FUNCTIONS AND REGULATION OF EXPRESSION OF A. TUMEFACIENS

本研究的目的是了解 根癌农杆菌毒力（vir）的表达 基因，并确定其基因产物在 细菌DNA转移和整合的整个过程 插入到植物的核基因组中。 体内和体外 将采用各种办法来解决监管方面的问题。 无论是在 体外转录-翻译和转录系统将是 开发 野生型和突变株的提取物 农杆菌将用于偶联转录- 翻译系统，以确定诱导的调控元件 vir基因的表达。 将E.大肠杆菌 在诱导型VIR启动子控制下的半乳糖苷酶将 用作模板DNA。 调控序列及其 最小长度将通过缺失诱变研究来确定。 生物化学和遗传学方法将用于识别和 研究阻遏物功能，如果有的话。 将纯化调节蛋白和RNA聚合酶， 开发一种体外转录系统， 确定vir基因表达的最低必要组分 和他们的规则。 纯化的蛋白质将用于研究 RNA聚合酶和/或调节蛋白的特异性结合 通过限制性位点保护测定将蛋白质与模板DNA结合， 脚印和其他方法。 这些研究报告将 有助于建立一个机制， vir基因表达 为了确定与vir基因相关的各种活动 产物，蛋白质将在E.大肠杆菌中， 土壤杆菌。 将检测virB操纵子的蛋白质， β-1导致4水解酶活性，因为显著的 衍生的氨基酸序列的区段之间的同源性 和动物溶菌酶的活性位点序列。 基因 融合程序将被用来确定是否作用的网站， 维雷基因产物存在于植物细胞内。 这项研究的长期目标是了解 微生物致病的分子机制。 所述vir基因 在赋予致病性方面起着核心作用， 土壤杆菌。 这些基因是如何被调控的 它们的蛋白质产物功能将极大地促进 了解致病性和基因转移处理器

The goal of this research is to understand the regulation of expression of the Agrobacterium tumefaciens virulence (vir) genes, and to define the roles of their gene products play in the overall process of transfer and integration of a bacterial DNA segment into the plant nuclear genome. In vivo and in vitro approaches will be used to address the regulatory aspects. Both in vitro transcription-translation and transcription systems will be developed. Extracts from wild-type and mutant strains of Agrobacterium will be used in the coupled transcription- translation system to define the regulatory elements in inducible expression of the vir genes. A fusion gene containing E. coli beta- galactosidase under the control of an inducible vir promoter will be used as template DNA. The regulatory sequences and their minimum length will be defined by deletion mutagenesis studies. Biochemical and genetic approaches will be used to identify and study repressor functions, if any. The regulatory proteins and RNA polymerase will be purified to develop an in vitro transcription system which will be used to define the minimum necessary components for vir gene expression and their regulation. The purified proteins will be used to study specific binding of RNA polymerase and/or of the regulatory proteins to template DNAs by restriction-site protection assays, foot-printing, and other methods. These studies will be instrumental in establishing a mechanism for the regulation of the vir gene expression. To determine the various activities associated with the vir gene products, the proteins will be overproduced in E. coli as well as in Agrobacterium. The proteins of the virB operon will be tested for beta-1 leads to 4 hydrolase activity because of significant homology between a segment of the derived amino acid sequence of virB1 and the active-site sequences of animal lysozymes. Gene fusion procedures will be used to determine if the site of action of virE gene products is within the plant cells. The long-term objective of this research is to understand the molecular mechanisms of microbial pathogenesis. The vir gene plays a central role in the confering pathogenicity to Agrobacterium. The knowledge of how these genes are regulated and their protein products function will greatly facilitate the understanding of pathogenicity and gene transfer processor