Nonrandom Intragenic Variations in Patterns of Codon Bias Implicate a Sequential Interplay Between Transitional Genetic Drift and Functional Amino Acid Selection

Although most codon third bases appear to be functionless, the synonymous codons so defined exhibit a strikingly nonrandom distribution (codon bias) within human and other genes. To examine this phenomenon further, we generated a database of DNA sequences encoding human transmembrane cell-surface receptor proteins. Using this database we show here that the guanine and cytosine content of codon third bases (GC3) varies intragenically with the nature of the specified receptor domains (transmembrane > extracellular > intracellular domains; p < 0.001), the phenotype of the encoded amino acids (hydrophobic > hydrophilic > neutral amino acids; p < 0.001), and the receptor affiliation of the transmembrane domain superfamily (G-protein- coupled receptors > receptor tyrosine kinases; p < 0.001). Within gene regions specifying transmembrane domains, GC3 declines as domain functionality becomes redundant with increasing hydrophobicity (p < 0.001). Codons containing the second-base cytosine (XCZ, which encodes neutral amino acids) are selectively depleted of third-base adenine content (A3: XCA codons) when encoding transmembrane domain residues, consistent with positive selection for transitional mutation of XCG to XTG (which encodes hydrophobic amino acids) rather than to the synonymous XCA. Supporting this XCG XTG mechanism of codon bias, the G3:A3 ratio of codons specifying the transmembrane amino acid glycine (GGZ) is intermediate between that of its functional homolog alanine (GCZ) and that of hydrophobic valine (GTZ), even though the C3:T3 ratios are similar. Conversely, nearest-neighbor analysis of third bases 5 to codons specifying valine and leucine (CTZ) confirms a significant difference in C3:T3 but not G3:A3 ratios (i.e., C3/G1 T3/G1 > C3/A1; p < 0.001), consistent with the functionally advantageous retention of hydrophobic residues. These data raise the possibility that patterns of intragenic codon bias reflect a balance between negative and positive selection, suggesting in turn that analysis of codon third-base usage may help to predict the functional significance of encoded products. Supplementary information: Current address: (K. Lin) College of Life Sciences, Beijing Normal University, Beijing 100875, China     

Correlations between the compositional properties of human genes,codon usage,and amino acid composition of proteins

Summary We have analyzed the correlation that exists between the GC levels of third and first or second codon position for about 1400 human coding sequences. The linear relationship that was found indicates that the large differences in GC level of third codon positions of human genes are paralleled by smaller differences in GC levels of first and second codon positions. Whereas third codon position differences correspond to very large differences in codon usage within the human genome, the first and second codon position differences correspond to smaller, yet very remarkable, differences in the amino acid composition of encoded proteins. Because GC levels of codon positions are linearly correlated with the GC levels of the isochores harboring the corresponding genes, both codon usage and amino acid composition are different for proteins encoded by genes located in isochores of different GC levels. Furthermore, we have also shown that a linear relationship with a unity slope and a correlation coefficient of 0.77 exists between GC levels of introns and exons from the 238 human genes currently available for this analysis. Introns are, however, about 5% lower in GC, on average, than exons from the same genes.     

DNA asymmetric strand bias affects the amino acid composition of mitochondrial proteins.

Variations in GC content between genomes have been extensively documented. Genomes with comparable GC contents can, however, still differ in the apportionment of the G and C nucleotides between the two DNA strands. This asymmetric strand bias is known as GC skew. Here, we have investigated the impact of differences in nucleotide skew on the amino acid composition of the encoded proteins. We compared orthologous genes between animal mitochondrial genomes that show large differences in GC and AT skews. Specifically, we compared the mitochondrial genomes of mammals, which are characterized by a negative GC skew and a positive AT skew, to those of flatworms, which show the opposite skews for both GC and AT base pairs. We found that the mammalian proteins are highly enriched in amino acids encoded by CA-rich codons (as predicted by their negative GC and positive AT skews), whereas their flatworm orthologs were enriched in amino acids encoded by GT-rich codons (also as predicted from their skews). We found that these differences in mitochondrial strand asymmetry (measured as GC and AT skews) can have very large, predictable effects on the composition of the encoded proteins.     

Compositional correlation and codon usage studies in Buchnera aphidicola

Compositional distributions in three different codon positions as well as codon usage biases of all available DNA sequences of Buchnera aphidicola genome have been analyzed. It was observed that GC levels among the three codon positions is I>II>III as observed in other extremely high AT rich organisms. B. aphidicola being an AT rich organism is expected to have A and/or T at the third positions of codons. Overall codon usage analyses indicate that A and/or T ending codons are predominant in this organism and some particular amino acids are abundant in the coding region of genes. However, multivariate statistical analysis indicates two major trends in the codon usage variation among the genes; one being strongly correlated with the GC contents at the third synonymous positions of codons, and the other being associated with the expression level of genes. Moreover, codon usage biases of the highly expressed genes are almost identical with the overall codon usage biases of all the genes of this organism. These observations suggest that mutational bias is the main factor in determining the codon usage variation among the genes in B. aphidicola.     

GC constituents and relative codon expressed amino acid composition in cyanobacterial phycobiliproteins

The genomic as well as structural relationship of phycobiliproteins (PBPs) in different cyanobacterial species are determined by nucleotides as well as amino acid composition. The genomic GC constituents influence the amino acid variability and codon usage of particular subunit of PBPs. We have analyzed 11 cyanobacterial species to explore the variation of amino acids and causal relationship between GC constituents and codon usage. The study at the first, second and third levels of GC content showed relatively more amino acid variability on the levels of G3 + C3 position in comparison to the first and second positions. The amino acid encoded GC rich level including G rich and C rich or both correlate the codon variability and amino acid availability. The fluctuation in amino acids such as Arg, Ala, His, Asp, Gly, Leu and Glu in α and β subunits was observed at G1C1 position; however, fluctuation in other amino acids such as Ser, Thr, Cys and Trp was observed at G2C2 position. The coding selection pressure of amino acids such as Ala, Thr, Tyr, Asp, Gly, Ile, Leu, Asn, and Ser in α and β subunits of PBPs was more elaborated at G3C3 position. In this study, we observed that each subunit of PBPs is codon specific for particular amino acid. These results suggest that genomic constraint linked with GC constituents selects the codon for particular amino acids and furthermore, the codon level study may be a novel approach to explore many problems associated with genomics and proteomics of cyanobacteria.     

Across bacterial phyla, distantly-related genomes with similar genomic GC content have similar patterns of amino acid usage

The GC content of bacterial genomes ranges from 16% to 75% and wide ranges of genomic GC content are observed within many bacterial phyla, including both gram negative and gram positive phyla. Thus, divergent genomic GC content has evolved repeatedly in widely separated bacterial taxa. Since genomic GC content influences codon usage, we examined codon usage patterns and predicted protein amino acid content as a function of genomic GC content within eight different phyla or classes of bacteria. We found that similar patterns of codon usage and protein amino acid content have evolved independently in all eight groups of bacteria. For example, in each group, use of amino acids encoded by GC-rich codons increased by approximately 1% for each 10% increase in genomic GC content, while the use of amino acids encoded by AT-rich codons decreased by a similar amount. This consistency within every phylum and class studied led us to conclude that GC content appears to be the primary determinant of the codon and amino acid usage patterns observed in bacterial genomes. These results also indicate that selection for translational efficiency of highly expressed genes is constrained by the genomic parameters associated with the GC content of the host genome.     

Comparative genome analysis of six malarial parasites using codon usage bias based tools

Codon usage bias (CUB) is an omnipresent phenomenon, which occurs in nearly all organisms. Previous studies of codon bias in Plasmodium species were based on a limited dataset. This study uses whole genome datasets for comparative genome analysis of six Plasmodium species using CUB and other related methods for the first time. Codon usage bias, compositional variation in translated amino acid frequency, effective number of codons and optimal codons are analyzed for P.falciparum, P.vivax, P.knowlesi, P.berghei, P.chabaudii and P.yoelli. A plot of effective number of codons versus GC3 shows their differential codon usage pattern arises due to a combination of mutational and translational selection pressure. The increased relative usage of adenine and thymine ending optimal codons in highly expressed genes of P.falciparum is the result of higher composition biased pressure, and usage of guanine and cytosine bases at third codon position can be explained by translational selection pressure acting on them. While higher usage of adenine and thymine bases at third codon position in optimal codons of P.vivax highlights the role of translational selection pressure apart from composition biased mutation pressure in shaping their codon usage pattern. The frequency of those amino acids that are encoded by AT ending codons are significantly high in P.falciparum due to action of high composition biased mutational pressure compared with other Plasmodium species. The CUB variation in the three rodent parasites, P.berghei, P.chabaudii and P.yoelli is strikingly similar to that of P.falciparum. The simian and human malarial parasite, P.knowlesi shows a variation in codon usage bias similar to P.vivax but on closer study there are differences confirmed by the method of Principal Component Analysis (PCA).

Abbreviations

CDS - Coding sequences, GC1 - GC composition at first site of codon, GC2 - GC composition at second site of codon, GC3 - GC composition at third site of codon, Ala - Alanine, Arg - Arginine, Asn - Asparagine, Asp - Aspartic acid, Cys - Cysteine, Gln - Glutamine Glu - Glutamic acid Gly - Glycine His - Histidine Ile - Isoleucine Leu - Leucine Lys - Lysine Met - Methionine Phe - Phenylalanine Pro - Proline Ser - Serine Thr - Threonine Trp - Tryptophan Tyr - Tyrosine Val - Valine.     

Base Composition Skews, Replication Orientation, and Gene Orientation in 12 Prokaryote Genomes

Variation in GC content, GC skew and AT skew along genomic regions was examined at third codon positions in completely sequenced prokaryotes. Eight out of nine eubacteria studied show GC and AT skews that change sign at the origin of replication. The leading strand in DNA replication is G-T rich at codon position 3 in six eubacteria, but C-T rich in two Mycoplasma species. In M. genitalium the AT and GC skews are symmetrical around the origin and terminus of replication, whereas its GC content variation has been shown to have a centre of symmetry elsewhere in the genome. Borrelia burgdorferi and Treponema pallidum show extraordinary extents of base composition skew correlated with direction of DNA replication. Base composition skews measured at third codon positions probably reflect mutational biases, whereas those measured over all bases in a sequence (or at codon positions 1 and 2) can be strongly affected by protein considerations due to the tendency in some bacteria for genes to be transcribed in the same direction that they are replicated. Consequently in some species the direction of skew for total genomic DNA is opposite to that for codon position 3. Received: 2 February 1998 / Accepted: 15 June 1998     

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.     

不具有3-碱基周期性的编码序列初探

对120个较短编码序列(＜1 200 bp)的Fourier频谱进行分析表明,3-碱基周期性在短编码序列中并不是绝对存在的.统计分析提示,编码序列有无3-碱基周期性与序列的碱基组成和分布、所编码蛋白质氨基酸的选用和顺序以及同义密码子的使用都有一定的关系.一般地,非周期-3序列中A+U含量高于G+C含量,周期-3序列的情况则相反;非周期-3序列中碱基在密码子三个位点上的分布比周期-3序列中的分布均匀;非周期-3序列密码子和氨基酸的使用偏向没有周期-3序列的大.在利用Fourier分析方法预测DNA序列中的基因和外显子时,应充分考虑到这些现象.     

Comparative analysis of the base composition and codon usages in fourteen mycobacteriophage genomes

To study the possible codon usage and base composition variation in the bacteriophages, fourteen mycobacteriophages were used as a model system here and both the parameters in all these phages and their plating bacteria, M. smegmatis had been determined and compared. As all the organisms are GC-rich, the GC contents at third codon positions were found in fact higher than the second codon positions as well as the first + second codon positions in all the organisms indicating that directional mutational pressure is strongly operative at the synonymous third codon positions. Nc plot indicates that codon usage variation in all these organisms are governed by the forces other than compositional constraints. Correspondence analysis suggests that: (i) there are codon usage variation among the genes and genomes of the fourteen mycobacteriophages and M. smegmatis, i.e., codon usage patterns in the mycobacteriophages is phage-specific but not the M. smegmatis-specific; (ii) synonymous codon usage patterns of Barnyard, Che8, Che9d, and Omega are more similar than the rest mycobacteriophages and M. smegmatis; (iii) codon usage bias in the mycobacteriophages are mainly determined by mutational pressure; and (iv) the genes of comparatively GC rich genomes are more biased than the GC poor genomes. Translational selection in determining the codon usage variation in highly expressed genes can be invoked from the predominant occurrences of C ending codons in the highly expressed genes. Cluster analysis based on codon usage data also shows that there are two distinct branches for the fourteen mycobacteriophages and there is codon usage variation even among the phages of each branch.     

Comparison of responses by bacteriophages and bacteria to pressures on the base composition of open reading frames

Differences in the base composition of genomes can occur because of GC pressure, purine-loading pressure (AG pressure) and RNY pressure, for which there are possible functional explanations, and because of the more abstract pressures exerted by individual bases. The graphical approach of Muto and Osawa was used to analyse how bacteriophages and bacteria balance potentially conflicting pressures on their genomes. Phages generally respond to AG pressure by increasing A while keeping T constant, and by decreasing C while keeping G constant. In contrast, bacteria generally increase both A and T, the former more so, and decrease both G and C, the latter more so. These differences largely occur at third codon positions, which are more responsive than first and second codon positions to AG pressure and GC pressure. Phages respond to AG pressure more in the third codon position than bacteria, whereas bacteria respond more in the first codon position than phages. Conversely, bacteria respond to GC pressure more in the third codon position than phages, whereas phages respond more in the first codon position than bacteria. As GC pressure increases, A is traded for C and AG pressure decreases; first and second codon positions, having more A than T, are most responsive to this negative effect of increased GC pressure; third positions either do not respond (phages) or respond weakly (bacteria). In a set of 48 phage-host pairs, degrees of purine loading were less correlated between phage and host than were GC percentages. These results suggest that pressures on conventional and genome phenotypes operate differentially in phages and bacteria, generating both general differences in base composition and specific differences characteristic of particular phage-host pairs. The reciprocal relationship between GC pressure and AG pressure implies that effects attributed to GC pressure may actually be due to AG pressure, and vice versa.     

A second regulatory gene, blaR1, encoding a potential penicillin-binding protein required for induction of beta-lactamase in Bacillus licheniformis.

A second regulatory locus (blaR1) required for the induction of beta-lactamase synthesis in Bacillus licheniformis 749 was cloned and sequenced. The gene was located on a 5.2-kilobase-pair SphI DNA fragment which also contained the beta-lactamase (blaP) and repressor (blaI) genes. Bacillus subtilis BD224 carrying these three genes synthesized beta-lactamase on exposure to cephalosporin C, whereas Escherichia coli HB101 carrying the genes did not show any detectable induction of the enzyme. An open reading frame of 1,803 bases was identified as the blaR1 gene by subcloning and DNA sequencing. The gene started 2 bases downstream of the termination codon of bla1 and was preceded by a putative Shine-Dalgarno sequence (AAGGA) with a spacing of 5 bases. The deduced blaR1 product (601 amino acids) had a molecular weight of 68,425. Five transmembrane regions were predicted from the hydrophobicity profile. The region around Phe-Ala-Pro-Ala-Ser-Thr-Tyr-Lys (amino acids 398 to 405), which appeared to be located outside the membrane, was homologous to the binding regions of penicillin-binding proteins, including the beta-lactamases. The segment of 22 amino acids from 400 to 421 showed more than 70% homology to the penicillin-binding region of PBP 2 of E. coli. The blaR1 gene encodes a potential penicillin receptor which is required for the induction of beta-lactamase in B. licheniformis 749.     

Amino acid and nucleotide recurrence in aligned sequences: synonymous substitution patterns in association with global and local base compositions

The tendency for repetitiveness of nucleotides in DNA sequences has been reported for a variety of organisms. We show that the tendency for repetitive use of amino acids is widespread and is observed even for segments conserved between human and Drosophila melanogaster at the level of >50% amino acid identity. This indicates that repetitiveness influences not only the weakly constrained segments but also those sequence segments conserved among phyla. Not only glutamine (Q) but also many of the 20 amino acids show a comparable level of repetitiveness. Repetitiveness in bases at codon position 3 is stronger for human than for D.melanogaster, whereas local repetitiveness in intron sequences is similar between the two organisms. While genes for immune system-specific proteins, but not ancient human genes (i.e. human homologs of Escherichia coli genes), have repetitiveness at codon bases 1 and 2, repetitiveness at codon base 3 for these groups is similar, suggesting that the human genome has at least two mechanisms generating local repetitiveness. Neither amino acid nor nucleotide repetitiveness is observed beyond the exon boundary, denying the possibility that such repetitiveness could mainly stem from natural selection on mRNA or protein sequences. Analyses of mammalian sequence alignments show that while the ‘between gene’ GC content heterogeneity, which is linked to ‘isochores’, is a principal factor associated with the bias in substitution patterns in human, ‘within gene’ heterogeneity in nucleotide composition is also associated with such bias on a more local scale. The relationship amongst the various types of repetitiveness is discussed.     

Mitochondrial cytochrome C oxidase subunit I of Manduca sexta and a comparison with other invertebrate genes

A cDNA encoding mitochondrial cytochrome c oxidase subunit I (mt COI) from Manduca sexta (Lepidoptera: Sphingidae) was cloned and sequenced. AT (adenine-thymine) content is high and codon usage is biased and likely reflects the role of mt COI in electron transport. The encoded protein is 514 amino acids long, contains seven invariant His residues observed in COIs in all organisms and would be predicted to be composed of 12 transmembrane regions.     

Mutation and Selection Cause Codon Usage and Bias in Mitochondrial Genomes of Ribbon Worms (Nemertea)

The phenomenon of codon usage bias is known to exist in many genomes and it is mainly determined by mutation and selection. To understand the patterns of codon usage in nemertean mitochondrial genomes, we use bioinformatic approaches to analyze the protein-coding sequences of eight nemertean species. Neutrality analysis did not find a significant correlation between GC12 and GC3. ENc-plot showed a few genes on or close to the expected curve, but the majority of points with low-ENc values are below it. ENc-plot suggested that mutational bias plays a major role in shaping codon usage. The Parity Rule 2 plot (PR2) analysis showed that GC and AT were not used proportionally and we propose that codons containing A or U at third position are used preferentially in nemertean species, regardless of whether corresponding tRNAs are encoded in the mitochondrial DNA. Context-dependent analysis indicated that the nucleotide at the second codon position slightly affects synonymous codon choices. These results suggested that mutational and selection forces are probably acting to codon usage bias in nemertean mitochondrial genomes.     

Reduction of wobble-position GC bases in Corynebacteria genes and enhancement of PCR and heterologous expression

Corynebacteria codon usage exhibits an overall GC content of 67%, and a wobble-position GC content of 88%. Escherichia coli, on the other hand has an overall GC content of 51%, and a wobble-position GC content of 55%. The high GC content of Corynebacteria genes results in an unfavorable codon preference for heterologous expression, and can present difficulties for polymerase-based manipulations due to secondary-structure effects. Since these characteristics are due primarily to base composition at the wobble-position, synthetic genes can, in principle, be designed to eliminate these problems and retain the wild-type amino acid sequence. Such genes would obviate the need for special additives or bases during in vitro polymerase-based manipulation and mutant host strains containing uncommon tRNA's for heterologous expression. We have evaluated synthetic genes with reduced wobble-position G/C content using two variants of the enzyme 2,5-diketo-D-gluconic acid reductase (2,5-DKGR A and B) from Corynebacterium. The wild-type genes are refractory to polymerase-based manipulations and exhibit poor heterologous expression in enteric bacteria. The results indicate that a subset of codons for five amino acids (alanine, arginine, glutamate, glycine and valine) contribute the greatest contribution to reduction in G/C content at the wobble-position. Furthermore, changes in codons for two amino acids (leucine and proline) enhance bias for expression in enteric bacteria without affecting the overall G/C content. The synthetic genes are readily amplified using polymerase-based methodologies, and exhibit high levels of heterologous expression in E. coli.     

Gene expression level shapes the amino acid usages in Prochlorococcus marinus MED4

Prochlorococcus species are the first example of free-living bacteria with reduced genome. Codon and amino acid usages bias of Prochlorococcus marinus MED4 was investigated using all protein coding genes having length greater than or equal to 100 amino acids. Correspondence analysis on relative synonymous codon usage (RSCU) values shows that there is no such influence of translational selection in shaping the codon usage variation among the genes in this organism. However, amino acid usages were markedly different between the highly and lowly expressed genes in this organism and in particular, GC rich amino acids were found to occur significantly higher in highly expressed genes than the lowly expressed genes. Comparative analysis of the homologous genes of Synechococcus sp. WH8102 and Prochlorococcus marinus MED4 shows that amino acids conservation in highly expressed genes is significantly higher than lowly expressed genes. Based on our results we concluded that conservation of GC rich amino acids in the highly expressed genes to its ancestor is the major source of variation in amino acid usages in the organism.