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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.

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.

26 mutants with increased sensitivity to the lethal effects of mitomycin C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Most of them were about 5-10 times more sensitive in terms of D37 values to MMC than were parental cells. 5 of the MMC-sensitive mutants isolated from independently mutagenized cell populations were further analyzed. They were highly sensitive to the killing by decarbamoyl (DC) MMC, a monofunctional derivative of MMC, but were not sensitive to ultraviolet radiation, X-rays, 4-nitroquinoline-1-oxide or methyl methanesulfonate. These 5 mutants were classified into at least 2 genetic complementation groups. The implication of these mutations in cross-link and mono-adduct repair of DNA damage induced by MMC and DCMMC is discussed.

A multiple mutagen-sensitive mutant (XUM1) of mouse T-cell lymphoma line, L5178Y, is hypersensitive to ionizing radiation, ultraviolet (UV) light, and cross-linking agents (such as mitomycin C). The frequency of transfection for XUM1 cells after exposure to calcium phosphate-coprecipitated pSV2neo DNA was more than 10(4)-fold less effective than that for Ltk-aprt- (LTA) cells. Other transfection methods (DEAE-dextran and polybrene-DMSO) were not effective for L5178Y and XUM1 cells. The transfection-proficient trait of LTA cells was demonstrated to be genetically dominant by examining the the transfection frequency in hybrid clones constructed between XUM1 and LTA cells. To circumvent the problem with XUM1, the LTA genes necessary for transformation processes were introduced into XUM1 cells by constructing hybrids between XUM1 and LTA cells irradiated with X-rays which causes directional chromosome elimination for hybrid cells. Four of 194 hybrid clones tested were transfection-proficient and hypersensitive to UV (XL102, XL107, XL215, and XL216). All four clones were not hypersensitive to X-rays or mitomycin C. The frequencies of transfection for XL102 and XL216 were nearly the same level as that for LTA cells. The efficiency of transfection for XL107 and XL215 was 10 to 100-fold lower than that for LTA cells.

We have isolated 56 UV-sensitive mutant clones from a mouse L51 T/t line of L5178Y cells by a cell suspension spotting method. Five mutants have also been isolated from L51 T/t and L5178Y cells by the method reported by Thompson and coworkers (22). We divided the mutants into two groups, "highly sensitive" and "moderately sensitive" mutants, according to their sensitivity to UV irradiation. Fifty-eight mutants were highly sensitive and three were moderately sensitive to UV. The reconstruction experiments indicate that more than 90% of highly sensitive mutants were recovered by the cell suspension spotting method. Frequencies of recovered mutants highly sensitive to UV increased with increasing dose of mutagens. Recovered mutant frequency reached 10(-2) after treatment with 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (survival 0.2%). Eight UV-sensitive mutants were divided into four complementation groups. These mutants were 2-6 times more sensitive to UV than parental L51 T/t cells in terms of D37 (dose required to reduce survival to 37%). Four representative UV-sensitive mutants which are classified into different complementation groups were examined for their sensitivity to killing by UV, 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), X-rays, and MNNG. All four classes of mutants were found to be cross-sensitive to UV, 4NQO, and MMC, but not sensitive to X-rays and MNNG.

The mutant mouse lymphoma cell M10, which is sensitive to methyl methanesulfonate and ionizing radiation, was compared with the parental L5178Y cells for mutation induction after gamma-irradiation. The rate of induced mutations to 6-thioguanine resistance in L5178Y cells was 2-3 X 10(-7) per R, as determined after exposures ranging from 25 to 500 R. The induced mutation frequency per unit dose per locus in M10 cells was about 4 times higher than that in L5178Y cells at the lower doses of exposure (25-75 R), but it declined sharply at the higher doses of gamma-rays (100-150 R). The rate of induced mutation per unit cell killing in M10 cells was nearly the same as that in L5178Y cells when they were compared at the levels of lower cell killing.

The mouse lymphoma L5178Y cell line and its radiosensitive variants M10 and LX830 were examined for DNA synthesis after X-irradiation. The dose-response curves show that the rates of DNA synthesis immediately after exposure are reduced in a dose-dependent fashion and that the extents of reduction in these 3 cell lines are similar to one another. But a difference was observed in the recovery of DNA synthesis with time of incubation. The recovered levels in M10 and LX830 cells were much higher than those in L5178Y cells at high doses of X-rays. These results are discussed in relation to radioresistant DNA synthesis in ataxia telangiectasia cells.

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.

Two-methyl-methanesulfonate-sensitive strains have been isolated, one of which, M10, was cross-sensitive to X-rays as reported before. Sensitivities of parental L5178Y, M10, and newly isolated MS-1 cells to various mutagens were examined. Mutagens tested were UV, X-rays, 4-nitroquinoline 1-oxide (4NQO), caffeine and alkylating agents; methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and mitomycin C (MMC). In terms of D37 values, M10 cells were 2.5-7 times more sensitive to EMS, MMC and 4NQO as well as to MMS and X-rays than were parental L5178Y cells, while the new mutant MS-1 was about 3 times more sensitive to MMS, EMS, MMC and caffeine than were parental cells. The characteristics in sensitivities of M10 cells to X-rays, alkylating agents and 4NQO resemble resemble some ataxia telangiectasia cells; and MS-1 cells to alkylating agents and caffeine are novel among mammalian cell mutants so far reported. Sensitivity of M10 cells to mutagens has so far been stable for one year, and that of MS-1 cells was stable for 6 months in continuous culture.

A mouse-cell mutant sensitive to methyl methanesulfonate (MMS), X-rays, ultraviolet light (UV), and crosslinking agents was selected using the replica plating and cell suspension spotting methods. This mutant (XUM1) is a mitomycin C-sensitive derivative of previously reported XU1, a mutant sensitive to MMS, X-rays and UV. Since XU1 is highly susceptible to the lethal effect of 4-nitroquinoline-1-oxide (4NQO), XUM1 is also hypersensitive to 4NQO. Growth inhibition area tests showed that low concentrations of mutagens were detected with the multiple mutagen-sensitive mutant XUM1. Hence XUM1 cells will be useful in detecting with high sensitivity a wide range of mutagens and carcinogens which mimic X-rays, UV and crosslinking agents.

Three X-ray-sensitive mutants (LX821, LX827 and LX830) have been isolated from mouse-lymphoma L5178Y cells. These mutants are much more sensitive to the lethal effects of ionizing radiation than the parental L5178Y cells but are as resistant to ultraviolet radiation as L5178Y cells. We have previously isolated a mutant M10 that is sensitive to methyl methanesulfonate (MMS) and cross sensitive to ionizing radiation and 4-nitroquinoline 1-oxide (4NQO). Unlike M10 cells, newly isolated mutants were not sensitive to MMS and were less sensitive to 4NQO. These results indicate that new mutants may be deficient in the repair of DNA damage specific to ionizing radiation. LX821 and LX827 cells were concomitantly resistant to 5-bromodeoxyuridine, whereas LX830 cells were not.