Ethyl Methanesulfonate Mutagenesis Research Articles

Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells.

The amino acid analog albizziin was used to isolate Chinese hamster ovary cell lines which overproduce asparagine synthetase. Mutants selected in a single step after ethyl methane sulfonate mutagenesis were approximately 10-fold more resistant to the drug than the parental lines and expressed 8- to 17-fold elevations in enzyme activity. The karyotypes of these lines show alterations such as breaks and translocations affecting the long arm of chromosome 1. Cell lines isolated in several steps by growth in progressively increasing concentrations of albizziin were more resistant to the drug and exhibited up to 300-fold enhancement of asparagine synthetase activity. The multistep albizziin-resistant cell lines usually had expanded chromosomal regions which stained somewhat homogeneously, often on the long arm of chromosome 1. These results suggest that resistance to albizziin in the multistep lines may be due to gene amplification.

Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells.

The amino acid analog albizziin was used to isolate Chinese hamster ovary cell lines which overproduce asparagine synthetase. Mutants selected in a single step after ethyl methane sulfonate mutagenesis were approximately 10-fold more resistant to the drug than the parental lines and expressed 8- to 17-fold elevations in enzyme activity. The karyotypes of these lines show alterations such as breaks and translocations affecting the long arm of chromosome 1. Cell lines isolated in several steps by growth in progressively increasing concentrations of albizziin were more resistant to the drug and exhibited up to 300-fold enhancement of asparagine synthetase activity. The multistep albizziin-resistant cell lines usually had expanded chromosomal regions which stained somewhat homogeneously, often on the long arm of chromosome 1. These results suggest that resistance to albizziin in the multistep lines may be due to gene amplification.

Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase.

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.

Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase.

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.

Human mutant cell lines with altered RNA polymerase II.

A human fibrosarcoma cell line, HT-1080-6TG-9AM, resistant to alpha-amanitin at concentrations up to 10 micrograms/ml, was isolated after ethylmethanesulfonate mutagenesis and stepwise selection. The mutation is stable and dominant. RNA polymerase II purified from the mutant cells showed an altered affinity for labeled alpha-amanitin and the sensitivity of the enzyme to the fungal toxin was decreased 50- to 100-fold. This functional test demonstrated that the biochemical basis for the resistance of the cells to alpha-amanitin is due to an alteration of RNA polymerase II.

Isolation and characterization of Streptococcus mutans mutants with altered cellular morphology or chain length.

A nitrosoguanidine-induced mutant, designated UAB90, of Streptococcus mutans PS14 (serotype c) strain UAB62, was identified on the basis of its unique colony morphology and isolated on brain heart infusion agar. Other mutants displaying similar colony morphologies on brain heart infusion agar were isolated after ethyl methane sulfonate mutagenesis of UAB62 and S. mutans 6715 (serotype g) strains UAB61 and UAB66, and these were found to exhibit abnormalities in cell morphology, chain length, or both. All mutants were examined further for (i) adherence and aggregation after overnight growth in medium containing sucrose, (ii) growth and aggregation in brain heart infusion broth and medium containing glucose, (iii) aggregation of nongrowing cells in the presence of 2 mg of sucrose per ml or 200 mug of dextran per ml, (iv) dextranase activity, and (v) ease of cell lysis. Mutants isolated included several with long chains of enlarged cocci, and two of these strains, UAB261 and UAB433, along with UAB90, were more susceptible to cell lysis than were their parents. UAB261, isolated from UAB62, maintained other parental characteristics, whereas UAB433, isolated from UAB66, lost its ability to aggregate in the presence of either sucrose or dextran. The "fragile" mutant UAB90 was particularly useful in the isolation of high-molecular-weight DNA for early gene cloning experiments by our laboratory. Two other cell morphology mutants, UAB272 from UAB66 and UAB289 from UAB61, did not lyse better than their parents, but both lacked measurable dextranase activity. A final mutant, UAB276 from UAB66, displayed only increased chain length without apparent cell morphology variations. Chains produced by this mutant were up to 10 times longer than those produced by UAB66. UAB276 lysed slightly less well than its parent but retained all other wild-type characteristics examined.

SELF‐STERILE AND MATURATION‐DEFECTIVE MUTANTS OF THE HOMOTHALLIC ALGA, CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE).1

ABSTRACTHomothallic sexual reproduction in Chlamydomonas monoica Strehlow culminated in the formation of mature, chloroform‐resistant zygospores (zygotes) in clonal culture. Early in the zygote maturation process, a distinctive “primary zygote wall” was released into the culture medium where it remained stable for at least several days. This wall appeared as a rigid, darkly‐outlined, and often multilayered structure, as viewed by phase contrast microscopy. From a sample, of 2500 individual clones isolated after ethyl methanesulfonate mutagenesis, five maturation‐defective strains (zym) produced abnormal zygotes which failed to release a primary zygote wall, failed to develop the normal reticulate zygospore wall, and disintegrated within five days. These strains were utilized to identify additional mutants which were sexually competent, but self‐sterile (het). Mixed cultures of the zym and het mutant strains were found to contain numerous, fully‐matured, chloroform‐resistant zygospores and discarded primary zygote walls. In combination, the two types of mutants provided a useful system for the selective recovery of heterozygous zygospores, thus facilitating genetic studies on a homothallic Chlamydomonas.

Cyclic AMP may not be involved in catabolite repression in Saccharomyes cerevisiae: evidence from mutants capable of utilizing it as an adenine source.

Mutants able to utilize 5'-AMP or cyclic AMP as the adenine source were isolated from an ade6 ade10 double mutant by ethyl methane sulfonate mutagenesis. A single amp1 mutation, primarily selected on 5'-AMP medium, confers the phenotype for utilization of exogenous 5'-AMP as the adenine source. From the ade6 ade10 amp1 triple mutant, a mutant able to utilize cyclic AMP was isolated, and the mutant phenotype was proven to be due to the simultaneous occurrence of triple mutations designated as cam1, cam2, and cam3. The cam3 mutation, but not cam1 or cam2, also confers the phenotype for utilizing 5'-AMP, the same phenotype as the amp1 mutation. All of these mutations are recessive to the respective wild-type counterparts. Cells having the ade6 ade10 amp1 cam1 cam2 cam3 genotype showed significant ability to take up exogenous cyclic AMP, whereas no differences were observed in cyclic AMP phosphodiesterase activity in comparison with that of the original strains used in the mutant isolation. Since glucose severely repressed galactokinase synthesis in the constitutive GAL81 mutant having the ade6 ade10 amp1 cam1 cam2 cam3 genotype, irrespective of the presence or absence of cyclic AMP in the medium, it was suggested that cyclic AMP is not involved in the mechanism of catabolite repression in Saccharomyces cerevisiae. It does, however, have a stimulative effect on the galactokinase synthesis in the GAL81 mutant in the absence of glucose.

Prolactin-deficient variants of GH3 rat pituitary tumor cells: linked expression of prolactin and another hormonally responsive protein in GH3 cells.

GH3 cells normally synthesize and secrete two pituitary polypeptide hormones, prolactin and growth hormone. From an ethyl methane sulfonate-mutagenized population, prolactin low-producing variants have been isolated at a frequency near 20%. Intracellular prolactin synthesis in the variants was reduced 40- to 100-fold compared to wild-type cells while growth hormone synthesis varied less than 2-fold. This decrease was paralleled by a decrease in intracellular preprolactin mRNA. Although reduced, prolactin synthesis was still repressible by glucocorticoids. There was a coordinate loss of expression of p21, a thyroid and glucocorticoid hormone-regulated protein, in GH3 cells, whereas the synthesis and regulation of other hormonally responsive proteins were unimpaired in the variants. Since p21 expression was coordinately regained in a high-producing prolactin revertant cell, expression of the two proteins is tightly coupled in GH3 cells. The stability of the low-producing phenotype differed among variants. One (B2) gave rise to revertants at about 20% frequency even after two rounds of subcloning, whereas another (B3) was more stable in that only 1 weak revertant was found in 47 subclones. The reversion frequency of B3 cells was also measured at less than 0.5%. Unmutagenized GH3 cells were phenotypically stable in that no prolactin-deficient variant was found among 57 subclones. Since variants were ony found after ethyl methane sulfonate mutagenesis, the DNA alkylating agent appears to have promoted an epigenetic change in pituitary gene expression.

Prolactin-deficient variants of GH3 rat pituitary tumor cells: linked expression of prolactin and another hormonally responsive protein in GH3 cells.

GH3 cells normally synthesize and secrete two pituitary polypeptide hormones, prolactin and growth hormone. From an ethyl methane sulfonate-mutagenized population, prolactin low-producing variants have been isolated at a frequency near 20%. Intracellular prolactin synthesis in the variants was reduced 40- to 100-fold compared to wild-type cells while growth hormone synthesis varied less than 2-fold. This decrease was paralleled by a decrease in intracellular preprolactin mRNA. Although reduced, prolactin synthesis was still repressible by glucocorticoids. There was a coordinate loss of expression of p21, a thyroid and glucocorticoid hormone-regulated protein, in GH3 cells, whereas the synthesis and regulation of other hormonally responsive proteins were unimpaired in the variants. Since p21 expression was coordinately regained in a high-producing prolactin revertant cell, expression of the two proteins is tightly coupled in GH3 cells. The stability of the low-producing phenotype differed among variants. One (B2) gave rise to revertants at about 20% frequency even after two rounds of subcloning, whereas another (B3) was more stable in that only 1 weak revertant was found in 47 subclones. The reversion frequency of B3 cells was also measured at less than 0.5%. Unmutagenized GH3 cells were phenotypically stable in that no prolactin-deficient variant was found among 57 subclones. Since variants were ony found after ethyl methane sulfonate mutagenesis, the DNA alkylating agent appears to have promoted an epigenetic change in pituitary gene expression.

Influence of experimental conditions and DNA repair ability on EMS-induced mutagenesis and DNA binding in Escherichia coli K12 : Comparison with mammalian cell mutagenesis

In continuation of previous experiments aimed at quantitatively comparing the magnitude of ethyl methanesulfonate (EMS)-induced DNA binding and mutagenesis in bacterial and mammalian cells, the present studies were undertaken to determine the influence of various experimental conditions on EMS-induced changes in E. coli K12/343/113. The sensitivity to EMS mutagenesis in this strain was assayed for various genetic endpoints, namely gal +, MTR r, NAL r, VAL r and arg +. EMS mutagenesis and DNA binding in the repair-proficient wild-type was, furthermore, compared to that in derivatives deficient in excision repair (Δ uvrB101) and post-replication repair ( recA13). Attempts were, finally, undertaken to determine the shape of the dose-response curve for the most sensitive genetic systems found, namely NAL r and VAL r mutations, at relatively low EMS-exposure conditions of 0–7 mM for 120 min at 37°C. From the present experiments and from previous results obtained under identical EMS treatment conditions in E. coli 343/113 and in the mammalian cell lines V79 and L5178Y, the following conclusions can be drawn: (i) At EMS exposure concentrations ranging from 2.5 to 50 mM and treatment periods of 120 min at 37°C in buffer pH 7.2, the levels of DNA alkylation at termination of the treatment are similar in E. coli and mammalian cells and increase from 1.7 × 10 −4 to 1.3 × 10 −3 ethylations per nucleotide. (ii) The presence of nutrients such as l-arginine, D-glucose and fetal calf serum, during EMS treatment of stationary E. coli cells does not influence the level of DNA alkylation but greatly increases EMS-induced mutation yields, probably because of the accumulation of these nutrients into the thereby leading to increased capability of genetic fixation and/or phenotypic expression of the premutational DNA changes. (iii) The mammalian cells are more sensitive to the killing actions of EMS than E. coli repair-proficient cells (survival at 10 mM of 12 and 100%, respectively), which indicates that the genetic and/or physiologic target for inactivation of colony-forming ability is larger in the mammalian cells than in bacteria, or alternatively that these cells have lower ability to repair EMS-induced DNA lesions. (iv) The 2 new mutation systems tested in the present series of experiments, namely NAL r and VAL r mutations appear to be quite comparable to those tested in mammalian cells, such as thioguanine resistance (HGPRT −) and BUdR resistance (TK −/−), in the sense that at low doses, i.e. EMS-exposure concentrations of up to 7 mM for 120 min at 37°C in buffer, pH 7.2, the induction kinetics in all systems are compatible with a linear relationship between dose and mutation frequency, with no indication of a threshold. In the case of NAL r in E. coli and HGPRT − in V79 mammalian cells the Lowest Effective Concentrations of EMS are 1–2 mM for treatment periods of 120 min at 37°C. It remains to be determined if the same EMS-induced DNA adducts are responsible in both cell types for the genetic effects observed; further comparative experiments are also required to assess the general suitability of bacteria in quantifying mutagenic and tumor-initiating potency of chemicals.

Increased levels of threonyl-tRNA synthetase in a borrelidin-resistant Chinese hamster ovary cell line.

The growth of Chinese hamster ovary cells in medium containing reduced concentrations of threonine is inhibited by borrelidin, a macrolide antibiotic. Borrelidin-resistant clones have been isolated after ethyl methanesulfonate mutagenesis. One clone, 1C-1, has a 3-fold increased level of threonyl-tRNA synthetase [L-threonine:tRNAThr ligase (AMP-forming), EC 6.1.1.3] as determined by both activity measurements and antiserum titrations. The levels of four other aminoacyl-tRNA synthetases and of tRNAThr are the same in strain 1C-1 and in the wild-type parent. The phenotype of increased threonyl-tRNA synthetase activity is recessive to wild type in cell hybrids.

Aminoglycoside-resistant mutants of Pseudomonas aeruginosa deficient in cytochrome d, nitrite reductase, and aerobic transport.

Two gentamicin-resistant mutants of Pseudomonas aeruginosa PAO 503 were selected after ethyl methane sulfonate mutagenesis. Mutant PAO 2403 had significantly increased resistance to aminoglycoside but not to other antibiotics. Mutant PAO 2402 showed a similar spectrum of resistance but of lower magnitude. Both mutants showed no detectable cytochrome d and had a high frequency of reversion to a fully wild-type phenotype. PAO 2403 had a marked decrease and PAO 2402 had a moderate decrease in nitrite reductase activity. Both mutants had reduced uptake of gentamicin and dihydrostreptomycin. Mutant PAO 2403 showed a general decrease in transport rate of cationic compounds, whereas mutant PAO 2402 had only deficient glucose transport. Both mutants showed enhanced rates of glutamine transport and no change in glutamic acid transport. Other components of electron transport and oxidative phosphorylation were normal. These mutants involve ferrocytochrome C551 oxidoreductase formed only on anaerobic growth but illustrate transport defects in aerobically grown cells.

Genetic analysis of temperature-sensitive embryogenesis mutants in Caenorhabditis elegans

A large set of temperature-sensitive ( ts) embryonic arrest mutants in the nematode Caenorhabditis elegans has been isolated. Following ethylmethane sulfonate mutagenesis, ts mutants were identified from 10,000 segregated clones by replica plating from 16 to 25°C. At least 54 independent ts developmental mutants showed embryonic arrest when the appropriate developmental stage was shifted up. These represent 28% of the independent developmental mutants from 294 ts clones from 155 mutagenized animals. Initial characterization of this subset of emb mutants is reported. Most of them arrest in middle or late embryogenesis with abnormal terminal phenotypes. Additional nonembryonic arrest stages (depending on shift-up regimen) suggest that many emb genes are also required for pre- or postembryonic development. For most of the emb mutants parental gene expression is sufficient for embryonic development at 25°C. Based on complementation analysis of 37 new emb mutants together with 32 isolated by other investigators, 25 new emb genes are reported. This brings the total of embryonic arrest genes defined in C. elegans to 54. The new genes have been mapped approximately. In the present set, 10 emb genes map in the middle of linkage group III. The frequency of occurrence of second alleles gives one estimate (by Poisson analysis) for the number of genes required for embryogenesis of about 200. A second estimate is obtained from the fraction of 28% emb mutants among all ts lethal mutants in the screen; for an estimated 2000 essential genes in C. elegans, this corresponds to 560 genes required for embryogenesis. A small subset of highly mutable genes is also described. Results of the present screen for emb mutants are compared to those of others. Several ts mutants gave complex linkage results (apparently not due to double mutations), suggesting chromosome rearrangements.

Isolation and preliminary characterization of bleomycin-resistant mutants from Chinese hamster ovary cells.

Stable mutants resistant to an anticancer antibiotic, bleomycin-A2, were selected in Chinese hamster ovary (CHO) cell either spontaneously or after ethylmethane sulfonate mutagenesis. Fluctuation analysis showed that bleomycin resistance occurs in CHO at a rate of 6.50--6.58 x 10(-7) mutations per cell per generation. Bleomycin-A2-resistant cell lines exhibited increased resistance to bleomycin analogs--bleomycin-A5, -B2, -B4, and pepleomycin. Colchicine, mitomycin C, and ultraviolet light irradiation inhibited colony formation equally in CHO cells and in bleomycin-resistant mutants. Cell-cell hybridization tests showed that bleomycin-resistance behaves as a dominant trait. Bleomycin-inactivating activity in the mutant cell extracts was three to fourfold higher than that in extracts of the parental CHO cell.

ICR-191 and ethyl methanesulfonate induced mutagenesis at the immunoglobulin locus in the Y5606 cultured myeloma cell line

The Y5606 mouse tumor synthesizing an IgG 3,λ immunoglobulin (Ig) was adapted to continuous growth in tissue culture. The spontaneous mutation rate at the Ig locus (∼3 × 10 −5/cell/generation) in this cell line was found to be less than that in other cultured mouse myeloma lines. Treatment with either ICR-191 or ethyl methanesulfonate (EMS) increased the mutation rate approx. 100-fold. Spontaneous and ICR-191 induced mutants were synthetic variants that is they synthesized either heavy (H) or light (L) chains alone instead of the H and L chains synthesized by the parent. Following EMS treatment assembly variants which were synthesizing structurally altered H chains were isolated in addition to synthetic variants. The assembly varriants appear to be a unique consequence of EMS mutagenesis.

Lyt-2-/Lyt 3- variants of a cloned cytolytic T cell line lack an antigen receptor functional in cytolysis.

To investigate the role of Lyt-2 and Thy-1 in cytolysis, we have generated, by ethyl methanesulfonate mutagenesis and selection, variants of the cloned cytolytic T lymphocyte line L3 that specifically lack either Lyt-2 or Thy-1. An analysis of these variants indicates that neither Lyt-2 nor Lyt-3 is responsible for the lethal hit, but suggests that Lyt-2 and/or Lyt-3 are required for an antigen receptor functional in cytolysis. The data also suggest that the expression of Lyt-3 on the cell surface is not independent of the expression of Lyt-2. Finally the data indicate the Thy-1 plays no role in cytolysis.

DNA polymerase alpha mutants from a Drosophila melanogaster cell line.

Aphidicolin, a tetracyclic diterpenoid antibiotic, is a specific inhibitor of DNA synthesis in vivo and DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) alpha of eukaryotic organisms. After ethyl methanesulfonate mutagenesis, we have recovered mutants of Drosophila melanogaster Schneider cell line no. 2 that grow at concentrations of aphidicolin that completely inhibit wild-type cells. The DNA polymerase alpha from one of these mutants, aph-10, is much more resistant to inhibition by the drug; the apparent Ki of the wild-type enzyme is 12 nM aphidicolin, whereas the apparent Ki of the aph-10 polymerase is more than 100 nM. (The apparent Km for dCTP is the same for both enzymes.) Another mutant, aph-13, overproduces DNA polymerase alpha at least 8-fold. The DNA polymerase of this mutant has the same apparent Km and Ki for dNTPs and aphidicolin as does wild-type polymerase.

Characterization of an autosomal rudimentary-shaped wing mutation in Drosophila melanogaster that affects pyrimidine synthesis.

A new autosomal mutation, rudimental (ral), which causes rudimentary-shaped wings in Drosophila melanogaster, has been isolated following ethyl methanesulfonate (EMS) mutagenesis. The wing phenotype of rudimental is identical to that of the X-linked rudimentary (r) mutation, which affects the first three enzymes in the pyrimidine biosynthetic pathway. The autosomal mutant maps very close to ebony (3-70.7) at 70.42 on the right arm of chromosome 3. Analysis of the enzyme activities of orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase) indicates that the rala26a allele has less than wild-type activity for both enzymes. This result is discussed in light of the fact that the OPRTase and ODCase activities are part of an enzyme complex, as are the carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which are encoded by the complex rudimentary locus. We suggest that rudimental is also a complex lucus.

An Escherichia coli mutant refractory to nitrosoguanidine mutagenesis.

A newly-isolated Escherichia coli mutant suffers only about 10% as many mutations as normal strains on exposure to nitrosoguanidine. The responsible mutation, inm-1, maps at approximately minute 79 in the current E. coli genetic map. The mutant is normal for overall growth, nitrosoguanidine lethality, spontaneous mutagenesis, ultraviolet light lethality and mutagenesis, ethyl methanesulfonate lethality and mutagenesis, and the adaptive repair induced by alkylating agents. The existence of this mutation proves that nitrosoguanidine mutagenesis is not merely the result of reactions between the chemical and DNA, but requires specific cellular function(s), and underscores the peculiarity of nitrosoguanidine as a mutagen.