Flow cytometric characterization of a Chinese hamster x man hybrid cell line retaining the human Y chromosome

Summary A Chinese hamster x man hybrid cell line (CH-Y-VII) was established which retains a free human Y chromosome. Exponentially growing CH-Y-VII cells were arrested with colcemid; metaphase chromosomes were isolated and stained with 33258 Hoechst (HO) plus Chromomycin A3 (CA3), or with ethidium bromide (EB). The HO/CA3-stained chromosomes were measured in a dual beam flow cytometer, and bivariate HO/CA3 flow karyotypes and univariate HO and CA3 flow karyotypes were established. EB-stained chromosomes were analyzed in a modified Becton Dickinson FACS-Sorter. For all three stains used, the human Y chromosome forms a separate peak in univariate flow karyotypes; the optimum resolution was obtained for the HO distribution. In the bivariate HO/CA3 flow karyotype, the peak for the human Y chromosome is completely separated from the Chinese hamster chromosomes.Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract number W-7405-ENG-48 with support from the Deutsche Forschungsgemeinschaft (Cr 60/3-1 and Wo 148/18)     

Replication pattern of a large homogenously staining chromosome region in antifolate-resistant Chinese hamster cell lines

We have investigated the replication pattern of a large, homogenously staining chromosome region (HSR) in two antifolate-resistant Chinese hamster cell lines. This region is believed to be the location of an amplified genetic sequence which includes at least the gene coding for dihydrofolate reductase and which may be present in as many as 200 copies. It is shown that the HSR in both cell lines is among the first chromosome regions to begin DNA synthesis after reversal of an early G1 block. In cells synchronized in the S period with hydroxyurea, it is also clear that the HSR in both cell lines begins replication at many sites within its length in early S. The replicons comprising the HSR therefore may respond to a common initiation signal in early S. In one cell line (A3), replication of the HSR requires, at most, 3 hours of a 7-hour S period; in a second line (MQ19), replication proceeds for approximately 5 hours. In neither line does replication of the HSR occur concomitantly with synthesis of characteristic late replicating regions. These results were confirmed in exponential cultures using a retroactive labeling technique. The significance of these findings is discussed with reference to the possible origin and arrangement of the amplified sequence in these two cell lines.     

Structure and dynamics of human interphase chromosome territories in vivo

A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5–10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture. Received: 10 November 1997 / Accepted: 24 November 1997     

Development of arm-specific and subtelomeric region-specific painting probes for Chinese hamster chromosomes and their utility in chromosome identification of Chinese hamster cell lines

Arm-specific and subtelomeric region-specific painting probes for Chinese hamster chromosomes have been generated by microdissection and use of the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). Fluorescence in situ hybridization (FISH) analyses using these probes demonstrated their specificity. These probes painted every chromosome arm and a total of 15 subtelomeric regions, namely, both ends of chromosomes 1, 2, 3, 4, and 8 and one end of chromosome arms 5q, 6q, 7q, 9p, and Xp. Many cryptic chromosomal rearrangements in the CHO-9 and V79 cell lines that were not detectable with whole chromosome paints could be recognized when these newly developed probes were used.     

An improved fixation method for chromosome preparation of Chinese hamster, Chinese hamster-human hybrid, and mouse cell lines

Lowering the proportion of acetic acid in the standard 1:3 acetic acid:methanol chromosome fixative used both during initial fixation and subsequent washing produced up to a 20-fold increase in the yield of intact metaphases from cultures of several permanent cell lines. Although this inhibited chromosome spreading, addition of various acetic acid-methanol mixtures immediately after the cell suspension was dropped onto slides increased the degree of spreading and resulted in well-spread, cytoplasm-free metaphases.     

Analysis of replication patterns in chromosomes of normal Chinese hamster and its cell lines

Replication patterns of the normal male Chinese hamster chromosomes and the three cell lines CHW, 1102 and 1103, were determined using fluorescent, plus Giemsa or acridine orange, techniques. The individual chromosomes or chromosomal segments were consistent in the replication patterns of normal Chinese hamster chromosomes and all the transformed cell lines. Late DNA replication was regularly identified in the long arm of the X chromosome, the entire Y chromosome, the short arms of chromosomes 6 and 7, and the paracentromeric regions of chromosomes 8, 9 and 10. A similar consistency was demonstrated in the large late replicating areas of chromosomes X and Y. Each cell line had specific marker chromosomes by which the cell line was identified and their replication patterns have been described. The chromosome analysis in cell line 1103 indicated that chromosomes 2, 3, 8 and 9 were more stable than others, of which chromosome 2 was extremely stable. The markers M4 and M5 in cell line 1103 are very interesting. The cytogenetic behaviour of marker M4 indicated a new phenomenon of translocation by simple association. The marker chromosome M5 indicated that inactivation spread to the early replicating distal region. These cell lines are very useful tools for studying replication patterns and providing a basic understanding of mammalian cytogenetics.     

Karyologic study of continuous cell lines. IV. Study of human cell lines using the C-method of staining chromosomes

With the aid of the C-method of chromosome staining marker chromosomes three classes of human continuous cell lines were studied: 1) HeLa and HeLa-like cell lines (HEp-2, U, KB); 2) non-HeLa cell lines, with type B mobility of glucose-6-phosphate-dehydrogenase (HOS, A-549, A-204); 3) lymphoblastoid cell lines (Raji, Namalva, L-101). Two C-marker chromosomes were observed in two investigated cell lines A-204 and KB, one C-marker chromosome was observed in HEp-2, HeLa, U, A-549, Namalva cell lines; C-markers were absent in HOS and L-101 cell lines. Y-chromosome was found in Raji, A-549 and L-101 cell lines. The C-method of chromosome staining is a simple method, promoting an intraspecific identification of human cell lines.     

Specific chromosome aberrations in senescent fibroblast cell lines derived from human embryos.

In senescent fibroblast cell lines derived from human embryos, the number of chromosome aberrations were found to increase rapidly. In addition to an increase in aneuploidy and polyploidy, a high frequency of dicentrics occurred, but the number of other chromosome abnormalities remained approximately constant. Banding revealed that many of the dicentrics appeared to be end-to-end fusions of whole chromosomes. The involvement of chromosomes was nonrandom. This "telomeric binding" may reflect a progressive decrease in the stability of telomeric sequences or associated enzymes which may also occur in vivo.     

Studies of mammalian chromosome replication. I. BrdU-induced differential staining patterns in interphase and metaphase chromosomes

The patterns of differential staining based on the effects of BrdU-substitution in chromosomal DNA have been examined in both metaphase chromosomes and prematurely condensed chromosomes (PCC) of interphase Chinese hamster cells. Results indicate that differential staining may be obtained in chromosomes from all stages of the cell cycle and correspond to the semi-conservation mode of DNA replication. Such fidelity of differential staining in both interphase and metaphase chromosomes suggests that components essential for induction of differential staining are present throughout the cell cycle and chromosomes may contain similar structures and organization throughout the cycle.     

Three-dimensional reconstruction of painted human interphase chromosomes: active and inactive X chromosome territories have similar volumes but differ in shape and surface structure

This study provides a three-dimensional (3D) analysis of differences between the 3D morphology of active and inactive human X interphase chromosomes (Xa and Xi territories). Chromosome territories were painted in formaldehyde-fixed, three-dimensionally intact human diploid female amniotic fluid cell nuclei (46, XX) with X-specific whole chromosome compositive probes. The colocalization of a 4,6-diamidino-2- phenylindole dihydrochloride-stained Barr body with one of the two painted X territories allowed the unequivocal discrimination of the inactive X from its active counterpart. Light optical serial sections were obtained with a confocal laser scanning microscope. 3D- reconstructed Xa territories revealed a flatter shape and exhibited a larger and more irregular surface when compared to the apparently smoother surface and rounder shape of Xi territories. The relationship between territory surface and volume was quantified by the determination of a dimensionless roundness factor (RF). RF and surface area measurements showed a highly significant difference between Xa and Xi territories (P < 0.001) in contrast to volume differences (P > 0.1). For comparison with an autosome of similar DNA content, chromosome 7 territories were additionally painted. The 3D morphology of the chromosome 7 territories was similar to the Xa territory but differed strongly from the Xi territory with respect to RF and surface area (P < 0.001).     

Specific staining of the human No. 1 chromosome in spermatozoa

Summary A new differential staining technique specific for the secondary constriction region of the human No. 1 chromosome makes it possible to study the transmission of this chromosome during mitosis and meiosis and to determine the meiotic non-disjunction frequency for chromosome No. 1 in ejaculated spermatozoa.

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Zusammenfassung Eine neue differentiale F?rbetechnik, die für die Sekund?rkonstriktion auf dem menschlichen Chromosom Nr. 1 spezifisch ist, erm?glicht es, die übertragung dieses Chromosoms w?hrend der Mitose und Meiose zu verfolgen und die meiotische Nondisjunktion-H?ufigkeit des Chromosomes Nr. 1 in ejaculierten Spermatozoen zu bestimmen.

     

Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines.     

Analysis of chromosome positions in the interphase nucleus of Chinese hamster cells by laser-UV-microirradiation experiments

Summary Unsynchronized cells of an essentially diploid strain of female Chinese hamster cells derived from lung tissue (CHL) were laser-UV-microirradiated (=257 nm) in the nucleus either at its central part or at its periphery. After 7–9 h postincubation with 0.5 mM caffeine, chromosome preparations were made in situ. Twenty-one and 29 metaphase spreads, respectively, with partial chromosome shattering (PCS) obtained after micro-irradiation at these two nuclear sites, were Q-banded and analyzed in detail. A positive correlation was observed between the frequency of damage of chromosomes and both their DNA content and length at metaphase. No significant difference was observed between the frequencies of damage obtained for individual chromosomes at either site of microirradiation. The frequency of joint damage of homologous chromosomes was low as compared to nonhomologous ones. Considerable variation was noted in different cells in the combinations of jointly shattered chromosomes. Evidence which justifies an interpretation of these data in terms of an interphase arrangement of chromosome territories is discussed. Our data strongly argue against somatic pairing as a regular event, and suggest a considerable variability of chromosome positions in different nuclei. However, present data do not exclude the possibility of certain non-random chromosomal arrangements in CHL-nuclei. The interphase chromosome distribution revealed by these experiments is compared with centromere-centromere, centromere-center and angle analyses of metaphase spreads and the relationship between interphase and metaphase arrangements of chromosomes is discussed.