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We show here that in the fungus Sordaria macrospora, the meiosis-specific HORMA-domain protein Hop1 is not essential for the basic early events of chromosome axis development, recombination initiation, or recombination-mediated homolog coalignment/pairing. In striking contrast, Hop1 plays a critical role at the leptotene/zygotene transition which is defined by transition from pairing to synaptonemal complex (SC) formation. During this transition, Hop1 is required for maintenance of normal axis structure, formation of SC from telomere to telomere, and development of recombination foci. These hop1Δ mutant defects are DSB dependent and require Sme4/Zip1-mediated progression of the interhomolog interaction program, potentially via a pre-SC role. The same phenotype occurs not only in hop1Δ but also in absence of the cohesin Rec8 and in spo76-1, a non-null mutant of cohesin-associated Spo76/Pds5. Thus, Hop1 and cohesins collaborate at this crucial step of meiotic prophase. In addition, analysis of 4 non-null mutants that lack this transition defect reveals that Hop1 also plays important roles in modulation of axis length, homolog-axis juxtaposition, interlock resolution, and spreading of the crossover interference signal. Finally, unexpected variations in crossover density point to the existence of effects that both enhance and limit crossover formation. Links to previously described roles of the protein in other organisms are discussed. Hop1 has been previously shown to display diverse meiotic functions in different organisms. This study identifies and characterizes the functions of Hop1 in the fungus Sordaria macrospora and shows that it plays a crucial role during the leptotene/zygotene transition and formation of the synaptonemal complex during meiotic prophase. [ABSTRACT FROM AUTHOR]

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

Repeat-induced point mutation (RIP) is a homology-dependent gene-silencing mechanism that introduces C:G-to-T:A transitions in duplicated DNA segments. Cis-duplicated sequences can also be affected by another mechanism called premeiotic recombination (PR). Both are active over the sexual cycle of some filamentous fungi, e.g., Neurospora crassa and Podospora anserina. During the sexual cycle, several developmental steps require precise nuclear movement and positioning, but connections between RIP, PR, and nuclear distributions have not yet been established. Previous work has led to the isolation of amil, the P. anserina ortholog of the Aspergillus nidulans apsA gene, which is required for nuclear positioning. We show here that amil is involved in nuclear distribution during the sexual cycle and that alteration of amil delays the fruiting-body development. We also demonstrate that amil alteration affects loss of transgene functions during the sexual cycle. Genetically linked multiple copies of transgenes are affected by RIP and PR much more frequently in an amil mutant cross than in a wild-type cross. Our results suggest that the developmental slowdown of the amil mutant during the period of RIP and PR increases time exposure to the duplication detection system and thus increases the frequency of RIP and PR.

It has been previously reported that, at the time of death, the Podospora anserina AS1-4 mutant strains accumulate specific deleted forms of the mitochondrial genome and that their life spans depend on two natural alleles (variants) of the rmp1 gene: AS1-4 rmp1-2 strains exhibit life spans strikingly longer than those of AS1-4 rmp1-1. Here, we show that rmp1 is an essential gene. In silico analyses of eight rmp1 natural alleles present in Podospora isolates and of the putative homologs of this orphan gene in other filamentous fungi suggest that rmp1 evolves rapidly. The RMP1 protein is localized in the mitochondrial and/or the cytosolic compartment, depending on cell type and developmental stage. Strains producing RMP1 without its mitochondrial targeting peptide are viable but exhibit vegetative and sexual defects.

Peroxins (PEX) are proteins required for peroxisome biogenesis. Mutations in PEX genes cause lethal diseases in humans, metabolic defects in yeasts, and developmental disfunctions in plants and filamentous fungi. Here we describe the first large-scale screening for suppressors of apex mutation. In Podospora anserina, pex2 mutants exhibit a metabolic defect [inability to grow on medium containing oleic acid (OA medium) as sole carbon source] and a developmental defect (inability to differentiate asci in homozygous crosses). Sixty-three mutations able to restore growth of pex2 mutants on OA medium have been analyzed. They fall in six loci (suol to suo6) and act as dominant, allele-nonspecific suppressors. Most suo mutations have pleiotropic effects in a pex2[+] background: formation of unripe ascospores (all loci except suo5 and suo6), impaired growth on OA medium (all loci except suo4 and suo6), or sexual defects (suo4). Using immunofluorescence and GFP staining, we show that peroxisome biogenesis is partially restored along with a low level of ascus differentiation in pex2 mutant strains carrying either the suo5 or the suo6 mutations. The data are discussed with respect to ss-oxidation of fatty acids, peroxisome biogenesis, and cell differentiation.

Meiotic silencing by unpaired DNA during meiosis affects all DNA homologous with the unpaired DNA. In Neurospora, the meiotic silencing phenomenon may lead to reproductive isolation of species.

The heterothallic fungus Podospora anserina has two mating-type alleles termed mat+ and mat-. The mat+ sequence contains one gene, FPR1, while mat- contains three genes: FMR1, SMR1, and SMR2. FPR1 and FMR1 are required for fertilization, which is followed by mitotic divisions of the two parental nuclei inside the female organ. This leads to the formation of plurinucleate cells containing a mixture of parental mat+ and mat- nuclei. Further development requires a recognition between mat+ and mat- nuclei before migration of the mat+ / mat- pairs into specialized hyphae in which karyogamy, meiosis, and ascospore formation take place. FPR1, FMR1, and SMR2 control this internuclear recognition step. Initial development of the dikaryotic stage is supposed to require SMR1; disruption of SMR1 results in barren perithecia. In a systematic search for suppressors restoring fertility, we isolated 15 suppressors--all of them mutations in the mating-type genes. These fmr1, smr2, and fpr1 mutants, as well as the strains disrupted for FMR1, SMR2, and FPR1, are weakly self-fertile. They are able to act as the male partner on a strain of the same mating type and give a mixture of biparental and uniparental progeny when crossed with a wild-type strain of opposite mating type. These observations lead us to propose that SMR2, FMR1, and FPR1 act as activators and repressors of fertilization and internuclear recognition functions.

During mitosis, from late prophase onward, sister chromatids are connected along their entire lengths by axis-linking chromatin/ structure bridges. During prometaphase/metaphase, these bridges ensure that sister chromatids retain a parallel, paranemic relation- ship, without helical coiling, as they undergo compaction. Bridges must then be removed during anaphase. Motivated by these find- ings, the present study has further investigated the process of ana- phase sister separation. Morphological and functional analyses of mammalian mitoses reveal a three-stage pathway in which inter- axis bridges play a prominent role. First, sister chromatid axes glob- ally separate in parallel along their lengths, with concomitant bridge elongation, due to intersister chromatin pushing forces. Sis- ter chromatids then peel apart progressively from a centromere to telomere region(s), step-by-step. During this stage, poleward spin- dle forces dramatically elongate centromere-proximal bridges, which are then removed by a topoisomerase IIα–dependent step. Finally, in telomere regions, widely separated chromatids remain invisibly linked, presumably by catenation, with final separation during anaphase B. During this stage increased separation of poles and/or chromatin compaction appear to be the driving force(s). Cohesin cleavage licenses these events, likely by allowing bridges to respond to imposed forces. We propose that bridges are not sim- ply removed during anaphase but, in addition, play an active role in ensuring smooth and synchronous microtubule-mediated sister separation. Bridges would thereby be the topological gatekeepers of sister chromatid relationships throughout all stages of mitosis. [ABSTRACT FROM AUTHOR]

Research was conducted to examine the characterization and cloning of an Ascobolus gene, masc1, encoding a protein with the primary structure of a DNA methyltransferase (MTase). The disruption of masc1 has no effect on viability or maintenance methylation but prevents the de novo methylation associated with methylation induced premeiotically process. Thus, Masc1, the gene product which is involved in de novo methylation, is likely to represent a novel type of a eukaryotic MTase.

The Podospora (P.) anserina car1 gene encodes a peroxisomal protein and is a functional part of the peroxisomal assembly factor 1 (PAF1) gene, one of the nine genes whose mutations can cause the cerebrohepatorenal Zellweger syndrome. Peroxisomes perform various specific functions in different eukaryotes and are responsible for some inherited diseases. Peroxisomes are needed in a specific stage of sexual development in P. anserina. In lower eukaryotes, a functional homolog of the PAF1 gene is present.

Mating type behavior in Podospora anserina was examined to determine mat+ and mat- nuclei compartmentalization factors and the impact of mating type locus. In vitro mutagenesis and transformation experiments yielded mat gene strains with interaction that indicated dikaryotic state development based on mat information. Other ascus information generated are selfishness for nucleus with mating-type mutation and karyogamy, meiosis and sporulation processes with absent opposite mating types.

Drug disruptions, light and immunofluorescence microscopy of homothallic, pseudohomothallic and heterothallic Ascomycete species to study the function of cytoskeleton in recognizing and separating nuclei during sexual cycle indicates that the relation between spindle pole body (SPB) and nuclear DNA may play a role in the recognition of nuclei from opposite mating types. Actin-myosin interaction is the key cytoskeletal factor that mediates normal sporulation following the migration of nuclear pairs. A orientation- and size-modified SPB segment separates from the nucleus to function as a cortical microtubule organizing center while the other SPB segment remains attached to the nucleus.

A study indicates the presence of a correlation between recombination nodules, chiasmata, meiotic recombination and sites where pairing begins in the Ascomycete Sordaria macrospora. The study examined the correlation between synaptonemal complex (SC) initiation sites and reciprocal meiotic changes among bivalents in the Ascomycete. The patterns of synapsis of meiotic prophase I in mutant and wild types were examined with the help of serial section reconstruction microscopy.

The application of the different recombination complexes in mediating the local destabilization of underlying chromosome axes is presented. The meiotic cohesin, Rec8 is shown to play very important auxiliary roles for meiotic and mitotic cohesion-associated components.

Studies conducted in budding yeast shows that crossovers (CO) and noncrossovers (NCO) designation occurs before stable strand exchange. The results throw new light on the regulation of meiotic recombination and its relationship to chromosome synapsis.