Institut Jacques Monod, UMR 7592 Dynamique du Génome et Evolution CNRS, Universités P. et M. Curie, D. Diderot, Paris, France
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Abstract |
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Key Words: Hoppel element P element transposable element introns Drosophila melanogaster
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Introduction |
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The P element is a class II transposable element flanked by 31-bp terminal inverted repeats (TIRs) (O'Hare and Rubin 1983). It transposes via a cut-and-paste mechanism (Kaufman and Rio 1992). The canonical full-length P element is 2,907 bp in length and contains four exons (O'Hare and Rubin 1983). In germ line cells, all four exons are required and encode an 87-kDa transposase (Karess and Rubin 1984; Rio, Laski, and Rubin 1986). In somatic cells, the third intron is retained and produces a 66-kDa truncated transposase (Rio, Laski, and Rubin 1986), which acts as a repressor of transposition (Robertson and Engels 1989; Misra and Rio 1990).
The P element was first isolated in Drosophila melanogaster (Bingham, Kidwell, and Rubin 1982), but further investigations led to the discovery of P homologs in numerous Drosophila species (for review, see Pinsker et al. 2001) and even in other genera like Scaptomyza (Simonelig and Anxolabéhère 1991). Sequences homologous to the P element have also been detected in other Diptera, like Musca domestica (Lee, Clark, and Kidwell 1999) and Lucilia cuprina (Perkins and Howells 1992), and have recently been detected in humans (Hagemann and Pinsker 2001). The study of P-element distribution reveals several discontinuities suggesting the occurrence of horizontal transfers or of losses of the element (Pinsker et al. 2001). These discontinuities are well illustrated in the melanogaster subgroup. No P sequences have been detected in the melanogaster species subgroup, except in D. melanogaster, which acquired the P element by horizontal transfer from D. willistoni approximately 50 years ago (Daniels et al. 1990). The D. melanogaster laboratory strains collected before this time are devoid of P elements, as are closely related species (Kidwell 1983; Anxolabéhère, Kidwell, and Periquet 1988). However, the melanogaster subgroup belongs to the Sophophora subgenus, in which P sequences are widely distributed, suggesting that P sequences were present in the common ancestor of these species. Thus, the absence of P homologs in the melanogaster subgroup could result either from their strong divergence beyond recognition by the methods available until recently, or from their loss from these genomes (Clark, Kim, and Kidwell 1998). To distinguish between these two hypotheses, we carried out an in silico search for P-homologous sequences in the D. melanogaster genomic sequence, which is devoid of any recently acquired P elements. Our search led to the detection of a sequence with a significant level of sequence similarity to P-element proteins. Moreover, this sequence shared several structural characteristics with the P element (e.g., TIR size, footprint arrangement), providing evidence for a functional relationship with the P-element family. In fact, this sequence belongs to the Hoppel-element family, which is known to be composed exclusively of short copies (Kurenova et al. 1990). These short copies are deleted copies of the large element that we described here. Structural data from the coding region of this full-length Hoppel show that it is devoid of the canonical P-element introns. To explain this feature, two alternative evolutionary scenarios will be discussed: (1) loss of introns as a result of the retrotranscription of a P-mRNA and (2) acquisition of introns.
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Materials and Methods |
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DNA Amplification, Cloning, and Sequencing
Total genomic DNA was extracted from 50 flies from each strain, as described by Junakovic, Caneva, and Balario (1984). For single fly extractions, the same protocol was followed. The sequences of the oligonucleotide primers used are given in table 1. Amplification was performed with 1.25 units of AmpliTaq DNA polymerase (Perkin) in a 25 µl volume, with 50 ng of genomic DNA and buffer adjusted to 1.5 mM of MgCl2. When screening species, the concentration of MgCl2 was adjusted to 2.5 mM. The reaction conditions were: 92°C for 5 min and 30 cycles of 92°C for 1 min, hybridization temperature (depending on the primer) for 30 s and 72°C for 1 min 30 s, followed by 10 min at 72°C. For the single fly amplifications with three primers (one forward and two reverse), the concentration of the forward primer was double that of the reverse primers. Polymerase chain reaction (PCR) products were separated in a 1% agarose gel or in a 1.5% agarose gel for single fly PCR products. Long PCR was performed with the Expand Long PCR system (Boehringer Mannheim), according to the supplier's recommendations.
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RNA Isolation and Reverse Transcriptase Amplification
Total RNA was isolated from the ovaries of 25 ISO1 flies by use of the RNeasy Mini Kit (Qiagen), which includes a DNase step. cDNA was obtained with the Omniscript RT (reverse transcriptase) Kit (Qiagen), by use of the oligo-dT primer and DNA amplification followed at the conditions described above.
Sequence Analysis
The GenBank accession numbers of all the sequences used in this study are shown in table 2. D. melanogaster genomic sequences were analyzed at the Berkeley Drosophila Genome Project (BDGP) Web site (http://www.fruitfly.org). Blast searches were performed using the WU-Blast package (http://www.fruitfly.org/blast/; W. Gish 1996, 2002; http://www.blast.wustl.edu) with the default parameter values. Putative promoters were identified by use of the Neural Network Promoter Prediction tool (http://www.fruitfly.org/seq_tools/promoter.html). Polyadenylation signals were identified by use of the Nucleotide Sequence Analysis tool in the GCG (1990) program (http://genomic.sanger.ac.uk/gf.gf.html).
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PMC(Si|) is the probability of nucleotide Si using the Markov chain parameters
. a
i,
i+1 reflects the probability of switching from state
i to state
i+1 (transition probability). Thus, the parameters of an HMM with n states are n2 transition probabilities and n sets of Markov chain parameters (also called emission probabilities).
We used an HMM with five states: coding exons in phases 1, 2, and 3, intron, and "terminal" sequence. A transition departing from a coding exon state can go to the next phase coding exon state (most probable), or the intronic state; it can also stay in the same state or go to the preceding coding state, to account for frameshifts. The terminal state represents the noncoding, non-intronic parts at the beginning and end of the sequences. A path in the HMM starts and ends in this state. For each state, the emission probabilities are simple second-order Markov chains.
The models were trained using the Baum-Welsch algorithm, an EM (expectation maximization) algorithm (Durbin et al. 1998) with labeled sequences of P elements. The starting point for the iterative Baum-Welsch algorithm was an HMM with random emission probabilities but fixed transition probabilities. Training estimates these emission probabilities and refines the transition probabilities, but the overall structure of the HMM is maintained. The training was repeated several times with different random starting points, and the estimate with the best likelihood was kept.
Given a nucleotide sequence S, we calculated the most probable state sequence using the Viterbi algorithm (Durbin et al. 1998). We also calculated the posterior probability of states, that is the probability that nucleotide i lies in a state k of the HMM: ). By calculating this for each nucleotide of S, we were able to plot the probability of state k along the sequence.
Multiple alignments were obtained with the PILEUP program of the GCG package (Madison, Wis.) using the default options.
Phylogenetic Analysis
Aligned sequences were analyzed by the Neighbor-Joining method in the PHYLO_WIN program (Galtier, Gouy, and Gautier 1996). PAM distance and global gap removal options were chosen for Neighbor-Joining analysis. Five hundred bootstrap replicates were performed.
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Results |
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The in silico analysis of the regions upstream and downstream of the P-like sequence revealed the presence of TIRs that are characteristic of class II transposable elements. These TIRs were 31 bp long and were flanked by 7-bp target site duplications (TSDs). The size of the hypothetical transposon is 3,408 bp. Another copy of the same length and sharing 99.5% identity was detected in the chromosomal site 38A (accession number AE003664). A BlastN search was carried out to detect other genomic copies of this transposon. At least 100 copies carrying deletions in their terminal or central region were found throughout the genome. A particular class of these dispersed and deleted sequences has been previously described and is called the 1360 or Hoppel-element family (Kholodilov et al. 1988; Kurenova et al. 1990). The Hoppel element is about 1,100 bp long and is entirely encompassed within the 3.4-kb element under investigation (fig. 1a). The only elements of the Hoppel family that have been described to date are internally deleted elements that do not contain large enough open reading frames (ORFs) to encode a transposase and are devoid of the P-like sequence. We propose that the 3.4-kb elements be included in the Hoppel family. It is noteworthy that the 3.4-kb sequences can also be found in Repbase Update (Jurka 2000) with the protoP identifier.
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Sequence Analysis of the 3.4-kb Hoppel Transposable Elements: Hoppel-1 and Hoppel-2
Sequence analysis of Hoppel-1 and Hoppel-2 elements has shown that TIRs can be extended up to 51 bp long. These 51-bp TIRs are composed of the 31-bp perfect TIRs cited previously and 20 additional base pairs that are not perfectly repeated: only 15 bp of the 20 are identical (fig. 1a). In addition, a 31-bp footprint of Hoppel element (see below) is present within the element at positions 507538. It consists of the first 17 bp of the 5' Hoppel TIR and the first 14 bp of the 3' TIR.
To determine the capability of the 3.4-kb Hoppel elements to encode a transposase, ORFs were sought. The result for Hoppel-1 is shown in figure 1b. Three consecutive large ORFs, separated by a single stop codon, were found in the central region of the transposon (frame +3). These ORFs correspond to the peptide that was similar to the P proteins found by TBlastN. Hoppel-2 presents the same ORFs, except for a frame shift in the first ORF at position 1019 (data not shown).
To locate the position of P-element introns in the Hoppel sequence, the following proteins were aligned with the region of the Hoppel-1 element that contains the three ORFs including the two stop codons: DbifM, Dhel, Spal2, Dmel, and DbifO. The alignment was performed by the PILEUP program, and the results are shown in figure 2. The similarity between P and Hoppel extends from the beginning of the first Hoppel ORF, which matches the middle of the P-transposases exon 1, to the end of the third Hoppel ORF, which matches the end of the third exon of the P transposases (fig. 2). The Hoppel matching region cannot be expanded upstream in any of the reading frames. Remarkably, the amino acid sequence of the Hoppel element does not present any discontinuity of the similarity with the splicing regions of exons 12 and 23 of the P transposases. Moreover, the two stop codons are not located in these regions. Thus, the P-element introns are not present in the Hoppel sequence.
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Another TBlastN analysis was carried out with the peptide generated from the three ORFs of Hoppel-1 (including the two stop codons). One of the output sequences presented the same coding capacity as the query (91% similarity), but without the two stop codons. Thus, it gives rise to an ORF of 1,746 nucleotides (fig. 1c). The Hoppel element corresponding to this sequence (AC010916.8 location unknown) is truncated in its 5' extremity up to the nucleotide 570. It will be called 5'-Hoppel. This sequence was not considered in the first TBlastN analysis because it is not a full-length copy.
Taken together, the sequence analyses for Hoppel-1, Hoppel-2 elements and 5'-Hoppel, suggest a coding structure for the Hoppel element (fig. 4). The putative protein is 582 amino acids long and has an estimated molecular weight of 67.3 kDa. It is 41% similar to the Scaptomyza pallida P transposase.
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Mobility of Hoppel-1 and Hoppel-2
To check the mobility of Hoppel-1 and 2, the insertional site polymorphism was investigated in 18 strains of D. melanogaster (listed in Materials and Methods). This polymorphism was investigated by PCR using primers based on flanking and internal sequences (table 1). DNA from 50 individuals from each strain was analyzed, corresponding to 100 insertion sites for each element. A first PCR was carried out with a primer pair corresponding to the flanking regions of Hoppel-1 or Hoppel-2 (H1F 5'H1F 3' for Hoppel-1 and H2F 5'H2F 3' for Hoppel-2). A second PCR was performed using the 5' flanking primer coupled with the HIr1 reverse primer internal to the element. If there is an insert, no amplification product should be obtained with the first PCR, because the expected band is too large to be amplified in the conditions used. However, a band should be obtained with the second PCR and the internal primer. On the contrary, if there is no insertion, an amplification product should be obtained only with the first PCR, corresponding to the target devoid of the Hoppel element. In all tested strains, the specific Hoppel-1 primers amplified a product only in the second PCR. Thus, a Hoppel-1 insertion is present in all of the tested strains. Furthermore, no insertional polymorphism is present in any of them. Fourteen of the 18 strains tested contained the Hoppel-2 insertion. Amplification products were seen for the two types of PCR in 10 cases, a finding that provides evidence for insertional polymorphism (table 3).
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Similarities Between Hoppel and P-Element Features
Full-length Hoppel elements were found in the D. melanogaster genome because their amino acid sequences were similar to those of P elements. To confirm that the Hoppel element is a member of the P-element family, a profile HMM was derived from a multiple alignment of P-element transposases (HMMER package [Eddy 1998]): Dmel, DbifM, DbifO, Dhel, Spal2, Spal18, Luci, and Kboc. The P-element profile was then aligned with the Hoppel protein deduced from 5'-Hoppel (data not shown). The E value resulting from the alignment was highly significant (
), strongly suggesting that the Hoppel protein is related to the P transposases. The profile HMM of the three leucine zipper motifs (LZ1, LZ2, and LZ3, according to their relative positions on the P element) and the helix-turn-helix motif (HTH) present on the P transposases were established with the same set of P amino acid sequences. Five amino acids on each side of these motifs, as described in Nouaud and Anxolabéhère (1997), were included in the profile construction. Three of the four profiles could be identified on the Hoppel protein (fig. 4) with E values of
,
, and
, respectively, for the LZ2, LZ3, and HTH profiles. The positions of the motifs in the Hoppel protein corresponded to the positions in the P proteins. It is noteworthy that the LZ2 motif overlaps with the HTH domain as in the P proteins, and that LZ1 was not found in the Hoppel protein. The latter finding was expected, because the region including this motif does not exist in this protein.
Three other features imply that the Hoppel element is closely related to the P-element family. First, the Hoppel element has perfect 31-bp TIRs like the P element (O'Hare and Rubin 1983); however, they do not share significant similarity with any P-element TIRs. Second, the Hoppel footprints were the same size as those of the P element :17 bp of the 5' extremity and 17 bp of the 3' extremity (Takasu-Ishikawa, Yoshihara, and Hotta 1992; Staveley et al. 1995). Third, the TSD is 7 bp long for the Hoppel element and 8 bp long for the P element. These three characteristics of the DNA sequences are functionally associated with the transposase for type II transposable elements.
The DNA and protein data described above confirm that the Hoppel element is closely related to the P family, although its structure in a single exon is different from the canonical P structure, which is formed by four exons.
To define the relationship between Hoppel and other members of the P-element family, a phylogenetic analysis was performed using the Neighbor-Joining method. For this study, distantly related P transposases were chosen: Dmel, DbifM, DbifO, Spal2, Luci, Musca, Homo, and the Hoppel protein. The phylogenetic tree resulting from the Neighbor-Joining method is shown in figure 6. Hoppel is located out of the cluster formed by the other P elements. Thus, given their phylogenetic distance and their structural difference (1 versus 4 exons), we propose that the Hoppel element be included in the P-element superfamily.
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Discussion |
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The Full-Length Hoppel Element Is a Member of the P-Element Superfamily
We present structural and functional evidence that the Hoppel element is related to the P-transposable elements and should be included in the P superfamily. Multiple alignments of P proteins and the Hoppel-1 peptide provided by three consecutive ORFs (fig. 2) show that the region of similarity extends from the middle of exon 1 to the end of exon 3 of the P proteins. Pairwise comparisons of P proteins with the Hoppel protein displayed similarity values of about 40%. Moreover, two LZ motifs and a HTH motif, both characteristic of P transposases, are present on the Hoppel protein. In addition, TBlastN searches in Repbase Update (all organisms section merged) using the Hoppel protein as a query, detected significant similarity only with P transposases.
Regarding their working modalities, the P and Hoppel elements share two main characteristics. First, the perfect TIRs of the two elements are the same length, 31 bp. However, the Hoppel TIRs can be expanded to 51-bp imperfect TIRs. This can be explained by an extension of TIRs in the Hoppel lineage conferring a transposition efficiency advantage. Indeed, TIRs are important cis-acting DNA sequences that are necessary for efficient transposition. The two fixation sites of the P transpsosase (a 20-bp region recognized by the transposase) are located 16 bp from the 5' TIR and 4 bp from the 3' TIR (Kaufman, Doll, and Rio 1989). These two sites have a consensus sequence of 10 bp that are in inverted orientation with respect to the ends of the P element. In the Hoppel element, the two closely located inverted structures (TIRs and transposase fixation sites) could have been assembled, forming a 51-bp imperfect inverted sequence.
The second characteristic shared by the two elements is the structure of their footprints, despite the absence of significant similarity between their TIRs. The excision of the P element is mediated by 17-nucleotide staggered cleavages at its ends, which are without precedent for all known transposase and restriction endonuclease cleavage sites determined to date (Beall and Rio 1997). These cleavages result in the P-element footprint characteristics. Most of the footprints resulting from germ-line excision are formed by 16 ± 1 bp of the 5' and 16 ± 1 bp of the 3' ends (Takasu-Ishikawa, Yoshihara, and Hotta 1992; Staveley et al. 1995). Several Hoppel footprints detected on the whole genome sequence correspond to similar structures like the perfect 34-bp-long P footprints (17 bp of 5' and 17 bp of 3' TIRs) (e.g., fig. 5). Moreover, P-element footprints often present short sequences separating the two P-element extremities called "fillers." The filler usually corresponds to part of the 5' or the 3' TIRs. Filler sequences are probably generated by replication-slippage-replication during the breakpoint repair of the donor site (Kurkulos et al. 1994). Several Hoppel footprints also present additional nucleotides (fig. 5). Thus, the excision of the Hoppel elements leaves footprints with the same structural characteristics as those of P. The 17-bp staggering of cleavage sites is specific to the DNA binding site and the endonuclease site of the transposase (Beall and Rio 1997). Thus, the similarity of their footprints suggests that the Hoppel and the P transposases were derived from a common origin.
Moreover, the TSDs are 8 bp and 7 bp long for the P and Hoppel elements, respectively. This TSD size is a characteristic of the endonuclease activity of the transposase that causes a staggered cleavage at the target site. This observation alone has no significance concerning the relationship of the two elements, but together with the common features shared by their proteins, it reinforces the hypothesis that these elements share common functional modalities.
The Presence/Absence of Introns in the P Superfamily Results from Intron Loss or Gain Events?
Despite having homologous coding sequences, the structures of the P and Hoppel elements differ in two main ways. First, the coding region corresponding to exon 0 and the first half of exon 1 of the P element is totally absent from the Hoppel protein. Thus, if the amino acid multiple alignments are read from the COOH terminal to the NH2 end, there is an abrupt breakpoint of the similarity in the middle of exon 1. Second, the Hoppel coding region does not present the introns that interrupt the P-coding sequence. An appealing hypothesis explain these two differences with a single event : the Hoppel coding sequence arose from the retrotranscription of a P-element processed mRNA. Retropseudogenes and retrogenes can be recognized by the following characteristics: the lack of introns found in their functional counterparts, the presence of a poly-A tail at their 3' end and the presence of flanking TSDs (Vanin 1985). In addition, the interruption of the retrotranscription before the 5' end of the mRNA can lead to the 5' truncated cDNA observed in numerous cases. Thus, the coding region of the Hoppel element was probably derived from a 5' truncated P-element mRNA. However, there is no evidence for the presence of a poly-A tail in the downstream region of the Hoppel coding sequence or of the TSDs in its vicinity. This is expected if the retrotranscription event occurred a very long time ago. Another explanation for the lack of poly-A tail in the retrotranscripts is that the reverse transcription was initiated within a 3' A-rich region upstream of the poly-A tail (Kleene et al. 1998). Remarkably, the 3' regions of all P elements are A-rich, making this process possible.
Under this hypothesis we propose the following retrotranscription scenario: a P-element mRNA is retrotranscribed in the germ line; this retrotranscription is initiated inside a deleted P element from the same family as the one that provided the P-processed mRNA. This could result from a P element homing process (Delattre, Anxolabéhère, and Coen 1995; Delattre, Tatout, and Coen 2000). In fact, the P-element transposase, which has affinity for the P sequence, may remain attached to the P-mRNA and target it to a deleted P element inside of which the reverse transcription takes place. Finally, the retrotranscript is interrupted in the middle of exon 1. Thus, the resulting sequence is formed by the partial reverse transcription of the coding regions of a P element nested inside the 5' and 3' cis-acting sequences of the host P-deleted element. Although common in vertebrates (Vanin 1985; Wilde 1986), processed pseudogenes are rare in Drosophila for protein-coding genes (Jeffs and Ashburner 1991). However, flies do possess retroelements that generate reverse-transcriptase enzymes and sequences derived from the reverse transcription of RNA (Finnegan 1989). Very few retrogenes have been described in the Drosophila genome (Neufeld, Carthew, and Rubin 1991), and if this hypothesis is true, the Hoppel element would be the first example of a retrotranscribed class II transposable element. However, the Hoppel element is not the only member of the P superfamily presenting these characteristics. A P sequence without the canonical P introns, exon 0, and TIRs was recently described in the human genome and called Phsa (Hagemann and Pinsker 2001). This sequence has two introns located inside the region corresponding to the P-element exon 1, but it does not present the two canonical P introns separating exons 12 and 23. According to the loss-of-intron hypothesis, either Hoppel and P elements form a monophyletic group and two independent retrotranscription events are required in the Hoppel and Phsa lineage, or Hoppel elements form a monophyletic group with Phsa, and a single retrotranscription event is required before their divergence, followed by a horizontal transfer of the Hoppel element from deuterostomians to protostomians.
An alternative hypothesis is that the absence of introns corresponds to the ancestral structure of P elements. In this case, the P-element canonical introns were inserted into the P lineage after the divergence of P and Hoppel elements. As described previously, the amino acids flanking the two P-element introns are conserved in the Hoppel protein, which implies that in the case of intron gain, splicing does not change the protein encoded by the P element compared to the intronless Hoppel sequence. Indeed, several examples of such intron insertions have been described in the literature. A first example is the insertion of 16 introns in the triose-phosphate isomerase gene from different species (Kwiatowski et al. 1995; Logsdon et al. 1995). Multiple alignments of the proteins encoded by these genes clearly show that the amino acids in the vicinity of the inserted introns are perfectly conserved. Within the P superfamily, two intron-insertion events have taken place without the addition or deletion of any amino acids; the first, in the exon 1 region of the Phsa sequence as mentioned above, and the second in exon 2 of a P element carried by the common ancestor of L. cuprina and M. domestica (Perkins and Howels 1992; Lee, Clark, and Kidwell 1999). Therefore, intron gain events without any changes in the transposase sequence are observed within the P-element lineage. According to this hypothesis, the lack of exon 0 in the Hoppel element sequence could result from the strong divergence of its 5' region from P sequences. Indeed, the 5' coding region of P elements is weakly conserved between species. Obviously, another possibility is the gain of the exon 0 in the P lineage. Three examples of new exons have been described in the domesticated P sequences of the montium subgroup: the first is a noncoding exon located upstream of exon 0, and the other two are additional exons 0 inserted either upstream of the canonical exon 0 or between the canonical exons 0 and 1 (Nouaud et al. 1999; Nouaud, Quesneville, and Anxolabéhère 2003). Additional data concerning the presence or absence of canonical P introns within the P superfamily are required to define the most parsimonious scenario.
The most closely related sequence to the P element in the D. melanogaster genome is the Hoppel element. However, the presence of the Hoppel element does not explain the absence of P-family sequences in the melanogaster subgroup. Indeed, the rate of divergence between them and the presence of Hoppel elements in species that do not belong to the melanogaster subgroup suggests that their split occurred before the radiation of this taxon. It is noteworthy that, beside the Hoppel sequence, no other kind of P sequence has been detected in the ISO1 genome, suggesting that this absence resulted from genetic drift or a deletion process as described in Petrov and Hartl (1998).
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Acknowledgements |
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Footnotes |
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