Dntf-2r, a Young Drosophila Retroposed Gene With Specific Male Expression Under Positive Darwinian Selection

Corresponding author: Manyuan Long, University of Chicago, 1101 E. 57th St., Chicago, IL 60637., mlong{at}midway.uchicago.edu (E-mail)

Communicating editor: M. A. F. NOOR

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A direct approach to investigating new gene origination is to examine recently evolved genes. We report a new gene in the Drosophila melanogaster subgroup, Drosophila nuclear transport factor-2-related (Dntf-2r). Its sequence features and phylogenetic distribution indicate that Dntf-2r is a retroposed functional gene and originated in the common ancestor of D. melanogaster, D. simulans, D. sechellia, and D. mauritiana, within the past 3–12 million years (MY). Dntf-2r evolved more rapidly than the parental gene, under positive Darwinian selection as revealed by the McDonald-Kreitman test and other evolutionary analyses. Comparative expression analysis shows that Dntf-2r is male specific whereas the parental gene, Dntf-2, is widely expressed in D. melanogaster. In agreement with its new expression pattern, the Dntf-2r putative promoter sequence is similar to the late testis promoter of ß2-tubulin. We discuss the possibility that the action of positive selection in Dntf-2r is related to its putative male-specific functions.

IT has been more than 3 decades since gene duplication was suggested to be a major source of evolutionary novelties (OHNO 1970 ). We are now able to examine this process in detail. Analyses of genome sequences have revealed high frequencies of gene duplicates in vertebrates, invertebrates, and plants (ARABIDOPSIS GENOME INITIATIVE 2000; BLANC et al. 2000 ; RUBIN et al. 2000 ; BALL and CHERRY 2001 ; LI et al. 2001 ; GU et al. 2002 ), which may suggest rapid evolution of novel functions. Our understanding of detailed molecular processes and underlying population evolutionary processes most often depends upon the direct observation of a young gene duplicate (LONG et al. 1999 ; LONG 2001 ; BETRAN and LONG 2002 ). Examples of genes recently arisen [<3 million years ago (MYA)] by several different mechanisms have been found in Drosophila: jingwei (jgw; LONG and LANGLEY 1993 ), Adh-Finnegan (BEGUN 1997 ), Sdic (NURMINSKY et al. 1998 , NURMINSKY et al. 2001 ), exuperantia 1 X copy (YI and CHARLESWORTH 2000 ), and Sphinx (WANG et al. 2002 ; for a more comprehensive review see LONG 2001 ). Jgw was created after a retroposition event that recruited duplicated exons of a neighboring gene. Sdic is the product of the fusion of two previously duplicated genes. Exuperantia 1 X copy is a transposition of a whole gene by ectopic recombination. In these examples, positive selection has been shown to have played a crucial role in their evolution (LONG and LANGLEY 1993 ; NURMINSKY et al. 1998 ; YI and CHARLESWORTH 2000 ; NURMINSKY et al. 2001 ; LLOPART et al. 2002 ).

Previous work (LONG and LANGLEY 1993 ) has revealed that retroposition can generate duplicates in Drosophila and leave clear evidence of the molecular process that generated the new gene copy from the parental copy. A newly derived retroposed gene can be identified by examining the hallmarks of retroposition (LI 1997 ): (1) one member of the pair is intronless in the coding region of similar sequence (new copy) while the other contains introns (parental copy); (2) the new copy contains a poly(A) tract; and (3) the new copy may still be flanked by short duplicate sequences. Properties 2 and 3 can be used to infer new retroposed genes if the parental genes do not contain introns. In a genome-wide analysis of retroduplicates, we inferred parental and derived copies by examining these fingerprints of retroposition process (BETRAN et al. 2002 ). At the threshold of >70% protein sequence identity, we identified 24 pairs of retroposition events with various ages (BETRAN et al. 2002 ). Here, we report the evolutionary analysis of one of these retroposed genes, Nuclear transport factor-2-related [Dntf-2r (CG10174)] and its parental gene, Dntf-2 (CG1740). Dntf-2, which contains three introns, is located on the X chromosome, whereas Dntf-2r, which contains no introns, is likely a recent retroposed copy of Dntf-2 and is located on the left arm of chromosome 2 in D. melanogaster. In addition, Dntf-2r contains a putative poly(A) tract at the end of the retroposed region (Fig 1). We determined the age of Dntf-2r by surveying its phylogenetic distribution using fluorescence in situ hybridization (FISH) experiments, genomic Southern blot, and PCR analyses. We found that Dntf-2r is present in only four species of Drosophila: D. melanogaster, D. simulans, D. sechellia, and D. mauritiana, suggesting that Dntf-2r originated recently, 3–12 MYA (LACHAISE et al. 1988 ). Analysis of polymorphism and divergence for the new and parental genes reveals that Dntf-2r is a new functional gene whose protein has been subject to both purifying and positive Darwinian selection. Analyses of expression patterns in D. melanogaster indicate that Dntf-2r is male specific, in contrast to the wide expression of Dntf-2.

View larger version (48K): In this window In a new window Download PPT slide   Figure 1. Alignment of the Dntf-2r gene with its parental gene Dntf-2 for D. melanogaster. The Dntf2r mRNA sequence in this species is shown in italics. The putative promoter, coding region, and poly(A) tract of Dntf2r are shown in boldface type.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Phylogenetic distribution of Dntf-2r:
The phylogenetic distribution of Dntf-2r was determined by FISH to polytene chromosomes, Southern analysis, and polymerase chain reactions on the species of the melanogaster subgroup: D. melanogaster, D. simulans, D. sechellia, D. mauritiana, D. yakuba, D. teissieri, D. erecta, and D. orena. A probe of 300 bp comprising the coding region of Dntf-2r was hybridized to polytene chromosomes of D. melanogaster, D. simulans, D. yakuba, and D. erecta following the WANG et al. 2000 protocol. The probe was synthesized by PCR from Dntf-2r and digoxygenin (DIG) labeled by random priming after gel purification of the specific band. For Southern analysis, 2 µg of genomic DNA from species of the melanogaster subgroup were digested and blotted to nylon membrane (SAMBROOK et al. 1989 ). Hybridizations and detection were carried out following the immunochemiluminescent protocol of Roche (Indianapolis). We did PCR with flanking and homologous primers for the Dntf-2r gene. Primer sequences were 5' GCAGGGCGCATTGTTCAG 3' and 5' CATACGCCTGCCAATACGAGT 3' to amplify Dntf-2r from its flanking region and 5' TTGTCCAGCAGTACTACGCC 3' and 5' AGCCACGAAGAGGGATCCTC 3' to amplify Dntf-2r from its coding region.

DNA samples and sequencing:
Single male fly genomic DNA was obtained using a Puregene kit. Dntf-2r and Dntf-2 were amplified by PCR from this genomic DNA. D. melanogaster samples come from a worldwide distribution: OK17, HG84, and Z(s)56 from Africa; yep3, yep18, yep25, Cof3, BLI5, cal4, y10, and y2 from Australia; 253.4, 253.27, 253.30, and 253.38 from Taiwan; Closs3, Closs10, Closs16, Closs19, and Seattle from the United States; Rio from Brazil; and Rinanga, Bdx, Besançon, Prunay, and Capri from France. Other stocks used were D. simulans from Florida (provided by J. Coyne), D. sechellia (provided by J. Coyne), D. mauritiana (163.1, LEMEUNIER and ASHBURNER 1976 ), and D. yakuba (115, LEMEUNIER and ASHBURNER 1976 ). Oligoprimers 5' ATCGGATCGGATTTCCCATAATCT 3'/5' TGCCGAGCTTGTTGTTATCATCTG 3' and 5' CTGGCGGCCCATTTTGTTGACA 3'/5' AGAAAAGTCGTCCCGAGCGAGGAA 3' were used to amplify Dntf-2 in D. melanogaster and D. yakuba (outgroup sequence; see below) and 5' TGCAGGGCGCATTGTTCAG 3' and 5' CATACGCCTGCCAATACGAGT 3' to amplify Dntf-2r in D. melanogaster, D. simulans, D. sechellia, and D. mauritiana, the four species where the gene is present. Haplotypes were obtained by direct sequencing for Dntf-2 since it is on the X chromosome. Nine alleles of D. melanogaster Dntf-2 were sequenced. For Dntf-2r, on the second chromosome, PCRs of individuals heterozygous at more than one site were cloned into a TOPO cloning vector (Invitrogen, San Diego) and one clone was sequenced to infer the haplotype. Only 1 allele randomly chosen in heterozygous individuals was analyzed, giving a total of 26 alleles. PCR products were sequenced directly after purification [QIAGEN (Valencia, CA) kit] on an ABI automated DNA sequencer (Applied Biosystems, Foster City, CA), using fluorescent DyeDeoxy terminator reagents.

Sequence analysis:
Sequences were aligned by means of Clustal W (THOMPSON et al. 1994 ). Polymorphism and divergence patterns were studied in Dntf-2 and Dntf-2r to determine the relative importance of natural selection and drift in the evolution of these genes.

Synonymous and nonsynonynous substitutions per site (K_S and K_A) were computed following GOLDMAN and YANG 1994 and YANG 1998 , using PAML 3.1 software (YANG 1997 ). This method accounts for transition/transversion bias (), for different base frequencies at different codon positions, and for the genetic code structure (GOLDMAN and YANG 1994 ; YANG 1998 ). A model of a single rate for all sites was specified ( = 0; YANG 1997 , YANG 1998 ). For the analysis, a tree with Dntf-2 of D. yakuba as outgroup (Fig 2A) was used. This tree was obtained by considering the age of the gene (see RESULTS) and the phylogenetic information (TING et al. 2000 ). K_A/K_S ratio differences in different lineages were tested using the maximum-likelihood ratio test. Log likelihoods of different models were compared with a ² distribution with as many degrees of freedom as the difference in number of variable parameters of the nested models (YANG 1998 ). Maximum-likelihood estimates of parameters for each branch (branch length and = K_A/ K_S) together with the estimate of can be used to calculate K_A and K_S per branch and construct nonsynonynous and synonymous trees.

View larger version (12K): In this window In a new window Download PPT slide   Figure 2. (A) Gene and species genealogy used in the analyses of KA/KS. (B) KS (left) and KA (right) divergence tree for Dntf-2r and Dntf-2 estimated under the "6 KA/KS ratio" model (Table 3). Estimated numbers of substitutions are shown in every branch.

, the average number of nucleotide differences per site between two random sequences (TAJIMA 1989 ), and _W, Watterson's estimate of from the number of segregating sites (WATTERSON 1975 ), were calculated. Both values estimate the equilibrium neutral parameter = 4N_eµ for autosomal loci and = 3N_eµ for X-linked loci, where N_e is the effective population size and µ is the neutral mutation rate. The difference between and _W (Tajima's D) reveals nonequilibrium conditions in the history of the sample. Tajima's D (TAJIMA 1989 ) was calculated and tested by 10,000 simulations using DNAsp 3.53 (ROZAS and ROZAS 1999 ). Fay and Wu's H test (FAY and WU 2000 ) was also applied to the polymorphism data of Dntf-2r. The H statistic was used to measure the excess of derived variants at high frequency, a hallmark of recent positive selection (FAY and WU 2000 ; OTTO 2000 ). Fay and Wu's H test was computed at http://crimp.lbl.gov/htest.html and tested by 10,000 simulations. Recombination rate (R per gene) for all those simulations was estimated from the data using DNAsp 3.53 (ROZAS and ROZAS 1999 ).

Under neutrality, intraspecific variation is correlated with interspecific divergence (KIMURA 1983 ). Deviations from this expectation can result from a number of causes including positive Darwinian selection (MCDONALD and KREITMAN 1991 ; NIELSEN 2001 ). We compared intraspecific variation with interspecific divergence at synonymous and replacement sites (MCDONALD and KREITMAN 1991 ). DNAsp 3.53 software was used to carry out this comparison (ROZAS and ROZAS 1999 ).

Expression analysis:
Tissues were homogenized and total RNA was prepared, as described by the QIAGEN protocol, from 200 males and females, 15 virgin females, 15 gonadectomized males, 100 testes plus accessory glands, and 100 testes of D. melanogaster. Gonadectomized males (males from which we removed testes and accessory glands), testes plus accessory glands, and testes were obtained by dissecting mature males in saline solution. After dissection, tissues were preserved in RNA-later solution (Ambion, Austin, TX) at -20° after soaking them at 4° overnight until they were processed. mRNA was prepared from the total RNA of 200 males and females following the QIAGEN protocol.

The full-length sequence of the D. melanogaster Dntf-2r transcript from testis was obtained by 5' and 3' rapid amplification of cDNA ends (RACE) experiments. Single-strand cDNA was synthesized from mRNA using Superscript (GIBCO-BRL, Gaithersburg, MD). Oligo(dT) was used to prime the synthesis of the 3' end of the cDNA. Oligo(dT) and the specific primers 5' TTGTCCAGCAGTACTACGCC 3' and 5' TCGTCCTTGGAAGACTAAAA 3' were used to PCR amplify the 3' end. The nested PCR product was subcloned and sequenced. Primer 5' AGCCACGAAGAGGGATCCTC 3' was used to synthesize the 5' end of the Dntf-2r cDNA. This cDNA was tailed with dCTP by using terminal transferase (GIBCO-BRL 5' race system). Oligo(dG) adaptors and the nested primers 5' TTGGGCTTCAGCAAAAAGAT 3' and 5' GGGGATCGTCATCGCATTT 3' were used to PCR amplify the 5' end of the cDNA (GIBCO-BRL 5' race system).

RT-PCR was conducted on total RNA from virgin females, gonadectomized males, testes plus accessory glands, and testes for Dntf-2r and Dntf-2. Analysis of expression of intronless genes (such as Dntf-2r) is challenging because genomic contamination can produce a band of the same size as that expected from the cDNA. Therefore, we digested possible contaminating DNA from the total RNA (DNase I amplification grade; GIBCO-BRL) and ran controls including DNA-digested total RNA without retrotranscriptase. Single-strand complementary DNA (cDNA) was synthesized using Superscript and oligo(dT) (GIBCO-BRL). RT-PCR was carried out using specific primers 5' TTGTCCAGCAGTACTACGCC 3'/5' AGCCACGAAGAGGGATCCTC 3' for Dntf-2r and 5' TTGTGCAGCAGTACTATGCG 3'/5' GGCCACAAAGAAGGTGCCTG 3' for Dntf-2.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Structure of Dntf-2r:
The complete D. melanogaster Dntf-2r transcript is given in Fig 1. The transcript consists of the retroposed regions and recruits seven additional nucleotides from its 5' flanking region and four nucleotides from its 3' flanking region. Unlike jingwei (LONG and LANGLEY 1993 ), Dntf-2r did not recruit any new coding region. Note that a nonconsensus polyadenylation signal must be used for this gene.

Phylogenetic distribution of Dntf-2r:
We dated the appearance of Dntf-2r by establishing which species have the duplication, using several complementary techniques. Fig 3A shows polytene in situ hybridization in D. melanogaster, D. simulans, D. yakuba, and D. erecta. Positive hybridization of Dntf-2r probe in band 2L36F is shown in D. melanogaster and D. simulans. Two additional signals (not shown) were observed in the D. melanogaster and D. simulans genome corresponding to Dntf-2 (X chromosome) and a lighter secondary signal (3R). Only two hybridization signals were observed in D. yakuba and D. erecta, in the X and 3R. Both signals are shown in D. erecta in addition to the lack of hybridization in 36F (2R in this species due to a pericentric inversion; see ASHBURNER 1989 ). Only Dntf-2 (X chromosome) hybridization is shown in D. yakuba.

View larger version (52K): In this window In a new window Download PPT slide   Figure 3. (A) Polytene in situ hybridization with a probe of Dntf-2r in D. melanogaster, D. simulans, D. yakuba, and D. erecta. (B) Southern blot hybridized with a probe of Dntf-2r. Species name is shown at top of each lane. EcoRI digest is shown. (C) Dntf-2r was amplified with flanking region primers. One band of the same size was obtained in D. melanogaster, D. sechellia, D. simulans, and D. mauritiana (Table 1). A shorter band was obtained for D. yakuba, D. teissieri, and D. erecta. This band was sequenced in D. yakuba, D. teissieri, and D. erecta. Sequences aligned well with the flanking sequence of Dntf-2r but the gene insert (new gene) was missing (Fig 4). (D) Dntf-2r was amplified with primers from the coding region. One band of the same size was obtained in D. melanogaster, D. sechellia, D. simulans, and D. mauritiana. No band of the expected size was obtained for D. yakuba, D. teissieri, and D. erecta.

Southern blot analysis (Fig 3B) shows extra strong bands in D. melanogaster, D. simulans, D. mauritiana, and D. sechellia corresponding to Dntf-2r. Fig 3C and Fig D, shows PCR with primers in the flanking and coding regions, respectively. A short product (lacking the Dntf-2r insertion) was obtained for D. yakuba, D. teissieri, and D. erecta (Fig 3C). The products from D. yakuba, D. teissieri, and D. erecta were sequenced. The sequence confirmed that this short fragment corresponds to the flanking region of Dntf-2r (Fig 4). In addition, primers in the coding region were unable to amplify Dntf-2r in D. yakuba, D. teissieri, and D. erecta (Fig 3D).

View larger version (54K): In this window In a new window Download PPT slide   Figure 4. Alignment of the flanking region of Dntf-2r of D. melanogaster with the short product (lacking the Dntf-2r insertion) obtained from D. yakuba, D. teissieri, and D. erecta. The first and last codons of Dntf-2r are shown in boldface type.

These data established that the distribution of Dntf-2r is limited to the four species in the D. melanogaster clade: D. melanogaster, D. simulans, D. sechellia, and D. mauritiana. Therefore, the Dntf-2r gene is between 3 and 12 million years old (i.e., the time length from the common ancestor of all D. melanogaster subgroup species to the four-species clade; see LACHAISE et al. 1988 ).

Sequence analysis:
Sequence variants in the coding region for Dntf-2 and Dntf-2r in related species are shown in Table 1, and Table 2 shows variants for the noncoding region of Dntf-2 in D. melanogaster.

Divergence analyses were carried out using the consensus sequence for D. melanogaster and two alleles (haplotypes 1 and 2) for D. mauritiana (Table 2). Log-likelihood values and maximum-likelihood estimates of the K_A/K_S ratio for each branch of the tree for Dntf-2r and Dntf-2 sequences (Fig 2A) under several models are given in Table 3. A free-ratio model (B) was first applied to the data (YANG 1998 ). This model with 23 parameters differs significantly from the one-ratio model (A) with 13 parameters (ln L_B = -935.41, ln L_A = -948.38; X²₍₁₀₎ = 2( ln L) = 25.94; P = 0.0038). Thus, we conclude that (K_A/K_S) differs among different branches of the tree. However, model B does not differ significantly from model C, the six-ratio model (X²₍₅₎ = 0.12; P > 0.05). So, the six-ratio model is the simplest model that still contains all the information from the free model. Fig 2B shows the estimated numbers of synonymous and replacement substitutions per branch under model C. Now that we know that there are differences in K_A/K_S ratios along the tree, we want to answer two questions. Is DNTF-2R evolving faster than DNTF-2? If so, is positive Darwinian selection acting in any of the Dntf-2r lineages? We compared different models to answer these questions.

Model A vs. C [X²₍₅₎ = 25.82; P = 0.0001], A vs. D [X²₍₁₎ = 11.6; P = 0.0007], and A vs. F [X²₍₂₎ = 19.56; P = 0.00006] tests reveal that DNTF-2 evolved much more slowly than DNTF-2R (K_A/K_S = 0.0499 vs. K_A/K_S = 0.5405 on average, respectively). Model C does not differ from model F [X²₍₃₎ = 6.26; P > 0.05], showing that K_A/K_S for Dntf-2 is on average very small: 0.0502. Thus, Dntf-2 evolved under strong purifying selection, suggesting high functional constraint. The significantly accelerated evolution of DNTF-2R (A vs. D and A vs. F) can be the result of two different phenomena: relaxation of selection or positive Darwinian selection for novel function after the duplication. Relaxation of selection would occur if the protein product translated from Dntf-2r is under less constraint than the protein from Dntf-2. However, if amino acid substitutions in a lineage occur faster than the neutral rate, K_A/K_S ratios will exceed 1, revealing the action of positive selection.

Relaxation of selective constraints and positive selection in Dntf-2r can be investigated by considering additional models. Model F is significantly more likely than both model D [X²₍₁₎ = 7.96; P = 0.0048] and model E [X²₍₂₎ = 12.48; P = 0.00195], and model G is more likely than model E [X²₍₁₎ = 10.92; P = 0.00095], revealing that K_A/K_S ratios are significantly <1 in some Dntf-2r lineages (12-1, 12-2, 9-10, 9-5, and 8-9). Thus, we see clear effects of purifying selection in these branches (K_A/K_S 0.33), indicating functional constraint for this gene. However, K_A/K_S ratios could be larger than or equal to one in segments 11-12, 11-3, 10-11, and 10-4 because model F is not a significant improvement over model G [X²₍₁₎ = 1.56; P > 0.05]—which does not support the action of positive selection. Significance of the other likelihood-ratio tests remains after correcting for multiple comparisons (Bonferroni correction, P < 0.005; SOKAL and ROHLF 1995 ).

We have shown that the K_A/K_S ratio that maximizes the likelihood is 0.33 in some Dntf-2r lineages and 1.0000 in others. However, these values do not discriminate between the alternatives of relaxation of selection or positive selection on Dntf-2r. This is because the boundary K_A/K_S > 1 sets a high threshold for testing positive selection (KREITMAN and AKASHI 1995 ; WYCKOFF et al. 2000 ). Only a part of the protein is likely to be susceptible to advantageous mutations while the remainder remains subject to purifying selection.

Polymorphism data (Table 1 and Table 2) were analyzed next. Levels of variation at synonymous and nonsynonymous sites and sites in noncoding regions were calculated (Table 4). The Dntf-2 sequence is variable only in noncoding regions, confirming the action of strong purifying selection on its coding region. Tajima's D for Dntf-2 was 0.7416 (P > 0.10); i.e., the frequency spectrum shows no deviation from neutrality.

We detected variation in the coding region for Dntf-2r in D. melanogaster (Table 1 and Table 4). Tajima's D for Dntf-2r was -0.45276 (P > 0.10) and Fay and Wu's H was -1.8338 (P = 0.0752; assuming the value of back mutation of 0.10 and recombination rate estimated for the data, 0.0344 per base, and using Dntf-2 of D. melanogaster as ancestral sequence). Although the frequency spectrum of Dntf-2r variation in the coding region shows no deviation from neutrality, the negative Fay and Wu's H is at a marginal level of significance, suggesting that the three derived sites (117, 239, and 303) are at high frequency. Thus, these derived alleles may have been driven by the effects of positive Darwinian selection. While these tests can detect selection, they have power to detect it only for a short time (0.5N generations in the favorable case of no recombination; SIMONSEN et al. 1995 ; FAY and WU 2000 ). This is equivalent to only 0.05 MY if we consider 10⁶ as the population size and 10 generations per year for Drosophila.

The McDonald-Kreitman test (MCDONALD and KREITMAN 1991 ), which considers both within- and between-species variation, was next performed for the Dntf-2r data. Table 5 shows the results for three comparisons with D. melanogaster. The comparisons (D. melanogaster vs. D. mauritiana, D. melanogaster vs. D. simulans, and D. melanogaster vs. D. sechellia) are not independent (see Fig 2). However, if we consider the comparison in which we have polymorphism data for both species (D. melanogaster vs. D. mauritiana), we see the most significant pattern (P = 0.0072). The significance remains after Bonferroni correction for multiple comparisons (P < 0.017; SOKAL and ROHLF 1995 ). We also made a comparison pooling all the independent information we have in the four species: divergence, mapped in every independent lineage from Fig 2B (R/S = 32/12), and polymorphism from Table 5 (R/S = 3/8). This test shows a highly significant excess of amino acid replacements (G = 7.648; P = 0.0057). The significantly higher ratios of replacement to silent substitutions compared to the ratios of replacement to silent polymorphic sites are consistent with positive selection in the Dntf-2r lineages.

Dntf-2r expression and promoter analysis:
RT-PCR results for Dntf-2r and Dntf-2 in different tissues of D. melanogaster are shown in Fig 5. We differentially amplified the two genes with specific primers. We observed that while Dntf-2 is expressed in all tissues studied, Dntf-2r is expressed only in testes.

View larger version (98K): In this window In a new window Download PPT slide   Figure 5. RT-PCR for (A) Dntf-2r and (B) Dntf-2 in D. melanogaster. Lane 1, PCR from female cDNA; lane 2, gonadectomized male cDNA; lane 3, testes plus accessory gland cDNA; lanes 4–6, the respective negative controls after DNA digestion; and lane 7, negative control of the PCR. (C) RT-PCR from testes cDNA for Dntf-2r and Dntf-2. Lane 1, testes cDNA; lane 2, negative control after DNA digestion; lane 3, the negative control of the PCR for Dntf-2r; lane 4, testes cDNA; lane 5, negative control after DNA digestion; and lane 6, negative control of the PCR for Dntf-2.

The ß2-tubulin gene has a late testis-specific promoter (MICHIELS et al. 1989 ). This promoter is known to exhibit only two elements: ß2-tubulin upstream element 1 (ß2UE1; 14 bp), which is essential for spermatocyte-specific expression, and a quantitative element (7 bp; MICHIELS et al. 1989 ). Interestingly, we identified a region of sequence similarity with these late testis-specific promoter elements of ß2-tubulin at -42 bp from the transcription initiation site of Dntf-2r in D. melanogaster. Dntf-2r putative promoter elements, upstream element (ATCAGC-TTAGCGGT -62) and quantitative element (GGATATT -42), have a 57% nucleotide identity with the ß2UE1 (ATC-GCAGTAGTCTA) and a 100% identity with the ß2-tubulin quantitative element (GGATATT) of D. hydei, respectively.

Examination of the flanking region of the insertion site in D. teissieri, D. yakuba, and D. erecta (outgroups lacking the insertion; Fig 4) reveals similarity to the 5' putative promoter sequence of Dntf-2r in D. melanogaster. The GGATATT putative quantitative element is present in these outgroup sequences as well as three nucleotides TAG of the putative Dntf-2r upstream element (see Fig 4). This would favor the hypothesis that Dntf-2r developed a new promoter with late testis expression after retroposition by acquiring only a few modifications to the preexisting 5' sequence.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We investigated evolution of a recently originated gene and its parental copy in D. melanogaster, Dntf-2r (CG10174) and Dntf-2 (CG1740). Sequence comparison revealed that the new gene was generated in a retroposition event. Recent work on the parental copy Dntf-2 in D. melanogaster revealed that this gene, playing a role in the nuclear transport of proteins with nuclear localization signals, is essential for the antimicrobial immune response (BHATTACHARYA and STEWARD 2002 ). This important role is in agreement with the strong sequence constraint on Dntf-2 that we observed: there was no variation in the coding region within D. melanogaster and the K_A/K_S ratio was 0.05 between D. melanogaster and D. yakuba.

On the other hand, there was no information on the function of Dntf-2r. Our sequence analyses of divergence and polymorphism for this gene, as well as our expression evidence, indicate that this gene may produce a functional protein. First, we observed that polymorphism is higher for synonymous than for replacement sites: _R/_S = 0.11 (Table 4), revealing the action of purifying selection. Second, K_A/K_S ratios for substitutions in Dntf-2r are on average significantly lower than unity (0.5), which is not consistent with the hypothesis that the gene is a pseudogene in many of the species. The K_A/K_S ratio of 0.5 for Dntf-2r is higher than that for Dntf-2. However, the McDonald-Kreitman test revealed a significant excess of amino acid substitutions, suggesting that the accelerated protein sequence evolution is likely a consequence of the action of positive Darwinian selection. Consistent with this interpretation, the Fay-Wu test, with an H statistic of marginal significance, gives a strong hint of an excess of high-frequency variants that would be coupled with the fixation of beneficial mutations. Thus, both purifying selection and adaptive evolution detected in these analyses argue that Dntf-2r encodes a protein, possibly with an evolving novel function. Dntf-2r provides new evidence for the role of positive Darwinian selection in the origin of new genes.

Whether or not a retroposed sequence recruits a new promoter is a critical step to its future fate. If a retroposed sequence integrates in a genomic region devoid of expression potential, it would be doomed to evolve into a pseudogene (JEFFS and ASHBURNER 1991 ). However, Dntf-2r has developed a tissue-specific expression pattern in D. melanogaster while Dntf-2 is widely expressed in this species (this work and BHATTACHARYA and STEWARD 2002 ). Consistent with these results, we observe that the 5' flanking region of Dntf-2r in D. melanogaster is similar to the ß2-tubulin promoter elements: ß2UE1, which is essential for spermatocyte-specific expression, and the quantitative element. Although Dntf-2r tissue expression remains to be analyzed in D. simulans, D. mauritiana, and D. sechellia, a similar pattern of expression may be possible, considering that the two putative promoter elements are 100% conserved in these species (data not shown). The Dntf-2r retroposed sequence, as expected from its mRNA origin, did not contain the promoter sequence of the parental gene. It instead recruited a novel 5' regulatory sequence from the insertion site, needing few mutations to become a promoter and leading to a testis-specific pattern of expression. The examination of the D. teissieri, D. yakuba, and D. erecta orthologous regions of the insertion site for Dntf-2r reveals an element similar to the putative promoter sequence of Dntf-2r in D. melanogaster with only a few substitutions. However, it is unclear if this previously existing sequence is a functional promoter for some unknown gene in the region or is just a random genomic sequence that happens to be similar to a promoter sequence. Given its high similarity to the promoter region of ß2-tubulin and its similar expression site (testis), it would be tempting to take the first possibility as a working hypothesis in further research. The promoter capabilities of the 5' preexisting sequence and the 5' region of Dntf-2r in D. melanogaster should be further investigated to test this hypothesis. In conclusion, the Dntf-2r gene is a chimera: the regulatory sequences and protein-coding regions originated from different sources.

An accelerated rate of evolution has been widely observed in some reproduction-related genes, probably due to competition among sperm from different males, female choice, and/or intersexual genomic conflict (EBERHARD 1985 ; METZ and PALUMBI 1996 ; TING et al. 1998 ; TSAUR et al. 1998 ; AGUADE 1999 ; WYCKOFF et al. 2000 ; SWANSON et al. 2001A , SWANSON et al. 2001B ). We can speculate that the detected positive Darwinian selection on Dntf-2r may be related to its newly evolved male-specific function(s). On the other hand, Dntf-2 is also expressed in the germline. The strong purifying selection on this parental gene could be a consequence of its expression in other tissues, a possibly different timing in germline expression, or a different function.

It is known that, in Drosophila, X inactivation occurs early in spermatogenesis (LIFSCHYTZ and LINDSLEY 1972 ). This implies that Dntf-2 (on the X chromosome) is inactivated at an early stage in spermatogenesis. Dntf-2r on an autosome and expressed in male germline stages could carry out newly evolved function(s) in spermatogenesis. Although this is a plausible interpretation, an alternative scenario ought to be discussed: Dntf-2r maintains the functions of its X-linked parental copy, Dntf-2, in male germline cells after X inactivation. In this scenario, the signature of positive selection observed in the Dntf-2r sequence must be explained by its being subject to a new set of selective pressures in a specific testis tissue despite maintaining the same function. However, this explanation would encounter a difficulty. X inactivation in Drosophila evolved before Dntf-2r originated, since there is evidence for X inactivation in D. pseudoobscura (LIFSCHYTZ and LINDSLEY 1972 ), which diverged from the melanogaster subgroup 40 MYA (POWELL 1997 ). LIFSCHYTZ and LINDSLEY 1972 observed that in the spermatocytes of D. pseudoobscura, the arm of the X homologous to the D. melanogaster X chromosome is heteropycnotic, whereas the other arm homologous to the right arm of the D. melanogaster second chromosome does not show heteropycnosis. Thus, there would be a long period (at least 30 MY) from the emergence of the X inactivation to the origin of Dntf-2r, during which the putative Dntf-2 function would be silenced in late male germline stages. Silencing of a previously existing function for such a long evolutionary period might not be tolerable. Thus, it is more parsimonious to assume that the Dntf-2r does not maintain the same function as its parental copy. The observed accelerated substitution in the protein encoded by Dntf-2r is more likely a consequence of positive selection for novel function.

	FOOTNOTES

Sequence data from this article have been deposited with EMBL/GenBank Data Libraries under accession nos. AY150763–65, AY150768, AY150770–73, AY150775–78, AY150780–87, AY150789–90, AY150792–93, AY150796–97, and AY301039, AY301040, AY301041, AY301042, AY301043, AY301044, AY301045, AY301046, AY301047, AY301048, AY301049, AY301050, AY301051, AY301052, AY301053, AY301054, AY301055, AY301056, AY301057, AY301058, AY301059, AY301060, AY301061.

	ACKNOWLEDGMENTS

We thank J. Coyne, P. Gibert, F. Lemeunier, and M.-L. Wu for providing Drosophila strains used in this work; Janice Spofford for critically reading the manuscript; and members of the Long lab, especially Kevin Thornton, for valuable discussions. We also thank two anonymous reviewers for their comments and corrections that improved the manuscript. This work was supported by grants from National Science Foundation (Career Award) and Packard Fellowship in Science and Engineering to M.L.

Manuscript received September 20, 2002; Accepted for publication March 4, 2003.

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