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Mutagenesis in multinucleate cells: the effects of N-methyl-N'-nitro-N-nitrosoguanidine on Phycomyces spores
Abstract:
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.
Insights
This study introduces a method using N-methyl-N'-nitro-N-nitrosoguanidine to create single-nucleus fungal cells, enabling the study of recessive mutations in Phycomyces. This technique improves the isolation of desired genetic mutations.
Area of Science:
- * Mycology
- * Molecular Biology
- * Genetics
Background:
- * Multinucleate cells, like Phycomyces spores, present challenges for isolating recessive mutants due to multiple gene copies.
- * Traditional mutagenesis methods are hindered by the presence of multiple nuclei, masking recessive traits.
Purpose of the Study:
- * To develop a method for isolating recessive mutants in multinucleate fungal cells.
- * To optimize mutagenesis conditions for Phycomyces using N-methyl-N itro-N-nitrosoguanidine.
- * To enable the study of recessive mutations by achieving functional uninucleate cells.
Main Methods:
- * Employing N-methyl-N itro-N-nitrosoguanidine (nitrosoguanidine) for nuclear killing in Phycomyces spores.
- * Selecting for survivors with a single functional nucleus after nitrosoguanidine treatment.
- * Utilizing heterokaryon spores and recessive markers for positive selection of uninucleate cells.
Main Results:
- * Nitrosoguanidine effectively reduces nuclei in some Phycomyces cells, allowing recessive mutation expression.
- * Approximately 35% of nitrosoguanidine-treated survivors achieve functional uninucleate status.
- * Optimal nitrosoguanidine mutagenesis conditions in Phycomyces differ from bacteria and yeast, with less emphasis on buffer pH.
Conclusions:
- * Nuclear killing with nitrosoguanidine is a viable strategy for isolating recessive mutants in multinucleate fungi.
- * The developed method facilitates the study of recessive mutations by enabling the isolation of functionally uninucleate cells.
- * Understanding mutagenesis kinetics (survival and mutation induction) is crucial for optimizing genetic studies in Phycomyces.