Activity coefficients of counterions in solutions of diethylaminoethyl dextran hydrochloride

Activity coefficients of counterions in solutions of diethylaminoethyl dextran hydrochloride have been determined. It has been observed that they increase with decreasing concentration of the polyelectrolyte. The experimental values have been compared with those calculated using Oosawa's theory of activity coefficients. The calculated values are higher than those observed, which suggests that the rodlike model on which Oosawa's theory is based is inadequate for the present case. Activity coefficients of counterions of some solutions containing NaCl and KC1, respectively, have also been determined. It has been found that the additivity rule for activity of counterions applies for these solutions.     

Equilibrium sedimentation of a polyelectrolyte in a density gradient of a low-molecular weight electrolyte. I. DNA in CsCl

Previous work had indicated that the molecular weight calculated from the equilibrium distribution of DNA in a CsCl density gradient appears to be one half the estimated value of the true molecular weight. In the present study, the sedimentation equilibrium of a polyclectrolyte in a density gradient generated by a low molecular weight electrolyte is treated in terms of the representation according to which a constant fraction of the counterions is considered permanently immobilized and the remaining fraction completely free. Equations for the preferential hydration and the molecular weight are obtained. The validity of the treatment is tested by applying it to experimental data on the equilibrium sedimentation of ?X 174 DNA in CsCl, and it is found that the molecular weight calculated in this way is in agreement with the accepted value for this molecule. Also, recalculation of published data on density gradient centrifugation of T2 DNA in CsCl according to the present treatment brings up the molecular weight to within the range of values given by other methods.     

Cyclic AMP-protein-DNA complex formation in mammalian cell-free systems.

A cytoplasmic protein fraction from KB and Chinese hamster ovary cells (CHO-Kl) was shown to bind in vitro to cAMP and subsequently to DNA-cellulose. This protein complex was not found in DE-52 purified CHO-K1 cAMP-dependent protein kinases. The complex appeared to exist as a small fraction of the total cAMP binding proteins, preferred native to denatured DNA and exhibited multiple sedimentation coefficients in glycerol gradients. This complex, after elution from the DNA cellulose column, was shown to have bound specifically to [3H]-cAMP which could be displaced by non-radioactive cAMP in competitive binding assays.     

Study of the self-diffusion coefficients of cations in the presence of an acidic polysaccharide

Self-diffusion coefficient measurements have been applied to the study of Na⁺, Ca⁺⁺, and Sr⁺⁺ in the presence of a linear acidic polysaccharide extracted from cartilage, i.e., chondroitin sulfate. A series of experimental determinations was made with and without supporting electrolytes, and an analysis of experiments involving the separation of the electrostatic interaction terms by an extension of Manning's theory produced results showing the preponderant nature of these electrostatic terms. The specificity of each type of ion or the influence of the pH can be considered merely as higher order corrections with respect to the preceding interactions. Counterion concentration ranges and pH ranges were determined, where either the alkaline-earth counterions become free and hence move with their normal diffusion rate, or these cations are associated with the polyelectrolyte molecule, thus giving a diffusion coefficient similar to that of the macromolecule, or the apparent diffusion coefficients vary between these two extreme diffusion rates as a function of the association equilibria. This variation can be expressed as a function of the linear charge density parameter ξ related to the structure of the polyelectrolyte, the value of which was determined and found in good agreement with published values.     

Salt dependent changes in structure and dynamics of circular single stranded DNA of filamentous phages of Escherichia coli

We have analyzed the static and dynamic behaviour of the circular single stranded DNA of the filamentous Escherichia coli phages F1 and M13mp8 in solution as a function of salt concentration using static and dynamic light scattering and sedimentation analysis in the analytical ultracentrifuge. We show by static light scattering that native and denatured single stranded DNA behave like a randomly coiled macromolecule at all salt concentrations used. The size of the native single stranded DNA is governed by the formation of secondary structures. While the radius of gyration decreases with increasing salt concentration the translational diffusion of the center-of-mass of native single stranded DNA and the sedimentation coefficient increase with increasing salt concentration in a biphasic manner. Below 100 mM monovalent cation concentration there is a strong dependence of the hydrodynamic parameters upon salt which is reduced approx. 3-fold at higher salt concentrations. We attribute the compaction of single stranded DNA by salt to electrostatic shielding and, in case of native single stranded DNA, secondary structure formation. Internal motions of the native single stranded DNA are observable at all salt concentrations and can be interpreted with a model of segmental diffusion of the elements of the polymer chain. The observed segmental diffusion coefficient of the native single stranded polynucleotide increases with increasing salt under the conditions investigated.     

Sedimentation and intrinsic viscosity behavior of PM2 bacteriophage DNA in alkaline solution

The sedimentation coefficient and intrinsic viscosity of nicked and closed circular PM2 bacteriophage DNA have been measured as a function of pH in the alkaline region. A gradual increase in the sidimentation coefficient, and a corresponding decrease in the intrinsic viscosity, are observed for the superhelical (closed) circle in the pH region from 10.5 to about 10.9. This has been tentatively interpreted in terms of the known dependence of sedimentation coefficient upon the number of superhelical turns. At slightly higher pH values, the curve passes through the minimum (sedimentation coefficient) and maximum (intrinsic viscosity) expected when the superhelical turns present at neutral pH are unwound by partial alkaline denaturation. Sedimentation studies of the relaxed (nicked) circular species have revealed the existence of DNA forms in the pH region from 11.27 to 11.37 which sediment considerably faster than the closed circle in the same pH region. These have been identified as partially denatured nicked circles, in which varying fractions of the duplex structure have undergone alkaline denaturation, but strand separation has not yet occurred. Varying fractions of a slower species, either undenatured or completely denatured nicked circles, are also observed in some of these experiments. A corresponding result is observed in the intrinsic viscosity vs. pH curve. When nicked circular PM2 DNA is exposed to various alkaline pH's, rapidly neutralized, and sedimented at neutral pH, the expected sharp transition from native to denatured (strand-separated) molecules is seen. However, a very narrow pH range is noted in which native and denatured forms coexist in a single experiment. The above experiments carried out upon the closed form also reveal a narrow pH range in which the bulk of the transition from native closed circles to the collapsed cyclic coil takes place, in acccord with an earlier study on a different DNA. This transition is shown never to be completely effected, however, as there is a fraction (7–8%)of the closed circles which renature to the native form, regardless of the alkaline pH employed. This same phenomenon was not observed in the case of artificially closed λb₂b₅c DNA circles. Possible explanations for some of the above results are discussed.     

Sedimentation coefficient of polyoma virus DNA

The sedimentation coefficient of the twisted circular form of polyoma virus DNA is calculated from the Kirkwood sedimentation–diffusion equation, the structure being assumed to be a rigid double superhelix. Agreement with the experimental sedimentation coefficient can be obtained, with the use of an experimental value for the number of superhelical turns, when the pitch of the superhelix is intermediate between its minimal and maximal possible values. Another model, which has been proposed for polyoma DNA at low ionic strengths, may be visualized as a superhelical structure wound about a torus. Calculations of sedimentation coefficients for this model agree qualitatively with experimental data at ionic strengths Below 10^?2M.     

Studies on Transformations of Hemophilus influenzae : II. The molecular weight of transforming DNA by sedimentation and diffusion measurements

The sedimentation and diffusion coefficients have been determined for Hemophilus influenzae transforming activity and DNA using P³²-labeled DNA. The methods employed the Spinco fixed boundary separation cell for measurements of the sedimentation coefficient and the Northrop-Anson diffusion cell to determine the diffusion coefficient. There was a very close correlation between the amount of DNA and transforming activity sedimented or diffused. The sedimentation coefficient (s_20°), for both biological activity and DNA was 27 and the diffusion coefficient (D_20°) 1 x 10^-8 cm²/sec. The molecular weight calculated from these coefficients gave a value of 16 million. There was no difference in the sedimentation coefficients for the two unlinked markers, streptomycin and erythromycin resistance, and the diffusion coefficients for single markers or the linked markers, streptomycin and cathomycin, were the same.     

Transport phenomena and ion binding in solutions of sodium polystyrenesulphonate

The results of transference and conductance measurements on solutions of sodium polystyrenesulpnonate, in the concentration range from 0.0025 to 0.075 monomolar, are presented. The fraction of free Counterions calculated from experimental data is found to he independent of molecular weight of the polyelectrolyte within the range of degree of polymerization from 200 to 2400. The experimental data, combined with the cell model, are used to calculate the electrostatic potential at a cylindrical surface Which surrounds the polyion and divides the counterions into bound and free. The same value for this potential is found for the whole concentration range investigated, which lends support to the assumption of cylindrical distribution of counterions in polyelectrolyte solutions.     

Sedimentation characteristics of DNA multiply crosslinked by a difunctional alkylating agent, mustard gas

It is confirmed from sedimentation studies that only a small fraction of diguaninyl derivatives formed on reaction on reactions of native DNA with mustard gas crosslink the two DNA chains. It is also apparent that heavily alkylated DNA can be studied in the denatured form in dimethyl sulfoxide without the gross degradation that results if denaturation is effected by alkali.     

IMMUNOCHEMICAL PROPERTIES OF NATIVE AND DENATURED HORSE SERUM GLOBULINS

1. The influence of guanidine hydrochloride on the denaturation and regeneration of Type I antipneumococcal horse serum globulin was determined by measurements of viscosity, diffusion, and sedimentation in the ultracentrifuge. In addition, the effect of NaCNS on the antibody globulin was studied. 2. Both the irreversibly denatured and the regenerated fractions were found to be precipitable by SI. The observed changes in combining ratio have been tentatively explained in terms of (a) changes in the mean molecular weight, or alternatively (b) an increase in the number of serologically active groups upon denaturation, followed by masking of the latter upon regeneration. Discounting a specific effect of NaCNS on either fraction, the extent of specific precipitation is of the same order of magnitude for native and irreversibly denatured antibody. 3. Quantitative precipitin titrations have been performed on rabbit antisera to native and irreversibly denatured horse antibody, and normal globulin GI, respectively. No significant differences in the antigenic activity of these proteins were found. Measurements of their cross-reactivity led to the conclusion that the native and irreversibly denatured fractions of antibody globulin are antigenically more closely related to each other than to the corresponding fractions of normal globulin, and vice versa.     

Effects of microorganisms on effective oxygen diffusion coefficients and solubilities in fermentation media

Effective oxygen diffusion coefficients and solubilities were measured for submerged cultures of Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. Both effective oxygen diffusion coefficients and solubilities were found to decrease with increasing cell concentrations in the fermentation media. Comparison of the experimental results of effective oxygen diffusion coefficients in fermentation media with values theoretically predicted on the assumption of unpenetrable microbial cells indicates that oxygen molecules diffuse through the cells during the diffusion process. Within the cell concentration range of typical submerged fermentations, the effective oxygen diffusion coefficient of the fermentation media can be described as D(e) = A(1)f + A(2)f(2). In this equation, fis the cell volume fraction and both A(1) and A(2) are functions of the shape of the cells and the ratio of effective oxygen diffusion coefficient in microbial cells to that in the medium.     

Effect of finite ionic size on the solution of the Poisson-Boltzmann equation: Application to the binding of divalent metal ions to DNA

The nonlinear Poisson-Boltzmann equation is solved for a cylindrical polyelectrolyte solution containing mono- and divalent counterions and monovalent coions. The finite size of the ions is taken into account by the introduction of the distances of closest approach between the ionic charges and the surface of the polyelectrolyte. The choice of these distances is based on the physicochemical properties of the polyelectrolyte and ions in solution. The effects of the finite ionic size on the distribution of the counterions around the polyelectrolyte and on the local ion concentration and the integrated charge fraction of the divalent cations in the vicinity of the polyelectrolyte are discussed. Theoretical predictions regarding the overall extent of binding and the extent of inner-sphere binding of divalent counterions to rodlike polyions are compared with the results of nmr studies of the binding of divalent metal ions to DNA.     

Immobilization of native and denatured DNA on Sephadex G200

An efficient method for the immobilization of DNA on Sephadex G200 in the presence of water soluble carboiimide is described and investigated in this paper. An increase in the extent of binding was observed when the incubation temperature of the DNA-Sephadex mixture was changed. It was found that native DNA immobilized to Sephadex with higher efficiency than denatured DNA. However, the stability of native DNA-Sephadex complex was about the same as that of denatured DNA-Sephadex. The size of DNA released by DNA-Sephadex after incubation of a suspension of the complex was the same as that of the DNA used for immobilization. The binding mechanism of DNA to Sephadex is discussed.     

Hydrodynamics of concentrated proteoglycan solutions

The dynamics of water transport in proteoglycan compartments has been studied in relation to osmotic flow (proteoglycan diffusion) and hydraulic permeability (proteoglycan sedimentation) in concentrated solutions of proteoglycan subunit and native proteoglycan aggregate isolated from Swarm rat chondrosarcoma. A central parameter that describes the kinetics of both types of water movement is the hydrodynamic frictional coefficient of water with proteoglycan. The frictional coefficient is markedly concentration dependent, increasing with increasing concentration, and highlights important structural features and types of organization of the proteoglycans in concentrated solutions. These include the requirements that proteoglycans in the extracellular matrix not to be immobilized but to have translational diffusive mobility and concentration gradients to be osmotically active, that chondroitin sulfate segmental mobility describing translational motion largely determines osmotic flow and hydraulic permeability of the proteoglycans, and that the proteoglycans exhibit an enhanced ability to resist flow as compared to other macromolecules. Additional dynamic studies suggest the formation of transient super-aggregate structures may occur at high concentrations which endows the proteoglycan subunit hydrodynamic properties similar to proteoglycan aggregate.     

DNA Polymerase Activity Associated with the Membrane Fraction of E. coli

Spheroplasts were disrupted with 0.2% Brij 58 and the separation of intact cells, spheroplasts, disrupted spheroplasts, fragmented membrane, and supernatant was performed on a linear 40~55% sucrose gradient. About half an amount of nucleic acid components was distributed in disrupted spheroplast fractions, while only a small amount of protein components was found in these fractions.

DNA polymerase in the fragmented membrane fraction incorporated ³H-TTP more rapidly than that in the supernatant fraction for the first 5 to 6 min, and then the incorporation rate decreased, while DNA polymerase in the supernatant fraction incorporated ³H-TTP linearly up to 20 min when native DNA was used as a primer. The former required native DNA as a primer and showed little activity towards denatured DNA, while the latter incorporated ³H-TTP at a similar rate to both the primer DNA’s.

DNA polymerase of the fragmented membrane fraction synthesized various sizes of DNA from short to a size of primer when native DNA was used as a primer, while when denatured DNA was used, products were only short. DNA polymerase of the supernatant fraction synthesized various sizes of DNA when both native and denatured DNA’s were used as primers.     

Limiting laws and counterion condensation in polyelectrolyte solutions: IV. The approach to the limit and the extraordinary stability of the charge fraction

The limiting laws for polyelectrolyte solutions developed in previous papers of this series have been amply confirmed by measurement. A surprising result of the accumulated data is that the limiting polyelectrolyte charge fraction (fraction of fixed charges uncompensated by condensed counterions in the limit of zero concentration), persists up to concentrations of 0.1 M or even higher. Here the theory is extended in a simple manner to finite concentrations, and the stability of the charge fraction is found to be firmly based on consequences of the long-range polyelectrolyte field. The associated counterions are assumed to translate freely in a region centered on the contour axis of the polyion. The numerical value of the free volume is determined self-consistently from the axial charge density of the polyelectrolyte and is used as the general framework within which specific binding effects are treated.     

Localisation and dynamics of sodium counterions around DNA in solution from molecular dynamics simulation

The localisation and dynamics of sodium counterions around the DNA duplex d(AGCGTACTAGTACGCT)₂ corresponding to the trp operator fragment used in the crystal structure of the half site complex has been studied by a 1.4 ns molecular dynamics simulation in explicit solvent. A continuous and well-defined counterion density is shown to be present around the minor groove, while density patches are found in the major groove in regions where DNA bending is observed. A residence time analysis reveals the dynamic nature of these distributions. The resulting picture agrees with previous theoretical and experimental studies of A-tract DNA sequences, and is consistent with the polyelectrolyte condensation model. Received: 12 August 1999 / Revised version: 11 November 1999 / Accepted: 7 December 1999     

Purification and properties of a novel DNA methyltransferase from cultured rice cells

DNA methyltransferase activity has been observed in a total crude homogenate of rice cells grown in suspension culture using either native plant DNA or, under the conditions used, the more responsive hemimethylated poly (dI-MedC).poly(dI-dC). Using the latter substrate we have purified an enzyme fraction 380-fold by salt extraction of chromatin, DEAE cellulose and phosphocellulose. This purified fraction showed enzyme activity only with poly (dI-MedC).poly(dI-dC) thus suggesting the occurrence in plants of a DNA methyltransferase specific for hemimethylated DNA. A Mr value of 54000 was calculated on the basis of the sedimentation coefficient which was determined by sucrose density gradient centrifugation. Apparent Km values for poly (dI-MedC).poly(dI-dC) and S-adenosyl-L-methionine were found to be 17 micrograms/ml and 2.6 microM, respectively.     

Cross-links in African swine fever virus DNA.

African swine fever virus DNA sediments in neutral sucrose density gradients as a single component with a sedimentation coefficient of 60S. In alkaline sucrose density gradients, this material shows two components with sedimentation coefficients of 85S and 95S, respectively. The sedimentation rate value of alkali-denatured virus DNA in neutral sucrose density gradients and the renaturation velocity of denatured DNA show that is reassociated much faster than expected from its genetic complexity. This behavior is compatible with the existence of interstrand cross-links in the molecule. We also present results which suggest that there are only a few such cross-links per molecule, that they are sensitive to S1 nuclease digestion, and that they are probably located next to the ends of the DNA.