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: 7905544
• 负责人: PETER J RUSSELL
• 金额: $ 9.85万
• 依托单位: REED COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7905544
• 关键词: Affect; Alleles; Animal Model; Aphids; Basic Science; Capsid; Chimeric Proteins; Collection; Development; Distal; Elements; Firefly Luciferases; Frequencies; Future; Genes; Genetic; Genetic Screening; Goals; Human; Infection Control; Integration Host Factors; LacZ Genes; Libraries; Luteovirus; Modeling; Modification; Molecular; Nucleotides; Organism; Plants; Plasmids; Principal Investigator; Process; Property; Proteins; Protocols documentation; RNA Viruses; Renilla Luciferases; Reporter; Reporting; Research; Research Design; Retroviridae; Role; Saccharomyces cerevisiae; Side; System; Terminator Codon; Testing; Tobacco Mosaic Virus; Translation Process; Translations; Virus; Yeasts; base; design; expectation; overexpression; programs; promoter; public health relevance; transmission process; vector; virus host interaction

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）：多种正义RNA病毒，包括一些哺乳动物逆转录病毒，利用终止密码子读通的翻译后退机制合成特异性融合蛋白。在大麦黄矮病毒（BYDV）中，读取是由靠近UAG止点的序列元件和下游几百个核苷酸的远端元件组合而成的。对于BYDV和大多数其他RNA病毒，宿主因子是否参与程序性终止密码子读取尚不清楚。长期目标是鉴定和表征参与BYDV编程读透的宿主基因。为了便于遗传方法，研究中使用的模式宿主生物是酵母。本研究的三个具体目标是：(1)建立酵母中BYDV编程读透的实验系统。单和双报告结构包括读透编程序列元件的设计，以证明读透发生在酵母。设计了类似的烟草花叶病毒（TMV）读透序列双报告结构，以测试影响烟草花叶病毒（BYDV）读透的基因是否也影响烟草花叶病毒的读透。(2)野生型酵母菌BYDV和TMV序列的终止密码子解读定量。使用(1)的双报告结构，读取效率将被量化。该结果将提示酵母是否是研究BYDV遗传的合适模型宿主。(3)酵母菌与BYDV编程读透相关基因的鉴定与表征。使用基于报告者的读透系统，筛选酵母过表达文库，以识别显着提高读透效率的基因。这样确定的基因将被详细地描述，包括测试它们的过度表达是否也会影响TMV的解读。同时，已知影响翻译终止、终止密码子读通或翻译保真度的酵母基因的作用将直接通过读通报告系统进行测试。公共卫生相关性：该项目涉及基础研究，旨在促进我们对RNA病毒与宿主相互作用的遗传学的理解。许多致病性RNA病毒感染人类。一般来说，从宿主方面更多地了解病毒与宿主的相互作用，有可能为旨在了解和控制致病性RNA病毒感染的研究提供信息。