Mutagen Ethyl Methanesulfonate Research Articles

Characterization of a Life-Extending Mutation in age-2, a New Aging Gene in Caenorhabditis elegans

We have generated a life-extending mutation, yw23, in Caenorhabditis elegans. The mutation is in what appears to be a new aging gene, which we have designated age-2. When homozygous, yw23 produces an increase of mean and maximum life span of about 20% over that of the wild-type strain, N2. Strain HG23 [age-2(yw23)] was obtained by screening for longer life spans among 430 lines of nematodes two generations after exposure to the mutagen ethylmethanesulfonate. Strain HG231 [age-2(yw23)] was obtained after a single out-crossing of HG23 to N2. When compared with N2, HG231 exhibits normal motility, slightly higher swimming rates, reduced fertility (especially at higher temperatures), somewhat longer development times, and a slightly larger size at the time of first egg laying. A Gompertz analysis suggests that HG231 extends life span by reducing the initial mortality rate. In genetic crosses, yw23 complements other known aging mutants in C. elegans genes-age-1, daf-2, spe-26, clk-1, clk-2, clk-3, and gro-1. A double-mutant strain, HG284, combining mutations in age-1 and age-2, lives longer than animals with individual mutations in either age-1 or age-2, and exhibits a longer life span at 25 degrees C than at 20 degrees C.

Analysis of mutagens with single cell gel electrophoresis, flow cytometry, and forward mutation assays in an isolated clone of Chinese hamster ovary cells

We investigated the induction of DNA strand breaks in the single cell gel electrophoresis (SCGE or comet) assay with whole cell clastogenicity measured with flow cytometric analysis in cells from an isolated clone of the Chinese hamster ovary (CHO) AS52 cell line. Under identical treatment conditions the responses were compared with forward mutation at gpt using 2-acetoxyacetylaminofluorene (2AAAF), ultraviolet radiation (UV) and ethyl methanesulfonate (EMS). Cytotoxicity for each agent was evaluated in the SCGE and forward mutation assays. Forward mutation was 4-10-fold more sensitive than DNA strand breaks detected in the SCGE assay. For 2AAAF and EMS, the kinetics of the induction of genetic damage were similar for the three assays, although there were differences in sensitivity. With UV, the induction kinetics of gpt mutation differed from that expressed by SCGE and flow cytometric analysis. With the chemical mutagens 2AAAF and EMS, there was a high correlation between the SCGE assay and forward mutation and also between the SCGE assay and flow cytometry. There was no significant correlation between flow cytometry and forward mutation. With UV, only the SCGE assay and flow cytometry were correlated. Agent-specific variations in the intragenomic distribution of DNA damage for each mutagen was measured in the SCGE assay.

Influence of alkyltransferase activity and chromosomal locus on mutational hotspots in Chinese hamster ovary cells.

High-density mutational spectra have been established for exon 3 of the gene encoding adenine phosphoribosyltransferase (APRT) of the Chinese hamster ovary (CHO) cell line derivative D422 and closely related and/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC-->AT transitions yielding a selectable APRT- phenotype) was estimated at 31 based on our own accumulated data base of 136 sequenced exon 3 mutations and on literature reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 "baseline" sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one comprising a cluster at nucleotides 1293-1309 and the other at nucleotide 1365. Collectively, the latter sites were about 10-fold more frequently mutated than the others. CHO cells are mer- as they lack the repair enzyme O6-methylguanidine methyltransferase (EC 2.1.1.63). In modified repair-proficient CHO cells, the distribution of mutations among all of the 31 sites was random, with only 3 of the 19 GC-->AT transitions in the above hotspots. To determine whether the distribution was locus-dependent, two independent lines carrying single copies of transfected APRT genes were generated from a derivative of D422 carrying a deletion in the endogenous APRT gene. Nucleotides 1293-1309 were again no longer preferentially mutated, but the site at nucleotide 1365 was still a hotspot. We conclude that mutational spectra in mer- cells are at least in part locus dependent and that some sequences are particularly susceptible to EMS mutagenesis and perhaps also to methyltransferase repair.

A C. elegans mutant screen based on antibody or histochemical staining

A method has been developed for isolating mutations in Caenorhabditis elegans that alter antibody or histochemical staining patterns. The basis for this method is a new procedure for making C. elegans permeable that does not kill the eggs contained within the uterus of gravid adult hermaphrodites. A mutagenized population of gravid hermaphrodites is made permeable and then stained with either an antibody or a histochemical stain. Animals that stain aberrantly are picked to individual petri plates and the eggs within the uterus of the stained mother hatch and establish a new genetic line. Antibody and histochemical stains are especially useful phenotypes because the staining pattern will usually directly reflect the gene expression pattern of the gene that codes for the antigen or enzyme. This method was used to isolate mutants that alter the expression of a mec-7 lacZ fusion gene. Transgenic animals that contained the mec-7 lacZ gene integrated into chromosome I were treated with the mutagen ethylmethanesulfonate, allowed to self-fertilize for two generations and then stained with X-gal or antibodies against β-galactosidase. Gravid animals that stained abnormally were picked to fresh petri plates and offspring were used to establish new mutant lines.

Characterization of spontaneous and induced mutations in SV40-transformed normal and ataxia telangiectasia cell lines.

Spontaneous and induced mutations at the HPRT locus were analyzed in one normal (MRC5CV1) and one ataxia telangiectasia (AT5BIVA) SV40-transformed cell line derived from male donors. Multiplex PCR and Southern analyses revealed a high frequency of spontaneous deletion mutations that may be a consequence of the SV40 transformation. Four mutagens (ethyl methanesulfonate, bromodeoxyuridine, bleomycin, adriamycin), which differ in their types of primary DNA lesions, caused specific patterns of mutations. By using fluorescence in situ hybridization (FISH) techniques, we were able to show that more than 90% of the AT5BIVA cells contained two X chromosomes with HPRT alleles, while in more than 90% of the MRC5CV1 cells genomic hemizygosity for the HPRT gene was found. Taking into account these findings we found that the AT5BIVA cell line possesses spontaneous hypermutability as well as hypersensitivity and hypermutability to bleomycin (BLM) and adriamycin (AM). Both mutagens induced deletion mutations in both cell lines, but more complex mutations and larger deletions were found in AT5BIVA cells.

Spectrum of spontaneous missense mutations causing cyclic AMP-resistance phenotypes in cultured S49 mouse lymphoma cells differs markedly from those of mutations induced by alkylating mutagens

Mutants of S49 mouse lymphoma cells resistant to cytolysis by analogs of cyclic AMP (cAMP) generally have missense mutations in the gene encoding the regulatory subunit of cAMP-dependent protein kinase. We have compared the mutations in 95 spontaneous isolates with those in 60 mutagen-induced isolates by sequence analysis of amplified cDNAs. Twenty-nine single basepair substitutions in 19 codons produced selectable phenotypes. The spontaneous mutant spectrum was dominated by a CpG transition hotspot in the codon for Arg334. This and other nearby CpG sites were found to be methylated in genomic S49 cell DNA by restriction enzyme analyses. Most of the remaining spontaneous mutants had either G-C-->C-G or T-A-->G-C transversions, which have been associated with damage caused by oxygen radicals. In contrast, the majority of mutants induced with the alkylating mutagens ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine had G-C-->A-T mutations at non-CpG sites; in addition, EMS induced several A-T-->G-C, A-T-->T-A, and G-C-->T-A substitutions. A single ICR191-induced mutant analyzed had a unique A-T-->G-C lesion. A number of spontaneous and mutagen-induced isolates had closely linked double or triple substitutions, and two isolates had tandem triple substitutions.

Chemically selected subclones of the CEM cell line demonstrate resistance to HIV-1 infection resulting from a selective loss of NF-kappa B DNA binding proteins.

To delineate cellular genes that are required for optimal HIV-1 infection, CEM cells were subjected to treatment with the chemical mutagen ethylmethanesulfonate (EMS) and subclones were selected based on their increased resistance to HIV-1 infection and reduced syncytium formation, despite relatively normal CD4 expression (20,000 to 25,000 receptors/cell). Two subclones with this phenotype demonstrated a diminished capacity of HIV-1 long terminal repeat-chloramphenicol acetyl transferase expression either after treatment with the protein kinase C activator PMA, or through Tat-mediated transactivation. In this study, we show that the cellular levels of the NF-kappa B DNA binding proteins (but not AP1 or SP1) are markedly reduced in these cell mutants both at the mRNA and protein levels, resulting in reduced nuclear localization of p50/p65 after PMA induction or treatment with the lymphokine TNF-alpha. Transient reconstitution with a plasmid expressing p50 resulted in partial recovery of PMA-inducible LTR-chloramphenicol acetyl transferase expression. These data suggest that, at least in the CEM T cell line, a selective reduction in the NF-kappa B DNA binding proteins is sufficient to curtail HIV-1 infection.

Human CYP2A6 activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): mutational specificity in the gpt gene of AS52 cells.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) is a potential human carcinogen that is known to be metabolized to DNA-reactive intermediates by the cytochromes P450. We have examined the nature of NNK's DNA damaging effects in a mammalian cell system expressing a specific human cytochrome P450 (2A6) and containing a target gene for mutagenesis. Human CYP2A6, which is known to activate NNK to a mutagen, was lipofected via a retroviral vector into the Chinese hamster ovary AS52 cell line, which contains the bacterial gpt gene and can be mutated to 6-thioguanine resistance. AS52 cells expressed negligible CYP2A6-specific coumarin 7-hydroxylase activity (0.7 pmol/mg protein/min), while a CYP2A6 transfected clone (AS52-E8) expressed 30 pmol/mg protein/min. Both cell lines were equally sensitive to the cytotoxic and mutagenic effects of the direct-acting mutagen ethylmethanesulfonate; however, only the AS52-E8 cells exhibited a dose-dependent increase in cytotoxicity and mutant frequency upon treatment with NNK. At the highest NNK dose (1200 micrograms/ml), the mutant frequency in AS52-E8 cells was 14-fold (339 x 10(-6)) greater than the spontaneous frequency of 24 x 10(-6). Ninty-eight mutant clones were isolated following NNK treatment. Based on PCR analysis, 21 clones contained deletions/rearrangements and 77 were putative point mutants. Sequencing potential point mutants showed that 81% contained G:C to A:T transitions. Four of six G:C to A:T hotspots were at the second G of the GGT motif, which is the motif and major mutation found in codon 12 of Ki-ras from NNK-induced lung tumors in strain A mice. Since NNK may be metabolized via different pathways to pyridyloxobutylate or methylate DNA, the data suggest that methylation damage causes the major mutagenic events in AS52-E8 cells when NNK is activated by human CYP2A6.

Downstream signal transduction defects that suppress transformation in two revertant cell lines expressing activated rat neu oncogene.

The neu gene encodes the transmembrane tyrosine kinase growth factor receptor, p185. To study neu-induced cellular transformation, we developed revertant cells from the neu-transformed NIH 3T3 cell line, B104-1-1, by treating the cells with the chemical mutagen ethyl methanesulfonate. The morphologically normal revertant cells were first selected by their ability either to attach to culture plates or to survive in the presence of the cytotoxic reagents colchicine or 5-fluoro-2-deoxyuridine. Two of the 21 candidate revertant cell lines isolated were further characterized and were found to lose their anchorage independence and ability to grow in 1% calf serum, indicating that they were nontransformed even though they still expressed p185 oncoprotein. The tyrosine residues of p185 in these two revertants were underphosphorylated, which may have contributed to their nontransformed status. In addition, these revertants also resisted transformation by neu and several additional oncogenes (H-ras, N-ras, v-mos, v-abl, and v-fos) as determined by focus forming assays. These results indicated that we had successfully developed, from neu-transformed cells, revertants exhibiting defective tyrosine phosphorylation of the neu oncoprotein. The results also suggested that neu and several other oncogenes may share common elements in their pathways for the induction of cellular transformation.

Occurrence of Temperature-Sensitive Phenotypic Plasticity in Chlorophyll-Deficient Mutants of Arabidopsis thaliana

A collection of 75 putative mutants with alterations in leaf pigmentation was visually selected from Arabidopsis thaliana plants (M(2) generation) grown at 26 degrees C from seeds treated with the mutagen ethylmethanesulfonate. Fifty-eight of the plants were found to have chlorophyll contents decreased by at least 10% from the parental Columbia ecotype. These plants were screened for chlorophyll content and the ratio of chlorophyll b/a after growth at 20 or 26 degrees C. Relative to the parental type, a significant number of individuals in which the chlorophyll-deficient phenotype was exacerbated at one of the growth temperatures were identified. We conclude that temperature-sensitive phenotypic plasticity for chlorophyll content is relatively common in mutant populations of higher plants.

Selection and Characterization of a Gibberellin-Deficient Mutant of Thlaspi arvense L.

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for reproductive development. Previous work in this laboratory has demonstrated that the bolting aspect (rapid stem growth) of reproductive development is mediated by gibberellins (GA). The present paper describes the selection and characterization of a mutant lacking the capacity for thermoinduced stem growth. Seeds of an inbred field pennycress line (CR(1)) were treated with the chemical mutagen ethyl methane sulfonate, germinated, and allowed to produce seed. Plants derived from these seeds were screened for reduced stem growth. A mutant line, EMS-141, in which the lack of stem growth can be fully overcome with exogenous GA(3), was selected for further analysis. Other phenotypic abnormalities exhibited by the mutant line include reduced petiole growth, slightly delayed floral initiation, and failure of flowers to develop fully. These are also reversed with exogenous GA(3). Evidence is presented indicating that all of the alterations in growth and development exhibited by EMS-141 are conferred by a recessive mutation of a single nuclear gene. Through quantitative analysis of endogenous GA and GA precursors and a comparison of the abilities of various compounds to restore normal growth when applied to plants of EMS-141, the physiological basis for the mutant phenotype was determined to be the result of highly reduced endogenous GA levels. Moreover, the affected site in GA biosynthesis appears to be the accumulation of ent-kaurene, probably at the level of ent-kaurene synthase. The relative abilities of exogenous GA and GA precursors to restore normal growth of petioles and stems are compared, and the results are used to make inferences on the functions of the two different pathways of GA metabolism that exist in field pennycress.

Chemically induced CD4 mutants of a human T cell line. Evidence for dissociation between binding of HIV I envelope and susceptibility to HIV I infection and syncytia formation.

Abstract This study describes the derivation of a series of mutants from the human leukemic cell line CEM using the frame shift mutagen Ethyl-methanesulfonate followed by negative selection with multiple treatments of OKT4A + C, and sorting into CD4-, CD4-dull, and CD4-intermediate mutants. These mutants express reduced CD4 levels ranging from 0 to 60% of the parental line. The mutants were analyzed by staining with a battery of CD4-specific mAb, by assessing their ability to bind soluble gp120, and by their ability to form syncytia after infection with cell-free HIV I virus and a gp160-vaccinia vector. Two groups of particularly interesting mutants were identified: (1) CD4-dull mutants expressing only 5 to 10% of the wild type surface CD4 density, which nevertheless were infectable by HIV I and produced as many syncytia and reverse transcriptase activity as the parental line after infection with gp160-vaccinia or cell free HIV I. (2) CD4-intermediate mutants (30 to 60% of parental CD4 level), which express CD4-epitopes required for interaction with the HIV I envelope protein, yet are markedly deficient in their ability to form syncytia after gp160-vaccinia or HIV I infection. Two of these mutants did form syncytia after transient reconstitution with a wild type CD4 containing vaccinia vector. Inasmuch as they were found to bind soluble gp120 with the same avidity as other, functionally normal, CD4-intermediate mutants, these human T cell mutants may have a reduced susceptibility to HIV I infection due to the absence of a "fusogenic component" or to a structural alteration in a region of the CD4 molecule not required for binding of the HIV I envelope, but for the subsequent fusion and entry process.

Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency.

GC3/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methanesulfonate, and three clones deficient in thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1. 45) activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS- c1 and TS- c3), whereas one clone (TS- c2) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2'-deoxyuridine 5'-monophosphate-binding capacity and with incorporation of 2'-deoxy[6-3H]uridine into DNA. In the presence of optimal thymidine, growth rates were only 5-18% that of the parental clone (GC3/c1), which grew equally well in thymidine-deficient or -replete medium. Analysis of poly(A)+ RNA showed normal levels of a 1.6-kilobase transcript in TS- c1 and TS- c2 but decreased levels (approximately 6% control) in TS- c3. Clone TS- c3 was 32-, 750-, and greater than 100,000-fold more resistant than the parental clone to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, and methotrexate, respectively. When inoculated into athymic nude mice, each TS- clone produced tumors, demonstrating continued ability to proliferate in vivo.

Enhanced constitutive expression of the 27-kDa heat shock proteins in heat-resistant variants from Chinese hamster cells.

Four heat-resistant variants were isolated after treatment of Chinese hamster lung cells with the mutagen ethyl methane sulfonate, followed by a single-step selection procedure consisting in a severe hyperthermic treatment of 4 h at 44 degrees C. The isolated clones had a stable resistant phenotype for at least 150 generations during which they showed a 5,000-fold increased survival to a 4-h treatment at 44 degrees C when compared to wild-type cells. Comparative two-dimensional electrophoretic analyses of proteins revealed that, like induced thermotolerant wild-type cells (i.e., cells induced to a transient physiological state of thermotolerance by a sublethal heat conditioning treatment administered 18 h before), the heat-resistant variants had, at normal temperature, an increased content of a heat-shock protein with Mr of 27,000 (HSP27). In three of the four heat-resistant variants, the increased content of HSP27 was correlated with a two-fold increase in the constitutive level of the mRNA encoding HSP27. Chinese hamster HSP27 is composed of three species that differ in their relative isoelectric point, among which the two most acidic forms are phosphoproteins. In both the heat-resistant variant and wild-type cells, heat shock induces a rapid enhancement of the phosphorylation of HSP27: maximal phosphorylation occurs within 10 min upon changing the incubation temperature from 35 degrees to 44 degrees C. A concomitant shift in silver-staining intensity is rapidly detectable between the three isoforms, which seems to indicate that the two phosphorylated species represent post-translational modifications of the more basic species. It is concluded that most likely the enhanced expression of HSP27 is linked to the resistant phenotype of the variants. The study provides supporting evidence that both the content and phosphorylation status of HSP27 are determining factors in the ability of cells to survive hyperthermic treatments.

Genetic Analysis of the Heterochromatin of Chromosome 3 in Drosophila Melanogaster. II. Vital Loci Identified through Ems Mutagenesis.

Chromosome 3 of Drosophila melanogaster contains the last major blocks of heterochromatin in this species to be genetically analyzed. Deficiencies of heterochromatin generated through the detachment of compound-3 chromosomes revealed the presence of vital loci in the heterochromatin of chromosome 3, but an extensive complementation analysis with various combinations of lethal and nonlethal detachment products gave no evidence of tandemly repeated vital genes in this region. These findings indicate that the heterochromatin of chromosome 3 is genetically similar to that of chromosome 2. A more thorough genetic analysis of the heterochromatic regions has been carried out using the chemical mutagen ethyl methanesulfonate (EMS). Seventy-five EMS-induced lethals allelic to loci uncovered by detachment-product deficiencies were recovered and tested for complementation. In total, 12 complementation groups were identified, ten in the heterochromatin to the left of the centromere and two to the right. All but two complementation groups in the left heterochromatic block could be identified as separate loci through deficiency mapping. The interallelic complementation observed between some EMS-induced lethals, as well as the recovery of a temperature-sensitive allele for each of the two loci, provided further evidence that single-copy, transcribed vital genes reside in the heterochromatin of chromosome 3. Cytological analysis of three detachment-product deficiencies provided evidence that at least some of the genes uncovered in this study are located in the most distal segments of the heterochromatin in both arms. This study provides a detailed genetic analysis of chromosome 3 heterochromatin and offers further information on the genetic nature and heterogeneity of Drosophila heterochromatin.

A mammalian mutant cell lacking detectable lanosterol 14 alpha-methyl demethylase activity.

A Chinese hamster ovary cell mutant, AR45, was selected for amphotericin B resistance after treatment with the mutagen ethyl methanesulfonate. The mutant is a cholesterol auxotroph with a deficiency in cholesterol biosynthesis. Whole cell experiments demonstrate that the mutant accumulates the C30 sterols, lanosterol and dihydrolanosterol, under culture conditions which promote active sterol biosynthesis. Metabolic studies show that the C29 sterol demethylation product of lanosterol, but not lanosterol itself, is actively converted to end product cholesterol by whole cells as well as by microsomal preparations derived from the mutant. Detectable amounts of several cytochromes can be observed spectrally in the AR45 demonstrating that it is not a general heme-deficient mutant. Collectively, these results characterize the AR45 mutant cells as being lanosterol 14 alpha-methyl demethylase-deficient. The cell line should prove useful in studying regulation of the demethylase enzyme and the putative endogenous regulatory oxysterol. It should also be a useful tool in the molecular cloning and elucidation of genetic properties of the demethylase.

Lethal sectoring is not the basis for EMS-induced pure mutant clones in Chinese hamster cells

Treatment of G1-synchronized mammalian cells with mutagenic agents which act on one strand of the DNA at a given site would be expected to produce colonies containing both mutant and wild-type cells (mosaic). We have observed that in addition to mosaic colonies, G1-synchronized Chinese hamster ovary cells which had been treated with the single-strand mutagen ethyl methanesulfonate (EMS), produced colonies in which all the cells lacked glucose-6-phosphate dehydrogenase activity. These completely mutant (pure) colonies could be derived from a potentially mosaic colony by the “death” of the wild-type cell after the first cell division, leaving only the glucose-6-phosphate dehydrogenase (G6PD)-deficient cell to grow into a colony (lethal sectoring). Using time-lapse photography to analyze cell lineages after EMS treatment, we find that cell lysis, cell release, cell migration, or proliferative failure of one lineage at the 2-cell stage accounts for only 20–25% of the pure mutant colonies observed. This result suggests that in the Chinese hamster cell there exists a mutational mechanism which fixes the mutation in both strands of the DNA before the next replication cycle following EMS treatment.

Factors relating to the aerotolerance of Spirillum volutans

A variant of the microaerophile Spirillum volutans capable of growth under an air atmosphere (21% oxygen) was obtained by sequential selection and stabilization of colonies able to grow on a casein hydrolysate – succinate – potassium metabisulfite medium under an atmosphere of 14, 16, 18, 20, and finally 21% O2. A small amount of bisulfite (0.0025%) was still required, however, for aerobic growth. Both the variant and the wild type lacked catalase activity, and they had the same levels of superoxide dismutase activity. However, the mutant possessed 13 times more donor: hydrogen-peroxide oxidoreductase (peroxidase) activity (0.008 IU) than the wild type (0.0006 IU). An aerotolerant mutant was also isolated in one step by treatment with the mutagenic agent ethyl methane sulfonate, followed by incubation under 21% O2; it was found to possess approximately three times the peroxidase activity of the wild type. This mutant lacked catalase activity and the level of superoxide dismutase activity it contained was comparable with the wild type.

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.