Mutagenesis vol. 18 no. 6 pp. 527-531,
November 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Germline mutations induced by N-nitroso-N-ethylurea do not affect the inserted copia retrotransposon in a Drosophila melanogaster wa mutant
Grup de Mutagènesi, Unitat de Genètica, Departament de Genètica i de Microbiologia, Facultat de Ciències, Edifici Cn, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
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Abstract |
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The white-apricot (wa) mutant of Drosophila melanogaster is characterized by a copia retrotransposon inserted in the second intron of the white locus. After germinal exposure to the alkylating agent N-ethyl-N-nitrosourea, we have obtained new phenotypes in the offspring, mainly lighter eye colour, but not revertants to the original phenotype. Subsequent genetic crosses showed that only 3 out of 13 new mutant phenotypes were allelic. Three white gene regions were analysed by Southern blot in order to determine the nature of the mutations. These three regions were the 5' regulatory region, the copia insertion site and the 3' coding region. The results obtained indicate that the treatment does not induce the total or partial excision of copia in the white locus. Two of the new allelic mutants present a 5' or 3' deletion in the white locus. The other new phenotypes seem to be caused by mutations being induced in other loci acting as modifiers, most of them located on the X chromosome.
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Introduction |
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It is well known that transposable elements (TE) may change their distribution in the genome, due to both excision and/or insertion of new copies. Thus, they could be considered a major cause of mutation (Rubin, 1983
; Harada et al., 1990). One of the most controversial subjects is the possibility of induction of TE mobilization due to external stress conditions (Arnault and Dufournel, 1994). Under stress conditions, changes in TE copy number have been shown to occur in several cases, including bacteria, yeast, cultured cells and protoplasts. Induction of TE transposition is often observed immediately after cell culture initiation. This may be due to some of the medium components that affect all the cells in this system in the same way (Rubin, 1983; Junakovic et al., 1988; Di Franco et al., 1992). Eeken and Sobels (1986) associated X-ray exposure with P transposition induction in Drosophila melanogaster and UV ray exposure has been proved to induce Ty transposition in Saccharomyces cerevisae and Tn1 transposition in Escherichia coli (Sankaranarayanan, 1986; Bradshaw and McEntee, 1989). Furthermore, ethyl methanesulfonate exposure is followed by Tc1 excision in Caenorhabditis elegans and transposition of Spm in Zea mays (Collins et al., 1987; Burr and Burr, 1988). However, lack of excision or transposition induced by other treatments have also been found, for example by Arnault et al. (1991), who did not detect mobilization of copia, mdg1, I or 412 after treatment with dichlorvos, hydrogen peroxide or ecdysterone. On the other hand, although phenotypic alterations, including total or partial reversions, have been described in the offspring of insertional mutants after exposure to N-ethyl-N-nitrosourea (ENU), none of them is due to excision of the inserted element. The effects are due to insertions, deletions or base changes in the TE or near the insertion point or in other regions of the same or other genes (Lacy et al., 1986; Osgood and Lacy, 1986; Pastink et al., 1988; Soriano et al., 1998). Some published work has focused on total genome studies by Southern blot or in situ hybridization on polytene chromosomes, whereas other studies are based on phenotypic analysis. Excision of the TE produces phenotypic reversion of the insertional mutant, but needs molecular confirmation, because a phenotypic reversion can be induced by different changes.
We thought that insertional mutants of the white locus of D.melanogaster might be an appropriate system for testing and analysing, at the molecular level, the effects of stress as an external factor on TE excision. In a previous paper (Soriano et al., 1998), we reported the results of an analysis of revertants obtained from the white-spotted-1 (wsp1) mutant of D.melanogaster, which has a B104 retrotransposon inserted in the regulatory region of the white locus. Following this work, we studied the mutant white-apricot (wa), which carries an insertion in the structural region instead of in the regulatory region, as in wsp1, at the molecular level. The TE studied is the copia retrotransposon inserted in the second intron of the white locus. In the present paper we describe the results obtained after germinal exposure of the wa mutant to the alkylating agent ENU, which has been proved to be an inducer of reversion of insertional mutants of D.melanogaster (Soriano et al., 1995, 1998).
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Materials and methods |
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Chemicals
N-nitroso-N-ethylurea (CAS no. 759-73-9) was supplied by Sigma Chemical Co. (St Louis, MO). ENU was dissolved in double-distilled water to the different concentrations used, just before the treatments.
Drosophila stocks
The following strains were used: (i) Canton-S, a wild-type strain (CS); (ii) C (1) DX, yf/Y carrying attached X chromosomes; (iii) white-apricot (wa); (iv) zeste (z1).
The wa strain was purchased from Carolina Biological Supply Co. (Burlington, NC). The other strains were obtained from the Umeå Drosophila Stock Center (Sweden). For a detailed description of the genetic markers and special chromosomes see Lindsley and Zimm (1992) or FlyBase (available at http//:flybase.bio.indiana.edu/).
Treatment procedure
Two-day-old males were treated by feeding for 24 h in glass filter special feeding units (1D3; Scott, Mainz, Germany) after 2 h of starvation. The mutagen was supplied at the desired concentrations in 5% (w/v) sucrose (Würgler et al., 1977). After treatment, the males were collected and crossed with C (1) DX, yf/Y virgin females. The offspring were scored for the presence of males with different eye colour from parent males, which could be the result of germinal mutations. Induced germinal mutants were maintained with C (1) DX, yf/Y virgin females and were subsequently used for the molecular analysis. Concurrent negative controls with water were performed for each experiment. All the crosses were carried out at 25 ± 1°C.
Genetic crosses
In order to determine if the molecular alterations were located in the white locus, appropriate crosses were carried out with wa females and male mutants isolated in the germinal chemical treatments. In the case of a mutation located in the white locus we expected that the heterozygous F1 females would show a different eye colour than wa. On the other hand, if the mutation was in another locus (a modifier locus, for example) then the wa phenotype would be restored by complementation with a female wild-type modifier locus dominant over the mutant locus of the male. Crosses with wild-type CS were also carried out to check the expected dominance.
Analysis of testes and Malpighian tubules
Because the white gene is also expressed in testes and Malpighian tubules, we decided to analyse these structures in males from the studied strains, in order to observe any phenotypical change produced by variation in expression of the white locus.
Abdomens of males were dissected in 0.85% NaCl and colour of the testes and Malpighian tubules were examined under a stereoscopic microscope. To obviate an age-dependent increase in pigmentation, only 5-day-old males were analysed.
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 deproteinization with chloroform. Genomic DNA (4–5 µg) was digested with suitable restriction enzymes (BamHI, HindIII and SacI) according to the supplier’s instructions (Roche Diagnostics GmbH, Mannheim, Germany). DNA fragments were separated by electrophoresis on a 0.8% agarose gel. Southern blotting on a positively charged nylon filter was carried out using the VacuGeneTM XL vacuum blotting system (Pharmacia Biotech, Uppsala, Sweden) according to the supplier’s instructions.
Hybridization probes
The probes were obtained by subcloning fragments of the plasmid pWP2 (kindly provided by Dr W.J.Gehring, Department of Cell Biology, Basel University, Basel, Switzerland). The probes were BamHI–SacI (BS), SalI–SalI (SS) and BamHI–BamHI (BB), corresponding to the insertion site of copia, the exons of the coding region downstream of the insertion site and the 5' regulatory region, with tissue-specific enhancers, respectively. According to the coordinates of Levis et al. (1982), they are located between positions +1383 and –601, –669 and –1530 and +4438 and +1383, respectively (see Figure 1). The probes BB and SS were cloned in pBluescript (Stratagene, La Jolla, CA) and BS in pTZ (Genescribe-Z; USB Corp., Cleveland, OH).
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Probes were labelled with digoxigenin-11-dUTP, either by random primed reaction using a DIG DNA Labelling and Detection Kit (Roche Diagnostics GmbH) or by amplification by PCR. The primers used in the PCR reaction were the universal sequencing primers, M13/pUC sequencing primer (New England Biolabs, Beverly, MA) and M13/pUC reverse sequencing primer (Promega Corp., Madison, WI), and the nucleotide mixture was the same as in the random primed reaction.
Hybridization and detection of genomic blots
The hybridization was performed overnight in a hybridization oven 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 bands, the DIG Luminescent Detection Kit (Roche Diagnostics GmbH) was used following the supplier’s instructions, except that in buffer 2, a 1% solution of powdered skimmed milk was added instead of blocking reagent and a lower concentration of substrate solution (3 µl disodium 3 (4-methoxy spiro{1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.1.3,7]decan}-4-yl) phenyl phos phate in 10 ml of buffer 3) was used.
Rehybridization
To reprobe the hybridized filters, the previous probes were removed by the following washing steps: (i) 10 min in 2x SSC at room temperature; (ii) 20 min in 0.2 N NaOH and 0.1% SDS at 50°C; (iii) 20 min in 0.1x SSC and 0.25 M Tris–HCl, pH 8.0, at room temperature; (iv) 5 min in Tris–EDTA buffer at room temperature.
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Results |
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Two-day-old wa males were treated with ENU at each concentration tested, 0.5, 1.0 and 5.0 mM. Over 150 males were treated at each concentration. At 24 h the mortality was null at the lowest concentration, one individual at the intermediate concentration and nine individuals at the highest concentration. Higher concentrations were not tested because ENU has proved to be highly toxic (Soriano et al., 1995
). The effect on fertility was not tested because the treatments were carried out in mass matings of treated males. Treated males at these non-toxic concentrations were mated with attached-X females [C (1) DX, yf/Y] and their male progeny were analysed for reversion events that had taken place in the parental germline. No revertants were obtained, but among the descendants three distinct new phenotypes of eye colour were detected: nearly white, lighter than wa (yellow) and darker than wa. Also, several cases of mosaicism in the colour of eyes were obtained (eyes of different colours or eyes with sectors of different colours).
Genetic crosses
Table I shows the scores of new phenotypes obtained in the male progeny after ENU treatment. All the males with new phenotypes were mated with attached-X females and, as can be seen in the table, a great number of males were sterile or their phenotypes were not passed on.
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The results of the crosses carried out to determine the allelism are shown in Table II. Nevertheless, the results of crosses with CS are not shown in the table, even though all the mutants were crossed with wild-type CS. The eye colour of all the female progeny was wild-type due to the dominance of such an allele, as can be expected.
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The F1 females of 10 crosses show the phenotype of complementation, apricot eye colour in crosses with wa, and, therefore, these mutations were not alleles of white. The female progeny of mutants Q01, Q69 and Q59 showed a lighter eye colour than the dominant allele wa, especially the former.
Since a pale yellow eye colour resembled the zeste phenotype, we also crossed some of these mutants with zeste-1 females. The heterozygous female offspring showed wild-type colour of eyes and therefore were not alleles of zeste (data not shown).
On the other hand, we also studied the phenotype of ocelles, Malpighian tubules and testis sheaths from mutant males since their colour is also regulated by the white locus (Lindsley and Zimm, 1992). The almost general feature is the lack of pigment in these organs, which seem to be transparent. The mutants whose eye colour was darker than wa, such as Q15 and Q70, also had darker organs than wa. The mutant Q59, likely to be allelic, showed testes with the same colour as wa. Two mutants, Q19 and Q71, had coloured organs as wa, in spite of an eye colour that showed complementation as with most of the other mutants.
Molecular analysis
In order to determine the integrity or the presence of induced mutations in the locus white or in the copia retrotransposon, molecular studies were carried out by Southern blot technique. To achieve this, as indicated above, we considered three regions in the locus white: the insertion site of copia, the exons of the coding region downstream of the insertion site and the 5' regulatory region, with tissue-specific enhancers. Two enzyme (BamHI and SacI) digestions and three probes (BB, BS and SS) were used to distinguish the three regions. In Figure 1 the elements needed to follow the steps of our experimental process are shown, such as the locus white, the restriction map, the probes and the coordinates of the physical map. The results obtained are shown in Figure 2.
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DNA BstEII digest as marker in the electrophoresis.A double digest with BamHI and SacI was hybridized with the probe BS. For the wa allele, a fragment of
7 kb is expected (2 kb of wild-type plus 5.4 kb from copia). The Southern blot allows detection of modifications in the copia insertion site, due to total or partial excision of the inserted element, because the insertion site is contained in it.
The same filter rehybridized with the probe SS was used to detect modifications in some part of the coding region. As in the wild-type, the expected length of the fragment hybridized in wa was 8 kb.
The HindIII digest hybridized with the probe BB was used to detect modifications in the control region and in the 5' end of the transcription region. Two fragments were expected to be detected in this case, the upstream fragment, size 6 kb, and the downstream fragment, of 5.4 kb, that 3' ends inside copia.
Neither total nor partial excision of copia was detected in either case (as can be seen in Figure 2A, the analysed samples of all mutants show a 7 kb band like the sample of wa). In the coding region, only the mutant Q01 has a detectable mutation in the locus white: a deletion that reduces the expected length of a detected fragment from 8 to 1.2 kb. Finally, the other mutation detected was in the 5' regulatory region, where the mutant Q69 showed a deletion of 1 kb, therefore reducing its length from 6 to 5 kb.
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Discussion |
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We used ENU as the alkylating agent due to the results obtained in a previous work (Soriano et al., 1998
). The most efficient agent tested in the production of revertant phenotypes from insertional mutants was ENU, when compared with other alkylating agents such as ethyl methanesulfonate (EMS) or methyl methanesulfonate (MMS), although these agents also have a high clastogenic potential (Soriano et al., 1995). In this respect, other authors have reported the higher mutagenic potential of ENU with respect to EMS and MMS (Vogel and Natarajan, 1979; Natarajan et al., 1984; Lee et al., 1989). Furthermore, it is known that ENU induces point mutations and short deletions (Pastink et al., 1988; Singer, 1990; Bronstein et al., 1991; Tong et al., 1997).
Despite not finding a linear dose–effect relationship between the treatment and the new observed phenotypes, Table I shows the highest number of new phenotypes that appeared after treatment with the highest ENU concentration. In all cases, individuals with mosaicism in the eyes or with no heritable colour were obtained in the offspring. This means that germinal post-meiotic cells were affected but mutations were fixed after several mitotic divisions and, therefore, not all the cells of an organism inherited the same genetic information (Lee, 1976). Similar phenotypes have also been obtained after ENU treatment from both wa (Osgood and Lacy, 1986) and w+ (Pastink et al., 1988).
One can expect that an alteration in the integrity of the inserted element may result in a phenotypic change, producing a total or partial phenotypic reversion, due to the following causes: (i) excision of the copia retrotransposon by homologous recombination between the two small direct repeated sequences flanking the long terminal repeats (LTRs), in which no copia signal is observed (Carbonare and Gehring, 1985); (ii) excision of the copia element by homologous recombination between the two LTRs, leaving the LTR flanked by two 5 bp direct repeat sequences in the host genome (Carbonare and Gehring, 1985; Zachar et al., 1985; Mount et al., 1988; Kurkulos et al., 1994); (iii) secondary insertions into copia that seem to interrupt or to interfere with the regulation signals of the element (Mount et al., 1988); (iv) mutations in modifier genes (Mount et al., 1988). In our cases, none of the mutants showed either total or partial excision of copia, including the Q15, Q59 and Q70 mutants, which show a partial phenotypic reversion (see Figure 2A). Therefore, the mutations induced by ENU treatment of these insertional mutants would probably be due to mutations at other sites in the white locus or to mutations in other loci acting as modifiers.
As can be seen in Figure 2B and C, and as noted in the results, the molecular analysis of the white locus by Southern blot shows alterations in only two of the mutants tested. The mutant Q01, with white eyes, presents a deletion of at least 7 kb long affecting the 3' end, which might include the last exon. This may be the cause of this hypomorphic phenotype, because a defective protein is produced. The mutant Q69, with pale yellow eyes, has a 1 kb long deletion in the 5' end, in the regulatory region. In this case the enhancer for eye colour, one of the multiple intensifying boxes localized between positions –312 and –1850 upstream of the +1 start point of transcription, may be affected, as described by Quian et al. (1992) (see Figure 1). This phenotype may be due to the low level of transcription. On the other hand, the existence of other mutations affecting the white locus cannot be ruled out. Nevertheless, point mutations or small deletions may be at the limit of detection of the Southern method, unless they affect the sequence recognized by restriction enzymes.
The results of the crosses carried out to determine the allelic relationships show that the Q01 mutant behaves as a white allele, as can be expected by the result of Southern analysis. The detected mutation in the white locus may produce the phenotype observed and therefore it may not be complemented by other genomic factors. The Q59 mutant, whose crosses give similar results to Q01, does not show differences in its restriction pattern. This could probably be due to a point mutation being small enough to not be detected by Southern blot. The Q69 mutant, bearing a 1 kb deletion affecting the 5' untranslated regulatory region with tissue-specific enhancers, evidently also behaves as a white allele.
The rest of the mutants studied at the molecular level may be considered as mutant alleles of modifier loci located on the X chromosome. Most of them were recessive, without effect in heterozygotes resulting from crosses with wa or CS females.
The only autosomic mutant was Q15. This mutation was inherited by 50% of the male progeny as dominant with a suppressor effect of wa, and the eye colour was darker in homozygotes.
We expanded the study to other organs than the eye because the white gene regulates not only eye colour but also the colour of ocelles, Malpighian tubules and the sheaths of testes by means of a set of boxes. As was demonstrated in Results and is shown in Table II, most of the new phenotypes behave as sex-linked modifiers. The mutants Q15 and Q70 may be modifiers of white, acting on all the tissue-specific enhancers darkening all the controlled tissues. Two other non-allelic mutants, Q19 and Q71, may only act on the enhancer of eye colour, since the other organs showed the wa colour. The allelic mutants Q01 and Q69 showed colourless phenotypes likely to be due to the deletions detected by Southern blot, located in the coding region or in the regulatory region, respectively. The other allelic mutant, Q59, not detectable by Southern blot, may be located in the control region, since the testes had the expected colour.
In view of these results we can conclude that the ENU treatment, under our experimental conditions, does not have any apparent effect on the biology of the copia retrotransposon inserted into the white locus in wa mutants, including mutants with partial phenotypic reversion. After treatment with this alkylating agent, neither total nor partial excision of copia was induced or detected in the modified phenotypes. Instead, some small alterations in the white locus were described. Of course, deletions may be induced by ENU, but they could also be spontaneous in origin, as suggested by Pastink et al. (1988). Furthermore, most of the new phenotypes found were caused by other loci than white itself, such as modifier loci. Almost all of them were located on the X chromosome. Therefore, a total or partial excision of a TE is not required for a phenotypic change, including reversion, in insertional mutants.
These results are in agreement with those described in Soriano et al. (1998). In the present work we have studied the effect of an alkylating agent on the copia retrotransposon inserted into an intron of the white locus, whereas in another case the effect of alkylating agents was studied over the retrotransposon B104 inserted in the 5' regulatory region of the white locus. The new phenotypes obtained in the present study were mainly due to the existence of a second site modifier acting on expression of the white gene or on different tissue-specific enhancers located upstream near the start point of transcription.
The results obtained in this work, together with others previously published (Soriano et al., 1995, 1998), clearly show that care must be taken in interpreting results obtained in mutation assays based on genetic systems with the white locus bearing transposable elements. It must be taken into account that the phenotypic changes induced by mutagens could be misinterpreted. These phenotypic changes, including total or partial reversion, are not mainly due to alterations of the inserted transposable element. Induced mutations affecting other loci may act as modifiers of genetic expression of the white locus carrying the insertional mutation. Furthermore, the mutation rate may be very high, due to the high number of target sequences that may be affected by the mutagen rather than to the instability of the transposable element.
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Acknowledgements |
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We thank M.McCarthy for her secretarial assistance. This work was supported by the Spanish Ministry of Education and Science (grant BOS2000-0329) and the Generalitat de Catalunya (grant SGR-00197-2002). E.B. was supported by a doctoral fellowship (FPI) from the Spanish Ministry of Education and Science.
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Notes |
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1To whom correspondence should be addressed. Tel: +34 93 581 3305; Fax: + 34 93 581 23 87; Email: oriol.cabre{at}uab.es
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