FUNCTIONS AND REGULATION OF EXPRESSION OF A. TUMEFACIENS

根癌农杆菌的功能和表达调控

• 批准号: 3292902
• 负责人: Anath DAS
• 金额: $ 11.21万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292902
• 关键词: Agrobacterium tumefaciens; DNA binding protein; bacterial genetics; bacterial proteins; binding proteins; crown gall; genetic transcription; genetic translation; gram negative bacteria; molecular oncology; mutant; neoplasm /cancer genetics; nucleic acid sequence; operon; plasmids; transposon /insertion element; virulence

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

这项研究的目的是了解 农杆菌毒力（VIR）的表达 基因，并定义其基因产品在 细菌DNA转移和整合的总体过程 细分进入植物核基因组。 体内和体外 方法将用于解决监管方面。 两者都在 体外转录翻译和转录系统将是 发达。 野生型和突变菌株的提取物 农杆菌将用于耦合转录 - 翻译系统来定义可诱导中的调节元素 VIR基因的表达。 一个含有大肠杆菌β-的融合基因 诱导型VIR启动子控制下的半乳糖苷酶将 用作模板DNA。 监管序列​​及其 最小长度将通过缺失诱变研究来定义。 生化和遗传方法将用于识别和 研究阻遏物功能（如果有）。 调节蛋白和RNA聚合酶将被纯化为 开发一种体外转录系统，将用于 定义VIR基因表达的最小必要组成部分 和他们的监管。 纯化的蛋白质将用于研究 RNA聚合酶和/或调节的特异性结合 蛋白质通过限制站点保护测定法到模板DNA， 脚印和其他方法。 这些研究将是 有助于建立调节机制 VIR基因表达。 确定与VIR基因相关的各种活动 产品，蛋白质将在大肠杆菌和大肠杆菌中过量生产 农杆菌。 VIRB操纵子的蛋白质将进行测试 β-1由于显着而导致4个水解酶活性 衍生氨基酸序列之间的同源性 virb1和动物溶菌酶的活跃位点序列。 基因 融合程序将用于确定是否 VIRE基因产物在植物细胞内。 这项研究的长期目标是了解 微生物发病机理的分子机制。 Vir基因 在赋予致病性中起着核心作用 农杆菌。 对这些基因的调节方式的了解 他们的蛋白质产品功能将极大地促进 了解致病性和基因转移处理器