Isolation and characterization of Chinese hamster cell line resistant to monofunctional alkylating agents

A clonal derivative of a Chinese hamster Don D-6 cell line resistant to methyl methane sulfonate (MMS) has been isolated following mutagenesis by ethyl methane sulfonate (EMS). The clone, designated as MMS^r-1, exhibited high resistance to killing by the monofunctional alkylating agents MMS and EMS. This characteristic had not been acquired by a transient adaptation to the alkylating agents but was found to be a stable heritable trait. MMS^r-1 was more sensitive to high-molecular-weight chemicals, such as colchicine and puromycin, than Don D-6. Both MMS^r-1 and its parental cells showed the same ability to take up radioactive MMS. The resistance of MMS^r-1 appears not to be due to altered uptake of MMS. The resistance was accompanied by low chromosomal aberration and sister chromatid exchange (SCE) induction but not by mutability. Protein synthesis inhibitors such as cycloheximide and puromycin reduced the resistance to the same level as that in Don D-6. SCE induction by MMS in this clone was not antagonized by the protein synthesis inhibitors, whereas mutagenesis was reversed to the normal parental cell level by these inhibitors. Aphidicolin, a DNA-synthesis inhibitor, exhibited no such effects. These results suggest that MMS^r-1 might have modified repair capacity, which can be normalized by treatment with the protein-synthesis inhibitors, for lethal DNA damage by monofunctional alkylating agents, and that SCE formation by the alkylating agents is closely correlated with chromosomal aberration and cell lethality.     

Isolation and characterization of Chinese hamster ovary cell mutants resistant to the amino acid analogβ-aspartyl hydroxamate

Chinese hamster ovary cell lines which are resistant to an amino acid analog, -aspartyl hydroxamate, have been isolated and characterized. Mutants resistant to 100–150 M -aspartyl hydroxamate arose from ethyl methane sulfonate-treated parental lines at frequencies of 3.4×10^–6 to 1.3 ×10^–7. The mutants fell into at least two genetic classes: 18% of the mutants behaved codominantly in hybrids, the others recessively. Complementation studies indicated that all the recessive mutants belonged to the same class. Mutants selected after one step of mutagenesis overproduce the enzyme asparagine synthetase constitutively with four- to sixfold increases in specific activities over the basal levels of the parental lines. -Aspartyl hydroxamate-resistant cell lines with up to 20-fold elevations in asparagine synthetase activity have been isolated after two steps of mutagenesis. In addition, highly resistant lines have been selected by long-term growth of a dominant mutant in increasing concentrations of the drug. Resistance in the latter appears to be due not only to overproduction of asparagine synthetase but also to an alteration in the affinity of the enzyme for -aspartyl hydroxamate.     

Isolation and characterization of a mutant Chinese hamster ovary cell line that is resistant to Chlamydia trachomatis infection at a novel step in the attachment process

Host factors involved in Chlamydia trachomatis pathogenesis were investigated by random chemical mutagenesis of Chinese hamster ovary (CHO-K1) cells followed by selection for clones resistant to chlamydial infection. A clonal mutant cell line, D4.1-3, refractory to infection by the C. trachomatis L2 serovar was isolated. The D4.1-3 cell line appears to be lacking in a previously undescribed temperature-dependent and heparin-resistant binding step that occurs subsequent to engagement of cell surface heparan sulfate by L2 elementary bodies. This novel binding step differentiates the lymphogranuloma venereum (LGV) serovar from other serovars and may contribute the different pathologies associated with LGV and non-LGV strains.     

Isolation and characterization of a Chinese hamster lung cell tryptophanyl-tRNA synthetase mutant

A novel type of temperature-sensitive protein synthetic mutant was isolated from V-79 Chinese hamster lung cells using an amino acid analog suicide selection. The expression of the temperature sensitive phenotype of the mutant is greatly affected by the concentration of tryptophan in the culture medium. In addition, the activity of tryptophanyltRNA synthetase is undetectable in cellfree extracts prepared from the mutant cells. The results suggest that the mutant has an alteration in the structural gene encoding tryptophanyl-tRNA synthetase.     

Isolation and characterization of a UV-sensitive hypermutable aphidicolin-resistant Chinese hamster cell line

Aphidicolin is a specific inhibitor of DNA polymerase and blocks DNA synthesis in vivo. The inhibition of purified -polymerase has been shown to be competitive with dCTP but not with the other three deoxynucleoside triphosphates (dNTPs). In order to study the various roles that the -polymerase might play in DNA replication and/or repair, we have attempted to isolate Chinese hamster V79 cells that are resistant to aphidicolin. Four resistant mutants were isolated from BrdU-black light- and UV-mutagenized cells. None of the mutants isolated contains an -polymerase that is resistant, in crude extract measurements, to aphidicolin. Three mutants isolated, however, were found to be resistant to araC. Two mutants tested were found to be sensitive to cytidine and have elevated levels of dCTP or all 4 dNTPs. These results indicate that they are nucleotide pool mutants instead of -polymerase mutants. One mutant, aph^r-4, is characterized by the following: (1) high level of dCTP; (2) thymidine (or CdR, UdR, auxotrophic; (3) sensitive to thymidine (and AdR, GdR); (4) slow-growing; (5) cytidine sensitive; (6) UV sensitive and hypermutable at the ouabain-resistant locus; and (7) a ninefold increase in frequency of chromatid gaps and breaks when cells are exposed to BrdU- containing medium. Revertants of aph^r-4 which are partially aphidicolin-resistant and retain the first three characteristics listed above, but not the others, have been isolated. The appearance of this type of revertant indicates that either aph^r- 4 or its revertant is a double mutant.     

Isolation and characterization of Chinese hamster cell mutants resistant to the cytotoxic effects of chromate

Stable mutants resistant to the toxic anion chromate have been isolated from a variety of Chinese hamster cell lines. The mechanism of chromate toxicity is not known, but it must involve internalization via the sulfate transport pathway. All mutant lines had a defective sulfate transport system, showing a 10-fold reduction in the rate of uptake of radioactive sulfate into the cell. The chromate resistance phenotype in CHO cell mutants behaved recessively in somatic cell hybrids; in other cell lines the Chr^r phenotype was partially expressed (codominant) in cell hybrids. Complementation analysis in cell hybrids between 18 different mutant pairs failed to reveal any complementation, indicating that chromate selects mutants primarily, if not exclusively, at a single gene locus.     

Isolation and characterization of glucose-6-phosphate dehydrogenase-deficient Chinese hamster cells derived from pure mutant colonies

Ten pure glucose-6-phosphate dehydrogenase (G6PD)-deficientmutants were isolated from colonies composed entirely of cellswhich lacked G6PD staining activity. These mutants were analyzedfor G6PD enzyme activity and the presence of immunologicallycross-reactive proteins using immunoblotting techniques andantiserum directed against bovine G6PD. Four mutants had nodetectable enzyme activity and did not contain protein whichproduces a detectable cross-reaction with G6PD antibody. Onemutant had residual enzyme activity and altered electrophoreticmobility but did not have detectable immunological crossreactivity.These results could be explained by either a DNA deletion orpoint mutational mechanism. On the other hand, five of the 10mutants analyzed had characteristics consistent with a pointmutation in the G6PD gene. All contain a protein which cross-reactswith the G6PD antibody and have the same subunit molecular weightas the parent cell's G6PD enzyme. Four of the five mutants hadresidual G6PD enzyme activity. Thus, the mechanism for the formationof pure mutants is not simply DNA deletion but is probably amore complex process involving the transfer of altered geneticinformation from one DNA strand to the other.     

Isolation and characterization of a novel mutant mouse cell line resistant to Newcastle disease virus: constitutive interferon production and enhanced interferon sensitivity

Summary In our attempt to isolate mutant cell lines resistant to Newcastle disease virus (NDV) we developed an improved procedure for enrichment of NDV-resistant cells from mouse FM3A cells and isolated a novel NDV-resistant mutant cell line, Had-2, with characteristics different from Had-1, a previously reported NDV-receptor-deficient mutant strain. Had-2 cells adsorbed NDV normally but the accumulation of viral mRNAs and proteins was inhibited. Had-2 cells had to be grown at higher cell densities in order to be NDV-resistant, and it was revealed that they did not exhibit NDV-resistance when grown at lower cell densities. A conditioned medium prepared from a culture of Had-2 cells grown at high cell density was able to make a low-density culture NDV-resistant. The activity of the conditioned medium to induce NDV-resistance was completely neutralized by addition of both anti interferon (IFN)- and anti IFN- antibodies, indicating that Had-2 cells were constitutively releasing IFNs, though their levels were rather low. Had-2 cells were also characterized by an increased sensitivity to IFNs as compared with the parental FM3A cells, since the conditioned medium containing IFNs did not render FM3A cells resistant to NDV.     

Isolation and characterization of a methylammonium resistant mutant of Neurospora crassa

Summary A mutant of Neurospora crassa has been isolated which is resistant to methylammonium, a structural analog of ammonium. In contrast to wild type, this mutant, mea-1, has derepressed nitrate reductase and nitrite reductase activities in the presence of ammonium. However, glutamine still represses these nitrate assimilation enzymes in mea-1. The nit-2 mutant was epistatic to mea-1 since the mea-1; nit-2 double mutant has the nit-2 mutant phenotype. In addition, mea-1; nit-2 double mutants cannot utilize ammonium as a nitrogen source. We suggest therefore that nit-2 and mea-1 loci play a role in ammonia/methylamine uptake.     

A temperature-sensitive DNA synthesis mutant isolated from the Chinese hamster ovary cell line

A temperature-sensitive DNA synthesis mutant, tsC8, was isolated from mutagenized Chinese hamster ovary cells by the fluorodeoxyuridine suicide technique. The tsC8 cells showed inhibition of DNA synthesis at the nonpermissive temperature (NPT) with little effect on initial levels of RNA and protein synthesis. Temperature-arrested tsC8 cells had G₁ or S DNA content and the temperature-sensitive (ts) period of the tsC8 cell cycle was the interval between the G₁/S border and the middle of the S period. The tsC8 cells were unable to enter the S phase when exposed to the NPT during the G₁ period of the cell cycle. When S phase tsC8 cells were shifted to the NPT, they incorporated [³H]thymidine at rates similar to the parental cell type for only 2 h, indicating ats defect in DNA synthesis. The tsC8 mutation is expressed in a recessive manner and is in a gene distinct from those affected in other DNA synthesis mammalian cell mutants.     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism III. Isolation and characterization of a mutant unable to convert IMP to AMP

The isolation and characterization of a new mutant of Chinese ovary cells (CHO-K1) is described. This mutant, Ade ^– H, has the following properties: (1) it forms a new genetic complementation group; (2) it specifically requires adenine for growth and will not grow on aminoimidazole carboxamide (AIC) or hypoxanthine; (3) it accumulates IMP; (4) it cannot synthesize adenine nucleotides; (5) its phenotype can be mimicked by treatment of CHO-K1 (the wild type parental strain) with hadacidin, an inhibitor of adenylosuccinate synthetase (E.C.6.3.4.4). Thus, the site of the defect in this mutant is presumed to involve the step in adenylate biosynthesis catalyzed by this enzyme. The usefulness of Ade ^– H for the study of regulation of purine biosynthesis in mammalian cells is discussed.     

Secondary mutation resistant to 7-ketocholesterol rescues a sterol metabolic defect in amphotericin B-resistant Chinese hamster cell line

Amphotericin B-resistant mutants isolated from Chinese hamster V79 cells (1) are defective in cholesterol synthesis and more sensitive to an oxygenated sterol analog, 7-ketocholesterol, than their parental cell line. We isolated 7-ketocholesterol-resistant mutants from an amphotericin B-resistant mutant, AMB^R-1. The 7-ketocholesterol-resistant mutants had regained increased level of free cholesterol, and they showed somewhat similar dose-response curves to amphotericin B as that of V79. Sterol synthesis from acetate, but not from mevalonate, in 7-ketocholesterol-resistant clones was threefold higher than that of AMB^R-1. 7-Ketocholesterol-resistant clone, unlike AMB^R-1, could form colonies in the presence of lipoprotein-depleted serum. The results are discussed in terms of probable change in the sterol biosynthetic pathway by the different lesions.     

A mutant of Chinese hamster ovary cells resistant toα-methyl- andα-difluoromethylornithine

We describe a mutant of Chinese hamster ovary cells which is resistant to elevated levels of -methylornithine and -difluoromethylornithine, reversible and enzymeactivated irreversible inhibitors, respectively, of the enzyme ornithine decarboxylase (ODC). The mutant cells have significantly elevated levels of enzyme activity compared to wildtype cells, but several of the physical parameters of the enzyme are completely normal: Michaelis-Menten parameter, K_m, affinity for the analog, and half-life. The temporal regulation of this activity in synchronized cells is not perturbed, and the suppression of ODC activity by the addition of putrescine is still observed. Indirect experiments suggest increased concentrations of ODC mRNA in the mutant cells.     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: Isolation and characterization of a mutant deficient in the activity of phosphoribosylaminoimidazole synthetase

A new purine-requiring mutant of Chinese hamster ovary cells (CHO-Kl) is described. This mutant, Ade^– G, grows on aminoimidazole carboxamide, hypoxanthine, or adenine. It complements all eight of our other previously described Ade^– mutants. Biochemical analysis of de novo purine synthesis in whole cells suggests that Ade^– G is capable of the first four reactions of de novo purine biosynthesis and that it synthesizes and accumulates phosphoribosylformylglycinamidine (FGAM). Direct enzyme assay in cell-free extracts confirms that Ade^– G is defective in phosphoribo-sylaminoimidazole synthetase activity and does not convert FGAM to phosphoribosylaminoimidazole (AIR), the next intermediate in the de novo biosynthetic pathway.Recipient of a Research Career Development Award (AM00044) from the National Institute of Arthritis, Metabolic and Digestive Diseases.     

Isolation and characterization of a CHO amino acid transport mutant resistant to melphalan (l-phenylalanine mustard)

A mutant of Chinese hamster ovary cells, CHY-2, was isolated on the basis of its reduced ability to grow on a limiting concentration of leucine and was found to be defective in uptake of leucine via the sodium-independent L system. Consistent with published reports that the L system can mediate melphalan uptake, the D₁₀ of the mutant for melphalan was increased threefold under conditions designed to limit drug uptake to the L system (brief exposure in sodium-free medium). Unlike a previously described melphalan-resistant CHO mutant (CH^r), CHY-2 displays no cross-resistance to colchicine or puromycin. It differs from a second melphalan-resistant CHO mutant, mel_r, in its sensitivity to melphalan in the presence of high Na⁺, and from a melphalan-resistant mouse leukemic cell in possessing normal levels of intracellular glutathione. Thus, CHY-2 represents a new melphalan-resistant mutant class. The effect of the CHY-2 mutation is pleiotropic, involving significant reductions in amino acid uptake via the L, A and Ly⁺ (but not ASC) systems. The primary defect is unknown; however, the mutant possesses normal intracellular concentrations of Na⁺ and K⁺ and normal membrane fluidity. The growth rate of the mutant in standard medium is greatly reduced (generation time of 60 h vs. 24 h), although it can be improved by the addition of a supplement containing high concentrations of leucine, proline, and peptides.     

Isolation and characterization of Chinese hamster ovary cell mutants with altered sensitivity to high doses of tunicamycin

A mutant, CTM422, resistant to low dose of tunicamycin (TM) was isolated from Chinese hamster ovary (CHO) cells, and it showed 7- to 10-fold higher resistance to TM than CHO. We further mutagenized CTM422, to isolate TM-high-resistant mutants which were resistant to about 100-fold higher dose of TM than CHO. The TM-high-resistant mutants (N101 and N102) acquired about 4-fold higher cross-resistance to 2-deoxy-D-glucose than CHO or CTM422, while both CHO and CTM422 showed similar sensitivity to 2-deoxy-D-glucose. TM-low-resistance appeared to be codominant, and TM-high-resistance was partially codominant against TM sensitivity, respectively. The transfer activity of N-acetylglucosamine from UDP-N-acetylglucosamine into the lipid fraction with CHO and CTM422 cell extracats was inhibited by TM to a similar extent, while the extract of N102 cells showed about 10-fold higher resistance to TM than CHO or CTM422.     

Isolation and characterization of interspecific heat-resistant hybrids between a temperature-sensitive Chinese hamster cell asparaginyl-tRNA synthetase mutant and normal human leukocytes: Assignment of humanasnS gene to chromosome 18

We isolated interspecific somatic cell hybrids between human peripheral leukocytes and a temperature-sensitive CHO cell line with a thermolabile asparaginyl-tRNA synthetase. The hybrids were selected at 39° C so as to require the expression of the human gene complementing the deficient CHO enzyme. In vitro heat-inactivation profiles of cell-free extracts from temperature-resistant hybrid cells indicate the presence of two forms of asparaginyl-tRNA synthetase. One form is very resistant to thermal inactivation, like the normal human enzyme, while the other form is very thermolabile, like the altered enzyme from the CHO parent. Hybrids and temperature-sensitive segregants derived from them were analyzed for the expression of known human chromosomal marker enzymes. The strong correlation between the expression of the human form of asparaginyl-tRNA synthetase and the presence of human chromosome 18 in hybrids suggests that the human gene, asnS,which corrects the heat-sensitive phenotype of the CHO asparaginyl-tRNA synthetase mutant, is located on chromosome 18.     

Cytogenetic characterization of the ionizing radiation-sensitive Chinese hamster mutant irs1

The X-ray-sensitive mutant V79 cell line irs1 was characterized with respect to chromosomal aberrations induced by ¹³⁷Cs, mitomycin C (MMC), and decarbamoyl mitomycin C (DCMMC). To measure chromosome damage induced at different cell cycle stages, irs1 and the parental V79-4 cell lines were pulse-labeled with bromodeoxyuridine (BrdUrd) at the time of exposure and harvested at various intervals corresponding to exposure in G₁, S, and G₂ phases of the cell cycle. Metaphase spreads were stained with an anti-BrdUrd antibody, followed by a fluorescein-conjugated second antibody. With propidium iodide as a counter stain, cells were scored for aberrations. Compared to the parental V79 cells, irs1 cells had: (1) greatly increased sensitivity to all 3 agents; (2) a high frequency of chromatid exchanges after exposure in each phase of the cell cycle; and (3) more sensitivity to the agent causing crosslinks (MMC) than its monofunctional analog (DCMMC). The finding of chromatid-type damage in cells exposed to ionizing radiation during G₁ is atypical of normal cells, but is similar to observations made in several mutant rodent cell lines and in ataxia telangiectasia cells. Our results suggest that the defect in irs1 cells can manifest itself as misrepair or misreplication during all phases of the cell cycle and leads to a high incidence of chromatid exchanges and deletions.     

Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell

A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma raysensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G ₁,early S, and late G ₂ phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D ₃₇ in the sensitive G ₁ period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage.