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Molecular characterization of the singed wings locus of Drosophila melanogaster

Abstract

Background

Hormones frequently guide animal development via the induction of cascades of gene activities, whose products further amplify an initial hormonal stimulus. In Drosophila the transformation of the larva into the pupa and the subsequent metamorphosis to the adult stage is triggered by changes in the titer of the steroid hormone 20-hydroxyecdysone. singed wings (swi) is the only gene known in Drosophila melanogaster for which mutations specifically interrupt the transmission of the regulatory signal from early to late ecdysone inducible genes.

Results

We have characterized singed wings locus, showing it to correspond to EG:171E4.2 (CG3095). swi encodes a predicted 68.5-kDa protein that contains N-terminal histidine-rich and threonine-rich domains, a cysteine-rich C-terminal region and two leucine-rich repeats. The SWI protein has a close homolog in D. melanogaster, defining a new family of SWI-like proteins, and is conserved in D. pseudoobscura. A lethal mutation, swit 476, shows a severe disruption of the ecdysone pathway and is a C>Y substitution in one of the two conserved CysXCys motifs that are common to SWI and the Drosophila Toll-4 protein.

Conclusions

It is not entirely clear from the present molecular analysis how the SWI protein may function in the ecdysone induced cascade. Currently all predictions agree in that SWI is very unlikely to be a nuclear protein. Thus it probably exercises its control of "late" ecdysone genes indirectly. Apparently the genetic regulation of ecdysone signaling is much more complex then was previously anticipated.

Background

Hormones often exert their effects on animal development via the induction of cascades of gene activities, whose products further amplify an initial hormonal stimulus. In Drosophila the transformation of the larva into the pupa and the subsequent metamorphosis to the adult stage is triggered by changes in the titer of the steroid hormone 20-hydroxyecdysone (referred to below as ecdysone). Ecdysteroids act through a regulatory cascade, first discovered by observations of changes in the morphology of the giant polytene chromosomes of larval salivary glands [1]. Transcribed genes in polytene chromosomes are often represented by puffs, providing an opportunity to visualize the process of hormonally triggered sequential gene activation.

The sharp increase in ecdysone titer in the late third instar larva leads to the rapid induction of a few "early" puffs. These regress after reaching their maximum size, and after a lag period of several hours, many "late" puffs become active. The drop in ecdysone concentration following puparium formation leads to the regression of these "late" puffs and the induction of novel genes characteristic of the "mid-prepupal" period; subsequently the prepupal ecdysone pulse re-induces many of the early and late puffs seen active earlier in late larval development [1, 2].

Ashburner and colleagues [1] proposed that ecdysone bound by a receptor protein directly induces the "early" puffs. The protein products of early puffs then both activate "late" puffs and repress the transcription of those genes that form the "early" puffs. The model also suggested that ecdysone directly represses the activation of the "late" puffs, preventing their premature induction by early puff products.

This model predicts that it should be possible to specifically disturb the sequence of puffing events by mutating the genes that are active as early puffs. Indeed, several mutations blocking the initiation of ecdysone-dependent puffing have been isolated. The affected genes include components of the functional ecdysone receptor, a heterodimer of the EcR and usp proteins [3, 4], as well as transcription factors encoded by the Broad-Complex (BR-C), which forms an early puff [5]. In addition, mutations in the βFTZ-F1 orphan receptor (which forms the 75D puff in mid-prepupae) impair the specific induction of early genes after the prepupal ecdysone pulse [6].

Surprisingly, only one mutation has been isolated that specifically disturbs the puffing sequence downstream of "early" puff induction in third instar larvae. This is the t467 allele of the swi (singed wings) complementation group. The swi-complementation group is localized in region 2B6 – 2B8 and includes two hypomorphic mutations and four recessive semi-lethal or lethal mutations. Survivors have faded (singed) wings and swollen abdomens. swit 467is fully lethal, most swit 467homozygotes die as pharate adults [7]. Third instar larvae homozygous for swit 467have normal sized salivary glands and polytene chromosomes. The induction and repression of "early" as well as "mid-prepupal" puffs in the polytene chromosomes of mutant flies remains unaffected by the mutation, but most of "late" puffs are either greatly reduced in size or completely absent [8].

Experiments using in vitro cultured larval salivary glands of swit 467/swit 467larvae showed that the premature removal of ecdysone from cultured glands partially rescues late puffing activity. These findings suggest that the product of the swi locus may control "late" puff induction and, in addition, that there are distinct pathways leading to early puff repression and late puff activation.

Interestingly, about 2.5% of swit 467homozygous larvae pupariate only in the anterior part of the body. This phenotype is reminiscent of one described by Rayle [9] for the flies bearing the halfway (hfw) mutation. Since this mutation was mapped to the same cytological interval as swi, it is very likely to have been allelic to swi (see Methods).

The peculiarity of the swi mutant phenotype made it of interest to characterize this gene at the molecular level. This we have done, showing it to correspond EG:171E4.2 (CG3095), encoding an uncharacterized protein that is similar to mammalian decorins and the Drosophila Toll-4 protein; this gene also has at least one close homolog in D. melanogaster and a homolog in D. pseudoobscura.

Results and discussion

Genetic complexity of the 2B6/B7-8 region

Our earlier data placed the swi locus in region 2B6/B7-8 of the X chromosome, between the proximal break points of Df(1)S39 and Dp(1;Y)y267g24.2Y [7]. This region corresponds to 1421800–1466900 bp of the Release 3.1 X chromosome sequence [10–12]. This interval is overlapped by the EDGP cosmids 73D1 and 9D2 (Fig. 1). Saturation mutagenesis of this region revealed only two loci [7]deep orange (dor) (see [13]) and singed wings (swi). Subsequently, Makunin and co-authors [14] identified two further genes (b6 and a6) (Fig. 1) and the computational analysis of the genomic sequence identified a further six [12] (Fig. 1). cDNA sequences are available for all of the genes predicted by sequence analysis, with the exception of CG14797 [15].

Figure 1
figure 1

Organization of 2B6/B7-8 region The region is situated between the break points of Df(1)S39 and the duplication Dp(1;Y)y267g24.2Y (shown by arrows). The shaded rectangles indicate the precision of rearrangement mapping. The restriction map of the region linked to the genomic sequence coordinates of FlyBase (in kb) is shown below. E-Eag I, N-Nhe I, B-Bam HI, X-Xba I, R-Eco R, H-Hpa I. In the case of those sites marked with an asterisk only ones relevant to this study are shown. The positions of genomic clones (indicated by dashed lines), genetic loci (shown with arrows) and pieces of genomic DNA used for transformation (cross-hatched lines) are presented under the restriction map. The genes a6 and b6 correspond to CG3771 and CG3100 respectively.

swi corresponds to EG:171E4.2

In order to narrow the list of possible candidate loci for swi a genomic library made from the DNA of swi+ flies in the CosPeR cosmid vector (see Methods) was screened using the DNA of the clone λ1487 as a probe. A 30 Kb clone (5581) was recovered and mapped to the genome (Fig. 1). Two lines independently transformed by this cosmid (5581.118 and 5581.4), each with a single independent autosomal insertion, were obtained and tested for complementation of swit 476. Neither of them was able to complement swit 476, implying that a6, b6, CG14798 and EG:9D2.4 are not swi.

To choose between remaining four loci we made a further set of transposons (Fig 1). SWI1 contained the very 3'-end of dor, a complete EG:171E4.4 gene and the 5'-half of EG:171E4.2. SWI3 overlapped the 3'-end of dor, and the full sequences of EG:171E4.4 and EG:171E4.2 while SWI4 included only EG:171E4.2. Five independent transformed lines with insertions of SWI1, seven lines containing insertions of SWI3 and sixteen lines with insertions of SWI4 were established. All insertions of SWI1 were autosomal; none complemented the swit 476mutation. On the contrary, all six autosomal insertions of SWI3 (Table 1) and all 16 insertions of SWI4 completely rescued this mutation (Table 2). These results unambiguously indicate that the swi gene is identical to EG:171E4.2.

Table 1 Complementation test of SWI3 lines.
Table 2 Complementation test of SWI4 lines.

To confirm this conclusion we had isolated the DNA from homozygous y1swit 476wpn/y1swit 47wpnthird instar larvae and sequenced the region spanning from bases 1425864 to 1431743 (Release 3 coordinates). There are nineteen differences between this sequence (deposited in EMBL-Bank as AJ626646) and the reference sequence (AE003421). Only one of these differences affects the predicted protein product of EG:171E4.2, a G to A transition at base 1428762, that would change Cys379 to Tyr379.

The transformation rescue of the mutant swi allele by a construct that includes only EG:171E4.2, and the fact that the mutant allele carries a very non-conservative amino acid substitution prove that swi corresponds to this predicted gene.

Analysis of swi

The 4.9 kb Hpa I-Hpa I fragment from SWI4 (Fig. 1) is capable of fully rescuing the swit 476phenotype; this fragment, therefore, must include not only the coding sequence of swi, but also all of its necessary regulatory sequences. The swi (EG:171E4.2) mRNA as judged from the longest found cDNA sequence (RE03173) spans the region from position 1426365 to 1429883. We suspect that the true length of the transcript is slightly greater, since neither a consensus Drosophila Initiator (INR) element nor any known Drosophila promoter element could be found in the immediate vicinity of RE03173 5'-end. At the same time there is a perfect TATA box consensus (TATAAA), at position 1426228 (seen with PromH, [16]) and CA and T nucleotides at positions 30 and 36 downstream of this, characteristic of INR elements [17] (Additional file 1). The latter suggests that swi may have a strong TATA promoter, initiating 107 bp upstream of RE03173 and that is consistent with the fact that only a 150 bp upstream region is required for swi transgene function.

We have performed BLAST search of the Drosophila pseudoobscura genomic sequence database with the sequence of the 4.9 kb Hpa I-Hpa I fragment. This identified a region (coordinates 5225–8898) of contig 2140 that aligns with the D. melanogaster sequence from coordinate 1426109 to 1429698 and a short region of contig 2139 complementary sequence that aligns with the very 3' end of D. melanogaster Hpa I-Hpa I fragment (Additional file 1). The alignment of D. melanogaster sequence with that of contig 2140 corresponds to almost the entire swi locus with only last 185-bp of the 3'-UTR missing. The degree of conservation between these sequences is not uniform: there is high nucleotide conservation (78%) in the central coding region of swi (coordinates 1428116 – 1429529) (Additional file 1A), but less (44%) elsewhere (Additional file 1). In addition, there are short highly conserved regions within the first intron (coordinates 1427144 – 1427515) (Additional file 1). This region includes a number of short, but highly conserved, motifs, often AT rich and resembling the consensus homeobox binding consensus sequence (NNATTA) [18]. These motifs may well have a regulatory function.

Properties of the SWI protein

Translation of the swi open reading frame (ORF) predicts a 611 amino acid protein with threonine and histidine rich N-terminal domains, two leucine rich repeats and a cysteine rich C-terminus (Fig. 2A). The predicted amino acid sequence of the Dpse\SWI protein has 71% identity and 73% similarity to that of its D. melanogaster homolog.

Figure 2
figure 2

The products of swi gene A. The structure of the swi gene; exons are shown as boxes and introns as angled lines. Regions of exons encoding various domains are shaded: the histidine-rich domain (HR, black box), the threonine-rich domain (TR, grey box), the leucine-rich repeats (LRR, chess-desk box) and the cysteine-rich region (CR, shaded box). B. The alignment of the SWI amino acid sequence with amino acid sequences of its closest simalogs. For the better representation the following color code is applied. Residues in common between all proteins are marked in red, the ones common only between SWI, SWI2 and Dpse\SWI in pink and the ones identical only among the Dpse \SWI, SWI, and Agam\SWI2 are marked in black. Brown marks the amino acids common only between Dpse \SWI, Agam \SWI2 and SWI2 proteins, dark blue – the ones common only between SWI, Agam \SWI2 and SWI2. The pairs of amino acids identical between SWI and Dpse \SWI, SWI2 and Agam \SWI2, Agam \SWI2 and Dpse \SWI, Dpse \SWI and SWI2, SWI and Agam \SWI2 are marked in green, turquoise, violet, grey and blue, respectively. The amino acids similar between all four proteins are underlined. The CysXCys motives are boxed. Note the conserved Cys-379 (marked by a triangle) that is substituted in swit 476mutant flies. The regions of the proteins with the highest similarity to decorins are marked by dotted boxes.

The predicted SWI protein was used to search public protein databases for known or predicted proteins of similar sequence. The sequences of the protein of the predicted CG14485 (TrEMBL:Q8SXT3) gene (e = 8e-24) and of the predicted Anopheles gambiae protein (agCP6178; protein_id:EAA11923.1) (e = 1e-14) showed significant similarity to that of SWI. It is also important to note that this proteins appeared to be more similar to each other than to SWI itself suggesting Q8SXT3 to be designated as SWI2, the second member of Drosophila SWI family of proteins, and EAA11923.1 as its Anopheles gambiae simalog Agam\SWI2.

The second large group of sequences showing much lower, but evident, similarity to SWI (e ~1e-07) consists of virtually all known homologs of decorin and biglycan proteins. These proteins are characteristic members of the Class I small leucine-reach proteoglycan protein family. These proteins possess a protein core substituted with a single glycosaminoglycan chain near their N-terminus [19]. The most salient feature of decorins and biglycans is the presence of 10 leucine-reach repeats flanked by cysteine-rich regions [20]. Despite the relatively small size of the protein core (~36 kDa), these proteins possess several distinct protein binding activities. Originally, they were shown to bind collagen fibers, playing a role in their assembly. More recently decorin and biglycan were also discovered to interact with the Transforming Growth Factor-β and Epidermal Growth Factor (EGF) receptors, participating in cellular proliferation control [19].

The third group of sequences similar to SWI contains two members of Toll receptor superfamily: Toll related protein Ae\Toll1B from Aedes aegypti (Prm = 7e-04) and Toll-4 (Prm = 9e-04) from Drosophila melanogaster. Both of these were discovered in course of the systematic characterization of Toll-like proteins [21, 22]. Although some members of Toll protein family are transmembrane receptors, with a well established role in innate host defense, no evidence is available for the function of either Ae\Toll1B or Toll-4 [21].

We have made all possible pairwise combinations and multiple alignments of these proteins. As expected, the best multiple alignment is between the SWI, Dpse\SWI, SWI2 and Agam\ SWI2 protein sequences. (Fig. 2B). Examination of this alignment shows that the C-terminal 211 amino acids of SWI are conserved between four proteins (20% identity; 52% similarity) with much higher homology between SWI and Dpse\SWI (90% identity). This alignment also clearly shows that Agam\SWI2 is more similar to SWI2 than to SWI.

The region of significant similarity between SWI and the decorin/biglycan family proteins is narrower, it is limited to 90 amino acids between positions 396–485 of SWI. In decorins this region is N-terminal (residues 54–141 of murine decorin). This region contains the very end of N-terminal cysteine-rich flanking region and the first two leucine-rich repeats [20]. The region of homology between the proteins does not correspond to the leucine-rich repeats of SWI. The two stretches of amino acids especially conserved between SWI and decorins are also well conserved between SWI, SWI2, Agam\SWI2 and Dpse\SWI (Fig. 2B). Interestingly enough, such conserved regions have no documented role in known decorin functions, implying that decorins might have some yet undiscovered properties that these proteins have in common with SWI.

The region of homology between the SWI and Ae\Toll1B or Toll-4 proteins is situated between positions 249–580 of the SWI amino acid sequence. This corresponds to positions 307–626 of Toll-4 and includes one of its N-terminal cysteine-rich flanking region and two leucine-rich repeats; this region of similarity does not overlap the Toll/IL-1R (TIR) domain of Toll4. This cytoplasmic homology domain is a key feature common of all Toll-like proteins and is indispensable for the activation of the antifungal response in flies by Toll and 18-wheeler (Toll-2) [23]. Comparison of SWI and Ae\Toll1B or Toll-4 sequences revealed two distinct conserved CysXCys motifs (Fig. 2B). Both of them are also conserved in SWI2, Agam\SWI2 and Dpse\SWI. The swit 476mutation affects one of the cysteines in the first CysXCys motif. These CysXCys motifs may be part of a yet uncharacterized functional protein domain (or two distinct but similar protein domains) indispensable for SWI activity and, perhaps, also important for Toll-4 function.

We have used a number of prediction programs in an attempt to learn more about the possible cellular location of the swi protein. von Heijne's algorithm [24], as implemented by PSORTII, indicates that the protein may have a cleavable signal sequence (residues 1–19) (a prediction confirmed by the TargetP Server v1.01 [25]. This is consistent with predictions from the Pastuer Institute's implementation of Claros and von Heijne's TopPred algorithm, which indicate a predominantly extracellular protein anchored by cytoplasmic N- and C-termini.

As an alternative approach we have searched the database of Drosophila protein interactions recently built on the basis of genome-wide yeast two-hybrid screening [26]. SWI was found to have only one interacting partner, the product of CG9025 gene. This interaction seems to be very specific and has a very high confidence score (0.9085 with 1.0 being the maximum). CG9025 is an uncharacterized protein similar to mammalian sex-determination protein encoded by Fem1b gene. CG9025 in turn has eight high confidence interactors from which all with known cellular localization are cytoplasmic. It is interesting to note that one of CG9025 interacting proteins, the product of TBPH (CG10327) has a putative RNA binding domain.

Conclusions

The present molecular analysis, unfortunately, throw remarkably little light on how the SWI protein may function in the ecdysone induced cascade. By far all predictions agree in that SWI is very unlikely to be a nuclear protein. Thus it probably exercises its control of "late" puffing indirectly. Clearly the genetic regulation of ecdysone signaling appears to be much more complex then was previously anticipated.

Methods

DNA clones and fly stocks

The phage genomic clones overlapping the 2B6/B7-8 region have been described by [10]. The coordinates of the FlyBase Drosophila genome annotation (Release 3.1) are used in this paper. The cosmid 73D1 was provided by F. Galibert. The genomic library made from DNA of isogenic y1; cn1bw1sp1 flies in the CosPeR cosmid vector, the generous gift of J. Tamkun. CosPeR contains P-element termini and the white gene as a marker, which allows the direct use of a clone for Drosophila transformation. The stock y1swit 476wpn/FM6B, carrying the lethal allele of singed wings, has been described earlier [7]. Lindsley and Zimm [27] designated an EMS-induced mutation swit 467as hfw7. However, complementation tests of the hfw1 allele described by Rayle [9] with any of the swi alleles was never done, since the hfw1 stock had been lost by the time any swi mutant allele was obtained. We use the swit 467allele from the Novosibirsk stock collection. The y1swit 476wpnmutant chromosome was also rebalanced over FM7, y93jsc8w oc pt B, P{w+mC, act::GFP=pActGFP}, a "green" balancer (the generous gift of J.-M. Reichhart) to make the y1swit 476wpn/FM7, y93jsc8w oc ptg B, P{w+mC, act::GFP=pActGFP} stock. This strain allows the easy selection of y1swit 476wpn/y1swit 476wpnlarvae as non-GFP animals.

DNA manipulations and sequence analysis

All general molecular methods were after Sambrook and co-authors [28]. Plasmid and cosmid DNA or PCR products were directly sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer). To amplify fragments of genomic DNA isolated from y1swit 476wpn/y1swit 476wpnflies the following pairs of primers were used:

Swi-m1 5'-CCGTGCGGCAGTATGACG-3', Swi-m2 5'-TCTGCAGGCTCGTTCGGT-3';

Swi-m3 5'-CAGCGAGTGCTCTGCTGC-3', Swi-m4 5'-GGCTTCGGCATGGAGTGG-3';

Swi-m5 5'-CCTCGTATCCTCACACGC-3', Swi-m6 5'-CTTCCTTCAGCGGCGCAT-3';

Swi-m7 5'-CAGGGCACATGGCTCCTG-3', Swi-m8 5'-GGCGTTCACCAGTCGAGT-3';

Swi-m9 5'-TGCCAGTGCAGGGAGCAC-3', Swi-m10 5'-TGGGGTAGCTCCTCCAGG-3';

Swi-m11 5'-CGAACTACGGCAGCTGCA-3', Swi-m12 5'-AGGGCAGATAGCCAGCCG-3';

Mut1 5'-GAACAGCCCGAGTATCTG-3', Mut2 5'-GTCGTAGGACACCTTGGT-3';

Mut3 5'-TACCTGGCAGGCAACAAG-3', Mut4 5'-CAACAGTGAGCCGTATCC-3';

Mut5 5'-TCGAGCGCGCACCTAAGT-3', Mut6 5'-ATCCAGAGGCCAGGACCT-3'.

Sequence analysis

The raw sequence data were analyzed using BLAST [29] and ClustalW [30] computer programs. Screening of Drosophila pseudoobscura genome sequence for homology to the swi sequence was done with BLAST at http://www.hgsc.bcm.tmc.edu/projects/drosophila/. To look for promoter elements, genomic DNA sequence corresponding to the region of the putative transcription start, i.e. the 5' end of the BDGP cDNA clone RE03173 (accession number AY070931) was visually inspected for sequences corresponding to the TATA, INR and DPE consensus sequences, as described by Kutach and Kadonaga [17]. In addition, the entire sequence of swi locus was analyzed with PromH program [16]. Unless specially stated the default parameters of all software programs were employed. To browse D. melanogaster genome annotations the Apollo (v1.3.4) software was used [31]. The parameters of protein primary structure were calculated using the ProtParam tool at [32] and a protein motif search was performed with InterProScan software [33]. To predict protein localization the following programs were used. PSORTII at [34], the TargetP Server v1.01 [25] at [35] and the Pastuer Institute's implementation of Claros and von Heijne's TopPred algorithm [36]. The Drosophila protein yeast two-hybrid interaction network [26] was searched at [37].

Transposon construction

To make the SWI1 construct the 4.4 kb Bam HI-Bam HI fragment from the λ1479 clone (Fig. 1) was inserted into the CaSpeR3 vector [38] digested with Bam HI. The SWI3 transposon was constructed as follows: The 9.4 kb Xba I-Eag I fragment from the cosmid 73D1 clone was ligated into pBluescript KS (Stratagene), digested with Xba I and Eag I, to give 73D1-XE-pBl-KS. The 73D1-XE-pBl-KS construct was further digested with Xba I and Nhe I and the resulting 8.5 kb Xba I-Nhe I fragment was directly inserted into the CaSpeR2 vector (Pirrotta, 1988), digested with Xba I. The SWI4 transposon was constructed in similar way: The 73D1-XE-pBl-KS construct was cut with Hpa I and the resulting 4.9 kb fragment was inserted into the CaSpeR4 vector [38], digested with the same restriction enzyme. All constructs were injected into the host strain y1, Df(1)w67c 23as described by Spradling [39].

Complementation analysis

To test complementation of the swit 476mutation by various transformed fragments of genomic DNA y1swit 476wpn/FM6B females were crossed to transformed y1, Df(1)w67c 23/Y males carrying an autosomal insertion of the genomic fragment under study. The rescue of the swi lethal phenotype was judged by the appearance of yellow males in the progeny.

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Acknowledgements

Work in Novosibirsk was supported by a grant from Molecular and Cellular Biology Programme of the Russian Academy of Sciences. Work in Cambridge was supported by The Royal Society and a MRC Programme Grant to M. Ashburner, D. Gubb and S. Russell. We are grateful to John Roote, David Gubb, and Steven Russell for the fruitful discussions and advice.

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Correspondence to Igor F Zhimulev.

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YBS performed molecular and genetic experiments, made bioinformatics studies, wrote the paper. TB made molecular and genetic experiments. ESB participated in the design of the study, made control cytogenetic experiments and participated in manuscript editing. MA edited the paper and provided guidance and funding for this project. IFZ conceived and supervised the project. All authors read and approved the final manuscript.

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12863_2004_187_MOESM1_ESM.pdf

Additional File 1: The alignment between the DNA sequence of the D. melanogaster swi gene and the sequence of D. pseudoobscura contigs 2140 and 2139. For Drosophila melanogaster DNA sequence the coordinates of the FlyBase genome annotation (Release 3.1) were used. Identical nucleotides are marked with asterisks. The putative TATA box and characteristic CA and T nucleotides at the 5' region of swi are shown in red font. The 5' and 3' UTRs are marked in green, the coding part of the swi DNA sequence is marked in red. Note that the region of the first intron contains the number of small well conserved motifs. Those containing the NNATTA sequence, characteristic of homeobox protein binding sites, are marked in blue the others are marked in black. The very 3' part of the locus could be aligned with the complementary sequence of the 2139 contig due to the two stretches of extremely conserved DNA. (PDF 33 KB)

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Schwartz, Y.B., Boykova, T., Belyaeva, E.S. et al. Molecular characterization of the singed wings locus of Drosophila melanogaster. BMC Genet 5, 15 (2004). https://doi.org/10.1186/1471-2156-5-15

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