The molecular mechanisms that regulate the expression of trypanosome genes will be studied. We will study the regulation of the expression of variant antigen genes (VAGs) in T. equiperdum, the mechanism of discontinuous transcription and the role of common U terminated sequences on RNA subsets. For the study of VAG expression the primary structure of a cloned genomic fragment containing 12Kb of DNA located 5' to the duplicated transposed active VAG will be determined and compared with the structure of the same fragment cloned from trypanosomes in which it is inactive. The chromatin structures of the active and inactive region will be examined for nuclease sensitivity and nucleosome organization. By in vivo pulse labeling in vitro nuclear run-on experiments, the primary VAG transcript will be identified and the transcription initiation site localized. The mechanism of and effects of simultaneous double VAG expression will be examined. The mechanism by which the 35 base mini-exon sequence is added to trypanosome mRNAs will be studied using nuclear extracts and whole cells. Whether it is put on by priming or by addition after transcription will be determined. These studies will be facilitated by use of a axzenic culture system for growth of the trypanosomes. Finally, we propose to study the mechanism of addition and the role of other sequences which are found on RNA subsets in trypanosomes. A detailed description of the mechanisms involved in VAG transcription and RNA metabolism in parasitic trypanosomes may lead to development of means of eradicating or controlling a major economic problem.
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