Control of chromosomal replication in relation to cell duplication

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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