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.     

Emi1 Class of Proteins Regulate Entry into Meiosis and the Meiosis I to Meiosis II Transition in Xenopus Oocytes

Xenopus oocytes are arrested at the G2/prophase boundary of meiosis I and enter meiosis in response to progesterone. A hallmark of meiosis is the absence of DNA replication between the successive cell division phases meiosis I (MI) and meiosis II (MII). After the MI-MII transition, Xenopus eggs are locked in metaphase II by the cytostatic factor (CSF) arrest to prevent parthenogenesis. Early Mitotic Inhibitor 1 (Emi1) maintains CSF arrest by inhibiting the ability of the Anaphase Promoting Complex (APC) to direct the destruction of cyclin B. To investigate whether Emi1 has an earlier role in meiosis, we injected Xenopus oocytes with neutralizing antibodies against Emi1 at G2/prophase and during the MI-MII transition. Progesterone-treated G2/prophase oocytes injected with anti-Emi1 antibody fail to activate Maturation Promoting Factor (MPF), a complex of cdc2/cyclin B, and the MAPK pathway, and do not undergo germinal vesicle breakdown (GVBD). Injection of purified ?90 cyclin B protein or blocking anti-Emi1 antibody with purified Emi1 protein rescues these meiotic processes in Emi1-neutralized oocytes. Acute inhibition of Emi1 in progesterone treated oocytes immediately after GVBD causes rapid loss of cdc2 activity with simultaneous loss of cyclin B levels and inactivation of the MAPK pathway. These oocytes decondense their chromosomes and enter a DNA replication phase instead of progressing to MII. Prior ablation of Cdc20, addition of methyl-ubiquitin, or addition of indestructible ?90 cyclin B rescues the MI-MII transition in Emi1 inhibited oocytes.     

Effects of several meiotic mutations on female meiosis in maize

A modified enzyme digestion technique of ovary isolation followed by staining and squash preparation has allowed us to observe female meiosis in normal maize meiotically dividing megaspore mother cells (MMCs). The first meiotic division in megasporogenesis of maize is not distinguishable from that in mi-crosporogenesis. The second female meiotic division is characterized as follows: (1) the two products of the first meiotic division do not simultaneously enter into the second meiotic division; as a rule, the chalazal-most cell enters division earlier than the micropylar one, (2) often the second of the two products does not proceed with meiosis, but degenerates, and (3) only a single haploid meiotic product of the tetrad remains alive, and this cell proceeds with three rounds of mitoses without any intervening cell wall formation to produce the eight-nucleate embryo sac. This technique has allowed us to study the effects of five meiotic mutations (aml, aml-pral, afdl, dsy *-9101, and dvl) on female meiosis in maize. The effects of the two alleles of the aml gene (aml and aml-pral) and of the afdl and dsy *-9101mutations are the same in both male and female meiosis. The aml allele prevents the entrance of MMCs into meiosis and meiosis is replaced by mitosis; the aml-pral permits MMCs to enter into meiosis, but their progress is stopped at early prophase I stages. The afdl gene is responsible for substitution of the first meiotic (reductional) division by an equational division including the segregation of sister chromatid centromeres at anaphase I. The dsy * -9101 gene exhibits abnormal chromosome pairing; paired homologous chromosomes are visible at pachytene, but only univalents are observed at diakinesis and metaphase I stages. These mutation specific patterns of abnormal meiosis are responsible for the bisexual sterility of these meiotic mutants. The abnormal divergent shape of the spindle apparatus and the resulting abnormal segregation of homologous chromosomes observed in micro-sporogenesis in plants homozygous for the dv1 mutation have not been found in meiosis of megasporogenesis. Only male sterility is induced by the dv1 gene in the homozygous condition. © 1993 Wiley-Liss, Inc.     

Interorganelle interactions and inheritance patterns of nuclei and vacuoles in budding yeast meiosis

Many of the mechanisms by which organelles are inherited by spores during meiosis are not well understood. Dramatic chromosome motion and bouquet formation are evolutionarily conserved characteristics of meiotic chromosomes. The budding yeast bouquet genes (NDJ1, MPS3, CSM4) mediate these movements via telomere attachment to the nuclear envelope (NE). Here, we report that during meiosis the NE is in direct contact with vacuoles via nucleus-vacuole junctions (NVJs). We show that in meiosis NVJs are assembled through the interaction of the outer NE-protein Nvj1 and the vacuolar membrane protein Vac8. Notably, NVJs function as diffusion barriers that exclude the nuclear pore complexes, the bouquet protein Mps3 and NE-tethered telomeres from the outer nuclear membrane and nuclear ER, resulting in distorted NEs during early meiosis. An increase in NVJ area resulting from Nvj1-GFP overexpression produced a moderate bouquet mutant-like phenotype in wild-type cells. NVJs, as the vacuolar contact sites of the nucleus, were found to undergo scission alongside the NE during meiotic nuclear division. The zygotic NE and NVJs were partly segregated into 4 spores. Lastly, new NVJs were also revealed to be synthesized de novo to rejoin the zygotic NE with the newly synthesized vacuoles in the mature spores. In conclusion, our results revealed that budding yeast nuclei and vacuoles exhibit dynamic interorganelle interactions and different inheritance patterns in meiosis, and also suggested that nvj1Δ mutant cells may be useful to resolve the technical challenges pertaining to the isolation of intact nuclei for the biochemical study of meiotic nuclear proteins.     

SHUGOSHINs and PATRONUS protect meiotic centromere cohesion in Arabidopsis thaliana

In meiosis, chromosome cohesion is maintained by the cohesin complex, which is released in a two‐step manner. At meiosis I, the meiosis‐specific cohesin subunit Rec8 is cleaved by the protease Separase along chromosome arms, allowing homologous chromosome segregation. Next, in meiosis II, cleavage of the remaining centromere cohesin results in separation of the sister chromatids. In eukaryotes, protection of centromeric cohesion in meiosis I is mediated by SHUGOSHINs (SGOs). The Arabidopsis genome contains two SGO homologs. Here we demonstrate that Atsgo1 mutants show a premature loss of cohesion of sister chromatid centromeres at anaphase I and that AtSGO2 partially rescues this loss of cohesion. In addition to SGOs, we characterize PATRONUS which is specifically required for the maintenance of cohesion of sister chromatid centromeres in meiosis II. In contrast to the Atsgo1 Atsgo2 double mutant, patronus T‐DNA insertion mutants only display loss of sister chromatid cohesion after meiosis I, and additionally show disorganized spindles, resulting in defects in chromosome segregation in meiosis. This leads to reduced fertility and aneuploid offspring. Furthermore, we detect aneuploidy in sporophytic tissue, indicating a role for PATRONUS in chromosome segregation in somatic cells. Thus, ploidy stability is preserved in Arabidopsis by PATRONUS during both meiosis and mitosis.     

ULTRASTRUCTURE OF MEIOSIS IN DASYA BAILLOUVIANA (RHODOPHYTA). II. PROMETAPHASE I—TELOPHASE II AND POST-DIVISION NUCLEAR BEHAVIOR1

This is the second of two papers which together are the first comprehensive ultrastructural report of meiosis in a red alga. Many details of the meiotic process in Dasya baillouviana (Gmelin) Montagne are the same as those reported previously for mitotic cells in ceramialian red algae, but several characteristics seem unique to meiotic cells. The nucleus and nucleolus of meiotic cells are larger than those of mitotic cells and large accumulations of smooth ER are often found at the division poles during meiosis 1. The function of the ER accumulations is unknown. Importantly, both interkinesis and a simultaneous division of two separate nuclei during meiosis II was demonstrated. These new observations fail to support earlier speculation on higher red algae for a “uninuclear” meiosis (both nuclear divisions within the same nuclear envelope). However, following meiosis II the four nuclei migrate centripetally and possibly fuse in the center of the tetrasporangium. This post-division nuclear maneuvering is not understood, but our interpretation accounts for the earlier and erroneous impression of “uninuclear” meiosis. Perhaps the most important aspect of meiosis observed in Dasya is its basic adherence to the pattern commonly seen in higher plants and animals. This conservatism of the meiotic process lends further skepticism to the belief that red algae are extremely “primitive” organisms, although they undoubtedly represent a very “ancient” group of eukaryotic plants.     

The pattern of zygotene and pachytene pairing in allotetraploid Aegilops species sharing the U genome

Allotetraploid Aegilops species sharing the U genome, Ae. columnaris (UUMM), Ae. ovata (UUMM), Ae. triaristata (UUMM), Ae. triuncialis (UUCC) and Ae. variabilis (UUSS), regularly form bivalents at metaphase I of meiosis. The pattern of zygotene and pachytene pairing was analyzed by whole-mount surface-spreading of synaptonemal complexes under the electron microscope. The data indicated that at the zygotene stage the chromosomes were almost exclusively associated as bivalents; only a few multivalents (7%) were observed. These observations are discussed in relation to mechanisms of diploidization of polyploid meiosis.     

鱼腥草花粉母细胞减数分裂特征及其育性研究

为深入了解鱼腥草花粉母细胞的减数分裂特征与花粉育性的关系,该研究采用卡宝品红染色法对2个鱼腥草居群花粉母细胞的减数分裂过程进行观察,并采用氯化三苯基四氮唑(TTC)染色法、I2-KI染色法、B-K培养基培养法及荧光显微镜观察法来检测鱼腥草花粉的活力及萌发率。结果发现:(1)鱼腥草减数分裂的进程与花序大小、花药颜色、花药长度均有密切的关系。(2)2个居群的鱼腥草中花粉母细胞减数分裂过程正常占88.2%,有11.8%的花粉母细胞减数分裂异常。(3)减数分裂异常表现在减数分裂过程中出现微核、落后染色体、染色体桥、不均等分离、多分体等现象,并发现在二分体阶段及单核花粉发育过程中存在细胞融合。(4)2个居群的鱼腥草花粉活力均不超过1.5%,花粉几乎不萌发。研究认为,鱼腥草花粉育性低的主要原因是单核花粉的发育过程异常,而非鱼腥草花粉母细胞减数分裂异常所致。     

The Drosophila hus1 gene is required for homologous recombination repair during meiosis

The checkpoint proteins, Rad9, Rad1, and Hus1 (9-1-1), form a complex which plays a central role in the DNA damage-induced checkpoint response. Previously, we demonstrated that Drosophila hus1 is essential for activation of the meiotic checkpoint elicited in double-strand DNA break (DSB) repair enzyme mutants. The hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in these repair enzymes, suggesting that hus1 plays a role independent of its meiotic checkpoint activity. In this study, we further analyzed the function of hus1 during meiosis and discovered that the synaptonemal complex (SC) disassembles abnormally in hus1 mutants. Oocyte nuclear and SC defects of hus1 mutants can be suppressed by blocking the formation of DSBs, implying that the hus1 oocyte nuclear defects depend upon DSBs. Interestingly, eliminating checkpoint activity through mutations in DmChk2 but not mei-41 suppress the oocyte nucleus and SC defects of hus1, suggesting that these processes are dependent upon DmChk2 checkpoint activity. Moreover, we showed that in hus1, DSBs that form during meiosis are not processed efficiently, and that this defect is not suppressed by a mutation in DmChk2. We found a genetic interaction between hus1 and the Drosophila brca2 homologue, which was shown to participate in DNA repair during meiosis. Together, our results imply that hus1 is required for repair of DSBs during meiotic recombination.     

CHROMOSOME DIFFERENTIATION IN SEVERAL SPECIES OF MELILOTUS

Shastry , Sishta V. S., William K. Smith , and Delmer C. Cooper . (U. Wisconsin, Madison.) Chromosome differentiation in several species of Melilotus. Amer. Jour. Bot. 47(8) : 613–621. Illus. 1960.—Two species of the section Eumelilotus (M. alba and M. officinalis), 2 of Micromelilotus (M. messanensis and M. segetalis), 2 F₁ hybrids (M. officinalis × M. alba and M. messanensis × M. segetalis), 2 autotetraploids (M. alba and M. officinalis), and 1 allotetraploid (M. officinalis × M. alba), were utilized during the course of this investigation. The 4 species and F₁ hybrids have 16 somatic chromosomes and the tetraploids have twice that number (32). The 2 Eumelilotus species are completely isolated because of seed failure after cross pollination. The F₁ hybrid (M. officinalis × M. alba), obtained elsewhere by the use of embryo-culture techniques, was intermediate between the parents in certain morphological characters and was ca. 75% pollenfertile whereas the parents approached complete fertility. No structural differences between the chromosomes were evident at pachytene. Disturbances which led to the reduced fertility occurred at later stages of meiosis. The Micromelilotus species are cross compatible, but the F₁ hybrid (M. messanensis × M. segetalis) is highly sterile. Despite chromosome structural differences of various types evident at pachytene, bivalents regularly occur at metaphase I. Irregular distribution of the chromosomes at later stages of meiosis leads to high sterility. Species which readily cross but produce a hybrid of very low fertility are likely to compound chromosomal structural differences, because of abnormalities in meiosis, in contrast with species that are completely incompatible.     

Comparative expression profiling reveals gene functions in female meiosis and gametophyte development in Arabidopsis

Megasporogenesis is essential for female fertility, and requires the accomplishment of meiosis and the formation of functional megaspores. The inaccessibility and low abundance of female meiocytes make it particularly difficult to elucidate the molecular basis underlying megasporogenesis. We used high‐throughput tag‐sequencing analysis to identify genes expressed in female meiocytes (FMs) by comparing gene expression profiles from wild‐type ovules undergoing megasporogenesis with those from the spl mutant ovules, which lack megasporogenesis. A total of 862 genes were identified as FMs, with levels that are consistently reduced in spl ovules in two biological replicates. Fluorescence‐assisted cell sorting followed by RNA‐seq analysis of DMC1:GFP‐labeled female meiocytes confirmed that 90% of the FMs are indeed detected in the female meiocyte protoplast profiling. We performed reverse genetic analysis of 120 candidate genes and identified four FM genes with a function in female meiosis progression in Arabidopsis. We further revealed that KLU, a putative cytochrome P450 monooxygenase, is involved in chromosome pairing during female meiosis, most likely by affecting the normal expression pattern of DMC1 in ovules during female meiosis. Our studies provide valuable information for functional genomic analyses of plant germline development as well as insights into meiosis.     

Meiosis VIII: Segregation matrix methods applied to meiotic drive in Drosophila melanogaster males with an SC 4-SC 8 inverted X

Two alternative matrix solutions of drive in Drosophila males with sc ⁴-sc ⁸ as studied by Peacock (1965) poses questions concerning meiosis in such flies. The two solutions are based primarily on the observations that sc ⁴-sc ⁸ males with low levels of X, Y non-disjunction produce twice as many female-as male offspring; that 50% of the sperm in this Drosophila line may be dysfunctional; that at all levels of non-disjunction studied, the difference between the % of female and male offspring produced by normal disjunction is about 33; and finally that, among non-disjunction types, nullo sperm werer covered more frequently than those with X and Y.The first matrix solution suggests a new model involving production of one infertile- and one fertile daughter cell at meiosis 1 in 1/3 of the cases and at meiosis 2 in the other 2/3. The second solution is already well known and would require the production of one fertile and one infertile secondary cyte at meiosis 1, in all cases. The unique feature of the first solution is that all four anaphase 2 poles (or genes) are present in primary spermatocytes as primordia. Two of these would predetermine fertility and two, infertility; and by assuming a random 2-by-2 assortment of these four poles at M1, together with physical binding of one of them with an X-or a Y chromatid, all known sc ⁴-sc ⁸ data as listed above, can be explained. The second model is necessarily more cumbersome, as it is difficult to account for a 2/1, female/male, ratio being produced at one cell division (meiosis 1).Discussed are possible applications of the segregation matrix method as a tool for determining degrees of freedom (complexity) in any biological system; implication of the models for segregation distorter (SD), for heterozygous translocations analyzed in Drosophila by Glass (1935) and by Zimmering (1955), and in other known or suspected cases of drive.     

The sporulation capable (sca) mutation of Saccharomyces cerevisiae is an allele of the SIR2 gene

Summary We have used the special properties of the spo13-1 mutation in order to study the regulation of yeast meiosis by the mating type loci. We have found that both the rme1-1 mutation and the sca mutation allow haploid meiosis in spo13-1 strains. Therefore, haploid meiosis is regulated in the same manner as diploid meiosis. Unlike rme1-1, the sca mutation allows meiosis through derepression of the silent mating type cassettes; sca strains can sporulate only because they express both MAT a and MAT information. We have found further that sca is an allele of SIR2, one of the genes involved in repression of the silent cassettes. Therefore, the RME1 gene is the only known candidate for a master negative regulator through which the MAT locus controls meiosis.     

Meiotic defects in the<Emphasis Type="Italic"> Arabidopsis</Emphasis><Emphasis Type="Italic"> rad50</Emphasis> mutant point to conservation of the MRX complex function in early stages of meiotic recombination

The Rad50, Mre11 and Xrs2/Nbs1 proteins, which form the highly conserved MRX complex, perform a wide range of functions concerning the maintenance and function of DNA in eukaryotes. These include recombination, DNA repair, replication, telomere homeostasis and meiosis. Notwithstanding the attention paid to this complex, the inviability of vertebrate rad50 and mre11 mutants has led to a relative lack of information concerning the role of these proteins in meiosis in higher eukaryotes. We have previously reported that Arabidopsis atrad50 mutant plants are viable and that atrad50 mutant plants are sterile. The present study reports an analysis of the causes of this sterility and the implication of the AtRad50 protein in meiosis. Both male and female gametogenesis are defective in the Arabidopsis atrad50 mutant and cytological observation of male meiosis indicates that in the absence of the AtRad50 protein, homologous chromosomes are unable to synapse. Finally, the atrad50 mutation leads to the destruction of chromosomes during meiosis. These phenotypes support a role for the Arabidopsis MRX complex in early stages of meiotic recombination.     

Analysis of Polo-like kinase Cdc5 in the meiosis recombination checkpoint

In meiosis, accumulation of recombination intermediates or defects in chromosome synapsis trigger checkpoint-mediated arrest in prophase I. Such 'checkpoints' are important surveillance mechanisms that ensure temporal dependence of cell cycle events. The budding yeast Polo-like kinase, Cdc5, has been identified as a key regulator of the meiosis I chromosome segregation pattern. Here we have analysed the role of Cdc5 in the recombination checkpoint and observed that Polo-like kinase is not required for checkpoint activation in yeast meiosis. Surprisingly, depletion of CDC5 in the Drad17 checkpoint-defective background resulted in nuclear fragmentation to levels even higher than that observed inDdmc1 Drad17 cells that bypass the checkpoint arrest despite accumulating DNA double-strand breaks. The spindle morphology of Cdc5-depleted cells included short, thick metaphase I spindles in mononucleate cells and disassembled spindles in binucleate and tetranucleate cells, although this phenotype does not appear to be the cause of the nuclear fragmentation. An exaggeration of chromosome synapsis defects occurred in Cdc5-depleted Drad17 cells and may contribute to the nuclear fragmentation phenotype. The analysis also uncovered a role for Cdc5 in maintaining spindle integrity in Ddmc1 Drad17 cells. Further analysis confirmed that adaptation to DNA damage does occur in meiosis and that CDC5 is required for this process. The cdc5-ad mutation that renders cells unable to adapt to DNA damage in mitosis did not affect checkpoint adaptation in meiosis, indicating that the mechanisms of checkpoint adaptation in mitosis and meiosis are not fully conserved.     

Transitions in the evolution of meiosis

Meiosis may have evolved gradually within the eukaryotes with the earliest forms having a one‐step meiosis. It has been speculated that the putative transition from a one‐step meiosis without recombination to one with recombination may have been stimulated by the invasion of Killer alleles. These imaginary selfish elements are considered to act prior to recombination. They prime for destruction (which occurs after cell division) the half of the cell on the opposite side of the meiotic spindle. Likewise the transition from one‐step to two‐step meiosis might have been stimulated by a subtly different sort of imaginary distorter allele, a SisterKiller. These are proposed to act after recombination. It has yet to be established that the presence of such distorter alleles could induce the transitions in question. To investigate these issues we have analysed the dynamics of a modifier (1) of recombination and (2) of the number of steps of meiosis, as they enter a population with one‐step meiosis. For the modifier of recombination, we find that invasion conditions are very broad and that persistence of Killer and modifier is likely through most parameter space, even when the recombination rate is low. However, if we allow a Killer element to mutate into one that is self‐tolerant, the modifier and the nonself‐tolerant alleles are typically both lost from the population. The modifier of the number of steps can invade if the SisterKiller acts at meiosis II. However, a SisterKiller acting at meiosis I, far from promoting the modifier’s spread, actually impedes it. In the former case the invasion is easiest if there is no recombination. The SisterKiller hypothesis therefore fails to provide a reasonable account of the evolution of two‐step meiosis with recombination. As before, the evolution of self‐tolerance on the part of the selfish element destroys the process. We conclude that the conditions under which SisterKillers promote the evolution of two‐step meiosis are very much more limited than originally considered. We also conclude that there is no universal agreement between ESS and modifier analyses of the same transitions.