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


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 


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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

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