Mechanisms of Chromosome Maintenance in Bacteria

我们的兴趣是了解DNA复制频率在细胞周期中是如何调节的。我们一直在使用质粒P1研究这一点，其拷贝数控制机制似乎在细菌中很常见。该机制涉及对起始蛋白合成的自体控制、通过二聚化使起始蛋白失活、通过分子伴侣将无活性二聚体转化为活性单体、起始剂结合至起始点中的多个位点以及通过使起始点失活的结合的起始剂使起始点配对。这些多种控制模式有助于通过增加具有少于平均拷贝数的细胞中的复制速率并降低具有太多质粒的细胞中的速率来将质粒拷贝数维持在窄的限度内。对质粒拷贝数的稳态的研究提供了一个如何在生物系统中调节波动的模型。 遗传学和荧光显微镜技术的最新发展表明，染色体复制和分离的过程在细菌和酵母中都有联系。我们已经发展了一种双标记技术，使用易于区分的荧光蛋白同时标记大肠杆菌的两个染色体位点。coli分离，并分析其分离模式。我们的前提是，将子染色体锚定到细胞两极的基因座将首先定位到一个极，因此有资格作为细菌着丝粒。我们正在将我们的研究扩展到霍乱弧菌染色体。这种细菌的基因组分为两条染色体。我们的兴趣是确定两条染色体复制的控制元件。这应该有助于了解染色体是如何维持的，以及它们的复制是否处于检查点控制之下，以确保细胞分裂前两条染色体的完全复制。两条染色体的位置正在通过上述双标记技术进行研究。

Our interest is to understand how the DNA replication frequency is adjusted in the cell cycle. We have been studying this using plasmid P1 whose mechanism of copy number control appears to be common in bacteria. The mechanism involves autogenous control on initiator protein synthesis, initiator protein inactivation by dimerization, conversion of inactive dimers to active monomers by molecular chaperones, initiator binding to multiple sites in the origin, and pairing of origins via the bound initiators that inactivates the origins. These multiple modes of control help to maintain plasmid copy number within narrow limits by increasing the rate of replication in cells that have fewer than average copy number and reducing the rate in cells that have too many plasmids. The study of homeostasis of plasmid copy number provides a model of how fluctuations can be adjusted in biological systems. Recent developments in genetic and fluorescent-based microscopic techniques have shown that the processes of chromosome replication and segregation are connected both in bacteria and in yeast. We have developed double-labeling techniques using easily distinguished fluorescent proteins to mark two chromosomal loci simultaneously in E. coli and analyze their segregation pattern. Our premise is that the locus that anchors daughter chromosomes to the cell poles will localize to a pole first, and thus qualify as a bacterial centromere. We are extending our studies to V. cholerae chromosomes. The genome of this bacterium is split into two chromosomes. Our interest is to identify the control elements of replication for the two chromosomes. This should help to understand how the chromosomes are maintained and whether their replication is under a check-point control that ensures complete replication of both the chromosomes before cell division. The location of the two chromosomes is being studied by the double-labeling technique described above.