Thymus Nucleohistone Research Articles

Effect of nickel(II) and tetraglycine on hydroxylation of the guanine moiety in 2′-deoxyguanosine, DNA, and nucleohistone by hydrogen peroxide

The purpose of this study was to determine whether the Ni(II)-tetraglycine complex system (NiG 4) that is known to disproportionate H 2O 2 at pH ≥ 8 can catalyze oxidation of the guanine residues in 2′-deoxyguanosine (dG), calf thymus DNA, and calf thymus nucleohistone (NH) by H 2O 2 at physiological pH. Incubation of dG with H 2O 2 in the presence of NiG 4 at 37°C, produced two effects: (a) formation of 8-hydroxy-2′-deoxyguanosine (8-OH-dG) and (b) decomposition of dG to 2,6-diamino-4-hydroxy-5-formamidopyrimidine and several low molecular weight unidentified products. The magnitude of both effects depended on incubation time (1–48h), H 2O 2 concentration (7.5–40 mM), NiG 4 concentration (0.1 or 1 mM), and pH (6.0–8.0). The effects were not detected below pH 6 and above pH 8.0. For 0.1 mM NiG 4 and 7.5 mM H 2O 2, production of 8-OH-dG from dG (0.75 mM) during 24 h at 37°C was significantly lower than from NH (1 mg/ml) or DNA (0.5 mg/ml), indicating possible specific effects that might be related to the strength of interaction of NiG 4 with dG, NH, or DNA. The results indicate production of hydroxyl radical or other oxidizing species in the reaction of H 2O 2 with NiG 4 at pH 7–8. Reactions like this may be relevant to the mechanisms of Ni(II)-mediated oxidative damage, observed in vitro and in vivo, which may contribute to the toxic and carcinogenic effects of this metal.

Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone

Hydroxyl radical induced formation of a DNA-protein cross-link involving cytosine and tyrosine in nucleohistone in buffered aqueous solution is reported. The technique of gas chromatography-mass spectrometry was used for this investigation. A gamma-irradiated aqueous mixture of cytosine and tyrosine was first investigated in order to obtain gas chromatographic-mass spectrometric properties of possible cytosine-tyrosine cross-links. One cross-link was observed, and its structure was identified as the product from the formation of a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. With the use of gas chromatography-mass spectrometry with selected-ion monitoring, this cytosine-tyrosine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone gamma-irradiated in N2O-saturated aqueous solution. The yield of this DNA-protein cross-link in nucleohistone was found to be a linear function of the radiation dose in the range of 100-500 Gy (J.kg-1). This yield amounted to 0.05 nmol.J-1. Mechanisms underlying the formation of the cytosine-tyrosine cross-link in nucleohistone were proposed to involve radical-radical and/or radical addition reactions of hydroxyl adduct radicals of cytosine and tyrosine moieties, forming a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. When oxygen was present in irradiated solutions, no cytosine-tyrosine cross-links were observed.

Structure of hydroxyl radical-induced DNA-protein crosslinks in calf thymus nucleohistone in vitro.

Hydroxyl radicals are known to produce DNA-protein crosslinks in chromatin in vivo and in vitro. Here we investigated DNA-protein crosslinks formed between aliphatic amino acids and thymine in calf thymus nucleohistone exposed predominantly to hydroxyl radicals in gamma-irradiated N2O-saturated aqueous solution. Aliphatic amino acids are the predominant types of amino acids in the core histones of calf thymus, and thus are likely to form crosslinks with DNA. For identification of the crosslinks we first investigated hydroxyl radical-induced crosslinking of thymine to aliphatic amino acids in model systems, i.e. an aqueous mixture of thymine and a single amino acid. Samples were analyzed for possible thymine-amino acid crosslinks by gas chromatography-mass spectrometry. Using this approach the structure of the crosslinks was elucidated, and information on their gas chromatographic and mass spectral properties was obtained. Gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS/SIM) was then used to identify DNA-protein crosslinks in acidic hydrolysates of calf thymus nucleohistone exposed to ionizing radiation in buffered aqueous solution. DNA-protein crosslinks involving thymine and the amino acids Gly, Ala, Val, Leu, Ile and Thr were identified. In some cases several isomers of the same crosslink were observed. The yield of the crosslinks was measured by GC-MS/SIM and was found to be a linear function of radiation dose in the range 49 to 436 Gy. The mechanism for the formation of these DNA-protein crosslinks is thought to involve hydrogen atom abstraction by hydroxyl radicals from the aliphatic amino acid followed by addition of the amino acid radical to the carbon(6)-position of thymine and subsequent oxidation of the adduct radical.

The use of fluorescence correlation spectroscopy to probe chromatin in the cell nucleus

All systems in thermodynamic equilibrium are subject to spontaneous fluctuations from equilibrium. For very small systems, the fluctuations can be made apparent, and can be used to study the behavior of the system without introducing any external perturbations. The mean squared amplitude of these fluctuations contains information about the absolute size of the system. The characteristic time of the fluctuation autocorrelation function contains kinetic information. In the experiments reported here, these concepts are applied to the binding equilibrium between ethidium bromide and DNA, a system where the fluorescence properties of the dye greatly enhance the effect of spontaneous fluctuations in the binding equilibrium. Preliminary experiments employ well-characterized DNA preparations, including calf thymus DNA, SV40 DNA, and calf thymus nucleohistone particles. Additional measurements are described which have been made in small regions of individual nuclei, isolated from green monkey kidney cells, observing as few as 5000 dye molecules. The data indicate that the strength of dye binding increases in nuclei isolated from cells which have been stimulated to enter the cell growth cycle. The viscosity of nuclear material is inferred to be between one and two orders of magnitude greater than that of water, and it decreases as the cells leave the resting state and enter the cell growth cycle. Washing the nuclei also lowers the viscosity. These experiments demonstrate that fluorescence correlation spectroscopy can provide information at the subnuclear level that is otherwise unavailable.

Reaction of Formaldehyde with Calf-Thymus Nucleohistone

The reactions of formaldehyde with calf thymus nucleohistone were analyzed in the following ways: measurement with fluorescamine of the decrease in primary amino groups resulting from hydroxymethylation and crosslinking reactions, measurement with dodecylsulphate-gel electrophoresis of formation of histone oligomers, measurement of fixation of histones to the DNA in nucleohistone, and measurement of changes in the circular dichroism spectrum in the region of 250--300 nm. In the presence of formaldehyde, the primary amino groups of histones decreased very rapidly, attaining an equilibrium within 60 min, and successively intermolecular crosslinks were also formed between histone molecules, the resulting dimers and oligomers being separable by dodecylsulfate-gel electrophoresis. Whereas the fixation reaction proceeded much more slowly. The extent of fixation could be measured more accurately by dodecylsulfate/sucrose centrifugation analysis than by sulfuric acid extraction. After removal of formaldehyde from the reaction mixture, the fraction of masked amino groups decreased, perhaps due to the reverse reaction, but the extent of fixation of histones continued to increase with time. No specificity was observed among five molecular species of histones in the fixation reaction. With increase in formaldehyde concentration, the ellipticity of nucleohistone decreased to a minimum with about 0.4% formaldehyde, and then increased.

Distribution of histone F1 on calf thymus nucleohistone DNA

A method of large-scale preparation of the histone F1-DNA complex by removing all other proteins from calf thymus nucleohistone was established. This involved gel filtration of nucleohistone through a column containing a band of sodium dodecyl sulfate. The F1-DNA complex obtained had the original amount of F1 and no other. The F1-DNA complex exhibited distinct two-step melting on thermal denaturation. The first step was apparently attributable to naked DNA regions and the second step, about 30 deg. C higher than the first step, to the regions covered with F1. Buoyant density experiments with the complex after fixation with formalin revealed that F1 was distributed fairly evenly over DNA fragments of an average molecular weight of about 4 × 10 6. Electron microscopic examination of the complex after various degrees of denaturation with formalin indicated that the longest stretch of unbound DNA was about 0·3 μm.

The action of staphylococcal nuclease (EC-number 3. 1. 4. 7.) on thymus nucleohistone (TNH) and on some nucleoprotamines.

The biphasic nature of the time course of the action of staphylococcal nuclease on thymus nucleohistone was confirmed by studying the hydrolysis of this nucleoprotein at various enzyme concentrations. The transition from the rapid first to the sluggish second phase of the time course was particularly distinct at the highest enzyme concentrations. The rapid initial phase of the hydrolysis curve leveled off sharply when between 60 and 65 per cent of the total TNH phosphorus had been converted to acid-soluble phosphorus compounds. The insoluble complexes of TNH with protamines were found to be very resistant against the action of staphylococcal nuclease. The time course of the action of staphylococcal nuclease on a commercial nucleoprotamine of salmon testicles was found to become very sluggish when between 35 and 40 per cent of its total phosphorus had been converted to acid-soluble phosphorus compounds. When nucleoprotamines prepared in the laboratory from the secreted sperm cell suspension of Brown Brook Trout were digested with staphylococcal nuclease, only between 15 and 20 per cent of the total phosphorus were cleaved to acid-soluble phosphorus compounds during the rapid phase of the nuclease action. The respective values for the phosphorus fractions available for magnesium-binding and those susceptible to the rapid cleavage by staphylococcal nuclease were found to be very similar.

Similarity of chromatin from different tissues.

Abstract DNA-protein complex was extracted from a variety of sources and the accessibility of the phosphate groups of the DNA determined by the addition of polylysine; excess unbound polylysine was estimated by its reaction with methyl orange. In chromatin from the livers of normal rats and from rats treated with dimethylnitrosamine, in calf thymus nucleohistone and in avian erythrocyte chromatin, 42 to 47% of the DNA phosphate groups are available for binding polylysine, whereas about 23% of those in DNA-protamine from fish sperm are accessible; however, in the latter case, the free DNA phosphate groups occur only in very small stretches. The binding of polylysine to the chromatin protein carboxyl groups was shown to be negligible. On addition of DNase I to hen erythrocyte chromatin, oligonucleotides and protein are released concurrently, as found previously for rat thymus chromatin (Itzhaki, 1971 b ). This suggests that in both types of chromatin the DNA is associated extensively with protein, though not fully neutralized. It is concluded that chromatin extracted from very diverse sources is structurally similar, despite large differences in non-histone protein content.

Polylysine binding to histone-bound regions in chromatin

Thermal denaturation of calf thymus nucleohistone, polylysine-nucleohistone and polylysine-pure DNA were studied. In calf thymus nucleohistone polylysine binds preferentially to the DNA regions originally bound by non-histone proteins or the gaps between adjacent histone-bound regions and regions covered by less basic half-molecules of histones. Polylysine binds to chromatin with only one half efficiency when compared to polylysine binding to pure DNA. Using polylysine titration to determine the fraction of free DNA regions in chromatin is questionable.

The action of pancreas deoxyribonuclease I (deoxyribonucleate oligonucleotidohydrolase, EC-number 3.1.4.5.) on calf thymus nucleohistone

The DNA-components of thymus nucleohistone 2 (TNH) are hydrolyzed by pancreas deoxyribonuclease I in the presence of Mg 2+-ions to mixtures of acid-soluble oligonucleotides and of cleavage nucleoproteins (c-NPs) 2 2 Abbreviations. Thymus nucleohistone, TNH; cleavage nucleoproteins, c-NPs; deoxyribonuclease, DNase. which contain the total amount of the acid-insoluble portion of the hydrolyzed DNA-components and almost the total amounts of the substrate proteins. The kinetics of this hydrolysis is characterized by a rapid initial phase up to hydrolysis degrees 3 3 The term “degree of hydrolysis,” as used in this investigation for the extent of the enzymatic cleavage of DNA-chains into smaller fragments, is defined as the ratio acid-soluble P formed: total P of the substrate. of approximately 50% which is followed by a much slower continuation of the enzymatic hydrolysis. During this phase, the c-NPs precipitate almost quantitatively. In contrast to the anionic nature of the substrate-TNH, in which the cationic groups amount to only 57%, of the total anionic groups, the corresponding ratio of the insoluble c-NPs is stoichiometric. The decrease of the phosphoryl groups of the nucleoproteins during the incubation with DNase I is accompanied by corresponding decreases of their magnesium-binding capacities. The rapid initial and the sluggish second phase of the DNase I-catalyzed hydrolysis of TNH was observed not only with dissolved substrate-TNH, but also with suspensions of the insoluble complexes of TNH with stoichiometric amounts of Mg 2+-ions or of diamines of low molecular weights. This made it unlikely that the sluggishness of the hydrolysis during the second phase could be explained as a consequence of the coarse dispersion of the insoluble c-NPs. On the other hand, homogenized suspensions of the insoluble c-NPs or of insoluble, stoichiometric complexes of TNH with polylysines were hydrolyzed by DNase I at much slower initial rates than those observed with the TNH-magnesium or the TNH-diamine-complexes. This suggests that the initial rapid phase of the DNase I-catalyzed cleavage of TNH involves predominantly those segments of the substrate-DNA-chains that are not directly bound to the cationic amino acid residues of its histone components. The mixture of the acid-insoluble DNA-components of the c-NPs (they account for 100% of the total P of the soluble c-NPs, but only for 75–85% of the total P of the insoluble c-NPs) was sedimented in a sucrose density gradient. Only one peak was obtained which coincided with that of a reference sample of yeast tRNA. The sedimentation pattern of the DNA-component of the c-NPs did not significantly change between hydrolysis degrees of 17 and 60%, except for the fact that the yields of the acid-insoluble polynucleotide fraction of the c-NPs decreased appreciably during the later phases of the enzymatic digestion owing to the slow degradation of the c-NPs. With regard to the distribution of the histone components of the substrate-TNH along its DNA-chains, it seems that “histone-bound” 4 4 The terms “histone-free” or “histone-bound” are used in this investigation as abbreviated designations of those DNA-phosphoryl groups which are not directly bound to cationic amino acid residues of the histone components or vice versa. DNA-segments of mutually similar chain lengths alternate with “histone-free” 4 segments of unknown individual chain lengths. The resistance of insoluble TNH-complexes with spermidine and spermine, respectively, against the action of DNase I was found to be unexpectedly high in view of the low molecular weights of these compounds and of the small number of cationic groups per molecule. These observations suggest the important role of structural features such as the chain lengths of the alkyl bridges connecting the pairs of amine groups for the stability of the complexes of a cationic polyelectrolyte with TNH.

ALTERATION OF HISTONES FROM MOUSE EPIDERMAL CELLS AFTER INCUBATION WITH ELASTASE AND HYALURONIDASE

Chromatin was isolated from whole mouse skin, mouse epidermal cells and mouse liver by standard procedures used for isolation of chromatin from other mammalian tissues. Chromatin from whole mouse skin or from mouse epidermal cells had not been isolated or characterized earlier. For the preparation of chromatin from mouse epidermal cells, the latter was separated from dermis by incubation for 30 min at 37°C in a solution containing the enzymes elastase and hyaluronidase. The relative proportions of the chromatin components, the T m and the ultraviolet absorption spectrum were all similar to that of chromatin from whole mouse skin which was not treated with enzymes and to other mammalian chromatin preparations. Electrophoresis of the histones from epidermal chromatin in polyacrylamide gels revealed the absence of histones F1, F3 and F2a2 and the appearance of a new band. Histones isolated from chromatin prepared from the whole mouse skin had a gel electrophoresis pattern virtually identical with histones isolated from mouse liver chromatin and to reported histone patterns from other mammalian tissues. The alterations in mouse epidermal histones are similar to reported changes in histones from calf thymus nucleohistone previously subjected to incubation at various temperatures. The enzymatic incubation technique can therefore not be used as a method of isolating unaltered mouse epidermal chromatin. The findings illustrate that very subtle chemical alterations can be induced by usual methods of tissue preparation and that these changes can only be detected by highly sensitive analytical techniques.

Vergleichende Untersuchungen über hydratationsabhängige DNS-Sekundärstrukturänderungen bei isolierter DNS, isoliertem Nukleohiston und DNS in situ / Comparative studies on hydration dependent changes of DNA secondary structure of isolated DNA, isolated nucleohistone and DNA in situ

Hydration dependent changes of DNA conformation of calf thymus DNA, Locusta sperm DNA, calf thymus and Locusta sperm nucleohistone and Locusta DNA in situ were studied by means of linear dichroic measurements. A comparison of the results leads to the following conclusion. The changes of DNA conformation in situ as a function of the degree of hydration may depend on the species specifity of (a) both DNA and protein of the complex, (b) the conformation of the complex and (c) the interactions between DNA and protein.

Triplet-triplet energy transfer from histone to DNA in deoxyribonucleohistone

1. The phosphorescence spectrum of calf thymus nucleohistone was found to be greatly different from the spectrum expected from its constituents, histone and DNA. This difference is attributed to energy transfer from the tyrosine residue of histone to the DNA bases. 2. Comparing the fluorescence and phosphorescence of dissociated nucleohistone at high ionic strength with those of associated one, it is concluded that the observed energy transfer occurs via triplet state (triplet-triplet energy transfer). 3. Energy transfer was observed also for reconstituted and denatured nucleohistone, but not for dissociated nucleohistone or for the mixture of an amino acid (tyrosine) and DNA. The occurrence of energy transfer in the denatured nucleohistone indicates that histone remains to be associated with each single strand of DNA molecule under the condition of the thermal denaturation. 4. From emission spectra obtained, a possible explanation is given of the structure of nucleohistone.

The molecular structure of nucleohistone (DNH)

X-ray diffraction patterns obtained from fibre specimens of calf thymus nucleohistone (DNH) have been used in experiments to investigate the role of histone proteins in the supercoil configuration of the nucleohistone complex. Reconstruction of the complex from purified DNA and mixtures of histone types was done by dialysis from 2M NaCl to 0.25M NaCl before sedimenting. Other procedures for reconstruction are also described. A variety of methods have been used in attempts to obtain individual types of histone for reconstruction. Thus far the mixture of histones f2 a2 and f 3 is the simplest for which true reconstruction has been obtained. Molecular models have been used to investigate the secondary structure likely to be adopted by histone f2 a1 for which the amino acid sequence is known. A model for the attachment of this histone to DNA is described.

Further Studies of a Thymus Nucleohistone-associated Protease

Abstract The endogenous proteolytic degradation of the histones of calf thymus nucleohistone has been followed with polyacrylamide disc gel electrophoresis and dansylation of amino-terminal amino acids. The proteolytic enzyme is tightly associated with nucleohistone, is essentially inactive below pH 7.0, is decreased in activity at lower ionic strengths, shows a rather specific bond cleavage for those histone fractions which are susceptible to its attack, and is inactivated by brief heat treatment (2 min at 90°). The susceptibility of the five histone fractions to proteolysis is critically dependent upon whether the histones form a complex or not with DNA. In the intact nucleohistone three groups of histone are almost or totally resistant to proteolytic attack while the lysine-rich histone and the sulfhydryl-containing histone fractions are rapidly attacked. If histones are freed from DNA, only the lysine-rich fraction is resistant to the protease; all other fractions are rapidly degraded. In general those tissues having a high cell turnover, e.g. thymus and intestinal mucosa, exhibit a greater rate of proteolysis of nucleohistone than is observed for other tissue. The proteolytic activity is reversibly inhibited by either the presence of sodium bisulfite or by storing the nucleohistone at low ionic strength (∼5 x 10-4).

Template activity of partially dehistonized nucleohistones for DNA dependent RNA polymerase.

Histone was removed partially from calf thymus nucleohistone, and free phosphate groups not bound to histones on DNA were estimated by the toluidine blue method. The template activities of nucleohistones containing different amounts of histone for DNA dependent RNA polymerase [EC 2.7.7.6] from E. coli B (H) revealed the fact that the amount of RNA synthesized by RNA polymerase is proportional to the amount of free phosphate groups in the template but not to the amount of removed histone. The nearest neighbour frequencies of the RNAs formed with these partially dehistonized nucleohistones using [32P)ATP and the base compositions were closely similar to each other. It was therefore suggested that histones distribute evenly along the DNA chains in the nucleohistone. Furthermore, there was found no remarkable difference in the inhibitory effect between lysine-rich and arginine-rich histones.

The viscosity of nucleohistone in urea

A large increase in the intrinsic viscosity of calf thymus nucleohistone is observed in urea solutions between 1.0 and 3.5 M. This phenomenon is due neither to dissociation of the nucleoprotein complex into free DNA, nor to formation of aggregates. It is proposed that the increase in intrinsic viscosity is primarily due to transition from the supercoiled conformation of nucleohistone to a more extended rod-like conformation, although a contribution from an increase in hydration of nucleohistone is not excluded.

Proteolytic contamination of calf thymus nucleohistone and its inhibition

Proteolytic contamination of calf thymus nucleohistone and its inhibition

FLUORESCENT ANTIBODY STUDIES OF CERTAIN DERMATOSES.

The fluorescent antibody technique was used to investigate the possible role of autoimmune factors in certain dermatoses. Serum, sections of cutaneous lesions, and specimens of uninvolved skin were studied. Control procedures, sera, and tissues were involved in the investigation. Specific antigen-antibody reaction was not detected in the skin of patients who had one of the following conditions: psoriasis, erythema multiforme bullosum, pemphigus vulgaris, and dermatitis herpetiformis. Antibodies for calf thymus nucleohistone extract (NHE) were detected in serum of two patients who had both psoriasis and arthropathy and another patient who had had severe cutaneous manifestations of psoriasis for 17 years. The significance of the presence of the antibody was postulated. Non-species-specific antinuclear antibodies were detected in sera of patients who had discoid lupus erythematosus. Specific yellow-green fluorescence was observed when the immune substance reacted with human epidermal cell nuclei and calf thymus nucleohistone extract. The conceivable importance of autoimmune factors in the pathogenesis of discoid lupus erythematosus was discussed. Antinuclear antibody was demonstrated in the serum of patients who had scleroderma. Tissue specificity was not noted with the technique employed. The results of this study further support the concept that an autoimmune reaction may be a factor in the development of enlarging annular lesions (erythema annulare centrifugum). A correlation could not be made between the presence of serum antinuclear antibodies, as determined by the fluorescent antibody technique, and abnormal serum protein fractions detected by electrophoresis.

The Masked Lipids of Nuclei

ABSTRACT The histochemistry of masked lipids of chromosomes has been investigated with the aid of paper chromatographic procedures. At least three tightly bound associations of phospholipid with protein have been identified in calf thymus nucleohistone. Hence it seems likely that the ‘spurious’ reactions of nuclei and of nucleohistone, especially after extraction with lipid solvents, is a true indication of the increased availability of these closely linked phospholipids. Moreover, not more than about 10% of the material extracted by hot pyridine from calf thymus was fatty, the rest being watersoluble. After treatment of plant cells with hot trichloroacetic acid had produced increased staining with methods for demonstrating lipids, fatty matter could be extracted from the tissues. This demonstrates that the cells which gave the ‘spurious’ reaction did contain lipid. Hence it seems probable that extraction with solvents does not remove all lipids but may make those that remain more availabel for staining, so giving rise to what has been considered to be a spurious reaction. The nature of the binding of lipid to protein and the relevance of such complexes to histochemistry and the composition of nuclei are discussed.