VACCINIA VIRUS BIOCHEMICAL GENETICS

痘病毒牛痘，这是活疫苗病毒中使用的， 消灭天花，是一种含有大双链DNA的病毒 一直以来都是用基因来理解基因组， 牛痘已经发现，对正确的包括牛痘基因至关重要 感染细胞的稳态细胞质增加。 我们的长期目标 牛痘方法的功能组织。 我们正在进行的 对一个基本病毒基因网络的遗传分析， 控制晚期病毒转录。 网络A18R，当 突变导致晚期病毒RNA和基因G2R水平的合成， 当其突变时导致晚期病毒蛋白质的合成减少。 在这个应用中，我们建议扩展这个网络的分析 并继续努力开发定向体外方法， 在必需牛痘中构建条件致死突变 基因. 具体目标是：1）优化和表征病毒 特异性体外转录系统，2）使用生化方案 和适当的突变体，表征A18R基因的产物， 确定其在感染中的作用，3）使用生物化学方案， 合适的突变体，表征G2R基因的产物， 确定其在感染中的作用，4）使用遗传方案，确定 基因A18R和G2R之间的关系，并寻找其他基因。 参与网络，和5）测试丙氨酸扫描诱变耦合 利用瞬时显性选择作为体外定向的方法， 构建牛痘的条件致死突变体。

The poxvirus vaccinia, which is the live vaccine virus used in the eradication of smallpox, is a large double stranded DNA containing virus that replicates in the has been to understand genome using a genetic vaccinia has uncovered be critical for proper includes vaccinia gene increased steady state cytoplasm of infected cells. Our long-range goal the functional organization of the vaccinia approach. Our ongoing genetic analysis of a network of essential viral genes which seem to control of late viral transcription. The network A18R, which when mutated leads to synthesis of levels of late viral RNA, and gene G2R, which when mutated leads to decreased synthesis of late viral proteins. In this application, we propose to extend the analysis of this network and continue our efforts to develop directed in vitro methods for construction of conditionally lethal mutations in essential vaccinia genes. The specific aims are: 1) optimize and characterize a virus specific in vitro transcription system, 2) using biochemical protocols and appropriate mutants, characterize the product of the A18R gene and determine its role in infection, 3) using biochemical protocols and appropriate mutants, characterize the product of the G2R gene and determine its role in infection, 4) using genetic protocols, determine .the relationship between genes A18R and G2R, and seek additional genes involved in the network, and 5) test alanine scanning mutagenesis coupled with transient dominant selection as a method for directed in vitro construction of conditionally lethal mutants of vaccinia.