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Genetics and Molecular Biology

Print version ISSN 1415-4757On-line version ISSN 1678-4685

Genet. Mol. Biol. vol.27 no.3 São Paulo  2004 



Characterization of two full-sized P elements from Drosophila sturtevanti and Drosophila prosaltans



Juliana Polachini de Castro; Claudia M.A. Carareto

Universidade Estadual Paulista, Departamento de Biologia, São José do Rio Preto, SP, Brazil





Previously, only partial P element sequences have been reported in the saltans group of Drosophila but in this paper we report two complete P element sequences from Drosophila sturtevanti and Drosophila prosaltans. The divergence of these sequences from the canonical P element of Drosophila melanogaster is about 31% at the nucleotide level. Phylogenetic analysis revealed that both elements belong to a clade of divergent sequences from the saltans and willistoni groups previously described by other authors.

Key words: D. sturtevanti, D. prosaltans, full-size P element, phylogeny.




The P elements were first discovered in Drosophila melanogaster because of their ability to induce hybrid dysgenesis (Kidwell et al., 1977). Autonomous P elements are 2.9 kb in length and have four open reading frames which encode two polypeptides, an 87 kDa transposase enzyme necessary for transposition (Rio et al., 1986) and a 66 kDa repressor protein (Robertson and Engels, 1989). Also required for transposition are the element termini, which include flanking 31-bp perfect inverted repeats (O'Hare and Rubin, 1983), 11-bp subterminal repeats and unique terminal sequences comprising approximately 150 bp (see Engels, 1989 for a review).

Sequences belonging to the P family are particularly common in the four principal species groups (melanogaster, obscura, saltans and willistoni) which make up the subgenus Sophophora (Daniels et al., 1990) but have also been described in drosophilid species such as Drosophila mediopunctata which is not part of the Sophophora subgenus (Loreto et al., 2001) and also in Scaptomyza pallida, a drosophilid which does not belong to the genus Drosophila (Anxolabéhère et al., 1985; Simonelig and Anxolabéhère, 1991, 1994). Transposable elements similar to the P elements of the Drosophilidae have also been isolated from members of a few other Diptera families, e.g. Lucilia cuprina from the Calliphoridae (Perkins and Howells, 1992), Musca domestica from the Muscidae (Lee et al., 1999) and seven species Anopheles from the Culicidae (Sarkar et al., 2003). More divergent and rudimentary sequences related to P-transposable elements have also been described using 'in silico' searches such as Hoppel (Reiss et al., 2003) and Proto P (Kapitonov and Jurka, 2003) for the Drosophila melanogaster genome and Phsa (Hagemann and Pinsker, 2001) for the human genome.

Phylogenetic studies based on nucleotide sequences (Clark and Kidwell, 1997; Hagemann et al., 1994, 1996; Silva and Kidwell, 2000) indicated that the more than 200 P element sequences obtained to date fall into 16 distinct clades or subfamilies (Figure 1). Four of these subfamilies have been well characterized. The canonical subfamily appears to be restricted to the sophophoran New World species groups saltans and willistoni (Clark et al., 1995), with the notable exception of Drosophila mediopunctata, which contains P elements due to horizontal transfer (Loreto et al., 2001). Three P element subfamilies (M-, O- and T-type) are found in the Old World obscura species group (Hagemann et al., 1992, 1994, 1996), with the T-type appearing to be restricted to the obscura lineage (Hagemann et al., 1998) while the M- and O-types also occur in the saltans and willistoni groups. A new subfamily, the K-type (restricted to the montium subgroup species), has recently been described by Nouaud et al. (2003).



The descriptions of the P element subfamilies in the saltans and willistoni species groups have been based so far mainly on partial sequences (Clark et al., 1995; Clark and Kidwell, 1997; Haring et al., 2000; Silva and Kidwell, 2000). The work described in this paper compared two complete sequences obtained from two different saltans subgroups (sturtevanti and saltans) to some of the complete sequences from different P element subfamilies as well as to a data set consisting of 80 partial consensus sequences provided by Clark and Kidwell (1997) in order to establish their phylogenetic relationships.


Materials and Methods

Fly stocks

We used Drosophila sturtevanti collected in the Mexican town of Matlapa and Drosophila prosaltans collected in the town of Eldorado in the Brazilian state of Rio Grande do Sul both strains having been recently derived from natural populations and subsequently maintained in the laboratory, under standard conditions.

DNA Amplification and Sequencing

Polymerase chain reactions were carried out using a total volume of 50 mL containing 100 ng of genomic DNA, 1 mM MgCl2, 400 mM of each deoxynucleotide, 0.5 mM of M-IR primer (5'CATAAGGTGGTCCCGTCG3', corresponding to nt. 14-31 and 2877-2894, within the TIRs - terminal inverted repeats - of the D. melanogaster canonical P element; Haring et al., 1995) and 2 units of Taq polymerase in 1x polymerase buffer. Amplification consisted of 30 cycles of 45 s denaturation at 94 °C, 45 s of primer annealing at 57 °C and 1.5 min of primer extension at 72 °C. The first cycle was preceded by a step of 7 min at 94 °C for denaturation and the last cycle was followed by a final extension at 72 °C for 10 min. The PCR products were cloned into a TOPO TA cloning vector (Invitrogen) and both strands of a randomly-chosen single clone were sequenced for each species. The primers Stu1369 (5'GTTCCGTATCG AGAC CCGA C3'), Stu22402 (5'AATGACGAAGACTC GTCGC G3'), Stu51091 (5' GGAAGCAACCAGTTTTCT TT3') and Stu61598 (5'CACATCAAACCAATCATTTA3') were designed based on the clone sequences and used to complete the sequencing of the element.

Sequence alignment and phylogenetic analysis

For alignment we used a set (Clark and Kidwell, 1997) of 80 partial (429 bp) P-element consensus sequences mapped to positions 1328-1757 in open reading frame 2 (ORF 2) of the canonical P element and the following full-length P element nucleotide sequences obtained from the literature: D. melanogaster p25.1 (O'Hare and Rubin, 1983); Drosophila nebulosa N10 (Lansman et al., 1987); Drosophila willistoni (Daniels et al., 1990); Drosophila guanche, Drosophila bifasciata and D. bifasciata jbifM3 (Hagemann et al., 1992); Drosophila ambigua T-type (Hagemann et al., 1998); Drosophila helvetica M-type (Haring et al., 2000); D. mediopunctata (Loreto et al., 2001); Scaptomyza pallida (Simonelig and Anxolabéhère, 1991) and M. domestica (Lee et al., 1999). The P element DNA sequences obtained by us in this study have been deposited in the GenBank under accession number AF530052 for D. sturtevanti and AF530053 for D. prosaltans.

Alignments of sequences were done by the CLUSTAL W program (Thompson et al., 1994) and the phylogenetic relationships between the P sequences constructed using the maximum parsimony method as implemented in PAUP program version 4.0b10 (Swofford, 1997). Branch support was calculated using bootstrap analysis with 500 replicates. The distance matrix was constructed according to the Kimura two-parameter model of nucleotide substitution (Kimura, 1980).


Results and Discussion

Sequence features

The M-IR primer corresponds to positions 14-31 and 2877-2894 of the canonical P element, and so cannot amplify the first and last 13 bp of a complete P element. The D. sturtevanti P element is 2829 bp and the D. prosaltans 2828 bp, if the TIRs of these elements are complete they should be 2854 bp for D. sturtevanti and 2855 bp for D. prosaltans, which is about 50 bp less than the D. melanogaster canonical P element. The alignment of the two sequences against the D. melanogaster sequence showed that the D. sturtevanti and D. prosaltans P elements are similar in structure and sequence to each other (88%) but strongly divergent from the D. melanogaster canonical P element (31% different). Table 1 shows the main differences between the alignments from which it can be seen that, in general, D. sturtevanti has the same deletions and insertions as the D. prosaltans.



Even though the TIRs were not completely sequenced, PCR amplification with primers specific to the TIR regions indicates that at least the second half of the TIRs are present and well conserved both in D. sturtevanti and D. prosaltans. However, the transposase binding sites, located at positions 48-68 and 2855-2871 in D. melanogaster (Kaufman et al., 1989), the TATA box and the 11-bp subterminal inverted repeats are not well conserved. In all four exons the translational reading frame is interrupted by stop codons and frameshift mutations, suggesting that these sequences do not encode a functional protein. Indeed, leucine-zipper and helix-turn-helix motifs were not detected, supporting the suggestion that these sequences might be non-autonomous in the genome.

A nucleotide differentiation and genetic difference matrix based on Kimura's two-parameter method was calculated for the full-length P element sequences from the literature and the two sequences described here (Table 2) and it was found that the D. sturtevanti and D. prosaltans sequences present an overall divergence of 31% as compared to the canonical sequences described in D. melanogaster, D. willistoni, D. mediopunctata and D. nebulosa.



Phylogenetic analysis

Phylogenetic analyses of P elements in the subgenus Sophophora (Clark et al., 1995, 1998; Clark and Kidwell, 1997; Silva and Kidwell, 2000) indicate the existence of multiple P element subfamilies in lineages of single species that apparently must have entered the genome at different times during the past (Lee et al., 1999; Haring et al., 2000). The aim of our phylogenetic analysis was to determine if the D. sturtevanti and D. prosaltans P element sequences belonged to some of the well-characterized P element subfamilies. Figure 1 summarizes the results of our phylogenetic analysis of P element sequences using parsimony, from which it can be seen that in 100% of bootstrap replicates the D. sturtevanti and D. prosaltans sequences clustered in Clark and Kidwell's (1997) F clade, which contains P element sequences of some other saltans group species as well as some willistoni group species.

Based on an internal portion of the P element exon 2, Clark et al. (1995) and Clark and Kidwell (1997) placed the P elements of saltans group in four different clades or subfamilies (A, E, F and O) and found that the divergence within the other three saltans-willistoni clades, excluding the canonical P element clade, ranges from 17% to 30% up to 46%. Our sequences belong to Clark and Kidwell's (1997) clade F and have a nucleotide divergence varying from 6% to 17% within this clade. Clark and Kidwell thought that the F subfamily may be under represented in the saltans group because they had sampled only a few sequences, although they did not discard the hypothesis that this low frequency could be due to the use of PCR primers for sampling. However, our data suggest that the F clade P element subfamily might be more widely distributed in the saltans group than previously believed because this P element was detected in two different species in spite of the fact that canonical sequences (Castro and Carareto, 2004) also existed in these genomes.



We are indebted to Drs. M.G. Kidwell and A.J. Holyoake who kindly provided advice and laboratory facilities at the University of Arizona and for their critical comments on earlier versions of the manuscript. We also thank J.C. Silva (University of Arizona, Tucson, USA) and V.L.S. Valente (Universidade Federal do Rio Grande do Sul, Departamento de Genética, Porto Alegre-RS, Brazil) for supplying the drosophilid strains used in this study and J.B. Clark and M.G. Kidwell for the sequences data set. This work was supported by the Brazilian agencies FAPESP (Grants n. 95/7192-2, 98/08734-1 and 00/11313-0 and a fellowship to J.P.C.) and CNPq.



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Associate Editor: Louis Bernard Klaczko



Correspondence to
Claudia M.A. Carareto
Universidade Estadual Paulista, Departamento de Biologia
Rua Cristóvão Colombo 2265
15054-000 São José do Rio Preto, SP, Brazil

Received: May 28, 2003;
Accepted: February 16, 2004.

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