Diversity of aspartate carbamoyltransferase genes of Trypanosoma cruzi.

The pyrimidine-biosynthetic (pyr) gene cluster, a tandem array of pyr1-pyr3-pyr6/5-pyr2(ACT)-pyr4 from the 5' terminus, encodes all the six enzymes of de novo pyrimidine biosynthesis and occurs as a polycistronic transcription unit in Trypanosoma cruzi. The gene encoding aspartate carbamoyltransferase (ACT), the second enzyme of the pathway, was characterised using a laboratory-reared Tulahuen strain and Tulahuen-derived clones of T. cruzi. Three loci with different restriction maps that contain ACT1, ACT2, and ACT3 were identified. ACT1 and ACT2 are involved in the pyr gene cluster on two different chromosomal DNA molecules of 1,000 and 800 kb, respectively, whereas ACT3 is linked with pyr4 alone. There are 29 nucleotide substitutions out of 981 positions in these three ACTs, yielding 13 amino acid replacements, and a deletion of triplet nucleotides in ACT1 entails a lack of single amino acid residue. Transcription of the three ACTs takes place in the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. Pulsed field gel electrophoresis and Southern blot analyses demonstrated that the cloned T. cruzi stocks, Y-02, CAN III/1, Sylvio-X10/4, and possibly Esmeraldo/3, also possess two complete sets of the pyr gene cluster including ACT, accompanied by additional incomplete clusters. These results suggest that the marked intra-species diversity in the copy number and chromosomal localisations of ACT and other pyr genes may have resulted from partial duplications and subsequent translocations of the polycistronic pyr gene cluster.