Showing papers on "Metaphase published in 1999"

Temporal and spatial control of cyclin B1 destruction in metaphase.

Abstract: The proteolysis of key regulatory proteins is thought to control progress through mitosis. Here we analyse cyclin B1 degradation in real time and find that it begins as soon as the last chromosome aligns on the metaphase plate, just after the spindle-assembly checkpoint is inactivated. At this point, cyclin B1 staining disappears from the spindle poles and from the chromosomes. Cyclin B1 destruction can subsequently be inactivated throughout metaphase if the spindle checkpoint is reimposed, and this correlates with the reappearance of cyclin B1 on the spindle poles and the chromosomes. These results provide a temporal and spatial link between the spindle-assembly checkpoint and ubiquitin-mediated proteolysis.

TIN2, a new regulator of telomere length in human cells.

Abstract: Telomeres are DNA-protein structures that cap linear chromosomes and are essential for maintaining genomic stability and cell phenotype. We identified a novel human telomere-associated protein, TIN2, by interaction cloning using the telomeric DNA-binding-protein TRF1 as a bait. TIN2 interacted with TRF1 in vitro and in cells, and co-localized with TRF1 in nuclei and metaphase chromosomes. A mutant TIN2 that lacks amino-terminal sequences effects elongated human telomeres in a telomerase-dependent manner. Our findings suggest that TRF1 is insufficient for control of telomere length in human cells, and that TIN2 is an essential mediator of TRF1 function.

The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.

Abstract: Publisher Summary This chapter presents detailed methods for the preparation of cytostatic factor (CSF) extracts and for performing spindle assembly reactions. It also describes methods for depleting specific components from extracts, an approach that has been used successfully to determine the contributions of both motor and nonmotor components to spindle assembly. It also describes methods for analyzing anaphase in vitro. Mature Xenopus eggs are arrested in metaphase of meiosis II by CSF, which is thought to be the product of the c-mos protooncogene. Sperm entry triggers a calcium spike that initiates a series of events leading to the destruction of CSF and exit from the meiosis II metaphase arrest. This calcium sensitivity of the CSF arrest is exploited in the preparation of extracts by use of the calcium chelator EGTA. The presence of EGTA in buffers results in extracts that maintain the CSF arrest but can be induced to exit the CSF arrest by addition of calcium. This convenient control of cell cycle state allows one to easily obtain in vitro spindles with replicated chromosomes.

Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4

Abstract: The condensin complex in frog extracts, containing two SMC (structural maintenance of chromosomes) and three non-SMC subunits, promotes mitotic chromosome condensation, and its supercoiling activity increases during mitosis by Cdc2 phosphorylation. Here, we report that fission yeast has the same five-member condensin complex, each of which is essential for mitotic condensation. The condensin complex was purified and the subunits were identified by microsequencing. Cnd1, Cnd2, and Cnd3, three non-SMC subunits showing a high degree of sequence conservation to frog subunits, are essential for viability, and their gene disruption leads to a phenotype indistinguishable from that observed in cut3-477 and cut14-208, known mutations in SMC4 and SMC2-like subunits. Condensin subunits tagged with GFP were observed to alter dramatically their localization during the cell cycle, enriched in the nucleus during mitosis, but cytoplasmic during other stages. This stage-specific alteration in localization requires mitosis-specific phosphorylation of the T19 Cdc2 site in Cut3. The T19 site is phosphorylated in vitro by Cdc2 kinase and shows the maximal phosphorylation in metaphase in vivo. Its alanine substitution mutant fails to suppress the temperature-sensitive phenotype of cut3-477, and shows deficiency in condensation, probably because Cut3 T19A remains cytoplasmic. Therefore, direct Cdc2 phosphorylation of fission yeast condensin may facilitate its nuclear accumulation during mitosis.

The disappearance of cyclin B at the end of mitosis is regulated spatially in Drosophila cells.

Abstract: We have followed the behaviour of a cyclin B–green fluorescent protein (GFP) fusion protein in living Drosophila embryos in order to study how the localization and destruction of cyclin B is regulated in space and time We show that the fusion protein accumulates at centrosomes in interphase, in the nucleus in prophase, on the mitotic spindle in prometaphase and on the microtubules that overlap in the middle of the spindle in metaphase In cellularized embryos, toward the end of metaphase, the spindle‐associated cyclin B–GFP disappears from the spindle in a wave that starts at the spindle poles and spreads to the spindle equator; when the cyclin B–GFP on the spindle is almost undetectable, the chromosomes enter anaphase, and any remaining cytoplasmic cyclin B–GFP then disappears over the next few minutes The endogenous cyclin B protein appears to behave in a similar manner These findings suggest that the inactivation of cyclin B is regulated spatially in Drosophila cells We show that the anaphase‐promoting complex/cyclosome (APC/C) specifically interacts with microtubules in embryo extracts, but it is not confined to the spindle in mitosis, suggesting that the spatially regulated disappearance of cyclin B may reflect the spatially regulated activation of the APC/C

Proper metaphase spindle length is determined by centromere proteins Mis12 and Mis6 required for faithful chromosome segregation

Abstract: High-fidelity chromosome transmission is fundamental in controlling the quality of the cell division cycle. The spindle pole-to-pole distance remains constant from metaphase to anaphase A. We show that fission yeast sister centromere-connecting proteins, Mis6 and Mis12, are required for correct spindle morphogenesis, determining metaphase spindle length. Thirty-five to sixty percent extension of metaphase spindle length takes place in mis6 and mis12 mutants. This may be due to incorrect spindle morphogenesis containing impaired sister centromeres or force unbalance between pulling by the linked sister kinetochores and kinetochore-independent pushing. The mutant spindle fully extends in anaphase, although it is accompanied by drastic missegregation by aberrant sister centromere separation. Hence, metaphase spindle length may be crucial for segregation fidelity. Suppressors of mis12 partly restore normal metaphase spindle length. In mis4 that is defective in sister chromatid cohesion, metaphase spindle length is also long, but anaphase spindle extension is blocked, probably due to the activated spindle checkpoint. Extensive missegregation is caused in mis12 only when Mis12 is inactivated from the previous M through to the following M, an effective way to avoid missegregation in the cell cycle. Mis12 has conserved homologs in budding yeast and filamentous fungi.

Arsenic Targets Tubulins to Induce Apoptosis in Myeloid Leukemia Cells

Abstract: Arsenic exhibits a differential toxicity to cancer cells. At a high concentration (>5 μm), As2O3 causes acute necrosis in various cell lines. At a lower concentration (0.5–5 μm), it induces myeloid cell maturation and an arrest in metaphase, leading to apoptosis. As2O3-treated cells have features found with both tubulin-assembling enhancers (Taxol) and inhibitors (colchicine). Prior treatment of monomeric tubulin with As2O3 markedly inhibits GTP-induced polymerization and microtubule formation in vitro but does not destabilize GTP-induced tubulin polymers. Cross-inhibition experiments indicate that As2O3 is a noncompetitive inhibitor of GTP binding to tubulin. These observations correlate with the three-dimensional structure of β-tubulin and suggest that the cross-linking of two vicinal cysteine residues (Cys-12 and Cys-213) by trivalent arsenic inactivates the GTP binding site. Furthermore, exogenous GTP can prevent As2O3-induced mitotic arrest.

Mutations in the Essential Spindle Checkpoint Gene bub1 Cause Chromosome Missegregation and Fail to Block Apoptosis in Drosophila

Abstract: We have characterized the Drosophila mitotic checkpoint control protein Bub1 and obtained mutations in the bub1 gene. Drosophila Bub1 localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element–induced, near-null mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants. We have also investigated the relationship between Bub1 and other kinetochore components. We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

Role for yeast inhibitor of apoptosis (IAP)-like proteins in cell division

Abstract: Inhibitors of apoptosis (IAPs) are a family of proteins that bear baculoviral IAP repeats (BIRs) and regulate apoptosis in vertebrates and Drosophila melanogaster. The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both encode a single IAP, designated BIR1 and bir1, respectively, each of which bears two BIRs. In rich medium, BIR1 mutant S. cerevisiae underwent normal vegetative growth and mitosis. Under starvation conditions, however, BIR1 mutant diploids formed spores inefficiently, instead undergoing pseudohyphal differentiation. Most spores that did form failed to survive beyond two divisions after germination. bir1 mutant S. pombe spores also died in the early divisions after spore germination and became blocked at the metaphase/anaphase transition because of an inability to elongate their mitotic spindle. Rather than inhibiting caspase-mediated cell death, yeast IAP proteins have roles in cell division and appear to act in a similar way to the IAPs from Caenorhabditis elegans and the mammalian IAP Survivin.

The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation

Abstract: The yeast and human SKP1 genes regulate the mitotic cell cycle but are not yet known to be required for meiosis. Nine Arabidopsis SKP1 homologues have been uncovered and are named ASK1 through ASK9. Here, we report the isolation and characterization of a male sterile Arabidopsis mutant and show that the mutant defect was caused by a Ds transposon insertion into the ASK1 gene. In the ask1-1 mutant, abnormal microspores exhibit a range of sizes. Furthermore, during mutant male meiosis, although homologous chromosome pairing appeared normal at metaphase I, chromosome segregation at anaphase I is unequal, and some chromosomes are abnormally extended. Therefore, in ask1-1, at least some homologues remain associated after metaphase I. In addition, immunofluorescence microscopy indicates that the mutant spindle morphology at both metaphase I and early anaphase I is normal; thus, the abnormal chromosome segregation is not likely caused by a spindle defect. Because the yeast Skp1p is required for targeting specific proteins for ubiquitin-mediated proteolysis, we propose that ASK1 controls homologue separation by degrading or otherwise removing a protein that is required directly or indirectly for homologue association before anaphase I.

Mapping EBNA-1 domains involved in binding to metaphase chromosomes.

Abstract: The Epstein-Barr virus (EBV) genome can persist in dividing human B cells as multicopy circular episomes. Viral episomes replicate in synchrony with host cell DNA and are maintained at a relatively constant copy number for a long time. Only two viral elements, the replication origin OriP and the EBNA-1 protein, are required for the persistence of viral genomes during latency. EBNA-1 activates OriP during the S phase and may also contribute to the partition and/or retention of viral genomes during mitosis. Indeed, EBNA-1 has been shown to interact with mitotic chromatin. Moreover, viral genomes are noncovalently associated with metaphase chromosomes. This suggests that EBNA-1 may facilitate the anchorage of viral genomes on cellular chromosomes, thus ensuring proper partition and retention. In the present paper, we have investigated the chromosome-binding activity of EBV EBNA-1, herpesvirus papio (HVP) EBNA-1, and various derivatives of EBV EBNA-1, fused to a variant of the green fluorescent protein. The results show that binding to metaphase chromosomes is a common property of EBV and HVP EBNA-1. Further studies indicated that at least three independent domains (CBS-1, -2, and -3) mediate EBNA-1 binding to metaphase chromosomes. In agreement with the anchorage model, two of these domains mapped to a region that has been previously demonstrated to be required for the long-term persistence of OriP-containing plasmids.

Direct imaging of DNA in living cells reveals the dynamics of chromosome formation.

Abstract: Individual chromosomes are not directly visible within the interphase nuclei of most somatic cells; they can only be seen during mitosis. We have developed a method that allows DNA strands to be observed directly in living cells, and we use it to analyze how mitotic chromosomes form. A fluorescent analogue (e.g., Cy5-dUTP) of the natural precursor, thymidine triphosphate, is introduced into cells, which are then grown on the heated stage of a confocal microscope. The analogue is incorporated by the endogenous enzymes into DNA. As the mechanisms for recognizing and removing the unusual residues do not prevent subsequent progress around the cell cycle, the now fluorescent DNA strands can be followed as they assemble into chromosomes, and segregate to daughters and granddaughters. Movies of such strands in living cells suggest that chromosome axes follow simple recognizable paths through their territories during G2 phase, and that late replicating regions maintain their relative positions as prophase chromosomes form. Quantitative analysis confirms that individual regions move little during this stage of chromosome condensation. As a result, the gross structure of an interphase chromosome territory is directly related to that of the prophase chromosome.

Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase

Abstract: During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes. We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion.

mini spindles: A gene encoding a conserved microtubule-associated protein required for the integrity of the mitotic spindle in Drosophila.

Abstract: We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.

Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90Rsk.

Abstract: Before fertilization, vertebrate eggs are arrested in metaphase of meiosis II by cytostatic factor (CSF), an activity that requires activation of the mitogen-activated protein kinase (MAPK) pathway. To investigate whether CSF arrest is mediated by the protein kinase p90Rsk, which is phosphorylated and activated by MAPK, a constitutively activated (CA) form of Rsk was expressed in Xenopus embryos. Expression of CA Rsk resulted in cleavage arrest, and cytological analysis showed that arrested blastomeres were in M phase with prominent spindles characteristic of meiotic metaphase. Thus, Rsk appears to be the mediator of MAPK-dependent CSF arrest in vertebrate unfertilized eggs.

The small GTP-binding protein rho regulates cortical activities in cultured cells during division

Abstract: We have investigated the role of the small GTP-binding protein Rho in cytokinesis by microinjecting an inhibitor, C3 ribosyltransferase, into cultured cells. Microinjection of C3 into prometaphase or metaphase normal rat kidney epithelial cells induced immediate and global cortical movement of actin toward the metaphase plate, without an apparent effect on the mitotic spindle. During anaphase, concentrated cortical actin filaments migrated with separating chromosomes, leaving no apparent concentration of actin filaments along the equator. Myosin II in injected epithelial cells showed a diffuse distribution throughout cell division. All treated, well-adherent cells underwent cleavage-like activities and most of them divided successfully. However, cytokinesis became abnormal, generating irregular ingressions and ectopic cleavage sites even when mitosis was blocked with nocodazole. The effects of C3 appeared to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibited irregular cortical ingression only when cells started to increase attachment during respreading, but managed to complete cytokinesis. Poorly adherent HeLa cells showed neither ectopic cleavage nor completion of cytokinesis. Our results indicate that Rho does not simply activate actin–myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex.

c-Myc Overexpression Uncouples DNA Replication from Mitosis

Abstract: c-myc has been shown to regulate G1/S transition, but a role for c-myc in other phases of the cell cycle has not been identified. Exposure of cells to colcemid activates the mitotic spindle checkpoint and arrests cells transiently in metaphase. After prolonged colcemid exposure, the cells withdraw from mitosis and enter a G1-like state. In contrast to cells in G1, colcemid-arrested cells have decreased G1 cyclin-dependent kinase activity and show hypophosphorylation of the retinoblastoma protein. We have found that overexpression of c-myc causes colcemid-treated human and rodent cells to become either apoptotic or polyploid by replicating DNA without chromosomal segregation. Although c-myc-induced polyploidy is not inhibited by wild-type p53 in immortalized murine fibroblasts, overexpression of c-myc in primary fibroblasts resulted in massive apoptosis of colcemid-treated cells. We surmise that additional genes are altered in immortalized cells to suppress the apoptotic pathway and allow c-myc-overexpressing cells to progress forward in the presence of colcemid. Our results also suggest that c-myc induces DNA rereplication in this G1-like state by activating CDK2 activity. These observations indicate that activation of c-myc may contribute to the genomic instability commonly found in human cancers.

Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase IIα localization and chromosome condensation

Abstract: Mechanisms of chromosome condensation and segregation during the first meiotic division are not well understood. Resolution of recombination events to form chiasmata is important, for it is chiasmata that hold homologous chromosomes together for their oppositional orientation on the meiotic metaphase spindle, thus ensuring their accurate segregation during anaphase I. Events at the centromere are also important in bringing about proper attachment to the spindle apparatus. This study was designed to correlate the presence and activity of two proteins at the centromeric heterochromatin, topoisomerase II alpha (TOP2A) and histone H3, with the processes of chromosome condensation and individualization of chiasmate bivalents in murine spermatocytes. We tested the hypothesis that phosphorylation of histone H3 is a key event instigating localization of TOP2A to the centromeric heterochromatin and condensation of chromosomes as spermatocytes exit prophase and progress to metaphase. Activity of topoisomerase II is required for condensation of chromatin at the end of meiotic prophase. Histone H3 becomes phosphorylated at the end of prophase, beginning with its phosphorylation at the centromeric heterochromatin in the diplotene stage. However, it cannot be involved in localization of TOP2A, since TOP2A is localized to the centromeric heterochromatin throughout most of meiotic prophase. This observation suggests a meiotic function for TOP2A in addition to its role in chromatin condensation. The use of kinase inhibitors demonstrates that phosphorylation of histone H3 can be uncoupled from meiotic chromosome condensation; therefore other proteins, such as those constituting metaphase-promoting factor, must be involved. These results define the timing of important meiotic events at the centromeric heterochromatin and provide insight into mechanisms of chromosome condensation for meiotic metaphase.

CENP-C is necessary but not sufficient to induce formation of a functional centromere.

Abstract: CENP-C is an evolutionarily conserved centromeric protein. We have used the chicken DT40 cell line to test the idea that CENP-C is sufficient as well as necessary for the formation of a functional centromere. We have compared the effects of disrupting the localization of CENP-C with those of inducibly overexpressing the protein. Removing CENP-C from the centromere causes disassembly of the centromere protein complex and blocks cells at the metaphase-anaphase junction. Overexpressed CENP-C is associated with an increase in errors of chromosome segregation and inhibits the completion of mitosis. However, the excess CENP-C does not disrupt the native centromeres detectably and does not associate with another conserved centromere protein, ZW10. The distribution of the excess CENP-C changes during the cell cycle. In metaphase, the excess CENP-C coats the chromosome arms. At the metaphase-anaphase transition, the excess CENP-C clusters, and during interphase it is present in large bodies which form around pre-existing centromeres which are also clustered. These results indicate that CENP-C is necessary but not sufficient for the formation of a functional centromere and suggest that the structure of CENP-C may be regulated during the cell cycle.

The maize homologue of the cell cycle checkpoint protein MAD2 reveals kinetochore substructure and contrasting mitotic and meiotic localization patterns.

Abstract: We have identified a maize homologue of yeast MAD2, an essential component in the spindle checkpoint pathway that ensures metaphase is complete before anaphase begins. Combined immunolocalization of MAD2 and a recently cloned maize CENPC homologue indicates that MAD2 localizes to an outer domain of the prometaphase kinetochore. MAD2 staining was primarily observed on mitotic kinetochores that lacked attached microtubules; i.e., at prometaphase or when the microtubules were depolymerized with oryzalin. In contrast, the loss of MAD2 staining in meiosis was not correlated with initial microtubule attachment but was correlated with a measure of tension: the distance between homologous or sister kinetochores (in meiosis I and II, respectively). Further, the tension-sensitive 3F3/2 phosphoepitope colocalized, and was lost concomitantly, with MAD2 staining at the meiotic kinetochore. The mechanism of spindle assembly (discussed here with respect to maize mitosis and meiosis) is likely to affect the relative contributions of attachment and tension. We support the idea that MAD2 is attachment-sensitive and that tension stabilizes microtubule attachments.

Human herpesvirus-8-encoded LNA-1 accumulates in heterochromatin- associated nuclear bodies.

Abstract: Subnuclear distribution of the human herpesvirus-8 (HHV-8)- encoded nuclear protein LNA-1 was analysed at high resolution in body cavity (BC) lymphoma-derived cell lines, in cell hybrids between BC cells and various human and mouse cells and in freshly infected K562 and ECV cell lines. Three-dimensional reconstruction of nuclei from optical sections and quantitative analysis of the distribution of LNA-1 fluorescence in relation to chromatin showed that LNA-1 associates preferentially with the border of heterochromatin in the interphase nuclei. This was further confirmed in the following systems: in endo- and exonuclease-digested nuclei, in human–mouse (BC-1–Sp2- 0) hybrids and on chromatin spreads. LNA-1 was found to bind to mitotic chromosomes at random. Epstein–Barr virus (EBV), but not HHV-8, was rapidly lost from mouse–human hybrid cells in parallel with the loss of human chromosomes. HHV-8 could persist on the residual mouse background for more than 8 months. In early human–mouse hybrids that contain a single fused nucleus, LNA-1 preferentially associates with human chromatin. After the gradual loss of the human chromosomes, LNA-1 becomes associated with the murine pericentromeric heterochromatin. In human–human hybrids derived from the fusion of the HHV-8-carrying BCBL-1 cells and the EBV-immortalized lymphoblastoid cell line IB4, LNA-1 did not co-localize with EBNA-1, EBNA-2, EBNA-5 or EBNA-6. LNA-1 was not associated with PML containing ND10 bodies either. DNase but not RNase or detergent treatment of isolated nuclei destroys LNA-1 bodies. In advanced apoptotic cells LNA- 1 bodies remain intact but are not included in the apoptotic bodies themselves.

Localization of single- and low-copy sequences on tomato synaptonemal complex spreads using fluorescence in situ hybridization (FISH).

Abstract: Fluorescence in situ hybridization (FISH) is a powerful means by which single- and low-copy DNA sequences can be localized on chromosomes. Compared to the mitotic metaphase chromosomes that are normally used in FISH, synaptonemal complex (SC) spreads (hypotonically spread pachytene chromosomes) have several advantages. SC spreads (1) are comparatively free of debris that can interfere with probe penetration, (2) have relatively decondensed chromatin that is highly accessible to probes, and (3) are about ten times longer than their metaphase counterparts, which permits FISH mapping at higher resolution. To investigate the use of plant SC spreads as substrates for single-copy FISH, we probed spreads of tomato SCs with two single-copy sequences and one low-copy sequence (ca. 14 kb each) that are associated with restriction fragment length polymorphism (RFLP) markers on SC 11. Individual SCs were identified on the basis of relative length, arm ratio, and differential staining patterns after combined propidium iodide (PI) and 4', 6-diamidino-2-phenylindole (DAPI) staining. In this first report of single-copy FISH to SC spreads, the probe sequences were unambiguously mapped on the long arm of tomato SC 11. Coupled with data from earlier studies, we determined the distance in micrometers, the number of base pairs, and the rates of crossing over between these three FISH markers. We also observed that the order of two of the FISH markers is reversed in relation to their order on the molecular linkage map. SC-FISH mapping permits superimposition of markers from molecular linkage maps directly on pachytene chromosomes and thereby contributes to our understanding of the relationship between chromosome structure, gene activity, and recombination.

Retention of the Bub3 checkpoint protein on lagging chromosomes

Abstract: Accurate chromosome segregation at mitosis is ensured both by the intrinsic fidelity of the mitotic machinery and by the operation of checkpoints that monitor chromosome-microtubule attachment. When unattached kinetochores are present, anaphase is delayed and the time available for chromosome-microtubule capture increases. Genes required for this delay first were identified in budding yeast (the MAD and BUB genes), but it is not yet known how the checkpoint senses unattached chromosomes or how it signals cell-cycle arrest. We report the isolation and analysis of a murine homologue of BUB3, a gene whose deletion abolishes mitotic checkpoint function in Saccharomyces cerevisiae. mBub3 belongs to a small gene family that has been highly conserved through evolution. By expressing recombinant proteins in insect cells, we show that mBub3, like yeast Bub3p, binds to Bub1 to form a complex with protein kinase activity. During prophase and prometaphase, preceding kinetochore-microtubule attachment, Bub3 localizes to kinetochores. High levels of mBub3 remain associated with lagging chromosomes but not with correctly aligned chromosomes during metaphase, consistent with a role for Bub3 in sensing microtubule attachment. Intriguingly, the number of lagging chromosomes with high Bub3 staining increases dramatically in cells treated with low (and pharmacologically relevant) concentrations of the chemotherapeutic taxol and the microtubule poison nocodazole.

Rapid visualization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs.

Abstract: The present study was aimed to facilitate karyotyping of human blastomeres using the metaphase-inducing factors present in unfertilized eggs. A rapid technique for karyotyping would have wide application in the field of preimplantation genetic diagnosis. When cryopreserved invitro matured bovine oocytes were fused with human blastomeres, the transferred human nuclei were forced into metaphase within a few hours. Eighty-seven human blastomeres from abnormal or arrested embryos were fused with bovine oocytes in a preclinical study. Fusion efficiency was 100%. In 21 of the hybrid cells, no trace of human chromatin was found. Of the remaining 66, 64 (97%) yielded chromosomes suitable for analysis. The method was used to karyotype embryos from two patients with maternal translocations. One embryo which was judged to be karyotypically normal was replaced in the first patient, resulting in one pregnancy with a normal fetus. None of the second patient’s embryos was diagnosed as normal, and hence none was transferred. The results of the present study demonstrated that the ooplasmic factors which induce and maintain metaphase in bovine oocytes can force transferred human blastomere nuclei into premature metaphase, providing the basis for a rapid method of karyotyping blastomeres from preimplantation embryos and, by implication, cells from other sources.

A mutant deubiquitinating enzyme (Ubp-M) associates with mitotic chromosomes and blocks cell division

Abstract: A new ubiquitin-processing protease (Ubp-M) has been identified in mammalian cells that is phosphorylated at the onset of mitosis and dephosphorylated during the metaphase/anaphase transition. The carboxyl-terminal domain of this 823-aa protein can be phosphorylated in vitro with either extracts of mitotic cells or purified cdc-2/cyclin B complexes. Recombinant Ubp-M is able to deubiquitinate histone H2A in vitro, and the phosphorylated form is also enzymatically active. Wild-type Ubp-M, transiently expressed as green fluorescent protein-fusion proteins, localizes in the cytoplasm of cultured cells, but mutant forms, lacking an active-site cysteine, associate closely with mitotic chromosomes during all stages of cell division and remain within the nucleus during the postmitotic period. Cells transfected with plasmids containing mutant Ubp-M genes stop dividing and eventually undergo apoptosis. Ubp-M may deubiquitinate one or more critical proteins that are involved in the condensation of mitotic chromosomes, possibly acting selectively on histones H2A and H2B, the major ubiquitinated proteins of chromatin.

Chromosomes as well as chromosomal subdomains constitute distinct units in interphase nuclei

Abstract: Fluorescence in situ hybridization has demonstrated that chromosomes form individual territories in interphase nuclei. However, this technique is not suitable to determine whether territories are mutually exclusive or interwoven. This notion, however, is essential for understanding functional organizations in the cell nucleus. Here, we analyze boundary areas of individual chromosomes during interphase using a sensitive method based on replication labeling and immunocytochemistry. Thymidine analogues IdUrd and CldUrd were incorporated during S-phase into DNA of Chinese Hamster fibroblasts. Cells labeled with IdUrd were fused with cells labeled with CldUrd. Fused nuclei contained both IdUrd or CldUrd labeled chromosomes. Alternatively, the two labels were incorporated sequentially during successive S-phases and segregated to separate chromosomes by culturing the cells one more cell cycle. Metaphase spreads showed IdUrd-, CldUrd- and unlabeled chromosomes. Some chromatids were divided sharply in differently labeled subdomains by sister chromatid exchanges. With both methods, confocal imaging of interphase nuclei revealed labeled chromosomal domains containing fiber-like structures and unlabeled areas. At various sites, fiber-like structures were embedded in other territories. Even so, essentially no overlap between chromosome territories or between subdomains within a chromosome was observed. These observations indicate that chromosome territories and chromosomal subdomains in G(1)-phase are mutually exclusive at the resolution of the light microscope.