Fragoso S P, Mattei D, Hines J C, Ray D, Goldenberg S
Instituto Oswaldo Cruz, Department of Bioquímica e Biologia Molecular, Rio de Janeiro, RJ, Brazil.
Mol Biochem Parasitol. 1998 Aug 1;94(2):197-204. doi: 10.1016/s0166-6851(98)00066-8.
Topoisomerases are enzymes that participate in many cellular functions involving topological manipulation of DNA strands. There are two types of topoisomerases in the cell: (a) type I topoisomerases; and (b) type II topoisomerases (topo II). Previously we have cloned and sequenced the gene encoding Trypanosoma cruzi topo II (TcTOP2). This study group has raised an antiserum against recombinant type II DNA topoisomerase (TctopoII) to study the expression of this gene during T. cruzi differentiation and to determine the cellular location of the enzyme. Western blot analysis showed that T. cruzi TctopoII is expressed in the replicative epimastigotes but not in the infective and non-replicative trypomastigotes. However, slot blot analysis of RNAs extracted from epimastigotes and metacyclic trypomastigotes showed that the mRNA encoding the enzyme is present in both developmental stages of the parasite. Confocal laser microscopy using the antiserum raised against recombinant TctopoII showed that the enzyme is located exclusively in the nucleus of the parasite. Similar results were obtained by immunofluorescence analysis of Crithidia fasciculata. However, monoclonal antisera against the corresponding enzyme extracted from C. fasciculata recognizes a kinetoplast protein in both T. cruzi and Crithidia.
拓扑异构酶是参与许多涉及DNA链拓扑操作的细胞功能的酶。细胞中有两种类型的拓扑异构酶：（a）I型拓扑异构酶；和（b）II型拓扑异构酶（topo II）。此前我们已克隆并测序了编码克氏锥虫topo II（TcTOP2）的基因。该研究小组制备了一种针对重组II型DNA拓扑异构酶（TctopoII）的抗血清，以研究该基因在克氏锥虫分化过程中的表达，并确定该酶的细胞定位。蛋白质印迹分析表明，克氏锥虫TctopoII在增殖性无鞭毛体中表达，但在感染性和非增殖性锥鞭毛体中不表达。然而，对从无鞭毛体和循环后期锥鞭毛体中提取的RNA进行斑点印迹分析表明，编码该酶的mRNA在寄生虫的两个发育阶段均存在。使用针对重组TctopoII制备的抗血清进行共聚焦激光显微镜检查表明，该酶仅位于寄生虫的细胞核中。通过对 fasciculata进行免疫荧光分析也获得了类似的结果。然而，针对从 fasciculata中提取的相应酶的单克隆抗血清在克氏锥虫和 fasciculata中均识别一种动质体蛋白。
