Fig. 8b. Complementation in S. pombe. The goal is to test whether the mutations m1 and m2, which produce the same phenotype, are in the same complementation group. Standard crosses are used to construct the two haploid strains in the top line, which cannot grow in the absence of adenine. These are mixed together on ME plates for 16-24 hours at 25°, then transferred to MM (minimal medium) containing a nitrogen source (but no adenine) and the stain, phloxine B. Under these conditions, only diploid colonies (resulting from mating of the two haploid strains; the ade6-M210 and ade6-M216 mutations complement each other [intragenic complementation]) can grow, but diploid colonies aren't very healthy and produce a lot of dead cells, which stain dark red with phloxine B (indicated by the dark red color outlining the diploid cells in the above diagram). If m1 and m2 are in the same complementation group (as indicated in the left-hand diploid cell), then the diploid cells will display the mutant phenotype. But if they are in different complementation groups (as indicated in the right-hand diploid cell), then they will be complemented by the corresponding wild-type genes and will display the wild-type phenotype. S. pombe diploid cells spontaneously tend to go through meiosis and sporulate. In the case of diploid cells bearing the mutually complementing ade6-M210 and ade6-M216 mutations, in most cases this will produce haploid spores that are either ade6-M210 or ade6-M216 and therefore cannot grow in the absence of adenine. But once in a while, an intragenic recombination event will occur between the ade6-M210 and ade6-M216 sites within the ade6 gene, resulting in one pair of spores that is ade6+ and another pair that is doubly mutant (both the M210 and M216 alleles in the same gene; no intragenic recombination occurs in this case). The ade6+ spores can grow in the absence of adenine, but they can be distinguished from true diploid colonies by their lighter red color. In the above diagram, cells that cannot grow in the absence of adenine are indicated by a green color.