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Genetics. Apr 2005; 169(4): 2047–2059.
PMCID: PMC1449584

Molecular Characterization and Chromosomal Distribution of Galileo, Kepler and Newton, Three Foldback Transposable Elements of the Drosophila buzzatii Species Complex

Abstract

Galileo is a foldback transposable element that has been implicated in the generation of two polymorphic chromosomal inversions in Drosophila buzzatii. Analysis of the inversion breakpoints led to the discovery of two additional elements, called Kepler and Newton, sharing sequence and structural similarities with Galileo. Here, we describe in detail the molecular structure of these three elements, on the basis of the 13 copies found at the inversion breakpoints plus 10 additional copies isolated during this work. Similarly to the foldback elements described in other organisms, these elements have long inverted terminal repeats, which in the case of Galileo possess a complex structure and display a high degree of internal variability between copies. A phylogenetic tree built with their shared sequences shows that the three elements are closely related and diverged ~10 million years ago. We have also analyzed the abundance and chromosomal distribution of these elements in D. buzzatii and other species of the repleta group by Southern analysis and in situ hybridization. Overall, the results suggest that these foldback elements are present in all the buzzatti complex species and may have played an important role in shaping their genomes. In addition, we show that recombination rate is the main factor determining the chromosomal distribution of these elements.

TRANSPOSABLE elements (TEs) are ancient components of genomes, which are present in both prokaryotes and eukaryotes (Berg and Howe 1989; Capy et al. 1998). In some cases, TEs make up an important fraction of the genome, like in humans or mice where they represent nearly half of the DNA content (Lander et al. 2001; Waterston et al. 2002). TEs have been considered to be intracellular parasites or selfish DNA (Doolittle and Sapienza 1980; Orgel and Crick 1980), which are maintained in the genomes simply due to their ability to replicate (Charlesworth et al. 1994). As a result of their parasitic activity, TEs have mainly a negative effect by producing deleterious mutations. However, an increasing body of evidence suggests that some of these elements may also have played an important role in the evolution of species (McDonald 1993; Wessler et al. 1995; Kidwell and Lisch 1997; Kazazian 2004).

TEs are classified into two major groups according to their mechanism of transposition (Finnegan 1989): class I elements or retrotransposons, which alternate DNA and RNA phases, and class II elements or DNA transposons, which are mobilized through a “cut-and-paste” mechanism. Foldback elements are a particular group of class II elements with some distinctive characteristics (Finnegan 1989; Capy et al. 1998). These elements were first described in Drosophila melanogaster and took their name from the FB (or Foldback) element of this species (Potter et al. 1980; Truett et al. 1981). Since then, similar elements have been subsequently described in several other organisms (Hoffman-Liebermann et al. 1985; Hankeln and Schmidt 1990; Yuan et al. 1991; Rebatchouk and Narita 1997; Adé and Belzile 1999; Cheng et al. 2000; Simmen and Bird 2000; Windsor and Waddell 2000; ceres et al. 2001).

The main structural features common to all foldback elements that distinguish them from other class II TEs are summarized in Figure 1 (Adé and Belzile 1999; Simmen and Bird 2000). First, foldback elements contain very long inverted terminal repeats (IRs) that usually extend along almost the entire element and are separated by a middle domain (M) of variable length and composition. As a consequence of this particular structure, when denatured, the two IRs of the element can fold back and pair, giving rise to very stable stem-loop secondary structures that inspired their name (Potter et al. 1980). The IRs have a modular organization with three possible sequence domains: the most external flanking domain (IR-FD); the outer domain (IR-OD), which includes several imperfect repeats in tandem; and the inner domain (IR-ID), which usually contains A-T-rich sequences (Figure 1). Surprisingly, no coding capability has been found in the vast majority of foldback transposons described and their mechanism of transposition is not fully understood (Capy et al. 1998). In D. melanogaster, FB transposition depends on the presence of the 4-kb NOF sequence, which is found in the M domain of ~10% of the elements (Harden and Ashburner 1990; Smith and Corces 1991). The NOF sequence encodes a 120-kD protein, but its function is still unknown (Templeton and Potter 1989; Harden and Ashburner 1990). More recently, the FARE2 foldback transposons of Arabidopsis thaliana have been predicted to harbor a transposase (Windsor and Waddell 2000).

Figure 1.
Schematic of the structure of foldback elements and their different domains. Open triangles represent the target-site duplications generated during insertion. L and R refer to the left and right sides of the elements, respectively. Arrows below the element ...

At the functional level, foldback elements are characterized by their capacity to induce genetic instability and recombination processes, leading to the generation of spontaneous mutations and chromosomal rearrangements in D. melanogaster (Levis et al. 1982; Bingham and Zachar 1989; Smith and Corces 1991). These processes appear to occur by ectopic recombination between different copies of the same element (Collins and Rubin 1984). Ectopic recombination events between sequences of the same element could also explain the great structural heterogeneity described in most elements of this family (Hoffman-Liebermann et al. 1985; Cheng et al. 2000; Windsor and Waddell 2000). The capacity of foldback elements to mediate these rearrangements is probably caused by the presence of long IRs, which have been demonstrated to be a source of genomic instability (Zhou et al. 2001). IRs have the ability to form secondary structures, which in turn stimulate the production of double-strand breaks and recombination (Lobachev et al. 1998).

Interestingly, Galileo, a transposon of Drosophila buzzatii, is implicated in the generation of two natural polymorphic inversions through ectopic recombination between copies of the element (ceres et al. 1999b; Casals et al. 2003). Moreover, the four inversion breakpoints have become hotspots for genetic instability and TE insertions (ceres et al. 2001; Casals et al. 2003). These insertions were mainly formed by class II elements, and three of them, Galileo, Kepler, and Newton, share sequence and structural similarities, including the presence of long inverted repeats, and were tentatively classified as foldback elements (ceres et al. 2001). Here, we have isolated additional copies of the three elements and carried out a detailed molecular characterization of all available copies. We have also studied the abundance and chromosomal distribution of these foldback-like elements in D. buzzatii and other species of the repleta group. These analyses allow us to estimate the age of the elements and to gain insight into the factors controlling their distribution in the chromosomes.

MATERIALS AND METHODS

Drosophila stocks:

Twenty-three lines of D. buzzatii and 12 lines of other Drosophila species were used (supplementary Table 1 at http://www.genetics.org/supplemental/). These lines were isolated from different natural populations, and for D. buzzatii they cover most of the species' geographical distribution. All but three D. buzzatii lines are homokaryotypic for five natural different chromosomal arrangements: 2 standard (2st), 2j, 2jz3, 2jq7, and 2y3 (supplementary Table 1 at http://www.genetics.org/supplemental/). Line s-1 segregates for the 4 standard (4st) and 4s chromosomal arrangements (Ruiz and Wasserman 1993). Lines j-23 and j-24 are homokaryotypic for inversion 5I and translocation t(5,1), respectively, which were induced by introgressive hybridization (Naveira and Fontdevila 1985).

PCR amplification:

PCR was carried out in a volume of 50 μl, including 100–200 ng of genomic DNA, 20 pmol of each primer, 200 μm dNTPs, 1.5 mm MgCl2, and 1–1.5 units of Taq DNA polymerase. To isolate new copies of the D. buzzatii foldback elements, primers G7 (5′-CCATACAACACATAGACTGGACA-3′) and G8 (5′-TCGTATTTGCTCGGGTTCTTACT-3′), corresponding to the IRs of Galileo (Figure 2), were used. Different PCRs with several combinations of these primers and cycling conditions of 30 rounds of 30 sec at 94°, 30 sec at 59°–62.5°, and 1 min 30 sec–2 min 30 sec at 72° were carried out from genomic DNA of lines st-2, st-3, st-4, j-7, and j-8. PCR products were gel purified and cloned into the pGEM-T vector (Promega, Madison, WI). For each fragment, 10–12 different clones were analyzed by restriction mapping and one of them, corresponding to the predominant organization, was finally sequenced.

Figure 2.
Schematic of the 23 foldback-like elements of D. buzzatii characterized in this study. (A) Galileo elements. (B) Kepler elements. (C) Newton elements. The different copies of each element are represented with respect to the longest copy found (top). The ...

Genomic library screening and Southern analysis:

Genomic library screening and Southern hybridizations were performed according to standard procedures (Sambrook et al. 1989). Probes were labeled by random primers with digoxygenin-11-dUTP under the conditions specified by the supplier (Roche). Hybridization was performed overnight in standard buffer with 50% formamide at 42° for intraspecific and 37° for interspecific hybridization. Stringency washes were performed with 0.1× SSC 0.1% SDS solution at 68° and 50° for intraspecific and interspecific hybridizations, respectively. Additional copies of the foldback elements were isolated by plaque hybridization of a λ genomic library of the j-1 line (ceres et al. 1999b) with a pGPE107.2.1.1 probe containing the entire Galileo-2 element (0.7 kb) (ceres et al. 1999b). Positive phages were analyzed by restriction mapping and Southern hybridization. DNA fragments of interest were then subcloned into the Bluescript II SK vector (Stratagene, La Jolla, CA) after gel purification and sequenced. Southern hybridizations to quantify foldback element abundance were carried out with 15 D. buzzatii lines representative of different chromosomal arrangements (st-1, st-3, st-7, st-10, j-2, j-9, j-19, j-23, j-24, jq7-1, jq7-4, jz3-6, jz3-7, y3-1, and s-1) and several lines from other Drosophila species (H84, J79, KO-2, MA-4, 1371.5, 1611.2, SD-12, D62C2B, 1451.0, SM-3, and UN-2) (supplementary Table 1 at http://www.genetics.org/supplemental/). Genomic DNA of these lines was digested with the restriction enzymes BamHI and HindIII. Southern blots were hybridized with a 1.1-kb probe containing approximately half of the Galileo-12 element. Probe was produced by PCR amplification with primers G19 (ceres et al. 2001) and E14 (5′-CACTAACCATACAACACATAG-3′) (Figure 2) from clone pGPE208 (Casals et al. 2003). Prior to the amplification, DNA from pGPE208 was digested with DraI to separate the IRs and to prevent the formation of secondary structures.

In situ hybridization:

Hybridization to the larval salivary gland polytene chromosomes was carried out according to the procedure described by Montgomery et al. (1987) using the same 1.1-kb Galileo probe as in the Southern hybridizations. Probe was labeled with biotin-16-dUTP (Roche) by nick translation. Intraspecific in situ hybridizations with D. buzzatii lines including all the chromosomal arrangements studied (st-1, j-2, j-23, j-24, jq7-4, jz3-6, jz3-7, y3-1, and s-1) were carried out at 37°. Interspecific hybridizations with the 12 lines from other Drosophila species (supplementary Table 1 at http://www.genetics.org/supplemental/) were carried out at 25°. When the line was polymorphic for different chromosomal arrangements, hybridization was performed over heterokaryotypes. Detection was carried out using the ABC-Elite kit from Vector Laboratories (Burlingame, CA). Chromosomal localization of the hybridization signals was determined using the cytological maps of D. buzzatii, D. koepferae, D. gouveai, and D. seriema (Ruiz and Wasserman 1993) and of D. antonietae and D. serido (Ruiz et al. 2000). The relative length of chromosomes and of the different chromosomal regions was estimated according to the number of bands included in them in D. repleta, the reference species of the group (Wharton 1942). Coincidence of TE insertion sites and the cytological localization of chromosomal inversion breakpoints described in D. buzzatii and the buzzatii species complex (supplementary Table 2 at http://www.genetics.org/supplemental/) was tested with the statistical method described by Zelentsova et al. (1999).

DNA sequencing and sequence analysis:

Sequences were obtained on an ABI 373 A (Perkin-Elmer, Norwalk, CT) automated DNA sequencer. Fragments cloned into Bluescript II SK or pGEM-T were sequenced using M13 universal forward and reverse primers. PCR products were gel purified using the Geneclean spin kit (Bio 101) and sequenced directly with the same primers used for amplification. Nucleotide sequences were analyzed using GeneToolLite software (BioTools). Similarity searches in the GenBank/EMBL databases were carried out using Blastx, Tblastx, and Fasta. Multiple sequence alignments were performed with ClustalW (Thompson et al. 1994), followed by analysis with the DnaSP version 4.0 program (Rozas et al. 2003). Phylogenetic analysis was performed using the PHYLIP software package (Felsenstein 1989).

RESULTS

New copies of D. buzzatii foldback elements:

We have isolated 10 additional copies of the three foldback elements Galileo, Kepler, and Newton through PCR amplification and genomic library screening. PCR amplification was carried out using primers G7 and G8, located in the IRs of the elements (Figure 2). Cloning and sequencing of the three most intense bands (0.6, 1.3, and 2.8 kb) resulted in four new foldback elements (Table 1 and Figure 2). The 0.6-kb fragment contained a small copy of Kepler (Kepler-4), whereas the 1.3-kb fragment was formed by partial copies of Galileo (Galileo-8) and Newton (Newton-3) consecutively arranged. The 2.8-kb fragment contained sequences homologous to Galileo IRs at both ends (Galileo-7), separated by an ISBu2 element (ceres et al. 2001) of 553 bp located 8 bp away from the left IR; an ISBu1 element (ceres et al. 2001) of 61 bp located 191 bp away from the right IR; and ~1 kb of unknown composition. Therefore, apparently only partial and chimeric elements were amplified by PCR, probably because the formation of secondary structures between the long IRs of complete elements hinders their PCR amplification (ceres et al. 2001).

TABLE 1
Summary of the main characteristics of the different copies of the foldback elements ofD. buzzatii

As an alternative approach, we also searched for new copies of the elements by screening a genomic library of line j-1 (ceres et al. 1999b) with a Galileo-2 probe. Many positive phages were obtained, and several of them were selected for further study by restriction enzyme analysis and Southern hybridization. Bands of interest from four of these phages (λj-1/1, λj-1/2, λj-1/3, and λj-1/4) were subcloned and partially sequenced. Overall, five new copies of Galileo and one copy of Kepler were found (Table 1 and Figure 2), together with some other TEs. λj-1/1 contained the Galileo-5 and Kepler-6 elements arranged in tandem. λj1-3 contained the Galileo-6 element only. λj1-4 consisted of two Galileo elements (Galileo-9 and -13) separated by ~11 kb, plus several copies of other TEs. Galileo-9 is flanked by a 446-bp fragment of BuT3 (ceres et al. 2001) and a BuT1 element of 932 bp (ceres et al. 2001), located 0.9 kb and 21 bp, respectively, away from the end of the IRs. The BuT1 element contains a 1731-bp insertion of an element homologous to the Tc-1-like transposon Paris of D. virilis (Petrov et al. 1995), which had not been previously described in D. buzzatii. Galileo-13 is inserted into an ISBu1 element of 894 bp, which is separated by 12 bp from a 103-bp fragment of an Osvaldo retrotransposon (Labrador and Fontdevila 1994). Finally, λj-1/2 included the Galileo-14 element, flanked at one side by an IsBu1 element (ceres et al. 2001) 79 bp from its left IR and a BuT6 element (Casals et al. 2003) immediately adjacent to the other side. The inclusion of truncated copies of foldback elements and several other TEs in most of these phages resembles the organization of D. melanogaster β-heterochromatin (Hoskins et al. 2002) and suggests a heterochromatic origin.

Structure of the foldback elements of D. buzzatii:

Altogether, we have analyzed 14 copies of Galileo, 6 of Kepler, and 3 of Newton (Table 1 and Figure 2). The main characteristic of the three types of elements is the presence of very long IRs, often spanning almost the entire element. In addition, these elements show the high degree of structural variability between and within copies, which is also typical of other foldback elements. Despite the differences between the multiple copies and the fact that many of them are incomplete, a consensus canonical structure for each of the elements can be inferred.

Galileo elements vary in size from 20 to 2304 bp with IRs from 8 to 1115 bp (Table 1) and show the structural domains previously described in other foldback elements (Figure 2). The most external part of Galileo IRs contains a 479-bp terminal region (IR-FD), which is relatively conserved in all copies. This domain is followed by up to three imperfect 136-bp tandem repeats, with an average identity of 91%, plus a fourth smaller incomplete repeat of 43 bp (IR-OD). Typically there are two to three repeats per IR and this number differs between both sides of the element. The IR-ID domain is extremely heterogeneous in length and sequence composition and is characterized by a high AT content (up to 75%). In Galileo-3 and -6, this region includes a 141-bp open reading frame (ORF) that shows similarity (34% amino acid identity) with the transposase of Hoppel, a P-like element of D. melanogaster (Reiss et al. 2003; Figure 2). Finally, several of the elements contain a central region of 81–158 bp that has not yet been found to form part of the IRs and can be considered to represent the M domain.

Kepler and Newton show a 90% nucleotide identity and differ from Galileo in that they apparently contain only IR-FD and M domains, but not tandem repeats (IR-OD) or IR-ID domains (Figure 2). The Kepler elements analyzed show considerable variability and include mostly partial copies, with sizes between 381 and 930 bp (Table 1). In the longest copy (Kepler-5), the IRs are formed by an IR-FD domain of ~346 bp flanking an M section of 254 bp. The two longest Newton elements (Newton-1 and -2) are almost identical, with IR-FD domains of 566–575 bp and an M segment of 363–378 bp. Neither ORFs coding for >100 amino acids nor sequence similarities with a transposase have been observed for these elements.

In addition to the structural similarity, the IRs of the three foldback elements of D. buzzatii also share a high degree of sequence homology. On average, the first ~600 bp of Galileo (IR-FD and part of the first tandem repeat), Kepler (IR-FD and most of M region), and Newton (IR-FD) have an ~73% nucleotide identity (ceres et al. 2001). In particular, the most terminal 40 bp are almost identical between them and a more internal region of 200 bp shows an ~86% mean pairwise nucleotide identity (Figure 2). Another common characteristic of Galileo, Kepler, and Newton is the generation of a 7-bp duplication of the target site during insertion. The comparison of the 19 flanking sequences of these elements (Table 1) suggests that their preferential insertion site has a consensus sequence of G16T16a10g10T15A18c9. Interestingly, this sequence is palindromic and the first two bases of each side are complementary to the ends of the elements (CA … TG).

Phylogenetic analysis of D. buzzatii foldback elements:

The structural and sequence similarity of Galileo, Kepler, and Newton suggests that they are related and belong to the same family of TEs. To determine the phylogenetic relationship among them, we built an unrooted tree by neighbor joining using the two homologous sequences of the IRs (totaling ~250 bp; Figure 2). Only the elements that contain both complete regions were included in the analysis and, when available, the two ends of the elements have been considered independently (Figure 3). The tree shows two highly divergent clades, one including the Galileo copies and the other one the copies of Kepler and Newton. Galileo copies show a high degree of variation and form several different subgroups. Kepler and Newton copies form two separate yet closely related monophyletic groups (Figure 3). In most cases, the two IRs of the same element group closely together, which indicates a higher similarity within than between elements (Casals et al. 2003).

Figure 3.
Neighbor-joining tree of the 251-bp-long homologous regions of the IRs of the Galileo, Kepler, and Newton elements described in this work. When available, a and b designate, respectively, the left and right half of the element according to Figure 2. Only ...

Abundance and chromosomal distribution of foldback elements in D. buzzatii:

To estimate the abundance of foldback elements, Southern blots of genomic DNA from 15 different D. buzzatii lines were carried out with a Galileo-12 probe, which contains sequences homologous to the three elements. Genomic DNA of each line was digested with restriction enzymes BamHI and HindIII, which do not have restriction sites in any of the 23 copies of Galileo, Kepler, and Newton reported here. The number of hybridization bands per line provides a minimum estimate of the number of Galileo-like TEs in the D. buzzatii genome and varies between 21 and 29, with an average of 26.7 (Table 2). No significant differences among the different lines were observed (χ2 = 3.63; d.f. = 14; P = 1.00).

TABLE 2
Observed insertions of foldback elements in lines ofD. buzzatii and other Drosophila species by Southern blot andin situ hybridization analysis

In situ hybridization to the polytene chromosomes of nine different D. buzzatii lines with the same Galileo-12 probe allowed us to examine the chromosomal distribution of these TEs. In all cases, several hybridization signals in the euchromatic portion of the chromosomes plus a strong staining in the centromeres were observed (Figure 4). The cytological localization of these signals is summarized in supplementary Table 3 at http://www.genetics.org/supplemental/. The average number of euchromatic signals per line was 56.1 (Table 2), and no significant differences among lines were observed (χ2 = 5.32; d.f. = 8; P = 0.72). In addition, we compared the distribution of these signals among chromosomes, pooling together the data of all D. buzzatii lines (Table 3). A very significant deviation between the observed number of signals in each chromosome and that expected according to a random distribution was found. This difference was mainly due to the accumulation of insertions in the dot chromosome 6 (Table 3). When this chromosome is excluded from the analysis, there are still significant differences in the number of signals among chromosomes, apparently due to an excess of insertions in chromosome 3 and a deficit of insertions in chromosome 5 (Table 3). However, no significant differences between the X chromosome and the autosomes were found (χ2 = 3.13; d.f. = 1; P = 0.08).

Figure 4.
In situ hybridization of a Galileo-12 probe to the salivary gland chromosomes of D. buzzatii lines jz3-7 (a), jq7-4 (b), s-1 (c), D. serido (d), D. antonietae (e), and D. stalkeri (f). Arrows indicate hybridization signals at the breakpoints of inversions ...
TABLE 3
Foldback element insertions observed in the chromosomes of six species of thebuzzatii cluster

We analyzed the intrachromosomal distribution of these elements by comparing the observed and expected number of signals in the distal, central, and proximal regions of the chromosomes. The distal and the proximal regions were defined as the 10% of chromosomal bands closer to the telomere and centromere, respectively, in the cytological map of the species (see materials and methods). Galileo elements clearly tend to accumulate in the proximal regions, as measured by both pooling the data of all chromosomes together (Table 4) and considering each individually (data not shown). Moreover, the insertions in the proximal regions and also in chromosome 6 are observed in most lines (relative frequency 0.64 and 0.66, respectively) whereas those insertions in the distal and central regions are usually found in a single line (relative frequency 0.15 and 0.16, respectively).

TABLE 4
Foldback element insertions observed in three chromosomal regions of six species of thebuzzatii cluster

Finally, we examined the distribution of the euchromatic Galileo insertions in five natural inversions of D. buzzatii. When the number of signals inside and outside the inverted region were compared, a significant association between the TE insertions and the chromosomal inversions was found (Table 5). The accumulation of insertions was especially clear in inversions 2q7 and 2y3 (Table 5). Conversely, the only inversion induced by introgressive hybridization (5I) did not show any association with insertions (χ2 = 1.00; d.f. = 1; P > 0.05). The Galileo element was located at the breakpoints of inversions 2j and 2q7 (Figure 4, a and b), as previously shown (ceres et al. 1999b, 2001; Casals et al. 2003), and at the proximal breakpoint of inversion 4s (Figure 4c). However, a global comparison of the chromosomal distribution of Galileo insertions and the cytological position of the breakpoints of the 16 natural polymorphic inversions described in D. buzzatii and 18 inversions induced by introgressive hybridization did not find a significant association (supplementary Table 4 at http://www.genetics.org/supplemental/).

TABLE 5
Foldback element insertions inside and outside of the inverted segment in five polymorphic inversions present in natural populations ofD. buzzatii

Abundance and chromosomal distribution of foldback elements in the buzzatii species complex:

Southern blot and in situ hybridization were also carried out in 10 additional species of the buzzatii complex (supplementary Table 1 at http://www.genetics.org/supplemental/) and two other species of the repleta group, D. mulleri (mulleri subgroup) and D. repleta (repleta subgroup). The number of bands produced by Southern hybridization in the species of the buzzatii cluster (D. seriema, D. koepferae, D. antonietae, D. serido, and D. gouveai) was similar to that seen in D. buzzatii, whereas fewer bands were observed in the species included in the martensis cluster (D. martensis, D. uniseta, D. venezolana, and D. starmeri) or in the stalkeri cluster (D. stalkeri). No bands could be observed in D. mulleri and D. repleta (Table 2).

In situ hybridization of the polytene chromosomes of these species (supplementary Table 3 at http://www.genetics.org/supplemental/) yielded results similar to the Southern analysis. Few hybridization signals were observed in the martensis and stalkeri cluster species, and all of them were restricted to chromosome 6 and the proximal regions of the other chromosomes (Figure 4f). In the species of the buzzatii cluster the number of signals and their inter- and intrachromosomal distribution were similar to those in D. buzzatii lines (Tables 24; Figure 4, d and e). First, signals tend to accumulate in the dot chromosome of all species (Table 3). When chromosome 6 is excluded from the analysis, there were still significant differences in the distribution of insertions between chromosomes in D. gouveai and D. koepferae (Table 3). In addition, there were significant differences in the number of insertions between the X chromosome and the autosomes in D. gouveai2 = 11.98; d.f. = 1; P < 0.001), D. koepferae2 = 9.72; d.f. = 1; P = 0.002), D. serido2 = 6.24; d.f. = 1; P = 0.012), and D. seriema2 = 4.58; d.f. = 1; P = 0.032). Second, in all cases TE insertions accumulated in the proximal regions of the chromosomes (Table 4). However, if the proximal regions are excluded from the analysis, no significant differences are detected between the X chromosome and the autosomes in any species, and there are significant differences among chromosomes only in D. buzzatii2 = 21.55; d.f. = 4; P < 0.001).

When the relationship between TE insertion sites and the cytological position of inversion breakpoints was examined, hybridization signals were found precisely at the two breakpoints of the 2a8 inversion of D. serido (Figure 4d). Overall, no significant association was observed between chromosomal inversion breakpoints and hybridization signals for the three species in which polymorphic inversions have been reported (D. koepferae, D. serido, and D. seriema; supplementary Table 4 at http://www.genetics.org/supplemental/). However, after taking into account the breakpoints of all the fixed and polymorphic inversions described in the buzzatii cluster species, there is a strong association with the location of foldback insertions (supplementary Table 4 at http://www.genetics.org/supplemental/).

DISCUSSION

Similarities of Galileo, Kepler, and Newton with other foldback elements:

The general organization and main features of the foldback elements described in other eukaryotic organisms are summarized in Figure 1. According to Rebatchouk and Narita (1997) and Simmen and Bird (2000), foldback elements can be classified in five types depending on the presence of different domains in the IRs. Galileo elements fall unambiguously into the type 4 class, since in most cases they include IR-FD, IR-OD, and IR-ID domains (Figure 2). It is noteworthy that the internal tandem repeats of Galileo IR-OD are larger than those of other foldback elements, which are usually 7–32 bp long. Kepler and Newton elements show only the IR-FD domain and M region (Figure 2) and can be tentatively classified as a new type 6 of foldback elements, although it is possible that all copies analyzed here are defective. This structural organization resembles that of the Hairpin elements described in A. thaliana, which have been classified as type 3 foldback elements due to the presence of AT-rich IRs (equivalent to the IR-ID of other foldback elements) (Adé and Belzile 1999). The IRs of Kepler and Newton show two blocks of high sequence similarity to the IR-FD and first tandem repeat of the IR-OD of Galileo (Figure 2). A similar situation in which foldback elements with an overall different organization contain homologous modules has been found in other organisms. For example, the IR-ODs of the type 1 elements SOFT1 and SOFT2 of tomato and the type 2 element SoFT3 described in potato show a high level of sequence similarity (Rebatchouk and Narita 1997). In A. thaliana, FARE1 and FARE2 elements share sequence and structural similarities at the terminal regions of the IR-OD, although the arrangement of the tandem repeats of these elements is more complex (Windsor and Waddell 2000).

In general, the mechanism of transposition of foldback elements and the proteins involved remain largely a mystery. Similarly, the copies of the three D. buzzatii foldback elements that we have characterized do not show any evidence of coding capacity or ORFs with homology to a known protein, with the exception of two Galileo elements bearing a segment with low similarity to the Hoppel transposase. The high level of structural heterogeneity described in these and other foldback elements (Hoffman-Liebermann et al. 1985; Cheng et al. 2000; Windsor and Waddell 2000; this work) suggests that all the elements found could be defective and have lost the transposase coding sequence. Alternatively, these elements could be mobilized through the proteins produced by other TEs or cellular processes (Rebatchouk and Narita 1997). It has been proposed that FARE2 elements of A. thaliana encode proteins that could also interact with homologous structures in FARE1 elements, which do not show coding capacity, and promote their mobilization (Windsor and Waddell 2000). In D. buzzatii, the similarities between the target insertion sites of the three foldback elements suggest that probably they are mobilized by the same mechanism of transposition. In addition, the conservation of certain regions of the IRs, especially the ends, could be indicative of a possible role in the recognition and binding of the transposase. Finally, it seems that the internal tandem repeats could play an important role in the recognition and binding of proteins. These sequences could be the target of the transposase, which first binds to them and then moves to the end of the element. A higher probability of mobilizing an element would be achieved by increasing the number of tandem repeats (Cheng et al. 2000).

Estimate of the age of D. buzzatii foldback elements:

The presence of some polymorphic Galileo, Kepler, and Newton insertions at the 2j and 2q7 inversion breakpoints (Table 1) indicates that these elements were active in D. buzzatii at least until recently. The age of TEs can be estimated by calculating the average pairwise nucleotide diversity among the different copies (Brookfield 1986; Kapitonov and Jurka 1996; Costas and Naveira 2000; Bowen and McDonald 2001). Table 6 shows an estimate of the time to coalescence of the Galileo, Kepler, and Newton copies, considering all the sequence information available for each of the copies and assuming an average synonymous substitution rate of Drosophila of 0.016 substitutions/nucleotide/million years (Li 1997). When the sequences of the three elements are considered separately, time to coalescence is 3–5 million years (Table 6). However, the divergence between different elements is much older, with a divergence time between Galileo and Newton of ~10 million years (Table 6). These estimates suggest that these elements are old components of the D. buzzatii genome but are subject to a considerable uncertainty. For instance, the nucleotide substitution rate of TE sequences might be higher than the Drosophila average, resulting in an overestimation of the age of the elements. Conversely, if parts of the elements are under selective constraint, this dating could be an underestimation.

TABLE 6
Nucleotide variation and age estimate for the three foldback elements ofD. buzzatii

An alternative method to estimate the age of transposable elements is based on their presence in other phylogenetically related species. The divergence time of the buzzatii cluster (where D. buzzatii belongs) from the other two clusters of this complex, the martensis and stalkeri clusters, is 5.8 and 6.2 million years, respectively (Russo et al. 1995; Rodríguez-Trelles et al. 2000). By Southern blot and in situ hybridization analysis (Table 2 and Figure 4), we have shown that Galileo elements are very abundant among the buzzatii cluster species. They are also present in the martensis and stalkeri cluster species, although in smaller numbers and seemingly located in the proximal regions of the chromosomes and in the dot chromosome only (Table 2 and Figure 4). Foldback elements were not detected in the more distantly related species D. mulleri (mulleri subgroup) and D. repleta (repleta subgroup), although this observation could be due to the fact that the sequence divergence of the elements makes them undetectable with the techniques used here. In summary, the interspecific distribution pattern of these foldback elements suggests that they were present before the radiation of the buzzatii complex, being transmitted vertically during the subsequent speciation events. No evidence of horizontal transmission was found here or in previous studies of the interspecific distribution of other foldback elements, such as that of the D. melanogaster FB element in the genus Drosophila (Silber et al. 1989) or the SoFT elements in the Solanum and Lycopersicon genus (Rebatchouk and Narita 1997). These results differ considerably from what has been observed in several class II transposons, which have been frequently transmitted horizontally between distant species (Capy et al. 1998).

Factors determining the chromosomal distribution of the elements:

Galileo, Kepler, and Newton elements are found in moderately high copy numbers in the genome of the buzzatii cluster species. These elements are not randomly distributed and clearly tend to accumulate in the proximal regions of the chromosomes (Table 4) and in the dot chromosome (Table 3). Two main factors have been proposed to account for the distribution of TEs in the genome: recombination rate and gene density. Recombination rate is expected to inversely affect TE abundance in two different ways. First, the reduction of recombination rate in certain regions will hinder the elimination of slightly deleterious insertions by natural selection (Hill and Robertson 1966; Gordo and Charlesworth 2001). Second, TEs inserted in regions of low recombination will probably avoid the generation of deleterious chromosomal rearrangements by ectopic recombination (Langley et al. 1988; Montgomery et al. 1991; Goldman and Lichten 1996). On the other hand, TEs could accumulate in regions of low gene density, where their insertion is less likely to cause deleterious effects.

Although very little is known about the genetic map of these species (Schafer et al. 1993), the situation is probably very similar to that of D. melanogaster, in which the recombination is reduced around the proximal and distal ends of the chromosomes and disappears completely in the dot chromosome (Charlesworth 1996). At the proximal end, recombination disappears in all chromosomes in a region spanning 0.3 to 1.1% of the chromosome and is reduced in the adjacent region of 5–14%; at the distal end, the recombination is reduced in all chromosomes (5.2–10.6% of the chromosome) and disappears only in a 1.7% region in the X chromosome (Charlesworth 1996). Drosophila species show differences both in genetic map length and in the variation of the recombination rate between chromosomal regions (True et al. 1996; ceres et al. 1999a). Considering all this, we defined the distal and the proximal regions as the 10% of chromosomal bands closer to the telomere and centromere, respectively, according to the available cytological maps of each species (Table 4). Thus, the accumulation of D. buzzattii foldback elements close to the centromeric regions and in the dot chromosome is consistent with the expected effect of recombination rate. The same chromosomal distribution pattern was described in the TEs of D. melanogaster (Bartolomé et al. 2002; Kaminker et al. 2002), where the elements tend to be localized in known regions of low or null recombination rate (Ashburner 1989; Charlesworth 1996). Previous studies also showed a high density of TEs in the heterochromatin of several Drosophila species (Charlesworth et al. 1994; Carmena and González 1995; Pimpinelli et al. 1995; Dimitri 1997; Junakovic et al. 1998; Dimitri et al. 2003). Finally, the colonization by four retrotransposons of the neo-Y chromosome of D. miranda is further evidence of the accumulation of TEs in low-recombination regions (Steinemann and Steinemann 1991, 1997; Bachtrog 2003). It has been proposed that regions with high-recombination rates contain a higher number of genes that could make the survival of TE insertions difficult. However, the accumulation of TEs in the dot chromosome, whose genic density is similar to that of other regions with high recombination (Charlesworth et al. 1992; Bartolomé and Maside 2004), contradicts this hypothesis. Interestingly, in both D. melanogaster (Bartolomé et al. 2002; Kaminker et al. 2002) and D. buzzatii (this work) TEs do not accumulate in telomeric regions, where the recombination rate is also known to be low (Charlesworth 1996). One possible explanation is that ectopic recombination is not reduced in these regions (Bartolomé et al. 2002), as has been found in subtelomeric regions in yeast (Haber et al. 1991). In addition, the low-recombination regions close to the telomere are generally shorter than those in the centromeric regions, and the presence of TE insertions in low-recombination regions appears to be related to the distance from the high-recombination region (Maside et al. 2001).

The distribution of TEs within chromosomal inversions provides additional support for the idea that recombination rate is the main factor determining the distribution of TEs in the genome (Eanes et al. 1992; Sniegowski and Charlesworth 1994). Recombination rate drops inside the inverted region in heterokaryotypes and this reduction extends for ~1 Mb from the breakpoints (Andolfatto et al. 2001). In D. buzzatii, foldback elements clearly tend to accumulate in the inverted regions (Table 5). Notably, this accumulation is most pronounced in the two inversions with the lowest frequency in natural populations. The majority of chromosomes carrying these inversions will be found in heterozygosis, and thus we expect a larger reduction of recombination than in inversions with higher frequency (Eanes et al. 1992). Furthermore, the recombination reduction is especially pronounced in the inversion breakpoints (Navarro et al. 1997), which could explain the presence of multiple TE insertions in the breakpoints of the 2j and 2q7 inversions (ceres et al. 2001; Casals et al. 2003). However, a tendency for certain TEs to insert inside each other could also be involved (ceres et al. 2001). Because of the accumulation of TEs within inversions, there is an excess of insertions on the second chromosome of D. buzzatii (significant after excluding the dot and the proximal chromosome regions), which is not observed in the other species.

The precise localization of a foldback element at one breakpoint of the 4s inversion of D. buzzatii (Figure 4c) and the two breakpoints of the 2a8 inversion of D. serido (Figure 4d), together with the implication of Galileo in the generation of D. buzzatii inversions 2j and 2q7 (ceres et al. 1999b; Casals et al. 2003), suggests that these elements could have played an important role in the genome evolution of the buzzatii species complex. Moreover, there is a high correlation between foldback elements insertion sites and the cytological location of the breakpoints of the chromosomal inversions of the buzzatii species complex (supplementary Table 4 at http://www.genetics.org/supplemental/). This kind of association has been interpreted as evidence supporting the implication of TEs in the generation of the inversions in the virilis group of Drosophila (Zelentsova et al. 1999; Evgen'ev et al. 2000). It has also been suggested that the breakpoints of the two D. buzzatii inversions characterized so far are genetically unstable regions, probably because of the presence of foldback elements (ceres et al. 2001; Casals et al. 2003). Studies of the chromosomal distributions of other TEs found in these species may help to corroborate this extent and to check if foldback elements are able to induce hotspots for TE insertions at other genomic regions.

Acknowledgments

We thank J. M. Ranz for initiating the in situ hybridization analyses, M. Puig for the information on Hoppel transposase homology, and M. Badal for helpful comments on the foldback elements structure. This work was supported by grant BMC2002-01708 from the Dirección General de Enseñanza Superior e Investigación Científica (Ministerio de Educación y Cultura, Spain) awarded to A.R. and a personal fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo (Brazil) proc.01/06373-6 to M.H.M.

Notes

Sequence data from this article have been deposited in the EMBL/GenBank Data Libraries under accession nos. AY756161, AY756162, AY756163, AY756164, AY756165, AY756166, AY756167, AY756168, AY756169, AY756170.

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