[Multiplicity of site-specific DNA-methyltransferases of the BstF5I restriction modification system from Bacillus stearothermophilus F5].
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Biomedical subjects
Publications and source records attributed to V V Gutorov.
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A new restriction endonuclease Sst12I belonging to the II type and recognizing the sequence 5'-CTGCAG-3' was isolated from the bacterial strain Streptomyces sp. St-12. The enzyme hydrolyzes DNA between adenine and guanine residues; thus, it is a true isoschizomer of restrictase PstI. In contrast to PstI, the restriction endonuclease Sst12I hydrolyses DNA both at 37 degrees and 55 degrees C and remains active after long-term storage.
Five antigen-positive samples isolated from patients with Crimean-Congo hemorrhagic fever (CCHF) and from Hyalomma marginatum ticks collected in the European part of Russia and three laboratory strains of CCHF isolated in Russia, Uzbekistan, and Tadjikistan were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Comparison of nucleotide sequences of fragments of CCHF virus genome S segment and phylogenetic analysis of Russian strains showed that all CCHF strains isolated from humans and H. marginatum circulating in Russia were closely related and differed essentially from CCHF variants from other regions. Strains isolated in Uzbekistan and Tadjikistan were most closely related to CCHF strains from China.
Complete S-segment nucleotide sequences of genomic RNA were determined for two Crimea-Congo hemorrhagic fever (CCHF) virus strains, i.e. LEIV 10145 Uz isolated from ticks in Uzbekistan, 1985, and LEIV 29223 Stv isolated from a patient in Stavropol region, 2000. It was established that the S-segment length is 1672 and 1674 nucleotides. Therefore, the initiating codon (for methionine) is located at positions 56-58; the length of translation frames for the nucleocapsid protein is 482 amino acid residues. Distinctions in the length of S-segment, as compared to other strains, are related only with the 5' and 3' non-coding regions. A comparison of the nucleotide and amino-acid sequences of S-segments of genome of the mentioned strains with the early published data showed that the CCHF virus strain isolated in Uzbekistan is mostly close to strains isolated in China, and that the strain isolated in Stavropol region forms, jointly with Drozdov strain isolated in the Astrakhan region, a separate branch in the phylogenetic tree.
HindIII and XhoI genome fragments of variola major virus strain India-1967 were inserted into the bacterial plasmids and cosmid. Sequencing and computer analysis of the region of HindIII M, L, and I DNA fragments of the virus studied have been carried out.
S-segment nucleotide sequences for two Crimean-Congo hemorrhagic fever (CCHF) virus strains isolated in the Rostov Region of Russia and in Bulgaria have been determined. Analysis of complete S-segment nucleotide sequences in the viral strains from different regions of the world has established that the CCHF virus strains isolated from ticks and human beings in different southern Russian regions in 1967 and 2000 are very closely genetically and they form an individual subgroup in the basic European genetic group. By the S-segment structure, the CCHF virus strain isolated in Bulgaria in 1978 belongs to the same genetic group as a representative of its second subgroup. Analysis of the S-segment 3'-noncoding region suggests that the CCHF virus circulating in Europe, Central Asia, and China may have originated from one global focus of infection, including several CCHF virus genovariants. During evolution, fragmental exchange apparently occurred in the S-segment 3'-noncoding region as a result of homological recombination.
Genes encoding virus-specific late proteins with molecular mass 36 kDa and 12 kDa were mapped in HindIII-P DNA fragment of vaccinia virus strain L-IVP by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. RNA origin site of the 36K protein was detected in HindIII-J fragment. Nucleotide sequences of these genes were determined. Amino acid sequences of the 36K and 12K polypeptides were compared with the protein bank PIR.
Sequencing and computer analysis of the variola major virus strain India-1967 (VAR-IND) genome segment (53,018 bp) from the right terminal region have been carried out. Fifty nine potential open reading frames (ORFs) of over 60 amino acid residues have been identified. Structure-function organization of VAR-IND DNA segment under study was compared with the previously reported sequences from the analogous genomic regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR) and variola virus strain Harvey (VAR-HAR). Multiple distinctions in the genetic map of VAR-IND from VAC-COP and VAC-WR have been revealed along with the high similarity to the corresponding VAR-HAR segment. Possible functions of the predicted viral proteins and the effect of their differences on the features of orthopoxviruses are discussed.
Comparative structural analysis of the 36K proteins of vaccinia, ectromelia, cowpox and variola viruses revealed that it is conservative among Orthopoxviruses. The possible role of this protein was suggested. A study of the synthesis kinetics of vaccinia and ectromelia virus 36K proteins established that they belong to late proteins. Electron microscopy of infected cells using protein A labelled with colloidal gold showed that the 36K protein is located in the viroplast and not coupled to virions or any cell organelles.
Computer analysis of variola major virus (VAR) genomic fragment bounded by open reading frames (ORFs) D1R and A33L which is 47,961 bp long revealed 46 potential ORFs. The VAR proteins were compared with the analogous proteins of vaccinia virus strain Copenhagen. The subunits of DNA-dependent RNA polymerase, as well as the transcription factors, mRNA capping enzymes, and proteins necessary for the virion morphogenesis proved to be highly conservative within orthopoxviruses. The most pronounced differences between the VAR genome fragment under study and the corresponding vaccinia virus fragment were revealed in the vicinity of the gene encoding the A-type inclusion body protein. The possible functions of the analyzed viral proteins are discussed.
Sequencing of variola virus (VAR) genome region of 43069 bp was carried out. This area contains 42 potential genes. Computer analysis of proteins coding for these viral genes was done. We compared VAR proteins with the those of vaccinia virus. The region studied is conservative for orthopoxviruses.
Genes I3 and A2 of the vaccinia virus strain L-IVP DNA were cloned into bacterial expressing vectors. The monospecific antisera to the expression products of these genes in E. coli were obtained. By means of immunochemical cross-analysis two polypeptides of equal electrophoretic mobility were found in the virion preparations in the band of the major envelope protein p35. The major of them is the product of gene I3, and the minor is encoded by gene A2.
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The epidemiological situation with cytomegalovirus infection in Siberia is still to be studied and serological diagnosis of human cytomegalovirus (HCMV) is not satisfactory. Two regions of HCMV genome (strain AD-169) have been examined for obtaining diagnostic reagents by expression cloning. Using polymerase chain reaction, fragments of gene of the immediate early protein (IE2) and of the region encoding for the entire hydrophilic part (1176 bp) of the large phospoprotein gene (pp150) have been obtained. Both fragments were cloned in bacterial vectors. Analysis of nucleotide sequence showed negligible substitutions in comparison with previously reported sequences for these genes.
MT-4 cell line is a continuous strain of human T lymphocytes expressing defective noninfective subviral HTLV-1 particles. A fragment of sequence encoding the p24 protein and gene for envelope protein (env) have been obtained from genomic DNA of this culture by polymerase chain reaction. Both HTLV-1 fragments were cloned in bacterial vectors, and the nucleotide sequence of these regions was determined. The cloned DNA fragment encoding the p24 has only four point nucleotide exchanges. Analysis of the env gene structure revealed that the sequence had several amino acid exchanges and two deletions (13 bp and 70 bp).