The Segregation of Bacterial Chromosomes to Daughter Cel

细菌染色体向子细胞的分离

• 批准号: 7338467
• 负责人: STUART AUSTIN
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7338467
• 关键词: Segregation; Bacterial; Chromosomes; Daughter; Cel

The bacterium Escherichia coli has a single, circular chromosome that is replicated and segregated with great precision to daughter cells during cell division. Replication proceeds bi-directionally from a single origin and terminates on the opposite side of the chromosome. The relative simplicity of this system and the limited number of cell components required for its propagation make it a model system for DNA replication and segregation in general.We have developed a P1 parS GFP-ParB system for localization by fluorescent microscopy of any desired locus on the E. coli chromosome in living cells. Using similar DNA recognition systems of different specificities, we can now label up to three chromosomal loci simultaneously, using three differently colored fluorescent proteins. The technique works well in living cells and allows us to follow the fate of chromosomal sequences through several generations by time-lapse microscopy. In addition, we have used the technique, in combination with flow cytometry, to determine the spatial distributions of given loci at defined points in the cell cycle in a cell population. This effort has been greatly augmented by collaboration with the laboratory of Flemming Hansen, the Technical university of Denmark. With him, have developed automated methods for the measurement of the positions of fluorescent foci in the cells that permits accurate measurement of thousands of cells from microscopic images. We are also developing rapid methods for the analysis of the large data sets that we are able to collect. These methods provide us with powerful tools for the investigation of the replication and segregation dynamics of the chromosome. So far, we have been able to disprove the currently popular model for chromosome segregation involving simultaneous segregation of the bulk of the DNA. Rather, we show clearly that DNA is segregated progressively as it is replicated. Our investigations are revealing unexpected features of DNA organization and motion, including the fact that the two arms of the circular chromosome lie in opposite halves of the resting cell. We are currently investigating many other aspects of the process, and hope to be able to derive a complete description of the segregation process in the near future. The visible properties of DNA replication and segregation need to be linked to the biochemical and structural properties of the macromolecules involved in the key events. To date, we have made significant progress in understanding the role of the SeqA protein that is involved in both replication and segregation of the chromosome. In collaboration with Dr. Alba Guarne (McMaster University) we have solved the crystal structure of the SeqA protein in a complex with its cognate DNA sequence. Using the structure as a guide, we have constructed mutant proteins and have determined their effects on DNA replication and segregation. These studies have lead us to a working model for the roles of SeqA that is currently being tested.

大肠杆菌有一个单一的圆形染色体，在细胞分裂过程中可以非常精确地复制和分离到子细胞。复制从单一起始点双向进行，并终止于染色体的另一侧。该系统相对简单，繁殖所需的细胞成分数量有限，使其成为DNA复制和分离的一般模型系统。我们开发了一种P1 PARS GFP-PARB系统，用于通过荧光显微镜在活细胞中定位大肠杆菌染色体上的任何所需位置。使用不同特异性的相似DNA识别系统，我们现在可以同时标记多达三个染色体位点，使用三种不同颜色的荧光蛋白。这项技术在活细胞中工作得很好，使我们能够通过延时显微镜跟踪几代人的染色体序列的命运。此外，我们还将该技术与流式细胞术相结合，用来确定细胞群体中细胞周期中特定位置的空间分布。通过与丹麦技术大学弗莱明·汉森的实验室合作，这一努力得到了极大的加强。与他一起，开发了测量细胞中荧光焦点位置的自动化方法，使从显微图像中准确测量数千个细胞成为可能。我们还在开发快速方法，用于分析我们能够收集的大型数据集。这些方法为我们研究染色体的复制和分离动力学提供了强有力的工具。到目前为止，我们已经能够反驳目前流行的染色体分离模型，该模型涉及同时分离大部分DNA。相反，我们清楚地表明，DNA在复制时是逐步分离的。我们的研究揭示了DNA组织和运动的意想不到的特征，包括环形染色体的两个臂位于静止细胞的相反一半的事实。我们目前正在调查这一过程的许多其他方面，并希望在不久的将来能够得出关于隔离过程的完整描述。DNA复制和分离的可见属性需要与关键事件中涉及的大分子的生化和结构属性相联系。到目前为止，我们在了解SEQA蛋白在染色体复制和分离中的作用方面取得了重大进展。在与Alba Guarne博士(麦克马斯特大学)的合作下，我们已经解决了SEQA蛋白与其同源DNA序列的复合体中的晶体结构。以该结构为指导，我们构建了突变蛋白，并确定了它们对DNA复制和分离的影响。这些研究使我们找到了SEQA作用的工作模型，目前正在进行测试。