GENETIC ANALYSIS OF RICKETTSIAL INTRACELLULAR PARASITISM

普氏立克次体是流行性斑疹伤寒的病原体， 一种专性细胞内寄生细菌， 在其真核宿主细胞的细胞质内，未被 液泡膜 本研究的总体目标是 增加我们对生物化学和遗传学的理解 参与成功的细胞内寄生的机制， 这个有机体。 以前的工作，采用重组DNA 技术，导致了一些R的隔离。普劳瓦泽基 基因. 本申请中描述的工作的主要目标是 使用DNA对这些基因和基因产物的表征 序列分析和随后的比较研究， 在大肠杆菌中发现的相应基因和基因产物 和/或真核系统。 其他基因，编码酶 三羧酸循环中，将被分离，以便 研究立克次体基因协同表达。 的 立克次体细胞表面蛋白质编码基因的克隆 也将大力推进。 的分离与鉴定 这些基因和基因产物将成为 随后关于立克次体识别和渗透的研究 它的宿主细胞 这笔赠款的一个主要目标是利用 从分离和表征中获得的知识 立克次氏体基因，以探索调控机制， 活立克次体 克隆的基因或适当的片段将被 用作探针来检测特定的信使RNA。 最后 工作将开始建立一个系统的直接遗传 分析了R. prowazekii。 这将涉及筛选低 帕森斯河天然质粒的普氏菌株 可能用作立克次体载体。 另外研究 旨在评估能力或R。prowazekii采取和 表达外源DNA。 这项工作应 了解立克次体的基因结构， 功能，并确定直接遗传操作的限制， 这种专性细胞内寄生物。

Rickettsiae prowazekii, the etiologic agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of its eucaryotic host cell, unbound by a vacuolar membrane. The overall goal of this research is to increase our understanding of the biochemical and genetic mechanisms involved in the successful intracellular parasitism by this organism. Previous work, employing recombinant DNA techniques, has led to the isolation of a number of R. prowazekii genes. A major goal of the work described in this application is the characterization of these genes and gene products using DNA sequence analysis and subsequent comparison studies with corresponding genes and gene products found in Escherichia coli and/or eucaryotic systems. Additional genes, coding for enzymes of the tricarboxylic acid cycle, will be isolated in order to investigate coordinate expression of rickettsial genes. The cloning of genes coding for proteins of the rickettsial cell surface will also be vigorously pursued. Isolation and characterization of these genes and gene products will form the foundation for subsequent studies on rickettsial recognition and penetration of its host cell. A major objective of this grant will be to use the knowledge gained from the isolation and characterization of rickettsial genes to probe the regulatory control mechanisms in viable rickettsiae. Cloned genes or appropriate fragments will be used as probes to detect specific messenger RNA. Finally, this work will begin to establish a system for the direct genetic analysis of R. prowazekii. This will involve screening low passaged R. prowazekii strains for naturally-occurring plasmids potentially useful as rickettsial vectors. In addition, studies designed to assess the ability or R. prowazekii to take-up and express exogenous DNA will also be performed. This work should provide an understanding of rickettsial gene structure and function and define the limits of direct genetic manipulation in this obligate intracellular parasite.