Flow dichroism of capsid DNA phages. II. Effect of DNA deletions and intercalating dyes

The flow linear dichroism of bacteriophage λ and its deletion mutants, λ b2 and λ b221, was determined. The hydrodynamic behavior of the three phages differed slightly, but the magnitude of the dichroism was substantially the same with 〈cos²θ_μp〉 = 0.364, 0.368, and 0.372, respectively. The dichroism of intercalating dyes combined with bacteriophage was used as a further probe of phage structure. The reduced dichroism from proflavin with T4 showed no change with time during the reaction, but the interpretation of the ligand dichroism is complicated by an alteration of the hydrodynamic behavior of the phage–dye complex relative to the phage alone. Ethidium with λ also produced a stable reduced dichroism, but the signal indicated an average orientation of intercalated dye that is different from the average base orientation. The reduced dichroism of ethidium changes with time as it penetrates λ b2, eventually approaching the dichroism of the nucleotide bases. The implication of these findings on the plausibility of various simple DNA packing models is discussed.     

Intrinsic viscosities of cyclic and linear lamda DNA

The ratio of the intrinsic viscosities of the linear and circular forms of λ DNA, [η]L /[η]c, has been measured as a function of ionic strength in the range [Na⁺] = 0.6. M–0.03MCorrections were made for the presence of uncyclizable linear contaminant in circular preparations. By combining data in the literature on the ionic strength dependence of linear DNA of various molecular weights with that obtained here, it was possible to determine the expansion parameter εL as a function of [Na⁺]. εL is defined by the relation 〈L²〉 = b²N^1+εL, where 〈L¹〉 is the mean-square end-to-end distance of a chain of N segments of length b. The empirical relation εL = 0.05 ? 0.11 log [Na⁺] for native NaDNA at 25°C is found. When εL = 0, [η]L /[η]c extrapolates to 1.6, in good agreement with the theoretical prediction of 1.55. As εL increases, [η]L /[η]c increases, in agreement with a theory of Bloomfield and Zimm.     

Precollapse of T7 DNA by spermidine at low ionic strength: A linear dichroism and intrinsic viscosity study

At low salt ([Na⁺] = 10^?3M), spermidine is capable of transforming DNA from a highly extended random coil to a compact particle. The transition takes place at a spermidine concentration of around 25 μM and the compact particle has been previously studied in considerable detail for several different DNAs. The objective of the present study is to see what effect, if any, spermidine has on T7 DNA conformation prior to collapse using flow dichroism and intrinsic viscosity. We conclude that increasing the spermidine concentration from 0 to the collapse transition point (above 20 μM) makes DNA increasingly nondraining. Furthermore, the persistence length dropped from 785 (±42) to 560 (±32) to 445 (±26) Å on increasing the ambient spermidine concentration from 0 to 1 to 10 μM. These results are in good agreement with counterion condensation theory and Odijk's theory of the electrostatic contribution to the persistence length of DNA. Nonetheless, it is concluded that counterion condensation is not entirely responsible for DNA collapse and that crosslinking promotes the transition to the compact state.     

Flow dichroism of deoxyribonucleic acid solutions

The flow dichroism of dilute DNA solutions (A₂₆₀ ≈ 0.1) has been studied in a Couette-type apparatus with the outer cylinder rotating and with the light path parallel to the cylinder axis. Shear gradients in the range of 5–160 sec.^?1 were studied. The DNA samples were whole, “half,” and “quarter” molecules of T4 bacteriophage DNA, and linear and circular λb₂b₅c, DNA. For the linear molecules, the fractional flow dichroism is a linear function of molecular weight. The dichroism for linear λ DNA is about 1.8 that of the circular molecule. For a given DNA, the dichroism is an approximately linear function of shear gradient, but with a slight upward curvature at low values of G, and some trend toward saturation at larger values of G. The fractional dichroism increases as the supporting electrolyte concentration decreases.     

Chromatin structure studied by linear dichroism at different salt concentrations

We have studied the linear dichroism (LD) of rat liver chromatin oriented by flow. Soluble chromatin, prepared by brief nuclease digestion, is found to exhibit a positive LD at low ionic strength (1 mM NaCl), with a constant LD/A over the absorption band centered at 260 nm (A, isotropic absorbance). Several previous dichroism studies on soluble chromatin have been performed on sonicated materials and have given negative LD, probably due to the presence of uncoiled DNA. The positive dichroism can be interpreted in terms of a supercoil of DNA in chromatin with a pitch angle larger than 55°, and is, for example, consistent with a model where the cylindrical nucleosome core particles are stacked face to face in the chromatin filament. In contrast to the nuclease-digested chromatin, sonicated chromatin was confirmed to exhibit negative LD. This difference can be attributed to a partial uncoiling of the linker regions between the nucleosomes due to the shearing. The structural transition of chromatin to a compact form can be observed as a reduction of the positive LD of the nuclease-digested chromatin to almost zero in 0.1 M NaCl or in 0.1 mM MgCl₂. This transition is due to a decreased electrostatic repulsion between negative phosphate groups on the DNA chain. In the case of Na⁺, this can be explained as a screening effect due to the bulk concentration of Na⁺. With Mg²⁺ a considerably stronger effect may indicate a more localized binding to the phosphates. At ionic strengths higher than 0.5M NaCl, the dissociation of the histones from DNA leads to uncoiling of chromatin. The change in LD during this process shows that histone H1 contributes only to a small degree to the coiling of the DNA chain, whereas histones H3 and H4 play the major role in the coiling.     

Light scattering and hydrodynamic properties of linear and circular bacteriophage lambda DNA

Low-angle light scattering, sedimentation velocity, and intrinsic viscosity measurements have been made on circular and linear forms of lambda (λ) bacteriophage DNA. Available equations, used to relate hydrodynamic parameters of both forms to the molecular weight, give relatively consistent values of particle weights which essentially agree with the light-scattering results. An average molecular weight of (34 ± 3) × 10⁶ for λ DNA was obtained in good agreement with literature values of (31–33) × 10⁶. The linear λ DNA has a larger root-mean-square radius than the circular molecule, when determined by light scattering, but the difference does not appear to be us large as expected from hydrodynamic data. The two forms also show significantly different angular distrbutions of scattered light intensities which agree only qualitatively with those derived from existing theory. The light-scattering results suggest that further experiments and modifications of available theories should be undertaken.     

Flow dichroism of DNA: a new apparatus and further studies

A new apparatus for the study of flow dichroism of macromolecules is described. The flow is down a long, narrow channel and an unpolarized light beam propagates along the flow direction. For a molecule such as DNA, in which the transition moments of the chromophores are perpendicular to the axis of orientation, an increase of absorbance is observed during flow. The apparatus is best suited for macromolecules which are readily orientable or at high shear gradients so that the extinction angle is close to 0°. The apparatus has the following advantages: dilute macromolecule solutions can be used; high shear gradients are easily obtained; only small volumes of solution are needed. The flow can be stopped rapidly so that relaxation times for disorientation can be studied. The flow dichorism of native, two-stranded DNA has been measured for the molecular weight range of 0.6 × 10⁶ to 125 × 10⁶, and for the shear gradient range (in aqueous solution at 25°C) from 200 sec^?1 to 21000 sec^?1. At a fixed gradient the dichroism increases with molecular weight, but the curve is concave downwards. At a given molecular weight the dichroism increases with increasing shear gradient, but the curve is concave downwards. When the solvent viscosity and temperature are varied, the dichroism is a function of η〈G〉/T showing that the orientation is due to hydro-dynamic shear stress and that the flexibility of DNA in a flow field is not due to local denaturation. The Zimm-Rouse theory with no parameters taken from flow optical data predicts the correct order of magnitude of the dichroism but the experimentally observed shear gradient and molecular weight dependence do not fit the theory. This is an expected result, since the theory is believed to be applicable only at small distortions and extensions of the macromolecule.     

Linear dichroism studies of nucleic acids. III. Reduced dichoism curves of DNA in ethanol–water and in poly(vinyl alcohol) films

The uv linear dichroism of calf thylus DNA has been studied at different degrees of orientation both in flow-oriented ethanol–water solutions and in a stretched aqueous host of poly(vinyl alcohol) (PVA). The reduced dichroism (LD^R) curves in the region 250–290 nm for DNA in PVA films at 75 and 100% relative humidity (r.h.) are in fair agreement with the curves calculated for the A- and B-forms of DNA, based on the fiber structures and the π-π* transitions of the free bases. This suggests that DNA adopts its A and B conformations in PVA at 75 and 100% r.h. In ethanol, on the other hand, a deviation from the A-form spectrum shows that the conformation of DNA in the solution can differ from the fiber structure. At shorter wavelenghts, a positive contribution to LD^R is explained in terms of an out-of-plane polarized n-π* transition.     

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.     

Helix-coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes

Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10^?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises T_m, the melting temperature of free base pairs. The linear dependence of T_m on log Na⁺ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na⁺ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs T_m′ remains constant at various ionic strengths. On the other hand, the T_m′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of T_m′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na⁺ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers T_m′. This explanation is supported by circular dichroism (CD) results.     

Trivalent counterion condensation on DNA measured by pulse gel electrophoresis

Pulse gel electrophoresis was used to measure the reduction of mobilities of λ-DNA-Hind III fragments ranging from 23.130 to 2.027 kilobase pairs in Tris borate buffer solutions mixed with either hexammine cobalt(III), or spermidine³⁺ trivalent counterions that competed with Tris⁺ and Na⁺ for binding onto polyion DNA. The normalized titration curves of mobility were well fit by the two-variable counterion condensation theory. The agreement between measured charge fraction neutralized and counterion condensation prediction was good over a relatively wide range of trivalent cation concentrations at several solution conditions (pH, ionic strength). The effect of ionic strength, trivalent cation concentration, counterion structure, and DNA length on the binding were discussed based on the experimental measurements and the counterion condensation theory. © 1996 John Wiley & Sons, Inc.     

Na+ effects on transition of DNA and polynucleotides of variable linear charge density.

A range of linear charge densities of the ordered and disordered forms of DNA or polynucleotides can be obtained experimentally by acid or alkaline titration, or by the investigation of unusual complexes involving protonated bases or three-stranded helices. The variation of melting temperatures with Na⁺ concentration for various of these systems is known and in some cases is complemented by structural and thermodynamic information. We have extended the condensation–screening theory of Manning [Biopolymers, 11 , 937–955 (1972)] to these systems. The stabilizing and destabilizing effects of Na⁺ (condensation and screening, respectively) and be independently varied, and the theory is successful in predicting the qualitative (in some cases, quanittative) behaviour that is observed. Comparison of theory and experiment indicates that the axial phosphate distance b for single-stranded polynucleotides increases with increasing pH. Values of the critical parameter ξ are obtained for the various polynucleotide structures. These values are essential for an understanding of ionic effects on charged ligand–polynucleotide interactions.     

Magnesium ion effect on the helix-coil transition of DNA

The effect of magnesium ions on the parameters of the DNA helix-coil transition has been studied for the concentration range 10^?6–10^?1M at the ionic strengths of 10^?3M Na⁺. Special attention has been given to the region of low ion concentrations and to the effect of polyvalent metallic impurities present in DNA. It has been shown that binding with Mg⁺⁺ increases the DNA stability, the effect being observed mainly in the concentration range 10^?6–10^?4M. At[Mg⁺⁺]>10^?2M the thermal stability of DNA starts to decrease. The melting range extends to concentrations ～10^?5M and then decreases to 7–8°C at the ion content of 10^?3M. Asymmetry of the melting curves is observed at low ionic strengths ([Na⁺] = 10^?3M) and [Mg⁺⁺] ? 10^?5M. The results, analyzed in terms of the statistical thermodynamic theory of double-stranded homopolymers melting in the presence of ligands, suggest that the effects observed might be due to the ion redistribution from denatured to native DNA. An experimental DNA–Mg⁺⁺ phase diagram has been obtained which is in good agreement with the theory. It has been shown that thermal denaturation of the system may be an efficient method for determining the ion-binding constants for both native and denatured DNA.     

Optico-hydrodynamic properties of high molecular weight DNA from steady-state flow birefringence and viscosity at extremely low velocity gradients

The flow birefringence, extinction angles, and shear-dependent viscosity over a velocity gradient range of approximately 0.1–3 sec^?1 have been obtained for T2 bacteriophage DNA at low concentration in neutral aqueous buffer. The data are found to be interpretable and self-consistent in terms of subchain dynamical theory, including hydrodynamic, interactions and excluded volume, and the parameters characterizing these phenomena are in good agreement with the results of other hydrodynamic experiments and theoretical calculations. No necessity for modification of the subchain model in terms of limited extensibility or internal viscosity is found for high molecular weight DNA at the velocity gradients studied, although the latter cannot be ruled out on the basis of the present data. The Kuhn statistical segment length is determined from the intrinsic optical anisotropy and is estimated as 930 Å. Implications of these findings and their relation to appropriate dynamical models for DNA are discussed.     

86 Characterization and differentiation of binding modes of water-soluble porphyrins at complexation with DNA

The influence of water-soluble cationic meso-tetra-(4?N-oxyethylpyridyl)porphyrin (H₂TOEPyP4) and it’s metallocomplexes with Ni, Cu, Co, and Zn on hydrodynamic and spectral behavior of DNA solutions has been studied by UV/Vis absorption and viscosity measurement. It was shown that the presence of planar porphyrins such as H₂TOEPyP4, NiTOEPyP4, and СuTOEPyP4 leads to an increase in viscosity at relatively small concentrations, and then decrease to stable values. Such behavior is explained by intercalation of these porphyrins in DNA structure because the intercalation mode involves the insertion of a planar molecule between DNA base pairs which results in a decrease in the DNA helical twist and lengthening of the DNA. Further decrease of viscosity is explained by the saturation intercalation sites and occurs outside the binding mode. But, in the case of porphyrins with axial ligands such as CoTOEPyP4 and ZnTOEPyP4, the hydrodynamic parameters decrease, which is explained by self-stacking of these porphyrins in DNA surface. This data are proved by spectral measurements. The results obtained from titration experiments were used for calculation of binding parameters: the binding constant K _b and the number of binding sites per base pair n. Obtained data reveal that K _b varies between 3.4 and 5.4?×?10⁶?M^?1 for a planar porphyrins, a range typical for intercalation mode interactions, and 5.6?×?10⁵?M^?1 and 1.8?×?10⁶?M^?1 for axial porphyrins. In addition, the exclusion parameter n also testifies that at intercalation, (n～2) the adjacent base pairs are removed to place the planar molecules, and for outside binders to pack on the surface needs too few places (n～0.5–1). It is apparent that the binding is somewhat stronger at intercalation. The viscometric and spectrophotometric measurements are in good agreement.     

Linear dichroism studies of nucleic acids. II. Calculation of reduced dichroism curves of A-and B-form DNA

We have calculated the uv linear dichroism for the A- and B-forms of DNA using π-π* transition moments and band components determined from the free DNA bases. The reduced dichroism (LD^R) as a function of wavelength is estimated is in the 220–300-nm region, for both the oriented-gas model and a simple exciton model. For B-form DNA, LD^R is obtained to ?1.48S (S being the orientation factor) over the whole wavelenth region by both models. For A-form DNA, LD^R is not constant, but changes monotonically from about ?1.15S at 220 nm to about ?1.35S to ?1.45S at 300 nm, depending on base combination and degree of interaction (?1.35S for the oriented gas). It is emphasized that a common assumption of a single “effective” transition moment of the principal band at 260 nm may not generally be made because of the extensive overlap of differently polarized bands. The possibility of using the reduced dichroism curve for characterizing the secondary structure of DNA is discussed.     

Ultraviolet circular dichroism of polyproline and oriented collagen

The ultraviolet absorption, linear dichroism, circular dichroism, and oriented circular dichroism of collagen are reported and the spectra are resolved into a self-consistent set of bands in accord with exciton theory. The parallel band at 200 nm has 40% of the π → π* intensity; the perpendicular band is placed at 189 nm yielding a splitting of 2700 cm^?1. The circular dichroism is resolved into two Gaussians at λ_∥ and λ_τ (rotational strengths +14 × 10^?40 and ?32 × 10^?40 esu². cm²) plus a large non-Gaussian (“helix”) band with ampplitude ?25,000° at 201 nm. These data appear to be in reasonably good accord with recent calculations. Measurements of the absorption, linear dichroism and circular dichroism of polyproline I and II are also reported and are resolved into their component bands. Polyproline I is in good accord with exciton theory, whereas polyproline II remains unsatisfactory.     

Dielectric saturation of the aqueous boundary layers adjacent to charged bilayer membranes

Summary The surface charge density resulting from the adsorption of hydrophobic anions of dipicrylamine onto dioleyl-lecithin bilayer membranes has been measured directly using a high field pulse method. The surface charge density increases linearly with adsorbate concentration in the water until electrostatic repulsion of impinging hydrophobic ions by those already adsorbed becomes appreciable. Then Gouy-Chapman theory predicts that surface charge density will increase sublinearly, with the power [z ⁺/(z ⁺+2)] of the adsorbate concentration, wherez ⁺ is the cation valence of the indifferent electrolyte screening the negatively charged membrane surface. The predicted 1/3 and 1/2 power laws for univalent and divalent cations, respectively, have been observed in these experiments using Na⁺, Mg⁺⁺, and Ba⁺⁺ ions. Gouy-Chapman theory predicts further that the change from linear to sublinear dependence takes place at a surface charge density governed by the static dielectric constant of water and the concentration of indifferent electrolyte. Quantitative agreement with experiment is obtained at electrolyte concentrations of 10^–4 m and 10^–3 m, but can be maintained at higher concentrations only if the aqueous dielectric constant is decreased. A transition field model is proposed in which the Gouy-Chapman theory is modified to take account of dielectric saturation of water in the intense electric fields adjacent to charged membrane surfaces.     

Electric and Flow Linear Dichroism of Unfolded and Condensed Chromatin: A Comparative Study at Low and Intermediate Ionic Strength

Abstract

Identical samples containing polynucleosomal chains of chicken erythrocyte (CE) and Ehrlich ascites tumour (EA) chromatin were studied under various ionic conditions with regard to electric linear dichroism (ELD) and flow linear dichroism (FLD). Both orientation techniques consistently confirmed that, in the limit of very low ionic strength and in the absence of multivalent cations, the reduced linear dichroism of chromatin is negative in the DNA-base absorption band, as expected for an extended zig-zag polynucleosomal conformation. With increasing electrolyte content, both ELD and FLD decreased drastically in amplitude, but in contrast to the ELD which remains negative in an intermediate range of low ionic strength (0.1–0.5 mM Mg²⁺) the FLD changes sign and becomes positive. The ELD and FLD amplitudes decrease with higher Mg²⁺ concentrations and FLD even vanishes in the region of 0.2–0.4 mM; both signals are positive above 0.4–0.5 mM Mg²⁺.

The origin of the dissimilarities between ELD and FLD observations is still not fully understood. Several possibilities are considered: ELD signals are more influenced than FLD by the presence of short chromatin chains, nucleosomes and small pieces of naked DNA, while FLD is more susceptible to the presence of large, easily orientable, scattering aggregates. Different preferred orientation directions of the chromatin fibre with respect to electric and hydrodynamic fields may also be involved. Finally, FLD and ELD probably “see” different features of the chromatin structure.     

Evolutionary conservation of DNA sequences expressed in sea urchin eggs and early embryos

Summary DNA sequence divergence measurements indicate thatStrongylocentrotus franciscanus is more distinct fromS. purpuratus andS. drobachiensis than these two species are from each other, in agreement with paleontological and morphological evidence. The evolutionary divergence of several classes of expressed DNA sequences was compared with that of total single-copy DNA. BetweenS. franciscanus andS. purpuratus the divergence of cDNA made from gastrula cytoplasmic poly(A)⁺ RNA is about half that of total single-copy DNA. Similar results were obtained for cDNA made from unfertilized egg poly(A)⁺ RNA. In contrast, sequences expressed in gastrula nuclear RNA have diverged almost as much as total single-copy DNA.