Heterogeneity of 5S RNA in fungal ribosomes.

Neurospora crassa has at least seven types of 5S RNA genes (alpha, beta, gamma, epsilon, delta, zeta, and eta) with different coding regions. A high resolution gel electrophoresis system was developed to separate minor 5S RNA's from the major 5S RNA (alpha). A study of several Neurospora crassa strains, four other species in the genus Neurospora, members of two closely related genera, and three distantly related genera demonstrated that 5S RNA heterogeneity is common among fungi. In addition, different 5S RNA's are present in Neurospora ribosomes. The finding that fungal ribosomes are structurally heterogeneous suggests that ribosomes may be functionally heterogeneous as well.

DNAs of the two mating-type alleles of Neurospora crassa are highly dissimilar.

The mating-type alleles A and a of Neurospora crassa control mating in the sexual cycle and function in establishing heterokaryon incompatibility in the vegetative cycle. The A and a alleles were cloned, and they were shown to encode both the sexual functions and vegetative incompatibility. The mating-type clones contain nonhomologous DNA segments that are flanked by common DNA sequences. Neurospora crassa and all heterothallic and pseudohomothallic Neurospora species contain a single copy of one mating-type sequence or the other within each haploid genome. The six known self-fertile homothallic isolates contain an A homolog, but only one species also contains a homologous sequences. Homothallism in these species is not due to mating-type switching, as it is in Saccharomyces cerevisiae.

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.

Analysis of the DNA-binding and dimerization activities of Neurospora crassa transcription factor NUC-1.

NUC-1, a positive regulatory protein of Neurospora crassa, controls the expression of several unlinked target genes involved in phosphorus acquisition. The carboxy-terminal end of the NUC-1 protein has sequence similarity to the helix-loop-helix family of transcription factors. Bacterially expressed and in vitro-synthesized proteins, which consist of the carboxy-terminal portion of NUC-1, bind specifically to upstream sequences of two of its target genes, pho2+ and pho-4+. These upstream sequences contain the core sequence, CACGTG, a target for many helix-loop-helix proteins. A large loop region (47 amino acids) separates the helix I and helix II domains. Mutations and deletion within the loop region did not interfere with the in vitro or in vivo functions of the protein. Immediately carboxy-proximal to the helix II domain, the NUC-1 protein contains an atypical zipper domain which is essential for function. This domain consists of a heptad repeat of alanine and methionine rather than leucine residues. Analysis of mutant NUC-1 proteins suggests that the helix II and the zipper domains are essential for the protein dimerization, whereas the basic and the helix I domains are involved in DNA binding. The helix I domain, even though likely to participate in dimer formation while NUC-1 is bound to DNA, is not essential for in vitro dimerization.

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by S1 nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the leu-5 mutation had been mapped before. We show that the leu-5 strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the leu-5 strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in leu-5 extracts, the leu-5 strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.

The structural gene for a phosphorus-repressible phosphate permease in Neurospora crassa can complement a mutation in positive regulatory gene nuc-1.

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.

Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA.

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.

Robust and efficient synthetic method for forming DNA microarrays.

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.

A cancer-associated, fast, homoarginine-sensitive electrophoretic form of serum alkaline phosphatase.

Serum from patients with a variety of cancers often (p = 70%) contains a characteristic electrophoretic form of alkaline phosphatase detectable by cellulose polyacetate electrophoresis. The lower prevalence of this electrophoretic form in presumably healthy individuals (p = 7%) and noncancer hospitalized patients (p = 30%) suggests that it may be useful as a diagnostic or monitory marker.

Premeiotic change of nucleolus organizer size in Neurospora.

We have investigated the heritability of nucleolus organizer region (NOR) size in Neurospora crassa. By pulsed-field gel electrophoresis, we followed in genetic crosses the size of the normal or "terminal" NORs and the size of a small interstitial NOR. Tetrad analysis revealed that changes in NOR size occur frequently in the sexual phase. Moreover, most size changes occurred in the period between fertilization and meiosis, although some changes occurred during and after meiosis. Unexpectedly, increases and decreases in NOR size were not equally frequent: decreases were more common. The NOR size changes generated during meiosis were not the result of unequal crossing over between NORs on homologous chromosomes.

Expansion and contraction of the nucleolus organizer region of Neurospora: changes originate in both proximal and distal segments.

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.

Genetic control of phosphorus assimilation in Neurospora crassa: dose-dependent dominance and recessiveness in constitutive mutants.

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.

Regulation of phosphate metabolism in Neurospora crassa: identification of the structural gene for repressible alkaline phosphatase.

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.

Sexual development genes of Neurospora crassa.

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.

Activator-independent gene expression in Neurospora crassa.

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.

Isolation and properties of selenomethionine-resistant mutants of Neurospora crassa.

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.

Regulation of phosphate metabolism in Neurospora crassa: isolation of mutants deficient in ther repressible alkaline phosphatase.

Mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase, but, unlike nuc-1 and nuc-2 mutants, are able to make the repressible acid phosphatase and the repressible phosphate permease under conditions of derepression (phosphate deprivation). The new mutants, called pho-2, map in Linkage Group V, and are unlinked to the putative control mutants, nuc-1, nuc-2-pcon(c), and preg(c). Three of the pho-2 mutants do not make detectable amounts of repressible alkaline phosphatase, but the fourth makes about 1% of the level found in wild type. The small amount of alkaline phosphatase made by this strain appears to be qualitatively similar or identical to the wild-type enzyme, as judged by electrophoretic mobility, heat stability, and titration with specific antibody to the wild-type enzyme. Several revertants of this strain have been examined in the same way, and the alkaline phosphatase of these strains also appears to be qualitatively normal. Reversion events can occur at, or near, the pho-2 locus, but also occur in at least two unlinked sites (suppressor mutations). One suppressor maps very close to nuc-1.

Insertional mutagenesis in Neurospora crassa: cloning and molecular analysis of the preg+ gene controlling the activity of the transcriptional activator NUC-1.

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.

Species-specific and mating type-specific DNA regions adjacent to mating type idiomorphs in the genus Neurospora.

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.