Mammalian genes as molecular clocks?

Summary In analyzing the silent nucleotide substitutions in some mammalian mitochondrial mRNA coding genes, we had found that the frequency of each of the four nucleotides in rat, mouse, and cow, but not in humans, is the same in the silent third codon position (Lanave C, Preparata G, Saccone C, Serio G (1984) J Mol Evol 20:86-93). Because our findings for these three species were compatible with a stationary Markov process for the evolution of nucleotide sequences, we applied such a model to calculate the effective evolutionary silent substitution rate (v_s) and the divergence times among the species. In this paper we have analyzed the first and second codon positions in the same mammalian mitochondrial genes. We found that in the first and second codon positions the human mitochondrial genes satisfy the stationarity conditions. This has allowed us to use the stochastic model mentioned above to calculate the divergence times among mouse, rat, cow, and human. Furthermore, we have analyzed the silent substitution rate in one nuclear gene for these four mammals. We found that in this gene the effective silent substitution rate is about 3 times lower than in mitochondrial genes, and that humans are in this case stationary with respect to the other three mammals in the third codon position as well. Application of our Markov model to this latter gene yields divergence times consistent with our previous determinations.     

Mammalian genes as molecular clocks?

In analyzing the silent nucleotide substitutions in some mammalian mitochondrial mRNA coding genes, we had found that the frequency of each of the four nucleotides in rat, mouse, and cow, but not in humans, is the same in the silent third codon position (Lanave C, Preparata G, Saccone C, Serio G (1984) J Mol Evol 20:86-93). Because our findings for these three species were compatible with a stationary Markov process for the evolution of nucleotide sequences, we applied such a model to calculate the effective evolutionary silent substitution rate (vs) and the divergence times among the species. In this paper we have analyzed the first and second codon positions in the same mammalian mitochondrial genes. We found that in the first and second codon positions the human mitochondrial genes satisfy the stationarity conditions. This has allowed us to use the stochastic model mentioned above to calculate the divergence times among mouse, rat, cow, and human. Furthermore, we have analyzed the silent substitution rate in one nuclear gene for these four mammals. We found that in this gene the effective silent substitution rate is about 3 times lower than in mitochondrial genes, and that humans are in this case stationary with respect to the other three mammals in the third codon position as well. Application of our Markov model to this latter gene yields divergence times consistent with our previous determinations.     

Synonymous substitution rates in Drosophila: Mitochondrial versus nuclear genes

Synonymous substitution rates in mitochondrial and nuclear genes of Drosophila were compared. To make accurate comparisons, we considered the following: (1) relative synonymous rates, which do not require divergence time estimates, should be used; (2) methods estimating divergence should take into account base composition; (3) only very closely related species should be used to avoid effects of saturation; (4) the heterogeneity of rates should be examined. We modified the methods estimating synonymous substitution numbers to account for base composition bias. By using these methods, we found that mitochondrial genes have 1.7–3.4 times higher synonymous substitution rates than the fastest nuclear genes or 4.5–9.0 times higher rates than the average nuclear genes. The average rate of synonymous transversions was 2.7 (estimated from the melanogaster species subgroup) or 2.9 (estimated from the obscura group) times higher in mitochondrial genes than in nuclear genes. Synonymous transversions in mitochondrial genes occurred at an approximately equivalent rate to those in the fastest nuclear genes. This last result is not consistent with the hypothesis that the difference in turnover rates between mitochondrial and nuclear genomes is the major factor determining higher synonymous substitution rates in mtDNA. We conclude that the difference in synonymous substitution rates is due to a combination of two factors: a higher transitional mutation rate in mtDNA and constraints on nuclear genes due to selection for codon usage. Received: 27 November 1996 / Accepted: 8 May 1997     

Evolution in bacteria: Evidence for a universal substitution rate in cellular genomes

Summary This paper constructs a temporal scale for bacterial evolution by tying ecological events that took place at known times in the geological past to specific branch points in the genealogical tree relating the 16S ribosomal RNAs of eubacteria, mitochondria, and chloroplasts. One thus obtains a relationship between time and bacterial RNA divergence which can be used to estimate times of divergence between other branches in the bacterial tree. According to this approach,Salmonella typhimurium andEscherichia coli diverged between 120 and 160 million years (Myr) ago, a date which fits with evidence that the chief habitats occupied now by these two enteric species became available that long ago.The median extent of divergence betweenS. typhimurium andE. coli at synonymous sites for 21 kilobases of protein-coding DNA is 100%. This implies a silent substitution rate of 0.7–0.8%/Myr—a rate remarkably similar to that observed in the nuclear genes of mammals, invertebrates, and flowering plants. Similarities in the substitution rates of eucaryotes and procaryotes are not limited to silent substitutions in protein-coding regions. The average substitution rate for 16S rRNA in eubacteria is about 1%/50 Myr, similar to the average rate for 18S rRNA in vertebrates and flowering plants. Likewise, we estimate a mean rate of roughly 1%/25 Myr for 5S rRNA in both eubacteria and eucaryotes.For a few protein-coding genes of these enteric bacteria, the extent of silent substitution since the divergence ofS. typhimurium andE. coli is much lower than 100%, owing to extreme bias in the usage of synonymous codons. Furthermore, in these bacteria, rates of amino acid replacement were about 20 times lower, on average, than the silent rate. By contranst, for the mammalian genes studied to date, the average replacement rate is only four to five times lower than the rate of silent substitution.     

Mutation-selection models of codon substitution and their use to estimate selective strengths on codon usage

Current models of codon substitution are formulated at the levels of nucleotide substitution and do not explicitly consider the separate effects of mutation and selection. They are thus incapable of inferring whether mutation or selection is responsible for evolution at silent sites. Here we implement a few population genetics models of codon substitution that explicitly consider mutation bias and natural selection at the DNA level. Selection on codon usage is modeled by introducing codon-fitness parameters, which together with mutation-bias parameters, predict optimal codon frequencies for the gene. The selective pressure may be for translational efficiency and accuracy or for fine-tuning translational kinetics to produce correct protein folding. We apply the models to compare mitochondrial and nuclear genes from several mammalian species. Model assumptions concerning codon usage are found to affect the estimation of sequence distances (such as the synonymous rate d(S), the nonsynonymous rate d(N), and the rate at the 4-fold degenerate sites d(4)), as found in previous studies, but the new models produced very similar estimates to some old ones. We also develop a likelihood ratio test to examine the null hypothesis that codon usage is due to mutation bias alone, not influenced by natural selection. Application of the test to the mammalian data led to rejection of the null hypothesis in most genes, suggesting that natural selection may be a driving force in the evolution of synonymous codon usage in mammals. Estimates of selection coefficients nevertheless suggest that selection on codon usage is weak and most mutations are nearly neutral. The sensitivity of the analysis on the assumed mutation model is discussed.     

Rates of synonymous substitution in plant nuclear genes

Summary The rate of synonymous nucleotide substitution in nuclear genes of higher plants has been estimated. The rate varies among genes by a factor of up to two, in a manner that is not immediately explicable in terms of base composition or codon usage bias. The average rate, in both monocots and dicots, is about four times higher than that in chloroplast genes. This leads to an estimated absolute silent substitution rate of 6 × 10^–9 substitutions per site per year that falls within the range of average rates (2–8 × 10^–9) seen in different mammalian nuclear genomes.     

Selection on Codon Usage for Error Minimization at the Protein Level

Given the structure of the genetic code, synonymous codons differ in their capacity to minimize the effects of errors due to mutation or mistranslation. I suggest that this may lead, in protein-coding genes, to a preference for codons that minimize the impact of errors at the protein level. I develop a theoretical measure of error minimization for each codon, based on amino acid similarity. This measure is used to calculate the degree of error minimization for 82 genes of Drosophila melanogaster and 432 rodent genes and to study its relationship with CG content, the degree of codon usage bias, and the rate of nucleotide substitution. I show that (i) Drosophila and rodent genes tend to prefer codons that minimize errors; (ii) this cannot be merely the effect of mutation bias; (iii) the degree of error minimization is correlated with the degree of codon usage bias; (iv) the amino acids that contribute more to codon usage bias are the ones for which synonymous codons differ more in the capacity to minimize errors; and (v) the degree of error minimization is correlated with the rate of nonsynonymous substitution. These results suggest that natural selection for error minimization at the protein level plays a role in the evolution of coding sequences in Drosophila and rodents.Reviewing Editor: Dr. Massimo Di Giulio     

Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species

A detailed comparison was made of codon usage of chloroplast genes with their host (nuclear) genes in the four angiosperm speciesOryza sativa, Zea mays, Triticum aestivum andArabidopsis thaliana. The average GC content of the entire genes, and at the three codon positions individually, was higher in nuclear than in chloroplast genes, suggesting different genomic organization and mutation pressures in nuclear and chloroplast genes. The results of Nc-plots and neutrality plots suggested that nucleotide compositional constraint had a large contribution to codon usage bias of nuclear genes inO. sativa, Z. mays, andT. aestivum, whereas natural selection was likely to be playing a large role in codon usage bias in chloroplast genomes. Correspondence analysis and chi-test showed that regardless of the genomic environment (species) of the host, the codon usage pattern of chloroplast genes differed from nuclear genes of their host species by their AU-richness. All the chloroplast genomes have predominantly A- and/or U-ending codons, whereas nuclear genomes have G-, C- or U-ending codons as their optimal codons. These findings suggest that the chloroplast genome might display particular characteristics of codon usage that are different from its host nuclear genome. However, one feature common to both chloroplast and nuclear genomes in this study was that pyrimidines were found more frequently than purines at the synonymous codon position of optimal codons.     

Codon usage in Aspergillus nidulans.

Summary Synonymous codon usage in genes from the ascomycete (filamentous) fungus Aspergillus nidulans has been investigated. A total of 45 gene sequences has been analysed. Multivariate statistical analysis has been used to identify a single major trend among genes. At one end of this trend are lowly expressed genes, whereas at the other extreme lie genes known or expected to be highly expressed. The major trend is from nearly random codon usage (in the lowly expressed genes) to codon usage that is highly biased towards a set of 19–20 optimal codons. The G+C content of the A. nidulans genome is close to 50%, indicating little overall mutational bias, and so the codon usage of lowly expressed genes is as expected in the absence of selection pressure at silent sites. Most of the optimal codons are C- or G-ending, making highly expressed genes more G+C-rich at silent sites.     

Analysis of nucleotide substitutions of mitochondrial DNAs in Drosophila melanogaster and its sibling species

To study the rate and pattern of nucleotide substitution in mitochondrial DNA (mtDNA), we cloned and sequenced a 975-bp segment of mtDNA from Drosophila melanogaster, D. simulans, and D. mauritiana containing the genes for three transfer RNAs and parts of two protein- coding genes, ND2 and COI. Statistical analysis of synonymous substitutions revealed a predominance of transitions over transversions among the three species, a finding differing from previous results obtained from a comparison of D. melanogaster and D. yakuba. The number of transitions observed was nearly the same for each species comparison, including D. yakuba, despite the differences in divergence times. However, transversions seemed to increase steadily with increasing divergence time. By contrast, nonsynonymous substitutions in the ND2 gene showed a predominance of transversions over transitions. Most transversions were between A and T and seemed to be due to some kind of mutational bias to which the A + T-rich mtDNA of Drosophila species may be subject. The overall rate of nucleotide substitution in Drosophila mtDNA appears to be slightly faster (approximately 1.4 times) than that of the Adh gene. This contrasts with the result obtained for mammals, in which the mtDNA evolves approximately 10 times faster than single-copy nuclear DNA. We have also shown that the start codon of the COI gene is GTGA in D. simulans and GTAA in D. mauritiana. These codons are different from that of D. melanogaster (ATAA).      

The rate of synonymous substitution in enterobacterial genes is inversely related to codon usage bias

Genes sequences from Escherichia coli, Salmonella typhimurium, and other members of the Enterobacteriaceae show a negative correlation between the degree of synonymous-codon usage bias and the rate of nucleotide substitution at synonymous sites. In particular, very highly expressed genes have very biased codon usage and accumulate synonymous substitutions very slowly. In contrast, there is little correlation between the degree of codon bias and the rate of protein evolution. It is concluded that both the rate of synonymous substitution and the degree of codon usage bias largely reflect the intensity of selection at the translational level. Because of the high variability among genes in rates of synonymous substitution, separate molecular clocks of synonymous substitution might be required for different genes.      

The factors dictating the codon usage variation among the genes in the genome of Burkholderia pseudomallei

Burkholderia pseudomallei is a recognized biothreat agent and the causative agent of melioidosis. Codon usage biases of all protein-coding genes (length greater than or equal to 300 bp) from the complete genome of B. pseudomallei K96243 have been analyzed. As B. pseudomallei is a GC-rich organism (68.5%), overall codon usage data analysis indicates that indeed codons ending in G and/or C are predominant in this organism. But multivariate statistical analysis indicates that there is a single major trend in the codon usage variation among the genes in this organism, which has a strong positively correlation with the expressivities of the genes. The majority of the lowly expressed genes are scattered towards the negative end of the major axis whereas the highly expressed genes are clustered towards the positive end. At the same time, from the results that there were two significant correlations between axis 1 coordinates and the GC, GC3s content at silent sites of each sequence, and clearly significant negatively correlations between the ‘Effective Number of Codons’ values and GC, GC3s content, we inferred that codon usage bias was affected by gene nucleotide composition also. In addition, some other factors such as the lengths of the genes as well as the hydrophobicity of genes also influence the codon usage variation among the genes in this organism in a minor way. At the same time, notably, 21 codons have been defined as ‘optimal codons’ of the B. pseudomallei. In summary, our work have provided a basic understanding of the mechanisms for codon usage bias and some more useful information for improving the expression of target genes in vivo and in vitro. Sheng Zhao and Qin Zhang contributed equally to this work.     

Mitochondrial DNA sequences of primates: Tempo and mode of evolution

Summary We cloned and sequenced a segment of mitochondrial DNA from human, chimpanzee, gorilla, orangutan, and gibbon. This segment is 896 bp in length, contains the genes for three transfer RNAs and parts of two proteins, and is homologous in all 5 primates. The 5 sequences differ from one another by base substitutions at 283 positions and by a deletion of one base pair. The sequence differences range from 9 to 19% among species, in agreement with estimates from cleavage map comparisons, thus confirming that the rate of mtDNA evolution in primates is 5 to 10 times higher than in nuclear DNA. The most striking new finding to emerge from these comparisons is that transitions greatly outnumber transversions. Ninety-two percent of the differences among the most closely related species (human, chimpanzee, and gorilla) are transitions. For pairs of species with longer divergence times, the observed percentage of transitions falls until, in the case of comparisons between primates and non-primates, it reaches a value of 45. The time dependence is probably due to obliteration of the record of transitions by multiple substitutions at the same nucleotide site. This finding illustrates the importance of choosing closely related species for analysis of the evolutionary process. The remarkable bias toward transitions in mtDNA evolution necessitates the revision of equations that correct for multiple substitutions at the same site. With revised equations, we calculated the incidence of silent and replacement substitutions in the two protein-coding genes. The silent substitution rate is 4 to 6 times higher than the replacement rate, indicating strong functional constraints at replacement sites. Moreover, the silent rate for these two genes is about 10% per million years, a value 10 times higher than the silent rate for the nuclear genes studied so far. In addition, the mean substitution rate in the three mitochondrial tRNA genes is at least 100 times higher than in nuclear tRNA genes. Finally, genealogical analysis of the sequence differences supports the view that the human lineage branched off only slightly before the gorilla and chimpanzee lineages diverged and strengthens the hypothesis that humans are more related to gorillas and chimpanzees than is the orangutan.Abbreviations mtDNA mitochondrial DNA - bp base pair - URF unidentified reading frame     

Mammalian mitochondrial DNA evolution: A comparison of the cytochrome b and cytochrome c oxidase II genes

The evolution of two mitochondrial genes, cytochrome b and cytochrome c oxidase subunit II, was examined in several eutherian mammal orders, with special emphasis on the orders Artiodactyla and Rodentia. When analyzed using both maximum parsimony, with either equal or unequal character weighting, and neighbor joining, neither gene performed with a high degree of consistency in terms of the phylogenetic hypotheses supported. The phylogenetic inconsistencies observed for both these genes may be the result of several factors including differences in the rate of nucleotide substitution among particular lineages (especially between orders), base composition bias, transition/transversion bias, differences in codon usage, and different constraints and levels of homoplasy associated with first, second, and third codon positions. We discuss the implications of these findings for the molecular systematics of mammals, especially as they relate to recent hypotheses concerning the polyphyly of the order Rodentia, relationships among the Artiodactyla, and various interordinal relationships.Correspondence to: R.L. Honeycutt     

Understanding molecular biology of codon usage in mitochondrial complex IV genes of electron transport system: Relevance to mitochondrial diseases

The mitochondrial cytochrome oxidase (CO) genes are involved in complex IV of the electron transport system, and dysfunction of CO genes leads to several diseases. However, no work has been reported on the codon usage pattern of these genes. We used bioinformatic methods to analyze the compositional properties and the codon usage pattern of the COI, COII, and COIII genes in fishes, birds, and mammals to understand the similarities and dissimilarities of codon usage in these genes, which gave an insight into the molecular biology of these genes. The effective number of codons (ENC) value of genes was high in different species of fishes, birds and mammals, which indicates that the codon bias of CO genes was low and the ENC values were significantly different among fishes, birds, and mammals, as revealed from the t test. The overall guanine and cytosine (GC) content in fishes, birds, and mammals was lower than 50% in all genes, indicating that the genes were AT-rich and significantly different among fishes, birds, and mammals. The TCA codon was overrepresented in fishes, birds, and mammals for the COI gene, in birds and mammals for the COII gene, but it was not overrepresented in others. Only three codons, namely CTA, CGA, and AAA, were overrepresented in all three groups for the COI, COII, and COIII genes, repectively. From the neutrality plot in fishes, birds, and mammals, it was observed that the slopes of the regression lines (regression coefficients) in the COI, COII, and COIII genes were <0.5, suggesting that natural selection played a major role, whereas mutation pressure played a minor role.     

Molecular Evolution of Duplicated Ray Finned Fish HoxA Clusters: Increased Synonymous Substitution Rate and Asymmetrical Co-divergence of Coding and Non-coding Sequences

In this study the molecular evolution of duplicated HoxA genes in zebrafish and fugu has been investigated. All 18 duplicated HoxA genes studied have a higher non-synonymous substitution rate than the corresponding genes in either bichir or paddlefish, where these genes are not duplicated. The higher rate of evolution is not due solely to a higher non-synonymous-to-synonymous rate ratio but to an increase in both the non-synonymous as well as the synonymous substitution rate. The synonymous rate increase can be explained by a change in base composition, codon usage, or mutation rate. We found no changes in nucleotide composition or codon bias. Thus, we suggest that the HoxA genes may experience an increased mutation rate following cluster duplication. In the non-Hox nuclear gene RAG1 only an increase in non-synonymous substitutions could be detected, suggesting that the increased mutation rate is specific to duplicated Hox clusters and might be related to the structural instability of Hox clusters following duplication. The divergence among paralog genes tends to be asymmetric, with one paralog diverging faster than the other. In fugu, all b-paralogs diverge faster than the a-paralogs, while in zebrafish Hoxa-13a diverges faster. This asymmetry corresponds to the asymmetry in the divergence rate of conserved non-coding sequences, i.e., putative cis-regulatory elements. These results suggest that the 5′ HoxA genes in the same cluster belong to a co-evolutionary unit in which genes have a tendency to diverge together. Reviewing Editor: Dr. Axel Meyer     

Intralineage variation in the pattern ofrbcL nucleotide substitution

Variation in chloroplastrbcL sequences was studied in representative species of four different lineages: the tribeRubieae (Rubiaceae), and the generaDrosera (Droseraceae),Nothofagus (Nothofagaceae) andIlex (Aquifoliaceae). Each lineage has its particular non-overlapping set ofrbcL polymorphic sites, indicating that common unconstrainedrbcL sites are not shared. Large differences in the rate and pattern of nucleotide substitution are observed among the four lineages. The genusIlex has the lowest rate of substitution, the lowest transition/transversion ratio, the lowest synonymous/replacement ratio and the lowest number of substitutions at the third codon position. An apparent relationship of these measures to the age of the lineages is observed. The A + T content and codon use among the four lineages are very similar and, apparently, cannot account for the observed differences in patterns of nucleotide substitution. However, the A + T content of the two bases immediately flanking the polymorphic sites is higher inIlex than in the other lineages. This could be correlated with the transversion/transition bias observed inIlex. The particularly low synonymous/replacement ratio found inIlex could also be explained by the small population sizes of species in this genus.     

Comparative Analysis of Codon Usage Patterns Among Mitochondrion, Chloroplast and Nuclear Genes in Triticum aestivum L.

In many organisms, the difference in codon usage patterns among genes reflects variation in local base compositional biases and the intensity of natural selection. In this study, a comparative analysis was performed to investigate the characteristics of codon bias and factors in shaping the codon usage patterns among mitochondrion, chloroplast and nuclear genes in common wheat （Triticum aestivum L.）. GC contents in nuclear genes were higher than that in mitochondrion and chloroplast genes. The neutrality and correspondence analyses indicated that the codon usage in nuclear genes would be a result of relative strong mutational bias, while the codon usage patterns of mitochondrion and chloroplast genes were more conserved in GC content and influenced by translation level. The Parity Rule 2 （PR2） plot analysis showed that pyrimidines were used more frequently than purines at the third codon position in the three genomes. In addition, using a new alterative strategy, 11, 12, and 24 triplets were defined as preferred codons in the mitochondrion, chloroplast and nuclear genes, respectively. These findings suggested that the mitochondrion, chloroplast and nuclear genes shared particularly different features of codon usage and evolutionary constraints.