After a single treatment with EMS the number of non-colony-forming cells increases for many generations in yeast populations

The course of lethal events occurring in populations of haploid Saccharomyces cerevisiae after DNA-damaging treatments was studied. After X-irradiation and after incubation with methyl methanesulfonate (MMS) populations recovered according to expectation, if one assumes successive dilution of killed cells by the proliferating survivors. However, populations treated with ethyl methanesulfonate (EMS) for many generations of proliferation contained more inviable cells than expected. This behaviour was not due to EMS or toxic reaction products remaining with the cells after treatment but to residual divisions of lethally mutated cells. In addition the data suggest that lethal fixations may occur in cells originating from later than the first generation after EMS treatment.     

Induction of specific-locus mutations in male mice by ethyl methanesulfonate (EMS)

Using a sequential mating procedure, the induction of specific-locus mutations by ethyl methanesulfonate (EMS) was reinvestigated in male mice. Doses of 175 mg/kg b.w. and 250 mg/kg b.w. of EMS induce gene mutations in the mating intervals 5-8 and 9-12 days post treatment. However, only the frequency of dominant lethal mutations increases with the dose, not the frequency of specific-locus mutations. This observation implies that with a higher dose of EMS a larger fraction of mutagenized spermatozoa and spermatids are selectively eliminated, leading to underestimation of the specific-locus mutation yield at high doses. EMS does not induce specific-locus mutations in spermatogonia.     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. II. Homozygous Effect of Polygenic Mutations

Polygenic mutations affecting viability were accumulated on the second chromosome of Drosophila melanogaster by treating flies with EMS in successive generations. The treated chromosomes were later made homozygous and tested for their effects on viability by comparison of the frequency of such homozygotes with that of other genotypes in the same culture. The treated wild-type chromosomes were kept heterozygous in Pm/+ males by mating individual males in successive generations to Cy/Pm females. The number of generations of accumulation was 1 to 30 generations, depending on the concentration of EMS. A similar experiment for spontaneous polygenic mutations was also conducted by accumulating mutations for 40 generations. The lower limit of the spontaneous mutation rate of viability polygenes is estimated to be 0.06 per second chromosome per generation, which is about 12 times as high as the spontaneous recessive lethal mutation rate, 0.005. EMS-induced polygenic mutations increase linearly with the number of treated generations and with the concentration of EMS. The minimum mutation rate of viability polygenes is about 0.017 per 10(-4)m, which is only slightly larger than the lethal rate of 0.013 per 10(-4) m. The maximum estimate of the viability reduction of a single mutant is about 6 to 10 percent of the normal viability. The data are consistent with a constant average effect per mutant at all concentrations, but this is about three times as high as that for spontaneous mutants. It is obvious that one can obtain only a lower limit for the mutation rate, since some mutants may have effects so near to zero that they cannot be detected. The possibility of measuring something other than the lower limit is discussed. The ratio of the load due to detrimental mutants to that caused by lethals, the D/L ratio, is about 0.2 to 0.3 for EMS-induced mutants, as compared to about 0.5 for spontaneous mutants. This is to be expected if EMS treatment produces a large fraction of small deletions and other chromosome rearrangements which are more likely to be lethal.     

Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster

Summary The present report describes the recovery and genetic characterization of mutant alleles at zygotic loci on the third chromosome ofDrosophila melanogaster which alter the morphology of the larval cuticle. We derived 12600 single lines from ethyl methane sulfonate (EMS)-treatedst e orrucuca chromosomes and assayed them for embryonic lethal mutations by estimating hatch rates of egg collections. About 7100 of these lines yielded at least a quarter of unhatched eggs and were then scored for embryonic phenotypes. Through microscopic examination of unhatched eggs 1772 lines corresponding to 24% of all lethal hits were classified as embryonic lethal. In 198 lines (2.7% of all lethal hits), mutant embryos showed distinct abnormalities of the larval cuticle. These embryonic visible mutants define 45 loci by complementation analysis. For 32 loci, more than one mutant allele was recovered, with an average of 5.8 alleles per locus. Complementation of all other mutants was shown by 13 mutants. The genes were localized on the genetic map by recombination analysis, as well as cytologically by complementation analysis with deficiencies. They appear to be randomly distributed along the chromosome. Allele frequencies and comparisons with deficiency phenotypes indicate that the 45 loci represent most, if not all, zygotic loci on the third chromosome, where lack of function recognizably affects the morphology of the larval cuticle.     

Analysis of mutations induced by alkylating compounds in Drosophila sperm. I. Character of visible changes induced by N-nitrosoethylurea and ethylmethanesulfonate in a succession of Drosophila generations]

Newly appearing hereditary traits were observed in several generations of Drosophila melanogaster: changes in eye and body colour, wing shape, number and shape of chaetae. The results obtained may be interpreted as the evidence for the realization of potential changes induced by NEU and EMS into visible mutations during 1-4th generations. X-irradiation (at a dose of 1000 r) of males from the first generation did not affect the realization of long-living potential changes induced by NEU.     

The Zuker collection: a resource for the analysis of autosomal gene function in Drosophila melanogaster

The majority of genes of multicellular organisms encode proteins with functions that are not required for viability but contribute to important physiological functions such as behavior and reproduction. It is estimated that 75% of the genes of Drosophila melanogaster are nonessential. Here we report on a strategy used to establish a large collection of stocks that is suitable for the recovery of mutations in such genes. From approximately 72,000 F(3) cultures segregating for autosomes heavily treated with ethyl methanesulfonate (EMS), approximately 12,000 lines in which the treated second or third chromosome survived in homozygous condition were selected. The dose of EMS induced an estimated rate of 1.2-1.5 x 10(-3) mutations/gene and predicts five to six nonessential gene mutations per chromosome and seven to nine alleles per locus in the samples of 6000 second chromosomes and 6000 third chromosomes. Due to mosaic mutations induced in the initial exposure to the mutagen, many of the lines are segregating or are now fixed for lethal mutations on the mutagenized chromosome. The features of this collection, known as the Zuker collection, make it a valuable resource for forward and reverse genetic screens for mutations affecting a wide array of biological functions.     

Induction of specific-locus and dominant lethal mutations in male mice by 1-methyl-1-nitrosourea (MNU)

1-Methyl-1-nitrosourea (MNU) induced specific-locus mutations in mice in all spermatogenic stages except spermatozoa. After intraperitoneal injection of 70 mg/kg body weight of MNU a high yield of specific-locus mutations was observed in spermatids (21.8 × 10⁻⁵ mutations per locus per gamete). The highest mutational yield was induced in differentiating spermatogonia. In 1954 offspring we observed 5 specific-locus mutants (44.8 × 10⁻ mutations per locus per gamete). In addition, 2 mosaics were recovered, which gave a combined mutation rate of 62.7 × 10⁻⁵. In A_s spermatogonia the mutation rate was 3.9 × 10⁻⁵. The same dose of 70 mg/kg of MNU induced dominant lethal mutations 5–48 days post treatment, mainly due to post-implantation loss in spermatids and spermatocytes. It is interesting to compare the induction pattern of mutations by MNU with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethylnitrosourea (ENU). Based on the different spermatogenic response of the induction of specific-locus mutations we can characterize the 4 mutagens in the following way: EMS = MMS ≠ MNU ≠ ENU.     

Relative Rates at Which Dominant-Lethal Mutations and Heritable Translocations Are Induced by Alkylating Chemicals in Postmeiotic Male Germ Cells of Mice

There is a close relationship between the rates at which dominant lethal mutations and heritable translocations are induced by ethyl methanesulfonate (EMS) or triethylenemelamine (TEM) in male postmeiotic germ cells. This relationship does not hold for isopropyl methanesulfonate (IMS), which induced only negligible frequencies of heritable translocations at doses that induced high levels of dominant lethal mutations. Nor does IMS behave like EMS and TEM in the degree to which eggs of different stocks of females repair premutational lesions that are carried in the sperm-large differences between stocks for IMS treatment and small differences for EMS or TEM treatment. These dissimilarities between IMS and the other two alkylating chemicals are postulated to be attributable to differences in the types of lesions present at the time of repair activity and to whether or not chromosomal aberrations are already fixed prior to postfertilization pronuclear DNA synthesis.     

The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells.

The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells clone K1-BH4 was studied at many EMS doses including the minimally lethal range (0-100 microng/ml) as well as the exponential killing portion (100-800 microng/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10(-6)(8.73+3.45 X), where 0 less than or equal to X less than or equal to 800 microng/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.     

Map positions of third chromosomal female sterile and lethal mutations of Drosophila melanogaster.

Chromosomal mutations induced by ethyl methanesulfonate (EMS) treatment can cause female sterility or maternal-effect lethality in Drosophila. EMS is particularly useful to researchers because it creates mutations independent of position effects. However, because researchers have little control over the chromosomal site of mutation, post-mutagenic genetic mapping is required to determine the cytological location of the mutation. To make a valuable set of mutants more useful to the research community, we have mapped the uncharacterized part of the female-sterile - maternal-effect lethal Tubingen collection. We mapped 49 female-sterile - maternal-effect lethal alleles and 72 lethal alleles to individual deficiency intervals on the third chromosome. In addition, we analyzed the phenotype of ovaries resulting from female sterile mutations. The observed phenotypes range from tumorous ovaries and early blocks in oogenesis, to later blocks, slow growth, blocks in stage 10, to apparently full development of the ovary. The mapping and phenotypic characterization of these 121 mutations provide the necessary information for the researcher to consider a specific mutant as a candidate for their gene of interest.     

A comparison of the effects of physical and chemical mutagens in sesame (Sesamum indicum L.)

Three sesame genotypes (Rama, SI 1666 and IC 21706) were treated with physical (γ-rays: 200 Gy, 400 Gy or 600 Gy) or chemical (ethyl methane sulphonate, EMS: 0.5%, 1.0%, 1.5% or 2.0%) mutagens and their mutagenic effectiveness and efficiency were estimated in the M (2) generation. The M (3) generation was used to identify the most effective mutagen and dose for induction of mutations. The average effectiveness of EMS was much higher than γ-rays. The lowest dose of γ-rays (200 Gy) and the lowest concentration of EMS (0.5%) showed the highest mutagenic efficiency in all genotypes. Analysis of the M (3) generation data based on parameters such as the variance ratio and the difference in residual variances derived from the model of Montalván and Ando indicated that 0.5% concentration of EMS was the most effective treatment for inducing mutations.     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. I. Recessive Lethal Mutations

The efficiency of the adult feeding method for EMS treatment in Drosophila melanogaster was studied by measuring the frequency of induced recessive lethals on the second chromosome. The treatment was most effective when mature spermatozoa or spermatids were treated and was much less effective on earlier stages. The number of mutations induced was proportional to the concentration except at the highest doses. The recessive lethal rate was estimated to be about 0.012 per second chromosome per 10(-4) M. In addition, about 0.004-0.005 recessive lethals per 10(-4) M were found in a later generation in chromosomes that had not shown the lethal effect in the previous generation. When the experiments are done in a consistent manner and gametes treated as mature sperm or spermatids are sampled, the results are highly reproducible. However, modifications of the procedure, such as starvation before EMS treatment, can considerably alter the effectiveness of the mutagen.     

Whole-genome effects of ethyl methanesulfonate-induced mutation on nine quantitative traits in outbred Drosophila melanogaster

We induced mutations in Drosophila melanogaster males by treating them with 21.2 mm ethyl methanesulfonate (EMS). Nine quantitative traits (developmental time, viability, fecundity, longevity, metabolic rate, motility, body weight, and abdominal and sternopleural bristle numbers) were measured in outbred heterozygous F3 (viability) or F2 (all other traits) offspring from the treated males. The mean values of the first four traits, which are all directly related to the life history, were substantially affected by EMS mutagenesis: the developmental time increased while viability, fecundity, and longevity declined. In contrast, the mean values of the other five traits were not significantly affected. Rates of recessive X-linked lethals and of recessive mutations at several loci affecting eye color imply that our EMS treatment was equivalent to approximately 100 generations of spontaneous mutation. If so, our data imply that one generation of spontaneous mutation increases the developmental time by 0.09% at 20 degrees and by 0.04% at 25 degrees, and reduces viability under harsh conditions, fecundity, and longevity by 1.35, 0.21, and 0.08%, respectively. Comparison of flies with none, one, and two grandfathers (or greatgrandfathers, in the case of viability) treated with EMS did not reveal any significant epistasis among the induced mutations.     

Effects of L-ascorbic acid on the mutagenicity of ethyl methanesulfonate in cultured mammalian cells.

The effects of L-ascorbic acid (AsA) on the mutations induced by ethyl methanesulfonate (EMS) were examined by means of the 6-thioguanine (6TG)-resistant mutation assay and chromosome aberration assay in cultured Chinese hamster V79 cells. When cells were treated with EMS at various concentrations in the presence of 100 micrograms/ml AsA, EMS-induced 6TG-resistant mutations were reduced about one third or one fourth. EMS-induced chromosome aberrations were also reduced by AsA. These reductions in the mutagenicity of EMS were also found when cells were treated with mixtures of AsA and EMS which had previously been incubated at 37.0 degrees C for 2 h. In pre- and post-treatments with AsA, however, the frequencies of EMS-induced mutations were not reduced, but rather increased markedly.     

Induction of specific-locus and dominant lethal mutations in male mice in the low dose range by methyl methanesulfonate (MMS)

Methyl methanesulfonate (MMS) induces specific-locus and dominant lethal mutations in spermatozoa and spermatids of mice. A dose of 15 mg/kg b.w. of MMS induces 9% dominant lethal mutations in the most sensitive germ-cell stages, corresponding to the mating intervals 5-8 and 9-12 days post treatment. A dose of 150 mg/kg b.w. of MMS in the same mating intervals induces 100% dominant lethal mutations. The sensitivity pattern for the induction of dominant lethal and specific-locus mutations is the same. In the mating interval 5-8 days a dose of 20 mg/kg b.w. of MMS induced 3.8 x 10(-5) mutations per locus per gamete. The yield of specific-locus and dominant lethal mutations in the low dose range increases proportionally with the dose. A dose given in 2, 4 or 5 fractions yields the same frequency of mutations as a single injection of the total dose. The additivity of small doses proves that the pre-mutational lesions are not or only partially repaired in these stages and that MMS is not or only partially detoxified. In addition, the frequency of dominant lethal and specific-locus mutations depends on the germ-cell stage.     

Desmutagenic and bio-antimutagenic activity of docosahexaenoic acid and eicosapentaenoic acid in cultured Chinese hamster V79 cells

The antimutagenic activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were examined by studying their effects on induction of 6-thioguanine (6TG)-resistant mutations by ethyl methanesulfonate (EMS) in cultured Chinese hamster V79 cells. DRA had a remarkable inhibitory effect against the cytotoxicity of EMS, when cells were simultaneously-treated with EMS, showing a blocking or scavenging activity of DHA in reduction of surviving fraction of cells. DHA had not so significant effect, when cells were treated before and after treatment with EMS. On the other hand, EPA had marked inhibiting effects against cytotoxicity of EMS, when cells were treated with EPA, before, simultaneous and after treatment with EMS. Against the induction of mutations by EMS, an antimutagenic activity of DHA was found when cells were pre-treated, simultaneously-treated or post-treated with DHA. EPA was also effective in reducing EMS-induced 6TG-resistant mutations when the cells were treated using the three different treatment procedures described above. The results suggest that in cultured Chinese hamster V79 cells, DHA and EPA may have both desmutagenic activity, which inactivates EMS chemically and/or enzymatically and bio-antimutagenic activity which suppresses mutation fixation after DNA is damaged by EMS.     

Analysis of mutation frequency following continuous and fractionated application of ethyleneimine and ethyl methanesulfonate to male sex cells of Drosophila melanogaster]

The increase in the frequency of recessive lethal sex-linked mutations induced by fractionated effect of ethylene imine (EI) an ethylmethanesulphonate (EMS) on mature sperm of Drosophila melanogaster was observed and compared uith prolonged treatment (8h) and with the additive effect. This effect of dose fractionation was observed in the case of the treatment of sperms in male gonads and in female spermathecas. The increase of the mutation frequency was noted by brood-pattern method after fractionated treatment of spermatocytes and spermatogonia only with EMS. This increase was not observed under the effect of EI on spermatocytes and spermatogonia because of the high sierilization activity of EI. Possible mechanisms of the effects observed 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.     

Effects of Dose on the Induction of Dominant-Lethal Mutations with Triethylenemelamine in Male Mice

Dose effects of triethylenemelamine (TEM) in the induction of dominant-lethal mutations were studied at the early spermatozoon, midspermatid and spermatocyte stages. The pattern of effects on spermatocytes, unlike midspermatids and early spermatozoa, indicated possible cytotoxic damage, so for the determination of TEM dose-response curves in the induction of genetic damage only the data for midspermatids and early spermatozoa were used. The TEM dose-effect curves for those two stages differ markedly from ethyl methanesulfonate (EMS) dose-effect curves. Beginning with the lowest doses at which significant effects are observed, there is a considerably more rapid increase in dominant-lethal effects with dose of EMS than TEM. Another marked difference between the two compounds is in the ratio of the genetically effective dose (as measured by dominant-lethal mutations) to the lethal dose. The ratio is 1:100 for TEM and only 1:3.5 for EMS; thus, TEM is mutagenic far below its toxic level. Obviously, these results have important implications not only for our understanding of the nature of chemical induction and recovery of chromosomal aberrations but also for the practical problems of evaluating the mutagenic effects of chemicals.     

Latheo, a New Gene Involved in Associative Learning and Memory in Drosophila Melanogaster, Identified from P Element Mutagenesis

Genetic dissection of learning and memory in Drosophila has been limited by the existence of ethyl methanesulfonate (EMS)-induced mutations in only a small number of X-linked genes. To remedy this shortcoming, we have begun a P element mutagenesis to screen for autosomal mutations that disrupt associative learning and/or memory. The generation of "P-tagged" mutant alleles will expedite molecular cloning of these new genes. Here, we describe a behavior-genetic characterization of latheoP1, a recessive, hypomorphic mutation of an essential gene. latheoP1 flies perform poorly in olfactory avoidance conditioning experiments. This performance deficit could not be attributed to abnormal olfactory acuity or shock reactivity-two task-relevant "peripheral" behaviors which are used during classical conditioning. Thus, the latheoP1 mutation appears to affect learning/memory specifically. Consistent with chromosomal in situ localization of the P element insertion, deficiencies of the 49F region of the second chromosome failed to complement the behavioral effect of the latheoP1 mutation. Further complementation analyses between latheoP1 and lethal alleles, produced by excision of the latheoP1 insert or by EMS or gamma-rays, in the 49F region mapped the latheo mutation to one vital complementation group. Flies heterozygous for latheoP1 and one of two EMS lethal alleles or one lethal excision allele also show the behavioral deficits, thereby demonstrating that the behavioral and lethal phenotypes co-map to the same locus.