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  • Articles  (13)
  • Oxford University Press  (13)
  • 2010-2014  (13)
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  • Nucleic Acids Research  (13)
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  • Biology  (13)
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  • Articles  (13)
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  • Oxford University Press  (13)
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  • 2010-2014  (13)
  • 2005-2009
  • 2000-2004
  • 1990-1994
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  • Biology  (13)
  • Computer Science
  • 1
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    Study of PcaV from Streptomyces coelicolor yields new insights into ligand-responsive MarR family transcription factors (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: MarR family proteins constitute a group of 〉12 000 transcriptional regulators encoded in bacterial and archaeal genomes that control gene expression in metabolism, stress responses, virulence and multi-drug resistance. There is much interest in defining the molecular mechanism by which ligand binding attenuates the DNA-binding activities of these proteins. Here, we describe how PcaV, a MarR family regulator in Streptomyces coelicolor, controls transcription of genes encoding β-ketoadipate pathway enzymes through its interaction with the pathway substrate, protocatechuate. This transcriptional repressor is the only MarR protein known to regulate this essential pathway for aromatic catabolism. In in vitro assays, protocatechuate and other phenolic compounds disrupt the PcaV–DNA complex. We show that PcaV binds protocatechuate in a 1:1 stoichiometry with the highest affinity of any MarR family member. Moreover, we report structures of PcaV in its apo form and in complex with protocatechuate. We identify an arginine residue that is critical for ligand coordination and demonstrate that it is also required for binding DNA. We propose that interaction of ligand with this arginine residue dictates conformational changes that modulate DNA binding. Our results provide new insights into the molecular mechanism by which ligands attenuate DNA binding in this large family of transcription factors.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    The Comparative Toxicogenomics Database: update 2013 (2012)
    Oxford University Press
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    Publication Date: 2012-12-20
    Description: The Comparative Toxicogenomics Database (CTD; http://ctdbase.org/ ) provides information about interactions between environmental chemicals and gene products and their relationships to diseases. Chemical–gene, chemical–disease and gene–disease interactions manually curated from the literature are integrated to generate expanded networks and predict many novel associations between different data types. CTD now contains over 15 million toxicogenomic relationships. To navigate this sea of data, we added several new features, including DiseaseComps (which finds comparable diseases that share toxicogenomic profiles), statistical scoring for inferred gene–disease and pathway–chemical relationships, filtering options for several tools to refine user analysis and our new Gene Set Enricher (which provides biological annotations that are enriched for gene sets). To improve data visualization, we added a Cytoscape Web view to our ChemComps feature, included color-coded interactions and created a ‘slim list’ for our MEDIC disease vocabulary (allowing diseases to be grouped for meta-analysis, visualization and better data management). CTD continues to promote interoperability with external databases by providing content and cross-links to their sites. Together, this wealth of expanded chemical–gene–disease data, combined with novel ways to analyze and view content, continues to help users generate testable hypotheses about the molecular mechanisms of environmental diseases.
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    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 3
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    Functional domains of the 50S subunit mature late in the assembly process (2014)
    Oxford University Press
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    Publication Date: 2014-03-13
    Description: Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages of assembly of the 50S subunit using Bacillus subtilis cells depleted of RbgA, a highly conserved GTPase. We found that RbgA-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins L16, L27, L28, L33a, L35 and L36. Using a novel pulse labeling/quantitative mass spectrometry technique, we show that this particle is physiologically relevant and is capable of maturing into a complete 50S particle. Cryo-electron microscopy and chemical probing revealed that the central protuberance, the GTPase associating region and tRNA-binding sites in this intermediate are unstructured. These findings demonstrate that key functional sites of the 50S subunit remain unstructured until late stages of maturation, preventing the incomplete subunit from prematurely engaging in translation. Finally, structural and biochemical analysis of a ribosome particle depleted of L16 indicate that L16 binding is necessary for the stimulation of RbgA GTPase activity and, in turn, release of this co-factor, and for conversion of the intermediate to a complete 50S subunit.
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    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 4
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    Sequence motifs associated with hepatotoxicity of locked nucleic acid--modified antisense oligonucleotides (2014)
    Oxford University Press
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    Publication Date: 2014-05-01
    Description: Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure–toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 ( Apoc3 ), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 ( Crtc2 ) or Glucocorticoid Receptor ( GR , NR3C1 ) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo . These results suggest in silico approaches can be utilized to establish structure–toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing.
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    Topics: Biology
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  • 5
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    BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle (2014)
    Oxford University Press
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    Publication Date: 2014-10-10
    Description: Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the two prominent pathways responsible for the repair of DNA double-strand breaks (DSBs). NHEJ is not restricted to a cell-cycle stage, whereas HR is active primarily in the S/G2 phases suggesting there are cell cycle-specific mechanisms that play a role in the choice between NHEJ and HR. Here we show NHEJ is attenuated in S phase via modulation of the autophosphorylation status of the NHEJ factor DNA-PKcs at serine 2056 by the pro-HR factor BRCA1. BRCA1 interacts with DNA-PKcs in a cell cycle-regulated manner and this interaction is mediated by the tandem BRCT domain of BRCA1, but surprisingly in a phospho-independent manner. BRCA1 attenuates DNA-PKcs autophosphorylation via directly blocking the ability of DNA-PKcs to autophosphorylate. Subsequently, blocking autophosphorylation of DNA-PKcs at the serine 2056 phosphorylation cluster promotes HR-required DNA end processing and loading of HR factors to DSBs and is a possible mechanism by which BRCA1 promotes HR.
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    Topics: Biology
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  • 6
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    Comparative RNA-Seq based dissection of the regulatory networks and environmental stimuli underlying Vibrio parahaemolyticus gene expression during infection (2014)
    Oxford University Press
    Details
    Publication Date: 2014-11-07
    Description: Vibrio parahaemolyticus is the leading worldwide cause of seafood-associated gastroenteritis, yet little is known regarding its intraintestinal gene expression or physiology. To date, in vivo analyses have focused on identification and characterization of virulence factors—e.g. a crucial Type III secretion system (T3SS2)—rather than genome-wide analyses of in vivo biology. Here, we used RNA-Seq to profile V. parahaemolyticus gene expression in infected infant rabbits, which mimic human infection. Comparative transcriptomic analysis of V. parahaemolyticus isolated from rabbit intestines and from several laboratory conditions enabled identification of mRNAs and sRNAs induced during infection and of regulatory factors that likely control them. More than 12% of annotated V. parahaemolyticus genes are differentially expressed in the intestine, including the genes of T3SS2, which are likely induced by bile-mediated activation of the transcription factor VtrB. Our analyses also suggest that V. parahaemolyticus has access to glucose or other preferred carbon sources in vivo , but that iron is inconsistently available. The V. parahaemolyticus transcriptional response to in vivo growth is far more widespread than and largely distinct from that of V. cholerae , likely due to the distinct ways in which these diarrheal pathogens interact with and modulate the environment in the small intestine.
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    Topics: Biology
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  • 7
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    High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model-based analyses of transposon-insertion sequencing data (2013)
    Oxford University Press
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    Publication Date: 2013-10-19
    Description: The coupling of high-density transposon mutagenesis to high-throughput DNA sequencing (transposon-insertion sequencing) enables simultaneous and genome-wide assessment of the contributions of individual loci to bacterial growth and survival. We have refined analysis of transposon-insertion sequencing data by normalizing for the effect of DNA replication on sequencing output and using a hidden Markov model (HMM)-based filter to exploit heretofore unappreciated information inherent in all transposon-insertion sequencing data sets. The HMM can smooth variations in read abundance and thereby reduce the effects of read noise, as well as permit fine scale mapping that is independent of genomic annotation and enable classification of loci into several functional categories (e.g. essential, domain essential or ‘sick’). We generated a high-resolution map of genomic loci (encompassing both intra- and intergenic sequences) that are required or beneficial for in vitro growth of the cholera pathogen, Vibrio cholerae. This work uncovered new metabolic and physiologic requirements for V. cholerae survival, and by combining transposon-insertion sequencing and transcriptomic data sets, we also identified several novel noncoding RNA species that contribute to V. cholerae growth. Our findings suggest that HMM-based approaches will enhance extraction of biological meaning from transposon-insertion sequencing genomic data.
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    Topics: Biology
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  • 8
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    High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs (2014)
    Oxford University Press
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    Publication Date: 2014-06-03
    Description: Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30–70% at high transfection efficiencies and ~2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ~1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9.
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    Topics: Biology
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  • 9
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    NCBI GEO: archive for functional genomics data sets--update (2012)
    Oxford University Press
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    Publication Date: 2012-12-20
    Description: The Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/ ) is an international public repository for high-throughput microarray and next-generation sequence functional genomic data sets submitted by the research community. The resource supports archiving of raw data, processed data and metadata which are indexed, cross-linked and searchable. All data are freely available for download in a variety of formats. GEO also provides several web-based tools and strategies to assist users to query, analyse and visualize data. This article reports current status and recent database developments, including the release of GEO2R, an R-based web application that helps users analyse GEO data.
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    Topics: Biology
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  • 10
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    Cell type-specific genomics of Drosophila neurons (2012)
    Oxford University Press
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    Publication Date: 2012-10-24
    Description: Many tools are available to analyse genomes but are often challenging to use in a cell type–specific context. We have developed a method similar to the isolation of nuclei tagged in a specific cell type (INTACT) technique [Deal,R.B. and Henikoff,S. (2010) A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev. Cell , 18 , 1030–1040; Steiner,F.A., Talbert,P.B., Kasinathan,S., Deal,R.B. and Henikoff,S. (2012) Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. Genome Res ., doi:10.1101/gr.131748.111], first developed in plants, for use in Drosophila neurons. We profile gene expression and histone modifications in Kenyon cells and octopaminergic neurons in the adult brain. In addition to recovering known gene expression differences, we also observe significant cell type–specific chromatin modifications. In particular, a small subset of differentially expressed genes exhibits a striking anti-correlation between repressive and activating histone modifications. These genes are enriched for transcription factors, recovering those known to regulate mushroom body identity and predicting analogous regulators of octopaminergic neurons. Our results suggest that applying INTACT to specific neuronal populations can illuminate the transcriptional regulatory networks that underlie neuronal cell identity.
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    Topics: Biology
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