Metaphase Nuclei Research Articles

Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization.

This report describes the use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations. Biotin-labeled DNA was hybridized to target chromosomes and subsequently rendered fluorescent by successive treatments with fluorescein-labeled avidin and biotinylated anti-avidin antibody. Human chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic DNA was used as a probe. Interspecies translocations were detected easily at metaphase. The human-specific fluorescence intensity from cell nuclei and chromosomes was proportional to the amount of target human DNA. Human Y chromosomes were fluorescently stained in metaphase and interphase nuclei by using a 0.8-kilobase DNA probe specific for the Y chromosome. Cells from males were 40 times brighter than those from females. Both Y chromosomal domains were visible in most interphase nuclei of XYY amniocytes. Human 28S ribosomal RNA genes on metaphase chromosomes were distinctly stained by using a 1.5-kilobase DNA probe.

Ultrastructure of cell fusion and premature chromosome condensation (PCC) of thymocyte nuclei in metaphase II mouse oocytes.

Following PEG (polyethylene glycol) treatment of ovulated metaphase II mouse oocytes aggregated with thymocytes, fusion of cell membranes occurs. Prerequisites for cell fusion are: close apposition of lectin-agglutinated (phytohemagglutinin-treated) membranes of both cells, formation of firm punctual adhesion sites, and expansion of adhesion sites over a certain area. Establishment of the firm cell-cell contact is associated with development of actin-like filaments along both of the adhering plasma membranes. Membrane fusion occurs at single or multiple sites, and is followed by internalization of thymocyte-oocyte membrane complexes decorated with actin filaments into the hybrid cell cytoplasm. A filamentous actin layer forms also along the inner surface of newly formed hybrid oocyte-thymocyte plasma membrane. Thymocyte nuclei incorporated into oocyte cytoplasm undergo nuclear envelope breakdown and premature chromosome condensation (PCC) leading, eventually, to formation of single chromatids complete with kinetochores. Concomitantly with chromatin condensation an extensive polymerization of microtubules starts in the center of the chromatin mass which leads to the formation of an apparently non-functional spindle-like structure.

Mitotic activity in cells of the wool follicle bulb.

Mitotic activity in the cells of the germinative region of wool follicle bulbs was quantified by using small (0.1-0.5 ml) intradermal doses of colchicine and selective staining of the metaphase-blocked nuclei using either crystal violet, iodine and eosin or haematoxylin and eosin. The number of metaphase nuclei present 3 h after colchicine administration increased with colchicine dose from 0 to 1 microgram and thereafter remained relatively constant up to 200 micrograms colchicine. The accumulation of metaphase nuclei was linear for up to 6 h after intradermal colchicine. The metaphase-blocking effect of intradermal colchicine was confined to a radius of less than 5 cm from the injection site, allowing a number of estimates of mitotic rates to be made over a small area of skin. Such estimates revealed little variation in mitotic activity over the midside region of the sheep, although there were substantial differences in follicle activity at different sites over the body. The technique is simple, allows serial or concurrent estimates of mitotic activity to be made in the same animal, and eliminates problems associated with intravenous colchicine administration. It was used to derive the relationship between follicle activity and fibre production after nutritional changes, and to define the time course of mitotic events after administration of the antimitotic defleecing agent cyclophosphamide.

MITOSIS AND CELL DIVISION IN CYLINDROCAPSA GEMINELLA (CHLOROPHYCEAE)1

ABSTRACTMitosis and cell division were studied in the green alga Cylindrocapsa geminella Wolle with transmission electron microscopy. Vegetative cells possess a parietal, lobed chloroplast, and a central pyrenoid. Prophase and metaphase nuclei are surrounded by 1–3 layers of perinuclear endoplasmic reticulum. At early prophase a small number of perinuclear microtubules (MTs) are present while at late prophase MTs are concentrated at the presumptive spindle poles. At the same time, MTs begin to appear in the nucleoplasm. Metaphase spindles are diamond‐shaped and centric. During telophase, centrioles migrate towards the center of the equatorial zone, presumably guided by a small group of perinuclear MTs. A second system of MTs develops in the equatorial plane, initially consisting of randomly orientated microtubular elements. Later they tend to run in a predominantly radial direction although a common MT focal point or organizing center is not apparent. The two centriole complexes remain at the center of the equatorial plane until well into interphase, facing each other across the newly formed transverse septum. Centrioles are associated with root templates and connecting fibers. The present observations corroborate the view that C. geminella does not form a true filament in the ulotrichalean or chaetophoralean sense, but rather consists of a row of autospores. Its affinity with other “pseudo‐filamentous” green algae and the Chlorococcales is discussed. The interpretation of the cytokinetic MTs in C. geminella as a phycoplast appears to be problematic.

DNA Content of Diffusely Infiltrative Carcinomas in the Stomach

DNA content of diffusely infiltrative carcinomas of 15 Japanese patients, ranging in age from 38 to 65 years, was determined by cytofluorometry, using paraffin sections which were available for histological examination. Sections were stained with 0.0025% propidium iodide. By measuring the fluorescence intensity of at least 50 mitotic cells, ploidy patterns of the cancers were determined, whereas a control of the diploid DNA content was set by measuring the fluorescence intensity of mitotic cells in the non-cancerous gastric mucosa; only metaphase nuclei in the sections were considered to have a whole amount of nuclear DNA in the cytofluorometric measurement. In the present study, it was found that most of the diffusely infiltrative carcinomas consisted of a heteroploid cell line. Their stem DNA contents ranged between 2.8C and 4.8C (2C corresponds to a diploid amount of nuclear DNA). Of 15 cancers, four comprised a mosaic cancer of two different heteroploid cell lines. We encountered only one carcinoma containing a diploid cell line. However, this cancer also contained a heteroploid cell line. Six cancers contained a polyploid cell population, the DNA content of which was double of the stem DNA content. In most cases, the cancer cells distributed in the mucosal layer were also heteroploid, thereby suggesting a heteroploid origin of the diffusely infiltrative carcinomas.

Culture of mouse germ cells isolated from fetal gonads

Mouse germ cells isolated from male or female genital ridges at 12 1 2 days post coitum were cultured at room temperature for up to 6 days, with [ 3H]thymidine present in the culture medium for either the first 24 h or the last 24 h of each culture period. Germ cells were also isolated 13 1 2 – 16 1 2 days post coitum and cultured for 24 h in the presence of [ 3H]thymidine. The proportion of cells in metaphase, and the proportion of labelled interphase and metaphase nuclei, was recorded. The labelling index declined from 13 1 2 days onwards, after development either in vivo or in vitro. No labelled metaphase plates were seen after 24 h in the presence of [ 3H]thymidine, suggesting that under these culture conditions the duration of the G2 phase exceeded 24 h. The results showed that the culture system, in spite of the low temperature, allowed the germ cells to replicate their DNA and undergo mitosis for up to 6 days. Addition of db-cAMP to the culture medium proved highly toxic to male germ cells, and did not markedly increase the proliferation rate of female germ cells.

Indirect immunofluorescence of microtubules in Dictyostelium discoideum: A study with polyclonal and monoclonal antibodies to tubulins

Amebae of D. discoideum on coverslips were fixed in situ with glutaraldehyde and permeabilized with Triton X-100. Of six antibodies tested, only a monoclonal antibody to yeast tubulin consistently gave bright fluorescence. Counterstaining with DAPI facilitated the identification of interphase and mitotic stages. Most microtubules (MTs) in interphase amebae emanated from a nucleus-associated centre that had a non-fluorescent core. Amebae in early stages of mitosis lacked cytoplasmic MTs almost entirely. The nascent spindle in prophase appeared as a brightly fluorescent dot, whereas the prometaphase spindle was a short rod. Spindles in metaphase and anaphase nuclei were more elongate, some consisting of several fluorescent lines. Astral MTs were prominent on spindles in anaphase and telophase. Asters are obviously converted to the interphase complex of MTs in post-mitotic cells, while the shaft-like remnant of the central spindle disappears. The cyclical changes in the MT system related to cell division resemble those observed in higher eukaryotes and probably reflect changes in the locomotory behavior of the amebae rather than changes in cell shape.

Changes in the karyotype of the cell line, DSIR-HA-1179, and a comparison with that of its parent insect, Heteronychus arator (F.) (Coleoptera: Scarabaeidae)

Eighteen months after establishment of the primary cell culture, 36 metaphase nuclei of cell line DSIR-HA-1179, derived from the black beetle, Heteronychus arator (F.), had between 32 and 43 chromo...

Mass isolation of plant chromosomes and nuclei

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.

Cell-cycle-dependent dissociation of histone H1 from chromatin in nuclei of P. polycephalum.

The dissociation curves of histone H1 from chromatin in interphase and metaphase nuclei from Physarum polycephalum have been determined using CaCl2 as dissociating agent. H1 is less strongly bound to metaphase chromosomes than to interphase chromatin. However, no differences could be detected in the binding of Hl to early S, late S or G2 phase chromatin. The number of CaCl2 molecules involved in binding one H1 molecule to chromatin was reduced from 5 in interphase to 4 in metaphase. The non-electrostatic contribution to the free-energy of binding was small in both cases. A comparison of the binding properties of H1 to sheared chromatin, native chromatin and metaphase chromosomes suggests that the electrostatic binding functions of H1 are completely satisfied within the nucleosome and that further electrostatic interactions are not involved in folding the nucleosomal fibre into the 300 A "solenoid" or the more tightly folded metaphase chromosome.

Quantitation of in situ hybridization of ribosomal ribonucleic acids to human diploid cells.

The hybridization of 5S and 28S ribosomal RNAs to human fibroblast and leukocyte cells was used as a model system to quantitative the technique of in situ hybridization for human diploid cell types. Quantitation consisted of counting (scoring) the number of grains formed over both interphase nuclei and metaphase chromosomes on slides after various hybridization procedures. The average number of grains/nucleus per slide was then used to determine hybridization percentages. As with nitrocellulose filter hybridizations the kinetics of in situ hybridizations can be fit with a single first-order rate-constant. However, the in situ hybridization rate was approximately 10 times slower than the corresponding filter hybridization rate. The efficiency of in situ hybridization was found to range between 5 and 15% for both leukocyte and fibroblast cell types and for both metaphase and interphase nuclei. Determination of the parameters of the in situ hybridization reaction of ribosomal RNAs to diploid chromosomes define the experimental conditions needed for the localization of single copy genes to diploid chromosomes.

Apparent absence of chromatin condensation in metaphase nuclei of Saprolegnia as revealed by mithramycin staining

Mithramycin is shown to be able to reveal details of heterochromatin organization in interphase and dividing nuclei of higher plants. It has been used to show that in hyphal tip nuclei of Saprolegnia ferax the chromatin does not undergo any detectable condensation until telophase when it appears to contract into two homogeneous polar masses. In contrast, presumptive meiotic prophase nuclei in antheridia did contain condensed chromatin. These results suggest that the chromatin of S. ferax does not undergo a normal cycle of condensation during the prophase and metaphase stages of the mitotic cycle. Mithramycin also has the potential to help preselect desired nuclei prior to electron microscope studies.

More evidence for replication-transcription-coupling in Physarum polycephalum.

Endogenous RNA polymerase activity of isolated nuclei from Physarum polycephalum was determined at high (400 mM KCl) and low (5--100 mM KCl) ionic strength. The activity of RNA polymerase B (alpha-amanitin-sensitive UMP incorporation) and of RNA polymerase A (plus C) (alpha-amanitin-resistant UMP incorporation) was compared in accurately sized nuclear samples derived from macroplasmodia at distinct points of the mitotic cycle. Minimum total RNA polymerase activity was detected in metaphase nuclei. A constant level of RNA polymerase B activity was detected at all other stages of the mitotic cycle, if nuclei were assayed at high ionic strength. However, a high level in S-phase, a low level in G2-phase and again a high level in early prophase were measured, if nuclei were assayed at low ionic strength. Inhibition of DNA synthesis by hydroxyurea in vivo had a selective and drastic effect on in vitro RNA polymerase activity of isolated nuclei derived from S-phase plasmodia, yielding up to 100% inhibition in early S-phase.

An analysis of spindle ultrastructure during prometaphase and metaphase of micronuclear division in Tetrahymena.

Mitotic micronuclei were isolated from Tetrahymena thermophila in a medium containing hexylene glycol and their ultrastructure was analyzed using thin section techniques. The two stages selected for analysis were early prometaphase and metaphase. A comparison of data from these two stages revealed several differences in nuclear morphology. Metaphase nuclei were longer, they contained more microtubules, and the distribution of microtubules at metaphase was different from that at early prometaphase. Increases in microtubules, which are a unique class of microtubules that can be distinguished from other classes on the basis of their close association to the nuclear membrane. Growth of peripheral sheath microtubules is thought to be significant because it could be the mechanical basis of nuclear elongation. Crossbridges were observed throughout the spindle between all classes of microtubules, but the exact function of these elements remains to be determined.

Changes in the generation cycle of duodenal crypt cells in chickens infected with Eimeria acervulina.

Changes in the duration of the progenitor cycle and its four phases were determined for duodenal crypt cells in chickens infected with Eimeria acervulina. Metaphase curves were constructed using percent labelled metaphase nuclei in duodenal crypt cells at short intervals after the injection of [3H]thymidine. The duration of the progenitor cycle and its four phases were calculated using the synthetic index and data obtained from the metaphase curves. The cycle time was reduced from 14 h in control birds to 10.2 h at 2 days and 10.6 h at 4 days postinfection. The change was attributable entirely to a reduction in G1 or the presynthetic phase. In addition, the population of dividing cells within each duodenal crypt was almost doubled in infected birds. These increases in cell production precedes all the histological changes observed earlier in the intestines of E. acervulina infected chickens. At least in this instance, changes in crypt morphology seems, therefore, to result from an induced change in the functional activity of the crypt.

Chromosome-mediated gene transfer between closely realted strains of cultured mouse cells.

Gene transfer between two closely related mouse cell lines has been carried out, using as the vector a cell-free preparation of metaphase chromosomes and nuclei. Distinction between gene transferents and revertants of the recipient mutant phenotype was achieved by the use of a donor strain carrying a mutationally altered (8-azaguanine-resistant) hypoxanthine-guanine phosphoribosyltransferase (HPRTase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8). The transferred HPRTase gene is initially unstable; in nonselective medium, it is lost at a rate of about 0.1 per cell per generation. Stabilization occurs as a rare event, with a frequency on the order of 1 X 10(-5) per cell per generation. The unstable state can be maintained for at least 200 generations through serial passages of the transferent in selective medium. Under the conditions of cultivation used in these experiments, the unstable HPRTase-positive cells are eventually replaced by the stable HPRTase-positive cells in the population.

Studies on the intranuclear distribution of human and mouse genomes and formation of human-mouse hybrid cells.

Autoradiographic examination of early hybrid cells formed by the fusion of 3H-thymidine labeled D98/AH2 cells and 3T3-4E cells revealed that human and mouse chromosomes are often separated within metaphase and interphase nuclei. Although the marked separation of human and mouse chromosomes progressively disappeared with succeeding cell division, the occurrence of sectored nuclei in 16-cell hybrid colonies and the labeling pattern of human chromosomes within separated metaphases indicate that separation of human and mouse chromosomes may persist through several mitoses. Fusion of 3H-thymidine labeled D98/AH2 cells and 3T3-4E cells was coupled with aminopterin selection to study other aspects of hybrid cell formation. Hybrid cells arose from heterokaryons by cell division. Nuclear fusion may have occurred in two out of several thousand heterokaryons examined. However, these could reflect the close apposition of adjacent nuclei. A large fraction (greater than 0.5) of D98/3T3 heterokaryons underwent at least one cell division. However, the number of hybrid colonies containing more than eight cells at ten days following fusion was about 0.03 of the total number of heterokaryons. Many hybrid colonies arrested growth before the 8-cell stage, and the cells in such colonies exhibited nuclear abnormalities.

Repeated structure of chromatin in metaphase nuclei of Physarum

Repeated structure of chromatin in metaphase nuclei of Physarum

DNA content of colchicine-induced endopolyploid nuclei in vicia faba L.

The relationship between ploidy level and nuclear DNA content was investigated by Feulgen microdensitometry in colchicine induced polyploid root-tip meristem cells of three Vicia species. Nuclear DNA content is expected to increase proportionately with the increase in ploidy, assuming DNA constancy. Our results showed that: (i) Absorption readings were significantly lower than expected for cells of higher ploidy levels. (ii) The deviation of the absorption readings obtained below those expected increased progressively with ploidy level. (iii) These trends occurred in absorption estimates of single metacentric chromosomes at metaphase in V. faba, metaphase nuclei in V. faba, and prophase nuclei in all three Vicia species. (iv) Loss of chromosomal DNA, as evidenced by a large and progressive increase in the frequency of occurrence of micronuclei in cells at higher ploidy level, was partly responsible for cell absorption estimates below expectation. (v) Variation in the density and in the absorbance of nuclear DNA may also have been a cause of absorption estimates below expectation. In our opinion, apart from the loss of chromosomal DNA to form micronuclei, it is unlikely that any real decrease in DNA content per genome occurred in cells at higher ploidy levels.

THE ULTRASTRUCTURE OF CRUCIFORM NUCLEAR DIVISION IN SOROSPHAERA VERONICAE (PLASMODIOPHOROMYCETE)

Vegetative nuclear divisions in cystosoral Plasmodia from the shoot system of Sorosphaera veronicae Schroeter were studied with standard transmission electron microscopy. Each metaphase nucleus forms a cruciform configuration as the persistent nucleolus elongates perpendicularly to chromatin aligned on the equatorial plate. The nuclear envelope remains intact during metaphase and anaphase. Each spindle pole consists of a fenestrated nuclear envelope with an exteriorly situated centriole and closely associated endoplasmic reticulum. Intranuclear membranous vesicles occur within metaphase and anaphase nuclei and are closely associated with chromatin and nuclear envelope. Microtubules pass from centrioles into the nucleus and are also attached to chromatin at kinetochores.