Joint research on regulation of chromosomal replication in bacteria

我们发现，在各种细菌中，dna基因及其结合序列（dnaA -box）在染色体复制起始区是保守的。本国际联合研究的目的是通过比较研究dna蛋白和dna -box在枯草芽孢杆菌和大肠杆菌中的作用，分析它们的功能保守性和多样性。在与Dr.Hansen和Dr.Atlung（丹麦技术大学）的合作下，我们成功地从枯草芽孢杆菌染色体的复制起始区分离出一个自主复制的序列。最小的基本片段包含两个由dna基因分隔的dna盒子区域。对ARS的两个区域要求和oriC质粒的低拷贝数与大肠杆菌oriC表现出的特征形成鲜明对比。与美国卫斯理大学（Wesleyan University）的firshein博士和柏林马克斯-普朗克研究所（Max-Planck Institute）的messer博士合作，对枯草芽孢杆菌oriC质粒的体外复制进行了进一步的研究，揭示了两个dna -box区域通过与它们结合的dna蛋白之间的相互作用而相互作用。与rasmussen博士（丹麦技术大学，丹麦）合作，我们分析了枯草芽孢杆菌细胞的细胞周期控制，发现染色体复制的起始时间与大肠杆菌细胞一样受到严格调节。发现枯草芽孢杆菌的DnaA和DnaB蛋白起控制作用。与阿隆索博士（柏林马克斯-普朗克研究所）合作，正在构建一种新的枯草芽孢杆菌突变细胞，其染色体复制独立于dnaA基因，目前正在进行中，以分析dnaA蛋白在体内起始调控中的确切作用。

We have found that dnaA gene and its binding sequences(DnaA-box)are conserved in the replication origin region of the chromosome among various bacteria. The aim of this international joint research was to analyze the functional conservation and diversity of DnaA protein and DnaA-boxes through comparative studies of their roles in Bacillus subtilis and Escherichia coli. In collaboration with Dr.Hansen and Dr.Atlung(Technical University of Denmark, Denmark), we have succeeded to isolate an autonomously replicating sequence from the replication origin region of the B.subtilis chromosome. The minimum essential fragment contained two DnaA-box regions separated by the dnaA gene. Requirements of two regions for ARS and the low copy number of the oriC plasmids are in sharp contrast to features exhibited by E. coli oriC. Further studies of replication of the B.subtilis oriC plasmids in vitro, in collaboration with Dr.Firshein (Wesleyan University, USA)and Dr.Messer(Max-Planck Institute, Berlin), revealed the interaction of the two DnaA-box regions through interaction between DnaA proteins bound to them. In collaboration with Dr.Rasmussen(Technical University of Denmark, Denmark), we have analyzed cell cycle control of B.subtilis cells and found that timing of initiation of chromosomal replication is strictly regulated like in E.coli cells. DnaA and DnaB proteins of B.subtilis were found to be responsible for the control. In collaboration with Dr.Alonso(Max-Planck Institute, Berlin), construction of a new B. subtilis mutant cell, in which chromosomal replication is independent of the dnaA gene, is now underway in order to analyze the precise role of DnaA protein in regulation of initiation in vivo.