Amino Acid Replacement Substitutions Research Articles

Gene Conversion and DNA Sequence Polymorphism in the Sex-Determination Gene fog-2 and Its Paralog ftr-1 in Caenorhabditis elegans

Gene conversion, a form of concerted evolution, bears enormous potential to shape the trajectory of sequence and functional divergence of gene paralogs subsequent to duplication events. fog-2, a sex-determination gene unique to Caenorhabditis elegans and implicated in the origin of hermaphroditism in this species, resulted from the duplication of ftr-1, an upstream gene of unknown function. Synonymous sequence divergence in regions of fog-2 and ftr-1 (excluding recent gene conversion tracts) suggests that the duplication occurred 46 million generations ago. Gene conversion between fog-2 and ftr-1 was previously discovered in experimental fog-2 knockout lines of C. elegans, whereby hermaphroditism was restored in mutant obligately outcrossing male-female populations. We analyzed DNA-sequence variation in fog-2 and ftr-1 within 40 isolates of C. elegans from diverse geographic locations in order to evaluate the contribution of gene conversion to genetic variation in the two gene paralogs. The analysis shows that gene conversion contributes significantly to DNA-sequence diversity in fog-2 and ftr-1 (22% and 34%, respectively) and may have the potential to alter sexual phenotypes in natural populations. A radical amino acid change in a conserved region of the F-box domain of fog-2 was found in natural isolates of C. elegans with significantly lower fecundity. We hypothesize that the lowered fecundity is due to reduced masculinization and less sperm production and that amino acid replacement substitutions and gene conversion in fog-2 may contribute significantly to variation in the degree of inbreeding and outcrossing in natural populations.

Hotspots of Biased Nucleotide Substitutions in Human Genes

Genes that have experienced accelerated evolutionary rates on the human lineage during recent evolution are candidates for involvement in human-specific adaptations. To determine the forces that cause increased evolutionary rates in certain genes, we analyzed alignments of 10,238 human genes to their orthologues in chimpanzee and macaque. Using a likelihood ratio test, we identified protein-coding sequences with an accelerated rate of base substitutions along the human lineage. Exons evolving at a fast rate in humans have a significant tendency to contain clusters of AT-to-GC (weak-to-strong) biased substitutions. This pattern is also observed in noncoding sequence flanking rapidly evolving exons. Accelerated exons occur in regions with elevated male recombination rates and exhibit an excess of nonsynonymous substitutions relative to the genomic average. We next analyzed genes with significantly elevated ratios of nonsynonymous to synonymous rates of base substitution (d N /d S) along the human lineage, and those with an excess of amino acid replacement substitutions relative to human polymorphism. These genes also show evidence of clusters of weak-to-strong biased substitutions. These findings indicate that a recombination-associated process, such as biased gene conversion (BGC), is driving fixation of GC alleles in the human genome. This process can lead to accelerated evolution in coding sequences and excess amino acid replacement substitutions, thereby generating significant results for tests of positive selection.

A multilocus assay reveals high nucleotide diversity and limited differentiation among Scandinavian willow grouse (Lagopus lagopus)

BackgroundThere is so far very little data on autosomal nucleotide diversity in birds, except for data from the domesticated chicken and some passerines species. Estimates of nucleotide diversity reported so far in birds have been high (~10-3) and a likely explanation for this is the generally higher effective population sizes compared to mammals. In this study, the level of nucleotide diversity has been examined in the willow grouse, a non-domesticated bird species from the order Galliformes, which also holds the chicken. The willow grouse (Lagopus lagopus) has an almost circumpolar distribution but is absent from Greenland and the north Atlantic islands. It primarily inhabits tundra, forest edge habitats and sub-alpine vegetation. Willow grouse are hunted throughout its range, and regionally it is a game bird of great cultural and economical importance.ResultsWe sequenced 18 autosomal protein coding loci from approximately 15–18 individuals per population. We found a total of 127 SNP's, which corresponds to 1 SNP every 51 bp. 26 SNP's were amino acid replacement substitutions. Total nucleotide diversity (πt) was between 1.30 × 10-4 and 7.66 × 10-3 (average πt = 2.72 × 10-3 ± 2.06 × 10-3) and silent nucleotide diversity varied between 4.20 × 10-4and 2.76 × 10-2 (average πS = 9.22 × 10-3 ± 7.43 × 10-4). The synonymous diversity is approximately 20 times higher than in humans and two times higher than in chicken. Non-synonymous diversity was on average 18 times lower than the synonymous diversity and varied between 0 and 4.90 × 10-3 (average πa = 5.08 × 10-4 ± 7.43 × 103), which suggest that purifying selection is strong in these genes. FST values based on synonymous SNP's varied between -5.60 × 10-4 and 0.20 among loci and revealed low levels of differentiation among the four localities, with an overall value of FST = 0.03 (95% CI: 0.006 – 0.057) over 60 unlinked loci. Non-synonymous SNP's gave similar results. Low levels of linkage disequilibrium were observed within genes, with an average r2 = 0.084 ± 0.110, which is expected for a large outbred population with no population differentiation. The mean per site per generation recombination parameter (ρ) was comparably high (0.028 ± 0.018), indicating high recombination rates in these genes.ConclusionWe found unusually high levels of nucleotide diversity in the Scandinavian willow grouse as well as very little population structure among localities with up to 1647 km distance. There are also low levels of linkage disequilibrium within the genes and the population recombination rate is high, which is indicative of an old panmictic population, where recombination has had time to break up any haplotype blocks. The non-synonymous nucleotide diversity is low compared with the silent, which is in agreement with effective purifying selection, possibly due to the large effective population size.

Signatures of Selection in the Human Olfactory Receptor OR5I1 Gene

The human olfactory receptor (OR) repertoire is reduced in comparison to other mammals and to other nonhuman primates. Nonetheless, this olfactory decline opens an opportunity for evolutionary innovation and improvement. In the present study, we focus on an OR gene, OR5I1, which had previously been shown to present an excess of amino acid replacement substitutions between humans and chimpanzees. We analyze the genetic variation in OR5I1 in a large worldwide human panel and find an excess of derived alleles segregating at relatively high frequencies in all populations. Additional evidence for selection includes departures from neutrality in allele frequency spectra tests but no unusually extended haplotype structure. Moreover, molecular structural inference suggests that one of the nonsynonymous polymorphisms defining the presumably adaptive protein form of OR5I1 may alter the functional binding properties of the OR. These results are compatible with positive selection having modeled the pattern of variation found in the OR5I1 gene and with a relatively ancient, mild selective sweep predating the "Out of Africa" expansion of modern humans.

Periodic Extinctions of Transposable Elements in Bacterial Lineages: Evidence from Intragenomic Variation in Multiple Genomes

Most previous work on the evolution of mobile DNA was limited by incomplete sequence information. Whole genome sequences allow us to overcome this limitation. I study the nucleotide diversity of prominent members of five insertion sequence families whose transposition activity is encoded by a single transposase gene. Eighteen among 376 completely sequenced bacterial genomes and plasmids carry between 3 and 20 copies of a given insertion sequence. I show that these copies generally show very low DNA divergence. Specifically, more than 68% of the transposase genes are identical within a genome. The average number of amino acid replacement substitutions at amino acid replacement sites is Ka = 0.013, that at silent sites is Ks = 0.1. This low intragenomic diversity stands in stark contrast to a much higher divergence of the same insertion sequences among distantly related genomes. Gene conversion among protein-coding genes is unlikely to account for this lack of diversity. The relation between transposition frequencies and silent substitution rates suggests that most insertion sequences in a typical genome are evolutionarily young and have been recently acquired. They may undergo periodic extinction in bacterial lineages. By implication, they are detrimental to their host in the long run. This is also suggested by the highly skewed and patchy distribution of insertion sequences among genomes. In sum, one can think of insertion sequences as slow-acting infectious diseases of cell lineages.

Evolutionary proteomics: Computer dating for proteins

Evolutionary proteomics: Computer dating for proteins

Isolation and characterization of cytochrome c from the marine copepod Tigriopus californicus

Mitochondrial energy production requires complex interactions among proteins encoded in both the nuclear and mitochondrial genomes. The intergenomic coevolution of interacting gene products has been previously suggested based on interspecific comparisons of cytochrome c (encoded by the nuclear CYC gene) and cytochrome c oxidase (partly encoded in the mitochondrial DNA by the COX1, COX2 and COX3 genes). In the intertidal copepod, Tigriopus californicus, non-synonymous substitutions in the COX1 gene have previously been found in interpopulation comparisons. In order to determine if CYC also shows interpopulation variation, this gene was isolated from a cDNA library using a degenerate primer/polymerase chain reaction approach. Characterization of a cDNA sequence and 25 genomic DNA sequences derived from four T. californicus populations yielded the following results: (1) the T. californicus CYC gene is interrupted by an intron that occurs at the same position as the intron found in vertebrate CYC genes; (2) there is extensive sequence variation within both the coding region and intron of this gene and the vast majority of this variation occurs between sequences drawn from geographically distinct populations; (3) the coding sequence variation includes a minimum of five amino acid replacement substitutions; (4) segregation of length variants among offspring in an interpopulation cross revealed genotypic ratios consistent with the proposed allelic nature of the CYC variants. These results demonstrate that the requisite genetic variation required for intergenomic coevolution exists in the CYC– COX system in T. californicus.

Cytochrome b evolution in birds and mammals: an evaluation of the avian constraint hypothesis.

Patterns of molecular evolution in birds have long been considered anomalous. Compared with other vertebrates, birds have reduced levels of genetic divergence between groups of similar taxonomic ranks for a variety of nuclear and mitochondrial markers. This observation led to the avian constraint hypothesis, which identifies increased functional constraint on avian proteins as the cause for the reduction in genetic divergence. Subsequent investigations provided additional support for the avian constraint hypothesis when rates of molecular evolution were found to be slower in birds than in mammals in a variety of independent calibrations. It is possible to test the avian constraint hypothesis as an explanation for this avian slowdown by comparing DNA sequence data from protein-coding regions in birds and homologous regions in mammals. The increased selective constraints should lead to a reduction in the proportion of amino acid replacement substitutions. To test for such a decrease, we calculated the numbers of amino acid replacement substitutions per replacement site (dN) and silent substitutions per silent site (dS) for the complete mitochondrial cytochrome b gene using 38 avian and 43 mammalian comparisons that were phylogenetically independent. We find that dN/dS is significantly smaller in birds than in mammals. This difference cannot be explained by differences in codon bias affecting dS values. We suggest that the avian slowdown can be explained, at least in part, by a decreased tolerance for amino acid substitutions in avian species relative to mammalian species.

Molecular evolution of the Amy multigenes in the subgenus Sophophora of Drosophila.

Molecular evolution of the Amy multigenes in Drosophila was investigated using PCR amplification. Twenty-five partial Amy sequences from 13 species belonging mainly to the subgenus Sophophora were determine, and a molecular phylogeny of the Amy genes in Drosophila was constructed, together with published Amy sequences. Clustering of species are mostly consistent with the traditional classification and that inferred from other genes. From sequence divergence between PCR products, several species, including D. elegans and D. fuyamai, were suggested to have multiple copies of the Amy genes. The loss of an intron took place at least three times after the Sophophora radiation. In order to investigate the mechanism of sequence evolution, the numbers of amino acid replacement and synonymous substitutions in five lineages were estimated. The heterogeneity in the relative numbers of synonymous and replacement substitutions among the lineages was found. Possible roles of selection in the sequence evolution of the Amy gene are discussed.

Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms.

Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions.

Sequence of the cDNA for the liver/non-muscle isoform of mouse cytochrome- c oxidase subunit VIII

We have isolated and sequenced the cDNA for the liver (L) or non-muscle isoform of mouse cytochrome- c oxidase subunit VIII (COX VIII-L). Comparison of deduced COX VIII-L protein sequences from three mammalian species indicated that the human gene has sustained more amino acid replacement substitutions than either the mouse or the cow. The most highly conserved regions of this subunit are the N-terminal presequence and the C-terminal domain of the mature protein.

The absence of ongoing immunoglobulin gene hypermutation suggests a distinct mechanism for c-myc mutation in endemic Burkitt's lymphoma.

Burkitt's lymphoma is a malignancy of mature, immunoglobulin (Ig)-bearing B cells characterized by translocation between c-myc and Ig gene loci. A role for the juxtaposed Ig genes in the mutation and deregulation of c-myc expression typical of endemic Burkitt's lymphoma (eBL) has been proposed, but never proven. Our objective was to determine whether Ig gene hypermutation is ongoing in eBL. We isolated Ig heavy-chain sequences from K962 eBL tumor cells using reverse transcription and polymerase chain reaction (PCR) amplification. The PCR product was ligated into Bluescript II vectors. Multiple subclones were sequenced and the variable regions were compared for evidence of ongoing Ig hypermutation. Six total single base substitutions were observed within four of the nine subclones studied. Four substitutions resulted in amino acid changes and two were silent. There was no clustering of mutations in hypervariable regions, or a high incidence of amino acid replacement or link substitutions, all of which are characteristic of Ig hypermutation. The observed mutations occurred at a rate consistent with Taq polymerase error. Our data indicate that in the eBL tumor sample K962, the mechanism underlying c-myc mutation is distinct from that which gives rise to Ig hypermutation.

Nucleotide substitution type dependence of generation time effect of molecular evolution.

Using DNA sequence data of 18 genes from 14 mammals, we analyzed how the average molecular evolution rate per year per site (Vy) depends on the generation time (g). (I) Assuming the relation Vy varies; is directly proportional to g(-alpha), the index of generation time effect, (alpha) was estimated to be about 0.14 for amino acid replacement substitutions (A), and about 0.32 for synonymous substitutions (S). (II) Assuming the relation Vy = V(m)g g-1 + V(e)y, where V(m)g and V(e)y are constant independent of g, the fraction, r(e) = V(e)y/Vy, of the mutation rate independent part (V(e)y) in the total evolution rate (Vy) was estimated under the assumptions of the star phylogeny and the constancy of the mutation rate per generation. r(e) was smallest for mouse with the shortest generation time among our analyzed species, and it was estimated to be about 0.57 for A and 0.31 for S. Both results do not support the view that Vy is equal to the neutral mutation rate per site both for A and for S. They are in line with the thesis that, at least for A and probably even for S, the molecular evolution rate is influenced by some causes other than the mutation rate, such as changing environment.

Phylogenetic Relationships among Members of the Coregoninae Inferred from Direct Sequencing of PCR-Amplified Mitochondrial DNA

We determined the DNA sequence of a portion of the mitochondrial cytochrome b gene for eight members of the Coregoninae (Salmonidae) from North America in an effort to elucidate phylogenetic relationships in the subfamily. DNA was prepared for sequencing by the polymerase chain reaction (PCR). Direct estimates of mitochondrial DNA (mtDNA) sequence divergence among taxa ranged from 0.0% between Arctic cisco (Coregonus autumnalis) from the Mackenzie River, Canada, and cisco (C. artedi) from the Laurentian Great Lakes to 5.8% between the inconnu (Stenodus leucichthys) and the round whitefish (Prosopium cylindraceum). As has been noted in other fish species and vertebrates in general, third position silent substitutions predominated over any other type of nucleotide change. No amino acid replacement substitutions were found among any of the eight taxa examined. Comparison of mtDNA sequence divergence estimates from this and other studies suggests that the radiation of the genera within the Coregoninae occurred relatively recently (2–6 million yr B.P.). Cladistic analysis of the mtDNA sequence data yields a hypothesis of relationships that supports previous genetic and morphological classifications of coregonines.

Amino acid substitution at the Adh locus of Drosophila is facilitated by small population size.

The number of amino acid replacement substitutions and that of synonymous substitutions are examined by using DNA sequences of the Adh locus of Drosophila. The ratio of replacement to synonymous substitutions is higher in sequence comparisons between species than in polymorphisms within species. The ratio for the between-species comparisons is highest in the Hawaiian group and lowest in the obscura group. These observations suggest that amino acid substitutions are facilitated by small population size. The result is in accord with the nearly neutral theory of molecular evolution.

Tandemly duplicated alpha globin genes of gibbon.

The alpha globin gene locus of the common gibbon (Hylobates lar) was isolated, and it contains two closely linked alpha globin genes that share the same arrangement as that found for the homologous genes of other catarrhine primates. The nucleotide sequences of the gibbon alpha globin genes were determined and compared with the alpha globin gene sequences from other primate species (human, chimpanzee, orangutan, baboon, and rhesus); the prosimian primate, galago, alpha A and alpha B globin genes provided the out-group for this analysis. The degree of divergence for both synonymous and nonsynonymous substitutions among the alpha globin genes are generally smaller for intraspecies than interspecies comparisons, which is indicative of concerted evolution between the paired alpha globin genes of each catarrhine species. The pattern of differences is suggestive of gene conversions. This possibility is supported by both site-by-site and branch-swapping parsimony analyses. The site-by-site parsimony analysis was also used to determine the 3' boundary of each catarrhine species-specific conversion. These alpha globin gene conversion events encompass most of the transcriptional region, including the promoter, exons, introns, and about one-half of the 3'-untranslated region. However, the paired gibbon alpha globin genes show the highest degree of divergence, differing by five nucleotides in their coding regions, three of which encode amino acid replacement substitutions. It appears that the last convergence event between the paired gibbon alpha globin genes is relatively ancient, having occurred approximately 11 million years ago. The remaining 3'-untranslated region and flanking DNAs show no evidence of involvement in any recent conversion event as the parsimony analysis of these sequences group all the alpha 1 globin genes of the different catarrhine species into one clade and all the alpha 2 globin gene of these species into a separate clade. The sequence comparison among the alpha globin genes of different primate species also provides interesting implications regarding the evolution and functional domains of the 3'-untranslated regions.

Adaptive protein evolution at the Adh locus in Drosophila

Proteins often differ in amino-acid sequence across species. This difference has evolved by the accumulation of neutral mutations by random drift, the fixation of adaptive mutations by selection, or a mixture of the two. Here we propose a simple statistical test of the neutral protein evolution hypothesis based on a comparison of the number of amino-acid replacement substitutions to synonymous substitutions in the coding region of a locus. If the observed substitutions are neutral, the ratio of replacement to synonymous fixed differences between species should be the same as the ratio of replacement to synonymous polymorphisms within species. DNA sequence data on the Adh locus (encoding alcohol dehydrogenase, EC 1.1.1.1) in three species in the Drosophila melanogaster species subgroup do not fit this expectation; instead, there are more fixed replacement differences between species than expected. We suggest that these excess replacement substitutions result from adaptive fixation of selectively advantageous mutations.

Why do tubulin gene families lack diversity in flagellate/ciliate protists?

Considerable amino acid sequence diversity is found among tubulin isotypes encoded by tubulin gene families in animal, higher plant, and fungal systems. In contrast, relatively little diversity is found among the isotypes produced by the gene families in a number of flagellate or ciliate protists. It is possible that proper assembly of the axoneme requires a homogeneous pool of tubulin subunits and that the axoneme thus provides a stringent selection against amino acid replacement substitutions among tubulin genes in these systems.

Detection of Intraspecific.DNA Sequence Variation in the Mitochondrial CytochromebGene of Atlantic Cod (Gadus morhua) by the Polymerase Chain Reaction

We determined the DNA sequence of a portion of the mitochondrial cytochrome b gene for 55 Atlantic cod (Gadus morhua) from Norway and from 10 locations within the Northern Cod complex and adjacent stocks off Newfoundland. DNA was prepared for sequencing by the polymerase chain reaction (PCR). Eleven variable nucleotide positions within a 298 base region defined 12 genotypes. Genotype proportions differed significantly between Newfoundland and Norwegian populations: the majority genotype among Newfoundland populations was present in a minority of Norwegian cod. Newfoundland cod showed less genotypic diversity than those from the eastern Atlantic: nine genotypes were found among all 10 Newfoundland populations, as compared with seven genotypes within the single Norwegian population. An exception was an overwintering, inshore Newfoundland population that showed four genotypes among five fish. As in other vertebrates, third position synonymous transitions predominate over other types of nucleotide changes. However, two amino acid replacement substitutions occur among cod, and the ratio of purine transitions to pyrimidine transitions is significantly higher than in other species. The existence of DNA sequence polymorphism permits the various hypotheses of the distribution and differentiation of Newfoundland cod stocks to be tested, and points to the utility of PCR technology in fishery genetics.

The generation of MHC class II gene polymorphism in the genus Mus

Nucleotide sequences of exon 2 from 46 alleles of the murine MHC class II Aβ gene derived from 12 rodent species were determined using the polymerase chain reaction (PCR). This exon (273 bp) exhibited extensive nucleotide sequence diversity that did not follow predicted phylogenetic relationships. The nucleotide sequence diversity of exon 2 was focussed predominantly in five discrete sub-domains which contained polymorphic sequence motifs. These sub-domains each encode a segment of the antigen binding site of MHC class II molecule. The alleles in each sub-domain were organized into homogeneous lineages by parsimony analysis. The alleles in these lineages were very stable over long evolutionary periods. Some alleles derived from mice and rats have the same sequence motifs, indicating that they have been retained as polymorphisms since prior to the separation of the genus Mus and Rattus (10 Myr). Our findings indicate that polymorphism of MHC class II genes are generated by a combination of two processes. The steady accumulation of point mutations has produced highly divergent polymorphic sequence motifs in each sub-domain. This diversity is extensively amplified by repeated intra-exonic recombinations that shuffle these polymorphic sequence motifs into many, novel, allelic combinations. These intra-exonic recombinational events do not seem to occur at a extraordinary rate, or at specific sites within the exon. The protein sequences encoded by the polymorphic sequence motifs differed on average in 39.3% of amino acids and 67.3% of these substitutions were non-conservative. These findings indicate that they encode protein segments in the antigen binding site with distinct chemical properties. Codons for the antigen binding site have accumulated more non-synonymous (amino acid replacement) substitutions than synonymous (silent) substitutions, whereas the opposite is true for the non-antigen binding site codons in exon 2. All these results suggest that diversity is selectively maintained within the antigen binding sites. We propose that a special mechanism (termed divergent allele advantage), together with overdominant selection, accounts for the maintenance of a large number of highly divergent alleles in natural mouse populations. The extraordinary conservation of sequence motifs suggest that purifying selection may operate on MHC genes to selectively maintain certain sequence motifs and possibly combinations of specific motifs.