Control of chromosomal replication in relation to cell duplication

1. 我们发现枯草芽孢杆菌染色体复制起始区的基因及其组织与大肠杆菌染色体的dna基因区域非常相似。这一发现导致了多种细菌中dna基因和含有dna蛋白结合序列的调控区域（DnaA-box区）的保守性的发现。大肠杆菌的otic区包含4个dna盒子，距离dna基因45kb，在进化过程中被认为是易位的。为了确定大肠杆菌型和B. subthis型哪一种类型更接近于祖先细菌的复制起源，我们选择了恶臭假单胞菌作为第二革兰氏阴性细菌，并与B. subthis进行了广泛的起源区核苷酸序列测定。dna上游序列显示，在p.p putida和b.s ubthis中，有一个与gida基因密切相关的dna -box区域被保守。三种细菌染色体的起源区域比较清楚地表明，除了大肠杆菌中约50kb的片段被反转，包括与gidia相关的oiic，祖先组织是非常保守的。大肠杆菌和亚伯氏杆菌之间的dna基因和dna -box区域的保存表明它们分别作为染色体复制起始所需的蛋白质和ozic，也在亚伯氏杆菌中起作用。我们通过体外诱变构建了一个温度敏感的突变体DnaA蛋白，并可以证明DnaA蛋白确实是B. subthis染色体复制起始所必需的。此外，dna蛋白的细胞含量决定了染色体起始的频率。我们从B.亚this染色体的复制起始区分离出ARS片段，并能够表征ARS功能所必需的结构。必需片段包含由dna基因分隔的两个dna盒区域。这两个dna盒区域本身都没有显示ARS活性。估计含有ARS的质粒（oricplasmid）的拷贝数为每条复制染色体1个。这些质粒不稳定，容易丢失或整合到染色体中。这些特征与大肠杆菌oric表现出的特征形成鲜明对比，大肠杆菌oric需要一个dna -box区域，并且可以在大肠杆菌细胞中存在多个副本。少

1. We have found that genes and their organization in the replication origin region of the Bacillus subtilis chromosome are remarkably similar to those of DnaA gene region of the Eschezichia coli chromosome. This finding led to the discovery of the conservation of DnaA gene and regulatory regions containing DnaA protein binding sequences (DnaA-box region) among various bacteria.2. The otic region of E. coli mihich contains 4 DnaA-boxes is located 45kb away from the DnaA gene and thought to be translocated during evolution. In order to determine which of the two types, the E. coli type or the B. subthis type, represents more closely the replication origin of the ancestral bacteria, we chose Pseudomonas putida as a second gram negative bacterium and determined nucleotide sequence of the origin region extensively in comparison with that of B. subthis. Nucleotide sequences upstream of DnaA gene revealed that an additional DnaA-box region which was linked closely to gida gene was conserved … More in P. putida and B. subthis. Comparison of the origin region of the three bacterial chromosomes shows clearly that the ancestral organization is remarkably conserved except that some 50kb fragment is inverted in E. coli including oiic linked to gida.3. Conservation of both in DnaA gene and DnaA-box regions between E. coli and B. subthis suggested that they function as a protein essential for initiation of chromosomal replication and as an ozic, respectively, also in B. subthis. We have constructed a temperature sensitive mutant DnaA protein by in vitro mutagenesis and could demonstrate that DnaA protein was indeed essential for initiation of chromosomal replication in B. subthis. Furthermore the cellular content of DnaA protein was shown to determine the frequency of initiation of the chromosome.4. We isolated ARS fragments from the replication origin region of the B. subthis chromosome and were able to characterize structures essential for ARS function. Essential fragment contained two DnaA-box regions separated by the DnaA gene. Neither one of the DnaA-box regions by itself showed ARS activity. The copy number of the ARS containing plasmid (oricplasmid) was estimated as one per replicating chromosome. These plasmids were unstable and tend to be lost or integrated into chromosome. These features of the B. subthis oricplasmid are in sharp contrast to those exhibited by E. coli oric which requires one DnaA-box region and can exist in multiple copies in the E. coli cell. Less