Showing papers on "Metaphase published in 1983"

Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components

Abstract: A cell-free preparation of the cytoplasm from activated eggs of Rana pipiens induces, in demembranated sperm nuclei of Xenopus laevis, formation of a nuclear envelope, chromatin decondensation, initiation of DNA synthesis, and chromosome condensation. Both soluble and particulate cytoplasmic constituents are required to initiate these processes in vitro. The observed changes resemble processes occurring during fertilization and the mitotic cycle in early amphibian embryos. Therefore, this cell-free system may be useful in biochemical analysis of the interactions of nucleus and cytoplasm that control nuclear behavior.

Architecture of metaphase chromosomes and chromosome scaffolds.

Abstract: We have developed procedures for depositing intact mitotic chromosomes and isolated residual scaffolds on electron microscope grids at controlled and reproducible levels of compaction. The chromosomes were isolated using a recently developed aqueous method. Our study has addressed two different aspects of chromosome structure. First, we present a method for improved visualization of radial chromatin loops in undisrupted mitotic chromosomes. Second, we have visualized a nonhistone protein residual scaffold isolated from nuclease-digested chromosomes under conditions of low salt protein extraction. These scaffolds, which have an extremely simple protein composition, are the size of chromosomes, are fibrous in nature, and are found to retain differentiated regions that appear to derive from the kinetochores and the chromatid axis. When our standard preparation conditions were used, the scaffold appearance was found to be very reproducible. If the ionic conditions were varied, however, the scaffold appearance underwent dramatic changes. In the presence of millimolar concentrations of Mg++ or high concentrations of NaCl, the fibrous scaffold protein network was observed to undergo a lateral aggregation or assembly into a coarse meshlike structure. The alteration of scaffold structure was apparently reversible. This observation is consistent with a model in which the scaffolding network plays a dynamic role in chromosome condensation at mitosis.

Pre-B cells in mouse bone marrow: immunofluorescence stathmokinetic studies of the proliferation of cytoplasmic mu-chain-bearing cells in normal mice.

Abstract: By using a technique that combines metaphase arrest with immunofluorescence labeling, the proliferation of specifically identified pre-B cells in mouse bone marrow has been analyzed under physiological conditions in vivo. Pre-B cells bearing cytoplasmic mu-chains and no surface mu-chains constituted 12% of marrow nucleated cells, or 27 X 10(5) cells/femur, whereas surface mu-bearing B lymphocytes totaled 33 X 10(5) cells/femur. Pre-B cells measured 7 to 14 micron in diameter, the small number seen in metaphase (1 to 2%) being large cells (greater than 10 microns). After vincristine injection, the metaphase incidence (Imet) of pre-B cells increased with cell size; a broad-dose range of vincristine gave similar Imet values. Mitoses were arrested for 4 hr with no apparent cell death. Linear regression analysis of the increase in Imet of pre-B cells 2 to 4 hr after vincristine revealed a rate of entry into mitosis of 6.3%/hr, relative to all pre-B cells (average compartment turnover time, 16 hr), and 15.3%/hr for the large proliferating pre-B cell subset. This represented a pre-B cell production of 1.3 X 10(5) cells/femoral shaft/hr or 0.5 X 10(8) cells/whole bone marrow organ/day, emphasizing the magnitude of B lymphocyte genesis in normal bone marrow. Combined with reported renewal rates for small pre-B cells and small B lymphocytes, these values form a kinetic model of B lymphocyte development. The results reveal an apparent overproduction of large pre-B cells, consistent with a speculative post-mitotic loss of some immature primary B lymphocytes.

Chromatin organization and restriction endonuclease activity on human metaphase chromosomes

Abstract: Human metaphase chromosomes were treated with Haelll, Hindlll, EcoRI, and Alul restriction endonucleases and subsequently stained with either Giemsa or ethidium bromide. The results obtained seemed to suggest that the structural organization of specific chromosome regions can play a primary role in determining the cytological effect after digestion with specific restriction endonucleases.

The role of calcium ions during mitosis. Calcium participates in the anaphase trigger.

Abstract: Calcium-containing solutions were microinjected into dividing PtK1 cells to assess the effect of calcium ion concentration on the morphology and physiology of the mitotic spindle. Solutions containing 50 μM or more CaCl2 are immediately and irreversibly toxic to PtK1 cells. Those containing 5–10 μM CaCl2 cause reversible reduction in spindle birefringence followed by normal anaphase and cytokinesis. Microinjection of 5 μM or less CaCl2 into anaphase PtK1 cells has no detectable effect on the rate or extent of chromosome movement. Metaphase cells tend to enter anaphase 4–5 min after injection with 1–10 μM CaCl2, compared with an average of 16 min after injection with calcium-free buffer. Reducing the intracellular calcium concentration by injection of EGTA-CaCl2 buffers increases the lag between injection and anaphase to 20 min or more. Microinjection of calcium solutions does not promote precocious chromatid separation in nocodazole-arrested metaphase cells, indicating that the increase in calcium concentration does not induce centromere separation directly. An increase in the concentration of free calcium ions during metaphase appears to stimulate the onset of anaphase. Such an increase, regulated by the cell itself, may contribute to the initiation of chromosome separation in mammalian cells.

Low angle x-ray diffraction studies of chromatin structure in vivo and in isolated nuclei and metaphase chromosomes

Abstract: Diffraction of x-rays from living cells, isolated nuclei, and metaphase chromosomes gives rise to several major low angle reflections characteristic of a highly conserved pattern of nucleosome packing within the chromatin fibers. We answer three questions about the x-ray data: Which reflections are characteristic of chromosomes in vivo? How can these reflections be preserved in vitro? What chromosome structures give rise to the reflections? Our consistent observation of diffraction peaks at 11.0, 6.0, 3.8, 2.7 and 2.1 nm from a variety of living cells, isolated nuclei, and metaphase chromosomes establishes these periodicities as characteristic of eukaryotic chromosomes in vivo. In addition, a 30-40- nm peak is observed from all somatic cells that have substantial amounts of condensed chromatin, and a weak 18-nm reflection is observed from nucleated erythrocytes. These observations provide a standard for judging the structural integrity of isolated nuclei, chromosomes, and chromatin, and thus resolve long standing controversy about the “tru” nature of chromosome diffraction. All of the reflection seen in vivo can be preserved in vitro provided that the proper ionic conditions are maintained. Our results show clearly that the 30-40-nm maximum is a packing reflection. The packing we observe in vivo is directly correlated to the side-by-side arrangement of 20- 30-nm fibers observed in thin sections of fixed and dehydrated cells and isolated chromosomes. This confirms that such packing is present in living cells and is not merely an artifact of electron microscopy. As expected, the packing reflection is shifted to longer spacings when the fibers are spread apart by reducing the concentration of divalent cations in vitro. Because the 18-, 11.0-, 6.0-, 3.8-, 2.7-, and 2.1-nm reflections are not affected by the decondensation caused by removal of divalent cations, these periodicities must reflect the internal structure of the chromaticn fibers.

Ionic changes in the mitotic apparatus at the metaphase/anaphase transition.

Abstract: We have employed a series of permeant, nontoxic, fluorescent probes to detect changes in ionic conditions within the mitotic apparatus of living endosperm cells of Haemanthus during the transition from metaphase to anaphase. Fluorescence emission intensity measurements from the spindle for chlorotetracycline (CTC) decline before the onset of anaphase, indicating a reduction in the amount of membrane-associated Ca2+ and suggesting an efflux of Ca2+ from membrane compartments into the spindle. Subsequent to the onset of anaphase, we observe increases in fluorescence with both 8-anilino-1-naphthalene sulfonate (ANS) and 3,3'-dipentyl 2,2'-dioxacarbocyanine (diO-C5(3)), sensitive to cationic and anionic charges at membrane surfaces, respectively. The increases with ANS and diO-C5(3) suggest that redistributions of ions within the spindle accompany anaphase motion. During the metaphase/anaphase transition, spindle membrane content remains constant, as evidenced by unchanging fluorescence with the hydrophobic probe, N-phenyl-1-naphthylamine (NPN). Shifts in emission intensity from the nonspindle cytoplasm or from the spindle poles do not accompany the changes in fluorescence we observe in the spindle, suggesting that any ionic fluxes responsible for the changes in fluorescence are restricted to the spindle domain.

Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation.

Abstract: Our simple instrumentation for generating a UV-microbeam is described UV microbeam irradiations of the central spindle in the pennate diatom Hantzschia amphioxys have been examined through correlated birefringence light microscopy and TEM. A precise correlation between the region of reduced birefringence and the UV-induced lesion in the microtubules (MTs) of the central spindle is demonstrated. The UV beam appears to dissociate MTs, as MT fragments were rarely encountered. The forces associated with metaphase and anaphase spindles have been studied via localized UV-microbeam irradiation of the central spindle. These spindles were found to be subjected to compressional forces, presumably exerted by stretched or contracting chromosomes. Comparisons are made with the results of other writers. These compressional forces caused the poles of a severed anaphase spindle to move toward each other and the center of the cell. As these poles moved centrally, the larger of the two postirradiational central spindle remnants elongated with a concomitant decrease in the length of the overlap. Metaphase spindles, in contrast, did not elongate nor lose their overlap region. Our interpretation is that the force for anaphase spindle elongation in Hantzschia is generated between half-spindles in the region of MT overlap.

Action of taxol on mitosis: modification of microtubule arrangements and function of the mitotic spindle in Haemanthus endosperm

Abstract: We have studied the effect of taxol on mitosis in Haemanthus endosperm. Immuno-Gold Stain (IGS), a new immunocytochemical method (17), was used to visualize microtubules (MTs) in the light microscope. Observations on MT arrangements were correlated with studies in vivo. Chromosome movements are affected in all stages of mitosis which progresses over at least 10(4) range of taxol concentrations. The three most characteristic effects on MTs are: (a) enhancement of the lateral associations between MTs, seen especially during the reorganization of the polar region of the spindle, (b) promotion of MT assembly, leading to the formation of additional MTs in the spindle and MT arrays in the cytoplasm, and (c) an increase in MT stability, demonstrated in their increased cold resistance. In this report, the emphasis is on the primary, immediate effects, occurring in the first 30 min of taxol action. Effects are detected after a few mins, are reversible, and are concentration/time dependent. The spindle and phragmoplast are remarkably modified due to the enhancement of lateral associations of MTs and the formation of abundant nonkinetochore and polar, asterlike MTs. The equatorial region of the interzone in anaphase may be entirely depleted of MTs, and the spindle may break perpendicular to the spindle axis. Mitosis is completed in these conditions, providing evidence for the motile autonomy of each half-spindle. Trailing chromosome arms in anaphase are often stretched and broken. Chromosome fragments are transported away from the polar regions, i.e., in the direction opposite to that expected (5, 6). This supplies the first direct evidence of pushing by elongating MTs in an anastral higher plant spindle. These observations draw attention to the relation between the lateral association of MT ends to assembly/disassembly and to the role of such an interaction in spindle function and organization.

Specific DNA sequence amplification in human neuroblastoma cells.

Abstract: Southern blot analysis of a number of EcoRI-digested human neuroblastoma DNAs has revealed the presence of a family of discrete restriction fragments, the majority of which are amplified in most, but not all, of the neuroblastoma cell lines tested. None of these sequences is abundantly present in DNA from other human tumors of different tissue origins, including several either known or presumed to contain amplified DNA. Hence, these sequences appear to be specifically amplified by neuroblastoma cells. Hybridization with metaphase chromosomes in situ has localized these sequences to either the homogeneously staining regions or double-minute chromosomes of different neuroblastoma cell lines, indicating that these chromosomal structures, although present in cell lines established from different patients, share many sequences and may have a common, but as yet unknown, function.

Microtubule‐containing detergent‐extracted cytoskeletons in sea urchin eggs from fertilization through cell division: Antitubulin immunofluorescence microscopy

Abstract: The microtubule-containing structures that appear in eggs during fertilization and cell division in the sea urchins Lytechinus variegatus and Arbacia punctulata were detected by antitubulin immunofluorescence microscopy of detergent extracted cytoskeletal preparations. The extraction buffer, which is composed of 0.55 mM MgCl2, 10 mM EGTA, 25 mM MES, 25% glycerol, 1% Nonidet P-40, and 25 μM PMSF, pH 6.7, allows for dramatically improved fluorescent images compared to those obtained using conventional staining procedures, with residual background staining being reduced to near zero. The immunofluorescent images obtained using this technique provide information on several motile events that occur during the first cell cycle. This technique demonstrates that all of the cytoplasmic microtubules are associated with the incorporated sperm's centrioles during female pronuclear migration. This changes during the centration of the male and female pronuclei at which time a monastral array of microtubules forms in the egg's cytoplasm. A large proportion of the monastral microtubules do not appear to be associated with the centrioles. At prophase and early metaphase, the centrioles are the dominant microtubule organizing centers (MTOCs) consistent with mitotic theories that the kinetochore catches, but does not initiate, microtubules. Observations of intercentriolar distances show that there are three stages of pole separation during the first cell cycle. The initial separation occurs during pronuclear centration, the second during the streak stage, and the final one during the late stages of mitosis. At telophase, polar microtubules appear to extend into the cortex supporting the cell surface at all regions except the presumptive cleavage site.

Selective digestion of human metaphase chromosomes by Alu I restriction endonuclease.

Abstract: Human cytological preparations have been digested with Alu I restriction endonuclease (Endo R Alu I) and subsequently stained with acridine orange. Metaphase chromosomes treated in this way revealed a clear-cut bright/ dull longitudinal differentiation. It has been postulated that these cytological findings could be related to the selective digestion, by Alu I, of all DNA with the exception of some heterochromatic, possibly satellite, fractions.

Mass isolation of plant chromosomes and nuclei

Abstract: A method is presented for mass isolation of metaphase chromosomes and nuclei from plant protoplasts. The isolation procedure was developed for both monocotyledonous and dicotyledonous species using wheat (Triticum monococcum) and poppy (Papaver somniferum) cell cultures. Metaphase chromosomes were isolated from partially synchronized mitotic protoplasts, while for the isolation of nuclei unsynchronized protoplasts were used. Light and electron-microscopic studies revealed that isolated chromosomes and nuclei preserved their intact morphology. A preliminary biochemical study of chromosomal proteins was made by polyacrylamide-gel electrophoresis. Because of the purity and high quantity of isolated chromosomes and nuclei, the given isolation procedure can supply useful material for structural and biochemical studies, and for genetic manipulation.

DNA density in mitotic and meiotic metaphase chromosomes of plants and animals.

Abstract: Studies of chromosome disposition at metaphase using serial thin-sectioning and three-dimensional reconstruction techniques have produced accurate estimates of the total volume of chromosomes per cell in 15 plant and two animal species. Comparing this character with the 4C DNA amount showed no indiction of systemic differences in DNA density between either organisms with widely different (greater than 200-fold) C values or different groups or organisms. For example, there was no significant difference between the density of DNA in somatic metaphase chromosomes of man (0.141 pg/micrometers3) and its mean in 14 angiosperm plant species (0.182 pg/micrometers3), or between four dicotyledons (0.180 pg/miocrometers3) and 10 monocotyledons (0.182 pg/micrometers3). However, evidence was found showing that DNA density can vary significantly within a species. Thus, although the total chromosome volume per cell was closely correlated (r greater than 0.97) with 4C DNA amount in somatic and meiotic cells, the density of DNA in metaphase chromosomes was significantly lower in meiocytes (0.131 pg/micrometers3) than in somatic metaphase cells (0.19 pg/micrometers3).

Evidence for the presence of inhibitors of mitotic factors during G1 period in mammalian cells.

Abstract: Our earlier studies indicated that the mitotic factors, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus oocytes, are preferentially associated with metaphase chromosomes and that they bind to chromatin as soon as they are synthesized during the G2 phase. In this study, we attempted to determine the fate of these factors as the cell completes mitosis and enters G1. Extracts from HeLa cells at different points during G1, S, and G2 periods were mixed with mitotic extracts in various proportions, incubated, and then injected into Xenopus oocytes to determine their maturation-promoting activity. The maturation-promoting activity of the mitotic extracts was neutralized by extracts of G1 cells during all stages of G1 but not by those of late S and G2 phase cells. Extracts of quiescent (G0) human diploid fibroblasts exhibited very little inhibitory activity. However, UV irradiation of G0 cells, which is known to cause decondensation of chromatin, significantly enhanced the inhibitory activity of extracts of these cells. These factors are termed inhibitors of mitotic factors (IMF). They seem to be activated, rather than newly synthesized, as the cell enters telophase when chromosomes begin to decondense. The IMF are nondialyzable, nonhistone proteins with a molecular weight of greater than 12,000. Since mitotic factors are known to induce chromosome condensation, it is possible that IMF, which are antagonistic to mitotic factors, may serve the reverse function of the mitotic factors, i.e., regulation of chromosome decondensation.

Mitotic architecture of the cell: the filament networks of the nucleus and cytoplasm.

Abstract: The skeletal framework of cells at the various stages of mitosis are prepared by extraction with nonionic detergent and examined by stereoscopic whole mount electron microscopy. The insoluble filament network remaining after the detergent-extraction and the depolymerization of microtubules is shown. The nonchromatin filament network of the nucleus, or nuclear matrix, becomes visible as the chromatin condenses at prophase. Filaments are associated with the chromosomes throughout mitosis. Parts of the chromosomes are associated with or are near the nuclear lamina at early stages. The nuclear lamina disappears at metaphase while chromosomes remain associated with filaments now continuous with the cytoplasmic network. Microtubules appear to be unnecessary for maintaining the chromosome position in these preparations since comparison of cells with and without microtubules shows no gross change in chromosome arrangement. The cellular filament network at metaphase and anaphase appears continuous from the plasma lamina to the chromosomes. The filament networks visualized here may be responsible for the prometaphase chromosome movement and participate in the formation of the midbody.

Non-random chromosome loss in PHA-stimulated lymphocytes from normal individuals

Abstract: 31773 lymphocyte metaphase cells from 280 karyotypically normal men aged 18-46 were examined for chromosome gain or loss. Chromosome loss was much more common than chromosome gain. Frequency of chromosome loss did not conform to a binomial distribution. There is a striking non-linear, inverse relationship between likelihood of loss and chromosome length. Chromosome gain shows a near binomial distribution between cells and no clear relationship to chromosome length. These facts indicate that the hypodiploid cells mostly arose as technical artefacts during slide preparation but that hyperdiploid cells were mainly due to non-disjunctional gain.

Preparation of centromeric heterochromatin by restriction endonuclease digestion of mouse L929 cells.

Abstract: When L929 cells in metaphase are digested with either Eco RI or Alu I, chromatin containing about 85% of the DNA is released. DNA from the Alu I- and Eco RI-resistant chromatin is enriched 6.8- and 3.7-fold, respectively, in satellite sequences. Analysis by electron microscopy of these digests reveals the existence of structures containing condensed heterochromatin and kinetochores. When these preparations are incubated with anticentromere serum from a human CREST scleroderma patient and then with rhodamine-conjugated antihuman IgG, fluorescence appears in the form of paired dots, the same pattern found in whole metaphase chromosomes. The fluorescent staining pattern, the electron microscopy, and the enrichment of satellite DNA sequences together support the conclusion that the Eco RI- and Alu I-resistant structures contain centromeres. We anticipate that these preparations will be useful in studies of the interactions between centromeric heterochromatin, kinetochores, and microtubules.

A dominantly inherited cytogenetic anomaly: A possible cell division mutant

Abstract: Short-term lymphocyte cultures from three unrelated patients showed an increased frequency of mitoses with separated centromeres and splayed chromatids in the presence of colcemid. We refer to this phenomenon as premature centromere division (PCD). In two of the three patients the frequency of PCD in lymphocytes decreased when colcemid was omitted prior to harvest but was still higher than controls, whereas in the third patient, the frequency appeared unchanged. Cultured fibroblasts from the latter patient exhibited increased tetraploidy and multinucleated cells. Transmission of the trait in the three families was compatible with autosomal dominant inheritance. Time lapse cinemicrographic studies on fibroblasts from one patient demonstrate a shortened metaphase time, suggesting that the separation of chromatids observed in this patient may indeed be premature. The nature of the mutation(s) and phenotype correlation if any is unknown.

Heterochromatin and karyotypic differentiation of some neotropical cactus-breeding species of the Drosophila repleta species group

Abstract: Metaphase chromosomes from neuroblasts of strains from both laboratory stocks and natural populations of D. serido, D. meridionalis, D. borborema and D. buzzatii have been studied using colcemid pretreatment, and air-drying followed by Giemsa staining. The two latter species both show a uniform ‘basic’ metaphase karyotype. D. serido and D. meridionalis, on the other hand, both include a number of different, geographically distinct, metaphase karyotypes involving differences in the major blocks of constitutive heterochromatin present on the sex chromosomes and/or the 6th chromosome (microchromosome). These chromosomal differences are largely due to the acquisition of extra heterochromatin though pericentric inversions appear to be responsible for some of the Y-chromosome variants in D. serido. Moreover, the cytological evidence demonstrates that populations of both species are far from continuous in distribution. The extent to which such cytological differences reflect the existence of subspecific or specific complexes with minimal morphological differentiation is under investigation.

The Spatial Order of Chromosomes in Root-Tip Metaphases of Aegilops umbellulata

Abstract: Twelve root-tip metaphase cells of the diploid grass Aegilops umbellulata Zhuk. (2n = 2x = 14) were reconstructed in three dimensions from electron micrographs of serial thin sections. In ten of these cells each individual chromosome in the nuclei was identified by morphological criteria. No evidence for somatic association of centromeres of homologous chromosomes was found. However, with use of computer analysis, evidence was found suggesting: (i) that the centromeres are not arranged randomly on the metaphase plate but are ordered in two haploid sets; and (ii) that the order within each set is that predicted by a model based on spatial association of pairs of corresponding arms of most similar size on heterologous chromosomes.

Low angle x-ray diffraction studies of HeLa metaphase chromosomes: effects of histone phosphorylation and chromosome isolation procedure.

Abstract: To test whether gross changes in chromatin structure occur during the cell cycle, we compared HeLa mitotic metaphase chromosomes and interphase nuclei by low angle x-ray diffraction. Interphase nuclei and metaphase chromosomes differ only in the 30-40-nm packing reflection, but not in the higher angle part of the x-ray diffraction pattern. Our interpretation of these results is that the transition to metaphase affects only the packing of chromatin fibers and not, to the resolution of our method, the internal structure of nucleosomes or the pattern of nucleosome packing within chromatin fibers. In particular, phosphorylation of histones H1 and H3 at mitosis does not affect chromatin fiber structure, since the same x-ray results are obtained whether or not histone dephosphorylation is prevented by isolating metaphase chromosomes in the presence of 5,5'-dithiobis(2-nitrobenzoate) or low concentrations of p-chloromercuriphenylsulfonate (ClHgPhSO3). We also compared metaphase chromosomes isolated by several different published procedures, and found that the isolation procedure can significantly affect the x-ray diffraction pattern. High concentrations of ClHgPhSO3 can also profoundly affect the pattern.