The splice leader addition domain represents an essential conserved motif for heterologous gene expression in B. malayi

摘要

Two promoters from the human filarial parasite Brugia malayi have been mapped in detail. The essential domains of both promoters lacked canonical eukaryotic core promoter motifs. However, the largest contiguous essential domain in both promoters flanked and included the splice leader addition site. These findings suggested that the region flanking the trans-splicing addition site might represent a conserved core domain in B. malayi promoters. To test this hypothesis, the putative promoters of 12 trans-spliced genes encoding ribosomal protein homologues from B. malayi were isolated and tested for activity in a B. malayi transient transfection system. Of the 12 domains examined, 11 produced detectable reporter gene activity. Mutant constructs of the six most active promoters were prepared in which the spliced leader acceptor site and the10 nt upstream and downstream of the site were deleted. All deletion constructs exhibited >90% reduction in reporter gene activity relative to their respective wild type sequences. A conserved pyrimidine-rich tract was located directly upstream from the spliced leader splice acceptor site which contained a conserved T residue located at position −3. Mutation of the entire polypyrimidine tract or the conserved T individually resulted in the loss of over 90% of reporter gene activity. In contrast, mutation of the splice acceptor site did not significantly reduce promoter activity. These data suggest that the region surrounding the splice acceptor site in the ribosomal promoters represents a conserved essential domain which functions independently of splice leader addition.