Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra (Barcelona), Spain
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Abstract |
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Key Words: genome evolution chromosomal inversions inversion breakpoints transposable elements hotspots Drosophila buzzatii
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Introduction |
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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 inversionsi.e., those effectively contributing to adaptation and/or evolution of Drosophila speciesand 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).
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Materials and Methods |
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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) 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).
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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 100200 ng of genomic DNA, 20 pmols of each primer, 200 µM dNTPs, 1.5 mM MgCl2, and 11.5 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).
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Results |
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By Southern hybridization with the pGPE205.1 probe, the sequences of jq7-4/1 homologous to the A region were identified in a 2 kb Bam HI-Hind III fragment (pGPE209), and the sequences of
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
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, 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
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).
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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.
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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.
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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 (; 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 (
= 0.0178) and those with the 2q7 inversion (
= 0). Based on the heterogeneity test of Kreitman and Hudson (1991), this difference is statistically significant (
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 (
2L = 24.78, df = 3, P < 0.001). Finally, the distal and proximal breakpoint insertions of inverted chromosomes show much higher nucleotide diversity (
= 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 (
2L = 10.45, df = 2, P < 0.01).
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Discussion |
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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).
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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.
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Acknowledgements |
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Footnotes |
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E-mail: alfredo.ruiz{at}uab.es.
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