The mushroom Coprinopsis cinerea is a classic experimental model for multicellular development in fungi because it grows on defined media, completes its life cycle in 2 weeks, produces some 10 8 synchronized meiocytes, and can be manipulated at all stages in development by mutation and transformation. The 37-megabase genome of C. cinerea was sequenced and assembled into 13 chromosomes. Meiotic recombination rates vary greatly along the chromosomes, and retrotransposons are absent in large regions of the genome with low levels of meiotic recombination. Single-copy genes with identifiable orthologs in other basidiomycetes are predominant in low-recombination regions of the chromosome. In contrast, paralogous multicopy genes are found in the highly recombining regions, including a large family of protein kinases (FunK1) unique to multicellular fungi. Analyses of P450 and hydrophobin gene families confirmed that local gene duplications drive the expansions of paralogous copies and the expansions occur in independent lineages of Agaricomycotina fungi. Gene-expression patterns from microarrays were used to dissect the transcriptional program of dikaryon formation (mating). Several members of the FunK1 kinase family are differentially regulated during sexual morphogenesis, and coordinate regulation of adjacent duplications is rare. The genomes of C. cinerea and Laccaria bicolor , a symbiotic basidiomycete, share extensive regions of synteny. The largest syntenic blocks occur in regions with low meiotic recombination rates, no transposable elements, and tight gene spacing, where orthologous single-copy genes are overrepresented. The chromosome assembly of C. cinerea is an essential resource in understanding the evolution of multicellularity in the fungi.

We examined the inheritance of 5-methylcytosine residues at a centromere-linked locus in the basidiomycete Coprinus cinereus. Although methylated and unmethylated tracts were inherited both mitotically and meiotically the lengths of these tracts were variable. This variation was not confined to any one phase of the life cycle of the organism, and it usually involved the simultaneous de novo methylation of at least four HpaII-MspI sites. We also found that the higher levels of methylation at this locus were transmitted through meiosis, regardless of the level of methylation of the homologous chromosome.Postreplicative methylation of DNA cytosine residues is a common feature of procaryotic and eucaryotic DNAs. In eucaryotes, cytosine methylation is believed to be a controlling mechanism in gene expression (for examples, see references 5 and 14) and development (7,8). Since most methylation in animals and fungi is at the nucleotide doublet CpG (2, 17), the isoschizomer pair of restriction endonucleases, HpaII and MspI, has provided a powerful tool for the study of the methylation patterns of specific DNA sequences (3, 18). These two enzymes recognize the same sequence (5'-CCGG-3'), but HpaII is sensitive to CpG methylation, while MspI is sensitive to CpC methylation (9).Coprinus cinereus is a particularly useful organism for the study of eucaryotic DNA methylation. This basidiomycete has a small nuclear genome size of 38,000 kilobase pairs (kb) (6), which facilitates molecular analyses. In addition, genetic studies and experimental manipulation of all phases of its life cycle are straightforward and simple. We have shown previously that the nuclear genome of this fungus is extensively methylated at the nucleotide doublet CpG (M. E. Zolan and P. J. Pukkila, in Molecular Genetics of Filamentous Fungi, in press). In that study, we also examined the inheritance of DNA methylation at a centromere-linked locus, termed 16-1, which is methylated in one geographical isolate (Okayama-7) but not in others. Tetrad analysis of progeny from a cross between Okayama-7 and PJP52 (which is unmethylated at 16-1) revealed that methylated tracts are inherited 2:2 during meiosis. That is, progeny that received the chromosome containing locus 16-1 from Okayama-7 were always methylated at that locus, and progeny that inherited locus 16-1 from PJP52 were not methylated at that locus. However, we observed altered lengths of methylated tracts among the methylated progeny. In this study, we asked whether these alterations are a direct consequence of the meiotic process, and we also asked whether the altered tracts resulting from de novo methylation are transmitted as simple Mendelian alleles.MATERIALS AND METHODS C. cinereus strains and growth. The two parents, Okayama-7 and PJP52, of MZ1, the first cross in this study, were described previously (4). The remainder of the strains used were progeny of MZ1 and are described in al. (4). To isolate oidia, 5 ml of sterile H20 was poured onto the surface of a confluent plate of a monokaryotic cultu...

Spo11, a type II topoisomerase, is likely to be required universally for initiation of meiotic recombination. However, a dichotomy exists between budding yeast and the animals Caenorhabditis elegans and Drosophila melanogaster with respect to additional roles of Spo11 in meiosis. In Saccharomyces cerevisiae, Spo11 is required for homolog pairing, as well as axial element (AE) and synaptonemal complex (SC) formation. All of these functions are Spo11 independent in C.elegans and D.melanogaster. We examined Spo11 function in a multicellular fungus, Coprinus cinereus. The C.cinereus spo11‐1 mutant shows high levels of homolog pairing and occasionally forms full‐length AEs, but no SC. In C.cinereus, Spo11 is also required for maintenance of meiotic chromosome condensation and proper spindle formation. Meiotic progression in spo11‐1 is aberrant; late in meiosis basidia undergo programmed cell death (PCD). To our knowledge, this is the first example of meiotic PCD outside the animal kingdom. Ionizing radiation can partially rescue spo11‐1 for both AE and SC formation and viable spore production, suggesting that the double‐strand break function of Spo11 is conserved and is required for these functions.

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In eubacteria, the recA gene has long been recognized as essential for homologous recombination and DNA repair. Recent work has identified recA homologs in archaebacteria and eukaryotes, thus emphasizing the universal role this gene plays in DNA metabolism. We have isolated and characterized two new recA homologs, one from the basidiomycete Coprinus cinereus and the other from the angiosperm Lycopersicon esculentum. Like the RAD51 gene of Saccharomyces cerevisiae, the Coprinus gene is highly induced by gamma irradiation and during meiosis. Phylogenetic analyses of eukarotic recA homologs reveal a gene duplication early in eukaryotic evolution which gave rise to two putatively monophyletic groups of recA-like genes. One group of 11 characterized genes, designated the rad51 group, is orthologous to the Saccharomyces RAD51 gene and also contains the Coprinus and Lycopersicon genes. The other group of seven genes, designated the dmc1 group, is orthologous to the Saccharomyces DMC1 gene. Sequence comparisons and phylogenetic analysis reveal extensive lineage- and gene-specific differences in rates of RecA protein evolution. Dmc1 consistently evolves faster than Rad51, and fungal proteins of both types, especially those of Saccharomyces, change rapidly, particularly in comparison to the slowly evolving vertebrate proteins. The Drosophila Rad51 protein has undergone remarkably rapid sequence divergence.