Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster: XII. The resistance factor rar-3: stage specificity

In earlier studies the recessive genetic factor rar-3 (3-49.8) of Drosophila melanogaster had been found to reduce the sensitivity of immature oocytes to the mutagenic action of X-rays. The present work was devoted to an extension of these studies to other germ-cell stages in both male and female and also somatic cells. The results show that, in the female, the effects of rar-3 are manifest in all germ-cell stages including gonia and nurse cells but not in mature oocytes. In the male germ-cell stages, rar-3 was without any measurable effect; maternal-effect studies were likewise negative. Somatic tissues were also unaffected. Futhermore, rar-3 was apparently not active in larval oogania. It is therefore concluded that the activity of rar-3 is switched on in oogonia during puparium formation or metamorphis and persists until before the formation of the mature oocyte.     

Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster: X. The resistance factor rar 3: genetics

In earlier work, immature oocytes of the irradiated population RÖI₄ of Drosophila melanpgaster were found to be radioresistant relative to those of the basic population RÖI and to those of the control population Berlin wild (+K). The resistance of RÖI₄ relative to RÖI was previously attributed to a hypothetical “factor” rar-3. In the present paper, evidence is presented to show that rar-3 is a single, recessive genetic factor, located on chromosome 3 at a map position of about 49.8. The action of rar-3 is apparently independent of that of rar-1 and rar-2, the factors already present in RÖI.     

I−R hybrid dysgenesis in Drosophila melanogaster: nature and site specificity of induced recessive lethals

This paper presents results of the genetic and cytological analysis of 144 sex-linked recessive lethals, plus 1 non-lethal. All of them were induced by I−R hybrid dysgenesis. This collection of mutants was pooled from experiments involving inducer chromosomes that differ in the chrosomal position of their I elements. Our results show that 30% of the recessive lethals are associated with chromosomal rearrangements which depend on the strength of the I−R interaction. These lethals are induced on both inducer- and reactive-origin chromosomes, and their frequency is dependent on the structure of the inducer chromosome used. The I−R-induced lethals occur along the entire length of the X chromosome. These sites probably correspond to specific loci which are more or less homologous with I. The complementation relationshups showed that some specific loci were more frequently involved in all the lethal mutations tested. The most sensitive loci are, in order of observation: l(1)J1, ct, f, ma¹ and m. Among induced recessive lethals considered to be point mutation, complementation tests showed that many of them are in fact multilocius deficiencies which can be detected only at the molecular level.

It seems that the production of I−R rearrangements (cytologically visible or not) may be the most important mechanism leading to lethal mutations. These mutations probably occur during the transposition of I elements, hence their importance from an evolutionary standpoint.     

Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster. VIII. The system of relative radioresistance in immature oocytes of the irradiated population ROI4

Prophase I oocytes of the irradiated population ROI4 of Drosophila melanogaster are radioresistant relative to those of a control population (+K). The system of relative radioresistance is apparently dose-modifying and can be described by Dose-Reduction Factors (DRFs). At least 3 constituent components of the system can be distinguished, as follows. The genetic factor rar-1 contributes to the system with respect to the induction of dominant (DRF = 1.31) and sex-linked recessive lethals (DRF = 1.31) in a way that is inhibited by caffeine. The factor rar-2, independently reduces both types of lethal to the same amount as does rar-1, but also affects the production of X-chromosome loss (DFR = 1.72). The results of several different approaches allow, as a working hypothesis, the interpretation that rar-2 reduces the association of heterologous, chiasmatic chromosomes in the chromocentre in time and/or space and thus minimizes the preconditions for the production of certain types of interchange and of non-disjunction. A third factor, rar-3, is postulated to contribute, independently from the others, to the system of relative radioresistance with respect to dominant lethals (DRF = 1.58), interchanges and non-disjunction (DRFs = 1.58), and sex-linked recessive lethals (DRF = 1.87).     

Studies on mutagen-sensitive strains of Drosophila melanogaster VIII. Further data on differences between Canton-S and ebony strains with respect to maternal effects for the X-ray induction of autosomal translocations and ring-X chromosome losses in mature spermatozoa

The influence of the maternal genotype (Canton-S, proficient in the repair of X-ray-induced chromosome breaks and ebony, less proficient in this regard) on the recovery of X-ray-induced autosomal (II–III) translocations and ring-X chromosome losses in mature spermatozoa was studied. In the first series of experiments, males carrying appropriate markers on their second and third chromosomes were irradiated and mated to Canton-S or ebony females and the frequencies of II–III translocations were determined. In the second series of experiments, males carrying ring-X chromosomes were irradiated in N₂ or in O₂, mated to Canton-S or ebony females and the frequencies of XO males were determined; additionally, under similar gas-treatment and radiation conditions, the pattern of egg-mortality was also assessed.

The data on translocations show that the yields are higher with ebony than with Canton-S females; these and earlier results on dominant lethals and sex-linked recessive lethals support the interpretation that the maternal repair system in the ebony strain is less proficient and more error-prone than that of the Canton-S strain.

Those on the losses of ring-X chromosomes demonstrate that (i) the absolute yields of XO males are lower with ebony than with Canton-S females irrespective of whether the parental males are irradiated in N₂ or in O₂; (ii) the exposure-frequency relationships are all linear, but the slopes are higher when the males are irradiated in O₂ and are consistent with an oxygen-enhancement-ratio of about 1.5 and (iii) the relationships between the logarithm of egg-survival and XO male frequency are also linear, but the slopes for the O₂ groups are lower than those for the N₂ groups (slope ratios of 0.86–0.87).

The finding that at given survival levels, the XO frequencies are lower in the O₂ than in the N₂ groups of both the Canton-S and ebony series viewed in the context of the mechanisms that have been postulated to explain the loss of ring-X chromosomes in irradiated mature spermatozoa permits the following interpretation for the observed results: (i) a higher proportion of potential XO zygotes is lost through dominant lethality in the O₂ groups than in the N₂ ones presumably because the chromosome breaks induced in O₂ are qualitatively different in the sense that they have higher probability to undergo reunions relative to restitution, compared with breaks induced under anoxia and (ii) this leads to lower than expected oxygen-enhancement ratios (i.e., expected on the basis of published data on sex-linked recessive lethals, another kind of genetic damage which shows a linear exposure-frequency relationship).     

Distribution of MR-induced sex-linked recessive lethal mutations in Drosophila melanogaster

In the ‘doubling-dose’ method currently used in genetic risk evaluation, two principle assumptions are made and these are: (1) there is proportionality between spontaneous and induced mutations and (2) the lesions that lead to spontaneous and induced mutations are essentially similar. The studies reported in this paper were directed at examining the validity of these two assumptions in Drosophila. An analysis was made of the distribution of sex-linked recessive lethals induced by MR, one of the well-studied mutator systems in Drosophila.

Appropriate genetic complementation tests with 15 defined X-chromosome duplications showed that MR-induced lethals occurred at many sites along the X-chromosome (in contrast to the known locus specificity of MR-induced visible-mutations); some, but not all these sites at which recessive lethals arose in the MR-system are the same as those known to be hot-spots for X-ray-induced lethals. With in situ hybridization we were able to demonstrate that a majority of MR-induced lethals is associated with a particular mobile DNA sequence, the P-element, i.e. they arose as a result of transposition.

The differences between the profiles of MR-induced and X-ray-induced recessive lethals, and the nature of MR-induced and X-ray-induced mutations, thus raise questions about the validity of the assumptions involved in the use of the ‘doubling-dose’ method.     

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.     

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.     

Distribution of MR-induced sex-linked recessove lethal mutations in Drosophila melanogaster

In the ‘doubling-dose’ method currently used in genetic risk evaluation, two principle assumptions are made and these are: (1) there is proportionality between spontaneous and induced mutations and (2) the lesions that lead to spontaneous and induced mutations are essentially similar. The studies reported in this paper were directed at examining the validity of these two assumptions in Drosophila. An analysis was made of the distribution of sex-linked recessive lethals induced by MR, one of the well-studied mutator systems in Drosophila.Appropriate genetic complementation tests with 15 defined X-chromosome duplications showed that MR-induced lethals occurred at many sites along the X-chromosome (in contrast to the known locus specificity of MR-induced visible-mutations); some, but not all these sites at which recessive lethals arose in the MR-system are the same as those known to be hot-spots for X-ray-induced lethals. With in situ hybridization we were able to demonstrate that a majority of MR-induced lethals is associated with a particular mobile DNA sequence, the P-element, i.e. they arose as a result of transposition.The differences between the profiles of MR-induced and X-ray-induced recessive lethals, and the nature of MR-induced and X-ray-induced mutations, thus raise questions about the validity of the assumptions involved in the use of the ‘doubling-dose’ method.     

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.     

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.     

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.     

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.     

Analysis of hexamethylphosphoramide (HMPA)-induced genetic alterations in relation to DNA damage and DNA repair in Drosophila melanogaster

This paper reports the results of a study on the mutagenic profile of HMPA in Drosophila melanogaster. HMPA produced all types of genetic damage tested for in post-meiotic cells of treated males; at the concentrations used, recessive lethals and ring-X losses were induced at significant rates while 2–3 translocations, entire and partial Y-chromosome losses only occurred at low rates. From a comparison with alkylation-induced mutational spectra, we note a number of peculiarities of HMPA mutagenesis:

1. (1) there is no storage effect on HMPA-induced translocations;

2. (2) the ratio of F₂-lethals: F₃-lethals varies from 6 : 1 to 9 : 1, indicating a low capacity of HMPA for delayed mutations;

3. (3) the use of the DNA-repair-deficient mei-9^L1 females instead of an excision-proficient control strain has no influence on the recovery of mutations )recessive lethals) induced in males;

4. (4) the high frequencies of chromosome loss (CL) induced by HMPA, which are mostly due to ring-X loss, leads us to speculate that one (or more) of its metabolites acts as a DNA-crosslinking agent. In experiments on maternal effects with mei-9^L1 females, there is a 20–40% reduction in the rates of induced CL. Conversely, with mei-41^D5 females, there is a weak increase in CL frequencies.

5. (5) HPLC analysis of DNA reacted with [¹⁴C]HMPA exhibits no methylation at the O⁶ or the N-7 of guanine. This finding, together with the observed inactivity of hexaethylphosphoramide (HEPA) in the recessive lethal assay, suggests that the formation of DNA-bound forms from HMPA may not be the result of simple methylation reactions. This conclusion is supported by the genetic data, i.e., the lack of a storage effect on HMPA-induced chromosome rearrangements.

Consistent with a hypothesis by Brodberg et al. (1983) to explain the action of cisplatin in Drosophila, comparisons of the spectra of genetic alterations produced by HMPA, A 139 (bifunctional) and Thio-TEPA (trifunctional) in the assay for chromosome loss suggest the involvement of two distinct mechanisms in the formation of ring-X loss by crosslinking agents. One pathway concerns induction of chromosome loss as a consequence of sister-chromatid exchanges (SCEs). The second mechanism may be due to DNA adducts or a single adduct responsible for both a fraction of CL and for induced partial Y-loss (PL). Inactivation of the mei-9⁺ function has two consequences: SCE-mediated ring-X loss frequency is lowered in mei-9 females in comparison to the repair-proficient control strain, while the opposite effect is indicated for that fraction of ring-X loss generated by the second mutational pathway. Additional complicating factors include the observation of a dual role of storage in our study: the proportion of SCE-related chromosome losses decreases with increasing time of storage, but that produced by the alternative pathway increases. Thus, the CL frequencies actually recorded under the heading ‘chromosome loss’ appear as the net result of two mechanisms counteracting each other in their effects.     

Testing of chloroquine and quinacrine for mutagenicity in Drosophila melanogaster

To extend the data on the mutagenic effects of intercalating agents in Drosophila melanogaster, chloroquine and quinacrine were tested for the induction of genetic damage in D. melanogaster males. Sex-linked recessive lethals and sex-chromosome loss induction were studied following treatment of adult males using a feeding technique. Our results show that both intercalating compounds increase significantly the frequency of sex-linked recessive lethals, but are unable to induce sex-chromosome loss in male germ cells under the conditions of testing.     

Genotoxicity of Rogor studied in the sex-linked recessive lethal test and wing, eye and female germ-line mosaic assays in Drosophila melanogaster

The genotoxic potential of Rogor (dimethoate), an anticholinesterase organophosphate insecticide, has been studied in the sex-linked recessive lethal test and the wing, eye and female germ-line mosaic assays in Drosophila melanogaster. Larvae of different instars carrying suitable recessive genetic markers on their first and third chromosomes were exposed to the LD50 or half of this dose for the entire larval life. The Basc technique was followed for the detection of the induction of sex-linked recessive lethals. The wings and eyes of the adult flies and the eggs laid by the heterozygous females were checked for the induction of mosaicism. It is concluded that Rogor induces sex-linked recessive lethals in immature male germ cells and is recombinogenic and/or mutagenic in both the somatic and the germ-line cells of Drosophila.     

Evidence of an essential difference between point mutations and chromosome breaks induced by triethylene melamine inDrosophila spermatozoa

Summary Storing of triethylene melamine-treated mature spermatozoa in untreated females was found to result in increased frequencies of both sex-linked recessive lethals and translocations involving the Y, II and III chromosomes. Frequencies of these mutations in effectively unstored spermatozoa were determined from progenies produced using sperm 2–4 days after treatment. The increase in translocation frequencies was on the order of 12-fold in progenies from sperm utilized 11–13 days after treatment when the sperm were stored at 25°C, and 3- to 6-fold when comparable sperm were stored at 12.5°C. Consistent but much smaller increases in frequencies of sex-linked lethals were found, with the increase in lethals tending to be correlated with relative increase in translocation frequency in a given experiment. On the assumption that sex-linked lethals related to chromosome breakage would be expected to increase in frequency in the same proportion as do translocations, approximate agreement was obtained when the proportions of breakage-related lethals among unstored lethals were estimated from the data in the four experimental series. The data are thus consistent with the hypothesis that chromosome breaks but not point mutations are realized during storage of treated spermatozoa. Possible interpretations of a differential effect of storage on treated chromosomes are discussed.Studies carried out while the author was a guest investigator at the Institute of Animal Genetics on sabbatical leave from the University of Minnesota.     

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.     

Effect of glyoxal pretreatment on radiation-induced genetic damage in Drosophila melanogaster

The effects of glyoxal and of glyoxal pretreatments on radiation-induced genetic damage were investigated in Drosophila melanogaster mature sperm, by means of sex-linked recessive and dominant lethality, reciprocal translocation and chromosome loss tests. In addition, the possible mutagenic effect of glyoxal was assessed in postmeiotic cells up to 7 days after treatment. The results obtained show: (1) the frequencies of recessive lethals after glyoxal treatment were within control values, (2) no clastogenic effect of glyoxal was observed, (3) glyoxal pretreatment did not modify the frequency of recessive lethals induced by X-rays, (4) after pretreatment with glyoxal a consistent, though not significant, increase was seen in the frequency of reciprocal translocations in 3 replicate experiments, (5) the yield of dominant lethals and of complete and partial chromosome loss induced by radiation was significantly increased by pretreatments with glyoxal. It is suggested that the increase of the frequency of genetic endpoints resulting from chromosome breakage, when glyoxal was administered prior to irradiation, could be ascribed to: (a) a sensitizing action of glyoxal to the clastogenic effect of ionizing radiation; (b) the formation of reactive species by the interaction of glyoxal with radiation; and/or (c) interference of glyoxal with the normal handling of radiation-induced lesions in mature postmeiotic male cells.