Analyzing expression of the calmodulin and ubiquitin-fusion genes of Trypanosoma cruzi using simultaneous, independent dual gene replacements.

We describe here a strategy for introducing simultaneous, independent gene replacements into the Trypanosoma cruzi chromosome. The goal of this study was to use two linear DNA fragments to simultaneously replace the CalA2 calmodulin and FUS1 ubiquitin-fusion genes with the neomycin resistance (neo(r)) and chloramphenicol acetyltransferase (CAT) genes, respectively. One clone (D6), of thirty G418-resistant clones analyzed, carried the desired dual gene replacement. CDNA sequence analysis indicated that the CAT mRNA was accurately trans-spliced using the previously identified FUS1 mini-exon addition site. However, DNA sequence analysis of the intergenic sequence immediately upstream of the neo(r) gene in clone D6 identified a mutation which altered the pattern of trans-splicing of the neo(r) mRNA. Possible effects of this mutation on 3' splice acceptor site selection are discussed.