STRUCTURE AND GENETIC CONTROL OF COLICINES

Colicines 的结构和遗传控制

• 批准号: 3124348
• 负责人: DONALD R HELINSKI
• 金额: $ 19.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-02-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3124348
• 关键词: Escherichia coli; R factors; ampicillin; bacterial DNA; bacterial genetics; bacteriophage lambda; cell free system; chemical structure function; circular DNA; colicines; electron microscopy; gel electrophoresis; gene dosage; gene expression; genetic manipulation; genetic promoter element; genetic recombination; genetic strain; molecular cloning; mutant; nuclear magnetic resonance spectroscopy; nucleic acid sequence; radiotracer; streptomycin

This proposal is concerned with a genetic and biochemical analysis of the regulation of initiation of replication of the antibiotic resistance plasmid R6K in Escherichia coli. This multi-copy plasmid is 38 kilobases in size and specifies resistance to the antibiotics ampicillin and streptomycin. Previous studies have defined a contiguous replication region that spans 4 kilobases in size. Within this region three origins of replication, designated Alpha, Beta and Gamma, have been identified and shown to function in vivo and in an in vitro replication system. In addition, a gene (pir) specifies a replication initiation protein (Pi) is located between the Gamma and Beta origins and is required for activity of all three origins. The Pi protein is multi-functional in that it exhibits positive and negative activity in the initiation of R6K replication and autoregulates the expression of the pir gene. The activity of the three replication origins also requires in cis seven 22 base pair direct repeats located in the Gamma-origin region. Molecular genetic and biochemical analysis will be directed at determining the role of the Pi protein in the regulation of initiation of plasmid R6K with emphasis on the nature of the interaction of the Pi protein with the direct repeat region, other segments of the R6K replicon, and E. coli replication proteins. The introduction of wild-type and mutant forms of the Pi protein and the direct repeats will be analyzed using gel electrophoresis, electron microscopy and NMR spectroscopy techniques. The effect of mutational changes in the direct repeat region and the Pi protein on the formation of RNA transcripts of the R6K replicons and replication also will be examined in vitro. An R6K gene product that directs the initiation of replication from the Beta-origin has been identified and will be characterized. In addition molecular genetic approaches will be directed at the mechanism of autoregulation of pir gene expression and the role of this autoregulation in plasmid copy number control. Finally, the role of E. coli host proteins in R6K replication will be explored by isolating and characterizing E. coli mutants that alter the replication properties of wild-type and mutant R6K plasmids. These experimental approaches are designed to elucidate the major components of the regulatory machinery and the nature of their interactions responsible for the control of the copy number of plasmid R6K, a member of a major group of plasmids characterized by the presence of direct repeats at a replication origin and a plasmid encoded replication protein.

这项建议涉及到遗传和生物化学分析， 抗生素耐药性复制起始的调节 质粒R6 K在大肠杆菌中。 这个多拷贝质粒是38个内切酶 并指定对抗生素氨苄青霉素和 链霉素。 以前的研究已经定义了一个连续的复制 在大小上跨越4个半音阶的区域。 在这一地区， 复制，指定为阿尔法，贝塔和伽马，已经确定， 显示在体内和体外复制系统中起作用。 在 此外，指定复制起始蛋白（Pi）的基因（pir）， 位于伽马和β原点之间， 三个起源 Pi蛋白是多功能的，因为它表现出 在R6 K复制起始中的阳性和阴性活性， 自动调节PIR基因的表达。 三人的活动 复制起点顺式也需要7个22碱基对的直接重复 位于伽玛射线源区 分子遗传与生物化学 分析将旨在确定Pi蛋白在其中的作用 质粒R6 K起始的调节，重点是 Pi蛋白与直接重复区、其他片段 R6 K复制子的表达，E. coli复制蛋白。 引入 野生型和突变形式的Pi蛋白和同向重复序列将是 使用凝胶电泳、电子显微镜和NMR分析 光谱技术。 突变的影响，在直接 重复区域和Pi蛋白对RNA转录物形成的影响 还将在体外检查R6 K复制子和复制。 R6 K基因 指导从β-起点开始复制的产物具有 已被识别并将被描述。 此外，分子遗传 pir基因自身调节机制的研究将是今后研究的重点 表达和这种自我调节在质粒拷贝数中的作用 控制 最后，对E. R6 K复制中的大肠杆菌宿主蛋白 将探讨通过分离和表征E.大肠杆菌突变体， 野生型和突变型R6 K质粒的复制特性。 这些 实验方法旨在阐明的主要组成部分， 监管机制及其相互作用的性质 为了控制质粒R6 K的拷贝数，主要的 一组质粒，其特征在于在一个位点存在直接重复序列， 复制起点和质粒编码的复制蛋白。