Concentration Of Calf Thymus DNA Research Articles

Determination of DNA by its enhancement effect of resonance light scattering by azur A

The interaction of azur A (AA) with DNA in aqueous medium has been studied by measurement of resonance light scattering (RLS), and accordingly a method for DNA determination is proposed with sensitivity at the ng level. At pH 3.29 and ionic strength 0.003, the RLS of AA was greatly enhanced by DNA characterized by the maximum scattering peaks at 398 and 472 nm. Mechanistic studies show that the interaction of AA with DNA involves the salting-out of AA encouraged by the electrostatic attraction of the positive charge of AA molecules with the negative charges of the DNA polyanion, and the salted-out AA molecules bind with DNA in a weak cooperative mode. Scatchard plots were constructed for the interaction equilibrium of AA with double-stranded DNA based on the RLS data, and it was found that each nucleotide residue could bind to four AA molecules with the binding constant being at the 10 5 level. The enhanced intensity of RLS is proportional to the concentration of calf thymus DNA and fish sperm DNA in the range 0–2.0 μg ml −1 if 3.0×10 −5 mol l −1 AA was employed. Four synthetic samples were determined satisfactorily.

Interaction of Metal Complexes of Bis(salicylidene)-ethylenediamine with DNA

The fluorescence spectral characteristics and interaction of several metal complexes of bis(salicylidene)-ethylenediamine (salen) with DNA are described. Among the complexes tested, Co-salen bound to DNA showed a marked decrease in the fluorescence intensity with a bathochromic shift of the excitation and emission peaks. A hypochromism in the UV absorption spectra was also observed. KI quenching and competitive binding to DNA between Co-salen and ethidium bromide were studied in connection with other experimental observations to show that the interactive model between Co-salen and DNA is an intercalative one. The pH and salt effect on the fluorescence properties was also investigated. The intrinsic binding constant and the binding site number were estimated to be 5.76 × 106 mol L-1 in base pairs and 0.058, respectively. A linear relationship between F/F0 and the concentration of calf thymus DNA covers 1.0 × 10-6 - 5 × 10-4 mol L-1, which can be utilized for determining traces of calf thymus DNA with a detection limit of 4.6 × 10-7 mol L-1 in base pairs.

Ethanol-Induced Condensation of Calf Thymus DNA Studied by Laser Light Scattering

Effect of ethanol on calf thymus DNA (CT-DNA) has been studied in the probe length scale range 3.2 ≤ qRg ≤ 6.3 in sodium chloride/sodium citrate buffer (10 mM) adjusted to pH = 7.0 with the CT-DNA concentration fixed at 2 × 10-8 M at room temperature (20 °C). The measured hydrodynamic radius Rh and radius of gyration Rg clearly indicate condensation behavior, and the plausible conformation has been inferred from the Rh/Rg ratio. A sharp conformational transition was observed at an ethanol/water concentration 40:60. The polydispersity index deduced from the width of particle size distribution shows a minima corresponding exactly to this concentration. The results are supported by UV absorption studies done at λ = 260 nm.

Determination of nanograms of nucleic acids by their enhancement effect on the resonance light scattering of the cobalt(II)/4-[(5-chloro-2-pyridyl)azo]-1,3-diaminobenzene complex.

Using a common spectrofluorometer to measure the intensity of resonance light-scattering, a method for determination of nucleic acids in the nanogram range has been developed. In the pH range 11.5-12.0, the resonance light-scattering of the binary complex of cobalt(II)/ 4-[(5-chloro-2-pyridyl)azo]-1,3-diaminobenzene (5-Cl-PADAB) is greatly enhanced by nucleic acids, with the maximum scattering peak located at 547.0 nm. The enhanced intensity of resonance light-scattering is in proportion to the concentration of calf thymus DNA in the range 0-400 ng/mL and to that of fish sperm DNA and yeast RNA in the range 0-300 ng/mL. The limits of detection are 1.4 ng/mL for calf thymus DNA, 0.8 ng/ mL for fish sperm DNA, and 1.3 ng/mL for yeast RNA. Precision at 200 ng/mL for the three nucleic acids is 1.9%, 2.0%, and 0.8%, respectively. Six synthetic samples were determined satisfactorily. Mechanism studies showed that the nature of the reaction is that the binary complex of Co(II)/5-Cl -PADAB reacts with single-stranded nucleic acid, and the enhancement effect of nucleic acids on the resonance light scattering of the binary complex is due to the stacking of the binary complex on nucleic acids, which act as a template.

Alteration of the aflatoxin cyclopentenone ring to a .delta.-lactone reduces intercalation with DNA and decreases formation of guanine N7 adducts by aflatoxin epoxides

The regio- and stereospecificity exhibited by reaction of aflatoxin B1 8,9-epoxide with DNA as well as the efficiency of reaction are remarkable and suggests that a specific orientation of bound epoxide facilitates formation of the transition state leading to guanine N7 adducts. We have compared aflatoxins B1 and B2 with aflatoxins G1 and G2 as to their binding with calf thymus DNA, d(ATGCAT)2, d(GCATGC)2, and plasmid pBR322. Aflatoxins B1 and B2 contain a cyclopentenone ring fused to the lactone ring of the coumarin. They have similar DNA association constants and intercalate with B-DNA, as demonstrated by NMR analysis of association with d(ATGCAT)2 and d(GCATGC)2, alteration of pBR322 electrophoretic mobility, and flow dichroism using linearly oriented calf thymus DNA. The less planar delta-lactone ring of aflatoxins G1 and G2 reduces DNA binding affinity by approximately 1 order of magnitude. Nevertheless, binding studies with d(ATGCAT)2 and d(GCATGC)2 suggest that aflatoxins G1 and G2 also bind B-DNA by intercalation. To establish the existence of a relationship between the association of these aflatoxins with DNA and adduct formation induced by aflatoxin epoxides, we compared the yield of guanine N7 adduct from aflatoxin B1 8,9-epoxide and from aflatoxin G1 9,10-epoxide at three concentrations of calf thymus DNA. As DNA concentration is decreased, two observations are made: (1) the number of adducts formed by either aflatoxin B1 8,9-epoxide or aflatoxin G1 9,10-epoxide is reduced with a concomitant increase in formation of the respective dihydrodiols, and (2) the ratio of adducts formed by aflatoxin G1 9,10-epoxide to those formed by an equivalent concentration of aflatoxin B1 8,9-epoxide decreases.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormone receptors from IM-9 cells but not HeLa cells bind to promoters of triiodothyronine-responsive genes

We have used thyroid hormone receptors from two different human cell lines to investigate receptor binding to the promoters of thyroid hormone-responsive genes. Receptors extracted from IM-9 cells or HeLa cells displayed virtually identical affinity and specificity for [ 125I]triiodothyronine binding. The cells expressed a c- erbAα gene in the same relative proportions as the receptor concentrations. Both receptors were bound to DNA-cellulose and could be displaced with increasing concentrations of calf thymus DNA or pBR322 DNA. Relative to pBR322 DNA (designated as 1), binding to the hGH gene promoter was 8.1 ± 1.1 using the IM-9 cell receptor. With the HeLa cell receptor relative binding was only 1.1 ± 0.2. Similar relative differences were obtained with the mouse glandular kallikrein gene, mGK-6. In heat stability studies the IM-9 cell receptor was more resistant to heat inactivation than the HeLa receptor. Triiodothyronine receptors with identical hormone binding patterns may require the presence of an unidentified factor(s) which allows correct recognition of regulation sequences within responsive genes.

Properties of Purified Calf Thymus Poly(Adenosine Diphosphate Ribose) Polymerase. Comparison of the DNA-Independent and the DNA-Dependent Enzyme

The physicochemical properties of the purified calf thymus poly(ADP-ribose) polymerase were investigated. The enzyme purified to homogeneity was shown to contain about 10% DNA on a weight basis and its activity to be DNA independent. After removing this fragment of DNA, called the sDNA fraction, the enzyme becomes DNA dependent. The activity of this enzyme preparation was entirely dependent on, and completely restored by, added calf thymus DNA or sDNA. However, the calf thymus DNA concentration needed was a hundred times higher than that of sDNA. The properties of the two enzyme preparations, DNA independent and DNA dependent, were essentially the same. They both reacted against the specific antibody obtained with the DNA-independent poly(ADP-ribose) polymerase. The pH optimum was around 8; the activity was stimulated by Mg2+, Mn2+ and Ca2+, and inhibited by high ionic strength, p-chloromercuribenzoate, ADP-ribose, AMP and polylysine. Nicotinamide, thymidine and NADP were shown to be competitive inhibitors. The enzymatic activity was stimulated by histone H1 when the ratio of DNA to histone H1 was 2. Histones H2A, H2B, H3 and H4 had little effect on the DNA-independent enzyme activity, but were strongly inhibitory for the DNA-dependent enzyme. This inhibitory effect could be reversed by allowing the DNA-dependent enzyme to react with the sDNA fraction before adding histone subfractions. The apparent Km for NAD of the DNA-dependent poly(ADP-ribose) polymerase was shown to vary with the DNA concentration. It was minimum when the amount of sDNA was 10% of that of the enzyme. The ratio of the apparent Km for sDNA to the enzyme concentration was constant at any enzyme concentration. The minimum estimation of the number of base pairs of sDNA required for maximal activation of one enzyme molecule was 16. For calf thymus DNA, this estimation was of 640. These results suggest that the activation of the enzyme needs the formation of some complex between the protein and a specific part of the DNA. This complex was preserved in the DNA-independent enzyme preparation.