FUNCTION OF A TRANSPOSABLE ELEMENT IN YEAST

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The sigma compostie element of S. cerevisiae, Ty sigma, is uniquely position specific for tRNA genes. Ty sigma is composed of an internal sequence of several kbp flanked by long terminal repeats of sigma, a 340 bp sequence. The structural studies conducted during the current project, which resulted in the discovery of Ty sigma, showed that both Ty sigma and sigma occur almost exclusively at the 5' ends of tRNA genes. Despite this unique position specificity, however, Ty sigma is a retrovirus- like transposable element which has both structural analogies and sequence homologies with Ty, copia, and spleen necrosis virus. The research in this proposal focuses on three objectives: 1. Characterization of Ty sigma transcription. An almost full- length transcript of Ty sigma, which is regulated by pheromones and which appears to be regulated by mating- type has been observed. The pattern of Ty sigma transcription after pheromone treatment and in different genetic backgrounds will be studied. Deletions from each end of Ty sigma will be used to sequence the element and then to create scanning mutations in order to define regions which are involved in regulating expression and determine whether they mediate positive or negative effects. 2. Identification of interactions which occur between the tRNA gene and Ty sigma. The effect of Ty sigma insertion on tRNA gene expression will be followed by measuring expression of the SUP2 tRNATyr gene associated with various recombinant configurations of Ty sigma. Expression of Ty sigma associated with transcriptionally active and inactive SUP2 genes will be determined. 3. Development of assay for Ty sigma transposition. Ty sigma will be over expressed and transposition into the genome or into a plasmid-borne tRNA gene target will be anlyzed. The roles that tRNA gene structural sequences and tRNA gene transcription play in position specificity can then be determined. The association between Ty sigma and tRNA genes offers a unique opportunity to extend the observations on regulatory interactions of transposable elements and nearby genes to a new class of genes and potentially different kinds of regulation. The position specificity of Ty sigma further suggests that these studies can make unique contributions to our understanding of retrotransposon integration mechanisms.