Radiosensitive mutants of Drosophila. VI. The effect of ultraviolet rays and methylmethane sulfonate on the viability and incidence of chromosome aberrations in the somatic cells of mutant mus(2)201G1

The mus(2)201G1 mutation determining high sensitivity to UV-rays and methyl methansulfonate (MMS) has been studied. The larvae of Drosophila of different age were treated with UV-rays and MMS. Lethality of organisms during the larvae and the pupa stages of the development, as well as the frequency of spontaneous and induced chromosome aberrations were registered. The mus(2)201G1 mutation was shown to determine high lethality of Drosophila during larvae and pupa stages as well as a high frequency of spontaneous and induced chromosome aberrations. The conclusion was made that chromosome aberrations are not the single reason for the death of the mutant flies after mutagenic treatment and that the function of the mus(2)201G1 gene is necessary for divided and undivided cells.     

The effect of dose and time on the induction of genetic alterations insaccharomyces cerevisiae by aminoacridines in the presence and absence of visible light irradiation in comparison with the dose-effect-curves of mutagens with other type of action

Summary Aminoacridines induce frameshift mutations and are photodynamically active, depending on whether visible light is absent or present. Therefore, a test system which allows to compare quantitatively the genetic effects of aminoacridines irradiated or unirradiated by visible light ought to be susceptible to the different DNA alterations which can be induced by these substances. For this reason in most experiments mitotic gene conversion and only in some selected experiments reverse mutation was chosen as the indicator of genetic activity. In contrast to mutation systems mitotic gene conversion has never shown a response specific to only some types of mutagens. The three aminoacridine derivatives used-acridine orange (AO), proflavine (PF), and acridine yellow (AY)—were successful in the induction of convertants at two different loci. No locus-specificity could be observed. The time-dependent induction of convertants proceeds quickly but soon reaches—especially after treatment without light—a saturation point. The dose/effect-curve after treatment in the dark has a slope increasing with increasing concentration. Irradiation with visible light results in a dose/effect-curve consisting of three parts. At first the increase of convertants is nearly linear extending one (AY) to three (AO) orders of magnitude. After that a saturation effect begins at the point at which an effectiveness of the acridines in the dark is apparent. At high concentrations an induction of convertants can again be observed which is nearly the same as that after treatment in the dark. To determine whether the dose/effect-curves obtained for gene conversion refer to similar curves for gene mutations after treatment with AO at the same locus not only gene conversions but also reverse mutations were scored for. AO-treatment in the dark is ineffective in inducing reverse mutations. Irradiation with visible light results in a dose/effect-curve beeing parallel only in its first part to the dose/effect-curve obtained for gene conversion, while in its second part a mutation frequency decline can be observed. Comparing the dose/effect-curves of AO resulting from the induction of gene conversion and gene mutation, and taking into account that no mutants can be induced by AO-treatment in the dark, the increase in convertants at high acridine-concentrations can be explained as an addition of light-dependent and light-independent effects. That means, in mutation systems at low concentrations of aminoacridines irradiation with visible light should cause transitions, transversions and microlesions, at intermediate concentrations frameshift lesions should begin to appear, and at very high concentrations nearly exclusively frameshift lesions should occur. The dose/effect-curves of aminoacridines compared with those of other mutagens are very complex. The dose/effect-curves of the mutagens of other type of action tested are linear in a double logarithmic scale, and parallel for induced gene conversion and induced gene mutation. These results indicate that the gene conversion ability of a given compound depends on its mutagenic property. That means, many mutagens may exert specific genetic effects not directly but mainly in indirect ways by leading to DNA damage, a situation for repair synthesis resulting as well in mutations as recombinations.     

Physiological modification of alkylating agent induced mutagenesis. II. Influence of the numbers of chromosome replicating forks and gene copies on the frequency of mutations induced in Escherichia coli.

The frequency of reversions induced in Escherichia coli K-12 trpA58 by any of five different monofunctional alkylating agents increased as the growth rate of the organism was raised prior to mutagen treatment. The increase in mutation frequency did not correlate with growth rate-dependent changes in cell area or total cellular protein and DNA. After treatment of cells with N-methyl-N-nitrosourea (MNUA), no growth rate-dependent change was observed in the total DNA alkylation or percentage of O6-methylguanine present in the DNA extracted. The frequency of reversions induced by one mutagen, methyl methanesulphonate (MMS), increased in proportion to the average number of trpA gene copies per cell, whereas the frequency of reversions induced by the other compounds was dependent on the average number of chromosome replicating forks per cell. This difference was attributed to the different ratios of DNA base alkylation products observed, formed after treatment with MMS, an SN2-type reagent, or after treatment with the SN1-type reagents ethyl methanesulphonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), MNUA and N-ethyl-N-nitrosourea (ENUA). Possible reasons for the dependence of mutation frequency on the number of replicating forks per cell are discussed.     

Adaptive response to alkylating agents in the Drosophila sex-linked recessive lethal assay

The adaptive response to alkylating agents was studied in Drosophila assays under various treatment procedures. Pre-treatment of males as well as treatment of females with low doses of EMS (0.05-0.1 mM) did not affect sex-linked recessive lethal (SLRL) rates induced by high doses of this mutagen (10 mM, various feeding duration) in mature sperm cells. Pre-treatment of males with a low dose of MMS (0.1 mM) enhanced mutagenesis induced by the high dose of EMS (10 mM) at different stages of spermatogenesis, the observed effects exceeding the additive action of both mutagens. On the contrary, larval pre-treatment with the adaptive dose of EMS (0.05 mM) resulted in resistance of their germ cells to higher doses of EMS (1 mM). Specifically, offspring production increased while dominant lethality in F(1) as well SLRL frequency in F(2) was significantly reduced as compared with the effects of larval exposure to the challenge dose. Under the conditions tested, the adaptive response of germ cells to alkylating agents was demonstrated in larvae, but not in adult flies.     

Quantitative Measurement of the Ability of Different Mutagens to Induce an Inherited Change in Phenotype to Allow Maltose Utilization in Suspension Cultures of the Soybean, GLYCINE MAX (L.) Merr

Using a newly developed plating system, we have measured cell survival and the frequencies of variation in an inherited trait after treatment of soybean cell suspensions with different mutagens: ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-Methyl-N'-nitro-N-nitroso-guanidine (MNNG), hycanthone (1-{[2-(diethylamino) ethyl] amino}-4-(hydroxymethyl)-9H-thioxanthen-9-one and ultraviolet light (UV).—The heritable variation selected for displays a phenotype of rapid growth on maltose as carbon source. The marker is stable in the absence of maltose, and prolonged growth of variant cells on sucrose has not shown reversions to slow growth. Doubling time in suspension cultures is decreased from 100 hr to ca. 30 hr by the mutation. Both wild-type and variant cells grow on sucrose with a 24-hr doubling time. Thus, lethality after mutagen treatment can be estimated rapidly by growth on sucrose, whereas variants are scored on maltose medium. The spontaneous frequency of variants was 1.2 x 10^-7; induced frequencies ranged from a low of 3.6 x 10^-5 for EMS to a high of 10^-3 for hycanthone. The high frequency of variants induced by hycanthone, a frame-shift mutagen, and the observation that UV induces variants in haploid cells with much higher frequency than in diploid cells suggests a recessive mutation.     

Increment kinetics of recessive lethal mutations and dominant lethals in offspring of Drosophila melanogaster on storage of methyl-methanesulfonate-treated sperm in females

The frequencies of sex-linked recessive lethal mutations in F1 males after feeding adult male Drosophila melanogaster with 0.25 and 0.5 mM methyl methanesulfonate (MMS) orally for 24 h increased approximately linearly with storage of the treated spermatozoa in females, whereas the number of hits of dominant lethals in the sperm after feeding 0.3 and 0.5 mM MMS increased approximately with the square of the storage time. Chromosome losses and mosaics in F1 males also increased with the dose of MMS to males, but their yields were too low to be analyzed quantitatively, only indicating a slight increase of chromosome loses and a slight decrease of mosaics with the time of storage of sperm. Maternal non-disjunctions (or chromosome losses), detected in F1 males, decreased with the dose of MMS to spermatozoa and their yield decreased with the time of storage of sperm of both MMS-treated and the control groups. A unitary model is proposed to explain the effect of storage on the dominant lethals and recessive lethal mutations.     

Characterization of MMS-sensitive mutants of Neurospora crassa

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer resistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways (as indicated by their ability to utilize DNA as the sole phosphate source in the growth medium). On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. The first group includes three mutants, mus-(SC3), mus-(SC13), and mus-(SC28). These are slow growers, insensitive to histidine with no apparent meiotic defects and may have reduced frequency of spontaneous mutation. In addition, their mycelial growth is sensitive to MMS but the conidial viability following MMS, UV or X-ray treatment appears normal or only slightly more sensitive than the wild-type. The second group includes only one mutant, mus-(SC15); its mycelial growth is very sensitive to MMS but the conidial survival following treatment with MMS or UV appears normal; however, the conidial survival following exposure to X-ray is significantly reduced. This mutant shows an increase (more than 10-fold) frequency of spontaneous mutation, but behaves normal like the wild-type with respect to fertility, growth rate and insensitivity to histidine. The third group includes mutants mus-(SC10), mus-(SC25), and mus-(SC29). These mutants are very sensitive to UV, X-rays and MMS and to histadine but have normal growth rates on minimal medium. Mutant mus-(SC10), but not mus-(SC25) and mus-(SC29), has an increased (11 ×) frequency of spontaneous mutation. On the basis of data presented, the MMS sensitivity of the first group of mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to a number of DNA-damaging agents such as MMS, UV and/or X-ray, high frequencies of spontaneous mutation (mutator effects) and defects in meiotic behavior.     

The relationship between DNA damage and mutation frequency in mammalian cell lines treated with N-nitroso-N-2-fluorenylacetamide

The induction of DNA single-strand breaks in C3H10T1/2 mouse fibroblasts and Chinese hamster ovary (CHO) cells by N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA) was demonstrated by the alkaline elution technique. Without metabolic activating system (i.e., rat liver S9 fraction), N-NO-2-FAA exhibits more direct and strong damaging effects on DNA than its parent compound, 2-FAA, at equal concentration in both cell lines. To compare the DNA-damaging potency of N-NO-2-FAA with other well-known carcinogens, such as benzo[a]pyrene, 2-nitrofluorene, and N-methyl-N'-nitrosoguanidine (MNNG), the order of potency is as follows: MNNG (5 microM) greater than N-NO-2-FAA (150 microM) greater than benzo[a]pyrene (20 microM) at equitoxic concentrations, LD37, in the same cell system. Another parallel experiment indicated that N-NO-2-FAA could disrupt the superhelicity of circular plasmid DNA (pBR 322) at a dose range of 0.1-50 mM; however, a complete conversion to form III linear DNA was found at the highest concentration (50 mM). After treatment with various concentrations of N-NO-2-FAA, ouabain resistance (ouar) was induced in C3H10T1/2 cells, while both ouar and 6-thioguanine resistance (6-TGr) were induced in CHO cells. The mutation frequency in the Na+/K+-ATPase locus in CHO cells (1.5 X 10(-6) mutants/microM) is higher than that in C3H10T1/2 cells (1.0 X 10(-6) mutants/microM). The maximal mutation frequency at the Na+/K+-ATPase gene locus was attained with 30 min of exposure in C3H10T1/2 cells, whereas the mutation frequency in CHO cells continued to increase up to 80 min of treatment. Similarly, the maximal mutation frequency at the HPRT locus also continued to increase up to 80 min of treatment. Finally, a linear plot of alkali-labile lesions versus 6-TGr mutations was obtained; but the same relationship was not observed in the case of ouar mutation.     

The effect of two chemical mutagens ENU and MMS on MR-mediated reversion of an insertion-sequence mutation in Drosophila melanogaster

The effects of two mutagens ENU and MMS characterized by different alkylation patterns have been studied on the reversion of an MR-induced singed mutation to wild-type. Reversion of this unstable singed mutation under the influence of MR is assumed to represent the removal or transposition of an insertion element. Since MR acts primarily in spermatogonia, the mutagens were fed to 1st instar larvae. Recessive lethal tests were carried out simultaneously to calibrate for the mutagenic effectiveness of the chemicals. For both powerful mutagens, it was observed that the frequency of reversion remained far below of what would have been expected on the basis of the mutagenic effectiveness, as registered in the lethal tests. Thus 1 mM ENU, 5 mM and 10 mM MMS did not affect the reversion frequency at all, and with 3 mM ENU only a doubling of the reversion frequency was observed, despite a 5-fold increase in the lethal frequency. The threshold at 1 mM EMU and the low effectiveness of 3 mM on the reversion process are taken as an indication that ENU affected the transposition process in an indirect manner, rather than the excision events themselves. The data obtained with Drosophila are consistent with the microbial observations in that mutation involving removal or transposition of an insertion element is not affected by mutagenic treatments. This finding may have consequences for the evaluation of induced genetic damage on the basis of the spontaneous load of genetic detriment in man.

An incidental observation was that non-MR Cy larvae exhibited greater sensitivity to the induction of recessive lethals by MMS than MR-individuals.     

Inducible DNA-repair systems in yeast: competition for lesions

DNA lesions may be recognized and repaired by more than one DNA-repair process. If two repair systems with different error frequencies have overlapping lesion specificity and one or both is inducible, the resulting variable competition for the lesions can change the biological consequences of these lesions. This concept was demonstrated by observing mutation in yeast cells (Saccharomyces cerevisiae) exposed to combinations of mutagens under conditions which influenced the induction of error-free recombinational repair or error-prone repair. Total mutation frequency was reduced in a manner proportional to the dose of 60Co-gamma- or 254 nm UV radiation delivered prior to or subsequent to an MNNG exposure. Suppression was greater per unit radiation dose in cells gamma-irradiated in O2 as compared to N2. A rad3 (excision-repair) mutant gave results similar to wild-type but mutation in a rad52 (rec-) mutant exposed to MNNG was not suppressed by radiation. Protein-synthesis inhibition with heat shock or cycloheximide indicated that it was the mutation due to MNNG and not that due to radiation which had changed. These results indicate that MNNG lesions are recognized by both the recombinational repair system and the inducible error-prone system, but that gamma-radiation induction of error-free recombinational repair resulted in increased competition for the lesions, thereby reducing mutation. Similarly, gamma-radiation exposure resulted in a radiation dose-dependent reduction in mutation due to MNU, EMS, ENU and 8-MOP + UVA, but no reduction in mutation due to MMS. These results suggest that the number of mutational MMS lesions recognizable by the recombinational repair system must be very small relative to those produced by the other agents. MNNG induction of the inducible error-prone systems however, did not alter mutation frequencies due to ENU or MMS exposure but, in contrast to radiation, increased the mutagenic effectiveness of EMS. These experiments demonstrate that in this lower eukaryote, mutagen exposure does not necessarily result in a fixed risk of mutation, but that the risk can be markedly influenced by a variety of external stimuli including heat shock or exposure to other mutagens.     

Dosage-response relationships for methyl methanesulfonate in Drosophila melanogaster spermatozoa: DNA methylation per nucleotide vs. sex-linked recessive lethal frequency

Two different mechanisms for mutagenesis following treatment with methyl methanesulfonate (MMS) are suggested from the dose-response curve that is best fit by the linear quadratic model where m = 0.130D + 0.038D2 (D = dose measured as alkylations per nucleotide X 10(3), APdN; m = percent sex-linked recessive lethals, SLRL). A predominant component of the dose-response curve at moderate to high dose is the quadratic component which is interpreted as the result of two single-strand breaks. The distribution of methyl adducts in vivo is consistent with the previously determined in vitro distribution of methyl adducts on DNA following treatment with MMS. With the use of HPLC, 82% of the 3H-labeled adducts are found on the N-7 of guanine. It has previously been shown by both in vitro studies and in vivo correlation with mutagenesis that the N-7 alkyl guanine is not itself a predominately genotoxic lesion; however, N-7 alkyl guanine destabilizes guanine resulting in an increased rate of hydrolysis producing apurinic sites. In data presented in this paper, the loss of labeled adducts is shown to be at a rate consistent with hydrolysis of the destabilized alkyl guanine. The apurinic site thus produced should be converted to single-strand breaks by AP endonucleases once sperm has fertilized the egg. Single-strand breaks are repaired by excision repair which is not error-prone; however, multiple breaks producing a proximity effect should lead to double-strand breaks that are repaired by an error-prone process. Mutations that are induced by a proximity effect would account for the quadratic term. It is hypothesized that a proximity effect is produced when two breaks are sufficiently close together to prevent using the complementary strand as a template. The linear component of the dose-response curve is probably due to alkylation of oxygens in the purine or pyrimidine ring leading to mispairing. However, due to the low frequency of ring-oxygen alkylation following treatment with MMS, this important genotoxic site is not the predominant one observed at experimental levels normally used in the laboratory. From the dose-response curve, it is calculated that at mutation frequencies of 10 times the spontaneous frequency or higher, the predominant mechanism is the multi-hit component; however, at mutation induced frequencies of 0.1 of the spontaneous frequency, which are levels more likely to be encountered in man's exposure to environmental mutagens, the dominant mechanism is the linear component.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytotoxicity of monofunctional alkylating agents methyl methanesulfonate and methyl-N′-nitro-N-nitrosoguanidine have different mechanisms of toxicity for 10T1/2 cells

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) are directly active alkylating agents that methylate cellular macromolecules by SN₁ and SN₂ mechanisms, respectively. These two chemicals produce similar types of alkylation products in DNA and a similar level of total alkylations on a molar basis, but strikingly different proportions of alkylations of ring oxygen atoms of purines and pyrimidines. Because of this attribute, they have been used in combination to attempt to determine which types of alkylation products are responsible for mutation, transformation, and toxicity. Studies have suggested that the mutation rates produced by these and similar chemicals in cells surviving toxicity correlate well with the number of methyl adducts at the O⁶ position of guanine, but that cytotoxicity (reduced colony-forming efficiency) does not correlate with any single adduct or with the total level of alkylation of DNA. In this study we have investigated the cytotoxic mechanisms of MNNG and MMS in synchronized 10T1/2 cells, using colony-forming ability as a measure of toxicity. Both MNNG and MMS cause dose-dependent reduction in the ability of 10T1/2 cells to produce colonies of more than 50 cells after 2 weeks in culture. MNNG is about 100-fold more toxic than MMS on a molar basis. As indicated by the inability of cells to exclude trypan blue, MMS kills a fraction of the population of treated 10T1/2 cells after a 30-min exposure; the fraction of cells that excludes trypan blue is correlated with dose of MMS and with colony-forming efficiency. Neither the fraction of cells that is permeable to trypan blue nor the relative colony-forming efficiency is affected by the phase of the cycle when 10T1/2 cells are treated with MMS. Furthermore, MMS toxicity for 10T1/2 cells is not potentiated by caffeine, MMS treatment does not delay progress of S phase, and cells that survive acute membrane toxicity complete the cell cycle without significant delay. In contrast, MNNG treatment produces toxicity that is maximal when 10T1/2 cells are exposed during the S phase and the effect of potentiated by caffeine. MNNG treatment delays DNA replication and this delay is reversed by caffeine. In sharp contrast to 10T1/2 cells treated with MMS. MNNG-treated cells are not made permeable to trypan blue, but are blocked in their ability to proliferate. These observations indicate that MNNG and MMS kill 10T1/2 cells by drastically different mechanisms, MNNG producing toxicity mainly by preventing chromosome replication and MMS producing toxicity mainly by damaging cell membranes.     

Mutagenesis induced by 5-bromouracil and methyl methane sulfonate: role of DNA polymerase I

A polA1 mutation in the DNA polymerase I gene of E. coli results in a drastic reduction of the frequency of mutagenesis induced by 5-bromo-2'-deoxyuridine (BUdR). Comparisons of the effect of a polA1 mutation on mutagenesis induced by methyl methane sulfonate (MMS), ultraviolet irradiation (UV) and 2-aminopurine (2-AP) demonstrated that a similar effect of a polA1 mutation is observed with MMS. This effect is much less marked with UV-and-2-AP-induced mutagenesis. It follows that DNA polymerase I plays a key role in the process of mutagenesis induced by BU and MMS. Bearing in mind that mutagenesis provoked by UV, MMS and BU involves participation of the accompanying induced error-prone system, the sources of the differences in requirement for DNA polymerase I are critically examined.     

Effect of storage and dose on MMS-induced deletions. Complementation analysis of X-chromosomal recessive lethals in the zeste-white and maroon-like regions of Drosophila melanogaster

The effect of storage on MMS-induced recessive lethals in the zeste-white (3A1-3C2) and the maroon-like (18F4-20F) regions was studied by complementation analysis. (1) Without any exception, all 52 mutants (from unstored spermatozoa) mapped in the zeste-white region were restricted to single complementation units. Furthermore, none of an additional 15 lethals, sampled from sperm that had been stored in females for 9-12 days, was associated with a deletion. (2) Of 34 mutations induced by 8.5 X 10(-2) mM MMS in the maroon-like (mal) region, 4 spanned 2 or more complementation units, and thus are considered to be deletions. A high dose of 2.5 mM MMS provided 55 lethals for analysis of which 4 were deletions. There was no evidence for any difference in the frequency of deletions as the MMS concentration was enhanced from 8.5 X 10(-2) mM to 2.5 mM. However, with storage, 47.1% lethals (16 of 34 mutants induced by 2.5 mM MMS) mapped in the mal region were found to involve large structural changes. (3) A high proportion of double mutants in both the zeste-white (z w) and the maroon-like regions was found among the chromosomes analyzed. These double mutants have one lethal positioned within the region studied and the other outside it. Clearly, the proportion of double mutants increased with dose, from 6.3 to 41.7% in z w and from 14.7 to 61.8% in the mal section. Apurinic sites in DNA reacted with MMS are considered as the likely primary lesions responsible for the storage effect on MMS-induced recessive lethals in the mal region. Thus, the ability of MMS to produce delayed deletion lethals seems to correlate with preference for alkylation of base nitrogens. An interesting aspect for further analysis is the apparent infrequency in the zeste-white region of alkylation-induced chromosomal breakage, as observed by various investigators for MMS, EMS and MNNG.     

Hybrid DNA Extension and Reciprocal Exchanges: Alternative Issues of an Early Intermediate during Meiotic Recombination?

Large heterologies in gene b2 strongly increase the frequencies of reciprocal exchanges on their left border, towards the high conversion end. In a previous study, we observed that heterozygous point mutations located in the high conversion end (region F) stimulate the reciprocal exchanges instigated by the large heterology 138. We have defined some properties of this stimulation. The effect does not depend on the nature of the large heterology used. It is effective only with point mutations located on the left side of the large heterology. It does not depend on the number of heterozygosities accumulated in region F. It is not specific on the location of point mutations in region F: it decreases from region F (left end) to region E (middle part of b2). It is correlated with the mismatch correction efficiencies of the point mutations used. It is not observed in the absence of a large heterology. Point mutation heterozygosities which stimulate reciprocal exchanges also decrease the frequency of HDNA formation in gene b2. We propose a model in which reciprocal exchanges on the one hand and hybrid DNA formation on the other hand correspond to alternative processings of a common recombination intermediate.     

Non-Locus-Specific Polygenes Giving Responses to Selection for Gene Conversion Frequencies in Ascobolus Immersus

Selection for higher and lower meiotic conversion frequencies was investigated in the fungus Ascobolus immersus. Strains carrying the same known gene conversion control factors, which have major effects on conversion frequencies at their specific target locus, sometimes gave significant differences in conversion frequency. Selection for high or low conversion frequencies at the w1-78 site was practiced for five generations, giving significant responses in both directions. These responses were due to polygenes, or genes of minor effect, not to new conversion control factors of major effect. Crosses of selected strains to strains with other mutations showed that the genes' effects were not specific to w1-78, but could affect conversion frequencies of another mutation, w1-3C1, at that locus and of two other loci, w-BHj and w9, which are unlinked to w1 or to each other. The proportional changes in gene conversion frequency due to selection varied according to the locus and site involved and according to the conversion control factor alleles present. There were differences of >/=277% in conversion frequency between ``high' and ``low' strains. Selection for conversion frequency had little effect on other features of conversion, such as the frequency of postmeiotic segregation or the relative frequencies of conversion to mutant or wild type.     

Effects of caffeine or EDTA post-treatment on EMS mutagenesis in soybean

Seeds of soybean cultivar LD4 were mutagenically treated with EMS (0.3, 0.5, 0.6, 0.9, 0.5 and 1.8%) for 3 h only or plus caffeine (50 mM) or EDTA (1 mM) post-treatment for 5 h. The experimental results indicated that: (1) of the different concentrations of EMS treatment, the M2 mutation frequency induced with 0.6% EMS was the highest (9.7%). When the EMS concentration was over 0.9%, the mutation frequency decreased rapidly. (2) Of the EMS treatments plus caffeine or EDTA post-treatment, the mutagenic effect of 0.6% EMS was the best for inducing morphological variations. Caffeine post-treatment decreased notably the mutation frequency of EMS treatment; when concentrations of EMS were very high (1.5% and 1.8%), mutation frequencies of EDTA post-treatment were still 5.0% and 4.88%, but no mutants were found in EMS treatment or plus caffeine post-treatment. (3) In the M2 mutation spectrum, 11 kinds of mutant types were observed in EMS treatment or plus caffeine or EDTA post-treatment. Relative frequencies of some mutant types (growth period, plant height, grain size, leaf shape and sterility, etc.) were similat among the three treatments, but EDTA post-treatment could change the relative frequencies of yield characteristics (number of pods and grains, grain weight/plant) induced by EMS treatment only.     

Survival and mutagenesis of bacteriophage T7 damaged by methyl methanesulfonate and ethyl methanesulfonate

We have examined survival and mutagenesis of bacteriophage T7 after exposure to the alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). It was found that although both alkylating agents caused increased reversion of specific T7 mutations, EMS caused a higher frequency of reversion than did MMS. Exposure of the host cells to ultraviolet light so as to induce the SOS system resulted in increased survival (Weigle reactivation) of T7 phage damaged with either EMS or MMS. However, after SOS induction of the host we did not detect an accompanying increase in mutation frequency measured as either reversion of specific T7 mutants or by generation of mutations in the T7 gene that codes for phage ligase. Neither mutation frequency nor survival of alkylated phage was affected by the umuD,C mutation in the Escherichia coli host nor by the presence of plasmid pKM101. This may mean that the mode of Weigle reactivation that is detected in T7 is not mutagenic in nature.     

Procarbazine-induced specific-locus mutations in male mice.

Procarbazine is used in drug-combination treatment of Hodgkin's disease. The specific locus method was used to test and confirm the ability of procarbazine to induce gene mutations in pre- and post-meiotic germ cells of male mice. The lowest dose of procarbazine that significantly increased the mutation frequency in As spermatogonia over the control frequency was 400 mg/kg (P = 0.003). The corresponding dose for the post-spermatogonial germ-cell stages was 600 mg/kg (P = 0.009). The dose--response was linear for the point estimates of the mutation frequencies after treatment of As spermatogonia with 0, 200, 400 and 600 mg/kg. The point estimate of the mutation frequency at the 800 mg/kg level was one-third of that expected from a linear extrapolation. Variation in mutation rates among the 7 loci between the lowest (a locus) and the highest (p locus) was 12-fold. Only 24% of procarbazine-induced specific locus mutations in As spermatogonia were lethal in the homozygous condition. From the mutation spectra and the viability tests, it is concluded that procarbazine-induced mutations may be mainly due to base-pair changes. Procarbazine-induced specific-locus mutations fulfilled the criteria for the estimation of the doubling dose, the dose necessary to induce as many mutations as occur spontaneously. The doubling dose of procarbazine in As spermatogonia of mice was 114 mg/kg. The therapeutic dose for procarbazine is about 215 mg/kg. If man and mouse were equally sensitive, this dose would induce 1.9 times as many mutations as arise spontaneously. From the incidence of patients with Hodgkin's disease (1 : 42 000) the calculated population dose of procarbazine is 5.12 micrograms/kg. Assuming equal sensitivity between the sexes we can calculate, for an estimated number of 30 000 genes, the induction of about 22 mutations per million children due to procarbazine treatment. The same number of induced mutations can be calculated if the risk of patients is used for the estimation of the genetic hazard.