Random spore analysis (RSA) allows many more spores to be examined than in tetrad analysis and thus facilitates rapid recombination mapping and strain construction. It’s easy to do in pombe because a simple enzyme treatment will kill remaining vegetative (unsporulated) cells and release the spores from their asci, but will have no effect on the spores themselves. However, when contemplating RSA, it is essential that all the classes of spores are viable when studying recombination frequencies, and also that double mutants may be unambiguously identified; otherwise tetrad analysis is essential. For example, it’s easy to use RSA to construct a ura- leu- double mutant, since this can be unambiguously distinguished from the ura- and leu- parental types, but it is not so straightforward to make a double temperature sensitive, unless you can reliably distinguish the phenotype of the double mutant from either parent (and if the mutants are synthetically lethal, the double mutant may be dead). There is no point in doing random spores and then having to do tetrads anyway. When in doubt, pull tetrads.
The RSA protocol is simple: Using a three day old cross check for the presence of asci under the light microscope. 1 ml of sterile distilled water is inoculated with a loopful of the cross, and glusalase (Dupont/NEN) is added to a final concentration of 0.5%. The mixture should be incubated overnight at 25-29°C or for at least 6 hours at 29°C. Glusalase is a crude snail gut enzyme that breaks down the ascus wall and kills vegetative cells. Between 200-1000 spores/plate can be plated out on YES Agar or selective medium. The plates are then incubated at an appropriate temperature until colonies form.
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Random spore analysis can also be useful for plasmid complementation assays. For example, assume you have a mammalian gene on a plasmid that is homologous to a pombe gene, and you want to determine whether it cross-complements. If you have a conditional mutation in the pombe gene (e.g., temperature sensitive), you can simply transform the plasmid into the haploid strain, and raise the temperature. But more commonly, all you will have is a lethal disruption mutation. In this case, you transform your plasmid (with a different marker than the disruption) into the heterozygous diploid, sporulate, and select or screen for growth on the appropriate plates. Plasmids are usually lost at a high rate in meiosis, but this method allows you to screen a large population of spores and recover the approximately 10% that contain the plasmid. You can then determine whether those that contain the plasmid also contain the disruption marker (if it all works perfectly, about 50% of them should). Click here for considerations on building plasmids for inter-species cross-complementation experiments.
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