Six new amino acid-auxotrophic markers for targeted gene integration and disruption in fission yeast

Abstract

Fission yeast Schizosaccharomyces pombe is amenable to genetics and is an excellent model system for studying eukaryotic cell biology. However, auxotrophic markers that can be used for both targeted gene integration and disruption are very limited. Here we performed a forward genetic screen in an effort to develop a new set of selectable markers for use in this yeast. Mutants that were auxotrophic for arginine, asparagine, cysteine, lysine, methionine and phenylalanine were isolated. Six genes were analyzed in detail and the mutations in the genes were identified. Among these six are three new genes: asn1 (+), cys2 (+) and pha2 (+) were required for biosynthesis of asparagine, cysteine and phenylalanine, respectively. New alleles of arg1 (+), lys3 (+) and met6 (+) were also identified. All of these genes proved to be suitable as selectable markers for targeted gene integration and disruption. We also showed that in Schizosaccharomyces pombe there are two apparent homologues of Saccharomyces cerevisiae MET2: the previously known met6 (+), and SPBC106.17c (named cys2 (+)). The cys2 mutation required cysteine rather than methionine. These new tools, specifically, new selectable markers, will be useful in further genetic and biological studies in fission yeast.