Origin and sequence of chromosome replication in Escherichia coli

Abstract

Two methods have been used to determine the origin and direction of chromosome replication in Escherichia coli: gradient of marker frequency and sequence of replication in synchronized cultures. In both cases, DNA-DNA hybridization was used to assay for gene dosage. A series of isogenic strains were made lysogenic for phage λ and for phage Mu-1, with phage Mu-1 in a different chromosomal location in each strain. In a first group of experiments, DNA from exponential cultures of the various strains was extracted, denatured, immobilized on filters and hybridized against a mixture of differentially labeled phage λ and phage Mu-1 DNA. This was done for several culture conditions. The ratio of hybridization Mu-1/λ gives a measurement of the dosage of the chromosome region where phage Mu-1 is integrated. A plot of this ratio versus map position reflects the marker frequency distribution. In another group of experiments, several of the strains were synchronized by amino-acid starvation, then restitution of essential amino acids. The cultures thus synchronized were density labeled with bromouracil and their DNA extracted at various times, separated by density on a CsCl gradient, and the amount of phage Mu-1 specific DNA present in each band was measured by DNA-DNA hybridization. The following conclusions have been reached. 1. (1) The origin of replication in near ilv, at 74 minutes on the standard genetic map of Escherichia coli. 2. (2) Replication proceeds simultaneously in the two opposite directions, at approximately the same velocity in both directions. 3. (3) This velocity is influenced, in a thymineless mutant, by the concentration of exogenous thymine. 4. (4) The two growing forks meet at a point diametrically opposed to the origin, near trp (25 min).