Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1987 Nov 25;15(22):9461–9469. doi: 10.1093/nar/15.22.9461

Rapid sequencing of cloned DNA using a transposon for bidirectional priming: sequence of the Escherichia coli K-12 avtA gene.

L Liu 1, W Whalen 1, A Das 1, C M Berg 1
PMCID: PMC306480  PMID: 2825136

Abstract

A new approach to determining the sequence of cloned DNA is described. Unique regions near each end of the transposable element gamma-delta provide a pair of "portable" primer-specific sites for bidirectional sequencing by the dideoxy chain termination method. A set of gamma-delta insertions positioned about 200 bp apart over the entire cloned DNA allowed us to determine the sequence of both strands in a single parental plasmid without subcloning. The avtA (alanine-valine transaminase) gene of E. coli K-12 was sequenced by this approach. Surprisingly, gamma-delta insertions downstream of the coding region were found to significantly reduce avtA expression. We suggest that these nondisruptive insertions probably change the DNA topology and thereby alter gene expression.

Full text

PDF
9461

Selected References

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

  1. Adachi T., Mizuuchi M., Robinson E. A., Appella E., O'Dea M. H., Gellert M., Mizuuchi K. DNA sequence of the E. coli gyrB gene: application of a new sequencing strategy. Nucleic Acids Res. 1987 Jan 26;15(2):771–784. doi: 10.1093/nar/15.2.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahmed A. A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli. Gene. 1985;39(2-3):305–310. doi: 10.1016/0378-1119(85)90328-2. [DOI] [PubMed] [Google Scholar]
  3. Barnes W. M., Bevan M., Son P. H. Kilo-sequencing: creation of an ordered nest of asymmetric deletions across a large target sequence carried on phage M13. Methods Enzymol. 1983;101:98–122. doi: 10.1016/0076-6879(83)01008-3. [DOI] [PubMed] [Google Scholar]
  4. Berg C. M., Whalen W. A., Archambault L. B. Role of alanine-valine transaminase in Salmonella typhimurium and analysis of an avtA::Tn5 mutant. J Bacteriol. 1983 Sep;155(3):1009–1014. doi: 10.1128/jb.155.3.1009-1014.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Drlica K. Biology of bacterial deoxyribonucleic acid topoisomerases. Microbiol Rev. 1984 Dec;48(4):273–289. doi: 10.1128/mr.48.4.273-289.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Guyer M. S. The gamma delta sequence of F is an insertion sequence. J Mol Biol. 1978 Dec 15;126(3):347–365. doi: 10.1016/0022-2836(78)90045-1. [DOI] [PubMed] [Google Scholar]
  8. Guyer M. S. Uses of the transposon gamma delta in the analysis of cloned genes. Methods Enzymol. 1983;101:362–369. doi: 10.1016/0076-6879(83)01027-7. [DOI] [PubMed] [Google Scholar]
  9. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Heller K., Mann B. J., Kadner R. J. Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli. J Bacteriol. 1985 Mar;161(3):896–903. doi: 10.1128/jb.161.3.896-903.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  12. Korneluk R. G., Quan F., Gravel R. A. Rapid and reliable dideoxy sequencing of double-stranded DNA. Gene. 1985;40(2-3):317–323. doi: 10.1016/0378-1119(85)90055-1. [DOI] [PubMed] [Google Scholar]
  13. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  14. Reed R. R., Young R. A., Steitz J. A., Grindley N. D., Guyer M. S. Transposition of the Escherichia coli insertion element gamma generates a five-base-pair repeat. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4882–4886. doi: 10.1073/pnas.76.10.4882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sancar A., Rupp W. D. Cloning of uvrA, lexC and ssb genes of Escherichia coli. Biochem Biophys Res Commun. 1979 Sep 12;90(1):123–129. doi: 10.1016/0006-291x(79)91598-5. [DOI] [PubMed] [Google Scholar]
  16. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  18. Stokes H. W., Hall B. G. Topological repression of gene activity by a transposable element. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6115–6119. doi: 10.1073/pnas.81.19.6115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wang B. M., Liu L., Groisman E. A., Casadaban M. J., Berg C. M. High frequency generalized transduction by miniMu plasmid phage. Genetics. 1987 Jun;116(2):201–206. doi: 10.1093/genetics/116.2.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wang M. D., Liu L., Wang B. M., Berg C. M. Cloning and characterization of the Escherichia coli K-12 alanine-valine transaminase (avtA) gene. J Bacteriol. 1987 Sep;169(9):4228–4234. doi: 10.1128/jb.169.9.4228-4234.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Whalen W. A., Berg C. M. Analysis of an avtA::Mu d1(Ap lac) mutant: metabolic role of transaminase C. J Bacteriol. 1982 May;150(2):739–746. doi: 10.1128/jb.150.2.739-746.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES