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

描述（由申请人提供）：酵母中的Ty1逆转录因子通过一种与感染较大真核生物的逆转录病毒有许多相似之处的机制转位。酵母为研究调控转位过程的宿主细胞因子提供了一个强大的遗传系统。Ty1转位过程本身对温度敏感。RTT105基因在过表达时克服高温缺陷的基因筛选中被鉴定出来。缺失分析证实RTT105是Ty1转位的正调控因子。RTT105在宿主细胞中的作用尚不清楚。定义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。RTT105基因是一个理想的候选者，让本科生参与定义未知基因的功能，使用从经典遗传筛选到蛋白质组学的技术。RTT105基因将被随机诱变，由此产生的突变文库筛选影响转位和细胞生长的突变，以确定该基因的功能重要区域。Rtt105蛋白纯化后用于制备多克隆抗血清。多克隆血清以及表位标记的Rtt105蛋白将用于研究Rtt105表达的动力学，并试图分离与Rtt105相互作用的宿主细胞因子。该多克隆抗体还将用于Rtt105蛋白的荧光显微镜研究，以确定蛋白表达的细胞定位。最后，RTT105-Tn3插入等位基因的表达将被表征，因为该等位基因已被证明可以增强Ty1转位。公共卫生相关性：酿酒酵母或贝克酵母是Ty1逆转录因子的宿主，其功能类似于人类免疫缺陷病毒（HIV）等人类病毒。已经克隆出一个调节Ty1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，以及它是如何调控Ty1逆转录因子的。

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.