NASU Institute of Molecular Biology and Genetics

Kiev, Ukraine

NASU Institute of Molecular Biology and Genetics

Kiev, Ukraine
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Stepanenko A.A.,NASU Institute of Molecular Biology and Genetics | Heng H.H.,Wayne State University
Mutation Research - Reviews in Mutation Research | Year: 2017

Transient and stable vector transfections have played important roles in illustrating the function of specific genes/proteins. The general assumption is that such a platform could effectively link a given gene/protein to gained phenotypes, revealing the mechanism of how a gene works. However, in reality, increased studies have surprisingly noticed some unexpected results. In this review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. A DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. To further address the common mechanisms of these unexpected findings, we will apply the genome theory of somatic evolution to explain stress-mediated system dynamics and the limitations of predicting the system behavior solely based on targeted genes. We conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes. Such analysis calls for more attention on the reduced specificities of gene-focused methodologies. © 2017 Elsevier B.V.

News Article | March 4, 2016

As political turmoil and conflict rock Ukraine, the country’s main scientific organization is in a bind. In January,  Parliament passed a law to modernize the ailing National Academy of Sciences of Ukraine (NASU). Yet an austerity budget imposed around the same time makes this impossible to achieve — at least this year. The resulting cuts to science funding threaten the jobs of young researchers in particular, who are best poised to revitalize the country’s failing economy. “We have an extraordinarily high number of potential young scientists who are ready to work for the welfare of the country,” says Liliya Hrynevych, who chairs the Ukrainian Parliament’s Committee on Science and Education and voted in favour of the modernizing law. “But without setting priorities for science and research, it will be impossible for Ukraine to become a strong and wealthy European nation.” The academy employs some 20,000 scientists across 120 research institutes. On 26 November, Parliament began to debate a “law of Ukraine on scientific and technical activity”, in an attempt to streamline and strengthen the organization, which was founded in the Soviet era. Long deemed outdated and resistant to modernization, the academy uses an opaque system to award funding, and many of its members are elderly, not least the 97-year-old metallurgist Boris Paton, who has run the NASU for decades. The law stipulates the creation of a science advisory council that includes foreign specialists, and an independent grant-giving agency. All NASU institutes will undergo an external evaluation to examine their productivity and efficiency, and overall, government science spending must increase from a current 0.3% of gross domestic product to at least 1.7% by fiscal year 2017 — near the European Union average. But before the law took effect, Ukraine passed its 2016 austerity budget, in the wake of widespread closure of mines and factories, inflation, debt and currency devaluation. The budget allocates a meagre 2.05 billion hryvnia (US$76 million) to the NASU — about 12% less than in 2015, continuing a trend of decline (see ‘Ailing academy’). The cutbacks are irreconcilable with the science law, says Hrynevych, who is campaigning in Parliament for a budget revision after the first quarter of 2016. The budget will leave the academy with scarcely enough to cover the scant salaries (about US$200 per month on average) paid to its administrative staff and scientists. “We won’t be able to buy any new equipment this year, and purchase of consumables will need to be reduced to a minimum,” says Anatoly Zagorodny, director of the Bogolyubov Institute for Theoretical Physics in Kiev and a vice-president of the academy. The fresh cuts, he says, will also force institutes to reduce staff — in some circumstances, by more than one-third — and to discontinue many areas of research, even though science is crucial to economic recovery, he adds. Young scientists are the least protected by existing labour laws and so will feel the brunt of the job cuts, says Irina Yehorchenko, a research fellow at the NASU’s Institute of Mathematics in Kiev. She and some of her colleagues launched a petition in December calling on the country’s president, Petro Poroshenko, to save Ukrainian science. “I, for one, might be able to find a postdoc position abroad,” says Oleksandr Skorokhod, a cell biologist at the NASU Institute of Molecular Biology and Genetics in Kiev who is chair of the academy’s Council of Young Scientists. “But I’d much rather stay and try to change the bad state of affairs in my country.” Ukrainian science has struggled to recover from Russia’s annexation of the Crimea peninsula in 2014. General consensus in the international community is that Crimea is still part of the Ukraine — the United Nations General Assembly declared invalid a March 2014 referendum in which voters in Crimea approved the peninsula’s secession from Ukraine. But all 22 Crimean institutes formerly run by the NASU are now under Russian control, and only a few of their 1,320 staff members have relocated to Ukraine-controlled territory. The academy lost access to its only research ship, the RV Professor Vodianytsky, three astronomical observatories in Nauchny, Katsiveli and Yevpatoria and the 204-year-old Nikitsky Botanical Garden near Yalta, on the Black Sea shore. The Ukrainian government, moreover, expects scientists in Ukraine to cut all ties with colleagues who stayed on the peninsula, says Hrynevych, because any collaboration would be viewed as legitimizing the Russian occupation. The armed conflict with pro-Russian militants in eastern Ukraine is also causing problems for scientists, especially in the country’s Donbas region. Some 12,000 scientists and university lecturers there — about 60% of the former staff of 26 research institutes and universities in the province — have moved to safe institutions in Kiev and elsewhere. But many evacuating scientists left behind equipment or lost irreplaceable research material. Marine, environmental and climate studies in the Black Sea region, mining-related geology and a variety of archaeological and historical research have all been hit hard, says Zagorodny.

Tatarskyy P.,NASU Institute of Molecular Biology and Genetics
TSitologiia i genetika | Year: 2010

Analysis of F2, F5 and MTHFR genes SNPs allelic variants in population of Ukraine. Polymorphic variants were analyzed in 172 unrelated individuals using PCR followed by RFLP analysis. Following genotypes have been identified: GG (97%), GA (3%) for F2 gene G20210A SNP, GG (96.5%), GA (3.5%) for F5 gene G1691A SNP and CC (49.5%), CT (43%), TT (7.5%) for MTHFR gene C677T SNP. Following combined genotypes have been detected. We observed 1.7% heterozygous carriers of MTHFR gene 677T SNP which were heterozygous for one of the alleles of F5 1691A or F2 20210A genes. On the other hand, the 7.5% MTHFR gene 677T SNP homozygous individuals carried wild type alleles only of F5 and F2 genes. None of the individuals was carrying F5 1691A and F2 20210A genes polymorphic variants simultaneously. The data about F2, F5 and MTHFR genes SNPs allelic frequencies in the population of Ukraine have been obtained. Thus, distribution of F2, F5 and MTHFR genotypes based on analysis of SNP in those three genes simultaneously has been detected.

BACKGROUND: The methylotrophic yeast, Hansenula polymorpha is an industrially important microorganism, and belongs to the best studied yeast species with well-developed tools for molecular research. The complete genome sequence of the strain NCYC495 of H. polymorpha is publicly available. Some of the well-studied strains of H. polymorpha are known to ferment glucose, cellobiose and xylose to ethanol at elevated temperature (45 - 50°C) with ethanol yield from xylose significantly lower than that from glucose and cellobiose. Increased yield of ethanol from xylose was demonstrated following directed metabolic changes but, still the final ethanol concentration achieved is well below what is considered feasible for economic recovery by distillation.RESULTS: In this work, we describe the construction of strains of H. polymorpha with increased ethanol production from xylose using an ethanol-non-utilizing strain (2EthOH-) as the host. The transformants derived from 2EthOH- overexpressing modified xylose reductase (XYL1m) and native xylitol dehydrogenase (XYL2) were isolated. These transformants produced 1.5-fold more ethanol from xylose than the original host strain. The additional overexpression of XYL3 gene coding for xylulokinase, resulted in further 2.3-fold improvement in ethanol production with no measurable xylitol formed during xylose fermentation. The best ethanol producing strain obtained by metabolic engineering approaches was subjected to selection for resistance to the known inhibitor of glycolysis, the anticancer drug 3-bromopyruvate. The best mutant selected had an ethanol yield of 0.3 g/g xylose and produced up to 9.8 g of ethanol/l during xylose alcoholic fermentation at 45°C without correction for ethanol evaporation.CONCLUSIONS: Our results indicate that xylose conversion to ethanol at elevated temperature can be significantly improved in H. polymorpha by combining methods of metabolic engineering and classical selection.

Stepanenko A.A.,NASU Institute of Molecular Biology and Genetics
T{combining double inverted breve}Sitologii{combining double inverted breve}a i genetika | Year: 2012

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MC-F10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different "immortalizing agents", oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor "oncogene and chromosome addiction", intra-/intertumor heterogeneity, and chromosome instability.

Blazhenko O.V.,NASU Institute of Molecular Biology and Genetics
Current Microbiology | Year: 2014

The Pichia guilliermondii GSH1 and GSH2 genes encoding Saccharomyces cerevisiae homologues of glutathione (GSH) biosynthesis enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, respectively, were cloned and deleted. Constructed P. guilliermondii Δgsh1 and Δgsh2 mutants were GSH auxotrophs, displayed significantly decreased cellular GSH+GSSG levels and sensitivity to tert-butyl hydroperoxide, hydrogen peroxide, and cadmium ions. In GSH-deficient synthetic medium, growths of Δgsh1 and Δgsh2 mutants were limited to 3-4 and 5-6 cell divisions, respectively. Under these conditions Δgsh1 and Δgsh2 mutants possessed 365 and 148 times elevated riboflavin production, 10.7 and 2.3 times increased cellular iron content, as well as 6.8 and 1.4 fold increased ferrireductase activity, respectively, compared to the wild-type strain. Glutathione addition to the growth medium completely restored the growth of both mutants and decreased riboflavin production, cellular iron content, and ferrireductase activity to the level of the parental strain. Cysteine also partially restored the growth of the Δgsh2 mutants, while methionine or dithiothreitol could not restore the growth neither of the Δgsh1, nor of the Δgsh2 mutants. Besides, it was shown that in GSH presence riboflavin production by both Δgsh1 and Δgsh2 mutants, similarly to that of the wild-type strain, depended on iron concentration in the growth medium. Furthermore, in GSH-deficient synthetic medium P. guilliermondii Δgsh2 mutant cells, despite iron overload, behaved like iron-deprived wild-type cells. Thus, in P. guilliermondii yeast, glutathione is required for proper regulation of both riboflavin and iron metabolism. © 2014 Springer Science+Business Media.

Filonenko V.V.,NASU Institute of Molecular Biology and Genetics
Biopolymers and Cell | Year: 2013

This review summarizes experimental data related to the studies of PI3K/mTOR/S6K signaling conducted at the department of cell signaling. Analysis of novel S6Ks protein-protein interactions provided valuable information for understanding molecular mechanisms of regulation of S6Ks functional activity and subcellular localization mediated by PKC, CK2 and ROC1 ubiquitin ligase. We discuss the identification and functional analysis of novel isoform of ribosomal protein S6 kinase - S6K2 and of mTOR kinase - mTORβ, as well as their oncogenic properties. Identification of CoA synthase responsible for last two steps in CoA biosynthesis and characterization of its interactions with S6K1 and other signaling molecules uncovere a potential link between mTOR/S6K signaling pathway and energy metabolism through regulation of CoA biosynthesis. The data concerning new molecular mechanisms of CoA synthase regulation are presented. © Institute of Molecular Biology and Genetics, NAS of Ukraine, 2013.

Kozyrovska N.O.,NASU Institute of Molecular Biology and Genetics
Biopolymers and Cell | Year: 2013

Plants are heavily populated by pro and eukaryotic microorganisms and represent therefore the tremendous complexity as a biological system. This system exists as an information processing entity with rather complex processes of communication, occurring throughout the individual plant. The plant cellular information processing network constitutes the foundation for processes like growth, defense, and adaptation to the environment. Up to date, the molecular mechanisms, underlying perception, transfer, analysis, and storage of the endogenous and environmental information within the plant, remain to be fully understood. The associated microorganisms and their investment in the information conditioning are often ignored. Endophytes as plant partners are indispensable integrative part of the plant system. Diverse endophytic microorganisms comprise «normal» microbiota that plays a role in plant immunity and helps the plant system to survive in the environment (providing assistance in defense, nutrition, detoxification etc.). The role of endophytic microbiota in the processing of information may be presumed, taking into account a plant-microbial co-evolution and empirical data. Since the literature are beginning to emerge on this topic, in this article, I review key works in the field of plant-endophytes interactions in the context of information processing and represent the opinion on their putative role in plant information web under defense and the adaptation to changed conditions. © Institute of Molecular Biology and Genetics, NAS of Ukraine, 2013.

Kropyvko S.V.,NASU Institute of Molecular Biology and Genetics
Biopolymers and Cell | Year: 2015

TKS4 scaffold protein is involved in formation of invadopodia, production of reactive oxygen species (ROS) by tumor cells and other cellular processes. Aim. To identify new TKS4 partners involved in the actin cytoskeleton rearrangements and endo-/exocytosis. Methods. The GST pull-down assay was used to identify the interaction. Results. We revealed that TKS4 SH3 domains interacted with the actin cytoskeleton reorganization proteins N-WASP and CR16, as well as with DNM2, SYNJ1 and OPHN1 which are involved in endo-/exocytosis. We also tested the WIP, WIRE, SHIP2, RhoU, RhoV and NUMB proteins, but their interaction with the SH3 domains of TKS4 was not found. Conclusions. The SH3 domains of TKS4 interact with N-WASP, DNM2, SYNJ1, OPHN1 and weakly with CR16 in vitro. © 2015 S. V. Kropyvko.

Malanchuk O.M.,NASU Institute of Molecular Biology and Genetics
Biopolymers and Cell | Year: 2015

Aim. Rictor is a component of the protein complex mTORC2 that is activated by growth factors and regulates cell growth, survival and migration. Here, we describe the development of the Rictor specific monoclonal antibody and characterize its suitability for various immunological assays. Methods. Hybridoma technology has been used for the monoclonal antibody production. Immunization was carried out with the recombinant N-terminal fragment of human Rictor expressed in E. coli as a GST-tagged fusion protein. Results. Specific monoclonal antibody (mAb) against Rictor has been developed. Conclusions. The generated mAb specifically recognizes the recombinant and endogenous Rictor and is suitable for Western blotting, immunoprecipi-tation, and immunofluorescence assays of mammalian cells. This mAb will be a useful tool for the investigations of a physiological role of Rictor as well as the function of the mTORC2 in general. © 2015 O. M. Malanchuk.

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