Use of a ura5 ^sup +^-lys7 ^sup +^ cassette to construct unmarked gene knock-ins in Schizosaccharomyces pombe

While the counterselectable Schizosaccharomyces pombe ura4 ^sup +^ gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4 ^sup +^ confers 5FOA-resistant (5FOA^sup R^) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOA^sup R^ mutants. Relative to the same approach using the homologous URA3 ^sup +^ gene in Saccharomyces cerevisiae, knock-ins in S. pombe are harder to identify due to a lower efficiency of homologous recombination and a relatively high background of spontaneous 5FOA^sup R^ colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we first determined that 5FOA^sup R^ strains carry mutations in either of two genes; ura4 ^sup +^ and ura5 ^sup +^. We then cloned the S. pombe ura5 ^sup +^ orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5 ^sup +^ together with the S. pombe lys7 ^sup +^ gene. Using this doubly marked cassette to disrupt the sck1 ^sup +^ kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4 ^sup -^ and ura5 ^sup -^ mutants by screening 5FOA^sup R^ colonies for the loss of the lys7 ^sup +^ marker. The utility of this system, especially when the phenotype for the strain carrying the knock-in allele is indistinguishable from that of the disruption strain, is borne out by the fact that ~95% of 5FOA^sup R^ colonies in our studies arose from background ura4 ^sup -^ and ura5 ^sup -^ mutations.[PUBLICATION ABSTRACT]