de Marval M G, Souto-Padron T, Gottesdiener K, Silva R, van der Ploeg L H, Rondinelli E
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil.
Biol Res. 1993;26(1-2):313-4.
To identify the members of the HSP70 and HSP60 families of Trypanosoma cruzi, we analysed 35S methionine epimastigote cells by two dimensional Western blot. At 29 degrees C, an HSP70 monoclonal antibody (anti-D. melanogaster) recognized eight isotypes. At least five of these were heat-induced. Polyclonal antibody against the 65 KDa antigen (anti-M. tuberculosis) recognized three isotypes with identical molecular weights, but different microliters. Only one isoform was heat induced. The cellular distribution of HSP70 and HSP60 was studied by immunoelectron microscopy. Anti-HSP70 reactive protein was localized in the cytoplasm, mitochondria and nucleus, while anti-HSP60 protein was found in the mitochondrion and in close association with the kinetoplast. To characterize the HSP60 gene and its proteins, we isolated a genomic T. cruzi clone encoding the HSP60 gene. T. cruzi HSP60 genes could be shown to be organized in 2100 nt tandem arrays. RELP in the HSP60 genes revealed that at least three different types of HSP60 genes were encoded in the T cruzi genome. The predicted open reading frame measured exhibits about 50% identity to other HSP60 described. Expression of these HSP60 genes could not be induced by 2 hours heat shock at 37 degrees C. Post-transcriptional mechanisms may be responsible for HSP60 induction in T. cruzi.
为了鉴定克氏锥虫HSP70和HSP60家族的成员，我们通过二维蛋白质免疫印迹分析了35S甲硫氨酸标记的上鞭毛体期细胞。在29℃时，一种HSP70单克隆抗体（抗黑腹果蝇）识别出8种同型。其中至少5种是热诱导型。针对65 kDa抗原的多克隆抗体（抗结核分枝杆菌）识别出3种分子量相同但微升数不同的同型。只有一种亚型是热诱导型。通过免疫电子显微镜研究了HSP70和HSP60的细胞分布。抗HSP70反应性蛋白定位于细胞质、线粒体和细胞核中，而抗HSP60蛋白则存在于线粒体中并与动基体紧密相连。为了表征HSP60基因及其蛋白，我们分离了一个编码HSP60基因的克氏锥虫基因组克隆。克氏锥虫HSP60基因可显示为以2100 nt串联阵列形式组织。HSP60基因中的RELP显示，克氏锥虫基因组中至少编码了三种不同类型的HSP60基因。预测的开放阅读框与其他已描述的HSP60约有50%的同一性。这些HSP60基因的表达在37℃下经2小时热休克不能被诱导。转录后机制可能是克氏锥虫中HSP60诱导的原因。
