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Use of site-specific recombination to regenerate selectable markers

Summary

A method which allows the repeated use of a single selectable marker in DNA transformations was demonstrated. This marker regeneration method employed portions of the Saccharomyces cerevisiae 2 μm circle plasmid: the inverted repeat sequences (FRTs), and the FLP gene whose product, a site-specific recombinase, catalyzes recombination events between FRTs. When FRTs were oriented as direct repeats and integrated into the genome of the yeast Pichia pastoris, FLP-mediated recombination resulted in the efficient and precise deletion of DNA located between the repeats. In the example described, the S. cerevisiae ARG4 gene, placed between a set of FRTs and integrated into Pichia in a prior transformation, was deleted by FLP, thereby regenerating an arginine-requiring phenotype in the P. pastoris strain.

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References

  1. Alani E, Cao L, Kleckner N (1987) A method for gene disruption that allows repeated use of Ura3 selection in the construction of multiply disrupted yeast strains. Genetics 116:541–545

  2. Babineau D, Vetter D, Andrews BJ, Gronostajski RM, Protean GA, Beatty LG, Sadowski PD (1985) The FLP protein of the 2-micron plasmid of yeast. J Biol Chem 260:12313–12319

  3. Beacham IR, Schweitzer BW, Warrick HM, Carbon J (1984) The nucleotide sequence of the yeast ARG4 gene. Gene 29:271–279

  4. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346

  5. Broach JR (1981) The yeast plasmid 2 μm circle. In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast Saccharomyces: Life cycle and inheritance. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 455–470

  6. Cox MM (1983) The FLP protein of the yeast 2 mu plasmid: expression of a eucaryotic genetic recombination system in E. coli. Proc Natl Acad Sci USA 80:4223–4227

  7. Cregg JM, Madden KR (1987) Development of yeast transformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris by gene disruption. In: Stewart GG, Russell I, Klein RD, Hiebsch RR (eds) Biological research on industrial yeasts. Vol 11. CRC Press, Boca Raton, Florida, pp 1–18

  8. Cregg JM, Madden KR (1988) Development of the methylotrophic yeast, Pichia pastoris, as a host system for the production of foreign proteins. Dev Ind Microbiol 29:33–41

  9. Cregg JM, Barringer KJ, Hessler AY, Madden KR (1985) Pichia pastoris as a host system for transformations. Mol Cell Biol 5:3376–3385

  10. Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9:1316–1323

  11. Digan ME, Tschopp J, Grinna L, Lair SV, Craig WS, Velicelebi G, Siegel R, Davis GR, Thill G (1988) Secretion of heterologous proteins from the methylotrophic yeast, Pichia pastoris. Dev Ind Microbiol 29:59–65

  12. Futcher AB (1988) The 2 gmm circle plasmid of Saccharomyces cerevisiae. Yeast 4:27–40

  13. Hartley J, Donelson JE (1980) Nucleotide sequence of the yeast plasmid. Nature 286:860–865

  14. Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933

  15. McLeod M, Volkert F, Broach J (1984) Components of the sitespecific recombination system encoded by the yeast plasmid 2-micron circle. Cold Spring Harbor Symp Quant Biol 49:779–787

  16. Preibisch G, Kleinhans U, Roggenkamp R, Hollenberg CP (1984) Recombinational properties of the Saccharomyces cerevisiae FLP gene expressed Escherichia coli. Curr Genet 8:439–448

  17. Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–210

  18. Rudolf H, Koenig-Rauseo I, Hinnen A (1985) One-step gene replacement in yeast by cotransformation. Gene 36:87–95

  19. Sadowski P (1986) Site-specific recombinases: changing partners and doing the twist. J Bacteriol 165:341–347

  20. Sauer B (1987) Functional expression of the ere-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol 7:2087–2096

  21. Senecoff JF, Cox MM (1986) Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing. J Biol Chem 261:7380–7386

  22. Som T, Armstrong KA, Volkert FC, Broach JR (1988) Autoregulation of 2 μm circle gene expression provides a model for maintenance of stable plasmid copy levels. Cell 52:27–37

  23. Southern EM (1975) Detection of specific sequences among DNA fragments separated by agarose gel electrophoresis. J Mol Biol 98:503–517

  24. Tschumper G, Carbon J (1982) Delta sequences and double symmetry in a yeast chromosomal replicator region. J Mol Biol 156:293–307

  25. Vetter D, Andrews BJ, Roberts-Beatty L, Sadowski PD (1983) Site-specific recombination of yeast 2-μm DNA in vitro. Proc Natl Acad Sci USA 80:7284–7288

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Author information

Correspondence to James M. Cregg.

Additional information

Communicated by C.P. Hollenberg

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Cregg, J.M., Madden, K.R. Use of site-specific recombination to regenerate selectable markers. Molec. Gen. Genet. 219, 320–323 (1989). https://doi.org/10.1007/BF00261194

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Key words

  • 2 μm plasmid
  • FLP gene
  • Pichia pastoris
  • Methylotrophic yeast