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 Ca(2+) 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.

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 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.

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.epigenetics ͉ meiosis ͉ MSUD ͉ Neurospora ͉ RNA interference C oenocytic organisms, in which nuclei coexist in a common cytoplasm, are probably at especially high risk from proliferation of detrimental retrotransposons. In the haploid ascomycete Neurospora crassa, several gene-silencing mechanisms exist to maintain its genome integrity. Quelling, which defends the organism during the vegetative phase, is an RNA interference (RNAi) system that suppresses the expression of transgenes occurring in more than one copy (1, 2). Another surveillance system, known as repeat-induced point mutation (RIP), is a premeiotic process that scans the genome for duplicated sequences and targets them for C to T mutations (3). A third silencing mechanism, named meiotic silencing by unpaired DNA, scans the genome and monitors the pairing of DNA segments with their homologs during meiotic prophase (4-6). This mechanism probably prevents the expression and transposition of invasive sequences, and serves the organism in its need to counter exogenous elements and perhaps to regulate endogenous elements. Deletion or extensive mutation in an RdRPencoding gene, sad-1 (suppressor of ascus dominance), reduces meiotic silencing to a low level. RdRP plays an important role in some RNAi systems (2). For example, if foreign nucleic acids trigger the production of aberrant RNA (aRNA), the singlestranded aRNA can be replicated into a double-stranded species (dsRNA) via the activity of an RdRP. The dsRNA is then processed into small interfering RNA (siRNA) duplexes by Dicer. The siRNAs subsequently guide the cleavage of mRNA via the RNA-induced silencing complex. The fact that an RdRP is required for meiotic silencing suggests that the synthesis of dsRNA, its amplification, or both, are essential for the process.We have now identified an additional gene, sad-2, which is also required for meiotic silencing. Dominant mutations (Sad-2) can suppress the meiotic silencing of unpaired loci with efficiency comparable to that of Sad-1. A Sad-2 mutation does not give any obvious abnormal phenotype during vegetative growth and, correspondingly, sad-2 ϩ mRNA can only...