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A gene unpaired during the meiotic homolog pairing stage in Neurospora generates a sequence-specific signal that silences the expression of all copies of that gene. This process is called Meiotic Silencing by Unpaired DNA (MSUD). Previously, we have shown that SAD-1, an RNA-directed RNA polymerase (RdRP), is required for MSUD. We isolated a second gene involved in this process, sad-2 . Mutated Sad-2 RIP alleles, like those of Sad-1 , are dominant and suppress MSUD. Crosses homozygous for Sad-2 are blocked at meiotic prophase. SAD-2 colocalizes with SAD-1 in the perinuclear region, where small interfering RNAs have been shown to reside in mammalian cells. A functional sad-2 + gene is necessary for SAD-1 localization, but the converse is not true. The data suggest that SAD-2 may function to recruit SAD-1 to the perinuclear region, and that the proper localization of SAD-1 is important for its activity.

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.

In Neurospora, a gene not paired with a homolog in prophase I of meiosis generates a signal that transiently silences all sequences homologous to it by a process called meiotic silencing by unpaired DNA (MSUD). Thus a deletion mutation in a heterozygous cross is formally “ascus-dominant” because its unpaired wild-type partner silences itself. We describe in detail the isolation of a mutation, Sad-1UV, that suppresses the dominance of various ascus-dominant mutations. Additional dominant, semidominant, and recessive Sad-1 alleles have been generated by RIP; the DNA of the dominant RIP alleles becomes methylated, but dim-2-dependent methylation is not necessary for silencing. The barrenness of homozygous Sad-1 crosses is not due to the failure to silence unpaired mating-type mat A-2 mat A-3 genes. Transcripts of sad-1+ can be detected during the sexual phase in a homozygous wild-type cross, indicating that the gene is expressed even if all DNA can pair normally. Meiotic silencing is confined to the ascus in which DNA is unpaired, and silencing does not spread to neighboring asci in a fruiting body of mixed genetic constitution.

The transcriptional activator NUC-1 controls the transcription of the genes for phosphorus acquisition enzymes, and its activity is regulated by the negative regulatory factors, PREG and PGOV In this report, we describe the cloning and molecular analysis of the preg+ gene. In Neurospora crassa, as in higher eukaryotes, transformation frequently results in nonhomologous integration of transforming DNA. Insertion of transforming DNA into host genes mutates the gene and provides a molecular tag for cloning it. We obtained two mutants that have an insertion in the preg+ and pgov+ genes, respectively, among 2 x 10(5) transformants. The preg+ gene was cloned by screening a Neurospora genomic DNA library with DNA sequences flanking the transforming DNA of the rescued plasmid. Northern analysis showed that the transcription of the preg+ gene is not regulated by phosphate. The carboxy-terminal half of PREG shows strong homology with Saccharomyces cerevisiae PHO80 whose function is analogous to that of PREG. The pregc mutations are located in the well conserved residues which may directly interact with the residues in the regulatory domain of NUC-1.

The Neurospora crassa Asm-1 + (ascospore maturation 1) gene encodes an abundant nucleus-localized protein required for formation of female structures and for ascospore maturation. Deletion mutants of Asm-1 + are "ascus-dominant," i.e., when crossed to wild type, neither Asm-1 + nor Asm-1 Δ spores mature. To explain this behavior, we considered three models: an effect of reduced dosage of the gene product, failure of internuclear communication, and failure of transvection (regulation dependent on pairing of alleles). We found that for proper regulation of subsequent sexual sporulation, Asm-1 + must be in proximity, probably paired, to its allelic counterpart in the zygote: i.e., transvection must occur. Disruption of pairing causes failure of ascospore progeny to mature. Transvection in Neurospora, unlike in Drosophila, occurs immediately before meiosis, and can be demonstrated between wild-type alleles.

A serine auxotroph of Neurospora requires exogenous serine to repress the arylsulfatase, suggesting that this enzyme is repressed by cysteine and not by methionine.

In wild-type Neurospora, alkaline phosphatase is made under conditions of phosphate limitation, but not conditions of phosphate sufficiency. Mutants at two unlinked loci, nuc-1 and nuc-2, do not make alkaline phosphatase under any conditions, while mutants at two quite closely linked loci, pcon and preg, make alkaline phosphatase even under conditions of phosphate sufficiency. pcon is extremely closely linked to nuc-2. nuc-2 and pregc (constitutive) mutants are recessive to their wild-type alleles in partial diploids as well as in heterokaryons, while pconc mutants are dominant or co-dominant. nuc-1 is epistatic to both pconc and pregc mutants. The implications of these findings for theories of metabolic control in eukaryotes are briefly discussed.

Trevithick, John R. (University of Wisconsin Medical School, Madison), and Robert L. Metzenberg. Molecular sieving by Neurospora cell walls during secretion of invertase isozymes. J. Bacteriol. 92: 1010–1015. 1966.—The secretion of invertase by young mycelia of Neurospora was studied. The process of secretion was found to be dependent upon growth. The results indicate that fractionation of light invertase, the monomer, from heavy invertase, the aggregated form, occurs at the cell wall. Neurospora strains wild type, crisp, osmotic, and the double mutant crisp osmotic were tested. An inverse relation exists between the fraction of the total invertase activity of the culture which the mold secretes into the medium and the degree of fractionation, defined as the ratio of the fraction of the invertase secreted into the medium that is light invertase to the fraction of the invertase remaining associated with the cells that is light invertase. The hypothesis is offered that the increased secretion of invertase and decreased degree of fractionation seen in osmotic mutants, and to a lesser extent in the other mutants, can be explained by an increased porosity of the cell wall.

Aryl sulfatase (arylsulfate sulfohydrolase, EC 3.1.6.1) was found to have multiple locations in Neurospora conidia. Some enzyme activity remained in the supernatant when a spore suspension was centrifuged or filtered. Part of the cell-bound activity could be detected by adding the assay ingredients to a suspension of intact spores (patent enzyme), and additional activity was only detectable when the spores were first treated to destroy their permeability barriers (cryptic enzyme). Such treatments include: disruption with an X-press, brief rinsing with chloroform or acetone, incubation at 60 C for 5 min, and incubation with phenethyl alcohol, nystatin, or ascosin. Part of the patent aryl sulfatase was inactivated by briefly acid treating the intact spores (no loss of conidial viability). This enzyme was considered to have a cell surface location. Some enzyme was acid-resistant in intact spores, but all of the enzyme was acid-sensitive in spores whose permeability barriers had been disrupted. The p H dependence, kinetic properties, and p -nitrophenyl sulfate uptake were investigated in acid-treated conidia. No aryl sulfatase was detected in ascospores. Young mycelia contained more aryl sulfatase than did conidia, but little, if any, was secreted into the growth medium. Cryptic activity was demonstrated in young mycelia by brief chloroform treatment or by rinsing the cells with 0.1 m acetate buffer. Enzyme activity in young mycelia was completely labile to acid treatment, as was cell viability.