TRANSCRIPTION CONTROL IN NUCLEAR POLYHEDROSIS VIRUS

本研究的长期目标是了解其机制 被核型多角体病毒用来控制它们的基因表达。 该病毒已被选为真核生物中基因表达的模型， 具有相对简单但又足够复杂的优点， 使用各种有趣的控制机制。 更好地理解 这种病毒基因表达的控制可能会揭示 在人类发育过程中发生的基因表达的控制， 在正常细胞发展成癌细胞的过程中。 本 该项目有两个主要的具体目标：1）确定病毒基因， 其赋予灰翅夜蛾（宿主）细胞α-鹅膏蕈碱抗性。 是 这些基因可能在从 宿主对α-鹅膏蕈碱敏感的早期病毒基因转录 聚合酶对晚期病毒的α-鹅膏蕈碱抗性转录的影响 基因. 这个特定的目标将通过消灭夜蛾来实现 细胞与克隆的病毒DNA片段，并测定转染的细胞 对α-鹅膏蕈碱的抗药性 (It已经很清楚病毒感染 非常迅速地赋予α-鹅膏蕈碱抗性）。 当相关基因 它们和它们的转录本将用限制性内切酶 定位、S1定位和北方印迹。这些基因也会 测序 2)第二个具体目标是比较一个 晚期出现的新的α-鹅膏蕈碱抗性RNA聚合酶 感染了宿主RNA聚合酶II 酶将被纯化 如果可能的话，使用针对它们的单克隆抗体。 如果这 证明是不切实际的，将通过常规方法纯化酶， 采用柱色谱法、液相色谱法和 超离心 酶的亚基结构将是 测定 如果它们看起来相同， 将研究酶亚基。任何病毒编码的基因 新酶中的亚基将被定位。

The long-term of objective of this research is to understand the mechanisms used by nuclear polyhedrosis viruses to control their gene expression. This virus has been chosen as a model for gene expression in eukaryotes; it has the advantage of being relatively simple and yet complex enough to employ a variety of interesting control mechanisms. A better understanding of the control of gene expression in this virus will probably shed light on the control of gene expression that occurs during human development and during the progression of a normal cell to a cancer cell. The present project contains two major specific aims: 1) to identify the viral gene(s) that confer(s) alpha-amanitin resistance on Spodoptera (host) cells. It is possible that these genes play a role in the switch from alpha-amanitin-sensitive transcription of early viral genes by host polymerase to the alpha-amanitin-resistant transcription of the late viral genes. This specific aim will be approached by transfecting Spodoptera cells with cloned viral DNA fragments and assaying the transfected cells for alpha-amanitin-resistance. (It is already clear that viral infection very rapidly confers alpha-amanitin-resistance.) When the relevant genes are identified, they and their transcripts will be mapped using restriction mapping, S1-mapping and Northern blotting. The genes will also be sequenced. 2) The second specific aim is to compare the structure of a novel alpha-amanitin-resistant RNA polymerase appearing in the late phase of infection with host RNA polymerase II. The enzymes will be purified using monoclonal antibodies directed against them, if possible. If this proves impractical, the enzymes will be purified by conventional means, employing column chromatography, liquid chromatography and ultracentrifugation. The subunit strucures of the enzymes will be determined. If they appear identical, possible charge differences in the enzyme subunits will be investigated. The genes for any virus-coded subunits in the novel enzyme will be mapped.