In Arabidopsis thaliana codon volatility scores reflect GC3 composition rather than selective pressure

ABSTRACT: BACKGROUND: Synonymous codon usage bias has typically been correlated with, and attributed to translational efficiency. However, there are other pressures on genomic sequence composition that can affect codon usage patterns such as mutational biases. This study provides an analysis of the codon usage patterns in Arabidopsis thaliana in relation to gene expression levels, codon volatility, mutational biases and selective pressures. RESULTS: We have performed synonymous codon usage and codon volatility analyses for all genes in the A. thaliana genome. In contrast to reports for species from other kingdoms, we find that neither codon usage nor volatility are correlated with selection pressure (as measured by dN/dS), nor with gene expression levels on a genome wide level. Our results show that codon volatility and usage are not synonymous, rather that they are correlated with the abundance of G and C at the third codon position (GC3). CONCLUSIONS: Our results indicate that while the A. thaliana genome shows evidence for synonymous codon usage bias, this is not related to the expression levels of its constituent genes. Neither codon volatility nor codon usage are correlated with expression levels or selective pressures but, because they are directly related to the composition of G and C at the third codon position, they are the result of mutational bias. Therefore, in A. thaliana codon volatility and usage do not result from selection for translation efficiency or protein functional shift as measured by positive selection.     

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.     

Vertebrate codon bias indicates a highly GC-rich ancestral genome

Two factors are thought to have contributed to the origin of codon usage bias in eukaryotes: 1) genome-wide mutational forces that shape overall GC-content and create context-dependent nucleotide bias, and 2) positive selection for codons that maximize efficient and accurate translation. Particularly in vertebrates, these two explanations contradict each other and cloud the origin of codon bias in the taxon. On the one hand, mutational forces fail to explain GC-richness (~ 60%) of third codon positions, given the GC-poor overall genomic composition among vertebrates (~ 40%). On the other hand, positive selection cannot easily explain strict regularities in codon preferences. Large-scale bioinformatic assessment, of nucleotide composition of coding and non-coding sequences in vertebrates and other taxa, suggests a simple possible resolution for this contradiction. Specifically, we propose that the last common vertebrate ancestor had a GC-rich genome (~ 65% GC). The data suggest that whole-genome mutational bias is the major driving force for generating codon bias. As the bias becomes prominent, it begins to affect translation and can result in positive selection for optimal codons. The positive selection can, in turn, significantly modulate codon preferences.     

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.     

Evolutionary Basis of Codon Usage and Nucleotide Composition Bias in Vertebrate DNA Viruses

Understanding the extent and causes of biases in codon usage and nucleotide composition is essential to the study of viral evolution, particularly the interplay between viruses and host cells or immune responses. To understand the common features and differences among viruses we analyzed the genomic characteristics of a representative collection of all sequenced vertebrate-infecting DNA viruses. This revealed that patterns of codon usage bias are strongly correlated with overall genomic GC content, suggesting that genome-wide mutational pressure, rather than natural selection for specific coding triplets, is the main determinant of codon usage. Further, we observed a striking difference in CpG content between DNA viruses with large and small genomes. While the majority of large genome viruses show the expected frequency of CpG, most small genome viruses had CpG contents far below expected values. The exceptions to this generalization, the large gammaherpesviruses and iridoviruses and the small dependoviruses, have sufficiently different life-cycle characteristics that they may help reveal some of the factors shaping the evolution of CpG usage in viruses. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Nicolas Galtier]     

Codon usage <Emphasis Type="BoldItalic">vis-a-vis</Emphasis> start and stop codon context analysis of three dicot species

To understand the variation in genomic composition and its effect on codon usage, we performed the comparative analysis of codon usage and nucleotide usage in the genes of three dicots, Glycine max, Arabidopsis thaliana and Medicago truncatula. The dicot genes were found to be A/T rich and have predominantly A-ending and/or T-ending codons. GC3s directly mimic the usage pattern of global GC content. Relative synonymous codon usage analysis suggests that the high usage frequency of A/T over G/C mononucleotide containing codons in AT-rich dicot genome is due to compositional constraint as a factor of codon usage bias. Odds ratio analysis identified the dinucleotides TpG, TpC, GpA, CpA and CpT as over-represented, where, CpG and TpA as under-represented dinucleotides. The results of (NcExp?NcObs)/NcExp plot suggests that selection pressure other than mutation played a significant role in influencing the pattern of codon usage in these dicots. PR2 analysis revealed the significant role of selection pressure on codon usage. Analysis of varience on codon usage at start and stop site showed variation in codon selection in these sites. This study provides evidence that the dicot genes were subjected to compositional selection pressure.     

Analysis of factors shaping codon usage in the mitochondrion genome of Oryza sativa

In this paper, the main factors shaping codon usage in the mitochondrion genome of rice were reported. Correspondence analysis, a commonly used multivariate statistical approach, was carried out to analyze synonymous codon usage bias. The results showed that the main trend was strongly correlated with the gene expression level assessed by the 'Codon Adaptation Index' value, a result that was confirmed by the distribution of genes along the first axis. From the results that there were two significant correlations between axis 1 coordinates and the GC, GC3s content at silent sites of each sequence, and clearly significant correlations between the 'Effective Number of Codons' values and GC, GC3s content, we inferred that codon usage bias was affected by gene nucleotide composition also. In addition, the hydrophobicity of each protein also played some roles in shaping codon usage in this organelle, which could be confirmed by the significant correlation between the positions of genes placed on the first axis and the hydrophobicity value of each protein. In summary, natural selection played a crucial role, nucleotide mutational bias and amino acid composition only in a minor way, in shaping codon usage in the mitochondrion genome of rice. Notably, 21 codons defined firstly as 'optimal codons' might provide some more useful information for gene engineering and/or evolution studying.     

葡萄基因组密码子使用偏好模式研究

根据完整基因组序列,运用多元统计分析和对应分析的方法,探讨了葡萄全基因组序列密码子的使用模式和影响密码子使用的各种可能因素。结果显示:葡萄密码子偏好性主要受到碱基差异(r=0.925)和自然选择(r=0.193)共同作用的影响,突变压力占了主导因素,自然选择的作用较小。同时基因长度和蛋白质疏水性也对密码子的偏好性有所影响。确定了葡萄的20个最优密码子。     

Rapid divergence of codon usage patterns within the rice genome

Background

Synonymous codon usage varies widely between genomes, and also between genes within genomes. Although there is now a large body of data on variations in codon usage, it is still not clear if the observed patterns reflect the effects of positive Darwinian selection acting at the level of translational efficiency or whether these patterns are due simply to the effects of mutational bias. In this study, we have included both intra-genomic and inter-genomic comparisons of codon usage. This allows us to distinguish more efficiently between the effects of nucleotide bias and translational selection.

Results

We show that there is an extreme degree of heterogeneity in codon usage patterns within the rice genome, and that this heterogeneity is highly correlated with differences in nucleotide content (particularly GC content) between the genes. In contrast to the situation observed within the rice genome, Arabidopsis genes show relatively little variation in both codon usage and nucleotide content. By exploiting a combination of intra-genomic and inter-genomic comparisons, we provide evidence that the differences in codon usage among the rice genes reflect a relatively rapid evolutionary increase in the GC content of some rice genes. We also noted that the degree of codon bias was negatively correlated with gene length.

Conclusion

Our results show that mutational bias can cause a dramatic evolutionary divergence in codon usage patterns within a period of approximately two hundred million years.The heterogeneity of codon usage patterns within the rice genome can be explained by a balance between genome-wide mutational biases and negative selection against these biased mutations. The strength of the negative selection is proportional to the length of the coding sequences. Our results indicate that the large variations in synonymous codon usage are not related to selection acting on the translational efficiency of synonymous codons.

     

Synonymous codon usage in Thermosynechococcus elongatus (cyanobacteria) identifies the factors shaping codon usage variation

Analysis of synonymous codon usage pattern in the genome of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 using multivariate statistical analysis revealed a single major explanatory axis accounting for codon usage variation in the organism. This axis is correlated with the GC content at third base of synonymous codons (GC3s) in correspondence analysis taking T. elongatus genes. A negative correlation was observed between effective number of codons i.e. Nc and GC3s. Results suggested a mutational bias as the major factor in shaping codon usage in this cyanobacterium. In comparison to the lowly expressed genes, highly expressed genes of this organism possess significantly higher proportion of pyrimidine-ending codons suggesting that besides, mutational bias, translational selection also influenced codon usage variation in T. elongatus. Correspondence analysis of relative synonymous codon usage (RSCU) with A, T, G, C at third positions (A3s, T3s, G3s, C3s, respectively) also supported this fact and expression levels of genes and gene length also influenced codon usage. A role of translational accuracy was identified in dictating the codon usage variation of this genome. Results indicated that although mutational bias is the major factor in shaping codon usage in T. elongatus, factors like translational selection, translational accuracy and gene expression level also influenced codon usage variation.     

Analysis of synonymous codon usage in <Emphasis Type="Italic">Zea mays</Emphasis>

It is important and meaningful to understand the codon usage pattern and the factors that shape codon usage of maize. In this study, trends in synonymous codon usage in maize have been firstly examined through the multivariate statistical analysis on 7402 cDNA sequences. The results showed that the genes positions on the primary axis were strongly negatively correlated with GC3s, GC content of individual gene and gene expression level assessed by the codon adaptation index (CAI) values, which indicated that nucleotide composition and gene expression level were the main factors in shaping the codon usage of maize, and the variation in codon usage among genes may be due to mutational bias at the DNA level and natural selection acting at the level of mRNA translation. At the same time, CDS length and the hydrophobicity of each protein were, respectively, significantly correlated with the genes locations on the primary axis, GC3s and CAI values. We infer that genes length and the hydrophobicity of the encoded protein may play minor role in shaping codon usage bias. Additional 28 codons ending with a G or C base have been defined as “optimal codons”, which may provide useful information for maize gene-transformation and gene prediction.     

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.     

Evolution of Base Composition and Codon Usage Bias in the Genus Flavivirus

The extent to which base composition and codon usage vary among RNA viruses, and the possible causes of this bias, is undetermined in most cases. A maximum-likelihood statistical method was used to test whether base composition and codon usage bias covary with arthropod association in the genus Flavivirus, a major source of disease in humans and animals. Flaviviruses are transmitted by mosquitoes, by ticks, or directly between vertebrate hosts. Those viruses associated with ticks were found to have a significantly lower G+C content than non-vector-borne flaviviruses and this difference was present throughout the genome at all amino acids and codon positions. In contrast, mosquito-borne viruses had an intermediate G+C content which was not significantly different from those of the other two groups. In addition, biases in dinucleotide and codon usage that were independent of base composition were detected in all flaviviruses, but these did not covary with arthropod association. However, the overall effect of these biases was slight, suggesting only weak selection at synonymous sites. A preliminary analysis of base composition, codon usage, and vector specificity in other RNA virus families also revealed a possible association between base composition and vector specificity, although with biases different from those seen in the Flavivirus genus. Received: 29 August 2000 / Accepted: 19 December 2000     

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.     

Genome-wide codon usage profiling of ocular infective Chlamydia trachomatis serovars and drug target identification

Chlamydia trachomatis (C.t) is a Gram-negative obligate intracellular bacteria and is a major causative of infectious blindness and sexually transmitted diseases. Among the varied serovars of this organism, A, B and C are reported as prominent ocular pathogens. Genomic studies of these strains shall aid in deciphering potential drug targets and genomic influence on pathogenesis. Hence, in this study we performed deep statistical profiling of codon usage in these serovars. The overall base composition analysis reveals that these serovars are over biased to AU than GC. Similarly, relative synonymous codon usage also showed preference towards A/U ending codons. Parity Rule 2 analysis inferred unequal distribution of AT and GC, indicative of other unknown factors acting along with mutational pressure to influence codon usage bias (CUB). Moreover, absolute quantification of CUB also revealed lower bias across these serovars. The effect of natural selection on CUB was also confirmed by neutrality plot, reinforcing natural selection under mutational pressure turned to be a pivotal role in shaping the CUB in the strains studied. Correspondence analysis (COA) clarified that, C.t C/TW-3 to show a unique trend in codon usage variation. Host influence analysis on shaping the codon usage pattern also inferred some speculative relativity. In a nutshell, our finding suggests that mutational pressure is the dominating factor in shaping CUB in the strains studied, followed by natural selection. We also propose potential drug targets based on cumulative analysis of strand bias, CUB and human non-homologue screening.     

Trends in the codon usage patterns of Chromohalobacter salexigens genes

Chromohalobacter salexigens, a Gammaproteobacterium belonging to the family Halomonadaceae, shows a broad salinity range for growth. In order to reveal the factors influencing architecture of protein coding genes in C. salexigens, pattern of synonymous codon usage bias has been investigated. Overall codon usage analysis of the microorganism revealed that C and G ending codons are predominantly used in all the genes which are indicative of mutational bias. Multivariate statistical analysis showed that the genes are separated along the first major explanatory axis according to their expression levels and their genomic GC content at the synonymous third positions of the codons. Both NC plot and correspondence analysis on Relative Synonymous Codon Usage (RSCU) indicates that the variation in codon usage among the genes may be due to mutational bias at the DNA level and natural selection acting at the level of mRNA translation. Gene length and the hydrophobicity of the encoded protein also influence the codon usage variation of genes to some extent. A comparison of the relative synonymous codon usage between 10% each of highly and lowly expressed genes determines 23 optimal codons, which are statistically over represented in the former group of genes and may provide useful information for salt-stressed gene prediction and gene-transformation. Furthermore, genes for regulatory functions; mobile and extrachromosomal element functions; and cell envelope are observed to be highly expressed. The study could provide insight into the gene expression response of halophilic bacteria and facilitate establishment of effective strategies to develop salt-tolerant crops of agronomic value.     

Genome-wide comparative analysis of the codon usage patterns in plants

Codon usage analysis has been a classical area of study for decades and is important for evolution, mRNA translation, and new gene discovery. Recently, genome sequencing has made it possible to perform studies of the entire genome in plant kingdoms. The base composition of the coding sequence, codon usage pattern, codon pairs, and related indicators of relative synonymous codon usage (RSCU), including the Fop, Nc, RSCU, CAI and GC contents, were analyzed. We found that the GC content of single-celled algae is the highest, whereas dicotyledons are the lowest. Moreover, the base composition of plants is similar within the same family. In addition, the GC content of the second base of the codon is lower than the first and third base. In conclusion, the codon usage characteristics are opposite in Gramineae, single-celled algae, fern and dicotyledon, moss, and Pinaceae. Furthermore, the degree of codon usage bias is decreasing with evolution. Therefore, we hypothesize that the lower the plants, the more that they must optimize codons and that higher plants no longer need to optimize codons.     

The Effect of Multiple Evolutionary Selections on Synonymous Codon Usage of Genes in the Mycoplasma bovis Genome

Mycoplasma bovis is a major pathogen causing arthritis, respiratory disease and mastitis in cattle. A better understanding of its genetic features and evolution might represent evidences of surviving host environments. In this study, multiple factors influencing synonymous codon usage patterns in M. bovis (three strains’ genomes) were analyzed. The overall nucleotide content of genes in the M. bovis genome is AT-rich. Although the G and C contents at the third codon position of genes in the leading strand differ from those in the lagging strand (p<0.05), the 59 synonymous codon usage patterns of genes in the leading strand are highly similar to those in the lagging strand. The over-represented codons and the under-represented codons were identified. A comparison of the synonymous codon usage pattern of M. bovis and cattle (susceptible host) indicated the independent formation of synonymous codon usage of M. bovis. Principal component analysis revealed that (i) strand-specific mutational bias fails to affect the synonymous codon usage pattern in the leading and lagging strands, (ii) mutation pressure from nucleotide content plays a role in shaping the overall codon usage, and (iii) the major trend of synonymous codon usage has a significant correlation with the gene expression level that is estimated by the codon adaptation index. The plot of the effective number of codons against the G+C content at the third codon position also reveals that mutation pressure undoubtedly contributes to the synonymous codon usage pattern of M. bovis. Additionally, the formation of the overall codon usage is determined by certain evolutionary selections for gene function classification (30S protein, 50S protein, transposase, membrane protein, and lipoprotein) and translation elongation region of genes in M. bovis. The information could be helpful in further investigations of evolutionary mechanisms of the Mycoplasma family and heterologous expression of its functionally important proteins.     

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。     

Analysis of synonymous codon usage in Shigella flexneri 2a strain 301 and other Shigella and Escherichia coli strains

In this study, we analysed synonymous codon usage in Shigella flexneri 2a strain 301 (Sf301) and performed a comparative analysis of synonymous codon usage patterns in Sf301 and other strains of Shigella and Escherichia coli. Although there was a significant variety in codon usage bias among different Sf301 genes, there was a slight but observable codon usage bias that could primarily be attributable to mutational pressure and translational selection. In addition, the relative abundance of dinucleotides in Sf301 was observed to be independent of the overall base composition but was still caused by differential mutational pressure; this also shaped codon usage. By comparing the relative synonymous codon usage values across different Shigella and E. coli strains, we suggested that the synonymous codon usage pattern in the Shigella genomes was strain specific. This study represents a comprehensive analysis of Shigella codon usage patterns and provides a basic understanding of the mechanisms underlying codon usage bias.