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

Abstract

In exponentially growing cultures of Escherichia coli strains carrying the dnaC28 mutation, DNA replication can be synchronized by temperature changes (R. L. Rodriguez, M. S. Dalbey, and C. I. Davern, J. Mol. Biol. 74:599-604, 1973). We used this synchronization procedure and DNA-DNA hybridization to develop a technique for the localization of cloned chromosomal fragments on the genetic map. Because of the bidirectional nature of replication in E. coli, our method gave two possible positions (one on each replication arm). However because of the precision obtained for each position (±1 map unit), the final mapping with various genetic techniques was greatly facilitated. Using this technique and a simple chromosomal mobilization test, we located at 93.2 ± 1 min a cloned DNA fragment carrying an extragenic suppressor of dnaA46, a thermosensitive mutation in the dnaA initiation gene. Further analysis showed that the groES (mopA) and groEL (mopB) genes, both located at 94.2 min on the standard map, were indeed carried by the cloned suppressor fragment.Replication in Escherichia coli initiates at a fixed site, oriC, located at 84 min on the genetic map (2, 38) and proceeds bidirectionally at a constant rate, terminating in the opposite region, terC, around 31 min (5, [17][18][19]. The dnaC gene product is implicated in initiation and elongation of DNA replication because thermosensitive mutant alleles affecting one or the other step have been isolated (6,31). 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).In the experiments reported here, we have taken advantage of this possibility of synchronizing replication to develop a technique, based also on DNA-DNA hybridization, which permits the mapping of cloned chromosomal DNA fragments. We used this technique to determine the chromosomal location of a cloned fragment able, when amplified, to suppress the thermosensitive DNA initiation defect caused by the dnaA46 mutation (12). MATERIALS AND METHODSStrains and plasmids. Two strains, derived from E. coli K-12 W1485 (1), were used for DNA pulse-labeling. LN681 is F-dnaC28 thyA deoB ( Media and buffers. Since growth in rich media did not allow sufficient incorporation, E medium (36) was used in [3H]thymidine pulse-labeling experiments. The salt solution was supplemented with 0.5% (wt/vol) glucose-0.5% (wt/vol) vitamin-free Casamino Acids (Difco Laboratories)-thiamine (1 ,ug/ml)-tryptophane (40 pug...

Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures

“…5 Fayet and Prere (11) to filters that were loaded with plasmids pBL13, pEMBL8+, pLN47, and pBS28 (see Table 1 for plasmid descriptions); and the radioactivity that was retained on each filter was measured. The sigmoidal curves were obtained by integration of the hybridization peak that was observed for each plasmid probe (11). Points for which no radioactivity was found were omitted.…”

“…The mapping procedure based on the determination of the replication time of defined DNA segments in synchronized cell culture has been described previously (11). The mapping procedure based on hybridization experiments of cloned DNA segments to the E. coli genomic library in phage X was as follows.…”

arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon

“…P1 transductions were done as described by Miller (28). The position of the mini-Tet insertion described here was initially mapped by DNA hybridization, in which synchronized LN681 cells were pulsed with [3H]thymidine, and samples removed at various times were probed with a plasmid containing the mini-Tet insertion, as described by Fayet and Prere (8).…”

“…The mini-Tet insertion was initially mapped to either -16 min or 68 min of the E. coli genetic map by using the synchronized cell DNA hybridization technique described by Fayet and Prere (8). To further verify and more precisely map the position of the mini-Tet insertion, the overlapping Kohara bacteriophages 7E10, 18F11, lOG5, 4H1, and 1OF3 (24), which carry DNA from the 16.7-min region of the E. coli physical map, were grown on strain DW114 to test their abilities to recombine the mini-Tet insertion.…”

Cloning, mapping, and sequencing of the gene encoding Escherichia coli quinoprotein glucose dehydrogenase

“…The gcd gene was localized on the chromosome by using the DnaC synchronization method (16) and by hybridization with DNA of several lambda bacteriophages from the fully characterized complete library from the E. coli chromosome reported by Kohara et al (24). Dot blots were prepared by spotting 5 ,ul of lambda stocks at 109 phages per ml on a strip of Hybond CE membrane (Amersham).…”