BACTERIAL CELL MEMBRANE--GROWTH AND CELL DIVISION

细菌细胞膜——生长和细胞分裂

• 批准号: 3284664
• 负责人: MOSELIO SCHAECHTER
• 金额: $ 30.62万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3284664
• 关键词: DNA binding protein; DNA footprinting; DNA methylation; DNA replication; DNA replication origin; Escherichia coli; Salmonella typhimurium; bacterial DNA; bacterial genetics; bacterial proteins; binding proteins; cell cycle; cell membrane; chromosome movement; electron microscopy; gel electrophoresis; genetic promoter element; membrane proteins; membrane structure; microorganism growth; mutant; protein biosynthesis; protein structure; radiotracer; receptor; synchronous cell division

Our long term objective is to understand how chromosomal segregation takes place in bacteria, i.e., what is the equivalent of mitosis in prokaryotes? We believe that such knowledge will be useful for a general understanding of cell function and, in particular, to help define new targets for antibacterial chemotherapy. This proposal is based on our observation that the replication origin of the Escherichia coli chromosome binds to the membrane for a defined period of time during the cell cycle. We have postulated that this time represent a biological clock during which the incipient progeny chromosomes become relegated to the two cell halves. During this period, origin DNA is hemimethylated, that is, it is newly replicated, but has not yet been modified by the major E. coli methylase, Dam. We have found a membrane protein we call Hob that binds uniquely to hemimethylated origin DNA. We have also delineated the gene for this protein, hob, to within a lambdal phage of the Kohara collection. The E. coli DNA inserted into this phage includes a gene, pcsA, involved in chromosome segregation. Cold sensitive mutants in pcsA cannot partition their chromosome and are defective in cell division. We wish to determine whether the Hob protein acts to anchor the replicative origin to the membrane (thus functioning as part of a bacterial kinetochore-equivalent). Because this is a novel topic, we must first carry out considerable biochemical and genetic groundwork. To this end, we will ask the following questions: What is the sequence of Hob? Are the hob and pcsA genes the same? Is hob essential? What are cytological features of hob mutants? We will then study the sequence in oriC that is recognized by Hob, the specificity of the interaction, and the state of aggregation of Hob in solution. Because the Hob protein is unlikely to function in isolation, we will determine what other proteins it interacts with by looking for extragenic suppressors of conditional hob mutations. We will determine the intracellular localization of the Hob protein and possible "suppressor" proteins. Our model makes a strong prediction about the time in the cell cycle when the Hob protein binds to the DNA. We will test this notion by in vivo footprinting techniques using cells synchronized in their DNA replication. This study will also reveal the DNA "occupancy time" of other proteins during the initiation and early replication of the E. coli chromosome. Ultimately, the function of the Hob protein must be studied by physiological means. We recognize that such studies are difficult and must rely on a firm biochemical and genetic knowledge of the system. Should we make sufficient progress during the period of this grant, we will study the activity and regulation of Hob during the cell cycle, in cultures growing at different rates, and in the presence of different amounts of oriC in the cell.

我们的长期目标是了解染色体分离是如何 在细菌中的位置，即原核生物中有丝分裂的等价物是什么？ 我们相信，这些知识将有助于对 ，特别是为了帮助定义新的目标 抗菌化疗。 这一建议是基于我们的观察，即复制起源 大肠埃希氏菌染色体与膜结合一段时间 在细胞周期中的时间。我们假设这一次代表着 一种生物钟，在此期间初生的后代染色体变成 降级到两个牢房的一半。在此期间，起源DNA是 半甲基化，即它是新复制的，但还没有被复制 被主要的大肠杆菌甲基酶Dam修饰。我们发现了一层膜 我们称为HOB的蛋白质，它唯一地与半甲基化来源的DNA结合。我们 我还把这种名为hob的蛋白质的基因描绘成了一个lambdal 小原收藏的噬菌体。插入到该噬菌体中的大肠杆菌DNA 包括一个基因，PCSA，与染色体分离有关。对冷敏感 PCSA中的突变体不能分割其染色体，并且在细胞中存在缺陷 组织。 我们希望确定HoB蛋白是否起到锚定复制体的作用 膜的起源(因此作为细菌的一部分 动粒等价物)。因为这是一个新奇的话题，我们必须首先 进行大量的生化和遗传基础工作。为此，我们 我会问以下问题：HOB的顺序是什么？是不是 HOB和PCSA基因相同吗？滚刀是必不可少的吗？什么是细胞学？ HOB突变体的特征？然后我们将研究Oric中的序列，即 被Hob识别、交互的特殊性以及 HOB在溶液中的聚集。 由于HoB蛋白不太可能在孤立的情况下发挥作用，我们将 通过寻找基因外基因来确定它与哪些其他蛋白质相互作用 条件性HOB突变的抑制者。我们将确定 HOB蛋白在细胞内的定位及其可能的“抑制子” 蛋白质。 我们的模型对细胞周期中的时间做出了强有力的预测 HoB蛋白与DNA结合。我们将在活体内测试这一概念 足迹技术使用的细胞与其DNA复制同步。 这项研究还将揭示其他蛋白质的dna“占用时间”。 在大肠杆菌染色体的启动和早期复制过程中。 最终，必须通过以下方法研究HoB蛋白的功能 生理手段。我们认识到，这种研究是困难的，必须 依靠扎实的生化和遗传系统知识。我们要不要 在这笔拨款期间取得足够进展，我们会研究 HOB在细胞周期中的活性及其调控 在不同的速率下，以及在不同数量的ORIC存在的情况下 手机。