This proposal describes an approach to exploring novel mechanisms of gene regulation in Leishmania, a Trypanosomatid protozoan and one of the most ancient of eukaryotic lineages. A number of lines of evidence suggest that transcription initiation may not be the major regulatory point for gene expression in Leishmania. An attractive alternative model might be that gene expression is modulated by regulation of trans-splicing. The maturation of Leishmania nuclear mRNA proceeds via an obligatory trans-splicing event in which a trimethyl-G capped miniexon is ligated to splice acceptor sites located close to the 5' end of each coding sequence in a polycistronic precursor RNA. Therefore, splice acceptor sites rather than promoters determine the 5' end of mature mRNAs. Trans-splicing would be an ideal point for the regulation of genes whose expression is restricted to one stage of the Leishmania lifecycle. Genes encoding promastigote-specific components of Leishmania flagella will be isolated. Promastigote specific genes will be characterized by sequence analysis and genomic clones will be isolated. Cis elements responsible for promastigote specific expression of flagellar genes will be identified and their mode of action determined. Sequences responsible for the stage-specific pattern of expression will be identified by transfecting chimeric constructs in which potential regulatory sequences flanking isolated stage-specific genes are appended to a reporter gene into both amastigote-like and promastigote forms. Mutant parasite lines lacking individual flagellar will be constructed to test for the functional importance of cloned genes. Analysis of mutant parasite lines will be coupled with ultrastructural and fluorescence microscopic examination to develop an understanding of the function of deleted genes. %%% Leishmania has two distinct stages in its life cycle with different hosts and has evolved a morphologically distinct form specialized for survival in each. The amastigote form is optimized for growth in the lysosomal compartment of mammalian macrophage and the promastigote form multiplies extracellularly in the gut of the sandfly vector. The unique properties of each stage must reflect the expression of stage-specific genes but as yet, little is known about how expression of such genes might be regulated. The results of this study should give greater insight into such stage-specific regulation of gene expression.
