Intensity fluctuation spectroscopy of deoxyribonucleic acid (DNA). II. Temperature profile of bacteriophage ϕ29-DNA

Intensity-fluctuation laser-scattering spectroscopy measurements have been reported for the DNA isolated from bacteriophage ?29. The cumulant method of data analysis gives well-defined weighted average decay frequencies. Available theoretical models are shown to yield good values for the diffusion coefficient and longest intramolecular relaxation time for low-molecular-weight DNA's but not for higher molecular weight species. The temperature dependence of the apparent diffusion coefficient exhibits drastic oscillations in the helix–coil transition region. The number of these oscillations is consistent with independent measurements of uv hyperchromicity measurements and suggests that ?29-DNA is composed of at least three regions of differing G + C content.     

Kinetics of cruciform formation and stability of cruciform structure in superhelical DNA

This is a study of the kinetics of formation of a cruciform structure from the longest palindromic sequence in plasmid pAO3 DNA. DNA was prepared so as to be free of cruciforms even in topoisomers whose negative superhelicity was great enough to induce cruciform formation. Samples of such DNA were incubated at various temperatures, the incubation time varying over a wide range. Then the state was frozen by chilling. Two-dimensional electrophoretic analysis made it possible to estimate the fraction of molecules that got the cruciform structure during incubation. Precautions were taken for electrophoresis conditions to rule out any spontaneous conformational changes within the palindromic region. The relaxation time at the midpoint of the transition ranged from 30 min at 30 C to 50 hrs at 20 C, both in 0.1SSC. An increase in the negative superhelical density by 0.01 led to a 500-fold reduction of the relaxation time at 30 C but had little effect at 20 C. The probability of cruciform formation has been examined as a function of temperature. It has been shown that the cruciform state is no longer the predominant one at elevated temperatures: the cruciformation probability drops to an insignificant value for all of the topoisomers involved. Data have been obtained suggesting that the cruciform formation at the major palindromic site is not the only structural transition possible in pAO3 DNA.     

Diffusion coefficients of segmentally flexible macromolecules with two subunits: a study of broken rods

A general treatment for the solution dynamics of segmentally flexible macromolecules having two subunits is presented. Bead modeling allows for a complete inclusion of hydrodynamic interactions in this treatment. The finite size of the beads is also considered, so that it is therefore possible to account properly for torsional motions of the subunits. Expressions for the components of the resistance matrix are derived. From them, the translational and rotational diffusion coefficients can be calculated. Distinction is made between hinged macromolecules, whose only internal motion is bending, and swivel-jointed macromolecules, for which torsions of the subunits are also allowed. Numerical results are presented for broken rods with the two types of flexibility. The effects of hydrodynamic interaction between arms of broken rods are about 25% for translation and under 10% for rotation. These findings give support to the treatments of Harvey, Wegener, and co-workers in which interactions were neglected. The rotational dynamics of hinged and swivel-jointed rods are compared. Although there are differences in the short-time behavior, the longest relaxation time is the same for the two cases. Finally, the validity of Wegener's rotational diffusion constants is discussed.     

Effect of ionic strength on the diffusion coefficient of chondroitin sulfate and heparin measured by quasielastic light scattering

The normal modes for a mixture of charged macromolecules and electrolyte solution are calculated. We derive a generalized Debye relaxation time and the apparent diffusion coefficient of the macroion, which is shown to increase from its Stokes value, obtained in excess of added salt, with decreasing ionic strength. We test our result with experimental data for macromolecules with different charge densities: heparin and chondroitin sulfate. Besides, we show for this latter molecule that while the diffusion coefficient is increased, the scattered intensity is decreased but not by the same factor. Our results are compared with other theories developed in quasielastic light scattering.     

Kinetics of Cruciform Formation and Stability of Cruciform Structure in Superhelical DNA

Abstract

This is a study of the kinetics of formation of a cruciform structure from the longest palindromic sequence in plasmid pA03 DNA. DNA was prepared so as to be free of cruciforms even in topoisomers whose negative superhelicity was great enough to induce cruciform formation. Samples of such DNA were incubated at various temperatures, the incubation time varying over a wide range. Then the state was frozen by chilling. Two-dimensional electrophoretic analysis made it possible to estimate the fraction of molecules that got the cruciform structure during incubation. Precautions were taken for electrophoresis conditions to rule out any spontaneous conformational changes within the palindromic region. The relaxation time at the midpoint of the transition ranged from 30 min at 30 C to 50 hrs at 20 C, both in 0.1SSC. An increase in the negative superhelical density by 0.01 led to a 500-fold reduction of the relaxation time at 30 C but had little effect at 20 C. The probability of cruciform formation has been examined as a function of temperature. It has been shown that the cruciform state is no longer the predominant one at elevated temperatures: the cruciformation probability drops to an insignificant value for all of the topoisomers involved. Data have been obtained suggesting that the cruciform formation at the major palindromic site is not the only structural transition possible in pA03 DNA.     

Interactions of spermidine and methylspermidine with DNA studied by nuclear magnetic resonance self-diffusion measurements.

The NMR pulsed field gradient self-diffusion method has been used to study the self-diffusion of the polyamine spermidine and the polyamine analog methylspermidine (completely N-methylated spermidine). The self-diffusion coefficient, D, was measured in solutions of calf thymus DNA prepared from nucleosome core particles (with an average length of 120 base pairs) as a function of the concentration ratio of polyamine to DNA phosphate. A study of the self-diffusion quotient, D/Do (where Do is the diffusion coefficient for free polyamine, not associated with DNA), in additions of spermidine and methyl-spermidine to solutions of NaDNA/NaCl, gave almost identical results with complete association of polyamine to DNA in the initial part of the titrations, indicating similar affinities for DNA. A large influence on the measured self-diffusion coefficients was detected for methylspermidine in NaDNA solutions with different concentrations of NaCl, which shows a considerable salt effect on the polyamine-DNA association. No notable differences in D/Do for methylspermidine were observed in competitive titrations of solutions of Li- and NaDNA, indicating that sodium and lithium ions behave similarly in their interactions with DNA. In titration experiments of methylspermidine into MgDNA solution, the results showed that the polyamine association is less effective than in the case of NaDNA, because of competition from magnesium binding to DNA. Comparisons with calculations based on the electrostatic Poisson-Boltzmann cell model were performed. It is suggested that the interaction is primarily of electrostatic nature, with no binding to specific sites on the DNA molecule.     

Transport properties of polymer solutions. A comparative approach

A variety of transport properties have been measured for solutions of the water soluble polymer poly(ethylene oxide)(PEO) with molecular weights ranging from 200 to 14,000, and volume fractions ranging from 0-80%. The transport properties are thermal conductivity, electrical conductivity at audio frequencies (in solutions containing dilute electrolyte), and water self-diffusion. These data, together with dielectric relaxation data previously reported, are amenable to analysis by the same mixture theory. The ionic conductivity and water self-diffusion coefficient, but not the thermal conductivity, are substantially smaller than predicted by the Maxwell and Hanai mixture relations, calculated using the known transport properties of pure liquid water. A 25% (by volume) solution of PEO exhibits an average dielectric relaxation frequency of the suspending water of one half that of pure water, with clear evidence of a distribution of relaxation times present. The limits of the cumulative distribution of dielectric relaxation times that are consistent with the data are obtained using a linear programming technique. The application of simple mixture theory, under appropriate limiting conditions, yields hydration values for the more dilute polymer solutions that are somewhat larger than values obtained from thermodynamic measurements.     

Some biophysical applications of motional contrast in n.m.r. microscopy

The principal advantage of the n.m.r. imaging method lies in the specific contrasts which are available. In this work we describe the use of velocity and diffusion contrast methods in biophysical applications and at microscopic spatial resolution. In the first example, involving water-protein interactions, the relationship between water self-diffusion and water concentration, as measured using pulsed gradient spin echo n.m.r., is shown. It is demonstrated that this relationship can be used to provide a water concentration image. The result is compared with the conventional proton density and transverse relaxation maps. The next example concerns the use of dynamic n.m.r. microscopy to obtain water diffusion and velocity maps for wheat grain in vivo. Finally we suggest how the method may be used in the study of polymer-water interactions in an unusual adjunct to conventional polymer self-diffusion studies.     

Nuclear Magnetic Resonance Transverse Relaxation Times of Water Protons in Skeletal Muscle

The observation of the spin-echo decay in a long time domain has revealed that there exist at least three different fractions of non- (or slowly) exchanging water in the rat gastrocnemius muscle. These fractions of water are characterized with different nuclear magnetic resonance (NMR) relaxation times and are identified with the different parts of tissue water. The water associated with the macromolecules was found to be approximately 8% of the total tissue water and not to exchange rapidly with the rest of the intracellular water. The transverse relaxation time (T₂) of the myoplasm is 45 ms which is roughly a 40-fold reduction from that of a dilute electrolyte solution. This fraction of water accounts for 82% of the tissue water. The reduced relaxation time is shown neither to be caused by fast exchange between the hydration and myoplasmic water nor by the diffusion of water across the local magnetic field gradients which arise from the heterogeneity in the sample. About 10% of the tissue water was resolved to be associated with the extracellular space, the relaxation time of which is approximately four times that of the myoplasm. Mathematical treatments of the proposed mechanisms which may be responsible for the reduction of tissue water relaxation times are given in this paper. The results of our study are consistent with the notion that the structure and/or motions of all or part of the cellular water are affected by the macromolecular interface and this causes a change in the NMR relaxation rates.     

Deconvolution can be used in electrooptic studies to correct for non-ideal electric excitation pulses only when the electric dipole moment of the studied molecules is predominantly induced

The electric field pulses used for most measurements of transient electrooptic properties such as birefringence and dichroism, are rectangular and assumed to be ideal, but in practice do all such pulses have non-zero rise and fall times. Claims have been made that this non-ideality may be taken into account by employing standard deconvolution techniques. We find that this approach yields exact results in the zero electric field limit when the electric dipole moment of the studied macromolecules is predominantly induced. However, for finite electric field strengths and/or macromolecules with partly or fully permanent electric dipole moments, we find that the deconvolution method yields erroneous estimates of the electrooptic relaxation times. When the decay time of the electric pulse and the electrooptic decay time are equal, and the system is operated in the Kerr domain, this systematic error for macromolecules with purely permanent electric dipole moment equals 37%. In a research field where the uncertainty of the reported relaxation times normally is assumed to be only a few percent this is an error that may seriously mislead unsuspecting users. We find that this systematic error can readily be avoided by employing standard numerical integration of a set of coupled first-order differential equations instead of the standard deconvolution techniques.     

Unwinding kinetics of cooperatively melting regions in DNA

Unwinding of a single cooperatively melting region of ColE1 DNA is investigated by a slow temperature jump in formamide–neutral buffer mixed solvent. The semilogarithmic plots of unwinding relaxation curves show a marked terminal linear region following the fast decay, which occurred within the temperature rise time (1 ～ 2 s). This longest relaxation is ascribed to the total unwinding of a single cooperatively melting region. The longest relaxation time, τ₁, is uniquely determined by the final equilibrium state and becomes shorter as the final temperature increases. Decrease in ionic strength makes τ₁ and its fractional amplitude increase, and the relaxation almost approaches single-exponential decay. The facts that (1) τ₁ of a single cooperatively melting region whose unwinding suffers larger frictional resistance does not always unwind more slowly, as was shown by the observations of τ₁'s of almost the same cooperatively melting region located at different positions on two linearized ColEl DNAs and of τ₁'s of two cooperatively melting regions on the same linearized ColEl DNA; (2) τ₁ has strong dependence on the equilibrium state after a temperature jump; and (3) the observed τ₁ is much longer than the expected time of the frictional barrier all demonstrate that the τ₁ is limited by chemical but not hydrodynamic processes. The detailed unwinding process of a single cooperatively melting region, elucidated by evidence of a negative apparent activation energy of the rewinding process and by extensive computer simulation of the equilibrium melting process, suggests that the local heterogeneity of G+C content in a cooperatively melting region, as well as its averaged G+C content, strongly affects its unwinding rate. The present study of a single cooperatively melting region is found to be useful to improve our understanding of the detailed mechanism of complex unwinding of large natural DNAs, in which many cooperatively melting regions unwind.     

Dynamics of drug-DNA interactions: a comparative temperature jump study of ellipticinium and 9-hydroxy ellipticinium

The temperature-jump method has been used to compare the binding of 2-N methyl ellipticinium (NME) and 2-N methyl 9 hydroxy ellipticinium (NMHE) to three natural DNA's of different AT/GC composition. The relaxation signals, analyzed by the Padé-Laplace method, are characterized by two distinct relaxation times, tau 1 and tau 2, respectively in the 1-4 ms and 20-80 ms range. In the case of the NMHE/DNA interaction, the slower relaxation time tau 2 depends on the DNA composition, as follows: tau 2 (Micrococcus lysodeikticus) greater than tau 2 (Calf thymus) greater than tau 2 (Clostridium perfringens). Contrary to NMHE, NME which does not possess an OH group at the C-9 position, shows no relaxation time dependence upon DNA base composition. The observation of two relaxation times indicates that the binding equilibria are associated with at least two distinct drug/DNA complexes (probably arising from two distinct DNA binding sites). Three kinetic models, involving the formation of a weak intermediate ionic complex, are given to explain the binding reaction between these cationic drugs and the DNA. They allow the determination of the four rate constants associated with the two binding steps and lead to equilibrium association constants in agreement with those obtained from spectroscopic studies. The validity of the models is discussed and it is shown that the best kinetic scheme, for either NMHE or NME, could be that in which the ionic step is not a prerequiste to intercalation. The kinetic results show that the residence time of 9 hydroxy ellipticinium is markedly increased in GC rich DNA's and this could be related to the higher in vitro and in vivo cytotoxic properties of 9 hydroxy substituted ellipticines.     

Concerning Measurements of the Self-Diffusion Coefficient of Water Molecules and Time of Proton Spin-Lattice Relaxation in Plant Tissues

Measurements of the coefficient of water molecules self-diffusion (D) and the time of spin-lattice relaxation (T ₁) in prosenchyme (elongated) plant cells, whose length significantly exceeding their transverse size, show that the orientation of plant tissues in the H ₀field significantly affects the measured parameters. We conclude that this effect should be taken into account in experiments on the measurement of self-diffusion coefficients and time of proton spin-lattice relaxation in plant tissues containing prosenchyme cells.     

Copper(II)-DNA denaturation. II. The model of DNA denaturation

In a continuing study of the denaturation of DNA as brought abought about by Cu(II) ions, results are presented for the dependence of T_m and τ (the terminal relaxation time) on ionic strength, pH, reactant concentrations, and temperature. Maximum stability of the double helix, as reflected by the longest relaxation times and highest T_m values, was observed between pH 5.3 and 6.2. Outside this range both T_m and τ decreased sharply. A second, faster relaxation time was deduced from the kinetic cureves. The apparent activation energies of the rapid and slow (“terminal”) relaxations were found to be 12 and 55 kcal/mole, respectively. Several lines of evidence led to the conclusions that (1) the rate-determining step in DNA denaturation, when occurring in the transition region, is determined by chemical events and (2) the interactions which are disrupted kinetically in the rate-determining step are those which account for the major portion of the thermal (T_m) stability of helical DNA.     

Direct cloning of cDNA inserts from λgt11 phage DNA into a plasmid vector by a novel and simple method

Bacteriophage λgt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for λgt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from λgt11 phage were cloned directly into the pBR322 plasmid vector, by-passing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage λ vectors when bacteria containing endogenous pBR322 are used as host cells.     

Effect of intramolecular energetic barriers on conformation-non-equilibrium states of protein globules in ensemble

Within the Blumenfeld's model interpretation theoretical investigations of conformation relaxation of the protein globule were carried out. Analytical expressions describing the relaxation time and cyclic frequency of enzymes synchronization by arbitrary number of metastable states and unequal probabilities of transitions between them were found. It was shown that inequality of activation energies affects the frequency and number of cycles of enzymes synchronous work to a considerably greater degree, than the relaxation time of initial disturbance. The number of macromolecules in the definite conformation state was evaluated and conditions of experimental observation of synchronization were discussed.     

Nucleosome structure relaxation during DNA unwrapping: Molecular dynamics simulation study

Effects of local relaxation of the nucleosome structure after DNA unwrapping from the histone octamer are considered in this paper. The influence of charge distribution in histones on the kinetics of DNA rewrapping was studied. It was shown that ionic environment rapidly stabilizes during relaxation simulation of the system by molecular dynamics. In the case of short relaxation, a rapid irreversible restoration of the structure, which is similar to a crystal one, occurs. In the case of longer relaxation, DNA rewrapping does not occur despite the absence of apparent differences in the ionic environment of DNA. The change in the quadrupole moment of the system during relaxation was shown.     

Dielectric increment and dielectric dispersion of solutions containing simple charged linear macromolecules. II. Experimental results with synthetic polyelectrolytes

The electric permittivity of aqueous solutions of different synthetic polyelectrolytes have been measured as a function of frequency in the range 5 kHz up to 100 MHz in the absence of added salt. Solutions of polymethacrylic acid and polyacrylic acid of different degrees of polymerization, both partially neutralized with NaOH, were investigated as well as solutions of Na-polystyrenesulphonate at different concentrations.For all systems a dispersion profile with two separated dispersion regions was obtained with a molecular weight dependent value of the static electric permittivity. The low frequency dispersion region was found to be characterized by a molecular weight dependent mean relaxation time while for the high frequency dispersion region both the mean relaxation time and the dielectric increment are molecular weight independent. It is shown that the reciprocal values of the specific increments and of the relaxation times depend linearly on the macromolecular concentration. Extrapolation of the corresponding quantities to infinite dilution was found to be possible. A comparison of these extrapolated values with calculated ones according to the previously derived theory also applicable to flexible macromolecules establishes that this theory describes satisfactorily the dielectric behaviour of the systems investigated.The conclusion is reached that the high frequency dispersion and relaxation can be attributed to fluctuations in the distribution of bound counterions along limited parts of the macromolecule. The relaxation time of the low frequency dispersion region seems to be essentially determined by the rotation of the complete molecule and the static electric permittivity can he explained in terms of fluctuations in the counterion density extending over the whole macromolecule.     

Self-diffusion of polymers in cartilage as studied by pulsed field gradient NMR

Pulsed field gradient (PFG) nuclear magnetic resonance (NMR) was used to investigate the self-diffusion behaviour of polymers in cartilage. Polyethylene glycol and dextran with different molecular weights and in different concentrations were used as model compounds to mimic the diffusion behaviour of metabolites of cartilage. The polymer self-diffusion depends extremely on the observation time: The short-time self-diffusion coefficients (diffusion time Delta approximately 15 ms) are subjected to a rather non-specific obstruction effect that depends mainly on the molecular weights of the applied polymers as well as on the water content of the cartilage. The observed self-diffusion coefficients decrease with increasing molecular weights of the polymers and with a decreasing water content of the cartilage. In contrast, the long-time self-diffusion coefficients of the polymers in cartilage (diffusion time Delta approximately 600 ms) reflect the structural properties of the tissue. Measurements at different water contents, different molecular weights of the polymers and varying observation times suggest that primarily the collagenous network of cartilage but also the entanglements of the polymer chains themselves are responsible for the observed restricted diffusion. Additionally, anomalous restricted diffusion was shown to occur already in concentrated polymer solutions.