Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Glicoproteínas , Nifurtimox/farmacología , Nitroimidazoles/farmacología , Proteínas Protozoarias , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Enfermedad de Chagas/parasitología , Resistencia a Medicamentos , Amplificación de Genes , Humanos , Cariotipificación , Ratones , Pruebas de Sensibilidad Parasitaria , Polimorfismo de Longitud del Fragmento de Restricción , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMEN
The iron-containing superoxide dismutase (FeSOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) enzymes constitutively expressed by the strictly anaerobic bacterium Desulfovibrio gigas were purified and characterized. The FeSOD, isolated as a homodimer of 22-kDa subunits, has a specific activity of 1,900 U/mg and exhibits an electron paramagnetic resonance (EPR) spectrum characteristic of high-spin ferric iron in a rhombically distorted ligand field. Like other FeSODs from different organisms, D. gigas FeSOD is sensitive to H(2)O(2) and azide but not to cyanide. The N-terminal amino acid sequence shows a high degree of homology with other SODs from different sources. On the other hand, D. gigas catalase has an estimated molecular mass of 186 +/- 8 kDa, consisting of three subunits of 61 kDa, and shows no peroxidase activity. This enzyme is very sensitive to H(2)O(2) and cyanide and only slightly sensitive to sulfide. The native enzyme contains one heme per molecule and exhibits a characteristic high-spin ferric-heme EPR spectrum (g(y,x) = 6.4, 5.4); it has a specific activity of 4,200 U/mg, which is unusually low for this class of enzyme. The importance of these two enzymes in the context of oxygen utilization by this anaerobic organism is discussed.
Asunto(s)
Catalasa/aislamiento & purificación , Desulfovibrio/enzimología , Superóxido Dismutasa/aislamiento & purificación , Secuencia de Aminoácidos , Aminoácidos/análisis , Anaerobiosis , Desulfovibrio/crecimiento & desarrollo , Electroforesis en Gel de Poliacrilamida , Magnetismo , Datos de Secuencia MolecularRESUMEN
The ribosomal RNA (rRNA) gene promoter was used to construct plasmid vectors that simultaneously express multiple exogenous genes in Trypanosoma cruzi. Vector pBSPANEO expresses neomycin phosphotransferase, and pPAGFPAN expresses both green fluorescent protein and neomycin phosphotransferase from a single promoter. Both vectors require the presence of the rRNA promoter for stable transfection; epimastigotes transfected with pPAGFPAN strongly fluoresced due to green fluorescent protein expression. Intact plasmids were rescued from the T. cruzi-transfected population after >8 mo of culture, indicating stable replication of these vectors. Vectors were integrated into the rRNA locus by homologous recombination and into other loci, presumably by illegitimate recombination. Parasites bearing tandem concatamers of plasmids were also found among the transfectants. Transfectants expressing green fluorescent protein showed a bright green fluorescence distributed throughout the cell. Fluorescence was also detected in amastigotes after infection of mammalian cells with transfected parasites, indicating that the rRNA promoter can drive efficient expression of these reporter genes in multiple life-cycle stages of the parasite. Expression of the heterologous genes was detected after passage in mice or in the insect vector. These vectors will be useful for the genetic dissection of T. cruzi biology and pathogenesis.
Asunto(s)
Regulación de la Expresión Génica , Kanamicina Quinasa/genética , Proteínas Luminiscentes/genética , Trypanosoma cruzi/genética , Animales , Southern Blotting , ADN Protozoario/genética , Electroforesis en Gel de Campo Pulsado , Vectores Genéticos , Proteínas Fluorescentes Verdes , Insectos Vectores/parasitología , Kanamicina Quinasa/metabolismo , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Plásmidos , Regiones Promotoras Genéticas/genética , ARN Ribosómico/genética , Pase Seriado , Transfección , Triatominae/parasitología , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/metabolismoRESUMEN
We have dissected the topoisomerase II gene of members of the two recently characterized subgroups of Trypanosoma cruzi to obtain further evidence to support this dichotomy of isolates in this important parasite. Pulsed field gel electrophoresis showed a striking heterogeneity in the molecular karyotypes of the strains analyzed. Southern analysis of these chromosome gels also showed heterogeneity in the size and number of chromosomes containing the topoisomerase II gene. Analysis of DNA restriction fragment length polymorphisms of the topoisomerase II gene also showed two principal patterns consistent with the two previously characterized groups. Finally, the sequences of portions of the topoisomerase II genes from members of the T. cruzi groups showed two distinct patterns, again consistent with the previous grouping of this parasite. Thus, this work clearly supports previous observations suggesting an ancient divergence of known T. cruzi isolates into two main branches.