Thermal denaturation of calf thymus DNA: existence of a GC-richer fraction

In 2.5 x 10(-4)M EDTA buffer, the derivative melting curve of calf thymus DNA shows a major band at 47 degrees with a shoulder at about 54 degrees . The fraction of melting area of this shoulder is about 13%. For reconstituted polylysine-calf thymus DNA complexes, in addition to the melting of free DNA regions at about 50 degrees (T(m)) there is another melting at about 106 degrees (T(m)) of polylysine-bound regions. The melting band of the complex at T(m) is not symmetrical. As more polylysine is bound to DNA the melting amplitude is diminished greatly on the major band at 47 degrees but only slightly on the shoulder at 54 degrees . The insensitivity of this shoulder appears to result from the existence of a 13% fraction of calf thymus DNA containing 55% GC. It is not favorably bound by polylysine. It remains in the supernatant after centrifugation and melts at about 54-56 degrees . This conclusion is further supported by two facts: the reconstitution method provides a condition for selective binding of polylysine to AT-rich DNA, and it yields a fully symmetric melting band at T(m) for complexes of polylysine with homogeneous bacterial DNA such as the one from M. luteus.     

Specific interaction of histone H1 with eukaryotic DNA.

The interaction of calf thymus histone H1 with homologous and heterologous DNA has been studied at different ionic strengths. It has been found that about 0.5 M NaCl histone H1, and its fragments N-H1 (residues 1-72) and C-H1 (residues 73-C terminal), precipitate selectively a small fraction of calf thymus DNA. This selective precipitation is preserved up to very high values (less than 2.0) of the input histone H1/DNA ratio. The percentage of DNA insolubilized by histone H1 under these ionic conditions is dependent upon the molecular weight of the nucleic acid, diminishing from 18% fro a Mw equals 1.0 x 10(7) daltons to 5% for a Mw equals 8.0 x 10(4) daltons. The base composition of the precipitated DNA is similar to that of the bulk DNA. Calf thymus histone H1 also selectively precipitates a fraction of DNA from other eukaryotes (herring, trout), but not from some prokaryotes (E. coli, phage gamma. On the other hand, at 0.5 M NaCl, the whole calf thymus DNA (but not E. coli DNA) presents a limited number of binding sites for histone H1, the saturation ratio histone H1 bound/total DNA being similar to that found in chromatin. A similar behavior is observed from the histone H1 fragments, N-H1 and C-H1, which bind to DNA in complementary saturation ratios. It is suggested that in eukaryotic organisms histone H1 molecules maintain specific interactions with certain DNA sequences. A fraction of such specific complexes could act as nucleation points for the high-order levels of chromatin organization.     

Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Cytotoxicity, DNA binding and localisation of novel bis-naphthalimidopropyl polyamine derivatives

Bis-naphthalimidopropyl spermidine (BNIPSpd), spermine (BNIPSpm) and oxa-spermine (BNIPOSpm) showed high in vitro cytotoxicity against human breast cancer MCF-7 cells with IC(50) values of 1.38, 2.91 and 8.45 microM, respectively. These compounds were found to effectively displace the intercalating agent ethidium bromide bound to the calf thymus DNA using fluorimetric methods (C(50) 0.08-0.12 microM) and their apparent equilibrium binding constants (K(app)) were calculated to be in the range of 10.5-18 x 10(7) M(-1). Furthermore, strong stabilisation of calf thymus DNA duplex in the presence of bis-naphthalimidopropyl polyamine derivatives (BNIPSpd, BNIPSpm and BNIPOSpm) was observed by UV spectrophotometric analysis (T(m)=93.3-97 degrees C compared with 75 degrees C for calf thymus DNA without drug). Because of their inherent fluorescence, these compounds were localised preferentially inside the nucleus as evidenced by their direct observation under the fluorescence microscope. The results obtained suggest that the cytotoxic activity of the bis-naphthalimidopropyl polyamines may be in part, caused by their effects on DNA.     

Cleavage of DNA by electrochemically activated MnIII and FeIII complexes of meso-tetrakis (N-methyl-4-pyridiniumyl)porphine

Electrochemical methods were used to activate MnIII and FeIII complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2TMPyP) to cause cleavage of pBR322 DNA and to study their interaction with sonicated calf thymus DNA. Electrochemical reduction of MnIIITMPyP and FeIIITMPyP (at low concentrations) in the presence of O2 was required to activate these complexes. However, FeIIITMPyP at 1 x 10(-6) M produced DNA strand breakage without being electrochemically reduced. At low concentrations, FeIITMPyP was more efficient at cleaving DNA than MnIITMPyP. Reduction of O2 at a platinum electrode also produced some cleavage but to a much smaller extent. The oxidized form of MnIIITMPyP (charge 5+) has higher affinity for sonicated calf thymus (CT) DNA than the reduced form (charge 4+), as determined by the negative shift in E degrees' for the voltammetric wave in the presence of DNA. Both forms of FeIIITMPyP (charge 4+) interact with DNA to about the same extent. Differential pulse voltammetry was used to determine binding constants (K) and binding-site sizes (s) of the interaction of these metalloporphyrins with sonicated CT DNA. The data were analyzed assuming both mobile and static equilibria. MnIIITMPyP binds to DNA (5 mM Tris, 50 mM NaCl, pH 7) with K = 5 (+/- 2) x 10(6) M-1, s = 3 bp (mobile) or K = 3.6 (+/- 0.3) x 10(6) M-1, s = 4 bp (static). FeIIITMPyP at that ionic strength caused DNA precipitation. At higher ionic strength (0.1 M Tris, 0.1 M NaCl, pH 7), FeIIITMPyP associates to DNA with K = 4.4 (+/- 0.2) x 10(4) M-1, s = 5 bp (mobile) or K = 1.9 (+/- 0.1) x 10(4) M-1, s = 6 bp (static).     

The fractionation of calf thymus DNA by multiple formation of sedimentable complexes with homologous lysine-rich histone KAP. Derivative melting curves and ultracentrifugation in CsCl concentration gradients

The process of fractionation of total calf thymus DNA using a step precipitation of DNA by means of increasing concentrations of the homologous histone KAP was investigated. In addition to the known fractions three so far undescribed ones/in thymus/,characterized by buoyant densities in CsCl equal 1.692, 1.706 and 1.728 g/ccm, were identified. Considerable amounts of preparations seriously enriched in individual satellite fractions were obtained. The ability of GC-rich satellite DNAs to form more soluble complexes with histone KAP is suggested as reason for the observed fractionation.     

Physicochemical characterization of a new glucocorticoid receptor

A new glucocorticoid-binding protein (Peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography was identified previously in the rats subjected to stress or treated with glucocorticoid (100 micrograms/100 g body wt.), while the 'classic' glucocorticoid receptor (Peak B) eluted with 0.07 M NaCl was found predominantly in untreated rats. The new glucocorticoid-binding protein, Peak C, was characterized by Scatchard analysis and competition with other steroids as a glucocorticoid receptor. The saturation curve of Peak C for dexamethasone was sigmoidal, whereas that of Peak B was hyperbolic. The Hill coefficient was 1.0 for Peak B and 3.1 for Peak C. These results show that Peak C has multiple binding sites. Peak C bound specifically to only natural or synthetic glucocorticoids, whereas Peak B bound not only to glucocorticoids but also to progesterone and aldosterone. Peak C was far more labile than Peak B, its binding activity decreasing 80% when it was incubated for 30 min at 25 degrees C. The molecular sizes of these two peaks (B and C) were similar, being about 90 000-100 000 as determined by Sepharose 6B column chromatography at high ionic strength (0.34 M KCl). The hormone-receptor complex of Peak C bound to rat liver chromatin specifically, but did not bind to calf thymus DNA. The complex of Peak B bound to not only the chromatin but also calf thymus DNA. Peak B reacted well with antiserum to the 'classic' glucocorticoid receptor, but Peak C did not react with this antiserum. These results indicate that Peak C is a different glucocorticoid receptor protein from Peak B, or classic glucocorticoid receptor, and plays physiologically important roles as a glucocorticoid receptor mediating the action of the hormone at a high level.     

Effects of diadenosine 5',5'-P1, P4-tetraphosphate and of adenosine 5'-triphosphate on the higher order structure of calf thymus DNA

The effective length and the hard core radius were calculated by scaled particle theory for high molecular weight calf thymus DNA in the presence of varying concentrations of diadenosine 5',5'-P1, P4-tetraphosphate and of adenosine 5'-triphosphate in aqueous millimolar NaCl. DNA became slightly more flexible in the presence of diadenosine 5',5'-P1, P4-tetraphosphate at concentrations of 10(-9)-10(-7) M. DNA was denatured in the presence of 5 X 10(-5) M adenosine triphosphate.     

DNA binding by cyclic adenosine 3',5'-monophosphate dependent protein kinase from calf thymus nuclei.

Cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase and proteins specifically binding cAMP have been extracted from calf thymus nuclei and analyzed for their abilities to bind to DNA. Approximately 70% of the cAMP-binding activity in the nucleus can be ascribed to a nuclear acidic protein with physical and biochemical characteristics of the regulatory (R) subunit of cAMP-dependent protein kinase. Several peaks of protein kinase activity and of cAMP-binding activity are resolved by affinity chromatography of nuclear acidic proteins on calf thymus DNA covalently linked to aminoethyl Sephrarose 4B. When an extensively purified protein kinase is subjected to chromatography on the DNA column in the presence of 10(-7) M cAMP, the R subunit of the kinase is eluted from the column at 0.05 M NaCl while the catalytic (C) subunit of the enzyme is eluted at 0.1-0.2 M NaCl. When chromatographed in the presence of histones, the R subunit is retained on the column and is eluted at 0.6-0.9 M NaCl. In the presence of cAMP, association of the C subunit with DNA is enhanced, as determined by sucrose density gradient centrifugation of DNA-protein kinase complexes. cAMP increases the capacity of the calf thymus cAMP-dependent protein kinase preparation to bind labeled calf thymus DNA, as determined by a technique employing filter retention of DNA-protein complexes. This protein kinase preparation binds calf thymus DNA in preference to salmon DNA, Escherichia coli DNA, or yeast RNA. Binding of protein kinases to DNA may be part of a mechanism for localizing cyclic nucleotide stimulated protein phosphorylation at specific sites in the chromatin.     

Physicochemical characterization of a new glucocorticoid receptor

A new glucocorticoid-binding protein (Peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography was identified previously in the rats subjected to stress or treated with glucocorticoid (100 μg/100 g body wt.), while the ‘classic’ glucocorticoid receptor (Peak B) eluted with 0.07 M NaCl was found predominantly in untreated rats. The new glucocorticoid-binding protein, Peak C, was characterized by Scatchard analysis and competition with other steroids as a glucocorticoid receptor. The saturation curve of Peak C for dexamethasone was sigmoidal, whereas that of Peak B was hyperbolic. The Hill coefficient was 1.0 for Peak B and 3.1 for Peak C. These results show that Peak C has multiple binding sites. Peak C bound specificially to only natural or synthetic glucocorticoids, whereas Peak B bound not only to glucocorticoids but also to progesterone and aldosterone. Peak C was far more labile than Peak B, its binding activity decreasing 80% when it was incubated for 30 min at 25°C. The molecular sizes of these two peaks (B and C) were similar, being about 90 000–100 000 as determined by Sepharose 6B column chromatography at high ionic strength (0.35 M KCl). The hormone-receptor complex of Peak C bound to rat liver chromatin specifically, but did not bind to calf thymus DNA. The complex of Peak B bound to not only the chromatin but also calf thymus DNA. Peak B reacted well with antiserum to the ‘classic’ glucocorticoid receptor, but Peak C did not react with this antiserum. These results indicate that Peak C is a different glucocorticoid receptor protein from Peak B, or classic glucocorticoid receptor, and plays physiologically important roles as a glucocorticoid receptor mediating the action of the hormone at a high level.     

Structural transition in chromatin induced by ions in solution

Structural transition in chromatin was measured as a function of counter ions in solution (NaCl or MgCl(2)) and of histones bound on the DNA. The addition of counter ions to aqueous solutions of chromatin, partially dehistonized chromatin, and DNA caused a drastic reduction in viscosity and a significant increase in sedimentation coefficient. Transitions occurred primarily at about 2 x 10(-3) M NaCl and 1 x 10(-5) M MgCl(2) and are interpreted as a change in structure of chromatin induced by tight binding of cations (Na(+) or Mg(++)) to DNA, either free or bound by histones, and is an intrinsic property of DNA rather than of the type of histone bound. At a given ionic condition, removal of histone H1 from chromatin had only a minor effect on the hydrodynamic properties of chromatin while removal of other histones caused a drastic change in these properties. An increase in the sedimentation coefficient of DNA was observed also for protamine. DNA complexes wherein the bound protein contains only unordered coil rather than the alpha-helices found in histones.     

Solution chemistry and DNA binding properties of MEN 10755, a novel disaccharide analogue of doxorubicin.

The behavior under physiological conditions of MEN 10755, a novel disaccharide analogue of doxorubicin, was investigated in detail by a variety of spectroscopic techniques including spectrophotometry, fluorescence, and (1)H NMR. The pH dependent properties of MEN 10755 were also analysed by spectrophotometry and potentiometry within the pH range 5--11. It is found that MEN 10755 behaves very similarly to doxorubicin and reproduces closely its pH dependent pattern. Like doxorubicin, MEN 10755 undergoes dimerization with a significantly smaller association constant. The interaction of MEN 10755 with calf thymus DNA was studied in detail. Spectrophotometric and fluorescence titrations of MEN 10755 with calf thymus DNA show spectral patterns almost identical to those obtained with doxorubicin implying that the binding mechanism and the stability of the resulting adducts are very similar. An apparent affinity constant of 1.2 x 10(6) was determined for the interaction of MEN 10755 with calf thymus DNA to be compared with the value of 3.3 x 10(6) measured for doxorubicin, under the same conditions. The effects of both anthracyclines on the thermal denaturation profiles of calf thymus DNA were also analyzed; both compounds turned out to stabilize to a similar extent the DNA double helix and to give rise to a characteristic two-step melting profile. The implications of the present results for the pharmacological activity and the mechanism of action of this novel and promising antitumor compound are discussed.     

Oxidative degradation of cationic metalloporphyrins in the presence of nucleic acids: a way to binding constants?

Mn(III) and Fe(III) complexes of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (M-TMePyP) and related hybrid molecules ("metalloporphyrin-ellipticine") were activated by potassium monopersulfate in the presence of variable calf thymus (CT) DNA and NaCl concentrations. Monitored by visible spectroscopy (Soret band), fast degradation of the free metalloprophyrin was observed while the DNA-bound form appeared protected. This direct quantitation of free versus bound metalloporphyrin ratios allowed determination of binding constants: Mn- and Fe-TMePyP respectively bind to CT DNA (5 mM phosphate buffer, 0.1 M NaCl, pH 7) with K = 3 X 10(4) and 1.2 X 10(4) M-1. Mn-TMePyP showed a greater affinity for poly[d(A-T)] (K = 1.2 X 10(5) M-1) than for poly[d(G-C)] (K = 0.2 X 10(4) M-1). This method allowed us access to the intrinsic DNA affinity of the metalloporphyrin moiety of the hybrid molecules "metalloporphyrin-ellipticine".     

A comparative study of DNA complexation with Mg(II) and Ca(II) in aqueous solution: major and minor grooves bindings

Although structural differences for the Mg-DNA and Ca-DNA complexes are provided in the solid state, such comparative study in aqueous solution has been less investigated. The aim of this study was to examine the bindings of Mg and Ca cations with calf thymus DNA in aqueous solution at physiological pH, using constant concentration of DNA (1.25 or 12.5 mM) and various concentrations of metal ions (2 microM-650 microM). Capillary electrophoresis, UV-visible, and Fourier transform infrared spectroscopic methods were used to determine the cation-binding modes, the binding constants, and DNA structural variations in aqueous solution. Direct Ca-PO(2) binding was evident by major spectral changes (shifting and splitting) of the backbone PO(2) asymmetric stretching at 1222 cm(-1) with K = 4.80 x 10(5) M(-1), whereas an indirect Mg-phosphate interaction occurred (due to the lack of shifting and splitting of the phosphate band at 1222 cm(-1)) with K = 5.6 x 10(4) M(-1). The metal-base bindings were directly for the Mg with K = 3.20 x 10(5) M(-1) and indirectly for the Ca cation with K = 3.0 x 10(4) M(-1). Both major and minor groove bindings were observed with no alteration of the B-DNA conformation.     

Interaction of metallopyrazoliumylporphyrins with calf thymus DNA.

The interaction of transition metal complexes of cationic porphyrins bearing five membered rings, meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (MPzP, M=Mn(III), Ni(II), Cu(II) or Zn(II)), with calf thymus DNA (ctDNA) has been studied. Metalloporphyrins NiPzP and CuPzP are intercalated into the 5'GC3' step of ctDNA. MnPzP is bound edge-on at the 5'TA3' step of the minor groove of ctDNA, while ZnPzP is bound face-on at the 5'TA3' step of the major groove of ctDNA. The binding constants of the metalloporphyrins to ctDNA range from 1.05x10(5) to 2.66x10(6) M(-1) and are comparable to those of other reported cationic porphyrins. The binding process of the metallopyrazoliumylporphyrins to ctDNA is endothermic and entropically driven. These results have revealed that the kind of central metal ions of metalloporphyrins influences the binding characteristics of the porphyrin to DNA.     

Intramolecular complex formation of poly(N-isopropylacrylamide) with human serum albumin

Complexation of human serum albumin (HSA) with poly(N-isopropylacrylamide) (PNIPA) ranging in molecular weight (M(PNIPA)) from 2.1 x 10(4) to 1.72 x 10(6) was studied in an aqueous system (pH 3) containing NaCl as a supporting salt. Dynamic light scattering, static light scattering, electrophoretic light scattering, and dialyzing techniques were used as the experimental tool in a suitable combination. The measurements were performed mainly at 25 degrees C and at 0.01 M NaCl as a function of mixing ratio (r(m), molar ratio of PNIPA to HSA). The results of DLS and ELS evidently demonstrated the formation of a water-soluble complex through mixing of HSA and PNIPA. A detailed analysis of SLS data with the aid of dialysis data revealed that the resulting complex is an "intramolecular" complex consisting of a PNIPA chain with several of bound HSA molecules. Both hydrodynamic radius (R(h)) and radius gyration (R(g)) of intramolecular complexes decreased as r(m) was increased. This result correlated well to the fact that the number (n) of bound proteins per polymer decreases with increasing r(m). The size and the molar mass of the complex became large depending on M(PNIPA), but the increase of M(PNIPA) led to a decrease in n at r(m) < 1. The increase in NaCl concentration from 0.01 to 0.3 M brought about the increase in the size and the molar mass of an intramolecular HSA-PNIPA complex prepared at r(m) = 1.1. This was found to be due to an increase of n. A similar trend was observed when temperature rose from 25 to 32 degrees C (close to lower critical solution temperature of PNIPA). However, the effect of temperature on the increase of was strong in comparison with that of ionic strength. On the basis of these results obtained, the complexation mechanism was discussed in detail.     

Structural heterogeneity of chromatin preparations at the level of DNA topology

The structural heterogeneity of calf thymus chromatin preparations was studied at the level of DNA topology by analysing the influence of ethidium bromide on the chromatin viscosity in deproteinizing medium. In 0.7 M NaCl the chromatin was separated into the fractions with linear DNA (3--36% in various preparations) and with supercoiled circular DNA (scc DNA), which differ from each other in their adhesive properties. Reduction of disulfide bonds in residual chromatin protein with 5% mercaptoethanol linearized scc DNA, present in chromatin preparations as nuclear matrix subunits containing some loops of scc DNA on the protein globule.     

Adsorption chromatography of nucleic acids on silicone-coated porous glass

Bovine liver tRNA was adsorbed on silicone-coated porous glass in 5 M NaCl, 10 mM Tris-HCl (pH 7.6) and fractionated by elution with decreasing NaCl concentrations. tRNAPro, tRNAVal, tRNAIle, tRNAThr, tRNASer, and tRNAPhe were eluted in this order. tRNA which had been digested with ribonuclease A was not adsorbed. Q beta RNA (adsorbed onto the glass in 5 M NaCl) was eluted with 1.5 M NaCl. RNA species in a crude rRNA fraction from Escherichia coli were separated into tRNA, 5S rRNA, and high molecular weight rRNA on siliconized porous glass. A half of calf thymus DNA was adsorbed on the glass in 5 M NaCl and the residual part passed through the column. The CD spectra showed that DNA and tRNA took the C-form and the A-form in 5 M NaCl, respectively. Therefore, the discrepancies of behavior of the DNA and RNA on siliconized porous glass may be related to the occurrence of these forms. The recovery of these nucleic acids from the column was 83-100%. Adsorption chromatography on siliconized porous glass may be a useful method for the separation of tRNA, rRNA, and mRNA.     

The effect of low ionic strength on the radiation chemistry and physical properties of calf thymus DNA

Studies of ultraviolet and circular dichroism spectra of aqueous solutions of calf thymus (CT) DNA confirm the tendency of DNA to change conformation at low ionic strength. The qualitative shape and transition width of 260 nm melting curves below 1 mM NaCl differed significantly from those previously published for DNA solutions containing 1 mM NaCl and above. Neutral aqueous solutions of CT DNA at low ionic strengths (0.1 mM-10 mM NaCl) were irradiated with low doses of gamma-rays. The melting temperature, Tm, of irradiated DNA samples increased below 1 mM NaCl suggesting interstrand crosslinking of the denatured DNA or formation of regions of more thermally stable DNA conformation. The magnitudes of these radiation responses were found to be a function of the time elapsed between salt concentration changes and irradiation as well as time after irradiation. These results are consistent with the hypothesis that the purine and pyrimidine base chromophores in double stranded DNA are sheltered from radical attack by the sugar phosphate backbone. Low dose radiation studies (0.8-8.0 Gy) of CT DNA in 1 mM NaCl and below showed a split dose and dose rate dependence for the sample melting curves.