Showing papers on "Metaphase published in 1970"

Identification of human chromosomes by DNA-binding fluorescent agents

Abstract: The distribution of DNA along metaphase chromosomes that are not excessively contracted can be visualized in the fluorescence microscope with the aid of fluorescent DNA-binding agents. Additional, characteristic details in the fluorescence patterns are obtained with fluorochromes that bind preferentially to certain chromosomal regions. The highly fluorescent alkylating agent quinacrine mustard (QM) effects discrete, fluorescent labeling of both plant and mammalian metaphase chromosomes, presumably by selective binding to guanine residues in DNA, and is also capable of intercalation in the DNA double helix. Chromosome regions fluorescing particularly strongly with QM have been demonstrated in human metaphase chromosomes 3, 13–15 and Y. A convenient measuring technique has been developed for the rapid and accurate recording of fluorescence patterns in human metaphase chromosomes. These photoelectric recordings of the fluorescence patterns contain far greater detail than can be seen by the human eye. The fluorescence patterns described are based on measurements of about 1,000 human metaphase chromosomes. This new technique of determining fluorescence patterns in human chromosomes should be particularly valuable for the identification of chromosomes 4–5 and the individual types in the 6–12 group. Individual, typical patterns also occur within the groups 13–15, 17–18, and 21–22.

Effect of colcemid on the locomotory behaviour of fibroblasts.

Abstract: Effects of metaphase inhibitors (colcemid, colchicine, vinblastine) on mouse and human embryonic, fibroblast-like cells growing on glass and on an oriented substrate (fish scale) were studied. All three inhibitors caused similar changes in the form of interphase cells and inhibited their directional locomotion. The effects of two inhibitors (colcemid and vinblastine) were found to be completely reversible. Microcinematographic studies have shown that the most conspicuous change of locomotory behaviour induced by colcemid was the disappearance of non-active stable parts of the cell edge; in normal cells only the leading part of the edge was actively moving, while in colcemid-treated cells all parts of the edge eventually became active. Activation of the whole edge made these cells unable to perform directional translocation. It is suggested that colcemid and other metaphase inhibitors prevent stabilization of the non-active state of the cell surface. The possible role of this suggested colcemid-sensitive stabilization mechanism in the normal locomotory behaviour of fibroblasts is discussed. Electron-microscopic examination has shown that microtubules disappeared from the cytoplasm of colcemid-treated, mouse, fibroblast-like cells. The formation of microtubules as the possible structural basis of the stabilization of the non-active state of the cell surface is discussed.

Effects of ultraviolet radiation on the mitotic cycle and DNA, RNA and protein synthesis in mammalian epidermis in vivo.

Abstract: — Acute effects of ultraviolet radiation on the mitotic cycle and macromolecular synthesis were investigated on hairless mouse epidermis in vivo. Colcemid was used to arrest mitoses in metaphase and thus allow more accurate mitotic counts. The radioactive tracers, TdR-3H, cytidine-3H, and the amino acids, histidine-3H and methionine-3H were used to examine DNA, RNA and protein synthesis, respectively. Using these techniques, we found that wavelengths shorter than 320 nm markedly inhibited mitosis, increased the basal cell turnover time and depressed DNA, RNA and protein synthesis within the first few hours post-irradiation. By 24hr, recovery and acceleration of these functions were in progress, reaching a peak by 48–72 hr and persisting though to a lesser degree for 7 days. This stage of acceleration was associated with epidermal hyperplasia and most likely represented post-injury cell renewal.

DNA repair and chromatid anomalies in mammalian cells exposed to 4-nitroquinoline 1-oxide

Abstract: The oncogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO) induces DNA-repair synthesis (unscheduled DNA synthesis) in diploid, aneuploid, normal and neoplastic human and Syrian-hamster cells. DNA-repair synthesis occurs in nuclei at G1, G2 and S-phase and in metaphase chromosomes of Syrian-hamster cells exposed to 4NQO. DNA-repair synthesis was separated from DNA-replication synthesis associated with chromosome replication at S-phase by arginine deprivation. The degree of [ 3 H]TdR incorporation into nuclear DNA is dependent on the dose of 4NQO (5·10 −8 to 1·10 −5 M ) and on the amount of DNA per cell. The time course of DNA-repair synthesis induced by 4NQO or UV was examined on non-dividing cells which were arrested by an arginine-deficient culture medium: an early occurring peak is followed by an abrupt decline at about 8 h post-treatment which is succeeded by a prolonged low level incorporation of [ 3 H]TdR. The effect of a completed and uncompleted repair synthesis on the flow of cells into S-phase and on the frequency of chromosome anomalies was studied on cells arrested by arginine deprivation and triggered to divide by addition of arginine. There appears to be a relationship between the degree of repair synthesis and on the other hand frequency of cells entering S-phase, incidence of metaphase plates with chromatid breaks, flow of cells from G2 into pro-metaphase and “uncoiling” of metaphase chromosomes or heterochromatic segments of interphase nuclei.

The spindle as a basal body distributor. A study in the meiosis of the male silkworm moth, Bombyx mori.

Abstract: The spindle of metazoan cells functions as a dual distributor that guarantees the accurate segregation of both chromosomes and centrioles (basal bodies). This combined mechanism may have evolved from the separate distribution devices for chromosomes and basal bodies found in Protozoa. Typical eupyrene spermatocytes of the silk moth were compared with atypical apyrene spermatocytes. ( a ) Long microtubules, persisting during centriolar movements, develop in the meiotic prophases in continuity with the centrioles ( b ) There is no longer a centriole-microtubule continuity at metaphase-anaphase ( c ) Spindles comprise microtubules and endoplasmic reticulum (ER). The microtubules form a barrel-shaped structure terminating far in front of the centrioles. The two types of spermatocytes differ in the structure of the ER, which is vesicular in the eupyrene and lamellar in the apyrene line. It is suggested that the ER influences the anaphase movement of chromosomes, ( d ) The chromosomes lack localized centromeres. Microtubules penetrate all along the polar faces of the eupyrene metaphase chromosomes. The apyrene chromosomes form irregular blocks that are penetrated by microtubules all around their periphery, ( e ) The centriole comprises 3 concentric zones. Typical changes in the centriole are correlated with changes in the cell cycle. Replication, de novo formation and disappearance of centrioles are manifestations of the state of flux of the microtubules.

Ultraviolet-Microbeam Irradiation of Newt-Cell Cytoplasm: Spindle Destruction, False Anaphase, and Delay of True Anaphase

Abstract: Mitotic tissue-culture cells of the newt Triturus viridescens were irradiated in various cytoplasmic regions with monochromatic or heterochromatic ultraviolet microbeams (conical, with 25° half-angle, 8 μ in diameter in focal plane). Depending on exposure, some spindles were destroyed (i.e., true anaphase was prevented), and this was sometimes followed by quasirosette formation and false anaphase. Efficiency of spindle destruction was not detectably affected by the presence of a centrosome or part of the formed metaphase spindle in the irradiated region. For 280-nm irradiation the exposure which destroyed 50% of spindles was $2\times 10^{12}$ in terms of incident photons per cell times the cell depth in microns. Direct irradiation of centrosomes did not impair their mutual attraction for kinetochores. In metaphase-irradiated cells which survived spindle destruction, delay of true anaphase was critically dependent on whether irradiation occurred before or after the arrival of the last kinetochore at the sp...

Mode of action of the cytostatic agent "ICRF 159".

Abstract: ‘ICRF 159’, a new cytostatic agent, blocks the entry of cultured human lymphocytes into mitosis and arrests dividing cells in prophase and early metaphase.

Observations on the fine structure and development of the spindle at mitosis and meiosis in a marine centric diatom (Lithodesmium undulatum). IV. The second meiotic division and conclusion.

Abstract: The second meiotic division is shown to be of critical importance for interpretation of structures and events already seen at other divisions. The development of flagellar bases at interkinesis is demonstrated in relation to precursor material seen to accumulate near each pole at meiosis I. The name ‘paracentrosome’ is suggested for this material, which is used up in forming the flagellar bases and spindle precursor. The spindle at late prophase, metaphase and telophase II is shown to resemble those of other divisions except that it is consistently smaller; this fact is numerically demonstrated from serial sections of metaphase II cells. The greatly reduced size and unusual shape of the polar plates present in addition to flagellar bases at metaphase II suggest that these are in a sense equivalent structures with a mutually competitive relation to the paracentrosome. Preliminary observations with the light microscope on the relatively large nuclei of oogonia during meiosis I have shown that the haploid chromosome number is not less than 19 nor more than 23 and that chromatid separation in relation to the kinetochores at anaphase I is normal; the bearing of these findings on interpretation of the spindle is discussed. Comparisons with other organisms are carried out in a preliminary way and the investigation ends with a resume of the more important externally visible events in male gametogenesis adjusted to a common time scale in the course of one day.

An Estimate of the Amount of Microtubule Protein in the Isolated Mitotic Apparatus

Abstract: The microtubule content of the isolated mitotic apparatus of sea-urchin eggs ( Arbacia punctulata has been investigated by electron microscopy. Cross-sections were made through asters or spindles of flat-embedded mitotic apparatuses of known mitotic stage and specific orientation in the block. Cross-sections between chromosomes and poles of five metaphase half-spindles revealed approximately 2000-2300 sectioned microtubules. The number was somewhat higher in three anaphase half-spindles examined, approximately 2400-2600. A method was devised for calculating the total number of microtubules in an aster, based upon the number of microtubules appearing in cross-sections. Application of this method to selected mitotic apparatuses enabled calculation of the total number of microtubules in metaphase mitotic apparatuses of average dimensions. Using a 13-protofilament model of the microtubule and existing data on possible monomer sizes and molecular weights, the total amount of microtubule protein in the isolated mitotic apparatus was calculated. The values obtained are in the range of about 1-2 x 10 -8 mg microtubule protein per isolated mitotic apparatus. These values are close to those reported for the 4-5s protein of the isolated mitotic apparatus, but are considerably lower than the amount of 22s protein. The results are discussed with respect to cellular factors which determine microtubule number, and the possible sources and origin of mitotic microtubule protein.

Whole-mount electron microscopy of the centromere region of metacentric and telocentric mammalian chromosomes.

Abstract: Whole-mount electron microscopy of metaphase mouse L-cells showed that in many cases the region of the centromere was located at one extreme end of the chromosome with no evidence for short arms of any significant size. In such telocentric chromosomes there was only a single area of chromatid association. By contrast, the region of the centromere in mouse metacentric chromosomes was much longer. When well dispersed, it was seen to consist of two distinct areas of chromatid association, each of which was comparable in length to that of the telocentric chromosomes. In the region between these two areas there was a decrease in the density of the chromatin fibers. A similar quadripartite morphology was seen in human, Chinese hamster, and sheep metacentric chromosomes. These findings are most simply interpreted by the proposal that the quadripartite centromere of the metacentric chromosomes has resulted from the fusion of two telocentric chromosomes with bipartite centromeres.

Observations on the fine structure and development of the spindle at mitosis and meiosis in a marine centric diatom (Lithodesmium Undulatum) : III. The Later Stages of Meiosis i in Male Gametogenesis

Abstract: Numerical information with respect to spindle microtubules in serial sections of 3 cells at metaphase of the first meiotic division has been compiled and illustrated sufficiently to supplement the previous accounts with respect to both longitudinal and transverse views. At the equator the microtubules of the spindle are united laterally into bundles, the number of bundles being of the same order as that encountered at a premeiotic mitosis, though there is slight variation from cell to cell. The number of microtubules is greater than that at mitosis in a normal spermatogonium, though the distribution in different parts of the spindle is qualitatively similar. Stages of anaphase have been illustrated for the first time and shown to involve at least types of physical forces mediating chromosome movements of various kinds. The later stages of cytokinesis have been timed and are illustrated from living cells and from sections. The relation of the spindle to the cord which unites the separating protoplasts towards the end of cytokinesis has been traced in outline. The cord itself is shown to contain micro- tubules in limited number and it is therefore not the whole spindle. The interpretation of these findings is discussed in a preliminary way.

Cell Division in Oedogonium II. Nuclear Division in O. Cardiacum

Abstract: Nuclear division in O. cardiacum is described. Before division, the nucleus enlarges considerably. At prophase, the nucleolus starts dispersing and kinetochores appear on the condensing chromatin, situated and oriented apparently at random in the nucleus. By prometaphase, the kinetochore pairs become aligned along the spindle axis before moving into the metaphase-plate configuration; this supports an earlier theory explaining metakinesis. During prophase and metaphase particularly, the nuclear envelope at the poles forms channels that extend for some distance into the cytoplasm; these may also bifurcate. The nucleolus disperses but remains in the intranuclear spindle throughout division as a loosely knit skein of granular material. The kinetochores have a complex structure, up to seven distinct layers being detectable; the kinetochore pairs split, and then migrate polewards at anaphase with the rest of the chromosome trailing behind. Large numbers of microtubules run from the kinetochore into evaginations of the nuclear envelope which increase in size during anaphase. The spindle grows in length considerably during anaphase, this coinciding with a proliferation of interzonal microtubules, first seen amongst the trailing chromosome arms. The nuclear envelope enclosing the spindle becomes severely stretched at this stage; it contracts closely around each of the daughter nuclei, isolating them from the rest of the spindle (including microtubules and the remains of the nucleolus). The spindle then collapses; the nuclei come together and then flatten against one another; between them, vesicles and other components of the septum collect amongst a large number of transversely oriented micro tubules.

Mitochondrial RNA Synthesis during Mitosis

Abstract: HeLa cells arrested in metaphase synthesized relatively normal amounts of mitochondrial RNA, while little RNA synthesis associated with the nucleus was detected. The RNA synthesized resembled the portion of mitochondrial RNA sensitive to ethidium bromide in interphase cells, with major peaks at 21, 12, and 4S. Unlike that in interphase cells, RNA synthesis in the mitoclhonidrial fraction of mitotic cells was completely inhibited by ethidium bromide.

Effects of certain nitrogen mustards upon the progression of cultured H.Ep. no. 2 cells through the cell cycle.

Abstract: The Colcemid block method with some modifications has been used to analyze the cell cycle of cultured H.Ep. No. 2 cells and to determine the effects of certain nitrogen mustards upon the progression of cells through the cycle. The results and conclusions are as follows: Analysis of the cell cycle of H.Ep. No. 2 cells under the described experimental conditions yielded the following values: T C, 26.1 hr; T G1, 14.4 hr; T S, 6.9 hr; T G2, 3.9 hr; and T M, 0.9 hr. Treatment with 2,2′-dichloro- N -methyldiethylamine did not interfere with the gross fixation of thymidine-methyl-3H into the acid-insoluble residue of cell nuclei, and hence supposedly not with the gross synthesis of DNA, during the concurrent cell cycle. Treatment with low but toxic doses of 2,2′-dichloro- N -methyldiethylamine did not interfere with the progression of cells through G1 and into S during the concurrent cell cycle. Continuous treatment with low but toxic doses of 2,2′-dichloro- N -methyldiethylamine had little effect upon the progression to mitosis of cells initially in G2 at the time of addition of the agent, but it retarded or inhibited the progression to mitosis of cells initially in G1 or S. Pulse treatment of cells in G1 and in S interfered with the subsequent progression of these cells from S to mitosis. The fact that cells treated with low doses of 2,2′-dichloro- N -methyldiethylamine eventually do progress to mitosis is consistent with the possibility of repair of sublethal damage. Most cells treated with low concentrations of 2,2′-dichloro- N -methyldiethylamine that reach metaphase are probably capable of dividing and initiating colonies. Upon treatment with higher concentrations the cells that reach metaphase are apparently nonviable, as determined by the cloning technique. Continuous treatment with two monofunctional nitrogen mustards (2-chloro- N , N -dimethylethylamine and 2-chloro- N , N -diethylethylamine) affect the progression of cells through the cycle in a manner similar to that of 2,2′-dichloro- N -methyldiethylamine, but higher concentrations of the agents are required. The results suggest that although cross-linking of DNA by polyfunctional alkylating agents might contribute to the toxic effects of these agents, cross-linking is not a prerequisite for toxicity by alkylating agents.

A Microsurgical Methodology for Human Cells in Vitro: Evolution and Applications

Abstract: A methodology for micromanipulating human cells of normal and malignant origin, in vitro, has evolved from the study of about 2000 HeLa, ERK (a subline of HeLa cells), and human embryonic lung cells during interphase and mitosis. It is now possible to microinject interphase cells with aqueous and nonaqueous fluids intracytoplasmically. Chromosomes from human embryonic lung metaphase cells have been transplanted. Chromosomes have been manipulated within mitotic human embryonic lung, ERK, and HeLa cells. Clones have been obtained from HeLa cells subjected to such manipulation. Predictable derangements of mitotic cells and their progeny have been obtained. Intranuclear injections of silicone oil, DNA, and sodium chloride solutions have been made with survival of the cells. HeLa cells have been cloned from such injected cells. Subcellular fractions have been introduced into the nuclei and cytoplasms of HeLa and human embryonic lung cells. The lung cells have been subjected to nuclear micropuncture in groups and a clone has been obtained. Virus suspensions have been introduced into the nuclei of HeLa cells without killing the cells. Applications of this methodology are discussed.

The immediate response of the quiescent centre to x‐rays

Abstract: SUMMARY Roots of Allium sativum and Zea mays were given 150 rads and 1800 rads of X-rays respectively. Cells of the quiescent centre respond at once by coining into mitosis although most are at G, before irradiation. There is a stimulation of DNA synthesis in the quiescent centre, but not elsewhere, immediately after irradiation. Some cells, however, undergo mitosis without having synthesized DNA and this results in prophase and metaphase nuclei without the normal 4C complement of DNA.

Identical Linear Order of Chromosomes in Both Gametes of the Acoel Turbellarian Polychoerus carmelensis: A Preliminary Note

Abstract: In an exceptionally well-defined first cleavage metaphase in a sectioned egg of Polychoerus carmelensis all 34 (2n) chromosomes were present in a single 8-micron section, and it was possible to identify the homologous chromosomes derived from the two parent gametes. Three groups of chromosomes from one parent, containing 7, 7, and 3 chromosomes, respectively, could be exactly matched by corresponding groups of homologues derived from the other gamete. The probability of such an ordered pattern occurring by chance is somewhat less than 1 in 248. The simplest explanation of this arrangement is that the 17 chromosomes contributed by the egg were in precisely the same linear order as those contributed by the spermatozoon. This suggests that during certain stages preceding first cleavage metaphase, the 17 members of each haploid set of chromosomes may have been attached, end-to-end, in a linear fashion, by highly specific bonds.

Dose-response curve for x-ray-induced translocations in mouse spermatogonia. I. Single doses.

Abstract: When mle mice were given single doses of X-rays and chromosomes at first meiotic metaphase were examined 8 or more weeks later, the incidences of cells with translocations after doses of 500, 600, 700 and 800 rad were 12.2, 14.0, 14.2 and 6.8% resp. This humped dose-response curve is in good agreement with other work.

In situ analysis of normal and abnormal patterns of the mitotic apparatus in cultured rat-kangaroo cells.

Abstract: Dividing cells in monolayers of the rat-kangaroo (Potorous tridactylis) cell line Pt-K1 have large spindles and are flat, thus making possible studies of interactions between the achromatic and chromatic parts of the mitotic apparatus during the cell cycle. At prophase, asters and centrioles seem to exert pressure on the nuclear membrane leading to its rupture and penetrance of the centrioles. Apparently, the long axis of the spindle is shorter than the nuclear diameter. What appears as persistent, large portions of the nuclear membrane were observed in some metaphase and anaphase cells. Such a condition might also indicate an arrested mitosis. The midbody, which was often bipartite, was found to be of a ribonucleoprotein nature. — Three-group metaphases were of common occurrence and might represent early stages of chromosome orientation preceding the final alignment of the chromosomes on the equatorial plate. They could also be an expression of an anomalous condition as a result of mitotic arrest during prometaphase owing to spindle inactivation or breakage, errors in centromere-spindle attachments, interference with chromosome movement, or a duplicated centriolar constitution. Most of these aberrations could be attributed to the flatness of dividing cells, which might also bring about the failure of centriole separation and spindle organization in prometaphase stages, as well as multipolar mitosis.De novo organization of half spindles might take place in cells with ruptured spindles. Anaphase cells showing signs of a previous three-group orientation were rare. — Multipolar mitoses were prevalent mainly in cells with high chromosome numbers. They were often star-shaped with the chromosomes oriented between opposite and adjacent poles, and rarely as end-to-end associations of spindles. Apparently, one or more centrioles might share a common polar region. Multipolar configurations have either a mono- or multinuclear origin. Nuclei usually enter division synchronously in binucleate cells and the spindles become organized between centrioles associated with individual or different nuclei.

Mitosis in the fungusBasidiobolus ranarum as revealed by electron microscopy

Abstract: Mitosis of nuclei in vegetative hyphae of the fungusBasidiobolus ranarum has been studied by electron microscopy. Cells fixed with glutaraldehyde and OsO4 were embedded in Vestopal. Sections were obtained of single cells whose mitotic status was known. Attention was paid to the behaviour of the microtubules, the nuclear envelope and the nucleolus. Nuclear division begins with the dilution and rearrangement of nucleolar material and the gradual breakdown of the nuclear envelope. At this stage the nucleus is surrounded by a sheet of closely packed microtubules. Some of these penetrate into the nucleus through gaps in the envelope. Dissolution of the envelope is followed or accompanied by the development of an extensive labyrinth of membranous cisternae which persists at the periphery of the division site through mitosis and probably contributes material to the envelopes of the daughter nuclei. The drum-shaped spindle of metaphase is composed of large numbers of microtubules aligned parallel to each other. Many of them are associated with chromosomes. Metaphase is soon followed by the movement of dense masses of nucleolar material and chromosomes to the poles of the division figure to form the socalled “end plates”. Microtubules extend into the end plates but not beyond. Neither centrioles nor “centriolar plaques” have been seen.

Meiosis in the Djungarian hamster. I. General pattern of male meiosis.

Abstract: A study of male meiosis has been carried out on air-dried testicular preparations from 10 adult Djungarian hamsters (Phodopus sungorus campbelli Th.). The general course of meiosis in this species seems to be similar to that in most of the mammals so far studied. A short description of the main meiotic stages from pachytene to metaphase II has been presented. The sex-chromosomes (X and Y) show end-to-end pairing and prereduction. Between pachytene and diplotene there probably exists a diffuse stage. The large size of nuclei, the presence of double, occasionally crossed thin chromatin threads with a small number of double chromatin knobs are typical for this stage. The sex-vesicle is still present here but it is not so distinct as at pachytene, probably due to the beginning of straightening of the sex-bivalent. The differential arm of the X chromosome shows allocycly. During diplotene-diakinesis this arm looks like a thin double thread which is several times longer than the pairing arm. At metaphase I the differential arm is more condensed but usually the X does not become metacentric as at metaphase of mitosis. The allocycly of the X has been observed already at pachytene. The light zone of the sex-vesicle is formed by a smooth coiled thread or band which in some of the nuclei looks like a loop or “tail” protruding from the dark zone. A possible relationship between the allocycly of the X chromosome arm and its active functional state during meiotic prophase is suggested.

Induced Tetraploidy in Vinca rosea Linn

Abstract: Tetraploidy was induced in Vinca rosea Linn. by treating seeds with. 0.4%/ of aqueous colchinine. By selfing the C1 plants, C2 progeny was raised. The tetraploid is characterised by possessing a stout stem and branches, broader and shorter leaves, larger stomata, slower rate of growth, larger pollen grains and high pollen sterility. The total alkaloid content is more in both generations of the tetraploid plants. Mitosis in the tetraploid is almost normal. Meiosis in both Cl and Cz plants is irregular. It is analysed statistically. The presence of univalents, micronuclei of different shape, spindle abnormalities followed by aggregation of chromosomes into groups at metaphase, and anaphase, irregular anaphase and formation of polyads have been frequently observed in the PMC's.The coefficient of correlation between the abnormalities at different stages of meiosis and pollen sterility has been determined. There is no significant correlation between these irregularities and pollen sterility.