Different patterns of evolution in alternative and constitutive coding regions of Drosophila alternatively spliced genes

Seven hundred and ninety Drosophila melanogaster genes, alternatively spliced in coding regions were considered together with their Drosophila pseudoobscura orthologs. It was found that nucleotide substitutions in alternative coding regions accumulate more intensively than in constitutive regions. Moreover, the evolutionary pattern of alternative regions depends on their inclusion mechanisms (use of alternative promoters, splicing sites or polyadenylation sites) significantly. The rate of synonymous substitutions varies is more dramatically than that of nonsynonymous substitutions. Nucleotide substitution patterns in different classes of alternative regions of mammalian and Drosophila genes have little in common.     

Neutral and Non-Neutral Evolution of Drosophila Mitochondrial DNA

To test hypotheses of neutral evolution of mitochondrial DNA (mtDNA), nucleotide sequences were determined for 1515 base pairs of the NADH dehydrogenase subunit 5 (ND5) gene in the mitochondrial DNA of 29 lines of Drosophila melanogaster and 9 lines of its sibling species Drosophila simulans. In contrast to the patterns for nuclear genes, where D. melanogaster generally exhibits much less nucleotide polymorphism, the number of segregating sites was slightly higher in a global sample of nine ND5 sequences in D. melanogaster (s = 8) than in the nine lines of D. simulans (s = 6). When compared to variation at nuclear loci, the mtDNA variation in D. melanogaster does not depart from neutral expectations. The ND5 sequences in D. simulans, however, show fewer than half the number of variable sites expected under neutrality when compared to sequences from the period locus. While this reduction in variation is not significant at the 5% level, HKA tests with published restriction data for mtDNA in D. simulans do show a significant reduction of variation suggesting a selective sweep of variation in the mtDNA in this species. Tests of neutral evolution based on the ratios of synonymous and replacement polymorphism and divergence are generally consistent with neutral expectations, although a significant excess of amino acid polymorphism within both species is localized in one region of the protein. The rate of mtDNA evolution has been faster in D. melanogaster than in D. simulans and the population structure of mtDNA is distinct in these species. The data reveal how different rates of mtDNA evolution between species and different histories of neutral and adaptive evolution within species can compromise historical inferences in population and evolutionary biology.     

Nucleotide Variation at the Gpdh Locus in the Genus Drosophila

The Gpdh locus was sequenced in a broad range of Drosophila species. In contrast to the extreme evolutionary constraint seen at the amino acid level, the synonymous sites evolve at rates comparable to those of other genes. Gpdh nucleotide sequences were used to infer a phylogenetic tree, and the relationships among the species of the obscura group were examined in detail. A survey of nucleotide polymorphism within D. pseudoobscura revealed no amino acid variation in this species. Applying a modified McDonald-Kreitman test, the amino acid divergence between species in the obscura group does not appear to be excessive, implying that drift is adequate to explain the patterns of amino acid change at this locus. In addition, the level of polymorphism at the Gpdh locus in D. pseudoobscura is comparable to that found at other loci, as determined by a Hudson-Kreitman-Aguade test. Thus, the pattern of nucleotide variation within and between species at the Gpdh locus is consistent with a neutral model.     

Codon Usage Bias and Base Composition of Nuclear Genes in Drosophila

The nuclear genes of Drosophila evolve at various rates. This variation seems to correlate with codon-usage bias. In order to elucidate the determining factors of the various evolutionary rates and codon-usage bias in the Drosophila nuclear genome, we compared patterns of codon-usage bias with base compositions of exons and introns. Our results clearly show the existence of selective constraints at the translational level for synonymous (silent) sites and, on the other hand, the neutrality or near neutrality of long stretches of nucleotide sequence within noncoding regions. These features were found for comparisons among nuclear genes in a particular species (Drosophila melanogaster, Drosophila pseudoobscura and Drosophila virilis) as well as in a particular gene (alcohol dehydrogenase) among different species in the genus Drosophila. The patterns of evolution of synonymous sites in Drosophila are more similar to those in the prokaryotes than they are to those in mammals. If a difference in the level of expression of each gene is a main reason for the difference in the degree of selective constraint, the evolution of synonymous sites of Drosophila genes would be sensitive to the level of expression among genes and would change as the level of expression becomes altered in different species. Our analysis verifies these predictions and also identifies additional selective constraints at the translational level in Drosophila.     

Nucleotide sequence of the Xdh region in Drosophila pseudoobscura and an analysis of the evolution of synonymous codons

The nucleotide sequence of the Xdh region of Drosophila pseudoobscura is presented. The Xdh gene structure and organization are compared with the homologous region in D. melanogaster. This locus is shown to have similar organization in the two species, although an additional intron and three insertion/deletion events are described for the D. pseudoobscura coding region. The encoded proteins are predicted to have very similar charges and hydrophobic/hydrophilic domains even though 11% of the amino acids are different. A gene 5' to Xdh, putative l(3)s12, is suggested from sequence similarity between the species. Synonymous differences at the Xdh locus between the two species are analyzed using a new method described in the preceding paper by Lewontin. This analysis shows that synonymous positions within the Xdh locus are evolving at very different rates, being dependent on level of codon redundancy. A comparison of synonymous divergence between D. melanogaster and D. pseudoobscura in five additional genes reveals variation in the level of synonymous substitution.      

Early duplication and functional diversification of the opsin gene family in insects

Recent analysis of the complete mosquito Anopheles gambiae genome has revealed a far higher number of opsin genes than for either the Drosophila melanogaster genome or any other known insect. In particular, the analysis revealed an extraordinary opsin gene content expansion, whereby half are long wavelength-sensitive (LW) opsin gene duplicates. We analyzed this genomic data in relationship to other known insect opsins to estimate the relative timing of the LW opsin gene duplications and to identify "missing" paralogs in extant species. The inferred branching patterns of the LW opsin gene family phylogeny indicate at least one early gene duplication within insects before the emergence of the orders Orthoptera, Mantodea, Hymenoptera, Lepidoptera, and Diptera. These data predict the existence of one more LW opsin gene than is currently known from most insects. We tested this prediction by using a degenerate PCR strategy to screen the hymenopteran genome for novel LW opsin genes. We isolated two LW opsin gene sequences from each of five bee species, Bombus impatiens, B. terrestris, Diadasia afflicta, D. rinconis, and Osmia rufa, including 1.1 to 1.2 kb from a known (LW Rh1) and 1 kb from a new opsin gene (LW Rh2). Phylogenetic analysis suggests that the novel hymenopteran gene is orthologous to A. gambiae GPRop7, a gene that is apparently missing from D. melanogaster. Relative rate tests show that LW Rh2 is evolving at a slower rate than LW Rh1 and, therefore, may be a useful marker for higher-level hymenopteran systematics. Site-specific rate tests indicate the presence of several amino acid sites between LW Rh1 and LW Rh2 that have undergone shifts in selective constraints after duplication. These sites and others are discussed in relationship to putative structural and functional differences between the two genes.     

Molecular evolution of sex-biased genes in Drosophila

Studies of morphology, interspecific hybridization, protein/DNA sequences, and levels of gene expression have suggested that sex-related characters (particularly those involved in male reproduction) evolve rapidly relative to non-sex-related characters. Here we report a general comparison of evolutionary rates of sex-biased genes using data from cDNA microarray experiments and comparative genomic studies of Drosophila. Comparisons of nonsynonymous/synonymous substitution rates (d(N)/d(S)) between species of the D. melanogaster subgroup revealed that genes with male-biased expression had significantly faster rates of evolution than genes with female-biased or unbiased expression. The difference was caused primarily by a higher d(N) in the male-biased genes. The same pattern was observed for comparisons among more distantly related species. In comparisons between D. melanogaster and D. pseudoobscura, genes with highly biased male expression were significantly more divergent than genes with highly biased female expression. In many cases, orthologs of D. melanogaster male-biased genes could not be identified in D. pseudoobscura through a Blast search. In contrast to the male-biased genes, there was no clear evidence for accelerated rates of evolution in female-biased genes, and most comparisons indicated a reduced rate of evolution in female-biased genes relative to unbiased genes. Male-biased genes did not show an increased ratio of nonsynonymous/synonymous polymorphism within D. melanogaster, and comparisons of polymorphism/divergence ratios suggest that the rapid evolution of male-biased genes is caused by positive selection.     

The Drosophila ninaE gene encodes an opsin

The Drosophila ninaE gene was isolated by a multistep protocol on the basis of its homology to bovine opsin cDNA. The gene encodes the major visual pigment protein (opsin) contained in Drosophila photoreceptor cells R1-R6. The coding sequence is interrupted by four short introns. The positions of three introns are conserved with respect to positions in mammalian opsin genes. The nucleotide sequence has intermittent regions of homology to bovine opsin coding sequences. The deduced amino acid sequence reveals significant homology to vertebrate opsins; there is strong conservation of the retinal binding site and two other regions. The predicted protein secondary structure strikingly resembles that of mammalian opsins. We conclude the Drosophila and vertebrate opsin genes are derived from a common ancestor.     

Evolutionary rearrangement of the amylase genomic regions between Drosophila melanogaster and Drosophila pseudoobscura

Two Drosophila pseudoobscura genomic clones have sequence similarity to the Drosophila melanogaster amylase region that maps to the 53CD region on the D. melanogaster cytogenetic map. The two clones with similarity to amylase map to sections 73A and 78C of the D. pseudoobscura third chromosome cytogenetic map. The complete sequences of both the 73A and 78C regions were compared to the D. melanogaster genome to determine if the coding region for amylase is present in both regions and to determine the evolutionary mechanism responsible for the observed distribution of the amylase gene or genes. The D. pseudoobscura 73A and 78C linkage groups are conserved with the D. melanogaster 41E and 53CD regions, respectively. The amylase gene, however, has not maintained its conserved linkage between the two species. These data indicate that amylase has moved via a transposition event in the D. melanogaster or D. pseudoobscura lineage. The predicted genes within the 73A and 78C regions show patterns of molecular evolution in synonymous and nonsynonymous sites that are consistent with previous studies of these two species.     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Functional diversification of lepidopteran opsins following gene duplication.

A comparative approach was taken for identifying amino acid substitutions that may be under positive Darwinian selection and are correlated with spectral shifts among orthologous and paralogous lepidopteran long wavelength-sensitive (LW) opsins. Four novel LW opsin fragments were isolated, cloned, and sequenced from eye-specific cDNAs from two butterflies, Vanessa cardui (Nymphalidae) and Precis coenia (Nymphalidae), and two moths, Spodoptera exigua (Noctuidae) and Galleria mellonella (Pyralidae). These opsins were sampled because they encode visual pigments having a naturally occurring range of lambda(max) values (510-530 nm), which in combination with previously characterized lepidopteran opsins, provide a complete range of known spectral sensitivities (510-575 nm) among lepidopteran LW opsins. Two recent opsin gene duplication events were found within the papilionid but not within the nymphalid butterfly families through neighbor-joining, maximum parsimony, and maximum likelihood phylogenetic analyses of 13 lepidopteran opsin sequences. An elevated rate of evolution was detected in the red-shifted Papilio Rh3 branch following gene duplication, because of an increase in the amino acid substitution rate in the transmembrane domain of the protein, a region that forms the chromophore-binding pocket of the visual pigment. A maximum likelihood approach was used to estimate omega, the ratio of nonsynonymous to synonymous substitutions per site. Branch-specific tests of selection (free-ratio) identified one branch with omega = 2.1044, but the small number of substitutions involved was not significantly different from the expected number of changes under the neutral expectation of omega = 1. Ancestral sequences were reconstructed with a high degree of certainty from these data. Reconstructed ancestral sequences revealed several instances of convergence to the same amino acid between butterfly and vertebrate cone pigments, and between independent branches of the butterfly opsin tree that are correlated with spectral shifts.     

Synonymous Substitutions in the Xdh Gene of Drosophila: Heterogeneous Distribution along the Coding Region

The Xdh (rosy) region of Drosophila subobscura has been sequenced and compared to the homologous region of D. pseudoobscura and D. melanogaster. Estimates of the numbers of synonymous substitutions per site (Ks) confirm that Xdh has a high synonymous substitution rate. The distributions of both nonsynonymous and synonymous substitutions along the coding region were found to be heterogeneous. Also, no relationship has been detected between Ks estimates and codon usage bias along the gene, in contrast with the generally observed relationship among genes. This heterogeneous distribution of synonymous substitutions along the Xdh gene, which is expression-level independent, could be explained by a differential selection pressure on synonymous sites along the coding region acting on mRNA secondary structure. The synonymous rate in the Xdh coding region is lower in the D. subobscura than in the D. pseudoobscura lineage, whereas the reverse is true for the Adh gene.     

Salmonid Opsin Sequences Undergo Positive Selection and Indicate an Alternate Evolutionary Relationship in Oncorhynchus

Positive selection can be demonstrated by statistical analysis when non-synonymous nucleotide substitutions occur more frequently than synonymous substitutions (dN>dS). This pattern of sequence evolution has been observed in the rhodopsin gene of cichlids. Mutations in opsin genes resulting in amino acid (AA) replacement appear to be associated with the evolution of specific color patterns and the evolution of courtship behaviors. Within fish, AA replacements in opsin proteins have improved vision at great depths and have occurred in deep-sea species. Salmonids experience diverse photic environments during their life history. Furthermore, sexual selection has resulted in species-specific male and female coloration during spawning. To look for evidence of positive selection in salmonid opsins, we sequenced the RH1, RH2, LWS, SWS1, and SWS2 genes from six Pacific salmon species as well as the Atlantic salmon. These salmonids include landlocked and migratory species and species that vary in their coloration during spawning. In each opsin gene comparison from all species sampled, traditional dN:dS analysis did not indicate positive selection. However, the more sensitive Creevey–McInerney statistical analysis indicates that RH1 and RH2 experienced positive selection early in the evolution and speciation of salmonids.     

DNA Sequence Evolution of the Amylase Multigene Family in Drosophila Pseudoobscura

The alpha-Amylase locus in Drosophila pseudoobscura is a multigene family of one, two or three copies on the third chromosome. The nucleotide sequences of the three Amylase genes from a single chromosome of D. pseudoobscura are presented. The three Amylase genes differ at about 0.5% of their nucleotides. Each gene has a putative intron of 71 (Amy1) or 81 (Amy2 and Amy3) bp. In contrast, Drosophila melanogaster Amylase genes do not have an intron. The functional Amy1 gene of D. pseudoobscura differs from the Amy-p1 gene of D. melanogaster at an estimated 13.3% of the 1482 nucleotides in the coding region. The estimated rate of synonymous substitutions is 0.398 +/- 0.043, and the estimated rate of nonsynonymous substitutions is 0.068 +/- 0.008. From the sequence data we infer that Amy2 and Amy3 are more closely related to each other than either is to Amy1. From the pattern of nucleotide substitutions we reason that there is selection against synonymous substitutions within the Amy1 sequence; that there is selection against nonsynonymous substitutions within the Amy2 sequence, or that Amy2 has recently undergone a gene conversion with Amy1; and that Amy3 is nonfunctional and subject to random genetic drift.     

Signatures of selection and gene conversion associated with human color vision variation

Trichromatic color vision in humans results from the combination of red, green, and blue photopigment opsins. Although color vision genes have been the targets of active molecular and psychophysical research on color vision abnormalities, little is known about patterns of normal genetic variation in these genes among global human populations. The current study presents nucleotide sequence analyses and tests of neutrality for a 5.5-kb region of the X-linked long-wave "red" opsin gene (OPN1LW) in 236 individuals from ethnically diverse human populations. Our analysis of the recombination landscape across OPN1LW reveals an unusual haplotype structure associated with amino acid replacement variation in exon 3 that is consistent with gene conversion. Compared with the absence of OPN1LW amino acid replacement fixation since divergence from chimpanzee, the human population exhibits a significant excess of high-frequency OPN1LW replacements. Our results suggest that subtle changes in L-cone opsin wavelength absorption may have been adaptive during human evolution.     

The role of gene conversion in determining sequence variation and divergence in the Est-5 gene family in Drosophila pseudoobscura.

Nucleotide sequences of eight Est-5A and Est-5C genes corresponding to previously sequenced Est-5B genes in Drosophila pseudoobscura were determined to compare patterns of polymorphism and divergence among members of this small gene family. The three esterase genes were also sequenced from D. persimilis and D. miranda for interspecific comparisons. The data provide evidence that gene conversion between loci contributes to polymorphism and to the homogenization of the Est5 genes. For Est-5B, which encodes one of the most highly polymorphic proteins in Drosophila, 12% of the segregating amino acid variants appear to have been introduced via gene conversion from other members of the gene family. Interlocus gene conversion can also explain high sequence similarity, especially at synonymous sites, between Est-5B and Est-5A. Tests of neutrality using interspecific comparisons show that levels of polymorphism conform to neutral expectations at each Est-5 locus. However, McDonald-Kreitman tests based on intraspecific gene comparisons indicate that positive selection on amino acids has accompanied Est-5 gene duplication and divergence in D. pseudoobscura.