Evolutionary change of codon usage for the histone gene family in Drosophila melanogaster and Drosophila hydei

The nucleotide divergence in the protein-coding region for replication-dependent and replication-independent histone 3 and 4 genes of Drosophila melanogaster and Drosophila hydei occurred mostly at the synonymous site. Therefore, the pattern of codon usage was analyzed in the two species, considering the genomic codon bias, which is proposed for estimating the genomic composition pressure in the protein-coding regions. The results indicated that the codon usage in the histone gene family could be explained mostly by the genomic codon bias. However, biases for Ala and Arg were commonly observed for the histone 3 and histone 4 gene families, and biases for Ser, Leu, and Glu were observed in a gene-specific manner. This suggests that both genomic codon bias and gene- or codon-specific bias are responsible for the nucleotide differentiation in the protein-coding region of the histone genes.     

Molecular evolution of the histone 3 multigene family in the Drosophila melanogaster species subgroup

Molecular evolution of the histone multigene family was studied by cloning and sequencing regions of the histone 3 gene in the Drosophila melanogaster species subgroup. Analysis of the nucleotide substitution pattern showed that in the coding region synonymous changes occurred more frequently to A or T in contrast to the GC-rich base composition, while in the 3' region the nucleotide substitutions were most likely in equilibrium. These results suggested that the base composition at the third codon position of the H3 gene, i.e., codon usage, has been changing to A or T in the Drosophila melanogaster species subgroup.     

Evolution of the GC content of the histone 3 gene in seven Drosophila species

The molecular evolution of the histone multigene family was studied by cloning and determining the nucleotide sequences of the histone 3 genes in seven Drosophila species, D. takahashii, D. lutescens, D. ficusphila, D. persimilis, D.pseudoobscura, D. americana and D. immigrans. CT repeats, a TATA box and an AGTG motif in the 5' region, and a hairpin loop and purine-rich motifs (CAA(T/G)GAGA) in the 3' region were conserved even in distantly related species. In D. hydei and D.americana, the GC content at the third codon position in the protein coding region was relatively low (49% and 45%), while in D. takahashii and D. lutescens it was relatively high (64% and 65%). The non- significant correlation between the GC contents in the 3' region and at the third codon position as well as the evidence of less constraint in the 3' region suggested that mutational bias may not be the major mechanism responsible for the biased nucleotide change at the third codon position or for codon usage bias.     

Variation in synonymous codon use and DNA polymorphism within the Drosophila genome

A strong negative correlation between the rate of amino-acid substitution and codon usage bias in Drosophila has been attributed to interference between positive selection at nonsynonymous sites and weak selection on codon usage. To further explore this possibility we have investigated polymorphism and divergence at three kinds of sites: synonymous, nonsynonymous and intronic in relation to codon bias in D. melanogaster and D. simulans. We confirmed that protein evolution is one of the main explicative parameters for interlocus codon bias variation (r(2) approximately 40%). However, intron or synonymous diversities, which could have been expected to be good indicators of local interference [here defined as the additional increase of drift due to selection on tightly linked sites, also called 'genetic draft' by Gillespie (2000)] did not covary significantly with codon bias or with protein evolution. Concurrently, levels of polymorphism were reduced in regions of low recombination rates whereas codon bias was not. Finally, while nonsynonymous diversities were very well correlated between species, neither synonymous nor intron diversities observed in D. melanogaster were correlated with those observed in D. simulans. All together, our results suggest that the selective constraint on the protein is a stable component of gene evolution while local interference is not. The pattern of variation in genetic draft along the genome therefore seems to be instable through evolutionary times and should therefore be considered as a minor determinant of codon bias variance. We argue that selective constraints for optimal codon usage are likely to be correlated with selective constraints on the protein, both between codons within a gene, as previously suggested, and also between genes within a genome.     

Codon usage bias covaries with expression breadth and the rate of synonymous evolution in humans, but this is not evidence for selection.

In numerous species, from bacteria to Drosophila, evidence suggests that selection acts even on synonymous codon usage: codon bias is greater in more abundantly expressed genes, the rate of synonymous evolution is lower in genes with greater codon bias, and there is consistency between genes in the same species in which codons are preferred. In contrast, in mammals, while nonequal use of alternative codons is observed, the bias is attributed to the background variance in nucleotide concentrations, reflected in the similar nucleotide composition of flanking noncoding and exonic third sites. However, a systematic examination of the covariants of codon usage controlling for background nucleotide content has yet to be performed. Here we present a new method to measure codon bias that corrects for background nucleotide content and apply this to 2396 human genes. Nearly all (99%) exhibit a higher amount of codon bias than expected by chance. The patterns associated with selectively driven codon bias are weakly recovered: Broadly expressed genes have a higher level of bias than do tissue-specific genes, the bias is higher for genes with lower rates of synonymous substitutions, and certain codons are repeatedly preferred. However, while these patterns are suggestive, the first two patterns appear to be methodological artifacts. The last pattern reflects in part biases in usage of nucleotide pairs. We conclude that we find no evidence for selection on codon usage in humans.     

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.     

Selection on Silent Sites in the Rodent H3 Histone Gene Family

Selection promoting differential use of synonymous codons has been shown for several unicellular organisms and for Drosophila, but not for mammals. Selection coefficients operating on synonymous codons are likely to be extremely small, so that a very large effective population size is required for selection to overcome the effects of drift. In mammals, codon-usage bias is believed to be determined exclusively by mutation pressure, with differences between genes due to large-scale variation in base composition around the genome. The replication-dependent histone genes are expressed at extremely high levels during periods of DNA synthesis, and thus are among the most likely mammalian genes to be affected by selection on synonymous codon usage. We suggest that the extremely biased pattern of codon usage in the H3 genes is determined in part by selection. Silent site G + C content is much higher than expected based on flanking sequence G + C content, compared to other rodent genes with similar silent site base composition but lower levels of expression. Dinucleotide-mediated mutation bias does affect codon usage, but the affect is limited to the choice between G and C in some fourfold degenerate codons. Gene conversion between the two clusters of histone genes has not been an important force in the evolution of the H3 genes, but gene conversion appears to have had some effect within the cluster on chromosome 13.     

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.     

Patterns of synonymous codon usage in Drosophila melanogaster genes with sex-biased expression

The nonrandom use of synonymous codons (codon bias) is a well-established phenomenon in Drosophila. Recent reports suggest that levels of codon bias differ among genes that are differentially expressed between the sexes, with male-expressed genes showing less codon bias than female-expressed genes. To examine the relationship between sex-biased gene expression and level of codon bias on a genomic scale, we surveyed synonymous codon usage in 7276 D. melanogaster genes that were classified as male-, female-, or non-sex-biased in their expression in microarray experiments. We found that male-biased genes have significantly less codon bias than both female- and non-sex-biased genes. This pattern holds for both germline and somatically expressed genes. Furthermore, we find a significantly negative correlation between level of codon bias and degree of sex-biased expression for male-biased genes. In contrast, female-biased genes do not differ from non-sex-biased genes in their level of codon bias and show a significantly positive correlation between codon bias and degree of sex-biased expression. These observations cannot be explained by differences in chromosomal distribution, mutational processes, recombinational environment, gene length, or absolute expression level among genes of the different expression classes. We propose that the observed codon bias differences result from differences in selection at synonymous and/or linked nonsynonymous sites between genes with male- and female-biased expression.     

Analysis of synonymous codon usage in H5N1 virus and other influenza A viruses

In this study, we calculated the codon usage bias in H5N1 virus and performed a comparative analysis of synonymous codon usage patterns in H5N1 virus, five other evolutionary related influenza A viruses and a influenza B virus. Codon usage bias in H5N1 genome is a little slight, which is mainly determined by the base compositions on the third codon position. By comparing synonymous codon usage patterns in different viruses, we observed that the codon usage pattern of H5N1 virus is similar with other influenza A viruses, but not influenza B virus, and the synonymous codon usage in influenza A virus genes is phylogenetically conservative, but not strain-specific. Synonymous codon usage in genes encoded by different influenza A viruses is genus conservative. Compositional constraints could explain most of the variation of synonymous codon usage among these virus genes, while gene function is also correlated to synonymous codon usages to a certain extent. However, translational selection and gene length have no effect on the variations of synonymous codon usage in these virus genes.     

Codon usage and amino acid usage influence genes expression level

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

Variable Rates of Evolution among Drosophila Opsin Genes

DNA sequences and chromosomal locations of four Drosophila pseudoobscura opsin genes were compared with those from Drosophila melanogaster, to determine factors that influence the evolution of multigene families. Although the opsin proteins perform the same primary functions, the comparisons reveal a wide range of evolutionary rates. Amino acid identities for the opsins range from 90% for Rh2 to more than 95% for Rh1 and Rh4. Variation in the rate of synonymous site substitution is especially striking: the major opsin, encoded by the Rh1 locus, differs at only 26.1% of synonymous sites between D. pseudoobscura and D. melanogaster, while the other opsin loci differ by as much as 39.2% at synonymous sites. Rh3 and Rh4 have similar levels of synonymous nucleotide substitution but significantly different amounts of amino acid replacement. This decoupling of nucleotide substitution and amino acid replacement suggests that different selective pressures are acting on these similar genes. There is significant heterogeneity in base composition and codon usage bias among the opsin genes in both species, but there are no consistent relationships between these factors and the rate of evolution of the opsins. In addition to exhibiting variation in evolutionary rates, the opsin loci in these species reveal rearrangements of chromosome elements.     

Hill-Robertson interference is a minor determinant of variations in codon bias across Drosophila melanogaster and Caenorhabditis elegans genomes

According to population genetics models, genomic regions with lower crossing-over rates are expected to experience less effective selection because of Hill-Robertson interference (HRi). The effect of genetic linkage is thought to be particularly important for a selection of weak intensity such as selection affecting codon usage. Consistent with this model, codon bias correlates positively with recombination rate in Drosophila melanogaster and Caenorhabditis elegans. However, in these species, the G+C content of both noncoding DNA and synonymous sites correlates positively with recombination, which suggests that mutation patterns and recombination are associated. To remove this effect of mutation patterns on codon bias, we used the synonymous sites of lowly expressed genes that are expected to be effectively neutral sites. We measured the differences between codon biases of highly expressed genes and their lowly expressed neighbors. In D. melanogaster we find that HRi weakly reduces selection on codon usage of genes located in regions of very low recombination; but these genes only comprise 4% of the total. In C. elegans we do not find any evidence for the effect of recombination on selection for codon bias. Computer simulations indicate that HRi poorly enhances codon bias if the local recombination rate is greater than the mutation rate. This prediction of the model is consistent with our data and with the current estimate of the mutation rate in D. melanogaster. The case of C. elegans, which is highly self-fertilizing, is discussed. Our results suggest that HRi is a minor determinant of variations in codon bias across the genome.     

Substitution rates in Drosophila nuclear genes: implications for translational selection

The relationships between synonymous and nonsynonymous substitution rates and between synonymous rate and codon usage bias are important to our understanding of the roles of mutation and selection in the evolution of Drosophila genes. Previous studies used approximate estimation methods that ignore codon bias. In this study we reexamine those relationships using maximum-likelihood methods to estimate substitution rates, which accommodate the transition/transversion rate bias and codon usage bias. We compiled a sample of homologous DNA sequences at 83 nuclear loci from Drosophila melanogaster and at least one other species of Drosophila. Our analysis was consistent with previous studies in finding that synonymous rates were positively correlated with nonsynonymous rates. Our analysis differed from previous studies, however, in that synonymous rates were unrelated to codon bias. We therefore conducted a simulation study to investigate the differences between approaches. The results suggested that failure to properly account for multiple substitutions at the same site and for biased codon usage by approximate methods can lead to an artifactual correlation between synonymous rate and codon bias. Implications of the results for translational selection are discussed.     

Codon bias in actin multigene families and effects on the reconstruction of phylogenetic relationships

Codon usage patterns and phylogenetic relationships in the actin multigene family have been analyzed for three dipteran species—Drosophila melanogaster, Bactrocera dorsalis, and Ceratitis capitata. In certain phylogenetic tree reconstructions, using synonymous distances, some gene relationships are altered due to a homogenization phenomenon. We present evidence to show that this homogenization phenomenon is due to codon usage bias. A survey of the pattern of synonymous codon preferences for I I actin genes from these three species reveals that five out of the six Drosophila actin genes show high degrees of codon bias as indicated by scaled ² values. In contrast to this, four out of the five actin genes from the other species have low codon bias values. A Monte Carlo contingency test indicates that for those Drosophila actin genes which exhibit codon bias, the patterns of codon usage are different compared to actin genes from the other species. In addition, the genes exhibiting codon bias also appear to have reduced rates of synonymous substitution. The homogenization phenomenon seen in terms of synonymous substitutions is not observed for nonsynonymous changes. Because of this homogenization phenomenon, trees constructed based on synonymous substitutions will be affected. These effects can be overt in the case of multigene families, but similar distortions may underlie reconstructions based on single-copy genes which exhibit codon usage bias.Correspondence to: M. He     

Synonymous Substitutions in the Xdh Gene of Drosophila: Heterogeneous Distribution along the Coding Region

The Xdh (rosy) region of Drosophila subobscura has been sequenced and compared to the homologous region of D. pseudoobscura and D. melanogaster. Estimates of the numbers of synonymous substitutions per site (Ks) confirm that Xdh has a high synonymous substitution rate. The distributions of both nonsynonymous and synonymous substitutions along the coding region were found to be heterogeneous. Also, no relationship has been detected between Ks estimates and codon usage bias along the gene, in contrast with the generally observed relationship among genes. This heterogeneous distribution of synonymous substitutions along the Xdh gene, which is expression-level independent, could be explained by a differential selection pressure on synonymous sites along the coding region acting on mRNA secondary structure. The synonymous rate in the Xdh coding region is lower in the D. subobscura than in the D. pseudoobscura lineage, whereas the reverse is true for the Adh gene.     

Genomic background drives the divergence of duplicated amylase genes at synonymous sites in Drosophila

In some Drosophila species, there are two types of greatly diverged amylase (Amy) genes (Amy clusters 1 and 2), each encoding active amylase isozymes. Cluster 1 is located at the middle of its chromosomal arm, and the region has a normal local recombination rate. However, cluster 2 is near the centromere, and this region is known to have a reduced recombination rate. Although nonsynonymous substitutions follow a molecular clock, synonymous substitutions were accelerated in cluster 2 after gene duplications. This resulted in a higher GC content at the third codon position (GC3) and codon usage bias in cluster 1, and lower GC3 content and codon usage bias in the cluster 2. However, no systematic difference in GC content was observed in the first and second codon positions or the 3'-flanking regions. Therefore, differences in local recombination rate rather than mutation bias might explain the divergence at synonymous sites between the two Amy clusters within species (Hill-Robertson effect). Alternatively, the different patterns and levels of expression between the two clusters may imply that the reduced expression level in cluster 2 caused by chromatin potentiation decreased the codon bias. Both of these hypotheses imply the importance of the genomic background as a driving force of divergence between non-tandemly duplicated genes.