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

描述(申请人提供)：酿酒酵母中的Ty1逆转录元件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进行转位。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。Ty1转座过程天生对温度敏感。RTT105基因是在筛选过表达时克服高温缺陷的基因时被发现的。缺失分析证实RTT105是Ty1转座的正调控因子。RTT105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。RTT105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，使用从经典基因筛查到蛋白质组学的各种技术。RTT105基因将被随机突变，并对产生的突变库进行筛选，以确定影响转座和细胞生长的突变，以确定该基因的功能重要区域。将纯化的Rtt105蛋白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蛋白质的荧光显微镜研究，以确定蛋白质表达的细胞定位。最后，将表征RTT105-Tn3插入等位基因的表达，因为该等位基因已被证明能增强Ty1转座。公共卫生相关性：酿酒酵母是Ty1逆转录元件的宿主，该逆转录元件在功能上类似于人类病毒，如人类免疫缺陷病毒(HIV)。一个调控Ty1的酵母基因已经被克隆，但它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方法研究该基因在宿主细胞中的功能，以及它如何被用来调节Ty1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.