Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Sep;85(17):6468–6472. doi: 10.1073/pnas.85.17.6468

Tn5tac1, a derivative of transposon Tn5 that generates conditional mutations.

W Y Chow 1, D E Berg 1
PMCID: PMC281994  PMID: 2842773

Abstract

Conditional lethal mutations are valuable for analyzing essential genes. We describe here a derivative of the bacterial transposon Tn5 called Tn5tac1 and its use in an innovative strategy for making mutations with conditional phenotypes. The 4.6-kilobase Tn5tac1 element contains a strong, regulatable, outward-facing promoter (Ptac) near one end and is polar on the expression of distal genes when the inducer of Ptac [isopropyl beta-D-thiogalactoside (IPTG)] is absent. Our results show that two unusual conditional mutant phenotypes can result from Tn5tac1 insertion in Escherichia coli: one is corrected by IPTG while the other is induced by IPTG. The broad host range of Tn5 and the conditional nature of these mutant phenotypes makes Tn5tac1 well suited for identifying essential genes in diverse bacterial species.

Full text

PDF
6470

Images in this article

Selected References

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

  1. Adhya S., Gottesman M. Promoter occlusion: transcription through a promoter may inhibit its activity. Cell. 1982 Jul;29(3):939–944. doi: 10.1016/0092-8674(82)90456-1. [DOI] [PubMed] [Google Scholar]
  2. Amann E., Brosius J. "ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli. Gene. 1985;40(2-3):183–190. doi: 10.1016/0378-1119(85)90041-1. [DOI] [PubMed] [Google Scholar]
  3. Bachmann B. J., Low K. B., Taylor A. L. Recalibrated linkage map of Escherichia coli K-12. Bacteriol Rev. 1976 Mar;40(1):116–167. doi: 10.1128/br.40.1.116-167.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balbás P., Soberón X., Merino E., Zurita M., Lomeli H., Valle F., Flores N., Bolivar F. Plasmid vector pBR322 and its special-purpose derivatives--a review. Gene. 1986;50(1-3):3–40. doi: 10.1016/0378-1119(86)90307-0. [DOI] [PubMed] [Google Scholar]
  5. Berg D. E., Schmandt M. A., Lowe J. B. Specificity of transposon Tn5 insertion. Genetics. 1983 Dec;105(4):813–828. doi: 10.1093/genetics/105.4.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berg D. E., Weiss A., Crossland L. Polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol. 1980 May;142(2):439–446. doi: 10.1128/jb.142.2.439-446.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. CAMPBELL A. Sensitive mutants of bacteriophage lambda. Virology. 1961 May;14:22–32. doi: 10.1016/0042-6822(61)90128-3. [DOI] [PubMed] [Google Scholar]
  8. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  9. Davis M. A., Simons R. W., Kleckner N. Tn10 protects itself at two levels from fortuitous activation by external promoters. Cell. 1985 Nov;43(1):379–387. doi: 10.1016/0092-8674(85)90043-1. [DOI] [PubMed] [Google Scholar]
  10. Groisman E. A., Castilho B. A., Casadaban M. J. In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1480–1483. doi: 10.1073/pnas.81.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Howe M. M. Prophage deletion mapping of bacteriophage Mu-1. Virology. 1973 Jul;54(1):93–101. doi: 10.1016/0042-6822(73)90118-9. [DOI] [PubMed] [Google Scholar]
  12. Jackson E. N., Yanofsky C. Internal deletions in the tryptophan operon of Escherichia coli. J Mol Biol. 1972 Nov 14;71(2):149–161. doi: 10.1016/0022-2836(72)90343-9. [DOI] [PubMed] [Google Scholar]
  13. Johnson R. C., Reznikoff W. S. DNA sequences at the ends of transposon Tn5 required for transposition. Nature. 1983 Jul 21;304(5923):280–282. doi: 10.1038/304280a0. [DOI] [PubMed] [Google Scholar]
  14. Kaback D. B., Oeller P. W., Yde Steensma H., Hirschman J., Ruezinsky D., Coleman K. G., Pringle J. R. Temperature-sensitive lethal mutations on yeast chromosome I appear to define only a small number of genes. Genetics. 1984 Sep;108(1):67–90. doi: 10.1093/genetics/108.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kohiyama M., Cousin D., Ryter A., Jacob F. Mutants thermosensibles d'Escherichia coli K 12. I. Isolement et caractérisation rapide. Ann Inst Pasteur (Paris) 1966 Apr;110(4):465–486. [PubMed] [Google Scholar]
  16. Lusky M., Botchan M. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature. 1981 Sep 3;293(5827):79–81. doi: 10.1038/293079a0. [DOI] [PubMed] [Google Scholar]
  17. Makris J. C., Nordmann P. L., Reznikoff W. S. Mutational analysis of insertion sequence 50 (IS50) and transposon 5 (Tn5) ends. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2224–2228. doi: 10.1073/pnas.85.7.2224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mitchell D. H., Reznikoff W. S., Beckwith J. R. Genetic fusions defining trp and lac operon regulatory elements. J Mol Biol. 1975 Apr 15;93(3):331–350. doi: 10.1016/0022-2836(75)90281-8. [DOI] [PubMed] [Google Scholar]
  19. Moir D., Stewart S. E., Osmond B. C., Botstein D. Cold-sensitive cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies. Genetics. 1982 Apr;100(4):547–563. doi: 10.1093/genetics/100.4.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nag D. K., Huang H. V., Berg D. E. Bidirectional chain-termination nucleotide sequencing: transposon Tn5seq1 as a mobile source of primer sites. Gene. 1988 Apr 15;64(1):135–145. doi: 10.1016/0378-1119(88)90487-8. [DOI] [PubMed] [Google Scholar]
  21. Phadnis S. H., Berg D. E. Identification of base pairs in the outside end of insertion sequence IS50 that are needed for IS50 and Tn5 transposition. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9118–9122. doi: 10.1073/pnas.84.24.9118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Sasakawa C., Carle G. F., Berg D. E. Sequences essential for transposition at the termini of IS50. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7293–7297. doi: 10.1073/pnas.80.23.7293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sasakawa C., Lowe J. B., McDivitt L., Berg D. E. Control of transposon Tn5 transposition in Escherichia coli. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7450–7454. doi: 10.1073/pnas.79.23.7450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Smith D. H., Berget P. B., King J. Temperature-sensitive mutants blocked in the folding or subunit assembly of the bacteriophage P22 tail-spike protein. I. Fine-structure mapping. Genetics. 1980 Oct;96(2):331–352. doi: 10.1093/genetics/96.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Studier F. W. Bacteriophage T7. Science. 1972 Apr 28;176(4033):367–376. doi: 10.1126/science.176.4033.367. [DOI] [PubMed] [Google Scholar]
  27. Ubben D., Schmitt R. A transposable promoter and transposable promoter probes derived from Tn1721. Gene. 1987;53(1):127–134. doi: 10.1016/0378-1119(87)90100-4. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Zumstein L., Wang J. C. Probing the structural domains and function in vivo of Escherichia coli DNA topoisomerase I by mutagenesis. J Mol Biol. 1986 Oct 5;191(3):333–340. doi: 10.1016/0022-2836(86)90130-0. [DOI] [PubMed] [Google Scholar]
  30. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES