Robert L. Metzenberg

Meiotic Silencing by Unpaired DNA

The silencing of gene expression by segments of DNA present in excess of the normal number is called cosuppression in plants and quelling in fungi. We describe a related process, meiotic silencing by unpaired DNA (MSUD). DNA unpaired in meiosis causes silencing of all DNA homologous to it, including genes that are themselves paired. A semidominant Neurospora mutant, Sad-1, fails to perform MSUD. Sad-1 suppresses the sexual phenotypes of many ascus-dominant mutants. MSUD may provide insights into the function of genes necessary for meiosis, including genes for which ablation in vegetative life would be lethal. It may also contribute to reproductive isolation of species within the genus Neurospora. The wild-type allele, sad-1(+), encodes a putative RNA-directed RNA polymerase.

Loss of growth polarity and mislocalization of septa in a Neurospora mutant altered in the regulatory subunit of cAMP-dependent protein kinase.

In filamentous fungi, growth polarity (i.e. hyphal extension) and formation of septa require polarized deposition of new cell wall material. To explore this process, we analyzed a conditional Neurospora crassa mutant, mcb, which showed a complete loss of growth polarity when incubated at the restrictive temperature. Cloning and DNA sequence analysis of the mcb gene revealed that it encodes a regulatory subunit of cAMP‐dependent protein kinase (PKA). Unexpectedly, the mcb mutant still formed septa when grown at the restrictive temperature, indicating that polarized deposition of wall material during septation is a process that is, at least in part, independent of polarized deposition during hyphal tip extension. However, septa formed in the mcb mutant growing at the restrictive temperature are mislocalized. Both polarized growth and septation are actin‐dependent processes, and a concentration of actin patches is observed at growing hyphal tips and sites where septa are being formed. In the mcb mutant growing at the restrictive temperature, actin patches are uniformly distributed over the cell cortex; however, actin patches are still concentrated at sites of septation. Our results suggest that the PKA pathway regulates hyphal growth polarity, possibly through organizing actin patches at the cell cortex.

Homothallic Sordariaceae from nature: The absence of strains containing only thea mating type sequence

Abstract An efficient procedure is described for the isolation of heterothallic and homothallic Sordariaceae from soil. New strains obtained by this method were classified to genus on the basis of ascospore morphology and ornamentation. Hetero- and homothallic Neurospora spp. were most often isolated from soils collected in the tropics, whereas Gelasinospora spp. were obtained from both tropical and temperate zone soils. The use of molecular probes on genomic DNA of the isolates showed that all of the new homothallic Neurospora strains contained a sequence similar to the A mating type sequence of N. crassa , but none contained a sequence similar to the a mating type. In contrast, all of the homothallic Gelasinospora strains contained sequences similar to both the A and a mating type sequences of N. crassa . Genomic DNA from type cultures of homothallic species of Gelasinospora ( G. calospora, G. reticulospora , and Gelasinospora S23), Sordaria ( S. fimicola and S. macrospora ), and Anixiella ( A. sublineata ) also contained sequences similar to the N. crassa A and a sequences. Unlike the homothallic Gelasinospora , heterothallic Gelasinospora isolates contained a sequence corresponding to either the A mating type or the a mating type, but not both. Hybridization with additional molecular probes was performed to characterize the new isolates further on the basis of restriction fragment length polymorphisms (RFLPs). Surprisingly, many of the homothallic strains could not be distinguished from one another either morphologically or on the basis of several molecular probes, although they were isolated from distinct geographical regions. This is in contrast to numerous RFLPs detected between heterothallic Gelasinospora and Neurospora isolates.

Positive control by the cys-3 locus in regulation of sulfur metabolism in Neurospora

Abstract Three enzymes of sulfur metabolism are simultaneously lost in the cys -3 mutant of Neurospora . Revertants of cys -3 have been selected and appear to arise from mutation within the original locus. The revertants can be grouped into at least three classes according to the amounts of the enzymic activities that are restored and by temperature sensitivity. The properties of the revertant strains indicate that the cys -3 mutant is not a large deletion, a super-repressor mutation, or an extreme polar mutant; nor does the cys -3 locus code for a polypeptide chain common to the three enzymes. We suggest that cys -3 is a regulatory gene which exerts positive control over enzyme synthesis.

Multiple Functions of mfa-1, a Putative Pheromone Precursor Gene of Neurospora crassa

ABSTRACT 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.

Nucleotide sequence of pho-4+, encoding a phosphate-repressible phosphate permease ofNeurospora crassa

The nucleotide (nt) sequence of the Neurospora crassa pho-4+ gene, which encodes a phosphate-repressible phosphate permease, has been determined. The gene specifies a protein of 590 amino acids (aa) and contains two introns. Two RNA transcripts of 3.3 and 2.4 kb have been identified, and transcription start points (tsp) and termination sites and/or processing sites have been located. The 3.3-kb message is initiated about 890 nt upstream from the tsp for the 2.4-kb transcript. A hydropathy profile of the aa sequence suggests ten to twelve membrane-spanning helices with a large hydrophilic domain between the eighth and ninth helices. This model for the predicted secondary structure of the protein is very similar to models proposed for other sequenced integral membrane proteins from both prokaryotes and eukaryotes. Since very few permease-encoding genes of eukaryotes have been examined in molecular detail, it will be of interest to compare the sequence of pho-4+ with those encoding other anion transport proteins, as they become available.

Molecular analysis of nuc-1+, a gene controlling phosphorus acquisition in Neurospora crassa.

In response to phosphorus starvation, Neurospora crassa makes several enzymes that are undetectable or barely detectable in phosphate-sufficient cultures. The nuc-1+ gene, whose product regulates the synthesis of these enzymes, was cloned and sequenced. The nuc-1+ gene encodes a protein of 824 amino acids with a predicted molecular weight of 87,429. The amino acid sequence shows homology with two yeast proteins whose functions are analogous to that of the NUC-1 protein. Two nuc-1+ transcripts of 3.2 and 3.0 kilobases were detected; they were present in similar amounts during growth at low or high phosphate concentrations. The nuc-2+ gene encodes a product normally required for NUC-1 function, and yet a nuc-2 mutation can be complemented by overexpression of the nuc-1+ gene. This implies physical interactions between NUC-1 protein and the negative regulatory factor(s) PREG and/or PGOV. Analysis of nuc-2 and nuc-1; nuc-2 strains transformed by the nuc-1+ gene suggests that phosphate directly affects the level or activity of the negative regulatory factor(s) controlling phosphorus acquisition.

A gene affecting the repression of invertase and trehalase in Neurospora

Growth of Neurospora on media containing mannose results in low activities of invertase and trehalase, whereas very high levels of these enzymes are attained during growth in galactose media. A mutant has been isolated which, under suitable conditions of culture, produces much larger amounts of these two enzymes than does the parental strain. Evidence is presented that the levels of enzymes are affected by a single gene alteration.

An upstream signal is required for in vitro transcription of Neurospora 5S RNA genes.

SummaryThe DNA sequences upstream of the 5S RNA genes inNeurospora crassa are largely different from one another, but share a short consensus sequence located in the segment 29 to 26 nucleotides preceding the transcribed region. Differences among flanking sequences do not appear to affect transcription. Deletion analysis indicates, however, that a DNA segment including the conserved “TATA box” is required for in vitro transcription of Neurospora 5S RNA genes.

Concerted evolution of dispersed Neurospora crassa 5S RNA genes: pattern of sequence conservation between allelic and nonallelic genes.

About 100 genes coding for 5S RNA in Neurospora crassa are dispersed throughout the genome (Selker et al., Cell 24:815-818, 1981; R. L. Metzenberg, J. N. Stevens, E. U. Selker, and E. Morzycka-Wroblewska, manuscript in preparation). The majority of them correspond to the most abundant species (alpha) of 5S RNA found in the cell. Gene conversion, gene transposition, or both may be responsible for the maintenance of sequence homogeneity (concerted evolution) of alpha-type 5S genes. To explore these possibilities, we isolated and characterized separate 5S regions from two distantly related laboratory strains of N. crassa. Restriction and sequence analyses revealed no differences in molecular location of allelic 5S genes between the two strains. However, the DNA sequences around the 5S genes are ca. 10% divergent. We concluded that transposition is not frequent enough to account for the concerted evolution of N. crassa alpha-5S genes. In contrast to sequence divergence in the flanking regions between the two strains, the 5S transcribed regions are identical (with one exception), suggesting that these genes are being corrected. We have found that flanking sequences of various N. crassa 5S genes within each strain are largely different. Thus, if the correction mechanism is based on gene conversion, it is limited to the transcribed regions of the genes. However, we did find a short region of consensus including the sequence TATA located 25 to 30 nucleotides preceding the position of transcription initiation. This region may be involved in the transcription of N. crassa 5S genes.