The role of SIR4 in Ty1 retrotransposition

Juniata College, Huntingdon PA

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Abstract

DESCRIPTION (provided by applicant): The Tyl retroelement of yeast transposes via a mechanism bearing many similarities to retroviruses that infect larger eukaryotes. Tyl is a good model system for studying viral mechanisms, as dissection of the Tyl transposition pathway is facilitated by the ease and flexibility of yeast molecular genetic techniques. The Tyl transposition process is inherently temperature sensitive. Deletion of SIR4, a silent information regulator gene, partially restores transposition at non-permissive temperature. SIR4 complexes with other Silent Information Regulator genes do transcriptionally regulate the mating loci and telomeres by creating a silenced chromatin region. SIR4 has also been shown to play a role in cellular aging. An allele of SIR4, called sir4-42, was isolated as a stress-resistant mutant that also demonstrates increased life span potential. The specific aims of this grant are directed towards the goal of determining the role of SIR4 in the transposition of Tyl. Experiments will focus on determining whether the increased transposition events occurring in a sir4 deletion strain at high temperature are true integration events, or are due to an increase in recombination of Tyl cDNA to the genome. The effects of sir4 on targeting will be assessed by cloning and sequencing transposition events. Parallel experiments will focus on determining if Sir4p co-purifies with Tyl Virus-Like Particles (VLPs), as well as the protein processing, cDNA production and integration steps of Tyl in sir4 mutant strains. SIR4 has been widely studied, and the effects on Tyl transposition of mutations in gene products known to interact with Sir4p or to be affected by sir4 mutations will also be determined. A simple genetic assay will be used to determine if high temperature affects silencing.