Potential targets for chemotherapy in microbial infections are the biochemical pathways which are unique to the pathogen. For the African trypanosomes, the obligatory synthesis of all messenger RNAs by dicontinuous transcription represents a target for such a strategy. The long term aim of this research is to analyze gene structure and discontinuous transcription in Trypanosoma brucei. The experiments described here will define protein-DNA interactions in the proximity of the start sites of transcription of a) the mini- exon) (5'-exon) and b) representative protein-coding genes (3'- exons). These two-classes of genes are representative of the two discrete genetic loci involved in discontinuous transcription. Gel- retardation and Exonuclease III-protection assays will be used to identify and characterize specific protein-DNA interactions on a cloned mini-exon gene incubated with crude nuclear extracts from T. brucei. Nuclear extracts enriched by chromatography for the specific DNA-binding proteins will be used in DNA footprinting assays to identify the nucleotides bound by the protein. The transcription initiation sites of active ubiquitin genes and the 117 VSG gene, two apparently different classes of 3'-exon, will be identified by cosmid cloning, the analysis of nascent RNA by nuclear run-on and nuclease-protection assays. Gel retardation assays will be used to characterize proteins binding to the transcription indication sites of these 3'-exons.
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