Molecular analysis of mutation at the hprt locus of Chinese hamster V79 cells induced by ethyl methanesulphonate and mitomycin C

Mutation at the hprt locus of Chinese hamster V79 cells were induced by treatment with ethyl methanesulphonate (EMS), considered primarily a point mutagen and mitomycin C (MMC), a potent clastogen. EMS gave a dose-dependent induction of mutants while MMC induced a poor mutagenic response. Mutations were analysed using Southern and Northern blotting.Analysis of 9 EMS-induced and 4 spontaneous mutants yielded no detectable alterations in the hprt locus after digestion of DNA with 6 restriction enzymes. Mutants without detectable changes carried presumptive point mutations. In contrast, 4 out of 12 MMC-induced mutants had detectable alterations. 2 of these appeared to have lost the entire hprt gene while the other 2had prodable partial deletions. For these 4 deletion mutants no hprt mRNA was detected. 3 MMC-induced and 1 EMS-induced mutants had reduced levels of hprt mRNA. All the other mutants showed normal levels of hprt mRNA and the message detected was always of the correct size.It is suggested that the poor mutagenic response induced by MMC may be due to the lethal nature of large deletions involving both the hemizygous hprt locus and adjacent essential genes. This may lead to an underestimate of the mutagenicity of clastogenic agents such as MMC in the V79 HPRT mutation assay.     

Isolation and characterization of spontaneously occurring mutations at the HPRT locus in V79 Chinese hamster cells

The aim of the present investigation was to screen for rare types of spontaneously occurring mutational events in order to provide information on the organization of the mammalian genome. For this purpose a hierarchical sequence of analyses is used with a first step utilizing a forward reverse mutation approach. The present paper deals with the characterization of 22 isolated mutants from 2 groups, 11 spontaneously appearing mutants and, in comparison, 11 ethyl methanesulfonate-induced mutants at the HPRT locus in V79 Chinese hamster cells, by means of reverse mutation analyses using selection with medium containing L-azaserine. Nine out of the 11 mutant clones of each group could be reverted either spontaneously or induced by treatments with ethyl nitrosourea (ENU), ICR191 or 5-azacytidine (5AC), which indicates that they were caused by point mutations. Two of the revertible mutant clones of spontaneous origin were found to be resistant to HAT but not HAsT medium. These 2 6TGrHATr mutants were the only mutants isolated which could be affected by 5AC with a significant increase in reversion frequency. Chromosome aberration analysis did not indicate any enhancement in aberration frequency in the X-chromosome by 5AC treatment. Studies on the mutagenicity at the OUA locus indicated that the 5AC- and ENU-induced mutation frequencies in these 2 mutants were comparable to the effects in the parent wild-type cell line. Their cellular incorporation of 3H-hypoxanthine was enhanced in the presence of aminopterin, but decreased with L-azaserine indicating that they were phosphoribosyl pyrophosphate (PRPP) mutants. On the basis of these results, it is hypothesized that reversion of these 2 6TGrHATr mutants may occur by a gene amplification mechanism and that this process may be facilitated by 5AC treatment.     

Molecular analysis of spontaneous and ethyl methanesulphonate-induced mutations of the hprt gene in hamster cells

Independent spontaneous or ethyl methanesulphonate (EMS)-induced mutants lacking HPRT enzyme activity were analysed for changes in hprt gene structure. Of 21 spontaneous mutants, 6 had total gene deletions, 2 had partial gene deletions, and 13 were indistinguishable from wild-type by Southern analysis. In contrast a sample of 23 EMS-induced mutants, each of which showed potentially interesting characteristics (e.g. high reversion frequency, X-chromosome rearrangement), showed no detectable hprt gene changes. RNA isolated from 59 mutants with presumptive point mutations (13 spontaneous, 46 EMS-induced) was analysed on dot blots for changes in the amount of hprt mRNA. A wide range of mRNA levels was found, from mutants with undetectable amounts to those with more than wild-type amounts. However, Northern blots of all these mutant RNAs revealed only one (EMS-induced) mutation with a change in hprt mRNA size. Taken with our previously-published data on these mutants, it is argued that they represent a broad range of mutational types, and that the hprt gene mutation system provides a sensitive means of distinguishing mutational spectra of different DNA-damaging agents.     

Effects of L-ascorbic acid on the mutagenicity of ethyl methanesulfonate in cultured mammalian cells.

The effects of L-ascorbic acid (AsA) on the mutations induced by ethyl methanesulfonate (EMS) were examined by means of the 6-thioguanine (6TG)-resistant mutation assay and chromosome aberration assay in cultured Chinese hamster V79 cells. When cells were treated with EMS at various concentrations in the presence of 100 micrograms/ml AsA, EMS-induced 6TG-resistant mutations were reduced about one third or one fourth. EMS-induced chromosome aberrations were also reduced by AsA. These reductions in the mutagenicity of EMS were also found when cells were treated with mixtures of AsA and EMS which had previously been incubated at 37.0 degrees C for 2 h. In pre- and post-treatments with AsA, however, the frequencies of EMS-induced mutations were not reduced, but rather increased markedly.     

Characterization of HAT- and HAsT-resistant HPRT mutant clones of V79 Chinese hamster cells.

HPRT mutant clones of V79 Chinese hamster cells, isolated after 6-thioguanine (6TG) selection, normally exhibit sensitivity to growth in medium containing the folic acid inhibitor aminopterin or the glutamine analogue L-azaserine (e.g., HAT or HAsT medium). However, it has been shown that some HPRT- clones are resistant to both HAT and HAsT medium. The present study was undertaken to investigate whether any common structural gene alteration exists for such 6TGr-HATr-HAsTr clones. Four clones were studied, 1 of spontaneous origin, 2 induced by a low dose of MNU and 1 EMS-induced. In contrast to wild-type cells and a mutant clone carrying a complete deletion of the HPRT gene, these 4 investigated 6TGr-HATr-HAsTr clones all showed an enhanced incorporation of exogenous 3H-hypoxanthine in the presence of aminopterin and L-azaserine suggesting that these clones carry mutations in the structural part of the HPRT gene. Sequence analysis of PCR-amplified HPRT cDNA from these mutants showed that the spontaneous and the 2 MNU-induced mutant clones lacked exon 4, while the EMS-induced mutant had a GC to AT transition in exon 6. Southern blot analysis of genomic DNA after digestion with BglII, EcoRI and PstI showed no changes in fragment patterns as compared to the wild type. Further sequence analysis of PCR-amplified genomic DNA using exon 4-specific primers showed that all these 3 mutants had an AT to GC or GC to AT transition in exon 4, but had no alterations in the splice sites of exon 4. Based on their characteristics of hypoxanthine incorporation, the present mutant clones fit the model for the proposed functional domains of the HPRT protein.     

Molecular analysis of mutations induced by N-ethyl-N-nitrosourea at the HPRT locus in mouse lymphoma cells

The molecular basis of 29 N-ethyl-N-nitrosourea (ENU)-induced HPRT-deficient mutants of mouse lymphoma cells (GRSL 13-2) was investigated using nucleic acid blot hybridization techniques. DNA from all 29 mutants showed normal restriction patterns on Southern blots when probed with HPRT cDNA, but 10 mutants differed from wild-type cells in their cytoplasmic HPRT mRNA level. In 5 mutants we found 10-25% of the normal amount of HPRT mRNA, whereas in another 5 mutants no HPRT mRNA could be detected at all. These mutants do not seem to be induced by hypermethylation of regulatory sequences of the HPRT gene, since they could not be reverted to an HPRT-proficient phenotype by treatment of the cells with 5-azacytidine.     

The nature of mutants induced by ionising radiation in cultured hamster cells. III. Molecular characterization of HPRT-deficient mutants induced by gamma-rays or alpha-particles showing that the majority have deletions of all or part of the hprt gene

DNA from 58 independent HPRT-deficient mutants of V79 hamster cells induced by ionising radiation was analysed by Southern blot hybridization to a full-length hamster hprt cDNA. About half of the gamma-ray-induced mutants (20/43) were apparently total gene deletions, because they lacked all functional hprt gene sequences hybridizing to the cDNA probe. Another 10 mutants showed various partial deletions and/or rearrangements of the hprt gene. The remaining 13 mutants showed no detectable change in comparison to the structure of the normal gene, which correlated well with previous characterization of these mutants indicating that most carry point mutations in the hprt gene. However, it is probable that some of these point mutations occurred spontaneously rather than being radiation-induced. A smaller number of alpha-particle induced mutants gave similar results: out of a total of 15 mutants, 6 appeared to be total gene deletions, 5 had partial deletions and/or rearrangements, and 4 had no detectable changes. Thus, 70% or more of radiation-induced HPRT-deficient mutants arise through large genetic changes, especially deletions of all or part of the hprt gene. This result is to be contrasted with data published previously by ourselves and others indicating that the majority of spontaneous and ethyl methanesulphonate-induced mutations of hprt and similar genes arise by point mutation.     

Azaguanine-resistant mutants induced by several mutagens in a neurospora heterokaryon.

This paper reports the development of a new system for the detection of forward mutations in a heterokaryon of Neurospora crassa. This system, designed to detect a wide variety of genetic alterations, is based upon the detection of 8-azaguanine-resistant mutants in an adenine-requiring heterokaryon carrying the aza-3 gene. Induction of mutations in the aza-3 gene was detected after treatment of conidia with ultraviolet light, X-rays, ethyl methanesulfonate (EMS), or the acridine mustard, ICR-170. The pattern of appearance of mutant colonies and various factors in mutant selection are discussed.     

Different modifications by vanillin in cytotoxicity and genetic changes induced by EMS and H2O2 in cultured Chinese hamster cells.

The modifying effects of vanillin on the cytotoxicity and 6-thioguanine (6TG)-resistant mutations induced by two different types of chemical mutagens, ethyl methanesulfonate (EMS) and hydrogen peroxide (H2O2), were examined using cultured Chinese hamster V79 cells. The effects of vanillin on H2O2-induced chromosome aberrations were also examined. Vanillin had a dose-dependent enhancing effect on EMS-induced cytotoxicity and 6TG-resistant mutations, when cells were simultaneously treated with vanillin. The post-treatment with vanillin during the mutation expression time of cells after treatment with EMS also showed an enhancement of the frequency of mutations induced by EMS. However, vanillin suppressed the cytotoxicity induced by H2O2 when cells were post-treated with vanillin after H2O2 treatment. Vanillin showed no change in the absence of activity of H2O2 to induce mutations. Post-treatment with vanillin also suppressed the chromosome aberrations induced by H2O2. The differential effects of vanillin were probably due to the quality of mutagen-induced DNA lesions and vanillin might influence at least two different kinds of cellular repair functions. The mechanisms by which vanillin enhances or suppresses chemical-induced cytotoxicity, mutations and chromosome aberrations are discussed.     

Molecular analysis of X-ray-induced mutants at the HPRT locus in V79 Chinese hamster cells

Spontaneous and X-ray-induced mutants at the hypoxanthine phosphoribosyl transferase (HPRT) locus have been isolated from V79 Chinese hamster cells and characterized at the biochemical and cytogenetic levels. Fourteen spontaneous and 24 X-ray-induced clones were azaguanine and thioguanine resistant, did not grow in HAT medium (AZRTGRHATS) and failed to incorporate significant levels of [14C]hypoxyanthine. Cytogenetic analysis of two spontaneous and eight X-ray-induced mutants revealed no major X chromosome rearrangements. In two induced mutants, one of which was hypotetraploid (mode 35-39) with 2 X chromosomes, the short arm of the chromosome (Xp) was slightly shorter than normal. A third mutant was hyperdiploid (mode 22-23) compared with the parental clone (mode 21). When compared with wild-type clones, no other cytogenetic changes were evident in the remaining mutants. Analysis at the DNA level using a Chinese hamster HPRT cDNA probe showed major deletion of HPRT sequences in two and partial deletion in another two induced mutants. In two of the mutants with deletions of HPRT sequences there was a visible shortening of the Xp arm. In the other six mutants two spontaneous and four induced) no karyotypic changes or alterations in restriction fragment patterns were detected suggesting that they carry small deletions or point mutations at the HPRT locus.     

A comparison of the genotoxicity of ethylnitrosourea and ethyl methanesulfonate in lacZ transgenic mice (Muta™Mouse)

We compared the induction of gene mutations and chromosomal aberrations by ethylating agents in lacZ transgenic mice (Muta™Mouse). Chromosomal aberrations were detected by the peripheral blood micronucleus assay. Gene mutations were detected in the lacZ transgene. A small amount of blood was sampled from a tail vessel during the expression time for fixation of gene mutations in vivo; this enabled us to detect and compare clastogenicity and gene mutations in the identical mouse. Single intraperitoneal injections of ENU (50–200 mg/kg) and EMS (100–400 mg/kg) strongly induced micronucleated reticulocytes (MN) detectable in peripheral blood 48 h after treatment. The maximum MN frequencies induced were 6.6% and 3.3% for ENU (100 mg/kg) and EMS (400 mg/kg), respectively (the control value was 0.3%). lacZ mutant frequency (MF) was analyzed in bone marrow and liver 7 days after treatment. Spontaneous MFs were 2.0–4.6x10⁻⁶. MF in bone marrow was increased by ENU to 3.4x10⁻⁵ at 200 mg/kg and induced by EMS to 1.8x10⁻⁵ at 400 mg/kg. In liver, however, both chemicals at their highest doses induced only slight increases in MF. The induction of both micronuclei and lacZ mutations in bone marrow by both ENU and EMS correlated better with O⁶-ethylguanine adducts than with N7-ethylguanine adducts. The mutants (19 for ENU and 12 for EMS) were subjected to DNA sequence analysis. Among EMS-induced mutations, 75% were GC to AT transitions, which were probably caused by O⁶-ethylguanine. Among ENU-induced mutations, in contrast, 40% occurred as AT base pair substitutions (6 AT to TA transversions and 2 AT to GC transitions) (no such mutations were induced by EMS). These results, together with the known reactivity of ENU to thymine suggest that thymine adducts play a significant role in the ENU mutagenesis.     

Molecular and classical genetic analyses of his-3 mutants of Neurospora crassa. I. Tests for allelic complementation and specific revertibility

A collection of 81 his-3 mutants of Neurospora crassa was analyzed in assays for allelic complementation and specific revertibility. In these studies, the linearity of the complementation map of the his-3 cistron (Webber, 1965) was confirmed and mutants were classified as complementing with non-polarized or polarized complementation patterns, or non-complementing. In the assays for spontaneous or induced revertibility, 89% (71/80) of the mutants reverted either spontaneously or after treatment with the chemical mutagens N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino) acridine dihydrochloride, nitrous acid or hydroxylamine. The frequency of revertible mutants among the non-polarized complementing mutants was 96% (45/47), and 79% (15/19) for the polarized complementing and 79% (11/14) for the non-complementing mutants. The results of these classical genetic assays for allelic complementation and specific revertibility suggest a correlation between complementation pattern and presumptive genetic alterations at the molecular level among his-3 mutants similar to that found with ad-3B mutants induced by nitrous acid (Malling and de Serres, 1967), ethyl methanesulfonate (Malling and de Serres, 1968), or ultraviolet (Kilbey et al., 1971).     

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.     

Isolation of antibiotic-resistant and antimetabolite-resistant mutants of Frankia strains EuI1c and Cc1.17

Antibiotic-resistant and antimetabolite-resistant mutants of the nitrogen-fixing symbiotic bacterium Frankia were isolated to provide strains with genetic backgrounds amenable to genetic analysis. The lethal and mutagenic effects of ethyl methanesulfonate (EMS) and UV light on four Frankia strains were investigated. UV irradiation or EMS treatment of strain EuI1c cells resulted in the formation of two different colony types: rough and smooth. The smooth colonies were conditional sporulation mutants. In the case of EMS-induced cells of strain Cc1.17, resistance to lincomycin, ampicillin, and 5-fluorouracil occurred at a frequency of 1 x 10(-5), 1 x 10(-5), and 4 x 10(-5), respectively. The lincomycin-resistant mutants produced a yellow-tan pigment that was released into the growth medium. Resistance to tetracycline and lincomycin with EMS-induced cells of strain EuI1c occurred at a frequency of 3.2 x 10(-3) and 4.7 x 10(-4), respectively. These strains will be useful for the development of genetic methods for Frankia.     

Analysis of repair processes by the determination of the induction of cell killing and mutations in two repair-deficient Chinese hamster ovary cell lines

Two UV sensitive DNA-repair-deficient mutants of Chinese hamster ovary cells (43-3B and 27-1) have been characterized. The sensitivity of these mutants to a broad spectrum of DNA-damaging agents: UV254nm, 4-nitroquinoline-1-oxide (4NQO), X-rays, bleomycin, ethylnitrosourea (ENU), ethyl methanesulphonate (EMS), methyl methanesulphonate (MMS) and mitomycin C (MMC) has been determined. Both mutants were not sensitive to X-rays and bleomycin. 43-3B was found to be sensitive to 4NQO, MMC and slightly sensitive to alkylating agents. 27-1 was sensitive only to alkylating agents. The results suggest the existence of two repair pathways for UV-induced cytotoxicity: one pathway which is also used for the removal of 4NQO and MMC adducts and a second pathway which is also used for the removal of alkyl adducts. Parallel to the toxicity, the induction of mutations at the HPRT and Na+/K+-ATPase loci was determined. The increased cytotoxicity to UV, MMC and 4NQO in 43-3B cells and the increased cytotoxicity to UV in 27-1 cells correlated with increased mutability. It was observed that the increase in mutation induction at the HPRT locus was higher than that at the Na+/K+-ATPase locus. As only point mutations give rise to viable mutants at the Na+/K+-ATPase locus the lower mutability at this locus suggests that defective excision repair increases the chance for deletions. Despite an increased cytotoxicity to ENU in 27-1 cells the mutation induction by ENU was the same in 27-1 and wild-type cells at both loci, which suggests that the mutations are mainly induced by directly miscoding adducts (e.g. O-6 alkylguanine), which cannot be removed by CHO cells. As EMS and MMS treatment of 27-1 cells caused an increase in mutation induction at the HPRT locus and a decrease at the Na+/K+-ATPase locus it indicates that these agents induce a substantial fraction of other mutagenic lesions, which can be repaired by wild-type cells. This suggests that O-6 alkylation is not the only mutagenic lesion after treatment with alkylating agents.     

Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation.

HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).     

Reversion of the 8-azaguanine resistant phenotype of variant Chinese hamster cells treated with alkylating agents and 5-brono-2'-deoxyuridine.

From cultures of V79 Chinese hamster cells, 10 independent clones of 8-azaguanine resistant cells were isolated and subcultured. Cells from all ten clones were resistant to 1 mg/ml levels of 8-azaguanine (8-AzG), contained less than 3% of the wild type levels of the enzyme, hypoxanthine guanine phosphoribosyl transferase (HGPRT), and were unable to grow in HAT medium. The ten clones were classified according to the conditions under which they reverted to the wild type phenotype. Clones in classes I and II reverted spontaneously with frequencies of 40-10(-5) and about 3-10(-5) respectively, and the reversion frequency was independent of the density of cells of all but one of the clones in the culture medium used. Class II clones evinced increased reversion frequencies with ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and to a lesser extent with 5-bromo-2'-deoxyuridine (budR), suggesting that these clones contained point mutations in a locus which controls HGPRT activity. The processes of reversion and toxicity appeared to be associated. Class III clones did not revert spontaneously or with BUdR and MNNG, but did revert with EMS. The reversion frequency of class I clones was not increased after treatment with EMS, MNNG or BUdR.     

Studies on temperature-sensitive mutants of Chinese hamster ovary cells affected in DNA synthesis

DNA synthesis in two mutants of Chinese hamster overy cells, ts 13A and ts 15C, which were temperature sensitive for growth, was found to be shut off rapidly at the nonpermissive temperature. The mutants did not complement each other and the ts lesion was not located on the X chromosome. Both isolates were found to be considerably more sensitive to the alkylating agents, ethylmethanesulfonate (EMS) and methylmethanesulfonate (MMS), as compared to the parental cells, but showed normal sensitivity to UV irradiation. The mutants also showed interesting differences in their response to EMS-induced mutation frequencies at the ouabain-resistant and thioguanine-resistant loci. At high survival (50%) the frequencies of mutations at these genetic loci were markedly low in the ts mutants as compared to the parental cells. In ts+ revertants isolated from the mutants, the ts phenotype and the increased sensitivity to EMS and MMS were affected simultaneously, indicating that both these characteristics resulted from a single genetic lesion.     

Relationship between specific alkylated bases and mutations at two gene loci induced by ethylnitrosourea and diethyl sulfate in CHO cells

DNA adduct formation and induction of mutations at 2 gene loci, hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and Na,K-ATPase, were determined simultaneously in Chinese hamster ovary (CHO) cells after treatment with 2 ethylating agents, ethylnitrosourea (ENU) or diethyl sulfate (DES). Doses of DES and ENU, which resulted in equal levels of O6-ethylguanine (O6-EtGua) and O4-ethylthymine (O4-EtThy) in the DNA, were found to induce very similar frequencies of 6-thioguanine-resistant (6-TGr) mutants. Formation of these DNA adducts might therefore be correlated with mutations induced at the HPRT locus. When, however, the same analysis was applied to ouabain-resistant (ouar) mutants, it was found that, at similar levels of O6-EtGua and O4-EtThy, DES induced many more ouar mutants than ENU. This result supports the notion that primary DNA lesions other than O6-EtGua and O4-EtThy are involved in the fixation of ENU- and DES-induced mutations at the Na,K-ATPase gene locus.     

Genome‐wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato

Genome‐wide mutations induced by ethyl methanesulfonate (EMS) and gamma irradiation in the tomato Micro‐Tom genome were identified by a whole‐genome shotgun sequencing analysis to estimate the spectrum and distribution of whole‐genome DNA mutations and the frequency of deleterious mutations. A total of ~370 Gb of paired‐end reads for four EMS‐induced mutants and three gamma‐ray‐irradiated lines as well as a wild‐type line were obtained by next‐generation sequencing technology. Using bioinformatics analyses, we identified 5920 induced single nucleotide variations and insertion/deletion (indel) mutations. The predominant mutations in the EMS mutants were C/G to T/A transitions, while in the gamma‐ray mutants, C/G to T/A transitions, A/T to T/A transversions, A/T to G/C transitions and deletion mutations were equally common. Biases in the base composition flanking mutations differed between the mutagenesis types. Regarding the effects of the mutations on gene function, >90% of the mutations were located in intergenic regions, and only 0.2% were deleterious. In addition, we detected 1 140 687 spontaneous single nucleotide polymorphisms and indel polymorphisms in wild‐type Micro‐Tom lines. We also found copy number variation, deletions and insertions of chromosomal segments in both the mutant and wild‐type lines. The results provide helpful information not only for mutation research, but also for mutant screening methodology with reverse‐genetic approaches.