tRNA Genes and Genetic Mobility in S. cerevisiae

酿酒酵母中的 tRNA 基因和遗传流动性

• 批准号: 0450159
• 负责人: Suzanne Sandmeyer
• 金额: --
• 依托单位: University of California-Irvine
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-15 至 2009-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0450159&HistoricalAwards=false
• 关键词: tRNA; Genes; Genetic; Mobility; S.

Transfer RNA genes (tDNA's) are important not only for the RNA's they encode, but also for their roles in genomic function and evolution. They have been identified as boundaries of heterochromatic DNA, repressors of pol II transcription, sites of genomic rearrangement and ectopic recombination, sites of pathogenic island insertion, and targets of long terminal repeat (LTR) retrotransposon integration. The Sandmeyer laboratory studies Ty3, a retroviruslike element in Saccharomyces cerevisiae that inserts at RNA polymerase III transcription initiation sites. It is known from an in vitro system that includes viruslike particles and a plasmid-borne target, that the pol III transcription factor TFIIIB bound to DNA is sufficient for integration in vitro. However, much remains to be learned about the relationship between this element, tDNA's, and their genomic contexts. This project has three goals: 1) Characterization of the Ty3 preintegration complex (PIC) and reconstitution of a defined in vitro Ty3 integration system. A central step in the Ty3 lifecycle is conversion of the VLP into the integration-competent cDNA-associated PIC. A PIC fraction will be isolated based on cDNA integrating activity. It will be characterized for Ty3 and host proteins and imaged using AFM. 2) Identification of specific interactions between the Ty3 PIC and its target. The minimal target requirement for in vitro integration of Ty3 is comprised of the TATA binding protein (TBP) and Brf1 bound to a RNA pol III promoter. The domains in the PIC and target which interact will be identified by testing for interactions between Ty3 proteins, particularly integrase, and Brf1. If those candidates do not interact, Ty3 will be mutagenized and a genetic screen will be used to identify mutants affected in position specificity. 3) Characterization of features of chromosomal targets of Ty3 integration. The relationship of in vivo chromosome-based transcriptional activity and Ty3 targeting will be addressed using a diverse subset of Pol III-transcribed genes. TFIIIB and TFIIIC and Pol III occupancy and transposition will be monitored using chromatin immunoprecipitation quantitative PCR, respectively. Retrotransposons and genes for tRNA's are major forces affecting mobilization in prokaryotic and eukaryotic genomes yet relatively little is actually understood about the ability of tRNA genes to act as delimiters of chromosomal regions or why retrotransposons prefer integration in some regions over others. This research will lead to a better understanding of these two interacting and dynamic genomic components. It will also be used by the PI to introduce summer high school students, undergraduate biology majors, and graduate students to the principles of yeast molecular biology and genomic research. UCI has an active Minority Scientist Program, and students from a wide range of educational institutions, including community colleges and medical schools, are recruited into this program and are eligible for this project. Data will be disseminated by publication and by posting a database summarizing the findings at a public website.

转移RNA基因(TDNA)不仅对它们编码的RNA很重要，而且对它们在基因组功能和进化中的作用也很重要。它们分别是异染色质DNA的边界、PolII转录抑制因子、基因组重排和异位重组位点、致病岛插入位点和长末端重复序列反转录转座子整合靶点。Sandmeyer实验室研究Ty3，这是酿酒酵母中的一种逆转录病毒元件，插入在RNA聚合酶III的转录起始点。从包括病毒样颗粒和质粒携带的靶点的体外系统中已知，与DNA结合的PolIII转录因子TFIIIB足以在体外整合。然而，关于该元件、tDNA和它们的基因组环境之间的关系仍有许多需要了解。该项目有三个目标：1)鉴定Ty3前整合复合体(PIC)并重建已确定的体外Ty3整合系统。Ty3生命周期中的一个中心步骤是将VLP转换为具有整合能力的cDNAs相关的PIC。根据cDNA整合活性分离出PIC组分。它将针对Ty3和宿主蛋白进行表征，并使用AFM进行成像。2)确定Ty3PIC与其靶标之间的特定相互作用。Ty3体外整合的最低靶向要求是由TATA结合蛋白(TBP)和与RNA PolIII启动子结合的Brf1组成。PIC和靶蛋白中相互作用的区域将通过测试Ty3蛋白，特别是整合酶与Brf1之间的相互作用来确定。如果这些候选基因没有相互作用，Ty3将被诱变，基因筛查将被用来识别受位置特异性影响的突变。3)整合了Ty3基因的染色体靶标的特征。体内基于染色体的转录活性和Ty3靶向的关系将使用POLIII转录基因的不同子集来解决。TFIIIB和TFIIIC以及POL III的占位和转座将分别用染色质免疫沉淀定量PCR进行监测。反转录转座子和tRNA的基因是影响原核和真核基因组动员的主要力量，但人们对tRNA基因作为染色体区域的分隔符的能力以及为什么反转录转座子在某些区域比其他区域更喜欢整合的了解相对较少。这项研究将有助于更好地理解这两个相互作用和动态的基因组成分。它还将被PI用来向夏季高中生、生物学专业本科生和研究生介绍酵母分子生物学和基因组研究的原理。UCI有一个活跃的少数族裔科学家计划，来自广泛教育机构的学生，包括社区大学和医学院，都被招募到这个计划中，并有资格参加这个项目。数据将通过出版物和在公共网站上张贴总结调查结果的数据库的方式传播。