Isolation of Neurospora crassa A mating type mutants by repeat induced point (RIP) mutation.
In the filamentous fungus, Neurospora crassa, mating type is regulated by a single locus with alternate alleles, termed A and a. The mating type alleles control entry into the sexual cycle, but during vegetative growth they function to elicit heterokaryon incompatibility, such that fusion of A and a hypha results in death of cells along the fusion point. Previous studies have shown that the A allele consists of 5301 bp and has no similarity to the a allele; it is found as a single copy and only within the A genome. The a allele is 3235 bp in length and it, too, is found as a single copy within the a genome. Within the A sequence, a single open reading frame (ORF) of 288 amino acids (mt A-1) is thought to confer fertility and heterokaryon incompatibility. In this study, we have used repeat induced point (RIP) mutation to identify functional regions of the A idiomorph. RIP mutations in mt A-1 resulted in the isolation of sterile, heterokaryon-compatible mutants, while RIP mutations generated in a region outside of mt A-1 resulted in the isolation of mutants capable of mating, but deficient in ascospore formation.
Glass
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Department of Botany and Biotechnology Laboratory, University of British Columbia, Vancouver, Canada.
Lee
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D000483:Alleles; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005800:Genes, Fungal; D049770:Genes, Mating Type, Fungal; D005816:Genetic Complementation Test; D008855:Microscopy, Electron, Scanning; D009492:Neurospora crassa / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D017354:Point Mutation; D014170:Transformation, Genetic
D004271:DNA, Fungal