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发生的 放置在细菌中，即，原核生物中有丝分裂的等价物是什么 我们认为，这些知识将有助于普遍了解 特别是帮助确定新的靶点， 抗菌化疗 这一建议是基于我们的观察，即复制起点的 大肠杆菌染色体在一段确定的时间内与细胞膜结合 在细胞周期中。 我们假设这一时间代表 一种生物钟，在此期间， 被降级到两个半细胞。 在此期间，原始DNA 半甲基化，也就是说，它是新复制的，但还没有被复制。 由E大调修改。coli甲基化酶、Dam. 我们发现了一层膜 我们称之为Hob的蛋白质，它唯一地与半甲基化的原始DNA结合。 我们 我还描绘了这种蛋白质的基因，hob，在一个基因组中， 小原收藏的噬菌体。 急诊插入噬菌体的大肠杆菌DNA 包括参与染色体分离的基因pcsA。 冷敏 pcsA中的突变体不能分配它们的染色体，并且在细胞中是有缺陷的。 师. 我们希望确定Hob蛋白是否起锚定复制基因的作用， 起源于膜（因此作为细菌的一部分） 动势舞蹈等效）。 因为这是一个新的话题，我们必须首先 进行大量的生物化学和遗传学基础研究。 为此我们 会问以下问题：滚刀的顺序是什么？ 是 hob和pcsA基因相同吗 炉灶必不可少吗？ 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， 由Hob识别，相互作用的特异性，以及 Hob在溶液中的聚集。 由于Hob蛋白不太可能单独发挥作用，我们将 通过寻找基因外的蛋白质来确定它与哪些其他蛋白质相互作用 条件性hob突变的抑制子。 康贝特人将以 Hob蛋白的细胞内定位和可能的"抑制因子" proteins. 我们的模型对细胞周期中的时间做出了强有力的预测， Hob蛋白会与DNA结合 我们将通过体内试验来验证这一概念。 使用DNA复制同步的细胞足迹技术。 这项研究还将揭示其他蛋白质的DNA“占用时间” 在E. coli染色体。 最终，Hob蛋白的功能必须通过以下方法来研究： 生理手段。 我们认识到，这种研究是困难的，必须 依赖于系统的坚实的生物化学和遗传知识。 我们应该 在此期间取得足够的进展，我们将研究 在细胞周期中，在培养物中生长的Hob的活性和调节 以不同的速率，并在存在不同量的oriC在 cell.