Heat-denatured Calf Thymus DNA Research Articles

The Effects of Polyamines on the Immunogenicity of Polynucleotides

Polyamines--putrescine, spermidine, and spermine--are small organic cations that are present in all living cells. Recent studies revealed that polyamines could provoke a left-handed Z-DNA conformation in poly(dA-dC).poly(dG-dT) and related alternating purine-pyrimidine sequences. In order to examine whether polyamine-induced Z-DNA conformation of poly(dA-dC).poly(dG-dT) is capable of eliciting anti-Z-DNA antibodies, we immunized rabbits with poly(dA-dC).poly(dG-dT) in the presence and absence of spermidine and spermine. Rabbits immunized with the polynucleotide alone produced antibodies reacting toward poly(dA-dC).poly(dG-dT) and heat-denatured calf thymus DNA (ssDNA). In contrast, immunization with poly(dA-dC).poly(dG-dT) complexed with spermidine or spermine produced antibodies reacting with Z-DNA in addition to those binding toward poly(dA-dC).poly(dG-dT) and ssDNA. Antibodies elicited by polynucleotide.polyamine complexes had no reactivity toward polyamines. Solution inhibition studies suggested that anti-poly(dA-dC).poly(dG-dT), anti-ssDNA and anti-Z-DNA antibodies are distinct populations that favor each one of these antigens. Our results suggest that natural polyamines are capable of altering the immunogenicity of polynucleotides by mechanisms involving the stabilization of Z-DNA conformation. This result may have implications in the recent findings of high levels of polyamines and anti-Z-DNA antibodies in the sera of lupus patients and autoimmune mice.

Adsorption of deoxyribonucleic acid and poly(cytidylic acid) · poly(inosinic acid) onto the poly(styrene) latex

Adsorption of native and heat-denatured calf thymus DNA ( M w = 2.5 × 10 5 and 4.5 × 10 6) and poly(cytidylic acid) · poly(inosinic acid) ((C) n · (I) n , M w = 1.5 × 10 5, and 7.7 × 10 5) duplex onto the negatively charged poly(styrene) latex was examined at 4°C with emphasis on the effect of the molecular chain length, the species of added salts (NaCl, CaCl 2, and MgCl 2), and their ionic strengths ((1–10) × 10 −3). The amount of adsorbed nucleic acids was determined photometrically from teh difference in the concentrations before and after mixing with the latex (average diameter, 471 nm; surface charge density, −3.52 μC/cm 2). For native DNA, the higher the molecular weights and ionic strengths were, the more was adsorbed, the effect of the added ions being Mg 2+ > Ca 2+ ⩾ Na +. For denaturated DNA, the adsorption behavior was qualitatively the same but the adsorbed amount was less. These trends were reversed for (C) n · (l) n , but the effect of added ions remained the same as that for DNA. The divalent cations play a role of a binder between two types of negative charges on nucleic acids and on latex.

A DNA primase from yeast. Purification and partial characterization.

A DNA primase activity has been purified from the budding yeast Saccharomyces. The resulting preparation was nearly homogeneous and was devoid of DNA and RNA polymerase activities. The primase activity cofractionated with a Mr 65,000 polypeptide in sedimentation and chromatography procedures, and the native molecular weight of the enzyme corresponded closely to this value suggesting that the primase or an active proteolytic fragment of the protein exists as a monomer. Both heat-denatured calf thymus DNA and poly(dT) could be utilized by the enzyme as templates. Primase exhibited an absolute requirement for divalent cations and for rATP on a poly(dT) template. Although it required the ribonucleotide to initiate primer chains, the enzyme could incorporate the deoxynucleotide into primers. The product of the primase-catalyzed reaction was an oligonucleotide of discrete length (11-13 nucleotides), and oligonucleotides that were apparently dimers of this unit length were also observed. Primers that were synthesized were virtually identical in size in both the presence and absence of dATP incorporation. Although the bulk of DNA primase activity was isolated as a "free" enzyme, a portion of cellular primase activity co-chromatographed with DNA polymerase suggesting an association between these enzymes similar to that found in several higher eukaryotes.

Interactionin vitro of the neurofilament triplet proteins from porcine spinal cord with natural RNA and DNA

Neurofilaments were isolated from porcine spinal cord and separated into their subunit proteins (68 Kd NFP, 145 Kd NFP, 200 Kd NFP) by ion exchange chromatography on DEAE-cellulose in 6 M urea. The individual proteins were reacted with total rRNA from Ehrlich ascites tumor cells and the reaction products analysed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. All three proteins interacted with rRNA with a preference for 18S rRNA. Competition experiments with native and heat-denatured calf thymus DNA showed that the affinities of the 68 Kd and 145 Kd NFPs were considerably higher for denatured DNA than for rRNA and that native DNA was only a weak competitor. The binding of the 200 Kd NFP to rRNA was unaffected by native and by denatured DNA. When denatured DNA was reacted with a mixture of the 68 Kd and 145 Kd NFPs, the two proteins interacted independently with the nucleic acid, giving rise to two different populations of deoxyribonucleoprotein particles. This segregation is the result of the cooperative interaction of the neurofilament proteins with single-stranded DNA. It could not be observed with rRNA or bacteriophage MS2 RNA. The results clearly show that the 68 Kd and 145 Kd NFPs are single-stranded RNA- and DNA-binding proteins, whereas the 200 Kd NFP seems to be only a single-stranded RNA-binding protein.

Interaction of copper(II) ions with the daunomycin-calf thymus deoxyribonucleic acid complex.

The interaction of Cu(II) with the native and heat-denatured DNA complexes of daunomycin (1) and N-(trifluoroacetyl)daunomycin (3) has been examined by using absorption and circular dichroism spectroscopies. At low rt, where rt is the input molar ratio of drug to DNA phosphate, Cu(II) interacts with the native and heat-denatured calf thymus DNA complex of daunomycin to form a ternary complex involving the aglycon portion of the antibiotic, Cu(II), and DNA. A Job plot of the titration involving Cu(II) and heat-denatured DNA shows that the Cu(II)-drug stoichiometry in the ternary complex is less than or equal to 1. Although the N-(trifluoroacetyl)daunomycin-native DNA complex does not form a ternary complex, the denatured DNA complex with the antibiotic does. Copper(II) titrations of the daunomycin-native DNA complex, at high rt, where both strongly and weakly bound antibiotic molecules are very likely present in solution result in the formation of both the ternary species as well as a binary complex involving only the metal ion and the antibiotic. The spectroscopic results indicate that in the ternary complex, the Cu(II) ion is bound to the unintercalated aglycon portion of the antibiotic and very likely also to the heterocyclic bases of DNA.

DNase A, a poly(dA) and poly(dT)-specific deoxyribonuclease from Achatina fulica. Further investigation on the specificity.

The degradation of heat-denatured and native calf thymus DNAs, poly(dA-dT), poly(dA-dC) . poly(dG-dT), and poly(dG-dC) by DNase A has been investigated with the main aim of providing background information for studying the specificity of the enzyme. The specificity of the DNase A was studied by determining the base compositions of 5'- and 3'-terminal nucleotides of oligonucleotides released by the enzyme. The 5'- and 3'-terminal nucleotide compositions were found to vary in the average chain length (Pn) range of 45 to 7 for degradations of heat-denatured calf thymus DNA at pH 4.0, 4.5, 5.0, and 5.5. When the heat-denatured DNA was digested at the optimal pH (pH 5.0), at the Pn = 45 dAdo was predominant (39%) and dThd was minor (9%) in 5'-terminals, whereas dThd was predominant (43%) and dAdo was minor (9%) in 3'-terminals. At Pn = 7, dAdo was even more predominant (49%) and there was very little dThd (7%) in 5'-terminals. No preference was seen in 3'-terminals. This finding indicates that change in the specificity takes place during digestion. The compositions of 5'- and 3'-terminal nucleotides of oligonucleotides released from poly(dA-dT) by exhaustive digestion of the enzyme showed pronounced preferences for dAdo in 5'-terminals (81%) and dThd in 3'-terminals (78%). The hexamer or above deoxyadenylate and the nonamer or above of thymidylate were good substrates of the enzyme. It can be concluded that DNase A is a novel cluster-specific DNase which recognizes and cleaves sequences of oligodeoxyadenylate and/or sequences of oligothymidylate.

Partial purification and characterization of poly(dC)-dependent DNA polymerase and its stimulating factor

Poly(dC)-dependent dGMP incorporating activity without a primer molecule and its stimulating factor were partially purified by successive column chromatographies. Polymerase activity that was highly dependent on the stimulating factor was separated from similar activity that was not stimulated by this factor by native DNA-cellulose column chromatography. The factor stimulating activity was strictly dependent on dGTP as substrate and incorporated dGMP into the 3′-OH terminus of poly(dC). However, no terminal deoxynucleotidyl transferase (EC 2.7.7.31) activity was detected in the preparation. The activity also responded to heat-denatured calf thymus DNA and poly(dT) as template, although to a lesser extent. The activity was inhibited by dideoxyGTP and N-ethylmaleimide, and was decreased significantly by aphidicolin and β-lapachon.

Three forms of DNA polymerase from Drosophila melanogaster embryos. Purification and properties.

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.

Studies on sheep kidney nuclease: I. An improved purification method and some properties

An improved purification method of the sheep kidney nuclease (nuclease SK) specific for single-strand nucleic acid, which includes extraction with 0.85% NaCl, treatment with DEAE-cellulose, fractionation with polyethylene glycol, phospho-cellulose chromatography, CM-Sephadex chromatography and phospho-cellulose rechromatography is described. The nuclease was purified approx. 390-fold. Identity was established by comparison with known properties. Molecular weight was estimated to be 52 000–53 000 by gel filtration on Sephadex G-100. The properties of the purified enzyme agreed well those reported previously. The purified enzyme hydrolyzed heat-denatured calf thymus DNA, yeast RNA and no hydrolytic activity for native calf thymus DNA, A 2′-pA, A 3′-pA, ADP, ATP, 5′-AMP and cyclic AMP.

RNA polymerase activities in liver and brain tissue of aging mice.

RNA polymerase activity has been measured in liver and brain of C57BL-6J mice to determine if a change in enzyme activity can be correlated with decreasing survivorship of the animals. The RNA polymerases in tissue homogenates were solubilized by treatment with a buffer of high ionic strength and resolved by DEAE-Sephadex chromatography. Enzyme activity was quantitated by measuring the incorporation of [3H]UMP into RNA using heat denatured calf thymus DNA as the template. Statistically significant differences in polymerase activities were not observed in liver tissue from 18-, 25-, and 29-month-old animals or in brain tissue from 23- to 31-month-old animals. These age groups span the period of most rapid decrease in survivors,ip in our colony of mice (from 93% to 16%). The evidence indicates that changes in liver or brain RNA polymerase activities are not correlated with the rapid decrease in survivorship of these animals.

RNA polymerase activities in heart tissue of aging mice

RNA polymerase activities have been measured in heart tissue of C57B1/6J mice to determine if any change can be correlated with decreasing survivorship of the animals. The RNA polymerases were solubilized by homogenization of the tissue in a buffer of high ionic strength. The multiple enzymes were resolved by differential elution from DEAE-Sephadex. Enzyme activity in the column fractions was quantitated by measuring the incorporation of [ 3H]UMP into RNA using heat denatured calf thymus DNA as the template. The data were normalized to tissue DNA levels as measured by the diphenylamine procedure. Statistically significant decrease in polymerase I and polymerase II activities were observed in animals ranging in age from 21 to 30 months. Polymerase III levels in these animals did not significantly differ. Since these age groups span the period of most rapid decline in survivorship in the colony of mice (from 97% to 25%), it suggests that the decreases in heart RNA polymerase activities may be related to the decreasing survivorship. Heart tissue from 30-month old animals that had experienced stress was also studied. In these animals the polymerase I activity increased, the polymerase II activity decreased, but the polymerase III activity remained constant when compared to non-stressed animals of the same age. The results demonstrate that these old animals are capable of increasing polymerase I activity in response to physiological stimuli. Collectively the data suggest that the level of each polymerase is regulated independently.

Chromatography of different forms of DNA on immobilized histone columns

Histones 1, 2A, 2B, 3, 4, and 5 from chicken erythrocytes have each been covalently immobilized on agarose beads and tested for their ability to bind DNA. Both calf thymus DNA and PM-2 viral DNA (form II, a nicked circle) bind to columns of immobilized histone at low ionic strength and can be eluted with 0.2–0.8 M NaCl, depending on the histone column. The strength of DNA binding by histone columns is in the order H5 > H1 > > H2 B ≈ H3 > H4 ≈ H2 A . About 70% of a heat-denatured calf thymus DNA sample and 100% of a supercoiled (form I) PM-2 DNA sample bind very tightly to histone columns and are released only by strong protein denaturants.

Templates for eukaryotic RNA polymerase II: Artefacts can produce an apparent preference for denatured DNA over native DNA

Three artefacts have been identified which can reverse the preference of wheat germ RNA Polymerase II for native over heat-denatured calf thymus DNA. Self-absorption during liquid scintillation counting, contamination of DNA with RNase, and the use of UTP-limiting substrate containing less UTP than ATP, CTP, or GTP all decrease measured enzyme activity on native DNA. Eliminating these effects causes a 20-fold increase in the ratio of synthesis on native DNA to synthesis on heat-denatured DNA.

Ribonucleases from Porcine Brain. Partial Purification and Properties1

1. An acid ribonuclease was partially purified from an acetone powder of porcine brain. This enzyme was an acidic protein with a molecular weight of aroung 70,000. It acted on yeast RNA optimally at about pH 5.9, yielding only a mixture of 3'-mononucleotides, and therefore appears to be an exonuclease. It did not hydrolyze heat-denatured calf thymus DNA or bis(rho-nitrophenyl) phosphate. It was fairly unstable to heat and acid. 2. An alkaline ribonuclease was partially purified from the same source simultaneously. This enzyme was a basic protein with a molecular weight of 25,000-26,000. It was a pyrimidine-specific endoribonuclease, and acted on yeast RNA optimmally at around pH 7.4. It did not hydrolyze heat-denatured calf thymus DNA or bis(rho-nitrophenyl) phosphate. It was fairly stable to heat and acid.

Reaction of cytidine with semicarbazide in the presence of bisulfite. A rapid modification specific for single-stranded polynucleotide.

Semicarbazide reacted rapidly with 5,6-dihydrocytidine-6-sulfonate, which was formed from cytidine by addition of bisulfite across the 5,6-double bond. The transaminated product, 5,6-dihydro-4-semicarbazido-2-ketotopyrimidine-6-sulfonate ribofuranoside, was identified by comparison with that formed by treatment of 4-semicarbazido-2-ketopyrimidine ribofuranoside with bisulfite. The progress of the transamination was monitored spectrophotometrically by use of a strong absorbance of the product in alkali. The reaction between cytidine and the semicarbazide-bisulfite mixture was optimal at pH 4.5. Complete transformation of cytidine into the product required only 5 min with the use of 3M semicarbazide-1M sodium bisulfite, pH 5.0, at the reaction temperature 37 degrees C. The product was stable in unbuffered solution but in phosphate buffers it underwent elimination of bisulfite to give 4-semicarbazido-2-ketopyrimidine ribofuranoside. The rate of the elimination at pH 7.0 and 37 degrees C increased proportionally with the increase of the phosphate concentration. Complete elimination was obtained by treatment with 1 M sodium phosphate for 2 h. When heat-denatured calf-thymus DNA was treated with 3 M semicarbazide-1 M bisulfite at 37 degrees C and pH 5.0 the transamination of reactive cytosine residues was completed by 10 min of incubation. At 20 degrees C, it required 85 min of incubation. Cytosine residues in native DNA did not react at all even by prolonged incubations. The modified DNA samples were further treated with a phosphate buffer at pH 7, producing 4-semicarbazido-2-ketopyrimidine residues in the DNA. Analysis of the base compositions of these samples by perchloric acid hydrolysis showed that the modification was selective to cytosine, which had been expected from studies with monomers. It also showed that the reactive cytosine residues in the denatured DNA, constitute about 80% of the total cytosine, which was consistent with the view that heat-denatured DNA still contains a considerable amount of secondary structure. The semicarbazide-bisulfite modification is expected to be a sensitive method to locate cytosine residues in single-stranded regions of polynucleotides.

The effect of bisulfite modification on the template activity of DNA for DNA polymerase I.

Cytosine residues of poly(C) and heat-denatured calf thymus DNA were transformed into 5,6-dihydrouracil-6-sulfonate (U(SO(-) (3))) residues by treatment with bisulfite. The poly(U(SO(-) (3))(2), C(3)) and poly(U(SO(-) (3))(9), C(1)) prepared did not form inter-base binding with either poly(A) or poly(I) as judged by the absence of hypochromicity in ultraviolet absorbance. U(SO(-) (3)) residues in the DNA inactivated it to serve as template for E.coli DNA polymerase I, while the template activity was restored by conversion of the U(SO(-) (3)) residues into U.

Effect of different sizes of polyacrylic acid on the structure and DNA template properties of isolated rat liver nuclei and soluble chromatin

1. 1. Polyacrylic acid is capable of releasing the DNA template restrictions on isolated rat liver nuclei or soluble chromatin when assayed with either DNA polymerase or RNA polymerase from Escherichia coli B. 2. 2. The ability of polyacrylic acids to release these template restrictions is dependent on their size. Polyacrylic acids of molecular weight 20 400 and 4680 were very effective; whereas polyacrylic acid with a molecular weight of 1260 was completely ineffective. 3. 3. The similarity of the effect of polyacrylic acid in releasing the template restrictions in both isolated nuclei and soluble chromatin indicates that this characteristic is not determined by the ability of the polymer to pass through the nuclear membrane. 4. 4. All sizes of polyacrylic acid were able to precipitate purified unfractionated calf thymus histones; however, the smallest polymer was nearly twice as effective as the larger ones (60 μg histone precipitated by 100 μg polyacrylic acid, mol. wt = 1260 ). 5. 5. The larger sizes of polyacrylic acid, but not the smallest size, caused an increase in nuclear volume and an optical clearing of isolated nuclei when examined at 600 nm. This phenomenon was accompanied by a release of DNA from the nuclei. 6. 6. The two large sizes of polyacrylic acid were able to protect high molecular weight DNA in nuclei from degradation when nuclear suspensions were incubated at 37 °C in the presence of 2.5 mM Mg 2+. 7. 7. DNA template characteristics of purified, heat-denatured calf thymus DNA could be completely inhibited by the addition of isolated calf thymus histones. The two larger sizes of polyacrylic acid were specifically able to reactivate the DNA template properties of these histone-inhibited DNA preparations.

Studies on the Autolysis of Aspergillus oryzae Part XI

Crystalline nuclease O obtained from autolyzed Aspergillus oryzae hydrolyzed heatdenatured calf thymus DNA 19 times faster than native DNA. Digestion of the heatdenatured DNA with an exess of the enzyme produced mono-, di- and tri-nucleotides with 5'-terminal phosphate, which amounted 3.4, 58.3 and 38.2%, respectively, of total degradation products. Hydrolysis of the native DNA with a sufficient amount of nuclease O produced mono-, di-, tri- and tetra-nucleotides with 5'-terminal phosphate, which amounted 1.9, 47.9, 36.7 and 13.6%, respectively, of total degradation products. Although nuclease O showed no strict base specificity on the native and heat-denatured DNA, diand tri-nucleotides in the digests were resistant to further hydrolysis by nuclease O. Native DNA was hydrolyzed by nuclease O through the mechanism of single strand break, which was shown by neutral and alkaline sucrose density gradient centrifugations.

Reaction of antinucleoside antibodies with human cells in vitro

Antibodies to purine nucleosides were induced in rabbits by immunization with nucleoside-protein conjugates. These antibodies were reactive with homologous conjugates and heat-denatured calf thymus DNA. Fixed human fibroblasts, stained with fluorescein-labeled antibodies, showed nuclear attachment of the antibody only during the S period, i.e., when DNA replication was occurring. This finding was established by a correlated study using 3H-TdR uptake and autoradiography. Antibody did not attach to metaphase chromosomes unless they were denatured by heat or alkali. Since the antibody does not attach to native DNA, it is concluded that human fibroblast chromosomal DNA is at least partially single stranded in the S phase of the cell cycle and at no other time. Fixation for longer than 5 min prevented antibody attachment.

Dynamic aspects of native DNA structure: Kinetics of the formaldehyde reaction with calf thymus DNA

In this paper we outline the use of formaldehyde as a chemical probe to study the dynamic structure of native calf thymus DNA at temperatures well below the melting transition zone. It is demonstrated that the over-all reaction can be formulated as an equilibrium conformational “opening” step, followed by a slow chemical reaction of formaldehyde with nucleotide amino-groups normally involved in interchain hydrogen bonding. The kinetics of the latter reaction are calibrated as a function of temperature, formaldehyde concentration, pH and NaClO 4 concentration, using heat-denatured calf thymus DNA (after correction for residual base-pairing) as a model for the “transiently-open” regions in native DNA. The initial rate of formylation of native DNA is found to be equal to the product of K conf and the initial rate of formylation of denatured DNA under the same conditions, where K conf represents the fraction of the total base-pairs open (and unstacked) under the particular experimental conditions. Thus, we obtain a direct measure of this parameter. By studying the temperature dependence of K conf, we show that the enthalpy of the co-operative opening process which precedes the formation of the formaldehyde adduct ( ΔH conf) is temperature independent and corresponds to a co-operative opening unit of 9 ±1 base-pairs over a temperature range from just below T m to temperatures at least 30 deg. C lower. Increasing the concentration of the helix destablizing agent, sodium perchlorate, markedly diminishes the temperature dependence of K conf, and thus also the magnitude of ( ΔH conf. A number of lines of evidence are invoked to show that this decrease in ΔH confis not associated with a diminution in the size of the co-operative opening unit, but rather with a decrease in the enthalpy of melting per base-pair. Finally, we consider the possible biological significance of transiently open segments of this length in otherwise native DNA.