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Although messenger RNA translation is tightly regulated to preserve protein synthesis and cellular homeostasis, chronic exposure to interferon‐γ (IFN‐γ) in several cancers can lead to tryptophan (Trp) shortage via the indoleamine‐2,3‐dioxygenase (IDO)‐ kynurenine pathway and therefore promotes the production of aberrant peptides by ribosomal frameshifting and tryptophan‐to‐phenylalanine (W>F) codon reassignment events (substitutants) specifically at Trp codons. However, the effect of Trp depletion on the generation of aberrant peptides by ribosomal mistranslation in gastric cancer (GC) is still obscure. Here, it is shows that the abundant infiltrating lymphocytes in EBV‐positive GC continuously secreted IFN‐γ, upregulated IDO1 expression, leading to Trp shortage and the induction of W>F substitutants. Intriguingly, the production of W>F substitutants in EBV‐positive GC is linked to antigen presentation and the activation of the mTOR/eIF4E signaling pathway. Inhibiting either the mTOR/eIF4E pathway or EIF4E expression counteracted the production and antigen presentation of W>F substitutants. Thus, the mTOR/eIF4E pathway exposed the vulnerability of gastric cancer by accelerating the production of aberrant peptides and boosting immune activation through W>F substitutant events. This work proposes that EBV‐positive GC patients with mTOR/eIF4E hyperactivation may benefit from anti‐tumor immunotherapy. [ABSTRACT FROM AUTHOR]

The genetic code is a set of instructions that determine how the information in our genetic material is translated into amino acids. In general, it is universal for all organisms, from viruses and bacteria to humans. However, in the last few decades, exceptions to this rule have been identified both in pro‐ and eukaryotes. In this review, we discuss the 16 described alternative eukaryotic nuclear genetic codes and observe theories of their appearance in evolution. We consider possible molecular mechanisms that allow codon reassignment. Most reassignments in nuclear genetic codes are observed for stop codons. Moreover, in several organisms, stop codons can simultaneously encode amino acids and serve as termination signals. In this case, the meaning of the codon is determined by the additional factors besides the triplets. A comprehensive review of various non‐standard coding events in the nuclear genomes provides a new insight into the translation mechanism in eukaryotes. [ABSTRACT FROM AUTHOR]

Abstract Although messenger RNA translation is tightly regulated to preserve protein synthesis and cellular homeostasis, chronic exposure to interferon‐γ (IFN‐γ) in several cancers can lead to tryptophan (Trp) shortage via the indoleamine‐2,3‐dioxygenase (IDO)‐ kynurenine pathway and therefore promotes the production of aberrant peptides by ribosomal frameshifting and tryptophan‐to‐phenylalanine (W>F) codon reassignment events (substitutants) specifically at Trp codons. However, the effect of Trp depletion on the generation of aberrant peptides by ribosomal mistranslation in gastric cancer (GC) is still obscure. Here, it is shows that the abundant infiltrating lymphocytes in EBV‐positive GC continuously secreted IFN‐γ, upregulated IDO1 expression, leading to Trp shortage and the induction of W>F substitutants. Intriguingly, the production of W>F substitutants in EBV‐positive GC is linked to antigen presentation and the activation of the mTOR/eIF4E signaling pathway. Inhibiting either the mTOR/eIF4E pathway or EIF4E expression counteracted the production and antigen presentation of W>F substitutants. Thus, the mTOR/eIF4E pathway exposed the vulnerability of gastric cancer by accelerating the production of aberrant peptides and boosting immune activation through W>F substitutant events. This work proposes that EBV‐positive GC patients with mTOR/eIF4E hyperactivation may benefit from anti‐tumor immunotherapy.

Abstract Background Cancer immunotherapy is implemented by identifying antigens that are presented on the cell surface of cancer cells and illicit T-cell response (Schumacher and Schreiber, Science 348:69–74, 2015; Waldman et al., Nat Rev Immunol 20:651–668, 2020; Zhang et al., Front Immunol 12:672,356, 2021b). Classical candidates of such antigens are the peptides resulting from genetic alterations and are named “neoantigen" (Schumacher and Schreiber, Science 348:69–74, 2015). Neoantigens have been widely catalogued across several human cancer types (Tan et al., Database (Oxford) 2020;2020b; Vigneron et al., Cancer Immun 13:15, 2013; Yi et al., iScience 24:103,107, 2021; Zhang et al., BMC Bioinformatics 22:40, 2021a). Recently, a new class of inducible antigens has been identified, namely Substitutants, that are produced as a result of aberrant protein translation (Pataskar et al., Nature 603:721–727, 2022). Main Catalogues of Substitutant expression across human cancer types, their specificity and association to gene expression signatures remain elusive for the scientific community's access. As a solution, we present ABPEPserver, an online database and analytical platform that can visualize a large-scale tumour proteomics analysis of Substitutant expression across eight tumour types sourced from the CPTAC database (Edwards et al., J Proteome Res 14:2707–2713, 2015). Functionally, ABPEPserver offers the analysis of gene-association signatures of Substitutant peptides, a comparison of enrichment between tumour and tumour-adjacent normal tissues, and a list of peptides that serve as candidates for immunotherapy design. ABPEPserver will significantly enhance the exploration of aberrant protein production in human cancer, as exemplified in a case study. Conclusion ABPEPserver is designed on an R SHINY platform to catalogue Substitutant peptides in human cancer. The application is available at https://rhpc.nki.nl/sites/shiny/ABPEP/ . The code is available under GNU General public license from GitHub ( https://github.com/jasminesmn/ABPEPserver ).

Termination of translation is essential but hinders applications of genetic code engineering, e.g., unnatural amino acids incorporation and codon randomization mediated saturation mutagenesis. Here, for the first time, it is demonstrated that E. coli Pth and ArfB together play an efficient translation termination without codon preference in the absence of class‐I release factors. By degradation of the targeted protein, both essential and alternative termination types of machinery are completely removed to disable codon‐dependent termination in cell extract. Moreover, a total of 153 engineered tRNAs are screened for efficient all stop‐codons decoding to construct a codon‐dependent termination defect in vitro protein synthesis with all 64 sense‐codons, iPSSC. Finally, this full sense genetic code achieves significant improvement in the incorporation of distinct unnatural amino acids at up to 12 positions and synthesis of protein encoding consecutive NNN codons. By decoding all information in nucleotides to amino acids, iPSSC may hold great potential in building artificial protein synthesis beyond the cell. [ABSTRACT FROM AUTHOR]

Fornicata, a lineage of a broader and ancient anaerobic eukaryotic clade Metamonada, contains diverse taxa that are ideally suited for evolutionary studies addressing various fundamental biological questions, such as the evolutionary trajectory of mitochondrion-related organelles (MROs), the transition between free-living and endobiotic lifestyles, and the derivation of alternative genetic codes. To this end, we conducted detailed microscopic and transcriptome analyses in a poorly documented strain of an anaerobic free-living marine flagellate, PCS, in the so-called CL3 fornicate lineage. Fortuitously, we discovered that the original culture contained two morphologically similar and closely related CL3 representatives, which doubles the taxon representation within this lineage. We obtained a monoeukaryotic culture of one of them and formally describe it as a new member of the family Caviomonadidae, Euthynema mutabile gen. et sp. nov. In contrast to previously studied caviomonads, the endobiotic Caviomonas mobilis and Iotanema spirale , E. mutabile possesses an ultrastructurally discernible MRO. We sequenced and assembled the transcriptome of E. mutabile , and by sequence subtraction, obtained transcriptome data from the other CL3 clade representative present in the original PCS culture, denoted PCS-ghost. Transcriptome analyses showed that the reassignment of only one of the UAR stop codons to encode Gln previously reported from I. spirale does not extend to its free-living relatives and is likely due to a unique amino acid substitution in I. spirale 's eRF1 protein domain responsible for termination codon recognition. The backbone fornicate phylogeny was robustly resolved in a phylogenomic analysis, with the CL3 clade amongst the earliest branching lineages. Metabolic and MRO functional reconstructions of CL3 clade members revealed that all three, including I. spirale , encode homologs of key components of the mitochondrial protein import apparatus and the ISC pathway, indicating the presence of a MRO in all of them. In silico evidence indicates that the organelles of E. mutabile and PCS-ghost host ATP and H2 production, unlike the cryptic MRO of I. spirale. These data suggest that the CL3 clade has experienced a hydrogenosome-to-mitosome transition independent from that previously documented for the lineage leading to Giardia. [ABSTRACT FROM AUTHOR]

Fornicata, a lineage of a broader and ancient anaerobic eukaryotic clade Metamonada, contains diverse taxa that are ideally suited for evolutionary studies addressing various fundamental biological questions, such as the evolutionary trajectory of mitochondrion-related organelles (MROs), the transition between free-living and endobiotic lifestyles, and the derivation of alternative genetic codes. To this end, we conducted detailed microscopic and transcriptome analyses in a poorly documented strain of an anaerobic free-living marine flagellate, PCS, in the so-called CL3 fornicate lineage. Fortuitously, we discovered that the original culture contained two morphologically similar and closely related CL3 representatives, which doubles the taxon representation within this lineage. We obtained a monoeukaryotic culture of one of them and formally describe it as a new member of the family Caviomonadidae, Euthynema mutabile gen. et sp. nov. In contrast to previously studied caviomonads, the endobiotic Caviomonas mobilis and Iotanema spirale, E. mutabile possesses an ultrastructurally discernible MRO. We sequenced and assembled the transcriptome of E. mutabile, and by sequence subtraction, obtained transcriptome data from the other CL3 clade representative present in the original PCS culture, denoted PCS-ghost. Transcriptome analyses showed that the reassignment of only one of the UAR stop codons to encode Gln previously reported from I. spirale does not extend to its free-living relatives and is likely due to a unique amino acid substitution in I. spirale’s eRF1 protein domain responsible for termination codon recognition. The backbone fornicate phylogeny was robustly resolved in a phylogenomic analysis, with the CL3 clade amongst the earliest branching lineages. Metabolic and MRO functional reconstructions of CL3 clade members revealed that all three, including I. spirale, encode homologs of key components of the mitochondrial protein import apparatus and the ISC pathway, indicating the presence of a MRO in all of them. In silico evidence indicates that the organelles of E. mutabile and PCS-ghost host ATP and H2 production, unlike the cryptic MRO of I. spirale. These data suggest that the CL3 clade has experienced a hydrogenosome-to-mitosome transition independent from that previously documented for the lineage leading to Giardia.

The genetic code has been proposed to be a ‘frozen accident,’ but the discovery of alternative genetic codes over the past four decades has shown that it can evolve to some degree. Since most examples were found anecdotally, it is difficult to draw general conclusions about the evolutionary trajectories of codon reassignment and why some codons are affected more frequently. To fill in the diversity of genetic codes, we developed Codetta, a computational method to predict the amino acid decoding of each codon from nucleotide sequence data. We surveyed the genetic code usage of over 250,000 bacterial and archaeal genome sequences in GenBank and discovered five new reassignments of arginine codons (AGG, CGA, and CGG), representing the first sense codon changes in bacteria. In a clade of uncultivated Bacilli, the reassignment of AGG to become the dominant methionine codon likely evolved by a change in the amino acid charging of an arginine tRNA. The reassignments of CGA and/or CGG were found in genomes with low GC content, an evolutionary force that likely helped drive these codons to low frequency and enable their reassignment.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating N ε - (o -azidobenzyloxycarbonyl)- l -lysine (o -Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o -Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype. • A designer amino acid was site-specifically introduced into the human λ light chain. • The bio-orthogonal azido chemistry was used for site-specific chemical conjugation. • Multiple positions useful for the conjugation were identified in the λ light chain. • Bispecific Fab dimers made of the κ and λ isotypes showed antagonistic activity. [ABSTRACT FROM AUTHOR]

Synthetic biology holds promise to revolutionize the life sciences and biomedicine via expansion of macromolecular diversity outside the natural chemical space. Use of non-canonical amino acids (ncAAs) via codon reassignment has found diverse applications in protein structure and interaction analysis, introduction of post-translational modifications, production of constrained peptides, antibody-drug conjugates, and novel enzymes. However, simultaneously encoding multiple ncAAs in vivo requires complex engineering and is sometimes restricted by the cell's poor uptake of ncAAs. In contrast the open nature of cell-free protein synthesis systems offers much greater freedom for manipulation and repurposing of the biosynthetic machinery by controlling the level and identity of translational components and reagents, and allows simultaneous incorporation of multiple ncAAs with non-canonical side chains and even backbones (N-methyl, D-, β-amino acids, α-hydroxy acids etc.). This review focuses on the two most used Escherichia coli-based cell-free protein synthesis systems; cell extract- and PURE-based systems. The former is a biological mixture with >500 proteins, while the latter consists of 38 individually purified biomolecules. We delineate compositions of these two systems and discuss their respective advantages and applications. Also, we dissect the translational components required for ncAA incorporation and compile lists of ncAAs that can be incorporated into polypeptides via different acylation approaches. We highlight the recent progress in using unnatural nucleobase pairs to increase the repertoire of orthogonal codons, as well as using tRNA-specific ribozymes for in situ acylation. We summarize advances in engineering of translational machinery such as tRNAs, aminoacyl-tRNA synthetases, elongation factors, and ribosomes to achieve efficient incorporation of structurally challenging ncAAs. We note that, many engineered components of biosynthetic machinery are developed for the use in vivo but are equally applicable to the in vitro systems. These are included in the review to provide a comprehensive overview for ncAA incorporation and offer new insights for the future development in cell-free systems. Finally, we highlight the exciting progress in the genomic engineering, resulting in E. coli strains free of amber and some redundant sense codons. These strains can be used for preparation of cell extracts offering multiple reassignment options.

We designed a novel growth controller regulated by feeding of an unnatural amino acid, Nε-benzyloxycarbonyl-l-lysine (ZK), using a specific incorporation system at a sense codon. This system is constructed by a pair of modified pyrrolisyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl). Although ZK is non-toxic for normal organisms, the growth of Escherichia coli carrying the ZK incorporation system was inhibited in a ZK concentration-dependent manner without causing rapid bacterial death, presumably due to generation of non-functional or toxic proteins. The extent of growth inhibition strongly depended on the anticodon sequence of the tRNApyl gene. Taking advantage of the low selectivity of PylRS for tRNApyl anticodons, we experimentally determined the most effective anticodon sequence among all 64 nucleotide sequences in the anticodon region of tRNApyl gene. The results suggest that the ZK-regulated growth controller is a simple, target-specific, environmental noise-resistant and titratable system. This technique may be applicable to a wide variety of organisms because the growth inhibitory effects are caused by “informational disturbance”, in which the highly conserved system for transmission of information from DNA to proteins is perturbed.

Genetic code deviations involving stop codons have been previously reported in mitochondrial genomes of several green plants (Viridiplantae), most notably chlorophyte algae (Chlorophyta). However, as changes in codon recognition from one amino acid to another are more difficult to infer, such changes might have gone unnoticed in particular lineages with high evolutionary rates that are otherwise prone to codon reassignments. To gain further insight into the evolution of the mitochondrial genetic code in green plants, we have conducted an in-depth study across mtDNAs from 51 green plants (32 chlorophytes and 19 streptophytes). Besides confirming known stop-to-sense reassignments, our study documents the first cases of sense-to-sense codon reassignments in Chlorophyta mtDNAs. In several Sphaeropleales, we report the decoding of AGG codons (normally arginine) as alanine, by tRNA(CCU) of various origins that carry the recognition signature for alanine tRNA synthetase. In Chromochloris, we identify tRNA variants decoding AGG as methionine and the synonymous codon CGG as leucine. Finally, we find strong evidence supporting the decoding of AUA codons (normally isoleucine) as methionine in Pycnococcus. Our results rely on a recently developed conceptual framework (CoreTracker) that predicts codon reassignments based on the disparity between DNA sequence (codons) and the derived protein sequence. These predictions are then validated by an evaluation of tRNA phylogeny, to identify the evolution of new tRNAs via gene duplication and loss, and structural modifications that lead to the assignment of new tRNA identities and a change in the genetic code. [ABSTRACT FROM AUTHOR]

The essential feature of the genetic code is the strict one-to-one correspondence between codons and amino acids. The canonical code consists of three stop codons and 61 sense codons that encode 20% of the amino acid repertoire observed in nature. It was originally designated as immutable and universal due to its conservation in most organisms, but sequencing of genes from the human mitochondrial genomes revealed deviations in codon assignments. Since then, alternative codes have been reported in both nuclear and mitochondrial genomes and genetic code engineering has become an important research field. Here, we review the most recent concepts arising from the study of natural non-standard genetic codes with special emphasis on codon re-assignment strategies that are relevant to engineering genetic code in the laboratory. Recent tools for synthetic biology and current attempts to engineer new codes for incorporation of non-standard amino acids are also reviewed in this article.

The standard genetic code (SGC) is central to molecular biology and its origin and evolution is a fundamental problem in evolutionary biology, the elucidation of which promises to reveal much about the origins of life. In addition, we propose that study of its origin can also reveal some fundamental and generalizable insights into mechanisms of molecular evolution, utilizing concepts from complexity theory. The first is that beneficial traits may arise by non-adaptive processes, via a process of “neutral emergence”. The structure of the SGC is optimized for the property of error minimization, which reduces the deleterious impact of point mutations. Via simulation, it can be shown that genetic codes with error minimization superior to the SGC can emerge in a neutral fashion simply by a process of genetic code expansion via tRNA and aminoacyl-tRNA synthetase duplication, whereby similar amino acids are added to codons related to that of the parent amino acid. This process of neutral emergence has implications beyond that of the genetic code, as it suggests that not all beneficial traits have arisen by the direct action of natural selection; we term these “pseudaptations”, and discuss a range of potential examples. Secondly, consideration of genetic code deviations (codon reassignments) reveals that these are mostly associated with a reduction in proteome size. This code malleability implies the existence of a proteomic constraint on the genetic code, proportional to the size of the proteome (P), and that its reduction in size leads to an “unfreezing” of the codon – amino acid mapping that defines the genetic code, consistent with Crick’s Frozen Accident theory. The concept of a proteomic constraint may be extended to propose a general informational constraint on genetic fidelity, which may be used to explain variously, differences in mutation rates in genomes with differing proteome sizes, differences in DNA repair capacity and genome GC content between organisms, a selective pressure in the evolution of sexual reproduction, and differences in translational fidelity. Lastly, the utility of the concept of an informational constraint to other diverse fields of research is explored.

Stop codon reassignments are widely distributed in prokaryotic, eukaryotic and organellar genomes, but are remarkably convergent in terms of the stop codons and amino acids reassigned. Strikingly, the identities of stop codon reassignments are closely matched to the properties of naturally occurring nonsense suppressor (NONS) tRNAs, suggesting that pre-existing nonsense suppression in an ancestral tRNA facilitated the occurrence of stop codon reassignments. Here this idea is expanded, by exploring the mechanism by which the gene duplication of tRNAs has occurred, leading to the reassignment of stop codons. Two types of stop codon reassignment are identified: those that necessitate a tRNA gene duplication, and those that do not because a single tRNA can recognize the reassigned stop codon and the canonical codon(s) for the cognate amino acid. Where tRNA gene duplication has occurred, this implies a multi-functional ancestral NONS tRNA, followed by adaptive mutation in the anticodon of one of the gene duplicates to become complementary to the stop codon, constituting a clear example of escape from adaptive conflict. The best exemplar is the UAA + UAG → gln reassignment, which has occurred 9 times independently in a diverse range of genomes, and appears to reflect the widespread occurrence of naturally occurring nonsense suppression of the UAA + UAG stop codons by glutamine tRNAs. Consideration of pre-existing tRNA functionality and the mechanism of gene duplication provide new insights into the process of stop codon reassignment. [ABSTRACT FROM AUTHOR]

The canonical genetic code ubiquitously translates nucleotide into peptide sequence with several alterations known in viruses, bacteria, mitochondria, plastids, and single-celled eukaryotes. A new hypothesis to explain genetic code changes, termed tRNA loss driven codon reassignment, has been proposed recently when the polyphyly of the yeast codon reassignment events has been uncovered. According to this hypothesis, the driving force for genetic code changes are tRNA or translation termination factor loss-of-function mutations or loss-of-gene events. The free codon can subsequently be captured by all tRNAs that have an appropriately mutated anticodon and are efficiently charged. Thus, codon capture most likely happens by near-cognate tRNAs and tRNAs whose anticodons are not part of the recognition sites of the respective aminoacyl-tRNA-synthetases. This hypothesis comprehensively explains the CTG codon translation as alanine in Pachysolen yeast together with the long known translation of the same codon as serine in Candida albicans and related species, and can also be applied to most other known reassignments. [ABSTRACT FROM AUTHOR]

mRNA decoding by tRNAs and tRNA charging by aminoacyl-tRNA synthetases are biochemically separated processes that nevertheless in general involve the same nucleotides. The combination of charging and decoding determines the genetic code. Codon reassignment happens when a differently charged tRNA replaces a former cognate tRNA. The recent discovery of the polyphyly of the yeast CUG sense codon reassignment challenged previous mechanistic considerations and led to the proposal of the so-calledtRNA loss driven codon reassignmenthypothesis. Accordingly, codon capture is caused by loss of a tRNA or by mutations in the translation termination factor, subsequent reduction of the codon frequency through reduced translation fidelity and final appearance of a new cognate tRNA. Critical for codon capture are sequence and structure of the new tRNA, which must be compatible with recognition regions of aminoacyl-tRNA synthetases. The proposed hypothesis applies to all reported nuclear and organellar codon reassignments. [ABSTRACT FROM PUBLISHER]

Background: Departures from the standard genetic code in eukaryotic nuclear genomes are known for only a handful of lineages and only a few genetic code variants seem to exist outside the ciliates, the most creative group in this regard. Most frequent code modifications entail reassignment of the UAG and UAA codons, with evidence for at least 13 independent cases of a coordinated change in the meaning of both codons. However, no change affecting each of the two codons separately has been documented, suggesting the existence of underlying evolutionary or mechanistic constraints. Results: Here, we present the discovery of two new variants of the nuclear genetic code, in which UAG is translated as an amino acid while UAA is kept as a termination codon (along with UGA). The first variant occurs in an organism noticed in a (meta)transcriptome from the heteropteran Lygus hesperus and demonstrated to be a novel insectdwelling member of Rhizaria (specifically Sainouroidea). This first documented case of a rhizarian with a non-canonical genetic code employs UAG to encode leucine and represents an unprecedented change among nuclear codon reassignments. The second code variant was found in the recently described anaerobic flagellate Iotanema spirale (Metamonada: Fornicata). Analyses of transcriptomic data revealed that I. spirale uses UAG to encode glutamine, similarly to the most common variant of a non-canonical code known from several unrelated eukaryotic groups, including hexamitin diplomonads (also a lineage of fornicates). However, in these organisms, UAA also encodes glutamine, whereas it is the primary termination codon in I. spirale. Along with phylogenetic evidence for distant relationship of I. spirale and hexamitins, this indicates two independent genetic code changes in fornicates. Conclusions: Our study documents, for the first time, that evolutionary changes of the meaning of UAG and UAA codons in nuclear genomes can be decoupled and that the interpretation of the two codons by the cytoplasmic translation apparatus is mechanistically separable. The latter conclusion has interesting implications for possibilities of genetic code engineering in eukaryotes. We also present a newly developed generally applicable phylogeny-informed method for inferring the meaning of reassigned codons. [ABSTRACT FROM AUTHOR]

Background: Cancer immunotherapy is implemented by identifying antigens that are presented on the cell surface of cancer cells and illicit T-cell response (Schumacher and Schreiber, Science 348:69-74, 2015; Waldman et al., Nat Rev Immunol 20:651-668, 2020; Zhang et al., Front Immunol 12:672,356, 2021b). Classical candidates of such antigens are the peptides resulting from genetic alterations and are named "neoantigen" (Schumacher and Schreiber, Science 348:69-74, 2015). Neoantigens have been widely catalogued across several human cancer types (Tan et al., Database (Oxford) 2020;2020b; Vigneron et al., Cancer Immun 13:15, 2013; Yi et al., iScience 24:103,107, 2021; Zhang et al., BMC Bioinformatics 22:40, 2021a). Recently, a new class of inducible antigens has been identified, namely Substitutants, that are produced as a result of aberrant protein translation (Pataskar et al., Nature 603:721-727, 2022). MAIN: Catalogues of Substitutant expression across human cancer types, their specificity and association to gene expression signatures remain elusive for the scientific community's access. As a solution, we present ABPEPserver, an online database and analytical platform that can visualize a large-scale tumour proteomics analysis of Substitutant expression across eight tumour types sourced from the CPTAC database (Edwards et al., J Proteome Res 14:2707-2713, 2015). Functionally, ABPEPserver offers the analysis of gene-association signatures of Substitutant peptides, a comparison of enrichment between tumour and tumour-adjacent normal tissues, and a list of peptides that serve as candidates for immunotherapy design. ABPEPserver will significantly enhance the exploration of aberrant protein production in human cancer, as exemplified in a case study., Conclusion: ABPEPserver is designed on an R SHINY platform to catalogue Substitutant peptides in human cancer. The application is available at https://rhpc.nki.nl/sites/shiny/ABPEP/ . The code is available under GNU General public license from GitHub ( https://github.com/jasminesmn/ABPEPserver )., (© 2023. The Author(s).)

The standard genetic code (SGC) assigns amino acids to codons in such a way that the impact of point mutations is reduced, this is termed ‘error minimization’ (EM). The occurrence of EM has been attributed to the direct action of selection, however it is difficult to explain how the searching of alternative codes for an error minimized code can occur via codon reassignments, given that these are likely to be disruptive to the proteome. An alternative scenario is that EM has arisen via the process of genetic code expansion, facilitated by the duplication of genes encoding charging enzymes and adaptor molecules. This is likely to have led to similar amino acids being assigned to similar codons. Strikingly, we show that if during code expansion the most similar amino acid to the parent amino acid, out of the set of unassigned amino acids, is assigned to codons related to those of the parent amino acid, then genetic codes with EM superior to the SGC easily arise. This scheme mimics code expansion via the gene duplication of charging enzymes and adaptors. The result is obtained for a variety of different schemes of genetic code expansion and provides a mechanistically realistic manner in which EM has arisen in the SGC. These observations might be taken as evidence for self-organization in the earliest stages of life. [ABSTRACT FROM AUTHOR]

The essential feature of the genetic code is the strict one-to-one correspondence between codons and amino acids. The canonical code consists of three stop codons and 61 sense codons that encode 20% of the amino acid repertoire observed in nature. It was originally designated as immutable and universal due to its conservation in most organisms, but sequencing of genes from the human mitochondrial genomes revealed deviations in codon assignments. Since then, alternative codes have been reported in both nuclear and mitochondrial genomes and genetic code engineering has become an important research field. Here, we review the most recent concepts arising from the study of natural non-standard genetic codes with special emphasis on codon re-assignment strategies that are relevant to engineering genetic code in the laboratory. Recent tools for synthetic biology and current attempts to engineer new codes for incorporation of non-standard amino acids are also reviewed in this article. [ABSTRACT FROM AUTHOR]

There have been two distinct phases of evolution of the genetic code: an ancient phase-prior to the divergence of the three domains of life, during which the standard genetic code was established-and a modern phase, in which many alternative codes have arisen in specific groups of genomes that differ only slightly from the standard code. Here we discuss the factors that are most important in these two phases, and we argue that these are substantially different. In the modern phase, changes are driven by chance events such as tRNA gene deletions and codon disappearance events. Selection acts as a barrier to prevent changes in the code. In contrast, in the ancient phase, selection for increased diversity of amino acids in the code can be a driving force for addition of new amino acids. The pathway of code evolution is constrained by avoiding disruption of genes that are already encoded by earlier versions of the code. The current arrangement of the standard code suggests that it evolved from a four-column code in which Gly, Ala, Asp, and Val were the earliest encoded amino acids. [ABSTRACT FROM AUTHOR]

standard genetic code (SGC) is central to molecular biology and its origin and evolution is a fundamental problem in evolutionary biology, the elucidation of which promises to reveal much about the origins of life. In addition, we propose that study of its origin can also reveal some fundamental and generalizable insights into mechanisms of molecular evolution, utilizing concepts from complexity theory. The first is that beneficial traits may arise by non-adaptive processes, via a process of "neutral emergence". The structure of the SGC is optimized for the property of error minimization, which reduces the deleterious impact of point mutations. Via simulation, it can be shown that genetic codes with error minimization superior to the SGC can emerge in a neutral fashion simply by a process of genetic code expansion via tRNA and aminoacyl-tRNA synthetase duplication, whereby similar amino acids are added to codons related to that of the parent amino acid. This process of neutral emergence has implications beyond that of the genetic code, as it suggests that not all beneficial traits have arisen by the direct action of natural selection; we term these "pseudaptations", and discuss a range of potential examples. Secondly, consideration of genetic code deviations (codon reassignments) reveals that these are mostly associated with a reduction in proteome size. This code malleability implies the existence of a proteomic constraint on the genetic code, proportional to the size of the proteome (P), and that its reduction in size leads to an "unfreezing" of the codon -- amino acid mapping that defines the genetic code, consistent with Crick's Frozen Accident theory. The concept of a proteomic constraint may be extended to propose a general informational constraint on genetic fidelity, which may be used to explain variously, differences in mutation rates in genomes with differing proteome sizes, differences in DNA repair capacity and genome GC content between organisms, a selective pressure in the evolution of sexual reproduction, and differences in translational fidelity. Lastly, the utility of the concept of an informational constraint to other diverse fields of research is explored. [ABSTRACT FROM AUTHOR]

The universal genetic code defines the translation of nucleotide triplets, called codons, into amino acids. In many Saccharomycetes a unique alteration of this code affects the translation of the CUG codon, which is normally translated as leucine. Most of the species encoding CUG alternatively as serine belong to the Candida genus and were grouped into a so-called CTG clade. However, the "Candida genus" is not a monophyletic group and several Candida species are known to use the standard CUG translation. The codon identity could have been changedin a single branch, the ancestor of the Candida, or toseveral branches independently leading to a polyphyletic alternative yeast codon usage (AYCU). In order to resolve themonophyly or polyphyly of theAYCU,weperformed a phylogenomics analysis of 26 motor and cytoskeletal proteins from 60 sequenced yeast species. By investigating the CUG codon positions with respect to sequence conservation at the respective alignment positions, we were able to unambiguously assign the standard code orAYCU.Quantitative analysis of the highly conserved leucine and serine alignment positions showedthat61.1%and 17%of the CUGcodons coding for leucine and serine, respectively, are at highly conserved positions, whereas only 0.6%and 2.3% of the CUG codons, respectively, are at positions conserved in the respective other amino acid. Plotting the codon usage onto the phylogenetic tree revealed the polyphyly of the AYCU with Pachysolen tannophilus and the CTG clade branching independently within a time span of 30-100 Ma. [ABSTRACT FROM AUTHOR]

Many fungi restructured their proteomes through incorporation of serine (Ser) at thousands of protein sites coded by the leucine (Leu) CUG codon. How these fungi survived this potentially lethal genetic code alteration and its relevance for their biology are not understood. Interestingly, the human pathogen Candida albicans maintains variable Ser and Leu incorporation levels at CUG sites, suggesting that this atypical codon assignment flexibility provided an effective mechanism to alter the genetic code. To test this hypothesis, we have engineered C albicans strains to misincorporate increasing levels of Leu at protein CUG sites. Tolerance to the misincorporations was very high, and one strain accommodated the complete reversion of CUG identity from Ser back to Leu. Increasing levels of Leu misincorporation decreased growth rate, but production of phenotypic diversity on a phenotypic array probing various metabolic networks, drug resistance, and host immune cell responses was impressive. Genome resequencing revealed an increasing number of genotype changes at polymorphic sites compared with the control strain, and 80% of Leu misincorporation resulted in complete loss of heterozygosity in a large region of chromosome V. The data unveil unanticipated links between gene translational fidelity, proteome instability and variability, genome diversification, and adaptive phenotypic diversity. They also explain the high heterozygosity of the C albicans genome and open the door to produce microorganisms with genetic code alterations for basic and applied research. [ABSTRACT FROM AUTHOR]

Despite the fact that the genetic code is known to vary between organisms in rare cases, it is believed that in the lifetime of a single cell the code is stable. We found Acetohalobium arabaticum cells grown on pyruvate genetically encode 20 amino acids, but in the presence of trimethylamine (TMA), A. arabaticum dynamically expands its genetic code to 21 amino acids including pyrrolysine (Pyl). A. arabaticum is the only known organism that modulates the size of its genetic code in response to its environment and energy source. The gene cassette pylTSBCD, required to biosynthesize and genetically encode UAG codons as Pyl, is present in the genomes of 24 anaerobic archaea and bacteria. Unlike archaeal Pyl-decoding organisms that constitutively encode Pyl, we observed that A. arabaticum controls Pyl encoding by down-regulating transcription of the entire Pyl operon under growth conditions lacking TMA, to the point where no detectable Pyl-tRNAPyl is made in vivo. Pyl-decoding archaea adapted to an expanded genetic code by minimizing TAG codon frequency to typically ∼5% of ORFs, whereas Pyl-decoding bacteria (∼20% of ORFs contain in-frame TAGs) regulate Pyl-tRNAPyl formation and translation of UAG by transcriptional deactivation of genes in the Pyl operon. We further demonstrate that Pyl encoding occurs in a bacterium that naturally encodes the Pyl operon, and identified Pyl residues by mass spectrometry in A. arabaticum proteins including two methylamine methyltransferases. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondrial genes code for additional proteins after +2 frameshifts by reassigning stops to code for amino acids, which defines overlapping genetic codes for overlapping genes. Turtles recode stops UAR→Trp and AGR→Lys (AGR→Gly in the marine Olive Ridley turtle, Lepidochelys olivacea). In Lepidochelys the +2 frameshifted mitochondrial Cytb gene lacks stops, open reading frames from other genes code for unknown proteins, and for regular mitochondrial proteins after frameshifts according to the overlapping genetic code. Lepidochelys’ inversion between proteins coded by regular and overlapping genetic codes substantiates the existence of overlap coding. ND4 differs among Lepidochelys mitochondrial genomes: it is regular in DQ486893; in NC_011516, the open reading frame codes for another protein, the regular ND4 protein is coded by the frameshifted sequence reassigning stops as in other turtles. These systematic patterns are incompatible with Genbank/sequencing errors and DNA decay. Random mixing of synonymous codons, conserving main frame coding properties, shows optimization of natural sequences for overlap coding; Ka/Ks analyses show high positive (directional) selection on overlapping genes. Tests based on circular genetic codes confirm programmed frameshifts in ND3 and ND4l genes, and predicted frameshift sites for overlap coding in Lepidochelys. Chelonian mitochondria adapt for overlapping gene expression: cloverleaf formation by antisense tRNAs with predicted anticodons matching stops coevolves with overlap coding; antisense tRNAs with predicted expanded anticodons (frameshift suppressor tRNAs) associate with frameshift-coding in ND3 and ND4l, a potential regulation of frameshifted overlap coding. Anaeroby perhaps switched between regular and overlap coding genes in Lepidochelys. [Copyright &y& Elsevier]

Abstract: The genetic encoding of synthetic or “non-natural” amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG “stop” triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites. [Copyright &y& Elsevier]

Abstract: We here report a deviant genetic code, in which AUA is read as methionine (Met) instead of isoleucine (Ile), in the green alga-derived plastid in the dinoflagellate Lepidodinium chlorophorum. Although L. chlorophorum cDNA sequences of 11 plastid-encoded genes were deposited in the GenBank database, the non-canonical usage of AUA in this dinoflagellate plastid has been overlooked prior to this study. We compared 11 plastid-encoded genes of L. chlorophorum with the corresponding genes of 17 green algal plastids. Intriguingly, AUA often occurred in the L. chlorophorum sequences at codon positions that are predominantly occupied by Met amongst the green algal sequences. Coincidentally, the L. chlorophorum sequences utilized few AUA codons at the positions predominantly occupied by Ile amongst the green algal sequences. These observations clearly indicated that both AUA and AUG encode Met, while AUU and AUC encode Ile, in the L. chlorophorum plastid. Despite the rapidly-evolving nature of L. chlorophorum plastid-encoded genes, our statistical tests incorporating the deviant code suggest no significant difference in amino acid composition among the L. chlorophorum plastid and the green algal plastids considered in this study. Finally, the possible evolutionary events required for the reassignment of AUA from Ile to Met in Lepitodinium plastids were discussed. [Copyright &y& Elsevier]

Background: The Hemichordata comprises solitary-living Enteropneusta and colonial-living Pterobranchia, sharing morphological features with both Chordata and Echinodermata. Despite their key role for understanding deuterostome evolution, hemichordate phylogeny is controversial and only few molecular data are available for phylogenetic analysis. Furthermore, mitochondrial sequences are completely lacking for pterobranchs. Therefore, we determined and analyzed the complete mitochondrial genome of the pterobranch Rhabdopleura compacta to elucidate deuterostome evolution. Thereby, we also gained important insights in mitochondrial tRNA evolution. Results: The mitochondrial DNA of Rhabdopleura compacta corresponds in size and gene content to typical mitochondrial genomes of metazoans, but shows the strongest known strand-specific mutational bias in the nucleotide composition among deuterostomes with a very GT-rich main-coding strand. The order of the proteincoding genes in R. compacta is similar to that of the deuterostome ground pattern. However, the protein-coding genes have been highly affected by a strand-specific mutational pressure showing unusual codon frequency and amino acid composition. This composition caused extremely long branches in phylogenetic analyses. The unusual codon frequency points to a selection pressure on the tRNA translation system to codon-anticodon sequences of highest versatility instead of showing adaptations in anticodon sequences to the most frequent codons. Furthermore, an assignment of the codon AGG to Lysine has been detected in the mitochondrial genome of R. compacta, which is otherwise observed only in the mitogenomes of some arthropods. The genomes of these arthropods do not have such a strong strand-specific bias as found in R. compacta but possess an identical mutation in the anticodon sequence of the tRNALys. Conclusion: A strong reversed asymmetrical mutational constraint in the mitochondrial genome of Rhabdopleura compacta may have arisen by an inversion of the replication direction and adaptation to this bias in the protein sequences leading to an enigmatic mitochondrial genome. Although, phylogenetic analyses of protein coding sequences are hampered, features of the tRNA system of R. compacta support the monophyly of Ambulacraria. The identical reassignment of AGG to Lysine in two distinct groups may have occurred by convergent evolution in the anticodon sequence of the tRNALys. [ABSTRACT FROM AUTHOR]

The discovery of the genetic code provided one of the basic foundations of modern molecular biology. Most organisms use the same genetic language, but there are also well-documented variations representing codon reassignments within specific groups of organisms (such as ciliates and yeast) or organelles (such as plastids and mitochondria). In addition, duality in codon function is known in the use of AUG in translation initiation and methionine insertion into internal protein positions as well as in the case of selenocysteine and pyrrolysine insertion (encoded by UGA and UAG, respectively) in competition with translation termination. Ambiguous meaning of CUG in coding for serine and leucine is also known. However, a recent study revealed that codons in any position within the open reading frame can serve a dual function and that a change in codon meaning can be achieved by availability of a specific type of RNA stem-loop structure in the 3′-untranslated region. Thus, duality of codon function is a more widely used feature of the genetic code than previously known, and this observation raises the possibility that additional recoding events and additional novel features have evolved in the genetic code. [ABSTRACT FROM AUTHOR]

The establishment of the genetic code remains elusive nearly five decades after the code was elucidated. The stereochemical hypothesis postulates that the code developed from interactions between nucleotides and amino acids, yet supporting evidence in a biological context is lacking. We show here that anticodons are selectively enriched near their respective amino acids in the ribosome, and that such enrichment is significantly correlated with the canonical code over random codes. Ribosomal anticodon-amino acid enrichment further reveals that specific codons were reassigned during code evolution, and that the code evolved through a two-stage transition from ancient amino acids without anticodon interaction to newer additions with anticodon interaction. The ribosome thus serves as a molecular fossil, preserving biological evidence that anticodon- amino acid interactions shaped the evolution of the genetic code. [ABSTRACT FROM AUTHOR]

Abstract: The high conservation of the genetic code and its fundamental role in genome decoding suggest that its evolution is highly restricted or even frozen. However, various prokaryotic and eukaryotic genetic code alterations, several alternative tRNA-dependent amino acid biosynthesis pathways, regulation of tRNA decoding by diverse nucleoside modifications and recent in vivo incorporation of non-natural amino acids into prokaryotic and eukaryotic proteins, show that the code evolves and is surprisingly flexible. The cellular mechanisms and the proteome buffering capacity that support such evolutionary processes remain unclear. Here we explore the hypothesis that codon misreading and reassignment played fundamental roles in the development of the genetic code and we show how a fungal codon reassignment is enlightening its evolution. [Copyright &y& Elsevier]

The standard genetic code (SGC) has a fundamental error-minimizing property which has been widely attributed to the action of selection. However, a clear mechanism for how selection can give rise to error minimization (EM) is lacking. A search through a space of alternate codes (code space) via codon reassignments would be required, to select a code optimized for EM. There are two commonly discussed mechanisms of codon reassignment; the Codon Capture mechanism, which proposes a loss of the codon during reassignment, and the Ambiguous Intermediate mechanism, which proposes that the codon underwent an ambiguous phase during reassignment. When searching of code space via the Codon Capture mechanism is simulated, an optimized genetic code can rarely be achieved (0–3.2% of the time) with most searches ending in failure. When code space is searched via the Ambiguous Intermediate mechanism, under constraints derived from empirical observations of codon reassignments from extant genomes, the searches also often end in failure. When a local minimum is avoided and optimization is achieved, 20–41 sequential improving codon reassignments are required. Furthermore, the structures of the optimized codes produced by these simulations differ from the structure of the SGC. These data are challenges for the Adaptive Code hypothesis to address, which proposes that the EM property was directly selected for, and suggests that EM is simply a byproduct of the addition of amino acids to the expanding code, as described by the alternative ‘Emergence’ hypothesis. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Many cases of nonstandard genetic codes are known in mitochondrial genomes. We carry out analysis of phylogeny and codon usage of organisms for which the complete mitochondrial genome is available, and we determine the most likely mechanism for codon reassignment in each case. Reassignment events can be classified according to the gain-loss framework. The “gain” represents the appearance of a new tRNA for the reassigned codon or the change of an existing tRNA such that it gains the ability to pair with the codon. The “loss” represents the deletion of a tRNA or the change in a tRNA so that it no longer translates the codon. One possible mechanism is codon disappearance (CD), where the codon disappears from the genome prior to the gain and loss events. In the alternative mechanisms the codon does not disappear. In the unassigned codon mechanism, the loss occurs first, whereas in the ambiguous intermediate mechanism, the gain occurs first. Codon usage analysis gives clear evidence of cases where the codon disappeared at the point of the reassignment and also cases where it did not disappear. CD is the probable explanation for stop to sense reassignments and a small number of reassignments of sense codons. However, the majority of sense-to-sense reassignments cannot be explained by CD. In the latter cases, by analysis of the presence or absence of tRNAs in the genome and of the changes in tRNA sequences, it is sometimes possible to distinguish between the unassigned codon and the ambiguous intermediate mechanisms. We emphasize that not all reassignments follow the same scenario and that it is necessary to consider the details of each case carefully. [ABSTRACT FROM AUTHOR]

Using a comparative genomics approach we demonstrate a negative correlation between the number of codon reassignments undergone by 222 mitochondrial genomes and the mitochondrial genome size, the number of mitochondrial ORFs, and the sizes of the large and small subunit mitochondrial rRNAs. In addition, we show that the TGA-to-tryptophan codon reassignment, which has occurred 11 times in mitochondrial genomes, is found in mitochondrial genomes smaller than those which have not undergone the reassignment. We therefore propose that mitochondrial codon reassignments occur in a wide range of phyla, particularly in Metazoa, due to a reduced “proteomic constraint” on the mitochondrial genetic code, compared to the nuclear genetic code. The reduced proteomic constraint reflects the small size of the mitochondrial-encoded proteome and allows codon reassignments to occur with less likelihood of lethality. In addition, we demonstrate a striking link between nonsense codon reassignments and the decoding properties of naturally occurring nonsense suppressor tRNAs. This suggests that natural preexisting nonsense suppression facilitated nonsense codon reassignments and constitutes a novel mechanism of genetic code change. These findings explain for the first time the identity of the stop codons and amino acids reassigned in mitochondrial and nuclear genomes. Nonsense suppressor tRNAs provided the raw material for nonsense codon reassignments, implying that the properties of the tRNA anticodon have dictated the identity of nonsense codon reassignments. [ABSTRACT FROM AUTHOR]

Abstract: The genetic code of nuclear genes in some ciliates was found to differ from that of other organisms in the assignment of UGA, UAG, and UAA codons, which are normally assigned as stop codons. In some ciliate species, the universal stop codons UAA and UAG instead encode glutamine. In some other ciliates, the universal stop codon UGA appears to be translated as cysteine or tryptophan. Eukaryotic release factor 1 (eRF1) is a key protein in stop codon recognition, thus, the protein is believed to play an important role in the stop codon reassignment in ciliates. We have cloned, sequenced, and analyzed the cDNA of eRF1 from four ciliate species of three different classes: Karyorelictea (Loxodes striatus), Heterotrichea (Blepharisma musculus), and Litostomatea (Didinium nasutum, Dileptus margaritifer). Phylogenetic analysis of these eRF1s supports the hypothesis that the genetic code in ciliates has deviated independently several times from the universal genetic code, and that different ciliate eRF1s may have undergone different processes to change the codon specificity. Using computational methods, we have also suggested areas on the surface of eRF1s that are important for stop codon recognition in ciliate eRF1s. [Copyright &y& Elsevier]

Mitochondria often use genetic codes different from the standard genetic code. Now that many mitochondrial genomes have been sequenced, these variant codes provide the first opportunity to examine empirically the processes that produce new genetic codes. The key question is: Are codon reassignments the sole result of mutation and genetic drift? Or are they the result of natural selection? Here we present an analysis of 24 phylogenetically independent codon reassignments in mitochondria. Although the mutation-drift hypothesis can explain reassignments from stop to an amino acid, we found that it cannot explain reassignments from one amino acid to another. In particular-and contrary to the predictions of the mutation-drift hypothesis-the codon involved in such a reassignment was not rare in the ancestral genome. Instead, such reassignments appear to take place while the codon is in use at an appreciable frequency. Moreover, the comparison of inferred amino acid usage in the ancestral genome with the neutral expectation shows that the amino acid gaining the codon was selectively favored over the amino acid losing the codon. These results are consistent with a simple model of weak selection on the amino acid composition of proteins in which codon reassignments are selected because they compensate for multiple slightly deleterious mutations throughout the mitochondrial genome. We propose that the selection pressure is for reduced protein synthesis cost: most reassignments give amino acids that are less expensive to synthesize. Taken together, our results strongly suggest that mitochondrial genetic codes evolve to match the amino acid requirements of proteins. [ABSTRACT FROM AUTHOR]