Mechanisms of Chromosome Maintenance in Bacteria

细菌染色体维持机制

• 批准号: 7049793
• 负责人: lichten
• 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/7049793
• 关键词: DNA binding protein; DNA replication; DNA replication origin; Escherichia coli; Vibrio cholerae; bacterial genetics; cell cycle; chromosome movement; fluorescence microscopy; gene expression; genetic promoter element; genetic regulatory element; nucleic acid repetitive sequence; nucleic acid structure; plasmids; protein binding; replicon; transcription factor

Controls of DNA replication and segregation of replicated DNA to daughter cells are essential for chromosome maintenance in all organisms. Subversion of these controls leads to cancer. Dr Chattoraj is studying these controls in E. coli and in V. cholerae, the latter having two chromosomes provides an opportunity to study coordination of replication and segregation in a genetically tractable bacterium. Unlike low copy number plasmids and many other bacteria, E. coli contains no homology to any known segregation system. Richard Fekete is trying to identify cis-acting sequences in the E. coli chromosome that would behave in a centromere-like manner. He has labeled different loci on the same E. coli chromosome using LacI-YFP and Lambda cI-CFP fusion proteins bound to arrays of their respective binding sites. Analysis of migration of a few such pairs of loci using fluorescence microscopy suggests a potential centromere site since it migrates towards the cell pole ahead of the other loci. He is attempting time-lapse photography to track the movement of the centromere to its final destination. He is also trying to identify centromere binding proteins. Preeti Srivastava is doing similar studies to follow the migration of the two cholera chromosomes.To study chromosome maintenance in V. cholerae, Ranajit Ghosh made a genomic library from which the origin of replication and potential "centromere" sequences for chromosome I were mined. He purified a chromosomal homolog of ParB, a centromere binding protein for plasmids, and developed antibody against the protein. This is being used to verify ParB binding to the candidate centromeric sequences in vivo using the ChIP assay. Role of ParB in chromosome stability is also in progress.We are close to understanding replication control of plasmid P1. Nilangshu Das with some help from a mathematician from Cambridge (UK), Johan Paulsson, has characterized control-defective initiator mutants that confer higher copy number to the plasmid. The properties of the mutants are best explained assuming initiators to be limiting for replication. Tatiana Venkova-Kanova is in the process of testing the applicability of the plasmid control models to the V. cholerae chromosome II whose origin has features similar to those of plasmid P1. The control of the cholera origin has to be also novel as, unlike P1, it involves a small RNA. The role of the small RNA is being studied by Debasish Pal.

DNA复制的控制和复制的DNA到子细胞的分离对于所有生物体中染色体的维持是必不可少的。破坏这些控制会导致癌症。Chattoraj博士正在E.在大肠杆菌和霍乱弧菌中，后者具有两条染色体，这为研究遗传上易处理的细菌中复制和分离的协调提供了机会。与低拷贝数质粒和许多其他细菌不同，E.大肠杆菌不含有与任何已知分离系统的同源性。理查德·费科特正试图在大肠杆菌中识别顺式作用序列。大肠杆菌染色体，这将表现在一个着丝粒样的方式。他在同一个E上标记了不同的位点。使用LacI-YFP和Lambda cI-CFP融合蛋白结合到其各自结合位点的阵列。使用荧光显微镜分析几对这样的位点的迁移表明一个潜在的着丝粒位点，因为它向细胞极迁移的其他位点之前。他正在尝试用延时摄影来追踪着丝粒到其最终目的地的运动。他也在尝试识别着丝粒结合蛋白。Preeti Srivastava正在做类似的研究，以跟踪两条霍乱染色体的迁移。为了研究霍乱弧菌的染色体维持，Ranajit Ghosh制作了一个基因组文库，从中挖掘出复制起点和I号染色体的潜在“着丝粒”序列。他纯化了ParB的染色体同源物，ParB是质粒的着丝粒结合蛋白，并开发了针对该蛋白的抗体。这被用于使用ChIP测定法在体内验证ParB与候选着丝粒序列的结合。ParB在染色体稳定性中的作用也在研究中，我们即将了解质粒P1的复制控制。Nilangshu Das在来自英国剑桥的数学家Johan Paulsson的帮助下，已经描述了控制缺陷型启动子突变体的特征，这些突变体赋予质粒更高的拷贝数。突变体的性质最好解释假设启动子是限制复制。Tatiana Venkova-Kanova正在测试质粒控制模型对霍乱弧菌染色体II的适用性，其起源具有与质粒P1相似的特征。霍乱起源的控制也必须是新颖的，因为与P1不同，它涉及小RNA。小RNA的作用正在由Debasish研究。