Synonymous and Nonsynonymous Substitutions in Genes from Gramineae: Intragenic Correlations

In this work, we have investigated the relationships between synonymous and nonsynonymous rates and base composition in coding sequences from Gramineae to analyze the factors underlying the variation in substitutional rates. We have shown that in these genes the rates of nucleotide divergence, both synonymous and nonsynonymous, are, to some extent, dependent on each other and on the base composition. In the first place, the variation in nonsynonymous rate is related to the GC level at the second codon position (the higher the GC₂ level, the higher the amino acid replacement rate). The correlation is especially strong with T₂, the coefficients being significant in the three data sets analyzed. This correlation between nonsynonymous rate and base composition at the second codon position is also detectable at the intragenic level, which implies that the factors that tend to increase the intergenic variance in nonsynonymous rates also affect the intragenic variance. On the other hand, we have shown that the synonymous rate is strongly correlated with the GC₃ level. This correlation is observed both across genes and at the intragenic level. Similarly, the nonsynonymous rate is also affected at the intragenic level by GC₃ level, like the silent rate. In fact, synonymous and nonsynonymous rates exhibit a parallel behavior in relation to GC₃ level, indicating that the intragenic patterns of both silent and amino acid divergence rates are influenced in a similar way by the intragenic variation of GC₃. This result, taken together with the fact that the number of genes displaying intragenic correlation coefficients between synonymous and nonsynonymous rates is not very high, but higher than random expectation (in the three data sets analyzed), strongly suggests that the processes of silent and amino acid replacement divergence are, at least in part, driven by common evolutionary forces in genes from Gramineae. Received: 2 July 1998 / Accepted: 18 April 1999     

Synonymous substitution rates in Drosophila: Mitochondrial versus nuclear genes

Synonymous substitution rates in mitochondrial and nuclear genes of Drosophila were compared. To make accurate comparisons, we considered the following: (1) relative synonymous rates, which do not require divergence time estimates, should be used; (2) methods estimating divergence should take into account base composition; (3) only very closely related species should be used to avoid effects of saturation; (4) the heterogeneity of rates should be examined. We modified the methods estimating synonymous substitution numbers to account for base composition bias. By using these methods, we found that mitochondrial genes have 1.7–3.4 times higher synonymous substitution rates than the fastest nuclear genes or 4.5–9.0 times higher rates than the average nuclear genes. The average rate of synonymous transversions was 2.7 (estimated from the melanogaster species subgroup) or 2.9 (estimated from the obscura group) times higher in mitochondrial genes than in nuclear genes. Synonymous transversions in mitochondrial genes occurred at an approximately equivalent rate to those in the fastest nuclear genes. This last result is not consistent with the hypothesis that the difference in turnover rates between mitochondrial and nuclear genomes is the major factor determining higher synonymous substitution rates in mtDNA. We conclude that the difference in synonymous substitution rates is due to a combination of two factors: a higher transitional mutation rate in mtDNA and constraints on nuclear genes due to selection for codon usage. Received: 27 November 1996 / Accepted: 8 May 1997     

Evolutionary Lability of Context-Dependent Codon Bias in Bacteria

In bacteria, synonymous codon usage can be considerably affected by base composition at neighboring sites. Such context-dependent biases may be caused by either selection against specific nucleotide motifs or context-dependent mutation biases. Here we consider the evolutionary conservation of context-dependent codon bias across 11 completely sequenced bacterial genomes. In particular, we focus on two contextual biases previously identified in Escherichia coli; the avoidance of out-of-frame stop codons and AGG motifs. By identifying homologues of E. coli genes, we also investigate the effect of gene expression level in Haemophilus influenzae and Mycoplasma genitalium. We find that while context-dependent codon biases are widespread in bacteria, few are conserved across all species considered. Avoidance of out-of-frame stop codons does not apply to all stop codons or amino acids in E. coli, does not hold for different species, does not increase with gene expression level, and is not relaxed in Mycoplasma spp., in which the canonical stop codon, TGA, is recognized as tryptophan. Avoidance of AGG motifs shows some evolutionary conservation and increases with gene expression level in E. coli, suggestive of the action of selection, but the cause of the bias differs between species. These results demonstrate that strong context-dependent forces, both selective and mutational, operate on synonymous codon usage but that these differ considerably between genomes. Received: 6 May 1999 / Accepted: 29 October 1999     

Why are translationally sub-optimal synonymous codons used in Escherichia coli?

Natural selection favors certain synonymous codons which aid translation in Escherichia coli, yet codons not favored by translational selection persist. We use the frequency distributions of synonymous polymorphisms to test three hypotheses for the existence of translationally sub-optimal codons: (1) selection is a relatively weak force, so there is a balance between mutation, selection, and drift; (2) at some sites there is no selection on codon usage, so some synonymous sites are unaffected by translational selection; and (3) translationally sub-optimal codons are favored by alternative selection pressures at certain synonymous sites. We find that when all the data is considered, model 1 is supported and both models 2 and 3 are rejected as sole explanations for the existence of translationally sub-optimal codons. However, we find evidence in favor of both models 2 and 3 when the data is partitioned between groups of amino acids and between regions of the genes. Thus, all three mechanisms appear to contribute to the existence of translationally sub-optimal codons in E. coli. Received: 18 July 2000 / Accepted: 17 April 2001     

Coding sequence divergence between two closely related plant species: Arabidopsis thaliana and Brassica rapa ssp. pekinensis

To characterize the coding-sequence divergence of closely related genomes, we compared DNA sequence divergence between sequences from a Brassica rapa ssp. pekinensis EST library isolated from flower buds and genomic sequences from Arabidopsis thaliana. The specific objectives were (i) to determine the distribution of and relationship between K _a and K _s, (ii) to identify genes with the lowest and highest K _a:K _s values, and (iii) to evaluate how codon usage has diverged between two closely related species. We found that the distribution of K _a:K _s was unimodal, and that substitution rates were more variable at nonsynonymous than synonymous sites, and detected no evidence that K _a and K _s were positively correlated. Several genes had K _a:K _s values equal to or near zero, as expected for genes that have evolved under strong selective constraint. In contrast, there were no genes with K _a:K _s >1 and thus we found no strong evidence that any of the 218 sequences we analyzed have evolved in response to positive selection. We detected a stronger codon bias but a lower frequency of GC at synonymous sites in A. thaliana than B. rapa. Moreover, there has been a shift in the profile of most commonly used synonymous codons since these two species diverged from one another. This shift in codon usage may have been caused by stronger selection acting on codon usage or by a shift in the direction of mutational bias in the B. rapa phylogenetic lineage.     

Codon Bias and Mutability in HIV Sequences

A survey of the patterns of synonymous codon preference in the HIV env gene reveals a correlation between the codon bias and the mutability requirements of different regions of the protein. At hypervariable regions in gp120 one finds a greater proportion of codons that tend to mutate nonsynonymously, but to a target that is similar in hydrophobicity and volume. We argue that this strategy results from a compromise between the selective pressure placed on the virus by the induced immune response, which favors amino acid substitutions in the complementarity determining regions, and the negative selection against missense mutations that violate structural constraints of the env protein. Received: 9 June 1997 / Accepted: 25 May 1998     

The Nonrandom Location of Synonymous Codons Suggests That Reading Frame-Independent Forces Have Patterned Codon Preferences

Biased codon usage is common in eukaryotic and prokaryotic genes. Evidence from Escherichia, Saccharomyces, and Drosophila indicates that it favors translational efficiency and accuracy. However, to date no functional advantages have been identified in the codon–anticodon interactions involving the most frequently used (preferred) codons. Here we present evidence that forces not related to the individual codon–anticodon interaction may be involved in determining which synonymous codons are preferred or avoided. We show that the ``off-frame' trinucleotide motif preferences inferrable from Drosophila coding regions are often in the same direction as Drosophila's ``in-frame' codon preferences, i.e., its codon usage. The off-frame preferences were inferred from the nonrandomness of the location of confamilial synonymous codons along coding regions—a pattern often described as a context dependence of nucleotide choice at synonymous positions or as codon-pair bias. We relied on randomizations of the location of confamilial codons that do not alter, and cannot be influenced by, the encoded amino acid sequences, codon usage, or base composition of the genes examined. The statistically significant congruency of in-frame and off-frame trinucleotide preferences suggests that the same kind of reading-frame-independent force(s) may also influence synonymous codon choice. These forces may have produced biases in codon usage that then led to the evolution of the translational advantages of these motifs as preferred codons. Under this scenario, tRNA pool size differences between preferred and nonpreferred codons initially were evolved to track the default overrepresentation of codons with preferred motifs. The motif preference hypothesis can explain the structuring of codon preferences and the similarities in the codon usages of distantly related organisms. Received: 10 November 1998 / Accepted: 23 February 1999     

Estimation of the Transition/Transversion Rate Bias and Species Sampling

The transition/transversion (ti/tv) rate ratios are estimated by pairwise sequence comparison and joint likelihood analysis using mitochondrial cytochrome b genes of 28 primate species, representing both the Strepsirrhini (lemurs and lories) and the Anthropoidea (monkeys, apes, and humans). Pairwise comparison reveals a strong negative correlation between estimates of the ti/tv ratio and the sequence distance, even when both are corrected for multiple substitutions. The maximum-likelihood estimate of the ti/tv ratio changes with the species included in the analysis. The ti/tv bias within the lemuriform taxa is found to be as strong as in the anthropoids, in contradiction to an earlier study which sampled only one lemuriform. Simulations show the surprising result that both the pairwise correction method and the joint likelihood analysis tend to overcorrect for multiple substitutions and overestimate the ti/tv ratio, especially at low sequence divergence. The bias, however, is not large enough to account for the observed patterns. Nucleotide frequency biases, variation of substitution rates among sites, and different evolutionary dynamics at the three codon positions can be ruled out as possible causes. The likelihood-ratio test suggests that the ti/tv rate ratios may be variable among evolutionary lineages. Without any biological evidence for such a variation, however, we are left with no plausible explanations for the observed patterns other than a possible saturation effect due to the unrealistic nature of the model assumed. Received: 1 October 1997 / Accepted: 29 September 1998     

Limitations of compositional approach to identifying horizontally transferred genes

Genes with atypical G+C content and pattern of codon usage in a certain genome are possibly of exotic origin, and this idea has been applied to identify horizontal events. In this way, it was postulated that a total of 755 genes in the E. coli genome are relics of horizontal events after the divergence of E. coli from the Salmonella lineage 100 million years ago (Lawrence and Ochman, 1998). In this paper we propose a new way to study sequence composition more thoroughly. We found that although the 755 genes differ in composition from other genes in the E. coli genome, the difference is minor. If we accepted that these genes are horizontally transferred, then (1) it would be more likely that they were transferred from genomes evolutionarily closely related to E. coli; but (2) the dating method used by Lawrence and Ochman (1997, 1998) largely underestimated the average age of introduced sequences in the E. coli genome, in particular, most of the 755 genes should be introduced into E. coli before, instead of after, the divergence of E. coli from the Salmonella lineage. Our study reveals that atypical G+C content and pattern of codon usage are not reliable indicators of horizontal gene transfer events. Received: 27 September 2000 / Accepted: 9 April 2001     

Multiple Substitutions Affect the Phylogenetic Utility of Cytochrome b and 12S rDNA Data: Examining a Rapid Radiation in Leporid (Lagomorpha) Evolution

Partial sequences of two mitochondrial genes, the 12S ribosomal gene (739 bp) and the cytochrome b gene (672 bp), were analyzed in hopes of reconstructing the evolutionary relationships of 11 leporid species, representative of seven genera. However, partial cytochrome b sequences were of little phylogenetic value in this study. A suite of pairwise comparisons between taxa revealed that at the intergeneric level, the cytochrome b gene is saturated at synonymous coding positions due to multiple substitution events. Furthermore, variation at the nonsynonymous positions is limited, rendering the cytochrome b gene of little phylogenetic value for assessing the relationships between leporid genera. If the cytochrome b data are analyzed without accounting for these two classes of nucleotides (i.e., synonymous and nonsynonymous sites), one may incorrectly conclude that signal exists in the cytochrome b data. The mitochondrial 12S rRNA gene, on the other hand, has not experienced excessive saturation at either stem or loop positions. Phylogenies reconstructed from the 12S rDNA data support hypotheses based on fossil evidence that African rock rabbits (Pronolagus) are outside of the main leporid stock and that leporids experienced a rapid radiation. However, the molecular data suggest that this radiation event occurred in the mid-Miocene several millions of years earlier than the Pleistocene dates suggested by paleontological evidence. Received: 23 April 1998 / Accepted: 14 May 1998     

Diversifying Selection Governs Sequence Polymorphism in the Major Adhesin Proteins FimA, PapA, and SfaA of Escherichia coli

Fimbriae or pili are essential adherence factors usually found in pathogenic bacteria to aid colonization of host cells. Three major structural pilin genes, fimA, sfaA, and papA, from Escherichia coli natural isolates were examined and nucleotide sequence data revealed elevated levels of both synonymous and nonsynonymous site variation at these loci. Examination of synonymous site variation shows a fivefold increase in fimA sites, relative to the housekeeping gene mdh; and similarly the sfaA and papA genes have increased synonymous sites variation relative to fimA. Nonsynonymous site variation is also elevated at all three loci but, in particular, at the papA locus (k _N= 0.44). The k _N/k _S ratio for the three genes are among the highest yet reported for E. coli genes. Regional variation in nucleotide polymorphism within each of the genes reveal hypervariable segments where nonsynonymous substitutions exceed synonymous substitutions. We propose that at the fimA, papA, and sfaA genes, diversifying selection has brought about the increase levels of polymorphism. Received: 7 August 1997 / Accepted: 8 March 1998     

Gene Expression, Amino Acid Conservation, and Hydrophobicity Are the Main Factors Shaping Codon Preferences in Mycobacterium tuberculosis and Mycobacterium leprae

Mycobacterium tuberculosis and Mycobacterium leprae are the ethiological agents of tuberculosis and leprosy, respectively. After performing extensive comparisons between genes from these two GC-rich bacterial species, we were able to construct a set of 275 homologous genes. Since these two bacterial species also have a very low growth rate, translational selection could not be so determinant in their codon preferences as it is in other fast-growing bacteria. Indeed, principal-components analysis of codon usage from this set of homologous genes revealed that the codon choices in M. tuberculosis and M. leprae are correlated not only with compositional constraints and translational selection, but also with the degree of amino acid conservation and the hydrophobicity of the encoded proteins. Finally, significant correlations were found between GC₃ and synonymous distances as well as between synonymous and nonsynonymous distances. Received: 30 October 1998 / Accepted: 16 August 1999     

The Evolution of Codon Preferences in Drosophila: A Maximum-Likelihood Approach to Parameter Estimation and Hypothesis Testing

Synonymous codon usage in related species may differ as a result of variation in mutation biases, differences in the overall strength and efficiency of selection, and shifts in codon preference—the selective hierarchy of codons within and between amino acids. We have developed a maximum-likelihood method to employ explicit population genetic models to analyze the evolution of parameters determining codon usage. The method is applied to twofold degenerate amino acids in 50 orthologous genes from D. melanogaster and D. virilis. We find that D. virilis has significantly reduced selection on codon usage for all amino acids, but the data are incompatible with a simple model in which there is a single difference in the long-term N _e, or overall strength of selection, between the two species, indicating shifts in codon preference. The strength of selection acting on codon usage in D. melanogaster is estimated to be |N _e s|≈ 0.4 for most CT-ending twofold degenerate amino acids, but 1.7 times greater for cysteine and 1.4 times greater for AG-ending codons. In D. virilis, the strength of selection acting on codon usage for most amino acids is only half that acting in D. melanogaster but is considerably greater than half for cysteine, perhaps indicating the dual selection pressures of translational efficiency and accuracy. Selection coefficients in orthologues are highly correlated (ρ= 0.46), but a number of genes deviate significantly from this relationship. Received: 20 December 1998 / Accepted: 17 February 1999     

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     

Unbiased estimation of symmetrical directional mutation pressure from protein-coding DNA

The most generally applicable procedure for obtaining estimates of the symmetrical, or strand-nonspecific, directional mutation pressure (μ_D) on protein-coding DNA sequences is to determine the G+C content at synonymous codon sites (P _syn), and to divide P _syn by twice the arithmetic mean of the G+C content at synonymous codon sites of a large number of randomly generated, synonymously coding DNA sequences (P _syn). Unfortunately, the original procedure yields biased estimates of P _syn and μ_D and is computationally expensive. We here present a fast procedure for estimating unbiased μ_D values. The procedure employs direct calculation of P _syn (≈P _syn) and two normalization procedures, one for P _syn≤P _syn and another for P _syn≥P _syn. The normalization removes a bias sometimes caused by codons specifying arginine, asparagine, isoleucine, and leucine. Consequently, comparison of protein-coding genes that are translated using different genetic codes is facilitated. Received: 5 May 1995 / Accepted: 30 November 1995     

Codon Usage Bias and tRNA Abundance in Drosophila

Codon usage bias of 1,117 Drosophila melanogaster genes, as well as fewer D. pseudoobscura and D. virilis genes, was examined from the perspective of relative abundance of isoaccepting tRNAs and their changes during development. We found that each amino acid contributes about equally and highly significantly to overall codon usage bias, with the exception of Asp which had very low contribution to overall bias. Asp was also the only amino acid that did not show a clear preference for one of its synonymous codons. Synonymous codon usage in Drosophila was consistent with ``optimal' codons deduced from the isoaccepting tRNA availability. Interestingly, amino acids whose major isoaccepting tRNAs change during development did not show as strong bias as those with developmentally unchanged tRNA pools. Asp is the only amino acid for which the major isoaccepting tRNAs change between larval and adult stages. We conclude that synonymous codon usage in Drosophila is well explained by tRNA availability and is probably influenced by developmental changes in relative abundance. Received: 5 December 1996 / Accepted: 14 June 1997     

Detection of Genes with Atypical Nucleotide Sequence in Microbial Genomes

Along the gene, nucleotides in various codon positions tend to exert a slight but observable influence on the nucleotide choice at neighboring positions. Such context biases are different in different organisms and can be used as genomic signatures. In this paper, we will focus specifically on the dinucleotide composed of a third codon position nucleotide and its succeeding first position nucleotide. Using the 16 possible dinucleotide combinations, we calculate how well individual genes conform to the observed mean dinucleotide frequencies of an entire genome, forming a distance measure for each gene. It is found that genes from different genomes can be separated with a high degree of accuracy, according to these distance values. In particular, we address the problem of recent horizontal gene transfer, and how imported genes may be evaluated by their poor assimilation to the host's context biases. By concentrating on the third- and succeeding first position nucleotides, we eliminate most spurious contributions from codon usage and amino-acid requirements, focusing mainly on mutational effects. Since imported genes are expected to converge only gradually to genomic signatures, it is possible to question whether a gene present in only one of two closely related organisms has been imported into one organism or deleted in the other. Striking correlations between the proposed distance measure and poor homology are observed when Escherichia coli genes are compared to Salmonella typhi, indicating that sets of outlier genes in E. coli may contain a high number of genes that have been imported into E. coli, and not deleted in S. typhi. Received: 16 January 2001 / Accepted: 30 August 2001     

A Survey of the Molecular Evolutionary Dynamics of Twenty-Five Multigene Families from Four Grass Taxa

We surveyed the molecular evolutionary characteristics of 25 plant gene families, with the goal of better understanding general processes in plant gene family evolution. The survey was based on 247 GenBank sequences representing four grass species (maize, rice, wheat, and barley). For each gene family, orthology and paralogy relationships were uncertain. Recognizing this uncertainty, we characterized the molecular evolution of each gene family in four ways. First, we calculated the ratio of nonsynonymous to synonymous substitutions (d _N/d _S) both on branches of gene phylogenies and across codons. Our results indicated that the d _N/d _S ratio was statistically heterogeneous across branches in 17 of 25 (68%) gene families. The vast majority of d _N/d _S estimates were <<1.0, suggestive of selective constraint on amino acid replacements, and no estimates were >1.0, either across phylogenetic lineages or across codons. Second, we tested separately for nonsynonymous and synonymous molecular clocks. Sixty-eight percent of gene families rejected a nonsynonymous molecular clock, and 52% of gene families rejected a synonymous molecular clock. Thus, most gene families in this study deviated from clock-like evolution at either synonymous or nonsynonymous sites. Third, we calculated the effective number of codons and the proportion of G+C synonymous sites for each sequence in each gene family. One or both quantities vary significantly within 18 of 25 gene families. Finally, we tested for gene conversion, and only six gene families provided evidence of gene conversion events. Altogether, evolution for these 25 gene families is marked by selective constraint that varies among gene family members, a lack of molecular clock at both synonymous and nonsynonymous sites, and substantial variation in codon usage. Received: 25 May 2000 / Accepted: 16 October 2000     

Synonymous and Nonsynonymous Substitution Rates in Diatoms: A Comparison Between Chloroplast and Nuclear Genes

Rates of synonymous and nonsynonymous nucleotide substitutions and codon usage bias (ENC) were estimated for a number of nuclear and chloroplast genes in a sample of centric and pennate diatoms. The results suggest that DNA evolution has taken place, on an average, at a slower rate in the chloroplast genes than in the nuclear genes: a rate variation pattern similar to that observed in land plants. Synonymous substitution rates in the chloroplast genes show a negative association with the degree of codon usage bias, suggesting that genes with a higher degree of codon usage bias have evolved at a slower rate. While this relationship has been shown in both prokaryotes and multicellular eukaryotes, it has not been demonstrated before in diatoms. Received: 3 June 1998 / Accepted: 11 August 1998     

A Translocated Mitochondrial Cytochrome b Pseudogene in Voles (Rodentia: Microtus)

A full-length cytochrome b pseudogene was found in rodents; it has apparently been translocated from a mitochondrion to the nuclear genome in the subfamily Arvicolinae. The pseudogene (ψcytb) differed from its mitochondrial counterpart at 201 of 1143 sites (17.6%) and by four indels. Cumulative evidence suggests that the pseudogene has been translocated to the nucleus. Phylogenetic reconstruction indicates that the pseudogene arose before the diversification of M. arvalis/M. rossiaemeridionalis from M. oeconomus, but after the divergence of the peromyscine/sigmodontine/arvicoline clades some ∼10 MYA. Published rates of divergence between mitochondrial genes and their nuclear pseudogenes suggest that the translocation of this mitochondrial gene to the nuclear genome occurred some 6 MYA, in agreement with the phylogenetic evidence. Received: 16 January 1998 / Accepted: 18 July 1998