Mutagenesis in multinucleate cells: the effects of N-methyl-N'-nitro-N-nitrosoguanidine on Phycomyces spores

Multinucleate cells, such as the spores of the fungus Phycomyces, are unsuitable for the isolation of recessive mutants. Nuclear killing by N-methyl-N'-nitro-N-nitrosoguanidine (henceforth nitrosoguanidine) eliminates all but one of the nuclei in some of the cells and allows the expression of recessive mutations. Even in the best conditions, only about 35% of the survivors have a single functional nucleus. Functionally uninucleate cells can be positively selected. This involves the exposure to nitrosoguanidine of the spores of a heterokaryon and selection for a recessive marker present in a small fraction of its nuclei. The optimal conditions for nitrosoguanidine mutagenesis in Phycomyces differ from those for bacteria and yeast. Buffer composition and pH are less important than in other organisms. Survival is an exponential function and mutation induction a linear function of the dose of the mutagen (concentration X time). Spore germination leads to an immediate increase in the number of gene copies per cell, thus further hindering the expression of recessive mutations; dominant mutations are then nearly always isolated in heterokaryotic form.