CONJUGATIVE MOBILIZATION OF PLASMID R1162

质粒 R1162 的接合动员

• 批准号: 2178775
• 负责人: RICHARD J MEYER
• 金额: $ 17.93万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178775
• 关键词: DNA binding protein; DNA replication; Escherichia coli; affinity chromatography; biochemical evolution; genetic manipulation; genetic mapping; genetic recombination; host organism interaction; microorganism conjugation; molecular cloning; mutant; nucleic acid sequence; open reading frames; plasmids; point mutation; protein biosynthesis; protein purification; protein sequence; protein transport; site directed mutagenesis; transcription factor; transposon /insertion element

The experiments outlined in this proposal are directed toward an understanding of the DNA processing reactions that occur during conjugative transfer of the broad host-range plasmid R1162. A model is proposed in which the origin of transfer (oriT) consists of two domains, one required for initial nicking of the DNA prior to transfer, and the other for subsequent recircularization of the transferred but overlapping regions of a large open reading frame (ORFI). Mutations will be isolated in oriT to identify and map the different functional domains. Proteins fragments of ORFT that are involved in different processing events of oriT will be purified, and their biochemical activities tested in vitro. Protein-DNA relaxation complex will be characterized to identify the processing intermediate related to this structure. DNA synthesis occurs from within oriT in cell extracts and is dependent on the plasmid genes for transfer. The origin and direction of this synthesis will be determined. ORFI overlaps rep2, a gene required for plasmid DNA replication. The genetic relationship between rep2 and ORFI will be clarified, and the involvement of the rep2 region in DNA synthesis from oriT will be assessed. Mutations in oriT will also be tested to see whether nicking is required for synthesis. Another mobilization protein, MobI, complements the rep2 region of ORFI in vivo, and inhibits the processing of single-stranded oriT DNA. MobI will be tested for support of DNA synthesis from oriT, and the target of this protein will be identified. Finally, the R1162 DNA strands that are transferred during conjugation will be identified by capturing this DNA in infectious particles.

本提案中概述的实验针对 了解在此期间发生的DNA处理反应 宽宿主范围质粒R1162的共轭转移。 模型是 提出的转移起源（orit）由两个域组成， 转移前DNA初始划分的必需品，而 其他用于随后转移但重叠的循环 大型开放式阅读框（ORFI）的区域。 突变将被隔离 在Orit中识别和映射不同的功能域。 蛋白质 ORIT的不同处理事件涉及的ORFT片段 将被纯化，并在体外测试其生化活性。 蛋白质-DNA松弛复合物将被表征以识别 处理与此结构相关的中间体。 DNA合成来自细胞提取物中的Orit内部，取决于 质粒基因转移。 此的起源和方向 将确定合成。 ORFI重叠Rep2，一个基因 质粒DNA复制。 REP2和ORFI之间的遗传关系 将澄清，Rep2区域参与DNA合成 将评估Orit。 ORIT中的突变也将进行测试以查看 合成是否需要划痕。 另一个动员蛋白， mobi，对体内ORFI的REP2区域进行补充，并抑制 单链orit DNA的处理。 MOBI将经过测试以获得支持 来自Orit的DNA合成，该蛋白的靶标将是 确定。 最后，在此期间转移的R1162 DNA链 通过在传染性中捕获此DNA来确定结合 颗粒。