Escherichia coli (E. coli) are commonly used as hosts for DNA cloning and sequencing. Upon transformation of E. coli with recombined vector carrying a gene of interest, the bacteria multiply the gene of interest while maintaining the integrity of its content. During the subcloning of a mouse genomic fragment into a plasmid vector, we noticed that the size of the insert increased significantly upon replication in E. coli. The sequence of the insert was determined and found to contain a novel DNA sequence within the mouse genomic insert. A BLAST search of GenBank revealed the novel sequence to be that of the Insertion Sequence 2 (IS2) element from E. coli that was likely inserted during replication in that organism. Importantly, a detailed search of GenBank shows that the IS2 is present within many eukaryotic nucleotide sequences, and in many cases, has been annotated as being part of the protein. The results of this study suggest that one must perform additional careful analysis of the sequence results using BLAST comparisons, and further verification of gene annotation before submission into the GenBank.
{"title":"Eukaryotic gene invasion by a bacterial mobile insertion sequence element IS2 during cloning into a plasmid vector.","authors":"Alireza G Senejani, Joann B Sweasy","doi":"10.1186/2041-9414-1-2","DOIUrl":"https://doi.org/10.1186/2041-9414-1-2","url":null,"abstract":"<p><p> Escherichia coli (E. coli) are commonly used as hosts for DNA cloning and sequencing. Upon transformation of E. coli with recombined vector carrying a gene of interest, the bacteria multiply the gene of interest while maintaining the integrity of its content. During the subcloning of a mouse genomic fragment into a plasmid vector, we noticed that the size of the insert increased significantly upon replication in E. coli. The sequence of the insert was determined and found to contain a novel DNA sequence within the mouse genomic insert. A BLAST search of GenBank revealed the novel sequence to be that of the Insertion Sequence 2 (IS2) element from E. coli that was likely inserted during replication in that organism. Importantly, a detailed search of GenBank shows that the IS2 is present within many eukaryotic nucleotide sequences, and in many cases, has been annotated as being part of the protein. The results of this study suggest that one must perform additional careful analysis of the sequence results using BLAST comparisons, and further verification of gene annotation before submission into the GenBank.</p>","PeriodicalId":53596,"journal":{"name":"Genome Integrity","volume":"1 1","pages":"2"},"PeriodicalIF":0.0,"publicationDate":"2010-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/2041-9414-1-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29164804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Prarthana Srikanth, Amit Roy Chowdhury, G. Low, Radha Saraswathy, Akira Fujimori, Birendranath Banerjee, Wilner Martinez-Lopez, M. Hande
Our cellular genome is susceptible to cytotoxic lesions which include single strand breaks and double strand breaks among other lesions. Ataxia telangiectasia mutated (ATM) protein was one of the first DNA damage sensor proteins to be discovered as being involved in DNA repair and as well as in telomere maintenance. Telomeres help maintain the stability of our chromosomes by protecting the ends from degradation. Cells from ataxia telangiectasia (AT) patients lack ATM and accumulate chromosomal alterations. AT patients display heightened susceptibility to cancer. In this study, cells from AT patients (called as AT-/- and AT+/- cells) were characterized for genome stability status and it was observed that AT-/- cells show considerable telomere attrition. Furthermore, DNA damage and genomic instability were compared between normal (AT+/+ cells) and AT-/- cells exhibiting increased frequencies of spontaneous DNA damage and genomic instability markers. Both AT-/- and AT+/- cells were sensitive to sodium arsenite (1.5 and 3.0 μg/ml) and ionizing radiation-induced (2 Gy, gamma rays) oxidative stress. Interestingly, telomeric fragments were detected in the comet tails as revealed by comet-fluorescence in situ hybridization analysis, suggestive of telomeric instability in AT-/- cells upon exposure to sodium arsenite or radiation. Besides, there was an increase in the number of chromosome alterations in AT-/- cells following arsenite treatment or irradiation. In addition, complex chromosome aberrations were detected by multicolor fluorescence in situ hybridization in AT-/- cells in comparison to AT+/- and normal cells. Telomere attrition and chromosome alterations were detected even at lower doses of sodium arsenite. Peptide nucleic acid – FISH analysis revealed defective chromosome segregation in cells lacking ATM proteins. The data obtained in this study substantiates the role of ATM in telomere stability under oxidative stress.
{"title":"Oxidative Damage Induced Telomere Mediated Genomic Instability in Cells from Ataxia Telangiectasia Patients","authors":"Prarthana Srikanth, Amit Roy Chowdhury, G. Low, Radha Saraswathy, Akira Fujimori, Birendranath Banerjee, Wilner Martinez-Lopez, M. Hande","doi":"10.11343/AMN.53.S45","DOIUrl":"https://doi.org/10.11343/AMN.53.S45","url":null,"abstract":"Our cellular genome is susceptible to cytotoxic lesions which include single strand breaks and double strand breaks among other lesions. Ataxia telangiectasia mutated (ATM) protein was one of the first DNA damage sensor proteins to be discovered as being involved in DNA repair and as well as in telomere maintenance. Telomeres help maintain the stability of our chromosomes by protecting the ends from degradation. Cells from ataxia telangiectasia (AT) patients lack ATM and accumulate chromosomal alterations. AT patients display heightened susceptibility to cancer. In this study, cells from AT patients (called as AT-/- and AT+/- cells) were characterized for genome stability status and it was observed that AT-/- cells show considerable telomere attrition. Furthermore, DNA damage and genomic instability were compared between normal (AT+/+ cells) and AT-/- cells exhibiting increased frequencies of spontaneous DNA damage and genomic instability markers. Both AT-/- and AT+/- cells were sensitive to sodium arsenite (1.5 and 3.0 μg/ml) and ionizing radiation-induced (2 Gy, gamma rays) oxidative stress. Interestingly, telomeric fragments were detected in the comet tails as revealed by comet-fluorescence in situ hybridization analysis, suggestive of telomeric instability in AT-/- cells upon exposure to sodium arsenite or radiation. Besides, there was an increase in the number of chromosome alterations in AT-/- cells following arsenite treatment or irradiation. In addition, complex chromosome aberrations were detected by multicolor fluorescence in situ hybridization in AT-/- cells in comparison to AT+/- and normal cells. Telomere attrition and chromosome alterations were detected even at lower doses of sodium arsenite. Peptide nucleic acid – FISH analysis revealed defective chromosome segregation in cells lacking ATM proteins. The data obtained in this study substantiates the role of ATM in telomere stability under oxidative stress.","PeriodicalId":53596,"journal":{"name":"Genome Integrity","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2009-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63683651","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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