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Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes

Nature Biotechnology volume 18, pages 97–100 (2000)Cite this article

Abstract

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene–operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1 , and Ty1) have been described, which bypass many limitations of in vivo systems2,3,4,5,6,7. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.

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Figure 1: Transposome formation.
Figure 2: DNA sequence analysis of candidate inserts indicate that they represent bona fide Tn5-like transposition events.
Figure 3: Southern blot analysis of transposition clones in P. vulgaris. Hpa I/EcoRV digests of genomic DNAs isolated from presumed transposition clones were subjected to Southern blot analysis using as probes 23-mer and 25-mer oligonucleotides that correspond to the two ends of the transposon.

References

  1. Berg, C.M. & Berg, D.E. in Escherichia coli and Salmonella cellular and molecular biology (eds Neidhardt, F.C. et al.) 2588–2612 (ASM Press, Washington, DC; 1996).

    Google Scholar 

  2. Goryshin, I.Y. & Reznikoff, W.S. Tn5 in vitro transposition . J. Biol. Chem. 273, 7367– 7374 (1998).

    CAS  Article  Google Scholar 

  3. Gwinn, M.L., Stellwagen, A.E., Craig, N.L., Tomb, J.F. & Smith, H.O. In vitro Tn7 mutagenesis of Haemophilus influenzae Rd and characterization of the role of atpA in transformation. J. Bacteriol. 179, 7315–7320 (1997).

    CAS  Article  Google Scholar 

  4. Haapa, S., Taira, S., Heikkinen, E. & Savilahti, H. An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications . Nucleic Acids Res. 27, 2777– 2784 (1999).

    CAS  Article  Google Scholar 

  5. Akerley, B.J., et al. Systematic identification of essential genes by in vitro Mariner mutagenesis. Proc. Natl. Acad. Sci. USA 95, 8927–8932 (1998).

    CAS  Article  Google Scholar 

  6. Devine, S.E. & Boeke, J.D. Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis. Nucleic. Acids Res. 22, 3765–3772 (1994).

    CAS  Article  Google Scholar 

  7. Braam, L.M. & Reznikoff, W.S. in Embryonic encyclopedia of life sciences (Nature Publishing Group, London, UK; 1999), available at www.els.net.

  8. Reznikoff, W.S. et al. Tn5: a molecular window on transposition. Biochem. Biophys. Res. Commun. In press.

  9. Wu, F., Goldberg, I. & Filutowicz, M. Roles of a 106-bp origin enhancer and Escherichia coliDnaA protein in replication of plasmid R6K. Nucleic Acids Res. 20, 811–817 ( 1992).

    CAS  Article  Google Scholar 

  10. Zhou, M., Bhasin, A. & Reznikoff, W.S. Molecular genetic analysis of transposase – end sequence recognition: cooperation of three adjacent base pairs in specific interactionwith a mutant Tn5transposase. J. Mol. Biol. 276, 913–925 (1998).

    CAS  Article  Google Scholar 

  11. Dover, W.J., Miller, J.F. & Ragsdale, C.W. High efficiency transformation of E. coliby high voltage electroporation. Nucleic Acids Res. 16, 6127–6145 (1988).

    Article  Google Scholar 

  12. Blattner, F.R. et al. The complete genome sequence of Escherichia coli K12 . Science 277, 1453–1462 (1997).

    CAS  Article  Google Scholar 

  13. Miller, J.H. Experiments in molecular genetics. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; (1972).

    Google Scholar 

  14. Thompson, J.R., Register, E., Curotto, J., Kurtz, M. & Kelley, R. An improved protocol for the preparation of yeast cells for transformation by electroporation. Yeast 14, 565–571 (1998).

    CAS  Article  Google Scholar 

  15. Grant, S.G., Jessee, J., Bloom, F.R. & Hanahan, D. Differential plasmid rescue from transgenic mouse DNAs into Escherichia colimethylation-restriction mutants. Proc. Natl. Acad. Sci. USA 87, 4645–4649 (1990).

    CAS  Article  Google Scholar 

  16. Oka, A., Sugisaki, H. & Takanami, M. Nucleotide sequence of the kanamycin resistant transposon Tn903. J. Mol. Biol. 147, 217– 226 (1981).

    CAS  Article  Google Scholar 

  17. Chang, A.C. & Cohen, S.N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134, 1141– 1156 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Wach, A., Brachat, A., Poehlmann, R. & Philippsen, P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808 (1994).

    CAS  Article  Google Scholar 

  19. Braam, L.A.M., Goryshin, I.Y. & Reznikoff, W.S. A mechanism for Tn5inhibition. J. Biol. Chem. 274, 86–92 (1999).

    CAS  Article  Google Scholar 

  20. Bolivar, F., et al. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95–113 (1977).

    CAS  Article  Google Scholar 

  21. Marmur, J. A procedure for the isolation of deoxyribonucleic acid from microorganisms . J. Mol. Biol. 3, 208– 218 (1961).

    CAS  Article  Google Scholar 

  22. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning: a laboratory manual 137–138 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; (1989).

    Google Scholar 

  23. Heiner, K.L., Hunkapiller, K.L., Chen, S.-M., Glass, J.I. & Chen, E.Y. Sequencing multimegabase-template DNA with BigDye terminator chemistry. Genome Res. 8 , 557–561 (1998).

    CAS  Article  Google Scholar 

  24. Reed, K.C. & Mann, D.A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13, 7207–7221 (1985).

    CAS  Article  Google Scholar 

  25. Goffeau, A. et al. Life with 6000 genes. Science 274, 563–567 (1996).

    Article  Google Scholar 

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Acknowledgements

We wish to thank Erich Moan and Joanne Decker for their technical assistance and input, Keith Derbyshire for communicating results prior to publication, and George Buriakov and Guy Plunkett for help with the E. coli insert analysis. Some of the reported experiments were supported by a grant (GM50692) from the National Institutes of Health to W.S.R. A preliminary report of the S. cerevisiae experiments can be found in Hoffman et al., Curr. Genet .35, 305 (1999).

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Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, 53706, WI

    Igor Y. Goryshin & William S. Reznikoff

  2. Epicentre Technologies, >1202 Ann St. , Madison, 53713, WI

    Jerry Jendrisak, Les M. Hoffman & Ronald Meis

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  1. Igor Y. Goryshin
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  2. Jerry Jendrisak
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  3. Les M. Hoffman
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Correspondence to William S. Reznikoff.

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Goryshin, I., Jendrisak, J., Hoffman, L. et al. Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat Biotechnol 18, 97–100 (2000). https://doi.org/10.1038/72017

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