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

Abstract

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, ura4- cells 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 kb HindIII 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.

The regulatory subunits of fission yeast protein phosphatase 2A (PP2A) affect cell morphogenesis, cell wall synthesis and cytokinesis

“…One step gene replacement (Rothstein 1983;Grimm et al 1988) was employed. The 0.7 kb EcoRV-Hind111 fragment in the coding region o f p a b l + was replaced with the 1.8 kb S. pombe ura4+ gene, and the resulting plasmid was linearized with Bam HI and Cla I double digestion, and introduced onto the chromosome of a diploid by homologous recombination.…”

Defining the minimal requirements for papilloma viral E6-mediated inhibition of human p53 activity in fission yeast

“…Where required, L-arginine was added to a ®nal concentration of 1 mg/ml (Van Hu el et al, 1992). The 5-Fluoroorotic acid (5-FOA): uracil ratio in the media was 1 mg/ml : 0.05 mg/ml, in accord with Grimm et al (1988). A constituent of the 5-FOA medium described by Grimm et al (1988) is yeast nitrogen base which contains thiamine.…”

“…The 5-Fluoroorotic acid (5-FOA): uracil ratio in the media was 1 mg/ml : 0.05 mg/ml, in accord with Grimm et al (1988). A constituent of the 5-FOA medium described by Grimm et al (1988) is yeast nitrogen base which contains thiamine. The relative concentration in this 5-FOA media is 0.2 mM and so nmt1+ promoter driven expression would be predicted to be minimal.…”

A ‘marker switch’ approach for targeted mutagenesis of genes in Schizosaccharomyces pombe

“…The 1.8 kb HindIII fragment of pCG1 (Grimm et al, 1988) pombe genome, so can not target recombination to a particular locus; thus, it is not likely to influence the frequency of targeting of constructs to the native locus. pDM1 is bluescript with an insert comprising the sequences from the BglII site 499 bp 5 of the plo1 + ORF to the HindIII site 818 bp 3 of plo1 + .…”