[Putative temperature sensitive riboswitchers in Salmonella genome].

Currently, a number of structurally and functionally different temperature-sensitive elements like as RNA thermometers which control a variety of biological processes of bacteria, including virulence, are known. Well-known RNA thermometers correspond to one long step-loop structure or few hairpins which can be matched or mismatched. Based on the computer and thermodynamical analysis of 25 isolates of Salmonella enterica with complete genome, algorithm and the criteria of search for putative RNA thermometers were developed. It will permit to perform the search of potential riboswitchers in genome of socially significant pathogens in the future. For S. enterica, in addition to well-known 4U RNA thermometer, four step-loop structures that may be new RNA thermometers were identified and two of them are localized in 5'-UTR of virulence regulators gltB and yaeQ. They correspond to necessary and sufficient conditions of RNA thermometer formation as far as these highly conservative structures are found in genome of all 25 isolates of S. enterica. Matched hairpins forming cruciform structure in supercoiled pUC8 plasmid were visualized by atomic force microscopy.

[Molecular technologies in detecting sensitive and isoniazid-resistant Mycobacterium tuberculosis].

Two types of techniques for detection of single nucleotide polymorphism in 315 codon of katG gene of MTB are developed. Isoniazid resistance of MTB is associated with point mutations in the mentioned codon. Two primer sets with additional competitive blocking primer containing 3'-terminal phosphate group (for elimination of unspecific amplification) allow detecting the most frequent point mutations AGC --> ACC and AGC --> AGA in 315 codon of katG gene. PCR with primer set of two primers one of which contains five LNA-monomers allows to determine an occurrence of any type from six known mutations in 315 codon of katG gene, i.e. to differentiate wild type and isoniazid-resistant MTB. Purity and structure of 17 bp long primers with LNA-modified nucleotides were characterized by time-of-flight MALDI-mass spectrometry. Duplex of 17 bp length formed by two complementary oligonucleotides with LNA-monomers was studied using melting.

[Visualization of bacteriophage T7 RNA-polymerase complexes with DNA template in the process of transcription elongation].

Complexes of bacteriophage T7 RNA polymerase (T7 RNAP) with a DNA template (which contains a promoter and internal terminator of T7 RNAP) in transcription elongation were visualized by atomic force microscopy (AFM). Images of specific stable complexes of T7 RNAP with a terminal fragment of DNA template located at a distance of 200 bp from the T7 RNAP promoter 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 nonspecific DNA-protein binding. Detailes of specific and nonspecific complex formation for the T7 RNAP--DNA system during initiation and transcription elongation are discussed.

[Visualization of RNA transcripts with atomic force microscopy].

RNA transcripts after transcription elongation with T7 RNA polymerase in vitro were visualized by using atomic force microscopy (AFM). Fragment of pGEMEX linear DNA with the length of 1414 nucleotide pairs carrying promoter and terminator of bacteriophage T7 was used as DNA template for transcription. Immobilized on the mica (AFM substrate) RNA transcripts formed rod-like condensed structures with the length of 122 +/- 10 nm and characteristic aspect ratio ca. 4.5-5. Problems of RNA immobilization onto mica for subsequent visualization by AFM are discussed.

[Amplification of proviral DNA of bovine immunodeficiency virus in experimentally infected animals].

The primer set for detection of bovine immunodeficiency virus (BIV) proviral DNA, retrovirus of unknown pathology, by standard polymerase chain reaction was developed. Amplicon of the expected size was detected in DNA from peripheral blood mononuclear cells of seropositive experimentally BIV infected sheep and cattle. Primers targeted the short fragment of BIV pol gene. Sequences of BIV proviral DNA extracted from BIV infected cell culture as well as from experimentally infected cows were taken for targeted pol BI BPX gene locus. Problems of BIV detection from the clinical material of the experimentally and naturally infected animals are discussed.

[Visualisation of cyanobacteria in aqueous solution by atomic force microscopy]. / Vizualizatsiia tsianobakterii u vodnomu rozchyni za dopomohoiu atomno-sylovoï mikroskopiï.

Methodical approaches for studying of living cells in aqueous solutions by atomic force microscopy (AFM) are demonstrated. Images of intact cyanobacteria Synechocystis PCC 6803 in TES buffer were captured in tapping mode using aminomodified mica as AFM substrate. Modification of freshly cleaved mica has been done in 3-aminopropyltri-ethoxysilane vapours. The average size of cyanobacteria was determined from AFM images. The linear size of Synechocystis PCC 6803 in TES buffer was equal to 70 x 90 nm and their height was about 20 nm. Possible causes of insufficiently high resolution of the cyanobacteria AFM images in aqueous solutions and possible ways for gaining molecular resolution in studies of structural, functional and micromechanical properties of living cells are discussed.

[Genome analysis and primer sets determination for listeria species detection and gene typing]. / Analiz henomu ta vyznachennia naboriv praimeriv dlia vyiavlennia i henotypuvannia listerii.

Computer analysis of listeria genome isolates of sequences from EMBL, GenBank, DDBJ data bases has been made. Variable and highly conservative (homology degree is 90-100% for all known isolates) genes loci iap and listeriolysin (cytolysin) gene locuses have been determined. Primer sets for detection and differentiation of Listeria species by polymerase chain reaction (PCR) were designed by computer and following thermodynamic analysis. Primer sets can provide detection of Listeria monocytogenes, L. ivanovii, L. seeligeri, L. grayi, L. innocua, L. welshimeri and detection of only pathogenic L. monocytogenes and Listeria species with listeriolysin (cytolysin) gene. Differentiation of 6 Listeria species can be done by means of two primer sets for iap and listeriolysin genes. Amplified fragments of listeria species DNA have different amplicon size. Primers melting temperatures selection allows one to carry out listeria species typing by multiplex PCR in a single tube.

[Study of microorganism genome DNA by atomic force microscopy]. / Vyvchennia genomnoï DNK mikroorganizmiv metodom atomno-sylovoï mikroskopiï.

The potential of atomic force microscopy (AFM) for the investigation of peculiarities of microorganisms genome structure is demonstrated. AFM images of phage lambda DNA linear molecules and supercoiled mica in buffer solution was imaged in air. New experimental method of DNA stretching based on using amino-modified mica with a decreased surface density of active amino-groups is proposed. Stretched molecules of phage lambda DNA were imaged by AFM.