Identification of interspecific translocation chromosomes in human-Chinese hamster hybrid cells.

Chromosome material of human and Chinese hamster origin can be stained differentially in human-Chinese hamster translocation chromosomes by use of the Giemsa-11 staining technique.     

The nature of 3 cytogenetic phenomena--delayed spiralization of regions of the metaphase chromosomes, delayed disruption of the telomeric links of chromosomes and the premature division of X-chromosome centromeres

The phenomena mentioned above in the title cannot be explained by the routine scheme: gene-protein-chromatin. These are presumably associated with the existence of a system of mitotic chromosome transformation independent of the cytoplasm.     

Delayed disruption of the telomeric links between chromosomes in experimentally induced cells with micronuclei exposed to 5-bromodeoxyuridine in the 1st S period after colcemid administration

Under a long-term administration of colcemid in the Chinese hamster cell culture some cells with micronuclei are seen to form. In the case of co-treatment with colcemid and 5-bromodeoxyuridine (5-BrdU) at metaphases of the first division of cells with micronuclei polycentric chromosomes were observed. These polycentric chromosomes occur due to delayed disruption of telomeric links, previously existing in the interphase. During colcemid treatment the cells pass through two S-periods: one in mononuclear cells, the other in cells with micronuclei. This phenomenon was tested according to the frequency of metaphases with dicentrics after 5-BrdU-treatment of cells at the first or second S-period or during the two cycles of chromosome replication. The 5-BrdU treatment during the first cycle or two cycles of replication resulted in the same frequency of cells with dicentrics--about 50%. The treatment with colcemid alone during two cycles of replication and administration 5-BrdU at the second S-period results in a considerably lower amount (%) of cells with dicentrics--about 10%. Thus, the delayed disruption of telomeric links between chromosomes may occur under the treatment with 5-BrdU at the first S-period after colcemid administration. It is also concluded that this phenomenon can be reproduced in cell with micronuclei when 5-BrdU is incorporated differentially in the sister chromatids.     

Bleomycin induces delayed instability of interstitial telomeric sequences in Chinese hamster ovary cells

We analyzed the behavior of interstitial telomeric sequences (ITSs) in the progeny of Chinese Hamster Ovary (CHO) cells exposed to the radiomimetic compound bleomycin (BLM) in order to determine if ITSs play some role in the long-term clastogenic effect of this antibiotic. To this end, CHO cells were treated with a single concentration of BLM (2.5μg/ml), and the frequency of unstable chromosomal aberrations was determined at several times after treatment (18h, and 6, 15 and 34/36 days) by using PNA-FISH with a pan-telomeric probe [(TTAGGG)n repeats]. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 6 days after treatment in BLM-exposed cultures vs. untreated cultures, although the yield of BLM-induced aberrations decreased on average five times 6 days after treatment compared with the one induced 18h after treatment. Moreover, no significant differences in the frequency of aberrations were observed between untreated and BLM-exposed cells at 15 or 34/36 days after treatment. These data indicate that, in terms of unstable aberrations, the in vitro clastogenic effect of BLM on CHO cells persists for at least 6 days but less than 15 days after exposure. In addition, we found that BLM induces ITSs instability, cytogenetically detectable as acentric fragments (18h after treatment) or additional (new) FISH signals (6 days after treatment). We propose that the delayed effect of BLM on ITSs mainly results from breakage of heterochromatic ITSs blocks and further insertion of these sequences at the sites of monochromatid breaks occurring at G2 phase of the cell cycle, since most of the additional FISH signals were present as single dots and located at interstitial sites of the involved chromosomes.     

Streptonigrin induces delayed chromosomal instability involving interstitial telomeric sequences in Chinese hamster ovary cells

We analyzed the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells exposed to the radiomimetic compound streptonigrin (SN), in order to determine whether interstitial telomeric sequences (ITSs) are involved in the long-term clastogenic effect of this antibiotic. CHO cells were treated with a single concentration of SN (100ng/ml), and the frequency of unstable chromosomal aberrations was determined at three times after treatment (18h, and 6 and 15 days) by using PNA-FISH with a pan-telomeric probe. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 6 days after treatment in SN-exposed cultures vs. untreated cultures. The percentage of damaged cells and the yield of SN-induced aberrations at 6 days after treatment increased on average twofold compared with the ones at 18h after treatment. Moreover, a significant decrease in the frequency of aberrations was observed in SN-exposed cells at 15 days after treatment, resulting in a frequency of aberrations significantly lower than the frequency of aberrations observed in the corresponding control cultures. These data indicate that SN induces delayed chromosomal instability in CHO cells, and that the in vitro clastogenic effect of this compound persists for at least 6 days but less than 15 days after treatment. In addition, we found that SN induces delayed ITSs instability, cytogenetically detectable as additional FISH signals and centromeric breaks involving dissociation of the telomeric signal 6 days after treatment. We propose that the delayed effect of SN on ITSs results from breakage of heterochromatic centromeric ITSs blocks and further insertion of these sequences at the sites of mono- or isochromatid breaks occurring at G2 or G1-S phases of the cell cycle, respectively, since most of the additional FISH signals were present as single or double dots, and located at interstitial sites of the involved chromosomes.     

Localization of genes on human chromosomes by studies of human-Chinese hamster somatic cell hybrids

Summary About 75 man-Chinese hamster hybrid clones were analysed for their human chromosome complement and simultaneously tested for human enzyme markers. Correlation of the presence of chromosomes and enzyme activity revealed assignments of the PGD linkage group to chromosome 1, ME₁, PGM₃ and IPO-B to 6, LDH-A to 11, LDH-B to 12 and IPO-A to 21.The assignment of PGM₃ puts the HL-A loci on chromosome 6. Segregation of the enzymes of the PGD linkage group was demonstrated in a clone which had retained a deleted chromosome 1. Subclones of this line indicate that the loci for PGD and PGM₁ are situated on the short arm or proximal part of the long arm of 1 and the locus for Pep-C on the long arm.

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Zusammenfassung Etwa 75 Hybrid-Zellklone Mensch/Chinesischer Hamster wurden in bezug auf den menschlichen Anteil ihres Chromosomensatzes analysiert und gleichzeitig auf menschliche Enzym-Marker untersucht. Die Korrelation zwischen Anwesenheit von Chromosomen und Enzym-Markern ließ die Folgerung zu, daß die PGD-Koppelungsgruppe auf Chromosom 1, ME₁, PGM₃ und IPO-B auf Nr. 6, LDH-A auf 11, LDH-B auf 12 und IPO-A auf Chromosom 21 gelegen ist.Die Lokalisation von PGM₃ läßt die Folgerung zu, daß auch die HL-A-loci auf Chromosom 6 lokalisiert sind. Aufspaltung der Enzyme der PGD-Koppelungsgruppe konnte an einem Klon dargestellt werden, der ein deletiertes Chromosom 1 enthielt. Die Subklone dieser Linie zeigen, daß die loci für PGD und PGM₁ auf dem kurzen Arm oder dem proximalen Teil des langen Arms von Chromosom Nr. 1 liegen, während der locus für Pep-C auf dem langen Arm gelegen ist.

     

High-resolution mapping of interstitial telomeric repeats in Syrian hamster metaphase chromosomes

Karyotype analysis of the Syrian hamster (Mesocricetus auratus) was performed after DAPI-banding of metaphase chromosomes obtained from cultivated skin fibroblasts of a newborn animal. Fluorescence in situ hybridization with telomeric FITC-conjugated peptide nucleic acid probe was applied to map interstitial blocks of (TTAGGG)(n) repeats. Strong fluorescence in situ hybridization signals corresponded to interstitial telomeric repeats in pericentromeric chromatin bands of chromosomes 2, 4, 14, 20, and X. High-resolution DAPI-banding allowed specifying the arrangement of bands in the pericentromeric regions of these chromosomes.     

Chinese hamster X mouse hybrid cells segregating mouse chromosomes and isozymes.

Hybrid cells are readily formed by fusing clonal Chinese hamster cells to fresh, noncultured, adult mouse spleen cells followed by isolation in selective medium. The vast majority of such hybrids retain Chinese hamster chromosomes and isozymes while segregating mouse chromosomes and isozymes. The growth, plating efficiency, ease of karyology, and rapid segregation of mouse markers allows linkage tests in primary clones. Analysis of 13 isozymes showed 12 to be asyntenic and on epair (PGD-PGM2) to be syntenic This system will allow extensive somatic cell hybrid gene mapping in the mouse and permit a comparison of human and mouse linkage relationships.     

Compositional properties of telomeric regions from human chromosomes.

We have investigated the GC levels of third codon position of genes localized in G- (Giemsa), R-(reverse) and T-(telomeric) bands of human metaphase chromosomes, as well as the hybridization of telomeric probes on fractionated human DNA. The first set of results shows much higher GC levels for genes localized in T-bands than in G- or R-bands (the latter being higher than the former). The second set of data shows that telomeric probes corresponding to T-bands hybridize on the GC-richest family (H3) of isochores, whereas telomeric probes corresponding to R-bands hybridize on GC-rich families H1 and H2; in agreement with these findings, the telomeric repeat common to all chromosomes hybridized on isochore families H1, H2 and H3.     

Predominant loss of hamster chromosomes in hybrids of somatic cells from the Dzungarian hamster and mice]

By means of the application of UV-inactivated Sendai virus interspecific hybrids of Dzungarian hamsterXmouse somatic cells were obtained in HAT selective medium. Karyotypic changes in these hybrid somatic cells were recorded during a 13 months' period. In the beginning each hybrid somatic cell contained 1 chromosome set of Dzungarian hamster and 1 mouse chromosome set. It was observed that throughout 13 months' of cultivation the elimination of Dzungarian hamster chromosomes prevailed over that of mouse chromosomes.     

Quantitative initiation of microtubule assembly by chromosomes from Chinese hamster ovary cells

The microtubule nucleating capacity of chromosomes was tested in vitro in lysates of Chinese hamster ovary cells. Colcemid-blocked mitotic cells were lysed with the detergent Triton X-100, incubated with exogenous porcine brain tubulin, attached to electron microscope grids and observed as whole-mounts. Under suitable conditions, greater than 98% of the chromosomes gave rise to microtubules at their kinetochore regions, thus unequivocally demonstrating that chromosomes are competent to initiate specifically microtubule formation. The average number of microtubules that polymerized onto a chromosome was 8 +/- 5, and greater than 36% of the chromosomes had between 10 and 19 microtubules per kinetochore region. We conclude that under the lysis conditions employed, virtually all the chromosomes retain their kinetochores, and that the kinetochores retain a substantial fraction of their microtubule nucleating capacity.     

Metabolic co-operation between biochemically variant hamster cells and heterokaryons formed between these cells and chick erythrocytes.

Observations on heterokaryons formed between multiple biochemically variant polyoma virus transformed hamster cells and chick erythrocytes indicate that gain of active preformed adenosine phosphoribosyl transferase and hypoxanthine/guanine phosphoribosyl transferase immediately endows the heterokaryons with the potential to act as donors for metabolic cooperation in these systems.     

Arginine-rich histones do not exchange between human and mouse chromosomes in hybrid cells

Following division of HeLa-3T3 heterokaryons, human and mouse chromosomes occupy distinct regions within the resulting hybrid nuclei. This favorable orientation of genomes has allowed us to determine whether histones exchange between chromosomes in vivo. Acrylamide gel electrophoresis of the proteins from HeLa cells labeled with ³H-arginine during S phase showed that the core histones were labeled preferentially, constituting 30% of the total cellular tritium and 50% of the label in a crude nuclear fraction. Autoradiographic analysis of cells formed by fusion of ³H-arginine-labeled HeLa cells and 3T3-4E cells showed that ³H-arginine-labeled proteins did not migrate between nuclei in heterokaryons; hybrid cells formed from such heterokaryons contained nuclei in which ³H proteins occupied a sector within the nucleus; “sectored nuclei” could persist for at least 4 days; and the unequal distribution of ³H proteins did not change during DNA synthesis. Electron microscopic examination of hybrid nuclei failed to reveal a physical partition between human and mouse chromosome sets. Sectored nuclei were also observed in synkaryons derived from ³H-arginine-labeled HeLa and unlabeled HeLa cells, indicating that the unequal distribution of ³H-arginine-labeled proteins in HeLa-3T3 hybrid cells did not result from species-specific binding of proteins and DNA. The persistent unequal distribution of ³H-arginine-labeled proteins within hybrid nuclei in the apparent absence of a barrier between mouse and human chromosomes indicates that histones, the principal ³H-arginine-labeled proteins, do not dissociate from DNA in vivo.     

A flow cytometric study of chromosomes from rat kangaroo and chinese hamster cells

Summary Chromosomes from rat kangaroo (PTK) and chinese hamster (CHV 79) cells have been prepared for quantitative flow-cytometric analysis. The preparation time was optimized down to 30 (PTK) and 40 min (CHV 79). DAPI was used as a AT-sensitive fluorescent dye to stain for monoparameter DNA measurements. Simultaneous two-parameter DNA-protein analysis was carried out with DAPI and SR 101 (as a general protein fluorochrome) in combination. The karyotype of the PTK cells with 13 (14) chromosomes was separated into 10 DNA peaks. The X-chromosome bearing the nucleolus organizer region generates a distinct peak. The karyotype of the CHV 79 cells with 22 chromosomes was separated into 15 peaks. The DNA profile obtained indicates a geometric grading of the chromosomal amount of AT components in the karyotype of this particular cell line. The simultaneous DNA-protein analysis performed show enough sensitivity of the instrument utilizing high power UV excitation illumination to discriminate the two color emission consisting of blue (DAPI) and red (SR 101) fluorescence. Color overlapping could be completely avoided. Additionally, the quality (number, location, and resolution of peaks) of the DNA distribution was not influenced by the simultaneous application of a second fluorescent stain. Fluorescence activated electronic sorting applied on chromosomal fluorescence distributions providing purified fractions of chromosomes for subsequent biochemical and biological determinations is discussed.     

Transfer of the human genes coding for thymidine kinase and galactokinase to Chinese hamster cells and human-Chinese hamster cell hybrids.

Cotransfer of two linked human genes, coding for the enzymes thymidine kinase (TK) and galactokinase (Gak) was demonstrated following incubation of Chinese hamster TK-deficient cells with isolated human chromosomes. The 5 colonies which were isolated all expressed a stable TK-positive phenotype. Cotransfer of the human genes coding for TK and Gak has also been observed in experiments in which isolated human chromosomes were incubated with TK-deficient human-Chinese hamster cell hybrids. These receipient hybrids had lost all human chromosomes at the time of incubation. From these experiments, four colonies were isolated, all expressing an unstable TK-positive phenotype. Using chromosome staining techniques, the presence of human chromosomes could not be demonstrated in either of the transformed clonal lines obtained with the Chinese hamster and the hybrid recipient cells. This indicates that incorporation of only the fragment of the human chromosome 17, bearing the genes for TK and Gak, has occurred in the recipient cells.