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 Ura4pts supports growth at 30°C but is unable to allow growth at 37°C in the absence of uracil when a single copy of the gene is integrated into the host chromosome. Using the ura4ts 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 ura4ts gene and fail to grow when replicated to 37°C in the absence of uracil. Copyright © 1999 John Wiley & Sons, Ltd.