CHROMOSOME & PLASMID SEGREGATION IN BACTERIA

染色体

• 批准号: 6180690
• 负责人: ANDREW WRIGHT
• 金额: $ 23.82万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180690
• 关键词: Escherichia coli; bacterial genetics; chimeric proteins; chromosome movement; gene mutation; genetic mapping; green fluorescent proteins; nucleic acid sequence; plasmids

DESCRIPTION (adapted from investigator's abstract): The long range goal of this project is to understand the mechanisms by which the chromosome and low copy plasmids P1 and F of E. coli segregate into daughter cells. Dr. Wright proposes two approaches to study this process. In the first, he will isolate mutants with defects in the segregation process in order to identify genes and gene products which are involved. He has developed a direct selection for such mutants which makes use of cells carrying derivatives of the low copy plasmids P1 and F which carry genes that can be selected against. Selection for simultaneous loss of both plasmids yields segregation mutants with defects in either plasmid segregation or in plasmid and chromosome segregation. The method will be used to isolate a range of mutations which will be characterized by mapping, sequence analysis and by function of the deduced gene products. This approach should allow him to define many of the components of the segregation machinery. In the second approach, he will use a derivative of Green Fluorescent Protein, fused to the lac repressor (LacI) which binds to lac operator DNA to study chromosome and plasmid behavior in living cells. By placing an array of lac operator sequences at different sites on the chromosome, he will be able to examine sites that are replicated at different times and that may be localized differently in the cell. He will examine plasmids P1 and F, carrying lac operator sites, in the same way. The resolution of the technique should allow him to determine when particular sites on the chromosome are duplicated, when plasmids are duplicated, when they separate from one another and where they become localized within the cell. He will utilize the same technique to characterize mutants isolated by the first approach. This approach should yield new information about the components of the segregation apparatus in bacteria which must include elements that specify position within the cell. Such positional information is crucial for proper growth and division in all cells. The present approach presents an opportunity to identify positional elements in bacteria, about which little is currently known.

描述（改编自研究者的摘要）：长期目标 该项目旨在了解染色体和低水平的机制 大肠杆菌的复制质粒 P1 和 F 分离到子细胞中。 赖特博士 提出了两种研究这一过程的方法。 首先，他将 分离分离过程中存在缺陷的突变体，以便识别 所涉及的基因和基因产物。 他开发了一种直接 利用携带以下衍生物的细胞来选择此类突变体 携带可选择基因的低拷贝质粒P1和F 反对。 选择同时丢失两个质粒的产量 质粒分离或质粒中有缺陷的分离突变体 和染色体分离。 该方法将用于隔离一系列 突变将通过作图、序列分析和 推导的基因产物的功能。 这种方法应该让他能够 定义了分离机械的许多组件。 在第二个 方法中，他将使用绿色荧光蛋白的衍生物，融合 与 lac 操纵基因 DNA 结合以研究染色体的 lac 阻遏蛋白 (LacI) 和活细胞中的质粒行为。 通过放置 lac 运算符数组 染色体上不同位点的序列，他将能够检查 在不同时间复制且可能本地化的站点 细胞内不同。 他将检查携带 lac 的质粒 P1 和 F 运营商站点，以同样的方式。 该技术的分辨率应 让他确定染色体上的特定位点何时 复制，当质粒复制时，当它们与一个质粒分离时 另一个以及它们在细胞内定位的位置。 他将利用 使用相同的技术来表征通过第一种方法分离的突变体。 这种方法应该会产生有关该组件的新信息 细菌中的分离装置必须包含指定的元素 细胞内的位置。 此类位置信息对于正确定位至关重要 所有细胞的生长和分裂。 本方法提出了一种 识别细菌中位置元素的机会，这方面的知识很少 目前已知。