Anti-topoisomerase II recognizes meiotic chromosome cores

At meiotic prophase the chromatin becomes arranged in loops on newly formed chromosome cores. The cores of homologous chromosomes become aligned in parallel and thus form the synaptonemal complex (SC), a structure found in the meiocytes of nearly all recombinationally competent, sexually reproducing organisms. We report that two polyclonal antibodies against topoisomerase II (topo II), which recognize the mitotic metaphase chromosome scaffold give, at pachytene, a positive immunocytological reaction with the chromatin and, predominantly, with the cores and centromeric regions of the paired chromosomes. It therefore appears that during meiotic prophase, topo II — a DNA-binding enzyme implicated in transient double-strand breaks, chromosome condensation, and anaphase separation — is associated with the chromatin and SCs of the pachytene and diplotene chromosomes.     

Über das Verhalten eines Ringchromosoms in der Mitose und Meiose von Antirrhinum majus L.

Summary The mitotic and meiotic behaviour of a ring-chromosome in Antirrhinum majus was analysed. 26.5% mitotic anaphases showed bridges demonstrating the occurrence of a crossing-over-like process in meristematic cells. From pachytene studies the ring-chromosome could be identified as chromosome 6.An attempt was made to derive the details of the crossing-over process from the various anaphase configurations in pollen mother cells with a heterozygous ring-rod-bivalent. The observed frequencies could only be brought in approximate correspondance with theoretical values by postulating (i) the occurrence of sister-strand and non-sister-strand crossing-over in certain quantitative combinations, and (ii) an unexplained loss or irricognizability of most double bridges in anaphase I.The frequency of plants heterozygous for the ring-chromosome in progenies after seifing was 16.8%. The rate of chromosome mutations in these progenies was not increased. Chromosomal aberrations resulting from meiotic disturbances in the ring plants are probably lost by gonal elimination of unbalanced chromosome sets.     

An indirect test for a role of the synaptonemal complex in the establishment of sister chromatid cohesiveness

The meiotic behavior of a special maize trisome was quantitatively observed at pachytene, metaphase I, anaphase I, prophase II, metaphase II and anaphase II. The data obtained are consistent with (but do not prove) the model that sister chromatid cohesiveness at anaphase I may be established during pachytene synapsis of the chromosome regions involved. The data suggest, however, that the normal prophase II maintenance of dyad integrity by cohesiveness of sister chromatid centromere regions does not depend upon prior synapsis of these regions, although monads separated from each other on the anaphase I spindle may be delivered to the same prophase II daughter nucleus. — The strands which some of the time connect sister chromatids which are separating equationally at anaphase I show a positive Feulgen staining reaction.     

The mitotic, polytene, and meiotic chromosomes of Drosophila ananassae.

The mitotic chromosome complement of D. ananassae consists of four structurally distinguishable submetacentric pairs and all four have been identified with their linkage groups. For the polytene chromosome complement of six arms representing the X, second and third chromosomes, an improved reference map has been constructed and used to describe selected cytogenetically useful rearrangements. In meiotic prophase of spermatocytes, chromosomes 2 and 3 form pachytene-diplotene bivalents whose arms may be associated by chiasmata in postdiplotene stages, but the X, Y and fourth chromosomes participate in a complex multivalent. No correlation was detected between meiotic chromosome behavior and specific genes that regulate crossing over in males. In male inversion heterozygotes having high levels of genetically monitored crossing over, no unequivocal evidence was found for formation of either pachytene inversion loops or anaphase bridges and fragments.     

Sister and non-sister chromatid U-type exchange in rye meiosis

Aberrant meiotic chromosome configurations in an experimental population of rye lines are known to result from spontaneous U-type exchanges during meiotic prophase. Both sister chromatid and non-sister chromatid exchanges occur and this study is concerned with the relative frequencies of sister and non-sister exchanges. The anaphase I observations reveal a marked excess of non-sister U-type exchange configurations and it is argued that this reflects an original excess of prophase non-sister U-type exchanges. This conclusion is discussed with special reference to the origin of meiotic U-type exchanges and their relation to regular crossover exchanges.     

Immunofluorescent synaptonemal complex analysis in azoospermic men

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.     

Slk19p is necessary to prevent separation of sister chromatids in meiosis I

BACKGROUND: A fundamental difference between meiotic and mitotic chromosome segregation is that in meiosis I, sister chromatids remain joined, moving as a unit to one pole of the spindle rather than separating as they do in mitosis. It has long been known that the sustained linkage of sister chromatids through meiotic anaphase I is accomplished by association of the chromatids at the centromere region. The localization of the cohesin Rec8p to the centromeres is essential for maintenance of sister chromatid cohesion through meiosis I, but the molecular basis for the regulation of Rec8p and sister kinetochores in meiosis remains a mystery. RESULTS: We show that the SLK19 gene product from Saccharomyces cerevisiae is essential for proper chromosome segregation during meiosis I. When slk19 mutants were induced to sporulate they completed events characteristic of meiotic prophase I, but at the first meiotic division they segregated their sister chromatids to opposite poles at high frequencies. The vast majority of these cells did not perform a second meiotic division and proceeded to form dyads (asci containing two spores). Slk19p was found to localize to centromere regions of chromosomes during meiotic prophase where it remained until anaphase I. In the absence of Slk19p, Rec8p was not maintained at the centromere region through anaphase I as it is in wild-type cells. Finally, we demonstrate that Slk19p appears to function downstream of the meiosis-specific protein Spo13p in control of sister chromatid behavior during meiosis I. CONCLUSIONS: Our results suggest that Slk19p is essential at the centromere of meiotic chromosomes to prevent the premature separation of sister chromatids at meiosis I.     

Studies in the Mitosis of Euglena I. On the Chromosome Cycle of Euglena viridis Ehrbg.

The chromosome cycle in the vegetative division of Euglena viridis was investigated. The seeming chromatin granules in the interphase nucleus are in reality thread structures, paired and very loosely twisted. Each component of the paired threads is called a chromatid, and consists of a fine thread of even thickness, the chromonema.
In the prophase, linear contraction and thickening of the chromatids occurs by means of the spiralization of them. In the later prophase, the coiled chromonema splits into two finer strands which show the plectonemic spiral. At the metaphase, the chromosomes are arranged in the form of an equatorial ring, encircling the median portion of the elongated endosome. Nearly all of the chromosomes have a submedian or a sub-terminal and a few of them have a terminal kinetochore. In the early anaphase, separation of the sister chromosomes takes place beginning at the kinetochore. The spindle fibres in the metaphase and anaphase were not observed. The two stranded spiral in the chromosomes is separated into distinct components by the uncoiling in the later telophase, and they are transformed, in the interphase nucleus, into the paired chromatids.     

A kinesin mutant with an atypical bipolar spindle undergoes normal mitosis

Motor proteins have been implicated in various aspects of mitosis, including spindle assembly and chromosome segregation. Here, we show that acentrosomal Arabidopsis cells that are mutant for the kinesin, ATK1, lack microtubule accumulation at the predicted spindle poles during prophase and have reduced spindle bipolarity during prometaphase. Nonetheless, all abnormalities are rectified by anaphase and chromosome segregation appears normal. We conclude that ATK1 is required for normal microtubule accumulation at the spindle poles during prophase and possibly functions in spindle assembly during prometaphase. Because aberrant spindle morphology in these mutants is resolved by anaphase, we postulate that mitotic plant cells contain an error-correcting mechanism. Moreover, ATK1 function seems to be dosage-dependent, because cells containing one wild-type allele take significantly longer to proceed to anaphase as compared with cells containing two wild-type alleles.     

Chromosome distribution: experiments on cell hybrids and in vitro.

Ostergren (1951) provided a simple explanation for both chromosome distribution in mitosis and chromosome segregation in meiosis, and more recently a molecular extension of his hypothesis has been possible. This report focuses on experimental tests of these ideas. Micromanipulation experiments on cell hybrids containing both meiotic and mitotic spindles demonstrate that differences in meiotic and mitotic chromosome behavior are determined by something intrinsic to the chromosome: meiotic chromosomes transferred to a mitotic spindle (or vice versa) behave just as they normally would. The molecular explanation postulates polarized growth or binding of microtubules at kinetochores. This has just been tested in vitro by McGill & Brinkley (1975) and by Telzer, Moses & Rosenbaum (1975), and their results are reviewed. In addition, a novel method for in vitro studies is described - mechanical demembranation of cells which is compatible with quite normal chromosome movement in anaphase. After addition of microtubule subunits to a demembranated prophase cell, chromosome orientation and movement toward an aster was observed for the first time in vitro. It is concluded that important aspects of chromosome distribution are probably understood at both the cellular and molecular levels, but final tests are still required. The outlook is hopeful indeed because the gaps in our knowledge are well known - the necessity of observations on prophase is a recurrent theme - and the means of filling the gaps are in hand.     

The Caenorhabditis elegans SCC-3 homologue is required for meiotic synapsis and for proper chromosome disjunction in mitosis and meiosis

The product of the Caenorhabditis elegans ORF F18E2.3 is homologous to the cohesin component Scc3p. By antibody staining the product of F18E2.3 is found in interphase and early meiotic nuclei. At pachytene it localizes to the axes of meiotic chromosomes but is no longer detectable on chromatin later in meiosis or in mitoses. Depletion of the gene product by RNAi results in aberrant mitoses and meioses. In meiosis, homologous pairing is defective during early meiotic prophase and at diakinesis there occur univalents consisting of loosely connected sister chromatids or completely separated sisters. The recombination protein RAD-51 accumulates in nuclear foci at higher numbers during meiotic prophase and disappears later than in wild-type worms, suggesting a defect in the repair of meiotic double-stranded DNA breaks. Embryos showing nuclei of variable size and anaphase bridges, indicative of mitotic segregation defects, are frequently observed. In the most severely affected gonads, nuclear morphology cannot be related to any specific stage. The cytological localization and the consequences of the lack of the protein indicate that C. elegans SCC-3 is essential for sister chromatid cohesion both in mitosis and in meiosis.     

Meiotic behavior of a tiny fragment chromosome that carries a transposed centromere

A supernumerary, tiny chromosome with a transposed centromere, in an apparently normal maize background, was observed through meiotic stages from pachytene through anaphase II. Departures from normal meiotic chromosome behavior were noted for this tiny chromosome. These included failure of the usual degree of condensation at pachytene, failure of synapsis, and most strikingly the ability of sister centromeres to interact with the spindle on schedule with the normal dyads at anaphase I, so that monads were commonly distributed to the poles for telophase I and then often lagged at anaphase II. Possible significance of the unusual behavior is discussed.     

Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line

The replication of chromosomes in meiosis is an important first step for subsequent chromosomal interactions that promote accurate disjunction in the first of two segregation events to generate haploid gametes. We have developed an assay to monitor DNA replication in vivo in mitotic and meiotic germline nuclei of the nematode Caenorhabditis elegans. Using mutants that affect the mitosis/meiosis switch, we show that meiotic S phase is at least twice as long as mitotic S phase in C. elegans germ cell nuclei. Furthermore, our assay reveals that different regions of the genome replicate at different times, with the heterochromatic-like X chromosomes replicating at a distinct time from the autosomes. Finally, we have exploited S-phase labeling to monitor the timing of progression through meiotic prophase. Meiotic prophase for oocyte production in hermaphrodites lasts 54-60 h. Further, we find that the duration of the pachytene sub-stage is modulated by the presence of sperm. On the other hand, meiotic prophase for sperm production in males is completed by 20-24 h. Possible sources for the sex-specific differences in meiotic prophase kinetics are discussed.     

Comparative analysis of female and male meiosis in three meiotic mutants of tomato.

Female meiosis was analysed in squash preparations of ovules from three meiotic mutants and wild-type plants of tomato. In the completely asynaptic mutant as6, chromosome pairing and chiasma formation were virtually absent in both sexes. In the partially asynaptic mutant asb, with intermediate levels of chromosome pairing at pachytene, there were a higher number of chiasmate chromosome arms in female meiosis than in male meiosis, whereas in the desynaptic mutant as5 there were normal levels of chromosome pairing at pachytene and a similar reduction in chiasma frequency in the two sexes. In wild-type tomato, we found slightly higher numbers of chiasmate chromosome arms in female meiosis than in male meiosis. We propose that the higher female chiasma frequencies in mutant asb and wild-type tomato result from a longer duration of female meiotic prophase. This would allow chromosomes more time to pair and recombine. It is possible that a longer duration of prophase I does not affect mutants as5 and as6, either because the meiotic defect acts before the pairing process begins (in as6) or because it acts at a later stage and involves chiasma maintenance (in as5).     

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.     

Chromosome condensation in mitosis and meiosis of rye (Secale cereale L.)

Structural investigation and morphometry of meiotic chromosomes by scanning electron microscopy (in comparison to light microscopy) of all stages of condensation of meiosis I + II show remarkable differences during chromosome condensation in mitosis and meiosis I of rye (Secale cereale) with respect to initiation, mode and degree of condensation. Mitotic chromosomes condense in a linear fashion, shorten in length and increase moderately in diameter. In contrast, in meiosis I, condensation of chromosomes in length and diameter is a sigmoidal process with a retardation in zygotene and pachytene and an acceleration from diplotene to diakinesis. The basic structural components of mitotic chromosomes of rye are "parallel fibers" and "chromomeres" which become highly compacted in metaphase. Although chromosome architecture in early prophase of meiosis seems similar to mitosis in principle, there is no equivalent stage during transition to metaphase I when chromosomes condense to a much higher degree and show a characteristic "smooth" surface. No indication was found for helical winding of chromosomes either in mitosis or in meiosis. Based on measurements, we propose a mechanism for chromosome dynamics in mitosis and meiosis, which involves three individual processes: (i) aggregation of chromatin subdomains into a chromosome filament, (ii) condensation in length, which involves a progressive increase in diameter and (iii) separation of chromatids.     

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity.     

Spo76p is a conserved chromosome morphogenesis protein that links the mitotic and meiotic programs.

Spo76p is conserved and related to the fungal proteins Pds5p and BIMD and the human AS3 prostate proliferative shutoff-associated protein. Spo76p localizes to mitotic and meiotic chromosomes, except at metaphase(s) and anaphase(s). During meiotic prophase, Spo76p assembles into strong lines in correlation with axial element formation. As inferred from spo76-1 mutant phenotypes, Spo76p is required for sister chromatid cohesiveness, chromosome axis morphogenesis, and chromatin condensation during critical transitions at mitotic prometaphase and meiotic midprophase. Spo76p is also required for meiotic interhomolog recombination, likely at postinitiation stage(s). We propose that a disruptive force coordinately promotes chromosomal axial compaction and destabilization of sister connections and that Spo76p restrains and channels the effects of this force into appropriate morphogenetic mitotic and meiotic outcomes.     

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.     

Nuclear division in the red algae Chondria nidifica and Chondria tenuissima

Cytological investigations are reported for two Chondria species, the Pacific species Chondria nidifica Harvey and Chondria tenuissima (Goodenough et Woodward) C. A. Agardh from the shore of the Marmara Sea in Istanbul. Nuclear division during mitosis and meiosis has been followed in somatic cells and in tetrasporangial mother cells respectively of diploid tetrasporic plants. The spherical interphase nucleus stains densely, showing many chromatin granules. Mitotic nuclei in the apical groove show a large number of chromosomes at metaphase; the chromosome number has been estimated at diakinesis to be 40 in both C. nidifica and C. tenuissima. The meiotic nuclei of tetraspore mother cells in prophase contain several relatively large nucleolar-derivatives in both species. The nucleolar derivatives disappear completely before the chromosomes begin to differentiate. In meiotic prophase the tetraspore mother cell enlarges from its original diameter. The period of the second meiotic anaphase seems to be extremely short in comparison with other nuclear phases. When the chromosomes reach the poles, they spread and subsequently form a relatively compact mass at telophase. The spindle has not been observed in C. tenuissima. Photographs are presented of nucleoli and nucleolar-derivatives in mitotic and meiotic divisions.