Molecular characterization of CPS II and ACT genes form Trypanosoma cruzi

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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.