Cation effects on the nonlinear electrical properties of DNA solutions

The nonlinear electrical properties of DNA solutions were measured when different monovalent cations were added to DNA. The influence of different parameters has been examined: fundamental frequency, field strength, and concentration. A linear relationship between the harmonic current I_h and the DNA concentration is shown, even for higher concentration values (400 mg/l.). The frequency dispersion of I_h has the same shape for all the cations and the low-frequency amplitude of I_h increases in the following order: Li⁺ < Na⁺ < K⁺ < NH < Cs⁺. The nonlinear polarizability values are compared with the linear ones determined using the very low field electric birefringence technique. Both linear and nonlinear values are of the same order of magnitude. It is thought that the nonlinear electrical property of high-molecular-weight DNA mainly results from the deformation of the DNA coils by the electric field.     

Electric birefringence of native DNA in an alternating field

The relaxation of the birefringence of native DNA in solution was investigated in a pulsed sine-wave electric field. Relaxation times were calculated from the degree of damping of the birefringence signal and were studied as a function of the strength and frequency of the applied field, the molecular weight of the DNA, and the viscosity and ionic strength of the solvent. Relaxation times decrease with increasing field strength. For high-molecular weight DNA (>10⁶ daltons), the relaxation times decreased with frequency and increased less than linearly with viscosity. For low-molecular-weight DNA (<6 × 10⁵ daltons), the relaxation times were independent of frequency, increased linearly with viscosity, and varied with the 1.65 ± 0.1 power of the molecular weight. The average birefringence of high-molecular-weight DNA decreased with frequency in 0.001M Na₂ EDTA plus NaOH, pH 7.0, but is much less frequency-dependent if the EDTA concentration is reduced tenfold, while the average birefringence of sonicated DNA increases in both solvents with increasing frequency.     

Electric birefringence of restriction enzyme fragments of DNA: optical factor and electric polarizability as a function of molecular weight

The electric birefringence of restriction enzyme fragments of DNA has been investigated as a function of DNA concentration, buffer concentration, and molecular weight, covering a molecular weight range from 80 to 4364 base pairs (bp) (6 × 10⁴–3 × 10⁶ daltons). The specific birefringence of the DNA fragments is independent of DNA concentration below 20 μg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent. At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field at which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law region the rise of the birefringence is accurately symmetrical with the decay for fragments ≤ 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated from a 1/E extrapolation of the high field birefringence data is ?0.028, independent of molecular weight; if a 1/E² extrapolation is used, the optical factor is ?0.023. The induced polarizability, calculated from the Kerr constant and the optical factor, is proportional to the square of the length of the DNA fragments, and inversely proportional to temperature. Saturation curves for DNA fragments ≤ 161 bp can be described by theoretical saturation curves for induced dipole orientation. The saturation curves of larger fragments are broadened, because of a polarization term which is approximately linear in E, possibly related to the saturation of the induced dipole in high electric fields. This “saturated induced dipole” is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp sample is decreased 6°C in an electric field of 8 kV/cm, because the lower charge density of the coil form of DNA makes it more stable in an electric field than the helix form.     

Electrophoretic mobility of lambda phage HIND III and HAE III DNA fragments in agarose gels: a detailed study

The electrophoretic mobility (μ) of DNA fragments from λ phage and ΦX 174, split by restriction enzyme to molecular lengths from 3 × 10² to 2.36 × 10⁴ base pairs, has been investigated in 0.6–4% agarose gels at various field strengths, ionic strengths, and temperatures. As already observed, μ is seen to be very sensitive to the field, increasing with field strength. The sensitivity increases with the molecular length of the DNA and decreases at high gel concentration. Our data are in qualitative agreement with recent theoretical predictions that concern the influence of the electric field on electrophoretic mobility. Mobility data have been extrapolated to zero field. This enables a comparison of our experimental results with theoretical predictions on the dependence of μ on the molecular weight of the DNA fragments. Our data fit, quite closely, a reptation model, where the tube path is described as a semiflexible entity with a persistence length equal to the pore diameter. The influence of the agarose concentration and the ionic strength of the buffer on the two parameters of the model—intrinsic electrophoretic mobility (μ₀) and the number of base pairs per element of the tube (g)—are well described by the model. The temperature dependence of the electrophoretic mobility, together with the influence of the agarose concentration on μ₀, indicate that the hydrodynamic drag is the leading frictional force on the DNA molecules in the gel.     

Electrophoresis in protein crystal: nonequilibrium molecular dynamics simulations

Electrophoresis of a mixture of NaCl and CaCl₂ in a lysozyme crystal is investigated using nonequilibrium molecular dynamics (MD) simulations. Upon exposure to an electric field, the stability of lysozyme is found to decrease slightly. This finding is demonstrated by increases in the root mean-square deviations of the heavy atoms of lysozyme, in the solvent-accessible surface area of hydrophobic residues, and in the number of hydrogen bonds between lysozyme and water. The solvent-accessible surface area of hydrophilic residues changes marginally, and the number of hydrogen bonds between lysozyme molecules decreases. Water molecules tend to align preferentially parallel to the electric field, and the dipole moment along the pore axis increases linearly with increasing field strength. Two pronounced layered structures are observed for Na⁺ and Ca²⁺ in the vicinity of protein surface, but only one enriched layer is observed for Cl⁻. The number distributions of all ions are nearly independent of the electric field. The water coordination numbers of all ions are smaller in the crystal than in aqueous bulk solution; however, the reverse is found for the Cl⁻ coordination numbers of cations. Both the water and the Cl⁻ coordination numbers are insensitive to the electric field. Ion diffusivities in the crystal are ∼2 orders of magnitude smaller than those in aqueous bulk solution. The drift velocities of ions increase proportionally to the electric field, particularly at high strengths, and depend on ionic charge and coordination with oppositely charged ions. Electrical current exhibits a linear relationship with the field strength. The zero-field electrical conductivity is estimated to be 0.56 S/m, which is very close to 0.61 S/m as predicted by the Nernst-Einstein equation.     

The dichroism of DNA in electric fields

We have studied the dichroism of various samples of calf thymus DNA (of molecular weight from 3 × 10⁵ to 7 × 10⁶) in pulsed electric fields. The results may be summarized as follows:

• 1 We find that calf thymus DNA behaves in electrical orientation as if it possessed a large permanent dipole moment. This apparent moment is sensitive to such effects as Mg⁺⁺ binding which lower the net charge on DNA.
• 2 The limiting dichroism at infinite field corresponds to an angle of at least 80% between the transition moments at 265 nm and the helix axis, and could be consistant with a number of known forms of DNA. This result is independent of DNA molecular weight. There is evidence that the conformation may be different in 80% ethanol.
• 3 The dichroism relaxation curves contain a component with a relaxation time of about 8 μsec, which is nearly independent of molecular weight, and a longest component which behaves either according to the Broersma theory for low-molecular-weight samples, or the Zimm-Rouse theory at high molecular weights.

     

Optimum Conditions for Electric Pulse-mediated Gene Transfer to Bacillus subtilis Cells

It was found that plasmid DNA (pUB 110) can be introduced into not only protoplasts but also intact cells of Bacillus subtilis by electric field pulses. The transformation of, B. subtilis using protoplasts results in an efficiency of 2.5 × 10⁴ transformants per μg of DNA, with a single pulse of 50 jisec with an initial electric field strength of 7kV/cm. Even transformation of intact B. subtilis cells results in a maximum efficiency of 1.5 × 10³ transformants per μg DNA, with a single pulse of 400 μsec with an initial electric field strength of 16kV/cm. The cell survival of protoplasts and intact cells was approximately 100% and 30%, respectively, under the conditions found to be optimal for the transformation process. Plasmid DNA isolated from pUB 110 containing transformants was indistinguishable from authentic preparations of pBU 110 on gel electrophoretic analysis.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

Interactive effects in 60-HZ electric-field exposure systems

The level of exposure of laboratory animals to 60-Hz electric fields is commonly specified in terms of the unperturbed field strength present before the introduction of experimental subjects and their cages. In the research reported in this paper, rats were housed in two parallel rows of 12.4 cm × 25.1 cm × 10.2 cm high plastic cages resting on the lower electrode of a parallel-plate exposure system, and the actual perturbed electric fields experienced by an experimental animal were investigated. The most important results are: 1) Reducing the spacing between the exposure electrodes from 8.7 to 1.7 times the height of a singly exposed rat model, while maintaining a constant unperturbed field strength, resulted in a 15% increase in the electric field at the highest point on the surface of the body and a 10% increase in the short-circuit current of the model. 2) For multiple animal exposures, increases of 10% in both the field at the highest point of the body and the short-circuit current were observed when the electrode spacing was reduced from 8.7 to 2.6 times the height of a rat. 3) Plastic cages caused 1 – 6% reductions in the electric field at the surface of the body, except very near the cage walls, where enhancements of more than 20% were observed. 4) When 16 rats were simultaneously exposed, the short-circuit current, I_s, of an individual subject of weight W (in g), that was surrounded on all sides by other rats of weight W, was reduced from the short-circuit current, I_u, measured with the same subject individually exposed as follows: during a 12 h light (sleeping) cycle, I_s/I_u = 1.00 – 0.0173W^1/2; during a 12 h night (awake) cycle, I_s/I_u = 1.00 – 0.0136W^1/2.     

Improved transformation of Pseudomonas putida KT2440 by electroporation

Summary An electric field-mediated transformation (i.e. electroporation) was performed to determine optimal conditions for P. putida transformation. The effects of culture age, electroporation buffer composition, electric field strength, pulse time constant and DNA concentration on transformation efficiency were examined. When plasmid DNA of 8 to 11 kb in size was used with an electroporation buffer containing 1 mM HEPES (pH 7.0), maximum transformation efficiency of 1.0 × 10⁷ transformants/g DNA was obtained at field strength of 12 kV/cm with pulse time of 2.5 millisecond. A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude. A linear relationship was observed between growth phase and transformation efficiency up to OD₆₀₀ = 2.0. This reliable and simple method should be useful for introduction of plasmid DNA into intact P. putida cells.     

Characterization of Bacteriophage S13 suN15 Single-Strand and Replicative-Form Deoxyribonucleic Acid

The deoxyribonucleic acid (DNA) of bacteriophage S13 was shown to be single-stranded by the criteria of reactivity with formaldehyde, dependence of optical density on ionic strength, broad temperature-absorbance profile, and lack of molar equivalence of the purine and pyrimidine bases. The DNA has a molecular weight of 1.8 × 10⁶ daltons, an S°₂₀ of 24.6 in SSC (0.15 m NaCl plus 0.015 m sodium citrate), and a buoyant density of 1.726 g/cc in CsCl. Electron microscopy showed the molecule to be circular. S13 replicative-form DNA was shown to be a double-stranded, circular molecule with a molecular weight of 3.5 × 10⁶ daltons, an S_[ill] of 20.7 in SSC, and a buoyant density in CsCl of 1.710 g/cc. The finding that S13 DNA is slightly more pyrimidine-rich than X174 DNA but is indistinguishable by all other parameters supports the close genetic relationship between the two bacteriophages.     

The free solution mobility of DNA

The free solution mobility of DNA has been measured by capillary electrophoresis in the two buffers most commonly used for DNA gel electrophoresis, Tris-borate-EDTA (TBE) and Tris-acetate-EDTA (TAE). The capillaries were coated with polymers of either of two novel acrylamide monomers, N-acryloylaminoethoxyethanol or N-acryloylaminopropanol, both of which are stable at basic pH and effectively eliminate the electroendosmotic mobility due to the capillary walls. The free solution mobility of DNA in TAE buffer was found to be (3.75 ± 0.04) × 10⁻⁴ cm² V⁻¹ s⁻¹ at 25°C, independent of DNA concentration, sample size, electric field strength, and capillary coating, and in good agreement with other values in the literature. The free solution mobility was independent of DNA molecular weight from ∼ 400 base pairs to 48.5 kilobase pairs, but decreased monotonically with decreasing molecular weight for smaller fragments. Surprisingly, the free solution mobility of DNA in TBE buffer was found to be (4.5 ± 0.1) × 10⁻⁴ cm² V⁻¹ s⁻¹, about 20% larger than observed in TAE buffer, presumably because of the formation of nonspecific borate-deoxyribose complexes. © 1997 John Wiley & Sons, Inc. Biopoly 42: 687–703, 1997     

Effects of DNA Topology on Transformation Efficiency of Bacillus subtilis ISW1214 by Electroporation

We report an investigation of electrotransformation by three different topological isomers, circular supercoiled (sc DNA), circular relaxed (cr DNA), and linearized (In DNA) forms of the plasmids pUB110 (4.5 kbp) and pBDR331T (12.6 kbp), of a Gram-positive bacterium, Bacillus subtilis ISW1214. Treatment of the sc DNA with calf thymus topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of sc DNA with restriction endonuclease linearized the DNA. The transformation with the sc DNA of pUB110 resulted in the maximum efficiency of (2.6±0.6) × 10⁵ transformants per μg DNA higher than that ((2.0±0.3) × 10⁴ transformants per μg DNA) for the cr DNA, using the DNA concentration of 20 μg/ml at an electric field strength of 7kV/cm and a capacitance of 10 μF with a single decayed pulse. The transformation efficiency (TE) for the In DNA was zero. The variations of TE for different topological forms of DNA reflected their relative stability in the host cells. The molecular efficiency (ME, transformants per molecule) for sc DNA was nearly one order of magnitude greater for the lower molecular size of pUB110 DNA than that for the higher molecular size of pBDR331T DNA.     

Nonlinear electrophoresis and focusing of macromolecules

Effects of nonlinear dependence drift velocity of (double-stranded) DNA vs. electric field strength were investigated. In comparatively weak fields, the molecular drift velocity is proportional to the external electric field, while in strong fields there is additional nonlinear component. This effect offers possibilities to manipulate the total drift velocity at will-the macromolecules of different size can be made to move in opposite directions in pulsed field gel electrophoresis.A new approach for focusing DNA molecules based on nonlinear electrophoresis and geometric trapping in electric fields is proposed. The focusing is carried out in an alternating nonuniform electric field, created by using a wedge gel with hyperbolic boundaries. It is shown that the fractions separated in such wedge retain their rectilinear shape.Gel electrophoresis experiments supported the possibility of a pronounced nonlinear focusing of DNA molecules. This nonlinear separation technique presents encouraging prospects for micromanipulating systems and also for preparative isolation of long DNA fragments and development of new separation methods for bacterial fingerprinting.     

Galvanotaxis of human granulocytes: electric field jump studies

The static and dynamic responses of human granulocytes to an electric field were investigated. The trajectories of the cells were determined from digitized pictures (phase contrast). The basic results are: (i) The track velocity is a constant as shown by means of the velocity autocorrelation function. (ii) The chemokinetic signal transduction/response mechanism is described in analogy to enzyme kinetics. The model predicts a single gaussian for the track velocity distribution density as measured. (iii) The mean drift velocity induced by an electric field, is the product of the mean track velocity and the polar order parameter. (iv) The galvanotactic dose-response curve was determined and described by using a generating function. This function is linear in E for E < E ₀ = 0.78 V/mm with a galvanotaxis coefficient K _G of (–0.22 V/mm)^–1 at 2.5 mM Ca⁺⁺. For E > E ₀ the galvanotactic response is diminished. This inhibition is described by a second term in the generating function (–K _G · K _I (E – E ₀)) with an inhibition coefficient K _I of 3.5 (v) The characteristic time involved in directed movement is a function of the applied electric field strength: about 30 s at low field strengths and below 10 s at high field strengths. The characteristic time is 32.4 s if the cells have to make a large change in direction of movement even at large field strength (E jump). (vi) The lag-time between signal recognition and cellular response was 8.3 s. (vii) The galvanotactic response is Ca⁺⁺ dependent. The granulocytes move towards the anode at 2.5 mM Ca⁺⁺ towards the cathode at 0.1 mM Ca⁺⁺. (viii) The directed movement of granulocytes can be described by a proportional-integral controler. Offprint requests to: H. Gruler     

Electro-optical properties of carboxymethylcellulose. I. Concentration and ionic strength dependence

The experimental conditions for studying the electro-optical properties of a natural, modified polyelectrolyte, carboxymethylcellulose (DS 1.3; DP 180) were determined. The transient Kerr effect was found to be a function of CMC concentration, field strength, and ionic strength, I. If the concentration and I were low enough (c < 20 mg.l^?1), saturation was obtained for field strengths of approximately 15 kV.cm^?1. The optical anisotropy was shown to be independent of I; the electrical anisotropy decreased sharply when I increased. These results are discussed in connection with polarization theories of polyelectrolytes. The molecular dimensions of carboxymethylcellulose, calculated from the birefringence kinetics, suggest that the molecule is a rigid rod.     

Optical Kerr effect in biopolymer solutions

The electric field in a single mode, YAG laser beam has been used to induce orientational birefringence in solutions of tobacco rattle virus, DNA, heparin, and hyaluronic acid. Using this laser in its “fixed-Q” mode, laser pulses were generated which persisted for up to 200 μsec in which the effective electric field vector rose to 5 kV cm^?1. The birefringence amplitudes so produced had a quadratic dependence on the effective field strength and thus obeyed Kerr's law. From the birefringence decay rates, relaxation times were determined which, by comparison with the birefringence induced by pulsed static electric fields revealed the biopolymer orientational origins of the effects. This indicated how these experiments can lead to the evaluation of particle geometry, the electronic contribution to electrical polarizabilities, and the optical polarizability of biopolymers in solution.     

Polyelectrolyte complexes of glycol chitosan with some polysaccharides. II. Electrical conductivity

The dc electrical conductivity of films of the polyelectrolyte complexes of glycol chitosan (GlChi) with the sodium salts of dextran sulfate (DS), carboxymethyl cellulose (CMC), polygalacturonic acid (GalUA)_n, and alginic acid (AlgA) was measured at temperatures above and below room temperature. The maximum field strength in the thinnest film used amounted to 3 × 10⁴ V/cm. A plot of normalized current against the reciprocal of the absolute temperature revealed two regions with different slopes, and activation energies in these two regions have been obtained for all the complexes. The activation energies in the high-temperature region vary from 0.85 to 1.18 eV and in the low-temperature region from 0 to 0.22 eV. Reasons are given to show that the conductivity is probably ionic. Near room temperature, the current–voltage relation is almost linear in the GlChi–DS complex, while in the other three complexes the current varies as a power n of the voltage with the value of n ranging from 1.7 to 2.5. A rise in temperatures causes an increase in the slope of the log I vs log V plot in GlChi–DS and GlChi–CMC complexes. The nonlinear current–voltage relation is ascribed to a space-charge-limited conductivity.     

Nonquasineutral current equilibria as elementary structures of plasma dynamics

A study is made of the fundamental features of current filaments with a nonzero electron vorticity Ω _e ≡ B − (c/e) ▿ × p _ee ≠ 0 and the corresponding Lagrangian invariant I _e . Such current structures can exist on spatial scales of up to ω _pi ⁻¹. It is shown that the dissipative stage of the plasma evolution and the violation of Thomson’s theorem on vorticity conservation in an electron fluid are of fundamental importance for the onset of electron current structures. A key role of the screening of electric and magnetic fields at distances on the order of the magnetic Debye radius r _B = B/(4πen _e )—the main property of such current structures in a Hall medium with σB/(en _e c) ≫ 1—is stressed. Since the minimum size of a vortex structure is the London length c/ω _pe , the structures under consideration correspond to the condition r _B > c/ω _pe or B ² > 4πn _e m _e c ², which leads to strong charge separation in the filament and relativistic electron drift. It is demonstrated that the specific energy content in current structures is high at a filament current of 10–15 kA: from 100 J/cm³ at a plasma density of 10¹⁴ cm⁻³ (the parameters of a lightning leader) to 10⁷ J/cm³ for a fully ionized atmospheric-pressure air. Estimates are presented showing that the Earth’s ionosphere, circumsolar space, and interstellar space are all Hall media in which current vortex structures can occur. A localized cylindrical equilibrium with a magnetic field reversal is constructed—an equilibrium that correlates with the magnetic structures observed in intergalactic space. It is shown that a magnetized plasma can be studied by using evolutionary equations for the electron and ion Lagrangian invariants I _e and I _i . An investigation is carried out of the evolution of a current-carrying plasma in a cylinder with a strong external magnetic field and with a longitudinal electron current turned on in the initial stage—an object that can serve as the simplest electrodynamic model of a tokamak. In this case, it is assumed that the plasma conductivity is low in the initial stage and then increases substantially with time. Based on the conservation of the integral momentum of the charged particles and electromagnetic field in a plasma cylinder within a perfectly conducting wall impenetrable by particles, arguments are presented in support of the generation of a radial electric field in a plasma cylinder and the production of drift ion fluxes along the cylinder axis. A hypothesis is proposed that the ionized intergalactic gas expands under the action of electromagnetic forces.     

Polarization of counterions in polyelectrolytes

A theory of the polarization of counterions bound to a polyion, such as a DNA, in low and high electric field strengths is developed using statistical mechanics of inhomogeneous systems. For low fields, one finds that the polarizability p is (Zq)² ρ0βL³/(12[1 + Lρ0σ(L, b, ζ, Z, I, ρ0)]J), where σ = ∫¹₀ (λ′ − λ₀ {dc(λ − λ′)/dλ}_{λ = λ0} dλ′J), Z and L are the valence and the length of the polyion, respectively, q is the proton charge, β = 1/k_BT, T is the temperature, k_B is the Boltzmann constant, I is the ionic strength, λ = x/L and λ₀ = x₀/L are scaled distances, x₀ is a reference point such that the inhomogeneous counterion density at x₀ is equal to ρ₀—the uniform density in the absence of an electric field E—and c(x) is the direct correlation function of the homogeneous counterion-polyion phase, which includes attractive and repulsive interactions. If Lσ(L, .) is much less than one, then the polarizability is proportional to L³. If the term Lσ(L, .) is much larger than one, the polarizability scales as L². The induced dipole moment saturates and its value is the same as that of Mandel-Manning theories. The onset of the saturation, however, depends critically on the direct correlation function and hence polyelectrolyte effects. In the formalism, the polarization of the counterions is the equilibrium response to an electric field provided E is less than E_saturated. A dynamical scheme that incorporates the fact that in high fields the bound counterions conduct is discussed. © 1996 John Wiley & Sons, Inc.