Novel mutations of the A mating-type idiomorph in Neurospora crassa

The mating-type idiomorphs of Neurospora crassa, A and a, function in the sexual cycle to initiate mating and meiosis (A x a is fertile) and during vegetative growth to prevent heterokaryosis (A + a is incompatible). The A and a idiomorphs encodeputative regulatory polypeptides of 288 amino acids (mt A-1) and 382 amino acids(mt a-1), respectively, that are thought to control the expression of mating-specificgenes and determine a self versus non-self mechanism of recognition. The present study is an attempt to isolate new mutants of the A mating-type locus to further delineate the functional domains of the mt A-1 ORF that controls these seemingly unrelated functions. Previously, all A mating-type mutants isolated using UV mutagenesis were sterile and heterokaryon compatible, the result of frame shift mutations within mt A-1. In an attempt to isolate fertile and heterokaryon compatible A mutants, mutational analysiswas performed on mating-type A strains using 6-N-hydroxylaminopurine (H.A.P.), achemical mutagen that produces GC-*AT base pair substitutions, in addition to UV. All of the mutants isolated with UV were heterokaryon compatible and sterile. Onemutant isolated with H.A.P., Am99, was heterokaryon compatible and sterile as themale, but fertile as the female, producing near normal numbers of asci and ascopsores, although reduced numbers of perithecia. The mutant phenotype segregated with the A mating-type in all of the progeny tested. Sequencing analysis of the mt A-1 ORF showed that the mutant phenotype was the result of a base pair substitution that changed Trp-86 to a stop codon that would result in a truncated polypeptide of 85 amino acids. A second mutant isolated with H.A.P., Am13, was heterokaryon compatible and sterile as the male, but produced near normal numbers of perithecia, but not ascospores, as the female. Sequencing analysis of the mt A-1 ORF of Am13 showed a frame shift mutation at amino acid 208 produced the mutant phenotype. The different phenotypes observed between Am99, Am13 and A wild-type strains can thus be ascribed to differences in function of the polypeptides encoded by their mt A-1 ORFs. A model for mt A-1 function is presented to account for the phenotypes of these mutants in comparison to A wild-type strains.Medicine, Faculty ofMedical Genetics, Department ofGraduat