Endonuclease banding of isolated mammalian metaphase chromosomes

Evidence is presented that endonuclease digestion of isolated, unfixed chromosomes results in the production of banding patterns similar to those produced by digestion of fixed, air-dried chromosomes. Mouse L cell chromosomes were isolated under acidic or relatively neutral pH conditions, exposed in situ (as wet mounts on glass slides) or in vitro (in suspension) to micrococcal nuclease, Alu I or Eco RI, treated with a buffered salt solution, and stained with Giemsa. After any of these endonuclease treatments in situ, the centromeric regions of the chromosomes were intensely stained, characteristic of the C-banding observed in fixed chromosomes exposed to the same treatments. Although the fixed chromosomes were morphologically well-preserved after endonuclease digestion, the morphology of chromosomes digested in situ was variable, ranging from normal to swollen to highly distorted chromosomes. In the latter, the endonucleases induced dispersion of non-C-band chromatin; however, C-bands were still apparent as condensed, differentially-stained regions. Exposure of isolated chromosomes to Alu I in vitro also resulted in well-defined C-banding and led to the extraction of about 70% of the chromosomal DNA. From these results, the mechanism of endonuclease-induced C-banding appears to involve the dispersion and extraction of digested chromatin.     

Isopycnic centrifugation of mammalian metaphase chromosomes in Nycodenz

Separation of chromosomes on the basis of buoyant density has proved difficult due to their high density and sensitivity to high ionic strength. Nycodenz is a new non-ionic gradient medium which offers significant advantages over previously described media for separation of chromosomes. The purpose of this study was to examine the banding characteristics of isolated mammalian metaphase chromosomes in Nycodenz gradients. The results indicate that chromosomes can be efficiently concentrated and separated from nuclei using Nycodenz gradients. Furthermore, chromosomes density-labeled with bromodeoxyuridine (BrdU) can be separated from unlabeled chromosomes in Nycodenz gradients. Nycodenz does not appear to alter chromosome morphology or protein complement. The introduction of Nycodenz represents a significant new tool for use in chromosome separation and purification.     

Responses of mammalian metaphase chromosomes to endonuclease digestion

Digestion of fixed metaphase chromosomes by endonucleases (micrococcal nuclease and DNase II) under optimal digestion conditions followed by Giemsa staining produces sharp banding patterns identical to G-bands. In ³H-thymidine labeled, synchronized metaphase cells of the chinese hamster (CHO line), the band induction is accompanied by the removal of DNA. The single strand specific nuclease S1 and DNase I do not produce such banding patterns.     

Differences in DNA composition along mammalian metaphase chromosomes

Denaturation of chromosomal DNA in situ can be achieved without disruption of chromosomal morphology by heating slides at 25–90° C in 10–95% formamide in SSC. The extent of denaturation is proportional to formamide concentration and temperature. Reassociation of denatured DNA is prevented with formaldehyde. — The DNA in the paracentromeric constrictions in human chromosomes 1, 9 and 16 denatures earlier than in any other regions, as shown by the red colour with acridine orange. When the temperature or formamide concentration is raised a red and green banding pattern emerges in which regions known to stain brightly with quinacrine mustard are red whereas other regions are green. The last regions to turn red are the short arms of some acrocentric chromosomes. Since A+T-rich DNA denatures before G+C-rich DNA, it is inferred that QM-bright areas are rich in A+T. Similar results are obtained with mouse and Microtus agrestis cells. — Reassociation of chromosomal DNA denatured by heat and formamide occurs if no formaldehyde is used. In human cells, kinetic studies on reassociation indicate that the highest degree of repetition is in the DNA of the distal half of the Y chromosome. Next in degree of repetition are the paracentromeric constrictions, the short arm regions of some of the acrocentric chromosomes, and all the centromeric regions. Highly repetitious DNA is found in all mouse centromeric regions except that of the Y chromosome. Constitutively heterochromatic segments of X and Y and the autosomal centromeric regions of Microtus agrestis also contain repetitious DNA. — It is proposed that differential base content and susceptibility to denaturation of DNA contribute to or at least accompany Q-, G- and R-banding. The degree of C-banding is related to repetitious DNA. The human Y chromosomal DNA is probably A+T-rich and exceptionally repetitious, exhibiting spontaneous reassociation under many experimental conditions.     

Radiation-induced double-strand scission of the DNA of mammalian metaphase chromosomes

The molecular weight distribution of double-stranded mammalian (hamster) DNA was determined by ultracentrifugation of isolated metaphase chromosomes previously layered onto sucrose gradients containing high salt concentrations to dissociate the protein and nucleic acid components. In untreated controls the distribution (as determined by counting the incorporated radioactivity in the resultant fractions) exhibited a peak at 225 X 10(6) daltons. Inclusion of mercaptoethanol and hydroxylamine into the gradients produced no significant change of these sedimentation patterns. Gamma-radiation-induced reduction in the number and weight average molecular weights was used to calculate a value of 1.05 X 10(11) double-strand breaks/gram rad, equivalent to about 600 eV/break. No significant difference was observed for chromosomes irradiated either before or after isolation from intact mitotic cells. Irradiation in the presence of cystamine resulted in at least a sevenfold reduction in the apparent double-strand scission. The observed sedimentation patterns were compared with those generated by a theoretical computer simulation of radiation-induced degradation which assumed random selection and breakage of molecules. These results suggested that at least 80 to 90 % of the isolated DNA was distributed approximately normally with a mean molecular weight of about 200 X 10(6) daltons and a standard deviation of about 50 X 10(6) daltons.     

Incorporation and replication of foreign metaphase chromosomes in cultured mammalian cells

It is shown that the dyes used to produce banding patterns on chromosomes, quinacrine and Giemsa, are bound to DNA, and not to non-histone protein, the other chromosomal component remaining after acetic acid fixation. Studies on fixed nuclei and on extracted DNA in gelatine films show that the amount of dye bound is not affected by whether the DNA is native or denatured, and is not directly related to the amount of DNA present. Quinacrine is bound to the DNA ionically. With Giemsa, a new magenta compound is formed in situ, consisting of two molecules of methylene blue and one of eosin; this compound is attached to the chromosome by hydrogen bonds. Both quinacrine and the magenta compound formed from Giemsa appear to be attached to DNA molecules at two separate points, and the available evidence suggests that the amount of dye bound is related to the concentration of the DNA. It is suggested that the dye molecules bridge longitudinally separated sites brought into close proximity by folding of the DNA, and that the spatial arrangement of sites in the chromosome is influenced by non-histone proteins. It is concluded that chromosome banding is thus a consequence of the reduction of dye binding in those regions where the DNA chains become sufficiently dispersed to prevent bridging by the dye molecules. Possible indirect effects of base composition and repetition on dye binding at certain chromosomal sites are discussed.     

The mechanism of G and C banding in mammalian metaphase chromosomes

Bands have been observed in the fixed mitotic chromosomes of Mus musculus L cell with no further treatment. These bands, which are very similar to G bands, can be seen by phase contrast microscopy, ultraviolet microscopy and Gold/Palladium shadowing. This indicates that the cause of banding is a differential distribution of chromatin. Alterations in chromatin morphology can be induced by post fixation treatments of the fixed chromosome. A model is constructed on this evidence which unifies the apparent variety in the techniques which are thought to induce G bands and explains the action of Giemsa stain. It is concluded that these treatments act by promoting the disruption of chromatin structure which is then reformed in the presence of the Giemsa stain, the Azure-B component of the stain acting in a manner similar to divalent cations. A new technique for inducing C bands is reported. The denaturation and differential reassociation of DNA is not a suitable explanation of the mechanism of this technique. The hypothesis put forward here, explaining G bands, also explains the induction of C bands as a result of a differential destruction of chromatin morphology. The differential distribution of chromatin that gives G bands is thought to be disrupted by a technique that maintains the more resistant morphology of the centromeric heterochromatin, C bands.     

X-ray diffraction from isolated metaphase chromosomes

Metaphase chromosomes were isolated from Chinese hamster fibroblasts. They were found to give the same characteristic diffraction pattern in the wet state as isolated nucleohistone but when dry did not give the reflections normally associated with dry nucleohistone. No higher structural order than that associated with the nucleohistone supercoil was detected.     

Poleward force at the kinetochore in metaphase depends on the number of kinetochore microtubules

To examine the dependence of poleward force at a kinetochore on the number of kinetochore microtubules (kMTs), we altered the normal balance in the number of microtubules at opposing homologous kinetochores in meiosis I grasshopper spermatocytes at metaphase with a focused laser microbeam. Observations were made with light and electron microscopy. Irradiations that partially damaged one homologous kinetochore caused the bivalent chromosome to shift to a new equilibrium position closer to the pole to which the unirradiated kinetochore was tethered; the greater the dose of irradiation, the farther the chromosome moved. The number of kMTs on the irradiated kinetochore decreased with severity of irradiation, while the number of kMTs on the unirradiated kinetochore remained constant and independent of chromosome-to-pole distance. Assuming a balance of forces on the chromosome at congression equilibrium, our results demonstrate that the net poleward force on a chromosome depends on the number of kMTs and the distance from the pole. In contrast, the velocity of chromosome movement showed little dependence on the number of kMTs. Possible mechanisms which explain the relationship between the poleward force at a kinetochore, the number of kinetochore microtubules, and the lengths of the kinetochore fibers at congression equilibrium include a "traction fiber model" in which poleward force producers are distributed along the length of the kinetochore fibers, or a "kinetochore motor-polar ejection model" in which force producers located at or near the kinetochore pull the chromosomes poleward along the kMTs and against an ejection force that is produced by the polar microtubule array and increases in strength toward the pole.     

Structure of the mammalian kinetochore

The structure of the mammalian trilaminar kinetocnore was investigated using stereo electron microscopy of chromosomes in hypotonie solutions which unraveled the chromosome but maintained microtubules. Mouse and Chinese hamster ovary cells were arrested in Colcemid and allowed to reform microtubules after Colcemid was removed. Recovered cells were then swelled, lysed or spread in hypotonic solutions which contained D₂O to preserve microtubules. The chromosomes were observed in thin and thick sections and as whole mounts using high voltage electron microscopy. Bundles of microtubules were seen directly attached to chromatin, indicating that the kinetochore outer layer represents a differential arrangement of chromatin, continuous with the body of the chromosome. In cells fixed without pretreatment, the outer layer could be seen to be composed of hairpin loops of chromatin stacked together to form a solid layer. The hypotonically-induced unraveling of the outer layer was found to be reversible, and the typical 300 nm thick disk reformed when cells were returned to isotonic solutions. Short microtubules, newly nucleated after Colcemid removal, were found not to be attached to the kinetochore outer layer, but were situated in the fibrous corona on the external surface of the outer layer. This was verified by observations of thick sections in stereo which made it possible to identify microtubule ends within the section. Thus, kinetochore microtubules are nucleated within the fibrous corona, and subsequently become attached to the outer layer. We dedicate this paper to Wolfgang Beermann on the occasion of his 60th birthday in appreciation of many years of friendship and his pioneering contributions in the field of chromosome biology     

Isolation and initial characterization of the mammalian midbody

Midbodies were isolated from synchronized cultures of Chinese hamster ovary (CHO) cells and their protein composition was studied by means of SDS PAGE. Gels of the midbodies included alpha and beta tubulins as major bands (approximately 30% of the total protein) and approximately 35 other bands, none of which constituted greater than 3.5% of the total protein. Extraction of the isolated midbodies with Sarkosyl NL-30- solubilized the midbody microtubules but left the central, dense matrix zone of the midbody intact. A protein doublet of approximately 115,000 mol wt was retained preferentially by the particulate fraction containing the matrix zones, indicating it to be a component of the matrix. The 115,000 mol wt doublet was also present in gels of isolated mitotic spindles from CHO cells. The overall protein composition of the isolated spindles was very similar to that of the isolated midbodies.     

Immunoelectronmicroscopy of metaphase chromosomes

Summary A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.     

Immunoelectronmicroscopy of metaphase chromosomes

A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.     

The kinetochore is part of the metaphase chromosome scaffold

We used antisera from patients with the CREST syndrome of scleroderma (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) to show that an antigenic component of the kinetochore present in metaphase chromosomes is also present in nonhistone chromosome scaffolds isolated following extensive digestion of the DNA and extraction of the bulk of chromosomal protein. All sera from 12 scleroderma CREST patients previously shown by immunofluorescence microscopy to have circulating antikinetochore antibodies recognise a protein of Mr 77,000 (CREST-77) in an immunoblotting assay. 9 of the 12 sera also recognise an antigen of Mr 110,000 (CREST-110). These proteins are present in isolated chromosomes and nonhistone scaffolds derived from them by two different procedures. Sera of five scleroderma CREST patients who are antikinetochore negative (by immunofluorescence) bind to neither protein in immunoblots. These data suggest that CREST-77 (and possibly CREST-110) is a component of the human kinetochore, and that the kinetochore is an integral part of the mitotic chromosome scaffolding.     

Organization within the mammalian kinetochore

The organization within the mammalian kinetochore was examined using whole-mount electron microscopic techniques on chromosomes digested with restriction enzymes or micrococcal nuclease. These preparations revealed that a portion of the kinetochore is highly resistant to nuclease digestion and can be visualized as a discrete structure. The relationship of this structure to the remainder of the chromosome suggests that it represents the outer kinetochore plate. The plate is composed of a series of fibrillar loops that are arranged in a parallel array along the plane of the plate. These fibers are 25–30 nm in diameter. The morphology, particulate substructure, and ultimate susceptibility to nuclease digestion suggest that these fibers contain DNA. A model is presented that suggests that the outer plate contains the apexes of chromatin loops that originate within the body of the primary constriction.