Mutagenesis, Vol. 15, No. 4, 357-359,
July 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Sequence analysis of the boundaries of the tandem duplication from the white-ivory mutant of Drosophila melanogaster and two chemically induced revertants
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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We have previously shown that revertants obtained from the white-ivory mutants of Drosophila melanogaster, both spontaneous and induced, have lost a DNA fragment of 2.9 kb that is duplicated in tandem in the white-ivory mutation. To prove the accuracy of the deletion in revertants obtained after treatment with alkylating agents, we have sequenced DNA fragments previously amplified by PCR. These fragments correspond to the ends of the remaining 2.9 kb copy of these revertants and the internal region of the junction of both copies, which constitutes the duplication in the white-ivory mutant. These sequences are compared with those from white-ivory mutants. Our results show slight differences from the published sequence of the white-ivory mutation and with the wild-type sequence of the white locus. The sequences of the two revertants analysed show that excision of the duplicated fragment is very precise. We hypothesize the mechanism of excision in terms of intrachromosomal recombination induced by double-strand break repair after treatment with alkylating agents.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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It is assumed that the white-ivory (wi) phenotype of Drosophila melanogaster is caused by a 2.9 kb tandem duplication (Karess and Rubin, 1982
) of the region located between positions +2795 and –173 of the white locus according to the coordinate system of Levis et al. (1982), as located by O'Hare et al. (1984). In addition, there is a 6 bp repeat at the 5'-end of the second copy of the duplication (O'Hare et al., 1984). As indicated in Figure 1a, the duplication covers from the first intron to the third exon.
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The use of the wi mutation has been proposed by several authors (Green et al., 1986
; Clements et al., 1990; Howe and Clements, 1990; Würgler and Kägi, 1991; Xamena et al., 1991; Batiste-Alentorn et al., 1994) to evaluate the genotoxic potential of chemicals, because flies with the wi mutation can revert to the wild-type phenotype both spontaneously and by chemical induction (Lewis, 1959; Bowman, 1965; Ryo et al., 1985; Green et al., 1986; Suárez et al., 1996). Nevertheless, the discrepancy between the results obtained in the wi eye spot test compared with both the mwh/flr and the w/w+ systems (Graf and Würgler, 1996; Ferreiro et al., 1997) and a lack of knowledge of the mechanism involved in the wi reversion would limit its application as a short-term test for mutagen identification. The test is based on phenotypical reversion to the wild-type red eye colour. This reversion has been attributed to precise excision of one of the copies of the duplication (Karess and Rubin, 1982; Green et al., 1986; Suárez et al., 1996) by a mechanism not yet well understood, although some authors, such as Bowman (1965) and Howe and Clements (1990), have proposed two different mechanisms for reversion of the wi allele. Thus, germinal reversion of a direct tandem duplication to the wild-type phenotype can take place by either interchromosomal meiotic crossing over or intrachromosomal mitotic exchange in the germline before meiosis. However, other possible mechanisms, such as gene conversion or unequal crossing-over, should be considered, since both processes may produce loss of a duplicated fragment (Becker, 1975; Karess and Rubin, 1982).
To determine the accuracy of the 2.9 kb deletion in the wi revertants, we have sequenced fragments corresponding to the internal junction of the duplicated sequence of the wi mutant and both ends of the characteristic 2.9 kb segment, in the wi mutant and in two independent revertants obtained after treatment with alkylating agents (Suárez et al., 1996).
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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We have used the strains ywi, obtained from the Umeå Drosophila Stock Center (Sweden), and ywiR5 and ywiR21, two wild-type eye colour revertants obtained in our laboratory after chemical treatment of ywi larvae with alkylating agents (Suárez et al., 1996
; Suárez, 1997). Revertant ywiR5 was obtained after treatment with methyl methanesulfonate (MMS) and revertant ywiR21 after treatment with N-nitroso-N-ethylurea (ENU). For a detailed description of the y and wi genetic markers, see Lindsley and Zimm (1992).
Both the 5'- and 3'-flanking sequences and the internal junction region of the duplicated sequence of the wi allele were previously analysed by PCR using primers wip1, wip2, wif3 and wif4 as described (Suárez et al., 1996; Figure 1b
). Before sequencing, the amplified regions were purified from the agarose gel using a GeneClean kit (Bio101 Inc., USA). These amplified fragments were directly sequenced using the following 5'-fluorescein-labelled primers: wis1, 5'-QAATTGTTTCAAAGAGCC-3'; wis2, 5'-QTGAATGCCCTTGCCTTTCG-3'.
The sequences were determined by automatic sequencing (ALF; Pharmacia Biotech, Sweden). Such sequences were aligned with the white locus sequence of D.melanogaster (accession no. X02974) by the NALIGN program (PCGENE; Intelligenetics Inc. and Genofit, Switzerland).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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In a previous work (Suárez et al., 1996
) we obtained several germinal revertants after treatment of wi flies with different alkylating agents. The molecular analysis, carried out by Southern blotting and PCR, showed that these revertants had lost one copy of the 2.9 kb tandemly repeated DNA sequence. Similar results were also observed in the intragenic tandem duplication f3N (Ishimaru et al., 1995). PCR amplifications from both the 5'- and 3'-boundary regions as well as the internal junction sequence of the duplicated region of the wi mutation were carried out but, as expected, amplification failed in the internal region of the revertants. To obtain more information about the molecular characteristics of these revertants and add light to the mechanistic basis, we sequenced stretches of the amplified DNA fragments from the wi mutant and from the two different revertants.
Figure 1b shows a diagram of the pairs of primers used to amplify some defined regions (the 5'-boundary, the internal region and the 3'-boundary) of the wi mutation and the sequencing primers used to obtain stretches of sequences of these regions, ranging from ~250 to ~500 bp.
Figure 2a shows a diagram of the studied regions of the white locus. Figure 2b presents the alignment of a few bases at the ends of flanking sequences of the Canton-S wild-type, the wi mutation and two of the revertants obtained from wi after treatment with MMS (ywiR5) or ENU (ywiR21). The studied sequences cover the three regions indicated above (Figure 2a): the 5'-boundary (from the 5'-flanking sequence 1A to the beginning of the first part of duplication 1B), the internal region (from the end of the first part of duplication 2A to the beginning of the second part of duplication 1B) and the 3'-boundary (from the end of the second part of duplication 2A to the 3'-flanking sequence 2B).
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The sequences of the 5'-boundary and internal region of wi are entered in the EMBL Nucleotide Sequence Database under accession nos Y17798 and Y17799, respectively. The 3'-boundary sequence was not submitted because no differences were apparent.
The stretches of sequences obtained were aligned with the white locus sequence available under accession no. X02974, taking the numbering of this as a reference (Figure 2b). Several polymorphisms can be observed when the Canton-S wild-type and wi alleles are compared. Thus, three differences in a single nucleotide at positions 8229 (C
G), 8384 (TC) and 8430 (TC) are observed. Both duplicated copies of the wi mutation show this last change in nt 39 from the start of the duplicated sequence (Figure 2b, 1B), indicating that this change was already present in the white locus prior to duplication. The wild-type also presents a direct repeat of the sequence AGATACT (C repeat) starting at 8236, but wi presents only one copy (Figure 2b, C). Such polymorphisms seem to be common in the white locus, mainly in the first intron (Miyashita and Langley, 1988).
The sequence GCATCT (M repeat) is directly repeated at the start of the second part of the duplication in the internal region, but present once at the start of the first part in the 5'-boundary (Figure 2b, M), as described by O'Hare et al. (1984). Taking this sequence as the reference point to localize the ends of the duplication, we found the first base (G) at position 8392 and the last base (A) at position 11372, instead of 8402 and 11025, respectively, as marked out by O'Hare in accession no. X02974. Thus, we have found that the duplicated segment is 2981 bp long, a value close to the length proposed in previous reports (Karess and Rubin, 1982; Green et al., 1986; Suárez et al., 1996).
The two revertants analysed show the same features as wi except for the M repeat at the 5'-end of the duplication. Thus, the single nucleotide differences and the C repeat upstream of the duplication agree with the unequivocal origin of these revertants from the wi stock used in this study.
The lack of the M repeat indicates that the reversion may be a consequence of a recombinational event instead of a true excision of one of the two copies, as is commonly considered in current works (Karess and Rubin, 1982; Ryo et al., 1985; Green et al., 1986; Suárez et al., 1996). Taking into account that our experiments were carried out with adult males arising from treated larvae, the mechanism should be intrachromosomal due to the presence of only one X chromosome in males. In this sense, intrachromosomal exchange between any pair of homologous points of the duplicated sequence could split off a fragment with the 3'- and the 5'-portions looping out between these points. It could also be considered as an unequal sister chromatid exchange after replication or slippage of the replication fork. This deletion restores the wild-type genotype and removes the M repeat.
Mitotic intrachromosomal recombination can be induced by a double-strand break (for a review see Osman and Subramani, 1998), as studied in detail, for example, between direct repeats (for a review see Lambert et al., 1999). In addition, a double-strand break made within pre- or post-replicated DNA or in the replication fork also induces repair that leads to recombination (for a review see Haber, 1999). Thus, once a double-strand break is produced in a repeated sequence, it can be repaired both by double-strand break repair and single-strand annealing, giving a recombinant with only one copy of the duplication. In the case of the wi mutation, where the two direct repeats are in tandem (separated only by the M repeat), the double-strand break induced by alkylating agents could be resolved by the above mechanism generating revertants with a very precise excision of one copy of the duplication.
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Acknowledgments |
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We thank M.McCarthy for her secretarial assistance. This investigation was supported by the Spanish Ministry of Education and Culture (grants nos SAF95-0813 from the CICYT and PB96-1138 from the DGES).
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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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Received on February 7, 2000; accepted on March 27, 2000.
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