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Pesquisa : D08.811.739.500 [Categoria DeCS]
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Id: lil-723107
Autor: Navidinia, Masoumeh; Peerayeh, Shahin Najar; Fallah, Fatemeh; Bakhshi, Bita; Sajadinia, Raheleh Sadat.
Título: Phylogenetic grouping and pathotypic comparison of urine and fecal Escherichia coli isolates from children with urinary tract infection
Fonte: Braz. j. microbiol;45(2):509-514, Apr.-June 2014. tab.
Idioma: en.
Resumo: The aim of this study was to investigate the phylogenetic background and to assess hlyD (involved in the secretion of haemolysin A) and intll (encoding a class 1 integrase) in Escherichia coli isolates derived from urinary and fecal specimens. A total of 200 E. coli isolates was collected from patients presenting with urinary tract infection (UTI) during September 2009 to September 2010 and screened for hlyD and intll genes by polymerase chain reaction (PCR). Phylogenetic analysis showed that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D) and that uropathogenic E. coli (UPEC) isolates mainly belong to groups B2 (54%) and D (34%) whereas group A (44%) and D (26%) are predominant among commensal E. coli isolates. In this study, hlyD was present in 26% of UPEC and 2% of commensal E. coli isolates. However, hemolytic activity was detected for 42% of UPEC and 6% of commensal E. coli isolates (p < 0.05). intll gene was more frequently expressed in UPEC (24%) in comparison with commensal E. coli isolates (12%). Resistance to aztreonam, co-trimoxazole and cefpodoxime were frequently found among UPEC isolates whereas commensal E. coli isolates were commonly resistant to co-trimoxazole, nalidixic acid and cefotaxime. Concluding, a considerable difference between UPEC and commensal E. coli isolates was observed regarding their phylogenetic groups, presence of class 1 integron and hlyD gene, hemolysin activity and resistance pattern. The detection of class 1 integrons and hlyD gene was higher among UPEC compared with commensal E. coli isolates. These findings may contribute for a better understanding of the factors involved in the pathogenesis of UPEC.
Descritores: Infecções por Escherichia coli/microbiologia
Escherichia coli/classificação
Fezes/microbiologia
Variação Genética
Filogenia
Infecções Urinárias/microbiologia
Urina/microbiologia
-Antibacterianos/farmacologia
Análise por Conglomerados
Farmacorresistência Bacteriana
Proteínas de Escherichia coli/genética
Escherichia coli/efeitos dos fármacos
Escherichia coli/genética
Escherichia coli/isolamento & purificação
Genótipo
Proteínas Hemolisinas/genética
Integrases/genética
Proteínas de Membrana Transportadoras/genética
Reação em Cadeia da Polimerase
Análise de Sequência de DNA
Limites: Criança
Pré-Escolar
Feminino
Seres Humanos
Masculino
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: BR1.1 - BIREME


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Id: lil-650121
Autor: Peña, Ángela; Yosa, Juvenal; Cuesta-Astroz, Yesid; Acevedo, Orlando; Lareo, Leonardo; García-Vallejo, Felipe.
Título: Influence of Mg2+ ions on the interaction between 3,5-dicaffeoylquinic acid and HTLV-I integrase / Influencia de los iones Mg+2 sobre la interacción entre el ácido 3,5- dicafeoilquínico y la integrasa del HTLV-I / Influência dos íons Mg2+ sobre a interação entre o ácido 3,5-dicafeoilquínico e a integrase do HTLV-I
Fonte: Univ. sci;17(1):5-15, Jan.-Apr. 2012. ilus, tab.
Idioma: en.
Resumo: Objective. Using molecular simulation, we studied the influence of Mg2+ ions on the binding mode of HTLV-I Integrase (IN) catalytic domain (modeled by homology) with the 3,5- Dicaffeoylquinic Acid (DCQA). HTLV-I Integrase homology model was built using template-like crystallographic data of the IN catalytic domain solved for Avian Sarcoma Virus (VSA, pdb: 1VSD). Materials and methods. In order to analyze the role of Mg2+ in the interaction or coupling between 3,5-DCQA and Integrase, three models were created: i) in the absence of Mg2+ ions, ii) with a Mg2+ ion coordinated at Asp15 and Asp72 and iii) model with two Mg2+ ions coordinated at Asp15-Asp72 and Asp72-Glu108. Coupling force and binding free energy between 3,5-DCQA and HTLV-I IN were assessed in the three models. Results. The lowest docking score and free energy binding were obtained for the second model. Mg2+ ion strongly affected the coupling of the inhibitor 3,5-DCQA with HTLV-I catalytic domain of Integrase, thus revealing a strong interaction in the ligand-protein complex regardless of the ligand-catalytic interaction sites for all three models. Conclusion. Altogether, these results strengthen the hypothesis that the presence of one Mg2+ ion could enhance the interaction in the complex by decreasing free energy, therefore increasing the affinity. Moreover, we propose 3,5-DCQA as an important pharmacophore in the rational design of new antiretroviral drugs.

Objetivo: Usando simulación molecular, estudiamos la influencia de los iones Mg2+ en la interacción del dominio catalítico de la integrasa HTLV-I (IN) (modelado por homología) con el ácido 3,5-Dicafeoilquínico (DCQA). Materiales y métodos. El modelo por homología de la HTLV-I IN fue construido usando como molde la estructura cristalina de la IN del virus del sarcoma aviar (VSA, pdb: 1VSD). Para analizar el rol de los iones Mg2+ en la interacción con el DCQA y la integrasa, tres modelos fueron creados: i) en ausencia de iones Mg2+, ii) con un ion Mg2+ coordinado con Asp15 y Asp72 y iii) con dos iones Mg2+ coordinados con Asp15-Asp72 y Asp72-Glu108. Las fuerzas de interacción y la energía libre de unión entre el DCQA y la HTLV-I IN fueron calculadas en los tres modelos. Resultados. El puntaje más bajo en el docking y la menor energía libre fueron obtenidos para el modelo con un solo ion de Mg2+. El Mg2+ afecta fuertemente el acoplamiento del inhibidor DCQA con el dominio catalítico de la HTLV-I IN, revelando una fuerte interacción entre el complejo ligando-proteína que es independiente del sitio catalítico de los tres modelos usados. Conclusión. Los resultados sugieren que la presencia de un ion de Mg² + podría incrementar la interacción en el complejo debido a la disminución de la energía libre, intensificando así la afinidad. Por lo que, proponemos al DCQA como farmacóforo para el diseño de drogas antiretrovirales.

Objetivo. Usando simulação molecular, estudamos a influência dos íons Mg2+ na interação do domínio catalítico da integrase HTLV-I (IN) (modelado por homologia) com o ácido 3,5-dicafeoilquínico (3,5-diCQA). Materiais e métodos. O modelo de homologia da HTLV-I IN foi construído utilizando como fôrma a estrutura cristalina da IN de vírus do sarcoma aviário (VSA, pdb: 1VSD). Para analisar o papel dos íons Mg2+ na interação com o 3,5-diCQA e a integrase, três modelos foram criados: i) na ausência de íons Mg2+, ii) com um íon Mg2+ coordenado com Asp15 e Asp72 e, iii) com dois íons Mg2+ coordenados com Asp15-Asp72 e Asp72-Glu108. As forças de interação e a energia livre de ligação entre o 3,5-diCQA e a HTLV-I IN foram calculadas nos três modelos. Resultados. A pontuação mais baixa no docking e a menor energia livre foram obtidas para o modelo com um único íon de Mg2+. O Mg2+ afeta fortemente o acoplamento do inibidor 3,5-diCQA com o domínio catalítico da HTLV-I IN, revelando uma forte interação entre o complexo ligando-proteína que é independente do sítio catalítico dos três modelos utilizados. Conclusão. Os resultados sugerem que a presença de um íon Mg2+ poderia aumentar a interação no complexo devido à diminuição da energia livre, aumentando assim a afinidade. Assim, propomos ao 3,5-diCQA como farmacóforo para a fabricação de medicamentos antirretrovirais.
Descritores: Antirretrovirais
Integrases
Magnésio
Limites: Seres Humanos
Responsável: CO332 - Facultad de Medicina


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Id: lil-634630
Autor: Ibar, M. P.; Vigo, G.; Piñeyro, P.; Caffer, M. I.; Quiroga, P.; Perfumo, C.; Centrón, D.; Giacoboni, G..
Título: Serovariedades de Salmonella enterica subespecie enterica en porcinos de faena y su resistencia a los antimicrobianos / Serovars of Salmonella enterica subspecies enterica and its antimicrobial resistance in slaughterhouse pigs
Fonte: Rev. argent. microbiol;41(3):156-162, jul.-sep. 2009. graf, tab.
Idioma: es.
Resumo: Se realizó un estudio para determinar la prevalencia de Salmonella y sus serovariedades en cerdos de faena, para evaluar sus perfiles de resistencia a los antimicrobianos y para conocer la presencia de integrones de clase 1 como posibles reservorios de resistencia. A partir de un total de 386 muestras de porcinos provenientes de cuatro frigoríficos de las provincias de Buenos Aires y de Santa Fe (Argentina), se identificaron 93 (24,1%) cepas de Salmonella enterica subespecie enterica, 52 (55,9%) de contenido cecal y 41 (44,1%) de nódulo linfático ileocecal. Se hallaron 13 serovariedades de S. enterica, las más prevalentes fueron S. Schwarzengrund, S. Heidelberg, S. subespecie I 6,8:e,h:-, S. Derby y S. Bredeney. Se probaron 15 antimicrobianos por el método de dilución en agar: amikacina, gentamicina, ciprofloxacina, cefalotina, cefotaxima, enrofloxacina, fosfomicina, polimixina-B, tetraciclina, cloranfenicol, estreptomicina, trimetoprima-sulfametoxazol, ampicilina, nitrofurantoína y ácido nalidíxico. Según se estableció mediante la determinación de la CIM, el 73% de las cepas de S. enterica subespecie enterica fueron sensibles a todos los antimicrobianos probados. Se observó resistencia a tetraciclina en 24 (25,8%) de las 93 cepas, a cloranfenicol en 22 (23,7%), a estreptomicina en 22 (23,7%) a trimetoprima-sulfametoxazol en 20 (21,5%), a ampicilina en 18 (19,4%), a nitrofurantoína en 3 (3,2%) y a ácido nalidíxico en 3 (3,2%). Algunos aislamientos de S. Typhimurium, S. Heildelberg, S. Derby y S. Orion presentaron multirresistencia y portaban el gen de la integrasa clase 1. Los mayores porcentajes de resistencia correspondieron a los antimicrobianos habitualmente utilizados en veterinaria y en las explotaciones porcinas.

A study was carried out in order to determine the prevalence of Salmonella and its serovars among porcine slaughterhouses, to evaluate the antimicrobial resistance profiles and to know the presence of class 1 integrons as possible reservoir of resistance. From a total of 386 samples from four porcine slaughterhouses of Buenos Aires and Santa Fe Provinces (Argentina), 93 (24,1%) Salmonella enterica subspecies enterica strains were identified, 52 (55,9%) from cecal contents and 41 (44,1%) from ileocecal lymph nodes. Thirteen serovars of S. enterica were found, the most prevalent were: S. Schwarzengrund, S. Heidelberg, S. subspecie I 6,8:e,h:-, S. Derby and S. Bredeney. Fifteen antimicrobials by the agar dilution method were tested: amikacin, gentamicin, ciprofloxacin, cephalotin, cefotaxime, enrofloxacin, fosfomycin, polimixin-B, tetracycline, chloramphenicol, streptomycin, trimethoprim-sulfamethoxazole, ampicillin, nitrofurantoin, and nalidixic acid. According to the CIM determination, 73% Salmonella enterica subspecies enterica strains were sensible to all the antimicrobials tested. Antimicrobial resistance was observed to tetracycline in 24 (25,8%) of 93 strains, to chloramphenicol in 22 (23,7%), to streptomycin in 22 (23,7%), to trimethoprim-sulfamethoxazole in 20 (21,5%), to ampicillin in 18 (19,4%), to nitrofurantoin in 3 (3,2%) and to nalidixic acid in 3 (3,2%). Some isolates of S. Typhimurium, S. Heidelberg, S. Derby, S. Orion showed multidrug resistance and carried the class 1 integrase gene. The highest percentage of resistance corresponded to the antimicrobials currently used in veterinary and porcine farms.
Descritores: Microbiologia de Alimentos
Carne/microbiologia
Salmonella enterica/isolamento & purificação
Sus scrofa/microbiologia
-Matadouros
Criação de Animais Domésticos
Argentina
Antibacterianos/administração & dosagem
Ceco/microbiologia
Reservatórios de Doenças
Farmacorresistência Bacteriana Múltipla
Conservação de Alimentos
Integrases/genética
Integrons/genética
Linfonodos/microbiologia
Refrigeração
Sorotipagem
Salmonella enterica/classificação
Salmonella enterica/efeitos dos fármacos
Salmonella enterica/genética
Limites: Animais
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: AR1.2 - Instituto de Investigaciónes Epidemiológicas


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Id: lil-562517
Autor: García Vallejo, Felipe; Cuesta Astroz, Yesid; Dominguez, Martha C; Sánchez, Adalberto; Salcedo Cifuentes, Mercedes; Díaz, Diane María; Quintana, Milton.
Título: Modelación molecular y variación estructural de las integrasas de dos retrovirus humanos: HTLV-I y VIH-1 / Molecular modeling and structural variation of two human retrovirus integrases: HTLV-I and HIV-1
Fonte: Salud UNINORTE;25(1):1-16, ene. 2009. ilus, tab, graf.
Idioma: es.
Resumo: Objetivo: Analizar las características moleculares y de variación de secuencias de las integrasas del HTLV-I y del VIH-1 y sus variantes poblacionales. Metodología: Análisis de secuencias y estructuras obtenidas de diferentes bases de datos; para ello se utilizaron programas computacionales de modelación de estructuras proteicas e identificación de sustituciones polimórficas en secuencias de aminoácidos de integrasas del HTLV-I y VIH-1 previamente reportadas. Materiales y métodos: Tanto la integrasa del HTLV-I como la del VIH-1 son proteínas compuestas por 288 residuos de aminoácidos. Se encontró un parecido de estructuras terciarias entre los dominios catalíticos de las IN de VIH-1, ASV y RSV con la del HTLV- I. A partir de 103 secuencias completas de la integrasa del VIH-1 se registraron, en 46 codones, un total de 53 sustituciones que se localizaron en diferentes posiciones de la proteína nativa; las más frecuentes fueron: N27G (32,1%), A265V (30,1%), L101I (31,1%) y T123A (27,0%). Ninguna de las sustituciones más frecuentemente encontradas generó un cambio en el plegamiento nativo de la correspondiente región. Conclusión: La estructura tridimensional del dominio central catalítico de la integrasa condicionaría su actividad y su relación con moléculas potencialmente inhibidoras. Las sustituciones observadas fueron neutrales sin alterar la estructura nativa. Los resultados obtenidos confirman que la integrasa es un nuevo y promisorio blanco para el desarrollo de terapias antirretrovirales más efectivas en el siglo XXI...

Objective: To analyze the molecular characteristics and aminoacid sequence variations of HTLV-I and of HIV-1 integrases and their population variants. Materials and methods: Data mining and analysis of integrase sequences and protein structure data bases by using appropriate software for modelling and search for polymorphic substitutions in HTLV-I and HIV-1 integrase amino acid sequences previously reported. Results: HTLV-I and HIV-1 integrases are proteins of 288 amino acid residues. Structural modeling of tertiary folding of HTLV-I integrase catalytic central domain's, showed closed structural characteristic with those of HIV-1, ASV and RSV. From 103 full amino acid sequences of HIV-1 integrase, 53 substitutions located in 46 different codons were recorded. The more frequents correspond to N27G (32,1%), L101I (31,1%), A265V (30,1%) and T123A (27,0%). None of these frequent substitutions introduced changes in the folding of HIV-1 native integrase. Conclusion: The tridimensional structure of central catalytic domain would influence the integrase activity and its relationship with potentially inhibitory molecules. Those observed aminoacid substitutions were neutral and do not alter the native protein structure. Our data confirm those previously published, and enable us to propose that IN is a new and promissory target for develop more effective antiviral therapies in the XXI century...
Descritores: Integrases
Modelos Moleculares
Conformação Proteica
Responsável: CO181.1 - Biblioteca General


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Id: lil-544566
Autor: Pinilla, Gladys; Muñoz, Liliana; Ruiz, Ariel Ivan; Chavarro, Bibiana; Cifuentes, Yolanda.
Título: Aislamiento de Staphylococcus epidermidis portador de integrón clase 1en un paciente con sepsis neonatal / Isolation of Staphylococcus epidermidis strain carrier of the class one integron in a septic neonatal patient
Fonte: Infectio;13(3):196-202, sept. 2009. ilus.
Idioma: es.
Resumo: Objetivo. Determinar la presencia y secuencia del integrón clase I en un aislamiento clínico de Staphylococcus epidermidis proveniente de un neonato con diagnóstico de sepsis. Materiales y métodos. En una cepa de S. epidermidis, aislada de una muestra de hemocultivo de un neonato, se realizaron las pruebas de identificación microbiológica, susceptibilidad antimicrobiana y la caracterización molecular de los genes aac6'-aph2'', mecA, el gen de la integrasa intI1 y el gen casete aac6'. Resultados. Se identificó el gen de la integrasa intl1 y mediante secuenciación de nucleótidos se encontró una homología del 78% con las secuencias reportadas en la base de datos del NCBI para bacterias Gram negativas. También se evidenció el casete genético aac6' presente dentro del integrón clase 1, el cual acetila los aminoglucósidos para conferir resistencia a este antibiótico; como parte de la caracterización molecular se encontró la presencia del gen (aac6'-aph2''), que codifica para una enzima bifuncional que inactiva a los aminoglucósidos, y el gen mecA que confiere resistencia a los ß-lactámicos. En las pruebas de susceptibilidad se encontró resistencia a ampicilina, oxacilina y gentamicina. Conclusiones. Se reporta por primera vez en Colombia, la secuencia del gen de la integrasa intl1 en un aislamiento de S. epidermidis, proveniente de un recién nacido con sepsis neonatal de la unidad de cuidados intensivos en un hospital de tercer nivel de Bogotá. Esta integrasa ha sido relacionada sólo en integrones clase 1, los cuales se han asociado a multirresistencia en aislamientos clínicos en bacterias Gram negativas. El hallazgo de este mecanismo de resistencia, en el presente estudio realizado en una bacteria Gram positiva, sugiere la relación con transferencia horizontal de genes de resistencia entre especies, efectuado mediante la...

Objective: To determine the presence and the sequence of the class one integron in a Staphylococcus epidermidis strain isolated from a septic neonatal patient. Materials and methods: The S. epidermidis strain was isolated from a blood culture of a newborn. The microbiological identification, test of sensitivity and molecular characterization of the integrase gene int/1, the cassette gene aac6; aac6'aph2'' resistant gene to aminoglycoside antibiotics and mecA gene resistant to ß-lactamics were realized. Results: The nucleotide sequence of the integrase gen intl1 in S. epidermidis is reported, which showed a 78% similarity to the reported sequence of the NCBI data base in Gram negative bacteria. The gene cassette aac6 aac6' (aminoglycoside acetilation) was also identified within the class one integron confering aminoglycoside resistance. Through molecular characterization we also found the aminoglycoside ß-lactamics resistance genes (aac6'aph2'y mecA'); the susceptibility tests showed resistance to ampicilin, oxacilin and gentamicin. Conclusions: The nucleotide sequence of the integrase gen int/1 is reported for the first time in Colombia in a S. epidermidis strain isolated from a septic neonatal patient, at the neonatal care unit of a third level hospital in Bogotá. This integrase in class one integron was reported, which has shown multiresistance association in clinical isolates in Gram negative bacteria. The resistance mechanism found during this approach realized in Gram negative bacteria showed the evidence of an interspecies horizontal transfer especially by gene transfer or by moving elements such as integrons and gene cassettes.The molecular character of the causing agent of sepsis is important for epidemiological control of the infection and treatment in the newborn.
Descritores: Acetilação
Aminoglicosídeos
Resistência Microbiana a Medicamentos
Integrases
Integrons
Staphylococcus epidermidis
-Sepse
Limites: Seres Humanos
Responsável: CO42.1 - Biblioteca Nacional de Salud José Celestino Mutis


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Id: lil-499172
Autor: Pedersen, Nina; Poulsen, Thomas Tuxen; Poulsen, Hans Skovgaard.
Título: Cre-loxP recombination vectors for promoter studies
Fonte: Electron. j. biotechnol;10(2):315-321, Apr. 15, 2007. ilus, graf.
Idioma: en.
Resumo: For promoter studies the cloning, subcloning and transfer to different plasmid vectors usually requires use of restriction enzymes and ligation reactions. One obstacle is the nucleotide polymorphisms of eukaryotic genomic DNA, which has the consequence that a sequence often differs from published sequences. Therefore sequencing, rigorous restriction enzyme analysis or introduction of suitable sites has to be performed prior to cloning and subcloning. In addition, conventional methods using restriction enzymes, insert purifications and ligations is expensive and labour demanding. We have developed a fast, efficient and inexpensive Cre recombinase-loxP based method, which allows cloning of promoter regions and subcloning of these into a variety of vectors in a restriction enzyme independent manner. We here demonstrate that expression of a number of reporter genes and a therapeutic gene from both a viral and 2 mammalian promoters cloned by this recombinase method have activities comparable to conventionally cloned plasmids.
Descritores: Clonagem Molecular/métodos
Genes Reporter
Vetores Genéticos
Integrases/genética
Terapia Genética/métodos
-Linhagem Celular
Enzimas de Restrição do DNA
Integrases/metabolismo
Neoplasias/genética
Neoplasias/terapia
Regiões Promotoras Genéticas
Recombinação Genética
Transfecção
Limites: Seres Humanos
Animais
Responsável: CL1.1 - Biblioteca Central


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Id: lil-491551
Autor: Jianmin, W; Ruihua, S; Lei, C; Liangjun, Y; Bo, C; Jing, S; Yuanbing, G; Ling, Z; Lin, C.
Título: Construction of engineered murine embryonic stem cells with conditional knockout of FGFR2 depending on Cre-loxP
Fonte: Biocell;30(2):269-278, ago. 2006. ilus.
Idioma: en.
Resumo: OBJECTIVE: To investigate the functions of Fibroblast Growth Factor Receptor-2 (FGFR2) at different stages of cell differentiation. The engineered murine embryonic stem (ES) cells with conditional knockout of FGFR2 were developed depending on Cre-loxP. METHODS: Cre-loxP system was used in a conditional targeting vector. The competent AM-1 bacteria, which expressed Cre-recombinase, was used to confirm the Cre-mediated deletion of the floxed exons 7 and 8 of FGFR2. The targeting vector was electroporated into the ES cells, and the transfected ES cells were screened with G418 and Ganciclovir. Finally, the ES clones with correct targeting events were identified by Southern Blot and PCR. RESULTS: The targeting vector with conditional knockout of murine FGFR2 was successfully constructed andconfirmed by PCR and digesti on analysis in bacteria. 86 ES clones were collected by selective culture with G418 and Ganciclovir. Four of the 86 ES clones were found containing the targeting gene sequence in genomic DNA proved by Southern Blot with a 5'-end flank probe. Two of the four ES clones had the correct targeting events that included the insertion of the targeting gene sequence in genomic DNA and were checked by Southern Blot with a 3'-end flanking probe. Finally, the insertion of loxP (loxP3) between exons 8 and 9 in genomic DNA was identified in one of the two ES clones by Southern Blot and PCR.CONCLUSION: FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.
Descritores: Células-Tronco Embrionárias/metabolismo
Estruturas Embrionárias/citologia
Marcação de Genes
Genoma/genética
Vetores Genéticos/genética
-Sequência de Bases
Integrases/genética
Integrases/metabolismo
Dados de Sequência Molecular
Recombinação Genética
Mapeamento por Restrição
Análise de Sequência de DNA
Limites: Animais
Camundongos
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: AR40.1 - Biblioteca de la Facultad de Ciencias Médicas de la UNCuyo


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Id: lil-490634
Autor: Vera, Jorge; Valenzuela, Beatriz; Roth, Mónica J; León, Óscar.
Título: Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking
Fonte: Biol. Res;41(1):69-80, 2008. ilus, graf.
Idioma: en.
Projeto: Fondecyt; . National Institutes of Health.
Resumo: Processing of viral DNA by retroviral integrase leaves a dinucleotide single-strand overhang in the unprocessed strand. Previous studies have stressed the importance of the 5' single-stranded (ss) tail in the integration process. To characterize the ss-tail binding site on M-MuLV integrase, we carried out crosslinking studies utilizing a disintegration substrate that mimics the covalent intermediate formed during integration. This substrate carried reactive groups at the 5' ss tail. A bromoacetyl derivative with a side chain of 6 A was crosslinked to the mutant IN 106-404, which lacks the N-terminal domain, yielding a crosslinked complex of 50 kDa. Treatment of IN 106-404 with N-ethylmaleimide (NEM) prevented crosslinking, suggesting that Cys209 was involved in the reaction. The reactivity of Cys209 was confirmed by crosslinking of a more specific derivative carrying maleimide groups that spans 8A approximately. In contrast, WT IN was not reactive, suggesting that the N-terminal domain modifies the reactivity of the Cys209 or the positioning of the crosslinker side chain. A similar oligonucleotide-carrying iodouridine at the 5'ss tail reacted with both IN 106-404 and WT IN upon UV irradiation. This reaction was also prevented by NEM, suggesting that the ss-tail positions near a peptide region that includes Cys209.
Descritores: DNA Viral/química
Integrases/genética
Vírus da Leucemia Murina de Moloney/enzimologia
Sequências Repetidas Terminais/genética
Integração Viral
-Sequência de Aminoácidos
Sequência de Bases
Sítios de Ligação/genética
Reagentes para Ligações Cruzadas
Cisteína
Integrases/química
Vírus da Leucemia Murina de Moloney/genética
Oligonucleotídeos/genética
Oligonucleotídeos/metabolismo
Limites: Animais
Tipo de Publ: Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Responsável: BR1.1 - BIREME



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BIREME/OPAS/OMS - Centro Latino-Americano e do Caribe de Informação em Ciências da Saúde