Departament de Genètica, Universitat de València, València, Spain
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Abstract |
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Introduction |
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The analyses of the distribution of transposable elements have shown practically as many different patterns as the number of studied elements. The most general scenario is the vertical transmission of the element to the progeny (e.g., see Stacey et al. 1986
; Eickbush and Eickbush 1995
). Frequently, the studies on transposable element distribution have shown that sequences homologous to an element present in a group of species are absent in closely related species (e.g., see de Frutos, Peterson, and Kidwell 1992
; Cizeron et al. 1998
). Two processes account for this incomplete distribution: stochastic loss can explain the absence of elements from closely related species (Engels 1981
), while horizontal transfer (Kidwell 1993
) can explain the presence of closely related elements in species relatively distant in evolutionary terms. Horizontal transfer is the most likely explanation in the case of the I factor and the P, mariner, hobo, and copia elements (Bucheton et al. 1986
; Daniels et al. 1990
; Maruyama and Hartl 1991
; Simmons 1992
; Jordan and McDonald 1998
). Phylogenetic and sequence analyses are also useful in studying the evolutionary history of the elements and their hosts (Lathe et al. 1995
; Sezutsu, Nitasaka, and Yamazaki 1995
; Booth, Ready, and Smith 1996
; Clark and Kidwell 1997
; Jordan and McDonald 1998
).
Bilbo is a non-long-terminal-repeat (non-LTR) retrotransposon characterized at sequence level in Drosophila subobscura. A rough estimate indicates that there are between 30 and 100 bilbo copies in the genome of this species (Blesa and Martínez-Sebastián 1997
). In this study, we analyzed the distribution of sequences homologous to the D. subobscura bilbo element in 52 species of the family Drosophilidae. Most of these are species from the obscura and melanogaster groups, as they are more closely related and better studied. We also analyzed the phylogenetic relationship of bilbo elements in species from the obscura group.
Drosophilidae encompasses two subfamilies, Steganinae, which is relatively small, and Drosophilidae, with some 35 to 40 genera assigned, with the genus Drosophila being the most studied and best characterized at genetic and systematic levels. The two major subgenera of Drosophila split no more recently than 30 MYA, and perhaps as early as 60 MYA. Subgenus Sophophora contains three major groups whose relationships are very well supported. The melanogaster group is closest to the obscura group, with the willistoni group being the oldest lineage. The relationships between members of the Drosophila subgenus have proven much more difficult to define (for a revision, see Powell 1997
). Radiations, genera, subgenera, groups, and subgroups of the species used in this work are shown in table 1
.
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Materials and Methods |
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Southern Blot Hybridization
Sequences homologous to bilbo in the Drosophilidae species were detected by the Southern blot technique. Total genomic DNA was prepared following the method of Junakovic, Caneva, and Ballario (1984)
. We digested approximately 1.7 µg of total genomic DNA from each species with 20 U of SacI restriction endonuclease for 16 h at 37°C. We quantified this digested DNA by fluorescence spectroscopy, and 1 µg was loaded in each lane. As a positive control, and to enable us to compare relative signal intensities among filters, we loaded 200 ng of SacI-digested total genomic DNA from third-instar larvae of D. subobscura strain H271. In this study, we used two probes cloned in the plasmid vector pUC18, named DsF112 and Sm0.7 (see fig. 1
). The nonradioactive DIG system of Roche Molecular Biochemicals was used for the random priming labeling and detection; an aliquot fraction of these probes was used with each filter.
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Isolation of bilbo Sequences
For the phylogenetic analysis of bilbo, we amplified DNA by the polymerase chain reaction (PCR) from D. subobscura, Drosophila madeirensis, Drosophila obscura, Drosophila ambigua, Drosophila bifasciata, Drosophila pseudoobscura, Drosophila pseudoobscura bogotana, Drosophila miranda, Drosophila persimilis, Drosophila affinis, Drosophila athabasca, Drosophila algonquin, Drosophila azteca, Drosophila narragansett, and Drosophila tolteca. The primers for the PCR were based on the sequence of the second and fourth regions of conserved amino acid residues in the reverse transcriptase (RT) domain from bilbo1 (after Xiong and Eickbush 1990
) (FRPISL [3316-5'-GACTCTAGACCAATAAGTCT-3'-3335] and GTPQGG [3719-5'- TAGTCTAGACACCGCCTTGCGGGG-3'-3696], respectively). The size of the amplified DNA fragment was 406 bp in bilbo1. We modified the sequence of the primers to create an XbaI cloning restriction site (underlined sequences in the primers). Amplification was carried out in 50-µl reactions that included 100 ng of genomic DNA, 100 pmol of each primer, 2.5 U of Taq DNA polymerase (Roche Molecular Biochemicals), and buffer supplied by the manufacturer. Amplification was conducted in a Perkin-Elmer GenAmp PCR System 2400 as follows: 10 cycles of 1 min at 94°C, 1 min at 49°C, and 1 min at 72°C, and 30 cycles of 30 s at 94°C, 30 s at 54°C, and 30 s at 72°C.
Five microliters of the PCR products were blotted and hybridized with DsF112 as a probe. The final wash was performed in 0.1 x SSC, 0.1% SDS at 60°C. This hybridization showed that in all species, the major PCR product, which had the expected size predicted from the bilbo1 sequence, corresponded to bilbo sequences (data not shown). The product of approximately 400 bp, which hybridized with DsF112, was purified from an agarose gel and cloned at the pUC18 XbaI restriction site. We performed single-strand sequencing of eight different clones with the fragment of the expected size for each of the following species: D. subobscura, D. obscura, D. bifasciata, D. pseudoobscura, D. miranda, D. azteca, and D. algonquin.
Phylogenetic Analysis
A multiple alignment was generated with the PileUp program (GCG software package, Madison, Wis.) using the sequences from the PCR product, excluding the PCR primers, plus the corresponding region from bilbo1 (called Subbilbo in the alignment) and DsF112 (SubF11). Sequences of the equivalent region of the elements TRIM from D. miranda (Steinemann and Steinemann 1991
) and LOA from D. silvestris (Felger and Hunt 1992
) were also included. We used an alignment based on amino acid sequences of bilbo1, TRIM, LOA, and a number of other Drosophila retrotransposons (unpublished data) to align TRIM and LOA with the bilbo sequences. The alignment comprised 51 sequences: 10 from D. subobscura (Subbilbo, SubF11, Sub1Sub8), 8 from D. obscura (Obs2Obs9), 6 from D. bifasciata (Bif1, Bif3, Bif4, Bif6, Bif7, Bif9), 8 from D. pseudoobscura (Pse1Pse8), 1 from D. miranda (Mir), 8 from D. algonquin (Alg2, Alg4Alg10), 8 from D. azteca (Azt1Azt6, Azt8, Azt9), 1 from the TRIM element, and 1 from the LOA element. Manual refinement was necessary mostly due to 33 one-base insertions scattered among the sequences. This alignment contains 431 positions and has been deposited at EMBL with accession number DS38321.
Phylogenetic trees were constructed using several methods: neighbor joining implemented in programs from MEGA (Kumar, Tamura and Nei 1993
) and the PHYLIP package (Felsenstein 1993
), maximum parsimony (MEGA), and maximum likelihood (PHYLIP). For the neighbor-joining clustering method (Saitou and Nei 1987
) in MEGA, input distances were estimated by the Kimura two-parameter model. In this analysis, LOA was used as an outgroup, and a bootstrap analysis was conducted with 500 pseudoreplicates and the "pairwise deletion" option for the gaps. Maximum parsimony (Fitch 1971
) in "branch and bound search" allowed us to obtain the most parsimonious tree with some clusters of sequences. Due to technical limitations, we performed this analysis using all of the elements from one species and one element from its closest species. We did this for all species except for D. algonquin and D. bifasciata. With the PHYLIP package, bootstrap analysis of 500 pseudoreplicates was performed with the programs SEQBOOT and DNADIST. Distances were estimated by the Kimura two-parameter model with a transition-to-transversion ratio of 1.0000 (which is the average value of the 51 sequences analyzed) and one category of substitution rates. Trees were constructed with the neighbor-joining method using LOA as an outgroup, and the program CONSENSE generated a consensus tree of the 500 replicates. To perform a maximum-likelihood analysis (DNAML program) (Felsenstein 1981
), we chose one sequence per species when all the sequenced clones constituted a monophyletic cluster according to the neighbor-joining and maximum-parsimony analysis. From each cluster, we selected the sequences with the smallest numbers of ambiguities and the shortest distances in relation to the other sequences according to an estimate by the Kimura two-parameter method carried out with MEGA. These sequences were as follows: D. subobscura, Subbilbo; D. obscura, Obs5; D. miranda, Mir; D. pseudoobscura, Pse4; D. azteca, Azt1; D. algonquin, Alg5, Alg7, and Alg2; and D. bifasciata, Bif9 and Bif4. We also included the sequences of the elements TRIM and LOA. We extracted the alignment for this analysis from the general one, and it contained 377 positions. Distances were estimated with a transition-to-transversion ratio of 1.0200 (the value that produced the maximum-likelihood tree estimated by neighbor-joining), and empirical base frequencies and one category of substitution rates were used. The DNAML program was run randomizing the input order of the sequences 10 times and with global rearrangements. LOA was used as an outgroup.
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Results and Discussion |
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When the Southern blot films from figure 2A and B are superimposed, common bands detected with both probes are revealed. These bands are indicated in the control lane (lane C) in figure 2C and D . The proportions of common and noncommon signals in other species are similar to those in D. subobscura.
No specific signal was detected in D. willistoni (fig. 2 , lane 16) and D. saltans (fig. 2 , lane 17) (fig. 2A and B ), even under low-stringency washes (data not shown).
In the melanogaster species group (fig. 2C and D, lanes 1830), we detected sequences homologous to one or the other probe in all species under medium-stringency wash conditions. Under high-stringency conditions (assayed only with DsF112 probe), the hybridization signal disappeared in all species except D. simulans (lane 19) and D. serrata (lane 30) (data not shown). The signals obtained with the Sm0.7 probe seemed weaker (fig. 2D ), perhaps because of the presence of elements truncated at their 5' ends.
The diversity of hybridization patterns that can be seen in the melanogaster group is greater than that in the obscura group. This is probably due to a higher sequence divergence between the probes and the bilbo elements in the melanogaster group.
We detected many sequences homologous to DsF112 in Scaptodrosophila dimorpha (lane 31) and in Scaptodrosophila lebanonensis (lane 32) under medium- stringency conditions (fig. 2C ). All of these signals disappeared under high-stringency washes (data not shown). In Scaptodrosophila latifasciaeformis (lane 33), only a single band, approximately 4 kb in size, was detected under medium- and high-stringency washes. No hybridization signal was detected with Sm0.7 in these three species (fig. 2D ).
The species of the Drosophila subgenus and D. pictiventris and D. busckii were only analyzed with DsF112 (fig. 2E, lanes 3449). This probe detected a single band (similar to that of D. simulans [lane 19] and D. serrata [lane 30] in the melanogaster group and to that of S. latifasciaeformis [lane 33]). Detection of these signals required longer exposure times (compare control lane [lane C] in fig. 2E with that of other filters in fig. 2 ). This signal was detected in some species under high- stringency conditions (data not shown).
In Zaprionus tuberculatus, Scaptomyza adusta, and Samoaia leonensis, we did not detect specific hybridization using DsF112 as a probe (data not shown); Sm0.7 was not used with these species.
Our Southern blot results show that most of the analyzed species bear sequences homologous to bilbo from D. subobscura. We obtained patterns of bands similar to those of other transposable elements (Stacey et al. 1986
; Daniels, Chovnick, and Boussy 1990
; Daniels et al. 1990
; Mizrokhi and Mazo 1990
; de Frutos, Peterson, and Kidwell 1992
; Di Franco, Galuppi, and Junakovic 1992
). Our data suggest an ancient origin for the elements of the bilbo family in Drosophila. In some species, even in some subgroups, the elements of this family have been lost or their similarity with the probes is not sufficient to allow their detection. There is also a noticeable correlation between signal intensity and phylogenetic distance, even within the species of the obscura group (see fig. 2B
).
Bilbo Elements in the D. obscura Species Group and Phylogenetic Analysis
The presence of elements homologous to bilbo1 in species other than D. subobscura encouraged us to perform a phylogenetic analysis. We restricted this analysis to the obscura species group. The PCR amplified DNA in all species of the obscura group, as expected from the Southern blot data.
Once we showed that the bilbo sequences could be amplified in all species of the obscura group, we cloned the PCR products and performed a single-strand sequencing of eight different clones from D. subobscura, D. obscura, D. bifasciata, D. pseudoobscura, D. miranda (only one sequence was obtained from this species), D. algonquin, and D. azteca, which are representative of the different subgroups within the obscura group (see table 1 ; D. algonquin belongs to the affinis subgroup). All of the sequences obtained in this study can be found in GenBank (accession numbers AF034267AF034315).
Two conclusions can be drawn from the neighbor- joining phylogram (fig. 3
). First, with the exception of D. algonquin, the sequences from each species are grouped in monophyletic clusters. Second, the elements of different species group together by species subgroups, that is to say, the elements of D. obscura group with those of D. subobscura (obscura subgroup), elements of D. miranda with D. pseudoobscura (pseudoobscura subgroup) and elements of D. algonquin with D. azteca (affinis subgroup) (Barrio, Latorre, and Moya 1994
). There is still some uncertainty with regard to the order of these subgroups in the phylogeny of the obscura group (Barrio and Ayala 1997
), but our neighbor-joining phylogram tree is in agreement with the most probable scenario. The lower bootstrap value, in the node [D. miranda, D. pseudoobscura], [D. algonquin, D. azteca], coincides with the major uncertainty on the phylogeny of the obscura group. In an exception to the concordance with the phylogeny of the group, the sequences from D. bifasciata are distantly related to other bilbo elements and phylogenetically closer to TRIM.
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Only a few transposable elements have been studied for which more than three or four sequences per species have been analyzed; in most of them, a polyphyletic or paraphyletic status between closer species has been shown. When sequences from two species are compared, it can happen that sequences of elements in each species are genealogically closer to heterospecific sequences than to homospecific copies. If this happen in both species, their status is polyphyletic; if it only happens in one species and not in the other, they are paraphyletic; and if all the copies of the element within each species are genealogically closer to one another than to any heterospecific copy, this is a case of reciprocal monophyly. The elements in which several copies of an element have been studied are the non-LTR retrotransposon Helena, studied in the virilis and melanogaster species groups (Petrov and Hartl 1997, 1998
), P elements in the Sophophora subgenus (Clark and Kidwell 1997
), and the endogenous retrovirus gypsy in the obscura species group (Vázquez-Manrique et al. 2000). Other studies with the I factor (Sezutsu, Nitasaka, and Yamazaki 1995
) and copia LTR retrotransposon (Csink and McDonald 1995
) in the melanogaster group and L1 element in the mouse (Cabot et al. 1997
) also showed polyphyly between species, but these studies were inconclusive in this respect because of the very few sequences isolated. Eickbush et al. (1997)
, working on R1 and R2, found that all copies were highly homogeneous within each species, most probably because of concerted evolution acting on the rRNA genes in which these elements specifically insert. Our neighbor-joining and maximum-parsimony analysis showed reciprocal monophyly in most of the studied species in relation to the obtained sequences of their bilbo elements. Only D. algonquin and D. azteca were paraphyletic. Taking into account all of our sequences, we obtained diversity, divergence, and a range of indels similar to the diversity, divergence, and range of indels shown in the studies cited above. Because the PCR technique cannot detect all of the sequences present in a genome, it is possible that this reciprocal monophyly is not real, but a product of the inherent limitation of the technique. A more exhaustive screening and analysis of bilbo elements in these species would be necessary to confirm or reject these results.
To corroborate the second conclusion drawn from the neighbor-joining analysis, an analysis with the maximum-likelihood method was performed (data not shown). The program examined 4,019 trees, with the best one having a log likelihood value of -2,826.16796. The topology of this tree agreed with the topology of the neighbor-joining tree, except in this case in which Alg5 and Alg7 became sister clades. This result supports the conclusion from the neighbor-joining analysis: the representative elements first group together in previously established phylogenetic species subgroups, and then these subgroups follow the phylogeny of the obscura group, which is indicative of vertical transmission of bilbo elements in species of the obscura group.
The bilbo Family and the bilbo-TRIM Superfamily
Our data suggest that the TRIM and bilbo elements probably diverged before the speciation of the obscura group. Drosophila miranda has the elements, bilbo (Mir) and TRIM, which differ 38.23% in their nucleotide sequences. The protein sequence predicted from these sequences contains most of the conserved amino acid residues in the RT of non-LTR retrotransposons. The bilbo sequence from D. miranda (Mir) and that from D. subobscura (SubF11) diverge 12.78% at the nucleotide level, while TRIM and SubF11 diverge 38.33%. We suggest, therefore, that bilbo1 and TRIM belong to two distinct families within a superfamily.
The Southern blot performed with genomic DNA from D. bifasciata shows that elements from this species are relatively distinct from those of the rest of the species from the obscura group. Taking into account the phylogeny of the obscura group (Barrio, Latorre, and Moya 1994
; Barrio and Ayala 1997
), our phylogenetic analysis suggests that the elements from D. bifasciata could belong to the TRIM family. We did not find bilbo elements in this species, which means that they either were not amplified in the PCR experiments or have been lost along the evolutionary history of this species.
The bilbo Family Has Been Vertically Transmitted in the Drosophilidae
The presence of bilbo elements in species derived from basal branches of the family Drosophilidae and in most of the species analyzed indicates that an ancestor of the bilbo family was present at the beginning of the Scaptodrosophila radiation. The descendants of this ancestral sequence have diverged during the speciation process, and meanwhile, some species have most probably lost them. The phylogenetic analysis suggests that, at least in the obscura group, these elements have been transmitted from parents to offspring since the appearance of this group.
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Acknowledgements |
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Footnotes |
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1 Laboratorio de Hematología y Oncología, Hospital Clínico Universitario, Valencia, Spain
2 Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias, València, Spain
3 Abbreviations: EN, endonuclease; LTR, long terminal repeat; ORF, open reading frame; PCR, polymerase chain reaction; RH, RNase H; RT, reverse transcriptase; UTR, untranslated region.
4 Keywords: bilbo elements
non-LTR retrotransposons
LINE-like elements
Drosophilidae
evolution
distribution
phylogeny
5 Address for correspondence and reprints: María J. Martínez-Sebastián, Departament de Genètica, Universitat de València, C/Dr. Moliner, 50, E-46100 Burjassot, València, Spain. maria.jose.martinez{at}uv.es
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literature cited |
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