Synonymous Codon Substitutions Research Articles

A Comparative Survey of the Frequency and Distribution of Polymorphism in the Genome of Xenopus tropicalis

Naturally occurring DNA sequence variation within a species underlies evolutionary adaptation and can give rise to phenotypic changes that provide novel insight into biological questions. This variation exists in laboratory populations just as in wild populations and, in addition to being a source of useful alleles for genetic studies, can impact efforts to identify induced mutations in sequence-based genetic screens. The Western clawed frog Xenopus tropicalis (X. tropicalis) has been adopted as a model system for studying the genetic control of embryonic development and a variety of other areas of research. Its diploid genome has been extensively sequenced and efforts are underway to isolate mutants by phenotype- and genotype-based approaches. Here, we describe a study of genetic polymorphism in laboratory strains of X. tropicalis. Polymorphism was detected in the coding and non-coding regions of developmental genes distributed widely across the genome. Laboratory strains exhibit unexpectedly high frequencies of genetic polymorphism, with alleles carrying a variety of synonymous and non-synonymous codon substitutions and nucleotide insertions/deletions. Inter-strain comparisons of polymorphism uncover a high proportion of shared alleles between Nigerian and Ivory Coast strains, in spite of their distinct geographical origins. These observations will likely influence the design of future sequence-based mutation screens, particularly those using DNA mismatch-based detection methods which can be disrupted by the presence of naturally occurring sequence variants. The existence of a significant reservoir of alleles also suggests that existing laboratory stocks may be a useful source of novel alleles for mapping and functional studies.

Precise manipulation of chromosomes in vivo enables genome-wide codon replacement.

We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed hierarchical conjugative assembly genome engineering (CAGE) to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects. Our methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.

The Synonymous V107V Mutation In Factor IX Is Not So Silent and May Cause Hemophilia B In Patients

AbstractAbstract 2197Hemophilia B is characterized by structural and functional defects in coagulation factor IX (FIX) caused by mutations in the F9 gene. Various mutations (nonsense, missense, etc.) are known to be associated with the disease, including a synonymous V107V mutation reported recently by Knobe and colleagues (Knobe et al., Hemophilia, 2008). However the mechanism by which this synonymous mutation contributes to the disease has not yet been elucidated. Earlier we have shown that synonymous codon substitutions in the mRNA of the multidrug resistance protein (MDR1) may change the conformation of the protein and result in altered functionality (Kimchi-Sarfaty et al., Science, 2008). Here we have performed in silico analyses of the synonymous codon substitution (GTGàGTA) leading to the V107V polymorphism and found that it may change the mRNA structure, stability, codon usage, and 3D structure of the encoded protein. We hypothesize that changes in codon usage might affect the rhythm of protein translation and thus result in slightly altered FIX conformation. In vitro analyses of FIX mRNA and protein expression supported our in silico analyses. The GTGàGTA (V107V) synonymous mutation results in reduced expression levels as well as an encoded protein with a slightly different conformation compared to wild-type FIX. These results show that the V107V polymorphism is not silent and might cause mild hemophilia B. This work sheds further light on ways in which synonymous mutations impact disease.The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination policy Disclosures:No relevant conflicts of interest to declare.

Identical Skin Toxins by Convergent Molecular Adaptation in Frogs

The Tree of Life is rife with adaptive convergences at all scales and biological levels of complexity. However, natural selection is not likely to result in the independent evolution of identical gene products. Here we report such a striking example of evolutionary convergence in the toxic skin secretions of two distantly related frog lineages. Caeruleins are important decapeptides in pharmacological and clinical research [1] and are commonly believed to represent a single evolutionary class of peptides [2-4]. Instead, our phylogenetic analyses combining transcriptome and genome data reveal that independently evolved precursor genes encode identical caeruleins in Xenopus and Litoria frogs. The former arose by duplication from the cholecystokinin (cck) gene, whereas the latter was derived from the gastrin gene. These hormone genes that are involved in many physiological processes diverged early in vertebrate evolution, after a segmental duplication during the Cambrian period. Besides implicating convergent mutations of the peptide-encoding sequence, recurrent caerulein origins entail parallel shifts of expression from the gut-brain axis to skin secretory glands. These results highlight extreme structural convergence in anciently diverged genes as an evolutionary mechanism through which recurrent adaptation is attained across large phylogenetic distances.

Comparative Genomics in Switchgrass Using 61,585 High‐Quality Expressed Sequence Tags

The development of genomic resources for switchgrass (Panicum virgatum L.), a perennial NAD+‐malic enzyme type C4 grass, is required to enable molecular breeding and biotechnological approaches for improving its value as a forage and bioenergy crop. Expressed sequence tag (EST) sequencing is one method that can quickly sample gene inventories and produce data suitable for marker development or analysis of tissue‐specific patterns of expression. Toward this goal, three cDNA libraries from callus, crown, and seedling tissues of ‘Kanlow’ switchgrass were end‐sequenced to generate a total of 61,585 high‐quality ESTs from 36,565 separate clones. Seventy‐three percent of the assembled consensus sequences could be aligned with the sorghum [Sorghum bicolor (L.) Moench] genome at a E‐value of <1 × 10−20, indicating a high degree of similarity. Sixty‐five percent of the ESTs matched with gene ontology molecular terms, and 3.3% of the sequences were matched with genes that play potential roles in cell‐wall biogenesis. The representation in the three libraries of gene families known to be associated with C4 photosynthesis, cellulose and β‐glucan synthesis, phenylpropanoid biosynthesis, and peroxidase activity indicated likely roles for individual family members. Pairwise comparisons of synonymous codon substitutions were used to assess genome sequence diversity and indicated an overall similarity between the two genome copies present in the tetraploid. Identification of EST–simple sequence repeat markers and amplification on two individual parents of a mapping population yielded an average of 2.18 amplicons per individual, and 35% of the markers produced fragment length polymorphisms.

SynPAM—A Distance Measure Based on Synonymous Codon Substitutions

Measuring evolutionary distances between DNA or protein sequences forms the basis of many applications in computational biology and evolutionary studies. Of particular interest are distances based on synonymous substitutions, since these substitutions are considered to be under very little selection pressure and therefore assumed to accumulate in an almost clock-like manner. SynPAM, the method presented here, allows the estimation of distances between coding DNA sequences based on synonymous codon substitutions. The problem of estimating an accurate distance from the observed substitution pattern is solved by maximum-likelihood with empirical codon substitution matrices employed for the underlying Markov model. Comparisons with established measures of synonymous distance indicate that SynPAM has less variance and yields useful results over a longer time range.

Differentiating between Near- and Non-Cognate Codons in Saccharomyces cerevisiae

BackgroundDecoding of mRNAs is performed by aminoacyl tRNAs (aa-tRNAs). This process is highly accurate, however, at low frequencies (10−3 – 10−4) the wrong aa-tRNA can be selected, leading to incorporation of aberrant amino acids. Although our understanding of what constitutes the correct or cognate aa-tRNA:mRNA interaction is well defined, a functional distinction between near-cognate or single mismatched, and unpaired or non-cognate interactions is lacking.Methodology/Principal FindingsMisreading of several synonymous codon substitutions at the catalytic site of firefly luciferase was assayed in Saccharomyces cerevisiae. Analysis of the results in the context of current kinetic and biophysical models of aa-tRNA selection suggests that the defining feature of near-cognate aa-tRNAs is their potential to form mini-helical structures with A-site codons, enabling stimulation of GTPase activity of eukaryotic Elongation Factor 1A (eEF1A). Paromomycin specifically stimulated misreading of near-cognate but not of non-cognate aa-tRNAs, providing a functional probe to distinguish between these two classes. Deletion of the accessory elongation factor eEF1Bγ promoted increased misreading of near-cognate, but hyperaccurate reading of non-cognate codons, suggesting that this factor also has a role in tRNA discrimination. A mutant of eEF1Bα, the nucleotide exchange factor for eEF1A, promoted a general increase in fidelity, suggesting that the decreased rates of elongation may provide more time for discrimination between aa-tRNAs. A mutant form of ribosomal protein L5 promoted hyperaccurate decoding of both types of codons, even though it is topologically distant from the decoding center.Conclusions/SignficanceIt is important to distinguish between near-cognate and non-cognate mRNA:tRNA interactions, because such a definition may be important for informing therapeutic strategies for suppressing these two different categories of mutations underlying many human diseases. This study suggests that the defining feature of near-cognate aa-tRNAs is their potential to form mini-helical structures with A-site codons in the ribosomal decoding center. An aminoglycoside and a ribosomal factor can be used to distinguish between near-cognate and non-cognate interactions.

Positive selection for unpreferred codon usage in eukaryotic genomes

BackgroundNatural selection has traditionally been understood as a force responsible for pushing genes to states of higher translational efficiency, whereas lower translational efficiency has been explained by neutral mutation and genetic drift. We looked for evidence of directional selection resulting in increased unpreferred codon usage (and presumably reduced translational efficiency) in three divergent clusters of eukaryotic genomes using a simple optimal-codon-based metric (Kp/Ku).ResultsHere we show that for some genes natural selection is indeed responsible for causing accelerated unpreferred codon substitution, and document the scope of this selection. In Cryptococcus and to a lesser extent Drosophila, we find many genes showing a statistically significant signal of selection for unpreferred codon usage in one or more lineages. We did not find evidence for this type of selection in Saccharomyces. The signal of positive selection observed from unpreferred synonymous codon substitutions is coincident in Cryptococcus and Drosophila with the distribution of upstream open reading frames (uORFs), another genic feature known to reduce translational efficiency. Functional enrichment analysis of genes exhibiting low Kp/Ku ratios reveals that genes in regulatory roles are particularly subject to this type of selection.ConclusionThrough genome-wide scans, we find recent selection for unpreferred codon usage at approximately 1% of genetic loci in a Cryptococcus and several genes in Drosophila. Unpreferred codons can impede translation efficiency, and we find that genes with translation-impeding uORFs are enriched for this selection signal. We find that regulatory genes are particularly likely to be subject to selection for unpreferred codon usage. Given that expression noise can propagate through regulatory cascades, and that low translational efficiency can reduce expression noise, this finding supports the hypothesis that translational efficiency may be suppressed in some cases to reduce stochastic noise in gene expression.

New Onto-Tools: Promoter-Express, nsSNPCounter and Onto-Translate

The Onto-Tools suite is composed of an annotation database and eight complementary, web-accessible data mining tools: Onto-Express, Onto-Compare, Onto-Design, Onto-Translate, Onto-Miner, Pathway-Express, Promoter-Express and nsSNPCounter. Promoter-Express is a new tool added to the Onto-Tools ensemble that facilitates the identification of transcription factor binding sites active in specific conditions. nsSNPCounter is another new tool that allows computation and analysis of synonymous and non-synonymous codon substitutions for studying evolutionary rates of protein coding genes. Onto-Translate has also been enhanced to expand its scope and accuracy by fully utilizing the capabilities of the Onto-Tools database. Currently, Onto-Translate allows arbitrary mappings between 28 types of IDs for 53 organisms. Onto-Tools are freely available at .

Structural and Spectroscopic Studies Shed Light on the Mechanism of Oxalate Oxidase

Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.

The odorant receptor repertoire of teleost fish

BackgroundVertebrate odorant receptors comprise three types of G protein-coupled receptors: the OR, V1R and V2R receptors. The OR superfamily contains over 1,000 genes in some mammalian species, representing the largest gene superfamily in the mammalian genome.ResultsTo facilitate an informed analysis of OR gene phylogeny, we identified the complete set of 143 OR genes in the zebrafish genome, as well as the OR repertoires in two pufferfish species, fugu (44 genes) and tetraodon (42 genes). Although the genomes analyzed here contain fewer genes than in mammalian species, the teleost OR genes can be grouped into a larger number of major clades, representing greater overall OR diversity in the fish.ConclusionBased on the phylogeny of fish and mammalian repertoires, we propose a model for OR gene evolution in which different ancestral OR genes or gene families were selectively lost or expanded in different vertebrate lineages. In addition, our calculations of the ratios of non-synonymous to synonymous codon substitutions among more recently expanding OR subgroups in zebrafish implicate residues that may be involved in odorant binding.

The “resurrection method” for modification of specific proteins in higher plants

We describe a new method designated “the resurrection method” by which a modified protein is expressed in higher plants in place of the original protein. The modified gene constructed by introducing synonymous codon substitutions throughout the original gene to prevent the sequence-specific degradation of its mRNA during RNA silencing is expressed while the expression of the original gene is suppressed. Here, we report the successful alteration of the biochemical properties of green fluorescent protein expressed in transgenic Nicotiana benthamiana, suggesting that this method could be useful for gene control in living plants.

Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro

The coding sequence for chloramphenicol acetyl transferase (CAT) contains several rare codons; three of them are ATA encoding isoleucine in positions 13, 84 and 119 of the amino acid sequence. Expression of CAT on Escherichia coli ribosomes in vitro results in mostly full-length product but also distinct smaller polypeptides from less than 3 kDa to over 20 kDa. As reported earlier, the smaller polypeptides are the predominant products, if translation is initiated with fluorophore-Met-tRNA f. All this translational pausing is eliminated when the first ATA codon is mutated to ATC, a frequently used codon for isoleucine in E. coli. Addition of large amounts of E. coli tRNA to the coupled transcription/translation reaction does not reduce the number of pause-site peptides seen in the expression of wild-type CAT. Thus we hypothesize that the mRNA structure may be an important determinant for translational pausing.

Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation

To investigate the possible influence of the local rates of translation on protein folding, 16 consecutive rare (in Escherichia coli) codons in the chloramphenicol acetyltransferase (CAT) gene have been replaced by frequent ones. Site-directed silent mutagenesis reduced the pauses in translation of CAT in E. coli S30 extract cell-free system and led to the acceleration of the overall rate of CAT protein synthesis. At the same time, the silently mutated protein (with unaltered protein sequence) synthesized in the E. coli S30 extract system was shown to possess 20% lower specific activity. The data suggest that kinetics of protein translation can affect the in vivo protein-folding pathway, leading to increased levels of protein misfolding.

Rapid Evolution of a Homeodomain: Evidence for Positive Selection

One often-noted feature of homeobox genes is the conservation of the homeodomain among orthologous genes from distantly related species. This sequence conservation is presumed to reflect functional conservation, which indeed has been demonstrated in several cases. We analyzed the evolution of an orphan homeobox gene, Pem, which is expressed preferentially in male and female reproductive tissue. Sequence analysis of 12 species of mice and rats indicated that the Pem gene has evolved at a remarkably high rate. The most rapidly evolving region of the Pem protein is the amino portion of the homeodomain, including the flexible N-terminal arm, helices I and II, and the linker regions between the helices. In contrast, the third helix, which is known to mediate base-specific DNA contacts in other homeodomains, is conserved in the Pem protein. Analysis of the ratio of nonsynonymous and synonymous codon substitution rates within the Pem homeodomain suggested that its divergence was driven by adaptive selection. The rate of nonsynonymous substitutions in Pem was higher than that of the sex-determination gene Sry, which also appears to have undergone directional selection over a short evolutionary period. Despite the rapid evolution of the Pem gene, we detected no Pem polymorphisms and observed no variation in the homeobox sequence among closely related Mus species. This suggests that purifying episodes followed phases in which selection pressure drove the rapid divergence of this locus. We propose that transcription factors that function in reproductive events can be subject to rapid adaptive selection.

Transient mutators: a semiquantitative analysis of the influence of translation and transcription errors on mutation rates.

A population of bacteria growing in a nonlimiting medium includes mutator bacteria and transient mutators defined as wild-type bacteria which, due to occasional transcription or translation errors, display a mutator phenotype. A semiquantitative theoretical analysis of the steady-state composition of an Escherichia coli population suggests that true strong genotypic mutators produce about 3 x 10(-3) of the single mutations arising in the population, while transient mutators produce at least 10% of the single mutations and more than 95% of the simultaneous double mutations. Numbers of mismatch repair proteins inherited by the offspring, proportions of lethal mutations and mortality rates are among the main parameters that influence the steady-state composition of the population. These results have implications for the experimental manipulation of mutation rates and the evolutionary fixation of frequent but nearly neutral mutations (e.g., synonymous codon substitutions).

Why the rate of silent codon substitutions is variable within a vertebrate's genome

Different genes within the murine genome are diverging at different rates. The rate of synonymous codon substitutions in these genes is related to their base composition. It is proposed that the variabilities of rate of mutation accumulation, of codon choice and of average GC content within vertebrate genomes are caused by differences between DNA synthesis in repair and replication, as far as the frequency and compositional bias of mutations introduced by these systems are concerned. DNA repair contributes substantially to the evolution of the DNA domains, which are actively repaired in germline cells and which correspond to regions available for transcription in these cells and to Giemsa-negative bands in stained chromosomes.

Modification of mRNA secondary structure and alteration of the expression of human interferon α1 in Escherichia coli

A plasmid (pNL015) was constructed to contain a human interferon αl (IFN-α1) gene under the transcriptional control of the Escherichia coli lipoprotein promoter. The E. coli cells harboring this plasmid produce 2.8 × 10 4 units/ml of IFN. Secondary structure analysis of the transcripts produced by pNL015 showed that the coding region could base pair with the Shine-Dalgarno (SD) region with a ΔG = −3.9 kcal/mol. A new plasmid pNLOOS was constructed by modifying pNL015 with an 11-bp deletion and a 2-bp insertion in the coding region, so that the SD region is not involved in the secondary structure. E. coli cells harboring pNL008 produce ten times more IFN activity than cells harboring pNL015. A series of experiments were carried out to show that the specific activities of IFN, differential rates of IFN transcription, protein degradation or mRNA degradation could not account for the difference observed in expression. A rigorous test on this model of translational inhibition was conducted by the construction of pNL017 with a single bp substitution which did not change the amino acid sequence of the IFN (synonymous codon substitution) but which increased the calculated energy of interaction with the SD sequence to ΔG = −10.8 kcal/mol. The E. coli cells harboring pNL017 produced no detectable IFN activity.