A Bacolovirus-induced RNA Polymerase

加州签名虫核多角体病毒的早期转录是由宿主RNA聚合酶II指导的，而晚期转录依赖于抗a-amanitin病毒诱导的聚合酶。杆状病毒作为产生大量有用蛋白的表达载体越来越重要。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，抗a-amanitin的，病毒诱导的RNA聚合酶将特别有价值，因为这是在杆状病毒表达载体中转录克隆基因的酶。这个项目的直接目标是绘制和描述编码这种酶的基因。具体目标是：1。在编码晚期特异性病毒RNA聚合酶的基因中分离具有病变的病毒突变体。这将分三步完成：a.获得缺乏晚期基因表达的温度敏感（ts）病毒突变体，这可以通过无法形成非封闭病毒粒子来证明。b.筛选这些ts突变体在离体细胞核非允许温度下无法进行抗a-amanitin晚期转录。c.通过纯化a - ts病毒诱导的聚合酶并在体外证明其为s- ts，从特异性靶1b中筛选阳性突变体。这也将最终证明，这确实是负责后期病毒转录的聚合酶。2. 特异靶1c的突变基因图谱。这将通过克隆病毒基因组片段来拯救野生型表型来实现。3. 对突变基因及其野生型对应基因进行测序，以定位导致ts表型的开放阅读框和病变。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体，那么我们分离出的ts突变体应该是在晚期特异性转录因子的基因中。这些也是非常有趣的，并将被映射和测序。

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.