A homologue of the yeast HOP1 gene is inactivated in the Arabidopsis meiotic mutant asy1

Synapsis of homologous chromosomes is a key event in meiosis as it is essential for normal chromosome segregation and is implicated in the regulation of crossover frequency. We have previously reported the identification and cytological characterisation of a T-DNA-tagged asynaptic mutant of Arabidopsis thaliana. We have demonstrated that this mutant, asy1, is defective in meiosis in both males and females. Cloning and nucleotide sequencing of the ASY1 gene has revealed that it encodes a polypeptide of 596 amino acids that exhibits similarity to the HOP1 gene of Saccharomyces cerevisiae, which is known to encode a protein essential for synaptonemal complex assembly and normal synapsis. Expression studies indicate that, in common with a number of other Arabidopsis meiotic genes, ASY1 exhibits low-level expression in a range of plant tissues. Southern analysis coupled with database searching has resulted in the identification of an ASY1 homologue, ASY2. Although asy1 exhibits a strong asynaptic phenotype, a residual low level of synapsis indicates that ASY1 and ASY2 may exhibit a low degree of functional redundancy. Received: 22 September 1999; in revised form: 18 October 1999 / Accepted: 18 October 1999     

A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast.

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.     

The 14-kDa dynein light chain-family protein Dlc1 is required for regular oscillatory nuclear movement and efficient recombination during meiotic prophase in fission yeast

A Schizosaccharomyces pombe spindle pole body (SPB) protein interacts in a two-hybrid system with Dlc1, which belongs to the 14-kDa Tctex-1 dynein light chain family. Green fluorescent protein-tagged Dlc1 accumulated at the SPB throughout the life cycle. During meiotic prophase, Dlc1 was present along astral microtubules and microtubule-anchoring sites on the cell cortex, reminiscent of the cytoplasmic dynein heavy chain Dhc1. In a dlc1-null mutant, Dhc1-dependent nuclear movement in meiotic prophase became irregular in its duration and direction. Dhc1 protein was displaced from the cortex anchors and the formation of microtubule bundle(s) that guide nuclear movement was impaired in the mutant. Meiotic recombination in the dlc1 mutant was reduced to levels similar to that in the dhc1 mutant. Dlc1 and Dhc1 also have roles in karyogamy and rDNA relocation during the sexual phase. Strains mutated in both the dlc1 and dhc1 loci displayed more severe defects in recombination, karyogamy, and sporulation than in either single mutant alone, suggesting that Dlc1 is involved in nuclear events that are independent of Dhc1. S. pombe contains a homolog of the 8-kDa dynein light chain, Dlc2. This class of dynein light chain, however, is not essential in either the vegetative or sexual phases.     

The Study of Joint Inheritance of Mutations Impairing the Structure of Meiotic Chromosomes in Rye Secale cereale L.

Genetic analysis has demonstrated that meiotic mutations mei8 (irregular condensation and fragmentation of meiotic chromosomes) andmei10 (chromosome overcompaction) are nonallelic. Mutation mei10 exhibits digenic inheritance (with a segregation ratio of 13 : 3) in the combinations of crosses studied. It is assumed that the phenotypic expression of mutation mei10is suppressed by the effect of recessive genelch1 or lch2 (long chromosomes), both of which have been revealed in one of the parental lines (Mc10). These genes determine weak condensation of meiotic chromosomes. In double mutantsmei8 mei10, the mutations are expressed independently of each other. Gene mei10 is linked with gene mei8(r^ = 36.8 ± 5.38%); genes lch1 and lch2 are not linked either with them or with each other. Taking into account the data on the linkage between genes mei10and sy10 and between mei8andsy10, the order of genes in the linkage group is shown to be the following: mei8–sy10–mei10.     

Mutation in mouse hei10, an e3 ubiquitin ligase, disrupts meiotic crossing over

Crossing over during meiotic prophase I is required for sexual reproduction in mice and contributes to genome-wide genetic diversity. Here we report on the characterization of an N-ethyl-N-nitrosourea-induced, recessive allele called mei4, which causes sterility in both sexes owing to meiotic defects. In mutant spermatocytes, chromosomes fail to congress properly at the metaphase plate, leading to arrest and apoptosis before the first meiotic division. Mutant oocytes have a similar chromosomal phenotype but in vitro can undergo meiotic divisions and fertilization before arresting. During late meiotic prophase in mei4 mutant males, absence of cyclin dependent kinase 2 and mismatch repair protein association from chromosome cores is correlated with the premature separation of bivalents at diplonema owing to lack of chiasmata. We have identified the causative mutation, a transversion in the 5′ splice donor site of exon 1 in the mouse ortholog of Human Enhancer of Invasion 10 (Hei10; also known as Gm288 in mouse and CCNB1IP1 in human), a putative B-type cyclin E3 ubiquitin ligase. Importantly, orthologs of Hei10 are found exclusively in deuterostomes and not in more ancestral protostomes such as yeast, worms, or flies. The cloning and characterization of the mei4 allele of Hei10 demonstrates a novel link between cell cycle regulation and mismatch repair during prophase I.     

Dominant enhancer effect of the meiotic mei4 mutant on recombination frequencies restricted to linkage group VI in Podospora anserina.

Summary A mutant which increases second division segregation (SDS) frequency of locus 110 (linkage group VI) was isolated. It was called mei4 because of its meiotic deficiency. The present paper deals with its effect on meiotic recombination when heterozygous. mei4 then only acts on linkage group VI. The SDS frequencies were increased for all markers used, except locus 5 located very close to the centromere. This quasi general enhancement results exclusively in an enlargement of map distance on linkage group VI's proximal part. Crosses involving three mutant genes allowed to check that the distances on the distal part were constant. This is due to a real lack of crossover frequency modification in this region and not to a change of chiasma interference. Among the seven linkage groups of Podospora anserina, group VI exhibits several other particularities concerning meiotic recombination, especially a lower positive chiasma interference and a more regular crossover distribution, suggesting a particular recombination regulation.Laboratoire associé n⁰ 86 du Centre National de la Recherche Scientifique     

Homolog pairing and meiotic progression in Coprinus cinereus

We have used fluorescence in situ hybridization to examine homolog pairing during the synchronous meiosis of the basidiomycete Coprinus cinereus. Using spread preparations of meiotic nuclei, we confirmed previous studies that showed that at 6 h post-karyogamy essentially all meiotic nuclei are in pachytene. We found that homolog pairing occurs rapidly after karyogamy, that a 1 Mb chromosome does not associate more quickly than a 2.5 Mb chromosome, and that interstitial, single-copy sites can associate stably prior to nucleolar fusion. Analysis of two probes for the same pair of homologs revealed that by 4 h after karyogamy each chromosome examined was at least partially paired in all meiotic cells. In addition, these studies showed that chromatin condensation increases after pairing and that chromatin shows stable compaction at pachytene. Received: 4 January 1999; in revised form: 22 June 1999 / Accepted: 20 July 1999     

Pathway analysis of radiation-sensitive meiotic mutants ofCoprinus cinereus

We have isolated 37 radiation-sensitive mutants of the basidiomyceteCoprinus cinereus. Each mutation is recessive, and the collection defines at least ten complementation groups for survival of gamma irradiation. Four complementation groups define the genesrad3, rad9, rad11 andrad12, which are required both for survival of gamma irradiation and for meiosis. Mutants in each of these four groups fail to complete meiosis and produce mushrooms with greatly reduced numbers of viable spores. Propidium iodide staining of meiotic nuclei showed a characteristic terminal appearance for each mutant: few cells of any of the meiotic mutants progress beyond prophase I, and both condensation and fragmentation or dispersal of meiotic chromatin are frequently observed. Scanning electron micrographs showed that the meiotic mutants make varying numbers (0–6) of basidiospore initials and that few of these initials develop into mature spores. When initials are present they are always symmetrically arrayed on the basidium, regardless of initial number. In quantitative measurements of gamma ray sensitivity, double mutants of every tested combination ofrad3, rad9, rad11 andrad12 consistently showed the same gamma ray sensitivity as the more sensitive single mutant parent of the cross. Therefore, these four genes are in the same pathway for the repair of gamma radiation damage, and this pathway also represents one or more functions essential for meiosis.     

Distribution and evolution of mobile elements in the virilis species group of Drosophila

The distributions of Penelope and Ulysses, two transposable elements that can induce hybrid dysgenesis, were studied in several species groups of Drosophila. No significant hybridization to Penelope and Ulysses probes was detected by Southern blot analyses of species outside the virilis group. In contrast, both element families have had a long residence in all species of the virilis species group, as indicated by their strong presence in the heterochromatic chromocenter. Except for D. kanekoi, D. lummei, and some strains of D. virilis, species of the group carry full-sized, and at least potentially functional, copies of both element families. Consistent with the occurrence of recent transposition, Penelope and Ulysses elements are located at different chromosomal sites in different geographical strains of the same species. A total of 79 Penelope and 47 Ulysses euchromatic insertion sites were localized to chromosomal subsections in species of the virilis group. Highly significant deviations from independence of the distributions of Penelope and Ulysses and previously established inversion breakpoints were documented, suggesting that these transposable elements may have played an important role in genomic reorganization and evolution of the virilis species group, which is especially rich in karyotypic variation. Received: 13 April 1999; in revised form: 20 July 1999 / Accepted: 27 July 1999     

Characterization of aLycopersicon esculentum xSolanum lycopersicoides somatic hybrid lacking a glutamate oxaloacetate transaminase isozyme

Morphological, cytological, isozyme and chloroplast DNA analyses were used to determine possible mechanism(s) for the loss of glutamate oxaloacetate transaminase-4 (GOT-4) isozyme activity in a somatic hybrid. Plant 204-1, derived by cell fusion between tomato (Lycopersicon esculentum) andSolanum lycopersicoides, was characterized for bothGot-4 and acid phosphatase-2 (Aps-2), two isozyme loci which are closely linked (recombination 2.5 cM). This hybrid was determined to be chimeric for bothGot-4 andAps-2. TheS. lycopersicoides plant used to provide cells for the fusion was determined to be heterozygous for bothGot-4 andAps-2. Only oneS. lycopersicoides allelic form ofAps-2 andGot-4 was found in plant 204-1. This observation indicated that either the alternative copy of theS. lycopersicoides chromosome region encodingGot-4 andAps-2 is deleted or the entire chromosome is absent. Plant 204-1 was cytologically determined to be aneuploid with approximately 62 chromosomes. Sixty-two somatic hybrids of separate callus origin were analysed for GOT-4 and a high proportion (27%) lacked theS. lycopersicoides form ofGot-4. The loss of this allele and the linkedAps allele most likely occurred in the suspension culture ofS. lycopersicoides used to provide cells for fusion.     

mei-41 is required for precocious anaphase in Drosophila females

This paper reports on a new role for mei-41 in cell cycle control during meiosis. This function is revealed by the requirement of mei-41 for the precocious anaphase observed in crossover-defective mutants. Normally in Drosophila oocytes, tension on the meiotic spindle causes a metaphase I arrest. This tension results because crossovers, and the resulting chiasmata, hold homologs together that are being pulled by kinetochore microtobules toward opposite spindle poles. In the absence of tension, such as in a recombination-defective mutant, metaphase arrest is not observed and meiosis proceeds through the two divisions. Here we show that in some recombination-defective mutants, the precocious anaphase requires the mei-41 gene product. For example, metaphase arrest is not observed in mei-218 mutants because of the severe reduction in crossing over. In mei-41 mei-218 double mutants, however, metaphase arrest was restored. The effect of mei-41 is dependent on double-strand break formation. Thus, in mutants that fail to initiate meiotic recombination the absence of mei-41 has no effect. Received: 15 October 1999; in revised form: 9 December 1999 / Accepted: 13 December 1999     

The effect of spo0 mutations on the expression of spo0A- and spo0F-lacZ fusions

Summary We have constructedspo0A-lacZ andspo0F-lacZ fusions with a temperate phage vector and have investigated howspo0 gene products are involved in the expression of each of these genes. The expression ofspo0A-lacZ andspo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo⁺ cells. This stimulation ofspo0A-lacZ was impaired by mutations in thespo0B, D, E, F orH genes but was not affected by mutations in thespo0J orK genes. Similar results were obtained with thespo0F-lacZ fusion. The effect of thespo0A mutation onspo0A-lacZ expression was characteristic: thespo0A-directed β-galactosidase activity found during vegetative growth was significantly enhanced in thespo0A mutant. This result suggests thatspo0A gene expression is autoregulated being repressed by its own gene product. Another remarkable observation was the effect of thesof-1 mutation, which is known to be aspo0A allele; it suppressed the sporulation deficiency ofspo0B, spo0D andspo0F mutants. Thespo0A-lacZ stimulation, which is impaired by any one of thesespo0 mutations, was restored by the additionalsof-1 mutation.     

Pathway analysis of radiation-sensitive meiotic mutants ofCoprinus cinereus

We have isolated 37 radiation-sensitive mutants of the basidiomyceteCoprinus cinereus. Each mutation is recessive, and the collection defines at least ten complementation groups for survival of gamma irradiation. Four complementation groups define the genesrad3, rad9, rad11 andrad12, which are required both for survival of gamma irradiation and for meiosis. Mutants in each of these four groups fail to complete meiosis and produce mushrooms with greatly reduced numbers of viable spores. Propidium iodide staining of meiotic nuclei showed a characteristic terminal appearance for each mutant: few cells of any of the meiotic mutants progress beyond prophase I, and both condensation and fragmentation or dispersal of meiotic chromatin are frequently observed. Scanning electron micrographs showed that the meiotic mutants make varying numbers (0–6) of basidiospore initials and that few of these initials develop into mature spores. When initials are present they are always symmetrically arrayed on the basidium, regardless of initial number. In quantitative measurements of gamma ray sensitivity, double mutants of every tested combination ofrad3, rad9, rad11 andrad12 consistently showed the same gamma ray sensitivity as the more sensitive single mutant parent of the cross. Therefore, these four genes are in the same pathway for the repair of gamma radiation damage, and this pathway also represents one or more functions essential for meiosis.     

The genetic control of sporulation in Saccharomyces. II. Dominance and complementation of mutants of meiosis and spore formation

Summary Heat-sensitive sporulation-deficient (spo) mutants ofS. cerevisiae may be either dominant or recessive. The number of loci which can mutate to thespo phenotype has been estimated to be 48±27 from complementation studies. Comparison of the wild type and mutants by light microscopy after exposure to sporulation medium at the restrictive temperature and Giemsa staining has shown that mutant populations can not complete the meiotic nuclear divisions.Supported by NSF grants GB-8564 and GB-27688, and the Wallace C. and Clara Abbott Memorial Fund from the University of Chicago.     

Absence of SUN1 and SUN2 proteins in Arabidopsis thaliana leads to a delay in meiotic progression and defects in synapsis and recombination

The movement of chromosomes during meiosis involves location of their telomeres at the inner surface of the nuclear envelope. Sad1/UNC‐84 (SUN) domain proteins are inner nuclear envelope proteins that are part of complexes linking cytoskeletal elements with the nucleoskeleton, connecting telomeres to the force‐generating mechanism in the cytoplasm. These proteins play a conserved role in chromosome dynamics in eukaryotes. Homologues of SUN domain proteins have been identified in several plant species. In Arabidopsis thaliana, two proteins that interact with each other, named AtSUN1 and AtSUN2, have been identified. Immunolocalization using antibodies against AtSUN1 and AtSUN2 proteins revealed that they were associated with the nuclear envelope during meiotic prophase I. Analysis of the double mutant Atsun1‐1 Atsun2‐2 has revealed severe meiotic defects, namely a delay in the progression of meiosis, absence of full synapsis, the presence of unresolved interlock‐like structures, and a reduction in the mean cell chiasma frequency. We propose that in Arabidopsis thaliana, overlapping functions of SUN1 and SUN2 ensure normal meiotic recombination and synapsis.     

An Arabidopsis homologue of the Drosophila meiotic gene Pelota

We have identified a plant homologue of the Drosophila meiotic gene Pelota in Arabidopsis thaliana (AtPelota1). This gene maps to chromosome 4 of Ara- bidopsis and is one of two Pelota homologues present in this plant. When the expression pattern of AtPelota1 was examined it was found to be expressed at similar levels in all plant tissues tested (whole plant, bud, stem, leaf and root). This expression pattern corresponds to that seen for some other Arabidopsis meiotic genes and their homologues. A search of the databases reveal that the AtPelota gene family is widespread with homologues present in higher and lower eukaryotes and archaebacteria, but not eubacteria. Received: 13 December 1999 / Accepted: 27 December 1999     

Ploidy reduction usingp-Fluorophenylalanine of fusion products ofSaccharomyces cerevisiae

Fusion of yeast protoplasts was induced in the presence of polyethylene glycol and Ca++ ions. Two auxotrophic complementingSaccharomyces cerevisiae mutant strains were used in fusion experiments. One diploid and several polyploid fusion products were selected by complementation in minimal medium. The assessment of ploidy has been based on the DNA content of the parental cells and fusion products, assayed with the diphenylamine method. Treating the fusion product cells withp-Fluorophenylalanine (p-FPA), parentalhis andleu markers could not be recovered. Instead, a strong reduction of polyploid fusion product cell DNA content was evident. The analysis of meiotic products after hybridizing one fusion product with a prototrophicSaccharomyces cerevisiae standard strain led to the recovery of thehis parental marker. Preliminary evidence thatp-Fluorophenylalanine could be used as a diploidizing agent towards polyploid strains ofSaccharomyces cerevisiae is reported.