45 results on '"Shagin DA"'
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2. Novel Klebsiella pneumoniae virulent bacteriophage KPPK108.1 capable of infecting the K108 serotype strains
- Author
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Evseev, PV, primary, Shneider, MM, additional, Mikhailova, YuV, additional, Shelenkov, AA, additional, Yanushevich, YuG, additional, Karlova, MG, additional, Moiseenko, AV, additional, Sokolova, OS, additional, and Shagin, DA, additional
- Published
- 2021
- Full Text
- View/download PDF
3. On the unpredictability of outcomes of immunotherapy and preventive immunization against COVID-19
- Author
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Chebotar, IV, primary and Shagin, DA, additional
- Published
- 2020
- Full Text
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4. The structure of Klebsiella pneumoniae K108 capsular polysaccharide is similar to Escherichia coli colanic acid.
- Author
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Kasimova AA, Shneider MM, Evseev PV, Shelenkov AA, Mikhailova YV, Miroshnikov KA, Chebotar IV, and Shagin DA
- Subjects
- Polysaccharides, Bacterial chemistry, Multigene Family, Klebsiella pneumoniae genetics, Klebsiella pneumoniae metabolism, Escherichia coli genetics, Escherichia coli metabolism
- Abstract
The clinical isolate of Klebsiella pneumoniae 1333/P225 was revealed as containing a KL108 K. pneumoniae K locus for capsule biosynthesis. The gene cluster demonstrated a high level of sequence and arrangement similarity with that of the E. coli colanic acid biosynthesis gene cluster. The KL108 gene cluster includes a gene of WcaD polymerase responsible for joining oligosaccharide K units into capsular polysaccharide (CPS), acetyltransferase, pyruvyltransferasefive and genes for glycosyltransferases (Gtrs), four of which have homologues in genetic units of the colanic acid synthesis. The fifth Gtr is specific to this cluster. The work involved the use of sugar analysis, Smith degradation and one- and two-dimensional
1 H and13 C NMR spectroscopy to establish the structure of the K108 CPS. The CPS repetitive K unit is composed of branched pentasaccharide with three monosaccharides in the backbone and a disaccharide side chain. The main chain is the same as for colanic acid but the side chain differs. Two bacteriophages infecting K. pneumoniae strain 1333/P225 were isolated and structural depolymerase genes were determined; depolymerases Dep108.1 and Dep108.2 were cloned, expressed and purified. It was demonstrated that both depolymerases specifically cleave the β-Glcp-(1→4)-α-Fucp linkage between K108 units in the CPS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)- Published
- 2023
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5. Involvement of a Phage-Encoded Wzy Protein in the Polymerization of K127 Units To Form the Capsular Polysaccharide of Acinetobacter baumannii Isolate 36-1454.
- Author
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Arbatsky NP, Kasimova AA, Shashkov AS, Shneider MM, Popova AV, Shagin DA, Shelenkov AA, Mikhailova YV, Yanushevich YG, Hall RM, Knirel YA, and Kenyon JJ
- Subjects
- Bacterial Capsules metabolism, Humans, Polymerization, Polysaccharides, Bacterial metabolism, Acinetobacter Infections, Acinetobacter baumannii chemistry, Acinetobacter baumannii genetics, Acinetobacter baumannii metabolism, Bacteriophages
- Abstract
A comprehensive understanding of capsular polysaccharide (CPS) diversity is critical to implementation of phage therapy to treat panresistant Acinetobacter baumannii infections. Predictions from genome sequences can assist identification of the CPS type but can be complicated if genes outside the K locus (CPS biosynthesis gene cluster) are involved. Here, the CPS produced by A. baumannii clinical isolate 36-1454 carrying a novel K locus, KL127, was determined and compared to other CPSs. KL127 differs from KL128 in only two of the glycosyltransferase ( gtr ) genes. The K127 unit in 36-1454 CPS was the pentasaccharide β-d-Glc p -(1→6)-d-β-Gal p NAc-(1→6)-α-d-Gal p -(1→6)-β-d-Glс p -(1→3)-β-d-Gal p NAc in which d-Glc p at position 4 replaces d-Gal p in K128, and the glycosyltransferases encoded by the different gtr genes form the surrounding linkages. However, although the KL127 and KL128 gene clusters encode nearly identical Wzy polymerases, the linkages between K units that form the CPS chains are different, i.e., β-d-Gal p NAc-(1→3)-d-Gal p in 36-1454 (K127) and β- d- Gal p NAc-(1→4)-d-Gal p in KZ-1093 (K128). The linkage between K127 units in 36-1454 is the same as the K-unit linkage in five known CPS structures, and a gene encoding a Wzy protein related to the Wzy of the corresponding K loci was found encoded in a prophage genome in the 36-1454 chromosome. Closely related Wzy proteins were encoded in unrelated phage in available KL127-carrying genomes. However, a clinical isolate, KZ-1257, carrying KL127 but not the prophage was found, and K127 units in the KZ-1257 CPS were β-d-Gal p NAc-(1→4)-d-Gal p linked, confirming that Wzy
KL127 forms this linkage and thus that the phage-encoded WzyPh1 forms the β-d-Gal p NAc-(1→3)-d-Gal p linkage in 36-1454. IMPORTANCE Bacteriophage therapy is an attractive innovative treatment for infections caused by extensively drug resistant Acinetobacter baumannii, for which there are few effective antibiotic treatments remaining. Capsular polysaccharide (CPS) is a primary receptor for many lytic bacteriophages, and thus knowledge of the chemical structures of CPS produced by the species will underpin the identification of suitable phages for therapeutic cocktails. However, recent research has shown that some isolates carry additional genes outside of the CPS biosynthesis K locus, which can modify the CPS structure. These changes can subsequently alter phage receptor sites and may be a method utilized for natural phage resistance. Hence, it is critical to understand the genetics that drive CPS synthesis and the extent to which genes outside of the K locus can affect the CPS structure.- Published
- 2022
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6. Specific Interaction of Novel Friunavirus Phages Encoding Tailspike Depolymerases with Corresponding Acinetobacter baumannii Capsular Types.
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Popova AV, Shneider MM, Arbatsky NP, Kasimova AA, Senchenkova SN, Shashkov AS, Dmitrenok AS, Chizhov AO, Mikhailova YV, Shagin DA, Sokolova OS, Timoshina OY, Kozlov RS, Miroshnikov KA, and Knirel YA
- Abstract
Acinetobacter baumannii is one of the most clinically important nosocomial pathogens. The World Health Organisation refers it to its «critical priority» category to develop new strategies for effective therapy. This microorganism is capable of producing structurally diverse capsular polysaccharides (CPSs), which serve as primary receptors for A. baumannii bacteriophages carrying polysaccharide-depolymerasing enzymes. In this study, eight novel bacterial viruses that specifically infect A. baumannii strains belonging to K2/K93, K32, K37, K44, K48, K87, K89 and K116 capsular types were isolated and characterized. The overall genomic architecture demonstrated that these viruses are representatives of the Friunavirus genus of the family Autographiviridae The linear double-stranded DNA phage genomes of 41,105-42,402 bp share high nucleotide sequence identity, except for genes encoding structural depolymerases or tailspikes which determine the host specificity. Deletion mutants lacking N-terminal domains of tailspike proteins were cloned, expressed and purified. The structurally defined CPSs of the phage bacterial hosts were cleaved with the specific recombinant depolymerases, and the resultant oligosaccharides that corresponded to monomers or/and dimers of the CPS repeats (K-units) were isolated. Structures of the derived oligosaccharides were established by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The data obtained showed that all depolymerases studied were glycosidases that cleave specifically the A. baumannii CPSs by the hydrolytic mechanism, in most cases, by the linkage between the K-units. IMPORTANCE Acinetobacter baumannii , a nonfermentative, Gram-negative, aerobic bacterium, is one of the most significant nosocomial pathogens. The pathogenicity of A. baumannii is based on the cooperative action of many factors, one of them being the production of capsular polysaccharides (CPSs) that surround bacterial cells with a thick protective layer. Polymorphism of the chromosomal capsule loci is responsible for the observed high structural diversity of the CPSs. In this study, we describe eight novel lytic phages which have different tailspike depolymerases (TSDs) determining the interaction of the viruses with corresponding A. baumannii capsular types (K-types). Moreover, we elucidate the structures of oligosaccharide products obtained by cleavage of the CPSs by the recombinant depolymerases. We believe that as the TSDs determine phage specificity, the diversity of their structures should be taken into consideration as selection criteria for inclusion of certain phage candidate to the cocktail designed to control A. baumannii with different K-types., (Copyright © 2020 Popova et al.)
- Published
- 2021
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7. [Increasing the Uniformity of Genome Fragment Coverage for High-Throughput Sequencing of Influenza A Virus].
- Author
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Mikhaylova YV, Shelenkov AA, Yanushevich YG, and Shagin DA
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- Polymerase Chain Reaction, Genome, Viral, High-Throughput Nucleotide Sequencing, Influenza A virus genetics
- Abstract
The high variability of the influenza A virus poses a significant threat to public health, therefore monitoring viral strains and studying their genetic properties are important tasks. One part of this monitoring includes sequencing of influenza A viruses of any subtype and analysis of their whole genomes, which is especially important in cases of interspecies adaptation and reassortment. High-throughput sequencing technologies have significantly extended the capabilities of influenza virus epidemiological surveillance. The preparation stages for next generation sequencing (NGS) of influenza A virus include whole genome amplification using one-step RT-PCR, the results of which vary greatly depending on the sample type and quality, that, in turn, affects the coverage of virus fragments and the sequencing results in general. In this work, we propose to supplement the aforementioned technique of whole genome amplification of influenza A virus with sequential suppression PCRs to obtain an even coverage of viral segments of different lengths, which allows sequencing of samples with lower read coverage without decreasing the sequencing quality.
- Published
- 2020
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8. Complete Genome Sequence of Acinetobacter baumannii Phage BS46.
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Popova AV, Shneider MM, Mikhailova YV, Shelenkov AA, Shagin DA, Edelstein MV, and Kozlov RS
- Abstract
Acinetobacter myovirus BS46 was isolated from sewage by J. S. Soothill in 1991. We have sequenced the genome of BS46 and found it to be almost unique. BS46 contains double-stranded DNA with a genome size of 94,068 bp and 176 predicted open reading frames. The gene encoding the tailspike that presumably possesses depolymerase activity toward the capsular polysaccharides of the bacterial host was identified., (Copyright © 2020 Popova et al.)
- Published
- 2020
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9. Structure of the K128 capsular polysaccharide produced by Acinetobacter baumannii KZ-1093 from Kazakhstan.
- Author
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Arbatsky NP, Kasimova AA, Shashkov AS, Shneider MM, Popova AV, Shagin DA, Shelenkov AA, Mikhailova YV, Yanushevich YG, Azizov IS, Edelstein MV, Hall RM, Kenyon JJ, and Knirel YA
- Subjects
- Acinetobacter baumannii genetics, Acinetobacter baumannii metabolism, Glycosyltransferases genetics, Glycosyltransferases metabolism, Kazakhstan, Multigene Family, Polysaccharides, Bacterial biosynthesis, Acinetobacter baumannii chemistry, Bacterial Capsules chemistry, Polysaccharides, Bacterial chemistry
- Abstract
The structure of the K128 capsular polysaccharide (CPS) produced by Acinetobacter baumannii isolate KZ-1093 from Kazakhstan was established by sugar analysis and Smith degradation along with 1D and 2D
1 H and13 C NMR spectroscopy. The CPS was found to consist of branched pentasaccharide repeating units containing only neutral sugars, and its composition and topology are closely related to those of the A. baumannii K116 CPS. The K128 and K116 oligosaccharide units differ in the linkage between the disaccharide side chain and the main chain, with a β-(1 → 6) linkage in K128 replacing a β-(1 → 4) linkage in K116. The linkages between the repeating units in the K128 and K116 CPSs are also different, with K128 units linked by β-d-GalpNAc-(1 → 4)-d-Galp, and β-d-GalpNAc-(1 → 3)-d-Galp linkages between K116 units. The KZ-1093 genome was sequenced and the CPS biosynthesis gene cluster at the chromosomal K locus was designated KL128. Consistent with the CPS structures, KL128 differs from KL116 in one glycosyltransferase gene and the gene for the Wzy polymerase. In KL128, the gtr200 glycosyltransferase gene replaces gtr76 in KL116, and Gtr200 was therefore assigned to the different β-d-GalpNAc-(1 → 6)-d-Galp linkage in K128. Similarly, the WzyK128 polymerase could be assigned to the β-d-GalpNAc-(1 → 4)-d-Galp linkage between the K128 units., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2019
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10. Acinetobacter baumannii K116 capsular polysaccharide structure is a hybrid of the K14 and revised K37 structures.
- Author
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Shashkov AS, Cahill SM, Arbatsky NP, Westacott AC, Kasimova AA, Shneider MM, Popova AV, Shagin DA, Shelenkov AA, Mikhailova YV, Yanushevich YG, Edelstein MV, Kenyon JJ, and Knirel YA
- Subjects
- Acinetobacter baumannii enzymology, Acinetobacter baumannii metabolism, Bacterial Capsules chemistry, Bacterial Capsules metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Carbohydrate Sequence, Evolution, Molecular, Genome, Bacterial, Glycosyltransferases metabolism, Multigene Family, Polysaccharides, Bacterial biosynthesis, Whole Genome Sequencing, Acinetobacter baumannii genetics, Glycosyltransferases genetics, Polysaccharides, Bacterial chemistry
- Abstract
The genome of Acinetobacter baumannii clinical isolate, MAR-303, recovered in Russia was sequenced and found to contain a novel gene cluster at the A. baumannii K locus for capsule biosynthesis. The gene cluster, designated KL116, included four genes for glycosyltransferases (Gtrs) and a gene for a Wzy polymerase responsible for joining oligosaccharide K units into the capsular polysaccharide (CPS). The arrangement of KL116 was a hybrid of previously described A. baumannii gene clusters, with two gtr genes and the wzy gene shared by KL37 and the two other gtr genes found in KL14. The structure of the K116 CPS was established by sugar analysis and Smith degradation, along with one- and two-dimensional
1 H and13 C NMR spectroscopy. The CPS is composed of branched pentasaccharide K units containing only neutral sugars, with three monosaccharides in the main chain and a disaccharide side chain. The K116 unit shares internal sugar linkages with the K14 and K37 units, corresponding to the presence of shared gtr genes in the gene clusters. However, the specific linkage formed by Wzy was discrepant between K116 and the previously reported K37 CPS produced by A. baumannii isolate NIPH146. The K37 structure was therefore revised in this study, and the corrected Wzy linkage found to be identical to the Wzy linkage in K116. The KL116, KL14 and KL37 gene clusters were found in genomes of a variety of A. baumannii strain backgrounds, indicating their global distribution., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2019
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11. Inactivation of the oprD porin gene by a novel insertion sequence ISPa195 associated with large deletion in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.
- Author
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Bocharova Y, Savinova T, Shagin DA, Shelenkov AA, Mayanskiy NA, and Chebotar IV
- Subjects
- Anti-Bacterial Agents pharmacology, Bacterial Proteins, Carbapenems pharmacology, Gene Components, Humans, Imipenem pharmacology, Meropenem metabolism, Microbial Sensitivity Tests, Pseudomonas Infections microbiology, Pseudomonas aeruginosa drug effects, beta-Lactamases, DNA Transposable Elements genetics, Drug Resistance, Multiple, Bacterial genetics, Porins genetics, Pseudomonas aeruginosa genetics, Sequence Deletion
- Abstract
Objectives: Alteration of the porin-encoding gene oprD by insertion sequences (ISs) is one mechanism conferring carbapenem resistance in Pseudomonas aeruginosa. Here we describe a carbapenem-resistant clinical P. aeruginosa isolate 36-989 harbouring a novel IS (ISPa195) in oprD., Methods: Minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the broth microdilution method. Carbapenemase activity was assessed using a MALDI-TOF/MS-based assay of meropenem hydrolysis. Efflux-dependent carbapenem resistance was evaluated using an assay with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The oprD gene and IS sequence were analysed by the Sanger method. Whole-genome sequencing was performed on an Illumina HiSeq 2500 platform., Results: Antimicrobial susceptibility testing demonstrated that P. aeruginosa 36-989 was resistant to imipenem (MIC=32mg/L) and meropenem (MIC=16mg/L). No carbapenemase activity was detected, however an efflux-mediated component of carbapenem resistance was revealed. A new IS element (ISPa195) was found in the oprD gene of P. aeruginosa 36-989. ISPa195 was 1190bp in length, belonging to the IS3 family, and contained two open reading frames that overlapped through a ribosomal slippage to translate the full-size transposase enzyme. There was an IS-associated 284-bp deletion in the oprD gene; no direct repeats at flanking regions of the IS were detected., Conclusion: The absence of direct repeats at flanking regions in combination with the IS-associated deletion distinguished ISPa195 from other ISs previously detected in oprD. Carbapenem resistance in P. aeruginosa 36-989 was conferred by a combination of oprD alteration and carbapenem efflux., (Copyright © 2019 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2019
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12. First whole genome sequencing of Russian isolate of Capnocytophaga canimorsus, opportunistic pathogen causing lethal sepsis.
- Author
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Shelenkov AA, Karan LS, Mihaylova YV, Yanushevich YG, Shipulin GA, and Shagin DA
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- Capnocytophaga isolation & purification, High-Throughput Nucleotide Sequencing, Humans, Male, Middle Aged, Molecular Sequence Annotation, Moscow, Sequence Analysis, DNA, Capnocytophaga genetics, Gram-Negative Bacterial Infections microbiology, Sepsis microbiology, Whole Genome Sequencing
- Abstract
Capnocytophaga canimorsus is a part of healthy oral flora of dogs and cats. However, when it is transmitted to human subjects via animal bites or scratches, it can cause severe complications like endocarditis or even lethal septic shock, especially in immunocompromised persons. In this study, we performed the first whole-genome sequencing on Illumina HiSeq platform of Russian isolate of C. canimorsus that have caused lethal sepsis in 51-old male from Moscow. We believe that the availability of genomic sequence and annotation for the given strain could be useful for future epidemiological surveillance studies in Russia and other countries., (Copyright © 2018. Published by Elsevier Ltd.)
- Published
- 2018
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13. Structure and gene cluster of the K125 capsular polysaccharide from Acinetobacter baumannii MAR13-1452.
- Author
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Arbatsky NP, Shneider MM, Dmitrenok AS, Popova AV, Shagin DA, Shelenkov AA, Mikhailova YV, Edelstein MV, and Knirel YA
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- Acinetobacter baumannii classification, Glycosyltransferases chemistry, Glycosyltransferases metabolism, Magnetic Resonance Spectroscopy, Polysaccharides, Bacterial biosynthesis, Structure-Activity Relationship, Sugars chemistry, Acinetobacter baumannii chemistry, Acinetobacter baumannii genetics, Multigene Family, Polysaccharides, Bacterial chemistry
- Abstract
A capsular polysaccharide (CPS) was isolated from strain MAR13-1452 of an emerging pathogen Acinetobacter baumannii and assigned type K125. The following structure of the CPS was established by sugar analysis, Smith degradation, and 1D and 2D
1 H and13 C NMR spectroscopy: Proteins encoded by the KL125 gene cluster in the genome of MAR13-1452, including three glycosyltransferases, were assigned roles in the biosynthesis of the K125 CPS., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2018
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14. Cloning and characterization of serpin from red king crab Paralithodes camtschaticus.
- Author
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Kostin NN, Bobik TV, Shurdova EM, Ziganshin RH, Surina EA, Shagin DA, Shagina IA, Knorre VD, Isaev VA, Rudenskaya GN, Gabibov AG, and Smirnov IV
- Subjects
- Animals, Arthropod Proteins metabolism, Cattle, Cloning, Molecular, Hemocytes metabolism, Immunity, Innate, Muscles metabolism, Myocardium metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Serpins metabolism, Trypsin Inhibitors isolation & purification, Anomura genetics, Arthropod Proteins genetics, Serpins genetics
- Abstract
Serpins are a family of serine protease inhibitors that are involved in numerous physiological processes and are known to regulate innate immunity pathways. To advance our understanding of their role in P. camtschaticus, a commercially significant species, we cloned and characterized a serpin from this species, designated serpin PC, that has anticoagulant and anticomplement effects on human blood. We found that serpin PC is a secreted protein with a typical serpin-like primary structure that is similar to other known crustacean serpins. Recombinant serpin PC was found to have inhibitory activity against R/K-specific bovine cationic trypsin. The reaction proceeds through the formation of a stable covalent complex of peptidase with P1 residue R383 of serpin PC. This interaction is characterized by a relatively high overall inhibition constant k
ass =(2.3 ± 0.7) × 106 M-1 s-1 and an SI of 4.7 ± 0.8. Protein localization by western blotting showed that serpin PC is present in the muscles and, to a lesser extent, the heart, whereas it is transcribed predominantly in hemocytes and the heart. Through peptidase activity profiling of hemocytes and plasma, we found that serpin PC inhibits at least two R/K-specific activities and showed that it inhibits phenoloxidase (PO) activity induction in hemocytes., (Copyright © 2018. Published by Elsevier Ltd.)- Published
- 2018
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15. [Suppression of NR0B2 gene in Clear Cell Renal Cell Carcinoma Is Associated with Hypermethylation of Its Promoter].
- Author
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Kudryavtseva AV, Nyushko KM, Zaretsky AR, Shagin DA, Sadritdinova AF, Fedorova MS, Savvateeva MV, Guvatova ZG, Pudova EA, Alekseev BY, Dmitriev AA, and Snezhkina AV
- Subjects
- Carcinoma, Renal Cell genetics, Carcinoma, Renal Cell pathology, DNA, Neoplasm genetics, Female, Humans, Kidney Neoplasms genetics, Kidney Neoplasms pathology, Male, Receptors, Cytoplasmic and Nuclear genetics, Tumor Suppressor Proteins genetics, Carcinoma, Renal Cell metabolism, DNA Methylation, DNA, Neoplasm metabolism, Down-Regulation, Gene Expression Regulation, Neoplastic, Kidney Neoplasms metabolism, Promoter Regions, Genetic, Receptors, Cytoplasmic and Nuclear biosynthesis, Tumor Suppressor Proteins biosynthesis
- Abstract
Clear cell renal cell carcinoma (ccRCC) is a common urologic malignancy. Understanding of the transcriptional regulation of oncogenes and tumor suppressor genes involved is critical for the development of the treatments for renal tumors. Using ccRCC subdivision of the TCGA dataset, we identified NR0B2 encoding orphan nuclear receptor as a tumor suppressor candidate in renal tissue. In independent cohort of primary renal tumors, quantitative PCR experiments confirmed significant suppression of NR0B2 mRNA in 86% of ccRCC samples studied. In 80% of these cases, we detected the hypermethylation of the NR0B2 pro-moter region. These results suggest that NR0B2 is a tumor suppressor gene in ccRCC, and that the hypermethylation of promoter region is the main mechanism of its downregulation.
- Published
- 2018
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16. [Transcription Factor SAP30 Is Involved in the Activation of NETO2 Gene Expression in Clear Cell Renal Cell Carcinoma].
- Author
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Snezhkina AV, Nyushko KM, Zaretsky AR, Shagin DA, Sadritdinova AF, Fedorova MS, Guvatova ZG, Abramov IS, Pudova EA, Alekseev BY, Dmitriev AA, and Kudryavtseva AV
- Subjects
- Carcinoma, Renal Cell genetics, Carcinoma, Renal Cell pathology, Female, Histone Deacetylases genetics, Humans, Kidney Neoplasms genetics, Kidney Neoplasms pathology, Male, Membrane Proteins genetics, Neoplasm Proteins genetics, Carcinoma, Renal Cell metabolism, Gene Expression Regulation, Neoplastic, Histone Deacetylases metabolism, Kidney Neoplasms metabolism, Membrane Proteins biosynthesis, Neoplasm Proteins metabolism, Up-Regulation
- Abstract
Clear cell renal cell carcinoma (ccRCC) is a common oncourological disease with a high mortality level. The incidence of this type of cancer is constantly increasing, while molecular mechanisms involved in the disease initiation and progression remain far from being fully understood. A problem of the search for novel markers is crucial for improvement of diagnosis and therapy of ccRCC. We have previously found that the disease is characterized by increased expression of the NETO2 gene. In the present study, we showed that isoform 1 (NM_018092.4) makes the main contribution to the upregulation of this gene. Using original CrossHub software, "The Cancer Genome Atlas" (TCGA) project data were analyzed to identify possible mechanisms of NETO2 gene activation in ccRCC. The absence of significant contribution of methylation to the increase of mRNA level of the gene was observed. At the same time, a number of genes encoding transcription factors, which could potentially regulate the expression of NETO2 in ccRCC, were identified. Three such genes (MYCBP, JMY, and SAP30) were selected for the further analysis of their mRNA levels in a set of ccRCC samples with quantitative PCR. We showed a significant increase in mRNA level of one of the examined genes, SAP30, and revealed its positive correlation with NETO2 gene expression. Thus, upregulation of NETO2 gene is first stipulated by the isoform 1 (NM_018092.4), and the probable mechanism of its activation is associated with the increased expression of SAP30 transcription factor.
- Published
- 2018
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17. Application of nonsense-mediated primer exclusion (NOPE) for preparation of unique molecular barcoded libraries.
- Author
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Shagin DA, Turchaninova MA, Shagina IA, Shugay M, Zaretsky AR, Zueva OI, Bolotin DA, Lukyanov S, and Chudakov DM
- Subjects
- ErbB Receptors genetics, High-Throughput Nucleotide Sequencing, Sequence Analysis, DNA, DNA Primers genetics, Gene Library, Polymerase Chain Reaction methods
- Abstract
Background: Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps., Results: Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification., Conclusion: We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.
- Published
- 2017
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18. A high-throughput assay for quantitative measurement of PCR errors.
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Shagin DA, Shagina IA, Zaretsky AR, Barsova EV, Kelmanson IV, Lukyanov S, Chudakov DM, and Shugay M
- Subjects
- Alleles, Analysis of Variance, Gene Library, Polymerase Chain Reaction methods, Polymorphism, Single Nucleotide, Sequence Analysis, DNA, High-Throughput Nucleotide Sequencing methods, Polymerase Chain Reaction standards
- Abstract
The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.
- Published
- 2017
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19. MAGERI: Computational pipeline for molecular-barcoded targeted resequencing.
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Shugay M, Zaretsky AR, Shagin DA, Shagina IA, Volchenkov IA, Shelenkov AA, Lebedin MY, Bagaev DV, Lukyanov S, and Chudakov DM
- Subjects
- Biomarkers, Tumor blood, Biomarkers, Tumor genetics, Databases, Genetic, Humans, Neoplasms genetics, RNA, Viral genetics, Sequence Analysis, DNA methods, Sequence Analysis, RNA methods, Computational Biology methods, High-Throughput Nucleotide Sequencing methods, Software
- Abstract
Unique molecular identifiers (UMIs) show outstanding performance in targeted high-throughput resequencing, being the most promising approach for the accurate identification of rare variants in complex DNA samples. This approach has application in multiple areas, including cancer diagnostics, thus demanding dedicated software and algorithms. Here we introduce MAGERI, a computational pipeline that efficiently handles all caveats of UMI-based analysis to obtain high-fidelity mutation profiles and call ultra-rare variants. Using an extensive set of benchmark datasets including gold-standard biological samples with known variant frequencies, cell-free DNA from tumor patient blood samples and publicly available UMI-encoded datasets we demonstrate that our method is both robust and efficient in calling rare variants. The versatility of our software is supported by accurate results obtained for both tumor DNA and viral RNA samples in datasets prepared using three different UMI-based protocols.
- Published
- 2017
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20. NON-INVASIVE MONITORING OF EGFR MUTATIONS IN NONSMALL CELL LUNG CANCER (NSCLC) DURING TARGETED THERAPY: STATE OF THE ART AND OUR EXPERIENCE.
- Author
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Chermenko PA, Zaretsky AR, Shagin DA, Breder VV, and Laktionov KK
- Subjects
- Adult, Aged, Aged, 80 and over, Carcinoma, Non-Small-Cell Lung blood, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung pathology, Circulating Tumor DNA blood, Circulating Tumor DNA genetics, ErbB Receptors blood, ErbB Receptors genetics, Female, Humans, Male, Middle Aged, Molecular Targeted Therapy, Mutation, Pilot Projects, Protein Kinase Inhibitors adverse effects, Carcinoma, Non-Small-Cell Lung drug therapy, Drug Resistance, Neoplasm genetics, Protein Kinase Inhibitors administration & dosage
- Abstract
Liquid biopsy is a promising approach to molecular tumor testing in the context of targeted therapy. During this pilot study we applied a high-sensitivity protocol for detection of tumor-derived mutations in circulating plasma DNA of EGFR-positive non-small cell lung cancer (NSCLC) patients during EGFR-TKI therapy. We showed that this protocol was well suited for dynamic monitoring during targeted therapy as well as for detection of acquired resistance mutations.
- Published
- 2016
21. A digestive prolyl carboxypeptidase in Tenebrio molitor larvae.
- Author
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Goptar IA, Shagin DA, Shagina IA, Mudrik ES, Smirnova YA, Zhuzhikov DP, Belozersky MA, Dunaevsky YE, Oppert B, Filippova IY, and Elpidina EN
- Subjects
- Amino Acid Sequence, Animals, Carboxypeptidases chemistry, Carboxypeptidases genetics, Hydrolysis, Larva enzymology, Larva genetics, Molecular Sequence Data, Prolyl Hydroxylases genetics, Prolyl Hydroxylases isolation & purification, Substrate Specificity, Tenebrio genetics, Carboxypeptidases isolation & purification, Digestive System enzymology, Prolyl Hydroxylases chemistry, Tenebrio enzymology
- Abstract
Prolyl carboxypeptidase (PRCP) is a lysosomal proline specific serine peptidase that also plays a vital role in the regulation of physiological processes in mammals. In this report, we isolate and characterize the first PRCP in an insect. PRCP was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor, using a three-step chromatography strategy, and it was determined that the purified enzyme was a dimer. The cDNA of PRCP was cloned and sequenced, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity and kinetic parameters of the enzyme were determined. The T. molitor PRCP participates in the hydrolysis of the insect's major dietary proteins, gliadins, and is the first PRCP to be ascribed a digestive function. Our collective data suggest that the evolutionary enrichment of the digestive peptidase complex in insects with an area of acidic to neutral pH in the midgut is a result of the incorporation of lysosomal peptidases, including PRCP., (Published by Elsevier Ltd.)
- Published
- 2013
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22. [Preparation of prokaryotic cDNA for high-throughput transcriptome analysis].
- Author
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Bogdanova EA, Shagina IA, Ianushevich IuG, Vagner LL, Luk'ianov SA, and Shagin DA
- Subjects
- Genome, Bacterial, Prokaryotic Cells chemistry, RNA, Untranslated chemistry, DNA, Complementary isolation & purification, Gene Expression Profiling, High-Throughput Screening Assays
- Abstract
High contents of non-coding RNA in total bacteria RNA complicates considerably transcriptome analysis using standard approaches like high-throughput sequencing, gene expression profiles, subtractive hybridization. We suggest a procedure of preparation of bacterial cDNA for transcriptomics that includes rRNA and tRNA depletion with preservation of relative abundance of coding sequences. The method is based on the second order hybridization kinetics and unique properties of Kanchatka crab duplex-specific nuclease. The method efficacy was demonstrated on a model experiments.
- Published
- 2011
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- View/download PDF
23. [Application of the duplex-specific nuclease for fast analysis of single nucleotide polymorphisms and detection of target DNA in complex PCR products].
- Author
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Shagina IA, Bogdanova EA, Al'tshuler IM, Luk'ianov SA, and Shagin DA
- Subjects
- Animals, Cell Cycle Proteins analysis, Cloning, Molecular, DNA chemistry, Humans, Nuclear Proteins analysis, Polymerase Chain Reaction, Receptors, Antigen, T-Cell analysis, Receptors, Antigen, T-Cell chemistry, Brachyura enzymology, DNA genetics, DNA Mutational Analysis methods, Deoxyribonucleases chemistry, Polymorphism, Single Nucleotide genetics
- Abstract
We have developed a simple method for fast analysis of single nucleotide polymorphisms and identification of target clones from cloned complex PCR products. The method utilizes Kamchatka crab duplex-specific nuclease and universal fluorescent probe and is alternative to laborious screening procedures using radioactive probes, restriction analysis followed by gel electrophoresis or expensive sequencing. The method efficacy was demonstrated in several model experiments.
- Published
- 2011
- Full Text
- View/download PDF
24. Normalization of full-length-enriched cDNA.
- Author
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Bogdanova EA, Barsova EV, Shagina IA, Scheglov A, Anisimova V, Vagner LL, Lukyanov SA, and Shagin DA
- Subjects
- Animals, Anomura enzymology, Anomura genetics, DNA, Complementary metabolism, DNA, Single-Stranded genetics, Deoxyribonucleotides metabolism, Electrophoresis, Agar Gel methods, Endonucleases metabolism, Nucleic Acid Denaturation, Nucleic Acid Hybridization, Polymerase Chain Reaction, Sequence Analysis, DNA, Complementary analysis, DNA, Complementary genetics, Gene Library
- Abstract
A well-recognized obstacle to efficient high-throughput analysis of cDNA libraries is the differential abundance of various transcripts in any particular cell type. Decreasing the prevalence of clones representing abundant transcripts before sequencing, using cDNA normalization, may significantly increase the efficacy of random sequencing and is essential for rare gene discovery. Duplex-specific nuclease (DSN) normalization allows the generation of normalized full-length-enriched cDNA libraries to permit a high gene discovery rate. The method is based on the unique properties of DSN from the Kamchatka crab and involves denaturation-reassociation of cDNA, degradation of the ds-fraction formed by abundant transcripts by DSN, and PCR amplification of the remaining ss-DNA fraction. The method has been evaluated in various plant and animal models.
- Published
- 2011
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- View/download PDF
25. A new set of markers for human identification based on 32 polymorphic Alu insertions.
- Author
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Mamedov IZ, Shagina IA, Kurnikova MA, Novozhilov SN, Shagin DA, and Lebedev YB
- Subjects
- Alleles, Chromosomes, Human genetics, Gene Frequency genetics, Genetic Loci genetics, Genetic Markers, Heterozygote, Humans, Polymerase Chain Reaction, Alu Elements genetics, Forensic Anthropology methods, Mutagenesis, Insertional genetics, Polymorphism, Genetic
- Abstract
A number of genetic systems for human genetic identification based on short tandem repeats or single nucleotide polymorphisms are widely used for crime detection, kinship studies and in analysis of victims of mass disasters. Here, we have developed a new set of 32 molecular genetic markers for human genetic identification based on polymorphic retroelement insertions. Allele frequencies were determined in a group of 90 unrelated individuals from four genetically distant populations of the Russian Federation. The mean match probability and probability of paternal exclusion, calculated based on population data, were 5.53 x 10(-14) and 99.784%, respectively. The developed system is cheap and easy to use as compared to all previously published methods. The application of fluorescence-based methods for allele discrimination allows to use the human genetic identification set in automatic and high-throughput formats.
- Published
- 2010
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- View/download PDF
26. Normalizing cDNA libraries.
- Author
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Bogdanov EA, Shagina I, Barsova EV, Kelmanson I, Shagin DA, and Lukyanov SA
- Subjects
- Base Sequence, Cloning, Molecular methods, DNA, Complementary metabolism, Deoxyribonucleases metabolism, Molecular Sequence Data, Oligonucleotides genetics, Oligonucleotides metabolism, Polymerase Chain Reaction methods, DNA, Complementary genetics, Gene Library
- Abstract
The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses., ((c) 2010 by John Wiley & Sons, Inc.)
- Published
- 2010
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27. Individual characterization of stably expanded T cell clones in ankylosing spondylitis patients.
- Author
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Mamedov IZ, Britanova OV, Chkalina AV, Staroverov DB, Amosova AL, Mishin AS, Kurnikova MA, Zvyagin IV, Mutovina ZY, Gordeev AV, Khaidukov SV, Sharonov GV, Shagin DA, Chudakov DM, and Lebedev YB
- Subjects
- Amino Acid Sequence, Clone Cells immunology, Flow Cytometry, Humans, Male, Middle Aged, Molecular Sequence Data, Perforin metabolism, Sequence Analysis, DNA, T-Lymphocytes chemistry, CD3 Complex chemistry, CD3 Complex genetics, CD3 Complex metabolism, Lymphocyte Activation, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell metabolism, Spondylitis, Ankylosing immunology, T-Lymphocytes immunology
- Abstract
Ankylosing spondylitis (AS) is commonly characterized by clonal expansions of T cells. However, these clonal populations are poorly studied and their role in disease initiation and progression remains unclear. Here, we performed mass sequencing of TCR V beta libraries to search for the expanded T cell clones for two AS patients. A number of clones comprising more than 5% of the corresponding TCR V beta family were identified in both patients. For the first time, expanded clones were shown to be stably abundant in blood samples of AS patients for the prolonged period (1.5 and 2.5 years for two patients, correspondingly). These clones were individually characterized in respect to their differentiation status using fluorescent cell sorting with CD27, CD28, and CD45RA markers followed by quantitative identification of each clone within corresponding fraction using real time PCR analysis. Stable clones differed in phenotype and several were shown to belong to the proinflammatory CD27 - /CD28 - population. Their potentially cytotoxic status was confirmed by staining with perforin-specific antibodies. Search for the TCR V beta CRD3 sequences homologous to the identified clones revealed close matches with the previously reported T cell clones from AS and reactive arthritis patients, thus supporting their role in the disease and proposing consensus TCR V beta CDR3 motifs for AS. Interestingly, these motifs were also found to have homology with earlier reported virus-specific CDR3 variants, indicating that viral infections could play role in development of AS.
- Published
- 2009
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28. DSN depletion is a simple method to remove selected transcripts from cDNA populations.
- Author
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Bogdanova EA, Shagina IA, Mudrik E, Ivanov I, Amon P, Vagner LL, Lukyanov SA, and Shagin DA
- Subjects
- Animals, Anomura genetics, Anomura metabolism, Anthozoa enzymology, Anthozoa genetics, DNA, Complementary genetics, Deoxyribonucleases genetics, Female, Humans, Nucleic Acid Hybridization, Phosphoric Diester Hydrolases genetics, Phosphoric Diester Hydrolases metabolism, Placenta enzymology, Placenta metabolism, Polymerase Chain Reaction, Polymorphism, Single Nucleotide, Anomura enzymology, DNA, Complementary metabolism, Deoxyribonucleases metabolism, Gene Library
- Abstract
A novel DSN-depletion method allows elimination of selected sequences from full-length-enriched cDNA libraries. Depleted cDNA can be applied for subsequent EST sequencing, expression cloning, and functional screening approaches. The method employs specific features of the kamchatka crab duplex-specific nuclease (DSN). This thermostable enzyme is specific for double-stranded (ds) DNA, and is thus used for selective degradation of ds DNA in complex nucleic acids. DSN depletion is performed prior to library cloning, and includes the following steps: target cDNA is mixed with excess driver DNA (representing fragments of the genes to be eliminated), denatured, and allowed to hybridize. During hybridization, driver molecules form hybrids with the target sequences, leading to their removal from the ss DNA fraction. Next, the ds DNA fraction is hydrolyzed by DSN, and the ss fraction is amplified using long-distance PCR. DSN depletion has been tested in model experiments.
- Published
- 2009
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29. Is crab duplex-specific nuclease a member of the Serratia family of non-specific nucleases?
- Author
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Anisimova VE, Shcheglov AS, Bogdanova EA, Rebrikov DV, Nekrasov AN, Barsova EV, Shagin DA, and Lukyanov SA
- Subjects
- Animals, Binding Sites, Molecular Structure, Mutagenesis, Site-Directed, Phylogeny, Protein Structure, Tertiary, Brachyura enzymology, Deoxyribonucleases chemistry, Deoxyribonucleases classification, Serratia enzymology
- Abstract
Kamchatka crab duplex-specific nuclease (Par_DSN) has been classified as a member of the family of DNA/RNA non-specific beta-beta-alpha metal finger (bba-Me-finger) nucleases, the archetype of which is the nuclease from Serratia marcescens. Although the enzyme under investigation seems to belong to the family of S. marcescens nucleases, Par_DSN exhibits a marked preference for double-stranded DNA as a substrate and this property is unusual for other members of this family. We have searched other Arthropod species and identified a number of novel Par_DSN homologs. A phylogenetic analysis demonstrates that the Par_DSN-like enzymes constitute a separate branch in the evolutionary tree of bba-Me-finger nucleases. Combining sequence analysis and site-directed mutagenesis, we found that Par_DSN and its homologs possess the nuclease domain that is slightly longer than that of classic Serratia relatives. The active site composition of Par_DSN is similar but not identical to that of classic Serratia nucleases. Based on these findings, we proposed a new classification of Par_DSN-like nucleases.
- Published
- 2008
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30. Isolation, characterization and molecular cloning of duplex-specific nuclease from the hepatopancreas of the Kamchatka crab.
- Author
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Anisimova VE, Rebrikov DV, Shagin DA, Kozhemyako VB, Menzorova NI, Staroverov DB, Ziganshin R, Vagner LL, Rasskazov VA, Lukyanov SA, and Shcheglov AS
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Brachyura genetics, Endonucleases chemistry, Endonucleases genetics, Molecular Sequence Data, Brachyura enzymology, Cloning, Molecular methods, Endonucleases isolation & purification, Hepatopancreas enzymology
- Abstract
Background: Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking., Results: Using degenerative primers and the rapid amplification of cDNA ends (RACE) procedure, we cloned the Duplex-Specific Nuclease (DSN) gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 - 7.5) and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact., Conclusion: We describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate specificity of DSN should prove valuable in certain molecular biology applications.
- Published
- 2008
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- View/download PDF
31. Normalization of full-length enriched cDNA.
- Author
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Bogdanova EA, Shagin DA, and Lukyanov SA
- Subjects
- Animals, DNA, Complementary metabolism, Deoxyribonucleases metabolism, DNA, Complementary analysis, DNA, Complementary genetics, Gene Library
- Abstract
Analysis of rare messages in cDNA libraries is extremely difficult due to the substantial variations in the abundance of different transcripts in cells and tissues. Therefore, for rare transcript searches and analyses, the generation of equalized (normalized) cDNA is essential. Several cDNA normalization methods have been developed since 1990. A number of these methods have been optimized for the normalization of full-length enriched cDNA, and used in various applications, including transcriptome analysis and functional screening of cDNA libraries. One such procedure (named DSN-normalization) is based on the unique properties of duplex-specific nuclease (DSN) from kamchatka crab and allows the generation of normalized cDNA libraries with a high gene discovery rate.
- Published
- 2008
- Full Text
- View/download PDF
32. [The Sse9I restriction-modification system: organization of genes and comparative analysis of proteins structures].
- Author
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Gonchar DA, Abdurashitov MA, Okhapkina SS, Shagin DA, Kileva EV, and Degtiarev SKh
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, DNA Restriction-Modification Enzymes genetics, Deoxyribonuclease EcoRI genetics, Deoxyribonucleases, Type II Site-Specific genetics, Gram-Positive Endospore-Forming Bacteria enzymology, Molecular Sequence Data, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Site-Specific DNA-Methyltransferase (Adenine-Specific) genetics, Site-Specific DNA-Methyltransferase (Adenine-Specific) isolation & purification, DNA Restriction-Modification Enzymes metabolism, Operon, Site-Specific DNA-Methyltransferase (Adenine-Specific) metabolism
- Abstract
A nucleotide sequence was established for the full-length Sporosarcina species 9D operon coding for enzymes of type II restriction-modification system Sse9I. These enzymes recognize the tetranucleotide DNA sequence 5'-AATT-3'. The operon was shown to consist of three genes that are situated with the order: sse9IC-sse9IR-sse9IM and are transcribed in the same direction. These genes encode the control protein (C.Sse9I), restriction endonuclease (R.Sse9I) and DNA-methyltransferase (M.Sse9I), respectively. A specific DNA sequence (C-box) presumably recognized by C-protein was found immediately upstream of sse9IC gene. The comparative analysis of amino acid sequences of C.Sse9I and R.Sse9I with those of relative proteins has been done. It was found that R.Sse9I revealed the most homology with the segments of R.MunI (5'-CAATTG-3') and R.EcoRI (5'-GAATTC-3'), where amino acid residues, responsible for recogniton of AATT core sequence are located. The sse9IR gene was cloned into the temperature-inducible expression vector, and recombinant Sse9I restriction endonuclease preparation was isolated.
- Published
- 2007
33. Decoding the fine-scale structure of a breast cancer genome and transcriptome.
- Author
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Volik S, Raphael BJ, Huang G, Stratton MR, Bignel G, Murnane J, Brebner JH, Bajsarowicz K, Paris PL, Tao Q, Kowbel D, Lapuk A, Shagin DA, Shagina IA, Gray JW, Cheng JF, de Jong PJ, Pevzner P, and Collins C
- Subjects
- Cell Line, Tumor, Chromosomes, Artificial, Bacterial metabolism, Chromosomes, Human, Female, Gene Expression Profiling methods, Genome, Human, Humans, In Situ Hybridization, Fluorescence, Molecular Sequence Data, Polymerase Chain Reaction, Reproducibility of Results, Breast Neoplasms genetics, Sequence Analysis, DNA methods, Transcription, Genetic
- Abstract
A comprehensive understanding of cancer is predicated upon knowledge of the structure of malignant genomes underlying its many variant forms and the molecular mechanisms giving rise to them. It is well established that solid tumor genomes accumulate a large number of genome rearrangements during tumorigenesis. End Sequence Profiling (ESP) maps and clones genome breakpoints associated with all types of genome rearrangements elucidating the structural organization of tumor genomes. Here we extend the ESP methodology in several directions using the breast cancer cell line MCF-7. First, targeted ESP is applied to multiple amplified loci, revealing a complex process of rearrangement and co-amplification in these regions reminiscent of breakage/fusion/bridge cycles. Second, genome breakpoints identified by ESP are confirmed using a combination of DNA sequencing and PCR. Third, in vitro functional studies assign biological function to a rearranged tumor BAC clone, demonstrating that it encodes anti-apoptotic activity. Finally, ESP is extended to the transcriptome identifying four novel fusion transcripts and providing evidence that expression of fusion genes may be common in tumors. These results demonstrate the distinct advantages of ESP including: (1) the ability to detect all types of rearrangements and copy number changes; (2) straightforward integration of ESP data with the annotated genome sequence; (3) immortalization of the genome; (4) ability to generate tumor-specific reagents for in vitro and in vivo functional studies. Given these properties, ESP could play an important role in a tumor genome project.
- Published
- 2006
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34. [Application of the duplex-specific nuclease preference method to the analysis of point mutations in human genes].
- Author
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Al'tshuler IM, Zhulidov PA, Bogdanova EA, Mudrik NN, and Shagin DA
- Subjects
- Alleles, Animals, Brachyura enzymology, DNA Primers, Genes, ras, Genetic Diseases, Inborn genetics, Humans, Point Mutation, DNA chemistry, Endonucleases chemistry, Genome, Human, Polymorphism, Single Nucleotide
- Abstract
A new modification of the single nucleotide polymorphism (SNP) analysis (DSNP, duplex-specific nuclease preference) method using the duplex-specific nuclease from the king crab was proposed. The method was used to study SNPs in the following human genes: kRAS, nRAS, hRAS, and p53, the genes of blood coagulation factor V, methyltetrahydrofolate reductase, prothrombin, and apolipoprotein E and a deletion in the BRCA1 gene. DSNP was shown to be useful for the estimation of the mutant allele content in DNA samples. A system for the simultaneous identification of several adjacent single-nucleotide polymorphisms in the kRAS gene was proposed. The approaches could be used to develop test systems for the detection of SNPs in human genes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.
- Published
- 2005
35. [A method for the preparation of normalized cDNA libraries enriched with full-length sequences].
- Author
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Zhulidov PA, Bogdanova EA, Shcheglov AS, Shagina IA, Vagner LL, Khazpekov GL, Kozhemiako VV, Luk'ianov SA, and Shagin DA
- Subjects
- Animals, Brachyura, Endonucleases chemistry, Humans, Nucleic Acid Hybridization, Polymerase Chain Reaction, DNA, Complementary chemistry, DNA, Single-Stranded chemistry, Gene Library
- Abstract
We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.
- Published
- 2005
- Full Text
- View/download PDF
36. [Spectral diversity among the members of the family of Green Fluorescent Protein in hydroid jellyfish (Cnidaria, Hydrozoa)].
- Author
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Ianushevich IuG, Shagin DA, Fradkov AF, Shakhbazov KS, Barsova EV, Gurskaia NG, Labas IuA, Matts MV, Luk'ianov kA, and Lul'ianov SA
- Subjects
- Amino Acid Sequence, Animals, Cloning, Molecular, Green Fluorescent Proteins genetics, Hydrozoa genetics, Molecular Sequence Data, Spectrometry, Fluorescence, Green Fluorescent Proteins chemistry, Hydrozoa chemistry
- Abstract
The cDNAs encoding the genes of new proteins homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria were cloned. Two green fluorescent proteins from one un-identified anthojellyfish, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent chromoprotein from another unidentified anthojellyfish were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.
- Published
- 2005
37. [A family of genes of multidomain free lectins from a planarian: structure, expression, and use as markers for regeneration monitoring ].
- Author
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Bogdanova EA, Barsova EV, Pun'kova NI, Britanova OV, Shagin DA, Gurskaia NG, Usman NIu, and Luk'ianov SA
- Subjects
- Amino Acid Sequence, Animals, Helminth Proteins metabolism, Immunohistochemistry, Introns, Lectins, C-Type metabolism, Microscopy, Electron, Transmission, Molecular Sequence Data, Planarians metabolism, Planarians ultrastructure, Regeneration genetics, Helminth Proteins genetics, Lectins, C-Type genetics, Planarians genetics, Regeneration physiology
- Abstract
A family of genes of the agamic race of planarian Girardia tigrina were described that encode proteins that belong to the superfamily of C-type lectins and were demonstrated to have a unique domain organization. The genes are differentially expressed in the planarian body. The protein products of at least two genes (scarf2 and gtlec1) are expressed in specifically differentiated gland cells of the planarian and secreted into the environment through long cell necks. A comparison of the results obtained by electron microscopy and immunohistochemistry with literature data allows the assignment of these cells to the group of adhesion glands. The observation of the regeneration of the cell necks in normal and artificial two-headed planaria indicated that the dorsoventral contact at the edge of the head part of the planarian body directs and maintains the growth of the gtLec1-producing cell necks during regeneration. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.
- Published
- 2004
- Full Text
- View/download PDF
38. GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity.
- Author
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Shagin DA, Barsova EV, Yanushevich YG, Fradkov AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers A, Nunez JM, Widder EA, Lukyanov SA, and Matz MV
- Subjects
- Animals, Bacterial Proteins genetics, Biotechnology, Cloning, Molecular, Crustacea genetics, DNA, Complementary metabolism, Evolution, Molecular, Green Fluorescent Proteins metabolism, Hydrozoa genetics, Luminescent Proteins genetics, Microscopy, Fluorescence, Molecular Sequence Data, Phylogeny, Protein Structure, Tertiary, Spectrophotometry, Green Fluorescent Proteins genetics, Multigene Family
- Abstract
Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.
- Published
- 2004
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- View/download PDF
39. Simple cDNA normalization using kamchatka crab duplex-specific nuclease.
- Author
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Zhulidov PA, Bogdanova EA, Shcheglov AS, Vagner LL, Khaspekov GL, Kozhemyako VB, Matz MV, Meleshkevitch E, Moroz LL, Lukyanov SA, and Shagin DA
- Subjects
- Animals, Aplysia genetics, Central Nervous System metabolism, DNA, Complementary genetics, Gene Library, Humans, Molecular Biology methods, Molecular Sequence Data, Muscle, Skeletal metabolism, Brachyura enzymology, DNA, Complementary metabolism, Deoxyribonucleases metabolism
- Abstract
We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.
- Published
- 2004
- Full Text
- View/download PDF
40. A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas.
- Author
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Shagin DA, Rebrikov DV, Kozhemyako VB, Altshuler IM, Shcheglov AS, Zhulidov PA, Bogdanova EA, Staroverov DB, Rasskazov VA, and Lukyanov S
- Subjects
- Animals, Cloning, Molecular methods, Liver enzymology, Models, Genetic, Molecular Sequence Data, Oligonucleotides chemical synthesis, Oligonucleotides metabolism, Pancreas enzymology, Substrate Specificity genetics, Anomura enzymology, Anomura genetics, Endonucleases genetics, Nucleic Acid Heteroduplexes genetics, Polymorphism, Single Nucleotide genetics
- Abstract
We have characterized a novel nuclease from the Kamchatka crab, designated duplex-specific nuclease (DSN). DSN displays a strong preference for cleaving double-stranded DNA and DNA in DNA-RNA hybrid duplexes, compared to single-stranded DNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is essentially higher than that for nonperfectly matched duplexes of the same length. Thus, DSN differentiates between one-nucleotide variations in DNA. We developed a novel assay for single nucleotide polymorphism (SNP) detection based on this unique property, termed "duplex-specific nuclease preference" (DSNP). In this innovative assay, the DNA region containing the SNP site is amplified and the PCR product mixed with signal probes (FRET-labeled short sequence-specific oligonucleotides) and DSN. During incubation, only perfectly matched duplexes between the DNA template and signal probe are cleaved by DSN to generate sequence-specific fluorescence. The use of FRET-labeled signal probes coupled with the specificity of DSN presents a simple and efficient method for detecting SNPs. We have employed the DSNP assay for the typing of SNPs in methyltetrahydrofolate reductase, prothrombin and p53 genes on homozygous and heterozygous genomic DNA.
- Published
- 2002
- Full Text
- View/download PDF
41. Altering electrical connections in the nervous system of the pteropod mollusc Clione limacina by neuronal injections of gap junction mRNA.
- Author
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Kelmanson IV, Shagin DA, Usman N, Matz MV, Lukyanov SA, and Panchin YV
- Subjects
- Animals, Brain cytology, Brain metabolism, Cell Communication drug effects, Cell Communication genetics, Ganglia, Invertebrate cytology, Ganglia, Invertebrate metabolism, Gap Junctions drug effects, Membrane Potentials drug effects, Membrane Potentials genetics, Molecular Sequence Data, Mollusca cytology, Mollusca physiology, Neural Pathways cytology, Neural Pathways metabolism, Neurons cytology, Neurons drug effects, Organ Culture Techniques, RNA, Messenger pharmacology, Brain growth & development, Connexins genetics, Ganglia, Invertebrate growth & development, Gap Junctions metabolism, Mollusca growth & development, Neural Pathways growth & development, Neurons metabolism
- Abstract
Neurons can communicate with each other either via exchange of specific molecules at synapses or by direct electrical connections between the cytoplasm of either cell [for review see Bruzzone et al. (1996) Eur. J. Biochem., 238, 1-27]. Although electrical connections are abundant in many nervous systems, little is known about the mechanisms which govern the specificity of their formation. Recent cloning of the innexins--gap junction proteins responsible for electrical coupling in invertebrates (Phelan et al. (1998) Trends Genet., 14, 348-349], has made it possible to study the molecular mechanisms of patterning of the electrical connections between individual neurons in model systems. Here we demonstrate that intracellular injection of mRNA encoding the molluscan innexin Panx1 (Panchin et al. 2000 Curr. Biol., 10, R473-R474) drastically alters the specificity of electrical coupling between identified neurons of the pteropod mollusc Clione limacina.
- Published
- 2002
- Full Text
- View/download PDF
42. Identification and characterization of a new family of C-type lectin-like genes from planaria Girardia tigrina.
- Author
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Shagin DA, Barsova EV, Bogdanova E, Britanova OV, Gurskaya N, Lukyanov KA, Matz MV, Punkova NI, Usman NY, Kopantzev EP, Salo E, and Lukyanov SA
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Calcium metabolism, Conserved Sequence, Exons, Helminth Proteins chemistry, Introns, Lectins, C-Type chemistry, Microtubules ultrastructure, Molecular Sequence Data, Mucus metabolism, Phylogeny, Planarians ultrastructure, Protein Sorting Signals, Protein Structure, Tertiary, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Lectins, C-Type genetics, Planarians genetics
- Abstract
A novel family of C-type lectin-like genes, denoted multidomain free lectin (MDFL), was identified in the freshwater planaria Girardia (Dugesia) tigrina. We cloned several genes that encode proteins comprising a signal peptide and a number of consecutive C-type lectin-like domains (CTLDs) interconnected by short linker stretches. Analyses of genomic organization, CTLD amino acid sequences, and the overall architecture of these proteins indicate that planarian proteins are a separate family of C-type lectin-like proteins. These genes are expressed in specifically differentiated gland cells of planaria and the corresponding proteins are excreted as components of the planarian body surface mucus.
- Published
- 2002
- Full Text
- View/download PDF
43. Regulation of average length of complex PCR product.
- Author
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Shagin DA, Lukyanov KA, Vagner LL, and Matz MV
- Subjects
- Bacteriophage lambda genetics, DNA chemistry, DNA, Complementary, DNA, Viral analysis, DNA, Viral chemistry, Particle Size, DNA analysis, Polymerase Chain Reaction methods
- Abstract
A method to achieve the preference towards longer products during PCR is described. The extent of this preference can be adjusted by slight variation of the PCR conditions. Being combined with the natural tendency of PCR to amplify shorter fragments more efficiently than longer ones, it allows one to regulate the average length of the complex PCR product over a very wide range to make it most suitable for further manipulations. The technique can be used for amplifying any complex DNA sample.
- Published
- 1999
- Full Text
- View/download PDF
44. [Cloning of region-specific genetic markers of planaria using a new method--ordered differential display].
- Author
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Mats MV, Shagin DA, Usman NIu, Bogdanova EA, Fradkov AF, Soboleva TA, and Luk'ianov SA
- Subjects
- Animals, Base Sequence, DNA Primers, DNA, Complementary, RNA, Messenger genetics, Reverse Transcriptase Polymerase Chain Reaction, Cloning, Molecular methods, Genetic Markers genetics, Planarians genetics
- Abstract
A new method for finding differentially expressed genes, termed ordered differential display of mRNAs (ODD), was used in the search for region-specific molecular markers of freshwater planarian Dugesia tigrina. In this method, the effect of selective suppression of a polymerase chain reaction (PCR) is used for the differential amplification of a pool of 3'-terminal cDNA fragments generated by digestion of cDNAs with a restriction endonuclease. In the resulting amplified cDNAs, every mRNA is represented by a cDNA fragment whose length is determined by the position of the restriction site nearest to the 3'-terminus. Subsequent PCR with primers 3'-extended by two random nucleotides allowed the amplification of 1/192 part of all cDNA molecules present in the sample. The comparison of the generated pools of cDNA molecules separated by PAGE leads to the identification of differentially expressed sequences. The systematic study of the total mRNA pool is achieved by the successive use of all possible combinations of extended primers. Some sequences preferentially expressed along the anterior-posterior axis of planarian were identified using ODD.
- Published
- 1998
45. [Cloning cDNA for the ha-SDGF gene from a Syrian hamster cell line with increased metastatic potential using subtractive hybridization].
- Author
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Gurskaia NG, Shagin DA, Luk'ianov KA, Vagner LL, Shtutman MS, Musatkina EA, Moinova EV, Tatosian AG, Luk'ianov SA, and Sverdlov ED
- Subjects
- Amino Acid Sequence, Amphiregulin, Animals, Cell Line, Transformed, Cloning, Molecular, Cricetinae, DNA, Complementary, EGF Family of Proteins, Humans, Mesocricetus, Molecular Sequence Data, Neoplasm Metastasis genetics, Nucleic Acid Hybridization, Rodentia, Sequence Homology, Amino Acid, Glycoproteins, Growth Substances genetics, Intercellular Signaling Peptides and Proteins
- Abstract
Using subtractive hybridization, a cDNA library containing over 50% of clones specific for a highly metastatic cell line was obtained from two hamster embryo fibroblast lines with different metastatic potentials. Most of the clones (83%) contained new sequences. One clone contained the ha-SDGF gene cDNA homologous to SDGF cDNA from rodents. The level of ha-SDGF mRNA expression was considerably higher in the highly metastatic cell line.
- Published
- 1996
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