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PLoS One. 2009; 4(11): e7883.
Published online Nov 18, 2009. doi:  10.1371/journal.pone.0007883
PMCID: PMC2775673

The Transposon Galileo Generates Natural Chromosomal Inversions in Drosophila by Ectopic Recombination

Robert DeSalle, Editor

Abstract

Background

Transposable elements (TEs) are responsible for the generation of chromosomal inversions in several groups of organisms. However, in Drosophila and other Dipterans, where inversions are abundant both as intraspecific polymorphisms and interspecific fixed differences, the evidence for a role of TEs is scarce. Previous work revealed that the transposon Galileo was involved in the generation of two polymorphic inversions of Drosophila buzzatii.

Methodology/Principal Findings

To assess the impact of TEs in Drosophila chromosomal evolution and shed light on the mechanism involved, we isolated and sequenced the two breakpoints of another widespread polymorphic inversion from D. buzzatii, 2z3. In the non inverted chromosome, the 2z3 distal breakpoint was located between genes CG2046 and CG10326 whereas the proximal breakpoint lies between two novel genes that we have named Dlh and Mdp. In the inverted chromosome, the analysis of the breakpoint sequences revealed relatively large insertions (2,870-bp and 4,786-bp long) including two copies of the transposon Galileo (subfamily Newton), one at each breakpoint, plus several other TEs. The two Galileo copies: (i) are inserted in opposite orientation; (ii) present exchanged target site duplications; and (iii) are both chimeric.

Conclusions/Significance

Our observations provide the best evidence gathered so far for the role of TEs in the generation of Drosophila inversions. In addition, they show unequivocally that ectopic recombination is the causative mechanism. The fact that the three polymorphic D. buzzatii inversions investigated so far were generated by the same transposon family is remarkable and is conceivably due to Galileo's unusual structure and current (or recent) transpositional activity.

Introduction

A sizable portion of eukaryotic and prokaryotic genomes is composed of transposable elements (TEs) with the potential to cause chromosomal rearrangements such as inversions, translocations and duplications [1][3]. These rearrangements however may be generated also by other processes that do not involve TEs (see below). Thus, the actual contribution of TEs to the evolutionary reorganization of genomes is unclear. One of the most frequent and widespread types of chromosomal rearrangements during evolution are inversions, which alter gene order often without changing total gene content [4]. Inversions are remarkably abundant in the genus Drosophila, both as intraspecific polymorphisms and as interspecific fixed differences [5], [6] and increasing evidence point to their prevalence in many other species, e.g. humans [7][11].

TEs can generate chromosomal inversions by intrachromosomal homologous recombination between two copies of the same TE family arranged in opposite orientation [12]. This mechanism is known as TE-mediated ectopic recombination or nonallelic homologous recombination (NAHR). TEs can also induce inversions as well as other types of rearrangements when two ends coming from different TE copies participate together in an aberrant transposition event. The outcome depends on the location and orientation of the two cooperating TE copies in the parental chromosome and the chromosomal site where they insert (Figure S1). If the two copies are located in sister chromatids or homologous chromosomes, the process is referred to as hybrid element insertion [13][15]. When the two copies are located at neighboring sites on the same chromatid, the mechanism is known as reversed ends transposition [16], [17]. Inversions can be also generated by two other mechanisms not involving TEs. One such mechanism is chromosomal breakage and repair by non-homologous end-joining (NHEJ). Double strand breaks (DSBs) are produced in many ways in all cells and the machinery to deal with these lesions is conserved from yeasts to vertebrates [18], [19]. When two or more DSBs occur simultaneously, repair by NHEJ may produce gross rearrangements if the joining takes place between previously unlinked DNA molecules [20]. Finally, inversions may result from ectopic recombination between other repeated sequences besides TEs, such as tRNA genes [21] or segmental duplications (SDs) [7], [8].

TE-mediated ectopic recombination has generated natural chromosomal inversions in bacteria [22][27] and some lineages have experienced an striking degree of rearrangement caused by this process [28][31]. Likewise, Ty-recombination mediated deletions, duplications, inversions and translocations have been found to occur in yeast [12], [32][34]. In mammals, long and short interspersed elements (LINEs and SINEs, respectively) have been implicated in the generation by ectopic recombination of 50 inversions fixed between humans and chimpanzees [35], [36]. In Drosophila, the evidence for the implication of TEs in the generation of inversions is limited. Two D. buzzatii polymorphic inversions, 2j and 2q7, were seemingly generated by ectopic recombination between copies of the transposon Galileo [37], [38]. In D. pseudoobscura, the polymorphic inversion Arrowhead and a number of fixed inversions have been also generated by ectopic recombination between 128-bp and 315-bp repeats, yet the nature of these repeats is obscure [39]. Inversion In(4)a of D. americana has been found to be flanked by copies of a new transposon and was likely generated by an intrachromosomal exchange between these repeats [40]. TEs have been found also at the breakpoints of two Anopheles gambiae inversions, 2Rd' and 2La, but the implication of these TEs in the origin of the inversions is circumstantial [41], [42].

Chromosomal breakage and repair by NHEJ is also a common mechanism for the generation of chromosomal inversions. This process may generate duplications flanking the inverted segment when one or both DSBs occur in a staggered manner [43]. In Drosophila, this process has been responsible for most of the inversions fixed between D. melanogaster and D. yakuba [43] as well as three D. melanogaster polymorphic inversions [44][46]. In addition, this mechanism likely generated several inversions fixed in other lineages where TEs were not detected at the breakpoints or when present were not involved in the origin of the inversion [47][50]. SDs represent a significant fraction of mammalian genomes and ectopic recombination between SDs seems to be a common mechanism inducing chromosomal inversions in these genomes. Six of the nine large pericentric inversion differences between the human and chimpanzee genomes have been associated with SDs [51] and there is a significant SD enrichment at the sites of breakpoints which occurred during primate evolution [52][58] although it is not clear whether ectopic recombination is always the cause for the co-location of SDs and breakpoints. Ectopic recombination between SDs is also responsible for the generation of chromosomal inversions in other groups, e.g insects [59].

The transposon Galileo was discovered in D. buzzatii and tentatively classified (along with two related elements named Newton and Kepler) as a Foldback-like element because of its long, internally repetitive, terminal inverted repeats (TIRs) and lack of coding capacity [60], [61]. We have recently shown that Galileo is a cut-and-paste transposon belonging to the P superfamily that is present in six of the 12 recently sequenced Drosophila genomes [62]. Galileo, Newton and Kepler show a high degree of nucleotide similarity (including the most terminal 40 bp that are almost identical) and produce 7-bp target site duplications (TSDs) with the same consensus sequence, GTAGTAC, which suggests that they are mobilized by the same transposase [61]. They should be considered only as different subfamilies of Galileo in the genome of D. buzzatii and will be denoted hereafter as GalileoG, GalileoN and GalileoK, respectively.

In order to increase our understanding of the mechanisms underlying the generation of Drosophila inversions in nature and test for an implication of transposable elements, here we isolated and characterized the breakpoints of another D. buzzatii polymorphic inversion, 2z3. This inversion arose on a chromosome carrying the 2j inversion, giving rise to arrangement 2jz3. The 2z3 segment encompasses about one third of chromosome 2 (~11 Mb) and overlaps the 2j segment so that the two inversions can not be separated by recombination [63] Thus, three chromosome 2 arrangements are commonly found in D. buzzatii natural populations, 2 standard (2st), 2j and 2jz3. Arrangement 2jz3 has a wide geographical distribution being present in natural populations of Argentina, Southern Brazil, Chile and the Old World [64], [65]. In 18 Argentina populations where arrangement 2jz3 is present, its relative frequencies range from 0.5 to 31.5% with an average of ~8% [65]. We choose to study this inversion in part because its proximal breakpoint was located at chromosomal band 2F1c [63] very near the site (2F1c-e) where the proboscipedia-Ultrabithorax portion of the Hox gene complex has been localized [66], [67]. We seek to determine the precise distance from the inversion breakpoint to the Hox genes and find out whether these genes were affected in any way by the inversion. The results show that copies of the transposon GalileoN are located at both inversion 2z3 breakpoints. The arrangement of TSDs and the chimeric nature of both GalileoN copies provide unequivocal evidence that this transposon generated inversion 2z3 by ectopic recombination. The 2z3 proximal breakpoint lies ~24 kb downstream of the proboscipedia gene in a poorly annotated region where two novel genes, Dlh and Mdp, have been discovered.

Results

Physical Mapping of the 2z3 Inversion Breakpoints in the D. buzzatii Genome

Previous cytological observations in D. buzzatii located the distal and proximal breakpoints of inversion 2z3 near chromosome 2 bands 2E4c and 2F1c, respectively [63], [68]. We used the BAC-based physical map of the D. buzzatii genome [69] and the available genome sequence of the related species D. mojavensis [70] to pinpoint the 2z3 distal breakpoint in the intergenic region between CG2046 and CG10326 (see Figure 1 left and Materials and Methods for details). A detailed physical map of the D. buzzatii chromosomal region encompassing the 2z3 proximal breakpoint had been constructed in a previous study [67] and one of the four BAC clones bearing the breakpoint (BAC 40C11) was already fully sequenced and annotated. We mapped the proximal breakpoint within the gene lodestar (lds) that had been tentatively annotated in that region of BAC 40C11 (see Figure 1 right and Materials and Methods). This annotation was put into question by the subsequent annotation of the D. mojavensis genome [70] and a close scrutiny of the region (see below) revealed the presence of two novel genes that we have named Dlh and Mdp. The 2z3 proximal breakpoint falls in the intergenic space between them.

Figure 1
Experimental strategy used for mapping the distal (left) and proximal (right) breakpoints of inversion 2z3.

Breakpoint Sequences in the Non-Inverted Chromosomes

Following previous sequence analyses of inversion breakpoints [37], [44], the distal and proximal breakpoint regions of 2z3 were designated as AB and CD in the non-inverted chromosomes (2st or 2j) and as AC and BD in the inverted chromosome (2jz3). Using primers designed in the D. mojavensis genome, we amplified and sequenced 1,022 bp of the distal breakpoint region (AB) between genes CG2046 and CG10326 in three 2st lines and five 2j lines from diverse geographic origins. In line st-1, the AB sequence comprises 281 bp of gene CG2046, 163 bp of gene CG10326 and the 578-bp intergenic region (Figure 2) including an (AT)23 microsatellite (272 bp away from the start codon of CG2046). No structural variation was found in the AB region between the eight non-inverted lines except for the number of repeats in the microsatellite (between 16 and 24).

Figure 2
Schematic representation of the structures found at the distal and proximal breakpoint of inversion 2z3 in D. buzzatii.

The proximal breakpoint (CD) was localized in the Dlh - Mdp intergenic region (Figure 2). In line st-1, the intergenic region between these two genes is 1,102-bp long and includes two TE fragments: a 296-bp fragment of GalileoN (element Galileo, subfamily Newton), and a 202-bp fragment of BuT5 (an unclassified D. buzzatii transposon [60]). The CD region was amplified by PCR and sequenced in seven non-inverted lines besides st-1. The CD sequence (1,771 bp) includes 238 bp of gene Dlh and 337 bp of gene Mdp. All seven lines contained the BuT5 fragment but only one (j-19) contained the GalileoN fragment.

Levels of nucleotide variation in the 2z3 breakpoint regions were estimated from the AB and CD sequences of the eight lines without the inversion (Figure 3 and Table S1). Overall, 2,422 bp were analyzed comprising 719 bp of coding sequence, 1,501 bp of non-coding sequence (introns and intergenic segments) and 202 bp of the BuT5 insertion. Coding and non-coding sequences were analyzed separately. Both the (AT)16–24 microsatellite and the polymorphic GalileoN insertion were excluded from the analysis. Besides this GalileoN insertion, one small insertion of 4 bp and 9 deletions (ranging in size from 1 to 64 nucleotides) were observed in the set of eight lines. Non-coding sequences contain 33 segregating sites (10 in AB and 23 in CD), coding sequences 12 and the BuT5 insertion six (Figure 3). Nucleotide diversity [71] values in the different regions are given in Table S1 and a neighbour-joining phylogenetic three built with the non-coding sequences of the single-copy breakpoint regions (ABCD) is shown in Figure 4.

Figure 3
Nucleotide polymorphism at the breakpoints of inversion 2z3 in inverted and non-inverted chromosomes.
Figure 4
Neighbour-joining phylogenetic tree of the ABCD breakpoint sequences excluding the (AT)16–24 microsatellite and the TE insertions.

Results can be summarized as follows. First, diversity level does not vary significantly between 2st and 2j chromosomes in AB and CD non coding regions (based on the heterogeneity test [72], χ2L = 1.69, df = 1, 0.5<P<0.1 and χ2L = 1.72, df = 1, 0.5<P<0.1, respectively). In addition, the lines of the two arrangements appear intermingled in the phylogenetic tree (Figure 4). Second, pooling the eight lines, diversity level of polymorphism of the CD non-coding region is more than two times higher than that of AB non-coding region (π = 0.01391 and π = 0.00567, respectively) and the difference is statistically significant (χ2L = 19.23, df = 1, P<0.001). This latter result was corroborated by computer simulations. Finally, the level of polymorphism is lower in coding sequences. Three of the sampled genes contain a total of 6 synonymous polymorphisms and 6 amino acid replacement polymorphisms whereas the fourth (CG10326) does not present any segregating sites (Figure 3). One of the replacement polymorphisms generates a stop codon in exon 2 of Dlh in one of the lines (Figure 3).

Breakpoint Sequences in the Inverted Chromosome

To isolate the AC and BD regions of the inverted arrangement, two 2jz3 lambda genomic libraries were screened with appropriate probes from regions C, D and B (see Materials and Methods). Two positive clones were isolated with probe C. In situ hybridization of these clones to 2j chromosomes produced an intense signal at the proximal breakpoint and weak additional signals in multiple sites. This indicates that these clones bear sequences from region C but do not span the complete distal breakpoint region (AC) and also that they contain repetitive sequences. Clone λz3-96 was selected for subcloning because its insert reached further away in direction to the breakpoint, and subclones containing the fragments located closest to the breakpoint were sequenced (Figure 1). This provided the sequence of region C and also repetitive sequences inserted at the breakpoint junction but not region A. The rest of the AC region was isolated by PCR using two primers, NR located at the end of the λz3-96 clone and AF anchored in gene CG2046 from region A (Figure 1). The resulting PCR product was sequenced (1,072 bp) and assembled together with the insert of clone λz3-96 to complete the sequence of the distal breakpoint AC.

Three positive clones were isolated with probe D. These clones produced an intense signal at the proximal breakpoint when hybridized to 2j chromosomes but also weak additional signals in multiple locations. This indicates that these clones bear sequences from region D but do not span the complete proximal breakpoint region (BD) and also that they contain repetitive sequences. Clone λz3-79 containing the longest insert was subcloned and subclones of interest sequenced confirming that it did not contain sequences from region B (Figure 1). Moreover, this time the remaining part of the sequence could not be amplified by PCR, so we screened the two 2z3 lambda libraries with a probe from region B. Three additional lambda clones were isolated and tested by PCR for the presence of the genes at each side of the breakpoint. Clone λz3-99 was chosen as it contained the genes CG10326 and Mdp, located in regions B and D, respectively. Southern blot analysis revealed that in λz3-99 clone these markers were separated by ~5 kb, therefore it was completely sequenced and the sequence of the proximal breakpoint (BD) was determined.

In total, we sequenced 4,067 bp and 12,520 bp from the distal (AC) and proximal (BD) breakpoint regions in a chromosome with the 2z3 inversion. Comparison of these sequences with the breakpoint regions in non-inverted chromosomes (AB and CD) allowed us to locate the precise site of the breakpoint junctions within the intergenic regions (Figure 2). This comparison also revealed that there are no fixed nucleotide substitutions between inverted and non-inverted chromosomes (Figure 3). In the phylogenetic tree the 2jz3 chromosome does not form a separate lineage and appears to be closest to the j-9 line (Figure 4), with which it shares the premature stop codon in Dlh exon 2 (Figure 3). Relatively large insertions were found at the AC (2,870 bp) and BD (4,786 bp) junctions that were not present in non-inverted chromosomes (Figure 2). These insertions are composed of several TE insertions, most of them similar to elements previously characterized in D. buzzatii [38], [60]. The detailed TE content of the breakpoint insertions is summarized in Table 1.

Table 1
Transposable elements found at the breakpoint regions of inversion 2z3 in D. buzzatii.

The 2,870-bp insertion in the AC junction comprises a copy of GalileoN (GalileoN-4) with two nested insertions: a copy of BuT5 (BuT5-7) flanked by 8-bp TSDs and a 261-bp copy of a LINE-like element (Figure 2). The latter copy has no apparent ORF and no significant sequence homology with described elements. We have classified this insertion as a partial LINE-like element because it shows a 41-bp long polyA tail and two flanking 13-bp TSDs. The 4,786-bp insertion in the BD junction comprises also a copy of GalileoN (GalileoN-5) with two other nested TE insertions (Figure 2): a copy of BuT4 (BuT4-3) flanked by 8-bp TSDs and a copy of BuT3 (BuT3-7) flanked also by 8-bp TSDs. BuT4 was previously classified as a Class II element of the hAT superfamily [60]. This is corroborated by the 87% nucleotide identity observed between this copy of BuT4 and Homo7, a hAT element recently described in D. mojavensis [73]. This copy of BuT4 includes a 1774-bp segment with a 87.7% identity to Homo7 transposase-encoding ORF.

The two GalileoN copies inserted at the breakpoint junctions (GalileoN-4 and GalileoN-5) have relatively long TIRs (Table 1) and are very similar to copies of the subfamily Newton previously described in D. buzzatii [60]. Upon insertion, Galileo generates 7-bp TSDs with the consensus sequence GTAGTAC [61], [62]. The 7-bp sequence flanking GalileoN-4 in region C (GTAGTAC) is the reverse and complementary version of the 7-bp sequence flanking GalileoN-5 in region D (GTACTAC). Likewise, the 7-bp sequence flanking GalileoN-4 in region A (GTACTAT) is the inverted and complementary version of that flanking GalileoN-5 in region B (ATAGTAC). Only one single copy of the 7-bp sequence GTACTAT is present at the distal breakpoint (AB) and one copy of the target sequence GTACTAC is found at the proximal breakpoint (CD) in the non-inverted chromosomes. This pattern of exchanged TSDs is consistent with ectopic recombination as the mechanism that generated the 2z3 inversion (see Discussion).

Two Novel Drosophila Genes

The proximal breakpoint of inversion 2z3 was located within BAC 40C11 in the genomic region between genes CG14899 and CG14290. This D. buzzatii chromosome 2 region had been tentatively annotated as containing a single five-exon gene orthologous to D. melanogaster lds [67]. However, only three of the five exons of the D. buzzatii gene model showed significant homology with Dmel\lds. We failed to corroborate the structure of the putative D. buzzatii lds gene by RT-PCR using primers anchored in exons 1 and 5. In addition, the sequencing and annotation of 12 Drosophila genomes [70] revealed that in D. mojavensis, the closest species to D. buzzatii, the lds ortholog is located in a distant chromosome 2 region casting doubts on the D. buzzatii annotation. These observations prompted a detailed comparative analysis of the 7.5-kb D. buzzatii region between genes CG14899 and CG14290 with the homologous regions in D. mojavensis and D. virilis and a search for RNA expression by RT-PCR (see Materials and Methods).

The results lead us to discard the lds annotation and discover two novel Drosophila genes, whose main characteristics are described in Table S2. In D. buzzatii, the gene that we have named MADF domain protein (Mdp) is composed of three exons and two introns with a total length of 794 bp (Table S2). The coding sequence is 651-bp long and encodes a 216-aa protein with a MADF domain (Figure S2). Mdp has been found also in D. mojavensis and D. virilis with a similar structure, although a somewhat longer coding sequence in D. virilis and a stop codon in position 142 of the third exon in D. mojavensis. As expected from the phylogenetic relationships, nucleotide identity and amino acid identity were higher with D. mojavensis (82.5% and 76.3%, respectively) than with D. virilis (70.4% and 60.9%, respectively). The overall codon-based Z-test of purifying selection shows highly significant results (Z = −10.15, P<10−6) and the ratio of synonymous to non-synonymous substitutions (Ka/Ks  = 0.22) shows a moderate degree of functional constraint. The second gene has been named DEAD-like helicase (Dlh) and in D. buzzatii it comprises four exons and three introns with a total length of 2,826 bp. The coding sequence is 1,554-bp long and encodes a 517-aa protein with a SNF2-related or DEAD-like helicase N-terminal domain and a DNA/RNA helicase C-terminal domain (Figure S3). This gene is also present in D. mojavensis with a similar structure, but could not be found in D. virilis (Table S2). Nucleotide identity of the coding sequence (76.8%) and amino acid identity of the protein (64.5%) support orthology. The estimated ratio Ka/Ks was relatively high (0.48), but significantly lower than 1 (Z = −5.56, P = 2×10−7) suggesting that this is a relatively fast evolving gene.

Discussion

Inversion 2z3 Was Generated by Ectopic Recombination between Galileo Copies

Many studies have shown the potential of TEs to induce chromosomal rearrangements in experimental Drosophila populations implicating retrotransposons (e.g. BEL, roo, Doc, and I) as well as transposons (e.g. P, hobo, and FB) [74]. In contrast, the evidence for the involvement of TEs in the generation of natural Drosophila inversions, i.e. those effectively contributing to adaptation and/or evolution of natural populations, is scarce (see Introduction). We have previously found that the cut-and-paste transposon Galileo was involved in the generation of two polymorphic inversions of D. buzzatii, 2j and 2q7 [37], [38]. Here we have isolated and sequenced the breakpoints of another polymorphic inversion of D. buzzatii, 2z3. Our results provide the most compelling evidence for the participation of Galileo in the generation of Drosophila inversions and for ectopic recombination as the responsible mechanism.

Several TE insertions were found at the breakpoint regions in the chromosome with the 2z3 inversion that were not present in non-inverted chromosomes (Table 1). Remarkably, only GalileoN was present at the two breakpoint junctions. This fact and the evidence presented below indicate that GalileoN is the element responsible for the generation of the 2z3 inversion. Two other TE insertions, BuT5 and LINE-like, were found nested within the GalileoN copy in the distal breakpoint and another two, BuT3 and BuT4, within the GalileoN copy in the proximal breakpoint. These four TE insertions are present at a single breakpoint junction only and each of them is flanked by identical direct TSDs. Thus, they are unlikely to be responsible for the generation of the inversion and are best interpreted as secondary colonizers of the breakpoint regions (see below). Another two TE fragments (BuT5 and GalileoN) are present in the proximal breakpoint region (but not in the junction) of non-inverted chromosomes and thus can not be involved in the generation of the inversion either.

Two processes can explain the induction of chromosomal inversions by TEs: ectopic recombination [12], [74] and aberrant transposition [13][17]. Ectopic recombination requires the presence in the parental chromosome of two homologous TE copies inserted in opposite orientation at different sites. After the inversion is generated, two chimeric TE copies are expected to be found flanking the inverted segment with their TSDs exchanged. On the other hand, two transposon copies may participate in an aberrant transposition event, by which a hybrid element formed by the 5′ end of one copy and the 3′ end of the other copy transposes to a new chromosomal site. The outcome of this process is an inversion flanked by two transposon copies in opposite orientation accompanied by deletions or duplications when the original copies were inserted at separate chromosomal sites (Figure S1). The lack of any deletions or duplications and the pattern of TSDs in the 2z3 breakpoints allow us to reject this latter possibility. However, we must consider the possibility of an aberrant transposition with the two original transposon copies located at the same chromosomal site (hybrid insertion model). The outcome in this case (Figure S1 A) is strikingly similar to that of ectopic recombination except for the fact that the two TE copies flanking the inversion are identical under the hybrid element insertion model but chimeric under the ectopic recombination [38].

The two GalileoN copies found in the 2z3 breakpoints (named GalileoN-4 and GalileoN-5) have similar sizes and structures, with relatively long TIRs and a middle segment oriented in opposite direction in the two copies, and show a high similarity with two other copies previously described (GalileoN-1 and GalileoN-2) [60], [61]. Each of the latter two copies was flanked by perfect 7-bp TSDs generated upon insertion. By contrast, the 7-bp duplications flanking the GalileoN copies at the 2z3 breakpoints are exchanged (Figure 2 and Results). In the non-inverted chromosomes, only one copy of the corresponding 7-bp target sequence is detected at each breakpoint (Figure 2). These observations are consistent with the presence of two GalileoN insertions in the parental chromosome and the generation of the 2z3 inversion by ectopic recombination between them, but does not rule out the hybrid element insertion model (see above). Further evidence was revealed by comparing the nucleotide sequence of the TIRs within and between GalileoN copies. GalileoN-1 and GalileoN-2 possess TIRs >99% identical within each copy but ~7% divergent between copies (Table 2). In contrast, GalileoN-4 and GalileoN-5 show TIRs that are ~6% divergent within each copy but >99% identical between copies (Table 2). These results suggest that both GalileoN-4 and GalileoN-5 are chimeric. A closer scrutiny of the four GalileoN copies revealed a striking pattern and led to the same conclusion (Figure 5). In 33 variable sites, from position 1 through 824, the nucleotide present in GalileoN-4 is identical to that in GalileoN-1 and the nucleotide present in GalileoN-5 is identical to that in GalileoN-2 (Figure 5 top). The situation is completely reversed for 20 variable sites from position 966 to the end of the element where the nucleotide present in GalileoN-4 is identical to that in GalileoN-2 while that in GalileoN-5 is identical to that in GalileoN-1 (Figure 5 top). Phylogenetic analyses of the four sequences carried out separately for the two portions of the element (Figure 5 bottom) and the maximum chi-square method (χ2 = 53.00, df = 1, P<1×10−7) [75], [76] corroborated the chimeric structure of GalileoN-4 and GalileoN-5. These observations provide strong support for the ectopic recombination model and suggest that the recombination event that gave rise to the 2z3 inversion took place within 141-bp of the middle segment between positions 825 and 965 of GalileoN (Figure 5). The absence of GalileoN insertions in the analyzed non-inverted chromosomes should be no surprise because insertions of actively transposing families are expected to be present at low population frequencies under transposition-selection balance [77], [78] and we sampled just a few non-inverted chromosomes.

Figure 5
Chimeric structure of the two GalileoN copies (GalileoN-4 and GalileoN-5) observed at the breakpoints of inversion 2z3.
Table 2
Nucleotide divergence between the TIRs of four GalileoN copies.

We can conclude that the three polymorphic inversions of D. buzzatii studied so far, 2j, 2q7 and 2z3, have been generated by the same TE family, Galileo, and very likely by the same molecular mechanism, ectopic recombination. In all three cases, after the generation of the inversion, many TE copies have accumulated at the breakpoint regions, which became hotspots for secondary TE insertions (Table 3). This accumulation is probably a consequence of the reduction of recombination in these regions [79], [80] that protects TE copies from being eliminated by deleterious ectopic exchanges [77], [78]. It is intriguing though that the 40 TE copies associated with inversion breakpoints in D. buzzatii belong to a limited set of nine TE families (Table 3). All of them but one (the LINE-like element in the distal breakpoint of inversion 2z3) are Class II elements: ISBu elements are Helitrons [81] and the remaining elements are cut-and-paste transposons [82]. This enrichment of breakpoint regions in specific TE families may be due (1) to the fact that these TE families were among the most transpositionally active elements in the D. buzzatii genome when the opportunity window for insertion was open, and/or (2) to insertional preference [83].

Table 3
Number of TE copies found in the breakpoint regions of three D. buzzatii polymorphic inversions.

Because many different TE families are able to induce chromosomal rearrangements in Drosophila [74], the question arises as to why the three polymorphic D. buzzatii inversions should be generated by the same TE family, namely Galileo. The frequency of ectopic recombination should increase with copy number and length, and this prediction is borne out by the data ([78], D. Petrov, personal communication). In the D. melanogaster genome, at least 121 TE families are present [84], [85]. A total of 996 copies from 81 families were annotated in the euchromatin of the sequenced genome (excluding the proximal 2 Mb where TEs regularly accumulate) and copy number per family varied between 1 and 124 with an average of 12.3 [84]. Although no detailed inventory of the TE families in the D. buzzatii genome is yet available, there is no ground for assuming a smaller number of families than in D. melanogaster. Galileo copy number per genome was estimated as 11.7 in the euchromatic distal-central region of chromosomes (i.e. excluding the dot and pericentromeric regions) [61]. The analogous figure for BuT5 is 11.4 copies per genome and lower values were estimated for another five D. buzzatii transposons [83]. In summary, Galileo copy number does not seem particularly high in the D. buzzatii genome, although more data is needed. Length of Galileo copies is not unusual either. The canonical copy is ~5.4 kb long [62] but most copies are non-autonomous and much shorter. Average length (± SD) of a combined sample of 23 non-autonomous copies of GalileoG, GalileoN and GalileoK is 953 bp (±640 bp) [61]. In D. melanogaster, the average length of the TE copies annotated by [84] was 2.9 kb.

Two characteristics of Galileo can explain its primary role in the generation of rearrangements by ectopic recombination: (1) its transpositional activity; and (2) its unusual structure. Galileo belongs to the P superfamily of TIR transposons and is likely to transpose by a cut-and-paste mechanism similar to that of the D. melanogaster P element [86], [87]. This transposition mechanism involves the binding of the transposase to the element TIRs and the excision of the element generating a DSB at the donor site followed by the integration of the element into a different chromosomal site. Hence DSBs produced during normal or aberrant transposition events may provide the initial step for ectopic recombination events. The accumulation of Galileo copies after the generation of inversions 2j and 2q7 (Table 3) indicates that Galileo is (or has been recently) active in the genome of D. buzzatii. Nevertheless, unless Galileo has an unusually high transposition rate, this explanation is insufficient because Galileo is not the only TE family transpositionally active in the D. buzzatii genome (at least another eight TE families must be active; Table 3).

The participation of Galileo in the generation of inversions may be also related to its unusual structure with up to 1.2-kb long TIRs [61], [62]. The two GalileoN copies involved in the generation of the 2z3 inversion have ~575 bp long TIRs separated by a ~350 bp long middle segment (Table 1). This kind of spaced inverted repeat sequences is well known to form stem-loop structures in single-stranded DNA or cruciform structures in double-stranded DNA and induce DSBs and rearrangements in a wide variety of organisms [88]-[93]. Generation of DSBs by these secondary structures may be due to the fact that they are substrates for nuclease cleavage or because they interrupt replication fork progression [94], [95]. In D. melanogaster, Foldback (FB) elements, which also present very long TIRs and induce secondary structures, are known to cause rearrangements at a high rate in the laboratory [96], [97]. We propose that the long TIRs of Galileo induce the formation of secondary structures and DSBs at high rate and this contributes to its unique capacity to generate chromosomal inversions. The fact that the recombination event that generated inversion 2z3 took place in the middle segment of GalileoN seems consistent with nuclease cleavage at the loop.

Functional Consequences of the 2z3 Inversion

Inversion 2z3 seems to have a recent origin as no fixed nucleotide substitution was observed in the breakpoint regions between non-inverted and inverted chromosomes (Figure 3). This is in clear contrast with the ~1 Myr and ~0.5 Myr old inversions 2j and 2q7 where 17 and 14 fixed nucleotide substitutions were observed, respectively [38], [60]. The monomorphism of the α-esterase5 gene in 2jz3 chromosomes is also consistent with a recent origin of inversion 2z3 [98]. In spite of being a very young inversion, 2z3 exhibits a widespread distribution in natural populations (see Introduction), suggesting that it must have a considerable selective value. In Argentina, the frequency of 2jz3 is significantly correlated with latitude, a putatively selective pattern [65]. Furthermore, selection component analyses and biometrical studies they all have detected significant effects of 2jz3 chromosomes [99][102]. One possible explanation for its adaptive advantage is provided by the position effect hypothesis, which proposes that the localization of the inversion breakpoints near or inside genes could affect their function or expression profile by disrupting their coding regions or causing changes in the promoter and regulatory regions [103], [104]. Another factor that could affect the expression of genes adjacent to the breakpoints is the presence of TEs in these regions as they have been shown to alter gene expression in different ways [103], [105], [106].

The 2z3 proximal breakpoint lies in a region previously sequenced where a gene named lodestar (lds) had been tentatively annotated [67]. A comparative analysis with other Drosophila genomes and expression experiments by RT-PCR discarded the lds annotation and has unveiled two novel genes flanking the inversion breakpoint, Dlh in region C and Mdp in region D. Three observations suggest that these two genes are fully functional. (i) In D. buzzatii, both genes are expressed throughout the whole life cycle, although they present slightly different expression patterns (results not shown). (ii) Their overall structure and encoded protein sequence are conserved in at least another Drosophila species (Table S2). (iii) Both genes are evolving under purifying selection with Ka/Ks ratios significantly different from 1 (strict neutrality). The relatively short intergenic region (796 bp) and the close proximity of the proximal breakpoint to the initiation codon of Dlh (118 bp) suggest that the inversion might be affecting the expression of Dlh and/or Mdp, a question that deserves further work.

In D. buzzatii, the Hox gene complex is split in three portions: proboscipedia (pb)-Ultrabithorax (Ubx), abdominalA (abdA)-AbdominalB (AbdB) and labial (lab) [66], [67]. We analyzed the breakpoints of inversion 2z3 in part because of the cytological vicinity of the 2z3 proximal breakpoint to the pb-Ubx portion of the Hox gene complex [63], [66], [67]. Our results show that the 2z3 proximal breakpoint lies outside of the Hox gene complex ~23.7-kb downstream of pb. The segment that separates the 2z3 proximal breakpoint from pb contains three genes, CG17836, CG14290 and Dlh. It seems unlikely that the 2z3 proximal breakpoint altered the regulatory sequences or the expression pattern of pb because the lab-pb split that took place much nearer the 3′ end of pb in the ancestor of the repleta group did not [67]. Nevertheless, the pb-Ubx portion of the Hox gene complex is located within the inverted segment and thus the 2z3 inversion relocates these genes to a much more distal region within chromosome 2. Whether this change in the chromatin environment has had any effect on the expression of Hox genes remains an open question.

Materials and Methods

Drosophila Stocks

Nine lines of D. buzzatii homokaryotypic for one of three different chromosome 2 arrangements (2st, 2j and 2jz3) were used. These lines were isolated from natural populations with different geographical origin: st-1, Carboneras (Spain); st-12, Trinkey (Australia); st-13, Mazán (Argentina); j-2, Carboneras (Spain); j-8, San Luis (Argentina); j-9, Quilmes (Argentina); j-13, Guaritas (Brazil); j-19, Ticucho (Argentina); and jz3-2, Carboneras (Spain). The stock of D. mojavensis (15081–1352.22, UC San Diego Drosophila Species Stock Center) comes from Santa Catalina Island (California) and is the stock used to sequence the D. mojavensis genome [70].

Probes and In Situ Hybridization

DNA from BAC and plasmid clones was extracted by alkaline lysis following standard protocols and used as probes for in situ hybridization. All remaining probes were produced by polymerase chain reaction (PCR) amplification of D. buzzatii or D. mojavensis genomic DNA with different primer pairs. Probes were labelled with biotin-16-dUTP (Roche) by random priming and hybridization to the larval salivary gland polytene chromosomes was carried out according to the procedure described [107]. Intraspecific in situ hybridizations with D. buzzatii lines and probes were carried out at 37°C while interspecific hybridizations of D. mojavensis probes to D. buzzatii polytene chromosomes were carried out at 25°C. Hybridization results were recorded as digital images captured with phase contrast Nikon Optiphot-2 microscope at 600× magnification and a Nikon Coolpix 4500 camera. Cytological localization of the hybridization signal was determined using the cytological maps of D. buzzatii [63], [69].

Physical Mapping of the Inversion Breakpoints

We searched the BAC-based physical map of the D. buzzatii genome [69] for clones located near the cytological breakpoints and selected eight clones from contig 961 mapping near the distal breakpoint, and seven clones from contig 968 mapping near the proximal breakpoint (Table S3). The fifteen BAC clones were hybridized to the salivary gland chromosomes of one line with the inversion (jz3-2) and one line without the inversion (j-9) to identify those clones containing a breakpoint (that should produce two hybridization signals in the first case and a single hybridization signal in the second). Three BAC clones from contig 961 (18L15, 15P22 and 15L20) were found to include the distal breakpoint (Figure S4A), and four clones from contig 968 (22N23, 22M06, 16A20, and 40C11) were found to contain the proximal breakpoint (Figure S4E).

Both ends of each BAC clone bearing the distal breakpoint were sequenced and the sequences mapped onto the genome sequence of D. mojavensis using BLASTN (Figure 1 left). The distal breakpoint was located in the overlapping region between the three D. buzzatii BAC clones, a segment ~50-kb long of D. mojavensis scaffold_6540 that corresponds to chromosome 2 [108]. To narrow down the position of the breakpoint we chose four genes within this segment (CG1193, CG14906, Adk3 and CG4674) and used them as probes for in situ hybridization to 2jz3 chromosomes (Table S4). The CG1193 probe (marker 1 in Figure 1 left) mapped at the distal breakpoint, outside the inversion, while the other three probes (markers 2, 3 and 4 in Figure 1) hybridized at the proximal breakpoint, indicating that they are located inside the inverted segment. As a result, we located the distal breakpoint in the 13-kb segment between genes CG1193 and CG14906 (markers 1 and 2 in Figure 1). Seven genes had been annotated in this segment of D. mojavensis chromosome 2 and we designed primers to amplify the intergenic region between each pair of genes in this species, as well as in D. buzzatii strains with and without inversion 2z3. Our rationale was that the intergenic region containing the distal breakpoint would amplify in D. mojavensis and in the line with the non-inverted chromosome, but not in the line carrying the inversion. In fact, all the intergenic segments were amplified in the three lines, except that between CG2046 and CG10326 (segment 9 in Figure 1 left) which failed to amplify in the line carrying the inversion. To corroborate this observation, PCR products amplified using the primers 8F-8R, 9F-9R and 10F-10R were used as in situ hybridization probes to chromosomes with the inversion, and they produced the expected results (Figure S4B, C and D). Therefore, the distal breakpoint of inversion 2z3 was located in the ~600-bp region between genes CG2046 and CG10326 of D. mojavensis.

One of the four BAC clones bearing the 2z3 proximal breakpoint (BAC 40C11) was already fully sequenced and annotated and a physical map of the region was built using sequence tagged sites (STSs) [67]. This map allowed us to locate the proximal breakpoint in the ~70-kb region of overlap between the four clones (Figure 1 right). Three STS markers generated in this region were amplified and hybridized to 2jz3 chromosomes, in order to further delimit the region which contains the proximal breakpoint (Figure 2 right). One marker (number 13 in Figure 1 right) hybridized to the distal breakpoint and therefore was located inside the inversion, whereas the other two (markers 14 and 15 in Figure 2 right) mapped on the region of the proximal breakpoint, indicating that they are located outside the inverted segment. As a result, the proximal breakpoint could be narrowed down to a 16-kb segment between genes CG17836 and CG2520 (markers 13 and 14 in Figure 1 right). Ten plasmid subclones from BAC 40C11 which cover this segment were also used for hybridization to inverted chromosomes (Figure S4F, G and H and Table S4), allowing us to locate the proximal breakpoint more precisely in the ~0.8-kb intergenic region between genes Dlh and Mdp (Figure 1 right).

Southern Blot and Screening of Genomic Libraries

Southern hybridization and library screenings were carried out by standard methods [109]. Three different probes amplified from D. buzzatii DNA: DF-DR (800 bp), CF-CR (337 bp) and BF-BR (505 bp) were used (Table S5). Probes were labelled by random priming with digoxigenin-11-dUTP under the conditions specified by the supplier (Roche). Hybridization was carried out overnight at 42°C in a standard hybridization solution (Roche). Stringency washes were performed with 0.5x SSC 0.1% SDS solution at 65°C. Two lambda genomic libraries were screened. One library was constructed with DNA derived from D. buzzatii line jz3-2 using the LambdaGEM-11 vector following manufacturer's instructions (Promega). The second lambda library was derived previously from D. buzzatii line jz3-4 [60] and was amplified using standard methods [109]. Two positive clones (λz3-91 and λz3-96) were recovered from the first library with probe CF-CR and six positive clones were recovered from the second library, three with probe DF-DR (λz3-77, λz3-79 and λz3-98) and three with probe BF-BR (λz3-99, λz3-102 and λz3-104). The span of each clone was determined through a combination of PCR, restriction mapping and Southern blotting. DNA fragments of interest from positive phages were subcloned into pBluescript II SK vector (Stratagene).

PCR Amplification

Polymerase chain reaction was carried out in a volume of 25 µl, including 50–100 ng of genomic DNA, 10 pmol of each primer, 100 µM dNTPs, 1x buffer and 1–1.5 units of Taq DNA polymerase. Temperature cycling conditions were 30 rounds of 30 s at 94°C; 30 s at the annealing temperature, and 30–60 s at 72°C, with annealing temperatures varying from 55 to 60°C depending on the primer pair. Sequences of oligonucleotide primers are given in Table S5.

RNA Extraction and RT-PCR Amplification

Total RNA was isolated from embryos, larvae, pupae, and adults of the D. buzzatii st-1 line using TRIzol (Invitrogen). Total RNA was treated with 1 unit of DNase I (Ambion) for 30 min at 37°C to eliminate DNA contamination. cDNA was synthesized from 1 µg of DNase I-treated RNA by using an oligo(dT) primer (Transcriptor First Strand cDNA Synthesis kit for RT-PCR, Roche). PCR reactions were performed as describe above. To differentiate the size of amplification products, both cDNA and st-1 genomic DNA were used as templates. RT-PCR products were sequenced and their sequences compared with those of genomic DNA to determine exon-intron boundaries (Figures S2 and S3).

DNA Sequencing and Sequence Analysis

Sequencing was performed in the Servei de Genòmica of the Universitat Autònoma de Barcelona, Macrogen Inc. (Seoul, Korea) and GATC Biotech (Konstanz, Germany). Fragments cloned into pBluescript II SK were sequenced with the M13 universal and reverse primers. PCR products were gel purified using QIAquick Gel Extraction Kit (Qiagen), and sequenced directly with the same primers used for amplification.

Sequences from different lines were aligned with MUSCLE 3.2 [110] and similarity searches in the GenBank/EMBL, Assembly/Alignment/Annotation of 12 related Drosophila species (http://rana.lbl.gov/drososphila/) and FlyBase databases were carried out using BLASTN [111]. Nucleotide variability was estimated by means of the number of segregating sites (S), and the nucleotide diversity (π, average number of pairwise differences per site) using DnaSP (version 4.50.3) software [112]. This software was also used to test for differences in nucleotide variability by means of computer simulations based on the coalescent process. Simulations were carried out given the number of segregating sites and analysing the nucleotide diversity (π) on the genealogy, fixing the options of no recombination to AB region and free recombination to CD region, because AB region mapped inside the 2j inversion. Interspecific nucleotide and amino acid similarities were estimated with MEGA 4 [113]. The ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) were estimated using Nei-Gojobori method and Jukes-Cantor distance. The null hypothesis that Ka/Ks  = 1 was tested by means of the Z-test of selection. Phylogenetic analyses were also conducted using MEGA 4.

Sequence data from this article have been deposited in the GenBank/EMBL Database Libraries under accession nos. GU132438-GU132454.

Supporting Information

Figure S1

Chromosomal inversions may be generated by transposons when two ends that are not part of the same transposon participate in an aberrant transposition event to a new site [13][17]. Target site duplications (TSD) are indicated by ○ or □ (cooperating TE copies) and Δ (new insertion site). (A) The two TE copies are located at the same site of sister chromatids or homologous chromosomes and share the same TSD (○). The result of the aberrant transposition is an inversion (segment BC) flanked by two TE copies. (B) The two TE copies are inserted at separate sites in the two homologous chromosomes and each has its own TSD (indicated by ○ and □). The aberrant transposition event produces an inversion (segment BC) and a deletion (segment D). (C) The two TE copies are arranged as in (B) but two different element ends are involved. The resulting chromosome carries an inversion (segment BC) and a duplication (segment D). (D) The two TE copies are inserted at separate sites on the same chromatid and each has its own TSD (indicated by ○ and □). The resulting chromosome has an inversion (segment BC) and a deletion (segment D).

(0.02 MB PDF)

Figure S2

Alignment of gene Mdp sequences in three Drosophila species. The aligned sequences are: positions 50294–51354 from D. buzzatii BAC clone 40C11 (accession number AY900632), positions 6137692–6136590 from D. mojavensis scaffold_6540 and positions 5807143–5806092 from D. virilis scaffold_12855. Yellow boxes indicate exons with the initial methionine and the final stop codon colored in orange and red, respectively. The premature stop codon found in the D. mojavensis sequence is also shown as a red box. Note that there are some parts of the sequence upstream of the coding region that are conserved in the different species suggesting that they may be part of the 5′ UTR or the regulatory regions of the gene. A putative polyA signal determined only on the basis of sequence conservation in the different species is included in a purple rectangle. The blue bar below the alignment indicates the 763-bp fragment amplified by RT-PCR and sequenced in D. buzzatii with primer pair DF-DR. The protein sequence encoded by the D. buzzatii gene is shown above the alignment. The residues enclosed in a green box correspond to the MADF domain found using InterProScan (http://www.ebi.ac.uk/Tools/InterProScan/).

(0.01 MB PDF)

Figure S3

Alignment of gene Dlh sequences in two Drosophila species. The aligned sequences are: positions 52175–55219 from D. buzzatii BAC clone 40C11 (accession number AY900632) and positions 6136143–6133352 from D. mojavensis scaffold_6540. This gene could not be found in the D. virilis genome sequence. Yellow boxes indicate exons with the initial methionine and the final stop codon colored in orange and red, respectively. Enclosed in a purple rectangle is the codon in the second exon of the gene that becomes a polymorphic premature stop codon in lines j-9 and jz3-1 by changing from TCA to TAA. No further upstream non-coding sequence could be included in the alignment because of the presence of a polymorphic GalileoN insertion in the st-1 line, from which the D. buzzatii BAC clone is derived. Bars below the alignment in different shades of blue indicate the three overlapping fragments amplified by RT-PCR and sequenced in D. buzzatii with primer pairs CF-CR (278 bp), CF-RT1R (609 bp) and RT2F-RT2R (1,011 bp). The protein sequence encoded by the D. buzzatii gene is shown above the alignment. The residues enclosed in a dark green box correspond to a SNF2-related or a DEAD-like helicase N-terminal domain and the aminoacids in a light green box correspond to a DNA/RNA helicase C-terminal domain. The protein domains have been analyzed using InterProScan ((http://www.ebi.ac.uk/Tools/InterProScan/).

(0.02 MB PDF)

Figure S4

In situ hybridization to D. buzzatii chromosomes carrying inversion 2z3 of BAC clones, plasmid clones and PCR probes coming from the distal breakpoint (A-D) and the proximal breakpoint (E-H). A: BAC clone 18L15; B: PCR fragment 10F-10R; C: PCR fragment 9F-9R; D: PCR fragment 8F-8R. E: BAC clone 40C11; F: plasmid clone 9F01; G: plasmid clone 8H04; H: plasmid clone 8D03. Arrows indicate hybridization signals.

(4.84 MB TIF)

Table S1

Nucleotide variability in non-inverted chromosomes. N  = number of chromosomal lines; m = number of compared nucleotides.

(0.05 MB PDF)

Table S2

Structure and similarities of two novel Drosophila genes: MADF domain protein (Mdp) and DEAD-like helicase (Dlh). NT  = nucleotide; AA  = amino acid.

(0.01 MB PDF)

Table S3

BAC clones used for in situ hybridization.

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Table S4

Plasmid clones used as probes for in situ hybridization to map the proximal breakpoint of the 2z3 inversion.

(0.01 MB PDF)

Table S5

Sequence of oligonucleotide primers used for PCR amplification.

(0.05 MB PDF)

Acknowledgments

We are grateful to A. Barbadilla and J. Rozas for help with statistical analysis of the nucleotide variation, and M. Cáceres for help with the experimental design.

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: This work was supported by grant BFU2005-02237 from the Ministerio de Educación y Ciencia (MEC, Spain) and grant BFU2008-04988 from the Ministerio de Ciencia e Innovación (MICINN, Spain) awarded to A.R. and by a post-doctoral fellowship from the Fundación Carolina (Spain) awarded to A.D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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