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[PMID]:29372656
[Au] Autor:Mikalkenas A; Ravoityte B; Tauraite D; Serviene E; Meskys R; Serva S
[Ad] Endereço:a Department of Biochemistry and Molecular Biology, Institute of Biosciences, Life Sciences Center , Vilnius University , Vilnius , Lithuania.
[Ti] Título:Conjugation of phosphonoacetic acid to nucleobase promotes a mechanism-based inhibition.
[So] Source:J Enzyme Inhib Med Chem;33(1):384-389, 2018 Dec.
[Is] ISSN:1475-6374
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.
[Mh] Termos MeSH primário: DNA Polimerase Dirigida por DNA/metabolismo
Inibidores Enzimáticos/farmacologia
Nucleotídeos/farmacologia
Ácido Fosfonoacéticos/farmacologia
[Mh] Termos MeSH secundário: Inibidores Enzimáticos/síntese química
Inibidores Enzimáticos/química
HIV-1/enzimologia
Estrutura Molecular
Vírus da Leucemia Murina de Moloney/enzimologia
Nucleotídeos/química
Ácido Fosfonoacéticos/síntese química
Ácido Fosfonoacéticos/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Enzyme Inhibitors); 0 (Nucleotides); EC 2.7.7.7 (DNA-Directed DNA Polymerase); N919E46723 (Phosphonoacetic Acid)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180212
[Lr] Data última revisão:
180212
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180127
[St] Status:MEDLINE
[do] DOI:10.1080/14756366.2017.1417275


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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]: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]:28381565
[Au] Autor:Liberatore RA; Mastrocola EJ; Powell C; Bieniasz PD
[Ad] Endereço:Aaron Diamond AIDS Research Center, New York, New York, USA.
[Ti] Título:Tetherin Inhibits Cell-Free Virus Dissemination and Retards Murine Leukemia Virus Pathogenesis.
[So] Source:J Virol;91(12), 2017 Jun 15.
[Is] ISSN:1098-5514
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The relative contributions of cell-free virion circulation and direct cell-to-cell transmission to retroviral dissemination and pathogenesis are unknown. Tetherin/Bst2 is an antiviral protein that blocks enveloped virion release into the extracellular milieu but may not inhibit cell-to-cell virus transmission. We developed live-cell imaging assays which show that tetherin does not affect Moloney murine leukemia virus (MoMLV) spread, and only minimally affects vesicular stomatitis virus (VSV) spread, to adjacent cells in a monolayer. Conversely, cell-free MLV and VSV virion yields and VSV spread to distal cells were dramatically reduced by tetherin. To elucidate the roles of tetherin and cell-free virions during viral dissemination and pathogenesis, we developed mice carrying an inducible human tetherin (hTetherin) transgene. While ubiquitous hTetherin expression was detrimental to the growth and survival of mice, restriction of hTetherin expression to hematopoietic cells gave apparently healthy mice. The expression of hTetherin in hematopoietic cells had little or no effect on the number of MoMLV-infected splenocytes and thymocytes. However, hTetherin expression significantly reduced cell-free plasma viremia and also delayed MoMLV-induced disease. Overall, these results suggest that MoMLV spread within hematopoietic tissues and cell monolayers involves cell-to-cell transmission that is resistant to tetherin but that virion dissemination via plasma is inhibited by tetherin and is required for full MoMLV pathogenesis. Retroviruses are thought to spread primarily via direct cell-to-cell transmission, yet many have evolved to counteract an antiviral protein called tetherin, which may selectively inhibit cell-free virus release. We generated a mouse model with an inducible tetherin transgene in order to study how tetherin affects retroviral dissemination and on which cell types its expression is required to do so. We first developed a novel live-cell imaging assay to demonstrate that while tetherin does indeed dramatically reduce cell-free virus spreading, it has little to no effect on direct cell-to-cell transmission of either vesicular stomatitis virus (VSV) or the retrovirus MoMLV. Using our transgenic mouse model, we found that tetherin expression on hematopoietic cells resulted in the specific reduction of MoMLV cell-free plasma viremia but not the number of infected hematopoietic cells. The delay in disease associated with this scenario suggests a role for cell-free virus in retroviral disease progression.
[Mh] Termos MeSH primário: Antígenos CD/metabolismo
Vírus da Leucemia Murina de Moloney/fisiologia
Infecções por Retroviridae/virologia
Vírus da Estomatite Vesicular Indiana/fisiologia
Internalização do Vírus
Liberação de Vírus
[Mh] Termos MeSH secundário: Animais
Antígenos CD/genética
Antígenos CD/farmacologia
Proteínas Ligadas por GPI/genética
Proteínas Ligadas por GPI/metabolismo
Proteínas Ligadas por GPI/farmacologia
Células-Tronco Hematopoéticas/metabolismo
Células-Tronco Hematopoéticas/virologia
Seres Humanos
Camundongos
Camundongos Transgênicos
Células NIH 3T3
Baço/citologia
Baço/virologia
Timócitos/virologia
Viremia
Vírion/metabolismo
Replicação Viral
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antigens, CD); 0 (BST2 protein, human); 0 (GPI-Linked Proteins)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170727
[Lr] Data última revisão:
170727
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170407
[St] Status:MEDLINE


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[PMID]:28323884
[Au] Autor:Baker SL; Hogg JR
[Ad] Endereço:Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
[Ti] Título:A system for coordinated analysis of translational readthrough and nonsense-mediated mRNA decay.
[So] Source:PLoS One;12(3):e0173980, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing premature termination codons, limiting the expression of potentially deleterious truncated proteins. This activity positions the pathway as a regulator of the severity of genetic diseases caused by nonsense mutations. Because many genetic diseases result from nonsense alleles, therapeutics inducing readthrough of premature termination codons and/or inhibition of NMD have been of great interest. Several means of enhancing translational readthrough have been reported to concomitantly inhibit NMD efficiency, but tools for systematic analysis of mammalian NMD inhibition by translational readthrough are lacking. Here, we introduce a system that allows concurrent analysis of translational readthrough and mRNA decay. We use this system to show that diverse readthrough-promoting RNA elements have similar capacities to inhibit NMD. Further, we provide evidence that the level of translational readthrough required for protection from NMD depends on the distance of the suppressed termination codon from the end of the mRNA.
[Mh] Termos MeSH primário: Códon sem Sentido/genética
Degradação do RNAm Mediada por Códon sem Sentido/genética
Biossíntese de Proteínas/genética
Estabilidade de RNA/genética
RNA Mensageiro/metabolismo
[Mh] Termos MeSH secundário: Sequência de Bases
Linhagem Celular Tumoral
Vírus da Febre do Carrapato do Colorado/genética
Epidermólise Bolhosa/genética
Doenças Genéticas Inatas/genética
Células HEK293
Células HeLa
Seres Humanos
Sequências Repetidas Invertidas/genética
Vírus da Leucemia Murina de Moloney/genética
Regiões Promotoras Genéticas/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Codon, Nonsense); 0 (RNA, Messenger)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170926
[Lr] Data última revisão:
170926
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170322
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0173980


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[PMID]:28160599
[Au] Autor:Döring J; Hurek T
[Ad] Endereço:Department of Microbe-Plant Interactions, CBIB (Center for Biomolecular Interactions Bremen), University of Bremen, PO Box 330440, D-28334 Bremen, Germany.
[Ti] Título:Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase.
[So] Source:Nucleic Acids Res;45(7):3967-3984, 2017 Apr 20.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Branchpoint nucleotides of intron lariats induce pausing of DNA synthesis by reverse transcriptases (RTs), but it is not known yet how they direct RT RNase H activity on branched RNA (bRNA). Here, we report the effects of the two arms of bRNA on branchpoint-directed RNA cleavage and mutation produced by Moloney murine leukemia virus (M-MLV) RT during DNA polymerization. We constructed a long-chained bRNA template by splinted-ligation. The bRNA oligonucleotide is chimeric and contains DNA to identify RNA cleavage products by probe hybridization. Unique sequences surrounding the branchpoint facilitate monitoring of bRNA purification by terminal-restriction fragment length polymorphism analysis. We evaluate the M-MLV RT-generated cleavage and mutational patterns. We find that cleavage of bRNA and misprocessing of the branched nucleotide proceed arm-specifically. Bypass of the branchpoint from the 2΄-arm causes single-mismatch errors, whereas bypass from the 3΄-arm leads to deletion mutations. The non-template arm is cleaved when reverse transcription is primed from the 3΄-arm but not from the 2΄-arm. This suggests that RTs flip ∼180° at branchpoints and RNases H cleave the non-template arm depending on its accessibility. Our observed interplay between M-MLV RT and bRNA would be compatible with a bRNA-mediated control of retroviral and related retrotransposon replication.
[Mh] Termos MeSH primário: Vírus da Leucemia Murina de Moloney/enzimologia
Mutação
Oligorribonucleotídeos/química
DNA Polimerase Dirigida por RNA/metabolismo
Transcrição Reversa
[Mh] Termos MeSH secundário: Clonagem Molecular
DNA/biossíntese
DNA Complementar/biossíntese
DNA Complementar/química
Oligorribonucleotídeos/isolamento & purificação
Clivagem do RNA
DNA Polimerase Dirigida por RNA/genética
Ribonuclease H/genética
Moldes Genéticos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Complementary); 0 (Oligoribonucleotides); 9007-49-2 (DNA); EC 2.7.7.49 (RNA-Directed DNA Polymerase); EC 3.1.26.4 (Ribonuclease H)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171005
[Lr] Data última revisão:
171005
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170205
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx073


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[PMID]:28066922
[Au] Autor:Guan R; Aiyer S; Cote ML; Xiao R; Jiang M; Acton TB; Roth MJ; Montelione GT
[Ad] Endereço:Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway, New Jersey, 08854.
[Ti] Título:X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition.
[So] Source:Proteins;85(4):647-656, 2017 Apr.
[Is] ISSN:1097-0134
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1-44) and an HHCC zinc-finger NTD (residues 45-105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1-105 (NTR ) and 8-105 (NTR ) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR packs as a dimer and NTR packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel ß-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three ß-strands form an extended ß-sheet with another ß-strand in the HHCC Zn binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647-656. © 2016 Wiley Periodicals, Inc.
[Mh] Termos MeSH primário: DNA Viral/química
Integrases/química
Vírus da Leucemia Murina de Moloney/química
Proteínas Virais/química
Dedos de Zinco
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Sítios de Ligação
Domínio Catalítico
Clonagem Molecular
Cristalografia por Raios X
DNA Viral/metabolismo
Escherichia coli/genética
Escherichia coli/metabolismo
Expressão Gênica
Integrases/genética
Integrases/metabolismo
Modelos Moleculares
Vírus da Leucemia Murina de Moloney/enzimologia
Ligação Proteica
Conformação Proteica em alfa-Hélice
Conformação Proteica em Folha beta
Domínios e Motivos de Interação entre Proteínas
Multimerização Proteica
Proteínas Recombinantes/química
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Alinhamento de Sequência
Homologia de Sequência de Aminoácidos
Proteínas Virais/genética
Proteínas Virais/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Viral); 0 (Recombinant Proteins); 0 (Viral Proteins); EC 2.7.7.- (Integrases)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170726
[Lr] Data última revisão:
170726
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170110
[St] Status:MEDLINE
[do] DOI:10.1002/prot.25245


  8 / 3581 MEDLINE  
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[PMID]:28063925
[Au] Autor:Chu W; Weerasekera A; Kim CH
[Ad] Endereço:Department of Chemistry and Biochemistry, California State University East Bay, Hayward, CA 94542, United States.
[Ti] Título:On the conformational stability of the smallest RNA kissing complexes maintained through two G·C base pairs.
[So] Source:Biochem Biophys Res Commun;483(1):39-44, 2017 Jan 29.
[Is] ISSN:1090-2104
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Two identical 5'GACG3' tetra-loop motifs with different stem sequences (called H2 and H3) are found in the 5' end region of Moloney Murine Leukemia Virus (MMLV) genomic RNA. They play important roles in RNA dimerization and encapsidation through two identical tetra-loops (5'GACG3') forming a loop-to-loop kissing complex, the smallest RNA kissing complex ever found in nature. We examined the effects of a loop-closing base pair as well as a stem sequence on the conformational stability of the kissing complex. UV melting analysis and gel electrophoresis were performed on eight RNA sequences mimicking the H2 and H3 hairpin tetra-loops with variation in loop-closing base pairs. Our results show that changing the loop-closing base pair from the wildtype (5'A·U3' for H3, 5'U·A3' for H2) to 5'G·C3'/5'C·G3' has significant effect on the stability of the kissing complexes: the substitution to 5'C·G3' significantly decreases both thermal and mechanical stability, while switching to the 5'G·C3' significantly increases the mechanical stability only. The kissing complexes with the wildtype loop-closing base pairs (5'A·U3' for H3 and 5'U·A3' for H2) show different stability when attached to a different stem sequence (H2 stem vs. H3 stem). This suggests that not only the loop-closing base pair itself, but also the stem sequence, affects the conformational stability of the RNA kissing complex.
[Mh] Termos MeSH primário: RNA Viral/química
RNA Viral/genética
[Mh] Termos MeSH secundário: Animais
Composição de Bases
Sequência de Bases
Sequências Repetidas Invertidas
Camundongos
Vírus da Leucemia Murina de Moloney/química
Vírus da Leucemia Murina de Moloney/genética
Conformação de Ácido Nucleico
Motivos de Nucleotídeos
Estabilidade de RNA
Termodinâmica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (RNA, Viral)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170605
[Lr] Data última revisão:
170605
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170109
[St] Status:MEDLINE


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[PMID]:27871370
[Au] Autor:Okano H; Katano Y; Baba M; Fujiwara A; Hidese R; Fujiwara S; Yanagihara I; Hayashi T; 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:Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase.
[So] Source:Enzyme Microb Technol;96:111-120, 2017 Jan.
[Is] ISSN:1879-0909
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4pol from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis.
[Mh] Termos MeSH primário: DNA Complementar/biossíntese
DNA Complementar/genética
RNA/análise
RNA/genética
[Mh] Termos MeSH secundário: Sequência de Bases
DNA Helicases/genética
DNA Polimerase Dirigida por DNA/genética
DNA Polimerase Dirigida por DNA/metabolismo
Estabilidade Enzimática
Expressão Gênica
Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/enzimologia
Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/genética
Vírus da Leucemia Murina de Moloney/enzimologia
Vírus da Leucemia Murina de Moloney/genética
Análise de Sequência com Séries de Oligonucleotídeos
Engenharia de Proteínas
RNA Helicases/genética
DNA Polimerase Dirigida por RNA/genética
DNA Polimerase Dirigida por RNA/metabolismo
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Temperatura Ambiente
Thermococcus/enzimologia
Thermococcus/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Complementary); 0 (Recombinant Proteins); 63231-63-0 (RNA); EC 2.7.7.49 (RNA-Directed DNA Polymerase); EC 2.7.7.7 (DNA-Directed DNA Polymerase); EC 3.6.4.- (DNA Helicases); EC 3.6.4.13 (RNA Helicases)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170530
[Lr] Data última revisão:
170530
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161123
[St] Status:MEDLINE


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[PMID]:27795446
[Au] Autor:Wang GZ; Goff SP
[Ad] Endereço:Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University, New York, New York, USA.
[Ti] Título:Transcriptional Silencing of Moloney Murine Leukemia Virus in Human Embryonic Carcinoma Cells.
[So] Source:J Virol;91(1), 2017 Jan 01.
[Is] ISSN:1098-5514
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Embryonic carcinoma (EC) cells are malignant counterparts of embryonic stem (ES) cells and serve as useful models for investigating cellular differentiation and human embryogenesis. Though the susceptibility of murine EC cells to retroviral infection has been extensively analyzed, few studies of retrovirus infection of human EC cells have been performed. We tested the susceptibility of human EC cells to transduction by retroviral vectors derived from three different retroviral genera. We show that human EC cells efficiently express reporter genes delivered by vectors based on human immunodeficiency virus type 1 (HIV-1) and Mason-Pfizer monkey virus (M-PMV) but not Moloney murine leukemia virus (MLV). In human EC cells, MLV integration occurs normally, but no viral gene expression is observed. The block to MLV expression of MLV genomes is relieved upon cellular differentiation. The lack of gene expression is correlated with transcriptional silencing of the MLV promoter through the deposition of repressive histone marks as well as DNA methylation. Moreover, depletion of SETDB1, a histone methyltransferase, resulted in a loss of transcriptional silencing and upregulation of MLV gene expression. Finally, we provide evidence showing that the lack of MLV gene expression may be attributed in part to the lack of MLV enhancer function in human EC cells. IMPORTANCE: Human embryonic carcinoma (EC) cells are shown to restrict the expression of murine leukemia virus genomes but not retroviral genomes of the lentiviral or betaretroviral families. The block occurs at the level of transcription and is accompanied by the deposition of repressive histone marks and methylation of the integrated proviral DNA. The host machinery required for silencing in human EC cells is distinct from that in murine EC cell lines: the histone methyltransferase SETDB1 is required, but the widely utilized corepressor TRIM28/Kap1 is not. A transcriptional enhancer element from the Mason-Pfizer monkey virus can override the silencing and promote transcription of chimeric proviral DNAs. The findings reveal novel features of human EC gene regulation not present in their murine counterparts.
[Mh] Termos MeSH primário: Inativação Gênica
Genoma Viral
HIV-1/genética
Células-Tronco Embrionárias Humanas/imunologia
Vírus dos Macacos de Mason-Pfizer/genética
Vírus da Leucemia Murina de Moloney/genética
Células-Tronco Neoplásicas/imunologia
[Mh] Termos MeSH secundário: Animais
Diferenciação Celular
Metilação de DNA
Genes Reporter
HIV-1/metabolismo
Histonas/genética
Histonas/imunologia
Especificidade de Hospedeiro
Células-Tronco Embrionárias Humanas/virologia
Seres Humanos
Vírus dos Macacos de Mason-Pfizer/metabolismo
Camundongos
Vírus da Leucemia Murina de Moloney/metabolismo
Células-Tronco Neoplásicas/virologia
Regiões Promotoras Genéticas
Metiltransferases de Proteína/antagonistas & inibidores
Metiltransferases de Proteína/genética
Metiltransferases de Proteína/imunologia
RNA Interferente Pequeno/genética
RNA Interferente Pequeno/metabolismo
Especificidade da Espécie
Transcrição Genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Histones); 0 (RNA, Small Interfering); EC 2.1.1.- (Protein Methyltransferases); EC 2.1.1.- (SETDB1 protein, human)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:171123
[Lr] Data última revisão:
171123
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161101
[St] Status:MEDLINE



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