TRANSCRIPTION CONTROL IN NUCLEAR POLYHEDROSIS VIRUS

本研究的长期目标是了解其作用机制。 被核型多角体病毒用来控制其基因表达。 这种病毒已被选为真核生物中基因表达的模型；它 的优点是相对简单，但又足够复杂， 采用各种有趣的控制机制。更好地理解 对这种病毒中基因表达的控制可能会揭示 在人类发育和发展过程中对基因表达的控制 在正常细胞向癌细胞发展的过程中。现在 该项目包含两大具体目标：1)确定病毒基因(S) 这使(S)α-氨基甲酸甘油酯对夜蛾(寄主)细胞产生了抗性。它是 这些基因可能在从 宿主对早期病毒基因的α-Amanitin敏感转录 聚合酶对晚期病毒α-Amanitin抗性转录的影响 基因。这一特定的目标将通过转基因Spotor tera来实现 克隆病毒DNA片段的细胞及其转基因细胞的检测 对阿尔法-阿曼尼汀的抗药性。(已经很明显，病毒感染 很快就会产生对阿尔法-阿曼尼丁的抗药性。)当相关基因 ，他们和他们的成绩单将使用限制 作图、S1-作图和Northern杂交。这些基因也将是 已排序。2)第二个具体目标是比较一个 晚期出现新的抗α-Amanitin RNA聚合酶 感染宿主RNA聚合酶II。这些酶将被提纯 如果可能的话，使用针对它们的单抗。如果这个 证明是不切实际的，这些酶将用传统的方法提纯， 采用柱层析、高效液相色谱和 超速离心法。这些酶的亚基结构将是 下定决心。如果它们看起来相同，则可能存在电荷差异 将对酶亚基进行研究。任何病毒的基因编码 这种新型酶的亚基将被绘制出来。

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.