5' SEQUENCES ESSENTIAL FOR TRANSSPLICING OF MSP (GP63) RNAS IN LEISHMANIA-CHAGASI

The 5' sequences essential for spliced leader (SL) addition to RNA enc oding the abundant Leishmania surface protease gp63 were determined. A DNA segment found upstream of all Leishmania chagasi gp63 genes was c loned in front of a luciferase gene, and luciferase activity was measu red in transiently transfected L. chagasi promastigotes. Two hundred a nd twenty bp of the upstream region was needed for optimal luciferase activity. Deletions and point mutations were placed in this segment to determine which nucleotides participate in the splicing events. A reg ion containing 87% pyrimidines located between 31 and 69 bp upstream o f the splice acceptor dinucleotide and containing three potential bran ch point A residues was highly beneficial for reported gene activity. In contrast, a 30-nt pyrimidine-rich sequence immediately upstream of the splice acceptor site did not by itself enhance luciferase activity . Luciferase activity was associated with the presence of trans-splice d luciferase mRNA. The results suggest that sequences 31-69 bp upstrea m of gp63 genes enhance gene expression through provision of signals f or efficient trans-splicing.