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 intactchromosomes in micronuclei. Such evidence was based on the re-expressionof metaphase chromosomes by micronuclei fused with whole cells.Three different experimental approaches were used; they differedonly in the criteria used to discriminate between the chromosomesof micronuclear origin and those present in the cell to whichmicronuclei were fused. These methods involved the expressionof: (a) human chromosomes in mouse cells, (b) radioactivelylabelled human chromosomes in unlabelled human lymphocytes and(c) Chinese hamster chromosomes in Chinese hamster cells withdifferentially stained chromosomes. With all methods, evidencefor the presence of one or a few chromosomes in micronucleiwas obtained.     

Chinese hamster × 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 one pair (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.     

Specifically altered metaphase chromosomes in antifolate-resistant Chinese hamster cells that overproduce dihydrofolate reductase

Specific karyotype alterations are described which occur in 16 different antifolate-resistant Chinese hamster sublines with increases in target enzyme dihydrofolate reductase activity ranging from 2.9- to 281-fold. Sublines were selected with either methotrexate or methasquin and exhibit a complex set of relationship between increase in resistance and increase in target enzyme activity. Drug resistant sublines generally contain one or the other of two types of abnormally banding chromosome regions as revealed by the Giemsa-banding method. In those cell lines with less than a 50-fold increase in dihydrofolate reductase activity there are long segments with distinctly abnormal band patterns. In sublines with greater than 85-fold increases in enzyme activity, cells contain a long, homogeneously staining region (HSR). HSRs comprise 2 to 5% of the total metaphase chromosome complement. The specific, abnormally banding regions are a consistent feature of the antifolate-resistant sublines. There is usually 1 per cell, preferentially located on the long arm of a chromosome #2. Results of experiments with cloned, HSR-containing cells maintained for about 2 years in antifolate-free growth medium indicate a quantitative relationship between length of the HSR and levels of drug resistance and dihydrofolate reductase activity; there was a parallel decline in these three parameters with time in culture.     

Expression of human hypoxanthine phosphoribosyl transferase in Chinese hamster cells treated with isolated human chromosomes

Chinese hamster cells deficient for the enzyme hypoxanthine phosphoribosyl transferase (HPRT) were incubated with isolated human metaphase chromosomes and 21 colonies were isolated in HAT medium. Three different types of cell lines were established from these clones. First, 4 cell lines had 10–30% of normal Chinese hamster HPRT activity with the same electrophoretic mobility as human HPRT. This HPRT activity remains detectable during at least 8 weeks of growth of the cells in nonselective medium. Second, 3 cell lines also had human-like HPRT with the same activity as the first type. This HPRT persists only if the cells are grown in HAT medium and disappears during 8 weeks of growth in nonselective medium. Third, other clones survived in HAT medium as well as in medium with 8-azaguanine. These cells had no detectable HPRT activity. Using differential chromosome staining techniques no recognizable human chromosome fragments were found in any of the cell lines.     

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 $\text{140 ± 25}\text{A}\text{?}$ in height, $\text{100 ± 20}\text{A}\text{?}$ in width, and $\text{54 ± 4}\text{A}\text{?}$ in thickness. The center-to-center distance of two units of a bipartite disk is about $\text{34 ± 3}\text{A}\text{?}$. The core of a disk appears to contain four substructures that measure about $\text{25 ± 5}\text{A}\text{?}$ in diameter. Through the center of the disk core there is an axial structure measuring about $\text{20 ± 5}\text{A}\text{?}$ in diameter, which spans and interconnects similar bipartite disks to form a continuous linear array. A structure measuring $\text{20 ± 4}\text{A}\text{?}$ 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.     

The stability of chromosomes in yeast

Summary CL mutants with high instability of chromosome III were UV-induced in haploid strain disomic for chromosome III. The obtained CL mutants can be divided into two groups: (1) CL2, CL3, CL7, CL11–L13 with elevated level of spontaneous inter- and intragenic recombination and (2) CL4, CL8 in which instability of chromosome III is not accompanied by elevation of mitotic recombination frequency. CL4 and CL8 mutants also show unstable maintenance of artificial minichromosomes with different chromosomal replicators and centromeric loci. The instability of chromosome III and minichromosomes in CL4 and CL8 is determined by two nonallelic genes designated ch14 and ch18. The role of ch14 and ch18 genes in mitotic chromosome transmission is discussed.     

Delayed mutation in Chinese hamster cells

The possibility was examined that mutational events can be delayed for more than one or two cell divisions following treatment of Chinese hamster cells with the DNA alkylating agent ethyl methane sulfonate. If mutations in mammalian cells are delayed, the proportion of mutant cells in colonies grown from single mutagen-treated cells will reflect the cell division at which the mutation is genetically fixed, i.e., a first division mutation yields a 1/2 mutant colony, a fifth division mutation produces a 1/32 mutant colony, etc. In the present study, replating of cells from single colonies grown for six to seven days after mutagen treatment resulted in the discrete ratios of glucose-6-phosphate dehydrogenase (G6PD)-deficient mutant to wild-type colonies expected for a delayed mutational process which produces mutations over at least 8–10 cell generations. Further, when cells from 7- to 10-day colonies, grown from ethyl methane sulfonate (EMS)-treated cells were replated into selective medium containing 6-thioguanine (6TG), the number of 6TG-resistant colonies obtained per flask was distributed over a very wide range, consistent with a mutational delay process. These results could not be explained by differences in the number of cells per colony or plating efficiency in selective medium. Assuming that the relative number of 6TG-resistant colonies per flask reflects the time of mutation, EMS treatment produced two groups of mutational events: one which occurred within the first five cell generations and another uniformly distributed over at least the next eight to nine divisions. These results support the conclusion that EMS induces mutants for at least 10–14 cell generations after treatment and raise the possibility that current methods to assess the mutagenic potential of an agent might lead to significant underestimation. The role of delayed mutation in the phenomenon of “mutation expression time” is also discussed.     

Branched-chain aminotransferase deficiency in Chinese hamster cells complemented by two independent genes on human chromosomes 12 and 19

Branched-chain aminotransferase (BCT) catalyzes the reversible transamination of the branched-chain -keto acids to the branched-chainl-amino acids. Since branched-chainl-amino acids (l-isoleucine,l-leucine, andl-valine) are essential for cell growth, cells which lack BCT were unable to proliferate in media containing -keto acids in place of the correspondingl-amino acids. CHW-1102, a Chinese hamster cell line, lacks BCT and does not grow in -keto acid media. Somatic cell hybrids were made by the fusion of CHW-1102 (HPRT^–) with several human cell lines and isolated on HAT medium. Growth assays of hybrid clones on -keto acid selection media independent of the HAT selection medium indicated two cell hybrid phenotypes: either (1) the hybrid clone, like the parental CHW-1102, could not utilize -keto acid media, or (2) the hybrid could proliferate on all three -keto acid media. The ability of hybrid cells to proliferate on -keto acid media correlated with the presence of either of two human genes which independently complemented the Chinese hamster deficiency. Two human genes, BCT1 assigned to chromosome 12 and BCT2 assigned to chromosome 19, were demonstrated to code for the expression of two molecular forms of BCT.A preliminary abstract of this work has been presented at the Human Gene Mapping Workshop V [Naylor, S.L. and Shows, T.B. (1979).Cytogenet. Cell Genet. 25:191–192].     

R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells

The mechanism responsible for immunoglobulin class switch recombination is unknown. Previous work has shown that class switch sequences have the unusual property of forming RNA-DNA hybrids when transcribed in vitro. Here we show that the RNA-DNA hybrid structure that forms in vitro is an R-loop with a displaced guanine (G)-rich strand that is single-stranded. This R-loop structure exists in vivo in B cells that have been stimulated to transcribe the gamma3 or the gamma2b switch region. The length of the R-loops can exceed 1 kilobase. We propose that this distinctive DNA structure is important in the class switch recombination mechanism     

Gene for M1 subunit of ribonucleotide reductase is amplified in hydroxyurea-resistant hamster cells

The hydroxyurea-resistant Chinese hamster cell line 600H has been shown to have greatly elevated quantities of ribonucleotide reductase. This increase in enzyme activity is due to an increased level of both the M1 and M2 subunit activities. The M1 subunit has been purified from the 600H cell line and shown to consist of a series of six protein spots with apparent molecular weights of 88,000 daltons, but with varying isoelectric points in the range of pH 6.5–7.0. Western blot analyses with antisera against the M1 and M2 proteins indicated that both subunit proteins are present in elevated quantities in the 600H cell line when compared to the wild-type V79 cell line. Southern blot analyses with genomic DNA from the series of stepwise-selected hydroxyurea-resistant cell lines leading to 600H showed that, in latter steps of selection, genomic sequences homologous to a mouse M1 cDNA have undergone a fivefold amplication. This was accompanied by a four-to eightfold increase in the single M1 homologous mRNA.     

The selection of Chinese hamster cells deficient in oxidative energy metabolism

A selection scheme based on the nutritional requirements of a previously described respiration-deficient Chinese hamster line has been used to isolate new mutants defective in oxidative energy metabolism. Three of the primary characteristics of this type of mutant are (1) a strict dependency on the continued presence of glucose for survival; (2) a drastic reduction in the rate of oxygen consumption; (3) an inhibition of Krebs cycle activity resulting in auxotrophy for asparagine and carbon dioxide. In the case of one cell line which was used (V79), up to 65% of the survivors of a selection were found to possess this phenotype after only one round of selection. By contrast, it proved much more difficult to obtain such mutants from another cell line (CCL16). A preliminary characterization of a number of these mutants is presented.     

Acetaldehyde-induced aneuploidy in cultured Chinese hamster cells

In this paper we report the cytogenetic activity of ethanol(EA) and its main metabolic derivative, acetaldehyde (AA) incultured Chinese hamster cells. AA induced an increase of aneuploidyand chromosome breaks and exchanges. EA only induced an increaseof achromatic lesions (gaps). However, following treatment withAA, the most notable effect was an increase of hypodiploid cellsand a parallel increase of multinucleated interphase cells,the frequency of hyperdiploid cells being considerably lower.A strong correlation (r = 0.93, P < 0.01) has been foundon comparing the frequency of hypodiploid cells to the frequencyof hyperdiploid plus multinucleated interphase cells. We concludethat there exists a higher chance of hypodiploid cells reachingthe second mitosis after treatment and that the main effectof AA is the induction of hypodiploidy rather than hyperdiploidyand chromosome aberrations. ¹To whom correspondence should be addressed     

The production and action of interferon in Chinese hamster cells.

The interferon system has been investigated in primary cell cultures established from Chinese hamster embryos and new born pups. Interferon synthesis was induced with Sindbis virus, ultraviolet inrradiated Newcastle disease virus (u.v.-NDV) and with polyriboinosine acid-polyribocytidylic acid complex [poly (rI). poly (rC)]. Only u.v.-NDV induced significant production of interferon, maximum amounts being produced in 'aged' cells. Its apparent mol. wt. was 25000. CHO-KI cells, an established line of Chinese hamster cells, did not synthesize interferon in response to viruses, but were sensitive to its action.     

Incorporation of isolated chromosomes and induction of hypoxanthine phosphoribosyltransferase in chinese hamster cells

Evidence is presented for the uptake of radioactive-labeled isolated Chinese hamster chromosomes following incubation with Chinese hamster cells. Metaphases were found which contained radioactive labeled chromosomes in a very low frequency, and in some of the labeled chromosomes only one chromatid was labeled. Incubation of hypoxanthine phosphoribosyltransferase (HPRT)-deficient Chinese hamster cells with chromosomes isolated from HPRT ⁺ Chinese hamster or human cells resulted in the appearance of HPRT⁺ cells. Clones derived from these cells were isolated in HA T medium. Cells in mitosis during incubation with the chromosomes yielded three times more HPRT⁺ clones than did cells in interphase. The intraspecies combination involving recipient cells and chromosomes from Chinese hamster origin yielded significantly higher numbers of HPRT⁺ clones than did the interspecies system using human chromosomes and Chinese hamster recipient cells (5×10^–5 and 6×10^–6 respectively). Electrophoresis of HPRT from Chinese hamster cells treated with human chromosomes revealed the pattern of the human enzyme.     

Osmotic stress variants in Chinese hamster cells

Stable variants resistant to hypertonic stress have been obtained from V79 cells by one-step selection in media supplemented with graded concentrations of NaCl. Such variants retain a potential for resistance when isolated and propagated in isotonic media. On replating in graded NaCl, a family of dose-response curves is obtained, rising in level of resistance according to the degree of hypertonicity used to isolate the variants initially. In hybrids between variants and sensitive cells, phenotypic expression of resistance to hypertonic NaCl is recessive. Stable variants can also be isolated by one-step selection in media made hypertonic with D-mannitol. Clonal sublines selected with mannitol, as well as those obtained with NaCl, are resistant to both types of hypertonic media. Fluctuation tests in media supplemented with NaCl show that resistance arises spontaneously and at random, with measured rates of variation that depend on the concentration of NaCl used for selection. Treatment of sensitive cells with 5-azacytidine increases the frequency of resistant variants in assays with high levels of added NaCl but is less effective when selection is performed at lower concentrations. Exposure to ethyl methane sulfonate has little or no effect on variant frequency.     

Valinomycin-induced apoptosis in Chinese hamster ovary cells

Accumulating evidence endorses that excessive K(+) efflux is an ionic mechanism underlying apoptosis both in neuronal and non-neuronal cells. K(+) channels play important roles in mediating the pro-apoptotic K(+) efflux. Chinese hamster ovary (CHO) cells have been widely used for gene transfection experiments. These cells lack detectable endogenous voltage-gated K(+) channels. We were interested in knowing whether the absence of endogenous K(+) channels would render wild-type CHO cells more resistant to apoptotic death. We also wished to determine if direct stimulation of K(+) efflux would trigger apoptosis in these cells. Exposing CHO cells to hypoxia (1% O(2)) or to a typical apoptotic insult of serum deprivation for up to 24h did not affect cell survival. On the other hand, the K(+) ionophore valinomycin caused substantial cell death within 12h of its application. Valinomycin-treated CHO cells underwent several apoptotic events, including phosphatidylserine (PS) membrane translocation, caspase-3 activation, and mitochondrial membrane depolarization during the first few hours of exposure. Reducing K(+) efflux by elevating extracellular K(+) concentrations noticeably attenuated valinomycin-induced cell death. This study reinforces a K(+) efflux-mediated apoptotic mechanism in CHO cells and may help to explain the unique feature of their higher tolerance to apoptosis.