Chinese Hamster Metaphase Chromosomes Research Articles

Fucosylated glycoproteins in Chinese hamster metaphase chromosomes.

Distribution of fucosylated proteins in Chinese hamster metaphase chromosomes was studied with a fluorescein isothiocyanate conjugated monofucosyl-specific lectin, Ulex europaeus agglutinin I (UEA I). In situ binding of UEA I showed that fucosylated chromosomal proteins are preferentially localized to two internal structural domains of metaphase chromosomes: the helically coiled substructure of chromatids, and the Q-band regions. Corresponding proteins were identified in Western blots of isolated metaphase chromosomes. Several proteins with molecular weights ranging from 33 to 195 kD were recognized by UEA I. These data suggest that a subset of chromosomal proteins are fucosylated; they may have a role in the structural organization of chromosomes.

Glycoproteins with N-acetylglucosamine and mannose residues in Chinese hamster metaphase chromosomes.

Distribution of glycosylated proteins in Chinese hamster metaphase chromosomes was studied with fluorescein isothiocyanate conjugated lectins. Three substructural domains with distinct glycoprotein compositions were identified. In situ binding of the lectins Wheat germ agglutinin (WGA) and Datura stramonium agglutinin (DSA) showed that chromosomal proteins containing N-acetylglucosamine residues preferentially localize to the surface domain and the helically coiled substructure of chromatids. In Western blots, digoxigenin conjugated WGA and DSA bound to several chromosomal proteins with molecular weight ranges of 45-220 kD and 66-220 kD, respectively. Binding of Galanthus nivalis agglutinin revealed that mannosylated chromosomal proteins are enriched at the surface and G/Q band domains, and their molecular weights range from 97 to 200 kD. The carbohydrate side chain structure of these mannosylated proteins must be quite specific, as after binding of another mannose-specific lectin, Concanavalin A, only a faint fluorescence was observed in metaphase chromosomes and only one protein band of 185 kD was weakly stained in Western blots. These data suggest that chromosomal glycoproteins containing N-acetylglucosamine and mannose residues play a role in the higher order structural organization of metaphase chromosomes.

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.

LOCALIZATION AND IDENTIFICATION OF GALACTOSE/N-ACETYLGALACTOSAMINE AND SIALIC ACID-CONTAINING PROTEINS IN CHINESE HAMSTER METAPHASE CHROMOSOMES

Four lectins were used to recognize galactose/N-acetyl-galactosamine (Gal/GalNAc) and sialic acid residues in proteins of Chinese hamster metaphase chromosomes.In situbinding pattern of a fluorescein isothiocyanate-labelled (Gal/GalNAc)-specific lectinSophora japonicaagglutinin (SJA) showed that chromosomal SJA-binding proteins are primarily localized to the helically coiled substructure of chromatids. Numerous SJA-binding proteins were identified in Western blots of chromosomal proteins, their molecular weights ranging from 26 to 200kDa. Another Gal/GalNAc-specific lectin, peanut agglutinin (PNA), with a slightly different sugar binding specificity, did not bind to Chinese hamster metaphase chromosomes, and in Western blots only two chromosomal protein bands were faintly stained. Thein situlabelling patterns of two sialic acid-specific lectins,Maackia amurensis(MAA) andSambucus nigra(SNA) agglutinins, both showed that the helically coiled substructure of chromatids is also enriched in sialylated proteins. In Western blot analysis 11 MAA-binding protein bands with molecular weights ranging from 54 to 215kDa were identified, while SNA only bound to one protein band of 67kDa. MAA and SNA are specific for α(2|ad3)- andα(2|ad6)-linked sialic acid residues, respectively. Thus, it is likely thatα(2|ad3)-linked sialic acid residues are more common in chromosomal proteins thanα(2|ad6)-linked sialic acid residues. These data suggest that Gal/GalNAc and sialic acid-containing glycoproteins exist in metaphase chromosomes and that these proteins may have a role in the formation of higher order metaphase chromosome structures.

Fine structure of hydrous chromosomes observed by low vacuum scanning electron microscopy.

Chinese hamster metaphase chromosomes were stained with platinum (Pt) blue and observed in the hydrous state with low vacuum scanning electron microscopes (LVSEM). The coiled structure of the chromatin fibers in the chromosomes was well recognized through the surrounding perichromosomal substances in the backscattered electron (BSE) mode at accelerating voltages of over 20 kV. Findings indicated that chromatin fibers in native chromosomes have a structure similar to the hierarchic coiled model. The present study also demonstrated that not only surface structures but also subsurface structures can be studied in the BSE mode of LVSEM, when the subsurface structures have been stained with heavy metal salts such as Pt blue.

Natural size classes in DNA from Chinese hamster metaphase chromosomes

DNA from isolated Chinese hamster ovary (CHO) metaphase chromosomes can be obtained in three different molecular weight classes. The two largest forms have sedimentation coefficients of 80 and 120 S at 7,500 rpm. Based on sedimentation and speed dependence analysis these have molecular weights of 220 million and above 5,000 million, and are thought to be analogs of DNA classes observed in a prior study of human metaphase chromosomes. An extract can be converted to primarily the 80 S form through alkaline pH treatment of metaphase DNA. The third class (45 S DNA) is formed as a result of metaphase chromosome exposure to the nuclease Bal31, and has a mass distribution analogous to the CHO replicon.

A comparative study on proteins released from metaphase chromosomes after digestion with restriction endonucleases and deoxyribonuclease I.

Extensive digestion of Chinese hamster metaphase chromosomes with Alu I, Hae III and Hinf I released up to 40 distinct chromosomal proteins. Some of the proteins released by Hae III or Hinf I were enriched in the protein moiety liberated by Alu I but several proteins released by Hae III were not released by Alu I digestion. The amount of chromosomal protein released by deoxyribonuclease I (DNase I) was comparable to that liberated by the three restriction enzymes so far tested, while only four abundant protein species were detectable in the protein moiety released by DNase I. Two of them with molecular weights of 58,000 and 50,000 were also released by the three restriction enzymes and are similar in size to those found previously in the core-like structure of histone-depleted chromosomes.

Nick-translation of metaphase chromosomes: in vitro labeling of nuclease-hypersensitive regions in chromosomes.

Chinese hamster metaphase chromosomes were labeled by nick-translation, which involved pretreatment of metaphase chromosomes with low levels of DNase I followed by incubation with DNA polymerase I and radioactively labeled nucleotides. The labeled DNA was located on nuclease-hypersensitive regions of the chromosomes, as suggested by the following observations. (i) The labeled DNA was hypersensitive to the subsequent DNase I digestion. (ii) The labeled DNA contained no nucleosomes. DNA reassociation kinetic analysis suggested that the labeled DNA was enriched in repetitive DNA sequences. Base composition analyses showed that the labeled DNA was highly enriched in guanine and adenine residues, suggesting that the nick-translation reaction was asymmetrical and the strand enriched in purine was preferentially translated. Autoradiographic analysis revealed that the label was distributed on every chromosome, but there was a lower grain density on the Y chromosome, which is heterochromatic and exhibits a relatively low level of gene activity. The locations of silver grains on the Y chromosomes were generally consistent with that revealed by the in situ hybridization using [3H]cDNA synthesized from the total Chinese hamster messenger RNA. These observations suggest that a specific subset of genomic DNA on active chromatin is the preferred site of the nick-translation.

Chinese hamster metaphase chromosomes isolated under physiological conditions: A partial characterization of associated non-histone proteins and protein cores

In a previous report [2] we have described a non-histone protein core which could be isolated from Chinese hamster metaphase chromosomes. This core structure maintained the overall morphology of the metaphase chromosome even after removal of all of the histones, together with many of the non-histone proteins and the bulk of the DNA. As part of our work on the characterization of these core structures, we have developed a novel procedure for the isolation of metaphase chromosomes which avoids the use of high pH buffers and hexylene glycol, as well as eliminating the numerous centrifugation and resuspension steps previously employed. Chromosome cores prepared by 2 M NaCl extraction and DNase I digestion from metaphase chromosomes isolated under these more gentle, quasi-physiological conditions, are shown to contain a relatively simple subset of non-histone proteins. One-dimensional SDS-polyacrylamide gel electrophoresis shows two major groups of polypeptides having molecular weights 48 000–52 000 and 65 000–72 000 D respectively, with similarities in mobilities to the nuclear pore complex-lamina polypeptides and tubulins. However, more detailed analysis by two-dimensional gel electrophoresis and peptide mapping has failed to detect these proteins. A 52 000 D polypeptide component of the core is tentatively identified as the intermediate filament protein vimentin. The in vivo significance of chromosome cores is discussed.

Dansyl chloride-stained nucleolar organizers and core-like structures in Chinese hamster metaphase chromosomes

When chromosomes containing both BrdU-substituted and unsubstituted regions were treated with hot NaH 2PO 4 at high or low pH and then stained with dansyl chloride, brightly fluorescent nucleolar organizer regions (NORs) and core-like structures were apparent in the chromosomes. These structures closely parallel the appearance of the same structures in silver-stained chromosomes. Since dansyl chloride is a protein-specific fluorochrome, the distribution of fluorescence suggests that the NORs and central zone of each chromatid contain higher concentrations of protein relative to other chromosome regions. The fluorescent core structures are interpreted to be artefacts of the NaH 2PO 4 pretreatment induced by changes in the concentration of chromatin (including protein) between the chromatin-dense center and more dispersed peripheral region of each chromatid.

Electron microscopy of silver-stained core-like structures in metaphase chromosomes.

Chinese hamster metaphase chromosomes, stained with ammoniacal silver and examined by electron microscopy, were covered with fine silver grains of variable size. In addition, many chromosomes contained linear aggregates of silver grains running continuously from one end of each chromatid to the other, forming a core-like structure. Extensive deposits of silver were observed over the nucleolar organizers and the centromeric regions, and the silver precipitate in the latter region appeared to be a localized differentiation of the core-like structure. The silver cores ranged from thin to thick and continuous to discontinuous elements in different chromosomes. This extreme variability suggests that these cores are not true structural components of chromosomes. The amount of silver deposit over any given chromosome region may simply reflect the concentration of chromatin in that region of the chromosome. The silver-stained core-like structure probably reflects an underlying difference in the concentration of chromatin in the central and peripheral region of each chromatid. Such differences in chromatin concentration may be induced during the prolonged hypotonic treatment required for the subsequent visualization of cores.

Reversible differential decondensation of unfixed Chinese hamster chromosomes induced by change in calcium ion concentration of the medium.

Differential decondensation of isolated unfixed Chinese hamster metaphase chromosomes was obtained by decreasing the calcium ion concentration in the surrounding medium. A banded appearance of the swollen chromosomes could be observed either directly by phase contrast microscopy or after glutaraldehyde fixation and staining. There was a gradual transition from homogeneously dense to banded and finally to extensively decondensed chromosomes. The patterns induced at different stages were similar to those observed on fixed chromosomes after standard banding procedures (i.e., G-, Cd-, Ag-NOR-staining). Chromosomes decondensation could be reversed by the addition of calcium ions to the medium. Ca ++-dependent reversible differential chromosome decondensation was not observed if the chromosomes were previously treated with 0.35 M NaCl. Chromosome regions which had incorporated BrdU into their DNA were more resistant to a decrease in calcium ion concentration than BrdU non-substituted regions.

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.

Chromosome bands and the subunit structure of Chinese hamster metaphase chromosomes.

Isolated Chinese hamster chromosomes dissociate into a series of specific chromatin subunits approximately the size of stainable chromosome bands upon reduction of the divalent ion concentration during or after isolation. At high pH the chromatin in some bands is differentially removable during chromosome isolation, leaving a banded chromosome with a pattern typical of most G-band procedures. This provides an alternate molecular mechanism to explain the production of banded chromosomes by a variety of staining procedures. These results also suggest an approach to chromatin fractionation, using metaphase chromosomes as a starting material.

Higher order structure of chromosomes

Isolated Chinese hamster metaphase chromosomes were resuspended in 4 M ammonium acetate and spread on a surface of distilled water or 0.15 to 0.5 M ammonium acetate. The DNA was released in the form of a regular series of rosettes connected by interrossette DNA. The mean length of the rosette DNA was 14 micron, similar to the mean length of 10 micron for chromomere DNA of Drosophila polytene chromosomes. The mean interrosette DNA was 4.2 micron. SDS gel electrophoresis of the chromosomal nonhistone proteins showed them to be very similar to nuclear nonhistone proteins except for the presence of more actin and tubulin. Nuclear matrix proteins were present in the chromosomes and may play a role in forming the rosettes. Evidence that the rosette pattern is artifactual versus the possibility that it represents a real organizational substructure of the chromosomes is reviewed.

A partial characterization of DNA fragments protected from nuclease degradation in histone depleted metaphase chromosomes of the Chinese hamster.

A small proportion (0.1-0.5%) of the total DNA content of native Chinese hamster metaphase chromosomes is protected from nucleolytic degradation following the removal of histones by extraction with either 0.2 N HCl or 2 M NaCl, and remains attached to the nonhistone protein core. Acid extraction followed by DNase I digestion leads to small fragments of 10-30 bases. Salt extraction followed by micrococcal nuclease digestion gives approx. 140 b.p. fragments which are undistinguishable in size from nucleosome core DNA fragments. Furthermore, DNase I treatment of salt extracted chromosomes gives DNA fragments containing single strands which are multiples of 10 bases in length, again characteristic of the nucleosome structure. Reassociation kinetics using the 32P-labelled 140 b.p. fragments as probes suggests they are enriched for rapidly reassociating sequences.

Identification of chick chromosomes in cell hybrids formed between chick erythrocytes and adenine-requiring mutants of Chinese hamster cells.

When chick erythrocytes were fused with adenine-requiring mutants of Chinese hamster cells of two different complementation classes and grown in adenine-free medium for 1 week or longer, mitotic cells were observed containing both chick and Chinese hamster metaphase chromosomes within a single cell. Stable hybrid clones were subsequently isolated that possessed, in addition to a complete Chinese hamster genome, a single specific chick chromosome. Hybrids resulting from fusions involving different adenine mutants retained different, identifiable chick chromosomes. Thus, the two chick genes for the endogenous synthesis of adenine were assigned to the respective chick chromosomes.

STRUCTURE OF CHROMOSOMES VISUALIZED BY SCANNING ELECTRON MICROSCOPY

Isolated metaphase chromosomes of Chinese hamster observed under the scanning electron microscope appear to have a distinct rough surface composed of chromonemata. The chromonema is a fine filament coated with nucleoprotein, which can be easily removed by hypotonic sodium citrate solution. The centromere, a narrowed, tight portion of the chromosome, is also multistranded. No membrane has been seen in metaphase chromosomes.

Separation of isolated Chinese hamster metaphase chromosomes into three size-groups

A method is described for the isolation of metaphase Chinese hamster chromosomes and their separation into three size-groups. DNA extracted from fractionated chromosomes does not show heterogeneity in thermal denaturation temperature or buoyant density. The separation technique has been used to demonstrate a high rate of DNA synthesis in small hamster chromosomes during late S phase.