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Niv Antonovsky - One of the best experts on this subject based on the ideXlab platform.
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point mutations in topoisomerase i alter the mutation spectrum in e coli and impact the emergence of drug resistance genotypes
Nucleic Acids Research, 2020Co-Authors: Amit Bachar, Elad Itzhaki, Shmuel Gleizer, Melina Shamshoom, Ron Milo, Niv AntonovskyAbstract:Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in Escherichia coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and Deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA topoisomerase I (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we find that point mutations in topA increase the rate of Sequence Deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short Sequence duplications, which are independent of existing Sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and leads to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.
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point mutations in topoisomerase i alter the mutation spectrum in e coli and impact the emergence of drug resistance genotypes
bioRxiv, 2019Co-Authors: Amit Bachar, Elad Itzhaki, Shmuel Gleizer, Melina Shamshoom, Ron Milo, Niv AntonovskyAbstract:Abstract Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in E. coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and Deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA Topoisomerase 1 (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we show that point mutations in topA increase the rate of Sequence Deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short Sequence duplications, which are independent of existing Sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and lead to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.
Charles E Samuel - One of the best experts on this subject based on the ideXlab platform.
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isolation of the interferon inducible rna dependent protein kinase pkr promoter and identification of a novel dna element within the 5 flanking region of human and mouse pkr genes
Virology, 1997Co-Authors: Kelli L Kuhen, Charles E SamuelAbstract:The RNA-dependent protein kinase (PKR) is inducible by interferon (IFN) and is implicated in the antiviral and antiproliferative actions of IFN. We have now isolated human genomic clones that contain the promoter region required for transcription of the Pkr gene. Transient transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the Pkr gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription of CAT. Sequence determination and Deletion analysis of the promoter region revealed an element (5'GGAAAACGAAACT3') involved in IFN inducibility that corresponds to the consensus Sequence of the IFN-stimulated response element (ISRE). Comparison of the promoter Sequence of the human Pkr gene to that of the mouse homolog identified a novel element (5'GGGAAGGCGGAGTCC3') immediately upstream of the ISRE element which so far is unique to the human and mouse Pkr gene promoters. We have designated this new motif as KCS, for kinase conserved Sequence. Deletion and substitution mutants of the Pkr promoter region showed that the ISRE element was required for transcriptional induction by type I IFN, whereas the KCS motif increased promoter activity mediated by the ISRE. Additional potential regulatory cis-elements were identified in the human Pkr promoter that are commonly associated with growth control regulation and differentiation. Other than the ISRE and novel KCS elements, the overall organization of potential binding sites for transcription factors was not well conserved between the IFN-inducible promoters of the human and mouse Pkr genes. The strict conservation of Sequence, distance, and position of KCS, relative to ISRE, together with mutagenesis results, suggest an important functional role for the newly recognized KCS motif.
Amit Bachar - One of the best experts on this subject based on the ideXlab platform.
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point mutations in topoisomerase i alter the mutation spectrum in e coli and impact the emergence of drug resistance genotypes
Nucleic Acids Research, 2020Co-Authors: Amit Bachar, Elad Itzhaki, Shmuel Gleizer, Melina Shamshoom, Ron Milo, Niv AntonovskyAbstract:Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in Escherichia coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and Deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA topoisomerase I (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we find that point mutations in topA increase the rate of Sequence Deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short Sequence duplications, which are independent of existing Sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and leads to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.
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point mutations in topoisomerase i alter the mutation spectrum in e coli and impact the emergence of drug resistance genotypes
bioRxiv, 2019Co-Authors: Amit Bachar, Elad Itzhaki, Shmuel Gleizer, Melina Shamshoom, Ron Milo, Niv AntonovskyAbstract:Abstract Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in E. coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and Deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA Topoisomerase 1 (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we show that point mutations in topA increase the rate of Sequence Deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short Sequence duplications, which are independent of existing Sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and lead to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.
Harold Gainer - One of the best experts on this subject based on the ideXlab platform.
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the 216 to 100 bp Sequence in the 5 flanking region of the oxytocin gene contains a cell type specific regulatory element for its selective expression in oxytocin magnocellular neurones
Journal of Neuroendocrinology, 2015Co-Authors: Raymond L Fields, Harold GainerAbstract:The oxytocin (OXT) gene is abundantly and highly selectively expressed in magnocellular neurones (MCNs) of the hypothalamic-neurohypophysial system. Previous DNA Sequence Deletion studies in vivo have shown that the −216- to −100-bp Sequence in the 5′-flanking region of the oxytocin gene was required for its cell-type specific expression in the rat supraoptic nucleus. In the present study, we test the coupled hypotheses that this −216- to −100-bp Sequence is responsible for (i) the selective expression of the OXT gene in OXT-MNCs and (ii) its selective repression in vasopressin (AVP)-MCNs. We show that, consistent with hypothesis 1, removal of the −216- to −100-bp Sequence from the OXT gene completely eliminates its expression in OXT-MCNs in vivo but, in contrast to the prediction of hypothesis 2, there was no appearance of OXT gene expression in AVP-MCNs. Taken together, these and other data demonstrate that the −216- to −100-bp Sequence in the 5′-flanking region of the oxytocin gene contains only an activator of transcription operating in the OXT-MCNs.
Gregory R. Dressler - One of the best experts on this subject based on the ideXlab platform.
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Expression of Pax2 in the intermediate mesoderm is regulated by YY1.
Developmental Biology, 2004Co-Authors: Gregory R. DresslerAbstract:Abstract The transcription factor Pax2 is essential for the development of the urogenital system. Pax2 expression can be detected by mouse embryonic day 8.5 (E8.5) in the region of intermediate mesoderm fated to become the nephric duct, pronephros, and mesonephros. Elements that direct Pax2 expression to nephrogenic precursor cells must be responding to positional information that controls nephrogenic fate. A 4.1-kb Pax2 promoter/enhancer fragment directs expression of a lacZ reporter to the nephrogenic region and the midbrain–hindbrain junction in transgenic mice. As kidney development proceeds, transgene expression is limited to the nephric duct and its derivatives, but not the metanephric mesenchyme. The early expression driven by the 4.1 promoter does not require the Pax2 protein, demonstrating that it receives positional information in the absence of a developing pro- or mesonephros. We have identified two DNAseI hypersensitive regions within this promoter, one at the start site of transcription initiation and a second approximately 2-kb upstream. Deletion of the more distal site significantly attenuates lacZ expression in the nephrogenic region but not in the midbrain–hindbrain region. DNA footprinting of this fragment revealed a highly conserved Sequence between mouse and human Pax2 promoter Sequences. Using fractionated nuclear extracts, we identified the transcription factor Yin Yang 1 (YY1) as the protein that binds to this conserved Sequence. Deletion of the YY1 element significantly attenuated expression of a full-length 4.1 promoter. Moreover, inclusion of this YY1 binding element significantly enhanced expression of a minimal Pax2 promoter/enhancer transgene in E12 embryos. These data point to a novel role for YY1 in the establishment of high level tissue-specific expression within the intermediate mesoderm.
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genomes developmental control expression of pax2 in the intermediate mesoderm is regulated by yy1
2004Co-Authors: Sanjeevkumar R. Patel, Gregory R. DresslerAbstract:AbstractThe transcription factor Pax2 is essential for the development of the urogenital system. Pax2 expression can be detected by mouseembryonic day 8.5 (E8.5) in the region of intermediate mesoderm fated to become the nephric duct, pronephros, and mesonephros. Elementsthat direct Pax2 expression to nephrogenic precursor cells must be responding to positional information that controls nephrogenic fate. A 4.1-kb Pax2 promoter/enhancer fragment directs expression of a lacZ reporter to the nephrogenic region and the midbrain–hindbrain junction intransgenic mice. As kidney development proceeds, transgene expression is limited to the nephric duct and its derivatives, but not themetanephric mesenchyme. The early expression driven by the 4.1 promoter does not require the Pax2 protein, demonstrating that it receivespositional information in the absence of a developing pro- or mesonephros. We have identified two DNAseI hypersensitive regions withinthis promoter, one at the start site of transcription initiation and a second approximately 2-kb upstream. Deletion of the more distal sitesignificantly attenuates lacZ expression in the nephrogenic region but not in the midbrain–hindbrain region. DNA footprinting of thisfragment revealed a highly conserved Sequence between mouse and human Pax2 promoter Sequences. Using fractionated nuclear extracts, weidentified the transcription factor Yin Yang 1 (YY1) as the protein that binds to this conserved Sequence. Deletion of the YY1 elementsignificantly attenuated expression of a full-length 4.1 promoter. Moreover, inclusion of this YY1 binding element significantly enhancedexpression of a minimal Pax2 promoter/enhancer transgene in E12 embryos. These data point to a novel role for YY1 in the establishment ofhigh level tissue-specific expression within the intermediate mesoderm.D 2003 Elsevier Inc. All rights reserved.
