Characterization of mutant alleles of RTT105 in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae, commonly known as baker's yeast, contains several retrotransposons within its genome, including the Ty1 element. There are many host genes that have been shown to regulate Ty1 retrotransposition, including RTT105; however, the host function of this gene is unknown. RTT105 was originally identified as a negative Ty1 regulator by the insertion of the bacterial transposon, Tn3, creating a disruption at the 212bp of the 627bp gene. The resulting mutant allele, rtt105::Tn3‐LacZ/Leu2, increases transposition of an endogenous Ty1 element. However, our lab isolated the RTT105 gene in a screen of genes that when over‐expressed, increased retrotransposition of a galactose inducible Ty1 element on a 2‐micron (high copy) plasmid. We have shown that a complete deletion of RTT105 results in dramatically less galactose‐induced Ty1 retrotransposition. From this, we hypothesize that the Tn3 allele does not nullify gene function but rather enhances it. Real‐time quantitative PCR and northern blots are being used to determine expression levels of the wild type and rtt105::Tn3‐LacZ/Leu2 allele from RNA extracts of the strains. Since the Tn3 insert is over 7kbp in size, disruption mutants at the same 212bp location are being made with auxotrophic markers varying in length. This will help determine whether expression of the N‐terminal region of RTT105 specifically inhibits transposition or whether sequences within Tn3 may be impacting RTT105 expression. The various insertion mutants will be assayed for retrotransposition and RNA extracts will be used to look at expression levels of the mutant rtt105 alleles. Additionally, we are purifying the Rtt105 protein for antibody production by expressing in E. coli. This research is supported by NIH grant GM074658‐01 to JBK.