Gene disruption in Schizosaccharomyces pombe using a temperature-sensitive Ura4p.
We have generated a temperature-sensitive form of the Ura4p protein from the fission yeast Schizosaccharomyces pombe. A single T-to-C mutation at nucleotide 782 (relative to the initiator ATG codon of ura4) changes the leucine residue at position 261 in Ura4p to a proline. The mutant Ura4p(ts) supports growth at 30 degrees C but is unable to allow growth at 37 degrees C in the absence of uracil when a single copy of the gene is integrated into the host chromosome. Using the ura4(ts) cassette for gene replacements simplifies the identification of transformants in which the disruption construct has undergone homologous integration into the host chromosome, as these individuals contain a single copy of the ura4(ts) gene and fail to grow when replicated to 37 degrees C in the absence of uracil.
Davis
K
K
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K.
Pateman
C
C
Davey
J
J
D019943:Amino Acid Substitution; D015139:Blotting, Southern; D004274:DNA, Recombinant / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D018628:Gene Dosage; D005800:Genes, Fungal / Q000235:genetics / Q000502:physiology; D016254:Mutagenesis, Insertional / Q000379:methods*; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000502:physiology; D029702:Schizosaccharomyces pombe Proteins; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D014170:Transformation, Genetic; D014498:Uracil / Q000378:metabolism
D004274:DNA, Recombinant; D005656:Fungal Proteins; D029702:Schizosaccharomyces pombe Proteins; C400810:ura4 protein, S pombe; D014498:Uracil