A DNA fragment containing the origin of replication of the Escherichia coli chromosome.

Abstract

ABSTRACJA 38 kilobase pair region of the Escherichia coli K12 chromosome containing the replication origin has been physically mapped with restriction endonucleases EcoRI and HindIII. Replication starts within or very near a 1.3 kilobase pair HindIII fragment in the middle of this region and proceeds outward in both directions with apparently equal speed. This pattern was observed in both dnaA and dnaC temperaturesensitive (ts) initiation mutants at the start of the synchronous round of replication which occurs after downshift from the nonpermissive to the permissive temperature. The origin of replication of the Escherichia coli chromosome is located in the vicinity of the iiv operon (1-3), most likely between the dnaA and bglA genes (4, 5). This would place it at approximately 82 min on the revised genetic map of E. coli (6). From this site, replication proceeds in both directions around the large, circular chromosome and terminates within the region diametrically opposite the origin (1, 2, 7, 8).The isolation of conditional mutants defective in initiation has led to the identification of several genes specifically involved in this process: dnaA and dnaC (see ref. 9 for review), dnaI (10), and dnaP (11). The dnaH initiation mutant described by Sakai et al. (12) apparently is a double mutant with the defect in initiation due to a dnaA lesion (13). The dnaA and dnaC loci were the first discovered and are the best characterized. Mutations and 10-ml aliquots taken at 1-or 2-min intervals for pulse labeling with 0.4 ml of [3H]thymidine (0.5 mCi/ml and 60 Ci/ mmol, Schwarz/Mann). Pulses were quenched by adding nonradioactive thymidine to 100 ,ug/ml and pouring the aliquot over ice in a centrifuge tube containing enough KCN to make its final concentration 10 mM.Total cellular DNA was purified as follows: Pelleted cells were resuspended in 0.4 ml of 20% sucrose/100 mM NaCl/10 mM Tris-HCI (pH 8.2), treated for 20 min on ice with 0.1 ml of a 4 mg/ml solution of lysozyme in 120 mM Tris-HCl (pH 8,2)/S0 mM EDTA, and lysed with Sarkosyl (0.4 ml of a 2.5% solution). The lysate was heated to 650 for 5 min, cooled, diluted with water to 7.7 ml, and digested for 4 hr at 370 with 0.1 ml of a 1 mg/ml solution of Pronase (Calbiochem). Solid KI was added to bring the volume of the lysate to 10 ml and a density gradient was formed by centrifugation in a Beckman 50 Ti rotor at 37,000 rpm for 40 hr at 200. The DNA, which banded in the center of the gradient, was collected and dialyzed against 20 mM Tris-HCl (pH 7.8)/0.2 mM EDTA.Restriction Endonuclease Digestion. Restriction endonucleases were purchased from Miles Laboratories and New England Biolabs. A standard reaction mixture was used for all digestions [20 mM Tris-HCI (pH 7.8)/10 mM MgCl2/10 mM NaCI/7 mM 2-mercaptoethanol/0.2 mM EDTA/0.1% gelatin]. Sufficient enzyme was added to give a limit digest within a 4-hr incubation period at 370. When the DNA was digested with more than one enzyme, all were added at the beginning of the digestion period.Gel Electrophoresis and Fluorograph...

Bacteriophage lambda carrying the Escherichia coli chromosomal region of the replication origin.

“…We cannot exclude the possibility, for instance, that a new replication origin was created during construction of the plasmid pMCR1 15. Nonetheless, the following facts support our conclusion. First, the E. coli chromosomal segment that was identified by Marsh and Worcel (28) to be the earliest to replicate during a cycle of synchronous replication almost exactly coincided with the left-most part of the EcoRI fragment of Xasn DNA (Fig. 3) The earlier observation of Hiraga (7) led him to propose that oriC might reside in the dnaA-bglB region (bglB previously designated bglA), but more recent data amend it to the locality consistent with the present result (35 (Figs.…”

“…The order of these cannot primarily be determined, but comparing our data (see Fig. 2) with those of Marsh and Worcel (28), in e, the transducing segment of Xasn DNA was mapped as shown in d. Combining the genetic evidence (see Fig. 1) with the cleavage map, the transducing markers were assigned to each segment as shown at the bottom.…”

Cell cycle-dependent transcription from the gid and mioC promoters of Escherichia coli

“…Replication of the Escherichia coli chromosome is normally initiated at a unique site, oriC, at 84 min on the E. coli genetic map and proceeds bidirectionally (7,27). The minimal oriC sequence essential for autonomous replication in the plasmid state has been delimited to a stretch of 245 bp between the gid and mioC genes (34).…”

Method for localization of cloned DNA fragments on the Escherichia coli chromosome

“…The dnaC2(Ts) and dnaC28(Ts) alleles impair initiation but not elongation. After a shift to nonpermissive temperature, initiation at oriC is blocked but ongoing rounds of replication proceed to completion (4,14,21,29). In addition, the thermal inactivation of the dnaC2 and dnaC28 altered gene products is reversible.…”

“…Such properties have been exploited to synchronize DNA replication by temperature changes in cultures of dnaC2 or dnaC28 strains. This in turn has allowed the autoradiographic demonstration of bidirectional replication (29), the physical mapping of the oriC region (21), the physical mapping of the terC region (4), and the analysis of initiation frequency control (14).…”