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隔离如何进行 放置在细菌中，即原核生物中有丝分裂的等效物是什么？ 我们认为，这样的知识对于一般理解将很有用 细胞功能，尤其是为了帮助定义新目标 抗菌化疗。 该提议是基于我们的观察，即复制起源 大肠杆菌染色体在定义的时期与膜结合 在细胞周期中的时间。 我们假设这次代表 生物时钟，在此期间，初期后代染色体变成 降级为两半。 在此期间，原点DNA为 半甲基化，即，它是新复制的，但尚未 由主要的大肠杆菌甲基酶大坝修饰。 我们发现了一个膜 我们称之为hob，它与半甲基化的起源DNA唯一结合。 我们 还将该蛋白质hob的基因描绘成羔羊 Kohara系列的噬菌体。 大肠杆菌DNA插入此噬菌体 包括参与染色体分离的基因PCSA。 冷敏感 PCSA中的突变体不能分割其染色体，并且在细胞中有缺陷 分配。 我们希望确定HOB蛋白是否作用于锚定复制 起源于膜（因此作为细菌的一部分起作用 kinetochore等效）。 因为这是一个新颖的话题，我们必须首先 进行大量的生化和遗传基础。 为此，我们 会问以下问题：HOB的顺序是什么？ 是 HOB和PCSA基因相同吗？ 霍布必不可少吗？ 什么是细胞学 霍布突变体的功能？ 然后，我们将研究Oric中的序列 通过HOB认可，互动的特异性和 HOB在解决方案中的聚集。 因为Hob蛋白不太可能孤立起作用，我们将 通过寻找外部化来确定与其他蛋白质相互作用的其他蛋白质 有条件霍布突变的抑制剂。 我们将确定 霍布蛋白的细胞内定位和可能的“抑制剂” 蛋白质。 我们的模型对细胞周期中的时间进行了强烈的预测 HOB蛋白与DNA结合。 我们将通过体内测试这个概念 使用在其DNA复制中同步的细胞进行足迹技术。 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，必须研究霍布蛋白的功能 生理手段。 我们认识到这样的研究很困难，必须 依靠系统的生化和遗传知识。 我们应该 在这笔赠款期间取得足够的进展，我们将研究 在细胞周期中，培养物的活动和调节 以不同的速度，在存在不同量的oric的情况下 细胞。