[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.

[Search for destruction factors of bacterial biofilms: comparison of phage properties in a group of Pseudomonas putida bacteriophages and specificity of their halo-formation products].

Comparison of Pseudomonas putida group of phages attributed to five species (af, phi15, phi27, phi2F, and pf16) with their common property of halo-formation (formation of lightening zones) around phage plaques was conducted. The halo around phage plaques appears as a result of reduction or disappearance of bacterial polysaccharide capsules. The concentration of viable bacteria remains unchanged within the halo. A comparison of specificities of halo-formation products from various phages was conducted by a simple method. These products were shown to be highly specific and inactive on other species of pseudomonads. Phage-resistant P. putida mutants scored with respect to various phages, which lost phage adsorption ability, were tolerant to the effect of halo-formation products in most cases. Apparently, the capsular polysaccharides, which serve as a substrate for depolymerases and are the primary phage receptors, may be often lost. Results of partial sequencing of the af phage genome revealed an open reading frame that encodes the enzyme transglycosylase similar rather to transglycosylases of oligotrophic bacteria belonging to different species than to lysozymes of other phages. Possibly, it is a polyfunctional enzyme combining functions of lysozyme and an enzyme that executes the penetration of phage particle across extracellular slime and capsule.

[Pseudomonas aeruginosa bacteriophage SN: 3D-reconstruction of the capsid and identification of surface proteins by electron microscopy].

The virulent P. aeruginosa bacteriophage SN belongs to the PB1-like species of the Myoviridae family. The comparatively small (66391 bp) DNA genome of this phage encodes 89 predicted open reading frames and the proteome involves more than 20 structural proteins. A 3D model of the phage capsid to approximately 18 A resolution reveals certain peculiarities of capsomer structure typical of only this bacteriophage species. In the present work recombinant structural proteins SN gp22 and gp29 were expressed and purified; and specific polyclonal antibodies were obtained. Immune-electron microscopy of purified phage SN using secondary gold-conjugated antibodies has revealed that gp29 forms a phage sheath, and gp22 decorates the capsid. Precise identification of multicopy major capsid proteins is essential for subsequent construction of gene-engineered phages bearing non-native peptides on their surfaces (phage display).

[Genome conservatism of phiKMV-like bacteriophages (T7 supergroup) active against Pseudomonas aeruginosa].

The T7-like phiKMV bacteriophage active on Pseudomonas aeruginosa was previously isolated by us and shown to have DNA resistant to many endonucleases. A loss of sensitive sites might be a consequence of a long phiKMV evolution on different hosts. To elucidate, whether this trait is shared by other similar phages, several new phiKMV-like phages were isolated from different sources and compared. All studied phiKMV-like phages formed three groups, insignificantly differing in the number and localization of endonuclease-sensitive DNA sites. This confirms that the present-day phages of this species have highly conserved genomes. Mutational "restoration" of the lost sites may be restricted by a lethal effect. The phiKMV-like phages were shown for the first time to increase the rate of in vitro accumulation of giant phiKZ-like phages of P. aeruginosa. This effect is characteristic only of phiKMV-like phages.

[Comparison of genomes of new gigantic Pseudomonas aeruginosa phages from native populations from different regions]. / Sravnenie genomov novykh gigantskikh fagov Pseudomonas aeruginosa iz priorodnykh populiatsii raznykh regionov.

To study the genome diversity of bacteriophages from geographically distant natural populations, new giant phi KZ-like Pseudomonas aeruginosa phages isolated in two different regions were compared with earlier known phages of three species (phi KZ, Lin68, EL). A broad spectrum of lytic activity was demonstrated for all phi KZ-like phages. Phages of the phi KZ species proved to be common in natural populations of various regions, while IL- and Lin68-related phages were extremely rare. Most phi KZ-related phages had unique DNA restriction patterns, but the differences between these were only minor, and the genomes did not contain nonhomologous fragments. The spectrum of capsid polypeptides proved to be conserved in each species, and was proposed as a character necessary and sufficient for express classification of phages with an accuracy of species. Phages isolated in different geographical regions showed no substantial difference. Some phages only slightly differing in DNA restriction pattern from phi KZ may be used to study the origin of phi KZ genes coding for orthologs of proteins of unrelated species (other phages, pathogenic bacteria, eukaryotes).

[Compact particles of double-stranded polyribonucleotides. I. Conditions for the formation of optically active compact double-stranded RNA particles]. / Kompaktnye chastitsy dvukhtsepochnykh poliribonuckleotidov. I. Usloviia formirovaniia opticheski aktivnykh kompaktnykh chastits dvukhtsepochechnoi RNK.

The conditions (mol. weight of PEG, ionic strength) of formation of double-stranded RNA (dsRNA) compact particles in water-salt solutions, containing poly(ethylene glycol) (PEG) have been determined. It has been shown, that in solutions of mild ionic strength (approximately 0,3) the compact particles of dsRNA are characterized by an intense positive CD-band (lambda 270 nm), but in solutions of high ionic strength (1,0-1,5)--by intense positive or negative CD-bands (lambda 270 nm). The heating of the solutions of a high ionic strength, containing the compact particles with negative CD-bands is accompanied by the change of sign of CD-band. The same effect is observed in the case of decrease of the ionic strength of the solutions. "Melting" of compact particles as revealed by the CD-method occurs prior to the melting of the secondary structure of dsRNA. These data show, that the intense CD-bands reflect the ordered arrangement of the chromophores of polynucleotide chain in compact particles. The reasons of change of the sign of the CD-bands are discussed.

[Properties of activated chromatin from rat liver shortly after partial hepatectomy]. / Svoistva aktivirovannogo khromatina iz pecheni krys v rannie sroki posle chastichnoi gepatéktomii.

The properties of rat liver chromatin 1, 2 and 6 hours after partial hepatectomy have been studied by means of cytochemical and biochemical methods. An increase in the accessibility of DNA to low molecular weight ligands, RNA--polymerase and RNAse I and also of the distances between nucleosomes and their heterogeneity in length on electron -- microscopic photographs has been found. Analysis of the isotherms of adsorption has revealed an increase in the number of binding sites for ethidium bromide on DNA and accordingly a decrease in the extent of the filling of the template with protein in activated chromatin. Two hours after partial hepatectomy rat liver chromatin does not differ in all parameters studied from control chromatin. Limited digestion of chromatin with DNAse I almost fully eliminates the difference between the fractions of activated and control chromatin in the number of binding sites for the ligands to the fractions resistent in these conditions to nuclease. A suggestion that the changes in the properties of chromatin upon activation are due to the change in the character of chromatin proteins interaction with DNA are discussed.

[Correlation between conformation distortion of the DNA sugar-phosphate backbone and high optical activity of its compact form]. / O sviazi mezhdu konformatsionnoi neodnorodnost'iu struktury dvukhtsepochechnoi DNK i vysokoi opticheskoi aktivnost'iu ee kompaktnoi formy.

Different physico-chemical methods (CD, ORD, small-angle X-ray diffraction, etc) were used for investigating the properties of the DNA compact particles formed in PEG-containing water-salt solutions. It has been shown that small-angle reflection, characteristic of the DNA compact particles, changes from 36.8 A (CPEG = 140 mg/ml) to 25 A (CPEG = 300 mg/ml). The maximal optical activity (the intense negative CD-band and optical rotation [alpha] = 60 000 degrees) are inherent properties of the DNA compact particles formed at CPEG 120--180 mg/ml. The high optical activity points to the twist of DNA chromophores through the DNA molecule resulting in a long-rang pitch (P approximately 2000A). Such macroscopic superhelical structure (diameter 40--30 A) is due to conformational distortion of the DNA double-helix with alternating "left" and "right" orientation of chromophoes. Disappearance of conformation distortion is accompanied by disappearance of the high optical activity of the DNA compact particles and results in a small-angle reflection of 25 A. Taking into account the reasons of formation of the optically-active DNA compact particles conditions are suggested to conserve high optical activity at CPEG equal to 400 mg/ml.

[Formation of the compact form of DNA in solution after reaction with spermidine]. / Obrazovanie kompaktnoi formy DNK v rastvore pri vzaimodeistvii so spermidinom.

The formation of compact particles from double-stranded DNA molecules in water-salt solutions containing spermidine was studied. It has been shown that in solutions of low ionic strength (0.01 M NaCl) DNA-spermidine complexes have the form of large particles which scatter UV-light. Electron micrographs show that such complexes formed at certain molar ratios spermidine/DNA may exist both as intermolecular aggregates and as toroidal particles 1500 A in diameter. The CD spectra of solutions containing DNA-spermidine complexes are characterized by the positive band (delta epsilon max = 10) at 265--270 nm. The appearance of the positive CD band may be caused by two factors: interaction between DNA and spermidine may lead to the alteration of the DNA secondary structure "in direction to A-form" or intermolecular aggregation, which may change the initial shape of the CD spectrum. The exclusion of spermidine molecules from DNA-spermidine complexes by Na+ ions in presence of poly(ethylene glycol) which occurs as the ionic strength increases from 0.01 to 0.3 does not lead to decompactization of DNA molecules but is accompained by the appearance of the intense negative CD band at 270 nm.

A compact from of methylated DNA is solutions containing poly (ethylene glycol).

Some peculiarities of compactization of double-stranded DNA molecules containing methylated nitrogen bases have been studied in water-salt solutions of PEG. It is shown that the methylation of N7-atoms of guanyl residues in original DNA molecules does not prevent the formation of DNA compact particles, but results in a decrease of the amplitude of the negative band in the CD spectrum of compact particles. The influence of N7-guanine methylation on the shape of the CD spectrum being the greater, the lower is the concentration of PEG. The dependence of the negative band amplitude in the CD spectrum on the content of methylated guanyl residues is practically the same for low-molecular weight DNA's from different sources. The observed decrease in the negative band amplitude is interpreted as a result of alterration of guanyl residue orientation relative to the helix axis which leads to diminished optical activity of the "microcrystalline" domains of compact particles. The evidence obtained suggests that changes in the secondary structure of DNA lead to considerable difference between CD spectra of compact particles of methlated DNA and psi-form of DNA. (The changes in the CD spectrum of the DNA compact particles occur also as a result of methylation of C5-atoms of cytosine residues). It is suggested that the negative band in the CD spectrum can be used a criterion for detection of negligible alterations in the DNA secondary structure.

[Compact form of DNA in solution. XII. Double-stranded polyribonucleotide compacting in the presence of polyethylene glycol]. / Kompaktnaia forma DNK v rastvore. XII. Kompaktizatsiia dvukhtsepochechnykh poliribonukleotidov v prisutstvii poliétilenglikolia.

Double-stranded polyribonucleotides (a replicative form of phage f2 RNA--dsRNA and poly(A) poly(U), can adopt a compact from in solutions, containing NaCl and poly(ethylene glycol) (PEG). According to electron-microscopic observations dsRNA compact particles have the form of disks or doughnuts 200--400 A in diameter. X-ray diffraction patterns from dense slurries of dsRNA compact particles show a reflection at a spacing of 35 A, which is indicative of the existance of ordered regions in compact particles. The intense positive CD band, which is characteristic of dsRNA and poly(A) poly(U) compact particles, presumably results from the ordered regions in the particles. Heating of the solution leads to the disappearance of the intense positive CD band, probably as a result of the destruction of the ordered structure of compact particles. Heat or acid denatured dsRNA molecules as well as single-stranded molecules of ribosomal RNA also form large particles in PEG-containing solutions. However, X-ray diffraction patterns from these particles do not show the 35 A reflection and the specific positive band is not present in their CD spectra, which indicates that such particles lack ordered internal structure. It is suggested that similar mechanism of compactization of double-stranded polynucleotides (DNA and RNA) exist, and compact particles may be divided into two families (psi+ and psi-), differing by the secondary structure of double-stranded polynucleotides, which form the particles.

A comparative X-ray diffraction and circular dichroism study of DNA compact particles formed in water-salt solutions, containing poly(ethylene glycol).

Comparative CD and X-ray diffraction studies of DNA compact particules which were obtained in PEG-containing water-salt solutions, have been carried out. Compact particles, formed from native DNA, produce a psi CD spectrum (characterized by a negative band at lambda-270 nm) and a small-angle X-ray diffraction pattern, which shows two reflections: I at 34-40 A and II at 80-90 A (together with its second-order reflection). Compact particules, formed from DNA molecules with partially disordered secondary structure, do not produce the psi CD spectrum and the reflection I, while the reflection II remains unchanged. It is suggested that the spacing of 34-40 A is associated with a side-by-side packing of DNA fragments in "microcrystallization' regions in compact particules and that such "microcrystallization' accounts for the generation of the psi CD spectrum.

[DNA compact form. VI. Changes of DNA secondary structure under conditions preceding its compaction in a solution]. / Kompaktnaia forma DNK v rastvore. VI. Izmenenie struktury dvukhtsepochechnoi DNK v usloviiakh, predshestvuiushchikh ee kompaktizatsii.

Optical and thermochemical properties of E. coli DNA molecules are compared in solutions containing poly(ethyleneglycol) (PEG) in concentrations at which compactization is not yet observed. It is shown that under conditions preceding DNA compactization (CPEG less than 60 mg/ml) changes in CD spectra occur which suggest that the secondary structure of some DNA fragments is altered. These changes of the secondary structure result from dehydration of DNA molecules in PEG-containing solutions. Electron micrographs of DNA molecules obtained under conditions preceding compactization suggest that under these conditions linear DNA molecules may form "four-stranded" fragments as well as double-stranded "loops".

[Structure of chromosomal deoxyribonucleoproteins. VII. Free dna in preparation of fragmented chromatin]. / Struktura khromosomnykh dezoksiribonukleoproteidov. VII. Svobodnaia DNK v preparatakh fragmentirovannogo khromatina

Chromatin which was hydrodynamically sheared in a low ionic strength buffer lacking divalent cations (mu = 0.005) contains a heterogeneous set of DNP particles but no molecules of free DNA. The main finding is that a transference of sheared chromatin to 1-2 mM MgCl2 or to 0.1-0.2M NaCl results in the appearance of completely free DNA molecules. A salt-induced rearrangementof DNA-bound histones, but not a partial loss of them is responsible for the observed phenomenon. Formation of free DNA molecules is accompanied by aggregation of the majority of remaining DNP particles. Percentage of free DNA molecules in the chromatin which was sheared to an average DNA length of approx. 400 base pairs is increased from zero in the initial DNP sample to 8-9% in 1 mM MgCl2 and further to 30-31% of the total DNA in 0.30 M NaCl, 2 mM MgCl2. Free DNA molecules in the sheared chromatin are observed not only upon isopycnic banding of formaldehyde-fixed DNP in CsCl gradients but also in non-ionic Metrizamide gradients with either fixed or unfixed DNP samples. Process of free DNA formation is a reversible one; its direction and the equilibrium state depend in particular on the ionic conditions of the medium. Percentage of free DNA molecules in the sheared chromatin at a given ionic strength of solution is strongly decreased upon an increase of the average length of DNA in the DNP particles. Several lines of evidence suggest that free DNA molecules are formed in the sheared chromatin as a result of cooperative rearrangements of histones in salt-induced DNP aggregates. A dynamical model of chromosomal fiber is proposed on the basis of the present and earlier experimental data [1]. According to the model histones are arranged on DNA in clusters separated by stretches of free DNA. A salt-induced migration of histones along or between DNP fibers can result in unification of different clusters, thereby generating longer stretches of free DNA, the total amount of free DNA being approximately constant. Possible in vivo significance of such a dynamical structure is discussed.