The Foldback-like Transposon Galileo Is Involved in the Generation of Two Different Natural Chromosomal Inversions of Drosophila buzzatii

Ferran Casals, Mario Cáceres1, and Alfredo Ruiz

Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra (Barcelona), Spain


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Chromosomal inversions are the most common type of genome rearrangement in the genus Drosophila. Although the potential of transposable elements (TEs) for generating inversions has been repeatedly demonstrated in the laboratory, little is known on their role in the generation of natural inversions, which are those effectively contributing to the adaptation and/or evolution of species. We have cloned and sequenced the two breakpoints of the polymorphic inversion 2q7 of D. buzzatii. The sequence analysis of the breakpoint regions revealed the presence in the inverted chromosomes of large insertions, formed by complex assemblies of transposons, that are absent from the chromosomes without the inversion. Among the transposons inserted, the Foldback-like element Galileo, that was previously found responsible of the generation of the widespread inversion 2j of D. buzzatii, is present at both 2q7 breakpoints and is the most likely inducer of the inversion. A detailed study of the nucleotide and structural variation in the breakpoint regions of six chromosomal lines with the 2q7 inversion detected no nucleotide differences between them, which suggests a monophyletic and recent origin. In contrast, a remarkable degree of structural variation was observed in the same six chromosomal lines. It thus appears that the two breakpoints of the inverted chromosomes have become genetically unstable hotspots, as was previously found for the 2j inversion breakpoints. The possibility that this instability is caused by structural properties of Foldback elements is discussed.

Key Words: genome evolution • chromosomal inversions • inversion breakpoints • transposable elements • hotspots • Drosophila buzzatii


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Since the discovery of transposable elements (TEs) in maize, their evolutionary potential for rearranging genomes has been repeatedly emphasized (Finnegan 1989; McDonald 1993). In the laboratory, both class I elements (RNA transposable elements) and class II elements (DNA transposable elements or transposons) have been shown to mediate the generation of deletions, duplications, inversions, and reciprocal translocations (Berg and Howe 1989), and several molecular mechanisms may be involved. The simplest manner in which TEs can induce rearrangements is by acting as substrates for ectopic recombination between homologous TE copies located in different sites of the genome (Petes and Hill 1988; Lim and Simmons 1994). However, several other more complex models, most of them involving aberrant transposition events of transposons, have been put forth and supported empirically (Gray 2000). In recent years a growing body of evidence has shown that TEs are implicated in the origin of natural chromosomal rearrangements in a wide range of organisms, including bacteria (Daveran-Mingot et al. 1998), yeasts (Kim et al. 1998), flies (Cáceres et al. 1999), and hominids (Schwartz et al. 1998).

Perhaps the most extraordinary and intensely studied case of chromosomal variation in eukaryotes is found in the Drosophila genus (Sperlich and Pfriem 1986; Krimbas and Powell 1992). More than half of the Drosophila species are polymorphic for paracentric inversions (Powell 1997), and interspecific comparisons of physical maps reveal an extreme rate of chromosomal rearrangement, with nearly four inversions fixed per lineage and Myr (Segarra et al. 1995; Ranz, Casals, and Ruiz 2001; González, Ranz, and Ruiz 2002). Studies in Drosophila were among the first to show the capacity of TEs to induce chromosomal rearrangements in the laboratory. The implicated TEs include the class I elements BEL, roo, Doc, and I, and the class II elements P, hobo, and FB (Lim and Simmons 1994). In contrast, relatively little is yet known about the origin of natural inversions—i.e., those effectively contributing to adaptation and/or evolution of Drosophila species—and their consequences at the molecular level.

Circumstantial evidence for the implication of TEs in the origin of Drosophila natural inversions was first gathered by in situ hybridization with probes of different TEs in D. melanogaster (Lyttle and Haymer 1992), D. willistoni (Regner et al. 1996), and the virilis species group (Zelentsova et al. 1999; Evgen'ev et al. 2000). However, the molecular characterization of inversion breakpoints yielded diverse or contradictory results. Initially, no traces of TEs were found in the breakpoints of the first two natural inversions characterized at the molecular level: the polymorphic inversion In(3L)Payne of D. melanogaster (Wesley and Eanes 1994) and an inversion fixed between D. melanogaster and D. suboscura (Cirera et al. 1995). Later, a new repetitive sequence (designated Odysseus) was detected at the distal breakpoint junction of inversion 2Rd' in the mosquito Anopheles arabiensis (Mathiopoulos et al. 1998), and a LINE-like retrotransposon was found near one of the breakpoints of inversion In(2L)t of D. melanogaster (Andolfatto, Wall, and Kreitman 1999). Finally, the clearest evidence to date of the role of TEs in the generation of Drosophila inversions was provided by the D. buzzatii widespread 2j inversion (Cáceres et al. 1999). In this case, both breakpoints contained large insertions made up of the Foldback-like element Galileo plus several other transposons, which have integrated within or near the original Galileo elements (Cáceres, Puig, and Ruiz 2001). The arrangement of target site duplications flanking the Galileo copies at each breakpoint strongly suggested that the inversion was generated by ectopic recombination between them (Cáceres et al. 1999). Furthermore, the low nucleotide variability in the breakpoint regions and the clustering of all 2j chromosomes in a single clade were consistent with the uniqueness of the recombination event and a monofiletic origin of the 2j inversion (Cáceres et al. 1999; Cáceres, Puig, and Ruiz 2001).

To test how common are these observations and provide further information on the origin of Drosophila natural inversions, we have cloned and sequenced the two breakpoints of another polymorphic inversion of D. buzzatii, 2q7. The isolation of the breakpoints of this inversion was made possible by the availability of several D. melanogaster markers that mapped by in situ hybridization close to its distal breakpoint (Ranz, Casals, and Ruiz 2001). The 2q7 inversion differs from the 2j inversion by three characteristics: (1) The 2j inversion is located in a middle position of chromosome 2. Inversion 2q7 overlaps with inversion 2j and is located more proximally, with one breakpoint near the centromere (Ruiz and Wasserman 1993);(2) inversion 2q7 arose on a 2j chromosome, giving rise to the 2jq7 arrangement, and is more recent than inversion 2j;(3) inversion 2j has a widespread distribution and is present at high frequencies in most D. buzzatii populations, whereas the 2jq7 arrangement has a much restricted geographical distribution and is usually present at rather low frequencies (<10%) (Hasson et al. 1995). Here we show that, despite these differences, the molecular structure of the breakpoint regions is strikingly similar in both inversions. As in the case of inversion 2j, the 2q7 inversion breakpoints contain large insertions made up of Galileo plus several other TEs, and they qualify as genetically unstable hotspots (Cáceres, Puig, and Ruiz 2001).


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Drosophila Stocks
Twenty-seven lines of D. buzzatii, one line of D. koepferae (KO-2, from Sierra San Luis, Argentina), one line of D. melanogaster (Canton S, 1611.2 from The National Drosophila Species Resource Center, Bowling Green, Ohio), and one of D. virilis (VIR-Tokyo, Japan) were used. The D. buzzatii lines are homokaryotipic for one of four different chromosome 2 arrangements: 2st, 2j, 2jz3, and 2jq7. They were isolated from different natural populations covering most of the distribution range of the species and their geographical origin is as follows: st-1, j-1, jz3-1 and jq7-1, Carboneras (Spain); st-3, Vipos (Argentina); st-4 and j-12, Guaritas (Brazil); st-5, Catamarca (Argentina); st-6 and j-16, Salta (Argentina); st-7, Termas de Rio Hondo (Argentina); st-8 and j-19, Ticucho (Argentina); st-9, j-22 and jz3-5, Trinkey (Australia); j-8, San Luis (Argentina); j-9, Quilmes (Argentina); j-10, Palo Labrado (Argentina); j-11, Los Negros (Bolivia); j-17 and jz3-4, Tilcara (Argentina); jq7-2, Mogan, Canary Islands (Spain); jq7-3, Caldetas (Spain); and jq7-4, jq7-5 and jq7-6, Otamendi (Argentina).

Probes and in Situ Hybridization
Three gene clones (Pli, Rb97D, and ro), two cosmid clones, and six P1 phages were used as probes for in situ hybridization to map the distal breakpoint of the 2q7 inversion. The Pli clone contains 1.6 kb from the 3' end of the Pli (Pellino) gene (Grosshans, Schnorrer, and Nusslein-Volhard 1999) and was obtained by polymerase chain reaction (PCR) amplification of D. melanogaster genomic DNA with appropriate primers (table 1) and cloning of the PCR product into the pGEM-T vector (Promega). All remaining clones come from D. melanogaster, except that containing the ro gene that comes from D. virilis, and were kindly provided by different authors (Rb97D and ro; see Ranz, Casals, and Ruiz 2001), the European Drosophila Genome Project (cosmids), or the Berkeley Drosophila Genome Project (P1 phages). In situ hybridization of DNA probes to the larval salivary gland chromosomes was carried out using the procedure described by Montgomery, Charlesworth, and Langley (1987). All probes were labeled with biotin-16-dUTP (Roche) by nick translation, and detection was carried out with the ABC-Elite Vector Laboratories kit. The D. melanogaster clones were hybridized to the chromosomes of D. melanogaster (Canton S) as control and to the chromosomes of a 2j line (j-1) and a 2jq7 line (jq7-4) of D. buzzatii for breakpoint mapping. The ro probe was hybridized to the chromosomes of D. virilis as control, as well as to the chromosomes of D. melanogaster and D. buzzatii. In addition, during the isolation of the breakpoint regions (fig. 1) {lambda} phages derived from D. buzzatii genomic libraries and their subclones were hybridized to the chromosomes of the j-1 and jq7-4 lines. Heterologous (interspecific) hybridizations were performed at 25°C, and homologous (intraspecific) hybridizations were completed at 37°C. Cytological localization of the hybridization signals was determined from the cytological maps of D. buzzatii (Ruiz and Wasserman 1993) and the photographic and electron microscopy maps of D. melanogaster (Lefevre 1976; Heino, Saura, and Sorsa 1994).


View this table:
[in this window]
[in a new window]
 
Table 1 Sequence of Oligonucleotide Primers Used for PCR Amplification.

 


View larger version (64K):
[in this window]
[in a new window]
 
FIG. 1. In situ hybridization of lambda clones {lambda}j-19/25, {lambda}j-19/27, {lambda}jq7-4/1, and {lambda}jq7-4/2 to the salivary gland chromosomes of D. buzzatii lines j-19 (2j arrangement) and jq7-4 (2jq7 arrangement). Arrows indicate hybridization signals corresponding to the 2q7 inversion breakpoint regions. Clones {lambda}j-19/25 and {lambda}j-19/27 produce a single hybridization signal on the 2j arrangement but two signals on the 2jq7 chromosomes, and contain the distal (AB) and proximal (CD) breakpoint regions, respectively. Clone {lambda}jq7-4/1 produces a strong signal on the 2jq7 arrangement and two signals on the 2j arrangement and encompasses the distal breakpoint region (AC). Clone {lambda}jq7-4/2 hybridizes strongly to the proximal breakpoint region in the 2jq arrangement and to the distal breakpoint on the 2j arrangement, and seems to contain at least part of the proximal breakpoint region (BD). The last two clones, {lambda}jq7-4/1 and {lambda}jq7-4/2, show additional hybridization signals at other chromosomal sites (including the centromere) owing to the presence of repetitive DNA

 
Construction and Screening of Genomic Libraries
A genomic library was constructed with DNA derived from the jq7-4 line of D. buzzatii using the LambdaGEM-11 vector according to the manufacturer's instructions (Promega). Briefly, high molecular weight genomic DNA was randomly fragmented by partial digestion with Sau 3A, electrophoresed on a 0.4% agarose gel, and fragments of 15–23 kb were recovered and ligated with the lambda vector arms. This library and a lambda genomic library of the j-19 line (Cáceres, Puig, and Ruiz 2001) were screened by plaque hybridization with the probes indicated in text. The j-19 library was previously amplified following the procedures described in Sambrook, Fritsch, and Maniatis (1989). DNA fragments of interest from positives phages were subcloned into Bluescript II SK vector (Stratagene) after restriction enzyme digestion and gel purification.

Southern Analysis
Southern hybridization was carried out according to standard procedures (Sambrook, Fritsch, and Maniatis 1989). Probes were labeled by random primer with digoxygenin-11-dUTP under the conditions specified by the supplier (Roche). The probes used were Pli (the Pli1-Pli2 D. melanogaster PCR product of 1.6 kb), AB (the A1-B1 D. buzzatii PCR product of 0.6 kb), and CD (the Cla I D. buzzatii restriction fragment of 0.9 kb; see below). Hybridization was carried out overnight in standard buffer with 50% formamide at 42°C for homologous probes (AB and CD) and at 37°C for heterologous probes (Pli). Stringency washes were performed with 0.1 x SSC 0.1% SDS solution at 68°C and 50°C for homologous and heterologous hybridizations, respectively.

PCR Amplification
Polymerase chain reaction testing was carried out in a volume of 50 µl, including 100–200 ng of genomic DNA, 20 pmols of each primer, 200 µM dNTPs, 1.5 mM MgCl2, 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 60 s at 72°C, with annealing temperature varying from 58° to 68°C depending on the primer pair used. Amplifications of the A1-B1 and C1-D1 fragments in D. koepferae were performed at 54°C. Sequences of oligonucleotide primers are given in table 1. Two primer pairs (A1-B1 and C1-D1) were used to amplify the inversion breakpoint regions (AB and CD) in D. buzzatii lines without the inversion (2st, 2j, and 2jz3) and D. koepferae. Different primer combinations (see table 1) were tested to amplify the inversion breakpoint regions (AC and BD) of lines with the 2q7 inversion (2jq7).

DNA Sequencing and Sequence Analysis
Sequences were obtained on an ABI 373 A (Perkin-Elmer) automated DNA sequencer. Fragments cloned into Bluescript II SK or pGEM-T were sequenced with M13 universal and reverse primers. The PCR products were gel-purified using the Geneclean Spin Kit (Bio 101) and sequenced directly with the same primers used for amplification. To estimate the nucleotide variation of the distal and proximal breakpoints region, the A1-B1 and C1-D1 PCR products were sequenced in a sample of nine 2st, 2j, or 2jz3 lines without the inversion. In addition, PCR products obtained with primer pairs A1-T1, B1-T4 (B1-T5 in line jq7-6), T3-C1, and T9-D1 were sequenced in all 2jq7 lines to determine the exact location of the insertions present in the inversion breakpoints and to estimate the nucleotide diversity. Nucleotide sequences were analyzed with the Wisconsin Package (Genetics Computer Group). BESTFIT was used to compare homologous sequences in different lines and to detect changes between them. Similarity searches in the GenBank/EMBL databases were carried out using BLASTX, TBLASTX, and FASTA. Nucleotide variation between the different D. buzzatii strains was obtained by multiple alignments of the sequences with ClustalW (Thompson, Higgins, and Gibson 1994), followed by analysis with the DnaSP version 3.51 software (Rozas and Rozas 1999).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
High-Resolution Mapping of the 2q7 Inversion Distal Breakpoint
Cytological studies localized the distal and proximal breakpoints of inversion 2q7 near bands D3a and G2f, respectively, of chromosome 2 of D. buzzatii (Ruiz and Wasserman 1993). Our detailed physical map of chromosome 2 in D. buzzatii and D. repleta (Ranz, Casals, and Ruiz 2001) allowed us to find two cosmids 109B4 and 58B10, which mapped in band D3d of D. buzzatii chromosome 2, outside inversion 2q7 and not far from its distal breakpoint (table 2). Seven additional available markers (one gene clone and six P1 phages) from the 95B-C region of D. melanogaster were therefore hybridized to 2jq7 chromosomes of D. buzzatii (table 2). All mapped very close to the distal breakpoint of inversion 2q7. Two of them, the gene Pli and the P1 phage DS05188, were located inside the inversion, whereas the remaining five P1 phages were located outside the inversion (table 2). The cytological position of these markers points to band D3c of D. buzzatii chromosome 2 as the precise site of the 2q7 distal breakpoint. To estimate the distance from Pli to the 2q7 distal breakpoint in D. buzzatii, we carried out a Southern hybridization of genomic DNA from 2j and 2jq7 chromosomal lines digested with the restriction enzymes Bam HI, Hind III, Pst I, and Sal I with a 1.6 kb Pli probe. In Bam HI, Pst I, and Sal I digestions, hybridization bands differed between the lines with and without the 2q7 inversion, indicating that Pli was very close to the breakpoint. Specifically, we located Pli and the distal breakpoint within a Sal I fragment of 6 kb in the 2j chromosome.


View this table:
[in this window]
[in a new window]
 
Table 2 Markers from D. melanogaster Hybridized to the Chromosomes of D. buzzatii for the High-Resolution Mapping of the 2q7 Inversion Distal Breakpoint and Their Position in Relation to the Inversion.

 
Isolation and Sequencing of the 2q7 Inversion Breakpoints
According to previous studies (Wesley and Eanes 1994; Cáceres et al. 1999), the distal and proximal breakpoint regions were designated as AB and CD in the non-inverted chromosomes (2st, 2j, or 2jz3) and as AC and BD in the inverted chromosomes (2jq7) (fig. 2). To isolate the distal breakpoint of inversion 2q7 in the non-inverted arrangement, ~75,000 phages of a lambda genomic library derived from a 2j line (j-19; Cáceres, Puig and Ruiz 2001) were screened with the Pli probe, and four positive clones were obtained. These four clones mapped by in situ hybridization to band D3c of 2j chromosomes and produced two hybridization signals at both 2q7 inversion breakpoints in 2jq7 chromosomes, indicating that they spanned the distal breakpoint region (AB). Figure 1 shows the hybridization pattern of one of the four clones, {lambda}j-19/25. A restriction map of this clone was made and several subclones were hybridized to chromosomes with and without the 2q7 inversion, locating the distal breakpoint first in a 6 kb Sal I-Sau 3A fragment (pGPE205) and then in a 1.9 kb Bam HI-Hind III fragment (pGPE205.1). The latter fragment and the adjacent 1 kb Hind III fragment (pGPE205.3) were completely sequenced (fig. 2).



View larger version (12K):
[in this window]
[in a new window]
 
FIG. 2. Experimental strategy for cloning the breakpoint regions of the 2q7 inversion. The breakpoint regions are designated AB and CD in the non-inverted chromosome (2j) and AC and BD in the inverted chromosome (2jq7). Lines above the map represent the lambda clones isolated during the cloning of the 2q7 breakpoints and some of the subclones derived from them. Thick segments of the lambda clones represent sequenced regions (sequence analysis is shown in fig. 3). Horizontal arrows represent the primers employed to complete the isolation of the BD region in the inverted chromosome. Long vertical arrows mark the location of the breakpoints and the limits of the insertions found at the breakpoints in the inverted chromosomes. Short vertical arrows limit other insertions found in the inverted chromosome and show its corresponding location in the non-inverted chromosome. Empty boxes represent genes sequenced totally (snRNA:U1) or partially (Pli). Cen: centromere; Tel: telomere. Restriction sites: B: Bam HI; C: Cla I; E: Eco RI; H: Hind III; S: Sal I; U: Sau 3A

 
To clone the proximal (AC) and distal (BD) breakpoints in the chromosome with the inversion, a lambda genomic library was constructed from a 2jq7 chromosomal line (jq7-4), and ~60,000 phages were screened with the AB subclone (pGPE205.1). Twenty-one positive phages were obtained. These phages were rescreened independently with A and B probes, and two phages—{lambda}jq7-4/1 and {lambda}jq7-4/2—that hybridized with pGPE205.2 and pGPE205.3, respectively, were selected (fig. 2). In situ hybridization of {lambda}jq7-4/1 produced an intense signal at the distal breakpoint on 2jq7 chromosomes but two signals at both breakpoints of 2j chromosomes (fig. 1), corroborating that it spanned the distal breakpoint region (AC). Likewise, hybridization of clone {lambda}jq7-4/2 produced intense signals at the proximal breakpoint (BD) on 2jq7 chromosomes and the distal breakpoint (AB) on 2j chromosomes (fig. 1), indicating that the clone came from the proximal breakpoint region. It is worthwhile to note, however, that both {lambda}jq7-4/1 and {lambda}jq7-4/2 phages produced additional weaker signals at different euchromatic sites and stained heavily the centromeres of all chromosomes (fig. 1), which suggests that both of them contain repetitive sequences.

By Southern hybridization with the pGPE205.1 probe, the sequences of {lambda}jq7-4/1 homologous to the A region were identified in a 2 kb Bam HI-Hind III fragment (pGPE209), and the sequences of {lambda}jq7-4/2 homologous to the B region were found in a 6-kb Hind III fragment (pGPE208) (fig. 2). These two fragments were subcloned and completely sequenced. In addition to sequences homologous to the single-copy A and B sequences found in non-inverted chromosomes, they contained sequences homologous to TEs already described in D. buzzatii (Cáceres, Puig, and Ruiz 2001). To complete the isolation of the distal breakpoint region (AC), three fragments contiguous with pGPE209 were cloned and sequenced: a 0.9 kb Hind III-Eco RI band (pGPE210.1), a 0.7 kb Eco RI-Cla I band (pGPE210.2), and a 1 kb Cla I band (pGPE210.3) (fig. 2). Sequence analysis revealed the presence of repetitive DNA in pGPE210.1 and pGPE210.2, and sequences homologous to D. melanogaster single-copy DNA, presumably from the C region, in pGPE210.2 and pGPE210.3. In the proximal breakpoint region (BD), the sequencing of the outermost 90 bp Hind III-Sau 3A fragment of {lambda}jq7-4/2 (pGPE211) (fig. 2) revealed that it contained only repetitive sequences similar to those in pGPE208. Thus, the D region in the inverted chromosome (2jq7) was obtained after the cloning and sequencing of the proximal breakpoint region (CD) in the non-inverted chromosome (2j) (see below) by PCR amplification with primers T9 and D1 (fig. 2). Sequencing of the PCR product of 671 bp showed that it contained repetitive sequences and single-copy DNA from D.

Finally, the proximal breakpoint region (CD) in the chromosome without the inversion (2j) was isolated by screening the j-19 lambda genomic library with pGPE210.3 (fig. 2). Five positive phages were obtained, and all of them were shown by in situ hybridization to span the CD breakpoint (fig. 1). A restriction map of one of them, {lambda}j-19/27, was made and the breakpoint was further located to a 6.5 kb Sal I-Sau 3A fragment (pGPE212) by in situ hybridization of different {lambda}j-19/27 subclones. This clone was partially sequenced, and 3.1 kb of single-copy sequence from the CD region were obtained.

Sequence Analysis of the Breakpoint Regions
Overall, we sequenced 2,933 bp and 3,095 bp of the AB and CD 2q7 breakpoint regions, respectively, in a 2j line (j-19), and 4,565 bp and 6,751 bp of the AC and BD 2q7 breakpoint regions in a 2jq7 line (jq7-4). Comparison of the breakpoint sequences in the non-inverted (AB and CD) and inverted (AC and BD) chromosomes allowed us to determine the exact boundaries of the inverted segment and to identify the repetitive sequences inserted at the breakpoint junctions (fig. 3).



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 3. Schematic diagram of the sequenced regions of the 2q7 inversion breakpoints in D. buzzatii lines j-19 (2j arrangement) and jq7-4 (2jq7 arrangement). Thick lines represent the single-copy A, B, C, and D sequences. Coding sequences of genes are represented as empty boxes, with 5' and 3' indicating their orientation. Transposable elements are represented as shaded boxes with pointed ends. The different copies of the known TEs have been numbered sequentially following the order of the copies previously described (Cáceres 2000; Cáceres, Puig, and Ruiz 2001). Some of the TE copies (BuT5-3, BuT5-4, Galileo-11, and one end of Kepler-5) are very small (see table 3) and are not drawn to scale. Target site duplications flanking several of the TEs are shown in boxes above them. Thick vertical arrows limit the insertions found at the A–C and B–D junctures. The total size of each insertion is given below. Horizontal arrows represent primers used for PCR amplification. Lines under the sequences indicate probes used for Southern hybridizations. Restriction sites: A: Xmn I; B: Bam HI; C: Cla I; D: Dra I; E: Eco RI; H: Hind III; S: Sal I; X: Xba I

 
In the line without the inversion (j-19), the genes snRNA:U1 and Pellino (Pli) were found flanking the distal breakpoint (AB), with the Lsp-1ß gene located also in the A region further away from the breakpoint. The proximal breakpoint (CD) was flanked by the CG1172 gene and by a tRNA-thr gene (fig. 3). This same A, B, C, and D single-copy sequences were present in the line with the inversion (jq7-4), but in an AC and BD arrangement, with B and C sequences in the inverted orientation (fig. 3). In addition, some small deletions were detected with respect to the j-19 chromosome, consisting of 23 nucleotides precisely located at the A-B junction and 45-bp located 89 bp away from the breakpoint in A. More importantly, several large insertions were found in the AC and BD breakpoint regions of the 2jq7 chromosome (fig. 3). In the distal breakpoint there is a 1.8-kb insertion just in the A-C juncture and a 387-bp insertion in the A region, between the Lsp-1ß and snRNA:U1 genes (fig. 3). In the proximal breakpoint region (BD) there are two insertions of 2.4 and 3 kb separated by 11 bp only (CTTGTTCCCAG), which corresponds to the first 11 nucleotides of the D sequence (fig. 3).

The sequences of all these large insertions showed high identity with a set of TEs previously found in D. buzzatii (Cáceres, Puig, and Ruiz 2001). The only exception is the 387-bp insertion in A that shows all the hallmarks of what may be considered a new transposon and has been designated BuT6, following Cáceres, Puig, and Ruiz (2001). This element is flanked by 8-bp target site duplications and ends with short inverted terminal repeats (ITRs) of 13 bp, similar to those of other Class II elements of the hAT superfamily (Calvi et al. 1991). The TE content of the remaining breakpoint insertions is summarized in table 3 and figure 3. Briefly, the 1.8-kb insertion in the A-C junction comprises a truncated copy of the transposon BuT5 and a Galileo element. The 2.4-kb insertion in the B-D junction is made up of three partial copies and one complete copy of BuT5, one copy of Kepler, and two small sequences corresponding to Galileo ends. Finally, the 3-kb D insertion contains one long Galileo copy inserted within a BuT3 element.


View this table:
[in this window]
[in a new window]
 
Table 3 Transposable Elements Found at the Breakpoint Regions of Inversion 2q7 in the jq7-4 Line of D. buzzatii.

 
Structural Variation of the Inversion 2q7 Breakpoint Regions
In addition to the sequencing of the inversion breakpoints in j-19 and jq7-4, the genomic structure of the breakpoint regions was investigated in 20 other lines without the 2q7 inversion (eight 2st lines, nine 2j lines, and three 2jz3 lines) and in five other lines with the 2q7 inversion. The techniques used include Southern blotting, PCR amplification, restriction mapping, and sequencing of PCR products. In the lines without the inversion, two PCR reactions were carried out with primer pairs A1-B1 and C1-D1 to amplify the distal and proximal breakpoint regions, respectively (see fig. 3). In most lines a PCR product of similar size to that generated by the reference line (j-19) was obtained, but st-8, st-9, j-9, j-11, j-12, and j-16 in the A1-B1 PCR and st-3 and j-16 in the C1-D1 PCR produced slightly different amplification bands containing small insertions and/or deletions. One line (jz3-4) failed to yield any A1-B1 product and by hybridization of Bam HI-Hind III digested DNA with the AB probe it was shown that it contains a 1.6 kb insertion (probably corresponding to an element of the Foldback family, as suggested by the failure of the PCR reaction). The same Southern hybridization also showed that none of the lines without the inversion examined (st-1, st-8, j-1, j-9, j-19, jz3-1) had the BuT6 insertion that was present in the A region of all 2jq7 lines (see below).

In the lines with the inversion, we performed Southern analysis with AB and CD probes (fig. 3) and PCR amplification of the distal (AC) and proximal (BD) breakpoint regions using different primer pairs (table 1 and fig. 3). In all the amplifications, at least one of the primers in each pair was anchored in single-copy DNA to avoid unspecific amplification. Results of informative PCR reactions and Southern analysis are shown in tables 4 and 5, respectively. In the distal breakpoint region (AC), the BuT6 insertion was found in all the 2jq7 lines and the same 1.8-kb insertion was present at exactly the same position in all 2jq7 lines, except in jq7-6. This line possess a 170-bp deletion in A, lacks two 19-bp and 66-bp BuT5-2 deletions present in the other 2jq7 lines, has an additional copy of the 13-bp right-end sequence of Galileo-10, and the AC insertion is ~3 kb larger (figs. 4 and 5). Further structural variability between the 2jq7 lines was detected in the proximal breakpoint (BD) region, where four out of five lines showed major differences with jq7-4. These differences, summarized in figures 4 and 5, basically are these: (1) the substitution of part of the left end of the BD insertion by a different BuT5 fragment (BuT5-7) and the lack of the BuT5-6 insertion in jq7-6, and (2) the absence of the Galileo-12 element and the presence of additional sequences in the central portion of the BD insertion in jq7-1, jq7-2, jq7-3, and jq7-6.


View this table:
[in this window]
[in a new window]
 
Table 4 PCR Amplification Analysis of the 2q7 Breakpoint Regions in Six 2jq7 Chromosomal Lines.

 

View this table:
[in this window]
[in a new window]
 
Table 5 Southern Hybridization Analysis of the 2q7 Breakpoint Regions in Six 2jq7 Chromosomal Lines.

 


View larger version (19K):
[in this window]
[in a new window]
 
FIG. 4. Structural variation at the 2q7 inversion breakpoint regions in six different 2jq7 lines. Partial structures inferred from Southern analysis, PCR amplification, and sequencing are shown only for those lines with differences when compared to line jq7-4. Dotted lines indicate segments where the exact structure is not known, and the total size of the insertions is given below. Structure in line jq7-4 is shown as reference. Symbols are as in figure 3

 


View larger version (30K):
[in this window]
[in a new window]
 
FIG. 5. Nucleotide polymorphism at the breakpoint regions of the inversion 2q7. For each region, nucleotide positions are numbered taking the breakpoints as start points. For convenience, the 23 nucleotides located between A and B regions in non-inverted chromosomes that have not been found in inverted chromosomes, are included in the A sequence. j-19 sequence is taken as reference for the A, B, C, and D regions, and jq7-4 is taken as reference for the breakpoint insertions. Positions with nucleotides identical to the reference sequence are indicated by a dot. Insertions and deletions are represented by minus and plus signs in the reference sequence, respectively, and a number in the line with the insertion or deletion indicating its size in nucleotides. Deletions including more than one position of the reference line are included in rectangles. Question marks indicate missing data. TE insertions found at the proximal and distal breakpoints in inverted chromosomes are indicated as i1 and i2, and the insertion found in region D as i3. Asterisks indicate positions with fixed nucleotide differences between inverted and non-inverted chromosomes

 
Nucleotide Variation at the 2q7 Breakpoints and Dating of the Inversion
To estimate the nucleotide variation at the breakpoint regions of the 2q7 inversion, we sequenced the PCR products obtained with primer pairs A1-B1 and C1-D1 in nine lines without the 2q7 inversion and those obtained with primer pairs A1-T1, T3-C1, B1-T4 (B1-T5 in jq7-6), and T9-D1 in all six 2jq7 lines (fig. 5). In addition, the A, B, C, and D regions were also sequenced in D. koepferae, a close relative of D. buzzatii. All the sequences of A, B, C, and D regions are located in non-coding regions, except for 37 nucleotides of A corresponding to the snRNA:U1 gene and 35 nucleotides of C corresponding to the CG1172 gene.

Overall, 86 and 21 segregating sites were found in single-copy DNA regions (ABCD) and the transposable elements sequences, respectively (table 6). In addition, excluding the large TE insertions, there were 11 small insertions (ranging from 1 to 32 nucleotides) and 22 deletions (ranging from 1 to 170 nucleotides) in the A, B, C, and D sequences, and five insertions (ranging from 1 to 66 nucleotides) in the transposable element sequences (fig. 5). Nucleotide diversity ({pi}; Nei 1987) values in the different regions are given in table 6. First, there is a sharp contrast in the nucleotide diversity of the single-copy DNA regions between the non-inverted chromosomes ({pi} = 0.0178) and those with the 2q7 inversion ({pi} = 0). Based on the heterogeneity test of Kreitman and Hudson (1991), this difference is statistically significant ({chi}2L = 29, df = 1, P < 0.001). Second, the level of polymorphism varies between the four single-copy DNA regions (ABCD) of non-inverted chromosomes, with region A exhibiting around four times more nucleotide variation than regions B, C, and D ({chi}2L = 24.78, df = 3, P < 0.001). Finally, the distal and proximal breakpoint insertions of inverted chromosomes show much higher nucleotide diversity ({pi} = 0.0125) than the adjacent single-copy sequences, and this value is more than four times higher in the distal breakpoint insertion than in the proximal breakpoint insertion ({chi}2L = 10.45, df = 2, P < 0.01).


View this table:
[in this window]
[in a new window]
 
Table 6 Nucleotide Variation at the Breakpoints Regions of the 2q7 Inversion.

 
No significant departures from the neutral model in the four single-copy regions were found with the Tajima (1989) and Fu and Li (1993) tests. Thus, we used the divergence in these regions between inverted and non-inverted chromosomes to date the origin of the 2q7 inversion (Hasson and Eanes 1996; Cáceres, Puig, and Ruiz 2001). The average numbers of nucleotide substitutions per site, dxy, and the net number of nucleotide substitutions per site, da, between inverted (2jq7) and non-inverted (2st, 2j, and 2jz3) chromosomes are 0.0155 and 0.0066, respectively (Nei 1987). The corresponding values for the comparison between D. buzzatii and D. koepferae, two species that diverged 4.2 MYA (Russo, Takezaki, and Nei 1995; Rodríguez-Trelles, Alarcón, and Fontdevila 2000), are 0.0748 and 0.0571. We have used as an estimate of the intraspecific nucleotide diversity the {pi} value for non-inverted chromosomes (table 6), because the low frequency of the 2q7 inversion and its endemic distribution make its overall contribution to the species diversity negligible. The same value was taken as an estimate for D. koepferae intraspecific diversity. A rate of 6.8 x 10-9 nucleotide substitutions per site and per year results, which indicates that the 2q7 inversion is 0.49 Myr old. If the A region, which displays a higher polymorphism level, is excluded from the calculations, a rate of 6.25 x 10-9 nucleotide substitutions per site and per year, and an age of 0.67 Myr for inversion 2q7 are obtained. Finally, we have used the amount of nucleotide diversity to estimate the age of the sampled 2jq7 alleles (Rozas et al. 1999; Cáceres, Puig, and Ruiz 2001). Given that no nucleotide polymorphism has been found in the single-copy DNA regions between the different 2jq7 chromosomes, an upper bound for the coalescence time of the 2jq7 alleles has been estimated assuming a single nucleotide change in one sequence ({pi} = 0.0003). Using the two previous rates of nucleotide substitution, this results in a maximum age for the sampled 2jq7 alleles between 20,000 years (including the A region) and 25,000 years (excluding the A region).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Two different models have been proposed to explain the induction of chromosomal inversions by TEs: the ectopic recombination model (Petes and Hill 1988; Lim and Simmons 1994) and the hybrid element insertion model (Gray 2000). Under the ectopic recombination model, inversions arise by homologous recombination between two copies of the same TE inserted in opposite orientation at different sites of the same chromosome. Thus, the TEs precede the generation of the inversion and serve as (perhaps passive) substrates for the recombination event. Both Class I and Class II elements may participate in ectopic recombination and the cellular machinery involved may be the same operative in regular meiotic recombination (Virgin and Bailey 1998). The outcome is an inversion flanked by a pair of TE copies that have their original target site duplications, aa and bb, exchanged to give the arrangement ab' and a'b (where a' and b' designate the inverted and complementary versions of sequences a and b, respectively). Transposable elements may also induce inversions by a different mechanism, called the hybrid element insertion model (Gray 2000). Under this model, a single TE insertion results, after DNA replication, in two TE copies inserted at the same site in sister chromatids. These two copies may participate in an aberrant transposition event, by which a hybrid element formed by one end of the first copy and the opposite end of the second copy transposes to a new chromosomal site. The outcome is an inversion of the segment between the two chromosomal sites flanked by two TE copies, which originated by DNA replication and are initially identical. The arrangement of the target site duplications flanking the TE copies will be the same as in the ectopic recombination model (see above). Thus, the outcomes of the two models are strikingly similar, except perhaps for the fact that the two TE copies involved in recombination may differ, yielding chimerical elements, whereas those resulting from the aberrant transposition of a hybrid element must be identical, at least shortly after the origin of the inversion.

The location of the insertions of 1.8 kb and 2.4 kb directly into the A-C and B-D junctures, respectively, points to their involvement in the origin of the inversion (see below). The other two insertions at the breakpoint regions are not directly involved in the generation of the 2q7 inversion and were seemingly caused by secondary TE invaders, as shown by the direct target site duplications flanking BuT6 in region A and BuT3-6 in region D. Among the inserted TEs, the Foldback-like transposon Galileo is the most likely inducer of the 2q7 inversion. One long copy of Galileo is found at the A-C junction (Galileo-10) and two small Galileo end fragments are found at the B-D junction (Galileo-11) (fig. 3). Galileo duplicates 7 nucleotides of the target site upon insertion (Cáceres, Puig, and Ruiz 2001), and as expected if Galileo originated the 2q7 inversion, the 7-bp sequence flanking Galileo-10 in region C (GAACAAG) is the inverted and complementary version of the 7-bp sequence flanking Galileo-11 in region D. The 7-bp sequence at the other end of Galileo-10 (GTTATAC) fits well with the consensus target sequence (G10T11a8g7T11A13c6) proposed for Galileo and two other D. buzzatii Foldback-like elements, Kepler and Newton (Cáceres 2000). This 7-bp sequence pertains to BuT5-2, which suggests that Galileo-10 inserted originally within a pre-existing BuT5 element. Neither the complementary 7-bp sequence nor the remaining fragment of BuT5-2 were found at the other end of Galileo-11 in region B, as would be expected for the ectopic recombination and hybrid element insertion models. However, because the BD region bears the largest number of insertions and structural changes, the most likely explanation is that a deletion removed the 7-bp duplication along with the BuT5-2 sequences. This deletion could also have removed the 23 nucleotides of the AB sequence that are not present in the chromosomes with the inversion.

Additional evidence supports the role of Galileo in the origin of the 2q7 inversion and suggests that ectopic recombination may be the actual mechanism involved: Galileo-10 seems to be a chimerical element that was probably generated by recombination between two slightly different Galileo copies. We have compared the sequence of the inverted terminal repeats (ITRs) at both ends of Galileo-10 with the corresponding sequences of Galileo-12 and two other Galileo copies described previously, Galileo-3 and Galileo-4 (Cáceres, Puig, and Ruiz 2001). The ITRs from the same element always exhibit much less sequence divergence than those of different elements (table 7). The exception is Galileo-10, whose ITRs show a degree of divergence similar to that observed between ITRs from different elements and six times higher than between ITRs belonging to the same element. Although the differences between the two ITRs of Galileo-10 may also have arisen by mutation, the mutation rate should be exceedingly high to account for such level of divergence. Thus the likely chimerical nature of Galileo-10 provides support for the ectopic recombination model. Similarly, the comparison between the long terminal repeats (LTR) of endogenous retroviruses (HERVs) and the target site duplications flanking them has been employed as an indicator of genomic rearrangements occurring through ectopic recombination in humans (Hughes and Coffin 2001).


View this table:
[in this window]
[in a new window]
 
Table 7 Divergence Between the Last 336 Nucleotides of the ITRs of Four Galileo Elements.

 
A high frequency of TE insertions and other structural changes has been detected at the 2q7 inversion breakpoints. This amount of structural variability is surprising if we consider that we have not found any nucleotide polymorphism in the 2jq7 chromosomes. The structural differences between the six studied lines should have arisen in a very short period of time, because the age of the alleles has been estimated to be less than 25,000 years. Both estimates of the age of the 2q7 inversion (0.49–0.67 Myr) and of the sampled alleles (20,000–25,000 years) are consistent with those calculated for the 2j inversion, ~1 Myr and 83,000 years, respectively (Cáceres, Puig, and Ruiz 2001), from which the 2q7 inversion arose. In addition, the lack of nucleotide polymorphism and the young age for the alleles are features consistent with the low frequencies of the inversion in the natural populations of D. buzzatii (Hasson et al. 1995). It is noteworthy that a similar degree of structural variability was found at the 2j inversion breakpoints (Cáceres, Puig, and Ruiz 2001), and thus the four inversion breakpoints sequenced in D. buzzatii have in common the presence of Galileo elements and the fact that they seem to be hotspots for TE insertions and other structural changes. It has been proposed that TEs should accumulate in low-recombination regions owing to a low rate of elimination by ectopic recombination (Charlesworth, Sniegowski, and Stephan 1994; Sniegowski and Charlesworth 1994), and the distribution of TEs in the recently sequenced genome of D. melanogaster (Adams et al. 2000) provides support for this hypothesis (Rizzon et al. 2002; Bartolomé, Maside, and Charlesworth 2002). Thus an accumulation of TEs is expected near the inversion breakpoints because of the reduction of recombination in these regions in inversion heterokaryotypes (Eanes, Wesley, and Charlesworth 1992; Sniegowski and Charlesworth 1994). This effect can certainly account in part for the TE clusters found at D. buzzatii inversion breakpoints (Cáceres, Puig, and Ruiz 2001, this work). However, we think that the particular properties of the Foldback-like Galileo elements may also be involved in the observed structural instability.

In D. melanogaster Foldback elements are known to give rise to genetic instability and promote different types of recombination processes (Levis, Collins, and Rubin 1982; Bingham and Zachar 1989; Smith and Corces 1991). These elements generate deletions, inversions, and reciprocal translocations at high frequencies, apparently through ectopic recombination (Collins and Rubin 1984); they show a high frequency of excision (Collins and Rubin 1983); and they usually harbor other TE insertions (Brierley and Potter 1985; Harden and Ashburner 1990). The capacity for generating rearrangements of these elements through recombination processes is probably increased by the presence of very long ITRs. Palindromic sequences, such as ITRs, have been demonstrated to be a source of genomic instability, causing different types of chromosomal rearrangements in a wide variety of organisms (Zhou, Akgün, and Jasin 2001). The number of rearrangements increases with the length of the ITRs and decreases with the size of the internal sequence, and it is probably related to its ability to form secondary structures and give rise to double-strand breaks (Lobachev et al. 1998). The size of the ITRs of the most complete described copies of Galileo varies between 683 bp (Galileo-3) and 1115 bp (Galileo-12) (Cáceres, Puig, and Ruiz 2001; this work) and, together with the length of the spacer DNA between them, are among those expected to produce the larger increases in the number of rearrangements. The capacity of D. melanogaster Foldback elements to form secondary structures when denatured was early demonstrated (Truett, Jones, and Potter 1981), and in the case of Galileo and related elements this ability is exemplified by the difficulties encountered during their amplification by PCR (Cáceres, Puig, and Ruiz 2001; this work).

The Galileo element of D. buzzatii has seemingly originated the two polymorphic inversions 2j and 2q7 of this species studied to date. Moreover, the four inversion breakpoints appear to have become genetically unstable regions and hotspots for the accumulation of TE insertions and other structural changes. These observations suggest that Galileo is an active element that may play an important role in the genome evolution of D. buzzatii and related species. Future studies of the chromosomal distribution of the TEs found at the inversion breakpoints will help to answer some questions regarding the copy number and distribution of these elements in the D. buzzatii genome, their association to other low recombination regions apart from inversion breakpoints, and the existence or not of similar hotspots in other chromosomal regions.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank E. Hasson and J. S. F. Barker for supplying D. buzzatii lines, and J. González and J. M. Ranz for advice and technical support. This work was supported by grant PB98-0900-C02-01 from the Dirección General de Enseñanza Superior e Investigación Científica (Ministerio de Educación y Cultura, Spain) awarded to A.R., and by a doctoral FI fellowship from the Universitat Autònoma de Barcelona awarded to F.C.


    Footnotes
 
1 Present address: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California. Back

E-mail: alfredo.ruiz{at}uab.es. Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 

    Adams, M. D., S. E. Celniker, and R. A. Holt, et al. (192 co-authors). 2000. The genome sequence of Drosophila melanogaster. Science 287:2185-2195.[Abstract/Free Full Text]

    Andolfatto, P., J. D. Wall, and M. Kreitman. 1999. Unusual haplotype structure at the proximal breakpoint of In(2L)t in a natural population of Drosophila melanogaster. Genetics 153:1297-1311.[Abstract/Free Full Text]

    Bartolomé, C., X. Maside, and B. Charlesworth. 2002. On the abundance and distribution of transposable elements in the genome of Drosophila melanogaster. Mol. Biol. Evol. 19:926-937.[Abstract/Free Full Text]

    Berg, D. E., and M. M. Howe. 1989. Mobile DNA. American Society for Microbiology, Washington, D.C.

    Bingham, P. M., and Z. Zachar. 1989. Retrotransposons and the FB transposon from Drosophila melanogaster. Pp. 485–502 in D. E. Berg and M. M. Howe, eds. Mobile DNA. American Society for Microbiology, Washington, D.C.

    Brierley, H. L., and S. S. Potter. 1985. Distinct characteristics of loop sequences of two Drosophila foldback transposable elements. Nucleic Acids Res. 13:485-500.[Abstract]

    Cáceres, M. 2000. Inversiones cromosómicas en Drosophila: origen molecular y significado evolutivo de su tamaño. Ph.D. thesis, Universitat Autònoma de Barcelona, Bellaterra (Barcelona), Spain.

    Cáceres, M., M. Puig, and A. Ruiz. 2001. Molecular characterization of two natural hotspots in the Drosophila genome induced by transposon insertions. Genome Res. 11:1353-1364.[Abstract/Free Full Text]

    Cáceres, M., J. M. Ranz, A. Barbadilla, M. Long, and A. Ruiz. 1999. Generation of a widespread Drosophila inversion by a transposable element. Science 285:415-418.[Abstract/Free Full Text]

    Calvi, B. R., T. J. Hong, S. D. Findley, and W. M. Gelbart. 1991. Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants: hobo, Activator, and Tam3. Cell 66:465-471.[ISI][Medline]

    Charlesworth, B., P. Sniegowski, and W. Stephan. 1994. The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371:215-220.[CrossRef][ISI][Medline]

    Cirera, S., J. M. Martín-Campos, C. Segarra, and M. Aguadé. 1995. Molecular characterization of the breakpoints of an inversion fixed between Drosophila melanogaster and D. subobscura. Genetics 139:321-326.[Abstract/Free Full Text]

    Collins, M., and G. M. Rubin. 1983. High-frequency precise excision of the Drosophila foldback transposable element. Nature 303:259-260.[CrossRef][ISI][Medline]

    Collins, M., and G. M. Rubin. 1984. Structure of chromosomal rearrangements induced by the FB transposable element in Drosophila. Nature 308:323-327.[CrossRef][ISI][Medline]

    Daveran-Mingot, M.-L., N. Campo, P. Ritzenthaler, and P. le Bourgeois. 1998. A natural large chromosomal inversion in Lactococcus lactis is mediated by homologous recombination between two insertion sequences. J. Bacteriol. 180:4834-4842.[Abstract/Free Full Text]

    Eanes, W. F., C. Wesley, and B. Charlesworth. 1992. Accumulation of P elements in minority inversions in natural populations of Drosophila melanogaster. Genet. Res. 59:1-9.[ISI][Medline]

    Evgen'ev, M. B., H. Zelentsova, H. Poluectova, G. T. Lyozin, V. Veleikodvorskaja, K. I. Pyatkov, L. A. Zhivotovsky, and M. G. Kidwell. 2000. Mobile elements and chromosomal evolution in the virilis group of Drosophila. Proc. Natl. Acad. Sci. USA 97:11337-11342.[Abstract/Free Full Text]

    Finnegan, D. J. 1989. Eukaryotic transposable elements and genome evolution. Trends Genet. 5:103-107.[CrossRef][ISI][Medline]

    Fu, Y.-X., and W.-H. Li. 1993. Statistical tests of neutrality of mutations. Genetics 133:693-709.[Abstract/Free Full Text]

    González, J., J. M. Ranz, and A. Ruiz. 2002. Chromosomal elements evolve at different rates in the Drosophila genome. Genetics 161:1137-1154.[Abstract/Free Full Text]

    Gray, Y. H. M. 2000. It takes two transposons to tango. Transposable-element mediated chromosomal rearrangements. Trends Genet. 16:461-468.[CrossRef][ISI][Medline]

    Grosshans, J., F. Schnorrer, and C. Nusslein-Volhard. 1999. Oligomerisation of tube and pelle leads to nuclear localisation of dorsal. Mech. Dev. 81:127-138.[CrossRef][ISI][Medline]

    Harden, N., and M. Ashburner. 1990. Characterization of the FOB-NOF transposable element of Drosophila melanogaster. Genetics 126:387-400.[Abstract/Free Full Text]

    Hasson, E., and W. F. Eanes. 1996. Contrasting histories of three gene regions associated with In(3L)Payne of Drosophila melanogaster. Genetics 144:1565-1575.[Abstract/Free Full Text]

    Hasson, E., C. Rodríguez, J. J. Fanara, H. Naveira, O. A. Reig, and A. Fontdevila. 1995. The evolutionary history of Drosophila buzzatii. XXVI. Macrogeographic patterns of inversion polymorphism in New World populations. J. Evol. Biol. 8:369-384.[ISI]

    Heino, T. I., A. O. Saura, and V. Sorsa. 1994. Maps of the salivary gland chromosomes of Drosophila melanogaster. Dros. Inform. Serv. 73:621-738.

    Hughes, J. F., and J. M. Coffin. 2001. Evidence for genomic rearrangements mediated by human endogenous retroviruses during primate evolution. Nat. Genet. 29:487-489.[CrossRef][ISI][Medline]

    Kim, J. M., S. Vanguri, J. D. Boeke, A. Gabriel, and D. F. Voytas. 1998. Transposable elements and genome organization: a comprehensive survey of retrotransposons revealed by the complete Saccharomyces cerevisiae genome sequence. Genome Res. 8:464-478.[Abstract/Free Full Text]

    Kreitman, M., and R. R. Hudson. 1991. Inferring the evolutionary histories of the Adh and Adh-dup loci in Drosophila melanogaster from patterns of polymorphism and divergence. Genetics 127:565-582.[Abstract/Free Full Text]

    Krimbas, C. B., and J. R. Powell. 1992. Drosophila inversion polymorphism. CRC Press, Boca Raton, Fla.

    Lefevre, G., Jr. 1976. A photoghraphyc representation and interpretation of the polytene chromosomes of Drosophila melanogaster salivary glands. Pp. 31–36 in M. Ashburner, H. L. Carson, and J. N. Thompson, eds. The genetics and biology of Drosophila. Academic Press, London.

    Levis, R., M. Collins, and G. M. Rubin. 1982. FB elements are the common basis for the instability of the wDZL and wc Drosophila mutations. Cell 30:551-565.[CrossRef][ISI][Medline]

    Lim, J. K., and M. J. Simmons. 1994. Gross chromosome rearrangements mediated by transposable elements in Drosophila melanogaster. Bioessays 16:269-275.[ISI][Medline]

    Lobachev, K. S., B. M. Shor, H. T. Tran, W. Taylor, J. D. Keen, M. A. Resnick, and D. A. Gordenin. 1998. Factors affecting inverted repeat stimulation of recombination and deletion in Saccharomyces cerevisiae. 1998. Genetics 148:1507-1524.[Abstract/Free Full Text]

    Lyttle, T. W., and D. S. Haymer. 1992. The role of the transposable element hobo in the origin of endemic inversions in wild populations of Drosophila melanogaster. Genetica 86:113-126.[ISI][Medline]

    Mathiopoulos, K. D., A. della Torre, V. Predazzi, V. Petrarca, and M. Coluzzi. 1998. Cloning of inversion breakpoints in the Anopheles gambiae complex traces a transposable element at the inversion junction. Proc. Natl. Acad. Sci. USA 95:12444-12449.[Abstract/Free Full Text]

    McDonald, J. F. 1993. Evolution and consequences of transposable elements. Curr. Opin. Genet. Dev. 3:855-864.[Medline]

    Montgomery, E., B. Charlesworth, and C. H. Langley. 1987. A test for role of natural selection in the stabilization of transposable element copy number in a population of Drosophila melanogaster. Genet. Res. 49:31-41.[ISI][Medline]

    Nei, M. 1987. Molecular evolutionary genetics. Columbia University Press, New York.

    Petes, T. D., and C. W. Hill. 1988. Recombination between repeated genes in microorganisms. Annu. Rev. Genet. 22:147-168.[CrossRef][ISI][Medline]

    Powell, J. R. 1997. Progress and prospects in evolutionary biology: the Drosophila model. Oxford University Press, New York.

    Ranz, J. M., F. Casals, and A. Ruiz. 2001. How malleable is the eukaryotic genome? Extreme rate of chromosomal rearrangement in the genus Drosophila. Genome Res. 11:230-239.[Abstract/Free Full Text]

    Regner, L. P., M. S. O. Pereira, C. E. V. Alonso, E. Abdelhay, and V. L. S. Valente. 1996. Genomic distribution of P elements in Drosophila willistoni and a search for their relationship with chromosomal inversions. J. Hered. 87:191-198.[Abstract]

    Rizzon, C., G. Marais, M. Gouy, and C. Biemont. 2002. Recombination rate and the distribution of transposable elements in the Drosophila melanogaster genome. Genome Res. 12:400-407.[Abstract/Free Full Text]

    Rodríguez-Trelles, F., L. Alarcón, and A. Fontdevila. 2000. Molecular evolution of the buzzatii complex (Drosophila repleta group): a maximum-likelihood approach. Mol. Biol. Evol. 17:1112-1122.[Abstract/Free Full Text]

    Rozas, J., and R. Rozas. 1999. DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15:174-175.[Abstract/Free Full Text]

    Rozas, J., C. Segarra, G. Ribó, and M. Aguadé. 1999. Molecular population genetics of the rp49 gene region in different chromosomal inversions of Drosophila subobscura. Genetics 151:189-202.[Abstract/Free Full Text]

    Ruiz, A., and M. Wasserman. 1993. Evolutionary cytogenetics of the Drosophila buzzatii species complex. Heredity 70:582-596.[ISI][Medline]

    Russo, C.A.M., N. Takezaki, and M. Nei. 1995. Molecular phylogeny and divergence times of drosophilid species. Mol. Biol. Evol. 4:406-425.

    Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Schwartz, A., D. C. Chan, L. G. Brown, R. Alagappan, D. Pettay, C. Disteche, B. McGillivray, A. de la Chapelle, and D. C. Page. 1998. Reconstructing hominid Y evolution: X-homologous block, created by X-Y transposition, was disrupted by Yp inversion through LINE-LINE recombination. Hum. Mol. Genet. 7:1-11.[Abstract/Free Full Text]

    Segarra, C., E. R. Lozovskaya, G. Ribó, M. Agaudé, and D. L. Hartl. 1995. P1 clones from Drosophila melanogaster as markers to study the chromosomal evolution of Muller's A element in two species of the obscura group of Drosophila. Chromosoma 104:129-136.[CrossRef][ISI][Medline]

    Smith, P. A., and V. G. Corces. 1991. Drosophila transposable elements: mechanisms of mutagenesis and interactions with the host genome. Adv. Genet. 29:229-300.[Medline]

    Sniegowski, P. D., and B. Charlesworth. 1994. Transposable element numbers in cosmopolitan inversions from a natural population of Drosophila melanogaster. Genetics 137:815-827.[Abstract/Free Full Text]

    Sperlich, D., and P. Pfriem. 1986. Chromosomal polymorphism in natural and experimental populations. Pp. 257–309 in M. Ashburner, H. L. Carson, and J. N. Thompson, eds. The genetics and biology of Drosophila. Vol. 3. Academic Press, London.

    Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585-595.[Abstract/Free Full Text]

    Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract]

    Truett, M. A., R. S. Jones, and S. S. Potter. 1981. Unusual structure of the FB family of transposable elements in Drosophila. Cell 24:753-763.[CrossRef][ISI][Medline]

    Virgin, J. B., and J. P. Bailey. 1998. The M26 hotspot of Schizosaccharomyces pombe stimulates meiotic ectopic recombination and chromosomal rearrangements. Genetics 149:1191-1204.[Abstract/Free Full Text]

    Wesley, C. S., and W. F. Eanes. 1994. Isolation and analysis of the breakpoint sequences of chromosome inversion In(3L) Payne in Drosophila melanogaster. Evolution 91:3132-3136.

    Zelentsova, H., H. Poluectova, L. Mnjoian, G. Lyozin, V. Veleikodvorskaja, L. Zhivotovsky, M. G. Kidwell, and M. B. Evgen'ev. 1999. Distribution and evolution of mobile elements in the virilis species group of Drosophila. Chromosoma 108:443-56.[CrossRef][ISI][Medline]

    Zhou, Z-H., E. Akgün, and M. Jasin. 2001. Repeat expansion by homologous recombination in the mouse germ line at palindromic sequences. Proc. Natl. Acad. Sci USA 98:8326-8333.[Abstract/Free Full Text]

Accepted for publication December 10, 2002.