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A I Poletaev

Publications and source records attributed to A I Poletaev.

36 records · Page 2Linked to original sources
PubMedDate not supplied

[Effect of deproteinization on the in situ chromatin staining with 7-aminoactinomycin D].

The possibility of use of 7-amino-actinomycin D (7aAMD)--fluorescent analog of actinomycin D--as a specific dye for DNA staining in the suspended cells was studied by means of laser flow-cytometry. The optimal conditions for staining were obtained: 7aAMD concentration 10(-5) M, pH 7, staining time 20 min, 37 degrees C, ionic strength 0.15 M Na+. In this case the fluorescent signal is proportional to the DNA amount and coefficient of variation is about 0.03. The influence of the stepwise extraction of the proteins from chromatin also was studied. In the course of the salt deproteinization the fluorescence intensity gradually rose thus showing the increase of the binding sides-number. The deproteinization of cells nuclei by 0.1 HCl increased the number of binding sites 2.5 times more. It was shown that the incubation of cells with RNAse at elevated ionic strength (0.3-0.7 M NaCl) leads to an additional increase of the cell fluorescence and produces no effect at low and normal ionic strength. The deproteinizing effect of RNAse and its possible mechanism is discussed.

Animals↗
PubMedDate not supplied

[Isolation of individual chromosomes and their use in studying the human and animal genome].

This mini-review considers the main achievements in the field of human and animal chromosome separation: technical problems of flow-karyotyping and flow-sorting, the types of cell lines and chromosome source used, different protocols of chromosome suspension preparation, fluorochromes for chromosome staining, efficiency of chromosome sorting and purity of the fractions. The use of fractions for creating chromosome-specific human gene libraries and mapping cloned genes and DNA fragment are also considered.

Animals↗
PubMedDate not supplied

[Flow cytometry and cell sorting: current state and prospects for use in molecular biology].

A survey of the specific features of flow cytometry, principals of instrumentation and main parameters of the modern cell sorting cytometers is given. Analytical capacities of flow cytometry as well as the main directions of its applications in cell biology, clinical diagnostics, immunology, biotechnology and molecular biology are considered. Also a possible future development of flow cytometry instrumentation and applications in molecular biology are briefly discussed.

Animals↗
PubMedDate not supplied

[Circular dichroism of DNA--dye complexes. II. Anisotropy of the long-wave circular dichroism effect and structure of the complex].

Anisotropy of torsional strength of the splitted electronic transition in the case of chromophore-chromophore interaction of dye molecules situated on the helical matrix was considered theoretically and as analytical expression for the value Rperpendicular/Rparallel was obtained. These theoretical results were compared with the experimental data obtained with DNA-proflavine, DNA-pyronine and DNA-acridine orange complexes oriented in multicappilar flow-cell. Studies of the optical effects (optical density and CD changes) due to orientation of these complexes showed that the acridine chromophores are not perpendicular with respect to the DNA axis (alpha D = 19--22 degrees). The DNA base pairs in complexes as assumed also are not perpendicular to the DNA axis, the inclination angle of their transition moments (for the band near 260 nm) being bigger than that of dye chromophores (24 degrees). These results indicate that under experimental conditions used by us no intercalation can be observed.

Acridines↗
PubMedDate not supplied

[Kinetics of the formation of AT-specific DNA-distamin complex].

Kinetics of DNA--distamin complex formation was studied by means of the stop-flow method. It has been found that the binding reaction is much faster than in the case with distamycin A and has only two stages. The differences in kinetic properties of the complexes of distamin and distamycin A with DNA has been interpreted as resulting from the formation of different bonds by protonated positively charged N-containing groups of ligands with negatively charged DNA phosphates.

Adenine↗
PubMedDate not supplied

[Kinetics of DNA-distaxin complex-formation].

Kinetics of DNA--distaxines complexes formation was studied spectrophotometrically by means of the stop--flow method. It has been found that increase of number of bulky pyrrolcarboxamide group in the row of distaxines: -0, -1, -2, - slows down the binding reactions and increases the number of their stages.

Animals↗
PubMedDate not supplied

[Secondary structure of RNA of influenza virus in free form and in ribonucleoprotein].

Large amounts of RNA and RNP isolated from influenza virus were obtained. This has allowed us to undertake detailed physical studies of the secondary structure of RNA of influenza virus in free form and in RNP. Analysis of CD spectrum and the hypochromic effect after thermal denaturation of RNA indicated that RNA in free form contains 58--62% double-stranded regions. By comparative studies of the secondary structure of RNA in RNP, it was estimated that 12--14% of the RNA exists in double-stranded form.

Hot Temperature↗
PubMedDate not supplied

[Formation kinetics of the AT-specific complex: DNA-distamycin A].

The kinetics of DNA-distamycin A complex formation and dissociation was studied by means of the stop-flow method. It has been found that the complex formation has at least five steps, the formation of AT-specific complex is limited by the dissociation of less specific intermediate complexes, then follows the statistical rearrangement of the ligand molecules and "fixation" of the specific complex with the longest lifetime and the biggest number of the additional H-bounds with the matrix. The energy of a single specific H-bond between the ligand molecule and AT-pair is equal to--1.05 kcal/mol.

Adenine↗
PubMedDate not supplied

[Structure of DNA complexes with regular polypeptides].

The conformation of some regular polypeptides: (Lys-Ala)50, (Lys-Ala2)37, (Lys-Ala2)26, (Lys-Ala3)18, (Lys3-Pro)29, (Orn3-Gly)28 was studied by means of CD. The complexes of these polypeptides with DNA were obtained by the methods of jump-dilution of a two-components mixture from 2 M NaCl to 0.05 M NaCl. The extent of DNA covering by the polypeptides was compared using binding isoterms of ethidium on DNA and DNA-polypeptide complex. The length, L, which polypeptides cover on DNA was estimated by means of energy transfer between the dyes absorbed on the complexes. The CD spectra of the complexes revealed a high sensitivity to changes of the environmental conditions. Small variations in the temperature and ionic strength produces marked changes in the CD spectra of the complexes. It was suggested that observed CD changes are due to both the structural relaxation of the complexes and the existence of liquid-crystal domains in solution.

Chemical Phenomena↗
PubMedDate not supplied

[Thermodynamic characteristics of nucleic acid complexes with silver ions].

By means of mixing reaction calorimetry the enthalpy of the complexes formation between Ag+ ions and DNA and dsRNA was measured. It was shown that Ag+ ions are able to form two types of complexes (I and II) with dsRNA. Using the method of the competitive reaction with chloride ions the stability constants of complex formation were obtained for dsRNA-Ag+ complexes for different temperatures. These measurements gave the delta H and delta S values for both complexes: delta HI = -74,9 +/- 7,1 kjouls/mol, delta SI = -100.0 +/- 25.0 jouls/mol deg; delta HII = -39,8 +/- 4,2 kjouls/mol, delta SII = +2 +/- 14 jouls/mol deg. The calorimetric results of delta H determination are the same within the limits of experimental errors. The enthalpy term of dsRNA-Ag+ complexes proved to bring the main contribution into the free energy of complex formation.

Animals↗
PubMedDate not supplied

[Quantitative processing of results of uniparametric fluorescent flow analysis of human chromosomes].

The proposed procedure of computer analysis of the flow karyotype data, obtained in human chromosomes studies, is able to provide information about the basic parameters of the karyotypes: the positions of the peaks (corresponding to the relative size of chromosomes), peaks areas (relative number of chromosomes in the sample), coefficients of variation (CV) of the peaks--possible differences between homologous chromosomes. The analysis is based on the assumption that all chromosomal components of the experimental distributions are normal (Gaussians). The algorithm of the analysis uses a combination of two approaches: truncation method and least squares method. As the flow data are "contaminated" by background components, special tools for filtering off the contaminating signals were designed including the original integral Fourier filtering procedure. This analysis is realized in a program package utilizing IBM-compatible PCs. The user is able to get the desired parameters for most chromosomes of the karyotype under study from univariate flow data: differences between particular homologous chromosomes, presence of chromosome aberrations, extra chromosomes, etc., since structural aberrations and chromosome number variation lead to specific changes of the parameters of chromosome-related components.

Algorithms↗
PubMedDate not supplied

[Uniparametric analysis of chromosomes from human fibroblasts using flow cytofluorometry].

Metaphase chromosomes from human diploid fibroblasts were isolated by the polyamine method and stained with different fluorochromes: propidium iodide, chromomycine A2, bisbensimide H33258, and DAPI. The fluorescently stained chromosomes were analyzed using a cell sorter ATC-3000 ("Bruker-Spectrospine"), and the flow histograms were investigated to identify the peaks of individual chromosomes and to choose the chromosomes which can be isolated to a high purity. High resolution was obtained with H33258 or DAPI. It was demonstrated that the peaks of chromosomes 13, 17, 20 are well distinguished from the adjacent peaks, so that these chromosomes can be sorted with a high purity. The peaks formed by chromosomes 5, 6, 8, 14, 15 are slightly overlapped by the adjacent peaks, and the sorting on the basis of monovariate analysis cannot provide sufficient purity of the sorted fractions (< 0.5). Chromosomes 1-2, 3-4, 7-X, 9-12, 16-18, 19, 21-22 cannot be discriminated for successful sorting of an individual chromosome type. The data obtained provided an experimental basis for choosing the chromosomes to be sorted with high purity for the subsequent cloning of their DNA.

Cells, Cultured↗
PubMedDate not supplied

[Circular dichroism of DNA complexes with dyes. III. Effect of latent optical activity and the structure of the complexes].

Circular dichroism anisotropy was studied both theoretically and experimentally for the complexes of DNA and dsRNA with dyes (proflavine, 2,7-di-t-butyl proflavine, "Hoechst-33258") and antibiotics (distamycin A, netropsin and olivomycin). Theoretical analysis showed that general features of CD anisotropy, revealed in the previous studies (CD components--delta epsilon parallel to and delta epsilon perpendicular--are ten times or more bigger than the CD-effect without orientation, and delta epsilon parallel to approximately 2 delta epsilon perpendicular) are due to the existence of a specific effect named "latent" optical activity (LOA). This effect can be observed in many cases of non-chiral symmetrical chromophores if they are oriented. The effect is due to the excitation of an electrical dipole transition and a perpendicular magnetic dipole transition (or quadrupole transition) of a molecule. The amplitude and the sign of the LOA-effect depends on the orientation of the chromophores with respect to the light beam; with a random orientation the mutual compensation of LOA-effects of different chromophores happens and no LOA-effect appears. The analitycal expressions relating the value of LOA-effect of the system with electronical characteristics of the chromophores and the geometrical parametra of their arrangement was obtained. The experimental data obtained for the oriented complexes of DNA and dsRNA with proflavine made it possible to determine an angle between the chromophore and the plane perpendicular to the DNA axis--gamma. For the calf thymus DNA gamma = = + 1.8 +/- 0.4 degrees, for the phage T2 DNA gamma = + 2.2 +/- 0.4 degrees, and for phage f2 dsRNA gamma=--3.5 +/- +/- 0.5 degrees. These results, obtained at relatively low concentrations of the bound proflavine (r approximately 0.01), are in accordance with the intercalating mode of the dye binding. A study of CD anisotropy of DNA complexes with other ligands showed that many different chromophores possess LOA-effect. This phenomenon can be used to obtain both spectroscopic and structural information about the systems similar to those reported here.

Acridines↗
PubMedDate not supplied

[Two-parameter flow fluorescence analysis of human chromosomes. Quantitative processing].

A procedure for the quantitative analysis of the results of fluorimetric studies on human chromosomes in a flow is described. The procedure enables one to simultaneously follow two parameters by using two fluorochromes, one of which is selective to the GC-, and the other, to AT base pairs of DNA. Thus, it becomes possible to derive from the experimentally obtained distributions an information on the relative DNA content in individual chromosomes, differences in DNA content between homologous chromosomes, the relative number of chromosomes of each type in the sample tested, as well as on the percent ratio of AT and GC base pairs in a particular chromosome. In addition, the procedure enables one to assign, with a high degree of accuracy, the peaks on experimental distributions to the objects of analysis chromosomes. The analysis cutting off the extreme combines the method of components with the least square method, as applied to the peaks of the Gaussian shape. The first step involves the filtration of the starting experimental data from contaminating signals from both the parts of degraded chromosomes and stained cytoplasm fragments, and from chromosome aggregates. The procedure is realized as a package of programs for IBM-compatible PC/AT computers (486 and later versions), which permits one to perform a comprehensive analysis of two-parameter distributions of chromosome fluorescence signals to solve a number of problems, such as the identification of chromosome aberrations in cell lines tested, quantitative comparison of distinguishing features of homologous chromosomes and whole chromosome sets.

Chromosomes, Human↗