VirB Mediated DNA and Protein Transfer from A. tumefaciens

VirB 介导的根癌农杆菌 DNA 和蛋白质转移

• 批准号: 9817149
• 负责人: Andrew Binns
• 金额: $ 51万
• 依托单位: University of Pennsylvania
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9817149&HistoricalAwards=false
• 关键词: VirB; Mediated; DNA; Protein; Transfer

Agrobacteria are bacteria that infect damaged plants. Bacterial DNA is transferred to the plant nucleus, and this process is used to move desired genes into plants in the process of genetic engineering. It is important to understand how this process occurs, as it represents a natural occurence of inter-kingdom DNA transfer, as well as of practical importance. The movement of DNA from Agrobacterium tumefaciens into plant cells depends upon the activities of the virulence (vir) genes of the Ti plasmid. Several of the Vir proteins are required for the production of a single-stranded DNA covalently attached to VirD2 at its 5' terminus. The long term objectives are to understand how the VirB complex assembles and functions. Towards this end, a variety of genetic and biochemical methods have been developed to study the interaction of VirB proteins with themselves and with transported substrates. Recent studies yielded the surprising result that the expression of a subset of the VirB proteins in the recipient vastly increased the efficiency of virB-mediated conjugal transfer of RSF1010 between agrobacteria. This result leads to several questions about the VirB proteins and recipient activity: a) what is the minimal complement of VirB proteins necessary for increased recipient activity, b) does the presence of RSF1010 in the recipient affect this activity and c) what is the generality of this phenomenon? The answer to these questions may allow the identification of a "core" VirB complex that could simplify genetic and biochemical studies, and may have important implications to plasmid transfer in general.In order to define the VirB complex and its activity, the interactions between the VirB proteins and themselves or the transported substrates must be understood.. In this project physical/biochemical analysis, utilizing chemical cross-linking and immunoprecipitation will be continued; yeast two hybrid system analysis will be carried out, looking specifically at the possibility that other VirB proteins - and possibly other unidentified proteins - interact with the VirB7-10 group; and saturation mutagenesis on the virB7-10 genes will be carried out to help define regions required for protein interaction and for virulence in general.

农杆菌是感染受损植物的细菌。 细菌DNA被转移到植物细胞核中，这一过程用于在基因工程过程中将所需基因转移到植物中。 了解这个过程是如何发生的是很重要的，因为它代表了王国间DNA转移的自然发生，以及实际的重要性。 DNA从根癌农杆菌进入植物细胞的运动取决于Ti质粒的毒力（vir）基因的活性。 几种Vir蛋白是产生在其5'末端共价连接至VirD 2的单链DNA所必需的。长期目标是了解VirB复合体的组装和功能。 为此，已经开发了各种遗传和生物化学方法来研究VirB蛋白与自身和与转运底物的相互作用。 最近的研究产生了令人惊讶的结果，即在受体中表达VirB蛋白的子集极大地提高了农杆菌之间virB介导的RSF 1010接合转移的效率。 该结果导致关于VirB蛋白和受体活性的几个问题：a）增加受体活性所需的VirB蛋白的最小补体是什么，B）受体中RSF 1010的存在是否影响该活性，以及c）该现象的一般性是什么？ 这些问题的答案可能会允许一个“核心”VirB复合物，可以简化遗传和生化研究的鉴定，并可能有重要的影响，质粒transfer一般为了定义VirB复合物及其活性，VirB蛋白和它们自己或运输底物之间的相互作用必须被理解。 在这个项目中，将继续进行物理/生物化学分析，利用化学交联和免疫沉淀;将进行酵母双杂交系统分析，特别研究其他VirB蛋白质-可能还有其他未鉴定的蛋白质-与VirB 7 -10组相互作用的可能性;并对virB 7 -10基因进行饱和诱变，以帮助确定蛋白质相互作用和一般毒力所需的区域。