Expression data from Pseudomonas aeruginosa phosphomimetic and phosphoablative AlgR
Ontology highlight
ABSTRACT:
ORGANISM(S): Pseudomonas aeruginosa PAO1 Pseudomonas aeruginosa
PROVIDER: GSE97245 | GEO | 2017/10/03
SECONDARY ACCESSION(S): PRJNA381068
REPOSITORIES: GEO
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Project description:Mechanisms governing Mycobacterium tuberculosis acid-fastness and its capacity to induce long-term infections remain unknown. Serine/Threonine phosphorylation represents an emerging theme allowing mycobacteria to adapt their cell envelope structure/composition in response to environmental changes. We addressed whether phosphorylation of KasB, a mycolic acid biosynthetic enzyme, modulates M. tuberculosis pathogenicity. Phosphorylation of KasB occurred at Thr334 and Thr336 in vitro and in mycobacteria. A mutant strain bearing an kasB_T334D/T336D allele, mimicking constitutive KasB phosphorylation, was generated by specialized linkage transduction. This resulted in shortened mycolic acids and the lack of trans-cyclopropanation. Structural/modeling analyses revealed Thr334 and Thr336 in the vicinity of the catalytic triad, implying that phosphorylation of these residues impaired KasB activity. Importantly, the phosphomimetic strain lost acid-fast staining and was more attenuated than a kasB deletion mutant in immunocompetent and immunodeficient mice. The absence of lung pathology and mortality infers this mutant to represent a valuable vaccine candidate. This work emphasizes the critical role of Ser/Thr kinase-dependent signaling in controlling mycolic acid elongation, acid-fastness, virulence and has important clinical implications for diagnosis of latent infections. Transcriptome of kasB null, phosphoablative, and phosphomimetic mutants compared to parental. Triplicate 10ml cultures of M. tuberculosis CDC1551 and kasB null, phosphoablative, and phosphomimetic mutants were grown to OD 1.0 and harvested for transcriptional profiling.
2014-04-01 | E-GEOD-47640 | biostudies-arrayexpress
Project description:Mechanisms governing Mycobacterium tuberculosis acid-fastness and its capacity to induce long-term infections remain unknown. Serine/Threonine phosphorylation represents an emerging theme allowing mycobacteria to adapt their cell envelope structure/composition in response to environmental changes. We addressed whether phosphorylation of KasB, a mycolic acid biosynthetic enzyme, modulates M. tuberculosis pathogenicity. Phosphorylation of KasB occurred at Thr334 and Thr336 in vitro and in mycobacteria. A mutant strain bearing an kasB_T334D/T336D allele, mimicking constitutive KasB phosphorylation, was generated by specialized linkage transduction. This resulted in shortened mycolic acids and the lack of trans-cyclopropanation. Structural/modeling analyses revealed Thr334 and Thr336 in the vicinity of the catalytic triad, implying that phosphorylation of these residues impaired KasB activity. Importantly, the phosphomimetic strain lost acid-fast staining and was more attenuated than a kasB deletion mutant in immunocompetent and immunodeficient mice. The absence of lung pathology and mortality infers this mutant to represent a valuable vaccine candidate. This work emphasizes the critical role of Ser/Thr kinase-dependent signaling in controlling mycolic acid elongation, acid-fastness, virulence and has important clinical implications for diagnosis of latent infections.
2014-04-01 | GSE47640 | GEO
Project description:Background: the transcription of tumor mutations from DNA into RNA has implications for biology, epigenetics and clinical practice. It is not clear if mutations are in general transcribed and, if so, at what proportion to the wild-type allele. Here, we examined the correlation between DNA mutation allele frequency and RNA mutation allele frequency. Methods: we sequenced the exome and transcriptome of tumor cell lines with large copy number variations, identified heterozygous single nucleotide mutations and absolute DNA copy number, and determined the corresponding DNA and RNA mutation allele fraction. Results: we found that 99% of the DNA mutations in expressed genes are expressed as RNA. Moreover, we found a high correlation between the DNA and RNA mutation allele frequency. Exceptions are mutations that cause premature termination codons and therefore activate nonsense-mediated decay. Beyond this, we did not find evidence of any wide-scale mechanism, such as allele-specific epigenetic silencing, preferentially promoting mutated or wild-type alleles. Conclusion: taken together, our data strongly suggest that genes are equally transcribed from all alleles, mutated and wild-type, and thus transcribed in proportion to their DNA allele frequency.
2014-03-05 | E-MTAB-2383 | biostudies-arrayexpress
Project description:In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Over-expression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Constitutive up-regulation of ERG11 is a major cause of resistance to fluconazole in clinical isolates of C. albicans, yet the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately up-regulated with ERG11 in a fluconazole resistant clinical isolate as compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug susceptible strain resulted in constitutive up-regulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans. Keywords: genome-wide expression profiling Clinical isolates S1 (susceptible) and S2 (resistant), genome strain SC5314, UPC2 disruption strains (UPC2M4A and UPC2M4B), UPC2 re-integrant strains of non-mutated UPC2 allele (UPC2M2K21A and UPC2M2K21B), and UPC2 re-integrant strains of mutated UPC2 allele (UPC2M2K31A and UPC2M2K31B) were grown for 3 hours until log phase. RNA was extracted for each isolate/strain. Experiments were performed in duplicate.
2008-11-04 | E-GEOD-11320 | biostudies-arrayexpress
Project description:The putative trancriptional regulator PA2449 was found to be essential for both glycine/serine metabolism and the production of phenazines in P. aeruignosa PAO1. We examined the regulon controlled by PA2449 via microarray analysis between wild-type P. aeruignosa PAO1 and the PA2449-null mutant P. aeruginosa PW5126. Both strains were obtained from the PA-two allele library (Univ. of Washington, Ref. Jacobs et al. 2003. PNAS 100, 14339).
2012-10-15 | GSE39044 | GEO
Project description:We report long-read sequencing results from the sox32 allele targeted with a multi-guide Cas9 RNP strategy. Embryos were cell-injected with Cas9 RNP programmed with 4 guides simultaneously targeting different portions of the sox32 gene. Genomic DNA was extracted from each embryo and the sox32 coding sequence and intron were amplified and sequenced. Six individual embryos were evaluated. We found that the majority of sequences were unique and most involved mutations confined to the target sites rather than larger lesions spanning two or more target sites. We also found that alleles accumulated mutations over several DNA replication cycles, which may in part explain the diversity of alleles that were generated. This study is the first to use high-throughput sequencing to evaluate of an allele of this size mutated by a multi-guide Cas9 RNP strategy and provides insights into the dynamics of CRISPR-Cas9 RNP gene editing in vivo.
2018-07-01 | GSE115233 | GEO
Project description:We tamoxifen treated 8-12 week old mice that had floxed alleles of the following: 1) both Apc alleles (giving rise to Apc truncation/inactivation); 2) both Cdx2 alleles (giving rise to Cdx2 inactivation; 3) one Braf allele, that upon Cre-mediated recombination gives a Braf V600E mutant allele (details below), and 4) the combination of both the Cdx2 alleles and the BrafV600E allele. All four of those groups also had a CDX2P-CreERT2 transgene that expresses Cre recombinase fused to a tamoxifen-regulated fragment of the estrogen receptor ligand binding domain. CreERT2 expression occurs only in tissues where the Cdx2 gene is expressed, which is almost exclusively in adult mouse cecum and colon epithelium. A fifth group of mice had the floxed Cdx2 alleles, but no CDX2P-CreERT2 gene. Treating the mice having CDX2P-CreERT2 with tamoxifen permits the Cre recombinase to enter the cell nucleus and recombine the Apc, Braf, and/or Cdx2 alleles containing loxP sequence elements. Mice were treated with intraperitoneal injection of tamoxifen dissolved in corn oil. Three mice per group were used. The control mice did not develop tumors or any morphological or histological changes in their epithelium, but their colons were used to create the 3 control samples. To obtain the BrafV600E allele we used a genetically engineered mouse line previously described by Dankort et al. (Genes Dev 2007, 21:379-84) that can express the BrafV600E mutant protein following Cre-mediated recombination. The Braf(CA) (Braf-Cre-activated) allele mice carry a gene-targeted allele of Braf, where Braf sequences from exons 15-18 are present in the normal mouse Braf intron 14, followed by a mutated exon 15 (carrying the V600E mutation). The exon 15-18 sequence element is flanked by loxP sites. In the absence of Cre-mediated recombination, the Braf(CA) allele expresses a wild type Braf protein. Following Cre-mediated recombination, the Braf exon 15-18 element is removed, and the Braf(CA) allele then encodes the Braf V600E protein (from the introduced mutated exon 15). RNA was purified from tumor or normal tissue, and targets for Affymetrix arrays were synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST arrays, which hold 41345 probe-sets, but we largely analyzed just those 25216 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with the Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the five groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which selects subsets of the probe-sets as differentially expressed between pairs of groups, as well as significant Cdx2-/- by Braf V600E interactions. It also gives the homologous human gene IDs we used for enrichment testing, which were 1-to-1 best homologs according to build 68 of NCBI's Homologene. A second supplementary sheet shows the data we enrichment tested after collapsing to distinct human homologs, joins of the results of tests with GSE4045 data and of tests with TCGA data to the mouse genes, and the intersections of selected genes in those data set with our gene selections in mouse. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform.
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