Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster: XI. The resistance factor rar-3: effects in inmature oocytes

The effects of the radioresistance factor rar-3 on the X-ray induction of various types of genetic damage in immature oocytes (about stage7) of Drosophila melanogaster were studied.

The dose-reduction factors previously postulated for rar-3 with respect to dominant lethals (1.58), sex-linked recessive lethals (1.87), non-disjunction of major chromosomes (1.58), and homologous interchanges (1.58)_were confirmed experimentally. It is concluded that all effects attributed arbitrarily to rar-3 are contributed by the single genetic factor rar-3.

No difference were found in quality of sex-linked recessive lethals (Y suppression, distribution over the X) induced in either rar-3 or rar-3⁺. Recombination frequencies were normal in unirradiated rar-3.     

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.     

Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster XVI. Adaptation to the mutagenic effects of X-rays in several experimental populations with irradiation histories

Selection responses of the laboratory stock Berlin wild (+60, +K) of Drosophila melanogaster to the mutagenic effects of high, accumulated exposures of X-rays were studied in several sub-populations with long irradiation histories. Interest was focussed on adaptive adjustments of mutation rates. In samples from the populations, radiosensitives of immature oocytes were tested, by using dominant lethals (A), X-chromosome losses (B) and sex-linked recessive lethals (C) as end-points of genetic radiation damage.

Populations RÖ II and RÖ V are similar to the previously studied population RÖ I and were exposed to 2100 R/generation, delivered to oocyte stages 6–14, mature sperm, and spermatids. RÖ II (first tests after 160 generations) is radioresistant relative to +60 (control). The resistance was characterized by dose-reduction factors (DRFs) of 1.72 (with respect to end-points A,B) and 1.53 (C), and these were similar to those previously obtained for RÖ I. The resistance of RÖ II was inherited semidominantly as was that of RÖ I, and the radiosensitivity of the hybrids RÖ I / RÖ II was similar to that of RÖ I and RÖ II with respect to end-points A and B. RÖ V did not become resistant within 25 generations of irradiation history (A).

Populations RÖ III (6000 R/generation) and RÖ IV (7000 R/generation) have histories of irradiations given to oogonia and spermatogonia. Radiosensitivities of immature oocytes of RÖ III did not differ from those of +K after 55 generations, but after 105 and 135 generations of irradiation history, DRFs of 1.2 (A) and 1.44 (B) were observed. RÖ IV was characterized in generations 55–135 by DRFs of 1.31 (A) and 1.72 (B).

Selection for relative radioresistance of immature oocytes was found (1) to be reproducible (RÖ II–RÖ V) (2) not to require genetic pre-adaptation (RÖ V), and (3) to be, in part, also achieved by ‘indirect’ selection (RÖ III, RÖ IV). It is concluded that mutation rates in populations are selectively adjusted to evolutionary requirements.     

Radiosensitivity of Drosophila lines. VII. The effect of the maternal genotype on the yield of recessive and dominant lethal mutations induced by the gamma irradiation of males

The effect of material repair on induction of paternal mutations was tested with radiosensitive rad(2)201G1 mutant. Basc males were irradiated at doses from 0 to 60 Gy of gamma-rays and mated to the radiosensitive mutant or control females. Frequencies of sex-linked recessive lethals and dominant lethals (induced in the paternal genome) were determined. With control females, the rate of recessive lethals increased linearly from 0 to 60 Gy. With rad(2)201G1 mutant, an increase in spontaneous and induced rates of paternal dominant lethals was observed; the rate of sex-linked recessive lethals increased non-linearly from 0 to 60 Gy.     

Effects of a chromosome-3 mutator gene on radiation-induced mutability in Drosophila melanogaster females

A series of X-irradiation experiments was carried out using Drosophila melanogaster females homozygous for a third chromosome mutator gene and females which had a similar genetic background except that the mutator-bearing third chromosomes were substituted by normal wild-type chromosomes. The mutator females had been previously shown by Gold and Green to manifest a higher level of radiation-induced mutability (as measured by the X-ray-induction of sex-linked recessive lethals) in their pre-meiotic germ cells compared to normal females at an exposure of 100 R. In the presence work, the sensitivity of the pre-meiotic germ cells of mutator and normal females to the X-ray induction (2000 R) of sex-linked recessive lethals was studied. In addition, experiments were conducted to examine the sensitivity of the immature (stage 7; prophase I of meiosis) oocytes of both kinds of females to the induction of dominant lethals, X-linked recessive lethals and X-chromosome losses. The result show that in pre-meiotic germ cells, the frequencies of radiation-induced recessive lethals are similar in both kinds of females. However, the proportion of these mutations that occur in clusters of size 3 and higher, is higher in mutator than in normal females. In stage-7 oocytes, the frequencies of radiation-induced dominant lethals and sex-linked recessive lethals were similar in both kinds of females. The X-loss frequencies however, were consistently higher in mutator females although statistical significance was obtained only at higher exposures (3000 and 3750 R) and not at lower ones (750-2250 R). Possible reasons for the discrepancy between the present results and those of Gold and Green with respect to pre-meiotic germ cells are discussed.     

Mutagenicity of hycanthone in Drosophila melanogaster

The schistosomicidal agent hycanthone was tested for mutagenicity in Drosophila melanogaster. The compound was administered either by injection into adult males or by larval feeding. The following types of genetic damage were measured:(1) complete and mosaic sex-linked recessive lethal mutations; (2) II–III translocations; and (3) dominant lethals.In postmeiotic germ cells, especially in late spermatids, a pronounced increase was found in the frequency of sex-linked recessive lethals, both completes and mosaics. By contrast, translocations and dominant lethals were not induced.     

The mutational spectrum of procarbazine in Drosophila melanogaster.

The antineoplastic agent Procarbazine was tested for the induction of genetic damage in Drosophila melanogaster. The compound was administered to adult males by oral application. The following types of genetic damage were measured: (1) sex-linked recessive lethals; (2) dominant lethals; (3) total and partial sex-chromosome loss; and (4) translocations. Procarbazine is highly mutagenic in causing recessive lethal mutations in all stages of spermatogenesis. In sperm a clear-cut concentration-effect relationship is not apparent, but in spermatids such a relationship is obtained for mutation induction at low levels of procarbazine exposure, while at high concentrations the induction of recessive lethals is not a function of concentration. A low induction of total sex-chromosome loss (X,Y) and dominant lethals was observed in metabolically active germ cells (spermatids), but procarbazine failed to produce well-defined breakage events, such as partial sex-chromosome loss (YL,YS) and II-III translocations. The results obtained in Drosophila melanogaster are discussed and compared with the mutational pattern reported in the mouse after procarbazine treatment.     

Effects of a small X-ray exposure to Drosophila melanogaster females on the recovery of genetic damage from X-irradiated males.

Wild-type (Oregon-K) Drosophila melanogaster males were X-irradiated and mated to Oster females (y sc^s1 In49sc⁸; bw; st p^p) that had received a 20 R X-ray exposure (Group MF) or no irradiation (group M). Mature spermatozoa of the irradiated males were sampled and the frequencies of dominant lethals, sex-linked recessive lethals and 2–3 translocations were measured. In the group in which the irradiated males were mated to irradiated females, the survival of eggs was significantly higher than in the group in which only the males were irradiated. However, there was no consistent and detectable difference between the two groups with respect to the frequencies of recessive lethals and translocations.The relatively higher egg survival in the MF group is amenable to an interpretation based on an inducible repair process in females that acts on radiation damage induced in spermatozoa but, such an explanation is inadequate to explain the other results. It is concluded that the observations considered together preclude a general and unifying interpretation based on a low-dose-X-ray-inducible genetic repair process in females (acting on damage in spermatozoa). Possible reasons for the discrepancy between the expectation of differences in response between the MF and M groups (in sex-linked lethal and translocation frequencies) and the observation of no consistent differences between them are discussed.     

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.     

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.     

Studies on mutagen-sensitive strains of Drosophila melanogaster I. The enhanced X-ray sensitivity of a mutant strain (ebony) which is UV-sensitive and also deficient in photorepair

Yegorova and colleagues (1978) showed that a mutant strain of Drosophila melanogaster (ebony) was more sensitive to UV-induced killing of embryos and also less proficient in photoreactivating (PR) ability than a wild-type (Canton-S) strain and that the genes governing UV sensitivity and PR ability were different and presumably located on the autosomes. The experiments reported in the present paper were designed to compare the patterns of sensitivity of these 2 strains and their hybrids to X-irradiation. The sensitivity of the larvae to the killing effects of X-irradiation, and of male and female germ-cell stages to the X-ray induction of genetic damage was studied.It was found that the larvae of the ebony strain are more sensitive to X-ray-induced killing than those of the Canton-S strain. The frequencies of radiation-induced dominant lethals and sex-linked recessive lethals are higher in spermatozoa sampled from ebony males than in those of Canton-S males. In spermatozoa sampled from hybrid males, the yields of dominant lethals are no higher than in those sampled from Canton-S males and do not seem to depend on the origin of the X-chromosome. There are no statistically significant differences between the ebony and Canton-S strains in the sensitivity of their spermatozoa to the induction of autosomal translocations.Stage-7 oocytes sampled from ebony females are more sensitive to the X-ray induction of dominant lethality than are those from Canton-S females; oocytes sampled from hybrid females manifest a level of sensitivity that is significantly lower than that in either parental strain. The frequencies of X-chromosome losses induced in in this germ-cell stage are significantly lower in ebony than in Canton-S females at least at the exposure level of 3000 R at which 3 experiments were carried out. There are no measurable differences in the amount of dominant lethality induced in stage-14 oocytes of ebony, Canton-S and hybrid females.When X-irradiated Berlin-K males are mated to ebony or Canton-S females, the yields of dominant lethals are higher when ebony females are used, showing that there is a “maternal effect” for this kind of damage. Such a maternal effect is also found for sex-linked recessive lethals (irradiated Muller-5 males mated to ebony or Canton-S females). However, when irradiated ring-X-chromosome-carrying males are mated to ebony or Canton-S females, the frequencies of paternal sex-chromosome losses (scored as XO males) are lower when ebony females are used.These results have been interpreted on the assumption that the ebony strain is homozygous for recessive, autosomal genes that confer increased radiosensitivity and that the Canton-S strain carries the normal, wild-type alleles for these genes. The higher yields of dominant and recessive lethals in mature spermatozoa and of dominant lethals in stage-7 oocytes are a consequence of an enhanced sensitivity to the mutagenic (in particular, to the chromosome-breaking) effects of X-irradiation and/or of defective repair of radiation-induced genetic damage. The lower yield of XO males from irradiated stage-7 oocytes of ebony females is probably a consequence of a defect in the repair of chromosome-breakage effects, resulting in the conversion of potential X losses in females into dominant lethals. The “maternal effects” for dominant lethals, sex-linked recessive lethals and for the loss of ring-X chromosomes are assumed to have a common causal basis, namely, a defective repair of chromosome-breakage events in the females of the ebony strain.     

The mutability of the minute loci of Drosophila melanogaster with ethyl methanesulfonate.

It has been suggested that the Minute loci of Drosophila melanogaster are the redundant structural loci for the transfer RNA's [31]. To inquire whether the Minute loci differed from other loci in their genetic organization we have determined the dose response curves for the induction of Minutes and sex-linked recessive lethals with ethyl methanesulfonate (EMS). There are approx. 67.75 +/- 9.35 Minute mutants induced for every 5000 recessive lethals induced in the genome and this relationship is independent of EMS dosage. This is in good agreement with the relative numbers of Minute and lethal loci in the genome. Because the target size of the average Minute locus is the same as that of the average locus capable of mutating to a lethal, these data do not support the view that the Minute loci are special in their genetic organization. Since Minute mutants can be scored in the F1 of mutagenized flies it is suggested that the induction of Minute mutants may provide a more rapid and economical means of assessing mutagenicity than do traditional screens for the induction of recessive lethals.     

Radioprotective effect of six chemicals against X-ray-induced genetic damage in D. melanogaster

In Drosophila melanogater six chemicals were tested for radioprotectiveeffect against X-ray-induced genetic damage such as sex-linked recessive lethals and autosomal translocations using Oster's ring-X chromosome stock. A 2-day brood pattern was followed to score the damage induced at different spermatogenic stages separately. In all cases the chemicals were injected before X-irradiation. 10-mM solution of reduced glutathione (GSH) provided statistically significant protection against sex-linked recessive lethals in all broods. In translocation tests this chemical reduced the frequency in all broods but the result is not statistically significant. Cysteamine (MEA) did not show any protective effect but the frequency of lethals was slightly reduced in the first and fourth broods. 2-Aminoethyl isothiuronium Br·HBr (AET) showed a statistically significant protective effect when the data of the replicate experiments were pooled. Negative results were obtained for 5-hydroxytryptamine (5-HT) in sex-linked lethal tests. Aminoethyl phosphorothioate (AEPT) reduced the frequencies of both sex-linked lethals and autosomal translocations in all broods consistently but the results are not statistically significant. In tests for both lethals and translocations the reduction was largest in the stages with highest radiosensitivity. N(3-Aminopropyl)aminoethyl phosphorothioate (3AP-AEPT) gave no protection.     

Mechanism of hycanthone mutagenesis: the induction of temperature-sensitive mutations in Drosophila melanogaster.

A total of 358 sex-linked recessive lethals induced by hycanthone methane sulphonate (HMS) were checked for temperature sensitivity, along with 239 EMS-induced lethals as a positive control. Only about 1-2% of the HMS-induced lethals were temperature-sensitive, in contrast to about 7-8% for EMS-induced lethals. This can be reasonably explained by assuming that in Drosophila, HMS mainly acts by inducing frame-shift mutations or deletions.     

Evidence for a mutagenic effect of the narcotic antagonist, naltrexone, in germ cells of Drosophila.

The narcotic antagonist, Naltrexone, was tested for mutagenicity in Drosophila. The frequency of sex-linked recessive lethals at a non-toxic dose of 10 mg/ml was 0.43% (42 lethals in 9697 X-chromosomes tested) and 0.16% (19/11536) in the controls. The difference is statistically significant (P less than 0.001). Results from large-scale experiments testing for chromosome breakage and nondisjunction were negative.     

I-R hybrid dysgenesis in Drosophila melanogaster. Use of in situ hybridization to show the association of I factor DNA with induced sex-linked recessive lethals.

The purpose of this paper is the genetic visualization by in situ hybridization of 130 sex-linked recessive lethals plus a non-lethal induced by I-R dysgenesis. This collection of lethals involves inducer strains which differ in the position of the I elements on the X chromosomes. The I-R interaction was strong. Our previous results have shown that about 30% of the induced recessive lethals are associated with cytologically visible chromosomal rearrangements. (1) The rearrangements induced by I-R-type hybrid dysgenesis often exhibit homology with the I factor at the level of one or both junction points, depending on the types of chromosome rearrangements. These results suggest that the chromosome rearrangements arise directly from the transposition of I elements. However, the breakpoints of some types of cytologically non-visible deficiencies and of 2 small cytologically visible deficiencies do not present detectable homology with the I factor. (2) The majority of rearrangements do not involve the I elements already present on the paternal X chromosome. (3) The hybridization signal distributions on the X chromosome are not uniform. They present peaks of various heights which may correspond to specific anchoring areas of copies of I in the course of integration. (4) The data presented here agree with the literature with respect to the mean number of copies of I per X chromosome and to the excess of copies of I at locus 1A. Two rearrangement formation mechanisms are envisaged: crossing-over and 'target' exchanges.     

Effect of low temperature on radiation-induced genetic damage in Drosophila melanogaster: response of motile sperm and late spermatids.

The response of fully mature motile sperm and late spermatids when challenged with X-radiation at 0 degrees C has been studied in sex-linked recessive lethals, II-III translocations and dominant lethality experiments. At 0 degrees C a significant increase in both mutagenic and clastogenic damage was detected compared to that obtained at 24 degrees C. Furthermore, the results of experiments performed with different postirradiation temperatures demonstrate that the low temperature during irradiation was the sole factor responsible for the observed increase. In the recessive lethal and translocation tests the response of late spermatids was higher than that shown by motile spermatozoa. As a whole, the results, which are rather similar to data reported on the effect of irradiation in oxygen of the same cell stages, suggest that the low temperature acted as a dose-modifying factor.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. II. Detection of qualitative differences between genetic damage induced by X-irradiation of mature spermatozoa in oxygenated and anoxic atmospheres through the use of the repair-deficient mutant mei-9a

Muller-5 males were irradiated with X-rays in nitrogen, in air or in oxygen (followed by nitrogen or oxygen post-treatments in the nitrogen and oxygen series) and were mated to females of a repair-proficient strain (mei+) or to those of a strain known to be deficient in excision repair of UV damage (in somatic cells). The latter strain, designated as mei-9a, is also known to be sensitive, in the larval stages, to the killing effects of UV, X-rays and to a number of chemical mutagens. The frequencies of sex-linked recessive lethals and autosomal translocations induced in the spermatozoa of males were determined and compared. The frequencies of sex-linked recessive lethals in the mei-9 control groups were consistently higher than in the mei+ groups. Irradiation in air or in nitrogen led to significantly higher yields of recessive lethals when the irradiated males were mated to mei-9 females, whereas, after irradiation in oxygen, the yields were similar with both kinds of female. No significant differences in the frequencies of reciprocal translocations were observed between the mei+ and mei-9 groups after irradiation of the males in nitrogen, in air or in oxygen. Likewise, no differential effects of the contrasting post-treatments (nitrogen versus oxygen), either for recessive lethals or for translocations, could be discerned. These results are considered to support the notion that the kinds of genetic damage induced in mature spermatozoa in air or in nitrogen are qualitatively similar (at least with respect to the component(s) that lead to the production of recessive lethal mutations), but clearly different when induced in an oxygen atmosphere. The enhanced yields of recessive lethals with mei-9 females (after irradiation of the males either in air or in nitrogen) has been interpreted on the assumption that the mei-9 mutant is also deficient for the repair of X-ray-induced, recessive lethal-generating premutational lesions. Possible reasons for the lack of differences between the mei+ and mei-9 groups with respect to translocation yields and for the absence of measurable differences in response between the contrasting post-treatments (after irradiation of the males in nitrogen) are discussed.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. VII. Effects of repair deficiency in males on X-ray-induced sex-linked recessive lethals in spermatozoa

The response of mature spermatozoa to the X-ray induction (500 R and 3000 R) of sex-linked recessive lethals was studied in Drosophila melanogaster males known to be deficient in excision- or post-replication repair of UV damage in somatic cells. The results show that the induced frequencies of recessive lethals in the excision-repair-deficient males (mei-9a and mei-9L1) are similar to those in the appropriate repair-proficient males (mei+ and Berlin-K). However, in the post-replication-repair-deficient males (w mus(1)101D1), these frequencies are significantly lower than in the comparable repair-proficient males (w) after 500 R, but not after 3000 R.