Programa de Pós-graduação em Ciências Biológicas
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Navegando Programa de Pós-graduação em Ciências Biológicas por Por Orientador "BATAUS, Luiz Artur Mendes"
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Item Caracterização do plasmídeo pVCM 04 extraídos de Salmonella enterica isolada de carcaçãs de frangos(Universidade Federal de Goiás, 2010-05-31) CARNEIRO, Lílian Carla; BATAUS, Luiz Artur Mendes; http://lattes.cnpq.br/5637230378599476A small cryptic plasmid isolated from Salmonella enterica Enteritidis called pVCM04 was sequenced and characterizated. pVCM04 is a 3583 pb circle molecule that showed no homology with other plasmids deposited in the GenBank. 12 ORF with more than 50 aminoacids were predicted using the ORF finder program. ORF1 and ORF2 showed homology with replication proteins of different plasmids. ORF 3-5 showed homology with mobilization proteins present in several plasmids; the others seven ORF showed no homology with genes deposited in GenBank. The pVCM04 possess a region with more than 500 pb that is not associated with none of the predicted proteins. This region is organizated in a G+C rich, A+T rich and two repeat direct sequences. The second repeat direct sequence contains a region of DnaA box connection (TTTACAC). This region is probably associated to the replication origin theta type. The phylogenetic relationship among replicase and mobilization deduced protein showed highest similarity of replicase proteins than mobilization proteins. Conjugation experiments showed that the pVCM04/pUC18 fusion not have a good ability to transfer, the plasmid stability test showed that the cells lost 60% of pVCM04/pUC18 on the first day of cultivation. The characterization suggests that the pVCM04 probably would be a cryptic plasmid from fusion of different ancestral plasmid.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.Item Detecção e caracterização molecular de talassemia alfa(Universidade Federal de Goiás, 2009-03-27) PENNA, Karlla Greick Batista Dias; BATAUS, Luiz Artur Mendes; http://lattes.cnpq.br/5637230378599476Alpha thalassemia is a syndrome resulting from disturbances in the synthesis of alpha globin chain that forms the tetramer of the hemoglobin molecule. Alpha thalassemia is classified into four types according to the number of alpha genes affected: silent carrier (-α/αα), alpha thalassemia trait (--/αα or -α/-α) Hemoglobin H disease (-- /-α), and fetalis hydrops (--/--). The decrease in synthesis of alpha globin causes inadequate production of hemoglobin resulting in hypochromic and microcytic anaemia. Also it causes accumulation of beta chains, inside the erythrocytes, resulting in formation of beta chain tetramer of hemoglobin called Hb H. Clinically the individual with thalassemia can be asymptomatic or present severe anemia. Asymptomatic forms of thalassemia, silent carrier and alpha thalassemia trait, are more difficult to diagnose because of the inclusions bodies of Hb H are not always present. In these situations it is necessary to research the molecular characterization of the genotype and confirming the presence of alpha thalassemia. This is mainly because the diagnosis by conventional methods, although important, is limited and imprecise. This study evaluated some of the traditional laboratory methods in the detection of alpha thalassemia and associated molecular characterization of the more prevalent deletion forms α3,7 and α4,2. For confirmation and characterization of alpha thalassemia, new oligonucleotides were designed. By conventional PCR technique, using 3.7F/KGB01 primers it was possible to detect the deletion α3,7, differentiating the normal genotype (αα/αα), the heterozygote (-α3,7/αα), and homozygous (-- α3,7/- α3,7). Although it was designed to detect the deletion α3,7, this primers also identified the deletion α4,2 when in homozigose (-α4,2/- α4,2). The primers KGB04/KGB05 detected the deletion α4,2, but without differentiating between the heterozygous and homozygous genotype. The most prevalent deletion founded was the α4,2 (20.0%) which represents 9.2% in the homozygous form (- α4,2/-α4,2). The deletion α3,7 in the heterozygous form was detected in 12.3% of patients. The data demonstrate that the importance of molecular detection for alpha thalassemia is not limited only to the definition of the genotype, but also confirmation of the presence in patients with abnormal erythrogram values, with regular erythrogram values, with values closed to the boundary values and in neonates.