RESUMO
A relatively rapid and efficient method for the extraction of chromosomal DNA from Mycobacterium paratuberculosis and other mycobacteria was developed. Approximately 25 to 50 micrograms of DNA could be extracted from 100 mg (wet weight) of cells, which was sufficient to perform several restriction endonuclease analyses from a single preparation. The DNA from five Mycobacterium species, including four strains of M. paratuberculosis and four strains of M. avium, was analyzed by this method. Digestion with the restriction endonucleases BstEII and PstI yielded the most definitive restriction patterns. For some strains, the restriction endonuclease analysis results were in agreement with the current identification of these organisms. The two strains of M. avium serotype 2 had identical fragment patterns. Similarly, the two strains of M. avium complex serotype 6 had identical fragment patterns. The three mycobactin-dependent M. paratuberculosis strains were very similar, whereas the mycobactin-independent M. paratuberculosis strain was more similar to the M. avium serotype 2 strains. Although many more cultures would need to be evaluated to determine correct groupings, the results of this study demonstrated the potential of restriction enzyme analysis for the differentiation of slowly growing mycobacteria.
Assuntos
DNA Bacteriano/análise , Mycobacterium avium/genética , Mycobacterium/genética , Animais , Cromossomos Bacterianos , Enzimas de Restrição do DNA , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Ágar , Micobactérias não Tuberculosas/genética , Paratuberculose/microbiologiaRESUMO
One- and two-dimensional gel electrophoresis of the solubilized mitochondrial proteins of bloodstream and procyclic trypomastigote Trypanosoma brucei rhodesiense and radiolabeling of proteins in the presence of cycloheximide were used to identify proteins synthesized in the trypanosome mitochondrion. The proteins which comprise the mitochondrion were found to be very similar in both bloodstream and procyclic trypomastigotes, but do differ in their level of synthesis. A protein putatively identified as subunit II of cytochrome oxidase (EC 1.9.3.1) was detected in mitochondria from both the procyclic and bloodstream organisms. The presence of this protein in bloodstream trypomastigotes and the overall similarity of protein content in the trypanosome mitochondria is noteworthy in view of the fact that bloodstream trypomastigotes have a repressed mitochondrion with no detectable tricarboxylic acid cycle or cytochrome electron transport chain.
Assuntos
Mitocôndrias/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Anticorpos Monoclonais , Cicloeximida/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel de Poliacrilamida , Mitocôndrias/efeitos dos fármacos , Peso Molecular , Proteínas/isolamento & purificação , Proteínas/metabolismo , Ratos , Ratos Endogâmicos , Trypanosoma brucei brucei/fisiologiaAssuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Antimicina A/farmacologia , Citocromos/metabolismo , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Glicerolfosfato Desidrogenase/antagonistas & inibidores , Cianeto de Potássio/farmacologia , Salicilamidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Hybridization studies of chromosomal DNA from leptospiral strains representing Leptospira interrogans, serogroups Sejroe and Pomona from cattle and swine were performed to determine the degree of homology among their DNA sequences. Chromosomal DNA isolated from leptospires of the Sejroe and Pomona serogroups was radiolabeled and used to probe DNA isolated from other strains in these serogroups. Serovars hardjo (hardjoprajitno), hardjo (hardjo-bovis), balcanica (1627 Burgas), pomona (pomona), and kennewicki (LT-1026) were probed to determine the degree of homology among their chromosomes. Serovars pomona and kennewicki were homologous to each other. They also had a high degree of homology with hardjo (hardjoprajitno) and, to a lesser extent, with hardjo (hardjo-bovis) strains. However, hardjoprajitno and hardjo-bovis had little homology to each other. Serovar balcanica had a high degree of homology with hardjo-bovis isolates, but little homology with hardjoprajitno. Although serologically indistinguishable, the reference strain hardjoprajitno was genetically dissimilar to hardjo-bovis strains isolated from North American cattle.
Assuntos
Doenças dos Bovinos/microbiologia , DNA Bacteriano/genética , Leptospira interrogans/genética , Doenças dos Suínos/microbiologia , Doença de Weil/veterinária , Animais , Bovinos , DNA Bacteriano/isolamento & purificação , Leptospira interrogans/classificação , Hibridização de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Sorotipagem , Especificidade da Espécie , Suínos , Doença de Weil/microbiologiaRESUMO
We have developed a rapid method for the isolation of leptospiral chromosomal DNA which yields DNA of a purity suitable for restriction endonuclease analysis. A small volume (15 to 20 ml) of an exponentially growing culture of leptospires yielded 2 to 4 micrograms of chromosomal DNA. In a 1-day protocol, the DNA was isolated, restricted with endonucleases, and fractionated on an agarose gel. Chromosomal DNA from dinger zones (visible subsurface zones of leptospiral growth) of first semisolid subcultures of field isolates was also isolated and characterized, thus greatly speeding up the diagnostic process.
