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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.

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.

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.

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.

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.

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.

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.

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.

Mutants called nuc-1C, constitutive for alkaline phosphatase synthesis, were isolated and mapped very close to nuc-1 mutants in which this enzyme is not expressed. nuc-1 is epistatic to nuc-1C. nuc-1C acts only if it is cis to normal nuc-1 function. The preparation of partial diploids heterozygous for various nuc-1 alleles is described; nuc-1C is dominant to nuc-1+, which in turn is dominant to nuc-1. In heterocaryons with nuc-1+, nuc-1C is dominant when it is present in high proportion, but essentially recessive if it is present in low proportions. In heterocaryons with nuc-1, nuc-1C is again dominant when present in high proportions, but in low proportions it "complements" to give essentially normal repressibility. A model of regulation consistent with these findings is presented.

In translocation OY321 of Neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group I. In crosses of Translocation x Translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to Normal. Genetic, cytological, and molecular evidence indicates that reversion is the result of meiotic crossing over between homologous displaced rDNA repeats. Marker linkages are wild type in these exceptional progeny. They differ from wild type, however, in retaining an interstitial block of rRNA genes which can be demonstrated cytologically by the presence of a second, small interstitial nucleolus and genetically by linkage of an rDNA restriction site polymorphism to the mating-type locus in linkage group I. The interstitial rDNA is more highly methylated than the terminal rDNA. The mechanism by which methylation enzymes distinguish between interstitial rDNA and terminal rDNA is unknown. Some hypotheses are considered.

Five additional mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase. The mutations in these strains map at a previously assigned locus on Linkage Group V designated pho-2 (Gleason and Metzenberg 1974). The five new mutants, as well as three previously isolated by Gleason and Metzenberg (1974), were examined for the presence of cross-reacting material to antibody prepared against purified wild-type enzyme. Two of the mutants produced high levels of cross-reacting material, thus providing evidence that the pho-2 locus includes the structural gene for the repressible alkaline phosphatase. Two revertants were obtained from one of the mutants that contained cross-reacting material. Neither revertant produced an enzyme that could be distinguished physicochemically from that of wild type. A method for measuring very low levels of repressible alkaline phosphatase in crude extracts is also described.

A mutant of Neurospora crassa, called UW-6, differs from wild type in being partially constitutive for synthesis of a species of alkaline phosphatase, and also for a species of phosphate permease that has a high affinity for phosphate at high pH. UW-6 is possibly allelic with a mutant called nuc-2 that was previously isolated by Ishikawa. nuc-2 has the converse phenotype, in that it cannot be derepressed for either of these two activities. UW-6 is co-dominant with its wild-type allele in heterokaryons and in partial diploids. An unlinked mutant, nuc-1, is like nuc-2 in that it fails to make the alkaline phosphatase or the permease referred to above. nuc-1 is epistatic to UW-6 in the double mutant. The control of phosphorus metabolism is discussed, and is compared with some other control systems in filamentous fungi.