Induced Mutation Frequency Research Articles

RecA-independent mutagenesis in Escherichia coli: effects of umuC and mucB mutations.

We have studied the effects of a umuC mutation on reversion of the lacZ19124 and lacZ19136 frameshift markers of Escherichia coli. Introduction of a umuC::Tn5 mutation into a strain carrying the lacZ19136 marker resulted in enhanced reversion by 9-aminoacridine and the acridine half-mustard ICR191, whereas reversion of the lacZ19124 marker was decreased (but not abolished) in a umuC strain. The reversion frequency of the lacZ19136 marker was decreased by the presence of plasmid pKM101, and further decreased by a derivative of pKM101 in which the mucB gene was inactivated by a Tn5 insertion. The reversion frequency of the lacZ19124 marker was relatively unchanged by either plasmid. Since both 9-aminoacridine and ICR191 mutagenesis of these strains is independent of the recA+ and lexA+ gene products, these results may suggest a broader role for the UmuC protein in regulating induced mutation frequencies than has previously been suspected. The contrasting effects of the umuC and mucB mutations on reversion of the lacZ19136 marker may suggest a copy number effect, or perhaps more likely that there are inherent (functional) differences between the UmuC and MucB proteins.

Isolation of protease-proficient, recombinase-deficient recA mutants of Escherichia coli K-12.

We isolated recA mutants with altered protease activity and then examined recombinase activity to determine whether the protease and recombinase functions of the RecA protein of Escherichia coli are separable. We found five mutants that had moderately strong constitutive RecA protease activity but no recombinase activity above the delta recA strain background, the first clear-cut examples of mutants of this class, designated Prtc Rec-. We also isolated 65 mutants that were protease-defective toward the LexA repressor and found that all of them were also recombinase deficient. Four of these mutants retained both partial recombinase activity and partial inducible protease activity. The recombinase-defective mutants were much more sensitive than the recA+ strain to crystal violet, kanamycin, and chloramphenicol, indicating altered membrane permeability. The recA (Prtc Rec-) mutants had a subtle alteration in protease specificity, all being defective in spontaneous induction of phages lambda imm434 and 21. They differed from Prtc Rec+ mutants of comparable or even weaker constitutive protease strength, all of which showed dramatic spontaneous induction of these prophages. However, treating a Prtc Rec- mutant with mitomycin C resulted in significant prophage induction. Thus, the RecA proteins of the Prtc Rec- mutants have constitutive protease activity toward the LexA repressor, but have only DNA damage-activable protease activity toward phage repressors. UV-induced mutagenesis from his to his+ was studied for one Prtc Rec- mutant, and induced mutation frequencies as high as those for the recA+ strain were found despite the absence of recombinase activity.

Analysis of trifluorothymidine-resistant (TFT r) mutants of L5178Y/TK +/− mouse lymphoma cells

Three classes of TFT r variants of L5178Y/TK +/− - 3.7.2C mouse lymphoma cells can be identified — large colony (λ), small colony (σ), and tiny colony (τ). The σ and λ mutants are detectable in the routine mutagenesis assay using soft agar cloning. The τ mutants are extremely slow growing and are quantitated only in suspension cloning in microwells. Variants of all three classes have been analyzed in the process of evaluating the usefulness of the thymidine kinase locus in L5178Y/TK +/− mouse lymphoma cells for detecting induced mutational damage. 150 of 152 variants from mutagen treated cultures and 163 of 168 spontaneous mutants were TFT r when rechallenged approximately 1 week after isolation (3 weeks after induction). All of the 41 mutants assayed for enzyme activity were TK-deficient. The σ and τ phenotypes were found to correlate with slow cellular growth rates (doubling time > 12 h), rather than from effects of the TFT selection or mutagen toxicity. Cytogenetic analysis of σ mutants approximately 3 weeks after induction shows an association between the σ phenotype and readily observable (at the 230–300 band level) chromosomal abnormalities (primarily translocations involving that chromosome 11 carrying the functional TK gene) in 30 of 51 induced mutants studied. Using an early clonal analysis of mutants (approximately 2 weeks after induction) 28 of 30 σ mutants showed chromosome 11 rearrangements. All λ mutants studied (17 of 17 evaluated 3 weeks after induction and 8 of 8 evaluated 2 weeks after induction) showed normal karyotypes (at the 230–300 band resolution level), including the chromosome 11s. These observations support the hypothesis that σ (and likely τ) mutants represent chromosomal mutations and λ mutants represent less extensive mutations affecting the TK locus. The inclusion of σ mutants in the total induced mutant frequency, as well as distinguishing them as a separate subpopulation of TK-deficient mutants, is, therefore, essential in obtaining maximum utility of the information provided by the L5178Y/TK +/− mouse lymphoma assay.

Mutation induction in Escherichia coli incubated in the reaction mixture of NADPH-dependent lipid peroxidation of rat-liver microsomes

Experiments were carried out to examine mutation induction in E. coli cells incubated in the reaction mixture of NADPH-dependent lipid peroxidation of microsomes isolated from rat liver. The results obtained were as follows: (1) Lipid peroxidation of microsomes occurred extensively on incubation with NADPH and Fe 2+. In the E. coli WP2 uvrA(pKM101) system, the mutation frequency to streptomycin resistance increased markedly when the cells were incuated in the reaction mixture of microsomal lipid peroxidation. The induced mutation frequencies were dependent on the extent of the lipid peroxidation. (2) It was also found that the mutations were induced at the same rate as in the case (1) when the cells added to the microsomal suspensions after the reactions due to short-lived free radicals had terminated.(3) The cytotoxicity of the lipid peroxidation products was larger in the DNA repair-defective mutant, E. coli SR18 ( uvrArecA) than the wild-type strain, SR749. From these results it is concluded that some DNA-damaging and mutagenic substances are indeed produced in the degradation process of peroxidized polyunsaturated fatty acids in liver microsomal lipids.

Evidence for linear response for the induction of mutations in human cells by x-ray exposures below 10 rads.

The induction of 6-thioguanine resistance (6TGR) was studied in continuous human lymphoblast cultures exposed to daily x-ray doses of 1, 2.5, 5, or 10 rads (1 rad = 0.01 gray) for periods up to 1 month. Samples were taken every 5 days for determinations of induced mutation frequency. Cells receiving daily doses of 1-10 rads showed a mutation frequency of 0.069 X 10(-6) 6TGR cell per rad, virtually identical to the value of 0.062 X 10(-6) 6TGR cell per rad determined for lymphoblasts receiving the same total dose of radiation in a single acute exposure. The effects of small daily fractions were additive, suggesting that doses as small as 1 rad are mutagenic in human lymphoblasts. Similar results were observed when resistance to trifluorothymidine, indicative of thymidine kinase deficiency, was used as a mutational marker. When 6TGR frequency was plotted against days of irradiation, a positive linear slope was observed for all dose groups. The values of these slopes were plotted against x-ray dose in rads/day to construct a dose-response relationship for 1-10 rads. A linear increase in mutation frequency was observed over this dose range, with no apparent threshold. The slope of this linear increase was 0.060 X 10(-6) 6TGR cell per rad. These results suggest that, for human lymphoblasts, the mutagenic risk of low doses of x-rays can be accurately estimated by linear extrapolation from high-dose effects.

VALINE-RESISTANCE, A POTENTIAL MARKER IN PLANT CELL GENETICS. II. OPTIMIZATION OF UV MUTAGENESIS AND SELECTION OF VALINE-RESISTANT COLONIES DERIVED FROM TOBACCO MESOPHYLL PROTOPLASTS

The induction and selection of valine-resistant mutants from haploid tobacco (Nicotiana tabacum L.) mesophyll protoplast-derived cells have been studied. Using cells from an original mutant plant obtained previously, we performed reconstruction experiments in order to determine the best conditions for the recovery of resistant cells among a population of sensitive cells. Optimal selective conditions were shown to depend on various factors including cell density, time of addition of valine and seasonal variations affecting the mother plants.-Using cell densities of approximately 10( 4) cells/ml, we defined efficient selective conditions: more than 25% of the putative mutant clones selected from UV-mutagenized protoplasts were reproducibly confirmed to be valine resistant. Further characterization of some regenerated mutant plants indicated that valine-resistance was associated with an uptake deficiency, as in the case of the original mutant plant of the Val(r)-2 line used for reconstruction experiments. Spontaneous mutation rates for valine-resistance were below accurately detectable levels, i.e., less than 10(-6) per cell per generation. Induced mutation frequency varied nonlinearily with UV dose from 10(-5) to 5 x 10(-4) resistant clones per surviving colony. Two independent loci (vr2 and vr3) were previously shown to be involved in valine-resistance due to amino acid uptake deficiency. Haploid tobacco plants were produced through anther culture from an F(1) double-heterozygous plant obtained from a cross between the original mutant plant and a wild-type plant. Study of the level of resistance to valine of protoplast-derived cells allowed the classification of these haploid plants in four types: sensitive, resistant and two intermediary resistant types believed to result from the presence of a mutant allele at only one of the two loci involved. The frequencies of UV-induced mutations in cells derived from haploid plants of one of the intermediary types were compared to those observed in wild-type cells. The results are considered in light of the amphidiploid structure of the tobacco genome.

SCE induction is uncoupled from mutation induction in mammalian cells following exposure to ethylnitrosourea (ENU).

It has been suggested not only that sister chromatid exchange (SCE) induction might serve as a qualitative indicator of mutagenesis, but also that induced SCE frequencies are linearly related to induced mutation frequencies. The consistency of the relationship between SCEs and mutations was tested in the present work. Confluent Chinese hamster ovary (CHO) cells were exposed to ethylnitrosourea (ENU) and then held at confluency for various times prior to initiation of SCE and mutation assays. Cells held at confluency are typically thought to be arrested in their progression through the cell cycle, so that "S-dependent" processes such as fixation of mutations and formation of SCEs will not occur, while DNA repair processes might continue to operate. If repair processes reduce the number of SCE-inducing lesions during the holding period and, hence, reduce the subsequently determined SCE frequencies, then mutation frequencies should similarly be reduced if SCEs and mutations are related. It was observed, however, that induced SCE frequencies decreased exponentially with holding time, while mutation frequencies remained constant. Qualitatively similar results were obtained in log-phase cells. Cell cycle analysis demonstrates that confluent CHO cells can cycle, and ways are considered in which this might affect SCE and mutation frequencies. It is concluded that the decline in SCE frequency (with time) cannot be attributed solely to the presence of cycling cells in confluent cultures. It appears, therefore, that at least some forms of ENU-induced DNA damage that can lead to SCE were repaired and as such are distinct from those forms that are mutagenic. Thus SCEs are not necessarily related to mutations, because the two events may represent manifestations of different forms of DNA damage. Whether or not this represents a universal phenomenon that would hold true for agents other than ENU remains to be determined.

Specific-locus mutation rates in the mouse following inhalation of ethylene oxide, and application of the results to estimation of human genetic risk

Male (101 × C3H)F 1 mice were exposed in an inhalation chamber to ethylene oxide (EtO) in air at a concentration of (generally) 255 ppm. After accumulating total exposures of 101 000 or 150 000 ppm.h in 16–23 weeks, the males were mated to T-stock females for a standard specific-locus mutation-rate study in which 71 387 offspring were observed. The spermatogonial stem-cell mutation rate at each exposure level, as well as the combined result, does not differ significantly from the historical control frequency. At the lower and higher exposure levels, the results rule out (at the 5% significance level) an induced frequency that is, respectively, 0.97 and 6.33 times the spontaneous rate; the combined results rule out a multiple of 1.64. The relationship between mouse spermatogonial stem-cell mutation rates and EtO-induced testis ethylations was compared with the relationship between Drosophila post-stem-cell mutation rates and sperm ethylations (Lee, 1980). The comparison does not rule out equal mutability per ethylation; but it cannot prove parallelism. An assessment of the mouse-Drosophila relationship will require a more efficient alkylator than EtO and the use of comparable germ-cell stages. More meaningful conclusions may be drawn by utilizing the data for direct estimation of human risk by expressing the induced mutation frequency that is ruled out (at the 5% significance level) as a multiple of control rate and extrapolating to human exposure levels. The probable absence of major stem-cell killing (and thus, possibly, cell selection) by EtO indicates that such extrapolation probably does not produce an underestimate. For a human exposure concentration of 0.1 ppm on working days during the reproductive lifespan, the mouse experimental results rule out (at the 5% significance level) an induced spermatogonial stem-cell gene mutation rate greater than 8% of the spontaneous rate; for 1.0 ppm, they rule out an induced rate roughly equal to the spontaneous rate. The induced rate for any one poststem-cell stage would have to be about 3 orders of magnitude higher than that for stem cells to constitute an equivalent risk.

The Genotoxicity of Alpha Particles in Human Embryonic Skin Fibroblasts

Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to alpha particles from 238Pu (LET at the cell surface was 100 keV/microns) and 250 kVp X rays. The survival curves resulting from exposure to alpha particles are exponential. The mean lethal dose, D0, is approximately 1.3 Gy for X rays and 0.25 Gy for alpha particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for alpha particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to alpha particles than to X rays.

Mutations in Salmonella typhimurium and inactivation of Bacillus subtilis transforming DNA induced by phenylhydroxylamine derivatives

Phenylhydroxylamine (PHA) and its derivatives such as monomethyl (2-Me, 3-Me, 4-Me) and dimethyl (2,3-diMe, 2,4-diMe, 2,5-diMe, 2,6-diMe, 3,4-diMe, 3,5-diMe) were tested for their mutagenicity and for their inducing ability to inactivate transforming DNA. All these compounds except PHA and 3,5-diMePHA were found to be mutagenic in Salmonella typhimurium TA100 even in the absence of S9 mix, and their mutagenic potency was in the order: 2,6-diMe- > 2,4-diMe- = 3,4-diMe- > 4-Me- > 2,3-diMe- = 2,5-diMe- > 2-Me]- = 3-MePHA. Besides mutagenicities, all the PHA derivatives except 2,6-diMePHA caused severe reductions in the activity of Bacillus subtilis transforming DNA. To establish the structure-activity relationship, we examined the correlation between these activities and the stabilities of the PHA derivatives, and the results indicated that the more chemically unstable the PHA derivatives were, the more active they were with respect to the mutations and to the inactivation of the transforming DNA. The mutagenic activity of 2,6-diMePHA was the sole exception, because it was most stable, but its induced mutation frequency was highest. From these results, we suggest that all the PHA derivatives, except 2,6-diMePHA, cause DNA damage through the generation of active molecular species, such as nitrenium ions, without any enzymatic activation, while 2,6-diMePHA requires further metabolic activation by bacterial enzymes to stimulate mutagenesis.

PHOTOBIOLOGICAL ACTIVITY OF 4‐METHYLPSORALEN and 4‐METHYL‐4‘,5’‐DIHYDROPSORALEN WITH RESPECT TO LETHAL and MUTAGENIC EFFECTS ON E. coli, and PROPHAGE INDUCTION

Abstract— The lethal and mutagenic effects on E. coli as well as the induction of prophage lambda were determined after treatment with 4‐methylpsoralen, 8‐methoxypsoralen, psoralen or 4‐methyl‐4′,5′‐dihydropsoralen and UV‐A irradiation. All psoralens used caused photokilling and photomutagenesis of strains H/r30R and Hs30R. 4‐Methylpsoralen was more efficient for killing and for the induced mutation than 8‐methoxypsoralen or psoralen in view of the dose modification factor. This finding can be explained by the methylation effect of psoralen. 4‐Methylpsoralen induced more mutation in Hs30R than in H/r30R. Monofunctional 4‐methyl‐4′,5′‐dihydropsoralen required much higher fluence than bifunctional psoralens to kill cells and to induce the mutation. When the induced mutation frequency was expressed as a function of survival, mutagenic efficiency ranked in the following order: 8‐methoxypsoralen > psoralen > 4‐methylpsoralen > 4‐methyl‐4′,5′‐dihydropsoralen. 4‐Methylpsoralen was 3–4‐fold less mutagenic than 8‐methoxypsoralen in this plot. Lytic growth of prophage in E. coli AB1157 (ñ) was induced by the treatment. When the bifunctional psoralens were used, the maximum induced fraction was larger than 20%. However, it was only 2% when 4‐methyl‐4′,5′‐dihydropsoralen was used.

Effects of furazolidone on the mutation ofVibrio cholerae cells to streptomycin resistance

Furazolidone induced the streptomycin-resistant (Str-r) forward mutation ofVibrio cholerae (classical) OGAWA 154 cells. The induced mutation frequency increased up to the furazolidone dose of 7.0 μg/ml×h and then gradually declined. Statistical analysis (t-test and variance analysis) revealed that the furazolidone-induced mutation ofV. cholerae cells at any of the doses studied was highly significant.

Recovery from Lethal and Mutagenic Damage during Postirradiation Holding and Low-Dose-Rate Irradiations of Cultured Hamster Cells

Survival and mutation to thioguanine resistance were measured in V79-4 hamster cells grown to plateau phase without refeeding and irradiated with 60Co gamma rays. The effects of low-dose-rate irradiation and of postirradiation holding on recovery from gamma-ray damage leading to these two responses were also studied. The responses of these plateau (extended G1)-phase cells to acute irradiation were similar to those we previously found for exponentially growing cells, including the linear relationship between induced mutant frequency and (log) surviving fraction. Irradiation at low dose rate (0.34 rad/min) considerably reduced both the lethal and mutagenic effects of given doses of gamma rays, but the linear mutation-survival relationship was approximately the same as for acute irradiation. In contrast, cells given a 5-hr holding period after acute irradiation showed the anticipated recovery from potentially lethal damage but no recovery from damage leading to mutation. These results are discussed in terms of previously proposed cellular repair processes (sublethal damage repair and potentially lethal damage repair) and the possibility that the radiation damage leading to lethality is different from mutagenic damage.

Influence of confluent holding time on UV light mutagenesis in human diploid fibroblasts

We have investigated the induction of mutants resistant to 6-thioguanine (6TG) following 254 nm ultraviolet light exposure of density-inhibited cultures of human diploid fibroblasts. Phenotypic expression of 6TG resistance was maximal within 9 days and remained stable through 19 days after irradiation. In reconstruction studies, complete recovery of 6TG-resistant mutants occurred at cell densities of up to 35 000 cells per 100-mm petri dish. The induced mutation frequency increased linearly with dose over the range of 3–9 J/m 2; the D 0 of the survival curve was 4.2 J/m 2. Delaying subculture to low density for 1.5–24 h after irradiation produced unexpected alterations in induced mutation frequencies. An increase in UV-induced mutations of approximately 3-fold was observed in cultures maintained in confluence for 3 h. This trend was reversed with longer holding times: the mutation frequency declined sharply in cultures held for 6 h compared to the 3-h value, and thereafter showed a steady and gradual diminution to background levels. These data suggest that the repair of potentíally mutagenic damage is a complex phenomenon which can lead to an increase or decrease in mutation frequency as a function of holding time. Although the decline in mutation frequency observed following longer holding intervals is consistent with the notion of an error-free process, we hypothesize that the increased mutation frequency produced by a short holding period reflects the existence of a cell-mediated process which enhances the mutagenic potential of at least some UV-induced DNA photoproducts.

The mutagenicity of a cyanide antidote, dimethylaminophenol, in Chinese hamster cells

The mutagenic activity of a cyanide antidote, dimethylaminophenol (DMAP), has been studied at the hypoxanthine-guanine-phosphoribosyltransferase (HPRT) locus in a line of Chinese hamster cells (V79), using induced resistance to 8-azaguanine as a marker for forward, point mutation. In a replating type of assay DMAP produced a dose-dependent increase in mutation frequency in suspension-treated cells, in the absence of any extrinsic metabolic activation system. The incorporation of a liver microsome preparation raised the concentration of DMAP required to induce mutation, but similar levels of induced mutation frequency (IMF) were produced.

Induction and expression of mutations at multiple drug-resistance marker loci in Chinese hamster ovary cells.

We have observed quantitative and qualitative differences in the mutability and mutagen-specificity of various drug-resistance marker loci in Chinese hamster ovary (CHO) cells, which suggest that mammalian gene loci may differ in their relative mutability by a given mutagenic agent. We have used the CHO-AT3-2 multiple-marker mutagenesis assay system to examine the dose-dependent induction and kinetics of expression of mutations at four well-characterized, drug-resistance marker loci, after treatment with chemical agents which produce various types of DNA damage. The CHO-AT3-2 subline allows simultaneous quantitation and direct comparison of induced mutation frequencies at the hgprt, oua (Na+/K+ ATPase), aprt, and tk loci. The agents tested in this study included ethyl methanesulfonate, methyl methanesulfonate, mitomycin C, ICR-191, benzo[a]pyrene, and dimethylnitrosamine. The expression kinetics and optimal expression times for each drug-resistance marker were determined in dose-response experiments in which cells from mutagen-treated populations were plated at 1-2-day intervals over a period of 10 days following mutagenesis. Comparison of induced mutation frequencies for each drug-resistance marker after mutagen treatments yielding equivalent cell survivals (equitoxic doses resulting in relative cell survivals of 0.37) revealed locus-specific differences in the relative mutagenicities of the agents tested. These results indicate that the apparent mutagenicity of a particular agent at a single genetic locus may not necessarily be an accurate indicator of that agent's mutagenic potential for the genome as a whole.

Induction of mutations by 5-fluorodeoxyuridine: A mechanism of self-potentiated drug resistance?

In medium containing concentrations of deoxycytidine that occur in vivo , 5-fluorodeoxyuridine induced mutation frequencies 6–90 fold greater than spontaneous mutant frequencies at two genetic loci in Chinese hamster cells. In medium lacking deoxycytidine, 5-fluorodeoxyuridine was more cytotoxic but induced no mutants. Hence, the effectiveness of cancer therapy with 5-fluorodeoxyuridine may be limited by self potentiated development of 5-fluorodeoxyuridine-resistant mutants and enhanced and prolonged by manipulating deoxycytidine metabolism.

Mutation frequencies in male mice and the estimation of genetic hazards of radiation in men.

Estimation of the genetic hazards of ionizing radiation in men is based largely on the frequency of transmitted specific-locus mutations induced in mouse spermatogonial stem cells at low radiation dose rates. The publication of new data on this subject has permitted a fresh review of all the information available. The data continue to show no discrepancy from the interpretation that, although mutation frequency decreases markedly as dose rate is decreased from 90 to 0.8 R/min (1 R = 2.6 x 10(-4) coulombs/kg) there seems to be no further change below 0.8 R/min over the range from that dose rate of 0.0007 R/min. Simple mathematical models are used to compute: (a) a maximum likelihood estimate of the induced mutation frequency at the low dose rates, and (b) a maximum likelihood estimate of the ratio of this to the mutation frequency at high dose rates in the range of 72 to 90 R/min. In the application of these results to the estimation of genetic hazards of radiation in man, the former value can be used to calculate a doubling dose--i.e, the dose of radiation that induces a mutation frequency equal to the spontaneous frequency. The doubling dose based on the low-dose-rate data compiled here is 110 R. The ratio of the mutation frequency at low dose rate to that at high dose rate is useful when it becomes necessary to extrapolate from experimental determinations, or from human data, at high dose rates to the expected risk at low dose rates. The ratio derived from the present analysis is 0.33.

Modern developments in mutagenesis

AbstractMutants of Penicillium chrysogenum and Streptomyces clavuligerus have been isolated on the basis of increased sensitivity to far ultraviolet light far (u.v). In the former case the strain was also sensitive to a wide range of other mutagens, including alkylating agents and ionising radiations, but proved not to be hypermutable by any of these, whereas in S. clavuligerus two strains were hypermutable relative to their wild‐ type progenitor when treated with far u.v. In addition, various environmental conditions, including liquid holding and treatment with caffeine, isoniazid and nalidixic acid, increased mutant yield. The results obtained clearly show that induced mutation frequency can be enhanced both by mutation of genes involved in DNA repair and by modification of environmental conditions during or after mutagenic treatment. The importance of this and other modern developments in mutagenesis for the design of rational industrial strain‐improvement programmes is discussed.

Methods and criteria for deciding whether specific-locus mutation-rate data in mice indicate a positive, negative, or inconclusive result

The binomial approximation of the UMPU (uniformly most powerful unbiased) test for the equality of 2 binomial proportions is shown to be a highly accurate and easily applied method for testing the hypothesis that a given mouse specific-locus mutation frequency is not higher than the spontaneous mutation frequency (43 mutations in 801 406 offspring, for males). Critical sample sizes have been calculated that show at a glance whether P < 0.05. The first hypothesis that the mutation frequency (induced + spontaneous) of treated mice is not higher than the spontaneous mutation frequency is combined with the second hypothesis that the induced mutation frequency of treated mice is no less than 4 times the historical-control mutation frequency to produce a multiple decision procedure with 4 possible decisions: inconclusive result, negative result, positive result, and weak mutagen. Critical sample sizes for the second hypothesis, also with P < 0.05, are combined with those for the first hypothesis into a grid that permits rapid evaluation of data according to these criterea. The justification for using these criteria in reaching decisions, assuming a high level of exposure has been given, is the practical necessity of rapidly determining which chemicals are potent mutagens. Positive results can become apparent in relatively small samples. Larger samples, of at least 11 166 offspring, are required to obtain a negative result. If samples of 18 000 are routinely collected (unless positive results are found earlier), 75% of tests of chemicals that are non-mutagens will give a negative result. If the question being asked is not whether a chemical induces gene mutations but, rather, whether the exposure received by humans causes any important risk from gene mutations, a much smaller sample size may be acceptable, under certain conditions. A comparison of the relative efficiencies of the specific-locus test (for gene mutations and small deficiencies) and the heritable-translocation test (for transmissible chromosome rearrangements), in detecting the same proportional increases over the spontaneous frequencies of their respective types of genetic damage, shows that less work is involved in reaching a conclusive result in the specific-locus test. Proposed specific-locus tests using biochemical markers are at a considerable statistical disadvantage compared with the standard test (using 7 visible markers) for which there is available a very large historical control showing a very low mutation rate.