MECHANISM OF PLASMID DNA TRANSFER IN BACTERIA

描述：（改编自研究者摘要） 本项目旨在阐明广寄主范围接合的机制 由革兰氏阴性菌的IncP质粒介导。 P质粒 携带抗生素抗性基因，并能够将DNA转移到 大多数革兰氏阴性菌以及革兰氏阳性菌和酵母菌。 最近 证据还表明IncP结合和 农杆菌介导的DNA向植物的转移。 IncP偶联的研究 将增强对基因交换机制的理解， P质粒作为遗传工具在多种医学重要领域的应用 细菌 当前提案的一个广泛目标是定义 必需转移蛋白和结合之间的结构相互作用 转移起源地内导致转移启动的位点 过程 oriT功能需要250个碱基对（bp）序列，并且 该位点需要两种质粒特异性蛋白质TraJ和TraI 在oriT启动转移的特异性切口。 具体目标 项目是使用定点突变来确定19个 bp反向重复序列和与切口位点相邻的序列，以及 将这些结构变化与蛋白质-DNA相互作用联系起来， 切刻复合物的必需TraJ和TraI蛋白及其 oriT中的结合位点。 第二个广泛的目标是定义转移基因， 基因产物负责广泛的宿主范围属性。 具体目标 分析这两种DNA引发酶蛋白在宿主范围内的作用， 构建专门产生一种或另一种的克隆，并使用 这些以补充引发酶缺失突变体转移到多种 革兰氏阴性菌，以确定两者结合中的作用 traI中由不同阅读框编码的基因。

DESCRIPTION: (Adapted from Investigator's Abstract) The long term goal of this project is to elucidate the mechanism of broad host range conjugation mediated by the IncP plasmids of gram negative bacteria. The P plasmids carry antibiotic resistance genes and are capable of transferring DNA to most gram negative bacteria as well as gram positives and yeast. Recent evidence also suggests a relationship between IncP conjugation and Agrobacterium mediated DNA transfer to plants. Study of IncP conjugation will enhance the understanding of gene exchange mechanisms and facilitate use of P plasmids as genetic tools in a wide variety of medically important bacteria. One broad goal of this current proposal is to define the structural interactions between essential transfer proteins and binding sites within the origin of transfer that lead to initiation of the transfer process. A 250 basepair(bp) sequence is required for oriT function, and two plasmid specific proteins, TraJ and TraI, are required for the site specific nicking that initiates transfer at oriT. Specific aims for this project are to use site directed mutagenesis to define the role of the 19 bp inverted repeat and the sequence adjacent to the nick site, and to correlate these structural changes with protein-DNA interactions between the essential TraJ and TraI proteins of the nicking complex and their binding sites in oriT. A second broad goal is to define transfer genes and gene products responsible for the broad host range property. Specific aims are to analyze the role of the two DNA primase proteins in host range by constructing clones that produce one or the other exclusively, and using these to complement a primase deletion mutant in transfer to a variety of gram negative bacteria, to determine the roles in conjugation of the two genes that are encoded by different reading frames in traI.