Cycloheximide resistance in Chinese hamster cells I. Spontaneous mutagenesis

Resistance to cycloheximide (CHM) was studied in cultured Chinese hamster cells. Concentrations of CHM above 5.10⁻⁷M were toxic for the cells. At concentrations above 9.10⁻⁷M no colonies were recovered in selective medium. 15 resistant clones of independent origin were isolated in selective medium containing 7.10⁻⁷M CHM. Resistance was stable when the cells were cultured under non-selective conditions. The spontaneously mutation rate was determined by the fluctuation test. Mutations to CHM resistance arose spontaneously. The spontaneous mutation rate to CHM resistance was about 10⁻⁵.     

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 ? 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/mm². 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/mm² 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.     

Induction of heat shock proteins in Chinese hamster ovary cells and development of thermotolerance by intermediate concentrations of puromycin

During 4 hr after puromycin (PUR: 20 micrograms/ml) treatment, the synthesis of three major heat shock protein families (HSPs: Mr = 110,000, 87,000, and 70,000) was enhanced 1.5-fold relative to that of untreated cells, as studied by one-dimensional gel electrophoresis. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed at 10(-3) isosurvival as a thermotolerance ratio (TTR) of either 2 or greater than 5 after heating at either 45.5 degrees C or 43 degrees C, respectively. However, thermotolerance was induced by only intermediate concentrations (3-30 micrograms/ml) of puromycin that inhibited protein synthesis by 15-80%; a high concentration of PUR (100 micrograms/ml) that inhibited protein synthesis by 95% did not induce either HSPs or thermotolerance. Also, thermotolerance was never induced by any concentration (0.01-10 micrograms/ml) of cycloheximide that inhibited protein synthesis by 5-94%. Furthermore, after PUR (20 micrograms/ml) treatment, the addition of cycloheximide (CHM: 10 micrograms/ml), at a concentration that reduces protein synthesis by 94%, inhibited both thermotolerance and synthesis of HSP families. Thus, thermotolerance induced by intermediate concentrations of PUR correlated with an increase in newly synthesized HSP families. This thermotolerance phenomenon was compared with another phenomenon termed heat resistance and observed when cells were heated at 43 degrees C in the presence of CHM or PUR immediately after a 2-hr pretreatment with CHM or PUR. Heat protection increased with inhibition of synthesis of both total protein and HSP families. Moreover, this heat protection decayed rapidly as the interval between pretreatment and heating increased to 1-2 hr, and did not have any obvious relationship to the synthesis of HSP families. Therefore, there are two distinctly different pathways for developing thermal resistance. The first is thermotolerance after intermediate concentrations of PUR treatment, and it requires incubation after treatment and apparently the synthesis of HSP families. The second is resistance to heat after CHM or PUR treatment immediately before and during heating at 43 degrees C, and it apparently does not require synthesis of HSP families. This second pathway not requiring the synthesis of HSP families also was observed by the increase in thermotolerance at 45.5 degrees C caused by heating at 43 degrees C after cells were incubated for 2-4 hr following pretreatment with an intermediate concentration of PUR.     

Mutagenesis by simian virus 40. I. detection of mutations in Chinese hamster cell lines using different resistance markers.

The mutagenic action of SV40 in permanent lines of Chinese hamster cells (CHO-K1 and V79) was investigated with the aid of different resistance markers. The markers studied had resistance to 8-azaguanine (25 and 30 mug/ml), aminopterin (3.3--5.5X10(-3) mug/ml), colchicine (6.5 and 7.0X10(-2) mug/ml) and 5-bromodeoxyuridine (50--120 mug/ml), respectively. After virus infection the mutation frequencies were increased by one (azaguanine, aminopterin) and two (colchicine) orders of magnitude as compared with spontaneous mutation frequencies. In contrast, it was not possible to enhance the frequency of mutation to BUdR resistance. On the other hand, the ability to proliferate in HAT medium was induced in three of five BUdR-resistant cell clones by infection with SV40. The resistance induced by SV40 was stable when isolated clones were cultured under non-selective conditions. Mechanisms are proposed that may be responsible for the mutagenic action of SV40.     

Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: involvement of heat shock proteins

After sodium arsenite (100 microM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one-dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000-fold increase in survival from 10(-6) to 10(-1) after 4 hr at 43 degrees C, and as a thermotolerance ratio (TTR) of 2-3 at 10(-3) isosurvival for heating at 45.5 degrees C. Cycloheximide (CHM: 10 micrograms/ml) or puromycin (PUR: 100 micrograms/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2-6 hr before heating and left on during heating caused a 10,000-100,000-fold enhancement of survival when cells were heated at 43 degrees C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43 degrees C after CHM treatment were much different than those manifesting resistance to 43 degrees C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45 degrees C after 5 hr of heating at 43 degrees C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45 degrees C after 5 hr of heating at 43 degrees C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43 degrees C or 45.5 degrees C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43 degrees C with little resistance at 45.5 degrees C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45 degrees C caused by heating at 43 degrees C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.     

Fluctuation analyses of spontaneous mutations to 6-thioguanine resistance in Chinese hamster ovary cells in culture

Fluctuation analyses of the spontaneous appearance of 6-thioguanine (TG)-resistant mutants in cultured Chinese hamster ovary (CHO) cells were performed to investigate (1) whether the resistance is induced by the selective agent or is the result of a mutation which occurs prior to the TG selection and (2) to estimate the spontaneous mutation rate at the hypoxanthine—guanine phosphoribosyl transferase (hgprt) locus. The potential problem of phenotypic delay was minimized by allowing an adequate expression time through maintenance of the cultures in a division-arrested, viable state. The results demonstrate that the TG-resistant (TG^r) cells arise randomly in the cultures, independently of the selective agent, which is consistent with spontaneous mutations. The average values for mutation rate ± standard deviation, based on 4 independent determinations and 2 methods of calculation, are 3.4 ± 1.2 × 10^?7 (median method) and 5.1 ± 1.8 × 10^?7 (mean method) mutants/cell/generation.     

Protection of Chinese hamster ovary cells from hyperthermic killing by cycloheximide or puromycin

Cycloheximide (CHM) and puromycin (PUR) were used at various concentrations up to maxima of 10 micrograms/ml and 100 micrograms/ml, respectively, which inhibited protein synthesis by 95% without any cytotoxicity. The drugs were added to the cells for a maximum period of 7 h, with various combinations for treatment before, during, and after heating. Maximum protection, i.e., a 10,000-fold increase in survival from 5 X 10(-6) to 5 X 10(-2) after 4 h at 43 degrees C, required both 1-2 h of treatment before heating and 1-2 h of treatment during heating. For treatments at 45.5 degrees C, the protection was less, i.e., a 100-fold increase in survival from 10(-5) to 10(-3). Little or no protection was observed if after treatment, the drug was removed before heating, or if the drug was added at the start of heating and left on for 5 min to 3 h after heating. For both drugs, the amount of protection increased as inhibition of protein synthesis increased. However, the amount of protection from the drugs was the same only at about 95% inhibition; at 60-85% inhibition, CHM afforded more protection than PUR. Therefore, the modes of action of the drugs might be common at high drug concentrations, but different when intermediate concentrations are used.     

Linear dose--response relationships after prolonged expression times in V-79 Chinese hamster cells.

The expression time for induced mutants resistant to 6-thioguanine, in V-79 Chinese hamster cells, was determined by respreading the cells in the selective medium, at various times after treatment. The length of the expression time for mutants induced by X-rays, ethyl methane sulphonate and ultraviolet irradiation was dose dependent. For the highest dose used this was 7 to 8 days, beyond which there was no further changes in mutant frequency. The dose-response relationship of these agents does not appear to deviate from linearity; this permits the calculation of mutation rate per unit dose. For X-rays this value was 1.35 - 10(-7) per rad per locus, for ethyl methane sulphonate, 2.2 - 10(-2) per mole per locus and for ultraviolet irradiation, 6.3 - 10(-6) per erg per mm2 per locus. The effectiveness of the 3 different mutagens for the induction of mutations was compared by calculating the increase in mutant frequency per unit of decrease in survival (Do). These increments in frequency were: 5.6 - 10(-5) for X-rays, 69.5 - 10(-5) for ethyl methane sulphonate and 16.1 - 10(-5) for ultraviolet irradiation.     

Correlation between specific DNA-methylation products and mutation induction at the HGPRT locus in Chinese hamster ovary cells

Suspension cultures of Chinese hamster ovary (CHO) cells were exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) and assayed for mutation induction (6-thioguanine resistance) and for specific DNA adducts. DNA methylation at the 1-, 3- and 7-positions of adenine, the 3-, O6- and 7-positions of guanine, and phosphate was detected in cultures exposed to MMS, while MNU produced 3- and 7-methyladenine, 3-methylcytosine, 3-, O6- and 7-methylguanine, O4-methylthymidine and methylated phosphodiesters. When mutations induced by MMS and MNU were compared by linear correlation analysis with levels of each of these adducts, only O6-methylguanine displayed a strong correlation with mutations (r = 0.879, p less than 0.001). The relationship between O6-methylguanine and induced mutations in CHO cells is similar to that previously reported in CHO cells for O6-ethylguanine and mutations (Heflich et al., 1982) and indicates that alkylation-induced mutations at the HGPRT locus in CHO cells are primarily associated with O6-alkylguanine formation.     

Spontaneous mutation rate to thioguanine resistance is decreased in polyploid hamster cells

The mutation rate to thioguanine resistance was 3.11 X 10(-6) in a near diploid V79 hamster cell line and 7.58 X 10(-8) in a near tetraploid derivative produced with colchicine. The specific activities of glucose-6-phosphate dehydrogenase and phosphoglycerate kinase of the tetraploid line were greater than that of the diploid which suggests that twice the number of active X chromosomes were present in the tetraploid. These results are compatible with the hypothesis that spontaneous variants resistant to thioguanine arise through mutation and chromosomal segregation, as has been suggested for induced mutations in tetraploid hamster cells.     

The relationship between transformation and somatic mutation in human and Chinese hamster cells

The frequencies of transformations of primary human and Chinese hamster fibroblasts have been compared with the spontaneous and induced frequencies of mutation for resistance to thioguanine and ouabain, and for ability to use fructose, using the carcinogens benzo (alpha) pyrene and urethane. Whereas the rates and frequencies of mutation were similar in the two cell systems, transformations to morphologically altered cells was observed only in hamster cells. The frequency of this latter transformation event in hamster cells was abour 10(3) greater than the frequencies of mutation in these cells. The morphologically altered cells formed in the above transformation process cannot grow in agar (aga-) and do not produce tumors when injected into animals. The frequency of transition of these latter cells to aga+ cells which produce tumors in animals is similar to the mutation-like events.     

The dose-response relationship for ultraviolet-light-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells.

Exposure of Chinese hamster ovary (CHO) cells clone K1BH4 to ultraviolet (UV) light at doses up to 86 ergs/mm2 did not significantly reduce cell survival, but UV doses of 86-648 ergs/mm2 produced an exponential cell killing. Observed mutation frequency ro 6-thioguannine resistance induced by UV increases approximately in proportion to increasing doses up to 260 ergs/mm2 in a range of 5-648 ergs/mm2 examined. The pooled data of mutation frequency f(X) as a function of dose X from 0-260 ergs/mm2 is adequately described by f(X)=10(-6) (13.6 + 2.04 X). That the UV-induced mutations to 6-thioguanine resistance affects the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus is supported by the observation that all randomly isolated drug-resistant colonies contained highly reduced or undetectable HGPRT activity.     

Platinum-induced mutations to 8-azaguanine resistance in Chinese hamster ovary cells.

6 platinum (Pt) compounds were compared in suspension cultured Chinese hamster ovary (CHO-S) cells with respect to their inhibition of growth, their reduction of cloning efficiency, and their induction of mutants resistant to 200 microM (30 micrograms/ml) 8-azaguanine (8-AG) and 3 mM ouabain (OUA), respectively. The toxicity of these compounds can be ranked by the medium concentrations which decrease suspension growth/or cloning efficiency by 50%: cis-Pt(NH3)2-Cl2 (0.9/1.5 microM) greater than Pt(SO4)2 + methylcobalamin (MeB-12) methylation product (20/10 microM) greater than K2PtCl4 (32/50 microM) = K2PtCl6 (34/50 microM) = MePtCl2-3 (60/50 microM) greater than Pt(SO4)2 (66/105 microM). Following 20 h exposures to concentrations which resulted in relative survivals of 80-2%, none of the foregoing compounds increased consistently the frequency of OUA(R) mutants above the spontaneous frequency (6.0 x 10(-6)). Parallel treatments with 800 microM (100 micrograms/ml) ethyl methanesulfonate (EMS) increased the OUA(R) mutant frequency 10--12-fold. Using 8-AG for mutant selection, dose-dependent increases of 5--7-fold above the spontaneous frequency (3--8 x 10(-5) were obtained with cis-Pt(NH3)2Cl2, Pt(S04)2, and the product from Pt(SO4)2 + MeB-12. Identical 20 h exposures to varying amounts of K2PtCl4, K2PtCl6, and MePtCl2-3 did not induce 8-AG(R) mutants. Optimal detection of Pt-induced 8-AG(R) mutants required 7 post-treatments, expression doublings in suspension culture. Under our selection conditions 8/8 spontaneous and 24/24 Pt-induced 8-AG(R) variants contained reduced hypoxanthine-guanine phosphoribosyl transferase (HGPRT) specific activities (means ranging from 3 to 11% of the parental CHO-S cells). When compared from linear plots of the 8-Ag(r) frequency against the initial medium concentration, cis-Pt(NH3)2Cl2 is 134 times and Pt(SO4)2 si 3.5 times more mutagenic than EMS. However, on a cell-survival basis EMS is 8--10-fold more mutagenic than these two Pt-compounds. 6-Thioguanine (10 microM) can be substituted for 8-AG to assay mutant induction by cis-Pt(NH3)2Cl2 and Pt(SO4)2 in CHO-S cells. The sensitivity of the CHO-S HGPRT locus for detecting mutagenesis by Pt complexes can be increased several fold by continuous subculture in the presence of these agents for 10--25 population doublings. By this procedure K2PtCl6 is seen to be weakly mutagenic and 20 microM Pt(SO4)2 produces 8-AG(R) mutants at frequencies requiring 7--8-fold higher concentrations when a fixed 20 h exposure is used.     

Alteration of cadmium-induced mutational spectrum by catalase depletion in Chinese hamster ovary-K1 cells.

Previously, we have demonstrated that cadmium acetate significantly induces hprt mutation frequency in Chinese hamster ovary (CHO)-K1 and that 3-amino-1,2,4-triazole (3AT), a catalase inhibitor, potentiates the mutagenicity of cadmium [Chem. Res. Toxicol. 9 (1996) 1360-1367]. In this study, we investigate the role of intracellular peroxide in the molecular nature of mutations induced by cadmium. Using 2',7'-dichlorofluorescin diacetate and fluorescence spectrophotometry, we have shown that cadmium dose-dependently increased the amounts of intracellular peroxide and the levels were significantly enhanced by 3AT. Furthermore, we have characterized and compared the hprt mutation spectra in 6-thioguanine-resistant mutants derived from CHO-K1 cells exposed to 4 microM of cadmium acetate for 4h in the absence and presence of 3AT. The mutation frequency induced by cadmium and cadmium plus 3AT was 11- and 16-fold higher than that observed in untreated populations (2.2 x 10(-6)), respectively. A total of 40 and 51 independent hprt mutants were isolated from cadmium and cadmium plus 3AT treatments for mRNA-polymerase chain reaction (PCR), genomic DNA-PCR and DNA sequencing analyses. 3AT co-administration significantly enhanced the frequency of deletions induced by cadmium. Cadmium induced more transversions than transitions. In contrast, 3AT co-administration increased the frequency of GC-->AT transitions and decreased the frequencies of TA-->AT and TA-->GC transversions. Together, the results suggest that intracellular catalase is important to prevent the formation of oxidative DNA damage as well as deletions and GC-->AT transitions upon cadmium exposure.     

Repeated doses of gamma rays induce resistance to N-methyl-N'-nitro-N-nitrosoguanidine in Chinese hamster cells

Chinese hamster V79 cells were preirradiated repeatedly with gamma rays and then exposed to ultraviolet (uv) light or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The cell killing and induction of mutation at the hypoxanthine-guanine phosphoribosyltransferase locus were examined following these treatments. Cells preirradiated with multiple fractions of gamma rays exhibit the same sensitivity to uv light as the control cells with respect to cell survival and mutation induction. Following treatment with MNNG, resistance to cell killing was observed along with a decreased frequency of mutations induced. These results indicate that the progeny of cells irradiated with multiple fractions of gamma rays could display subsequent changes in sensitivity to lethal and mutagenic effects of additional treatment with DNA-damaging agents.     

Enhancing effects of cytosine arabinoside on ethyl methanesulfonate-induced 6-thioguanine resistance mutations in Chinese hamster V79 cells

We studied the synergistic enhancement effects of two chemicals which are different in their mechanism of action on DNA in cells. The test chemicals used were ethyl methanesulfonate (EMS) as an alkylating agent and cytosine arabinoside (Ara-C) as an analogue of cytidine. For determination of mutagenesis we measured the induction of resistance to 6-thioguanine (6-TG) in Chinese hamster V79 cells. EMS had a strong mutagenic effect on V79 cells, but for Ara-C the results were less clear. In this study, Ara-C had no detectable effect in inducing mutation up to a concentration of 5 X 10(-4) M. The mutation frequency of combined treatment with EMS and Ara-C was significantly higher than that obtained with EMS alone. These results indicate that Ara-C had an enhancing effect on mutations induced by EMS.     

Mammalian cell genetics. 3. Characterization of x-ray-induced forward mutations in Chinese hamster cell cultures

X-irradiation induces forward mutations from 8-azaguanine sensitvity to resistance in Chinese hamster cells in culture. At this locus the number of induced mutations increases non-linearly with X-ray exposure. The mutation rate increase from 4.2·10⁻⁷ per locus per R with 200 R to 1.8·10⁻⁶ per locus per R with 1200 R. Several factors including cell density markedly influence the mutational yield. Reversion tests using specific chemical mutagens on 72 randomly isolated, azaguanine-resistant mutants suggest that both point mutations and chromosome deletions might have occurred in the hamster cells after exposure to ionizing radiation.     

Measurement of spontaneous mutation rates at the Na+/K+ ATPase locus (ouabain resistance) of human fibroblasts using improved growth conditions

The mutation rate for the Na+/K+ ATPase locus (ouabain resistance, OuaR) in mammalian cells in culture has been reported to be 10-100-fold lower than the mutation rate of other gene loci in culture, such as the hypoxanthine phosphoribosyl transferase (HPRT) locus. Determination of the mutation rate to ouabain resistance is sensitive to culture conditions and the concentration of ouabain used to select mutants. Our improved growth conditions for human cells have permitted absolute cloning efficiencies of 70-90% and population doubling times of 16-17 h with both normal human diploid fibroblasts, KD, and their chemically induced neoplastic derivative, Hut-11A. Ouabain at 10(-7) M was found to be adequate to select for resistant (OuaR) mutants with an absolute recovery efficiency of 54-102%. Under these conditions, the mutation rates to ouabain resistance for human cells were measured and found to be 1-8.5 X 10(-7)/cell/generation for KD cells and 6-13 X 10(-7)/cell/generation for Hut-11A cells. These rates are 5-25 times higher than previously reported for human cells. Improved growth and the use of a lower concentration of ouabain for selection may allow for the increased recovery of OuaR mutants and an improved estimate of the mutation rate at this locus, which is only 2-10-fold less than the mutation rate at the HPRT locus in the same cells.     

X-ray induced mutation in Syrian hamster fetal cells

Transabdominal X-rays are a risk factor for childhood leukemia, and X-ray exposure of mouse fetuses has led to increases in both mutations and initiated tumors in offspring. However, fetal sensitivity and dose-response characteristics with regard to transplacental mutagenesis by X-rays have never been quantified. In the current experiment, pregnant Syrian hamsters at day 12 of gestation were irradiated with 300-kV X-rays. Twenty-four hours later, the fetuses were removed and their cells were allowed a 5 day expression time in culture. They were then seeded for colony formation and also for mutation selection by 6-thioguanine (6-TG). Mutation frequency was linear over the entire dose range, 10-600 R. The average induced 6-TG mutant frequency was 4.7 x 10(-7) per R. These results suggest that fetal cells are highly sensitive to induction of mutations by X-rays, and that a no-effect threshold is not likely. The 10 R dose caused a 25-fold increase in mutation frequency over the historical control, 45 x 10(-7) versus 1.8 x 10(-7), an increase per R of 2.5-fold. Increased risk of childhood cancer related to obstetrical transabdominal X-ray has also been estimated at 2.5-fold per R. Thus, our results are consistent with mutation contributing to this effect.     

Characterization of ultraviolet light-induced ouabain-resistant mutations in Chinese hamster cells.

Ouabain-resistant mutations in Chinese hamster cells have been quantitatively characterized. The mutation frequencies were found to be induced curvilinearly with treatments of increasing doses of ultraviolet light (UV). For the range of UV doses tested (5--20 J/m(2)), the observed frequency, Y, as a function of UV dose X, follows a curvilinear function, Y = (-28 + 13.37 X--1.52X(2) + 0.08X(3)) . 10(-6). The frequencies of UV-induced mutations were directly correlated with cell survival, indicating a similar causal relationship between cell killing and mutation induction. Under the same experimental conditions, X-rays induced 6--thioguanine-, but not ouabain-, resistant mutations. UV-induced ouabain-resistant (ouar) mutants exhibit a selection disadvantage. Their phenotypic expressions are modifiable by various agents. Wild type and 16 ouar mutants were compared with respect to their sensitivity to ouabain inhibition of 86Rb uptake by whole cells. All the ouar mutants assayed are less sensitive to the drug than are wild-type cells. In the absence of ouabain, the Na+--K+--ATPase activities can be significantly higher or lower than that of the wild-type cells.