Chromosomes Of Chinese Hamster Cells Research Articles

The effect of cyclamate and saccharin on the chromosomes of a Chinese hamster cell line.

Two artificial sweeteners, cyclamate and saccharin, were studied as to their effect on the chromosomes of Chinese hamster cells in vitro. An incidence of chromosome breaks and gaps, significantly increased over the control level, was found in the treated experimental groups. A certain dose relationship of the disturbances induced was indicated. The distribution over the karyotype of the aberrations was nonrandom and the pattern of breaks and gaps in the treated groups coincided largely with that in the controls.

Dual Control of Replication Timing: STOCHASTIC ONSET BUT PROGRAMMED COMPLETION OF MAMMALIAN CHROMOSOME DUPLICATION

In mammalian cells, DNA replication proceeds according to a precise temporal order during the S phase, but how this program is controlled remains poorly understood. We analyzed the replication-dependent bromodeoxyuridine banding of chromosomes in Chinese hamster cells treated with the spindle poison nocodazole. In these cells, nocodazole induces a transient mitotic arrest, followed by DNA re-replication without intervening cell division. Nuclear fragmentation is often observed in tetraploid derivatives, and previous studies suggest that replication timing of chromosomes could be affected when they are segregated into different micronuclei. Here we show that the onset of replication is frequently asynchronous on individual chromosomes during the re-replication process. Moreover, fluorescence in situ hybridization analysis revealed that replication synchrony is equally altered in fragmented and non-fragmented nuclei, indicating that asynchronous onset of replication is not dependent on physical separation of the chromosomes into isolated compartments. We also show that the ordered program of replication is always preserved along individual chromosomes. Our results demonstrate that the onset of replication of individual chromosomes in the same nuclear compartment can be uncoupled from the time of S-phase entry and from the programmed replication of chromosome sub-domains, revealing that multi-level controls contribute to establish replication timing in mammalian cells.

Localization of amplified CAD genes on rearranged chromosomes of Chinese hamster cells

Chinese hamster cell lines carrying an amplified CAD region were selected from V79,B7 cells by their resistance to N-phosphonacetyl-L-aspartate (PALA). In one of the selected cell lines, SP PALA (inf1) (supR) L, an acrocentric chromosome with abnormally elongated q arms was identified as a marker for the PALA-resistant phenotype. The marker chromosome carried a homogeneously staining region close to a telomeric nucleolar organizer region. In the same region, localization of amplified CAD sequences was demonstrated by in situ hybridization. The marker chromosome was found to undergo extensive rearrangements. In particular, dicentric chromosomes, occurring with an unusually high incidence, were found to originate from end-fusion of two homologous marker chromosomes.

New evidence for the presence of chromosomes in micronuclei of human and Chinese hamster cells.

The aim of this work was to demonstrate the presence of intact chromosomes in micronuclei. Such evidence was based on the re-expression of metaphase chromosomes by micronuclei fused with whole cells. Three different experimental approaches were used; they differed only in the criteria used to discriminate between the chromosomes of micronuclear origin and those present in the cell to which micronuclei were fused. These methods involved the expression of: (a) human chromosomes in mouse cells, (b) radioactively labelled human chromosomes in unlabelled human lymphocytes and (c) Chinese hamster chromosomes in Chinese hamster cells with differentially stained chromosomes. With all methods, evidence for the presence of one or few chromosomes in micronuclei was obtained.

The Diethyldithiocarbamate Concentration Effects and Interactions with Other Cytotoxic Agents on Chinese Hamster Cells (V79)

A metal chelator, diethyldithiocarbamate (DDC) perturbs the chromosome condensation processes in dividing cells. The length of the metaphase chromosomes in Chinese hamster cells (V79) treated with 17.2 micrograms/ml of DDC for 2 hr is about half of that in untreated cells. However, concentrations of 1.7 microgram or 172 micrograms/ml DDC apparently do not produce this effect. DDC at 17.2 micrograms/ml also disrupts spindle fibers. Penicillamine, EDTA, EGTA, and diamide show no effect on chromosome condensation. Bleomycin, but not mitomycin and cisplatin, added simultaneously with DDC can prevent the DDC effect on chromosomes. The cytotoxic effect of increasing concentrations of DDC to V79 cells incubated at 37 degrees C exhibits a similar biphasic response. This concentration biphasic toxic effect is not altered when the cells are treated with DDC in combination with radiation, heat, or other cytotoxic drugs. These observations suggest that the different effects of DDC concentrations on chromosome condensation should be considered as one important modification factor for DDC related toxicity.

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.

Evidence of nucleosomes in situ and their organization in chromatin and chromosomes of Chinese hamster cells

Freeze-fracture studies of intact metaphase Chinese hamster cells indicate that the chromosomes are composed of compactly aligned, flat and hexagonally shaped bipartite disks. The overall dimensions of a bipartite disk are about 140 ± 25 A ̊ in height, 100 ± 20 A ̊ in width, and 54 ± 4 A ̊ in thickness. The center-to-center distance of two units of a bipartite disk is about 34 ± 3 A ̊ . The core of a disk appears to contain four substructures that measure about 25 ± 5 A ̊ in diameter. Through the center of the disk core there is an axial structure measuring about 20 ± 5 A ̊ in diameter, which spans and interconnects similar bipartite disks to form a continuous linear array. A structure measuring 20 ± 4 A ̊ in diameter is wound around the edges of the disk core and interconnects with the next bipartite disk. The connecting strand is parallel to the axial structure. In addition, there appear to be bridgings between the curled structure and disk core. The chromatin of intact interphase cell nuclei shows similar bipartite disks with dimensions and features similar to those of metaphase chromosomes. The bipartite disks are aligned in a linear array in both chromatin and chromosomes.

Separation of large quantities of chinese hamster chromosomes by velocity sedimentation at unit gravity followed by flow sorting (FACS II)

Separation of large quantities of isolated metaphase chromosomes of Chinese hamster cells was performed by velocity sedimentation at unit gravity in a specially designed sedimentation chamber. This simple and easy technique results in chromosome fractions of relatively high purity as determined by flow cytometry and microscopy. Up to 10 10 chromosomes can be processed depending upon the size of the sedimentation device, and enrichments up to 10 times of individual chromosomes were achieved. In addition, further chromosome purification was performed by fluorescence activated flow sorting using fractions, pre-enriched at unit gravity. The flow sorted chromosomal fractions were pure according to flow cytometric analyses. The combination of l g sedimentation and flow-sorting opens the possibility for preparative chromosome sorting by reducing the flow sorting time considerably.

Mutagenicity studies

An antiulcer drug, 4-(2-carboxyethyl) phenyl trans 4-aminomethylcyclohexane carboxylate hydrochloride (DV-1006), was studied for mutagenicity using bacterial systems, in vitro and in vivo cytogenetics, and dominant lethal tests. No mutagenicity of DV-1006 was observed either in the rec-assay on Bacillus subtilis or in the Salmonella/microsome test (Ames test). In in vitro cytogenetics, DV-1006 had no effects on the chromosomes of chinese hamster cells at cytotoxic doses. Rats were treated singly or on 5 consecutive days orally with dose levels of 16, 160, or 1600 mg DV-1006/kg for detecting cytogenetic effects in vivo. As a result, no increase of the incidence of chromosomal aberrations in bone marrow cells was observed in any group of DV-1006. A single or 5 daily oral administration of DV-1006 (16 or 1600 mg/kg) to male mice and subsequent mating for 8 weeks produced no dominant lethal mutational effects. These results show that DV-1006 has no mutagenic potential.

UV-induced unscheduled DNA synthesis and its distribution in mitotic chromosomes of Chinese hamster cells

Two established chemical mutagens—ethylmethanesulphonate (EMS) and triethylenemelamine (TEM)—were tested for the ability to induce chromosome aberrations in mouse spermatogonia. While not a single aberration was detected following the EMS treatment, a low frequency of translocations and fragments was found in the TEM groups. These findings are in agreement with the data obtained with the specific locus mutation test as applied to male mouse premeiotic germ cells but contrast with the effectiveness of these chemicals in breaking chromosomes in male mouse postmeiotic germ cells. A differential sensitivity of post- and premeiotic germ cells to any kind of genetic damage by these chemical mutagens is most likely to be the correct interpretation of all the data. However, it is also suggested that a high proportion of translocations induced in spermatogonia by chemical mutagens may not be detectable by present methods.

Distribution of UV-induced unscheduled DNA synthesis in metaphase chromosomes of Chinese hamster cells

Summary Unscheduled DNA synthesis (USD) occurred in metaphase chromosomes of cultured Chinese hamster cells after ultraviolet light (UV) irradiation. When the chromosomes were labeled by UV-induced USD in metaphase, the number of grains was in proportion to the amount of chromosomal DNA and the grain densities were approximately equal all over the segments of chromosomes.

Banding and chromatid separation in Chinese hamster chromosomes.

IN general, a chromosome exhibiting its banding pattern does not permit any internal helix to be seen, and vice versa. During studies with chromosomes of Chinese hamster cells, cultivated in vitro, it was possible to correlate G banding and spiral structure with the spatial relation between sister chromatids. G banding of human chromosomes was first described by Sumner et al.1 and later was demonstrated in the Chinese hamster chromosomes by Radabli and Kriukova2. By a hypotonic treatment, the spiralisation of the chromosomes was demonstrated by Ohnuki3 in man and later by Kato and Yoshida4 in the Chinese hamster.

The effect of X-rays on labelling pattern of M 1 and M 2 chromosomes in Chinese hamster cells

Chinese hamster cells (line CHEF-125) were cultured for 1 or 4 h in the presence of tritiated thymidine ( 3HTdR). Immediately after the end of the treatment with 3HTdR or 4 h afterwards, some cultures were irradiated with X-rays, while others served as controls. Analysis of colchicine-C anaphase of M 1 and M 2 cells showed that: ( a) in the M 1 chromosomes the X-rays produced a significant departure from a 1:1 ratio of the number of silver grains counted on the sister chromatids; and ( b) in the M 2 chromosomes the X-rays increased significantly the frequency of sister-chromatid exchanges and of isolabelling regions. Subchromatid exchanges involving a single polynucleotide strand may be induced by X-rays. These exchanges would cause inequality of labelling over M 1 sister chromatids and isolabelling in the M 2 chromosomes.

Autoradiographic study on the synthesis of nuclear proteins. VI. The data on the turnover rate of nuclear proteins and formation of the protein component in chromosomes of Chinese hamster cells cultured in vitro.

Experiments were carried out on cultures of Chinese hamster fibroblasts. Cells were short-labelled with H3-Iysine or H3-tryptophane. The kinetics of silver grain number over interphase nuclei, metaphases, and individual chromosomes of the first pair were studied. — The grain count distribution over chromosomes of different groups and over sister-chromatids was analyzed. The data presented allow us to conclude that: 1. There is equal distribution of nuclear and chromosomal proteins between daughter nuclei and sister-chromatids, resp., during each mitotic cycle. 2. The synthesis of chromosome proteins, in general, and nonhistone proteins, in particular, takes place at all stages of the cycle. 3. At each stage of the cycle, proteins are incorporated into chromosomes of different groups simultaneously and at equal rate. The rate of this process in S and G2 is two times that in G1.

Reutilization of Presumably Degraded Radioactive Label from a Chromosomal Fraction of Homologous and Heterologous Cells

Summary(1) A method for producing a suspension of pure mitotic chromosomes of Chinese hamster cells with insignificant contamination by interphase nuclei or their fragments is described. The yield obtained is high and the chromosome morphology is well preserved. (2) H3 thymidine label from such chromosomes became specifically incorporated into the chromosomes of fresh Chinese hamster cells and of human leucocyte cultures. No gross alteration in the chromosomes of the receptor cells occurred, and uptake of label was inhibited by the presence of added carrier thymidine in the medium, in an amount insufficient to inhibit DNA synthesis. Therefore, most of the incorporation appears to have involved previous degradation of the DNA at least to the level of free nucleotide.The authors gratefully acknowledge the helpful suggestions of Dr. Theodore T. Puck, Dr. Sherman M. Weissman, and Dr. Gordon Allen, as well as the technical assistance of Mrs. Gloria E. Johnson.

The fine structure of the kinetochore of a mammalian cell in vitro.

The chromosomes of Chinese hamster cells were examined with the electron microscope and the following observations were made concerning the structure and organization of the kinetochore. — The kinetochore consists of a dense core 200–300 A in diameter surrounded hy a less dense zone 200–600 A wide. The dense core consists of a pair of axial fibrils 50–80 A in diameter which may be coiled together in a cohelical manner. The less dense zone about the axial elements is composed of numerous microfibrils which loop out at right angles to the axial fibrils. Together the structures comprise a lampbrush-like filament which extends along the surface of each chromatid. Some sections suggested that two such filaments may be present on each chromatid. The fine structure of kinetochores associated with spindle filaments was essentially the same as those free of filaments. The structure and organization of the kinetochore of these mammalian cells was compared to that of lampbrush chromosomes of certain amphibian oocytes, dipteran polytene chromosome puffs, and the synaptinemal complex seen during meiotic prophase.

Further Observations on the Chromosomes of Chinese Hamster Cells in Tissue Culture<xref ref-type="fn" rid="FN1">2</xref>

The chromosome complements of 3 Chinese hamster cell strains and a methylcholanthrene-treated subline varied during observation periods of 12 to 22 months. The complements were formed basically from multiples of the haploid constitution, and in 1 strain the normal diploid complement persisted as the predominant pattern for many months. Abnormalities of varying types began to appear soon after a culture was initiated, and in some sublines particular abnormal chromosome patterns persisted for many months, while in others variation was continuous. Except in the rare, markedly polyploid and partially disintegrated metaphases, the normal morphology of the individual chromosomes was usually maintained. Thus the metaphase chromosomes could nearly always be identified, though exact similarity to the normal types was not found when deletions resulted in partial loss of chromosome arms. Telocentric chromosomes, not present in the normal diploid complement, were a feature of most strains and in some cases could be related to centromeric misdivision with persistence of 1 or 2 arms as functioning chromosomes. Other aberrant chromosome forms could not be identified with normal chromosome types. The changes in chromosome complements in these nonclonal cultures were fluctuating events unassociated with obvious changes in cultural or morphological appearances. A characteristic chromosome pattern with an unusual chromosome form appeared in a methylcholanthrene-treated subline of 1 cell strain, 5 months after exposure to the carcinogen. Subsequently, however, this characteristic pattern disappeared, and the chromosome complement of the treated subline did not differ significantly from the untreated parent cell strain.