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  1 / 8319 MEDLINE  
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[PMID]:28466789
[Au] Autor:Rodic A; Blagojevic B; Zdobnov E; Djordjevic M; Djordjevic M
[Ad] Endereço:Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000, Belgrade, Serbia.
[Ti] Título:Understanding key features of bacterial restriction-modification systems through quantitative modeling.
[So] Source:BMC Syst Biol;11(Suppl 1):377, 2017 02 24.
[Is] ISSN:1752-0509
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: Restriction-modification (R-M) systems are rudimentary bacterial immune systems. The main components include restriction enzyme (R), which cuts specific unmethylated DNA sequences, and the methyltransferase (M), which protects the same DNA sequences. The expression of R-M system components is considered to be tightly regulated, to ensure successful establishment in a naïve bacterial host. R-M systems are organized in different architectures (convergent or divergent) and are characterized by different features, i.e. binding cooperativities, dissociation constants of dimerization, translation rates, which ensure this tight regulation. It has been proposed that R-M systems should exhibit certain dynamical properties during the system establishment, such as: i) a delayed expression of R with respect to M, ii) fast transition of R from "OFF" to "ON" state, iii) increased stability of the toxic molecule (R) steady-state levels. It is however unclear how different R-M system features and architectures ensure these dynamical properties, particularly since it is hard to address this question experimentally. RESULTS: To understand design of different R-M systems, we computationally analyze two R-M systems, representative of the subset controlled by small regulators called 'C proteins', and differing in having convergent or divergent promoter architecture. We show that, in the convergent system, abolishing any of the characteristic system features adversely affects the dynamical properties outlined above. Moreover, an extreme binding cooperativity, accompanied by a very high dissociation constant of dimerization, observed in the convergent system, but absent from other R-M systems, can be explained in terms of the same properties. Furthermore, we develop the first theoretical model for dynamics of a divergent R-M system, which does not share any of the convergent system features, but has overlapping promoters. We show that i) the system dynamics exhibits the same three dynamical properties, ii) introducing any of the convergent system features to the divergent system actually diminishes these properties. CONCLUSIONS: Our results suggest that different R-M architectures and features may be understood in terms of constraints imposed by few simple dynamical properties of the system, providing a unifying framework for understanding these seemingly diverse systems. We also provided predictions for the perturbed R-M systems dynamics, which may in future be tested through increasingly available experimental techniques, such as re-engineering R-M systems and single-cell experiments.
[Mh] Termos MeSH primário: Enzimas de Restrição-Modificação do DNA/metabolismo
Escherichia coli/enzimologia
Modelos Biológicos
[Mh] Termos MeSH secundário: Enzimas de Restrição-Modificação do DNA/biossíntese
Enzimas de Restrição-Modificação do DNA/química
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo
Escherichia coli/genética
Escherichia coli/imunologia
Escherichia coli/metabolismo
Multimerização Proteica
Estrutura Quaternária de Proteína
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (DNA Restriction-Modification Enzymes); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific); EC 3.1.21.4 (GATATC-specific type II deoxyribonucleases)
[Em] Mês de entrada:1712
[Cu] Atualização por classe:171223
[Lr] Data última revisão:
171223
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170504
[St] Status:MEDLINE
[do] DOI:10.1186/s12918-016-0377-x


  2 / 8319 MEDLINE  
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[PMID]:29034815
[Au] Autor:Laczmanski L; Lwow F; Osina A; Kepska M; Laczmanska I; Witkiewicz W
[Ad] Endereço:1 Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Science, Wroclaw, Poland.
[Ti] Título:Association of the vitamin D receptor FokI gene polymorphism with sex- and non-sex-associated cancers: A meta-analysis.
[So] Source:Tumour Biol;39(10):1010428317727164, 2017 Oct.
[Is] ISSN:1423-0380
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Currently higher morbidity and mortality rates are observed in cancer diseases, especially sex-dependent cancers. A positive role of endogenous vitamin D concentration in cancer diseases has been reported in many publications. Furthermore, there has been observed a relationship between serum vitamin D and testosterone concentrations in an elderly Caucasian population carrying the vitamin D receptor FokI gene polymorphism. The aim of this study was to investigate whether the vitamin D receptor FokI polymorphism is associated with cancerogenesis in sex-dependent cancers. The MEDLINE and ResearchGate databases were used to search for articles up to January 2017, and 96 articles concerning the FokI polymorphism were chosen. Odds ratios with 95% confidence intervals were used to assess the strength of associations between polymorphisms of vitamin D receptor and cancer risk in the described populations. The fixed-effects model and the DerSimonian-Laird random-effects model (with weights based on the inverse variance) were used to calculate summary odds ratios, and both within- and between-study variation were considered. Generally, the F variant reduces the risk of cancer by 4% (odds ratio = 0.96, p value = 0.0057). This effect is particularly evident in female sex-associated cancers (odds ratio = 0.96, 95% confidence interval: 0.93-0.99, p value = 0.0259), but it is not observed in non-sex-associated cancers. Polymorphism FokI is associated with breast and ovarian cancers.
[Mh] Termos MeSH primário: Desoxirribonucleases de Sítio Específico do Tipo II/genética
Neoplasias/genética
Polimorfismo Genético/genética
Receptores de Calcitriol/genética
[Mh] Termos MeSH secundário: Alelos
Estudos de Casos e Controles
Grupo com Ancestrais do Continente Europeu/genética
Feminino
Predisposição Genética para Doença/genética
Genótipo
Seres Humanos
Masculino
Razão de Chances
Fatores de Risco
Vitamina D/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; META-ANALYSIS
[Nm] Nome de substância:
0 (Receptors, Calcitriol); 0 (VDR protein, human); 1406-16-2 (Vitamin D); EC 3.1.21.- (endodeoxyribonuclease FokI); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171020
[Lr] Data última revisão:
171020
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171017
[St] Status:MEDLINE
[do] DOI:10.1177/1010428317727164


  3 / 8319 MEDLINE  
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[PMID]:28934493
[Au] Autor:Sasnauskas G; Tamulaitiene G; Tamulaitis G; Calyseva J; Laime M; Rimseliene R; Lubys A; Siksnys V
[Ad] Endereço:Institute of Biotechnology, Vilnius University, Sauletekio av. 7, LT-10257 Vilnius, Lithuania.
[Ti] Título:UbaLAI is a monomeric Type IIE restriction enzyme.
[So] Source:Nucleic Acids Res;45(16):9583-9594, 2017 Sep 19.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Type II restriction endonucleases (REases) form a large and highly diverse group of enzymes. Even REases specific for a common recognition site often vary in their oligomeric structure, domain organization and DNA cleavage mechanisms. Here we report biochemical and structural characterization of the monomeric restriction endonuclease UbaLAI, specific for the pseudosymmetric DNA sequence 5'-CC/WGG-3' (where W = A/T, and '/' marks the cleavage position). We present a 1.6 Å co-crystal structure of UbaLAI N-terminal domain (UbaLAI-N) and show that it resembles the B3-family domain of EcoRII specific for the 5'-CCWGG-3' sequence. We also find that UbaLAI C-terminal domain (UbaLAI-C) is closely related to the monomeric REase MvaI, another enzyme specific for the 5'-CCWGG-3' sequence. Kinetic studies of UbaLAI revealed that it requires two recognition sites for optimal activity, and, like other type IIE enzymes, uses one copy of a recognition site to stimulate cleavage of a second copy. We propose that during the reaction UbaLAI-N acts as a handle that tethers the monomeric UbaLAI-C domain to the DNA, thereby helping UbaLAI-C to perform two sequential DNA nicking reactions on the second recognition site during a single DNA-binding event. A similar reaction mechanism may be characteristic to other monomeric two-domain REases.
[Mh] Termos MeSH primário: Desoxirribonucleases de Sítio Específico do Tipo II/química
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo
[Mh] Termos MeSH secundário: Cristalografia por Raios X
DNA/química
DNA/metabolismo
Clivagem do DNA
Desoxirribonucleases de Sítio Específico do Tipo II/genética
Modelos Moleculares
Domínios Proteicos
Especificidade por Substrato
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
9007-49-2 (DNA); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171024
[Lr] Data última revisão:
171024
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170922
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx634


  4 / 8319 MEDLINE  
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[PMID]:28911108
[Au] Autor:Skowron PM; Anton BP; Czajkowska E; Zebrowska J; Sulecka E; Krefft D; Jezewska-Frackowiak J; Zolnierkiewicz O; Witkowska M; Morgan RD; Wilson GG; Fomenkov A; Roberts RJ; Zylicz-Stachula A
[Ad] Endereço:Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland.
[Ti] Título:The third restriction-modification system from Thermus aquaticus YT-1: solving the riddle of two TaqII specificities.
[So] Source:Nucleic Acids Res;45(15):9005-9018, 2017 Sep 06.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Two restriction-modification systems have been previously discovered in Thermus aquaticus YT-1. TaqI is a 263-amino acid (aa) Type IIP restriction enzyme that recognizes and cleaves within the symmetric sequence 5'-TCGA-3'. TaqII, in contrast, is a 1105-aa Type IIC restriction-and-modification enzyme, one of a family of Thermus homologs. TaqII was originally reported to recognize two different asymmetric sequences: 5'-GACCGA-3' and 5'-CACCCA-3'. We previously cloned the taqIIRM gene, purified the recombinant protein from Escherichia coli, and showed that TaqII recognizes the 5'-GACCGA-3' sequence only. Here, we report the discovery, isolation, and characterization of TaqIII, the third R-M system from T. aquaticus YT-1. TaqIII is a 1101-aa Type IIC/IIL enzyme and recognizes the 5'-CACCCA-3' sequence previously attributed to TaqII. The cleavage site is 11/9 nucleotides downstream of the A residue. The enzyme exhibits striking biochemical similarity to TaqII. The 93% identity between their aa sequences suggests that they have a common evolutionary origin. The genes are located on two separate plasmids, and are probably paralogs or pseudoparalogs. Putative positions and aa that specify DNA recognition were identified and recognition motifs for 6 uncharacterized Thermus-family enzymes were predicted.
[Mh] Termos MeSH primário: Proteínas de Bactérias/genética
Desoxirribonucleases de Sítio Específico do Tipo II/genética
Motivos de Nucleotídeos
Plasmídeos/metabolismo
Thermus/enzimologia
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Proteínas de Bactérias/metabolismo
Clonagem Molecular
Clivagem do DNA
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo
Escherichia coli/genética
Escherichia coli/metabolismo
Expressão Gênica
Isoenzimas/genética
Isoenzimas/metabolismo
Peso Molecular
Plasmídeos/química
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Alinhamento de Sequência
Homologia de Sequência de Aminoácidos
Especificidade por Substrato
Thermus/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Isoenzymes); 0 (Recombinant Proteins); EC 3.1.21.- (endodeoxyribonuclease TaqII); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific)
[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/gkx599


  5 / 8319 MEDLINE  
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[PMID]:28911102
[Au] Autor:Nath S; Somyajit K; Mishra A; Scully R; Nagaraju G
[Ad] Endereço:Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
[Ti] Título:FANCJ helicase controls the balance between short- and long-tract gene conversions between sister chromatids.
[So] Source:Nucleic Acids Res;45(15):8886-8900, 2017 Sep 06.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The FANCJ DNA helicase is linked to hereditary breast and ovarian cancers as well as bone marrow failure disorder Fanconi anemia (FA). Although FANCJ has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), the molecular mechanism underlying the tumor suppressor functions of FANCJ remains obscure. Here, we demonstrate that FANCJ deficient human and hamster cells exhibit reduction in the overall gene conversions in response to a site-specific chromosomal DSB induced by I-SceI endonuclease. Strikingly, the gene conversion events were biased in favour of long-tract gene conversions in FANCJ depleted cells. The fine regulation of short- (STGC) and long-tract gene conversions (LTGC) by FANCJ was dependent on its interaction with BRCA1 tumor suppressor. Notably, helicase activity of FANCJ was essential for controlling the overall HR and in terminating the extended repair synthesis during sister chromatid recombination (SCR). Moreover, cells expressing FANCJ pathological mutants exhibited defective SCR with an increased frequency of LTGC. These data unravel the novel function of FANCJ helicase in regulating SCR and SCR associated gene amplification/duplications and imply that these functions of FANCJ are crucial for the genome maintenance and tumor suppression.
[Mh] Termos MeSH primário: Proteína BRCA1/genética
Fatores de Transcrição de Zíper de Leucina Básica/genética
Cromátides/química
DNA/genética
Proteínas de Grupos de Complementação da Anemia de Fanconi/genética
Reparo de DNA por Recombinação
[Mh] Termos MeSH secundário: Animais
Proteína BRCA1/metabolismo
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo
Células CHO
Linhagem Celular Tumoral
Cromátides/metabolismo
Cricetulus
DNA/metabolismo
Quebras de DNA de Cadeia Dupla
Desoxirribonucleases de Sítio Específico do Tipo II/farmacologia
Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo
Pontos de Checagem da Fase G2 do Ciclo Celular
Regulação da Expressão Gênica
Recombinação Homóloga/efeitos dos fármacos
Seres Humanos
Mutação
Osteoblastos/citologia
Osteoblastos/efeitos dos fármacos
Osteoblastos/metabolismo
Ligação Proteica
Proteínas de Saccharomyces cerevisiae/farmacologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (BACH1 protein, human); 0 (BRCA1 Protein); 0 (BRCA1 protein, human); 0 (Basic-Leucine Zipper Transcription Factors); 0 (Fanconi Anemia Complementation Group Proteins); 0 (Saccharomyces cerevisiae Proteins); 9007-49-2 (DNA); EC 3.1.21.- (SCEI protein, S cerevisiae); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific)
[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/gkx586


  6 / 8319 MEDLINE  
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[PMID]:28911000
[Au] Autor:Chen X; Li WF; Wu X; Zhang HC; Chen L; Zhang PY; Liu LY; Ma D; Chen T; Zhou L; Xu Y; Zhou MT; Tang KF
[Ad] Endereço:Institute of Translational Medicine.
[Ti] Título:Dicer regulates non-homologous end joining and is associated with chemosensitivity in colon cancer patients.
[So] Source:Carcinogenesis;38(9):873-882, 2017 Sep 01.
[Is] ISSN:1460-2180
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:DNA double-strand break (DSB) repair is an important mechanism underlying chemotherapy resistance in human cancers. Dicer participates in DSB repair by facilitating homologous recombination. However, whether Dicer is involved in non-homologous end joining (NHEJ) remains unknown. Here, we addressed whether Dicer regulates NHEJ and chemosensitivity in colon cancer cells. Using our recently developed NHEJ assay, we found that DSB introduction by I-SceI cleavage leads to Dicer upregulation. Dicer knockdown increased SIRT7 binding and decreased the level of H3K18Ac (acetylated lysine 18 of histone H3) at DSB sites, thereby repressing the recruitment of NHEJ factors to DSB sites and inhibiting NHEJ. Dicer overexpression reduced SIRT7 binding and increased the level of H3K18Ac at DSB sites, promoting the recruitment of NHEJ factors to DSBs and moderately enhancing NHEJ. Dicer knockdown and overexpression increased and decreased, respectively, the chemosensitivity of colon cancer cells. Dicer protein expression in colon cancer tissues of patients was directly correlated with chemoresistance. Our findings revealed a function of Dicer in NHEJ-mediated DSB repair and the association of Dicer expression with chemoresistance in colon cancer patients.
[Mh] Termos MeSH primário: Neoplasias do Colo/tratamento farmacológico
Neoplasias do Colo/genética
RNA Helicases DEAD-box/fisiologia
Reparo do DNA por Junção de Extremidades/genética
Resistência a Medicamentos Antineoplásicos/genética
Ribonuclease III/fisiologia
[Mh] Termos MeSH secundário: Animais
RNA Helicases DEAD-box/genética
Quebras de DNA de Cadeia Dupla
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo
Feminino
Técnicas de Silenciamento de Genes
Células HCT116
Células HEK293
Histonas/metabolismo
Seres Humanos
Estimativa de Kaplan-Meier
Camundongos
Camundongos Endogâmicos BALB C
Camundongos Nus
Transplante de Neoplasias
RNA Interferente Pequeno/genética
Ribonuclease III/genética
Proteínas de Saccharomyces cerevisiae/metabolismo
Sirtuínas/genética
Sirtuínas/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Histones); 0 (RNA, Small Interfering); 0 (SIRT7 protein, human); 0 (Saccharomyces cerevisiae Proteins); EC 3.1.21.- (AI4 protein, S cerevisiae); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific); EC 3.1.26.3 (DICER1 protein, human); EC 3.1.26.3 (Ribonuclease III); EC 3.5.1.- (Sirtuins); EC 3.6.4.13 (DEAD-box RNA Helicases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171003
[Lr] Data última revisão:
171003
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170916
[St] Status:MEDLINE
[do] DOI:10.1093/carcin/bgx059


  7 / 8319 MEDLINE  
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[PMID]:28825727
[Au] Autor:Kakui Y; Rabinowitz A; Barry DJ; Uhlmann F
[Ad] Endereço:Chromosome Segregation Laboratory, The Francis Crick Institute, London, UK.
[Ti] Título:Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast.
[So] Source:Nat Genet;49(10):1553-1557, 2017 Oct.
[Is] ISSN:1546-1718
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The eukaryotic genome consists of DNA molecules far longer than the cells that contain them. They reach their greatest compaction during chromosome condensation in mitosis. This process is aided by condensin, a structural maintenance of chromosomes (SMC) family member. The spatial organization of mitotic chromosomes and how condensin shapes chromatin architecture are not yet fully understood. Here we use chromosome conformation capture (Hi-C) to study mitotic chromosome condensation in the fission yeast Schizosaccharomyces pombe. This showed that the interphase landscape characterized by small chromatin domains is replaced by fewer but larger domains in mitosis. Condensin achieves this by setting up longer-range, intrachromosomal DNA interactions, which compact and individualize chromosomes. At the same time, local chromatin contacts are constrained by condensin, with profound implications for local chromatin function during mitosis. Our results highlight condensin as a major determinant that changes the chromatin landscape as cells prepare their genomes for cell division.
[Mh] Termos MeSH primário: Adenosina Trifosfatases/fisiologia
Montagem e Desmontagem da Cromatina/fisiologia
Cromossomos Fúngicos/ultraestrutura
Proteínas de Ligação a DNA/fisiologia
Complexos Multiproteicos/fisiologia
Proteínas de Schizosaccharomyces pombe/fisiologia
Schizosaccharomyces/genética
[Mh] Termos MeSH secundário: Adenosina Trifosfatases/genética
Sequência de Bases
Cromatina/ultraestrutura
Montagem e Desmontagem da Cromatina/genética
Imunoprecipitação da Cromatina
DNA Fúngico/genética
Proteínas de Ligação a DNA/genética
Desoxirribonucleases de Sítio Específico do Tipo II
Interfase
Mitose
Complexos Multiproteicos/genética
Schizosaccharomyces/ultraestrutura
Proteínas de Schizosaccharomyces pombe/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Chromatin); 0 (DNA, Fungal); 0 (DNA-Binding Proteins); 0 (Multiprotein Complexes); 0 (Schizosaccharomyces pombe Proteins); 0 (condensin complexes); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific); EC 3.1.21.4 (GATC-specific type II deoxyribonucleases); EC 3.6.1.- (Adenosine Triphosphatases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171024
[Lr] Data última revisão:
171024
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170822
[St] Status:MEDLINE
[do] DOI:10.1038/ng.3938


  8 / 8319 MEDLINE  
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[PMID]:28817121
[Au] Autor:Renfer E; Technau U
[Ad] Endereço:Department of Molecular Evolution and Development, Faculty of Life Sciences, University of Vienna, Vienna, Austria.
[Ti] Título:Meganuclease-assisted generation of stable transgenics in the sea anemone Nematostella vectensis.
[So] Source:Nat Protoc;12(9):1844-1854, 2017 Sep.
[Is] ISSN:1750-2799
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The sea anemone Nematostella vectensis is a model system used by a rapidly growing research community for comparative genomics, developmental biology and ecology. Here, we describe a microinjection procedure for creating stable transgenic lines in Nematostella based on meganuclease (I-SceI)-assisted integration of a transgenic cassette into the genome. The procedure describes the preparation of the reagents, microinjection of the transgenesis vector and the husbandry of transgenic animals. The microinjection setup differs from those of previously published protocols by the use of a holding capillary mounted on an inverted fluorescence microscope. In one session of injections, a single researcher can microinject up to 1,300 zygotes with a reporter construct digested with the meganuclease I-SceI. Under optimal conditions, fully transgenic heterozygous F1 animals can be obtained within 4-5 months of the injections, with a germ-line transmission efficiency of ∼3%. The method is versatile and, after a short training phase, can be carried out by any researcher with basic training in molecular biology. Flexibility of construct design enables this method to be used for numerous applications, including the functional dissection of cis-regulatory elements, subcellular localization of proteins, detection of protein-binding partners, ectopic expression of genes of interest, lineage tracing and cell-type-specific reporter gene expression.
[Mh] Termos MeSH primário: Animais Geneticamente Modificados/genética
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo
Engenharia Genética/métodos
Microinjeções/métodos
Proteínas de Saccharomyces cerevisiae/metabolismo
Anêmonas-do-Mar/genética
[Mh] Termos MeSH secundário: Animais
Animais Geneticamente Modificados/fisiologia
Clonagem Molecular
Desenho de Equipamento
Microinjeções/instrumentação
Anêmonas-do-Mar/fisiologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Saccharomyces cerevisiae Proteins); EC 3.1.21.- (SCEI protein, S cerevisiae); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170830
[Lr] Data última revisão:
170830
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170818
[St] Status:MEDLINE
[do] DOI:10.1038/nprot.2017.075


  9 / 8319 MEDLINE  
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[PMID]:28756139
[Au] Autor:Nelson RS; Valadon P
[Ad] Endereço:Department of Molecular Biology, Antibody Design Labs, 11175 Flintkote Ave Suite B, San Diego, CA 92121, USA.
[Ti] Título:A universal phage display system for the seamless construction of Fab libraries.
[So] Source:J Immunol Methods;450:41-49, 2017 Nov.
[Is] ISSN:1872-7905
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:The construction of Fab phage libraries requires the cloning of domains from both the light and the heavy chain of antibodies. Despite the advent of powerful strategies such as splicing-by-overlap extension PCR, obtaining high quality libraries with excellent coverage remains challenging. Here, we explored the use of type IIS restriction enzymes for the seamless cloning of Fab libraries. We analyzed human, murine and rabbit germline antibody repertoires and identified combinations of restriction enzymes that exhibit very few or no recognition sites in the antibody sequences. We describe three phagemid vectors, pUP-22Hb, pUP-22Mc and pUP-22Rc, which were employed for cloning the Fab repertoire of these hosts using BsmBI and SapI (human) or SapI alone (mouse and rabbit). Using human serum albumin as a model immunization, we built a mouse/human chimeric Fab library and a mouse Fab library in a single step ligation and successfully panned multiple cognate antibodies. The overall process is highly scalable and faster than PCR-based techniques, with a Fab insertion success rate of around 80%. By using carefully chosen overhangs on each end of the antibody domains, this approach paves the way to the universal, sequence- and vector-independent cloning and reformatting of antibody libraries.
[Mh] Termos MeSH primário: Técnicas de Visualização da Superfície Celular
Clonagem Molecular
Fragmentos Fab das Imunoglobulinas/genética
Biblioteca de Peptídeos
Albumina Sérica/imunologia
[Mh] Termos MeSH secundário: Animais
Bacteriófagos/genética
Bacteriófagos/metabolismo
Desoxirribonucleases de Sítio Específico do Tipo II/genética
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo
Epitopos
Feminino
Vetores Genéticos
Seres Humanos
Imunização
Fragmentos Fab das Imunoglobulinas/imunologia
Fragmentos Fab das Imunoglobulinas/metabolismo
Camundongos Endogâmicos BALB C
Coelhos
Albumina Sérica/administração & dosagem
Albumina Sérica Humana
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (ALB protein, human); 0 (Epitopes); 0 (Immunoglobulin Fab Fragments); 0 (Peptide Library); 0 (Serum Albumin); EC 3.1.21.- (endodeoxyribonuclease SapI); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific); EC 3.1.21.4 (endodeoxyribonuclease BsmBI); ZIF514RVZR (Serum Albumin, Human)
[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:170731
[St] Status:MEDLINE


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[PMID]:28703698
[Au] Autor:Deng CL; Zhang QY; Chen DD; Liu SQ; Qin CF; Zhang B; Ye HQ
[Ad] Endereço:1​CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
[Ti] Título:Recovery of the Zika virus through an in vitro ligation approach.
[So] Source:J Gen Virol;98(7):1739-1743, 2017 Jul.
[Is] ISSN:1465-2099
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:In this study, an in vitro ligation method was developed to assemble a full-length infectious cDNA clone of the Zika virus (ZIKV). Four contiguous cDNA subclones covering the complete ZIKV genome were constructed with unique BglI restriction sites at the ends of each fragment. The BglI restriction sites only allow in vitro ligation to happen between interconnecting restriction sites from adjacent cDNA fragments, resulting in an intact full-length cDNA of ZIKV. RNA transcripts derived from the full-length cDNA were infectious. The recombinant virus replicated as efficiently as the wild-type virus with similar growth kinetics and plaque morphologies in Vero and C6/36 cells. Both viruses were inhibited by NITD008 treatment. This in vitro ligation method will facilitate manipulation of the viral genome through genetic modifications of four separated subclones of ZIKV for the rapid and rational development of candidate vaccines and viral replication study.
[Mh] Termos MeSH primário: Clonagem Molecular/métodos
DNA Complementar/genética
DNA Viral/genética
RNA Viral/genética
Zika virus/genética
[Mh] Termos MeSH secundário: Adenosina/análogos & derivados
Adenosina/farmacologia
Aedes
Animais
Antivirais/farmacologia
Linhagem Celular
Cercopithecus aethiops
Cricetinae
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo
Genoma Viral/genética
Células Vero
Zika virus/efeitos dos fármacos
Zika virus/isolamento & purificação
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antiviral Agents); 0 (DNA, Complementary); 0 (DNA, Viral); 0 (NITD008); 0 (RNA, Viral); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific); EC 3.1.21.4 (GCCNNNNNGGC-specific type II deoxyribonucleases); K72T3FS567 (Adenosine)
[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:170714
[St] Status:MEDLINE
[do] DOI:10.1099/jgv.0.000862



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