Molecular characterization of CPS II and ACT genes form Trypanosoma cruzi

本研究的目的是研究锥虫CRUZI基因编码氨甲酰磷酸合成酶II(CPS II)和天冬氨酸氨基甲酰基转移酶(ACT)这两个从头合成嘧啶核苷酸的第一和第二酶。从头合成嘧啶是大多数生物体所必需的，最初的三种酶，CPS II，ACT和Hihyorotase，在包括哺乳动物在内的高等真核生物中以多功能蛋白CAD的形式表达。另一方面，我们证明，在克鲁兹锥虫中，CPS II和ACT基因在其基因组中独立编码，并同时定位在两条不同的染色体上。到目前为止，还没有关于锥虫基因组中密切相关基因的分布以及这些基因的转录系统的报道。从这些观点出发，我们研究了克鲁兹毛滴虫CPS II和ACT基因的基因组结构。用CPS II和ACT特异的探针筛选该寄生虫的基因组文库，并对获得的阳性克隆进行比对，以检测它们之间的重叠区域。结果表明，CPS II基因位于ACT基因上游10kb处。此外，我们还获得了这些基因的两个不同的限制性内切酶图谱，分别对应于每个等位基因，反映了锥虫基因组的可塑性。有趣的是，我们还发现了与CPSII无关的ACT基因，这表明在克鲁兹毛滴虫中存在ACT的同源基因(S)或假基因(S)。

The purpose of this study was to characterize the Trypanosoma cruzi genes encoding carbamoyl-phosphate synthetase II (CPS II) and aspartate carbamoyltransferase (ACT), the first and second enzymes of de novo pyrimidine nucleotide biosynthesis. De novo pyrimidine synthesis is essential in most of organisms and the initial three enzymes, CPS II,ACT,and hihydroorotase, are expressed as a multi-functional protein, CAD,in higher eukaryotes including mammals. On the other hand, we demonstrated that, in Trypanosoma cruzi, CPS II and ACT genes were encoded independently in its genome and localized simultaneously on two different chromosomes. As far as we know, there has been no report about a distribution of closely related genes and a transcriptional system of such genes in trypanosomal genome. From these views, we have examined the genomic structure of the CPS II and ACT genes in T.cruzi. A genomic library from the parasite was screened by using CPS II- and ACT-specific probes, and positive clones obtained were aligned so as to detect an overlapping region between them. The results showed that the CPS II gene was located at 10 kb upstream of the ACT gene. In addition, we have obtained two different restriction maps of these genes, corresponding to each allele and reflecting the plasticity of trypanosome genome. Interestingly, we have also found the ACT genes which are not linked to CPS II,suggesting the existence of homologue(s) or pseudogene(s) of ACT in T.cruzi.