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  1 / 22541 MEDLINE  
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[PMID]:28456965
[Au] Autor:Ling KY; Cheow LF; Quake SR; Burkholder WF; Messerschmidt DM
[Ad] Endereço:Developmental Epigenetics and Disease Laboratory, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
[Ti] Título:Single Cell Restriction Enzyme-Based Analysis of Methylation at Genomic Imprinted Regions in Preimplantation Mouse Embryos.
[So] Source:Methods Mol Biol;1605:171-189, 2017.
[Is] ISSN:1940-6029
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The methylation of cytosines in DNA is a fundamental epigenetic regulatory mechanism. During preimplantation development, mammalian embryos undergo extensive epigenetic reprogramming, including the global erasure of germ cell-specific DNA methylation marks, to allow for the establishment of the pluripotent state of the epiblast. However, DNA methylation marks at specific regions, such as imprinted gene regions, escape this reprogramming process, as their inheritance from germline to soma is paramount for proper development. To study the dynamics of DNA methylation marks in single blastomeres of mouse preimplantation embryos, we devised a new approach-single cell restriction enzyme analysis of methylation (SCRAM). SCRAM allows for reliable, fast, and high-throughput analysis of DNA methylation states of multiple regions of interest from single cells. In the method described below, SCRAM is specifically used to address loss of DNA methylation at genomic imprints or other highly methylated regions of interest.
[Mh] Termos MeSH primário: Blastocisto/enzimologia
Metilação de DNA
Enzimas de Restrição do DNA/metabolismo
Análise de Célula Única/métodos
[Mh] Termos MeSH secundário: 5-Metilcitosina/metabolismo
Animais
Blastocisto/química
Blastômeros/química
Blastômeros/enzimologia
Epigênese Genética
Feminino
Impressão Genômica
Camundongos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
6R795CQT4H (5-Methylcytosine); EC 3.1.21.- (DNA Restriction Enzymes)
[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:170501
[St] Status:MEDLINE
[do] DOI:10.1007/978-1-4939-6988-3_12


  2 / 22541 MEDLINE  
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[PMID]:27770358
[Au] Autor:Bilichak A; Kovalchuk I
[Ad] Endereço:Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, AB, Canada. a.bilichak@gmail.com.
[Ti] Título:Analysis of Global Genome Methylation Using the Cytosine-Extension Assay.
[So] Source:Methods Mol Biol;1456:73-79, 2017.
[Is] ISSN:1940-6029
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:DNA methylation is a reversible covalent chemical modification of DNA intended to regulate chromatin structure and gene expression in a cell- and tissue-specific manner and in response to the environment. Cytosine methylation is predominantly occurring in plants, and cytosine nucleotides in plants can be methylated at symmetrical (CpG and CpHpG) and nonsymmetrical sites. Although there exists a number of various methods for the detection of cytosine methylation, most of them are either laborious or expensive or both. Here, we describe a quick inexpensive method for the analysis of global genome methylation using a cytosine-extension assay. The assay can be used for the analysis of the total level of CpG, CpHpG, and CpHpH methylation in a given sample of plant DNA.
[Mh] Termos MeSH primário: Citosina/metabolismo
Metilação de DNA
Epigenômica/métodos
Genoma
[Mh] Termos MeSH secundário: Ilhas de CpG
Enzimas de Restrição do DNA/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
8J337D1HZY (Cytosine); EC 3.1.21.- (DNA Restriction Enzymes)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180112
[Lr] Data última revisão:
180112
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161023
[St] Status:MEDLINE


  3 / 22541 MEDLINE  
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[PMID]:29072133
[Au] Autor:Iqbal MUN; Khan TA
[Ad] Endereço:Department of Physiology, University of Karachi, Karachi, Pakistan.
[Ti] Título:Association between Vitamin D receptor (Cdx2, Fok1, Bsm1, Apa1, Bgl1, Taq1, and Poly (A)) gene polymorphism and breast cancer: A systematic review and meta-analysis.
[So] Source:Tumour Biol;39(10):1010428317731280, 2017 Oct.
[Is] ISSN:1423-0380
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The purpose of this systemic review and meta-analysis was to examine the relationship between VDR gene polymorphisms and breast cancer. Literature was searched through PubMed database, Google scholar, and the web of knowledge from December 2015 to January 2017 and consists of 34 studies (26,372 cases and 32,883 controls). All statistical measures were done using STATA version 11.2. The heterogeneity among studies was tested using I statistics. Mantel-Haenszel method and DerSimonian-Laird method were used to combine data from studies using both random-effect model and fixed-effect model, respectively. Potential publication bias was evaluated by Egger's test. Sensitivity analysis was also performed to evaluate the quality and consistency in results. The results of this meta-analysis revealed that VDR gene polymorphisms (Bsm1 bb vs BB; SOR = 1.18, 95% CI = 1.054-1.322, Apa1 aa vs AA; SOR = 1.18, 95% CI = 0.87-1.59, Poly (A) LL vs SS; SOR = 1.41, 95% CI = 1.06-1.88, Fok1 ff + Ff vs FF; SOR = 1.25, 95% CI = 0.896-1.759, Apa1 aa+Aa vs AA; SOR = 1.13, 95% CI = 0.95-1.35, Poly (A) LL + LS vs SS; SOR = 1.19, 95% CI = 1.00-1.43, Poly (A) L vs S; SOR = 1.18, 95% CI = 1.03-1.35) are associated with the breast cancer. Cdx2, Bgl1, and Taq1 do not show association with breast cancer. Thus, the finding of this meta-analysis concluded that VDR Bsm1, Apa1, Fok1, and Poly (A) gene polymorphisms may be susceptible for breast cancer development.
[Mh] Termos MeSH primário: Neoplasias da Mama/genética
Estudos de Associação Genética
Predisposição Genética para Doença
Receptores de Calcitriol/genética
[Mh] Termos MeSH secundário: Neoplasias da Mama/patologia
Enzimas de Restrição do DNA/genética
Feminino
Seres Humanos
Polimorfismo Genético
[Pt] Tipo de publicação:JOURNAL ARTICLE; META-ANALYSIS; REVIEW
[Nm] Nome de substância:
0 (Receptors, Calcitriol); 0 (VDR protein, human); EC 3.1.21.- (DNA Restriction Enzymes)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171102
[Lr] Data última revisão:
171102
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171027
[St] Status:MEDLINE
[do] DOI:10.1177/1010428317731280


  4 / 22541 MEDLINE  
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[PMID]:29040308
[Au] Autor:Krefft D; Papkov A; Zylicz-Stachula A; Skowron PM
[Ad] Endereço:Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, Gdansk, Poland.
[Ti] Título:Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene.
[So] Source:PLoS One;12(10):e0186633, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes' expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified 'codon randomization' strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.
[Mh] Termos MeSH primário: Proteínas de Bactérias/metabolismo
Códon/química
Enzimas de Restrição do DNA/metabolismo
Metiltransferases/metabolismo
RNA Mensageiro/química
Thermus thermophilus/enzimologia
[Mh] Termos MeSH secundário: Proteínas de Bactérias/genética
Composição de Bases
Sequência de Bases
Clonagem Molecular
Códon/metabolismo
Enzimas de Restrição do DNA/genética
Estabilidade Enzimática
Escherichia coli/enzimologia
Escherichia coli/genética
Expressão Gênica
Genes Sintéticos
Temperatura Alta
Cinética
Metiltransferases/genética
Conformação de Ácido Nucleico
Regiões Promotoras Genéticas
RNA Mensageiro/genética
RNA Mensageiro/metabolismo
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Relação Estrutura-Atividade
Thermus thermophilus/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Codon); 0 (RNA, Messenger); 0 (Recombinant Proteins); EC 2.1.1.- (Methyltransferases); EC 3.1.21.- (DNA Restriction Enzymes)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171031
[Lr] Data última revisão:
171031
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171018
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0186633


  5 / 22541 MEDLINE  
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[PMID]:28890334
[Au] Autor:Billon P; Bryant EE; Joseph SA; Nambiar TS; Hayward SB; Rothstein R; Ciccia A
[Ad] Endereço:Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032, USA.
[Ti] Título:CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.
[So] Source:Mol Cell;67(6):1068-1079.e4, 2017 Sep 21.
[Is] ISSN:1097-4164
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.
[Mh] Termos MeSH primário: Proteínas Associadas a CRISPR/genética
Sistemas CRISPR-Cas
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
Códon de Terminação
Edição de Genes/métodos
Inativação Gênica
[Mh] Termos MeSH secundário: Animais
Arabidopsis/genética
Arabidopsis/metabolismo
Proteínas Associadas a CRISPR/metabolismo
Códon sem Sentido
Biologia Computacional
Enzimas de Restrição do DNA/genética
Enzimas de Restrição do DNA/metabolismo
Bases de Dados Genéticas
Regulação Fúngica da Expressão Gênica
Regulação Neoplásica da Expressão Gênica
Regulação da Expressão Gênica de Plantas
Células HEK293
Seres Humanos
Camundongos
Neoplasias/genética
Neoplasias/metabolismo
Polimorfismo de Fragmento de Restrição
RNA Guia/genética
RNA Guia/metabolismo
Ratos
Saccharomyces cerevisiae/genética
Saccharomyces cerevisiae/metabolismo
Transfecção
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (CRISPR-Associated Proteins); 0 (Codon, Nonsense); 0 (Codon, Terminator); 0 (RNA, Guide); EC 3.1.21.- (DNA Restriction Enzymes)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171116
[Lr] Data última revisão:
171116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170912
[St] Status:MEDLINE


  6 / 22541 MEDLINE  
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[PMID]:28854738
[Au] Autor:Toliusis P; Zaremba M; Silanskas A; Szczelkun MD; Siksnys V
[Ad] Endereço:Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Sauletekio al. 7, LT-10257, Vilnius, Lithuania.
[Ti] Título:CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases.
[So] Source:Nucleic Acids Res;45(14):8435-8447, 2017 Aug 21.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5'-GCCGC-3' site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes.
[Mh] Termos MeSH primário: Proteínas de Bactérias/metabolismo
RNA Helicases DEAD-box/metabolismo
Clivagem do DNA
Enzimas de Restrição do DNA/metabolismo
DNA/metabolismo
[Mh] Termos MeSH secundário: Trifosfato de Adenosina/metabolismo
Sequência de Bases
Biocatálise
Corynebacterium glutamicum/enzimologia
DNA/genética
DNA Circular/genética
DNA Circular/metabolismo
Modelos Genéticos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (DNA, Circular); 8L70Q75FXE (Adenosine Triphosphate); 9007-49-2 (DNA); EC 3.1.21.- (DNA Restriction Enzymes); EC 3.6.4.13 (DEAD-box RNA Helicases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171023
[Lr] Data última revisão:
171023
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170901
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx580


  7 / 22541 MEDLINE  
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[PMID]:28854737
[Au] Autor:Nagamalleswari E; Rao S; Vasu K; Nagaraja V
[Ad] Endereço:Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
[Ti] Título:Restriction endonuclease triggered bacterial apoptosis as a mechanism for long time survival.
[So] Source:Nucleic Acids Res;45(14):8423-8434, 2017 Aug 21.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Programmed cell death (PCD) under certain conditions is one of the features of bacterial altruism. Given the bacterial diversity and varied life style, different PCD mechanisms must be operational that remain largely unexplored. We describe restriction endonuclease (REase) mediated cell death by an apoptotic pathway, beneficial for isogenic bacterial communities. Cell death is pronounced in stationary phase and when the enzyme exhibits promiscuous DNA cleavage activity. We have elucidated the molecular mechanism of REase mediated cell killing and demonstrate that released nutrients from dying cells support the growth of the remaining cells in the population. These findings illustrate a new intracellular moonlighting role for REases which are otherwise established host defence arsenals. REase induced PCD appears to be a cellular design to replenish nutrients for cells undergoing starvation stress and the phenomenon could be wide spread in bacteria, given the abundance of restriction-modification (R-M) systems in the microbial population.
[Mh] Termos MeSH primário: Apoptose
Proteínas de Bactérias/metabolismo
Enzimas de Restrição do DNA/metabolismo
Escherichia coli/enzimologia
[Mh] Termos MeSH secundário: Adaptação Fisiológica
Proteínas de Bactérias/genética
Western Blotting
Morte Celular
Enzimas de Restrição do DNA/genética
Enzimas de Restrição-Modificação do DNA/genética
Enzimas de Restrição-Modificação do DNA/metabolismo
Escherichia coli/citologia
Escherichia coli/genética
Regulação Bacteriana da Expressão Gênica
Viabilidade Microbiana
Microscopia Confocal
Densidade Demográfica
Crescimento Demográfico
Reação em Cadeia da Polimerase Via Transcriptase Reversa
Fatores de Tempo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (DNA Restriction-Modification Enzymes); EC 3.1.21.- (DNA Restriction Enzymes)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171023
[Lr] Data última revisão:
171023
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170901
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx576


  8 / 22541 MEDLINE  
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[PMID]:28666410
[Au] Autor:Yuan Y; Bayer PE; Scheben A; Chan CK; Edwards D
[Ad] Endereço:School of Biological Sciences, the University of Western Australia, Perth, WA, Australia.
[Ti] Título:BioNanoAnalyst: a visualisation tool to assess genome assembly quality using BioNano data.
[So] Source:BMC Bioinformatics;18(1):323, 2017 Jun 30.
[Is] ISSN:1471-2105
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: Reference genome assemblies are valuable, as they provide insights into gene content, genetic evolution and domestication. The higher the quality of a reference genome assembly the more accurate the downstream analysis will be. During the last few years, major efforts have been made towards improving the quality of genome assemblies. However, erroneous and incomplete assemblies are still common. Complementary to DNA sequencing technologies, optical mapping has advanced genomic studies by facilitating the production of genome scaffolds and assessing structural variation. However, there are few tools available to comprehensively examine misassemblies in reference genome sequences using optical map data. RESULTS: We present BioNanoAnalyst, a software package to examine genome assemblies based on restriction endonuclease cut sites and optical map data. A graphical user interface (GUI) allows users to assess reference genome sequences on different computer platforms without the requirement of programming knowledge. The zoom function makes visualisation convenient, while a GFF3 format output file gives an option to directly visualise questionable assembly regions by location and nucleotides following import into a local genome browser. CONCLUSIONS: BioNanoAnalyst is a tool to identify misassemblies in a reference genome sequence using optical map data. With the reported information, users can rapidly identify assembly errors and correct them using other software tools, which could facilitate an accurate downstream analysis.
[Mh] Termos MeSH primário: Genômica
Interface Usuário-Computador
[Mh] Termos MeSH secundário: Cromossomos Humanos Par 1/genética
Cromossomos Humanos Par 1/metabolismo
Enzimas de Restrição do DNA/metabolismo
Genoma Humano
Seres Humanos
Internet
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
EC 3.1.21.- (DNA Restriction Enzymes)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171107
[Lr] Data última revisão:
171107
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170702
[St] Status:MEDLINE
[do] DOI:10.1186/s12859-017-1735-4


  9 / 22541 MEDLINE  
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[PMID]:28654677
[Au] Autor:Fomenkov A; Sun Z; Dila DK; Anton BP; Roberts RJ; Raleigh EA
[Ad] Endereço:Research Department, New England Biolabs, Ipswich, MA, United States of America.
[Ti] Título:EcoBLMcrX, a classical modification-dependent restriction enzyme in Escherichia coli B: Characterization in vivo and in vitro with a new approach to cleavage site determination.
[So] Source:PLoS One;12(6):e0179853, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Here we characterize the modification-dependent restriction enzyme (MDE) EcoBLMcrX in vivo, in vitro and in its genomic environment. MDE cleavage of modified DNAs protects prokaryote populations from lethal infection by bacteriophage with highly modified DNA, and also stabilizes lineages by reducing gene import when sparse modification occurs in the wrong context. The function and distribution of MDE families are thus important. Here we describe the properties of EcoBLMcrX, an enzyme of the E. coli B lineage, in vivo and in vitro. Restriction in vivo and the genome location of its gene, ecoBLmcrX, were determined during construction and sequencing of a B/K-12 hybrid, ER2566. In classical restriction literature, this B system was named r6 or rglAB. Like many genome defense functions, ecoBLmcrX is found within a genomic island, where gene content is variable among natural E. coli isolates. In vitro, EcoBLMcrX was compared with two related enzymes, BceYI and NhoI. All three degrade fully cytosine-modified phage DNA, as expected for EcoBLMcrX from classical T4 genetic data. A new method of characterizing MDE specificity was developed to better understand action on fully-modified targets such as the phage that provide major evolutionary pressure for MDE maintenance. These enzymes also cleave plasmids with m5C in particular motifs, consistent with a role in lineage-stabilization. The recognition sites were characterized using a site-ranking approach that allows visualization of preferred cleavage sites when fully-modified substrates are digested. A technical constraint on the method is that ligation of one-nucleotide 5' extensions favors G:C over A:T approximately five-fold. Taking this bias into account, we conclude that EcoBLMcrX can cleave 3' to the modified base in the motif Rm5C|. This is compatible with, but less specific than, the site reported by others. Highly-modified site contexts, such as those found in base-substituted virulent phages, are strongly preferred.
[Mh] Termos MeSH primário: Enzimas de Restrição do DNA/metabolismo
Escherichia coli/metabolismo
[Mh] Termos MeSH secundário: Enzimas de Restrição do DNA/genética
Escherichia coli/genética
Mutação
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
EC 3.1.21.- (DNA Restriction Enzymes)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170920
[Lr] Data última revisão:
170920
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170628
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0179853


  10 / 22541 MEDLINE  
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[PMID]:28624296
[Au] Autor:Petell CJ; Loiseau G; Gandy R; Pradhan S; Gowher H
[Ad] Endereço:Department of Biochemistry, Purdue University, West Lafayette, IN 47907, United States.
[Ti] Título:A refined DNA methylation detection method using MspJI coupled quantitative PCR.
[So] Source:Anal Biochem;533:1-9, 2017 Sep 15.
[Is] ISSN:1096-0309
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:DNA methylation is a highly conserved epigenetic modification with critical roles ranging from protection against phage infection in bacteria to the regulation of gene expression in mammals. DNA methylation at specific sequences can be measured by using methylation dependent or sensitive restriction enzymes coupled to semi- or quantitative PCR (MD-qPCR). This study reports a refined MD-qPCR method for detecting gain or loss of DNA methylation at specific sites through the specific use of MspJI or HpaII, respectively. By employing varying concentrations of DNA with methylation ranging from 0 to 100%, our data provide evidence that compared to HpaII, MspJI increases the sensitivity and accuracy of detecting relative DNA methylation gains by MD-qPCR. We also show that the MspJI-coupled MD-qPCR can accurately determine the percent gain in DNA methylation at the Sall4 enhancer and is more sensitive than HpaII in detecting relative gains in DNA methylation at the Oct4 proximal enhancer during embryonic stem cell (ESC) differentiation. The high specificity and sensitivity of this targeted approach increases its potential as a diagnostic tool to detect relatively smaller gains in DNA methylation at specific sites from limited amounts of sample.
[Mh] Termos MeSH primário: Diferenciação Celular/genética
Metilação de DNA/genética
Enzimas de Restrição do DNA/genética
Epigênese Genética
[Mh] Termos MeSH secundário: Células-Tronco Embrionárias/metabolismo
Elementos Facilitadores Genéticos
Regulação da Expressão Gênica
Seres Humanos
Fator 3 de Transcrição de Octâmero/genética
Reação em Cadeia da Polimerase em Tempo Real
Fatores de Transcrição/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Octamer Transcription Factor-3); 0 (POU5F1 protein, human); 0 (SALL4 protein, human); 0 (Transcription Factors); EC 3.1.21.- (DNA Restriction Enzymes)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170804
[Lr] Data última revisão:
170804
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170619
[St] Status:MEDLINE



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