[Stimulation of DNA molecules association with amphiphilic derivatives of 1,3-diazaadamantane containing hydrophobic side chanins].

Earlier, a new class of compounds--amphiphilic derivatives of 1,3-diazaadamantanes, capable of facilitating the strand exchange in the system of short oligonucleotides was revealed. Longer hydrophobic side chains of 1,3-diazaadamantanes promoted stronger acceleration of the reaction. In this study, interaction with DNA of two 1,3-diazaadamantane derivatives containing different side chains was investigated by use of optical methods. Concentration of the investigated 1,3-diazaadamantans micelles formation were determined by the means of monitoring fluorescence intensity enhancement of 1-anilinonaphtalene-8-sulphonate probe; as well as the ranges of concentrations where the compounds/water mixtures existed as true solutions. 1,3-diazaadamantanes affinity to DNA was determined with Fluorescent Intercalator Displacement (FID) approach. Significant increase in hydrodynamic volume of short DNA hairpins in the complexes with 1,3-diazaadamantanes was revealed by estimation of the fluorescence polarization of ethidium bromide probe bound to the hairpins. Intermolecular association of DNA hairpins upon binding with 1,3-diazaadamantans was confirmed by Förster resonance energy transfer in system of an equimolar mixture of fluorescently labeled with Cy-3 and Cy-5 hairpins. In this study, the number of positive charges at 1,3-diazaadamantane derivatives containing side chains of different lengths was demonstrated to affect their affinity to DNA, whereas longer length of the hydrophobic side chains ensured more efficient interaction between the DNA duplexes that may facilitate, in particular, DNA strand exchange.

Binding, annealing and strand exchange between oligonucleotides in different sites of the RecA protein filament.

The efficiency of single-stranded (ss) oligonucleotides binding at the secondary site of the RecA protein filament is demonstrated to depend on the strandedness of DNA bound at the primary site. When the primary site is occupied by a ss-oligonucleotide, the binding of another ss-oligonucleotide at the secondary site is characterized by higher affinity and a lower rate of dissociation than is the case when the primary site is occupied by a double-stranded oligonucleotide. In contrast to a DNA strand exchange reaction suppressed by a heterologous oligonucleotide bound at the secondary site of the RecA filament, the occupation of the secondary site by a heterologous oligonucleotide does not prevent renaturation between the oligonucleotides bound at the primary site and complementary oligonucleotides from solution demonstrating that the binding of a DNA strand in the secondary site is not a necessary intermediate step in RecA-promoted DNA renaturation.

Horizontal spread of mer operons among gram-positive bacteria in natural environments.

Horizontal dissemination of the genes responsible for resistance to toxic pollutants may play a key role in the adaptation of bacterial populations to environmental contaminants. However, the frequency and extent of gene dissemination in natural environments is not known. A natural horizontal spread of two distinct mercury resistance (mer) operon variants, which occurred amongst diverse Bacillus and related species over wide geographical areas, is reported. One mer variant encodes a mercuric reductase with a single N-terminal domain, whilst the other encodes a reductase with a duplicated N-terminal domain. The strains containing the former mer operon types are sensitive to organomercurials, and are most common in the terrestrial mercury-resistant Bacillus populations studied in this work. The strains containing the latter operon types are resistant to organomercurials, and dominate in a Minamata Bay mercury-resistant Bacillus population, previously described in the literature. At least three distinct transposons (related to a class II vancomycin-resistance transposon, Tn1546, from a clinical Enterococcus strain) and conjugative plasmids are implicated as mediators of the spread of these mer operons.

Periodicity in recA protein-DNA complexes.

The reaction of guanine residues with dimethylsulfate was studied for complexes of recA protein with fluorescent dye tagged double stranded oligonucleotides. The patterns of dimethylsulfate modification obtained demonstrate a similarity of DNA states in the complexes with recA protein formed as a result of recA promoted strand exchange and renaturation reactions. The guanine modification efficiency varies periodically as a function of the base position along the oligonucleotide axis, with a period of 3 nucleotides. This effect suggests that the arrangement of recA monomers along the oligonucleotide is strictly ordered, and the dimethylsulfate reactivity of a guanine residue depends on the site of its binding in a recA monomer.

[Identification and expression of plasmid genes participating in the synthesis of microcin C51]. / Identifikatsiia i ékspressiia plazmidnykh genov, uchastvuiushchikh v sinteze mikrotsina C51.

A structural gene of heptapeptide, which is a component of the microcin C51 molecule, was identified by hybridization of plasmid DNA fragments and a mixture of synthesized oligonucleotides with the sequence corresponding to that of amino acids in the peptide. Sequence analysis of the structural gene of microcin peptide and its promoter region was performed. The data obtained indicate that the peptide of microcin C51 is synthesized on ribosomes. Four polypeptides of 67, 39, 16, and 14 kDa were identified using the system of minicells. These polypeptides are specified by a DNA fragment responsible for microcin synthesis and immunity in a producer cell. Apparently, three of these polypeptides with molecular masses of 67, 39, and 16 kDa are responsible for microcin production and immunity. The 67 kDa polypeptide is involved in the expression of immunity to microcin and, probably, in microcin production.

[Characteristics of hybrid genes coding functionally-active secretory metalloproteinases from bacilli]. / Kharakteristika gibridnykh genov, kodiruiushchikh funktsional'no- aktivnye sekretornye metalloproteinazy batsill.

A set of different hybrid genes encoding functionally active enzymes was obtained by homologous recombination between the fragments of related Bacillus amyloliquefaciens and Bacillus brevis metalloprotease genes cloned in plasmid vector in tandem orientation. The nucleotide sequences of hybrid genes were analyzed. It was demonstrated that the presence even of short homologous regions is sufficient for effective recombination in Bacillus cells.

Efficient interaction of recA protein with fluorescent dye-labeled oligonucleotides.

Some fluorescein derivatives attached to the 5'-end of oligonucleotides stimulate recA protein-oligonucleotide binding. The complex formation at near stoichiometric DNA/protein ratios is demonstrated for 18-bases-long oligonucleotides. The complexes with dye-labeled oligonucleotides are shown to be active in the reaction of homologous strand exchange. The strand exchange reaction in the presence of adenosine-5'-O-(3-thiotriphosphate) proceeds with the formation of a stable complex of recA protein with the double stranded oligonucleotide, which is a product of the strand exchange. The displaced single-stranded oligonucleotide is shown to be bound weakly.

[Interaction of double-stranded DNA in the Escherichia coli RecA protein system with modified oligonucleotides containing an alkylated terminal group]. / Vzaimodeistvie dvunitevoi DNK v sisteme RecA-belka Escherichia coli s modifitsirovannym oligonukleotidom, soderzhashchim alkiliruiushchim alkiliruiushchuiu kontsevuiu gruppu.

We studied modification of double-stranded DNA with chemically active homologous oligonucleotide derivatives carrying an alkylating 4-[N-2-chloroethyl-N-methylamino)benzyl]aminogroup in DNA recombination reaction promoted by E. coli RecA protein. It was shown that this chemical group does not prevent the formation of a complex between RecA protein and oligonucleotide as well as binding of this complex to double-stranded DNA. Formation of cross-links between DNA and oligonucleotide derivatives was observed at oligonucleotide lengths of no less than 30. The supercoiled form of the plasmid was alkylated with a higher efficacy than relaxed or linear form. In spite of homology between oligonucleotide and a certain region of double-stranded DNA (pBR322), cross-links were observed throughout the DNA length including nonhomologous regions. The causes of such behavior are discussed.

Electron microscopy visualization of oligonucleotide binding to duplex DNA via triplex formation.

Using biotinylated oligonucleotides and streptavidin as a marker, we have visualized, with the help of electron microscopy, the triplex formation. We used the natural homopurine-homopyrimidine sequence from human papillomavirus 16 cloned within a plasmid. Under conditions favouring the formation of pyrimidine-purine-pyrimidine triplex the corresponding pyrimidine oligonucleotide formed a complex with the insert and this complex was detected by electron microscopy. Similarly, under conditions favouring the formation of pyrimidine-purine-purine triplex the corresponding purine oligonucleotide formed a stable complex detected by electron microscopy. In both cases the complexes we observed exhibited remarkable sequence specificity. Near 80% of DNA molecules carried the streptavidin marker in the correct position and very few cases of non-specific binding were detected. We conclude that the triplex mode of recognition may provide very efficient sequence-specific markers for electron microscopy of DNA.

Amino acid sequence pattern in the regulatory peptides.

The essential properties of the primary structure of regulatory peptides, i.e. amino acid residues and their combinations, which are characteristic of the whole population of regulatory peptides, have been revealed using statistical methodology. These properties are as follows: increased content of certain residues (Gly, Pro, Phe, Arg, Tyr, Met and Trp) as well as an increased rate of occurrence of certain pairs of residue as compared with proteins, a random sequence of residues and "nonregulatory" peptides. By representing regulatory peptides as a sequence of hydrophobic (2 types) and hydrophilic (3 types) segments, the pattern for alternation of these segments in regulatory peptides has been determined. The segments were classified into 5 types according to the peculiarities of mutual localization of hydrophobic and hydrophilic residues within the primary structure of regulatory peptides. As compared with proteins, "nonregulatory" peptides and a random sequence of segments, regulatory peptides were characterized by an increased frequency of 4 particular pairs of segments among 12 theoretically possible pairs. These 4 pairs are fragments of the periodic segment sequence with periods of 4 segments. The revealed pattern indicates that there exists a general principle of the regulatory peptide primary structure organization and possibly a common type of the regulatory peptides flexible peptide chain folding at the ligand-receptor complex formation.

[Interaction with DNA and aggregation properties of C-terminal fragment of RecA protein]. / Vzaimodeistvie s DNK i agregatsionnye svoistva C-kontsevogo fragmenta belka RecA.

C-terminal fragment of Escherichia coli RecA protein 150 amino acids residues in length--the product of RecA protein BrCN-hydrolysis--was isolated by single stranded DNA-cellulose chromatography. In low salt buffer this fragment tightly bounds with single and double stranded DNAs. Aggregational properties of the fragment are similar to such of native protein: the fragment oligomerises in low salt buffer and precipitates in the presence of Mg2+. Double stranded DNA in the last case coprecipitates with the fragment, forming a complex which is stable at higher salt concentration, like the complexes with a native RecA protein. These results indicate that the C-terminal half of RecA protein participates in DNA binding and intersubunits interaction of RecA protein.