Base de dados : MEDLINE
Pesquisa : G05.360.340.024.340.465 [Categoria DeCS]
Referências encontradas : 2113 [refinar]
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  1 / 2113 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


  2 / 2113 MEDLINE  
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[PMID]:28977654
[Au] Autor:Shepherd TR; Du L; Liljeruhm J; Samudyata; Wang J; Sjödin MOD; Wetterhall M; Yomo T; Forster AC
[Ad] Endereço:Department of Cell and Molecular Biology, Uppsala University, Uppsala 751 36, Sweden.
[Ti] Título:De novo design and synthesis of a 30-cistron translation-factor module.
[So] Source:Nucleic Acids Res;45(18):10895-10905, 2017 Oct 13.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.
[Mh] Termos MeSH primário: Genes Sintéticos
Biossíntese de Proteínas
Proteínas Ribossômicas/genética
[Mh] Termos MeSH secundário: Plasmídeos/genética
Transcrição Genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Ribosomal Proteins)
[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:171005
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx753


  3 / 2113 MEDLINE  
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[PMID]:28911123
[Au] Autor:Lubock NB; Zhang D; Sidore AM; Church GM; Kosuri S
[Ad] Endereço:Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA.
[Ti] Título:A systematic comparison of error correction enzymes by next-generation sequencing.
[So] Source:Nucleic Acids Res;45(15):9206-9217, 2017 Sep 06.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Gene synthesis, the process of assembling gene-length fragments from shorter groups of oligonucleotides (oligos), is becoming an increasingly important tool in molecular and synthetic biology. The length, quality and cost of gene synthesis are limited by errors produced during oligo synthesis and subsequent assembly. Enzymatic error correction methods are cost-effective means to ameliorate errors in gene synthesis. Previous analyses of these methods relied on cloning and Sanger sequencing to evaluate their efficiencies, limiting quantitative assessment. Here, we develop a method to quantify errors in synthetic DNA by next-generation sequencing. We analyzed errors in model gene assemblies and systematically compared six different error correction enzymes across 11 conditions. We find that ErrASE and T7 Endonuclease I are the most effective at decreasing average error rates (up to 5.8-fold relative to the input), whereas MutS is the best for increasing the number of perfect assemblies (up to 25.2-fold). We are able to quantify differential specificities such as ErrASE preferentially corrects C/G transversions whereas T7 Endonuclease I preferentially corrects A/T transversions. More generally, this experimental and computational pipeline is a fast, scalable and extensible way to analyze errors in gene assemblies, to profile error correction methods, and to benchmark DNA synthesis methods.
[Mh] Termos MeSH primário: Técnicas de Química Sintética/normas
DNA/síntese química
Genes Sintéticos
Sequenciamento de Nucleotídeos em Larga Escala
[Mh] Termos MeSH secundário: Benchmarking
DNA/genética
Desoxirribonuclease I/genética
Desoxirribonuclease I/metabolismo
Proteínas de Escherichia coli/genética
Proteínas de Escherichia coli/metabolismo
Proteína MutS de Ligação de DNA com Erro de Pareamento/genética
Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo
Oligodesoxirribonucleotídeos/química
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Escherichia coli Proteins); 0 (Oligodeoxyribonucleotides); 9007-49-2 (DNA); EC 3.1.21.1 (Deoxyribonuclease I); EC 3.6.1.3 (MutS DNA Mismatch-Binding Protein); EC 3.6.1.3 (MutS protein, E coli)
[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:170916
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx691


  4 / 2113 MEDLINE  
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[PMID]:28578326
[Au] Autor:Yu D; Du Z; Li W; Chen H; Ye S; Hoffman AR; Cui J; Hu JF
[Ad] Endereço:Stem Cell and Cancer Center, First Hospital, Jilin University, Changchun, China.
[Ti] Título:Targeting Jurkat T Lymphocyte Leukemia Cells by an Engineered Interferon-Alpha Hybrid Molecule.
[So] Source:Cell Physiol Biochem;42(2):519-529, 2017.
[Is] ISSN:1421-9778
[Cp] País de publicação:Switzerland
[La] Idioma:eng
[Ab] Resumo:BACKGROUND/AIMS: Adult T-cell leukemia/lymphoma (ATL) is a very aggressive T cell malignancy that carries a poor prognosis, primarily due to its resistance to chemotherapy and to life-threatening infectious complications. Interferon-alpha (IFNα) has been used in combination with the anti-retroviral drug zidovudine to treat patients with ATL. However, the efficacy of long-term therapy is significantly limited due to the systemic toxicity of IFNα. METHODS: We utilized phage display library screening to identify short peptides that specifically bind to Jurkat T lymphocyte leukemia cells. By fusing the Jurkat-binding peptide to the C-terminus of IFNα, we constructed an engineered chimeric IFNα molecule (IFNP) for the treatment of ATL. RESULTS: We found that IFNP exhibited significantly higher activity than wild type IFNα in inhibiting the growth of leukemia cells and inducing cell blockage at the G0/G1 phase. The synthetic IFNP molecule exerted its antitumor activity by upregulating the downstream genes involved in the STAT1 pathway and in apoptosis. Using a cell receptor binding assay, we showed that this Jurkat-binding peptide facilitated the binding affinity of IFNα to the cell surface type I IFN receptor. CONCLUSION: The isolated Jurkat-binding peptide significantly potentiates the therapeutic activity of IFNα in T lymphocyte leukemia cells. The engineered IFNP molecule may prove to a novel antitumor approach in the treatment of patients with ATL.
[Mh] Termos MeSH primário: Interferon-alfa/genética
Leucemia-Linfoma de Células T do Adulto/genética
Peptídeos/genética
[Mh] Termos MeSH secundário: Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem
Apoptose/efeitos dos fármacos
Linhagem Celular Tumoral
Genes Sintéticos/genética
Engenharia Genética
Seres Humanos
Interferon-alfa/administração & dosagem
Interferon-alfa/efeitos adversos
Células Jurkat
Leucemia-Linfoma de Células T do Adulto/patologia
Leucemia-Linfoma de Células T do Adulto/terapia
Biblioteca de Peptídeos
Peptídeos/administração & dosagem
Fator de Transcrição STAT1/biossíntese
Fator de Transcrição STAT1/genética
Linfócitos T/efeitos dos fármacos
Linfócitos T/patologia
Zidovudina/administração & dosagem
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Interferon-alpha); 0 (Peptide Library); 0 (Peptides); 0 (STAT1 Transcription Factor); 0 (STAT1 protein, human); 4B9XT59T7S (Zidovudine)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:171109
[Lr] Data última revisão:
171109
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170605
[St] Status:MEDLINE
[do] DOI:10.1159/000477601


  5 / 2113 MEDLINE  
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[PMID]:28525738
[Au] Autor:van der Sloot A; Tyers M
[Ad] Endereço:Institute for Research in Immunology and Cancer, University of Montreal, Montreal, QC H3C 3J7, Canada. Electronic address: am.van.der.sloot@umontreal.ca.
[Ti] Título:Synthetic Genomics: Rewriting the Genome Chromosome by Chromosome.
[So] Source:Mol Cell;66(4):441-443, 2017 May 18.
[Is] ISSN:1097-4164
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The Synthetic Yeast Genome Project (Sc2.0) consortium has recently published a collection of seven reports on the design, assembly, and in vivo characterization of five entirely synthetic yeast chromosomes that set the stage for de novo construction of synthetic custom-engineered eukaryotes.
[Mh] Termos MeSH primário: DNA Fúngico/genética
Genes Sintéticos
Genoma Fúngico
Genômica/métodos
Proteínas de Saccharomyces cerevisiae/genética
Saccharomyces cerevisiae/genética
[Mh] Termos MeSH secundário: DNA Fúngico/metabolismo
Bases de Dados Genéticas
Regulação Fúngica da Expressão Gênica
Genótipo
Fenótipo
Saccharomyces cerevisiae/metabolismo
Proteínas de Saccharomyces cerevisiae/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Fungal); 0 (Saccharomyces cerevisiae Proteins)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170906
[Lr] Data última revisão:
170906
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170520
[St] Status:MEDLINE


  6 / 2113 MEDLINE  
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[PMID]:28525578
[Au] Autor:Hirosawa M; Fujita Y; Parr CJC; Hayashi K; Kashida S; Hotta A; Woltjen K; Saito H
[Ad] Endereço:Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
[Ti] Título:Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.
[So] Source:Nucleic Acids Res;45(13):e118, 2017 Jul 27.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information.
[Mh] Termos MeSH primário: Sistemas CRISPR-Cas
Edição de Genes/métodos
MicroRNAs/genética
[Mh] Termos MeSH secundário: Regiões 5' não Traduzidas
Elementos Alu
Proteínas de Bactérias/genética
Proteínas de Bactérias/metabolismo
Diferenciação Celular
Técnicas de Cocultura
Endonucleases/genética
Endonucleases/metabolismo
Genes de Troca
Genes Sintéticos
Proteínas de Fluorescência Verde/genética
Células HeLa
Seres Humanos
Células-Tronco Pluripotentes Induzidas/citologia
Células-Tronco Pluripotentes Induzidas/metabolismo
MicroRNAs/metabolismo
Neurônios/citologia
Neurônios/metabolismo
RNA Guia/genética
RNA Guia/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (5' Untranslated Regions); 0 (Bacterial Proteins); 0 (MicroRNAs); 0 (RNA, Guide); 0 (enhanced green fluorescent protein); 147336-22-9 (Green Fluorescent Proteins); EC 3.1.- (Cas9 endonuclease Streptococcus pyogenes); EC 3.1.- (Endonucleases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171011
[Lr] Data última revisão:
171011
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170520
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx309


  7 / 2113 MEDLINE  
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[PMID]:28500881
[Au] Autor:Manaresi E; Conti I; Bua G; Bonvicini F; Gallinella G
[Ad] Endereço:Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
[Ti] Título:A Parvovirus B19 synthetic genome: sequence features and functional competence.
[So] Source:Virology;508:54-62, 2017 Aug.
[Is] ISSN:1096-0341
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Central to genetic studies for Parvovirus B19 (B19V) is the availability of genomic clones that may possess functional competence and ability to generate infectious virus. In our study, we established a new model genetic system for Parvovirus B19. A synthetic approach was followed, by design of a reference genome sequence, by generation of a corresponding artificial construct and its molecular cloning in a complete and functional form, and by setup of an efficient strategy to generate infectious virus, via transfection in UT7/EpoS1 cells and amplification in erythroid progenitor cells. The synthetic genome was able to generate virus with biological properties paralleling those of native virus, its infectious activity being dependent on the preservation of self-complementarity and sequence heterogeneity within the terminal regions. A virus of defined genome sequence, obtained from controlled cell culture conditions, can constitute a reference tool for investigation of the structural and functional characteristics of the virus.
[Mh] Termos MeSH primário: Genes Sintéticos
Genoma Viral
Infecções por Parvoviridae/virologia
Parvovirus B19 Humano/genética
RNA Viral/síntese química
[Mh] Termos MeSH secundário: Seres Humanos
Parvovirus B19 Humano/fisiologia
RNA Viral/genética
RNA Viral/metabolismo
Replicação Viral
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (RNA, Viral)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170818
[Lr] Data última revisão:
170818
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170514
[St] Status:MEDLINE


  8 / 2113 MEDLINE  
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[PMID]:28499033
[Au] Autor:Lau YH; Stirling F; Kuo J; Karrenbelt MAP; Chan YA; Riesselman A; Horton CA; Schäfer E; Lips D; Weinstock MT; Gibson DG; Way JC; Silver PA
[Ad] Endereço:Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, 5th Floor, Boston, MA 02115, USA.
[Ti] Título:Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA.
[So] Source:Nucleic Acids Res;45(11):6971-6980, 2017 Jun 20.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The ability to rewrite large stretches of genomic DNA enables the creation of new organisms with customized functions. However, few methods currently exist for accumulating such widespread genomic changes in a single organism. In this study, we demonstrate a rapid approach for rewriting bacterial genomes with modified synthetic DNA. We recode 200 kb of the Salmonella typhimurium LT2 genome through a process we term SIRCAS (stepwise integration of rolling circle amplified segments), towards constructing an attenuated and genetically isolated bacterial chassis. The SIRCAS process involves direct iterative recombineering of 10-25 kb synthetic DNA constructs which are assembled in yeast and amplified by rolling circle amplification. Using SIRCAS, we create a Salmonella with 1557 synonymous leucine codon replacements across 176 genes, the largest number of cumulative recoding changes in a single bacterial strain to date. We demonstrate reproducibility over sixteen two-day cycles of integration and parallelization for hierarchical construction of a synthetic genome by conjugation. The resulting recoded strain grows at a similar rate to the wild-type strain and does not exhibit any major growth defects. This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification.
[Mh] Termos MeSH primário: DNA Bacteriano/genética
Engenharia Genética/métodos
Salmonella typhimurium/genética
[Mh] Termos MeSH secundário: Códon
Genes Bacterianos
Genes Sintéticos
Genoma Bacteriano
Leucina/genética
Viabilidade Microbiana
Reprodutibilidade dos Testes
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Codon); 0 (DNA, Bacterial); GMW67QNF9C (Leucine)
[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:170513
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx415


  9 / 2113 MEDLINE  
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[PMID]:28277678
[Au] Autor:Milazzo L; Tognaccini L; Howes BD; Sinibaldi F; Piro MC; Fittipaldi M; Baratto MC; Pogni R; Santucci R; Smulevich G
[Ad] Endereço:Dipartimento di Chimica "Ugo Schiff", Università di Firenze , Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy.
[Ti] Título:Unravelling the Non-Native Low-Spin State of the Cytochrome c-Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only.
[So] Source:Biochemistry;56(13):1887-1898, 2017 Apr 04.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH ion.
[Mh] Termos MeSH primário: Monóxido de Carbono/química
Cardiolipinas/química
Citocromos c/química
Histidina/química
Metionina/química
[Mh] Termos MeSH secundário: Animais
Cardiolipinas/metabolismo
Clonagem Molecular
Citocromos c/genética
Citocromos c/metabolismo
Escherichia coli/genética
Escherichia coli/metabolismo
Expressão Gênica
Genes Sintéticos
Cavalos
Ligações de Hidrogênio
Miocárdio/química
Ligação Proteica
Dobramento de Proteína
Estrutura Secundária de Proteína
Desdobramento de Proteína
Proteínas Recombinantes/química
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Temperatura Ambiente
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Cardiolipins); 0 (Recombinant Proteins); 4QD397987E (Histidine); 7U1EE4V452 (Carbon Monoxide); 9007-43-6 (Cytochromes c); AE28F7PNPL (Methionine)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170515
[Lr] Data última revisão:
170515
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170310
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.6b01281


  10 / 2113 MEDLINE  
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[PMID]:28204586
[Au] Autor:Trubitsyna M; Michlewski G; Finnegan DJ; Elfick A; Rosser SJ; Richardson JM; French CE
[Ad] Endereço:Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK.
[Ti] Título:Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.
[So] Source:Nucleic Acids Res;45(10):e89, 2017 Jun 02.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.
[Mh] Termos MeSH primário: Elementos de DNA Transponíveis
Proteínas de Ligação a DNA/genética
Mutagênese Insercional
Plasmídeos/metabolismo
Transposases/genética
[Mh] Termos MeSH secundário: Sequência de Bases
Clonagem Molecular
Proteínas de Ligação a DNA/metabolismo
Eletroporação
Escherichia coli/genética
Escherichia coli/metabolismo
Genes Sintéticos
Células HEK293
Células HeLa
Seres Humanos
Sequências Repetidas Invertidas
Lipídeos/química
Plasmídeos/química
Análise de Sequência de DNA
Transfecção
Transposases/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA Transposable Elements); 0 (DNA-Binding Proteins); 0 (Lipids); 0 (Lipofectamine); 0 (mariner transposases); EC 2.7.7.- (Transposases)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170912
[Lr] Data última revisão:
170912
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170217
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx113



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