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. 1984 May;158(2):636–643. doi: 10.1128/jb.158.2.636-643.1984

Method for determining whether a gene of Escherichia coli is essential: application to the polA gene.

C M Joyce, N D Grindley
PMCID: PMC215477  PMID: 6233260

Abstract

We have developed a general method for determining whether a gene of Escherichia coli is essential for viability. The method requires cloned DNA spanning the gene in question and a reasonably detailed genetic and physical map of the cloned segment. Using this information, one constructs a deletion of the target gene in vitro. For convenience, the deletion can be marked by an antibiotic resistance gene. A DNA segment containing the deletion is then cloned onto an att delta phage lambda vector. Integration of this phage, by homologous recombination at the target locus, and subsequent excision provide an efficient route for crossing the marked deletion onto the bacterial chromosome. Failure to delete the target gene indicates either that the resulting deletion was not viable or that the desired recombinational event did not take place. The use of prophage excision to generate the deletion allows one to estimate the fraction of deletion-producing events by analysis of the other product of the excision, the phage produced on induction of the prophage. In this way one can determine whether failure to recover a particular chromosomal deletion was due to its never having been formed, or, once formed, to its failure to survive. Applying this method to the polA gene, we found that polA is required for growth on rich medium but not on minimal medium. We repeated the experiment in the presence of plasmids carrying functional fragments of the polA gene, corresponding to the 5'-3' exonuclease and the polymerase-3'-5' exonuclease portions of DNA polymerase I. Surprisingly, either of these fragments, in the absence of the other, was sufficient to allow growth on rich medium.

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Selected References

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

  1. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev. 1983 Jun;47(2):180–230. doi: 10.1128/mr.47.2.180-230.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brutlag D., Atkinson M. R., Setlow P., Kornberg A. An active fragment of DNA polymerase produced by proteolytic cleavage. Biochem Biophys Res Commun. 1969 Dec 4;37(6):982–989. doi: 10.1016/0006-291x(69)90228-9. [DOI] [PubMed] [Google Scholar]
  4. Cameron J. R., Panasenko S. M., Lehman I. R., Davis R. W. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3416–3420. doi: 10.1073/pnas.72.9.3416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clements M. B., Syvanen M. Isolation of polA mutation that affects transposition of insertion sequences and transposons. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):201–204. doi: 10.1101/sqb.1981.045.01.032. [DOI] [PubMed] [Google Scholar]
  7. Cooper P. Excision-repair in mutants of Escherichia coli deficient in DNA polymerase I and/or its associated 5' leads to 3' exonuclease. Mol Gen Genet. 1977 Jan 7;150(1):1–12. doi: 10.1007/BF02425319. [DOI] [PubMed] [Google Scholar]
  8. DiNardo S., Voelkel K. A., Sternglanz R., Reynolds A. E., Wright A. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell. 1982 Nov;31(1):43–51. doi: 10.1016/0092-8674(82)90403-2. [DOI] [PubMed] [Google Scholar]
  9. Grindley N. D., Joyce C. M. Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7176–7180. doi: 10.1073/pnas.77.12.7176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grindley N. D., Kelley W. S. Effects of different alleles of the E. coli K12 pol A gene on the replication of non-transferring plasmids. Mol Gen Genet. 1976 Feb 2;143(3):311–318. doi: 10.1007/BF00269409. [DOI] [PubMed] [Google Scholar]
  11. Gutterson N. I., Koshland D. E., Jr Replacement and amplification of bacterial genes with sequences altered in vitro. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4894–4898. doi: 10.1073/pnas.80.16.4894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guyer M. S., Reed R. R., Steitz J. A., Low K. B. Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):135–140. doi: 10.1101/sqb.1981.045.01.022. [DOI] [PubMed] [Google Scholar]
  13. Heijneker H. L., Ellens D. J., Tjeerde R. H., Glickman B. W., van Dorp B., Pouwels P. H. A mutant of Escherichia coli K12 deficient in the 5'-3' exonucleolytic activity of DNA polymerase I. II. Purification and properties of the mutant enzyme. Mol Gen Genet. 1973 Jul 31;124(1):83–96. doi: 10.1007/BF00267167. [DOI] [PubMed] [Google Scholar]
  14. Joyce C. M., Grindley N. D. Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1830–1834. doi: 10.1073/pnas.80.7.1830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Joyce C. M., Grindley N. D. Identification of two genes immediately downstream from the polA gene of Escherichia coli. J Bacteriol. 1982 Dec;152(3):1211–1219. doi: 10.1128/jb.152.3.1211-1219.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Joyce C. M., Kelley W. S., Grindley N. D. Nucleotide sequence of the Escherichia coli polA gene and primary structure of DNA polymerase I. J Biol Chem. 1982 Feb 25;257(4):1958–1964. [PubMed] [Google Scholar]
  17. Kelley W. S., Chalmers K., Murray N. E. Isolation and characterization of a lambdapolA transducing phage. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5632–5636. doi: 10.1073/pnas.74.12.5632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kelley W. S., Joyce C. M. Genetic characterization of early amber mutations in the Escherichia coli polA gene and purification of the amber peptides. J Mol Biol. 1983 Mar 15;164(4):529–560. doi: 10.1016/0022-2836(83)90049-9. [DOI] [PubMed] [Google Scholar]
  19. Kelly R. B., Cozzarelli N. R., Deutscher M. P., Lehman I. R., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. XXXII. Replication of duplex deoxyribonucleic acid by polymerase at a single strand break. J Biol Chem. 1970 Jan 10;245(1):39–45. [PubMed] [Google Scholar]
  20. Kingsbury D. T., Helinski D. R. Temperature-sensitive mutants for the replication of plasmids in Escherichia coli: requirement for deoxyribonucleic acid polymerase I in the replication of the plasmid ColE 1 . J Bacteriol. 1973 Jun;114(3):1116–1124. doi: 10.1128/jb.114.3.1116-1124.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Klenow H., Henningsen I. Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Proc Natl Acad Sci U S A. 1970 Jan;65(1):168–175. doi: 10.1073/pnas.65.1.168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Konrad E. B., Lehman I. R. A conditional lethal mutant of Escherichia coli K12 defective in the 5' leads to 3' exonuclease associated with DNA polymerase I. Proc Natl Acad Sci U S A. 1974 May;71(5):2048–2051. doi: 10.1073/pnas.71.5.2048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lehman I. R., Chien J. R. Persistence of deoxyribonucleic acid polymerase I and its 5'--3' exonuclease activity in PolA mutants of Escherichia coli K12. J Biol Chem. 1973 Nov 25;248(22):7717–7723. [PubMed] [Google Scholar]
  24. Lehman I. R., Uyemura D. G. DNA polymerase I: essential replication enzyme. Science. 1976 Sep 10;193(4257):963–969. doi: 10.1126/science.781842. [DOI] [PubMed] [Google Scholar]
  25. Oka A., Sugisaki H., Takanami M. Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol. 1981 Apr 5;147(2):217–226. doi: 10.1016/0022-2836(81)90438-1. [DOI] [PubMed] [Google Scholar]
  26. Olivera R. M., Bonhoeffer E. Replication of Escherichia coli requires DNA polymerase I. Nature. 1974 Aug 9;250(5466):513–514. doi: 10.1038/250513a0. [DOI] [PubMed] [Google Scholar]
  27. Wahl A. F., Hockensmith J. W., Kowalski S., Bambara R. A. Alternative explanation for excision repair deficiency caused by the polAex1 mutation. J Bacteriol. 1983 Aug;155(2):922–925. doi: 10.1128/jb.155.2.922-925.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Weinert T. A., Schaus N. A., Grindley N. D. Insertion sequence duplication in transpositional recombination. Science. 1983 Nov 18;222(4625):755–765. doi: 10.1126/science.6314502. [DOI] [PubMed] [Google Scholar]

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