Tuesday, November 26, 2024
11/26/2024
DESCRIPTION (provided by applicant): The Ty1 retroelement in the yeast Saccharomyces cerevisiae transposes via a mechanism bearing many similarities to retroviruses which infect larger eukaryotes. Yeast provides a powerful genetic system for studying host cell factors that regulate the transposition process. The Ty1 transposition process is inherently temperature sensitive. The RTT105 gene was identified in a screen for genes that over-come the high temperature defect when overexpressed. Deletion analysis confirmed that RTT105 is a positive regulator of Ty1 transposition. The function of RTT105 in the host cell is unknown. Defining the function of uncharacterized genes requires systematic and integrated genetic, molecular, and proteomic analysis. The RTT105 gene is an ideal candidate to involve undergraduate students in the process of defining the function of an unknown gene, using techniques ranging from classical genetic screens to proteomics. The RTT105 gene will be randomly mutagenized, and the resulting mutant library screened for mutations that effect transposition and cell growth to define the functionally important regions of the gene. Rtt105 protein will be purified and used to make a polyclonal antiserum. The polyclonal serum, as well as epitope-tagged Rtt105 protein, will be used to study the kinetics of Rtt105 expression and to attempt to isolate host cell factors that interact with Rtt105. The polyclonal antibody will also be used for fluorescence microscopy studies of Rtt105 protein to determine cellular localization of protein expression. Finally, the expression of an RTT105-Tn3 insertion allele will be characterized, as this allele has been shown to enhance Ty1 transposition. Public Health Relevance: Saccharomyces cerevisiae, or Baker's yeast, is host to the Ty1 retroelement, which is functionally similar to human viruses such as the Human Immunodeficiency Virus (HIV). A yeast gene has been cloned that regulates Ty1, but its function in the host yeast cell is unknown. This study aims to use genetics and proteomic approaches to study the function of the gene in the host cell and how it is used to regulate the Ty1 retroelement. Public Health Relevance: Project Narrative. Saccharomyces cerevisiae, or Baker's yeast, is host to the Ty1 retroelement, which is functionally similar to human viruses such as the Human Immunodeficiency Virus (HIV). A yeast gene has been cloned that regulates Ty1, but its function in the host yeast cell is unknown. This study aims to use genetics and proteomic approaches to study the function of the gene in the host cell and how it is used to regulate the Ty1 retroelement.
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