Interspecific comparative molecular analyses of transposed genes and their flanking regions can help to elucidate the time, direction, and mechanism of gene transposition. In the Drosophila melanogaster genome, three Larval serum protein 1 (Lsp1) genes (, ß and ) are present and each of them is located on a different chromosome, suggesting multiple transposition events. We have characterized the molecular organization of Lsp1 genes in D. buzzatii, a species of the Drosophila subgenus and in D. pseudoobscura, a species of the Sophophora subgenus. Our results show that only two Lsp1 genes (ß and ) exist in these two species. The same chromosomal localization and genomic organization, different from that of D. melanogaster, is found in both species for the Lsp1ß and Lsp1 genes. Overall, at least two duplicative and two conservative transpositions are necessary to explain the present chromosomal distribution of Lsp1 genes in the three Drosophila species. Clear evidence for implication of snRNA genes in the transposition of Lsp1ß in Drosophila has been found. We suggest that an ectopic exchange between highly similar snRNA sequences was responsible for the transposition of this gene. We have also identified the putative cis-acting regulatory regions of these genes, which seemingly transposed along with the coding sequences.

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SEQUENCE analysis of genomes has revealed that gene transposition contributes significantly to the reorganization of eukaryotic genomes. Gene transposition refers to the movement of relatively small genomic segments, containing one or a few genes, from one chromosomal position to another. This movement may be accompanied or not by the duplication of the genomic segment, two processes that may be denoted as duplicative and conservative transposition, respectively. In nematodes, gene transposition seems to be the most frequent kind of genome rearrangement (COGHLAN and WOLFE 2002) whereas duplications of chromosomal segments encompassing a few genes followed by differential gene loss is a common cause of gene order changes in yeasts (LLORENTE et al. 2000; FISCHER et al. 2001). In plants, repeated rounds of large-scale genome duplication followed by selective gene loss are the main factors in genome evolution. Chromosomal rearrangements were thought to be only a minor factor in the divergence of plant genomes (KU et al. 2000). However, when more detailed comparisons were performed, many chromosomal rearrangements were found. For example, the comparison of genome sequences of rice to orthologous regions from other grass species revealed that numerous local rearrangements, including transpositions of single genes to different chromosomes, have occurred (BENNETZEN and MA 2003). Transposition has also played a significant role in the evolution of the mammalian genome. Segmental duplications originated from the duplicative transposition of small portions of chromosomal material represent 5% of the human genome (EICHLER 2001; LANDER et al. 2001; BAILEY et al. 2002) and at least 1.2% of the mouse genome (CHEUNG et al. 2003). Some of the duplicated segments in the human genome are associated with rapid gene innovation and chromosomal rearrangement in the genomes of man and the great apes (SAMONTE and EICHLER 2001; ARMENGOL et al. 2003; LOCKE et al. 2003).

In Drosophila, segmental duplications seem to be rare in comparison to the number found in mammalian genomes (LANDER et al. 2001; CELNIKER et al. 2002). Also, detailed analyses by in situ hybridization show that the gene content of chromosomal elements is generally conserved and suggest that gene transpositions are relatively scarce (GONZáLEZ et al. 2002; RANZ et al. 2003) in relation to paracentric inversions, which have been traditionally considered as the chief type of chromosomal change (KRIMBAS and POWELL 1992; POWELL 1997). However, recent sequence analyses comparing five different Drosophila species point to similar numbers of inversions and gene transpositions (BERGMAN et al. 2002) and a number of new genes originated by retroposition has been unveiled (BETRáN et al. 2002).

Diverse molecular mechanisms (sometimes poorly understood) may be responsible for gene transposition events. A common mechanism for gene transposition is retroposition, which implies reverse transcription of RNA and insertion of the resulting cDNA into a different genome site. In humans, the long interspersed element (LINE) L1 often associates 3' flanking DNA as a read-through transcript and carries the non-L1 sequence to a new genomic location, a process termed L1-mediated transduction (MORAN et al. 1999; LANDER et al. 2001). Seemingly the LINE machinery can also act in trans to cellular RNA substrates giving rise to the trans-mobilization of genomic DNA, processed pseudogenes, and occasionally new functional genes (ESNAULT et al. 2000; BETRáN et al. 2002; EJIMA and YANG 2003; LONG et al. 2003). Another mechanism of transposition is transposon-mediated excision and insertion of genomic segments. For instance, in D. melanogaster, Folback elements flanking relatively large genomic segments are able to transport these segments to sites far away in the genome, forming the so-called "giant transposons" (CHIA et al. 1985; LOVERING et al. 1991). Excision and insertion of these giant transposons is mediated by homologous recombination involving the Foldback sequences at the transposon termini. Transposable elements seem to be implicated also in the origin of segmental duplications in humans. Duplication junctions have been found to be enriched for Alu short interspersed element sequences with a significant proportion of all segmental duplications ending within Alu sequences (BAILEY et al. 2003). This observation suggests Alu-Alu homologous recombination as the most likely mechanism for these rearrangements. A similar mechanism has previously been shown to generate small duplications, deletions, and inversions in diverse organisms.

Larval serum protein 1 (Lsp1) genes provide one of the few examples of gene transposition in the genus Drosophila. In D. melanogaster, each of the three Lsp1 genes is located on a different chromosome: Lsp1 in chromosome X, Lsp1ß in chromosomal arm 2L, and Lsp1 in chromosomal arm 3L (ROBERTS and EVANS-ROBERTS 1979; SMITH et al. 1981). These chromosomal arms correspond to Muller's elements A, B, and D, respectively (Table 1). BROCK and ROBERTS (1983) mapped Lsp1 genes by in situ hybridization in nine other species of the Sophophora subgenus (including D. pseudoobscura) and five species of the Drosophila subgenus (including D. hydei as a representative species of the repleta group). In the melanogaster subgroup species they were able to localize the three Lsp1 genes: Lsp1 on element A, Lsp1ß on element B, and Lsp1 on element D. In all the other species, both Lsp1 and Lsp1ß hybridized to the same polytene band of Muller's element E, suggesting a gene exchange between elements. No hybridization of the Lsp1 gene was observed although -like proteins were detected with specific antibodies. To determine Lsp1 gene number, BROCK and ROBERTS (1983) performed Southern analyses and concluded that at least two genes, one -like and one ß-like, were present in all the species analyzed. Their data also suggested that the ancestor of the genus Drosophila probably had its Lsp1 genes on element E. Recently, we localized by in situ hybridization Lsp1 in chromosome 2 (Muller's element E) of D. repleta and D. buzzatii (GONZáLEZ et al. 2002; RANZ et al. 2003), corroborating their results.

View this table: In this window In a new window   TABLE 1 Chromosomal homologies between D. melanogaster, D. pseudoobscura, and D. buzzatii, the three Drosophila species analyzed in this study (from POWELL 1997)

 
In this work, Lsp1 genes and their flanking sequences have been cloned and sequenced in D. buzzatii, a species belonging to the repleta group of the Drosophila subgenus, which diverged from D. melanogaster 40–62 MYA (BEVERLY and WILSON 1984; RUSSO et al. 1995). In addition, the genome sequence of D. pseudoobscura (available at http://www.hgsc.bcm.tmc.edu) has been searched and Lsp1 genes have been annotated in this species, which diverged from D. melanogaster 30 MYA (THROCKMORTON 1975) and belongs to the same subgenus. The aims of this study are: (i) to determine beyond doubt the number and localization of Lsp1 genes in these two Drosophila species because Southern analyses and in situ hybridization results for members of gene families may be misleading (BACHTROG and CHARLESWORTH 2003); (ii) to ascertain the number and type of transposition events undergone by Lsp1 genes during the evolution of the genus Drosophila; (iii) to uncover the molecular mechanism of transposition, in particular to test the hypothesis of an involvement of transposable elements; and (iv) to identify putative regulatory sequences of Lsp1 genes and determine whether these regulatory sequences transposed along with the coding sequences or were recruited ex novo at the new chromosomal location.

Drosophila stocks:

Two lines of D. buzzatii were used: line j-19 from Ticucho (Argentina) is fixed for chromosomal arrangement 2j and line jq⁷-4 from Otamendi (Argentina) is fixed for arrangement 2jq⁷. The genome sequence of D. pseudoobscura comes from inbred line MV2-25 (http://www.hgsc.bcm.tmc.edu).

Screening of genomic libraries:

Two different lambda genomic libraries were screened by plaque hybridization: the j-19 library (CáCERES et al. 2001) and the jq⁷-4 library (CASALS et al. 2003). Both libraries were previously amplified as described in SAMBROOK et al. (1989). DNA from positive phages was digested and the resulting fragments were subcloned into Bluescript II SK vector (Stratagene, La Jolla, CA) after gel purification. The j-19 library was screened with a 0.7-kb BamHI fragment of the D. melanogaster Lsp1 gene (BROCK and ROBERTS 1983). Six positive phages were recovered and one of them, j-19/8, was partially sequenced and found to contain the 5' region of Lsp1ß (Figure 1a). Another lambda phage, j-19/25, containing the 3' region of this gene had been previously isolated in our laboratory (CASALS et al. 2003). None of the six positive phages contained Lsp1. To clone this gene, the j-19 library was screened with a 0.6-kb HindIII-SalI fragment of D. buzzatii Lsp1ß (Figure 1a). Nine positive phages were identified but again none of them included Lsp1. To clone D. buzzatii Lsp1, a different library (jq⁷-4) was screened with the same fragment of D. melanogaster Lsp1 previously used to screen the j-19 library. Five positive phages were identified and one of them, jq⁷-4/12, contained the 3' end of the Lsp1 gene. To clone the other end of the gene, a 0.75-kb SalI-ClaI fragment of D. buzzatii Lsp1 was used to screen the same library (Figure 2a). Seventeen positive phages were recovered and one of them, jq⁷-4/27, included almost the entire Lsp1 gene.

View larger version (16K): In this window In a new window Download PPT slide   FIGURE 1.— Genome organization of the Lsp1ß region in D. buzzatii, D. pseudoobscura, and D. melanogaster. Arrows indicate the start point and direction of transcription. (a) Annotation and chromosomal localization of the 9464 bp sequenced in D. buzzatii. Restriction map includes EcoRI (E), HindIII (H), and SalI (S) sites. The -phages used to sequence this region are represented as lines: solid lines are sequenced regions and broken lines are regions cloned but not sequenced. The short thick segment represents the probe used to screen the D. buzzatii libraries. (b) Annotation of the homologous region in D. pseudoobscura. (c) Fragment of the AE003745 genomic clone of D. melanogaster where those genes flanking Lsp1ß in D. buzzatii and D. pseudoobscura are located. The chromosomal localization of these genes is also given. Note that in this species Lsp1ß is not present between CG12492 and Pellino. (d) Genomic organization and chromosomal localization of the Lsp1ß region in D. melanogaster. This region is included in the genomic clone AE003588 of this species.

 

View larger version (14K): In this window In a new window Download PPT slide   FIGURE 2.— Genome organization of the Lsp1 region in D. buzzatii, D. pseudoobscura, and D. melanogaster (for symbols see legend for Figure 1). (a) Annotation and chromosomal localization of the 10,817 bp sequenced in D. buzzatii. Restriction map includes EcoRI (E), HindIII (H), SalI (S), and ClaI (C) sites. (b) Annotation of the homologous region in D. pseudoobscura. Note that Lsp1 is nested within Sema-1a gene. (c) Fragment of the AE003621 genomic clone of D. melanogaster containing Sema-1a gene. Note that Lsp1 is not present within the first intron. (d) Fragment of D. melanogaster AE003467 genomic clone that contains Lsp1.

 

Southern analysis:

Southern hybridization was performed as described in SAMBROOK et al. (1989). Probes were labeled by random primer with digoxygenin-11-dUTP and hybridization was carried out overnight in standard buffer with 50% formamide at 42° for homologous probes and at 37° for heterologous probes. Stringency washes were performed with 2x SSC 0.1% SDS and 0.1x SSC 0.1% SDS solutions at 68° and 50° for homologous and heterologous hybridizations, respectively.

DNA sequencing and sequence analysis:

Sequences were obtained with an ABI 373 A (Perkin Elmer, Norwalk, CT) automated DNA sequencer using M13 universal forward and reverse primers. A few internal primers were also designed when necessary. Nucleotide sequences were assembled using GeneToolLite software (BioTools). Similarity searches in the GenBank/EMBL and in the Drosophila pseudoobscura genome project (available at http://www.hgsc.bcm.tmc.edu) databases were performed using blastn and fasta3 programs. Multiple sequence alignments were obtained with ClustalW (THOMPSON et al. 1994) and DiAlign (MORGENSTERN 1999). PAML software (YANG 1997) was used to estimate the number of synonymous (d_S) and nonsynonymous (d_N) substitutions per site. This software avoids using reconstructed ancestral sequences to estimate d_S and d_N for lineages in a phylogeny by using a maximum likelihood approach. Different codon-based likelihood models that allow for different d_N/d_S ratios among evolutionary lineages can be devised. The models can then be compared to test the neutral prediction that the d_N/d_S ratio is identical among lineages (YANG and BIELAWSKI 2000).

In situ hybridization:

In situ hybridization of DNA probes was carried out as described in MONTGOMERY et al. (1987). Hybridization temperature was 37°. Probes were labeled with biotin-16-dUTP by nick translation and detection was done using the ABC-Elite kit from Vector Laboratories (Burlingame, CA). Hybridization signals were localized using the cytological maps of D. buzzatii (RUIZ and WASSERMAN 1993) and photographs were taken with a phase contrast Nikon Optiphot-2 microscope at x600 magnification.

Lsp1 gene number:

Screening of two genomic libraries of D. buzzatii with different probes allowed us to isolate clones containing the Lsp1ß and Lsp1 genes (see MATERIALS AND METHODS). To determine the number of Lsp1 genes present in the D. buzzatii genome, a Southern analysis was performed (see Figure S1 available as online supplementary material at http://www.genetics.org/supplemental/). Genomic DNA from D. buzzatii and D. melanogaster (as control) was digested with EcoRI and HindIII and hybridized with the same three probes used to screen the D. buzzatii libraries (see MATERIALS AND METHODS). The D. melanogaster Lsp1 probe hybridized to 6- and 4.1-kb fragments of D. melanogaster genomic DNA corresponding to Lsp1 and Lsp1ß, respectively (BROCK and ROBERTS 1983). In D. buzzatii this probe hybridized to a single fragment of 3.1 kb corresponding to Lsp1ß. The D. buzzatii Lsp1ß probe hybridized to 6- and 4.1-kb fragments of D. melanogaster corresponding to Lsp1 and Lsp1ß, respectively (BROCK and ROBERTS 1983), and a 3.1-kb fragment of D. buzzatii genomic DNA corresponding to Lsp1ß. Finally, the D. buzzatii Lsp1 probe hybridized to the 4.1-kb fragment corresponding to Lsp1ß and to 1.8- and 1.4-kb fragments both corresponding to Lsp1 of D. melanogaster (BROCK and ROBERTS 1983) and also with a 3.1-kb fragment corresponding to Lsp1ß and a 1-kb fragment corresponding to Lsp1 of D. buzzatii. Overall, the results of Southern analyses indicated that only two genes, Lsp1ß and Lsp1, are present in the D. buzzatii genome. No Lsp1 gene was detected by Southern analysis in the D. buzzatii genome.

Similarity searches against the D. pseudoobscura genome sequence database were carried out (Table 2). Two sequences with significant similarity were found with Lsp1ß. The most similar sequence, that included in contig 815_contig 5737, was considered to correspond to the D. pseudoobscura Lsp1ß ortholog. When the database was searched with the coding sequence of Lsp1 as query the same two hits were recovered. This time the most similar sequence included in contig 1500_contig 3546 was considered to correspond to the D. pseudoobscura ortholog of Lsp1. No new sequences were identified when D. melanogaster Lsp1 sequence was used as query, the only significant hits being those previously identified as Lsp1ß and Lsp1. We conclude that only Lsp1ß and Lsp1 are present in the D. pseudoobscura genome.

View this table: In this window In a new window   TABLE 2 Results of the blast search against the D. pseudoobscura genome database as of August 28, 2003, using as queries the coding sequences of Lsp1, Lsp1ß, and Lsp1

 

Organization of the Lsp1ß and Lsp1 genomic regions in D. buzzatii and D. pseudoobscura:

In D. buzzatii, the Lsp1ß gene was localized by in situ hybridization, using the j-19/25 phage as probe to polytene band D3c of chromosome 2 (see Figure S2 at http://www.genetics.org/supplemental/). This chromosome is homologous to chromosomal arm 3R of D. melanogaster (Muller's element E; Table 1). Overall, 9464 bp including the entire D. buzzatii Lsp1ß gene and its flanking regions were sequenced (accession no. AY561258). In this species, Lsp1ß is flanked by CG12492 and Pellino (Figure 1a). Between Lsp1ß and Pellino there is a snRNA:U1 gene, orthologous to snRNA:U1:95Ca of D. melanogaster.

In D. pseudoobscura, the Lsp1ß gene is included within contig 815_contig 5737, which has been putatively assigned to chromosome XR, homologous to D. melanogaster chromosomal arm 3L (Muller's element D; Table 1). As in D. buzzatii, Lsp1ß is flanked by CG12492 and Pellino but in D. pseudoobscura there are three snRNA:U1 genes between Lsp1ß and Pellino (Figure 1b). In D. melanogaster, CG12492, snRNA:U1:95Ca, and Pellino are localized in chromosomal arm 3R (Figure 1c) while Lsp1ß is located at 21D-E in chromosomal arm 2L (Figure 1d). The alignments of D. pseudoobscura and D. melanogaster genome sequences, available at http://pipeline.lbl.gov/pseudo/, show that Lsp1ß and its flanking genes are in fact included in contig 7891_contig 7492 assigned to chromosome 2 (Muller's element E; Table 1). We concluded that this must be the correct localization on the basis of the fact that CG12492 and Pellino are found on element E in both D. melanogaster and D. buzzatii.

In situ hybridization of the jq⁷-4/27 phage, containing the entire Lsp1 coding sequence, to the polytene chromosomes of D. buzzatti allowed us to map this gene to band C2g of chromosome 3 (see Figure S2 at http://www.genetics.org/supplemental/). This chromosome is homologous to chromosomal arm 2L of D. melanogaster (Muller's element B; Table 1). In total, 10,817 nucleotides including the entire Lsp1 gene and its flanking regions have been sequenced in D. buzzatii (accession no. AY561259). Upstream of Lsp1, 4.5 kb from the ATG codon, there is a Leucyl transfer RNA (Leu-tRNA) gene (Figure 2a).

In D. pseudoobscura, Lsp1 is found within contig 1500_contig 3546, which belongs to chromosome 4, homologous to chromosomal arm 2L of D. melanogaster (Muller's element B; Table 1). Analysis of the D. pseudoobscura genomic sequence revealed that 4.9 kb upstream from Lsp1 there is an Aspartic acid transfer RNA (Asp-tRNA). Both Lsp1 and Asp-tRNA are nested inside the first intron of the gene Sema-1a (Figure 2b). In both D. pseudoobscura and D. buzzatii, we detected eight short sequences, 28 to 59 nucleotides long, scattered along the 5-kb region upstream of the Lsp1 coding sequence, that are highly conserved in D. melanogaster (86–97% nucleotide identity). Another four short sequences, 41–55 nucleotides long, also highly conserved between the three species (91–98% of nucleotide identity), were found downstream of Lsp1 in D. pseudoobscura and D. buzzatii (Figure 2). The conservation in number and relative position of these 12 highly conserved noncoding sequences flanking Lsp1 in both D. buzzatii and D. pseudoobscura led us to conclude that the molecular organization of this gene region is the same in the two species; i.e., Lsp1 is nested within an intron of Sema-1a in both cases. On the other hand, in D. melanogaster (Figure 2, c and d), the Sema-1a gene is located in band 29E1-3 on chromosomal arm 2L (Muller's element B; Table 1) and inside its first intron there is also an Asp-tRNA gene (Figure 2c) but not the Lsp1 gene, which is located on chromosomal arm 3L (Muller's element D; Table 1).

Molecular structure of Lsp1ß and Lsp1 genes in D. buzzatii and D. pseudoobscura:

In the two species, both Lsp1ß and Lsp1 are made up of two exons separated by a small intron (Table 3). The alignment of D. buzzatii, D. pseudoobscura, and D. melanogaster nucleotide sequences shows a 79.4% nucleotide identity for Lsp1ß and 75.7% nucleotide identity for Lsp1. For both genes, the intron is placed in the same precise site in the three species and has a similar length (63–71 nucleotides for Lsp1ß and 57–65 nucleotides for Lsp1) but shows little nucleotide conservation other than the donor (G·T·^A/_G·A·G·^T/_C) and acceptor (C·A·G) splice sites (DELANEY et al. 1986).

View this table: In this window In a new window   TABLE 3 Molecular structure of Lsp1 genes in D. buzzatii and D. pseudoobscura compared to those of D. melanogaster

 
Both Lsp1ß and Lsp1 possess a TATA box, which is localized in D. buzzatii and D. pseudoobscura at the same nucleotide position as in D. melanogaster (–32 to –26). Sequence similarity extends for several nucleotides on either side of the TATA box (Figure 3). The 5'-UTR of D. buzzatii Lsp1ß has 89 bp, the first 21 nucleotides being identical to those in D. melanogaster and D. pseudoobscura. The 5'-UTR of D. buzzatii Lsp1 is 68 bp long and 17 of the first 21 nucleotides are identical to those in D. melanogaster and D. pseudoobscura. In both cases, another block with significant sequence similarity is found further downstream (Figure 3). A polyadenylation signal (AATAAA) is located 100 bp downstream from the stop codon in Lsp1ß and Lsp1. In Lsp1ß, but not in Lsp1, the sequence around this signal is highly conserved in the three species (Table 4a). Apart from this, no other conserved sequences have been found in the 3' region of Lsp1 genes.

View larger version (44K): In this window In a new window Download PPT slide   FIGURE 3.— ClustalW alignment of 5'-UTR and close upstream sequences of Lsp1 in D. melanogaster (Dm) and Lsp1ß and Lsp1 in D. melanogaster, D. pseudoobscura (Dp), and D. buzzatii (Db). Conserved regions are included in rectangles. Asterisks show nucleotides identical in the seven sequences and boldface type indicates the most common nucleotide in each position. TATA box is underlined; +1 shows the first nucleotide of the 5'-UTR. The first AUG codon is in boldface type and is indicated by dots below the sequence.

 

View this table: In this window In a new window   TABLE 4 Conserved sequences between D. melanogaster, D. pseudoobscura, and D. buzzatii in the 3'-UTR (a) and 5' region (b, c, and d) of Lsp1ß and in the 5' region (e, f, and g) of Lsp1

 
We searched for putative regulatory sequences in the 5' regions of Lsp1 genes in D. buzzatii and D. pseudoobscura following the criteria devised by BERGMAN and KREITMAN (2001). Comparison of the 5' ends of Lsp1ß led to the identification of three conserved sequences starting at sites –189, –126, and –683 of D. buzzatii (Table 4, b, c, and d). Another three conserved sequences starting at sites –379, –181, and –71 of D. buzzatii were found in the 5' region of Lsp1 (Table 4, e, f, and g).

Molecular evolution of the Lsp1 genes:

The coding sequence of Lsp1ß and Lsp1 from D. buzzatii, D. melanogaster, and D. pseudoobscura and that of Lsp1 from D. melanogaster were aligned using ClustalW. The number of synonymous and nonsynonymous substitutions per site for pairwise comparisons between the three species were estimated using maximum likelihood methods (YANG 1997). The d_N/d_S ratios for Lsp1ß (0.0226–0.0449) are similar to those for Lsp1 (0.0209–0.0512). Comparisons between Lsp1 of D. melanogaster with Lsp1ß (0.0470–0.0545) and Lsp1 (0.0367–0.0386) of the other two species also yielded similar results (Table 5). Overall, the d_N/d_S ratios were low, suggesting a relatively high degree of functional constraint of these genes in the three species analyzed.

View this table: In this window In a new window   TABLE 5 Maximum likelihood estimates of the number of synonymous and nonsynonymous substitutions per site in the coding regions of Lsp1 genes for pairwise comparisons among D. melanogaster, D. pseudoobscura, and D. buzzatii

 
Two different methods, neighbor joining and the unweighted pair group method using arithmetic averages (UPGMA), were used to construct phylogenetic trees using PHYLIP software (FELSENSTEIN 1989). In addition to the seven above-mentioned sequences, we included in the trees those of arylphorin, an Lsp1-like gene of Calliphora vicina (NAUMANN and SCHELLER 1991), and Lsp2 of D. melanogaster (ADAMS et al. 2000). Alternative models for the evolution of the Lsp genes were then tested using maximum likelihood methods (YANG 1997; BIELAWSKI and YANG 2003). First we tested for the constancy of evolution rates by comparing both trees: that produced with the UPGMA method, which assumes a molecular clock, and that built with the neighbor-joining method, assuming no clock. In both cases a single d_N/d_S ratio for all lineages was considered. The difference between the likelihood of both trees was significant (2l = 32.64; 7 d.f.; P < 0.005) indicating that the model assuming no clock provides a significantly better fit to the data (Figure 4). We then tested for homogeneity in the d_N/d_S ratio between lineages by comparing the model assuming no clock and a single d_N/d_S ratio with a free-ratio model, which assumes an independent d_N/d_S ratio for each lineage. The result was significant (2l = 83.18; 14 d.f.; P < 0.0001) indicating that the d_N/d_S ratios are heterogeneous among lineages. Finally we compared a model assuming several d_N/d_S ratios (one background ratio and one ratio for each of the lineages leading to the Lsp1 genes in D. melanogaster) with the single-ratio model. Again the difference was statistically significant (2l = 21.86; 3 d.f.; P < 0.005) indicating a better fit to the data for the model assuming several d_N/d_S ratios.

View larger version (19K): In this window In a new window Download PPT slide   FIGURE 4.— Neighbor-joining tree of Lsp1 genes from D. melanogaster, D. pseudoobscura, and D. buzzatii species. Arylphorin from C. vicina and Lsp2 from D. melanogaster are also included. The tree was constructed with an alignment of the coding sequence of the genes.

 

Lsp1 gene number:

In D. melanogaster the LSP-1 protein is made up of three subunits encoded by three Lsp1 genes: Lsp1, Lsp1ß and Lsp1 (ROBERTS and EVANS-ROBERTS 1979). However, in D. buzzatii, a species of the Drosophila subgenus, and in D. pseudoobscura, a species of the Sophophora subgenus, only two Lsp1 genes seem to be present: Lsp1ß and Lsp1. Two different genomic libraries of D. buzzatii have been screened using as probes a fragment of D. melanogaster Lsp1 and a fragment of D. buzzatii Lsp1ß or Lsp1. Overall, four different library screenings have been carried out and in every case all positive clones contained either Lsp1ß or Lsp1. No Lsp1 gene was found. To corroborate this result, genomic DNA was digested with restriction enzymes and hybridized with the same three probes used to screen the libraries and again the results were in agreement with the existence of only two genes, Lsp1ß and Lsp1, in the D. buzzatii genome. Also, the D. pseudoobscura genome has been recently sequenced to approximately sevenfold coverage and is available at http://www.hgsc.bcm.tmc.edu. Similarity searches against this database have been performed, allowing us to annotate Lsp1ß and Lsp1 but not Lsp1. Thus, our results indicate that Lsp1 is not present in D. buzzatii or D. pseudoobscura. The most parsimonious explanation for this observation is that the duplicative transposition that gave rise to Lsp1 took place in the lineage leading to D. melanogaster after the divergence of the D. pseudoobscura lineage 30 MYA (but see below). The fact that D. melanogaster Lsp1 is not dosage compensated although it is X linked is in agreement with a recent duplicative transposition onto this chromosome (ROBERTS and EVANS-ROBERTS 1979).

BROCK and ROBERTS (1983) reported that in eight species of the Drosophila and Sophophora subgenera, Lsp1 and Lsp1ß map to the same polytene band in chromosomal element E except in D. pseudoobscura, where they observed an extra signal in element B (see Table 1). They concluded that there were at least two genes, one -like and one ß-like, given that Lsp1 could not be localized by in situ hybridization. Contrasting results have been obtained in this work. We have unambiguously localized Lsp1ß to chromosomal element E and Lsp1 gene to element B in both D. buzzatii and D. pseudoobscura. According to our results the extra signal in D. pseudoobscura element B likely corresponds to Lsp1. A plausible explanation for their results is that Lsp1 and Lsp1ß probes were cross-hybridizing to Lsp1ß as is suggested by the fact that the signals obtained with the Lsp1ß probe were stronger than those obtained with the Lsp1 probes (BROCK and ROBERTS 1983). Likewise, our previous in situ hybridization of D. melanogaster Lsp1 to D. buzzatii (and D. repleta) chromosomes (GONZáLEZ et al. 2002; RANZ et al. 2003) must be reinterpreted as due to cross-hybridization with Lsp1ß (see Figure S2 at http://www.genetics.org/supplemental/).

Larval serum proteins belong to the hemocyanin superfamily and are thought to act as storage proteins that provide amino acids and energy during nonfeeding periods of immature or adult development (BURMESTER et al. 1998). In D. melanogaster LSP-1 is a heterohexamer of randomly associated subunits encoded by Lsp1, Lsp1ß, and Lsp1 genes. The lack of one subunit in D. buzzatii and D. pseudoobscura does not imply that the LSP-1 protein will not be functional. In fact, it has been reported that an inbred stock of D. melanogaster lacking the -chain is viable under laboratory conditions, suggesting that a subunit specific function for the LSP-1 monomers does not exist (BROCK and ROBERTS 1980). The polypeptides encoded by Lsp1ß and Lsp1 of D. melanogaster, D. pseudoobscura, and D. buzzatii and by Lsp1 of D. melanogaster were aligned with the ClustalW algorithm (see Figure S3 at http://www.genetics.org/supplemental/). These seven sequences have a similar length (772–789 aa), 49% of the amino acids are identical, and 33% of the amino acid substitutions are conservative or semiconservative. However, different subunits can accumulate different specific amino acids and this could allow the organism to modulate the availability of these amino acids in different developmental processes (MASSEY et al. 1997). The three subunits of the LSP-1 protein of D. melanogaster, as well as similar proteins in other Diptera, are enriched in aromatic amino acids (BURMESTER et al. 1998). Aromatic residues are thought to serve as precursors for quinones, which play a role in cuticle hardening during metamorphosis (BURMESTER et al. 1998). The polypeptides coded by Lsp1 and Lsp1ß are also enriched in methionine but not those encoded by Lsp1. The same pattern is observed in D. pseudoobscura and D. buzzatii. The role of methionine in Drosophila development is not clear (MASSEY et al. 1997). Gene duplication is considered a major force in gene family expansion and gene innovation. After duplication, one copy may become silenced (nonfunctionalization) or assume a novel function (neofunctionalization) or both copies may split the multiple functions of the ancestral gene (subfunctionalization; LYNCH and CONERY 2000). Gene amplification of highly expressed functions often lead to highly conserved paralogs in microbial genomes (HOOPER and BERG 2003). The latter seems to be the case of the Lsp1 genes in Drosophila. The protein coded by these genes is accumulated to high levels by feeding larvae (MASSEY et al. 1997) and gene amplification has not resulted in different discernible functions.

Genome organization and number of transposition events:

In both D. buzzatii and D. pseudoobscura, Lsp1ß is flanked by CG12492 and Pellino genes on chromosomal element E (Figure 1, a and b). The homologous region in D. melanogaster, where CG12492 and Pellino are found, is also located on element E. This region shows no sequence similarity to any Lsp1 gene whatsoever (Figure 1c). In D. melanogaster, Lsp1ß is located on element B (Figure 1d). In the three species, an snRNA:U1 is closely linked to the Lsp1ß gene. Given that D. buzzatii and D. pseudoobscura, which belong to two different subgenera separated by 80–124 MYR (BEVERLY and WILSON 1984; SPICER 1988), show the same genomic organization for Lsp1ß, this is likely to be the ancestral organization of the genus and therefore Lsp1ß would have conservatively transposed from element E to element B in the lineage leading to D. melanogaster. The maximum size of the transposed region is 5.7 kb and the only protein-coding gene included is Lsp1ß.

In D. buzzatii and D. pseudoobscura, Lsp1 is flanked by a tRNA and both genes are located inside the first intron of Sema-1a on element B (Figure 2, a and b). In D. melanogaster, Sema-1a is located on element B while Lsp1 is located on element D (Figure 2, c and d). Since the genomic organization of Lsp1 is the same in D. buzzatii as in D. pseudoobscura, this is likely to be the ancestral organization of the genus and thus the Lsp1 gene would have conservatively transposed from element B to element D in the lineage leading to D. melanogaster. The maximum size of the transposed region is 5.9 kb and includes the putative regulatory regions located upstream of the gene. Another transposition event where the nested organization was the ancestral one had been previously reported in Drosophila (NEUFELD et al. 1991).

Given that Lsp1ß and Lsp1 are present in the three species studied in different chromosomes, the duplicative transposition originating these two genes must have occurred before the divergence of these species (40–62 MYA). The chromosomal localization of the ancestral Lsp1 gene in the genus Drosophila is unknown. There is no reason to believe that this chromosome was element E as suggested by BROCK and ROBERTS (1983) because in both D. buzzatii and D. pseudoobscura Lsp1ß is located on element E and Lsp1 is located on element B. Therefore, the localization of the ancestral Lsp1 gene could be any of these two elements. In any case, to explain the current localization of these two genes in D. melanogaster at least two conservative transpositions are needed (see above). Another duplicative transposition gave rise to D. melanogaster Lsp1. The absence of this gene in D. pseudoobscura indicates that this transposition likely occurred after the divergence between D. melanogaster and D. pseudoobscura. However, the neighbor-joining tree of Lsp1 sequences (Figure 4) suggests that this transposition occurred before the divergence between D. melanogaster and D. pseudoobscura. If this were the case, this gene would have been lost in the D. pseudoobscura lineage. Overall, at least two duplicative and two conservative transpositions are needed to explain the present localization of Lsp1 genes (Figure 5).

View larger version (18K): In this window In a new window Download PPT slide   FIGURE 5.— Phylogenetic relationships and karyotypic organization of D. melanogaster and D. buzzatii. The present chromosomal localization of Lsp1 genes in D. melanogaster and D. buzzatii is shown. At least two duplicative and two conservative transpositions are required to explain the evolution of these genes from an ancestral Lsp1 gene.

 

Mechanism of transposition:

As stated before, the position of the unique intron of Lsp1ß and Lsp1 and the 5' putative regulatory sequences are conserved in the three species analyzed. This allows us to rule out retroposition (BETRáN et al. 2002) as the mechanism of transposition. The analysis of the flanking regions of both genes provides no evidence that transposition had been mediated by transposable elements (TEs). A single TE copy was found in the original position of Lsp1ß in D. melanogaster (Figure 1c) but there are no indications of its involvement in the transposition. However, taking into account the divergence time between the species analyzed, mobile elements could have played a role in the origin of the transposition and then be lost by deletion or excision. The most striking feature of Lsp1 genes in the three species analyzed is that they are very close to snRNA or tRNA genes. Both are repetitive genes that are scattered in the genome. However, the probability that Lsp1 genes are close to snRNA or tRNA genes just by chance seems very low. A recent review of D. melanogaster genome sequence-Release 3 has found 290 tRNA genes and only 28 snRNAs in the euchromatin (MISRA et al. 2002), i.e., 1 tRNA gene/0.4 Mb and 1 snRNA gene/4.3 Mb of euchromatic DNA. Repeated genes, e.g., tRNA and ribosomal protein genes, have been previously implied in the origin of chromosomal rearrangements in yeasts (SZANKASI et al. 1986; KELLIS et al. 2003).

We propose that an ectopic exchange between snRNA:U1 sequences mediated the Lsp1ß transposition in Drosophila. Three observations indicate that snRNA:U1 genes are implied in this transposition. First, they are present in the original location of chromosomal element E as well as in the destination site of element B. Second, snRNA:U1 genes present a high level of nucleotide identity among the three species (98%) suggesting that they might act as substrates for ectopic exchanges. Finally, the snRNA:U1 gene represents the downstream boundary of the transposed chromosomal segment (Figure 1), which would be expected if ectopic recombination actually took place. To our knowledge, snRNA genes have not been previously implicated in the generation of rearrangements. Nevertheless, there is no reason why they could not act as substrates for ectopic recombination in a manner similar to that of tRNAs in yeasts or Alu sequences in humans. As a matter of fact, the mechanism for gene transposition in Drosophila could be quite similar to that originating segmental duplications in humans (see Figure 6 in BAILEY et al. 2003).

The evidence for the implication of tRNA genes in the transposition of Lsp1 is weak. The tRNA genes found near Lsp1 in D. buzzatii and D. pseudoobscura belong to a different isoacceptor type and no tRNA gene was found near Lsp1 in D. melanogaster. In addition these tRNAs lie outside the transposed chromosomal segment. Therefore, an involvement of tRNA genes in the transposition of Lsp1 is unlikely and in this case the mechanism remains uncertain.

5' noncoding conserved sequences:

Transposition implies the localization of the transposed gene in a new genomic environment. The probability of success of a transposition should be higher when it includes the regulatory regions than when it does not. To be functional, in the latter case the transposed gene would need to recruit new regulatory regions (BETRáN et al. 2002). Comparative sequence analysis allows the identification of conserved DNA sequences in noncoding regions that are considered putative cis-acting regulatory elements (BERGMAN and KREITMAN 2001). Comparison of the 5' ends of Lsp1, Lsp1ß and Lsp1 genes of D. melanogaster led to the identification of two such conserved sequences (DELANEY et al. 1986). These sequences are also conserved in Lsp1ß (Table 4, b and c) and Lsp1 (Table 4, e and f) in the three species analyzed. These two regions are more conserved between Lsp1 and Lsp1ß than between Lsp1ß and Lsp1, which is in agreement with the origin of Lsp1 from a duplication of Lsp1ß (SMITH et al. 1981). Two conserved sequences not previously described in D. melanogaster have been also identified. One is exclusive of Lsp1ß, the other is exclusive of Lsp1 (Table 4, d and g), and both are highly conserved in the three species analyzed. All conserved sequences except that located at –181 of D. buzzatii Lsp1 fulfill the requirements used by BERGMAN and KREITMAN (2001) to identify noncoding conserved blocks. The mean size of these five blocks is 13 bp, similar to the modal size of 11 bp reported by these authors, and three of them are included in the conserved blocks that they have described. They follow the pattern described for cis-regulatory elements in Drosophila, i.e., highly conserved sequences separated by unalignable gaps (BERGMAN and KREITMAN 2001).

The 5'-untranslated regions of the RNA show two highly conserved sequences common to the three Lsp1 genes in the three species analyzed (Figure 3). Although the significance of these homologies in the 5'-UTR is not clear, it has been suggested that the synthesis of LSP-1 protein may be under translational control (POWELL et al. 1984). All the signals necessary for the correct tissue and temporal specificity expression of Lsp1 and Lsp1ß in D. melanogaster lie 1650 and 2250 bp upstream from these two genes, respectively (JOWETT 1985; DAVIES et al. 1986). In this work we have analyzed more than 2 kb upstream from Lsp1ß and Lsp1 genes and we are quite confident that the majority if not all the cis-acting regulatory sequences have been identified. The fact that the transposition of Lsp1 genes included not only the genes but also the regulatory regions has probably played a role in the success of these transpositions.

Molecular evolution:

Maximum likelihood methods of phylogenetic inference were used to test alternative models for the evolution of Lsp1 genes (YANG 1997). Figure 4 shows the phylogenetic tree that provides a significantly better fit to the data. The tree is in agreement with the appearance of Lsp1 from a duplication of Lsp1ß in the Sophophora subgenus.

The ratio of synonymous (silent, d_S) to nonsynonymous (amino acid changing, d_N) substitution rates is a measure of selective pressure on a protein: if amino acid changes are neutral d_N/d_S = 1, if they are mostly deleterious d_N/d_S < 1, and if they offer a selective advantage d_N/d_S > 1 (YANG and BIELAWSKI 2000; BIELAWSKI and YANG 2003). A model assuming free d_N/d_S ratios for different lineages provided a significantly better fit to the data than a model with a single ratio for all lineages. This led us to test if a model assuming four different d_N/d_S ratios, one background ratio and one ratio for each of the three lineages leading to D. melanogaster Lsp1 genes, was significantly different from the single-ratio model. According to our observations, the Lsp1 genes have transposed in these three lineages, and we wanted to test if the change of chromosomal location has affected the molecular evolution of these genes. Again the results were significant, indicating that the model assuming four different d_N/d_S ratios fits better to the data. The estimates obtained for the d_N/d_S ratios in this model were 0.0518 for the background ratio and 0.0769, 0.0598, and 0.0231 for the lineages leading to Lsp1, Lsp1ß, and Lsp1 of D. melanogaster, respectively. These results suggest that changes in the selection regime (degree of functional constraint) are associated with Lsp1 transpositions. However d_N/d_S ratios do not show a consistent pattern, being higher than the background ratio for Lsp1 and lower than the background ratio for Lsp1. In addition, the background ratio is itself heterogeneous, indicating that other factors besides transposition have an effect on the molecular evolution of Lsp1 genes.

We thank L. Sánchez (Centro de Investigaciones Biológicas, Consejo Superior de Invesigaciones Cientifícas, Madrid) for providing the D. melanogaster Lsp1 clone and E. Hasson (Universidad de Buenos Aires) for D. buzzatii stocks. We also thank B. Negre for technical advice and helpful discussion. This work was supported by grant BMC2002-01708 from the Dirección General de Investigación (Ministerio de Ciencia y Tecnología, Spain) awarded to A.R.

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY561258 and AY561259.

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