FUNCTIONS AND REGULATION OF EXPRESSION OF A. TUMEFACIENS

本研究的目的是为了了解生物多样性的调节 根癌农杆菌毒力(VIR)的表达 基因，并确定它们的基因产物在 细菌DNA转移整合的全过程 进入植物核基因组的片段。体内和体外 将使用各种方法来解决监管方面的问题。两个都在 体外转录-翻译和转录系统将是 发展起来的。野生型和突变型菌株的提取物 农杆菌将用于偶联转录- 翻译系统以定义可诱导中的调节要素 VIR基因的表达。一种含有大肠杆菌β-的融合基因- 在可诱导VIR启动子控制下的半乳糖苷酶将 作为模板DNA。调控序列及其相互作用 最小长度将通过删除突变研究来确定。 将使用生化和遗传方法来识别和 研究抑制物功能，如果有的话。 调节蛋白和RNA聚合酶将被提纯以 开发一种体外转录系统，将用于 确定VIR基因表达的最低必需成分 以及他们的规定。纯化的蛋白质将用于研究 RNA聚合酶和/或调节性蛋白的特异性结合 通过限制酶切位点保护分析将蛋白质转化为DNA模板， 脚印等方法。这些研究将是 有助于建立一种监管机制 VIR基因的表达。 以确定与VIR基因相关的各种活性 产品中，蛋白质将在大肠杆菌中以及在 农杆菌。VIRB操纵子的蛋白质将被测试 β-1导致4水解酶活性，因为显著 衍生的氨基酸序列的一段之间的同源性 和动物溶菌酶的活性部位序列。基因 聚变程序将被用来确定行动地点是否 VIRE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。VIR基因 在赋予致病力的过程中起着核心作用 农杆菌。关于这些基因如何被调控的知识 它们的蛋白质产物功能将极大地促进 对致病性和基因转移处理器的理解

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