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[PMID]: 29422501 [Au] Autor: Collopy LC; Ware TL; Goncalves T; Í Kongsstovu S; Yang Q; Amelina H; Pinder C; Alenazi A; Moiseeva V; Pearson SR; Armstrong CA; Tomita K [Ad] Endereço: Chromosome Maintenance Group, UCL Cancer Institute, University College London, London, WC1E 6DD, UK. [Ti] Título: LARP7 family proteins have conserved function in telomerase assembly. [So] Source: Nat Commun;9(1):557, 2018 02 08. [Is] ISSN: 2041-1723 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: Understanding the intricacies of telomerase regulation is crucial due to the potential health benefits of modifying its activity. Telomerase is composed of an RNA component and reverse transcriptase. However, additional factors required during biogenesis vary between species. Here we have identified fission yeast Lar7 as a member of the conserved LARP7 family, which includes the Tetrahymena telomerase-binding protein p65 and human LARP7. We show that Lar7 has conserved RNA-recognition motifs, which bind telomerase RNA to protect it from exosomal degradation. In addition, Lar7 is required to stabilise the association of telomerase RNA with the protective complex LSm2-8, and telomerase reverse transcriptase. Lar7 remains a component of the mature telomerase complex and is required for telomerase localisation to the telomere. Collectively, we demonstrate that Lar7 is a crucial player in fission yeast telomerase biogenesis, similarly to p65 in Tetrahymena, and highlight the LARP7 family as a conserved factor in telomere maintenance. [Mh] Termos MeSH primário: Proteínas Nucleares/genética Fosfoproteínas/genética Proteínas de Protozoários/genética RNA Fúngico/genética DNA Polimerase Dirigida por RNA/genética RNA/genética Ribonucleoproteínas/genética Schizosaccharomyces/genética Telomerase/genética [Mh] Termos MeSH secundário: Motivos de Aminoácidos Sequência Conservada Expressão Gênica Seres Humanos Isoenzimas/genética Isoenzimas/metabolismo Proteínas Nucleares/metabolismo Fosfoproteínas/metabolismo Ligação Proteica Proteínas de Protozoários/metabolismo RNA/metabolismo Estabilidade de RNA RNA Fúngico/metabolismo DNA Polimerase Dirigida por RNA/metabolismo Ribonucleoproteínas/metabolismo Schizosaccharomyces/metabolismo Telomerase/metabolismo Telômero/química Telômero/ultraestrutura Tetrahymena thermophila/genética Tetrahymena thermophila/metabolismo [Pt] Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T [Nm] Nome de substância: 0 (Isoenzymes); 0 (Larp7 protein, human); 0 (Nuclear Proteins); 0 (Pdd1 protein, Tetrahymena); 0 (Phosphoproteins); 0 (Protozoan Proteins); 0 (RNA, Fungal); 0 (Ribonucleoproteins); 0 (Ter1 telomerase subunit, S pombe); 63231-63-0 (RNA); EC 2.7.7.49 (RNA-Directed DNA Polymerase); EC 2.7.7.49 (Telomerase) [Em] Mês de entrada: 1803 [Cu] Atualização por classe: 180307 [Lr] Data última revisão: 180307 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 180210 [St] Status: MEDLINE [do] DOI: 10.1038/s41467-017-02296-4

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[PMID]: 29111407 [Au] Autor: Chahorm K; Prakitchaiwattana C [Ad] Endereço: Department of Food Technology, Faculty of Science, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand. [Ti] Título: Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods. [So] Source: Int J Food Microbiol;264:46-52, 2018 Jan 02. [Is] ISSN: 1879-3460 [Cp] País de publicação: Netherlands [La] Idioma: eng [Ab] Resumo: The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 10 to 10 CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 10 CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively. [Mh] Termos MeSH primário: Eletroforese em Gel de Gradiente Desnaturante/métodos Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos Vibrio alginolyticus/genética Vibrio cholerae/genética Vibrio mimicus/genética Vibrio parahaemolyticus/genética [Mh] Termos MeSH secundário: Primers do DNA/genética Microbiologia de Alimentos/métodos Técnicas de Amplificação de Ácido Nucleico/métodos Reação em Cadeia da Polimerase/métodos DNA Polimerase Dirigida por RNA Vibrio alginolyticus/classificação Vibrio alginolyticus/isolamento & purificação Vibrio cholerae/classificação Vibrio cholerae/isolamento & purificação Vibrio mimicus/classificação Vibrio mimicus/isolamento & purificação Vibrio parahaemolyticus/classificação Vibrio parahaemolyticus/isolamento & purificação [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (DNA Primers); EC 2.7.7.49 (RNA-Directed DNA Polymerase) [Em] Mês de entrada: 1802 [Cu] Atualização por classe: 180226 [Lr] Data última revisão: 180226 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 171108 [St] Status: MEDLINE

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[PMID]: 29319937 [Au] Autor: Food and Drug Administration, HHS. [Ti] Título: Medical Devices; Clinical Chemistry and Clinical Toxicology Devices; Classification of the Reagents for Molecular Diagnostic Instrument Test Systems. Final order. [So] Source: Fed Regist;82(247):61162-3, 2017 Dec 27. [Is] ISSN: 0097-6326 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: The Food and Drug Administration (FDA or we) is classifying the reagents for molecular diagnostic instrument test systems into class I (general controls). We are taking this action because we have determined that classifying the device into class I (general controls) will provide a reasonable assurance of safety and effectiveness of the device. We believe this action will also enhance patients' access to beneficial innovative devices, in part by reducing regulatory burdens. [Mh] Termos MeSH primário: Testes de Química Clínica/classificação Testes de Química Clínica/instrumentação Segurança de Equipamentos/classificação Indicadores e Reagentes/classificação Biologia Molecular/classificação Biologia Molecular/instrumentação Kit de Reagentes para Diagnóstico/classificação [Mh] Termos MeSH secundário: DNA Polimerase Dirigida por DNA/classificação Seres Humanos Ácidos Nucleicos/classificação Nucleotídeos/classificação DNA Polimerase Dirigida por RNA/classificação [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (Indicators and Reagents); 0 (Nucleic Acids); 0 (Nucleotides); 0 (Reagent Kits, Diagnostic); EC 2.7.7.49 (RNA-Directed DNA Polymerase); EC 2.7.7.7 (DNA-Directed DNA Polymerase) [Em] Mês de entrada: 1801 [Cu] Atualização por classe: 180122 [Lr] Data última revisão: 180122 [Sb] Subgrupo de revista: T [Da] Data de entrada para processamento: 180111 [St] Status: MEDLINE

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[PMID]: 29232693 [Au] Autor: Rai SK; Sangesland M; Lee M; Esnault C; Cui Y; Chatterjee AG; Levin HL [Ad] Endereço: Section on Eukaryotic Transposable Elements, Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (NIH), Bethesda, Maryland, United States of America. [Ti] Título: Host factors that promote retrotransposon integration are similar in distantly related eukaryotes. [So] Source: PLoS Genet;13(12):e1006775, 2017 12. [Is] ISSN: 1553-7404 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements. [Mh] Termos MeSH primário: Fatores Hospedeiros de Integração/genética Recombinação Genética Retroelementos/genética Sequências Repetidas Terminais/genética Ubiquitina-Proteína Ligases/genética [Mh] Termos MeSH secundário: Reparo do DNA/genética Eucariotos/genética Regulação Fúngica da Expressão Gênica Integrases/genética Regiões Promotoras Genéticas DNA Polimerase Dirigida por RNA/genética Retroviridae/genética Saccharomyces cerevisiae/genética Proteínas de Saccharomyces cerevisiae/genética Schizosaccharomyces/genética [Pt] Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL [Nm] Nome de substância: 0 (Integration Host Factors); 0 (Retroelements); 0 (Saccharomyces cerevisiae Proteins); 0 (Spp382 protein, S cerevisiae); EC 2.3.2.27 (Rhp18 protein, S pombe); EC 2.3.2.27 (Ubiquitin-Protein Ligases); EC 2.7.7.- (Integrases); EC 2.7.7.49 (RNA-Directed DNA Polymerase); EC 2.7.7.49 (reverse transcriptase Ty3, S cerevisiae) [Em] Mês de entrada: 1801 [Cu] Atualização por classe: 180111 [Lr] Data última revisão: 180111 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 171213 [St] Status: MEDLINE [do] DOI: 10.1371/journal.pgen.1006775

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[PMID]: 28967961 [Au] Autor: Baba M; Kakue R; Leucht C; Rasor P; Walch H; Ladiges D; Bell C; Kojima K; Takita T; Yasukawa K [Ad] Endereço: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. [Ti] Título: Further increase in thermostability of Moloney murine leukemia virus reverse transcriptase by mutational combination. [So] Source: Protein Eng Des Sel;30(8):551-557, 2017 Aug 01. [Is] ISSN: 1741-0134 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: We previously generated a highly thermostable triple variant of Moloney murine leukemia virus reverse transcriptase, MM3 (E286R/E302K/L435R), by introducing positive charges by site-directed mutagenesis at positions that have been implicated in the interaction with template-primer (Yasukawa et al., (2010) J. Biotechnol., 150, 299-306). In this study, we attempted to further increase the thermostability of MM3. Twenty-nine mutations were newly designed, focusing on the number of surface charge, stabilization of hydrophobic core, and introduction of salt bridge. The corresponding 29 single variants were produced in Escherichia coli and characterized for activity and stability. Six mutations (A32V, L41D, L72R, I212R, L272E and W388R) were selected as the candidates for further stabilize MM3. Fifteen multiple variants were designed by combining two or more of the six mutations with the MM3 mutations, produced and characterized. The sextuple variant MM3.14 (A32V/L72R/E286R/E302K/W388R/L435R) exhibited higher thermostability than MM3. [Mh] Termos MeSH primário: Vírus da Leucemia Murina de Moloney/genética Mutagênese Sítio-Dirigida/métodos DNA Polimerase Dirigida por RNA/genética Proteínas Recombinantes/genética Proteínas Virais/genética [Mh] Termos MeSH secundário: Estabilidade Enzimática Escherichia coli/genética Temperatura Alta Vírus da Leucemia Murina de Moloney/enzimologia DNA Polimerase Dirigida por RNA/química DNA Polimerase Dirigida por RNA/metabolismo Proteínas Recombinantes/química Proteínas Recombinantes/metabolismo Proteínas Virais/química Proteínas Virais/metabolismo [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (Recombinant Proteins); 0 (Viral Proteins); EC 2.7.7.49 (RNA-Directed DNA Polymerase) [Em] Mês de entrada: 1710 [Cu] Atualização por classe: 171010 [Lr] Data última revisão: 171010 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 171003 [St] Status: MEDLINE [do] DOI: 10.1093/protein/gzx046

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[PMID]: 28911121 [Au] Autor: Agudo R; Calvo PA; Martínez-Jiménez MI; Blanco L [Ad] Endereço: Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Cantoblanco, E-28049 Madrid, Spain. [Ti] Título: Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase. [So] Source: Nucleic Acids Res;45(15):9046-9058, 2017 Sep 06. [Is] ISSN: 1362-4962 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: We have developed a straightforward fluorometric assay to measure primase-polymerase activity of human PrimPol (HsPrimPol). The sensitivity of this procedure uncovered a novel RNA-dependent DNA priming-polymerization activity (RdDP) of this enzyme. In an attempt to enhance HsPrimPol RdDP activity, we constructed a smart mutant library guided by prior sequence-function analysis, and tested this library in an adapted screening platform of our fluorometric assay. After screening less than 500 variants, we found a specific HsPrimPol mutant, Y89R, which displays 10-fold higher RdDP activity than the wild-type enzyme. The improvement of RdDP activity in the Y89R variant was due mainly to an increased in the stabilization of the preternary complex (protein:template:incoming nucleotide), a specific step preceding dimer formation. Finally, in support of the biotechnological potential of PrimPol as a DNA primer maker during reverse transcription, mutant Y89R HsPrimPol rendered up to 17-fold more DNA than with random hexamer primers. [Mh] Termos MeSH primário: Substituição de Aminoácidos Bioensaio DNA Primase/genética DNA Polimerase Dirigida por DNA/genética Enzimas Multifuncionais/genética Engenharia de Proteínas/métodos DNA Polimerase Dirigida por RNA/genética RNA/genética [Mh] Termos MeSH secundário: Arginina/química Arginina/metabolismo Clonagem Molecular DNA Primase/metabolismo Primers do DNA/síntese química Primers do DNA/química DNA Polimerase Dirigida por DNA/metabolismo Escherichia coli/genética Escherichia coli/metabolismo Corantes Fluorescentes/química Fluorometria/métodos Expressão Gênica Biblioteca Gênica Seres Humanos Enzimas Multifuncionais/metabolismo Mutação Oligonucleotídeos/química Oligonucleotídeos/metabolismo Compostos Orgânicos/química Ligação Proteica Multimerização Proteica RNA/metabolismo DNA Polimerase Dirigida por RNA/metabolismo Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos Tirosina/química Tirosina/metabolismo [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (DNA Primers); 0 (Fluorescent Dyes); 0 (Multifunctional Enzymes); 0 (Oligonucleotides); 0 (Organic Chemicals); 163795-75-3 (SYBR Green I); 42HK56048U (Tyrosine); 63231-63-0 (RNA); 94ZLA3W45F (Arginine); EC 2.7.7.- (DNA Primase); EC 2.7.7.- (PrimPol protein, human); EC 2.7.7.49 (RNA-Directed DNA Polymerase); EC 2.7.7.7 (DNA-Directed DNA Polymerase) [Em] Mês de entrada: 1710 [Cu] Atualização por classe: 171012 [Lr] Data última revisão: 171012 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170916 [St] Status: MEDLINE [do] DOI: 10.1093/nar/gkx633

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[PMID]: 28845978 [Au] Autor: Alenko A; Fleming AM; Burrows CJ [Ad] Endereço: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States. [Ti] Título: Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8-Oxo-7,8-dihydroguanine and A and G opposite 5-Guanidinohydantoin and Spiroiminodihydantoin Diastereomers. [So] Source: Biochemistry;56(38):5053-5064, 2017 Sep 26. [Is] ISSN: 1520-4995 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: Reactive oxygen species, both endogenous and exogenous, can damage nucleobases of RNA and DNA. Among the nucleobases, guanine has the lowest redox potential, making it a major target of oxidation. Although RNA is more prone to oxidation than DNA is, oxidation of guanine in RNA has been studied to a significantly lesser extent. One of the reasons for this is that many tools that were previously developed to study oxidation of DNA cannot be used on RNA. In the study presented here, the lack of a method for seeking sites of modification in RNA where oxidation occurs is addressed. For this purpose, reverse transcription of RNA containing major products of guanine oxidation was used. Extension of a DNA primer annealed to an RNA template containing 8-oxo-7,8-dihydroguanine (OG), 5-guanidinohydantoin (Gh), or the R and S diastereomers of spiroiminodihydantoin (Sp) was studied under standing start conditions. SuperScript III reverse transcriptase is capable of bypassing these lesions in RNA inserting predominantly A opposite OG, predominantly G opposite Gh, and almost an equal mixture of A and G opposite the Sp diastereomers. These data should allow RNA sequencing of guanine oxidation products by following characteristic mutation signatures formed by the reverse transcriptase during primer elongation past G oxidation sites in the template RNA strand. [Mh] Termos MeSH primário: Guanidinas/química Guanina/análogos & derivados Guanosina/análogos & derivados Hidantoínas/química RNA/química Transcrição Reversa Compostos de Espiro/química [Mh] Termos MeSH secundário: Adenina/química Guanina/química Guanosina/química Guanosina/genética Cinética Oxirredução DNA Polimerase Dirigida por RNA/química DNA Polimerase Dirigida por RNA/metabolismo Estereoisomerismo [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (Guanidines); 0 (Hydantoins); 0 (Spiro Compounds); 0 (guanidinohydantoin); 0 (spiroiminodihydantoin); 12133JR80S (Guanosine); 5614-64-2 (8-hydroxyguanine); 5Z93L87A1R (Guanine); 63231-63-0 (RNA); EC 2.7.7.49 (RNA-Directed DNA Polymerase); JAC85A2161 (Adenine) [Em] Mês de entrada: 1710 [Cu] Atualização por classe: 171010 [Lr] Data última revisão: 171010 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170829 [St] Status: MEDLINE [do] DOI: 10.1021/acs.biochem.7b00730

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[PMID]: 28820243 [Au] Autor: Sexton AN; Wang PY; Rutenberg-Schoenberg M; Simon MD [Ad] Endereço: Department of Molecular Biophysics & Biochemistry, Yale University , New Haven, Connecticut 06511, United States. [Ti] Título: Interpreting Reverse Transcriptase Termination and Mutation Events for Greater Insight into the Chemical Probing of RNA. [So] Source: Biochemistry;56(35):4713-4721, 2017 Sep 05. [Is] ISSN: 1520-4995 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given the variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely nonoverlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments. [Mh] Termos MeSH primário: Fibroblastos/metabolismo DNA Polimerase Dirigida por RNA/metabolismo RNA/química [Mh] Termos MeSH secundário: Animais Células Cultivadas Regulação Enzimológica da Expressão Gênica Camundongos Mutação DNA Polimerase Dirigida por RNA/química [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 63231-63-0 (RNA); EC 2.7.7.49 (RNA-Directed DNA Polymerase) [Em] Mês de entrada: 1709 [Cu] Atualização por classe: 171021 [Lr] Data última revisão: 171021 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170819 [St] Status: MEDLINE [do] DOI: 10.1021/acs.biochem.7b00323

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[PMID]: 28778390 [Au] Autor: Yasukawa K; Iida K; Okano H; Hidese R; Baba M; Yanagihara I; Kojima K; Takita T; Fujiwara S [Ad] Endereço: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. Electronic address: yasukawa@kais.kyoto-u.ac.jp. [Ti] Título: Next-generation sequencing-based analysis of reverse transcriptase fidelity. [So] Source: Biochem Biophys Res Commun;492(2):147-153, 2017 Oct 14. [Is] ISSN: 1090-2104 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: In this study, we devised a simple and rapid method to analyze fidelity of reverse transcriptase (RT) using next-generation sequencing (NGS). The method comprises a cDNA synthesis reaction from standard RNA with a primer containing a tag of 14 randomized bases and the RT to be tested, PCR using high-fidelity DNA polymerase, and NGS. By comparing the sequence of each read with the reference sequence, mutations were identified. The mutation can be identified to be due to an error introduced by either cDNA synthesis, PCR, or NGS based on whether the sequence reads with the same tag contain the same mutation or not. The error rates in cDNA synthesis with Moloney murine leukemia virus (MMLV) RT thermostable variant MM4 or the recently developed 16-tuple variant of family B DNA polymerase with RT activity, RTX, from Thermococcus kodakarensis, were 0.75-1.0 × 10 errors/base, while that in the reaction with the wild-type human immunodeficiency virus type 1 (HIV-1) RT was 2.6 × 10 errors/base. Overall, our method could precisely evaluate the fidelity of various RTs with different reaction conditions in a high-throughput manner without the use of expensive optics and troublesome adaptor ligation. [Mh] Termos MeSH primário: DNA Complementar/genética HIV-1/enzimologia Sequenciamento de Nucleotídeos em Larga Escala/métodos Vírus da Leucemia Murina de Moloney/enzimologia DNA Polimerase Dirigida por RNA/genética Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos Thermococcus/enzimologia [Mh] Termos MeSH secundário: Sequência de Bases DNA Polimerase Dirigida por DNA/genética Transcriptase Reversa do HIV/genética HIV-1/genética Vírus da Leucemia Murina de Moloney/genética DNA Polimerase Dirigida por RNA/química Thermococcus/genética [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (DNA, Complementary); EC 2.7.7.- (reverse transcriptase, Human immunodeficiency virus 1); EC 2.7.7.49 (HIV Reverse Transcriptase); EC 2.7.7.49 (RNA-Directed DNA Polymerase); EC 2.7.7.7 (DNA-Directed DNA Polymerase) [Em] Mês de entrada: 1709 [Cu] Atualização por classe: 170925 [Lr] Data última revisão: 170925 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170806 [St] Status: MEDLINE

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[PMID]: 28606835 [Au] Autor: Nouroz F; Noreen S; Khan MF; Ahmed S; Heslop-Harrison JSP [Ad] Endereço: Department of Biology, University of Leicester, UK; Department of Botany, Hazara University Mansehra, Pakistan. Electronic address: faisalnouroz@hu.edu.pk. [Ti] Título: Identification and characterization of mobile genetic elements LINEs from Brassica genome. [So] Source: Gene;627:94-105, 2017 Sep 05. [Is] ISSN: 1879-0038 [Cp] País de publicação: Netherlands [La] Idioma: eng [Ab] Resumo: Among transposable elements (TEs), the LTR retrotransposons are abundant followed by non-LTR retrotransposons in plant genomes, the lateral being represented by LINEs and SINEs. Computational and molecular approaches were used for the characterization of Brassica LINEs, their diversity and phylogenetic relationships. Four autonomous and four non-autonomous LINE families were identified and characterized from Brassica. Most of the autonomous LINEs displayed two open reading frames, ORF1 and ORF2, where ORF1 is a gag protein domain, while ORF2 encodes endonuclease (EN) and a reverse transcriptase (RT). Three of four families encoded an additional RNase H (RH) domain in pol gene common to 'R' and 'I' type of LINEs. The PCR analyses based on LINEs RT fragments indicate their high diversity and widespread occurrence in tested 40 Brassica cultivars. Database searches revealed the homology in LINE sequences in closely related genera Arabidopsis indicating their origin from common ancestors predating their separation. The alignment of 58 LINEs RT sequences from Brassica, Arabidopsis and other plants depicted 4 conserved domains (domain II-V) showing similarity to previously detected domains. Based on RT alignment of Brassica and 3 known LINEs from monocots, Brassicaceae LINEs clustered in separate clade, further resolving 4 Brassica-Arabidopsis specific families in 2 sub-clades. High similarities were observed in RT sequences in the members of same family, while low homology was detected in members across the families. The investigation led to the characterization of Brassica specific LINE families and their diversity across Brassica species and their cultivars. [Mh] Termos MeSH primário: Brassica/genética Genoma de Planta Elementos Nucleotídeos Longos e Dispersos [Mh] Termos MeSH secundário: Sequência de Aminoácidos Brassica/classificação Mineração de Dados Fases de Leitura Aberta Filogenia DNA Polimerase Dirigida por RNA/química Alinhamento de Sequência [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: EC 2.7.7.49 (RNA-Directed DNA Polymerase) [Em] Mês de entrada: 1708 [Cu] Atualização por classe: 170807 [Lr] Data última revisão: 170807 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170614 [St] Status: MEDLINE

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