Ethyl Methanesulfonate Treatment Research Articles

Induction of dominant lethals with ethyl methane-sulfonate in male germ cells of mulberry silkworm, Bombyx mori L.

Sensitivity of male germ cells in the mulberry silkworm, Bombyx mori L., to ethyl methanesulfonate (EMS) was determined by treating newly emerged 5th-instar larvae, and 2-day- and 7-day-old pupae with 3 concentrations, 0.05, 0.1 and 0.15%, of the mutagen. The frequency of dominant-lethal mutations induced by EMS treatment was used as the parameter for the study. Spermatids and spermatozoa were markedly sensitive to EMS. Statistical analysis confirmed that differences in respect of percentage of egg hatch among the 3 different treatments as well as the interactions between the 3 factors, e.g. stages, hatchability and EMS treatment, were highly significant.

FITNESS EFFECTS OF EMS-INDUCED MUTATIONS ON THE X CHROMOSOME OF DROSOPHILA MELANOGASTER. I. VIABILITY EFFECTS AND HETEROZYGOUS FITNESS EFFECTS

Drosophila melanogaster X chromosomes were mutagenized by feeding males sucrose solutions containing ethyl methanesulfonate (EMS); the concentrations of EMS in the food were 2.5 mM, 5.0 mM, and 10.0 mM. Chromosomes were exposed to the mutagen up to three times by treating males in succeeding generations. After treatment, the effective exposures were 2.5, 5.0, 7.5, 10.0, 15.0, and 30.0 mM EMS. X chromosomes treated in this manner were tested for effects on fitness in both hemizygous and heterozygous conditions, and for effects on viability in hemizygous and homozygous conditions. In addition, untreated X chromosomes were available for study. The viability and heterozygous fitness effects are presented in this paper, and the hemizygous fitness effects are discussed in the accompanying one (MITCHELL and SIMMONS 1977). Hemizygous and homozygous viability effects were measured by segregation tests in vial cultures. For hemizygous males, viability was reduced 0.5 percent per mM EMS treatment; for homozygous females, it was reduced 0.7% per mM treatment. The decline in viability appeared to be a linear function of EMS dose. The viabilities of males and females were strongly correlated. Heterozygous fitness effects were measured by monitoring changes in the frequencies of treated and untreated X chromosomes in discrete generation populations which, through the use of an X-Y translocation, maintained them only in heterozygous condition. Flies that were heterozygous for a treated chromosome were found to be 0.4% less fit per mM EMS than flies heterozygous for an untreated one.

The genetics of curly, a dominant, sex-linked mutant of Culex tritaeniorhynchus.

Curly , a new wing mutant isolated following ethyl methanesulfonate treatment of the mosquito Culex tritaeniorhynchus , is described. Genetic experiments indicate that the mutant is dominant and sex-linked (linkage group I) but is lethal when homozygous. The gene sequence is dt-Cy-wre-M and the recombination frequencies are as follows: dt-Cy = 4.6%, Cy-wre = 21.1%, and wre-M = 16.0%.

Temperature-sensitive mutant of Schizosaccharomyces pombe exhibiting enhanced radiation sensitivity

A conditional lethal and radiation-sensitive mutant of Schizosaccharomyces pombe is described in which both characteristics result from a single gene mutation. Confirmation of the pleiotropic nature of this mutant was obtained by tetrad analysis and by testing the radiation sensitivity of a large number of revertants that grew normally at the restrictive temperature. The colony-forming ability of the mutant after ultraviolet radiation, gamma radiation, and ethyl methane sulfonate treatment is considerably altered by the post-treatment incubation temperature, showing higher survival at 25 than at 30degreesC. The radiosensitivity of the mutant is also influenced by the stage of growth. The difference in radiation sensitivity between the wild type and mutant is greater when log-phase cultures are compared. The characteristics of this mutant suggest that it is defective in a step common to both deoxyribonucleic acid replication and repair.

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.

Genetic complementation in hybrid cells derived from mutagen-induced mouse clones deficient in HGPRT activity

Cultured mouse clonal cells, H-5, were treated with two different mutagens, ethyl methanesulfonate (EMS) and N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). Then two selective procedures using 8-azaguanine (8-AZ) or 6-thioguanine (6-TG) were compared in an effort to isolate hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-deficient cells containing different gene alterations. While many 8-AZ resistant cells were induced by EMS treatment, considerably more 6-TG resistant cells were induced by the same treatment. MNNG treatment induced many 8-AZ resistant mutants but induced hardly any 6-TG resistant mutants. After a fusion experiment of 91 sets involving 13 HGPRT-deficient mouse clones, 7 of which were resistant to 8 AZ and 6 of which were resistant to 6TG with subsequent selection on HAT medium, complementation occurred only in those hybrid mixtures formed between 8-AZ- and 6-TG-resistant clones, while it did not occur at all in hybrid mixtures formed between different 8-AZ-resistant clones and mixtures formed between different 6-TG-resistant clones. The clonally isolated HGPRT-positive cells, characterized by tetraploid karyology, had an apparent activity of HGPRT ranging from 25 to 30% of that of the wild-type parental cells. Heat-inactivation of HGPRT at 65 °C revealed that HGPRT from wild-type cells was heat stable and HGPRT from some 8-AZ-resistant clones were heat labile, while HGPRT from hybrid cells had intermediate stability. These results indicate that there would be alterations in the structural gene of HGPRT in the 8-AZ- or 6-TG-resistant mutants, and also that two selective procedures with 8-AZ or 6-TG alone can isolate different alterations in the structural gene of HGPRT. Moreover, this indicates that some of these gene alterations were mutually complementary. It is most likely that there would be at least 3 cistrons in the locus responsible for HGPRT activity in the mouse cells.

Effect of recurrent mutagen seed treatments on mutation frequency and combining ability for forage yield in pearl millet ( Pennisetum americanum (L.) K. Schum.)

The effects of 3 cycles of recurrent seed treatment with thermal neutrons (TN), ethyl methane sulfonate (EMS), and diethyl sulfate (DS) on mutant frequency and combining ability in pearl millet ( Pennisetum americanum (L.) K. Schum., formerly P. typhoides ) were studied for eleven years. TN seed treatments gave the highest percentages of M 1 striped plants, the lowest percentage of M 1 selfed seed set, and the highest M 2 frequency of chlorophyll-deficient seedlings of the mutagens tested. Combining a low dose of EMS or DS with high and low TN treatments generally increased these effects. Normal lines, which looked like the controls, selected from lines subjected to 3 cycles of mutagen treatment, were compared with controls in 3 × 3 or larger Design II hybrid matings in 9 × 9 forage-yield trials. The 1,637 singlecrosses between normal lines from mutagen treatment failed to exceed the 825 control singlecrosses in average forage yield or highest forage yield. These results suggest that pearl millet has many specific-yield genes, none of which exerts a very great effect on yield. Genetic variances estimated from the Design II diallels were not significantly altered by mutagen seed treatment. EMS treatments increased the percentage of non-additive genetic variance over that in the control and TN treatments, but the failure of any of the 486 hybrids from EMS lines to outyield the best control hybrid suggested that these variance estimates might not be significant. The study suggests that attempts to improve the combining ability of inbred lines of pearl millet by mutagen treatment without several cycles of recurrent selection are not likely to succeed.

Light sensitivity of ethyl methanesulfonate treatment in Hordeum vulgare

Light sensitivity of ethyl methanesulfonate treatment in Hordeum vulgare

The effect of ethyl methanesulfonate treatment on floret sterility and chlorophyll mutation rate in barley

Early effects of ethyl methanesulfonate (EMS) on five barley cultivars (Clipper, G. I. 3576, Proctor, Ketch and Prior) with contrasting yield and adaptation characteristics were investigated. Considerable increases in the M1 sterility and the frequency and the spectrum of M2-chlorophyll mutations were noted. The highest sterility and mutation frequencies were obtained with treated Proctor and Prior cultivars whereas with Clipper, both these responses were minimal.

Light sensitivity of ethyl methanesulfonate treatment in Hordeum vulgare

Light sensitivity of ethyl methanesulfonate treatment in Hordeum vulgare

Comparison of somatic mutation rates induced in tradescantia by chemical and physical mutagens

Summary When genes for flower color and other phenotypic characteristics are present in a Heterozygous condition in Tradescantia plants, they can be used as genetic markers for the study of induced mutation. The procedure used is to expose cuttings containing young flower buds to chemical or physical mutagens. 1 to 3 weeks later the resultant mutant pink or colorless cells can be seen in mature flowers, and after relatively simple scoring procedures somatic mutation rates can be calculated. Dose-response curves for ethyl methanesulfonate (EMS) and 1,2-dibromoethane (DBE) were obtained and compared with existing X-ray dose-response curves. Two clones, 02 and 4430 were exposed to known levels (from 5–250 ppm) of EMS in gaseous form for periods of from 1–19 h. After exposure to X-rays pink mutation rates showed similar sensitivities for both clones but the rate for colorless was much higher for clone 4430 and the slope much steeper. After treatment with EMS both colorless and pink rates were appreciably higher for clone 4430 than for clone 02. Preliminary data show similar differences in clonal sensitivity following exposures to DBE. The large differences in mutation rates between loci and between clones after chemical and physical mutagen treatments are not understood at present. Possibly differences in mutation rates between the two clones after EMS treatment may be associated with a larger amount of heterochromatin or a greater number of SAT-chromosomes in clone 4430. The genetic basis for the phenotypic changes (pink and colorless) is not definitely known but may be associated with chromosome breakage, gene mutation, chromosome non-disjunction, or somatic crossing over.

Effect of caffeine on chromosome damage induced by chemical mutagens and ionizing radiation in Vicia faba and Secale cereale

The potentiating effect of caffeine on chemically induced chromosome aberrations was studied in Vicia root tips exposed to maleic hydrazide (MH), difunctional alkylating agent diepoxybutane (DEB) and two monofunctional ones, N-ethyl-N- nitrosourea (ENU) and methyl methanesulphonate. Aberrations induced by MH and DEB were strongly potentiated by caffeine post-treatments; the effect was noted even when the time between the mutagen and caffeine treatment was considerably prolonged. The data obtained suggest that cells exposed to these mutagens are most sensitive to caffeine during their first passage through the S-phase. Induction of aberrations by ethyl methanesulfonate (EMS) and ENU were also highly potentiated by caffeine post-treatment. However, following EMS treatment the potentiating effect of caffeine was observed already after 20 h of recovery whilst after ENU treatment this effect was delayed up to 30 h. The yields of aberrations incluced by X- and γ-rays in Vicia were not significantly increased by caffeine pre- or post-treatments. This was true both for the damage induced in seeds by X- and γ-rays and for chromatid-type aberrations induced by X-rays in root lips. In contrast to Vicia the yield of aberrations induced by X-rays in rye seeds was significantly increased by caffeine post-treatment.

Nuclear and extranuclear mutations in yeast induced by ethyl methanesulfonate.

When zero-point mutations were induced in the yeastSaccharomyces cerevisiae using ethyl methanesulfonate (EMS) no differences were found in the frequency of auxotrophic mutants formed by a short and a prolonged treatment of the agent at equal survival level. The expression of a part of the mutations induced by a prolonged EMS treatment was delayed by one or two division cycles. The total frequency of auxotrophs due to both the zero point and delayed mutations, however, is still considerably lower than the frequency of auxotrophs induced by a prolonged treatment of EMS in some bacterial species. Both the prolonged and short EMS treatment induces in yeast also extranuclear respiration-deficient (RD) mutants at a relatively high frequency; in wild strains at equal survival level the prolonged treatment produces a higher number of RD mutants than the short one. In strain which is more susceptible to the lethal EMS effect than wild strain the number of RD mutants produced by the agent is much higher than in the wild strain. The results support the assumption of the different DNA arrangement in yeast nuclei and mitochondria and indicate the possible effect of repair mechanisms during the induction of mutations causing the respiration deficiency.

Temperature-sensitive mutations in Drosophila melanogaster: XI. Male sterile mutants of the Y chromosome

Eight temperature-sensitive ( ts) male sterile mutations have been induced by ethyl methanesulfonate treatment of Y chromosomes derived from a selected temperature-resistant Amherst wild-type stock of Drosophila melanogaster. Males carrying such mutated Y chromosomes (Y ts ) are sterile when raised at 29°C but fertile when reared at 22°C. Complementation tests of the mutants with Y chromosome fragments, deletions, and inter se localized all eight to the long arm of the chromosome in four different complementation groups. When Y ts -bearing males, reared to adulthood at 22°C, were subjected to a 48-hr regimen at 29°C and mated to fresh virgin females daily, a significant reduction in fertility resulted 5 days after initiation of 29°C treatments. This period of sterility was transient (48–72-hr duration) and corresponded to a temperature-sensitive period (TSP) of spermatogenesis during the primary spermatocyte stage. A more precise definition of the TSP utilized exposure of subadult males to 29°C at selected developmental periods during which only certain germ cell stages are present. Upon eclosion adult males were subjected to a similar schedule of consecutive matings of 12-hr duration in order to detect any delay in the appearance of fertility. Different ts males could be distinguished by the resultant pattern of sterility, and the TSP of different mutations thus localized to either primary spermatocyte or immediately post-meiotic stage. Associated with Y ts -mediated sterility, spermiogenesis is defective at restrictive temperature as evidenced by the production of nonmotile sperm and a failure to transfer such sperm to the female during copulation. In addition, electron microscopy detected a variety of ultrastructural abnormalities, including defects of axoneme formation, irregularities of Nebenkern derivative development, and failures of separation from the syncitial state or mature cyst with subsequent degeneration.

Extra-chromosomal mutability determined by a nuclear gene locus in arabidopsis

Two recessive mutations isolated after ethyl methanesulfonate treatment produce variegation when homozygous with excellent penetrance and variable expressivity. The variegation involves different degrees of pigmentation and morphology of the leaves as well as fertility. The altered phenotypes observed in the mutants display non-mendelian inheritance though the primary cause of the alteration is the mutation at a chromosomal locus ( chm) in linkage group 3. The two mutants seem allelic but non-identical. The results of the genetic analysis are consistent with the interpretation that a nuclear mutator locus is inducing several different type of hereditary alterations in the plastome. The plastome mutations reveal involvement of the plastome in the control of its own morphology as well as in cellular and organ differentiation. The system indicates that though the plastome is endowed with a considerable autonomy in some functions its mutability is subject also to nuclear control. The informational content of the 20–80 plastids per cell is apparently identical. The occurrence of cells with different types of plastids reveals that “epistasis” is not of major importance among the plastids within a single cell. The independent expression and inheritance of color variegation from morphological alterations indicate a separate localization (absence of linkage) of the pertaining genetic elements. The predominantly joint appearance of other phenes point to the existence of pleiotropy or linkage within the plastids.

Grain yield mutations induced by ethyl methanesulfonate treatment of oat seeds

Arias J. and Frey K.J. Grain yield mutations induced by ethyl methanesulfonate treatment of oat seeds. Radiation Botany 13, 73-85, 1973.-Populations of oat lines derived from seeds that either were treated or not treated with ethyl methanesulfonate (EMS) were evaluated for mutations that were suitable for direct yield improvement. Field experiments were carried out with equal numbers of check and normal appearing M2-derived lines for each of 16 cultivars: nine Corn Belt, five non Corn Belt, one tetraploid, and one diploid. The M2-derived lines from cultivars were generated from treatment of seeds with a 0·12 m solution of EMS for 4 hr, and the check lines were bulks from individual plants of the same cultivar. For a mutagen-derived line to be considered a deviant for grain yield, it had to have either a higher or a lower yield than the highest or lowest line, respectively, found in the check population. Using this definition, there were 160 mutagen-derived lines with negative deviations and one with a positive deviation for grain yield among 1141 lines tested. The mutagen-derived line with improved yield was 12 per cent higher than the highest check line from its parent cultivar, but it was considerably lower than several of the highest yielding check lines from other cultivars. The genetic variabilities for grain yield in mutagen-derived populations from the 16 cultivars were 2–10 times larger than those in corresponding check populations. Simultaneously there was a 17 per cent reduction in the overall yield mean of the mutant lines. All check populations showed significant genetic variation among lines for grain yield, which shows that these cultivars were not genetically homogenous for this trait. Seemingly, this variation could be utilized to improve yield by intracultivar selection.

Mutational response of rice seeds to ethyl methanesulfonate and busulfan in relation to chromosome duplication

At various times after soaking of rice seeds they were treated with ethyl methanesulfonate (EMS) and its bifunctional agent 1,4-di(methanesulfonoxy)butane (busulfan). After 60-h soaked seeds had been treated for 2 h with EMS and busulfan, the LD 50 was estimated as 4·10 −4 M for the former, and 1.3·10 −5 M for the latter. In EMS- or busulfan-treated materials field survival was reduced to a marked extent at the S stage, and the maximal lethality was attained at the late S and/or early G 2 phase. After EMS treatment the mutation frequency was very high in late G 1, decreased suddenly at middle S, increased again markedly at late S and/or early G 2 and subsequently decreased. On the other hand, busulfan produced the mutation only in the cells that were in the process of DNA synthesis. Concerning the mutation spectrum the tigrina mutant appeared only in treatments made in the G 1 phase. In the light of these results, the mutagenesis of EMS and busulfan is discussed.

The isolation and characterization of a Saccharomyces mutant deficient in Δ 1-pyrroline-5-carboxylate dehydrogenase activity

1. Clones failing to grow on proline were isolated after ethylmethane-sulfonate treatment of a glutamate auxotroph of Saccharomyces, originally capable of utilizing proline as principal nitrogen source. 2. Most of the non-utilizers of proline were cytochromeless. One, however, possessed normal cytochrome levels but lacked Δ 1-pyrroline-5-carboxylate dehydrogenase activity. This is the first isolate of this lesion in Saccharomyces.

A characterization of repair mechanisms operating subsequent to genetic damage induced by ethyl methanesulfonate and gamma radiation in Bracon hebetor

Summary This investigation utilized females of the parasitic waspHabrobracon juglandis(Ashmead) = Bracon hebetor Say to study the biological and biochemical processes involved in the modification of genetic damage induced by a chemical and a physical mutagen, ethyl methanesulfonate (EMS) and gamma radiation, respectively. The hatchability of methaphase-prophase oocytes improved when EMS-injected females were temporarily prevented from ovipositing by withholding hosts. Storage following 2000 R gamma radiation also resulted in improved hatchability. Repair processes appear to take place during storage of mature oocytes. Mature oocytes ready for deposition are halted in metaphase I of meiosis. Unfertilized eggs routinely haploid males. In this organism the oviposition process, rather than sperm penetration, is considered the developmental stimulus. Storage without hosts following mutagenic treatment postpones development and apparently provides optimum conditions for repair. If, however, the mitotic divisions of cleavage begin immediately following mutagenic treatment, then the repair processes have not had sufficient time to correct genetic damage and cannot do so during development. This was reflected in the higher hatchability of stored oocytes. The physical and chemical environment of the egg during storage were modified and conditions necessary for repair were determined. Inhibition studies following EMS treatment demonstrated that low temperature, low oxygen concentration, 2,4-dinitrophenol and reduced glutathione inhibited repair. Radiation repair was also inhibited by low oxygen, low temperature and dinitrophenol. Deoxyadenosine, chloramphenicol, copper sulfate, and two reducing agents were not effective inhibitors. Negative results were also obtained with two enzyme inhibitors,p-hydroxymercuribenzoate, and N-ethylamaleimide. An identical “error-correcting mechanism” may be involved in the repair of radiation and chemically induced genetic damage.

Dosimetry of the chemical mutagen ethyl methanesulfonate in spermatozoan DNA from Drosophila melanogaster

Summary A method for determining the chemical dose of ethyl methanesulfonate (EMS) to the DNA of Drosophila melanogaster sperm cells has been developed. Males fed as larvae on media containing [ Me - 3 H]thymidine were fed0.025 M , 0.0125 M and 0.008 M concentrationd of [I- 14 C]ethyl methanesulfonate ([ 14 ]EMS) as adults. Our feeding procedures and concentrations were chosen so as to duplicate as nearly as possible the treatments other workers have used. The amount of 3 H per sperm cell was determined by microscopically counting sperm cells, washing them with cold trichloroacetic acid (TCA) onto a MIllipore filter, and counting radioactivity with a liquid scintillation spectrometer. 14 C and 3 H counts were determined for oligodeoxynucleotides released from labeled sperm DNA by electrophoretically purified deoxyribonuclease. From these two counts, the specific activity of [ 14 C]EMS and the amount of 3 H per cell, the number of ethylations per sperm cell DNA was found to range from 1.10 7 to 3.10 8 . Estimates for the number of nucleotides per sperm cell DNA of Drosophila are found from the literature to range from 1.6· 10 8 to 4.5 · 10 8 . It has, therefore, been concluded that the concentrations of [ 14 C]EMS used in these experiments were able to ethylate between 3% and 100% of the nucleotides in the sperm cell DNA of Drosophila, assuming one ethylation per nucleotide. The results of our chemical dosimetry experiments are also discussed in relation to comparative mutagenesis studies with other genetically well studied organisms, such as the mouse. On the basis of the extensive DNA ethylation found in Drosophila sperm combined with previously published reports on sex-linked recessive lethal frequencies and production of mosaics following EMS treatment of Drosophila, it is hypothesized that ethylation of a rare site or combination of sites in the sperm DNA is responsible for the mutagenic action of EMS. Previous models models suggesting the production of mutations by incorrect base pairing of 7-ethylguanine following treatment by EMS cannot explain mutagenesis in Drosophila.