Genetic Analysis of Escherichia coli lac Permease Biogenesis

MANOIL，ColinMCB-9905048大肠埃希氏菌乳胶渗透酶生物遗传分析该项目有两个总体目标。第一个是开发一种在细菌中发现基因的程序，该程序基于识别基因标记蛋白质的亚细胞位置或生化活动。出口的蛋白质被选择性地标记上63个氨基酸的插入序列，这允许免疫学检测和快速纯化。这些标签是通过染色体插入新构建的转座子而产生的。筛选菌种库，寻找产生具有所需性质的标记蛋白的个体，例如定位到特定膜或特定酶活性，然后对相应的标记基因进行序列鉴定。该项目的第二个总体目标是分析之前发现的一些突变是如何使大肠杆菌乳胶通透酶失活的，以及细胞如何对这些突变蛋白做出反应。具体地说，这些实验的目的是，1，确定几种不同的突变是否通过对膜插入、折叠或功能的主要影响而改变乳胶通透酶的关键区域(细胞质片段C3)来使蛋白质失活；2，确定几种已知的应激反应或新的应激反应是否由突变的乳胶渗透酶诱导。这项研究将使用功能基因组学方法来开发鉴定细菌蛋白质的生物学功能和细胞内定位的策略。其他实验将研究细菌蛋白质的一个重要子类--完整的膜蛋白是如何通过突变改变而改变的，以及这些改变的蛋白质如何影响细胞生理。这两种方法都将有助于加深对微生物生命所需的细胞过程的整合的理解。

Manoil, ColinMCB-9905048Abstract - Genetic Analysis of Escherichia coli lac permease biogenesisThe project has two general objectives. The first is to develop a procedure for gene discovery in bacteria which is based on identifying the subcellular locations or biochemical activities of genetically tagged proteins. Exported proteins are selectively tagged with a 63 amino acid inserted sequence which permits immunological detection and rapid purification. The tags are generated by chromosomal insertion of a newly constructed transposon. Pools of strains are screened for individuals producing tagged proteins with a desired property, such as localization to a particular membrane or a particular enzymatic activity, followed by sequence identification of corresponding tagged genes. The second general objective of the project is to analyze how a number of previously identified mutations inactivate E. coli lac permease, and how the cell responds to such mutant proteins. Specifically, the experiments aim, 1, to determine whether several different mutations altering a key region of lac permease (cytoplasmic segment C3) inactivate the protein by primary effects on membrane insertion, folding, or function, and 2, to determine whether several known stress responses, or a new stress response, are induced by mutant lac permeases. This research will use a functional genomics approach to develop strategies for identifying the biological function and intracellular localization of bacterial proteins. Additional experiments will examine how an important subclass of bacterial proteins, integral membrane proteins, are altered by mutational changes, as well as how such altered proteins affect cell physiology. Both of these approaches will contribute to an increased understanding of the integration of cellular processes required for microbial life.