FUNCTION AND REGULATION OF A TUMEFACIENS VIRULENCE GENES

根癌病菌毒力基因的功能与调控

• 批准号: 2178822
• 负责人: Anath DAS
• 金额: $ 16.54万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178822
• 关键词: Agrobacterium tumefaciens; DNA binding protein; DNA directed RNA polymerase; DNA topoisomerases; Escherichia coli; bacterial DNA; bacterial genetics; bacterial proteins; crown gall; gene expression; gene mutation; genetic manipulation; genetic mapping; genetic transcription; laboratory rabbit; microorganism genetics; molecular cloning; neoplastic transformation; nucleic acid sequence; phosphorylation; site directed mutagenesis; tissue /cell culture; transcription factor; transfection; virulence

The goal of the proposed research is to understand the mechanism of DNA transfer from Agrobacterium to plant cells. The role of the virulence (vir) genes in the DNA transfer process and the mechanism of transcription regulation of the vir genes will be investigated. Two regulatory proteins, VirA and VirG, in conjunction with a plant phenolic acetosyringone control vir gene expression. The signal molecules (plant phenolics) generated by a susceptible host plant induce modification of the regulatory proteins presumably via phosphorylation leading to the activation of the pathogenic machinery of the bacterium. Mutational studies will be performed to identify the signal processing and transcriptional activator domains of VirA and VirG. The effect of these mutations on protein phosphorylation will be investigated. An in vitro transcription system will be developed to study the role of protein phosphorylation in regulation of vir gene expression and to elucidate the mechanism of transcription activation. An intermediate in DNA transfer from bacteria to plant cells is a single- stranded (ss) DNA complexed with proteins (T-complex). Formation of the ssDNA is catalyzed by the VirD2 endonuclease and requires the presence of VirD1. In vivo studies will be performed to determine the mechanism of VirD1-VirD2 endonuclease action. Deletion mutagenesis studies will be performed to define the function domains of the VirD2 endonuclease. Both VirD1 and VirD2 presumably form covalent protein-DNA complexes. The amino acids that participate in the formation of these complexes will be identified. The role of the other VirD proteins in DNA transfer and integration will be studied. Biochemical studies will be performed to determine the composition of the T-complex and to stud its interaction with bacterial membranes. Mutations in vir genes will be used to identify the gene products that participate in these reactions. An in vitro system will be developed to identify Vir and other proteins required for DNA transfer to plant cells. Using assays that can distinguish between DNA transfer and DNA integration, the role of different Vir proteins in these reactions will be investigated. The long term goal of this study is to understand the mechanism of agrobacterial pathogenicity. The vir genes play a central role in this process. Elucidation of the mechanism of expression of these genes and understanding the functions of the encoded gene products are vital towards a better understanding of mechanisms bacteria employ for their pathogenic action.

拟议的研究的目的是了解DNA的机制 从农杆菌转移到植物细胞。 毒力的作用 （VIR）DNA转移过程中的基因和转录机理 将研究VIR基因的调节。 两种调节蛋白，即Vira和Virg，与植物结合 酚类乙酰酮控制VIR基因表达。 信号分子 （植物酚类）由易感宿主植物产生的诱导修饰 大概是通过磷酸化导致调节蛋白的 细菌的致病机制的激活。 突变 将进行研究以确定信号处理和 VIRA和VIRG的转录激活域。 这些效果 将研究蛋白质磷酸化的突变。 体外 将开发转录系统以研究蛋白质的作用 在调节VIR基因表达中的磷酸化并阐明 转录激活的机理。 DNA从细菌到植物细胞转移的中间体是唯一的 滞留的（SS）DNA与蛋白质复合（T-复合物）。 形成 ssDNA被VIRD2核酸内切酶催化，需要存在 vird1。 将进行体内研究以确定 vird1-vird2核酸内切酶动作。 缺失诱变研究将是 执行以定义VIRD2内核酸酶的功能域。 两个都 VIRD1和VIRD2大概会形成共价蛋白-DNA复合物。 氨基 参与这些复合物形成的酸将是 确定。 其他Vird蛋白在DNA转移和 将研究集成。 将进行生化研究以确定 T型复合物，并与细菌膜相互作用。 突变 在Vir基因中，将用于识别参与的基因产品 这些反应。 将开发一个体外系统来识别VIR和 DNA转移到植物细胞所需的其他蛋白质。 使用该测定 可以区分DNA转移和DNA整合， 将研究这些反应中不同的VIR蛋白。 这项研究的长期目标是了解 农杆菌致病性。 VIR基因在此起着核心作用 过程。 阐明这些基因表达机理和 了解编码基因产品的功能至关重要 更好地理解细菌用于病原的机制 行动。