The Ty1 elements of yeast are retrotransposons that encode structural proteins, protease, integrase, and reverse transcriptase. Like retroviruses, these products are formed by transcription of polycistronic messages and post-translational processing events such as protease cleavages and phosphorylation. Although Ty1 RNA represents a large fraction of total RNA in the cell, transposition events are rare, presumably due to a mechanism of regulation called transpositional dormancy. The main objective of this proposal is to identify and characterize the cellular genes involved in transpositional dormancy. These genes are identified by mutations that cause a hypertranspositional phenotype. The PI has used a novel reporter gene construct to screen for such mutations and at present over 160 mutations have been isolated. The initial focus will be on characterizing a subset of these which affect transposition at a post-transcriptional level rather than those mutations that increase Ty1 transcription rates. Genetic characterization of the mutants will include complementation test, dominance relationships, and several genetic and molecular screens to determine the level at which the mutation affects the transposition process. Selected genes will be cloned by complementation of the hypertranspositional phenotype. Steps in the formation of Ty1-like particles, such as protease activation (a rate-limiting step) and VLP formation will be studied in mutant and wild- type cells to identify new mechanisms of transpositional regulation.
