Biomedical subjects
Publications and source records attributed to P O Vardevanian.
Melting temperature and GC content were evaluated for DNA of some nitrogen-fixing bacteria of Rhizobium leguminosarum and Onobrychis spp. (Adans). The degree of homology between strains of the same species was determined. A combination of thermal denaturing and molecular hybridization can serve as a rapid test for evaluating the genome homology of the organisms compared.
We treat theoretically conformational transitions in DNA-ligand complexes allowing for the existence of different binding parameters of the ligand to different DNA conformations. The parameters of binding are determined from the best fit of the theory to experimental data for the difference between transition point (Tm) and the width of transition curve (delta T) for the complexes and for naked DNA. The analysis shows that Ethidium Bromide (EB) and Actinomycin D (AMD) each may form at least five types of complexes: three types (one "strong" and two "weak") with helix DNA and two types ("strong" and "weak") with single-stranded DNA. The parameters of the complexes have been obtained. Some testable experimental predictions of the theory are also discussed.
The effect of ligand interacting with native DNA by two types on the parameters of helix-coil transition in homopolymers is considered using the most probable distribution method (Yu.S. Lazurkin et al., Biopolymers 1970). It is shown that at a small relative concentration of ligand the melting enthalpy (delta H) of DNA may be obtained from the universal formula which contains only values directly known from the experiments. It is shown that the formula for the change of melting temperature and width of melting range depending on the total ligand concentration in solution is converted into the corresponding formulae which are defined for the case when only one type of interaction of ligand and DNA is considered.
The enthalpy of helix-coil transition of DNA (delta H) is determined from the experiments on DNA melting with ligands by means of absolutely general formula, which contains only values directly known from the experiment (M.D. Frank-Kamenetskii, and A.T. Karapetian, Mol. Biol. USSR 6, 621 (1972)) with the combination of the "area" method (P.O. Vardevanian, et al., Biophysica 28, 130 (1983)). The experimentally obtained data show that delta H depends on both concentration of Na+ in solution and GC-content of DNA and is of high accuracy.
A highly active fraction of rat liver oligonucleosome DNA has been isolated and studied by means of thermal denaturation after induction by amino acid mixture or hydrocortisone. A considerable redistribution of DNA content has been shown in sucrose gradient fractions during these forms of induction. The changes are revealed in melting temperature, differential melting profile of DNA, isolated from actively transcribed chromatine fractions. Analysis of melting profiles shows changes of GC content of oligonucleosome DNA, suggesting that there are differences in activation during two studied forms of induction.
The changes of RNA-polymerase activity of hepatocytes nuclei, chromatin circular dichroism (CD) spectra and differential melting curves of hepatocytes of rats which were injected hydrocortisone or amino acid mixture intraperitoneally have been studied. Actinomycin D reduces essentially RNA-polymerase activity and maximum of CD spectra. Under the influence of hydrocortisone or amino acid mixture the inhibiting action of antibiotic is taken off but the RNA-polymerase activity and maximum of CD spectra don't achieve the meanings found under the influence of inductors only. The antibiotic injection changes essentially the chromatin melting parameters. A conclusion has been drawn that the amino acid mixture and hydrocortisone effect the transcription process affecting the different levels of chromatin structure.
It was shown that injections of an amino acid mixture essentially increase the number of nuclease-sensitive regions of chromatin and its active fraction (Mg2+-soluble fraction), while hydrocortisone increases the amount of the latter, which is less sensitive to the effect of DNAase II. Genome activation during nonhormonal induction after administration of the amino acid mixture or during hydrocortisone injection reflects in different ways on the parameters of melting of chromatin active fractions and on the relative content of its protein fractions.
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The results of investigations of the electrokinetic properties of cell nuclei in various functional states were summarized. A positive correlation between the value of the electrostatic charge on the nuclear membrane surface and the functional state of the genetic apparatus upon activation was established. It was shown that the value of the electrokinetic potential reflects differences in the mechanisms of action of biologically active substances, and that this may be related to the peculiarity of their action on both membrane surface and the cell genome. It is concluded that the electrokinetic properties of the nuclear membrane reflect changes occurring in the genome of the eukaryotic cell nucleus, and from the value of surface charge density, the extent of involvement of the genome under various influences can be determined.
The interaction of ethidium bromide with single-stranded synthetic and natural polynucleotides at high temperatures (t = 70 degrees C) and low pH values (pH 3.0) was studied. The isotherms of adsorption of ethidium bromide on single-stranded DNA were obtained. Two modes of binding of single-stranded DNA, strong and weak, were revealed. The values of the corresponding constants of interaction of this ligand and the number of bases per one binding site were determined.
The interaction of ethidium bromide with calf thymus DNA was investigated by the method of differential pulse voltammetry. It was found that ethidium bromide binds with DNA in several ways. Corresponding values of the constants and the number of binding sites were determined. The intercalation, semi-intercalation, and electrostatic mechanisms of interaction were shown. The results obtained are in good agreement with the data obtained by spectroscopic (absorption and fluorimetric) methods.
Ligand binding with nucleic acids is described in frames of the theory of random processes. It is shown that the probabilistic description of binding of a ligand to nucleic acid allows one to describe not only the kinetics of changes in the number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. A general analysis of absorption isotherms and the kinetics of ligand binding with nucleic acids allows one to determine the rate constants of formation and decomposition of the ligand-nucleic acid complex. A comparison of the results obtained with the case of low fillings is conducted.
There were investigated changes of electrokinetic potential (EKP) of hepatocyte nuclei during hydrocortisone induction and activation induced with amino acids mixture. An increase of RNA-polymerase activity was shown to correlate with EKP value. Actinomycin D essentially decreased this potential. Injection of hydrocortisone or amino acids mixture under the suppression of transcription with actinomycin D removed the inhibitor action, but the potential value did not reach that under the action of the activators only. Induction with hydrocortisone increased the potential by various degrees for the nuclei of different size. The results obtained suggest that EKP of the nuclei carries the information about functional condition of the genetic apparatus and can be a valuable and handy instrument of definition of its physiological activity.
It was shown that there existed a correlative connection between total clip activity and circular dichroism relative ellipticity. It is supposed that the parameters of total clip activity also may carry information about DNA conformation in the complexes with histones.
A method for determining the nucleotide content of DNA blocks from areas under the melting curve is suggested. It is shown that this method is more simple and the total melting curve is decomposed not to the Gauss components, but it is considered as as superposition of the melting curves of individual blocks. Application of the method for the determination of calf thymus DNA is discussed.
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The theory of the cooperative transition of DNA.ligand complexes have been developed, in which a model was implied where the multimodal ligands simultaneously interacted with DNA. Obtained formula express the dependence of the experimentally estimated values of the changes of transition point and width of transition on the concentration of the ligands. These expressions make possible to obtain the thermodynamic parameters of the interaction of the multimodal ligands with DNA (binding constants, number of base pairs corresponding to one binding site of DNA etc.) by comparing the theoretical and the experimental data.