Codon Bias and Mutability in HIV Sequences

A survey of the patterns of synonymous codon preference in the HIV env gene reveals a correlation between the codon bias and the mutability requirements of different regions of the protein. At hypervariable regions in gp120 one finds a greater proportion of codons that tend to mutate nonsynonymously, but to a target that is similar in hydrophobicity and volume. We argue that this strategy results from a compromise between the selective pressure placed on the virus by the induced immune response, which favors amino acid substitutions in the complementarity determining regions, and the negative selection against missense mutations that violate structural constraints of the env protein. Received: 9 June 1997 / Accepted: 25 May 1998     

An Evaluation of Measures of Synonymous Codon Usage Bias

Synonymous codons are not generally used at equal frequencies, and this trend is observed for most genes and organisms. Several methods have been proposed and used to estimate the degree of the nonrandom use of the different synonymous codons. The estimates obtained by these methods, however, show different levels of both precision and dispersion when coding regions of a finite number of codons are under analysis. Here, we present a study, based on computer simulation, of how the different methods proposed to evaluate the nonrandom use of synonymous codons are affected by the length of the coding region analyzed. The results show that some of these methods are heavily influenced by the number of codons and that the comparison of codon usage bias between coding regions of different lengths shows a methodological bias under different conditions of nonrandom use of synonymous codons. The study of the dispersion of the estimates obtained by the different methods gives, on the other hand, an indication of the methods to be applied to compare values of codon usage bias among coding regions of equivalent length. Received: 10 September 1997 / Accepted: 23 March 1998     

Synonymous Nucleotide Divergence and Saturation: Effects of Site-Specific Variations in Codon Bias and Mutation Rates

The synonymous divergence between Escherichia coli and Salmonella typhimurium is explained in a model where there is a large variation between mutation rates at different nucleotide sites in the genome. The model is based on the experimental observation that spontaneous mutation rates can vary over several orders of magnitude at different sites in a gene. Such site-specific variation must be taken into account when studying synonymous divergence and will result in an apparent saturation below the level expected from an assumption of uniform rates. Recently, it has been suggested that codon preference in enterobacteria has a very large site-specific variation and that the synonymous divergence between different species, e.g., E. coli and Salmonella, is saturated. In the present communication it is shown that when site-specific variation in mutation rates is introduced, there is no need to invoke assumptions of saturation and a large variability in codon preference. The same rate variation will also bring average mutation rates as estimated from synonymous sequence divergence into numerical agreement with experimental values. Received: 10 July 1998 / Accepted: 20 August 1998     

Codon Usage Bias and tRNA Abundance in Drosophila

Codon usage bias of 1,117 Drosophila melanogaster genes, as well as fewer D. pseudoobscura and D. virilis genes, was examined from the perspective of relative abundance of isoaccepting tRNAs and their changes during development. We found that each amino acid contributes about equally and highly significantly to overall codon usage bias, with the exception of Asp which had very low contribution to overall bias. Asp was also the only amino acid that did not show a clear preference for one of its synonymous codons. Synonymous codon usage in Drosophila was consistent with ``optimal' codons deduced from the isoaccepting tRNA availability. Interestingly, amino acids whose major isoaccepting tRNAs change during development did not show as strong bias as those with developmentally unchanged tRNA pools. Asp is the only amino acid for which the major isoaccepting tRNAs change between larval and adult stages. We conclude that synonymous codon usage in Drosophila is well explained by tRNA availability and is probably influenced by developmental changes in relative abundance. Received: 5 December 1996 / Accepted: 14 June 1997     

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     

Recent Selection on Synonymous Codon Usage in Drosophila

Evidence from a variety of sources indicates that selection has influenced synonymous codon usage in Drosophila. It has generally been difficult, however, to distinguish selection that acted in the distant past from ongoing selection. However, under a neutral model, polymorphisms usually reflect more recent mutations than fixed differences between species and may, therefore, be useful for inferring recent selection. If the ancestral state is preferred, selection should shift the frequency distribution of derived states/site toward lower values; if the ancestral is unpreferred, selection should increase the number of derived states/site. Polymorphisms were classified as ancestrally preferred or unpreferred for several genes of D. simulans and D. melanogaster. A computer simulation of coalescence was employed to derive the expected frequency distributions of derived states/site under various modifications of the Wright–Fisher neutral model, and distributions of test statistics (t and Mann–Whitney U) were derived by appropriate sampling. One-tailed tests were applied to transformed frequency data to assess whether the two frequency distributions deviated from neutral expectations in the direction predicted by selection on codon usage. Several genes from D. simulans appear to be subject to recent selection on synonymous codons, including one gene with low codon bias, esterase-6. Selection may also be acting in D. melanogaster. Received: 15 April 1998 / Accepted: 13 May 1999     

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

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

Synonymous Codon Choices in the Extremely GC-Poor Genome of Plasmodium falciparum: Compositional Constraints and Translational Selection

We have analyzed the patterns of synonymous codon preferences of the nuclear genes of Plasmodium falciparum, a unicellular parasite characterized by an extremely GC-poor genome. When all genes are considered, codon usage is strongly biased toward A and T in third codon positions, as expected, but multivariate statistical analysis detects a major trend among genes. At one end genes display codon choices determined mainly by the extreme genome composition of this parasite, and very probably their expression level is low. At the other end a few genes exhibit an increased relative usage of a particular subset of codons, many of which are C-ending. Since the majority of these few genes is putatively highly expressed, we postulate that the increased C-ending codons are translationally optimal. In conclusion, while codon usage of the majority of P. falciparum genes is determined mainly by compositional constraints, a small number of genes exhibit translational selection. Received: 10 November 1998 / Accepted: 28 January 1999     

The Role of Context-Dependent Mutations in Generating Compositional and Codon Usage Bias in Grass Chloroplast DNA

Abstract The influence of local base composition on mutations in chloroplast DNA (cpDNA) is studied in detail and the resulting, empirically derived, mutation dynamics are used to analyze both base composition and codon usage bias. A 4 × 4 substitution matrix is generated for each of the 16 possible flanking base combinations (contexts) using 17,253 noncoding sites, 1309 of which are variable, from an alignment of three complete grass chloroplast genome sequences. It is shown that substitution bias at these sites is correlated with flanking base composition and that the A+T content of these flanking sites as well as the number of flanking pyrimidines on the same strand appears to have general influences on substitution properties. The context-dependent equilibrium base frequencies predicted from these matrices are then applied to two analyses. The first examines whether or not context dependency of mutations is sufficient to generate average compositional differences between noncoding cpDNA and silent sites of coding sequences. It is found that these two classes of sites exist, on average, in very different contexts and that the observed mutation dynamics are expected to generate significant differences in overall composition bias that are similar to the differences observed in cpDNA. Context dependency, however, cannot account for all of the observed differences: although silent sites in coding regions appear to be at the equilibrium predicted, noncoding cpDNA has a significantly lower A+T content than expected from its own substitution dynamics, possibly due to the influence of indels. The second study examines the codon usage of low-expression chloroplast genes. When context is accounted for, codon usage is very similar to what is predicted by the substitution dynamics of noncoding cpDNA. However, certain codon groups show significant deviation when followed by a purine in a manner suggesting some form of weak selection other than translation efficiency. Overall, the findings indicate that a full understanding of mutational dynamics is critical to understanding the role selection plays in generating composition bias and sequence structure.     

Intron Length and Codon Usage

The correlation was shown between the length of introns and the codon usage of the coding sequences of the corresponding genes, which in some cases can be related to the level of gene expression. The link is positive in the unicellular organisms, i.e., genes with the longer introns show the higher bias of codon usage. It is most pronounced in baker's yeast, where it is definitely related to the level of gene expression—genes with the higher level of expression have the longer introns. The correlation is inverted in multicellular organisms as compared to unicellular ones. Some organisms, however, do not show the link. The presence or absence of the link does not seem to be related to the GC percent of the coding sequences. Received: 7 December 1999 / Accepted: 10 May 2000     

Codon Usage by Transposable Elements and Their Host Genes in Five Species

We compared the codon usage of sequences of transposable elements (TEs) with that of host genes from the species Drosophila melanogaster, Arabidopsis thaliana, Caenorhabditis elegans, Saccharomyces cerevisiae, and Homo sapiens. Factorial correspondence analysis showed that, regardless of the base composition of the genome, the TEs differed from the genes of their host species by their AT-richness. In all species, the percentage of A + T on the third codon position of the TEs was higher than that on the first codon position and lower than that in the noncoding DNA of the genomes. This indicates that the codon choice is not simply the outcome of mutational bias but is also subject to selection constraints. A tendency toward higher A + T on the third position than on the first position was also found in the host genes of A. thaliana, C. elegans, and S. cerevisiae but not in those of D. melanogaster and H. sapiens. This strongly suggests that the AT choice is a host-independent characteristic common to all TEs. The codon usage of TEs generally appeared to be different from the mean of the host genes. In the AT-rich genomes of Arabidopsis thaliana, Caenorhabditis elegans, and Saccharomyces cerevisiae, the codon usage bias of TEs was similar to that of weakly expressed genes. In the GC-rich genome of D. melanogaster, however, the bias in codon usage of the TEs clearly differed from that of weakly expressed genes. These findings suggest that selection acts on TEs and that TEs may display specific behavior within the host genomes. Received: 2 May 2001 / Accepted: 29 October 2001     

A Survey of Codon and Amino Acid Frequency Bias in Microbial Genomes Focusing on Translational Efficiency

Unequal use of synonymous codons has been found in several prokaryotic and eukaryotic genomes. This bias has been associated with translational efficiency. The prevalence of this bias across lineages is currently unknown. Here, a new method (GCB) to measure codon usage bias is presented. It uses an iterative approach for the determination of codon scores and allows the computation of an index of codon bias suitable for interspecies comparison. A server to calculate GCB-values of individual genes as well as a list of compiled results are available at . The method was applied to complete bacterial genomes. The relation of codon usage bias with amino acid composition and the choice of stop codons were determined and discussed.     

Heterogeneity in Codon Usage in the Flatworm Schistosoma mansoni

Synonymous codon choices vary considerably among Schistosoma mansoni genes. Principal components analysis detects a single major trend among genes, which highly correlates with GC content in third codon positions and exons, but does not discriminate among putatively highly and lowly expressed genes. The effective number of codons used in each gene, and its distribution when plotted against GC₃, suggests that codon usage is shaped mainly by mutational biases. The GC content of exons, GC₃, 5′, 3′, and flanking (5′+ 3′+ introns) regions are all correlated among them, suggesting that variations in GC content may exist among different regions of the S. mansoni genome. We propose that this genome structure might be among the most important factors shaping codon usage in this species, although the action of selection on certain sequences cannot be excluded. Received: 10 March 1997 / Accepted: 27 June 1997     

Codon Bias is a Major Factor Explaining Phage Evolution in Translationally Biased Hosts

The size and diversity of bacteriophage populations require methodologies to quantitatively study the landscape of phage differences. Statistical approaches are confronted with small genome sizes forbidding significant single-phage analysis, and comparative methods analyzing full phage genomes represent an alternative but they are of difficult interpretation due to lateral gene transfer, which creates a mosaic spectrum of related phage species. Based on a large-scale codon bias analysis of 116 DNA phages hosted by 11 translationally biased bacteria belonging to different phylogenetic families, we observe that phage genomes are almost always under codon selective pressure imposed by translationally biased hosts, and we propose a classification of phages with translationally biased hosts which is based on adaptation patterns. We introduce a computational method for comparing phages sharing homologous proteins, possibly accepted by different hosts. We observe that throughout phages, independently from the host, capsid genes appear to be the most affected by host translational bias. For coliphages, genes involved in virion morphogenesis, host interaction and ssDNA binding are also affected by adaptive pressure. Adaptation affects long and small phages in a significant way. We analyze in more detail the Microviridae phage space to illustrate the potentiality of the approach. The small number of directions in adaptation observed in phages grouped around ϕX174 is discussed in the light of functional bias. The adaptation analysis of the set of Microviridae phages defined around ϕMH2K shows that phage classification based on adaptation does not reflect bacterial phylogeny. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Why are translationally sub-optimal synonymous codons used in Escherichia coli?

Natural selection favors certain synonymous codons which aid translation in Escherichia coli, yet codons not favored by translational selection persist. We use the frequency distributions of synonymous polymorphisms to test three hypotheses for the existence of translationally sub-optimal codons: (1) selection is a relatively weak force, so there is a balance between mutation, selection, and drift; (2) at some sites there is no selection on codon usage, so some synonymous sites are unaffected by translational selection; and (3) translationally sub-optimal codons are favored by alternative selection pressures at certain synonymous sites. We find that when all the data is considered, model 1 is supported and both models 2 and 3 are rejected as sole explanations for the existence of translationally sub-optimal codons. However, we find evidence in favor of both models 2 and 3 when the data is partitioned between groups of amino acids and between regions of the genes. Thus, all three mechanisms appear to contribute to the existence of translationally sub-optimal codons in E. coli. Received: 18 July 2000 / Accepted: 17 April 2001     

Trends in Codon and Amino Acid Usage in Thermotoga maritima

The usage of synonymous codons and the frequencies of amino acids were investigated in the complete genome of the bacterium Thermotoga maritima using a multivariate statistical approach. The GC3 content of each gene was the most prominent source of variation of codon usage. Surprisingly the usage of UGU and UGC (synonymous triplets coding for Cys, the least frequent amino acid in this species) was detected as the second most prominent source of variation. However, this result is probably an artifact due to the very low frequency of Cys together with the nonbiased composition of this genome. The third trend was related to the preferential usage of a subset of codons among highly expressed genes, and these triplets are presumed to be translationally optimal. Concerning the amino acid usage, the hydropathy level of each protein (and therefore the frequency of charged residues) was the main trend, while the second factor was related to the frequency of usage of the smaller residues, suggesting that the cell economy strongly influences the architecture of the proteins. The third axis of the analysis discriminated the usage of Phe, Tyr, Trp (aromatic residues) plus Cys, Met, and His. These six residues have in common the property of being the preferential targets of reactive oxygen species, and therefore the anaerobic condition of T. maritima is an important factor for the amino acid frequencies. Finally, the Cys content of each protein was the fourth trend. Received: 22 June 2001 / Accepted: 1 October 2001     

Phylogenetic Analysis of Evolutionary Relationships of the Planctomycete Division of the Domain Bacteria Based on Amino Acid Sequences of Elongation Factor Tu

Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique 11-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum-likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria. It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions. Received: 21 August 2000 / Accepted: 8 January 2001     

Lineage Specificity of the Evolutionary Dynamics of the mtDNA D-Loop Region in Rodents

This paper reports an intraorder study on the D-loop-containing region of the mitochondrial DNA in rodents. A complete multialignment of this region is not feasible with the exception of some conserved regions. The comparative analysis of 25 complete rodent sequences from 23 species plus one lagomorph has revealed that only the central domain (CD), a conserved region of about 80 bp in the extended termination-associated sequences (ETAS) domain, adjacent to the CD, the ETAS1, and conserved sequence block (CSB) 1 blocks are present in all rodent species, whereas the presence of CSB2 and CSB3 is erratic within the order. We have also found a conserved region of 90 bp located between tRNAPro and ETAS1 present in fat dormouse, squirrel, guinea pig, and rabbit. Repeated sequences are present in both the ETAS and the CSB domain, but the repeats differ in length, copy number, and base composition in different species. The potential use of the D-loop for evolutionary studies has been investigated; the presence/absence of conserved blocks and/or repeated sequences cannot be used as a reliable phylogenetic marker, since in some cases they may be shared by distantly related organisms but not by close ones, while in other ones a relationship between tree topology and presence/absence of such motifs is observed. Better results can be obtained by the use of the CD, which, however, due to its reduced size, when used for tracing a phylogenetic tree, shows some nodes with low statistical support. Received: 26 February 2001 / Accepted: 6 June 2001     

Multiple Mechanosensitive Ion Channels from Escherichia coli,Activated at Different Thresholds of Applied Pressure

Mechanosensitive ion channels from Escherichia coli were studied in giant proteoliposomes reconstituted from an inner membrane fraction, or in giant round cells in which the outer membrane and the cell wall had been disrupted by a lysozyme-EDTA treatment and a mild osmotic shock. Patch-clamp experiments revealed the presence in these two preparations of an array of different conductances (100 to 2,300 pS in 0.1 m KCl) activated by stretch. The electrical activity induced by stretch in the native membrane was complex, due to the activation of several different conductances. In contrast, patches of proteoliposomes generally contained clusters of identical conductances, which differed from patch to patch. These experiments are consistent with the notion that these different conductances correspond to different proteins in the plasma membrane of E. coli, which segregate into clusters of identical channels on dilution involved in reconstitution in proteoliposomes. These conductances could be grouped into three subfamilies of poorly selective channels. In both preparations, the higher the conductance, the higher was the negative pressure needed for activation. We discuss the putative role of these channels as parts of a multicomponent osmoregulatory system. Received: 23 May 1995/Revised: 31 January 1996