ASSESSMENT OF PHEROMONE PRODUCTION AND RESPONSE IN FISSION YEAST BY A HALO TEST OF INDUCED SPORULATION

We describe a rapid, sensitive and semi-quantitative plate assay for monitoring pheromone activity in the fission yeast Schizosaccharomyces pombe. It is based on the observation that meiosis requires stimulation by pheromone and exploits diploid strains that will only sporulate after addition of exogenous pheromone. The tester strains are heterozygous for mating type, are non-switching, and are mutated in one of the early subfunctions (either mat1-Me or mat1-Pc), so that meiosis is only induced after exposure to exogenous pheromone (M-factor or P-factor, respectively). Pheromone activity is assessed as an iodine-positive halo of sporulation surrounding the pheromone source, and the width of the halo is related to the amount of pheromone being produced. The assay is sufficiently sensitive to monitor the low amount of M-factor produced by an M mam1 strain, and its sensitivity towards P-factor is greatly increased by using a hyper-sensitive tester strain lacking the Sxa2 protease that is believed to degrade this pheromone. We also demonstrate that the production of P-factor is very much stimulated by exposure of P cells to M-factor.