[Study of elongation complexes for T7 RNA polymerase].

Complexes of bacteriophage T7 RNA polymerase with a DNA template for transcription elongation were visualized by atomic force microscopy. Images for complexes of T7 RNA polymerase with terminal fragments of DNA template were obtained for single molecules. Complexes of a single DNA template molecule with several T7 RNA polymerase molecules corresponding to stages of initiation, elongation and termination of transcription were visualized under the elimination of unspecific DNA-protein binding. Immobilized on the amino mica RNA transcripts form rod-like condensed structures. Detailes of specific and unspecific complex formation for the T7 RNA polymerase-DNA system during initiation and transcription elongation are discussed.

[Characterization of oligonucleotides with LNA-monomers for PCR detection of point mutations in mycobacteria tuberculosis genome].

Point mutations associated with isoniazid resistance in Mycobacterium tuberculosis (MTB) have been analyzed in codon 315 of the katG gene by conventional polymerase chain reaction (PCR) using primers containing locked nucleic acid (LNA) modified nucleotides. Purity and structure of primers containing 5 LNA monomers of 17 nucleotides in length were characterized by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and a 17-mer duplex formed by two complementary oligonucleotides was characterized by the method of thermal denaturation. The duplex containing five LNA monomers per each strand was characterized by a higher melting temperature than it was expected using extrapolation of theoretical calculation for nucleotide modification of one strand of the duplex. Detection of any of six possible mutations in katG codon 315 (i.e. discrimination between sensitive and resistant MTB) requires just one PCR employing a set of two primers with one LNA-modified primer; this is an important advantage of oligonucleotides containing LNA over unmodified nucleotides: employment of multiplex PCR would require up to 12 primers. Problems of control of oligonucleotide modification by LNA monomers are discussed.

[Detection of wild type isolates and point mutations in genome of isoniazid-resistant Mycobacteria tuberculosis].

The isoniazid resistance of mycobacteria tuberculosis (MBT) is associated with point mutations in the codon 315 of katG gene of MBT. The two PCR-techniques for detection of point mutations in codon 315 have been developed. A use of two sets of primers comprising the additional competitive blocking primer with 3'-terminal phosphate group (in order to eliminate unspecific amplification) allowed to identify most frequent point mutations AGC-->ACC and AGC-->AGA in the codon 315. PCR with a set of two primers one of which contained 5 LNA-monomers allowed to discriminate between wild type and isoniazid-resistant MBT isolates; any of 6 known mutations in codon 315 of kafG gene being detected. A structure and purity of the LNA-containing oligonucleotides (length of 17 nucleotides) was characterized by MALDI-TOF mass spectrometry; the duplex formed by two LNA-containing complementary oligonucleotides (length of 17 b.p.) was anlyzed by thermal denaturation.

[Species-specific detection of Mycobacterium tuberculosis complex].

The routine polymerase chain reaction (PCR) was used for the species-specific detection of related Mycobacterium tuberculosis complex (MTC) strains characterized by a high level of nucleotide sequence homology. Nucleotide sequences for the 16S rRNA, rpoB, gyrB genes of MTC strains, which were potential markers for their genotyping, were analyzed. The constructed phylogenetic trees confirmed the accuracy of the current taxonomic classification of MTC. Five sets of primers were developed for the species-specific detection of the mycobacteria M. tuberculosis, M. canettii, M. microti, M. bovis, and M. caprae. It has been shown that PCR by means of the primers having oscillating 3'-terminal nucleotides (complementary DNA of only a certain mycobacterial type) may be used to genotype MTC in the correct choice of the temperature of primer annealing.

[Distribution of potentially hairpin-loop structures in the genome of bovine retroviruses].

Inverted repeats which can form hairpin-loop structures in the genomic RNA and cruciform structures in the proviral DNA of bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) have been determined. Localization diagrams have been made up for hairpins (one of the elements of signaling genome function). The retroviruses BLV and BIV, about 8.5 kbp in length, are characterized by the varying quantitative and qualitative composition of hairpin-loop structures. The BLV and BIV genomes have been found to have 7 and 18 hairpins with energy (-deltaG) of more than 10 kcal/mol, respectively. Furthermore, in the BIV genome, there are 3 thermodynamically stable (i.e. detectable on model systems in vitro) hairpins (with the loop up to 6 nucleotides), two of them are perfect. But thermodynamically stable hairpins have not been found in the BLV genome.

[Visualization of elongation complexes for t7 Rna polymerase by atomic force microscopy].

Complexes of bacteriophage T7 RNA polymerase (RNAP) with a DNA template for transcription elongation were visualized by atomic force microscopy (AFM). Fragment of pGEMEX linear DNA with length of 1414 bp carrying promot- er and terminator of bacteriophage T7 was DNA template for transcription. Promoter and terminator are located unsymmetrically on the ends of DNA template. Images of stable complexes of T7 RNAP with terminal fragments of DNA template were obtained for single molecules. Complexes of a single DNA template molecule with 2-3 T7 RNAP molecules corresponding to stages of initiation, elongation and termination of transcription were visualized under the elimination of non-specific DNA-protein binding. Also complexes of DNA, RNAP and RNA transcripts were imaged. Our results suggest that the initial stage is the formation of complex between T7 RNAP and terminal fragment of DNA template. Because promoter is localized near DNA terminus, it makes impossible an ommision of the promoter site.

[Markers for species-specific detection of bacteria from Bacillus cereus group].

rpoB and gyr genes (and their fragments) of chromosomal DNA of bacteria from Bacillus cereus group - B. anthracis, B. cereus, and B. thuringiensis - which are the potential markers for their genotyping were sequenced and phylogenetic trees were constructed. Sets of primers for species-specific detection of B. anthracis, B. cereus, and B. thuringiensis by multiplex polymerase chain reaction were designed. Also primers sets, which allow to differentiate strains of B. anthracis with various plasmid profiles (containing both plasmids (pXO1+, pXO2+), and without one (pXO1+, pXO2- or pXO1-, pXO2+) or both plasmids (pXO1-, pXO2-), determining pathogenic characteristics of the strains, were developed. For multiplex PCR primer sets were optimized on the annealing temperature of primers and amplicon length. Itwas shown that phylogenetic tree can be applied as an indicator of reliability and accuracy of taxonomical classification of microorganisms' species and subspecies. Comparison of pXO1 and pXO2 plasmid sequences of B. anthracis showed that these plasmids contain 18 and 4 palindrome sequences respectively which can potentially form thermodynamically stable hairpin-loop structures.

[Computerized analysis of inverted repeats in Mycobacterium tuberculosis genome]. / Komp'iuternyi analiz invertirovannykh povtorov v genome bakterii tuberkuleza.

Thermodynamic stable inverted repeats, capable of stabilizing nuclease-influenced mRNA, have been determined for M. tuberculosis slowly growing isolates H37Rv and CDC1551 with the completely sequenced genome. The genome of laboratory strain H37Rv may contain 50 pin structures formed by inverted repeats, whose stem varies in length from 11 to 28 nucleotide pairs (n.p.), the loop size being equal to 4-5 nucleotides and free energy deltaG=-15.2 to -56.2 kcal/mol. The genome of strain CDC1551 (clinical isolate) having a high level of virulence contains 47 pins. Each of the two isolates contains 8 long inverted repeats with a length of 48-62 nucleotides and free energy deltaG=-38.9 to -56.2 kcal/mol; of these, 6 pins are completely similar. At the same time in the genome of strain CDC1551, in contrast to that of strain H37Rv, a highly stable pin 58 nucleotides long is localized at 5'-end. The localization of the highly stable pin with deltaG=-53.9 kcal/mol in the area of 5'-end of isolate CDC1551 may lead to a different degree of stabilization of RNA-transcripts in M. tuberculosis CDC1551 in comparison with isolate H37Rv. This may, in its turn, serve as one of the reasons of differences in the virulence of strains. The possibility of the formation of pins with stems of 11 n.p. and longer has been visually cofirmed with the use of atomic power microscopy. The possibility of the participation of the secondary structures formed by pins in the protection of mRNA of slowly growing pathogenic M. tuberculosis from the degrading activity of RNases is discussed.

[Typing of cattle leukemia virus circulating in the Ukraine]. / Tipirovanie virusa leikoza krupnogo rogatogo skota, tsirkuliruiushchego v Ukraine.

Bovine leucosis virus (BLV), circulating in the Ukrainian territory, was characterized through the definition of its subspecies affiliation. The pro-viral BLV DNA was isolated from peripheral-blood lymphocytes of naturally-HIV-infected black-variegate animals taken from leucosis-affected farms in the Kharkov Region. The env-gene fragment of pro-viral DNA was amplified, sequenced and analyzed after the amplicon had been treated by three restriction enzymes, i.e. BamH I, Bcl I and Pvu II. According to the analysis of restriction-fragments' length polymorphism, the Ukrainian BLV isolate can be classified as belonging to the Australian subspecies, i.e. to one of the 3 known subspecies. Multiple alignment and phylogenetic analysis of the env-gene fragment of BLV isolates from the EMBL database showed that evolutionally the Ukrainian isolate is distantly located from the isolates' clusters of the Belgian, Japanese and Australian subspecie and has the biggest quantity (4) of non-coinciding nucleotides for the analyzed highly conservative locus of the BLV env-gene with a length of 444 pair of nucleotides.

[Development and approbation of polymerase chain reaction for detection of pathogen in Listeria infection]. / Razrabotka i aprobatsiia metoda polimeraznoi tsepnoi reaktsii dlia detektsii vozbuditelia listerioznoi infektsii.

A system of primers for detection of Listeria by polymerase chain reaction (PCR) has been developed. Specificity and sensitivity of the method was evaluated by analyses of reference clinical and abiotic samples. Clinical trials of PCR were carried by detecting latent (asymptomatic) carriers of Listeria among healthy women of epidemiologically significant professions. PCR is characterized by numerous advantages in comparison with the traditional bacteriological analysis.

[Development and testing of polymerase chain reaction method for detection of meningococcal infection pathogen]. / Razrabotka i aprobatsiia metoda polimeraznoi tsepnoi reaktsii dlia detektsii vozbuditelia meningokokkovoi infektsii.

A system of primers has been developed for the diagnosis of meningococcal infection has been developed on the basis of the polymerase chain reaction (PCR). Specificity and sensitivity of detection of N. meningitidis has been evaluated in analysis of the model clinical specimens. The proposed test system was tried in detection of N. meningitidis in nasopharyngeal discharge specimens from patients with acute nasopharyngitis and normal subjects from the focus of meningococcal infection.