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Sample records for regulatory sequence common

  1. Regulatory sequence analysis tools.

    PubMed

    van Helden, Jacques

    2003-07-01

    The web resource Regulatory Sequence Analysis Tools (RSAT) (http://rsat.ulb.ac.be/rsat) offers a collection of software tools dedicated to the prediction of regulatory sites in non-coding DNA sequences. These tools include sequence retrieval, pattern discovery, pattern matching, genome-scale pattern matching, feature-map drawing, random sequence generation and other utilities. Alternative formats are supported for the representation of regulatory motifs (strings or position-specific scoring matrices) and several algorithms are proposed for pattern discovery. RSAT currently holds >100 fully sequenced genomes and these data are regularly updated from GenBank.

  2. Population sequencing of two endocannabinoid metabolic genes identifies rare and common regulatory variants associated with extreme obesity and metabolite level

    PubMed Central

    2010-01-01

    Background Targeted re-sequencing of candidate genes in individuals at the extremes of a quantitative phenotype distribution is a method of choice to gain information on the contribution of rare variants to disease susceptibility. The endocannabinoid system mediates signaling in the brain and peripheral tissues involved in the regulation of energy balance, is highly active in obese patients, and represents a strong candidate pathway to examine for genetic association with body mass index (BMI). Results We sequenced two intervals (covering 188 kb) encoding the endocannabinoid metabolic enzymes fatty-acid amide hydrolase (FAAH) and monoglyceride lipase (MGLL) in 147 normal controls and 142 extremely obese cases. After applying quality filters, we called 1,393 high quality single nucleotide variants, 55% of which are rare, and 143 indels. Using single marker tests and collapsed marker tests, we identified four intervals associated with BMI: the FAAH promoter, the MGLL promoter, MGLL intron 2, and MGLL intron 3. Two of these intervals are composed of rare variants and the majority of the associated variants are located in promoter sequences or in predicted transcriptional enhancers, suggesting a regulatory role. The set of rare variants in the FAAH promoter associated with BMI is also associated with increased level of FAAH substrate anandamide, further implicating a functional role in obesity. Conclusions Our study, which is one of the first reports of a sequence-based association study using next-generation sequencing of candidate genes, provides insights into study design and analysis approaches and demonstrates the importance of examining regulatory elements rather than exclusively focusing on exon sequences. PMID:21118518

  3. Coordinate cytokine regulatory sequences

    DOEpatents

    Frazer, Kelly A.; Rubin, Edward M.; Loots, Gabriela G.

    2005-05-10

    The present invention provides CNS sequences that regulate the cytokine gene expression, expression cassettes and vectors comprising or lacking the CNS sequences, host cells and non-human transgenic animals comprising the CNS sequences or lacking the CNS sequences. The present invention also provides methods for identifying compounds that modulate the functions of CNS sequences as well as methods for diagnosing defects in the CNS sequences of patients.

  4. RSAT: regulatory sequence analysis tools.

    PubMed

    Thomas-Chollier, Morgane; Sand, Olivier; Turatsinze, Jean-Valéry; Janky, Rekin's; Defrance, Matthieu; Vervisch, Eric; Brohée, Sylvain; van Helden, Jacques

    2008-07-01

    The regulatory sequence analysis tools (RSAT, http://rsat.ulb.ac.be/rsat/) is a software suite that integrates a wide collection of modular tools for the detection of cis-regulatory elements in genome sequences. The suite includes programs for sequence retrieval, pattern discovery, phylogenetic footprint detection, pattern matching, genome scanning and feature map drawing. Random controls can be performed with random gene selections or by generating random sequences according to a variety of background models (Bernoulli, Markov). Beyond the original word-based pattern-discovery tools (oligo-analysis and dyad-analysis), we recently added a battery of tools for matrix-based detection of cis-acting elements, with some original features (adaptive background models, Markov-chain estimation of P-values) that do not exist in other matrix-based scanning tools. The web server offers an intuitive interface, where each program can be accessed either separately or connected to the other tools. In addition, the tools are now available as web services, enabling their integration in programmatic workflows. Genomes are regularly updated from various genome repositories (NCBI and EnsEMBL) and 682 organisms are currently supported. Since 1998, the tools have been used by several hundreds of researchers from all over the world. Several predictions made with RSAT were validated experimentally and published.

  5. Formation of Regulatory Modules by Local Sequence Duplication

    PubMed Central

    Nourmohammad, Armita; Lässig, Michael

    2011-01-01

    Turnover of regulatory sequence and function is an important part of molecular evolution. But what are the modes of sequence evolution leading to rapid formation and loss of regulatory sites? Here we show that a large fraction of neighboring transcription factor binding sites in the fly genome have formed from a common sequence origin by local duplications. This mode of evolution is found to produce regulatory information: duplications can seed new sites in the neighborhood of existing sites. Duplicate seeds evolve subsequently by point mutations, often towards binding a different factor than their ancestral neighbor sites. These results are based on a statistical analysis of 346 cis-regulatory modules in the Drosophila melanogaster genome, and a comparison set of intergenic regulatory sequence in Saccharomyces cerevisiae. In fly regulatory modules, pairs of binding sites show significantly enhanced sequence similarity up to distances of about 50 bp. We analyze these data in terms of an evolutionary model with two distinct modes of site formation: (i) evolution from independent sequence origin and (ii) divergent evolution following duplication of a common ancestor sequence. Our results suggest that pervasive formation of binding sites by local sequence duplications distinguishes the complex regulatory architecture of higher eukaryotes from the simpler architecture of unicellular organisms. PMID:21998564

  6. Formation of regulatory modules by local sequence duplication.

    PubMed

    Nourmohammad, Armita; Lässig, Michael

    2011-10-01

    Turnover of regulatory sequence and function is an important part of molecular evolution. But what are the modes of sequence evolution leading to rapid formation and loss of regulatory sites? Here we show that a large fraction of neighboring transcription factor binding sites in the fly genome have formed from a common sequence origin by local duplications. This mode of evolution is found to produce regulatory information: duplications can seed new sites in the neighborhood of existing sites. Duplicate seeds evolve subsequently by point mutations, often towards binding a different factor than their ancestral neighbor sites. These results are based on a statistical analysis of 346 cis-regulatory modules in the Drosophila melanogaster genome, and a comparison set of intergenic regulatory sequence in Saccharomyces cerevisiae. In fly regulatory modules, pairs of binding sites show significantly enhanced sequence similarity up to distances of about 50 bp. We analyze these data in terms of an evolutionary model with two distinct modes of site formation: (i) evolution from independent sequence origin and (ii) divergent evolution following duplication of a common ancestor sequence. Our results suggest that pervasive formation of binding sites by local sequence duplications distinguishes the complex regulatory architecture of higher eukaryotes from the simpler architecture of unicellular organisms.

  7. RSAT 2015: Regulatory Sequence Analysis Tools.

    PubMed

    Medina-Rivera, Alejandra; Defrance, Matthieu; Sand, Olivier; Herrmann, Carl; Castro-Mondragon, Jaime A; Delerce, Jeremy; Jaeger, Sébastien; Blanchet, Christophe; Vincens, Pierre; Caron, Christophe; Staines, Daniel M; Contreras-Moreira, Bruno; Artufel, Marie; Charbonnier-Khamvongsa, Lucie; Hernandez, Céline; Thieffry, Denis; Thomas-Chollier, Morgane; van Helden, Jacques

    2015-07-01

    RSAT (Regulatory Sequence Analysis Tools) is a modular software suite for the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, appropriate to genome-wide data sets like ChIP-seq, (ii) transcription factor binding motif analysis (quality assessment, comparisons and clustering), (iii) comparative genomics and (iv) analysis of regulatory variations. Nine new programs have been added to the 43 described in the 2011 NAR Web Software Issue, including a tool to extract sequences from a list of coordinates (fetch-sequences from UCSC), novel programs dedicated to the analysis of regulatory variants from GWAS or population genomics (retrieve-variation-seq and variation-scan), a program to cluster motifs and visualize the similarities as trees (matrix-clustering). To deal with the drastic increase of sequenced genomes, RSAT public sites have been reorganized into taxon-specific servers. The suite is well-documented with tutorials and published protocols. The software suite is available through Web sites, SOAP/WSDL Web services, virtual machines and stand-alone programs at http://www.rsat.eu/.

  8. RSAT 2015: Regulatory Sequence Analysis Tools

    PubMed Central

    Medina-Rivera, Alejandra; Defrance, Matthieu; Sand, Olivier; Herrmann, Carl; Castro-Mondragon, Jaime A.; Delerce, Jeremy; Jaeger, Sébastien; Blanchet, Christophe; Vincens, Pierre; Caron, Christophe; Staines, Daniel M.; Contreras-Moreira, Bruno; Artufel, Marie; Charbonnier-Khamvongsa, Lucie; Hernandez, Céline; Thieffry, Denis; Thomas-Chollier, Morgane; van Helden, Jacques

    2015-01-01

    RSAT (Regulatory Sequence Analysis Tools) is a modular software suite for the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, appropriate to genome-wide data sets like ChIP-seq, (ii) transcription factor binding motif analysis (quality assessment, comparisons and clustering), (iii) comparative genomics and (iv) analysis of regulatory variations. Nine new programs have been added to the 43 described in the 2011 NAR Web Software Issue, including a tool to extract sequences from a list of coordinates (fetch-sequences from UCSC), novel programs dedicated to the analysis of regulatory variants from GWAS or population genomics (retrieve-variation-seq and variation-scan), a program to cluster motifs and visualize the similarities as trees (matrix-clustering). To deal with the drastic increase of sequenced genomes, RSAT public sites have been reorganized into taxon-specific servers. The suite is well-documented with tutorials and published protocols. The software suite is available through Web sites, SOAP/WSDL Web services, virtual machines and stand-alone programs at http://www.rsat.eu/. PMID:25904632

  9. RSAT 2015: Regulatory Sequence Analysis Tools.

    PubMed

    Medina-Rivera, Alejandra; Defrance, Matthieu; Sand, Olivier; Herrmann, Carl; Castro-Mondragon, Jaime A; Delerce, Jeremy; Jaeger, Sébastien; Blanchet, Christophe; Vincens, Pierre; Caron, Christophe; Staines, Daniel M; Contreras-Moreira, Bruno; Artufel, Marie; Charbonnier-Khamvongsa, Lucie; Hernandez, Céline; Thieffry, Denis; Thomas-Chollier, Morgane; van Helden, Jacques

    2015-07-01

    RSAT (Regulatory Sequence Analysis Tools) is a modular software suite for the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, appropriate to genome-wide data sets like ChIP-seq, (ii) transcription factor binding motif analysis (quality assessment, comparisons and clustering), (iii) comparative genomics and (iv) analysis of regulatory variations. Nine new programs have been added to the 43 described in the 2011 NAR Web Software Issue, including a tool to extract sequences from a list of coordinates (fetch-sequences from UCSC), novel programs dedicated to the analysis of regulatory variants from GWAS or population genomics (retrieve-variation-seq and variation-scan), a program to cluster motifs and visualize the similarities as trees (matrix-clustering). To deal with the drastic increase of sequenced genomes, RSAT public sites have been reorganized into taxon-specific servers. The suite is well-documented with tutorials and published protocols. The software suite is available through Web sites, SOAP/WSDL Web services, virtual machines and stand-alone programs at http://www.rsat.eu/. PMID:25904632

  10. Evolution of cis-regulatory sequence and function in Diptera.

    PubMed

    Wittkopp, P J

    2006-09-01

    Cis-regulatory sequences direct patterns of gene expression essential for development and physiology. Evolutionary changes in these sequences contribute to phenotypic divergence. Despite their importance, cis-regulatory regions remain one of the most enigmatic features of the genome. Patterns of sequence evolution can be used to identify cis-regulatory elements, but the power of this approach depends upon the relationship between sequence and function. Comparative studies of gene regulation among Diptera reveal that divergent sequences can underlie conserved expression, and that expression differences can evolve despite largely similar sequences. This complex structure-function relationship is the primary impediment for computational identification and interpretation of cis-regulatory sequences. Biochemical characterization and in vivo assays of cis-regulatory sequences on a genomic-scale will relieve this barrier.

  11. Transcriptome Sequencing from Diverse Human Populations Reveals Differentiated Regulatory Architecture

    PubMed Central

    Lappalainen, Tuuli; Henn, Brenna M.; Kidd, Jeffrey M.; Yee, Muh-Ching; Grubert, Fabian; Cann, Howard M.; Snyder, Michael; Montgomery, Stephen B.; Bustamante, Carlos D.

    2014-01-01

    Large-scale sequencing efforts have documented extensive genetic variation within the human genome. However, our understanding of the origins, global distribution, and functional consequences of this variation is far from complete. While regulatory variation influencing gene expression has been studied within a handful of populations, the breadth of transcriptome differences across diverse human populations has not been systematically analyzed. To better understand the spectrum of gene expression variation, alternative splicing, and the population genetics of regulatory variation in humans, we have sequenced the genomes, exomes, and transcriptomes of EBV transformed lymphoblastoid cell lines derived from 45 individuals in the Human Genome Diversity Panel (HGDP). The populations sampled span the geographic breadth of human migration history and include Namibian San, Mbuti Pygmies of the Democratic Republic of Congo, Algerian Mozabites, Pathan of Pakistan, Cambodians of East Asia, Yakut of Siberia, and Mayans of Mexico. We discover that approximately 25.0% of the variation in gene expression found amongst individuals can be attributed to population differences. However, we find few genes that are systematically differentially expressed among populations. Of this population-specific variation, 75.5% is due to expression rather than splicing variability, and we find few genes with strong evidence for differential splicing across populations. Allelic expression analyses indicate that previously mapped common regulatory variants identified in eight populations from the International Haplotype Map Phase 3 project have similar effects in our seven sampled HGDP populations, suggesting that the cellular effects of common variants are shared across diverse populations. Together, these results provide a resource for studies analyzing functional differences across populations by estimating the degree of shared gene expression, alternative splicing, and regulatory genetics

  12. Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones.

    PubMed

    Wolf, Kyle J; Emerson, Ryan O; Pingel, Jeanette; Buller, R Mark; DiPaolo, Richard J

    2016-01-01

    Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4+ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRβ chains, and a Foxp3-GFP reporter. We purified CD4+Foxp3- and CD4+Foxp3+ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRβ chains. We identified >7,000 different TCRβ sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4+ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus. PMID:27100298

  13. Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones

    PubMed Central

    Emerson, Ryan O.; Pingel, Jeanette; Buller, R. Mark; DiPaolo, Richard J.

    2016-01-01

    Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4+ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRβ chains, and a Foxp3-GFP reporter. We purified CD4+Foxp3- and CD4+Foxp3+ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRβ chains. We identified >7,000 different TCRβ sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4+ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus. PMID:27100298

  14. Differences in regulatory sequences of naturally occurring JC virus variants.

    PubMed Central

    Martin, J D; King, D M; Slauch, J M; Frisque, R J

    1985-01-01

    The regulatory region was sequenced for DNAs representative of seven independent isolates of JC virus, the probable agent of progressive multifocal leukoencephalopathy. The isolates included an oncogenic variant (MAD-4), an antigenic variant (MAD-11), and two different isolates derived from the urine (MAD-7) and from the brain (MAD-8) of the same patient. The representative DNAs were molecularly cloned directly from diseased brain tissue and from human fetal glial cells infected with the corresponding isolated viruses. The regulatory sequences of these DNAs were compared with those of the prototype isolate, MAD-1, sequenced previously (R. J. Frisque, J. Virol. 46:170-176, 1983). We found that the regulatory region of JC viral DNA is highly variable due to complex alterations of the previously described 98-base-pair repeat of MAD-1 DNA. On the basis of these alterations, there are two general types of JC virus. There were no consistent alterations in regulatory sequences which could distinguish brain tissue DNAs from tissue culture DNAs. Furthermore, for each isolate except MAD-1 (R. J. Frisque, J. Virol. 46:170-176, 1983), the regulatory regions of brain tissue and tissue culture DNAs were not identical. The arrangement, sequence, or both of potential regulatory elements (TATA sequence, GGGXGGPuPu, tandem repeats) of JC viral DNAs are sufficiently different from those in other viral and eucaryotic systems that they may effect the unique properties of this slow virus. PMID:2981353

  15. Identification of cis-regulatory sequence variations in individual genome sequences.

    PubMed

    Worsley-Hunt, Rebecca; Bernard, Virginie; Wasserman, Wyeth W

    2011-01-01

    Functional contributions of cis-regulatory sequence variations to human genetic disease are numerous. For instance, disrupting variations in a HNF4A transcription factor binding site upstream of the Factor IX gene contributes causally to hemophilia B Leyden. Although clinical genome sequence analysis currently focuses on the identification of protein-altering variation, the impact of cis-regulatory mutations can be similarly strong. New technologies are now enabling genome sequencing beyond exomes, revealing variation across the non-coding 98% of the genome responsible for developmental and physiological patterns of gene activity. The capacity to identify causal regulatory mutations is improving, but predicting functional changes in regulatory DNA sequences remains a great challenge. Here we explore the existing methods and software for prediction of functional variation situated in the cis-regulatory sequences governing gene transcription and RNA processing.

  16. Identification of cis-regulatory sequence variations in individual genome sequences

    PubMed Central

    2011-01-01

    Functional contributions of cis-regulatory sequence variations to human genetic disease are numerous. For instance, disrupting variations in a HNF4A transcription factor binding site upstream of the Factor IX gene contributes causally to hemophilia B Leyden. Although clinical genome sequence analysis currently focuses on the identification of protein-altering variation, the impact of cis-regulatory mutations can be similarly strong. New technologies are now enabling genome sequencing beyond exomes, revealing variation across the non-coding 98% of the genome responsible for developmental and physiological patterns of gene activity. The capacity to identify causal regulatory mutations is improving, but predicting functional changes in regulatory DNA sequences remains a great challenge. Here we explore the existing methods and software for prediction of functional variation situated in the cis-regulatory sequences governing gene transcription and RNA processing. PMID:21989199

  17. Modeling DNA sequence-based cis-regulatory gene networks.

    PubMed

    Bolouri, Hamid; Davidson, Eric H

    2002-06-01

    Gene network analysis requires computationally based models which represent the functional architecture of regulatory interactions, and which provide directly testable predictions. The type of model that is useful is constrained by the particular features of developmentally active cis-regulatory systems. These systems function by processing diverse regulatory inputs, generating novel regulatory outputs. A computational model which explicitly accommodates this basic concept was developed earlier for the cis-regulatory system of the endo16 gene of the sea urchin. This model represents the genetically mandated logic functions that the system executes, but also shows how time-varying kinetic inputs are processed in different circumstances into particular kinetic outputs. The same basic design features can be utilized to construct models that connect the large number of cis-regulatory elements constituting developmental gene networks. The ultimate aim of the network models discussed here is to represent the regulatory relationships among the genomic control systems of the genes in the network, and to state their functional meaning. The target site sequences of the cis-regulatory elements of these genes constitute the physical basis of the network architecture. Useful models for developmental regulatory networks must represent the genetic logic by which the system operates, but must also be capable of explaining the real time dynamics of cis-regulatory response as kinetic input and output data become available. Most importantly, however, such models must display in a direct and transparent manner fundamental network design features such as intra- and intercellular feedback circuitry; the sources of parallel inputs into each cis-regulatory element; gene battery organization; and use of repressive spatial inputs in specification and boundary formation. Successful network models lead to direct tests of key architectural features by targeted cis-regulatory analysis. PMID

  18. Detection of Weakly Conserved Ancestral Mammalian RegulatorySequences by Primate Comparisons

    SciTech Connect

    Wang, Qian-fei; Prabhakar, Shyam; Chanan, Sumita; Cheng,Jan-Fang; Rubin, Edward M.; Boffelli, Dario

    2006-06-01

    Genomic comparisons between human and distant, non-primatemammals are commonly used to identify cis-regulatory elements based onconstrained sequence evolution. However, these methods fail to detectcryptic functional elements, which are too weakly conserved among mammalsto distinguish from nonfunctional DNA. To address this problem, weexplored the potential of deep intra-primate sequence comparisons. Wesequenced the orthologs of 558 kb of human genomic sequence, coveringmultiple loci involved in cholesterol homeostasis, in 6 nonhumanprimates. Our analysis identified 6 noncoding DNA elements displayingsignificant conservation among primates, but undetectable in more distantcomparisons. In vitro and in vivo tests revealed that at least three ofthese 6 elements have regulatory function. Notably, the mouse orthologsof these three functional human sequences had regulatory activity despitetheir lack of significant sequence conservation, indicating that they arecryptic ancestral cis-regulatory elements. These regulatory elementscould still be detected in a smaller set of three primate speciesincluding human, rhesus and marmoset. Since the human and rhesus genomesequences are already available, and the marmoset genome is activelybeing sequenced, the primate-specific conservation analysis describedhere can be applied in the near future on a whole-genome scale, tocomplement the annotation provided by more distant speciescomparisons.

  19. An evolutionary constraint: strongly disfavored class of change in DNA sequence during divergence of cis-regulatory modules.

    PubMed

    Cameron, R Andrew; Chow, Suk Hen; Berney, Kevin; Chiu, Tsz-Yeung; Yuan, Qiu-Autumn; Krämer, Alexander; Helguero, Argelia; Ransick, Andrew; Yun, Mirong; Davidson, Eric H

    2005-08-16

    The DNA of functional cis-regulatory modules displays extensive sequence conservation in comparisons of genomes from modestly distant species. Patches of sequence that are several hundred base pairs in length within these modules are often seen to be 80-95% identical, although the flanking sequence cannot even be aligned. However, it is unlikely that base pairs located between the transcription factor target sites of cis-regulatory modules have sequence-dependent function, and the mechanism that constrains evolutionary change within cis-regulatory modules is incompletely understood. We chose five functionally characterized cis-regulatory modules from the Strongylocentrotus purpuratus (sea urchin) genome and obtained orthologous regulatory and flanking sequences from a bacterial artificial chromosome genome library of a congener, Strongylocentrotus franciscanus. As expected, single-nucleotide substitutions and small indels occur freely at many positions within the regulatory modules of these two species, as they do outside the regulatory modules. However, large indels (>20 bp) are statistically almost absent within the regulatory modules, although they are common in flanking intergenic or intronic sequence. The result helps to explain the patterns of evolutionary sequence divergence characteristic of cis-regulatory DNA.

  20. Discovery of posttranscriptional regulatory RNAs using next generation sequencing technologies.

    PubMed

    Gelderman, Grant; Contreras, Lydia M

    2013-01-01

    Next generation sequencing (NGS) has revolutionized the way by which we engineer metabolism by radically altering the path to genome-wide inquiries. This is due to the fact that NGS approaches offer several powerful advantages over traditional methods that include the ability to fully sequence hundreds to thousands of genes in a single experiment and simultaneously detect homozygous and heterozygous deletions, alterations in gene copy number, insertions, translocations, and exome-wide substitutions that include "hot-spot mutations." This chapter describes the use of these technologies as a sequencing technique for transcriptome analysis and discovery of regulatory RNA elements in the context of three main platforms: Illumina HiSeq, 454 pyrosequencing, and SOLiD sequencing. Specifically, this chapter focuses on the use of Illumina HiSeq, since it is the most widely used platform for RNA discovery and transcriptome analysis. Regulatory RNAs have now been found in all branches of life. In bacteria, noncoding small RNAs (sRNAs) are involved in highly sophisticated regulatory circuits that include quorum sensing, carbon metabolism, stress responses, and virulence (Gorke and Vogel, Gene Dev 22:2914-2925, 2008; Gottesman, Trends Genet 21:399-404, 2005; Romby et al., Curr Opin Microbiol 9:229-236, 2006). Further characterization of the underlying regulation of gene expression remains poorly understood given that it is estimated that over 60% of all predicted genes remain hypothetical and the 5' and 3' untranslated regions are unknown for more than 90% of the genes (Siegel et al., Trends Parasitol 27:434-441, 2011). Importantly, manipulation of the posttranscriptional regulation that occurs at the level of RNA stability and export, trans-splicing, polyadenylation, protein translation, and protein stability via untranslated regions (Clayton, EMBO J 21:1881-1888, 2002; Haile and Papadopoulou, Curr Opin Microbiol 10:569-577, 2007) could be highly beneficial to metabolic

  1. Enhanced Regulatory Sequence Prediction Using Gapped k-mer Features

    PubMed Central

    Mohammad-Noori, Morteza; Beer, Michael A.

    2014-01-01

    Abstract Oligomers of length k, or k-mers, are convenient and widely used features for modeling the properties and functions of DNA and protein sequences. However, k-mers suffer from the inherent limitation that if the parameter k is increased to resolve longer features, the probability of observing any specific k-mer becomes very small, and k-mer counts approach a binary variable, with most k-mers absent and a few present once. Thus, any statistical learning approach using k-mers as features becomes susceptible to noisy training set k-mer frequencies once k becomes large. To address this problem, we introduce alternative feature sets using gapped k-mers, a new classifier, gkm-SVM, and a general method for robust estimation of k-mer frequencies. To make the method applicable to large-scale genome wide applications, we develop an efficient tree data structure for computing the kernel matrix. We show that compared to our original kmer-SVM and alternative approaches, our gkm-SVM predicts functional genomic regulatory elements and tissue specific enhancers with significantly improved accuracy, increasing the precision by up to a factor of two. We then show that gkm-SVM consistently outperforms kmer-SVM on human ENCODE ChIP-seq datasets, and further demonstrate the general utility of our method using a Naïve-Bayes classifier. Although developed for regulatory sequence analysis, these methods can be applied to any sequence classification problem. PMID:25033408

  2. Mutations of epigenetic regulatory genes are common in thymic carcinomas.

    PubMed

    Wang, Yisong; Thomas, Anish; Lau, Christopher; Rajan, Arun; Zhu, Yuelin; Killian, J Keith; Petrini, Iacopo; Pham, Trung; Morrow, Betsy; Zhong, Xiaogang; Meltzer, Paul S; Giaccone, Giuseppe

    2014-12-08

    Genetic alterations and etiology of thymic epithelial tumors (TETs) are largely unknown, hampering the development of effective targeted therapies for patients with TETs. Here TETs of advanced-stage patients enrolled in a clinical trial of molecularly-guided targeted therapies were employed for targeted sequencing of 197 cancer-associated genes. Comparative sequence analysis of 78 TET/blood paired samples obtained from 47 thymic carcinoma (TC) and 31 thymoma patients revealed a total of 86 somatic non-synonymous sequence variations across 39 different genes in 33 (42%) TETs. TCs (62%; 29/47) showed higher incidence of somatic non-synonymous mutations than thymomas (13%; 4/31; p < 0.0001). TP53 was the most frequently mutated gene in TETs (n = 13; 17%), especially in TCs (26%), and was associated with a poorer overall survival (p < 0.0001). Genes in histone modification [BAP1 (n = 6; 13%), SETD2 (n = 5; 11%), ASXL1 (n = 2; 4%)], chromatin remodeling [SMARCA4 (n = 2; 4%)], and DNA methylation [DNMT3A (n = 3; 7%), TET2 (n = 2; 4%), WT1 (n = 2; 4%)] pathways were recurrently mutated in TCs, but not in thymomas. Our results suggest a potential disruption of epigenetic homeostasis in TCs, and a substantial difference in genetic makeup between TCs and thymomas. Further investigation is warranted into the roles of epigenetic dysregulation in TC development and its potential for targeted therapy.

  3. Mutations of epigenetic regulatory genes are common in thymic carcinomas

    PubMed Central

    Wang, Yisong; Thomas, Anish; Lau, Christopher; Rajan, Arun; Zhu, Yuelin; Killian, J. Keith; Petrini, Iacopo; Pham, Trung; Morrow, Betsy; Zhong, Xiaogang; Meltzer, Paul S.; Giaccone, Giuseppe

    2014-01-01

    Genetic alterations and etiology of thymic epithelial tumors (TETs) are largely unknown, hampering the development of effective targeted therapies for patients with TETs. Here TETs of advanced-stage patients enrolled in a clinical trial of molecularly-guided targeted therapies were employed for targeted sequencing of 197 cancer-associated genes. Comparative sequence analysis of 78 TET/blood paired samples obtained from 47 thymic carcinoma (TC) and 31 thymoma patients revealed a total of 86 somatic non-synonymous sequence variations across 39 different genes in 33 (42%) TETs. TCs (62%; 29/47) showed higher incidence of somatic non-synonymous mutations than thymomas (13%; 4/31; p < 0.0001). TP53 was the most frequently mutated gene in TETs (n = 13; 17%), especially in TCs (26%), and was associated with a poorer overall survival (p < 0.0001). Genes in histone modification [BAP1 (n = 6; 13%), SETD2 (n = 5; 11%), ASXL1 (n = 2; 4%)], chromatin remodeling [SMARCA4 (n = 2; 4%)], and DNA methylation [DNMT3A (n = 3; 7%), TET2 (n = 2; 4%), WT1 (n = 2; 4%)] pathways were recurrently mutated in TCs, but not in thymomas. Our results suggest a potential disruption of epigenetic homeostasis in TCs, and a substantial difference in genetic makeup between TCs and thymomas. Further investigation is warranted into the roles of epigenetic dysregulation in TC development and its potential for targeted therapy. PMID:25482724

  4. Properties of Sequence Conservation in Upstream Regulatory and Protein Coding Sequences among Paralogs in Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Richardson, Dale N.; Wiehe, Thomas

    Whole genome duplication (WGD) has catalyzed the formation of new species, genes with novel functions, altered expression patterns, complexified signaling pathways and has provided organisms a level of genetic robustness. We studied the long-term evolution and interrelationships of 5’ upstream regulatory sequences (URSs), protein coding sequences (CDSs) and expression correlations (EC) of duplicated gene pairs in Arabidopsis. Three distinct methods revealed significant evolutionary conservation between paralogous URSs and were highly correlated with microarray-based expression correlation of the respective gene pairs. Positional information on exact matches between sequences unveiled the contribution of micro-chromosomal rearrangements on expression divergence. A three-way rank analysis of URS similarity, CDS divergence and EC uncovered specific gene functional biases. Transcription factor activity was associated with gene pairs exhibiting conserved URSs and divergent CDSs, whereas a broad array of metabolic enzymes was found to be associated with gene pairs showing diverged URSs but conserved CDSs.

  5. Finding the most significant common sequence and structure motifs in a set of RNA sequences.

    PubMed Central

    Gorodkin, J; Heyer, L J; Stormo, G D

    1997-01-01

    We present a computational scheme to locally align a collection of RNA sequences using sequence and structure constraints. In addition, the method searches for the resulting alignments with the most significant common motifs, among all possible collections. The first part utilizes a simplified version of the Sankoff algorithm for simultaneous folding and alignment of RNA sequences, but maintains tractability by constructing multi-sequence alignments from pairwise comparisons. The algorithm finds the multiple alignments using a greedy approach and has similarities to both CLUSTAL and CONSENSUS, but the core algorithm assures that the pairwise alignments are optimized for both sequence and structure conservation. The choice of scoring system and the method of progressively constructing the final solution are important considerations that are discussed. Example solutions, and comparisons with other approaches, are provided. The solutions include finding consensus structures identical to published ones. PMID:9278497

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

    NASA Astrophysics Data System (ADS)

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

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

  7. Nucleotide sequence and temporal expression of a baculovirus regulatory gene.

    PubMed

    Guarino, L A; Summers, M D

    1987-07-01

    The nucleotide sequence of a trans-activating regulatory gene (IE-1) of the baculovirus Autographa californica nuclear polyhedrosis virus has been determined. This gene encodes a protein of 581 amino acids with a predicted molecular weight of 66,856. A DNA fragment containing the entire coding sequence of IE-1 was inserted downstream of an RNA promoter. Subsequent cell-free transcription and translation directed the synthesis of a single peptide with an apparent molecular weight of 70,000. Quantitative S1 nuclease analysis indicated that IE-1 was maximally synthesized during a 1-h virus adsorption period and that steady-state levels of IE-1 message were maintained during the first 24 h of infection. Northern blot hybridization indicated that several late transcripts which overlap the IE-1 gene were transcribed from both strands. The precise locations of the 5' and 3' ends of these overlapping transcripts were mapped using S1 nuclease. The overlapping transcripts were grouped in two transcriptional units. One unit was composed of IE-1 and overlapping gamma transcripts which initiated upstream of IE-1 and terminated downstream of IE-1. The other unit, transcribed from the opposite strand, consisted of gamma transcripts with coterminal 5' ends and extended 3' ends. The shorter, more abundant transcripts in this unit overlapped 30 to 40 bases of IE-1 at the 3' end, while the longer transcripts overlapped the entire IE-1 gene. Transcription of several early A. californica nuclear polyhedrosis virus genes, in addition to 39K, was shown to be trans-activated by IE-1, indicating that IE-1 may have a central role in the regulation of beta-gene expression. PMID:16789264

  8. Massive contribution of transposable elements to mammalian regulatory sequences.

    PubMed

    Rayan, Nirmala Arul; Del Rosario, Ricardo C H; Prabhakar, Shyam

    2016-09-01

    Barbara McClintock discovered the existence of transposable elements (TEs) in the late 1940s and initially proposed that they contributed to the gene regulatory program of higher organisms. This controversial idea gained acceptance only much later in the 1990s, when the first examples of TE-derived promoter sequences were uncovered. It is now known that half of the human genome is recognizably derived from TEs. It is thus important to understand the scope and nature of their contribution to gene regulation. Here, we provide a timeline of major discoveries in this area and discuss how transposons have revolutionized our understanding of mammalian genomes, with a special emphasis on the massive contribution of TEs to primate evolution. Our analysis of primate-specific functional elements supports a simple model for the rate at which new functional elements arise in unique and TE-derived DNA. Finally, we discuss some of the challenges and unresolved questions in the field, which need to be addressed in order to fully characterize the impact of TEs on gene regulation, evolution and disease processes. PMID:27174439

  9. Massive contribution of transposable elements to mammalian regulatory sequences.

    PubMed

    Rayan, Nirmala Arul; Del Rosario, Ricardo C H; Prabhakar, Shyam

    2016-09-01

    Barbara McClintock discovered the existence of transposable elements (TEs) in the late 1940s and initially proposed that they contributed to the gene regulatory program of higher organisms. This controversial idea gained acceptance only much later in the 1990s, when the first examples of TE-derived promoter sequences were uncovered. It is now known that half of the human genome is recognizably derived from TEs. It is thus important to understand the scope and nature of their contribution to gene regulation. Here, we provide a timeline of major discoveries in this area and discuss how transposons have revolutionized our understanding of mammalian genomes, with a special emphasis on the massive contribution of TEs to primate evolution. Our analysis of primate-specific functional elements supports a simple model for the rate at which new functional elements arise in unique and TE-derived DNA. Finally, we discuss some of the challenges and unresolved questions in the field, which need to be addressed in order to fully characterize the impact of TEs on gene regulation, evolution and disease processes.

  10. Sequence similarity network reveals common ancestry of multidomain proteins.

    PubMed

    Song, Nan; Joseph, Jacob M; Davis, George B; Durand, Dannie

    2008-04-01

    We address the problem of homology identification in complex multidomain families with varied domain architectures. The challenge is to distinguish sequence pairs that share common ancestry from pairs that share an inserted domain but are otherwise unrelated. This distinction is essential for accuracy in gene annotation, function prediction, and comparative genomics. There are two major obstacles to multidomain homology identification: lack of a formal definition and lack of curated benchmarks for evaluating the performance of new methods. We offer preliminary solutions to both problems: 1) an extension of the traditional model of homology to include domain insertions; and 2) a manually curated benchmark of well-studied families in mouse and human. We further present Neighborhood Correlation, a novel method that exploits the local structure of the sequence similarity network to identify homologs with great accuracy based on the observation that gene duplication and domain shuffling leave distinct patterns in the sequence similarity network. In a rigorous, empirical comparison using our curated data, Neighborhood Correlation outperforms sequence similarity, alignment length, and domain architecture comparison. Neighborhood Correlation is well suited for automated, genome-scale analyses. It is easy to compute, does not require explicit knowledge of domain architecture, and classifies both single and multidomain homologs with high accuracy. Homolog predictions obtained with our method, as well as our manually curated benchmark and a web-based visualization tool for exploratory analysis of the network neighborhood structure, are available at http://www.neighborhoodcorrelation.org. Our work represents a departure from the prevailing view that the concept of homology cannot be applied to genes that have undergone domain shuffling. In contrast to current approaches that either focus on the homology of individual domains or consider only families with identical domain

  11. Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences.

    PubMed

    De Jaeger, Geert; Scheffer, Stanley; Jacobs, Anni; Zambre, Mukund; Zobell, Oliver; Goossens, Alain; Depicker, Ann; Angenon, Geert

    2002-12-01

    Over the past decade, several high value proteins have been produced in different transgenic plant tissues such as leaves, tubers, and seeds. Despite recent advances, many heterologous proteins accumulate to low concentrations, and the optimization of expression cassettes to make in planta production and purification economically feasible remains critical. Here, the regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) of common bean (Phaseolus vulgaris) were evaluated for producing heterologous proteins in dicotyledonous seeds. The murine single chain variable fragment (scFv) G4 (ref. 4) was chosen as model protein because of the current industrial interest in producing antibodies and derived fragments in crops. In transgenic Arabidopsis thaliana seed stocks, the scFv under control of the 35S promoter of the cauliflower mosaic virus (CaMV) accumulated to approximately 1% of total soluble protein (TSP). However, a set of seed storage promoter constructs boosted the scFv accumulation to exceptionally high concentrations, reaching no less than 36.5% of TSP in homozygous seeds. Even at these high concentrations, the scFv proteins had antigen-binding activity and affinity similar to those produced in Escherichia coli. The feasibility of heterologous protein production under control of arc5-I regulatory sequences was also demonstrated in Phaseolus acutifolius, a promising crop for large scale production.

  12. Genetic Diagnosis Using Whole Exome Sequencing in Common Variable Immunodeficiency

    PubMed Central

    Maffucci, Patrick; Filion, Charles A.; Boisson, Bertrand; Itan, Yuval; Shang, Lei; Casanova, Jean-Laurent; Cunningham-Rundles, Charlotte

    2016-01-01

    Whole exome sequencing (WES) has proven an effective tool for the discovery of genetic defects in patients with primary immunodeficiencies (PIDs). However, success in dissecting the genetic etiology of common variable immunodeficiency (CVID) has been limited. We outline a practical framework for using WES to identify causative genetic defects in these subjects. WES was performed on 50 subjects diagnosed with CVID who had at least one of the following criteria: early onset, autoimmune/inflammatory manifestations, low B lymphocytes, and/or familial history of hypogammaglobulinemia. Following alignment and variant calling, exomes were screened for mutations in 269 PID-causing genes. Variants were filtered based on the mode of inheritance and reported frequency in the general population. Each variant was assessed by study of familial segregation and computational predictions of deleteriousness. Out of 433 variations in PID-associated genes, we identified 17 probable disease-causing mutations in 15 patients (30%). These variations were rare or private and included monoallelic mutations in NFKB1, STAT3, CTLA4, PIK3CD, and IKZF1, and biallelic mutations in LRBA and STXBP2. Forty-two other damaging variants were found but were not considered likely disease-causing based on the mode of inheritance and/or patient phenotype. WES combined with analysis of PID-associated genes is a cost-effective approach to identify disease-causing mutations in CVID patients with severe phenotypes and was successful in 30% of our cohort. As targeted therapeutics are becoming the mainstay of treatment for non-infectious manifestations in CVID, this approach will improve management of patients with more severe phenotypes. PMID:27379089

  13. Genetic Diagnosis Using Whole Exome Sequencing in Common Variable Immunodeficiency.

    PubMed

    Maffucci, Patrick; Filion, Charles A; Boisson, Bertrand; Itan, Yuval; Shang, Lei; Casanova, Jean-Laurent; Cunningham-Rundles, Charlotte

    2016-01-01

    Whole exome sequencing (WES) has proven an effective tool for the discovery of genetic defects in patients with primary immunodeficiencies (PIDs). However, success in dissecting the genetic etiology of common variable immunodeficiency (CVID) has been limited. We outline a practical framework for using WES to identify causative genetic defects in these subjects. WES was performed on 50 subjects diagnosed with CVID who had at least one of the following criteria: early onset, autoimmune/inflammatory manifestations, low B lymphocytes, and/or familial history of hypogammaglobulinemia. Following alignment and variant calling, exomes were screened for mutations in 269 PID-causing genes. Variants were filtered based on the mode of inheritance and reported frequency in the general population. Each variant was assessed by study of familial segregation and computational predictions of deleteriousness. Out of 433 variations in PID-associated genes, we identified 17 probable disease-causing mutations in 15 patients (30%). These variations were rare or private and included monoallelic mutations in NFKB1, STAT3, CTLA4, PIK3CD, and IKZF1, and biallelic mutations in LRBA and STXBP2. Forty-two other damaging variants were found but were not considered likely disease-causing based on the mode of inheritance and/or patient phenotype. WES combined with analysis of PID-associated genes is a cost-effective approach to identify disease-causing mutations in CVID patients with severe phenotypes and was successful in 30% of our cohort. As targeted therapeutics are becoming the mainstay of treatment for non-infectious manifestations in CVID, this approach will improve management of patients with more severe phenotypes. PMID:27379089

  14. Sequence-based model of gap gene regulatory network

    PubMed Central

    2014-01-01

    Background The detailed analysis of transcriptional regulation is crucially important for understanding biological processes. The gap gene network in Drosophila attracts large interest among researches studying mechanisms of transcriptional regulation. It implements the most upstream regulatory layer of the segmentation gene network. The knowledge of molecular mechanisms involved in gap gene regulation is far less complete than that of genetics of the system. Mathematical modeling goes beyond insights gained by genetics and molecular approaches. It allows us to reconstruct wild-type gene expression patterns in silico, infer underlying regulatory mechanism and prove its sufficiency. Results We developed a new model that provides a dynamical description of gap gene regulatory systems, using detailed DNA-based information, as well as spatial transcription factor concentration data at varying time points. We showed that this model correctly reproduces gap gene expression patterns in wild type embryos and is able to predict gap expression patterns in Kr mutants and four reporter constructs. We used four-fold cross validation test and fitting to random dataset to validate the model and proof its sufficiency in data description. The identifiability analysis showed that most model parameters are well identifiable. We reconstructed the gap gene network topology and studied the impact of individual transcription factor binding sites on the model output. We measured this impact by calculating the site regulatory weight as a normalized difference between the residual sum of squares error for the set of all annotated sites and for the set with the site of interest excluded. Conclusions The reconstructed topology of the gap gene network is in agreement with previous modeling results and data from literature. We showed that 1) the regulatory weights of transcription factor binding sites show very weak correlation with their PWM score; 2) sites with low regulatory weight are

  15. Genome-Wide Identification of Regulatory Sequences Undergoing Accelerated Evolution in the Human Genome

    PubMed Central

    Dong, Xinran; Wang, Xiao; Zhang, Feng; Tian, Weidong

    2016-01-01

    Accelerated evolution of regulatory sequence can alter the expression pattern of target genes, and cause phenotypic changes. In this study, we used DNase I hypersensitive sites (DHSs) to annotate putative regulatory sequences in the human genome, and conducted a genome-wide analysis of the effects of accelerated evolution on regulatory sequences. Working under the assumption that local ancient repeat elements of DHSs are under neutral evolution, we discovered that ∼0.44% of DHSs are under accelerated evolution (ace-DHSs). We found that ace-DHSs tend to be more active than background DHSs, and are strongly associated with epigenetic marks of active transcription. The target genes of ace-DHSs are significantly enriched in neuron-related functions, and their expression levels are positively selected in the human brain. Thus, these lines of evidences strongly suggest that accelerated evolution on regulatory sequences plays important role in the evolution of human-specific phenotypes. PMID:27401230

  16. Genome-Wide Identification of Regulatory Sequences Undergoing Accelerated Evolution in the Human Genome.

    PubMed

    Dong, Xinran; Wang, Xiao; Zhang, Feng; Tian, Weidong

    2016-10-01

    Accelerated evolution of regulatory sequence can alter the expression pattern of target genes, and cause phenotypic changes. In this study, we used DNase I hypersensitive sites (DHSs) to annotate putative regulatory sequences in the human genome, and conducted a genome-wide analysis of the effects of accelerated evolution on regulatory sequences. Working under the assumption that local ancient repeat elements of DHSs are under neutral evolution, we discovered that ∼0.44% of DHSs are under accelerated evolution (ace-DHSs). We found that ace-DHSs tend to be more active than background DHSs, and are strongly associated with epigenetic marks of active transcription. The target genes of ace-DHSs are significantly enriched in neuron-related functions, and their expression levels are positively selected in the human brain. Thus, these lines of evidences strongly suggest that accelerated evolution on regulatory sequences plays important role in the evolution of human-specific phenotypes.

  17. On the Concept of Cis-regulatory Information: From Sequence Motifs to Logic Functions

    NASA Astrophysics Data System (ADS)

    Tarpine, Ryan; Istrail, Sorin

    The regulatory genome is about the “system level organization of the core genomic regulatory apparatus, and how this is the locus of causality underlying the twin phenomena of animal development and animal evolution” (E.H. Davidson. The Regulatory Genome: Gene Regulatory Networks in Development and Evolution, Academic Press, 2006). Information processing in the regulatory genome is done through regulatory states, defined as sets of transcription factors (sequence-specific DNA binding proteins which determine gene expression) that are expressed and active at the same time. The core information processing machinery consists of modular DNA sequence elements, called cis-modules, that interact with transcription factors. The cis-modules “read” the information contained in the regulatory state of the cell through transcription factor binding, “process” it, and directly or indirectly communicate with the basal transcription apparatus to determine gene expression. This endowment of each gene with the information-receiving capacity through their cis-regulatory modules is essential for the response to every possible regulatory state to which it might be exposed during all phases of the life cycle and in all cell types. We present here a set of challenges addressed by our CYRENE research project aimed at studying the cis-regulatory code of the regulatory genome. The CYRENE Project is devoted to (1) the construction of a database, the cis-Lexicon, containing comprehensive information across species about experimentally validated cis-regulatory modules; and (2) the software development of a next-generation genome browser, the cis-Browser, specialized for the regulatory genome. The presentation is anchored on three main computational challenges: the Gene Naming Problem, the Consensus Sequence Bottleneck Problem, and the Logic Function Inference Problem.

  18. PAZAR: a framework for collection and dissemination of cis-regulatory sequence annotation

    PubMed Central

    Portales-Casamar, Elodie; Kirov, Stefan; Lim, Jonathan; Lithwick, Stuart; Swanson, Magdalena I; Ticoll, Amy; Snoddy, Jay; Wasserman, Wyeth W

    2007-01-01

    PAZAR is an open-access and open-source database of transcription factor and regulatory sequence annotation with associated web interface and programming tools for data submission and extraction. Curated boutique data collections can be maintained and disseminated through the unified schema of the mall-like PAZAR repository. The Pleiades Promoter Project collection of brain-linked regulatory sequences is introduced to demonstrate the depth of annotation possible within PAZAR. PAZAR, located at , is open for business. PMID:17916232

  19. The genetic architecture of autism spectrum disorders (ASDs) and the potential importance of common regulatory genetic variants.

    PubMed

    Saffen, David

    2015-10-01

    Currently, there is great interest in identifying genetic variants that contribute to the risk of developing autism spectrum disorders (ASDs), due in part to recent increases in the frequency of diagnosis of these disorders worldwide. While there is nearly universal agreement that ASDs are complex diseases, with multiple genetic and environmental contributing factors, there is less agreement concerning the relative importance of common vs rare genetic variants in ASD liability. Recent observations that rare mutations and copy number variants (CNVs) are frequently associated with ASDs, combined with reduced fecundity of individuals with these disorders, has led to the hypothesis that ASDs are caused primarily by de novo or rare genetic mutations. Based on this model, large-scale whole-genome DNA sequencing has been proposed as the most appropriate method for discovering ASD liability genes. While this approach will undoubtedly identify many novel candidate genes and produce important new insights concerning the genetic causes of these disorders, a full accounting of the genetics of ASDs will be incomplete absent an understanding of the contributions of common regulatory variants, which are likely to influence ASD liability by modifying the effects of rare variants or, by assuming unfavorable combinations, directly produce these disorders. Because it is not yet possible to identify regulatory genetic variants by examination of DNA sequences alone, their identification will require experimentation. In this essay, I discuss these issues and describe the advantages of measurements of allelic expression imbalance (AEI) of mRNA expression for identifying cis-acting regulatory variants that contribute to ASDs. PMID:26335735

  20. Molecular sled sequences are common in mammalian proteins

    PubMed Central

    Xiong, Kan; Blainey, Paul C.

    2016-01-01

    Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins. PMID:26857546

  1. Molecular sled sequences are common in mammalian proteins.

    PubMed

    Xiong, Kan; Blainey, Paul C

    2016-03-18

    Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins. PMID:26857546

  2. RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences.

    PubMed

    Brule, C E; Dean, K M; Grayhack, E J

    2016-01-01

    The identification and analysis of sequences that regulate gene expression is critical because regulated gene expression underlies biology. RNA-ID is an efficient and sensitive method to discover and investigate regulatory sequences in the yeast Saccharomyces cerevisiae, using fluorescence-based assays to detect green fluorescent protein (GFP) relative to a red fluorescent protein (RFP) control in individual cells. Putative regulatory sequences can be inserted either in-frame or upstream of a superfolder GFP fusion protein whose expression, like that of RFP, is driven by the bidirectional GAL1,10 promoter. In this chapter, we describe the methodology to identify and study cis-regulatory sequences in the RNA-ID system, explaining features and variations of the RNA-ID reporter, as well as some applications of this system. We describe in detail the methods to analyze a single regulatory sequence, from construction of a single GFP variant to assay of variants by flow cytometry, as well as modifications required to screen libraries of different strains simultaneously. We also describe subsequent analyses of regulatory sequences.

  3. Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences

    NASA Technical Reports Server (NTRS)

    Li, X.; Eastman, E. M.; Schwartz, R. J.; Draghia-Akli, R.

    1999-01-01

    Relatively low levels of expression from naturally occurring promoters have limited the use of muscle as a gene therapy target. Myogenic restricted gene promoters display complex organization usually involving combinations of several myogenic regulatory elements. By random assembly of E-box, MEF-2, TEF-1, and SRE sites into synthetic promoter recombinant libraries, and screening of hundreds of individual clones for transcriptional activity in vitro and in vivo, several artificial promoters were isolated whose transcriptional potencies greatly exceed those of natural myogenic and viral gene promoters.

  4. Sequence evidence for common ancestry of eukaryotic endomembrane coatomers.

    PubMed

    Promponas, Vasilis J; Katsani, Katerina R; Blencowe, Benjamin J; Ouzounis, Christos A

    2016-03-02

    Eukaryotic cells are defined by compartments through which the trafficking of macromolecules is mediated by large complexes, such as the nuclear pore, transport vesicles and intraflagellar transport. The assembly and maintenance of these complexes is facilitated by endomembrane coatomers, long suspected to be divergently related on the basis of structural and more recently phylogenomic analysis. By performing supervised walks in sequence space across coatomer superfamilies, we uncover subtle sequence patterns that have remained elusive to date, ultimately unifying eukaryotic coatomers by divergent evolution. The conserved residues shared by 3,502 endomembrane coatomer components are mapped onto the solenoid superhelix of nucleoporin and COPII protein structures, thus determining the invariant elements of coatomer architecture. This ancient structural motif can be considered as a universal signature connecting eukaryotic coatomers involved in multiple cellular processes across cell physiology and human disease.

  5. Sequence evidence for common ancestry of eukaryotic endomembrane coatomers.

    PubMed

    Promponas, Vasilis J; Katsani, Katerina R; Blencowe, Benjamin J; Ouzounis, Christos A

    2016-01-01

    Eukaryotic cells are defined by compartments through which the trafficking of macromolecules is mediated by large complexes, such as the nuclear pore, transport vesicles and intraflagellar transport. The assembly and maintenance of these complexes is facilitated by endomembrane coatomers, long suspected to be divergently related on the basis of structural and more recently phylogenomic analysis. By performing supervised walks in sequence space across coatomer superfamilies, we uncover subtle sequence patterns that have remained elusive to date, ultimately unifying eukaryotic coatomers by divergent evolution. The conserved residues shared by 3,502 endomembrane coatomer components are mapped onto the solenoid superhelix of nucleoporin and COPII protein structures, thus determining the invariant elements of coatomer architecture. This ancient structural motif can be considered as a universal signature connecting eukaryotic coatomers involved in multiple cellular processes across cell physiology and human disease. PMID:26931514

  6. Sequence evidence for common ancestry of eukaryotic endomembrane coatomers

    PubMed Central

    Promponas, Vasilis J.; Katsani, Katerina R.; Blencowe, Benjamin J.; Ouzounis, Christos A.

    2016-01-01

    Eukaryotic cells are defined by compartments through which the trafficking of macromolecules is mediated by large complexes, such as the nuclear pore, transport vesicles and intraflagellar transport. The assembly and maintenance of these complexes is facilitated by endomembrane coatomers, long suspected to be divergently related on the basis of structural and more recently phylogenomic analysis. By performing supervised walks in sequence space across coatomer superfamilies, we uncover subtle sequence patterns that have remained elusive to date, ultimately unifying eukaryotic coatomers by divergent evolution. The conserved residues shared by 3,502 endomembrane coatomer components are mapped onto the solenoid superhelix of nucleoporin and COPII protein structures, thus determining the invariant elements of coatomer architecture. This ancient structural motif can be considered as a universal signature connecting eukaryotic coatomers involved in multiple cellular processes across cell physiology and human disease. PMID:26931514

  7. Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence

    PubMed Central

    Gordon, Kacy L.; Arthur, Robert K.; Ruvinsky, Ilya

    2015-01-01

    Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements. PMID:26020930

  8. Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence.

    PubMed

    Gordon, Kacy L; Arthur, Robert K; Ruvinsky, Ilya

    2015-05-01

    Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements.

  9. Poster — Thur Eve — 50: Common Regulatory Non-Compliances and How to Avoid Them

    SciTech Connect

    Heimann, M.

    2014-08-15

    The Accelerators and Class II Facilities Division (ACFD) of the Canadian Nuclear Safety Commission (CNSC), is responsible for the oversight of radiotherapy facilities containing Class II prescribed equipment in Canada. Over the past several years, ACFD has been performing compliance inspections of Class II nuclear facilities across the country (medical and otherwise), and in that time, has issued several hundred corrective actions to licensees due to non-compliance with regulatory requirements. Recently, a study was done to determine the most common regulatory non-compliances. The purpose of this poster presentation is to disseminate information to the licensee community about the nature of these non-compliances, and how they can be avoided by licensees in the future.

  10. COMMON WARM DUST TEMPERATURES AROUND MAIN-SEQUENCE STARS

    SciTech Connect

    Morales, Farisa Y.; Werner, M. W.; Bryden, G.; Stapelfeldt, K. R.; Rieke, G. H.; Su, K. Y. L.

    2011-04-01

    We compare the properties of warm dust emission from a sample of main-sequence A-type stars (B8-A7) to those of dust around solar-type stars (F5-K0) with similar Spitzer Space Telescope Infrared Spectrograph/MIPS data and similar ages. Both samples include stars with sources with infrared spectral energy distributions that show evidence of multiple components. Over the range of stellar types considered, we obtain nearly the same characteristic dust temperatures ({approx}190 K and {approx}60 K for the inner and outer dust components, respectively)-slightly above the ice evaporation temperature for the inner belts. The warm inner dust temperature is readily explained if populations of small grains are being released by sublimation of ice from icy planetesimals. Evaporation of low-eccentricity icy bodies at {approx}150 K can deposit particles into an inner/warm belt, where the small grains are heated to T{sub dust} {approx} 190 K. Alternatively, enhanced collisional processing of an asteroid belt-like system of parent planetesimals just interior to the snow line may account for the observed uniformity in dust temperature. The similarity in temperature of the warmer dust across our B8-K0 stellar sample strongly suggests that dust-producing planetesimals are not found at similar radial locations around all stars, but that dust production is favored at a characteristic temperature horizon.

  11. The Intolerance of Regulatory Sequence to Genetic Variation Predicts Gene Dosage Sensitivity

    PubMed Central

    Wang, Quanli; Halvorsen, Matt; Han, Yujun; Weir, William H.; Allen, Andrew S.; Goldstein, David B.

    2015-01-01

    Noncoding sequence contains pathogenic mutations. Yet, compared with mutations in protein-coding sequence, pathogenic regulatory mutations are notoriously difficult to recognize. Most fundamentally, we are not yet adept at recognizing the sequence stretches in the human genome that are most important in regulating the expression of genes. For this reason, it is difficult to apply to the regulatory regions the same kinds of analytical paradigms that are being successfully applied to identify mutations among protein-coding regions that influence risk. To determine whether dosage sensitive genes have distinct patterns among their noncoding sequence, we present two primary approaches that focus solely on a gene’s proximal noncoding regulatory sequence. The first approach is a regulatory sequence analogue of the recently introduced residual variation intolerance score (RVIS), termed noncoding RVIS, or ncRVIS. The ncRVIS compares observed and predicted levels of standing variation in the regulatory sequence of human genes. The second approach, termed ncGERP, reflects the phylogenetic conservation of a gene’s regulatory sequence using GERP++. We assess how well these two approaches correlate with four gene lists that use different ways to identify genes known or likely to cause disease through changes in expression: 1) genes that are known to cause disease through haploinsufficiency, 2) genes curated as dosage sensitive in ClinGen’s Genome Dosage Map, 3) genes judged likely to be under purifying selection for mutations that change expression levels because they are statistically depleted of loss-of-function variants in the general population, and 4) genes judged unlikely to cause disease based on the presence of copy number variants in the general population. We find that both noncoding scores are highly predictive of dosage sensitivity using any of these criteria. In a similar way to ncGERP, we assess two ensemble-based predictors of regional noncoding importance

  12. [Identification and mapping of cis-regulatory elements within long genomic sequences].

    PubMed

    Akopov, S B; Chernov, I P; Vetchinova, A S; Bulanenkova, S S; Nikolaev, L G

    2007-01-01

    The publication of the human and other metazoan genome sequences opened up the possibility for mapping and analysis of genomic regulatory elements. Unfortunately, experimental data on genomic positions of such sequences as enhancers, silencers, insulators, transcription terminators, and replication origins are very limited, especially at the whole genome level. As most genomic regulatory elements (e.g., enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements in silico is often ambiguous. Therefore, the development of high-throughput experimental approaches for identification and mapping of genomic functional elements is highly desirable. In this review we discuss novel approaches to high-throughput experimental identification of mammalian genomes cis-regulatory elements which is a necessary step toward the complete genome annotation. PMID:18240562

  13. Multiple Cis-Acting Sequences Contribute to Evolved Regulatory Variation for Drosophila Adh Genes

    PubMed Central

    Fang, X. M.; Brennan, M. D.

    1992-01-01

    Drosophila affinidisjuncta and Drosophila hawaiiensis are closely related species that display distinct tissue-specific expression patterns for their homologous alcohol dehydrogenase genes (Adh genes). In Drosophila melanogaster transformants, both genes are expressed at high levels in the larval and adult fat bodies, but the D. affinidisjuncta gene is expressed 10-50-fold more strongly in the larval and adult midguts and Malpighian tubules. The present study reports the mapping of cis-acting sequences contributing to the regulatory differences between these two genes in transformants. Chimeric genes were constructed and introduced into the germ line of D. melanogaster. Stage- and tissue-specific expression patterns were determined by measuring steady-state RNA levels in larvae and adults. Three portions of the promoter region make distinct contributions to the tissue-specific regulatory differences between the native genes. Sequences immediately upstream of the distal promoter have a strong effect in the adult Malpighian tubules, while sequences between the two promoters are relatively important in the larval Malpighian tubules. A third gene segment, immediately upstream of the proximal promoter, influences levels of the proximal Adh transcript in all tissues and developmental stages examined, and largely accounts for the regulatory difference in the larval and adult midguts. However, these as well as other sequences make smaller contributions to various aspects of the tissue-specific regulatory differences. In addition, some chimeric genes display aberrant RNA levels for the whole organism, suggesting close physical association between sequences involved in tissue-specific regulatory differences and those important for Adh expression in the larval and adult fat bodies. PMID:1644276

  14. Close Sequence Comparisons are Sufficient to Identify Humancis-Regulatory Elements

    SciTech Connect

    Prabhakar, Shyam; Poulin, Francis; Shoukry, Malak; Afzal, Veena; Rubin, Edward M.; Couronne, Olivier; Pennacchio, Len A.

    2005-12-01

    Cross-species DNA sequence comparison is the primary method used to identify functional noncoding elements in human and other large genomes. However, little is known about the relative merits of evolutionarily close and distant sequence comparisons, due to the lack of a universal metric for sequence conservation, and also the paucity of empirically defined benchmark sets of cis-regulatory elements. To address this problem, we developed a general-purpose algorithm (Gumby) that detects slowly-evolving regions in primate, mammalian and more distant comparisons without requiring adjustment of parameters, and ranks conserved elements by P-value using Karlin-Altschul statistics. We benchmarked Gumby predictions against previously identified cis-regulatory elements at diverse genomic loci, and also tested numerous extremely conserved human-rodent sequences for transcriptional enhancer activity using reporter-gene assays in transgenic mice. Human regulatory elements were identified with acceptable sensitivity and specificity by comparison with 1-5 other eutherian mammals or 6 other simian primates. More distant comparisons (marsupial, avian, amphibian and fish) failed to identify many of the empirically defined functional noncoding elements. We derived an intuitive relationship between ancient and recent noncoding sequence conservation from whole genome comparative analysis, which explains some of these findings. Lastly, we determined that, in addition to strength of conservation, genomic location and/or density of surrounding conserved elements must also be considered in selecting candidate enhancers for testing at embryonic time points.

  15. INCLUSive: a web portal and service registry for microarray and regulatory sequence analysis

    PubMed Central

    Coessens, Bert; Thijs, Gert; Aerts, Stein; Marchal, Kathleen; De Smet, Frank; Engelen, Kristof; Glenisson, Patrick; Moreau, Yves; Mathys, Janick; De Moor, Bart

    2003-01-01

    INCLUSive is a suite of algorithms and tools for the analysis of gene expression data and the discovery of cis-regulatory sequence elements. The tools allow normalization, filtering and clustering of microarray data, functional scoring of gene clusters, sequence retrieval, and detection of known and unknown regulatory elements using probabilistic sequence models and Gibbs sampling. All tools are available via different web pages and as web services. The web pages are connected and integrated to reflect a methodology and facilitate complex analysis using different tools. The web services can be invoked using standard SOAP messaging. Example clients are available for download to invoke the services from a remote computer or to be integrated with other applications. All services are catalogued and described in a web service registry. The INCLUSive web portal is available for academic purposes at http://www.esat.kuleuven.ac.be/inclusive. PMID:12824346

  16. Using RSAT to scan genome sequences for transcription factor binding sites and cis-regulatory modules.

    PubMed

    Turatsinze, Jean-Valery; Thomas-Chollier, Morgane; Defrance, Matthieu; van Helden, Jacques

    2008-01-01

    This protocol shows how to detect putative cis-regulatory elements and regions enriched in such elements with the regulatory sequence analysis tools (RSAT) web server (http://rsat.ulb.ac.be/rsat/). The approach applies to known transcription factors, whose binding specificity is represented by position-specific scoring matrices, using the program matrix-scan. The detection of individual binding sites is known to return many false predictions. However, results can be strongly improved by estimating P value, and by searching for combinations of sites (homotypic and heterotypic models). We illustrate the detection of sites and enriched regions with a study case, the upstream sequence of the Drosophila melanogaster gene even-skipped. This protocol is also tested on random control sequences to evaluate the reliability of the predictions. Each task requires a few minutes of computation time on the server. The complete protocol can be executed in about one hour.

  17. Anti-Sigma Factors in E. coli: Common Regulatory Mechanisms Controlling Sigma Factors Availability

    PubMed Central

    Treviño-Quintanilla, Luis Gerardo; Freyre-González, Julio Augusto; Martínez-Flores, Irma

    2013-01-01

    In bacteria, transcriptional regulation is a key step in cellular gene expression. All bacteria contain a core RNA polymerase that is catalytically competent but requires an additional σ factor for specific promoter recognition and correct transcriptional initiation. The RNAP core is not able to selectively bind to a given σ factor. In contrast, different σ factors have different affinities for the RNAP core. As a consequence, the concentration of alternate σ factors requires strict regulation in order to properly control the delicate interplay among them, which favors the competence for the RNAP core. This control is archived by different σ/anti-σ controlling mechanisms that shape complex regulatory networks and cascades, and enable the response to sudden environmental cues, whose global understanding is a current challenge for systems biology. Although there have been a number of excellent studies on each of these σ/anti-σ post-transcriptional regulatory systems, no comprehensive comparison of these mechanisms in a single model organism has been conducted. Here, we survey all these systems in E. coli dissecting and analyzing their inner workings and highlightin their differences. Then, following an integral approach, we identify their commonalities and outline some of the principles exploited by the cell to effectively and globally reprogram the transcriptional machinery. These principles provide guidelines for developing biological synthetic circuits enabling an efficient and robust response to sudden stimuli. PMID:24396271

  18. TOUCAN 2: the all-inclusive open source workbench for regulatory sequence analysis

    PubMed Central

    Aerts, Stein; Van Loo, Peter; Thijs, Gert; Mayer, Herbert; de Martin, Rainer; Moreau, Yves; De Moor, Bart

    2005-01-01

    We present the second and improved release of the TOUCAN workbench for cis-regulatory sequence analysis. TOUCAN implements and integrates fast state-of-the-art methods and strategies in gene regulation bioinformatics, including algorithms for comparative genomics and for the detection of cis-regulatory modules. This second release of TOUCAN has become open source and thereby carries the potential to evolve rapidly. The main goal of TOUCAN is to allow a user to come to testable hypotheses regarding the regulation of a gene or of a set of co-regulated genes. TOUCAN can be launched from this location: . PMID:15980497

  19. BayesPI-BAR: a new biophysical model for characterization of regulatory sequence variations.

    PubMed

    Wang, Junbai; Batmanov, Kirill

    2015-12-01

    Sequence variations in regulatory DNA regions are known to cause functionally important consequences for gene expression. DNA sequence variations may have an essential role in determining phenotypes and may be linked to disease; however, their identification through analysis of massive genome-wide sequencing data is a great challenge. In this work, a new computational pipeline, a Bayesian method for protein-DNA interaction with binding affinity ranking (BayesPI-BAR), is proposed for quantifying the effect of sequence variations on protein binding. BayesPI-BAR uses biophysical modeling of protein-DNA interactions to predict single nucleotide polymorphisms (SNPs) that cause significant changes in the binding affinity of a regulatory region for transcription factors (TFs). The method includes two new parameters (TF chemical potentials or protein concentrations and direct TF binding targets) that are neglected by previous methods. The new method is verified on 67 known human regulatory SNPs, of which 47 (70%) have predicted true TFs ranked in the top 10. Importantly, the performance of BayesPI-BAR, which uses principal component analysis to integrate multiple predictions from various TF chemical potentials, is found to be better than that of existing programs, such as sTRAP and is-rSNP, when evaluated on the same SNPs. BayesPI-BAR is a publicly available tool and is able to carry out parallelized computation, which helps to investigate a large number of TFs or SNPs and to detect disease-associated regulatory sequence variations in the sea of genome-wide noncoding regions. PMID:26202972

  20. Human and mouse ABCA1 comparative sequencing and transgenesis studies identify regulatory elements

    SciTech Connect

    Qiu, Yang; Cavelier, L.; Chiu, Sally; Rubin, Edward; Cheng, Jan-Fang

    2000-08-01

    The expression of ABCA1, a major participant in apolipoprotein mediated cholesterol efflux is highly regulated by a variety of factors including intracellular cholesterol concentration. To analyze its genomic organization and identify those sequences involved in its regulation we sequenced and compared approximately 200 Kb of orthologous DNA from mice and humans containing the ABCA1 gene and significant flanking DNA. The comparison revealed a variety of mouse human conserved sequences including 50 conserved ABCA1 exons over 147Kb of human and 124Kb of mouse genomic DNA as well as multiple mouse human conserved noncoding sequences. Using as a criteria for identifying putative regulatory elements in non-coding sequence, human and mouse sequences that were &62;75% identical for over 120 bp were screened for resulting in the identification of 34 elements. The two most highly conserved human mouse noncoding elements (CNS1: 88% identity over 498 bp, CNS2: 81% identity over 214 bp)! were also highly conserved in the ABCA1 genes of rats, dogs, cows, rabbits and pigs. Two independent studies have demonstrated that the DNA segments containing CNS2 function in vitro as a sterol response promoter. Support for the inclusion of major ABCA1 regulatory elements in the human genomic sequence examined was the demonstration that mice containing a human BAC transgene containing sequences exclusively from the analyzed interval, expressed human ABCA1 in a tissue distribution mimicking expression of endogenous mouse ABC1. These studies using a comparative genomic approach has characterized the structure of the human and mouse ABCA1 genes and has helped identify sequences participating in its expression.

  1. Partitioning Heritability of Regulatory and Cell-Type-Specific Variants across 11 Common Diseases

    PubMed Central

    Gusev, Alexander; Lee, S. Hong; Trynka, Gosia; Finucane, Hilary; Vilhjálmsson, Bjarni J.; Xu, Han; Zang, Chongzhi; Ripke, Stephan; Bulik-Sullivan, Brendan; Stahl, Eli; Ripke, Stephan; Neale, Benjamin M.; Corvin, Aiden; Walters, James T.R.; Farh, Kai-How; Holmans, Peter A.; Lee, Phil; Bulik-Sullivan, Brendan; Collier, David A.; Huang, Hailiang; Pers, Tune H.; Agartz, Ingrid; Agerbo, Esben; Albus, Margot; Alexander, Madeline; Amin, Farooq; Bacanu, Silviu A.; Begemann, Martin; Belliveau, Richard A.; Bene, Judit; Bergen, Sarah E.; Bevilacqua, Elizabeth; Bigdeli, Tim B.; Black, Donald W.; Børglum, Anders D.; Bruggeman, Richard; Buccola, Nancy G.; Buckner, Randy L.; Byerley, William; Cahn, Wiepke; Cai, Guiqing; Campion, Dominique; Cantor, Rita M.; Carr, Vaughan J.; Carrera, Noa; Catts, Stanley V.; Chambert, Kimberly D.; Chan, Raymond C.K.; Chen, Ronald Y.L.; Chen, Eric Y.H.; Cheng, Wei; Cheung, Eric F.C.; Chong, Siow Ann; Cloninger, C. Robert; Cohen, David; Cohen, Nadine; Cormican, Paul; Craddock, Nick; Crowley, James J.; Curtis, David; Davidson, Michael; Davis, Kenneth L.; Degenhardt, Franziska; Del Favero, Jurgen; DeLisi, Lynn E.; Demontis, Ditte; Dikeos, Dimitris; Dinan, Timothy; Djurovic, Srdjan; Donohoe, Gary; Drapeau, Elodie; Duan, Jubao; Dudbridge, Frank; Durmishi, Naser; Eichhammer, Peter; Eriksson, Johan; Escott-Price, Valentina; Essioux, Laurent; Fanous, Ayman H.; Farrell, Martilias S.; Frank, Josef; Franke, Lude; Freedman, Robert; Freimer, Nelson B.; Friedl, Marion; Friedman, Joseph I.; Fromer, Menachem; Genovese, Giulio; Georgieva, Lyudmila; Gershon, Elliot S.; Giegling, Ina; Giusti-Rodrguez, Paola; Godard, Stephanie; Goldstein, Jacqueline I.; Golimbet, Vera; Gopal, Srihari; Gratten, Jacob; Grove, Jakob; de Haan, Lieuwe; Hammer, Christian; Hamshere, Marian L.; Hansen, Mark; Hansen, Thomas; Haroutunian, Vahram; Hartmann, Annette M.; Henskens, Frans A.; Herms, Stefan; Hirschhorn, Joel N.; Hoffmann, Per; Hofman, Andrea; Hollegaard, Mads V.; Hougaard, David M.; Ikeda, Masashi; Joa, Inge; Julià, Antonio; Kahn, René S.; Kalaydjieva, Luba; Karachanak-Yankova, Sena; Karjalainen, Juha; Kavanagh, David; Keller, Matthew C.; Kelly, Brian J.; Kennedy, James L.; Khrunin, Andrey; Kim, Yunjung; Klovins, Janis; Knowles, James A.; Konte, Bettina; Kucinskas, Vaidutis; Kucinskiene, Zita Ausrele; Kuzelova-Ptackova, Hana; Kähler, Anna K.; Laurent, Claudine; Keong, Jimmy Lee Chee; Lee, S. Hong; Legge, Sophie E.; Lerer, Bernard; Li, Miaoxin; Li, Tao; Liang, Kung-Yee; Lieberman, Jeffrey; Limborska, Svetlana; Loughland, Carmel M.; Lubinski, Jan; Lnnqvist, Jouko; Macek, Milan; Magnusson, Patrik K.E.; Maher, Brion S.; Maier, Wolfgang; Mallet, Jacques; Marsal, Sara; Mattheisen, Manuel; Mattingsdal, Morten; McCarley, Robert W.; McDonald, Colm; McIntosh, Andrew M.; Meier, Sandra; Meijer, Carin J.; Melegh, Bela; Melle, Ingrid; Mesholam-Gately, Raquelle I.; Metspalu, Andres; Michie, Patricia T.; Milani, Lili; Milanova, Vihra; Mokrab, Younes; Morris, Derek W.; Mors, Ole; Mortensen, Preben B.; Murphy, Kieran C.; Murray, Robin M.; Myin-Germeys, Inez; Mller-Myhsok, Bertram; Nelis, Mari; Nenadic, Igor; Nertney, Deborah A.; Nestadt, Gerald; Nicodemus, Kristin K.; Nikitina-Zake, Liene; Nisenbaum, Laura; Nordin, Annelie; O’Callaghan, Eadbhard; O’Dushlaine, Colm; O’Neill, F. Anthony; Oh, Sang-Yun; Olincy, Ann; Olsen, Line; Van Os, Jim; Pantelis, Christos; Papadimitriou, George N.; Papiol, Sergi; Parkhomenko, Elena; Pato, Michele T.; Paunio, Tiina; Pejovic-Milovancevic, Milica; Perkins, Diana O.; Pietilinen, Olli; Pimm, Jonathan; Pocklington, Andrew J.; Powell, John; Price, Alkes; Pulver, Ann E.; Purcell, Shaun M.; Quested, Digby; Rasmussen, Henrik B.; Reichenberg, Abraham; Reimers, Mark A.; Richards, Alexander L.; Roffman, Joshua L.; Roussos, Panos; Ruderfer, Douglas M.; Salomaa, Veikko; Sanders, Alan R.; Schall, Ulrich; Schubert, Christian R.; Schulze, Thomas G.; Schwab, Sibylle G.; Scolnick, Edward M.; Scott, Rodney J.; Seidman, Larry J.; Shi, Jianxin; Sigurdsson, Engilbert; Silagadze, Teimuraz; Silverman, Jeremy M.; Sim, Kang; Slominsky, Petr; Smoller, Jordan W.; So, Hon-Cheong; Spencer, Chris C.A.; Stahl, Eli A.; Stefansson, Hreinn; Steinberg, Stacy; Stogmann, Elisabeth; Straub, Richard E.; Strengman, Eric; Strohmaier, Jana; Stroup, T. Scott; Subramaniam, Mythily; Suvisaari, Jaana; Svrakic, Dragan M.; Szatkiewicz, Jin P.; Sderman, Erik; Thirumalai, Srinivas; Toncheva, Draga; Tooney, Paul A.; Tosato, Sarah; Veijola, Juha; Waddington, John; Walsh, Dermot; Wang, Dai; Wang, Qiang; Webb, Bradley T.; Weiser, Mark; Wildenauer, Dieter B.; Williams, Nigel M.; Williams, Stephanie; Witt, Stephanie H.; Wolen, Aaron R.; Wong, Emily H.M.; Wormley, Brandon K.; Wu, Jing Qin; Xi, Hualin Simon; Zai, Clement C.; Zheng, Xuebin; Zimprich, Fritz; Wray, Naomi R.; Stefansson, Kari; Visscher, Peter M.; Adolfsson, Rolf; Andreassen, Ole A.; Blackwood, Douglas H.R.; Bramon, Elvira; Buxbaum, Joseph D.; Brglum, Anders D.; Cichon, Sven; Darvasi, Ariel; Domenici, Enrico; Ehrenreich, Hannelore; Esko, Tõnu; Gejman, Pablo V.; Gill, Michael; Gurling, Hugh; Hultman, Christina M.; Iwata, Nakao; Jablensky, Assen V.; Jönsson, Erik G.; Kendler, Kenneth S.; Kirov, George; Knight, Jo; Lencz, Todd; Levinson, Douglas F.; Li, Qingqin S.; Liu, Jianjun; Malhotra, Anil K.; McCarroll, Steven A.; McQuillin, Andrew; Moran, Jennifer L.; Mortensen, Preben B.; Mowry, Bryan J.; Nthen, Markus M.; Ophoff, Roel A.; Owen, Michael J.; Palotie, Aarno; Pato, Carlos N.; Petryshen, Tracey L.; Posthuma, Danielle; Rietschel, Marcella; Riley, Brien P.; Rujescu, Dan; Sham, Pak C.; Sklar, Pamela; St. Clair, David; Weinberger, Daniel R.; Wendland, Jens R.; Werge, Thomas; Daly, Mark J.; Sullivan, Patrick F.; O’Donovan, Michael C.; Ripke, Stephan; O’Dushlaine, Colm; Chambert, Kimberly; Moran, Jennifer L.; Kähler, Anna K.; Akterin, Susanne; Bergen, Sarah; Magnusson, Patrik K.E.; Neale, Benjamin M.; Ruderfer, Douglas; Scolnick, Edward; Purcell, Shaun; McCarroll, Steve; Sklar, Pamela; Hultman, Christina M.; Sullivan, Patrick F.; Kähler, Anna K.; Hultman, Christina M.; Purcell, Shaun M.; McCarroll, Steven A.; Daly, Mark; Pasaniuc, Bogdan; Sullivan, Patrick F.; Neale, Benjamin M.; Wray, Naomi R.; Raychaudhuri, Soumya; Price, Alkes L.

    2014-01-01

    Regulatory and coding variants are known to be enriched with associations identified by genome-wide association studies (GWASs) of complex disease, but their contributions to trait heritability are currently unknown. We applied variance-component methods to imputed genotype data for 11 common diseases to partition the heritability explained by genotyped SNPs (hg2) across functional categories (while accounting for shared variance due to linkage disequilibrium). Extensive simulations showed that in contrast to current estimates from GWAS summary statistics, the variance-component approach partitions heritability accurately under a wide range of complex-disease architectures. Across the 11 diseases DNaseI hypersensitivity sites (DHSs) from 217 cell types spanned 16% of imputed SNPs (and 24% of genotyped SNPs) but explained an average of 79% (SE = 8%) of hg2 from imputed SNPs (5.1× enrichment; p = 3.7 × 10−17) and 38% (SE = 4%) of hg2 from genotyped SNPs (1.6× enrichment, p = 1.0 × 10−4). Further enrichment was observed at enhancer DHSs and cell-type-specific DHSs. In contrast, coding variants, which span 1% of the genome, explained <10% of hg2 despite having the highest enrichment. We replicated these findings but found no significant contribution from rare coding variants in independent schizophrenia cohorts genotyped on GWAS and exome chips. Our results highlight the value of analyzing components of heritability to unravel the functional architecture of common disease. PMID:25439723

  2. Partitioning heritability of regulatory and cell-type-specific variants across 11 common diseases.

    PubMed

    Gusev, Alexander; Lee, S Hong; Trynka, Gosia; Finucane, Hilary; Vilhjálmsson, Bjarni J; Xu, Han; Zang, Chongzhi; Ripke, Stephan; Bulik-Sullivan, Brendan; Stahl, Eli; Kähler, Anna K; Hultman, Christina M; Purcell, Shaun M; McCarroll, Steven A; Daly, Mark; Pasaniuc, Bogdan; Sullivan, Patrick F; Neale, Benjamin M; Wray, Naomi R; Raychaudhuri, Soumya; Price, Alkes L

    2014-11-01

    Regulatory and coding variants are known to be enriched with associations identified by genome-wide association studies (GWASs) of complex disease, but their contributions to trait heritability are currently unknown. We applied variance-component methods to imputed genotype data for 11 common diseases to partition the heritability explained by genotyped SNPs (hg(2)) across functional categories (while accounting for shared variance due to linkage disequilibrium). Extensive simulations showed that in contrast to current estimates from GWAS summary statistics, the variance-component approach partitions heritability accurately under a wide range of complex-disease architectures. Across the 11 diseases DNaseI hypersensitivity sites (DHSs) from 217 cell types spanned 16% of imputed SNPs (and 24% of genotyped SNPs) but explained an average of 79% (SE = 8%) of hg(2) from imputed SNPs (5.1× enrichment; p = 3.7 × 10(-17)) and 38% (SE = 4%) of hg(2) from genotyped SNPs (1.6× enrichment, p = 1.0 × 10(-4)). Further enrichment was observed at enhancer DHSs and cell-type-specific DHSs. In contrast, coding variants, which span 1% of the genome, explained <10% of hg(2) despite having the highest enrichment. We replicated these findings but found no significant contribution from rare coding variants in independent schizophrenia cohorts genotyped on GWAS and exome chips. Our results highlight the value of analyzing components of heritability to unravel the functional architecture of common disease. PMID:25439723

  3. The PAZAR database of gene regulatory information coupled to the ORCA toolkit for the study of regulatory sequences

    PubMed Central

    Portales-Casamar, Elodie; Arenillas, David; Lim, Jonathan; Swanson, Magdalena I.; Jiang, Steven; McCallum, Anthony; Kirov, Stefan; Wasserman, Wyeth W.

    2009-01-01

    The PAZAR database unites independently created and maintained data collections of transcription factor and regulatory sequence annotation. The flexible PAZAR schema permits the representation of diverse information derived from experiments ranging from biochemical protein–DNA binding to cellular reporter gene assays. Data collections can be made available to the public, or restricted to specific system users. The data ‘boutiques’ within the shopping-mall-inspired system facilitate the analysis of genomics data and the creation of predictive models of gene regulation. Since its initial release, PAZAR has grown in terms of data, features and through the addition of an associated package of software tools called the ORCA toolkit (ORCAtk). ORCAtk allows users to rapidly develop analyses based on the information stored in the PAZAR system. PAZAR is available at http://www.pazar.info. ORCAtk can be accessed through convenient buttons located in the PAZAR pages or via our website at http://www.cisreg.ca/ORCAtk. PMID:18971253

  4. First Complete Genome Sequence of Bean common mosaic necrosis virus from East Timor

    PubMed Central

    Maina, Solomon; Edwards, Owain R.; de Almeida, Luis; Ximenes, Abel

    2016-01-01

    We present here the first complete Bean common mosaic necrosis virus (BCMNV) genomic sequence isolated from virus-infected common bean (Phaseolus vulgaris) in East Timor, and compare it with six complete BMCNV genomes from the Netherlands, and one each from the United States, Tanzania, and an unspecified country. It most resembled the Netherlands strain NL-8 genome. PMID:27688343

  5. First Complete Genome Sequence of Bean common mosaic necrosis virus from East Timor.

    PubMed

    Maina, Solomon; Edwards, Owain R; de Almeida, Luis; Ximenes, Abel; Jones, Roger A C

    2016-01-01

    We present here the first complete Bean common mosaic necrosis virus (BCMNV) genomic sequence isolated from virus-infected common bean (Phaseolus vulgaris) in East Timor, and compare it with six complete BMCNV genomes from the Netherlands, and one each from the United States, Tanzania, and an unspecified country. It most resembled the Netherlands strain NL-8 genome. PMID:27688343

  6. New gene-derived simple sequence repeat markers for common bean (Phaseolus vulgaris L.).

    PubMed

    Blair, Matthew W; Hurtado, Natalia; Sharma, Prem

    2012-07-01

    Common bean is an important and diverse crop legume with several wild relatives that are all part of the Phaseoleae tribe of tropical crop legumes. Sequence databases have been a good source of sequences to mine for simple sequence repeats (SSRs). The objective of this research was to evaluate 14 sequence collections from common bean for SSRs and to evaluate the diversity of the polymorphic microsatellites derived from these collections. SSRs were found in 10 of the GenBank sequence collections with an average of 11.3% of sequences containing microsatellite motifs. The most common motifs were based on tri- and dinucleotides. In a marker development programme, primers were designed for 125 microsatellites which were tested on a panel of 18 common bean genotypes. The markers were named as part of the bean microsatellite-database (BMd) series, and the average polymorphism information content was 0.404 for polymorphic markers and predicted well the genepool structure of common beans and the status of the wild and cultivated accessions that were included in the study. Therefore, the BMd series of microsatellites is useful for multiple studies of genetic relatedness and as anchor markers in future mapping of wide crosses in the species.

  7. Regulatory regions of two transport operons under nitrogen control: nucleotide sequences.

    PubMed Central

    Higgins, C F; Ames, G F

    1982-01-01

    We have determined the nucleotide sequences of the regulatory regions from two amino acid transport operons from Salmonella typhimurium: dhuA, which regulates the histidine transport operon, and argTr, which regulates argT, the gene encoding the lysine-arginine-ornithine-binding protein, LAO. The promoter for the histidine transport operon has been identified from the sequence change in the promoter-up mutation dhuA1. Neither regulatory region has any of the features typical of the regulatory regions of the amino acid biosynthetic operons, indicating that regulation of at least these transport genes does not involve a transcription attenuation mechanism. We have identified three interesting features, present in both of these sequences, which may be of importance in the regulation of these and other operons: a "stem-loop-foot" structure, a region of specific homology, and a mirror symmetry. The region of mirror symmetry may be a protein recognition site important is regulating expression of these and other operons in response to nitrogen availability. Mirror symmetry as a structure for DNA-protein interaction sites has not been proposed previously. PMID:7041112

  8. Analysis of mammalian gene batteries reveals both stable ancestral cores and highly dynamic regulatory sequences

    PubMed Central

    Ettwiller, Laurence; Budd, Aidan; Spitz, François; Wittbrodt, Joachim

    2008-01-01

    Background Changes in gene regulation are suspected to comprise one of the driving forces for evolution. To address the extent of cis-regulatory changes and how they impact on gene regulatory networks across eukaryotes, we systematically analyzed the evolutionary dynamics of target gene batteries controlled by 16 different transcription factors. Results We found that gene batteries show variable conservation within vertebrates, with slow and fast evolving modules. Hence, while a key gene battery associated with the cell cycle is conserved throughout metazoans, the POU5F1 (Oct4) and SOX2 batteries in embryonic stem cells show strong conservation within mammals, with the striking exception of rodents. Within the genes composing a given gene battery, we could identify a conserved core that likely reflects the ancestral function of the corresponding transcription factor. Interestingly, we show that the association between a transcription factor and its target genes is conserved even when we exclude conserved sequence similarities of their promoter regions from our analysis. This supports the idea that turnover, either of the transcription factor binding site or its direct neighboring sequence, is a pervasive feature of proximal regulatory sequences. Conclusions Our study reveals the dynamics of evolutionary changes within metazoan gene networks, including both the composition of gene batteries and the architecture of target gene promoters. This variation provides the playground required for evolutionary innovation around conserved ancestral core functions. PMID:19087242

  9. V1R promoters are well conserved and exhibit common putative regulatory motifs

    PubMed Central

    Stewart, Robert; Lane, Robert P

    2007-01-01

    Background The mouse vomeronasal organ (VNO) processes chemosensory information, including pheromone signals that influence reproductive behaviors. The sensory neurons of the VNO express two types of chemosensory receptors, V1R and V2R. There are ~165 V1R genes in the mouse genome that have been classified into ~12 divergent subfamilies. Each sensory neuron of the apical compartment of the VNO transcribes only one of the repertoire of V1R genes. A model for mutually exclusive V1R transcription in these cells has been proposed in which each V1R gene might compete stochastically for a single transcriptional complex. This model predicts that the large repertoire of divergent V1R genes in the mouse genome contains common regulatory elements. In this study, we have characterized V1R promoter regions by comparative genomics and by mapping transcription start sites. Results We find that transcription is initiated from ~1 kb promoter regions that are well conserved within V1R subfamilies. While cross-subfamily homology is not evident by traditional methods, we developed a heuristic motif-searching tool, LogoAlign, and applied this tool to identify motifs shared within the promoters of all V1R genes. Our motif-searching tool exhibits rapid convergence to a relatively small number of non-redundant solutions (97% convergence). We also find that the best motifs contain significantly more information than those identified in controls, and that these motifs are more likely to be found in the immediate vicinity of transcription start sites than elsewhere in gene blocks. The best motifs occur near transcription start sites of ~90% of all V1R genes and across all of the divergent subfamilies. Therefore, these motifs are candidate binding sites for transcription factors involved in V1R co-regulation. Conclusion Our analyses show that V1R subfamilies have broad and well conserved promoter regions from which transcription is initiated. Results from a new motif-finding algorithm, Logo

  10. A pan-cancer modular regulatory network analysis to identify common and cancer-specific network components.

    PubMed

    Knaack, Sara A; Siahpirani, Alireza Fotuhi; Roy, Sushmita

    2014-01-01

    Many human diseases including cancer are the result of perturbations to transcriptional regulatory networks that control context-specific expression of genes. A comparative approach across multiple cancer types is a powerful approach to illuminate the common and specific network features of this family of diseases. Recent efforts from The Cancer Genome Atlas (TCGA) have generated large collections of functional genomic data sets for multiple types of cancers. An emerging challenge is to devise computational approaches that systematically compare these genomic data sets across different cancer types that identify common and cancer-specific network components. We present a module- and network-based characterization of transcriptional patterns in six different cancers being studied in TCGA: breast, colon, rectal, kidney, ovarian, and endometrial. Our approach uses a recently developed regulatory network reconstruction algorithm, modular regulatory network learning with per gene information (MERLIN), within a stability selection framework to predict regulators for individual genes and gene modules. Our module-based analysis identifies a common theme of immune system processes in each cancer study, with modules statistically enriched for immune response processes as well as targets of key immune response regulators from the interferon regulatory factor (IRF) and signal transducer and activator of transcription (STAT) families. Comparison of the inferred regulatory networks from each cancer type identified a core regulatory network that included genes involved in chromatin remodeling, cell cycle, and immune response. Regulatory network hubs included genes with known roles in specific cancer types as well as genes with potentially novel roles in different cancer types. Overall, our integrated module and network analysis recapitulated known themes in cancer biology and additionally revealed novel regulatory hubs that suggest a complex interplay of immune response, cell

  11. A Pan-Cancer Modular Regulatory Network Analysis to Identify Common and Cancer-Specific Network Components

    PubMed Central

    Knaack, Sara A; Siahpirani, Alireza Fotuhi; Roy, Sushmita

    2014-01-01

    Many human diseases including cancer are the result of perturbations to transcriptional regulatory networks that control context-specific expression of genes. A comparative approach across multiple cancer types is a powerful approach to illuminate the common and specific network features of this family of diseases. Recent efforts from The Cancer Genome Atlas (TCGA) have generated large collections of functional genomic data sets for multiple types of cancers. An emerging challenge is to devise computational approaches that systematically compare these genomic data sets across different cancer types that identify common and cancer-specific network components. We present a module- and network-based characterization of transcriptional patterns in six different cancers being studied in TCGA: breast, colon, rectal, kidney, ovarian, and endometrial. Our approach uses a recently developed regulatory network reconstruction algorithm, modular regulatory network learning with per gene information (MERLIN), within a stability selection framework to predict regulators for individual genes and gene modules. Our module-based analysis identifies a common theme of immune system processes in each cancer study, with modules statistically enriched for immune response processes as well as targets of key immune response regulators from the interferon regulatory factor (IRF) and signal transducer and activator of transcription (STAT) families. Comparison of the inferred regulatory networks from each cancer type identified a core regulatory network that included genes involved in chromatin remodeling, cell cycle, and immune response. Regulatory network hubs included genes with known roles in specific cancer types as well as genes with potentially novel roles in different cancer types. Overall, our integrated module and network analysis recapitulated known themes in cancer biology and additionally revealed novel regulatory hubs that suggest a complex interplay of immune response, cell

  12. Identification of cis-acting repressive sequences within the negative regulatory element of human immunodeficiency virus type 1.

    PubMed Central

    Lu, Y C; Touzjian, N; Stenzel, M; Dorfman, T; Sodroski, J G; Haseltine, W A

    1990-01-01

    The negative regulatory element of human immunodeficiency virus type 1 is a 260-nucleotide-long sequence that decreases the rate of RNA transcription initiation specified by the long terminal repeat. This region has the potential to bind several cellular transcription factors. Here it is shown that sequences which recognize the NFAT-1 and USF cellular transcription factors contribute to this negative regulatory effect. The sequences within the negative regulatory element which resemble the AP-1 site and the URS do not negatively regulate human immunodeficiency virus long terminal repeat transcription initiation. PMID:2398545

  13. kmer-SVM: a web server for identifying predictive regulatory sequence features in genomic data sets

    PubMed Central

    Fletez-Brant, Christopher; Lee, Dongwon; McCallion, Andrew S.; Beer, Michael A.

    2013-01-01

    Massively parallel sequencing technologies have made the generation of genomic data sets a routine component of many biological investigations. For example, Chromatin immunoprecipitation followed by sequence assays detect genomic regions bound (directly or indirectly) by specific factors, and DNase-seq identifies regions of open chromatin. A major bottleneck in the interpretation of these data is the identification of the underlying DNA sequence code that defines, and ultimately facilitates prediction of, these transcription factor (TF) bound or open chromatin regions. We have recently developed a novel computational methodology, which uses a support vector machine (SVM) with kmer sequence features (kmer-SVM) to identify predictive combinations of short transcription factor-binding sites, which determine the tissue specificity of these genomic assays (Lee, Karchin and Beer, Discriminative prediction of mammalian enhancers from DNA sequence. Genome Res. 2011; 21:2167–80). This regulatory information can (i) give confidence in genomic experiments by recovering previously known binding sites, and (ii) reveal novel sequence features for subsequent experimental testing of cooperative mechanisms. Here, we describe the development and implementation of a web server to allow the broader research community to independently apply our kmer-SVM to analyze and interpret their genomic datasets. We analyze five recently published data sets and demonstrate how this tool identifies accessory factors and repressive sequence elements. kmer-SVM is available at http://kmersvm.beerlab.org. PMID:23771147

  14. Cloning and nucleotide sequence of luxR, a regulatory gene controlling bioluminescence in Vibrio harveyi.

    PubMed Central

    Showalter, R E; Martin, M O; Silverman, M R

    1990-01-01

    Mutagenesis with transposon mini-Mulac was used previously to identify a regulatory locus necessary for expression of bioluminescence genes, lux, in Vibrio harveyi (M. Martin, R. Showalter, and M. Silverman, J. Bacteriol. 171:2406-2414, 1989). Mutants with transposon insertions in this regulatory locus were used to construct a hybridization probe which was used in this study to detect recombinants in a cosmid library containing the homologous DNA. Recombinant cosmids with this DNA stimulated expression of the genes encoding enzymes for luminescence, i.e., the luxCDABE operon, which were positioned in trans on a compatible replicon in Escherichia coli. Transposon mutagenesis and analysis of the DNA sequence of the cloned DNA indicated that regulatory function resided in a single gene of about 0.6-kilobases named luxR. Expression of bioluminescence in V. harveyi and in the fish light-organ symbiont Vibrio fischeri is controlled by density-sensing mechanisms involving the accumulation of small signal molecules called autoinducers, but similarity of the two luminescence systems at the molecular level was not apparent in this study. The amino acid sequence of the LuxR product of V. harveyi, which indicates a structural relationship to some DNA-binding proteins, is not similar to the sequence of the protein that regulates expression of luminescence in V. fischeri. In addition, reconstitution of autoinducer-controlled luminescence in recombinant E. coli, already achieved with lux genes cloned from V. fischeri, was not accomplished with the isolation of luxR from V. harveyi, suggesting a requirement for an additional regulatory component. PMID:2160932

  15. Resequencing of the common marmoset genome improves genome assemblies and gene-coding sequence analysis

    PubMed Central

    Sato, Kengo; Kuroki, Yoko; Kumita, Wakako; Fujiyama, Asao; Toyoda, Atsushi; Kawai, Jun; Iriki, Atsushi; Sasaki, Erika; Okano, Hideyuki; Sakakibara, Yasubumi

    2015-01-01

    The first draft of the common marmoset (Callithrix jacchus) genome was published by the Marmoset Genome Sequencing and Analysis Consortium. The draft was based on whole-genome shotgun sequencing, and the current assembly version is Callithrix_jacches-3.2.1, but there still exist 187,214 undetermined gap regions and supercontigs and relatively short contigs that are unmapped to chromosomes in the draft genome. We performed resequencing and assembly of the genome of common marmoset by deep sequencing with high-throughput sequencing technology. Several different sequence runs using Illumina sequencing platforms were executed, and 181 Gbp of high-quality bases including mate-pairs with long insert lengths of 3, 8, 20, and 40 Kbp were obtained, that is, approximately 60× coverage. The resequencing significantly improved the MGSAC draft genome sequence. The N50 of the contigs, which is a statistical measure used to evaluate assembly quality, doubled. As a result, 51% of the contigs (total length: 299 Mbp) that were unmapped to chromosomes in the MGSAC draft were merged with chromosomal contigs, and the improved genome sequence helped to detect 5,288 new genes that are homologous to human cDNAs and the gaps in 5,187 transcripts of the Ensembl gene annotations were completely filled. PMID:26586576

  16. Resequencing of the common marmoset genome improves genome assemblies and gene-coding sequence analysis.

    PubMed

    Sato, Kengo; Kuroki, Yoko; Kumita, Wakako; Fujiyama, Asao; Toyoda, Atsushi; Kawai, Jun; Iriki, Atsushi; Sasaki, Erika; Okano, Hideyuki; Sakakibara, Yasubumi

    2015-11-20

    The first draft of the common marmoset (Callithrix jacchus) genome was published by the Marmoset Genome Sequencing and Analysis Consortium. The draft was based on whole-genome shotgun sequencing, and the current assembly version is Callithrix_jacches-3.2.1, but there still exist 187,214 undetermined gap regions and supercontigs and relatively short contigs that are unmapped to chromosomes in the draft genome. We performed resequencing and assembly of the genome of common marmoset by deep sequencing with high-throughput sequencing technology. Several different sequence runs using Illumina sequencing platforms were executed, and 181 Gbp of high-quality bases including mate-pairs with long insert lengths of 3, 8, 20, and 40 Kbp were obtained, that is, approximately 60× coverage. The resequencing significantly improved the MGSAC draft genome sequence. The N50 of the contigs, which is a statistical measure used to evaluate assembly quality, doubled. As a result, 51% of the contigs (total length: 299 Mbp) that were unmapped to chromosomes in the MGSAC draft were merged with chromosomal contigs, and the improved genome sequence helped to detect 5,288 new genes that are homologous to human cDNAs and the gaps in 5,187 transcripts of the Ensembl gene annotations were completely filled.

  17. Whole-genome sequencing to understand the genetic architecture of common gene expression and biomarker phenotypes

    PubMed Central

    Wood, Andrew R.; Tuke, Marcus A.; Nalls, Mike; Hernandez, Dena; Gibbs, J. Raphael; Lin, Haoxiang; Xu, Christopher S.; Li, Qibin; Shen, Juan; Jun, Goo; Almeida, Marcio; Tanaka, Toshiko; Perry, John R. B.; Gaulton, Kyle; Rivas, Manny; Pearson, Richard; Curran, Joanne E.; Johnson, Matthew P.; Göring, Harald H. H.; Duggirala, Ravindranath; Blangero, John; Mccarthy, Mark I.; Bandinelli, Stefania; Murray, Anna; Weedon, Michael N.; Singleton, Andrew; Melzer, David; Ferrucci, Luigi; Frayling, Timothy M

    2015-01-01

    Initial results from sequencing studies suggest that there are relatively few low-frequency (<5%) variants associated with large effects on common phenotypes. We performed low-pass whole-genome sequencing in 680 individuals from the InCHIANTI study to test two primary hypotheses: (i) that sequencing would detect single low-frequency–large effect variants that explained similar amounts of phenotypic variance as single common variants, and (ii) that some common variant associations could be explained by low-frequency variants. We tested two sets of disease-related common phenotypes for which we had statistical power to detect large numbers of common variant–common phenotype associations—11 132 cis-gene expression traits in 450 individuals and 93 circulating biomarkers in all 680 individuals. From a total of 11 657 229 high-quality variants of which 6 129 221 and 5 528 008 were common and low frequency (<5%), respectively, low frequency–large effect associations comprised 7% of detectable cis-gene expression traits [89 of 1314 cis-eQTLs at P < 1 × 10−06 (false discovery rate ∼5%)] and one of eight biomarker associations at P < 8 × 10−10. Very few (30 of 1232; 2%) common variant associations were fully explained by low-frequency variants. Our data show that whole-genome sequencing can identify low-frequency variants undetected by genotyping based approaches when sample sizes are sufficiently large to detect substantial numbers of common variant associations, and that common variant associations are rarely explained by single low-frequency variants of large effect. PMID:25378555

  18. Conserved regulatory elements of the promoter sequence of the gene rpoH of enteric bacteria

    PubMed Central

    Ramírez-Santos, Jesús; Collado-Vides, Julio; García-Varela, Martin; Gómez-Eichelmann, M. Carmen

    2001-01-01

    The rpoH regulatory region of different members of the enteric bacteria family was sequenced or downloaded from GenBank and compared. In addition, the transcriptional start sites of rpoH of Yersinia frederiksenii and Proteus mirabilis, two distant members of this family, were determined. Sequences similar to the σ70 promoters P1, P4 and P5, to the σE promoter P3 and to boxes DnaA1, DnaA2, cAMP receptor protein (CRP) boxes CRP1, CRP2 and box CytR present in Escherichia coli K12, were identified in sequences of closely related bacteria such as: E.coli, Shigella flexneri, Salmonella enterica serovar Typhimurium, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae. In more distant bacteria, Y.frederiksenii and P.mirabilis, the rpoH regulatory region has a distal P1-like σ70 promoter and two proximal promoters: a heat-induced σE-like promoter and a σ70 promoter. Sequences similar to the regulatory boxes were not identified in these bacteria. This study suggests that the general pattern of transcription of the rpoH gene in enteric bacteria includes a distal σ70 promoter, >200 nt upstream of the initiation codon, and two proximal promoters: a heat-induced σE-like promoter and a σ70 promoter. A second proximal σ70 promoter under catabolite-regulation is probably present only in bacteria closely related to E.coli. PMID:11139607

  19. Modelling Human Regulatory Variation in Mouse: Finding the Function in Genome-Wide Association Studies and Whole-Genome Sequencing

    PubMed Central

    Schmouth, Jean-François; Bonaguro, Russell J.; Corso-Diaz, Ximena; Simpson, Elizabeth M.

    2012-01-01

    An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and β-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant–harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation. PMID:22396661

  20. A common philosophy and FORTRAN 77 software package for implementing and searching sequence databases.

    PubMed

    Claverie, J M

    1984-01-11

    I present a common philosophy for implementing the EMBL and GENBANK (BBN-Los Alamos) nucleic acid sequence databases, as well as the National Biological Foundation (Dayhoff) protein sequence database. The associated FORTRAN 77 fully transportable software package includes: 1) modules for implementing each of these databases from the initial magnetic tape file, 2) modules performing a fast mnemonic access, 3) modules performing key-string access and allowing the definition of user-specific database subsets, 4) a common probe searching module allowing the stacking of multiple combined search requests over the databases. This software is particularly suitable for 32-bit mini/microcomputers but would eventually run on 16-bit computers.

  1. Transposable elements: an abundant and natural source of regulatory sequences for host genes.

    PubMed

    Rebollo, Rita; Romanish, Mark T; Mager, Dixie L

    2012-01-01

    The fact that transposable elements (TEs) can influence host gene expression was first recognized more than 50 years ago. However, since that time, TEs have been widely regarded as harmful genetic parasites-selfish elements that are rarely co-opted by the genome to serve a beneficial role. Here, we survey recent findings that relate to TE impact on host genes and remind the reader that TEs, in contrast to other noncoding parts of the genome, are uniquely suited to gene regulatory functions. We review recent studies that demonstrate the role of TEs in establishing and rewiring gene regulatory networks and discuss the overall ubiquity of exaptation. We suggest that although individuals within a population can be harmed by the deleterious effects of new TE insertions, the presence of TE sequences in a genome is of overall benefit to the population. PMID:22905872

  2. Genome Sequencing of Autism-Affected Families Reveals Disruption of Putative Noncoding Regulatory DNA

    PubMed Central

    Turner, Tychele N.; Hormozdiari, Fereydoun; Duyzend, Michael H.; McClymont, Sarah A.; Hook, Paul W.; Iossifov, Ivan; Raja, Archana; Baker, Carl; Hoekzema, Kendra; Stessman, Holly A.; Zody, Michael C.; Nelson, Bradley J.; Huddleston, John; Sandstrom, Richard; Smith, Joshua D.; Hanna, David; Swanson, James M.; Faustman, Elaine M.; Bamshad, Michael J.; Stamatoyannopoulos, John; Nickerson, Deborah A.; McCallion, Andrew S.; Darnell, Robert; Eichler, Evan E.

    2016-01-01

    We performed whole-genome sequencing (WGS) of 208 genomes from 53 families affected by simplex autism. For the majority of these families, no copy-number variant (CNV) or candidate de novo gene-disruptive single-nucleotide variant (SNV) had been detected by microarray or whole-exome sequencing (WES). We integrated multiple CNV and SNV analyses and extensive experimental validation to identify additional candidate mutations in eight families. We report that compared to control individuals, probands showed a significant (p = 0.03) enrichment of de novo and private disruptive mutations within fetal CNS DNase I hypersensitive sites (i.e., putative regulatory regions). This effect was only observed within 50 kb of genes that have been previously associated with autism risk, including genes where dosage sensitivity has already been established by recurrent disruptive de novo protein-coding mutations (ARID1B, SCN2A, NR3C2, PRKCA, and DSCAM). In addition, we provide evidence of gene-disruptive CNVs (in DISC1, WNT7A, RBFOX1, and MBD5), as well as smaller de novo CNVs and exon-specific SNVs missed by exome sequencing in neurodevelopmental genes (e.g., CANX, SAE1, and PIK3CA). Our results suggest that the detection of smaller, often multiple CNVs affecting putative regulatory elements might help explain additional risk of simplex autism. PMID:26749308

  3. Rapid evolution of cis-regulatory sequences via local point mutations

    NASA Technical Reports Server (NTRS)

    Stone, J. R.; Wray, G. A.

    2001-01-01

    Although the evolution of protein-coding sequences within genomes is well understood, the same cannot be said of the cis-regulatory regions that control transcription. Yet, changes in gene expression are likely to constitute an important component of phenotypic evolution. We simulated the evolution of new transcription factor binding sites via local point mutations. The results indicate that new binding sites appear and become fixed within populations on microevolutionary timescales under an assumption of neutral evolution. Even combinations of two new binding sites evolve very quickly. We predict that local point mutations continually generate considerable genetic variation that is capable of altering gene expression.

  4. Annotation and sequence diversity of transposable elements in common bean (Phaseolus vulgaris).

    PubMed

    Gao, Dongying; Abernathy, Brian; Rohksar, Daniel; Schmutz, Jeremy; Jackson, Scott A

    2014-01-01

    Common bean (Phaseolus vulgaris) is an important legume crop grown and consumed worldwide. With the availability of the common bean genome sequence, the next challenge is to annotate the genome and characterize functional DNA elements. Transposable elements (TEs) are the most abundant component of plant genomes and can dramatically affect genome evolution and genetic variation. Thus, it is pivotal to identify TEs in the common bean genome. In this study, we performed a genome-wide transposon annotation in common bean using a combination of homology and sequence structure-based methods. We developed a 2.12-Mb transposon database which includes 791 representative transposon sequences and is available upon request or from www.phytozome.org. Of note, nearly all transposons in the database are previously unrecognized TEs. More than 5,000 transposon-related expressed sequence tags (ESTs) were detected which indicates that some transposons may be transcriptionally active. Two Ty1-copia retrotransposon families were found to encode the envelope-like protein which has rarely been identified in plant genomes. Also, we identified an extra open reading frame (ORF) termed ORF2 from 15 Ty3-gypsy families that was located between the ORF encoding the retrotransposase and the 3'LTR. The ORF2 was in opposite transcriptional orientation to retrotransposase. Sequence homology searches and phylogenetic analysis suggested that the ORF2 may have an ancient origin, but its function is not clear. These transposon data provide a useful resource for understanding the genome organization and evolution and may be used to identify active TEs for developing transposon-tagging system in common bean and other related genomes. PMID:25071814

  5. Insights from exome sequencing in common and rare human endocrine disorders

    PubMed Central

    Dauber, Andrew; de Bruin, Christiaan

    2016-01-01

    Exome sequencing has emerged in recent years as a rapid and effective tool for the elucidation of genetic defects underlying both rare and common human disease. Increased availability and decreased costs of next generation sequencing has enabled researchers worldwide to use this approach not only in individual patients with rare diseases, but also to screen larger cohorts or populations for genetic determinants of disease. Within the field of endocrinology, exome sequencing has led to significant advancements in our understanding of numerous disorders including adrenal disease, growth and pubertal disorders, type 2 diabetes, as well as a multitude of rare genetic syndromes with prominent endocrine involvement. In this review, we aim to provide an overview of these recent new insights and discuss the role that exome sequencing is expected to play in endocrine research and clinical practice in the coming years. PMID:25963271

  6. Variable Genome Sequences of the Murine Pneumotropic Virus (Polyomaviridae) Regulatory Region Isolated from an Infected Mouse Tissue Viral Suspension

    PubMed Central

    Libbey, Jane E.

    2016-01-01

    The murine pneumotropic virus genome, isolated from an infected murine tissue homogenate, was sequenced to completion. The lungs, liver, spleen, and kidneys were the source of the tissue homogenate in order to mirror the heterogeneity of the virus population in vivo. The regulatory region sequence was found to be highly variable. PMID:27231357

  7. Comprehensive Functional Analyses of Expressed Sequence Tags in Common Wheat (Triticum aestivum)

    PubMed Central

    Manickavelu, Alagu; Kawaura, Kanako; Oishi, Kazuko; Shin-I, Tadasu; Kohara, Yuji; Yahiaoui, Nabila; Keller, Beat; Abe, Reina; Suzuki, Ayako; Nagayama, Taishi; Yano, Kentaro; Ogihara, Yasunari

    2012-01-01

    About 1 million expressed sequence tag (EST) sequences comprising 125.3 Mb nucleotides were accreted from 51 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including abiotic stresses and pathogen challenges in common wheat (Triticum aestivum). Expressed sequence tags were assembled with stringent parameters after processing with inbuild scripts, resulting in 37 138 contigs and 215 199 singlets. In the assembled sequences, 10.6% presented no matches with existing sequences in public databases. Functional characterization of wheat unigenes by gene ontology annotation, mining transcription factors, full-length cDNA, and miRNA targeting sites were carried out. A bioinformatics strategy was developed to discover single-nucleotide polymorphisms (SNPs) within our large EST resource and reported the SNPs between and within (homoeologous) cultivars. Digital gene expression was performed to find the tissue-specific gene expression, and correspondence analysis was executed to identify common and specific gene expression by selecting four biotic stress-related libraries. The assembly and associated information cater a framework for future investigation in functional genomics. PMID:22334568

  8. Complete genome sequence of a dahlia common mosaic virus isolate from New Zealand.

    PubMed

    Hadfield, James; Linderme, Daphné; Shepherd, Dionne N; Bezuidenhout, Marion; Lefeuvre, Pierre; Martin, Darren P; Varsani, Arvind

    2011-12-01

    Dahlia mosaic disease of the ornamental flowering plant Dahlia is caused by two caulimoviruses, dahlia mosaic virus (DMV) and dahlia common mosaic virus (DCMV). We used a rolling-circle amplification method to amplify, clone and determine for the first time the full genome sequence of a DCMV isolate from New Zealand (DCMV-NZ). Within the 7949-bp circular double-stranded retro-transcribing DCMV-NZ DNA, we identified six putative open reading frames, typical of all genomes in the family Caulimoviridae. The availability of the complete DCMV sequence provides a reference genome against which all others can be compared. PMID:21960043

  9. Development of taxon-specific sequences of common wheat for the detection of genetically modified wheat.

    PubMed

    Iida, Mayu; Yamashiro, Satomi; Yamakawa, Hirohito; Hayakawa, Katsuyuki; Kuribara, Hideo; Kodama, Takashi; Furui, Satoshi; Akiyama, Hiroshi; Maitani, Tamio; Hino, Akihiro

    2005-08-10

    Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests.

  10. SNPs in putative regulatory regions identified by human mouse comparative sequencing and transcription factor binding site data

    SciTech Connect

    Banerjee, Poulabi; Bahlo, Melanie; Schwartz, Jody R.; Loots, Gabriela G.; Houston, Kathryn A.; Dubchak, Inna; Speed, Terence P.; Rubin, Edward M.

    2002-01-01

    Genome wide disease association analysis using SNPs is being explored as a method for dissecting complex genetic traits and a vast number of SNPs have been generated for this purpose. As there are cost and throughput limitations of genotyping large numbers of SNPs and statistical issues regarding the large number of dependent tests on the same data set, to make association analysis practical it has been proposed that SNPs should be prioritized based on likely functional importance. The most easily identifiable functional SNPs are coding SNPs (cSNPs) and accordingly cSNPs have been screened in a number of studies. SNPs in gene regulatory sequences embedded in noncoding DNA are another class of SNPs suggested for prioritization due to their predicted quantitative impact on gene expression. The main challenge in evaluating these SNPs, in contrast to cSNPs is a lack of robust algorithms and databases for recognizing regulatory sequences in noncoding DNA. Approaches that have been previously used to delineate noncoding sequences with gene regulatory activity include cross-species sequence comparisons and the search for sequences recognized by transcription factors. We combined these two methods to sift through mouse human genomic sequences to identify putative gene regulatory elements and subsequently localized SNPs within these sequences in a 1 Megabase (Mb) region of human chromosome 5q31, orthologous to mouse chromosome 11 containing the Interleukin cluster.

  11. Suffix tree searcher: exploration of common substrings in large DNA sequence sets

    PubMed Central

    2014-01-01

    Background Large DNA sequence data sets require special bioinformatics tools to search and compare them. Such tools should be easy to use so that the data can be easily accessed by a wide array of researchers. In the past, the use of suffix trees for searching DNA sequences has been limited by a practical need to keep the trees in RAM. Newer algorithms solve this problem by using disk-based approaches. However, none of the fastest suffix tree algorithms have been implemented with a graphical user interface, preventing their incorporation into a feasible laboratory workflow. Results Suffix Tree Searcher (STS) is designed as an easy-to-use tool to index, search, and analyze very large DNA sequence datasets. The program accommodates very large numbers of very large sequences, with aggregate size reaching tens of billions of nucleotides. The program makes use of pre-sorted persistent "building blocks" to reduce the time required to construct new trees. STS is comprised of a graphical user interface written in Java, and four C modules. All components are automatically downloaded when a web link is clicked. The underlying suffix tree data structure permits extremely fast searching for specific nucleotide strings, with wild cards or mismatches allowed. Complete tree traversals for detecting common substrings are also very fast. The graphical user interface allows the user to transition seamlessly between building, traversing, and searching the dataset. Conclusions Thus, STS provides a new resource for the detection of substrings common to multiple DNA sequences or within a single sequence, for truly huge data sets. The re-searching of sequence hits, allowing wild card positions or mismatched nucleotides, together with the ability to rapidly retrieve large numbers of sequence hits from the DNA sequence files, provides the user with an efficient method of evaluating the similarity between nucleotide sequences by multiple alignment or use of Logos. The ability to re

  12. Using mitochondrial nucleotide sequences to investigate diversity and genealogical relationships within common carp (Cyprinus carpio L.).

    PubMed

    Thai, B T; Burridge, C P; Pham, T A; Austin, C M

    2005-02-01

    Direct sequencing of mitochondrial DNA (mtDNA) D-loop (745 bp) and MTATPase6/MTATPase8 (857 bp) regions was used to investigate genetic variation within common carp and develop a global genealogy of common carp strains. The D-loop region was more variable than the MTATPase6/MTATPase8 region, but given the wide distribution of carp the overall levels of sequence divergence were low. Levels of haplotype diversity varied widely among countries with Chinese, Indonesian and Vietnamese carp showing the greatest diversity whereas Japanese Koi and European carp had undetectable nucleotide variation. A genealogical analysis supports a close relationship between Vietnamese, Koi and Chinese Color carp strains and to a lesser extent, European carp. Chinese and Indonesian carp strains were the most divergent, and their relationships do not support the evolution of independent Asian and European lineages and current taxonomic treatments.

  13. Comparisons of the Distribution of Nucleotides and Common Sequences in Deoxyribonucleic Acid from Selected Bacteriophages

    PubMed Central

    Skalka, A.; Hanson, P.

    1972-01-01

    Results from comparisons of deoxyribonucleic acid (DNA) from several classes of bacteriophages suggest that most phage chromosomes contain either a homogeneous distribution of nucleotides or are made up of a few, rather large segments of different quanine plus cytosine (G + C) contents which are internally homogeneous. Among those temperate phages tested, most contained segmented DNA. Comparisons of sequence similarities among segments from lambdoid phage DNA species revealed the following order in relatedness to λ: 82 (and 434) > 21 > 424 > φ80. Most common sequences are found in the highest G + C segments, which in λ contain head and tail genes. Hybridization tests with λ and 186 or P2 DNA species verified that the lambdoids and 186 and P2 belong to two distinct groups. There are fewer homologous sequences between the DNA species of coliphages λ and P2 or 186 than there are between the DNA species of coliphage λ and salmonella phage P22. PMID:4553679

  14. Upstream regulatory regions required to stabilize binding to the TATA sequence in an adenovirus early promoter.

    PubMed

    Garcia, J; Wu, F; Gaynor, R

    1987-10-26

    Of the five early adenovirus promoters, the early region 3 (E3) promoter is one of the most strongly induced by the E1A protein. To identify cellular proteins involved in both the basal and E1A-induced transcriptional regulation of the E3 promoter, DNase I footprinting using partially purified Hela cell extracts was performed. Four regions of the E3 promoter serve as binding domains for cellular proteins. These regions are found between -156 to -179 (site IV), -83 to -103 (site III), -47 to -67 (site II), and -16 to -37 (site I), relative to the start of transcription. Examination of the DNA sequences in each binding domain suggests that site III likely serves as a binding site for activator protein 1 (AP-1), site II for the cyclic AMP regulatory element binding protein (CREB), and site I for a TATA binding factor. The factors binding to either site II or III were sufficient to stabilize binding to the TATA sequence (site I). Mutagenesis studies indicated that both sites II and III, in addition to site I, are needed for complete basal and E1A-induced transcription. These results suggest that multiple cellular factors are involved in both the basal and E1A-induced transcriptional regulation of the E3 promoter, and that either of two upstream regions are capable of stabilizing factor binding to the TATA sequence.

  15. Application of high-throughput sequencing to measure the performance of commonly used selective cultivation methods for the foodborne pathogen Campylobacter.

    PubMed

    Oakley, Brian B; Morales, Cesar A; Line, J Eric; Seal, Bruce S; Hiett, Kelli L

    2012-02-01

    Campylobacter is an important foodborne human pathogen, which has traditionally been studied using a variety of selective cultivation methods. Here we use next-generation sequencing to ask the following: (i) how selective are commonly used Campylobacter cultivation methods relative to the initial sample and (ii) how do the specificity and sensitivity of these methods compare with one another? To answer these questions, we used 16S rRNA tagged-pyrosequencing to sequence directly from a pooled fecal sample representing a c. 16,000 bird poultry flock and compared these data to exhaustive sequencing of colonies formed after plating. We compared five commonly used media [Cefex, Cape Town, modified cefoperazone charcoal deoxycholate agar (mCCDA), Campy-Line agar (CLA), and Campy-CVA agar (CVA)], two incubation atmospheres (10% CO(2), 5% O(2), 85% N(2) and 10% CO(2), 10% H(2), 80% N(2)), and two incubation temperatures (37 and 42 °C). Analysis of 404,104 total sequence reads, including 19 472 total fecal reads, revealed Campylobacter represented only a small proportion (< 0.04%) of sequences present in the feces, but 88-97% of sequences from each media type. Incubation atmosphere had little effect on recovery, but a significant difference in media specificity (more non-Campylobacter OTUs; P = 0.028) was found at 42 vs. 37 °C. The most common non-Campylobacter sequence type was Proteus, which ranged from 0.04% of sequences (mCCDA) to 10.8% (Cape Town). High-throughput sequencing provides a novel and powerful approach to measure the performance of selective media, which remain widely used for research and regulatory purposes.

  16. Mining the LIPG allelic spectrum reveals the contribution of rare and common regulatory variants to HDL cholesterol.

    PubMed

    Khetarpal, Sumeet A; Edmondson, Andrew C; Raghavan, Avanthi; Neeli, Hemanth; Jin, Weijun; Badellino, Karen O; Demissie, Serkalem; Manning, Alisa K; DerOhannessian, Stephanie L; Wolfe, Megan L; Cupples, L Adrienne; Li, Mingyao; Kathiresan, Sekar; Rader, Daniel J

    2011-12-01

    Genome-wide association studies (GWAS) have successfully identified loci associated with quantitative traits, such as blood lipids. Deep resequencing studies are being utilized to catalogue the allelic spectrum at GWAS loci. The goal of these studies is to identify causative variants and missing heritability, including heritability due to low frequency and rare alleles with large phenotypic impact. Whereas rare variant efforts have primarily focused on nonsynonymous coding variants, we hypothesized that noncoding variants in these loci are also functionally important. Using the HDL-C gene LIPG as an example, we explored the effect of regulatory variants identified through resequencing of subjects at HDL-C extremes on gene expression, protein levels, and phenotype. Resequencing a portion of the LIPG promoter and 5' UTR in human subjects with extreme HDL-C, we identified several rare variants in individuals from both extremes. Luciferase reporter assays were used to measure the effect of these rare variants on LIPG expression. Variants conferring opposing effects on gene expression were enriched in opposite extremes of the phenotypic distribution. Minor alleles of a common regulatory haplotype and noncoding GWAS SNPs were associated with reduced plasma levels of the LIPG gene product endothelial lipase (EL), consistent with its role in HDL-C catabolism. Additionally, we found that a common nonfunctional coding variant associated with HDL-C (rs2000813) is in linkage disequilibrium with a 5' UTR variant (rs34474737) that decreases LIPG promoter activity. We attribute the gene regulatory role of rs34474737 to the observed association of the coding variant with plasma EL levels and HDL-C. Taken together, the findings show that both rare and common noncoding regulatory variants are important contributors to the allelic spectrum in complex trait loci. PMID:22174694

  17. Beyond Junk-Variable Tandem Repeats as Facilitators of Rapid Evolution of Regulatory and Coding Sequences

    PubMed Central

    Gemayel, Rita; Cho, Janice; Boeynaems, Steven; Verstrepen, Kevin J.

    2012-01-01

    Copy Number Variations (CNVs) and Single Nucleotide Polymorphisms (SNPs) have been the major focus of most large-scale comparative genomics studies to date. Here, we discuss a third, largely ignored, type of genetic variation, namely changes in tandem repeat number. Historically, tandem repeats have been designated as non functional “junk” DNA, mostly as a result of their highly unstable nature. With the exception of tandem repeats involved in human neurodegenerative diseases, repeat variation was often believed to be neutral with no phenotypic consequences. Recent studies, however, have shown that as many as 10% to 20% of coding and regulatory sequences in eukaryotes contain an unstable repeat tract. Contrary to initial suggestions, tandem repeat variation can have useful phenotypic consequences. Examples include rapid variation in microbial cell surface, tuning of internal molecular clocks in flies and the dynamic morphological plasticity in mammals. As such, tandem repeats can be useful functional elements that facilitate evolvability and rapid adaptation. PMID:24704980

  18. Neuronal precursor-specific activity of a human doublecortin regulatory sequence.

    PubMed

    Karl, Claudia; Couillard-Despres, Sebastien; Prang, Peter; Munding, Matthias; Kilb, Werner; Brigadski, Tanja; Plötz, Sonja; Mages, Wolfgang; Luhmann, Heiko; Winkler, Jürgen; Bogdahn, Ulrich; Aigner, Ludwig

    2005-01-01

    The doublecortin (DCX) gene encodes a 40-kDa microtubule-associated protein specifically expressed in neuronal precursors of the developing and adult CNS. Due to its specific expression pattern, attention was drawn to DCX as a marker for neuronal precursors and neurogenesis, thereby underscoring the importance of its promoter identification and promoter analysis. Here, we analysed the human DCX regulatory sequence and confined it to a 3.5-kb fragment upstream of the ATG start codon. We demonstrate by transient transfection experiments that this fragment is sufficient and specific to drive expression of reporter genes in embryonic and adult neuronal precursors. The activity of this regulatory fragment overlapped with the expression of endogenous DCX and with the young neuronal markers class III beta-tubulin isotype and microtubule-associated protein Map2ab but not with glial or oligodendroglial markers. Electrophysiological data further confirmed the immature neuronal nature of these cells. Deletions within the 3.5-kb region demonstrated the relevance of specific regions containing transcription factor-binding sites. Moreover, application of neurogenesis-related growth factors in the neuronal precursor cultures suggested the lack of direct signalling of these factors on the DCX promoter construct. PMID:15663475

  19. Development of polymorphic expressed sequence tag-single sequence repeat markers in the common Chinese cuttlefish, Sepiella maindroni.

    PubMed

    Li, R H; Lu, S K; Zhang, C L; Song, W W; Mu, C K; Wang, C L

    2014-01-01

    The common Chinese cuttlefish (Sepiella maindroni) is one of the popular edible cephalopod consumed across Asia. To facilitate the population genetic investigation of this species, we developed fourteen polymorphic microsatellite makers from expressed sequence tags of S. maindroni. The number of alleles at each locus ranged from 6 to 10 with an average of 7.9 alleles per locus. The ranges of observed and expected heterozygosity were from 0.615 to 0.962 and 0.685 to 0.888, respectively. Four loci were found deviated significantly from Hardy-Weinberg equilibrium. The polymorphism information content ranged from 0.638 to 0.833. These polymorphic microsatellite loci will be helpful for the population genetic, genetic linkage map, and other genetic studies of S. maindroni. PMID:25117305

  20. Global Mapping of Open Chromatin Regulatory Elements by Formaldehyde-Assisted Isolation of Regulatory Elements Followed by Sequencing (FAIRE-seq).

    PubMed

    Bianco, Stéphanie; Rodrigue, Sébastien; Murphy, Bruce D; Gévry, Nicolas

    2015-01-01

    Genetic information is organized in a complex structure composed of DNA and proteins together designated chromatin. Chromatin plays a dynamic role in transcriptional processes in that alteration of the interaction between its components results in the deregulation of cellular transcriptional program. Modification of epigenetic marks, variation in the precise positioning of nucleosomes, and consequent mobilization of nucleosomes regulate the access of various transcriptional factors to its underlying DNA template. Nucleosome-depleted regions, also designated open chromatin domains, are associated with active DNA regulatory elements, including promoters, enhancers, silencers, and insulators. Here, we describe the protocol of a rapid and simple technique entitled FAIRE (formaldehyde-assisted isolation of regulatory elements). Combined with high-throughput sequencing (FAIRE-seq), this procedure allows isolation of nucleosome-free regions and their mapping along the genome, thereby providing a global view of cell-specific regulatory elements.

  1. Common DNA sequences with potential for detection of genetically manipulated organisms in food.

    PubMed

    MacCormick, C A; Griffin, H G; Underwood, H M; Gasson, M J

    1998-06-01

    Foods produced by genetic engineering technology are now appearing on the market and many more are likely to emerge in the future. The safety aspects, regulation, and labelling of these foods are still contentious issues in most countries and recent surveys highlight consumer concerns about the safety and labelling of genetically modified food. In most countries it is necessary to have approval for the use of genetically manipulated organisms (GMOs) in the production of food. In order to police regulations, a technology to detect such foods is desirable. In addition, a requirement to label approved genetically modified food would necessitate a monitoring system. One solution is to 'tag' approved GMOs with some form of biological or genetic marker, permitting the surveillance of foods for the presence of approved products of genetic engineering. While non-approved GMOs would not be detected by such a surveillance, they might be detected by a screen for DNA sequences common to all or most GMOs. This review focuses on the potential of using common DNA sequences as detection probes for GMOs. The identification of vector sequences, plant transcription terminators, and marker genes by PCR and hybridization techniques is discussed.

  2. Sequencing of SCN5A identifies rare and common variants associated with cardiac conduction

    PubMed Central

    Magnani, Jared W.; Brody, Jennifer A.; Prins, Bram P.; Arking, Dan E.; Lin, Honghuang; Yin, Xiaoyan; Liu, Ching-Ti; Morrison, Alanna C.; Zhang, Feng; Spector, Tim D.; Alonso, Alvaro; Bis, Joshua C.; Heckbert, Susan R.; Lumley, Thomas; Sitlani, Colleen M.; Cupples, L. Adrienne; Lubitz, Steven A.; Soliman, Elsayed Z.; Pulit, Sara L.; Newton-Cheh, Christopher; O'Donnell, Christopher J.; Ellinor, Patrick T.; Benjamin, Emelia J.; Muzny, Donna M.; Gibbs, Richard A.; Santibanez, Jireh; Taylor, Herman A.; Rotter, Jerome I.; Lange, Leslie A.; Psaty, Bruce M.; Jackson, Rebecca; Rich, Stephen S.; Boerwinkle, Eric; Jamshidi, Yalda; Sotoodehnia, Nona

    2014-01-01

    Background The cardiac sodium channel SCN5A regulates atrioventricular and ventricular conduction. Genetic variants in this gene are associated with PR and QRS intervals. We sought to further characterize the contribution of rare and common coding variation in SCN5A to cardiac conduction. Methods and Results In the Cohorts for Heart and Aging Research in Genomic Epidemiology Targeted Sequencing Study (CHARGE), we performed targeted exonic sequencing of SCN5A (n=3699, European-ancestry individuals) and identified 4 common (minor allele frequency >1%) and 157 rare variants. Common and rare SCN5A coding variants were examined for association with PR and QRS intervals through meta-analysis of European ancestry participants from CHARGE, NHLBI’s Exome Sequencing Project (ESP, n=607) and the UK10K (n=1275) and by examining ESP African-ancestry participants (N=972). Rare coding SCN5A variants in aggregate were associated with PR interval in European and African-ancestry participants (P=1.3×10−3). Three common variants were associated with PR and/or QRS interval duration among European-ancestry participants and one among African-ancestry participants. These included two well-known missense variants; rs1805124 (H558R) was associated with PR and QRS shortening in European-ancestry participants (P=6.25×10−4 and P=5.2×10−3 respectively) and rs7626962 (S1102Y) was associated with PR shortening in those of African ancestry (P=2.82×10−3). Among European-ancestry participants, two novel synonymous variants, rs1805126 and rs6599230, were associated with cardiac conduction. Our top signal, rs1805126 was associated with PR and QRS lengthening (P=3.35×10−7 and P=2.69×10−4 respectively), and rs6599230 was associated with PR shortening (P=2.67×10−5). Conclusions By sequencing SCN5A, we identified novel common and rare coding variants associated with cardiac conduction. PMID:24951663

  3. Sequence analysis of the myosin regulatory light chain gene of the vestimentiferan Riftia pachyptila.

    PubMed

    Ravaux, J; Hassanin, A; Deutsch, J; Gaill, F; Markmann-Mulisch, U

    2001-01-24

    We have isolated and characterized a cDNA (DNA complementary to RNA) clone (Rf69) from the vestimentiferan Riftia pachyptila. The cDNA insert consists of 1169 base pairs. The aminoacid sequence deduced from the longest reading frame is 193 residues in length, and clearly characterized it as a myosin regulatory light chain (RLC). The RLC primary structure is described in relation to its function in muscle contraction. The comparison with other RLCs suggested that Riftia myosin is probably regulated through its RLC either by phosphorylation like the vertebrate smooth muscle myosins, and/or by Ca2+-binding like the mollusk myosins. Riftia RLC possesses a N-terminal extension lacking in all other species besides the earthworm Lumbricus terrestris. Aminoacid sequence comparisons with a number of RLCs from vertebrates and invertebrates revealed a relatively high identity score (64%) between Riftia RLC and the homologous gene from Lumbricus. The relationships between the members of the myosin RLCs were examined by two phylogenetic methods, i.e. distance matrix and maximum parsimony. The resulting trees depict the grouping of the RLCs according to their role in myosin activity regulation. In all trees, Riftia RLC groups with RLCs that depend on Ca2+-binding for myosin activity regulation. PMID:11223252

  4. Stable intronic sequence RNAs have possible regulatory roles in Drosophila melanogaster.

    PubMed

    Pek, Jun Wei; Osman, Ismail; Tay, Mandy Li-Ian; Zheng, Ruther Teo

    2015-10-26

    Stable intronic sequence RNAs (sisRNAs) have been found in Xenopus tropicalis, human cell lines, and Epstein-Barr virus; however, the biological significance of sisRNAs remains poorly understood. We identify sisRNAs in Drosophila melanogaster by deep sequencing, reverse transcription polymerase chain reaction, and Northern blotting. We characterize a sisRNA (sisR-1) from the regena (rga) locus and show that it can be processed from the precursor messenger RNA (pre-mRNA). We also document a cis-natural antisense transcript (ASTR) from the rga locus, which is highly expressed in early embryos. During embryogenesis, ASTR promotes robust rga pre-mRNA expression. Interestingly, sisR-1 represses ASTR, with consequential effects on rga pre-mRNA expression. Our results suggest a model in which sisR-1 modulates its host gene expression by repressing ASTR during embryogenesis. We propose that sisR-1 belongs to a class of sisRNAs with probable regulatory activities in Drosophila.

  5. Stable intronic sequence RNAs have possible regulatory roles in Drosophila melanogaster

    PubMed Central

    Osman, Ismail; Tay, Mandy Li-Ian; Zheng, Ruther Teo

    2015-01-01

    Stable intronic sequence RNAs (sisRNAs) have been found in Xenopus tropicalis, human cell lines, and Epstein-Barr virus; however, the biological significance of sisRNAs remains poorly understood. We identify sisRNAs in Drosophila melanogaster by deep sequencing, reverse transcription polymerase chain reaction, and Northern blotting. We characterize a sisRNA (sisR-1) from the regena (rga) locus and show that it can be processed from the precursor messenger RNA (pre-mRNA). We also document a cis-natural antisense transcript (ASTR) from the rga locus, which is highly expressed in early embryos. During embryogenesis, ASTR promotes robust rga pre-mRNA expression. Interestingly, sisR-1 represses ASTR, with consequential effects on rga pre-mRNA expression. Our results suggest a model in which sisR-1 modulates its host gene expression by repressing ASTR during embryogenesis. We propose that sisR-1 belongs to a class of sisRNAs with probable regulatory activities in Drosophila. PMID:26504165

  6. Sequence variation in the androgen receptor gene is not a common determinant of male sexual orientation

    SciTech Connect

    Macke, J.P.; Nathans, J.; King, V.L. ); Hu, N.; Hu, S.; Hamer, D.; Bailey, M. ); Brown, T. )

    1993-10-01

    To test the hypothesis that DNA sequence variation in the androgen receptor gene plays a causal role in the development of male sexual orientation, the authors have (1) measured the degree of concordance of androgen receptor alleles in 36 pairs of homosexual brothers, (2) compared the lengths of polyglutamine and polyglycine tracts in the amino-terminal domain of the androgen receptor in a sample of 197 homosexual males and 213 unselected subjects, and (3) screened the entire androgen receptor coding region for sequence variation by PCR and denaturing gradient-gel electrophoresis (DGGE) and/or single-strand conformation polymorphism analysis in 20 homosexual males with homosexual or bisexual brothers and one homosexual male with no homosexual brothers, and screened the amino-terminal domain of the receptor for sequence variation in an additional 44 homosexual males, 37 of whom had one or more first- or second-degree male relatives who were either homosexual or bisexual. These analyses show that (1) homosexual brothers are as likely to be discordant as concordant for androgen receptor alleles; (2) there are no large-scale differences between the distributions of polyglycine or polyglutamine tract lengths in the homosexual and control groups; and (3) coding region sequence variation is not commonly found within the androgen receptor gene of homosexual men. The DGGE screen identified two rare amino acid substitutions, ser[sup 205] -to-arg and glu[sup 793]-to-asp, the biological significance of which is unknown. 32 refs., 2 figs., 2 tabs.

  7. Common regulatory control of CTP synthase enzyme activity and filament formation

    PubMed Central

    Noree, Chalongrat; Monfort, Elena; Shiau, Andrew K.; Wilhelm, James E.

    2014-01-01

    The ability of enzymes to assemble into visible supramolecular complexes is a widespread phenomenon. Such complexes have been hypothesized to play a number of roles; however, little is known about how the regulation of enzyme activity is coupled to the assembly/disassembly of these cellular structures. CTP synthase is an ideal model system for addressing this question because its activity is regulated via multiple mechanisms and its filament-forming ability is evolutionarily conserved. Our structure–function studies of CTP synthase in Saccharomyces cerevisiae reveal that destabilization of the active tetrameric form of the enzyme increases filament formation, suggesting that the filaments comprise inactive CTP synthase dimers. Furthermore, the sites responsible for feedback inhibition and allosteric activation control filament length, implying that multiple regions of the enzyme can influence filament structure. In contrast, blocking catalysis without disrupting the regulatory sites of the enzyme does not affect filament formation or length. Together our results argue that the regulatory sites that control CTP synthase function, but not enzymatic activity per se, are critical for controlling filament assembly. We predict that the ability of enzymes to form supramolecular structures in general is closely coupled to the mechanisms that regulate their activity. PMID:24920825

  8. The genome sequence of the emerging common midwife toad virus identifies an evolutionary intermediate within ranaviruses.

    PubMed

    Mavian, Carla; López-Bueno, Alberto; Balseiro, Ana; Casais, Rosa; Alcamí, Antonio; Alejo, Alí

    2012-04-01

    Worldwide amphibian population declines have been ascribed to global warming, increasing pollution levels, and other factors directly related to human activities. These factors may additionally be favoring the emergence of novel pathogens. In this report, we have determined the complete genome sequence of the emerging common midwife toad ranavirus (CMTV), which has caused fatal disease in several amphibian species across Europe. Phylogenetic and gene content analyses of the first complete genomic sequence from a ranavirus isolated in Europe show that CMTV is an amphibian-like ranavirus (ALRV). However, the CMTV genome structure is novel and represents an intermediate evolutionary stage between the two previously described ALRV groups. We find that CMTV clusters with several other ranaviruses isolated from different hosts and locations which might also be included in this novel ranavirus group. This work sheds light on the phylogenetic relationships within this complex group of emerging, disease-causing viruses.

  9. The Genome Sequence of the Emerging Common Midwife Toad Virus Identifies an Evolutionary Intermediate within Ranaviruses

    PubMed Central

    Mavian, Carla; López-Bueno, Alberto; Balseiro, Ana; Casais, Rosa; Alcamí, Antonio

    2012-01-01

    Worldwide amphibian population declines have been ascribed to global warming, increasing pollution levels, and other factors directly related to human activities. These factors may additionally be favoring the emergence of novel pathogens. In this report, we have determined the complete genome sequence of the emerging common midwife toad ranavirus (CMTV), which has caused fatal disease in several amphibian species across Europe. Phylogenetic and gene content analyses of the first complete genomic sequence from a ranavirus isolated in Europe show that CMTV is an amphibian-like ranavirus (ALRV). However, the CMTV genome structure is novel and represents an intermediate evolutionary stage between the two previously described ALRV groups. We find that CMTV clusters with several other ranaviruses isolated from different hosts and locations which might also be included in this novel ranavirus group. This work sheds light on the phylogenetic relationships within this complex group of emerging, disease-causing viruses. PMID:22301140

  10. Understanding the Effects of Users' Behaviors on Effectiveness of Different Exogenous Regulatory Common Pool Resource Management Institutions

    NASA Astrophysics Data System (ADS)

    Madani, K.; Dinar, A.

    2013-12-01

    Tragedy of the commons is generally recognized as one of the possible destinies for common pool resources (CPRs). To avoid the tragedy of the commons and prolonging the life of CPRs, users may show different behavioral characteristics and use different rationales for CPR planning and management. Furthermore, regulators may adopt different strategies for sustainable management of CPRs. The effectiveness of different regulatory exogenous management institutions cannot be evaluated through conventional CPR models since they assume that either users base their behavior on individual rationality and adopt a selfish behavior (Nash behavior), or that the users seek the system's optimal solution without giving priority to their own interests. Therefore, conventional models fail to reliably predict the outcome of CPR problems in which parties may have a range of behavioral characteristics, putting them somewhere in between the two types of behaviors traditionally considered. This work examines the effectiveness of different regulatory exogenous CPR management institutions through a user-based model (as opposed to a system-based model). The new modeling framework allows for consideration of sensitivity of the results to different behavioral characteristics of interacting CPR users. The suggested modeling approach is applied to a benchmark groundwater management problem. Results indicate that some well-known exogenous management institutions (e.g. taxing) are ineffective in sustainable management of CPRs in most cases. Bankruptcy-based management can be helpful, but determination of the fair level of cutbacks remains challenging under this type of institution. Furthermore, some bankruptcy rules such as the Constrained Equal Award (CEA) method are more beneficial to wealthier users, failing to establish social justice. Quota-based and CPR status-based management perform as the most promising and robust regulatory exogenous institutions in prolonging the CPR's life and

  11. Safety and regulatory review of dyes commonly used as excipients in pharmaceutical and nutraceutical applications.

    PubMed

    Pérez-Ibarbia, Leire; Majdanski, Tobias; Schubert, Stephanie; Windhab, Norbert; Schubert, Ulrich S

    2016-10-10

    Color selection is one of the key elements of building a strong brand development and product identity in the pharmaceutical industry, besides to prevent counterfeiting. Moreover, colored pharmaceutical dosage forms may increase patient compliance and therapy enhancement. Although most synthetic dyes are classified as safe, their regulations are stricter than other classes of excipients. Safety concerns have increased during the last years but the efforts to change to natural dyes seem to be not promising. Their instability problems and the development of "non-toxic" dyes is still a challenge. This review focuses specifically on the issues related to dye selection and summarizes the current regulatory status. A deep awareness of toxicological data based on the public domain, making sure the compliance of standards for regulation and safety for successful product development is provided. In addition, synthetic strategies are provided to covalently bind dyes on polymers to possibly overcome toxicity issues. PMID:27531552

  12. Co-ordinate expression of the two threonyl-tRNA synthetase genes in Bacillus subtilis: control by transcriptional antitermination involving a conserved regulatory sequence.

    PubMed Central

    Putzer, H; Gendron, N; Grunberg-Manago, M

    1992-01-01

    In Bacillus subtilis, two genes, thrS and thrZ, encode distinct threonyl-tRNA synthetase enzymes. Normally, only the thrS gene is expressed. Here we show that either gene, thrS or thrZ, is sufficient for normal cell growth and sporulation. Reducing the intracellular ThrS protein concentration induces thrZ expression in a dose-compensatory manner. Starvation for threonine simultaneously induces thrZ and stimulates thrS expression. The 5'-leader sequences of thrS and thrZ contain, respectively, one and three transcription terminators preceded by a conserved sequence. We show that this sequence is essential for the regulation of thrS via a transcriptional antitermination mechanism. We propose that both genes, thrS and thrZ, are regulated by the same mechanism such that the additional regulatory domains present before thrZ account for its non-expression. In contrast to Escherichia coli, structurally similar regulatory domains, i.e. the consensus sequence preceding a terminator structure, are found in the leader regions of most aminoacyl-tRNA synthetase genes of Gram-positive bacteria. This suggests that they are regulated by a common mechanism. Images PMID:1379177

  13. Construction and Analysis of an Integrated Regulatory Network Derived from High-Throughput Sequencing Data

    PubMed Central

    Cheng, Chao; Yan, Koon-Kiu; Hwang, Woochang; Qian, Jiang; Bhardwaj, Nitin; Rozowsky, Joel; Lu, Zhi John; Niu, Wei; Alves, Pedro; Kato, Masaomi; Snyder, Michael; Gerstein, Mark

    2011-01-01

    We present a network framework for analyzing multi-level regulation in higher eukaryotes based on systematic integration of various high-throughput datasets. The network, namely the integrated regulatory network, consists of three major types of regulation: TF→gene, TF→miRNA and miRNA→gene. We identified the target genes and target miRNAs for a set of TFs based on the ChIP-Seq binding profiles, the predicted targets of miRNAs using annotated 3′UTR sequences and conservation information. Making use of the system-wide RNA-Seq profiles, we classified transcription factors into positive and negative regulators and assigned a sign for each regulatory interaction. Other types of edges such as protein-protein interactions and potential intra-regulations between miRNAs based on the embedding of miRNAs in their host genes were further incorporated. We examined the topological structures of the network, including its hierarchical organization and motif enrichment. We found that transcription factors downstream of the hierarchy distinguish themselves by expressing more uniformly at various tissues, have more interacting partners, and are more likely to be essential. We found an over-representation of notable network motifs, including a FFL in which a miRNA cost-effectively shuts down a transcription factor and its target. We used data of C. elegans from the modENCODE project as a primary model to illustrate our framework, but further verified the results using other two data sets. As more and more genome-wide ChIP-Seq and RNA-Seq data becomes available in the near future, our methods of data integration have various potential applications. PMID:22125477

  14. Isolation of expressed sequences from the region commonly deleted in Velo-cardio-facial syndrome

    SciTech Connect

    Sirotkin, H.; Morrow, B.; DasGupta, R.

    1994-09-01

    Velo-cardio-facial syndrome (VCFS) is a relatively common autosomal dominant genetic disorder characterized by cleft palate, cardiac abnormalities, learning disabilities and a characteristic facial dysmorphology. Most VCFS patients have interstitial deletions of 22q11 of 1-2 mb. In an effort to isolate the gene(s) responsible for VCFS we have utilized a hybrid selection protocol to recover expressed sequences from three non-overlapping YACs comprising almost 1 mb of the commonly deleted region. Total yeast genomic DNA or isolated YAC DNA was immobilized on Hybond-N filters, blocked with yeast and human ribosomal and human repetitive sequences and hybridized with a mixture of random primed short fragment cDNA libraries. Six human short fragment libraries derived from total fetus, fetal brain, adult brain, testes, thymus and spleen have been used for the selections. Short fragment cDNAs retained on the filter were passed through a second round of selection and cloned into lambda gt10. cDNAs shown to originate from the YACs and from chromosome 22 are being used to isolate full length cDNAs. Three genes known to be present on these YACs, catechol-O-methyltransferase, tuple 1 and clathrin heavy chain have been recovered. Additionally, a gene related to the murine p120 gene and a number of novel short cDNAs have been isolated. The role of these genes in VCFS is being investigated.

  15. Comprehensive analysis of hydrogen bonds in regulatory protein DNA-complexes: in search of common principles.

    PubMed

    Mandel-Gutfreund, Y; Schueler, O; Margalit, H

    1995-10-20

    A systematic analysis of hydrogen bonds between regulatory proteins and their DNA targets is presented, based on 28 crystallographically solved complexes. All possible hydrogen bonds were screened and classified into different types: those that involve the amino acid side-chains and DNA base edges and those that involve the backbone atoms of the molecules. For each interaction type, all bonds were characterized and a statistical analysis was performed to reveal significant amino acid-base interdependence. The interactions between the amino acid side-chains and DNA backbone constitute about half of the interactions, but did not show any amino acid-base correlation. Interactions via the protein backbone were also observed, predominantly with the DNA backbone. As expected, the most significant pairing preference was demonstrated for interactions between the amino acid side-chains and the DNA base edges. The statistically significant relationships could mostly be explained by the chemical nature of the participants. However, correlations that could not be trivially predicted from the hydrogen bonding potential of the residues were also identified, like the preference of lysine for guanine over adenine, or the preference of glutamic acid for cystosine over adenine. While Lys x G interactions were very frequent and spread over various families, the Glu x C interactions were found mainly in the basic helix-loop-helix family. Further examination of the side-chain-base edge contacts at the atomic level revealed a trend of the amino acids to contact the DNA by their donor atoms, preferably at position W2 in the major groove. In most cases it seems that the interactions are not guided simply by the presence of a required atom in a specific position in the groove, but that the identity of the base possessing this atom is crucial. This may have important implications in molecular design experiments.

  16. IgH sequences in common variable immune deficiency reveal altered B cell development and selection**

    PubMed Central

    Roskin, Krishna M.; Simchoni, Noa; Liu, Yi; Lee, Ji-Yeun; Seo, Katie; Hoh, Ramona A.; Pham, Tho; Park, Joon H.; Furman, David; Dekker, Cornelia L.; Davis, Mark M.; James, Judith A.; Nadeau, Kari C.; Cunningham-Rundles, Charlotte; Boyd, Scott D.

    2015-01-01

    Common variable immune deficiency (CVID) is the most common symptomatic primary immune deficiency, affecting ∼1 in 25,000 persons. These patients suffer from impaired antibody responses, autoimmunity, and susceptibility to lymphoid cancers. To explore the cellular basis for these clinical phenotypes, we conducted high-throughput DNA sequencing of immunoglobulin heavy chain gene rearrangements from 93 CVID patients and 105 control subjects and sorted naïve and memory B cells from 13 of the CVID patients and 10 of the control subjects. CVID patients showed abnormal VDJ rearrangement and abnormal formation of complementarity determining region 3 (CDR3). We observed decreased selection against antibodies with long CDR3 regions in memory repertoires and decreased V gene replacement, offering possible mechanisms for increased patient autoreactivity. Our data indicate that patient immunodeficiency might derive both from decreased diversity of the naïve B cell pool and decreased somatic hypermutation in memory repertoires. CVID patients also exhibited abnormal clonal expansion of unmutated B cells relative to controls. Although impaired B cell germinal center activation is commonly viewed as causative in CVID, these data indicate that CVID B cells diverge from controls as early as the pro-B cell stage and suggest possible explanations for the increased incidence of autoimmunity, immunodeficiency, and lymphoma CVID patients. PMID:26311730

  17. DNA sequence and translational product of a new nodulation-regulatory locus: syrM has sequence similarity to NodD proteins.

    PubMed Central

    Barnett, M J; Long, S R

    1990-01-01

    Rhizobium meliloti nodulation (nod) genes are expressed when activated by trans-acting proteins in the NodD family. The nodD1 and nodD2 gene products activate nod promoters when cells are exposed to plant-synthesized signal molecules. Alternatively, the same nod promoters are activated by the nodD3 gene when nodD3 is carried in trans along with a closely linked global regulatory locus, syrM (symbiotic regulator) (J. T. Mulligan and S. R. Long, Genetics 122:7-18, 1989). In this article we report the nucleotide sequence of a 2.6-kilobase SphI fragment from R. meliloti SU47 containing syrM. Expression from this locus was confirmed by using in vitro transcription-translation assays. The open reading frame encoded a protein of either 33 or 36 kilodaltons whose sequence shows similarity to NodD regulatory proteins. Images PMID:2361944

  18. The effectiveness of common thermo-regulatory behaviours in a cool temperate grasshopper.

    PubMed

    Harris, Rebecca M B; McQuillan, Peter; Hughes, Lesley

    2015-08-01

    Behavioural thermoregulation has the potential to alleviate the short-term impacts of climate change on some small ectotherms, without the need for changes to species distributions or genetic adaptation. We illustrate this by measuring the effect of behaviour in a cool temperate species of grasshopper (Phaulacridium vittatum) over a range of spatial and temporal scales in laboratory and natural field experiments. Microhabitat selection at the site scale was tested in free-ranging grasshoppers and related to changing thermal quality over a daily period. Artificial warming experiments were then used to measure the temperature at which common thermoregulatory behaviours are initiated and the subsequent reductions in body temperature. Behavioural means such as timing of activity, choice of substrates with optimum surface temperatures, shade seeking and postural adjustments (e.g. stilting, vertical orientation) were found to be highly effective at maintaining preferred body temperature. The maximum voluntarily tolerated temperature (MVT) was determined to be 44°C±0.4°C, indicating the upper bounds of thermal flexibility in this species. Behavioural thermoregulation effectively enables small ectotherms to regulate exposure to changing environmental temperatures and utilize the spatially and temporally heterogeneous environments they occupy. Species such as the wingless grasshopper, although adapted to cool temperate conditions, are likely to be well equipped to respond successfully to coarse scale climate change.

  19. The effectiveness of common thermo-regulatory behaviours in a cool temperate grasshopper.

    PubMed

    Harris, Rebecca M B; McQuillan, Peter; Hughes, Lesley

    2015-08-01

    Behavioural thermoregulation has the potential to alleviate the short-term impacts of climate change on some small ectotherms, without the need for changes to species distributions or genetic adaptation. We illustrate this by measuring the effect of behaviour in a cool temperate species of grasshopper (Phaulacridium vittatum) over a range of spatial and temporal scales in laboratory and natural field experiments. Microhabitat selection at the site scale was tested in free-ranging grasshoppers and related to changing thermal quality over a daily period. Artificial warming experiments were then used to measure the temperature at which common thermoregulatory behaviours are initiated and the subsequent reductions in body temperature. Behavioural means such as timing of activity, choice of substrates with optimum surface temperatures, shade seeking and postural adjustments (e.g. stilting, vertical orientation) were found to be highly effective at maintaining preferred body temperature. The maximum voluntarily tolerated temperature (MVT) was determined to be 44°C±0.4°C, indicating the upper bounds of thermal flexibility in this species. Behavioural thermoregulation effectively enables small ectotherms to regulate exposure to changing environmental temperatures and utilize the spatially and temporally heterogeneous environments they occupy. Species such as the wingless grasshopper, although adapted to cool temperate conditions, are likely to be well equipped to respond successfully to coarse scale climate change. PMID:26267501

  20. Reprint of: The effectiveness of common thermo-regulatory behaviours in a cool temperate grasshopper.

    PubMed

    Harris, Rebecca M B; McQuillan, Peter; Hughes, Lesley

    2015-12-01

    Behavioural thermoregulation has the potential to alleviate the short-term impacts of climate change on some small ectotherms, without the need for changes to species distributions or genetic adaptation. We illustrate this by measuring the effect of behaviour in a cool temperate species of grasshopper (Phaulacridium vittatum) over a range of spatial and temporal scales in laboratory and natural field experiments. Microhabitat selection at the site scale was tested in free-ranging grasshoppers and related to changing thermal quality over a daily period. Artificial warming experiments were then used to measure the temperature at which common thermoregulatory behaviours are initiated and the subsequent reductions in body temperature. Behavioural means such as timing of activity, choice of substrates with optimum surface temperatures, shade seeking and postural adjustments (e.g. stilting, vertical orientation) were found to be highly effective at maintaining preferred body temperature. The maximum voluntarily tolerated temperature (MVT) was determined to be 44 °C ± 0.4 °C, indicating the upper bounds of thermal flexibility in this species. Behavioural thermoregulation effectively enables small ectotherms to regulate exposure to changing environmental temperatures and utilize the spatially and temporally heterogeneous environments they occupy. Species such as the wingless grasshopper, although adapted to cool temperate conditions, are likely to be well equipped to respond successfully to coarse scale climate change. PMID:26615722

  1. CLAMMS: a scalable algorithm for calling common and rare copy number variants from exome sequencing data

    PubMed Central

    Packer, Jonathan S.; Maxwell, Evan K.; O’Dushlaine, Colm; Lopez, Alexander E.; Dewey, Frederick E.; Chernomorsky, Rostislav; Baras, Aris; Overton, John D.; Habegger, Lukas; Reid, Jeffrey G.

    2016-01-01

    Motivation: Several algorithms exist for detecting copy number variants (CNVs) from human exome sequencing read depth, but previous tools have not been well suited for large population studies on the order of tens or hundreds of thousands of exomes. Their limitations include being difficult to integrate into automated variant-calling pipelines and being ill-suited for detecting common variants. To address these issues, we developed a new algorithm—Copy number estimation using Lattice-Aligned Mixture Models (CLAMMS)—which is highly scalable and suitable for detecting CNVs across the whole allele frequency spectrum. Results: In this note, we summarize the methods and intended use-case of CLAMMS, compare it to previous algorithms and briefly describe results of validation experiments. We evaluate the adherence of CNV calls from CLAMMS and four other algorithms to Mendelian inheritance patterns on a pedigree; we compare calls from CLAMMS and other algorithms to calls from SNP genotyping arrays for a set of 3164 samples; and we use TaqMan quantitative polymerase chain reaction to validate CNVs predicted by CLAMMS at 39 loci (95% of rare variants validate; across 19 common variant loci, the mean precision and recall are 99% and 94%, respectively). In the Supplementary Materials (available at the CLAMMS Github repository), we present our methods and validation results in greater detail. Availability and implementation: https://github.com/rgcgithub/clamms (implemented in C). Contact: jeffrey.reid@regeneron.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26382196

  2. SNP marker development for linkage map construction, anchoring of the common bean whole genome sequence and genetic research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our objectives were to identify SNP DNA markers based on a diverse set of common bean cultivars via next generation sequencing technologies; to develop Illumina Infinium BeadChip assays containing SNPs with high polymorphism within and between common bean market classes, to create high density genet...

  3. Cis-regulatory sequence variation and association with Mycoplasma load in natural populations of the house finch (Carpodacus mexicanus)

    PubMed Central

    Backström, Niclas; Shipilina, Daria; Blom, Mozes P K; Edwards, Scott V

    2013-01-01

    Characterization of the genetic basis of fitness traits in natural populations is important for understanding how organisms adapt to the changing environment and to novel events, such as epizootics. However, candidate fitness-influencing loci, such as regulatory regions, are usually unavailable in nonmodel species. Here, we analyze sequence data from targeted resequencing of the cis-regulatory regions of three candidate genes for disease resistance (CD74, HSP90α, and LCP1) in populations of the house finch (Carpodacus mexicanus) historically exposed (Alabama) and naïve (Arizona) to Mycoplasma gallisepticum. Our study, the first to quantify variation in regulatory regions in wild birds, reveals that the upstream regions of CD74 and HSP90α are GC-rich, with the former exhibiting unusually low sequence variation for this species. We identified two SNPs, located in a GC-rich region immediately upstream of an inferred promoter site in the gene HSP90α, that were significantly associated with Mycoplasma pathogen load in the two populations. The SNPs are closely linked and situated in potential regulatory sequences: one in a binding site for the transcription factor nuclear NFYα and the other in a dinucleotide microsatellite ((GC)6). The genotype associated with pathogen load in the putative NFYα binding site was significantly overrepresented in the Alabama birds. However, we did not see strong effects of selection at this SNP, perhaps because selection has acted on standing genetic variation over an extremely short time in a highly recombining region. Our study is a useful starting point to explore functional relationships between sequence polymorphisms, gene expression, and phenotypic traits, such as pathogen resistance that affect fitness in the wild. PMID:23532859

  4. MoD Tools: regulatory motif discovery in nucleotide sequences from co-regulated or homologous genes.

    PubMed

    Pavesi, Giulio; Mereghetti, Paolo; Zambelli, Federico; Stefani, Marco; Mauri, Giancarlo; Pesole, Graziano

    2006-07-01

    Understanding the complex mechanisms regulating gene expression at the transcriptional and post-transcriptional levels is one of the greatest challenges of the post-genomic era. The MoD (MOtif Discovery) Tools web server comprises a set of tools for the discovery of novel conserved sequence and structure motifs in nucleotide sequences, motifs that in turn are good candidates for regulatory activity. The server includes the following programs: Weeder, for the discovery of conserved transcription factor binding sites (TFBSs) in nucleotide sequences from co-regulated genes; WeederH, for the discovery of conserved TFBSs and distal regulatory modules in sequences from homologous genes; RNAProfile, for the discovery of conserved secondary structure motifs in unaligned RNA sequences whose secondary structure is not known. In this way, a given gene can be compared with other co-regulated genes or with its homologs, or its mRNA can be analyzed for conserved motifs regulating its post-transcriptional fate. The web server thus provides researchers with different strategies and methods to investigate the regulation of gene expression, at both the transcriptional and post-transcriptional levels. Available at http://www.pesolelab.it/modtools/ and http://www.beacon.unimi.it/modtools/.

  5. Definition of regulatory sequence elements in the promoter region and the first intron of the myotonic dystrophy protein kinase gene.

    PubMed

    Storbeck, C J; Sabourin, L A; Waring, J D; Korneluk, R G

    1998-04-10

    Myotonic dystrophy is the most common inherited adult neuromuscular disorder with a global frequency of 1/8000. The genetic defect is an expanding CTG trinucleotide repeat in the 3'-untranslated region of the myotonic dystrophy protein kinase gene. We present the in vitro characterization of cis regulatory elements controlling transcription of the myotonic dystrophy protein kinase gene in myoblasts and fibroblasts. The region 5' to the initiating ATG contains no consensus TATA or CCAAT box. We have mapped two transcriptional start sites by primer extension. Deletion constructs from this region fused to the bacterial chloramphenicol acetyltransferase reporter gene revealed only subtle muscle specific cis elements. The strongest promoter activity mapped to a 189-base pair fragment. This sequence contains a conserved GC box to which the transcription factor Sp1 binds. Reporter gene constructs containing a 2-kilobase pair first intron fragment of the myotonic dystrophy protein kinase gene enhances reporter activity up to 6-fold in the human rhabdomyosarcoma myoblast cell line TE32 but not in NIH 3T3 fibroblasts. Co-transfection of a MyoD expression vector with reporter constructs containing the first intron into 10 T1/2 fibroblasts resulted in a 10-20-fold enhancement of expression. Deletion analysis of four E-box elements within the first intron reveal that these elements contribute to enhancer activity similarly in TE32 myoblasts and 10 T1/2 fibroblasts. These data suggest that E-boxes within the myotonic dystrophy protein kinase first intron mediate interactions with upstream promoter elements to up-regulate transcription of this gene in myoblasts.

  6. Sox2 regulatory region 2 sequence works as a DNA nuclear targeting sequence enhancing the efficiency of an exogenous gene expression in ES cells

    SciTech Connect

    Funabashi, Hisakage; Takatsu, Makoto; Saito, Mikako; Matsuoka, Hideaki

    2010-10-01

    Research highlights: {yields} SV40-DTS worked as a DTS in ES cells as well as other types of cells. {yields} Sox2 regulatory region 2 worked as a DTS in ES cells and thus was termed as SRR2-DTS. {yields} SRR2-DTS was suggested as an ES cell-specific DTS. -- Abstract: In this report, the effects of two DNA nuclear targeting sequence (DTS) candidates on the gene expression efficiency in ES cells were investigated. Reporter plasmids containing the simian virus 40 (SV40) promoter/enhancer sequence (SV40-DTS), a DTS for various types of cells but not being reported yet for ES cells, and the 81 base pairs of Sox2 regulatory region 2 (SRR2) where two transcriptional factors in ES cells, Oct3/4 and Sox2, are bound (SRR2-DTS), were introduced into cytoplasm in living cells by femtoinjection. The gene expression efficiencies of each plasmid in mouse insulinoma cell line MIN6 cells and mouse ES cells were then evaluated. Plasmids including SV40-DTS and SRR2-DTS exhibited higher gene expression efficiency comparing to plasmids without these DTSs, and thus it was concluded that both sequences work as a DTS in ES cells. In addition, it was suggested that SRR2-DTS works as an ES cell-specific DTS. To the best of our knowledge, this is the first report to confirm the function of DTSs in ES cells.

  7. Different regulatory sequences control creatine kinase-M gene expression in directly injected skeletal and cardiac muscle.

    PubMed Central

    Vincent, C K; Gualberto, A; Patel, C V; Walsh, K

    1993-01-01

    Regulatory sequences of the M isozyme of the creatine kinase (MCK) gene have been extensively mapped in skeletal muscle, but little is known about the sequences that control cardiac-specific expression. The promoter and enhancer sequences required for MCK gene expression were assayed by the direct injection of plasmid DNA constructs into adult rat cardiac and skeletal muscle. A 700-nucleotide fragment containing the enhancer and promoter of the rabbit MCK gene activated the expression of a downstream reporter gene in both muscle tissues. Deletion of the enhancer significantly decreased expression in skeletal muscle but had no detectable effect on expression in cardiac muscle. Further deletions revealed a CArG sequence motif at position -179 within the promoter that was essential for cardiac-specific expression. The CArG element of the MCK promoter bound to the recombinant serum response factor and YY1, transcription factors which control expression from structurally similar elements in the skeletal actin and c-fos promoters. MCK-CArG-binding activities that were similar or identical to serum response factor and YY1 were also detected in extracts from adult cardiac muscle. These data suggest that the MCK gene is controlled by different regulatory programs in adult cardiac and skeletal muscle. Images PMID:8423791

  8. The Effects of Sequence Variation on Genome-wide NRF2 Binding—New Target Genes and Regulatory SNPs

    PubMed Central

    Kuosmanen, Suvi M.; Viitala, Sari; Laitinen, Tuomo; Peräkylä, Mikael; Pölönen, Petri; Kansanen, Emilia; Leinonen, Hanna; Raju, Suresh; Wienecke-Baldacchino, Anke; Närvänen, Ale; Poso, Antti; Heinäniemi, Merja; Heikkinen, Sami; Levonen, Anna-Liisa

    2016-01-01

    Transcription factor binding specificity is crucial for proper target gene regulation. Motif discovery algorithms identify the main features of the binding patterns, but the accuracy on the lower affinity sites is often poor. Nuclear factor E2-related factor 2 (NRF2) is a ubiquitous redox-activated transcription factor having a key protective role against endogenous and exogenous oxidant and electrophile stress. Herein, we decipher the effects of sequence variation on the DNA binding sequence of NRF2, in order to identify both genome-wide binding sites for NRF2 and disease-associated regulatory SNPs (rSNPs) with drastic effects on NRF2 binding. Interactions between NRF2 and DNA were studied using molecular modelling, and NRF2 chromatin immunoprecipitation-sequence datasets together with protein binding microarray measurements were utilized to study binding sequence variation in detail. The binding model thus generated was used to identify genome-wide binding sites for NRF2, and genomic binding sites with rSNPs that have strong effects on NRF2 binding and reside on active regulatory elements in human cells. As a proof of concept, miR-126–3p and -5p were identified as NRF2 target microRNAs, and a rSNP (rs113067944) residing on NRF2 target gene (Ferritin, light polypeptide, FTL) promoter was experimentally verified to decrease NRF2 binding and result in decreased transcriptional activity. PMID:26826707

  9. Mining expressed sequence tags of rapeseed (Brassica napus L.) to predict the drought responsive regulatory network.

    PubMed

    Shamloo-Dashtpagerdi, Roohollah; Razi, Hooman; Ebrahimie, Esmaeil

    2015-07-01

    It is of great significance to understand the regulatory mechanisms by which plants deal with drought stress. Two EST libraries derived from rapeseed (Brassica napus) leaves in non-stressed and drought stress conditions were analyzed in order to obtain the transcriptomic landscape of drought-exposed B. napus plants, and also to identify and characterize significant drought responsive regulatory genes and microRNAs. The functional ontology analysis revealed a substantial shift in the B. napus transcriptome to govern cellular drought responsiveness via different stress-activated mechanisms. The activity of transcription factor and protein kinase modules generally increased in response to drought stress. The 26 regulatory genes consisting of 17 transcription factor genes, eight protein kinase genes and one protein phosphatase gene were identified showing significant alterations in their expressions in response to drought stress. We also found the six microRNAs which were differentially expressed during drought stress supporting the involvement of a post-transcriptional level of regulation for B. napus drought response. The drought responsive regulatory network shed light on the significance of some regulatory components involved in biosynthesis and signaling of various plant hormones (abscisic acid, auxin and brassinosteroids), ubiquitin proteasome system, and signaling through Reactive Oxygen Species (ROS). Our findings suggested a complex and multi-level regulatory system modulating response to drought stress in B. napus. PMID:26261397

  10. The nucleotide sequences of several tRNA genes from rat mitochondria: common features and relatedness to homologous species.

    PubMed Central

    Cantatore, P; De Benedetto, C; Gadaleta, G; Gallerani, R; Kroon, A M; Holtrop, M; Lanave, C; Pepe, G; Quagliariello, C; Saccone, C; Sbisa, E

    1982-01-01

    We have determined the nucleotide sequences of thirteen rat mt tRNA genes. The features of the primary and secondary structures of these tRNAs show that those for Gln, Ser, and f-Met resemble, while those for Lys, Cys, and Trp depart strikingly from the universal type. The remainder are slightly abnormal. Among many mammalian mt DNA sequences, those of mt tRNA genes are highly conserved, thus suggesting for those genes an additional, perhaps regulatory, function. A simple evolutionary relationship between the tRNAs of animal mitochondria and those of eukaryotic cytoplasm, of lower eukaryotic mitochondria or of prokaryotes, is not evident owing to the extreme divergence of the tRNA sequences in the two groups. However, a slightly higher homology does exist between a few animal mt tRNAs and those from prokaryotes or from lower eukaryotic mitochondria. PMID:7099963

  11. Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans

    PubMed Central

    2012-01-01

    Background Non-coding DNA in and around the human Amyloid Precursor Protein (APP) gene that is central to Alzheimer’s disease (AD) shares little sequence similarity with that of appb in zebrafish. Identifying DNA domains regulating expression of the gene in such situations becomes a challenge. Taking advantage of the zebrafish system that allows rapid functional analyses of gene regulatory sequences, we previously showed that two discontinuous DNA domains in zebrafish appb are important for expression of the gene in neurons: an enhancer in intron 1 and sequences 28–31 kb upstream of the gene. Here we identify the putative transcription factor binding sites responsible for this distal cis-acting regulation, and use that information to identify a regulatory region of the human APP gene. Results Functional analyses of intron 1 enhancer mutations in enhancer-trap BACs expressed as transgenes in zebrafish identified putative binding sites of two known transcription factor proteins, E4BP4/ NFIL3 and Forkhead, to be required for expression of appb. A cluster of three E4BP4 sites at −31 kb is also shown to be essential for neuron-specific expression, suggesting that the dependence of expression on upstream sequences is mediated by these E4BP4 sites. E4BP4/ NFIL3 and XFD1 sites in the intron enhancer and E4BP4/ NFIL3 sites at −31 kb specifically and efficiently bind the corresponding zebrafish proteins in vitro. These sites are statistically over-represented in both the zebrafish appb and the human APP genes, although their locations are different. Remarkably, a cluster of four E4BP4 sites in intron 4 of human APP exists in actively transcribing chromatin in a human neuroblastoma cell-line, SHSY5Y, expressing APP as shown using chromatin immunoprecipitation (ChIP) experiments. Thus although the two genes share little sequence conservation, they appear to share the same regulatory logic and are regulated by a similar set of transcription factors. Conclusion The

  12. Identification of novel craniofacial regulatory domains located far upstream of SOX9 and disrupted in Pierre Robin sequence.

    PubMed

    Gordon, Christopher T; Attanasio, Catia; Bhatia, Shipra; Benko, Sabina; Ansari, Morad; Tan, Tiong Y; Munnich, Arnold; Pennacchio, Len A; Abadie, Véronique; Temple, I Karen; Goldenberg, Alice; van Heyningen, Veronica; Amiel, Jeanne; FitzPatrick, David; Kleinjan, Dirk A; Visel, Axel; Lyonnet, Stanislas

    2014-08-01

    Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS.

  13. Identification of novel craniofacial regulatory domains located far upstream of SOX9 and disrupted in Pierre Robin sequence

    PubMed Central

    Gordon, Christopher T.; Attanasio, Catia; Bhatia, Shipra; Benko, Sabina; Ansari, Morad; Tan, Tiong Y.; Munnich, Arnold; Pennacchio, Len A.; Abadie, Véronique; Temple, I. Karen; Goldenberg, Alice; van Heyningen, Veronica; Amiel, Jeanne; FitzPatrick, David; Kleinjan, Dirk A.; Visel, Axel; Lyonnet, Stanislas

    2015-01-01

    Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions and duplications within a ~2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ~1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harbouring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple non-coding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS. PMID:24934569

  14. Cloning and Characterization of 5′ Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

    PubMed Central

    Tang, Guiying; Xu, Pingli; Liu, Wei; Liu, Zhanji; Shan, Lei

    2015-01-01

    LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5′ flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5′ terminal and/or 3′ terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65bp proximal promoter region and the 52bp 5′ UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements. PMID:26426444

  15. Development of molecular markers linked to disease resistance genes in common bean based on whole genome sequence.

    PubMed

    Meziadi, Chouaïb; Richard, Manon M S; Derquennes, Amandine; Thareau, Vincent; Blanchet, Sophie; Gratias, Ariane; Pflieger, Stéphanie; Geffroy, Valérie

    2016-01-01

    Common bean (Phaseolus vulgaris) is the most important grain legume for direct human consumption in the world, particularly in developing countries where it constitutes the main source of protein. Unfortunately, common bean yield stability is constrained by a number of pests and diseases. As use of resistant genotypes is the most economic and ecologically safe means for controlling plant diseases, efforts have been made to genetically characterize resistance genes (R genes) in common bean. Despite its agronomic importance, genomic resources available in common bean were limited until the recent sequencing of common bean genome (Andean genotype G19833). Besides allowing the annotation of Nucleotide Binding-Leucine Rich Repeat (NB-LRR) encoding gene family, which is the prevalent class of disease R genes in plants, access to the whole genome sequence of common bean can be of great help for intense selection to increase the overall efficiency of crop improvement programs using marker-assisted selection (MAS). This review presents the state of the art of common bean NB-LRR gene clusters, their peculiar location in subtelomeres and correlation with genetically characterized monogenic R genes, as well as how the availability of the whole genome sequence can boost the development of molecular markers for MAS.

  16. Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.)

    PubMed Central

    2011-01-01

    Background Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean. Results Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes. Conclusions The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the

  17. Monte Carlo Simulations of the Post-Common-Envelope White-Dwarf Main-Sequence Binary Population

    SciTech Connect

    Camacho, Judit; Torres, Santiago; Garcia-Berro, Enrique; Schreiber, Matthias R.

    2010-12-22

    We present a detailed Monte Carlo simulator of the population of binary systems within the solar neighborhood. We have used the most up-to-date stellar evolutionary models, a complete treatment of the Roche lobe overflow episode, as well as a full implementation of the orbital evolution of the binary system. Preliminary results are presented for the population of white-dwarf main-sequence binaries, resulting from a common envelope episode. We also study the role played by the binding energy parameter, {lambda}, and by the common envelope efficiency, {alpha}{sub CE}. Finally, results are compared with the population of identified white-dwarf main-sequence binaries.

  18. Use of a Drosophila Genome-Wide Conserved Sequence Database to Identify Functionally Related cis-Regulatory Enhancers

    PubMed Central

    Brody, Thomas; Yavatkar, Amarendra S; Kuzin, Alexander; Kundu, Mukta; Tyson, Leonard J; Ross, Jermaine; Lin, Tzu-Yang; Lee, Chi-Hon; Awasaki, Takeshi; Lee, Tzumin; Odenwald, Ward F

    2012-01-01

    Background: Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs. Results: We have generated a Drosophila genome-wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. Conclusions: cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process. Developmental Dynamics 241:169–189, 2012. © 2011 Wiley Periodicals, Inc. Key findings A genome-wide catalog of Drosophila conserved DNA sequence clusters. cis-Decoder discovers functionally related enhancers. Functionally related enhancers share balanced sequence element copy numbers. Many enhancers function during multiple phases of development. PMID:22174086

  19. The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics of regulatory sequences

    PubMed Central

    Kensche, Philip Reiner; Hoeijmakers, Wieteke Anna Maria; Toenhake, Christa Geeke; Bras, Maaike; Chappell, Lia; Berriman, Matthew; Bártfai, Richárd

    2016-01-01

    In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-associated processes and it is central to the control of gene expression. For Plasmodium falciparum, a causative agent of human malaria, the nucleosome positioning profile of regulatory regions deserves particular attention because of their extreme AT-content. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit by demarcating landmark sites (transcription/translation start and end sites). In addition, our analysis provides strong indications for the function of positioned nucleosomes in splice site recognition. Transcription start sites (TSSs) are bordered by a small nucleosome-depleted region, but lack the stereotypic downstream nucleosome arrays, highlighting a key difference in chromatin organization compared to model organisms. Furthermore, we observe transcription-coupled eviction of nucleosomes on strong TSSs during intraerythrocytic development and demonstrate that nucleosome positioning and dynamics can be predictive for the functionality of regulatory DNA elements. Collectively, the strong nucleosome positioning over splice sites and surrounding putative transcription factor binding sites highlights the regulatory capacity of the nucleosome landscape in this deadly human pathogen. PMID:26578577

  20. The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics of regulatory sequences.

    PubMed

    Kensche, Philip Reiner; Hoeijmakers, Wieteke Anna Maria; Toenhake, Christa Geeke; Bras, Maaike; Chappell, Lia; Berriman, Matthew; Bártfai, Richárd

    2016-03-18

    In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-associated processes and it is central to the control of gene expression. For Plasmodium falciparum, a causative agent of human malaria, the nucleosome positioning profile of regulatory regions deserves particular attention because of their extreme AT-content. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit by demarcating landmark sites (transcription/translation start and end sites). In addition, our analysis provides strong indications for the function of positioned nucleosomes in splice site recognition. Transcription start sites (TSSs) are bordered by a small nucleosome-depleted region, but lack the stereotypic downstream nucleosome arrays, highlighting a key difference in chromatin organization compared to model organisms. Furthermore, we observe transcription-coupled eviction of nucleosomes on strong TSSs during intraerythrocytic development and demonstrate that nucleosome positioning and dynamics can be predictive for the functionality of regulatory DNA elements. Collectively, the strong nucleosome positioning over splice sites and surrounding putative transcription factor binding sites highlights the regulatory capacity of the nucleosome landscape in this deadly human pathogen.

  1. Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model.

    PubMed

    Smith, Robin P; Taher, Leila; Patwardhan, Rupali P; Kim, Mee J; Inoue, Fumitaka; Shendure, Jay; Ovcharenko, Ivan; Ahituv, Nadav

    2013-09-01

    Despite continual progress in the cataloging of vertebrate regulatory elements, little is known about their organization and regulatory architecture. Here we describe a massively parallel experiment to systematically test the impact of copy number, spacing, combination and order of transcription factor binding sites on gene expression. A complex library of ∼5,000 synthetic regulatory elements containing patterns from 12 liver-specific transcription factor binding sites was assayed in mice and in HepG2 cells. We find that certain transcription factors act as direct drivers of gene expression in homotypic clusters of binding sites, independent of spacing between sites, whereas others function only synergistically. Heterotypic enhancers are stronger than their homotypic analogs and favor specific transcription factor binding site combinations, mimicking putative native enhancers. Exhaustive testing of binding site permutations suggests that there is flexibility in binding site order. Our findings provide quantitative support for a flexible model of regulatory element activity and suggest a framework for the design of synthetic tissue-specific enhancers. PMID:23892608

  2. Targeted genomic sequencing of follicular dendritic cell sarcoma reveals recurrent alterations in NF-κB regulatory genes.

    PubMed

    Griffin, Gabriel K; Sholl, Lynette M; Lindeman, Neal I; Fletcher, Christopher D M; Hornick, Jason L

    2016-01-01

    Follicular dendritic cell sarcoma is a rare mesenchymal neoplasm with a variable and unpredictable clinical course. The genetic alterations that drive tumorigenesis in follicular dendritic cell sarcoma are largely unknown. One recent study performed BRAF sequencing and found V600E mutations in 5 of 27 (19%) cases. No other recurrent genetic alterations have been reported. The aim of the present study was to identify somatic alterations in follicular dendritic cell sarcoma by targeted sequencing of a panel of 309 known cancer-associated genes. DNA was isolated from formalin-fixed paraffin-embedded tissue from 13 cases of follicular dendritic cell sarcoma and submitted for hybrid capture-based enrichment and massively parallel sequencing with the Illumina HiSeq 2500 platform. Recurrent loss-of-function alterations were observed in tumor suppressor genes involved in the negative regulation of NF-κB activation (5 of 13 cases, 38%) and cell cycle progression (4 of 13 cases, 31%). Loss-of-function alterations in the NF-κB regulatory pathway included three cases with frameshift mutations in NFKBIA and two cases with bi-allelic loss of CYLD. Both cases with CYLD loss were metastases and carried concurrent alterations in at least one cell cycle regulatory gene. Alterations in cell cycle regulatory genes included two cases with bi-allelic loss of CDKN2A, one case with bi-allelic loss of RB1, and one case with a nonsense mutation in RB1. Last, focal copy-number gain of chromosome 9p24 including the genes CD274 (PD-L1) and PDCD1LG2 (PD-L2) was noted in three cases, which represents a well-described mechanism of immune evasion in cancer. These findings provide the first insight into the unique genomic landscape of follicular dendritic cell sarcoma and suggest shared mechanisms of tumorigenesis with a subset of other tumor types, notably B-cell lymphomas.

  3. Complete Chloroplast Genome Sequence of Tartary Buckwheat (Fagopyrum tataricum) and Comparative Analysis with Common Buckwheat (F. esculentum).

    PubMed

    Cho, Kwang-Soo; Yun, Bong-Kyoung; Yoon, Young-Ho; Hong, Su-Young; Mekapogu, Manjulatha; Kim, Kyung-Hee; Yang, Tae-Jin

    2015-01-01

    We report the chloroplast (cp) genome sequence of tartary buckwheat (Fagopyrum tataricum) obtained by next-generation sequencing technology and compared this with the previously reported common buckwheat (F. esculentum ssp. ancestrale) cp genome. The cp genome of F. tataricum has a total sequence length of 159,272 bp, which is 327 bp shorter than the common buckwheat cp genome. The cp gene content, order, and orientation are similar to those of common buckwheat, but with some structural variation at tandem and palindromic repeat frequencies and junction areas. A total of seven InDels (around 100 bp) were found within the intergenic sequences and the ycf1 gene. Copy number variation of the 21-bp tandem repeat varied in F. tataricum (four repeats) and F. esculentum (one repeat), and the InDel of the ycf1 gene was 63 bp long. Nucleotide and amino acid have highly conserved coding sequence with about 98% homology and four genes--rpoC2, ycf3, accD, and clpP--have high synonymous (Ks) value. PCR based InDel markers were applied to diverse genetic resources of F. tataricum and F. esculentum, and the amplicon size was identical to that expected in silico. Therefore, these InDel markers are informative biomarkers to practically distinguish raw or processed buckwheat products derived from F. tataricum and F. esculentum.

  4. The Effects of Common Knowledge Construction Model Sequence of Lessons on Science Achievement and Relational Conceptual Change

    ERIC Educational Resources Information Center

    Ebenezer, Jazlin; Chacko, Sheela; Kaya, Osman Nafiz; Koya, Satya Kiran; Ebenezer, Devairakkam Luke

    2010-01-01

    The purpose of this study was to investigate the effects of the Common Knowledge Construction Model (CKCM) lesson sequence, an intervention based both in conceptual change theory and in Phenomenography, a subset of conceptual change theory. A mixed approach was used to investigate whether this model had a significant effect on 7th grade students'…

  5. Common 5S rRNA variants are likely to be accepted in many sequence contexts

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; D'Souza, Lisa M.; Lee, Youn-Hyung; Fox, George E.

    2003-01-01

    Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The

  6. Draft Genome Sequence of Catellicoccus marimammalium, a Novel Species Commonly Found in Gull Feces

    EPA Science Inventory

    Catellicoccus marimammalium is a relatively uncharacterized Gram-positive, facultative anaerobe with potential utility as an indicator of waterfowl fecal contamination. Here we report an annotated draft genome sequence that suggests this organism may be a symbiotic gut microbe.

  7. A Catalog of Regulatory Sequences for Trait Gene for the Genome Editing of Wheat

    PubMed Central

    Makai, Szabolcs; Tamás, László; Juhász, Angéla

    2016-01-01

    Wheat has been cultivated for 10000 years and ever since the origin of hexaploid wheat it has been exempt from natural selection. Instead, it was under the constant selective pressure of human agriculture from harvest to sowing during every year, producing a vast array of varieties. Wheat has been adopted globally, accumulating variation for genes involved in yield traits, environmental adaptation and resistance. However, one small but important part of the wheat genome has hardly changed: the regulatory regions of both the x- and y-type high molecular weight glutenin subunit (HMW-GS) genes, which are alone responsible for approximately 12% of the grain protein content. The phylogeny of the HMW-GS regulatory regions of the Triticeae demonstrates that a genetic bottleneck may have led to its decreased diversity during domestication and the subsequent cultivation. It has also highlighted the fact that the wild relatives of wheat may offer an unexploited genetic resource for the regulatory region of these genes. Significant research efforts have been made in the public sector and by international agencies, using wild crosses to exploit the available genetic variation, and as a result synthetic hexaploids are now being utilized by a number of breeding companies. However, a newly emerging tool of genome editing provides significantly improved efficiency in exploiting the natural variation in HMW-GS genes and incorporating this into elite cultivars and breeding lines. Recent advancement in the understanding of the regulation of these genes underlines the needs for an overview of the regulatory elements for genome editing purposes. PMID:27766102

  8. Using RSAT oligo-analysis and dyad-analysis tools to discover regulatory signals in nucleic sequences.

    PubMed

    Defrance, Matthieu; Janky, Rekin's; Sand, Olivier; van Helden, Jacques

    2008-01-01

    This protocol explains how to discover functional signals in genomic sequences by detecting over- or under-represented oligonucleotides (words) or spaced pairs thereof (dyads) with the Regulatory Sequence Analysis Tools (http://rsat.ulb.ac.be/rsat/). Two typical applications are presented: (i) predicting transcription factor-binding motifs in promoters of coregulated genes and (ii) discovering phylogenetic footprints in promoters of orthologous genes. The steps of this protocol include purging genomic sequences to discard redundant fragments, discovering over-represented patterns and assembling them to obtain degenerate motifs, scanning sequences and drawing feature maps. The main strength of the method is its statistical ground: the binomial significance provides an efficient control on the rate of false positives. In contrast with optimization-based pattern discovery algorithms, the method supports the detection of under- as well as over-represented motifs. Computation times vary from seconds (gene clusters) to minutes (whole genomes). The execution of the whole protocol should take approximately 1 h.

  9. Family-based association test using both common and rare variants and accounting for directions of effects for sequencing data.

    PubMed

    Chung, Ren-Hua; Tsai, Wei-Yun; Martin, Eden R

    2014-01-01

    Current family-based association tests for sequencing data were mainly developed for identifying rare variants associated with a complex disease. As the disease can be influenced by the joint effects of common and rare variants, common variants with modest effects may not be identified by the methods focusing on rare variants. Moreover, variants can have risk, neutral, or protective effects. Association tests that can effectively select groups of common and rare variants that are likely to be causal and consider the directions of effects have become important. We developed the Ordered Subset - Variable Threshold - Pedigree Disequilibrium Test (OVPDT), a combination of three algorithms, for association analysis in family sequencing data. The ordered subset algorithm is used to select a subset of common variants based on their relative risks, calculated using only parental mating types. The variable threshold algorithm is used to search for an optimal allele frequency threshold such that rare variants below the threshold are more likely to be causal. The PDT statistics from both rare and common variants selected by the two algorithms are combined as the OVPDT statistic. A permutation procedure is used in OVPDT to calculate the p-value. We used simulations to demonstrate that OVPDT has the correct type I error rates under different scenarios and compared the power of OVPDT with two other family-based association tests. The results suggested that OVPDT can have more power than the other tests if both common and rare variants have effects on the disease in a region.

  10. Novel phytochrome sequences in Arabidopsis thaliana: Structure, evolution, and differential expression of a plant regulatory photoreceptor family

    SciTech Connect

    Sharrock, R.A.; Quail, P.H. )

    1989-01-01

    Phytochrome is a plant regulatory photoreceptor that mediates red light effects on a wide variety of physiological and molecular responses. DNA blot analysis indicates that the Arabidopsis thaliana genome contains four to five phytochrome-related gene sequences. The authors have isolated and sequenced cDNA clones corresponding to three of these genes and have deduced the amino acid sequence of the full-length polypeptide encoded in each case. One of these proteins (phyA) shows 65-80% amino acid sequence identity with the major, etiolated-tissue phytochrome apoproteins described previously in other plant species. The other two polypeptides (phyB and phyC) are unique in that they have low sequence identity with each other, with phyA, and with all previously described phytochromes. The phyA, phyB, and phyC proteins are of similar molecular mass, have related hydropathic profiles, and contain a conserved chromophore attachment region. However, the sequence comparison data indicate that the three phy genes diverged early in plant evolution, well before the divergence of the two major groups of angiosperms, the monocots and dicots. The steady-state level of the phyA transcript is high in dark-grown A. thaliana seedlings and is down-regulated by light. In contrast, the phyB and phyC transcripts are present at lower levels and are not strongly light-regulated. These findings indicate that the red/far red light-responsive phytochrome photoreceptor system in A. thaliana, and perhaps in all higher plants, consists of a family of chromoproteins that are heterogeneous in structure and regulation.

  11. Spatio-temporal sequence of cross-regulatory events in root meristem growth.

    PubMed

    Scacchi, Emanuele; Salinas, Paula; Gujas, Bojan; Santuari, Luca; Krogan, Naden; Ragni, Laura; Berleth, Thomas; Hardtke, Christian S

    2010-12-28

    A central question in developmental biology is how multicellular organisms coordinate cell division and differentiation to determine organ size. In Arabidopsis roots, this balance is controlled by cytokinin-induced expression of SHORT HYPOCOTYL 2 (SHY2) in the so-called transition zone of the meristem, where SHY2 negatively regulates auxin response factors (ARFs) by protein-protein interaction. The resulting down-regulation of PIN-FORMED (PIN) auxin efflux carriers is considered the key event in promoting differentiation of meristematic cells. Here we show that this regulation involves additional, intermediary factors and is spatio-temporally constrained. We found that the described cytokinin-auxin crosstalk antagonizes BREVIS RADIX (BRX) activity in the developing protophloem. BRX is an auxin-responsive target of the prototypical ARF MONOPTEROS (MP), a key promoter of vascular development, and transiently enhances PIN3 expression to promote meristem growth in young roots. At later stages, cytokinin induction of SHY2 in the vascular transition zone restricts BRX expression to down-regulate PIN3 and thus limit meristem growth. Interestingly, proper SHY2 expression requires BRX, which could reflect feedback on the auxin responsiveness of SHY2 because BRX protein can directly interact with MP, likely acting as a cofactor. Thus, cross-regulatory antagonism between BRX and SHY2 could determine ARF activity in the protophloem. Our data suggest a model in which the regulatory interactions favor BRX expression in the early proximal meristem and SHY2 prevails because of supplementary cytokinin induction in the later distal meristem. The complex equilibrium of this regulatory module might represent a universal switch in the transition toward differentiation in various developmental contexts.

  12. Oxytocin receptor gene sequences in owl monkeys and other primates show remarkable interspecific regulatory and protein coding variation.

    PubMed

    Babb, Paul L; Fernandez-Duque, Eduardo; Schurr, Theodore G

    2015-10-01

    The oxytocin (OT) hormone pathway is involved in numerous physiological processes, and one of its receptor genes (OXTR) has been implicated in pair bonding behavior in mammalian lineages. This observation is important for understanding social monogamy in primates, which occurs in only a small subset of taxa, including Azara's owl monkey (Aotus azarae). To examine the potential relationship between social monogamy and OXTR variation, we sequenced its 5' regulatory (4936bp) and coding (1167bp) regions in 25 owl monkeys from the Argentinean Gran Chaco, and examined OXTR sequences from 1092 humans from the 1000 Genomes Project. We also assessed interspecific variation of OXTR in 25 primate and rodent species that represent a set of phylogenetically and behaviorally disparate taxa. Our analysis revealed substantial variation in the putative 5' regulatory region of OXTR, with marked structural differences across primate taxa, particularly for humans and chimpanzees, which exhibited unique patterns of large motifs of dinucleotide A+T repeats upstream of the OXTR 5' UTR. In addition, we observed a large number of amino acid substitutions in the OXTR CDS region among New World primate taxa that distinguish them from Old World primates. Furthermore, primate taxa traditionally defined as socially monogamous (e.g., gibbons, owl monkeys, titi monkeys, and saki monkeys) all exhibited different amino acid motifs for their respective OXTR protein coding sequences. These findings support the notion that monogamy has evolved independently in Old World and New World primates, and that it has done so through different molecular mechanisms, not exclusively through the oxytocin pathway. PMID:26025428

  13. Oxytocin receptor gene sequences in owl monkeys and other primates show remarkable interspecific regulatory and protein coding variation.

    PubMed

    Babb, Paul L; Fernandez-Duque, Eduardo; Schurr, Theodore G

    2015-10-01

    The oxytocin (OT) hormone pathway is involved in numerous physiological processes, and one of its receptor genes (OXTR) has been implicated in pair bonding behavior in mammalian lineages. This observation is important for understanding social monogamy in primates, which occurs in only a small subset of taxa, including Azara's owl monkey (Aotus azarae). To examine the potential relationship between social monogamy and OXTR variation, we sequenced its 5' regulatory (4936bp) and coding (1167bp) regions in 25 owl monkeys from the Argentinean Gran Chaco, and examined OXTR sequences from 1092 humans from the 1000 Genomes Project. We also assessed interspecific variation of OXTR in 25 primate and rodent species that represent a set of phylogenetically and behaviorally disparate taxa. Our analysis revealed substantial variation in the putative 5' regulatory region of OXTR, with marked structural differences across primate taxa, particularly for humans and chimpanzees, which exhibited unique patterns of large motifs of dinucleotide A+T repeats upstream of the OXTR 5' UTR. In addition, we observed a large number of amino acid substitutions in the OXTR CDS region among New World primate taxa that distinguish them from Old World primates. Furthermore, primate taxa traditionally defined as socially monogamous (e.g., gibbons, owl monkeys, titi monkeys, and saki monkeys) all exhibited different amino acid motifs for their respective OXTR protein coding sequences. These findings support the notion that monogamy has evolved independently in Old World and New World primates, and that it has done so through different molecular mechanisms, not exclusively through the oxytocin pathway.

  14. High resolution sequencing and modeling identifies distinct dynamic RNA regulatory strategies

    PubMed Central

    Rabani, Michal; Raychowdhury, Raktima; Jovanovic, Marko; Rooney, Michael; Stumpo, Deborah J.; Hacohen, Nir; Schier, Alexander F.; Blackshear, Perry J.; Friedman, Nir; Amit, Ido; Regev, Aviv

    2014-01-01

    Summary Cells control dynamic transitions in transcript levels by regulating transcription, processing and/or degradation through an integrated regulatory strategy. Here, we combine RNA metabolic labeling, rRNA-depleted RNA-seq, and DRiLL, a novel computational framework, to quantify the level, editing sites, and transcription, processing and degradation rates of each transcript at a splice junction resolution during the LPS response of mouse dendritic cells. Four key regulatory strategies, dominated by RNA transcription changes, generate most temporal gene expression patterns. Non-canonical strategies that also employ dynamic posttranscriptional regulation control only a minority of genes, but provide unique signal processing features. We validate Tristetraprolin (TTP) as a major regulator of RNA degradation in one non-canonical strategy. Applying DRiLL to the regulation of non-coding RNAs and to zebrafish embryogenesis demonstrates its broad utility. Our study provides a new quantitative approach to discover transcriptional and post-transcriptional events that control dynamic changes in transcript levels using RNA-Seq data. PMID:25497548

  15. De novo assembly and characterization of the spleen transcriptome of common carp (Cyprinus carpio) using Illumina paired-end sequencing.

    PubMed

    Li, Guoxi; Zhao, Yinli; Liu, Zhonghu; Gao, Chunsheng; Yan, Fengbin; Liu, Bianzhi; Feng, Jianxin

    2015-06-01

    Common carp (Cyprinus carpio) is one of the most important aquacultured species of the family Cyprinidae, and breeding this species for disease resistance is becoming more and more important. However, at the genome or transcriptome levels, study of the immunogenetics of disease resistance in the common carp is lacking. In this study, 60,316,906 and 75,200,328 paired-end clean reads were obtained from two cDNA libraries of the common carp spleen by Illumina paired-end sequencing technology. Totally, 130,293 unique transcript fragments (unigenes) were assembled, with an average length of 1400.57 bp. Approximately 105,612 (81.06%) unigenes could be annotated according to their homology with matches in the Nr, Nt, Swiss-Prot, COG, GO, or KEGG databases, and they were found to represent 46,747 non-redundant genes. Comparative analysis showed that 59.82% of the unigenes have significant similarity to zebrafish Refseq proteins. Gene expression comparison revealed that 10,432 and 6889 annotated unigenes were, respectively, up- and down-regulated with at least twofold changes between two developmental stages of the common carp spleen. Gene ontology and KEGG analysis were performed to classify all unigenes into functional categories for understanding gene functions and regulation pathways. In addition, 46,847 simple sequence repeats (SSRs) were detected from 35,618 unigenes, and a large number of single nucleotide polymorphism (SNP) and insertion/deletion (INDEL) sites were identified in the spleen transcriptome of common carp. This study has characterized the spleen transcriptome of the common carp for the first time, providing a valuable resource for a better understanding of the common carp immune system and defense mechanisms. This knowledge will also facilitate future functional studies on common carp immunogenetics that may eventually be applied in breeding programs.

  16. [Examination of processed vegetable foods for the presence of common DNA sequences of genetically modified tomatoes].

    PubMed

    Kitagawa, Mamiko; Nakamura, Kosuke; Kondo, Kazunari; Ubukata, Shoji; Akiyama, Hiroshi

    2014-01-01

    The contamination of processed vegetable foods with genetically modified tomatoes was investigated by the use of qualitative PCR methods to detect the cauliflower mosaic virus 35S promoter (P35S) and the kanamycin resistance gene (NPTII). DNA fragments of P35S and NPTII were detected in vegetable juice samples, possibly due to contamination with the genomes of cauliflower mosaic virus infecting juice ingredients of Brassica species and soil bacteria, respectively. Therefore, to detect the transformation construct sequences of GM tomatoes, primer pairs were designed for qualitative PCR to specifically detect the border region between P35S and NPTII, and the border region between nopaline synthase gene promoter and NPTII. No amplification of the targeted sequences was observed using genomic DNA purified from the juice ingredients. The developed qualitative PCR method is considered to be a reliable tool to check contamination of products with GM tomatoes.

  17. [Examination of processed vegetable foods for the presence of common DNA sequences of genetically modified tomatoes].

    PubMed

    Kitagawa, Mamiko; Nakamura, Kosuke; Kondo, Kazunari; Ubukata, Shoji; Akiyama, Hiroshi

    2014-01-01

    The contamination of processed vegetable foods with genetically modified tomatoes was investigated by the use of qualitative PCR methods to detect the cauliflower mosaic virus 35S promoter (P35S) and the kanamycin resistance gene (NPTII). DNA fragments of P35S and NPTII were detected in vegetable juice samples, possibly due to contamination with the genomes of cauliflower mosaic virus infecting juice ingredients of Brassica species and soil bacteria, respectively. Therefore, to detect the transformation construct sequences of GM tomatoes, primer pairs were designed for qualitative PCR to specifically detect the border region between P35S and NPTII, and the border region between nopaline synthase gene promoter and NPTII. No amplification of the targeted sequences was observed using genomic DNA purified from the juice ingredients. The developed qualitative PCR method is considered to be a reliable tool to check contamination of products with GM tomatoes. PMID:25743587

  18. Application of whole genome and RNA sequencing to investigate the genomic landscape of common variable immunodeficiency disorders.

    PubMed

    van Schouwenburg, Pauline A; Davenport, Emma E; Kienzler, Anne-Kathrin; Marwah, Ishita; Wright, Benjamin; Lucas, Mary; Malinauskas, Tomas; Martin, Hilary C; Lockstone, Helen E; Cazier, Jean-Baptiste; Chapel, Helen M; Knight, Julian C; Patel, Smita Y

    2015-10-01

    Common Variable Immunodeficiency Disorders (CVIDs) are the most prevalent cause of primary antibody failure. CVIDs are highly variable and a genetic causes have been identified in <5% of patients. Here, we performed whole genome sequencing (WGS) of 34 CVID patients (94% sporadic) and combined them with transcriptomic profiling (RNA-sequencing of B cells) from three patients and three healthy controls. We identified variants in CVID disease genes TNFRSF13B, TNFRSF13C, LRBA and NLRP12 and enrichment of variants in known and novel disease pathways. The pathways identified include B-cell receptor signalling, non-homologous end-joining, regulation of apoptosis, T cell regulation and ICOS signalling. Our data confirm the polygenic nature of CVID and suggest individual-specific aetiologies in many cases. Together our data show that WGS in combination with RNA-sequencing allows for a better understanding of CVIDs and the identification of novel disease associated pathways. PMID:26122175

  19. Nucleotide sequence and cloning in Bacillus subtilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT.

    PubMed Central

    Takagi, M; Takada, H; Imanaka, T

    1990-01-01

    The regulatory gene (degT) from Bacillus stearothermophilus NCA1503 which enhanced production of extracellular alkaline protease (Apr) was cloned in Bacillus subtilis with pTB53 as a vector. When B. subtilis MT-2 (Npr- [deficiency of neutral protease] Apr+) was transformed with the recombinant plasmid, pDT145, the plasmid carrier produced about three times more alkaline protease than did the wild-type strain. In contrast, when B. subtilis DB104 (Npr- Apr-) was used as a host, the transformant with pDT145 could not exhibit any protease activity. After construction of the deletion plasmids, DNA sequencing was done. A large open reading frame was found, and nucleotide sequence analysis showed that the degT gene was composed of 1,116 bases (372 amino acid residues, molecular weight of 41,244). A Shine-Dalgarno sequence was found nine bases upstream from the open reading frame. A B. subtilis strain carrying degT showed the following pleiotropic phenomena: (i) enhancement of production of extracellular enzymes such as alkaline protease and levansucrase, (ii) repression of autolysin activity, (iii) decrease of transformation efficiency for B. subtilis (competent cell procedure), (iv) altered control of sporulation, (v) loss of flagella, and (vi) abnormal cell division. When B. stearothermophilus SIC1 was transformed with the recombinant plasmid carrying degT, the transformants exhibited abnormal cell division. These phenomena are similar to those of the phenotypes of degSU(Hy) (hyperproduction), degQ(Hy), and degR mutants of B. subtilis. However, the amino acid sequence of the degT product (DegT) is different from those of the reported gene products. Furthermore, DegT includes a hydrophobic core region in the N-terminal portion (amino acid numbers 50 to 160), a consensus sequence for a DNA binding region (amino acid numbers 160 to 179), and a region homologous to transcription activator proteins (amino acid numbers 351 to 366). We discuss the possibility that the membrane

  20. A 454 sequencing approach for large scale phylogenomic analysis of the common emperor scorpion (Pandinus imperator).

    PubMed

    Roeding, Falko; Borner, Janus; Kube, Michael; Klages, Sven; Reinhardt, Richard; Burmester, Thorsten

    2009-12-01

    In recent years, phylogenetic tree reconstructions that rely on multiple gene alignments that had been deduced from expressed sequence tags (ESTs) have become a popular method in molecular systematics. Here, we present a 454 pyrosequencing approach to infer the transcriptome of the Emperor scorpion Pandinus imperator. We obtained 428,844 high-quality reads (mean length=223+/-50 b) from total cDNA, which were assembled into 8334 contigs (mean length 422+/-313 bp) and 26,147 singletons. About 1200 contigs were successfully annotated by BLAST and orthology search. Specific analyses of eight distinct hemocyanin sequences provided further proof for the quality of the 454 reads and the assembly process. The P. imperator sequences were included in a concatenated alignment of 149 orthologous genes of 67 metazoan taxa that covers 39,842 amino acids. After removal of low-quality regions, 11,168 positions were employed for phylogenetic reconstructions. Using Bayesian and maximum likelihood methods, we obtained strongly supported monophyletic Ecdysozoa, Arthropoda (excluding Tardigrada), Euarthropoda, Pancrustacea and Hexapoda. We also recovered the Myriochelata (Chelicerata+Myriapoda). Within the chelicerates, Pycnogonida form the sister group of Euchelicerata. However, Arachnida were found paraphyletic because the Acari (mites and ticks) were recovered as sister group of a clade comprising Xiphosura, Scorpiones and Araneae. In summary, we have shown that 454 pyrosequencing is a cost-effective method that provides sufficient data and coverage depth for gene detection and multigene-based phylogenetic analyses. PMID:19695333

  1. Nucleotide sequence of the regulatory locus controlling expression of bacterial genes for bioluminescence.

    PubMed Central

    Engebrecht, J; Silverman, M

    1987-01-01

    Production of light by the marine bacterium Vibrio fischeri and by recombinant hosts containing cloned lux genes is controlled by the density of the culture. Density-dependent regulation of lux gene expression has been shown to require a locus consisting of the luxR and luxI genes and two closely linked divergent promoters. As part of a genetic analysis to understand the regulation of bioluminescence, we have sequenced the region of DNA containing this control circuit. Open reading frames corresponding to luxR and luxI were identified; transcription start sites were defined by S1 nuclease mapping and sequences resembling promoter elements were located. Images PMID:3697093

  2. Sequence-based introgression mapping identifies candidate white mold tolerance genes in common bean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    White mold disease, caused by the necrotrophic fungus Sclerotinia sclerotiorum (Lib.) de Bary, is a major pathogen of common bean (Phaseolus vulgaris L.). More than 20 QTL were reported using multiple bi-parental populations. To study the disease in more detail, advanced back-cross populations seg...

  3. The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

    SciTech Connect

    Aklujkar, Muktak; Krushkal, Julia; DiBartolo, Genevieve; Lapidus, Alla; Land, Miriam L.; Lovley, Derek R.

    2008-12-01

    Background: The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences. Results: The experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recently in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes. Conclusion: The genomic evidence suggests that metabolism, physiology and regulation of gene expression in G. metallireducens may be dramatically different from other Geobacteraceae.

  4. Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons.

    PubMed

    Kim, Eun Hye; Lee, Hwan Young; Yang, In Seok; Jung, Sang-Eun; Yang, Woo Ick; Shin, Kyoung-Jin

    2016-05-01

    The next-generation sequencing (NGS) method has been utilized to analyze short tandem repeat (STR) markers, which are routinely used for human identification purposes in the forensic field. Some researchers have demonstrated the successful application of the NGS system to STR typing, suggesting that NGS technology may be an alternative or additional method to overcome limitations of capillary electrophoresis (CE)-based STR profiling. However, there has been no available multiplex PCR system that is optimized for NGS analysis of forensic STR markers. Thus, we constructed a multiplex PCR system for the NGS analysis of 18 markers (13CODIS STRs, D2S1338, D19S433, Penta D, Penta E and amelogenin) by designing amplicons in the size range of 77-210 base pairs. Then, PCR products were generated from two single-sources, mixed samples and artificially degraded DNA samples using a multiplex PCR system, and were prepared for sequencing on the MiSeq system through construction of a subsequent barcoded library. By performing NGS and analyzing the data, we confirmed that the resultant STR genotypes were consistent with those of CE-based typing. Moreover, sequence variations were detected in targeted STR regions. Through the use of small-sized amplicons, the developed multiplex PCR system enables researchers to obtain successful STR profiles even from artificially degraded DNA as well as STR loci which are analyzed with large-sized amplicons in the CE-based commercial kits. In addition, successful profiles can be obtained from mixtures up to a 1:19 ratio. Consequently, the developed multiplex PCR system, which produces small size amplicons, can be successfully applied to STR NGS analysis of forensic casework samples such as mixtures and degraded DNA samples. PMID:26799314

  5. A variant in the sonic hedgehog regulatory sequence (ZRS) is associated with triphalangeal thumb and deregulates expression in the developing limb

    PubMed Central

    Furniss, Dominic; Lettice, Laura A.; Taylor, Indira B.; Critchley, Paul S.; Giele, Henk; Hill, Robert E.; Wilkie, Andrew O.M.

    2008-01-01

    A locus for triphalangeal thumb, variably associated with pre-axial polydactyly, was previously identified in the zone of polarizing activity regulatory sequence (ZRS), a long range limb-specific enhancer of the Sonic Hedgehog (SHH) gene at human chromosome 7q36.3. Here, we demonstrate that a 295T>C variant in the human ZRS, previously thought to represent a neutral polymorphism, acts as a dominant allele with reduced penetrance. We found this variant in three independently ascertained probands from southern England with triphalangeal thumb, demonstrated significant linkage of the phenotype to the variant (LOD = 4.1), and identified a shared microsatellite haplotype around the ZRS, suggesting that the probands share a common ancestor. An individual homozygous for the 295C allele presented with isolated bilateral triphalangeal thumb resembling the heterozygous phenotype, suggesting that the variant is largely dominant to the wild-type allele. As a functional test of the pathogenicity of the 295C allele, we utilized a mutated ZRS construct to demonstrate that it can drive ectopic anterior expression of a reporter gene in the developing mouse forelimb. We conclude that the 295T>C variant is in fact pathogenic and, in southern England, appears to be the most common cause of triphalangeal thumb. Depending on the dispersal of the founding mutation, it may play a wider role in the aetiology of this disorder. PMID:18463159

  6. Evolutionary origin of a novel gene expression pattern through co-option of the latent activities of existing regulatory sequences.

    PubMed

    Rebeiz, Mark; Jikomes, Nick; Kassner, Victoria A; Carroll, Sean B

    2011-06-21

    Spatiotemporal changes in gene expression underlie many evolutionary novelties in nature. However, the evolutionary origins of novel expression patterns, and the transcriptional control elements ("enhancers") that govern them, remain unclear. Here, we sought to explore the molecular genetic mechanisms by which new enhancers arise. We undertook a survey of closely related Drosophila species to identify recently evolved novel gene expression patterns and traced their evolutionary history. Analyses of gene expression in a variety of developing tissues of the Drosophila melanogaster species subgroup revealed high rates of expression pattern divergence, including numerous evolutionary losses, heterochronic shifts, and expansions or contractions of expression domains. However, gains of novel expression patterns were much less frequent. One gain was observed for the Neprilysin-1 (Nep1) gene, which has evolved a unique expression pattern in optic lobe neuroblasts of Drosophila santomea. Dissection of the Nep1 cis-regulatory region localized a newly derived optic lobe enhancer activity to a region of an intron that has accumulated a small number of mutations. The Nep1 optic lobe enhancer overlaps with other enhancer activities, from which the novel activity was co-opted. We suggest that the novel optic lobe enhancer evolved by exploiting the cryptic activity of extant regulatory sequences, and this may reflect a general mechanism whereby new enhancers evolve.

  7. Enhancer sequence variants and transcription-factor deregulation synergize to construct pathogenic regulatory circuits in B-cell lymphoma.

    PubMed

    Koues, Olivia I; Kowalewski, Rodney A; Chang, Li-Wei; Pyfrom, Sarah C; Schmidt, Jennifer A; Luo, Hong; Sandoval, Luis E; Hughes, Tyler B; Bednarski, Jeffrey J; Cashen, Amanda F; Payton, Jacqueline E; Oltz, Eugene M

    2015-01-20

    Most B-cell lymphomas arise in the germinal center (GC), where humoral immune responses evolve from potentially oncogenic cycles of mutation, proliferation, and clonal selection. Although lymphoma gene expression diverges significantly from GC B cells, underlying mechanisms that alter the activities of corresponding regulatory elements (REs) remain elusive. Here we define the complete pathogenic circuitry of human follicular lymphoma (FL), which activates or decommissions REs from normal GC B cells and commandeers enhancers from other lineages. Moreover, independent sets of transcription factors, whose expression was deregulated in FL, targeted commandeered versus decommissioned REs. Our approach revealed two distinct subtypes of low-grade FL, whose pathogenic circuitries resembled GC B or activated B cells. FL-altered enhancers also were enriched for sequence variants, including somatic mutations, which disrupt transcription-factor binding and expression of circuit-linked genes. Thus, the pathogenic regulatory circuitry of FL reveals distinct genetic and epigenetic etiologies for GC B-cell transformation.

  8. Next-generation sequencing of common osteogenesis imperfecta-related genes in clinical practice

    PubMed Central

    Árvai, Kristóf; Horváth, Péter; Balla, Bernadett; Tobiás, Bálint; Kató, Karina; Kirschner, Gyöngyi; Klujber, Valéria; Lakatos, Péter; Kósa, János P.

    2016-01-01

    Next generation sequencing (NGS) is a rapidly developing area in genetics. Utilizing this technology in the management of disorders with complex genetic background and not recurrent mutation hot spots can be extremely useful. In this study, we applied NGS, namely semiconductor sequencing to determine the most significant osteogenesis imperfecta-related genetic variants in the clinical practice. We selected genes coding collagen type I alpha-1 and-2 (COL1A1, COL1A2) which are responsible for more than 90% of all cases. CRTAP and LEPRE1/P3H1 genes involved in the background of the recessive forms with relatively high frequency (type VII and VIII) represent less than 10% of the disease. In our six patients (1–41 years), we identified 23 different variants. We found a total of 14 single nucleotide variants (SNV) in COL1A1 and COL1A2, 5 in CRTAP and 4 in LEPRE1. Two novel and two already well-established pathogenic SNVs have been identified. Among the newly recognized mutations, one results in an amino acid change and one of them is a stop codon. We have shown that a new full-scale cost-effective NGS method can be developed and utilized to supplement diagnostic process of osteogenesis imperfecta with molecular genetic data in clinical practice. PMID:27335225

  9. SNP discovery in common bean by restriction-associated DNA (RAD) sequencing for genetic diversity and population structure analysis.

    PubMed

    Valdisser, Paula Arielle M R; Pappas, Georgios J; de Menezes, Ivandilson P P; Müller, Bárbara S F; Pereira, Wendell J; Narciso, Marcelo G; Brondani, Claudio; Souza, Thiago L P O; Borba, Tereza C O; Vianello, Rosana P

    2016-06-01

    Researchers have made great advances into the development and application of genomic approaches for common beans, creating opportunities to driving more real and applicable strategies for sustainable management of the genetic resource towards plant breeding. This work provides useful polymorphic single-nucleotide polymorphisms (SNPs) for high-throughput common bean genotyping developed by RAD (restriction site-associated DNA) sequencing. The RAD tags were generated from DNA pooled from 12 common bean genotypes, including breeding lines of different gene pools and market classes. The aligned sequences identified 23,748 putative RAD-SNPs, of which 3357 were adequate for genotyping; 1032 RAD-SNPs with the highest ADT (assay design tool) score are presented in this article. The RAD-SNPs were structurally annotated in different coding (47.00 %) and non-coding (53.00 %) sequence components of genes. A subset of 384 RAD-SNPs with broad genome distribution was used to genotype a diverse panel of 95 common bean germplasms and revealed a successful amplification rate of 96.6 %, showing 73 % of polymorphic SNPs within the Andean group and 83 % in the Mesoamerican group. A slightly increased He (0.161, n = 21) value was estimated for the Andean gene pool, compared to the Mesoamerican group (0.156, n = 74). For the linkage disequilibrium (LD) analysis, from a group of 580 SNPs (289 RAD-SNPs and 291 BARC-SNPs) genotyped for the same set of genotypes, 70.2 % were in LD, decreasing to 0.10 %in the Andean group and 0.77 % in the Mesoamerican group. Haplotype patterns spanning 310 Mb of the genome (60 %) were characterized in samples from different origins. However, the haplotype frameworks were under-represented for the Andean (7.85 %) and Mesoamerican (5.55 %) gene pools separately. In conclusion, RAD sequencing allowed the discovery of hundreds of useful SNPs for broad genetic analysis of common bean germplasm. From now, this approach provides an excellent panel

  10. ‘In silico expression analysis’, a novel PathoPlant web tool to identify abiotic and biotic stress conditions associated with specific cis-regulatory sequences

    PubMed Central

    Machens, Fabian; Brill, Yuri; Romanov, Artyom; Bülow, Lorenz; Hehl, Reinhard

    2014-01-01

    Using bioinformatics, putative cis-regulatory sequences can be easily identified using pattern recognition programs on promoters of specific gene sets. The abundance of predicted cis-sequences is a major challenge to associate these sequences with a possible function in gene expression regulation. To identify a possible function of the predicted cis-sequences, a novel web tool designated ‘in silico expression analysis’ was developed that correlates submitted cis-sequences with gene expression data from Arabidopsis thaliana. The web tool identifies the A. thaliana genes harbouring the sequence in a defined promoter region and compares the expression of these genes with microarray data. The result is a hierarchy of abiotic and biotic stress conditions to which these genes are most likely responsive. When testing the performance of the web tool, known cis-regulatory sequences were submitted to the ‘in silico expression analysis’ resulting in the correct identification of the associated stress conditions. When using a recently identified novel elicitor-responsive sequence, a WT-box (CGACTTTT), the ‘in silico expression analysis’ predicts that genes harbouring this sequence in their promoter are most likely Botrytis cinerea induced. Consistent with this prediction, the strongest induction of a reporter gene harbouring this sequence in the promoter is observed with B. cinerea in transgenic A. thaliana. Database URL: http://www.pathoplant.de/expression_analysis.php. PMID:24727366

  11. 'In silico expression analysis', a novel PathoPlant web tool to identify abiotic and biotic stress conditions associated with specific cis-regulatory sequences.

    PubMed

    Bolívar, Julio C; Machens, Fabian; Brill, Yuri; Romanov, Artyom; Bülow, Lorenz; Hehl, Reinhard

    2014-01-01

    Using bioinformatics, putative cis-regulatory sequences can be easily identified using pattern recognition programs on promoters of specific gene sets. The abundance of predicted cis-sequences is a major challenge to associate these sequences with a possible function in gene expression regulation. To identify a possible function of the predicted cis-sequences, a novel web tool designated 'in silico expression analysis' was developed that correlates submitted cis-sequences with gene expression data from Arabidopsis thaliana. The web tool identifies the A. thaliana genes harbouring the sequence in a defined promoter region and compares the expression of these genes with microarray data. The result is a hierarchy of abiotic and biotic stress conditions to which these genes are most likely responsive. When testing the performance of the web tool, known cis-regulatory sequences were submitted to the 'in silico expression analysis' resulting in the correct identification of the associated stress conditions. When using a recently identified novel elicitor-responsive sequence, a WT-box (CGACTTTT), the 'in silico expression analysis' predicts that genes harbouring this sequence in their promoter are most likely Botrytis cinerea induced. Consistent with this prediction, the strongest induction of a reporter gene harbouring this sequence in the promoter is observed with B. cinerea in transgenic A. thaliana. DATABASE URL: http://www.pathoplant.de/expression_analysis.php. PMID:24727366

  12. Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family.

    PubMed

    Dai, Qi; Ren, Aiming; Westholm, Jakub O; Duan, Hong; Patel, Dinshaw J; Lai, Eric C

    2015-01-01

    Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain ("BEN-solo" factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional

  13. Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family

    PubMed Central

    Dai, Qi; Ren, Aiming; Westholm, Jakub O.; Duan, Hong; Patel, Dinshaw J.

    2015-01-01

    Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain (“BEN-solo” factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional

  14. Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein

    PubMed Central

    Lee, Chang Hoon; Melchers, Mark; Wang, Hongsheng; Torrey, Ted A.; Slota, Rebecca; Qi, Chen-Feng; Kim, Ji Young; Lugar, Patricia; Kong, Hee Jeong; Farrington, Lila; van der Zouwen, Boris; Zhou, Jeff X.; Lougaris, Vassilios; Lipsky, Peter E.; Grammer, Amrie C.; Morse, Herbert C.

    2006-01-01

    Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein–tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6. PMID:16380510

  15. Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein.

    PubMed

    Lee, Chang Hoon; Melchers, Mark; Wang, Hongsheng; Torrey, Ted A; Slota, Rebecca; Qi, Chen-Feng; Kim, Ji Young; Lugar, Patricia; Kong, Hee Jeong; Farrington, Lila; van der Zouwen, Boris; Zhou, Jeff X; Lougaris, Vassilios; Lipsky, Peter E; Grammer, Amrie C; Morse, Herbert C

    2006-01-23

    Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein-tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5' sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.

  16. Training in High-Throughput Sequencing: Common Guidelines to Enable Material Sharing, Dissemination, and Reusability

    PubMed Central

    Schiffthaler, Bastian

    2016-01-01

    The advancement of high-throughput sequencing (HTS) technologies and the rapid development of numerous analysis algorithms and pipelines in this field has resulted in an unprecedentedly high demand for training scientists in HTS data analysis. Embarking on developing new training materials is challenging for many reasons. Trainers often do not have prior experience in preparing or delivering such materials and struggle to keep them up to date. A repository of curated HTS training materials would support trainers in materials preparation, reduce the duplication of effort by increasing the usage of existing materials, and allow for the sharing of teaching experience among the HTS trainers’ community. To achieve this, we have developed a strategy for materials’ curation and dissemination. Standards for describing training materials have been proposed and applied to the curation of existing materials. A Git repository has been set up for sharing annotated materials that can now be reused, modified, or incorporated into new courses. This repository uses Git; hence, it is decentralized and self-managed by the community and can be forked/built-upon by all users. The repository is accessible at http://bioinformatics.upsc.se/htmr. PMID:27309738

  17. Genome sequence and genetic diversity of the common carp, Cyprinus carpio.

    PubMed

    Xu, Peng; Zhang, Xiaofeng; Wang, Xumin; Li, Jiongtang; Liu, Guiming; Kuang, Youyi; Xu, Jian; Zheng, Xianhu; Ren, Lufeng; Wang, Guoliang; Zhang, Yan; Huo, Linhe; Zhao, Zixia; Cao, Dingchen; Lu, Cuiyun; Li, Chao; Zhou, Yi; Liu, Zhanjiang; Fan, Zhonghua; Shan, Guangle; Li, Xingang; Wu, Shuangxiu; Song, Lipu; Hou, Guangyuan; Jiang, Yanliang; Jeney, Zsigmond; Yu, Dan; Wang, Li; Shao, Changjun; Song, Lai; Sun, Jing; Ji, Peifeng; Wang, Jian; Li, Qiang; Xu, Liming; Sun, Fanyue; Feng, Jianxin; Wang, Chenghui; Wang, Shaolin; Wang, Baosen; Li, Yan; Zhu, Yaping; Xue, Wei; Zhao, Lan; Wang, Jintu; Gu, Ying; Lv, Weihua; Wu, Kejing; Xiao, Jingfa; Wu, Jiayan; Zhang, Zhang; Yu, Jun; Sun, Xiaowen

    2014-11-01

    The common carp, Cyprinus carpio, is one of the most important cyprinid species and globally accounts for 10% of freshwater aquaculture production. Here we present a draft genome of domesticated C. carpio (strain Songpu), whose current assembly contains 52,610 protein-coding genes and approximately 92.3% coverage of its paleotetraploidized genome (2n = 100). The latest round of whole-genome duplication has been estimated to have occurred approximately 8.2 million years ago. Genome resequencing of 33 representative individuals from worldwide populations demonstrates a single origin for C. carpio in 2 subspecies (C. carpio Haematopterus and C. carpio carpio). Integrative genomic and transcriptomic analyses were used to identify loci potentially associated with traits including scaling patterns and skin color. In combination with the high-resolution genetic map, the draft genome paves the way for better molecular studies and improved genome-assisted breeding of C. carpio and other closely related species.

  18. Molecular identification of the traditional herbal medicines, Arisaematis Rhizoma and Pinelliae Tuber, and common adulterants via universal DNA barcode sequences.

    PubMed

    Moon, B C; Kim, W J; Ji, Y; Lee, Y M; Kang, Y M; Choi, G

    2016-01-01

    Methods to identify Pinelliae Tuber and Arisaematis Rhizoma are required because of frequent reciprocal substitution between these two herbal medicines and the existence of several closely related plant materials. As a result of the morphological similarity of dried tubers, correct discrimination of authentic herbal medicines is difficult by conventional methods. Therefore, we analyzed DNA barcode sequences to identify each herbal medicine and the common adulterants at a species level. To verify the identity of these herbal medicines, we collected five authentic species (Pinellia ternata for Pinelliae Tuber, and Arisaema amurense, A. amurense var. serratum, A. erubescens, and A. heterophyllum for Arisaematis Rhizoma) and six common adulterant plant species. Maturase K (matK) and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) genes were then amplified using universal primers. In comparative analyses of two DNA barcode sequences, we obtained 45 species-specific nucleotides sufficient to identify each species (except A. erubescens with matK) and 28 marker nucleotides for each species (except P. pedatisecta with rbcL). Sequence differences at corresponding positions of the two combined DNA barcodes provided genetic marker nucleotides that could be used to identify specimens of the correct species among the analyzed medicinal plants. Furthermore, we generated a phylogenetic tree showing nine distinct groups depending on the species. These results can be used to authenticate Pinelliae Tuber and Arisaematis Rhizoma from their adulterants and to identify each species. Thus, comparative analyses of plant DNA barcode sequences identified useful genetic markers for the authentication of Pinelliae Tuber and Arisaematis Rhizoma from several adulterant herbal materials. PMID:26909979

  19. Molecular identification of the traditional herbal medicines, Arisaematis Rhizoma and Pinelliae Tuber, and common adulterants via universal DNA barcode sequences.

    PubMed

    Moon, B C; Kim, W J; Ji, Y; Lee, Y M; Kang, Y M; Choi, G

    2016-02-19

    Methods to identify Pinelliae Tuber and Arisaematis Rhizoma are required because of frequent reciprocal substitution between these two herbal medicines and the existence of several closely related plant materials. As a result of the morphological similarity of dried tubers, correct discrimination of authentic herbal medicines is difficult by conventional methods. Therefore, we analyzed DNA barcode sequences to identify each herbal medicine and the common adulterants at a species level. To verify the identity of these herbal medicines, we collected five authentic species (Pinellia ternata for Pinelliae Tuber, and Arisaema amurense, A. amurense var. serratum, A. erubescens, and A. heterophyllum for Arisaematis Rhizoma) and six common adulterant plant species. Maturase K (matK) and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) genes were then amplified using universal primers. In comparative analyses of two DNA barcode sequences, we obtained 45 species-specific nucleotides sufficient to identify each species (except A. erubescens with matK) and 28 marker nucleotides for each species (except P. pedatisecta with rbcL). Sequence differences at corresponding positions of the two combined DNA barcodes provided genetic marker nucleotides that could be used to identify specimens of the correct species among the analyzed medicinal plants. Furthermore, we generated a phylogenetic tree showing nine distinct groups depending on the species. These results can be used to authenticate Pinelliae Tuber and Arisaematis Rhizoma from their adulterants and to identify each species. Thus, comparative analyses of plant DNA barcode sequences identified useful genetic markers for the authentication of Pinelliae Tuber and Arisaematis Rhizoma from several adulterant herbal materials.

  20. Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins During in vitro Culture Attenuation.

    PubMed

    Lehmann, Jason S; Corey, Victoria C; Ricaldi, Jessica N; Vinetz, Joseph M; Winzeler, Elizabeth A; Matthias, Michael A

    2016-02-01

    Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12-25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis.

  1. Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins during in vitro Culture Attenuation

    PubMed Central

    Lehmann, Jason S.; Corey, Victoria C.; Ricaldi, Jessica N.; Vinetz, Joseph M.; Winzeler, Elizabeth A.; Matthias, Michael A.

    2016-01-01

    Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12–25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis. PMID:26711524

  2. Improving the safety of viral DNA vaccines: development of vectors containing both 5' and 3' homologous regulatory sequences from non-viral origin.

    PubMed

    Martinez-Lopez, A; Encinas, P; García-Valtanen, P; Gomez-Casado, E; Coll, J M; Estepa, A

    2013-04-01

    Although some DNA vaccines have proved to be very efficient in field trials, their authorisation still remains limited to a few countries. This is in part due to safety issues because most of them contain viral regulatory sequences to driving the expression of the encoded antigen. This is the case of the only DNA vaccine against a fish rhabdovirus (a negative ssRNA virus), authorised in Canada, despite the important economic losses that these viruses cause to aquaculture all over the world. In an attempt to solve this problem and using as a model a non-authorised, but efficient DNA vaccine against the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV), we developed a plasmid construction containing regulatory sequences exclusively from fish origin. The result was an "all-fish vector", named pJAC-G, containing 5' and 3' regulatory sequences of β-acting genes from carp and zebrafish, respectively. In vitro and in vivo, pJAC-G drove a successful expression of the VHSV glycoprotein G (G), the only antigen of the virus conferring in vivo protection. Furthermore, and by means of in vitro fusion assays, it was confirmed that G protein expressed from pJAC-G was fully functional. Altogether, these results suggest that DNA vaccines containing host-homologous gene regulatory sequences might be useful for developing safer DNA vaccines, while they also might be useful for basic studies.

  3. A meta-analysis of caloric restriction gene expression profiles to infer common signatures and regulatory mechanisms.

    PubMed

    Plank, Michael; Wuttke, Daniel; van Dam, Sipko; Clarke, Susan A; de Magalhães, João Pedro

    2012-04-01

    Caloric restriction, a reduction in calorie intake without malnutrition, retards age-related degeneration and extends lifespan in several organisms. CR induces multiple changes, yet its underlying mechanisms remain poorly understood. In this work, we first performed a meta-analysis of microarray CR studies in mammals and identified genes and processes robustly altered due to CR. Our results reveal a complex array of CR-induced changes and we re-identified several genes and processes previously associated with CR, such as growth hormone signalling, lipid metabolism and immune response. Moreover, our results highlight novel associations with CR, such as retinol metabolism and copper ion detoxification, as well as hint of a strong effect of CR on circadian rhythms that in turn may contribute to metabolic changes. Analyses of our signatures by integrating co-expression data, information on genetic mutants, and transcription factor binding site analysis revealed candidate regulators of transcriptional modules in CR. Our results hint at a transcriptional module involved in sterol metabolism regulated by Srebf1. A putative regulatory role of Ppara was also identified. Overall, our conserved molecular signatures of CR provide a comprehensive picture of CR-induced changes and help understand its regulatory mechanisms.

  4. Discovery of common Asian copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing.

    PubMed

    Park, Hansoo; Kim, Jong-Il; Ju, Young Seok; Gokcumen, Omer; Mills, Ryan E; Kim, Sheehyun; Lee, Seungbok; Suh, Dongwhan; Hong, Dongwan; Kang, Hyunseok Peter; Yoo, Yun Joo; Shin, Jong-Yeon; Kim, Hyun-Jin; Yavartanoo, Maryam; Chang, Young Wha; Ha, Jung-Sook; Chong, Wilson; Hwang, Ga-Ram; Darvishi, Katayoon; Kim, Hyeran; Yang, Song Ju; Yang, Kap-Seok; Kim, Hyungtae; Hurles, Matthew E; Scherer, Stephen W; Carter, Nigel P; Tyler-Smith, Chris; Lee, Charles; Seo, Jeong-Sun

    2010-05-01

    Copy number variants (CNVs) account for the majority of human genomic diversity in terms of base coverage. Here, we have developed and applied a new method to combine high-resolution array comparative genomic hybridization (CGH) data with whole-genome DNA sequencing data to obtain a comprehensive catalog of common CNVs in Asian individuals. The genomes of 30 individuals from three Asian populations (Korean, Chinese and Japanese) were interrogated with an ultra-high-resolution array CGH platform containing 24 million probes. Whole-genome sequencing data from a reference genome (NA10851, with 28.3x coverage) and two Asian genomes (AK1, with 27.8x coverage and AK2, with 32.0x coverage) were used to transform the relative copy number information obtained from array CGH experiments into absolute copy number values. We discovered 5,177 CNVs, of which 3,547 were putative Asian-specific CNVs. These common CNVs in Asian populations will be a useful resource for subsequent genetic studies in these populations, and the new method of calling absolute CNVs will be essential for applying CNV data to personalized medicine.

  5. Complete subunit sequences, structure and evolution of the 6 x 6-mer hemocyanin from the common house centipede, Scutigera coleoptrata.

    PubMed

    Kusche, Kristina; Hembach, Anne; Hagner-Holler, Silke; Gebauer, Wolfgang; Burmester, Thorsten

    2003-07-01

    Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure-function relationship and evolution. S. coleoptrata hemocyanin is a 6 x 6-mer composed of four distinct subunit types that occur in an approximate 2 : 2 : 1 : 1 ratio and are 49.5-55.5% identical. The cDNA of a fifth, highly diverged, putative hemocyanin was identified that is not included in the native 6 x 6-mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilopoda and Diplopoda more than 420 million years ago. In contrast to the situation in the Crustacea and Chelicerata, the substitution rates among the myriapod hemocyanin subunits are highly variable. Phylogenetic analyses do not support a common clade of Myriapoda and Hexapoda, whereas there is evidence in favor of monophyletic Mandibulata.

  6. Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts.

    PubMed

    Griffin, Bryan D; Nagy, Eva

    2011-06-01

    Recombinant fowl adenoviruses (FAdVs) have been successfully used as veterinary vaccine vectors. However, insufficient definitions of the protein-coding and non-coding regions and an incomplete understanding of virus-host interactions limit the progress of next-generation vectors. FAdVs are known to cause several diseases of poultry. Certain isolates of species FAdV-C are the aetiological agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS). In this study, we report the complete 45667 bp genome sequence of FAdV-4 of species FAdV-C. Assessment of the protein-coding potential of FAdV-4 was carried out with the Bio-Dictionary-based Gene Finder together with an evaluation of sequence conservation among species FAdV-A and FAdV-D. On this basis, 46 potentially protein-coding ORFs were identified. Of these, 33 and 13 ORFs were assigned high and low protein-coding potential, respectively. Homologues of the ancestral adenoviral genes were, with few exceptions, assigned high protein-coding potential. ORFs that were unique to the FAdVs were differentiated into high and low protein-coding potential groups. Notable putative genes with high protein-coding capacity included the previously unreported fiber 1, hypothetical 10.3K and hypothetical 10.5K genes. Transcript analysis revealed that several of the small ORFs less than 300 nt in length that were assigned low coding potential contributed to upstream ORFs (uORFs) in important mRNAs, including the ORF22 mRNA. Subsequent analysis of the previously reported transcripts of FAdV-1, FAdV-9, human adenovirus 2 and bovine adenovirus 3 identified widespread uORFs in AdV mRNAs that have the potential to act as important translational regulatory elements.

  7. Targeted re-sequencing analysis of 25 genes commonly mutated in myeloid disorders in del(5q) myelodysplastic syndromes

    PubMed Central

    Fernandez-Mercado, Marta; Burns, Adam; Pellagatti, Andrea; Giagounidis, Aristoteles; Germing, Ulrich; Agirre, Xabier; Prosper, Felipe; Aul, Carlo; Killick, Sally; Wainscoat, James S.; Schuh, Anna; Boultwood, Jacqueline

    2013-01-01

    Interstitial deletion of chromosome 5q is the most common chromosomal abnormality in myelodysplastic syndromes. The catalogue of genes involved in the molecular pathogenesis of myelodysplastic syndromes is rapidly expanding and next-generation sequencing technology allows detection of these mutations at great depth. Here we describe the design, validation and application of a targeted next-generation sequencing approach to simultaneously screen 25 genes mutated in myeloid malignancies. We used this method alongside single nucleotide polymorphism-array technology to characterize the mutational and cytogenetic profile of 43 cases of early or advanced del(5q) myelodysplastic syndromes. A total of 29 mutations were detected in our cohort. Overall, 45% of early and 66.7% of advanced cases had at least one mutation. Genes with the highest mutation frequency among advanced cases were TP53 and ASXL1 (25% of patients each). These showed a lower mutation frequency in cases of 5q- syndrome (4.5% and 13.6%, respectively), suggesting a role in disease progression in del(5q) myelodysplastic syndromes. Fifty-two percent of mutations identified were in genes involved in epigenetic regulation (ASXL1, TET2, DNMT3A and JAK2). Six mutations had allele frequencies <20%, likely below the detection limit of traditional sequencing methods. Genomic array data showed that cases of advanced del(5q) myelodysplastic syndrome had a complex background of cytogenetic aberrations, often encompassing genes involved in myeloid disorders. Our study is the first to investigate the molecular pathogenesis of early and advanced del(5q) myelodysplastic syndromes using next-generation sequencing technology on a large panel of genes frequently mutated in myeloid malignancies, further illuminating the molecular landscape of del(5q) myelodysplastic syndromes. PMID:23831921

  8. Regulatory function of conserved sequences upstream of the long-wave sensitive opsin genes in teleost fishes.

    PubMed

    Tam, Kevin J; Watson, Corey T; Massah, Shabnam; Kolybaba, Addie M; Breden, Felix; Prefontaine, Gratien G; Beischlag, Timothy V

    2011-11-01

    Vertebrate opsin genes often occur in sets of tandem duplicates, and their expression varies developmentally and in response to environmental cues. We previously identified two highly conserved regions upstream of the long-wave sensitive opsin (LWS) gene cluster in teleosts. This region has since been shown in zebrafish to drive expression of LWS genes in vivo. In order to further investigate how elements in this region control opsin gene expression, we tested constructs encompassing the highly conserved regions and the less conserved portions upstream of the coding sequences in a promoter-less luciferase expression system. A ∼4500 bp construct of the upstream region, including the highly-conserved regions Reg I and Reg II, increased expression 100-fold, and successive 5' deletions reduced expression relative to the full 4.5 Kb region. Gene expression was highest when the transcription factor RORα was co-transfected with the proposed regulatory regions. Because these regions were tested in a promoter-less expression system, they include elements able to initiate and drive transcription. Teleosts exhibit complex color-mediated adaptive behavior and their adaptive significance has been well documented in several species. Therefore these upstream regions of LWS represent a model system for understanding the molecular basis of adaptive variation in gene regulation of color vision.

  9. Variant upstream regulatory region sequences differentially regulate human papillomavirus type 16 DNA replication throughout the viral life cycle.

    PubMed

    Hubert, Walter G

    2005-05-01

    While the central role of the viral upstream regulatory region (URR) in the human papillomavirus (HPV) life cycle has been well established, its effects on viral replication factor expression and plasmid replication of HPV type 16 (HPV16) remain unclear. Some nonprototypic variants of HPV16 contain altered URR sequences and are considered to increase the oncogenic risk of infections. To determine the relationship between viral replication and variant URRs, hybrid viral genomes were constructed with the replication-competent HPV16 prototype W12 and analyzed in assays which recapitulate the different phases of normal viral replication. The establishment efficiencies of hybrid HPV16 genomes differed about 20-fold among European prototypes and variants from Africa and America. Generally, European and African genomes exhibited the lowest replication efficiencies. The high replication levels observed with American variants were primarily attributable to their efficient expression of the replication factors E1 and E2. The maintenance levels of these viral genomes varied about fivefold, which correlated with their respective establishment phenotypes and published P(97) activities. Vegetative DNA amplification could also be observed with replicating HPV16 genomes. These results indicate that efficient E1/E2 expression and elevated plasmid replication levels during the persistent stage of infection may comprise a risk factor in HPV16-mediated oncogenesis.

  10. A cis-regulatory sequence from a short intergenic region gives rise to a strong microbe-associated molecular pattern-responsive synthetic promoter.

    PubMed

    Lehmeyer, Mona; Hanko, Erik K R; Roling, Lena; Gonzalez, Lilian; Wehrs, Maren; Hehl, Reinhard

    2016-06-01

    The high gene density in Arabidopsis thaliana leaves only relatively short intergenic regions for potential cis-regulatory sequences. To learn more about the regulation of genes harbouring only very short upstream intergenic regions, this study investigates a recently identified novel microbe-associated molecular pattern (MAMP)-responsive cis-sequence located within the 101 bp long intergenic region upstream of the At1g13990 gene. It is shown that the cis-regulatory sequence is sufficient for MAMP-responsive reporter gene activity in the context of its native promoter. The 3' UTR of the upstream gene has a quantitative effect on gene expression. In context of a synthetic promoter, the cis-sequence is shown to achieve a strong increase in reporter gene activity as a monomer, dimer and tetramer. Mutation analysis of the cis-sequence determined the specific nucleotides required for gene expression activation. In transgenic A. thaliana the synthetic promoter harbouring a tetramer of the cis-sequence not only drives strong pathogen-responsive reporter gene expression but also shows a high background activity. The results of this study contribute to our understanding how genes with very short upstream intergenic regions are regulated and how these regions can serve as a source for MAMP-responsive cis-sequences for synthetic promoter design.

  11. A cis-regulatory sequence from a short intergenic region gives rise to a strong microbe-associated molecular pattern-responsive synthetic promoter.

    PubMed

    Lehmeyer, Mona; Hanko, Erik K R; Roling, Lena; Gonzalez, Lilian; Wehrs, Maren; Hehl, Reinhard

    2016-06-01

    The high gene density in Arabidopsis thaliana leaves only relatively short intergenic regions for potential cis-regulatory sequences. To learn more about the regulation of genes harbouring only very short upstream intergenic regions, this study investigates a recently identified novel microbe-associated molecular pattern (MAMP)-responsive cis-sequence located within the 101 bp long intergenic region upstream of the At1g13990 gene. It is shown that the cis-regulatory sequence is sufficient for MAMP-responsive reporter gene activity in the context of its native promoter. The 3' UTR of the upstream gene has a quantitative effect on gene expression. In context of a synthetic promoter, the cis-sequence is shown to achieve a strong increase in reporter gene activity as a monomer, dimer and tetramer. Mutation analysis of the cis-sequence determined the specific nucleotides required for gene expression activation. In transgenic A. thaliana the synthetic promoter harbouring a tetramer of the cis-sequence not only drives strong pathogen-responsive reporter gene expression but also shows a high background activity. The results of this study contribute to our understanding how genes with very short upstream intergenic regions are regulated and how these regions can serve as a source for MAMP-responsive cis-sequences for synthetic promoter design. PMID:26833485

  12. Common interruptions in the repeating tripeptide sequence of non-fibrillar collagens: sequence analysis and structural studies on triple-helix peptide models.

    PubMed

    Thiagarajan, Geetha; Li, Yingjie; Mohs, Angela; Strafaci, Christopher; Popiel, Magdalena; Baum, Jean; Brodsky, Barbara

    2008-02-22

    Interruptions in the repeating (Gly-X1-X2)(n) amino acid sequence pattern are found in the triple-helix domains of all non-fibrillar collagens, and perturbations to the triple-helix at such sites are likely to play a role in collagen higher-order structure and function. This study defines the sequence features and structural consequences of the most common interruption, where one residue is missing from the tripeptide pattern, Gly-X1-X2-Gly-AA(1)-Gly-X1-X2, designated G1G interruptions. Residues found within G1G interruptions are predominantly hydrophobic (70%), followed by a significant amount of charged residues (16%), and the Gly-X1-X2 triplets flanking the interruption are atypical. Studies on peptide models indicate the degree of destabilization is much greater when Pro is in the interruption, GP, than when hydrophobic residues (GF, GY) are present, and a rigid Gly-Pro-Hyp tripeptide adjacent to the interruption leads to greater destabilization than a flexible Gly-Ala-Ala sequence. Modeling based on NMR data indicates the Phe residue within a GF interruption is located on the outside of the triple helix. The G1G interruptions resemble a previously studied collagen interruption GPOGAAVMGPO, designated G4G-type, in that both are destabilizing, but allow continuation of rod-like triple helices and maintenance of the single residue stagger throughout the imperfection, with a loss of axial register of the superhelix on both sides. Both kinds of interruptions result in a highly localized perturbation in hydrogen bonding and dihedral angles, but the hydrophobic residue of a G4G interruption packs near the central axis of the superhelix, while the hydrophobic residue of a G1G interruption is located on the triple-helix surface. The different structural consequences of G1G and G4G interruptions in the repeating tripeptide sequence pattern suggest a physical basis for their differential susceptibility to matrix metalloproteinases in type X collagen.

  13. Integrative analysis of time course microarray data and DNA sequence data via log-linear models for identifying dynamic transcriptional regulatory networks.

    PubMed

    Choi, Hyung-Seok; Kim, Youngchul; Cho, Kwang-Hyun; Park, Taesung

    2013-01-01

    Since eukaryotic transcription is regulated by sets of Transcription Factors (TFs) having various transcriptional time delays, identification of temporal combinations of activated TFs is important to reconstruct Transcriptional Regulatory Networks (TRNs). Our methods combine time course microarray data, information on physical binding between the TFs and their targets and the regulatory sequences of genes using a log-linear model to reconstruct dynamic functional TRNs of the yeast cell cycle and human apoptosis. In conclusion, our results suggest that the proposed dynamic motif search method is more effective in reconstructing TRNs than the static motif search method.

  14. OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences.

    PubMed Central

    Murzin, A G

    1993-01-01

    A novel folding motif has been observed in four different proteins which bind oligonucleotides or oligosaccharides: staphylococcal nuclease, anticodon binding domain of asp-tRNA synthetase and B-subunits of heat-labile enterotoxin and verotoxin-1. The common fold of the four proteins, which we call the OB-fold, has a five-stranded beta-sheet coiled to form a closed beta-barrel. This barrel is capped by an alpha-helix located between the third and fourth strands. The barrel-helix frameworks can be superimposed with r.m.s. deviations of 1.4-2.2 A, but no similarities can be observed in the corresponding alignment of the four sequences. The nucleotide or sugar binding sites, known for three of the four proteins, are located in nearly the same position in each protein: on the side surface of the beta-barrel, where three loops come together. Here we describe the determinants of the OB-fold, based on an analysis of all four structures. These proposed determinants explain how very different sequences adopt the OB-fold. They also suggest a reinterpretation of the controversial structure of gene 5 ssDNA binding protein, which exhibits some topological and functional similarities with the OB-fold proteins. PMID:8458342

  15. Flanking regulatory sequences of the locus encoding the murine GDNF receptor, c-ret, directs lac Z (beta-galactosidase) expression in developing somatosensory system.

    PubMed

    Sukumaran, M; Waxman, S G; Wood, J N; Pachnis, V

    2001-11-01

    RET forms the catalytic component within the receptor complex that transmits signals from the GDNF family of neurotrophic factors. To study the mechanisms regulating the cell-type specific expression of this gene, we have cloned and characterised the murine c-ret locus. A cosmid contig comprising approximately 60 kb of the mouse genome encompassing the entire structural gene and flanking sequences have been isolated and the transcription initiation site identified and promoter characterised. The murine c-ret promoter lacks a TATA initiation motif and has GC enriched DNA sequences reminiscent of CpG islands. Analysis of transgenic mice lines bearing the Lac Z (beta-galactosidase) reporter gene under the control of 5' flanking sequences show modularity in the organisation of cis-regulatory domains within the locus. Cloned 5' flanking sequences comprise a distal regulatory domain directing Lac Z expression at the primitive streak, lateral mesoderm and facial ganglia and a proximal sensory neurones specific regulatory domain inducing Lac Z expression primarily within the developing somatosensory system. The spatial and temporal progression of transgene expression precisely recapitulates endogenous gene expression in developing sensory ganglia including its induction in postnatal Isolectin B4 binding nociceptive neurones. PMID:11747074

  16. Complete genome sequence of a common midwife toad virus-like ranavirus associated with mass mortalities in wild amphibians in the Netherlands.

    PubMed

    van Beurden, Steven J; Hughes, Joseph; Saucedo, Bernardo; Rijks, Jolianne; Kik, Marja; Haenen, Olga L M; Engelsma, Marc Y; Gröne, Andrea; Verheije, M Helene; Wilkie, Gavin

    2014-12-24

    A ranavirus associated with mass mortalities in wild water frogs (Pelophylax spp.) and other amphibians in the Netherlands since 2010 was isolated, and its complete genome sequence was determined. The virus has a genome of 107,772 bp and shows 96.5% sequence identity with the common midwife toad virus from Spain.

  17. Complete Genome Sequence of a Common Midwife Toad Virus-Like Ranavirus Associated with Mass Mortalities in Wild Amphibians in the Netherlands

    PubMed Central

    Hughes, Joseph; Saucedo, Bernardo; Rijks, Jolianne; Kik, Marja; Haenen, Olga L. M.; Engelsma, Marc Y.; Gröne, Andrea; Verheije, M. Helene; Wilkie, Gavin

    2014-01-01

    A ranavirus associated with mass mortalities in wild water frogs (Pelophylax spp.) and other amphibians in the Netherlands since 2010 was isolated, and its complete genome sequence was determined. The virus has a genome of 107,772 bp and shows 96.5% sequence identity with the common midwife toad virus from Spain. PMID:25540340

  18. The complete sequence of the human CD79b (Ig{beta}/B29) gene: Identification of a conserved exon/intron organization, immunoglobulin-like regulatory regions, and allelic polymorphism

    SciTech Connect

    Hashimoto, S.; Chiorazzi, N.; Gregersen, P.K. |

    1994-12-31

    We determined the complete genomic sequence of the human CD79b (Ig{beta}/B29) gene. The CD79b gene product is associated with the membrane immunoglobulin signaling complex which is composed of immunoglobulin (Ig) itself, associated in a noncovalent fashion with CD79b and a second polypeptide chain, CD79a (Ig{alpha}/mb1). The sequence and exon/intron organization of the human and mouse CD79b genes are highly similar. The gene organization suggests that some variant forms of CD79b may arise by virtue of alternative splicing of mRNA. In addition, a number of conserved regulatory sequences commonly found in Ig genes are present in sequences which flank the human CD79b gene. Some of these sequences are distinct from those found in the CD79a promoter. These differences may explain why transcription of CD79b, but not CD79a, is observed in plasma cells. A new Taq 1 restriction fragment length polymorphism is described that is not associated with any structural polymorphisms of the expressed CD79b polypeptide. 13 refs., 3 figs., 1 tab.

  19. Fully automated segmentation and tracking of the intima media thickness in ultrasound video sequences of the common carotid artery.

    PubMed

    Ilea, Dana E; Duffy, Caoimhe; Kavanagh, Liam; Stanton, Alice; Whelan, Paul F

    2013-01-01

    The robust identification and measurement of the intima media thickness (IMT) has a high clinical relevance because it represents one of the most precise predictors used in the assessment of potential future cardiovascular events. To facilitate the analysis of arterial wall thickening in serial clinical investigations, in this paper we have developed a novel fully automatic algorithm for the segmentation, measurement, and tracking of the intima media complex (IMC) in B-mode ultrasound video sequences. The proposed algorithm entails a two-stage image analysis process that initially addresses the segmentation of the IMC in the first frame of the ultrasound video sequence using a model-based approach; in the second step, a novel customized tracking procedure is applied to robustly detect the IMC in the subsequent frames. For the video tracking procedure, we introduce a spatially coherent algorithm called adaptive normalized correlation that prevents the tracking process from converging to wrong arterial interfaces. This represents the main contribution of this paper and was developed to deal with inconsistencies in the appearance of the IMC over the cardiac cycle. The quantitative evaluation has been carried out on 40 ultrasound video sequences of the common carotid artery (CCA) by comparing the results returned by the developed algorithm with respect to ground truth data that has been manually annotated by clinical experts. The measured IMT(mean) ± standard deviation recorded by the proposed algorithm is 0.60 mm ± 0.10, with a mean coefficient of variation (CV) of 2.05%, whereas the corresponding result obtained for the manually annotated ground truth data is 0.60 mm ± 0.11 with a mean CV equal to 5.60%. The numerical results reported in this paper indicate that the proposed algorithm is able to correctly segment and track the IMC in ultrasound CCA video sequences, and we were encouraged by the stability of our technique when applied to data captured under

  20. Regulatory Mechanisms That Prevent Re-initiation of DNA Replication Can Be Locally Modulated at Origins by Nearby Sequence Elements

    PubMed Central

    Richardson, Christopher D.; Li, Joachim J.

    2014-01-01

    Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, origins re-initiate with diverse efficiencies when these mechanisms are disabled, and this diversity cannot be explained by differences in the efficiency or timing of origin initiation during normal S phase replication. This observation raises the possibility of an additional layer of replication control that can differentially regulate re-initiation at distinct origins. We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs). We have mapped the two RIPs to ∼60 bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 reassociates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Our findings identify a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are compromised. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some regions of the genome

  1. Large-scale evaluation of common variation in regulatory T cell-related genes and ovarian cancer outcome.

    PubMed

    Charbonneau, Bridget; Moysich, Kirsten B; Kalli, Kimberly R; Oberg, Ann L; Vierkant, Robert A; Fogarty, Zachary C; Block, Matthew S; Maurer, Matthew J; Goergen, Krista M; Fridley, Brooke L; Cunningham, Julie M; Rider, David N; Preston, Claudia; Hartmann, Lynn C; Lawrenson, Kate; Wang, Chen; Tyrer, Jonathan; Song, Honglin; deFazio, Anna; Johnatty, Sharon E; Doherty, Jennifer A; Phelan, Catherine M; Sellers, Thomas A; Ramirez, Starr M; Vitonis, Allison F; Terry, Kathryn L; Van Den Berg, David; Pike, Malcolm C; Wu, Anna H; Berchuck, Andrew; Gentry-Maharaj, Aleksandra; Ramus, Susan J; Diergaarde, Brenda; Shen, Howard; Jensen, Allan; Menkiszak, Janusz; Cybulski, Cezary; Lubiłski, Jan; Ziogas, Argyrios; Rothstein, Joseph H; McGuire, Valerie; Sieh, Weiva; Lester, Jenny; Walsh, Christine; Vergote, Ignace; Lambrechts, Sandrina; Despierre, Evelyn; Garcia-Closas, Montserrat; Yang, Hannah; Brinton, Louise A; Spiewankiewicz, Beata; Rzepecka, Iwona K; Dansonka-Mieszkowska, Agnieszka; Seibold, Petra; Rudolph, Anja; Paddock, Lisa E; Orlow, Irene; Lundvall, Lene; Olson, Sara H; Hogdall, Claus K; Schwaab, Ira; du Bois, Andreas; Harter, Philipp; Flanagan, James M; Brown, Robert; Paul, James; Ekici, Arif B; Beckmann, Matthias W; Hein, Alexander; Eccles, Diana; Lurie, Galina; Hays, Laura E; Bean, Yukie T; Pejovic, Tanja; Goodman, Marc T; Campbell, Ian; Fasching, Peter A; Konecny, Gottfried; Kaye, Stanley B; Heitz, Florian; Hogdall, Estrid; Bandera, Elisa V; Chang-Claude, Jenny; Kupryjanczyk, Jolanta; Wentzensen, Nicolas; Lambrechts, Diether; Karlan, Beth Y; Whittemore, Alice S; Culver, Hoda Anton; Gronwald, Jacek; Levine, Douglas A; Kjaer, Susanne K; Menon, Usha; Schildkraut, Joellen M; Pearce, Celeste Leigh; Cramer, Daniel W; Rossing, Mary Anne; Chenevix-Trench, Georgia; Pharoah, Paul D P; Gayther, Simon A; Ness, Roberta B; Odunsi, Kunle; Sucheston, Lara E; Knutson, Keith L; Goode, Ellen L

    2014-04-01

    The presence of regulatory T cells (Treg) in solid tumors is known to play a role in patient survival in ovarian cancer and other malignancies. We assessed inherited genetic variations via 749 tag single-nucleotide polymorphisms (SNP) in 25 Treg-associated genes (CD28, CTLA4, FOXP3, IDO1, IL10, IL10RA, IL15, 1L17RA, IL23A, IL23R, IL2RA, IL6, IL6R, IL8, LGALS1, LGALS9, MAP3K8, STAT5A, STAT5B, TGFB1, TGFB2, TGFB3, TGFBR1, TGRBR2, and TGFBR3) in relation to ovarian cancer survival. We analyzed genotype and overall survival in 10,084 women with invasive epithelial ovarian cancer, including 5,248 high-grade serous, 1,452 endometrioid, 795 clear cell, and 661 mucinous carcinoma cases of European descent across 28 studies from the Ovarian Cancer Association Consortium (OCAC). The strongest associations were found for endometrioid carcinoma and IL2RA SNPs rs11256497 [HR, 1.42; 95% confidence interval (CI), 1.22-1.64; P = 5.7 × 10(-6)], rs791587 (HR, 1.36; 95% CI, 1.17-1.57; P = 6.2 × 10(-5)), rs2476491 (HR, = 1.40; 95% CI, 1.19-1.64; P = 5.6 × 10(-5)), and rs10795763 (HR, 1.35; 95% CI, 1.17-1.57; P = 7.9 × 10(-5)), and for clear cell carcinoma and CTLA4 SNP rs231775 (HR, 0.67; 95% CI, 0.54-0.82; P = 9.3 × 10(-5)) after adjustment for age, study site, population stratification, stage, grade, and oral contraceptive use. The rs231775 allele associated with improved survival in our study also results in an amino acid change in CTLA4 and previously has been reported to be associated with autoimmune conditions. Thus, we found evidence that SNPs in genes related to Tregs seem to play a role in ovarian cancer survival, particularly in patients with clear cell and endometrioid epithelial ovarian cancer. PMID:24764580

  2. Large-Scale Evaluation of Common Variation in Regulatory T Cell-Related Genes and Ovarian Cancer Outcome

    PubMed Central

    Charbonneau, Bridget; Moysich, Kirsten B.; Kalli, Kimberly R.; Oberg, Ann L.; Vierkant, Robert A.; Fogarty, Zachary C.; Block, Matthew S.; Maurer, Matthew J.; Goergen, Krista M.; Fridley, Brooke L.; Cunningham, Julie M.; Rider, David N.; Preston, Claudia; Hartmann, Lynn C.; Lawrenson, Kate; Wang, Chen; Tyrer, Jonathan; Song, Honglin; deFazio, Anna; Johnatty, Sharon E.; Doherty, Jennifer A.; Phelan, Catherine M.; Sellers, Thomas A.; Ramirez, Starr M.; Vitonis, Allison F.; Terry, Kathryn L.; Van Den Berg, David; Pike, Malcolm C.; Wu, Anna H.; Berchuck, Andrew; Gentry-Maharaj, Aleksandra; Ramus, Susan J.; Diergaarde, Brenda; Shen, Howard; Jensen, Allan; Menkiszak, Janusz; Cybulski, Cezary; Lubiński, Jan; Ziogas, Argyrios; Rothstein, Joseph H.; McGuire, Valerie; Sieh, Weiva; Lester, Jenny; Walsh, Christine; Vergote, Ignace; Lambrechts, Sandrina; Despierre, Evelyn; Garcia-Closas, Montserrat; Yang, Hannah; Brinton, Louise A.; Spiewankiewicz, Beata; Rzepecka, Iwona K.; Dansonka-Mieszkowska, Agnieszka; Seibold, Petra; Rudolph, Anja; Paddock, Lisa E.; Orlow, Irene; Lundvall, Lene; Olson, Sara H.; Hogdall, Claus K.; Schwaab, Ira; du Bois, Andreas; Harter, Philipp; Flanagan, James M.; Brown, Robert; Paul, James; Ekici, Arif B.; Beckmann, Matthias W.; Hein, Alexander; Eccles, Diana; Lurie, Galina; Hays, Laura E.; Bean, Yukie T.; Pejovic, Tanja; Goodman, Marc T.; Campbell, Ian; Fasching, Peter A.; Konecny, Gottfried; Kaye, Stanley B.; Heitz, Florian; Hogdall, Estrid; Bandera, Elisa V.; Chang-Claude, Jenny; Kupryjanczyk, Jolanta; Wentzensen, Nicolas; Lambrechts, Diether; Karlan, Beth Y.; Whittemore, Alice S.; Culver, Hoda Anton; Gronwald, Jacek; Levine, Douglas A.; Kjaer, Susanne K.; Menon, Usha; Schildkraut, Joellen M.; Pearce, Celeste Leigh; Cramer, Daniel W.; Rossing, Mary Anne; Chenevix-Trench, Georgia; Pharoah, Paul D.P.; Gayther, Simon A.; Ness, Roberta B.; Odunsi, Kunle; Sucheston, Lara E.; Knutson, Keith L.; Goode, Ellen L.

    2014-01-01

    The presence of regulatory T cells (Tregs) in solid tumors is known to play a role in patient survival in ovarian cancer and other malignancies. We assessed inherited genetic variations via 749 tag SNPs in 25 Treg-associated genes (CD28, CTLA4, FOXP3, IDO1, IL10, IL10RA, IL15, 1L17RA, IL23A, IL23R, IL2RA, IL6, IL6R, IL8, LGALS1, LGALS9, MAP3K8, STAT5A, STAT5B, TGFB1, TGFB2, TGFB3, TGFBR1, TGRBR2, and TGFBR3) in relation to ovarian cancer survival. We analyzed genotype and overall survival in 10,084 women with invasive epithelial ovarian cancer, including 5,248 high-grade serous, 1,452 endometrioid, 795 clear cell, and 661 mucinous carcinoma cases of European descent across 28 studies from the Ovarian Cancer Association Consortium (OCAC). The strongest associations were found for endometrioid carcinoma and IL2RA SNPs rs11256497 [HR=1.42, 95% CI: 1.22–1.64; p=5.7 × 10−6], rs791587 [HR=1.36, 95% CI:1.17–1.57; p=6.2 × 10−5], rs2476491 [HR=1.40, 95% CI: 1.191.64; p=5.6 × 10−5], and rs10795763 [HR=1.35, 95% CI: 1.17–1.57; p=7.9 × 10−5], and for clear cell carcinoma and CTLA4 SNP rs231775 [HR=0.67, 95% CI: 0.54–0.82; p=9.3 × 10−5] after adjustment for age, study site, population stratification, stage, grade, and oral contraceptive use. The rs231775 allele associated with improved survival in our study also results in an amino acid change in CTLA4 and previously has been reported to be associated with autoimmune conditions. Thus, we found evidence that SNPs in genes related to Tregs appear to play a role in ovarian cancer survival, particularly in patients with clear cell and endometrioid EOC. PMID:24764580

  3. Immunity related genes in dipterans share common enrichment of AT-rich motifs in their 5' regulatory regions that are potentially involved in nucleosome formation

    PubMed Central

    Hernandez-Romano, Jesus; Carlos-Rivera, Francisco J; Salgado, Heladia; Lamadrid-Figueroa, Hector; Valverde-Garduño, Veronica; Rodriguez, Mario H; Martinez-Barnetche, Jesus

    2008-01-01

    Background Understanding the transcriptional regulation mechanisms in response to environmental challenges is of fundamental importance in biology. Transcription factors associated to response elements and the chromatin structure had proven to play important roles in gene expression regulation. We have analyzed promoter regions of dipteran genes induced in response to immune challenge, in search for particular sequence patterns involved in their transcriptional regulation. Results 5' upstream regions of D. melanogaster and A. gambiae immunity-induced genes and their corresponding orthologous genes in 11 non-melanogaster drosophilid species and Ae. aegypti share enrichment in AT-rich short motifs. AT-rich motifs are associated with nucleosome formation as predicted by two different algorithms. In A. gambiae and D. melanogaster, many immunity genes 5' upstream sequences also showed NFκB response elements, located within 500 bp from the transcription start site. In A. gambiae, the frequency of ATAA motif near the NFκB response elements was increased, suggesting a functional link between nucleosome formation/remodelling and NFκB regulation of transcription. Conclusion AT-rich motif enrichment in 5' upstream sequences in A. gambiae, Ae. aegypti and the Drosophila genus immunity genes suggests a particular pattern of nucleosome formation/chromatin organization. The co-occurrence of such motifs with the NFκB response elements suggests that these sequence signatures may be functionally involved in transcriptional activation during dipteran immune response. AT-rich motif enrichment in regulatory regions in this group of co-regulated genes could represent an evolutionary constrained signature in dipterans and perhaps other distantly species. PMID:18613977

  4. Genetic polymorphism study of regulatory B cell molecules and cellular immunity function in an adult patient with Common Variable Immunodeficiency

    PubMed Central

    Sarantopoulos, A; Tselios, K; Skendros, P; Bougiouklis, D; Theodorou, I; Boura, P

    2008-01-01

    A 43 year old female patient presented for recurrent bacterial lower respiratory infections. A research for immunodeficiency status revealed total hypogammaglobulinemia, reduced IgG1, IgG2, IgG3 subclass levels, and low number of B lymphocytes (CD19+). Common Variable Immunodeficiency (CVID) 11.2 category was diagnosed according to recent criteria of primary immunodeficiencies (PID). Further immunological study consisting of genetic polymorphism of genes relating to differentiation, activation and function of B cells (ICOS, BAFF receptor BCMA and TACI) was performed, which did not reveal any related mutations. T cell parameters and Th1/Th2 cytokine network did not show any disturbances. It is postulated that probable endstage B cell differentiation defects should be investigated. The patient receives IVIGs replacement thereafter and the rate and severity of infections have significantly improved. PMID:18923749

  5. Creation of cis-regulatory elements during sea urchin evolution by co-option and optimization of a repetitive sequence adjacent to the spec2a gene.

    PubMed

    Dayal, Sandeep; Kiyama, Takae; Villinski, Jeffrey T; Zhang, Ning; Liang, Shuguang; Klein, William H

    2004-09-15

    The creation, preservation, and degeneration of cis-regulatory elements controlling developmental gene expression are fundamental genome-level evolutionary processes about which little is known. Here, we identify critical differences in cis-regulatory elements controlling the expression of the sea urchin aboral ectoderm-specific spec genes. We found multiple copies of a repetitive sequence element termed RSR in genomes of species within the Strongylocentrotidae family, but RSRs were not detected in genomes of species outside Strongylocentrotidae. spec genes in Strongylocentrotus purpuratus are invariably associated with RSRs, and the spec2a RSR functioned as a transcriptional enhancer and displayed greater activity than did spec1 or spec2c RSRs. Single-base pair differences at two cis-regulatory elements within the spec2a RSR increased the binding affinities of four transcription factors, SpCCAAT-binding factor at one element and SpOtx, SpGoosecoid, and SpGATA-E at another. The cis-regulatory elements to which these four factors bound were recent evolutionary acquisitions that acted to either activate or repress transcription, depending on the cell type. These elements were found in the spec2a RSR ortholog in Strongylocentrotus pallidus but not in RSR orthologs of Strongylocentrotus droebachiensis or Hemicentrotus pulcherrimus. Our results indicated that a dynamic pattern of cis-regulatory element evolution exists for spec genes despite their conserved aboral ectoderm expression.

  6. Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate.

    PubMed

    Mangold, Elisabeth; Böhmer, Anne C; Ishorst, Nina; Hoebel, Ann-Kathrin; Gültepe, Pinar; Schuenke, Hannah; Klamt, Johanna; Hofmann, Andrea; Gölz, Lina; Raff, Ruth; Tessmann, Peter; Nowak, Stefanie; Reutter, Heiko; Hemprich, Alexander; Kreusch, Thomas; Kramer, Franz-Josef; Braumann, Bert; Reich, Rudolf; Schmidt, Gül; Jäger, Andreas; Reiter, Rudolf; Brosch, Sibylle; Stavusis, Janis; Ishida, Miho; Seselgyte, Rimante; Moore, Gudrun E; Nöthen, Markus M; Borck, Guntram; Aldhorae, Khalid A; Lace, Baiba; Stanier, Philip; Knapp, Michael; Ludwig, Kerstin U

    2016-04-01

    Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO. PMID:27018475

  7. Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate

    PubMed Central

    Mangold, Elisabeth; Böhmer, Anne C.; Ishorst, Nina; Hoebel, Ann-Kathrin; Gültepe, Pinar; Schuenke, Hannah; Klamt, Johanna; Hofmann, Andrea; Gölz, Lina; Raff, Ruth; Tessmann, Peter; Nowak, Stefanie; Reutter, Heiko; Hemprich, Alexander; Kreusch, Thomas; Kramer, Franz-Josef; Braumann, Bert; Reich, Rudolf; Schmidt, Gül; Jäger, Andreas; Reiter, Rudolf; Brosch, Sibylle; Stavusis, Janis; Ishida, Miho; Seselgyte, Rimante; Moore, Gudrun E.; Nöthen, Markus M.; Borck, Guntram; Aldhorae, Khalid A.; Lace, Baiba; Stanier, Philip; Knapp, Michael; Ludwig, Kerstin U.

    2016-01-01

    Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10−2). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10−5; ORallelic = 2.46 [95% CI 1.6–3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10−9). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO. PMID:27018475

  8. Post-common envelope binaries from SDSS - VII. A catalogue of white dwarf-main sequence binaries

    NASA Astrophysics Data System (ADS)

    Rebassa-Mansergas, A.; Gänsicke, B. T.; Schreiber, M. R.; Koester, D.; Rodríguez-Gil, P.

    2010-02-01

    We present a catalogue of 1602 white-dwarf-main-sequence (WDMS) binaries from the spectroscopic Sloan Digital Sky Survey Data Release 6 (SDSS DR6). Among these, we identify 440 as new WDMS binaries. We select WDMS binary candidates by template fitting all 1.27 million DR6 spectra, using combined constraints in both χ2 and signal-to-noise ratio. In addition, we use Galaxy Evolution Explorer (GALEX) and UKIRT Infrared Sky Survey (UKIDSS) magnitudes to search for objects in which one of the two components dominates the SDSS spectrum. We use a decomposition/fitting technique to measure the effective temperatures, surface gravities, masses and distances to the white dwarfs, as well as the spectral types and distances to the companions in our catalogue. Distributions and density maps obtained from these stellar parameters are then used to study both the general properties and the selection effects of WDMS binaries in the SDSS. A comparison between the distances measured to the white dwarfs and the main-sequence companions shows dsec > dwd for approximately one-fifth of the systems, a tendency already found in our previous work. The hypothesis that magnetic activity raises the temperature of the inter-spot regions in active stars that are heavily covered by cool spots, leading to a bluer optical colour compared to inactive stars, remains the best explanation for this behaviour. We also make use of SDSS-GALEX-UKIDSS magnitudes to investigate the distribution of WDMS binaries, as well as their white-dwarf effective temperatures and companion star spectral types, in ultraviolet to infrared colour space. We show that WDMS binaries can be very efficiently separated from single main-sequence stars and white dwarfs when using a combined ultraviolet, optical and infrared colour selection. Finally, we also provide radial velocities for 1068 systems measured from the NaI λλ8183.27, 8194.81 absorption doublet and/or the Hα emission line. Among the systems with multiple SDSS

  9. Relation between mRNA expression and sequence information in Desulfovibrio vulgaris: Combinatorial contributions of upstream regulatory motifs and coding sequence features to variations in mRNA abundance

    SciTech Connect

    Wu, Gang; Nie, Lei; Zhang, Weiwen

    2006-05-26

    ABSTRACT-The context-dependent expression of genes is the core for biological activities, and significant attention has been given to identification of various factors contributing to gene expression at genomic scale. However, so far this type of analysis has been focused whether on relation between mRNA expression and non-coding sequence features such as upstream regulatory motifs or on correlation between mRN abundance and non-random features in coding sequences (e.g. codon usage and amino acid usage). In this study multiple regression analyses of the mRNA abundance and all sequence information in Desulfovibrio vulgaris were performed, with the goal to investigate how much coding and non-coding sequence features contribute to the variations in mRNA expression, and in what manner they act together...

  10. Sequence variations in the transcriptional regulatory region and intron1 of HLA-DQB1 gene and their linkage in southern Chinese ethnic groups.

    PubMed

    Xu, Yunping; Hu, Qingsong; Liu, Zehuan; Shen, Yang; Liu, Xiaoyi; Lin, Bin; Wu, Yuping; Chen, Shangwu; Xu, Anlong

    2005-08-01

    Sequence polymorphism in the transcriptional regulatory region extending to intron1 (DQRRI1) of HLA-DQB1 gene, and their haplotypic distributions were investigated in southern Chinese populations. We cloned and sequenced a 1.1-kb segment containing the transcriptional regulatory region, exon1, and partial intron1 of HLA-DQB1 gene of 37 individuals from nine different ethnic groups in southern China. A high-density map of 162 polymorphisms, including 152 single nucleotide polymorphisms (SNPs) and 10 insertion-deletion polymorphisms, was revealed. By comparing these data with SNPs deposited in dbSNP database in National Center for Biotechnology Information and polymorphisms that have been reported, 66 polymorphisms were firstly reported. A total of 16 different haplotypes were detected based on these 162 polymorphisms. The distribution of 16 alleles of DQRRI1 as well as their linkage with DQB1 exon2 alleles was also investigated based on the population study and phylogenetic analysis. Tight linkage between these two regions were discovered, as each of DQB1*02, DQB1*03, DQB1*04, DQB1*05, and DQB1*06 alleles was seen to be linked with specific DQRRI1 allele. Our study showed different pattern of transcriptional regulatory region, exon1, and intron1 of different DQB1 alleles.

  11. Monte Carlo simulations of post-common-envelope white dwarf + main sequence binaries: The effects of including recombination energy

    NASA Astrophysics Data System (ADS)

    Zorotovic, M.; Schreiber, M. R.; García-Berro, E.; Camacho, J.; Torres, S.; Rebassa-Mansergas, A.; Gänsicke, B. T.

    2014-08-01

    Context. Detached white dwarf + main sequence (WD+MS) post-common-envelope binaries (PCEBs) are perhaps the most suitable objects for testing predictions of close-compact binary-star evolution theories, in particular, common-envelope (CE) evolution. Consequently, the population of WD+MS PCEBs has been simulated by several authors in the past and the predictions have been compared with the observations. However, most of those theoretical predictions did not take into account the possible contributions to the envelope ejection from additional sources of energy (mostly recombination energy) stored in the envelope. Aims: Here we update existing binary population models of WD+MS PCEBs by assuming that in addition to a fraction αCE of the orbital energy, a fraction αrec of the recombination energy available within the envelope contributes to ejecting the envelope. Methods: We performed Monte Carlo simulations of 107 MS+MS binaries for 9 different combinations of αCE and αrec using standard assumptions for the initial primary mass function, binary separations, and initial-mass-ratio distribution and evolved these systems using the publicly available binary star evolution (BSE) code. Results: Including a fraction of the recombination energy leads to a clear prediction of a large number of long orbital period (≳10 days) systems mostly containing high-mass WDs. The fraction of systems with He-core WD primaries (MWD ≲ 0.5 M⊙) increases with the CE efficiency and the existence of very low-mass He WDs (≲0.3 M⊙) is only predicted for high values of the CE efficiency, i.e. αCE ≳ 0.5. All models predict on average longer orbital periods for PCEBs containing C/O-core WDs (MWD ≳ 0.5 M⊙) than for PCEBs containing He WDs. This effect increases with increasing values of both efficiencies, i.e., αCE and αrec. Longer periods after the CE phase are also predicted for systems containing more massive secondary stars. The initial-mass-ratio distribution affects the

  12. Sequence variants in SLC16A11 are a common risk factor for type 2 diabetes in Mexico.

    PubMed

    Williams, Amy L; Jacobs, Suzanne B R; Moreno-Macías, Hortensia; Huerta-Chagoya, Alicia; Churchhouse, Claire; Márquez-Luna, Carla; García-Ortíz, Humberto; Gómez-Vázquez, María José; Burtt, Noël P; Aguilar-Salinas, Carlos A; González-Villalpando, Clicerio; Florez, Jose C; Orozco, Lorena; Haiman, Christopher A; Tusié-Luna, Teresa; Altshuler, David

    2014-02-01

    Performing genetic studies in multiple human populations can identify disease risk alleles that are common in one population but rare in others, with the potential to illuminate pathophysiology, health disparities, and the population genetic origins of disease alleles. Here we analysed 9.2 million single nucleotide polymorphisms (SNPs) in each of 8,214 Mexicans and other Latin Americans: 3,848 with type 2 diabetes and 4,366 non-diabetic controls. In addition to replicating previous findings, we identified a novel locus associated with type 2 diabetes at genome-wide significance spanning the solute carriers SLC16A11 and SLC16A13 (P = 3.9 × 10(-13); odds ratio (OR) = 1.29). The association was stronger in younger, leaner people with type 2 diabetes, and replicated in independent samples (P = 1.1 × 10(-4); OR = 1.20). The risk haplotype carries four amino acid substitutions, all in SLC16A11; it is present at ~50% frequency in Native American samples and ~10% in east Asian, but is rare in European and African samples. Analysis of an archaic genome sequence indicated that the risk haplotype introgressed into modern humans via admixture with Neanderthals. The SLC16A11 messenger RNA is expressed in liver, and V5-tagged SLC16A11 protein localizes to the endoplasmic reticulum. Expression of SLC16A11 in heterologous cells alters lipid metabolism, most notably causing an increase in intracellular triacylglycerol levels. Despite type 2 diabetes having been well studied by genome-wide association studies in other populations, analysis in Mexican and Latin American individuals identified SLC16A11 as a novel candidate gene for type 2 diabetes with a possible role in triacylglycerol metabolism. PMID:24390345

  13. Sequence variants in SLC16A11 are a common risk factor for type 2 diabetes in Mexico.

    PubMed

    Williams, Amy L; Jacobs, Suzanne B R; Moreno-Macías, Hortensia; Huerta-Chagoya, Alicia; Churchhouse, Claire; Márquez-Luna, Carla; García-Ortíz, Humberto; Gómez-Vázquez, María José; Burtt, Noël P; Aguilar-Salinas, Carlos A; González-Villalpando, Clicerio; Florez, Jose C; Orozco, Lorena; Haiman, Christopher A; Tusié-Luna, Teresa; Altshuler, David

    2014-02-01

    Performing genetic studies in multiple human populations can identify disease risk alleles that are common in one population but rare in others, with the potential to illuminate pathophysiology, health disparities, and the population genetic origins of disease alleles. Here we analysed 9.2 million single nucleotide polymorphisms (SNPs) in each of 8,214 Mexicans and other Latin Americans: 3,848 with type 2 diabetes and 4,366 non-diabetic controls. In addition to replicating previous findings, we identified a novel locus associated with type 2 diabetes at genome-wide significance spanning the solute carriers SLC16A11 and SLC16A13 (P = 3.9 × 10(-13); odds ratio (OR) = 1.29). The association was stronger in younger, leaner people with type 2 diabetes, and replicated in independent samples (P = 1.1 × 10(-4); OR = 1.20). The risk haplotype carries four amino acid substitutions, all in SLC16A11; it is present at ~50% frequency in Native American samples and ~10% in east Asian, but is rare in European and African samples. Analysis of an archaic genome sequence indicated that the risk haplotype introgressed into modern humans via admixture with Neanderthals. The SLC16A11 messenger RNA is expressed in liver, and V5-tagged SLC16A11 protein localizes to the endoplasmic reticulum. Expression of SLC16A11 in heterologous cells alters lipid metabolism, most notably causing an increase in intracellular triacylglycerol levels. Despite type 2 diabetes having been well studied by genome-wide association studies in other populations, analysis in Mexican and Latin American individuals identified SLC16A11 as a novel candidate gene for type 2 diabetes with a possible role in triacylglycerol metabolism.

  14. Sequence variants in SLC16A11 are a common risk factor for type 2 diabetes in Mexico

    PubMed Central

    2014-01-01

    Performing genetic studies in multiple human populations can identify disease risk alleles that are common in one population but rare in others1, with the potential to illuminate pathophysiology, health disparities, and the population genetic origins of disease alleles. We analyzed 9.2 million single nucleotide polymorphisms (SNPs) in each of 8,214 Mexicans and Latin Americans: 3,848 with type 2 diabetes (T2D) and 4,366 non-diabetic controls. In addition to replicating previous findings2–4, we identified a novel locus associated with T2D at genome-wide significance spanning the solute carriers SLC16A11 and SLC16A13 (P=3.9×10−13; odds ratio (OR)=1.29). The association was stronger in younger, leaner people with T2D, and replicated in independent samples (P=1.1×10−4; OR=1.20). The risk haplotype carries four amino acid substitutions, all in SLC16A11; it is present at ≈50% frequency in Native American samples and ≈10% in East Asian, but rare in European and African samples. Analysis of an archaic genome sequence indicated the risk haplotype introgressed into modern humans via admixture with Neandertals. The SLC16A11 mRNA is expressed in liver, and V5-tagged SLC16A11 protein localizes to the endoplasmic reticulum. Expression of SLC16A11 in heterologous cells alters lipid metabolism, most notably causing an increase in intracellular triacylglycerol levels. Despite T2D having been well studied by genome-wide association studies (GWAS) in other populations, analysis in Mexican and Latin American individuals identified SLC16A11 as a novel candidate gene for T2D with a possible role in triacylglycerol metabolism. PMID:24390345

  15. Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing.

    PubMed

    Crampton, Mollee; Sripathi, Venkateswara R; Hossain, Khwaja; Kalavacharla, Venu

    2016-01-01

    Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar ("Sierra") using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation. PMID:27199997

  16. Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing

    PubMed Central

    Crampton, Mollee; Sripathi, Venkateswara R.; Hossain, Khwaja; Kalavacharla, Venu

    2016-01-01

    Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar (“Sierra”) using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation. PMID:27199997

  17. Common variable immunodeficiency, impaired neurological development and reduced numbers of T regulatory cells in a 10-year-old boy with a STAT1 gain-of-function mutation.

    PubMed

    Kobbe, Robin; Kolster, Manuela; Fuchs, Sebastian; Schulze-Sturm, Ulf; Jenderny, Jutta; Kochhan, Lothar; Staab, Julia; Tolosa, Eva; Grimbacher, Bodo; Meyer, Thomas

    2016-07-25

    Recently, gain-of-function (GOF) mutations in the gene encoding signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis (CMC). This case report describes a 10-year-old boy presenting with signs of common variable immunodeficiency (CVID), failure to thrive, impaired neurological development, and a history of recurrent mucocutaneous Candida infections. Sequencing of the STAT1 gene identified a heterozygous missense mutation in exon 7 encoding the STAT1 coiled-coil domain (c.514T>C, p.Phe172Leu). In addition to hypogammaglobulinemia with B-cell deficiency, and a low percentage of Th17 cells, immunological analysis of the patient revealed a marked depletion of forkhead-box P3(+)-expressing regulatory T cells (Tregs). In vitro stimulation of T cells from the patient with interferon-α (IFNα) and/or IFNɣ resulted in a significantly increased expression of STAT1-regulated target genes such as MIG1, IRF1, MX1, MCP1/CCL2, IFI-56K, and CXCL10 as compared to IFN-treated cells from a healthy control, while no IFNα/ɣ-mediated up-regulation of the FOXP3 gene was found. These data demonstrate that the STAT1 GOF mutation F172L, which results in impaired stability of the antiparallel STAT1 dimer conformation, is associated with inhibited Treg cell development and neurological symptoms.

  18. Common variable immunodeficiency, impaired neurological development and reduced numbers of T regulatory cells in a 10-year-old boy with a STAT1 gain-of-function mutation.

    PubMed

    Kobbe, Robin; Kolster, Manuela; Fuchs, Sebastian; Schulze-Sturm, Ulf; Jenderny, Jutta; Kochhan, Lothar; Staab, Julia; Tolosa, Eva; Grimbacher, Bodo; Meyer, Thomas

    2016-07-25

    Recently, gain-of-function (GOF) mutations in the gene encoding signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis (CMC). This case report describes a 10-year-old boy presenting with signs of common variable immunodeficiency (CVID), failure to thrive, impaired neurological development, and a history of recurrent mucocutaneous Candida infections. Sequencing of the STAT1 gene identified a heterozygous missense mutation in exon 7 encoding the STAT1 coiled-coil domain (c.514T>C, p.Phe172Leu). In addition to hypogammaglobulinemia with B-cell deficiency, and a low percentage of Th17 cells, immunological analysis of the patient revealed a marked depletion of forkhead-box P3(+)-expressing regulatory T cells (Tregs). In vitro stimulation of T cells from the patient with interferon-α (IFNα) and/or IFNɣ resulted in a significantly increased expression of STAT1-regulated target genes such as MIG1, IRF1, MX1, MCP1/CCL2, IFI-56K, and CXCL10 as compared to IFN-treated cells from a healthy control, while no IFNα/ɣ-mediated up-regulation of the FOXP3 gene was found. These data demonstrate that the STAT1 GOF mutation F172L, which results in impaired stability of the antiparallel STAT1 dimer conformation, is associated with inhibited Treg cell development and neurological symptoms. PMID:27063510

  19. A re-sequencing based assessment of genomic heterogeneity and fast neutron-induced deletions in a common bean cultivar

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A small fast neutron mutant population has been established from Phaseolus vulgaris cv. Red Hawk. We leveraged the available P. vulgaris genome sequence and high throughput next generation DNA sequencing to examine the genomic structure of five Phaseolus vulgaris cv. Red Hawk fast neutron mutants wi...

  20. Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence.

    PubMed Central

    Kirwan, Angie F; Bibby, Ashley C; Mvilongo, Thierry; Riedel, Heimo; Burke, Thomas; Millis, Sherri Z; Parissenti, Amadeo M

    2003-01-01

    The N-terminal pseudosubstrate site within the protein kinase Calpha (PKCalpha)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCalpha-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940-8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCalpha-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Ralpha(39-177): this protein contained the full regulatory domain of human PKCalpha fused to glutathione S-transferase (GST), but lacked amino acids 1-38 (including the pseudosubstrate sequence) and amino acids 178-270 (including the C2 region)]. GST-Ralpha(39-177) significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCalpha holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33-86 within GST-Ralpha(39-177) dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159-242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of

  1. On universal common ancestry, sequence similarity, and phylogenetic structure: the sins of P-values and the virtues of Bayesian evidence

    PubMed Central

    2011-01-01

    Background The universal common ancestry (UCA) of all known life is a fundamental component of modern evolutionary theory, supported by a wide range of qualitative molecular evidence. Nevertheless, recently both the status and nature of UCA has been questioned. In earlier work I presented a formal, quantitative test of UCA in which model selection criteria overwhelmingly choose common ancestry over independent ancestry, based on a dataset of universally conserved proteins. These model-based tests are founded in likelihoodist and Bayesian probability theory, in opposition to classical frequentist null hypothesis tests such as Karlin-Altschul E-values for sequence similarity. In a recent comment, Koonin and Wolf (K&W) claim that the model preference for UCA is "a trivial consequence of significant sequence similarity". They support this claim with a computational simulation, derived from universally conserved proteins, which produces similar sequences lacking phylogenetic structure. The model selection tests prefer common ancestry for this artificial data set. Results For the real universal protein sequences, hierarchical phylogenetic structure (induced by genealogical history) is the overriding reason for why the tests choose UCA; sequence similarity is a relatively minor factor. First, for cases of conflicting phylogenetic structure, the tests choose independent ancestry even with highly similar sequences. Second, certain models, like star trees and K&W's profile model (corresponding to their simulation), readily explain sequence similarity yet lack phylogenetic structure. However, these are extremely poor models for the real proteins, even worse than independent ancestry models, though they explain K&W's artificial data well. Finally, K&W's simulation is an implementation of a well-known phylogenetic model, and it produces sequences that mimic homologous proteins. Therefore the model selection tests work appropriately with the artificial data. Conclusions For K

  2. RegTransBase - A Database Of Regulatory Sequences and Interactionsin a Wide Range of Prokaryotic Genomes

    SciTech Connect

    Kazakov, Alexei E.; Cipriano, Michael J.; Novichkov, Pavel S.; Minovitsky, Simon; Vinogradov, Dmitry V.; Arkin, Adam; Mironov, AndreyA.; Gelfand, Mikhail S.; Dubchak, Inna

    2006-07-01

    RegTransBase, a manually curated database of regulatoryinteractions in prokaryotes, captures the knowledge in publishedscientific literature using a controlled vocabulary. Although a number ofdatabases describing interactions between regulatory proteins and theirbinding sites are currently being maintained, they focus mostly on themodel organisms Escherichia coli and Bacillus subtilis, or are entirelycomputationally derived. RegTransBase describes a large number ofregulatory interactions reported in many organisms and contains varioustypes of experimental data, in particular: the activation or repressionof transcription by an identified direct regulator; determining thetranscriptional regulatory function of a protein (or RNA) directlybinding to DNA (RNA); mapping or prediction of binding site for aregulatory protein; characterization of regulatory mutations. Currently,the RegTransBase content is derived from about 3000 relevant articlesdescribing over 7000 experiments in relation to 128 microbes. It containsdata on the regulation of about 7500 genes and evidence for 6500interactions with 650 regulators. RegTransBase also contains manuallycreated position weight matrices (PWM) that can be used to identifycandidate regulatory sites in over 60 species. RegTransBase is availableat http://regtransbase.lbl.gov.

  3. Specific cis-acting sequence for PHO8 expression interacts with PHO4 protein, a positive regulatory factor, in Saccharomyces cerevisiae.

    PubMed Central

    Hayashi, N; Oshima, Y

    1991-01-01

    The PHO8 gene of Saccharomyces cerevisiae encodes repressible alkaline phosphatase (rALPase; EC 3.1.3.1). The rALPase activity of the cells is two to three times higher in medium containing a low concentration of Pi than in high-Pi medium due to transcription of PHO8. The Pi signals are conveyed to PHO8 by binding of PHO4 protein, a positive regulatory factor, to a promoter region of PHO8 (PHO8p) under the influence of the PHO regulatory circuit. Deletion analysis of PHO8p DNA revealed two separate regulatory regions required for derepression of rALPase located at nucleotide positions -704 to -661 (distal region) and -548 to -502 (proximal region) and an inhibitory region located at -421 to -289 relative to the translation initiation codon. Gel retardation experiments showed that a beta-galactosidase-PHO4 fusion protein binds to a 132-bp PHO8p fragment bearing the proximal region but not to a 226-bp PHO8 DNA bearing the distal region. The fusion protein also binds to a synthetic oligonucleotide having the same 12-bp nucleotide sequence as the PHO8p DNA from positions -536 to -525. The 132-bp PHO8p fragment, connected at position -281 of the 5' upstream region of a HIS5'-'lacZ fused gene, could sense Pi signals in vivo, but a 20-bp synthetic oligonucleotide having the same sequence from -544 to -525 of the PHO8p DNA could not. Linker insertions in the PHO8p DNA indicated that the 5-bp sequence 5'-CACGT-3' from positions -535 to -531 is essential for binding the beta-galactosidase-PHO4 fusion protein and for derepression of rALPase. Images PMID:1990283

  4. Ubiquitous and gene-specific regulatory 5' sequences in a sea urchin histone DNA clone coding for histone protein variants.

    PubMed Central

    Busslinger, M; Portmann, R; Irminger, J C; Birnstiel, M L

    1980-01-01

    The DNA sequences of the entire structural H4, H3, H2A and H2B genes and of their 5' flanking regions have been determined in the histone DNA clone h19 of the sea urchin Psammechinus miliaris. In clone h19 the polarity of transcription and the relative arrangement of the histone genes is identical to that in clone h22 of the same species. The histone proteins encoded by h19 DNA differ in their primary structure from those encoded by clone h22 and have been compared to histone protein sequences of other sea urchin species as well as other eukaryotes. A comparative analysis of the 5' flanking DNA sequences of the structural histone genes in both clones revealed four ubiquitous sequence motifs; a pentameric element GATCC, followed at short distance by the Hogness box GTATAAATAG, a conserved sequence PyCATTCPu, in or near which the 5' ends of the mRNAs map in h22 DNA and lastly a sequence A, containing the initiation codon. These sequences are also found, sometimes in modified version, in front of other eukaryotic genes transcribed by polymerase II. When prelude sequences of isocoding histone genes in clone h19 and h22 are compared areas of homology are seen to extend beyond the ubiquitous sequence motifs towards the divergent AT-rich spacer and terminate between approximately 140 and 240 nucleotides away from the structural gene. These prelude regions contain quite large conservative sequence blocks which are specific for each type of histone genes. Images PMID:7443547

  5. Is There a Place for Asia in the K-12 Social Studies Curriculum? A Survey of Requirements and Common Course Sequences in the 50 States, 1987-1990.

    ERIC Educational Resources Information Center

    Martin, Roberta; Gronewold, Sue

    A nationwide survey of state education departments was conducted in order to learn the extent to which study of Asia is included in precollegiate education. It was found that there is a place for Asia in the curriculum. A course in world history, world geography, and/or world cultures is reportedly offered as part of the common course sequence in…

  6. Whole-Genome Sequencing Confirms that Burkholderia pseudomallei Multilocus Sequence Types Common to Both Cambodia and Australia Are Due to Homoplasy

    PubMed Central

    De Smet, Birgit; Mayo, Mark; Theobald, Vanessa; Kham, Chun; Heng, Seiha; Thong, Phe; Holden, Matthew T. G.; Peacock, Sharon J.; Spratt, Brian G.; Jacobs, Jan A.; Vandamme, Peter; Currie, Bart J.

    2014-01-01

    Burkholderia pseudomallei isolates with shared multilocus sequence types (STs) have not been isolated from different continents. We identified two STs shared between Australia and Cambodia. Whole-genome analysis revealed substantial diversity within STs, correctly identified the Asian or Australian origin, and confirmed that these shared STs were due to homoplasy. PMID:25392354

  7. Deciphering the molecular mechanisms underlying the binding of the TWIST1/E12 complex to regulatory E-box sequences.

    PubMed

    Bouard, Charlotte; Terreux, Raphael; Honorat, Mylène; Manship, Brigitte; Ansieau, Stéphane; Vigneron, Arnaud M; Puisieux, Alain; Payen, Léa

    2016-06-20

    The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers. PMID:27151200

  8. Deciphering the molecular mechanisms underlying the binding of the TWIST1/E12 complex to regulatory E-box sequences

    PubMed Central

    Bouard, Charlotte; Terreux, Raphael; Honorat, Mylène; Manship, Brigitte; Ansieau, Stéphane; Vigneron, Arnaud M.; Puisieux, Alain; Payen, Léa

    2016-01-01

    The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers. PMID:27151200

  9. Draft Genome Sequence of Escherichia coli 26R 793, a Plasmid-Free Recipient Strain Commonly Used in Conjugation Assays

    PubMed Central

    Hurley, Daniel; McGrath, Kathleen; Bai, Li; Hächler, Herbert; Stephan, Roger

    2016-01-01

    Here, we report the draft genome sequence of the lactose-negative, rifampin-resistant, Escherichia coli strain 26R 793. This isolate has been widely used in conjugation experiments as a general recipient strain. PMID:27795232

  10. De novo sequencing of root transcriptome reveals complex cadmium-responsive regulatory networks in radish (Raphanus sativus L.).

    PubMed

    Xu, Liang; Wang, Yan; Liu, Wei; Wang, Jin; Zhu, Xianwen; Zhang, Keyun; Yu, Rugang; Wang, Ronghua; Xie, Yang; Zhang, Wei; Gong, Yiqin; Liu, Liwang

    2015-07-01

    Cadmium (Cd) is a nonessential metallic trace element that poses potential chronic toxicity to living organisms. To date, little is known about the Cd-responsive regulatory network in root vegetable crops including radish. In this study, 31,015 unigenes representing 66,552 assembled unique transcripts were isolated from radish root under Cd stress based on de novo transcriptome assembly. In all, 1496 differentially expressed genes (DEGs) consisted of 3579 transcripts were identified from Cd-free (CK) and Cd-treated (Cd200) libraries. Gene Ontology and pathway enrichment analysis indicated that the up- and down-regulated DEGs were predominately involved in glucosinolate biosynthesis as well as cysteine and methionine-related pathways, respectively. RT-qPCR showed that the expression profiles of DEGs were in consistent with results from RNA-Seq analysis. Several candidate genes encoding phytochelatin synthase (PCS), metallothioneins (MTs), glutathione (GSH), zinc iron permease (ZIPs) and ABC transporter were responsible for Cd uptake, accumulation, translocation and detoxification in radish. The schematic model of DEGs and microRNAs-involved in Cd-responsive regulatory network was proposed. This study represents a first comprehensive transcriptome-based characterization of Cd-responsive DEGs in radish. These results could provide fundamental insight into complex Cd-responsive regulatory networks and facilitate further genetic manipulation of Cd accumulation in root vegetable crops. PMID:26025544

  11. De novo sequencing of root transcriptome reveals complex cadmium-responsive regulatory networks in radish (Raphanus sativus L.).

    PubMed

    Xu, Liang; Wang, Yan; Liu, Wei; Wang, Jin; Zhu, Xianwen; Zhang, Keyun; Yu, Rugang; Wang, Ronghua; Xie, Yang; Zhang, Wei; Gong, Yiqin; Liu, Liwang

    2015-07-01

    Cadmium (Cd) is a nonessential metallic trace element that poses potential chronic toxicity to living organisms. To date, little is known about the Cd-responsive regulatory network in root vegetable crops including radish. In this study, 31,015 unigenes representing 66,552 assembled unique transcripts were isolated from radish root under Cd stress based on de novo transcriptome assembly. In all, 1496 differentially expressed genes (DEGs) consisted of 3579 transcripts were identified from Cd-free (CK) and Cd-treated (Cd200) libraries. Gene Ontology and pathway enrichment analysis indicated that the up- and down-regulated DEGs were predominately involved in glucosinolate biosynthesis as well as cysteine and methionine-related pathways, respectively. RT-qPCR showed that the expression profiles of DEGs were in consistent with results from RNA-Seq analysis. Several candidate genes encoding phytochelatin synthase (PCS), metallothioneins (MTs), glutathione (GSH), zinc iron permease (ZIPs) and ABC transporter were responsible for Cd uptake, accumulation, translocation and detoxification in radish. The schematic model of DEGs and microRNAs-involved in Cd-responsive regulatory network was proposed. This study represents a first comprehensive transcriptome-based characterization of Cd-responsive DEGs in radish. These results could provide fundamental insight into complex Cd-responsive regulatory networks and facilitate further genetic manipulation of Cd accumulation in root vegetable crops.

  12. Differentiated evolutionary relationships among chordates from comparative alignments of multiple sequences of MyoD and MyoG myogenic regulatory factors.

    PubMed

    Oliani, L C; Lidani, K C F; Gabriel, J E

    2015-10-16

    MyoD and MyoG are transcription factors that have essential roles in myogenic lineage determination and muscle differentiation. The purpose of this study was to compare multiple amino acid sequences of myogenic regulatory proteins to infer evolutionary relationships among chordates. Protein sequences from Mus musculus (P10085 and P12979), human Homo sapiens (P15172 and P15173), bovine Bos taurus (Q7YS82 and Q7YS81), wild pig Sus scrofa (P49811 and P49812), quail Coturnix coturnix (P21572 and P34060), chicken Gallus gallus (P16075 and P17920), rat Rattus norvegicus (Q02346 and P20428), domestic water buffalo Bubalus bubalis (D2SP11 and A7L034), and sheep Ovis aries (Q90477 and D3YKV7) were searched from a non-redundant protein sequence database UniProtKB/Swiss-Prot, and subsequently analyzed using the Mega6.0 software. MyoD evolutionary analyses revealed the presence of three main clusters with all mammals branched in one cluster, members of the order Rodentia (mouse and rat) in a second branch linked to the first, and birds of the order Galliformes (chicken and quail) remaining isolated in a third. MyoG evolutionary analyses aligned sequences in two main clusters, all mammalian specimens grouped in different sub-branches, and birds clustered in a second branch. These analyses suggest that the evolution of MyoD and MyoG was driven by different pathways.

  13. Differentiated evolutionary relationships among chordates from comparative alignments of multiple sequences of MyoD and MyoG myogenic regulatory factors.

    PubMed

    Oliani, L C; Lidani, K C F; Gabriel, J E

    2015-01-01

    MyoD and MyoG are transcription factors that have essential roles in myogenic lineage determination and muscle differentiation. The purpose of this study was to compare multiple amino acid sequences of myogenic regulatory proteins to infer evolutionary relationships among chordates. Protein sequences from Mus musculus (P10085 and P12979), human Homo sapiens (P15172 and P15173), bovine Bos taurus (Q7YS82 and Q7YS81), wild pig Sus scrofa (P49811 and P49812), quail Coturnix coturnix (P21572 and P34060), chicken Gallus gallus (P16075 and P17920), rat Rattus norvegicus (Q02346 and P20428), domestic water buffalo Bubalus bubalis (D2SP11 and A7L034), and sheep Ovis aries (Q90477 and D3YKV7) were searched from a non-redundant protein sequence database UniProtKB/Swiss-Prot, and subsequently analyzed using the Mega6.0 software. MyoD evolutionary analyses revealed the presence of three main clusters with all mammals branched in one cluster, members of the order Rodentia (mouse and rat) in a second branch linked to the first, and birds of the order Galliformes (chicken and quail) remaining isolated in a third. MyoG evolutionary analyses aligned sequences in two main clusters, all mammalian specimens grouped in different sub-branches, and birds clustered in a second branch. These analyses suggest that the evolution of MyoD and MyoG was driven by different pathways. PMID:26505406

  14. Propionibacterium acnes: Disease-Causing Agent or Common Contaminant? Detection in Diverse Patient Samples by Next-Generation Sequencing

    PubMed Central

    Friis-Nielsen, Jens; Vinner, Lasse; Hansen, Thomas Arn; Richter, Stine Raith; Fridholm, Helena; Herrera, Jose Alejandro Romero; Lund, Ole; Brunak, Søren; Izarzugaza, Jose M. G.; Mourier, Tobias; Nielsen, Lars Peter

    2016-01-01

    Propionibacterium acnes is the most abundant bacterium on human skin, particularly in sebaceous areas. P. acnes is suggested to be an opportunistic pathogen involved in the development of diverse medical conditions but is also a proven contaminant of human clinical samples and surgical wounds. Its significance as a pathogen is consequently a matter of debate. In the present study, we investigated the presence of P. acnes DNA in 250 next-generation sequencing data sets generated from 180 samples of 20 different sample types, mostly of cancerous origin. The samples were subjected to either microbial enrichment, involving nuclease treatment to reduce the amount of host nucleic acids, or shotgun sequencing. We detected high proportions of P. acnes DNA in enriched samples, particularly skin tissue-derived and other tissue samples, with the levels being higher in enriched samples than in shotgun-sequenced samples. P. acnes reads were detected in most samples analyzed, though the proportions in most shotgun-sequenced samples were low. Our results show that P. acnes can be detected in practically all sample types when molecular methods, such as next-generation sequencing, are employed. The possibility of contamination from the patient or other sources, including laboratory reagents or environment, should therefore always be considered carefully when P. acnes is detected in clinical samples. We advocate that detection of P. acnes always be accompanied by experiments validating the association between this bacterium and any clinical condition. PMID:26818667

  15. Integration of small RNAs, degradome and transcriptome sequencing in hyperaccumulator Sedum alfredii uncovers a complex regulatory network and provides insights into cadmium phytoremediation.

    PubMed

    Han, Xiaojiao; Yin, Hengfu; Song, Xixi; Zhang, Yunxing; Liu, Mingying; Sang, Jiang; Jiang, Jing; Li, Jihong; Zhuo, Renying

    2016-06-01

    The hyperaccumulating ecotype of Sedum alfredii Hance is a cadmium (Cd)/zinc/lead co-hyperaccumulating species of Crassulaceae. It is a promising phytoremediation candidate accumulating substantial heavy metal ions without obvious signs of poisoning. However, few studies have focused on the regulatory roles of miRNAs and their targets in the hyperaccumulating ecotype of S. alfredii. Here, we combined analyses of the transcriptomics, sRNAs and the degradome to generate a comprehensive resource focused on identifying key regulatory miRNA-target circuits under Cd stress. A total of 87 721 unigenes and 356 miRNAs were identified by deep sequencing, and 79 miRNAs were differentially expressed under Cd stress. Furthermore, 754 target genes of 194 miRNAs were validated by degradome sequencing. A gene ontology (GO) enrichment analysis of differential miRNA targets revealed that auxin, redox-related secondary metabolism and metal transport pathways responded to Cd stress. An integrated analysis uncovered 39 pairs of miRNA targets that displayed negatively correlated expression profiles. Ten miRNA-target pairs also exhibited negative correlations according to a real-time quantitative PCR analysis. Moreover, a coexpression regulatory network was constructed based on profiles of differentially expressed genes. Two hub genes, ARF4 (auxin response factor 4) and AAP3 (amino acid permease 3), which might play central roles in the regulation of Cd-responsive genes, were uncovered. These results suggest that comprehensive analyses of the transcriptomics, sRNAs and the degradome provided a useful platform for investigating Cd hyperaccumulation in S. alfredii, and may provide new insights into the genetic engineering of phytoremediation. PMID:26801211

  16. Characterization of the cis elements in the proximal promoter regions of the anthocyanin pathway genes reveals a common regulatory logic that governs pathway regulation

    PubMed Central

    Zhu, Zhixin; Wang, Hailong; Wang, Yiting; Guan, Shan; Wang, Fang; Tang, Jingyu; Zhang, Ruijuan; Xie, Lulu; Lu, Yingqing

    2015-01-01

    Cellular activities such as compound synthesis often require the transcriptional activation of an entire pathway; however, the molecular mechanisms underlying pathway activation have rarely been explained. Here, the cis regulatory architecture of the anthocyanin pathway genes targeted by the transcription factor (TF) complex including MYB, bHLH, and WDR was systematically analysed in one species and the findings extended to others. In Ipomoea purpurea, the IpMYB1-IpbHLH2-IpWDR1 (IpMBW) complex was found to be orthologous to the PAP1-GL3-TTG1 (AtPGT) complex of Arabidopsis thaliana, and interacted with a 7-bp MYB-recognizing element (MRE) and a 6-bp bHLH-recognizing element (BRE) at the proximal promoter region of the pathway genes. There was little transcription of the gene in the absence of the MRE or BRE. The cis elements identified experimentally converged on two syntaxes, ANCNNCC for MREs and CACN(A/C/T)(G/T) for BREs, and our bioinformatic analysis showed that these were present within anthocyanin gene promoters in at least 35 species, including both gymnosperms and angiosperms. For the anthocyanin pathway, IpMBW and AtPGT recognized the interspecific promoters of both early and later genes. In A. thaliana, the seed-specific TF complex (TT2, TT8, and TTG1) may regulate all the anthocyanin pathway genes, in addition to the proanthocyanidin-specific BAN. When multiple TF complexes in the anthocyanin pathway were compared, the cis architecture played a role larger than the TF complex in determining the variation in promoter activity. Collectively, a cis logic common to the pathway gene promoters was found, and this logic is essential for the trans factors to regulate the pathway. PMID:25911741

  17. Full Mitochondrial Genome Sequence of the Sugar Beet Wireworm Limonius californicus (Coleoptera: Elateridae), a Common Agricultural Pest

    PubMed Central

    New, Daniel D.; Robison, Barrie D.; Rashed, Arash; Hohenlohe, Paul; Forney, Larry; Rashidi, Mahnaz; Wilson, Cathy M.; Settles, Matthew L.

    2016-01-01

    We report here the full mitochondrial genome sequence of Limonius californicus, a species of click beetle that is an agricultural pest in its larval form. The circular genome is 16.5 kb and contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. PMID:26798113

  18. High-Throughput Sequencing Reveals H2O2 Stress-Associated MicroRNAs and a Potential Regulatory Network in Brachypodium distachyon Seedlings

    PubMed Central

    Lv, Dong-Wen; Zhen, Shoumin; Zhu, Geng-Rui; Bian, Yan-Wei; Chen, Guan-Xing; Han, Cai-Xia; Yu, Zi-Tong; Yan, Yue-Ming

    2016-01-01

    Oxidative stress in plants can be triggered by many environmental stress factors, such as drought and salinity. Brachypodium distachyon is a model organism for the study of biofuel plants and crops, such as wheat. Although recent studies have found many oxidative stress response-related proteins, the mechanism of microRNA (miRNA)-mediated oxidative stress response is still unclear. Using next generation high-throughput sequencing technology, the small RNAs were sequenced from the model plant B. distachyon 21 (Bd21) under H2O2 stress and normal growth conditions. In total, 144 known B. distachyon miRNAs and 221 potential new miRNAs were identified. Further analysis of potential new miRNAs suggested that 36 could be clustered into known miRNA families, while the remaining 185 were identified as B. distachyon-specific new miRNAs. Differential analysis of miRNAs from the normal and H2O2 stress libraries identified 31 known and 30 new H2O2 stress responsive miRNAs. The expression patterns of seven representative miRNAs were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, which produced results consistent with those of the deep sequencing method. Moreover, we also performed RT-qPCR analysis to verify the expression levels of 13 target genes and the cleavage site of 5 target genes by known or novel miRNAs were validated experimentally by 5′ RACE. Additionally, a miRNA-mediated gene regulatory network for H2O2 stress response was constructed. Our study identifies a set of H2O2-responsive miRNAs and their target genes and reveals the mechanism of oxidative stress response and defense at the post-transcriptional regulatory level. PMID:27812362

  19. Existence of microsatellites in expressed sequence tags of common carp ( Cyprinus carpio L.) available in GenBank dbEST database

    NASA Astrophysics Data System (ADS)

    Hu, Jingjie; Wang, Xiaolong; Hu, Xiaoli; Bao, Zhenmin

    2006-01-01

    Common carp expressed sequence tags (ESTs) were analyzed for the existence of microsatellites, or simple sequence repeats (SSRs). In the NCBI dbEST database, a total of 10612 sequences were registered before December 31, 2004. A complete search of 2 6 nucleotide microsatellites resulted in the identification of 513 SSR-containing ESTs, accounting for 4.8% of the total. Cluster analysis indicated that 73 sequences of SSR-containing ESTs fell into 27 groups and the remaining 440 ESTs were indenpendent. A total of 467 unique SSR-containing ESTs were identified. These EST-SSRs contained a variety of simple sequence types, and di- and tri-nucleotide repeats were the most abundant, accounting for 42.1% and 27.9% of the whole, respectively. Of the dinucleotide repeats, CA/TG was the most abundant, followed by GA/TC. BLASTx search showed that 38.1% of the SSR loci could be associated with genes or proteins of known or unknown function. BLASTx searches of SSR-containing ESTs also showed high frequencies (98/179) of hits on zebrafish sequences.

  20. Existence of microsatellites in expressed sequence tags of common carp ( Cyprinus carpio L.) available in GenBank dbEST database

    NASA Astrophysics Data System (ADS)

    Jingjie, Hu; Xiaolong, Wang; Xiaoli, Hu; Zhenmin, Bao

    2006-01-01

    Common carp expressed sequence tags (ESTs) were analyzed for the existence of microsatellites, or simple sequence repeats (SSRs). In the NCBI dbEST database, a total of 10612 sequences were registered before December 31, 2004. A complete search of 2-6 nucleotide microsatellites resulted in the identification of 513 SSR-containing ESTs, accounting for 4.8% of the total. Cluster analysis indicated that 73 sequences of SSR-containing ESTs fell into 27 groups and the remaining 440 ESTs were indenpendent. A total of 467 unique SSR-containing ESTs were identified. These EST-SSRs contained a variety of simple sequence types, and di- and tri-nucleotide repeats were the most abundant, accounting for 42.1% and 27.9% of the whole, respectively. Of the dinucleotide repeats, CA/TG was the most abundant, followed by GA/TC. BLASTx search showed that 38.1% of the SSR loci could be associated with genes or proteins of known or unknown function. BLASTx searches of SSR-containing ESTs also showed high frequencies (98/179) of hits on zebrafish sequences.

  1. Structural analysis of the regulatory elements of the type-II procollagen gene. Conservation of promoter and first intron sequences between human and mouse.

    PubMed Central

    Vikkula, M; Metsäranta, M; Syvänen, A C; Ala-Kokko, L; Vuorio, E; Peltonen, L

    1992-01-01

    Transcription of the type-II procollagen gene (COL2A1) is very specifically restricted to a limited number of tissues, particularly cartilages. In order to identify transcription-control motifs we have sequenced the promoter region and the first intron of the human and mouse COL2A1 genes. With the assumption that these motifs should be well conserved during evolution, we have searched for potential elements important for the tissue-specific transcription of the COL2A1 gene by aligning the two sequences with each other and with the available rat type-II procollagen sequence for the promoter. With this approach we could identify specific evolutionarily well-conserved motifs in the promoter area. On the other hand, several suggested regulatory elements in the promoter region did not show evolutionary conservation. In the middle of the first intron we found a cluster of well-conserved transcription-control elements and we conclude that these conserved motifs most probably possess a significant function in the control of the tissue-specific transcription of the COL2A1 gene. We also describe locations of additional, highly conserved nucleotide stretches, which are good candidate regions in the search for binding sites of yet-uncharacterized cartilage-specific transcription regulators of the COL2A1 gene. PMID:1637314

  2. A site-specific, single-copy transgenesis strategy to identify 5' regulatory sequences of the mouse testis-determining gene Sry.

    PubMed

    Quinn, Alexander; Kashimada, Kenichi; Davidson, Tara-Lynne; Ng, Ee Ting; Chawengsaksophak, Kallayanee; Bowles, Josephine; Koopman, Peter

    2014-01-01

    The Y-chromosomal gene SRY acts as the primary trigger for male sex determination in mammalian embryos. Correct regulation of SRY is critical: aberrant timing or level of Sry expression is known to disrupt testis development in mice and we hypothesize that mutations that affect regulation of human SRY may account for some of the many cases of XY gonadal dysgenesis that currently remain unexplained. However, the cis-sequences involved in regulation of Sry have not been identified, precluding a test of this hypothesis. Here, we used a transgenic mouse approach aimed at identifying mouse Sry 5' flanking regulatory sequences within 8 kb of the Sry transcription start site (TSS). To avoid problems associated with conventional pronuclear injection of transgenes, we used a published strategy designed to yield single-copy transgene integration at a defined, transcriptionally open, autosomal locus, Col1a1. None of the Sry transgenes tested was expressed at levels compatible with activation of Sox9 or XX sex reversal. Our findings indicate either that the Col1a1 locus does not provide an appropriate context for the correct expression of Sry transgenes, or that the cis-sequences required for Sry expression in the developing gonads lie beyond 8 kb 5' of the TSS.

  3. An AT-rich sequence in human common fragile site FRA16D causes fork stalling and chromosome breakage in S. cerevisiae.

    PubMed

    Zhang, Haihua; Freudenreich, Catherine H

    2007-08-01

    Common fragile sites are regions of human chromosomes prone to breakage. Fragile site FRA16D spans the WWOX/FOR tumor suppressor gene and has been linked to cancer-causing deletions and translocations. Using a genetic assay in yeast, we found that a short AT-rich region (Flex1) within FRA16D increases chromosome fragility, whereas three other sequences within FRA16D do not. To our knowledge, this is the first identification of a sequence element within a common fragile site that increases chromosome fragility. The fragility of Flex1 was exacerbated by the absence of Rad52 or the presence of hydroxyurea. Flex1 contains a polymorphic AT repeat predicted to form a DNA structure, and two-dimensional gel analysis showed accumulation of stalled replication forks at the Flex1 sequence that was dependent on AT length. Our data suggest that the FRA16D Flex1 sequence causes increased chromosome breakage by forming secondary structures that stall replication fork progression.

  4. Uncommon HLA alleles identified by hemizygous ultra-high Sanger sequencing: haplotype associations and reconsideration of their assignment in the Common and Well-Documented catalogue.

    PubMed

    Voorter, Christina E M; Groeneweg, Mathijs; Groeneveld, Lisette; Tilanus, Marcel G J

    2016-02-01

    Although the number of HLA alleles still increases, many of them have been reported being uncommon. This is partly due to lack of full length gene sequencing, especially for those alleles belonging to an allele ambiguity in which the first discovered allele has been assigned as the most frequent one. As members of the working group on Common and Well Documented (CWD) alleles and since we implemented full length group-specific sequencing as standard method routinely, we have investigated the presence of presumably rare alleles in our collection of HLA typing data. We identified 50 alleles, that were not previously encountered as Common or Well Documented. Sixteen of them should be added to the CWD catalogue, since we encountered them in 5 or more unrelated individuals. Another 11 could be added, based upon our results and the data present in the IMGT database and the rare allele section of the allele frequencies database. Furthermore, tight associations were observed between several different alleles even at the level of synonymous and non-coding sequences. In addition, in several cases the uncommon allele was found to be more frequent than its common counterpart.

  5. De novo Sequencing of the Leaf Transcriptome Reveals Complex Light-Responsive Regulatory Networks in Camellia sinensis cv. Baijiguan.

    PubMed

    Wu, Quanjin; Chen, Zhidan; Sun, Weijiang; Deng, Tingting; Chen, Mingjie

    2016-01-01

    Tea plants (Camellia sinensis L.) possess high genetic diversity that is important for breeding. One cultivar, Baijiguan, exhibits a yellow leaf phenotype, reduced chlorophyll (Chl) content, and aberrant chloroplast structures under high light intensity. In contrast, under low light intensity, the flush shoot from Baijiguan becomes green, the Chl content increases significantly, and the chloroplasts exhibit normal structures. To understand the underlying molecular mechanisms for these observations, we performed de novo transcriptome sequencing and digital gene expression (DGE) profiling using Illumina sequencing technology. De novo transcriptome assembly identified 88,788 unigenes, including 1652 transcription factors from 25 families. In total, 1993 and 2576 differentially expressed genes (DEGs) were identified in Baijiguan plants treated with 3 and 6 days of shade, respectively. Gene Ontology (GO) and pathway enrichment analyses indicated that the DEGs are predominantly involved in the ROS scavenging system, chloroplast development, photosynthetic pigment synthesis, secondary metabolism, and circadian systems. The light-responsive gene POR (protochlorophyllide oxidoreductase) and transcription factor HY5 were identified. Quantitative real-time PCR (qRT-PCR) analysis of 20 selected DEGs confirmed the RNA-sequencing (RNA-Seq) results. Overall, these findings suggest that high light intensity inhibits the expression of photosystem II 10-kDa protein (PsbR) in Baijiguan, thus affecting PSII stability, chloroplast development and chlorophyll biosynthesis.

  6. De novo Sequencing of the Leaf Transcriptome Reveals Complex Light-Responsive Regulatory Networks in Camellia sinensis cv. Baijiguan

    PubMed Central

    Wu, Quanjin; Chen, Zhidan; Sun, Weijiang; Deng, Tingting; Chen, Mingjie

    2016-01-01

    Tea plants (Camellia sinensis L.) possess high genetic diversity that is important for breeding. One cultivar, Baijiguan, exhibits a yellow leaf phenotype, reduced chlorophyll (Chl) content, and aberrant chloroplast structures under high light intensity. In contrast, under low light intensity, the flush shoot from Baijiguan becomes green, the Chl content increases significantly, and the chloroplasts exhibit normal structures. To understand the underlying molecular mechanisms for these observations, we performed de novo transcriptome sequencing and digital gene expression (DGE) profiling using Illumina sequencing technology. De novo transcriptome assembly identified 88,788 unigenes, including 1652 transcription factors from 25 families. In total, 1993 and 2576 differentially expressed genes (DEGs) were identified in Baijiguan plants treated with 3 and 6 days of shade, respectively. Gene Ontology (GO) and pathway enrichment analyses indicated that the DEGs are predominantly involved in the ROS scavenging system, chloroplast development, photosynthetic pigment synthesis, secondary metabolism, and circadian systems. The light-responsive gene POR (protochlorophyllide oxidoreductase) and transcription factor HY5 were identified. Quantitative real-time PCR (qRT-PCR) analysis of 20 selected DEGs confirmed the RNA-sequencing (RNA-Seq) results. Overall, these findings suggest that high light intensity inhibits the expression of photosystem II 10-kDa protein (PsbR) in Baijiguan, thus affecting PSII stability, chloroplast development and chlorophyll biosynthesis. PMID:27047513

  7. Paleoecology of the Devonian-Mississippian black-shale sequence in eastern Kentucky with an atlas of some common fossils

    SciTech Connect

    Barron, L.S.; Ettensohn, F.R.

    1981-04-01

    The Devonian-Mississippian black-shale sequence of eastern North America is a distinctive stratigraphic interval generally characterized by low clastic influx, high organic production in the water column, anaerobic bottom conditions, and the relative absence of fossil evidence for biologic activity. The laminated black shales which constitute most of the black-shale sequence are broken by two major sequences of interbedded greenish-gray, clayey shales which contain bioturbation and pyritized micromorph invertebrates. The black shales contain abundant evidence of life from upper parts of the water column such as fish fossils, conodonts, algae and other phytoplankton; however, there is a lack of evidence of benthic life. The rare brachiopods, crinoids, and molluscs that occur in the black shales were probably epiplanktic. A significant physical distinction between the environment in which the black sediments were deposited and that in which the greenish-gray sediments were deposited was the level of dissolved oxygen. The laminated black shales point to anaerobic conditions and the bioturbated greenish-gray shales suggest dysaerobic to marginally aerobic-dysaerobic conditions. A paleoenvironmental model in which quasi-estuarine circulation compliments and enhances the effect of a stratified water column can account for both depletion of dissolved oxygen in the bottom environments and the absence of oxygen replenishment during black-shale deposition. Periods of abundant clastic influx from fluvial environments to the east probably account for the abundance of clays in the greenish-gray shale as well as the small amounts of oxygen necessary to support the depauparate, opportunistic, benthic faunas found there. These pulses of greenish-gray clastics were short-lived and eventually were replaced by anaerobic conditions and low rates of clastic sedimentation which characterized most of black-shale deposition.

  8. Comparison of Muscle Onset Activation Sequences between a Golf or Tennis Swing and Common Training Exercises Using Surface Electromyography: A Pilot Study.

    PubMed

    Vasudevan, John M; Logan, Andrew; Shultz, Rebecca; Koval, Jeffrey J; Roh, Eugene Y; Fredericson, Michael

    2016-01-01

    Aim. The purpose of this pilot study is to use surface electromyography to determine an individual athlete's typical muscle onset activation sequence when performing a golf or tennis forward swing and to use the method to assess to what degree the sequence is reproduced with common conditioning exercises and a machine designed for this purpose. Methods. Data for 18 healthy male subjects were collected for 15 muscles of the trunk and lower extremities. Data were filtered and processed to determine the average onset of muscle activation for each motion. A Spearman correlation estimated congruence of activation order between the swing and each exercise. Correlations of each group were pooled with 95% confidence intervals using a random effects meta-analytic strategy. Results. The averaged sequences differed among each athlete tested, but pooled correlations demonstrated a positive association between each exercise and the participants' natural muscle onset activation sequence. Conclusion. The selected training exercises and Turning Point™ device all partially reproduced our athletes' averaged muscle onset activation sequences for both sports. The results support consideration of a larger, adequately powered study using this method to quantify to what degree each of the selected exercises is appropriate for use in both golf and tennis. PMID:27403454

  9. Comparison of Muscle Onset Activation Sequences between a Golf or Tennis Swing and Common Training Exercises Using Surface Electromyography: A Pilot Study.

    PubMed

    Vasudevan, John M; Logan, Andrew; Shultz, Rebecca; Koval, Jeffrey J; Roh, Eugene Y; Fredericson, Michael

    2016-01-01

    Aim. The purpose of this pilot study is to use surface electromyography to determine an individual athlete's typical muscle onset activation sequence when performing a golf or tennis forward swing and to use the method to assess to what degree the sequence is reproduced with common conditioning exercises and a machine designed for this purpose. Methods. Data for 18 healthy male subjects were collected for 15 muscles of the trunk and lower extremities. Data were filtered and processed to determine the average onset of muscle activation for each motion. A Spearman correlation estimated congruence of activation order between the swing and each exercise. Correlations of each group were pooled with 95% confidence intervals using a random effects meta-analytic strategy. Results. The averaged sequences differed among each athlete tested, but pooled correlations demonstrated a positive association between each exercise and the participants' natural muscle onset activation sequence. Conclusion. The selected training exercises and Turning Point™ device all partially reproduced our athletes' averaged muscle onset activation sequences for both sports. The results support consideration of a larger, adequately powered study using this method to quantify to what degree each of the selected exercises is appropriate for use in both golf and tennis.

  10. Comparison of Muscle Onset Activation Sequences between a Golf or Tennis Swing and Common Training Exercises Using Surface Electromyography: A Pilot Study

    PubMed Central

    Shultz, Rebecca; Fredericson, Michael

    2016-01-01

    Aim. The purpose of this pilot study is to use surface electromyography to determine an individual athlete's typical muscle onset activation sequence when performing a golf or tennis forward swing and to use the method to assess to what degree the sequence is reproduced with common conditioning exercises and a machine designed for this purpose. Methods. Data for 18 healthy male subjects were collected for 15 muscles of the trunk and lower extremities. Data were filtered and processed to determine the average onset of muscle activation for each motion. A Spearman correlation estimated congruence of activation order between the swing and each exercise. Correlations of each group were pooled with 95% confidence intervals using a random effects meta-analytic strategy. Results. The averaged sequences differed among each athlete tested, but pooled correlations demonstrated a positive association between each exercise and the participants' natural muscle onset activation sequence. Conclusion. The selected training exercises and Turning Point™ device all partially reproduced our athletes' averaged muscle onset activation sequences for both sports. The results support consideration of a larger, adequately powered study using this method to quantify to what degree each of the selected exercises is appropriate for use in both golf and tennis. PMID:27403454

  11. CRISPR multitargeter: a web tool to find common and unique CRISPR single guide RNA targets in a set of similar sequences.

    PubMed

    Prykhozhij, Sergey V; Rajan, Vinothkumar; Gaston, Daniel; Berman, Jason N

    2015-01-01

    Genome engineering has been revolutionized by the discovery of clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated system genes (Cas) in bacteria. The type IIB Streptococcus pyogenes CRISPR/Cas9 system functions in many species and additional types of CRISPR/Cas systems are under development. In the type II system, expression of CRISPR single guide RNA (sgRNA) targeting a defined sequence and Cas9 generates a sequence-specific nuclease inducing small deletions or insertions. Moreover, knock-in of large DNA inserts has been shown at the sites targeted by sgRNAs and Cas9. Several tools are available for designing sgRNAs that target unique locations in the genome. However, the ability to find sgRNA targets common to several similar sequences or, by contrast, unique to each of these sequences, would also be advantageous. To provide such a tool for several types of CRISPR/Cas system and many species, we developed the CRISPR MultiTargeter software. Similar DNA sequences in question are duplicated genes and sets of exons of different transcripts of a gene. Thus, we implemented a basic sgRNA target search of input sequences for single-sgRNA and two-sgRNA/Cas9 nickase targeting, as well as common and unique sgRNA target searches in 1) a set of input sequences; 2) a set of similar genes or transcripts; or 3) transcripts a single gene. We demonstrate potential uses of the program by identifying unique isoform-specific sgRNA sites in 71% of zebrafish alternative transcripts and common sgRNA target sites in approximately 40% of zebrafish duplicated gene pairs. The design of unique targets in alternative exons is helpful because it will facilitate functional genomic studies of transcript isoforms. Similarly, its application to duplicated genes may simplify multi-gene mutational targeting experiments. Overall, this program provides a unique interface that will enhance use of CRISPR/Cas technology.

  12. CRISPR MultiTargeter: A Web Tool to Find Common and Unique CRISPR Single Guide RNA Targets in a Set of Similar Sequences

    PubMed Central

    Prykhozhij, Sergey V.; Rajan, Vinothkumar; Gaston, Daniel; Berman, Jason N.

    2015-01-01

    Genome engineering has been revolutionized by the discovery of clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated system genes (Cas) in bacteria. The type IIB Streptococcus pyogenes CRISPR/Cas9 system functions in many species and additional types of CRISPR/Cas systems are under development. In the type II system, expression of CRISPR single guide RNA (sgRNA) targeting a defined sequence and Cas9 generates a sequence-specific nuclease inducing small deletions or insertions. Moreover, knock-in of large DNA inserts has been shown at the sites targeted by sgRNAs and Cas9. Several tools are available for designing sgRNAs that target unique locations in the genome. However, the ability to find sgRNA targets common to several similar sequences or, by contrast, unique to each of these sequences, would also be advantageous. To provide such a tool for several types of CRISPR/Cas system and many species, we developed the CRISPR MultiTargeter software. Similar DNA sequences in question are duplicated genes and sets of exons of different transcripts of a gene. Thus, we implemented a basic sgRNA target search of input sequences for single-sgRNA and two-sgRNA/Cas9 nickase targeting, as well as common and unique sgRNA target searches in 1) a set of input sequences; 2) a set of similar genes or transcripts; or 3) transcripts a single gene. We demonstrate potential uses of the program by identifying unique isoform-specific sgRNA sites in 71% of zebrafish alternative transcripts and common sgRNA target sites in approximately 40% of zebrafish duplicated gene pairs. The design of unique targets in alternative exons is helpful because it will facilitate functional genomic studies of transcript isoforms. Similarly, its application to duplicated genes may simplify multi-gene mutational targeting experiments. Overall, this program provides a unique interface that will enhance use of CRISPR/Cas technology. PMID:25742428

  13. Whole transcriptome sequencing identifies BCOR internal tandem duplication as a common feature of clear cell sarcoma of the kidney

    PubMed Central

    Astolfi, Annalisa; Melchionda, Fraia; Perotti, Daniela; Fois, Maura; Indio, Valentina; Urbini, Milena; Genovese, Chiara Giusy; Collini, Paola; Salfi, Nunzio; Nantron, Marilina; D'Angelo, Paolo; Spreafico, Filippo; Pession, Andrea

    2015-01-01

    Purpose Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor that is frequently difficult to distinguish among other childhood renal tumors due to its histological heterogeneity. This work evaluates genetic abnormalities carried by a series of CCSK samples by whole transcriptome sequencing (WTS), to identify molecular biomarkers that could improve the diagnostic process. Methods WTS was performed on tumor RNA from 8 patients with CCSK. Bioinformatic analysis, with implementation of a pipeline for detection of intragenic rearrangements, was executed. Sanger sequencing and gene expression were evaluated to validate BCOR internal tandem duplication (ITD). Results WTS did not identify any shared SNVs, Ins/Del or fusion event. Conversely, analysis of intragenic rearrangements enabled the detection of a breakpoint within BCOR transcript recurrent in all samples. Three different in-frame ITD in exon15 of BCOR, were detected. The presence of the ITD was confirmed on tumor DNA and cDNA, and resulted in overexpression of BCOR. Conclusion WTS coupled with specific bioinformatic analysis is able to detect rare genetic events, as intragenic rearrangements. ITD in the last exon of BCOR is recurrent in all CCSK samples analyzed, representing a valuable molecular marker to improve diagnosis of this rare childhood renal tumor. PMID:26516930

  14. Functional relevance of specific interactions between herpes simplex virus type 1 ICP4 and sequences from the promoter-regulatory domain of the viral thymidine kinase gene.

    PubMed Central

    Imbalzano, A N; Shepard, A A; DeLuca, N A

    1990-01-01

    The herpes simplex virus (HSV) type 1 immediate-early regulatory protein ICP4 is required for induced expression of HSV early and late genes, yet the mechanism by which this occurs is not known. We examined the promoter and flanking sequences of the HSV early gene that encodes thymidine kinase for the ability to interact specifically with ICP4 in gel retardation assays. Protein-DNA complexes containing ICP4 were observed with several distinct regions flanking the tk promoter. cis-Acting elements that interact with cellular transcription factors were apparently not required for these interactions to form. Purified ICP4 formed protein-DNA complexes with fragments from these regions, and Southwestern (DNA-protein blot) analysis indicated that the interaction between ICP4 and these sequences can be direct. None of the tk sequences that interact with ICP4 contains a consensus binding site for ICP4 (S. W. Faber and K. W. Wilcox, Nucleic Acids Res. 14:6067-6083, 1986), reflecting the ability of ICP4 to interact with more than one DNA sequence. A mutated ICP4 protein expressed from the viral genome that retains the ability to bind to a consensus binding site but does not bind specifically to the identified sites flanking the tk promoter results in induced transcription of the tk gene. These data support hypotheses for ICP4-mediated transactivation of the tk promoter in Vero cells that do not require the intrinsic ability of ICP4 to bind specifically in or near the promoter of the tk gene. Images PMID:2159535

  15. Transposable Elements and DNA Methylation Create in Embryonic Stem Cells Human-Specific Regulatory Sequences Associated with Distal Enhancers and Noncoding RNAs.

    PubMed

    Glinsky, Gennadi V

    2015-05-07

    Despite significant progress in the structural and functional characterization of the human genome, understanding of the mechanisms underlying the genetic basis of human phenotypic uniqueness remains limited. Here, I report that transposable element-derived sequences, most notably LTR7/HERV-H, LTR5_Hs, and L1HS, harbor 99.8% of the candidate human-specific regulatory loci (HSRL) with putative transcription factor-binding sites in the genome of human embryonic stem cells (hESC). A total of 4,094 candidate HSRL display selective and site-specific binding of critical regulators (NANOG [Nanog homeobox], POU5F1 [POU class 5 homeobox 1], CCCTC-binding factor [CTCF], Lamin B1), and are preferentially located within the matrix of transcriptionally active DNA segments that are hypermethylated in hESC. hESC-specific NANOG-binding sites are enriched near the protein-coding genes regulating brain size, pluripotency long noncoding RNAs, hESC enhancers, and 5-hydroxymethylcytosine-harboring regions immediately adjacent to binding sites. Sequences of only 4.3% of hESC-specific NANOG-binding sites are present in Neanderthals' genome, suggesting that a majority of these regulatory elements emerged in Modern Humans. Comparisons of estimated creation rates of novel TF-binding sites revealed that there was 49.7-fold acceleration of creation rates of NANOG-binding sites in genomes of Chimpanzees compared with the mouse genomes and further 5.7-fold acceleration in genomes of Modern Humans compared with the Chimpanzees genomes. Preliminary estimates suggest that emergence of one novel NANOG-binding site detectable in hESC required 466 years of evolution. Pathway analysis of coding genes that have hESC-specific NANOG-binding sites within gene bodies or near gene boundaries revealed their association with physiological development and functions of nervous and cardiovascular systems, embryonic development, behavior, as well as development of a diverse spectrum of pathological conditions

  16. Transposable Elements and DNA Methylation Create in Embryonic Stem Cells Human-Specific Regulatory Sequences Associated with Distal Enhancers and Noncoding RNAs

    PubMed Central

    Glinsky, Gennadi V.

    2015-01-01

    Despite significant progress in the structural and functional characterization of the human genome, understanding of the mechanisms underlying the genetic basis of human phenotypic uniqueness remains limited. Here, I report that transposable element-derived sequences, most notably LTR7/HERV-H, LTR5_Hs, and L1HS, harbor 99.8% of the candidate human-specific regulatory loci (HSRL) with putative transcription factor-binding sites in the genome of human embryonic stem cells (hESC). A total of 4,094 candidate HSRL display selective and site-specific binding of critical regulators (NANOG [Nanog homeobox], POU5F1 [POU class 5 homeobox 1], CCCTC-binding factor [CTCF], Lamin B1), and are preferentially located within the matrix of transcriptionally active DNA segments that are hypermethylated in hESC. hESC-specific NANOG-binding sites are enriched near the protein-coding genes regulating brain size, pluripotency long noncoding RNAs, hESC enhancers, and 5-hydroxymethylcytosine-harboring regions immediately adjacent to binding sites. Sequences of only 4.3% of hESC-specific NANOG-binding sites are present in Neanderthals’ genome, suggesting that a majority of these regulatory elements emerged in Modern Humans. Comparisons of estimated creation rates of novel TF-binding sites revealed that there was 49.7-fold acceleration of creation rates of NANOG-binding sites in genomes of Chimpanzees compared with the mouse genomes and further 5.7-fold acceleration in genomes of Modern Humans compared with the Chimpanzees genomes. Preliminary estimates suggest that emergence of one novel NANOG-binding site detectable in hESC required 466 years of evolution. Pathway analysis of coding genes that have hESC-specific NANOG-binding sites within gene bodies or near gene boundaries revealed their association with physiological development and functions of nervous and cardiovascular systems, embryonic development, behavior, as well as development of a diverse spectrum of pathological conditions

  17. Transvection in the Drosophila Abd-B domain: extensive upstream sequences are involved in anchoring distant cis-regulatory regions to the promoter.

    PubMed Central

    Sipos, L; Mihály, J; Karch, F; Schedl, P; Gausz, J; Gyurkovics, H

    1998-01-01

    The Abd-B gene, one of the three homeotic genes in the Drosophila bithorax complex (BX-C), is required for the proper identity of the fifth through the eighth abdominal segments (corresponding to parasegments 10-14) of the fruitfly. The morphological difference between these four segments is due to the differential expression of Abd-B, which is achieved by the action of the parasegment-specific cis-regulatory regions infra-abdominal-5 (iab-5), -6, -7 and -8. The dominant gain-of-function mutation Frontabdominal-7 (Fab-7) removes a boundary separating two of these cis-regulatory regions, iab-6 and iab-7. As a consequence of the Fab-7 deletion, the parasegment 12- (PS12-) specific iab-7 is ectopically activated in PS11. This results in the transformation of the sixth abdominal segment (A6) into the seventh (A7) in Fab-7 flies. Here we report that point mutations of the Abd-B gene in trans suppress the Fab-7 phenotype in a pairing-dependent manner and thus represent a type of transvection. We show that the observed suppression is the result of trans-regulation of the defective Abd-B gene by the ectopically activated iab-7. Unlike previously demonstrated cases of trans-regulation in the Abd-B locus, trans-suppression of Fab-7 is sensitive to heterozygosity for chromosomal rearrangements that disturb homologous pairing at the nearby Ubx locus. However, in contrast to Ubx, the transvection we observed in the Abd-B locus is insensitive to the allelic status of zeste. Analysis of different deletion alleles of Abd-B that enhance trans-regulation suggests that an extensive upstream region, different from the sequences required for transcription initiation, mediates interactions between the iab cis-regulatory regions and the proximal Abd-B promoter. Moreover, we find that the amount of DNA deleted in the upstream region is roughly proportional to the strength of trans-interaction, suggesting that this region consists of numerous discrete elements that cooperate in tethering

  18. RNA sequencing and functional analysis implicate the regulatory role of long non-coding RNAs in tomato fruit ripening

    PubMed Central

    Zhu, Benzhong; Yang, Yongfang; Li, Ran; Fu, Daqi; Wen, Liwei; Luo, Yunbo; Zhu, Hongliang

    2015-01-01

    Recently, long non-coding RNAs (lncRNAs) have been shown to play critical regulatory roles in model plants, such as Arabidopsis, rice, and maize. However, the presence of lncRNAs and how they function in fleshy fruit ripening are still largely unknown because fleshy fruit ripening is not present in the above model plants. Tomato is the model system for fruit ripening studies due to its dramatic ripening process. To investigate further the role of lncRNAs in fruit ripening, it is necessary and urgent to discover and identify novel lncRNAs and understand the function of lncRNAs in tomato fruit ripening. Here it is reported that 3679 lncRNAs were discovered from wild-type tomato and ripening mutant fruit. The lncRNAs are transcribed from all tomato chromosomes, 85.1% of which came from intergenic regions. Tomato lncRNAs are shorter and have fewer exons than protein-coding genes, a situation reminiscent of lncRNAs from other model plants. It was also observed that 490 lncRNAs were significantly up-regulated in ripening mutant fruits, and 187 lncRNAs were down-regulated, indicating that lncRNAs could be involved in the regulation of fruit ripening. In line with this, silencing of two novel tomato intergenic lncRNAs, lncRNA1459 and lncRNA1840, resulted in an obvious delay of ripening of wild-type fruit. Overall, the results indicated that lncRNAs might be essential regulators of tomato fruit ripening, which sheds new light on the regulation of fruit ripening. PMID:25948705

  19. RNA sequencing and functional analysis implicate the regulatory role of long non-coding RNAs in tomato fruit ripening.

    PubMed

    Zhu, Benzhong; Yang, Yongfang; Li, Ran; Fu, Daqi; Wen, Liwei; Luo, Yunbo; Zhu, Hongliang

    2015-08-01

    Recently, long non-coding RNAs (lncRNAs) have been shown to play critical regulatory roles in model plants, such as Arabidopsis, rice, and maize. However, the presence of lncRNAs and how they function in fleshy fruit ripening are still largely unknown because fleshy fruit ripening is not present in the above model plants. Tomato is the model system for fruit ripening studies due to its dramatic ripening process. To investigate further the role of lncRNAs in fruit ripening, it is necessary and urgent to discover and identify novel lncRNAs and understand the function of lncRNAs in tomato fruit ripening. Here it is reported that 3679 lncRNAs were discovered from wild-type tomato and ripening mutant fruit. The lncRNAs are transcribed from all tomato chromosomes, 85.1% of which came from intergenic regions. Tomato lncRNAs are shorter and have fewer exons than protein-coding genes, a situation reminiscent of lncRNAs from other model plants. It was also observed that 490 lncRNAs were significantly up-regulated in ripening mutant fruits, and 187 lncRNAs were down-regulated, indicating that lncRNAs could be involved in the regulation of fruit ripening. In line with this, silencing of two novel tomato intergenic lncRNAs, lncRNA1459 and lncRNA1840, resulted in an obvious delay of ripening of wild-type fruit. Overall, the results indicated that lncRNAs might be essential regulators of tomato fruit ripening, which sheds new light on the regulation of fruit ripening.

  20. A common mechanism for the enhancement of mRNA 3' processing by U3 sequences in two distantly related lentiviruses.

    PubMed

    Graveley, B R; Gilmartin, G M

    1996-03-01

    The protein coding regions of all retroviral pre-mRNAs are flanked by a direct repeat of R-U5 sequences. In many retroviruses, the R-U5 repeat contains a complete core poly(A) site-composed of a highly conserved AAUAAA hexamer and a GU-rich downstream element. A mechanism that allows for the bypass of the 5' core poly(A) site and the exclusive use of the 3' core poly(A) site must therefore exist. In human immunodeficiency virus type 1 (HIV-1), sequences within the U3 region appear to play a key role in poly(A) site selection. U3 sequences are required for efficient 3' processing at the HIV-1 poly(A) site both in vivo and in vitro. These sequences serve to promote the interaction of cleavage and polyadenylation specificity factor (CPSF) with the core poly(A) site. We have now demonstrated the presence of a functionally analogous 3' processing enhancer within the U3 region of a distantly related lentivirus, equine infectious anemia virus (EIAV). U3 sequences enhanced the processing of the EIAV core poly(A) site sevenfold in vitro. The U3 sequences also enhanced the stability of CPSF binding at the core poly(A) site. Optimal processing required the TAR RNA secondary structure that resides within the R region 28 nucleotides upstream of the AAUAAA hexamer. Disruption of TAR reduced processing, while compensatory changes that restored the RNA structure also restored processing to the wild-type level, suggesting a position dependence of the U3-encoded enhancer sequences. Finally, the reciprocal exchange of the EIAV and HIV U3 regions demonstrated the ability of each of these sequences to enhance both 3' processing and the binding of CPSF in the context of the heterologous core poly(A) site. The impact of U3 sequences upon the interaction of CPSF at the core poly(A) site may therefore represent a common strategy for retroviral poly(A) site selection. PMID:8627681

  1. SNP Assay Development for Linkage Map Construction, Anchoring Whole-Genome Sequence, and Other Genetic and Genomic Applications in Common Bean

    DOE PAGES

    Song, Qijian; Jia, Gaofeng; Hyten, David L.; Jenkins, Jerry; Hwang, Eun-Young; Schroeder, Steven G.; Osorno, Juan M.; Schmutz, Jeremy; Jackson, Scott A.; McClean, Phillip E.; et al

    2015-08-28

    A total of 992,682 single-nucleotide polymorphisms (SNPs) was identified as ideal for Illumina Infinium II BeadChip design after sequencing a diverse set of 17 common bean (Phaseolus vulgaris L) varieties with the aid of next-generation sequencing technology. From these, two BeadChips each with >5000 SNPs were designed. The BARCBean6K_1 BeadChip was selected for the purpose of optimizing polymorphism among market classes and, when possible, SNPs were targeted to sequence scaffolds in the Phaseolus vulgaris 14× genome assembly with sequence lengths >10 kb. The BARCBean6K_2 BeadChip was designed with the objective of anchoring additional scaffolds and to facilitate orientation of largemore » scaffolds. Analysis of 267 F2 plants from a cross of varieties Stampede × Red Hawk with the two BeadChips resulted in linkage maps with a total of 7040 markers including 7015 SNPs. With the linkage map, a total of 432.3 Mb of sequence from 2766 scaffolds was anchored to create the Phaseolus vulgaris v1.0 assembly, which accounted for approximately 89% of the 487 Mb of available sequence scaffolds of the Phaseolus vulgaris v0.9 assembly. A core set of 6000 SNPs (BARCBean6K_3 BeadChip) with high genotyping quality and polymorphism was selected based on the genotyping of 365 dry bean and 134 snap bean accessions with the BARCBean6K_1 and BARCBean6K_2 BeadChips. The BARCBean6K_3 BeadChip is a useful tool for genetics and genomics research and it is widely used by breeders and geneticists in the United States and abroad.« less

  2. SNP Assay Development for Linkage Map Construction, Anchoring Whole-Genome Sequence, and Other Genetic and Genomic Applications in Common Bean.

    PubMed

    Song, Qijian; Jia, Gaofeng; Hyten, David L; Jenkins, Jerry; Hwang, Eun-Young; Schroeder, Steven G; Osorno, Juan M; Schmutz, Jeremy; Jackson, Scott A; McClean, Phillip E; Cregan, Perry B

    2015-08-28

    A total of 992,682 single-nucleotide polymorphisms (SNPs) was identified as ideal for Illumina Infinium II BeadChip design after sequencing a diverse set of 17 common bean (Phaseolus vulgaris L) varieties with the aid of next-generation sequencing technology. From these, two BeadChips each with >5000 SNPs were designed. The BARCBean6K_1 BeadChip was selected for the purpose of optimizing polymorphism among market classes and, when possible, SNPs were targeted to sequence scaffolds in the Phaseolus vulgaris 14× genome assembly with sequence lengths >10 kb. The BARCBean6K_2 BeadChip was designed with the objective of anchoring additional scaffolds and to facilitate orientation of large scaffolds. Analysis of 267 F2 plants from a cross of varieties Stampede × Red Hawk with the two BeadChips resulted in linkage maps with a total of 7040 markers including 7015 SNPs. With the linkage map, a total of 432.3 Mb of sequence from 2766 scaffolds was anchored to create the Phaseolus vulgaris v1.0 assembly, which accounted for approximately 89% of the 487 Mb of available sequence scaffolds of the Phaseolus vulgaris v0.9 assembly. A core set of 6000 SNPs (BARCBean6K_3 BeadChip) with high genotyping quality and polymorphism was selected based on the genotyping of 365 dry bean and 134 snap bean accessions with the BARCBean6K_1 and BARCBean6K_2 BeadChips. The BARCBean6K_3 BeadChip is a useful tool for genetics and genomics research and it is widely used by breeders and geneticists in the United States and abroad.

  3. SNP Assay Development for Linkage Map Construction, Anchoring Whole-Genome Sequence, and Other Genetic and Genomic Applications in Common Bean

    SciTech Connect

    Song, Qijian; Jia, Gaofeng; Hyten, David L.; Jenkins, Jerry; Hwang, Eun-Young; Schroeder, Steven G.; Osorno, Juan M.; Schmutz, Jeremy; Jackson, Scott A.; McClean, Phillip E.; Cregan, Perry B.

    2015-08-28

    A total of 992,682 single-nucleotide polymorphisms (SNPs) was identified as ideal for Illumina Infinium II BeadChip design after sequencing a diverse set of 17 common bean (Phaseolus vulgaris L) varieties with the aid of next-generation sequencing technology. From these, two BeadChips each with >5000 SNPs were designed. The BARCBean6K_1 BeadChip was selected for the purpose of optimizing polymorphism among market classes and, when possible, SNPs were targeted to sequence scaffolds in the Phaseolus vulgaris 14× genome assembly with sequence lengths >10 kb. The BARCBean6K_2 BeadChip was designed with the objective of anchoring additional scaffolds and to facilitate orientation of large scaffolds. Analysis of 267 F2 plants from a cross of varieties Stampede × Red Hawk with the two BeadChips resulted in linkage maps with a total of 7040 markers including 7015 SNPs. With the linkage map, a total of 432.3 Mb of sequence from 2766 scaffolds was anchored to create the Phaseolus vulgaris v1.0 assembly, which accounted for approximately 89% of the 487 Mb of available sequence scaffolds of the Phaseolus vulgaris v0.9 assembly. A core set of 6000 SNPs (BARCBean6K_3 BeadChip) with high genotyping quality and polymorphism was selected based on the genotyping of 365 dry bean and 134 snap bean accessions with the BARCBean6K_1 and BARCBean6K_2 BeadChips. The BARCBean6K_3 BeadChip is a useful tool for genetics and genomics research and it is widely used by breeders and geneticists in the United States and abroad.

  4. Avian sarcoma virus gag and env gene structural protein precursors contain a common amino-terminal sequence.

    PubMed

    Ficht, T A; Chang, L J; Stoltzfus, C M

    1984-01-01

    The initiation site for translation of the avian sarcoma virus glycoprotein precursor, Pr63env, has been determined by analyzing the amino-terminal peptides of Pr63env and the polyprotein precursor Pr76gag encoded by the viral gag gene. The acceptor splice junction used to form the env gene mRNA has also been identified. Hybrid-selected virus-specific mRNAs were translated in vitro in the presence of either L-[35S]methionine to label at every methionine residue or L-[35S]methionine-tRNAMeti to label specifically at the amino-terminal methionine residues. Tryptic peptide maps of Pr63env labeled at every methionine residue contain all of the peptides, plus one additional peptide, present in the map of Pr57env, a nonglycosylated env-encoded polypeptide of molecular weight 57,000 immunoprecipitated from tunicamycin-treated cells. Specific amino-terminal labeling of the in vitro-synthesized polypeptides showed that the peptide missing from Pr57env corresponds to the amino-terminal tryptic peptide of Pr63env, which is removed in vivo as part of the amino-terminal signal peptide. Comparison of the amino-terminal tryptic peptides of Pr63env and Pr76gag showed that they are identical. In contrast, the chymotryptic amino-terminal peptides of Pr76gag and Pr63env are not identical. The location of the acceptor-splice junction in the env mRNA of the Prague A strain of avian sarcoma virus was determined by mung bean nuclease mapping to be at nucleotide 5,078. Fusion of the gag and env gene sequences during splicing results in use of the same AUG codon to initiate synthesis of Pr76gag and Pr63env. This sequence is contained within the 397-nucleotide 5' terminal leader that is spliced to the body of the env mRNA. The possible significance of these results for the regulation of avian sarcoma virus synthesis and translation is discussed.

  5. ICO amplicon NGS data analysis: a Web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS Junior next-generation sequencing.

    PubMed

    Lopez-Doriga, Adriana; Feliubadaló, Lídia; Menéndez, Mireia; Lopez-Doriga, Sergio; Morón-Duran, Francisco D; del Valle, Jesús; Tornero, Eva; Montes, Eva; Cuesta, Raquel; Campos, Olga; Gómez, Carolina; Pineda, Marta; González, Sara; Moreno, Victor; Capellá, Gabriel; Lázaro, Conxi

    2014-03-01

    Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.

  6. Identification of sequence similarity between 60 kDa and 70 kDa molecular chaperones: evidence for a common evolutionary background?

    PubMed Central

    Flores, A I; Cuezva, J M

    1997-01-01

    Recent findings support the premise that chaperonins (60 kDa stress-proteins) and alpha-subunits of F-type ATPases (alpha-ATPase) are evolutionary related protein families. Two-dimensional gel patterns of synthesized proteins in unstressed and heat-shocked embryonic Drosophila melanogaster SL2 cells revealed that antibodies raised against the alpha-subunit of the F1-ATPase complex from rat liver recognize an inducible p71 member of the 70 kDa stress-responsive protein family. Molecular recognition of this stress-responsive 70 kDa protein by antibodies raised against the F1-ATPase alpha-subunit suggests the possibility of partial sequence similarity within these ATP-binding protein families. A multiple sequence alignment between alpha-ATPases and 60 kDa and 70 kDa molecular chaperones is presented. Statistical evaluation of sequence similarity reveals a significant degree of sequence conservation within the three protein families. The finding suggests a common evolutionary origin for the ATPases and molecular chaperone protein families of 60 kDa and 70 kDa, despite the lack of obvious structural resemblance between them. PMID:9065788

  7. Genetic divergence analysis of the Common Barn Owl Tyto alba (Scopoli, 1769) and the Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile using COI sequence.

    PubMed

    Colihueque, Nelson; Gantz, Alberto; Rau, Jaime Ricardo; Parraguez, Margarita

    2015-01-01

    In this paper new mitochondrial COI sequences of Common Barn Owl Tyto alba (Scopoli, 1769) and Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile are reported and compared with sequences from other parts of the World. The intraspecific genetic divergence (mean p-distance) was 4.6 to 5.5% for the Common Barn Owl in comparison with specimens from northern Europe and Australasia and 3.1% for the Short-eared Owl with respect to samples from north America, northern Europe and northern Asia. Phylogenetic analyses revealed three distinctive groups for the Common Barn Owl: (i) South America (Chile and Argentina) plus Central and North America, (ii) northern Europe and (iii) Australasia, and two distinctive groups for the Short-eared Owl: (i) South America (Chile and Argentina) and (ii) north America plus northern Europe and northern Asia. The level of genetic divergence observed in both species exceeds the upper limit of intraspecific comparisons reported previously for Strigiformes. Therefore, this suggests that further research is needed to assess the taxonomic status, particularly for the Chilean populations that, to date, have been identified as belonging to these species through traditional taxonomy. PMID:26668551

  8. Genetic divergence analysis of the Common Barn Owl Tyto alba (Scopoli, 1769) and the Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile using COI sequence.

    PubMed

    Colihueque, Nelson; Gantz, Alberto; Rau, Jaime Ricardo; Parraguez, Margarita

    2015-01-01

    In this paper new mitochondrial COI sequences of Common Barn Owl Tyto alba (Scopoli, 1769) and Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile are reported and compared with sequences from other parts of the World. The intraspecific genetic divergence (mean p-distance) was 4.6 to 5.5% for the Common Barn Owl in comparison with specimens from northern Europe and Australasia and 3.1% for the Short-eared Owl with respect to samples from north America, northern Europe and northern Asia. Phylogenetic analyses revealed three distinctive groups for the Common Barn Owl: (i) South America (Chile and Argentina) plus Central and North America, (ii) northern Europe and (iii) Australasia, and two distinctive groups for the Short-eared Owl: (i) South America (Chile and Argentina) and (ii) north America plus northern Europe and northern Asia. The level of genetic divergence observed in both species exceeds the upper limit of intraspecific comparisons reported previously for Strigiformes. Therefore, this suggests that further research is needed to assess the taxonomic status, particularly for the Chilean populations that, to date, have been identified as belonging to these species through traditional taxonomy.

  9. Genetic divergence analysis of the Common Barn Owl Tyto alba (Scopoli, 1769) and the Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile using COI sequence

    PubMed Central

    Colihueque, Nelson; Gantz, Alberto; Rau, Jaime Ricardo; Parraguez, Margarita

    2015-01-01

    Abstract In this paper new mitochondrial COI sequences of Common Barn Owl Tyto alba (Scopoli, 1769) and Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile are reported and compared with sequences from other parts of the World. The intraspecific genetic divergence (mean p-distance) was 4.6 to 5.5% for the Common Barn Owl in comparison with specimens from northern Europe and Australasia and 3.1% for the Short-eared Owl with respect to samples from north America, northern Europe and northern Asia. Phylogenetic analyses revealed three distinctive groups for the Common Barn Owl: (i) South America (Chile and Argentina) plus Central and North America, (ii) northern Europe and (iii) Australasia, and two distinctive groups for the Short-eared Owl: (i) South America (Chile and Argentina) and (ii) north America plus northern Europe and northern Asia. The level of genetic divergence observed in both species exceeds the upper limit of intraspecific comparisons reported previously for Strigiformes. Therefore, this suggests that further research is needed to assess the taxonomic status, particularly for the Chilean populations that, to date, have been identified as belonging to these species through traditional taxonomy. PMID:26668551

  10. Identification of co-expression gene networks, regulatory genes and pathways for obesity based on adipose tissue RNA Sequencing in a porcine model

    PubMed Central

    2014-01-01

    Background Obesity is a complex metabolic condition in strong association with various diseases, like type 2 diabetes, resulting in major public health and economic implications. Obesity is the result of environmental and genetic factors and their interactions, including genome-wide genetic interactions. Identification of co-expressed and regulatory genes in RNA extracted from relevant tissues representing lean and obese individuals provides an entry point for the identification of genes and pathways of importance to the development of obesity. The pig, an omnivorous animal, is an excellent model for human obesity, offering the possibility to study in-depth organ-level transcriptomic regulations of obesity, unfeasible in humans. Our aim was to reveal adipose tissue co-expression networks, pathways and transcriptional regulations of obesity using RNA Sequencing based systems biology approaches in a porcine model. Methods We selected 36 animals for RNA Sequencing from a previously created F2 pig population representing three extreme groups based on their predicted genetic risks for obesity. We applied Weighted Gene Co-expression Network Analysis (WGCNA) to detect clusters of highly co-expressed genes (modules). Additionally, regulator genes were detected using Lemon-Tree algorithms. Results WGCNA revealed five modules which were strongly correlated with at least one obesity-related phenotype (correlations ranging from -0.54 to 0.72, P < 0.001). Functional annotation identified pathways enlightening the association between obesity and other diseases, like osteoporosis (osteoclast differentiation, P = 1.4E-7), and immune-related complications (e.g. Natural killer cell mediated cytotoxity, P = 3.8E-5; B cell receptor signaling pathway, P = 7.2E-5). Lemon-Tree identified three potential regulator genes, using confident scores, for the WGCNA module which was associated with osteoclast differentiation: CCR1, MSR1 and SI1 (probability scores respectively 95.30, 62.28, and

  11. A Common Missense Variant in the Glucokinase Regulatory Protein Gene (GCKR) Is Associated with Increased Plasma Triglyceride and C-Reactive Protein but Lower Fasting Glucose Concentrations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OBJECTIVE-Using the genome-wide-association approach, we recently identified the glucokinase regulatory protein gene (GCKR, rs780094) region as a novel quantitative trait locus for plasma triglyceride concentration in Europeans. Here, we sought to study the association of GCKR variants with metaboli...

  12. Analysis of the coding-complete genomic sequence of groundnut ringspot virus suggests a common ancestor with tomato chlorotic spot virus.

    PubMed

    de Breuil, Soledad; Cañizares, Joaquín; Blanca, José Miguel; Bejerman, Nicolás; Trucco, Verónica; Giolitti, Fabián; Ziarsolo, Peio; Lenardon, Sergio

    2016-08-01

    Groundnut ringspot virus (GRSV) and tomato chlorotic spot virus (TCSV) share biological and serological properties, so their identification is carried out by molecular methods. Their genomes consist of three segmented RNAs: L, M and S. The finding of a reassortant between these two viruses may complicate correct virus identification and requires the characterization of the complete genome. Therefore, we present for the first time the complete sequences of all the genes encoded by a GRSV isolate. The high level of sequence similarity between GRSV and TCSV (over 90 % identity) observed in the genes and proteins encoded in the M RNA support previous results indicating that these viruses probably have a common ancestor. PMID:27260536

  13. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans

    PubMed Central

    Khachatoorian, Careen; Judelson, Howard S.

    2015-01-01

    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies. PMID:26716454

  14. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

    PubMed

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S

    2015-01-01

    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies. PMID:26716454

  15. HLA-F coding and regulatory segments variability determined by massively parallel sequencing procedures in a Brazilian population sample.

    PubMed

    Lima, Thálitta Hetamaro Ayala; Buttura, Renato Vidal; Donadi, Eduardo Antônio; Veiga-Castelli, Luciana Caricati; Mendes-Junior, Celso Teixeira; Castelli, Erick C

    2016-10-01

    Human Leucocyte Antigen F (HLA-F) is a non-classical HLA class I gene distinguished from its classical counterparts by low allelic polymorphism and distinctive expression patterns. Its exact function remains unknown. It is believed that HLA-F has tolerogenic and immune modulatory properties. Currently, there is little information regarding the HLA-F allelic variation among human populations and the available studies have evaluated only a fraction of the HLA-F gene segment and/or have searched for known alleles only. Here we present a strategy to evaluate the complete HLA-F variability including its 5' upstream, coding and 3' downstream segments by using massively parallel sequencing procedures. HLA-F variability was surveyed on 196 individuals from the Brazilian Southeast. The results indicate that the HLA-F gene is indeed conserved at the protein level, where thirty coding haplotypes or coding alleles were detected, encoding only four different HLA-F full-length protein molecules. Moreover, a same protein molecule is encoded by 82.45% of all coding alleles detected in this Brazilian population sample. However, the HLA-F nucleotide and haplotype variability is much higher than our current knowledge both in Brazilians and considering the 1000 Genomes Project data. This protein conservation is probably a consequence of the key role of HLA-F in the immune system physiology.

  16. Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

    PubMed Central

    Ch'ang, L Y; Yang, W K; Myer, F E; Yang, D M

    1989-01-01

    Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs. Images PMID:2542587

  17. Common methods for fecal sample storage in field studies yield consistent signatures of individual identity in microbiome sequencing data

    PubMed Central

    Blekhman, Ran; Tang, Karen; Archie, Elizabeth A.; Barreiro, Luis B.; Johnson, Zachary P.; Wilson, Mark E.; Kohn, Jordan; Yuan, Michael L.; Gesquiere, Laurence; Grieneisen, Laura E.; Tung, Jenny

    2016-01-01

    Field studies of wild vertebrates are frequently associated with extensive collections of banked fecal samples—unique resources for understanding ecological, behavioral, and phylogenetic effects on the gut microbiome. However, we do not understand whether sample storage methods confound the ability to investigate interindividual variation in gut microbiome profiles. Here, we extend previous work on storage methods for gut microbiome samples by comparing immediate freezing, the gold standard of preservation, to three methods commonly used in vertebrate field studies: lyophilization, storage in ethanol, and storage in RNAlater. We found that the signature of individual identity consistently outweighed storage effects: alpha diversity and beta diversity measures were significantly correlated across methods, and while samples often clustered by donor, they never clustered by storage method. Provided that all analyzed samples are stored the same way, banked fecal samples therefore appear highly suitable for investigating variation in gut microbiota. Our results open the door to a much-expanded perspective on variation in the gut microbiome across species and ecological contexts. PMID:27528013

  18. Common methods for fecal sample storage in field studies yield consistent signatures of individual identity in microbiome sequencing data.

    PubMed

    Blekhman, Ran; Tang, Karen; Archie, Elizabeth A; Barreiro, Luis B; Johnson, Zachary P; Wilson, Mark E; Kohn, Jordan; Yuan, Michael L; Gesquiere, Laurence; Grieneisen, Laura E; Tung, Jenny

    2016-01-01

    Field studies of wild vertebrates are frequently associated with extensive collections of banked fecal samples-unique resources for understanding ecological, behavioral, and phylogenetic effects on the gut microbiome. However, we do not understand whether sample storage methods confound the ability to investigate interindividual variation in gut microbiome profiles. Here, we extend previous work on storage methods for gut microbiome samples by comparing immediate freezing, the gold standard of preservation, to three methods commonly used in vertebrate field studies: lyophilization, storage in ethanol, and storage in RNAlater. We found that the signature of individual identity consistently outweighed storage effects: alpha diversity and beta diversity measures were significantly correlated across methods, and while samples often clustered by donor, they never clustered by storage method. Provided that all analyzed samples are stored the same way, banked fecal samples therefore appear highly suitable for investigating variation in gut microbiota. Our results open the door to a much-expanded perspective on variation in the gut microbiome across species and ecological contexts. PMID:27528013

  19. In planta analysis of a cis-regulatory cytokinin response motif in Arabidopsis and identification of a novel enhancer sequence.

    PubMed

    Ramireddy, Eswarayya; Brenner, Wolfram G; Pfeifer, Andreas; Heyl, Alexander; Schmülling, Thomas

    2013-07-01

    The phytohormone cytokinin plays a key role in regulating plant growth and development, and is involved in numerous physiological responses to environmental changes. The type-B response regulators, which regulate the transcription of cytokinin response genes, are a part of the cytokinin signaling system. Arabidopsis thaliana encodes 11 type-B response regulators (type-B ARRs), and some of them were shown to bind in vitro to the core cytokinin response motif (CRM) 5'-(A/G)GAT(T/C)-3' or, in the case of ARR1, to an extended motif (ECRM), 5'-AAGAT(T/C)TT-3'. Here we obtained in planta proof for the functionality of the latter motif. Promoter deletion analysis of the primary cytokinin response gene ARR6 showed that a combination of two extended motifs within the promoter is required to mediate the full transcriptional activation by ARR1 and other type-B ARRs. CRMs were found to be over-represented in the vicinity of ECRMs in the promoters of cytokinin-regulated genes, suggesting their functional relevance. Moreover, an evolutionarily conserved 27 bp long T-rich region between -220 and -193 bp was identified and shown to be required for the full activation by type-B ARRs and the response to cytokinin. This novel enhancer is not bound by the DNA-binding domain of ARR1, indicating that additional proteins might be involved in mediating the transcriptional cytokinin response. Furthermore, genome-wide expression profiling identified genes, among them ARR16, whose induction by cytokinin depends on both ARR1 and other specific type-B ARRs. This together with the ECRM/CRM sequence clustering indicates cooperative action of different type-B ARRs for the activation of particular target genes. PMID:23620480

  20. Subgroup C avian metapneumovirus (MPV) and the recently isolated human MPV exhibit a common organization but have extensive sequence divergence in their putative SH and G genes.

    PubMed

    Toquin, D; de Boisseson, C; Beven, V; Senne, D A; Eterradossi, N

    2003-08-01

    The genes encoding the putative small hydrophobic (SH), attachment (G) and polymerase (L) proteins of the Colorado isolate of subgroup C avian pneumovirus (APV) were entirely or partially sequenced. They all included metapneumovirus (MPV)-like gene start and gene end sequences. The deduced Colorado SH protein shared 26.9 and 21.7 % aa identity with its counterpart in human MPV (hMPV) and APV subgroup A, respectively, but its only significant aa similarities were to hMPV. Conserved features included a common hydrophobicity profile with an unique transmembrane domain and the conservation of most extracellular cysteine residues. The Colorado putative G gene encoded several ORFs, the longer of which encoded a 252 aa long type II glycoprotein with aa similarities to hMPV G only (20.6 % overall aa identity with seven conserved N-terminal residues). The putative Colorado G protein shared, at best, 21.0 % aa identity with its counterparts in the other APV subgroups and did not contain the extracellular cysteine residues and short aa stretch highly conserved in other APVs. The N-terminal end of the Colorado L protein exhibited 73.6 and 54.9 % aa identity with hMPV and APV subgroup A, respectively, with four aa blocks highly conserved among Pneumovirus: Phylogenetic analysis performed on the nt sequences confirmed that the L sequences from MPVs were genetically related, whereas analysis of the G sequences revealed that among MPVs, only APV subgroups A, B and D clustered together, independently of both the Colorado isolate and hMPV, which shared weak genetic relatedness at the G gene level.

  1. Common chromosomal fragile site FRA16D sequence: identification of the FOR gene spanning FRA16D and homozygous deletions and translocation breakpoints in cancer cells.

    PubMed

    Ried, K; Finnis, M; Hobson, L; Mangelsdorf, M; Dayan, S; Nancarrow, J K; Woollatt, E; Kremmidiotis, G; Gardner, A; Venter, D; Baker, E; Richards, R I

    2000-07-01

    Fluorescence in situ hybridization of a tile path of DNA subclones has previously enabled the cyto-genetic definition of the minimal DNA sequence which spans the FRA16D common chromosomal fragile site, located at 16q23.2. Homozygous deletion of the FRA16D locus has been reported in adenocarcinomas of stomach, colon, lung and ovary. We have sequenced the 270 kb containing the FRA16D fragile site and the minimal homozygously deleted region in tumour cells. This sequence enabled localization of some of the tumour cell breakpoints to regions which contain AT-rich secondary structures similar to those associated with the FRA10B and FRA16B rare fragile sites. The FRA16D DNA sequence also led to the identification of an alternatively spliced gene, named FOR (fragile site FRA16D oxidoreductase), exons of which span both the fragile site and the minimal region of homozygous deletion. In addition, the complete DNA sequence of the FRA16D-containing FOR intron reveals no evidence of additional authentic transcripts. Alternatively spliced FOR transcripts (FOR I, FOR II and FOR III) encode proteins which share N-terminal WW domains and differ at their C-terminus, with FOR III having a truncated oxidoreductase domain. FRA16D-associated deletions selectively affect the FOR gene transcripts. Three out of five previously mapped translocation breakpoints in multiple myeloma are also located within the FOR gene. FOR is therefore the principle genetic target for DNA instability at 16q23.2 and perturbation of FOR function is likely to contribute to the biological consequences of DNA instability at FRA16D in cancer cells. PMID:10861292

  2. Rearrangements of the transcriptional regulatory networks of metabolic pathways in fungi.

    PubMed

    Lavoie, Hugo; Hogues, Hervé; Whiteway, Malcolm

    2009-12-01

    Growing evidence suggests that transcriptional regulatory networks in many organisms are highly flexible. Here, we discuss the evolution of transcriptional regulatory networks governing the metabolic machinery of sequenced ascomycetes. In particular, recent work has shown that transcriptional rewiring is common in regulons controlling processes such as production of ribosome components and metabolism of carbohydrates and lipids. We note that dramatic rearrangements of the transcriptional regulatory components of metabolic functions have occurred among ascomycetes species.

  3. Cis-regulatory mutations in human disease

    PubMed Central

    2009-01-01

    Cis-acting regulatory sequences are required for the proper temporal and spatial control of gene expression. Variation in gene expression is highly heritable and a significant determinant of human disease susceptibility. The diversity of human genetic diseases attributed, in whole or in part, to mutations in non-coding regulatory sequences is on the rise. Improvements in genome-wide methods of associating genetic variation with human disease and predicting DNA with cis-regulatory potential are two of the major reasons for these recent advances. This review will highlight select examples from the literature that have successfully integrated genetic and genomic approaches to uncover the molecular basis by which cis-regulatory mutations alter gene expression and contribute to human disease. The fine mapping of disease-causing variants has led to the discovery of novel cis-acting regulatory elements that, in some instances, are located as far away as 1.5 Mb from the target gene. In other cases, the prior knowledge of the regulatory landscape surrounding the gene of interest aided in the selection of enhancers for mutation screening. The success of these studies should provide a framework for following up on the large number of genome-wide association studies that have identified common variants in non-coding regions of the genome that associate with increased risk of human diseases including, diabetes, autism, Crohn's, colorectal cancer, and asthma, to name a few. PMID:19641089

  4. Lung Parenchymal Signal Intensity in MRI: A Technical Review with Educational Aspirations Regarding Reversible Versus Irreversible Transverse Relaxation Effects in Common Pulse Sequences.

    PubMed

    Mulkern, Robert; Haker, Steven; Mamata, Hatsuho; Lee, Edward; Mitsouras, Dimitrios; Oshio, Koichi; Balasubramanian, Mukund; Hatabu, Hiroto

    2014-03-01

    Lung parenchyma is challenging to image with proton MRI. The large air space results in ~l/5th as many signal-generating protons compared to other organs. Air/tissue magnetic susceptibility differences lead to strong magnetic field gradients throughout the lungs and to broad frequency distributions, much broader than within other organs. Such distributions have been the subject of experimental and theoretical analyses which may reveal aspects of lung microarchitecture useful for diagnosis. Their most immediate relevance to current imaging practice is to cause rapid signal decays, commonly discussed in terms of short T2 (*) values of 1 ms or lower at typical imaging field strengths. Herein we provide a brief review of previous studies describing and interpreting proton lung spectra. We then link these broad frequency distributions to rapid signal decays, though not necessarily the exponential decays generally used to define T2 (*) values. We examine how these decays influence observed signal intensities and spatial mapping features associated with the most prominent torso imaging sequences, including spoiled gradient and spin echo sequences. Effects of imperfect refocusing pulses on the multiple echo signal decays in single shot fast spin echo (SSFSE) sequences and effects of broad frequency distributions on balanced steady state free precession (bSSFP) sequence signal intensities are also provided. The theoretical analyses are based on the concept of explicitly separating the effects of reversible and irreversible transverse relaxation processes, thus providing a somewhat novel and more general framework from which to estimate lung signal intensity behavior in modern imaging practice. PMID:25228852

  5. Lung Parenchymal Signal Intensity in MRI: A Technical Review with Educational Aspirations Regarding Reversible Versus Irreversible Transverse Relaxation Effects in Common Pulse Sequences

    PubMed Central

    MULKERN, ROBERT; HAKER, STEVEN; MAMATA, HATSUHO; LEE, EDWARD; MITSOURAS, DIMITRIOS; OSHIO, KOICHI; BALASUBRAMANIAN, MUKUND; HATABU, HIROTO

    2014-01-01

    Lung parenchyma is challenging to image with proton MRI. The large air space results in ~l/5th as many signal-generating protons compared to other organs. Air/tissue magnetic susceptibility differences lead to strong magnetic field gradients throughout the lungs and to broad frequency distributions, much broader than within other organs. Such distributions have been the subject of experimental and theoretical analyses which may reveal aspects of lung microarchitecture useful for diagnosis. Their most immediate relevance to current imaging practice is to cause rapid signal decays, commonly discussed in terms of short T2* values of 1 ms or lower at typical imaging field strengths. Herein we provide a brief review of previous studies describing and interpreting proton lung spectra. We then link these broad frequency distributions to rapid signal decays, though not necessarily the exponential decays generally used to define T2* values. We examine how these decays influence observed signal intensities and spatial mapping features associated with the most prominent torso imaging sequences, including spoiled gradient and spin echo sequences. Effects of imperfect refocusing pulses on the multiple echo signal decays in single shot fast spin echo (SSFSE) sequences and effects of broad frequency distributions on balanced steady state free precession (bSSFP) sequence signal intensities are also provided. The theoretical analyses are based on the concept of explicitly separating the effects of reversible and irreversible transverse relaxation processes, thus providing a somewhat novel and more general framework from which to estimate lung signal intensity behavior in modern imaging practice. PMID:25228852

  6. Vision from next generation sequencing: multi-dimensional genome-wide analysis for producing gene regulatory networks underlying retinal development, aging and disease.

    PubMed

    Yang, Hyun-Jin; Ratnapriya, Rinki; Cogliati, Tiziana; Kim, Jung-Woong; Swaroop, Anand

    2015-05-01

    Genomics and genetics have invaded all aspects of biology and medicine, opening uncharted territory for scientific exploration. The definition of "gene" itself has become ambiguous, and the central dogma is continuously being revised and expanded. Computational biology and computational medicine are no longer intellectual domains of the chosen few. Next generation sequencing (NGS) technology, together with novel methods of pattern recognition and network analyses, has revolutionized the way we think about fundamental biological mechanisms and cellular pathways. In this review, we discuss NGS-based genome-wide approaches that can provide deeper insights into retinal development, aging and disease pathogenesis. We first focus on gene regulatory networks (GRNs) that govern the differentiation of retinal photoreceptors and modulate adaptive response during aging. Then, we discuss NGS technology in the context of retinal disease and develop a vision for therapies based on network biology. We should emphasize that basic strategies for network construction and analyses can be transported to any tissue or cell type. We believe that specific and uniform guidelines are required for generation of genome, transcriptome and epigenome data to facilitate comparative analysis and integration of multi-dimensional data sets, and for constructing networks underlying complex biological processes. As cellular homeostasis and organismal survival are dependent on gene-gene and gene-environment interactions, we believe that network-based biology will provide the foundation for deciphering disease mechanisms and discovering novel drug targets for retinal neurodegenerative diseases. PMID:25668385

  7. Vision from next generation sequencing: Multi-dimensional genome-wide analysis for producing gene regulatory networks underlying retinal development, aging and disease

    PubMed Central

    Yang, Hyun-Jin; Ratnapriya, Rinki; Cogliati, Tiziana; Kim, Jung-Woong; Swaroop, Anand

    2015-01-01

    Genomics and genetics have invaded all aspects of biology and medicine, opening uncharted territory for scientific exploration. The definition of “gene” itself has become ambiguous, and the central dogma is continuously being revised and expanded. Computational biology and computational medicine are no longer intellectual domains of the chosen few. Next generation sequencing (NGS) technology, together with novel methods of pattern recognition and network analyses, has revolutionized the way we think about fundamental biological mechanisms and cellular pathways. In this review, we discuss NGS-based genome-wide approaches that can provide deeper insights into retinal development, aging and disease pathogenesis. We first focus on gene regulatory networks (GRNs) that govern the differentiation of retinal photoreceptors and modulate adaptive response during aging. Then, we discuss NGS technology in the context of retinal disease and develop a vision for therapies based on network biology. We should emphasize that basic strategies for network construction and analyses can be transported to any tissue or cell type. We believe that specific and uniform guidelines are required for generation of genome, transcriptome and epigenome data to facilitate comparative analysis and integration of multi-dimensional data sets, and for constructing networks underlying complex biological processes. As cellular homeostasis and organismal survival are dependent on gene-gene and gene-environment interactions, we believe that network-based biology will provide the foundation for deciphering disease mechanisms and discovering novel drug targets for retinal neurodegenerative diseases. PMID:25668385

  8. Exploiting Illumina sequencing for the development of 95 novel polymorphic EST-SSR markers in common vetch (Vicia sativa subsp. sativa).

    PubMed

    Liu, Zhipeng; Liu, Peng; Luo, Dong; Liu, Wenxian; Wang, Yanrong

    2014-05-05

    The common vetch (Vicia sativa subsp. sativa), a self-pollinating and diploid species, is one of the most important annual legumes in the world due to its short growth period, high nutritional value, and multiple usages as hay, grain, silage, and green manure. The available simple sequence repeat (SSR) markers for common vetch, however, are insufficient to meet the developing demand for genetic and molecular research on this important species. Here, we aimed to develop and characterise several polymorphic EST-SSR markers from the vetch Illumina transcriptome. A total number of 1,071 potential EST-SSR markers were identified from 1025 unigenes whose lengths were greater than 1,000 bp, and 450 primer pairs were then designed and synthesized. Finally, 95 polymorphic primer pairs were developed for the 10 common vetch accessions, which included 50 individuals. Among the 95 EST-SSR markers, the number of alleles ranged from three to 13, and the polymorphism information content values ranged from 0.09 to 0.98. The observed heterozygosity values ranged from 0.00 to 1.00, and the expected heterozygosity values ranged from 0.11 to 0.98. These 95 EST-SSR markers developed from the vetch Illumina transcriptome could greatly promote the development of genetic and molecular breeding studies pertaining to in this species.

  9. Common and rare von Willebrand factor (VWF) coding variants, VWF levels, and factor VIII levels in African Americans: the NHLBI Exome Sequencing Project

    PubMed Central

    Johnsen, Jill M.; Auer, Paul L.; Morrison, Alanna C.; Jiao, Shuo; Wei, Peng; Haessler, Jeffrey; Fox, Keolu; McGee, Sean R.; Smith, Joshua D.; Carlson, Christopher S.; Smith, Nicholas; Boerwinkle, Eric; Kooperberg, Charles; Nickerson, Deborah A.; Rich, Stephen S.; Green, David; Peters, Ulrike; Cushman, Mary

    2013-01-01

    Several rare European von Willebrand disease missense variants of VWF (including p.Arg2185Gln and p.His817Gln) were recently reported to be common in apparently healthy African Americans (AAs). Using data from the NHLBI Exome Sequencing Project, we assessed the association of these and other VWF coding variants with von Willebrand factor (VWF) and factor VIII (FVIII) levels in 4468 AAs. Of 30 nonsynonymous VWF variants, 6 were significantly and independently associated (P < .001) with levels of VWF and/or FVIII. Each additional copy of the common VWF variants encoding p.Thr789Ala or p.Asp1472His was associated with 6 to 8 IU/dL higher VWF levels. The VWF variant encoding p.Arg2185Gln was associated with 7 to 13 IU/dL lower VWF and FVIII levels. The type 2N-related VWF variant encoding p.His817Gln was associated with 17 IU/dL lower FVIII level but normal VWF level. A novel, rare missense VWF variant that predicts disruption of an O-glycosylation site (p.Ser1486Leu) and a rare variant encoding p.Arg2287Trp were each associated with 30 to 40 IU/dL lower VWF level (P < .001). In summary, several common and rare VWF missense variants contribute to phenotypic differences in VWF and FVIII among AAs. PMID:23690449

  10. DNA Sequence Variants in the Five Prime Untranslated Region of the Cyclooxygenase-2 Gene Are Commonly Found in Healthy Dogs and Gray Wolves.

    PubMed

    Safra, Noa; Hayward, Louisa J; Aguilar, Miriam; Sacks, Benjamin N; Westropp, Jodi L; Mohr, F Charles; Mellersh, Cathryn S; Bannasch, Danika L

    2015-01-01

    The aim of this study was to investigate the frequency of regional DNA variants upstream to the translation initiation site of the canine Cyclooxygenase-2 (Cox-2) gene in healthy dogs. Cox-2 plays a role in various disease conditions such as acute and chronic inflammation, osteoarthritis and malignancy. A role for Cox-2 DNA variants in genetic predisposition to canine renal dysplasia has been proposed and dog breeders have been encouraged to select against these DNA variants. We sequenced 272-422 bases in 152 dogs unaffected by renal dysplasia and found 19 different haplotypes including 11 genetic variants which had not been described previously. We genotyped 7 gray wolves to ascertain the wildtype variant and found that the wolves we analyzed had predominantly the second most common DNA variant found in dogs. Our results demonstrate an elevated level of regional polymorphism that appears to be a feature of healthy domesticated dogs.

  11. DNA Sequence Variants in the Five Prime Untranslated Region of the Cyclooxygenase-2 Gene Are Commonly Found in Healthy Dogs and Gray Wolves

    PubMed Central

    Safra, Noa; Hayward, Louisa J.; Aguilar, Miriam; Sacks, Benjamin N.; Westropp, Jodi L.; Mohr, F. Charles; Mellersh, Cathryn S.; Bannasch, Danika L.

    2015-01-01

    The aim of this study was to investigate the frequency of regional DNA variants upstream to the translation initiation site of the canine Cyclooxygenase-2 (Cox-2) gene in healthy dogs. Cox-2 plays a role in various disease conditions such as acute and chronic inflammation, osteoarthritis and malignancy. A role for Cox-2 DNA variants in genetic predisposition to canine renal dysplasia has been proposed and dog breeders have been encouraged to select against these DNA variants. We sequenced 272–422 bases in 152 dogs unaffected by renal dysplasia and found 19 different haplotypes including 11 genetic variants which had not been described previously. We genotyped 7 gray wolves to ascertain the wildtype variant and found that the wolves we analyzed had predominantly the second most common DNA variant found in dogs. Our results demonstrate an elevated level of regional polymorphism that appears to be a feature of healthy domesticated dogs. PMID:26244515

  12. Rare, Low-Frequency, and Common Variants in the Protein-Coding Sequence of Biological Candidate Genes from GWASs Contribute to Risk of Rheumatoid Arthritis

    PubMed Central

    Diogo, Dorothée; Kurreeman, Fina; Stahl, Eli A.; Liao, Katherine P.; Gupta, Namrata; Greenberg, Jeffrey D.; Rivas, Manuel A.; Hickey, Brendan; Flannick, Jason; Thomson, Brian; Guiducci, Candace; Ripke, Stephan; Adzhubey, Ivan; Barton, Anne; Kremer, Joel M.; Alfredsson, Lars; Sunyaev, Shamil; Martin, Javier; Zhernakova, Alexandra; Bowes, John; Eyre, Steve; Siminovitch, Katherine A.; Gregersen, Peter K.; Worthington, Jane; Klareskog, Lars; Padyukov, Leonid; Raychaudhuri, Soumya; Plenge, Robert M.

    2013-01-01

    The extent to which variants in the protein-coding sequence of genes contribute to risk of rheumatoid arthritis (RA) is unknown. In this study, we addressed this issue by deep exon sequencing and large-scale genotyping of 25 biological candidate genes located within RA risk loci discovered by genome-wide association studies (GWASs). First, we assessed the contribution of rare coding variants in the 25 genes to the risk of RA in a pooled sequencing study of 500 RA cases and 650 controls of European ancestry. We observed an accumulation of rare nonsynonymous variants exclusive to RA cases in IL2RA and IL2RB (burden test: p = 0.007 and p = 0.018, respectively). Next, we assessed the aggregate contribution of low-frequency and common coding variants to the risk of RA by dense genotyping of the 25 gene loci in 10,609 RA cases and 35,605 controls. We observed a strong enrichment of coding variants with a nominal signal of association with RA (p < 0.05) after adjusting for the best signal of association at the loci (penrichment = 6.4 × 10−4). For one locus containing CD2, we found that a missense variant, rs699738 (c.798C>A [p.His266Gln]), and a noncoding variant, rs624988, reside on distinct haplotypes and independently contribute to the risk of RA (p = 4.6 × 10−6). Overall, our results indicate that variants (distributed across the allele-frequency spectrum) within the protein-coding portion of a subset of biological candidate genes identified by GWASs contribute to the risk of RA. Further, we have demonstrated that very large sample sizes will be required for comprehensively identifying the independent alleles contributing to the missing heritability of RA. PMID:23261300

  13. Push it to the limit: Characterizing the convergence of common sequences of basis sets for intermolecular interactions as described by density functional theory

    NASA Astrophysics Data System (ADS)

    Witte, Jonathon; Neaton, Jeffrey B.; Head-Gordon, Martin

    2016-05-01

    With the aim of systematically characterizing the convergence of common families of basis sets such that general recommendations for basis sets can be made, we have tested a wide variety of basis sets against complete-basis binding energies across the S22 set of intermolecular interactions—noncovalent interactions of small and medium-sized molecules consisting of first- and second-row atoms—with three distinct density functional approximations: SPW92, a form of local-density approximation; B3LYP, a global hybrid generalized gradient approximation; and B97M-V, a meta-generalized gradient approximation with nonlocal correlation. We have found that it is remarkably difficult to reach the basis set limit; for the methods and systems examined, the most complete basis is Jensen's pc-4. The Dunning correlation-consistent sequence of basis sets converges slowly relative to the Jensen sequence. The Karlsruhe basis sets are quite cost effective, particularly when a correction for basis set superposition error is applied: counterpoise-corrected def2-SVPD binding energies are better than corresponding energies computed in comparably sized Dunning and Jensen bases, and on par with uncorrected results in basis sets 3-4 times larger. These trends are exhibited regardless of the level of density functional approximation employed. A sense of the magnitude of the intrinsic incompleteness error of each basis set not only provides a foundation for guiding basis set choice in future studies but also facilitates quantitative comparison of existing studies on similar types of systems.

  14. Push it to the limit: Characterizing the convergence of common sequences of basis sets for intermolecular interactions as described by density functional theory.

    PubMed

    Witte, Jonathon; Neaton, Jeffrey B; Head-Gordon, Martin

    2016-05-21

    With the aim of systematically characterizing the convergence of common families of basis sets such that general recommendations for basis sets can be made, we have tested a wide variety of basis sets against complete-basis binding energies across the S22 set of intermolecular interactions-noncovalent interactions of small and medium-sized molecules consisting of first- and second-row atoms-with three distinct density functional approximations: SPW92, a form of local-density approximation; B3LYP, a global hybrid generalized gradient approximation; and B97M-V, a meta-generalized gradient approximation with nonlocal correlation. We have found that it is remarkably difficult to reach the basis set limit; for the methods and systems examined, the most complete basis is Jensen's pc-4. The Dunning correlation-consistent sequence of basis sets converges slowly relative to the Jensen sequence. The Karlsruhe basis sets are quite cost effective, particularly when a correction for basis set superposition error is applied: counterpoise-corrected def2-SVPD binding energies are better than corresponding energies computed in comparably sized Dunning and Jensen bases, and on par with uncorrected results in basis sets 3-4 times larger. These trends are exhibited regardless of the level of density functional approximation employed. A sense of the magnitude of the intrinsic incompleteness error of each basis set not only provides a foundation for guiding basis set choice in future studies but also facilitates quantitative comparison of existing studies on similar types of systems.

  15. Genetic structure of the widespread and common Mediterranean bryophyte Pleurochaete squarrosa (Brid.) Lindb. (Pottiaceae) - evidence from nuclear and plastidic DNA sequence variation and allozymes.

    PubMed

    Grundmann, Michael; Ansell, Stephen W; Russell, Stephen J; Koch, Marcus A; Vogel, Johannes C

    2007-02-01

    The Mediterranean Basin as one the world's most biologically diverse regions provides an interesting area for the study of plant evolution and spatial structure in plant populations. The dioecious moss Pleurochaete squarrosa is a widespread and common bryophyte in the Mediterranean Basin. Thirty populations were sampled for a study on molecular diversity and genetic structure, covering most major islands and mainland populations from Europe and Africa. A significant decline in nuclear and chloroplast sequence and allozyme variation within populations from west to east was observed. While DNA sequence data showed patterns of isolation by distance, allozyme markers did not. Instead, their considerable interpopulation genetic differentiation appeared to be unrelated to geographic distance. Similar high values for coefficients of gene diversity (G(ST)) in all data sets provided evidence of geographic isolation and limited gene flow among populations (i) within islands, (ii) within mainland areas, and (iii) between islands and mainland. Notably, populations in continental Spain are strongly genetically isolated from all other investigated areas. Surprisingly, there was no difference in gene diversity and G(ST) between islands and mainland areas. Thus, we conclude that large Mediterranean islands may function as 'mainland' for bryophytes. This hypothesis and its implication for conservation biology of cryptogamic plants warrant further investigation. While sexually reproducing populations were found all over the Mediterranean Basin, high levels of multilocus linkage disequilibrium provide evidence of mainly vegetative propagation even in populations where sexual reproduction was observed. PMID:17284206

  16. Draft Genome Sequence of Textile Azo Dye-Decolorizing and -Degrading Pseudomonas aeruginosa Strain PFK10, Isolated from the Common Effluent Treatment Plant of the Ankleshwar Industrial Area of Gujarat, India

    PubMed Central

    Faldu, P. R.; Kothari, V. V.; Kothari, C. R.; Rawal, C. M.; Domadia, K. K.; Patel, P. A.; Bhimani, H. D.; Raval, V. H.; Parmar, N. R.; Nathani, N. M.; Koringa, P. G.; Joshi, C. G.

    2014-01-01

    Here, we report the draft genome sequence of Pseudomonas aeruginosa strain PFK10, isolated from the common effluent treatment plant (CETP) of the Ankleshwar industrial area of Gujarat, India. The 6.04-Mb draft genome sequence of strain PFK10 provides information about the genes encoding enzymes that enable the strain to decolorize and degrade textile azo dye. PMID:24503984

  17. [Mosaic expression of the lacZ reporter-gene under control of 5'-regulatory sequences of the alpha-S1-casein gene in transgenic mice].

    PubMed

    Serova, I A; Andreeva, L E; Khaĭdarova, N V; Dias, L P; Dvorianchikov, G A; Burkov, I A; Baginskaia, N V

    2009-01-01

    Phenomenon of mosaic expression at cellular level is widely presented in tissues and organs of transgenic animals. The communication is concerned a study of the mosaics in transgenic mice carrying the lacZ reporter-gene under control of the bovine and goat alpha-S1-casein genes with 5'-flanked sequences of various ex-tent: pCLZ1--721bp, pCLZ2-- 2001 bp and pCLZ3 3409 bp constructs. Five transgenic founders were generated by injection of the recombinant DNA into zygotes: pCLZ 1 - N 16, pCLZ2 - N 37 and pCLZ3 N 7, N 36, and N 48. Positive for J3-galactosidase activity cells were detected in lactating mammary glands of all transgenic females, however, distribution of the positive cells was variable. We observed two types of mosaics: clonal or "lobule" type with positive cells filling the whole of the globule or stochastic type with single positive cells scattered over one or different lobules. Two types of mosaics were characteristic of all the transgenic animals, although, females carrying the pCLZ2 transgene showed "lobule" type more often than transgenic animals with the transgenes pCLZ and pCLZ3. It is suggested that the stochastic type of mosaics occurs in the cells at terminal stage of differentiation, whereas the type arises from positive for P-galactosidase proliferating precursors. Analysis of the inheritance of the transgenes in different lines demonstrated that the pCLZl transgene was inserted in the X-chromosome of the founder whereas the other two localized in autosomes. Localization of the pCLZl transgene in the X-chromosome did not influence the mosaicism; it was similar to that of transgenic animals carrying the transgenes in autosomes. Ectopic expression of the reporter-gene was detected in mandibular glands from the offsprings of the founders N 16 and N 37 only, as well as in atrezed follicles in N 37. The weak ectopic expression saggests that the 5 S-flanked regulatory sequences used in the constructs are able to provide perfect tissue

  18. Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

    NASA Astrophysics Data System (ADS)

    Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

    2000-05-01

    The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

  19. Interferon regulatory factor 4b (IRF4b) in Japanese flounder, Paralichthys olivaceus: Sequencing, ubiquitous tissue distribution and inducible expression by poly(I:C) and DNA virus.

    PubMed

    Liu, Dahai; Chen, Jinjing; Zhang, Haiyan; Hu, Mengzhu; Lou, Huimin; Liu, Qiuming; Zhang, Shicui; Hu, Guobin

    2016-09-01

    Interferon regulatory factor 4 (IRF4) in mammals is known to be critical in regulation of development and functions of lymphomyeloid cell lineages. Recent studies have demonstrated its involvement in immune responses to bacterial and viral challenges in teleosts. In this study, an IRF4 gene was cloned from Japanese flounder (Paralichthys olivaceus) and its expression in response to polyinosinic:polycytidylic acid [poly(I:C)] and lymphocystis disease virus (LCDV) stimulations was studied in vivo. The cloned gene spans over 5.9 kb, comprises eight exons and seven introns and encodes a putative protein of 456 amino acids. The deduced amino acid sequence possesses a conserved DNA-binding domain (DBD), an IRF-association domain (IAD) and a nuclear localization signal (NLS). Phylogenetic analysis clustered it into the teleost IRF4b clade and, thus, it was named Paralichthys olivaceus (Po)IRF4b. The constitutive expression of PoIRF4b transcripts was detectable in all examined organs, with highest levels found in lymphomyeloid-rich tissues. They were induced by both poly(I:C) and LCDV with a similar inducibility in immune or non-immune organs. Two waves of induced expression of PoIRF4b were observed with the two stimuli during a 7-day time course in the immune organs, with the early-phase induction being stronger. The maximum increases of PoIRF4b transcript levels ranged from 1.3 to 4.0-fold and appeared at day 1-5 post-injection depending on different organs and stimuli. In both stimulation cases, the strongest induction was detected in spleen and the weakest in muscle. These results indicate that PoIRF4b may participate in regulation of immune responses of flounders to both RNA and DNA virus infections. PMID:27084058

  20. Barley Grain Maturation and Germination: Metabolic Pathway and Regulatory Network Commonalities and Differences Highlighted by New MapMan/PageMan Profiling Tools1[W][OA

    PubMed Central

    Sreenivasulu, Nese; Usadel, Björn; Winter, Andreas; Radchuk, Volodymyr; Scholz, Uwe; Stein, Nils; Weschke, Winfriede; Strickert, Marc; Close, Timothy J.; Stitt, Mark; Graner, Andreas; Wobus, Ulrich

    2008-01-01

    Plant seeds prepare for germination already during seed maturation. We performed a detailed transcriptome analysis of barley (Hordeum vulgare) grain maturation, desiccation, and germination in two tissue fractions (starchy endosperm/aleurone and embryo/scutellum) using the Affymetrix Barley1 GeneChip. To aid data evaluation, Arabidopsis thaliana MapMan and PageMan tools were adapted to barley. The analyses allow a number of conclusions: (1) Cluster analysis revealed a smooth transition in transcription programs between late seed maturation and germination within embryo tissues, but not in the endosperm/aleurone. (2) More than 12,000 transcripts are stored in the embryo of dry barley grains, many of which are presumably activated during germination. (3) Transcriptional activation of storage reserve mobilization events occurs at an early stage of germination, well before radicle protrusion. (4) Key genes of gibberellin (GA) biosynthesis are already active during grain maturation at a time when abscisic acid peaks suggesting the formation of an endogenous store of GA in the aleurone. This GA probably acts later during germination in addition to newly synthesized GA. (5) Beside the well-known role of GA in gene activation during germination spatiotemporal expression data and cis-element searches in homologous rice promoters confirm an equally important gene-activating role of abscisic acid during this developmental period. The respective regulatory webs are linked to auxin and ethylene controlled networks. In summary, new bioinformatics PageMan and MapMan tools developed in barley have been successfully used to investigate in detail the transcriptome relationships between seed maturation and germination in an important crop plant. PMID:18281415

  1. Characterization of trimethoprim-sulfamethoxazole resistance genes and their relatedness to class 1 integron and insertion sequence common region in gram-negative bacilli.

    PubMed

    Shin, Hae Won; Lim, Jinsook; Kim, Semi; Kim, Jimyung; Kwon, Gye Cheol; Koo, Sun Hoe

    2015-01-01

    Trimethoprim-sulfamethoxazole (TMP-SMX) has been used for the treatment of urinary tract infections, but increasing resistance to TMP-SMX has been reported. In this study, we analyzed TMP-SMX resistance genes and their relatedness with integrons and insertion sequence common regions (ISCRs) in uropathogenic gram-negative bacilli. Consecutive nonduplicate TMP-SMX nonsusceptible clinical isolates of E. coli, K. pneumoniae, Acinetobacter spp., and P. aeruginosa were collected from urine. The minimal inhibitory concentration was determined by Etest. TMP-SMX resistance genes (sul and dfr), integrons, and ISCRs were analyzed by PCR and sequencing. A total of 45 E. coli (37.8%), 15 K. pneumoniae (18.5%), 12 Acinetobacter spp. (70.6%), and 9 Pseudomonas aeruginosa (30.0%) isolates were found to be resistant to TMP-SMX. Their MICs were all over 640. In E. coli and K. pneumoniae, sul1 and dfr genes were highly prevalent in relation with integron1. The sul3 gene was detected in E. coli. However, in P. aeruginosa and Acinetobacter spp., only sul1 was prevalent in relation with class 1 integron; however, dfr was not detected and sul2 was less prevalent than in Enterobacteriaceae. ISCR1 and/or ISCR2 were detected in E. coli, K. pneumoniae, and Acinetobacter spp. but the relatedness with TMP-SMX resistance genes was not prominent. ISCR14 was detected in six isolates of E. coli. In conclusion, resistance mechanisms for TMP-SMX were different between Enterobacteriaceae and glucose non-fermenting gram-negative bacilli. Class 1 integron was widely disseminated in uropathogenic gram-negative baciili, so the adoption of prudent use of antimicrobial agents and the establishment of a surveillance system are needed.

  2. Characterization of trimethoprim-sulfamethoxazole resistance genes and their relatedness to class 1 integron and insertion sequence common region in gram-negative bacilli.

    PubMed

    Shin, Hae Won; Lim, Jinsook; Kim, Semi; Kim, Jimyung; Kwon, Gye Cheol; Koo, Sun Hoe

    2015-01-01

    Trimethoprim-sulfamethoxazole (TMP-SMX) has been used for the treatment of urinary tract infections, but increasing resistance to TMP-SMX has been reported. In this study, we analyzed TMP-SMX resistance genes and their relatedness with integrons and insertion sequence common regions (ISCRs) in uropathogenic gram-negative bacilli. Consecutive nonduplicate TMP-SMX nonsusceptible clinical isolates of E. coli, K. pneumoniae, Acinetobacter spp., and P. aeruginosa were collected from urine. The minimal inhibitory concentration was determined by Etest. TMP-SMX resistance genes (sul and dfr), integrons, and ISCRs were analyzed by PCR and sequencing. A total of 45 E. coli (37.8%), 15 K. pneumoniae (18.5%), 12 Acinetobacter spp. (70.6%), and 9 Pseudomonas aeruginosa (30.0%) isolates were found to be resistant to TMP-SMX. Their MICs were all over 640. In E. coli and K. pneumoniae, sul1 and dfr genes were highly prevalent in relation with integron1. The sul3 gene was detected in E. coli. However, in P. aeruginosa and Acinetobacter spp., only sul1 was prevalent in relation with class 1 integron; however, dfr was not detected and sul2 was less prevalent than in Enterobacteriaceae. ISCR1 and/or ISCR2 were detected in E. coli, K. pneumoniae, and Acinetobacter spp. but the relatedness with TMP-SMX resistance genes was not prominent. ISCR14 was detected in six isolates of E. coli. In conclusion, resistance mechanisms for TMP-SMX were different between Enterobacteriaceae and glucose non-fermenting gram-negative bacilli. Class 1 integron was widely disseminated in uropathogenic gram-negative baciili, so the adoption of prudent use of antimicrobial agents and the establishment of a surveillance system are needed. PMID:25348695

  3. Push it to the limit: Characterizing the convergence of common sequences of basis sets for intermolecular interactions as described by density functional theory.

    PubMed

    Witte, Jonathon; Neaton, Jeffrey B; Head-Gordon, Martin

    2016-05-21

    With the aim of systematically characterizing the convergence of common families of basis sets such that general recommendations for basis sets can be made, we have tested a wide variety of basis sets against complete-basis binding energies across the S22 set of intermolecular interactions-noncovalent interactions of small and medium-sized molecules consisting of first- and second-row atoms-with three distinct density functional approximations: SPW92, a form of local-density approximation; B3LYP, a global hybrid generalized gradient approximation; and B97M-V, a meta-generalized gradient approximation with nonlocal correlation. We have found that it is remarkably difficult to reach the basis set limit; for the methods and systems examined, the most complete basis is Jensen's pc-4. The Dunning correlation-consistent sequence of basis sets converges slowly relative to the Jensen sequence. The Karlsruhe basis sets are quite cost effective, particularly when a correction for basis set superposition error is applied: counterpoise-corrected def2-SVPD binding energies are better than corresponding energies computed in comparably sized Dunning and Jensen bases, and on par with uncorrected results in basis sets 3-4 times larger. These trends are exhibited regardless of the level of density functional approximation employed. A sense of the magnitude of the intrinsic incompleteness error of each basis set not only provides a foundation for guiding basis set choice in future studies but also facilitates quantitative comparison of existing studies on similar types of systems. PMID:27208948

  4. Comparative inter-strain sequence analysis of the putative regulatory region of murine psychostimulant-regulated gene GNB1 (G protein beta 1 subunit gene).

    PubMed

    Kitanaka, Nobue; Kitanaka, Junichi; Walther, Donna; Wang, Xiao-Bing; Uhl, George R

    2003-08-01

    We isolated a cDNA clone from a murine genomic library of C57BL/6 strain, carrying 13.8 kb of nucleotides including exon 1 of heterotrimeric GTP-binding protein beta 1 subunit gene (genetic symbol, GNB1) and 10.6 kb of the 5' flanking region. Sequence comparison with GNB1 gene locus from 129Sv strain revealed a 0.2% divergence in a 13.2 kb common region between these two strains. The divergence consisted of eight single nucleotide polymorphisms, three insertions and one deletion, with 129Sv used as the reference. The exon 1 and the putative regulation elements, such as cyclic AMP response element, AP1, AP2, Sp1 and nuclear factor-kappa B recognition sites, were perfectly conserved. The expression of GNB1 mRNA was significantly increased in mouse striatum 2 h after single methamphetamine administration with an approximately 150% expression level compared with the basal level. In contrast, no change in the expression level was observed in the cerebral cortex. After the chronic methamphetamine treatment regimen, the expression level of GNB1 mRNA did not change in any brain regions examined. These results suggest (1) that the 5' flanking nucleotide sequence of GNB1 gene was strictly conserved for its possible contribution to the same change in the expression level between the mouse strains in response to psychostimulants and (2) that the initial process of development of behavioral sensitization appeared to occur parallel to the significant increase in the expression level of GNB1 gene in the mouse striatum. PMID:14631649

  5. Regulatory RNAs

    PubMed Central

    Vazquez-Anderson, Jorge; Contreras, Lydia M

    2013-01-01

    RNAs have many important functional properties, including that they are independently controllable and highly tunable. As a result of these advantageous properties, their use in a myriad of sophisticated devices has been widely explored. Yet, the exploitation of RNAs for synthetic applications is highly dependent on the ability to characterize the many new molecules that continue to be discovered by large-scale sequencing and high-throughput screening techniques. In this review, we present an exhaustive survey of the most recent synthetic bacterial riboswitches and small RNAs while emphasizing their virtues in gene expression management. We also explore the use of these RNA components as building blocks in the RNA synthetic biology toolbox and discuss examples of synthetic RNA components used to rewire bacterial regulatory circuitry. We anticipate that this field will expand its catalog of smart devices by mimicking and manipulating natural RNA mechanisms and functions. PMID:24356572

  6. Regulation of Synthesis of the Branched-Chain Amino Acids and Cognate Aminoacyl-Transfer Ribonucleic Acid Synthetases of Escherichia coli: a Common Regulatory Element

    PubMed Central

    Jackson, Julius; Williams, L. S.; Umbarger, H. E.

    1974-01-01

    Regulation of isoleucine, valine, and leucine biosynthesis and isoleucyl-, valyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined in two mutant strains of Escherichia coli. One mutant was selected for growth resistance to the isoleucine analogue, ketomycin, and the other was selected for growth resistance to both trifluoroleucine and valine. Control of the synthesis of the branched-chain amino acids by repression was altered in both of these mutants. They also exhibited altered control of formation of isoleucyl-tRNA synthetase (EC 6.1.15, isoleucine:sRNA ligase, AMP), valyl-tRNA synthetase (EC 6.1.1.9, valine:sRNA ligase, AMP), and leucyl-tRNA synthetase (EC 6.1.1.4, leucine:sRNA ligase, AMP). These results suggest the existence of a common element for the control of these two classes of enzymes in Escherichia coli. PMID:4612020

  7. Dynamic control of nutrient-removal from industrial wastewater in a sequencing batch reactor, using common and low-cost online sensors.

    PubMed

    Dries, Jan

    2016-01-01

    On-line control of the biological treatment process is an innovative tool to cope with variable concentrations of chemical oxygen demand and nutrients in industrial wastewater. In the present study we implemented a simple dynamic control strategy for nutrient-removal in a sequencing batch reactor (SBR) treating variable tank truck cleaning wastewater. The control system was based on derived signals from two low-cost and robust sensors that are very common in activated sludge plants, i.e. oxidation reduction potential (ORP) and dissolved oxygen. The amount of wastewater fed during anoxic filling phases, and the number of filling phases in the SBR cycle, were determined by the appearance of the 'nitrate knee' in the profile of the ORP. The phase length of the subsequent aerobic phases was controlled by the oxygen uptake rate measured online in the reactor. As a result, the sludge loading rate (F/M ratio), the volume exchange rate and the SBR cycle length adapted dynamically to the activity of the activated sludge and the actual characteristics of the wastewater, without affecting the final effluent quality. PMID:26901715

  8. Dynamic control of nutrient-removal from industrial wastewater in a sequencing batch reactor, using common and low-cost online sensors.

    PubMed

    Dries, Jan

    2016-01-01

    On-line control of the biological treatment process is an innovative tool to cope with variable concentrations of chemical oxygen demand and nutrients in industrial wastewater. In the present study we implemented a simple dynamic control strategy for nutrient-removal in a sequencing batch reactor (SBR) treating variable tank truck cleaning wastewater. The control system was based on derived signals from two low-cost and robust sensors that are very common in activated sludge plants, i.e. oxidation reduction potential (ORP) and dissolved oxygen. The amount of wastewater fed during anoxic filling phases, and the number of filling phases in the SBR cycle, were determined by the appearance of the 'nitrate knee' in the profile of the ORP. The phase length of the subsequent aerobic phases was controlled by the oxygen uptake rate measured online in the reactor. As a result, the sludge loading rate (F/M ratio), the volume exchange rate and the SBR cycle length adapted dynamically to the activity of the activated sludge and the actual characteristics of the wastewater, without affecting the final effluent quality.

  9. Sequence and Expression Analysis of Interferon Regulatory Factor 10 (IRF10) in Three Diverse Teleost Fish Reveals Its Role in Antiviral Defense

    PubMed Central

    Xu, Qiaoqing; Jiang, Yousheng; Wangkahart, Eakapol; Zou, Jun; Chang, Mingxian; Yang, Daiqin; Secombes, Chris J.; Nie, Pin; Wang, Tiehui

    2016-01-01

    Background Interferon regulatory factor (IRF) 10 was first found in birds and is present in the genome of other tetrapods (but not humans and mice), as well as in teleost fish. The functional role of IRF10 in vertebrate immunity is relatively unknown compared to IRF1-9. The target of this research was to clone and characterize the IRF10 genes in three economically important fish species that will facilitate future evaluation of this molecule in fish innate and adaptive immunity. Molecular Characterization of IRF10 in Three Fish Species In the present study, a single IRF10 gene was cloned in grass carp Ctenopharyngodon idella and Asian swamp eel Monopterus albus, and two, named IRF10a and IRF10b, in rainbow trout Oncorhynchus mykiss. The fish IRF10 molecules share highest identities to other vertebrate IRF10s, and have a well conserved DNA binding domain, IRF-associated domain, and an 8 exon/7 intron structure with conserved intron phase. The presence of an upstream ATG or open reading frame (ORF) in the 5’-untranslated region of different fish IRF10 cDNA sequences suggests potential regulation at the translational level, and this has been verified by in vitro transcription/translation experiments of the trout IRF10a cDNA, but would still need to be validated in fish cells. Expression Analysis of IRF10 In Vivo and In Vitro Both trout IRF10 paralogues are highly expressed in thymus, blood and spleen but are relatively low in head kidney and caudal kidney. Trout IRF10b expression is significantly higher than IRF10a in integumentary tissues i.e. gills, scales, skin, intestine, adipose fin and tail fins, suggesting that IRF10b may be more important in mucosal immunity. The expression of both trout IRF10 paralogues is up-regulated by recombinant IFN-γ. The expression of the IRF10 genes is highly induced by Poly I:C in vitro and in vivo, and by viral infection, but is less responsive to peptidoglycan and bacterial infection, suggesting an important role of fish IRF10

  10. PCR detection and DNA sequence analysis of the regulatory region of lymphotropic papovavirus in peripheral blood mononuclear cells of an immunocompromised rhesus macaque

    NASA Technical Reports Server (NTRS)

    Lednicky, John A.; Halvorson, Steven J.; Butel, Janet S.

    2002-01-01

    A lymphotropic papovavirus (LPV) archetypal regulatory region was amplified from DNA from the blood of an immunocompromised rhesus monkey. We believe this is the first nonserological evidence of LPV infection in rhesus monkeys.

  11. A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements.

    PubMed

    Henry, Kelli F; Kawashima, Tomokazu; Goldberg, Robert B

    2015-06-01

    Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean (Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we use site-directed mutagenesis experiments in transgenic tobacco globular-stage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. A homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.

  12. Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the Pisum sativum (pea) chlorophyl a/b binding protein gene AB80: identification of a repeated heptamer motif.

    PubMed

    Argüello, G; García-Hernández, E; Sánchez, M; Gariglio, P; Herrera-Estrella, L; Simpson, J

    1992-05-01

    Two protein factors binding to the regulatory region of the pea chlorophyl a/b binding protein gene AB80 have been identified. One of these factors is found only in green tissue but not in etiolated or root tissue. The second factor (denominated ABF-2) binds to a DNA sequence element that contains a direct heptamer repeat TCTCAAA. It was found that presence of both of the repeats is essential for binding. ABF-2 is present in both green and etiolated tissue and in roots and factors analogous to ABF-2 are present in several plant species. Computer analysis showed that the TCTCAAA motif is present in the regulatory region of several plant genes. PMID:1303797

  13. Integration of bioinformatics and synthetic promoters leads to the discovery of novel elicitor-responsive cis-regulatory sequences in Arabidopsis.

    PubMed

    Koschmann, Jeannette; Machens, Fabian; Becker, Marlies; Niemeyer, Julia; Schulze, Jutta; Bülow, Lorenz; Stahl, Dietmar J; Hehl, Reinhard

    2012-09-01

    A combination of bioinformatic tools, high-throughput gene expression profiles, and the use of synthetic promoters is a powerful approach to discover and evaluate novel cis-sequences in response to specific stimuli. With Arabidopsis (Arabidopsis thaliana) microarray data annotated to the PathoPlant database, 732 different queries with a focus on fungal and oomycete pathogens were performed, leading to 510 up-regulated gene groups. Using the binding site estimation suite of tools, BEST, 407 conserved sequence motifs were identified in promoter regions of these coregulated gene sets. Motif similarities were determined with STAMP, classifying the 407 sequence motifs into 37 families. A comparative analysis of these 37 families with the AthaMap, PLACE, and AGRIS databases revealed similarities to known cis-elements but also led to the discovery of cis-sequences not yet implicated in pathogen response. Using a parsley (Petroselinum crispum) protoplast system and a modified reporter gene vector with an internal transformation control, 25 elicitor-responsive cis-sequences from 10 different motif families were identified. Many of the elicitor-responsive cis-sequences also drive reporter gene expression in an Agrobacterium tumefaciens infection assay in Nicotiana benthamiana. This work significantly increases the number of known elicitor-responsive cis-sequences and demonstrates the successful integration of a diverse set of bioinformatic resources combined with synthetic promoter analysis for data mining and functional screening in plant-pathogen interaction. PMID:22744985

  14. Integration of Bioinformatics and Synthetic Promoters Leads to the Discovery of Novel Elicitor-Responsive cis-Regulatory Sequences in Arabidopsis1[C][W][OA

    PubMed Central

    Koschmann, Jeannette; Machens, Fabian; Becker, Marlies; Niemeyer, Julia; Schulze, Jutta; Bülow, Lorenz; Stahl, Dietmar J.; Hehl, Reinhard

    2012-01-01

    A combination of bioinformatic tools, high-throughput gene expression profiles, and the use of synthetic promoters is a powerful approach to discover and evaluate novel cis-sequences in response to specific stimuli. With Arabidopsis (Arabidopsis thaliana) microarray data annotated to the PathoPlant database, 732 different queries with a focus on fungal and oomycete pathogens were performed, leading to 510 up-regulated gene groups. Using the binding site estimation suite of tools, BEST, 407 conserved sequence motifs were identified in promoter regions of these coregulated gene sets. Motif similarities were determined with STAMP, classifying the 407 sequence motifs into 37 families. A comparative analysis of these 37 families with the AthaMap, PLACE, and AGRIS databases revealed similarities to known cis-elements but also led to the discovery of cis-sequences not yet implicated in pathogen response. Using a parsley (Petroselinum crispum) protoplast system and a modified reporter gene vector with an internal transformation control, 25 elicitor-responsive cis-sequences from 10 different motif families were identified. Many of the elicitor-responsive cis-sequences also drive reporter gene expression in an Agrobacterium tumefaciens infection assay in Nicotiana benthamiana. This work significantly increases the number of known elicitor-responsive cis-sequences and demonstrates the successful integration of a diverse set of bioinformatic resources combined with synthetic promoter analysis for data mining and functional screening in plant-pathogen interaction. PMID:22744985

  15. CRISPRs of Enterococcus faecalis and E. hirae isolates from pig feces have species-specific repeats but share some common spacer sequences.

    PubMed

    Katyal, Isha; Chaban, Bonnie; Ng, Beata; Hill, Janet E

    2013-07-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) are currently a topic of interest in microbiology due to their role as a prokaryotic immune system. Investigations of CRISPR distribution and characterization to date have focused on pathogenic bacteria, while less is known about CRISPR in commensal bacteria, where they may have a significant role in the ecology of the microbiota of humans and other animals, and act as a recorder of interactions between bacteria and viruses. A combination of PCR and sequencing was used to determine prevalence and distribution of CRISPR arrays in Enterococcus faecalis and Enterococcus hirae isolates from the feces of healthy pigs. Both type II CRISPR-Cas and Orphan CRISPR (without Cas genes) were detected in the 195 isolates examined. CRISPR-Cas was detected in 52 (46/88) and 42 % (45/107) E. faecalis and E. hirae isolates, respectively. The prevalence of Orphan CRISPR arrays was higher in E. faecalis isolates (95 %, 84/88) compared with E. hirae isolates (49 %, 53/107). Species-specific repeat sequences were identified in Orphan CRISPR arrays, and 42 unique spacer sequences were identified. Only two spacers matched previously characterized pig virome sequences, and many were apparently derived from chromosomal sequences of enterococci. Surprisingly, 17 (40 %) of the spacers were detected in both species. Shared spacer sequences are evidence of a lack of species specificity in the agents and mechanisms responsible for integration of spacers, and the abundance of spacer sequences corresponding to bacterial chromosomal sequences reflects interspecific interactions within the intestinal microbiota.

  16. Exploration of noncoding sequences in metagenomes.

    PubMed

    Tobar-Tosse, Fabián; Rodríguez, Adrián C; Vélez, Patricia E; Zambrano, María M; Moreno, Pedro A

    2013-01-01

    Environment-dependent genomic features have been defined for different metagenomes, whose genes and their associated processes are related to specific environments. Identification of ORFs and their functional categories are the most common methods for association between functional and environmental features. However, this analysis based on finding ORFs misses noncoding sequences and, therefore, some metagenome regulatory or structural information could be discarded. In this work we analyzed 23 whole metagenomes, including coding and noncoding sequences using the following sequence patterns: (G+C) content, Codon Usage (Cd), Trinucleotide Usage (Tn), and functional assignments for ORF prediction. Herein, we present evidence of a high proportion of noncoding sequences discarded in common similarity-based methods in metagenomics, and the kind of relevant information present in those. We found a high density of trinucleotide repeat sequences (TRS) in noncoding sequences, with a regulatory and adaptive function for metagenome communities. We present associations between trinucleotide values and gene function, where metagenome clustering correlate with microorganism adaptations and kinds of metagenomes. We propose here that noncoding sequences have relevant information to describe metagenomes that could be considered in a whole metagenome analysis in order to improve their organization, classification protocols, and their relation with the environment. PMID:23536879

  17. Exploration of noncoding sequences in metagenomes.

    PubMed

    Tobar-Tosse, Fabián; Rodríguez, Adrián C; Vélez, Patricia E; Zambrano, María M; Moreno, Pedro A

    2013-01-01

    Environment-dependent genomic features have been defined for different metagenomes, whose genes and their associated processes are related to specific environments. Identification of ORFs and their functional categories are the most common methods for association between functional and environmental features. However, this analysis based on finding ORFs misses noncoding sequences and, therefore, some metagenome regulatory or structural information could be discarded. In this work we analyzed 23 whole metagenomes, including coding and noncoding sequences using the following sequence patterns: (G+C) content, Codon Usage (Cd), Trinucleotide Usage (Tn), and functional assignments for ORF prediction. Herein, we present evidence of a high proportion of noncoding sequences discarded in common similarity-based methods in metagenomics, and the kind of relevant information present in those. We found a high density of trinucleotide repeat sequences (TRS) in noncoding sequences, with a regulatory and adaptive function for metagenome communities. We present associations between trinucleotide values and gene function, where metagenome clustering correlate with microorganism adaptations and kinds of metagenomes. We propose here that noncoding sequences have relevant information to describe metagenomes that could be considered in a whole metagenome analysis in order to improve their organization, classification protocols, and their relation with the environment.

  18. Genetic analysis of bristle loss in hybrids between Drosophila melanogaster and D. simulans provides evidence for divergence of cis-regulatory sequences in the achaete-scute gene complex.

    PubMed

    Skaer, N; Simpson, P

    2000-05-01

    The two closely related species of Drosophila, D. melanogaster and D. simulans, display an identical bristle pattern on the notum, but hybrids between the two are lacking a variable number of bristles. We show that the loss is temperature-dependent and provide evidence for two periods of temperature sensitivity. A first period of heat sensitivity occurs during larval development and corresponds to the time when the prepattern of expression of genes whose products activate achaete-scute in the proneural clusters preceding bristle precursor formation is established. A second period of cold sensitivity corresponds to the time of emergence of the bristle precursor cells and the maintenance of their neural fate, a process requiring high levels of Achaete-Scute. Expression of achaete-scute at these two critical periods depends on cis-regulatory elements of the achaete-scute complex (AS-C). The differences between males, which have only one copy of the X-linked AS-C from D. simulans, and females, which have copies from both parental species, are compared, together with the effects of crossing in different rearrangements of the D. melanogaster AS-C that delete regulatory and/or coding sequences. We provide evidence that bristle loss in the hybrids may result from a decrease in the level of transcription at the AS-C and argue that interaction between trans-acting factors and cis-regulatory elements within the AS-C has diverged between the two species.

  19. Commonly-occurring polymorphisms in the COMT, DRD1 and DRD2 genes influence different aspects of motor sequence learning in humans.

    PubMed

    Baetu, Irina; Burns, Nicholas R; Urry, Kristi; Barbante, Girolamo Giovanni; Pitcher, Julia B

    2015-11-01

    Performing sequences of movements is a ubiquitous skill that involves dopamine transmission. However, it is unclear which components of the dopamine system contribute to which aspects of motor sequence learning. Here we used a genetic approach to investigate the relationship between different components of the dopamine system and specific aspects of sequence learning in humans. In particular, we investigated variations in genes that code for the catechol-O-methyltransferase (COMT) enzyme, the dopamine transporter (DAT) and dopamine D1 and D2 receptors (DRD1 and DRD2). COMT and the DAT regulate dopamine availability in the prefrontal cortex and the striatum, respectively, two key regions recruited during learning, whereas dopamine D1 and D2 receptors are thought to be involved in long-term potentiation and depression, respectively. We show that polymorphisms in the COMT, DRD1 and DRD2 genes differentially affect behavioral performance on a sequence learning task in 161 Caucasian participants. The DRD1 polymorphism predicted the ability to learn new sequences, the DRD2 polymorphism predicted the ability to perform a previously learnt sequence after performing interfering random movements, whereas the COMT polymorphism predicted the ability to switch flexibly between two sequences. We used computer simulations to explore potential mechanisms underlying these effects, which revealed that the DRD1 and DRD2 effects are possibly related to neuroplasticity. Our prediction-error algorithm estimated faster rates of connection strengthening in genotype groups with presumably higher D1 receptor densities, and faster rates of connection weakening in genotype groups with presumably higher D2 receptor densities. Consistent with current dopamine theories, these simulations suggest that D1-mediated neuroplasticity contributes to learning to select appropriate actions, whereas D2-mediated neuroplasticity is involved in learning to inhibit incorrect action plans. However, the

  20. A CRE/ATF-like site in the upstream regulatory sequence of the human interleukin 1 beta gene is necessary for induction in U937 and THP-1 monocytic cell lines.

    PubMed Central

    Gray, J G; Chandra, G; Clay, W C; Stinnett, S W; Haneline, S A; Lorenz, J J; Patel, I R; Wisely, G B; Furdon, P J; Taylor, J D

    1993-01-01

    Transfection of U937 and THP-1 cells with a recombinant plasmid, pIL1(4.0kb)-CAT, containing 4 kb of the interleukin 1 beta (IL-1 beta) gene upstream regulatory sequence resulted in inducer-dependent expression of chloramphenicol acetyltransferase activity. Treatment of the transfected cells with various combinations of the inducers lipopolysaccharide, phorbol myristate acetate, and dibutyryl cyclic AMP upregulated the IL-1 beta promoter. In U937 and THP-1 cells, maximum stimulation of both the endogenous IL-1 beta gene and pIL1(4.0kb)-CAT transfectants was observed following treatment with the combination of inducing agents lipopolysaccharide-phorbol myristate acetate-dibutyryl cyclic AMP. This combination of inducing agents was used to identify and study, at the molecular level, some of the regulatory elements necessary for induction of the IL-1 beta gene. A series of 5' deletion derivatives of the upstream regulatory sequence were used in transient transfection assays to identify an 80-bp fragment located between -2720 and -2800 bp upstream of the mRNA start site that was required for induction. Exonuclease III mapping, electrophoretic mobility shift assays (EMSA), and DNA sequence analysis of this region were used to identify a transcription factor binding sequence which contained a potential cyclic AMP response element (CRE/ATF)- and NF-kappa B-like binding site. Site-directed mutagenesis of the CRE/ATF-like site resulted in the loss of binding of a specific factor or factors as determined by EMSA. The loss of binding activity directly correlated with a loss of approximately 75% of promoter activity as determined in transient transfection assays. As determined by EMSA, the factor binding to the CRE/ATF-like site was present in nuclear extracts prepared from both uninduced and induced THP-1 and U937 cells. However, the intensity of the band appeared to be increased when nuclear extracts from induced cells were used. In contrast to the CRE/ATF mutation, which

  1. Cofunctional Subpathways Were Regulated by Transcription Factor with Common Motif, Common Family, or Common Tissue

    PubMed Central

    Su, Fei; Shang, Desi; Xu, Yanjun; Feng, Li; Yang, Haixiu; Liu, Baoquan; Su, Shengyang; Chen, Lina; Li, Xia

    2015-01-01

    Dissecting the characteristics of the transcription factor (TF) regulatory subpathway is helpful for understanding the TF underlying regulatory function in complex biological systems. To gain insight into the influence of TFs on their regulatory subpathways, we constructed a global TF-subpathways network (TSN) to analyze systematically the regulatory effect of common-motif, common-family, or common-tissue TFs on subpathways. We performed cluster analysis to show that the common-motif, common-family, or common-tissue TFs that regulated the same pathway classes tended to cluster together and contribute to the same biological function that led to disease initiation and progression. We analyzed the Jaccard coefficient to show that the functional consistency of subpathways regulated by the TF pairs with common motif, common family, or common tissue was significantly greater than the random TF pairs at the subpathway level, pathway level, and pathway class level. For example, HNF4A (hepatocyte nuclear factor 4, alpha) and NR1I3 (nuclear receptor subfamily 1, group I, member 3) were a pair of TFs with common motif, common family, and common tissue. They were involved in drug metabolism pathways and were liver-specific factors required for physiological transcription. In short, we inferred that the cofunctional subpathways were regulated by common-motif, common-family, or common-tissue TFs. PMID:26688819

  2. Development of 65 novel polymorphic cDNA-SSR markers in common vetch (Vicia sativa subsp. sativa) using next generation sequencing.

    PubMed

    Chung, Jong-Wook; Kim, Tae-Sung; Suresh, Sundan; Lee, Sok-Young; Cho, Gyu-Taek

    2013-07-16

    Vetch (Vicia sativa L.) is one of the most important annual forage legumes in the World due to its multiple uses (i.e., hay, grain, silage and green manure) and high nutritional value. However, detrimental cyanoalanine toxins in its plant parts including seeds and its vulnerability to hard winter conditions are currently reducing the agronomic values of vetch varieties. Moreover, the existence in the public domain of very few genomic resources, especially molecular markers, has further hampered breeding efforts. Polymorphic simple sequence repeat markers from transcript sequences (cDNA; simple sequence repeat [SSR]) were developed for Vicia sativa subsp. sativa. We found 3,811 SSR loci from 31,504 individual sequence reads, and 300 primer pairs were designed and synthesized. In total, 65 primer pairs were found to be consistently scorable when 32 accessions were tested. The numbers of alleles ranged from 2 to 19, frequency of major alleles per locus were 0.27-0.87, the genotype number was 2-19, the overall polymorphism information content (PIC) values were 0.20-0.86, and the observed and expected heterozygosity values were 0.00-0.41 and 0.264-0.852, respectively. These markers provide a useful tool for assessing genetic diversity, population structure, and positional cloning, facilitating vetch breeding programs.

  3. Complete Genome Sequence of Klebsiella pneumoniae Strain ATCC 43816 KPPR1, a Rifampin-Resistant Mutant Commonly Used in Animal, Genetic, and Molecular Biology Studies.

    PubMed

    Broberg, Christopher A; Wu, Weisheng; Cavalcoli, James D; Miller, Virginia L; Bachman, Michael A

    2014-09-25

    Klebsiella pneumoniae is an urgent public health threat due to the spread of carbapenem-resistant strains causing serious, and frequently fatal, infections. To facilitate genetic, molecular, and immunological studies of this pathogen, we report the complete chromosomal sequence of a genetically tractable, prototypical strain used in animal models.

  4. Border sequences of Medicago truncatula CLE36 are specifically cleaved by endoproteases common to the extracellular fluids of Medicago and soybean.

    PubMed

    Djordjevic, Michael A; Oakes, Marie; Wong, Chui E; Singh, Mohan; Bhalla, Prem; Kusumawati, Lucia; Imin, Nijat

    2011-08-01

    CLE (CLAVATA3/ESR-related) peptides are developmental regulators that are secreted into the apoplast. Little is known about the role of the sequences that flank CLE peptides in terms of their biological activity or how they are targeted by proteases that are known to liberate the final active CLE peptides from their precursor sequences. The biological activity of Medicago truncatula CLE36, which possesses broadly conserved border sequences flanking the putative final active CLE36 peptide product, was assessed. Using in vitro root growth assays and an in vitro root and callus formation assay it is shown that CLE36 peptides of different lengths possess differential biological activities. Using mass spectrometry, Glycine max and Medicago extracellular fluids were each shown to possess an endoproteolytic activity that recognizes and cleaves at border sequences in a synthetic 31 amino acid CLE36 'propeptide bait' to liberate biologically active peptide products. Inhibitor studies suggest that a subtilisin, in combination with a carboxypeptidase, liberated and trimmed CLE36, respectively, to form biologically relevant 11-15 amino acid cleavage products. The 15 amino acid cleavage product is more biologically potent on Arabidopsis than shorter or longer CLE peptides. In situ hybridization shows that the soybean orthologue of CLE36 (GmCLE34) is expressed in the provascular tissue. The results suggest that secreted subtilisins can specifically recognize the border sequences of CLE36 propeptides and liberate biologically active cleavage products. These secreted proteases may affect the stability and biological activity of CLE peptides in the apoplast or be involved in CLE36 processing.

  5. RNA Sequencing of Laser-Capture Microdissected Compartments of the Maize Kernel Identifies Regulatory Modules Associated with Endosperm Cell Differentiation[OPEN

    PubMed Central

    Zhan, Junpeng; Thakare, Dhiraj; Ma, Chuang; Lloyd, Alan; Nixon, Neesha M.; Arakaki, Angela M.; Burnett, William J.; Logan, Kyle O.; Wang, Dongfang; Wang, Xiangfeng; Drews, Gary N.; Yadegari, Ramin

    2015-01-01

    Endosperm is an absorptive structure that supports embryo development or seedling germination in angiosperms. The endosperm of cereals is a main source of food, feed, and industrial raw materials worldwide. However, the genetic networks that regulate endosperm cell differentiation remain largely unclear. As a first step toward characterizing these networks, we profiled the mRNAs in five major cell types of the differentiating endosperm and in the embryo and four maternal compartments of the maize (Zea mays) kernel. Comparisons of these mRNA populations revealed the diverged gene expression programs between filial and maternal compartments and an unexpected close correlation between embryo and the aleurone layer of endosperm. Gene coexpression network analysis identified coexpression modules associated with single or multiple kernel compartments including modules for the endosperm cell types, some of which showed enrichment of previously identified temporally activated and/or imprinted genes. Detailed analyses of a coexpression module highly correlated with the basal endosperm transfer layer (BETL) identified a regulatory module activated by MRP-1, a regulator of BETL differentiation and function. These results provide a high-resolution atlas of gene activity in the compartments of the maize kernel and help to uncover the regulatory modules associated with the differentiation of the major endosperm cell types. PMID:25783031

  6. Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo.

    PubMed

    Eskin, Julian A; Rankova, Aneliya; Johnston, Adam B; Alioto, Salvatore L; Goode, Bruce L

    2016-03-01

    Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1's functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles.

  7. High density genome wide genotyping-by-sequencing and association identifies common and low frequency SNPs, and novel candidate genes influencing cow milk traits

    PubMed Central

    Ibeagha-Awemu, Eveline M.; Peters, Sunday O.; Akwanji, Kingsley A.; Imumorin, Ikhide G.; Zhao, Xin

    2016-01-01

    High-throughput sequencing technologies have increased the ability to detect sequence variations for complex trait improvement. A high throughput genome wide genotyping-by-sequencing (GBS) method was used to generate 515,787 single nucleotide polymorphisms (SNPs), from which 76,355 SNPs with call rates >85% and minor allele frequency ≥1.5% were used in genome wide association study (GWAS) of 44 milk traits in 1,246 Canadian Holstein cows. GWAS was accomplished with a mixed linear model procedure implementing the additive and dominant models. A strong signal within the centromeric region of bovine chromosome 14 was associated with test day fat percentage. Several SNPs were associated with eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, CLA:9c11t and gamma linolenic acid. Most of the significant SNPs for 44 traits studied are novel and located in intergenic regions or introns of genes. Novel potential candidate genes for milk traits or mammary gland functions include ERCC6, TONSL, NPAS2, ACER3, ITGB4, GGT6, ACOX3, MECR, ADAM12, ACHE, LRRC14, FUK, NPRL3, EVL, SLCO3A1, PSMA4, FTO, ADCK5, PP1R16A and TEP1. Our study further demonstrates the utility of the GBS approach for identifying population-specific SNPs for use in improvement of complex dairy traits. PMID:27506634

  8. High density genome wide genotyping-by-sequencing and association identifies common and low frequency SNPs, and novel candidate genes influencing cow milk traits.

    PubMed

    Ibeagha-Awemu, Eveline M; Peters, Sunday O; Akwanji, Kingsley A; Imumorin, Ikhide G; Zhao, Xin

    2016-01-01

    High-throughput sequencing technologies have increased the ability to detect sequence variations for complex trait improvement. A high throughput genome wide genotyping-by-sequencing (GBS) method was used to generate 515,787 single nucleotide polymorphisms (SNPs), from which 76,355 SNPs with call rates >85% and minor allele frequency ≥1.5% were used in genome wide association study (GWAS) of 44 milk traits in 1,246 Canadian Holstein cows. GWAS was accomplished with a mixed linear model procedure implementing the additive and dominant models. A strong signal within the centromeric region of bovine chromosome 14 was associated with test day fat percentage. Several SNPs were associated with eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, CLA:9c11t and gamma linolenic acid. Most of the significant SNPs for 44 traits studied are novel and located in intergenic regions or introns of genes. Novel potential candidate genes for milk traits or mammary gland functions include ERCC6, TONSL, NPAS2, ACER3, ITGB4, GGT6, ACOX3, MECR, ADAM12, ACHE, LRRC14, FUK, NPRL3, EVL, SLCO3A1, PSMA4, FTO, ADCK5, PP1R16A and TEP1. Our study further demonstrates the utility of the GBS approach for identifying population-specific SNPs for use in improvement of complex dairy traits. PMID:27506634

  9. Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo

    PubMed Central

    Eskin, Julian A.; Rankova, Aneliya; Johnston, Adam B.; Alioto, Salvatore L.; Goode, Bruce L.

    2016-01-01

    Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1’s functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles. PMID:26764093

  10. Multiple regulatory mechanisms of hepatocyte growth factor expression in malignant cells with a short poly(dA) sequence in the HGF gene promoter.

    PubMed

    Sakai, Kazuko; Takeda, Masayuki; Okamoto, Isamu; Nakagawa, Kazuhiko; Nishio, Kazuto

    2015-01-01

    Hepatocyte growth factor (HGF) expression is a poor prognostic factor in various types of cancer. Expression levels of HGF have been reported to be regulated by shorter poly(dA) sequences in the promoter region. In the present study, the poly(dA) mononucleotide tract in various types of human cancer cell lines was examined and compared with the HGF expression levels in those cells. Short deoxyadenosine repeat sequences were detected in five of the 55 cell lines used in the present study. The H69, IM95, CCK-81, Sui73 and H28 cells exhibited a truncated poly(dA) sequence in which the number of poly(dA) repeats was reduced by ≥5 bp. Two of the cell lines exhibited high HGF expression, determined by reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The CCK-81, Sui73 and H28 cells with shorter poly(dA) sequences exhibited low HGF expression. The cause of the suppression of HGF expression in the CCK-81, Sui73 and H28 cells was clarified by two approaches, suppression by methylation and single nucleotide polymorphisms in the HGF gene. Exposure to 5-Aza-dC, an inhibitor of DNA methyltransferase 1, induced an increased expression of HGF in the CCK-81 cells, but not in the other cells. Single-nucleotide polymorphism (SNP) rs72525097 in intron 1 was detected in the Sui73 and H28 cells. Taken together, it was found that the defect of poly(dA) in the HGF promoter was present in various types of cancer, including lung, stomach, colorectal, pancreas and mesothelioma. The present study proposes the negative regulation mechanisms by methylation and SNP in intron 1 of HGF for HGF expression in cancer cells with short poly(dA).

  11. Deep Sequencing-Based Transcriptome Analysis Reveals the Regulatory Mechanism of Bemisia tabaci (Hemiptera: Aleyrodidae) Nymph Parasitized by Encarsia sophia (Hymenoptera: Aphelinidae)

    PubMed Central

    Wang, Ran; Li, Fei; Zhang, Fan; Wang, Su

    2016-01-01

    The whitefly Bemisia tabaci is a genetically diverse complex with multiple cryptic species, and some are the most destructive invasive pests of many ornamentals and crops worldwide. Encarsia sophia is an autoparasitoid wasp that demonstrated high efficiency as bio-control agent of whiteflies. However, the immune mechanism of B. tabaci parasitization by E. sophia is unknown. In order to investigate immune response of B. tabaci to E. Sophia parasitization, the transcriptome of E. sophia parasitized B. tabaci nymph was sequenced by Illumina sequencing. De novo assembly generated 393,063 unigenes with average length of 616 bp, in which 46,406 unigenes (15.8% of all unigenes) were successfully mapped. Parasitization by E. sophia had significant effects on the transcriptome profile of B. tabaci nymph. A total of 1482 genes were significantly differentially expressed, of which 852 genes were up-regulated and 630 genes were down-regulated. These genes were mainly involved in immune response, development, metabolism and host signaling pathways. At least 52 genes were found to be involved in the host immune response, 33 genes were involved in the development process, and 29 genes were involved in host metabolism. Taken together, the assembled and annotated transcriptome sequences provided a valuable genomic resource for further understanding the molecular mechanism of immune response of B. tabaci parasitization by E. sophia. PMID:27332546

  12. The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit, and characterization of transcriptional and post-transcriptional regulatory sequences.

    PubMed

    Shima, D T; Kuroki, M; Deutsch, U; Ng, Y S; Adamis, A P; D'Amore, P A

    1996-02-16

    We describe the genomic organization and functional characterization of the mouse gene encoding vascular endothelial growth factor (VEGF), a polypeptide implicated in embryonic vascular development and postnatal angiogenesis. The coding region for mouse VEGF is interrupted by seven introns and encompasses approximately 14 kilobases. Organization of exons suggests that, similar to the human VEGF gene, alternative splicing generates the 120-, 164-, and 188-amino acid isoforms, but does not predict a fourth VEGF isoform corresponding to human VEGF206. Approximately 1. 2 kilobases of 5'-flanking region have been sequenced, and primer extension analysis identified a single major transcription initiation site, notably lacking TATA or CCAT consensus sequences. The 5'-flanking region is sufficient to promote a 7-fold induction of basal transcription. The genomic region encoding the 3'-untranslated region was determined by Northern and nuclease mapping analysis. Investigation of mRNA sequences responsible for the rapid turnover of VEGF mRNA (mRNA half-life, <1 h) (Shima, D. T. , Deutsch, U., and D'Amore, P. A. (1995) FEBS Lett. 370, 203-208) revealed that the 3'-untranslated region was sufficient to trigger the rapid turnover of a normally long-lived reporter mRNA in vitro. These data and reagents will allow the molecular and genetic analysis of mechanisms that control the developmental and pathological expression of VEGF.

  13. Multiple ESBL-Producing Escherichia coli Sequence Types Carrying Quinolone and Aminoglycoside Resistance Genes Circulating in Companion and Domestic Farm Animals in Mwanza, Tanzania, Harbor Commonly Occurring Plasmids.

    PubMed

    Seni, Jeremiah; Falgenhauer, Linda; Simeo, Nabina; Mirambo, Mariam M; Imirzalioglu, Can; Matee, Mecky; Rweyemamu, Mark; Chakraborty, Trinad; Mshana, Stephen E

    2016-01-01

    The increased presence of extended-spectrum beta-lactamase (ESBL)-producing bacteria in humans, animals, and their surrounding environments is of global concern. Currently there is limited information on ESBL presence in rural farming communities worldwide. We performed a cross-sectional study in Mwanza, Tanzania, involving 600 companion and domestic farm animals between August/September 2014. Rectal swab/cloaca specimens were processed to identify ESBL-producing Enterobacteriaceae. We detected 130 (21.7%) animals carrying ESBL-producing bacteria, the highest carriage being among dogs and pigs [39.2% (51/130) and 33.1% (43/130), respectively]. The majority of isolates were Escherichia coli [93.3% (125/134)] and exotic breed type [OR (95%CI) = 2.372 (1.460-3.854), p-value < 0.001] was found to be a predictor of ESBL carriage among animals. Whole-genome sequences of 25 ESBL-producing E. coli were analyzed for phylogenetic relationships using multi-locus sequence typing (MLST) and core genome comparisons. Fourteen different sequence types were detected of which ST617 (7/25), ST2852 (3/25), ST1303 (3/25) were the most abundant. All isolates harbored the bla CTX-M-15 allele, 22/25 carried strA and strB, 12/25 aac(6')-lb-cr, and 11/25 qnrS1. Antibiotic resistance was associated with IncF, IncY, as well as non-typable plasmids. Eleven isolates carried pPGRT46-related plasmids, previously reported from isolates in Nigeria. Five isolates had plasmids exhibiting 85-99% homology to pCA28, previously detected in isolates from the US. Our findings indicate a pan-species distribution of ESBL-producing E. coli clonal groups in farming communities and provide evidence for plasmids harboring antibiotic resistances of regional and international impact.

  14. Circuitry and dynamics of human transcription factor regulatory networks

    PubMed Central

    Neph, Shane; Stergachis, Andrew B.; Reynolds, Alex; Sandstrom, Richard; Borenstein, Elhanan; Stamatoyannopoulos, John A.

    2012-01-01

    SUMMARY The combinatorial cross-regulation of hundreds of sequence-specific transcription factors defines a regulatory network that underlies cellular identity and function. Here we use genome-wide maps of in vivo DNaseI footprints to assemble an extensive core human regulatory network comprising connections among 475 sequence-specific transcription factors, and to analyze the dynamics of these connections across 41 diverse cell and tissue types. We find that human transcription factor networks are highly cell-selective and are driven by cohorts of factors that include regulators with previously unrecognized roles in control of cellular identity. Moreover, we identify many widely expressed factors that impact transcriptional regulatory networks in a cell-selective manner. Strikingly, in spite of their inherent diversity, all cell type regulatory networks independently converge on a common architecture that closely resembles the topology of living neuronal networks. Together, our results provide the first description of the circuitry, dynamics, and organizing principles of the human transcription factor regulatory network. PMID:22959076

  15. Sequence analysis of serotype-specific synthesis regions II of Haemophilus influenzae serotypes c and d: evidence for common ancestry of capsule synthesis in Pasteurellaceae and Neisseria meningitidis.

    PubMed

    Lâm, Thiên-Trí; Claus, Heike; Frosch, Matthias; Vogel, Ulrich

    2011-06-01

    Sequencing of yet unknown Haemophilus influenzae serotype c (Hic) and d (Hid) capsule synthesis regions II revealed four (ccs1-4) and five (dcs1-5) open reading frames, respectively. The inferred gene functions were in line with capsular polysaccharide structures. One or more proteins encoded by the Hic capsule synthesis region II showed similarity to Actinobacillus pleuropneumoniae serotype 1 and Actinobacillus suis K1 enzymes. Orthologues to the complete operon were observed in Actinobacillus minor strain 202, where even the gene order was conserved. Furthermore, Ccs4 was related to the capsule O-acetyltransferase of Neisseria meningitidis serogroup W-135. For the Hid locus, similarities to Hie, Mannheimia haemolytica A1 and N. meningitidis serogroup A were identified and the succession of genes was similar in the different species. The resemblance of genes and gene organization found for Hic and Hid with other species suggested horizontal gene transfer during capsule evolution across the bacterial classes.

  16. Swine Leukocyte Antigen (SLA) class I allele typing of Danish swine herds and identification of commonly occurring haplotypes using sequence specific low and high resolution primers.

    PubMed

    Pedersen, Lasse Eggers; Jungersen, Gregers; Sorensen, Maria Rathmann; Ho, Chak-Sum; Vadekær, Dorte Fink

    2014-12-15

    The swine major histocompatibility complex (MHC) genomic region (SLA) is extremely polymorphic comprising high numbers of different alleles, many encoding a distinct MHC class I molecule, which binds and presents endogenous peptides to circulating T cells of the immune system. Upon recognition of such peptide-MHC complexes (pMHC) naïve T cells can become activated and respond to a given pathogen leading to its elimination and the generation of memory cells. Hence SLA plays a crucial role in maintaining overall adaptive immunologic resistance to pathogens. Knowing which SLA alleles that are commonly occurring can be of great importance in regard to future vaccine development and the establishment of immune protection in swine through broad coverage, highly specific, subunit based vaccination against viruses such as swine influenza, porcine reproductive and respiratory syndrome virus, vesicular stomatitis virus, foot-and-mouth-disease virus and others. Here we present the use of low- and high-resolution PCR-based typing methods to identify individual and commonly occurring SLA class I alleles in Danish swine. A total of 101 animals from seven different herds were tested, and by low resolution typing the top four most frequent SLA class I alleles were those of the allele groups SLA-3*04XX, SLA-1*08XX, SLA-2*02XX, and SLA-1*07XX, respectively. Customised high resolution primers were used to identify specific alleles within the above mentioned allele groups as well as within the SLA-2*05XX allele group. Our studies also suggest the most common haplotype in Danish pigs to be Lr-4.0 expressing the SLA-1*04XX, SLA-2*04XX, and SLA-3*04XX allele combination.

  17. Common Cold

    MedlinePlus

    ... nose, coughing - everyone knows the symptoms of the common cold. It is probably the most common illness. In ... avoid colds. There is no cure for the common cold. For relief, try Getting plenty of rest Drinking ...

  18. Fast and Efficient Cloning of Cis-Regulatory Sequences for High-Throughput Yeast One-Hybrid Analyses of Transcription Factors.

    PubMed

    Kelemen, Zsolt; Przybyla-Toscano, Jonathan; Tissot, Nicolas; Lepiniec, Loïc; Dubos, Christian

    2016-01-01

    Yeast one-hybrid (Y1H) assay has been proven to be a powerful technique to characterize in vivo the interaction between a given transcription factor (TF), or its DNA-binding domain (DBD), and target DNA sequences. Comprehensive characterization of TF/DBD and DNA interactions should allow designing synthetic promoters that would undoubtedly be valuable for biotechnological approaches. Here, we use the ligation-independent cloning system (LIC) in order to enhance the cloning efficiency of DNA motifs into the pHISi Y1H vector. LIC overcomes important limitations of traditional cloning technologies, since any DNA fragment can be cloned into LIC compatible vectors without using restriction endonucleases, ligation, or in vitro recombination. PMID:27557765

  19. Interstitial telomeric sequences in human chromosomes cluster with common fragile sites, mutagen sensitive sites, viral integration sites, cancer breakpoints, proto-oncogenes and breakpoints involved in primate evolution

    SciTech Connect

    Adekunle, S.S.A.; Wyandt, H.; Mark, H.F.L.

    1994-09-01

    Recently we mapped the telomeric repeat sequences to 111 interstitial sites in the human genome and to sites of gaps and breaks induced by aphidicolin and sister chromatid exchange sites detected by BrdU. Many of these sites correspond to conserved fragile sites in man, gorilla and chimpazee, to sites of conserved sister chromatid exchange in the mammalian X chromosome, to mutagenic sensitive sites, mapped locations of proto-oncogenes, breakpoints implicated in primate evolution and to breakpoints indicated as the sole anomaly in neoplasia. This observation prompted us to investigate if the interstitial telomeric sites cluster with these sites. An extensive literature search was carried out to find all the available published sites mentioned above. For comparison, we also carried out a statistical analysis of the clustering of the sites of the telomeric repeats with the gene locations where only nucleotide mutations have been observed as the only chromosomal abnormality. Our results indicate that the telomeric repeats cluster most with fragile sites, mutagenic sensitive sites and breakpoints implicated in primate evolution and least with cancer breakpoints, mapped locations of proto-oncogenes and other genes with nucleotide mutations.

  20. A Xenopus laevis gene encoding EF-1 alpha S, the somatic form of elongation factor 1 alpha: sequence, structure, and identification of regulatory elements required for embryonic transcription.

    PubMed

    Johnson, A D; Krieg, P A

    1995-01-01

    Transcription of the Xenopus laevis EF-1 alpha S gene commences at the mid-blastula stage of embryonic development and then continues constitutively in all somatic tissues. The EF-1 alpha S promoter is extremely active in the early Xenopus embryo where EF-1 alpha S transcripts account for as much as 40% of all new polyadenylated transcripts. We have isolated the Xenopus EF-1 alpha S gene and used microinjection techniques to identify promoter elements responsible for embryonic transcription. These in vivo expression studies have identified an enhancer fragment, located approximately 4.4 kb upstream of the transcription start site, that is required for maximum expression from the EF-1 alpha S promoter. The enhancer fragment contains both an octamer and a G/C box sequence, but mutation studies indicate that the octamer plays no significant role in regulation of EF-1 alpha S expression in the embryo. The presence of a G/C element in the enhancer and of multiple G/C boxes in the proximal promoter region suggests that the G/C box binding protein, Sp1, plays a major role in the developmental regulation of EF-1 alpha S promoter activity. PMID:8565334

  1. Cis-regulatory hairpin-shaped mRNA encoding a reporter protein: catalytic sensing of nucleic acid sequence at single nucleotide resolution.

    PubMed

    Narita, Atsushi; Ogawa, Kazumasa; Sando, Shinsuke; Aoyama, Yasuhiro

    2007-01-01

    DNA sensing at a single nucleotide resolution is achieved using a hairpin-shaped, unmodified (unlabeled) RNA probe or the precursor double-stranded DNA (dsDNA) in a prokaryotic cell-free translation medium. The molecular-beacon-like probe consists of a loop region that is complementary to the target sequence and a stem composed of a ribosome-binding site (RBS) and its docking domain; the RBS is followed by the gene for a reporter protein such as luciferase or beta-galactosidase. Target binding at the loop opens the hairpin to make RBS accessible by the ribosome to start translation of the reporter protein. This sensing system is signal amplifying by virtue of catalytic DNA-to-RNA transcription when using a dsDNA probe, catalytic RNA-to-protein translation, catalytic signal transduction by the enzymatic reaction of the translated reporter protein and, in the presence of RNase H, catalytic or even irreversible translation-activation of the target-probe heteroduplex. Preparation of a probe takes 1-3 d and gene sensing using the probe takes 1-3 h.

  2. The regulatory network of cluster-root function and development in phosphate-deficient white lupin (Lupinus albus) identified by transcriptome sequencing.

    PubMed

    Wang, Zhengrui; Straub, Daniel; Yang, Huaiyu; Kania, Angelika; Shen, Jianbo; Ludewig, Uwe; Neumann, Günter

    2014-07-01

    Lupinus albus serves as model plant for root-induced mobilization of sparingly soluble soil phosphates via the formation of cluster-roots (CRs) that mediate secretion of protons, citrate, phenolics and acid phosphatases (APases). This study employed next-generation sequencing to investigate the molecular mechanisms behind these complex adaptive responses at the transcriptome level. We compared different stages of CR development, including pre-emergent (PE), juvenile (JU) and the mature (MA) stages. The results confirmed that the primary metabolism underwent significant modifications during CR maturation, promoting the biosynthesis of organic acids, as had been deduced from physiological studies. Citrate catabolism was downregulated, associated with citrate accumulation in MA clusters. Upregulation of the phenylpropanoid pathway reflected the accumulation of phenolics. Specific transcript expression of ALMT and MATE transporter genes correlated with the exudation of citrate and flavonoids. The expression of transcripts related to nucleotide degradation and APases in MA clusters coincided with the re-mobilization and hydrolysis of organic phosphate resources. Most interestingly, hormone-related gene expression suggested a central role of ethylene during CR maturation. This was associated with the upregulation of the iron (Fe)-deficiency regulated network that mediates ethylene-induced expression of Fe-deficiency responses in other species. Finally, transcripts related to abscisic acid and jasmonic acid were upregulated in MA clusters, while auxin- and brassinosteroid-related genes and cytokinin receptors were most strongly expressed during CR initiation. Key regulations proposed by the RNA-seq data were confirmed by quantitative real-time polymerase chain reaction (RT-qPCR) and some physiological analyses. A model for the gene network regulating CR development and function is presented. PMID:24635386

  3. Common Cold

    MedlinePlus

    ... News & Events Volunteer NIAID > Health & Research Topics > Common Cold Skip Website Tools Website Tools Print this page ... Help people who are suffering from the common cold by volunteering for NIAID clinical studies on ClinicalTrials. ...

  4. Accelerated Evolution of Conserved Noncoding Sequences in theHuman Genome

    SciTech Connect

    Prambhakar, Shyam; Noonan, James P.; Paabo, Svante; Rubin, EdwardM.

    2006-07-06

    Genomic comparisons between human and distant, non-primatemammals are commonly used to identify cis-regulatory elements based onconstrained sequence evolution. However, these methods fail to detect"cryptic" functional elements, which are too weakly conserved amongmammals to distinguish from nonfunctional DNA. To address this problem,we explored the potential of deep intra-primate sequence comparisons. Wesequenced the orthologs of 558 kb of human genomic sequence, coveringmultiple loci involved in cholesterol homeostasis, in 6 nonhumanprimates. Our analysis identified 6 noncoding DNA elements displayingsignificant conservation among primates, but undetectable in more distantcomparisons. In vitro and in vivo tests revealed that at least three ofthese 6 elements have regulatory function. Notably, the mouse orthologsof these three functional human sequences had regulatory activity despitetheir lack of significant sequence conservation, indicating that they arecryptic ancestral cis-regulatory elements. These regulatory elementscould still be detected in a smaller set of three primate speciesincluding human, rhesus and marmoset. Since the human and rhesus genomesequences are already available, and the marmoset genome is activelybeing sequenced, the primate-specific conservation analysis describedhere can be applied in the near future on a whole-genome scale, tocomplement the annotation provided by more distant speciescomparisons.

  5. Clays, common

    USGS Publications Warehouse

    Virta, R.L.

    1998-01-01

    Part of a special section on the state of industrial minerals in 1997. The state of the common clay industry worldwide for 1997 is discussed. Sales of common clay in the U.S. increased from 26.2 Mt in 1996 to an estimated 26.5 Mt in 1997. The amount of common clay and shale used to produce structural clay products in 1997 was estimated at 13.8 Mt.

  6. Evidence for a common evolutionary rate in metazoan transcriptional networks.

    PubMed

    Carvunis, Anne-Ruxandra; Wang, Tina; Skola, Dylan; Yu, Alice; Chen, Jonathan; Kreisberg, Jason F; Ideker, Trey

    2015-01-01

    Genome sequences diverge more rapidly in mammals than in other animal lineages, such as birds or insects. However, the effect of this rapid divergence on transcriptional evolution remains unclear. Recent reports have indicated a faster divergence of transcription factor binding in mammals than in insects, but others found the reverse for mRNA expression. Here, we show that these conflicting interpretations resulted from differing methodologies. We performed an integrated analysis of transcriptional network evolution by examining mRNA expression, transcription factor binding and cis-regulatory motifs across >25 animal species, including mammals, birds and insects. Strikingly, we found that transcriptional networks evolve at a common rate across the three animal lineages. Furthermore, differences in rates of genome divergence were greatly reduced when restricting comparisons to chromatin-accessible sequences. The evolution of transcription is thus decoupled from the global rate of genome sequence evolution, suggesting that a small fraction of the genome regulates transcription.

  7. Student Commons

    ERIC Educational Resources Information Center

    Gordon, Douglas

    2010-01-01

    Student commons are no longer simply congregation spaces for students with time on their hands. They are integral to providing a welcoming environment and effective learning space for students. Many student commons have been transformed into spaces for socialization, an environment for alternative teaching methods, a forum for large group meetings…

  8. The miRNAs and their regulatory networks responsible for pollen abortion in Ogura-CMS Chinese cabbage revealed by high-throughput sequencing of miRNAs, degradomes, and transcriptomes.

    PubMed

    Wei, Xiaochun; Zhang, Xiaohui; Yao, Qiuju; Yuan, Yuxiang; Li, Xixiang; Wei, Fang; Zhao, Yanyan; Zhang, Qiang; Wang, Zhiyong; Jiang, Wusheng; Zhang, Xiaowei

    2015-01-01

    Chinese cabbage (Brassica rapa ssp. pekinensis) is one of the most important vegetables in Asia and is cultivated across the world. Ogura-type cytoplasmic male sterility (Ogura-CMS) has been widely used in the hybrid breeding industry for Chinese cabbage and many other cruciferous vegetables. Although, the cause of Ogura-CMS has been localized to the orf138 locus in the mitochondrial genome, however, the mechanism by which nuclear genes respond to the mutation of the mitochondrial orf138 locus is unclear. In this study, a series of whole genome small RNA, degradome and transcriptome analyses were performed on both Ogura-CMS and its maintainer Chinese cabbage buds using deep sequencing technology. A total of 289 known miRNAs derived from 69 families (including 23 new families first reported in B. rapa) and 426 novel miRNAs were identified. Among these novel miRNAs, both 3-p and 5-p miRNAs were detected on the hairpin arms of 138 precursors. Ten known and 49 novel miRNAs were down-regulated, while one known and 27 novel miRNAs were up-regulated in Ogura-CMS buds compared to the fertile plants. Using degradome analysis, a total of 376 mRNAs were identified as targets of 30 known miRNA families and 100 novel miRNAs. A large fraction of the targets were annotated as reproductive development related. Our transcriptome profiling revealed that the expression of the targets was finely tuned by the miRNAs. Two novel miRNAs were identified that were specifically highly expressed in Ogura-CMS buds and sufficiently suppressed two pollen development essential genes: sucrose transporter SUC1 and H (+) -ATPase 6. These findings provide clues for the contribution of a potential miRNA regulatory network to bud development and pollen engenderation. This study contributes new insights to the communication between the mitochondria and chromosome and takes one step toward filling the gap in the regulatory network from the orf138 locus to pollen abortion in Ogura-CMS plants from a mi

  9. The miRNAs and their regulatory networks responsible for pollen abortion in Ogura-CMS Chinese cabbage revealed by high-throughput sequencing of miRNAs, degradomes, and transcriptomes

    PubMed Central

    Wei, Xiaochun; Zhang, Xiaohui; Yao, Qiuju; Yuan, Yuxiang; Li, Xixiang; Wei, Fang; Zhao, Yanyan; Zhang, Qiang; Wang, Zhiyong; Jiang, Wusheng; Zhang, Xiaowei

    2015-01-01

    Chinese cabbage (Brassica rapa ssp. pekinensis) is one of the most important vegetables in Asia and is cultivated across the world. Ogura-type cytoplasmic male sterility (Ogura-CMS) has been widely used in the hybrid breeding industry for Chinese cabbage and many other cruciferous vegetables. Although, the cause of Ogura-CMS has been localized to the orf138 locus in the mitochondrial genome, however, the mechanism by which nuclear genes respond to the mutation of the mitochondrial orf138 locus is unclear. In this study, a series of whole genome small RNA, degradome and transcriptome analyses were performed on both Ogura-CMS and its maintainer Chinese cabbage buds using deep sequencing technology. A total of 289 known miRNAs derived from 69 families (including 23 new families first reported in B. rapa) and 426 novel miRNAs were identified. Among these novel miRNAs, both 3-p and 5-p miRNAs were detected on the hairpin arms of 138 precursors. Ten known and 49 novel miRNAs were down-regulated, while one known and 27 novel miRNAs were up-regulated in Ogura-CMS buds compared to the fertile plants. Using degradome analysis, a total of 376 mRNAs were identified as targets of 30 known miRNA families and 100 novel miRNAs. A large fraction of the targets were annotated as reproductive development related. Our transcriptome profiling revealed that the expression of the targets was finely tuned by the miRNAs. Two novel miRNAs were identified that were specifically highly expressed in Ogura-CMS buds and sufficiently suppressed two pollen development essential genes: sucrose transporter SUC1 and H+-ATPase 6. These findings provide clues for the contribution of a potential miRNA regulatory network to bud development and pollen engenderation. This study contributes new insights to the communication between the mitochondria and chromosome and takes one step toward filling the gap in the regulatory network from the orf138 locus to pollen abortion in Ogura-CMS plants from a mi

  10. Common File Formats.

    PubMed

    Mills, Lauren

    2014-03-21

    An overview of the many file formats commonly used in bioinformatics and genome sequence analysis is presented, including various data file formats, alignment file formats, and annotation file formats. Example workflows illustrate how some of the different file types are typically used.

  11. COMMON LISP: The language

    SciTech Connect

    Steele, G.L. Jr.

    1984-01-01

    This book describes COMMON LISP,which is becoming the industry and government standard AI language. Topics covered include the following: data types; scope and extent; type specifiers; program structure; predicates; control structure; macros; declarations; symbols; packages; numbers; characters; sequences; lists; hash tables; arrays; strings; structures; the evaluator; streams; input/output; file system interface; and errors.

  12. Specific sequences commonly found in the V3 domain of HIV-1 subtype C isolates affect the overall conformation of native Env and induce a neutralization-resistant phenotype independent of V1/V2 masking.

    PubMed

    Salomon, Aidy; Krachmarov, Chavdar; Lai, Zhong; Honnen, William; Zingman, Barry S; Sarlo, Julie; Gorny, Miroslaw K; Zolla-Pazner, Susan; Robinson, James E; Pinter, Abraham

    2014-01-01

    Primary HIV-1 isolates are relatively resistant to neutralization by antibodies commonly induced after infection or vaccination. This is generally attributed to masking of sensitive epitopes by the V1/V2 domain and/or glycans situated at various positions in Env. Here we identified a novel masking effect mediated by subtype C-specific V3 sequences that contributes to the V1/V2-independent and glycan-independent neutralization resistance of chimeric and primary Envs to antibodies directed against multiple neutralization domains. Positions at several conserved charged and hydrophobic sites in the V3 crown and stem were also shown to affect neutralization phenotype. These results indicated that substitutions typically present in subtype C and related V3 sequences influence the overall conformation of native Env in a way that occludes multiple neutralization targets located both within and outside of the V3 domain, and may reflect an alternative mechanism for neutralization resistance that is particularly active in subtype C and related isolates.

  13. Common cold

    MedlinePlus

    ... been tried for colds, such as vitamin C, zinc supplements, and echinacea. Talk to your health care ... nih.gov/pubmed/22962927 . Singh M, Das RR. Zinc for the common cold. Cochrane Database of Systematic ...

  14. Phylogenetic structure and evolution of regulatory genes and integrases of P2-like phages.

    PubMed

    Nilsson, Hanna; Cardoso-Palacios, Carlos; Haggård-Ljungquist, Elisabeth; Nilsson, Anders S

    2011-07-01

    The phylogenetic relationships and structural similarities of the proteins encoded within the regulatory region (containing the integrase gene and the lytic-lysogenic transcriptional switch genes) of P2-like phages were analyzed, and compared with the phylogenetic relationship of P2-like phages inferred from four structural genes. P2-like phages are thought to be one of the most genetically homogenous phage groups but the regulatory region nevertheless varies extensively between different phage genomes.   The analyses showed that there are many types of regulatory regions, but two types can be clearly distinguished; regions similar either to the phage P2 or to the phage 186 regulatory regions. These regions were also found to be most frequent among the sequenced P2-like phage or prophage genomes, and common in phages using Escherichia coli as a host. Both the phylogenetic and the structural analyses showed that these two regions are related. The integrases as well as the cox/apl genes show a common monophyletic origin but the immunity repressor genes, the type P2 C gene and the type 186 cI gene, are likely of different origin. There was no indication of recombination between the P2-186 types of regulatory genes but the comparison of the phylogenies of the regulatory region with the phylogeny based on four structural genes revealed recombinational events between the regulatory region and the structural genes. Less common regulatory regions were phylogenetically heterogeneous and typically contained a fusion of genes from distantly related or unknown phages and P2-like genes.

  15. Phylogenetic structure and evolution of regulatory genes and integrases of P2-like phages

    PubMed Central

    Nilsson, Hanna; Cardoso-Palacios, Carlos; Haggård-Ljungquist, Elisabeth; Nilsson, Anders S.

    2011-01-01

    The phylogenetic relationships and structural similarities of the proteins encoded within the regulatory region (containing the integrase gene and the lytic–lysogenic transcriptional switch genes) of P2-like phages were analyzed, and compared with the phylogenetic relationship of P2-like phages inferred from four structural genes. P2-like phages are thought to be one of the most genetically homogenous phage groups but the regulatory region nevertheless varies extensively between different phage genomes.   The analyses showed that there are many types of regulatory regions, but two types can be clearly distinguished; regions similar either to the phage P2 or to the phage 186 regulatory regions. These regions were also found to be most frequent among the sequenced P2-like phage or prophage genomes, and common in phages using Escherichia coli as a host. Both the phylogenetic and the structural analyses showed that these two regions are related. The integrases as well as the cox/apl genes show a common monophyletic origin but the immunity repressor genes, the type P2 C gene and the type 186 cI gene, are likely of different origin. There was no indication of recombination between the P2–186 types of regulatory genes but the comparison of the phylogenies of the regulatory region with the phylogeny based on four structural genes revealed recombinational events between the regulatory region and the structural genes. Less common regulatory regions were phylogenetically heterogeneous and typically contained a fusion of genes from distantly related or unknown phages and P2-like genes. PMID:23050214

  16. Expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene under control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice.

    PubMed

    Burkov, I A; Serova, I A; Battulin, N R; Smirnov, A V; Babkin, I V; Andreeva, L E; Dvoryanchikov, G A; Serov, O L

    2013-10-01

    Expression of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected "protective or enhancer effect" from the MAR element on the hGM-CSF gene expression was not observed.

  17. Qualitative De Novo Analysis of Full Length cDNA and Quantitative Analysis of Gene Expression for Common Marmoset (Callithrix jacchus) Transcriptomes Using Parallel Long-Read Technology and Short-Read Sequencing

    PubMed Central

    Uno, Yasuhiro; Uehara, Shotaro; Inoue, Takashi; Murayama, Norie; Onodera, Jun; Sasaki, Erika; Yamazaki, Hiroshi

    2014-01-01

    The common marmoset (Callithrix jacchus) is a non-human primate that could prove useful as human pharmacokinetic and biomedical research models. The cytochromes P450 (P450s) are a superfamily of enzymes that have critical roles in drug metabolism and disposition via monooxygenation of a broad range of xenobiotics; however, information on some marmoset P450s is currently limited. Therefore, identification and quantitative analysis of tissue-specific mRNA transcripts, including those of P450s and flavin-containing monooxygenases (FMO, another monooxygenase family), need to be carried out in detail before the marmoset can be used as an animal model in drug development. De novo assembly and expression analysis of marmoset transcripts were conducted with pooled liver, intestine, kidney, and brain samples from three male and three female marmosets. After unique sequences were automatically aligned by assembling software, the mean contig length was 718 bp (with a standard deviation of 457 bp) among a total of 47,883 transcripts. Approximately 30% of the total transcripts were matched to known marmoset sequences. Gene expression in 18 marmoset P450- and 4 FMO-like genes displayed some tissue-specific patterns. Of these, the three most highly expressed in marmoset liver were P450 2D-, 2E-, and 3A-like genes. In extrahepatic tissues, including brain, gene expressions of these monooxygenases were lower than those in liver, although P450 3A4 (previously P450 3A21) in intestine and P450 4A11- and FMO1-like genes in kidney were relatively highly expressed. By means of massive parallel long-read sequencing and short-read technology applied to marmoset liver, intestine, kidney, and brain, the combined next-generation sequencing analyses reported here were able to identify novel marmoset drug-metabolizing P450 transcripts that have until now been little reported. These results provide a foundation for mechanistic studies and pave the way for the use of marmosets as model animals

  18. RNA Sequencing Analysis of the msl2msl3, crl, and ggps1 Mutants Indicates that Diverse Sources of Plastid Dysfunction Do Not Alter Leaf Morphology Through a Common Signaling Pathway

    PubMed Central

    Luesse, Darron R.; Wilson, Margaret E.; Haswell, Elizabeth S.

    2015-01-01

    Determining whether individual genes function in the same or in different pathways is an important aspect of genetic analysis. As an alternative to the construction of higher-order mutants, we used contemporary expression profiling methods to perform pathway analysis on several Arabidopsis thaliana mutants, including the mscS-like (msl)2msl3 double mutant. MSL2 and MSL3 are implicated in plastid ion homeostasis, and msl2msl3 double mutants exhibit leaves with a lobed periphery, a rumpled surface, and disturbed mesophyll cell organization. Similar developmental phenotypes are also observed in other mutants with defects in a range of other chloroplast or mitochondrial functions, including biogenesis, gene expression, and metabolism. We wished to test the hypothesis that the common leaf morphology phenotypes of these mutants are the result of a characteristic nuclear expression pattern that is generated in response to organelle dysfunction. RNA-Sequencing was performed on aerial tissue of msl2msl3 geranylgeranyl diphosphate synthase 1 (ggps1), and crumpled leaf (crl) mutants. While large groups of co-expressed genes were identified in pairwise comparisons between genotypes, we were only able to identify a small set of genes that showed similar expression profiles in all three genotypes. Subsequent comparison to the previously published gene expression profiles of two other mutants, yellow variegated 2 (var2) and scabra3 (sca3), failed to reveal a common pattern of gene expression associated with superficially similar leaf morphology defects. Nor did we observe overlap between genes differentially expressed in msl2msl3, crl, and ggps1 and a previously identified retrograde core response module. These data suggest that a common retrograde signaling pathway initiated by organelle dysfunction either does not exist in these mutants or cannot be identified through transcriptomic methods. Instead, the leaf developmental defects observed in these mutants may be achieved

  19. Clinical, cytogenetic, and molecular outcomes in a series of 66 patients with Pierre Robin sequence and literature review: 22q11.2 deletion is less common than other chromosomal anomalies.

    PubMed

    Gomez-Ospina, Natalia; Bernstein, Jonathan A

    2016-04-01

    Pierre Robin sequence (PRS) is an important craniofacial anomaly that can be seen as an isolated finding or manifestation of multiple syndromes. 22q11.2 deletion and Stickler syndrome are cited as the two most common conditions associated with PRS, but their frequencies are debated. We performed a retrospective study of 66 patients with PRS and reviewed their genetic testing, diagnoses, and clinical findings. The case series is complemented by a comprehensive literature review of the nature and frequency of genetic diagnosis in PRS. In our cohort 65% of patients had associated anomalies; of these, a genetic diagnosis was established in 56%. Stickler syndrome was the most common diagnosis, comprising approximately 11% of all cases, followed by Treacher Collins syndrome (9%). The frequency of 22q11.2 deletion was 1.5%. Chromosome arrays, performed for 72% of idiopathic PRS with associated anomalies, revealed two cases of 18q22→qter deletion, a region not previously reported in association with PRS. A review of the cytogenetic anomalies identified in this population supports an association between the 4q33-qter, 17q24.3, 2q33.1, and 11q23 chromosomal loci and PRS. We found a low frequency of 22q11.2 deletion in PRS, suggesting it is less commonly implicated in this malformation. Our data also indicate a higher frequency of cytogenetic anomalies in PRS patients with associated anomalies, and a potential new link with the 18q22→qter locus. The present findings underscore the utility of chromosomal microarrays in cases of PRS with associated anomalies and suggest that delaying testing for apparently isolated cases should be considered.

  20. Common-Cause Failure Analysis in Event Assessment

    SciTech Connect

    Dana L. Kelly; Dale M. Rasmuson

    2008-09-01

    This paper describes the approach taken by the U. S. Nuclear Regulatory Commission to the treatment of common-cause failure in probabilistic risk assessment of operational events. The approach is based upon the Basic Parameter Model for common-cause failure, and examples are illustrated using the alpha-factor parameterization, the approach adopted by the NRC in their Standardized Plant Analysis Risk (SPAR) models. The cases of a failed component (with and without shared common-cause failure potential) and a component being unavailable due to preventive maintenance or testing are addressed. The treatment of two related failure modes (e.g., failure to start and failure to run) is a new feature of this paper. These methods are being applied by the NRC in assessing the risk significance of operational events for the Significance Determination Process (SDP) and the Accident Sequence Precursor (ASP) program.

  1. Finding regulatory elements and regulatory motifs: a general probabilistic framework

    PubMed Central

    van Nimwegen, Erik

    2007-01-01

    Over the last two decades a large number of algorithms has been developed for regulatory motif finding. Here we show how many of these algorithms, especially those that model binding specificities of regulatory factors with position specific weight matrices (WMs), naturally arise within a general Bayesian probabilistic framework. We discuss how WMs are constructed from sets of regulatory sites, how sites for a given WM can be discovered by scanning of large sequences, how to cluster WMs, and more generally how to cluster large sets of sites from different WMs into clusters. We discuss how 'regulatory modules', clusters of sites for subsets of WMs, can be found in large intergenic sequences, and we discuss different methods for ab initio motif finding, including expectation maximization (EM) algorithms, and motif sampling algorithms. Finally, we extensively discuss how module finding methods and ab initio motif finding methods can be extended to take phylogenetic relations between the input sequences into account, i.e. we show how motif finding and phylogenetic footprinting can be integrated in a rigorous probabilistic framework. The article is intended for readers with a solid background in applied mathematics, and preferably with some knowledge of general Bayesian probabilistic methods. The main purpose of the article is to elucidate that all these methods are not a disconnected set of individual algorithmic recipes, but that they are just different facets of a single integrated probabilistic theory. PMID:17903285

  2. Combinatorial Gene Regulatory Functions Underlie Ultraconserved Elements in Drosophila

    PubMed Central

    Warnefors, Maria; Hartmann, Britta; Thomsen, Stefan; Alonso, Claudio R.

    2016-01-01

    Ultraconserved elements (UCEs) are discrete genomic elements conserved across large evolutionary distances. Although UCEs have been linked to multiple facets of mammalian gene regulation their extreme evolutionary conservation remains largely unexplained. Here, we apply a computational approach to investigate this question in Drosophila, exploring the molecular functions of more than 1,500 UCEs shared across the genomes of 12 Drosophila species. Our data indicate that Drosophila UCEs are hubs for gene regulatory functions and suggest that UCE sequence invariance originates from their combinatorial roles in gene control. We also note that the gene regulatory roles of intronic and intergenic UCEs (iUCEs) are distinct from those found in exonic UCEs (eUCEs). In iUCEs, transcription factor (TF) and epigenetic factor binding data strongly support iUCE roles in transcriptional and epigenetic regulation. In contrast, analyses of eUCEs indicate that they are two orders of magnitude more likely than the expected to simultaneously include protein-coding sequence, TF-binding sites, splice sites, and RNA editing sites but have reduced roles in transcriptional or epigenetic regulation. Furthermore, we use a Drosophila cell culture system and transgenic Drosophila embryos to validate the notion of UCE combinatorial regulatory roles using an eUCE within the Hox gene Ultrabithorax and show that its protein-coding region also contains alternative splicing regulatory information. Taken together our experiments indicate that UCEs emerge as a result of combinatorial gene regulatory roles and highlight common features in mammalian and insect UCEs implying that similar processes might underlie ultraconservation in diverse animal taxa. PMID:27247329

  3. Combinatorial Gene Regulatory Functions Underlie Ultraconserved Elements in Drosophila.

    PubMed

    Warnefors, Maria; Hartmann, Britta; Thomsen, Stefan; Alonso, Claudio R

    2016-09-01

    Ultraconserved elements (UCEs) are discrete genomic elements conserved across large evolutionary distances. Although UCEs have been linked to multiple facets of mammalian gene regulation their extreme evolutionary conservation remains largely unexplained. Here, we apply a computational approach to investigate this question in Drosophila, exploring the molecular functions of more than 1,500 UCEs shared across the genomes of 12 Drosophila species. Our data indicate that Drosophila UCEs are hubs for gene regulatory functions and suggest that UCE sequence invariance originates from their combinatorial roles in gene control. We also note that the gene regulatory roles of intronic and intergenic UCEs (iUCEs) are distinct from those found in exonic UCEs (eUCEs). In iUCEs, transcription factor (TF) and epigenetic factor binding data strongly support iUCE roles in transcriptional and epigenetic regulation. In contrast, analyses of eUCEs indicate that they are two orders of magnitude more likely than the expected to simultaneously include protein-coding sequence, TF-binding sites, splice sites, and RNA editing sites but have reduced roles in transcriptional or epigenetic regulation. Furthermore, we use a Drosophila cell culture system and transgenic Drosophila embryos to validate the notion of UCE combinatorial regulatory roles using an eUCE within the Hox gene Ultrabithorax and show that its protein-coding region also contains alternative splicing regulatory information. Taken together our experiments indicate that UCEs emerge as a result of combinatorial gene regulatory roles and highlight common features in mammalian and insect UCEs implying that similar processes might underlie ultraconservation in diverse animal taxa. PMID:27247329

  4. Sequence analysis of chromatin immunoprecipitation data for transcription factors

    PubMed Central

    Fraenkel, Ernest

    2013-01-01

    Chromatin immunoprecipitation (ChIP) experiments allow the location of transcription factors to be determined across the genome. Subsequent analysis of the sequences of the identified regions allows binding to be localized at a higher resolution than can be achieved by current high-throughput experiments without sequence analysis, and may provide important insight into the regulatory programs enacted by the protein of interest. In this chapter we review the tools, workflow, and common pitfalls of such analyses, and recommend strategies for effective motif discovery from these data. PMID:20827592

  5. Preserving sequence annotations across reference sequences

    PubMed Central

    2014-01-01

    Background Matching and comparing sequence annotations of different reference sequences is vital to genomics research, yet many annotation formats do not specify the reference sequence types or versions used. This makes the integration of annotations from different sources difficult and error prone. Results As part of our effort to create linked data for interoperable sequence annotations, we present an RDF data model for sequence annotation using the ontological framework established by the OBO Foundry ontologies and the Basic Formal Ontology (BFO). We defined reference sequences as the common domain of integration for sequence annotations, and identified three semantic relationships between sequence annotations. In doing so, we created the Reference Sequence Annotation to compensate for gaps in the SO and in its mapping to BFO, particularly for annotations that refer to versions of consensus reference sequences. Moreover, we present three integration models for sequence annotations using different reference assemblies. Conclusions We demonstrated a working example of a sequence annotation instance, and how this instance can be linked to other annotations on different reference sequences. Sequence annotations in this format are semantically rich and can be integrated easily with different assemblies. We also identify other challenges of modeling reference sequences with the BFO. PMID:25093075

  6. Regulatory Anatomy

    PubMed Central

    2015-01-01

    This article proposes the term “safety logics” to understand attempts within the European Union (EU) to harmonize member state legislation to ensure a safe and stable supply of human biological material for transplants and transfusions. With safety logics, I refer to assemblages of discourses, legal documents, technological devices, organizational structures, and work practices aimed at minimizing risk. I use this term to reorient the analytical attention with respect to safety regulation. Instead of evaluating whether safety is achieved, the point is to explore the types of “safety” produced through these logics as well as to consider the sometimes unintended consequences of such safety work. In fact, the EU rules have been giving rise to complaints from practitioners finding the directives problematic and inadequate. In this article, I explore the problems practitioners face and why they arise. In short, I expose the regulatory anatomy of the policy landscape. PMID:26139952

  7. Regulatory Physiology

    NASA Technical Reports Server (NTRS)

    Lane, Helen W.; Whitson, Peggy A.; Putcha, Lakshmi; Baker, Ellen; Smith, Scott M.; Stewart, Karen; Gretebeck, Randall; Nimmagudda, R. R.; Schoeller, Dale A.; Davis-Street, Janis

    1999-01-01

    As noted elsewhere in this report, a central goal of the Extended Duration Orbiter Medical Project (EDOMP) was to ensure that cardiovascular and muscle function were adequate to perform an emergency egress after 16 days of spaceflight. The goals of the Regulatory Physiology component of the EDOMP were to identify and subsequently ameliorate those biochemical and nutritional factors that deplete physiological reserves or increase risk for disease, and to facilitate the development of effective muscle, exercise, and cardiovascular countermeasures. The component investigations designed to meet these goals focused on biochemical and physiological aspects of nutrition and metabolism, the risk of renal (kidney) stone formation, gastrointestinal function, and sleep in space. Investigations involved both ground-based protocols to validate proposed methods and flight studies to test those methods. Two hardware tests were also completed.

  8. A genomic regulatory network for development

    NASA Technical Reports Server (NTRS)

    Davidson, Eric H.; Rast, Jonathan P.; Oliveri, Paola; Ransick, Andrew; Calestani, Cristina; Yuh, Chiou-Hwa; Minokawa, Takuya; Amore, Gabriele; Hinman, Veronica; Arenas-Mena, Cesar; Otim, Ochan; Brown, C. Titus; Livi, Carolina B.; Lee, Pei Yun; Revilla, Roger; Rust, Alistair G.; Pan, Zheng jun; Schilstra, Maria J.; Clarke, Peter J C.; Arnone, Maria I.; Rowen, Lee; Cameron, R. Andrew; McClay, David R.; Hood, Leroy; Bolouri, Hamid

    2002-01-01

    Development of the body plan is controlled by large networks of regulatory genes. A gene regulatory network that controls the specification of endoderm and mesoderm in the sea urchin embryo is summarized here. The network was derived from large-scale perturbation analyses, in combination with computational methodologies, genomic data, cis-regulatory analysis, and molecular embryology. The network contains over 40 genes at present, and each node can be directly verified at the DNA sequence level by cis-regulatory analysis. Its architecture reveals specific and general aspects of development, such as how given cells generate their ordained fates in the embryo and why the process moves inexorably forward in developmental time.

  9. Complete Genome Sequence of the Filamentous Fungus Aspergillus westerdijkiae Reveals the Putative Biosynthetic Gene Cluster of Ochratoxin A

    PubMed Central

    Chakrabortti, Alolika; Li, Jinming

    2016-01-01

    Ochratoxin A (OTA) is a common mycotoxin that contaminates food and agricultural products. Sequencing of the complete genome of Aspergillus westerdijkiae, a major producer of OTA, reveals more than 50 biosynthetic gene clusters, including a putative OTA biosynthetic gene cluster that encodes a dozen of enzymes, transporters, and regulatory proteins. PMID:27635003

  10. Complete Genome Sequence of the Filamentous Fungus Aspergillus westerdijkiae Reveals the Putative Biosynthetic Gene Cluster of Ochratoxin A.

    PubMed

    Chakrabortti, Alolika; Li, Jinming; Liang, Zhao-Xun

    2016-01-01

    Ochratoxin A (OTA) is a common mycotoxin that contaminates food and agricultural products. Sequencing of the complete genome of Aspergillus westerdijkiae, a major producer of OTA, reveals more than 50 biosynthetic gene clusters, including a putative OTA biosynthetic gene cluster that encodes a dozen of enzymes, transporters, and regulatory proteins. PMID:27635003

  11. LOESS correction for length variation in gene set-based genomic sequence analysis

    PubMed Central

    Aboukhalil, Anton; Bulyk, Martha L.

    2012-01-01

    Motivation: Sequence analysis algorithms are often applied to sets of DNA, RNA or protein sequences to identify common or distinguishing features. Controlling for sequence length variation is critical to properly score sequence features and identify true biological signals rather than length-dependent artifacts. Results: Several cis-regulatory module discovery algorithms exhibit a substantial dependence between DNA sequence score and sequence length. Our newly developed LOESS method is flexible in capturing diverse score-length relationships and is more effective in correcting DNA sequence scores for length-dependent artifacts, compared with four other approaches. Application of this method to genes co-expressed during Drosophila melanogaster embryonic mesoderm development or neural development scored by the Lever motif analysis algorithm resulted in successful recovery of their biologically validated cis-regulatory codes. The LOESS length-correction method is broadly applicable, and may be useful not only for more accurate inference of cis-regulatory codes, but also for detection of other types of patterns in biological sequences. Availability: Source code and compiled code are available from http://thebrain.bwh.harvard.edu/LM_LOESS/ Contact: mlbulyk@receptor.med.harvard.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22492312

  12. Making the Common Good Common

    ERIC Educational Resources Information Center

    Chase, Barbara

    2011-01-01

    How are independent schools to be useful to the wider world? Beyond their common commitment to educate their students for meaningful lives in service of the greater good, can they educate a broader constituency and, thus, share their resources and skills more broadly? Their answers to this question will be shaped by their independence. Any…

  13. Effects of gene regulatory reprogramming on gene expression in human and mouse developing hearts.

    PubMed

    Hsu, Chih-Hao; Ovcharenko, Ivan

    2013-01-01

    Lineage-specific regulatory elements underlie adaptation of species and play a role in disease susceptibility. We compared functionally conserved and lineage-specific enhancers by cross-mapping 5042 human and 6564 mouse heart enhancers. Of these, 79 per cent are lineage-specific, lacking a functional orthologue. Heart enhancers tend to cluster and, commonly, there are multiple heart enhancers in a heart locus providing a regulatory stability to the locus. We observed little cross-clustering, however, between lineage-specific and functionally conserved heart enhancers suggesting regulatory function acquisition and development in loci previously lacking heart activity. We also identified 862 human-specific heart enhancers: 417 featuring sequence conservation with mouse (class II) and 445 with neither sequence nor function conservation (class III). Ninety-eight per cent of class III enhancers were deleted from the mouse genome, and we estimated a similar-sized enhancer gain in the human lineage. Human-specific enhancers display no detectable decrease in the negative selection pressure and are strongly associated with genes partaking in the heart regulatory programmes. The loss of a heart enhancer could be compensated by activity of a redundant heart enhancer; however, we observed redundancy in only 15 per cent of class II and III enhancer loci indicating a large-scale reprogramming of the heart regulatory programme in mammals.

  14. 47 CFR 27.10 - Regulatory status.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 2 2011-10-01 2011-10-01 false Regulatory status. 27.10 Section 27.10 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Applications and Licenses § 27.10 Regulatory status. The following rules apply...

  15. 47 CFR 27.10 - Regulatory status.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 2 2014-10-01 2014-10-01 false Regulatory status. 27.10 Section 27.10 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Applications and Licenses § 27.10 Regulatory status. The following rules apply...

  16. 47 CFR 27.10 - Regulatory status.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 2 2013-10-01 2013-10-01 false Regulatory status. 27.10 Section 27.10 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Applications and Licenses § 27.10 Regulatory status. The following rules apply...

  17. 47 CFR 27.10 - Regulatory status.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Regulatory status. 27.10 Section 27.10 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Applications and Licenses § 27.10 Regulatory status. The following rules apply...

  18. 47 CFR 27.10 - Regulatory status.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 2 2012-10-01 2012-10-01 false Regulatory status. 27.10 Section 27.10 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Applications and Licenses § 27.10 Regulatory status. The following rules apply...

  19. 47 CFR 69.727 - Regulatory relief.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 3 2013-10-01 2013-10-01 false Regulatory relief. 69.727 Section 69.727 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES (CONTINUED) ACCESS CHARGES Pricing Flexibility § 69.727 Regulatory relief. (a) Phase I relief. Upon satisfaction of the Phase...

  20. 47 CFR 69.727 - Regulatory relief.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 3 2011-10-01 2011-10-01 false Regulatory relief. 69.727 Section 69.727 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES (CONTINUED) ACCESS CHARGES Pricing Flexibility § 69.727 Regulatory relief. (a) Phase I relief. Upon satisfaction of the Phase...

  1. 47 CFR 69.727 - Regulatory relief.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 3 2012-10-01 2012-10-01 false Regulatory relief. 69.727 Section 69.727 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES (CONTINUED) ACCESS CHARGES Pricing Flexibility § 69.727 Regulatory relief. (a) Phase I relief. Upon satisfaction of the Phase...

  2. 47 CFR 69.727 - Regulatory relief.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 3 2014-10-01 2014-10-01 false Regulatory relief. 69.727 Section 69.727 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES (CONTINUED) ACCESS CHARGES Pricing Flexibility § 69.727 Regulatory relief. (a) Phase I relief. Upon satisfaction of the Phase...

  3. 47 CFR 69.727 - Regulatory relief.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 3 2010-10-01 2010-10-01 false Regulatory relief. 69.727 Section 69.727 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES (CONTINUED) ACCESS CHARGES Pricing Flexibility § 69.727 Regulatory relief. (a) Phase I relief. Upon satisfaction of the Phase...

  4. Pleasure: the common currency.

    PubMed

    Cabanac, M

    1992-03-21

    At present as physiologists studying various homeostatic behaviors, such as thermoregulatory behavior and food and fluid intake, we have no common currency that allows us to equate the strength of the motivational drive that accompanies each regulatory need, in terms of how an animal or a person will choose to satisfy his needs when there is a conflict between two or more of them. Yet the behaving organism must rank his priorities and needs a common currency to achieve the ranking (McFarland & Sibly, 1975, Phil. Trans. R. Soc. Lond. 270 Biol 265-293). A theory is proposed here according to which pleasure is this common currency. The perception of pleasure, as measured operationally and quantitatively by choice behavior (in the case of animals), or by the rating of the intensity of pleasure or displeasure (in the case of humans) can serve as such a common currency. The tradeoffs between various motivations would thus be accomplished by simple maximization of pleasure. In what follows, the scientific work arising recently on this subject, with be reviewed briefly and our recent experimental findings will be presented. This will serve as the support for the theoretical position formulated in this essay.

  5. Pleasure: the common currency.

    PubMed

    Cabanac, M

    1992-03-21

    At present as physiologists studying various homeostatic behaviors, such as thermoregulatory behavior and food and fluid intake, we have no common currency that allows us to equate the strength of the motivational drive that accompanies each regulatory need, in terms of how an animal or a person will choose to satisfy his needs when there is a conflict between two or more of them. Yet the behaving organism must rank his priorities and needs a common currency to achieve the ranking (McFarland & Sibly, 1975, Phil. Trans. R. Soc. Lond. 270 Biol 265-293). A theory is proposed here according to which pleasure is this common currency. The perception of pleasure, as measured operationally and quantitatively by choice behavior (in the case of animals), or by the rating of the intensity of pleasure or displeasure (in the case of humans) can serve as such a common currency. The tradeoffs between various motivations would thus be accomplished by simple maximization of pleasure. In what follows, the scientific work arising recently on this subject, with be reviewed briefly and our recent experimental findings will be presented. This will serve as the support for the theoretical position formulated in this essay. PMID:12240693

  6. The limits of regulatory toxicology

    SciTech Connect

    Carrington, Clark D.; Bolger, P. Michael

    2010-03-01

    The Acceptable Daily Intake (ADI) has been used by regulatory and public health organizations (e.g., the U.S. Food and Drug and Administration, and the World Health Organization) for chemicals for more than 50 years. The ADI concept was also initially employed at the U.S. Environmental Protection Agency at its inception in 1971, although with the adoption of newer terminology, it later became known as the Reference Dose (RfD). It is clear from the literature that both were first devised as instruments of regulatory policy. In the intervening years, it has become common to use language that implies that these standards are statements of scientific fact. Similarly, some of the discretionary or default values that are used to derive regulatory standards are represented as scientific assumptions when in fact they also represent regulatory policy. This confusion impedes both the best use of the available science and informed public participation in policy making. In addition, the misconception of the ADI or the RfD as statements of scientific fact may impede the consideration of alternative means to reduce exposure to chemicals that may be harmful, including regulatory measures that do not involve prescribing a regulatory concentration limit.

  7. Plant nitrogen regulatory P-PII genes

    DOEpatents

    Coruzzi, Gloria M.; Lam, Hon-Ming; Hsieh, Ming-Hsiun

    2001-01-01

    The present invention generally relates to plant nitrogen regulatory PII gene (hereinafter P-PII gene), a gene involved in regulating plant nitrogen metabolism. The invention provides P-PII nucleotide sequences, expression constructs comprising said nucleotide sequences, and host cells and plants having said constructs and, optionally expressing the P-PII gene from said constructs. The invention also provides substantially pure P-PII proteins. The P-PII nucleotide sequences and constructs of the

  8. The common ancestry of life

    PubMed Central

    2010-01-01

    Background It is common belief that all cellular life forms on earth have a common origin. This view is supported by the universality of the genetic code and the universal conservation of multiple genes, particularly those that encode key components of the translation system. A remarkable recent study claims to provide a formal, homology independent test of the Universal Common Ancestry hypothesis by comparing the ability of a common-ancestry model and a multiple-ancestry model to predict sequences of universally conserved proteins. Results We devised a computational experiment on a concatenated alignment of universally conserved proteins which shows that the purported demonstration of the universal common ancestry is a trivial consequence of significant sequence similarity between the analyzed proteins. The nature and origin of this similarity are irrelevant for the prediction of "common ancestry" of by the model-comparison approach. Thus, homology (common origin) of the compared proteins remains an inference from sequence similarity rather than an independent property demonstrated by the likelihood analysis. Conclusion A formal demonstration of the Universal Common Ancestry hypothesis has not been achieved and is unlikely to be feasible in principle. Nevertheless, the evidence in support of this hypothesis provided by comparative genomics is overwhelming. Reviewers this article was reviewed by William Martin, Ivan Iossifov (nominated by Andrey Rzhetsky) and Arcady Mushegian. For the complete reviews, see the Reviewers' Report section. PMID:21087490

  9. Indexing Similar DNA Sequences

    NASA Astrophysics Data System (ADS)

    Huang, Songbo; Lam, T. W.; Sung, W. K.; Tam, S. L.; Yiu, S. M.

    To study the genetic variations of a species, one basic operation is to search for occurrences of patterns in a large number of very similar genomic sequences. To build an indexing data structure on the concatenation of all sequences may require a lot of memory. In this paper, we propose a new scheme to index highly similar sequences by taking advantage of the similarity among the sequences. To store r sequences with k common segments, our index requires only O(n + NlogN) bits of memory, where n is the total length of the common segments and N is the total length of the distinct regions in all texts. The total length of all sequences is rn + N, and any scheme to store these sequences requires Ω(n + N) bits. Searching for a pattern P of length m takes O(m + m logN + m log(rk)psc(P) + occlogn), where psc(P) is the number of prefixes of P that appear as a suffix of some common segments and occ is the number of occurrences of P in all sequences. In practice, rk ≤ N, and psc(P) is usually a small constant. We have implemented our solution and evaluated our solution using real DNA sequences. The experiments show that the memory requirement of our solution is much less than that required by BWT built on the concatenation of all sequences. When compared to the other existing solution (RLCSA), we use less memory with faster searching time.

  10. Common Cause Failure Modeling

    NASA Technical Reports Server (NTRS)

    Hark, Frank; Britton, Paul; Ring, Rob; Novack, Steven D.

    2015-01-01

    Common Cause Failures (CCFs) are a known and documented phenomenon that defeats system redundancy. CCFS are a set of dependent type of failures that can be caused by: system environments; manufacturing; transportation; storage; maintenance; and assembly, as examples. Since there are many factors that contribute to CCFs, the effects can be reduced, but they are difficult to eliminate entirely. Furthermore, failure databases sometimes fail to differentiate between independent and CCF (dependent) failure and data is limited, especially for launch vehicles. The Probabilistic Risk Assessment (PRA) of NASA's Safety and Mission Assurance Directorate at Marshall Space Flight Center (MFSC) is using generic data from the Nuclear Regulatory Commission's database of common cause failures at nuclear power plants to estimate CCF due to the lack of a more appropriate data source. There remains uncertainty in the actual magnitude of the common cause risk estimates for different systems at this stage of the design. Given the limited data about launch vehicle CCF and that launch vehicles are a highly redundant system by design, it is important to make design decisions to account for a range of values for independent and CCFs. When investigating the design of the one-out-of-two component redundant system for launch vehicles, a response surface was constructed to represent the impact of the independent failure rate versus a common cause beta factor effect on a system's failure probability. This presentation will define a CCF and review estimation calculations. It gives a summary of reduction methodologies and a review of examples of historical CCFs. Finally, it presents the response surface and discusses the results of the different CCFs on the reliability of a one-out-of-two system.

  11. Common Cause Failure Modeling

    NASA Technical Reports Server (NTRS)

    Hark, Frank; Britton, Paul; Ring, Rob; Novack, Steven D.

    2016-01-01

    Common Cause Failures (CCFs) are a known and documented phenomenon that defeats system redundancy. CCFS are a set of dependent type of failures that can be caused by: system environments; manufacturing; transportation; storage; maintenance; and assembly, as examples. Since there are many factors that contribute to CCFs, the effects can be reduced, but they are difficult to eliminate entirely. Furthermore, failure databases sometimes fail to differentiate between independent and CCF (dependent) failure and data is limited, especially for launch vehicles. The Probabilistic Risk Assessment (PRA) of NASA's Safety and Mission Assurance Directorate at Marshal Space Flight Center (MFSC) is using generic data from the Nuclear Regulatory Commission's database of common cause failures at nuclear power plants to estimate CCF due to the lack of a more appropriate data source. There remains uncertainty in the actual magnitude of the common cause risk estimates for different systems at this stage of the design. Given the limited data about launch vehicle CCF and that launch vehicles are a highly redundant system by design, it is important to make design decisions to account for a range of values for independent and CCFs. When investigating the design of the one-out-of-two component redundant system for launch vehicles, a response surface was constructed to represent the impact of the independent failure rate versus a common cause beta factor effect on a system's failure probability. This presentation will define a CCF and review estimation calculations. It gives a summary of reduction methodologies and a review of examples of historical CCFs. Finally, it presents the response surface and discusses the results of the different CCFs on the reliability of a one-out-of-two system.

  12. The last common bilaterian ancestor

    NASA Technical Reports Server (NTRS)

    Erwin, Douglas H.; Davidson, Eric H.

    2002-01-01

    Many regulatory genes appear to be utilized in at least superficially similar ways in the development of particular body parts in Drosophila and in chordates. These similarities have been widely interpreted as functional homologies, producing the conventional view of the last common protostome-deuterostome ancestor (PDA) as a complex organism that possessed some of the same body parts as modern bilaterians. Here we discuss an alternative view, in which the last common PDA had a less complex body plan than is frequently conceived. This reconstruction alters expectations for Neoproterozoic fossil remains that could illustrate the pathways of bilaterian evolution.

  13. Spectral sum rules and search for periodicities in DNA sequences

    NASA Astrophysics Data System (ADS)

    Chechetkin, V. R.

    2011-04-01

    Periodic patterns play the important regulatory and structural roles in genomic DNA sequences. Commonly, the underlying periodicities should be understood in a broad statistical sense, since the corresponding periodic patterns have been strongly distorted by the random point mutations and insertions/deletions during molecular evolution. The latent periodicities in DNA sequences can be efficiently displayed by Fourier transform. The criteria of significance for observed periodicities are obtained via the comparison versus the counterpart characteristics of the reference random sequences. We show that the restrictions imposed on the significance criteria by the rigorous spectral sum rules can be rationally described with De Finetti distribution. This distribution provides the convenient intermediate asymptotic form between Rayleigh distribution and exact combinatoric theory.

  14. Fungal Genes in Context: Genome Architecture Reflects Regulatory Complexity and Function

    PubMed Central

    Noble, Luke M.; Andrianopoulos, Alex

    2013-01-01

    Gene context determines gene expression, with local chromosomal environment most influential. Comparative genomic analysis is often limited in scope to conserved or divergent gene and protein families, and fungi are well suited to this approach with low functional redundancy and relatively streamlined genomes. We show here that one aspect of gene context, the amount of potential upstream regulatory sequence maintained through evolution, is highly predictive of both molecular function and biological process in diverse fungi. Orthologs with large upstream intergenic regions (UIRs) are strongly enriched in information processing functions, such as signal transduction and sequence-specific DNA binding, and, in the genus Aspergillus, include the majority of experimentally studied, high-level developmental and metabolic transcriptional regulators. Many uncharacterized genes are also present in this class and, by implication, may be of similar importance. Large intergenic regions also share two novel sequence characteristics, currently of unknown significance: they are enriched for plus-strand polypyrimidine tracts and an information-rich, putative regulatory motif that was present in the last common ancestor of the Pezizomycotina. Systematic consideration of gene UIR in comparative genomics, particularly for poorly characterized species, could help reveal organisms’ regulatory priorities. PMID:23699226

  15. Internationalization of regulatory requirements.

    PubMed

    Juillet, Y

    2003-02-01

    The aim of harmonisation of medicines regulatory requirements is to allow the patient quicker access to new drugs and to avoid animal and human duplications. Harmonisation in the European Union (EU) is now completed, and has led to the submission of one dossier in one language study leading to European marketing authorizations, thanks in particular to efficacy guidelines published at the European level. With the benefit of the European experience since 1989, more than 40 guidelines have been harmonised amongst the EU, Japan and the USA through the International Conference on Harmonisation (ICH). ICH is a unique process gathering regulators and industry experts from the three regions. Its activity is built on expertise and trust. The Common Technical Document (CTD), an agreed common format for application in the three regions, is a logical follow-up to the ICH first phase harmonising the content of the dossier. The CTD final implementation in July 2003 will have considerable influence on the review process and on the exchange of information in the three regions.

  16. Realistic artificial DNA sequences as negative controls for computational genomics

    PubMed Central

    Caballero, Juan; Smit, Arian F. A.; Hood, Leroy; Glusman, Gustavo

    2014-01-01

    A common practice in computational genomic analysis is to use a set of ‘background’ sequences as negative controls for evaluating the false-positive rates of prediction tools, such as gene identification programs and algorithms for detection of cis-regulatory elements. Such ‘background’ sequences are generally taken from regions of the genome presumed to be intergenic, or generated synthetically by ‘shuffling’ real sequences. This last method can lead to underestimation of false-positive rates. We developed a new method for generating artificial sequences that are modeled after real intergenic sequences in terms of composition, complexity and interspersed repeat content. These artificial sequences can serve as an inexhaustible source of high-quality negative controls. We used artificial sequences to evaluate the false-positive rates of a set of programs for detecting interspersed repeats, ab initio prediction of coding genes, transcribed regions and non-coding genes. We found that RepeatMasker is more accurate than PClouds, Augustus has the lowest false-positive rate of the coding gene prediction programs tested, and Infernal has a low false-positive rate for non-coding gene detection. A web service, source code and the models for human and many other species are freely available at http://repeatmasker.org/garlic/. PMID:24803667

  17. Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae.

    PubMed Central

    Goodman, S D; Scocca, J J

    1988-01-01

    DNA segments from Neisseria gonorrhoeae, cloned and propagated in Escherichia coli, were tested for the ability to competitively inhibit gonococcal transformation. The nucleotide sequences of active segments were determined and compared; these sequences contained the sequence 5' GCCGTCTGAA 3' in common. Subcloning studies confirmed the identity of this sequence as the gonococcal DNA recognition site. The three instances of the recognition sequence isolated from N. gonorrhoeae chromosomal DNA contain the sequence in the immediate neighborhood of its inverted repeat. Because a single copy of the sequence functions as a recognition site, the inverted duplication is not required for specific binding. The dyad symmetric arrangements of the chromosomal recognition sequences may form stable stem-loop structures that can function as terminators or attenuators of transcription. These inverted repeats are located at the boundaries of long open reading frames. The recognition sequence also constitutes part of two other probable terminators of gonococcal genes. We conclude that the signal for recognition of transforming DNA by gonococci is a frequent component of transcriptional terminator sequences. This regulatory function might account for the origin and maintenance of recognition sequences in the chromosomes of Gram-negative transformable bacteria. PMID:3137581

  18. Genomics in the land of regulatory science.

    PubMed

    Tong, Weida; Ostroff, Stephen; Blais, Burton; Silva, Primal; Dubuc, Martine; Healy, Marion; Slikker, William

    2015-06-01

    Genomics science has played a major role in the generation of new knowledge in the basic research arena, and currently question arises as to its potential to support regulatory processes. However, the integration of genomics in the regulatory decision-making process requires rigorous assessment and would benefit from consensus amongst international partners and research communities. To that end, the Global Coalition for Regulatory Science Research (GCRSR) hosted the fourth Global Summit on Regulatory Science (GSRS2014) to discuss the role of genomics in regulatory decision making, with a specific emphasis on applications in food safety and medical product development. Challenges and issues were discussed in the context of developing an international consensus for objective criteria in the analysis, interpretation and reporting of genomics data with an emphasis on transparency, traceability and "fitness for purpose" for the intended application. It was recognized that there is a need for a global path in the establishment of a regulatory bioinformatics framework for the development of transparent, reliable, reproducible and auditable processes in the management of food and medical product safety risks. It was also recognized that training is an important mechanism in achieving internationally consistent outcomes. GSRS2014 provided an effective venue for regulators andresearchers to meet, discuss common issues, and develop collaborations to address the challenges posed by the application of genomics to regulatory science, with the ultimate goal of wisely integrating novel technical innovations into regulatory decision-making.

  19. Target activation by regulatory RNAs in bacteria

    PubMed Central

    Papenfort, Kai; Vanderpool, Carin K.

    2015-01-01

    Bacterial small regulatory RNAs (sRNAs) are commonly known to repress gene expression by base pairing to target mRNAs. In many cases, sRNAs base pair with and sequester mRNA ribosome-binding sites, resulting in translational repression and accelerated transcript decay. In contrast, a growing number of examples of translational activation and mRNA stabilization by sRNAs have now been documented. A given sRNA often employs a conserved region to interact with and regulate both repressed and activated targets. However, the mechanisms underlying activation differ substantially from repression. Base pairing resulting in target activation can involve sRNA interactions with the 5′ untranslated region (UTR), the coding sequence or the 3′ UTR of the target mRNAs. Frequently, the activities of protein factors such as cellular ribonucleases and the RNA chaperone Hfq are required for activation. Bacterial sRNAs, including those that function as activators, frequently control stress response pathways or virulence-associated functions required for immediate responses to changing environments. This review aims to summarize recent advances in knowledge regarding target mRNA activation by bacterial sRNAs, highlighting the molecular mechanisms and biological relevance of regulation. PMID:25934124

  20. Raising the estimate of functional human sequences.

    PubMed

    Pheasant, Michael; Mattick, John S

    2007-09-01

    While less than 1.5% of the mammalian genome encodes proteins, it is now evident that the vast majority is transcribed, mainly into non-protein-coding RNAs. This raises the question of what fraction of the genome is functional, i.e., composed of sequences that yield functional products, are required for the expression (regulation or processing) of these products, or are required for chromosome replication and maintenance. Many of the observed noncoding transcripts are differentially expressed, and, while most have not yet been studied, increasing numbers are being shown to be functional and/or trafficked to specific subcellular locations, as well as exhibit subtle evidence of selection. On the other hand, analyses of conservation patterns indicate that only approximately 5% (3%-8%) of the human genome is under purifying selection for functions common to mammals. However, these estimates rely on the assumption that reference sequences (usually ancient transposon-derived sequences) have evolved neutrally, which may not be the case, and if so would lead to an underestimate of the fraction of the genome under evolutionary constraint. These analyses also do not detect functional sequences that are evolving rapidly and/or have acquired lineage-specific functions. Indeed, many regulatory sequences and known functional noncoding RNAs, including many microRNAs, are not conserved over significant evolutionary distances, and recent evidence from the ENCODE project suggests that many functional elements show no detectable level of sequence constraint. Thus, it is likely that much more than 5% of the genome encodes functional information, and although the upper bound is unknown, it may be considerably higher than currently thought.

  1. Regulatory gene networks and the properties of the developmental process

    NASA Technical Reports Server (NTRS)

    Davidson, Eric H.; McClay, David R.; Hood, Leroy

    2003-01-01

    Genomic instructions for development are encoded in arrays of regulatory DNA. These specify large networks of interactions among genes producing transcription factors and signaling components. The architecture of such networks both explains and predicts developmental phenomenology. Although network analysis is yet in its early stages, some fundamental commonalities are already emerging. Two such are the use of multigenic feedback loops to ensure the progressivity of developmental regulatory states and the prevalence of repressive regulatory interactions in spatial control processes. Gene regulatory networks make it possible to explain the process of development in causal terms and eventually will enable the redesign of developmental regulatory circuitry to achieve different outcomes.

  2. Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits

    PubMed Central

    Ravel, Catherine; Fiquet, Samuel; Boudet, Julie; Dardevet, Mireille; Vincent, Jonathan; Merlino, Marielle; Michard, Robin; Martre, Pierre

    2014-01-01

    The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression. PMID:25429295

  3. Evidence for a common evolutionary rate in metazoan transcriptional networks

    PubMed Central

    Carvunis, Anne-Ruxandra; Wang, Tina; Skola, Dylan; Yu, Alice; Chen, Jonathan; Kreisberg, Jason F; Ideker, Trey

    2015-01-01

    Genome sequences diverge more rapidly in mammals than in other animal lineages, such as birds or insects. However, the effect of this rapid divergence on transcriptional evolution remains unclear. Recent reports have indicated a faster divergence of transcription factor binding in mammals than in insects, but others found the reverse for mRNA expression. Here, we show that these conflicting interpretations resulted from differing methodologies. We performed an integrated analysis of transcriptional network evolution by examining mRNA expression, transcription factor binding and cis-regulatory motifs across >25 animal species, including mammals, birds and insects. Strikingly, we found that transcriptional networks evolve at a common rate across the three animal lineages. Furthermore, differences in rates of genome divergence were greatly reduced when restricting comparisons to chromatin-accessible sequences. The evolution of transcription is thus decoupled from the global rate of genome sequence evolution, suggesting that a small fraction of the genome regulates transcription. DOI: http://dx.doi.org/10.7554/eLife.11615.001 PMID:26682651

  4. Environmental regulatory update table

    SciTech Connect

    Brown, K.J.; Langston, M.E.; Tucker, C.S.; Reed, R.M.

    1987-06-01

    The Environmental Regulatory Update Table provides information on regulatory initiatives of interest to DOE operations and contractor staff with environmental management responsibilities. The table is updated each month with information from the Federal Register and other sources, including direct contact with regulatory agencies. Each table entry provides a chronological record of the rulemaking process for that initiative with an abstract and a projection of further action.

  5. Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core.

    PubMed Central

    Morris, J F; Hromas, R; Rauscher, F J

    1994-01-01

    The myeloid zinc finger gene 1, MZF1, encodes a transcription factor which is expressed in hematopoietic progenitor cells that are committed to myeloid lineage differentiation. MZF1 contains 13 C2H2 zinc fingers arranged in two domains which are separated by a short glycine- and proline-rich sequence. The first domain consists of zinc fingers 1 to 4, and the second domain is formed by zinc fingers 5 to 13. We have determined that both sets of zinc finger domains bind DNA. Purified, recombinant MZF1 proteins containing either the first set of zinc fingers or the second set were prepared and used to affinity select DNA sequences from a library of degenerate oligonucleotides by using successive rounds of gel shift followed by PCR amplification. Surprisingly, both DNA-binding domains of MZF1 selected similar DNA-binding consensus sequences containing a core of four or five guanine residues, reminiscent of an NF-kappa B half-site: 1-4, 5'-AGTGGGGA-3'; 5-13, 5'-CGGGnGAGGGGGAA-3'. The full-length MZF1 protein containing both sets of zinc finger DNA-binding domains recognizes synthetic oligonucleotides containing either the 1-4 or 5-13 consensus binding sites in gel shift assays. Thus, we have identified the core DNA consensus binding sites for each of the two DNA-binding domains of a myeloid-specific zinc finger transcription factor. Identification of these DNA-binding sites will allow us to identify target genes regulated by MZF1 and to assess the role of MZF1 as a transcriptional regulator of hematopoiesis. Images PMID:8114711

  6. Automated conserved non-coding sequence (CNS) discovery reveals differences in gene content and promoter evolution among grasses

    PubMed Central

    Turco, Gina; Schnable, James C.; Pedersen, Brent; Freeling, Michael

    2013-01-01

    Conserved non-coding sequences (CNS) are islands of non-coding sequence that, like protein coding exons, show less divergence in sequence between related species than functionless DNA. Several CNSs have been demonstrated experimentally to function as cis-regulatory regions. However, the specific functions of most CNSs remain unknown. Previous searches for CNS in plants have either anchored on exons and only identified nearby sequences or required years of painstaking manual annotation. Here we present an open source tool that can accurately identify CNSs between any two related species with sequenced genomes, including both those immediately adjacent to exons and distal sequences separated by >12 kb of non-coding sequence. We have used this tool to characterize new motifs, associate CNSs with additional functions, and identify previously undetected genes encoding RNA and protein in the genomes of five grass species. We provide a list of 15,363 orthologous CNSs conserved across all grasses tested. We were also able to identify regulatory sequences present in the common ancestor of grasses that have been lost in one or more extant grass lineages. Lists of orthologous gene pairs and associated CNSs are provided for reference inbred lines of arabidopsis, Japonica rice, foxtail millet, sorghum, brachypodium, and maize. PMID:23874343

  7. Definition of a GC-rich motif as regulatory sequence of the human IL-3 gene: coordinate regulation of the IL-3 gene by CLE2/GC box of the GM-CSF gene in T cell activation.

    PubMed

    Nishida, J; Yoshida, M; Arai, K; Yokota, T

    1991-03-01

    The human IL-3 gene, located on chromosome 5, contains several cis-acting DNA sequences, i.e. CLE (conserved lymphokine element) and a GC-rich region, similar to the GM-CSF gene. To investigate the role of these elements, the 5' flanking region of the IL-3 gene was attached to a bacterial chloramphenicol acetyltransferase (CAT) gene. The fusion plasmids were analyzed by an in vitro transcription system using Jurkat cell nuclear extract prepared from cells stimulated with phorbol-12-myristate-13-acetate and calcium ionophore (PMA/A23187), introduced into Jurkat cells, expressed transiently, and stimulated by co-transfection of human T cell leukemia virus type I (HTLV-I) encoded transactivator, p40tax. The GC-rich region enhanced TATA-dependent transcription in the in vitro transcription system and also strongly responded to p40tax stimulation in the in vivo cotransfection assay. Using this GC-rich region as a probe, we identified a constitutive DNA-protein complex, alpha, whose binding specificity correlates with transcription activity. However, this element is not sufficient for the expression of the IL-3 gene in response to T cell activation signals (PMA/A23187) and no sequence was found within the IL-3 gene which mediates the response to PMA/A23187. The enhancer sequence which responds to T cell activation signals may be located outside the IL-3 gene and may be shared by other lymphokines, possibly by GM-CSF. We propose that the GM-CSF enhancer (CLE2/GC box) which mediates the response to T cell activation signals may stimulate the expression of the IL-3 gene. PMID:2049340

  8. Evolutionary forces act on promoter length: identification of enriched cis-regulatory elements.

    PubMed

    Kristiansson, Erik; Thorsen, Michael; Tamás, Markus J; Nerman, Olle

    2009-06-01

    Transcription factors govern gene expression by binding to short DNA sequences called cis-regulatory elements. These sequences are typically located in promoters, which are regions of variable length upstream of the open reading frames of genes. Here, we report that promoter length and gene function are related in yeast, fungi, and plants. In particular, the promoters for stress-responsive genes are in general longer than those of other genes. Essential genes have, on the other hand, relatively short promoters. We utilize these findings in a novel method for identifying relevant cis-regulatory elements in a set of coexpressed genes. The method is shown to generate more accurate results and fewer false positives compared with other common procedures. Our results suggest that genes with complex transcriptional regulation tend to have longer promoters than genes responding to few signals. This phenomenon is present in all investigated species, indicating that evolution adjust promoter length according to gene function. Identification of cis-regulatory elements in Saccharomyces cerevisiae can be done with the web service located at http://enricher.zool.gu.se.

  9. Interpreting the biological relevance of bioinformatic analyses with T-DNA sequence for protein allergenicity.

    PubMed

    Harper, B; McClain, S; Ganko, E W

    2012-08-01

    Global regulatory agencies require bioinformatic sequence analysis as part of their safety evaluation for transgenic crops. Analysis typically focuses on encoded proteins and adjacent endogenous flanking sequences. Recently, regulatory expectations have expanded to include all reading frames of the inserted DNA. The intent is to provide biologically relevant results that can be used in the overall assessment of safety. This paper evaluates the relevance of assessing the allergenic potential of all DNA reading frames found in common food genes using methods considered for the analysis of T-DNA sequences used in transgenic crops. FASTA and BLASTX algorithms were used to compare genes from maize, rice, soybean, cucumber, melon, watermelon, and tomato using international regulatory guidance. Results show that BLASTX for maize yielded 7254 alignments that exceeded allergen similarity thresholds and 210,772 alignments that matched eight or more consecutive amino acids with an allergen; other crops produced similar results. This analysis suggests that each nontransgenic crop has a much greater potential for allergenic risk than what has been observed clinically. We demonstrate that a meaningful safety assessment is unlikely to be provided by using methods with inherently high frequencies of false positive alignments when broadly applied to all reading frames of DNA sequence.

  10. MISAE: a new approach for regulatory motif extraction.

    PubMed

    Sun, Zhaohui; Yang, Jingyi; Deogun, Jitender S

    2004-01-01

    The recognition of regulatory motifs of co-regulated genes is essential for understanding the regulatory mechanisms. However, the automatic extraction of regulatory motifs from a given data set of the upstream non-coding DNA sequences of a family of co-regulated genes is difficult because regulatory motifs are often subtle and inexact. This problem is further complicated by the corruption of the data sets. In this paper, a new approach called Mismatch-allowed Probabilistic Suffix Tree Motif Extraction (MISAE) is proposed. It combines the mismatch-allowed probabilistic suffix tree that is a probabilistic model and local prediction for the extraction of regulatory motifs. The proposed approach is tested on 15 co-regulated gene families and compares favorably with other state-of-the-art approaches. Moreover, MISAE performs well on "corrupted" data sets. It is able to extract the motif from a "corrupted" data set with less than one fourth of the sequences containing the real motif.

  11. metagene Profiles Analyses Reveal Regulatory Element’s Factor-Specific Recruitment Patterns

    PubMed Central

    Samb, Rawane; Lemaçon, Audrey; Bilodeau, Steve; Droit, Arnaud

    2016-01-01

    ChIP-Sequencing (ChIP-Seq) provides a vast amount of information regarding the localization of proteins across the genome. The aggregation of ChIP-Seq enrichment signal in a metagene plot is an approach commonly used to summarize data complexity and to obtain a high level visual representation of the general occupancy pattern of a protein. Here we present the R package metagene, the graphical interface Imetagene and the companion package similaRpeak. Together, they provide a framework to integrate, summarize and compare the ChIP-Seq enrichment signal from complex experimental designs. Those packages identify and quantify similarities or dissimilarities in patterns between large numbers of ChIP-Seq profiles. We used metagene to investigate the differential occupancy of regulatory factors at noncoding regulatory regions (promoters and enhancers) in relation to transcriptional activity in GM12878 B-lymphocytes. The relationships between occupancy patterns and transcriptional activity suggest two different mechanisms of action for transcriptional control: i) a “gradient effect” where the regulatory factor occupancy levels follow transcription and ii) a “threshold effect” where the regulatory factor occupancy levels max out prior to reaching maximal transcription. metagene, Imetagene and similaRpeak are implemented in R under the Artistic license 2.0 and are available on Bioconductor. PMID:27538250

  12. metagene Profiles Analyses Reveal Regulatory Element's Factor-Specific Recruitment Patterns.

    PubMed

    Joly Beauparlant, Charles; Lamaze, Fabien C; Deschênes, Astrid; Samb, Rawane; Lemaçon, Audrey; Belleau, Pascal; Bilodeau, Steve; Droit, Arnaud

    2016-08-01

    ChIP-Sequencing (ChIP-Seq) provides a vast amount of information regarding the localization of proteins across the genome. The aggregation of ChIP-Seq enrichment signal in a metagene plot is an approach commonly used to summarize data complexity and to obtain a high level visual representation of the general occupancy pattern of a protein. Here we present the R package metagene, the graphical interface Imetagene and the companion package similaRpeak. Together, they provide a framework to integrate, summarize and compare the ChIP-Seq enrichment signal from complex experimental designs. Those packages identify and quantify similarities or dissimilarities in patterns between large numbers of ChIP-Seq profiles. We used metagene to investigate the differential occupancy of regulatory factors at noncoding regulatory regions (promoters and enhancers) in relation to transcriptional activity in GM12878 B-lymphocytes. The relationships between occupancy patterns and transcriptional activity suggest two different mechanisms of action for transcriptional control: i) a "gradient effect" where the regulatory factor occupancy levels follow transcription and ii) a "threshold effect" where the regulatory factor occupancy levels max out prior to reaching maximal transcription. metagene, Imetagene and similaRpeak are implemented in R under the Artistic license 2.0 and are available on Bioconductor. PMID:27538250

  13. High-Throughput Sequencing Technologies

    PubMed Central

    Reuter, Jason A.; Spacek, Damek; Snyder, Michael P.

    2015-01-01

    Summary The human genome sequence has profoundly altered our understanding of biology, human diversity and disease. The path from the first draft sequence to our nascent era of personal genomes and genomic medicine has been made possible only because of the extraordinary advancements in DNA sequencing technologies over the past ten years. Here, we discuss commonly used high-throughput sequencing platforms, the growing array of sequencing assays developed around them as well as the challenges facing current sequencing platforms and their clinical application. PMID:26000844

  14. Nuclear Regulatory Commission Semiannual Regulatory Agenda

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-20

    ... agenda on April 26, 2010 (75 FR 21960). For this edition of the NRC's regulatory agenda, the most... publication of the last NRC semiannual agenda on April 26, 2010 (75 FR 21960). Within each group, the rules... regulations to improve the control over the distribution of source material to exempt persons and to...

  15. No Common Opinion on the Common Core

    ERIC Educational Resources Information Center

    Henderson, Michael B.; Peterson, Paul E.; West, Martin R.

    2015-01-01

    According to the three authors of this article, the 2014 "EdNext" poll yields four especially important new findings: (1) Opinion with respect to the Common Core has yet to coalesce. The idea of a common set of standards across the country has wide appeal, and the Common Core itself still commands the support of a majority of the public.…

  16. Contamination of sequence databases with adaptor sequences

    SciTech Connect

    Yoshikawa, Takeo; Sanders, A.R.; Detera-Wadleigh, S.D.

    1997-02-01

    Because of the exponential increase in the amount of DNA sequences being added to the public databases on a daily basis, it has become imperative to identify sources of contamination rapidly. Previously, contaminations of sequence databases have been reported to alert the scientific community to the problem. These contaminations can be divided into two categories. The first category comprises host sequences that have been difficult for submitters to manage or control. Examples include anomalous sequences derived from Escherichia coli, which are inserted into the chromosomes (and plasmids) of the bacterial hosts. Insertion sequences are highly mobile and are capable of transposing themselves into plasmids during cloning manipulation. Another example of the first category is the infection with yeast genomic DNA or with bacterial DNA of some commercially available cDNA libraries from Clontech. The second category of database contamination is due to the inadvertent inclusion of nonhost sequences. This category includes incorporation of cloning-vector sequences and multicloning sites in the database submission. M13-derived artifacts have been common, since M13-based vectors have been widely used for subcloning DNA fragments. Recognizing this problem, the National Center for Biotechnology Information (NCBI) started to screen, in April 1994, all sequences directly submitted to GenBank, against a set of vector data retrieved from GenBank by use of key-word searches, such as {open_quotes}vector.{close_quotes} In this report, we present evidence for another sequence artifact that is widespread but that, to our knowledge, has not yet been reported. 11 refs., 1 tab.

  17. Mapping the Shh long-range regulatory domain

    PubMed Central

    Anderson, Eve; Devenney, Paul S.; Hill, Robert E.; Lettice, Laura A.

    2014-01-01

    Coordinated gene expression controlled by long-distance enhancers is orchestrated by DNA regulatory sequences involving transcription factors and layers of control mechanisms. The Shh gene and well-established regulators are an example of genomic composition in which enhancers reside in a large desert extending into neighbouring genes to control the spatiotemporal pattern of expression. Exploiting the local hopping activity of the Sleeping Beauty transposon, the lacZ reporter gene was dispersed throughout the Shh region to systematically map the genomic features responsible for expression activity. We found that enhancer activities are retained inside a genomic region that corresponds to the topological associated domain (TAD) defined by Hi-C. This domain of approximately 900 kb is in an open conformation over its length and is generally susceptible to all Shh enhancers. Similar to the distal enhancers, an enhancer residing within the Shh second intron activates the reporter gene located at distances of hundreds of kilobases away, suggesting that both proximal and distal enhancers have the capacity to survey the Shh topological domain to recognise potential promoters. The widely expressed Rnf32 gene lying within the Shh domain evades enhancer activities by a process that may be common among other housekeeping genes that reside in large regulatory domains. Finally, the boundaries of the Shh TAD do not represent the absolute expression limits of enhancer activity, as expression activity is lost stepwise at a number of genomic positions at the verges of these domains. PMID:25252942

  18. Mapping the Shh long-range regulatory domain.

    PubMed

    Anderson, Eve; Devenney, Paul S; Hill, Robert E; Lettice, Laura A

    2014-10-01

    Coordinated gene expression controlled by long-distance enhancers is orchestrated by DNA regulatory sequences involving transcription factors and layers of control mechanisms. The Shh gene and well-established regulators are an example of genomic composition in which enhancers reside in a large desert extending into neighbouring genes to control the spatiotemporal pattern of expression. Exploiting the local hopping activity of the Sleeping Beauty transposon, the lacZ reporter gene was dispersed throughout the Shh region to systematically map the genomic features responsible for expression activity. We found that enhancer activities are retained inside a genomic region that corresponds to the topological associated domain (TAD) defined by Hi-C. This domain of approximately 900 kb is in an open conformation over its length and is generally susceptible to all Shh enhancers. Similar to the distal enhancers, an enhancer residing within the Shh second intron activates the reporter gene located at distances of hundreds of kilobases away, suggesting that both proximal and distal enhancers have the capacity to survey the Shh topological domain to recognise potential promoters. The widely expressed Rnf32 gene lying within the Shh domain evades enhancer activities by a process that may be common among other housekeeping genes that reside in large regulatory domains. Finally, the boundaries of the Shh TAD do not represent the absolute expression limits of enhancer activity, as expression activity is lost stepwise at a number of genomic positions at the verges of these domains. PMID:25252942

  19. Transcriptome and genome sequencing uncovers functional variation in humans

    PubMed Central

    Lappalainen, Tuuli; Sammeth, Michael; Friedländer, Marc R; ‘t Hoen, Peter AC; Monlong, Jean; Rivas, Manuel A; Gonzàlez-Porta, Mar; Kurbatova, Natalja; Griebel, Thasso; Ferreira, Pedro G; Barann, Matthias; Wieland, Thomas; Greger, Liliana; van Iterson, Maarten; Almlöf, Jonas; Ribeca, Paolo; Pulyakhina, Irina; Esser, Daniela; Giger, Thomas; Tikhonov, Andrew; Sultan, Marc; Bertier, Gabrielle; MacArthur, Daniel G; Lek, Monkol; Lizano, Esther; Buermans, Henk PJ; Padioleau, Ismael; Schwarzmayr, Thomas; Karlberg, Olof; Ongen, Halit; Kilpinen, Helena; Beltran, Sergi; Gut, Marta; Kahlem, Katja; Amstislavskiy, Vyacheslav; Stegle, Oliver; Pirinen, Matti; Montgomery, Stephen B; Donnelly, Peter; McCarthy, Mark I; Flicek, Paul; Strom, Tim M; Lehrach, Hans; Schreiber, Stefan; Sudbrak, Ralf; Carracedo, Ángel; Antonarakis, Stylianos E; Häsler, Robert; Syvänen, Ann-Christine; van Ommen, Gert-Jan; Brazma, Alvis; Meitinger, Thomas; Rosenstiel, Philip; Guigó, Roderic; Gut, Ivo G; Estivill, Xavier; Dermitzakis, Emmanouil T

    2013-01-01

    Summary Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of mRNA and miRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project – the first uniformly processed RNA-seq data from multiple human populations with high-quality genome sequences. We discovered extremely widespread genetic variation affecting regulation of the majority of genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on cellular mechanisms of regulatory and loss-of-function variation, and allowed us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome. PMID:24037378

  20. Plant nitrogen regulatory P-PII polypeptides

    DOEpatents

    Coruzzi, Gloria M.; Lam, Hon-Ming; Hsieh, Ming-Hsiun

    2004-11-23

    The present invention generally relates to plant nitrogen regulatory PII gene (hereinafter P-PII gene), a gene involved in regulating plant nitrogen metabolism. The invention provides P-PII nucleotide sequences, expression constructs comprising said nucleotide sequences, and host cells and plants having said constructs and, optionally expressing the P-PII gene from said constructs. The invention also provides substantially pure P-PII proteins. The P-PII nucleotide sequences and constructs of the invention may be used to engineer organisms to overexpress wild-type or mutant P-PII regulatory protein. Engineered plants that overexpress or underexpress P-PII regulatory protein may have increased nitrogen assimilation capacity. Engineered organisms may be used to produce P-PII proteins which, in turn, can be used for a variety of purposes including in vitro screening of herbicides. P-PII nucleotide sequences have additional uses as probes for isolating additional genomic clones having the promoters of P-PII gene. P-PII promoters are light- and/or sucrose-inducible and may be advantageously used in genetic engineering of plants.

  1. Variation in Vertebrate Cis-Regulatory Elements in Evolution and Disease

    PubMed Central

    Douglas, Adam Thomas; Hill, Robert E

    2014-01-01

    Much of the genetic information that drives animal diversity lies within the vast non-coding regions of the genome. Multi-species sequence conservation in non-coding regions of the genome flags important regulatory elements and more recently, techniques that look for functional signatures predicted for regulatory sequences have added to the identification of thousands more. For some time, biologists have argued that changes in cis-regulatory sequences creates the basic genetic framework for evolutionary change. Recent advances support this notion and show that there is extensive genomic variability in non-coding regulatory elements associated with trait variation, speciation and disease. PMID:25764334

  2. Genetic landscape of APOE in human longevity revealed by high-throughput sequencing.

    PubMed

    Ryu, Seungjin; Atzmon, Gil; Barzilai, Nir; Raghavachari, Nalini; Suh, Yousin

    2016-04-01

    Apolipoprotein E (APOE) gene has been the most replicated longevity-associated gene in humans. Two common APOE alleles are either significantly depleted (ε4 allele) or enriched (ε2 allele) in long-lived individuals as compared to controls. We performed high-throughput sequencing analysis of exons and 2kb proximal promoter of APOE in 450 centenarians and 500 controls of Ashkenazi Jewish decent. We found two common regulatory variants, rs405509 (p=0.006) and rs769449 (p=0.036), that were significantly depleted in centenarians. Genotyping analysis of rs7412 and rs429358 showed significant enrichment of ε2 allele (p=0.003) and ε2/ε3 genotype (p=0.005), and significant depletion of ε3/ε4 genotype (p=0.005) in centenarians. Our findings support the hypothesis that variants in both coding and regulatory regions of APOE may contribute to longevity in humans. PMID:26930295

  3. Responsive Bureaus, Equity, and Regulatory Negotiation: An Empirical View

    ERIC Educational Resources Information Center

    Langbein, Laura I.

    2002-01-01

    Rulemaking agencies commonly delegate the implementation and enforcement of rules to affected parties, but they rarely delegate rulemaking authority. Regulatory negotiation is an example of this uncommon behavior. Compared to conventional rulemaking, regulatory negotiation is thought to be an attempt to make bureaucracy more responsive to affected…

  4. Potential use of DNA barcodes in regulatory science: applications of the Regulatory Fish Encyclopedia.

    PubMed

    Yancy, Haile F; Zemlak, Tyler S; Mason, Jacquline A; Washington, Jewell D; Tenge, Bradley J; Nguyen, Ngoc-Lan T; Barnett, James D; Savary, Warren E; Hill, Walter E; Moore, Michelle M; Fry, Frederick S; Randolph, Spring C; Rogers, Patricia L; Hebert, Paul D N

    2008-01-01

    The use of a DNA-based identification system (DNA barcoding) founded on the mitochondrial gene cytochrome c oxidase subunit I (COI) was investigated for updating the U.S. Food and Drug Administration Regulatory Fish Encyclopedia (RFE; http://www.cfsan.fda.gov/-frf/rfe0.html). The RFE is a compilation of data used to identify fish species. It was compiled to help regulators identify species substitution that could result in potential adverse health consequences or could be a source of economic fraud. For each of many aquatic species commonly sold in the United States, the RFE includes high-resolution photographs of whole fish and their marketed product forms and species-specific biochemical patterns for authenticated fish species. These patterns currently include data from isoelectric focusing studies. In this article, we describe the generation of DNA barcodes for 172 individual authenticated fish representing 72 species from 27 families contained in the RFE. These barcode sequences can be used as an additional identification resource. In a blind study, 60 unknown fish muscle samples were barcoded, and the results were compared with the RFE barcode reference library. All 60 samples were correctly identified to species based on the barcoding data. Our study indicates that DNA barcoding can be a powerful tool for species identification and has broad potential applications.

  5. Hebbian learning for olfactory sequences.

    PubMed

    Johnson, Andrew J; Cauchi, Laura; Miles, Christopher

    2013-06-01

    The present paper explores the generality of the Hebb repetition effect to the learning of olfactory sequences in order to assess commonality of memory functioning across sensory modalities. Participants completed a serial-order reconstruction task comprising sequences of four olfactory stimuli. Following presentation of each sequence, participants were re-presented with the odours and were required to reconstruct their order of presentation. Surreptitious re-presentation of the repeated sequence occurred on every third trial. This order reconstruction task produced a serial-position function comprising recency only for both the non-repeated and the repeated sequences. Importantly, serial-order reconstruction for the repeated odour sequence produced improved performance for that sequence compared to the non-repeated sequences. This observation of a Hebb repetition effect for olfactory sequences further supports the proposition that sequential learning can operate amodally.

  6. Regulatory guidance document

    SciTech Connect

    1994-05-01

    The Office of Civilian Radioactive Waste Management (OCRWM) Program Management System Manual requires preparation of the OCRWM Regulatory Guidance Document (RGD) that addresses licensing, environmental compliance, and safety and health compliance. The document provides: regulatory compliance policy; guidance to OCRWM organizational elements to ensure a consistent approach when complying with regulatory requirements; strategies to achieve policy objectives; organizational responsibilities for regulatory compliance; guidance with regard to Program compliance oversight; and guidance on the contents of a project-level Regulatory Compliance Plan. The scope of the RGD includes site suitability evaluation, licensing, environmental compliance, and safety and health compliance, in accordance with the direction provided by Section 4.6.3 of the PMS Manual. Site suitability evaluation and regulatory compliance during site characterization are significant activities, particularly with regard to the YW MSA. OCRWM`s evaluation of whether the Yucca Mountain site is suitable for repository development must precede its submittal of a license application to the Nuclear Regulatory Commission (NRC). Accordingly, site suitability evaluation is discussed in Chapter 4, and the general statements of policy regarding site suitability evaluation are discussed in Section 2.1. Although much of the data and analyses may initially be similar, the licensing process is discussed separately in Chapter 5. Environmental compliance is discussed in Chapter 6. Safety and Health compliance is discussed in Chapter 7.

  7. Uncovering drug-responsive regulatory elements

    PubMed Central

    Luizon, Marcelo R; Ahituv, Nadav

    2016-01-01

    Nucleotide changes in gene regulatory elements can have a major effect on interindividual differences in drug response. For example, by reviewing all published pharmacogenomic genome-wide association studies, we show here that 96.4% of the associated single nucleotide polymorphisms reside in noncoding regions. We discuss how sequencing technologies are improving our ability to identify drug response-associated regulatory elements genome-wide and to annotate nucleotide variants within them. We highlight specific examples of how nucleotide changes in these elements can affect drug response and illustrate the techniques used to find them and functionally characterize them. Finally, we also discuss challenges in the field of drug-responsive regulatory elements that need to be considered in order to translate these findings into the clinic. PMID:26555224

  8. Motif co-regulation and co-operativity are common mechanisms in transcriptional, post-transcriptional and post-translational regulation.

    PubMed

    Van Roey, Kim; Davey, Norman E

    2015-01-01

    A substantial portion of the regulatory interactions in the higher eukaryotic cell are mediated by simple sequence motifs in the regulatory segments of genes and (pre-)mRNAs, and in the intrinsically disordered regions of proteins. Although these regulatory modules are physicochemically distinct, they share an evolutionary plasticity that has facilitated a rapid growth of their use and resulted in their ubiquity in complex organisms. The ease of motif acquisition simplifies access to basal housekeeping functions, facilitates the co-regulation of multiple biomolecules allowing them to respond in a coordinated manner to changes in the cell state, and supports the integration of multiple signals for combinatorial decision-making. Consequently, motifs are indispensable for temporal, spatial, conditional and basal regulation at the transcriptional, post-transcriptional and post-translational level. In this review, we highlight that many of the key regulatory pathways of the cell are recruited by motifs and that the ease of motif acquisition has resulted in large networks of co-regulated biomolecules. We discuss how co-operativity allows simple static motifs to perform the conditional regulation that underlies decision-making in higher eukaryotic biological systems. We observe that each gene and its products have a unique set of DNA, RNA or protein motifs that encode a regulatory program to define the logical circuitry that guides the life cycle of these biomolecules, from transcription to degradation. Finally, we contrast the regulatory properties of protein motifs and the regulatory elements of DNA and (pre-)mRNAs, advocating that co-regulation, co-operativity, and motif-driven regulatory programs are common mechanisms that emerge from the use of simple, evolutionarily plastic regulatory modules. PMID:26626130

  9. A regulatory role for Staphylococcus aureus toxin-antitoxin system PemIKSa.

    PubMed

    Bukowski, Michal; Lyzen, Robert; Helbin, Weronika M; Bonar, Emilia; Szalewska-Palasz, Agnieszka; Wegrzyn, Grzegorz; Dubin, Grzegorz; Dubin, Adam; Wladyka, Benedykt

    2013-01-01

    Toxin-antitoxin systems were shown to be involved in plasmid maintenance when they were initially discovered, but other roles have been demonstrated since. Here we identify and characterize a novel toxin-antitoxin system (pemIKSa) located on Staphylococcus aureus plasmid pCH91. The toxin (PemKSa) is a sequence-specific endoribonuclease recognizing the tetrad sequence U↓AUU, and the antitoxin (PemISa) inhibits toxin activity by physical interaction. Although the toxin-antitoxin system is responsible for stable plasmid maintenance our data suggest the participation of pemIKSa in global regulation of staphylococcal virulence by alteration of the translation of large pools of genes. We propose a common mechanism of reversible activation of toxin-antitoxin systems based on antitoxin transcript resistance to toxin cleavage. Elucidation of this mechanism is particularly interesting because reversible activation is a prerequisite for the proposed general regulatory role of toxin-antitoxin systems.

  10. Regulatory principles governing Salmonella and Yersinia virulence

    PubMed Central

    Erhardt, Marc; Dersch, Petra

    2015-01-01

    Enteric pathogens such as Salmonella and Yersinia evolved numerous strategies to survive and proliferate in different environmental reservoirs and mammalian hosts. Deciphering common and pathogen-specific principles for how these bacteria adjust and coordinate spatiotemporal expression of virulence determinants, stress adaptation, and metabolic functions is fundamental to understand microbial pathogenesis. In order to manage sudden environmental changes, attacks by the host immune systems and microbial competition, the pathogens employ a plethora of transcriptional and post-transcriptional control elements, including transcription factors, sensory and regulatory RNAs, RNAses, and proteases, to fine-tune and control complex gene regulatory networks. Many of the contributing global regulators and the molecular mechanisms of regulation are frequently conserved between Yersinia and Salmonella. However, the interplay, arrangement, and composition of the control elements vary between these closely related enteric pathogens, which generate phenotypic differences leading to distinct pathogenic properties. In this overview we present common and different regulatory networks used by Salmonella and Yersinia to coordinate the expression of crucial motility, cell adhesion and invasion determinants, immune defense strategies, and metabolic adaptation processes. We highlight evolutionary changes of the gene regulatory circuits that result in different properties of the regulatory elements and how this influences the overall outcome of the infection process. PMID:26441883

  11. Quantitative Sequencing for the Determination of Kdr-type Resistance Allele (V419L, L925I, I936F) Frequencies in Common Bed Bug (Hemiptera: Cimicidae) Populations Collected from Israel.

    PubMed

    Palenchar, Daniel J; Gellatly, Kyle J; Yoon, Kyong Sup; Mumcuoglu, Kosta Y; Shalom, Uri; Clark, J Marshall

    2015-09-01

    Human bed bug infestations have dramatically increased worldwide since the mid-1990s. A similar phenomenon was also observed in Israel since 2005, when infestations were reported from all over the country. Two single nucleotide polymorphisms (V419L and L925I) in the bed bug voltage-sensitive sodium channel confer kdr-type resistance to pyrethroids. Using quantitative sequencing (QS), the resistance allele frequencies of Israeli bed bug populations from across the country were determined. Genomic DNA was extracted from samples of 12 populations of bed bugs collected from Israel and DNA fragments containing the V419L or L925I and I936F mutations sites were PCR amplified. The PCR products were analyzed by QS and the nucleotide signal ratios calculated and used to predict the resistance allele frequencies of the unknown populations. Results of the genetic analysis show that resistant nucleotide signals are highly correlated to resistance allele frequencies for both mutations. Ten of the 12 tested populations had 100% of the L925I mutation and 0% of the V419L mutation. One population was heterogeneous for the L925I mutation and had 0% of the V419L mutation and another population was heterogeneous for the V419L mutation and had 100% of the L925I mutation. I936F occurred only at low levels. These results indicate that bed bugs in Israel are genetically resistant to pyrethroids. Thus, pyrethroids should only be used for bed bug management with caution using effective application and careful monitoring procedures. Additionally, new and novel-acting insecticides and nonchemical means of controlling bed bugs should be explored. PMID:26336243

  12. Quantitative Sequencing for the Determination of Kdr-type Resistance Allele (V419L, L925I, I936F) Frequencies in Common Bed Bug (Hemiptera: Cimicidae) Populations Collected from Israel.

    PubMed

    Palenchar, Daniel J; Gellatly, Kyle J; Yoon, Kyong Sup; Mumcuoglu, Kosta Y; Shalom, Uri; Clark, J Marshall

    2015-09-01

    Human bed bug infestations have dramatically increased worldwide since the mid-1990s. A similar phenomenon was also observed in Israel since 2005, when infestations were reported from all over the country. Two single nucleotide polymorphisms (V419L and L925I) in the bed bug voltage-sensitive sodium channel confer kdr-type resistance to pyrethroids. Using quantitative sequencing (QS), the resistance allele frequencies of Israeli bed bug populations from across the country were determined. Genomic DNA was extracted from samples of 12 populations of bed bugs collected from Israel and DNA fragments containing the V419L or L925I and I936F mutations sites were PCR amplified. The PCR products were analyzed by QS and the nucleotide signal ratios calculated and used to predict the resistance allele frequencies of the unknown populations. Results of the genetic analysis show that resistant nucleotide signals are highly correlated to resistance allele frequencies for both mutations. Ten of the 12 tested populations had 100% of the L925I mutation and 0% of the V419L mutation. One population was heterogeneous for the L925I mutation and had 0% of the V419L mutation and another population was heterogeneous for the V419L mutation and had 100% of the L925I mutation. I936F occurred only at low levels. These results indicate that bed bugs in Israel are genetically resistant to pyrethroids. Thus, pyrethroids should only be used for bed bug management with caution using effective application and careful monitoring procedures. Additionally, new and novel-acting insecticides and nonchemical means of controlling bed bugs should be explored.

  13. Effect of temperature and solvent composition on acid dissociation equilibria, I: Sequenced (s)(s)pKa determination of compounds commonly used as buffers in high performance liquid chromatography coupled to mass spectroscopy detection.

    PubMed

    Padró, Juan M; Acquaviva, Agustín; Tascon, Marcos; Gagliardi, Leonardo G; Castells, Cecilia B

    2012-05-01

    A new automated and rapid potentiometric method for determining the effect of organic-solvent composition on pK(a) has been developed. It is based on the measurements of pH values of buffer solutions of variable solvent compositions using a combined glass electrode. Additions of small volumes of one precisely thermostated solution into another, both containing exactly the same analytical concentrations of the buffer components, can produce continuous changes in the solvent composition. Two sequences of potential measurements, one of increasing and the other of decreasing solvent content, are sufficient to obtain the pK(a) values of the acidic compound within the complete solvent-composition range in about 2h. The experimental design, procedures, and calculations needed to convert the measured pH into the thermodynamic pK(a) values are thoroughly discussed. This rapid and automated method allows the systematic study of the effect of solvent compositions and temperatures on the pK(a). It has been applied to study the dissociation constants of two monoprotic acids: formic acid and triethylamine:HCl in acetonitrile/water mixtures within the range from 0 to 90% (v/v) at temperatures between 20°C and 60°C. These volatile compounds are frequently used to control the pH of the mobile phase in HPLC, especially in methods coupled to mass-spectrometry detection. The obtained pK(a) values are in excellent agreement with those previously reported. The results were fitted to empirical functions between pK(a) and temperature and composition. These equations, which can be used to estimate the pK(a) of these substances at any composition and temperature, would be highly useful in practical work during chromatographic method development.

  14. Single-Nucleotide Mutations in FMR1 Reveal Novel Functions and Regulatory Mechanisms of the Fragile X Syndrome Protein FMRP

    PubMed Central

    Suhl, Joshua A.; Warren, Stephen T.

    2015-01-01

    Fragile X syndrome is a monogenic disorder and a common cause of intellectual disability. Despite nearly 25 years of research on FMR1, the gene underlying the syndrome, very few pathological mutations other than the typical CGG-repeat expansion have been reported. This is in contrast to other X-linked, monogenic, intellectual disability disorders, such as Rett syndrome, where many point mutations have been validated as causative of the disorder. As technology has improved and significantly driven down the cost of sequencing, allowing for whole genes to be sequenced with relative ease, in-depth sequencing studies on FMR1 have recently been performed. These studies have led to the identification of novel variants in FMR1, where some of which have been functionally evaluated and are likely pathogenic. In this review, we discuss recently identified FMR1 variants, the ways these novel variants cause dysfunction, and how they reveal new regulatory mechanisms and functionalities of the gene. PMID:26819560

  15. 3 CFR - Regulatory Review

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... been learned since that time. Far more is now known about regulation—not only about when it is... interests of future generations; identify methods of ensuring that regulatory review does not produce...

  16. Assessing the regulatory picture

    SciTech Connect

    Not Available

    1994-02-01

    This article addresses the safety of the nation's drinking water supply and discusses compliance of the Clean Water Act. Right now, the shape of the regulatory future is uncertain. The results of the D-DBP regulatory negotiation are imminent. Congress is ready to begin debating reauthorization of the Safe Drinking Water Act, and utilities are trying to comply with the regulations while trying not to price water out of the reach of some of their customers.

  17. NRC regulatory initiatives

    SciTech Connect

    Johnson, T.C.

    1989-11-01

    The US Nuclear Regulatory Commission (NRC) is addressing several low-level waste disposal issues that will be important to waste generators and to States and Compacts developing new disposal capacity. These issues include Greater-Than-Class C (GTCC) waste, mixed waste, below regulatory concern (BRC) waste, and the low-level waste data base. This paper discusses these issues and their current status.

  18. Transphyletic conservation of developmental regulatory state in animal evolution

    PubMed Central

    Royo, José Luis; Maeso, Ignacio; Irimia, Manuel; Gao, Feng; Peter, Isabelle S.; Lopes, Carla S.; D'Aniello, Salvatore; Casares, Fernando; Davidson, Eric H.; Garcia-Fernández, Jordi; Gómez-Skarmeta, José Luis

    2011-01-01

    Specific regulatory states, i.e., sets of expressed transcription factors, define the gene expression capabilities of cells in animal development. Here we explore the functional significance of an unprecedented example of regulatory state conservation from the cnidarian Nematostella to Drosophila, sea urchin, fish, and mammals. Our probe is a deeply conserved cis-regulatory DNA module of the SRY-box B2 (soxB2), recognizable at the sequence level across many phyla. Transphyletic cis-regulatory DNA transfer experiments reveal that the plesiomorphic control function of this module may have been to respond to a regulatory state associated with neuronal differentiation. By introducing expression constructs driven by this module from any phyletic source into the genomes of diverse developing animals, we discover that the regulatory state to which it responds is used at different levels of the neurogenic developmental process, including patterning and development of the vertebrate forebrain and neurogenesis in the Drosophila optic lobe and brain. The regulatory state recognized by the conserved DNA sequence may have been redeployed to different levels of the developmental regulatory program during evolution of complex central nervous systems. PMID:21844364

  19. Transphyletic conservation of developmental regulatory state in animal evolution.

    PubMed

    Royo, José Luis; Maeso, Ignacio; Irimia, Manuel; Gao, Feng; Peter, Isabelle S; Lopes, Carla S; D'Aniello, Salvatore; Casares, Fernando; Davidson, Eric H; Garcia-Fernández, Jordi; Gómez-Skarmeta, José Luis

    2011-08-23

    Specific regulatory states, i.e., sets of expressed transcription factors, define the gene expression capabilities of cells in animal development. Here we explore the functional significance of an unprecedented example of regulatory state conservation from the cnidarian Nematostella to Drosophila, sea urchin, fish, and mammals. Our probe is a deeply conserved cis-regulatory DNA module of the SRY-box B2 (soxB2), recognizable at the sequence level across many phyla. Transphyletic cis-regulatory DNA transfer experiments reveal that the plesiomorphic control function of this module may have been to respond to a regulatory state associated with neuronal differentiation. By introducing expression constructs driven by this module from any phyletic source into the genomes of diverse developing animals, we discover that the regulatory state to which it responds is used at different levels of the neurogenic developmental process, including patterning and development of the vertebrate forebrain and neurogenesis in the Drosophila optic lobe and brain. The regulatory state recognized by the conserved DNA sequence may have been redeployed to different levels of the developmental regulatory program during evolution of complex central nervous systems.

  20. Common Cause Failure Modeling

    NASA Technical Reports Server (NTRS)

    Hark, Frank; Britton, Paul; Ring, Robert; Novack, Steven

    2015-01-01

    Space Launch System (SLS) Agenda: Objective; Key Definitions; Calculating Common Cause; Examples; Defense against Common Cause; Impact of varied Common Cause Failure (CCF) and abortability; Response Surface for various CCF Beta; Takeaways.

  1. cis-regulatory region analysis using BEARR.

    PubMed

    Vega, Vinsensius Berlian

    2006-01-01

    Genome-wide studies are fast becoming the norm, partly fueled by the availability of genome sequences and the feasibility of high-throughput experimental platforms, e.g., microarrays. An important aspect in any genome-wide studies is determination of regulatory relationships, believed to be primarily transacted through transcription factor binding to DNA. Identification of specific transcription factor binding sites in the cis-regulatory regions of genes makes it possible to list direct targets of transcription factors, model transcriptional regulatory networks, and mine other associated datasets for relevant targets for experimental and clinical manipulation. We have developed a web-based tool to assist biologists in efficiently carrying out the analysis of genes from studies of specific transcription factors or otherwise. The batch extraction and analysis of cis-regulatory regions (BEARR) facilitates identification, extraction, and analysis of regulatory regions from the large amount of data that is typically generated in genome-wide studies. This chapter highlights features and serves as a tutorial for using this publicly available software. The URL is http://giscompute.gis.a-star.edu.sg/~vega/BEARR1.0/. PMID:16888354

  2. Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

    SciTech Connect

    Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

    2003-12-31

    Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involved in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.

  3. ChIP-Seq-Annotated Heliconius erato Genome Highlights Patterns of cis-Regulatory Evolution in Lepidoptera.

    PubMed

    Lewis, James J; van der Burg, Karin R L; Mazo-Vargas, Anyi; Reed, Robert D

    2016-09-13

    Uncovering phylogenetic patterns of cis-regulatory evolution remains a fundamental goal for evolutionary and developmental biology. Here, we characterize the evolution of regulatory loci in butterflies and moths using chromatin immunoprecipitation sequencing (ChIP-seq) annotation of regulatory elements across three stages of head development. In the process we provide a high-quality, functionally annotated genome assembly for the butterfly, Heliconius erato. Comparing cis-regulatory element conservation across six lepidopteran genomes, we find that regulatory sequences evolve at a pace similar to that of protein-coding regions. We also observe that elements active at multiple developmental stages are markedly more conserved than elements with stage-specific activity. Surprisingly, we also find that stage-specific proximal and distal regulatory elements evolve at nearly identical rates. Our study provides a benchmark for genome-wide patterns of regulatory element evolution in insects, and it shows that developmental timing of activity strongly predicts patterns of regulatory sequence evolution.

  4. Overview Article: Identifying transcriptional cis-regulatory modules in animal genomes

    PubMed Central

    Suryamohan, Kushal; Halfon, Marc S.

    2014-01-01

    Gene expression is regulated through the activity of transcription factors and chromatin modifying proteins acting on specific DNA sequences, referred to as cis-regulatory elements. These include promoters, located at the transcription initiation sites of genes, and a variety of distal cis-regulatory modules (CRMs), the most common of which are transcriptional enhancers. Because regulated gene expression is fundamental to cell differentiation and acquisition of new cell fates, identifying, characterizing, and understanding the mechanisms of action of CRMs is critical for understanding development. CRM discovery has historically been challenging, as CRMs can be located far from the genes they regulate, have few readily-identifiable sequence characteristics, and for many years were not amenable to high-throughput discovery methods. However, the recent availability of complete genome sequences and the development of next-generation sequencing methods has led to an explosion of both computational and empirical methods for CRM discovery in model and non-model organisms alike. Experimentally, CRMs can be identified through chromatin immunoprecipitation directed against transcription factors or histone post-translational modifications, identification of nucleosome-depleted “open” chromatin regions, or sequencing-based high-throughput functional screening. Computational methods include comparative genomics, clustering of known or predicted transcription factor binding sites, and supervised machine-learning approaches trained on known CRMs. All of these methods have proven effective for CRM discovery, but each has its own considerations and limitations, and each is subject to a greater or lesser number of false-positive identifications. Experimental confirmation of predictions is essential, although shortcomings in current methods suggest that additional means of validation need to be developed. PMID:25704908

  5. Common Career Technical Core: Common Standards, Common Vision for CTE

    ERIC Educational Resources Information Center

    Green, Kimberly

    2012-01-01

    This article provides an overview of the National Association of State Directors of Career Technical Education Consortium's (NASDCTEc) Common Career Technical Core (CCTC), a state-led initiative that was created to ensure that career and technical education (CTE) programs are consistent and high quality across the United States. Forty-two states,…

  6. Functional evolution of a cis-regulatory module.

    PubMed

    Ludwig, Michael Z; Palsson, Arnar; Alekseeva, Elena; Bergman, Casey M; Nathan, Janaki; Kreitman, Martin

    2005-04-01

    Lack of knowledge about how regulatory regions evolve in relation to their structure-function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out a functional genetic complementation analysis of a eukaryotic cis-regulatory module-the even-skipped stripe 2 enhancer-from four Drosophila species. The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species. Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression. Our findings have implications for understanding enhancer structure-function, mechanisms of speciation and computational identification of regulatory modules.

  7. SRD: a Staphylococcus regulatory RNA database

    PubMed Central

    Sassi, Mohamed; Augagneur, Yoann; Mauro, Tony; Ivain, Lorraine; Chabelskaya, Svetlana; Hallier, Marc; Sallou, Olivier; Felden, Brice

    2015-01-01

    An overflow of regulatory RNAs (sRNAs) was identified in a wide range of bacteria. We designed and implemented a new resource for the hundreds of sRNAs identified in Staphylococci, with primary focus on the human pathogen Staphylococcus aureus. The “Staphylococcal Regulatory RNA Database” (SRD, http://srd.genouest.org/) compiled all published data in a single interface including genetic locations, sequences and other features. SRD proposes novel and simplified identifiers for Staphylococcal regulatory RNAs (srn) based on the sRNA's genetic location in S. aureus strain N315 which served as a reference. From a set of 894 sequences and after an in-depth cleaning, SRD provides a list of 575 srn exempt of redundant sequences. For each sRNA, their experimental support(s) is provided, allowing the user to individually assess their validity and significance. RNA-seq analysis performed on strains N315, NCTC8325, and Newman allowed us to provide further details, upgrade the initial annotation, and identified 159 RNA-seq independent transcribed sRNAs. The lists of 575 and 159 sRNAs sequences were used to predict the number and location of srns in 18 S. aureus strains and 10 other Staphylococci. A comparison of the srn contents within 32 Staphylococcal genomes revealed a poor conservation between species. In addition, sRNA structure predictions obtained with MFold are accessible. A BLAST server and the intaRNA program, which is dedicated to target prediction, were implemented. SRD is the first sRNA database centered on a genus; it is a user-friendly and scalable device with the possibility to submit new sequences that should spread in the literature. PMID:25805861

  8. Structure of Proximal and Distant Regulatory Elements in the Human Genome

    NASA Astrophysics Data System (ADS)

    Ovcharenko, Ivan

    Clustering of multiple transcription factor binding sites (TFBSs) for the same transcription factor (TF) is a common feature of cis-regulatory modules in invertebrate animals, but the occurrence of such homotypic clusters of TFBSs (HCTs) in the human genome has remained largely unknown. To explore whether HCTs are also common in human and other vertebrates, we used known binding motifs for vertebrate TFs and a hidden Markov model-based approach to detect HCTs in the human, mouse, chicken, and fugu genomes, and examined their association with cis-regulatory modules. We found that evolutionarily conserved HCTs occupy nearly 2% of the human genome, with experimental evidence for individual TFs supporting their binding to predicted HCTs. More than half of promoters of human genes contain HCTs, with a distribution around the transcription start site in agreement with the experimental data from the ENCODE project. In addition, almost half of 487 experimentally validated developmental enhancers contain them as well - a number more than 25-fold larger than expected by chance. We also found evidence of negative selection acting on TFBSs within HCTs, as the conservation of TFBSs is stronger than the conservation of sequences separating them. The important role of HCTs as components of developmental enhancers is additionally supported by a strong correlation between HCTs and the binding of the enhancer-associated co-activator protein p300. Experimental validation of HCT-containing elements in both zebrafish and mouse suggest that HCTs could be used to predict both the presence of enhancers and their tissue specificity, and are thus a feature that can be effectively used in deciphering the gene regulatory code. In conclusion, our results indicate that HCTs are a pervasive feature of human cis-regulatory modules and suggest that they play an important role in gene regulation in the human and other vertebrate genomes.

  9. Nucleotide-sequence of a canine oral papillomavirus containing a long noncoding region.

    PubMed

    Isegawa, N; Ohta, M; Shirasawa, H; Tokita, H; Yamaura, A; Simizu, B

    1995-07-01

    The DNA genome of a canine oral papillomavirus (COPV) was completely sequenced and found to consist of 8607 base pairs, which were the longest of all known papillomaviruses (PVs). Its organization was similar to that of other PVs except that it lacked early gene 5 (E5) and possessed a unique long noncoding region (L-NCR) between the end of the early genes and the beginning of the late genes. COPV also possessed a short noncoding region (S-NCR) which contained a putative upper regulatory region (URR), which is commonly found in PVs. The L-NCR did not show any similarity to known PV DNAs nor other DNA sequences in the GenBank database. Nucleotide sequence analysis of COPV showed that it was closely related to human papillomavirus type 1 (HPV 1) and animal PVs associated with cutaneous lesions in rabbit, European elk, deer and cow as we reported previously. PMID:21552821

  10. Identification of two homologous mitochondrial DNA sequences, which bind strongly and specifically to a mitochondrial protein of Paracentrotus lividus.

    PubMed Central

    Roberti, M; Mustich, A; Gadaleta, M N; Cantatore, P

    1991-01-01

    Using a combination of band shift and DNasel protection experiments, two Paracentrotus lividus mitochondrial sequences, able to bind tightly and selectively to a mitochondrial protein from sea urchin embryos, have been found. The two sequences, which compete with each other for binding to the protein, are located in two genome regions which are thought to contain regulatory signals for mitochondrial replication and transcription. A computer analysis suggests that the sequence TTTTRTANNTCYYATCAYA, common to the two binding regions, is the minimal recognition signal for the binding to the protein. We discuss the hypothesis that the protein binding capacity of these two sequences is involved in the control of sea urchin mtDNA replication during developmental stages. Images PMID:1956785

  11. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  12. Transcription factor trapping by RNA in gene regulatory elements.

    PubMed

    Sigova, Alla A; Abraham, Brian J; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M; Guo, Yang Eric; Jangi, Mohini; Giallourakis, Cosmas C; Sharp, Phillip A; Young, Richard A

    2015-11-20

    Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs.

  13. Recurrent Somatic Mutations in Regulatory Regions of Human Cancer Genomes

    PubMed Central

    Melton, Collin; Reuter, Jason A.; Spacek, Damek V.; Snyder, Michael

    2015-01-01

    Aberrant regulation of gene expression in cancer can promote survival and proliferation of cancer cells. Here we integrate TCGA whole genome sequencing data of 436 patients from eight cancer subtypes with ENCODE and other regulatory annotations to identify point mutations in regulatory regions. We find evidence for positive selection of mutations in transcription factor binding sites, consistent with these sites regulating important cancer cell functions. Using a novel method that adjusts for sample- and genomic locus-specific mutation rate, we identify recurrently mutated sites across cancer patients. Mutated regulatory sites include known sites in the TERT promoter and many novel sites, including a subset in proximity to cancer genes. In reporter assays, two novel sites display decreased enhancer activity upon mutation. These data demonstrate that many regulatory regions contain mutations under selective pressure and suggest a larger role for regulatory mutations in cancer than previously appreciated. PMID:26053494

  14. Identification of altered cis-regulatory elements in human disease.

    PubMed

    Mathelier, Anthony; Shi, Wenqiang; Wasserman, Wyeth W

    2015-02-01

    It has long been appreciated that variations in regulatory regions of genes can impact gene expression. With the advent of whole-genome sequencing (WGS), it has become possible to begin cataloging these noncoding variants. Evidence continues to accumulate linking clinical cases with cis-regulatory element disruption in a wide range of diseases. Identifying variants is becoming routine, but assessing their impact on regulation remains challenging. Bioinformatics approaches that identify variations functionally altering transcription factor (TF) binding are increasingly important for meeting this challenge. We present the current state of computational tools and resources for identifying the genomic regulatory components (cis-regulatory regions and TF binding sites, TFBSs) controlling gene transcriptional regulation. We review how such approaches can be used to interpret the potential disease causality of point mutations and small insertions or deletions. We hope this will motivate further the development of methods enabling the identification of etiological cis-regulatory variations.

  15. 75 FR 8770 - Self-Regulatory Organizations; Financial Industry Regulatory Authority, Inc.; Notice of Filing of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-25

    ... ``execution of such transactions, and in particular of purchases , in the common purchase and sale account may...), 74 FR 23912 (May 21, 2009) (Notice of Filing; File No. SR-FINRA-2009-028). FINRA will announce the implementation date of the proposed rule change in a Regulatory Notice to be published no later than 90...

  16. Negative feedback buffers effects of regulatory variants

    PubMed Central

    Bader, Daniel M; Wilkening, Stefan; Lin, Gen; Tekkedil, Manu M; Dietrich, Kim; Steinmetz, Lars M; Gagneur, Julien

    2015-01-01

    Mechanisms conferring robustness against regulatory variants have been controversial. Previous studies suggested widespread buffering of RNA misexpression on protein levels during translation. We do not find evidence that translational buffering is common. Instead, we find extensive buffering at the level of RNA expression, exerted through negative feedback regulation acting in trans, which reduces the effect of regulatory variants on gene expression. Our approach is based on a novel experimental design in which allelic differential expression in a yeast hybrid strain is compared to allelic differential expression in a pool of its spores. Allelic differential expression in the hybrid is due to cis-regulatory differences only. Instead, in the pool of spores allelic differential expression is not only due to cis-regulatory differences but also due to local trans effects that include negative feedback. We found that buffering through such local trans regulation is widespread, typically compensating for about 15% of cis-regulatory effects on individual genes. Negative feedback is stronger not only for essential genes, indicating its functional relevance, but also for genes with low to middle levels of expression, for which tight regulation matters most. We suggest that negative feedback is one mechanism of Waddington's canalization, facilitating the accumulation of genetic variants that might give selective advantage in different environments. PMID:25634765

  17. Ranking and Sequencing Model

    2009-08-13

    This database application (commonly called the Supermodel) provides a repository for managing critical facility/project information, allows the user to subjectively an objectively assess key criteria , quantify project risks, develop ROM cost estimates, determine facility/project end states, ultimately performing risk-based modeling to rank facilities/project based on risk, sequencing project schedules and provides an optimized recommended sequencing/scheduling of these projects which maximize the S&M cost savings to perform closure projects which benefit all stakeholders.

  18. Regulatory non-coding RNAs: revolutionizing the RNA world.

    PubMed

    Huang, Biao; Zhang, Rongxin

    2014-06-01

    The majority of the genomic DNA sequence in mammalian and other higher organisms can be transcribed into abundant functional RNA transcripts, especially regulatory non-coding RNAs (ncRNAs) that are expressed in a developmentally and species-specific regulated manner. Here, we review various regulatory non-coding RNAs, including regulatory small non-coding RNAs (sncRNAs) and long non-coding RNAs (lncRNAs), and summarize two and eight kinds of distinct modes of action for sncRNAs and lncRNAs respectively, by which functional ncRNAs mediate the regulation of intracellular events.

  19. Viral complement regulatory proteins.

    PubMed

    Rosengard, A M; Ahearn, J M

    1999-05-01

    The inactivation of complement provides cells and tissues critical protection from complement-mediated attack and decreases the associated recruitment of other inflammatory mediators. In an attempt to evade the host immune response, viruses have evolved two mechanisms to acquire complement regulatory proteins. They can directly seize the host cell complement regulators onto their outer envelope and/or they can produce their own proteins which are either secreted into the neighboring intercellular space or expressed as membrane-bound proteins on the infected host cell. The following review will concentrate on the viral homologues of the mammalian complement regulatory proteins, specifically those containing complement control protein (CCP) repeats. PMID:10408371

  20. Rationales for regulatory activity

    SciTech Connect

    Perhac, R.M.

    1997-02-01

    The author provides an outline which touches on the types of concerns about risk evaluation which are addressed in the process of establishing regulatory guides. Broadly he says regulatory activity serves three broad constituents: (1) Paternalism (private risk); (2) Promotion of social welfare (public risks); (3) Protection of individual rights (public risks). He then discusses some of the major issues encountered in reaching a decision on what is an acceptable level of risk within each of these areas, and how one establishes such a level.

  1. Prediction of functional regulatory SNPs in monogenic and complex disease

    PubMed Central

    Zhao, Yiqiang; Clark, Wyatt T.; Mort, Matthew; Cooper, David N.; Radivojac, Predrag; Mooney, Sean D.

    2013-01-01

    Next-Generation Sequencing (NGS) technologies are yielding ever-higher volumes of human genome sequence data. Given this large amount of data, it has become both a possibility and a priority to determine how disease-causing single nucleotide polymorphisms (SNPs) detected within gene regulatory regions (rSNPs) exert their effects on gene expression. Recently, several studies have explored whether disease-causing polymorphisms have attributes that can distinguish them from those that are neutral, attaining moderate success at discriminating between functional and putatively neutral regulatory SNPs. Here, we have extended this work by assessing the utility of both SNP-based features (those associated only with the polymorphism site and the surrounding DNA) and Gene-based features (those derived from the associated gene in whose regulatory region the SNP lies) in the identification of functional regulatory polymorphisms involved in either monogenic or complex disease. Gene-based features were found to be capable of both augmenting and enhancing the utility of SNP-based features in the prediction of known regulatory mutations. Adopting this approach, we achieved an AUC of 0.903 for predicting regulatory SNPs. Finally, our tool predicted 225 new regulatory SNPs with a high degree of confidence, with 105 of the 225 falling into linkage disequilibrium blocks of reported disease-associated GWAS SNPs. PMID:21796725

  2. Cis- and Trans-Regulatory Mechanisms of Gene Expression in the ASJ Sensory Neuron of Caenorhabditis elegans

    PubMed Central

    González-Barrios, María; Fierro-González, Juan Carlos; Krpelanova, Eva; Mora-Lorca, José Antonio; Pedrajas, José Rafael; Peñate, Xenia; Chavez, Sebastián; Swoboda, Peter; Jansen, Gert; Miranda-Vizuete, Antonio

    2015-01-01

    The identity of a given cell type is determined by the expression of a set of genes sharing common cis-regulatory motifs and being regulated by shared transcription factors. Here, we identify cis and trans regulatory elements that drive gene expression in the bilateral sensory neuron ASJ, located in the head of the nematode Caenorhabditis elegans. For this purpose, we have dissected the promoters of the only two genes so far reported to be exclusively expressed in ASJ, trx-1 and ssu-1. We hereby identify the ASJ motif, a functional cis-regulatory bipartite promoter region composed of two individual 6 bp elements separated by a 3 bp linker. The first element is a 6 bp CG-rich sequence that presumably binds the Sp family member zinc-finger transcription factor SPTF-1. Interestingly, within the C. elegans nervous system SPTF-1 is also found to be expressed only in ASJ neurons where it regulates expression of other genes in these neurons and ASJ cell fate. The second element of the bipartite motif is a 6 bp AT-rich sequence that is predicted to potentially bind a transcription factor of the homeobox family. Together, our findings identify a specific promoter signature and SPTF-1 as a transcription factor that functions as a terminal selector gene to regulate gene expression in C. elegans ASJ sensory neurons. PMID:25769980

  3. Performance comparison of Next Generation sequencing platforms.

    PubMed

    Erguner, Bekir; Ustek, Duran; Sagiroglu, Mahmut S

    2015-01-01

    Next Generation DNA Sequencing technologies offer ultra high sequencing throughput for very low prices. The increase in throughput and diminished costs open up new research areas. Moreover, number of clinicians utilizing DNA sequencing keeps growing. One of the main concern for researchers and clinicians who are adopting these platforms is their sequencing accuracy. We compared three of the most commonly used Next Generation Sequencing platforms; Ion Torrent from Life Technologies, GS FLX+ from Roche and HiSeq 2000 from Illumina.

  4. Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes.

    PubMed

    Nakagawa, So; Niimura, Yoshihito; Gojobori, Takashi; Tanaka, Hiroshi; Miura, Kin-ichiro

    2008-02-01

    Understanding regulatory mechanisms of protein synthesis in eukaryotes is essential for the accurate annotation of genome sequences. Kozak reported that the nucleotide sequence GCCGCC(A/G)CCAUGG (AUG is the initiation codon) was frequently observed in vertebrate genes and that this 'consensus' sequence enhanced translation initiation. However, later studies using invertebrate, fungal and plant genes reported different 'consensus' sequences. In this study, we conducted extensive comparative analyses of nucleotide sequences around the initiation codon by using genomic data from 47 eukaryote species including animals, fungi, plants and protists. The analyses revealed that preferred nucleotide sequences are quite diverse among different species, but differences between patterns of nucleotide bias roughly reflect the evolutionary relationships of the species. We also found strong biases of A/G at position -3, A/C at position -2 and C at position +5 that were commonly observed in all species examined. Genes with higher expression levels showed stronger signals, suggesting that these nucleotides are responsible for the regulation of translation initiation. The diversity of preferred nucleotide sequences around the initiation codon might be explained by differences in relative contributions from two distinct patterns, GCCGCCAUG and AAAAAAAUG, which implies the presence of multiple molecular mechanisms for controlling translation initiation.

  5. Common Interventional Radiology Procedures

    MedlinePlus

    ... of common interventional techniques is below. Common Interventional Radiology Procedures Angiography An X-ray exam of the ... into the vertebra. Copyright © 2016 Society of Interventional Radiology. All rights reserved. 3975 Fair Ridge Drive • Suite ...

  6. How Common Is the Common Core?

    ERIC Educational Resources Information Center

    Thomas, Amande; Edson, Alden J.

    2014-01-01

    Since the introduction of the Common Core State Standards for Mathematics (CCSSM) in 2010, stakeholders in adopting states have engaged in a variety of activities to understand CCSSM standards and transition from previous state standards. These efforts include research, professional development, assessment and modification of curriculum resources,…

  7. Automated Identification of Nucleotide Sequences

    NASA Technical Reports Server (NTRS)

    Osman, Shariff; Venkateswaran, Kasthuri; Fox, George; Zhu, Dian-Hui

    2007-01-01

    STITCH is a computer program that processes raw nucleotide-sequence data to automatically remove unwanted vector information, perform reverse-complement comparison, stitch shorter sequences together to make longer ones to which the shorter ones presumably belong, and search against the user s choice of private and Internet-accessible public 16S rRNA databases. ["16S rRNA" denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] In STITCH, a template 16S rRNA sequence is used to position forward and reverse reads. STITCH then automatically searches known 16S rRNA sequences in the user s chosen database(s) to find the sequence most similar to (the sequence that lies at the smallest edit distance from) each spliced sequence. The result of processing by STITCH is the identification of the most similar well-described bacterium. Whereas previously commercially available software for analyzing genetic sequences operates on one sequence at a time, STITCH can manipulate multiple sequences simultaneously to perform the aforementioned operations. A typical analysis of several dozen sequences (length of the order of 103 base pairs) by use of STITCH is completed in a few minutes, whereas such an analysis performed by use of prior software takes hours or days.

  8. EPAct Transportation Regulatory Activities

    SciTech Connect

    2011-11-21

    The U.S. Department of Energy's (DOE) Vehicle Technologies Program manages several transportation regulatory activities established by the Energy Policy Act of 1992 (EPAct), as amended by the Energy Conservation Reauthorization Act of 1998, EPAct 2005, and the Energy Independence and Security Act of 2007 (EISA).

  9. The regulatory horizon

    NASA Technical Reports Server (NTRS)

    Cook, ED

    1987-01-01

    The author briefly discusses the FAA's position as it relates to cockpit resource management. For example, if Cockpit Resource Management (CRM) is a positive concept, why isn't everyone required to implement it? The regulatory practice of the FAA is discussed and questions and answers are presented.

  10. The New Common School.

    ERIC Educational Resources Information Center

    Glenn, Charles L.

    1987-01-01

    Horace Mann's goal of creating a common school that brings our society's children together in mutual respect and common learning need not be frustrated by residential segregation and geographical separation of the haves and have-nots. Massachusetts' new common school vision boasts a Metro Program for minority students, 80 magnet schools, and…

  11. Knowledge representation for commonality

    NASA Technical Reports Server (NTRS)

    Yeager, Dorian P.

    1990-01-01

    Domain-specific knowledge necessary for commonality analysis falls into two general classes: commonality constraints and costing information. Notations for encoding such knowledge should be powerful and flexible and should appeal to the domain expert. The notations employed by the Commonality Analysis Problem Solver (CAPS) analysis tool are described. Examples are given to illustrate the main concepts.

  12. Canonical Commonality Analysis.

    ERIC Educational Resources Information Center

    Leister, K. Dawn

    Commonality analysis is a method of partitioning variance that has advantages over more traditional "OVA" methods. Commonality analysis indicates the amount of explanatory power that is "unique" to a given predictor variable and the amount of explanatory power that is "common" to or shared with at least one predictor variable. This paper outlines…

  13. Identification of transcriptional regulatory nodes in soybean defense networks using transient co-transactivation assays

    PubMed Central

    Wang, Yongli; Wang, Hui; Ma, Yujie; Du, Haiping; Yang, Qing; Yu, Deyue

    2015-01-01

    Plant responses to major environmental stressors, such as insect feeding, not only occur via the functions of defense genes but also involve a series of regulatory factors. Our previous transcriptome studies proposed that, in addition to two defense-related genes, GmVSPβ and GmN:IFR, a high proportion of transcription factors (TFs) participate in the incompatible soybean-common cutworm interaction networks. However, the regulatory mechanisms and effects of these TFs on those induced defense-related genes remain unknown. In the present work, we isolated and identified 12 genes encoding MYB, WRKY, NAC, bZIP, and DREB TFs from a common cutworm-induced cDNA library of a resistant soybean line. Sequence analysis of the promoters of three co-expressed genes, including GmVSPα, GmVSPβ, and GmN:IFR, revealed the enrichment of various TF-binding sites for defense and stress responses. To further identify the regulatory nodes composed of these TFs and defense gene promoters, we performed extensive transient co-transactivation assays to directly test the transcriptional activity of the 12 TFs binding at different levels to the three co-expressed gene promoters. The results showed that all 12 TFs were able to transactivate the GmVSPβ and GmN:IFR promoters. GmbZIP110 and GmMYB75 functioned as distinct regulators of GmVSPα/β and GmN:IFR expression, respectively, while GmWRKY39 acted as a common central regulator of GmVSPα/β and GmN:IFR expression. These corresponding TFs play crucial roles in coordinated plant defense regulation, which provides valuable information for understanding the molecular mechanisms involved in insect-induced transcriptional regulation in soybean. More importantly, the identified TFs and suitable promoters can be used to engineer insect-resistant plants in molecular breeding studies. PMID:26579162

  14. Increasing the dynamic control space of mammalian transcription devices by combinatorial assembly of homologous regulatory elements from different bacterial species.

    PubMed

    Bacchus, William; Weber, Wilfried; Fussenegger, Martin

    2013-01-01

    Prokaryotic transcriptional regulatory elements are widely utilized building blocks for constructing regulatory genetic circuits adapted for mammalian cells and have found their way into a broad range of biotechnological applications. Prokaryotic transcriptional repressors, fused to eukaryotic transactivation or repression domains, compose the transcription factor, which binds and adjusts transcription from chimeric promoters containing the repressor-specific operator sequence. Escherichia coli and Chlamydia trachomatis share common features in the regulatory mechanism of the biosynthesis of l-tryptophan. The repressor protein TrpR of C. trachomatis regulates the trpRBA operon and the TrpR of E. coli regulates the trpEDCBA operon, both requiring l-tryptophan as a co-repressor. Fusion of these bacterial repressors to the VP16 transactivation domain of Herpes simplex virus creates synthetic transactivators that could bind and activate chimeric promoters, assembled by placing repressor-specific operator modules adjacent to a minimal promoter, in an l-tryptophan-adjustable manner. Combinations of different transactivator and promoter variants from the same or different bacterial species resulted in a multitude of regulatory systems where l-tryptophan regulation properties, background noise, and maximal gene expression levels were significantly diverse. Different l-tryptophan analogues showed diverse regulatory capacity depending on the promoter/transactivator combination. We believe the systems approach to rationally choose promoters, transactivators and inducer molecules, to obtain desired and predefined genetic expression dynamics and control profiles, will significantly advance the design of new regulatory circuits as well as improving already existing ones. PMID:23178502

  15. Automatic Command Sequence Generation