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DnaA基因区的基因及其组织非常相似。这一发现导致了DnaA基因和含有DnaA蛋白结合序列的调控区（DnaA-box区）在不同细菌中的保守性的发现. E.大肠杆菌中含有4个DnaA盒，位于DnaA基因的45 kb处，被认为是在进化过程中易位的。为了确定这两种类型中的哪一种，E. coli型或B型。亚此类型的革兰氏阴性菌更接近于原始细菌的复制起点，我们选择恶臭假单胞菌作为第二种革兰氏阴性菌，并与B进行比较，广泛测定了其复制起点区的核苷酸序列。subthis. DnaA基因上游的核苷酸序列分析表明，与gida基因紧密连锁的DnaA-box区域是保守的 ...更多信息 恶臭假单胞菌和B. subthis.比较三种细菌染色体的起源区域，发现除了在E. coli，包括与gida连接的oiic. DnaA基因和DnaA-box区在大肠杆菌中的保守性均较好。coli和B.这表明它们在B中也分别作为染色体复制起始所必需的蛋白质和ozic起作用。subthis.我们已经构建了一个温度敏感的突变体DnaA蛋白在体外诱变，并可以证明，DnaA蛋白确实是必不可少的启动染色体复制在B。subthis.此外，DnaA蛋白的细胞含量被证明决定染色体起始的频率.我们从B的复制起始区分离ARS片段。subthis染色体，并能够表征ARS功能所必需的结构。必需片段含有两个由DnaA基因分隔的DnaA-box区。没有一个DnaA盒区域本身显示ARS活性。含ARS质粒（oricplasmid）的拷贝数估计为每个复制染色体一个。这些质粒不稳定，容易丢失或整合到染色体中。这些特点的B。subthis oricplasmid与E. colioric需要一个DnaA-box区域，在大肠杆菌中可以多拷贝存在。coli细胞。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