Two N-hydroxylaminopurines are highly mutagenic in the ad-3 forward-mutation test in growing cultures of heterokaryon 12 of Neurospora crassa

3 purine analogs were tested for their mutagenic activities in the ad-3 forward-mutation test in heterokaryon 12 (H-12) of Neurospora crassa. In growing cultures of H-12, the N-hydroxylaminopurines 2-amino-6-N-hydroxylaminopurine (AHA) and 6-N-hydroxylaminopurine (HAP) are potent and strong mutagens, respectively, whereas 2-aminopurine (AP) is a weak mutagen. AHA and HAP are about equally mutagenic at low doses, but AHA is more mutagenic than HAP at high doses. Despite their potent mutagenicity in growing cultures, AHA and HAP are not mutagenic in nongrowing conidia under the conditions of our experiments. AHA is the most potent mutagen tested in the ad-3 forward-mutation test in N. crassa. At the highest dose tested (30 micrograms/ml), it gave an ad-3 mutant frequency of 0.7 X 10(-2), about a 12,000-fold increase over the average spontaneous ad-3 mutant frequency. The potent mutagenicity of AHA may make it (and possibly HAP) especially useful for obtaining specific-locus mutations in other organisms.     

Influence of N2 or O2 on two alkylating agents in Neurospora crassa.

Previous studies have indicated that the alkylating agent, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine dihydrochloride (ICR-170), induces much more killing and mutation in conidia of Neurospora crassa treated in an atmosphere of N2 than in an atmosphere of O2. It was desirable to determine if a similar effect--more killing and mutation in N2 than in O2--could be observed with two other known alkylating agents, beta-propiolactone (BPL) and ethyl methanesulfonate (EMS), in the same test system. Conidia of a heterokaryotic strain of N. crassa were bubbled with N2 or O2 during treatment with BPL or EMS. Forward-mutation was measured in the ad-3 region by a direct method. The results indicate that N2 or O2 do not influence the lethal and mutagenic activities of BPL or EMS during treatment of conidia. Hence the influence of N2 or O2 on the lethal and mutagenic activites of ICR-170 is different from the influence of these gases on BPL or EMS using the ad-3 test system in N. crassa.     

Effect of the uvs-2 allele of Neurospora crassa on the mutagenic potency of two N-hydroxylaminopurines and 2-aminopurine in the ad-3 forward-mutation test

The mutagenic potencies of 3 purine analogs were determined in the ad-3 forward-mutation test in growing cultures of heterokaryon 59 (H-59), a nucleotide excision repair-deficient (uvs-2/uvs-2) 2-component heterokaryon of Neurospora crassa. Two N-hydroxylaminopurines, 2-amino-6-N-hydroxylaminopurine (AHA) and 6-N-hydroxylaminopurine (HAP), were potent and strong mutagens, respectively, whereas 2-aminopurine (AP) was a moderate mutagen. Dose-response curves showed that AHA and HAP were about equally mutagenic at low doses but that AHA was more mutagenic than HAP at high doses. Comparison of these results in H-59 with our earlier results in heterokaryon 12 (H-12) of N. crassa, which is identical to H-59 except for being DNA-repair-proficient (uvs-2+/uvs-2+), shows that the defect in nucleotide excision repair due to uvs-2 has little or no effect on the mutagenic potencies of these 3 purine analogs. Therefore, the nucleotide excision-repair pathway in N. crassa that is deficient in H-59 does not appear to have a major role in the repair of pre-mutational lesions induced by these 3 purine analogs. On the other hand, based on the controls of these experiments, the frequency of spontaneous ad-3 mutants was 4 greater in H-59 than in H-12. This result suggests that the nucleotide excision-repair pathway in N. crassa that is inactivated by the uvs-2 mutation has a major role in the repair of lesions that would lead to spontaneous mutation at the ad-3+ region if they were not repaired.     

Use of the spot, plate and suspension test systems for the detection of the mutagenicity of environmental agents and chemical carcinogens in Neurospora crassa.

N23 and N24, selected from hundreds of ad-3 mutants, have been used as testers for the spot, plate and suspension tests in Neurospora crassa. These two testers are highly sensitive to mutagens and are revertible by a specific group of chemicals. N23 can be reverted from an adenine-dependence to adenine-independence by agents which cause base-pair substitutions whereas N24 can be reverted by frameshift mutagens. Studies described here show that spot, plate and suspension tests using testers N23 and N24 are satisfactory substitutes for the ad-3 forward-mutation system for screening the mutagenic activity of environmental agents and chemical carcinogens in N. crassa.     

Mutagenic potency and specificity of procarbazine in the ad-3 forward-mutation test in growing cultures of heterokaryon 12 of Neurospora crassa.

Procarbazine (Natulan) was tested for its mutagenic potency and specificity in the ad-3 forward-mutation test in heterokaryon 12 (H-12) of Neurospora crassa. In these experiments, procarbazine was a weak mutagen when present in growing cultures but nonmutagenic when conidial suspensions (nongrowing conidia) were treated. A total of 208 ad-3 mutants recovered after exposure of growing cultures of H-12 to 1 mg of procarbazine/ml, and 2 ad-3 mutants of spontaneous origin, were characterized genetically. These tests distinguish among gene/point mutations (ad-3R) at the ad-3A or ad-3B locus, multilocus deletion mutations ([ad-3]IR) covering one or more loci in the ad-3 and immediately adjacent regions, and 3 different classes of multiple-locus mutations: gene/point ad-3 mutations with a recessive lethal mutation elsewhere in the genome (ad-3R + RL), gene/point mutations with a closely linked recessive lethal mutation (ad-3R + RLCL), and multilocus deletion mutations with a closely linked recessive lethal mutation ([ad-3]IR + RLCL). All of the procarbazine-induced ad-3 mutants resulted from gene/point mutations; 92.2% (200/217) resulted from gene/point mutations at the ad-3A or ad-3B locus, and 3.7% (8/217) resulted from gene/point mutations with a recessive lethal mutation elsewhere in the genome. Identical percentages (15.4% [20/130] and 15.4% [12/78]) of the sigma ad-3BR and sigma ad-3AR mutants were leaky, and a high percentage (71.5% [93/130]) of the sigma ad-3BR mutants had nonpolarized complementation patterns. These results indicate that procarbazine-induced ad-3 mutants of Neurospora crassa are composed solely of gene/point mutations (ad-3R) that resulted, predominantly or exclusively, from base-pair substitutions. The Neurospora specific-locus data on procarbazine-induced ad-3 mutants are compared with data from similar experiments with the mouse using the morphological specific-locus assay; marked similarities were found between the mutagenic effects of procarbazine in the 2 specific-locus assays.     

Mutagenic activities of aflatoxin B 1 and G 1 in Neurospora crassa

Summary The mutagenicity and mutagenic specificity of aflatoxin B₁ and G₁ were studied with the adenine-3 (ad-3) test system of Neurospora crassa. Aflatoxin B₁ and G₁ failed to show mutagenicity in resting conidia, but both agents were mutagenic in growing vegetative cultures. The frequencies of ad-3 mutants induced by aflatoxin B₁ and G₁ (40g/ml) were 70.7x10^-6 survivors and 9.6x10^-6 survivors, respectively. Since aflatoxin B₁ gave a 177-fold increase over the spontaneous mutation frequency it is a rather potent mutagen, whereas aflatoxin G₁ gave only a 24-fold increase and so is only moderately mutagenic.Genetic analyses of ad-3 mutants induced by aflatoxin B₁ and G₁ indicate that both agents induce a low frequency of multilocus deletions. The spectra of point mutations at the ad-3A and ad-3B loci induced by aflatoxin B₁ and G₁ are not distinguishable from each other. Hence both agents probably induce the same relative frequencies of genetic alterations. The frequencies of leakiness, allelic complementation, and classes of complementation patterns among the ad-3 mutants induced by both agents are higher than the frequencies among ICR-170-induced mutants and somewhat lower than those among NA- and AP-induced mutants. The results of reversion tests with NA, MNNG, and ICR-170 indicate that in addition to multilocus deletion, aflatoxin B₁-induced ad-3 mutants consist of frameshifts, base-pair transitions, and possibly other types of intragenic alterations.     

Comparison of the spectra of genetic damage in formaldehyde-induced ad-3 mutations between DNA repair-proficient and -deficient heterokaryons of Neurospora crassa.

The mutagenic effects of formaldehyde (FA) have been compared in DNA repair-proficient (heterokaryon 12) and DNA repair-deficient (heterokaryon 59) two-component heterokaryons of Neurospora crassa. The data from forward-mutation experiments were used to compare the spectra of FA-induced specific-locus mutations at two closely linked loci in the adenine-3 (ad-3) region and on the FA-induced inactivation of heterokaryotic conidia. Previous studies have demonstrated that specific-locus mutations at these two loci result from five major genotypic classes, namely two classes of gene/point mutations (ad-3A(R) and ad-3B(R)), and three classes of multilocus deletion mutations ([ad-3A](IR), [ad-3B](IR), and [ad-3A ad-3B](IR)). Genetic analysis of ad-3 mutants recovered from both heterokaryons after FA treatment demonstrates that predominantly gene/point mutations were found in H-12 (93.2% ad-3(R), 6.8% [ad-3](IR)) and a significantly higher frequency of multilocus deletion mutations in H-59 (62.8% ad-3(R), 37.0% [ad-3](IR)). The data from our experiments with FA on H-12 demonstrate and confirm the data from other assays that FA is a weak mutagen in this DNA repair-proficient strain. However, the data from our experiments with the DNA repair-deficient strain H-59 demonstrate that comparable concentrations of FA cause more pronounced inactivation of heterokaryotic conidia and, at the highest concentration tested, about a 35-fold higher frequency of ad-3 mutations. In addition, FA induced a 5.4-fold higher frequency of ad-3 mutations resulting from multilocus deletion mutation in H-59 than in H-12. Based on our earlier studies with X-ray-induced multilocus deletion mutations, it is this class of FA-induced ad-3 mutations that might be most expected to show deleterious heterozygous effects. The implications of the present data base from our experiments with Neurospora are that the mutagenic (and possibly the carcinogenic) effect of FA exposure might well vary in different human population subgroups.     

Comparison of toxicity and mutagenicity of methylnitrosourea, methylnitronitrosoguanidine and ICR-191 among human lymphoblast lines

The toxic and mutagenic effects of the alkylating agents methylnitrosourea (MNU) and methylnitronitrosoguanidine (MNNG) and of the frameshift mutagen, ICR-191 were compared among 3 human diploid lymphoblast lines, MIT-2, WI-L2 and GM 130. The MIT-2 and WI-L2 lines were both sensitive to the toxic and mutagenic effects of all 3 agents tested. The WI-L2 line was more sensitive to the toxic effects of MNU and MNNG than the MIT-2 line, while it was somewhat less sensitive to the mutagenic effects of these alkylating agents. The GM 130 line was strikingly resistant to both the toxic and mutagenic effects of the alkylating agents. The order of sensitivity to the toxic effect of ICR-191 was MIT-2 > WI-L2 > GM 130, while the order of sensitivity to the mutagenic effects of this frameshift mutagen was GM 130 > MIT-2 > WI-L2. These results point to the importance of accounting possible variations in mutability among individuals when extrapolating from any single mutagenicity assay for human risk assessment.     

Mutagenicity of neocarzinostatin in Neurospora crassa

Neocarzinostatin (NCS) is an acidic, single-chain polypeptide of 109 amino acids that has shown some antitumor activity in clinical trials. NCS is mutagenic in recA+ strains of Escherichia coli, but not in recA strains; on the other hand, a defect in the nucleotide-excision-repair pathway has no effect on the mutagenicity of NCS in E. coli. Similar results are seen in mammalian cells. Excision-repair-deficient xeroderma pigmentosum (XP) cells repair NCS-induced DNA damage at the same rate as repair-proficient XP heterozygotes, and X-ray-sensitive ataxia telangiectasia fibroblasts are also sensitive to NCS. I have investigated the mutagenicity of NCS in the ad-3 forward-mutation test in nucleotide excision-repair-sufficient and -deficient heterokaryons of Neurospora crassa. Resting conidia from a repair-sufficient strain, H-12, and a nucleotide-excision-repair-deficient strain (uvs-2) H-59, were exposed to NCS. These conidia were assayed for survival and ad-3 forward mutation. The results show that H-59 is more sensitive to the killing and mutagenic activities of NCS than is H-12. These data indicate, in contrast to E. coli and mammalian cells, that the nucleotide-excision-repair pathway of N. crassa does repair NCS-induced lesions. In other experiments, ad-3 mutants induced by NCS in H-59 were characterized to determine the spectrum of NCS-induced mutation. The results show that NCS induces both intracistronic mutations and multilocus deletions in H-59.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. IV. Comparison of dose-response curves for MNNG, 4NQO and ICR-170 induced inactivation and mutation-induction

The genetic effects of MNNG, 4NQO and ICR-170 have been compared on 5 different UV-sensitive strains and a standard wild-type strain of Neurospora crassa with regard to inactivation and the induction of forward-mutations at the ad-3A and ad-3B loci. Whereas all UV-sensitive strains (upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) are more sensitive to inactivation by MNNG and ICR-170 than wild-type, only uvs-5 shows survival comparable to wild-type after 4NQO treatment, all other strains are more sensitive to 4NQO. In contrast to the effects on inactivation, a wide variety of effects were found for the induction of ad-3A and ad-3B mutations: higher forward-mutation frequencies than were found in wild-type were obtained after treatment with MNNG or 4NQO for upr-1 and uvs-2, no significant increase over the spontaneous mutation frequency was found with uvs-3 after MNNG, 4NQO or ICR-170 treatment; mutation frequencies comparable to that found in wild-type were obtained with uvs-6 after MNNG, 4NQO or ICR-170 treatment and with upr-1 after ICR-170 treatment. Lower forward-mutation frequencies than were found in wild-type were obtained with uvs-2 after ICR-170 treatment and with uvs-5 after MNNG, 4NQO or ICR-170 treatment. These data clearly show that the process of forward-mutation at the ad-3A and ad-3B loci is under genetic control by mutations at other loci (e.g. upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) and that the effect is markedly mutagen-dependent.     

2-Amino-N6-hydroxyadenine induces gene/point mutations and multiple-locus mutations, but not multilocus deletion mutations, in the ad-3 region of a two-component heterokaryon of Neurospora crassa.

The mutagenicity of 2-amino-N6-hydroxyadenine (AHA) has been studied in Neurospora crassa by treating a two-component heterokaryon (H-12) and recovering specific-locus mutations induced in the ad-3 region. This assay system permits the identification of ad-3A and/or ad-3B mutants resulting from gene/point mutations, multilocus deletion mutations, and multiple-locus mutations of various genotypes, involving one or both loci. Genetic characterization of the ad-3 mutants recovered from experiments with AHA in H-12 shows that 98.9% (270/273) of the ad-3 mutants are gene/point mutations (ad-3R), 1.1% (3/270) are unknowns, and none is a multilocus deletion mutation (ad-3IR). Among the gene/point mutations, 3.3% (9/273) are multiple-locus mutations (gene/point mutations with a closely-linked recessive lethal mutation [ad-3R + RLCL]). Another 25.3% (69/273) are multiple-locus mutations with a recessive lethal mutation located elsewhere in the genome [ad-3R + RL]. Heterokaryon tests for allelic complementation among the ad-3BR mutants showed that 90.8% (139/153) of the mutants were complementing, and 20.3% (31/153) were leaky. In addition, 32.5% (38/117) of the ad-3AR mutants were leaky. These data are consistent with the hypothesis that AHA produces specific-locus mutations in the ad-3 region of N. crassa by base-pair substitution. The data from the present experiments are compared with the data for 2-aminopurine (2AP)-induced ad-3 mutants in H-12 (de Serres and Brockman, 1991). Whereas, 2AP is a weak mutagen in H-12, AHA is extremely potent (Brockman et al., 1987). In contrast with 2AP, AHA induces ad-3 mutants exclusively by gene/point mutation in H-12. We conclude that whereas AHA induces ad-3 mutants predominantly by AT to GC base-pair transitions, 2AP induces ad-3 mutants by a wide variety of mechanisms including: (1) AT to GC and GC to AT base-pair transitions, (2) frameshift mutations, (3) other, as yet unidentified, intragenic alterations, (4) small multilocus deletion mutations, and (5) multiple-locus ad-3R mutations with closely linked recessive lethal mutations.     

Effect of the homokaryotic state of the uvs-2 allele in Neurospora crassa on formaldehyde-induced killing and ad-3 mutation

Formaldehyde was tested for its killing and mutagenic activities in the ad-3 forward-mutation test in Neurospora crassa. The test was conducted in 3 two-component heterokaryons (dikaryons) of N. crassa in order to determine the effect of the uvs-2 allele, which causes a defect in nucleotide excision repair, on formaldehyde-induced killing and the induction of ad-3 mutants. These dikaryons were homokaryotic for uvs-2+ (H-12), homokaryotic for usv-2 (H-59), and heterokaryotic for uvs-2 (H-71). Formaldehyde induced killing and ad-3 mutants in H-12, but the presence of uvs-2 in the homokaryotic state (H-59) resulted in a 9-fold increase in killing and a 40-fold increase in the induction of ad-3 mutants. This increased sensitivity to formaldehyde-induced killing and mutation conferred by uvs-2 in the homokaryotic state (H-59 vs. H-12) is similar to that noted by others in Escherichia coli. Salmonella typhimurium and Saccharomyces cerevisiae. The dikaryon heterokaryotic for uvs-2 (H-71) has the same sensitivity to formaldehyde-induced ad-3 mutation as H-12, indicating that uvs-2 is recessive to uvs-2+.     

Effect of N2 on the mutagenic and lethal activities of ICR-170 in Neurospora crassa

The nature of the N₂ effect for ICR-170, i.e., the greater mutagenic and lethal activities of this agent in the presence of N₂ than O₂, has been studied at the ad-3 region of Neurospora crassa. The characteristics of the N₂ effect for ICR-170 were that (1) the N₂ effect with ICR-170 was displayed in conidia when N₂ was administered during, but not before or after, ICR-170 treatment, (2) the highly increased mutagenic and lethal activities of ICR-170 in the presence of N₂ were due to an anoxic condition rather than to the presence of N₂ per se, (3) the high killing activity of ICR-170 under N₂ was due largely to increased cytoplasmic inactivation, (4) the N₂ effect was a general phenomenon at the ad-3 region of N. crassa, and (5) the highly ICR-170-induced mutation in conidia under N₂ was attributable to an actual enhancement in the mutagenic activity of ICR-170 rather than to selective killing. With regard to the mechanisms of the N₂ effect with ICR-170, results indicate that this effect (1) was not due to more extracellular oxidative degradation of ICR-170 molecules in the presence of O₂, or to a greater uptake of ICR-170 by conidia under N₂, but (2) was due to the inhibition of conidial respiration under an anoxic environment.     

Mutation induction by rodent liver microsomal metabolities of aflatoxins B1 and G1 in Neurospora crassa.

The mutagenic activities of aflatoxins B1 and G1 were studied in the ad-3 test system of Neurospora crassa by treatment of conidia with aflatoxin and liver homogenate for 2 h. No significant increase in the ad-3 mutation frequency over the spontaneous frequency was observed when either aflatoxin or mammalian liver homogenate was omitted from the test system. The ad-3 mutation frequencies increased to between 29 and 87/10(6) survivors, which is a 73- to 217-fold increase over the average spontaneous ad-3 mutation frequency (0.4/10(6) survivors), after conidia of N. crassa were treated with 0.67 mM aflatoxin B1, hamster liver homogenate, and a NADPH generating system. A 9- to 15-fold increase in the mutation frequency over the spontaneous mutation frequency was found when 0.67 mM of aflatoxin G1 instead of aflatoxin B1 was used in the test system. Treatment of conidia with 0.44 mM aflatoxin B1 mice liver homogenate and a NADPH generating system caused a small, but significant increase in the ad-3 mutation frequencies. No significant increase in the mutation frequency was found when a single sample of human liver homogenate was used in the test system. These studies show that metabolic activation is necessary for the expression of the mutagenic activity of aflatoxins B1 and G1 in N. crassa.     

DNA Sequences of Frameshift and Other Mutations Induced by Icr-170 in Yeast

ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses. Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations. The sequence results indicate that ICR-170 primarily induces G.C additions at sites containing monotonous runs of three G.C base pairs. However, some (Formula: see text) sites within the CYC1 gene were not mutated by ICR-170. Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G.C base pairs.     

Ionizing radiation-induced mutagenesis: radiation studies in Neurospora predictive for results in mammalian cells.

Ionizing radiation was the first mutagen discovered and was used to develop the first mutagenicity assay. In the ensuing 70+ years, ionizing radiation became a fundamental tool in understanding mutagenesis and is still a subject of intensive research. Frederick de Serres et al. developed and used the Neurospora crassa ad-3 system initially to explore the mutagenic effects of ionizing radiation. Using this system, de Serres et al. demonstrated the dependence of the frequency and spectra of mutations induced by ionizing radiation on the dose, dose rate, radiation quality, repair capabilities of the cells, and the target gene employed. This work in Neurospora predicted the subsequent observations of the mutagenic effects of ionizing radiation in mammalian cells. Modeled originally on the mouse specific-locus system developed by William L. Russell, the N. crassa ad-3 system developed by de Serres has itself served as a model for interpreting the results in subsequent systems in mammalian cells. This review describes the primary findings on the nature of ionizing radiation-induced mutagenesis in the N. crassa ad-3 system and the parallel observations made years later in mammalian cells.     

Induction of temperature-sensitive 6-thioguanine-resistant mutants as a measure of base-pair substitution mutations in Chinese hamster ovary cells

A method which allows quantification of the frequency of temperature-sensitive (ts) 6-thioguanine-resistant mutants of Chinese hamster ovary cells is described. These mutants, as well as non-ts type of mutant, contain altered hypoxanthine-guanine phosphoribosyl transferase (HGPRT) enzyme activity. The frequency of these altered enzyme mutants allows estimation of the fraction of total mutagenic events in the hgprt gene which results from base-pair substitution and thus provides a measure of the type of lesions induced by mutagenic agents. With an alkylating agent, ethyl methanesulfonate, 16-22% of the total induced mutants show these altered protein phenotypes, while none were found with the putative frameshift mutagen, ICR-191.     

Mutation Induction by Difunctional Alkylating Agents in NEUROSPORA CRASSA

The genetic characterization of ad-3 mutants of Neurospora crassa induced by two carcinogenic difunctional akylating agents, 1,2,4,5-diepoxypentane (DEP) and 1,2,7,8-diepoxyoctane (DEO), has shown that point mutations at the ad-3B locus have similar complementation patterns. In addition to the induction of point mutations, DEP induces a low frequency (7.5%) of multilocus deletions, whereas DEO induces an extremely high frequency (42.0%). The distribution of the different classes of ad-3 mutants and the frequency of multilocus deletion mutants among DEP-induced mutants are not significantly different from those induced by the monofunctional alkylating agents EI, EMS and ICR-177 at comparable forward-mutation frequencies. Moreover, the frequencies of DEP-induced ad-3B mutants showing allelic completion or having nonpolarized complementation patterns are similar to those of ad-3B mutants induced by monofunctional agents. It is suggested, therefore, that the mechanism of mutation-induction by DEP in N. crassa is similar to that of monofunctional alkylating agents. Mutation-induction by DEO probably results both from the mechanism of action of monofunctional alkylating agents and from inter-strand cross-linkage of the DNA molecular by the two functional epoxy groups.     

Effects of pH on the mutagenicity of sodium azide in Neurospora crassa and Salmonella typhimurium

Sodium azide at various pH values did not cause a significant increase in the frequency of forward mutation above the control frequency at the adenine-3 (ad-3) region in resting conidia and in conidia from growing cultures of heterokaryons 12 and 59 of Neurospora crassa. Conidia from ad-3 mutants were plated with sodium azide at various pH values, and no obvious increase in reverse mutation above the controls was observed. Data are presented showing that sodium azide at pH 3 is inactivating conidia by interacting with the cytoplasma rather than the nucleus, and this may be the primary reasons that no mutation at the ad-3 region was detected. The dependence of sodium azide mutagenicity on pH was investigated in histidine-requiring mutants of Salmonella typhimurium using a suspension test. There were no significant differences in the reversion frequencies among the pH values (3-8) tested. Thus, no pH dependence is associated with sodium azide mutagenicity, nor are growth and/or DNA replication required for mutagenicity by sodium azide, in S. typhimurium.     

Induction of Cycloheximide-Resistant Mutants in Saccharomyces cerevisiae with N-Methyl-N′-Nitro-N-Nitrosoguanidine and ICR-170

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cycloheximide-resistant mutations in Saccharomyces cerevisiae, but few, if any, resistant mutants are induced by the acridine mustard ICR-170. Cycloheximide sensitivity in yeast is associated with the ribosome, and treatment with the antibiotic at concentrations of 2 mug/ml results in complete inhibition of protein synthesis. Missense mutations induced by MNNG probably lead to the loss of cycloheximide binding sites on the ribosome, resulting in resistance to the antibiotic without altering the activity of the organelle in protein synthesis. ICR-170, however, induced primarily frameshift mutations that would alter ribosome structural integrity, resulting in cell death rather than resistance. ICR-170 and MNNG are both mutagenic in a system in which base-pair substitution and frameshift mutations can be detected. These results indicate that cycloheximide resistance in S. cerevisiae, like streptomycin and spectinomycin resistance in Escherichia coli, can be induced by base-pair substitution mutagens but not by frameshift mutagens such as ICR-170.