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 t his study was to use two linear DNA fragments to simultaneously replac e the CalA2 calmodulin and FUS1 ubiquitin-fusion genes with the neomyc in resistance (neo(r)) and chloramphenicol acetyltransferase (CAT) gen es, respectively. One clone (D6), of thirty G418-resistant clones anal yzed, carried the desired dual gene replacement. CDNA sequence analysi s indicated that the CAT mRNA was accurately trans-spliced using the p reviously identified FUS1 mini-exon addition site. However, DNA sequen ce analysis of the intergenic sequence immediately upstream of the neo (r) gene in clone D6 identified a mutation which altered the pattern o f trans-splicing of the neo(r) mRNA. Possible effects of this mutation on 3' splice acceptor site selection are discussed.