Mutation-selection models of codon substitution and their use to estimate selective strengths on codon usage

Current models of codon substitution are formulated at the levels of nucleotide substitution and do not explicitly consider the separate effects of mutation and selection. They are thus incapable of inferring whether mutation or selection is responsible for evolution at silent sites. Here we implement a few population genetics models of codon substitution that explicitly consider mutation bias and natural selection at the DNA level. Selection on codon usage is modeled by introducing codon-fitness parameters, which together with mutation-bias parameters, predict optimal codon frequencies for the gene. The selective pressure may be for translational efficiency and accuracy or for fine-tuning translational kinetics to produce correct protein folding. We apply the models to compare mitochondrial and nuclear genes from several mammalian species. Model assumptions concerning codon usage are found to affect the estimation of sequence distances (such as the synonymous rate d(S), the nonsynonymous rate d(N), and the rate at the 4-fold degenerate sites d(4)), as found in previous studies, but the new models produced very similar estimates to some old ones. We also develop a likelihood ratio test to examine the null hypothesis that codon usage is due to mutation bias alone, not influenced by natural selection. Application of the test to the mammalian data led to rejection of the null hypothesis in most genes, suggesting that natural selection may be a driving force in the evolution of synonymous codon usage in mammals. Estimates of selection coefficients nevertheless suggest that selection on codon usage is weak and most mutations are nearly neutral. The sensitivity of the analysis on the assumed mutation model is discussed.     

Variation in synonymous codon use and DNA polymorphism within the Drosophila genome

A strong negative correlation between the rate of amino-acid substitution and codon usage bias in Drosophila has been attributed to interference between positive selection at nonsynonymous sites and weak selection on codon usage. To further explore this possibility we have investigated polymorphism and divergence at three kinds of sites: synonymous, nonsynonymous and intronic in relation to codon bias in D. melanogaster and D. simulans. We confirmed that protein evolution is one of the main explicative parameters for interlocus codon bias variation (r(2) approximately 40%). However, intron or synonymous diversities, which could have been expected to be good indicators of local interference [here defined as the additional increase of drift due to selection on tightly linked sites, also called 'genetic draft' by Gillespie (2000)] did not covary significantly with codon bias or with protein evolution. Concurrently, levels of polymorphism were reduced in regions of low recombination rates whereas codon bias was not. Finally, while nonsynonymous diversities were very well correlated between species, neither synonymous nor intron diversities observed in D. melanogaster were correlated with those observed in D. simulans. All together, our results suggest that the selective constraint on the protein is a stable component of gene evolution while local interference is not. The pattern of variation in genetic draft along the genome therefore seems to be instable through evolutionary times and should therefore be considered as a minor determinant of codon bias variance. We argue that selective constraints for optimal codon usage are likely to be correlated with selective constraints on the protein, both between codons within a gene, as previously suggested, and also between genes within a genome.     

Effects of gene expression on molecular evolution in Arabidopsis thaliana and Arabidopsis lyrata

We analyzed the complete genome sequence of Arabidopsis thaliana and sequence data from 83 genes in the outcrossing A. lyrata, to better understand the role of gene expression on the strength of natural selection on synonymous and replacement sites in Arabidopsis. From data on tRNA gene abundance, we find a good concordance between codon preferences and the relative abundance of isoaccepting tRNAs in the complete A. thaliana genome, consistent with models of translational selection. Both EST-based and new quantitative measures of gene expression (MPSS) suggest that codon preferences derived from information on tRNA abundance are more strongly associated with gene expression than those obtained from multivariate analysis, which provides further support for the hypothesis that codon bias in Arabidopsis is under selection mediated by tRNA abundance. Consistent with previous results, analysis of protein evolution reveals a significant correlation between gene expression level and amino acid substitution rate. Analysis by MPSS estimates of gene expression suggests that this effect is primarily the result of a correlation between the number of tissues in which a gene is expressed and the rate of amino acid substitution, which indicates that the degree of tissue specialization may be an important determinant of the rate of protein evolution in Arabidopsis.     

Rates of nucleotide substitution and mammalian nuclear gene evolution. Approximate and maximum-likelihood methods lead to different conclusions

Rates and patterns of synonymous and nonsynonymous substitutions have important implications for the origin and maintenance of mammalian isochores and the effectiveness of selection at synonymous sites. Previous studies of mammalian nuclear genes largely employed approximate methods to estimate rates of nonsynonymous and synonymous substitutions. Because these methods did not account for major features of DNA sequence evolution such as transition/transversion rate bias and unequal codon usage, they might not have produced reliable results. To evaluate the impact of the estimation method, we analyzed a sample of 82 nuclear genes from the mammalian orders Artiodactyla, Primates, and Rodentia using both approximate and maximum-likelihood methods. Maximum-likelihood analysis indicated that synonymous substitution rates were positively correlated with GC content at the third codon positions, but independent of nonsynonymous substitution rates. Approximate methods, however, indicated that synonymous substitution rates were independent of GC content at the third codon positions, but were positively correlated with nonsynonymous rates. Failure to properly account for transition/transversion rate bias and unequal codon usage appears to have caused substantial biases in approximate estimates of substitution rates.     

Evolution of amino-acid sequences and codon usage on the Drosophila miranda neo-sex chromosomes

We have studied patterns of DNA sequence variation and evolution for 22 genes located on the neo-X and neo-Y chromosomes of Drosophila miranda. As found previously, nucleotide site diversity is greatly reduced on the neo-Y chromosome, with a severely distorted frequency spectrum. There is also an accelerated rate of amino-acid sequence evolution on the neo-Y chromosome. Comparisons of nonsynonymous and silent variation and divergence suggest that amino-acid sequences on the neo-X chromosome are subject to purifying selection, whereas this is much weaker on the neo-Y. The same applies to synonymous variants affecting codon usage. There is also an indication of a recent relaxation of selection on synonymous mutations for genes on other chromosomes. Genes that are weakly expressed on the neo-Y chromosome appear to have a faster rate of accumulation of both nonsynonymous and unpreferred synonymous mutations than genes with high levels of expression, although the rate of accumulation when both types of mutation are pooled is higher for the neo-Y chromosome than for the neo-X chromosome even for highly expressed genes.     

Evolutionary rates and expression level in Chlamydomonas

In many biological systems, especially bacteria and unicellular eukaryotes, rates of synonymous and nonsynonymous nucleotide divergence are negatively correlated with the level of gene expression, a phenomenon that has been attributed to natural selection. Surprisingly, this relationship has not been examined in many important groups, including the unicellular model organism Chlamydomonas reinhardtii. Prior to this study, comparative data on protein-coding sequences from C. reinhardtii and its close noninterfertile relative C. incerta were very limited. We compiled and analyzed protein-coding sequences for 67 nuclear genes from these taxa; the sequences were mostly obtained from the C. reinhardtii EST database and our C. incerta EST data. Compositional and synonymous codon usage biases varied among genes within each species but were highly correlated between the orthologous genes of the two species. Relative rates of synonymous and nonsynonymous substitution across genes varied widely and showed a strong negative correlation with the level of gene expression estimated by the codon adaptation index. Our comparative analysis of substitution rates in introns of lowly and highly expressed genes suggests that natural selection has a larger contribution than mutation to the observed correlation between evolutionary rates and gene expression level in Chlamydomonas.     

Mutational Bias and Translational Selection Shaping the Codon Usage Pattern of Tissue-Specific Genes in Rice

The regulatory mechanisms of determining which genes specifically expressed in which tissues are still not fully elucidated, especially in plants. Using internal correspondence analysis, I first establish that tissue-specific genes exhibit significantly different synonymous codon usage in rice, although this effect is weak. The variability of synonymous codon usage between tissues accounts for 5.62% of the total codon usage variability, which has mainly arisen from the neutral evolutionary forces, such as GC content variation among tissues. Moreover, tissue-specific genes are under differential selective constraints, inferring that natural selection also contributes to the codon usage divergence between tissues. These findings may add further evidence in understanding the differentiation and regulation of tissue-specific gene products in plants.     

Widespread positive selection in synonymous sites of mammalian genes

Evolution of protein sequences is largely governed by purifying selection, with a small fraction of proteins evolving under positive selection. The evolution at synonymous positions in protein-coding genes is not nearly as well understood, with the extent and types of selection remaining, largely, unclear. A statistical test to identify purifying and positive selection at synonymous sites in protein-coding genes was developed. The method compares the rate of evolution at synonymous sites (Ks) to that in intron sequences of the same gene after sampling the aligned intron sequences to mimic the statistical properties of coding sequences. We detected purifying selection at synonymous sites in approximately 28% of the 1,562 analyzed orthologous genes from mouse and rat, and positive selection in approximately 12% of the genes. Thus, the fraction of genes with readily detectable positive selection at synonymous sites is much greater than the fraction of genes with comparable positive selection at nonsynonymous sites, i.e., at the level of the protein sequence. Unlike other genes, the genes with positive selection at synonymous sites showed no correlation between Ks and the rate of evolution in nonsynonymous sites (Ka), indicating that evolution of synonymous sites under positive selection is decoupled from protein evolution. The genes with purifying selection at synonymous sites showed significant anticorrelation between Ks and expression level and breadth, indicating that highly expressed genes evolve slowly. The genes with positive selection at synonymous sites showed the opposite trend, i.e., highly expressed genes had, on average, higher Ks. For the genes with positive selection at synonymous sites, a significantly lower mRNA stability is predicted compared to the genes with negative selection. Thus, mRNA destabilization could be an important factor driving positive selection in nonsynonymous sites, probably, through regulation of expression at the level of mRNA degradation and, possibly, also translation rate. So, unexpectedly, we found that positive selection at synonymous sites of mammalian genes is substantially more common than positive selection at the level of protein sequences. Positive selection at synonymous sites might act through mRNA destabilization affecting mRNA levels and translation.     

Determinants of DNA sequence divergence betweenEscherichia coli andSalmonella typhimurium: Codon usage,map position,and concerted evolution

Summary The nature and extent of DNA sequence divergence between homologous proteincoding genes fromEscherichia coli andSalmonella typhimurium have been examined. The degree of divergence varies greatly among genes at both synonymous (silent) and nonsynonymous sites. Much of the variation in silent substitution rates can be explained by natural selection on synonymous codon usage, varying in intensity with gene expression level. Silent substitution rates also vary significantly with chromosomal location, with genes nearoriC having lower divergence. Certain genes have been examined in more detail. In particular, the duplicate genes encoding elongation factor Tu,tufA andtufB, fromS. typhimurium have been compared to theirE. coli homologues. As expected these very highly expressed genes have high codon usage bias and have diverged very little between the two species. Interestingly, these genes, which are widely spaced on the bacterial chromosome, also appear to be undergoing concerted evolution, i.e., there has been exchange between the loci subsequent to the divergence of the two species.Presented at the NATO Advanced Research Workshop on Genome Organization and Evolution, held in Spetses, Greece, September 1990     

Molecular population genetics of herbivore-induced protease inhibitor genes in European aspen (Populus tremula L., Salicaceae)

Plants defend themselves against the attack of natural enemies by using an array of both constitutively expressed and induced defenses. Long-lived woody perennials are overrepresented among plant species that show strong induced defense responses, whereas annual plants and crop species are underrepresented. However, most studies of plant defense genes have been performed on annual or short-lived perennial weeds or crop species. Here I use molecular population genetic methods to survey six wound-inducible protease inhibitors (PIs) in a long-lived woody, perennial plant species, the European aspen (Populus tremula), to evaluate the likelihood of either recurrent selective sweeps or balancing selection maintaining amino acid polymorphisms in these genes. The results show that none of the six PI genes have reduced diversities at synonymous sites, as would be expected in the presence of recurrent selective sweeps. However, several genes show some evidence of nonneutral evolution such as enhanced linkage disequilibrium and a large number of high-frequency-derived mutations. A group of at least four Kunitz trypsin inhibitor genes appear to have experienced elevated levels of nonsynonymous substitutions, indicating allelic turnover on an evolutionary timescale. One gene, TI1, has enhanced levels of intraspecific polymorphism at nonsynonymous sites and also has an unusual haplotype structure characterized by two divergent haplotypes occurring at roughly equal frequencies in the sample. One haplotype has very low levels of intraallelic nucleotide diversity, whereas the other haplotype has levels of diversity comparable to other genes in P. tremula. Patterns of sequence diversity at TI1 do not fit a simple model of either balancing selection or recurrent selective sweeps. This suggests that selection at TI1 is more complex, possibly involving allelic cycling.     

Patterns of synonymous codon usage in Drosophila melanogaster genes with sex-biased expression

The nonrandom use of synonymous codons (codon bias) is a well-established phenomenon in Drosophila. Recent reports suggest that levels of codon bias differ among genes that are differentially expressed between the sexes, with male-expressed genes showing less codon bias than female-expressed genes. To examine the relationship between sex-biased gene expression and level of codon bias on a genomic scale, we surveyed synonymous codon usage in 7276 D. melanogaster genes that were classified as male-, female-, or non-sex-biased in their expression in microarray experiments. We found that male-biased genes have significantly less codon bias than both female- and non-sex-biased genes. This pattern holds for both germline and somatically expressed genes. Furthermore, we find a significantly negative correlation between level of codon bias and degree of sex-biased expression for male-biased genes. In contrast, female-biased genes do not differ from non-sex-biased genes in their level of codon bias and show a significantly positive correlation between codon bias and degree of sex-biased expression. These observations cannot be explained by differences in chromosomal distribution, mutational processes, recombinational environment, gene length, or absolute expression level among genes of the different expression classes. We propose that the observed codon bias differences result from differences in selection at synonymous and/or linked nonsynonymous sites between genes with male- and female-biased expression.     

Determinants of substitution rates in mammalian genes: expression pattern affects selection intensity but not mutation rate

To determine whether gene expression patterns affect mutation rates and/or selection intensity in mammalian genes, we studied the relationships between substitution rates and tissue distribution of gene expression. For this purpose, we analyzed 2,400 human/rodent and 834 mouse/rat orthologous genes, and we measured (using expressed sequence tag data) their expression patterns in 19 tissues from three development states. We show that substitution rates at nonsynonymous sites are strongly negatively correlated with tissue distribution breadth: almost threefold lower in ubiquitous than in tissue-specific genes. Nonsynonymous substitution rates also vary considerably according to the tissues: the average rate is twofold lower in brain-, muscle-, retina- and neuron-specific genes than in lymphocyte-, lung-, and liver-specific genes. Interestingly, 5' and 3' untranslated regions (UTRs) show exactly the same trend. These results demonstrate that the expression pattern is an essential factor in determining the selective pressure on functional sites in both coding and noncoding regions. Conversely, silent substitution rates do not vary with expression pattern, even in ubiquitously expressed genes. This latter result thus suggests that synonymous codon usage is not constrained by selection in mammals. Furthermore, this result also indicates that there is no reduction of mutation rates in genes expressed in the germ line, contrary to what had been hypothesized based on the fact that transcribed DNA is more efficiently repaired than nontranscribed DNA.     

The Selective Advantage of Synonymous Codon Usage Bias in Salmonella

The genetic code in mRNA is redundant, with 61 sense codons translated into 20 different amino acids. Individual amino acids are encoded by up to six different codons but within codon families some are used more frequently than others. This phenomenon is referred to as synonymous codon usage bias. The genomes of free-living unicellular organisms such as bacteria have an extreme codon usage bias and the degree of bias differs between genes within the same genome. The strong positive correlation between codon usage bias and gene expression levels in many microorganisms is attributed to selection for translational efficiency. However, this putative selective advantage has never been measured in bacteria and theoretical estimates vary widely. By systematically exchanging optimal codons for synonymous codons in the tuf genes we quantified the selective advantage of biased codon usage in highly expressed genes to be in the range 0.2–4.2 x 10⁻⁴ per codon per generation. These data quantify for the first time the potential for selection on synonymous codon choice to drive genome-wide sequence evolution in bacteria, and in particular to optimize the sequences of highly expressed genes. This quantification may have predictive applications in the design of synthetic genes and for heterologous gene expression in biotechnology.     

Selection on Silent Sites in the Rodent H3 Histone Gene Family

Selection promoting differential use of synonymous codons has been shown for several unicellular organisms and for Drosophila, but not for mammals. Selection coefficients operating on synonymous codons are likely to be extremely small, so that a very large effective population size is required for selection to overcome the effects of drift. In mammals, codon-usage bias is believed to be determined exclusively by mutation pressure, with differences between genes due to large-scale variation in base composition around the genome. The replication-dependent histone genes are expressed at extremely high levels during periods of DNA synthesis, and thus are among the most likely mammalian genes to be affected by selection on synonymous codon usage. We suggest that the extremely biased pattern of codon usage in the H3 genes is determined in part by selection. Silent site G + C content is much higher than expected based on flanking sequence G + C content, compared to other rodent genes with similar silent site base composition but lower levels of expression. Dinucleotide-mediated mutation bias does affect codon usage, but the affect is limited to the choice between G and C in some fourfold degenerate codons. Gene conversion between the two clusters of histone genes has not been an important force in the evolution of the H3 genes, but gene conversion appears to have had some effect within the cluster on chromosome 13.     

Codon usage in regulatory genes in Escherichia coli does not reflect selection for ''rare'' codons.

It has often been suggested that differential usage of codons recognized by rare tRNA species, i.e. "rare codons", represents an evolutionary strategy to modulate gene expression. In particular, regulatory genes are reported to have an extraordinarily high frequency of rare codons. From E. coli we have compiled codon usage data for highly expressed genes, moderately/lowly expressed genes, and regulatory genes. We have identified a clear and general trend in codon usage bias, from the very high bias seen in very highly expressed genes and attributed to selection, to a rather low bias in other genes which seems to be more influenced by mutation than by selection. There is no clear tendency for an increased frequency of rare codons in the regulatory genes, compared to a large group of other moderately/lowly expressed genes with low codon bias. From this, as well as a consideration of evolutionary rates of regulatory genes, and of experimental data on translation rates, we conclude that the pattern of synonymous codon usage in regulatory genes reflects primarily the relaxation of natural selection.     

Selection on Synonymous Sites for Increased Accessibility around miRNA Binding Sites in Plants

Synonymous codons are widely selected for various biological mechanisms in both prokaryotes and eukaryotes. Recent evidence suggests that microRNA (miRNA) function may affect synonymous codon choices near miRNA target sites. To better understand this, we perform genome-wide analysis on synonymous codon usage around miRNA target sites in four plant genomes. We observed a general trend of increased site accessibility around miRNA target sites in plants. Guanine-cytosine (GC)-poor codons are preferred in the flank region of miRNA target sites. Within-genome analyses show significant variation among miRNA targets in species. GC content of the target gene can partly explain the variation of site accessibility among miRNA targets. miRNA targets in GC-rich genes show stronger selection signals than those in GC-poor genes. Gene's codon usage bias and the conservation level of miRNA and its target also have some effects on site accessibility, but the expression level of miRNA or its target and the mechanism of miRNA activity do not contribute to site accessibility differences among miRNA targets. We suggest that synonymous codons near miRNA targets are selected for efficient miRNA binding and proper miRNA function. Our results present a new dimension of natural selection on synonymous codons near miRNA target sites in plants, which will have important implications of coding sequence evolution.     

Gene expression intensity shapes evolutionary rates of the proteins encoded by the vertebrate genome

Natural selection leaves its footprints on protein-coding sequences by modulating their silent and replacement evolutionary rates. In highly expressed genes in invertebrates, these footprints are seen in the higher codon usage bias and lower synonymous divergence. In mammals, the highly expressed genes have a shorter gene length in the genome and the breadth of expression is known to constrain the rate of protein evolution. Here we have examined how the rates of evolution of proteins encoded by the vertebrate genomes are modulated by the amount (intensity) of gene expression. To understand how natural selection operates on proteins that appear to have arisen in earlier and later phases of animal evolution, we have contrasted patterns of mouse proteins that have homologs in invertebrate and protist genomes (Precambrian genes) with those that do not have such detectable homologs (vertebrate-specific genes). We find that the intensity of gene expression relates inversely to the rate of protein sequence evolution on a genomic scale. The most highly expressed genes actually show the lowest total number of substitutions per polypeptide, consistent with cumulative effects of purifying selection on individual amino acid replacements. Precambrian genes exhibit a more pronounced difference in protein evolutionary rates (up to three times) between the genes with high and low expression levels as compared to the vertebrate-specific genes, which appears to be due to the narrower breadth of expression of the vertebrate-specific genes. These results provide insights into the differential relationship and effect of the increasing complexity of animal body form on evolutionary rates of proteins.     

Coalescent processes and relaxation of selective constraints leading to contrasting genetic diversity at paralogs AtHVA22d and AtHVA22e in Arabidopsis thaliana

Duplicate loci offer a very powerful system for understanding the complicated genome structure and adaptive evolution of a gene family. In this study, the genetic variation at paralogs AtHVA22d and AtHVA22e, members of an ABA- and stress-inducible gene family, is examined in the selfing Arabidopsis thaliana. Population genetic analysis indicates contrasting levels of nucleotide diversity at overall exon sequence and nonsynonymous sites between AtHVA22d (pi = 0.00337, pi(rep) = 0.00158) and AtHVA22e (pi = 0.00054, pi(rep) = 0.00023). The fact of Ka/Ks ratios significantly less than 1 in all sequences indicates that both genes are functional and subjected to purifying selection. In addition, rooted at barley HVA22, accelerated evolution is detected at replacement changes in the AtHVA22d locus, indicating relaxation of purifying selection after gene duplication. However, relative rate tests reveal no deviation from the neutrality at synonymous sites between the two paralogs. Based on clock-like evolution, the rate of synonymous substitution is estimated at 1.83 x 10(-9) substitutions per site per year; and the divergence of the two paralogs is traced to 90 MYA, coinciding with a period of the diversification of angiosperms. Given no codon usage bias in both genes, natural selection alone cannot account for the 6.4-fold differences in the nucleotide variation at synonymous sites between the two paralogs. Random processes resulting in different coalescence times, 3.65 MYA at AtHVA22d vs. 1.20 MYA at AtHVA22e, may have predominantly contributed to the evident differences of the genetic diversity. Partially nonoverlapping modes of expression between the two functional paralogs suggest a subfunctionalization hypothesis for explaining the fates of duplicate loci.     

No evidence for tissue-specific adaptation of synonymous codon usage in humans

It has been proposed that the synonymous codon usage of human tissue-specific genes was under selective pressure to modulate the expression of proteins by codon-mediated translational control (Plotkin, J. B., H. Robins, and A. J. Levine. 2004. Tissue-specific codon usage and the expression of human genes. Proc. Natl. Acad. Sci. USA 101:12588-12591.) To test this model, we analyzed by internal correspondence analysis the codon usage of 2,126 human tissue-specific genes expressed in 18 different tissues. We confirm that synonymous codon usage differs significantly between the tissues. However, the effect is very weak: the variability of synonymous codon usage between tissues represents only 2.3% of the total codon usage variability. Moreover, this variability is directly linked to isochore-scale (>100 kb) variability of GC-content that affect both coding and introns or intergenic regions. This demonstrates that variations of synonymous codon usage between tissue-specific genes expressed in different tissues are due to regional variations of substitution patterns and not to translational selection.