Heteroallelism at the het-c locus contributes to sexual dysfunction in outcrossed strains of Neurospora tetrasperma.

Neurospora tetrasperma is naturally heterokaryotic, with cells possessing haploid nuclei of both a and A mating types. As a result, isolates are self-fertile (pseudohomothallic). Occasional homokaryotic ascospores and conidia arise, however, and they produce strains that are self-sterile and must outcross to complete sexual reproduction. Invariably, laboratory crosses employing sibling a and A strains from the same parental heterokaryon restore the pseudohomothallic, heterokaryotic state. In contrast, outcrosses employing a and A strains from different wild isolates typically result in sexual dysfunction. Diverse sexual dysfunction types have been observed, ranging from complete sterility to reduced viability. We report that one type of dysfunction, characterized by spontaneous loss of the heterokaryotic state upon ascospore germination, can result from the interaction of incompatible alleles at heterokaryon incompatibility loci. Specifically, we demonstrate that homoallelism at the het-c locus in N. tetrasperma is required for heterokaryon stability. Heterokaryon incompatibility therefore provides an obstacle to outcrossing in this species, an observation with important implications for fungal life-cycle evolution.

Analysis of the pdx-1 (snz-1/sno-1) region of the Neurospora crassa genome: correlation of pyridoxine-requiring phenotypes with mutations in two structural genes.

We report the analysis of a 36-kbp region of the Neurospora crassa genome, which contains homologs of two closely linked stationary phase genes, SNZ1 and SNO1, from Saccharomyces cerevisiae. Homologs of SNZ1 encode extremely highly conserved proteins that have been implicated in pyridoxine (vitamin B6) metabolism in the filamentous fungi Cercospora nicotianae and in Aspergillus nidulans. In N. crassa, SNZ and SNO homologs map to the region occupied by pdx-1 (pyridoxine requiring), a gene that has been known for several decades, but which was not sequenced previously. In this study, pyridoxine-requiring mutants of N. crassa were found to possess mutations that disrupt conserved regions in either the SNZ or SNO homolog. Previously, nearly all of these mutants were classified as pdx-1. However, one mutant with a disrupted SNO homolog was at one time designated pdx-2. It now appears appropriate to reserve the pdx-1 designation for the N. crassa SNZ homolog and pdx-2 for the SNO homolog. We further report annotation of the entire 36,030-bp region, which contains at least 12 protein coding genes, supporting a previous conclusion of high gene densities (12,000-13,000 total genes) for N. crassa. Among genes in this region other than SNZ and SNO homologs, there was no evidence of shared function. Four of the genes in this region appear to have been lost from the S. cerevisiae lineage.

Allelic diversity at the het-c locus in Neurospora tetrasperma confirms outcrossing in nature and reveals an evolutionary dilemma for pseudohomothallic ascomycetes.

Vegetative cells of the filamentous ascomycete Neurospora tetrasperma are typically heterokaryotic, possessing haploid nuclei of both A and a mating types. As a consequence, N. tetrasperma is self-fertile. This life cycle, referred to as pseudohomothallism, clearly derives from true heterothallism of the type exhibited by related species such as N. crassa. Occasional homokaryotic, single-mating-type (heterothallic) isolates occur; in the laboratory, such strains can be outcrossed. The potential for outcrossing in N. tetrasperma raises the question of how this organism avoids heterokaryon incompatibility. Heterokaryon incompatability in vegetatively growing fungi is controlled by multiple loci. Two strains must be identical at each het locus (11 in N. crassa) to form a stable heterokaryon. Prior to the present survey, it seemed plausible that N. tetrasperma avoids heterokaryon incompatibility by maintaining compatible allele combinations through continual selfing. A survey of het-c variation among wild-type isolates in this study demonstrated that N. tetrasperma outcrosses in nature and that such matings can result in incompatible combinations of het-c alleles. Whereas individual wild-type isolates are invariably homoallelic for het-c, closely related strains may possess functionally different het-c alleles, which predate the origin of N. tetrasperma. Therefore, pseudohomothallic ascomycetes such as N. tetrasperma face an apparent evolutionary dilemma: the benefits of outcrossing must be balanced against the fact that matings can produce unstable heterokaryons and disrupt the pseudohomothallic life cycle.

Large-scale comparison of fungal sequence information: mechanisms of innovation in Neurospora crassa and gene loss in Saccharomyces cerevisiae.

We report a large-scale comparison of sequence data from the filamentous fungus Neurospora crassa with the complete genome sequence of Saccharomyces cerevisiae. N. crassa is considerably more morphologically and developmentally complex than S. cerevisiae. We found that N. crassa has a much higher proportion of "orphan" genes than S. cerevisiae, suggesting that its morphological complexity reflects the acquisition or maintenance of novel genes, consistent with its larger genome. Our results also indicate the loss of specific genes from S. cerevisiae. Surprisingly, some of the genes lost from S. cerevisiae are involved in basic cellular processes, including translation and ion (especially calcium) homeostasis. Horizontal gene transfer from prokaryotes appears to have played a relatively modest role in the evolution of the N. crassa genome. Differences in the overall rate of molecular evolution between N. crassa and S. cerevisiae were not detected. Our results indicate that the current public sequence databases have fairly complete samples of gene families with ancient conserved regions, suggesting that further sequencing will not substantially change the proportion of genes with homologs among distantly related groups. Models of the evolution of fungal genomes compatible with these results, and their functional implications, are discussed.

Suppressed recombination and a pairing anomaly on the mating-type chromosome of Neurospora tetrasperma.

Neurospora crassa and related heterothallic ascomycetes produce eight homokaryotic self-sterile ascospores per ascus. In contrast, asci of N. tetrasperma contain four self-fertile ascospores each with nuclei of both mating types (matA and mata). The self-fertile ascospores of N. tetrasperma result from first-division segregation of mating type and nuclear spindle overlap at the second meiotic division and at a subsequent mitotic division. Recently, Merino et al. presented population-genetic evidence that crossing over is suppressed on the mating-type chromosome of N. tetrasperma, thereby preventing second-division segregation of mating type and the formation of self-sterile ascospores. The present study experimentally confirmed suppressed crossing over for a large segment of the mating-type chromosome by examining segregation of markers in crosses of wild strains. Surprisingly, our study also revealed a region on the far left arm where recombination is obligatory. In cytological studies, we demonstrated that suppressed recombination correlates with an extensive unpaired region at pachytene. Taken together, these results suggest an unpaired region adjacent to one or more paired regions, analogous to the nonpairing and pseudoautosomal regions of animal sex chromosomes. The observed pairing and obligate crossover likely reflect mechanisms to ensure chromosome disjunction.

The nop-1 gene of Neurospora crassa encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins.

Opsins are a class of retinal-binding, seven transmembrane helix proteins that function as light-responsive ion pumps or sensory receptors. Previously, genes encoding opsins had been identified in animals and the Archaea but not in fungi or other eukaryotic microorganisms. Here, we report the identification and mutational analysis of an opsin gene, nop-1, from the eukaryotic filamentous fungus Neurospora crassa. The nop-1 amino acid sequence predicts a protein that shares up to 81.8% amino acid identity with archaeal opsins in the 22 retinal binding pocket residues, including the conserved lysine residue that forms a Schiff base linkage with retinal. Evolutionary analysis revealed relatedness not only between NOP-1 and archaeal opsins but also between NOP-1 and several fungal opsin-related proteins that lack the Schiff base lysine residue. The results provide evidence for a eukaryotic opsin family homologous to the archaeal opsins, providing a plausible link between archaeal and visual opsins. Extensive analysis of Deltanop-1 strains did not reveal obvious defects in light-regulated processes under normal laboratory conditions. However, results from Northern analysis support light and conidiation-based regulation of nop-1 gene expression, and NOP-1 protein heterologously expressed in Pichia pastoris is labeled by using all-trans [3H]retinal, suggesting that NOP-1 functions as a rhodopsin in N. crassa photobiology.

Identification of the first fungal annexin: analysis of annexin gene duplications and implications for eukaryotic evolution.

Annexin homologues have been found in animals, plants, and distinct protist lineages. We report the identification of the first fungal annexin, encoded by the anx14 gene of the filamentous ascomycete Neurospora crassa. Annexins have a complex evolutionary history and exhibit a large number of gene duplications and gene losses in various taxa, including the complete loss of annexin sequences from another ascomycete, the budding yeast Saccharomyces cerevisiae. Surprisingly, the N. crassa annexin homologue is most closely related to the annexin homologue of the slime mold Dictyostelium discoideum, suggesting a phylogenetic link between cellular slime molds and true fungi. Both of these annexin homologues are closely related to the family of annexin homologues present in animals, an observation consistent with the existence of the animal-fungal clade. These data further suggest that the gene duplications that generated the family of annexin sequences present in animals, fungi, and slime molds began prior to the divergence of these taxa.

Expressed sequences from conidial, mycelial, and sexual stages of Neurospora crassa.

In the Neurospora Genome Project at the University of New Mexico, expressed sequence tags (ESTs) corresponding to three stages of the life cycle of the filamentous fungus Neurospora crassa are being analyzed. The results of a pilot project to identify expressed genes and determine their patterns of expression are presented. 1,865 partial complementary DNA (cDNA) sequences for 1,409 clones were determined using single-pass sequencing. Contig analysis allowed the identification of 838 unique ESTs and 156 ESTs present in multiple cDNA clones. For about 34% of the sequences, highly or moderately significant matches to sequences (of known and unknown function) in the NCBI database were detected. Approximately 56% of the ESTs showed no similarity to previously identified genes. Among genes with assigned function, about 43.3% were involved in metabolism, 32.9% in protein synthesis and 8.4% in RNA synthesis. Fewer were involved in defense (6%), cell signalling (3.4%), cell structure (3.4%) and cell division (2.6%).

Phylogenetic analysis of heterothallic Neurospora species.

We examined the phylogenetic relationships among five heterothallic species of Neurospora using restriction fragment polymorphisms derived from cosmid probes and sequence data from the upstream regions of two genes, al-1 and frq. Distance, maximum likelihood, and parsimony trees derived from the data support the hypothesis that strains assigned to N. sitophila, N. discreta, and N. tetrasperma form respective monophyletic groups. Strains assigned to N. intermedia and N. crassa, however, did not form two respective monophyletic groups, consistent with a previous suggestion based on analysis of mitochondrial DNAs that N. crassa and N. intermedia may be incompletely resolved sister taxa. Trees derived from restriction fragments and the al-1 sequence position N. tetrasperma as the sister species of N. sitophila. None of the trees produced by our data supported a previous analysis of sequences in the region of the mating type idiomorph that grouped N. crassa and N. sitophila as sister taxa, as well as N. intermedia and N. tetrasperma as sister taxa. Moreover, sequences from al-1, frq, and the mating-type region produced different trees when analyzed separately. The lack of consensus obtained with different sequences could result from the sorting of ancestral polymorphism during speciation or gene flow across species boundaries, or both.

Pseudohomothallism and evolution of the mating-type chromosome in Neurospora tetrasperma.

Ascospores of Neurospora tetrasperma normally contain nuclei of both mating-type idiomorphs (a and A), resulting in self-fertile heterokaryons (a type of sexual reproduction termed pseudohomothallism). Occasional homokaryotic self-sterile strains (either a or A) behave as heterothallics and, in principle, provide N. tetrasperma with a means for facultative outcrossing. This study was conceived as an investigation of the population biology of N. tetrasperma to assess levels of intrastrain heterokaryosis (heterozygosity). The unexpected result was that the mating-type chromosome and autosomes exhibited very different patterns of evolution, apparently because of suppressed recombination between mating-type chromosomes. Analysis of sequences on the mating-type chromosomes of wild-collected self-fertile strains revealed high levels of genetic variability between sibling A and a nuclei. In contrast, sequences on autosomes of sibling A and a nuclei exhibited nearly complete homogeneity. Conservation of distinct haplotype combinations on A and a mating-type chromosomes in strains from diverse locations further suggested an absence of recombination over substantial periods of evolutionary time. The suppression of recombination on the N. tetrasperma mating-type chromosome, expected to ensure a high frequency of self fertility, presents an interesting parallel with, and possible model for studying aspects of, the evolution of mammalian sex chromosomes.

Superoxide dismutase (sod-1) null mutants of Neurospora crassa: oxidative stress sensitivity, spontaneous mutation rate and response to mutagens.

Enzymatic superoxide-dismutase activity is believed to be important in defense against the toxic effects of superoxide. Although superoxide dismutases are among the best studied proteins, numerous questions remain concerning the specific biological roles of the various superoxide-dismutase types. In part, this is because the proposed damaging effects of superoxide are manifold, ranging from inactivation of certain metabolic enzymes to DNA damage. Studies with superoxide-deficient mutants have proven valuable, but surprisingly few such studies have been reported. We have constructed and characterized Neurospora crassa mutants that are null for sod-1, the gene that encodes copper-zinc superoxide dismutase. Mutant strains are sensitive to paraquat and elevated oxygen concentrations, and they exhibit an increased spontaneous mutation rate. They appear to have near wild-type sensitive to near- and far-UV, heat shock and gamma-irradiation. Unlike the equivalent Saccharomyces cerevisiae mutant and the sodA sodB double mutant of Escherichia coli, they do not exhibit aerobic auxotrophy. These results are discussed in the context of an attempt to identify consensus phenotypes among superoxide dismutase-deficient mutants. N. crassa sod-1 null mutant strains were also employed in genetic and subcellular fractionation studies. Results support the hypothesis that a single gene (sod-1), located between Fsr-12 and leu-3 on linkage group I, is responsible for most or all CuZn superoxide dismutase activity in this organism.

Structure, exon pattern, and chromosome mapping of the gene for cytosolic copper-zinc superoxide dismutase (sod-1) from Neurospora crassa.

A 4.8-kilobase BamHI-HindIII fragment encoding the entire Neurospora crassa CuZn superoxide dismutase gene (herein designated sod-1) was isolated from a genomic library using two 60-base deoxyoligonucleotide probes corresponding to the published N. crassa amino acid sequence. The nucleotide sequence of the gene encodes an amino acid sequence matching the published protein sequence at 152 of 153 positions. Codon preference shows an unusually strong bias such that only 32 of the possible 61 codons are used, with no codons ending in A. Codon usage is that of highly expressed N. crassa genes. The gene contains three introns, none of which corresponds to any of the introns previously identified in the human gene. Analysis of the intron positions provides support for the hypothesis that CuZn superoxide dismutases evolved by gene duplication and fusion followed by the addition of exons encoding an N-terminal beta-hairpin and a zinc-binding subdomain. The N. crassa gene has an intron mapping to amino acid residue 114 in a sequence-conserved region of the protein whereas the human gene has an intron mapping to a similar but not identical position at residue 118. The discordant position of these introns suggests that one of them was inserted relatively recently. The first N. crassa intron contains a sequence that is similar to the transcriptional regulatory site, UAS1, of the yeast CYC1 (iso-1-cytochrome c) gene and to a putative UAS from the yeast manganese superoxide dismutase gene. A 10-nucleotide portion of this region also matches exactly a sequence in intron 2 of the con-10 gene of N. crassa. sod-1 was mapped to the left arm of chromosome I by following the segregation of a restriction fragment length polymorphism in a sexual cross. Although results indicate that there is a single gene for cytosolic CuZn superoxide dismutase, two additional, perhaps distantly related, sequences were identified that hybridized weakly to both oligonucleotide probes.

Evidence for three differentially regulated catalase genes in Neurospora crassa: effects of oxidative stress, heat shock, and development.

Genetic and biochemical studies demonstrated that Neurospora crassa possesses three catalases encoded by three separate structural genes. The specific activities of the three enzymes varied in response to superoxide-mediated stress, heat shock, and development. The three loci, which we designated cat-1, cat-2, and cat-3, map to the right arms of chromosomes III, VII, and III, respectively. The cat-1-encoded enzyme (designated Cat-1; estimated molecular weight, 315,000; pI 5.2) was the predominant catalase in rapid-growth mycelium, and its activity was substantially increased in paraquat-treated and heat-shocked mycelium. Cat-2 (Mw, 165,000; pI 5.4) was absent from rapid-growth mycelium but present at low levels in conidia and stationary-phase mycelium. It was the predominant catalase in extracts derived from mycelium that had been heat shocked for 2 h. Cat-3 (Mw, 340,000; pI 5.5) was the predominant catalase in extracts from mature conidia.

Isolation of gene fusions (soi::lacZ) inducible by oxidative stress in Escherichia coli.

Mu dX phage was used to isolate three gene fusions to the lacZ gene (soi::lacZ; soi for superoxide radical inducible) that were induced by treatment with superoxide radical anion generators such as paraquat and plumbagin. The induction of beta-galactosidase in these fusion strains with the superoxide radical generating agents required aerobic metabolism. Hyperoxygenation (i.e., bubbling of cultures with oxygen gas) also induced the fusions. On the other hand, hydrogen peroxide did not induce the fusions at concentrations that are known to invoke an adaptive response. Introduction of oxyR, htpR, or recA mutations did not affect the induction. Two of the fusion strains exhibited increased sensitivity to paraquat but not to hydrogen peroxide. The third fusion strain showed no increased sensitivity to either agent. All three fusions were located in the 45- to 61-min region of the Escherichia coli chromosome.

Human copper-zinc superoxide dismutase complements superoxide dismutase-deficient Escherichia coli mutants.

An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide. Expression of the evolutionarily unrelated eukaryotic CuZn superoxide dismutase in the sodAsodB E. coli mutant results in a wild-type phenotype with respect to aerobic growth on minimal medium and in resistance to paraquat and hydrogen peroxide. This supports the hypothesis that superoxide dismutation is the in vivo function of these proteins. Analysis of the growth of sodAsodB cells containing plasmids encoding partially active CuZn superoxide dismutases, produced by in vitro mutagenesis, shows a correlation between cell growth and enzyme activity. Thus, the sodAsodB strain provides a controlled selection for varying levels of superoxide dismutase activity.

Toxicity and mutagenicity of plumbagin and the induction of a possible new DNA repair pathway in Escherichia coli.

Actively growing Escherichia coli cells exposed to plumbagin, a redox cycling quinone that increases the flux of O2- radicals in the cell, were mutagenized or killed by this treatment. The toxicity of plumbagin was not found to be mediated by membrane damage. Cells pretreated with plumbagin could partially reactivate lambda phage damaged by exposure to riboflavin plus light, a treatment that produces active oxygen species. The result suggested the induction of a DNA repair response. Lambda phage damaged by H2O2 treatment were not reactivated in plumbagin-pretreated cells, nor did H2O2-pretreated cells reactivate lambda damaged by treatment with riboflavin plus light. Plumbagin treatment did not induce lambda phage in a lysogen, nor did it cause an increase in beta-galactosidase production in a dinD::Mu d(lac Ap) promoter fusion strain. Cells pretreated with nonlethal doses of plumbagin showed enhanced survival upon exposure to high concentrations of plumbagin, but were unchanged in their susceptibility to far-UV irradiation. polA and recA mutants were not significantly more sensitive than wild type to killing by plumbagin. However, xth-1 mutants were partially resistant to plumbagin toxicity. It is proposed that E. coli has an inducible DNA repair response specific for the type of oxidative damage generated during incubation with plumbagin. Furthermore, this response appears to be qualitatively distinct from the SOS response and the repair response induced by H2O2.

Genomic analysis of phase I and II Coxiella burnetii with restriction endonucleases.

Restriction endonuclease-digested DNAs from several isolates of phase I and phase II Coxiella burnetii were compared using agarose gel electrophoresis and soft-laser scanning densitometry. Our results demonstrate that the two phases are, as previously assumed, alternative phases of the same organism. Although the restriction endonuclease digestion revealed genetic differences between clonal isolates of phase I and phase II C. burnetii Nine Mile strain, these differences do not appear to be related to antigenic phase variation. However, analyses of the fragment patterns generated by restriction enzyme digestion suggest potential grouping of the different isolates.

Distribution and evolutionary significance of mitochondrial plasmids in Neurospora spp.

A mitochondrial plasmid line in the fungal genus Neurospora is geographically widely distributed and occurs in isolates of at least two species. On the basis of characterization with restriction endonucleases, it is apparent that plasmids from isolates of Neurospora tetrasperma are more closely related to one another than to an evolutionarily homologous plasmid from Neurospora intermedia; N. tetrasperma plasmids from Surinam and Hawaii differed from one another only slightly by our analysis, whereas the plasmid from N. intermedia (Fiji) exhibited substantial restriction site divergence from all N. tetrasperma plasmids. We believe these observations strengthen the presumption that four-spored isolates of Neurospora spp. represent a natural taxonomic grouping (N. tetrasperma). The plasmids from N. tetrasperma and N. intermedia (Fiji), although clearly related to each other as shown by hybridization studies, exhibited no detectable homology with either of two additional plasmid lines from isolates of Neurospora spp. Nor did they exhibit homology with the mitochondrial genome. Despite this lack of homology among three distinct plasmid lines, all the plasmids may possess a common mode of replication.