Melphalan, a second chemical for which specific-locus mutation induction in the mouse is maximum in early spermatids.

Melphalan (MLP), a bifunctional alkylating agent structurally related to the highly mutagenic chemical chlorambucil (CHL), was found to induce high frequencies of specific-locus mutations in postspermatogonial germ cells of the mouse, and to be one of only a few chemicals that is also mutagenic in spermatogonial stem cells. Productivity patterns following MLP exposures resembled those that had been found for CHL. Mutation rates in successive male germ-cell stages were measured at three MLP-exposure levels in a total of 95,375 offspring. While the induced (experimental minus historical-control) mutation rate is relatively low in stem-cell spermatogonia (1.2 x 10(-5) per locus at a weighted-mean exposure of 7.3 mg/kg), it is about 5 times higher in poststem-cell stages overall, and peaks at 26.7 x 10(-5) per locus in early spermatids at a weighted-mean exposure of only 5.7 mg/kg. This "type-2 pattern" of mutation yield (Russell et al., 1990), i.e., peak sensitivity in early spermatids, has heretofore been found for only one other chemical, CHL. Mutation-rate data earlier reported for CHL (Russell et al., 1989) were augmented in the present study for comparison with MLP-induced rates. Because of the greater toxicity of MLP, average exposures used for this chemical were only about one-half of those for CHL. When MLP and CHL mutation rates are extrapolated to equimolar doses, they appear very similar for poststem-cell stages overall. However, in the case of CHL, a somewhat higher proportion of the mutations is induced in early spermatids than in the case of MLP.     

Review of the mutagenicity/genotoxicity of butylated hydroxytoluene.

Butylated hydroxytoluene (BHT) is an effective, widely used, low cost antioxidant. A host of studies examining the potential of BHT to cause point mutations have been published. They include in vitro studies on various bacterial species and strains and on various types of mammalian cell lines as well as in vivo studies on Drosophila melanogaster, silk worms and also the mouse specific locus test (involving long-term exposure). Together these studies convincingly show the absence of a potential for BHT to cause point mutations. A great number of studies on many cell types and species have also been carried out to examine the potential of BHT to cause chromosome aberrations. In vitro studies have been published using plant cells and the WI-38, CHL, CHO, and V79 mammalian cell lines. In vivo studies have been carried out on somatic and/or germ cells of Drosophila melanogaster, rats and mice. Nearly all studies, especially those using validated test systems, indicate that BHT lacks clastogenic potential. In vitro studies on bacterial, yeast and various mammalian cell lines including DON, CHO, CHL cells and primary hepatocytes demonstrate the absence of interactions with or damage to DNA. Taking all the existing data into account, the weight of evidence suggests that BHT does not represent a relevant mutagenic/genotoxic risk to man.     

Analysis of chromosomal rearrangements induced by postmeiotic mutagenesis with ethylnitrosourea in zebrafish

Mutations identified in zebrafish genetic screens allow the dissection of a wide array of problems in vertebrate biology. Most screens have examined mutations induced by treatment of spermatogonial (premeiotic) cells with the chemical mutagen N-ethyl-N-nitrosourea (ENU). Treatment of postmeiotic gametes with ENU induces specific-locus mutations at a higher rate than premeiotic regimens, suggesting that postmeiotic mutagenesis protocols could be useful in some screening strategies. Whereas there is extensive evidence that ENU induces point mutations in premeiotic cells, the range of mutations induced in postmeiotic zebrafish germ cells has been less thoroughly characterized. Here we report the identification and analysis of five mutations induced by postmeiotic ENU treatment. One mutation, snh(st1), is a translocation involving linkage group (LG) 11 and LG 14. The other four mutations, oep(st2), kny(st3), Df(LG 13)(st4), and cyc(st5), are deletions, ranging in size from less than 3 cM to greater than 20 cM. These results show that germ cell stage is an important determinant of the type of mutations induced. The induction of chromosomal rearrangements may account for the elevated frequency of specific-locus mutations observed after treatment of postmeiotic gametes with ENU.     

Mutational signatures of non‐homologous and polymerase theta‐mediated end‐joining in embryonic stem cells

Cells employ potentially mutagenic DNA repair mechanisms to avoid the detrimental effects of chromosome breaks on cell survival. While classical non‐homologous end‐joining (cNHEJ) is largely error‐free, alternative end‐joining pathways have been described that are intrinsically mutagenic. Which end‐joining mechanisms operate in germ and embryonic cells and thus contribute to heritable mutations found in congenital diseases is, however, still largely elusive. Here, we determined the genetic requirements for the repair of CRISPR/Cas9‐induced chromosomal breaks of different configurations, and establish the mutational consequences. We find that cNHEJ and polymerase theta‐mediated end‐joining (TMEJ) act both parallel and redundant in mouse embryonic stem cells and account for virtually all end‐joining activity. Surprisingly, mutagenic repair by polymerase theta (Pol θ, encoded by the Polq gene) is most prevalent for blunt double‐strand breaks (DSBs), while cNHEJ dictates mutagenic repair of DSBs with protruding ends, in which the cNHEJ polymerases lambda and mu play minor roles. We conclude that cNHEJ‐dependent repair of DSBs with protruding ends can explain de novo formation of tandem duplications in mammalian genomes.     

Transmitted mutational events induced in mouse germ cells following acrylamide or glycidamide exposure

An increase in the germ line mutation rate in humans will result in an increase in the incidence of genetically determined diseases in subsequent generations. Thus, it is important to identify those agents that are mutagenic in mammalian germ cells. Acrylamide is water soluble, absorbed and distributed in the body, chemically reactive with nucleophilic sites, and there are known sources of human exposure. Here we review all seven published studies that assessed the effectiveness of acrylamide or its active metabolite, glycidamide, in inducing transmitted reciprocal translocations or gene mutations in the mouse. Major conclusions were (a) acrylamide is mutagenic in spermatozoa and spermatid stages of the male germ line; (b) in these spermatogenic stages acrylamide is mainly or exclusively a clastogen; (c) per unit dose, i.p. exposure is more effective than dermal exposure; and (d) per unit dose, glycidamide is more effective than acrylamide. Since stem cell spermatogonia persist and may accumulate mutations throughout the reproductive life of males, assessment of induced mutations in this germ cell stage is critical for the assessment of genetic risk associated with exposure to a mutagen. The two specific-locus mutation experiments which studied the stem cell spermatogonial stage yielded conflicting results. This discrepancy should be resolved. Finally, it is noted that no experiments have studied the mutagenic potential of acrylamide to increase the frequency of transmitted mutational events following exposure in the female germ line.     

Transmitted mutational events induced in mouse germ cells following acrylamide or glycidamide exposure

An increase in the germ line mutation rate in humans will result in an increase in the incidence of genetically determined diseases in subsequent generations. Thus, it is important to identify those agents that are mutagenic in mammalian germ cells. Acrylamide is water soluble, absorbed and distributed in the body, chemically reactive with nucleophilic sites, and there are known sources of human exposure. Here we review all seven published studies that assessed the effectiveness of acrylamide or its active metabolite, glycidamide, in inducing transmitted reciprocal translocations or gene mutations in the mouse. Major conclusions were (a) acrylamide is mutagenic in spermatozoa and spermatid stages of the male germ line; (b) in these spermatogenic stages acrylamide is mainly or exclusively a clastogen; (c) per unit dose, i.p. exposure is more effective than dermal exposure; and (d) per unit dose, glycidamide is more effective than acrylamide. Since stem cell spermatogonia persist and may accumulate mutations throughout the reproductive life of males, assessment of induced mutations in this germ cell stage is critical for the assessment of genetic risk associated with exposure to a mutagen. The two specific-locus mutation experiments which studied the stem cell spermatogonial stage yielded conflicting results. This discrepancy should be resolved. Finally, it is noted that no experiments have studied the mutagenic potential of acrylamide to increase the frequency of transmitted mutational events following exposure in the female germ line.     

Evaluation of mutagenic effects of hyperbaric oxygen (HBO) in vitro. II. Induction of oxidative DNA damage and mutations in the mouse lymphoma assay

We recently showed that treatment of V79 cells with hyperbaric oxygen (HBO) efficiently induced DNA effects in the comet assay and chromosomal damage in the micronucleus test (MNT), but did not lead to gene mutations at the hprt locus. Using the comet assay in conjunction with bacterial formamidopyrimidine DNA glycosylase (FPG protein), we now provide indirect evidence that the same treatment leads to the induction of 8-oxoguanine, a premutagenic oxidative DNA base modification in V79 and mouse lymphoma (L5178Y) cells. We also demonstrate that HBO efficiently induces mutations in the mouse lymphoma assay (MLA). Exposure of L5178Y cells to HBO (98% O(2); 3bar) for 2h caused a clear mutagenic effect in the MLA, which was further enhanced after a 3h exposure. As this mutagenic effect was solely due to the strong increase of small colony (SC) mutants, we suggest that HBO causes mutations by induction of chromosomal alterations. Molecular characterization of induced SC mutants by loss of heterozygosity (LOH) analysis showed an extensive loss of functional tk sequences similar to the pattern found in spontaneous SC mutants. This finding confirmed that the majority of HBO-induced mutants is actually produced by a clastogenic mechanism. The induction of point mutations as a consequence of induced oxidative DNA base damage seems to be of minor importance.     

Comparison of the genetic effects of equimolar doses of ENU and MNU: while the chemicals differ dramatically in their mutagenicity in stem-cell spermatogonia, both elicit very high mutation rates in differentiating spermatogonia

Mutagenic, reproductive, and toxicity effects of two closely related chemicals, ethylnitrosourea (ENU) and methylnitrosourea (MNU), were compared at equimolar and near-equimolar doses in the mouse specific-locus test in a screen of all stages of spermatogenesis and spermiogenesis. In stem-cell spermatogonia (SG), ENU is more than an order of magnitude more mutagenic than MNU. During post-SG stages, both chemicals exhibit high peaks in mutation yield when differentiating spermatogonia (DG) and preleptotene spermatocytes are exposed. The mutation frequency induced by 75mgMNU/kg during this peak interval is, to date, the highest induced by any single-exposure mutagenic treatment - chemical or radiation - that allows survival of the exposed animal and its germ cells, producing an estimated 10 new mutations per genome. There is thus a vast difference between stem cell and differentiating spermatogonia in their sensitivity to MNU, but little difference between these stages in their sensitivity to ENU. During stages following meiotic metaphase, the highest mutation yield is obtained from exposed spermatids, but for both chemicals, that yield is less than one-quarter that obtained from the peak interval. Large-lesion (LL) mutations were induced only in spermatids. Although only a few of the remaining mutations were analyzed molecularly, there is considerable evidence from recent molecular characterizations of the marker genes and their flanking chromosomal regions that most, if not all, mutations induced during the peak-sensitive period did not involve lesions outside the marked loci. Both ENU and MNU treatments of post-SG stages yielded significant numbers of mutants that were recovered as mosaics, with the proportion being higher for ENU than for MNU. Comparing the chemicals for the endpoints studied and additional ones (e.g., chromosome aberrations, toxicity to germ cells and to animals, teratogenicity) revealed that while MNU is generally more effective, the opposite is true when the target cells are SG.     

Preferential induction of AT-TA transversion, but not deletions, by chlorambucil at the hisG428 site of Salmonella typhimurium TA102.

The chemotherapeutic agent chlorambucil effectively induces deletion mutations in mouse germ cells. The possibility that this chemical also effectively induces deletion mutations in bacterial DNA was examined using Ames Salmonella tester strains. Chlorambucil was mutagenic only to strains TA102 (hisG428, rfa/pKM101) and YG2975 (hisG46, rfa/pKM101) when S9 mix was absent. Since strain TA102 can detect short deletions, the mutational changes of TA102 induced by this agent without S9 mix were directly determined by the DNA sequencing technique. It turned out that chlorambucil did not induce deletion mutations but preferentially induced AT-TA transversions at the hisG428 site of plasmid pAQ1 of strain TA102. These results caution that the positive results induced by chlorambucil in mutagenicity tests do not necessarily mean the occurrence of deletions.     

Introducing defined chromosomal rearrangements into the mouse genome

Chromosomal rearrangements have been instrumental in genetic studies in Drosophila. Visibly marked deficiencies (deletions) are used in mapping studies and region-specific mutagenesis screens by providing segmental haploidy required to uncover recessive mutations. Marked recessive lethal inversions are used as balancer chromosomes to maintain recessive lethal mutations and to maintain the integrity of mutagenized chromosomes. In mice, studies on series of radiation-induced deletions that surround several visible mutations have yielded invaluable functional genomic information in the regions analyzed. However, most regions of the mouse genome are not accessible to such analyses due to a lack of marked chromosomal rearrangements. Here we describe a method to generate defined chromosomal rearrangements using the Cre--loxP recombination system based on a published strategy [R. Ramirez-Solis, P. Liu, and A. Bradley, (1995) Nature 378, 720--724]. Various types of rearrangements, such as deletions, duplications, inversions, and translocations, can be engineered using this strategy. Furthermore, the rearrangements can be visibly marked with coat color genes, providing essential reagents for large-scale recessive genetic screens in the mouse. The ability to generate marked chromosomal rearrangements will help to elevate the level of manipulative mouse genetics to that of Drosophila genetics.     

Accuracy of intrachromosomal gene conversion in mouse cells.

Results of several recent studies suggest that homologous recombination and related processes in mammalian cells are highly mutagenic. We have examined the products of intrachromosomal gene conversion events that encompassed the last intron of the chicken thymidine kinase gene. Following plasmid rescue and DNA sequencing, we find no mutations associated with twenty conversion events representing 5380 total base pairs of which 2414 base pairs are intron sequence. Based on these studies we conclude that intrachromosomal gene conversion in mouse cells is not a highly mutagenic process but rather it operates with fidelity.     

Mutagenic activity of dioxydine]

A previous evaluation of mutagenic activity of some drugs and perspective substances is carried out using indicator microorganisms. The mutagenicity of dioxydine, a drag with discovered antibacterial activity, is investigated. Dioxydine is shown to induce reversions in mutant of Salmonella typhimurium TA-1950, the indicator strain which demonstrates mutagenic activity of agents, producing mutations of base pair substitution type. Dioxydine proved to affect logariphmiically growing bacterial culture with great activity. Mutageni effect of dioxydine is not modified itself in microsomal oxidation system in vitro. Some data concerning participation of excision reparation enzyme (uvr-B+ gene product) in repair of lethal damages induced by dioxydine, have been obtained. The dioxydine ability to cause bacterial gene mutations in host mediated assay as well as dominant and recessive sex-linked lethal mutations in Drosophila is demonstrated. Dioxydine is capable of inducing chromosome aberrations in bone marrow cells and dominant lethal mutations in mouse germ cells.     

Female-specific mutagenic response of mice to hycanthone

Male and female gametogeneses differ markedly in all mammals. While male germ cells are continuously being produced from stem cells throughout the reproductive life span, the number of female germ cells is fixed during prenatal development and, soon after birth, all of the oocytes are arrested in a modified diplotene, or dictyate, stage. Following puberty, dictyate oocytes are hormonally triggered to mature either singly or in groups, resulting in ovulation and the completion of the first meiotic division. It has been hypothesized that female mice are more susceptible to dominant lethal effects of intercalating agents than male mice because oocyte chromosomes, which are arrested in a diffuse state, are generally more accessable to intercalation than are the more condensed chromosomes present within most male germ cell stages. This hypothesis was further tested using the intercalating agent hycanthone methane-sulfonate. Effects of hycanthone were studied in maturing and primordial oocytes and in male germ cells throughout spermatogenesis. No induction of dominant lethality was observed for treated males while a significant increase in embryonic death, expressed around the time of implantation, was observed in females that mated within 4.5 days after treatment. These effects were the result of dominant lethal mutations induced in maturing oocytes and not of maternal toxicity as indicated by the presence of chromosomal aberrations observed at first-cleavage metaphase of zygotes obtained from treated females. These results add support to the hypothesis that certain intercalating chemicals, which are not mutagenic to male mice, may be mutagenic to females and point to a need for more in-depth studies of female-specific mutagenesis.     

Chlornaphazine and chlorambucil induce dominant lethal mutations in male mice

Two antineoplastic agents, chlornaphazine (CN) and chlorambucil (CHL), were tested for the induction of dominant lethal mutations in male mice. Both compounds are nitrogen mustard derivatives and have been shown to be genotoxic in a variety of organisms. CN was administered intraperitoneally to DBA/2J male mice at a dosage of 0, 500, 1000, or 1500 mg/kg body weight (bw). Immediately following treatment, each male was mated at 4-day intervals to two virgin C57BL/6J females. CHL was administered intraperitoneally to C3H/HeJ and DBA/2J males at a dosage of 0, 2.5, or 5.0 mg/kg bw. These males were mated at weekly intervals to two virgin T-stock females. CN and CHL clearly induced dominant lethal mutations. CN induced dominant lethal effects in all post-meiotic germ-cell stages of treated DBA males, with a clear dose-response relationship. The results with CHL-treated DBA males indicated that all post-meiotic germ-cell stages, except late-spermatids, were affected by CHL treatment, while in C3H males, CHL induced dominant lethal effects in all post-meiotic germ-cell stages. A dose-response relationship was also observed with CHL in C3H male mice. In the present experiments, regardless of the agent or the mouse strain used, spermatids appeared to be the germ-cell stage most sensitive to dominant lethal induction.     

Mutatect: a mouse tumour model for detecting radiation-induced mutations in vivo

A new mouse model (Mutatect) that permits detection of mutations at the hprt (hypoxanthine phosphoribosyltransferase) locus is described. It is highly sensitive to detection of mutants induced by clastogenic agents such as ionizing radiation. MN-11 cells are grown as a subcutaneous tumour in C57BL/6 mice for a period of 2 weeks, during which time they can be exposed to mutagenic treatments. Cells taken from the animal are cultured ex vivo and 6-thioguanine (6-TG)-resistant mutant clones can be readily identified and scored. This model system may have special utility for detecting multi-locus deletion events (chromosomal mutations) induced by high LET forms of radiation that might be encountered in space.     

Mouse genetic models for aneuploidy induction in germ cells

Rodents have been successfully used as models to identify risks of chemical exposures or age to aneuploidy induction in germ cells, which may be transmitted to the progeny. For this administration in vivo as well as exposures to in vitro maturing germ cells have been useful. Genetic models involving mice with structural chromosomal rearrangements and transgenic animals have the potential to model conditions predisposing to aneuploidy in one or both sexes, and in this way to identify potential targets for aneugens and gender-effects. The review provides an overview of mouse genetic models for aneuploidy induction in mammalian germ cells and discusses perspectives for combining genetic with experimental approaches in aneuploidy research.     

Evaluation of the database on mutant frequencies and DNA sequence alterations of vermilion mutations induced in germ cells of Drosophila shows the importance of a neutral mutation detection system

The vermilion gene in Drosophila has extensively been used for the molecular analysis of mutations induced by chemicals in germ cells in vivo. The gene is located on the X-chromosome and is a useful target for the study of mutagenesis since all types of mutations are generated. We have critically evaluated this system with respect to sensitivity for mutation induction and selectivity for different types of mutations, using a database of more than 600 vermilion mutants induced in postmeiotic male germ cells by 18 mutagens. From most of these mutants the mutation has been analysed. These data showed 336 base substitutions, 96 intra-locus DNA rearrangements and 78 multi-locus deletions (MLD). Mutants containing a MLD were either heterozygous sterile or homozygous and hemizygous lethal. The distribution of both basepair (bp) changes and intra-locus rearrangements over the coding region of the vermilion gene was uniform with no preferences concerning 5' or 3' regions, certain exons, splice sites, specific amino acid changes or nonsense mutations. Possible hotspots for base substitutions seem to be related to the type of DNA damage rather than to the vermilion system. Gene mutations other than bp changes were examined on sequence characteristics flanking the deletion breakpoints. Induction frequencies of vermilion mosaic mutants were, in general, higher than those of vermilion complete mutants, suggesting that persistent lesions are the main contributors to the molecular spectra. Comparison of induction frequencies of vermilion mutants and sex-linked recessive lethal (SLRL) mutants for the 18 mutagens showed that the sensitivity of the vermilion gene against a mutagenic insult is representative for genes located on the X-chromosome. The effect of nucleotide excision repair (NER) on the formation of SLRL mutants correlated with an increase of transversions in the vermilion spectra under NER deficient conditions. Furthermore, the clastogenic potency of the mutagens, i.e., the efficiency to induce chromosomal-losses vs. SLRL forward mutations, shows a positive correlation with the percentage of DNA deletions in the molecular spectra of vermilion mutants.     

Meiosis and retrotransposon silencing during germ cell development in mice

In mammals, germ cells derive from the pluripotent cells that are present early in embryogenesis, and then differentiate into male sperm or female eggs as development proceeds. Fusion between an egg and a sperm at fertilization allows genetic information from both parents to be transmitted to the next generation, and produces a pluripotent zygote to initiate the next round of embryogenesis. Meiosis is a central event in this self-perpetuating cycle that creates genetic diversity by generating new combinations of existing genetic alleles, and halves the number of chromosomes in the developing male and female germ cells to allow chromosome number to be maintained through successive generations. The developing germ cells also help to maintain genetic and chromosomal stability through the generations by protecting the genome from excessive de novo mutation. Several mouse mutants have recently been characterised whose germ cells exhibit defects in silencing the potentially mutagenic endogenous retroviruses and other retrotransposons that are prevalent in mammalian genomes, and these germ cells also exhibit defects in progression through meiosis. Here we review how mouse germ cells develop and proceed through meiosis, how mouse germ cells silence endogenous retroviruses and other retrotransposons, and discuss why silencing of endogenous retroviruses and other retrotransposons may be required for meiotic progression in mice.     

Frequency and nature of specific-locus mutations induced in female mice by radiations and chemicals: a review.

The inducibility of heritable mutations in female mammals has been measured in the mouse specific-locus test (SLT). For radiation-induced mutations, a large body of data has been accumulated that includes information about biological and physical factors that influence mutation yields. However, relatively few SLT studies in females have been conducted with chemicals to date. A single estimate of the spontaneous mutation rate in oocytes, 6/536,207, has been derived as the most appropriate one to subtract from experimental rates. This rate is highly significantly below the spontaneous mutation rate in males. Mutations recovered from females mutagenized at any time after about the 12th day post-conception are induced in non-dividing cells. In adult females, most oocytes are arrested in small follicles; maturation from this stage to ovulation takes several weeks. High-dose-rate radiations are more mutagenic in mature and maturing oocytes than in spermatogonia of the male; on the other hand, no clearly induced mutations have been recovered from irradiated arrested oocytes. Efficient repair processes have been invoked to explain the latter finding as well as the upward-curving dose-effect relation for acute irradiation, and the fact that dose protraction drastically reduces mutation yield from mature and maturing oocytes. The dose-protraction effect is much greater than that found in spermatogonia. Radiation-induced mutation rates in embryonic, fetal, and newborn females are overall lower than those in the mature and maturing oocytes of adults. A dose-protraction effect has also been demonstrated at an early developmental stage when the nuclear morphology of mouse oocytes most resembles that of the human. Of only 5 chemicals so far explored for their effect in oocytes, 2 (ethylnitrosourea, ENU, and triethylenemelamine, TEM), and possibly a third (procarbazine hydrochloride, PRC), are mutagenic--with at least one of these (ENU) mutagenic in arrested as well as maturing oocytes. However, the mutation rate is, in each case, lower than for treated male germ cells. By contrast, ENU-induced mutation yield for the maternal genome of the zygote is an order of magnitude higher than that for the zygote's paternal genome or for spermatogonia. A high proportion of mutants derived from chemical treatment of oocytes (including the oocyte genome in zygotes) are mosaics, probably owing to lesions affecting only 1 strand of the DNA. A characteristic of specific-locus mutations induced in oocytes is that they include a considerably higher percentage of large (multi-locus) lesions (LLs) than do mutations induced in spermatogonia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation induction by okadaic acid, a protein phosphatase inhibitor, in CHL cells, but not in S. typhimurium.

Okadaic acid (OA) is a specific and strong inhibitor of protein phosphatase 1 and 2A present in eukaryotes, and a potent promoter of carcinogenesis in mouse skin. In this study, we examined the mutagenicity of OA. OA did not induce mutations in S. typhimurium TA100 and TA98, with or without a microsomal metabolic activation system. However, it was strongly mutagenic to Chinese hamster lung (CHL) cells without a microsomal activation system, as shown using diphtheria toxin (DT) resistance (DT^r) as a selective marker. Treatment of CHL cells with OA at 17.5 ng/ml induced 164 DT^r mutants per 10⁶ survivors. A plot of the mutation frequency against the OA concentration gave a concave curve, and the mutant frequency was calculated to be 5500/10⁶ survivors/μg, with OA in the dose range of 10–15 ng/ml. This value was about 680 times that of ethyl methanesulfonate (EMS), and comparable to that of 2-amino-N⁶-hydroxyadenine, one of the strongest knowon mutgens. Elongation factor 2 (EF-2) obtained from 4 DT^r clones was not ADP-ribosylated by DT fragment A. PCR-direct sequencing revealed that the hot spot of EF-2 for EMS mutagenesis in CHO-K1 cells, the first letter of codon 717, was not a t spot for OA mutagenesis in CHL cells.