Biochemistry (Moscow) (v.80, #11)

Mechanisms of apoptosis by M. A. Savitskaya; G. E. Onishchenko (1393-1405).
Nearly 15 types of programmed cell death (PCD) have been identified to date. Among them, apoptosis is the most common and well-studied type of PCD. In this review, we discuss different apoptotic pathways in which plasma membrane and membrane organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, and nucleus play the pivotal role. Data concerning caspase cascades involved in these mechanisms are described. Various apoptosis induction mechanisms are analyzed and compared. The close relations between them and the possibility of switching from one pathway to another are demonstrated. In most cases, the result of these pathways is mitochondrial membrane permeabilization and/or caspase activation. These two events are closely linked and serve as the central point of integration of the apoptotic cell death pathways.
Keywords: apoptosis; necrosis; caspase pathways; membrane organelles

Aging epigenetics: Accumulation of errors or realization of a specific program? by V. V. Ashapkin; L. I. Kutueva; B. F. Vanyushin (1406-1417).
Aging in mammals is known to be accompanied by a progressive loss of methylated cytosines from DNA. This loss is tissue-specific to a certain extent and affects mainly repeated sequences, transposable elements, and intergenic genome parts. Age-dependent DNA hypomethylation is correlated with and perhaps partly caused by a diminished activity of DNA methyltransferases. Along with the global DNA demethylation during aging, hypermethylation of certain genes occurs. On the whole-genome scale, an age-dependent hypermethylation is typical for genes associated with promoter CG islands, whereas hypomethylation mostly affects CG-poor genes, besides the repeated sequences, transposable elements, and intergenic genome parts mentioned above. The methylation levels of certain CG sites display strict correlation to age and thus could be used as a molecular marker to predict biological age of cells, tissues, and organisms. Epigenetic cell reprogramming, such as induced pluripotent stem cell production, leads to complete resetting of their epigenetic age.
Keywords: epigenetics; aging; DNA methylation; epigenetic age; epigenetic reprogramming

Mitochondrial matrix processes by I. O. Mazunin; S. A. Levitskii; M. V. Patrushev; P. A. Kamenski (1418-1428).
Mitochondria possess their own genome that, despite its small size, is critically important for their functioning, as it encodes several dozens of RNAs and proteins. All biochemical processes typical for bacterial and nuclear DNA are described in mitochondrial matrix: replication, repair, recombination, and transcription. Commonly, their mechanisms are similar to those found in bacteria, but they are characterized by several unique features. In this review, we provide an overall description of mitochondrial matrix processes paying special attention to the typical features of such mechanisms.
Keywords: mitochondria; mtDNA; nucleoid; transcription; replication; repair; recombination

Small noncoding 6S RNAs of bacteria by O. Y. Burenina; D. A. Elkina; R. K. Hartmann; T. S. Oretskaya; E. A. Kubareva (1429-1446).
Small noncoding RNAs (ncRNAs) are non-translated transcripts with lengths below 300 nucleotide residues. Regulation of cellular processes under the influence of these ncRNAs is the most various in eukaryotic cells, but numerous ncRNAs are also found in bacteria. One of the best-known small prokaryotic ncRNAs is 6S RNA–it has been detected in all branches of bacteria. Due to their conserved secondary structure including a large central “loop” flanked by long double-helical arms, 6S RNAs can bind holoenzymes of RNA polymerase (RNAP) and inhibit their activity. This inhibits transcription of many genes. According to data of comparative transcriptome analysis, the 6S RNA-dependent regulation of transcription affects the expression level of hundreds of genes involved in various cellular processes. 6S RNA has the unique feature of serving as a transcription template for the synthesis of short product RNAs (pRNAs) complementary to the central part of the molecule. The length and abundance of pRNAs vary depending on the physiological status of the cell. The synthesis of pRNAs is of great importance because it releases RNAP and provides reversibility of the inhibition. A similar mechanism has been described for the noncoding mouse B2 RNA that inhibits the activity of RNAP II. This finding can be taken as evidence for the common evolutionary origin of the ncRNA-dependent regulation of RNAP and its immense significance for cells. This review summarizes the state of knowledge about the main features and functions of 6S RNAs from various bacterial species with a special focus on the peculiarities of pRNA synthesis. The majority of functional insights on 6S RNAs have been gained for E. coli 6S RNA as the best-studied model system.
Keywords: noncoding RNAs; transcription regulation; RNA polymerase; 6S RNA

Regulation of flagellar gene expression in Bacteria by I. A. Osterman; Yu. Yu. Dikhtyar; A. A. Bogdanov; O. A. Dontsova; P. V. Sergiev (1447-1456).
The flagellum of a bacterium is a supramolecular structure of extreme complexity comprising simultaneously both a unique system of protein transport and a molecular machine that enables the bacterial cell movement. The cascade of expression of genes encoding flagellar components is closely coordinated with the steps of molecular machine assembly, constituting an amazing regulatory system. Data on structure, assembly, and regulation of flagellar gene expression are summarized in this review. The regulatory mechanisms and correlation of the process of regulation of gene expression and flagellum assembly known from the literature are described.
Keywords: flagella; motility; bacteria; molecular machine; transcriptional control

Mitochondrial fission and fusion by M. V. Patrushev; I. O. Mazunin; E. N. Vinogradova; P. A. Kamenski (1457-1464).
Mitochondria are key cellular organelles responsible for many different functions. The molecular biology of mitochondria is continuously subject to comprehensive studies. However, detailed mechanisms of mitochondrial biogenesis are still unclear. Fusion and fission are among the most enigmatic processes connected with mitochondria. On the other hand, it has been shown that these events are of great biological importance for functioning of living cells. In this review, we summarize existing molecular data on mitochondrial dynamics and discuss possible biological functions of fusion and fission of these organelles.
Keywords: mitochondria; fusion; fission

StructAlign, a program for alignment of structures of DNA–protein complexes by Ya. V. Popov; A. A. Galitsyna; A. V. Alexeevski; A. S. Karyagina; S. A. Spirin (1465-1468).
Comparative analysis of structures of complexes of homologous proteins with DNA is important in the analysis of DNA–protein recognition. Alignment is a necessary stage of the analysis. An alignment is a matching of amino acid residues and nucleotides of one complex to residues and nucleotides of the other. Currently, there are no programs available for aligning structures of DNA–protein complexes. We present the program StructAlign, which should fill this gap. The program inputs a pair of complexes of DNA double helix with proteins and outputs an alignment of DNA chains corresponding to the best spatial fit of the protein chains.
Keywords: structural bioinformatics; DNA–protein complexes; alignment; web interface

Stages of cell cannibalism–entosis–in normal human keratinocyte culture by A. S. Garanina; L. A. Khashba; G. E. Onishchenko (1469-1477).
Entosis is a type of cell cannibalism during which one cell penetrates into another cell and usually dies inside it. Researchers mainly pay attention to initial and final stages of entosis. Besides, tumor cells in suspension are the primary object of studies. In the present study, we investigated morphological changes of both cells-participants of entosis during this process. The substrate-dependent culture of human normal keratinocytes HaCaT was chosen for the work. A combination of light microscopy and scanning electron microscopy was used to prove that one cell was completely surrounded by the plasma membrane of another cell. We investigated such “cell-in-cell” structures and described the structural and functional changes of both cells during entosis. The outer cell nucleus localization and shape were changed. Gradual degradation of the inner cell nucleus and of the junctions between the inner and the outer cells was revealed. Moreover, repeated redistribution of the outer cell membrane organelles (Golgi apparatus, lysosomes, mitochondria, and autophagosomes), rearrangement of its cytoskeleton, and change in the lysosomal, autophagosomal, and mitochondrial state in both entotic cells were observed during entosis. On the basis of these data, we divided entosis into five stages that make it possible to systematize description of this type of cell death.
Keywords: entosis; stages of entosis; cell cannibalism; cell organelles

A rapid and cost-effective method for DNA extraction from archival herbarium specimens by A. A. Krinitsina; T. V. Sizova; M. A. Zaika; A. S. Speranskaya; A. P. Sukhorukov (1478-1484).
Here we report a rapid and cost-effective method for the extraction of total DNA from herbarium specimens up to 50-90-year-old. The method takes about 2 h, uses AMPure XP magnetic beads diluted by PEG-8000-containing buffer, and does not require use of traditional volatile components like chloroform, phenol, and liquid nitrogen. It yields up to 4 μg of total nucleic acid with high purity from about 30 mg of dry material. The quality of the extracted DNA was tested by PCR amplification of 5S rRNA and rbcL genes (nuclear and chloroplast DNA markers) and compared against the traditional chloroform/isoamyl alcohol method. Our results demonstrate that the use of the magnetic beads is crucial for extraction of DNA suitable for subsequent PCR from herbarium samples due to the decreasing inhibitor concentrations, reducing short fragments of degraded DNA, and increasing median DNA fragment sizes.
Keywords: DNA extraction; herbarium; PCR; 5S rRNA ; rbcL ; genomic markers; sequence

Structure of intergenic spacer IGS1 of ribosomal operon from Schistidium mosses by I. A. Milyutina; E. A. Ignatova; M. S. Ignatov; D. V. Goryunov; A. V. Troitsky (1485-1491).
The structure of the intergenic spacer 1 (IGS1) of the ribosomal operon from 12 species of Schistidium mosses was studied. In the IGS1 sequences of these species, three conserved regions and two areas of GCand A-enriched repeats were identified. All of the studied mosses have a conserved pyrimidine-enriched motif at the 5′-end of IGS1. Species-specific nucleotide substitutions and insertions were found in the conserved areas. The repeated units contain single nucleotide substitutions that make unique the majority of repeated units. The positions of such repeats in IGS1 are species-specific, but their number can vary within the species and among operons of the same specimen. The comparison of IGS1 sequences from the Schistidium species and from representatives of ten other moss genera revealed the presence of common conserved motifs with similar localization. Presumably, these motifs are elements of termination of the pre-rRNA transcription and processing of rRNA.
Keywords: rDNA; IGS; intergenic spacer; conserved motifs; mosses

Primary structure of 28S rRNA gene confirms monophyly of free-living heterotrophic and phototrophic apicomplexans (Alveolata) by K. V. Mikhailov; D. V. Tikhonenkov; J. Janouškovec; A. Y. Diakin; M. V. Ofitserov; A. P. Mylnikov; V. V. Aleshin (1492-1499).
Phylogenetic analysis of large subunit ribosomal RNA (LSU rRNA or 28S rRNA) gene sequences from free-living predatory flagellates Colpodella angusta, Voromonas pontica, and Alphamonas edax (Apicomplexa) confirms their close relationship with chromerids Chromera velia and Vitrella brassicaformis, which possess a functional photosynthetic plastid. Together these organisms form a sister group to parasitic apicomplexans (coccidians and gregarines, or sporozoans sensu lato). This result agrees with the previous conclusion on monophyly of colpodellids and chromerids (chrompodellids) based on phylogenomic data. The revealed relationships demonstrate a complex pattern of acquisition, loss, or modification of plastids and transition to parasitism during alveolate evolution.
Keywords: molecular phylogenetics; ribosomal RNA genes; apicomplexans; Sporozoa; heterotrophic flagellates; plastids; evolution of parasitism

Modeling interactions of erythromycin derivatives with ribosomes by A. V. Shishkina; T. M. Makarova; A. G. Tereshchenkov; G. I. Makarov; G. A. Korshunova; A. A. Bogdanov (1500-1507).
Using a method of static simulation, a series of erythromycin A analogs was designed with aldehyde functions introduced instead of one of the methyl substituents in the 3′-N-position of the antibiotic that was potentially capable of forming a covalent bond with an amino group of one of the nucleotide residues of the 23S rRNA in the ribosomal exit tunnel. Similar interaction is observed for antibiotics of the tylosin series, which bind tightly to the large ribosomal subunit and demonstrate high antibacterial activity. Binding of novel erythromycin derivatives with the bacterial ribosome was investigated with the method of fluorescence polarization. It was found that the erythromycin analog containing a 1-methyl-3oxopropyl group in the 3′-N-position demonstrates the best binding. Based on the ability to inhibit protein biosynthesis, it is on the same level as erythromycin, and it is significantly better than desmethyl-erythromycin. Molecular dynamic modeling of complexes of the derivatives with ribosomes was conducted to explain the observed effects.
Keywords: ribosomal tunnel; macrolides; erythromycin; modeling

Induction of secondary carotenogenesis in new halophile microalgae from the genus Dunaliella (Chlorophyceae) by A. E. Solovchenko; E. A. Selivanova; K. A. Chekanov; R. A. Sidorov; N. V. Nemtseva; E. S. Lobakova (1508-1513).
We report on the effects of high light irradiance (480 μmol quanta/(m2·s)) and salinity (160 and 200 g/liter NaCl) on culture growth as well as on cell lipid pigment and fatty acid (FA) composition in three novel strains of halophile microalga from the genus Dunaliella. Based on the ITS1–5.8S rRNA–ITS2 sequence and on the capability of accumulation of secondary (uncoupled from the photosynthetic apparatus) β-carotene, the strains Dunaliella sp. BS1 and BS2 were identified as D. salina and Dunaliella sp. R5 as D. viridis. Under conditions optimal for growth, chlorophylls and primary carotenoids (mainly lutein) dominated the pigment profile of all investigated strains. The main FA were represented by unsaturated C18 FA typical of thylakoid membrane structural lipids. In all studied cells, stressors caused a decline in chlorophylls and an increase in unsaturated C16 and C18 FA associated with reserve lipids. The carotenogenic species D. salina demonstrated 10-fold increase in carotenoids accompanied by a decline in lutein and a drastic increase in β-carotene (up to 75% of total carotenoids). In D. viridis, only 1.5-fold increase in carotenoid content took place, the ratio of major carotenoids remaining essentially unchanged. The role of the carotenogenic response in mechanisms of protection against photooxidative damage is discussed in view of halophile microalgae stress tolerance and application of the new Dunaliella strains for biotechnological production of β-carotene.
Keywords: carotenoids; chlorophylls; fatty acids; molecular identification; photoacclimation; salt tolerance; stress

Metagenomic analyses of white sea picoalgae: First data by T. A. Belevich; L. V. Ilyash; I. A. Milyutina; M. D. Logacheva; D. V. Goryunov; A. V. Troitsky (1514-1521).
Picoalgae (defined as cells smaller than 2-3 μm) include members of diverse taxonomic groups. They are an important constituent of marine plankton and ice biota and play a significant ecological role in biogeochemical cycles. Despite their importance, the true extent of their diversity has only recently been uncovered by molecular surveys. The diversity of picoeukaryotes has not yet been studied in the White Sea, which is a unique marine environment combining features of temperate and Arctic seas. Here, we investigated the taxonomic composition of eukaryotic picoalgae in ice and under-ice water at a station located in the Kandalaksha Bay of the White Sea. We applied metagenomic survey using Illumina sequencing. Eight main algae phyla, namely, Chlorophyta, Katablepharidophyta, Haptophyta, Dinophyta, Cercozoa, Bacillariophyta, Cryptophyta, and Ochrophyta were identified. The genera Paraphysomonas and Micromonas and the order Pedinellales were most numerous in plankton; the genera Paraphysomonas, Micromonas, and Metopion were most abundant in ice. The number of “rare” phylotypes was 80 in under-ice water and 112 in ice. Some taxa of nanoand microalgae are identified for the first time in the White Sea phytoplankton. Our data provide a basis for further research of tiny phototrophs in the Russian Arctic.
Keywords: seasonal ice; under-ice plankton; picoalgae; metagenomic survey; White Sea

Moss phylogeny reconstruction using nucleotide pangenome of complete Mitogenome sequences by D. V. Goryunov; B. E. Nagaev; M. Yu. Nikolaev; A. V. Alexeevski; A. V. Troitsky (1522-1527).
Stability of composition and sequence of genes was shown earlier in 13 mitochondrial genomes of mosses (Rensing, S. A., et al. (2008) Science, 319, 64-69). It is of interest to study the evolution of mitochondrial genomes not only at the gene level, but also on the level of nucleotide sequences. To do this, we have constructed a “nucleotide pangenome” for mitochondrial genomes of 24 moss species. The nucleotide pangenome is a set of aligned nucleotide sequences of orthologous genome fragments covering the totality of all genomes. The nucleotide pangenome was constructed using specially developed new software, NPG-explorer (NPGe). The stable part of the mitochondrial genome (232 stable blocks) is shown to be, on average, 45% of its length. In the joint alignment of stable blocks, 82% of positions are conserved. The phylogenetic tree constructed with the NPGe program is in good correlation with other phylogenetic reconstructions. With the NPGe program, 30 blocks have been identified with repeats no shorter than 50 bp. The maximal length of a block with repeats is 140 bp. Duplications in the mitochondrial genomes of mosses are rare. On average, the genome contains about 500 bp in large duplications. The total length of insertions and deletions was determined in each genome. The losses and gains of DNA regions are rather active in mitochondrial genomes of mosses, and such rearrangements presumably can be used as additional markers in the reconstruction of phylogeny.
Keywords: mosses; phylogeny; pangenome; mitochondrial genome