Mestrado em Ciências Biológicas (ICB)
URI Permanente para esta coleção
Navegar
Navegando Mestrado em Ciências Biológicas (ICB) por Por Orientador "BATAUS, Luiz Artur Mendes"
Agora exibindo 1 - 2 de 2
Resultados por página
Opções de Ordenação
Item Caracterização Molecular Do Plasmídeo pLK39 Extraído De Uma Bactéria Endofítica Isolada De Solanum Lycocarpum(Universidade Federal de Goiás, 2002-02-28) OLIVEIRA, Vera Lúcia Cardoso de; BATAUS, Luiz Artur Mendes; http://lattes.cnpq.br/5637230378599476The characterization of the plasmid pLK39.was the aim of work.pLK39 was isolated from a endophytic Gram-negative, bactéria isolated from leaves form Solanum lycocarpum (Lobeira). In order to obtain a selection marker which would characterization in Escherichia coli the kanamycin, resistence gene from pUC4K. was subcloned into the PstIsite of pLK39. The characterization of PLK39 was basedon studies of stability, incompatibility, determination of the copy number and through DNA sequencing. The stability was carried out in Escherichia coli XL10 cells. Cells transformed with pLK39 were inoculated in Lúria broth containing Kanamycin (50μg/ml) during24 hours. After 24 hours of incubation one sample of the culture was in medim with and without selective pressure, and inoculated in Lúria broth without pressure médium, for 240 generations. After 240 generations 81,5% of cells maintained the plasmid, showing that pLK39is highly stable in Escherichia coli to make the incompatibility test, cells of Escherichia coli XL10 were co-transformed with pLK39/pUC18; pBR322 and pLK39/pACYC184. After 240 generations, the plasmid pLK39 iof detected in all systems used, showing the compatibility of pLK39 with the plasmids tested. The copý number pLK39 was estimated by the intensity of plasmidial bands in agarose gel, electrophoresis using a photodocumentation and analysis system (KODAK EDAS). The copý number of pLK39 was estimated as 25 copies per cell. PLK39 was digested with Sau3AI restriction and subcloned into the BamHI site of plasmid pUC18. The recombinant clones were sequenced and 1637 pb reading fragment was obtained. This sequence showed high homology with pSW200, pSW100, pEC3, pUCD5000 and pBERT, which were isolated from Gram-negative bacteria. This sequence possesses two possible genes. The ORF I showed high homology with mobB gene from plasmid pSW200 (96%), pSW100 (96%), pEC3 (94%), pUCD5000 (94%) and pBERT (87%). The ORF II showed homology with mobD gene from plasmids pSW200 (92%), pSW100 (90%), pEC3 (90%) and PUCD5000 (89%).Item Sequência completa e caracterização do plasmídeo crípico pVCM1 isolado de Salmonella enterica(Universidade Federal de Goiás, 2009-03-26) PENIDO, Ana Flávia Batista; BATAUS, Luiz Artur Mendes; http://lattes.cnpq.br/5637230378599476Samonella spp are Gram negatives bactérias belonging to Enterobacteriaceae family. S. enterica comprise about 2.500 sorovars. These sorovars can infect a broad range, including poultry, cattle, swins and humans, and are agent causative of salmonellosis an important public health issue worldwide. Small multicopy plasmids are frequently isolated from Gram negatives and Gram positives bacterias. In Salmonella, low molecular weight plasmids are found on 10% of Salmonella strains and their biological functions are unknown. However, many plasmids in Salmonella control important properties, such as, virulence factors, heavy metals and antibiotics resistance, and utilizations of alternative carbon sources. The pVCM1 plasmid was extracted from one strain of Samonella enteretidis isolated from broilers carcass. The strains were grown in liquid or solid Luria-Bertani broth at 37 °C. The plasmid was purified, separated on 1% agarose electrophoresis and visualized by ethidium bromide staining for analysis. Plasmid was digested with EcoRI enzyme and subcloned in the pUC18 vector.The plasmidal stability was evaluated, inoculating E. coli cells transformated with pVCM1 plasmid (cloned in pUC18) in liquid Luria-bertani broth supplemented with ampicillin. The pVCM1 was stable after 240 generations. The total DNA sequence of plasmid pVCM1 has 1981 pb. Genbank search resulted that pVCM1 showed 99% of identity with pB and 92% with pJ, which were isolated from Salmonella enteretidis. Only one ORF founded in pVCM1 showed significative similarity with others proteins of GenBank. The protein encoded by this ORF showed homology to Rep proteins of plasmids that replicates by rolling-circle mechanism. The pVCM1 posses three impotents elements: rep gene, single strand origin (SSO) and inverted repeat sequences. Such elements are importants for the rolling circle replication, suggesting that pVCM1 use this mechanism. The rep gene was amplified and cloned in the pGEMT-easy vector, but the heterologous expression of Rep protein wasn t gotten successfully.