Phylogenetic estimation under codon models can be biased by codon usage heterogeneity

In theory, codon models that account for the dependence of nucleotide substitutions between codon positions as well as differences between synonymous and non-synonymous changes best describe the sequence evolution in protein coding genes. However, in practice we know little about the degree to which violations of the assumptions of codon model-based estimates occur, and how significant these artifacts may be. In nucleotide-based phylogenies from first and second codon positions in a concatenated plastid gene data set, two distantly related taxa--dinoflagellate and haptophyte plastids--were robustly grouped together. This artifactual grouping is attributed to the parallel heterogeneity in leucine (Leu) and serine (Ser) codon usages in the data set. Here, by using this data set, we demonstrated that codon-based phylogenetic estimations are seriously biased, robustly uniting the dinoflagellate and haptophyte plastids into a monophyletic clade, when the model assumption of homogeneity of codon composition was violated. Our results suggest that similar phylogenetic artifacts may occur via codon usage heterogeneity in any amino acids in codon model-based estimations. We advise that homogeneity in codon usage across taxa in a data set be confirmed before codon model-based phylogenetic estimation is attempted.     

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.     

Adjustment of the tRNA population to the codon usage in chloroplasts.

In chloroplasts there is a correlation between the amounts of tRNAs specific for a given amino acid and the codons specifying this amino acid. Furthermore, for the amino acids coded for by more than one codon, the population of isoaccepting tRNAs is adjusted to the frequency of synonymous codons used in chloroplast protein genes. A comparison by two-dimensional gel electrophoresis of the tRNA populations extracted from chloroplasts and from chloroplast polysomes shows that all chloroplast tRNAs are involved in protein biosynthesis.     

Structural features of multiple nifH-like sequences and very biased codon usage in nitrogenase genes of Clostridium pasteurianum.

The structural gene (nifH1) encoding the nitrogenase iron protein of Clostridium pasteurianum has been cloned and sequenced. It is located on a 4-kilobase EcoRI fragment (cloned into pBR325) that also contains a portion of nifD and another nifH-like sequence (nifH2). C. pasteurianum nifH1 encodes a polypeptide (273 amino acids) identical to that of the isolated iron protein, indicating that the smaller size of the C. pasteurianum iron protein does not result from posttranslational processing. The 5' flanking region of nifH1 or nifH2 does not contain the nif promoter sequences found in several gram-negative bacteria. Instead, a sequence resembling the Escherichia coli consensus promoter (TTGACA-N17-TATAAT) is present before C. pasteurianum nifH2, and a TATAAT sequence is present before C pasteurianum nifH1. Codon usage in nifH1, nifH2, and nifD (partial) is very biased. A preference for A or U in the third position of the codons is seen. nifH2 could encode a protein of 272 amino acid residues, which differs from the iron protein (nifH1 product) in 23 amino acid residues (8%). Another nifH-like sequence (nifH3) is located on a nonadjacent EcoRI fragment and has been partially sequenced. C. pasteurianum nifH2 and nifH3 may encode proteins having several amino acids that are conserved in other proteins but not in C. pasteurianum iron protein, suggesting a possible role for the multiple nifH-like sequences of C. pasteurianum in the evolution of nifH. Among the nine sequenced iron proteins, only the C. pasteurianum protein lacks a conserved lysine residue which is near the extended C terminus of the other iron proteins. The absence of this positive charge in the C. pasteurianum iron protein might affect the cross-reactivity of the protein in heterologous systems.     

The evolution of biased codon and amino acid usage in nematode genomes

Despite the degeneracy of the genetic code, whereby different codons encode the same amino acid, alternative codons and amino acids are utilized nonrandomly within and between genomes. Such biases in codon and amino acid usage have been demonstrated extensively in prokaryote genomes and likely reflect a balance between the action of mutation, selection, and genetic drift. Here, we quantify the effects of selection and mutation drift as causes of codon and amino acid-usage bias in a large collection of nematode partial genomes from 37 species spanning approximately 700 Myr of evolution, as inferred from expressed sequence tag (EST) measures of gene expression and from base composition variation. Average G + C content at silent sites among these taxa ranges from 10% to 63%, and EST counts range more than 100-fold, underlying marked differences between the identities of major codons and optimal codons for a given species as well as influencing patterns of amino acid abundance among taxa. Few species in our sample demonstrate a dominant role of selection in shaping intragenomic codon-usage biases, and these are principally free living rather than parasitic nematodes. This suggests that deviations in effective population size among species, with small effective sizes among parasites, are partly responsible for species differences in the extent to which selection shapes patterns of codon usage. Nevertheless, a consensus set of optimal codons emerges that is common to most taxa, indicating that, with some notable exceptions, selection for translational efficiency and accuracy favors similar sets of codons regardless of the major codon-usage trends defined by base compositional properties of individual nematode genomes.     

Codon usage divergence of homologous vertebrate genes and codon usage clock

Summary This paper is concerned with the divergence of synonymous codon usage and its bias in three homologous genes within vertebrate species. Genetic distances among species are described in terms of synonymous codon usage divergence and the correlation is found between the genetic distances and taxonomic distances among species under study. A codon usage clock is reported in alphaglobin and beta-globin. A method is developed to define the synonymous codon preference bias and it is observed that the bias changes considerably among species.     

A role for tRNA modifications in genome structure and codon usage

Transfer RNA (tRNA) gene content is a differentiating feature of genomes that contributes to the efficiency of the translational apparatus, but the principles shaping tRNA gene copy number and codon composition are poorly understood. Here, we report that the emergence of two specific tRNA modifications shaped the structure and composition of all extant genomes. Through the analysis of more than 500 genomes, we identify two kingdom-specific tRNA modifications as major contributors that separated archaeal, bacterial, and eukaryal genomes in terms of their tRNA gene composition. We show that, contrary to prior observations, genomic codon usage and tRNA gene frequencies correlate in all kingdoms if these two modifications are taken into account and that presence or absence of these modifications explains patterns of gene expression observed in previous studies. Finally, we experimentally demonstrate that human gene expression levels correlate well with genomic codon composition if these identified modifications are considered.     

The effect of context on synonymous codon usage in genes with low codon usage bias.

The effect of neighbouring bases on the usage of synonymous codons in genes with low codon usage bias in yeast and E. coli is examined. The codon adaptation index is employed to identify a group of genes in each organism with low codon usage bias, which are likely to be weakly expressed. A similar pattern is found in complementary sequences with respect to synonymous usage of A vs G or of U vs C. It is suggested that this may reflect an effect of context on mutation rates in weakly expressed genes.     

Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes

By considering the nucleotide sequence of several highly expressed coding regions in bacteriophage MS2 and mRNAs from Escherichia coli, it is possible to deduce some rules which govern the selection of the most appropriate synonymous codons NNU or NNC read by tRNAs having GNN, QNN or INN as anticodon. The rules fit with the general hypothesis that an efficient in-phase translation is facilitated by proper choice of degenerate codewords promoting a codon-anticodon interaction with intermediate strength (optimal energy) over those with very strong or very weak interaction energy. Moreover, codons corresponding to minor tRNAs are clearly avoided in these efficiently expressed genes. These correlations are clearcut in the normal reading frame but not in the corresponding frameshift sequences +1 and +2. We hypothesize that both the optimization of codon-anticodon interaction energy and the adaptation of the population to codon frequency or vice versa in highly expressed mRNAs of E. coli are part of a strategy that optimizes the efficiency of translation. Conversely, codon usage in weakly expressed genes such as repressor genes follows exactly the opposite rules. It may be concluded that, in addition to the need for coding an amino acid sequence, the energetic consideration for codon-anticodon pairing, as well as the adaptation of codons to the tRNA population, may have been important evolutionary constraints on the selection of the optimal nucleotide sequence.     

Essential factors determining codon usage in ubiquitin genes

Summary Ubiquitin is ubiquitous in all eukaryotes and its amino acid sequence shows extreme conservation. Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers. The nucleotide sequences coding for 13 ubiquitin genes from 11 species reported so far have been compiled and analyzed. The G+C content of codon third base reveals a positive linear correlation with the genome G+C content of the corresponding species. The slope strongly suggests that the overall G+C content of codons of polyubiquitin genes clearly reflects the genome G+C content by AT/GC substitutions at the codon third position. The G+C content of ubiquitin codon third base also shows a positive linear correlation with the overall G+C content of coding regions of compiled genes, indicating the codon choices among synonymous codons reflect the average codon usage pattern of corresponding species. On the other hand, the monoubiquitin gene, which is different from the polyubiquitin gene in gene organization, gene expression, and function of the encoding protein, shows a different codon usage pattern compared with that of the polyubiquitin gene. From comparisons of the levels of synonymous substitutions among ubiquitin repeats and the homology of the amino acid sequence of the tail of monomeric ubiquitin genes, we propose that the molecular evolution of ubiquitin genes occurred as follows: Plural primitive ubiquitin sequences were dispersed on genome in ancestral eukaryotes. Some of them situated in a particular environment fused with the tail sequence to produce monomeric ubiquitin genes that were maintained across species. After divergence of species, polyubiquitin genes were formed by duplication of the other primitive ubiquitin sequences on different chromosomes. Differences in the environments in which ubiquitin genes are embedded reflect the differences in codon choice and in gene expression pattern between poly- and monomeric ubiquitin genes.     

Clonorchis sinensis: codon usage in nuclear genes

Codon usage in Clonorchis sinensis was analyzed using 12,515 codons from 38 coding sequences. Total GC content was 49.83%, and GC1, GC2 and GC3 contents were 56.32%, 43.15% and 50.00%, respectively. The effective number of codons converged at 51-53 codons. When plotted against total GC content or GC3, codon usage was distributed in relation to GC3 biases. Relative synonymous codon usage for each codon revealed a single major trend, which was highly correlated with GC content at the third position when codons began with A or U at the first two positions. In codons beginning with G or C base at the first two positions, the G or C base rarely occurred at the third position. These results suggest that codon usage is shaped by a bias towards G or C at the third base, and that this is affected by the first and second bases.     

Gradients in nucleotide and codon usage along Escherichia coli genes

The usage of codons and nucleotide combinations varies along genes and systematic variation causes gradients in usage. We have studied such gradients of nucleotides and nucleotide combinations and their immediate context in Escherichia coli. To distinguish mutational and selectional effects, the genes were subdivided into three groups with different codon usage bias and the gradients of nucleotide usage were studied in each group. Some combinations that can be associated with a propensity for processivity errors show strong negative gradients that become weaker in genes with low codon bias, consistent with a selection on translational efficiency. One of the strongest gradients is for third position G, which shows a pervasive positive gradient in usage in most contexts of surrounding bases.     

Measure of synonymous codon usage diversity among genes in bacteria

Background  

In many bacteria, intragenomic diversity in synonymous codon usage among genes has been reported. However, no quantitative attempt has been made to compare the diversity levels among different genomes. Here, we introduce a mean dissimilarity-based index (Dmean) for quantifying the level of diversity in synonymous codon usage among all genes within a genome.     

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.     

Synonymous codon usage variation among Giardia lamblia genes and isolates.

The pattern of codon usage in the amitochondriate diplomonad Giardia lamblia has been investigated. Very extensive heterogeneity was evident among a sample of 65 genes. A discrete group of genes featured unusual codon usage due to the amino acid composition of their products: these variant surface proteins (VSPs) are unusually rich in Cys and, to a lesser extent, Gly and Thr. Among the remaining 50 genes, correspondence analysis revealed a single major source of variation in synonymous codon usage. This trend was related to the extent of use of a particular subset of 21 codons which are inferred to be those which are optimal for translation; at one end of this trend were genes expected to be expressed at low levels with near random codon usage, while at the other extreme were genes expressed at high levels in which these optimal codons are used almost exclusively. These optimal codons all end in C or G so G + C content at silent sites varies enormously among genes, from values around 40%, expected to reflect the background level of the genome, up to nearly 100%. Although VSP genes are occasionally extremely highly expressed, they do not, in general, have high frequencies of optimal codons, presumably because their high expression is only intermittent. These results indicate that natural selection has been very effective in shaping codon usage in G. lamblia. These analyses focused on sequences from strains placed within G. lamblia "assemblage A"; a few sequences from other strains revealed extensive divergence at silent sites, including some divergence in the pattern of codon usage.