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. 2005 Dec;171(4):1719–1727. doi: 10.1534/genetics.105.041699

A Novel Chimeric Gene, siren, With Retroposed Promoter Sequence in the Drosophila bipectinata Complex

Masafumi Nozawa 1, Tadashi Aotsuka 1, Koichiro Tamura 1,1
PMCID: PMC1456098  PMID: 16143626

Abstract

Retrotransposons often produce a copy of host genes by their reverse transcriptase activity operating on host gene transcripts. Since transcripts normally do not contain promoter, a retroposed gene copy usually becomes a retropseudogene. However, in Drosophila bipectinata and a closely related species we found a new chimeric gene, whose promoter was likely produced by retroposition. This chimeric gene, named siren, consists of a tandem duplicate of Adh and a retroposed fragment of CG11779 containing the promoter and a partial intron in addition to the first exon. We found that this unusual structure of a retroposed fragment was obtained by retroposition of nanos, which overlaps with CG11779 on the complementary strand. The potential of retroposition to produce a copy of promoter and intron sequences in the context of gene overlapping was demonstrated.

GENE duplication and exon shuffling are the most important mechanisms for generation of new genes during genome evolution (reviewed by Brown 1999). Gene duplication produces two identical copies of an existing gene, which provides opportunities for one of them to accumulate mutations and acquire a new function and eventually become a new gene, while the other copy retains the original function supplied by the single-copy gene before duplication (Ohno 1970; Kimura and Ohta 1974). Because of the random nature of mutation, the protein-coding sequence of a duplicated gene is usually disrupted with time and the birth of a new functional gene has been thought to be rather rare event (Ohta and Kimura 1971). However, a recent study suggested that duplicated genes are almost as likely to acquire a new function as to be lost through acquisition of mutations that compromise the function of genes after genome duplications (Nadeau and Sankoff 1997). In general, many genes belonging to the same multigene family or gene superfamily are thought to result from gene duplications (see Graur and Li 2000 for review). Therefore, the cumulative contribution of gene duplication to genome evolution seems to be substantial. In addition to the accumulation of mutations, exon (or domain) shuffling can also produce a new gene by rearranging functional domains among duplicated and/or preexisting genes (Gilbert 1978). Many genes sharing common functional domains in different combinations (e.g., Rubin et al. 2000; Li et al. 2001) indicate a substantial contribution of exon shuffling to the diversity of genes and gene functions in genomes. Long et al. (1995) estimated that at least 19% of exons in eukaryotic genes have experienced exon shuffling in their evolutionary histories.

In contrast to the observed indirect evidence of gene duplication and exon shuffling, there have been only a few opportunities to explore the processes and mechanisms of gene duplication and exon shuffling. Among them, recent studies suggest that retrotransposons and retroposons contribute to gene duplication and exon shuffling by means of reverse transcription of expressed genes and integration of the reverse transcripts into new genomic positions (e.g., McCarrey and Thomas 1987; Noyce et al. 1997; Betrán and Long 2003). Since a retroposed gene copy does not contain the promoter sequence that resides in the untranscribed region, it will be inactive and eventually become a retropseudogene unless a promoter sequence is newly recruited (Long et al. 2003). For this reason, functional retrogenes often show a chimeric structure between a retroposed coding sequence and the part of the preexisting gene that supplied the promoter sequence (Long and Langley 1993; Long et al. 1999; Wang et al. 2002; Nisole et al. 2004; Sayah et al. 2004; Jones et al. 2005). Because the chance that a retroposed sequence will acquire a promoter sequence from a preexisting gene without damage to its function is expected to be very low, the potential of a retroposed sequence to become a new functional gene must be quite limited (see Graur and Li 2000). In other words, the mechanism for obtaining a promoter sequence is critical to the generation of functional retrogenes.

In this study, we report a novel chimeric gene consisting of a retroposed fragment of CG11779 (annotated in the Drosophila melanogaster genome of Release 3.2) and a tandem duplicate of the alcohol dehydrogenase (Adh) gene in species of the D. bipectinata complex. We named it siren after Greek myths, in which the Siren is a chimera of a human and a bird. Interestingly, sequence homology and expression pattern suggested that the promoter sequence of siren was carried by the retroposed CG11779 fragment. This unexpected structure was produced by retroposition of nanos, which overlaps with CG11779 on the complementary strand. We show a potential of retroposition to produce a copy of promoter as well as protein-coding sequences, when genes are overlapping.

MATERIALS AND METHODS

Fly species and sequence data:

For D. parabipectinata of the D. bipectinata complex, we determined, using molecular cloning techniques, a nucleotide sequence of a genomic region (17,499 bp) including the Adh gene and a new gene (siren) discovered in this study (DDBJ/EMBL/GenBank accession no. AB194414) and another sequence (17,580 bp) including CG11779 and nanos that overlap each other on the opposite strands (accession no. AB194415). To clarify the gene structures of these genes, cDNA sequences for siren, Adh, CG11779, and nanos were determined by using RT-PCR (Powell et al. 1987) and 5′- and 3′-RACE (Frohman et al. 1988) techniques. Partial nucleotide sequences (∼4.6–6.3 kb), including siren, were also determined for D. bipectinata, D. malerkotliana (D. m. malerkotliana and D. m. pallens), and D. pseudoananassae (D. p. nigrens and D. p. pseudoananassae) belonging to the D. bipectinata complex of the D. ananassae subgroup by using PCR techniques (accession nos. AB194416–AB194420). In addition, we determined partial nucleotide sequences of Adh and CG11779/nanos for all these species as well as for D. ananassae, D. varians, and D. merina of the D. ananassae subgroup for comparative sequence analyses (accession nos. AB194421–AB194436). The nucleotide sequences of Adh and CG11779/nanos regions for D. melanogaster (genome sequence of Release 3.2) were downloaded from GenBank.

The fly stocks of D. bipectinata, D. malerkotliana (D. m. malerkotliana and D. m. pallens), D. merina, D. parabipectinata, and D. varians were maintained at Tokyo Metropolitan University, and those of D. ananassae (stock no. 14024-0371.0), D. p. nigrens (stock no. 14024-0411.0), and D. p. pseudoananassae (stock no. 14024-0421.0) were originally obtained from the National Drosophila Species Stock Center at Bowling Green State University and maintained in Tokyo Metropolitan University.

DNA cloning, PCR, and sequencing:

We constructed a genomic library for D. parabipectinata with λEMBL3 phage vector (Promega, Madison, WI ) and obtained clones containing Adh and/or siren. The obtained clones were subcloned into plasmid vector pUC19 according to Henikoff (1984). The nucleotide sequences were determined by using BigDye Terminator v3.1 cycle sequencing kit and ABI Prism 377 DNA Sequencer (Applied Biosystems, Foster City, CA). We also constructed a SuperCos1-cosmid (Stratagene, La Jolla, CA) library to obtain CG11779/nanos clones and determined the nucleotide sequence by a shot-gun sequencing method in the DNA sequencing center (National Institute of Genetics). The standard PCR method was used to determine the genomic DNA sequences for siren, Adh, and CG11779/nanos for other species. The nucleotide sequences of these PCR products were determined by the direct sequencing method or via cloning in the cases of heterozygotes. For the latter cases, we followed Hanahan's (1985) procedures for cloning with pUC118 or pUC119 plasmid vector and determined the nucleotide sequences for multiple clones to correct PCR errors.

The template total RNA for RT-PCR and 5′- and 3′-RACE was extracted from 5–10 individuals by Boom et al.'s (1990) method with modifications for complete DNA removal. ThermoScript RT-PCR system (Invitrogen, San Diego) and 5′-RACE system ver.2.0 (Invitrogen) were used for RT-PCR and 3′-RACE and for 5′-RACE, respectively. The nucleotide sequences of these PCR products were determined by the direct sequencing method or via cloning as described above.

The RT-PCR was also used for detecting the presence or absence of transcripts at larval, pupal, and adult stages in male and female individuals to examine the expression pattern of siren, Adh, and CG11779 genes. For the case of Adh, the template cDNA for 10 μl of PCR mixture was reverse transcribed from 10 ng of total RNA, whereas it was obtained from 100 ng of total RNA for siren1, siren2, and CG11779. Ribosomal protein L32 (RpL32) gene was used as a control to ensure the efficiency of RT-PCR. Since the Adh gene has two different promoters (Savakis et al. 1986), the corresponding types of transcripts were discriminated by specific primers. Detailed information for the cloning probes and PCR primers is described in the supplementary material at http://www.genetics.org/supplemental/.

Genetic linkage analyses:

The relative chromosomal locations among siren, Adh, and CG11779/nanos were examined by genetic linkage analyses among siren1 (upstream locus for tandem duplicates of siren), Adh, and CG11779/nanos loci using B133 and CJB162 strains of D. bipectinata, which are homozygous for different alleles from each other in all three loci. A single virgin female of CJB162 and a single male of B133 were crossed in a culture vial for 7 days and a single pair of their F1 progeny was then crossed in the same manner. From each individual of the pair of F1 progeny and 96 (48 female and 48 male) F2 progenies, we extracted total DNA and amplified DNA fragments by PCR for each gene. The PCR products were digested by MboI for siren1 and CG11779/nanos or by HaeII for Adh and agarose-gel electrophoresed to distinguish the alleles. Accession numbers for the nucleotide sequences of siren1, Adh, and CG11779/nanos are AB194437–AB194439 and AB194440–AB194442 for B133 and CJB162 strains of D. bipectinata, respectively.

Sequence analyses:

To examine the homology between the genomic sequences obtained, we performed dot-plot analyses using the Nucleic Acid Dot Plots program (http://www.vivo.colostate.edu/molkit/). The parameter set of a 15-bp window size and a 1-bp mismatch limit was used. For detailed comparisons between the homologous parts identified, a mismatch limit of 3 bp was also used.

A molecular phylogenetic tree for siren and Adh was constructed by the minimum-evolution method (Rzhetsky and Nei 1992) with synonymous distances estimated by the modified Nei-Gojobori method (Zhang et al. 1998) with a transition/transversion ratio equal to 2 and a Jukes-Cantor correction (Jukes and Cantor 1969) for multiple hits. The 256 homologous codons between siren and Adh were used. The statistical confidence for each branch in the tree was evaluated by the bootstrap method (Felsenstein 1985) with 1000 replications. We used the MEGA3 program (Kumar et al. 2004) for these analyses.

RESULTS

An insertional nucleotide sequence in the D. parabipectinata genome:

We determined the nucleotide sequence of the Adh gene and its flanking region (17,499 bp) in the D. parabipectinata genome. This fragment was longer than its counterpart in the D. melanogaster genome sequence by 3943 bp. A dot plot of these homologous sequences indicates that the difference is largely attributed to a single insertional sequence (∼4.6 kb) in the D. parabipectinata sequence, and >300 short (mostly <10 bp) indels are required to fill up the rest of the difference (∼700 bp) (Figure 1A). Within this insertional sequence, two Adh homologous parts are clearly shown, suggesting that duplications involving the Adh sequence were responsible for the origin of the insertional sequence. The alternative possibility of a deletion in the D. melanogaster sequence is unlikely, since the insertional sequence does not exist in the sequence of an outgroup species (accession no. AADE01001152 for D. pseudoobscura). A dot plot of the insertional sequence with itself reveals the detailed structure, i.e., a duplication of an ∼1.5-kb segment including the Adh homologous sequence and a tandem direct repeat of a 180-bp unit length between the duplicates (Figure 1B). This structure was found in all other species of the D. bipectinata complex at the same location, while the number of repeat units varies among species (data not shown). The repeat sequence was not at all identified in the D. melanogaster whole-genome sequence (Release 3.2) by the standard BLAST search procedure (Altschul et al. 1990).

Figure 1.

Figure 1.

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Dot plot for Adh and its upstream region of D. parabipectinata. (A) Comparison of the genomic fragment including Adh and its upstream region between D. parabipectinata (horizontal axis) and D. melanogaster (vertical axis). The position of Adh is indicated by the open box and the 4.6-kb insertional sequence in the D. parabipectinata sequence is surrounded by the dashed line. (B) Comparison of the insertional sequence (position 1138–5957 in the D. parabipectinata genomic fragment) with itself. The parameter values used are a window size equal to 15 bp and a mismatch limit equal to 1 bp.

A new chimeric gene:

Within the duplicated 1.5-kb segments, both GENSCAN (Burge and Karlin 1997) and Genie (Kulp et al. 1996) gene-finding programs predicted the same open reading frames (ORFs). Using RT-PCR and 5′- and 3′-RACE techniques, we determined the complete cDNA sequences for both ORFs and confirmed the exon-intron structure of the genes corresponding to these two ORFs. These two genes were virtually two identical copies of the same gene. BLASTP (Altschul et al. 1990) showed that the second and third exons of this gene are homologous to the second and third exons of the Adh gene, respectively, at the translated amino-acid sequence level, as well as the nucleotide sequence level shown in Figure 1A. The remaining first exon showed the best homology to the first exon of CG11779 in the D. melanogaster genome at the translated amino-acid sequence level. Therefore, this gene is a chimera consisting of the first exon of CG11779 and the second and third exons of Adh. It should be noted that the encoded siren protein is also a chimera containing the 31-amino-acid N-terminal sequence of CG11779 coupled onto the entire Adh protein sequence. As mentioned, we named it siren (siren1 and siren2 corresponding to the upstream and downstream loci, respectively) after Greek myths, following another chimeric gene, sphinx, found in D. melanogaster (Wang et al. 2002). In the D. melanogaster genome sequence, CG11779 overlaps with another gene, nanos, on the complementary strand. We confirmed the same structure in the D. parabipectinata genome as well. A dot plot of the siren and CG11779/nanos sequences in the D. parabipectinata genome shows that the homology includes the 5′-untranscribed region, the first exon, and a part of the first intron of CG11779, which correspond to a part (mainly the 3′-untranslated region) of the third exon of nanos (Figure 2). The homology of siren to Adh starts after the CG11779/nanos homologous part and includes the second and third exons (Figure 2). Although the boundaries of these distinct parts are rather ambiguous due to substantial gaps (>600 bp in total) and base substitutions (Figure 2), it is clear that the introns of CG11779 and Adh are joined to be the first intron of siren, in which the donor and accepter sites are provided by CG11779 and Adh, respectively (Figure 3). The amino-acid sequence at the beginning of the second exon of siren is considerably modified by a 12-bp indel and base substitutions compared to the original second exon of Adh (Figure 3).

Figure 2.

Figure 2.

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Dot plots of siren and CG11779/nanos (blue dots) and siren and Adh (red dots) sequences of D. parabipectinata. The dot-plot parameters were a window size equal to 15 bp and a mismatch limit equal to 3 bp. (Top) The exon-intron structures deduced from cDNA sequences for siren, (left) for CG11779/nanos, and (right) for Adh, where solid and open boxes indicate exons and introns, respectively.

Figure 3.

Figure 3.

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Nucleotide and amino-acid sequence alignments for the exon-intron boundaries of siren in D. parabipectinata. (A) The boundary between the first exon and the first intron of siren1 and siren2 with its homologous part of CG11779. (B) The boundary between the first intron and the second exon of siren1 and siren2 with its homologous part of Adh. Dashes represent alignment gaps and each dot represents the nucleotide identical with that in the topmost sequence. The donor (A) and acceptor (B) sites of the first intron of siren and their counterparts in CG11779 and Adh, respectively, are surrounded by open boxes.

Chromosomal location of siren, Adh, and CG11779/nanos:

The relative chromosomal locations among siren1, Adh, and CG11779/nanos were examined by the genetic linkage analyses (Table 1). Since the F1 progeny were heterozygous for all three loci irrespective of their gender, these loci were identified to be autosomal. As expected by the physical distance between siren1 and Adh at the nucleotide sequence level (∼13 kb), these two loci showed a perfect linkage. On the other hand, the distribution of the observed numbers of F2 genotypes gave an excellent goodness of fit (Inline graphic) with the expected values under independent assortment of siren1 (or Adh) and CG11779/nanos without linkage. The results show that CG11779/nanos belongs to a different linkage group from siren and Adh; i.e., CG11779/nanos is located on a different chromosome or at least in a chromosomal region distant from siren and Adh.

TABLE 1.

The observed and expected numbers of individuals for each genotype in F2 progeny of B133 and CJB162 strains of D. bipectinata

No. of individuals
Genotype:a a1a1b1b1 a1a1b1b2 a1a1b2b2 a1a2b1b1 a1a2b1b2 a1a2b2b2 a2a2b1b1 a2a2b1b2 a2a2b2b2
a, siren1; b, Adh
    Observed 24 0 0 0 51 0 0 0 21
    Expectedb 24.0 0.0 0.0 0.0 48.0 0.0 0.0 0.0 24.0
a, siren1 (or Adh); b, CG11779/nanos
    Observed 7 11 6 14 24 13 4 9 8
    Expectedc 6.0 12.0 6.0 12.0 24.0 12.0 6.0 12.0 6.0
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a

The genotypes of B133 and CJB162 were a1a1b1b1 and a2a2b2b2, respectively.

b

The expected numbers of F2 individuals under complete linkage between two loci.

c

The expected numbers of F2 individuals under random association between two loci.

Expression patterns of siren, Adh, and CG11779:

The transcription patterns of siren1, siren2, Adh, and CG11779 for D. parabipectinata were examined by RT-PCR at larval, pupal, and adult stages and in female and male individuals separately (Figure 4). The transcription of siren1 and siren2 was detected only at pupal and adult stages in male individuals. This pattern was virtually identical with that of CG11779 but distinct from the sex-independent and pupa-depressed pattern of both types of Adh transcripts. In addition, the amount of transcripts for Adh was substantially larger than that for siren and CG11779 (data not shown). To equalize the production of RT-PCR for the sake of experimental convenience, we used 10 times more total RNA for siren and CG11779 than for Adh. These results indicate that the promoter sequence of siren originated from CG11779.

Figure 4.

Figure 4.

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Agarose-gel electrophoresis for RT-PCR products, showing expression patterns of siren1, siren2, Adh, CG11779, and RpL32 (control) genes at larval, pupa, or adult developmental stage in female or male individuals of D. parabipectinata. Transcripts from the distal (Adh-d) and proximal (Adh-p) promoters of the Adh gene were separately examined.

Molecular evolution of siren:

A phylogenetic tree for siren1, siren2, and Adh sequences was constructed to infer the evolutionary relationships among these genes (Figure 5). It is clear that a duplication of Adh occurred after the divergence of the D. bipectinata complex from other species of the D. ananassae subgroup to generate the main part of siren (solid circle in Figure 5). An insertion of the CG11779/nanos-derived sequence must have occurred during the same evolutionary period, since the inserted sequence was found in the species of the D. bipectinata complex but not in their outgroup species. However, given that the average synonymous distance between siren and Adh (0.25 ± 0.03) was very close to the distance between siren and CG11779 (0.23 ± 0.10), whether the duplication of Adh occurred before or after the insertion of the CG11779/nanos sequence is unclear. Rearrangements of the Adh and CG11779/nanos-derived parts were thought to occur subsequently to produce ancestral single-copy siren, and a tandem duplication of the ancestral siren occurred finally to produce siren1 and siren2 before the speciation within the D. bipectinata complex (open circle in Figure 5).

Figure 5.

Figure 5.

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Minimum-evolution tree for siren and Adh sequences. The solid and open circles indicate the duplications of Adh and ancestral siren, respectively.

Rates of synonymous (dS) and nonsynonymous (dN) substitution were computed to examine functional constraints on siren1 and siren2 among species of the D. bipectinata complex (Table 2). The average dN/dS ratios are significantly lower than unity for siren1 and siren2 as well as for Adh, suggesting that both copies of siren are under functional constraints at the amino-acid sequence level. However, the dS values for siren1 and siren2 are higher than that for Adh. This is because the synonymous rate for Adh is reduced by a selective constraint for high expression efficiency (Shields et al. 1988). The higher synonymous rate for siren suggests that the selective constraint on siren is much weaker than that on Adh.

TABLE 2.

The average numbers of synonymous (dS) and nonsynonymous (dN) substitutions per site (±standard error) and the dN/dS ratios for siren1, siren2, and Adh among species of the D. bipectinata complex

Gene dN dS dN/dS
Adh 0.003 ± 0.002 0.038 ± 0.009 0.084*
siren1a 0.005 ± 0.003 0.121 ± 0.016 0.046*
siren2a 0.009 ± 0.003 0.122 ± 0.016 0.075*
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*

P (dN/dS = 1) ≪ 0.001.

a

Only for the Adh homologous part (256 codons).

DISCUSSION

The siren gene includes two distinct components, i.e., almost a whole part of Adh and a part of CG11779/nanos. Since siren is located close to Adh (Figure 1A and Table 1), it is quite natural to assume that the Adh homologous part was created by a simple tandem duplication due to unequal crossing over or unequal sister-chromatid exchange (Brown 1999 for review) similar with other Adh duplicated genes found around Adh in Drosophila (e.g., Fisher and Maniatis 1985; Schaeffer and Aquadro 1987).

On the other hand, given that CG11779/nanos is located on a distant chromosomal region from siren as indicated by the genetic linkage analysis (Table 1), the CG11779/nanos homologous part is hardly explained by tandem duplication. Retroposition seems to be the best candidate to explain small-scale gene duplications into such distant genomic location (see Graur and Li 2000 for review). The essence of retroposition is the replication of parasitic retrotransposons and retroposons and involves reverse transcription of their own transcripts and integration of the reverse transcripts into the host genome (Moran et al. 1996). However, the reverse transcriptase that catalyzes this reaction occasionally operates on non-self transcripts, resulting in duplications of an expressed host gene into a genomic location irrelevant to the location of the parent gene (e.g., McCarrey and Thomas 1987; Noyce et al. 1997; Esnault et al. 2000; Wei et al. 2001; Betrán and Long 2003). Actually, Ohshima et al. (2003) reported that 87% of retropseudogenes were located on chromosomes different from their parental genes in the human genome. The CG11779/nanos homologous part of siren fulfills this condition.

Undergoing transcription, reverse transcription, and integration, the resultant retrogenes automatically acquire the following peculiarities: (1) absence of intron, (2) addition of a poly(A) tract, and (3) addition of flanking short direct repeats (Tokunaga et al. 1985). Absence of intron seems to be consistently observable after long evolutionary time, whereas the latter two characteristics are often masked by base substitutions, insertions, and deletions that superimpose on them with time. For instance, of 24 retrogenes identified in the D. melanogaster genome by the definition of a different chromosomal location from the parent gene and the absence of intron, only the youngest gene retains the direct repeats and the four youngest genes retain a degenerated poly(A) tract (Betrán et al. 2002).

In the case of siren, none of these characteristics were recognized for retroposition of CG11779. Especially, it is quite unlikely that the nonexon sequences (the 5′-untranscribed region and the first intron) were included in the retroposed sequence. Alternatively, retroposition of nanos can produce this sequence, since the entire CG11779 homologous part of siren is included in the third exon of nanos on the complementary strand (Figure 6). This is a plausible scenario in the context of the expression pattern: nanos mRNA is localized in germline cells (e.g., Forbes and Lehmann 1998), where the activity of retrotransposons is generally enhanced (Zhao and Bownes 1998; Kogan et al. 2003). Positive associations between the levels of transcription and transposition frequencies in male germline cells has been demonstrated for the copia retrotransposon in D. melanogaster (Pasyukova et al. 1997). If this is the case, we can expect absence of intron and presence of a poly(A) tract with respect to the gene structure of nanos rather than that of CG11779. Unfortunately, it is impossible to examine the former condition, since only the single exon of nanos is involved in the current structure of siren. In the D. bipectinata sequence, corresponding to the poly(A) tract of nanos, a 9-bp poly(T) tract was identified on the complementary strand at the position exactly matching with the polyadenylation site of nanos pre-mRNA (Figures 6C and 7). This gives clear evidence that a nanos mRNA was retroposed to be a part of siren. Although a similar poly(T) tract was not identified in other species, it is likely that such sequences have been already blurred by base substitution, insertion, and deletion as the date of the hypothesized retroposition is estimated to be old enough; the average dS value between siren and CG11779 is 0.23, which seems to be at the borderline for identifying the footprint of poly(A) tract (Betrán et al. 2002). Although the length of the current CG11779/nanos homologous part in siren is much shorter than the expected full length of the nanos transcript, it is known that the majority of retrotransposons and retropseudogenes are 5′ truncated (Maestre et al. 1995; Pavlíček et al. 2002; Farley et al. 2004). Alternatively, it is also plausible that the rest of the expected nanos-derived sequence was lost by deletions and/or destroyed by the tandem duplication of Adh if it occurred later. A number of gaps identified around the boundary between the CG11779/nanos and Adh homologous parts (Figure 2) indicate that many deletions and insertions have actually occurred during the evolution of siren.

Figure 6.

Figure 6.

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Schematic of the generation of siren. The exons of CG11779, nanos, and Adh (including their homologous parts in siren) are represented by solid, shaded, and hatched boxes, respectively, whereas introns are represented by open boxes. (A) CG11779 and nanos overlap each other on the complementary strands. (B) When a mature nanos mRNA is reverse transcribed and integrated into the genome, (C) the resultant insert sequence (surrounded by the dashed line) is expected to contain the CG11779 promoter sequence and an upstream poly(T) tract corresponding to the poly(A) tract of the nanos mRNA.

Figure 7.

Figure 7.

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Sequence alignment of the upstream region of siren1 and the corresponding region of CG11779/nanos for D. bipectitnata. The polyadenylation site (arrowhead) of the nanos transcript was deduced from the cDNA sequence, at which the poly(A) tract (lowercase characters) is expected to be appended. Identical nucleotides between siren and CG11779/nanos sequences are indicated by vertical lines.

Retropositions of exon-intron structure of a gene via reverse transcription of its overlapping gene have been also reported by Courseaux and Nahon (2001) and Ejima and Yang (2003) in primate genomes. However, a remarkable aspect found in this study is that the retroposition of nanos provided the promoter sequence of CG11779 for siren. This is strongly supported by the fact that the expression pattern of siren is the same as that of CG11779 but distinct from the alternative candidate, Adh (Figure 4). In the current point of view, a retroposed gene copy will normally become a retropseudogene due to a lack of promoter and it can survive as a functional gene only when it recruits a new promoter sequence. In fact, retrosequence-derived genes were often found as a chimera with a preexisting gene that supplied the promoter (Brosius 2003; Long et al. 2003 for reviews). Jingwei (Long and Langley 1993; Long et al. 1999; Wang et al. 2000) and TRIMCyp (Nisole et al. 2004; Sayah et al. 2004) are the best paradigms of such chimeric genes. Although siren is also a chimeric gene, the promoter sequence is provided by the retroposed sequence rather than by the preexisting gene. This is contrary to the current viewpoint and reveals an unexpected potential of retroposition to generate new genes, when genes are overlapping.

It has been shown recently that a certain fraction of genes has overlapping genes in various organisms. In mammals, many sense-antisense transcript (SAT) pairs produced from the same locus have been found, e.g., 5880 (22%) of 26,741 transcripts in humans (Chen et al. 2004). For 65% of them, however, either sense or antisense transcript is nuclear and poly(A) minus, which strongly restricts retroposition. Among the rest, only 30% have the configuration of head-to-head overlapping (Veeramachaneni et al. 2004), where a promoter sequence is involved in the overlapping part. Consequently, ∼600 genes remain as potential targets of the promoter retroposition. In mouse, SATs were found in 4962 (15%) of 33,360 transcripts (Kiyosawa et al. 2003), and 1886 of them were identified to form a coding/coding pair (Kiyosawa et al. 2005). Since the frequency of head-to-head overlapping is 37% (Veeramachaneni et al. 2004), 700 genes can be the candidate. Analyzing the D. melanogaster genome sequence data (Release 3.2), we obtained that 1863 (13.8%) of the 13,472 annotated protein-coding genes have at least one overlapping protein-coding gene on the complementary strand, which is consistent with Misra et al. (2002). After the deduction for the non-head-to-head overlapping fraction, 222 genes remain as the candidate. A similar extent of gene overlapping is also observed in plants. In Arabidopsis thaliana, 2680 (8.9%) of 29,993 transcripts are generated from overlapping protein-coding genes, of which 370 genes are in a head-to-head overlapping configuration (Wang et al. 2005). In rice, 1060 (5.2%) protein-coding genes have an overlapping protein-coding gene (Osato et al. 2003). When antisense transcripts are retroposed, the chance of the resultant sequences surviving as a new gene is possibly higher than in the case of the usual retroposition of sense transcripts. This is because intron sequences facilitate exon shuffling between retroposed and genomic preexisting genes without disturbing their protein-coding frames and because the promoter is essential for transcription—which is the case in siren.

The identification of retrogenes commonly relies on two major definitions, i.e., (1) a distant chromosomal location from the parent gene and (2) the absence of an intron (Betrán et al. 2002). As siren does not meet the latter criterion, any gene in a genome that fulfills only the former criterion can also be a candidate of retrogene when its parent gene has an overlapping gene. Consequently, the significance of retroposition as a mechanism of new gene generation may be greater than appreciated previously. In this study, we have reported a new chimeric gene named siren and shown a further potential of retroposition to generate new genes.

Acknowledgments

We greatly appreciate the reading of the nucleotide sequences of cosmid clones by Toshiro Aigaki, Yuji Kohara, and Tadasu Shin-I. We are also grateful to Yoshihiro Kawahara, Shigeyuki Koshikawa, and Kazumi Yumita for supporting our experiments. We also thank Shigeru Saito, Claire T. Saito, Ryoko D. Segawa, and Norikazu Kitamura for valuable comments on the manuscript. This work was supported by grants from the Japan Society for the Promotion of Science to T.A. (12375002 and 15255006) and K.T.

Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under accession nos. AB194414–AB194442.

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