[56] Molecular genetic analysis of fission yeast Schizosaccharomyces pombe

Publisher Summary This chapter describes techniques concerned with classical and molecular genetics, cell biology, and biochemistry that can be used with Schizosaccharomyces pombe . Conjugation and sporulation cannot take place in S. pombe except under conditions of nutrient starvation. ME medium is generally used for genetic crosses. To cross two strains, a loopful of h – and a loopful of h + are mixed together on a ME plate. The cross is left to dry and is then incubated below 30°, as conjugation is severely reduced above this temperature. Fully formed four-spore asci can be seen after 2–3 days of incubation. A 2 day-old cross is usually used for tetrad analysis. Using a 3-day-old cross, one can check for the presence of asci under the light microscope. Random spore analysis allows many more spores to be examined than in tetrad analysis, and in this way recombination mapping and strain construction can be carried out. Diploid cells arise spontaneously in most S. pombe strains, this characteristic can be used to isolate homozygous diploids of any strain. For mutagenesis of yeast strains, ethylmethane sulfonate (EMS) and nitrosoguanidine is used.