Evolutionary patterns of codon usage in the chloroplast gene rbcL

In this study we reconstruct the evolution of codon usage bias in the chloroplast gene rbcL using a phylogeny of 92 green-plant taxa. We employ a measure of codon usage bias that accounts for chloroplast genomic nucleotide content, as an attempt to limit plausible explanations for patterns of codon bias evolution to selection- or drift-based processes. This measure uses maximum likelihood-ratio tests to compare the performance of two models, one in which a single codon is overrepresented and one in which two codons are overrepresented. The measure allowed us to analyze both the extent of bias in each lineage and the evolution of codon choice across the phylogeny. Despite predictions based primarily on the low G + C content of the chloroplast and the high functional importance of rbcL, we found large differences in the extent of bias, suggesting differential molecular selection that is clade specific. The seed plants and simple leafy liverworts each independently derived a low level of bias in rbcL, perhaps indicating relaxed selectional constraint on molecular changes in the gene. Overrepresentation of a single codon was typically plesiomorphic, and transitions to overrepresentation of two codons occurred commonly across the phylogeny, possibly indicating biochemical selection. The total codon bias in each taxon, when regressed against the total bias of each amino acid, suggested that twofold amino acids play a strong role in inflating the level of codon usage bias in rbcL, despite the fact that twofolds compose a minority of residues in this gene. Those amino acids that contributed most to the total codon usage bias of each taxon are known through amino acid knockout and replacement to be of high functional importance. This suggests that codon usage bias may be constrained by particular amino acids and, thus, may serve as a good predictor of what residues are most important for protein fitness.     

Comparative Analysis of Codon Usage Bias and Codon Context Patterns between Dipteran and Hymenopteran Sequenced Genomes

Background

Codon bias is a phenomenon of non-uniform usage of codons whereas codon context generally refers to sequential pair of codons in a gene. Although genome sequencing of multiple species of dipteran and hymenopteran insects have been completed only a few of these species have been analyzed for codon usage bias.

Methods and Principal Findings

Here, we use bioinformatics approaches to analyze codon usage bias and codon context patterns in a genome-wide manner among 15 dipteran and 7 hymenopteran insect species. Results show that GAA is the most frequent codon in the dipteran species whereas GAG is the most frequent codon in the hymenopteran species. Data reveals that codons ending with C or G are frequently used in the dipteran genomes whereas codons ending with A or T are frequently used in the hymenopteran genomes. Synonymous codon usage orders (SCUO) vary within genomes in a pattern that seems to be distinct for each species. Based on comparison of 30 one-to-one orthologous genes among 17 species, the fruit fly Drosophila willistoni shows the least codon usage bias whereas the honey bee (Apis mellifera) shows the highest bias. Analysis of codon context patterns of these insects shows that specific codons are frequently used as the 3′- and 5′-context of start and stop codons, respectively.

Conclusions

Codon bias pattern is distinct between dipteran and hymenopteran insects. While codon bias is favored by high GC content of dipteran genomes, high AT content of genes favors biased usage of synonymous codons in the hymenopteran insects. Also, codon context patterns vary among these species largely according to their phylogeny.     

Evolutionary Patterns of Codon Usage in the Chloroplast Gene rbcL

In this study we reconstruct the evolution of codon usage bias in the chloroplast gene rbcL using a phylogeny of 92 green-plant taxa. We employ a measure of codon usage bias that accounts for chloroplast genomic nucleotide content, as an attempt to limit plausible explanations for patterns of codon bias evolution to selection- or drift-based processes. This measure uses maximum likelihood-ratio tests to compare the performance of two models, one in which a single codon is overrepresented and one in which two codons are overrepresented. The measure allowed us to analyze both the extent of bias in each lineage and the evolution of codon choice across the phylogeny. Despite predictions based primarily on the low G+C content of the chloroplast and the high functional importance of rbcL, we found large differences in the extent of bias, suggesting differential molecular selection that is clade specific. The seed plants and simple leafy liverworts each independently derived a low level of bias in rbcL, perhaps indicating relaxed selectional constraint on molecular changes in the gene. Overrepresentation of a single codon was typically plesiomorphic, and transitions to overrepresentation of two codons occurred commonly across the phylogeny, possibly indicating biochemical selection. The total codon bias in each taxon, when regressed against the total bias of each amino acid, suggested that twofold amino acids play a strong role in inflating the level of codon usage bias in rbcL, despite the fact that twofolds compose a minority of residues in this gene. Those amino acids that contributed most to the total codon usage bias of each taxon are known through amino acid knockout and replacement to be of high functional importance. This suggests that codon usage bias may be constrained by particular amino acids and, thus, may serve as a good predictor of what residues are most important for protein fitness. Present address (Joshua T. Herbeck): JBP Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA 02543, USA     

The 'effective number of codons' used in a gene

A simple measure is presented that quantifies how far the codon usage of a gene departs from equal usage of synonymous codons. This measure of synonymous codon usage bias, the 'effective number of codons used in a gene', Nc, can be easily calculated from codon usage data alone, and is independent of gene length and amino acid (aa) composition. Nc can take values from 20, in the case of extreme bias where one codon is exclusively used for each aa, to 61 when the use of alternative synonymous codons is equally likely. Nc thus provides an intuitively meaningful measure of the extent of codon preference in a gene. Codon usage patterns across genes can be investigated by the Nc-plot: a plot of Nc vs. G + C content at synonymous sites. Nc-plots are produced for Homo sapiens, Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, Dictyostelium discoideum, and Drosophila melanogaster. A FORTRAN77 program written to calculate Nc is available on request.     

Selected codon usage bias in members of the class Mollicutes

Mollicutes are parasitic microorganisms mainly characterized by small cell sizes, reduced genomes and great A and T mutational bias. We analyzed the codon usage patterns of the completely sequenced genomes of bacteria that belong to this class. We found that for many organisms not only mutational bias but also selection has a major effect on codon usage. Through a comparative perspective and based on three widely used criteria we were able to classify Mollicutes according to the effect of selection on codon usage. We found conserved optimal codons in many species and study the tRNA gene pool in each genome. Previous results are reinforced by the fact that, when selection is operative, the putative optimal codons found match the respective cognate tRNA. Finally, we trace selection effect backwards to the common ancestor of the class and estimate the phylogenetic inertia associated with this character. We discuss the possible scenarios that explain the observed evolutionary patterns.     

Missing something? Codon aversion as a new character system in phylogenetics

Although many studies have documented codon usage bias in different species, the importance of codon usage in a phylogenetic framework remains largely unknown. We demonstrate that a phylogenetic signal is present in the codon usage and non‐usage biases of 17 717 orthologues evaluated across 72 tetrapod species using a simple parsimony analysis of a binary matrix of codon characters. Phylogenies estimated using stop codons were more congruent with previous hypotheses than phylogenies based on any other single codon or a combination of codons. Although each codon is present in every species, specific genes have different codon preferences and may or may not use every possible codon. This observation allowed us to map the pattern of codon usage and non‐usage across the topology. These results suggest that codon usage is phylogenetically conserved across shallow and deep levels within tetrapods.     

Codon usage bias in prokaryotic pyrimidine-ending codons is associated with the degeneracy of the encoded amino acids

Synonymous codons are unevenly distributed among genes, a phenomenon termed codon usage bias. Understanding the patterns of codon bias and the forces shaping them is a major step towards elucidating the adaptive advantage codon choice can confer at the level of individual genes and organisms. Here, we perform a large-scale analysis to assess codon usage bias pattern of pyrimidine-ending codons in highly expressed genes in prokaryotes. We find a bias pattern linked to the degeneracy of the encoded amino acid. Specifically, we show that codon-pairs that encode two- and three-fold degenerate amino acids are biased towards the C-ending codon while codons encoding four-fold degenerate amino acids are biased towards the U-ending codon. This codon usage pattern is widespread in prokaryotes, and its strength is correlated with translational selection both within and between organisms. We show that this bias is associated with an improved correspondence with the tRNA pool, avoidance of mis-incorporation errors during translation and moderate stability of codon-anticodon interaction, all consistent with more efficient translation.     

Codon usage bias: causative factors,quantification methods and genome‐wide patterns: with emphasis on insect genomes

Codon usage bias refers to the phenomenon where specific codons are used more often than other synonymous codons during translation of genes, the extent of which varies within and among species. Molecular evolutionary investigations suggest that codon bias is manifested as a result of balance between mutational and translational selection of such genes and that this phenomenon is widespread across species and may contribute to genome evolution in a significant manner. With the advent of whole‐genome sequencing of numerous species, both prokaryotes and eukaryotes, genome‐wide patterns of codon bias are emerging in different organisms. Various factors such as expression level, GC content, recombination rates, RNA stability, codon position, gene length and others (including environmental stress and population size) can influence codon usage bias within and among species. Moreover, there has been a continuous quest towards developing new concepts and tools to measure the extent of codon usage bias of genes. In this review, we outline the fundamental concepts of evolution of the genetic code, discuss various factors that may influence biased usage of synonymous codons and then outline different principles and methods of measurement of codon usage bias. Finally, we discuss selected studies performed using whole‐genome sequences of different insect species to show how codon bias patterns vary within and among genomes. We conclude with generalized remarks on specific emerging aspects of codon bias studies and highlight the recent explosion of genome‐sequencing efforts on arthropods (such as twelve Drosophila species, species of ants, honeybee, Nasonia and Anopheles mosquitoes as well as the recent launch of a genome‐sequencing project involving 5000 insects and other arthropods) that may help us to understand better the evolution of codon bias and its biological significance.     

Comparative genome sequence analysis of Sulfolobus acidocaldarius and 9 other isolates of its genus for factors influencing codon and amino acid usage

In the present study, major constraints for codon and amino acid usage of Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodali, Sulfolobus islandis and 6 other isolates from islandicus species of genus Sulfolobus were investigated. Correspondence analysis revealed high significant correlation between the major trend of synonymous codon usage and gene expression level, as assessed by the “Codon Adaptation Index” (CAI). There is a significant negative correlation between Nc (Effective number of codons) and CAI demonstrating role of codon bias as an important determinant of codon usage. The significant correlation between major trend of synonymous codon usage and GC3s (G + C at third synonymous position) indicated dominant role of mutational bias in codon usage pattern. The result was further supported from SCUO (synonymous codon usage order) analysis. The amino acid usage was found to be significantly influenced by aromaticity and hydrophobicity of proteins. However, translational selection which causes a preference for codons that are most rapidly translated by current tRNA with multiple copy numbers was not found to be highly dominating for all studied isolates. Notably, 26 codons that were found to be optimally used by genes of S. acidocaldarius at higher expression level and its comparative analysis with 9 other isolates may provide some useful clues for further in vivo genetic studies on this genus.     

Codon usage bias covaries with expression breadth and the rate of synonymous evolution in humans, but this is not evidence for selection.

In numerous species, from bacteria to Drosophila, evidence suggests that selection acts even on synonymous codon usage: codon bias is greater in more abundantly expressed genes, the rate of synonymous evolution is lower in genes with greater codon bias, and there is consistency between genes in the same species in which codons are preferred. In contrast, in mammals, while nonequal use of alternative codons is observed, the bias is attributed to the background variance in nucleotide concentrations, reflected in the similar nucleotide composition of flanking noncoding and exonic third sites. However, a systematic examination of the covariants of codon usage controlling for background nucleotide content has yet to be performed. Here we present a new method to measure codon bias that corrects for background nucleotide content and apply this to 2396 human genes. Nearly all (99%) exhibit a higher amount of codon bias than expected by chance. The patterns associated with selectively driven codon bias are weakly recovered: Broadly expressed genes have a higher level of bias than do tissue-specific genes, the bias is higher for genes with lower rates of synonymous substitutions, and certain codons are repeatedly preferred. However, while these patterns are suggestive, the first two patterns appear to be methodological artifacts. The last pattern reflects in part biases in usage of nucleotide pairs. We conclude that we find no evidence for selection on codon usage in humans.     

Analysis of synonymous codon usage bias in Chlamydia

Chlamydiae are obligate intracellular bacterial pathogens that cause ocular and sexuallytransmitted diseases,and are associated with cardiovascular diseases.The analysis of codon usage mayimprove our understanding of the evolution and pathogenesis of Chlamydia and allow reengineering of targetgenes to improve their expression for gene therapy.Here,we analyzed the codon usage of C.muridarum,C.trachomatis(here indicating biovar trachoma and LGV),C.pneumoniae,and C.psittaci using the codonusage database and the CUSP(Create a codon usage table)program of EMBOSS(The European MolecularBiology Open Software Suite).The results show that the four genomes have similar codon usage patterns,with a strong bias towards the codons with A and T at the third codon position.Compared with Homosapiens,the four chlamydial species show discordant seven or eight preferred codons.The ENC(effectivenumber of codons used in a gene)-plot reveals that the genetic heterogeneity in Chlamydia is constrained bythe G+C content,while translational selection and gene length exert relatively weaker influences.Moreover,mutational pressure appears to be the major determinant of the codon usage variation among the chlamydialgenes.In addition,we compared the codon preferences of C.trachomatis with those of E.coli,yeast,adenovirus and Homo sapiens.There are 23 codons showing distinct usage differences between C.trachomatisand E.coli,24 between C.trachomatis and adenovirus,21 between C.trachomatis and Homo sapiens,butonly six codons between C.trachomatis and yeast.Therefore,the yeast system may be more suitable for theexpression of chlamydial genes.Finally,we compared the codon preferences of C.trachomatis with those ofsix eukaryotes,eight prokaryotes and 23 viruses.There is a strong positive correlation between the differ-ences in coding GC content and the variations in codon bias(r=0.905,P<0,001).We conclude that thevariation of codon bias between C.trachomatis and other organisms is much less influenced by phylogeneticlineage and primarily determined by the extent of disparities in GC content.     

Novel third-letter bias in Escherichia coli codons revealed by rigorous treatment of coding constraints

A novel bias in codon third-letter usage was found in Escherichia coli genes with low fractions of "optimal codons", by comparing intact sequences with control random sequences. Third-letter usage has been found to be biased according to preference in codon usage and to doublet preference from the following first letter. The present study examines third-letter usage in the context of the nucleotide sequence when these preferences are considered. In order to exclude any influence by these factors, the random sequences were generated such that the amino acid sequence, codon usage, and the doublet frequency in each gene were all preserved. Comparison of intact sequences with these randomly generated sequences reveals that third letters of codons show a strong preference for the purine/pyrimidine pattern of the next codons: purine (R) is preferred to pyrimidine (Y) at the third site when followed by an R-Y-R codon, and pyrimidine is preferred when followed by an R-R-Y, an R-Y-Y or a Y-R-Y codon. This bias is probably related to interactions of tRNA molecules in the ribosome.     

General Rules for Optimal Codon Choice

Different synonymous codons are favored by natural selection for translation efficiency and accuracy in different organisms. The rules governing the identities of favored codons in different organisms remain obscure. In fact, it is not known whether such rules exist or whether favored codons are chosen randomly in evolution in a process akin to a series of frozen accidents. Here, we study this question by identifying for the first time the favored codons in 675 bacteria, 52 archea, and 10 fungi. We use a number of tests to show that the identified codons are indeed likely to be favored and find that across all studied organisms the identity of favored codons tracks the GC content of the genomes. Once the effect of the genomic GC content on selectively favored codon choice is taken into account, additional universal amino acid specific rules governing the identity of favored codons become apparent. Our results provide for the first time a clear set of rules governing the evolution of selectively favored codon usage. Based on these results, we describe a putative scenario for how evolutionary shifts in the identity of selectively favored codons can occur without even temporary weakening of natural selection for codon bias.     

同义密码子使用模式对蛋白产物表达及构象形成的影响*

鉴于遗传密码子的简并性能够将基因遗传信息的容量提升,同义密码子使用偏嗜性得以在生物体的基因组中广泛存在。虽然同义密码子之间碱基的变化并不能导致氨基酸种类的改变,在研究mRNA半衰期、编码多肽翻译效率及肽链空间构象正确折叠的准确性和翻译等这一系列过程中发现,同义密码子使用的偏嗜性在某种程度上通过精微调控翻译机制体现其遗传学功能。同义密码子指导tRNA在翻译过程中识别核糖体的速率变化是由氨基酸的特定顺序决定,并且在新生多肽链合成时,蛋白质共翻译转运机制同时调节其空间构象的正确折叠从而保证蛋白的正常生物学功能。某些同义密码子使用偏嗜性与特定蛋白结构的形成具有显著相关性,密码子使用偏嗜性一旦改变将可能导致新生多肽空间构象出现错误折叠。结合近些年来国内外在此领域的研究成果,阐述同义密码子使用偏嗜性如何发挥精微调控翻译的生物学功能与作用。     

Genome-wide analysis of codon usage bias patterns in an enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> F18 strain

Enterogenic Escherichia coli (ETEC) F18 strains are the main pathogenic bacteria causing severe diarrhea in humans and domestic animals. However, the information about synonymous codon usage pattern of ETEC F18 genome remains unclear. We conducted a genome-wide analysis of synonymous codon usage patterns in the ETEC F18 strain SRA: SAMN02471895. After filtering of the complete genome sequence, 4327 coding sequences were analyzed using multivariate statistical methods to calculate synonymous codon usage patterns and to evaluate the influence of various factors in shaping the codon usage. The mean GC content was 51.38%, with a slight preference for G/C-ending codons. Twenty-two codons were determined as ‘‘optimal codons”. ENC plots showed some of the genes were on or close to the expected curve, while only points with low-ENC values were below the curve. PR2 analysis showed that GC and AT were not used proportionally, suggesting major roles for mutational pressure and natural selection in shaping usage. Neutrality plots showed a significant correlation between GC12 and GC3, suggesting that mutational pressure is responsible for nucleotide composition in shaping the strength of codon usage. Translational selection was the main factor shaping the codon usage pattern of ETEC F18 genome, while other factors such as protein length, GRAVY and ARO values also influenced codon usage to some extent. We analyzed the codon usage pattern systematically and identified the factors shaping codon usage bias in the ETEC F18 genome. Such information further elucidates the mechanisms of synonymous codon usage bias and provides the basis of molecular genetic engineering and evolutionary studies.     

Codon usage bias and base composition in MHC genes in humans and common chimpanzees

 Codon bias and base composition in major histocompatibility complex (MHC) sequences have been studied for both class I and II loci in Homo sapiens and Pan troglodytes. There is low to moderate codon bias for the MHC of humans and chimpanzees. In the class I loci, the same level of moderate codon bias is seen for HLA-B, HLA-C, Patr-A, Patr-B, and Patr-C, while at HLA-A the level of codon bias is lower. There is a correlation between codon usage bias and G+C content in the A and B loci in humans and chimps, but not at the C locus. To examine the effect of diversifying selection on codon bias, we subdivided class I alleles into antigen recognition site (ARS) and non-ARS codons. ARS codons had lower bias than non-ARS codons. This may indicate that the constraint of codon bias on nucleotide substitution may be selected against in ARS codons. At the class II loci, there are distinct differences between alpha and beta chain genes with respect to codon usage, with the beta chain genes being much more biased. Species-specific differences in base composition were seen in exon 2 at the DRB1 locus, with lower GC content in chimpanzees. Considering the complex evolutionary history of MHC genes, the study of codon usage patterns provides us with a better understanding of both the evolutionary history of these genes and the evolution of synonymous codon usage in genes under natural selection. Received: 2 April 1998 / Revised: 2 September 1998     

Codon usage in plant genes.

We have examined codon bias in 207 plant gene sequences collected from Genbank and the literature. When this sample was further divided into 53 monocot and 154 dicot genes, the pattern of relative use of synonymous codons was shown to differ between these taxonomic groups, primarily in the use of G + C in the degenerate third base. Maize and soybean codon bias were examined separately and followed the monocot and dicot codon usage patterns respectively. Codon preference in ribulose 1,5 bisphosphate and chlorophyll a/b binding protein, two of the most abundant proteins in leaves was investigated. These highly expressed are more restricted in their codon usage than plant genes in general.     

Phylogenetic utility of elongation factor-1 alpha in noctuoidea (Insecta: Lepidoptera): the limits of synonymous substitution

To test its phylogenetic utility, nucleotide sequence variation in a 1,240-bp fragment of the elongation factor-1 alpha (EF-1 alpha) gene was examined in 49 moth species representing the major groups of the superfamily Noctuoidea. Both parsimony and distance analyses supported the monophyly of nearly all groups for which there are clear morphological synapomorphies. Clades of subfamily rank and lower, probably mid-Tertiary and younger, were strongly supported. The third codon position contains 88% of variable sites, and approaches saturation at approximately 20% sequence divergence, possibly due to among-site rate heterogeneity and composition bias; higher divergences occur only in association with shifts in composition. Surprisingly, the few nonsynonymous changes appear no more phylogenetically reliable than synonymous changes. Signal strength for basal divergences is weak and fails to improve with character weighting; thus, dense taxon sampling is probably needed for strong inference from EF-1 alpha regarding deeper splits in Noctuoidea (probably early Tertiary). EF-1 alpha synonymous changes show promise for phylogeny reconstruction within Noctuidae and other groups of Tertiary age.