A rationale for the symmetries by base substitutions of degeneracy in the genetic code

The first symmetry by base substitutions of degeneracy in the genetic code was described by Rumer (1966) and the other symmetries were identified later by Jestin (2006) and Jestin and Soulé (2007). Here, a rationale accounting for these symmetries is reported. The number of non-synonymous substitutions over the replicated coding sequence is written as a function of the substitution matrix, whose elements are the number of substitutions from any codon to any other codon. The p-adic distance used as a similarity measure and applied to this matrix is shown to be biologically relevant. The rationale indicates that symmetries by base substitutions of degeneracy in the genetic code are symmetries of the measures of the number of non-synonymous substitutions for sets of synonymous codons.     

On Error Minimization in a Sequential Origin of the Standard Genetic Code

Distances between amino acids were derived from the polar requirement measure of amino acid polarity and Benner and co-workers' (1994) 74-100 PAM matrix. These distances were used to examine the average effects of amino acid substitutions due to single-base errors in the standard genetic code and equally degenerate randomized variants of the standard code. Second-position transitions conserved all distances on average, an order of magnitude more than did second-position transversions. In contrast, first-position transitions and transversions were about equally conservative. In comparison with randomized codes, second-position transitions in the standard code significantly conserved mean square differences in polar requirement and mean Benner matrix-based distances, but mean absolute value differences in polar requirement were not significantly conserved. The discrepancy suggests that these commonly used distance measures may be insufficient for strict hypothesis testing without more information. The translational consequences of single-base errors were then examined in different codon contexts, and similarities between these contexts explored with a hierarchical cluster analysis. In one cluster of codon contexts corresponding to the RNY and GNR codons, second-position transversions between C and G and transitions between C and U were most conservative of both polar requirement and the matrix-based distance. In another cluster of codon contexts, second-position transitions between A and G were most conservative. Despite the claims of previous authors to the contrary, it is shown theoretically that the standard code may have been shaped by position-invariant forces such as mutation and base content. These forces may have left heterogeneous signatures in the code because of differences in translational fidelity by codon position. A scenario for the origin of the code is presented wherein selection for error minimization could have occurred multiple times in disjoint parts of the code through a phyletic process of competition between lineages. This process permits error minimization without the disruption of previously useful messages, and does not predict that the code is optimally error-minimizing with respect to modern error. Instead, the code may be a record of genetic process and patterns of mutation before the radiation of modern organisms and organelles. Received: 28 July 1997 / Accepted: 23 January 1998     

Estimation of DNA Sequence Context-dependent Mutation Rates Using Primate Genomic Sequences

It is understood that DNA and amino acid substitution rates are highly sequence context-dependent, e.g., C --> T substitutions in vertebrates may occur much more frequently at CpG sites and that cysteine substitution rates may depend on support of the context for participation in a disulfide bond. Furthermore, many applications rely on quantitative models of nucleotide or amino acid substitution, including phylogenetic inference and identification of amino acid sequence positions involved in functional specificity. We describe quantification of the context dependence of nucleotide substitution rates using baboon, chimpanzee, and human genomic sequence data generated by the NISC Comparative Sequencing Program. Relative mutation rates are reported for the 96 classes of mutations of the form 5' alphabetagamma 3' --> 5' alphadeltagamma 3', where alpha, beta, gamma, and delta are nucleotides and beta not equal delta, based on maximum likelihood calculations. Our results confirm that C --> T substitutions are enhanced at CpG sites compared with other transitions, relatively independent of the identity of the preceding nucleotide. While, as expected, transitions generally occur more frequently than transversions, we find that the most frequent transversions involve the C at CpG sites (CpG transversions) and that their rate is comparable to the rate of transitions at non-CpG sites. A four-class model of the rates of context-dependent evolution of primate DNA sequences, CpG transitions > non-CpG transitions approximately CpG transversions > non-CpG transversions, captures qualitative features of the mutation spectrum. We find that despite qualitative similarity of mutation rates among different genomic regions, there are statistically significant differences.     

Transitions,transversions, and the molecular evolutionary clock

Summary Nucleotide substitutions in the form of transitions (purine-purine or pyrimidine-pyrimidine interchanges) and transversions (purine-pyrimidine interchanges) occur during evolution and may be complied by aligning the sequences of homologous genes. Referring to the genetic code tables, silent transitions take place in third positions of codons in family boxes and two-codon sets. Silent transversions in third positions occur only in family boxes, except for AC transversions between AGR and CGR arginine codons (R=A or G). Comparisons of several protein genes have been made, and various subclasses of transitional and transversional nucleotide substitutions have been compiled. Considerable variations occur among the relative proportions of transitions and transversions. Such variations could possibly be caused by mutator genes, favoring either transitions or, conversely, transversions, during DNA replication. At earlier stages of evolutionary divergence, transitions are usually more frequent, but there are exceptions. No indication was found that transversions usually originate from multiple substitutions in transitions.     

The Role of Context-Dependent Mutations in Generating Compositional and Codon Usage Bias in Grass Chloroplast DNA

Abstract The influence of local base composition on mutations in chloroplast DNA (cpDNA) is studied in detail and the resulting, empirically derived, mutation dynamics are used to analyze both base composition and codon usage bias. A 4 × 4 substitution matrix is generated for each of the 16 possible flanking base combinations (contexts) using 17,253 noncoding sites, 1309 of which are variable, from an alignment of three complete grass chloroplast genome sequences. It is shown that substitution bias at these sites is correlated with flanking base composition and that the A+T content of these flanking sites as well as the number of flanking pyrimidines on the same strand appears to have general influences on substitution properties. The context-dependent equilibrium base frequencies predicted from these matrices are then applied to two analyses. The first examines whether or not context dependency of mutations is sufficient to generate average compositional differences between noncoding cpDNA and silent sites of coding sequences. It is found that these two classes of sites exist, on average, in very different contexts and that the observed mutation dynamics are expected to generate significant differences in overall composition bias that are similar to the differences observed in cpDNA. Context dependency, however, cannot account for all of the observed differences: although silent sites in coding regions appear to be at the equilibrium predicted, noncoding cpDNA has a significantly lower A+T content than expected from its own substitution dynamics, possibly due to the influence of indels. The second study examines the codon usage of low-expression chloroplast genes. When context is accounted for, codon usage is very similar to what is predicted by the substitution dynamics of noncoding cpDNA. However, certain codon groups show significant deviation when followed by a purine in a manner suggesting some form of weak selection other than translation efficiency. Overall, the findings indicate that a full understanding of mutational dynamics is critical to understanding the role selection plays in generating composition bias and sequence structure.     

Sequence Evolution of Drosophila Mitochondrial DNA

We have compared nucleotide sequences of corresponding segments of the mitochondrial DNA (mtDNA) molecules of Drosophila yakuba and Drosophila melanogaster, which contain the genes for six proteins and seven tRNAs. The overall frequency of substitution between the nucleotide sequences of these protein genes is 7.2%. As was found for mtDNAs from closely related mammals, most substitutions (86%) in Drosophila mitochondrial protein genes do not result in an amino acid replacement. However, the frequencies of transitions and transversions are approximately equal in Drosophila mtDNAs, which is in contrast to the vast excess of transitions over transversions in mammalian mtDNAs. In Drosophila mtDNAs the frequency of C----T substitutions per codon in the third position is 2.5 times greater among codons of two-codon families than among codons of four-codon families; this is contrary to the hypothesis that third position silent substitutions are neutral in regard to selection. In the third position of codons of four-codon families transversions are 4.6 times more frequent than transitions and A----T substitutions account for 86% of all transversions. Ninety-four percent of all codons in the Drosophila mtDNA segments analyzed end in A or T. However, as this alone cannot account for the observed high frequency of A----T substitutions there must be either a disproportionately high rate of A----T mutation in Drosophila mtDNA or selection bias for the products of A----T mutation. --Consideration of the frequencies of interchange of AGA and AGT codons in the corresponding D. yakuba and D. melanogaster mitochondrial protein genes provides strong support for the view that AGA specifies serine in the Drosophila mitochondrial genetic code.     

Hypothetical stages in the mutational evolution of the present-day genetic code from its RNY,comma-free ancestor

The primitive comma-free genetic code may have had 16 triplets of the form RNY, where R = purine, N = purine or pyrimidine, and Y = pyrimidine, specifying eight (present-day) amino acids. Calculations reveal that in this primitive code all transition changes (A?G, C?U) are either silent or missense i.e. result in the same or another one of these particular eight amino acids. There are no single transitions to non-RNY codons. Single transversions in the primitive codons can, individually, generate new (present-day) codons for four or eight amino acids. Present-day glutamine, tryptophan and stop (UGA, UAA, UAG) codons cannot be so derived., by single transversions, from any of the eight primitive codons. The modern initiation codons, AUG and GUG, can however be generated by both C → G and U → G single transversions in primitive codons. Overall, a total of 32 modern sense codons, not represented in the primitive RNY code, can be derived from this code by single transversions. Many modern codons, including all those not generated by single transversions in the primitive code, can also be produced by either of the two types of frameshift possible in runs of U- or C-rich primitive codons. Present-day stop codons are generated by +1 (-2) type frameshifts in U-rich primitive runs; AUG and GUG initiation codons are produced by the other type, +2 (-1), frameshifts in U-rich runs.     

Use of Chou-Fasman amino acid conformational parameters to analyze the organization of the genetic code and to construct protein genealogies

Summary Chou-Fasman parameters, measuring preferences of each amino acid for different conformational regions in proteins, were used to obtain an amino acid difference index of conformational parameter distance (CPD) values. CPD values were found to be significantly lower for amino acid exchanges representing in the genetic code transitions of purines, GA than for exchanges representing either transitions of pyrimidines, CU, or transversions of purines and pyrimidines. Inasmuch as the distribution of CPD values in these non GA exchanges resembles that obtained for amino acid pairs with double or triple base differences in their underlying codons, we conclude that the genetic code was not particularly designed to minimize effects of mutation on protein conformation. That natural selection minimizes these changes, however, was shown by tabulating results obtained by the maximum parsimony method for eight protein genealogies with a total occurrence of 4574 base substitutions. At the beginning position of the codons GA transitions were in very great excess over other base substitutions, and, conversely, CU transitions were deficient. At the middle position of the codons only fast evolving proteins showed an excess of GA transitions, as though selection mainly preserved conformation in these proteins while weeding out mutations affecting chemical properties of functional sites in slow evolving proteins. In both fast and slow evolving proteins the net direction of transitions and transversions was found to be from G beginning codons to non-G beginning codons resulting in more commonly occurring amino acids, especially alanine with its generalized conformational properties, being replaced at suitable sites by amino acids with more specialized conformational and chemical properties. Historical circumstances pertaining to the origin of the genetic code and the nature of primordial proteins could account for such directional changes leading to increases in the functional density of proteins.In order to further explore the course of protein evolution, a modified parsimony algorithm was developed for constructing protein genealogies on the basis of minimum CPD length. The algorithm's ability to judge with finer discrimination that in protein evolution certain pathways of amino acid substitution should occur more readily than others was considered a potential advantage over strict maximum parsimony. In developing this CPD algorithm, the path of minimum CPD length through intermediate amino acids allowed by the genetic code for each pair of amino acids was determined. It was found that amino acid exchanges representing two base changes have a considerably lower average CPD value per base substitution than the amino acid exchanges representing single base changes. Amino acid exchanges representing three base changes have yet a further marked reduction in CPD per base change. This shows how extreme constraining effects of stabilizing selection can be circumvented, for by way of intermediate amino acids almost any amino acid can ultimately be substituted for another without damage to an evolving protein's conformation during the process.     

Challenges of the genetic code for exploring sequence space in directed protein evolution

Directed protein evolution is the most versatile method for studying protein structure-function relationships, and for tailoring a protein's properties to the needs of industrial applications. In this review, we performed a statistical analysis on the genetic code to study the extent and consequence of the organization of the genetic code on amino acid substitution patterns generated in directed evolution experiments. In detail, we analyzed amino acid substitution patterns caused by (a) a single nucleotide (nt) exchange at each position of all 64 codons, and (b) two subsequent nt exchanges (first and second nt, first and third nt, second and third nt). Additionally, transitions and transversions mutations were compared at the level of amino acid substitution patterns. The latter analysis showed that single nucleotide substitution in a codon generates only 39.5% of the natural diversity on the protein level with 5.2-7 amino acid substitutions per codon. Transversions generate more complex amino acid substitution patterns (increased number and chemically more diverse amino acid substitutions) than transitions. Simultaneous nt exchanges at both first and second nt of a codon generates very diverse amino acid substitution patterns, achieving 83.2% of the natural diversity. The statistical analysis described in this review sets the objectives for novel random mutagenesis methods that address the consequences of the organization of the genetic code. Random mutagenesis methods that favor transversions or introduce consecutive nt exchanges can contribute in this regard.     

Neighboring base composition is strongly correlated with base substitution bias in a region of the chloroplast genome

Nucleotide sequence from a region of the chloroplast genome is presented for 12 species spanning four subfamilies of the grass family. The region contains the coding sequence for the rbcL gene and the intergenic spacer between the gene coding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase rbcL and the photosystem I gene psal. This intergenic spacer contains a pseudogene for rpl23 as well as two noncoding segments with different A+T contents. Using the sequence of rbcL a chloroplast phylogeny of this family was constructed by parsimony. Variable sites of the two noncoding segments were traced onto the phylogeny to study the dynamics of base substitution. This was also performed for the fourfold-degenerate sites of the rbcL gene. A wide variation in transversion/transition is observed between the two noncoding segments and between the noncoding DNA and the fourfold-degenerate sites of rbcL This variation is correlated with regional A+T content. As regional A+T content decreases, the ratio of transversions to transitions also decreases. Substitutions were then scored in relation to neighboring base composition. The composition of the two bases immediately flanking each substitution is highly correlated with the transversion/transition bias. When both the 5 and 3 flanking bases are an A or a T, transversions are observed 2.2 times as frequently as transitions. When either or both neighbors are a C or a G, the opposite trend is found; transitions are observed 1.5 times more frequently than transversions. Correspondence to: Brian R. Morton     

Challenges of the genetic code for exploring sequence space in directed protein evolution

Directed protein evolution is the most versatile method for studying protein structure–function relationships, and for tailoring a protein's properties to the needs of industrial applications. In this review, we performed a statistical analysis on the genetic code to study the extent and consequence of the organization of the genetic code on amino acid substitution patterns generated in directed evolution experiments. In detail, we analyzed amino acid substitution patterns caused by (a) a single nucleotide (nt) exchange at each position of all 64 codons, and (b) two subsequent nt exchanges (first and second nt, first and third nt, second and third nt). Additionally, transitions and transversions mutations were compared at the level of amino acid substitution patterns. The latter analysis showed that single nucleotide substitution in a codon generates only 39.5% of the natural diversity on the protein level with 5.2–7 amino acid substitutions per codon. Transversions generate more complex amino acid substitution patterns (increased number and chemically more diverse amino acid substitutions) than transitions. Simultaneous nt exchanges at both first and second nt of a codon generates very diverse amino acid substitution patterns, achieving 83.2% of the natural diversity. The statistical analysis described in this review sets the objectives for novel random mutagenesis methods that address the consequences of the organization of the genetic code. Random mutagenesis methods that favor transversions or introduce consecutive nt exchanges can contribute in this regard.     

Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM-3 heme domain

Directed evolution represents a versatile tool to tailor enzyme properties to needs in industrial applications and to understand structure-function relationships. Genetic diversity is commonly generated using error-prone PCR. Exploration of sequence space by random mutagenesis strongly favors transitions when enzyme-based mutagenesis methods are employed (Wong, T. S., Zhurina, D., Schwaneberg, U., Comb. Chem. High Throughput Screen. 2006, 9, 271-288). The genetic code has been organized in a manner that limits chemical diversity when a single transition mutation occurs in a codon (Wong, T. S., Roccatano, D., Schwaneberg, U., Biocatal. Biotransformation 2006, in press). Are transitions more beneficial than transversions for adapting biocatalysts to non-natural process conditions? In a statistical analysis performed with the Mutagenesis Assistant Program (MAP), we compared the consequences of transition and transversion bias on amino acid substitution patterns of the P450 BM-3 heme domain. For the analysis, we used a recently introduced benchmarking system consisting of a protein structure indicator, an amino acid diversity indicator with a codon diversity coefficient, and a chemical diversity indicator. A detailed analysis for the P450 BM-3 heme domain showed that an ideal transversion bias generates more diverse amino acid substitution patterns with a significantly different chemical composition than an ideal transition bias. Emphasis is given on the theoretical analysis with a brief discussion on potential implication of transition and transversion bias in directed evolution experiments.     

Exploring the energy landscape of the genetic code

New insights into the arrangement of the genetic code table, based on the analysis of the physico-chemical properties of its molecular constituents, are reported in this paper. It will be demonstrated that the code has a twofold symmetry that is not apparent from the conventional code table, but becomes apparent when the codon-anticodon energies are listed for each triplet. The evolutionary development of the current code based on single base replacement mutations (transitions) from an 'iso-energetic' degenerated subset of 16 of the 64 codons is discussed. The energy landscape of all 64 codons is presented. A detailed analysis of the energy changes due to mutations in the 3rd, 1st or 2nd position of a codon reveals that the modern genetic code is highly robust. Changes come in small discrete steps that can be quantified in relation to the thermal noise of the system. The relation of the individual codon to its neighbours in the rearranged codon table can be completely understood based on thermodynamic considerations.     

Hydrogen peroxide induces G:C to TA and G:C to C:G transversions in the supF gene of Escherichia coli

A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe³⁺/EDTA complex and propagated in E. coli host cells that had been induced for SOS functions by ultraviolet irradiation. The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide. The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA. Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:CC:G (28 cases) and G:CT:A (26 cases) transversions predominating. Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5-PuGA-3 was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5PyGN3. G:CT:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine. The origin of the G:CC:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide. Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168. Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis.     

Molecular basis of spontaneous mutation at the aprt locus of hamster cells

Mutations occurring spontaneously at the hamster aprt locus were examined at the base-pair level by amplifying target sequences using the polymerase chain reaction and then directly sequencing the double-stranded products. In a collection of 89 sequenced genes, all types of mutations were found, with transitions (mostly G.C to A.T) constituting the largest class (35%), transversions accounting for 27%, and small deletions/duplications for 25%. Simple base substitutions were distributed throughout the aprt structural gene with few sites having recurring mutations and G.C base-pairs being the predominant substitution target. Small deletions, on the other hand, were not distributed so evenly, being concentrated in a region of aprt rich in short direct and inverted repeat sequences. The base substitutions were predominantly missense, while about 10% produced nonsense codons. Splice junctions, and start and stop codons were also significant targets for mutation. No alterations were detected in three aprt-deficient strains after sequencing all exons and substantial upstream and downstream regions.     

Characteristics of Nucleotide Substitution in the Hepatitis C Virus Genome: Constraints on Sequence Change in Coding Regions at Both Ends of the Genome

Comparison of complete genome sequences for different variants of hepatitis C virus (HCV) reveals several different constraints on sequence change. Synonymous changes are suppressed in coding regions at both 5′ and 3′ ends of the genome. No evidence was found for the existence of alternative reading frames or for a lower mutation frequency in these regions. Instead, suppression may be due to constraints imposed by RNA secondary structures identified within the core and NS5b genes. Nonsynonymous substitutions are less frequent than synonymous ones except in the hypervariable region of E2 and, to a lesser extent, in E1, NS2, and NS5b. Transitions are more frequent than transversions, particularly at the third position of codons where the bias is 16:1. In addition, nucleotide substitutions may not occur symmetrically since there is a bias toward G or C at the third position of codons, while T ↔ C transitions were twice as frequent as A ↔ G transitions. These different biases do not affect the phylogenetic analysis of HCV variants but need to be taken into account in interpreting sequence change in longitudinal studies. Received: 9 September 1996 / Accepted: 20 April 1997     

On the information content of the genetic code

In living organisms 20 amino acids along with the terminator value(s) are encoded by 64 codons giving a degeneracy of the codons as described by the genetic code. A basic theoretical problem of genetic codes is to explain the particular distribution of degeneracies of partitions involved in the codes. In this work the degeneracy problem is considered in the framework of information theory. It is shown by direct numerical evaluation of a certain degeneracy information function associated with the genetic code that the degeneracy of the codes is observed to be related to the optimization of this function.     

Phase-dependent nucleotide substitution in protein-coding sequences

It is well known that due to the degeneracy of genetic code, most of the silent substitutions appear in the third codon position, so the mutation frequency of the third codon position is much higher than that of the first two positions. However, it remains unknown whether the directionality of point mutation in three codon positions is similar or not. In this paper, through analyzing 15 sets of orthologous genes, it is revealed that most of the substitution types are significantly different between any two codon positions, especially between the 2nd and the 3rd phases. Furthermore, the average frequencies of each type of substitution calculated from the fifteen sets of orthologous genes are similar to those identified in single nucleotide polymorphisms (SNPs) of human and mouse genome. The present analyses suggest that the nucleotide substitution in protein-coding sequences is not only context-dependent (so called neighboring-nucleotide effects), but also phase-dependent, which is of significance to improving the prevalent nucleotide-evolution models.     

Evolution of the mitochondrial genetic code III. Reassignment of CUN codons from leucine to threonine during evolution of yeast mitochondria

Summary Yeast mitochondria use UUR as the sole leucine codons. CUN, universal leucine codons, are read as threonine by aberrant threonine tRNA with anticodon sequence (UAG).The reassignment of CUN codons to threonine during yeast mitochondrial evolution could have proceeded by the disappearance of CUN codons from the reading frames of messenger RNA, through mutation mainly to UUR leucine codons as a result of AT pressure. We suggest that this was accompanied by a loss of leucine-accepting ability of tRNA Leu(UAG). This tRNA could have then acquired threonine-accepting activity through the appearance of an additional threonyl-tRNA synthetase. CUN codons that subsequently appeared from mutations of various other codons would have been translated as threonine. This change in the yeast mitochondrial genetic code is likely to have evolved through a series of nondisruptive nucleotide substitutions that produced no widespread replacement of leucine by threonine in proteins as a consequence.     

Accepted mutations in a gene family: Evolutionary diversification of duplicated DNA

Summary We report and compare the DNA sequences of 14 silkmoth (Antheraea polyphemus) chorion genes, derived from either cDNA or chromosomal DNA clones. Seven of these genes are members of the A multigene family, and seven are members of the B family. Where available, the previously reported (Jones and Kafatos 1980) intronic and extragenic flanking DNA sequences are also considered. Closely related sequences are compared, revealing the types of spontaneous mutations that were fixed during paralogous evolution. Segmental mutations (i.e. mutations other than substitutions) are nearly always interpretable as small duplications or deletions. related to small direct repeats. Segmental mutations are strongly constrained in the coding regions, although they do occur. Nucleotide substitutions also appear to be under selective constraints: relatively few substitutions leading to amino acid replacements are accepted, silent substitutions leading to some codons (especially purine-terminated ones) are disfavored, and different compositional biases are maintained in different parts of the sequences. Other sequence differences can be interpreted as indicative of neutral drift, including most differences in non-coding regions and most T/C transitions in third-base positions. In the non-coding regions, which are thought to be only loosely constrained by selection, transitions are observed more frequently than might be expected: they account for 52% of all substitutions, and they appear to be favored two to threefold over transversions when allowance is made for the skewed base composition of these regions.