Diploid cells arise spontaneously in most S. pombe strains probably as a result of endoreplication. This characteristic can be used to isolate homozygous diploids of any strain. The strain is streaked out to single colonies on YEP (YE + Phloxin B) agar and incubated until colonies form. Diploids can be identified as clones that stain dark red with Phloxin B and which contain large cells upon microscopic examination. These diploids can undergo a diploid mitotic cycle and when starved of nutrients can conjugate with diploid cells of the opposite mating type to form a tetraploid zygote. This can be sporulated to generate four diploid spores, although aberrant segregation of chromosomes can occur during the tetraploid meiosis.
Homothallic strains also generate diploid cells which can switch mating type and therefore be induced to undergo meiosis. The cells can directly produce azygotic asci without mating and thus can be unambiguously identified. (They can also mate with each other)
Sporulating diploids can be isolated by crossing h- and h+ haploids with complementary growth requirements, for example using strains with the markers leu1-32 h- and a ura4-d18 h+. At various time-points after crossing (e.g. 8, 12, 24 hours), when conjugation has occurred, a loopful of the cross is streaked out onto minimal medium which will only allow growth of a conjugated diploid. However, since mating is rapidly followed by meiosis and sporulation, many of the colonies growing on selective media will be prototrophic haploid segregants. To circumvent this problem, ade6-M210 and ade6-M216 mutations are commonly used. ade6-M216 colonies are light pink and ade6-M210 dark pink on plates of YE medium or EMM containing 10mg/L adenine because of accumulation of a red adenine precursor. On media containing adenine in excess of 10mg/L the red colour is not observed. Diploid cells containing both mutations grow in the absence of adenine and the colonies formed are white due to intragenic complementation between the two ade6 alleles. Because the alleles are tightly linked and there is infrequent gene conversion, spores generated by meiosis are unlikely to be adenine prototrophs and will not form colonies on the selective media.
Sporulating diploids are very unstable and will generate spores if they enter stationary phase from minimal medium. They can be maintained on yeast extract medium which inhibits sporulation. Alternatively, non sporulating diploids can be derived by crossing over at the mating type locus which leads to homozygosis at the mating type locus. These arise fairly frequently and can be screened for by replica plating onto malt extract and looking for non iodine positive colonies which are not undergoing sporulation. Alternatively, Mat1-B102 can be used. This strain is defective in the Mat1+ gene required for meiosis, but functions normally as an h- . So, you can cross it to an h+ strain and sporulate, then you can cross to an h- strain if you want stable diploids.
Sometimes it is required to cross a diploid and a haploid strain. Such a cross yields less than 10% spore viability and most of the segregants are slow growing because of aneuploidy. Since S. pombe has only three linkage groups, normal haploid and diploid segregants can be obtained at a reasonable frequency from random spores; they are easily identified since they grow well.
Protocol for generating an h+/h- sporulating diploid
1) cross an ade6-M210 strain to an ade6-M216 strain on EMM-Glutamate. Put plate 29 oC. These are the KG2 and KG3 strains we have in the lab.
Minimal glutamate (EMMG Used for sporulating diploids in liquid).
As EMM. Replace NH4Cl with 1 g/l sodium glutamate (5.91 mM).
2) 8h, 12h and 24h after mixing the cells, take a loop-full and streak on minimal medium lacking adenine (EMM -ade). Incubate this plate at 32 oC.
3) Pick colonies as soon as they appear on the EMM -ade plate (typically after 2-4 days, If diploids are left too long on the EMM -ade plate they will undergo sporulation) and streak on YE (no supplements). Put the YE plate to grow at 32 oC for two days.
4) There will usually be two types of colonies on the YE plate: larger, white colonies (these are diploids) and smaller, red colonies (these are haploid colonies ade-which grew from spores generated when the diploid reached stationary phase on the EMM -ade plate and underwent sporulation, they are red because YE contains low levels of adenine and ade- cells will grow but accumulate a red metabolic precursor ). Check under the microscope to make sure that cells in the darker colonies have a larger size. Patch the diploids on YES and replica plate the YE plate on EMM-glutamate. Check after 2-3 days that the diploids picked are sporulating.
Remember to propagate the diploid on YES, which inhibites sporulation.
If the diploid is to be used for
deletion or tagging of a gene it is a good idea to dissect a couple of
tetrads beforehand to make sure that no recessive mutation is present in