An mre11 mutant of Coprinus cinereus has defects in meiotic chromosome pairing, condensation and synapsis

The rad11 gene of the basidiomycete Coprinus cinereus is required for the completion of meiosis and for survival after gamma irradiation. We have cloned the rad11 gene and shown that it is a homolog of MRE11, a gene required for meiosis and DNA repair in numerous organisms. The expression of C. cinereus mre11 is induced during prophase I of meiosis and following gamma irradiation. The gene encodes a predicted polypeptide of 731 amino acids, and the mre11-1 (rad11-1) mutation is a single base pair change that results in a stop codon after amino acid 315. The mre11-1 mutant shows enhanced sensitivity to ionizing radiation, but no enhanced sensitivity to UV radiation. It shows a delay in fruitbody formation and a reduction in the number of mushrooms formed per dikaryon. The mre11-1 mutant also has several meiotic defects. Pachytene chromatin condensation is disrupted, and although some meiotic cells appear to achieve metaphase I condensation, no further meiotic progression is observed. The mre11-1 mutant also fails to undergo proper chromosome synapsis; neither axial elements nor mature synaptonemal complexes are complete. Finally, meiotic homolog pairing is reduced in the mre11-1 mutant. Thus, in C. cinereus, Mre11 is required for meiotic DNA metabolism.     

White-cap mutants and meiotic apoptosis in the basidiomycete Coprinus cinereus

Among many white-cap mutants of Coprinus cinereus, four distinct classes have been identified cytologically. Mutants of one class progress through meiosis normally but fail to sporulate; the defect is post-meiotic and it triggers apoptosis in the tetrad stage. Mutants of the other three classes have defects in meiotic prophase and these are: (1) those that assemble synaptonemal complexes (SCs) normally; (2) those that assemble axial elements (AEs) but not SCs; and (3) those that assemble neither AEs nor SCs even though the chromosomes are condensed and also paired. All three meiotic mutant classes arrest at meiotic metaphase I and the arrest triggers meiosis-specific apoptosis showing characteristic chromatin condensation, DNA fragmentation as shown by the TUNEL assay, cytoplasmic shrinkage, and finally total DNA degradation. Apoptosis is very cell-type specific; it occurs only in the basidia while the neighboring somatic cells are perfectly healthy and the mushroom continues to develop and mature with very few basidiospores produced. The meiotic apoptosis in C. cinereus is under strict cell cycle control rather than at any time after defect; apoptosis is triggered only after entry to meiotic metaphase. It is intriguing to note that C. cinereus has two checkpoints for arrest and entry to apoptosis: one is meiotic at the metaphase I spindle checkpoint regardless of the time of defects, and one is post-meiotic at the tetrad stage. This is in striking contrast to multiple checkpoint arrests and entries to meiotic apoptosis found in the mouse.     

The control of meiosis progression in the fungus Coprinus cinereus by light/dark cycles

Meiosis progression in Coprinus cinereus is controlled by light/dark cycles. Light is essential to propel basidia into karyogamy and light intensity determines the timing of meiotic events. The higher the light intensities, the faster the fruiting bodies enter karyogamy. The critical period when light has this influence is between 16 and 6 h before karyogamy. The control is highly stage specific. A 3-h dark period is essential for a Java dikaryon and the Japanese A(mut)B(mut) homokaryon to enter meiotic metaphase; without it the fruit body is permanently arrested at diffused diplotene. This arrest is light intensity-dependent (>20 hlx) and temperature-dependent (e.g., 27 degrees C). The placement of the dark period is very stage specific; it has no effect when placed before karyogamy stage. A dikaryon of London origin is light blind and able to complete meiosis under continuous high light regime. Fruiting bodies arrested under a continuous high light can be rescued by a 3-h dark treatment, but there is always an 8-h lag time to enter meiotic metaphase. It is possible that the dark effect signals cellular processes leading to division events. Cytological studies of arrested fruiting bodies showed that chromosomes are normal in meiotic prophase through pachytene and diplotene, but are unable to undergo chromosome condensation. Genetic crosses between a monokaryon of Java stock J6;5.4 and a monokaryon BL55 or H5 of London stock showed that light-blindness is dominant, and is controlled by a single Mendelian gene.     

Chromosome dynamics in rad12 mutants of Coprinus cinereus

We have characterized the phenotypes of three rad12 mutants of the basidiomycete Coprinus cinereus, which were isolated on the basis of sensitivity to ionizing radiation. Electron microscopic studies of meiotic nuclear spreads showed that all three rad12 mutants are defective in chromosomal synapsis. For rad12-1 and rad12-4, very limited assembly of the synaptonemal complex occurs. The phenotype of rad12-15 is less severe and longer stretches of synapsed chromosomes are formed. However, for all three alleles mutant nuclei arrest in a diffuse state with little synaptonemal complex structure. Observations made of spreads of acridine orange-stained meiotic nuclei correlated with the electron microscopic data. In rad12 strains, chromosomes condense but do not pair, and they later arrest in a decondensed state; very few rad12 cells enter metaphase I. Homozygous dikaryons of rad12 mutants produce fruiting bodies with significantly fewer basidiospores than are found in wild-type dikaryons. The viability of these spores is greatly reduced: all spores produced by rad12-1 and rad12-4 mushrooms fail to germinate, while only 16% of rad12-15 spores are viable. Recombination within the tract of the ribosomal RNA gene repeats was not significantly different in the mutants when compared with a wild-type congenic control. Quantitative measurements of oidial survival indicate that all three rad12 alleles are sensitive to gamma radiation but insensitive to UV radiation relative to wild-type strains.     

Homologous pairing is reduced but not abolished in asynaptic mutants of yeast

In situ hybridization was used to examine chromosome behavior at meiotic prophase in the rad50S, hop1, rad50, and spo11 mutants of Saccharomyces cerevisiae, which are defective in chromosome synapsis and meiotic recombination. Painting of chromosomes I and III revealed that chromosome condensation and pairing are reduced in these mutants. However, there is some residual pairing in meiosis, suggesting that homologue recognition is independent of synaptonemal complex formation and recombination. Association of homologues was observed in the rad50, rad50S, and spo11 mutants, which are defective in the formation or processing of meiotic double-strand breaks. This indicates that double- strand breaks are not an essential component of the meiotic homology searching mechanism or that there exist additional or alternative mechanisms for locating homologues.     

Genetic collection of meiotic mutants of rye Secale cereale L

Genetic collection of meiotic mutants of winter rye Secale cereale L. (2n = 14) was created. Mutations were detected in inbred F2 generations after self-fertilization of the F1 hybrids, obtained by individual crossing of rye plants (cultivar Vyatka) or weedy rye with plants from autofertile lines. The mutations cause partial or complete plant sterility and are maintained in collection in a heterozygous state. Genetic analysis accompanied by cytogenetic study of meiosis has revealed six mutation types. (1) Nonallelic asynaptic mutations sy1 and sy9 caused the formation of only axial chromosome elements in prophase and anaphase. The synaptonemal complexes (SCs) were absent, the formation of the chromosome "bouquet" was impaired, and all chromosomes were univalent in meiotic metaphase I in 96% (sy1) and 67% (sy2) of cells. (2) Weak asynaptic mutation sy3, which hindered complete termination of synapsis in prophase II. Subterminal asynaptic segments were always observed in the SC, and at least one pair of univalents was present in metaphase I, but the number of cells with univalents did not exceed 2%. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19, which caused partially nonhomologous synapsis: change in pairing partners and fold-back chromosome synapsis in prophase I. In metaphase I, the number of univalents varied and multivalents were observed. (4) Mutation mei6, which causes the formation of ultrastructural protrusions on the lateral SC elements, gaps and branching of these elements. (5) Allelic mutations mei8 and mei10, which caused irregular chromatin condensation along chromosomes in prophase I, sticking and fragmentation of chromosomes in metaphase I. (6) Allelic mutations mei5 and mei10, which caused chromosome hypercondensation, defects of the division spindle formation, and random arrest of cells at different meiotic stages. However, these mutations did not affect the formation of microspore envelopes even around the cells, whose development was blocked at prophase I. Analysis of cytological pictures of meiosis in double rye mutants reveled epistatic interaction in the mutation series sy9 > sy1 > sy3 > sy19, which reflects the order of switching these genes in the course of meiosis. The expression of genes sy2 and sy19 was shown to be controlled by modifier genes. Most meiotic mutations found in rye have analogs in other plant species.     

Production and Characterization of Radiation-Sensitive Meiotic Mutants of Coprinus Cinereus

We have isolated four gamma-ray-sensitive mutants of the basidiomycete Coprinus cinereus. When homozygous, two of these (rad 3-1 and rad 9-1) produce fruiting bodies with very few viable basidiospores, the products of meiosis in this organism. A less radiation-sensitive allele of RAD 3, rad 3-2, causes no apparent meiotic defect in homozygous strains. Quantitative measurements of oidial survival of rad 3-1; rad 9-1 double mutants compared to the single mutants indicated that rad 3-1 and rad 9-1 mutants are defective in the same DNA repair pathway. In the few viable basidiospores that are produced by these two strains, essentially normal levels of meiotic recombination can be detected. None of the mutants exhibits increased sensitivity to UV radiation. Cytological examination of meiotic chromosomes from mutant and wild-type fruiting bodies showed that rad 3-1 homozygous strains fail to condense and pair homologous chromosomes during prophase I. Although rad 9-1 strains are successful at chromosome pairing, meiosis is usually not completed in these mutants.     

Abnormal meiosis in bisporic strains of white button mushroom Agaricus bisporus (Lange) imbach

A formerly developed method of microspreading of mushroom basidial nuclei was applied to study meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The anomalous phenotypes in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After partial veil breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

Abnormal meiosis in bisporic mutants of white button mushroom Agaricus bisporus (Lange) Imbach

A formerly developed method of obtaining spread preparations of mushroom basidial nuclei was applied to study of meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The phenotypes of disruptions in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After indusium breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

Semisterile meiotic mutant <Emphasis Type="Italic">sy11</Emphasis> with heterologous chromosome synapsis in rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

A study was made of the expression and inheritance of the sy11 mutation, which alters homologous chromosome synapsis in meiotic prophase I of rye. The abnormal phenotype proved to be determined by a recessive allele of a single sy11 gene. Univalents and multivalents were observed in homozygotes for the mutant allele. Analysis of the synaptonemal complex revealed a combination of homologous and nonhomologous synapsis in the mutant. The nonhomologous synapsis frequency significantly decreased in the course of meiotic prophase I in the mutant. The number of chiasmata per bivalent in metaphase I was 1.1 ± 0.01 versus 1.8 ± 0.01 in wild-type plants, and the number of univalents was 2.7 ± 0.06 versus 0.5 ± 0.05 in wild-type plants. As a result, a broad range of abnormalities was observed at subsequent stages of meiosis and led to the formation of defective microspores. Mutant plants were semisterile.     

Meiotic chromosome missegregation during apyrene meiosis in the gypsy moth,Lymantria dispar,is preceded by an aberrant prophase I

The gypsy moth, Lymantria dispar, produces two structurally and genetically distinct types of spermatozoa. The eupyrene spermatozoa are genetically haploid and structurally typical. The apyrene spermatozoa are anucleate and structurally different from eupyrene spermatozoa. To understand further the events contributing to meiotic chromosome missegregation in apyrene spermatocytes, we examined the progression of meiosis in these cells with respect to their eupyrene counterparts. Chromosomal bouquet formation and fusion of nucleolar organizing regions are disrupted in apyrene nuclei. In addition, the chromatin of apyrene nuclei is prematurely and extremely condensed compared with that of eupyrene nuclei. An antibody to the conserved synaptonemal complex protein 3 (SCP3) labeled eupyrene pachytene chromosomes, but not apyrene pachytene chromosomes. In addition, apyrene meiotic spindles are missing a subset of microtubules, which likely include kinetochore microtubules. Because the condensation behavior of meiotic chromatin in apyrene spermatocytes deviates from that of eupyrene spermatocytes, we examined the appearance and distribution of the phosphorylated form of histone H3, but no significant differences in histone H3 phosphorylation were found between apyrene and eupyrene spermatocytes. We argue that because a pachytene checkpoint is not initiated in apyrene spermatocytes, this system may provide a way to understand better the underlying biochemical connections between pairing, recombination, synapsis, kinetochore assembly and segregation of chromosomes during meiosis in a higher eukaryote.     

Meiotic chromosome interactions in inbred autotetraploid rye (Secale cereale).

Electron microscopy of whole-mount surface-spread synaptonemal complex complements and conventional light microscopy of chromosomes at first metaphase of meiosis were used to compare the relative frequencies of pairing configurations at the two stages in inbred autotetraploid rye (Secale cereale L.). Statistical tests showed significantly fewer multivalents at first metaphase than expectations based on random initiation of synapsis at each telomeric site within each group of four homologues. Direct observations of synaptic behaviour of chromosomes showed that this deviation is due primarily to a preponderance of bivalents during zygotene and pachytene. It is also the result of a significant drop in multivalent frequency from meiotic prophase to metaphase I, which is attributable both to a lack of chiasmata with which to consolidate multivalents and inhibition of chiasma formation in synaptonemal complex segments of multivalents that are nonhomologous.     

Stage-specific damage to synaptonemal complexes and metaphase chromosomes induced by X rays in male mouse germ cells

Synaptonemal complexes reveal mutagen-induced effects in germ cell meiotic chromosomes. This study was aimed at characterizing relationships between damage to synaptonemal complexes and metaphase I chromosomes following radiation exposure at various stages of spermatogenesis. Male mice were irradiated with doses of 0, 2, or 4 Gy, and spermatocytes were harvested at times consistent with earlier exposures as spermatogonial stem cells, preleptotene cells (premeiotic DNA synthesis), or meiotic prophase cells. After stem-cell exposure, twice as many rearrangements were observed in synaptonemal complexes as in metaphase I chromosomes. Irradiation during premeiotic DNA synthesis resulted in dose-related increases in synaptonemal complex breakage and rearrangements (including novel forms) and in metaphase chromosomal aberrations. Following prophase exposure, various types and levels of damage to synaptonemal complexes and metaphase chromosomes were observed. Irradiation of zygotene cells led to high frequencies of chromosome multivalents in metaphase I without a correspondingly high level of damage in preceding prophase synaptonemal complexes. Thus irradiation of premeiotic and meiotic cells results in variable relationships between damage to synaptonemal complexes and metaphase chromosomes. Interpretations of these relationships are based upon what is known about both radiation clastogenesis and the structural/temporal relationships between synaptonemal complexes at prophase and chromosomes at metaphase I of meiosis.     

Synapsis with and without recombination in the male meiosis of the leaf-footed bug <Emphasis Type="Italic">Holhymenia rubiginosa</Emphasis> (Coreidae,Heteroptera)

In organisms with chiasmatic meiosis two different relationships have been described between crossing over and synapsis: in one group of organisms synapsis depends on the initiation of meiotic recombination while in the other group it is independent of this initiation. These patterns have been observed mainly in organisms where all meiotic bivalents in the set have similar behaviors. In some heteropteran insects a pair of chromosomes named m chromosomes is known to behave differently from autosomes regarding synapsis and recombination. Here we used immunodetection of a synaptonemal complex component and acid-fixed squashes to investigate the conduct of the small m chromosome pair during the male meiosis in the coreid bug Holhymenia rubiginosa. We found that the m chromosomes form a synaptonemal complex during pachytene, but they are not attached by a chiasma in diakinesis. On the other hand, the autosomal bivalents synapse and recombine regularly. The co-existence of these variant chromosome behaviors during meiosis I add further evidence to the absence of unique patterns regarding the interdependence of synapsis and recombination.     

A Role for SUMO in meiotic chromosome synapsis

During meiotic prophase, homologous chromosomes engage in a complex series of interactions that ensure their proper segregation at meiosis I. A central player in these interactions is the synaptonemal complex (SC), a proteinaceous structure elaborated along the lengths of paired homologs. In mutants that fail to make SC, crossing over is decreased, and chromosomes frequently fail to recombine; consequently, many meiotic products are inviable because of aneuploidy. Here, we have investigated the role of the small ubiquitin-like protein modifier (SUMO) in SC formation during meiosis in budding yeast. We show that SUMO localizes specifically to synapsed regions of meiotic chromosomes and that this localization depends on Zip1, a major building block of the SC. A non-null allele of the UBC9 gene, which encodes the SUMO-conjugating enzyme, impairs Zip1 polymerization along chromosomes. The Ubc9 protein localizes to meiotic chromosomes, coincident with SUMO staining. In the zip1 mutant, SUMO localizes to discrete foci on chromosomes. These foci coincide with axial associations, where proteins involved in synapsis initiation are located. Our data suggest a model in which SUMO modification of chromosomal proteins promotes polymerization of Zip1 along chromosomes. The ubc9 mutant phenotype provides the first evidence for a cause-and-effect relationship between sumoylation and synapsis.     

Molecular assembly of meiotic proteins Asy1 and Zyp1 and pairing promiscuity in rye (Secale cereale L.) and its synaptic mutant sy10

Assembly of two orthologous proteins associated with meiotic chromosome axes in Arabidopsis thaliana (Asy1 and Zyp1) was studied immunologically at meiotic prophase of meiosis of wild-type rye (Secale cereale) and its synaptic mutant sy10, using antibodies derived from A. thaliana. The temporal and spatial expression of the two proteins were similar in wild-type rye, but with one notable difference. Unlike A. thaliana, in which foci of the transverse filament protein Zyp1 appear to linearize commensurately with synapsis, linear tracts of Asy1 and Zyp1 protein form independently at leptotene and early zygotene of rye and coalign into triple structures resembling synaptonemal complexes (SCs) only at later stages of synapsis. The sy10 mutant used in this study also forms spatially separate linear tracts of Asy1 and Zyp1 proteins at leptotene and early zygotene, and these coalign but do not form regular triple structures at midprophase. Electron microscopy of spread axial elements reveals extensive asynapsis with some exchanges of pairing partners. Indiscriminate SCs support nonhomologous chiasma formation at metaphase I, as revealed by multi-color fluorescence in situ hybridization enabling reliable identification of all the chromosomes of the complement. Scrutiny of chiasmate associations of chromosomes at this stage revealed some specificity in the associations of homologous and nonhomologous chromosomes. Inferences about the nature of synapsis in this mutant were drawn from such observations.     

Multiple roles of Spo11 in meiotic chromosome behavior

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.     

Meiosis in Coprinus. VIII. A time-course study of the fusion and division of the spindle pole body during meiosis

The time-course study of meiosis in the fungus Coprinus cinereus (C. lagopus) by electron microscopy reveals that two monoglobular spindle pole bodies (SPB's) of prekaryogamy nuclei come together during karyogamy and are fused. The fusion SPB of postkaryogamy nucleus persists through zygotene and pachytene as evidenced by the presence of axial components and synaptonemal complexes. At early diplotene, the SPB divides. The divided SPB takes on a diglobular form, which grows in size to form two daughter SPB's. These separate and move to opposite poles at metaphase I.