Institut de Genetique Humaine, Montpellier, France
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Abstract |
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Introduction |
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Sampling procedures concerning the number of retroelements and the number of species to be analyzed must be carefully considered. Indeed, mistakes in the interpretation of the relationships between elements may result from the mixture of (1) orthologous sequences (from the same element lineage) when one or few randomly sampled elements from a broad spectrum of taxa are analyzed or (2) paralogous sequences (from different elements lineages) which are not phylogenetically comparable when a large sampling of elements from a single species is examined (Capy, Anxolabehere, and Langin 1994
).
Phylogenetic relationships for partial Gypsy sequences from the eight species of the melanogaster subgroup (Lachaise et al. 1988
) were examined in the context of the phylogeny of their hosts. These partial sequences concern the integrase domain (int) of Gypsy (fig. 1
). The integrase protein is required for the integration of the provirus into the host genome. Hence, integrase is crucial for the replication of Gypsy and its interaction with the host genome because it specifies the DNA target site preferences.
Our results demonstrate that two main Gypsy lineages exist within the melanogaster subgroup. The distribution of these lineages among the species suggests a complex pattern of vertical and horizontal transfers.
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Materials and Methods |
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PCR Amplification, Cloning, and Sequencing
Three different pairs of primers were designed from conserved regions based on alignments of published sequences for the Gypsy elements from D. melanogaster (M12927; Marlor, Parkhurst and Corces 1986
), D. subobscura (X72390; Alberola and de Frutos 1996
), and D. virilis (M38438; Mizrokhi and Mazo 1991
). Primers were as follows (using the D. melanogaster Gypsy position numbers): primer A (plus strand), positions 489513; primer B (minus strand), positions 66916667; primer C (plus strand), positions 10051029; primer D (minus strand), positions 65336509; primer E (plus strand), positions 47184740; and primer F (minus strand), positions 51225103. A first round of amplification was performed using primers A and B in order to amplify an extra large Gypsy fragment (XL PCR; fig. 1 ). Reaction volumes were 50 µl and contained 2.5 U Taq Plus polymerase (Stratagene, La Jolla, Calif.), 5 µl Taq Plus low-salt buffer, 200 nM each dNTP, 400 nM each primer, and approximately 50 ng template DNA. Reaction parameters were as follows: 95°C for 5 min, followed by 20 cycles of 93°C for 30 s, 60°C for 30 s, and 72°C for 7 min, followed by 72°C for 10 min. One tenth of the reaction products were assayed on 1% agarose. A second round of amplification (int PCR) was performed from 1 µl of the XL PCR products using primers E and F in order to amplify the integrase domain. Reaction volumes were same as for XL PCR, except that 2.5 U of Pfu polymerase (Stratagene) and Pfu buffer was used. Reaction parameters were as follows: 95°C for 5 min, followed by 25 cycles of 93°C for 30 s, 55°C for 30 s, and 72°C for 1 min, followed by 72°C for 20 min. This product was cloned into the pCR-Script Amp SK(+) cloning vector according to the manufacturer's instructions (Stratagene).
The XL PCR and the following int PCR did not give any positive result for the D. simulans, D. mauritiana, and D. sechellia species. New PCRs were performed using primers C and D in order to amplify a large Gypsy fragment (L PCR; fig. 1 ). Reaction volumes were same as for XL PCR. Reaction parameters were as follows: 95°C for 5 min, followed by 2 cycles of 93°C for 1 min, 58°C for 30 s, and 72°C for 6 min, followed by 20 cycles of 93°C for 30 s, 55°C for 30 s, and 72°C for 7 min, followed by 72°C for 10 min. A second round of amplification was then performed from 1 µl of each L PCR product using primers E and F in order to amplify the int domain. These products were then purified and cloned into the pCR-Script Amp SK(+).
At least four clones from each species were sequenced in both directions using the reverse and forward M13 primers with an Applied Biosystems 373A automated sequencer according to the manufacturer's protocols.
Data Analyses
A multiple alignment of the nucleic acid sequences was performed using CLUSTAL X (Thompson et al. 1997
). Jukes-Cantor nucleotide distances were estimated using Distances (GCG, version 10.0). Phylogenetic analyses were done using the bootstrap neighbor-joining tree method (1,000 replicates) as implemented in CLUSTAL X, the maximum-likelihood method as implemented in the program PUZZLE, version 4.0 (Strimmer and von Haeseler 1997
), and the maximum-parsimony method using the PAUPSearch and PAUPDisplay programs as implemented in GCG, version 10.0 (Swofford 1991
). Diverge (GCG, version 10.0) was used to estimate the numbers of synonymous (Ks) and nonsynonymous (Ka) substitutions per site between two sequences coding for proteins (Li 1993
). Trees based on sequence data were obtained using Treeview (Rod Page, http://taxonomy.zoology.gla.ac.uk/rod). The sequences reported in this paper have been deposited in the EMBL database (accession numbers AJ279868AJ279900).
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Results and Discussion |
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Eight clones from D. melanogaster were obtained: four were issued from XL PCR and four were issued from L PCR. They were all identical, and this is why only one sequence (me) appears in the subsequent analyses. Only two clones have deletions: one from D. mauritiana (ma1), which contains two deletions of 21 bp and 22 bp, and one from D. sechellia (se3), which contains a 20-bp deletion. These deletions inactivate ma1 and se3 copies. The other sequences encode a polypeptide of 120 amino acids (fig. 2 ).
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Two Gypsy Lineages Exist in the melanogaster Subgroup Species
The fact that me clusters with [t, y, o, e12] is the first hint of disagreement between the Drosophila and Gypsy phylogenies. Such inconsistency suggests horizontal transfer events. However, it was shown that disagreements between phylogenies can also be explained by other evolutionary processes, such as faster evolution in certain lineages and/or ancestral polymorphism (Capy, Anxolabehere, and Langin 1994
). In order to test the possibility of horizontal transfers of Gypsy, we estimated the Jukes-Cantor nucleotide distances between int sequences from the eight species and compared them with the Jukes-Cantor distances between the 3' untranslated regions of R1 sequences (Eickbush et al. 1995
). R1 is a non-LTR retroelement found in many insects (Eickbush et al. 1995
). Comparison with R1 is useful for two reasons: (1) it was shown that R1 evolves at rates similar to those of nuclear genes in the melanogaster species subgroup (Eickbush et al. 1995
); (2) moreover, its replication involves, like Gypsy, a reverse transcriptase enzyme which is known to be error-prone. By comparing the distances values of int and R1, we can test horizontal versus vertical transfer: if int is vertically transmitted and evolves at the same rate as R1, the nucleotide distances for all pairwise comparisons of int and R1 from the eight species should be equal.
Results are presented in figure 4 . Pairwise distances of int and R1 from D. simulans, D. sechellia, and D. mauritiana are roughly equal. However, int distances between the [si, se, ma] and [me, t, y, o, e12] clusters are larger than the R1 distances, whereas int distances between species from the [me, t, y, o, e12] cluster are smaller than R1 distances. This result is the second hint of disagreement between the Drosophila and Gypsy phylogenies and rules out the presence of an ancestral polymorphism in the common ancestor of the melanogaster subgroup species. This strongly suggests horizontal transfers between D. erecta, D. orena, D. teissieri, D. yakuba, and D. melanogaster.
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Conclusions |
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Interestingly, the absence of GypB and the presence of GypA within the simulans complex species correlates with its biogeographic history. Moreover, the fact that we found only two defective sequences, ma1 and se3, in this screening for Gypsy elements in the melanogaster subgroup and that Ks/Ka values between [si, se, ma] sequences and the other sequences are relatively high suggest that these elements are active within the melanogaster subgroup. It would be worth knowing if the Gypsy elements from other species are potentially infectious like their counterpart in D. melanogaster (Kim et al. 1994
; Song et al. 1994
; Teysset et al. 1998
). Gypsy is normally repressed in D. melanogaster by a host gene called flamenco, which controls the transposition and infective properties of Gypsy (Bucheton 1995
). We do not yet know the status of flamenco in the other species, but it would be useful to determine if the alleles of genes homologous to flamenco are permissive or restrictive for Gypsy expression in species other than D. melanogaster in order to gain further insights into the evolutionary history of the relationship between endogenous retroviruses and their hosts.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: EnRV, endogenous retroviruses; ExRV, exogenous retroviruses; int, integrase; LTR, long terminal repeat.
2 Keywords: Gypsy,
endogenous retrovirus
Drosophila,
evolution
phylogeny
3 Address for correspondence and reprints: Christophe Terzian, Institut de Genetique HumaineCentre National de la Recherche Scientifique, 141 rue de la Cardonille, F-34396 Montpellier cedex, France. E-mail: christophe.terzian{at}igh.cnrs.fr
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literature cited |
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