Impact of bias discrepancy and amino acid usage on estimates of the effective number of codons used in a gene, and a test for selection on codon usage

The effective number of codons (Nc) used in a gene is one of the most commonly used measures of synonymous codon usage bias, owing much of its popularity to the fact that it is species independent and that simulation studies have shown that it is less dependent of gene length than other measures. In this paper I provide a clear and practically meaningful definition of bias discrepancy (BD; when the degree of codon bias varies within a degeneracy class). Moreover I evaluate the impact of BD and amino acid usage on estimates of Nc. It is shown that both factors have a significant effect on accuracy and precision. Both amino acid usage and BD influence accuracy considerably, especially in short genes. Finally, I demonstrate how the definition of bias discrepancy can be applied to investigate if codon usage is influenced by selection and I discuss this test in relation to the incongruous literature that exists for Buchnera sp. APS and Borrelia burgdorferi.     

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.     

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.     

普通野生稻线粒体蛋白质编码基因密码子使用偏好性的分析

以普通野生稻(Oryza rufipogon Griff.)线粒体基因组为对象,分析其蛋白质编码基因的密码子使用特征及与亚洲栽培稻(O. sativa L.)的差异,探讨其密码子偏性形成的影响因素和进化过程。结果显示:普通野生稻线粒体基因组编码序列第1、第2和第3位碱基的GC含量依次为49.18%、42.67%和40.86%;有效密码子数(Nc)分布于45.32～61.00之间,其密码子偏性较弱; Nc值仅与GC_3呈显著相关,密码子第3位的碱基组成对密码子偏性影响较大;第1向量轴上显示9.91%的差异,其与GC_3s、Nc、密码子偏好指数(CBI)和最优密码子使用频率(Fop)的相关性均达到显著水平;而GC_3和GC₁₂的相关性未达到显著水平。因此,普通野生稻线粒体基因组密码子的使用偏性主要受自然选择压力影响而形成。本研究确定了21个普通野生稻线粒体基因组的最优密码子,大多以A或T结尾,与叶绿体密码子具有趋同进化,但是与核基因组具有不同的偏好性。同义密码子相对使用度(RSCU)、PR2偏倚分析和中性绘图分析显示,普通野生稻线粒体基因功能和其密码子使用密切相关,且线粒体密码子使用在普通野生稻、粳稻(O. sativa L. subsp. japonica Kato)和籼稻(O. sativa L. subsp.indica Kato)内具有同质性。     

普通野生稻线粒体蛋白质编码基因密码子使用偏好性的分析

以普通野生稻（Oryza rufipogon Griff.）线粒体基因组为对象,分析其蛋白质编码基因的密码子使用特征及与亚洲栽培稻（O.sativa L.）的差异,探讨其密码子偏性形成的影响因素和进化过程。结果显示：普通野生稻线粒体基因组编码序列第1、第2和第3位碱基的GC含量依次为49.18%、42.67%和40.86%;有效密码子数（Nc）分布于45.32~61.00之间,其密码子偏性较弱;Nc值仅与GC₃呈显著相关,密码子第3位的碱基组成对密码子偏性影响较大;第1向量轴上显示9.91%的差异,其与GC_3s、Nc、密码子偏好指数（CBI）和最优密码子使用频率（Fop）的相关性均达到显著水平;而GC₃和GC₁₂的相关性未达到显著水平。因此,普通野生稻线粒体基因组密码子的使用偏性主要受自然选择压力影响而形成。本研究确定了21个普通野生稻线粒体基因组的最优密码子,大多以A或T结尾,与叶绿体密码子具有趋同进化,但是与核基因组具有不同的偏好性。同义密码子相对使用度（RSCU）、PR2偏倚分析和中性绘图分析显示,普通野生稻线粒体基因功能和其密码子使用密切相关,且线粒体密码子使用在普通野生稻、粳稻（O.sativa L.subsp.japonica Kato）和籼稻（O.sativa L.subsp.indica Kato）内具有同质性。     

The 'effective number of codons' revisited

Frank Wright [Gene 87 (1990) 23] derived a formula for calculation of a quantity termed the 'effective number of codons' (Nc) based on codon homozygosities. This quantity is a number between 20 and 61 and tells to what degree the codon usage in a gene is biased, i.e., it approaches 20 codons for the extremely biased genes, and approaches 61 for the genes where all possible codons are used with no preference. Among the different measures of codon bias Nc is considered the most useful and has found widespread use in papers dealing with codon usage phenomena. In this paper, the mathematical behaviours of codon homozygosities and Nc are evaluated, using Escherichia coli as the model organism. The results indicate that the classical formula for calculation of Nc could appropriately be substituted under circumstances, where there is bias discrepancy, i.e., when one amino acid (or more) within a degeneracy group is associated with strong codon bias while at the same time others in the same degeneracy group have little bias. An alternative estimator, termed Nc, is proposed and tested against Nc, and performs better when there is such bias discrepancy.     

Comparative study on factors influencing the codon and amino acid usage in Lactobacillus sakei 23K and 13 other lactobacilli

In this study, major factors shaping codon and amino acid usage variation Lactobacillus sakei 23K were investigated. It included 13 other Lactobacillus species for a comparative analysis. The correspondence analysis (COA) showed that in 13 species the major trend of synonymous codon usage was highly correlated with gene expression level as assessed by the “Codon Adaptation Index” (CAI) values. In addition, Nc (effective number of codons) plot, SCUO (synonymous codon usage order) plot and correlation analyses showed that the base composition and mutational bias have dominant role in the codon usage variation. However, the translational selection for genes at higher expression level, where more frequent synonymous codons correspond to more abundant cognate transfer RNAs (tRNAs), was not found to be similar in all species. The study also showed that the amino acid usage in these species was significantly (P < 0.01) influenced by hydrophobicity and aromaticity of proteins. Furthermore, 24 codons that were found to be optimally used by L. sakei and its comparative study with 13 Lactobacillus species might provide some useful information in their further study of molecular evolution and genetic engineering.     

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.     

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.     

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。     

栽培大豆和野生大豆线粒体基因组密码子使用偏性的比较分析

为分析栽培大豆和野生大豆线粒体基因组的密码子使用特征差异,该文以其线粒体基因组编码序列为研究对象,比较其密码子偏性形成的影响因素和演化过程。结果表明:(1)栽培大豆和野生大豆线粒体基因组编码区的GC含量分别为44.56%和44.58%,说明栽培大豆和野生大豆线粒体编码基因均富含A/T碱基。(2)栽培大豆和野生大豆线粒体基因组密码子第1位、第2位GC含量平均值与第3位GC含量的相关性均呈极显著水平,说明突变在其密码子偏性形成中的作用不可忽略; PR2-plot分析显示,在同义密码子第3位碱基的使用频率上,嘌呤低于嘧啶; Nc-plot分析中Nc比值位于-0.1～0.2区间的基因数占总基因数的95%以上;突变和选择等多重因素共同作用影响了大豆线粒体基因组编码序列密码子使用偏性的形成。(3)有20、21个密码子分别被确定为栽培大豆和野生大豆线粒体基因组编码序列的最优密码子,其中除丝氨酸TCC密码子外均以A或T结尾。综上结果认为,栽培大豆线粒体密码子偏性的形成受选择的影响要高于野生大豆,这可能是栽培大豆由野生大豆经长期人工栽培驯化的结果。     

Gene expression,nucleotide composition and codon usage bias of genes associated with human Y chromosome

Analysis of codon usage pattern is important to understand the genetic and evolutionary characteristics of genomes. We have used bioinformatic approaches to analyze the codon usage bias (CUB) of the genes located in human Y chromosome. Codon bias index (CBI) indicated that the overall extent of codon usage bias was low. The relative synonymous codon usage (RSCU) analysis suggested that approximately half of the codons out of 59 synonymous codons were most frequently used, and possessed a T or G at the third codon position. The codon usage pattern was different in different genes as revealed from correspondence analysis (COA). A significant correlation between effective number of codons (ENC) and various GC contents suggests that both mutation pressure and natural selection affect the codon usage pattern of genes located in human Y chromosome. In addition, Y-linked genes have significant difference in GC contents at the second and third codon positions, expression level, and codon usage pattern of some codons like the SPANX genes in X chromosome.     

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.     

毕赤酵母的密码子用法分析

通过分析Pichia pastoris的28个蛋白编码基因的同义密码子使用情况并计算该酵母的密码子用法,首次确定出P.pastoris的19个高表达优越密码子。这些结果经与已知的Saccharomyces cerevisiae及Kluyveromyces lactis的密码子用法基本相似,但在氨基酸谷氨酸的密码子选择上截然相反,提示这可能属于P.pastoris所偏爱的密码子用法。     

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 codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications.

A simple, effective measure of synonymous codon usage bias, the Codon Adaptation Index, is detailed. The index uses a reference set of highly expressed genes from a species to assess the relative merits of each codon, and a score for a gene is calculated from the frequency of use of all codons in that gene. The index assesses the extent to which selection has been effective in moulding the pattern of codon usage. In that respect it is useful for predicting the level of expression of a gene, for assessing the adaptation of viral genes to their hosts, and for making comparisons of codon usage in different organisms. The index may also give an approximate indication of the likely success of heterologous gene expression.     

SCUMBLE: a method for systematic and accurate detection of codon usage bias by maximum likelihood estimation

The genetic code is degenerate—most amino acids can be encoded by from two to as many as six different codons. The synonymous codons are not used with equal frequency: not only are some codons favored over others, but also their usage can vary significantly from species to species and between different genes in the same organism. Known causes of codon bias include differences in mutation rates as well as selection pressure related to the expression level of a gene, but the standard analysis methods can account for only a fraction of the observed codon usage variation. We here introduce an explicit model of codon usage bias, inspired by statistical physics. Combining this model with a maximum likelihood approach, we are able to clearly identify different sources of bias in various genomes. We have applied the algorithm to Saccharomyces cerevisiae as well as 325 prokaryote genomes, and in most cases our model explains essentially all observed variance.     

Codon usage and amino acid usage influence genes expression level

Highly expressed genes in any species differ in the usage frequency of synonymous codons. The relative recurrence of an event of the favored codon pair (amino acid pairs) varies between gene and genomes due to varying gene expression and different base composition. Here we propose a new measure for predicting the gene expression level, i.e., codon plus amino bias index (CABI). Our approach is based on the relative bias of the favored codon pair inclination among the genes, illustrated by analyzing the CABI score of the Medicago truncatula genes. CABI showed strong correlation with all other widely used measures (CAI, RCBS, SCUO) for gene expression analysis. Surprisingly, CABI outperforms all other measures by showing better correlation with the wet-lab data. This emphasizes the importance of the neighboring codons of the favored codon in a synonymous group while estimating the expression level of a gene.     

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.