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

酵母中 Ty1 转座调节因子 RTT105 的分子功能

• 批准号: 7515838
• 负责人: JILL B KEENEY
• 金额: $ 18.28万
• 依托单位: JUNIATA COLLEGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-20 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7515838
• 关键词: Alleles; Cell Survival; Cells; Characteristics; Classification; Defect; Epitopes; Eukaryota; Eukaryotic Cell; Fluorescence Microscopy; Fungal Genes; Fungal Genome; Genes; Genetic; Genetic Screening; HIV; High temperature of physical object; Human Virus; Immune Sera; Indium; Investigation; Kinetics; Libraries; Molecular; Molecular Genetics; Mutagenesis; Mutation; Open Reading Frames; Process; Protein Overexpression; Proteins; Proteomics; Public Health; Range; Reporting; Research Personnel; Retroelements; Retroviridae; Role; Saccharomyces cerevisiae; Serum; Students; System; Techniques; Temperature; Yeasts; cell growth; deletion analysis; deletion library; gene function; high throughput screening; mutant; polyclonal antibody; protein expression

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.

描述（由申请人提供）：酿酒酵母中的Ty1逆转录因子通过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。 Ty1 转座过程本质上是温度敏感的。 RTT105 基因是在筛选能够克服过度表达时高温缺陷的基因时发现的。缺失分析证实RTT105是Ty1转座的正调节因子。 RTT105 在宿主细胞中的功能尚不清楚。定义未表征基因的功能需要系统且集成的遗传、分子和蛋白质组分析。 RTT105 基因是让本科生使用从经典遗传筛选到蛋白质组学等技术来定义未知基因功能的过程的理想候选基因。 RTT105 基因将被随机诱变，并筛选产生的突变体库，筛选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。 Rtt105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 Rtt105 蛋白将用于研究 Rtt105 表达动力学，并尝试分离与 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，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可以增强 Ty1 转座。公共健康相关性：酿酒酵母或面包酵母是 Ty1 逆转录因子的宿主，其功能与人类免疫缺陷病毒 (HIV) 等人类病毒相似。调节 Ty1 的酵母基因已被克隆，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方法来研究该基因在宿主细胞中的功能以及它如何用于调节Ty1逆转录因子。