Studies on three mutagen-sensitive mutants of mouse L5178Y cells. I. Characterization of the hybrids between L5178Y and mutagen-sensitive mutants

Three mutagen-sensitive mutants, MS-1, M10 and Q31, have been isolated from mouse L5178Y cells. MS-1 cells are sensitive to methyl methanesulfonate (MMS), M10 cells are cross-sensitive to X-rays, MMS and 4-nitroquinoline 1-oxide (4NQO), and Q31 cells are cross-sensitive to UV and 4NQO. Lines resistant to 6-thioguanine (TGr) and 5-bromo-2'-deoxyuridine (BUr) were isolated from L5178Y and these three mutagen -sensitive mutants. All the TGr lines were sensitive to 5-bromo-2'-deoxyuridine and HAT medium and all the BUr lines were sensitive to 6-thioguanine and HAT medium. The hybrids homozygous for the mutagen-sensitive markers showed nearly the same sensitivity to UV, 4NQO, X-rays and MMS as their parental TGr and BUr lines. The hybrids constructed by fusing L5178Y BUr and TGr lines from each of MS-1, M10 and Q31 displayed the normal UV, X-ray and MMS resistancy of L5178Y cells. Thus the UV-, X-ray- and MMS-sensitive markers in MS-1, M10 and Q31 were recessive in somatic cell hybrids. The 4NQO-sensitive phenotype, however, behaved codominantly in somatic cell hybrids.     

UV- and X-ray-sensitive double mutants of mouse L5178Y cells are synergistically more sensitive to 4-nitroquinoline-1-oxide than is either of the single mutants

The X-ray-sensitive mutant M10 and the UV-sensitive mutant Q31 of mouse lymphoma L5178Y cells are both sensitive to killing by 4-nitroquinoline-1-oxide (4NQO). Since cell hybridization experiments showed that the 4NQO sensitivities in M10 and Q31 cells behaved as codominant traits (Shiomi et al., 1982c), it is not possible to determine by complementation test whether the M10 and the Q31 mutations responsible for 4NQO sensitivities are allelic. We have obviated this difficulty by selecting double mutants that are sensitive to both X-rays and UV. From X-ray-sensitive M10 cells, two UV-sensitive mutants (XU 1 and XU 2) were isolated by a cell-suspension spotting method. XU 1 and XU 2 were found to belong to the same complementation group as Q31 (group I). Double mutants XU 1 and XU 2 were 30-37-fold more sensitive to 4NQO than parental L5178Y cells, whereas the single mutants M10 and Q31 were only 6-8-fold more sensitive to 4NQO than L5178Y cells in terms of D10 values (dose required to reduce survival to 10%). These results show that the M10-Q31-double mutations enhance 4NQO sensitivity synergistically, indicating that the M10 and the Q31 mutations relevant to 4NQO sensitivities are non-allelic. The implications of this finding are discussed.     

DNA repair in a UV-sensitive mutant of of mouse cell line

A UV-sensitive mutant, Q31, isolated from mouse-lymphoma L5178Y cells, was studied for excision and post-replication rerpairs. A nearly equal number of UV endonuclease-sensitive sites was induced by UV in L5178Y, Q31, and human Raji cells. L5178Y cells irradiated with 10 J/m² removed 18% of sensitive sites from DNA during incubation for 24 h, and Q31 cells removed 3% of the sites, a fraction less than the limit of detection, whereas Raji cells eliminated about 60% of the sites. These results indicate that mouse-lymphoma cells are capable of excision repair to a limited extend as compared with human cells and that mutant Q31 cells are essentially devoid of dimer excision. The newly synthesized DNA was of smaller size in UV-irradiated and unirradiated Q31 cells than that in the corresponding L5178Y cells, but the DNAs in both strains increased to comparable sizes after a 2-h chase.     

Mutation induction by tritiated water and effects of deuterium oxide in cultured mouse leukemia cells

Induction of mutation to 6-thioguanine resistance was studied in L5178Y mouse leukemia cells after exposure to low-dose-rate gamma rays or tritiated water at dose rates of approximately 0.025 to 0.4 Gy/hr for 20 hr in the presence or absence of 45% (v/v) deuterium oxide. The effect of acute gamma-ray exposure was also examined. A higher frequency of induced mutations was observed after tritium beta rays than after gamma rays, both at equivalent doses and cell survival. Deuterium oxide enhanced the mutation induced by gamma rays and tritium beta rays but did not affect the survival-mutation correlation of the two radiations.     

Chromosomal hypersensitivity in mutant M10 and Q31 mouse cells exposed to ultraviolet radiation (UV) and 4-nitroquinoline-1-oxide (4NQO)

2 mutant mouse cells M10 and Q31 were examined for chromosomal aberrations induced by ultraviolet radiation (UV) and 4-nitroquinoline-1-oxide (4NQO), as compared with mouse lymphoma L5178Y cells. Q31 cells are UV- and 4NQO-sensitive cells isolated from L5178Y cells. M10 cells are similar but are sensitive to ionizing radiation and 4NQO. After treatment with UV or 4NQO, chromatid-type aberrations in these cell strains were induced more frequently in the first mitotic cells, at late fixation times. After UV exposure (2.4 J/m2), the maximal frequencies of chromatid-type breaks in Q31 cells were about 5 times higher than in L5178Y cells. In M10 cells such breaks were only as frequent as in L5178Y cells. After 4NQO treatment (50 ng/ml) the frequencies of chromatid-type breaks in M10 and Q31 cells were significantly higher than in L5178Y cells. From these results and those of previous studies (Takahashi et al., 1982), M10 cells may be considered hypersensitive to gamma-rays and 4NQO, but not to UV, and thus react similarly to L5178Y cells. The hypersensitivity of M10 cells to 4NQO may result from a defect in the ionizing-radiation repair mechanism as has been suggested to occur in ataxia telangiectasia (AT) cells. Q31 cells are hypersensitive to UV and 4NQO, but not to gamma-rays. Q31 cells may be considered to be deficient in a UV-like repair pathway. In conclusion, characteristics of murine M10 and Q31 cells are compared with those of human AT and xeroderma pigmentosum (XP) cells.     

A mutational assay system for L5178Y mouse lymphoma cells, using hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) -deficiency as marker. The occurrence of a long expression time for mutations induced by X-rays and EMS.

The development of a system for the detection of somatic cell mutation to hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) (EC 2.4.2.8) deficiency in L5178Y mouse lymphoma cells is described. The selection of mutant cells was not influenced by the concentration of the selective agent 6-thioguanine (6-TG). In addition, all the mutants selected, spontaneous as well as induced ones, showed a complete loss of HGPRT activity. In reconstruction experiments, in which mutant cells were mixed with wild-type cells, the recovery of mutant cells was only markedly influenced when wild-type cells were seeded in a cell density ten times higher than the one, 5-10(4) cells/ml, used in subsequent induction experiments. X-irradiation and treatment with ethyl methanesulfonate (EMS) increased in the mutation rate above the spontaneous background. A clear-cut dose-dependent mutagenic effect after exposure to X-rays was measured. The rate of induced mutations at the HGPRT locus in lymphoma cells was 1-3-10(-7) per R, as determined after exposures of 200, 300, 400, 500 and 600 R. The time the cells needed to express their mutations was much longer than 48 h. Further study of this phenomenon showed that the optimal expression time for TGr-resistant mutants in L5178Y cells was 6 to 7 days. No indication for a dose-dependent effect on the optimal expression of the mutants was found.     

A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells

Microtitration methods for assaying cell survival and mutation frequency to ouabain resistance, 6-thioguanine resistance and 1-β-d-arabinofuranosyl cytosine resistance in L5178Y mouse lymphoma cells were compared to the standard agar cloning technique. The two methods gave essentially similar results for untreated cells, and after treatment with ethyl methanesulphonate and 4-nitroquinoline 1-oxide. Potential advantages of the microtitration method as a routine assay system are discussed.     

The mutagenic potency of 1,8-dinitropyrene in cultured mouse lymphoma cells

Although non-toxic, 1,8-dinitropyrene (1,8-DNP) was mutagenic for mouse lymphoma L5178Y cells when assayed for induced resistance to 6-thioguanine, methotrexate, ouabain and 1-β-D-arabinofuranosyl cytosine. In bacteria, nitropyrenes are potent inducers of frame-shift mutations, and the induction of ouabain-resistant mutants, believed to be due to base-pair substitutions, suggests that the mechanism of action may be different in mouse cells and bacteria. Long treatment times were required to detect 1,8-DNP-induced mutants in L5178Y cells, suggesting the possibility of an inducible activation system. 4-Nitroquinoline 1-oxide was both toxic and mutagenic to these same 4 mutation assays after short (2 h) treatment times. The dilemma that exists when comparing the mutagenic potential of test chemicals when concentration of mutagen, treatment times and toxicity are markedly different, is discussed.     

Studies on three mutagen-sensitive mutants of mouse L5178Y cells. II. Complementation analyses between two methyl methanesulfonate-sensitive mutants and between two 4-nitroquinoline-1-oxide-sensitive mutants

Three mutagen-sensitive mutants, MS-1, M10 and Q31, were isolated from mouse L5178Y cells. MS-1 cells are sensitive to methyl methanesulfonate (MMS), M10 cells are cross-sensitive to X-rays, MMS and 4-nitroquinoline-1-oxide (4NQO); and Q31 cells are cross-sensitive to UV and 4NQO. MMS-, X-ray- and UV-sensitive markers in these mutants behaved recessively in hybrids between pairs of these mutants as in hybrids between L5178Y and these mutants as reported before (Shiomi et al., 1982b). Complementation analyses were carried out by forming hybrids between two MMS-sensitive mutants (MS-1 and M10) and between two 4NQO-sensitive mutants (M10 and Q31). MMS and 4NQO survivals were measured in these hybrid cells. MS-1 and M10 were found to belong to different complementation groups for MMS-sensitive phenotypes. The hybrid clones between M10 and Q31 were as sensitive to 4NQO as each of the mutants, indicating codominance of 4NQO sensitivity in these mutants. The hybrids constructed with L5178Y and three mutants were stable as to their chromosome constitution for 100 days of cultivation without selective pressure. From the segregation studies on these hybrids, it is concluded that neither the X-ray-sensitive mutation in M10 nor the UV-sensitive mutation in Q31 is located on the X chromosome.     

Characteristics of γ-ray-induced chromosomal aberrations in mutagen-sensitive mutants of L5178Y cells

We have examined the chromosomal radiosensitivities of an ionizing-radiation- and MMS-sensitive mutant (M10), and a UV- and 4NQO-sensitive mutant (Q31), isolated from mouse lymphoma L5178Y cells, with regard to killing effects. In the first mitoses after 100 R γ-irradiations, it was found that M10 cells were highly radiosensitive in terms of chromosomal aberrations accompanying longer mitotic delay (3 h); the frequencies of both chromatid-type and chromosome-type aberrations were, respectively, about 7 and 4 times higher than that of wild-type L5178Y cells. Furthermore, chromatid exchanges, particularly triradials, isochromatid breaks with sister union, and chromatid gaps and breaks were markedly enhanced at G₁ phase of M10 cells. In contrast, the chromosomal radiosensitivity of Q31 cells after 100 R irradiation was similar to that of L5178Y cells. On the other hand, spontaneous aberration frequencies (overall breaks per cell) of M10 and Q31 cells were, respectively, 5.1 and 2.2 times higher than that of wild-type L5178Y cells. The chromosomal hypersensitivity to γ-rays in M10 cells is discussed in the light of knowledge obtained from ataxia telangiectasia cells.     

UV-induced mutagenesis at the hypoxanthine—guanine phosphoribosyl transferase locus in two L5178Y mouse lymphoma cell strains with different UV sensitivities

Two strains of L5178Y mouse lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S), differ markedly in their sensitivity to 254 nm UV radiation (D₀ = 0.7 and 5.5 J/m²; n = 6.0 and 2.0 for LY-R and LY-S cells, respctively). In this study, the frequency o hypoxanthine-guanine-phosporibosyl-transferase-deficient mutants was determined, using 6-thioguanine (TG) as a selective agent, in populations of LY-R and LY-S cells exposed to various fluences of UV radiation. The spontaneous mutation frequency for LY-R cells was (3.7 ± 0.6) × 10^?5 TG^r mutants per viable cell, and the UV induction rate was (2.2 ± 0.8) × 10^?4 TG^r mutants per viable cell, per J/m². Both spontaneous and induced mutantion frequencies were much lower for LY-S cells. The sopntaneous mutation frequency for these cells were too low to make its measurement practicable ( < 0.0013 × 10^?5 TG^r mutants per viable cell). Mutation induction rate was (4.2 ± 2.2) × 10^?7 TG^r mutants per viable cell, per J/m². These differences in mutability do not appear to be due to gene duplication in LY-S cells, or to selective growth disadvantage of LY-S-derived TG-resistant mutants. Possible mechanisms underlying the differences in mutability of LY-R and LY-S cells are considered.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

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.     

Mutation induction by different dose rates of gamma rays in radiation-sensitive mutants of mouse leukemia cells

Induction of cell killing and mutation to 6-thioguanine resistance was examined in a radiation-sensitive mutant strain LX830 of mouse leukemia cells following gamma irradiation at dose rates of 30 Gy/h (acute), 20 cGy/h (low dose rate), and 6.2 mGy/h (very low dose rate). LX830 cells were hypersensitive to killing by acute gamma rays. A slight but significant increase was observed in cell survival with decreasing dose rate down to 6.2 mGy/h, where the survival leveled off above certain total doses. The cells were also hypersensitive to mutation induction compared to the wild type. The mutation frequency increased linearly with increasing dose for all dose rates. No significant difference was observed in the frequency of induced mutations versus total dose at the three different dose rates so that the mutation frequency in LX830 cells at 6.2 mGy/h was not significantly different from that for moderate or acute irradiation.     

The effect of caffeine on post-replication repair and survival in two L5178Y cell lines with different sensitivities to UV irradiation

2 Strains of murine lymphoma L5178Y cells that varied from the point of view of sensitivity to UV irradiation (mean lethal doses: 3.6 and 8.5 J/m2 for L5178Y-R and L5178Y-S cells, respectively) also differed with respect to sensitization by caffeine. L5178Y-S cells were sensitized to UV irradiation by 0.75 mM caffeine, whereas in the same conditions L5178Y-R cells were not sensitized. Sedimentation analysis of the newly synthesized DNA indicated UV-induced gap formation in L5178Y-S cells only. The subsequent gap filling was inhibited by caffeine. Exposure to UV irradiation induced no gaps in L5178Y-R cells. However, when caffeine was added immediately after irradiation, DNA with reduced molecular weight was found in irradiated cells of both strains after a 2-h chase. On the other hand, caffeine inhibited elongation of undamaged DNA strands in neither of the 2 cell strains.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.     

DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation

The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.     

Establishment of a dose-response relationship for reverse mutation at the HPRT (hypoxanthine guanine phosphoribosyl transferase) locus in L5178Y mouse lymphoma cells

L5178 mouse lymphoma cells were treated with the mismatching agent 6-hydroxy-aminopurine (HAP), a base analogue known to produce forward and reverse mutations in bacteria. Mutants with the phenotype deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT) were selected in 6-thioguanine (TG)-containing medium and isolated. Reverse mutations to Hhe HPRT-proficient phenotype oc occuredd both spontaneously and after treatment with ethyl nitrosourea (ENU), which suggested that the initial HAP treatment had induced point mutations at the HPRT locus.

Reconstruction experiments, in which a small number of wild-type cells together with different numbers of mutant cells were seeded in HAT medium, indicated that densities up to 10⁶ cells per ml can be used for the selection of revertants. Optimal expression of induced revertants was obtained two days after treatment.

The dose-response relationship for induction of reverse mutations by ENU appears not to deviate from linearity. The highest revertant frequency observed was 3.3 × 10⁻⁵ at an ENU concentration of 1 mM. The spontaneous reversion frequency per generation — based on 3 spontaneous revertants — was estimated to be 1.3 × 10⁻⁹. All revertants were indistinguishable from the parental wild-type line with respect to the activity as well as the electrophoretic mobility of HPRT.     

X-ray-induced mutation to 6-thioguanine resistance in cultured human diploid fibroblasts

X-ray induced mutation to 6-thioguanine (6TG)-resistance was studied in early passage cultures of human diploid fibroblasts.The appearance of phenotypic induced mutants in irradiated cell populations was linearly related to the number of post-irradiation cell doublings and to the duration of the growth period prior to mutant selection; the maximum yield of X-ray induced mutants was observed when cells surviving radiation had completed 3–4 doublings (6–7 days growth_in non-selective medium.The maximum induced mutation frequency was linearly related to X-ray dose and the mutation rate was estimated to be 3.1 · 10^?7 mutations per viable cell per rad.The data obtained for X-ray induced mutations in cultured human diploid fibroblasts were compared with (a) similar experimental data obtained with established cell cultures and (b) theoretical predictions of X-ray mutation rates in human germ cells.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. I. Five different genes participate in the formation of baseline sister-chromatid exchanges and spontaneous chromosomal aberrations

In a search for cell mutants that show an increase or a decrease in the frequency of baseline sister-chromatid exchanges (SCEs) or spontaneous chromosomal aberrations (CAs), large numbers of mutagen-sensitive clones previously isolated from mouse lymphoma L5178Y cells were analyzed. In addition to two SCE mutants (ES 4 and AC 12) previously reported, three other mutants were identified as an SCE mutant. An ethyl methanesulfonate-sensitive mutant ES 2 and an alkylating agent-sensitive mutant MS 1 exhibited, respectively, 1.4-fold and 1.8-fold higher baseline SCE frequencies than did the parental L5178Y. In contrast, M10, which is sensitive to X-ray and 4-nitroquinoline 1-oxide, showed a reduced frequency of baseline SCEs (0.65-fold). These 5 mutants including ES 4 and AC 12 had 3--9-fold increases in spontaneous CA frequencies. Measurement of baseline SCE formation in inter-mutant hybrids revealed that M10 mutation is dominant, MS 1 and ES 4 mutations are semidominant, and ES 2 and AC 12 mutations are recessive. Because SCE frequencies in hybrids formed between pairs of 4 mutants (ES 2, MS 1, ES 4 and AC 12) were significantly lower than those in the tetraploid mutant cells, these 4 mutants probably belong to different complementation groups. Since M10 behaved dominantly with respect to SCE phenotype, it was not possible to determine by complementation test whether it belongs to a different group from the other mutants. However, the finding that M10 is complemented by other mutants for EMS sensitivity indicates that the M10 mutation is different from the other mutations. From these results, it is concluded that at least 4 different genes participate in the formation of high levels of baseline SCEs. The defects in ES 2, MS 1, ES 4, and AC 12 produce common lesions responsible for the formation of both SCEs and CAs. In contrast, the defect in M10 is associated with a high increase in spontaneous CA frequency, but conversely associated with a decrease in baseline SCE frequency. This suggests that M10 is defective in the process involved in the formation of baseline SCEs.