Summary
Replication of pSC101 was analyzed by using DNA-DNA hybridization and alkaline sucrose gradient centrifugation. Mutants of thednaA 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 indnaA mutants at 42°C whereas active incorporation into plasmid DNA took place indnaA + strains under the same conditions. The effect of thednaA mutation was grater on plasmid DNA synthesis than on host chromosomal DNA synthesis. The numbers of copies of pSC101 per chromosome in wild type anddnaA strains, grown at 30°C, were about 8 and 2, respectively. These results indicate that thednaA gene product is required for the replication of pSC101 DNA.
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Cohen, S.N., Chang, A.C.Y.: Recirculation and autonomous replication of a sheared R-factor DNA segment inEscherichia coli transformants. Proc. nat. Acad. Sci. (Wash.)70, 1293–1297 (1973)
Cohen, S.N., Chang, A.C.Y., Boyer, H.W., Helling, R.B.: Construction of biologically functional bacterial plasmids in vitro. Proc. nat. Acad. Sci. (Wash.)70, 3240–3244 (1973)
Cohen, S.N., Chang, A.C.Y., Hsu, L.: Nonchromosomal antibiotic resistance in bacteria: genetic transformation ofEscherichia coli by R-factor DNA. Proc. nat. Acad. Sci. (Wash.)69, 2110–2114 (1972)
Collins, J., Williams, P., Helinski, D.R.: Plasmid ColEl DNA replication inEscherichia coli strains temperature-sensitive for DNA replication. Molec. gen. Genet.136, 273–289 (1975)
Denhardt, D.T.: A membrane filter technique for the detection of complementary DNA. Biochem. biophys. Res. Commun.23, 641–646 (1966)
Goebel, W.: The influence ofdnaA anddnaC mutations on the initiation of plasmid DNA replication. Biochem biophys. Res. Commun.51, 1000–1007 (1973)
Guerry, P., LeBlanc, D.J., Falkow, S.: General method for the isolation of plasmid deoxyribonucleic acid. J. Bact116, 1064–1066 (173)
Hashimoto, T., Sekiguchi, M.: Isolation of temperature-sensitive mutants of R plasmid by in vitro mutagenesis with hydroxylamine. J. Bact.127, 1561–1563 (1976)
Hirota, Y., Ryter, A., Jacob, F.: Thermosensitive mutants ofE. coli affected in the process of DNA synthesis and cellular division. Cold Spr. Harb. Symp. quant. Biol.33, 677–693 (1968)
Kretschmer, P.J., Chang, A.C.Y., Cohen, S.N.: Indirect selection of bacterial plasmid lacking identical phenotipic properties. J. Bact.124, 225–231 (1975)
Taketo, A.: Sensitivity ofEscherichia coli to viral nucleic acid VI. Capacity ofdna mutants and DNA polymerase-less mutants for multiplication of ΦA and ΦX174. Molec. gen Genet.122, 15–22 (1973)
Timmis, K., Cabello, F., Cohen, S.N.: Utilization of two distinct modes of replication by a hybrid plasmid constructed in vivo from separate replicons. Proc. nat. Acad. Sci. (Wash.)71, 4556–4560 (1974)
Warnaar, S.O., Cohen, J.A.: A quantitative assay for DNA-DNA hybrids using membrane filters. Biochem. biophys. Res. Commun.24, 554–558 (1966)
Wechsler, J.A., Gross, J.D.:Escherichia coli mutants temperaturesensitive for DNA synthesis. Molec. gen. Genet.113, 273–284 (1971)
Wickner, S., Hurwitz, J.: Conversion of ΦX174 vital DNA to double-stranded form by purifiedEscherichia coli proteins. Proc. nat. Acad. Sci. (Wash.).71, 4120–4142 (1974)
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Communicated by B.A. Bridges
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Hasunuma, K., Sekiguchi, M. Replication of plasmid pSC101 inEscherichia coli K12: Requirement fordnaA function. Molec. Gen. Genet. 154, 225–230 (1977). https://doi.org/10.1007/BF00571277
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DOI: https://doi.org/10.1007/BF00571277