Showing papers on "Metaphase published in 1990"

A view of interphase chromosomes.

Abstract: Metaphase chromosomes are dynamically modified in interphase. This review focuses on how these structures can be modified, and explores the functional mechanisms and significance of these changes. Current analyses of genes often focus on relatively short stretches of DNA and consider chromatin conformations that incorporate only a few kilobases of DNA. In interphase nuclei, however, orderly transcription and replication can involve highly folded chromosomal domains containing hundreds of kilobases of DNA. Specific "junk" DNA sequences within selected chromosome domains may participate in more complex levels of chromosome folding, and may index different genetic compartments for orderly transcription and replication. Three-dimensional chromosome positions within the nucleus may also contribute to phenotypic expression. Entire chromosomes are maintained as discrete, reasonably compact entities in the nucleus, and heterochromatic coiled domains of several thousand kilobases can acquire unique three-dimensional positions in differentiated cell types. Some aspects of neoplasia may relate to alterations in chromosome structure at several higher levels of organization.

Interphase and metaphase resolution of different distances within the human dystrophin gene

Abstract: Fluorescence in situ hybridization makes possible direct visualization of single sequences not only on chromosomes, but within decondensed interphase nuclei, providing a potentially powerful approach for high-resolution (1 Mb and below) gene mapping and the analysis of nuclear organization. Interphase mapping was able to extend the ability to resolve and order sequences up to two orders of magnitude beyond localization on banded or unbanded chromosomes. Sequences within the human dystrophin gene separated by less than 100 kb to 1 Mb were visually resolved at interphase by means of standard microscopy. In contrast, distances in the 1-Mb range could not be ordered on the metaphase chromosome length. Analysis of sequences 100 kb to 1 Mb apart indicates a strong correlation between interphase distance and linear DNA distance, which could facilitate a variety of gene-mapping efforts. Results estimate chromatin condensation up to 1 Mb and indicate a comparable condensation for different cell types prepared by different techniques.

Lamins A and C bind and assemble at the surface of mitotic chromosomes.

Abstract: To study a possible interaction of nuclear lamins with chromatin, we examined assembly of lamins A and C at mitotic chromosome surfaces in vitro. When a postmicrosomal supernatant of metaphase CHO cells containing disassembled lamins A and C is incubated with chromosomes isolated from mitotic Chinese hamster ovary cells, lamins A and C undergo dephosphorylation and uniformly coat the chromosome surfaces. Furthermore, when purified rat liver lamins A and C are dialyzed with mitotic chromosomes into a buffer of physiological ionic strength and pH, lamins A and C coat chromosomes in a similar fashion. In both cases a lamin-containing supramolecular structure is formed that remains intact when the chromatin is removed by digestion with micrococcal nuclease and extraction with 0.5 M KCl. Lamins associate with chromosomes at concentrations approximately eightfold lower than the critical concentration at which they self-assemble into insoluble structures in the absence of chromosomes, indicating that chromosome surfaces contain binding sites that promote lamin assembly. These binding sites are destroyed by brief treatment of chromosomes with trypsin or micrococcal nuclease. Together, these data suggest the existence of a specific lamin-chromatin interaction in cells that may be important for nuclear envelope reassembly and interphase chromosome structure.

Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes.

Abstract: A procedure for mapping small DNA probes directly on banded human chromosomes by fluorescence in situ hybridization has been developed. This procedure allows for the simultaneous visualization of banded chromosomes and hybridization signal without overlaying two separate photographic images. This method is simple and rapid, requires only a typical fluorescence microscope, has proven successful with DNA probes as small as 1 kilobase, is applicable for larger probes, and will greatly facilitate mapping the vast number of probes being generated to study genetic disease and define the human genome. Human metaphase chromosomes were prepared from phytohemagglutinin-stimulated lymphocyte cultures synchronized with bromodeoxyuridine and thymidine. Probes were labeled with biotin-dUTP, and the hybridization signal was amplified by immunofluorescence. Chromosomes were stained with both propidium iodide and 4',6-diamidino-2-phenylindole (DAPI), producing R- and Q-banding patterns, respectively, allowing unambiguous chromosome and band identification while simultaneously visualizing the hybridization signal. Thirteen unique DNA segments have been localized to the long arm of chromosome 11 by using this technique, and localization of 10 additional probes by using radioactive in situ hybridization provides a comparison between the two procedures. These DNA segments have been mapped to all long-arm bands on chromosome 11 and in regions associated with neoplasias and inherited disorders.

Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts.

Abstract: Global Ca2+ transients have been observed to precede nuclear envelope breakdown and the onset of anaphase in Swiss 3T3 fibroblasts in 8% (vol/vol) FBS. The occurrence of these Ca2+ transients was dependent on intracellular stores. These Ca2+ transients could be (a) abolished by serum removal without halting mitosis, and (b) eliminated by increasing intracellular Ca2+ buffering capacity through loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffer, via the tetra(acetoxymethyl) ester, without hindering the transition into anaphase. Microinjection of sufficient concentrations of BAPTA buffer could block nuclear envelope breakdown. Pulses of Ca2+ generated by flash photolysis of intracellularly trapped nitr-5, a "caged" Ca2+, could precipitate precocious nuclear envelope breakdown in prophase cells. In metaphase cells, photochemically generated Ca2+ pulses could cause changes in the appearance of the chromosomes, but the length of time required for cells to make the transition from metaphase to anaphase remained essentially unchanged regardless of whether a Ca2+ pulse was photoreleased during metaphase. The results from these photorelease experiments were not dependent on the presence of serum in the medium. Discharging intracellular Ca2+ stores with ionomycin in the presence of 1.8 mM extracellular Ca2+ doubled the time for cells to pass from late metaphase into anaphase, whereas severe Ca2+ deprivation by treatment with ionomycin in EGTA-containing medium halted mitosis. Our results collectively indicate that Ca2+ is actively involved in nuclear envelope breakdown, but Ca2+ signals are likely unnecessary for the metaphase-anaphase transition in Swiss 3T3 fibroblasts. Additional studies of intracellular Ca2+ concentrations in mitotic REF52 and PtK1 cells revealed that Ca2+ transients are not observed at all mitotic stages in all cells. The absence of observable global Ca2+ transients, where calcium buffers can block and pulses of Ca2+ can advance mitotic stages, may imply that the relevant Ca2+ movements are too local to be detected.

Rapid metaphase and interphase detection of radiation-induced chromosome aberrations in human lymphocytes by chromosomal suppression in situ hybridization.

Abstract: Chromosomal in situ suppression (CISS)-hybridization of biotinylated phage DNA-library inserts from sorted human chromosomes was used to decorate chromosomes 1 and 7 specifically from pter to qter and to detect structural aberrations of these chromosomes in irradiated human peripheral lymphocytes. In addition, probe pUC1.77 was used to mark the Iq12 subregion in normal and aberrant chromosomes 1. Low LET radiation (60Co-γ-rays; 1.17 and 1.33 MeV) of lymphocyte cultures was performed with various doses (D = 0, 2, 4, 8 Gy) 5 h after stimulation with phytohaemagglutinin. Irradiated cells were cultivated for an additional 67 h before Colcemid arrested metaphase spreads were obtained. Aberrations of the specifically stained chromosomes, such as deletions, dicentrics, and rings, were readily scored after in situ hybridization with either the 1q12 specific probe or DNA-library inserts. By the latter approach, translocations of the specifically stained chromosomes could also be reliably assessed. A linear increase of the percentage of specifically stained aberrant chromosomes was observed when plotted as a function of the square of the dose D. A particular advantage of this new approach is provided by the possibility to delineate numerical and structural chromosome aberrations directly in interphase nuclei. These results indicate that cytogenetic monitoring of ionizing radiation may be considerably facilitated by CISS-hybridization.

Injection of anticentromere antibodies in interphase disrupts events required for chromosome movement at mitosis.

Abstract: We have used autoantibodies to probe the function of three human centromere proteins in mitosis. These antibodies recognize three human polypeptides in immunoblots: CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD). Purified anticentromere antibodies (ACA-IgG) disrupt mitosis when introduced into tissue culture cells during interphase. We have identified two execution points for antibody inhibition. Antibodies injected into the nucleus greater than or equal to 3 h before mitosis prevent the chromosomes from undergoing normal prometaphase movements in the subsequent mitosis. Antibodies injected in the nucleus during late G2 cause cells to arrest in metaphase. Surprisingly, antibodies introduced subsequent to the beginning of prophase do not block mitosis. These results suggest that the CENP antigens are involved in two essential interphase events that are required for centromere action in mitosis. These may include centromere assembly coordinate with the replication of alpha-satellite DNA at the end of S phase and the structural maturation of the kinetochore that begins at prophase.

Mutations at the asp locus of Drosophila lead to multiple free centrosomes in syncytial embryos, but restrict centrosome duplication in larval neuroblasts.

Abstract: Mutations at abnormal spindle result in abnormally long and wavy microtubules in the meiotic spindles of males. Some of these spindles have a single pole and take the form of unopposed hemi-spindles. Unfertilised eggs produced by homozygous asp females may have either no nuclei, or a small number of large nuclei, consistent with there also being an effect upon female meiosis. Such eggs also display free centrosomes and independent arrays of microtubules. Embryos that have this phenotype are also present among the progeny of fertilised homozygous asp females, together with embryos that undergo varying degrees of aberrant morphogenesis, developing a variety of abnormal cuticle patterns. This latter category shows asynchronous mitoses prior to cellularisation, and has abnormal arrays of spindle microtubules. Such embryos can develop large areas that are either devoid of or have a reduced number of nuclei, in which there are centrosomes that have dissociated from the mitotic spindles. Neuroblasts in the brains of homozygous asp larvae display a high mitotic index, and have condensed chromosomes aligned as if blocked at metaphase. Immunostaining reveals that many cells contain a single centrosome connected to the metaphase chromosomes by microtubules in a hemi-spindle-like structure.

A unified model of eukaryotic chromosomes.

Abstract: A revised model of DNA packaging into chromosomes is presented. Its features are consistent with observed structural dimensions and the molecular periodicities related to transcription, replication and matrix attachment domains. The transitions between euchromatic, heterochromatic and metaphase states are explained simply. Molecular and physical properties of chromosomal bands, and their correlation with specific DNA sequence motifs are discussed.

Immunofluorescence study of the action of navelbine, vincristine and vinblastine on mitotic and axonal microtubules.

Abstract: Among the various non-naturally-occurring Vinca alkaloid compounds, nor-anhydro-vinblastine (Navelbine, NVB) exhibits in preliminary clinical studies broader anti-tumor activity and lower neurotoxicity than vinblastine (VBL) and vincristine (VCR). The action of these 3 Vinca alkaloids on axonal and mitotic microtubules has been studied experimentally in a specific model, the tectal plate anlage of mouse embryos at the earliest stages of neuronal differentiation. Post-implantation embryos were cultured in toto in a medium containing increasing concentrations of drugs. Microtubules were stained using immunofluorescence with a tubulin-specific polyclonal antibody in semi-thin sections after embedding in high-molecular-weight polyethylene glycol. All drugs induced depolymerization of mitotic interpolar microtubules and cell metaphase block at the same concentration. Increasing the concentrations led to progressive depolymerization of kinetochore microtubules. However, NVB was the only drug to induce complete microtubule depolymerization. The activity of the 3 compounds on axonal microtubules was identical: depolymerization of a labile pool of microtubules. This was observed at higher concentrations with NVB than with the 2 other Vinca alkaloids. Our results show that, in this model, NVB is as active on mitotic microtubules as VCR and VBL, and less active on axonal microtubules. None of the 3 drugs modified microtubule length but all appeared to induce disruption of the labile microtubule pool without altering the stable pool.

A monoclonal antibody to a mitotic microtubule-associated protein blocks mitotic progression.

Abstract: A monoclonal antibody raised against mitotic spindles isolated from CHO cells ([CHO1], Sellitto, C., and R. Kuriyama. 1988. J. Cell Biol. 106:431-439) identifies an epitope that resides on polypeptides of 95 and 105 kD and is localized in the spindles of diverse organisms. The antigen is distributed throughout the spindle at metaphase but becomes concentrated in a progressively narrower zone on either side of the spindle midplane as anaphase progresses. Microinjection of CHO1, either as an ascites fluid or as purified IgM, results in mitotic inhibition in a stage-specific and dose-dependent manner. Parallel control injections with nonimmune IgMs do not yield significant mitotic inhibition. Immunofluorescence analysis of injected cells reveals that those which complete mitosis display normal localization of CHO1, whereas arrested cells show no specific localization of the CHO1 antigen within the spindle. Immunoelectron microscopic images of such arrested cells indicate aberrant microtubule organization. The CHO1 antigen in HeLa cell extracts copurifies with taxol-stabilized microtubules. Neither of the polypeptides bearing the antigen is extracted from microtubules by ATP or GTP, but both are approximately 60% extracted with 0.5 M NaCl. Sucrose gradient analysis reveals that the antigens sediment at approximately 11S. The CHO 1 antigen appears to be a novel mitotic MAP whose proper distribution within the spindle is required for mitosis. The properties of the antigen(s) suggest that the corresponding protein(s) are part of the mechanism that holds the antiparallel microtubules of the two interdigitating half spindles together during anaphase.

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes.

Abstract: Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.

Ca2+/calmodulin-dependent protein kinase II: localization in the interphase nucleus and the mitotic apparatus of mammalian cells.

Abstract: Indirect immunofluorescence was used to determine the distribution of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in rat embryo fibroblast 3Y1 cells, rat C6 glioma cells, and human epidermoid carcinoma KB cells. During interphase at growing phase, CaM kinase II was localized diffusely in the cytoplasm and in the nucleus. In the nucleus, the enzyme was localized within the whole nuclear matrix in which the enzyme was specially concentrated in nucleoli. During mitosis, CaM kinase II was found to be a dynamic component of the mitotic apparatus, particularly present at microtubule-organizing centers. In metaphase and anaphase, CaM kinase II was observed at centrosomes and between the spindle poles. During telophase, CaM kinase II was condensed as a bright fluorescent dot at the midzone of the intercellular bridge between two daughter cells, while tubulin was found at each side of the midbody. Colchicine, a microtubule inhibitor, disorganized the tubulin- and CaM kinase II specific fluorescent structure of mitotic 3Y1 cells. In cold-treated cells, CaM kinase II was localized predominantly at centrosomes. The localization of CaM kinase II in the cell nucleus and the mitotic apparatus suggests that the enzyme may play a role in the cell cycle progression of mammalian cells.

Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos.

Abstract: The spatial and temporal dynamics of diploid chromosome organization, microtubule arrangement, and the state of the nuclear envelope have been analyzed in syncytial blastoderm embryos of Drosophila melanogaster during the transition from prophase to metaphase, by three-dimensional optical sectioning microscopy. Time-lapse, three-dimensional data recorded in living embryos revealed that congression of chromosomes (the process whereby chromosomes move to form the metaphase plate) at prometaphase occurs as a wave, starting at the top of the nucleus near the embryo surface and proceeding through the nucleus to the bottom. The time-lapse analysis was augmented by a high-resolution analysis of fixed embryos where it was possible to unambiguously trace the three-dimensional paths of individual chromosomes. In prophase, the centromeres were found to be clustered at the top of the nucleus while the telomeres were situated at the bottom of the nucleus or towards the embryo interior. This polarized centromere-telomere orientation, perpendicular to the embryo surface, was preserved during the process of prometaphase chromosome congression. Correspondingly, breakdown of the nuclear envelope started at the top of the nucleus with the mitotic spindle being formed at the positions of the partial breakdown of the nuclear envelope. Our observation provide an example in which nuclear structures are spatially organized and their functions are locally and coordinately controlled in three dimensions.

Stability of microtubule attachment to metaphase kinetochores in PtK1 cells.

Abstract: Kinetochore microtubules are known to be differentially stable to a variety of microtubule depolymerization agents compared to the non-kinetochore polar microtubules, but the dynamics of microtubule attachment to the kinetochore is currently controversial. We have examined the stability of kinetochore microtubules in metaphase PtK1 spindles at 23 degrees C when microtubule assembly is abruptly blocked with the drug nocodazole. Metaphase cells were incubated in medium containing 34 microM nocodazole for various times before fixation and processing either for immunofluorescence light microscopy or serial-section electron microscopy. Microtubules not associated with kinetochore fibers disappeared completely in less than 1 min. Kinetochore fibers persisted and shortened, as the spindle poles moved close to the chromosomes over a 10–20 min interval. During this shortening process, the number of kinetochore microtubules decreased slowly. The mean number of kinetochore microtubules was 24 +/− 5 in control cells and zero in cells incubated with nocodazole for 20 min. The half-time of microtubule attachment to the kinetochore was approximately 7.5 min. These results show that when microtubule assembly is blocked, kinetochore microtubules shorten more slowly and persist about 10 times longer than the labile polar microtubules. If kinetochore microtubules shorten by tubulin dissociation at their plus-ends like the non-kinetochore polar microtubules, then the microtubule surface lattice must be able to translocate through the kinetochore attachment site without frequent detachment occurring.

p34cdc2 kinase is localized to distinct domains within the mitotic apparatus.

Abstract: Antibodies to both the C-terminal and the N-terminal regions of the 34 kd serine-threonine specific protein kinase, p34cdc2, were used to study the distribution of this protein in dividing cells and isolated chromosomes of the Indian muntjac. p34cdc2 was found to be present throughout the cytoplasm of dividing cells. In addition, a portion of cellular p34cdc2 was localized to the centrosome, kinetochore, and intercellular bridge and along kinetochore-to-pole microtubules during cell division. Tubulin-denuded metaphase kinetochores retained their association with p34cdc2. The detection of p34cdc2 within a variety of domains of the mitotic apparatus, in addition to the previous reported association with the centrosome [Bailly et al., EMBO J. 8:3985-3995, 1989; Raibowol et al., Cell 57:393-401, 1989] suggests that p34cdc2 may play a role in events associated with anaphases A and B as well as with the transition between interphase and mitosis.

Centriole and centrosome cycle in the early Drosophila embryo.

Abstract: Centriole and centrosome cycles were examined by indirect immunofluorescence and electron microscopy techniques in the early Drosophila embryo. The centrosomes, which are already divided at interphase, appear as compact spheres during prophase and metaphase, expand and flatten from anaphase to telophase and split into two units in late telophase. Centriole separation starts in late metaphase, becomes evident in anaphase and increases during telophase. Procentrioles appear during the following interphase.

The receptors for prolactin and growth hormone are localized in the same region of human chromosome 5

Abstract: Prolactin receptor (PRLR) and growth hormone receptor (GHR) are encoded by members of a gene family containing regions of identical sequences. To determine their chromosomal locations, cDNA probes for these genes were used. Analysis of hybridization to several somatic cell hybrids, together with hybridization in situ to metaphase chromosomes, resulted in the assignment of the loci for both receptors to human chromosome 5 in the region p13----p14. Thus, these proteins may be encoded by a cluster of related genes that evolved from a common ancestral gene, in a manner consonant with that of their ligands.

UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.

Abstract: Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation.

Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and L1 primers.

Abstract: Specific amplification of human sequences of up to several kb length has recently been accomplished in man-hamster and man-mouse somatic hybrid cell DNA by IRS-PCR (interspersed repetitive sequence — polymerase chain reaction). This approach is based on oligonucleotide primers that anneal specifically to human Alu- or L1-sequences and allows the amplification of any human sequences located between adequately spaced, inverted Alu- or L1-blocks. Here, we demonstrate that probe pools generated from two somatic hybrid cell lines by Alu- and L1-PCR can be used for chromosome painting in normal human lymphocyte metaphase spreads by chromosomal in situ suppression (CISS-) hybridization. The painted chromosomes and chromosome subregions directly represent the content of normal and deleted human chromosomes in the two somatic hybrid cell lines. The combination of IRS-PCR and CISS-hybridization will facilitate and improve the cytogenetic analysis of somatic hybrid cell panels, in particular, in cases where structurally aberrant human chromosomes or human chromosome segments involved in interspecies translocations cannot be unequivocally identified by classical banding techniques. Moreover, this new approach will help to generate probe pools for the specific delineation of human chromosome subregions for use in cytogenetic diagnostics and research without the necessity of cloning.

Prophase of meiosis in human spermatocytes analysed by EM microspreading in infertile men and their controls and comparisons with human oocytes.

Abstract: Observations at the electron microscope (EM) level have been made on 1883 primary spermatocytes from 40 chromosomally normal subfertile men and 566 spermatocytes from 10 fertile controls, using the technique of microspreading. Spermatocytes of infertile men in general showed greater indications of degeneration including higher levels of background silver deposition, nucleolar organising region — XY associations, fragmentation of synaptonemal complexes and overproduction of XY excrescences. A few oligospermic men also showed an immature morphology of the XY pair and/or a reduced extent of XY synapsis. Dissociation of the sex chromosome axes at prophase was found to occur with a much lower frequency than that recorded for separated X and Y chromosomes at metaphase I. In a single spermatocyte, synaptonemal complex formation was observed between Xqter and Yqter, a situation that could enable rare XqYq interchange. A proteinaceous stalked body exists on the Y axis towards its non-pairing end; this structure might have a functional relationship with the gene for spermatogenesis, (AZF), located at the euchromatin/heterochromatin interface. Compared with human oocytes, spermatocytes show fewer anomalies of synapsis, i.e. asynapsed segments or whole axes, non-homologous associations, interchanges, interlocks. These latter data agree well with findings from the mouse.

A comparison of the effects of various spindle toxins on metaphase arrest and formation of micronuclei in cell-suspension cultures ofNicotiana plumbaginifolia.

Abstract: The effects of the spindle toxins colchicine, oryzalin and amiprophos-methyl (APM) on metaphase arrest, chromosome scattering, and on the induction and yield of micronuclei were compared in suspension cells ofNicotiana plumbaginifolia (kanamycin-resistant “Doba” line). The inhibition of spindle formation is stronger with oryzalin and APM than with colchicine, which resulted in a more efficient accumulation of meta-phases with well-scattered chromosomes, allowing the isolation of single chromosomes. Further, APM and oryzalin treatments resulted in a higher frequency of micro-nucleated cells and greater yield of micronuclei than after colchicine treatment. The different actions of the chemicals on the functioning of the spindle, development of nuclear membranes around the chromosomes, formation of micronuclei and fusion of micronuclei, resulting in restitution nuclei, are discussed.

Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.

Abstract: After ovulation mammalian oocytes arrest in second meiotic metaphase. We asked whether the microtubules that comprise the meiotic spindle of mouse oocytes were stable or were undergoing rapid cycles of assembly and disassembly. Porcine brain tubulin, derivatized with biotin or x-rhodamine [5- (and -6)-carboxy-x-rhodamine], was microinjected into living oocytes. Biotinylated tubulin incorporated into the meiotic spindle to apparent equilibrium within 15 min. To assess quantitatively the rates of disassembly and assembly of the microtubules, small domains within the spindles of oocytes injected with x-rhodamine-tubulin were photobleached and their recovery was analyzed by digital imaging microscopy. Fluorescence recovery in the spindles was rapid and extensive, plateauing to an average of 83% at 4 min. The calculated half-time for turnover of the spindle microtubules was 77 sec. In contrast, fluorescence recovery of the spindle midbodies in telophase oocytes was much more limited, averaging approximately 22% at 4 min. These data indicate that most microtubules within the arrested metaphase spindle of the mouse oocyte undergo rapid cycles of assembly and disassembly. Microtubules of the telophase midbody are more stable.