Biochemistry (Moscow) (v.72, #12)

Ab Ovo Usque Ad Mala by A. S. Spirin (1281-1283).

From birth to christening by A. S. Antonov (1284-1288).
A brief history of comparative studies of nucleic acids for systematic purposes is given. These studies were initiated by a group of Moscow State University scientists headed by A. N. Belozersky. Based mostly on comparative DNA studies, some main dogmas of a new branch of systematics were gradually developed. In Russia, this new branch of systematics is called “genosystematics”. Some of the main results obtained by genosystematics since its birth (1957) and up to its “christening” (1974) are described.
Keywords: genosystematics; phenosystematics; DNA; systematics; evolution of organisms

The pioneering data on base composition and pyrimidine sequences in DNA of pro-and eukaryotes are considered, and their significance for the origin of genosystematics is discussed. The modern views on specificity and functional role of enzymatic DNA methylation in eukaryotes are described. DNA methylation controls all genetic functions and is a mechanism of cellular differentiation and gene silencing. A model of regulation of DNA replication by methylation is suggested. Adenine DNA methylation in higher eukaryotes (higher plants) was first observed, and it was established that one and the same gene can be methylated at both cytosine and adenine moieties. Thus, there are at least two different and seemingly interdependent DNA methylation systems present in eukaryotic cells. The first eukaryotic adenine DNA-methyltransferase is isolated from wheat seedlings and described: the enzyme methylates DNA with formation of N6-methyladenine in the sequence TGATCA → TGm6ATCA. It is found that higher plants have endonucleases that are dependent on S-adenosyl-L-methionine (SAM) and sensitive to DNA methylation status. Therefore, as in bacteria, plants seem to have a restriction-modification (R-M) system. A system of conjugated up-and down-regulation of SAM-dependent endonucleases by SAM modulations is found in plants. Revelation of an essential role of DNA methylation in regulation of genetic processes is a fundament of materialization of epigenetics and epigenomics.
Keywords: genosystematics; DNA base composition; DNA-methylation; N6-methyladenine; 5-methylcytosine; cancer; replication; aging; evolution; plant endonucleases; epigenetics

Phylogenetic systematics of microorganisms inhabiting thermal environments by A. V. Lebedinsky; N. A. Chernyh; E. A. Bonch-Osmolovskaya (1299-1312).
Thermal habitats harbor specialized communities of thermophilic microorganisms, primarily prokaryotes. This review considers modern systematics of prokaryotes and the place of thermophilic archaea and bacteria in it. Among the existing hierarchical classifications of prokaryotes, the bulk of attention is given to the one accepted in the current second edition of “Bergey’s Manual of Systematic Bacteriology”, which is primarily based on 16S rRNA phylogeny and phenotypic properties of the organisms. Analysis of the genomics data shows that they on the whole agree with the 16S rRNA-based system, although revealing the significance of the evolutionary role of lateral transfer, duplication, and loss of genes. According to the classification elaborated in the current edition of “Bergey’s Manual”, the prokaryotes currently culturable under laboratory conditions are distributed among 26 phyla, two of which belong to the domain Archaea and 24 to the domain Bacteria. Six phyla contain exclusively thermophiles, and eleven phyla contain thermophiles along with mesophiles, thermophiles being usually separated phylogenetically and representing high-level taxa (classes, orders). In light of the data on the topology of the 16S rRNA-based phylogenetic tree and some other data, this review discusses the probable hyperthermophilic nature of the universal common ancestor.
Keywords: genosystematics; phylogenetic systematics; hierarchical classification; prokaryotes; archaea; bacteria; thermophiles

Do we need many genes for phylogenetic inference? by V. V. Aleshin; A. V. Konstantinova; K. V. Mikhailov; M. A. Nikitin; N. B. Petrov (1313-1323).
Fifty-six nuclear protein coding genes from Taxonomically Broad EST Database and other databases were selected for phylogenomic-based examination of alternative phylogenetic hypotheses concerning intergroup relationship between multicellular animals (Metazoa) and other representatives of Opisthokonta. The results of this work support sister group relationship between Metazoa and Choanoflagellata. Both of these groups form the taxon Holozoa along with the monophyletic Ichthyosporea or Mesomycetozoea (a group that includes Amoebidium parasiticum, Sphaeroforma arctica, and Capsaspora owczarzaki). These phylogenetic hypotheses receive high statistical support both when utilizing whole alignment and when only 5000 randomly selected alignment positions are used. The presented results suggest subdivision of Fungi into Eumycota and lower fungi, Chytridiomycota. The latter form a monophyletic group that comprises Chytridiales + Spizellomycetales + Blastocladiales (Batrachochytrium, Spizellomyces, Allomyces, Blastocladiella), contrary to the earlier reports based on the analysis of 18S rRNA and a limited set of protein coding genes. The phylogenetic distribution of genes coding for a ubiquitin-fused ribosomal protein S30 implies at least three independent cases of gene fusion: in the ancestors of Holozoa, in heterotrophic Heterokonta (Oomycetes and Blastocystis), and in the ancestors of Cryptophyta and Glaucophyta. Ubiquitin-like sequences fused with ribosomal protein S30 outside of Holozoa are not FUBI orthologs. Two independent events of FUBI replacement by the ubiquitin sequence were detected in the lineage of C. owczarzaki and in the monophyletic group of nematode worms Tylenchomorpha + Cephalobidae. Bursaphelenchus xylophilus (Aphelenchoidoidea) retains a state typical of the rest of the Metazoa. The data emphasize the fact that the reliability of phylogenetic reconstructions depends on the number of analyzed genes to a lesser extent than on our ability to recognize reconstruction artifacts.
Keywords: molecular evolution; homoplasy; origin of multicellular organisms; secondary structure of SSU rRNA; Tylenchida

Phylogeny of flowering plants by the chloroplast genome sequences: in search of a “lucky gene” by M. D. Logacheva; A. A. Penin; T. H. Samigullin; C. M. Vallejo-Roman; A. S. Antonov (1324-1330).
One of the most complicated remaining problems of molecular-phylogenetic analysis is choosing an appropriate genome region. In an ideal case, such a region should have two specific properties: (i) results of analysis using this region should be similar to the results of multigene analysis using the maximal number of regions; (ii) this region should be arranged compactly and be significantly shorter than the multigene set. The second condition is necessary to facilitate sequencing and extension of taxons under analysis, the number of which is also crucial for molecular phylogenetic analysis. Such regions have been revealed for some groups of animals and have been designated as “lucky genes”. We have carried out a computational experiment on analysis of 41 complete chloroplast genomes of flowering plants aimed at searching for a “lucky gene” for reconstruction of their phylogeny. It is shown that the phylogenetic tree inferred from a combination of translated nucleotide sequences of genes encoding subunits of plastid RNA polymerase is closest to the tree constructed using all protein coding sites of the chloroplast genome. The only node for which a contradiction is observed is unstable according to the different type analyses. For all the other genes or their combinations, the coincidence is significantly worse. The RNA polymerase genes are compactly arranged in the genome and are fourfold shorter than the total length of protein coding genes used for phylogenetic analysis. The combination of all necessary features makes this group of genes main candidates for the role of “lucky gene” in studying phylogeny of flowering plants.
Keywords: molecular phylogeny; angiosperms; gene sampling; chloroplast genome; plastid RNA polymerase

Genetic structure of the Salvelinus genus chars from reservoirs of the Kuril Islands by E. A. Shubina; E. V. Ponomareva; O. F. Gritsenko (1331-1348).
Genetic resemblance of chars Salvelinus alpinus krasheninnikovi (Salvelinus malma krasheninnikovi) of 35 samples collected in five Kuril Islands—Shumshu, Paramushir, Onekotan, Iturup, and Kunashir—has been studied by the PCR-RAPD method. In the limits of each island, both resident isolates and anadromous forms give strictly supported clusters distinct from samples from the other islands. The samples from five islands form three superclusters: the first from Kunashir and Iturup Islands, the second from Paramushir and Onekotan Islands, and the third from Shumshu Island. The possible reasons for genetic similarity of resident and anadromous forms of Dolly Varden chars inhabiting reservoirs of a definite island are considered (the founder effect, homing, limited migration).
Keywords: PCR-RAPD; Dolly Varden chars; Kuril Islands; cluster analysis

To solve some systematic questions as well as to study genetic variability and evolutionary relationships in two groups of fish belonging to the Mugilid (Mugilidae) and Cyprinid (Cyprinidae) families, we have used restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) fragments amplified in polymerase chain reaction. The analysis of three mtDNA fragments of 7220 bp total length of six Mugilid species has shown that Mediterranean Liza aurata, L. ramada, L. saliens, and Chelon labrosus form a common cluster, L. aurata and C. labrosus being the closest relatives, whereas L. haematocheilus (syn. C. haematocheilus) of the Sea of Japan forms a sister group to the Mediterranean cluster. It was found that Chelon and Liza genera are paraphyletic, and therefore their division into two genera is unnatural and they should be synonymized. According to priority, Liza species should be ascribed to Chelon genus. Mugil cephalus is the most distant compared to the rest of the species studied. The level of genetic divergence between allopatric samples of M. cephalus from the Sea of Japan and the Mediterranean Sea has proved to be very high—4.5% of nucleotide substitutions. The analysis of four mtDNA fragments of 9340 bp total length of six Cyprinid species has shown that L. waleckii is the most genetically distant. Pseudaspius leptocephalus is a sister group to Tribolodon species. All Tribolodon species form a common cluster with T. sachalinensis as a root. The remaining species form two branches, one of which includes T. nakamurai and T. brandtii, another one combines T. hakonensis and a new form of Tribolodon revealed that is close to T. hakonensis by its mtDNA (2.4% of nucleotide substitutions). This new form might be an independent species.
Keywords: Mugilidae; Cyprinidae; Tribolodon ; PCR-RFLP; mtDNA; phylogeny

Molecular systematics of Lachancea yeasts by E. S. Naumova; E. V. Serpova; G. I. Naumov (1356-1362).
This work presents the results of molecular-genetic investigation of a new yeast genus, Lachancea Kurtzman (2003). Analysis of rRNA sequences and molecular karyotyping have shown genetic homogeneity of the genus Lachancea. Yeasts of this genus have an identical haploid number of chromosomes equal to eight, whereas limiting chromosome sizes significantly differ in various species. The largest range of chromosome bands was registered in L. cidri strains (400–2800 kb), while the smallest was found in L. waltii (1400–2800 kb). The intra- and interspecies polymorphism of Lachancea chromosomes is discussed.
Keywords: Lachancea yeasts; 26S rRNA; 5.8S-ITS; chromosomal DNA; molecular karyotyping; phylogenetic analysis

The presence in proteins of amino acid residues that change in concert during evolution is associated with keeping constant the protein spatial structure and functions. As in the case with morphological features, correlated substitutions may become the cause of homoplasies—the independent evolution of identical non-homological adaptations. Our data obtained on model phylogenetic trees and corresponding sets of sequences have shown that the presence of correlated substitutions distorts the results of phylogenetic reconstructions. A method for accounting for co-evolving amino acid residues in phylogenetic analysis is proposed. According to this method, only a single site from the group of correlated amino acid positions should remain, whereas other positions should not be used in further phylogenetic analysis. Simulations performed have shown that replacement on the average of 8% of variable positions in a pair of model sequences by coordinately evolving amino acid residues is able to change the tree topology. The removal of such amino acid residues from sequences before phylogenetic analysis restores the correct topology.
Keywords: phylogenetic analysis; correlated substitutions of amino acid residues; homoplasies

Contribution of genosystematics to current concepts of phylogeny and classification of bryophytes by A. V. Troitsky; M. S. Ignatov; V. K. Bobrova; I. A. Milyutina (1368-1376).
This paper is a survey of the current state of molecular studies on bryophyte phylogeny. Molecular data have greatly contributed to developing a phylogeny and classification of bryophytes. The previous traditional systems of classification based on morphological data are being significantly revised. New data of the authors are presented on phylogeny of Hypnales pleurocarpous mosses inferred from nucleotide sequence data of the nuclear DNA internal transcribed spacers ITS1-2 and the trnL-F region of the chloroplast genome.
Keywords: molecular phylogenetics; genosystematics; hepatics; liverworts; hornworts; pleurocarpous ITS1-2; trnL-F

Modern approaches in DNA-based species identification are considered. Long used methods of species identification in procaryotes (G + C ratio, 16S rRNA nucleotide sequence, DNA-DNA hybridization) have recently been supplemented by the method of multilocus sequence analysis based on comparison of nucleotide sequences of fragments of several genes. Species identification in eukaryotes also employs one or two standard short fragments of the genome (known as DNA-barcodes). Potential benefits of new approaches and some difficulties during their practical realization are discussed.
Keywords: DNA species-specificity; species identification; DNA barcoding; prokaryotes; eukaryotes

Antioxidants specifically addressed to mitochondria have been studied for their ability to decelerate aging of organisms. For this purpose, a project has been established with participation of several research groups from Belozersky Institute of Physico-Chemical Biology and some other Russian research institutes as well as two groups from the USA and Sweden, with support by the “Mitotechnology” company founded by “RAInKo” company (O. V. Deripaska and Moscow State University). This paper summarizes the first results of the project and estimates its prospects. Within the framework of the project, antioxidants of a new type (SkQ) were synthesized comprising plastoquinone (an antioxidant moiety), a penetrating cation, and decane or pentane linker. Using planar bilayer phospholipid membranes, we selected SkQ derivatives with the highest penetrating ability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decylrhodamine 19 (SkQR1), and methylplastoquinonyl-decyl-triphenylphosphonium (SkQ3). Anti-and prooxidant properties of these substances and also of ubiquinone and ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested on isolated mitochondria. Micromolar concentrations of cationic quinones are found to be very strong prooxidants, but in lower (submicromolar) concentrations they display antioxidant activity. The antioxidant activity decreases in the series SkQ1 = SkQR1 > SkQ3 > MitoQ, so the window between the anti-and prooxidant effects is smallest for MitoQ. SkQ1 is rapidly reduced by complexes I and II of the mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Extremely low concentrations of SkQ1 and SkQR1 completely arrest the H2O2-induced apoptosis in human fibroblasts and HeLa cells (for SkQ1 C 1/2 = 1·10−9 M) Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In mice, SkQ1 decelerates the development of three types of accelerated aging (progeria) and also of normal aging, and this effect is especially demonstrative at early stages of aging. The same pattern is shown in invertebrates (drosophila and daphnia). In mammals, the effect of SkQs on aging is accompanied by inhibition of development of such age-related diseases as osteoporosis, involution of thymus, cataract, retinopathy, etc. SkQ1 manifests a strong therapeutic action on some already developed retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM skQ1, vision is recovered in 50 of 66 animals who became blind because of retinopathy. SkQ1-containing drops instilled in the early stage of the disease prevent the loss of sight in rabbits with experimental uveitis and restore vision to animals that had already become blind. A favorable effect is also achieved in experimental glaucoma in rabbits. Moreover, the pretreatment of rats with 0.2 nmol SkQ1 per kg body weight significantly decreases the H2O2-induced arrhythmia of the isolated heart. SkQ1 strongly reduces the damaged area in myocardial infarction or stroke and prevents the death of animals from kidney infarction. In p53−/− mice, SkQ1 decreases the ROS level in the spleen cells and inhibits appearance of lymphomas which are the main cause of death of such animals. Thus, it seems reasonable to perform clinical testing of SkQ preparations as promising drugs for treatment of age-related and some other severe diseases of human and animals.
Keywords: aging; mitochondria; reactive oxygen species; SkQs; antioxidants