Codon usage in Tetrahymena and other ciliates

Codon usage in ciliates was examined by analyzing the coding regions of 22 ciliate genes corresponding to a total of 26,142 nucleotides (8,714 codons). It was found that Tetrahymena, Paramecium and the hypotrichs (Oxytricha and Stylonychia) differed in which synonymous codons were used most frequently by their genes. In fact, the codon choices in highly expressed Tetrahymena genes were more similar to those of yeast genes than those of Paramecium genes. The ciliates do not appear to have unusually strong biases in codon usage frequency when compared to other protists such as yeast. The analysis of the Tetrahymena genes indicated that genes which are highly expressed during normal cell growth have a stronger bias towards using the "preferred" codons than those expressed at lower levels during growth or for brief periods during processes such as conjugation. This conforms to what is found in other protists.     

Patterns of codon usage in two ciliates that reassign the genetic code: Tetrahymena thermophila and Paramecium tetraurelia

We used the recently sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia to analyze the codon usage patterns in both organisms; we have analyzed codon usage bias, Gln codon usage, GC content and the nucleotide contexts of initiation and termination codons in Tetrahymena and Paramecium. We also studied how these trends change along the length of the genes and in a subset of highly expressed genes. Our results corroborate some of the trends previously described in Tetrahymena, but also negate some specific observations. In both genomes we found a strong bias toward codons with low GC content; however, in highly expressed genes this bias is smaller and codons ending in GC tend to be more frequent. We also found that codon bias increases along gene segments and in highly expressed genes and that the context surrounding initiation and termination codons are always AT rich. Our results also suggest differences in the efficiency of translation of the reassigned stop codons between the two species and between the reassigned codons. Finally, we discuss some of the possible causes for such translational efficiency differences.     

Cloning and characterization of the gene encoding the highly expressed ribosomal protein l3 of the ciliated protozoan Tetrahymena thermophila. Evidence for differential codon usage in highly expressed genes

We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features characteristic of ribosomal protein genes in Tetrahymena. The codon usage of the L3 gene is highly biased. A thorough analysis of codon usage in Tetrahymena genes revealed that genes could be categorized into two classes according to codon usage bias. Class A comprises r-protein genes and a number of other highly expressed genes. Class B comprises weakly expressed genes such as the conjugation induced CnjB and CnjC genes, but surprisingly, this class also contains abundantly expressed genes such as the genes encoding the surface antigens SerH3 and SerH1. Codon usage is slightly more restricted in class A than in class B, but both classes exhibit distinct and different codon usage biases. Class A genes preferentially use C and U in the silent third codon positions, whereas class B genes preferentially use A and U in the silent third codon positions. The analysis suggests that two different strategies have been employed for optimization of codon usage in the A+T-rich genome of Tetrahymena.     

Tandem Stop Codons in Ciliates That Reassign Stop Codons

Tandem stop codons are extra stop codons hypothesized to be present downstream of genes to act as a backup in case of read-through of the real stop codon. Although seemingly absent from Escherichia coli, recent studies have confirmed the presence of such codons in yeast. In this paper we will analyze the genomes of two ciliate species—Paramecium tetraurelia and Tetrahymena thermophila—that reassign the stop codons TAA and TAG to glutamine, for the presence of tandem stop codons. We show that there are more tandem stop codons downstream of both Paramecium and Tetrahymena genes than expected by chance given the base composition of the downstream regions. This excess of tandem stop codons is larger in Tetrahymena and Paramecium than in yeast. We propose that this might be caused by a higher frequency of stop codon read-through in these species than in yeast, possibly because of a leaky termination machinery resulting from stop codon reassignment.     

Codon usage in regulatory genes in Escherichia coli does not reflect selection for ''rare'' codons.

It has often been suggested that differential usage of codons recognized by rare tRNA species, i.e. "rare codons", represents an evolutionary strategy to modulate gene expression. In particular, regulatory genes are reported to have an extraordinarily high frequency of rare codons. From E. coli we have compiled codon usage data for highly expressed genes, moderately/lowly expressed genes, and regulatory genes. We have identified a clear and general trend in codon usage bias, from the very high bias seen in very highly expressed genes and attributed to selection, to a rather low bias in other genes which seems to be more influenced by mutation than by selection. There is no clear tendency for an increased frequency of rare codons in the regulatory genes, compared to a large group of other moderately/lowly expressed genes with low codon bias. From this, as well as a consideration of evolutionary rates of regulatory genes, and of experimental data on translation rates, we conclude that the pattern of synonymous codon usage in regulatory genes reflects primarily the relaxation of natural selection.     

Consequences of stop codon reassignment on protein evolution in ciliates with alternative genetic codes

Tetrahymena thermophila and Paramecium tetraurelia are ciliates that reassign TAA and TAG from stop codons to glutamine codons. Because of the lack of full genome sequences, few studies have concentrated on analyzing the effects of codon reassignment in protein evolution. We used the recently sequenced genome of these species to analyze the patterns of amino acid substitution in ciliates that reassign the code. We show that, as expected, the codon reassignment has a large impact on amino acid substitutions in closely related proteins; however, contrary to expectations, these effects also hold for very diverged proteins. Previous studies have used amino acid substitution data to calculate the minimization of the genetic code; our results show that because of the lasting influence of the code in the patterns of substitution, such studies are tautological. These different substitution patterns might affect alignment of ciliate proteins, as alignment programs use scoring matrices based on substitution patterns of organisms that use the standard code. We also show that glutamine is used more frequently in ciliates than in other species, as often as expected based on the presence of the 2 new reassigned codons, indicating that the frequencies of amino acids in proteomes is mostly determined by neutral processes based on their number of codons.     

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.     

Ciliates use both variant and universal genetic codes: evidence of omnipotent eRF1s in the class Litostomatea

Stop codon reassignments have occurred very frequently in ciliates. In some ciliate species, the universal stop codons UAA and UAG are translated into glutamine, while in some other species, the universal stop codon UGA appears to be translated into cysteine or tryptophan. The class Litostomatea has been hypothesized to be the only group of ciliates using the universal genetic code. However, the hypothesis was based on a statistical analysis of quite small sequence dataset which was insufficient to elucidate the codon usage of the class among such highly deviated phylum. In this study, together with the updated database sequence analysis for the class, we approached the problem of stop codon usage by examining the capacity of the translation termination factor eRF1 for recognizing stop codons. Using in vivo assay systems in budding yeast, we estimated the activity of eRF1 from two litostome species Didinium nasutum and Dileptus margaritifer. The results clearly showed that Didinium and Dileptus eRF1s efficiently recognize all three stop codons. This is the first experimental evidence that strongly supports the hypothesis that litostome ciliates use universal genetic code.     

Evidence for a trade-off between translational efficiency and splicing regulation in determining synonymous codon usage in Drosophila melanogaster

In Drosophila melanogaster, synonymous codons corresponding to the most abundant cognate tRNAs are used more frequently, especially in highly expressed genes. Increased use of such "optimal" codons is considered an adaptation for translational efficiency. Need it always be the case that selection should favor the use of a translationally optimal codon? Here, we investigate one possible confounding factor, namely, the need to specify information in exons necessary to enable correct splicing. As expected from such a model, in Drosophila many codons show different usage near intron-exon boundaries versus exon core regions. However, this finding is in principle also consistent with Hill-Robertson effects modulating usage of translationally optimal codons. However, several results support the splice model over the translational selection model: 1) the trends in codon usage are strikingly similar to those in mammals in which codon usage near boundaries correlates with abundance in exonic splice enhancers (ESEs), 2) codons preferred near boundaries tend to be enriched for A and avoid C (conversely those avoided near boundaries prefer C rather than A), as expected were ESEs involved, and 3) codons preferred near boundaries are typically not translationally optimal. We conclude that usage of translationally optimal codons usage is compromised in the vicinity of splice junctions in intron-containing genes, to the effect that we observe higher levels of usage of translationally optimal codons at the center of exons. On the gene level, however, controlling for known correlates of codon bias, the impact on codon usage patterns is quantitatively small. These results have implications for inferring aspects of the mechanism of splicing given nothing more than a well-annotated genome.     

Analysis of Genomic G + C Content, Codon Usage, Initiator Codon Context and Translation Termination Sites In Tetrahymena Thermophila

In recent years, the amount of molecular sequencing data from Tetrahymena thermophila has dramatically increased. We analyzed G + C content, codon usage, initiator codon context and stop codon sites in the extremely A + T rich genome of this ciliate. Average G + C content was 38% for protein coding regions, 21% for 5' non-coding sequences, 19% for 3' non-coding sequences, 15% for introns, 19% for micronuclear limited sequences and 17% for macronuclear retained sequences flanking micronuclear specific regions. The 75 available T. thermophila protein coding sequences favored codons ending in T and, where possible, avoided those with G in the third position. Highly expressed genes were relatively G + C-rich and exhibited an extremely biased pattern of codon usage while developmentally regulated genes were more A + T-rich and showed less codon usage bias. Regions immediately preceding Tetrahymena translation initiator codons were generally A-rich. For the 60 stop codons examined, the frequency of G in the end + 1 site was much higher than expected whereas C never occupied this position.     

Are codon usage patterns in unicellular organisms determined by selection-mutation balance?

Strongly biased codon usage is common in unicellular organisms, particularly in highly expressed genes. The bias is most simply explained as a balance between selection and mutation, with selection favouring those codons which are more efficiently translated. In a review Ikemura (1985) has proposed four rules for predicting which codons will be preferred, based on the properties of the transfer RNAs responsible for translating messenger RNA into protein. In this paper codon usage in E. coli and yeast is re-examined using the recent compilation of Maruyama et al. (1986). The codon adaptation index of Sharp and Li (1986a) is used as a measure of gene expression to investigate the importance of this factor. It is found that Ikemura's rules successfully predict preferred codons for yeast, but that two of them work less well for E. coli, and it is suggested that some of the apparent bias in weakly expressed genes of E. coli may be due to contextual effects on mutation rates.     

Synonymous codon usage variation among Giardia lamblia genes and isolates.

The pattern of codon usage in the amitochondriate diplomonad Giardia lamblia has been investigated. Very extensive heterogeneity was evident among a sample of 65 genes. A discrete group of genes featured unusual codon usage due to the amino acid composition of their products: these variant surface proteins (VSPs) are unusually rich in Cys and, to a lesser extent, Gly and Thr. Among the remaining 50 genes, correspondence analysis revealed a single major source of variation in synonymous codon usage. This trend was related to the extent of use of a particular subset of 21 codons which are inferred to be those which are optimal for translation; at one end of this trend were genes expected to be expressed at low levels with near random codon usage, while at the other extreme were genes expressed at high levels in which these optimal codons are used almost exclusively. These optimal codons all end in C or G so G + C content at silent sites varies enormously among genes, from values around 40%, expected to reflect the background level of the genome, up to nearly 100%. Although VSP genes are occasionally extremely highly expressed, they do not, in general, have high frequencies of optimal codons, presumably because their high expression is only intermittent. These results indicate that natural selection has been very effective in shaping codon usage in G. lamblia. These analyses focused on sequences from strains placed within G. lamblia "assemblage A"; a few sequences from other strains revealed extensive divergence at silent sites, including some divergence in the pattern of codon usage.     

Studies on codon usage in Entamoeba histolytica

Codon usage bias of Entamoeba histolytica, a protozoan parasite, was investigated using the available DNA sequence data. Entamoeba histolytica having AT rich genome, is expected to have A and/or T at the third position of codons. Overall codon usage data analysis indicates that A and/or T ending codons are strongly biased in the coding region of this organism. However, multivariate statistical analysis suggests that there is a single major trend in codon usage variation among the genes. The genes which are supposed to be highly expressed are clustered at one end, while the majority of the putatively lowly expressed genes are clustered at the other end. The codon usage pattern is distinctly different in these two sets of genes. C ending codons are significantly higher in the putatively highly expressed genes suggesting that C ending codons are translationally optimal in this organism. In the putatively lowly expressed genes A and/or T ending codons are predominant, which suggests that compositional constraints are playing the major role in shaping codon usage variation among the lowly expressed genes. These results suggest that both mutational bias and translational selection are operational in the codon usage variation in this organism.     

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 the primary structure of mRNA from Escherichia coli: occurrence of nucleotides on the 3'-side of the codon

The occurrence of nucleotides of the 3' side of codons has been determined in highly and weakly expressed genes from Escherichia coli. It was found that the usage of some amino acid codons in highly expressed genes was site specific, depending on the base 3' to the codon. The role of the 3' nucleotide as a modulator of codon translation effectiveness is discussed. The rules of synonymous codon usage in relation to the 3' flanking nucleotide have been established for highly expressed genes. For example, if a triplet next to the lysine codon starts with guanosine, lysine is preferably encoded by AAA and not by AAG (P less than 10(-8), while of cytidine is 3' to the lysine codon, AAG is preferred over AAA (P less than 0.001). These rules are observed in highly and absent in weakly expressed mRNAs and can be used in the chemical synthesis of genes designed for expression in E. coli.     

Regularities of the nucleotide sequence at the 5'-end of the codon in Escherichia coli genes

The frequencies of occurrence of nucleotides at the 5' side of codons have been determined in highly and weakly expressed genes from E. coli. Significant constraints on the nucleotide 5' to some codons were found in highly expressed genes. Certain rules of synonymous codon usage depending on the amino acid 3' of the codon were established. E. g., codon possessing quanosine in the third position (NNG) are preferred over NNA if the next amino acid is lysine (P less than 10(-5)). On the other hand, rules of synonymous codon usage in relation to 5' flanking nucleotide were found. For example, when coding for aspartic acid, GAC codon is preferred over GAU (P less than 0.001) if uridine is 5' to codon and on the contrary GAU is favoured (P less than 0.0001) if quanosine is at the 5' side of aspartic acid codon. These rules can be used in the chemical synthesis of genes designed for expression in E. coli.     

Ultradian clocks in eukaryotic microbes: from behavioural observation to functional genomics

Period homeostasis is the defining characteristic of a biological clock. Strict period homeostasis is found for the ultradian clocks of eukaryotic microbes. In addition to being temperature-compensated, the period of these rhythms is unaffected by differences in nutrient composition or changes in other environmental variables. The best-studied examples of ultradian clocks are those of the ciliates Paramecium tetraurelia and Tetrahymena sp. and of the fission yeast, Schizosaccharomyces pombe. In these single cell eukaryotes, up to seven different parameters display ultradian rhythmicity with the same, species- and strain-specific period. In fission yeast, the molecular genetic analysis of ultradian clock mechanisms has begun with the systematic analysis of mutants in identified candidate genes. More than 40 "clock mutants" have already been identified, most of them affected in components of major regulatory and signalling pathways. These results indicate a high degree of complexity for a eukaryotic clock mechanism. BioEssays 22:16-22, 2000.     

Codon optimizer: a freeware tool for codon optimization

Selection plays a major role in the determination of codon usage in all organisms studied so far. In highly expressed genes, a narrow set of codons is used and these codons correspond to the more abundant tRNA species. This minimizes the risk of tRNA depletion during translation. In fact, the codons in a gene may be true bottlenecks, especially in cases where foreign genes are expressed in a host in which the usage of codons in highly expressed genes does not resemble the usage of codons in the species from which the foreign gene originates. In such cases, it has been shown that substitution of rare codons in the introduced gene may increase the yield dramatically. In addition, replacement of rare codons might decrease the chance of misincorporation and protect the protein from premature turnover. Here, a piece of software is announced that calculates a codon-optimized sequence of any gene based on knowledge of highly expressed genes of a host. In addition, it calculates the codon adaptation index of the gene and identifies internal type II restriction sites of the optimized sequence. The program runs under Windows and is available as freeware for use in academia.     

Polypeptide release factor eRF1 from Tetrahymena thermophila: cDNA cloning, purification and complex formation with yeast eRF3.

The first cDNA for the translational release factor eRF1 of ciliates was cloned from Tetrahymena thermophila. The coding frame contained one UAG and nine UAA codons that are reassigned for glutamine in Tetrahymena. The deduced protein sequence is 57% identical to human eRF1. The recombinant Tetrahymena eRF1 purified from a yeast expression system was able to bind to yeast eRF3 as do other yeast or mammalian eRF1s as a prerequisite step for protein termination. The recombinant Tetrahymena eRF1, nevertheless, failed to catalyze polypeptide termination in vitro with rat or Artemia ribosomes, at least in part, due to less efficient binding to the heterologous ribosomes. Stop codon specificity and phylogenetic significance of Tetrahymena eRF1 are discussed from the conservative protein feature.     

The ciliate Euplotes octocarinatus expresses two polypeptide release factors of the type eRF1

Amplification of macronuclear DNA of the ciliate Euplotes octocarinatus revealed the presence of two genes encoding putative polypeptide release factors (RFs) of the codon specific class-I type. They are named eRF1a and eRF1b, respectively. cDNA amplification revealed that both eRF1 genes are expressed. Determination of their copy numbers showed that they are similarly amplified to a level of about 27,000. The deduced protein sequences of the two genes are 57 and 58% identical with human eRF1 and 79% identical to each other. The gene encoding eRF1b possesses three in-frame UGA codons. This codon is known to encode cysteine in Euplotes; only UAA and UAG are used as stop codons in this organism. The primary structure of the two release factors is analyzed and compared with the primary structure of other eukaryotic release factors including the one of Tetrahymena thermophila which uses only UGA as a stop codon. eRF1a and eRF1b of Euplotes as well as eRF1 of Tetrahymena differ from human eRF1 and other class-I release factors of eukaryotes in a domain recently proposed to be responsible for codon recognition. Based on the changes which we observe in this region and the differential use of the stop codons in these two ciliates we predict the amino acids participating in stop codon recognition in eRF1 release factors.