MEMBRANE SUBSTRUCTURE AND CELL DIVISION IN E. COLI

所有细胞都表现出生化反应，这些反应只发生在 在细胞膜内的特定位置。 根本 问题是为什么反应发生在一个地方，而不是一个地方。 在其他地方看起来相当的位置。 两个最高有效 大肠杆菌中的这些过程是相关的 细胞分裂：形成隔分裂平面，以及 染色体分离到子细胞。 远程 目的是了解分子机制， 细胞控制这些事件的位点特异性。 这项建议 旨在确定E.大肠杆菌的贡献 这个机制。 将对以前证明的方法进行改进 膜囊泡可以分成更广泛的类别 经典的内/外膜定义。 这些方法 包括通过电荷分离（囊泡电泳通过 稀释琼脂糖）和大小（通过Sephacryl- Slooo。 将开发其他技术，包括快速 基于凝集素亲和力的测定和分级分离。 这些方法将有助于回答以下问题： 蛋白质具有特定的序列， 囊泡亚群;主要的细胞分裂蛋白相互作用 在“分隔子域”中彼此相连;染色体是否 与特定的膜结构相互作用 第一个问题 将通过测量融合蛋白的位置来解决， 报告酶碱性磷酸酶 的余留问题 将解决：免疫印迹定位蛋白质在 野生型和突变型细菌的囊泡; 青霉素结合蛋白质类;基因鉴定 X174裂解蛋白E;和核酸杂交以检测DNA- 囊泡协会，使用oriC和mini-F质粒作为 实验探测器 从长远来看，了解E。大肠杆菌产生并维持 以及这种结构如何 细胞分裂的基础， 生物化学过程在空间上是受限制的， 这种排列调节细胞的生长和分裂， 可以抑制这些过程以控制细菌感染。

All cells exhibit biochemical reactions which occur exclusively at specific locations within their membranes. The fundamental question is why the reactions occur at one site instead of a seemingly equivalent location elsewhere. The two most significant of these processes in the bacterium Escherichia coli are related to cell division: formation of the septal division plane, and chromosomal segregation to daughter cells. The long-range objective is to understand the molecular mechanism by which the cell controls the site-specificity of these events. This proposal is designed to determine how the membrane of E. coli contributes to this mechanism. Improvements will be made in methods which previously demonstrated that membrane vesicles can be separated into classes more extensive than the classical inner/outer membrane definition. These methods include separation by charge (electrophoresis of vesicles through dilute agarose) and by size (chromatography through Sephacryl- SlOOO. Additional techniques will be developed, including rapid assays and fractionation based on lectin affinity. These methods will help answer the following questions: do proteins have specific sequences which direct them to different vesicle sub-populations; do major cell division proteins interact with one another in "septation subdomains;" and does the chromosome interact with a particular membrane structure? The first question will be addressed by surveying the location of proteins fused to the reporter enzyme, alkaline phosphatase. The remaining questions will be addressed by: immunoblot localization of proteins in vesicles of wild-type and mutant bacteria; localization of penicillin-binding proteins; genetic characterization using the X174 lysis protein E; and nucleic acid hybridization to detect DNA- vesicle associations, using oriC and mini-F plasmids as experimental probes. In the long term, knowing how E. coli creates and maintains organization within its membrane and how that structure contributes to cell division may form the basis for understanding how biochemical processes are spatially restricted, how this arrangement regulates cell growth and division, and how these processes may be inhibited to control bacterial infections.