3D Chromosome/Replisome Positioning in Bacterial Cells

细菌细胞中的 3D 染色体/复制体定位

• 批准号: 7016341
• 负责人: LUCILLE SHAPIRO
• 金额: $ 38.82万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7016341
• 关键词: DNA binding protein; DNA replication; bacterial DNA; bacterial genetics; bioimaging /biomedical imaging; cell cycle; chromosome movement; cytoplasm; fluorescence microscopy; immunoprecipitation; three dimensional imaging /topography

DESCRIPTION (provided by applicant): This is a program of collaborative research between engineers and physicists at Stanford University together with specialists in advanced microscopy to determine the three dimensional (3D) configuration of the chromosome and the replisome in the bacterial cell as the cell cycle progresses. Specific aims are: 1. To precisely define the dynamic 3D organization of the chromosome within the non-replicating bacterial cell (the Caulobacter swarmer cell) and during the cell cycle while the chromosome is being replicated. To do this, we will perform high-resolution 3D imaging by EM tomography and soft X-ray tomography to locate and map chromosomal loci in the cell. We will also perform time-lapse fluorescent microscopy tracking of the same loci in living cells. 2. To identify the proteins that mediate the spatial deployment of both replicating and non-replicating chromosomes by (a) determining the effect of mutations in proteins known to be involved in chromosome organization, (b) carrying out an automated high throughput screen for mutants that mislocalize discrete chromosomal loci, (c) determining the effect of cell structure by examining the chromosome organization in long filamentous cells, (d) modeling and analyzing the chromosome movement taking account of the hydrodynamic properties of the cytoplasm, and (e) determining if the actin-like MreB protein directly or indirectly binds to DNA at different times in the cell cycle using chromosome immunoprecipitation assays. 3. To define the spatial deployment of the replisome (replication factory) in the Caulobacter celt during its assembly at the cell pole and its movement during DNA replication. To do this, we will (a) use high resolution (20-100 nm) imaging by EM tomography and soft X-ray tomography, (b) use total internal reflection (TIR) microscopy to determine if the moving replisome follows an axial or a spiral path on its way to the cell division plane, and (c) use tomographic imaging to determine if the replisome co-positions with and follows the path of the MreB spiral that is deployed along the long axis of the cell.

描述（由申请人提供）：这是斯坦福大学工程师与物理学家之间的合作研究计划，以及高级显微镜专家，以确定染色体的三维（3D）配置，随着细胞周期的进展，细菌细胞中的三维（3D）配置。具体目的是：1。精确定义非复制细菌细胞内染色体的动态3D组织（Caulobacter蜂窝细胞）以及在复​​制染色体时细胞周期期间。为此，我们将通过EM断层扫描和软X射线断层扫描进行高分辨率3D成像，以定位和映射细胞中的染色体基因座。我们还将对活细胞中同一基因座进行延时荧光显微镜跟踪。 2. To identify the proteins that mediate the spatial deployment of both replicating and non-replicating chromosomes by (a) determining the effect of mutations in proteins known to be involved in chromosome organization, (b) carrying out an automated high throughput screen for mutants that mislocalize discrete chromosomal loci, (c) determining the effect of cell structure by examining the chromosome organization in long filamentous （d）通过考虑细胞质的流体动力特性进行建模和分析染色体运动，以及（e）确定使用染色体免疫沉淀分子在细胞周期中直接或间接地在细胞周期中直接或间接与DNA结合。 3。定义在花椰菜凯尔特（Caulobacter Celt）在细胞极点组装过程中的重置体（复制工厂）的空间部署，并在DNA复制过程中移动。 To do this, we will (a) use high resolution (20-100 nm) imaging by EM tomography and soft X-ray tomography, (b) use total internal reflection (TIR) microscopy to determine if the moving replisome follows an axial or a spiral path on its way to the cell division plane, and (c) use tomographic imaging to determine if the replisome co-positions with and follows the path of the MreB spiral that is deployed along the long细胞的轴。