FIG. 1.

Effect of the dnaA747 allele on the relative gene dosage of oriC-proximal DNA sequences relatively to terC-proximal DNA sequences. Strains carry a chromosomal Tn10dTc insertion located either in the proximity of oriC (Tn10-ori; zid-1257::Tn10dTc) or in the proximity of terC (Tn10-ter; zdi-6798::Tn10dTc). (A) Southern blot analysis of HpaI-digested chromosomal DNA hybridized with labeled pTS1 plasmid. Cells were grown overnight at 28°C in nutrient broth (NB), diluted 1 to 200, and incubated at 37°C in NB until an optical density at 600 nm of 0.4 was reached. Chromosomal DNA was extracted as described previously (3). DNA fragments were separated by electrophoresis on an agarose gel and transferred to a nylon membrane by capillarity under alkaline conditions (25). The membrane was hybridized to plasmid pTS1 DNA labeled with [α-³²P]CTP by nick translation (25). Plasmid pTS1 is a pUC18 (New England Biolabs) derivative carrying a 5.3-kb DNA insert which includes a Tn10dTc element (2.9 kb) and sequences from the region flanking the argV locus of S. typhimurium (4). In the experiment described above, pTS1 DNA hybridizes to a 2-kb fragment from the internal portion of Tn10dTc (band I) and to an 8-kb fragment from the argV region (band II). (The size of the latter fragment is reduced in a strain carrying an argV deletion [4]. Additional signals result from hybridization to the ends of Tn10dTc and to the Ap^r gene of the MudA element.) The strains used were MA786 (lane 1), MA923 (lane 2), MA785 (lane 3), and MA922 (lane 4). (B) Quantification of the relative gene dosage between oriC-proximal and terC-proximal Tn10dTc insertions. For each lane, the hybridization signal of band I was normalized to the signal of band II by using a 400S PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). The oriC/terC ratio was calculated for both dnaA⁺ and dnaA747 strains by dividing normalized signals corresponding to the Tn10-ori insertion by normalized signals corresponding to the Tn10-ter insertion. (C) Model in which the hisR locus is amplified relative to the his locus in a wild-type strain but not in the dnaA747 mutant at 37°C. Reduction of the hisR gene dosage in a dnaA strain is responsible for deattenuation of his operon transcription. The positions of the different loci are indicated in centisomes.