GENETIC ANALYSIS OF RICKETTSIA PROWAZEKII

普氏立克次体是流行性斑疹伤寒的病原体， 人类的虱传疾病。了解这种强制性疾病的生物学 细胞内的细菌寄生虫，以及有助于其 毒力受到与生长相关的困难的阻碍， 鸡蛋和细胞培养物中的立克次体， 在真核细胞内的生长条件，以及缺乏遗传 系统 立克次体突变体几乎不存在， 机制尚未确定。 因此，为了调查 R的一些基本属性。prowazekii，我们选择了 应用重组DNA技术，克隆了R.普氏DNA进入 大肠杆菌宿主。 通过这种方式，立克次体基因、基因产物和 监管机制可以在一个高度 可管理的系统。 克隆库的建立将使用多种 质粒和粘粒载体以及设计用于改变大小的克隆方法 和克隆DNA的拷贝数。 这样，克隆的概率 任何特定的基因都被增强。 克隆库将由以下人员筛选： 免疫学方法检测立克次体抗原的存在。 在 此外，将采用直接选择技术分离立克次体 与已知E.大肠杆菌营养缺陷型突变。 立克次体 筛选在E.大肠杆菌包括细胞质酶， 膜结合活性和R. prowazekii表面抗原 克隆 表达立克次体活性将提供基因产物， 生化研究和精确定义的R.普氏DNA片段 立克次体基因结构和表达的研究。

Rickettsia prowazekii is the etiologic agent of epidemic typhus, a louseborne disease of man. Understanding the biology of this obligate intracellular bacterial parasite, and the factors which contribute to its virulence has been hampered by the difficulties associated with growing rickettsiae in eggs and cell cultures, the impossibility of controlling growth conditions within a eukaryotic cell, and the absence of a genetic system. Rickettsial mutants are practically nonexistent and gene transfer mechanisms have not been identified. Accordingly, in order to investigate some of the fundamental attributes of R. prowazekii, we have elected to apply recombinant DNA techniques and clone R. prowazekii DNA into Escherichia coli hosts. In this way rickettsial genes, gene products, and regulatory mechanisms can be examined and manipulated in a highly manageable system. Clone banks will be established using a variety of plasmid and cosmid vectors and cloning methods designed to vary the size and copy number of the cloned DNA. In this way, the probability of cloning any specific gene is enhanced. Clone banks will be screened by immunological methods to detect the presence of rickettsial antigens. In addition, direct selection techniques will be used to isolate rickettsial genes complementing known E. coli auxotrophic mutations. The rickettsial genes to be screened for expression in E. coli include cytoplasmic enzymes, membrane-bound activities and R. prowazekii surface antigens. Clones expressing rickettsial activities will provide gene products for biochemical studies and precisely defined R. prowazekii DNA segments for studies on rickettsial gene structure and expression.