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

While the counterselectable Schizosaccharomyces pombe ura4(+) gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4(+) confers 5FOA-resistant (5FOA(R)) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOA(R) mutants. Relative to the same approach using the homologous URA3(+) 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(R) colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we first determined that 5FOA(R) strains carry mutations in either of two genes; ura4(+) and ura5(+). We then cloned the S. pombe ura5(+) orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5(+) together with the S. pombe lys7(+) gene. Using this doubly marked cassette to disrupt the sck1(+) kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4(-) and ura5(-) mutants by screening 5FOA(R) colonies for the loss of the lys7(+) 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(R) colonies in our studies arose from background ura4(-) and ura5(-) mutations.

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