The molecular function of RTT105, a regulator of Ty1 transposition in yeast

描述（由申请人提供）：酵母酿酒酵母中的Ty 1逆转录元件通过与感染较大真核生物的逆转录病毒具有许多相似性的机制转座。酵母为研究调节转座过程的宿主细胞因子提供了强大的遗传系统。Ty 1转座过程固有地对温度敏感。RTT 105基因是在筛选过表达时克服高温缺陷的基因中鉴定的。缺失分析证实RTT 105是Ty 1转座的正调控因子。RTT 105在宿主细胞中的功能尚不清楚。确定未表征基因的功能需要系统和综合的遗传，分子和蛋白质组学分析。RTT 105基因是一个理想的候选者，可以让本科生参与到定义未知基因功能的过程中，使用从经典遗传筛选到蛋白质组学的技术。将RTT 105基因随机诱变，并筛选所得突变体文库中影响转座和细胞生长的突变，以确定基因的功能重要区域。Rtt 105蛋白将被纯化并用于制备多克隆抗血清。多克隆血清，以及表位标记的Rtt 105蛋白，将用于研究Rtt 105表达的动力学，并试图分离与Rtt 105相互作用的宿主细胞因子。多克隆抗体也将用于Rtt 105蛋白的荧光显微镜研究，以确定蛋白表达的细胞定位。最后，RTT 105-Tn 3插入等位基因的表达将被表征，因为该等位基因已显示增强Ty 1转座。公共卫生相关性：酿酒酵母（Saccharomyces cerevisiae）或面包酵母（Baker's yeast）是Ty 1逆转录元件的宿主，Ty 1逆转录元件在功能上与人类病毒（如人类免疫缺陷病毒（HIV））相似。已经克隆了调节Ty 1的酵母基因，但其在宿主酵母细胞中的功能尚不清楚。本研究旨在利用遗传学和蛋白质组学的方法来研究该基因在宿主细胞中的功能，以及它是如何被用来调节Ty 1逆转录元件。

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.