A Bacolovirus-induced RNA Polymerase

杆状病毒诱导的RNA聚合酶

• 批准号: 9117186
• 负责人: Robert Weaver
• 金额: --
• 依托单位: University of Kansas Main Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-15 至 1996-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9117186&HistoricalAwards=false
• 关键词: Bacolovirus; induced; RNA; Polymerase

Early transcription in the nuclear polyhedrosis virus of Autographa californica is directed by the host RNA polymerase II, while late transcription depends on an a-amanitin-resistant, virus-induced polymerase. The baculoviruses are increasingly important as expression vectors for producing large quantities of useful proteins. An understanding of baculovirus gene expression will probably lead to better vectors. In particular, an understanding of the unique, a-amanitin-resistant, virus-induced RNA polymerase would be especially valuable, since this is the enzyme that transcribes the cloned genes in baculovirus expression vectors. The immediate goal of this project is to map and characterize the genes encoding this enzyme. The specific aims are: 1. Isolating viral mutants with lesions in the genes encoding the late-specific viral RNA polymerase. This will be accomplished in three steps: a. Obtaining temperature sensitive (ts) viral mutants that are deficient in late gene expression, as evidenced by failure to form non-occluded virions. b. Screening these ts mutants for the inability to carry out a-amanitin-resistnt late transcription at the non-permissive temperature in isolated nuclei. c. Screening the positive mutants from specific aim 1b for a ts virus-induced polymerase by purifying the enzyme and showing that it is ts in vitro. This would also demonstrate conclusively that this is indeed the polymerase responsible for late viral transcription. 2. Mapping the mutant genes from specific aim 1c. This will be done by rescue of the wild-type phenotype with cloned fragments of the viral genome. 3. Sequencing the mutant genes and their wild-type counterparts to locate open reading frames and the lesions responsible for the ts phenotype. In the unlikely event that we cannot find mutants in the genes for the virus-induced RNA polymerase itself, the ts mutants we do isolate should be in the genes for late-specific transcription factors. These are also of great interest, and will be mapped and sequenced.

核型多角体病毒的早期转录是由宿主RNA聚合酶II控制的，而晚期转录依赖于a-Amanitin抗性的病毒诱导的聚合酶。杆状病毒作为表达载体在生产大量有用蛋白方面发挥着越来越重要的作用。对杆状病毒基因表达的了解可能会带来更好的载体。特别是，了解独特的、a-Amanitin抗性的、病毒诱导的RNA聚合酶将特别有价值，因为这是转录杆状病毒表达载体中克隆基因的酶。这个项目的直接目标是绘制和表征编码这种酶的基因。其具体目的是：1.分离编码晚期特异性病毒RNA聚合酶基因中有损伤的病毒突变体。这将通过三个步骤完成：a.获得温度敏感型(Ts)病毒突变体，该突变体在晚期基因表达方面存在缺陷，证明无法形成非封闭的病毒粒子。对这些ts突变体进行筛选，发现它们不能在不允许的温度下在分离的核中进行a-Amanitin晚期转录。C.通过对TS病毒诱导的聚合酶进行纯化，筛选出特异性靶标1b的阳性突变体，并证明该酶在体外是TS。这也将确凿地证明，这确实是负责晚期病毒转录的聚合酶。2.从特异性靶标1c定位突变基因。这将通过用病毒基因组的克隆片段挽救野生型表型来实现。3.对突变基因和相应的野生型基因进行测序，以定位开放阅读框和与ts表型有关的病变。如果我们在病毒诱导的RNA聚合酶本身的基因中找不到突变，那么我们分离的ts突变应该在晚期特异转录因子的基因中。这些也是非常感兴趣的，并将被绘制和排序。