Replication of plasmid pSC101 inEscherichia coli K12: Requirement fordnaA function

Abstract

Replication of pSC101 was analyzed by using DNA-DNA hybridization and alkaline sucrose gradient centrifugation. Mutants of the dnaA gene were tested for their capacity to replicate pSC101 DNA at a non-permissive temperature. Only a small amount of radioactive precursor was incorporated into pSC101 DNA in dnaA mutants at 42 degrees C whereas active incorporation into plasmid DNA took place in dnaA+ strain under the same conditions. The effect of the dnaA mutation was grater on plasmid DNA synthesis than on host chromosomal DNA synthesis. The numbers of copies of pSC101 per chromosome in wild type and dnaA strains, grown at 30 degrees C, were about 8 and 2, respectively. These results indicate that the dna A gene product is required for the replication of pSC101 DNA.

New pSC101-derivative cloning vectors with elevated copy numbers

“…The qPCR method yielded a copy number of 16.5 plasmids/chromosome for pBR322, consistent with a range of 15-20 plasmids per chromosome (Lee et al, 2006;LinChao and Bremer, 1986). In addition, pWSK29 was found to have a copy number of 6.7 plasmids/chromosome, closely matching previous measurements for pSC101 of ∼6 plasmids/ chromosome (Cabello et al, 1976;Hasunuma and Sekiguchi, 1977). Measurements of the high copy number mutants showed the repAE93K mutation resulted in a copy number of 27 plasmids/chromosome.…”

“…pSC101-derivative plasmids are also stably maintained owing to the presence of the par locus (Tucker et al, 1984). pSC101-derivative plasmids replicate at a relatively low copy number (<8 copies/cell) (Cabello et al, 1976;Hasunuma and Sekiguchi, 1977) and numerous cloning vectors based on this replicon have been reported (Hashimoto-Gotoh et al, 2000;Hasnain and Thomas, 1986;Hoang et al, 1999;Lerner and Inouye, 1990;Phillips, 1999;Stoker et al, 1982;Takeshita et al, 1987;Wang and Kushner, 1991).…”

Coexpression of UmuD' with UmuC suppresses the UV mutagenesis deficiency of groE mutants

“…1). Previously we had observed that expression of umuDC from a high-copynumber plasmid (pBR322) would partially suppress the nonmutability of the groE strains (6 suppression because of increased gene dosage, we assayed UV mutagenesis in strains in which the umuDC or umuD'C genes were expressed from a relatively low-copy-number plasmid (pSC101, with approximately 8 copies per chromosome) (14). Under these conditions, the nonmutability phenotype of groEL100 and groES30 derivatives of AB1157 is only slightly suppressed by a plasmid encoding umuDC, pSE115 (Fig.…”

A dnaA box can functionally substitute for the priming signals in the oriV of the broad host‐range plasmid RSF 1010

“…These results suggest that DnaA protein can participate in the initiation event of the oriVaSFlOlo-dnaA box in pYTAI, especially in the priming reaction which may be directed by the inserted dnaA box. It has been reported that plasmid pSCIO1, the replication origin of which has a dnaA box, reduces its copy number when it is in a dnaA temperature-sensitive mutant even at the permissive temperature [25]. This inability of replication of the pYTA1 in the dnaA46 mutant seems to be similar to the case of pSC101.…”