Preparation and Properties of Nanoparticles, tRNA-Bivalent Metal Cation (Me2+) Complexes, and Prospects of Their Practical Use.

The patterns of formation of RNA nanoparticles (NPs) during thermal cycling of bacterial total tRNA in the presence of cations Ca2+, Mn2+, Ni2+, Zn2+, Co2+, and Cu2+ were studied. The optimal conditions for the production of NPs were found, and it was revealed that their size depends on the ratio of the concentrations of Me2+ and tRNA. The concentration of reagents for obtaining NPs of small size (from 5 to 100 nm) was selected. It was shown that tRNA-based nanoparticles can comprise short (20-50 nt) ribooligonucleotides, including aptamers and siRNAs. The stability of NPs during storage in buffer solutions of various composition was studied. It was found that the initial suspensions of NPs are quite stable, but they are rapidly destroyed in PBS buffer (pH 7.4). A simple and effective stabilizer (polyarginine) was found, the additives of which ensure the preservation of nanoparticles in PBS buffer for more than 5 h. Nanoparticles modified with the stabilizer are resistant to blood serum nucleases and can be used for transfection.

The structure-forming role of magnesium pyrophosphate in the formation of DNA-containing microparticles during PCR.

This work is devoted to studying the mechanisms of formation of DNA-containing microparticles (MPs) during PCR. It was found that pyrophosphate, a byproduct of DNA synthesis, and magnesium cations are required for their formation, as evidenced by the results of biochemical and electron microscopy studies.

[Condensed DNA particles formed in PCR with plasmid DNA: electron microscopy study].

An electron microscopy study of large-sized DNA microparticles produced in PCR with different gene-specific primers and plasmid DNAs is described. DNA microspheres of two distinct types were revealed in the all studied samples, namely smooth moderately electron-dense microspheres, and highly electron-dense particles with large thorns and offshoots. Singular microspheres have the average diameter of 1 mum, and their aggregates were up to 3 mum in dimensions. In addition, rare so-called three-dimensional net-like structures with various size (up to several micrometers) were observed. They consisted of different amounts of DNA nanoparticles, having the special compact topology. In some studied samples the discs (nanodiscs) of several dozens nm in thickness and up to 3 mum in diameter were revealed. It was shown that the quantity of net-like structures and nanodiscs sharply increases in asymmetric PCR. We also observed DNA nanowires of different length and thickness, nanodots, nanoparticles in the form of shits of paper as well as electron-dense spherical nanoparticles of big size. Aqueous suspensions of DNA microparticles were heated at 94 degrees C for 5 min and analyzed by electron microscopy. It was shown that microspheres in heated suspensions underwent partial melting; they lost a part of DNA, therefore details of their structure (ultrastructure) can be recognized. At the some time numerous tangles of nanowires appeared. Molecular mechanisms of the DNA micro- and nanoparticles formation are discussed.

[Micro- and nanoparticles of condensed DNA formed in a PCR with yeast genomic DNA as a template. Electron microscopy data].

It has been previously found that in a PCR with yeast genomic DNA as a template, microparticles of condensed DNA are formed in the presence of KlenTaq polymerase. In the present work, the study of these microparticles was continued using electron microscopy. It was shown that along with standard electron-dense microspheres, microspheres of a low electron density with a few thorns or without any thorns are formed. Various types of nanoparticles were detected in the samples: nanowires, dot-like electron-absorbing particles (nanodots), and compact nanoparticles (nanoscales) of different shape and size. It was found that increasing the number of PCR cycles above the optimum leads to an abrupt rise in the amount of nanoparticles in the PCR mixture. Suspensions of microparticles after quick (5 min) heating at 94 degrees C were examined. The partial melting of the microspheres in the heated samples was established: they lost part of the DNA and decreased in size; simultaneously, abundant clusters of nanowires appeared. The effect of nuclease S1 on the DNA of microspheres was studied. The molecular mechanisms of the formation of micro- and nanoparticles are discussed.

[Microparticles from coupled DNA formed in the process of polymerase chain reaction].

DNA microparticle formation in the course of a polymerase chain reaction (PCR) is reported. PCR with gene-specific and partially complementary primers and yeast genomic DNA as a template was shown to yield spherical DNA-composed microparticles as well as their aggregates and conglomerates, along with routine linear DNA. Microparticles were formed at late PCR stages and could be easily identified by the reaction with fluorescently labeled oligonucleotide primers or by staining of the PCR mixture with fluorescent dyes (acridine orange, propidium iodide or DAPI). According to the data of epifluorescent and electron microscopy, the microparticle size varied from 500 nm to 3-4 microm and the particles were multimeric star-shaped spheres or aggregates formed by several fused microspheres. Some properties of the microspheres were studied. It was found that the Mg2+ cations comprising the PCR buffer played a key role in the formation of microparticles and the stabilization of their structures.

The primary structure and characteristics of the ISAfe600, an insertion sequence from Acidithiobacillus ferrooxidans strains.

A new IS-like element (604 bp) was revealed in the genome of several Acidithiobacillus ferrooxidans strains isolated from diverse biotopes. It includes 26-bp imperfectly matched terminal inverted repeats (TIRs), similar in structure to the TIRs of ISAfel insertion element. The 60-bp DNA fragment adjacent to the right TIR (TIRR) exhibits pronounced homology with the similarly located DNA fragments in ISAfel and IST445, as well as with the internal fragment of ISAfel encoding the transposase gene (nucleotides from 254 to 311 bp). The central section of ISAfe600 is unique and exhibits no homology with any prokaryotic DNA. A duplication of 8 bp of the target DNA was found in the ISAfe600 insertion site. One to four copies of ISAfe600 were revealed by Southern hybridization in the genome of A. ferrooxidans strains studied. The number of ISAfe600 copies varies depending on the growth conditions (energy substrate). Since the open reading frames big enough to encode transposase are not presert in the structure of ISAfe600, it may be a deficient IS element; its translocation is possibly achieved under control of the ISAfel transposase.

[Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: application in PCR].

The procedure of obtaining DNA-containing cell envelopes ("micromummies") of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.

[Rapid and efficient extraction of soluble proteins from gram-negative microorganisms without disruption of cell walls].

The ability of buffer solutions containing low concentrations of nonionic detergents (Triton X-100, Tween 20, Brij 58, and Lubrol PX) and the anionic detergent sodium deoxycholate, as well as mixtures of these detergents with chaeotropes (urea and guanidine hydrochloride), to extract intracellular proteins of Gram-negative microorganisms (Escherichia coli and Pseudomonas aeruginosa) was studied. It was established that the solutions containing Triton X-100 and sodium deoxycholate and the mixtures of these detergents with urea are the most effective. It was shown that the extraction of proteins from bacterial cells under the studied conditions is not accompanied by a release of DNA into solution but is associated with extraction of low-molecular RNAs. The level of protein extraction reaches 80%. No disruption of the bacterial cell wall occurs during the extraction, and proteins probably permeate through meshes of the murein network. The efficiencies of our buffer mixtures are close to or higher than that of the commercial reagent CelLytic B (Sigma, United States). The practical uses of the chaeotropic mixtures developed are discussed.

[Fluorescence microscopic study of the microorganisms treated with chaotropic agents].

The yeasts Saccharomyces cerevisiae and Pichia pastoris and the bacteria Micrococcus luteus, Bacillus subtilis, and Anaerobacter polyendosporus have been treated with the chaotropic agents guanidine hydrochloride and guanidine thiocyanate and certain detergents and studied using fluorescence microscopy. Studies with the use of fluorochromes that can selectively stain nucleic acids (diamidino-2-phenylindole (DAPI), propidium iodide, and acridine orange) show that treatment of the bacterial and yeast cells at 37 degrees C for 3-5 h induces a release of DNA from the cytoplasm and its accumulation in the cellular zone, known as ectoplasm, located between the cell wall and the remainder of the cytoplasm (called endoplasm) in the form of one or several large granules. After treating the cells with the chaotropic agents at 100 degrees C for 5-6 min, the DNA is diffusively distributed over the ectoplasm. The fluorochromes used do not allow the detection of RNA. These findings are in agreement with previous data obtained from electron microscopic study of thin cell sections. After 33 PCR cycles, a considerable portion of DNA leaves the cells; as a result, they show a low level of diffusive fluorescence when stained with DAPI. When endospores of B. subtilis are treated with the chaotropic agents, they become highly permeable to the fluorochromes. Fluorescence microscopic study of such endospores shows that they contain DNA in the central part of their cores.

[Alternative splicing of pre-mRNA encoding the Musca domestica latrophilin-like protein(LLP): primary structures of four spliced forms of mRNA and their protein products].

An internal DNA fragment (approximately 2000 bp) homologous to the conserved regions of genes encoding latrophilin-like proteins (LLPs) was obtained by the PCR technique using degenerate primers to these gene regions. The gene-specific primers were synthesized based on the results of sequencing of the isolated fragment, and all overlapping cDNA fragments of the llp gene encoding the Musca domestica LLP were obtained by the rapid amplification of cDNA 5'- and 3'-ends (5'- and 3'-RACE). Four alternatively spliced mRNAs were found while sequencing the obtained cDNA fragments. Two long mRNAs (approximately 6000 nt) differ in the structures of both the sites encoding signal peptides and 5'-terminal untranslated regions. They encode large proteins (approximately 1800 aa), whose domain organization is similar to that of mammalian latrophilins. Each deduced protein contains a domain with seven transmembrane regions followed by an extended cytoplasmic C-terminal domain. Two other mRNA forms are derived from these long mRNAs; they encode proteins severly truncated at their C-termini (approximately 900 aa). They are composed of only three transmembrane regions and a short unique cytoplasmic C-terminal domain (23 aa). The limitations and drawbacks of the existing 3'-RACE techniques found during study of the long alternatively spliced cDNAs are analyzed, and ways for overcoming these difficulties are proposed.

[Interaction of chromosomal and plasmid DNA in Acidithiobacillus ferrooxidans strains adapted to different oxidation substrates]. / Vzaimodeistvie khromosomnoi i plazmidnoi DNK u shtammov Acidithiobacillus ferrooxidans, adaptirovannykh k raznym substratam okisleniia.

Restriction analysis of plasmids pTFK1 and pTFK2 of the Acidithiobacillus ferrooxidans strain TFBk was carried out, and the sizes of these plasmids were determined (13.5 and 30 kb, respectively). A macrorestriction map was built for plasmid pTFK1. DNA-DNA hybridization revealed that the plasmids contained homologous nucleotide sequences. Plasmid pTFK2 labeled with 32P was used as a probe for Southern hybridization with blots of XbaI-generated fragments of the chromosomal DNA of A. ferrooxidans strains grown on a medium containing Fe2+ or adapted to different oxidation substrates. Low-intensity hybridization signals were observed for many fragments of the chromosomal DNA of the strains studied. In the process of adaptation to new oxidation substrates, the localization of bands producing the low-intensity hybridization signals changed in a number of cases. Certain fragments of the chromosomal DNA of the strains adapted to different oxidation substrates produced strong hybridization signals with pTFK2. The data obtained are discussed in terms of the possible role of IST elements and plasmids in the adaptation of A. ferrooxidans to new energy substrates, microevolution, and strain polymorphism.

[Changes in the fine structure of microbial cells induced by chaotropic salts]. / Izmenenie tonkoi struktury kletok mikroorganizmov pod vozgeistviem solei-khaotropov.

The electron microscopic examination of the thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37 degrees C for 3-5 h or at 100 degrees C for 5-6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.

[Mrakia curviuscula sp. Nov.--a new species of psychrophilic yeasts from forest substrates]. / Mrakia curviuscula sp. Nov.--novyi vid psikhrofil'nykh drozhzhei iz lesnykh substratov.

Seven strains with similar characteristics from the laboratory collection of yeasts isolated from forest substrates collected in the central part of European Russia corresponded to none of the known yeast species. Based on the study of their life cycle, physiological characteristics, and the nucleotide composition of DNA and taking into account the data of PCR analysis with universal primers, the strains were ascribed to a new psychrophilic yeast species, Mrakia curviuscula sp. nov.