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.     

Induction and complementation of lysine auxotrophs in Saccharomyces

Four chemical agents, EMS EMS: Ethyl methanesulfonate; MNNG: N-methyl-N\t'-nitro-N\t'-nitrosoguanidine; NA: Nitrous acid; ICR-170: 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl) aminopropylamino] acridine 2 HCl; UV: Ultra violet radiation. , MNNG, NA, ICR-170, as well as UV were used to induce mutations in the wild-type haploid strain X2180-1B (α) of Saccharomyces. A total of 2053 (EMS, 427; MNNG, 444; NA, 469; ICR-170, 456; UV, 257) lysine-requiring mutant clones were isolated from many independent treatments and by nystatin enrichment technique. Mutants were classified into various functional groups on the basis of complementation analysis with 14 tester strains (lys 1 to lys 15 except lys 3). Of the clones analyzed, the number of isolates unable to complement with a given tester strain ranged from 2 for lys 5 to 918 for lys 4. Three of the mutually complementing lysine loci (lys 1, lys 2, and lys 4) accounted together for over 85% of the mutant clones whereas lys 6, lys 7, lys 8, and lys 14 had less than 10 noncomplementing isolates each. Mutants for lys 4 were most frequent with all of the mutagens tested except with NA in which case the mutants for lys 2 were most frequent. A total of 56 isolates failed to complement with lys 10, lys 11, and lys 12. Similarly, 47 isolates failed to complement with lys 9 and lys 13 simultaneously. Only 44 isolates complemented with all of the tester strains used.     

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.     

Effects of hydroxylamine and N-Methyl-N′-Nitro-N-nitrosoguanidine in Mimulus cardinalis (Scrophulariaceae): Survival curves and dominant mutants

Summary The lethal and mutagenic effects of hydroxylamine (HA) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were investigated in the higher plant Mimulus cardinalis. MNNG was found to be more toxic than HA. The shapes of the survival curves obtained at different concentrations of HA and MNNG are interpreted on the basis of decreased biological activity of the solution to increased age of solution. Based on the appearance of chlorophyll-deficient mutants, MNNG is mutagenic in Mimulus. No albinos were detected in HA treated plants. A total of 67 putative mutants were isolated in the mutation spectra of HA and MNNG treated plants. The frequency of mutants induced by HA and MNNG are different. MNNG is mutagenic at 1/10 the concentration of HA in inducing putative mutations in M ₁ plants.A portion of this work will be submitted by the senior author to the Faculty of Miami University in partial fulfillment of the Doctor of Philosophy degree.     

Genetic analysis of purple adenine (ad-3) mutants induced by methyl methanesulfonate in Neurospora crassa

The mutagenicity of methyl methanesulfonate (MMS) was studied in a genetically marked two-component heterokaryon of Neurospora crassa. Types of genetic alterations detectable in this system are (I) point mutations in the ad-3A and ad-3B loci; (2) multilocus (chromosome) deletions in the ad-3 region, and (3) recessive lethal mutations in the whole genome. Study of the inactivation kinetics of the heterokaryotic and homokaryotic conidial fractions has made it possible to distinguish between nuclear and cytoplasmic inactivation.Forward mutations in the ad-3 region induced by MMS in the heterokaryotic fraction of conidia were obtained by a direct method with the following results: (I) The overall ad-3 forward mutation frequency increases in proportion to the 1.91 power of the concentration of MMS. (2) The forward mutation frequency of point mutations at the ad-3A and ad-3B loci increases in proportion to the 1.68 power of the concentration. (3) The forward mutation frequency of chromosome deletions in the ad-3 region increases more than exponentially with increasing concentrations of MMS. (4) After treatment for 300 min with 20 mM MMS, 15.5% of the ad-3 mutations are multilocus deletions. Tests for genotype and allelic complementation of the point mutations showed that (I) the ratio between ad-3B and ad-3A mutants was 1.75, (2) 52.1% of the ad-3B mutants showed allelic complementation, with 39.2% non-polarized and 12.9% polarized complementation patterns and 47.9% noncomplementing mutants, and (3) both the ratio between point mutations in the ad-3A and ad-3B loci and the spectrum of complementation patterns among the ad-3B mutants were independent of MMS concentration.     

Two classes of Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents

Summary Six mutant strains of Bacillus subtilis hypersensitive to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were shown to be deficient in the adaptive response to MNNG and termed ada mutants (Morohoshi and Munakata 1985). All the mutations mapped between the attSPO2 and lin loci on the chromosome. The mutant and wild-type (ada ⁺) cells contained similar constitutive levels of O⁶-methylguanine-DNA methyltransferase activity. Pretreatment with low concentrations of MNNG increased the activity about nine-fold in the ada ⁺ cells, while it uniformly decreased the activity in the ada cells. The pretreatment of three mutants (ada-3, ada-4, and ada-6) as well as ada ⁺, augumented the activity of methylpurine-DNA glycosylase and rendered the cells resistant to the lethal and mutagenic effects of N-propyl- or N-butyl-N-nitro-N-nitrosoguanidine. With the rest of the mutant strains (ada-1, ada-2, and ada-5), neither of such responses was elicited by the pretreatment. Thus, the former ada strains seem to have a defect in the gene specifically involved in the induction of the methyltransferase, while the latter ada strains have a defect in the gene controlling the adaptive response as a whole.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - ENNG N-ethyl-N-nitro-N-nitrosoguanidine - PNNG N-propyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - MMS methyl methanesulphonate     

Induction of Mendelian and non-Mendelian streptomycin resistant mutants during the synchronous cell cycle of Chlamydomonas reinhardtii

Summary In synchronized cultures of Chlamydomonas reinhardtii N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was found to selectively mutate replicating forms of nuclear DNA. This conclusion was based on the 15- to 30-fold increase in the recovery of MNNG induced Mendelian streptomycin resistant mutants (sr-1) which was correlated with mutagenesis during the period of nuclear DNA replication. No concomitant increase in the recovery of non-Mendelian streptomycin resistant mutants (sr-2) occurred during this same period.Mutagenesis at the time of chloroplast DNA replication, however, resulted in a 1.5- to 1.6-fold increase in the recovery of both sr-1 and sr-2 induced mutants.     

Molecular analysis of complex human cell populations: mutational spectra of MNNG and ICR-191

We describe a method to identify and enumerate mutants at the nucleotide level in complex cell populations. Several thousand different mutants were induced at the HPRT locus in human lymphoblastoid cultures by either MNNG, an alkylating agent, or by ICR-191, a substituted acridine. HPRT mutants were selected en masse by resistance to 6-thioguanine. The most frequent mutations (hotspots) in HPRT exon 3 were determined by a combination of denaturing gradient gel electrophoresis and polymerase chain reaction. MNNG predominantly produced GC----AT transitions at nucleotides in a GGGGGG sequence, while ICR-191 produced both +1 frameshifts in the same GGGGGG sequence and +1 frameshifts in a CCC sequence.     

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.     

Isolation and mapping of Escherichia coli K12 mutants defective in Tn9 transposition

Summary Five mutants (called tnm) of Escherichia coli with impaired ability for transposition of Tn9 were isolated after treatment with ethyl methanesulfonate (EMS) or N-methyl-N-nitro-N-nitrosoguanidine (NG).The map locations of the tnm mutations were deterimined by a combination of Hfr matings, F episome complementation and P1 transductional mapping. The data obtained show that the five tnm mutations are located near 91 min on the Escherichia coli linkage map and are cotransducible with the metA marker with a frequency of 3%–4%. Introduction of F plasmids containing this region complements the Tnm^- phenotype for the two mutants tested i.e. tnm-1 and tnm-2 are recessive in tnm ⁺/tnm^-merodiploids.     

Mutagenicity of actinomycin D in Neurospora crassa.

Actinomycin D is known to bind to native DNA and is widely used as an antineoplastic agent and inhibitor of DNA-dependent RNA and protein synthesis. We report here the induction by actinomycin D of purple adenine-requiring mutants (ad-3) in wild-type Neurospora crassa. A significant increase in the frequency of ad-3 mutants was evident when the organism was grown vegetatively in the presence of actinomycin D; the mutation frequency was at least 3.6 per 106 survivors. The actinomycin D-induced ad-3 mutants were 29% ad-3A and 71% ad-3B. The ad-3B mutants were classed by complementation pattern as 25% nonpolarized complementing; 14% polarized complementing; and 61% noncomplementing. The spectrum of complementation types of the actinomycin D-induced mutants most closely parallels that of mutants induced by ICR-170, known to induce base-pair insertions or deletions, or that of X ray-induced or spontaneous mutants. It is significantly different from spectra seen following treatment with nitrous acid or N-methyl-N'-nitro-N-nitrosoguanidine, agents known to induce mainly base-pair substitutions.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. VI. Genetic characterization of ad-3 mutants provides evidence for qualitative differences in the spectrum of genetic alterations between wild-type and nucleotide excision-repair-deficient strains

Genetic characterization of ad-3B mutants induced in wild-type and UV-sensitive strains has revealed qualitative differences between the spectra of genetic alterations at the molecular level. Ad-3B mutants induced in the two nucleotide excision-repair-deficient strains upr-1 and uvs-2 (Worthy and Epler, 1973) had significantly lower frequencies of nonpolarized complementation patterns and higher frequencies of noncomplementing mutants than ad-3B mutants induced in the wild-type strain in samples induced by either UV, gamma-rays, 4NQO or MNNG. In these same samples ad-3B mutants induced in uvs-4, uvs-5 or uvs-6 did not differ significantly from those induced in the wild-type strain. After ICR-170 treatment, ad-3B mutants induced in the UV-sensitive strains did not differ significantly from those induced in wild-type. The comparisons in the present and previous studies demonstrate that the process of mutation-induction in the ad-3 region is under the control of other loci that not only alter mutant recovery quantitatively (de Serres, 1980; Schüpbach and de Serres, 1981; Inoue et al., 1981a, b) but also qualitatively. These data have important implications for comparative chemical mutagenesis, since the spectrum of genetic alterations produced by a given agent can be modified markedly as a result of defects in DNA repair.     

Mutagenic specificity of N′-methyl-N′-nitro-N-nitrosoguanidine in the gpt gene on a chromosome of Chinese hamster ovary cells and of Escherichia coli cells

Summary DNA base sequence changes induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis have been determined for the Escherichia coli gpt gene stably incorporated in a chromosome of Chinese hamster ovary cells and in the chromosome of both growing and starving E. coli cells, instead of on a plasmid as in most previous studies. In the three cases, nearly all mutations were G: C to A: T transitions, with a 2-to 4-fold higher mutation rate, compared to other sites, at guanines flanked on the 5 side by another guanine. Mutagenic hot spots in these experiments were less prominent than in published results for MNNG mutagenesis of gpt and of other genes. A suggested explanation involves repair of O⁶meG. At low levels of mutagenic products, most are repaired and even small differences in the repair rates leads to large differences in the relative amounts of residual O⁶meG at various sites; in contrast, at high levels of mutagenic products there is little effect of repair on the distribution.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - O⁶meG O⁶-methylguanine - N7meG N7-methylguanine - CHO Chinese hamster ovary     

Lethal and mutagenic effects of nitrosoguanidine on synchronizedChlamydomonas

Summary The lethal and mutagenic effects of 5 g/ml N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were maximal during the nuclear S-period of synchronously grownChlamydomonas reinhardtii. This was revealed by a 50% drop in survival and a 50- to 100-fold increase in the recovery of slow-growth mutants (up to 40% of the survivors) which were first recognized as small colonies on agar medium. Partial characterization of these isolates revealed about 50% to be stable on subculture, and several were demonstrated to be either acetate-dependent, dark-lethal (light-dependent), or acetate-sensitive mutants. There was no significant increase of lethality or of slow-growth mutants correlated with treatment during the chloroplast DNA replication phase of the cell-cycle.The results of genetic analysis with 13 mutants induced during the nuclear S-period were consistent with their nuclear origin. These analyses were hampered by the high proportion of lethality among the progeny of most crosses.It is concluded that the enhanced mutant induction among nuclear S-phase cells may indicate preferential mutagenesis of replication fork DNA and induction of multiple-closely-linked mutations, as in some bacteria. Consequently, forC. reinhardtii, caution should be exercised in drawing relationships between abnormal behavioral and biochemical phenotypes in MNNG-induced mutants.     

Genetics of somatic mammalian cells. IX. Quantitation of mutagenesis by physical and chemical agents

The system previously described for inducing single gene mutations in Chinese hamster cells has been extended to produce additional auxotrophic mutants. An improved method for quantitating the efficiency of single gene mutation to specific auxotrophies has been developed. Mutagenesis in the forward direction has been measured after treatment of these cells with ethyl methanesulfonate, N-methyl-N¹-nitro-N-nitrosoguanidine, hydroxylamine, an acridine mustard (ICR-191), caffeine and ultraviolet- and X-irradiation. For each agent, the single cell survival curve and the efficiency of chromatid breakage and rearrangement were measured. Similar measurements were also carried out with a water-soluble carcinogen N-nitrosomethylurea, which was shown to be effective in producing auxotrophic, somatic mutations. These results offer promise of illuminating the relationships between cell killing, chromosomal aberration, single gene mutations and carcinogenesis produced by various agents. The methods described can be used in routine testing of drugs, food additives, and environmental pollutants for mutagenic action in mammalian cells in vitro.     

Isolation of mutants of Clostridium pasteurianum

Twenty-nine independent mutants of Clostridium pasteurianum ATCC 6013, including several auxotrophs and UV resistants, have been isolated and characterized. The protoplast formation and regeneration procedure of Minton and Morris (1983) has also been successfully tried with some of these newly obtained mutants. The availability of these mutants together with the possibility of protoplast formation and regeneration will be useful for the development of a genetic exchange system in this species.Abbreviations CFU colony forming unit - DCCP dicyclohexyl carbodiamide - EMS ethylmethane sulfonate - IB isotonic buffer - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PEG polyethylene glycol - UV ultraviolet     

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.     

The influence of colicinogenic plasmids ColIb-P9, ColIa-CA53 and ColV-K30 on the repair, mutagenesis and induction of colicin E1 synthesis

Summary The presence of colicinogenic plasmids ColIb-P9 and ColIa-CA53 in E. coli K-12 cells, wild-type with respect to repair, enhanced the survival of cells after UV irradiation and increased the frequency of UV-induced argE3 and his-4 reversions, while the presence of ColV-K30 negatively affected repair and mutagenesis. The plasmid ColIb-P9 showed a UV-protective effect in E. coli cells carrying mutations in genes uvrA, uvrB, uvrC, polA, recB, recF, though in none of the mutants did cell survival reach the wild-type level. The effect of ColIb-P9 on mutagenesis did not depend on the uvrA or recB genes. The plasmids' protective effect and the enhancement of mutagenesis depended on the recA ⁺ lexA⁺ genotype. The frequency of 2-aminopurine-induced mutations was not affected by ColIb-P9 or ColV-K30. The presence of ColIb-P9 decreased the ability of ColEl-carrying cells to induce colicin E1 synthesis caused by DNA-damaging agents: UV, MNNG, mitomycin C, whereas ColV-K30 increased the percentage of colicin E1-producing cells. These plasmid effects on the level of induction of colicin E1 synthesis were not observed in the case of induction caused by chloramphenicol which did not depend on the products of recA and lexA genes.Abbreviations AP 2-aminopurine - MNNG N-methyl-N-nitro-N-nitrosoguanidine - ICS induction of colicin synthesis - CM chlorampheniol - MC mitomycin C     

Molecular analysis of the aidD6: : Mu dl (bla lac) fusion mutation of Escherichia coli K12

Summary In this report we present genetic and biochemical evidence indicating that the aidD6: : Mu dl (bla lac) fusion is an insertion of Mu dl (bla lac) into the alkB coding sequence. We describe the phenotypic effects resulting from this mutation and compare them with the effects of alkB22, alkA and ada mutations. We also constructed an alkA alkB double mutant and compared its phenotype with that of the single mutant strains. The observation that the methyl methanesulfonate (MMS) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) resistance of the double mutant is approximately at the level predicted from the additive sensitivity of each of the single mutants suggests that these two gene products act in different pathways of DNA repair.     

Induction of petite mutations during germination and outgrowth of Saccharomyces cerevisiae ascospores.

The germination and outgrowth of Saccharomyces cerevisiae ascospores were studied by determining the sensitivity of the ascospores to the action of chemical mutagens. Survival of the ascospores after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was low during the first 2 h of germination and then increased and remained constant. Survival of the ascospores after 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine-2HC1 (ICR-170) treatment was constant from 0 to 5 h, but as the ascospores completed outgrowth at 6 h they became more sensitive to killing by ICR-170. Survival of the ascospores remained high during treatment with 2-methoxy-6-chloro-9-(3-[ethyl-2-hydroxyethyl]aminopropylamino)acridine-2HC1 (ICR-170-OH) or 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide. The main classes of mutations screened for were petites and auxotrophs. The induction of petites and auxotrophs by MNNG was independent of the stage of germination and outgrowth treated. Petite induction by ICR-170 was dependent upon the stage of germination and outgrowth treated. The early hours of germination (0 to 3 h) were not sensitive to petite induction. However, there was maximal petite induction at 5 h into germination and outgrowth, followed by a decline. During this same time period, ICR-170 induced less than 1% auxotrophic colonies. This finding is very unusual because ICR-170 induced 15% auxotrophic colonies in starved log-phase cultures of S. cerevisiae. The acridine ICR-170-OH induced no mutations during germination and outgrowth of the ascospores. Ethidium bromide induced petites, and the petite frequency became maximal at 5 h of germination and outgrowth, a result similar to that obtained with ICR-170.