Calf Thymus DNA In Solution Research Articles

Rheology of semi-dilute solutions of calf-thymus DNA

We study the rheology of semi-dilute solutions of the sodium salt of calf-thymus DNA in the linear and nonlinear regimes. The frequency response data can be fitted very well to the hybrid model with two dominant relaxation times $\tau_{\circ}$ and $\tau_{1}$. The ratio $\frac{\tau_{\circ}}{\tau_{1}} \sim$ 5 is seen to be fairly constant on changing the temperature from 20$^{\circ}$C to 30$^{\circ}$C. The shear rate dependence of viscosity can be fitted to the Carreau model.

Probing irradiated DNA with the perturbed angular correlation method.

The technique of perturbed angular correlations of gamma rays has been used to study the effects of radiation on DNA molecules. The samples are buffered solutions of calf thymus DNA exposed to various doses (0-80 Gy) of gamma rays. Indium-111 is used as a probe. Rotational correlation times, tau(c), a parameter measuring the flexibility of a macromolecule, are obtained that show a dependence on radiation dose.

Interaction of Topotecan, DNA Topoisomerase I Inhibitor, with Double-Stranded Polydeoxyribonucleotides. 2. Formation of Topotecan–DNA Complex Containing Several DNA Molecules

This study is a continuation of a series of papers dealing with topotecan interaction with double-stranded polydeoxyribonucleotides. We showed earlier that topotecan molecules form dimers in solution at concentration above 10–5(per base pair). Topotecan interaction with calf thymus DNA in solutions of low ionic strength was studied by fluorescence, circular dichroism, and linear flow dichroism. The data obtained indicate that topotecan forms two types of complex with DNA, DNA molecules combining with each other during formation of one of these complexes. The association constant of two topotecan-filled DNA molecules with each other was estimated at 104M–1(per base pair) in 1 mM sodium cacodylate buffer, pH 6.8, at 20°C. A possibility of modulation of DNA topoisomerase I activity by topotecan due to complexation with several sites of a supercoiled DNA molecule is discussed.

Human neuronal tau promoting the melting temperature of DNA

The hyperchromic effect of ultraviolet spectroscopy shows that adding recombinant human neuronal tau to the solution of calf thymus DNA will promote the melting temperature (T m) from 67°C to 81°C. Similar result has been detected when adding tau to plasmid p Bluescript-II SK, by raising Tm from 75°C to 85°C. The kinetics of thermal denaturation of DNA with tau is much slower than that of control. It suggests that tau may stabilize the double helix conformation of DNA.

Interaction of alkaline–earth metal ions with calf thymus DNA. Volume and compressibility effects in diluted aqueous solutions

The binding of Mg 2+, Ca 2+, Sr 2+ and Ba 2+ ions to calf thymus DNA in solutions has been investigated by ultrasonic and densimetric techniques. The obtained parameters, the apparent molar volume, Φ V, and the apparent molar adiabatic compressibility, Φ K S , are very sensitive to hydration of investigated molecules. The interaction between the cations and DNA is accompanied by overlapping their hydration shells and consequently releasing the water molecules from hydration shells to bulk state. The change in the hydration is reflected in the measured parameters, Φ V and Φ K S . The magnitude of these hydration changes is determined by the position of the cation relative to DNA atomic groups involved in the binding, and thus can characterize the structure of cation–DNA complexes. The values of the dehydration effects of the binding, ΔΦ V and ΔΦ K S , correspond to two direct or higher number of indirect contacts between calf thymus DNA and the cations.

Synthesis, spectroscopic, electrochemical and antibacterial studies of new Ru(II) 1,10-phenanthroline complexes containing aryldiazopentane-2,4-dione as co-ligand

Mononuclear and dinuclear Ru(II) complexes of 1,10-phenanthroline containing aryldiazopentane-2,4-diones (L1H–L3H2) as co-ligands were prepared and characterised using IR, 1H NMR, UV/Vis spectra in addition to their elemental analysis and FAB mass spectral data. A representative ligand L2H2 and its complex [Ru2L2(phen)4]2+ (phen=1,10-phenanthroline) were characterised also by 1H–1H COSY, TOCSY and 1H–13C HMBC spectral data. Electrochemical behaviour of the complexes was studied in acetonitrile solution and showed irreversible RuII/RuIII redox couples. Luminescence and UV/Vis spectral properties of the complexes in the presence and absence of buffered solutions of calf thymus DNA were also compared. Antibacterial activity of the complexes has been evaluated against Pseudomonas aeruginosa.

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.

In vitro formation of DNA adducts by cisplatin, lobaplatin and oxaliplatin in calf thymus DNA in solution and in cultured human cells.

Two interesting representatives of a new generation of platinum-based cytostatic drugs that are currently being tested in clinical trials are lobaplatin [1,2-diaminomethylcyclobutane platinum(II) lactate] and oxaliplatin [1,2-diaminocyclohexane platinum (II) oxalate]. Since little is known about the DNA adduct formation of these compounds, we studied their formation in DNA in vitro in calf thymus DNA and in cells. The major adducts formed in vitro were the Pt-GG and Pt-AG intrastrand crosslinks. The latter adducts could be detected using a recently developed 32P-postlabelling method. Using both this assay and atomic absorption spectroscopy, it was shown that there is a substantially higher rate of the in vitro adduct formation by cisplatin, compared with lobaplatin and oxaliplatin. Platinum concentrations required to obtain 90% cell kill during a 2 h incubation of A2780 cells were 15 microM for cisplatin and oxaliplatin and 22 microM for lobaplatin. Using an antiserum originally raised against cisplatin-treated DNA, we were also able to detect platinum-DNA adducts induced by lobaplatin and oxaliplatin. Maximal nuclear staining for all three compounds was observed after a 4 h post-incubation period. The nuclear staining level induced by cisplatin was about 10-fold higher than after lobaplatin and oxaliplatin treatment. GG and AG adducts, measured by 32P-postlabelling, also showed maximum levels at about 4 h after treatment. Relative GG peak levels were 4:1:3 for cisplatin, lobaplatin and oxaliplatin, respectively. The ratios of GG over AG intrastrand crosslinks in the A2780 cells were not significantly different for the various compounds. In conclusion, the 32P-postlabelling technique has been shown to be appropriate for adduct analysis, not only for the classical Pt compounds cisplatin and carboplatin but also for novel platinum compounds like lobaplatin and oxaliplatin. Results indicated large differences in reactivity of the latter compounds to DNA in vitro, compared with cisplatin. This difference was smaller in cells, suggesting enhancement of adduct formation by certain cellular mechanisms and/or compounds. From these studies, no conclusions can be drawn with respect to the cytotoxicity of the different Pt-GG and Pt-AG intrastrand crosslinks formed by these compounds.

Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye

The fluorescence characteristics of ethidium bromide (Eb) complexed to calf thymus DNA have been examined using fluorescence lifetime analysis for a range of DNA (effective nucleotide concentration) to Eb molar ratios. Control of both temperature and ion concentration is necessary for reproducible analyses. Eb complexed to double stranded DNA has a maximum fluorescence lifetime of 23 ns and is easily distinguishable from a fluorescence lifetime value of 1.67 ns corresponding to unbound Eb. In a solution of calf thymus DNA containing excess Eb a binding equilibrium is reached, and this corresponds to one Eb molecule for every five nucleotides. With increasing amounts of unbound Eb, the fluorescence lifetime of the DNA-Eb complex decreases with a concomitant drop in the steady state fluorescence intensity, without a change in the amount of Eb bound to DNA. It is concluded that unbound Eb, acting via a quenching mechanism, shortens the fluorescence lifetime of bound Eb and consequently decreases the overall fluorescence intensity. This means that a different approach is necessary: time-resolved fluorescence spectroscopy directly distinguishes between a decrease in fluorescence intensity due to quenching by an excess of unbound Eb from that due to a decrease in Eb binding to double-stranded DNA. These studies suggest that techniques which measure total steady state fluorescence intensity of bound Eb in order to infer relative amounts of double-stranded DNA must be interpreted with caution. For such assays to be valid it is essential that no unbound Eb be present; otherwise a variable correction factor is required to account for unbound Eb.

Effects of DNA Polymer Length on Its Adsorption to Soils

Three different DNA fragments ranging size from 2.69 kbp (1.75 MDa) to 23 kbp (14.95 MDa) were used as tracers to study the adsorption of polydisperse solutions of calf thymus DNA to eight model soils. The adsorption of the three tracers to all soils was described by the Freundlich adsorption model, with adsorption coefficients (K) ranging from 1.1 for acid-washed sand to over 300 for one soil. An inverse relationship between tracer size and K was observed with six of the eight soils, indicating that smaller fragments are sorbed preferentially versus larger fragments in these soils. No significant correlation between K and the organic carbon contents, clay contents, pHs, or cation exchange capacities of the model soils was observed.

Eukaryotic DNAs in Solution Contain Characteristic Components of Tertiary Structure

For natural eukaryotic DNA in solution, we suggest the existence of secondary-helix components superponed to parts of the DNA double helices. In a previous report we found, for calf thymus DNA in solution and of different mean molar mass Mr, an electrostatically driven rise of the hydrodynamically operative contour length of the double helix. This result was derived from Mr-dependent systematic deviations from the almost but not exactly linear plots of intrinsic viscosity [eta] as a function of 1/cs1/2 (cs = Na+ concentration) accurately determined by a titration technique [K.G. and K.E.R., Nucl. Acids Res. 8, 2807 (1980)]. In order to discriminate between DNA elongation contributions caused by secondary or by tertiary structure effects, respective measurements have now been extended to different temperatures for two eukaryotic and two prokaryotic DNA species. The slope of the curves obtained for the (apparent) gradual elongation effect as a function of temperature is negative for the eukaryotic DNAs investigated and is smaller and positive for the prokaryotic species, thus revealing different underlying main elongation mechanisms. We propose that, for the eukaryotic DNA samples, an electrostatically driven partial abolition of tertiary structure components is responsible for the prevailing part of the DNA elongation effect measured. (A helix elongation of this type may be the result of an abolition of an apparent helix shortening as realized in a very high degree on formation of nucleosome chains or in a less degree by DNA molecules with a respective evolutionarily fitted tertiary structure). For the smaller effects of prokaryotic DNA species something like a base breathing seems to dominate. Recent literature results support such an interpretation.

WAVELENGTH DEPENDENT FORMATION OF THYMINE DIMERS AND (6‐4)PHOTOPRODUCTS IN DNA BY MONOCHROMATIC ULTRAVIOLET LIGHT RANGING FROM 150 TO 365 nm

We investigated the wavelength dependence of cyclobutane thymine dimer and (6-4)photoproduct induction by monochromatic UV in the region extending from 150 to 365 nm, using an enzyme-linked immunosorbent assay with two monoclonal antibodies. Calf thymus DNA solution was irradiated with 254-365 nm monochromatic UV from a spectrograph, or with 220-300 nm monochromatic UV from synchrotron radiation. Thymine dimers and (6-4)photoproducts were fluence-dependently induced by every UV below 220 nm extending to 150 nm under dry condition. We detected the efficient formation of both types of damage in the shorter UV region, as well as at 260 nm, which had been believed to be the most efficient wavelength for the formation of UV lesions. The action spectra for the induction of thymine dimers and (6-4)photoproducts were similar from 180 to 300 nm, whereas the action spectrum values for thymine dimer induction were about 9- and 1.4-fold or more higher than the values for (6-4)photoproduct induction below 160 nm and above 313 nm, respectively.

5-Formyldeoxyuridine: a new type of DNA damage induced by ionizing radiation and its mutagenicity to Salmonella strain TA102

An aqueous solution of calf thymus DNA was irradiated by 60Co γ-rays and modified nucleosides produced in DNA were analyzed by high-pressure liquid chromatography coupled with a photodiode array UV detector. A new product with UV absorption maxima at 230 nm and 280 nm was observed. The structure of this compound was proposed to be 5-formyldeoxyuridine (f 5dU) based on the mass spectrum of its trimethylsilyl derivative (M +, m/z 472) and the structure was confirmed by chemical synthesis. The yield of f 5dU ( 2.4 10 4 dT/krad ) in DNA was of roughly the same order as that of 8-hydroxydeoxyguanosine and 5-hydroxymethyldeoxyuridine. Free f 5dU was mutagenic to Salmonella typhimurium strain TA102: therefore f 5dU incorporated into DNA may induce mutations.

Electrochemically controlled binding of ethidium to calf thymus DNA at a carbon-paste / ethidium tetracyanoquinodimethane electrode

Ethidium tetracyanoquinodimethane, Et-TCNQ, carbon paste composite electrodes have been fabricated and studied in contact with aqueous solution. Low current efficiencies for the potential-dependent release of ethidium cations from the electrode surface were observed. In the presence of calf thymus DNA in solution, potential-dependent intercalation of ethidium from the electrode surface was demonstrated by detection of the fluorescence of the intercalated ion. The maximum fluorescence intensity was linear with DNA concentration over the range from 3 to 150 ppm calf thymus DNA.

DNA modifications as a means of detecting the irradiation of wheat

Irradiation produces a variety of modifications in DNA which, if detectable, would provide a means of determining whether food containing DNA had been irradiated. This paper presents initial attempts to detect thymidine glycol, a known DNA irradiation product. Two methods have been employed, a fluorescence assay and an enzyme immunoassay. The reliability of the fluorescence assay was first tested by using it to determine the radiolytic yields of thymidine glycol in the much simpler system of aqueous thymidine solutions saturated with either N 2O or N 2O/O 2(4:1). HPLC was employed to independently assess the radiolytic yields of thymidine glycol in these systems. Results from the fluorescence (values from HPLC experiments given in brackets) gave G-values for glycol of 0.11(0.07) μmol J -1 and 0.16(0.07) μmol J -1 in N 2O and N 2O/O 2 respectively. The fluorescence assay has also been applied to determine G(thymidine glycol) produced on irradiating aqueous solutions of calf thymus DNA, values of 0.03 μmol J -1 and 0.07 μmol J -1 being found in N 2O and N 2O/O 2 saturated solutions respectively. Techniques have been developed to enable DNA to be extracted from small samples of wheat. The preparation of antibodies specific for thymidine glycol-DNA required for the immunoassay is described. DNA from wheat has been tested both by the fluorimetric test and immunoassay. In both cases DNA from unirradiated wheat appeared to contain significant amounts of thymidine glycol. The fluorimetric assay showed increasing levels of glycol with increasing doses of radiation (0–1 kGy). No such correlation was apparent in the immunoassay, this is most probably due to the unsuitability of the initially selected antibody, which although recognising DNA treated with OsO 4 to produce intramolecular thymidine glycol, failed to interact with irradiated calf thymus DNA. Antibodies from other clones are now being screened in an endeavour to find one more suitable.

Effects of ionising radiation on deoxyribonucleic acid. Part 3. The effect of iodoacetamide

Exposure of frozen aqueous solutions of calf thymus DNA to 60Co γ-rays at 77 K gave equal yields of electron-gain centres localised on thymine (T–) and electron-loss centres localised on guanine (G+) as judged by e.s.r. spectroscopy. In the presence of iodoacetamide (ICH2CONH2) electron capture by the additive to form ˙CH2CONH2 and I– occurred in competition with electron capture by thymine. The yield of G+ remained unaffected as estimated from e.s.r. spectra using computer simulation. On annealing, the e.s.r. features for ˙CH2CONH2 were lost at ca. 190 K, with the simultaneous appearance of signals that were assigned to DNA radicals. Analysis for chemical damage using plasmid DNA (pBR 322), irradiated under similar conditions, revealed an increase in the number of single strand-breaks and approximately no change in the number of double strand-breaks arising from the presence of iodoacetamide. Various interpretations of the e.s.r. spectra of the secondary DNA radicals are offered, the most probable being that at least one of these species is formed by hydrogen-atom abstraction from the sugar moiety. We suggest that the increase in single strand-breaks occurs because, on warming, the trapped ˙CH2CONH2 radicals react with DNA to give sugar radicals, at least one of which leads to strand breaks. The observation that the sugar radicals are lost at temperatures below those at which the DNA radicals detected by e.s.r. normally decay, offers an explanation of why sugar radical intermediates have not been detected in our previous studies.

Synthesis and spectral studies on palladium and platinum complexes with mono- and bidentate N-donor organic ligands and their interaction with calf thymus DNA in solution

Palladium(II) and platinum(II) complexes of the types PdLX 2, PdL 2X 2, PtL 2X 2 and the Pt(IV) complexes PtLX 2Y 2, PtL 2X 2Y′ 2 (where L = mono- or bidentate organic ligand containing nitrogen donor atoms; X = Cl or Br; Y = Br and Y′ = OH) have been synthesized and characterized by elemental analysis, IR and X-ray photoelectron spectral data. The Pd 3 d 5/2 binding energies indicate that the 8-aminoquinoline ligand is a better electron donor to the metal than other ligands studied. The Cl 2 p 3/2 binding energies in the square planar pd(II) complexes are observed in the range 198.0–199.56 eV. The ν(PdCl) vibrations ( ca 340 and 320 cm −1) corresponding to two cis-Cl ligands were observed in the IR spectra. Binding through probably N-7 of the guanine residue and the phosphate oxygen in a chelate form is implied from UV difference spectral data.

Effects of lonising radiation on deoxyribonucleic acid. Part 2. The influence of nitroimidazole drugs on the course of radiation damage to aqueous deoxyribonucleic acid

Exposure of frozen aqueous solutions of calf-thymus DNA to 60Co γ-irradiation at 77 K gave electron-gain centres localised on thymine (T–) and electron-loss centres localised on guanine (G+), in approximately equal yields as judged from their e.s.r. spectra. The presence of metronidazole or misonidazole resulted in a marked reduction in the yield of T–, the drug anions being detected in their place. The yield of G+ was not enhanced, in contrast to the results of others. Similar treatment of frozen aqueous solutions of plasmid DNA (pBR 322) was shown by gel electrophoresis to result in extensive single- and double-strand breaks in the absence of drug. Inclusion of metronidazole significantly reduced the number of strand breaks, double-strand breaks being reduced by a proportionately greater amount. An explanation is offered for this. At the molecular level these drugs clearly protect DNA from radiation damage (at least in terms of radical yield and strand breaks), despite the fact that they are known to be efficient radiosensitisers at the cellular level. These observations are discussed with regard to the various proposals for the mechanism of action of these drugs.

Effects of ionizing radiation on deoxyribonucleic acid and related systems. Part 1. The rôle of oxygen

Exposure of aqueous solutions of calf-thymus DNA to 60Co γ-rays at 77 K results in the formation of equal yields of G+ and T– centres, detected by e.s.r. spectroscopy. In the presence of oxygen, O2– ions are also formed at the expense of T–, the primary yield of G+ centres being unaffected. On annealing above 77 K the e.s.r. features for O2– ions were initially modified as a result of changes in solvation, and then, at ca. 193 K, they were lost irreversibly. A large growth in signals due to RO2· radicals was observed simultaneously. These radicals are formed from T– anions or the protonated form, TH, and probably also indirectly from G+ radicals. In parallel experiments, under comparable conditions, we have analysed for single and double stand breaks using plasmid DNA (pBR 322). Both single and double strand breaks persist under conditions in which G+ and T– are the only detectable primary radical products. The presence of oxygen causes only a slight increase in damage to DNA.

Interaction of anthracycline antibiotics with biopolymers: 7. Equilibrium binding studies on the interaction of iremycin and DNA

We report spectrophotometric equilibrium studies of both the self-association of the new antibiotic iremycin and of its binding to calf thymus DNA in solution (ionic strength 0.2 M; pH 6.0). Iremycin forms dimers in this solution with a dimerization constant K 4=(1.19 ± 0.10) × 10 3 M −1. This equilibrium is taken into account in the evaluation of the interaction of iremycin with DNA. The binding behaviour can be completely described by a single binding mechanism of monomeric iremycin to DNA with allowance both for neighbour exclusion and for cooperativity of interaction. The three intrinsic binding parameters for the homogeneous model were determined simultaneously by a least squares fit of the original titration data: equilibrium constant of cooperative binding K = (2.72 ± 0.66) × 10 5 M −1 cooperativity parameter σ=0.38±3.27 ± 0.32. The binding parameters of iremycin and adriamycin and their microbial activities are compared.