Bovine leukemia virus pre-miRNA genes' polymorphism.

MicroRNAs (miRNAs) are small non-coding RNAs with a wide distribution in nature among the living things. They play a key role both in normal signaling pathways and in pathological ones. Bovine leukemia virus (BLV) is an oncogenic retrovirus of Deltaretrovirus genus causing persistent infection in its natural hosts - cattle, zebu and water buffalo with diverse clinical manifestations through the defeat of B-lymphocytes (B-cells). Ten BLV encoded miRNAs (further miRs-B) transcribed from five different pre-miRNA (further pre-miR-B) genes are abundantly detected in BLV infected B-cells. Here we report about several alleles of each of pre-miRs-B' genes, some of which have a highly significant association with an increase or a decrease of the number of leukocytes (WBCs - white blood cells) in BLV-infected cows.

[The role of Piwi nuclear localization in the differentiation and proliferation of germline stem cells].

The Piwi protein and its orthologs are considered as the key components of the piRNA machinery implicated in transcriptional silencing of transposons. Неre, we show that nuclear localization of the Piwi protein is required not only for transposon repression, but also for proper differentiation of germline stem cells (GSCs). piwi^(Nt) mutation that causes loss of nuclear Piwi and its retention in the cytoplasm leads to the accumulation of undifferentiated GSC-like cells. The analysis of piwi^(Nt) mutation in combination with a bam gene mutation blocking GSC differentiation shows that the loss of nuclear Piwi decreases GSC proliferation rate. This is accompanied by the accumulation of DNA double-strand breaks in GSCs that may be caused by transposition events. Here, for the first time a set of transposons repressed by Piwi in GSCs and surrounding niche cells has been identified. The present study together with our previous data show that nuclear and cytoplasmic Piwi can regulate different stages of the functioning of germinal cells: cytoplasmic Piwi is sufficient to maintain GSCs, while nuclear Piwi localization is necessary for their proper proliferation and differentiation.

[Effect of divalent metal ions on DNA conformational transitions in water-ethanol solutions].

The influence of different MgCl2 and MnCl2 concentrations on DNA conformational transitions in water-ethanol solutions was studied. It was shown that the presence of magnesium ions in solution at a concentration of 5 x 10(-4) M did not influence the decrease in the size of DNA without change in its persistent length at an alcohol concentration of about 17 % v/v. In contrast, manganese ions prevent this change in DNA parameters. At sufficiently high ethanol concentrations, the compaction of DNA followed by its precipitation takes place, which is accompanied by an increase of scattering in solution. As the concentration of Mg2+ and Mn2+ in solution increases, this process is observed at lower ethanol concentrations.

[Investigation of conformational changes of DNA molecule caused by gamma-irradiation in water-ethanol solutions]. / Issledovanie konformatsionnykh izmenenii molekuly DNK v vodno-étanol'nykh rastvorakh, vyzvannykh vozdeistviem gamma-izlucheniia.

The intrinsic viscosity, optical anisotropy and spectral properties of DNA molecule gamma-irradiated with the doses of 10, 20 and 30 Gy in water-ethanol solutions with ethanol concentrations 0-6 mol/l are investigated in the work. Specific volume of DNA at all doses used shows a complex non-monotone dependence on the ethanol content with a peculiarity at the alcohol concentration corresponding to the destruction of water structure in the mixed solvent (so-called, critical concentration, 3.5 mol/l). Ethanol presence at the concentrations below the critical one protects macromolecule from the radiation action. At the alcohol concentrations larger the critical an inversion of the dose dependence of the DNA specific volume is observed. At that the equilibrium rigidity and secondary structure of macromolecule do not change noticeably. The results obtained indicate a significant role of the solvent structure in radiation damage of DNA molecule.

[A comparison of effects of agents stabilizing or destroying the water structure on conformational changes of a DNA molecule in solutions exposed to gamma-irradiation]. / Sravnenie vliianiia soedinenii, stabiliziruiushchikh i razrushaiushchikh strukturu vody, na konformatsionnye izmeneniia molekuly DNK pri gamma-obluchenii ee rastvorov.

It was shown that gamma-irradiation of water-ethanol and water-n-propanol solutions of DNA with doses of 10-30 Gy leads to a fall of the specific volume of the macromolecule upon the cooperative transition at a critical concentration of alcohol in solution at which the destroying of water structure by a nonelectrolyte occurs.

An extended hydrophobic interactive surface of Yersinia pestis Caf1M chaperone is essential for subunit binding and F1 capsule assembly.

A single polypeptide subunit, Caf1, polymerizes to form a dense, poorly defined structure (F1 capsule) on the surface of Yersinia pestis. The caf-encoded assembly components belong to the chaperone-usher protein family involved in the assembly of composite adhesive pili, but the Caf1M chaperone itself belongs to a distinct subfamily. One unique feature of this subfamily is the possession of a long, variable sequence between the F1 beta-strand and the G1 subunit binding beta-strand (FGL; F1 beta-strand to G1 beta-strand long). Deletion and insertion mutations confirmed that the FGL sequence was not essential for folding of the protein but was absolutely essential for function. Site-specific mutagenesis of individual residues identified Val-126, in particular, together with Val-128 as critical residues for the formation of a stable subunit-chaperone complex and the promotion of surface assembly. Differential effects on periplasmic polymerization of the subunit were also observed with different mutants. Together with the G1 strand, the FGL sequence has the potential to form an interactive surface of five alternating hydrophobic residues on Caf1M chaperone as well as in seven of the 10 other members of the FGL subfamily. Mutation of the absolutely conserved Arg-20 to Ser led to drastic reduction in Caf1 binding and surface assembled polymer. Thus, although Caf1M-Caf1 subunit binding almost certainly involves the basic principle of donor strand complementation elucidated for the PapD-PapK complex, a key feature unique to the chaperones of this subfamily would appear to be capping via high-affinity binding of an extended hydrophobic surface on the respective single subunits.

[The role of ethanol during gamma-irradiation of water-salt DNA solutions]. / Rol' étanola v protsesse gamma-oblucheniia vodno-solevykh rastvorov DNK.

In this work we had assumed to clarify the role of the water structure in the process of the radiation damage of DNA. It is known that the aliphatic alcohols stabilize the water structure until the critical concentration. In this connection we have analyzed the changes of the long- and short-distance interactions in the DNA irradiated in the water-ethanol solutions with various concentration of the ethanol and ions of metal. It was shown that as the water structure becomes more stable the conformational damages in the DNA are decreasing and finally at the some concentration of the alcohol in the irradiated solution the damages disappear. By the achieving of the alcohol concentration which lead to the destruction of the water structure the radiation results in the same changes of the considered parameters as in the case of the DNA irradiated in the water-salt solution with ethanol. The analyses of the experimental data allow us to conclude that the radiation destroys the structure of the water and thus helps the positive ions from the solution to come nearer to the DNA, to say, the radiation reduce intramolecular electrostatic interactions. This concept allows us to explain the observed changes of intrinsic viscosity and the difference in the polarizabilities of the DNA in the process of the radiation damage.

Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system.

A vector-transformation system is described that permits replacement of a portion of the single rRNA operon of the archaeon Halobacterium halobium with a homologous fragment from a vector-borne gene. The vector construct contains three functional sections: (i) an entire H. halobium rRNA operon with two selective mutations in the 23S rRNA gene, the substitutions of A----G at position 1159 conferring resistance to thiostrepton and C----U at position 2471 conferring resistance to anisomycin; (ii) the complete pHSB1 plasmid from Halobacterium sp. SB3, which interferes with vector maintenance in the transformed halobacterial cells; and (iii) a segment of the pBR322 plasmid that permits vector replication in Escherichia coli. Transformation of H. halobium with the vector plasmid generates cells resistant to both anisomycin and thiostrepton that can be selected for, and discriminated from spontaneous mutants, by a two-step selection procedure. After transformation, the plasmid recombines homologously with the chromosome so that the plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, and the transforming plasmid is lost. Eventually, this leads to a homogeneous population of the mutant ribosomes in the cell. Other mutations that are engineered in the vector-borne rRNA sequences can be transferred to the chromosomal rRNA operon concomitantly with the selective markers. The system has considerable potential for ribosomal engineering.

[Acriflavine binding and DNA stability]. / Sviazyvanie akriflavina i stabil'nost' DNK.

The influence of acriflavine in the process of exposing DNA solution to gamma-radiation is studied. Acriflavine being actively bound to DNA is demonstrated not to protect DNA molecule from radiation damage. No radiation-induced variation in acriflavine-DNA binding degree is discovered. Acriflavine protective properties are revealed when the concentration of acriflavine is essentially high. This confirms our early results according to which only free ligands in solution protect DNA from radiation damage.