Arginine-rich Histones Research Articles

A study of the interaction between ethidium bromide and rye chromatin: comparison with calf thymus chromatin

We studied the interaction of ethidium bromide with rye and calf thymus chromatin. Both types of chromatin have the same dye accessibility, which is about 50% of that of DNA. From this result we conclude that the molecular structure of these two chromatins is similar. For rye, the extraction of H1 produces no change in the binding of ethidium bromide. The subsequent extraction of H2A and H2B produces a 14% increase in the binding, and the removal of H3 and H4, another 54% increase. At this stage, the number of binding sites is still less than that of DNA. This is presumably due to the presence of some tightly bound non-histones. Thus, the arginine-rich histones and the tightly bound non-histones are most responsible for limiting the binding of ethidium bromide to rye chromatin.

Human leukaemic cells. Determination of trace elements in nucleic acids and histones by neutron-activation analyses.

Trace metals were measured by neutron-activation analyses in purified nucleic acids and histone(s) of lymphocytes from patients with acute lymphocytic leukaemia or infectious mononucleosis and from normal donor DNA isolated from lymphocytes of a patient with infectious mononucleosis and a normal donor showed a high a high content of Cr2+, Sb2+, Fe2+, and Zn2+, whereas DNA of lymphoblasts from a patient with acute lymphocytic leukaemia had a lower content of these trace metals, but the Co2+ content was 20-fold higher than in DNA or normal donor lymphocytic cells. Total histones from leukaemic cells had higher contents of most of the trace metals except for Zn2+, which was present in lesser concentration than in histones from normal donor lymphocytic cells. Lysine-rich (F1) histones showed lower contents of Cr2+, Sb2+ and Co2+, whereas arginine-rich (F3) histones had significantly higher contents of these trace metals. These observations may be of interest in that F3 histones more effectively inhibit RNA synthesis in human lymphocytic cells than do other species of histones.

Chromatin proteins from normal, vegetalized, and animalized sea urchin embryos

The chemical composition of the chromatin, the fractional content of histones and nonhistone chromatin proteins (NHP), and the biosynthesis of these proteins in normal, vegetalized, and animalized embryos of the sea urchin Strongylocentrotus droebachiensis at the blastula, mesenchyme blastula, and gastrula stages have been studied. The amount of the NHP in the chromatin from normal and vegetalized embryos increases during early embryonic development while that in animalized embryos remains without change at the mesenchyme blastula stage and then decreases. During development the histone content in all three cases slightly decreases. Polyacrylamide gel electrophoresis reveals that both fractional composition of histones and their biosynthesis in normal, vegetalized, and animalized embryos display no differences. During development, however, some changes occur, so that the relative amount of histones F1 and F2a2 increases, F2b decreases, while F3 and F2a1 remains constant. Histone F1 at the blastula stage consists of two subfractions while at the gastrula stage it consists of three subfractions. The histone F2a1 consists of one and two, respectively. Histone F3 at all stages is made up of three subfractions; histone F2b is made up of two; and the histone F2a2 is electrophoretically homogeneous. Specific radioactivity of the arginine-rich histones F3 and F2a1 tends to increase during development, while that of moderately lysine-rich histones F2b and F2a2 does not change, and that of the lysine-rich histone F1 decreases. The NHP in normal, vegetalized, and animalized embryos at different developmental stages consist of 17 fractions that can be separated by isoelectrofocusing within the 4.5–8.8 pH range. Quantitative changes have been observed in the fractions focused at pH 4.5–6.1 during development and in normal and modified embryos at the gastrula stage.

Effect of estrogen on gene expression in the chick oviduct. Studies on the initiation of RNA synthesis on chromatin in vitro.

Studies on the effect of the temperature requirement for RNA chain initiation by Escherichia coli RNA polymerase on DNA, chromatin, and reconstituted chromatin were carried out in order to better understand the nature of the initiation process. Varying the temperature or ionic strength during preincubations had little effect on the formation of stable preinitiation complexes (RS complexes) between RNA polymerase and chromatin. This observation was in marked ocntrast to similar studies performed on native DNA and indicates that initiation sites for RNA synthesis on chromatin are different from those on double-stranded DNA and resemble more closely initiation of RNA synthesis on single-stranded DNA. These results suggest that the local unwinding of the initiation region which is required for RNA chain initiation on native DNA may not be a prerequisite for RNA initiation on chromatin. The results of studies on reconstituted chromatin devoid of different classes of chromatin proteins demonstrate that both histone and nonhistone fractions are essential in maintaining the characteristics inherent to initiation of RNA synthesis on chromatin. Removal of either moderatley lysine-rich histone or arginine-rich histone fractions led to the complete loss of the characteristic "chromatin type" initiation pattern for RNA synthesis, whereas, removing lysine-rich (Fi) histone had no effect. Single strand specific and double strand specific antibodies to DNA and a single strand specific nuclease were used as probes to examine the structure of chromatin sites for initiation of RNA synthesis. Their effects on RNA synthesis suggest that initiation sites on chromatin are not located in freely accessible single-stranded or double-stranded regions of DNA.

The organization of histones and DNA in chromatin: Evidence for an arginine-rich histone kernel

We have examined the role played by various histones in the organization of the DNA of the nucleosome, using staphylococcal nuclease as a probe of DNA conformation. When this enzyme attacks chromatin, a series of fragments evenly spaced at 10 base pair intervals is generated, reflecting the histone-DNA interactions within the nucleosome structure. To determine what contribution the various histones make to DNA organization, we have studied the staphylococcal nuclease digestion patterns of complexes of DNA with purified histones. Virtually all possible combinations of homogeneous histones were reconstituted onto DNA. Exhaustive digestion of a complex containing the four histones H2A, H2B, H3, and H4 yields a DNA fragment pattern very similar to that of whole chromatin. The only other combinations of histones capable of inducing chromatin-like DNA organization are H2A/H2B/H4 and those mixtures containing both H3 and H4. From an examination of the kinetics of digestion of H3/H4 reconstitutes, we conclude that although the other histones have a role in DNA organization within the nucleosome, the arginine-rich histone pair, H3/H4, can organize DNA segments the length of the nucleosome core in the absence of all other histones.

Arginine-rich histone synthesis and acetylation in WI 38 cells stimulated to proliferate

Confluent WI 38 human diploid fibroblasts were stimulated to proliferate by a change of medium containing 10% fresh serum. Synthesis and acetylation of arginine-rich histones in contactinhibited cells (G 0 stage) and in cells 60 min after serum stimulation (G 1 stage) as well as in exponentially growing cells were investigated using a method for isolation of nuclei which has been demonstrated to result in optimal recovery of arginine-rich fractions. Pulse-labelling with [ 3H]arginine-[ 3H]lysine or [ 3H]acetate was combined with treatment with either actinomycin D or cycloheximide in order to investigate whether histone turnover and acetylation following serum stimulation are controlled at the transcriptional and/or at the translational level. Results revealed that the rate of 3H-labeled amino acid incorporation into arginine-rich histone fractions is higher in G 0 than in G 1 cells and is sensitive to both actinomycin D and cycloheximide. On the other hand histone acetylation proceeds at similar rates in both G 0 and G 1 cells und is not affected by either inhibitor. Differences in the extractability of arginine-rich histones with 0.25 M HCl are also reported; H 4 was found to be more resistant to HCl extraction than any other fraction in G 0, G 1 or exponential populations. However fraction H 3 is extracted more rapidly from G 0 cells. These differences are presumed to reflect cell-cycle dependent modifications in the binding of histone fractions in chromatin.

Lysyl hydroxylase. Further purification and characterization of the enzume from chick embryos and chick embryo cartilage

A purification of up to 4000-fold is reported for lysyl hydroxylase (EC 1.14.11.4) from extract of chick-embryo homogenate and one of about 300fold from extract of chick-embryo cartilage. Multiple forms of the enzyme were observed during purification from whole chick embryos. In gel filtration the elution positions of the two main forms corresponded to average molecular weights of about 580 000 and 220 000. These two forms could also be clearly separated in hydroxyapatite chromatography. In addition, some enzyme activity was always eluted between the two main peaks both in gel filtration and in hydroxyapatite chromatography. The presence of the two main forms was also observed when purifying enzyme from chick embryo cartilage. Both forms of the enzyme hydroxylated lysine in arginine-rich histone, which does not contain any -X-Lys-Gly- sequence. No difference was found between the enzyme from whole chick embryos and from chick embryo cartilage in this respect. Lysyl hydroxylase was found to have affinity for concanavalin A, indicating the presence of some carbohydrate residues in the enzyme molecule. Lysyl and prolyl hydroxylase activities increased when the chick embryo homogenate was assayed in the presence of lysolecithin. Preincubation of the homogenate either with lysolecithin or with Triton X-100 increased lysyl hydroxylase activity in the homogenate, and in the 15 000 × g and 150 000 × g supernatants, suggesting thai the increase in the enzyme activity was due to liberation of the enzyme from the membranes. Divalent cations were found to inhibit the activity of lysyl and prolyl hydroxylases in vitro. An inhibition of about 50% was achieved with 15 mM calcium, 60 μM copper and 3 μM zinc concentrations. The mode of inhibition was tested with Cu 2+, and was found to be competitive with Fe 2+.

Release of chymotrypsin-like esterase from isolated rat peritoneal mast cells by arginine-rich calf thymus histone.

Release of chymotrypsin-like esterase from isolated rat peritoneal mast cells by arginine-rich and lysine-rich calf thymus histones was investigated. The arginine-rich histone released the esterase, but the lysine-rich histone did not release the esterase from the mast cells. The isolated mast cells spontaneously released the esterase gradually. The chymotrypsin-like esterase released from the mast cells is considered to digest the destructed material in tissues in a state of chronic destruction, such as chronically inflamed tissues and malignant tumours.

Interactions between arginine-rich histones and deoxyribonucleic acids. II. Circular dichroism.

Circular dichroism (CD) was used to investigate the conformations of arginine-rich histones, H3 (III or f3) and H4 (IV or f2a1), and DNA in the complexes prepared by four different methods: (A) NaCl gradient dialysis with urea; (B) NaCl gradient dialysis without urea; (C) direct mixing in 2.5 x 10(-4) M EDTA, pH 8.0; and (D) direct mixing in 0.01 M sodium phosphate, pH 7.0. Using the CD spectrum of native chromatin as a criterion to judge the closeness of a complex to its native state, it was observed that a complex made by direct mixing at low ionic strength (methods C and D) is better than the ones made by NaCl gradient dialysis with or without urea (methods A and B). It is explained as a result of lack of ordered secondary structures in histones due to the presence of urea in method A or due to nonspecific aggregation in NaCl without urea (method B). Compared with all the earlier reports in literature on the CD of histone-DNA complexes, the CD spectra of arginine-rich histone-DNA complexes prepared by methods C and D are closest to that of native chromatin both in shape and in amplitude. These results imply (a) that arginine-rich histones play an important role in maintaining the conformation of chromatin and (b) that the binding of these two histones to DNA prepared by methods C and D are close to that in native chromatin. Noticeable variation in conformation of free and bound histone and histone-bound DNA has also been observed in histone H3 with one or two cysteine residues, and in reduced or oxidized state even when the complexes were prepared and examined in the same condition. CD spectra of arginine-rich histones in 0.01 M phosphates, pH 7.0, indicate the presence of alpha-helix which could be responsible for a favorable binding of the less basic regions of these histones to DNA under this condition as demonstrated by thermal denaturation (Yu, S. .S, Li H. J., and Shih, T. Y. (1976), Bio-chemistry, the preceding paper in this issue). To preserve or generate alpha-helical structures in histones seems to be a critical step in reconstituting good histone-DNA complexes.

Characterization of soluble nucleohistone and histone from cotyledons of germinating seeds of Pinus pinea

Soluble nucleohistone and histones have been isolated from cotyledons of Pinus pinea; this is the first such isolation from any gymnosperm tissue. The nucleohistone isolated was similar in composition and physical characteristics to that of chromatin reported for comparable angiosperm tissues at similar stages of growth. Histones, purified by gel electrophoresis, possessed eight subfractions which are tentatively identified as follows in order of increasing electrophoretic mobility: very lysine-rich F1 (fractions 1–4), arginine-rich F3 (fraction 5), arginine-rich F2a2 (fraction 6) and F2b (fraction 7), as well as moderately arginine-rich F2a1 (fraction 8). Nucleohistone from days 15 to 22 of germination, a period of cell elongation in cotyledons, possessed greater amounts of histone, acidic protein, and RNA relative to DNA than day-14 'mitotically active' nucleohistone. Histone acetylation from sodium acetate, methylation from s-adenosyl-L-methionine, and phosphorylation from inorganic phosphate proceeded at low rates in isolated nuclei. Phosphorylation from [32P]ATP occurred to a greater extent; very lysine-rich F1 and arginine-rich F3 histones were phosphorylated to the greatest degree while F2a1 was phosphorylated least.

Ultrastructural visualization of Cabot rings in pernicious anemia.

Using the ammoniacal silver stain, Cabot rings were identified in peripheral blood erythrocytes from patients with severe untreated pernicious anemia. Ultrastructural studies of these erythrocytes showed silver deposits in partial loops and figure-eight forms, indicating that arginine rich histone may be a prominent component of the Cabot ring.

Amino acid composition of sperm histones in the house cricket Acheta domesticus.

Histones were isolated from late spermatids and spermatozoa of the house cricket Acheta domesticus, and the individual histone fractions were separated by electrophoresis on polyacrylamide-urea gels. The stained gels were cut so as to isolate the different histone fractions, and the amino acid compositions were determined using the technique of Houston (Houston, L.L.: Anal. Biochem. 44, 81-88 (1971). Five of the histones had amino acid compositions resembling those for the histones of calf thymus and were thus identified as fractions F1, F3, F2a2, F2b, and F2al. Another protein (SH) located exclusively in the late spermatids and spermatozoa was found to be basic and histone-like. It is a protein containing relatively high amounts of arginine (12.6%) and low amounts of lysine (7.6%), and, as a result, it has a low ratio of lysine-arginine (0.6). Other noteworthy features are its high contents of serine, glutamic acid, and glycine. It is arginine rich histone and in this regard resembles other such proteins, but it does contain unique features which distinguish it from all previously described histones.

Brain histone kinase: The structure, the substrate specificity and the mechanism of action

Cyclic adenosine 3′,5′-monophosphate-dependent histone kinase (ATP protein phosphotransferase, EC 2.7.1.37) was obtained from pig brain. The enzyme was purified 1140 times. It was homogeneous on polyacrylamide gel with electrophoresis and gel filtration. The established molecular weight of the enzyme is 120,000. The histone kinase dissociates into catalytic and regulatory subunits (molecular weight 40,000 and 90,000, respectively). With chromatography on DEAE-cellulose in the presence of excess cyclic AMP the homogeneous catalytic subunit was obtained. Using polylysylsepharose, which contains immobilized 8-(γ-carboxypropylthio)-cyclic AMP with affinity chromatography the homogeneous regulatory subunit was isolated. The methods permit us to obtain also individual catalytic and regulatory subunits of the histone kinase from partially purified preparations of the enzyme. Subunits of the histone kinase were homogeneous with polyacrylamide gel electrophoresis in the presence of SDS. The histone kinase had a high specificity of action according to lysine-rich histones F 1, F 2a2 and F 2b. Arginine-rich histones and other known protein substrates of cyclic AMP-dependent protein kinases (casein, E. coli, RNA polymerase, etc.) were not phosphorylated by this enzyme. The kinetics of phosphorylation of histones F 1, F 2a2, F 2b was investigated and corresponding sites of phosphorylation were determined in primary structures of these histones: Ser-38 of histone F 1, Ser-19 of histone F 2a2, Ser-14 and Ser-36 of histone F 2b. Phosphorylated serine residues were localized in all cases in sequence X-Y-Ser, where X is always a lysine or arginine residue, Y — an acidic or neutral amino acid. Using various original ATP and cyclic AMP analogs containing reactive groups, the functional topography of the active sites of catalytic and regulatory subunits active sites was investigated. Specific regions of ATP binding in the active centre of the catalytic subunit were determined and the imidazole ring of histidine was suggested as the catalytic group participating in the phosphotransferase reaction. The existence of at least three parts in the regulatory centre of histone kinase was suggested in which binding of a negatively charged hydroxyl group of the cyclophosphate system, the nucleophilic locus for binding of 2′-hydroxyl group of the ribose ring and the proton-donor group, which may take part in the interaction with the exo-amino group of the adenine base occurs. On this basis, the contribution of separate groups of molecule of the cyclic nucleotide to the enzyme activation was studied. The investigation of the primary structure of different subfractions of F 1 histone, the chemical modification reaction of F 1 histone and PMR spectra of native and phosphorylated Ser-38 molecules of F 1 histone in the complex with DNA permitted us to suggest that during the F 1 histone interaction with DNA the role of two cationic loci (15–36) and (107–212) of the F 1 histone molecule is determined by the realization of typical electrostatic contacts. The ionic interaction of one of the cationic loci, probably the cationic part, (15–36) is destroyed by the incorporation of phosphate group on Ser-38 of F 1 histone molecule. The phosphorylation on Ser-38 F 1 histone leads to the disorganization of the secondary structure of the central segment and leads to its more free movement in the complex with the polyanion template. PMR data demonstrated that the phosphorylation on Ser-38 residue of F 1 histone led to the weakening of the histone-DNA binding and influenced the secondary structure and protein-protein interactions in the nucleoproteid complex. Using the cytochemical approach the influence of the histone kinase on physico-chemical properties of chromatin was investigated. The hepatocyte treatment by the histone kinase led to the increase of acridine orange binding. The increase of acridine orange binding to DNP was the same both at 2.5 hr after the operation and after the histone kinase treatment of hepatocytes without operation: i.e., the cell treatment by the histone kinase had the same effect as partial hepatectomy. Thus, the phosphorylation of lysine-rich histones by the exogenous histone kinase led to the appearance of activated parts of genome and the stimulation of the transcription process.

Regulation of ribonucleic acid synthesis by polyamines. Reversal by spermine of inhibition by methylglyoxal bis(guanylhydrazone) of ribonucleic acid synthesis and histone acetylation in rabbit heart.

The relationship between polyamines and RNA synthesis was studied by considering the action of spermine on histone acetylation in perfused heart. In addition, the effect of methylglyoxal bis(guanylhydrazone), inhibitor of putrescine-activated S-adenosylmethionine decarboxylase activity, on RNA and polyamine specific radioactivity and on acetylation of histone fractions was also investigated in perfused heart. Different concentrations of spermine and/or methylglyoxas bis(guanylhydrazone) were injected into the heart, 15 min after beginning the perfusion. The results demonstrate that spermine stimulates the specific radioactivity of RNA of subcellular fractions. Acetylation of the arginine-rich histone fractions, involved in the regulation of RNA transcription, is enhanced by spermine. The perfusion with methylglyoxal bis(guanylhydrazone) causes a decrease in the specific radioactivity of polyamines and RNA, and in acetylation of histone fractions. However, spermine is able to reverse the methylglyoxal bis(guanylhydrazone) inhibition when injected simultaneously. From these results we may assume a possible role for spermine in the regulation of RNA transcription.

A very rapidly migrating f1 histone associated with gene-sized pieces of DNA in the macronucleus of Oxytricha sp.

Histones bound to gene-sized pieces of DNA in the macronucleus of Oxytricha sp. consist of five main fractions: f1, f2a1, f2a2, f2b and f3. Although the slightly lysine-rich and the arginine-rich histones in Oxytricha macronuclei are similar to comparable fractions in vertebrates, the lysine-rich f1 fraction differs. Oxytricha f1 is unique among eukaryotes in that it migrates faster than f2a1 and is located nearest the cathode in polyacrylamideurea gels.

Interactions of C-reactive protein with the complement system. II. C-reactive protein-mediated consumption of complement by poly-L-lysine polymers and other polycations.

Cationic homopolymers of poly-L-lysine were found to activate complement (C) via C-reactive protein (CRP) and deplete C3 and C5 as well as early-acting C components. Maximum C consumption was obtained with polymers of 2,000-8,000 daltons; polymers of 1,700, 11,000, and 23,000 daltons were intermediate in reactivity, while L-lysine, lysyl-L-lysine, tetra-L-lysine, and polymers of 70,000-400,000 daltons lacked significant C-consuming activity. Naturally occurring polycations which consumed C in the presence of CRP included myelin basic proteins, cationic proteins of rabbit leukocytes, and both lysine- and arginine-rich histones; poly-L-arginine polymers of 17,000 but not 65,000 daltons also were C-consuming. Polycations without such reactivity included poly-L-orithine (5,000 and 165,000 daltons), egg white and human lysozymes, and Polybrene. The polycations which failed to induce C consumption via CRP, inhibited its consumption by both active polycations and by C-polysaccharide (CPS). The relative inhibitory capacity of phosphorylcholine and polycations in CPS- and polycations-CRP systems was consistent with the concept that phosphate esters and polycations react at the same or an overlapping combining site. The ability of certain polycations to activate C via CRP increases the potential for initiation of host reactions via C. The capacity of other polycations to inhibit C activation via CRP introduces a potential for physiologic or pharmacologic manipulation. These considerations would seem to expand the potential role of CRP in the initiation and modulation of the inflammatory response.

Chemical and physical properties of fractionated chromatin.

Sonicated chicken reticulocyte chromatin was fractionated into transcriptionally active and transcriptionally repressed components. The active fraction is 8% of the whole chromatin but contains 70% of the newly synthesized chromosomal RNA. This RNA has five times as many hemoglobin RNA sequences as does the RNA in the repressed fraction. The amount of the active fraction in the chromatins of several tissues correlates with their synthetic activity. The molecular weight of the DNA of the repressed fraction is approximately twice that of the active fraction. Moreover, the configuration of repressed chromatin is much more compact, consistent with a much larger sedimentation constant. The transcriptionally active fraction displays a 6 degrees lower melting profile and is highly susceptible to DNase I relative to the repressed fraction. The active fraction contains twice as much non-histone protein and 15% less histone than the repressed fraction and is lacking the lysine-rich and much of the arginine-rich histones.

Hydroxylation of lysyl residues in lysine-rich and arginine-rich histones by lysyl hydroxylase in vitro

Lysine-rich and arginine-rich histones were examined as substrates for lysyl hydroxylase. Both proteins are known to be rich in lysyl residues, and lysine-rich histone also contains -X-Lys-Gly-sequences, whereas no such sequences are found in the arginine-rich histone. Both histones were found to be hydroxylated by lysyl hydroxylase, and the time courses of the hydroxylation reactions with these substrates were linear for at least 60 min. The K m values observed where 3 · 10 −6 for heal-denatured lysine-rich histone and 6 · 10 −6 M for heal-denatured arginine-rich histone. Heat-denatured lysine-rich histone was hydroxylated at a higher rate than non-denatured both at 37 and 25°C. No such phenomenon was found, however, when arginine-rich histone was examined as a substrate. Furthermore, at 37°C lysine-rich histone was a better substrate for lysyl hydroxylase then arginine-rich histone, but this relationship was reversed at 25°C. The synthesis of hydroxylysine observed with arginine-rich histone indicates that the lysyl hydroxylase preparation used in these experiments catalyzes the synthesis of hydroxylysine not only in the sequence -X-Lys-Gly-, but also in some other sequences. Certain collagen polypeptide chains are known to contain one hydroxylysyl residue in a sequence other than -X-Lys-Gly-, and the present results may explain this finding.

Origin and composition of Cabot rings in pernicious anemia.

Cytochemical studies were performed on erythrocytes containing Cabot rings from the peripheral blood of two patients with severe untreated pernicious anemia. These studies demonstrated that the Cabot ring contained arginine-rich histone and non-hemoglobin iron. Structures that may represent precursors of Cabot rings were found in stippled late intermediate marrow megaloblasts. The Cabot ring may result in part from abnormalities in metabolism of both iron and arginine-rich histone that are known to occur in pernicious anemia.

Partial characterization of a histone acetyltransferase from trout testis.

Histone acetyltransferase activity of trout testis was studied both in intact nuclei, and in high salt nuclear extracts. With intact nuclei, the distribution of incorporated [14C]acetate in the various histones was similar to that observed in vivo; the arginine-rich histones H3 and H4 showed the highest specific activities, and lower amounts of label were detected in histones H2a and H2b. Histone H1 incorporated little or no label. Acetyltransferase activity could be detected in purified, sheared chromatin after the addition of MgCl2 or KCl, suggesting that the enzyme is bound to chromatin. Treatment of nuclei with 0.4 M NaCl caused the dissociation of acetyltransferase activity. Most of this solubilized activity failed to bind to DEAE Sephadex and behaved as a high molecular weight heterogeneous complex on Sephadex G-100, suggesting that the enzyme is present as an aggregate with other proteins in the extract. The pH optimum of this preparation was approximately 8.5, and the enzyme showed a preference for histones H3 and H4 as substrates.