patricia; Williams, Jack; Aly, Ahmed S. I.; Kappe, Stefan H. I.

    2008-01-01

    The malaria parasite sporozoite transmission stage develops and differentiates within parasite oocysts on the Anopheles mosquito midgut. Successful inoculation of the parasite into a mammalian host is critically dependent on the sporozoite's ability to first infect the mosquito salivary glands. Remarkable changes in tissue infection competence are observed as the sporozoites transit from the midgut oocysts to the salivary glands. Our microarray analysis shows that compared to oocyst sporozoites, salivary gland sporozoites upregulate expression of at least 124 unique genes. Conversely, oocyst sporozoites show upregulation of at least 47 genes (upregulated in oocyst sporozoites [UOS genes]) before they infect the salivary glands. Targeted gene deletion of UOS3, encoding a putative transmembrane protein with a thrombospondin repeat that localizes to the sporozoite secretory organelles, rendered oocyst sporozoites unable to infect the mosquito salivary glands but maintained the parasites' liver infection competence. This phenotype demonstrates the significance of differential UOS expression. Thus, the UIS-UOS gene classification provides a framework to elucidate the infectivity and transmission success of Plasmodium sporozoites on a whole-genome scale. Genes identified herein might represent targets for vector-based transmission blocking strategies (UOS genes), as well as strategies that prevent mammalian host infection (UIS genes). PMID:18710954

  6. Genome-wide prediction of transcriptional regulatory elements of human promoters using gene expression and promoter analysis data

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    Kim Seon-Young

    2006-07-01

    Full Text Available Abstract Background A complete understanding of the regulatory mechanisms of gene expression is the next important issue of genomics. Many bioinformaticians have developed methods and algorithms for predicting transcriptional regulatory mechanisms from sequence, gene expression, and binding data. However, most of these studies involved the use of yeast which has much simpler regulatory networks than human and has many genome wide binding data and gene expression data under diverse conditions. Studies of genome wide transcriptional networks of human genomes currently lag behind those of yeast. Results We report herein a new method that combines gene expression data analysis with promoter analysis to infer transcriptional regulatory elements of human genes. The Z scores from the application of gene set analysis with gene sets of transcription factor binding sites (TFBSs were successfully used to represent the activity of TFBSs in a given microarray data set. A significant correlation between the Z scores of gene sets of TFBSs and individual genes across multiple conditions permitted successful identification of many known human transcriptional regulatory elements of genes as well as the prediction of numerous putative TFBSs of many genes which will constitute a good starting point for further experiments. Using Z scores of gene sets of TFBSs produced better predictions than the use of mRNA levels of a transcription factor itself, suggesting that the Z scores of gene sets of TFBSs better represent diverse mechanisms for changing the activity of transcription factors in the cell. In addition, cis-regulatory modules, combinations of co-acting TFBSs, were readily identified by our analysis. Conclusion By a strategic combination of gene set level analysis of gene expression data sets and promoter analysis, we were able to identify and predict many transcriptional regulatory elements of human genes. We conclude that this approach will aid in decoding

  7. Systematic analysis of essential genes reveals important regulators of G protein signaling.

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    Cappell, Steven D; Baker, Rachael; Skowyra, Dorota; Dohlman, Henrik G

    2010-06-11

    The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify additional signaling components, we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly represented. Included in this group are members of the Skp, Cullin, F box (SCF) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCF(Cdc4) acts together with the Cdc34 ubiquitin-conjugating enzyme at the level of the G protein; promotes degradation of the G protein alpha subunit, Gpa1, in vivo; and catalyzes Gpa1 ubiquitination in vitro. These insights to the G protein signaling network reveal the essential genome as an untapped resource for identifying new components and regulators of signal transduction pathways. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  8. Analysis of prostate-specific antigen transcripts in chimpanzees, cynomolgus monkeys, baboons, and African green monkeys.

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    James N Mubiru

    Full Text Available The function of prostate-specific antigen (PSA is to liquefy the semen coagulum so that the released sperm can fuse with the ovum. Fifteen spliced variants of the PSA gene have been reported in humans, but little is known about alternative splicing in nonhuman primates. Positive selection has been reported in sex- and reproductive-related genes from sea urchins to Drosophila to humans; however, there are few studies of adaptive evolution of the PSA gene. Here, using polymerase chain reaction (PCR product cloning and sequencing, we study PSA transcript variant heterogeneity in the prostates of chimpanzees (Pan troglodytes, cynomolgus monkeys (Macaca fascicularis, baboons (Papio hamadryas anubis, and African green monkeys (Chlorocebus aethiops. Six PSA variants were identified in the chimpanzee prostate, but only two variants were found in cynomolgus monkeys, baboons, and African green monkeys. In the chimpanzee the full-length transcript is expressed at the same magnitude as the transcripts that retain intron 3. We have found previously unidentified splice variants of the PSA gene, some of which might be linked to disease conditions. Selection on the PSA gene was studied in 11 primate species by computational methods using the sequences reported here for African green monkey, cynomolgus monkey, baboon, and chimpanzee and other sequences available in public databases. A codon-based analysis (dN/dS of the PSA gene identified potential adaptive evolution at five residue sites (Arg45, Lys70, Gln144, Pro189, and Thr203.

  9. Comprehensive Definition of the SigH Regulon of Mycobacterium tuberculosis Reveals Transcriptional Control of Diverse Stress Responses.

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    Jared D Sharp

    Full Text Available Expression of SigH, one of 12 Mycobacterium tuberculosis alternative sigma factors, is induced by heat, oxidative and nitric oxide stresses. SigH activation has been shown to increase expression of several genes, including genes involved in maintaining redox equilibrium and in protein degradation. However, few of these are known to be directly regulated by SigH. The goal of this project is to comprehensively define the Mycobacterium tuberculosis genes and operons that are directly controlled by SigH in order to gain insight into the role of SigH in regulating M. tuberculosis physiology. We used ChIP-Seq to identify in vivo SigH binding sites throughout the M. tuberculosis genome, followed by quantification of SigH-dependent expression of genes linked to these sites and identification of SigH-regulated promoters. We identified 69 SigH binding sites, which are located both in intergenic regions and within annotated coding sequences in the annotated M. tuberculosis genome. 41 binding sites were linked to genes that showed greater expression following heat stress in a SigH-dependent manner. We identified several genes not previously known to be regulated by SigH, including genes involved in DNA repair, cysteine biosynthesis, translation, and genes of unknown function. Experimental and computational analysis of SigH-regulated promoter sequences within these binding sites identified strong consensus -35 and -10 promoter sequences, but with tolerance for non-consensus bases at specific positions. This comprehensive identification and validation of SigH-regulated genes demonstrates an extended SigH regulon that controls an unexpectedly broad range of stress response functions.

  10. Comprehensive Definition of the SigH Regulon of Mycobacterium tuberculosis Reveals Transcriptional Control of Diverse Stress Responses.

    Science.gov (United States)

    Sharp, Jared D; Singh, Atul K; Park, Sang Tae; Lyubetskaya, Anna; Peterson, Matthew W; Gomes, Antonio L C; Potluri, Lakshmi-Prasad; Raman, Sahadevan; Galagan, James E; Husson, Robert N

    2016-01-01

    Expression of SigH, one of 12 Mycobacterium tuberculosis alternative sigma factors, is induced by heat, oxidative and nitric oxide stresses. SigH activation has been shown to increase expression of several genes, including genes involved in maintaining redox equilibrium and in protein degradation. However, few of these are known to be directly regulated by SigH. The goal of this project is to comprehensively define the Mycobacterium tuberculosis genes and operons that are directly controlled by SigH in order to gain insight into the role of SigH in regulating M. tuberculosis physiology. We used ChIP-Seq to identify in vivo SigH binding sites throughout the M. tuberculosis genome, followed by quantification of SigH-dependent expression of genes linked to these sites and identification of SigH-regulated promoters. We identified 69 SigH binding sites, which are located both in intergenic regions and within annotated coding sequences in the annotated M. tuberculosis genome. 41 binding sites were linked to genes that showed greater expression following heat stress in a SigH-dependent manner. We identified several genes not previously known to be regulated by SigH, including genes involved in DNA repair, cysteine biosynthesis, translation, and genes of unknown function. Experimental and computational analysis of SigH-regulated promoter sequences within these binding sites identified strong consensus -35 and -10 promoter sequences, but with tolerance for non-consensus bases at specific positions. This comprehensive identification and validation of SigH-regulated genes demonstrates an extended SigH regulon that controls an unexpectedly broad range of stress response functions.

  11. Sequential analysis of the numerical Stroop effect reveals response suppression.

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    Cohen Kadosh, Roi; Gevers, Wim; Notebaert, Wim

    2011-09-01

    Automatic processing of irrelevant stimulus dimensions has been demonstrated in a variety of tasks. Previous studies have shown that conflict between relevant and irrelevant dimensions can be reduced when a feature of the irrelevant dimension is repeated. The specific level at which the automatic process is suppressed (e.g., perceptual repetition, response repetition), however, is less understood. In the current experiment we used the numerical Stroop paradigm, in which the processing of irrelevant numerical values of 2 digits interferes with the processing of their physical size, to pinpoint the precise level of the suppression. Using a sequential analysis, we dissociated perceptual repetition from response repetition of the relevant and irrelevant dimension. Our analyses of reaction times, error rates, and diffusion modeling revealed that the congruity effect is significantly reduced or even absent when the response sequence of the irrelevant dimension, rather than the numerical value or the physical size, is repeated. These results suggest that automatic activation of the irrelevant dimension is suppressed at the response level. The current results shed light on the level of interaction between numerical magnitude and physical size as well as the effect of variability of responses and stimuli on automatic processing. (c) 2011 APA, all rights reserved.

  12. Early transcriptome analyses of Z-3-Hexenol-treated zea mays revealed distinct transcriptional networks and anti-herbivore defense potential of green leaf volatiles.

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    Jurgen Engelberth

    Full Text Available Green leaf volatiles (GLV, which are rapidly emitted by plants in response to insect herbivore damage, are now established as volatile defense signals. Receiving plants utilize these molecules to prime their defenses and respond faster and stronger when actually attacked. To further characterize the biological activity of these compounds we performed a microarray analysis of global gene expression. The focus of this project was to identify early transcriptional events elicited by Z-3-hexenol (Z-3-HOL as our model GLV in maize (Zea mays seedlings. The microarray results confirmed previous studies on Z-3-HOL -induced gene expression but also provided novel information about the complexity of Z-3-HOL -induced transcriptional networks. Besides identifying a distinct set of genes involved in direct and indirect defenses we also found significant expression of genes involved in transcriptional regulation, Ca(2+-and lipid-related signaling, and cell wall reinforcement. By comparing these results with those obtained by treatment of maize seedlings with insect elicitors we found a high degree of correlation between the two expression profiles at this early time point, in particular for those genes related to defense. We further analyzed defense gene expression induced by other volatile defense signals and found Z-3-HOL to be significantly more active than methyl jasmonate, methyl salicylate, and ethylene. The data presented herein provides important information on early genetic networks that are activated by Z-3-HOL and demonstrates the effectiveness of this compound in the regulation of typical plant defenses against insect herbivores in maize.

  13. Transcript profiling in the barley mildew pathogen Blumeria graminis by serial analysis of gene expression (SAGE).

    Science.gov (United States)

    Thomas, Stephen W; Glaring, Mikkel A; Rasmussen, Søren W; Kinane, Julia T; Oliver, Richard P

    2002-08-01

    The fungal pathogen Blumeria graminis f. sp. hordei develops on the barley leaf via distinct, morphologically well-defined stages. After landing on a host plant, the conidia rapidly germinate to form a primary germ tube. Subsequently, an appressorial germ tube emerges from the conidium and differentiates an appressorium from which penetration of the host cell wall is attempted. We have used serial analysis of gene expression to provide a measurement of messenger RNA contents in ungerminated conidia, during conidial germination, and during appressorium formation. The resulting data provide a resource for the characterization of changes in transcript accumulation during early development of B. graminis.

  14. A Study on the application of Data Mining Methods in the analysis of Transcripts

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    Luis Raunheitte

    2012-06-01

    Full Text Available Schools always had an essential role in the formation of students' intellect; however, the constant incorporation of knowledge to improve techniques and technologies used in the production of goods and services has caused a major demand for highly qualified professionals and, in order to meet that need, the teaching process must understand and adapt to the profile of the students. The transcript is the most used document to measure the performance of a student. Its digital storage combined with data mining methodologies can contribute not only to the analysis of performances, but also to the identification of significant information about student

  15. Analysis of global gene expression in Brachypodium distachyon reveals extensive network plasticity in response to abiotic stress.

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    Henry D Priest

    Full Text Available Brachypodium distachyon is a close relative of many important cereal crops. Abiotic stress tolerance has a significant impact on productivity of agriculturally important food and feedstock crops. Analysis of the transcriptome of Brachypodium after chilling, high-salinity, drought, and heat stresses revealed diverse differential expression of many transcripts. Weighted Gene Co-Expression Network Analysis revealed 22 distinct gene modules with specific profiles of expression under each stress. Promoter analysis implicated short DNA sequences directly upstream of module members in the regulation of 21 of 22 modules. Functional analysis of module members revealed enrichment in functional terms for 10 of 22 network modules. Analysis of condition-specific correlations between differentially expressed gene pairs revealed extensive plasticity in the expression relationships of gene pairs. Photosynthesis, cell cycle, and cell wall expression modules were down-regulated by all abiotic stresses. Modules which were up-regulated by each abiotic stress fell into diverse and unique gene ontology GO categories. This study provides genomics resources and improves our understanding of abiotic stress responses of Brachypodium.

  16. Association genetics and transcriptome analysis reveal a gibberellin-responsive pathway involved in regulating photosynthesis.

    Science.gov (United States)

    Xie, Jianbo; Tian, Jiaxing; Du, Qingzhang; Chen, Jinhui; Li, Ying; Yang, Xiaohui; Li, Bailian; Zhang, Deqiang

    2016-05-01

    Gibberellins (GAs) regulate a wide range of important processes in plant growth and development, including photosynthesis. However, the mechanism by which GAs regulate photosynthesis remains to be understood. Here, we used multi-gene association to investigate the effect of genes in the GA-responsive pathway, as constructed by RNA sequencing, on photosynthesis, growth, and wood property traits, in a population of 435 Populus tomentosa By analyzing changes in the transcriptome following GA treatment, we identified many key photosynthetic genes, in agreement with the observed increase in measurements of photosynthesis. Regulatory motif enrichment analysis revealed that 37 differentially expressed genes related to photosynthesis shared two essential GA-related cis-regulatory elements, the GA response element and the pyrimidine box. Thus, we constructed a GA-responsive pathway consisting of 47 genes involved in regulating photosynthesis, including GID1, RGA, GID2, MYBGa, and 37 photosynthetic differentially expressed genes. Single nucleotide polymorphism (SNP)-based association analysis showed that 142 SNPs, representing 40 candidate genes in this pathway, were significantly associated with photosynthesis, growth, and wood property traits. Epistasis analysis uncovered interactions between 310 SNP-SNP pairs from 37 genes in this pathway, revealing possible genetic interactions. Moreover, a structural gene-gene matrix based on a time-course of transcript abundances provided a better understanding of the multi-gene pathway affecting photosynthesis. The results imply a functional role for these genes in mediating photosynthesis, growth, and wood properties, demonstrating the potential of combining transcriptome-based regulatory pathway construction and genetic association approaches to detect the complex genetic networks underlying quantitative traits. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights

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

    Science.gov (United States)

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

    2016-01-01

    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. PMID:26857372

  18. Network analysis reveals a causal role of mitochondrial gene activity in atherosclerotic lesion formation.

    Science.gov (United States)

    Vilne, Baiba; Skogsberg, Josefin; Foroughi Asl, Hassan; Talukdar, Husain Ahammad; Kessler, Thorsten; Björkegren, Johan L M; Schunkert, Heribert

    2017-12-01

    Mitochondrial damage and augmented production of reactive oxygen species (ROS) may represent an intermediate step by which hypercholesterolemia exacerbates atherosclerotic lesion formation. To test this hypothesis, in mice with severe but genetically reversible hypercholesterolemia (i.e. the so called Reversa mouse model), we performed time-resolved analyses of mitochondrial transcriptome in the aortic arch employing a systems-level network approach. During hypercholesterolemia, we observed a massive down-regulation (>28%) of mitochondrial genes, specifically at the time of rapid atherosclerotic lesion expansion and foam cell formation, i.e. between 30 and 40 weeks of age. Both phenomena - down-regulation of mitochondrial genes and lesion expansion - were largely reversible by genetically lowering plasma cholesterol (by >80%, from 427 to 54 ± 31 mg/L) at 30 weeks. Co-expression network analysis revealed that both mitochondrial signature genes were highly connected in two modules, negatively correlating with lesion size and supported as causal for coronary artery disease (CAD) in humans, as expression-associated single nucleotide polymorphisms (eSNPs) representing their genes overlapped markedly with established disease risk loci. Within these modules, we identified the transcription factor estrogen related receptor (ERR)-α and its co-factors PGC1-α and -β, i.e. two members of the peroxisome proliferator-activated receptor γ co-activator 1 family of transcription regulators, as key regulatory genes. Together, these factors are known as major orchestrators of mitochondrial biogenesis and antioxidant responses. Using a network approach, we demonstrate how hypercholesterolemia could hamper mitochondrial activity during atherosclerosis progression and pinpoint potential therapeutic targets to counteract these processes. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Investigating Microbial Activity in Diazotrophic Methane Seep Sediment via Transcript Analysis and Single-Cell FISH-NanoSIMS

    Science.gov (United States)

    Dekas, A. E.; Connon, S. A.; Chadwick, G.; Orphan, V. J.

    2012-12-01

    pattern of transcripts recovered in the mesocosm experiments. This analysis revealed 15N enrichment in free-living (i.e. non-ANME associated) members of the Desulfobulbaceae in 15N2 incubations with methane, while free-living Desulfosarcina/Desulfococcus cells, as well as nearly 40 unidentified DAPI-stained cells, were not 15N enriched. However, further NanoSIMS analyses of DSB in a variety of incubation conditions suggests that this enrichment may be due to N sharing between the ANME and DSB while in tight physical association, and then subsequent dissociation, rather than nitrogen fixation by the DSB. If true, this is an excellent example of the potential pitfalls of single cell stable isotope labeling experiments, and potential false positives due to the recycling of labeled material between (even transiently) closely associated symbionts. This work highlights both the utility of transcript analysis as a hypothesis-generator for direct analyses of microbial activity via stable isotope labeling, as well as the need to contextualize labeling experiments with investigations of microbial community structure.

  20. Transcriptional regulation of the outer membrane porin gene ompW reveals its physiological role during the transition from the aerobic to the anaerobic lifestyle of Escherichia coli

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    Minfeng eXiao

    2016-05-01

    Full Text Available Understanding bacterial physiology relies on elucidating the regulatory mechanisms and cellular functions of those differentially expressed genes in response to environmental changes. A widespread Gram-negative bacterial outer membrane protein OmpW has been implicated in the adaptation to stresses in various species. It is recently found to be present in the regulon of the global anaerobic transcription factor FNR and ArcA in E. coli. However, little is known about the physiological implications of this regulatory disposition. In this study, we demonstrate that transcription of ompW is indeed mediated by a series of global regulators involved in the anaerobiosis of E. coli. We show that FNR can both activate and repress the expression of ompW through its direct binding to two distinctive sites, -81.5 and -126.5 bp respectively, on ompW promoter. ArcA also participates in repression of ompW under anaerobic condition, but in an FNR dependent manner. Additionally, ompW is also subject to the regulation by CRP and NarL which senses the availability and types of carbon sources and respiration electron acceptors in the environment respectively, implying a role of OmpW in the carbon and energy metabolism of E. coli during its anaerobic adaptation. Molecular docking reveals that OmpW can bind fumarate, an alternative electron acceptor in anaerobic respiration, with sufficient affinity. Moreover, supplement of fumarate or succinate which belongs to the C4-dicarboxylates family of metabolite, to E. coli culture rescues OmpW-mediated colicin S4 killing. Taken together, we propose that OmpW is involved in anaerobic carbon and energy metabolism to mediate the transition from aerobic to anaerobic lifestyle in E. coli.

  1. Isotope analysis reveals foraging area dichotomy for atlantic leatherback turtles.

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    Stéphane Caut

    Full Text Available BACKGROUND: The leatherback turtle (Dermochelys coriacea has undergone a dramatic decline over the last 25 years, and this is believed to be primarily the result of mortality associated with fisheries bycatch followed by egg and nesting female harvest. Atlantic leatherback turtles undertake long migrations across ocean basins from subtropical and tropical nesting beaches to productive frontal areas. Migration between two nesting seasons can last 2 or 3 years, a time period termed the remigration interval (RI. Recent satellite transmitter data revealed that Atlantic leatherbacks follow two major dispersion patterns after nesting season, through the North Gulf Stream area or more eastward across the North Equatorial Current. However, information on the whole RI is lacking, precluding the accurate identification of feeding areas where conservation measures may need to be applied. METHODOLOGY/PRINCIPAL FINDINGS: Using stable isotopes as dietary tracers we determined the characteristics of feeding grounds of leatherback females nesting in French Guiana. During migration, 3-year RI females differed from 2-year RI females in their isotope values, implying differences in their choice of feeding habitats (offshore vs. more coastal and foraging latitude (North Atlantic vs. West African coasts, respectively. Egg-yolk and blood isotope values are correlated in nesting females, indicating that egg analysis is a useful tool for assessing isotope values in these turtles, including adults when not available. CONCLUSIONS/SIGNIFICANCE: Our results complement previous data on turtle movements during the first year following the nesting season, integrating the diet consumed during the year before nesting. We suggest that the French Guiana leatherback population segregates into two distinct isotopic groupings, and highlight the urgent need to determine the feeding habitats of the turtle in the Atlantic in order to protect this species from incidental take by

  2. Mitochondrial Genome Analysis Reveals Historical Lineages in Yellowstone Bison.

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    David Forgacs

    Full Text Available Yellowstone National Park is home to one of the only plains bison populations that have continuously existed on their present landscape since prehistoric times without evidence of domestic cattle introgression. Previous studies characterized the relatively high levels of nuclear genetic diversity in these bison, but little is known about their mitochondrial haplotype diversity. This study assessed mitochondrial genomes from 25 randomly selected Yellowstone bison and found 10 different mitochondrial haplotypes with a haplotype diversity of 0.78 (± 0.06. Spatial analysis of these mitochondrial DNA (mtDNA haplotypes did not detect geographic population subdivision (FST = -0.06, p = 0.76. However, we identified two independent and historically important lineages in Yellowstone bison by combining data from 65 bison (defined by 120 polymorphic sites from across North America representing a total of 30 different mitochondrial DNA haplotypes. Mitochondrial DNA haplotypes from one of the Yellowstone lineages represent descendants of the 22 indigenous bison remaining in central Yellowstone in 1902. The other mitochondrial DNA lineage represents descendants of the 18 females introduced from northern Montana in 1902 to supplement the indigenous bison population and develop a new breeding herd in the northern region of the park. Comparing modern and historical mitochondrial DNA diversity in Yellowstone bison helps uncover a historical context of park restoration efforts during the early 1900s, provides evidence against a hypothesized mitochondrial disease in bison, and reveals the signature of recent hybridization between American plains bison (Bison bison bison and Canadian wood bison (B. b. athabascae. Our study demonstrates how mitochondrial DNA can be applied to delineate the history of wildlife species and inform future conservation actions.

  3. Quantitative flux analysis reveals folate-dependent NADPH production

    Science.gov (United States)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  4. Comparative functional genomic analysis of two Vibrio phages reveals complex metabolic interactions with the host cell

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    Dimitrios Skliros

    2016-11-01

    Full Text Available Sequencing and annotation was performed for two giant double stranded DNA bacteriophages, φGrn1 and φSt2 of the Myoviridae family, considered to be of great interest for phage therapy against Vibrios in aquaculture live feeds. In addition, phage-host metabolic interactions and exploitation was studied by transcript profiling of selected viral and host genes. Comparative genomic analysis with other giant Vibrio phages was also performed to establish the presence and location of homing endonucleases highlighting distinct features for both phages. Phylogenetic analysis revealed that they belong to the schizoT4like clade. Although many reports of newly sequenced viruses have provided a large set of information, basic research related to the shift of the bacterial metabolism during infection remains stagnant. The function of many viral protein products in the process of infection is still unknown. Genome annotation identified the presence of several viral ORFs participating in metabolism, including a Sir2/cobB (sirtuin protein and a number of genes involved in auxiliary NAD+ and nucleotide biosynthesis, necessary for phage DNA replication. Key genes were subsequently selected for detail study of their expression levels during infection. This work suggests a complex metabolic interaction and exploitation of the host metabolic pathways and biochemical processes, including a possible post-translational protein modification, by the virus during infection.

  5. Dynamic allostery in the methionine repressor revealed by force distribution analysis.

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    Wolfram Stacklies

    2009-11-01

    Full Text Available Many fundamental cellular processes such as gene expression are tightly regulated by protein allostery. Allosteric signal propagation from the regulatory to the active site requires long-range communication, the molecular mechanism of which remains a matter of debate. A classical example for long-range allostery is the activation of the methionine repressor MetJ, a transcription factor. Binding of its co-repressor SAM increases its affinity for DNA several-fold, but has no visible conformational effect on its DNA binding interface. Our molecular dynamics simulations indicate correlated domain motions within MetJ, and quenching of these dynamics upon SAM binding entropically favors DNA binding. From monitoring conformational fluctuations alone, it is not obvious how the presence of SAM is communicated through the largely rigid core of MetJ and how SAM thereby is able to regulate MetJ dynamics. We here directly monitored the propagation of internal forces through the MetJ structure, instead of relying on conformational changes as conventionally done. Our force distribution analysis successfully revealed the molecular network for strain propagation, which connects collective domain motions through the protein core. Parts of the network are directly affected by SAM binding, giving rise to the observed quenching of fluctuations. Our results are in good agreement with experimental data. The force distribution analysis suggests itself as a valuable tool to gain insight into the molecular function of a whole class of allosteric proteins.

  6. Construction of a public CHO cell line transcript database using versatile bioinformatics analysis pipelines.

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    Oliver Rupp

    Full Text Available Chinese hamster ovary (CHO cell lines represent the most commonly used mammalian expression system for the production of therapeutic proteins. In this context, detailed knowledge of the CHO cell transcriptome might help to improve biotechnological processes conducted by specific cell lines. Nevertheless, very few assembled cDNA sequences of CHO cells were publicly released until recently, which puts a severe limitation on biotechnological research. Two extended annotation systems and web-based tools, one for browsing eukaryotic genomes (GenDBE and one for viewing eukaryotic transcriptomes (SAMS, were established as the first step towards a publicly usable CHO cell genome/transcriptome analysis platform. This is complemented by the development of a new strategy to assemble the ca. 100 million reads, sequenced from a broad range of diverse transcripts, to a high quality CHO cell transcript set. The cDNA libraries were constructed from different CHO cell lines grown under various culture conditions and sequenced using Roche/454 and Illumina sequencing technologies in addition to sequencing reads from a previous study. Two pipelines to extend and improve the CHO cell line transcripts were established. First, de novo assemblies were carried out with the Trinity and Oases assemblers, using varying k-mer sizes. The resulting contigs were screened for potential CDS using ESTScan. Redundant contigs were filtered out using cd-hit-est. The remaining CDS contigs were re-assembled with CAP3. Second, a reference-based assembly with the TopHat/Cufflinks pipeline was performed, using the recently published draft genome sequence of CHO-K1 as reference. Additionally, the de novo contigs were mapped to the reference genome using GMAP and merged with the Cufflinks assembly using the cuffmerge software. With this approach 28,874 transcripts located on 16,492 gene loci could be assembled. Combining the results of both approaches, 65,561 transcripts were identified

  7. Heart morphogenesis gene regulatory networks revealed by temporal expression analysis.

    Science.gov (United States)

    Hill, Jonathon T; Demarest, Bradley; Gorsi, Bushra; Smith, Megan; Yost, H Joseph

    2017-10-01

    During embryogenesis the heart forms as a linear tube that then undergoes multiple simultaneous morphogenetic events to obtain its mature shape. To understand the gene regulatory networks (GRNs) driving this phase of heart development, during which many congenital heart disease malformations likely arise, we conducted an RNA-seq timecourse in zebrafish from 30 hpf to 72 hpf and identified 5861 genes with altered expression. We clustered the genes by temporal expression pattern, identified transcription factor binding motifs enriched in each cluster, and generated a model GRN for the major gene batteries in heart morphogenesis. This approach predicted hundreds of regulatory interactions and found batteries enriched in specific cell and tissue types, indicating that the approach can be used to narrow the search for novel genetic markers and regulatory interactions. Subsequent analyses confirmed the GRN using two mutants, Tbx5 and nkx2-5 , and identified sets of duplicated zebrafish genes that do not show temporal subfunctionalization. This dataset provides an essential resource for future studies on the genetic/epigenetic pathways implicated in congenital heart defects and the mechanisms of cardiac transcriptional regulation. © 2017. Published by The Company of Biologists Ltd.

  8. Complex structure of the DNA-binding domain of AdpA, the global transcription factor in Streptomyces griseus, and a target duplex DNA reveals the structural basis of its tolerant DNA sequence specificity.

    Science.gov (United States)

    Yao, Ming Dong; Ohtsuka, Jun; Nagata, Koji; Miyazono, Ken-Ichi; Zhi, Yuehua; Ohnishi, Yasuo; Tanokura, Masaru

    2013-10-25

    AdpA serves as the global transcription factor in the A-factor regulatory cascade, controlling the secondary metabolism and morphological differentiation of the filamentous bacterium Streptomyces griseus. AdpA binds to over 500 operator regions with the consensus sequence 5'-TGGCSNGWWY-3' (where S is G or C, W is A or T, Y is T or C, and N is any nucleotide). However, it is still obscure how AdpA can control hundreds of genes. To elucidate the structural basis of this tolerant DNA recognition by AdpA, we focused on the interaction between the DNA-binding domain of AdpA (AdpA-DBD), which consists of two helix-turn-helix motifs, and a target duplex DNA containing the consensus sequence 5'-TGGCGGGTTC-3'. The crystal structure of the AdpA-DBD-DNA complex and the mutant analysis of AdpA-DBD revealed its unique manner of DNA recognition, whereby only two arginine residues directly recognize the consensus sequence, explaining the strict recognition of G and C at positions 2 and 4, respectively, and the tolerant recognition of other positions of the consensus sequence. AdpA-DBD confers tolerant DNA sequence specificity to AdpA, allowing it to control hundreds of genes as a global transcription factor.

  9. The crystal structures of apo and cAMP-bound GlxR from Corynebacterium glutamicum reveal structural and dynamic changes upon cAMP binding in CRP/FNR family transcription factors.

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    Philip D Townsend

    Full Text Available The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition.

  10. Genome-wide analysis of coordinated transcript abundance during seed development in different Brassica rapa morphotypes.

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    Basnet, Ram Kumar; Moreno-Pachon, Natalia; Lin, Ke; Bucher, Johan; Visser, Richard G F; Maliepaard, Chris; Bonnema, Guusje

    2013-12-01

    Brassica seeds are important as basic units of plant growth and sources of vegetable oil. Seed development is regulated by many dynamic metabolic processes controlled by complex networks of spatially and temporally expressed genes. We conducted a global microarray gene co-expression analysis by measuring transcript abundance of developing seeds from two diverse B. rapa morphotypes: a pak choi (leafy-type) and a yellow sarson (oil-type), and two of their doubled haploid (DH) progenies, (1) to study the timing of metabolic processes in developing seeds, (2) to explore the major transcriptional differences in developing seeds of the two morphotypes, and (3) to identify the optimum stage for a genetical genomics study in B. rapa seed. Seed developmental stages were similar in developing seeds of pak choi and yellow sarson of B. rapa; however, the colour of embryo and seed coat differed among these two morphotypes. In this study, most transcriptional changes occurred between 25 and 35 DAP, which shows that the timing of seed developmental processes in B. rapa is at later developmental stages than in the relat