RESUMO
A cDNA clone derived from the Trypanosoma cruzi alpha-tubulin gene was isolated and sequenced (Tc alpha tub; L37345). Tc alpha tub revealed an 87.79 percent and an 85.36 percent identity with the DNA sequence of T. brucei and Leishmania, respectively. This clone was used to study, by Northern blots, alpha-tubulin gene expression in epimastigotes, cell-cultured derived trypomastigotes and extracellular amastigotes. alpha-tubulin MRNA levels were the same in epimastigotes and trypomastigotes, however, there was a drastic decrease in amastigotes. This clone could be useful to elucidate the regulatory mechanisms of alpha-tubulin gene expression during the differentiation of T. cruzi
Assuntos
Animais , Clonagem Molecular , DNA Complementar/isolamento & purificação , Expressão Gênica/genética , Nucleotídeos/genética , RNA Mensageiro/análise , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Trypanosoma cruzi/genética , Tubulina (Proteína)/genéticaRESUMO
Eleven antigenic clones were isolated from a Trypanosoma cruzi cDNA expression library using a pool of Chagasic sera from Venezuelan patients. These clones were tested with 26 Chagasic sera and 11 sera from patients with other diseases. One clone (Clone 2) reacted with the majority of the Chagasic sera and did not react with the non-Chagasic sera. Restriction enzyme digestion of Clone 2 DNA showed that it contained an insert of 5.2 kb. In a Southern blot of a pulse field gel electrophoresis, Clone 2 hybridized to three T. cruzi chromosomal fragments. By Northern blot analyses, it was observed that the clone hybridizes to a major T. cruzi RNA band of 0.97 kb and to two minor bands of 4.8 and 6.3 kb. The expression of these three RNAs was higher in trypomastigotes than in epimastigotes or spheromastigotes. Specific antibodies isolated against the beta-galactosidase-Clone 2 fusion protein expressed in E. coli reacted with a 28 kilodalton protein in T. cruzi