[Evaluation of biochips for the rifampin resistance detection of M. tuberculosis in strains isolated at the Novosibirsk and Tomsk regions].

Recently in Russia biochips for rifampin resistance detection of M. tuberculosis were developed. To investigate the conformity between rifampin resistance results determined both by the routinely used absolute concentration method and USING the biochips, 272 DNA samples of M. tuberculosis isolated from TB patients at Novosibirsk and Tomsk regions in 2000-2005 were analyzed. The biochip can detect 30 mutations in rpoB gene. The mutations were also tested using the single stranded conformational polymorphism method (SSCP). In addition, 60 DNAs were randomly sampled and sequenced. The results of rifampin resistance detection using biochip and absolute concentration methods were congruent in 86% cases, and were different when analyzed samples consisted of the susceptible and resistant strains of M. tuberculosis mixture. The most frequent mutations in the rpoB gene were S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). In 94% of rifampin resistant strains, there was also resistance to isoniazid. Therefore, in Siberia the rifampin resistance is the reliable marker for MDR strains of M. tuberculosis, and biochips can be used also for their detection. To hybridize with biochip the fluorescent-labeled single-stranded DNAs were routinely synthesized by two PCR, and intermediary product after the first PCR should be transferred into another tube. The last stage included high risk of cross-contamination. To exclude the risk, primer concentrations and temperature-time profile of PCR reactions were improved, and both PCR were combined in one tube. The two methods were congruent in 100%. The one tube method would be especially attractive for the routine PCR laboratory.

[Study of a role of artificial mycobacterial particles in the realization of immunological processes in experimental animal tuberculosis].

The protective properties of artificial mycobacterial particles versus BCG vaccine were studied in laboratory animals with experimental tuberculosis. The findings of the decreased rate of a tuberculous process and on the increased mean life span in animals inoculated with M. bovis suggest that immunization of guinea-pigs with mycobacterial particles promotes the enhanced development of antituberculous immunity in the animals. The paper proposes a promising method for designing artificial immunogens, the high-polymer antigenic structures that imitates mycobacterial particles.

[Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates].

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.

[Differentiation of the Mycobacterium tuberculosis W-Beijing family strains from the Russian Federation by the VNTR-typing].

Recent phylogenetic studies allowed the Mycobacterium tuberculosis complex to be divided into a number of the strain families. The W-Beijing family is one of most widespread M. tuberculosis variants frequently causing epidemic outbreaks. This family is genetically homogenous and conserved so that ETR A, B, C, D, E - typing is insufficient for the W-Beijing differentiation. All W-Beijing isolates have common profile (42435). This led to the false clustering in the molecular epidemiology study, especially in the region of predominance of the W-Beijing family. In this investigation we searched for the VNTR loci with high evolution rate, which were polymorphic in the W-Beijing genome. Eleven VNTR-loci were assayed in the DNA panel of 99 M. tuberculosis isolates from the tuberculosis patients in North-West and West-Siberian regions of Russia during the period from 2000 to 2001. Ninety nine strains of M. tuberculosis were divided into 74 VNTR-types, 51 isolates of the W-Beijing family were subdivided into 30 VNTR-types. The Hunter-Gudson index (HGDI) for all studied loci (ETR-A, ETR-C, ETR-E, V, V2, V3, V4, V5, V6, V10, V11) was close to one of the IS6110 RFLP indices being "the gold standard" of the M. tuberculosis complex genotyping. The V2, V3 loci located in the sequences of the PPE gene family, were highly polymorphic and more discriminative then others (HGDI is about 0.8). The congruence between the IS6110 RFLP-typing and 11 loci VNTR-typing was measured during genotyping for 23 isolates of the W-Beijing family. The isolates were divided into 9 genotypes by the IS6110 RFLP and into 13 variants by the VNTR-typing. The profiles correlation coefficient was 0.767689 that reflected the differences in the rate and type of the given genome target evolution.

[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.

[Molecular and genetic characterization of rifampicin- and/or izoniazid-resistent strains of M. tuberculosis, isolated in Novosibirsk and Tomsk regions].

The purpose of the work was to study rifampicin- and izoniazid-resistent strains of M. tuberculosis, circulating in Western Siberia, by VNTR and IS6110 typing. The authors also studied genetic causes of resistance to these antibiotics and undertook a search of new VNTR loci, displaying polymorphism in genomes of closely related clonally-disseminated variants of Mycobacterium tuberculosis in W-Beijing family model analysis.

Inserting foreign peptides into the major coat protein of bacteriophage M13.

Foreign DNA fragments were inserted into filamentous phage gene VIII to create hybrid B-proteins with foreign sequences in the amino terminus. The hybrid proteins are incorporated into the virions which retain viability and infectivity. Virions with hybrid B-proteins have the same contour length and the same number of B-protein molecules as virions with natural B-proteins. It was shown that for one of hybrid B-proteins the position of the processing site had changed.

[Structure-activity organization of leukocytic interferon]. / Strukturno-funktsional'naia organizatsiia leikotsitarnogo interferona.

The recent experimental data on structure-function organization of leucocyte interferon are reviewed. The antiviral activity of the interferon molecule is supposed to be determined by more than two conformational centres.

[The mutagenic activity of N4-(4-aminobutoxy)-2'-deoxycytidine-5'-triphosphate]. / O metagennoi aktivnosti N4-(4-aminobutoksi)-2'-dezoksitsitidin-5'- trifosfata.

We have shown that deoxycytidine-5'-triphosphate modified by O-(4-aminobutyl)hydroxylamine in the pyrimidine ring, is effectively incorporated into DNA synthesizing in vitro, replacing deoxythymidine-5'-triphosphate or deoxycytidine-5'-triphosphate and inducing A-->G and G-->A transitions, respectively. UV spectroscopy and NMR spectroscopy have shown that the modified cytidine-5'-triphosphate is identical to N4-(4-aminobutoxy)-2'-deoxycytidine-5'-triphosphate. When the modified deoxycytidine-5'-triphosphate was inserted into DNA in vitro by DNA polymerase I of E. coli Klenow fragment, retardation sites correlating with poly-A sites (when the modified triphosphate replaced deoxythymidine-5'-triphosphate) or with poly-G sites (when it replaced deoxycytidine-5'-triphosphate) were revealed. Our data show high mutagenic effect of the modified deoxycytidine-5'-triphosphate inserted into DNA, allowing us to recommend this compound for localized static mutagenesis.

[Introduction of statistical mutations into strictly defined areas of the genome using a modified oligonucleotide]. / Vvedenie statisticheskikh mutatsii v strogo opredelennyi raion genoma s pomoshch'iu modifitsirovannogo oligonukleotida.

A method for inducing mutations in the short region of a gene is suggested. The method involves oligonucleotide modification by hydroxylamine derivative, in vitro enzymatic synthesis of double-stranded DNA using modified oligonucleotide as a primer and selection of mutant colonies using the starting unmodified oligonucleotide as a probe.

[Study of the mutagenic properties of oligonucleotides containing and internucleotide pyrophosphate bond using a mutation system based on phage M13]. / Issledovanie mutagennykh svoistv oligonukleotidov, soderzhashchikh mezhnukleotidnoe pirofosfatnoe zveno, c pomoshch'iu mutatsionnoi sistemy na osnove faga M13.

Mutagenic properties of oligonucleotides with pyrophosphate internucleotide bond was studied. It was shown that the pyrophosphate bond in the oligo structure does not induce mutations but promotes a more efficient induction of marker deletions predetermined by the nucleotide sequence as compared to the native oligonucleotide. Marker deletion induction proceeds according to the repair mechanism as homozygotes dominate in the mutant generation.

[The use of filamentous phage M13 in protein engineering]. / Ispol'zovanie nitchatogo faga M13 dlia belkovoi inzhenerii.

M13B1 vector based on the filamentous phage M13 has been constructed. M13B1 phage carries the gene of resistance to ampicillin and contains the unique site of recognition for BamHI restriction endonuclease in gene VIII coding for the major coat protein. BamHI restriction site has been inserted into the gene of the major coat protein by means of oligonucleotide directed mutagenesis. The synthetic DNA fragment coding for the model peptides has been inserted through BamHI site into the M13B1 DNA. The possibility of inserting foreign peptides into the N-terminus at maintaining the viability of hybrid phages has been shown. The differences in specificity of the recombinant phage maturation have been determined by analysing the amino acid sequence of B-protein.

[Study of the mechanism of mutagenesis directed by phosphotriester analogs of oligonucleotides. The role of repair in mutagenesis]. / Issledovanie mekhanizmov mutageneza, napravlennogo fosfotriéfirnymi analogami oligonukleotidov. Rol' reparatsii v zakreplenii mutatsii.

The mutation system has been suggested in an effort to test insertion and deletion mutants by changing the Lac-phenotype of bacterial colonies transformed by mutant DNA. This system also makes possible to determine heterozygotes and homozygotes among the mutants. The yield of mutants in shown to depend on the structure of the DNA heteroduplex region. The yield of deletion mutants is greater than that of insertion mutants. Heterozygotes prevail in mutant colonies (greater than 90%).

[Mutagenesis, directed by artificial oligonucleotide analogs. I. Participation of ribonucleotide fragments in DNA replication and repair]. / Mutagenez, napravlennyi neprirodnymi analogami oligonukleotidov. I. Uchastie ribonukleotidnykh zven'ev v reparatsii i replikatsii DNK.

The mutation system has been developed to study the mutagenic properties of modified oligonucleotide analogs. The mutagenic properties of oligonucleotides containing one ribonucleotide have been examined. The presence of a ribonucleotide is shown not to induce any mutations. But when the oligonucleotide induces two marker deletions detached by 6 nucleotides they may be repaired separately, in this case the deletion bordering with the ribonucleotide is predominantly repaired.

[Primary structure of the mutant genes of human leukocyte interferon alpha2]. / Opredelenie pervichnoi struktury mutantnykh genov leikotsitarnogo interferona alpha2 cheloveka.

Nucleotide sequences of 10 mutant genes of human leukocyte interferon alpha 2 (IFN) with the use of 4 oligonucleotide primers containing ethyl substituents at phosphate groups were determined. To design primer sequences, an approach based on the local similarity profile of the IFN gene and M13mp7 vector DNA is described.

[Antiviral activity of mutant interferons. A new approach to the study of structural-functional organization of interferons]. / Antivirusnoe deistvie mutantnykh interferonov. Novyi podkhod k issledovaniiu strukturno-funktsional'noi organizatsii interferonov.

The developed approach to investing the structure-functional organization of interferon has been developed consisting in: 1) fusing genes of interferon and alpha-peptide of beta-galactosidase, the resultant protein having the interferon properties and being determined by the beta-galactosidase alpha-complementation test; 2) constructing mutant genes of interferon by the localized mutagenesis; 3) determining the mutant interferon activity; 4) deducing the amino acid sequence of mutant interferon by sequencing mutant genes; 5) analyzing structure-functional organization of interferon. In accordance with this approach, ten mutant interferons with up to 15 changes of amino acid substitutions are obtained and their antiviral activity is determined. The role of some amino acid residues in antiviral activity of interferon alpha 2 is revealed.

[Mutagenesis, induced by phosphotriester analogs of oligonucleotides and directed to the cleavage site of double-spiral DNA]. / Mutagenez, indutsirovannyi fosfotriéfirnymi analogami oligonukleotidov i napravlennyi na mesta razryva dvuspiral'noi DNK.

The mutagenic properties of phosphotriester analogues revealed in course of interaction with linearized plasmid DNA were studied. The plasmid-based model system permitting one to test reliably the induced mutations is proposed. The efficiency of mutagenesis was shown to depend on the length of the oligonucleotide-mutagen and the genotype of the transformed Escherichia coli strain. The possible mechanisms involved in mutagenesis are discussed.

[Expression in Escherichia coli of the human leukocyte interferon alpha2 gene fused with N-terminal fragment of beta-galactosidase]. / Ekspressiia v Escherichia coli gena leikotsitarnogo alpha2-interferona cheloveka, transliatsionno slitogo s N-kontsevym fragmentom beta-galaktozidazy.

Genes for leucocyte interferon and alpha-donor of galactosidase were fused by deletion mutagenesis or by site-directed mutagenesis. In both cases the fused protein was expressed. The protein having an antiviral activity of leucocyte interferon was easily detected in bacteria and solutions by the reaction of beta-galactosidase alpha-complementation and retained the antigenic determinants of interferon and beta-galactosidase. The use of fused proteins for optimization of gene expression and for the analysis of interferon structure-function relationship is discussed.

[Study of the effect of chicken embryo homogenate on the growth and metastasis of sarcoma of Pliss in rats]. / Issledovanie vliianiia gomogenata kyrinykh émbrionov na rost i metastazirovanie sarkomy plissa u krys.

The effects of injection of 6 day-old homogenate of chicken embryo tissue on sarcoma of Pliss have been investigated. It was found to drastically inhibit said malignancy in rats and to increase their survival. Histological study revealed extensive necrosis of tumor tissue as well as formation of fibro-vascular structures showing characteristic leukocyte-macrophageal infiltration in the treated animals unlike untreated controls and those injected with cyclophosphamide. A link between activation of cellular immunity and antitumor effects of embryonal tissue homogenate is suggested.

[Molecular genetic characteristics of rifampicin-resistant Mycobacterium tuberculosis isolates in Novosibirsk]. / Molekuliarno-geneticheskaia kharakteristika ustoichivykh k rifampitsinu izoliatov Mycobacterium tuberculosis, vydelennykh v Novosibirske.

Forty rifampicin-resistant clinical isolates from patients living in Novosibirsk were studied. Six alleles earlier described in the literature were identified by the sequencing technique. The frequency of mutations in the studied samples slightly differs from that earlier reported for other geographic regions: 21 (52.5%) strains carried the mutated codon TTG in position 531 (Ser-->Leu), 7 (17.5%) had GTC in position 516 (Asp-->Val) and 2 (5%) had the GAC substitution in position 526 (His-->Asp), which is prevalent elsewhere. Sequence analysis revealed no mitations in 5 (12.5%) of the 40 isolates although this isolate was repeatedly resistant to rifampicin. VNTR-typing targeted to tandem repeats (ETR A, B, C, D, and E) was carried out to establish a genetic relationship for rifampicin-resistant isolates. Nine genetic types with VNTR-profiles termed as 12322, 32122, 32123, 32124, 32125, 32522, 23524, 12223, 22222, 33433 were revealed. There was no strict correlation between the type of mutation in the rpoB gene and the VNTR-type, which reflects different rates of evolution and the level of selective pressure on these genetic targets. The isolates of VNTR-types 32123 and 32125 with mutations in codon 531, and type 32122 in codons 531, 526, 516 showed a high clustering. This is likely to reflect the recent transmission and clonal dissemination of the epidemic strains of Mycobacterium tuberculosis. Thus, mutations in the rpoB gene did not reduce the virulence and transmissivity of these clones. Twenty-six of 27 clinical isolates selected by rifampicin-resistance were also resistant to isoniazid, which confirms the known fact that rifampicin-resistance may be used as a marker of isoniazid-resistance.