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Bimolecular fluorescence complementation (BiFC) is based on the complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are united by interactions between proteins covalently linked to them. We have successfully applied BiFC in Neurospora crassa using two genes involved in meiotic silencing by unpaired DNA (MSUD) and observed macromolecular complex formation involving only SAD-1 proteins, only SAD-2 proteins, and mixtures of SAD-1 and SAD-2 proteins.

In Neurospora crassa, pairing of homologous DNA segments is monitored during meiotic prophase I. Any genes not paired with a homolog, as well as any paired homologs of that gene, are silenced during the sexual phase by a mechanism known as meiotic silencing by unpaired DNA (MSUD). Two genes required for MSUD have been described previously: sad-1 (suppressor of ascus dominance), encoding an RNA-directed RNA polymerase, and sad-2, encoding a protein that controls the perinuclear localization of SAD-1. Inactivation of either sad-1 or sad-2 suppresses MSUD. We have now shown that MSUD is also suppressed by either of two Spore killer strains, Sk-2 and Sk-3. These were both known to contain a haplotype segment that behaves as a meiotic drive element in heterozygous crosses of killer × sensitive. Progeny ascospores not carrying the killer element fail to mature and are inviable. Crosses homozygous for either of the killer haplotypes suppress MSUD even though ascospores are not killed. The killer activity maps to the same 30-unit-long region within which recombination is suppressed in killer × sensitive crosses. We suggest that the region contains a suppressor of MSUD.

Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes—more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca21 signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes.

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.

Complexity in and around the A and a mating type idiomorphs in Neurospora crassa was examined. Six sets of transcripts surrounding the idiomorphs were identified by Northern analysis. Several different patterns of regulation were observed. The two pairs of transcripts closest to the centromere-proximal idiomorph flanks (variable regions) exhibited mating type-specific size differences. DNA sequence was obtained for the region surrounding the four transcripts which showed mating type-specific size and expression. One of these pairs encoded amino acid sequences highly similar to a domain present in the plasma membrane ATPase. A mutant allele of one of these genes was induced by repeat-induced point mutation (RIP), which resulted in altered perithecial development and the elimination of ascospore production. These transcripts comprise a cluster of genes which may be involved in the control of mating and sexual development in N. crassa.

In an analysis of 22 of the roughly 100 dispersed 5S rRNA genes in Neurospora crassa, a methylated 5S rRNA pseudogene, Ψ63, was identified. We characterized the Ψ63 region to better understand the control and function of DNA methylation. The 120-bp 5S rRNA-like region of Ψ63 is interrupted by a 1.9-kb insertion that has characteristics of sequences that have been modified by repeat-induced point mutation (RIP). We found sequences related to this insertion in wild-type strains of N. crassa and other Neurospora species. Most showed evidence of RIP; but one, isolated from the N. crassa host of Ψ63, showed no evidence of RIP. A deletion from near the center of this sequence apparently rendered it incapable of participating in RIP with the related full-length copies. The Ψ63 insertion and the related sequences have features of transposons and are related to the Fot1 class of fungal transposable elements. Apparently Ψ63 was generated by insertion of a previously unrecognized Neurospora transposable element into a 5S rRNA gene, followed by RIP. We name the resulting inactivated Neurospora transposon PuntRIP1 and the related sequence showing no evidence of RIP, but harboring a deletion that presumably rendered it defective for transposition, dPunt.

This report describes the identification, cloning, and molecular analysis of Asm-1 + (Ascospore maturation 1), the Neurospora crassa homologue of the Aspergillus nidulans stuA (stunted A) gene. The Asm-1 + gene is constitutively transcribed and encodes an abundant, nucleus-localized 68.5-kD protein. The protein product of Asm-1 + (ASM-1), contains a potential DNA-binding motif present in related proteins from A. nidulans (StuA), Candida albicans (EFGTF-I), and Saccharomyces cerevisiae (Phd1 and Sok2). This motif is related to the DNA binding motif of the Swi4/Mbpl/Res family of transcription factors that control the cell cycle. Deletion of Asm-1 + destroys the ability to make protoperithecia (female organs), but does not affect male-specific functions. We propose that the APSES domain (ASM-1, Phdl, StuA, EFGTF-1, and Sok2) defines a group of proteins that constitute a family of related transcription factors involved in the control of fungal development.

To study, in vivo, potential P i –water oxygen exchange catalyzed by each of two high-affinity P i symporters of Neurospora crassa, we have developed methods for the purification of P i from whole-cell extracts and the subsequent derivatization of P i for analysis by GC–MS. We have also modified a published procedure for the preparation of 18 O-P i . However, the high background rate of transport-independent oxygen exchange, determined by monitoring the appearance of 18 O-P i in cells incubated in the presence of H 2 18 O, masks detection of any transport-dependent oxygen exchange which may occur. The rate of intracellular P i –water oxygen exchange is 4.36 nmol 18 O incorporated into P i per second per milligram of cell protein. The 18 O isotopic distribution of the intracellular P i closely resembles that predicted for random exchange of a single P i oxygen with that of water per enzymatic event. Based upon the observed isotopic enrichment, we calculate that the bulk intracellular P i pool must undergo an average of one oxygen exchange per phosphate ion about every 3 s.

In response to phosphorus limitation, the fungus Neurospora crassa synthesizes a number of enzymes that function to bring more phosphate into the cell. The NUC-2 protein appears to sense the availability of phosphate and transmits the signal downstream to the regulatory pathway. The nuc-2+ gene has been cloned by its ability to restore growth of a nuc-2 mutant under restrictive conditions of high pH and low phosphate concentration. We mapped the cloned gene to the right arm of linkage group II, consistent with the chromosomal position of the nuc-2 mutation as determined by classical genetic mapping. The nuc-2' open reading frame is interrupted by five introns and codes for a protein of 1066 amino acid residues. Its predicted amino acid sequence has high similarity to that of its homolog in Saccharomyces cerevisiae, PHO81. Both proteins contain six ankyrin repeats, which have been implicated in the cyclin-dependent kinase inhibitory activity of PHO81. The phenotypes of a nuc-2 mutant generated by repeat-induced point mutation and of a strain harboring a UV-induced nuc-2 allele are indistinguishable. Both are unable to grow under the restrictive conditions, a phenotype which is to some degree temperature dependent. The nuc-2+ gene is transcriptionally regulated. A 15-fold increase in the level of the nuc-2+ transcript occurs in response to a decrease in exogenous phosphate concentration.

Peleg, Y., Addison, R., Aramayo, R., and Metzenberg, R. L. 1996. Translocation of Neurospora crassa transcription factor NUC-1 into the nucleus is induced by phosphorus limitation. Fungal Genetics and Biology 20, 185–191. NUC-1, a basic helix–loop–helix zipper protein, activates the expression of several genes involved in phosphorus acquisition in Neurospora crassa. In the present study we investigated whether posttranscriptional mechanisms control the activity of NUC-1. The NUC-1 level was higher (up to fivefold) in wild-type cells grown at low external phosphate concentration and in mutant strains expressing the phosphorus acquisition genes constitutively than in a wild-type strain grown at high external phosphate concentration. Using indirect immunofluorescence we demonstrated that NUC-1 is localized at least predominantly in the cytosol when wild-type N. crassa is grown with an adequate supply of phosphate, whereas NUC-1 is largely concentrated in the nucleus upon limitation of external phosphate. In mutant strains expressing the phosphorus acquisition genes constitutively, NUC-1 localization was also primarily in the nucleus. Thus, subcellular compartmentation of regulatory proteins is an important mechanism in regulating gene expression in filamentous fungi.

A transgenic position effect that causes activator-independent gene expression has been described previously for three Neurospora crassa phosphate-repressible genes. We report analogous findings for two additional positively regulated genes, qa-2 + and ars-1 +, indicating that such position effects are not limited to genes involved in phosphorus metabolism. In addition, we have characterized a number of mutants that display activator-independent gene expression. Each of these mutants contains a chromosomal rearrangement with one breakpoint located in the 5’-upstream region of the affected gene. This suggests that the rearrangements are associated with activator-independent gene expression and that these cis-acting mutations may represent a position effect similar to that responsible for rendering some transgenes independent of their transcriptional activators. We suggest that positively regulated genes in N. crassa are normally held in a transcriptionally repressed state by a cis-acting mechanism until specifically activated. Disruption of this cis-acting mechanism, either by random integration of a gene by transformation or by chromosomal rearrangement, renders these genes independent or partly independent of the transcriptional activator on which they normally depend.

Neurospora crassa and related ascomycetes such as Podospora anserina exist in two mating types, encoded in a unique region of one chromosome. Classical genetic analysis outlined the nature of the questions and provided important materials for further work. In the mating type region, there is little DNA sequence resemblance between the two mating types. They are, therefore, called idiomorphs rather than alleles. There are no silent copies of these sequences in the genome, so mating type switching is impossible. Cloning, sequence analysis, and complementation studies involving these idiomorphs has begun to shed light on their function. One of the idiomorphs contains three reading frames; one is essential for fertilization and fruiting body formation and the other two are involved in post-fertilization functions including ascus and ascospore formation. In various species of the genus Neurospora, the centromere-proximal flank of the idiomorphs is highly variable in DNA sequence among species, and in some cases, between mating types. The similarities and differences in these flanking sequences allow some conclusions to be drawn about the possible phylogenetic relationship of these species. Key words: Neurospora, ascomycetes, mating, evolution, compatibility, HMG proteins.

Mating type idiomorphs control mating and subsequent sexual development in Neurospora crassa and were previously shown to be well conserved in other Neurospora species. The centromere-proximal flanks of the A and a idiomorphs, but not the distal flanks from representative heterothallic, pseudohomothallic, and homothallic Neurospora species contain apparent species-specific and/or mating type-specific sequences adjacent to the well-conserved idiomorphs. The variable flank is bordered by regions that are highly homologous in all species. The sequence of approximately 1 kb immediately flanking the conserved idiomorphs of each species was determined. Sequence identity between species ranged from 20% (essentially unrelated) to > 90%. By contrast, the mt-A1 gene shows 88-98% identity. Sequence and hybridization data also show that the centromere-proximal flanks are very different between the two mating types for N. intermedia, N. discreta, and N. tetrasperma, but not for N. sitophila and N. crassa. The data suggest a close evolutionary relationship between several of the species; this is suppported by phylogenetic analysis of their respective mt-A1 genes. The origin of the variable regions adjacent to the evolutionarily conserved mating type idiomorphs is unknown.

The filamentous fungus Neurospora crassa possesses two nonhomologous high-affinity phosphate permeases, PHO-4 and PHO-5. We have isolated separate null mutants of these permeases, allowing us to study the remaining active transporter in vivo in terms of phosphate uptake and sensitivity to inhibitors. The specificity for the cotransported cation differs for PHO-4 and PHO-5, suggesting that these permeases employ different mechanisms for phosphate translocation. Phosphate uptake by PHO-4 is stimulated 85-fold by the addition of Na+, which supports the idea that PHO-4 is a Na(+)-phosphate symporter. PHO-5 is unaffected by Na+ concentration but is much more sensitive to elevated pH than is PHO-4. Presumably, PHO-5 is a H(+)-phosphate symporter. Na(+)-coupled symport is usually associated with animal cells. The finding of such a system in a filamentous fungus is in harmony with the idea that the fungal and animal kingdoms are more closely related to each other than either is to the plant kingdom.

In Neurospora, transformation of spheroplasts is quite efficient and usually occurs with the transforming DNA integrated at ectopic sites in the chromosome. However, only a small fraction of the spheroplasts is actually competent for transformation. To distinguish whether the limitation to competence is at the level of the plasma membrane or at the level of the nucleus, we performed experiments in which heterocaryotic spheroplasts were required to integrate two different plasmids in one transformation procedure. The cotransformants were then analyzed to determine into which nucleus or nuclei the separate plasmids had integrated. Results of such experiments confirm that successful ectopic transformation in Neurospora crassa requires a competent nucleus. The integration patterns of the two separate plasmids indicate that the availability of appropriate chromosomal sites for ectopic integration may be an aspect of nuclear competence.

The filamentous fungus Neurospora crassa undergoes a complex program of sexual development to form a fruiting body composed of several kinds of specialized tissue. Subtractive hybridization was used to isolate genes that are expressed preferentially during this sexual phase. Many such sexual development (sdv) genes were identified in a cosmid library of Neurospora genomic DNA. Fourteen of the sdv genes were subcloned, and their expression in mutant strains and under crossing and vegetative growth conditions was examined. All of the regulated transcripts were less abundant (and in many cases not detectable) in strains grown under vegetative (high nitrogen) conditions, suggesting that nitrogen starvation is required for their synthesis. The expression of most of the sdv genes also required a functional A mating type product, even under crossing growth conditions, suggesting that this product functions as a master control in sexual development. To determine if the products of the sdv genes play essential roles in the sexual cycle, a reverse-genetic approach (based on RIP (repeat-induced point mutation)-mediated gene disruptions) was used to create mutations in the genes. A mutant strain (asd-1) with a recessive crossing defect (apparently caused by the RIP process) was isolated; in this strain, early development is normal and may asci are formed, but ascospores are never delineated. A second recessive mutant strain (asd-2) was apparently created by ectopic integration of the transforming DNA into a gene required for the sexual process; in this strain the sexual process was blocked at an early stage, and the ascogeneous tissue underwent little development.

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.

Previously we have shown that the nucleolus organizer region (NOR) of Neurospora crassa changes size frequently during the premeiotic portion of the sexual phase. Here, we have investigated whether these changes in size originate only in specific regions of the NOR, or are distributed throughout the NOR. In two special strains of Neurospora, the NOR was divided into proximal and distal segments. In the first, the NOR was divided by a translocation breakpoint and, in the second, the NOR was divided by a meiotic crossover point. The two strains were crossed individually to normal sequence tester strains and the sizes of the proximal and distal segments were followed by pulsed-field gel electrophoresis. The analysis of progeny from both crosses indicates that the events affecting NOR size are not limited to a specific region of the NOR. Additionally, we have obtained evidence that the rDNA of N. crassa can undergo unequal sister chromatid exchange.

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.

The mating-type locus of the haploid filamentous fungus Neurospora crassa is a regulatory region that controls entry into the sexual cycle and prevents formation of mixed mating-type heterokaryons in the vegetative phase. The locus consists of alternative sequences called A and a. The A mating-type DNA sequence of Neurospora crassa is composed of a region of 5301 base pairs that has little similarity to the sequence present at the mating-type locus in an a mating-type strain. However, the sequences flanking the mating-type locus in the A haploid and a haploid genome are essentially identical. The region of the A mating-type sequence required for expression of the heterokaryon incompatibility and sexual functions has been localized to a single open reading frame (ORF) encoding a polypeptide of 288 amino acids. Sequence analysis of sterile, heterokaryon-compatible mutants reveals frameshift mutations in this same ORF. The putative 288-amino acid product has a region of similarity to the MAT alpha 1 polypeptide of Saccharomyces cerevisiae.

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.

AS a new graduate student of Herschel Mitchell at Caltech in 1951, I soon had occasion to visit Horowitz's office. I immediately noticed a newspaper clipping taped to the door. “It is a Privilege to be Living in the Same Century as Horowitz,” gushed the title. The article was, as it turned out, about a recent concert by Vladimir Horowitz, but it prepared me for the wry Horowitz sense of humor, always delivered deadpan. Later, there was Thanksgiving dinner at his house with his wife, Pearl, his son and daughter, and his mother. Norm sat down to the piano and we all sang old chestnuts. Vladimir he was not. But full of food and drink as we were and enthralled with our own wine-enhanced voices, it was a perfect evening. I left knowing Norm Horowitz and I had begun a lifelong friendship.

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.

Self-incompatible het-6 OR/het-6 PA partial diploids of Neurospora crassa were selected from a cross involving the translocation strain, T(IIL → IIIR)AR18, and a normal sequence strain. About 25% of the partial diploids exhibited a marked increase in growth rate after 2 weeks, indicating that “escape” from het-6 incompatibility had occurred. Near isogenic tester strains with different alleles (het-6 OR and het-6 PA) were constructed and used to determine that 80 of 96 escape strains tested were het-6 PA, retaining the het-6 allele found in the normal-sequence LGII position; 16 were het-6 OR, retaining the allele in the translocated position. Restriction fragment length polymorphisms in 45 escape strains were examined with probes made from cosmids that spanned the translocated region. Along with electrophoretic analysis of chromosomes from three escape strains, RFLPs showed that escape is associated with deletion of part of one or the other of the duplicated DNA segments. Deletions ranged in size from ~70 kbp up to putatively the entire 270-kbp translocated region but always included a 35-kbp region wherein we hypothesize het-6 is located. The deletion spectrum at het-6 thus resembles other cases where mitotic deletions occur such as of tumor suppressor genes and of the hprt gene (coding for hypoxanthine-guanine phosphoribosyl-transferase) in humans.

Species of the genus Neurospora have been much studied as model systems for understanding metabolic pathways and their regulation, the processes and consequences of sexuality, and the natural history and evolution of a representative fungal genus. These subjects have been extensively reviewed in the literature, and there seems to be no urgent need to update them. The initiation and progression of events in fruiting bodies and asci has been the subject of a recent careful and scholarly review, to which the reader is enthusiastically referred (Raju, 1992). By contrast, the subject of nutrition of perithecia of developing asci, and of communication between nuclei as judged by dominance and by success or failure of complementation in the developing perithecial system has received little recent discussion. Much of the knowledge exists as unpublished bits of information, some of it from the laboratory of the author, or as minor observations made in connection with more substantial findings on other subjects. Such information is nonetheless useful in thinking about how development of fruiting bodies occurs. This review will be unabashedly eclectic, inferential, and anecdotal.

We have used a technique referred to as "sheltered RIP" (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa.

Previously we have shown that the nucleolus organizer region (NOR) of Neurospora crassa displays frequent size changes during crosses. In these initial studies, we observed that decreases in NOR size are far more common than increases. Here, we have investigated the inheritance of NOR size in a strain with an unusually small NOR. We call this strain SNO for small nucleolus organizer. We found that progeny that inherit their rDNA from SNO receive either an NOR that is larger than that of SNO or, rarely, the same size, but never an NOR that is smaller than that of SNO. The number of progeny that inherit their NOR from SNO is not significantly different from the number that inherit their NOR from the other parent in the cross. This argues against the idea that the failure to find progeny with NORs smaller than that of SNO is due to inviability of spores carrying such an NOR, or that it is due to unconscious bias by the experimenter against isolating such spores. These results can most easily be explained by a combination of unequal sister chromatid exchanges in the rDNA, or sister chromatid conversion, coupled with selection against nuclei harboring small NORs during the premeiotic phase of the Neurospora life cycle. Other, less conventional, explanations are also possible, such as "directed" increase in the target NOR without corresponding loss at some other NOR.

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.

In the heterothallic species Neurospora crassa, strains of opposite mating type, A and a, must interact to give the series of events resulting in fruiting body formation, meiosis, and the generation of dormant ascospores. The mating type of a strain is specified by the DNA sequence it carries in the mating type region; strains that are otherwise isogenic can mate and produce ascospores. The DNA of the A and a regions have completely dissimilar sequences. Probing DNA from strains of each mating type with labelled sequences from the A and the a regions has shown that, unlike in Saccharomyces cerevisiae, only a single copy of a mating type sequence is present in a haploid genome. The failure to switch is explainable by the physical absence of DNA sequences characteristic of the opposite mating type. While the mating type sequences must be of the opposite kind for mating to occur in the sexual cycle, two strains of opposite mating type cannot form a stable heterokaryon during vegetative growth; instead, they fuse abortively to give a heterokaryon incompatibility reaction, which results in death of the cells along the fusion line. The DNA sequences responsible for this reaction are coextensive with those sequences in the A and a regions which are necessary to initiate fruiting body formation. The genus Neurospora also includes homothallic species--ones in which a single haploid nucleus carries all the information necessary to form fruiting bodies, undergo meiosis, and produce new haploid spores. One such species, N. terricola, contains one copy each of the A and the a sequences within each haploid genome.(ABSTRACT TRUNCATED AT 250 WORDS)

The Neurospora crassa phosphate-repressible alkaline phosphatase-encoding gene pho -2 + was cloned and its nucleotide sequence was determined. An open reading frame was found that contains four introns and encodes a putative protein of 555 amino acids. ‘Activator-independent expression’ of ectopically integrated pho -2 + was observed, as noted before for ectopically integrated pho -4 + .

The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.

van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. This was unexpected because the nuc-1 host already contained a resident van+ gene. Plasmids carrying van+ complemented a nuc-2 mutation as well. Probing of RNA from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control by nuc-1+. Probing of the same RNAs with a cosmid clone, containing approximately 15 kilobases of upstream and downstream DNA, revealed no other detectable phosphorus-regulated transcripts within this 40-kilobase region of the chromosome.

The organization of the ribosomal DNA (rDNA) repeat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, . tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11 + 0.21 kb; standard error = 0.038; coefficient of variation (C.V.) = 2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5' end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.

The regulation of serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and methyltetrahydropteroylpolyglutamate:homocysteine methyltransferase was investigated in Neurospora crassa . Adding choline to the medium decreased the specific activity of these enzymes. Methionine potentiated the choline effect, but, when added alone, was without effect. Neither choline, methionine, nor S -adenosylmethionine appears to be the immediate corepressor of synthesis of these enzymes. Several nonallelic mutants were examined for the enzymes of methionine methyl group synthesis. The formate-requiring mutant for lacks serine hydroxymethyltransferase. The methionine-requiring mutants me -1 and me -8 lack, respectively, the reductase and the methyltransferase. The methionine-requiring mutants me -1, me -6 (folate polyglutamate synthetase deficient) and me -8 were found to have significantly higher serine hydroxymethyltransferase specific activities than did the wild-type strain.

When N. crassa is starved for phosphate the rate of synthesis of total RNA, as measured by the incorporation of uridine, rapidly dclines, attaining a value of 2% of the control after 4 h. Synthesis of ribosomal RNA (rRNA), measured by direct hybridization to ribosomal DNA covalently coupled to diazobenzyloxymethyl (DBM) paper, also declines to a value 3%–4% that of the control after 4 h of phosphate starvation. Measurement of rRNA synthesis in regulatory mutant strains expressing “phosphorus-family” enzymes indicates that two of these mutant strains, pgovc12 and nuc-1, respond differently to phosphate starvation from the response in wild-type or the other five mutant strains. The results suggest that the wild-type products of the regulatory loci, pgov+ and nuc-1+ may have a role in regulating rRNA synthesis as well as “phosphorus family” enzymes.

In Neurospora crassa, the phosphate-metabolizing enzymes are made during phosphate starvation, but not under phosphate sufficiency. The synthesis of these enzymes is controlled by three regulatory genes: pcon-nuc-2, preg and nuc-1. pcon-nuc-2 and preg are closely linked. A model of the hierarchical relationships among these regulatory genes is presented. Studies of double mutants and revertants confirm several predictions of the model. It has been found that nuc-2 (null) and pconc (constitutive) mutations reside in the same cistron. pregc (constitutive) mutations are epistatic to nuc-2 mutations. nuc-1 (null) mutations are epistatic to all others.