On the origin of the genetic code: signatures of its primordial complementarity in tRNAs and aminoacyl-tRNA synthetases.

If the table of the genetic code is rearranged to put complementary codons face-to-face, it becomes apparent that the code displays latent mirror symmetry with respect to two sterically different modes of tRNA recognition. These modes involve distinct classes of aminoacyl-tRNA synthetases (aaRSs I and II) with recognition from the minor or major groove sides of the acceptor stem, respectively. We analyze the anticodon pairs complementary to the face-to-face codon couplets. Taking into account the invariant nucleotides on either side (5' and 3'), we consider the risk of anticodon confusion and subsequent erroneous aminoacylation in the ancestral coding system. This logic leads to the conclusion that ribozymic precursors of tRNA synthetases had the same two complementary modes of tRNA aminoacylation. This surprising case of molecular mimicry (1) shows a key potential selective advantage arising from the partitioning of aaRSs into two classes, (2) is consistent with the hypothesis that the two aaRS classes were originally encoded by the complementary strands of the same primordial gene and (3) provides a 'missing link' between the classic genetic code, embodied in the anticodon, and the second, or RNA operational, code that is embodied mostly in the acceptor stem and is directly responsible for proper tRNA aminoacylation.

Epigenetic changes and repositioning determine the evolutionary fate of duplicated genes.

Consideration of epigenetic silencing, perhaps by DNA methylation, led to an epigenetic complementation (EC) model for evolution by gene duplication (Rodin and Riggs (2003) J. Mol. Evol., 56, 718-729). This and subsequent work on genome-wide analyses of gene duplicates in several eukaryotic species pointed to a fundamental link between localization in the genome, epigenetic regulation of expression, and the evolutionary fate of new redundant gene copies, which can be either non- or neo-functionalization. Our main message in this report is that repositioning of a new duplicate to an ectopic site epigenetically alters its expression pattern, and concomitantly the rate and direction of mutations. Furthermore, comparison of syntenic vs. non-syntenic pairs of gene duplicates of different age unambiguously indicates that repositioning saves redundant gene duplicates from pseudogenization and hastens their evolution towards a new development-time and tissue-specific pattern of function.

Cancerous hyper-mutagenesis in p53 genes is possibly associated with transcriptional bypass of DNA lesions.

The database of tumor-associated p53 base substitutions includes about 5% of tumors with two or more base substitutions. These multiplet base substitutions in one tumor are evidence for hyper-mutagenesis. Our retrospective analysis of this database indicates that most multiplets arise from a single transient hyper-mutagenic event in one cell that subsequently proliferated into a clonal tumor. The hyper-mutagenesis, 1.8 x 10(-4) substitutions per base pair, is detected as multiple mutations in p53 genes of tumors. It requires one strongly tumorigenic p53 substitution, usually missense, called the driver mutation. The occurrence frequencies of ancillary base substitutions, those that hitch-hike along with the driver mutation, are independent of their amino acid coding properties. In this respect, they act like neutral mutations. In support of this neutrality, we find that the frequency distribution of hitch-hiking CpG transitions along the p53 exons, their mutational spectrum, approximates the spontaneous pre-selection mutational spectrum of most human tissues and is correlated with the mutational spectrum of p53 pseudogenes in mammalian germ cells. The driver substitutions of multiplets predominantly originate along the transcribed strand while the ancillary substitutions tend to originate along the non-transcribed strand. This data is consistent with a model of time-dependent mutagenesis in non-dividing stem cells for generating multiple strand-asymmetric p53 mutations in tumors. By transcriptional bypass of DNA lesions with concomitant misincorporation, transcriptional mutagenesis generates a transient mutant p53 mRNA. The associated mutant p53 protein could allow the host cell a growth advantage, release from G1-arrest. Then, during subsequent DNA replication and misreading of the same lesion, the damaged base along the transcribed DNA strand would serve as the origin of the p53 base substitution that drives the hyper-mutagenic event leading to tumors with multiple p53 mutations.

Human lung cancer and p53: the interplay between mutagenesis and selection.

It is an almost consensus opinion that the major carcinogenic risk of tobacco smoke is in its direct mutagenic action on DNA of cancer-related genes. The key data supposedly linking smoke-induced mutations to lung cancer were obtained from the adduct spectrum of the p53 tumor suppressor gene. Results of our analysis of p53 mutations compiled from the International Agency for Research on Cancer p53 database (April 1999 update) and from the literature point to a different causative link. Our new analytical tests focused on complementary base substitutions and showed that it is strand-specific repair of primary lesions and site-specific selection of the resultant mutations that determine the lung cancer-specific hot spots of G:C to T:A transversions along the p53 gene and also their increased abundance in lung tissues as compared with smoke-inaccessible tissues. However, on each of the two strands of p53 DNA, our tests revealed no significant difference between smokers and nonsmokers, either in the frequency of different types of mutations or in the frequency of their occurrence along the p53 gene. Moreover, in both smokers and nonsmokers, there was the same frequency of lung tumors with silent p53 mutations. Accordingly, we offer here a selection-based explanation of why lung cancers with nonsilent p53 mutations are more common in smokers than in nonsmokers. We conclude that physiological stresses (not necessarily genotoxic) aggravated by smoking are the leading risk factor in the p53-associated etiology of lung cancer.

CpG transition strand asymmetry and hitch-hiking mutations as measures of tumorigenic selection in shaping the p53 mutation spectrum.

By the genetic code, the average protein perturbation expected from a CpG-->TpG transition is strand-specific and smallest when it originates with the C on the transcribed (noncoding) strand. To distinguish the effects of selection from mutagenesis, we measured strand asymmetry for CpG-->TpG transitions fixed in active p53 genes and pseudogenes during vertebrate evolution, and for p53 genes from human tumors with one (singlet) and two (doublet) p53 point mutations. Mutagenesis appears to generate the transitions symmetrically while selection usually acts asymmetrically being most sensitive to the larger protein perturbations. Tumorigenic selection acting on the central domain of the p53 gene appears exceptional in that it often senses gain of function amino acid substitutions whose altered function is unrelated to degree of protein perturbation. In doublets, the selection on some gain of function substitutions is relaxed as evidenced by a return to the transition strand symmetry.

Strand asymmetry of CpG transitions as indicator of G1 phase-dependent origin of multiple tumorigenic p53 mutations in stem cells.

In dividing cells, expression of mutations is DNA strand symmetric. Of all mutations originating de novo in nondividing cells, only those in the transcribed (noncoding) strand are immediately expressed in mRNA and protein. In contrast, any new mutation in the nontranscribed (coding) strand remains unexpressed until the cells enter S phase and begin proliferation. This previously unrecognized difference enables us to examine the cell cycle-dependent origin of multiple tumorigenic mutations in stem cells. The human p53 gene, which acts as a gatekeeper in the control of G1 to S phase transition, was chosen for the analysis. Of all multiple mutations contained in p53 databases, we have tested in detail CpG transitions. Three features of CpG sites dictate this choice: C --> T transitions at methylated mCpG are the direct product of mC deamination and are replication-independent; it is easy to identify the strand bearing a primary mC --> T event because C --> T on the transcribed strand appears as G --> A on the nontranscribed strand; and CpG transitions are the most frequent (as both singular and multiple occurrences) tumor-related p53 mutations. The origin of double nonsilent CpG transitions in nondividing cells predicts a significant excess of the heterostrand (C --> T, G --> A) doublets over the homostrand (C --> T, C --> T and G --> A, G --> A) doublets. For p53, we found such an excess. Based on this result, along with the results of three other tests reported here, we conclude that the majority of multiple p53 mutations from human tumors occurred in quiescent stem cells.

Four primordial modes of tRNA-synthetase recognition, determined by the (G,C) operational code.

In distinction to single-stranded anticodons built of G, C, A, and U bases, their presumable double-stranded precursors at the first three positions of the acceptor stem are composed almost invariably of G-C and C-G base pairs. Thus, the "second" operational RNA code responsible for correct aminoacylation seems to be a (G,C) code preceding the classic genetic code. Although historically rooted, the two codes were destined to diverge quite early. However, closer inspection revealed that two complementary catalytic domains of class I and class II aminoacyl-tRNA synthetases (aaRSs) multiplied by two, also complementary, G2-C71 and C2-G71 targets in tRNA acceptors, yield four (2 x 2) different modes of recognition. It appears therefore that the core four-column organization of the genetic code, associated with the most conservative central base of anticodons and codons, was in essence predetermined by these four recognition modes of the (G,C) operational code. The general conclusion follows that the genetic code per se looks like a "frozen accident" but only beyond the "2 x 2 = 4" scope. The four primordial modes of tRNA-aaRS recognition are amenable to direct experimental verification.

Two types of aminoacyl-tRNA synthetases could be originally encoded by complementary strands of the same nucleic acid.

The lack of even a marginal similarity between the two aminoacyl-tRNA synthetase (aaRS) classes suggests their independent origins (Eriani et al., 1990; Nagel and Doolittle, 1991). Yet, this independence is a puzzle inconsistent with the common origin of transfer RNAs, the coevolutionary theory of the genetic code (Wong, 1975, 1981) and other associated data and ideas. We present here the results of antiparallel 'class I versus class II' comparisons of aaRSs within their signature sequences. The two main HIGH- and KMSKS-containing motifs of class I appeared to be complementary to the class II motifs 2 and 1, respectively. The above sequence complementarity along with the mirror-image between crystal structures of complexes formed by the opposite aaRSs and their cognate tRNAs (Ruff et al., 1991), and the generally mirror ('head-to-tail') mapping of the basic functional sites in the sequences of aaRSs from the opposite two classes led us to conclude that these two synthetases emerged synchronously as complementary strands of the same primordial nucleic acid. This conclusion, combined with the hypothesis of tRNA concerted origin (Rodin et al., 1993a,b), may explain many intriguing features of aaRSs and favor the elucidation of the origin of the genetic code.

[A comparative computer analysis of thermodynamic parameters of transport RNA secondary structure]. / Sravnitel'nyi komp'iuternyi analiz termodinamicheskikh parametrov vtorichnoi struktury transportnykh RNK.

Thermodynamic parameters of the cloverleaf secondary structure of tRNAs of several major taxons (archaebacteria, eubacteria, eukaryotes, chloroplasts, and mitochondria) were subjected to computer analysis. The distribution of free-energy values was close to normal in all groups (except for the mitochondrial tRNA). In the organisms existing under extreme environmental conditions, the stability of the tRNA secondary structure was higher. Comparative analysis of teh frequency of ¿quasi-complementary¿ GU pairs in stems of randomly generated and real sequences demonstrated preferential fixation of such pairs in the contexts with the most favorable thermodynamics parameters. Noncanonic pairs in tRNA stems set limits on the presence of highly mutable CG dinucleotides. Moreover, the effect of noncanonic pairs other than GU on the frequency of such dinucleotides is far more pronounced than that of GU pairs.

Transposable elements and the penetrance of quantitative characters in Drosophila melanogaster.

We wanted to determine whether there is a correlation between the quantitative character, the penetrance of the loss of humeral bristles in scute lines, and the distribution of transposable genetic elements in their genomes. We derived 18 isogenic lines with penetrance ranging between 2.8% and 92.0% from six mutant lines. The localization of the transposable elements (TEs) P, mdg1, Dm412, copia, gypsy and B104 was determined in all isogenic derivatives by in situ hybridization. The total number of the TE sites over all lines was 180. A comparison of the distribution of the TEs in the isogenic lines revealed the location of sites typical of lines with similar penetrance, no matter which parental line was involved. The results obtained suggest that such typical sites appear to tag the genome regions where the polygenes affecting the character in question are most likely to be found.

[The coevolutionary aspects of the genome and mobile elements]. / Nekotorye aspekty koévoliustii genoma i mobil'nykh élementov.

Under investigation is congruent variation of movable elements, the "genome parasites" (GP), and genome proper, as the "host". Several differential equations are studied that describe the following situations. 1. GP is able to insert in free sites only, existing in autonomous stage (outside of the genome) limited time only. 2. GP inserts in free sites only, being able to exist autonomously relatively long time. 3. GP inserts in both free and occupied sites ("molecular memory"), existing autonomously relatively short time, its prototype being Alu-like repetitions in mammals. 4. GP inserts in both free and occupied sites, and is able to live autonomously, its prototype being retrovirus. In is presumed that the genome is tolerant to "selfish" breeding of GP as long as proportion of invaded sites does not exceed some critical value. It is shown that co-evolutionary complication of GP--from the simplest one that is able to insert in free sites only, through acquiring terminal repetitions ("molecular memory"), up to complicated entities existing outside of genome--accompanied by alteration of selective co-evolutionary limitations on genome size: limitations from above--no limitations--limitations from below. Thus, genetical movable elements can be considered as intrinsic factor of progressive, co-evolutionary stipulated complication of the genome.

[Theoretical analysis of some features of compensatory substitution in stem regions of transport RNA]. / Teoreticheskii analiz nekotorykh osobennostei kompensatornykh zamen v steblevykh uchastkakh transportnykh RNK.

Some families of isoacceptor tRNAs were studies using VOSTORG software package for phylogenetic analysis. While analysing the evolutionary trees in was shown that the fixation of double substitutions AU<-->GC in spiral double-stranded regions passes two stages with intermediate station, such as non-canonical base pairs. In all the families studied double substitutions can be explained not only by passages through slightly deleterious GU pairs. Comparative analysis of B2-like repeats shown the absence of compensatory substitutions in the parts which are homologous to RNA stem regions. The data obtained let us assume that compensatory substitutions in the RNA helical regions represent an expressive example of non-directed adaptive molecular coevolution.

[Mobile element distribution and quantitative character penetrance in the scute mutants of Drosophila melanogaster induced in the system of P-M hybrid dysgenesis]. / Raspredelenie mobil'nykh élementov i penetrantnost' kolichestvennykh priznakov v liniiakh mutantov scute Drosophila melanogaster, indutsirovannykh v sisteme P-M-gibridnogo disgeneza.

A series of new scute mutants noted for reduction in one pair of bristles (humerals), but different penetrance with respect to this character, were induced in the system of P--M hybrid dysgenesis. In situ hybridization technique was used to determine the sites of the MEs pertaining to six families in 6 scute mutants and 18 isogenic lined derived from those. Through calculating the similarity indices, similarity with respect to the pattern of total distribution of all MEs examined was estimated for the lines. Results obtained favour the correlation between penetrance and ME localization. The "ME pattern-penetrance" dependence was verified using the isogenic lines. The matrix of similarity indices and the similarity tree were constructed for the mutant lines following comparative analysis of distribution of 132 hybridization sites. The structure of the tree gives in a number (3-4) of blocks made up by isogenic lines of different origin. The rank of stocks following merely ordering them as regards penetrance and the structure of both the tree and the matrix run together pretty well; what's more, compact blocks of labelled sites answer to the groups of stocks of close penetrance. Our results suggest that penetrance with respect to the number of numerals should directly depend on the peculiarities of ME localization in the genomes of mutant lines.

[Adaptive significance of the limited gene expression of the immune system]. / Ob adaptivnom znachenii ogranichennoi ékspressii genov immunnoi sistemy.

Evolutionary development of immunity multigene systems seems to involve host-parasite molecular coevolution, as evidenced by comparison of metasoans and unicellular organisms in fixation rates of neutral and adaptive point mutations per one pair of corresponding genes. The estimations we attempted revealed that organisms with maximal restriction of Ig-like gene repertoire expression by an individual immune cell are more adaptive in formation of competent antigen-induced immune response, provided that the magnitude of potential repertoire of functional Ig-like receptors exceeds lymphoid cell population size. In contrast, when phage and bacteria populations are in coevolution, there is no need to limit the repertoire of expressive genes encoding bacteria surface receptor proteins which are recognized by a phage adsorption system. Other hypotheses of evolutionary premises and stimulus of immune genes expression limitation (allelic, isotypic etc.) are critically discussed.

[Theoretical analysis of the correspondence between the stages of evolutionary divergence and the functioning of multigene families of the immune system]. / Teoreticheskii analiz sootvetstviia mezhdu étapami évoliutsionnoi divergentsii i funktsionirovaniia mul'tigennykh semeistv immunnoi sistemy.

By means of phylogenetic comparison of main immunoglobulin-like multigene families (MF) and of different C-domain sequences representing them, the order of divergences of these MF in the process of evolution of the system was analysed. The order proved to be in good agreement with the ordered fashion of MF's involvement into recombinational rearrangements during the immune cell development. Indeed, according to topology of the tree for 34 C-domains, all MHC antigens are separated from the rest of Ig-like sequences, at first. Secondly, beta-chain of T-receptor is branched off the main stem of divergences, then there is a serium of divergences of CH-domains for different Ig-classes, and at last, kappa- and lambda-chains are separated. It is mu-chain of IgM which first of all branched off within an each group of isofunctional CH-sequences in the tree (CH1, CH2, CH3, CH4). The same order of events could be seen in ontogeny of the immune system. The results suggest that the appearance of a new MF of Ig-like type (or a new cluster of genetic segments within the MF) in the course of evolution could mean the addition of a new step in the regulatory system of immunologic gene expression already involved. In other words, this is unique example of recapitulation at the molecular-genetical level.