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A putative pheromone precursor gene of Neurospora crassa , mfa-1 (which encodes mating factor a -1), was identified as the most abundant clone in starved mycelial and perithecial cDNA libraries. Northern analysis demonstrated high mfa-1 expression in all mating type a tissues and suggested low expression levels in mat A tissues. The mfa-1 gene was expressed as an approximately 1.2-kb transcript predicted to encode a 24-residue peptide, followed by a long 3′ untranslated region (3′ UTR). The predicted MFA1 sequence showed 100% sequence identity to PPG2 of Sordaria macrospora and structural similarity (a carboxy-terminal CAAX motif) to many hydrophobic fungal pheromone precursors. Mutants with a disrupted open reading frame (ORF) in which the critical cysteine residue had been changed to a nonprenylatable residue, tyrosine (YAAX mutants), were isolated, as were mfa-1 mutants with intact ORFs but multiple mutations in the 3′ noncoding region (CAAX mutants). The 3′ UTR is required for the full range of mfa-1 gene activity. Both classes of mutants showed delayed and reduced vegetative growth (which was suppressed by supplementation with a minute amount [30 μM] of ornithine, citrulline, or arginine), as well as aberrant sexual development. When crossed as female parents to wild-type males, the CAAX and YAAX mutants showed greatly reduced ascospore production. No ascospores were produced in homozygous mfa-1 crosses. As males, YAAX mat a mutants were unable to attract wild-type mat A trichogynes (female-specific hyphae) or to initiate sexual development, while CAAX mat a mutants were able to mate and produce sexual progeny despite their inability to attract mat A trichogynes. In the mat A background, both CAAX and YAAX mutants showed normal male fertility but defective vegetative growth and aberrant female sexual development. Thus, the mfa-1 gene appears to have multiple roles in N. crassa development: (i) it encodes a hydrophobic pheromone with a putative farnesylated and carboxymethylated C-terminal cysteine residue, required by mat a to attract trichogynes of mat A ; (ii) it is involved in female sexual development and ascospore production in both mating types; and (iii) it functions in vegetative growth of both mating types.

Trevithick, John R. (University of Wisconsin Medical School, Madison), Robert L. Metzenberg and Donald F. Costello. Genetic alteration of pore size and other properties of the Neurospora cell wall. J. Bacteriol. 92:1016-1020. 1966.-Several properties of the cell walls of wild type and the osmotic mutant of Neurospora crassa have been examined. The peameability of the isolated cell walls to polyethylene glycol and dextran polymers of different molecular weights was investigated by the volume of distribution technique. The exclusion thresholds were evaluated by a statistical treatment. The molecular weights corresponding to these thresholds for wild type and osmotic were approximately 4,750 and 18,500, respectively; these values are significantly different. The cell walls of osmotic appeared to be thinner, more easily broken, and more easily compressed to ribbonlike shapes, whereas those of wild type were tubular and strong. Chemical analysis showed that osmotic walls had roughly a 30-fold higher galactosamine-glucosamine ratio than did wild type. It is proposed that the osmotic mutant has a cell wall with abnormally large pores, and that this may account for the increased rate of egress of invertase and the decreased fractionation of light from heavy invertase in this strain.