SEARCH · PubMed Health

Results for “Metaphase chromosomes”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1978

Selective digestion of mouse metaphase chromosomes.

Metaphase chromosomes prepared from colcemid-treated mouse L929 cells by non-ionic detergent lysis exhibit distinct heterochromatic centromere regions and associated kinetochores when viewed by whole mount electron microscopy. Deoxyribonuclease I treatment of these chromosomes results in the preferential digestion of the chromosomal arms leaving the centromeric heterochromatin and kinetochores apparently intact. Enrichment in centromere material after DNase I digestion was quantitated by examining the increase in 10,000 X g pellets of the 1.691 g/cc satellite DNA relative to main band DNA. This satellite species has been localized at the centromeres of mouse chromosomes by in situ hybridization. From our analysis it was determined that DNase I digestion results in a five to six-fold increase in centromeric material. In contrast to the effect of DNase I, micrococcal nuclease was found to be less selective in its action. Digestion with this enzyme solubilized both chromosome arms and centromeres leaving only a small amount of chromatin and intact kinetochores.

Animals↗

PubMed1978

Localization of Drosophila nasutoides satellite DNAs in metaphase chromosomes.

Metaphase chromosomes of D. nasutoides were hybridized in situ with 3H-cRNA synthesized from the four satellites which make up 50--60% of the total DNA of this species. All four satellites were localized in the large, metacentric, heterochromatic chromosome four. They did not, however, appear to hybridize to centromeric or other constitutive heterochromatin, nor did they, with the exception of satellite I, seem to hybridize in the specific regions of chromosome four which, on the basis of C, Q, and H banding and AT contents, were predicted to contain some of these satellites.--Comparison of grain patterns with the results of fluorescent staining indicated that satellite-bearing heterochromatin was not always associated with other fractions of constitutive heterochromatin in interphase nuclei and was, at least partially, decondensed in some larger nuclei.

Animals↗

PubMed1984

Development and use of metaphase chromosome flow-sorting methodology to obtain recombinant phage libraries enriched for parts of the human X chromosome.

Metaphase chromosomes isolated from human lymphoblastoid cell lines containing structurally abnormal X chromosomes have been stained with the bisbenzimidazole dye Hoechst 33258 and analyzed on a FACS II flow system equipped with a 5-W all-lines argon ion laser. The chromosomal fluorescence has been highly resolved at flow rates of 1,000-3,000 chromosomes per second. With the goal of obtaining recombinant DNA libraries from parts of the human X chromosome, fluorescence populations enriched for a dicentric X (Xpter- greater than Xq24::Xq24-greater than Xpter) chromosome and an isochromosome of the long arm of the X [i(Xq)] have been identified. The dicentric X chromosome has been resolved as a discrete peak in the fluorescence flow histogram. In contrast, the fluorescence intensity of the isochromosome is indistinguishable from that of chromosomes 3 and 4. Recombinant DNA libraries from the flow-sorted chromosomes have been constructed in the lambda phage, Charon 21A, and consist of 1.6 X 10(5) and 0.7 X 10(5) plaque-forming units in the case of the dicentric X and the isochromosome, respectively. Ninety percent of the phage in both recombinant libraries contain inserts which hybridize to highly repetitive human DNA sequences. The recombinant phage library from the flow-sorted dicentric X chromosome, which could be assigned to a discrete fluorescence peak, has been further characterized and shows at least a tenfold enrichment for X chromosome-specific DNA sequences as determined by Southern blot hybridization of cloned fragments.

Bacteriophage lambda↗

PubMed1981

[Identification of isolated metaphase chromosomes. II. A comparison with the recognizability of chromosomes in metaphase plates].

The metaphase chromosomes (MC) isolated from the Chinese hamster cells were identified with the aid of differential staining (G-bands). It was shown that differences in the relative recognizability of MC in metaphase plates and after their isolation are determined by changes in composition of isolated MC, rather than by those in staining capacity of MC after their isolation. The frequencies of identified MC are constant and independent upon the type of MC preparations and relation between identified and unidentified MC in certain preparations. At allows to apply the described method for the analysis of chromosome fractionation, using changes in frequencies of identified MC as a criterion of efficiency of the fractionation method. Possible ways of increasing the recognizability level of isolated MC are discussed.

Animals↗

PubMed1995

A postprophase topoisomerase II-dependent chromatid core separation step in the formation of metaphase chromosomes.

Metaphase chromatids are believed to consist of loops of chromatin anchored to a central scaffold, of which a major component is the decatenatory enzyme DNA topoisomerase II. Silver impregnation selectively stains an axial element of metaphase and anaphase chromatids; but we find that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes. Inhibition of topoisomerase II prevents this separation, and also prevents the contraction of chromatids that occurs when metaphase is arrested. Immunolocalization of topoisomerase II alpha reveals chromatid cores analogous to those seen with silver staining. We conclude that the chromatid cores in early mitosis form a single structure, constrained by DNA catenations, which must separate before metaphase chromatids can be resolved.

Animals↗

PubMed1976

[Ultrastructure of the centrometric regions of mouse chromosomes in metaphase chromosomes and interphase nuclei].

The chromatin ultrastructure was studied in the centromeric region of mitotic chromosomes and in interphase nuclei of mouse cells after differential staining on C-band. A new method is suggested to study centromeric region of chromosomes treated by the Giemsa banding technique. Fibers of chromosomes appeared to be packed denser in the centromeric regions of mitotic chromosomes than in arms. The disposition of chromatin fibers in the centromeric chromocentres of interphase nuclei is the same as in the centromeric regions of mitotic chromosomes.

Animals↗

PubMed2004

Preimplantation diagnosis of repeated miscarriage due to chromosomal translocations using metaphase chromosomes of a blastomere biopsied from 4- to 6-cell-stage embryos.

OBJECTIVE: To evaluate the safety and accuracy of karyotyping the blastomere chromosomes at metaphase in the natural cell cycle for preimplantation diagnosis. DESIGN: A pilot study. SETTING: A private infertility clinic and a university laboratory. PATIENT(S): Eleven patients undergoing IVF and preimplantation diagnosis. INTERVENTION(S): Intact human embryos at the 4- to 6-cell stage and human-mouse heterokaryons were cultured and checked hourly for disappearance of the nuclear envelope. After it disappeared, the metaphase chromosomes were analyzed by fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): Percentage of analyzable metaphase plates and safety and accuracy of the method. RESULT(S): The success rate of electrofusion to form human-mouse heterokaryons was 87.1% (27/31), and analyzable chromosomes were obtained from 77.4% (24/31) of the heterokaryons. On the other hand, disappearance of the nuclear envelope occurred in 89.5% (17/19) of the human embryos and it began earlier than that in the heterokaryons. Analyzable chromosomes were obtained and their translocation sites were identified in all blastomeres biopsied from the 17 embryos. After the biopsy, 67.0% of the embryos could develop to the blastocyst stage. CONCLUSION(S): The natural cell cycle method reported herein requires frequent observation, but it is safe, with no artificial effects on the chromosomes and without loss of or damage to blastomeres, which occurred with the electrofusion method. Using the natural cell cycle method, we could perform preimplantation diagnosis with nearly 100% accuracy.

Abortion, Habitual↗

PubMed1987

Metaphase chromosome folds and X-inactivation.

To evaluate the suggested nonrandom folding of Xq13-q21 (center of Barr body condensation) of the inactivated X chromosomes, metaphases from nine subjects with or without X chromosome abnormalities (eight females and one male) were investigated with RBG-staining. A significant increase (p less than .05) in the number of arm folds (Xq13-q21) of the late-replicating X chromosome, particularly in early to mid-metaphase, was observed in four of eight females. Therefore, the stage of chromosome contraction was an important factor with more folds observed at the centromeres and in longer chromosomes in early metaphase compared with mid- to late metaphase. X chromosome folds were present in cells of subjects treated with or without bromodeoxyuridine. While our study agrees with the relationship of Xq13-q21 fold with the X-inactivation center, the correlation of extended chromosomes and folding limits this method as a sole indicator of X-inactivation in routine mid-metaphase, but is useful in the analysis of early metaphase chromosomes.

Dosage Compensation, Genetic↗

PubMed1992

Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes.

Unfixed metaphase chromosome preparations from human lymphocyte cultures were immunofluorescently labelled using antibodies to defined histone epitopes. Both mouse monoclonal antibody HBC-7, raised against the N-terminal region of H2B, and rabbit serum R5/12, which recognizes H4 acetylated at Lys-12, gave non-uniform labelling patterns, whereas control antibodies against total histone fractions H4 and H1 produced homogeneous fluorescence. HBC-7 bound approximately uniformly to the bulk of the chromosomes, but the major heterochromatic domains of chromosomes 1, 9, 15, 16 and the Y showed significantly brighter fluorescence. Serum R5/12 indicated an overall reduction in acetylation of H4 in metaphase chromosomes compared with interphase nuclei, although some specific chromosomal locations had considerably elevated acetylation levels. Acetylation levels in the major heterochromatic domains appeared extremely low. To investigate further the differences noted in heterochromatin labelling, metaphases from cultures grown in the presence of various agents known to induce undercondensation of the major heterochromatic domains were similarly immunolabelled. Decondensed heterochromatin no longer exhibited higher than normal immunofluorescence levels with HBC-7. The higher resolution afforded by "stretching" the centromeric heterochromatin of chromosomes 1, 9 and 16 confirmed the low level of H4 acetylation in these domains. We consider the implications of these observations in relation to chromatin conformation and activity.

Acetylation↗

PubMed1985

Effects of sulphydryl reagents on the structure of dehistonized metaphase chromosomes.

Dehistonized metaphase chromosomes lose their apparent axial organization (the 'scaffold') and sediment more slowly following exposure to beta-mercaptoethanol (BME). We have subsequently treated BME chromosomes with reagents that oxidize protein sulphydryls to disulphides, and found that if calcium is also present during the oxidation an apparently similar axial structure is restored following dehistonization, as seen by microscopic examination. In general, however, we do not find that oxidation restores the higher sedimentation rate of dehistonized control chromosomes. Analysis of residual core protein in dehistonized chromosomes by sodium dodecyl sulphate/polyacrylamide gel electrophoresis fails to detect any differences in polypeptide composition related to the state of oxidation or to the presence or absence of visible axial organization. Combining our results with those of other workers, we conclude that the axial structure evident in dehistonized metaphase chromosomes is maintained, at least partially, by inter-protein cross-linking, although in vivo this may not be via simple disulphide bridges. Additional factors, which we have not yet characterized, but which possibly include heavy metal ions, appear to be involved in the axial organization existing in vivo.

Animals↗

PubMed1982

High-resolution scanning electron microscopy of human metaphase chromosomes.

Human metaphase chromosomes, prepared for light microscopy were examined by scanning electron microscopy. Use of an osmium impregnation technique eliminated the need for sputter-coating and allowed high-resolution visualization of uncoated specimens. Chromosomes were of three-dimensional cylindrical profile, with well-defined chromatids and centromeres. Prior to Giemsa-banding a smooth surface morphology was observed. Relaxation of chromosome integrity by Giemsa-banding pretreatment allowed resolution of several orders of chromosome structure not previously demonstrated by scanning electron microscopy. The observed organization of the chromatin fibres allowed parallels to be drawn with the radial loop model of chromosome construction as described by Marsden and Laemmli.

Chromosome Banding↗

PubMed1978

Transfer of genetic information via isolated amphibian metaphase chromosomes.

The metaphase chromosome transfer system of McBride and Ozer (1973) has been adapted to a haploid, euploid, frog cell line. Genes coding for a deoxypyrimidine kinase and an enzyme responsible for a thymidine-specific saturable transport system have each been transferred at frequencies between 10(-6) and 10(-5) transferents per cell treated. Revertants for each of these two genes were observed at frequencies between 10(-8) and 10(-7) revertants per cell tested. Selfing controls showed no transferents. Two colonies were obtained in which cotransfer of both genes may have occurred. Activities of the transferred genes were assayed by incorporation of [3H]thymidine into alkali-stable, acid-precipitable material. Growth properties of 13 transferents in various media were also determined and presence of the appropriate enzymes inferred. These transferents were tested for stability early (25 generations) after transfer and were found to be stable. All 13 transferents possess the normal haploid number of chromosomes (n = 13) with no cytologically detectable chromosomal fragments.

Animals↗

PubMed1984

Restriction enzyme banding of mouse metaphase chromosomes.

Fixed metaphase chromosomes from mouse strain RIII embryos or A9 cells were treated with a restriction endonuclease, followed by Giemsa staining. Alu I, Hinf I, or Mbo I treatment produced a C-band pattern, and Eco RII or Hae III produced a G-band plus C-band pattern. Ava II and Bst NI each produced a G-band pattern, but on most chromosomes only a small segment of each C-band, adjacent to the centromere, was stained. These tiny residual C-bands may contain a minor satellite located adjacent to the major satellite clusters.

Animals↗

PubMed1979

Scanning electron microscopy of uncoated human metaphase chromosomes.

Human metaphase chromosomes were processed with a 3% glutaraldehydetannic acid technique and examined in a scanning electron microscope at 20 kV either without added metal coating or with 2 nm of sputtered gold coating. Several substrates--aluminum mnium foil, silver mirror deposit and sputtered gold-provided good conductive backgrounds for chromosomal spreads. Silver mirror deposit was the best conductive substrate tested. This method should prove to be a useful tool for monitoring the three-dimensional morphology of mitotic chromosomes with the possibility of studying various banding techniques, chromosomal uncoiling and secondary constrictions currently being examined in chromosomal studies.

Aluminum↗

PubMed1983

Chromatin organization and restriction endonuclease activity on human metaphase chromosomes.

Human metaphase chromosomes were treated with HaeIII, HindIII, EcoRI, and AluI 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.

Azure Stains↗

PubMed1995

High-resolution FEISEM detection of DNA centromeric probes in HeLa metaphase chromosomes.

HeLa metaphase chromosome spreads were hybridized with centromeric biotinylated DNA probes and detected with gold-conjugated anti-biotin antibodies. Chromosomes were observed by an in-lens field emission scanning electron microscope (FEISEM), which permits detection of biological samples without any coating. DNA probes were well localized in the centromeric region of chromosomes and there was clear discrimination between 10 nm fibers that hybridized to DNA probes and those that did not hybridize. This approach shows that in situ hybridization can be directly visualized at the FEISEM level by evaluating only secondary electron emission, which allows physical localization of the hybridized probe with high resolution so that backscatter detection represents only a control. Because chromosomes maintain the 10-nm fiber organization after in situ hybridization procedures, our data suggest that this fiber represents the lowest order of chromatin arrangement that permits transitory DNA denaturation.

Centromere↗

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↗

PubMed1980

Silver stained core-like structures in chinese hamster metaphase Chromosomes.

Chinese hamster metaphase chromosomes, subjected to prolonged hypotonic pretreatment and subsequently stained with ammoniacal silver, contained a darkly-stained core-like structure in each chromatid, surrounded by a halo of dispersed chromatin which was pale yellow to brown in color. The core was variable in its appearance, ranging from a continuous linear configuration to a spiral structure or a discontinuous, particulate structure. Within the centromeric regions, the cores frequently appeared more intensely stained than elsewhere in the chromosome. The nucleolus organizers also stained darkly and appeared to be attached to the core-like structures. It remains to be determined whether the cores represent a real component of metaphase chromosome structure, or whether they are artifacts resulting from abnormal chromatin aggregation arising at the time of chromosome preparation.

Animals↗