STRUCTURE AND GENETIC CONTROL OF COLICINES

这项建议涉及到对人的遗传和生化分析 抗生素耐药性复制启动的调控 质粒R6K在大肠杆菌中的表达。该多拷贝质粒大小为38kb。 大小，并指定对抗生素氨苄西林和 链霉素。以前的研究已经定义了连续复制 大小为4千碱基的区域。在这个地区有三个发源地 复制，命名为Alpha、Beta和Gamma，已被识别并 被证明在体内和体外复制系统中起作用。在……里面 此外，一个基因(Pir)指定复制起始蛋白(PI)是 位于伽马和贝塔起源之间，是以下活动所必需的 这三个来源都是。PI蛋白是多功能的，因为它表现出 启动R6K复制的正负活性和 自动调节pir基因的表达。三者的活动 复制起点还需要在顺式基因中有7个22碱基对的直接重复 位于伽马起源区域。分子遗传学与生物化学 分析将针对确定PI蛋白在体内的作用。 R6K起始的调控，重点是R6K的性质 PI蛋白与直接重复区、其他片段的相互作用 R6K复制子和大肠杆菌复制蛋白。介绍了 PI蛋白的野生型和突变型以及直接重复序列将是 用凝胶电泳法、电子显微镜和核磁共振技术进行分析 光谱学技术。直接基因突变的影响 重复区域和PI蛋白对RNA转录本形成的影响 R6K复制子和复制也将在体外进行检测。R6K基因 指导从Beta源开始复制的产品具有 已经确定并将被确定为特征。此外，分子遗传学 研究方向将针对pir基因的自动调节机制。 这种自我调节在质粒拷贝数中的表达及其作用 控制力。最后，大肠杆菌宿主蛋白在R6K复制中的作用 将通过分离和鉴定可改变 野生型和突变型R6K质粒的复制特性。这些 实验方法被设计用来阐明 监管机制及其相互作用的性质 为了控制质粒R6K的拷贝数，一个主要的成员 一组以存在直接重复序列为特征的质粒组 复制起点和编码复制蛋白的质粒。

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.