SITE-SPECIFIC DNA INVERSION IN MORAXELLA LACUNATA

DNA重排，包括缺失、倒置、转座或 插入，已被证明控制基因的表达和发育 众多系统，从整合/切除噬菌体 Lambda对哺乳动物基因片段重排的影响 免疫球蛋白基因。相对简单的DNA重排研究 在原核生物中不仅揭示了控制，例如， 微生物病原体表面抗原的时相变化 赫氏疏螺旋体、奈瑟氏淋病和鼠伤寒沙门氏菌 也为理解基因表达的调控提供了模型。 在高等生物体中。本文件中描述的工作的目标 建议是定义特定部位DNA的分子细节 空腔莫拉氏菌的倒置系统。在M.lacunata中，一个使役 治疗人类结膜炎和角膜炎的药物，染色体倒位 片段调控两个4型菌毛蛋白基因的表达。初步 对M.lacunata倒置系统的描述表明， 利用了一种新的位点特异性DNA倒置机制。这个 这个项目的具体目标是确定蛋白质和DNA 组件，并描述这些元素如何 相互作用，形成一个富有成效的突触复合体。三步走的方法 将采取以下措施来实现这些目标：1)定义必需基因 以及体内和体外重组反应的DNA位点 亚克隆的插入和缺失/替换突变分析 反转区，识别的重组因子的过度表达， 以及开发用于反转反应的已定义的体外系统； 2)描述基本组件之间的相互作用 使用核酸酶/化学保护/干扰分析的转化系统； 3)阐明重组反应的分子细节 重组中间体的生化/物理分析，以及 重组酶和重组位点的突变分析。

DNA rearrangements, involving deletion, inversion, transposition, or insertion, have been shown to control gene expression and development in numerous systems ranging from integration/excision of bacteriophage lambda to rearrangement of the gene segments of the mammalian immunoglobulin genes. the study of relatively simple DNA rearrangements in prokaryotes not only reveals the mechanisms controlling, for example, phase variation of surface antigens in microbial pathogens such as Borrelia hermsii, Neisseria gonorrhea, and Salmonella typhimurium but also provides models for understanding the regulation of gene expression in higher organisms. The objective of the work described in this proposal is to define the molecular details of the site-specific DNA inversion system of Moraxella lacunata. In M. lacunata, a causative agent in human conjunctivitis and keratitis, inversion of a chromosomal segment regulates the expression of two type 4 pilin genes. Preliminary characterization of the M. lacunata inversion system suggests that a novel mechanism for site-specific DNA inversion is utilized. The specific goals of this project are to determine the protein and DNA components of this inversion system and characterize how these elements interact to form a productive synaptic complex. A three step approach will be taken to accomplish these goals: 1) Define the essential genes and DNA sites for the recombination reaction in vivo and in vitro by insertion and deletion/substitution mutagenesis analyses of the subcloned inversion region, over-expression of identified recombination factors, and development of a defined in vitro system for the inversion reaction; 2) Characterize the interactions among the essential components of the inversion system using nuclease/chemical protection/interference assays; 3) Elucidate the molecular details of the recombination reaction using biochemical/physical assays of recombination intermediates, and mutational analyses of the recombinase and recombination sites.