This report focuses on the application of enzymatic digestion techniques in electron microscopy to the study of the nuclear spindle poles of Ascomycetes and more specifically presents observations concerning the cytochemical characteristics of the plaques. Evidence is presented indicating that the plaques contain proteins digestable by pronase and trypsin (more than by pepsin), perhaps a low amount of RNA and that they also contain DNA as shown by their sensitivity to DNase and their Feulgen-positive reaction.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.

BIMD of Aspergillus nidulans belongs to a highly conserved protein family implicated, in filamentous fungi, in sister-chromatid cohesion and DNA repair. We show here that BIMD is chromosome associated at all stages, except from late prophase through anaphase, during mitosis and meiosis, and is involved in several aspects of both programs. First, bimD + function must be executed during S through M. Second, in bimD6 germlings, mitotic nuclear divisions and overall cellular program occur more rapidly than in wild type. Thus, BIMD, an abundant chromosomal protein, is a negative regulator of normal cell cycle progression. Third, bimD6 reduces the level of mitotic interhomolog recombination but does not alter the ratio between crossover and noncrossover outcomes. Moreover, bimD6 is normal for intrachromosomal recombination. Therefore, BIMD is probably not involved in the enzymology of recombinational repair per se . Finally, during meiosis, staining of the Sordaria ortholog Spo76p delineates robust chromosomal axes, whereas BIMD stains all chromatin. SPO76 and bimD are functional homologs with respect to their roles in mitotic chromosome metabolism but not in meiosis. We propose that BIMD exerts its diverse influences on cell cycle progression as well as chromosome morphogenesis and recombination by modulating chromosome structure.

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.

Antisuppressor mutations in the eEF1A gene of Podospora anserina were previously shown to impair ascospore formation, to drastically increase life span, and to permit the development of the Crippled Growth degenerative process. Here, we show that eEF1A controls ascospore formation through accuracy level maintenance. Examination of antisuppressor mutant perithecia reveals two main cytological defects, mislocalization of spindle and nuclei and nuclear death. Antisuppression levels are shown to be highly dependent upon both the mutation site and the suppressor used, precluding any correlation between antisuppression efficiency and severity of the sporulation impairment. Nevertheless, severity of ascospore differentiation defect is correlated with resistance to paromomycin. We also show that eEF1A controls fruiting body formation and longevity through a mechanism(s) different from accuracy control. In vivo, GFP tagging of the protein in a way that partly retains its function confirmed earlier cytological observation; i.e., this factor is mainly diffuse within the cytosol, but may transiently accumulate within nuclei or in defined regions of the cytoplasm. These data emphasize the fact that the translation apparatus exerts a global regulatory control over cell physiology and that eEF1A is one of the key factors involved in this monitoring.

Homeobox-containing genes are widely described among eukaryotic species other than filamentous ascomycetes. We describe here the isolation and characterization of the first homeobox gene (pah1) identified in a filamentous ascomycete. It encodes a putative protein of 610 amino acids containing a typical homeodomain with 60 amino acids. Deletion of the pah1 gene enhances the number of male gametes (microconidia), whereas overexpression of pah1 results in a decrease in microconidia. These results led us to suppose that pah1 may be a repressor of genes involved in the microconidiation process. Moreover, pah1 is involved in hyphal branching and possibly in the development of female organs.

The Podospora anserina ami1-1 mutant was identified as a male-sterile strain. Microconidia (which act as male gametes) form, but are anucleate. Paraphysae from the perithecium beaks are also anucleate when ami1-1 is used as the female partner in a cross. Furthermore, in crosses heterozygous for ami1-1, some crozier cells are uninucleate rather than binucleate. In addition to these nuclear migration defects, which occur at the transition between syncytial and cellular states, ami1-1 causes abnormal distribution of the nuclei in both mycelial filaments and asci. Finally, an ami1-1 strain bearing information for both mating types is unable to self-fertilize. The ami1 gene is an orthologue of the Aspergillus nidulans apsA gene, which controls nuclear positioning in filaments and during conidiogenesis (at the syncytial/cellular transition). The ApsA and AMI1 proteins display 42% identity and share structural features. The apsA gene complements some ami1-1 defects: it increases the percentage of nucleate microconidia and restores self-fertility in an ami1-1 mat+ (mat-) strain. The latter effect is puzzling, since in apsA null mutants sexual reproduction is quite normal. The functional differences between the two genes are discussed with respect to their possible history in these two fungi, which are very distant in terms of evolution.

Meiotic chromosomes have been studied for many years, in part because of the fundamental life processes they represent, but also because meiosis involves the formation of homolog pairs, a feature which greatly facilitates the study of chromosome behavior. The complex events involved in homolog juxtaposition necessitate prolongation of prophase, thus permitting resolution of events that are temporally compressed in the mitotic cycle. Furthermore, once homologs are paired, the chromosomes are connected by a specific structure: the synaptonemal complex. Finally, interaction of homologs includes recombination at the DNA level, which is intimately linked to structural features of the chromosomes. In consequence, recombination-related events report on diverse aspects of chromosome morphogenesis, notably relationships between sisters, development of axial structure, and variations in chromatin status. The current article reviews recent information on these topics in an historical context. This juxtaposition has suggested new relationships between structure and function. Additional issues were addressed in a previous chapter (551).

The synaptonemal complex (SC) is a prominent and evolutionaly well conserved structure which is strictly meiotic. Several evidences from mutant phenotypes support the hypothesis that recombination and SC formation are mutually interdependent processes. Moreover, the SC recombination nodules correspond in number and location to the crossing-over events. However, recent data confirm that SC formation does not require initiation of recombination, and several observations indicate that full synapsis is not required for recombination. The potential roles played by the SC will be discussed in the following framework: First, although not required for homology recognition, the SC could promote interhomolog interactions in situations where the normal processes have failed (interlocking, heterologous pairing, etc.); Second, polymerization of the SC components might permit the recombination process to progress by modulating the number and localisation of reciprocal versus nonreciprocal exchanges (i.e. interference) and; Third, the SC may play an important role in meiotic chromosome structure and especially in inter-sister interactions.

We show that pyruvate decarboxylase (PDC) 8- to 10-nm-diameter filaments, first described in vegetative cells ofNeurospora crassa,are ubiquitously present in filamentous fungi. The cellular arrangement of these structures was examined over the entire sexual cycle of the ascomycetesN. crassa, N. tetraesperma, Podospora anserina,andSordaria macrospora.PDC-filaments were found associated with the cortical microtubule array of asci and ascospores and absent from the vicinity of spindles and spindle pole bodies. Nocodazole-induced depolymerization of the cortical microtubules results in the loss of PDC-filaments. Moreover, aS. macrosporamutant defective in cortical MT distribution shows abnormal PDC organization. Neurospora asci generated on various metabolic conditions, which modify the presence and relative abundance of PDC-filaments in vegetative cells, have identical patterns of subcellular distribution of these structures. AN. crassamutant (snowflake) that accumulates giant bundles of PDC-filaments during vegetative growth, shows normal distribution of the filaments during ascogenesis. Thus, the regulation conditioning the presence and supramolecular assembly of PDC-filaments in Neurospora differs between vegetative and sexual cells. Taken together, these results suggest that PDC in filamentous fungi may perform two functions, intervening as an enzyme in vegetative metabolism and as a structural protein associated with the cytoskeleton during sexual development.

The leptotene/zygotene transition of meiosis, as defined by classical cytological studies, is the period when homologous chromosomes, already being discernible individualized entities, begin to be close together or touching over portions of their lengths. This period also includes the bouquet stage: Chromosome ends, which have already become integral components of the inner nuclear membrane, move into a polarized configuration, along with other nuclear envelope components. Chromosome movements, active or passive, also occur. The detailed nature of interhomologue interactions during this period, with special emphasis on the involvement of chromosome ends, and the overall role for meiosis and recombination of chromosome movement and, especially, the bouquet stage are discussed.

The Podospora anserina cro1 gene was identified as a gene required for sexual sporulation. Crosses homozygous for the cro1‐1 mutation yield fruiting bodies which produce few asci due to the formation of giant plurinucleate cells instead of dikaryotic cells after fertilization. This defect does not impair karyogamy, but meioses of the resultant polyploid nuclei are most often abortive. Cytological studies suggest that the primary defect of the mutant is its inability to form septa between the daughter nuclei after each mitosis, a step specific for normal dikaryotic cell divisions. The cro1‐1 mutant would thus be unable to leave the syncytial vegetative state while abiding by the meiotic programme. cro1‐1 also shows defects in ascospore germination and growth rate. GFP‐tagging of the CRO1 protein reveals that it is a cytosolic protein mainly expressed at the beginning of the dikaryotic stage and at the time of ascospore maturation. The CRO1 protein exhibits significant similarity to the SHE4 protein, which is required for asymmetric mating‐type switching in budding yeast cells. Thus, a gene involved in asymmetric cell divisions in a unicellular organism plays a key role at the transition between the syncytial (vegetative) state and the cellular (sexual) state in a filamentous fungus.

Molecular mechanisms determining methylation patterns in eukaryotic genomes still remain unresolved. We have characterized, in Ascobolus, a gene for de novo methylation. This novel eukaryotic gene, masc1, encodes a protein that has all motifs of the catalytic domain of eukaryotic C5-DNA-methyltransferases but is unique in that it lacks a regulatory N-terminal domain. The disruption of masc1 has no effect on viability or methylation maintenance but prevents the de novo methylation of DNA repeats, which takes place after fertilization, through the methylation induced premeiotically (MIP) process. Crosses between parents harboring the masc1 disruption are arrested at an early stage of sexual reproduction, indicating that the activity of Masc1, the product of the gene, is crucial in this developmental process.

Tom70 and Mdm10 are mitochondrial outer membrane proteins. Tom70 is implicated in the import of proteins from the cytosol into the mitochondria in Saccharomyces cerevisiae and Neurospora crassa. Mdm10 is involved in the morphology and distribution of mitochondria in S. cerevisiae. Here we report on the characterization of the genes encoding these proteins in the filamentous fungus Podospora anserina. The two genes were previously genetically identified through a systematic search for nuclear suppressors of a degenerative process displayed by the AS1-4 mutant. The PaTom70 protein shows 80% identity with its N. crassa homolog. The PaMdm10 protein displays 35.9% identity with its S. cerevisiae homolog, and cytological analyses show that the PaMDM10-1 mutant exhibits giant mitochondria, as does the S. cerevisiae mdm10-1 mutant. Mutations in PaTOM70 and PaMDM10 result in the accumulation of specific deleted mitochondrial genomes during the senescence process of the fungus. The phenotypic properties of the single- and double-mutant strains suggest a functional relationship between the Tom70 and Mdm10 proteins. These data emphasize the role of the mitochondrial outer membrane in the stability of the mitochondrial genome in an obligate aerobe, probably through the import process.

The car1 gene of the filamentous fungus Podospora anserina was cloned by complementation of a mutant defective for caryogamy (nuclear fusion), a process required for sexual sporulation. This gene encodes a protein that shows similarity to the mammalian PAF1 protein (Zellweger syndrome). Besides sequence similarity, the two proteins share a transmembrane domain and the same type of zinc finger motif. A combination of molecular, physiological, genetical, and ultrastructural approaches gave evidence that the P. anserina car1 protein is actually a peroxisomal protein. This study shows that peroxisomes are required at a specific stage of sexual development, at least in P. anserina, and that a functional homolog of the PAF1 gene is present in a lower eucaryote.

In wild-type crosses of the filamentous ascomycete Podospora anserina, after fertilization, only nuclei of opposite mating type can form dikaryons that undergo karyogamy and meiosis, producing biparental progeny. To determine the role played by the mating type in these steps, the four mat genes were mutagenized in vitro and introduced into a strain deleted for its mat locus. Genetic and cytological analyses of these mutant strain, crossed to each other and to wild type, showed that mating-type information is required for recognition of nuclear identity during the early steps of sexual reproduction. In crosses with strain carrying a mating-type mutation, two unusual developmental patterns were observed: monokaryotic cells, resulting in haploid meiosis, and uniparental dikaryotic cells providing, after karyogamy and meiosis, a uniparental progeny. Altered mating-type identity leads to selfish behavior of the mutant nucleus: it migrates alone or paired, ignoring its wild-type partner in all mutant x wild-type crosses. This behavior is nucleus-autonomous because, in the same cytoplasm, the wild-type nuclei form only biparental dikaryons. In P. anserina, mat genes are thus required to ensure a biparental dikaryotic state but appear dispensable for later stages, such as meiosis and sporulation.

In heterothallic filamentous ascomycetes, two nuclei of opposite mating type must recognize one another in a plurinucleate cell to form a pair prior to karyogamy. In pseudohomothallic species, two nuclei of opposite mating type must also pair after meiosis to form a binucleate spore. We have examined the cytoskeletal involvement in nuclear pairings by immunofluorescence and drug disruption, using heterothallic and pseudohomothallic species, as well as species without defined mating type (homothallic). Nuclei of species with defined mating type have spindle pole bodies which react with chromatin stains; those of homothallic species do not. The reactivity is seen only in interphase, not during nuclear divisions; thus, the DNA concerned is nuclear and not organellar. From light and immunofluorescence microscopy, the DNA is located at the nuclear face of the spindle pole body (SPB). We suggest that the DNA-SPB association may be involved in the recognition of self and nonself between nuclei of opposite mating types. Nuclei which cooperate in cell formation during ascus development or sporulation are placed in close proximity by the arrangement of spindles during the division preceeding cell formation; after division, each nuclear pair remains linked by intertwined microtubule asters. Nuclear pairs must migrate before binucleate spore formation. Drug disruptions established that actin-myosin interaction was the most important cytoskeletal factor in normal spore production. The ascomycete SPB shows unexpected flexibility in form and location during development. Prior to sporulation the outer plaque shows extensive modification in size and orientation. The modified portion detaches from the nucleus and acts as a cortical microtubule organizing center, while the rest of the spindle pole body remains at the nucleus.