Molecular and General Genetics MGG

, Volume 215, Issue 1, pp 81–86 | Cite as

Genetic engineering of Schizosaccharomyces pombe: A system for gene disruption and replacement using the ura4 gene as a selectable marker

  • Christian Grimm
  • Jürg Kohli
  • Johanne Murray
  • Kinsey Maundrell
Article

Summary

A system is described for gene disruption and replacement in Schizosaccharomyces pombe based on the homologous selectable marker, ura4, the structural gene for orotidine-5′-phosphate decarboxylase. The presence of a single copy of the wild-type gene can rescue a ura4 auxotrophic mutant. Furthermore, ura4cells can be selected for in the presence of 5-fluoroorotic acid (5-FOA). This allows a convenient means of selecting for both forward and backward mutations. The sequence of a 1.8 kb HindIII fragment which contains the functional gene is reported. It encodes a single open reading frame of 264 amino acids which shows considerable conservation with the orotidine-5′-phosphate (OMP) decarboxylases from other organisms. The ura4 transcript is approximately 850 nucleotides long. It begins 51 bp upstream of the protein coding sequence and is unusual in that transcription termination occurs at or very close to the translational stop codon. To facilitate the use of ura4 in gene disruption experiments we have also constructed a novel strain of S. pombe called ura4-D18, in which the 1.8 kbHindIII fragment has been deleted from the chromosome. Using a combination of this strain and vectors containing ura4 as a selectable marker, we present a general method for targeting recombination events to the chromosomal locus under investigation.

Key words

Schizosaccharomyces pombe Gene disruption Gene replacement ura4 sequence ura4 deletion 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Amstutz H, Munz P, Heyer W-D, Leupold U, Kohli J (1985) Concerted evolution of tRNA genes: Intergenic conversion between three unlinked serine tRNA genes in Schizosaccharomyces pombe. Cell 40:879–886Google Scholar
  2. Bach M-L (1987) Cloning and expression of the OMP decarboxy-lase gene URA4 from Schizosaccharomyces pombe. Curr Genet 12:527–534Google Scholar
  3. Beach D, Nurse P (1981) High frequency transformation of the fission yeast Schizosaccharomyces pombe. Nature 290:140–142Google Scholar
  4. Beach D, Piper M, Nurse P (1982) Construction of a Schizosaccharomyces pombe gene bank in a yeast bacterial shuttle vector and its use to isolate genes by complementation. Mol Gen Genet 187:326–329Google Scholar
  5. 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 197:345–346Google Scholar
  6. Chattoo BB, Sherman F, Atubalis DA, Fiellstedt TA, Mehnert D, Ogur M (1979) Selection of lys2 mutants of the yeast S. cerevisiae by the utilization of α-aminoadipate. Genetics 93:51–65Google Scholar
  7. Cherayil B, Krupp G, Schuchert P, Char S, Söll D (1987) The RNA component of Schizosaccharomyces pombe RNase P are essential for cell viability. Gene 60:157–161Google Scholar
  8. Clarke L, Carbon J (1978) Functional expression of cloned yeast DNA in Escherichia coli: specific complementation of argino succinate lyase (argH) mutations. J Mol Biol 120:517–532Google Scholar
  9. Grimm C, Kohli J (1988) Observations on integrative transformation in Schizosaccharomyces pombe. Mol Gen Genet 215:87–93Google Scholar
  10. Gutz H, Heslot H, Leupold U, Loprieno N (1974) Schizosaccharomyces pombe, p395–446. In: King RD (ed) Handbook of genetics, vol. 1. Plenum, New YorkGoogle Scholar
  11. Hayles J, Aves S, Nurse P (1986) suc1 is an essential gene involved in both the cell cycle and growth in fission yeast. EMBO J 5:3373–3379Google Scholar
  12. Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359Google Scholar
  13. Heyer W-D, Sipiczki M, Kohli J (1986) Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element. Mol Cell Biol 6:80–89Google Scholar
  14. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact cells treated with alkali cations. J Bacteriol 153:487–493Google Scholar
  15. Losson R, Lacroute F (1983) Plasmids carrying the yeast OMP decarboxylase structural and regulatory genes: transcription regulation in a foreign environment. Cell 32:371–377Google Scholar
  16. Maundrell K, Nurse P, Schönholzer F, Schweingruber ME (1985) Cloning and characterization of two genes restoring acid phosphatase activity in pho1 mutants of Schizosaccharomyces pombe. Gene 39:223–230Google Scholar
  17. Rose M, Grisafi P, Boststein D (1984) Structure and function of the yeast URA3 gene: expression in E. coli. Gene 29:113–124Google Scholar
  18. Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211Google Scholar
  19. Sanger F, Coulson AR, Barrell BG, Smith AJH, Roe BA (1980) Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143:161–178Google Scholar
  20. Struhl K (1983) The new yeast genetics. Nature 305:391–397Google Scholar
  21. Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Christian Grimm
    • 1
  • Jürg Kohli
    • 1
  • Johanne Murray
    • 2
  • Kinsey Maundrell
    • 3
  1. 1.Institute of General MicrobiologyUniversity of BernBernSwitzerland
  2. 2.School of Biological SciencesUniversity of SussexBrightonUK
  3. 3.Centro di RicercheSCLAVOSienaItaly

Personalised recommendations