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.     

Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster. IV. The genetics of the relative radioresistance in stage-7 oocytes of the irradiated population RO I

The genetic system that controls the relative radioresistance in an irradiated laboratory population of Drosophila melanogaster (RÖ I) was studied. Comparisons were made between an unirradiated control population (+60, +K), the population RÖ I (after 227–333 generations of irradiation at 2100 R per generation), the sub-population RÖ I₀ (derived from RÖ I after 260 generations of irradiation and kept without irradiation for up to 74 generations), the F₁ hybrids +60/RÖ I, various homo- and heterozygous carriers of the 3 major chromosomes of RÖ I and +60, respectively, in combination with suitable balancers, and several chromosome substitution stocks of +K and RÖ I. The criteria used to assess the magnitude of radiosensitivity were dominant lethals, X-chromosome loss, and sex-linked recessive lethals induced in stage-7 oocytes at various exposure levels of X-irradiation.The data show that the radioresistance in RÖ I is controlled by a stable and homozygous genetic system. The system is semidominant. With respect to the induction of dominant lethals and sex-linked recessive lethals, the relative resistance is mainly contributed by chromosomes I and II. The effects of the two chromosomes are additive, each contributing about half the relative resistance. Resistance to the X-ray induction of X-chromosome loss is solely contributed by chromosome II.The findings suggest that at least 2 different and independent mechanisms are involved in determining the resistance of the RÖ I population.     

Mutagenic effect of different types of irradiation on the sex cells of male mice. X. Frequency of recessive lethal mutations and reciprocal translocations in the spermatogonia of mice subjected to fractional gamma-irradiation]

The frequency of recessive lethal mutations and reciprocal translocations was investigated in spermatogonia of CBA male mice which were thrice gamma-irradiated at doses of 300 r with 28 days intervals. The rate of induced recessive lethals was estimated 1) by comparison of embryos survival between the irradiated and control groups in mating of the F1 males with their daughters, and 2) by estimation the frequency of males heterozygotes for recessive lethals in the first generation. In the first case the frequency of recessive lethals was 2,8 +/- 0,8-10(-4) per r per gamete (for the pre- and post-implantation death) and 1,6 +/- 0,1-10(-4) per r per gamete (for the pre- and post-implantation death) and 1,6 +/- 0,1-10(-4) per r per gamete in the second case. The frequency of heterozygotes for reciprocal translocations in the first generations of males was 3,1 +/- 0,9-10(-5) per r per gamete.     

Accumulation of lethals in highly selected lines of Drosophila melanogaster

Summary Four synthetic lines of D. melanogaster selected for low sternopleural bristle number for 50 generations were screened for lethals on chromosome III when their mean score equalled 2.5. Each line originated from a cross between line M (previously selected for the same trait during 130 generations) and a different unselected cage population. Line M was already known to carry a recessive lethal on chromosome III affecting the selected trait, such that the bristle score of the lethal heterozygote was lower than that of the viable homozygote. Tests revealed 18 lethals, 15 of these present in at least two lines. Each line carried from 10 to 16 lethals. All lines carried groups of lethals present on the same chromosome, and at least six lethals in each line were included in such an association with a frequency of 0.18 or higher. It appears that the lethal affecting bristle score in line M has protected a segment of chromosome III from natural selection and that the remaining 14 lethals have accumulated later in that line.     

Influence of the MR (mutator) factor on X-ray-induced genetic damage

The genetical effects induced by MR, in the progeny of outcrossed MR-males, include very high frequencies of visible and lethal mutations and chromosome aberrations. The hypothesis is that MR causes breaks at specific sites in the DNA where, subsequently, insertion sequences become integrated. To examine whether there exists an interaction between breaks and radiation induced lesions, MRh12/Cy males were crossed to Berlin K females and the male progeny from this cross carrying the MR or Cy chromosome were irradiated. The frequencies of X-linked recessive lethals and II-III translocations were determined. Non-irradiated MR and non-MR (Cy) male progeny were used in concurrent controls. The results show that the frequencies of II-III translocations in the MR-containing males is not significantly higher than in the controls. However, with regard to the production of recessive lethal mutations a clear synergism between MRh12 and X-irradiation was observed.     

Experimental studies on mating systems and genetic load in Onoclea sensibilis L. (Aspleniaceae: Athyrioideae)

Sporophytes from natural populations of Onoclea sensibilis L. from eastern North America and Japan were investigated to determine their mating system and the frequency of recessive deleterious genes (genetic load). Morphological and physiological criteria in the gametophyte generation indicate that this species has a high probability of intergametophytic mating in nature. All sporophytes exhibited some degree of heterozygosity for genetic load, expressed either as zygotic lethals or leaky lethals. Effects of antheridogen on gametophyte sex ratios and polyembryony are described.     

Mutagenicity of 7,12-dimethyl[a]anthracene and some other aromatic mutagens in Drosophila melanogaster

The optimal conditions for mutagenesis studies with DMBA and some other aromatic carcinogens in Drosophila were investigated in detail. The results presented in this paper indicate the following.The mutagenic effectiveness of DMBA is dependent on the route of administration, injection being far more effective when compared with feeding.The choice of the solvent is a crucial experimental condition. DMBA, when dissolved in oil/DMF, is ineffective whereas a special fat emulsion of DMBA gives high mutation frequencies.There appears to be an extreme strain dependence in the mutagenicity of DMBA. Mutagenic effectiveness in strain Berlin-K was rather low, whereas Oregon-K and Karsnäs-60 proved to be very susceptible to DMBA.Under the conditions of test, DMBA did not induce loss of a ring-X chromosome and did not produce recessive lethal mutations in such a chromosome.DMBA did not produce 2–3 translocations to any significant extent.An increase in DMBA-induced recessive lethal mutations was found upon storage of treated sperm with an optimal storage time of 4–10 days.DMBA is efficient in the production of delayed recessive lethal mutations in strain Berlin-K. Twice as many lethals were recovered with the F3 generation as compared with those in F2. In strain Oregon-K, where the frequency of F2 lethals was much higher than in strain Berlin-K, the ratio of F3/F2 lethals was clearly lower.Enzyme induction with phenobarbital reduces the mutagenic effectiveness of DMBAWith TMBA, similar strain differences in sensitivity were observed as those found for DMBA. Whereas TMBA was not mutagenic in Berlin-K, considerable mutagenicity was observed in Oregon-K and Karsnäs-60.Injection of carcinogenic polycyclic aromatic hydrocarbons and aromatic amines, when dissolved in special fat emulsions, enhances the mutagenic effectiveness of some compounds (DMBA, TMBA, DA and AcO-AAF), but this procedure does not always solve the problems-pertinent to these classes of promutagens in Drosophila.     

Farm-grade chlorpyrifos (Durmet) is genotoxic in somatic and germ-line cells of Drosophila.

The mutagenic potential of Durmet, a farm-grade formulation of chlorpyrifos, was studied in the Drosophila wing mosaic and sex-linked recessive lethal tests. Larvae of the 2nd or 3rd instar carrying suitable recessive genetic markers on chromosome 3 were exposed to different concentrations of the insecticide and the frequency of induction of mutant mosaic spots on the wings was noted. The Basc technique was followed to study the induction of sex-linked recessive lethals. On the basis of the frequency of induction of mosaic wing spots and sex-linked recessive lethals, it is concluded that Durmet is genotoxic in somatic cells as well as germ cells of Drosophila.     

A quick method for testing recessive lethal damage with a diploid strain of Aspergillus nidulans.

A simple method capable of detecting recessive lethal damage in a diploid strain of Aspergillus nidulans is described. The method scores the recessive lethals on the 1st, the 3rd and the 5th chromosomes, which represent about 40% of the total map of A. nidulans. Two examples of induced lethals, with ultraviolet irradiation and methyl methanesulfonate are shown. The frequency of lethals may reach 36% of the total population with UV irradiation.     

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.     

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.     

The genotoxicity of the anti-cancer drug mitoxantrone in somatic and germ cells of Drosophila melanogaster.

The novel antineoplastic drug mitoxantrone was studied for its genotoxic effects in Drosophila melanogaster. In male germ cells, the clinical preparation Novantrone, the dihydrochloride salt of mitoxantrone, did not induce sex-linked recessive lethal mutations in feeding and injection experiments with adult flies, although statistically the results were inconclusive rather than truly negative. However, the free base mitoxantrone was weakly, but significantly genotoxic in this test (0.14% lethals/mM exposure concentration); this is most probably the result of prolonged exposure. On the other hand, both forms of mitoxantrone assayed were clearly genotoxic in the somatic mutation and recombination test of the wing. This test assays the cells of the proliferating imaginal wing discs of larvae. Depending on the feeding method used, the overall clone induction frequency was in the range of about 2-6 x 10(-5) per cell and cell generation and per mM exposure dose. Correction of these frequencies according to mean clone size led to slightly higher estimates (by about 5-25% higher). Although the majority of the clone induction events are due to mitotic recombination, a significant proportion can be attributed to mutational events (gene and chromosome mutations). The genotoxicity of mitoxantrone seems to depend mainly on impaired DNA synthesis in cycling cells owing to the compound's ability to inhibit topoisomerase II by intercalation into DNA.     

Autosomal recessive lethal mutations in two mutator stocks of Drosophila ananassae.

The frequency of recessive lethals in the 2nd chromosome was examined in two mutator stocks of Drosophila ananassae, ca and ca; px. They are characterized respectively by possessing an extrachromosomal clastogenic mutator in males, and by the retrotransposon "tom", which induces Om mutability only in females. The frequencies of recessive lethal mutations in the 2nd chromosome among progenies from males and females of the ca; px stock are 0.35 and 0.34 percent, respectively. Similarity of these frequencies indicates that tom does not induce recessive lethals in females. In contrast to the ca; px stock, the frequency of recessive lethals in males of the ca mutator stock was estimated to be 1.54 percent for the 2nd chromosome. No visible mutants except Minutes were recovered. Some recessive lethals derived from ca stock males were associated with chromosomal rearrangements. Being consistent with its high rate of Minute mutation it was demonstrated that the ca clastogenic mutator also induced recessive lethals.     

Clan Lethals and Inter-Year Allelism in Death Valley DROSOPHILA PSEUDOOBSCURA

A chromosome 2 lethal allelism rate of about 3% was found in the 1974 population of D. pseudoobscura in Death Valley, California. This rate was significantly higher than allelism rates in other Southern California populations. The Death Valley population was sampled again in 1975 and 1977, with allelism rates of 1% and 0.5%, respectively. In 1974, several lethals were in high frequencies (about 1%), a pattern that reappeared in 1975 and 1977. However, none of the lethals in high frequency one year were in high frequency another year; the particular lethal alleles present in this ephemeral population appear to be due to their random presence in the flies which refound the population every winter. The results for the Death Valley population are compared with a Japanese population of D. melanogaster in which lethals in high frequency one year are also in high frequency in succeeding years and with earlier work on chromosome 3 of D. pseudoobscura, which showed a lower lethal frequency and higher allelism rate.     

Trenimon-induced sex chromosome loss in Drosophila: different dose-responses for ring-X loss as compared to rod-X loss and Y-marker loss

Drosophila melanogaster males carrying either a ring- or a rod-shaped X-chromosome were injected or fed with Trenimon (triaziquone) at concentrations ranging from 5 X 10(-5) to 2 X 10(-2) mM. The F1 generation was assayed for the occurrence of total sex chromosome loss and of Y-chromosome markers. Sex-linked recessive lethal tests were carried out simultaneously. The data show that significant induction of ring-X loss occurs already at very low treatment concentrations (5 X 10(-5) -10(-4) mM) whereas rod-X loss or Y-marker loss is only seen at 2-5 X 10(-3) mM and higher. Induction of sex-linked recessive lethals is observed from 10(-4) -10(-3) mM on. These results add to existing evidence that loss of ring-X chromosomes, induced by some chemicals, may proceed by a mechanism different from the kind of events leading to chromosome breakage, as measured by rod-X loss and Y-marker loss.     

Induction of sex-linked recessive lethals and autosomal translocations by beta-propiolactone in Drosophila: influence of the route of administration on mutagenic activity.

Beta-propiolactone (BPL) was tested for the induction of sex-linked recessive lethals and autosomal translocations in Drosophila melanogaster. The compound was administered to adult males either by oral application or by abdominal injection. When injected, BPL was a potent inducer of sex-linked recessive lethals. When BPL was given by feeding, its mutagenic activity was detectable only when the flies were starved and when the BPL-containing solutions were renewed several times. Nevertheless, the recessive-lethal frequency was one order of magnitude higher with injection. This difference in effects is attributed to (1) rapid decomposition of the compound in aqueous feeding solutions, and to (2) rapid degradation in vivo which restricts the activity of BPL mainly to the site of application. These data are compared with other studies in which both routes of application were applied. BPL induced translocations in stored spermatozoa when injected, but not when fed. This finding seems a logical consequence of (1) the difference in effectiveness of the two routes of application for BPL, and (2) the existence of different LECs for mutation induction (recessive lethals) and for chromosome breakage (translocations). In Drosophila, the breakage capacity of BPL was one order of magnitude lower than that of MMS, when a comparison was made on the basis of equal recessive-lethal frequencies.     

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.     

Inbreeding depression maintained by recessive lethal mutations interacting with stabilizing selection on quantitative characters in a partially self‐fertilizing population

The bimodal distribution of fitness effects of new mutations and standing genetic variation, due to early‐acting strongly deleterious recessive mutations and late‐acting mildly deleterious mutations, is analyzed using the Kondrashov model for lethals (K), with either the infinitesimal model for selfing (IMS) or the Gaussian allele model (GAM) for quantitative genetic variance under stabilizing selection. In the combined models (KIMS and KGAM) high genomic mutation rates to lethals and weak stabilizing selection on many characters create strong interactions between early and late inbreeding depression, by changing the distribution of lineages selfed consecutively for different numbers of generations. Alternative stable equilibria can exist at intermediate selfing rates for a given set of parameters. Evolution of quantitative genetic variance under multivariate stabilizing selection can strongly influence the purging of nearly recessive lethals, and sometimes vice versa. If the selfing rate at the purging threshold for quantitative genetic variance in IMS or GAM alone exceeds that for nearly recessive lethals in K alone, then in KIMS and KGAM stabilizing selection causes selective interference with purging of lethals, increasing the mean number of lethals compared to K; otherwise, stabilizing selection causes selective facilitation in purging of lethals, decreasing the mean number of lethals.     

Genetic Damage at Specific Gene Loci in Drosophila with Betatron X-Rays

The mutation rate was determined for mature sperm at eight specific gene loci on the third chromosome of Drosophila melanogaster using the low ion density radiations of 22 Mev betatron X-rays. A dose of 3000 rads of betatron X-rays produced a mutation rate of 4.36 x 10^-8 per rad/locus. Among the mutations observed, 66% were recessive lethals and 34% viable when homozygous. Only one of the 24 viable mutations was associated with a chromosome aberration. Among the 47 recessive lethals, no two-break aberrations were detected in 48.9% of the lethals, deletions were associated with 42.2%, inversions with 6.7% and translocations with 2.2%.—When these genetic results are compared to those for 250 KV X-rays, the mutation rate for betatron treatments was slightly lower (.76), the recessive lethal rate among induced mutations was higher, and the chromosome aberrations among lethal mutations were slightly lower than with 250 KV X-rays. Although the two types of irradiations differ by an ion density of approximately ten, the amount and types of inheritable genetic damage induced by the two radiations in mature sperm were not significantly different.     

Response to Selection for Mating Speed and Changes in Gene Arrangement Frequencies in Descendants from a Single Population of DROSOPHILA PSEUDOOBSCURA

Heritability estimates, based on 19 generations of selection for fast and slow mating speed, were not significantly different from zero at the 0.05 level in any replicate of selected lines in a population of flies descended from the Mather population in California. Only the combined heritability estimate of approximately 2% was significant. This indicated that very little additive genetic variance was present in the base population and that strong directional selection for rapid mating may have occurred in the previous history of the local population at Mather and/or during its many generations of laboratory propagation. Frequencies of third chromosome gene arrangements were monitored during the course of selection. Balancing selection, unrelated to that imposed for mating speed, and genetic drift appeared to be the major factors causing changes in chromosome frequencies. Present differences in adaptive value of third chromosome variants in nature may be associated with nonadditive effects on mating speed, as well as effects on other components of fitness.