Mechanisms of Chromosome Maintenance in Bacteria

在所有生物体中，控制DNA复制和将复制的DNA分离到子细胞是维持染色体的必要条件。破坏这些控制就会导致癌症。Chattoraj博士正在研究大肠杆菌和霍乱弧菌的这些控制，后者有两条染色体，这为研究一种遗传易感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，大肠杆菌与任何已知的分离系统都没有同源性。Richard Fekete正试图在大肠杆菌染色体中找出类似着丝粒的顺式作用序列。他利用LacI-YFP和Lambda cI-CFP融合蛋白结合到各自结合位点的阵列上，对同一大肠杆菌染色体上的不同位点进行了标记。利用荧光显微镜对一些这样的基因座对的迁移进行分析，表明了一个潜在的着丝点位置，因为它比其他基因座先向细胞极点迁移。他正在尝试用延时摄影来追踪着丝粒到达最终目的地的运动。他还试图鉴定着丝粒结合蛋白。Preeti Srivastava正在进行类似的研究，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建立了一个基因组文库，从中挖掘了1号染色体的复制起源和潜在的“着丝粒”序列。他纯化了质粒的着丝粒结合蛋白ParB的染色体同源物，并开发了针对该蛋白的抗体。这是用来验证ParB结合候选着丝粒序列在体内使用ChIP试验。ParB在染色体稳定性中的作用也在研究中。我们已经接近了解质粒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，描述了具有控制缺陷的启动突变体，这种突变体赋予质粒更高的拷贝数。突变体的特性最好的解释是假设启动者对复制有限制。Tatiana Venkova-Kanova正在测试质粒控制模型对起源与质粒P1相似的霍乱弧菌染色体II的适用性。霍乱起源的控制也必须是新颖的，因为与P1不同，它涉及一种小RNA。Debasish Pal正在研究小RNA的作用。

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.