Search for the new potential RNA thermometers in the genome of Salmonella enterica.

Currently, a number of structurally and functionally different temperature-sensitive elements such as RNA thermometers which control a variety of biological processes in bacteria, including virulence, are known. Based on computer and thermodynamic analysis of completely sequenced genomes of 25 Salmonella enterica isolates, the algorithm and criteria for the search of potential RNA thermometers were developed. It will make it possible to carry out the search for potential riboswitches in the genome of other socially important pathogens. For S. enterica, apart from the known 4U RNA thermometer, four hairpin-loop structures were identified which may probably act as additional RNA thermometers. They satisfy the necessary and sufficient conditions for formation of RNA thermometers and are highly conservative uncanonical structures, since these highly conservative structures were found in the genome of all 25 isolates of S. enterica. The hairpins forming a cruciform structure in the supercoiled pUC8 DNA were visualized by atomic force microscopy.

Potential thermosensitive riboswitches in the genome of Salmonella.

Currently, a number of structurally and functionally different thermosensitive elements, such as structurally and functionally different RNA thermometers, for controlling a variety of biological processes in bacteria, including virulence are known. These well-known RNA thermometers are structures, whether matched or mismatched, which are represented by either a single stretched hairpin structure or a few hairpins. Based on computer and thermodynamic analyses of 25 isolates of Salmonella enterica with complete genome, we have developed an algorithm and criteria to search for potential RNA thermometers, which will enable us to undertake a future search for potential riboswitches in the genomes of other socially significant pathogens. In addition to the well-known 4U RNA thermometer, another four hairpin-loop structures have been identified in S. enterica as new potential RNA thermometers and two of them are localized in 5'-UTR of virulence regulators gltB and yaeQ. They are highly conserved noncanonical structures and correspond to the necessary and sufficient conditions for forming RNA thermometers, since they are found in each of the 25 S. enterica genome isolates. We analyzed the thermosensitive motif in the pXO1 plasmid of Bacillus anthracis-an anthrax-causative pathogen-and visualized matched hairpins that form a cruciform structure in pUC8 supercoiled plasmid by atomic force microscopy.

[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.

[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.

[Compaction of single molecules of supercoiled DNA immobilized on amino mica: from duplex to minitoroidal and spheroidal conformations].

Supercoiled DNA pGEMEX with a length of 3993 nucleotides was immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica) and visualized by atomic force microscopy. Plectonemically supercoiled DNA molecules and molecules with an extremely high level of compaction were visualized on modified amino mica, which was characterized by increased surface charge density. It was found that the length of the superhelix axis decreases two and four times to form superhelix axes of the second and third orders as the DNA compaction level increases because of the twice folding of DNA molecules. In this case, the length of the superhelix axis decreases from L approximately 470 nm to L approximately 140 nm (which corresponds to 10% contour length of a relaxed molecule on assumption of B-DNA) to form minitoroids and spheroids of approximately 50 nm diameter. Note that the previously reported experimentally measured length of the superhelix axis was equal to 35% contour length of the relaxed DNA molecule at the maximal density of the superhelix. Our data show that the significant decrease in the length of superhelix axis and the compaction of single supercoiled DNA molecules to the level of spheroids and minitoroids are caused by the screening of negatively charged DNA phosphate groups by positively charged amino groups of the modified amino mica because of its high surface charge density and increased hydrophobicity compared with standard amino mica.

[Compaction of single supercoiled DNA molecules adsorbed onto amino mica].

A model of possible conformational transitions of supercoiled DNA in vitro in the absence of proteins under the conditions of increasing degree of compaction was developed. A 3993-bp pGEMEX supercoiled DNA immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica with a hydrophobicity higher than that of standard amino mica) was visualized by atomic force microscopy in air. On the modified amino mica, which has an increased density of surface positive charges, single molecules with an extremely high degree of compaction were visualized in addition to plectonemic DNA molecules. As the degree of DNA supercoiling increased, the length of the first-order superhelical axis of molecules decreased from 570 to 370 nm, followed by the formation of second- and third-order superhelical axes about 280 and 140 nm long, respectively. The compaction of molecules ends with the formation of minitoroids about 50 nm in diameter and molecules of spherical shape. It was shown that the compaction of single supercoiled DNA molecules immobilized on amino mica to the level of minitoroids and spheroids is due to the shielding of mutually repulsing negatively charged phosphate groups of DNA by positively charged amino groups of the amino mica, which has a high charge density of its surface.

[Functionalization of aminomodified probes for atomic force microscopy].

Probes for atomic force microscopy functionalized by bovine serum albumin were obtained, which may be used for molecular recognition studies. The procedure of the modification and functionalization of probes includes three stages. First, amino probes are obtained by modification in vapors of amino silane derivative. Then a homobifunctional amino reactive cross-linker is covalently linked to surface amino groups of the amino probe. And finally, the probe with the covalently attached cross-linker is functionalized by bovine serum albumin molecules. The probes obtained were characterized at different stages of the modification by atomic force microscopy: the adhesion force and the work of adhesion force were determined from histograms. The modification of probe surface was confirmed by visualization of bovine serum albumin and supercoiled pGEMEX DNA molecules immobilized on the amino mica and amino mica modified by cross-linker.

[S-DNA is oversupercoiled DNA with 1.94-2.19 A rise per base pair along duplex axis].

Supercoiled pGEMEX DNA with length of 3993 nucleotides was immobilized on four substrates (freshly cleaved mica, standard amino mica, modified amino mica with increased and decreased surface charge density compared with standard amino mica) and it was visualized by atomic force microscopy (AFM) in air. Plectonomically supercoiled DNA molecules as well as single molecules with extremely high level of compaction (i.e. molecules with significantly higher superhelix density values on comparison with previously experimentally measured and theoretically investigated ones) were visualized on modified amino mica which was characterized by increased surface charge density. Distance between base pairs along duplex axis was determined by measurements of contour length of single oversupercoiled DNA molecules. Determined rise per base pair was varied from 1.94 to 2.19 A. These compressed supercoiled DNA molecules like a spring with decreased rise/base pair on comparison with well-known DNA forms were called new DNA form--S-DNA. A model of S-DNA was built. Formation of the S-DNA molecules was suggested to be an intermediate stage on the compaction of the single supercoiled DNA molecules up to the spheroids and minitoroids. Oversupercoiling and further compression of the supercoiled DNA molecules was shown to cause by high surface charge density of amino mica on which DNA molecules were immobilized.

[The visualization of amplicons after polymerase chain reaction].

Linear DNA molecules amplified by the polymerase chain reaction were visualized by atomic force microscopy. The measured contour length of the PCR product of 1414 bp sequence was 435 +/- 15 nm. Considering that the calculated value of the distance between the nucleotides along the duplex axis is 0.31 nm, it was assumed that linear DNA molecules on the surface of mica, which serve as a support in the atomic force microscopy method, are in the A form. The influence of surface properties of the mica and the sample drying procedure on the conformation of adsorbed DNA molecules is discussed. Possible reasons for the Gaussian distribution of the contour length of the synthesized amplicon are considered.

[Species-specific detection of Proteus vulgaris and Proteus mirabilis by the polymerase chain reaction].

Sets of primers for the species-specific detection of P. mirabilis and P. vulgaris by the polymerase chain reaction (PCR) were developed. As targets for these primers beta-lactamase and 16S rRNA gene fragments were chosen on the basis of the multiple leveling of the sequences of the DNA of all known P. mirabilis and P. vulgaris isolates. For differential detection oligonucleotides were selected in such a way that primers, specific for P. vulgaris, contained the non-paired nucleotide for P. mirabilis isolate at the 3'-end, and all other nucleotides were complementary to the beta-lactamase gene fragment. Primers, specific for gene 16S rRNA of P. mirabilis, contained the non-paired nucleotide for P. vulgaris isolates at the 3'-end. Standard PCR was carried out for 6 P. mirabilis and P. vulgaris strains. The use of PCR species-specific primers to P. vulgaris DNA made it possible to amplify the DNA fragment of the expected length only for P. vulgaris isolates, while the result of PCR for P. mirabilis was negative. PCR with primers specific to P. mirabilis permitted the detection of amplicon sized 101 nucleotides pairs only for P. mirabilis strains. These primers were optimized so as to use them in the specific differentiation of closely related P. mirabilis and P. vulgaris species by multiplex PCR. Genus-specific primers permitted the detection of bacterial gyrB gene of the genus Proteus were developed also.

[PCR-based detection of proviral DNA of bovine immunodeficiency virus].

A set of primers was developed to detect by polymerase chain reaction (PCR) the proviral DNA of bovine immunodeficiency virus (BIV). A short fragment of 101 bp BIV gene was selected as a target for primers; sequences of proviral DNA isolated from both a cell culture with BIV and from lymphocytes of an experimentally infected animal were known for the fragment. An amplicon of an expected size was detected by standard PCR in a transformed cell series of bovine testicles with Florida 112 BIV DNA, and in a plasmid DNA with a cloned proviral DNA of R29 BIV. Described in the paper are the results of a theoretical comparison of primers used in the detection of BIV by PCR. The presence of non-complementary nucleotides in the set of "primer-single stranded amplicon" was shown to bring about false positive results in the detection of BIV by PCR. No 1500 bp PCR product was detected after PCR with a synthesized pair of primers and with 100% homology for all known BIV isolates complementary to env gene. Finally, the issue of how to detectVIR in clinical samples obtained from experimentally and naturally infected is discussed.

[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.

[Identification of hypervariable and conservative fragments of Listeria genome]. / Identifikatsiia gipervariabel'nykh i konservativnykh fragmentov genoma listerii.

The computer analysis revealed hypervariable and highly conservative fractions in the genes of Gram-positive bacteria of the Listeria genus. As a result of analysis of gene iap coding protein p60, PCR based test systems for detection of 6 Listeria species, L. monocytogenes, L. seeligeri, L. ivanovii, L. innocua, L. grayi and L. welshimeri have been developed. Species-specific and conservative gene fragments coding Listeria pathogenicity factors, listeriolysin and cytolysin, were detected. The sets of primers for detection and gene typing of L. monocytogenes, L. seeligeri and L. ivanovii containing cytolysin have been made. The gene typing of Listeria may be carried out in one reaction with the use of multiplex PCR: amplified fragments for different Listeria species differ in the length of the amplified product. The developed sets of primers have a 95-100% degree of homology and may be recommended for the detection and gene typing of Listeria.

[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.