Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1980 Aug;143(2):561–568. doi: 10.1128/jb.143.2.561-568.1980

Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region.

A Platz, B M Sjöberg
PMCID: PMC294315  PMID: 7009552

Abstract

Recombinant plasmids containing all or part of the genetic region of Escherichia coli coding for the two subunits of ribonucleoside diphosphate reductase (proteins B1 and B2) were constructed with the aid of the multicopy plasmid pBR322. Two of these plasmids (pPS1 and pPS2) appeared to carry both a regulator and the complete structural information for the enzyme and, after transformation of E. coli, directed a 10- to 20-fold overproduction of both proteins B1 and B2. The other plasmids (pPS101 and pPS201) carried structural information for only protein B2. Cells carrying pPS1 and pPS2 showed a 5- to 500-fold increased resistance against the drug hydroxyurea. This establishes that in E. coli the inhibition of deoxyribonucleic acid synthesis by hydroxyurea is fully explained by its action on ribonucleotide reductase.

Full text

PDF
561

Images in this article

564Image
on p.564
566Image
on p.566

Selected References

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

  1. Armstrong K. A., Hershfield V., Helinski D. R. Gene cloning and containment properties of plasmid Col E1 and its derivatives. Science. 1977 Apr 8;196(4286):172–174. doi: 10.1126/science.322277. [DOI] [PubMed] [Google Scholar]
  2. Bernard H. U., Helinski D. R. Use of the lambda phage promoter PL to promote gene expression in hybrid plasmid cloning vehicles. Methods Enzymol. 1979;68:482–492. doi: 10.1016/0076-6879(79)68037-0. [DOI] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Betlach M. C., Boyer H. W. Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene. 1977;2(2):75–93. doi: 10.1016/0378-1119(77)90074-9. [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. Brown N. C., Canellakis Z. N., Lundin B., Reichard P., Thelander L. Ribonucleoside diphosphate reductase. Purification of the two subunits, proteins B1 and B2. Eur J Biochem. 1969 Jul;9(4):561–573. doi: 10.1111/j.1432-1033.1969.tb00646.x. [DOI] [PubMed] [Google Scholar]
  6. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [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., Freedberg W. B., Lin E. C. Genetic control of L-alpha-glycerophosphate system in Escherichia coli. J Mol Biol. 1968 Feb 14;31(3):371–387. doi: 10.1016/0022-2836(68)90415-4. [DOI] [PubMed] [Google Scholar]
  9. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ehrenberg A., Reichard P. Electron spin resonance of the iron-containing protein B2 from ribonucleotide reductase. J Biol Chem. 1972 Jun 10;247(11):3485–3488. [PubMed] [Google Scholar]
  11. Engström Y., Eriksson S., Thelander L., Akerman M. Ribonucleotide reductase from calf thymus. Purification and properties. Biochemistry. 1979 Jul 10;18(14):2941–2948. doi: 10.1021/bi00581a004. [DOI] [PubMed] [Google Scholar]
  12. Eriksson S., Sjöberg B. M., Hahne S. Ribonucleoside diphosphate reductase from Escherichia coli. An immunological assay and a novel purification from an overproducing strain lysogenic for phage lambdadnrd. J Biol Chem. 1977 Sep 10;252(17):6132–6138. [PubMed] [Google Scholar]
  13. Fuchs J. A., Karlström H. O. A mutant of Escherichia coli defective in ribonucleosidediphosphate reductase. 2. Characterization of the enzymatic defect. Eur J Biochem. 1973 Feb 1;32(3):457–462. [PubMed] [Google Scholar]
  14. Fuchs J. A., Karlström H. O. Mapping of nrdA and nrdB in Escherichia coli K-12. J Bacteriol. 1976 Dec;128(3):810–814. doi: 10.1128/jb.128.3.810-814.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fuchs J. A., Karlström H. O., Warner H. R., Reichard P. Defective gene product in dnaF mutant of Escherichia coli. Nat New Biol. 1972 Jul 19;238(81):69–71. doi: 10.1038/newbio238069a0. [DOI] [PubMed] [Google Scholar]
  16. Gellert M., Mizuuchi K., O'Dea M. H., Itoh T., Tomizawa J. I. Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4772–4776. doi: 10.1073/pnas.74.11.4772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guerry P., LeBlanc D. J., Falkow S. General method for the isolation of plasmid deoxyribonucleic acid. J Bacteriol. 1973 Nov;116(2):1064–1066. doi: 10.1128/jb.116.2.1064-1066.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Haggerty D. M., Scheif R. F. Location in bacteriophage lamdba DNA of cleavage sites of the site-specific endonuclease from Bacillus amyloliquefaciens H. J Virol. 1976 May;18(2):659–663. doi: 10.1128/jvi.18.2.659-663.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Krakoff I. H., Brown N. C., Reichard P. Inhibition of ribonucleoside diphosphate reductase by hydroxyurea. Cancer Res. 1968 Aug;28(8):1559–1565. [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. LURIA S. E., BURROUS J. W. Hybridization between Escherichia coli and Shigella. J Bacteriol. 1957 Oct;74(4):461–476. doi: 10.1128/jb.74.4.461-476.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neuhard J., Thomassen E. Turnover of the deoxyribonucleoside triphosphates in Escherichia coli 15 T during thymine starvation. Eur J Biochem. 1971 May 11;20(1):36–43. doi: 10.1111/j.1432-1033.1971.tb01359.x. [DOI] [PubMed] [Google Scholar]
  23. Perricaudet M., Tiollais P. Defective bacteriophage lambda chromosome, potential vector for DNA fragments obtained after cleavage by Bacillus amyloliquefaciens endonuclease (BamI). FEBS Lett. 1975 Aug 1;56(1):7–11. doi: 10.1016/0014-5793(75)80099-8. [DOI] [PubMed] [Google Scholar]
  24. Rosenkranz H. S., Garro A. J., Levy J. A., Carr H. S. Studies with hydroxyurea. I. The reversible inhibition of bacterial DNA synthesis and the effect of hydroxyurea on the bactericidal action of streptomycin. Biochim Biophys Acta. 1966 Mar 21;114(3):501–515. [PubMed] [Google Scholar]
  25. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sjöberg B. M., Reichard P., Gräslund A., Ehrenberg A. The tyrosine free radical in ribonucleotide reductase from Escherichia coli. J Biol Chem. 1978 Oct 10;253(19):6863–6865. [PubMed] [Google Scholar]
  27. Sugino A., Peebles C. L., Kreuzer K. N., Cozzarelli N. R. Mechanism of action of nalidixic acid: purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4767–4771. doi: 10.1073/pnas.74.11.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thelander L. Physicochemical characterization of ribonucleoside diphosphate reductase from Escherichia coli. J Biol Chem. 1973 Jul 10;248(13):4591–4601. [PubMed] [Google Scholar]
  29. Thelander L., Reichard P. Reduction of ribonucleotides. Annu Rev Biochem. 1979;48:133–158. doi: 10.1146/annurev.bi.48.070179.001025. [DOI] [PubMed] [Google Scholar]
  30. Thomas M., Davis R. W. Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease. J Mol Biol. 1975 Jan 25;91(3):315–328. doi: 10.1016/0022-2836(75)90383-6. [DOI] [PubMed] [Google Scholar]
  31. Ullrich A., Shine J., Chirgwin J., Pictet R., Tischer E., Rutter W. J., Goodman H. M. Rat insulin genes: construction of plasmids containing the coding sequences. Science. 1977 Jun 17;196(4296):1313–1319. doi: 10.1126/science.325648. [DOI] [PubMed] [Google Scholar]
  32. Wigler M., Sweet R., Sim G. K., Wold B., Pellicer A., Lacy E., Maniatis T., Silverstein S., Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. doi: 10.1016/0092-8674(79)90093-x. [DOI] [PubMed] [Google Scholar]
  33. Worcel A., Burgi E. Properties of a membrane-attached form of the folded chromosome of Escherichia coli. J Mol Biol. 1974 Jan 5;82(1):91–105. doi: 10.1016/0022-2836(74)90576-2. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES