Kinetoplastid protozoa such as Leishmania spp., Trypanosoma brucei and Trypanosoma cruzi cause a variety of diseases in humans and livestock. The parasites generate mature messenger RNA by the addition of a common 39-nucleotide sequence (termed the mini-exon) to protein-encoding precursor RNAs in a trans-splicing reaction. Trans-splicing has not been identified in the mammalian hosts of these parasites, therefore the process is parasite-specific and a selective target for anti-parasite attacks. The long term aim of this research is to understand common mechanisms of gene expression in these pathogenic protozoa. Because the mini-exon is synthesized as a short, discrete precursor (termed medRNA) in all these species, the research in this proposal is directed to understanding the synthesis of medRNA (in the model organism Leishmania tarentolae). Essential promoter element(s) of the mini-exon gene will be defined. The upstream region of a cloned mini-exon gene, which has been marked with a 40-bp tag, will be subjected 1) to linker-scanning mutagenesis and 2) point mutation analysis of the -67/-58 region. Mutated genes contained on the stable transfection vector pX will be introduced into L. tarentolae by electroporation. Transcription of the episomal genes will be monitored by Northern blotting, nuclease protection, primer extension and nascent RNA analyses. Mutated genes will also be used as templates, in the gel mobility-shift assay, to define the sites of protein binding. Proteins that bind to the essential -67/-58 region will also be characterized by two complementary methods. DNA-binding protein sequence will be inferred from cDNA clones that have been isolated by virtue of their binding a """"""""-67/58"""""""" double-stranded oligonucleotide. Native protein(s) will also be purified from nuclear extracts by column chromatography. Purification of the protein(s) will be monitored by the gel mobility-shift assay. The proposed experiments will define the essential upstream promoter element of the Leishmania mini-exon gene. It will also characterize potential transcription factors that associate with the essential element of this important, parasite-specific gene.
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