Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
Navegar
4 resultados
Resultados da Pesquisa
Item Integração de pAN7- 1 no genoma de Magnaporthe grisea mediada por enzima de restrição(Fitopatologia Brasileira, 2006-05) Marchi, Carlos E.; Brommonschenkel, Sérgio H.; Queiroz, Marisa V. de; Mizubuti, Eduardo S. G.Visando explorar a mutagênese insercional em Magnaporthe grisea, foram avaliadas a transformação dos protoplastos obtidos após adequação do protocolo e a eficiência da integração de pAN7-1 no genoma do ascomiceto na presença da enzima de restrição Hind III. Os protoplastos de M. grisea I-22 foram prontamente transformados para a resistência à higromicina. Quando o vetor linearizado com Hind III foi usado para transformar o fungo na presença de Hind III, a eficiência de transformação foi 1,1 a 8,1 vezes superior ao tratamento sem a adição da enzima. No geral, a melhor concentração de Hind III foi 5 unidades/reação de transformação. Tal concentração promoveu a produção média de 332 transformantes/µg de pAN7-1/107 protoplastos. A presença do gene de seleção hph no genoma de 18 indivíduos resistentes à higromicina foi confirmada por PCR.Item Electrophoretic characterization ofaspergillus nidulans strains with chromosomal duplications(Genetics and Molecular Biology, 2000-06) Queiroz, Marisa V. de; Pizzirani-Kleiner, Aline Aparecida; Azevedo, João LúcioPulsed-field gel electrophoresis was used to characterize strains ofAspergillus nidulans with a chromosomal duplication Dp(I-II). Morphologically deteriorated and improved variants of these strains were also analyzed. The electrophoretic karyotype demonstrated that in two duplicated strains (A and B) the 4.2 Mb band, which corresponds to chromosome II, was absent and a new band was observed. Hybridization studies using the uapA (chromosome I) and wA (chromosome II) genes demonstrated that the new band corresponded to chromosome II plus the duplicated segment of chromosome I. The size of the chromosomal duplication was approximately 1.0 Mb. Analysis of the chromosomal bands of a morphologically improved strain showed that the duplicated segment of chromosome I was completely lost. The morphologically deteriorated variants V9 and V17 had the same karyotype as the duplicated strains. However, the deteriorated variant V5 lost part of chromosome I and had a rearrangement involving chromosome V. This rearrangement may have resulted from the mutagenic treatment used to obtain the genetic markers. Pulsed-field gel electrophoresis was found to be an excellent tool for locating chromosomal rearrangements.Item Molecular characterization and expression profile of pectin-lyase-encoding genes from Penicillium griseoroseum(Canadian Journal of Microbiology, 2006) Bazzolli, Denise S.; Ribon, Andrea O. B.; Queiroz, Marisa V. de; Araujo, Elza F. dePenicillium griseoroseum has been studied by our group because of its good pectinase production. Attempts have been done to clone pectinolytic genes, aiming to obtain pectinase-overproducing strains for industrial purposes. Here, two genes coding for pectin lyase were isolated from the P. griseoroseum genome. The plg1 gene has an open reading frame of 1341 bp coding for a putative protein of 374 amino acids with a calculated molecular mass of 40.1 kDa. The plg2 gene is characterized by an open reading frame of 1400 nucleotides and codes for a polypeptide of 383 amino acids. The plg1 gene 5'-flanking region contains putative binding sites for the transcription factors involved in regulation by ambient pH and catabolite repression. The primary structure of Plg1 and Plg2 proteins showed a relatively high homology (varying between 32.4% and 74.8%) to fungal pectin lyases characterized to date. Southern blotting analysis revealed that both genes are present as single copies in the fungus genome. Expression studies revealed a differing pattern of gene expression of plg1 and plg2 when mycelium was cultivated on medium containing different pectic components. Citric pectin followed by apple pectin were the carbon sources that best induced plg1 expression, and transcripts were detected from 24 to 76 h. The expression of the plg2 gene was monitored by reverse transcriptase--polymerase chain reaction, since Northern analysis failed to detect hybridization signals. The differential expression of these genes may provide means for the fungus to adapt to various growth conditions.Item Easy detection of green fluorescent protein multicopy transformants in Penicillium griseoroseum(Genetics and Molecular Research, 2004-12-21) Lopes, Francis J.F.; Araújo, Elza F. de; Queiroz, Marisa V. dePenicillium griseoroseum, a deuteromycete fungus producer of pectinolytic enzymes, was transformed with a gene encoding for green fluorescent protein (GFP). The selection of transformants was based on the homologous nitrate reductase gene (niaD). Protoplasts of a P. griseoroseum Nia mutant (PG63) were co-transformed with the plasmids pNPG1 and pAN52-1-GFP. The plasmid pNPG-1 carries the homologous niaD gene and pAN52-1-GFP carries the SGFP-TYG version of GFP. The highest transformation efficiency (102 transformants/µg of pNPG1) resulted from the utilization of equimolar amounts of transforming and co-transforming vectors. Analysis of pAN52-1-GFP insertions into the genomic DNA of the transformants revealed single and multiple copy integrations. The transformants possessing a single copy of the gfp gene showed a low level of fluorescence, whereas multicopy transformants displayed strong fluorescence under visualization with fluorescent light. The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.