[Mechanisms of HIV-1 drug resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors].

Global AIDS epidemics caused by human immunodeficiency virus type 1 (HIV-1) has existed for more than 25 years and involved more than 2 million newly infected people annually. The obstacle in combating the global epidemics is a rapid evolution of the virus by the selection of drug resistance mutations. In this review, we have summarized scientific achievements in the field of reverse transcriptase drug resistance to licensed antiviral drugs--nucleoside (NRTI) and non-nucleoside (NNRTI) inhibitors. Principal mechanisms of their antiviral action, major drug resistance mutations, and molecular aspects of classic resistance mechanisms of HIV to NRTIs and NNRTIs are described. Recently discovered role of RNase H activity in development of drug resistance to reverse transcriptase inhibitors is a focus for the detailed discussion. New dual resistance mechanism to NRTIs and NNRTIs associated with the reverse transcriptase mutations in the C-terminal region, which includes RNase H and connection domains, is analyzed. Comprehensive analysis of the factors affecting the HIV drug resistance is important for the understanding molecular mechanisms of resistance for the improvement of drug design and anti-HIV therapy.

[Somatic mutations of the P53 gene in stomach cancer].

The paper deals with a study of p53 gene somatic mutations in tumor cell genomes from patients with stomach cancers of different histological patterns. It used sequential and molecular cloning methods. The former involved amplicones characterized by abnormal volatility following SSCP analysis of plasmids from 9 tumors. Replacement nucleotides were identified in 4 tumors (intestinal--2, diffuse--2). Among 8 mutations were 1 single-nucleotide deletion in codon-249 with shifting sensing frame and one targeted mutation. Five of the former were missens-mutations which caused amino acid replacement while the other two silent mutations did not. Exon-assisted analysis of p53 ("wild") gene identified cells with stable structure in each tumor (1 mutation--2; 3 mutations--2 including genuinely-paired mutations in 1 exon). All mutations occurred in structurally and functionally important codons. Our evidence corroborated earlier data of SSCP analysis on tumor cell presence in populations with variable p53 genomes.

[RT-PCR-based methods for identification and typing of infectious hemopoietic necrosis virus in salmons].

A RT-PCR method has been developed to diagnose infectious hemopoietic necrosis virus (IHNV) in salmons. The authors show it possible to use the method for viral shedding in both a cell culture and a clinical sample from infected fishes. Genotyping of IHNV strains originating from North America, Europe, and Russia, by using the restriction fragment length polymerase analysis, has revealed that 10 of them belong to 3 existing genogroups (U, M, and L). Three Russian isolates are assigned into a separate subgroup. Phylogenetic analysis of several isolates has confirmed that viral strains from Katchatka belong to the North American U-genogroup whereas 3 Russian isolates from the continental zone of the country make up a separate subgroup within the same genogroup.

[Genetic polymorphism of clinical Mycobacterium tuberculosis strains circulating in the territory of Novosibirsk Region].

Mycobacterium tuberculosis strains isolated from patients treated at TB dispensary branches in different districts of Novosibirsk were studied by genetic analysis. The below molecular methods were used: 1. PCR with random primers; 2. A method based on variable number of tandem repeats in loci; 3. IS6110 inverse PCR. Thirty-five samples of genome DNA of M. tuberculosis isolated were analyzed. Each of the 3 methods detected the main group of isolates, which comprised 61.8% of closely related strains revealed by method 1, 75.8%--by method 2, and 74.3%--by method 3. The remaining clusters were represented by 1 to 4 strains. The data obtained denote a relative homogeneity of M. tuberculosis strains circulating in Novosibirsk Region. No interplay was detected between the clustering of isolates and the presence or absence of mutation in genes conditioning the resistance to antibiotics.

[Genomic and phenotypic analyses of microorganisms isolated from the sediments of Lake Baikal].

Genetic and biochemical methods and morphological examination were used to study microorganisms isolated from samples of deep drilling of the Lake Baikal bottom sediments. Based on blot hybridization patterns, the strains investigated were divided into several groups according to the degree of homology of their genomic DNA. Morphological, biochemical, and ultrastructural characteristics of bacterial strains are described, and their compliance with the genomic analysis data is demonstrated.

[DNA technology for diagnosis and characterization of agricultural animal pathogenic viruses]. / DNK-tekhnologii v diagnostike i kharakterizatsii virusnykh patogenov sel'skokhoziaistvennykh zhivotnykh.

Use of recombinant DNA for the development of diagnostic and therapeutic and preventive drugs became one of the priority trends in modern experimental veterinary. This paper discusses modern methods of virus analysis based on the DNA technologies: restriction mapping, nucleic acid hybridization, and polymerase chain reaction. Examples of utilization of these methods for clinical diagnosis and research of animal viruses are offered.

[Structure of over lapping region of ICP18.5 and gB genes of type 1 bovine herpesvirus strain TK-A]. / Struktura oblasti perekryvaniia genov ICP18.5 i gB gerpesvirusa krupnogo rogatogo skota 1-go tipa, shtamm TK-A.

Nucleotide sequence of DNA fragment coding 3'-terminal of ICO 18.5 gene that overlaps the regulatory region and 5'-terminal of open reading frame of gB gene of bovine herpesvirus (BHV-1, subtype 1.3), strain TK-A, was determined. Comparative analysis of the sequence with the corresponding DNA regions of BHV-1, subtype 1.1, equine herpesviruses of the first and fourth types, and porcine pseudorabies virus was performed.

[Identification and characterization of strains of Bacillus thuringiensis by genomic fingerprinting using biotinylated phage M13 DNA]. / Identifikatsiia i pasportizatsiia shtammov Bacillus thuringiensis metodom genomnoi daktiloskopii s ispol'zovaniem biotinilirovannoi DNK faga M13]

Genomic DNA of the entomopathogenic bacterium Bacillus thuringiensis was analyzed by the genomic fingerprinting technique. The biotin-labeled single-stranded DNA of the phage M13 was used as a marker of hypervariable sequences. A procedure for analyzing the differentiation among various Bacillus thuringiensis strains was developed. Characteristic patterns of fingerprints were obtained for several strains, the main representatives of subspecies that are most frequently used in the manufacture of bacterial insecticides, such as subsp. thuringiensis, subsp. kurstaki, and subsp. galleriae. Because no essential differences were revealed in band patterns upon comparing fingerprints of crystal-producing bacterial strains with those of acrystallic mutants, it was assumed that the loss of crystal-producing ability in the insect pathogen Bacillus thuringiensis is not connected with significant rearrangement of its genome.

Detection of spring viremia of carp virus isolates by hybridization with non-radioactive probes and amplification by polymerase chain reaction.

For detection of spring viremia of carp virus (SVCV) DNA probes have been constructed using the reverse transcription-polymerase chain reaction (RT-PCR) amplification technique and cDNA cloning in plasmid and phage vectors. The specific primers for amplification of SVCV M and G genes were chosen and synthesized. Studies were carried out to establish the sensitivity and specificity of viral RNA detection in infected cell culture and pathogenic material from fish by the use of non-radioactive probes and RT-PCR. The efficiency of amplification with primers, complementary to the genome of the reference Fijan strain, was estimated in RT-PCR experiments with two SVCV strains. Under the same conditions, the quantity of PCR products amplified from the M2 strain was less than that from the ZL4 strain, which implies that the latter is more similar to the reference European SVCV Fijan isolate. Using DNA probes and dot-blot hybridization, SVCV was tested in samples taken from different organs of artificially infected carp with clinical signs of acute disease. The virus could be detected most reliably in fish brain. In most cases the hybridization signal was positive with samples having a viral titer of not less than 10(5) TCID50/g.

[Detection of the hog cholera virus using the polymerase chain reaction]. / Vyiavlenie virusa klassicheskoi chumy svinei s pomoshch'iu polimeraznoi tsepnoi reaktsii.

A rapid and highly sensitive method for detecting hog cholera virus (HCV) based on a reverse transcription of the polymerase chain reaction (RT-PCR) is developed. Primers complementary to the most homologous sites of virus genome in an area coding the precursor for glycoproteins gp44/gp48 are selected. Detection of the virus in pathological material by the RT-PCR showed that use of these primers in amplification allows detection of different HCV strains.

[Identification and classification of strains of microorganisms using genomic fingerprinting with biotinylated phage M13 DNA]. / Identifikatsiia i pasportizatsiia shtammov mikroorganizmov metodom genomnoi daktiloskopii s ispol'zovaniem biotinilirovannoi DNK faga M13.

To analyze DNA polymorphisms of various bacterial strains, a nonradioactive variant of the genomic fingerprinting method was developed. The method was based on the application of biotin-labeled single-stranded phage M13 DNA as a probe. Characteristic patterns of fingerprints obtained by MvaI, HaeIII, and HinfI restriction enzymes are presented for several species of bacilli and other bacteria. The advantages of this method in microbiology for the identification and characterization of different microbial strains are shown.

[Detection of the spring viremia of carp virus by hybridization with nonradioactive DNA probes]. / Obnaruzhenie virusa vesennei viremii karpa gibridizatsiei s neradioaktivnymi DNK-zondami.

In order to detect spring viraemia of carp virus, DNA probes have been constructed using reverse transcription-PCR amplification technique and cDNA cloning in plasmid and phage vectors. The sensitivity and specificity of viral RNA detection was assessed using nonradioactive probes in infected FHM cell culture and in tissues from dead fish. Viral RNA was more frequently detected in the brain and gill than in the abdominal organs.

[Detection of bovine infectious rhinotracheitis virus by hybridization using nonradioactive DNA-probes]. / Vyiavlenie virusa infektsionnogo rinotrakheita krupnogo rogatogo skota gibridizatsiei s neradioaktivnymi DNK-zondami.

Biotin-labeled DNA probes for infectious bovine rhinotracheitis virus also known as bovine herpesvirus-1 (BHV-1) have been developed. The procedure is based on dot-blot hybridization using biotin-labeled bacteriophage M13 and plasmid probes containing cloned PstI and EcoRI-PstI restriction fragments of viral genome. The probes obtained were used to detect viral nucleic acids in specimens of bovine spermatic fluid or nasal swabs of calves. The method is simple and rapid, taking less than 24 h, and is highly specific and sensitive, this recommending it for practical veterinary.

[Preparation of a specific immunogen based on bacteriophage M13]. / Poluchenie spetsificheskogo immunogena na osnove bakteriofaga M13.

Earlier we developed an expression vector on the basis of bacteriophage M13 allowing the exposure of short peptides on the virion surface. It was used to obtain a recombinant phage carrying the antigenic determinant of HIVI gag protein p17. This phage was tested as immunogen in rabbits. It was shown by ELISA that Ig against the fusion phage reacted with the 17-kDa core protein of the virus and with its polyprotein precursor p55 on strips activated by the transfer of HIVI viral proteins. These data may be used in vaccine development.

[Use of non-radioactive biotin-labelled probes for detecting hepatitis A virus]. / Ispol'zovanie neradioaktivnykh biotin-mechennykh zondov dlia detektsii virusa gepatita A.

The biotin-labeled DNA probes were constructed on the basis of the hybrid bacteriophage M13nip 9 single-stranded DNA containing the fragments of the hepatitis A viral cDNA. The probes were biotin treated by chemical modification of the DNA by the peraminating reagent or photochemically. The labeled DNA probes were used in molecular hybridization experiments with the nuclear acids fixed on the nitrocellulose filters. The biotin treated DNA was determined by the avidin-gold colloid conjugate with the subsequent physical silver amplification or by the streptavidin-alkaline phosphatase conjugate. The sensitivity of both probes was identical and permitted the determination of 5 x 10(-11)-5 x 10(-12) g of the control DNA and 10(-9) g of the hepatitis A virus. The developed test systems were used for detection of the viral RNA in blood from patients.

[Behavior of the large fragment of DNA polymerase I (the Klenow fragment) during fractionation of a cell-free extract of E. coli MRE-600]. / Povedenie bol'shogo fragmenta DNK-polimerazy I (fragmenta Klenova) pri fraktsionirovanii beskletochnogo ékstrakta E. coli MRE-600.

Distribution of the DNA polymerase I large fragment (Klenow fragment) was studied during fractionation of the E. coli MRE-600 cell-free extract with polyethylenimine. On the basis of the results obtained a simple procedure is proposed that enables the Klenow fragment to be obtained as a coproduct of DNA polymerase I, RNA polymerase, polynucleotide phosphorylase, nucleotide kinases with acetokinase and nucleoside deoxy-ribosyltransferase in the framework of a combined technological scheme.

[Isolation and properties of immobilized alkaline phosphatase from E. coli]. / Poluchenie i izuchenie svoistv immobilizovannoi shchelochnoi fosfatazy E. coli.

Preparations of alkaline phosphatase from E. coli, immobilized on Sepharose, with a specific activity of 40-60 U/g wet weight were obtained. The immobilized enzyme was stable up to 50 degrees C; at higher temperatures it was inactivated. At 70 degrees most of the activity was lost for 1 h. The substrate (AMP) stabilized the enzyme. In the temperature range from 30 to 40 degrees C activation of the enzyme was observed, especially pronounced in the presence of the substrate. The pH optimum of the immobilized enzyme activity (7.8-8.2) is shifted towards the acid region, as compared to the soluble enzyme (8.0-8.6). The kinetic parameters for inhibition by the reaction product were determined using the integral Michaelis-Menten equation. KmAMP was found to be higher in case of the immobilized enzyme as compared to the soluble one (5.02 X 10(-4) M and 1.85 X 10(-5) M, respectively), which seems to be associated with diffusion limitations.

[Properties of phosphodiesterase from Vipera lebetina venom immobilized on Agarose]. / Izuchenie svoistv fosfodiesterazy iada giurzy, immobilizovannoi na agaroze.

Preparations of phosphodiesterase from Vipera lebetina venom immobilized on agarose were obtained. The kinetic properties for the hydrolyses of various substrates of soluble and immobilized phosphodiesterase, e. g. effects of pH, temperature, substrate concentrations, etc., were compared. The values of Km, V and activation energy for the substrate hydrolyses were determined.