The Impact of Message Mutation on the Fitness of a Genetic Code

The Standard Genetic Code is organized such that similar codons encode similar amino acids. One explanation suggested that the Standard Code is the result of natural selection to reduce the fitness ``load' that derives from the mutation and mistranslation of protein-coding genes. We review the arguments against the mutational load-minimizing hypothesis and argue that they need to be reassessed. We review recent analyses of the organization of the Standard Code and conclude that under cautious interpretation they support the mutational load-minimizing hypothesis. We then present a deterministic asexual model with which we study the mode of selection for load minimization. In this model, individual fitness is determined by a protein phenotype resulting from the translation of a mutable set of protein-coding genes. We show that an equilibrium fitness may be associated with a population with the same genetic code and that genetic codes that assign similar codons to similar amino acids have a higher fitness. We also show that the number of mutant codons in each individual at equilibrium, which determines the strength of selection for load minimization, reflects a long-term evolutionary balance between mutations in messages and selection on proteins, rather than the number of mutations that occur in a single generation, as has been assumed by previous authors. We thereby establish that selection for mutational load minimization acts at the level of an individual in a single generation. We conclude with comments on the shortcomings and advantages of load minimization over other hypotheses for the origin of the Standard Code. Received: 4 April 2001 / Accepted: 22 October 2001     

A Novel Theory on the Origin of the Genetic Code: A GNC-SNS Hypothesis

We have previously proposed an SNS hypothesis on the origin of the genetic code (Ikehara and Yoshida 1998). The hypothesis predicts that the universal genetic code originated from the SNS code composed of 16 codons and 10 amino acids (S and N mean G or C and either of four bases, respectively). But, it must have been very difficult to create the SNS code at one stroke in the beginning. Therefore, we searched for a simpler code than the SNS code, which could still encode water-soluble globular proteins with appropriate three-dimensional structures at a high probability using four conditions for globular protein formation (hydropathy, α-helix, β-sheet, and β-turn formations). Four amino acids (Gly [G], Ala [A], Asp [D], and Val [V]) encoded by the GNC code satisfied the four structural conditions well, but other codes in rows and columns in the universal genetic code table do not, except for the GNG code, a slightly modified form of the GNC code. Three three-amino acid systems ([D], Leu and Tyr; [D], Tyr and Met; Glu, Pro and Ile) also satisfied the above four conditions. But, some amino acids in the three systems are far more complex than those encoded by the GNC code. In addition, the amino acids in the three-amino acid systems are scattered in the universal genetic code table. Thus, we concluded that the universal genetic code originated not from a three-amino acid system but from a four-amino acid system, the GNC code encoding [GADV]-proteins, as the most primitive genetic code. Received: 11 June 2001 / Accepted: 11 October 2001     

Relationships Among Isoacceptor tRNAs Seems to Support the Coevolution Theory of the Origin of the Genetic Code

A new method for looking at relationships between nucleotide sequences has been used to analyze divergence both within and between the families of isoaccepting tRNA sets. A dendrogram of the relationships between 21 tRNA sets with different amino acid specificities is presented as the result of the analysis. Methionine initiator tRNAs are included as a separate set. The dendrogram has been interpreted with respect to the final stage of the evolutionary pathway with the development of highly specific tRNAs from ambiguous molecular adaptors. The location of the sets on the dendrogram was therefore analyzed in relation to hypotheses on the origin of the genetic code: the coevolution theory, the physicochemical hypothesis, and the hypothesis of ambiguity reduction of the genetic code. Pairs of 16 sets of isoacceptor tRNAs, whose amino acids are in biosynthetic relationships, occupied contiguous positions on the dendrogram, thus supporting the coevolution theory of the genetic code. Received: 4 May 1998 / Accepted: 11 July 1998     

Physicochemical Optimization in the Genetic Code Origin as the Number of Codified Amino Acids Increases

We have assumed that the coevolution theory of genetic code origin (Wong JT, Proc Natl Acad Sci USA 72:1909–1912, 1975) is essentially correct. This theory makes it possible to identify at least 10 evolutionary stages through which genetic code organization might have passed prior to reaching its current form. The calculation of the minimization level of all these evolutionary stages leads to the following conclusions. (1) The minimization percentages increased linearly with the number of amino acids codified in the codes of the various evolutionary stages when only the sense changes are considered in the analysis. This seems to favor the physicochemical theory of genetic code origin even if, as discussed in the paper, this observation is also compatible with the coevolution theory. (2) For the first seven evolutionary stages of the genetic code, this trend is less clear and indeed is inverted when we consider the global optimisation of the codes due to both sense changes and synonymous changes. This inverse correlation between minimization percentages and the number of amino acids codified in the codes of the intermediate stages seems to favor neither the physicochemical nor the stereochemical theories of genetic code origin, as it is in the early and intermediate stages of code development that these theories would expect minimization to have played a crucial role, and this does not seem to be the case. However, these results are in agreement with the coevolution theory, which attributes a role to the physicochemical properties of amino acids that, while important, is nevertheless subordinate to the mechanism which concedes codons from the precursor amino acids to the product amino acids as the primary factor determining the evolutionary structuring of the genetic code. The results are therefore discussed in the context of the various theories proposed to explain genetic code origin. Received: 25 October 1998 / Accepted: 19 February 1999     

Genetic Code Variations in Mitochondria: tRNA as a Major Determinant of Genetic Code Plasticity

Characteristic features of tRNA such as the anticodon sequence and modified nucleotides in the anticodon loop are thought to be crucial effectors for promoting or restricting codon reassignment. Our recent findings on basepairing rules between anticodon and codon in various metazoan mitochondria suggest that the complete loss of a codon is not necessarily essential for codon reassignment to take place. We postulate that a possible competition between two tRNAs with cognate anticodon sequences towards the relevant codon to be varied has a potential role in codon reassignment. Our proposition can be viewed as an expanded version of the codon capture theory proposed by Osawa and Jukes (J Mol Evol 28: 271–278, 1989). Received: 28 December 2000 / Accepted: 12 March 2001     

The Robust Statistical Bases of the Coevolution Theory of Genetic Code Origin

A paper (Amirnovin R, J Mol Evol 44:473–476, 1997) seems to undermine the validity of the coevolution theory of genetic code origin by shedding doubt on the connection between the biosynthetic relationships between amino acids and the organization of the genetic code, at a time when the literature on the topic takes this for granted. However, as a few papers cite this paper as evidence against the coevolution theory, and to cast aside all doubt on the subject, we have decided to reanalyze the statistical bases on which this theory is founded. We come to the following conclusions: (1) the methods used in the above referred paper contain certain mistakes, and (2) the statistical foundations on which the coevolution theory is based are extremely robust. We have done this by critically appraising Amirnovin's paper and suggesting an alternative method based on the generation of random codes which, along with the method reported in the literature, allows us to evaluate the significance, in the genetic code, of different sets of amino acid pairs in biosynthetic relationships. In particular, by using this method and after building up a certain set of amino acid pairs reflecting the expectations of the coevolution theory, we show that the presence of this set in the genetic code would be obtained, purely by chance, with a probability of 6 × 10⁻⁵. This observation seems to provide particularly strong support to the coevolution theory. Received: 28 June 1999 / Accepted: 23 October 1999     

An Analysis of the Metabolic Theory of the Origin of the Genetic Code

A computer program was used to test Wong's coevolution theory of the genetic code. The codon correlations between the codons of biosynthetically related amino acids in the universal genetic code and in randomly generated genetic codes were compared. It was determined that many codon correlations are also present within random genetic codes and that among the random codes there are always several which have many more correlations than that found in the universal code. Although the number of correlations depends on the choice of biosynthetically related amino acids, the probability of choosing a random genetic code with the same or greater number of codon correlations as the universal genetic code was found to vary from 0.1% to 34% (with respect to a fairly complete listing of related amino acids). Thus, Wong's theory that the genetic code arose by coevolution with the biosynthetic pathways of amino acids, based on codon correlations between biosynthetically related amino acids, is statistical in nature. Received: 8 August 1996 / Accepted: 26 December 1996     

How Mitochondria Redefine the Code

Annotated, complete DNA sequences are available for 213 mitochondrial genomes from 132 species. These provide an extensive sample of evolutionary adjustment of codon usage and meaning spanning the history of this organelle. Because most known coding changes are mitochondrial, such data bear on the general mechanism of codon reassignment. Coding changes have been attributed variously to loss of codons due to changes in directional mutation affecting the genome GC content (Osawa and Jukes 1988), to pressure to reduce the number of mitochondrial tRNAs to minimize the genome size (Anderson and Kurland 1991), and to the existence of transitional coding mechanisms in which translation is ambiguous (Schultz and Yarus 1994a). We find that a succession of such steps explains existing reassignments well. In particular, (1) Genomic variation in the prevalence of a codon's third-position nucleotide predicts relative mitochondrial codon usage well, though GC content does not. This is because A and T, and G and C, are uncorrelated in mitochondrial genomes. (2) Codons predicted to reach zero usage (disappear) do so more often than expected by chance, and codons that do disappear are disproportionately likely to be reassigned. However, codons predicted to disappear are not significantly more likely to be reassigned. Therefore, low codon frequencies can be related to codon reassignment, but appear to be neither necessary nor sufficient for reassignment. (3) Changes in the genetic code are not more likely to accompany smaller numbers of tRNA genes and are not more frequent in smaller genomes. Thus, mitochondrial codons are not reassigned during demonstrable selection for decreased genome size. Instead, the data suggest that both codon disappearance and codon reassignment depend on at least one other event. This mitochondrial event (leading to reassignment) occurs more frequently when a codon has disappeared, and produces only a small subset of possible reassignments. We suggest that coding ambiguity, the extension of a tRNA's decoding capacity beyond its original set of codons, is the second event. Ambiguity can act alone but often acts in concert with codon disappearance, which promotes codon reassignment. Received: 26 October 2000 / Accepted: 19 January 2001     

The Level and Landscape of Optimization in the Origin of the Genetic Code

We consider a model of the origin of genetic code organization incorporating the biosynthetic relationships between amino acids and their physicochemical properties. We study the behavior of the genetic code in the set of codes subject both to biosynthetic constraints and to the constraint that the biosynthetic classes of amino acids must occupy only their own codon domain, as observed in the genetic code. Therefore, this set contains the smallest number of elements ever analyzed in similar studies. Under these conditions and if, as predicted by physicochemical postulates, the amino acid properties played a fundamental role in genetic code organization, it can be expected that the code must display an extremely high level of optimization. This prediction is not supported by our analysis, which indicates, for instance, a minimization percentage of only 80%. These observations can therefore be more easily explained by the coevolution theory of genetic code origin, which postulates a role that is important but not fundamental for the amino acid properties in the structuring of the code. We have also investigated the shape of the optimization landscape that might have arisen during genetic code origin. Here, too, the results seem to favor the coevolution theory because, for instance, the fact that only a few amino acid exchanges would have been sufficient to transform the genetic code (which is not a local minimum) into a much better optimized code, and that such exchanges did not actually take place, seems to suggest that, for instance, the reduction of translation errors was not the main adaptive theme structuring the genetic code.     

The Genetic Code Is One in a Million

Statistical and biochemical studies of the genetic code have found evidence of nonrandom patterns in the distribution of codon assignments. It has, for example, been shown that the code minimizes the effects of point mutation or mistranslation: erroneous codons are either synonymous or code for an amino acid with chemical properties very similar to those of the one that would have been present had the error not occurred. This work has suggested that the second base of codons is less efficient in this respect, by about three orders of magnitude, than the first and third bases. These results are based on the assumption that all forms of error at all bases are equally likely. We extend this work to investigate (1) the effect of weighting transition errors differently from transversion errors and (2) the effect of weighting each base differently, depending on reported mistranslation biases. We find that if the bias affects all codon positions equally, as might be expected were the code adapted to a mutational environment with transition/transversion bias, then any reasonable transition/transversion bias increases the relative efficiency of the second base by an order of magnitude. In addition, if we employ weightings to allow for biases in translation, then only 1 in every million random alternative codes generated is more efficient than the natural code. We thus conclude not only that the natural genetic code is extremely efficient at minimizing the effects of errors, but also that its structure reflects biases in these errors, as might be expected were the code the product of selection. Received: 25 July 1997 / Accepted: 9 January 1998     

The Historical Factor: The Biosynthetic Relationships Between Amino Acids and Their Physicochemical Properties in the Origin of the Genetic Code

Two forces are in general, hypothesized to have influenced the origin of the organization of the genetic code: the physicochemical properties of amino acids and their biosynthetic relationships. In view of this, we have considered a model incorporating these two forces. In particular, we have studied the optimization level of the physicochemical properties of amino acids in the set of amino acid permutation codes that respects the biosynthetic relationships between amino acids. Where the properties of amino acids are represented by polarity and molecular volume we obtain indetermination percentages in the organization of the genetic code of approximately 40%. This indicates that the contingent factor played a significant role in structuring the genetic code. Furthermore, this result is in agreement with the genetic code coevolution hypothesis, which attributes a merely ancillary role to the properties of amino acids while it suggests that it was their biosynthetic relationships that organized the code. Furthermore, this result does not favor the stereochemical models proposed to explain the origin of the genetic code. On the other hand, where the properties of amino acids are represented by polarity alone, we obtain an indetermination percentage of at least 21.5%. This might suggest that the polarity distances played an important role and would therefore provide evidence in favor of the physicochemical hypothesis of genetic code origin. Although, overall, the analysis might have given stronger support to the latter hypothesis, this did not actually occur. The results are therefore discussed in the context of the different theories proposed to explain the origin of the genetic code. Received: 10 September 1996 / Accepted: 3 March 1997     

The Chemical Basis of Membrane Bioenergetics

All organisms rely on chemiosmotic membrane systems for energy transduction; the great variety of participating proteins and pathways can be reduced to a few universal principles of operation. This chemical basis of bioenergetics is reviewed with respect to the origin and early evolution of life. For several of the cofactors which play important roles in bioenergetic reactions, plausible prebiotic sources have been proposed, and it seems likely that these cofactors were present before elaborate protein structures. In particular, the hydrophobic quinones require only a membrane-enclosed compartment to yield a minimum chemiosmotic system, since they can couple electron transport and proton translocation in a simple way. It is argued that the central features of modern bioenergetics, such as the coupling of redox reactions and ion translocation at the cytoplasmic membrane, probably are ancient features which arose early during the process of biogenesis. The notion of a thermophile root of the universal phylogenetic tree has been discussed controversially, nevertheless, thermophiles are interesting model organisms for reconstructing the origin of chemiosmotic systems, since they are often acidophiles and anaerobic respirers exploiting iron–sulfur chemistry. This perspective can help to explain the prominent role of iron–sulfur proteins in extant biochemistry as well as the origin of both respiration and proton extrusion within the context of a possible origin of life in the vicinity of hot vents. Received: 6 June 2001 / Accepted: 16 October 2001     

Phenylalanine-Binding RNAs and Genetic Code Evolution

We isolated RNAs by selection–amplification, selecting for affinity to Phe–Sepharose and elution with free l-phenylalanine. Constant sequences did not contain Phe condons or anticodons, to avoid any possible confounding influence on initially randomized sequences. We examined the eight most frequent Phe-binding RNAs for inclusion of coding triplets. Binding sites were defined by nucleotide conservation, protection, and interference data. Together these RNAs comprise 70% of the 105 sequenced RNAs. The K _D for the strongest sites is ≈50 μM free amino acid, with strong stereoselectivity. One site strongly distinguishes free Phe from Trp and Tyr, a specificity not observed previously. In these eight Phe-binding RNAs, Phe codons are not significantly associated with Phe binding sites. However, among 21 characterized RNAs binding Phe, Tyr, Arg, and Ile, containing 1342 total nucleotides, codons are 2.7-fold more frequent within binding sites than in surrounding sequences in the same molecules. If triplets were not specifically related to binding sites, the probability of this distribution would be 4.8 × 10⁻¹¹. Therefore, triplet concentration within amino acid binding sites taken together is highly likely. In binding sites for Arg, Tyr, and Ile cognate codons are overrepresented. Thus Arg, Tyr, and Ile may be amino acids whose codons were assigned during an era of direct RNA–amino acid affinity. In contrast, Phe codons arguably were assigned by another criterion, perhaps during later code evolution.     

Membrane Fusion and Rupture in Liposomes: Effect of Biodegradable pH-Sensitive Surfactants

Biodegradable pH-sensitive surfactants (BPS) are a unique family of easily metabolized compounds that demonstrate pH-dependent surface activity. These agents, in combination with other delivery systems, have demonstrated effects in enhancing transnucleic acid activity. The increased activity has been hypothesized to occur from a release of endosomal contents. Simply, the BPS delivery system containing nucleic acids enters the cell through an endocytotoic process. It encounters an acidic pH and becomes surface active leading to defects in the endosomal membrane. In the current study, an in vitro model membrane was used to better understand the liposome defect mechanisms that BPS elicit. Using this system, it is shown that BPS can induce both liposome fusion and rupture depending upon the pH and mole ratio of BPS to membrane lipids. Futhermore, liposome fusion induced by BPS was dependent on the total numbers of liposome particles while rupture was independent of interacting liposome particles. The generated data indicate that BPS agents act differently from other typical surface active agents and fuosgenic compounds. Instead of facilitating membrane fusion through the hexagonal II phase, BPS appeared to contribute and participate in the membrane fusion at different stages. Received: 18 February 1998/Revised: 14 July 1998     

Directionally Evolving Genetic Code: The UGA Codon from Stop to Tryptophan in Mitochondria

For the comprehensive analyses of deviant codes in protistan mitochondria (mt), we sequenced about a 1.1-kb region of a mitochondrial (mt) gene, the cytochrome c oxidase subunit I (coxI) in two chlorarachniophytes, the filose amoeba Euglypha rotunda, the cryptomonad Cryptomonas ovata, the prymnesiophyte (haptophyte) Diacronema vlkianum (Pavlovales), and the diatom Melosira ambigua. As a result of this analysis, we noticed that the UGA codon is assigned to tryptophan (Trp) instead of being a signal for translational termination in two chlorarachniophytes and in E. rotunda. The same type of deviant code was reported previously in animals, fungi, ciliates, kinetoplastids, Chondrus crispus (a red alga), Acanthamoeba castellanii (an amoeboid protozoon), and three of the four prymnesiophyte orders with the exception of the Pavlovales. A phylogenetic analysis based on the COXI sequences of 56 eukaryotes indicated that the organisms bearing the modified code, UGA for Trp, are not monophyletic. Based on these studies, we propose that the ancestral mitochondrion was bearing the universal genetic code and subsequently reassigned the codon to Trp independently, at least in the lineage of ciliates, kinetoplastids, rhodophytes, prymnesiophytes, and fungi. We also discuss how this codon was directionally captured by Trp tRNA. Received: 26 January 1998 / Accepted: 24 April 1998     

A Blind Empiricism Against the Coevolution Theory of the Origin of the Genetic Code

Ronneberg et al. (Proc Natl Acad Sci USA 97:13690–13695, 2000) recently suggested abandoning the coevolution theory of genetic code origin on the basis of two pieces of evidence. They (1) criticize the use of several pairs of amino acids in a precursor–product relationship to support this theory and (2) suggest a new set of codes in which to investigate the statistical bases of the coevolution theory, reaching the conclusion that this theory is not statistically validated in this set. In this paper I critically analyze the robustness of these conclusions. Observations and arguments lead to the belief that the pairs of amino acids in a precursor–product relationship originally used by the coevolution theory are such, or may at least be interpreted as such, and are therefore a manifestation of this theory. Furthermore, the new set of codes that Ronneberg et al. suggest is open to criticism and is thus substituted by the set of amino acid permutation codes, in which even the pairs of amino acids they favor end up by supporting the coevolution theory. Overall, the analysis seems to show that the paper by Ronneberg et al. is of minor scientific value while the coevolution theory seems to be one of the best theories at our disposal for explaining the evolutionary organisation of the genetic code and is, contrary to their claims, statistically well validated. Received: 21 February 2001 / Accepted: 22 May 2001     

The First Sexual Lineage and the Relevance of Facultative Sex

Models for the origin of the sex incorporate either obligate or facultative sexual cycles. The relevance of each assumption to the ancestral sexual population can be examined by surveying the sexual cycles of eukaryotes, and by determining the first lineage to diverge after sexuality evolved. Two protistan groups, the parabasalids and the oxymonads, have been suggested to be early-branching sexual lineages. A maximum-likelihood analysis of elongation factor-1α sequences shows that the parabasalids diverged prior to the oxymonads and thus represent the earliest sexual lineage of eukaryotes. Since both of these protist lineages and most other eukaryotes are facultatively sexual, it is likely that the common ancestor of all known eukaryotes was facultatively sexual as well. This finding has important implications for the ``Best-Man hypothesis' and other models for the origin of sex. Received: 21 August 1998 / Accepted: 26 December 1998     

The Apical Membrane Glycocalyx of MDCK Cells

The microenvironment near the apical membrane of MDCK cells was studied by quantitation of the fluorescence of wheat germ agglutin attached to fluorescein (WGA). WGA was shown to bind to sialic acid residues attached to galactose at the α-2,3 position in the glycocalyx on the apical membrane. Young MDCK cells (5–8 days after splitting) showed a patchy distribution of WGA at stable sites that returned to the same locations after removal of sialic acid residues by neuraminidase treatment. Other lectins also showed stable binding to patches on the apical membrane of young cells. The ratio of WGA fluorescence emission at two excitation wavelengths was used to measure near-membrane pH. The near-membrane pH was markedly acidic to the pH 7.4 bathing solution in both young and older cells (13–21 days after splitting). Patches on the apical membrane of young cells exhibited a range of near-membrane pH values with a mean ±sem of 6.86 ± 0.04 (n= 121) while the near-membrane pH of older cells was 6.61 ± 0.04 (n= 120) with a uniform WGA distribution. We conclude that the distribution of lectin binding sites in young cells reflects the underlying nonrandom location of membrane proteins in the apical membrane and that nonuniformities in the pH of patches may indicate regional differences in membrane acid-base transport as well as in the location of charged sugars in the glycocalyx. Received: 15 December 1999/Revised: 16 March 2000