Deoxyribonucleic Acid Solutions Research Articles

Viscosity measurements on dilute aqueous calf thymus deoxyribonucleic acid solutions

The viscosity of DNA solutions at low solute concentrations was found to be time dependent. Viscosity data obtained in a Couette viscometer at low rates of shear have been extrapolated to the time of preparation of the dilute DNA solutions. The zero-time intrinsic viscosities were found to increase with decreasing added salt concentration. In the absence of added salt the data obtained suggest that the DNA molecules are fully extended in solution.

Flow birefringence in deoxyribonucleic acid solutions — II. Effect of thermal denaturation and ionic strength of the solution on the structure of DNA macromolecules : Ye. V. Frisman, V. I. Vorob'ev, L. V. Shchagina and N. K. Yanovskaya, Vysokomol. soyed. 5: No. 4, 622–627, 1963

Flow birefringence in deoxyribonucleic acid solutions — II. Effect of thermal denaturation and ionic strength of the solution on the structure of DNA macromolecules : Ye. V. Frisman, V. I. Vorob'ev, L. V. Shchagina and N. K. Yanovskaya, Vysokomol. soyed. 5: No. 4, 622–627, 1963

Flow birefringence in deoxyribonucleic acid solutions—II. Effect of thermal denaturation and the ionic strength of the solution on the structure of DNA macro-molecules

Flow birefringence in deoxyribonucleic acid solutions—II. Effect of thermal denaturation and the ionic strength of the solution on the structure of DNA macro-molecules

Influence of deoxyribonucleic acid on the intermolecular structure of water.

SEVERAL years ago Jacobson1 directed attention to the fact that the helical structure of deoxyribonucleic acid (DNA) proposed by Watson and Crick2 showed a remarkable relationship to the intermolecular structure of liquid water, which has been postulated to be quasi-crystalline and only slightly different from ice3. Jacobson proposed that when DNA is dissolved in water, an increased ordering of the water structure results and further suggested this increased ordering of the intermolecular water structure accounts for the high viscosity and dielectric constant of DNA solutions. In later work Jacobson et al.4 studied the proton resonance spectra of DNA solutions and interpreted the results of their experiments as offering further support of the above-mentioned proposals. Jacobson5 then made a detailed study of the dielectric constants of DNA solutions and interpreted the results as further evidence of stabilization of the normal water structure by the formation of hydration shells with diameters at least one-quarter the length of the dissolved macromolecules; he also cited certain X-ray studies6 for further support of his hypothesis. The sodium salt of deoxyribonucleic acid (sodium thymonucleate) was used in all Jacobson's experimental work.

Nonaqueous solutions of DNA: Factors determining the stability of the helical configuration in solution

The disorganization of the aqueous helical configuration of deoxyribonucleic acid (DNA) by a number of structurally related organic solvents has been investigated. In all the solvents investigated, denaturation is accompanied by 35–50% increase in absorbance (at 259 mμ) and a decrease in optical rotation (at 436 mμ) of 200–350 °. The following observations have been made: ( a) The effectiveness of the denaturant increases with both chain length and increasing hydrocarbon content. Thus ethyl and propyl alcohols were found to be more effective than methanol. ( b) The alkyl-substituted solvents N,N′-dimethylformamide, dimethyl sulfoxide, and tetramethylurea are the most effective denaturants among the various solvents employed. The midpoints of the denaturation transition due to these solvents in the presence of 1–5 × 10 −2 M salt, range from 19 to 27 mole % (57–62 vol.%), and the changes produced in optical rotation (at 436 mμ) upon denaturation are of the order of −300 to −350 °. ( c) N,N′-dimethylformamide is a more effective DNA denaturant than formamide. ( d) Increasing the hydroxyl content of the solvent, on the other hand, had no significant effect; the denaturation midpoints in methanol-water and ethylene glycol-water mixtures (in the presence of 0.5–5 × 10 −2 M salt) occur at 80 ± 1 mole % (90 ± 2 vol.%) of the nonaqueous component. These observations demonstrate the importance of hydrophobic forces and argue against the assignment of the stability of the aqueous configuration of DNA solely to hydrogen bonds. Except in dimethyl sulfoxide, DNA denaturation by the organic solvents employed could be largely reversed by water in the presence of electrolyte.

Metal ions as stabilizers or destabilizers of the deoxyriboucleic acid structure.

RECENTLY there has been considerable interest in the interaction of metal ions with nucleic acids and polynucleotides, primarily as a consequence of the fact that the hydrogen-bonded structure of these macro-molecules collapses in solution in the absence of metallic salts1–4. It is perhaps of equal importance that some metal ions are as effective in destroying the native structure of deoxyribonucleic acid (DNA) as others are in maintaining it. Such destabilization has previously remained largely undetected because investigations of metal interactions with DNA have generally been carried out in approximately 0.1 M solution of an electrolyte such as sodium chloride, in which the destabilization effects are masked5. However, the double-stranded helical configuration of DNA is stable in solutions containing as low as 10−3 M concentration of electrolyte6. In the present work, various divalent metal ions have been added in 10−4 M concentration to solutions of calf-thymus DNA (5 × 10−5 M in phosphate) in 5 × 10−3 M sodium nitrate. Changes in optical density at 260 mµ ara then observed in a Beckman DU spectrophotometer on heating these solutions at the rate of 1° per min. to 90° and then cooling at the same rate7. The conversion of the low optical density solution to the high optical density solution is correlated with the transformation of the double-stranded helix to single strands, and the mean temperature of transition (Tm) is a measure of the stability of the molecule7.

Relaxation enhancement by paramagnetic ion binding in deoxyribonucleic acid solutions.

THE effect of paramagnetic ions on proton relaxation times depends not only on their concentration but also on the binding of the ion to other molecules which may be present in the same solution. By measuring longitudinal relaxation times (T1) by nuclear magnetic resonance we have compared the proton relaxation-rates at constant ion concentration with and without deoxyribonucleic acid (DNA) present. In this way it was possible to detect binding and to distinguish between interior and exterior DNA binding sites for different transition metal ions.

Nonaqueous solutions of DNA. Reversible and irreversible denaturation in methanol

Deoxyribonucleic acid (DNA) undergoes denaturation in methanol-water mixtures. The region of solvent composition in which this transition occurs depends on the ionic strength and temperature but is little influenced by the average nucleotide composition of the DNA. As judged by both macromolecular and optical criteria, the disruption of secondary structure is almost complete, yet methanol denaturation is rapidly and readily reversible by water. The structural basis of this reversibility differs from that controlling the reversibility of partial denaturation in aqueous solution. An irreversible thermal transition has also been found to occur in methanol-rich solvents. The products of this transition may be reconverted to native DNA by water.

Uptake of thymidine and synthesis of deoxyribonucleic acid in mouse ascites cells.

RECENT studies of the synthesis of deoxyribonucleic acid have suggested that the radiation-sensitive period may occur at a stage in the cell-cycle before the polymerization of new deoxyribonucleic acid begins, that is, during the G1 period1–3. It therefore appeared to be of interest to study, in more detail, the assimilation of the precursors into cells and their subsequent incorporation into deoxyribonucleic acid. With this object we have examined the uptake of thymidine labelled with tritium into mouse Ehrlich and Landschutz ascites cells in vitro, measuring the total activity present in the cell and the activity incorporated into deoxyribonucleic acid, as a function of time. The distribution of the precursor among the cells of the population was determined by autoradiography. 2 ml. aliquots of suspension containing about 108 cells were incubated at 37° in a medium containing 50 per cent ascitic fluid and 50 per cent fortified Hanks' medium4 (5 gm. glucose/l.). At the end of the incubation the cells were washed five times with ice-cold Hanks' medium and finally suspended in 4 ml. of the medium. Autoradiographs were prepared in the usual manner, the cells being air dried, fixed with 2 per cent acetic acid or with 45 per cent acetic acid in alcohol and washed before the application of the stripping film. Deoxyribonucleic acid was prepared from 2 ml. of the cell suspension using the detergent method of Kay, Simmons and Dounce5, the final product being dissolved in 2 ml. of water. The solutions of deoxyribonucleic acid and the cell suspensions were homogenized by treatment with ultrasonics before plating on aluminium planchettes. Determinations of tritium activity were made using a windowless flow counter working in the proportional region with an efficiency of about 15 per cent. Deoxyribonucleic acid contents of the cell suspensions and acid solutions were determined using the modified diphenylamine method of Burton6.

Chemical effects of ionizing radiation on deoxyribonucleic acid in dilute aqueous solution.

WHEN purine and pyrimidine nucleotides in dilute aqueous solution are irradiated with ionizing radiation they are destroyed by two pathways1. The minor pathway is attack upon the sugar moiety followed by release of free base1. Details of the mode of sugar attack have not been established; but possible reactions have been suggested by Daniels et al. 2. The major pathway is attack of the base moiety which with purines in the absence of oxygen yields the corresponding formamido-pyrimidine nucleotide3. When pyrimidine nucleotides are irradiated in the absence of oxygen sugar phosphate is liberated, but there is no ultra-violet absorbing product arising from base attack. When oxygen is present during irradiation G-values (molecules per 100 eV.) for nucleotide destruction are increased 2–3 times4. For purine nucleotides the increased degradation in oxygen is almost entirely an augmented base attack, but no ultra-violet product arising from it is found4. When pyrimidines are irradiated in the presence of oxygen hydroxy-hydroperoxides are formed5. G-values for destruction of pyrimidines in the presence of oxygen are greater than those for purines1,6 and suggest that when deoxyribonucleic acid is irradiated pyrimidines will be destroyed to a greater extent. Extrapolation of the effects of radiation upon nucleotides to effects upon deoxyribonucleic acid is complicated in an uncertain way by the influence of inter-nucleotide linkages and hydrogen bonding of base pairs. This communication reports the amounts of base and sugar destroyed when aqueous solutions of calf thymus deoxyribonucleic acid are irradiated.

Sedimentation of deoxyribonucleic acid isolated under low hydrodynamic shear.

BACTERIOPHAGE deoxyribonucleic acid in solution was recently demonstrated to be degraded by moderate hydrodynamic shear gradients1. This observation provoked the question whether the 6–8,000,000 molecular weight, usually observed in purified preparations of deoxyribonucleic acid from animal or bacterial sources, is in any way indicative of the size of the molecules of the acid in the living cell, since in none of the common methods used for purifying deoxyribonucleic acid are precautions taken to minimize shearing.

Effect of ultra-violet light on the shape and size of deoxyribonucleic acid.

SEVERAL investigators1,2 have found that the viscosity of solutions of deoxyribonucleic acid is reduced by irradiation with ultra-violet light of 254 mµ. This change could be the result either of main-chain scission which reduces the length of the molecules or of an increase in flexibility which allows the molecule to take up a more coiled configuration and thereby reduces the volume occupied in solution, as measured by the radius of gyration.

Influence of Counterion Association on the Configurational Stability of Deoxyribonucleic Acid in Solution

THERE is strong evidence that the double stranded helix proposed by Crick and Watson on the basis of the X-ray diffraction photograph of deoxyribonucleic acid fibres1 is present also in solution2.

The influence of deoxyribonucleic acid on ultraviolet and visible light absoption of berberine

1. 1. Extinction of deoxyribonucleic acid (DNA)-berberine mixtures in the range of wavelengths from 2300 to 5000 A. has been measured and compared to the extinction values of the single components. 2. 2. Upon mixing berberine with a DNA solution, the maximum of berberine at 3475 A. shifts to 3500 A. and the height of the extinction maximum markedly diminishes. This diminution is dependent upon the DNA concentration used. It is, however, also dependent on the degree of polymerization of the DNA used, when the concentration remains unchanged. Similar but less pronounced changes also occur in the region of the extinction maximum of berberine at 4250 A., which shifts to 4300 A. upon mixing with DNA. 3. 3. These spectral changes are believed to indicate an interaction between DNA and berberine.

Denaturation or dissociation of deoxyribonucleic acid in dilute solution

Ultracentrifugal analysis of dilute solutions of deoxyribonucleic acid at low ionic strength shows that the sedimentation boundary is composed of two molecular species at pH values lower than 5. The proportion of each component varies from 100 to 0% in the pH range from 5 to 4. Combination of the sedimentation and viscometric data at pH7 and 4 indicates that one component is constituted of native molecules and the other one of denatured material. The sedimentation pattern proves also that the denaration process is “all-or-none” in these conditions.

Molecular weights and physical properties of deoxyribonucleic acid.

IN the measurements of the molecular weight from light scattering and of other physical properties of deoxyribonucleic acid, an extraordinary variety of results has been obtained1, even with samples prepared from a single source, namely, calf thymus. To some extent this might be accounted for as a result of fractionation of the material resulting from its physical heterogeneity, which has been demonstrated by Shooter and Butler2, and by Schachman et al. 3. It may also arise from small amounts of protein impurity, which have been shown4 to affect the physical properties to a marked extent. In order to reduce the sources of discrepancy, it has been thought desirable to determine (1) light scattering, (2) intrinsic viscosity, (3) sedimentation coefficients, on identical solutions of calf thymus deoxyribonucleic acid treated in various ways. It has been found that centrifuging as a means of clarifying the solutions is made much more effective by reducing the concentration from 0.1 to 0.04 per cent. Table 1 shows the effect of centrifuging for 18 hr. at 20,000g at these two concentrations. The difference must be due to the greater viscosity of the stronger solution. The light-scattering molecular weight, especially of the more turbid samples, is markedly reduced by centrifuging in this way at the lower concentration, without any great change of either the intrinsic viscosity or of the sedimentation coefficients. This must mean that the component which gives rise to high turbidities in the unclarified solutions does not contribute appreciably to the viscosity and sedimentation coefficient. That the high turbidities of the unclarified solutions is not due to deoxyribonucleic acid has been demonstrated by the fact that the action of deoxyribonuclease on a clarified solution (J40) with molecular weight 4×106 reduced the light scattering to 20 per cent of its original value ; while on a turbid solution (J38), having an apparent molecular weight of 17×106, the light scattering value was reduced by only 40 per cent, that is, to 10×106.

Formation of hydroperoxides from nucleic acids by irradiation with x-rays in aqueous systems.

IN the course of our studies of the chemical effects of X-rays on nucleic acids in aqueous solutions1, we have had occasion to measure the amounts of peroxide produced. Two methods were employed: (a) the reaction with titanium sulphate reagent2 and spectrophotometric measurement of the resulting complex at 405 mµ ; (b) the oxidation of potassium iodide3 and determination of the liberated iodine (I3 −) by the absorption at 353 mµ. It was found that, in solutions of thymus deoxyribonucleic acid and also of yeast ribonucleic acid irradiated in the presence of oxygen, the yields of total peroxide, as determined by the iodide method, were always greater than those of hydrogen peroxide, estimated by titanium sulphate. This observation can be explained by the presence of organic hydroperoxides in the irradiated nucleate solutions, since it is known that hydro-peroxides will oxidize iodide ions, but do not give, in general, the coloured complex with the titanium sulphate reagent.

Effect of urea on the viscosity of deoxyribonucleic acid solutions

AbstractThe effect of urea and sodium chloride on the viscosity of DNA has been examined at very low concentrations and rates of shear in a Couette viscometer. An increase of apparent intrinsic viscosity occurs in urea, but at higher concentrations of the polymer a reduction of viscosity is observed. When treated with urea, the intrinsic viscosity of the nucleic acid becomes lowered by sodium chloride. The results are consistent with a dimeric hydrogen‐bonded structure of the DNA molecule, which undergoes scission in the presence of urea to form more flexible fragments.

The effect of heat and x-rays on deoxyribonucleic acid

The effect of heating solutions of deoxyribonucleic acid on the sedimentation coefficient distribution and viscosity has been observed. There is in general a decrease in molecular weight, but the mode of preparation of the samples influences to some extent the degree of degradation and the distribution of size and shape of the fragments produced. Since an increased susceptibility to heat can be induced by X-irradiation of the solutions, the findings are in accordance with the view that degradation results from the presence of breaks in the polynucleotide strands. When DNA is irradiated in the solid state much higher doses are required to produce changes comparable with those obtained by irradiating dilute solutions, and a dose of 10 6r gives rise to marked aggregation.

Evidence from light-scattering studies for a dimeric structure for deoxyribonucleic acid in solution.

The investigation of the size and shape of the sodium salt of deoxyribonucleic acid (DNA) in solution has attracted much attention both because of its inherent interest and its great biological importance. But the complications of its polyelectrolyte character and its very great size, both of which contribute to the marked intermolecular interactions, make the interpretation of the experimental results difficult. The least ambiguous method, light scattering, indicates that the nucleic acid derived from calf thymus is best regarded as a stiffened random coil with a molecular weight of about 6 x 106, and using the formulae appropriate to coiling polymer chains, Doty, Reichmann, Rice & Thomas (1954) have shown that the results from measurements of viscosity and sedimentation are consistent with this value. Crick & Watson (1953, 1954) have proposed recently that the analytical data for DNA and the X-ray diffraction diagrams by Franklin & Gosling (1953) obtained forDNA fibres can best be explained by a model in which two strands of nucleic acid intertwine to form a double helix held together by hydrogen bonds between the amino and hydroxyl groups of the purines and pyrimidines. X-ray diagrams indicating the presence of such structures have also been obtained from DNA in native materials (Wilkins, Stokes & Wilson, 1953). Since it is unlikely that such a specific structure can be formed in the process of producing the fibres, it follows on this hypothesis that DNA in solution must already be associated as a double molecule. To test this hypothesis we examined by light scattering the size and shape of DNA in concentrations of urea which we had previously shown to dissociate hydrogen-bonded aggregates of dyes (Alexander & Stacey, 1952) and ofpolyvinyl alcohol (Stacey & Alexander, 1954a). The dissociation by 4M urea of the haemoglobin molecule into two halves by the breaking of hydrogen bonds was clearly demonstrated in the ultracentrifuge by Steinhardt (1938). The results ofpreliminary experiments (Stacey & Alexander, 1954b) indicated that there was a dissociation of the DNA molecule, although it appeared to retain its linear dimensions. Greenstein & Jenrette (1941) had observed that urea decreases the viscosity ofsolution ofDNA, and this observation was confirmed by Conway & Butler (1952), who interpreted this change as a loss of the rigidity of the DNA molecule and also found indications for a halving of the molecular weight. The ionization of the amino groups of cytosine and adenine which are involved in the hydrogen bonds should also produce a dissociation of the twin spiral structure, and for this reason the effect of adding acid on the size and shape of DNA has also been examined. Cecil & Ogston (1948) found in the ultracentrifuge that in solutions of nucleic acid acidified to pH 3*5 a second heterogeneous component appeared with a lower sedimentation constant and increased rate ofboundary spreading, but Doty, Bunce & Reichmann (1953) have shown that it is possible to titrate DNA to pH 2-6 and back to pH 5-8 without degradation. From the change in the angular distribution of the light scattering they concluded that the well-known decrease in viscosity on acidification was due to the contraction of the coil to a much more compact structure. Pouyet & Sadron (1954) have challenged this interpretation because they find no change in the light scattering on adding acid to pH 3-8 and suggest DNA is rather more rigid. We have studied the light scattering of nucleic acid from herring sperm, which appears to have the same properties as the more extensively investigated DNA from calf thymus.