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.     

Induced mutagenesis of plasmid and chromosomal genes inserted into plasmid DNA. I. The mutagenic action of radiation

This paper describes the results of treating plasmid DNA in vitro with mutagens, to obtain mutations both in plasmid genes and chromosomal genes comprised within the plasmid, thus avoiding disorganization characteristic of in vivo mutagenesis. The model system is represented by DNA of RSF2124 responsible for colicine E1 synthesis and resistance to ampicillin. Col- mutants were looked for after exposure to UV- and gamma-irradiation. The lethal effect was estimated as inactivation of the ampicillin resistance marker. After reisolation from mutant transformant of the plasmid DNA, the novel character and resistance to ampicillin proved to retain in the course of subsequent transformations and passages of transformed colonies, suggesting the mutational nature of the changes. Exposure of RSF2124 to short-wave UV-irradiation (lambda = 254 nm) produced a pronounced mutagenic effect: the relative quantity of Col- mutants under optimal conditions of mutagenesis increased about 10 times. In the case of W-reactivation (additional UV-irradiation of C600 wild type cells) of lethal lesions, a 95% reliable increase in mutagenic effect was observed. Significant enhancement of mutagenesis (about 4-fold) was detected when only recipient cells were exposed to low doses of UV (the so-called indirect UV mutagenesis). Thus, with regard to W- and indirect UV mutagenesis, the plasmid DNA behaves like DNA of temperate phages which suggests their evolutionary relationship. Treatment of plasmid DNA with acridine orange prior to UV, only protected from lethal lesions. Gamma-irradiation (60Co) at the dose producing 100-fold inactivation, increased the yield of Col- mutants by one order of magnitude. The presence of RSF2124 plasmid in a cell does not affect its UV sensitivity.     

Evaluation of effects of gamma-irradiation at low doses on repair and meiotic recombination mutants of Drosophila melanogaster

A comparative study of the effects of gene mutations mus209, mus309, mei-41 and rad54 of Drosophila melanogaster on the sensitivity to low-level exposure of different duration was carried out. Taken into account was the survival rate at different stages of ontogeny, female fecundity, the frequency of dominant lethal mutations (DLM) and the DNA damage. mei-41 and rad-54 mutants were most sensitive to the action of low dose radiation (80 mGy) in terms of survival and DLM. However, at the level of DNA damage, an increased radiosensitivity is observed only at larger doses of low intensity irradiation. Based on these observations, we can conclude about the importance of repair and its genes in the formation of the effect of low level doses of ionizing radiation in Drosophila.     

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.     

Genetic effects induced by nickel sulfate in germline and somatic cells of WR mice

We examined the effects of nickel sulfate at doses 0.5 to 5.0 mg/kg (LD50) on the frequency of dominant lethal mutations and two-strand DNA breaks (TSBs) in germline cells and on an increase in frequency in gene mutations W(y) in pigment cells of first-generation mice. The results indicated that spermatogenesis stages most sensitive to nickel sulfate (at a dose of 1.0 mg/kg) are spermatozoids, early spermatids, late spermatocytes, and stem spermatogonia. No statistically significant increase in the total TSB level was detected in spermatozoids 4 weeks after exposure. At the same time, a significant (P < 0.05) increase in percentage of cells with an extremely high level of DNA fragmentation (supposedly apoptotic cells) was observed upon exposure at a dose of 0.5 mg/kg. Nickel sulfate at doses of 5.0 and 1.0 mg/kg induced a marked increase in the c-kit gene expression in pigment cells of heterozygous first-generation WR mice as compared to control (P < 0.001). It was shown that the nonobservable adverse effect level (NOAEL) of nickel sulfate on the dominant lethal mutation frequency and gene mutations was 1/200 LD50, while the lowest observable adverse effect level (LOAEL) was 1/100 LD50.     

Modification of the repair processes in chemical mutagenesis in Drosophila melanogaster]

The effect of 1,4-dihydroisonicotinic acid derivative (1,4-DHINA), glutapyrone, on the frequency of sex-linked recessive lethals (SLRL), which were induced by ethyl methanesulfonate (EMS) in spermatozoa, was studied in Drosophila males and females under different exposure conditions. This test was used to analyze repair processes in EMS mutagenesis. Glutapyrone manifested the protective effect after precultivation of males at the stage of larvae and females with glutapyrone. This preparation was shown to have no effect on the frequency of EMS-induced mutations under other conditions (precultivation and subsequent cultivation of mature males with glutapyrone). These data confirmed a key role of indirect mechanisms responsible for the effect of the examined antimutagen. Glutapyrone may be assumed to improve the synthesis or functioning of enzymes involved in the repair of O6-ethylguanine. After adaptive treatment of Drosophila with low doses of the mutagen, glutapyrone increased the fertility of parents but did not suppress chemical mutagenesis.     

Studies on the mutagenic effect of contraceptive drugs. I. Induction of dominant lethal mutations in female mice

30 adult virgin female mice (2 strains) received either high or low doses of Anovlar or Lyndiol oral contraceptives and were tested for induction of dominant lethal mutations. The pregnant mice were dissected on Day 14 of pregnancy and total implantations, early deaths, late deaths, and corpora lutea were counted in each pregnancy. A significant reduction in fertile mating (p .025) was found in 1 strain of those who received the high dose of Lyndiol (10 times that of the low dose, which is physiologically equivalent to the human dose). This dose also increased the number of dead implants in both strains which resulted in higher estimates of dominant lethal mutations. It is concluded that when Lyndiol and Anovlar were given at the physiological dose level to control ovulation in mice, the frequency of dominant lethal mutations was not increased above the control level.     

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

Spontaneous and EMS-induced mutations were accumulated for several generations on the second chromosome of Drosophila melanogaster by keeping this chromosome heterozygous under conditions of minimal natural selection. This article reports studies of heterozygous effects of these mutants.--Both lethal and mildly deleterious mutants have a deleterious heterozygous effect. There was no discernible difference between heterozygotes in which all the mutants were on one chromosome and those where the mutants were distributed over both homologs; thus the coupling-repulsion effect of MUKAI and YAMAZAKI (1964, 1968) is not confirmed. The spontaneous polygenic mutants have a dominance of 0.4 to 0.5, and the same value is found at very low EMS doses. However, the value at higher EMS doses is only about half as high. Since the low doses have a large fraction of spontaneous mutants, the dominance of EMS mutants is less, in the range 0.1 to 0.3, but still larger than for lethals.     

Mutagenicity tests with metrifonate in Drosophila melanogaster

The organophosphorus insecticide metrifonate (O,O-dimethyl(1-hydroxy-2,2,2-trichloroethyl)phosphonate), also known as trichlorfon or Dipterex ®, was tested for its ability to induce sex-linked and autosomal recessive lethal mutations in Drosophila melanogaster. There was no evidence of an increase in the frequency of lethal mutations after feeding adult males with metrifonate, but the high toxicity of the compound meant that only low concentrations could be used in this test system.     

On the relationship between the frequency of two types of lethal-mutation and X-ray doses in drosophila

The dose-frequency relationship for each of 2 types of lethal mutations, fractional- and whole-lethal, was obtained using X-rays on Drosophila melanogaster. The results show that fractional-lethal mutations are induced by X-rays, and also that the proportion of fractional-lethal mutations in the total of mutations tends to decrease with increasing doses, namely, 61% at 0 R, 47% at 500 R, 37% at 1000 R and 20% at 2000 R. The same tendency is observed with visible mutations.In order to consider the problems related to the above results, the relationship between the true frequency and the observed frequency of the induced lethal mutations is discussed, taking into consideration the existence of the ontensible whole-lethal and the ontensible normal.     

Characteristic Features of Induced Mutagenesis in Hybrid Dysgenesis Systems of Drosophila melanogaster

The mutagenic effect of low-dose gamma-irradiation was studied inDrosophila melanogaster systems of hybrid dysgenesis by estimating polytene chromosome rearrangements, recombination frequency, and viability at the embryonic and postembryonic developmental stages. A dose of gamma-irradiation which had no effect detectable by routine interstrain cross proved to significantly reduce the number of recombinants in the H–E and P–M systems and mortality at postembryonic stages. However, this combined effect was obtained if irradiation followed trasposition, i.e., it depended on the application sequence of the mutagenic factors. The reverse order of the mutagenic treatment led to summation of the effects: as compared to either control, the frequencies of the dominant lethal mutations as well as the larval and pupal mortality in F₂ increased significantly (at the level of 99.9%). This allowed us to estimate the contribution of extremely low-dose gamma-irradiation into the mutagenic effect, which was impossible under routine conditions.     

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.     

The action of mutagens on MS2 phage and on its infective RNA. V. Kinetics of the chemical and functional changes of the genome under hydroxylamine treatment

The kinetics of chemical and functional changes induced in the genome of bacteriophage MS2 by hydroxylamine under the conditions of predominant modification of either cytidine (pH 5.0) or uridine (pH 8.0) have been studied.Comparison of the kinetics of chemical modifications of monomeric nucleotides with those of bacteriophage inactivation at pH 8.0 and 5.0 made it possible to estimate the effective number of exposed cytidine and uridine residues in the intra-phage RNA (B_eff^c and B_eff^u). The B_eff^u was close to that expected and increased from 70 to 130 as the temperature rose from 0 to 30°. The B_eff^c was much greater than that expected on the basis of the results with the monomer, suggesting that side reactions are involved in the inactivation of the phage at pH 5.0.A significant increase of the frequency of mutation occurs only under the conditions of predominant modification of cytidine (pH 5.0) at 0°. No such effect was observed at 30°. This was probably due to the increased contribution of inactivating side reactions. The effect of hydroxylamine on the phage under the conditions of predominant uridine modification (pH 8.0) did not lead to an increase in frequency of mutants.Incubation of the intact phage in acetate buffer resulted in considerable inactivation and mutations. Inactivation was inhibited by magnesium ions. Incubation at pH 5.5, of the phage inactivated by hydroxylamine treatment at pH 8.0, resulted in a considerable increase of the inefectivity with no effect on the frequency of mutants. The infectivity and the mutation frequency of the phage treated with hydroxylamine at pH 5.0 did not change as a result of incubation at pH 4.0 after the removal of the reagent.     

Effect of gamma radiation, cis-diamminedichloroplatinum and its derivates on the Escherichia coli cell survival and potentiality for adaptive response

The sensitivity to the lethal effect of gamma-rays, cis- and trans-diamminedichloroplatinum (DDP), cis- and trans-iminoethers of DDP (IE) was compared in two groups of E. coli--K12 and B. In all experiments, cells of wild types appeared to be most resistant to these agents. gamma-Resistant and gamma-sensitivity/hypersensitive strains occupy an intermediate position according to their sensitivity to cis-DDP derivatives. In almost all the cases, both single and especially double mutants defective for the systems of nucleotide excision repair, recombination repair, and inducible SOS-repair are most sensitive to DDP derivatives. The data obtained show that in E. coli the repair of lethal lesions after cis-DDP action is more complicated than after gamma-irradiation. Of DDP derivatives cis-DDP is most effective, while trans-DDP is less effective, and cis- and trans-IE are considerably less effective, respectively. It is shown that the effects of ionizing radiation in low doses (more than 10 different regimes), or of treatment with cis-DDP in low concentrations do not change the survival of E. coli after their respective effects in high doses. In other words, under the effect of ionizing radiation and cis-DDP no adaptive response for the lethal action was found in E. coli.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. IV. Comparison of dose-response curves for MNNG, 4NQO and ICR-170 induced inactivation and mutation-induction

The genetic effects of MNNG, 4NQO and ICR-170 have been compared on 5 different UV-sensitive strains and a standard wild-type strain of Neurospora crassa with regard to inactivation and the induction of forward-mutations at the ad-3A and ad-3B loci. Whereas all UV-sensitive strains (upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) are more sensitive to inactivation by MNNG and ICR-170 than wild-type, only uvs-5 shows survival comparable to wild-type after 4NQO treatment, all other strains are more sensitive to 4NQO. In contrast to the effects on inactivation, a wide variety of effects were found for the induction of ad-3A and ad-3B mutations: higher forward-mutation frequencies than were found in wild-type were obtained after treatment with MNNG or 4NQO for upr-1 and uvs-2, no significant increase over the spontaneous mutation frequency was found with uvs-3 after MNNG, 4NQO or ICR-170 treatment; mutation frequencies comparable to that found in wild-type were obtained with uvs-6 after MNNG, 4NQO or ICR-170 treatment and with upr-1 after ICR-170 treatment. Lower forward-mutation frequencies than were found in wild-type were obtained with uvs-2 after ICR-170 treatment and with uvs-5 after MNNG, 4NQO or ICR-170 treatment. These data clearly show that the process of forward-mutation at the ad-3A and ad-3B loci is under genetic control by mutations at other loci (e.g. upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) and that the effect is markedly mutagen-dependent.     

Genetic effects of cosmic radiation in Drosophila melanogaster]

To examine possible effects of space radiation on living organism, we have analyzedtwo types of mutations, sex-linked recessive lethal mutations and somatic mutations, in fruit fly of the species Drosophila melanogaster. Drosophila strains used were wild type strains and a radiation-sensitive strain mei-41. Two different developmental stages of samples were sent into space; young adult males to analyze sex-linked recessive lethal mutations and about 30hr-old larvae to detect somatic mutations in wing epidermal cells. For wild type and mei-41 strains each, about 200 adult male flies and about 6,000 larvae were loaded on space shuttle Endeavour. The male flies returned from space were mated to virgin female flies of a tester strain, and the presence of the lethal mutations was analyzed at F2 generation. The frequencies of sex-linked recessive lethal mutations in flight groups were 2 and 3 times higher for wild type Canton-S and mei-4 1, respectively, than those in ground control groups. Most larvae sent to space emerged as adult flies within about 10 days after the landing. The presence of wing-hair somatic mutations, which give morphological change in hairs growing on the surface of wing epidermal cells, was analyzed under microscope. In wild type strain Muller-5, the frequency of wing hair mutant spots in flight group was about 1.5-fold higher than that in ground control, and in Canton-S-derived wild type strain the frequencies were similar between the two groups. By contrast, for mei-41 strain the mutation frequency was lower in flight group than in control group. The observed higher frequency of lethal mutations in the flight group might be due to a possibility that radiation effects on reproductive cells could be greatly enhanced under micro gravity. However, if this would be the case, we do not have appropriate explanation for the apparent absence of such synergistic effects on somatic wing-hair mutation system.     

Protein denaturation during heat shock and related stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase inactivation in mouse cells

In an attempt to question the toxic effect of heat shock and related stress, we have studied the activity of reporter enzymes during stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase were synthesized in mouse and Drosophila cells after transfection of the corresponding genes. Both enzymes are rapidly inactivated during hyperthermia. The corresponding polypeptides are not degraded but become insoluble even in the presence of non-ionic detergents. The heat inactivation is more dramatic in vivo within the living cell than in vitro, in a detergent-free crude cell lysate. The extent of enzyme inactivation at a given temperature depends on the cell type in which the enzyme is expressed. Luciferase is inactivated at lower temperatures within Drosophila cells than within mouse cells, whereas beta-galactosidase is inactivated at higher temperatures in E. coli than in mouse cells. A "priming" heat shock confers a transient increased resistance (thermotolerance) of cells against a second "challenging" heat shock. Enzyme inactivation during heat shock or exposure of the cells to ethanol is attenuated in heat shock-primed cells. A comparable thermoprotection is raised by a priming heat shock for both luciferase activity and protein synthesis. Thus, the study of reporter enzyme inactivation is a promising tool for understanding the molecular basis of the toxicity of heat shock and related stress as well as the mechanisms leading to thermotolerance.     

Mutagenic repair in Escherichia coli. VIII. Effect of gyrB mutations on ultraviolet light mutagenesis

Introduction into Escherichia coli WP2 bacteria of a mutation in the gyrB locus previously shown to reduce the degree of chromosomal superhelicity caused a small decrease in the frequency of UV-induced mutations to streptomycin resistance (but not significantly) and to tryptophan independence (mostly ochre suppressors) in excision repair-proficient bacteria. It did not influence the 'broth effect' or the rate or extent of 'mutation frequency decline' of suppressor mutations. In an excision-deficient (uvrA 155) background the yield of UV-induced streptomycin-resistant mutations was lower in gyrB bacteria at all doses; the yield of tryptophan-independent mutations was slightly lower at low doses and slightly higher at high doses. In both excision-proficient and -deficient bacteria the yield of UV-induced mutations to rifampicin resistance was apparently lower in gyrB mutants but this could be due at least in part to a hypersensitivity of some Rifr gyrB bacteria to UV. The number of spontaneous tryptophan-independent mutations was lower in gyrB bacteria but this was almost certainly due to their poorer viability on tryptophan-limiting plates and not to a lower spontaneous mutation rate. In a temperature-sensitive presumed gyrase-deficient strain a small decrease in mutant yield at low doses was observed following incubation at restrictive temperature before UV. This was ascribed to an enhancement of excision repair. Our failure to find any significant effect of gyrB mutations does not support the hypothesis that hairpin formation (which should be dependent on a high degree of superhelicity) is involved in determining the 'broth effect', 'mutation frequency decline' or the probability that a mutation will occur spontaneously. Dramatic effects of superhelicity on UV mutagenesis also seem to be unlikely.     

Buthionine sulfoximine mediated enhancement of γ-radiation induced mutation frequency in Drosophila melanogaster

Experiments were carried out to investigate whether or not depletion of the glutathione (GSH) level in Drosophila melanogaster larvae with buthionine sulfoximine (BSO) treatment can result in the modulation of the frequency of sex-linked recessive lethal (SLRL) mutations induced by γ-radiation. Third instar larvae were fed on BSO for 24 h before exposure to 10 Gy γ-radiation. Immediately after this the larvae were divided into two batches, which were used to determining the GSH level and the induction of SLRL mutations respectively. The results obtained suggest that the depletion of the GSH level with BSO can lead to an enhancement in the frequency of SLRL mutations (significant at the 5% level). In a subsequent experiment in which adult Drosophila melanogaster male flies were fed on BSO for 72 h before irradiation, a significant increase was observed in the incidence of SLRL mutations.     

Drosophila nociceptors mediate larval aversion to dry surface environments utilizing both the painless TRP channel and the DEG/ENaC subunit, PPK1

A subset of sensory neurons embedded within the Drosophila larval body wall have been characterized as high-threshold polymodal nociceptors capable of responding to noxious heat and noxious mechanical stimulation. They are also sensitized by UV-induced tissue damage leading to both thermal hyperalgesia and allodynia very similar to that observed in vertebrate nociceptors. We show that the class IV multiple-dendritic(mdIV) nociceptors are also required for a normal larval aversion to locomotion on to a dry surface environment. Drosophila melanogaster larvae are acutely susceptible to desiccation displaying a strong aversion to locomotion on dry surfaces severely limiting the distance of movement away from a moist food source. Transgenic inactivation of mdIV nociceptor neurons resulted in larvae moving inappropriately into regions of low humidity at the top of the vial reflected as an increased overall pupation height and larval desiccation. This larval lethal desiccation phenotype was not observed in wild-type controls and was completely suppressed by growth in conditions of high humidity. Transgenic hyperactivation of mdIV nociceptors caused a reciprocal hypersensitivity to dry surfaces resulting in drastically decreased pupation height but did not induce the writhing nocifensive response previously associated with mdIV nociceptor activation by noxious heat or harsh mechanical stimuli. Larvae carrying mutations in either the Drosophila TRP channel, Painless, or the degenerin/epithelial sodium channel subunit Pickpocket1(PPK1), both expressed in mdIV nociceptors, showed the same inappropriate increased pupation height and lethal desiccation observed with mdIV nociceptor inactivation. Larval aversion to dry surfaces appears to utilize the same or overlapping sensory transduction pathways activated by noxious heat and harsh mechanical stimulation but with strikingly different sensitivities and disparate physiological responses.