The use of the method of anomalous time dependence of viscosity for registration of changes in the nucleoide conformation. Part 1

The sensitivity of the method of anomalous time dependence of viscosity to changes in the conformation of DNA-protein complexes (such as nucleoide) by the action of increased temperature (33, 70 and 85 degrees C) and the combined action of temperature and Na+, Cl- ions on lysates of Escherichia coli AB1157 cells has been studied. The optimal conditions of the cell lysis was determined on the basis of the curve parameters of the anomalous time dependence of viscosity.     

Registration of changes in the nucleoide conformation based on the analysis of the time dependence of viscosity

The sensitivity of the method of anomalous time dependence of viscosity to changes in the conformation of DNA-protein complexes (such as nucleoide) by the action of (1) nalidixic acid on cells and (2) one- and two-charged ions Na+, Cl-, Cu2+, Ca2+, Mg2+, Zn2+, SO4(2-) on Escherichia coli cells has been studied. The data obtained suggest that the method anomalous time dependence of viscosity is highly sensitive to the conformation of high-molecular DNA-protein complexes. It was shown that the method anomalous time dependence of viscosity can be used to register cell sensitivity to changes in the concentration of one- and two-charged ions in suspension.     

Permeability of the membrane of the Escherichia coli envelope for ethidium bromide

The interaction of ethidium bromide, a fluorescent dye, with Escherichia coli cells was studied. The envelope of intact cells was shown to be impermeable for ethidium bromide molecules. The dye penetrated however into E. coli spheroplasts. The barrier properties of the cell envelope against ethidium bromide were ruptured if the cells were treated with EDTA. The results suggest that the outer membrane serves as a principal barrier against penetration of ethidium bromide inside the cells while the cytoplasmic membrane of E. coli is permeable for the dye.     

Fluorescence microscopy of DNA-protein structures from osmotically lysed mitochondria of yeast and tobacco

Summary The mitochondrial nucleoid is a compact structure composed of DNA and protein. By fluorescence microscopy, decondensation of the nucleoids was observed when yeast and tobacco mitochondria were osmotically lysed and subjected to an electric field. Structures stained with ethidium bromide were seen moving toward either the anode or the cathode. Since the movement of deproteinized DNA is toward the anode, the structures moving toward the cathode represent DNA-protein complexes with a net positive charge. Nucleoid decondensation and unfolding of the DNA probably resulted from the removal of weakly bound proteins; yet high-affinity basic proteins were evidently retained yielding cationic DNA-protein structures. Some of the positively charged structures were observed to break, presumably at single-stranded DNA regions, releasing negatively charged particles. The DNA-protein structures were complex branching forms larger than the unit genome, suggesting that multigenomic, concatemeric DNA is present within the mitochondria.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EtBr ethidium bromide - HMG high-mobility group - mt-genome mitochondrial genome - mt-nucleoid mitochondrial nucleoid - PFGE pulsed-field gel electrophoresis - pt-nucleoid plastid nucleoid - ssDNA single-stranded DNA     

Effect of static magnetic field on E. coli cells and individual rotations of ion-protein complexes

The effect of week static magnetic fields on Escherichia coli K12 AB1157 cells was studied by the method of anomalous viscosity time dependencies (AVTD). The AVTD changes were found when E. coli cells were exposed to static fields within the range from 0 to 110 microT. The dependence of the effect on the magnetic flux density had several extrema. These results were compared with theoretical predictions of the ion interference mechanism. This mechanism links the dissociation probability of ion--protein complexes to parameters of magnetic fields. The mechanism was extended to the case of rotating complexes. Calculations were made for several ions of biological relevance. The results of simulations for Ca(2+), Mg(2+), and Zn(2+) showed a remarkable consistency with experimental data. An important condition for this consistency was that all complexes rotate with the same speed approximately 18 revolutions per second (rps). This suggests that the rotation of the same carrier for all ion--protein complexes may be involved in the mechanism of response to the magnetic field. We believe that this carrier is DNA.     

Cell-density dependent effects of low-dose ionizing radiation on E. coli cells

The changes in genome conformational state (GCS) induced by low-dose ionizing radiation in E. coli cells were measured by the method of anomalous viscosity time dependence (AVTD) in cellular lysates. Effects of X-rays at doses 0.1 cGy--1 Gy depended on post-irradiation time. Significant relaxation of DNA loops followed by a decrease in AVTD. The time of maximum relaxation was between 5-80 min depending on the dose of irradiation. U-shaped dose response was observed with increase of AVTD in the range of 0.1-4 Gy and decrease in AVTD at higher doses. No such increase in AVTD was seen upon irradiation of cells at the beginning of cell lysis while the AVTD decrease was the same. Significant differences in the effects of X-rays and gamma-rays at the same doses were observed suggesting a strong dependence of low-dose effects on LET. Effects of 0.01 cGy gamma-rays were studied at different cell densities during irradiation. We show that the radiation-induced changes in GCS lasted longer at higher cell density as compared to lower cell density. Only small amount of cells were hit at this dose and the data suggest cell-to-cell communication in response to low-dose ionizing radiation. This prolonged effect was also observed when cells were irradiated at high cell density and diluted to low cell density immediately after irradiation. These data suggest that cell-to-cell communication occur during irradiation or within 3 min post-irradiation. The cell-density dependent response to low-dose ionizing radiation was compared with previously reported data on exposure of E. coli cells to electromagnetic fields of extremely low frequency and extremely high frequency (millimeter waves). The body of our data show that cells can communicate in response to electromagnetic fields and ionizing radiation, presumably by reemission of secondary photons in infrared-submillimeter frequency range.     

Sensitivity of Escherichia coli cell membranes to various classes of detergents

The mechanisms of interaction between non-ionic or cationic surfactants with Escherichia coli K-12 cell membranes were studied using an approach based on the registration of changes in the membrane permeability to ethidium bromide, a fluorescent dye for nucleic acids. Triton X-100, a non-ionic detergent, was shown to exert no effect on the permeability of intact cell membranes. Triton X-100 interacted with the bacteria only after treatment with EDTA, a complexing agent for bivalent cations. Cetyltrimethyl ammonium bromide increased the permeability to ethidium bromide and the action of this cationic detergent did not require the pretreatment with the complexing agent. SDS, an anionic detergent, damaged E. coli K-12 and this could be registered by the lowering of intensity of light scattering by the bacterial suspension. The surface charge of E. coli K-12 cells was shown to influence the interaction of ionic detergents with bacterial cell membranes. Its variation by changing the pH of the incubation medium did not make E. coli K-12 sensitive to Triton X-100.     

HasF, a TolC-homolog of Serratia marcescens, is involved in energy-dependent efflux

A tolC-like gene (hasF) was identified upon scanning the incomplete database of the S. marcescens genome. This gene was amplified using PCR and cloned in the pUC18 vector to yield pUCHF. Sequencing of the S. marcescens tolC-like hasF gene and subsequent amino acid sequence prediction revealed approximately 80% amino acid homology with the Escherichia coli TolC. A tolC-deficient strain of E. coli (BL923) containing pUCHF/hasF was analyzed for susceptibility to fluoroquinolones (ciprofloxacin, norfloxacin, and ofloxacin), chloramphenicol, sodium dodecyl sulfate (SDS), and ethidium bromide. Antibiotic susceptibility assays of the E. coli tolC-deficient mutant BL923 demonstrated a 64-fold increase in resistance to SDS and ethidium bromide upon introduction of the S. marcescens tolC-like hasF gene. No change was observed for susceptibility to fluoroquinolones and chloramphenicol. Ethidium bromide accumulation assays performed using E. coli BL923:pUCHF established the role of the S. marcescens hasF gene product in proton gradient-dependent efflux.     

Cell-to-cell communication in response of E. coli cells at different phases of growth to low-intensity microwaves

Effects of millimeter waves (MMW) at the frequency of 51.755 GHz were studied in logarithmic and stationary E. coli cells at various cell densities. The changes in the genome conformational state (GCS) were analyzed by the method of anomalous viscosity time dependence (AVTD). Before lysis, the cells were adjusted to the cell density of 4x10(7) cells/ml and all AVTD measurements were run at this cell density. Stationary cells responded to MMW by increase in AVTD, while the same MMW exposure decreased AVTD in logarithmic cells. MMW effects depended on cell density during exposure and were stronger for stationary cells. The observed dependence on cell density suggested a cell-to-cell communication between cells during exposure to microwaves. Decrease in power density (PD) resulted in more striking differences between responses at different cell densities. The data provided evidence that intercellular communication in response to MMW depended on cell status and PD of microwaves. The MMW effects were studied in more detail at low intensity of 10(-17) W/cm(2) in the range of cell densities 4x10(7) to 8x10(8) cells/ml. The obtained sigmoid-like dependence of MMW effect on cell density saturated at approximately 5x10(8) cells/ml. The dependence of MMW effect on cell density was very similar in this study and in previous studies with weak extremely low frequency (ELF) electromagnetic fields (EMF). The data suggested that cell-to-cell communication might be involved in response of cells to weak EMF of various frequency ranges.     

Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus

NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.     

Comparative analysis of mechanisms of the modification of microorganism viability under the effect of UHF heating and hyperthermia]

In experiments with models of isogenic Escherichia coli strains, a comparative study was made of the effect of SHF of electromagnetic field and hyperthermia. The survival rate of bacteria was determined and, simultaneously, injuries to genetic supramolecular structures were registered through measuring the anomalous time dependence of cell lysate viscosity. The combined effects of the mixture of these factors with H2O2 microconcentrations were studied. The differences observed in their realization were attributed to the different mechanisms of action of these factors on the repair enzyme systems of the studied cells. It has been found that the effect of microwaves on microorganisms causes much severer damages to DNA that hyperthermia does.     

The binding of polyamines and of ethidium bromide to tRNA.

The binding of spermidine and ethidium bromide to mixed tRNA and phenylalanine tRNA has been studied under equilibrium conditions. The numbers and classes of binding sites obtained have been compared to those found in complexes isolated by gel filtration a low ionic strength. The latter complexes contain 10-11 moles of either spermidine or ethidium per mole of tRNA; either cation is completely displaceable by the other. In ethidium complexes, the first 2-3 moles are bound in fluorescent binding sites; the remaining 7-8 molecules bind in non-fluorescent form. At least one of the binding sites for spermidine appears similar to a binding site for fluorescent ethidium. Similar results are found with E. coli formylmethionine tRNA. Spermine, in excess of 18-20 moles per mole tRNA, causes precipitation of the complex. Putrescine does not form isolable complexes with yeast tRNA and displaces ethidium less readily from preformed ethidium-tRNA complexes. Under equilibrium conditions, in the absence of Mg++, there are 16-17 moles of spermidine bound per mole of tRNA as determined by equilibrium dialysis. Of these, 2-3 bind with a Ksence of 9 mM Mg++, the total number of binding sites is decreased slightly and there appears to be only one class of sites with a Ka = 600 M(-1). Quantitatively similar results are obtained for the binding of spermidine to yeast phenylalanine tRNA. When the interaction between ethidium bromide and mixed tRNA is studied by equilibrium dialysis or spectrophotometric titration, two classes of binding sites are obtained: 2-3 molecules bind with an average Ka = 6.6 x 10(5) M(-1) and 14-15 molecules bind with an average Ka = 4.1 x 10(4) M(-1). Spermidine, spermine, and Mg++ compete effectively for both classes of ethidium sites and have the effect of reducing the apparent binding constants for ethidium. When the binding of ethidium is studied by fluorometry, there are 3-4 highly fluorescent sites per tRNA. These sites are also affected by spermidine, spermine and Mg++. Putrescine has little effect on any of the classes of binding sites. These data are consistent with those found under non-equilibrium conditions. They suggest that polyamines bind to fairly specific regions of tRNA and may be involved in the maintenance of certain structural features of tRNA.     

Dependence of mammalian DNA replication on DNA supercoiling. I. Effects of ethidium bromide on DNA synthesis in permeable Chinese hamster ovary cells.

Chinese hamster ovary cells labelled with [14C]thymidine were made permeable, incubated with various concentrations of the intercalating dye ethidium bromide, and centrifuged through neutral sucrose gradients. The gradient profiles of these cells were qualitatively similar to those obtained by centrifuging DNA from untreated, lysed permeable cells through gradients containing ethidium bromide. The sedimentation distance of DNA had a biphasic dependence on the concentration of ethidium bromide, suggesting that the dye altered the amount of DNA supercoiling in situ. The effect of ethidium bromide intercalation on incorporation of [3H]dTMP into acid-precipitable material in an in vitro DNA synthesis mixture was measured. The incorporation of [3H]dTMP was unaffected by less than 1 microgram/ml of ethidium bromide, enhanced up to two-fold by 1--10 microgram/ml, and inhibited by concentrations greater than 10 micrograms/ml. Alkaline sucrose gradient analysis revealed a higher percentage of small DNA fragments (6--20 S) in the cells treated with 2 micrograms/ml ethidium bromide than in control cells. These fragments attained parental size within the same time as the fragments in control cells. In cells treated with 2 micrograms/ml ethidium bromide, a significant fraction of newly synthesized DNA resulted from new starts, whereas in untreated cells practically none of the newly synthesized DNA resulted from new starts. These results suggest that relaxation of DNA supercoiled structures ahead of the replication fork generates spurious initiations of DNA synthesis and that in intact cells the rate of chain elongation is limited by supercoiled regions ahead of the growing point.     

A comparison of fluorochromes for direct viable counts by image analysis

Total direct and direct viable counts of fresh and injured cultures of Escherichia coli were determined by image analysis in preparations stained with acridine orange, ethidium bromide and 4',6-diamidino-2-phenyl indole (DAPI). Cells stained with DAPI were not detected by image analysis. Fresh cultures stained with acridine orange or ethidium bromide gave comparable counts. Injured E. coli stained with ethidium bromide gave higher counts that with acridine orange. Injured cultures stained with acridine orange contain high proportions of green cells which are less easily detected than red cells in image analysis. In certain cases it may be better to use ethidium bromide, which stains all cells red, for direct viable counts by image analysis.     

Chromium-induced cross-linking of nuclear proteins and DNA

The in vivo cross-linking of proteins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 was studied. DNA-protein complexes were assayed by high speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and by electrophoretic identification of proteins associated with DNA-containing pellets. Further evidence of DNA-protein complexes, not dissociable in this buffer, was obtained by CsCl gradient centrifugation. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to chromium salt for at least 4 h, and the amount of DNA-protein complexes increased with longer incubation times. Complex formation occurred only with chromium salt concentrations of 200 microM or greater, and maximal cross-linking was effected at 5 mM. Immunotransfer methodology employing antibodies to nuclear matrix fraction and lamins was used to identify some of the polypeptides comprising the cross-linked complexes. These studies indicated specificity of chromium-induced complex formation within the nuclear protein fractions assayed. Our results document the ability of chromate to produce specific DNA-protein cross-links in living cells.     

Supercoiled DNA folded by nonhistone proteins in cultured mouse carcinoma cells.

Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with gamma-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.     

Dynamic and static chromatin

It was found that the dependence of the viscosity of calf thymus chromatin dispersions and human leukocytes on ethidium bromide concentration had two peaks indicative of domains with circular supercoiled DNA and varying resistance to ultrasound in the cells and isolated chromatin. The hypothesis of V. D. Paponov and P. S. Gromov (Bull. Exp. Biol. Med., N5, 590, 1985) on the transformation of static relations of nucleosome DNA-containing nuclei into dynamic, after chromatin exposure to ultrasound due to DNA linearization in chromatin domains possessing circular supercoiled DNA, has been confirmed.     

Ethidium bromide resistance in a rat liver epithelial cell line: Association with enhanced drug efflux

Ethidium bromide-resistant cell strains were obtained by continuous selection of an adult rat liver-derived cell line (ARL6T) grown in the continuous presence of 200 ngl ml ethidium bromide. Comparison of resistant strains and parental (sensitive) cells was made for uptake and binding of ethidium bromide, visualized as fluorescent ethidium bromide-nucleic acid complexes. Although uptake of ethidium bromide was similar in parental and resistant cells, efflux kinetics were markedly different. Over a three-hour period, parental (sensitive) cells maintained fluorescence following a short ethidium bromide pulse (100 g/ ml ethidium bromide). In contrast, ethidium bromide-resistant cell lines eliminated photographically detectable fluorescent complexes within three hours following pulse exposure to ethidium bromide. The rapid elimination of ethidium bromide fluorescent complexes in all (5) resistant cell strains examined supports an efflux mechanism as contributing to the resistance of ethidium bromide cytotoxicity in these cells.Abbreviations EtBr ethidium bromide - HBSS Hanks' balanced salt solution     

Copper(II) complexes with N,N'-dialkyl-1,10-phenanthroline-2,9-dimethanamine: synthesis, characterization, DNA-binding thermodynamical and kinetic studies

Copper(II) complexes (Cu-L, L=N,N'-dialkyl-1,10-phenanthroline-2,9-dimethanamine) were synthesized and characterized by elemental analyses, IR spectra and conductance measurement. The interaction of the copper(II) complex with calf thymus DNA was studied by means of UV melting experiments, fluorescence spectra and circular dichroic spectra. Using ethidium bromide as a fluorescence probe, the binding mode of the complexes Cu-L with calf-thymus DNA was studied spectroscopically. The results indicate that the complexes Cu-L perhaps interact with calf-thymus DNA by both intercalative and covalent binding. Kinetics of binding of the cupric complexes to DNA was studied for the first time using ethidium bromide as a fluorescence probe with stopped-flow spectrophotometer under pseudo-first-order condition. The stronger binding of two steps in the process of the complexes Cu-L interacting with DNA was observed, and the probable interaction process was discussed in detail. The corresponding k(obs) and E(a) of binding to DNA (where k(obs) is the observed pseudo-first-order rate constant, E(a) is the observed energy of activation) were obtained.     

The effects of the microwaves on E. coli cells depend on oxygen concentration and static magnetic field

The effects of non-thermal microwaves (MW), 10(-4) and 10(-10) W/cm(2), on conformation of nucleoids in E. coli cells were analyzed by the method of anomalous viscosity time dependence (AVTD). MW exposure was performed at different values of static magnetic field and concentration of oxygen, 8-90 microT, and 2.3-7.8 mg/l, respectively. It was shown, that slight changes in both static magnetic field and oxygen concentration result in significant changes of MW effects up to their disappearance. It was established, that changes in static magnetic field affected significantly the time kinetics of the MW effects. The obtained data provide further evidence for strong dependence of the effects of non-thermal microwaves on physical parameters of exposure and physiological factors. These dependences should be taken into account in replication studies. The obtained results encourage further investigation of possible modulation of non-thermal MW effects by additional electromagnetic fields.