A mutation in the FHA domain of Coprinus cinereus Nbs1 Leads to Spo11-independent meiotic recombination and chromosome segregation.

Nbs1, a core component of the Mre11-Rad50-Nbs1 complex, plays an essential role in the cellular response to DNA double-strand breaks (DSBs) and poorly understood roles in meiosis. We used the basidiomycete Coprinus cinereus to examine the meiotic roles of Nbs1. We identified the C. cinereus nbs1 gene and demonstrated that it corresponds to a complementation group previously known as rad3. One allele, nbs1-2, harbors a point mutation in the Nbs1 FHA domain and has a mild spore viability defect, increased frequency of meiosis I nondisjunction, and an altered crossover distribution. The nbs1-2 strain enters meiosis with increased levels of phosphorylated H2AX, which we hypothesize represent unrepaired DSBs formed during premeiotic replication. In nbs1-2, there is no apparent induction of Spo11-dependent DSBs during prophase. We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation. In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type. Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea.

BACKGROUND: The transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development. RESULTS: We evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom. CONCLUSIONS: We implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea.

Global gene expression in Coprinopsis cinerea meiotic mutants reflects checkpoint arrest.

The basidiomycete Coprinopsis cinerea is well-suited to studies of meiosis because meiosis progresses synchronously in 10 million cells within each mushroom cap. Approximately 20% of C. cinerea genes exhibit changing expression during meiosis, but meiosis and mushroom development happen concurrently and therefore differentially expressed genes might not be directly involved in meiotic processes. By using microarrays, we examined global gene expression across a meiotic time course in two mutants in which meiosis arrests but mushrooms develop normally. Genes differentially expressed in the mutants compared with the wild type are likely to be involved in meiosis and sporulation as opposed to mushroom development. In rad50-1, which arrests in late prophase, RNA abundance for a group of early meiotic genes remains high, whereas the expression of a group of late meiotic genes is never induced. In contrast, in msh5-22 (which fails to undergo premeiotic DNA replication), both early and late meiotic genes are underexpressed relative to wild type at late meiotic time points as the cells die. Genes that are differentially expressed relative to wild type in both mutants are particularly strong candidates for playing roles in meiosis and sporulation.

The spread of a transposon insertion in Rec8 is associated with obligate asexuality in Daphnia.

Although transitions from sexual to asexual reproduction are thought to have important evolutionary consequences, little is known about the mechanistic underpinnings of these changes. The cyclical parthenogen Daphnia pulex is a powerful model in which to address these issues because female-limited meiosis suppression can be transmitted to sexual individuals via males, providing the opportunity for genetic dissection of the trait. A previous study identified genomic regions differentiating obligately asexual females from their sexual counterparts, and a candidate gene within one such region, encoding the meiotic cohesin Rec8, is the subject of this investigation. The D. pulex genome contains three Rec8 loci, all of which are quite polymorphic. However, at one of the loci, all obligately asexual clones carry an allele containing an identical upstream insertion of a transposable element as well as a frameshift mutation, both of which are completely absent from sexual lineages. The low level of variation within the insertion allele across all asexual lineages suggests that this element may be in the process of spreading through the species, and abrogation or modification of Rec8 function is possibly responsible for converting meiotically reproducing lineages into obligate asexuals.

Analysis of the Basidiomycete Coprinopsis cinerea reveals conservation of the core meiotic expression program over half a billion years of evolution.

Coprinopsis cinerea (also known as Coprinus cinereus) is a multicellular basidiomycete mushroom particularly suited to the study of meiosis due to its synchronous meiotic development and prolonged prophase. We examined the 15-hour meiotic transcriptional program of C. cinerea, encompassing time points prior to haploid nuclear fusion though tetrad formation, using a 70-mer oligonucleotide microarray. As with other organisms, a large proportion (∼20%) of genes are differentially regulated during this developmental process, with successive waves of transcription apparent in nine transcriptional clusters, including one enriched for meiotic functions. C. cinerea and the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe diverged ∼500-900 million years ago, permitting a comparison of transcriptional programs across a broad evolutionary time scale. Previous studies of S. cerevisiae and S. pombe compared genes that were induced upon entry into meiosis; inclusion of C. cinerea data indicates that meiotic genes are more conserved in their patterns of induction across species than genes not known to be meiotic. In addition, we found that meiotic genes are significantly more conserved in their transcript profiles than genes not known to be meiotic, which indicates a remarkable conservation of the meiotic process across evolutionarily distant organisms. Overall, meiotic function genes are more conserved in both induction and transcript profile than genes not known to be meiotic. However, of 50 meiotic function genes that were co-induced in all three species, 41 transcript profiles were well-correlated in at least two of the three species, but only a single gene (rad50) exhibited coordinated induction and well-correlated transcript profiles in all three species, indicating that co-induction does not necessarily predict correlated expression or vice versa. Differences may reflect differences in meiotic mechanisms or new roles for paralogs. Similarities in induction, transcript profiles, or both, should contribute to gene discovery for orthologs without currently characterized meiotic roles.

Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus).

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.

Meiotic cytogenetics in Coprinus cinereus.

The basidiomycete fungus Coprinus cinereus has naturally synchronous meiosis and is amenable to analysis using an array of well-developed genetic and molecular tools. In this chapter, we explain in detail the two methods most commonly employed for C. cinereus, staining of intact gill segments and chromosome spreads, with an example of the application of each. We describe iron-hematoxylin staining of intact gill segments for the brightfield examination of meiotic progression, and the use of surface spreads and fluorescence in situ hybridization (FISH) to investigate meiotic chromosome pairing. Gill segments can alternatively be stained with DAPI for the determination of meiotic stage, or propidium iodide for the quantitation of nuclear DNA content, and the chromosome fixation and spreading techniques used for FISH are also suitable for immunolocalization studies of chromosomal proteins.

Methods for meiotic chromosome preparation, immunofluorescence, and fluorescence in situ hybridization in Daphnia pulex.

The genus Daphnia has an intriguing reproductive mode of cyclical parthenogenesis. This reproductive mode has been studied for centuries, but cytogenetic information is lacking due to technical limitations of classical methods. We have developed methods for the preparation and examination of meiotic chromosomes of Daphnia pulex from oocytes and spermatocytes. Oocyte chromosome preparations are obtained by isolating individual oocytes after the release of yolk granules from the ovary using pressure and capillary action. Spermatocyte chromosomes are prepared using a conventional squash method. Cryosectioning is an easy and fast way to prepare sections. We also illustrate the application of immunofluorescence staining against alpha tubulin, as well as fluorescence in situ hybridization (FISH) using the intergenic spacer of ribosomal DNA or single-copy cosmid clones.

Meiotic localization of Mre11 and Rad50 in wild type, spo11-1, and MRN complex mutants of Coprinus cinereus.

The Mre11-Rad50-Nbs1 (MRN) complex is required for numerous cellular processes that involve interactions with DNA double-strand breaks. For the majority of these processes, the MRN complex is thought to act as a unit, with each protein aiding the activity of the others. We have examined the relationship between Mre11 and Rad50 during meiosis in the basidiomycete Coprinus cinereus (Coprinopsis cinerea), investigating to what extent activities of Mre11 and Rad50 are interdependent. We showed that mre11-1 is epistatic to rad50-1 with respect to the time of meiotic arrest, indicating that Mre11 activity facilitates the diffuse diplotene arrest of rad50 mutants. Anti-Mre11 and anti-Rad50 antibodies were used to examine MRN complex localization in a wild-type strain and in spo11, mre11, and rad50 mutants. In wild type, numbers of Mre11 and Rad50 foci peaked at time points corresponding to leptotene and early zygotene. In the spo11-1 mutant, which is defective in meiotic double-strand break formation, foci accumulated throughout prophase I. Of seven MRN mutants examined, only two rad50 strains exhibited Mre11 and Rad50 foci that localized to chromatin, although Mre11 protein was found in the cell for all of them. Analysis of predicted mutant structures showed that stable localization of Mre11 and Rad50 does not depend upon a wild-type hook-proximal coiled coil, but does require the presence of the Rad50 ATPase/adenylate cyclase domains. We found that Mre11 and Rad50 were interdependent for binding to meiotic chromosomes. However, the majority of foci observed apparently contained only one of the two proteins. Independent Mre11 and Rad50 foci might indicate disassociation of the complex during meiosis or could reflect independent structural roles for the two proteins in meiotic chromatin.

Coprinus cinereus rad50 mutants reveal an essential structural role for Rad50 in axial element and synaptonemal complex formation, homolog pairing and meiotic recombination.

The Mre11/Rad50/Nbs1 (MRN) complex is required for eukaryotic DNA double-strand break (DSB) repair and meiotic recombination. We cloned the Coprinus cinereus rad50 gene and showed that it corresponds to the complementation group previously named rad12, identified mutations in 15 rad50 alleles, and mapped two of the mutations onto molecular models of Rad50 structure. We found that C. cinereus rad50 and mre11 mutants arrest in meiosis and that this arrest is Spo11 dependent. In addition, some rad50 alleles form inducible, Spo11-dependent Rad51 foci and therefore must be forming meiotic DSBs. Thus, we think it likely that arrest in both mre11-1 and the collection of rad50 mutants is the result of unrepaired or improperly processed DSBs in the genome and that Rad50 and Mre11 are dispensable in C. cinereus for DSB formation, but required for appropriate DSB processing. We found that the ability of rad50 mutant strains to form Rad51 foci correlates with their ability to promote synaptonemal complex formation and with levels of stable meiotic pairing and that partial pairing, recombination initiation, and synapsis occur in the absence of wild-type Rad50 catalytic domains. Examination of single- and double-mutant strains showed that a spo11 mutation that prevents DSB formation enhances axial element (AE) formation for rad50-4, an allele predicted to encode a protein with intact hook region and hook-proximal coiled coils, but not for rad50-1, an allele predicted to encode a severely truncated protein, or for rad50-5, which encodes a protein whose hook-proximal coiled-coil region is disrupted. Therefore, Rad50 has an essential structural role in the formation of AEs, separate from the DSB-processing activity of the MRN complex.

The Coprinus cinereus adherin Rad9 functions in Mre11-dependent DNA repair, meiotic sister-chromatid cohesion, and meiotic homolog pairing.

Mitotic sister-chromatid cohesion (SCC) is known to depend in part on conserved proteins called adherins, which although necessary for SCC are not themselves localized between sister chromatids. We have examined mitotic DNA-repair and meiotic chromosome behavior in the Coprinus cinereus adherin mutant rad9-1. Genetic pathway analysis established that Rad9 functions in an Mre11-dependent pathway of DNA repair. Using fluorescence in situ hybridization, we found that the rad9-1 mutant is defective in the establishment of meiotic homolog pairing at both interstitial and subtelomeric sites but in the maintenance of pairing at only interstitial loci. To determine the role of Rad9 in meiotic SCC, we hybridized nuclear spreads simultaneously with a homolog-specific probe and a probe that recognizes both members of a homologous pair. We found that Rad9 is required for wild-type levels of meiotic SCC, and that nuclei showing loss of cohesion were twice as likely also to fail at homolog pairing. To ask whether the contribution of Rad9 to homolog pairing is solely in the establishment of SCC, we examined a rad9-1;msh5-22 double mutant, in which premeiotic DNA replication is inhibited. The msh5-22 mutation partially suppressed the deleterious effects of the rad9-1 mutation on homolog pairing; however, pairing in the double mutant still was significantly lower than in the msh5-22 single mutant control. Because the role of Rad9 in homolog pairing is not obviated by the absence of a sister chromatid, we conclude that adherins have one or more early meiotic functions distinct from the establishment of cohesion.

The art and design of genetic screens: filamentous fungi.

In the 1940s, screens for metabolic mutants of the filamentous fungus Neurospora crassa established the fundamental, one-to-one relationship between a gene and a specific protein, and also established fungi as important genetic organisms. Today, a wide range of filamentous species, which represents a billion years of evolutionary divergence, is used for experimental studies. The developmental complexity of these fungi sets them apart from unicellular yeasts, and allows the development of new screens that enable us to address biological questions that are relevant to all eukaryotes.