Mutagenesis, Vol. 14, No. 2, 187-192,
March 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Molecular analysis of mutants obtained by treatment with alkylating agents in a quadruplicated white-ivory strain of Drosophila melanogaster
Grup de Mutagènesi, Unitat de Genètica, Departament de Genètica i de Microbiologia, Edifici Cn, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
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Abstract |
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The use of a white-ivory (wi) strain of Drosophila melanogaster carrying four copies of this allele, (wi)4, has proved to be useful in detecting somatic mutation in genotoxicity testing. Nevertheless, until now very little information exists about the nature of the genetic effects detected in such a strain. This work presents molecular data on the changes that have taken place in different germinal mutants obtained after treatment with alkylating agents. Three different phenotypes were obtained: wild-type red eyes, dark red eyes and eyes lighter than (wi)4. Our results show that, in at least one of the four copies of the allele, the wild-type red eye phenotypes are due to a precise excision of the 2.96 kb duplicated region characteristic of the wi allele. These data agree with previous results obtained in a strain carrying only a single copy of the wi allele. The dark red eye mutants analysed seemed to be generated as a cluster and all proved to be caused by deletions at the 3'-end of the duplicated wi region in two of the copies of the (wi)4 genome. Finally, the light eye mutants (obtained at high frequencies) failed to show alterations at the molecular level, although we cannot discard the possibility that they might have originated by the loss of some of the wi copies of the (wi)4 strain.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Both in vivo and in vitro, somatic assays are extensively used to identify potentially genotoxic agents. Among the different assays developed in Drosophila melanogaster, Green et al. (1986) proposed an assay to detect somatic mutations which uses a quadruplication of the white-ivory gene as a target. This assay is based on detecting reversion to the wild phenotype which involves switches in the ommatidia colour from yellow to red.
The white-ivory (wi) mutant was originally isolated as a cluster of males by Sturtevant in 1918 (Lindsley and Zimm, 1992
) and it can revert to the wild-type in both the somatic and germinal lines. Karess and Rubin (1982) reported that wi mutation originated from a 2.96 kb tandem duplication located in the structural region of the white locus. Reversion of the wi allele seemed to be associated with loss of the characteristic duplicated fragment (Karess and Rubin, 1982; Green et al., 1986; Suárez et al., 1996), by a mechanism not yet well understood. Several hypothesis have been postulated to explain the mechanism(s) responsible for the reversion, such as intrachromosomal recombination (Karess and Rubin, 1982; Howe and Clements, 1990), mispairing of duplicated sequence(s) at the replication fork (Howe and Clements, 1990) and gene conversion or unequal crossing over (Karess and Rubin, 1982).
Hypothetically, the quadruplicated strain (wi)4 obtained by Green et al. (1986) should be more sensitive to the action of mutagens than the original wi strain due to its larger target. Thus, it can be assumed that reversion in only one of the four copies can produce the phenotypic switch to the wild-type. Several studies confirm that (wi)4 shows a higher sensitivity to mutagens than the wi allele (Green et al., 1986; Howe and Clements, 1990), however, the increase in reversion was not as high as might be expected given the number of copies (Howe and Clements, 1990). The suitability of this assay in genotoxicity testing has been largely proved by the evaluation of a wide number of agents (Würgler and Kägi, 1991; Xamena et al., 1991; Batiste-Alentorn et al., 1994, 1995; Ferreiro et al., 1995; Consuegra et al., 1996; Graf and Würgler, 1996).
In a previous paper (Suárez et al., 1996) we confirmed the precise excision of the 2.96 kb duplicated DNA sequence in different revertants of a wi strain. Taking into acount that the quadruplicated wi allele is used as a mutation test, we planned to carry out a study of germline mutations in a (wi)4 strain in order to increase our knowledge about this system. Our interest was mainly to analyse at the molecular level the different revertant and mutant phenotypes obtained from a wi quadruplicated strain after treatment with alkylating agents, searching for other chromosomal rearrangements than reversion of the duplication itself.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Drosophila stocks
We have used the following strains: (i) Canton-S (CS), a wild-type strain; (ii) white-one (w1), w1; (iii) white-ivory quadruplicated (wi)4, C(1) DX y f/y2 Dp (1:1:1:1) wi. The CS strain was obtained from the Umeå Drosophila Stock Center (Sweden), the w1 strain was purchased from the Carolina Biological Supply Co. (Burlington, NC), while the (wi)4 strain was kindly supplied by Prof. K.Fujikawa (Takedo Chemical Industries Ltd, Japan). For a detailed description of the genetic markers and special chromosomes, see Lindsley and Zimm (1992).
Chemical treatment and obtention of mutants
In order to obtain some germinal mutant lines for their subsequent molecular analysis, we treated (wi)4 larvae aged 24 or 48 h with the selected mutagens. For the treatments, the larvae were washed off the standard culture bottles, filtered and seeded in vials containing 9 ml of Drosophila Instant Medium (Carolina Biological Supply Co., Burlington, NC) and 9 ml of the mutagen solution. The mutagens used in these experiments were: ethyl methanesulfonate (EMS, CAS no. 62-50-0), methyl methanesulfonate (MMS, CAS no. 66-27-3) and N-nitroso-N-ethylurea (ENU, CAS no. 759-73-9), supplied by Sigma Chemical Co. (St Louis, MO). Just before the treatments, all compounds were dissolved in double-distilled water to the different concentrations used. The larvae were kept in these vials until adults and the emergent males were collected and crossed with C(1)DX y f virgin females carrying attached X chromosomes. The offspring were scored by eye under a dissection microscope for the presence of males with a different eye colour from the (wi)4 phenotype. All the induced germinal revertants and mutants were individually maintained with C(1)DX y f virgin females and were the object of the molecular analysis. All the crosses were carried out at 25 ± 1°C.
Quantitative measurement of red-eye pigments
To classify the induced revertants, we used a quantitative measurement of the red eye pigments following the protocol described by Ashburner (1989). Twenty five heads of 6-day-old males were used and the relative absorbance was calculated as the ratio of the absorbance of the mutant (A485-R) with respect to the absorbance of the wild-type (A485-CS), corrected for the absorbance of the white-1 (A485-w1) strain, as follows:
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Hybridization probes
The BglII–BglII (pwBB), the HindIII–BamHI (pwHB) and the SalI–SalI (pwSS2) fragments of the white locus were used as probes in the Southern blot experiments. According to the coordinates of Levis et al. (1982), they are located between positions +6163 and +4776, +3171 and +1383 and –1530 and –3045, respectively (see Figure 1). The plasmids containing these fragments were obtained from the pwp2 plasmid (kindly provided by Dr W.J.Gerhing, Basel University), as described in Suárez et al. (1996). To label the probes, the cloned fragments were simultaneously amplified and labelled by PCR using universal sequencing primers [M13/pUc sequencing primer 1 (New England Biolabs Inc., Beverly, MA); M13/pUc reverse sequencing primer 2 (Promega Corp., Madison, WI)] and digoxigenin-11-dUTP (Boehringer-Mannheim, Mannheim, Germany).
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DNA analysis by Southern blot
Genomic DNA extractions were carried out from 0.2 g of adult flies as described by Piñol et al. (1988), except that a phenol deproteinization step was added before deproteinizing with chloroform. Genomic DNA (5 µg) was digested with BglII and SacI according to the supplier's instructions (Boehringer-Mannheim) and the DNA fragments were separated by electrophoresis on a 0.8% agarose gel. Southern blotting on a positively charged nylon filter (Boehringer-Mannheim) was carried out by using the VaccuGeneTM XL vacuum blotting system (Pharmacia Biotech, Sweden) according to the supplier's instructions.
Hybridization
The hybridization was performed in a hybridization oven FDH-12 (Ecogen, Spain) at 65°C in 0.25 M Na2HPO4, 1 mM EDTA, 10% SDS and 1% powdered skimmed milk (pH 7.5). The post-hybridization washes were carried out twice in 20 mM Na2HPO4, 1 mM EDTA and 1% SDS for 15 min at 65°C. To detect hybridization, the DIG Luminescent Detection Kit (Boehringer-Mannheim) was used following the supplier's instructions, except for buffer 2, where 1% powdered skimmed milk was added instead of the blocking reagent, and a lower concentration of substrate solution (3 µl CDP-StarTM in 10 ml of buffer 3) was used.
Rehybridization of the filters
To reprobe the hybridized filters, the previous probes were removed by the following washing steps: 10 min in 2x SSC at room temperature, 20 min in 0.2 N NaOH and 0.1% SDS at 50°C, 20 min in 0.25 M Tris–HCl, pH 8.0, and 0.1x SSC at room temperature and 5 min in TE at room temperature.
PCR analysis
A heminested PCR reaction was used to amplify the region where the duplicated fragment in the white-ivory allele is located. In the first 10 cycle reaction, the wip1 and wif4 PCR primers were used, while in the second 25 cycle reaction wip1 and a new internal PCR primer, WBSrev, were used (see Figure 1
). The sequences of these primers were as follows: wip1, 5'-AACAACATTGCTGGGTGTCT-3'; wif4, 5'-CTGGATCCTGAGTCCAGTCC-3'; wBSrev, 5'-GTTAGGGGAGCCGATAAAGAG-3'
For this reaction, 0.5 µg of DNA was amplified in a volume of 25 µl, containing 1x PCR reaction buffer (50 mM KCl, 10 mM Tris–HCl, pH 8.4, 0.01% gelatine), 2.5 mM MgCl2, 125 µM each dNTP, 0.25 µM each primer and 1 U Taq DNA polymerase (Gibco BRL Life Technologies, Gaithersburg, MN) using a Programmable Thermal Controler (PTC-100; MJ Research Inc., USA). The first reaction was carried out, after preincubation for 3 min at 94°C, for 10 cycles at 94°C for 1 min, 60°C for 45 s and 72°C for 2 min, followed by 5 min at 72°C. The second reaction was carried out, after preincubation for 3 min at 94°C, for 25 cycles at 94°C for 30 s, 62°C for 30 s and 72°C for 2 min 30 s, followed by 5 min at 72°C.
White-ivory quadruplicated revertants and mutants were also analysed by PCR amplifying both the flanking DNA sequences and the central junction sequence of the duplicated region of the white-ivory mutation (see Figure 1), as described in Suárez et al. (1996). Amplified fragments were detected by 1% agarose gel electrophoresis after staining with ethidium bromide.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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In a previous study, we found that in eight independent revertants obtained from wi flies treated with alkylating agents, the phenotypic reversion observed was due to the loss of one of the copies of the 2.96 kb DNA tandem duplicated fragment (Suárez et al., 1996
) characteristic of the wi mutation, as shown in Figure 1. These results agree with those obtained by Karess and Rubin (1982) and Green et al. (1986), who also reported that three germinal revertants obtained from a (wi)4 strain after chemical treatment showed excision of the tandem duplicated DNA fragment in at least one of the four copies. In order to analyse induced reversion in the quadruplicated wi strain and to increase our information about this point, we have obtained a number of individuals with an induced mutant phenotype following chemical treatment. The aim of this analysis was to find out whether the reversion represents the only phenotype produced, as well as to determine whether genomic changes other than excision of one copy of the duplication can be produced. The treatments were applied to the first and second instar larvae of the (wi)4 strain using three different strong mutagens, the alkylating agents EMS (0.25–2 mM), ENU (0.25–1 mM) and MMS (0.25–1 mM).
The males which emerged from these treatments were crossed with C(1)DX y f virgin females and their offspring were scored to detect males with wild-type red eye colour in both eyes. Surprisingly, in addition to the expected red eye males, which supposedly originated by reversion, males with light and dark red eye colour were also obtained. Different revertants or new phenotype lines were established on the basis of eye colour. From the 12 red eyed males recovered it was only possible to establish 10 different revertant lines to be maintained with C(1)DX y f females (Table I), since the other two were sterile. From these red eyed lines, seven showed a dark red eye colour (R61–R67) and were found in the same culture bottle, therefore a possible common premeiotic origin of these mutants could be considered.
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Males with light eye colour appeared with high frequency and it was possible to establish 27 different lines. From these, 18 were found in the same culture bottle and they could be considered a cluster. It is remarkable that all these mutants were induced by chemical treatment, since no germinal light eye colour mutants were obtained from 15 684 flies scored in the control. There is no description of these light eye colour phenotypes in the literature and only a description of light spots in (wi)4 eyes when the larvae were treated with different chemicals has been reported (Xamena et al., 1991
).
In order to classify the revertants obtained, a quantitative measurement of the red eye pigments from the different revertants was carried out and the results are indicated in Table I. The results obtained were unexpected. Wild-type phenotypic revertants showed red eye pigment values half of those obtained in the wild strain and the dark phenotype revertants showed 10-fold lower values than those from the CS strain. Since the wild-type revertants were derived from a strain with a quadruplicated white locus carrying a white-ivory allele in each copy, it may be that the white-ivory protein from the remaining wi copies interferes with the wild protein, either in the transport of the eye pigments or in their precursors. Thus, although reversion to the wild-type of one of the four copies of wi produced wild-type protein, the values of total red eye pigments could be lower than the CS strain, due to the action of the wi mutant protein from the rest of the copies.
Molecular analysis of germinal revertants was carried out to establish whether, in our reversion experiments, the wild eye colour phenotype was associated with complete loss of the 2.96 kb DNA duplicated segment of the white-ivory allele in at least one of the copies of the white locus of the (wi)4 strain.
Figure 1 shows a restriction map of the white locus from the CS wild-type strain and from the white-ivory mutant, indicating the restriction sites and the probes used in the Southern blot experiments (pwBB, pwHB and pwSS2). The pwHB probe was used to detect variations in the length of the 2.96 kb duplicated segment in the white-ivory allele, while the other probes, pwBB and pwSS2, were used to detect variation in the 5'-regulatory and 3'-structural regions, respectively.
Figure 2a shows the changes that take place in the region where the duplication in the (wi)4 strain is located. The 10 revertant lines (three with red eye colour and seven from the cluster with dark red eye colour) as well as the CS and (wi)4 strains were analysed by Southern blotting after double digestion with BglII and SacI. Hybridization with the pwHB probe shows a 5.4 kb fragment in the CS strain (lane 1) and a 8.3 kb fragment in the (wi)4 strain (lane 2). Red eye colour revertants (lanes 3, 4 and 12) show two bands of 8.3 and 5.4 kb, while the dark red eye colour mutants (lanes 5–11) show three bands of 8.3, 7.2 and 6.3 kb. The higher intensity of the 8.3 kb band is due to the presence of four or less copies in each genome.
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BstEII restriction fragments visible on the electrophoresis gel before blotting.Figure 2b
presents the results of the rehybridization of the previous filter with the pwBB probe, corresponding to the regulatory region of the wi locus. This figure shows a 1.4 kb band in all lanes, suggesting that no changes were induced in this region. When the filter was rehybridized with the pwSS2 probe from the structural region of the wi locus (Figure 2c), a unique 8.0 kb fragment was obtained in all the samples (from the last SacI site in the third exon to the first SacI site of the white locus). Both results are in accordance with those expected from the physical map of the white locus (Levis et al., 1982). The results obtained in the Southern blot experiments with the different germline revertants indicate that the three independent red eye colour revertants had lost the 2.96 kb duplicated fragment in at least one of the four copies of the (wi)4 strain. On the other hand, the dark red eye colour revertant cluster seems to present changes in at least two of the copies of the wi locus (7.2 and 6.3 kb bands). In all cases some of the copies remain intact (8.3 kb band). Our results obtained with the red eye colour revertants agree with the previous data obtained by Green et al. (1986), who reported loss of the duplicated fragment in at least one of the copies of the quadruplicated white locus when three germinal-induced revertants obtained after chemical treatment were analysed. No description about the dark red eye colour revertants has previously been published.
To confirm the Southern blot results, an amplification of the region where the duplicated fragment is located was carried out in the wild-type revertants as well as in the CS and (wi)4 strains as controls. In this experiment, a heminested PCR was performed using the wip1/wif4 primer pair in the first amplification, while the wip1/wBSrev primer pair was used in the second amplification (see Figure 1 for the location of PCR primers). Heminested PCR amplification results are shown in Figure 3, where a 3.2 kb fragment was obtained in the CS strain (lane 3) and in the three red eye colour revertants (lanes 5, 6 and 14). Nevertheless, no band was obtained in the (wi)4 strain (lane 4) nor in the dark red eye colour revertant cluster (lanes 7–13) due to the constraint of Taq DNA polymerase, which is not capable of amplifying the expected 6.2 kb fragment. These results agree with the Southern blot data and confirm that the three red eye colour revertants have lost the tandem duplicated fragment in at least one of the copies of the white locus.
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In all the strains and revertants tested, no differences were obtained when the boundary sequences at the ends of the duplicated region were amplified (see Figure 1
). Nevertheless, when the central region with the junction site of the duplicated sequence was amplified, amplification was obtained only in the (wi)4 strain and in all the revertants (Figure 4), confirming that at least one copy of the wi locus carrying the tandem duplication remains in all the revertants (red and dark red eye colour). In contrast, in the CS strain the central region was unable to be amplified and only the 5' and 3' boundaries were amplified, as expected.
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To analyse the lesions in the cluster of the dark red eye colour revertants observed in the Southern blot experiments, the 7.2 (B1) and 6.3 kb (B2) bands from BglII and SacI double-digested DNA from R64 were cut out from the agarose gel and purified separately. The DNA from B1 and B2 were amplified to analyse the boundaries and the internal region characteristics of the white-ivory allele, and these results are presented in Figure 5
. As shown in the figure, the DNA from the B1 and B2 bands amplify both the initial 697 bp fragment and the internal junction fragment. Nevertheless, they fail to amplify the final 424 bp fragment. The absence of this amplification may be due to a deletion of the DNA to be amplified. These data, together with the results of the Southern blot experiments, support our opinion that the dark red eye colour revertants have a deletion of ~1 and 2 kb in two of the copies of the white locus at the 3'-end of the duplicated region, because each one of the bands represents one of the copies of the white gene.
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The lines established from the males with light eye colour were also analysed, using the Southern blot analysis as a first approximation. Thus, DNA from 27 of these lines was double-digested with BglII and SacI restriction endonucleases and hybridized with the pwHB probe, which detects variations in the length of the duplicated fragment characteristic of the white-ivory allele. Figure 6
shows the results of the hybridization from 10 of these lines with an independent origin, indicating that a 5.4 kb band was obtained from the CS strain as expected from the wild-type white locus sequence (O'Hare et al., 1984). On the other hand, both the (wi)4 strain and the light eye colour lines show a 8.3 kb band suggesting that no excision of the duplicated fragment occured in these mutants. The other regions from the white gene were also analysed by Southern blot, using pwBB and pwSS2, indicating that there were no apparent changes in these regions (data not shown).
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Since there is no dose compensation for the wi mutant and, therefore, the number of copies is directly related to the increase in eye pigmentation, it seems that the light eye coloured mutants could be either due to the loss of some of the four copies of wi or to a mutation in other loci that could affect eye colour expression. In fact, and to support the second hypothesis, mutations in modifier loci have been shown to be the origin of phenotypic changes in different mutants of the white locus obtained after treatment with alkylating agents (Soriano et al., 1998
). In summary, our results show that the red colour phenotypes obtained after chemical treatment of (wi)4 flies are due to excision of the 2.96 kb DNA tandem duplicated fragment in some of the four copies of the wi allele, indicating a true reversion. These results agree with previous data obtained in the analysis of other stocks with a single copy of wi. Moreover, some apparent reverted phenotypes could probably be due to other changes affecting the duplicated region, such as a deletion. On the other hand, the appearance of the light eye colour phenotype is not related to any changes in the duplicated region of the wi alleles and could be due to the loss of complete copies or to second mutations in modifier loci.
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Acknowledgments |
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This investigation was supported in part by the Spanish Ministry of Education and Culture (grants nos: SAF95-0813, CICYT; PB96-1138, DGES) and by the Generalitat de Catalunya (CIRIT, SGR95-0161). We also thank M.McCarthy for her secretarial assistance.
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Notes |
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1 To whom correspondence should be addressed. Tel: + 34 93 581 27 31; Fax: + 34 93 581 23 87; Email: noel.xamena{at}uab.es
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References |
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Received on May 20, 1998; accepted on November 11, 1998.
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