Chinese Hamster Cell Mutants Research Articles

Characteristics of an HPRT-deficient chinese hamster cell line.

A mutant Chinese hamster cell line was selected from wild-type CHW cells in 30 <i>µ</i>g/ml 8-azaguanine, after exposure for 2 h to 10<sup>–3</sup> M methyl methanesulphonate. This line was subsequently cloned and one clone, isolated in 10 <i>µ</i>g/ml 8-azaguanine (CHW-1102), was selected for further study. CHW-1102 showed stability of resistance to 8-azaguanine after six months of growth in nonselective medium and an LD<sub>50</sub> to 8-azaguanine of between 10 and 20 <i>µ</i>g/ml, compared to 2.5 <i>µ</i>g/ml for the wild-type CHW cell line. The doubling time in culture was found to be 13 h, compared to a doubling time of 12 h for CHW. Biochemical studies showed that the mutant cell line CHW-1102 had a specific activity of HPRT of about 1–2 nmol/mg protein per hour compared to a specific activity of the wild type of 200 nmol/mg protein per hour. The spontaneous reversion rate from 8-azaguanine resistance to 8-azaguanine sensitivity was 4.23 × 10–8/locus per generation. Both the parental line (CHW) and the mutant (CHW-1102) have a modal chromosome number of 22 and a relatively stable karyotype. Both lines show some chromosome rearrangement and, in particular, the presence of one long acrocentric marker chromosome, which consists largely of material from chromosomes 6 and 8. Much of the distal part of the long arm of the X chromosome, which is heterochromatic, has been lost in both CHW and CHW-1102. So far as can be determined, no autosomal material, except perhaps the paracentromeric region of chromosome 6, is missing in CHW-1102, although even this material may be represented by a small marker chromosome M<sub>2</sub>. Both cell lines show characteristics that are of value for the study of mammalian cell genetics, and CHW-1102 represents a useful addition to the stock of mutant mammalian cell lines.

A temperature-sensitive function in a Chinese hamster line affecting DNA synthesis.

AbstractA temperature‐sensitive (ts) mutant of Chinese hamster cells (K12) is blocked at a step late in G1, required for the initiation of DNA synthesis. The ts lesion is recessive in interspecific hybrids.

Recent Developments with the L5178Y TK Heterozygote Mutagen Assay System

Recent Developments with the L5178Y TK Heterozygote Mutagen Assay System

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.

Genetics of somatic mammalian cells. XVII. Induction and isolation of Chinese hamster cell mutants requiring serine.

AbstractThe induction, isolation and some of the properties of serine‐requiring mutants of Chinese hamster ovary cells have been described. These cells promise to be useful in biochemical genetic studies and cancer research.

Inhibition of repair of UV-damaged DNA by caffeine and mutation induction in Chinese hamster cells

The effect of caffeine on UV-irradiated Chinese hamster cells in vitro was studied on the cellular and molecular levels. Caffeine (1 m M) was shown to decrease the colony-forming ability and the frequencies of spontaneous and UV-induced mutations in Chinese hamster cells. The effect of caffeine in reducing the frequency of UV-induced mutations was demonstrated only if caffeine was present in the culture medium during the first post-irradiation cell division. Using alkaline sucrose gradient centrifugation, both parental and newly synthesized DNA in UV-irradiated and unirradiated cells were studied in the presence and absence of caffeine. Caffeine affected the sedimentation profile of DNA synthesized in UV-irradiated cells but not in unirradiated cells. Caffeine had no apparent effect on the incorporation of [ 3H]-thymidine into DNA of control or UV-irradiated cells, nor on the small amount of excision of UV-induced pyrimidine dimers. These results may be interpreted by a hypothesis that caffeine inhibits a certain S-phase specific, post-replication, dark-repair mechanism. The hamster and perhaps other rodent cells exposed to low doses of UV are capable of DNA replication, by-passing the non-excised pyrimidine dimers. This postulated repair process probably involves de novo DNA synthesis to seal the gaps in the nascent strand. This repair may be also responsible for the enzymatic production of mutations.

Conditionally lethal mutations in Chinese hamster cells. I. Isolation of a temperature-sensitive line and its investigation by cell cycle studies.

AbstractThe isolation of a temperature sensitive cell line from the Chinese hamster line CCL39 of the American Type Culture Collection is described. At the nonpermissive temperature (39°C) the cells become attached to the surface of tissue culture dishes, but no microscopically observable colonies are formed upon prolonged incubation. Exposure to the high temperature for more than 24 hours leads to an almost complete loss in viability. A karyotypic analysis showed that this new line has lost one of the medium‐sized metacentric chromosomes, although no proof is available so far to show that this loss is not simply coincidental.In nonsynchronized cultures transferred to 39°C DNA synthesis stops first, RNA synthesis shortly thereafter, while protein synthesis (turnover) continues for a longer time. After such a shift the cell number increases by less than 15% as measured with the Coulter counter.Studies with synchronized cultures give the following results: (1) one round of DNA synthesis can occur at 39°C when the cells are released from serum starvation or a hydroxyurea block, or when mitotic cells are placed at 39°C; (2) the entry of cells into metaphase of mitosis at 39°C is almost normal when the preceding time interval at 39°C is only eight hours (release of cells from G1/S boundary), but considerably reduced when the cells spend an additional 12 to 15 hours at 39°C in G1 (release from serum starvation).Infection by SV40 virus temporarily induces DNA synthesis after it has come to a stop at the nonpermissive temperature, but cells permanently transformed by SV40 still exhibit the temperature‐sensitive phenotype.

Non-linkage of induced mutations in Chinese hamster cells.

The relatively rapid loss of human chromosomes from human-rodent somatic cell hybrids has allowed the determination of linkage relationships between several human genes1–4. Cells that have segregated out most of the human chromosomes are analysed for the presence or absence of particular human gene products; when two gene products are always found to be retained together, they are assumed to be linked. Little has been done to extend these genetic techniques to cell hybrids formed between two different mutants of the same cell line. A linkage analysis would provide a valuable means of interpreting the gene function altered in such mutants. The principal obstacle to such an approach has been the fact that homospecific cell hybrids are rather stable, losing chromosomes at only a low rate5–7. Nevertheless, by using suitably marked strains, it is possible to select rare segregants from a homospecific hybrid population7,8. I have applied such a system to test for linkage between several chemically induced mutations in a Chinese hamster cell line.

Mutagenesis in cultured mammalian cells: II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ṡ 10 −7 per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm 2. This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm 2 resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.

Effect of caffeine on the induction of mutations in Chinese hamster cells by ultraviolet light

Effect of caffeine on the induction of mutations in Chinese hamster cells by ultraviolet light

Effects of caffeine on the induction of mutations in Chinese hamster cells by ultraviolet light

Effects of caffeine on the induction of mutations in Chinese hamster cells by ultraviolet light

Genetics of somatic mammalian cells. IX. Quantitation of mutagenesis by physical and chemical agents.

AbstractThe system previously described for inducing single gene mutations in Chinese hamster cells has been extended to produce additional auxotrophic mutants. An improved method for quantitating the efficiency of single gene mutation to specific auxotrophies has been developed. Mutagenesis in the forward direction has been measured after treatment of these cells with ethyl methanesulfonate, N‐methyl‐N1‐nitro‐N‐nitrosoguanidine, hydroxylamine, an acridine mustard (ICR‐191), caffeine and ultraviolet‐ and X‐irradiation. For each agent, the single cell survival curve and the efficiency of chromatid breakage and rearrangement were measured. Similar measurements were also carried out with a water‐soluble carcinogen N‐nitrosomethylurea, which was shown to be effective in producing auxotrophic, somatic mutations. These results offer promise of illuminating the relationships between cell killing, chromosomal aberration, single gene mutations and carcinogenesis produced by various agents. The methods described can be used in routine testing of drugs, food additives, and environmental pollutants for mutagenic action in mammalian cells in vitro.

Isolation and characterization of thymidine transport mutants of Chinese hamster cells

In this paper a new system of checking mutation frequencies of the incorporation variety for cells in culture was presented. The mutation rate of a mutation in the thymidine incorporating system was determined by the fluctuation test to be 2.6 × 10 −4 mutants/cell/generation. A cell line mutant for thymidine transport was isolated and studied by autoradiographic staining technique. Thymidine kinase was assayed in normal and Tp − cell lines and both were found to have substantial enzyme activity. Incorporation studies were performed to demonstrate the specific and facilitative nature of thymidine uptake by cells. From the results presented, it is clear that the thymidine incorporation system of Chinese hamster cells suffers a high rate of spontaneous mutation. Some of the mutations in the thymidine incorporation system of these cells are at the level of the thymidine transport system.

Complementation analysis on virus-fused Chinese hamster cells with nutritional markers.

Cell fusion experiments have been carried out with Chinese hamster cell mutants with different nutritional growth requirements. Conditions have been devised in which approximately 1 to 2 percent of the cell population remaining after fusion are fused, hybrid cells. The all-or-none nature of the genetic markers employed and the extremely low reversion rates insure that no contamination of the hybrid population with parental forms occurs. Hybrids between glycine- and hypoxanthine-requiring mutants are prototrophic, which indicates that both mutations are recessive. Hybrids between a glycine-deficient mutant and a singlestep mutant which requires glycine, hypoxanthine, and thymidine are relieved of the glycine dependency, an indication that the two loci associated with glycine dependence are different. This mutation to the triple-supplement requirement as well as a proline deficiency were also shown to be recessive mutations. The system appears applicable to a variety of genetic problems.

Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro.

Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro.

Genetics of somatic mammalian cells, VII. Induction and isolation of nutritional mutants in Chinese hamster cells.

Genetics of somatic mammalian cells, VII. Induction and isolation of nutritional mutants in Chinese hamster cells.

Genetics of somatic mammalian cells. IV. Properties of Chinese hamster cell mutants with respect to the requirement for proline.

Genetics of somatic mammalian cells. IV. Properties of Chinese hamster cell mutants with respect to the requirement for proline.