Analysis of mitochondrial protein‐coding genes of Antheraea assamensis: Muga silkworm of Assam

To understand the synonymous codon usage pattern in mitochondrial genome of Antheraea assamensis, we analyzed the 13 mitochondrial protein‐coding genes of this species using a bioinformatic approach as no work was reported yet. The nucleotide composition analysis suggested that the percentages of A, T, G,and C were 33.73, 46.39, 9.7 and 10.17, respectively and the overall GC content was 19.86, that is, lower than 50% and the genes were AT rich. The mean effective number of codons of mitochondrial protein‐coding genes was 36.30 and it indicated low codon usage bias (CUB). Relative synonymous codon usage analysis suggested overrepresented and underrepresented codons in each gene and the pattern of codon usage was different among genes. Neutrality plot analysis revealed a narrow range of distribution for GC content at the third codon position and some points were diagonally distributed, suggesting both mutation pressure and natural selection influenced the CUB.     

An evolutionary perspective on synonymous codon usage in unicellular organisms

Summary Observed patterns of synonymous codon usage are explained in terms of the joint effects of mutation, selection, and random drift. Examination of the codon usage in 165Escherichia coli genes reveals a consistent trend of increasing bias with increasing gene expression level. Selection on codon usage appears to be unidirectional, so that the pattern seen in lowly expressed genes is best explained in terms of an absence of strong selection. A measure of directional synonymous-codon usage bias, the Codon Adaptation Index, has been developed. In enterobacteria, rates of synonymous substitution are seen to vary greatly among genes, and genes with a high codon bias evolve more slowly. A theoretical study shows that the patterns of extreme codon bias observed for someE. coli (and yeast) genes can be generated by rather small selective differences. The relative plausibilities of various theoretical models for explaining nonrandom codon usage are discussed.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Evidence for purifying selection against synonymous mutations in mammalian exonic splicing enhancers

Silent sites in mammals have classically been assumed to be free from selective pressures. Consequently, the synonymous substitution rate (Ks) is often used as a proxy for the mutation rate. Although accumulating evidence demonstrates that the assumption is not valid, the mechanism by which selection acts remain unclear. Recent work has revealed that the presence of exonic splicing enhancers (ESEs) in coding sequence might influence synonymous evolution. ESEs are predominantly located near intron-exon junctions, which may explain the reduced single-nucleotide polymorphism (SNP) density in these regions. Here we show that synonymous sites in putative ESEs evolve more slowly than the remaining exonic sequence. Differential mutabilities of ESEs do not appear to explain this difference. We observe that substitution frequency at fourfold synonymous sites decreases as one approaches the ends of exons, consistent with the existing SNP data. This gradient is at least in part explained by ESEs being more abundant near junctions. Between-gene variation in Ks is hence partly explained by the proportion of the gene that acts as an ESE. Given the relative abundance of ESEs and the reduced rates of synonymous divergence within them, we estimate that constraints on synonymous evolution within ESEs causes the true mutation rate to be underestimated by not more than approximately 8%. We also find that Ks outside of ESEs is much lower in alternatively spliced exons than in constitutive exons, implying that other causes of selection on synonymous mutations exist. Additionally, selection on ESEs appears to affect nonsynonymous sites and may explain why amino acid usage near intron-exon junctions is nonrandom.     

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.     

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.     

Codon usage in mitochondrial genomes: distinguishing context-dependent mutation from translational selection

We analyze the frequencies of synonymous codons in animal mitochondrial genomes, focusing particularly on mammals and fish. The frequencies of bases at 4-fold degenerate sites are found to be strongly influenced by context-dependent mutation, which causes correlations between pairs of neighboring bases. There is a pattern of excess of certain dinucleotides and deficit of others that is consistent across large numbers of species, despite the wide variation of single-nucleotide frequencies among species. In many bacteria, translational selection is an important influence on codon usage. In order to test whether translational selection also plays a role in mitochondria, we need to control for context-dependent mutation. Selection for translational accuracy can be detected by comparison of codon usage in conserved and variable sites in the same genes. We give a test of this type that works in the presence of context-dependent mutation. There is very little evidence for translational accuracy selection in the mitochondrial genes considered here. Selection for translational efficiency might lead to preference for codons that match the limited repertoire of anticodons on the mitochondrial tRNAs. This is difficult to detect because the effect would usually be in the same direction in comparable to codon families and so would not cause an observable difference in codon usage between families. Several lines of evidence suggest that this type of selection is weak in most cases. However, we found several cases where unusual bases occur at the wobble position of the tRNA, and in these cases, some evidence for selection on codon usage was found. We discuss the way that these unusual cases are associated with codon reassignments in the mitochondrial genetic code.     

The Effect of Mutation and Selection on Codon Adaptation in Escherichia coli Bacteriophage

Studying phage codon adaptation is important not only for understanding the process of translation elongation, but also for reengineering phages for medical and industrial purposes. To evaluate the effect of mutation and selection on phage codon usage, we developed an index to measure selection imposed by host translation machinery, based on the difference in codon usage between all host genes and highly expressed host genes. We developed linear and nonlinear models to estimate the C→T mutation bias in different phage lineages and to evaluate the relative effect of mutation and host selection on phage codon usage. C→T-biased mutations occur more frequently in single-stranded DNA (ssDNA) phages than in double-stranded DNA (dsDNA) phages and affect not only synonymous codon usage, but also nonsynonymous substitutions at second codon positions, especially in ssDNA phages. The host translation machinery affects codon adaptation in both dsDNA and ssDNA phages, with a stronger effect on dsDNA phages than on ssDNA phages. Strand asymmetry with the associated local variation in mutation bias can significantly interfere with codon adaptation in both dsDNA and ssDNA phages.     

Evolution in Hawaiian cave-adapted isopods (Oniscidea: Philosciidae): vicariant speciation or adaptive shifts?

Diatom plastid genes are examined with respect to codon adaptation and rates of silent substitution (Ks). It is shown that diatom genes follow the same pattern of codon usage as other plastid genes studied previously. Highly expressed diatom genes display codon adaptation, or a bias toward specific major codons, and these major codons are the same as those in red algae, green algae, and land plants. It is also found that there is a strong correlation between Ks and variation in codon adaptation across diatom genes, providing the first evidence for such a relationship in the algae. It is argued that this finding supports the notion that the correlation arises from selective constraints, not from variation in mutation rate among genes. Finally, the diatom genes are examined with respect to variation in Ks among different synonymous groups. Diatom genes with strong codon adaptation do not show the same variation in synonymous substitution rate among codon groups as the flowering plant psbA gene which, previous studies have shown, has strong codon adaptation but unusually high rates of silent change in certain synonymous groups. The lack of a similar finding in diatoms supports the suggestion that the feature is unique to the flowering plant psbA due to recent relaxations in selective pressure in that lineage.     

The proportion of all point mutations which are unacceptable: an estimate based on hemoglobin amino acid and nucleotide sequences.

Statistical analysis of the distribution of 156 kinds of human hemoglobin beta (Hbbeta) chain variants suggests that mutations are essentially random in their location. Thus differential fitness, not differential mutability, is the principal source of nonrandom distribution of interspecies differences in Hbbeta amino acid sequence. Similar analyses of both the location and the kind of interspecies differences detected among primates support this viewpoint and lead us to estimate that at least 95% of all amino acid subsitutions,i.e., nonsynonymous mutations, in Hbbeta are functionally unacceptable in homozygous state. Through the combined use of this estimate and the number of nonsynonymous and synonymous substitutions per nucleotide site inferred from comparisons of entire human and rabbit HbbetamRNA nucleotide sequences, we calculate (a) approximately 70% of synonymous Hbbeta mutations are adaptively undersirable and (b) the mutation rate underlying all changes is lesser than or equal to 10(-8) nucleotide substitutions per nucleotide site per year. Apart from such calculations, analyses of nucleotide patterns in HbbetamRNA as well as in rat preproinsulin mRNA reinforce the notion that a large portion of synonymous mutations are functionally unacceptable and rendered so by selective constraint, at a pretranslational level, of the abundance of particular nucleotide doublets such as CpG.     

Mutation bias is the driving force of codon usage in the Gallus gallus genome

Synonymous codons are used with different frequencies both among species and among genes within the same genome and are controlled by neutral processes (such as mutation and drift) as well as by selection. Up to now, a systematic examination of the codon usage for the chicken genome has not been performed. Here, we carried out a whole genome analysis of the chicken genome by the use of the relative synonymous codon usage (RSCU) method and identified 11 putative optimal codons, all of them ending with uracil (U), which is significantly departing from the pattern observed in other eukaryotes. Optimal codons in the chicken genome are most likely the ones corresponding to highly expressed transfer RNA (tRNAs) or tRNA gene copy numbers in the cell. Codon bias, measured as the frequency of optimal codons (Fop), is negatively correlated with the G + C content, recombination rate, but positively correlated with gene expression, protein length, gene length and intron length. The positive correlation between codon bias and protein, gene and intron length is quite different from other multi-cellular organism, as this trend has been only found in unicellular organisms. Our data displayed that regional G + C content explains a large proportion of the variance of codon bias in chicken. Stepwise selection model analyses indicate that G + C content of coding sequence is the most important factor for codon bias. It appears that variation in the G + C content of CDSs accounts for over 60% of the variation of codon bias. This study suggests that both mutation bias and selection contribute to codon bias. However, mutation bias is the driving force of the codon usage in the Gallus gallus genome. Our data also provide evidence that the negative correlation between codon bias and recombination rates in G. gallus is determined mostly by recombination-dependent mutational patterns.     

Synonymous codon bias is not caused by mutation bias in G+C-rich genes in humans

It is has been suggested that synonymous codon bias is a consequence of mutation bias in mammals. We tested this hypothesis in humans using single-nucleotide polymorphism data. We found a pattern of polymorphism which was inconsistent with the mutation bias hypothesis in G+C-rich genes. However, the data were consistent with the action of natural selection or biased gene conversion. Similar patterns of polymorphism were also observed in noncoding DNA, suggesting that natural selection or biased gene conversion may affect large tracts of the human genome.     

Synonymous and nonsynonymous rate variation in nuclear genes of mammals

A maximum likelihood approach was used to estimate the synonymous and nonsynonymous substitution rates in 48 nuclear genes from primates, artiodactyls, and rodents. A codon-substitution model was assumed, which accounts for the genetic code structure, transition/transversion bias, and base frequency biases at codon positions. Likelihood ratio tests were applied to test the constancy of nonsynonymous to synonymous rate ratios among branches (evolutionary lineages). It is found that at 22 of the 48 nuclear loci examined, the nonsynonymous/synonymous rate ratio varies significantly across branches of the tree. The result provides strong evidence against a strictly neutral model of molecular evolution. Our likelihood estimates of synonymous and nonsynonymous rates differ considerably from previous results obtained from approximate pairwise sequence comparisons. The differences between the methods are explored by detailed analyses of data from several genes. Transition/transversion rate bias and codon frequency biases are found to have significant effects on the estimation of synonymous and nonsynonymous rates, and approximate methods do not adequately account for those factors. The likelihood approach is preferable, even for pairwise sequence comparison, because more-realistic models about the mutation and substitution processes can be incorporated in the analysis. Received: 17 May 1997 / Accepted: 28 September 1997     

Substitution rates in Drosophila nuclear genes: implications for translational selection

The relationships between synonymous and nonsynonymous substitution rates and between synonymous rate and codon usage bias are important to our understanding of the roles of mutation and selection in the evolution of Drosophila genes. Previous studies used approximate estimation methods that ignore codon bias. In this study we reexamine those relationships using maximum-likelihood methods to estimate substitution rates, which accommodate the transition/transversion rate bias and codon usage bias. We compiled a sample of homologous DNA sequences at 83 nuclear loci from Drosophila melanogaster and at least one other species of Drosophila. Our analysis was consistent with previous studies in finding that synonymous rates were positively correlated with nonsynonymous rates. Our analysis differed from previous studies, however, in that synonymous rates were unrelated to codon bias. We therefore conducted a simulation study to investigate the differences between approaches. The results suggested that failure to properly account for multiple substitutions at the same site and for biased codon usage by approximate methods can lead to an artifactual correlation between synonymous rate and codon bias. Implications of the results for translational selection are discussed.     

An Evaluation of Measures of Synonymous Codon Usage Bias

Synonymous codons are not generally used at equal frequencies, and this trend is observed for most genes and organisms. Several methods have been proposed and used to estimate the degree of the nonrandom use of the different synonymous codons. The estimates obtained by these methods, however, show different levels of both precision and dispersion when coding regions of a finite number of codons are under analysis. Here, we present a study, based on computer simulation, of how the different methods proposed to evaluate the nonrandom use of synonymous codons are affected by the length of the coding region analyzed. The results show that some of these methods are heavily influenced by the number of codons and that the comparison of codon usage bias between coding regions of different lengths shows a methodological bias under different conditions of nonrandom use of synonymous codons. The study of the dispersion of the estimates obtained by the different methods gives, on the other hand, an indication of the methods to be applied to compare values of codon usage bias among coding regions of equivalent length. Received: 10 September 1997 / Accepted: 23 March 1998     

Synonymous substitution-rate constants in Escherichia coli and Salmonella typhimurium and their relationship to gene expression and selection pressure

Based on the differences in synonymous codon use between E. coli and S. typhimurium, the synonymous substitution rates can be estimated. In contrast to previous studies on the substitution rates in these two organisms, we use a kinetic model that explicitly takes the selection bias into account. The selection pressure on synonymous codons for a particular amino acid can be calculated from the observed codon bias. This offers a unique opportunity to study systematically the relationship between substitution-rate constants and selection pressure. The results indicate that the codon bias in these organisms is determined by a mutation-selection balance rather than by stabilizing selection. A best fit to the data implies that the mutation rate constant increases about threefold in genes at low expression levels relative to those that are highly expressed.Correspondence to: O.G. Berg     

The genomic rate of adaptive amino acid substitution in Drosophila

The proportion of amino acid substitutions driven by adaptive evolution can potentially be estimated from polymorphism and divergence data by an extension of the McDonald-Kreitman test. We have developed a maximum-likelihood method to do this and have applied our method to several data sets from three Drosophila species: D. melanogaster, D. simulans, and D. yakuba. The estimated number of adaptive substitutions per codon is not uniformly distributed among genes, but follows a leptokurtic distribution. However, the proportion of amino acid substitutions fixed by adaptive evolution seems to be remarkably constant across the genome (i.e., the proportion of amino acid substitutions that are adaptive appears to be the same in fast-evolving and slow-evolving genes; fast-evolving genes have higher numbers of both adaptive and neutral substitutions). Our estimates do not seem to be significantly biased by selection on synonymous codon use or by the assumption of independence among sites. Nevertheless, an accurate estimate is hampered by the existence of slightly deleterious mutations and variations in effective population size. The analysis of several Drosophila data sets suggests that approximately 25% +/- 20% of amino acid substitutions were driven by positive selection in the divergence between D. simulans and D. yakuba.     

Inference of site frequency spectra from high-throughput sequence data: quantification of selection on nonsynonymous and synonymous sites in humans

Sequencing errors and random sampling of nucleotide types among sequencing reads at heterozygous sites present challenges for accurate, unbiased inference of single-nucleotide polymorphism genotypes from high-throughput sequence data. Here, we develop a maximum-likelihood approach to estimate the frequency distribution of the number of alleles in a sample of individuals (the site frequency spectrum), using high-throughput sequence data. Our method assumes binomial sampling of nucleotide types in heterozygotes and random sequencing error. By simulations, we show that close to unbiased estimates of the site frequency spectrum can be obtained if the error rate per base read does not exceed the population nucleotide diversity. We also show that these estimates are reasonably robust if errors are nonrandom. We then apply the method to infer site frequency spectra for zerofold degenerate, fourfold degenerate, and intronic sites of protein-coding genes using the low coverage human sequence data produced by the 1000 Genomes Project phase-one pilot. By fitting a model to the inferred site frequency spectra that estimates parameters of the distribution of fitness effects of new mutations, we find evidence for significant natural selection operating on fourfold sites. We also find that a model with variable effects of mutations at synonymous sites fits the data significantly better than a model with equal mutational effects. Under the variable effects model, we infer that 11% of synonymous mutations are subject to strong purifying selection.     

A low rate of simultaneous double-nucleotide mutations in primates

The occurrence of double-nucleotide (doublet) mutations is contrary to the normal assumption that point mutations affect single nucleotides. Here we develop a new method for estimating the doublet mutation rate and apply it to more than a megabase of human-chimpanzee-baboon genomic DNA alignments and more than a million human single-nucleotide polymorphisms. The new method accounts for the effect of regional variation in evolutionary rates, which may be a confounding factor in previous estimates of the doublet mutation rate. Furthermore we determine sequence context effects by using sequence comparisons over a variety of lineage lengths. This approach yields a new estimate of the doublet mutation rate of 0.3% of the singleton rate, indicating that doublet mutations are far rarer than previously thought. Our results suggest that doublet mutations are unlikely to have caused the correlation between synonymous and nonsynonymous substitution rates in mammals, and also show that regional variation and sequence context effects play an important role in primate DNA sequence evolution.     

Codon Substitution in Evolution and the "Saturation" of Synonymous Changes

A mathematical model for codon substitution is presented, taking into account unequal mutation rates among different nucleotides and purifying selection. This model is constructed by using a 61 X 61 transition probability matrix for the 61 nonterminating codons. Under this model, a computer simulation is conducted to study the numbers of silent (synonymous) and amino acid-altering (nonsynonymous) nucleotide substitutions when the underlying mutation rates among the four kinds of nucleotides are not equal. It is assumed that the substitution rates are constant over evolutionary time, the codon frequencies being in equilibrium, and, thus, the numbers of synonymous and nonsynonymous substitutions both increase linearly with evolutionary time. It is shown that, when the mutation rates are not equal, the estimate of synonymous substitutions obtained by F. Perler, A. Efstratiadis, P. Lomedico, W. Gilbert, R. Kolodner and J. Dodgson's "Percent Corrected Divergence" method increases nonlinearly, although the true number of synonymous substitutions increases linearly. It is, therefore, possible that the "saturation" of synonymous substitutions observed by Perler et al. is due to the inefficiency of their method to detect all synonymous substitutions.     

Genome-wide selection on codon usage at the population level in the fungal model organism Neurospora crassa

Many organisms exhibit biased codon usage in their genome, including the fungal model organism Neurospora crassa. The preferential use of subset of synonymous codons (optimal codons) at the macroevolutionary level is believed to result from a history of selection to promote translational efficiency. At present, few data are available about selection on optimal codons at the microevolutionary scale, that is, at the population level. Herein, we conducted a large-scale assessment of codon mutations at biallelic sites, spanning more than 5,100 genes, in 2 distinct populations of N. crassa: the Caribbean and Louisiana populations. Based on analysis of the frequency spectra of synonymous codon mutations at biallelic sites, we found that derived (nonancestral) optimal codon mutations segregate at a higher frequency than derived nonoptimal codon mutations in each population; this is consistent with natural selection favoring optimal codons. We also report that optimal codon variants were less frequent in longer genes and that the fixation of optimal codons was reduced in rapidly evolving long genes/proteins, trends suggestive of genetic hitchhiking (Hill-Robertson) altering codon usage variation. Notably, nonsynonymous codon mutations segregated at a lower frequency than synonymous nonoptimal codon mutations (which impair translational efficiency) in each N. crassa population, suggesting that changes in protein composition are more detrimental to fitness than mutations altering translation. Overall, the present data demonstrate that selection, and partly genetic interference, shapes codon variation across the genome in N. crassa populations.