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PubMed1985

Light and scanning electron microscopy of the same human metaphase chromosomes.

A technique has been developed to examine the same G-banded human metaphase chromosomes, first in the light microscope and then in the scanning electron microscope (SEM). A structural involvement in chromosome banding was confirmed by a positional correlation between the G-positive bands observed in the light microscope and the circumferential grooves between the quaternary coils of the metaphase chromosomes, observed in the SEM. In further support of this the regions between the grooves showed a positional relationship with the G-negative or reverse (R) bands. The examination of slightly extended metaphase chromosomes in the light microscope demonstrated that the G-banding pattern corresponded to that described by the Paris nomenclature for metaphase chromosomes. The arrangement of the circumferential grooves of the same chromosomes, observed in the SEM, was shown to relate to that described by the Paris nomenclature for prometaphase chromosomes. Therefore, using the SEM it is possible to demonstrate the details of prometaphase banding in metaphase chromosomes.

Chromosome Banding↗

PubMed1992

Automatic assessment of the quality of G-banded metaphases. A preliminary study.

Assessment of the quality of cytogenetic preparations is important for 1) a general quality control of the cytogenetic laboratory and 2) for determining the quality of individual cytogenetic analyses. It is generally accepted that good preparations allow a more detailed search for structural abnormalities than poor preparations. As part of a comprehensive study on automated assessment of slide quality we have developed a simple algorithm for automated measurement of metaphase resolution. During semi-automated karyotyping with the Magiscan chromosome analysis system (Joyce-Loebl) three measurements related to resolution are automatically extracted 1) the total number of dark bands of the chromosomes of the metaphase (TB), 2) the normalized average length of the chromosomes (NL), and 3) the average "thickness" of the chromosomes (T). The algorithm TB x NL/T has been tested on 35 metaphases of various quality and compared with visual assessment of the same 35 metaphases. The results indicate that the automatic assessment of metaphase resolution is superior to the visual.

Chromosome Banding↗

PubMed2002

Three-dimensional helical coiling structures and band patterns of hydrous metaphase chromosomes observed by low vacuum scanning electron microscopy.

Helical coiling structures and band patterns of hydrous metaphase chromosomes were documented three-dimensionally by low vacuum scanning electron microscopy (SEM). Fixed or unfixed isolated Chinese hamster metaphase chromosomes were stained with platinum blue (Pt blue) and observed in the backscattered electron mode for low vacuum SEM without any hypotonic treatment or drying processes. Fibrous structures were shown both in the fixed and unfixed hydrous chromosomes; helical chromatid coils and their subcoils were clarified especially in the fixed chromosomes having contrasting alternative bands of light and darkness, while the translucent perichromosomal matrix and compact fibrous structures were recognized in the unfixed chromosomes. The helical coils were more clearly represented in a loosened chromatid of metaphase chromosomes. Treatment with a tris-HCl buffer solution and Pt blue staining in a hydrous condition successfully produced banding patterns similar to G-bands on metaphase chromosomes. These banded chromosomes observed by low vacuum SEM were also analyzed stereoscopically by field emission SEM after critical point drying. These findings indicate that: 1) native or unfixed chromosomes maintain the compact arrangement of high-order helical structures covered with the peri-chromosomal matrix; 2) helical coiling appearances of chromatids frequently observed in previous papers might be caused by loosening of the final level of the high-order structure of the metaphase chromosome; and 3) banding patterns might be produced by the rearrangement or reorganization of chromatin fibers at the 30 nm fiber level after the extraction of some chromosomal components including the peri- or intra-chromosomal materials during the banding procedure.

Animals↗

PubMed2003

Telomere dynamics in myelodysplastic syndrome determined by telomere measurement of marrow metaphases.

Myelodysplastic syndrome (MDS), which is known to be a preleukemic state, is a heterogeneous entity characterized by ineffective hematopoiesis and dysplastic morphological features. Most MDS patients show erosive telomeric repeats (TTAGGG)(n), without up-regulation of telomerase activity, suggesting that telomere shortening may be linked to cellular senescence in MDS. We measured telomere length in samples from 13 MDS patients and 8 healthy volunteers, based on telomere signals of individual chromosomes, using digital images of metaphases after quantitative fluorescence in situ hybridization (Q-FISH) with peptide nucleic acid probes and compared the results with results obtained with the standard method of determining terminal restriction fragment (TRF) length. In healthy volunteers, we found a significant correlation between TRF length and telomere fluorescence signals detected by Q-FISH, and a relatively wide distribution of fluorescence telomere signals was demonstrated in every sample. In contrast, we found no linear correlation between TRF length and telomere fluorescence signals in MDS, and most MDS patients showed weak telomere fluorescence signals, corresponding to short telomeres, with a narrow range compared with normal subjects. TRF length represented telomere DNA in whole marrow cells, whereas telomere fluorescence signals by Q-FISH represented only marrow metaphases corresponding to MDS-derived cells. Metaphases from most MDS patients showed homogeneous telomere shortening, irrespective of the presence of cytogenetic abnormality. In contrast, marrow metaphases from normal individuals showed a relatively wide range of telomere signals in each metaphase, indicating that in MDS cells, telomere shortening mechanisms that normally exist might be dysregulated. Therefore, analysis of telomere distribution as well as average telomere length detected by Q-FISH might be useful to clarify the telomere dynamics of MDS cells.

Adult↗

PubMed2005

[A Novel Method for Increasing the Number of Metaphase Cells Cultured in vitro.].

To increase the number of metaphase cells cultured in vitro, two bovine fibroblast cell lines (YFF and CNF) were frozen at 4 degrees for different length of time prior to colchicine treatment , and then chromosomal specimen were prepared. The percentage of metaphase cells was examined under conditions above. Using this method, the variation rate of karyotype of YFF and CNF subcultured up to passage 20 were also analyzed. It was found that the percentage of YFF and CNF metaphase cells in treatment group were significantly higher than that in control group (p0.05), and the number of YFF and CNF metaphase cells obtained in 20 h treatment group were increased more than 6 fold as many as that in control group (p0.01), 31.7% and 40.2% vs 4.7% and 6.4% ,respectively. These data suggest that the method of freezing at 4 degrees could be used for increasing the number of metaphase cells in vitro conveniently, and analyzing the variation rate of karyotype of cultured cells efficiently.

Animals↗

PubMed2005

[Morphological observation of metaphase I arrested oocytes in IVF-ET cycles].

OBJECTIVE: To observe morphological characteristics and in-vitro maturation ratio of all metaphase I arrested oocytes in IVF-ET cycles. METHODS: In two IVF-ET cycles, maturation culture in vitro was performed and we evaluated morphological characteristics of all oocytes arrested in metaphase I. RESULTS: Metaphase I arrested oocytes presented with uniform cytoplasm, compact zone and no space between oocyte and zone. Incompact association was observed between granule cell and zone without radiated structures. There was no markedly alterations in morphological characteristics after 24, 48, 72 hours of maturation culture, respectively. All oocytes were arrested in Metaphase I and no polar growed. CONCLUSION: All metaphase I arrested oocytes possess of unique morphological characteristics and fail to mature following current culture assay in IVF-ET cycles.

Adult↗

PubMed1991

[The nucleolus organizer regions of the metaphase chromosomes in cambial cell populations of mouse and rat placenta].

The number of silver-stained nucleolus organizing regions (Ag-NORs) was counted in metaphase plates of the fetal part of placenta of mice and rats and in the tissues of their embryos. On day 9 of mouse gestation and on day 12 of rat gestation, up to 82% of metaphases in the fetal part of placenta have the highest possible number of chromosomes with Ag-NORs (9-10 for mice and 5-6 for rats). In the later embryogenesis (day 10 for mice and day 14 for rats), a great number of metaphases have either no Ag-NORs (34.9% for mice, 17.9% for rats), or only 1-2 Ag-NOR-chromosomes (17.4% for mice and 14.5% for rats). But in the tissues of embryos being on the same embryonic stages the changes in frequency of metaphases with different numbers of Ag-NOR-chromosomes have proved to be less obvious. In the differentiated polyploid placenta cells the number of chromosomes with Ag-NORs can be considerably lower than in diploid ones. It is proposed that the changes in frequency of metaphases with different numbers of Ag-NOR-chromosomes may reflect the regulation of ribosomal gene transcription in the embryogenesis, in particular, in differentiation of cambial cell populations of rodent placenta.

Animals↗

PubMed1985

Phosphorylation of histone H1 through the cell cycle of Physarum polycephalum. 24 sites of phosphorylation at metaphase.

H1 phosphorylation has been studied through the precise nuclear division cycle of Physarum polycephalum. The number of sites of phosphorylation of Physarum H1 is very much larger than the number of sites reported for mammalian H1 molecules which is consistent with the larger molecular weight of Physarum H1. At metaphase all of the Physarum H1 molecules contain 20-24 phosphates. Immediately following metaphase, these metaphase-phosphorylated H1 molecules undergo rapid dephosphorylation to give an intermediate S phase set of phosphorylated H1 molecules containing 9-16 phosphates. Progressing into S phase newly synthesized H1 is phosphorylated and eventually merges with the old dephosphorylated H1 to give a ladder of bands 1-20. By the end of S phase or early G2 phase, there is a ladder of bands 1-16 all of which undergo phosphate turnover. Further into G2 phase the bands move to higher states of phosphorylation, and by prophase all of the H1 molecules contain 15-24 phosphates which increases to 20-24 phosphates at metaphase. These results support the proposals that H1 phosphorylation is an important factor in the process of chromosome condensation through G2 phase, prophase to metaphase.

Alkaline Phosphatase↗

PubMed1984

Roles of Ca ions and ooplasmic factors in the resumption of metaphase-arrested meiosis in Rana pipiens oocytes.

Fully grown amphibian oocytes are arrested at the diplotene stage of meiosis. When they undergo meiotic maturation, meiosis resumes and the oocyte chromosomes condense to metaphase. During this period, the oocyte cytoplasm develops 'chromosome condensation activity' (CCA), the ability to induce the formation of metaphase chromosomes from nuclei transplanted into the oocytes. The cytoplasm also produces 'maturation promoting factor' (MPF), the substance that induces meiotic maturation when injected into oocytes. Also, before meiosis is arrested again at the 2nd metaphase, the cytoplasm develops 'cytostatic factor' (CSF), the substance that causes metaphase arrest when injected into zygotes. Since CSF-arrested zygotes have properties similar to those of metaphase-arrested oocytes, including the potential to resume cell cycle activities, CSF appears to be a genuine factor that causes meiotic arrest. Following oocyte activation, meiosis is completed and the chromosomes decondense to form a pronucleus. During this period, the oocyte cytoplasm loses its CCA and develops 'chromosome decondensation activity' (CDA), the ability to decondense the chromatin of injected nuclei. Concomitantly, MPF and CSF disappear. Both MPF and CSF are inactivated by Ca2+ ions, in vitro. The sensitivity of CCA to Ca2+ and the requirement for Ca2+ during the development of CDA have also been demonstrated in vitro by incubating demembranated sperm nuclei in cell-free preparations from unactivated or activated oocytes. Preparations made from unactivated oocytes in the presence of EGTA exhibit CCA, whereas those made in the absence of EGTA, as well as those made from activated eggs, exhibit CDA. Unactivated ooplasmic preparations made using EGTA lose CCA and develop CDA when Ca2+ ions are added to them. However, at low Ca2+ concentrations CCA is sustained and, when unactivated and activated preparations are mixed, is able to overcome CDA. Therefore, it is likely that at low intracellular Ca2+ levels in unactivated oocytes, CSF is stable and CCA predominates over CDA, thus preventing oocyte chromosomes from decondensing. However, when Ca2+ levels are elevated during oocyte activation, CSF disappears and CCA is replaced by CDA. This change in cytoplasmic activities may allow meiosis to resume.

Animals↗

PubMed1979

Age-dependent variability of ribosomal RNA-gene activity in man as determined from frequencies of silver staining nucleolus organizing regions on metaphase chromosomes of lymphocytes and fibroblasts.

Frequencies of silver staining nucleolus organizing regions (NORs) have been determined in lymphocytes and fibroblasts from very young and from aged persons. Since silver staining of NORs is associated with activity of ribosomal RNA-genes, we used this approach to investigate a possible inactivation of these genes during aging. Our lymphocyte data are based on a study per age-group of 220 metaphases from 10 subjects. Although in both age-groups modal numbers of silver staining chromosomes per metaphase had similar ranges over the subjects, the frequency of metaphases containing the maximal number of staining chromosomes was in the old age-group (80--89 years) significantly lower than in the young age-group (less than 1 year old). In fibroblasts, of which 75 metaphases from 4 subjects were included per age-group, differences were more pronounced. Modal numbers of silver staining chromosomes were for the aged persons (69--83 years) lower than for the young children (less than 1 year old). Highly significant differences were observed between both groups in frequency of metaphases containing the maximal number of positively reacting acrocentric chromosomes and, more in general, in frequencies of silver staining D- and G-group chromosomes, the lower frequencies being found in the old age-group. We propose the term NOR-junctions as distinct from satellite associations for arrangements of acrocentric chromosomes which after silver staining are visibly connected at their NORs. The number of acrocentric chromosomes involved in lymphocyte NOR-junctions of aged people was significantly higher than the number of joined acrocentrics in young children. The frequency of these NOR-junctions themselves, irrespective of the number of chromsomes involved, was higher for aged persons than for young children, although this difference appeared to be statistically not significantly higher than in fibroblasts. Also based on qualitative observations from our study we discuss tcehnical and biological problems of our approach to study cell aging in vivo by means of silver staining of NORs. We conclude that in man, reflected by the difference in frequencies of silver staining NORs between young and aged persons, a rather extensive loss of ribosomal RNA-gene activity may occur during aging.

Aged↗

PubMed1973

Contrast between the environmental pH dependencies of prophasing and nuclear membrane formation in interphase-metaphase cells.

In Chinese hamster Don cells, fusion of an interphase cell with a metaphase cell resulted either in prophasing of the interphase nucleus, including loss of the nuclear envelope (NE), or in the formation of a double membrane around the metaphase chromosomes. Only one of these phenomena occurred in a given interphase-metaphase (I-M) binucleate cell. At pH 7.4, there was about an equal probability that either event could occur amongst the population of I-M cells. The effect of pH changes in the medium containing the fused cells was examined. At pH 6.6, prophasing was the predominant event; at pH 8.0, membrane formation predominated. It was found that the rate of progression of a mononucleate cell from G(2) to metaphase was appreciably faster at pH 6.6 than at pH 8.0. Conversely, the progression from metaphase to G(1) was faster at pH 8.0 than at pH 6.6. These results with the mononucleate cells strengthen the hypothesis that structural changes in I-M cells are reflections of normal mitotic phenomena. Additional evidence for this hypothesis was produced by electron microscope examination after direct fixation in chrom-osmium. The double membrane around the chromosomes of the I-M cell was indistinguishable from the normal NE. The results obtained by varying the pH of the medium containing the fused cells provide an indication that disruption or formation of the NE of Don cells depends on the balance reached between disruptive and formative processes.

Animals↗

PubMed1976

Nucleosomes in metaphase chromosomes.

Previous studies of the structure of metaphase chromosomes have relied heavily on electron micrography and have revealed the existence of a 10-nm unit fiber that is thought to generate the native 23-30-nm fiber by higher order folding. The structural relationship of these metaphase fibers to the interphase fiber remains obscure. Recent studies on the digestion of interphase chromatin have revealed the existence of a regularly repeating subunit of DNA and histone, the nucleosome that generates the appearance of 10-nm beads connected by a short fiber of DNA seen on electron micrographs. It was therefore of interest to probe the structure of the metaphase chromosome for the presence of nucleosomal subunits. To this end metaphase chromosomes were prepared from colchicine-arrested cultures of mouse L-cells and were subjected to digestion with stayphylococcal nuclease. Comparison of the early and limit digestion products of metaphase chromosomes with those obtained from interphase nuclei indicates that although significant morphologic changes occur within the chromatin fiber during mitosis, the basic subunit structure of the chromatin fiber is retained by the mitotic chromosome.

Cell Nucleus↗

PubMed1976

Template activity of unfixed metaphase chromosomes.

Unfixed metaphase and non-metaphase cells were tested for their template activity with RNA polymerase. A device was used which disrupts the cell membrane by centrifugation, and which also ensures that the cells do not continue with their mitotic cycle during the transcription process. The template activity of acid/methanol fixed cells was also tested. None of the unfixed metaphase cells transcribed RNA whereas most of the non-metaphase cells did. In contrast, using fixed cells both classes of cells were transcribed equally well and to a much greater extent. It was concluded that metaphase chromosomes in vivo cannot act as templates for RNA synthesis.

Cell Line↗

PubMed2026

DNA-FISH Metaphase Spreads to Distinguish Extrachromosomal DNA from Homogeneously Staining Regions in Human Cancer Cell Lines.

UNLABELLED: Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells. SUMMARY: We report a DNA-FISH metaphase spread protocol that visually detects locus copy number and location within the genome. This approach enables single-cell resolution of amplification states, specifically in cancer cell lines containing extrachromosomal DNA and homogeneously staining regions.

Journal Article↗

PubMed1988

Effect of metabolic inhibitors on sucrose-induced metaphase spindle elongation and spindle recovery.

Hyperosmotic sucrose treatment of metaphase PtK-1 cells has been shown to produce a reversible concentration-dependent effect on spindle elongation linked to a functional alteration in the connection of the chromosome to the spindle (Pover et al.: European Journal of Cell Biology 39:366-372, 1985). Spindle elongation, similar to that which occurs at anaphase B, is thought to be driven by the compression stored in the form of microtubule curvature in the nonkinetochore (nkMT) population of microtubules at metaphase (Snyder et al.: European Journal of Cell Biology 35:62-69, 1984 and 39:373-379, 1985). Addition of metabolic inhibitors to Ham's F-12 salts with deoxyglucose (D/F-12 medium) containing 0.4 M sucrose and 1 mM DNP does not within statistical error affect the rate and extent of sucrose-induced spindle elongation; rates and extents are 60-75% of normal anaphase B motions. Electron microscopic analysis of metaphase cells treated with D/F-12 medium and 0.4 M sucrose with 1 mM DNP demonstrates that spindle microtubules lose curvature and become straight in appearance, typical of microtubule organization in untreated anaphase cells. Sucrose-treated cells released into D/F-12 medium show a rapid reduction in spindle length; however, cells treated with either 0.4 M sucrose or 0.4 M sucrose and 1 mM DNP-containing D/F-12 medium and released into DNP-containing D/F-12 medium do not exhibit a significant reduction in spindle length. Electron microscopic analysis links changes in spindle length with microtubule/kinetochore associations. These data suggest that energy required for the initial phases of spindle elongation during anaphase is preloaded into the mitotic spindle by metaphase and does not require additional energy to be expressed as examined by sucrose-induced spindle elongation in the presence of metabolic inhibitors. Second, energy is required to make or maintain (or both) functional chromosome associations with the spindle as measured by reduction in spindle length following sucrose removal.

Animals↗

PubMed1981

Automated selection of metaphase cells by quality.

The performance of metaphase-finding systems could be improved if they were able to determine the quality of the cells detected. This paper discusses the extent to which this may be realized by the introduction of a metaphase-quality parameter. Data obtained from 300 cells were statistically analyzed. Seventeen features were measured and nine visual properties were determined for each cell. Discriminant analysis and regression analysis were used to extract those features and visual properties which contribute to assessment of metaphase quality. Rather low correlations were found between the selected measured features and visual properties. A quality-parameter based on a linear combination of cluster projections, areas and perimeters was found to account for 64% of the variation between visual and measured quality indicators. In addition, an increase in the predictive value for finding usable metaphases from 28-68% was achieved.

Cell Separation↗

PubMed1987

Enhancement of concurrent yield of spermatogonial, meiotic I, and meiotic II metaphase chromosomes from Chinese hamster testes.

A procedure to enhance the concurrent yield of large numbers of spermatogonial, meiotic I, and meiotic II metaphases from Chinese hamster testes has been developed. Animals were injected intraperitoneally with 5, 10, 20, or 40 mg of colchicine/kg and killed 3 hr after the treatment. Both testes from each animal were excised; one testis was minced, and germ cells were isolated by using 0.1% trypsin solution; the other testis was minced, and germ cells were isolated by using a conventional procedure. Chromosomal preparations were made with a standard air-drying technique. Examination of slides revealed that with the trypsin-isolation procedure the concurrent yields of spermatogonial, meiotic I, and meiotic II metaphases were significantly higher (P less than .05) than the yields obtained with the conventional procedure. Furthermore, 40 mg of colchicine/kg produced maximum numbers of all three types of germ cell metaphases. At this maximum concentration, metaphases were stable with no evidence of structural aberrations, and the frequency of hyperploidy was not significantly different (P greater than .05) from hypoploidy.

Animals↗

PubMed1994

The proteolysis-dependent metaphase to anaphase transition: calcium/calmodulin-dependent protein kinase II mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs.

It has been shown, using spindles assembled in vitro in extracts containing CSF (the cytostatic factor responsible for arresting unfertilized vertebrate eggs at metaphase), that onset of anaphase requires Ca(2+)-dependent activation of the ubiquitin-dependent proteolytic pathway that destroys both mitotic cyclins and an unknown protein responsible for metaphase arrest (Holloway et al., 1993, Cell, 73, 1382-1402). We showed recently that Ca2+/calmodulin-dependent protein kinase II (CaM KII) activates the ubiquitin-dependent cyclin degradation pathway in CSF extracts (Lorca et al., 1993, Nature, 366, 270-273), but did not investigate its possible effect on sister chromatid segregation. In this work we identify CaM KII as the only target of Ca2+ in inducing anaphase in CSF extracts, and further show that transition to anaphase does not require the direct phosphorylation of metaphase spindle components by CaM KII. A possible interpretation of the above results could have been that the ubiquitin-dependent degradation pathway is required for onset of anaphase only when spindles are clamped at metaphase due to CSF activity, and not in the regular cell cycle that occurs in the absence of CSF activity. We ruled out this possibility by showing that competitive inhibition of the ubiquitin-dependent degradation pathway still prevents the onset of anaphase in cycling extracts that lack CSF and do not require Ca2+ for sister chromatid separation.

Anaphase↗