Characterization of the unusual transcription machinery in trypanosomes may lead to new chemotherapeutic approaches as well as provide tools for the development of genetic systems to dissect the biology and pathogenesis of these parasites. We have begun to characterize apparent promoters for the T. cruzi SL RNA gene and rRNA genes. Both drive expression of exogenous genes in transfected plasmids, although the rRNA promoter is significantly stronger than the SL RNA gene promoter. We have identified critical cis-acting elements in both promoters, and demonstrated that each binds a specific factor(s) in T. cruzi nuclear extracts.
The specific aims of this proposal are: 1. To clone genes encoding transcription factors that bind cis-acting promoter elements of the T. cruzi SL and rRNA genes using the yeast one- hybrid system, phage display selection, or biochemical strategies. 2. To characterize the interaction of these putative transcription factors with the known promoter elements by DNA footprinting, by the development of in vitro transcription systems, and by functional analysis of protein domains. 3. To confirm and characterize additional apparent cis-acting promoter elements that we have observed in the SL RNA and rRNA transcription promoter regions by site-directed mutagenesis of the relevant sequences. 4. To design these gene promoters into an exogenously regulated T. cruzi gene expression system and use it to express parasite genes and gene specific antisense ribozymes to probe gene function in this parasite. The long term goals of this work are two-fold: 1) to use the information derived from these studies to develop a regulated gene expression system that will permit manipulation of the genetic complement of T. cruzi, and facilitate the investigation of the biology and pathogenesis of this parasite; and 2) to identify features of the T. cruzi transcriptional apparatus that vary sufficiently from analogous mammalian systems to permit development of alternative therapeutic strategies. Although the work proposed herein is critical to these long range objectives, their ultimate realization is probably well beyond the scope of this proposal.
