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. 1986 Mar;165(3):951–957. doi: 10.1128/jb.165.3.951-957.1986

Cloning and characterization of the polC region of Bacillus subtilis.

R W Ott, M H Barnes, N C Brown, A T Ganesan
PMCID: PMC214521  PMID: 2936731

Abstract

The polC gene of Bacillus subtilis is defined by five temperature-sensitive mutations and the 6-(p-hydroxyphenylazo)-uracil (HPUra) resistance mutation azp-12. Biochemical evidence suggests that polC codes for the 160-kilodalton DNA polymerase III. A recombinant plasmid, p154t, was isolated and found to contain the azp-12 marker and one end of the polC gene (N. C. Brown and M. H. Barnes, J. Cell. Biochem. 78 [Suppl.]: 116, 1983). The azp-12 marker was localized to a 1-kilobase DNA segment which was used as a probe to isolate recombinant lambda phages containing polC region sequences. A complete polC gene was constructed by in vitro ligation of DNA segments derived from two of the recombinant phages. The resulting plasmid, pRO10, directed the synthesis of four proteins of 160, 76, 39, and 32 kilodaltons in Escherichia coli maxicells. Recombination-deficient (recE) B. subtilis PSL1 containing pRO10 produced an HPUra-resistant polymerase III activity which was lost when the strain was cured of pRO10. In vivo, the HPUra resistance of the plasmid-encoded polymerase III appeared to be recessive to the resident HPUra-sensitive polymerase III enzyme.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Appleyard R K. Segregation of New Lysogenic Types during Growth of a Doubly Lysogenic Strain Derived from Escherichia Coli K12. Genetics. 1954 Jul;39(4):440–452. doi: 10.1093/genetics/39.4.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes M. H., Brown N. C. Antibody to B. subtilis DNA polymerase III: use in enzyme purification and examination of homology among replication-specific DNA polymerases. Nucleic Acids Res. 1979 Mar;6(3):1203–1219. doi: 10.1093/nar/6.3.1203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bazill G. W., Gross J. D. Effect of 6-(p-hydroxyphenyl)-azouracil on B. subtilis DNA polymerases. Nat New Biol. 1972 Nov 15;240(98):82–83. doi: 10.1038/newbio240082a0. [DOI] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Clements J. E., D'Ambrosio J., Brown N. C. Inhibition of Bacillus subtilis deoxyribonucleic acid polymerase III by phenylhydrazinopyrimidines. Demonstration of a drug-induced deoxyribonucleic acid-enzyme complex. J Biol Chem. 1975 Jan 25;250(2):522–526. [PubMed] [Google Scholar]
  7. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cozzarelli N. R., Low R. L. Mutational alteration of Bacillus subtilis DNA polymerase 3 to hydroxyphenylazopyrimidine resistance: polymerase 3 is necessary for DNA replication. Biochem Biophys Res Commun. 1973 Mar 5;51(1):151–157. doi: 10.1016/0006-291x(73)90521-4. [DOI] [PubMed] [Google Scholar]
  9. Dedonder R. A., Lepesant J. A., Lepesant-Kejzlarová J., Billault A., Steinmetz M., Kunst F. Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Appl Environ Microbiol. 1977 Apr;33(4):989–993. doi: 10.1128/aem.33.4.989-993.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donnelly C. E., Sonenshein A. L. Promoter-probe plasmid for Bacillus subtilis. J Bacteriol. 1984 Mar;157(3):965–967. doi: 10.1128/jb.157.3.965-967.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ehrlich S. D. DNA cloning in Bacillus subtilis. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1433–1436. doi: 10.1073/pnas.75.3.1433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  13. Haldenwang W. G., Banner C. D., Ollington J. F., Losick R., Hoch J. A., O'Connor M. B., Sonenshein A. L. Mapping a cloned gene under sporulation control by inserttion of a drug resistance marker into the Bacillus subtilis chromosome. J Bacteriol. 1980 Apr;142(1):90–98. doi: 10.1128/jb.142.1.90-98.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Karamata D., Gross J. D. Isolation and genetic analysis of temperature-sensitive mutants of B. subtilis defective in DNA synthesis. Mol Gen Genet. 1970;108(3):277–287. doi: 10.1007/BF00283358. [DOI] [PubMed] [Google Scholar]
  15. Love E., D'Ambrosio D., Brown N. C. Mapping of the gene specifying DNA polymerase III of Bacillus subtilis. Mol Gen Genet. 1976 Mar 30;144(3):313–321. doi: 10.1007/BF00341730. [DOI] [PubMed] [Google Scholar]
  16. Low R. L., Rashbaum S. A., Cozzarelli N. R. Purification and characterization of DNA polymerase III from Bacillus subtilis. J Biol Chem. 1976 Mar 10;251(5):1311–1325. [PubMed] [Google Scholar]
  17. Mackenzie J. M., Neville M. M., Wright G. E., Brown N. C. Hydroxyphenylazopyrimidines: characterization of the active forms and their inhibitory action on a DNA polymerase from Bacillus subtilis. Proc Natl Acad Sci U S A. 1973 Feb;70(2):512–516. doi: 10.1073/pnas.70.2.512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Meselson M., Yuan R. DNA restriction enzyme from E. coli. Nature. 1968 Mar 23;217(5134):1110–1114. doi: 10.1038/2171110a0. [DOI] [PubMed] [Google Scholar]
  19. Ostroff G. R., Pène J. J. Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA. J Bacteriol. 1983 Nov;156(2):934–936. doi: 10.1128/jb.156.2.934-936.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Piggot P. J., Hoch J. A. Revised genetic linkage map of Bacillus subtilis. Microbiol Rev. 1985 Jun;49(2):158–179. doi: 10.1128/mr.49.2.158-179.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rapoport G., Klier A., Billault A., Fargette F., Dedonder R. Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis. Mol Gen Genet. 1979 Oct 3;176(2):239–245. doi: 10.1007/BF00273218. [DOI] [PubMed] [Google Scholar]
  22. Riva S., van Sluis C., Mastromei G., Attolini C., Mazza G., Polsinelli M., Falaschi A. A new mutant of Bacillus subtilis altered in the initiation of chromosome replication. Mol Gen Genet. 1975;137(3):185–202. doi: 10.1007/BF00333015. [DOI] [PubMed] [Google Scholar]
  23. Sancar A., Rupert C. S. Determination of plasmid molecular weights from ultraviolet sensitivities. Nature. 1978 Mar 30;272(5652):471–472. doi: 10.1038/272471a0. [DOI] [PubMed] [Google Scholar]
  24. Spizizen J. TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. Proc Natl Acad Sci U S A. 1958 Oct 15;44(10):1072–1078. doi: 10.1073/pnas.44.10.1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. TAKAHASHI I. Genetic transduction in Bacillus subtilis. Biochem Biophys Res Commun. 1961 Jun 28;5:171–175. doi: 10.1016/0006-291x(61)90104-8. [DOI] [PubMed] [Google Scholar]
  26. TAKAHASHI I. Transducing phages for Bacillus subtilis. J Gen Microbiol. 1963 May;31:211–217. doi: 10.1099/00221287-31-2-211. [DOI] [PubMed] [Google Scholar]
  27. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  28. Vrooman M. J., Barnes M. H., Brown N. C. Bacillus subtilis dnaF: a mutation of the gene specifying the structure of DNA polymerase III. Mol Gen Genet. 1978 Sep 8;164(3):335–339. doi: 10.1007/BF00333165. [DOI] [PubMed] [Google Scholar]
  29. Wright G. E., Brown N. C. Inhibitors of Bacillus subtilis DNA polymerase III. 6-Anilinouracils and 6-(alkylamino)uracils. J Med Chem. 1980 Jan;23(1):34–38. doi: 10.1021/jm00175a007. [DOI] [PubMed] [Google Scholar]
  30. Wright G. E., Gambino J. J. Quantitative structure-activity relationships of 6-anilinouracils as inhibitors of Bacillus subtilis DNA polymerase III. J Med Chem. 1984 Feb;27(2):181–185. doi: 10.1021/jm00368a013. [DOI] [PubMed] [Google Scholar]

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