The objective of this proposal is to elucidate the molecular basis of antigenic variation in African trypanosomes, the causative agents of sleeping sickness. DNA sequences and regulatory factors which participate in selective transcription of trypanosome variant surface antigen (VSA) genes during antigenic variation in bloodstream forms and during development between mammalian and insect infective stages will be characterized. Evidence suggests that VSA gene expression requires (1) controlling regions whose location and nature have yet to be determined and (2) a telomere site capable of accepting a duplicated VSA gene copy (ELC) or containing a VSA gene which can be activated without duplication (NDA). The immediate goal is to identify and characterize these genomic regions and locate and sequence the boundaries which define them. (1) The VSA expression site will be defined by an ongoing cycle of genomic and cDNA restriction mapping and cloning of sequences from expressor and non-expressor variant antigen types (VATs) and their procyclic counterparts. DNA sequence analysis of relevant clones will be performed as required. (2) The role of a 35-nucleotide spliced leader sequence (SLS) found 5' to all VSA mRNAs and which may define the 5' boundary of the expression site will be characterized. Analysis of non-VSA cDNA clones containing the SLS in T. brucei and other trypanosomids, will determine the functional basis for conservation of SLS in the trypanosomidae. (3) Sequences which regulate transcription initiation in trypanosomes will be identified for certain genes, including Alpha and Beta tublin. These provide a basis for comparing (a) transcription initiation sequences between VSA and other gene families and (b) consensus sequences beween parasite and host. Regulatory sequences will be used to analyze and develop in vitro and in vivo systems for studying parasite gene expression. (4) In vitro and in vivo system for analyzing putative regulatory sequences in a biologically relevant context will be sought. Neither genetic nor gene transformation systems for parasitic protozoa exist which allow functional analysis of putative regulatory sequences. In vitro transcription system will defeine requirements for faithful in vitro transcription initiation. DNA transformation in trypanosomes will be studied using genetically engineered drug resistance and other selective markers. Unique features of gene regulation will provide insights for developing strategies for disease control and eradication. Where feasible extrapolations of findings to epidemiological and clinical application will be made.
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