GLAD-PCR Assay of R(5mC)GY Sites in the Regulatory Region of Tumor-Suppressor Genes Associated with Gastric Cancer.

At early stages of carcinogenesis, the regulatory regions of some tumor suppressor genes become aberrantly methylated at RCGY sites, which are substrates of DNA methyltransferase Dnmt3. Identification of aberrantly methylated sites in tumor DNA is considered to be the first step in the development of epigenetic PCR test systems for early diagnosis of cancer. Recently, we have developed a GLAD-PCR assay, a method for detecting the R(5mC)GY site in the genome position of interest even at significant excess of DNA molecules with a non-methylated RCGY site in this location. The aim of the present work is to use the GLAD-PCR assay to detect the aberrantly methylated R(5mC)GY sites in the regulatory regions of tumor suppressor genes (brinp1, bves, cacna2d3, cdh11, cpeb1, epha7, fgf2, galr1, gata4, hopx, hs3st2, irx1, lrrc3b, pcdh10, rprm, runx3, sfrp2, sox17, tcf21, tfpi2, wnt5a, zfp82, and znf331) in DNA samples obtained from gastric cancer (GC) tissues. The study of the DNA samples derived from 29 tumor and 25 normal gastric tissue samples demonstrated a high diagnostic potential of the selected RCGY sites in the regulatory regions of the irx1, cacna2d3, and epha7 genes; the total indices of sensitivity and specificity for GC detection being 96.6% and 100%, respectively.

[Application of GLAD-PCR Assay for Study on DNA Methylation in Regulatory Regions of Some Tumor-Suppressor Genes in Lung Cancer].

Hypermethylation of the gene regulatory regions are common for many cancer diseases. In this work we applied GLAD-PCR assay for identificating of the aberrantly methylated RCGY sites in the regulatory regions of some downregulated genes in tissue samples of lung cancer (LC). This list includes EFEMP1, EPHA5, HOXA5, HOXA9, LHX1, MYF6, NID2, OTX1, PAX9, RARB, RASSF1A, RXRG, SIX6, SKOR1 and TERT genes. The results of DNA samples from 40 cancer and 25 normal lung tissues showed a good diagnostic potential of selected RCGY sites in regulatory regions of MYF6, SIX6, RXRG, LHX1, RASSF1A and TERT genes with relatively high sensitivity (80.0 %) and specificity (88.0 %) of LC detection in tumor DNA.

[Application of GLAD-PCR analysis for the methylation sites detection in the regulatory areas of tumor-suppressor genes ELMo1 and EsR1 in colorectal cancer].

Aberrant methylation of regulation regions of tumorsuppressor genes is showed for many cancer diseases. In course of this modification an enzyme DNMT3 methylates RCGY sites in CpG-islands of regulation regions producing R(5mC)GY sites. Earlier we developed GLAD-PCR assay to determine R(5mC)GY site in a definite position of human genome. In this work we have applied GLAD-PCR assay to determine R(5mC)GY sites in regulation regions of ESR1 and ELMO1 tumor-suppressor genes. We have studied a fragment of first exon of ELMO1 gene and a part of ESR1 promoter region in DNA preparations from malignant cell line SW837 and colorectal tumor samples. We have checked four sites in each region and found two highly methylated sites: GCGC in first exon of ELMO1 gene and GCGT in promoter region of ESR1 gene. Site GCGT is weakly methylated in healthy tissues and more methylated in the most of colorectal samples. Site GCGC is not methylated in healthy tissues and significantly methylated in 60% of colorectal samples. A possibility to use GLAD-PCR assay for cancer diagnostics is discussed.

[Crimean-Congo hemorrhagic fever virus in Stavropol region in 2011].

The genetic analysis of the Crimean-Congo hemorrhagic fever (CCHF) virus circulating in Stavropol region during 2011 year was suggested. A total of 14 RNA isolates from the Crimean hemorrhagic fever patients were genetically typed. The genetic analysis of the CCHF virus stains based on M-segment sequences (positions 2607-2932) supported the circulation of the genotype Europe 1 in the Stavropol region of Russia. In addition to previously known lineage STV-ROS, the second lineage VLG/ROS was observed in Stavropol region.

[Design of deoxyribozymes for inhibition of influenza A virus].

Influenza A viruses take a significant place in human and animal pathology causing epidemics and epizootics. Therefore, the development of new antiflu drugs has become more and more urgent. Deoxyribozymes can be considered as promising antiviral agents due to their ability to efficiently and highly specifically cleave RNA molecules. In this study, a number ofgenomic sequences of the most relevant influenza A virus subtypes, H5N1, H3N2, and H1N1, were analyzed. Conservative regions were revealed in five the least variable segments of the fragmented viral RNA genome, and potential sites of their cleavage with "10-23" deoxyribozymes were determined. 46 virus-specific 33-mer deoxyribozymes with the general structure of 5'N8AGGCTAGCTACAACGAN9 were designed and synthesized. Screening of the antiviral activity of these agents in conjugation with lipofectin on the Madin-Darby Canine Kidney cells infected with highly pathogenic avian influenza virus A/chicken/Kurgan/05/2005 (H5N1) revealed 17 deoxyribozymes, which suppressed the titer of virus cytopathicity by more than 2.5 IgTCID50/mL (i.e. the virus neutralization index was more than 300), with five of them suppressing the virus titer by a factor of 1000 and more. The most active deoxyribozymes appeared to be specific to segment 5 of the influenza A virus genome, which encoded nucleoprotein (NP).

[Recombinant DNA-methyltransferase M1.Bst19I from Bacillus stearothermophilus 19: purification, propeties and amino acids sequence analysis].

The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5'-GCATC-3') in Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW, that carries a tandem of thermo inducible promoters P(R)/P(L) from phage lambda. Highly purified enzyme has been isolated by chromatography on various resins from E. coli cells where it accumulates in a soluble form. Study of M1.Bst19I properties has revealed that enzyme has a temperature optimum 50 degrees C and demonstrates the maximum activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5'-GCATC-3'. Kinetic parameters of M1.Bst19I DNA methylation reaction have been determined as follows: Km for lambda DNA is 0.68 +/- 0.07 microM, Km for S-adenosil-L-methionine is 2.02 +/- 0.31 microM. Catalytical constant (kcat) is 1.8 +/- 0.05 min(-1). Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and others alpha-N6-DNA methyltransferases has allowed to suppose a presence of two types of the enzymes containing a triplet ATG or ATC in the recognition sequence.

Substrate specificity and biochemical properties of M3.BstF5I DNA methyltransferase from the BstF5I restriction-modification system.

Optimal conditions for DNA methylation by the M3.BstF5I enzyme from Bacillus stearothermophilus and kinetic parameters of lambda phage DNA modification and that of a number of oligonucleotide substrates are established. Comparison of M1.BstF5I and M3.BstF5I kinetic parameters revealed that with similar temperature optima and affinity for DNA, M3.BstF5I has nearly fourfold lower turnover number (0.24 min(-1)) and modifies the hemimethylated recognition site with lower efficiency under optimal conditions than the unmethylated one. In contrast to another three methylases of the BstF5I restriction-modification system, the M3.BstF5I enzyme is able to optionally modify the noncanonical 5'-GGATC-3' DNA sequence with a rate more than one order of magnitude lower than the methylation rate of the canonical 5'-GGATG-3' recognition site.

Recombinant DNA-methyltransferase M1.BspACI from Bacillus psychrodurans AC: purification and properties.

A restriction-modification system from Bacillus psychrodurans AC (recognition sequence 5'-CCGC-3') comprises two DNA methyltransferases: M1.BspACI and M2.BspACI. The bspACIM1 gene was cloned in the pJW2 vector and expressed in Escherichia coli cells. High-purity M1.BspACI preparation has been obtained by chromatography on different carriers. M1.BspACI has a temperature optimum of 30°C and demonstrates maximum activity at pH 8.0. M1.BspACI modifies the first cytosine in the recognition sequence 5'-CCGC-3'. The kinetic parameters of M1.BspACI DNA methylation are as follows: K(m) for phage λ DNA is 0.053 µM and K(m) for S-adenosyl-L-methionine is 5.1 µM. The catalytic constant (k(cat)) is 0.095 min(-1).

[Design of oligonucleotide inhibitors of the human DNA-methyltransferase 1].

Mammalian DNA methyltransferase 1 (Dnmt1) is responsible for copying DNA methylation patterns during cell division. A number of studies demonstrate that Dnmt1 plays an important role in carcinogenesis, that causes, in particular, significant interest in searching for specific inhibitors of this enzyme. In the present study, with the purpose of design of oligonucleotide inhibitors of human Dnmt1, a number of single-, double-stranded and hairpin DNA-structures, containing canonical or modified enzyme recognition site 5'-CG were constructed on the basis of uniform 22 b sequence. It was shown, that such structural features as C:A-mismatch, phosphorothioates and hairpin are capable to incrementally increase oligonucleotide affinity to Dnmt1. The improvement of inhibitor properties were also achieved by substitution of target cytosine with 5,6-dihydro-5-azacytosine, 5-methyl-2-pyrimidinone and 6-methyl-pyrrolo-[2,3-d]-2-pyrimidinone. The concentrations of the most efficient oligonucleotides caused 50% inhibition of methylation of 1 microM conventional DNA substrate, polymer poly(dI-dC) * poly(dI-dC), were about 10(-7) M. In the equal in vitro conditions the constructed oligonucleotide inhibitors demonstrated much stronger effect compared to known inhibitors of Dnmt1, which were used as controls.

[Isolation and study of the immunobiological properties of the recombinant proteins of measles virus].

Recombinant proteins rN (nucleocapsid) and rH/Nh (hemagglutinin) of the measles virus strain NovO/96 of genotype A were obtained. The immunobiological properties of the proteins were studied in the reaction with a panel of positive and negative sera. Mice of the line Balb/C were immunized with recombinant proteins and native antigen of the measles virus strain NovO/96 in order to obtain hyperimmune serum and its analysis using ELISA (enzyme-linked immunosorbent assay) and PRN (plaque reduction neutralization). The hyperimmune serum against recombinant proteins and native antigen of the measles virus strain NovO/96 were found to be highly active in ELISA. The antibodies against the proteins rN and rH/Nh were found to be able to neutralize the virus in titer 1:13.5 and 1:22.9, respectively. The neutralization titer of the antibodies generated against native antigen of the measles virus strain NovO/96 was 1:25.7.

Study of measles virus recombinant proteins and their immunobiological properties.

Recombinant proteins rN (nucleocapsid) and rH/Nh (hemagglutinin) of the measles virus strain NovO/96 of genotype A were obtained. The immunobiological properties of the proteins were studied in the reaction with a panel of positive and negative sera. BALB/c mice were immunized with recombinant proteins and native antigen of the measles virus strain NovO/96 in order to obtain hyperimmune serum and its analysis using ELISA (enzyme-linked immunosorbent assay) and PRN (plaque reduction neutralization). The hyperimmune sera against recombinant proteins and native antigen of the measles virus strain NovO/96 were found to be highly active in ELISA. The antibodies against the proteins rN and rH/Nh were found to be capable of neutralizing the virus in titer 1 : 13.5 and 1 : 22.9, respectively. The neutralization titer of the antibodies generated against native antigen of the measles virus strain NovO/96 was 1 : 25.7.

M.BstF5I-2 and M.BstF5I-4 DNA methyltransferases from BstF5I restriction-modification system of Bacillus stearothermophilus F5.

The BstF5I restriction-modification system from Bacillus stearothermophilus F5 includes four site-specific DNA methyltransferases, thus differing from all known restriction-modification systems. Here we demonstrated for the first time that one bacterial cell can possess two pairs of methylases with identical substrate specificities (methylases BstF5I-1 and BstF5I-3 recognize GGATG, whereas methylases BstF5I-2 and BstF5I-4 recognize CATCC) that modify adenine residues on both DNA strands. Different chromatographic methods provide homogenous preparations of methylases BstF5I-2 and BstF5I-4. We estimated the principal kinetic parameters of the reaction of transfer of methyl group from the donor S-adenosyl-L-methionine to the recognition site 5;-CATCC-3; catalyzed by BstF5I-2 and BstF5I-4 DNA [N6-adenine]-methyltransferases from the BstF5I restriction-modification system.

[The unique FauI restriction-modification system: cloning and comparative analysis of protein structure]. / Unikal'naia sistema restriktsii-modifikatsii FauI: klonirovanie i sravnitel'nyi analiz struktury belkov.

The nucleotide sequence was established for the full-length Flavobacterium aquatile operon coding for the FauI restriction-modification system. The operon is unusual in structure and has the gene order control protein gene-DNA methyltransferase A gene-restriction endonuclease gene-DNA methyltransferase B gene, other than in the known analogs. The genes are similarly oriented and overlap. On evidence of sequence analysis, both methyltransferases are C5 enzymes, the control protein is similar to that of other restriction-modification systems, and restriction endonuclease is low-homologous to other enzymes cleaving the DNA upper strand in position 4 or 5 relative to the recognition site.

[Comparison of the homologous SfeI and LlaBI restriction-modification systems]. / Sravnitel'nyi analiz gomologichnykh sistem restriktsii-modifikatsii SfeI i LlaBI.

A fragment containing the SfeI restriction-modification system (RMS) operon was cloned from a Streptococcus faecalis SE72 plasmid. Nucleotide sequence analysis revealed its high (99.2%) homology to the operon for Lactococcus lactis subsp. cremoris W56 LlaBI RMS recognizing the same site, 5'-CTRYAG-3'. A substantial difference was that SfeI RMS operon had an additional 198-bp fragment and a larger gene for the putative control protein. No homology was observed between operon-flanking sequences of the two closely related species, suggesting horizontal transfer of the operon.

[M.BstF5I-4, the forth DNA methyltransferase of BstF5I restriction-modification system from Bacillus stearothermophilus F5]. / M.BstF5I-4--chetvertaia DNK-metiltransferaza sistemy restriktsii-modifikatsii BstF5I iz Bacillus stearothermophilus F5.

The fourth DNA-methyltransferase of the BstF5I restriction-modification (RM) system from Bacillus stearothermophilus F5 (M.BstF5I-4) was discovered, which modifies the adenine residue within the upper strand of the recognition site 5'-GGATG-3'/5'-CATCC-3'. Thus, unlike other known RM systems, the BstF5I RM system comprises four genes encoding DNA-methyltransferases, three of which possess the same substrate specificity and methylate adenine within the 5'-GGATG sequence. The English version of the paper.

[Comparative study of the M.Bstf5I-1 and M.BstF5I-3 DNA methyltransferases from the Bacillus stearothermophilus F5 restriction-modification system]. / Sravnitel'noe izuchenie svoistv DNK-metiltransferaz M.BstF5I-1 i M.BstF5I-3 sistemy restriktsii-modifikatsii BstF5I iz Bacillus stearothermophilus F5.

The BstF5I restriction-modification system from Bacillus stearothermophilus F5, unlike all known restriction-modification systems, contains three genes encoding DNA methyltransferases. In addition to revealing two DNA methylases responsible for modification of adenine in different DNA strands, it has been first shown that one bacterial cell has two DNA methylases, M.BstF5I-1 and M.BstF5I-3, with similar substrate specificity. The boundaries of the gene for DNA methyltransferase M.BstF5I-1 have been verified. The bstF5IM-1 gene was cloned in pJW and expressed in Escherichia coli. Homogeneous samples of M.BstF5I-1 and M.BstF5I-3 were obtained by chromatography with different sorbents. The main kinetic parameters have been determined for M.BstF5I-1 and M.BstF5I-3, both modifying adenine in the recognition site 5'-GGATG-3'.

[Preparation of single-chained antibodies to surface glycoprotein E from tick-borne encephalitis virus]. / Poluchenie odnotsepochechnykh antitel k poverkhnostnomu glikoproteinu E virusa kleshchevogo éntsefalita.

A single-stranded antibody gene scFv was designed on the base of cDNA fragments of genes coding for variable domains of heavy and light chains of MAb E6B to tick-borne encephalitis glycoprotein E. High production of stable soluble scFv was reproduced in Escherichia coli cells. Recombinant antibodies bound to antiidiotypical antibodies to initial MAb E6B and to recombinant virus protein E. Competitive analysis showed that single-stranded antibodies inhibited reaction between MAb E6B and protein E. These results confirm the formation of scFv with the original antigen-binding specificity towards tick-borne encephalitis virus glycoprotein E.

Cloning and characterization of the gene encoding M.FauI DNA methyltransferase.

The enzymes of the FauI restriction-modification system from the Flavobacterium aquatile strain recognize the non-palindromic sequence 5'-CCCGC-3'/3'-GG-GCG-5'. We have cloned the gene encoding the DNA modifying component of this system and determined its nucleotide sequence. The deduced amino acid sequence contains ten conserved motifs characteristic for [cytosine-5] DNA methyltransferases. Part of the gene sequence that encodes the putative target recognizing domain of the M.FauI shows some homology with the downstream region, thus indicating that duplication of the DNA segment was probably involved in the gene evolution.