Molecular dosimetry of the mutagen ethyl methanesulfonate in Drosophila melanogaster spermatozoa: linear relation of DNA alkylation per sperm cell (dose) to sex-linked recessive lethals.

The dosage-response curve for EMS was determined with dose measured as ethylations of DNA per sperm cell, and response measured as the relative frequency of sex-linked recessive lethals induced in sperm cells of Drosophila melanogaster. Dose can be converted to ethylations per nucleotide of DNA by dividing ethylations of DNA per sperm cell by 3 X 10(8) nucleotides per sperm cell. Adult males were exposed to equal amounts of either [3H]EMS for determining dose or nonlabeled EMS for determining mutational response. By feeding EMS for 24 h in a concentration of 25 mM, a high dose of 1.4 X 10(-2) ethylations per nucleotide was observed. With 1.4% of the nucleotides ethylated, 57% of the X-chromosomes were hemizygously viable; therefore, ethylation per se is not very efficient in inducing mutations. The relative frequency of mutations increased linearly with the dose from a dose of 2.1 X 10(-4) to 1.4 X 10(-2) ethylations per nucleotide. No threshold was apparent, and the statistical limits of the exponent, 1.0 +/- 0.1, excluded an exponent as high as 1.2. This linear relation suggests no change in mechanism of mutagenesis occurs from low to high dose in Drosophila. A nonlinear relation was found between exposure and dose; when exposure was increased by a factor of 250 (from 0.1 to 25 mM EMS in the feeding medium) dose was increased by a factor of only 68. By extrapolating down from our lowest dose of 2.1 X 10(-4) ethylations per nucleotide with an observed frequency of 0.55% +/- 0.08% sex-linked recessive lethals, we estimate the doubling dose for sex-linked recessive lethals to be 4 X 10(-5) ethylations per nucleotide.     

Estimation of the effects of chemical mutagens: Lessons from radiation genetics

Years of work with ionizing radiations have given us a wealth of data on radiation-induced mutations. These data, which have given insights regarding the mutational processes, should form the background for all mutagenesis work. In chemical mutagenesis, as in radiation mutagenesis, it is important to know the shape of the dose-effect curve in order to make further interpretations and calculations. It is also important to be on the constant alert for new relations that can be explored.     

Mutagen sensitivity of Drosophila melanogaster. I. Isolation and preliminary characterization of a methyl methanesulphonate-sensitive strain

Sensitivity to the monofunctional alkylating agent methyl methanesulfonate (MMS) has been tested as a selection technique to isolate mutant strains which can provide insights into the genetic control of DNA replication, DNA repair and recombination in the complex eucaryote, Drosophila melanogaster. The successful isolation of an X-linked MMS-sensitive strain, mut^s, has suggested that mutagen sensitivity is a feasible methodology for the selection of mutant strains of Drosophila which will be useful in the genetic and biochemical analysis of these cellular functions. Preliminary characterization of this mutant strain indicates that: (A) it is extremely sensitive to killing by MMS; (B) it is more mutable by MMS than the parent wildtype strain; and (C) it appears to possess mutator gene activity.     

Induction of dominant lethals with ethyl methane-sulfonate in male germ cells of mulberry silkwork, 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.     

Cytogenetic and dominant lethal studies on captan.

Possible mutagenic activity of captan was investigated by in vitro and in vivo cytogenetic studies and by the dominant lethal study in mice. In vitro cytogenetic study with cultured human diploid cells revealed a significant increase in the frequency of cells showing stickiness and a severe mitotic inhibition at concentrations of 3.0 and 4.0 microgram of captan per ml. although no chromosomal aberrations were observed. In in vivo cytogenetic study, no chromosomal aberrations were induced in the bone marrow cells of rats treated orally with captan at a single dose of 500, 1000 or 2000 mg/kg or at five consecutive doses of 200, 400 or 800 mg/kg/day. Dominant lethal study also failed to show any mutation induction after treatment of male mice with daily oral dose of 200 or 600 mg of captan per kg bw for five days.     

The effect of storage on the frequency of dominant lethals in Drosophila melanogaster

Summary Ethylenimine, ethyl methanesulfonate and formaldehyde have been shown to produce a storage effect in dominant lethality in Drosophila melanogaster.     

The nature of reverse mutations in Drosophila melanogaster

A selection system for the screening of reversions has been constructed and used to test reversions of lethals located in the proximal region of the X chromosome of Drosophila and of Kpn mutations.Spontaneous and induced reversions have been screened, X-rays and ethyl methanesulphonate (EMS) being the mutagens used in the induction experiments.No genuine back-mutation was found in 6·10⁵ gametes scored. Sterile reversions of all four lethals tested were obtained. Their frequency suggested that at least in three of the lethals the sterile reversions represented “escapers” of the lethal effect rather than true revertants.Three fertile reversions of l^x4 were found and analyzed. All three were autosomal suppressors, located on the second chromosome, allelic to each other, dominant in males and recessive in females.One fertile reversion of l^3DES was found to be an X-linked suppressor. It is suggested that this suppressor is a Y-suppressed lethal, showing a V-type position effect, resulting from an aberration included in the proximal heterochromatin of the X chromosome.Reversions of Kpn were obtained at a similar rate to that found in previous reports²².The absence of true back-mutants in our experiments, in contrast to findings in previous reports, is discussed. From the existing literature on spontaneous and induced back-mutations in Drosophila melanogaster it appears that for several mutations the rates of forward and back-mutation are of the same order of magnitude. It is suggested that reported cases of back-mutations represent mainly inter- and intrachromosomal recombination in duplicated regions rather than mutational events and that the frequency of true back-mutation in Drosophila is usually of an order of magnitude, similar to that known for microorganisms and fungi.     

Mutagen-sensitive mutants in Drosophila melanogaster: effects on premutational damage.

Drosophila melanogaster males from a Basc stock were mutagenized with either X-rays, ethyl methanesulfonate (EMS), or nitrogen mustard (HN2). Groups of identically treated males were crossed to different types of female. Sex-linked recessive lethals were scored as a genetic end point. The females used were homozygous for X-chromosomal mutations (mus(1)101D1, mus(1)104D1, mei-9 or mei-41D5) which lead to defective DNA repair and which increase the mutagen sensitivity of larvae. Females from a white stock with normal DNA repair capacities served as controls. The premutational lesions induced in mature sperm are only processed after insemination by the maternal enzyme systems present in the oocytes. Differences in the efficiency of the processing of lesions can lead to maternal effects on the frequency of mutations recovered from mutagenized sperm. It was found that, with the exception of mus(1)104D1, all mutants analysed significantly modify the mutation fixation of one or more types of premutational lesions. The most drastic effect is found with the mus(1)101D1 stock in which HN2-induced DNA cross-links do not lead to sex-linked recessive lethals. It is assumed that mus(1)101D1 is defective in an early step of DNA cross-link repair. Our first set of data clearly demonstrates that the study of maternal effects in Drosophila is an efficient tool to analyse the in vivo function of repair mutations on chemically induced mutagenesis.     

Isolation and characterization of temperature-sensitive mutants in a Chinese hamster cell line

A replica plating method was used for the isolation of temperature-sensitive (ts) mutants after treatment of Chinese hamster cells with ethyl methanesulfonate (EMS). No significant increase in ts mutants was found after this treatment. The limitations and advantages of the replicating procedure to detect such differences, as well as an alternative method, are discussed.Mutants isolated were classified into two general groups—density-dependent and clear-cut—as measured by survival at low and high cell densities at the restrictive temperature. The density-dependent mutants may be truly “leaky”, losing a metabolite to the medium at an excessive rate at the restrictive temperature. On the other hand, the one clear-cut mutant analyzed extensively dies at a rate determined by its ability to utilize one or more components from the medium. It shows an inverse density relationship in rate of death, as inferred from rates of macromolecular synthesis, as opposed to its growth rate at the permissive temperature.