Comparative analysis of the base biases at the gene terminal portions in seven eukaryote genomes

Adenine nucleotides have been found to appear preferentially in the regions after the initiation codons or before the termination codons of bacterial genes. Our previous experiments showed that AAA and AAT, the two most frequent second codons in Escherichia coli, significantly enhance translation efficiency. To determine whether such a characteristic feature of base frequencies exists in eukaryote genes, we performed a comparative analysis of the base biases at the gene terminal portions using the proteomes of seven eukaryotes. Here we show that the base appearance at the codon third positions of gene terminal regions is highly biased in eukaryote genomes, although the codon third positions are almost free from amino acid preference. The bias changes depending on its position in a gene, and is characteristic of each species. We also found that bias is most outstanding at the second codon, the codon after the initiation codon. NCN is preferred in every genome; in particular, GCG is strongly favored in human and plant genes. The presence of the bias implies that the base sequences at the second codon affect translation efficiency in eukaryotes as well as bacteria.     

The Effects of Mutation and Natural Selection on Codon Bias in the Genes of Drosophila

Codon bias varies widely among the loci of Drosophila melanogaster, and some of this diversity has been explained by variation in the strength of natural selection. A study of correlations between intron and coding region base composition shows that variation in mutation pattern also contributes to codon bias variation. This finding is corroborated by an analysis of variance (ANOVA), which shows a tendency for introns from the same gene to be similar in base composition. The strength of base composition correlations between introns and codon third positions is greater for genes with low codon bias than for genes with high codon bias. This pattern can be explained by an overwhelming effect of natural selection, relative to mutation, in highly biased loci. In particular, this correlation is absent when examining fourfold degenerate sites of highly biased genes. In general, it appears that selection acts more strongly in choosing among fourfold degenerate codons than among twofold degenerate codons. Although the results indicate regional variation in mutational bias, no evidence is found for large scale regions of compositional homogeneity.     

Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes.

Codon usage data has been compiled for 110 yeast genes. Cluster analysis on relative synonymous codon usage revealed two distinct groups of genes. One group corresponds to highly expressed genes, and has much more extreme synonymous codon preference. The pattern of codon usage observed is consistent with that expected if a need to match abundant tRNAs, and intermediacy of tRNA-mRNA interaction energies are important selective constraints. Thus codon usage in the highly expressed group shows a higher correlation with tRNA abundance, a greater degree of third base pyrimidine bias, and a lesser tendency to the A+T richness which is characteristic of the yeast genome. The cluster analysis can be used to predict the likely level of gene expression of any gene, and identifies the pattern of codon usage likely to yield optimal gene expression in yeast.     

Inferring Adaptive Codon Preference to Understand Sources of Selection Shaping Codon Usage Bias

Alternative synonymous codons are often used at unequal frequencies. Classically, studies of such codon usage bias (CUB) attempted to separate the impact of neutral from selective forces by assuming that deviations from a predicted neutral equilibrium capture selection. However, GC-biased gene conversion (gBGC) can also cause deviation from a neutral null. Alternatively, selection has been inferred from CUB in highly expressed genes, but the accuracy of this approach has not been extensively tested, and gBGC can interfere with such extrapolations (e.g., if expression and gene conversion rates covary). It is therefore critical to examine deviations from a mutational null in a species with no gBGC. To achieve this goal, we implement such an analysis in the highly AT rich genome of Dictyostelium discoideum, where we find no evidence of gBGC. We infer neutral CUB under mutational equilibrium to quantify “adaptive codon preference,” a nontautologous genome wide quantitative measure of the relative selection strength driving CUB. We observe signatures of purifying selection consistent with selection favoring adaptive codon preference. Preferred codons are not GC rich, underscoring the independence from gBGC. Expression-associated “preference” largely matches adaptive codon preference but does not wholly capture the influence of selection shaping patterns across all genes, suggesting selective constraints associated specifically with high expression. We observe patterns consistent with effects on mRNA translation and stability shaping adaptive codon preference. Thus, our approach to quantifying adaptive codon preference provides a framework for inferring the sources of selection that shape CUB across different contexts within the genome.     

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.     

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.     

Analysis of synonymous codon usage in 11 human bocavirus isolates

Human Bocavirus (HBoV) is a novel virus which can cause respiratory tract disease in infants or children. In this study, the codon usage bias and the base composition variations in the available 11 complete HBoV genome sequences have been investigated. Although, there is a significant variation in codon usage bias among different HBoV genes, codon usage bias in HBoV is a little slight, which is mainly determined by the base compositions on the third codon position and the effective number of codons (ENC) value. The results of correspondence analysis (COA) and Spearman's rank correlation analysis reveals that the G + C compositional constraint is the main factor that determines the codon usage bias in HBoV and the gene's function also contributes to the codon usage in this virus. Moreover, it was found that the hydrophobicity of each protein and the gene length are also critical in affecting these viruses’ codon usage, although they were less important than that of the mutational bias and the genes’ function. At last, the relative synonymous codon usage (RSCU) of 44 genes from these 11 HBoV isolates is analyzed using a hierarchical cluster method. The result suggests that genes with same function yet from different isolates are classified into the same lineage and it does not depend on geographical location. These conclusions not only can offer an insight into the codon usage patterns and gene classification of HBoV, but also may help in increasing the efficiency of gene delivery/expression systems.     

Predicting gene expression level from codon usage bias

The "expression measure" of a gene, E(g), is a statistic devised to predict the level of gene expression from codon usage bias. E(g) has been used extensively to analyze prokaryotic genome sequences. We discuss 2 problems with this approach. First, the formulation of E(g) is such that genes with the strongest selected codon usage bias are not likely to have the highest predicted expression levels; indeed the correlation between E(g) and expression level is weak among moderate to highly expressed genes. Second, in some species, highly expressed genes do not have unusual codon usage, and so codon usage cannot be used to predict expression levels. We outline a simple approach, first to check whether a genome shows evidence of selected codon usage bias and then to assess the strength of bias in genes as a guide to their likely expression level; we illustrate this with an analysis of Shewanella oneidensis.     

Predicting Gene Expression Level from Relative Codon Usage Bias: An Application to Escherichia coli Genome

We present an expression measure of a gene, devised to predictthe level of gene expression from relative codon bias (RCB).There are a number of measures currently in use that quantifycodon usage in genes. Based on the hypothesis that gene expressivityand codon composition is strongly correlated, RCB has been definedto provide an intuitively meaningful measure of an extent ofthe codon preference in a gene. We outline a simple approachto assess the strength of RCB (RCBS) in genes as a guide totheir likely expression levels and illustrate this with an analysisof Escherichia coli (E. coli) genome. Our efforts to quantitativelypredict gene expression levels in E. coli met with a high levelof success. Surprisingly, we observe a strong correlation betweenRCBS and protein length indicating natural selection in favourof the shorter genes to be expressed at higher level. The agreementof our result with high protein abundances, microarray dataand radioactive data demonstrates that the genomic expressionprofile available in our method can be applied in a meaningfulway to the study of cell physiology and also for more detailedstudies of particular genes of interest.     

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.     

Preferential codon usage in genes

We present a method which permits comparison of the preferential use of degenerate codons within any gene. The method makes use of the triplet frequencies in the noncoding frames to assess whether a preference is specific to the reading frame. Preference is given a statistical meaning by use of the analysis of variance coupled to Duncan's multiple range test.Preferential use of degenerate codons is gene-specific and independent of gene size. The data suggest that any correlation between codon frequency distribution and tRNA levels is unreliable. In those animal genes examined, codons ending in C or G are preferred; in animal viruses tested, codons ending in U or A are preferred. Similarly, the bacterial genes and the genes of single-stranded DNA phages that we analyzed differed from each other as well as from eukaryotic genes in the third base of the codon.     

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.     

Selection on codon usage in Drosophila americana

Synonymous codons are not used at random, significantly influencing the base composition of the genome. The selection-mutation-drift model proposes that this bias reflects natural selection in favor of a subset of preferred codons. Previous estimates in Drosophila of the intensity of selective forces involved seem too large to be reconciled with theoretical predictions of the level of codon bias. This probably results from confounding effects of the demographic histories of the species concerned. We have studied three species of the virilis group of Drosophila, which are more likely to satisfy the assumptions of the evolutionary models. We analyzed the patterns of polymorphism and divergence in a sample of 18 genes and applied a new method for estimating the intensity of selection on synonymous mutations based on the frequencies of unpreferred mutations among polymorphic sites. This yielded estimates of selection intensities (N(e)s) of the order of 0.65, which is more compatible with the observed levels of codon bias. Our results support the action of both selection and mutational bias on codon usage bias and suggest that codon usage and genome base composition in the D. americana lineage are in approximate equilibrium. Biased gene conversion may also contribute to the observed patterns.     

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.     

Codon bias and gene expression.

The frequencies with which individual synonymous codons are used to code their cognate amino acids is quite variable from genome to genome and within genomes, from gene to gene. One particularly well documented codon bias is that associated with highly expressed genes in bacteria as well as in yeast; this is the so-called major codon bias. Here, it is suggested that the major codon bias is not an arrangement for regulating individual gene expression. Instead, the data suggest that this codon bias, which is correlated with a corresponding bias of tRNA abundance, is a global arrangement for optimizing the growth efficiency of cells. On the practical side, it is suggested that heterologous gene expression is not as sensitive to codon bias as previously thought, but that it is quite sensitive to other characteristics of the heterologous gene.     

Composition strand asymmetries in prokaryotic genomes: mutational bias and biased gene orientation

Most prokaryotic genomes display strand compositional asymmetries, but the reasons for these biases remain unclear. When the distribution of gene orientation is biased, as it often is, this may induce a bias in composition, as codon frequencies are not identical. We show here that this effect can be estimated and removed, and that the residual base skews are the highest at third base codon positions and lower at first and second positions. This strongly suggests that compositional asymmetries result from 1) a replication-related mutational bias that is filtered through selective pressure and/or from 2) an uneven distribution of gene orientation. In most cases, the mutational bias alters the codon usage and amino acid frequencies of the leading and the lagging strand. However, these features are not ubiquitous amongst prokaryotes, and the biological reasons for them remain to be found.     

Species Based Synonymous Codon Usage in Fusion Protein Gene of Newcastle Disease Virus

Newcastle disease is highly pathogenic to poultry and many other avian species. However, the Newcastle disease virus (NDV) has also been reported from many non-avian species. The NDV fusion protein (F) is a major determinant of its pathogenicity and virulence. The functionalities of F gene have been explored for the development of vaccine and diagnostics against NDV. Although the F protein is well studied but the codon usage and its nucleotide composition from NDV isolated from different species have not yet been explored. In present study, we have analyzed the factors responsible for the determination of codon usage in NDV isolated from four major avian host species. The F gene of NDV is analyzed for its base composition and its correlation with the bias in codon usage. Our result showed that random mutational pressure is responsible for codon usage bias in F protein of NDV isolates. Aromaticity, GC3s, and aliphatic index were not found responsible for species based synonymous codon usage bias in F gene of NDV. Moreover, the low amount of codon usage bias and expression level was further confirmed by a low CAI value. The phylogenetic analysis of isolates was found in corroboration with the relatedness of species based on codon usage bias. The relationship between the host species and the NDV isolates from the host does not represent a significant correlation in our study. The present study provides a basic understanding of the mechanism involved in codon usage among species.     

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     

Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency.

The variation in base composition at the three codon sites in relation to gene expressivity, the latter estimated by the Codon Adaptation Index, has been studied in a sample of 1371 Escherichia coli genes. Correlation and regression analyses show that increasing expression levels are accompanied by higher frequencies of base G at first, of base A at second and of base C at third codon positions. However, correlation between expressivity and base compositional biases at each codon site was only significant and positive at first codon position. The preference for G-starting codons as gene expression level increases is discussed in terms of translational optimization.     

Codon bias as a factor in regulating expression via translation rate in the human genome

We study the interrelations between tRNA gene copy numbers, gene expression levels and measures of codon bias in the human genome. First, we show that isoaccepting tRNA gene copy numbers correlate positively with expression-weighted frequencies of amino acids and codons. Using expression data of more than 14,000 human genes, we show a weak positive correlation between gene expression level and frequency of optimal codons (codons with highest tRNA gene copy number). Interestingly, contrary to non-mammalian eukaryotes, codon bias tends to be high in both highly expressed genes and lowly expressed genes. We suggest that selection may act on codon bias, not only to increase elongation rate by favoring optimal codons in highly expressed genes, but also to reduce elongation rate by favoring non-optimal codons in lowly expressed genes. We also show that the frequency of optimal codons is in positive correlation with estimates of protein biosynthetic cost, and suggest another possible action of selection on codon bias: preference of optimal codons as production cost rises, to reduce the rate of amino acid misincorporation. In the analyses of this work, we introduce a new measure of frequency of optimal codons (FOP'), which is unaffected by amino acid composition and is corrected for background nucleotide content; we also introduce a new method for computing expected codon frequencies, based on the dinucleotide composition of the introns and the non-coding regions surrounding a gene.