A Bacolovirus-induced RNA Polymerase
A Bacolovirus-induced RNA Polymerase
批准号:
9117186
负责人:
Robert Weaver
金额:
$0.0万
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
项目状态:
已结题
起止时间:
1992-02-15 至 1996-07-31
中文摘要
苜蓿银纹夜蛾核型多角体病毒的早期转录由宿主RNA聚合酶II指导,而晚期转录依赖于α-鹅膏蕈碱抗性的病毒诱导的聚合酶。杆状病毒作为生产大量有用蛋白质的表达载体越来越重要。对杆状病毒基因表达的理解可能会导致更好的载体。特别是,了解独特的,α-鹅膏蕈碱抗性,病毒诱导的RNA聚合酶将是特别有价值的,因为这是转录杆状病毒表达载体中的克隆基因的酶。该项目的直接目标是绘制和表征编码这种酶的基因。具体目标是:1.分离在编码晚期特异性病毒RNA聚合酶的基因中具有损伤的病毒突变体。这将通过三个步骤实现:a.获得晚期基因表达缺陷的温度敏感性(ts)病毒突变体,如不能形成非封闭病毒体所证明的。B.筛选这些ts突变体,以确定其在分离的细胞核中在非允许温度下无法进行α-鹅膏蕈碱抗性晚期转录。C.通过纯化ts病毒诱导的聚合酶,筛选特异性目的1b的阳性突变体,并在体外显示其为ts。这也将最终证明这确实是负责晚期病毒转录的聚合酶。2.定位特定目标1c的突变基因。这将通过用病毒基因组的克隆片段拯救野生型表型来完成。3.对突变基因及其野生型对应基因进行测序,以定位开放阅读框和导致ts表型的病变。万一我们不能在病毒诱导的RNA聚合酶基因中找到突变体,我们分离的ts突变体应该在晚期特异性转录因子基因中。这些也是非常有趣的,将被绘制和排序。
英文摘要
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.
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BIOLOGICAL PEST CONTROL OF INSECT PESTS THAT THREATEN TREE HEALTH
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批准号:BB/L011891/1
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项目类别:Research Grant
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资助金额:$34.07万
-
财政年份:2014
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负责人:Robert Weaver
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SBIR Phase II: Dynamic Broadband Wireless Networks
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批准号:1058597
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项目类别:Standard Grant
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资助金额:$49.99万
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Enhancing resistance to existing and emerging insect pests of UK cereals
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资助金额:$24.82万
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财政年份:2007
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依托单位:
Research Initiation Award: Parallel Language and Application Development for Irregular Computations
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批准号:9410032
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项目类别:Continuing Grant
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资助金额:$9.98万
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财政年份:1994
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负责人:Robert Weaver
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批准号:8750848
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项目类别:Standard Grant
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资助金额:$3.65万
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财政年份:1987
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负责人:Robert Weaver
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依托单位:
国内基金
海外基金
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