*Department of Organismic and Evolutionary Biology, Harvard University;
Zoologisches Institut and
Biochemisches Institut, Universität Zürich, Zürich, Switzerland
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Abstract |
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Introduction |
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A second possibility is that both copies of the duplicated gene retain the original function. Under this scenario, the two genes remain under the same selective constraints and should show similar patterns of molecular evolution. The maintenance of two redundant genes is not expected to be stable over evolutionary time unless accompanied by some breaking of symmetry, such as the partitioning of gene function (Force et al. 1999
; Krakauer and Nowak 1999
). On a shorter timescale, two redundant genes may persist if there is a selective advantage to having multiple copies of the same gene, as is proposed for the Drosophila melanogaster metallothionein gene, Mtn. Metallothioneins play an important role in the detoxification and intracellular regulation of heavy metals. Polymorphism for tandem duplication of Mtn has been found in D. melanogaster, and flies with the duplication show increased levels of Mtn expression (Lange, Langley, and Stephan 1990
; Theodore, Ho, and Maroni 1991
). The increased expression may be favored in environments exposed to heavy metal pollution over recent human history (Lange, Langley, and Stephan 1990
).
A third possibility is that one gene copy may retain the original function while the other evolves a new function through changes to its amino acid sequence and/or expression pattern. In such a case, the gene adopting a new function is expected to experience selective pressures different from those experienced by the original gene. This is exemplified by the Adh and Adhr genes of Drosophila. The Adh gene product performs a well-known enzymatic function as an alcohol dehydrogenase. The function of the Adhr product is unknown, although considerable evidence suggests that it is not an alcohol dehydrogenase (reviewed by Ashburner 1998
). Conservation of the Adhr coding sequence between D. melanogaster and Drosophila pseudoobscura, however, implies strong selective constraints and a functional role (Schaeffer and Aquadro 1987
). Consistent with the above expectation, Adh and Adhr show different patterns of interspecific divergence and also differ in levels of codon bias (Schaeffer and Aquadro 1987
; Albalat, Marfany, and Gonzalez-Duarte 1994
). A more striking example of a gene duplication leading to a gene of novel function is the D. melanogaster Sdic gene. Sdic arose by a duplication of the cytoplasmic dynien intermediate chain gene, Cdic, followed by fusion to the 5' end of the AnnX gene and other rearrangements to produce a functional open reading frame (Nurminsky et al. 1998
). Sdic has evolved a sperm-specific pattern of expression and appears to have become fixed rapidly in D. melanogaster due to the action of positive selection in a "selective sweep" (Nurminsky et al. 1998
).
In this paper, we describe the molecular evolution of a newly identified sperm-specific gene, ocnus (ocn), and two related genes, janusA (janA) and janusB (janB). The three genes are arranged in tandem over a genomic region of less than 2.5 kb and appear to be the result of two separate duplication events. The janB gene also shows sperm-specific expression, while janA has two major alternatively spliced forms, one specific to the male germ line and the other showing a more general pattern of expression in both males and females (Yanicostas, Vincent, and Lepesant 1989
). Patterns of molecular evolution in these genes may reflect their role in male fertility. It has been suggested that genes influencing male reproductive traits evolve rapidly, and this "faster male" hypothesis may provide a partial explanation for Haldane's rule in species where males are the heterogametic sex (Wu and Davis 1993
; Presgraves and Orr 1998
). A previous study comparing genes between D. melanogaster and either Drosophila simulans or D. pseudoobscura that included janA and janB found a higher ratio of nonsynonymous to synonymous substitution rates in genes with sex-related functions than in genes with metabolic functions (Civetta and Singh 1998
). Here, we used protein-encoding sequences of the ocn, janA, and janB genes in the D. melanogaster species subgroup to examine the pattern of duplication within this genomic region and to compare selective constraints among the three genes.
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Materials and Methods |
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Proteins were analyzed by electrophoresis on an acetic acid/urea/polyacrylamide gel with 17% polyacrylamide and 6.25 M urea. The gel was stained with Coomassie brilliant blue. For sequence analysis, bands were transferred to a polyvinylidene difluoride membrane by electroblotting with 0.01 M acetic acid. The membrane was stained with amido black, and the appropriate band was cut out. Sequence analysis was carried out on a Model 477 sequencer (Applied Biosystems Inc., Foster City, Calif.) according to the manufacturer's recommendations.
Fly Stocks and Genomic DNA Preparation
A laboratory stock of the Canton S strain of D. melanogaster was used for all subsequent experiments. For D. simulans, Drosophila yakuba, and Drosophila teissieri, we used isofemale stocks derived from wild-caught flies. Drosophila sechellia, Drosophila mauritiana, Drosophila erecta, and Drosophila orena stocks were obtained from the Drosophila Species Stock Center (Bowling Green, Ohio). Genomic DNA was prepared from individual male flies by homogenization, followed by a 2-h incubation at 37°C in buffer (0.2 M sucrose, 0.1 M NaCl, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA [pH 8.0], 0.5% Triton X-100) containing 1% sarkosyl and 50 µg/ml Proteinase K. After incubation, the homogenate was extracted twice with phenol : chloroform and once with chloroform. DNA was precipitated in 100% ethanol, washed in 70% ethanol, vacuum-dried, and resuspended in 1 x TE (pH 8.0).
PCR Cloning and DNA Sequencing
Unless otherwise noted, PCR and RT-PCR reagents were supplied by Life Technologies (Gaithersburg, Md.). Approximately 100 ng of genomic DNA template was amplified for 25 cycles (94°C for 1 min, 55°C for 1 min, 72°C for 3 min) in a 50-µl reaction containing 1 x PCR buffer, 2.5 mM magnesium chloride, 125 µM each dNTP, 100 ng each primer, and 1 U Taq DNA polymerase. The primers used for amplification were ja1 (5'-GTATCTGGTCACATTGCTGGAC-3'), ja2(5'-GCAAAGCTACAGACTAACTGC-3'), jb1(5'-GCAGTTAGTCTGTAGCTTTGC-3'), jb2(5'-CC GAA AAG AAAC TGG TA TGAACGG-3'), oc1(5'-CCGTTCATACCAGTTTCTTTTCGG-3'), and oc2 (5'-GGCAAGATGATGTTGTAATGCTGG-3'). For D. melanogaster, D. simulans, and D. teissieri, the janA, janB, and ocn genes were amplified separately using the primer pairs ja1-ja2, jb1-jb2, and oc1-oc2, respectively. For D. yakuba, janA was amplified with the ja1-ja2 primers, and the janB-ocn region was amplified as a 1.6-kb fragment using the jb1-oc2 primers. For D. mauritiana, D. erecta, and D. orena, the entire janA-janB-ocn 2.4-kb genomic region was amplified using the primers ja1-oc2. For D. sechellia, amplification with the ja1-ja2 primers was unsuccessful; however, we were able to amplify the 1.6-kb genomic region containing the janB and ocn genes using the primers jb1-oc2. PCR products were cloned with the TOPO TA Cloning kit (Invitrogen, Carlsbad, Calif.). Plasmid DNA was purified following the alkaline lysis protocol of Sambrook, Fritsch, and Maniatis (1989, pp. 1.251.28), with an additional 100% chloroform extraction performed before ethanol precipitation. Approximately 200 ng of plasmid DNA template was used per sequencing reaction using the Dye Terminator cycle sequencing kit (Applied Biosystems Inc.). Gene-specific PCR primers (listed above) and universal M13 forward and reverse primers were used as sequencing primers. An additional sequencing primer was designed specific to the D. yakuba ocn sequence (5'-CTGGTTAGGCCGTGCATGTG-3'). Sequencing gels were run on an ABI 373 automated sequencer. DNA was sequenced on both strands, and at least two independent clones were sequenced for each gene in each species. Additional independent clones were sequenced when necessary to resolve ambiguities.
RNA Preparation and RT-PCR
Total RNA was prepared from whole adult flies, adult body segments, or hand-dissected tissues using TRIzol reagent and following the manufacturer's protocol. In all cases, adult flies were collected 68 days after eclosion. For whole flies, separate RNA preparations were made using 10 flies of each sex. An additional 10 adult males were sectioned into head, thorax, and abdomen segments, with each segment being used for a separate RNA preparation. The testes and midguts from 4050 adult males were isolated by hand dissection. These tissues were stored in RNAlater solution (Ambion, Austin, Tex.) prior to RNA extraction. First-strand cDNA was synthesized from approximately 500 ng of total RNA in a 20-µl reaction containing: 1 x first-strand buffer, 3 µg random primers, 0.1 M DTT, 1 U RNasin (Promega, Madison, Wis.), and 200 U Superscript II reverse transcriptase. The reaction was incubated for 1 h at 37°C and then heated to 65°C for 10 min. Five microliters of each cDNA reaction was used for PCR in a 50-µl reaction under the same conditions given above for genomic DNA amplification. As a control to ensure that sufficient cDNA was present, two PCR reactions were performed on each cDNA preparation. The two reactions were identical except for the primers. The first reaction contained primers specific to the ocn gene, while the second reaction contained primers specific to the actin gene, Act5C (GenBank accession number K00667). The ocn primers (oc1 and oc2) are expected to amplify a cDNA product of 489 bp; the Act5C primers (5'-GTGACGAAGAAGTTGCTGCTC-3' and 5'-ATCTGCTGGAAGGTGGACGAC-3') are expected to amplify a product of 1,063 bp. Following amplification, PCR products were separated on a 1% agarose gel and visualized under UV light by ethidium bromide staining.
Sequence Analysis
Protein-encoding sequences were aligned by first aligning amino acid sequences with the CLUSTAL X program (Thompson et al. 1997
), then back-translating the aligned amino acids into codons. Slight adjustments to improve the alignment of the codons were made by eye. Gene trees were constructed by maximum parsimony using PAUP* (Swofford 2000) with 1,000 bootstrap replicates. Maximum-likelihood analyses of substitution rates were performed using the PAML software package (Yang 2000
). Transition/transversion rates and nucleotide frequencies at the three codon positions were estimated separately for each gene except in the combined analysis, where the average values from all three genes were used for both the combined data and the individual genes.
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Results |
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ocn Expression Pattern
Since the ocn protein was originally isolated due to its abundance in testis nuclear extracts and shares significant homology with the testis-specific janB protein, it seemed likely that ocn expression was also specific to testes. To test this hypothesis, we examined the pattern of ocn expression using RT-PCR. Our results indicate that ocn is expressed in males, but not in females (fig. 3
). Males were further dissected into body segments, and a strong band of ocn product was obtained from cDNA prepared from the male abdomen (fig. 3
). Further dissection of the abdomen into testes and midguts indicated that the abdominal ocn expression was specific to the testes (fig. 3
). There appeared to be a very faint band corresponding to the expected ocn product in the "head" lane of figure 3
. This suggests that there may be low levels of ocn expression in the male head. The fact that ESTs corresponding to the ocn sequence were identified from a combined male and female head cDNA library by the BDGP (Rubin et al. 2000) supports this possibility. A band of a different size appears in the "thorax" lane of figure 3
. We believe this represents a nonspecific product, as the band is not distinct and was not present in other amplifications from the male thorax (not shown).
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Finally, we tested for the presence of heterogeneous among genes. In this case, the null model, M0, was applied to the combined data of all three genes. This model assumed a constant
for all sites, for all lineages, and for all genes. The alternative model allowed each gene to have a different
, estimated by applying M0 to each gene separately under the same parameters used for the combined data. Since the individual gene likelihoods were additive, their sum could be compared with the likelihood of the combined data. Because the D. sechellia janA sequence was unavailable, we assumed it to be identical to that of D. simulans. This was a conservative assumption for the purposes of our test, because it assumed a branch length of zero between D. sechellia and D. simulans with an equal number of synonymous and nonsynonymous substitutions (zero). Our results indicated that the gene-specific
model offers a significantly better fit to the data than M0 (2
= 57.1, df = 2, P < 0.001). Thus, we conclude that there is highly significant variation in
among the janA, janB, and ocn genes. The model of a separate
for each gene was also significantly better than models in which any two of the three genes shared a common
(data not shown).
To compare the rates of evolution of the janA, janB, and ocn genes with those of genes that do not have reproductive function, we calculated for other protein-encoding genes with sequences available from species throughout the D. melanogaster species subgroup: Cu-Zn superoxide dismutase (Sod), alpha-amylase proximal (Amy-p) and distal (Amy-d), and Adh. All four of these genes encode proteins with metabolic function, and all four have smaller
values than janA, janB, or ocn (fig. 7
). In all cases,
was significantly lower than that of janA as determined by the test described above (Sod: 2
= 42.4, df = 2, P < 0.001; Amy-p: 2
= 108.7, df = 2, P < 0.001; Amy-d: 2
= 87.6, df = 2, P < 0.001; Adh: 2
= 58.2, df = 2, P < 0.001).
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Discussion |
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While the above duplication narrative is most parsimonious with respect to the sequence-based phylogenetic tree, it requires two separate instances of intron gain/loss, because all of the janB sequences (including that of D. pseudoobscura) contain a first intron that is not present in any of the janA or ocn sequences. One possibility is that an intron inserted into the ancestral janB gene following the first duplication event and was lost in the ocn lineage following the second duplication. Alternatively, the first intron might have been present in the ancestral janA sequence and subsequently lost in both janA and ocn following duplication. In Drosophila, several examples indicate that parallel loss of introns may be relatively common (Anderson, Carew, and Powell 1993
; Da Lage, Wegnez, and Cariou 1996
). Our tree is also consistent with two intron insertions in the D. pseudoobscura and D. melanogaster species subgroup janB gene lineages, but we consider this less likely because it requires parallel intron insertion into the same location of the coding region. Since the 3' end of the janA transcript overlaps with the 5' end of janB (fig. 2
), it is likely that the janB sequence faces additional selective constraints required for proper processing of the janA transcript. Such constraints would not apply to ocn and may account for the divergence in intron-exon structure between janB and ocn.
Although none of the proteins encoded by janA, janB, or ocn contain recognizable structural motifs that suggest a molecular function, it is likely that they are involved in chromatin packaging in sperm nuclei. The predicted molecular weights of the janA, janB, and ocn proteins in D. melanogaster are 15.22, 15.86, and 16.89 kDa, respectively. All three proteins are basic, containing 18%21% positively charged amino acid residues. The fraction of basic residues in each protein is similar to that found in Drosophila histones. In addition, the ocn protein shows a migration pattern similar to histones H2 and H3 when separated by gel electrophoresis (fig. 1 ). Both ocn mRNA and protein are abundant in the testes of mature males, and the strong conservation between the janB and ocn 5' UTRs suggests that ocn translation is restricted to the postmeiotic stages of spermatogenesis.
Maximum-likelihood testing of different models of selective constraint indicates that rates of synonymous and nonsynonymous substitution within the janA-ocn region are best explained by assigning a different parameter to each of the three genes. There is strong statistical support for differences in
among all three genes, with the highest rate in janB and the lowest in janA. Since
is the ratio of dN to dS, differences in
among genes may result from differences in either of these quantities. For example, the higher
of janB could be the result of either an increased dN or a decreased dS relative to janA and ocn. Our results indicate that the differences in
among janA, janB, and ocn are due primarily to differences in dN (table 1
). Furthermore, if the increased
was the result of reduced synonymous substitution rates due to purifying selection on synonymous codon sites, we would expect genes with higher
to show greater levels of codon bias. Two measures of codon bias reveal that there is no positive correlation between
and codon bias (table 1
). In fact, we find the opposite: genes with higher values of
show lower levels of codon bias. Thus, we conclude that the differences in
among these genes are not the result of reduced rates of synonymous substitution caused by increased purifying selection against unpreferred codons.
The likelihood analyses presented in table 3
assume a tree topology of the D. melanogaster species subgroup identical to that of the janB gene in figure 6
. This tree contains no polytomies, and each node is supported by a bootstrap of 77%. An identical tree is predicted for the D. melanogaster species subgroup when data from the three genes are combined. Tree topologies based on ocn and janA, however, are more ambiguous and contain several polytomies (fig. 6
). To investigate whether the tree topology might affect our results, we repeated the above analyses using topologies suggested by either ocn or janA. Consistent with previous reports (Yang et al. 2000
), we found that the use of reasonable, alternate tree topologies had a negligible effect on the results and did not alter their statistical significance (not shown).
Comparison of genes from a group of closely related species, such as the D. melanogaster species subgroup used here, allows for powerful statistical tests to detect differences in the rates of evolution among genes. Using this method, we find significant differences in evolutionary rates both among paralogous genes located within a 2.4-kb region of the genome and between genes with reproductive and metabolic function. Although the sample size is small due to the lack of sequence availability for many species of the D. melanogaster species subgroup, these results are consistent with those of Civetta and Singh (1998)
, who found a higher ratio of nonsynonymous to synonymous substitution rates in genes with a sex-related function than in genes with developmental or metabolic function. Our results lend support to the "faster male" hypothesis (Wu and Davis 1993
) and suggest that rapid evolution of genes involved in male fertility may play a role in reproductive isolation between species. The increased rate of molecular evolution observed for janA, janB, and ocn could be the result of either positive selection for amino acid replacements or relaxed selective constraints. It is generally not possible to distinguish between these two possibilities through interspecific comparison of protein-encoding sequences. One exception to this occurs when there is strong diversifying selection, such as with antigenic proteins of infectious pathogens (Fitch et al. 1997
; Yang et al. 2000
), resulting in a value of
significantly greater than 1. Such cases, however, appear to be quite rare (Endo, Ikeo, and Gojobori 1996
). Further analysis of patterns of intraspecific variation in the janA-ocn region will allow the application of more powerful statistical tests (e.g., Hudson, Kreitman, and Aguadé 1987
; McDonald and Kreitman 1991
) to determine if positive selection has played a role in the molecular evolution and functional divergence of this duplicated gene family.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Keywords: janus,
ocnus,
gene duplication
sperm protein
Drosophila melanogaster
species subgroup
2 Address for correspondence and reprints: John Parsch, Department of Organismic and Evolutionary Biology, Harvard University Biological Laboratories, 16 Divinity Avenue, Cambridge, Massachusetts 02138-2020. jparsch{at}oeb.harvard.edu
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