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Current Genetics

, Volume 11, Issue 3, pp 217–225 | Cite as

Cloning and expression on a multicopy vector of five invertase genes of Saccharomyces cerevisiae

  • Stefan Hohmann
  • Friedrich K. Zimmermann
Original Articles

Summary

Six unlinked loci for invertase structural genes are known in the yeast Saccharomyces cerevisiae: SUC1-SUC5 and SUC7. These genes are similar in structure and expression but not identical. Different yeast strains possess none, one or several of these genes.

We have isolated the genes SUC1-SUC5, subcloned them into the multicopy vector YEp24 and compared the expression of the five SUC genes in one recipient strain.

SUC2 was isolated by transformation of a suc0 strain with a gene pool and complementation to sucrose fermentation. SUC4 was cloned from a minipool of chromosomal fragments which were shown to contain SUC4 by Southern hybridization. SUC1, SUC3 and SUC5 were isolated using the method of plasmid eviction. A plasmid containing regions flanking SUC4 was integrated next to these SUC genes. The plasmid together with the SUC genes were then cut out of the chromosome using an appropriate restriction endonuclease.

The length of chromosomal DNA fragments containing the different SUC genes were 4.8 kb for SUC1, 5.2 kb for SUC2, 4.8 kb for SUC3, 12.8 kb for SUC4 and 17.2 kb for SUC5.

Fragments containing the complete SUC genes and the sequences controlling their expression were subcloned into YEp24 and transformed into a strain without any active invertase gene. Invertase activity of transformants was measured after growth repressing (8% glucose) and derepressing (2% raffinose) conditions. As expected from results with strains carrying the individual SUC genes in a chromosomal location, the SUC genes were expressed to a different extent.

Key words

Saccharomyces cerevisiae Gene cloning Invertase genes Multicopy vector 

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References

  1. Beggs JD (1978) Nature 275:104–108Google Scholar
  2. Birnboim HC (1983) Methods Enzymol 100:243–255Google Scholar
  3. Bolivar F, Backman K (1979) Methods Enzymol 68:245–267Google Scholar
  4. Botstein D, Davis RW (1982) The molecular biology of the yeast Saccharomyces. Cold Spring Harbor Lab, pp 607–636Google Scholar
  5. Broach JR, Strathern JN, Hicks JB (1979) Gene 8:121–133Google Scholar
  6. Carlson M, Osmond BC, Botstein D (1981a) Genetics 98:25–40Google Scholar
  7. Carlson M, Osmond BC, Botstein D (1981b) Genetics 98:41–54Google Scholar
  8. Carlson M, Botstein D (1982) Cell 28:145–154Google Scholar
  9. Carlson M, Botstein D (1983) Mol Cell Biol 3:351–359Google Scholar
  10. Carlson M Celenza J, Eng FJ (1985) Mol Cell Biol 5:2894–2902Google Scholar
  11. Clewell DB (1972) J Bacteriol 110:667–679Google Scholar
  12. Cohen SN, Chang ACY, Hsu L (1972) Proc Natl Acad Sci USA 69:2110–2114Google Scholar
  13. Futcher AB, Cox BS (1984) J Bacteriol 157:283–290Google Scholar
  14. Gascón S, Ottolenghi P (1967) CR Trav Lab Carlsberg 36:85–93Google Scholar
  15. Gáscon S, Lampen JO (1968) J Biol Chem 243:1567–1572Google Scholar
  16. Goldstein A Lampen JO (1975) Methods Enzymol 42:504–511Google Scholar
  17. Grossmann MK (1979) Thesis phD TH DarmstadtGoogle Scholar
  18. Grossmann MK, Zimmermann FK (1979) Mol Gen Genet 175:223–229Google Scholar
  19. Kaiser CA, Botstein D (1986) Mol Cell Biol 6:2382–2391Google Scholar
  20. Lehle L, Cohen RE, Ballou CE (1979) J Biol Chem 254:12209–12218Google Scholar
  21. Mortimer R, Hawthorne D (1966) Genetics 53:165–173Google Scholar
  22. Nasmyth KA, Reed SI (1980) Proc Natl Acad Sci USA 77:2119–2123Google Scholar
  23. Nasmyth KA, Tatchell K (1980) Cell 19:753–764Google Scholar
  24. Ottolenghi P (1971) CR Trav Lab Carlsberg 38:213–221Google Scholar
  25. Parra F, Herrero P, Moreno F, Gascon S (1980) FEBS Lett 118:330–332Google Scholar
  26. Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) J Mol Biol 113:237–251Google Scholar
  27. Rodriguez L, Lampen JO, MacKay VL (1981) Mol Cell Biol 1:469–474Google Scholar
  28. Sarokin L, Carlson M (1984) Mol Cell Biol 4:2750–2757Google Scholar
  29. Sarokin L, Carlson M (1985) Nucleic Acid Res 13:6089–6113Google Scholar
  30. Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76:1035–1039Google Scholar
  31. Taussig R, Carlson M (1983) Nucleic Acid Res 11:1943–1954Google Scholar
  32. Tautz D, Renz M (1983) Anal Biochem 132:14–19Google Scholar
  33. Wahl GM, Stern M, Stark GR (1979) Proc Natl Acad Sci USA 76:3683–3687Google Scholar
  34. Winge O, Roberts C (1952) CR Trav Lab Carlsberg 25:141–171Google Scholar
  35. Winston F, Chumley F, Fink GR (1983) Methods Enzymol 101:211–227Google Scholar
  36. Zamenhoff S (1957) Methods Enzymol 3:696–704Google Scholar
  37. Zimmermann FK (1975) Mutat Res 31:71–86Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • Stefan Hohmann
    • 1
  • Friedrich K. Zimmermann
    • 1
  1. 1.Institut für Mikrobiologie, Technische Hochschule DarmstadtDarmstadtGermany

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