Laboratory of mechanisms of gene expression
The Laboratory of Mechanisms of Gene Expression was organized in May of 2007 on the basis of an independent research group under the same name existed since December 2001.
Since the very beginning the main focus of our research is studies of basic mechanisms of transcription in eukaryotes. The functional complementations in vivo of certain subunits of nuclear RNA polymerases I—III among evolutionary remote species (yeast, drosophila, man) has been first demonstrated (Gene, 1994, 147: 63–69; Mol. Cell. Biol., 1995, 15: 4702–4710) and functional relatedness of small subunits of RNA polymerases in Archaea and Eucarya was established (Russ. J. Bioorg. Chem., 1997, 23: 100–106;J. Biol. Chem., 1999, 274: 8421–8427).
The first comprehensive studies of exchangeability in vivo (in the cells of yeast Saccharomyces cerevisiae) of the subunits of nuclear RNA polymerases I, II, and III between evolutionary distant species (Schizosaccharomyces pombe and Homo sapiens) have been performed, whereby many of the components of the basal transcription apparatus of the fission yeasts were cloned and thoroughly characterized (J. Mol. Biol., 2000, 295: 1119—1127; Mol. Biol. (Mosk), 2002, 36: 3—26;Nucleic Acids Res., 2006, 34: 3615—3624). As a part of the the International Project on total sequencing of the Sch. pombe genome in three loci of its chromosome I the primary structure gaps were closed and the nucleotide sequence continuity was restored with clones pYUK71, pYUL23 and pYUG7 isolated in our laboratory (Nature, 2002, 415: 871—880; see also S. pombe genetic map). In the collaborative work with French scientists from ESBS (Strasbourg) was found that one of the subunits of human RNA polymerase II is encoded by a multigene family which expression give rise to at least three protein isoforms (BMC Mol. Biol., 2001, 2:14). Four independent genes encoding various variants of this hRPB11 subunit were revealed (Russ. J. Bioorg. Chem., 2004, 30: 561—565). Coevolution of Homo sapiens POLR2J and PMS2 genes, both located on chromosome 7 and encoding two essential components of such important molecular processes of the living cell as transcription and DNA mismatch repair, was shown: both these gene families undergone multiple rounds of duplication events started in the hominoid ancestor less than 20 million years ago (Dokl. Biochem. Biophys., 2006, 408: 175—179). Moreover, according to data obtained the evolution of these two gene families includes stages specific for humans (Russ. J. Genet., 2010, 46: 1112-1114; Biochemistry, 2011, 76: 976-980; Cell & Tissue Biology, 2013, 7: 314-319).
Mapping on the human chromosome 7 several genes that are important for primate evolution: four POLR2J paralogues (J1–J4) and sixteen PMS2 paralogues (PMS2, ψ0, ψ1–ψ14). Different stages (1, 2 and 3) of POLR2J amplification are shown; two of them are specific for Homo sapiens (marked with pink circles). Mya — million years ago.
|Georgij Shpakovski, D.Sc||depart. firstname.lastname@example.org, |
|Valerij Klykov, Ph.D.||s. r. email@example.com|
|Elena Shematorova, Ph.D.||s. r. firstname.lastname@example.org, |
|Dmitry Shpakovski||j. r. email@example.com, |
|Ivan Slovokhotov||j. r. firstname.lastname@example.org, |
|Galina Proshkina, Ph.D.||s. r. f.|
|Andrey Aralov, Ph.D.||r. f.||Baruh238@mail.ru|
|Sergej Proshkin||j. r. f.|
(2018). i-Clamp phenoxazine for the fine tuning of DNA i-motif stability. Nucleic Acids Res. 46 (6), 2751–2764 [+]
Non-canonical DNA structures are widely used for regulation of gene expression, in DNA nanotechnology and for the development of new DNA-based sensors. I-motifs (iMs) are two intercalated parallel duplexes that are held together by hemiprotonated C-C base pairs. Previously, iMs were used as an accurate sensor for intracellular pH measurements. However, iM stability is moderate, which in turn limits its in vivo applications. Here, we report the rational design of a new substituted phenoxazine 2'-deoxynucleotide (i-clamp) for iM stabilization. This residue contains a C8-aminopropyl tether that interacts with the phosphate group within the neighboring chain without compromising base pairing. We studied the influence of i-clamp on pH-dependent stability for intra- and intermolecular iM structures and found the optimal positions for modification. Two i-clamps on opposite strands provide thermal stabilization up to 10-11°C at a pH of 5.8. Thus, we developed a new modification that shows significant iM-stabilizing effect both at strongly and mildly acidic pH and increases iM transition pH values. i-Clamp can be used for tuning iM-based pH probes or assembling extra stable iM structures for various applications.ID:2074
(2017). A key enzyme of animal steroidogenesis can function in plants enhancing their immunity and accelerating the processes of growth and development. BMC Plant Biol. 17 (Suppl 1), 189 [+]
The initial stage of the biosynthesis of steroid hormones in animals occurs in the mitochondria of steroidogenic tissues, where cytochrome P450SCC (CYP11A1) encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone - the general precursor of all the steroid hormones, starting with progesterone. This stage is missing in plants where mitochondrial cytochromes P450 (the mito CYP clan) have not been found. Generating transgenic plants with a mitochondrial type P450 from animals would offer an interesting option to verify whether plant mitochondria could serve as another site of P450 monooxygenase reaction for the steroid hormones biosynthesis.ID:1982
(2017). Different roles of glutathione in copper and zinc chelation in Brassica napus roots. Plant Physiol. Biochem. 118, 333–341 [+]
We investigated the specific features of copper and zinc excess action on the roots of canola (Brassica napus L.) plants. Copper rapidly accumulated in canola root cells and reached saturation during several hours of treatment, whereas the root zinc content increased relatively slowly. Excessive copper and zinc entry inside the cell resulted in significant cell damage, as evidenced by alterations in plasmalemma permeability and decreases in cellular enzymatic activity. Zinc excess specifically damaged root hair cells, which correlated with a pronounced elevation of their labile zinc level. In vitro, we showed that reduced glutathione (GSH) readily reacted with copper ions to form complexes with blocked sulfhydryl groups. In contrast, zinc ions were ineffective as glutathione blockers, and glutathione molecules did not lose their specific chemical activity in the presence of Zn2+ ions. The effect of copper and zinc excess on the glutathione pool in canola root cells was analysed by a combination of biochemical determination of total and oxidized glutathione contents and fluorescent staining of free reduced glutathione with monochlorobimane dye. Excess copper led to dose-dependent diminution of free reduced glutathione contents in the root cells, which could not be explained by the loss of total cellular glutathione or its oxidation. In contrast, we observed little effect of much higher intracellular zinc concentrations on the free reduced glutathione content. We concluded that GSH plays an important role in copper excess, but not zinc excess chelation, in canola root cells.ID:1981
(2017). Synthesis of oligonucleotides containing novel G-clamp analogue with C8-tethered group in phenoxazine ring: Implication to qPCR detection of the low-copy Kemerovo virus dsRNA. Bioorg. Med. Chem. 25 (14), 3597–3605 [+]
Nowadays modified oligonucleotides are widely used in diagnostics and as novel therapeutics. Introduction of modified or unnatural residues into oligonucleotides allows fine tuning of their binding properties to complementary nucleic acids and leads to improved stability both in vitro and in vivo. Previously it was demonstrated that insertion of phenoxazine nucleotides with various groups in C9-position into oligonucleotides leads to a significant increase of duplex stability with complementary DNA and RNA. Here the synthesis of a novel G-clamp nucleoside analogue (G(8AE)-clamp) bearing 2-aminoethyl tether at C8-atom is presented. Introduction of such modified residues into oligonucleotides lead to enhanced specificity of duplex formation towards complementary DNA and RNA targets with increased thermal and 3'-exonuclease stability. According to CD-spectroscopy studies G(8AE)-clamp does not substantially disrupt helix geometry. Primers containing G(8AE)-clamp demonstrated superior sensitivity in qPCR detection of dsRNA of Kemerovo virus in comparison to native oligonucleotides.ID:1819
(2014). Mitochondria as a Possible Place for Initial Stages of Steroid Biosynthesis in Plants. Journal of Stress Physiology & Biochemistry 10 (4), 85–97 [+]
With the aim of thorough comparison of steroidogenic systems of plants and animals, transgenic plants of Solanaceae family expressing CYP11A1 cDNA encoding cytochrome P450SCC of mammalian mitochondria were further analysed. Positive effect of CYP11A1 on resistance of the transgenic tobacco plants to the infection by fungal phytopathogene Botrytis cinerea was for the first time detected. Subtle changes in mitochondria of the transgenic Nicotiana tabacum plants expressing mammalian CYP11A1 cDNA were demonstrated by transmissive electron microscopy. The main components of the electron transfer chain of plant mitochondria were for the first time cloned and characterized. It was established that plants from the Solanacea family (tomato, tobacco and potato) contain two different genes with similar exon-intron structures (all contain 8 exons) encoding mitochondrial type ferredoxins (MFDX), and one gene for mitochondrial ferredoxin reductase (MFDXR). The results obtained point out on profound relatedness of electron transfer chains of P450-dependent monooxygenases in mammalian and plant mitochondria and support our previous findings about functional compatability of steroidogenic systems of Plantae and Animalia.ID:1493
(2013). Genetic engineering strategies for enhancing tomato resistance to fungal and bacterial pathogens. Russian Journal of Plant Physiology 60 (6), 721–732 [+]
The classification and detailed overview of the currently known effective strategies used to increase the resistance of tomato (Solanum lycopersicum L., syn. Lycopersicon esculentumMill.) plants to infectious fungal and bacterial diseases by genetic engineering approaches are presented. Modern data on the mechanisms of the protective effect of heterologous genes on the enhancement of transgenic tomato resistance to fungal and bacterial pathogens are discussed.
(2013). [Novel complexes of gene expression and their role in the appearance and evolution of the genus Homo]. Tsitologiia 55 (3), 172–7 [+]
Using genetic (yeast two-hybrid system) and biochemical (co-precipitation of proteins from cellular lysates) approaches, we have performed a whole-genome wide search for interacting partners of the previously described by us variants of hRPB11 subunit of human RNA polymerase II - hRPB1 1balpha, hRPB11calpha and hRPB1 1bbeta, hRPB 11cbeta - in fetal brain and Jurkat cell line libraries. In consequence, the main spectrum of the protein partners of these human specific isoforms of the RNA polymerase II subunit hRPB 11 (POLR2J) has been established. Functional characteristics of the uncovered protein partners of hRPB 11balpha and hRPB 11calpha isoforms clearly indicate that these isoforms, similarly to the main (major) subunit hRPB11a, are components of the distinct transcription complexes participating not only in the transcription of the specific DNA matrices, but involving also in the later stages of mRNA biogenesis. The RNA polymerase I-III common subunit hRPB6 (POLR2F) and basal component of the exon-exon junction complex Y14 (RBM8A) have been found among the protein partners of the isoforms hRPB 11bbeta and hRPB 11cbeta together with a number of proteins involved in the biogenesis of microRNAs, including a novel, not previously described variant of the microRNA processing nuclease DROSHA, which indicates the existence of a special coordination between processes of transcription and RNA interference in the nuclei of human cells.ID:1492
(2011). A minor isoform of the human RNA polymerase II subunit hRPB11 (POLR2J) interacts with several components of the translation initiation factor eIF3. Biochemistry Mosc. 76 (8), 976–80 [+]
Using the yeast two-hybrid (YTH) system we have uncovered interaction of the hRPB11cα minor isoform of Homo sapiens RNA polymerase II hRPB11 (POLR2J) subunit with three different subunits of the human translation initiation factor eIF3 (hEIF3): eIF3a, eIF3i, and eIF3m. One variant of eIF3m identified in the study is the product of translation of alternatively spliced mRNA. We have named a novel isoform of this subunit eIF3mβ. By means of the YTH system we also have shown that the new eIF3mβ isoform interacts with the eIF3a subunit. Whereas previously described subunit eIF3mα (GA17) has clear cytoplasmic localization, the novel eIF3mβ isoform is detected predominantly in the cell nucleus. The discovered interactions of the hRPB11cα isoform with several hEIF3 subunits demonstrate a new type coordination between transcription and the following (downstream) stages of gene expression (such as mRNA transport from nucleus to the active ribosomes in cytoplasm) in Homo sapiens and point out the possibility of existence of nuclear hEIF3 subcomplexes.ID:1486
(2010). [PSM2 and POLR2J gene families as molecular markers of the higher primate evolution]. Genetika 46 (9), 1254–7 [+]
We have studied the molecular evolution of two gene families specific for primates: POLR2J of the transcription system and PMS2 of the MMR repair system. The appearance and improvement of the genetic structure in each of the families was shown to strongly correlate with the main stages of the higher primates biological evolution. Our results indicate that the PSM2 and POLR2J genes can serve as helpful and reliable molecular markers of anthropogenesis.ID:1491
(2009). [Construction and characteristics of transgenic tobacco Nicotiana tabacum L. plants expressing CYP11A1 cDNA encoding cytochrome P450scc]. Genetika 45 (9), 1217–24 [+]
In steroidogenic animal tissues cytochrome P450scc catalizes the conversion of cholesterol into pregnenolone, a common metabolic precursor of all steroid hormones. To study the possibility of functioning of mammalian cytochrome P450scc in plants and the mechanism of its integration in the plant steroidogenic system, transgenic plants of tobacco Nicotiana tabacum L. were developed carrying cDNA of CYP11A1 encoding cytochrome P450scc of bovine adrenal cortex. Pregnenolone, a product of the reaction catalyzed by cytochrome P450scc, was discovered in the steroid-containing fraction of transgenic plants. Transgenic plants are characterized by a reduced period of vegetative development (early flowering and maturation of bolls) and increased productivity. The contents of soluble protein and carbohydrates in leaves and seeds of transgenic plants are essentially higher than the contents of these components in leaves and seeds of control plants.ID:1488
(2009). [Characteristics of steroid metabolism in transgenic Nicotiana tabacum plants bearing the CYP11A1 cDNA of cytochrome P450(SCC) from the bovine adrenal cortex]. Bioorg. Khim. 36 (2), 241–50 [+]
In the mitochondria of animal steroidogenic tissues, cytochrome P450(SCC), encoded by the CYP11A1 gene, catalyzes the conversion of cholesterol into pregnenolone - the general precursor of all steroid hormones. In this work, we study the steroid metabolism in transgenic tobacco plants carrying the CYP11A1 cDNA cytochrome P450(SCC)from the bovine adrenal cortex. The transgenic plants under investigation markedly surpass the control wild-type plants by size and are characterized by a shortened period of vegetative growth (by rapid flowering); their leaves contain pregnenolone - the product of a reaction catalyzed by cytochrome P450(SCC). The level of progesterone in transgenic tobacco leaves is higher than in the control plants of the wild type. The seeds of the transgenic plants contain less (24R)-brassinosteroids than the wild-type tobacco plants. The results obtained indicate that the synthesis of an active P450(SCC) cytochrome in transgenic Nicotiana tabacum plants has a profound effect on steroid metabolism and is responsible for the specific phenotypic features of transgenic plants bearing CYP11A1 cDNA.ID:1490
- (2006). Human PMS2 gene family: origin, molecular evolution, and biological implications. Dokl. Biochem. Biophys. 408 (5), 175–179 ID:61
(2006). Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III. Nucleic Acids Res. 34 (13), 3615–24 [+]
RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53. Currently sequenced eukaryotic genomes revealed significant homology to these seventeen subunits in Fungi, Animals, Plants and Amoebozoans. Except for subunit Rpc31, this also extended to the much more distantly related genomes of Alveolates and Excavates, indicating that the complex subunit organization of RNA polymerase III emerged at a very early stage of eukaryotic evolution. The Sch.pombe subunits were expressed in S.cerevisiae null mutants and tested for growth. Ten core subunits showed heterospecific complementation, but the two largest catalytic subunits (Rpc1 and Rpc2) and all five RNA polymerase III-specific subunits (Rpc82, Rpc53, Rpc37, Rpc34 and Rpc31) were non-functional. Three highly conserved RNA polymerase III-specific domains were found in the twelve-subunit core structure. They correspond to the Rpc17-Rpc25 dimer, involved in transcription initiation, to an N-terminal domain of the largest subunit Rpc1 important to anchor Rpc31, Rpc34 and Rpc82, and to a C-terminal domain of Rpc1 that presumably holds Rpc37, Rpc53 and their Rpc11 partner.ID:60
(2004). New genes on human chromosome 7: bioinformatic analysis of a gene cluster from the POLR2J family. Bioorg. Khim. 30 (6), 621–5 [+]
Four independent genes encoding various variants of the hRPB11 subunit of Homo sapiens RNA polymerase II were revealed in human chromosome 7. Three genes (POLR2J1, POLR2J2, and POLR2J3) form a cluster of total length 214530 bp in the genetic locus 7q22.1 on the long arm of chromosome 7 (contig NT_007933). The fourth gene (POLR2J4, 31040 bp) was localized in the cytogenetic locus 7p13 of the short arm of chromosome 7 (contig NT_007819). An analysis enabled us to refine dissimilar experimental data on the mapping of the hRPB11 subunit gene on chromosome 7. In particular, the presence of three sites of its localization according to data on hybridization with fluorescent-labeled probes (the FISH method) was explained. It was established that, upon the expression of the four human POLR2J genes, at least 14 types of mature mRNAs encoding somewhat differing hRPB11 isoforms can be synthesized. Eleven of these mRNAs were revealed (as full-length copies or clearly identifiable fragments) in the available databases of expressed sequence tags and cDNAs. The most probable scheme of origination of the multiple genes of the POLR2J family, as a result of three consecutive segmented duplications increasing in size, was proposed and substantiated. On the basis of the scheme, some assumptions on the pathways of evolution of separate human genes and the mechanisms of generation of protein diversity in higher eukaryotes were made. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.ID:59
(2002). The genome sequence of Schizosaccharomyces pombe. Nature 415 (6874), 871–80 [+]
We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.ID:58
(2000). Functional conservation of RNA polymerase II in fission and budding yeasts. J. Mol. Biol. 295 (5), 1119–27 [+]
The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits.ID:57
Progesterone as a very ancient bioregulator of plant cells (2017-11-29)
The compatibility in vivo of even the most specific components of biosynthesis systems of steroid hormones in Plantae and Animalia was demonstrated for the first time. By increasing the level of the endogenic progesterone in the specially engineered CYP11A1 transgenic tobacco and tomato plants, we were able to accelerate the processes of growth and development and enhance the plants’ resistance to biotic and abiotic stresses. The formation of the above-noted successful (desirable) phenotypes of transgenic Solanaceae plants expressing mammalian cytochrome P450scc (CYP11A1) cDNA implies that progesterone can be considered as a very ancient bioregulator of plant cells and the first real hormone common to plants and animals. The results indicate a definite similarity of the steroid compounds biosynthesis and steroid regulatory systems of plants and animals and can be used in new biotechnologies for agriculture and pharmacology.
Novel nucleotide modifications for stabilization of the canonical and non-canonical secondary structures of nucleic acids (2017-11-29)
New phenoxazine-based nucleotide modifications for the stabilizing of canonical (G8AE-clamp) and non-canonical (i-Clamp) secondary structures of nucleic acids were developed. G8AE-clamp modification was shown to considerably stabilize nucleic acid duplexes and primers containing G8AE-clamp demonstrated superior sensitivity in qPCR detection of dsRNA of Kemerovo virus in natural isolates as compared with common oligonucleotides. To date i-clamp modification reveals the highest i-motif-stabilizing effect within the broad acidic pH range and could be used for tuning iM-based nanodevices such as pH sensors, molecular motor, hydrogels, delivery systems, etc.