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     

Genetic evolution and codon usage analysis of NKX-2.5 gene governing heart development in some mammals

NKX-2.5 gene is responsible for cardiac development and its targeted disruption apprehends cardiac development at the linear heart tube stage. Bioinformatic analysis was employed to investigate the codon usage pattern and dN/dS of mammalian NKX-2.5 gene. The relative synonymous codon usage analysis revealed variation in codon usage and two synonymous codons namely ATA (Ile) and GTA (Val) were absent in NKX-2.5 gene across selected mammalian species suggesting that these two codons were possibly selected against during evolution. Parity rule 2 analysis of two and four fold amino acids showed CT bias whereas six-fold amino acids revealed GA bias. Neutrality analysis suggests that selection played a prominent role while mutation had a minor role. The dN/dS analysis suggests synonymous substitution played a significant role and it negatively correlated with p-distance of the gene. Purifying natural selection played a dominant role in the genetic evolution of NKX-2.5 gene in mammals.     

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.     

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 Codon Usage for Error Minimization at the Protein Level

Given the structure of the genetic code, synonymous codons differ in their capacity to minimize the effects of errors due to mutation or mistranslation. I suggest that this may lead, in protein-coding genes, to a preference for codons that minimize the impact of errors at the protein level. I develop a theoretical measure of error minimization for each codon, based on amino acid similarity. This measure is used to calculate the degree of error minimization for 82 genes of Drosophila melanogaster and 432 rodent genes and to study its relationship with CG content, the degree of codon usage bias, and the rate of nucleotide substitution. I show that (i) Drosophila and rodent genes tend to prefer codons that minimize errors; (ii) this cannot be merely the effect of mutation bias; (iii) the degree of error minimization is correlated with the degree of codon usage bias; (iv) the amino acids that contribute more to codon usage bias are the ones for which synonymous codons differ more in the capacity to minimize errors; and (v) the degree of error minimization is correlated with the rate of nonsynonymous substitution. These results suggest that natural selection for error minimization at the protein level plays a role in the evolution of coding sequences in Drosophila and rodents.Reviewing Editor: Dr. Massimo Di Giulio     

A general model of codon bias due to GC mutational bias

Background

In spite of extensive research on the effect of mutation and selection on codon usage, a general model of codon usage bias due to mutational bias has been lacking. Because most amino acids allow synonymous GC content changing substitutions in the third codon position, the overall GC bias of a genome or genomic region is highly correlated with GC3, a measure of third position GC content. For individual amino acids as well, G/C ending codons usage generally increases with increasing GC bias and decreases with increasing AT bias. Arginine and leucine, amino acids that allow GC-changing synonymous substitutions in the first and third codon positions, have codons which may be expected to show different usage patterns.

Principal Findings

In analyzing codon usage bias in hundreds of prokaryotic and plant genomes and in human genes, we find that two G-ending codons, AGG (arginine) and TTG (leucine), unlike all other G/C-ending codons, show overall usage that decreases with increasing GC bias, contrary to the usual expectation that G/C-ending codon usage should increase with increasing genomic GC bias. Moreover, the usage of some codons appears nonlinear, even nonmonotone, as a function of GC bias. To explain these observations, we propose a continuous-time Markov chain model of GC-biased synonymous substitution. This model correctly predicts the qualitative usage patterns of all codons, including nonlinear codon usage in isoleucine, arginine and leucine. The model accounts for 72%, 64% and 52% of the observed variability of codon usage in prokaryotes, plants and human respectively. When codons are grouped based on common GC content, 87%, 80% and 68% of the variation in usage is explained for prokaryotes, plants and human respectively.

Conclusions

The model clarifies the sometimes-counterintuitive effects that GC mutational bias can have on codon usage, quantifies the influence of GC mutational bias and provides a natural null model relative to which other influences on codon bias may be measured.     

Relationships Among Stop Codon Usage Bias, Its Context, Isochores, and Gene Expression Level in Various Eukaryotes

It is well known that stop codons play a critical role in the process of protein synthesis. However, little effort has been made to investigate whether stop codon usage exhibits biases, such as widely seen for synonymous codon usage. Here we systematically investigate stop codon usage bias in various eukaryotes as well as its relationships with its context, GC3 content, gene expression level, and secondary structure. The results show that there is a strong bias for stop codon usage in different eukaryotes, i.e., UAA is overrepresented in the lower eukaryotes, UGA is overrepresented in the higher eukaryotes, and UAG is least used in all eukaryotes. Different conserved patterns for each stop codon in different eukaryotic classes are found based on information content and logo analysis. GC3 contents increase with increasing complexity of organisms. Secondary structure prediction revealed that UAA is generally associated with loop structures, whereas UGA is more uniformly present in loop and stem structures, i.e., UGA is less biased toward having a particular structure. The stop codon usage bias, however, shows no significant relationship with GC3 content and gene expression level in individual eukaryotes. The results indicate that genomic complexity and GC3 content might contribute to stop codon usage bias in different eukaryotes. Our results indicate that stop codons, like synonymous codons, exhibit biases in usage. Additional work will be needed to understand the causes of these biases and their relationship to the mechanism of protein termination. [Reviewing Editor: Dr. Manyuan Long]     

Synonymous Codon Usage, Accuracy of Translation, and Gene Length in Caenorhabditis elegans

In many unicellular organisms, invertebrates, and plants, synonymous codon usage biases result from a coadaptation between codon usage and tRNAs abundance to optimize the efficiency of protein synthesis. However, it remains unclear whether natural selection acts at the level of the speed or the accuracy of mRNAs translation. Here we show that codon usage can improve the fidelity of protein synthesis in multicellular species. As predicted by the model of selection for translational accuracy, we find that the frequency of codons optimal for translation is significantly higher at codons encoding for conserved amino acids than at codons encoding for nonconserved amino acids in 548 genes compared between Caenorhabditis elegans and Homo sapiens. Although this model predicts that codon bias correlates positively with gene length, a negative correlation between codon bias and gene length has been observed in eukaryotes. This suggests that selection for fidelity of protein synthesis is not the main factor responsible for codon biases. The relationship between codon bias and gene length remains unexplained. Exploring the differences in gene expression process in eukaryotes and prokaryotes should provide new insights to understand this key question of codon usage. Received: 18 June 2000 / Accepted: 10 November 2000     

Evidence from simulation studies for selective constraints on the codon usage of the Angiosperm psbA gene

The codon usage of the Angiosperm psbA gene is atypical for flowering plant chloroplast genes but similar to the codon usage observed in highly expressed plastid genes from some other Plantae, particularly Chlorobionta, lineages. The pattern of codon bias in these genes is suggestive of selection for a set of translationally optimal codons but the degree of bias towards these optimal codons is much weaker in the flowering plant psbA gene than in high expression plastid genes from lineages such as certain green algal groups. Two scenarios have been proposed to explain these observations. One is that the flowering plant psbA gene is currently under weak selective constraints for translation efficiency, the other is that there are no current selective constraints and we are observing the remnants of an ancestral codon adaptation that is decaying under mutational pressure. We test these two models using simulations studies that incorporate the context-dependent mutational properties of plant chloroplast DNA. We first reconstruct ancestral sequences and then simulate their evolution in the absence of selection on codon usage by using mutation dynamics estimated from intergenic regions. The results show that psbA has a significantly higher level of codon adaptation than expected while other chloroplast genes are within the range predicted by the simulations. These results suggest that there have been selective constraints on the codon usage of the flowering plant psbA gene during Angiosperm evolution.     

Chloroplast DNA codon use: Evidence for selection at the psb A locus based on tRNA availability

Codon use in the three sequenced chloroplast genomes (Marchantia, Oryza, and Nicotiana) is examined. The chloroplast has a bias in that codons NNA and NNT are favored over synonymous NNC and NNG codons. This appears to be a consequence of an overall high A + T content of the genome. This pattern of codon use is not followed by the psb A gene of all three genomes and other psb A sequences examined. In this gene, the codon use favors NNC over NNT for twofold degenerate amino acids. In each case the only tRNA coded by the genome is complementary to the NNC codon. This codon use is similar to the codon use by chloroplast genes examined from Chlamydomonas reinhardtii. Since psb A is the major translation product of the chloroplast, this suggests that selection is acting on the codon use of this gene to adapt codons to tRNA availability, as previously suggested for unicellular organisms.     

Codon usage patterns in cytochrome oxidase I across multiple insect orders

Synonymous codon usage bias is determined by a combination of mutational biases, selection at the level of translation, and genetic drift. In a study of mtDNA in insects, we analyzed patterns of codon usage across a phylogeny of 88 insect species spanning 12 orders. We employed a likelihood-based method for estimating levels of codon bias and determining major codon preference that removes the possible effects of genome nucleotide composition bias. Three questions are addressed: (1) How variable are codon bias levels across the phylogeny? (2) How variable are major codon preferences? and (3) Are there phylogenetic constraints on codon bias or preference? There is high variation in the level of codon bias values among the 88 taxa, but few readily apparent phylogenetic patterns. Bias level shifts within the lepidopteran genus Papilio are most likely a result of population size effects. Shifts in major codon preference occur across the tree in all of the amino acids in which there was bias of some level. The vast majority of changes involves double-preference models, however, and shifts between single preferred codons within orders occur only 11 times. These shifts among codons in double-preference models are phylogenetically conservative.     

亚心型扁藻叶绿体psbA基因的克隆及序列分析

psbA基因是叶绿体基因组中一个重要的光调控基因,编码光和系统Ⅱ反应中心的D1蛋白。根据叶绿体基因组序列高度保守的特性,利用菜茵衣藻（Chlamydomonasreinhardtii）psbA基因的保守序列（基因登录号：HQ667991．1）设计引物,采用PCR步移的方法从亚心型扁藻（Platymonassubcordiformis）基因组DNA中克隆到psbA基因全长（基因登录号：KF528742）。序列分析表明,亚心型扁藻psbA基因全长1939bp,编码区长度为1062bp,推导编码353个氨基酸,包括4个赖氨酸残基。有效密码子数显示脚删基因具有明显的密码子偏好性,并且偏好使用以A／T结尾的密码子。相对同义密码子使用度表明25个密码子在编码使用时具有偏好性,其中20个密码子以A／T碱基结尾,占到80％。其终止密码子使用了TAG。     

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.     

The Usage of Codons Which are Similar to Stop Codons in the Genomes of Xylella fastidiosa and Xanthomonas citri

During the evolution of living organisms, a natural selection event occurs toward the optimization of their genomes regarding the usage of codons. During this process which is known as codon bias, a set of preferred codons is naturally defined in the genome of a given organism, since there are 61 possible codons (plus 3 stop codons) to 20 amino acids. Such event leads to optimization of metabolic cellular processes such as translational efficiency, RNA stability and energy saving. Although we know why, we do not know how exactly a set of preferred codons for each amino acid is defined for a given genome considering that the usage frequency of each synonymous codons is peculiar to each organism. In order to help answering this question, we analyzed the usage frequency of codons which are similar to stop codons, since a minor mutation on these codons may lead to a stop codon into the open reading frame compromising the protein expression as a result. We found a reduced use of those codons in Xanthomomas axonopodis pv. citri which presents an optimized genome regarding codon usage. On the other hand, such codons are more often used in Xylella fastidiosa, which does not seem to have established codon preferences as previously shown. Our results support that a set of preferred codons is not randomly selected and propose new ideas to the field warranting further experiments in this regard.     

Synonymous codon usage of the VP2 gene of a very virulent infectious bursal disease virus isolate serial passaged in chicken embryos

Three very virulent infectious bursal disease virus (vvIBDV) strains were isolated from a single farm and shown to be phylogenetically related to the vvIBDV isolate UK661. In this study, a comparative analysis of the synonymous codon usage in the hypervariable region of theVP2 (vVP2) gene of the vvIBDV strains was done on viruses serially passaged in chicken embryos. Sequencing demonstrated that codons change during the serial passage in the vVP2 gene of the viruses. Nine codon mutations resulted in amino acids changes. The amino acid changes were I256V, I296L 6in isolate XA1989, A222P, I242V, Q253H, I256V in isolate XA1998, and Q253H, I256V, I296L in isolate XA2004. Three of the nine amino acid changes occurred at residue 256. The codons of the amino acids A232, N233, I234, T269, T283 and H338 changed to the synonymous codons in XA1989 after the 16th passage, in XA1998 after the 24th passage and in XA2004 22nd passage viruses. These mutations change the key amino acid residues Q253H and I256V in the domains which are essential for its virulence, and the synonymous codons were observed compared to classical virulent IBDV. The results indicated that the codon changes during the serial passage comprised of synonymous codon usage in the vVP2 gene of IBDV, and this synonymous codon bias was correlated with pathotypes. The extent of synonymous codon usage bias in the IBDV-vVP2 gene maybe influence the gene expression level and secondary structure of protein as well as hydrophobicity, therefore the results provide useful perspectives for evolution and understanding of the pathogenesis of IBDV.     

樟树叶绿体基因组密码子偏好性分析

为分析樟树(Cinnamomum camphora)叶绿体基因组密码子偏好性使用模式,该研究利用CodonW、EMBOSS、R语言等软件和程序,对53条樟树叶绿体基因组密码子使用模式及偏好性进行了系统分析。结果表明:樟树叶绿体基因的有效密码子数(ENC)在36.82～59.30之间,表明密码子的偏好性较弱。相对同义密码子使用度(RSCU)分析发现RSCU>1的密码子有32个,其中28个以A、U结尾,表明第3位密码子偏好使用A和U碱基。中性绘图分析发现GC₃与GC₁₂的相关性不显著,回归曲线斜率为0.049,说明密码子偏好性主要受到自然选择的影响。ENC-plot分析发现大部分基因落在曲线的下方,同样表明选择是影响密码子偏好性的主要因素。该研究发现共有9个密码子(UUU、CUU、UCA、ACA、UAU、AAU、GAU、UGA、GGA)被鉴定为樟树叶绿体基因组的最优密码子。     

紫花苜蓿叶绿体基因组密码子偏好性分析

为分析紫花苜蓿叶绿体基因组密码子偏好性的使用模式,该文以紫花苜蓿叶绿体基因组中筛选到的49条蛋白质编码序列为研究对象,利用CodonW、CUSP、CHIPS、SPSS等软件对其密码子的使用模式和偏好性进行研究。结果表明:(1)紫花苜蓿叶绿体基因的第3位密码子的平均GC含量为26.44%,有效密码子数(ENC)在40.6～51.41之间,多数密码子的偏好性较弱。(2)相对同义密码子使用度(RSCU)分析发现,RSCU>1 的密码子数目有30个,以A、U结尾的有29个,说明了紫花苜蓿叶绿体基因组A或U出现的频率较高。(3)中性分析发现,GC3与 GC12的相关性不显著,表明密码子偏性主要受自然选择的影响; ENC-plot 分析发现一部分基因落在曲线的下方及周围,表明突变也影响了部分密码子偏性的形成。此外,有17个密码子被鉴定为紫花苜蓿叶绿体基因组的最优密码子。紫花苜蓿叶绿体基因组的密码子偏好性可能受自然选择和突变的共同作用。该研究将为紫花苜蓿叶绿体基因工程的开展和目标性状的遗传改良奠定基础。     

scnRCA: A Novel Method to Detect Consistent Patterns of Translational Selection in Mutationally-Biased Genomes

Codon usage bias (CUB) results from the complex interplay between translational selection and mutational biases. Current methods for CUB analysis apply heuristics to integrate both components, limiting the depth and scope of CUB analysis as a technique to probe into the evolution and optimization of protein-coding genes. Here we introduce a self-consistent CUB index (scnRCA) that incorporates implicit correction for mutational biases, facilitating exploration of the translational selection component of CUB. We validate this technique using gene expression data and we apply it to a detailed analysis of CUB in the Pseudomonadales. Our results illustrate how the selective enrichment of specific codons among highly expressed genes is preserved in the context of genome-wide shifts in codon frequencies, and how the balance between mutational and translational biases leads to varying definitions of codon optimality. We extend this analysis to other moderate and fast growing bacteria and we provide unified support for the hypothesis that C- and A-ending codons of two-box amino acids, and the U-ending codons of four-box amino acids, are systematically enriched among highly expressed genes across bacteria. The use of an unbiased estimator of CUB allows us to report for the first time that the signature of translational selection is strongly conserved in the Pseudomonadales in spite of drastic changes in genome composition, and extends well beyond the core set of highly optimized genes in each genome. We generalize these results to other moderate and fast growing bacteria, hinting at selection for a universal pattern of gene expression that is conserved and detectable in conserved patterns of codon usage bias.     

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.