Genetics of Barley yellow dwarf virus stop-codon readthrough in yeast

酵母中大麦黄矮病毒终止密码子通读的遗传学

• 批准号: 7069437
• 负责人: PETER J RUSSELL
• 金额: $ 21.53万
• 依托单位: REED COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7069437
• 关键词: RNA virus; Saccharomyces cerevisiae; fungal genetics; genetic library; genetic regulation; genetic screening; genetic translation; high throughput technology; host organism interaction; nucleic acid sequence; phenotype; plant virus; plasmids; tobacco mosaic virus; virus genetics

DESCRIPTION (provided by applicant): A variety of positive-sense RNA viruses, including some mammalian retroviruses, use the translation receding mechanism of stop-codon readthrough to make specific fusion proteins. In Barley yellow dwarf virus (BYDV), readthrough is programmed by a combination of a sequence element proximal to the UAG stop, and a distal element several hundred nucleotides downstream. The involvement of host factors in programmed stop codon readthrough is unknown for BYDV and for most other RNA viruses. The objective in the long term is to identify and characterize host genes involved in BYDV programmed readthrough. To facilitate a genetic approach, the model host organism used in the study is yeast. The three specific aims of the study are: (1) Development of the experimental system for studying BYDV programmed readthrough in yeast. Single- and double-reporter constructs including the readthrough programming sequence elements are designed to demonstrate that readthrough occurs in yeast. Similar double-reporter constructs with a Tobacco mosaic virus (TMV) readthrough sequence are designed to test whether genes that affect BYDV readthrough also affect TMV readthrough. (2) Quantification of stop codon readthrough of BYDV and TMV sequences in wild-type yeast. Using the double-reporter constructs of (1), readthrough efficiencies will be quantified. The results will indicate whether yeast is an suitable model host for studying the genetics of BYDV readthrough. (3) Identification and characterization of yeast genes involved with BYDV programmed readthrough. Using the reporter-based readthrough system, a yeast overexpression library will be screened to identify genes that increase readthrough efficiency significantly. Genes so identified will be characterized in detail, including testing whether their overexpression also influences TMV readthrough. In parallel, the effect of yeast genes known to affect translation termination, stop codon readthrough, or translation fidelity will be tested directly with the readthrough reporter system. Public health relevance: The project involves basic research designed to advance our understanding of the genetics of RNA virus-host interactions. A number of pathogenic RNA viruses infect humans. Knowing more about virus-host interactions from the host side has the potential, in general, of informing research designed to understand and control infections by pathogenic RNA viruses.

描述（由申请人提供）：多种阳性RNA病毒，包括一些哺乳动物逆转录病毒，使用止回蛋白的翻译回收机理来制作特定的融合蛋白。在大麦黄色矮人病毒（BYDV）中，通过接近UAG停止的序列元件的组合和下游几百个核苷酸的远端元素来编程。 BYDV和大多数其他RNA病毒的宿主因素参与编程的终止密码子读取尚不清楚。从长远来看，目的是识别和表征与BYDV编程通知有关的寄主基因。为了促进遗传学方法，研究中使用的宿主生物是酵母。该研究的三个具体目的是：（1）开发用于研究BYDV编程的酵母菌的实验系统。包括读取编程序列元素在内的单个和双重旋转器构造旨在证明在酵母中出现通读。具有烟草镶嵌病毒（TMV）读取序列的类似的双重变形蛋白构建体旨在测试影响BYDV读取的基因是否也影响TMV读取。 （2）定量野生型酵母中BYDV和TMV序列的终止密码子读取。使用（1）的双重旋转构建体，将量化效率。结果将表明酵母是否是研究BYDV读取的遗传学的合适模型宿主。 （3）鉴定和表征与BYDV编程的读取有关的酵母基因。使用基于记者的读取系统，将筛选酵母过表达库，以识别可显着提高通读效率的基因。如此鉴定的基因将被详细表征，包括测试它们的过表达是否还会影响TMV读取。同时，将直接使用ReadThrough Reporter系统直接测试已知会影响翻译终止，停止密码子读取或翻译保真度的酵母基因的效果。公共卫生相关性：该项目涉及旨在促进我们对RNA病毒宿主相互作用遗传学的理解的基础研究。许多致病性RNA病毒感染了人类。一般来说，从宿主侧了解更多关于病毒宿主相互作用的病毒 - 宿主相互作用的潜力是，旨在通过致病性RNA病毒理解和控制感染的研究。