Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases.

Codon usage data for 56 Bacillus subtilis genes show that synonymous codon usage in B. subtilis is less biased than in Escherichia coli, or in Saccharomyces cerevisiae. Nevertheless, certain genes with a high codon bias can be identified by correspondence analysis, and also by various indices of codon bias. These genes are very highly expressed, and a general trend (a decrease) in codon bias across genes seems to correspond to decreasing expression level. This, then, may be a general phenomenon in unicellular organisms. The unusually small effect of translational selection on the pattern of codon usage in lowly expressed genes in B. subtilis yields similar dinucleotide frequencies among different codon positions, and on complementary strands. These patterns could arise through selection on DNA structure, but more probably are largely determined by mutation. This prevalence of mutational bias could lead to difficulties in assessing whether open reading frames encode proteins.     

Codon usage in highly expressed genes of Haemophillus influenzae and Mycobacterium tuberculosis: translational selection versus mutational bias

Biases in the codon usage and base compositions at three codon sites in different genes of A+T-rich Gram-negative bacterium Haemophillus influenzae and G+C-rich Gram-positive bacterium Mycobacterium tuberculosis have been examined to address the following questions: (1) whether the synonymous codon usage in organisms having highly skewed base compositions is totally dictated by the mutational bias as reported previously (Sharp, P.M., Devine, K.M., 1989. Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do `prefer' optimal codons. Nucleic Acids Res. 17, 5029–5039), or is also controlled by translational selection; (2) whether preference of G in the first codon positions by highly expressed genes, as reported in Escherichia coli (Gutierrez, G., Marquez, L., Marin, A., 1996. Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency. Nucleic Acids Res. 24, 2525–2527), is true in other bacteria; and (3) whether the usage of bases in three codon positions is species-specific. Result presented here show that even in organisms with high mutational bias, translational selection plays an important role in dictating the synonymous codon usage, though the set of optimal codons is chosen in accordance with the mutational pressure. The frequencies of G-starting codons are positively correlated to the level of expression of genes, as estimated by their Codon Adaptation Index (CAI) values, in M. tuberculosis as well as in H. influenzae in spite of having an A+T-rich genome. The present study on the codon preferences of two organisms with oppositely skewed base compositions thus suggests that the preference of G-starting codons by highly expressed genes might be a general feature of bacteria, irrespective of their overall G+C contents. The ranges of variations in the frequencies of individual bases at the first and second codon positions of genes of both H. influenzae and M. tuberculosis are similar to those of E. coli, implying that though the composition of all three codon positions is governed by a selection-mutation balance, the mutational pressure has little influence in the choice of bases at the first two codon positions, even in organisms with highly biased base compositions.     

Codon usage in mitochondrial genomes: distinguishing context-dependent mutation from translational selection

We analyze the frequencies of synonymous codons in animal mitochondrial genomes, focusing particularly on mammals and fish. The frequencies of bases at 4-fold degenerate sites are found to be strongly influenced by context-dependent mutation, which causes correlations between pairs of neighboring bases. There is a pattern of excess of certain dinucleotides and deficit of others that is consistent across large numbers of species, despite the wide variation of single-nucleotide frequencies among species. In many bacteria, translational selection is an important influence on codon usage. In order to test whether translational selection also plays a role in mitochondria, we need to control for context-dependent mutation. Selection for translational accuracy can be detected by comparison of codon usage in conserved and variable sites in the same genes. We give a test of this type that works in the presence of context-dependent mutation. There is very little evidence for translational accuracy selection in the mitochondrial genes considered here. Selection for translational efficiency might lead to preference for codons that match the limited repertoire of anticodons on the mitochondrial tRNAs. This is difficult to detect because the effect would usually be in the same direction in comparable to codon families and so would not cause an observable difference in codon usage between families. Several lines of evidence suggest that this type of selection is weak in most cases. However, we found several cases where unusual bases occur at the wobble position of the tRNA, and in these cases, some evidence for selection on codon usage was found. We discuss the way that these unusual cases are associated with codon reassignments in the mitochondrial genetic code.     

Synonymous codon usage in Lactococcus lactis: mutational bias versus translational selection

In this study codon usage bias of all experimentally known genes of Lactococcus lactis has been analyzed. Since Lactococcus lactis is an AT rich organism, it is expected to occur A and/or T at the third position of codons and detailed analysis of overall codon usage data indicates that A and/or T ending codons are predominant in this organism. However, multivariate statistical analyses based both on codon count and on relative synonymous codon usage (RSCU) detect a large number of genes, which are supposed to be highly expressed are clustered at one end of the first major axis, while majority of the putatively lowly expressed genes are clustered at the other end of the first major axis. It was observed that in the highly expressed genes C and T ending codons are significantly higher than the lowly expressed genes and also it was observed that C ending codons are predominant in the duets of highly expressed genes, whereas the T endings codons are abundant in the quartets. Abundance of C and T ending codons in the highly expressed genes suggest that, besides, compositional biases, translational selection are also operating in shaping the codon usage variation among the genes in this organism as observed in other compositionally skewed organisms. The second major axis generated by correspondence analysis on simple codon counts differentiates the genes into two distinct groups according to their hydrophobicity values, but the same analysis computed with relative synonymous codon usage values could not discriminate the genes according to the hydropathy values. This suggests that amino acid composition exerts constraints on codon usage in this organism. On the other hand the second major axis produced by correspondence analysis on RSCU values differentiates the genes into two groups according to the synonymous codon usage for cysteine residues (rarest amino acids in this organism), which is nothing but a artifactual effect induced by the RSCU values. Other factors such as length of the genes and the positions of the genes in the leading and lagging strand of replication have practically no influence in the codon usage variation among the genes in this organism.     

A mutational analysis of Caenorhabditis elegans in space

The International Caenorhabditis elegans Experiment First Flight (ICE-First) was a project using C. elegans as a model organism to study the biological effects of short duration spaceflight (11 days in the International Space Station). As a member of the ICE-First research team, our group focused on the mutational effects of spaceflight. Several approaches were taken to measure mutational changes that occurred during the spaceflight including measurement of the integrity of poly-G/poly-C tracts, determination of the mutation frequency in the unc-22 gene, analysis of lethal mutations captured by the genetic balancer eT1(III;V), and identification of alterations in telomere length. By comparing the efficiency, sensitivity, and convenience of these methods, we deduced that the eT1 balancer system is well-suited for capturing, maintaining and recovering mutational events that occur over several generations during spaceflight. In the course of this experiment, we have extended the usefulness of the eT1 balancer system by identifying the physical breakpoints of the eT1 translocation and have developed a PCR assay to follow the eT1 chromosomes. C. elegans animals were grown in a defined liquid media during the spaceflight. This is the first analysis of genetic changes in C. elegans grown in the defined media. Although no significant difference in mutation rate was detected between spaceflight and control samples, which is not surprising given the short duration of the spaceflight, we demonstrate here the utility of worms as an integrating biological dosimeter for spaceflight.     

The mutational structure of metabolism in Caenorhabditis elegans

A properly functioning organism must maintain metabolic homeostasis. Deleterious mutations degrade organismal function, presumably at least in part via effects on metabolic function. Here we present an initial investigation into the mutational structure of the Caenorhabditis elegans metabolome by means of a mutation accumulation experiment. We find that pool sizes of 29 metabolites vary greatly in their vulnerability to mutation, both in terms of the rate of accumulation of genetic variance (the mutational variance, VM) and the rate of change of the trait mean (the mutational bias, ΔM). Strikingly, some metabolites are much more vulnerable to mutation than any other trait previously studied in the same way. Although we cannot statistically assess the strength of mutational correlations between individual metabolites, principal component analysis provides strong evidence that some metabolite pools are genetically correlated, but also that there is substantial scope for independent evolution of different groups of metabolites. Averaged over mutation accumulation lines, PC3 is positively correlated with relative fitness, but a model in which metabolites are uncorrelated with fitness is nearly as good by Akaike's Information Criterion.     

Spontaneous mutational variation for body size in Caenorhabditis elegans

We measured the impact of new mutations on genetic variation for body size in two independent sets of C. elegans spontaneous mutation-accumulation (MA) lines, derived from the N2 strain, that had been maintained by selfing for 60 or 152 generations. The two sets of lines gave broadly consistent results. The change of among-line genetic variation between cryopreserved controls and the MA lines implied that broad sense heritability increased by 0.4% per generation. Overall, MA reduced mean body size by approximately 0.1% per generation. The genome-wide rate for mutations with detectable effects on size was estimated to be approximately 0.0025 per haploid genome per generation, and their mean effects were approximately 20%. The proportion of mutations that increase body size was estimated by maximum likelihood to be no more than 20%, suggesting that the amount of mutational variation available for selection for increased size could be quite small. This hypothesis was supported by an artificial selection experiment on adult body size, started from a single highly inbred N2 individual. We observed a strongly asymmetrical response to selection of a magnitude consistent with the input of mutational variance observed in the MA experiment.     

Proteome composition and codon usage in spirochaetes: species-specific and DNA strand-specific mutational biases

The genomes of the spirochaetes Borrelia burgdorferi and Treponema pallidum show strong strand-specific skews in nucleotide composition, with the leading strand in replication being richer in G and T than the lagging strand in both species. This mutation bias results in codon usage and amino acid composition patterns that are significantly different between genes encoded on the two strands, in both species. There are also substantial differences between the species, with T.pallidum having a much higher G+C content than B. burgdorferi. These changes in amino acid and codon compositions represent neutral sequence change that has been caused by strong strand- and species-specific mutation pressures. Genes that have been relocated between the leading and lagging strands since B. burgdorferi and T.pallidum diverged from a common ancestor now show codon and amino acid compositions typical of their current locations. There is no evidence that translational selection operates on codon usage in highly expressed genes in these species, and the primary influence on codon usage is whether a gene is transcribed in the same direction as replication, or opposite to it. The dnaA gene in both species has codon usage patterns distinctive of a lagging strand gene, indicating that the origin of replication lies downstream of this gene, possibly within dnaN. Our findings strongly suggest that gene-finding algorithms that ignore variability within the genome may be flawed.     

Selection against males in Caenorhabditis elegans under two mutational treatments

Within populations with mixed mating systems, selfing is expected to be favoured over outcrossing unless a countervailing process such as severe inbreeding depression is present. In this study, we consider the relationship between the expression of deleterious alleles and the maintenance of outcrossing in the nematode species, Caenorhabditis elegans. This species is characterized by an androdioecious breeding system composed of males at low frequency and self-fertilizing hermaphrodites that can only outcross via males. Here, we find that experimentally increasing the mutational load in four different isogenic wild isolates using 10 generations of Ethylmethane sulphonate (EMS) and UV irradiation mutagenesis significantly diminishes the cost of males. Males are maintained at higher frequencies in mutagenized versus non-mutagenized populations. Nevertheless, males still tend to be driven to low frequencies within isolates that are known to be prone to lose males. Further, we determine the viability effects of a single round of mutagen exposure and find that, for EMS, outcrossing overcomes the almost completely recessive and nearly lethal effects generated. We briefly interpret our results in light of current evolutionary theory of outcrossing rates.     

Transposable elements, mutational correlations, and population divergence in Caenorhabditis elegans

Transposable element activity is thought to be responsible for a large portion of all mutations, but its influence on the evolution of populations has not been well studied. Using mutation accumulation experiments with the nematode Caenorhabditis elegans, we investigated the impact of transposable element activity on the production of mutational variances and covariances. The experiments involved the use of two mutator strains (RNAi-deficient mutants) that are characterized by high levels of germline transposition, as well as the Bristol N2 strain, which lacks germline transposition. We found that transposition led to an increase in mutational heritabilities, as well as to the intensification of correlation patterns observed in the absence of transposition. No mutational trade-offs were detected and mutations generally had a deleterious effect on components of fitness. We also tested whether the pattern of mutational covariation could be used to predict observed patterns of population divergence in this species. Using 15 natural populations, we found that population divergence of C. elegans in multivariate phenotypic space occurred in directions only partially concordant with mutation, and thus other evolutionary factors, such as natural selection and genetic drift, must be acting to produce divergence within this species. Our results suggest that mutations induced by mobile elements in C. elegans are similar to other spontaneous mutations with respect to their contribution to the microevolution of quantitative traits.     

The Role of eIF1 in Translation Initiation Codon Selection in Caenorhabditis elegans

The selection of a proper AUG start codon requires the base-pairing interactions between the codon on the mRNA and the anticodon of the initiator tRNA. This selection process occurs in a pre-initiation complex that includes multiple translation initiation factors and the small ribosomal subunit. To study how these initiation factors are involved in start codon recognition in multicellular organisms, we isolated mutants that allow the expression of a GFP reporter containing a non-AUG start codon. Here we describe the characterization of mutations in eif-1, which encodes the Caenorhabditis elegans translation initiation factor 1 (eIF1). Two mutations were identified, both of which are substitutions of amino acid residues that are identical in all eukaryotic eIF1 proteins. These residues are located in a structural region where the amino acid residues affected by the Saccharomyces cerevisiae eIF1 mutations are also localized. Both C. elegans mutations are dominant in conferring a non-AUG translation initiation phenotype and lead to growth arrest defects in homozygous animals. By assaying reporter constructs that have base changes at the AUG start codon, these mutants are found to allow expression from most reporters that carry single base changes within the AUG codon. This trend of non-AUG mediated initiation was also observed previously for C. elegans eIF2β mutants, indicating that these two factors play a similar role. These results support that eIF1 functions in ensuring the fidelity of AUG start codon recognition in a multicellular organism.TRANSLATION initiation is thought to be one of the most complex cellular processes in eukaryotes. It involves at least 12 translation initiation factors (eIFs) comprising over 30 polypeptides (Pestova et al. 2007). These factors bring together an initiator methionyl tRNA (Met-tRNAi), the small ribosomal subunit, and a mRNA to form a 48S initiation complex. An important role performed by this complex is to select an AUG codon to initiate translation of the mRNA. Since the first AUG at the 5′ end of most mRNAs is selected as the start site, it is believed that the initiation complex scans for an AUG start codon as it moves from the 5′-capped end of the mRNA toward the 3′ end, as proposed in the ribosomal scanning model (Kozak 1978; Kozak 1989). The recognition of the AUG start codon is mediated by the anticodon of the Met-tRNAi, and the matching base-pairing interactions between the codon of the mRNA and the anticodon determine the site of initiation (Cigan et al. 1988). These base-pairing interactions are essential, but are likely not the only components required for accurately selecting the correct AUG start codon. Numerous initiation factors along with base-pairing interactions have been shown to aid in the AUG recognition process (Pestova et al. 2007).Translation initiation factors involved in start codon selection fidelity were first identified through genetic studies performed in the yeast Saccharomyces cerevisiae. Mutant strains with a modified His4 gene that had an AUU instead of an AUG at the native start site were selected for the ability to survive on media lacking histidine (Donahue et al. 1988; Castilho-Valavicius et al. 1990). These mutants were found to be able to produce the His4 protein by using a downstream inframe UUG codon (the third codon within the His4 coding region) as the translation start site. Further analyses determined that non-AUG initiation occurred mostly from a UUG codon and not significantly from other codons (Huang et al. 1997). These mutants defined five genetic loci and were named sui1-sui5 (suppressor of initiation codon) on the basis of their ability to initiate translation at a non-AUG codon.The sui1 suppressors were found to have missense mutations in eIF1. These missense mutations showed semidominant or codominant properties in non-AUG translation initiation while deletion of the eIF1 gene led to lethality in yeast (Yoon and Donahue 1992). eIF1 is a highly conserved protein with a size of approximately 12 kDa that plays a vital role in multiple translation initiation steps. eIF1 is incorporated into a multifactor complex that includes eIF1A, eIF3, and eIF5 and stimulates the recruiting of the ternary complex (consisting of eIF2 · GTP and the charged Met-tRNAi) to the small ribosomal subunit to form the 43S pre-initiation complex (Singh et al. 2004). eIF1 acts synergistically with eIF1A to promote continuous ribosomal scanning for AUG codons by stabilizing an open conformation that allows mRNA to pass through the complex (Maag et al. 2005; Cheung et al. 2007; Passmore et al. 2007). It also mediates the assembly of the ribosomal initiation complex at the AUG start codon (Pestova et al. 1998). eIF1 dissociates from the complex upon recognition of the AUG codon and this dissociation is necessary to trigger a series of conformational changes leading to the translation elongation phase (Algire et al. 2005). Consistent with these roles, sui1 mutations reduce the affinity of eIF1 for the ribosome and cause premature release of eIF1 at non-AUG codons (Cheung et al. 2007). Other sui mutations support the involvement of four additional genes in translation initiation fidelity in yeast. Mutations have been isolated in the heterotrimeric eIF2 as SUI2 (α-subunit) (Cigan et al. 1989), SUI3 (β-subunit) (Donahue et al. 1988), and SUI4 (γ-subunit) (Huang et al. 1997), and a mutation in eIF5 corresponds to the SUI5 mutant (Huang et al. 1997).However, the genetic studies that identified these translation fidelity mutants were conducted only in yeast. It is not known if there are similar mechanisms regulating translation initiation fidelity in multicellular organisms. To address this question, we designed a genetic system to isolate C. elegans mutants that have reduced fidelity in AUG start codon selection (Zhang and Maduzia 2010). Mutants were selected on the basis of their ability to express a GFP reporter that contains a GUG codon in place of its native translation start site. Here we report the characterization of two mutants that have mutations in eIF1. Unlike yeast sui1 mutants, which preferred the UUG codon, these mutants are capable of using a subset of non-AUG codons for translation initiation. Our results are consistent with eIF1 playing a role in the fidelity of AUG codon selection, perhaps by discriminating base-pairing interactions between the codon and anticodon during start-site selection.     

Codon usage in plant genes.

We have examined codon bias in 207 plant gene sequences collected from Genbank and the literature. When this sample was further divided into 53 monocot and 154 dicot genes, the pattern of relative use of synonymous codons was shown to differ between these taxonomic groups, primarily in the use of G + C in the degenerate third base. Maize and soybean codon bias were examined separately and followed the monocot and dicot codon usage patterns respectively. Codon preference in ribulose 1,5 bisphosphate and chlorophyll a/b binding protein, two of the most abundant proteins in leaves was investigated. These highly expressed are more restricted in their codon usage than plant genes in general.     

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

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

Codon usage in Pseudomonas aeruginosa.

We have generated a codon usage table for Pseudomonas aeruginosa. Codon usage in P. aeruginosa is extremely biased. In contrast to E. coli and yeast, P. aeruginosa preferentially uses those codons within a synonymous codon group with the strongest predicted codon-anticodon interaction. We were unable to correlate a particular codon usage pattern with predicted levels of mRNA expressivity. The choice of a third base reflects the high guanine plus cytosine content of the P. aeruginosa genome (67.2%) and cytosine is the preferred nucleotide for the third codon position.     

Codon usage in Entamoeba histolytica.

The codon usage of 10 E. histolytica genes comprising 4455 codons was analysed. The codon usage revealed an extremely biased use of synonymous codons with a preference for NNU (44%) and NNA (41.4%) codons. Codons CGG (arg), AGG (arg) and CCG (pro) were absent in the E. histolytica genes examined. The codon usage of E. histolytica resembled that of Plasmodium falciparum.     

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.     

Codon usage in Giardia lamblia.

A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons. Codon usage revealed a biased use of synonymous codons with a preference for NNC codons (42.1%). The codon usage of G. lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e. Trypanosoma brucei (r = 0.434) and Plasmodium falciparum (r = -0.31). These observations are consistent with the view that G. lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.     

Codon usage and gene expression.

The hypothesis that codon usage regulates gene expression at the level of translation is tested. Codon usage of Escherichia coli and phage lambda is compared by correspondence analysis, and the basis of this hypothesis is examined by connecting codon and tRNA distributions to polypeptide elongation kinetics. Both approaches indicate that if codon usage was random tRNA limitation would only affect the rarest tRNA species. General discrimination against their cognate codons indicates that polypeptide elongation rates are maintained constant. Thus, differences in expression of E. coli genes are not a consequence of their variable codon usage. The preference of codons recognized by the most abundant tRNAs in E. coli genes encoding abundant proteins is explained by a constraint on the cost of proof-reading.     

Synonymous codon usage in Escherichia coli: selection for translational accuracy

In many organisms, selection acts on synonymous codons to improve translation. However, the precise basis of this selection remains unclear in the majority of species. Selection could be acting to maximize the speed of elongation, to minimize the costs of proofreading, or to maximize the accuracy of translation. Using several data sets, we find evidence that codon use in Escherichia coli is biased to reduce the costs of both missense and nonsense translational errors. Highly conserved sites and genes have higher codon bias than less conserved ones, and codon bias is positively correlated to gene length and production costs, both indicating selection against missense errors. Additionally, codon bias increases along the length of genes, indicating selection against nonsense errors. Doublet mutations or replacement substitutions do not explain our observations. The correlations remain when we control for expression level and for conflicting selection pressures at the start and end of genes. Considering each amino acid by itself confirms our results. We conclude that selection on synonymous codon use in E. coli is largely due to selection for translational accuracy, to reduce the costs of both missense and nonsense errors.