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PubMed2004

Driving change: the evolution of alternative genetic codes.

Pioneering studies in the 1960s that elucidated the genetic code suggested that all extant forms of life use the same genetic code. This early presumption has subsequently been challenged by the discovery of deviations of the universal genetic code in prokaryotes, eukaryotic nuclear genomes and mitochondrial genomes. These studies have revealed that the genetic code is still evolving despite strong negative forces working against the fixation of mutations that result in codon reassignment. Recent data from in vitro, in vivo and in silico comparative genomics studies are revealing significant, previously overlooked links between modified nucleosides in tRNAs, genetic code ambiguity, genome base composition, codon usage and codon reassignment.

Animals↗

PubMed1994

On the relationship between preferred termination codon contexts and nonsense suppression in human cells.

The nucleotide sequences 3' to the translational termination codons in a collection of human genes have been analysed for evidence of a preferred 3' context for natural UAG codons. The aim was to see whether human UAG contexts can be related to the recent demonstration of the effects of 3' context on nonsense suppression in human cells. Since mammalian genomes are known to consist of a patchwork of blocks of sequences or 'isochores' with different G+C contents, the collection of genes was split into 5 classes containing genes with similar frequencies of G+C at the 3rd position of synonymous codons. This analysis revealed that the frequency of bases 3' to UAG varies with the G+C frequency of the gene, and that these changes were mirrored by changes in the patterns of bases in GN and AGN strings. The identity of the next 3' base appears therefore to be determined by genome wide changes in G+C composition, rather than selection to maintain a particular tetranucleotide stop signal. These findings argue strongly that the failure to find bias in the patterns of bases used in human coding sequences is an insensitive guide for the existence of codon usage or codon context effects during translation in human cells.

Base Composition↗

PubMed1980

DNA sequences from the str operon of Escherichia coli.

The str operon at 72 min on the Escherichia coli chromosome contains genes for ribosomal proteins (r-proteins) S12 (str or rpsL) and S7 (rpsG) and elongation factors G (fus) and Tu (tufA). The sequence of the entire S12 gene, the S12-S7 intercistronic region, and the beginning of the S7 gene is reported. Also, the sequence of the end of the S7 gene, the S7-G intercistronic region, and the beginning of the elongation fractor G gene is reported. The S12-S7 intercistronic region is 96 base pairs long, in contrast to other intercistronic regions in r-protein operons which have been found to vary from 3 to 66 base pairs. The S7-G intercistronic region is only 27 bases long, supporting the previous conclusion that r-protein and elongation factor genes are co-transcribed. A comparison of translation initiation sites of the S12 and S7 genes, and other examples of co-transcribed r-protein genes, reveals no obvious features that could account for equimolar synthesis of all r-proteins. The codon usage in the S12 and S7 genes follows the pattern observed in other r-protein genes; that is, there is a highly preferential usage of codons recognized by the most abundant of isoaccepting tRNA species. This pattern could reflect the cell's need for efficient translation or minimal errors, or both, in r-protein synthesis.

Amino Acid Sequence↗

PubMed1983

The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe.

We have cloned and sequenced the alcohol dehydrogenase gene of the fission yeast Schizosaccharomyces pombe. The gene was isolated by transformation and complementation of a Saccharomyces cerevisiae strain which lacked functional alcohol dehydrogenase with an S. pombe gene bank constructed in the autonomously replicating yeast plasmid YEp13. Southern hybridization analysis indicates that S. pombe contains only one alcohol dehydrogenase gene. The structural region of the gene is 50% homologous to the alcohol dehydrogenase encoding genes of the budding yeast S. cerevisiae. The gene exhibits a very strong codon usage bias; with the set of predominantly used codons generally resembling that which S. cerevisiae employs preferentially. All of the differences in codon usage bias between S. pombe and S. cerevisiae are in the direction of greater G + C content in S. pombe codons. It is argued that this observation supports the hypothesis that selection toward uniform codon-anticodon binding energies contributes to codon usage bias and that the optimum binding energy is, on the average, higher in S. pombe than S. cerevisiae.

Alcohol Dehydrogenase↗

PubMed2005

Molecular evolution in the Drosophila melanogaster species subgroup: frequent parameter fluctuations on the timescale of molecular divergence.

Although mutation, genetic drift, and natural selection are well established as determinants of genome evolution, the importance (frequency and magnitude) of parameter fluctuations in molecular evolution is less understood. DNA sequence comparisons among closely related species allow specific substitutions to be assigned to lineages on a phylogenetic tree. In this study, we compare patterns of codon usage and protein evolution in 22 genes (>11,000 codons) among Drosophila melanogaster and five relatives within the D. melanogaster subgroup. We assign changes to eight lineages using a maximum-likelihood approach to infer ancestral states. Uncertainty in ancestral reconstructions is taken into account, at least to some extent, by weighting reconstructions by their posterior probabilities. Four of the eight lineages show potentially genomewide departures from equilibrium synonymous codon usage; three are decreasing and one is increasing in major codon usage. Several of these departures are consistent with lineage-specific changes in selection intensity (selection coefficients scaled to effective population size) at silent sites. Intron base composition and rates and patterns of protein evolution are also heterogeneous among these lineages. The magnitude of forces governing silent, intron, and protein evolution appears to have varied frequently, and in a lineage-specific manner, within the D. melanogaster subgroup.

Amino Acid Substitution↗

PubMed2001

Inferring parameters of mutation, selection and demography from patterns of synonymous site evolution in Drosophila.

Selection acting on codon usage can cause patterns of synonymous evolution to deviate considerably from those expected under neutrality. To investigate the quantitative relationship between parameters of mutation, selection, and demography, and patterns of synonymous site divergence, we have developed a novel combination of population genetic models and likelihood methods of phylogenetic sequence analysis. Comparing 50 orthologous gene pairs from Drosophila melanogaster and D. virilis and 27 from D. melanogaster and D. simulans, we show considerable variation between amino acids and genes in the strength of selection acting on codon usage and find evidence for both long-term and short-term changes in the strength of selection between species. Remarkably, D. melanogaster shows no evidence of current selection on codon usage, while its sister species D. simulans experiences only half the selection pressure for codon usage of their common ancestor. We also find evidence for considerable base asymmetries in the rate of mutation, such that the average synonymous mutation rate is 20-30% higher than in noncoding regions. A Bayesian approach is adopted to investigate how accounting for selection on codon usage influences estimates of the parameters of mutation.

Animals↗

PubMed2006

[Expression and secretion of human bone morphogenetic protein-7 in Pichia pastoris].

The synonymous codons are used in a highly non-random manner in hosts of widely divergent species, which is termed "codon usage bias". Several reports suggest that codon usage bias sometimes frustrate attempts to express high levels of exogenous genes. In this study, we attempted to express mature peptide of human bone morphogenetic protein-7(hBMP7), with optimized codons in P. pastoris expression system. Three low-usage ARG codons (CGG or CGA) of gene fragment coding the mature peptide of hBMP7 have been successfully converted into P. pastoris-preferred ARG codons (AGA) by overlap extension PCR-based multiple-site-directed mutagenesis for a high level expression of hBMP7 mature peptide. The present results showed that the production level (25.45 mg/L) of codon-optimized hbmp7 had a remarkably improvement of 4.6-fold relative to that (5.5 mg/L) of non-codon-optimized hbmp7. Furthermore, a strain haboring multi-copy of codon-optimized hbmp7 expression cassette was screened, and showed a increased level of expression with 2-fold more potent than the single-copy one. The recombinant hBMP7 mature peptide were produced as a 18 kD monomer proteins, and were easily purified from culture supernatants by using ion-exchange chromatography. Functional assay demonstrated that rhBMP7 could induce ectopic cartilage formation, although its inductive ability was much less active than CHO cell-derived hBMP7.

Animals↗

PubMed1999

Evidence for genetic drift in endosymbionts (Buchnera): analyses of protein-coding genes.

Buchnera, the bacterial endosymbionts of aphids, undergo severe population bottlenecks during maternal transmission through their hosts. Previous studies suggest an increased effect of drift within these strictly asexual, small populations, resulting in an increased fixation of slightly deleterious mutations. This study further explores sequence evolution in Buchnera using three approaches. First, patterns of codon usage were compared across several homologous Escherichia coli and Buchnera loci, in order to test the prediction that selection for the use of optimal codons is less effective in small populations. A chi 2-based measure of codon bias was developed to adjust for the overall A + T richness of silent positions in the endosymbionts. In contrast to E. coli homologues, adaptive codon bias across Buchnera loci is markedly low, and patterns of codon usage lack a strong relationship with gene expression level. These data suggest that codon usage in Buchnera has been shaped largely by mutational pressure and drift rather than by selection for translational efficiency. One exception to the overall lack of bias is groEL, which is known to be constitutively overexpressed in Buchnera and other endosymbionts. Second, relative-rate tests show elevated rates of sequence evolution of numerous protein-coding loci across Buchnera, compared to E. coli. Finally, consistently higher ratios of nonsynonymous to synonymous substitutions in Buchnera loci relative to the enteric bacteria strongly suggest the accumulation of nonsynonymous substitutions in endosymbiont lineages. Combined, these results suggest a decreased effectiveness of purifying selection in purging endosymbiont populations of slightly deleterious mutations, particularly those affecting codon usage and amino acid identity.

Animals↗

PubMed1986

Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase.

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.

Amino Acid Sequence↗

PubMed1995

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 11 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 chi 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.

Actins↗

PubMed1994

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.

Animals↗

PubMed2004

Genomic choice of codons in 16 microbial species.

We study the codon usage over whole set of ORFs of 16 unicellular microbial species: eight archaebacteria, seven eubacteria, and one eukarya. We first try to define, for each species, the neutral expected codon usage to better approach subsequently the influence of selection. Overlapping triplets counted from the complete DNA genomic sequence and mean amino acid composition of ORFs allow us to build satisfying expected codon usage for each species. Within species deviation from this neutral model is then studied through Correspondence Analysis and characterization with bias index, N(C)' (effective number of codons reported to neutral model). Our results are compared to previously published ones for three species and let appear good agreement in spite of very different methods. We thus propose set of codons probably preferred by selection for nine other species. In the four last species, no clear preference can be evidenced. Finally, we characterize variation of codon usage over functional categories. We propose that the high degree of bias of proteins involved in translation, ribosomal structure and biogenesis has a positive influence on overexpression of the corresponding genes under optimum growth conditions and is a negative regulator of the same genes when amino acids become limited resources.

Base Composition↗

PubMed2002

Codon optimization improves heterologous expression of a Schistosoma mansoni cDNA in HEK293 cells.

Differences in codon usage can seriously hamper the expression of cloned cDNAs in heterologous systems. In this study, we show that the expression of a cloned Schistosoma mansoni cDNA in cultured HEK293 cells was dramatically increased by rewriting a portion of the cDNA according to human preferred codon usage, suggesting that codon optimization is a valuable strategy for improving the heterologous expression of helminth sequences. We further describe a simple modification of a recursive PCR-based method, which allows the rewriting of long stretches of DNA sequence in a single PCR reaction. This method can be used to optimize the codon usage of virtually any DNA from helminths and other parasites.

Animals↗

PubMed2003

Patterns of context-dependent codon biases.

The association of codon context and codon usage was studied in seven bacteria as well as Schizosaccharomyces pombe and Encephalitozoon cuniculi. The association is strongest in magnitude closest to the codons of interest but there is apparently no rule about which of the two contexts is generally strongest associated to codon usage. In all bacterial species and in the intron-rich Sch. pombe it was furthermore observed from plots of chi2 versus N that the wobble positions of codons in the proximity cause regular peaks both upstream and downstream. This observation is discussed in relation to a possible effect of mutational pressure on the association of codon usage and codon context. Absence of peaks corresponding to the wobble positions in the intron-poor En. cuniculi, and presence in Sch. pombe, may indicate that the role of introns in the context-dependent codon bias is negligible.

Animals↗

PubMed2003

[Codon optimization and expression in Pichia pastoris of E2 gene of classical swine fever virus].

Codon bias was one of the important parameter which influence heterogenous gene expression, optimizing codon sequence could improve expression level of heterogenous gene. In the preview study, wildtype E2 gene was expressed poorly in Pichia pastoris, in order to improve the expression level of E2 gene in Pichia pastoris, the low usage codons of E2 gene were mutated into high usage codons in Pichia pastoris by directed-mutagenesis based on PCR. The result showed that, compared with the results reported in preview study, the expression level of E2 gene in Pichia pastoris was improved observably by substituting 24 low usage codons of E2 gene for the high usage synonymous codons. It suggested the stragety to improve the expression of E2 gene in Pichia pastoris by codon optimization was successful.

Base Sequence↗

PubMed2006

Correlation of mRNA expression and protein abundance affected by multiple sequence features related to translational efficiency in Desulfovibrio vulgaris: a quantitative analysis.

The modest correlation between mRNA expression and protein abundance in large-scale data sets is explained in part by experimental challenges, such as technological limitations, and in part by fundamental biological factors in the transcription and translation processes. Among various factors affecting the mRNA-protein correlation, the roles of biological factors related to translation are poorly understood. In this study, using experimental mRNA expression and protein abundance data collected from Desulfovibrio vulgaris by DNA microarray and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) proteomic analysis, we quantitatively examined the effects of several translational-efficiency-related sequence features on mRNA-protein correlation. Three classes of sequence features were investigated according to different translational stages: (i) initiation, Shine-Dalgarno sequences, start codon identity, and start codon context; (ii) elongation, codon usage and amino acid usage; and (iii) termination, stop codon identity and stop codon context. Surprisingly, although it is widely accepted that translation initiation is the rate-limiting step for translation, our results showed that the mRNA-protein correlation was affected the most by the features at elongation stages, i.e., codon usage and amino acid composition (5.3-15.7% and 5.8-11.9% of the total variation of mRNA-protein correlation, respectively), followed by stop codon context and the Shine-Dalgarno sequence (3.7-5.1% and 1.9-3.8%, respectively). Taken together, all sequence features contributed to 15.2-26.2% of the total variation of mRNA-protein correlation. This study provides the first comprehensive quantitative analysis of the mRNA-protein correlation in bacterial D. vulgaris and adds new insights into the relative importance of various sequence features in prokaryotic protein translation.

Bacterial Proteins↗

PubMed1991

Influence of the codon following the initiation codon on the expression of the lacZ gene in Saccharomyces cerevisiae.

A set of 32 different codons were introduced in a lacZ expression vector (pPTK400) immediately 3' from the AUG initiation codon. Expression of the lacZ gene was determined in Saccharomyces cerevisiae by measuring the amount of beta-galactosidase fusion protein using immuno-gel electrophoresis. A 5.3-fold difference in expression was found among the various constructs. It was found that there was no preference for a certain nucleotide in any position of the second codon and there was no distinct correlation between the level of tRNA corresponding to any particular second codon and expression. No correlation could be found between the local secondary structure and expression. When the overall codon usage in yeast and the codon usage in the second position of the mRNA is compared, there is no obvious significant difference in preference. This indicates that in yeast, in contrast to Escherichia coli, the codon choice at the beginning of the mRNA does not deviate from the one further downstream and is determined by the requirements for optimal translation elongation. Important determinants of the optimal context for an initiation codon in yeast therefore must be located mainly 5' from this codon.

Amino Acid Sequence↗

PubMed2002

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.

Animals↗