Prometaphase Chromosomes Research Articles

The Pre-Metaphase Stretch: A Re-Examination

Pre-metaphase stretch is a term first coined by the preeminent cell biologist Sally Hughes-Schrader in 1950 to describe an elongation of prometaphase chromosomes observed in the primary spermatocytes of phasmid insects and praying mantids. Research from many groups since Hughes-Schrader’s initial observation has revealed reasons for both how and why chromosomes might elongate prior to metaphase. In this review, we describe Hughes-Schrader’s initial findings and discuss how recent work illuminates and provides some mechanistic explanation for this long-ago observed phenomenon.

Comparative Cytology of Female Meiosis I Among Drosophila Species.

The physical connections established by recombination are normally sufficient to ensure proper chromosome segregation during female Meiosis I. However, nonexchange chromosomes (such as the Muller F element or “dot” chromosome in D. melanogaster) can still segregate accurately because they remain connected by heterochromatic tethers. A recent study examined female meiosis in the closely related species D. melanogaster and D. simulans, and found a nearly twofold difference in the mean distance the obligately nonexchange dot chromosomes were separated during Prometaphase. That study proposed two speculative hypotheses for this difference, the first being the amount of heterochromatin in each species, and the second being the species’ differing tolerance for common inversions in natural populations. We tested these hypotheses by examining female meiosis in 12 additional Drosophila species. While neither hypothesis had significant support, we did see 10-fold variation in dot chromosome sizes, and fivefold variation in the frequency of chromosomes out on the spindle, which were both significantly correlated with chromosome separation distances. In addition to demonstrating that heterochromatin abundance changes chromosome behavior, this implies that the duration of Prometaphase chromosome movements must be proportional to the size of the F element in these species. Additionally, we examined D. willistoni, a species that lacks a free dot chromosome. We observed that chromosomes still moved out on the meiotic spindle, and the F element was always positioned closest to the spindle poles. This result is consistent with models where one role of the dot chromosomes is to help organize the meiotic spindle.

Quantitative Proteomics of the Mitotic Chromosome Scaffold Reveals the Association of BAZ1B with Chromosomal Axes*[S

In mitosis, chromosomes achieve their characteristic shape through condensation, an essential process for proper segregation of the genome during cell division. A classical model for mitotic chromosome condensation proposes that non-histone proteins act as a structural framework called the chromosome scaffold. The components of the chromosome scaffold, such as DNA topoisomerase IIα (TOP2A) and structural maintenance of chromosomes protein 2 (SMC2), are necessary to generate stable mitotic chromosomes; however, the existence of this scaffold remains controversial. The aim of this study was to determine the protein composition of the chromosome scaffold. We used the DT40 chicken cell line to isolate mitotic chromosomes and extract the associated protein fraction, which could contain the chromosome scaffold. MS revealed a novel component of the chromosome scaffold, bromodomain adjacent to zinc finger 1B (BAZ1B), which was localized to the mitotic chromosome axis. Knocking out BAZ1B caused prophase delay because of altered chromosome condensation timing and mitosis progression errors, and the effect was aggravated if BAZ1A, a BAZ1B homolog, was simultaneously knocked out; however, protein composition of prometaphase chromosomes was normal. Our results suggest that BAZ1 proteins are essential for timely chromosome condensation at mitosis entry. Further characterization of the functional role of BAZ1 proteins would provide new insights into the timing of chromosome condensation.

Localización de satélites y cromosomas NOR para la interpretación del cariotipo de Sesbania virgata (Papilionoideae, Sesbanieae) de dos poblaciones americanas

Antecedentes: Los estudios citogenéticos en el género Sesbania muestran desacuerdo entre los investigadores sobre el número preciso y posición de las constricciones secundarias y satélites, así como su relación con la organización del nucleolo. La carencia de esta información dificulta la realización de estudios de citogenética comparada y evolución cromosómica confiables en el género.Preguntas: ¿Dónde se ubican las constricciones secundarias y satélites en los cromosomas de Sesbania virgata ? ¿Estas regiones participan activamente en la organización nucleolar?Especie en estudio: Sesbania virgata (Cav.) Pers.Sitio y año de estudio: Municipio de Tlacotalpan, México y Provincia de Salta, Argentina, 2016.Métodos: Se aplicó una técnica de extendido en superficie y secado al aire para obtener cromosomas en prometafase y metafase típica a partir de meristemos radiculares.Resultados: Cada población exhibió un cariotipo diferente y solo dos constricciones secundarias asociadas a macrosatélites en los brazos cortos del par cromosómico más pequeño y no en brazos largos como fue sugerido por otros autores. La inclusión de las constricciones secundarias y satélites en el nucleolo de células en prometafase permitió corroborar su participación activa en la formación de éste. Esta información se utilizó para reevaluar la posición de las regiones del organizador nucleolar “NOR”.Conclusiones: Nuestros resultados concuerdan con el punto de vista predominante sobre la ubicación de los “NOR” en los brazos cortos de especies vegetales, particularmente en leguminosas. Además, dado que las poblaciones bajo estudio se encuentran geográficamente aisladas sugerimos que se favorece un proceso activo de especiación manifestado en los dos citotipos encontrados cuyas diferencias se atribuyen a cambios en la proporción de brazos de los cromosomas satelitales.

An improved method for inducing prometaphase chromosomes in plants

BackgroundDetailed karyotyping using metaphase chromosomes in melon (Cucumis melo L.) remains a challenge because of their small chromosome sizes and poor stainability. Prometaphase chromosomes, which are two times longer and loosely condensed, provide a significantly better resolution for fluorescence in situ hybridization (FISH) than metaphase chromosomes. However, suitable method for acquiring prometaphase chromosomes in melon have been poorly investigated.ResultsIn this study, a modified Carnoy’s solution II (MC II) [6:3:1 (v/v) ethanol: acetic acid: chloroform] was used as a pretreatment solution to obtain prometaphase chromosomes. We demonstrated that the prometaphase chromosomes obtained using the MC II method are excellent for karyotyping and FISH analysis. We also observed that a combination of MC II and the modified air dry (ADI) method provides a satisfactory meiotic pachytene chromosome preparation with reduced cytoplasmic background and clear chromatin spreads. Moreover, we demonstrated that pachytene and prometaphase chromosomes of melon and Abelia × grandiflora generate significantly better FISH images when prepared using the method described. We confirmed, for the first time, that Abelia × grandiflora has pairs of both strong and weak 45S ribosomal DNA signals on the short arms of their metaphase chromosomes.ConclusionThe MC II and ADI method are simple and effective for acquiring prometaphase and pachytene chromosomes with reduced cytoplasm background in plants. Our methods provide high-resolution FISH images that can help accelerate molecular cytogenetic research in plants.

Condensation patterns of prophase/prometaphase chromosome are correlated with H4K5 histone acetylation and genomic DNA contents in plants.

Mitotic prophase chromosome condensation plays an essential role in nuclear division being therefore regulated by highly conserved mechanisms. However, degrees of chromatin condensation in prophase-prometaphase cells may vary along the chromosomes resulting in specific condensation patterns. We examined different condensation patterns (CPs) of prophase and prometaphase chromosomes and investigated their relationship with genome size and distribution of histone H4 acetylated at lysine 5 (H4K5ac) in 17 plant species. Our results showed that most species with small genomes (2C < 5 pg) (Arachis pusilla, Bixa orellana, Costus spiralis, Eleutherine bulbosa, Indigofera campestris, Phaseolus lunatus, P. vulgaris, Poncirus trifoliata, and Solanum lycopersicum) displayed prophase chromosomes with late condensing terminal regions that were highly enriched in H4K5ac, and early condensing regions with apparently non-acetylated proximal chromatin. The species with large genomes (Allium cepa, Callisia repens, Araucaria angustifolia and Nothoscordum pulchellum) displayed uniformly condensed and acetylated prophase/prometaphase chromosomes. Three species with small genomes (Eleocharis geniculata, Rhynchospora pubera, and R. tenuis) displayed CP and H4K5ac labeling patterns similar to species with large genomes, whereas a forth species (Emilia sonchifolia) exhibited a gradual chromosome labeling, being more acetylated in the terminal regions and less acetylated in the proximal ones. The nucleolus organizer chromatin was the only chromosomal region that in prometaphase or metaphase could be hyperacetylated, hypoacetylated or non-acetylated, depending on the species. Our data indicate that the CP of a plant chromosome complement is influenced but not exclusively determined by nuclear and chromosomal DNA contents, whereas the CP of individual chromosomes is clearly correlated with H4K5ac distribution.

Knock-down of Pre-mRNA Splicing Factor Prp19 Causes Chromosome Misalignment and Prometaphase Arrest

To verify the role of the newly identified mitotic regulator candidate pre-mRNA processing factor 19 (Prp19) in mitosis and to clarify its underlying mechanism. FACS analyses with propidium iodide (PI) staining were performed to evaluate the effect of Prp19 knockdown on cell cycle distribution. To further clarify the role of Prp19 in mitosis, the effect of Prp19 depletion was monitored by time-lapse imaging of HeLa/GFP-H2B cells. Cold treatment experiment was used to examine the effect of Prp19 knockdown on the attachment of microtubules and kinetochores. To evaluate the effect of Prp19 knockdown on cell apoptosis, the control and Prp19-knockdown cells were analyzed by FACS with annexin V-FITC/-PI double staining. Furthermore, Western blot analysis of cleaved caspase-3 and PARP was also performed. Prp19 knockdown causesd mitotic arrest. Time-lapse imaging analysis showed that depletion of Prp19 in HeLa cells results in prometaphase arrest and chromosome misalignment. Cold treatment experiment showed that attachment between kinetochore and microtubule was impaired by Prp19 knockdown. Moreover, the depletion of Prp19 leaded to cell apoptosis in cancer cells. Prp19 is a key regulator of mitotic progression, and its inhibition may provide a new strategy for anti-cancer therapy.

Low Miki Expression in Myelodysplastic Syndromes Results in Polynuclear Cell Formation through Decondensation of Chromosomes in Prolonged Prometaphase

Polynuclear cells (PNCs) are routinely observed in the bone marrow of MDS patients. They are binuclear, trinuclear or even multinuclear cells with or without micronuclei, the underlying molecular mechanisms for the production of which are largely unknown. Because loss of the long arm of chromosome 7 (7q-) was reported to be associated with the presence of a higher frequency of PNCs, gene(s) preventing bone marrow cells from carrying such nuclear abnormalities may be located at 7q.We previously identified three candidate anti-myeloid tumor suppressor genes, namely Samd9, Samd9L and Miki, from the microdeletion in the 7q21 band frequently detected in JMML patients. SAMD9L-deficient mice develop MDS resembling human diseases associated with 7q-, most likely through enhancement of cytokine signals (Nagamachi et al., Cancer Cell 2013).Miki (mitotic kinetics regulator) translocates from the Golgi apparatus to mitotic centrosomes coincident with the disappearance of the Golgi body after poly-ADP-ribosylation (PARsylation). Miki is indispensable for centrosome maturation [the rapid increase of pericentriolar materials (PCM) during prophase and prometaphase], which is required for the production of robust mitotic spindles to move chromosomes promptly (Ozaki et al., Mol. Cell 2012). Consequently, as observed by time-lapse imaging of HeLa cells expressing histone H2A-GFP, downregulation of Miki by siRNA markedly prolonged the duration of prometaphase to more than several hours (normally around 15 minutes). Chromosomes were scarcely able to align and cells exited from prometaphase either by cell death or by decondensation of each chromosome. In the latter, cells with decondensed chromosomes then fused with one another within 30 minutes to form cells with relatively large nuclei, resulting in PNCs containing various sizes of nuclei including micronuclei. Indeed, reduction of Miki in HeLa cells by siRNA increased the frequency of PNCs from less than 0.5% to 4.5%.To test whether the chaotic chromosome decondensation in prometaphase causes the accumulation of PNCs observed in MDS, we initially used five cell lines derived from MDS associated with 7q-. PARsylated Miki was barely detectable in these cell lines and we found more cells at prometaphase than at metaphase (the ratio of prometa:meta in the lines ranged from 1.7:1 to 5.7:1). In contrast, in seven cell lines expressing PARsylated Miki at high levels, mitotic cells in prometaphase were found less frequently or at roughly the same frequency as those in metaphase (prometa:meta ratio 0.6:1 to 1.3:1). PNCs in five cell lines harboring 7q-were also more frequent (5.9 - 10.2%) than in the seven cell lines expressing high PARsylated Miki (0.8 - 2.4%). In addition, when we reduced Miki expression levels by shRNA in K562 cells, which express PARsylated Miki at high levels, the prometa:meta ratio increased from 1.1:1 to 3.8:1 and PNCs increased from 0.8% to 8.5%. This suggests that, as in HeLa cells, low expression levels of Miki cause prolongation of prometaphase and increase PNCs in blood cells.Fresh bone marrow preparations from 37 patients with MDS were examined to determine whether Miki mRNA-expression levels influence the prometaphase:metaphase ratio and the frequency of PNCs. We found a strong negative correlation (R=-0.59, p<0.01) between Miki mRNA expression levels in mononuclear cells of bone marrow samples and the prometa:meta ratio. We also found a moderate negative correlation (R=-0.4, p<0.05) between PNC frequencies and Miki mRNA expression levels. In addition, there was a strong positive correlation between prometa:meta ratios and PNC frequencies (R=-0.56, P<0.01).In conclusion, lack of one allele of the Miki gene due to 7q-reduces PARsylated Miki, resulting in the increase of PNCs through decondensation of chromosomes in prolonged prometaphase. This may contribute to poor outcome of MDS associated with 7q-through increased chromosome instability. DisclosuresNo relevant conflicts of interest to declare.

Karyotype analysis of Lablab purpureus (L.) Sweet using fluorochrome banding and fluorescence in situ hybridisation with rDNA probes

She Ch.-W., Jiang X.-H. (2015): Karyotype analysis of Lablab purpureus (L.) Sweet using fluorochrome banding and fluorescence in situ hybridisation with rDNA probes. Czech J. Genet. Plant Breed., 51: 110–116. The mitotic chromosomes of Lablab purpureus (L.) Sweet were characterised using sequential combined pro pidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI) (CPD) staining and fluorescence in situ hybridisation (FISH) with 5S and 45S rDNA probes. The detailed karyotype of this species was established using prometaphase chromosomes. After CPD staining, CPD and DAPI + bands were shown simultaneously. CPD bands occurred in the proximal regions of the long arms of all chromosome pairs and at all 45S rDNA sites, while the DAPI + bands appeared in all centromeres. FISH with rDNA probes revealed one 5S locus and eight 45S loci. The single 5S locus was interstitially located on the long arms of the shortest chromosome pair. Among the 45S loci, two large loci were located in the secondary constrictions of the short arms of two chromosome pairs; six small or minimal loci were proximally located on the short or long arms of six chromosome pairs. Each prometaphase chromosome pair could be identified using the CPD and DAPI + bands, the rDNA-FISH signals in combination with the chromosome measurements and condensation patterns. The karyotype was formulated as 2 n = 2x = 22 = 14m (2SAT) + 6sm + 2st (2SAT), and the asymmetry indices, CI, A 1, A2, As K%, AI and the Stebbins category were 38.23 ± 7.06, 0.36, 0.31, 61.99, 5.68 and 2B, respectively.

Chromosome segregation regulation in human zygotes: altered mitotic histone phosphorylation dynamics underlying centromeric targeting of the chromosomal passenger complex.

Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal phosphorylation dynamics of histone H2A at T120 (H2ApT120) by Bub1 kinase and subsequent recruitment of Shugoshin, but phosphorylation of histone H3 at threonine 3 (H3pT3) by Haspin failed to show the expected centromeric enrichment on metaphase chromosomes in the zygote. Human cleavage stage embryos show high levels of chromosomal instability. What causes this high error rate is unknown, as mechanisms used to ensure proper chromosome segregation in mammalian embryos are poorly described. In this study, we investigated the pathways regulating CPC targeting to the inner centromere in human embryos. We characterized the distribution of the CPC in relation to activity of its two main centromeric targeting pathways: the Bub1-H2ApT120-Sgo-CPC and Haspin-H3pT3-CPC pathways. The study was conducted between May 2012 and March 2014 on human surplus embryos resulting from in vitro fertilization treatment and donated for research. In zygotes, nuclear envelope breakdown was monitored by time-lapse imaging to allow timed incubations with specific inhibitors to arrest at prometaphase and metaphase, and to interfere with Haspin and Aurora B/C kinase activity. Functionality of the targeting pathways was assessed through characterization of histone phosphorylation dynamics by immunofluorescent analysis, combined with gene expression by RT-qPCR and immunofluorescent localization of key pathway proteins. Immunofluorescent analysis of the CPC subunit Inner Centromere Protein revealed the pool of stably bound CPC proteins was not strictly confined to the inner centromere of prometaphase chromosomes in human zygotes, as observed in later stages of preimplantation development and somatic cells. Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal phosphorylation dynamics of histone H2A at T120 (H2ApT120) by Bub1 kinase and subsequent recruitment of Shugoshin. However, phosphorylation of histone H3 at threonine 3 (H3pT3) by Haspin kinase failed to show the expected centromeric enrichment on metaphase chromosomes in the zygote, but not at later stages. Inhibition of Haspin revealed this activity to be essential for proper mitotic checkpoint complex activation in human zygotes, thus demonstrating an active mitotic checkpoint under normal conditions. Abolishment of H3pT3 during zygotic prometaphase further shows that centromeric H2ApT120 alone is not sufficient for proper shugoshin and CPC localization. As the removal of H3pT3 from the chromosome arms during prometaphase normally contributes to further centromeric enrichment of the CPC in somatic cells, CPC targeting may be less accurate in human zygotes. Owing to ethical limitations, tripronuclear zygotes were used in functional experiments. Although these represent the best available models, it is unknown if they are completely representative for dipronuclear zygotes. In addition, further research is needed to determine to what extent the differences we observed in H3T3 phosphorylation dynamics and CPC localization affect chromosome attachment. In the zygote, paternal and maternal chromosomes coming from two separate pronuclei, and with contrasting epigenetic signatures, need to be aligned on a single metaphase plate. Our results suggest that adaptations in mechanisms regulating CPC targeting exist in the human zygote, to ensure symmetric recruitment despite the epigenetic asymmetry between maternal and paternal chromosomes. This adaptation may come at a price regarding chromosome segregation fidelity. This study was funded by the Portuguese Fundação para a Ciência e Tecnologia and the Netherlands Organization for Scientific Research. The authors have no conflicts of interest to declare.

Investigations in vitro on the behaviour of chromosomes and the mitotic apparatus in endosperm cells of Haemanthus katherinae Baker treated with oleander glycosides

The effect of oleander glycosides on dividing endosperm cells of &lt;i&gt;Haemanthus katherine&lt;/i&gt; was investigated in vitro. The disturbances in the successive cell division phases were analysed in reference to cytokinesis. A strong tendency was noted to restitution nuclei formation in all phases of mitosis, and particularly in prophase. The observed chromosome pattern is the result of disturbances in prometaphase and anaphase chromosome movements owing to disturbances in the function of 'the 'mitotic spindle. It is probable that oleander glycosides inhibit formatiom of 'the microtubules of the mitotic spindle and disorganize the already formed spindle. They cause minor disturbances in cytokinesis, although frequently cell plates arise in quite unexpected places. The results of the present study are compared with those obtained in the case of root meristematic cells (Tarkowska, 1971a, b).

A simple method for cell culture of ‘Nemo’ ocellaris clownfish (Amphiprion ocellaris, Cuvier 1830)

Worldwide, the ‘Nemo’ ocellaris clownfish (Amphiprion ocellaris, Cuvier 1830) is one of the top three most exported ornamental fishes. It also served as a subject in various fields of study except for cell culture. This first report described a simple explant method for culturing cells from the vertebra of ocellaris clownfish. The fish was first anesthetised with iced cold water and decapitated. The body trunk was disinfected in isopropanol and washed in sterile PBS. The vertebra was aseptically excised, washed two times in PBS and minced in the dissection solution (PBS containing 250 IU/mL penicillin, 250 µg/mL streptomycin, 50 µg/mL gentamycin and 2.5 µg/mL amphotericin-B). Then, the vertebral biopsies were washed three more times in PBS before being seeded in 25 cm2 culture flasks containing 1.5 mL of RPMI-1640 supplemented with 20% FBS. A small amount of CO2 was injected into the flask before it was tightly capped and incubated at 28°C in the regular incubator. When the monolayer reached 40–50% confluence, the vertebral biopsies were dislodged together with the medium to initiate a new primary culture. The cell monolayer was subcultured with short, cold 0.05% trypsin. The Nemo cell line was grown in the medium containing 15% FBS. The cell line at passage 4 had the population doubling time of 39.6 h and the cell line at passage 5 could be cryopreserved with 80% viability. This simple and reliable explant method has been applied successfully to culture cells of both marine and freshwater fishes for the prometaphase chromosome preparation.

Chromosome congression explained by nanoscale electrostatics

Nanoscale electrostatic microtubule disassembly forces between positively charged molecules in kinetochores and negative charges on plus ends of microtubules have been implicated in poleward chromosome motions and may also contribute to antipoleward chromosome movements. We propose that chromosome congression can be understood in terms of antipoleward nanoscale electrostatic microtubule assembly forces between negatively charged microtubule plus ends and like-charged chromosome arms, acting in conjunction with poleward microtubule disassembly forces. Several other aspects of post-attachment prometaphase chromosome motions, as well as metaphase oscillations, are consistently explained within this framework.

Chromatin-induced spindle assembly plays an important role in metaphase congression of silkworm holocentric chromosomes

The kinetochore plays important roles in cell cycle progression. Interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell and to segregate the sister chromatids into daughter cells in mitosis. The chromosome passenger complex (CPC), composed of the Aurora B kinase and its regulatory subunits INCENP, Survivin, and Borealin, plays multiple roles in these chromosomal events. In the genome of the silkworm, Bombyx mori, which has holocentric chromosomes, the CPC components and their molecular interactions were highly conserved. In contrast to monocentric species, however, the silkworm CPC co-localized with the chromatin-driven spindles on the upper side of prometaphase chromosomes without forming bipolar mitotic spindles. Depletion of the CPC by RNAi arrested the cell cycle progression at prometaphase and disrupted the microtubule network of the chromatin-driven spindles. Interestingly, depletion of mitotic centromere-associated kinesin (MCAK) recovered formation of the microtubule network but did not overcome the cell cycle arrest at prometaphase. These results suggest that the CPC modulates the chromatin-induced spindle assembly and metaphase congression of silkworm holocentric chromosomes.

Is intracellular pH a clock for mitosis?

Experiments have shown that the intracellular pH of many cells rises to a maximum at the onset of mitosis, subsequently decreasing 0.3 to 0.5 pH units by the end of mitosis. This result, and observations that tubulin net charge depends strongly on pH, may be critical for microtubule (MT) dynamics during mitosis. In vivo studies demonstrate that MT dynamics is sensitive to pH, with MT growth favored by higher pH values. Therefore it seems likely that the shift from the dominance of microtubule growth during prophase, and to a lesser extent during prometaphase, to a parity between MT polymerization and depolymerization during metaphase chromosome oscillations is a consequence of gradually decreasing intracellular pH during mitosis. Thus the timing and sequencing of prophase, prometaphase, and metaphase chromosome motions may be understood as an increase in the MT disassembly to assembly probability ratio resulting from a continuously declining intracellular pH.

Nondisjunctional Segregations in Drosophila Female Meiosis I Are Preceded by Homolog Malorientation at Metaphase Arrest

The model of Drosophila female meiosis I was recently revised by the discovery that chromosome congression precedes metaphase I arrest. Use of the prior framework to interpret data from meiotic mutants led to the conclusion that chromosome segregation errors (nondisjunction, NDJ) occurred when nonexchange chromosomes moved out on the spindle in a maloriented configuration and became trapped there at metaphase arrest. The discovery that congression returns nonexchange chromosomes to the metaphase plate invalidates this interpretation and raises the question of what events actually do lead to NDJ. To address this, we have assayed an allelic series of ald (mps1) meiotic mutants that complete congression at wild-type rates, but have widely varying NDJ rates in an otherwise isogenic background, as well as a nod mutant background that primarily undergoes loss of chromosome 4. Using genetic assays to measure NDJ rates, and FISH assays to measure chromosome malorientation rates in metaphase-arrested oocytes, shows that these two rates are highly correlated across ald mutants, suggesting that malorientation during congression commits these chromosomes to eventually nondisjoin. Likewise, the rate of chromosome loss observed in nod is similar to the rate at which these chromosomes fail to associate with the main chromosome mass. Together these results provide a proximal mechanism for how these meiotic mutants cause NDJ and chromosome loss and improve our understanding of how prometaphase chromosome congression relates to anaphase chromosome segregation.

Structure of the nucleoli in domestic cattle spermatocytes

The work was aimed at determining the number and morphology of nucleoli in the prophase of the first meiotic division in domestic cattle males. The use of AgNO₃ staining, commonly applied in cytogenetics for the identification of nucleolar organiser regions, made it possible to identify nucleoli in first-order spermatocytes. One nucleolus was identified in each analysed cell. Considerable morphological differentiation of the nucleoli during the prophase of the first meiotic division, particularly in leptotene, unobserved in other farm animal species, was noticed. Dark-hued grain-like structures were found within the disintegrating nucleoli, corresponding approximately or exactly to the number of the nucleolar organiser regions in the domestic cattle karyotype. Dark areas were identified in the selected prometaphase chromosomes. Their number corresponded with the number of active NORs defined in the domestic cattle karyotype.

Structure of the nucleoli in domestic cattle spermatocytes

The work was aimed at determining the number and morphology of nucleoli in the prophase of the first meiotic division in domestic cattle males. The use of AgNO 3 staining, commonly applied in cytogenetics for the identification of nucleolar organiser regions, made it possible to identify nucleoli in first-order spermatocytes. One nucleolus was identified in each analysed cell. Considerable morphological differentiation of the nucleoli during the prophase of the first meiotic division, particularly in leptotene, unobserved in other farm animal species, was noticed. Dark-hued grain-like structures were found within the disintegrating nucleoli, corresponding approximately or exactly to the number of the nucleolar organiser regions in the domestic cattle karyotype. Dark areas were identified in the selected prometaphase chromosomes. Their number corresponded with the number of active NORs defined in the domestic cattle karyotype.

Integration of Linkage and Chromosome Maps of Red Clover (Trifolium pratense L.)

Red clover (Trifolium pratense L.) is a forage legume and an allogamous diploid plant (2n = 14; 440 Mb). Here, we examine the 7 prometaphase chromosomes of red clover using fluorescence in situ hybridization (FISH) with ribosomal RNA sequences, pericentromeric and telomeric repeats, as well as bacterial artificial chromosome (BAC) clones. Position of hybridization signals and chromosome condensation patterns were quantified by the help of the chromosome image analysis system ver. 4.0 (CHIAS IV). Fourteen BAC clones belonging to linkage groups (LG) 1–7 hybridized to individual chromosomes 4, 2, 6, 5, 1, 7, and 3, respectively. Quantitative analysis of FISH mapping and chromosome analysis using CHIAS IV allowed us to construct a quantitative idiogram that constitutes the comprehensive chromosome map of red clover. Chromosomal positions of the 26S rDNA locus were detected at a heterozygous locus on chromosome 6 in the variety HR, and polymorphisms of rDNA loci were observed in other varieties, although chromosomal positions of some BAC clones did not vary among HR and other varieties. These results demonstrate chromosomal collinearity among allogamous red clover varieties. This integration of genetic linkage and quantitative chromosome maps should provide valuable insight into allogamous legume genetics.

Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase

Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore-microtubule attachments (K-fibers) on sister chromatids that have attached to opposite spindle poles (bi-oriented)1. In many instances, phosphorylation correlates with K-fiber destabilization2–7. Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes compared to bi-oriented chromosomes, which have stable attachments2, 8. Paradoxically, however, these kinases also target to prometaphase chromosomes that have not yet established spindle attachments and it is therefore unclear how kinetochore-microtubule interactions can be stabilized when kinase levels are high. Here we show that generation of stable K-fibers depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibers are destabilized and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the phosphorylation of Aurora B and Plk1 kinetochore substrates as well as Plk1 recruitment to kinetochores. Consistent with increased substrate phosphorylation, we find that chemical inhibition of Aurora or Plk1 restores K-fibers in B56-PP2A depleted cells. Our findings reveal that PP2A, an essential tumor suppressor9, tunes the balance of phosphorylation to promote chromosome-spindle interactions during cell division.