Genetic analysis of two spored asci produced by the spo3 mutant of Saccharomyces

Summary Ascospores present in two-spored asci formed by spo3-1 diploids at a semipermissive temperature (30°C) represent random inclusion of haploid genomes into ascospores and exhibit normal viability, meiotic chromosome segregation and recombination. Genetic analysis and ultrastructural studies indicate that the function encoded by the spo3 locus is specifically required for the enclosure of the products of meiosis in prospore walls.Suppored by NSF grant GB-27688, the Wallace C. and Clara Abbott Memorial Fund from the University of Chicago, and Cancer Center Grant CCRC IIIB3.     

A meiosis-specific protein kinase, Ime2, is required for the correct timing of DNA replication and for spore formation in yeast meiosis

 In this report we study the regulation of premeiotic DNA synthesis in Saccharomyces cerevisiae. DNA replication was monitored by fluorescence-activated cell sorting analysis and by analyzing the pattern of expression of the DNA polymerase α-primase complex. Wild-type cells and cells lacking one of the two principal regulators of meiosis, Ime1 and Ime2, were compared. We show that premeiotic DNA synthesis does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. At late meiotic times, ime2Δ diploids exhibit an additional round of DNA synthesis. Furthermore, we show that in wild-type cells the B-subunit of DNA polymerase α is phosphorylated during premeiotic DNA synthesis, a phenomenon that has previously been reported for the mitotic cell cycle. Moreover, the catalytic subunit and the B-subunit of DNA polymerase α are specifically degraded during spore formation. Phosphorylation of the B-subunit does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. In addition, we show that Ime2 is not absolutely required for commitment to meiotic recombination, spindle formation and nuclear division, although it is required for spore formation. Received: 20 February 1996 / Accepted: 7 June 1996     

The Role of Radiation (rad) Genes in Meiotic Recombination in Yeast

In yeast, the functions controlled by radiation-repair genes RAD6, RAD50, RAD52 and RAD57 are essential for normal meiosis; diploids with lesions in these genes either fail to sporulate (rad6) or sporulate but produce inviable spores (rad50, 52, 57). Since RAD genes may control aspects of DNA metabolism, we attempted to define more precisely the role of each gene in meiosis, especially with regard to possible roles in premeiotic DNA replication and recombination. We constructed diploids singly homozygous for each of the four rad mutations, heteroallelic at his1 and heterozygous for a recessive canavanine-resistance marker. Each strain was exposed to sporulation-inducing conditions and monitored for (1) completion of mitotic cell cycles, (2) cell viability, (3) utilization of acetate for mass increases, (4) premeiotic DNA synthesis, (5) intragenic recombination at his1, and (6) formation of viable haploid spores. Control strains heterozygous for the rad mutations completed mitosis, metabolized acetate, replicated their DNA, and showed typically high levels of gene conversion and viable-spore formation. The mutant diploids also completed mitosis, utilized acetate, and carried out premeiotic DNA replication. The mutants, however, showed little or no meiotic gene conversion. The rad50, 52 and 57 strains sporulated, but the spores were inviable. The rad6 strain did not sporulate. The rad50, 52 and 57 strains exhibited viability losses that coincided with the period of DNA synthesis, but not with later meiotic events; the rad6 strain did not lose viability. We propose that the normal functions specified by RAD50, 52 and 57 are not essential for either the initial or terminal steps in meiosis, but are required for successful recombination. The rad6 strain may be recombination-defective, or it may fail to progress past DNA replication in the overall sequence leading to formation and recovery of meiotic recombinants.     

Persistence of histone H2AX phosphorylation after meiotic chromosome synapsis and abnormal centromere cohesion in poly (ADP-ribose) polymerase (Parp-1) null oocytes

In spite of the impact of aneuploidy on human health little is known concerning the molecular mechanisms involved in the formation of structural or numerical chromosome abnormalities during meiosis. Here, we provide novel evidence indicating that lack of PARP-1 function during oogenesis predisposes the female gamete to genome instability. During prophase I of meiosis, a high proportion of Parp-1^(−/−) mouse oocytes exhibit a spectrum of meiotic defects including incomplete homologous chromosome synapsis or persistent histone H2AX phosphorylation in fully synapsed chromosomes at the late pachytene stage. Moreover, the X chromosome bivalent is also prone to exhibit persistent double strand DNA breaks (DSBs). In striking contrast, such defects were not detected in mutant pachytene spermatocytes. In fully-grown wild type oocytes at the germinal vesicle stage, PARP-1 protein associates with nuclear speckles and upon meiotic resumption, undergoes a striking re-localization towards spindle poles as well as pericentric heterochromatin domains at the metaphase II stage. Notably, a high proportion of in vivo matured Parp-1^(−/−) oocytes show lack of recruitment of the kinetochore-associated protein BUB3 to centromeric domains and fail to maintain metaphase II arrest. Defects in chromatin modifications in the form of persistent histone H2AX phosphorylation during prophase I of meiosis and deficient sister chromatid cohesion during metaphase II predispose mutant oocytes to premature anaphase II onset upon removal from the oviductal environment. Our results indicate that PARP-1 plays a critical role in the maintenance of chromosome stability at key stages of meiosis in the female germ line. Moreover, in the metaphase II stage oocyte PARP-1 is required for the regulation of centromere structure and function through a mechanism that involves the recruitment of BUB3 protein to centromeric domains.     

Effects of the RAD52 Gene on Recombination in SACCHAROMYCES CEREVISIAE

Effects of the rad52 mutation in Saccharomyces cerevisiae on meiotic, γ-ray-induced, UV-induced and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Both intra and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the his1–1/his1–315 and trp5–2/trp5–48 heteroalleles. Gene-centromere recombination also was not observed in rad52/rad52 diploids. No γ-ray- or UV-induced intragenic mitotic recombination is seen in rad52/rad52 diploids. The rate of spontaneous mitotic recombination is lowered five-fold at the his1–1/his1–315 and leu1–c/leu1–12 heteroalleles. Spontaneous reversion rates of both his1–1 and his1–315 were elevated 10 to 20 fold in rad52/rad52 diploids.—The RAD52 gene function is required for spontaneous mitotic recombination, UV- and γ-ray-induced mitotic recombination and meiotic recombination.     

Genetics of CHLAMYDOMONAS REINHARDTII Diploids I. Isolation and Characterization and Meiotic Segregation Pattern of a Homozygous Diploid

A strain of Chlamydomonas reinhardtii has been investigated which, when mated with known wild-types, produces very few viable germination products and transmits its Mendelian markers to more than half of those products. Cytogenetic observations, fluorometric measurements of DNA and genetic data all suggest that the strain, d mt^-ery-M3a sr-u-1 is a stable homozygous diploid. This strain has twice as many nuclear chromatin bodies at metaphase and twice as much DNA as its haploid progenitor, and the phenotypes of its meiotic progeny are consistent with predictions based on triploid meiosis. Data from crosses involving d mt^-ery-M3a sr-u-1 and from crosses involving hybrid diploids indicate that the frequency of second division segregation increases in triploid zygotes and that mitotic segregation following triploid meiosis is a frequent event which may more often result from mitotic recombination than from chromosome loss.     

Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium

Background

Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes.

Results

Analysis of the cell cycle stages revealed that 91.2 ± 0.2% of confluent cells were at the G0/G1 phase and 54.1 ± 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors.

Conclusions

Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells.     

Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.     

Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II

In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes.     

Structure of the Ring Cleavage Product of 1-Hydroxy-2-Naphthoate, an Intermediate of the Phenanthrene-Degradative Pathway of Nocardioides sp. Strain KP7

1-Hydroxy-2-naphthoate (compound I) is a metabolite of the phenanthrene-degradative pathway in Nocardioides sp. strain KP7. This singly hydroxylated aromatic compound is cleaved by 1-hydroxy-2-naphthoate dioxygenase. In this study, the structure of the ring cleavage product generated by the action of homogeneous 1-hydroxy-2-naphthoate dioxygenase was determined upon separation by high-performance liquid chromatography at pH 2.5 by using nuclear magnetic resonance (NMR) and mass spectroscopic techniques. The ring cleavage product at this pH existed in equilibrium between two forms, 2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound III) and 2,2-dihydroxy-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound IV). After the pH of the solution was raised to 7.5, the structure of the major species became (E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II; common name, trans-2′-carboxybenzalpyruvate), which was in equilibrium with compound III. Direct monitoring of the enzymatic formation of the ring cleavage product by ¹H-NMR in a deuterated potassium phosphate buffer (pH 7.5) detected only compound II as a product, and the proton on carbon 3 of compound II was not exchanged with deuterium. Thus, compound II is likely to be the first stable product of dioxygenation of 1-hydroxy-2-naphthoate.     

TDM1 Regulation Determines the Number of Meiotic Divisions

Cell cycle control must be modified at meiosis to allow two divisions to follow a single round of DNA replication, resulting in ploidy reduction. The mechanisms that ensure meiosis termination at the end of the second and not at the end of first division are poorly understood. We show here that Arabidopsis thaliana TDM1, which has been previously shown to be essential for meiotic termination, interacts directly with the Anaphase-Promoting Complex. Further, mutations in TDM1 in a conserved putative Cyclin-Dependant Kinase (CDK) phosphorylation site (T16-P17) dominantly provoked premature meiosis termination after the first division, and the production of diploid spores and gametes. The CDKA;1-CYCA1.2/TAM complex, which is required to prevent premature meiotic exit, phosphorylated TDM1 at T16 in vitro. Finally, while CYCA1;2/TAM was previously shown to be expressed only at meiosis I, TDM1 is present throughout meiosis. These data, together with epistasis analysis, lead us to propose that TDM1 is an APC/C component whose function is to ensure meiosis termination at the end of meiosis II, and whose activity is inhibited at meiosis I by CDKA;1-TAM-mediated phosphorylation to prevent premature meiotic exit. This provides a molecular mechanism for the differential decision of performing an additional round of division, or not, at the end of meiosis I and II, respectively.     

ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature

To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allele of Pat1 [Pat1(L95A), designated pat1-as2] that can be used to generate synchronous meiotic cultures at physiological temperature. In pat1-as2 meiosis, chromosomes segregate with higher fidelity, and spore viability is higher than in pat1-114 meiosis, although recombination is lower by a factor of 2–3 in these mutants than in starvation-induced pat1⁺ meiosis. Addition of the mat-Pc gene improved chromosome segregation and spore viability to nearly the level of starvation-induced meiosis. We conclude that pat1-as2 mat-Pc cells offer synchronous meiosis with most tested properties similar to those of wild-type meiosis.     

The CYCLIN-A CYCA1;2/TAM Is Required for the Meiosis I to Meiosis II Transition and Cooperates with OSD1 for the Prophase to First Meiotic Division Transition

Meiosis halves the chromosome number because its two divisions follow a single round of DNA replication. This process involves two cell transitions, the transition from prophase to the first meiotic division (meiosis I) and the unique meiosis I to meiosis II transition. We show here that the A-type cyclin CYCA1;2/TAM plays a major role in both transitions in Arabidopsis. A series of tam mutants failed to enter meiosis II and thus produced diploid spores and functional diploid gametes. These diploid gametes had a recombined genotype produced through the single meiosis I division. In addition, by combining the tam-2 mutation with AtSpo11-1 and Atrec8, we obtained plants producing diploid gametes through a mitotic-like division that were genetically identical to their parents. Thus tam alleles displayed phenotypes very similar to that of the previously described osd1 mutant. Combining tam and osd1 mutations leads to a failure in the prophase to meiosis I transition during male meiosis and to the production of tetraploid spores and gametes. This suggests that TAM and OSD1 are involved in the control of both meiotic transitions.     

The molecular features of chromosome pairing at meiosis: the polyploid challenge using wheat as a reference

During meiosis, chromosome numbers are halved, leading to haploid gametes, a process that is crucial for the maintenance of a stable genome through successive generations. The process for the accurate segregation of the homologues starts in pre-meiosis as each homologue is replicated and the respective products are held together as two sister chromatids via specific cohesion proteins. At the start of meiosis, each chromosome must recognise its homologue from amongst all the chromosomes present in the nucleus and then associate or pair with that homologue. This process of homologue recognition in meiosis is more complicated in polyploids because of the greater number of related chromosomes. Despite the presence of these related chromosomes, for polyploids such as wheat to produce viable gametes, they must behave as diploids during meiosis with only true homologues pairing. In this review, the relationship between the Ph1 cyclin-dependent kinase (CDK)-like genes in wheat and the CDK2 genes in mammals and their involvement in controlling this process at meiosis is examined.     

Pushing the (nuclear) envelope into meiosis

A recent study shows that a short isoform of a mammalian nuclear lamin is important for homologous chromosome interactions during meiotic prophase in mice.Meiosis is the specialized cell division process required for sexual reproduction. As cells enter meiotic prophase, a relatively long period preceding the two chromosome divisions, nuclei and chromosomes undergo remodeling to promote interactions between homologous chromosomes. Each chromosome must find and identify its unique partner within the volume of the nucleus, a process that obviously involves large-scale chromosome movements.Over 100 years ago, cytological analysis of meiotic cells revealed a unique chromosome configuration termed the meiotic ''bouquet'', in which chromosome ends seem to be attached to the nuclear periphery, frequently in a tight cluster. The presence of the bouquet was found to coincide with the stage during which homologous chromosomes undergo pairing and synapsis. This was the first indication that interactions between the chromosomes and the nuclear envelope might be important for meiotic pairing. More recent analysis in diverse model systems has revealed that the bouquet is a consequence of interactions between chromosomes and cytoskeletal elements - microtubules or actin cables - via a protein bridge that spans the nuclear envelope. A study recently published in PLOS Genetics [1] has shed further light on the role of the nuclear lamina in meiotic progression by studying the role of a meiosis-specific isoform of a nuclear lamin protein.In metazoans the nuclear envelope is fortified by the nuclear lamina, a meshwork of intermediate filament proteins (lamins) and associated proteins that underlies the inner nuclear membrane. The lamina confers structural rigidity to nuclei and also interacts with a wide variety of nucleoplasmic, transmembrane and chromosome-associated proteins. The composition of the lamina in metazoans shows tissue-specific variability and developmental regulation. Most differentiated mammalian cells express both A-type lamins (lamins A and C, which are generated by alternative splicing of the LMNA gene) and B-type lamins (encoded by two different genes), whereas some invertebrates express only a single lamin protein. Stem cells typically lack A-type lamins, which are also dispensable for early development in mice.Among the nuclear envelope components that interact with lamins are LINC (linker of nucleoskeleton and cytoskeleton) complexes. These versatile networks involve a pair of SUN/KASH proteins that bridge both membranes of the nuclear envelope. SUN domain proteins traverse the inner membrane, with their amino termini projecting into the nucleus and their SUN domains in the lumen between the two membranes. Their partners have membrane-spanning regions adjacent to their carboxy-terminal KASH domains, short peptides that bind to the SUN domains. Using a variety of interaction modules, LINC complexes create connections between nuclear structures such as the lamina or chromosomes and cytoskeletal elements such as actin filaments or microtubules. Throughout the eukaryotes, they have essential roles in diverse processes, including the positioning and migration of nuclei within cells and anchorage of centrosomes to the nuclear envelope. During meiosis, specific LINC complexes are recruited to interact with chromosomes through the expression of meiosis-specific proteins that bind to telomeres or, less frequently, to other specialized loci [2]. These connections, probably in conjunction with meiosis-specific modifications to the cytoskeleton and motor proteins, lead to large-scale chromosome motions that facilitate homologous chromosome pairing. These movements involve dramatic motion of the LINC proteins within the nuclear membrane, sometimes involving movements of up to several micrometers that occur within a few seconds [3]. This stands in sharp contrast to the behavior of some of the same protein complexes in somatic or premeiotic cells, in which they show highly constrained motion and minimal turnover [3].In the new PLOS Genetics study [1], groups led by Manfred Alsheimer and Ricardo Benavente, both of the University of Würzburg, have now engineered a disruption of an exon in the mouse LMNA gene that is specific to the meiotic isoform lamin C2 to generate C2-deficient mice (C2^-/- mice). These collaborators have previously provided important insights into the regulation and functions of cell-type specific lamin isoforms, particularly during meiosis. Using antibodies, they characterized the lamin isoforms present in rat spermatocytes [4]. Immunolocalization revealed that a truncated isoform of lamin C (lamin C2) was localized in a patchy pattern along the nuclear envelope, along with a short B-type lamin (lamin B3) [4]. Because these short isoforms lack domains implicated in interactions between lamin subunits, they and others proposed that these proteins might form a more flexible network. This idea was supported by experiments in which meiosis-specific lamin C2 was ectopically expressed in fibroblasts and found to be more mobile within the nuclear envelope than full-length lamin C [5]. Expression of lamin C2 also resulted in aberrant localization of Sun1 in these cells. The collaborators also demonstrated that spermatogenesis was disrupted in Lmna^-/- mice, although oocyte meiosis was not obviously perturbed [6]. Although defects in meiosis-specific processes were observed in the knockout mice, it was not possible to rule out an indirect effect of lamin depletion in somatic cells on meiosis in spermatocytes, prior to the new study.An important feature of the new research [1] is that the C2^-/- mice show normal expression of all other A-type lamins. The C2^-/- males recapitulate the meiotic failure seen in Lmna^-/- mice. Nevertheless, their chromosomes frequently fail to synapse and they engage in heterologous associations or show aberrant telomere-telomere interactions; all of these defects are rare in wild-type spermatocytes. As a result of extensive apoptosis and failure of sperm maturation, the males are completely infertile. However, females are fertile, despite some evidence for pairing defects in C2^-/- oocytes.These sex-specific differences in the effects of lamin C2 loss are somewhat surprising. They could in part reflect differential implementation of meiotic checkpoints, which cull defective spermatocytes more ruthlessly than oocytes [7]. However, analysis of homologous pairing and synapsis in the C2^-/- mutant mice also revealed more severe defects in males. Both male and female mice lacking Sun1 protein are completely sterile and show synaptic failure during meiotic prophase [8]. This suggests that LINC-mediated chromosome dynamics are essential for homolog interactions during meiosis in both sexes. The milder defects caused by loss of lamin C2 in both male and female meiosis suggest that it has a less direct role in mediating chromosome movement than Sun1. This is consistent with the idea that expression of short lamin isoforms during meiosis acts primarily to increase the mobility of proteins within the nuclear envelope, relative to somatic cells. It seems likely that the dynamics of pairing, synapsis and recombination differ dramatically between spermatocytes, which are produced continually during the adult life of the male, and oocytes, which undergo meiotic prophase during fetal development. Such differences might render male meiosis more sensitive to changes in nuclear envelope organization or dynamics.The modifications made to the mouse nuclear envelope during meiosis are likely to be conserved in concept, if not in detail, in other taxa. As mentioned above, the isoforms and expression patterns of lamin proteins have diverged rapidly among the metazoa, as have the structures and functions of LINC complexes. For example, amphibians lack lamin C (and lamin C2), suggesting that its meiotic role in mammals is a recent innovation. Furthermore, the mouse Sun1 protein has a C2H2 zinc finger lacking in primate orthologs, which might suggest that it has evolved a distinct way to connect with meiotic chromosomes. It is thus not currently clear which aspects of meiotic lamina remodeling in mice can be extrapolated to other species.In Caenorhabditis elegans, meiotic chromosome dynamics are probably mediated by post-translational modification of the amino-terminal (nucleoplasmic) domain of sun-1 [9]. It is not yet known how this modification contributes to the function of the meiotic LINC complex. Direct observation has indicated that the motion of LINC complexes within the nuclear envelope becomes much less constrained as cells enter meiosis [3]. Phosphorylation of sun-1 may weaken interactions between the LINC complexes and the lamina to increase their mobility within the nuclear envelope, and/or promote interactions between LINC complexes to create high load-bearing aggregates of these proteins necessary to drive chromosome movement. It is not currently known whether the lamina itself is modified in C. elegans meiotic nuclei, but it is easy to imagine that phosphorylation could also be used to tweak protein-protein interactions within the lamina to optimize its properties during meiosis and other specialized cellular processes. It is likely that metazoans have evolved a wide range of mechanisms to modify their nuclear envelopes to meet the special demands of meiotic prophase.Homologous chromosome pairing remains one of the most mysterious aspects of meiosis. This new work in mice [1] adds an important piece of the puzzle by illuminating how the nuclear lamina can be modified to facilitate meiotic chromosome dynamics. To understand this process will clearly require looking beyond the chromosomes, and even beyond the nucleus, to the cellular networks connected by LINC complexes.     

DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1’s primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.     

Cytological and morphology characteristics of natural microsporogenesis within Camellia oleifera

Camellia oleifera is believed to exhibit a complex intraspecific polyploidy phenomenon. Abnormal microsporogenesis can promote the formation of unreduced gametes in plants and lead to sexual polyploidy, so it is hypothesized that improper meiosis probably results in the formation of natural polyploidy in Camellia oleifera. In this study, based on the cytological observation of meiosis in pollen mother cells (PMCs), we found natural 2n pollen for the first time in Camellia oleifera, which may lead to the formation of natural polyploids by sexual polyploidization. Additionally, abnormal cytological behaviour during meiosis, including univalent chromosomes, extraequatorial chromosomes, early segregation, laggard chromosomes, chromosome stickiness, asynchronous meiosis and deviant cytokinesis (monad, dyads, triads), was observed, which could be the cause of 2n pollen formation. Moreover, we confirmed a relationship among the length–width ratio of flower buds, stylet length and microsporogenesis. This result suggested that we can immediately determine the microsporogenesis stages by phenotypic characteristics, which may be applicable to breeding advanced germplasm in Camellia oleifera.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01002-5.     

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.     

The behaviour of kinetochore microtubules during meiosis in the fungusSaprolegnia

Summary InSaprolegnia, kinetochore microtubules persist throughout the mitotic nuclear cycle but, whilst present at leptotene, they disappear coincidently with the formation of synaptonemal complexes at pachytene and reform at metaphase I. In some other fungi chromosomal segregation is random in meiosis and non-random in mitosis. The attachment of chromosomes to persistent kinetochore microtubules in mitosis, but not meiosis, inSaprolegnia provides a plausible explanation for such behaviour. At metaphase I each bivalent is connected to the spindle by 2 laterally paired kinetochore microtubules whereas at metaphase II (as in mitosis) each univalent bears only one kinetochore microtubule, thus showing that all kinetochores are fully active at all stages of meiosis.