Arginine-rich Histones Research Articles

Studies of chromatin from normal and crown gall tumor tissue cultures of tobacco

The T m, the DNA: histone: residual protein ratios, RNA-polymerase activity and template activity of chromatin from normal and bacteria-free crown gall tumor tissue cultures of tobacco (cv. Wisconsin 38) were very similar. Template activity of unsheared chromatin, which was about one-tenth that of DNA, was nearly doubled on shearing. In comparison with normal tissue, the tumor tissue was much lower in soluble and chromatin-associated RNase, DNase and protease activity. The amounts of lysine-rich (F I), moderately lysinerich (F II) and arginine-rich (F III) histone fractions, respectively, were 92, 5·3 and 2·8% for the normal tissue, and 87, 10·2 and 2·9% for the tumor tissue. No significant differences between normal and tumor tissue were noted in the amino acid composition of the histone or non-histone protein fractions. It is suggested that cellular transformation by crown gall bacteria may not involve derepression of the host genome. The integration of the bacterial genome into the host genome (demonstrated elsewhere) could confer on the host cell the ability to produce auxin, cytokinin and other limiting factors, and thus transform it into a tumor cell.

Histone synthesis and phosphorylation in regenerating rat liver

Histone biosynthesis, measured by the incorporation of [ 14C]lysine, increases considerably during the regeneration of rat liver. This increase precedes the peak of DNA replication. At the time of maximum DNA synthesis, arginine-rich (F3) and very lysine-rich (F1) histones incorporate labeled lysine several times more rapidly than do fractions F2a and F2b. The incorporation of [ 32P]orthophosphate into the individual histone fractions increases most rapidly within the first 6 h after hepatectomy. Maximal incorporation in fractions F1 and F2a, occurs in about 12 h, and then declines slightly or remains unchanged until the end of the experiment (36 h). The moderately lysine-rich histone F2b incorporates phosphate rapidly during the first 6 h, declining to a slower increase between 12 and 36 h. The maximal phosphate incorporation into the arginine-rich F3 histone occurs at the time of DNA synthesis (24 h). A comparison of the relative rates of incorporation of 32P and [ 14C]lysine revealed a period of intensive phosphorylation of histones F2b, F1 and F2a during the first 6 h after hepatectomy. The arginine-rich histone F3 did not show an increase. During the period of intensive DNA replication, however, all of the histones are phosphorylated proportionately to their rates of biosynthesis.

Studies on deoxyribonucleoprotein structure. Redistribution of proteins in mixtures of deoxyribonucleoproteins, DNA and RNA.

The addition of tRNA to chromatin gel (initial insoluble deoxyribonucleoprotein preparation) in the presence of 1 mM MgCl2 or of 40 mM NaCl results in the transfer of the very lysine‐rich histone (F1) and of some nonhistone proteins to the tRNA, whereas all other histones remain in the deoxyribonucleoprotein. This result provides a new general method for the mild removal of histone F1 from the chromatin gel. The addition of tRNA to chromatin gel in the absence of Mg2+ and Na+ results not only in the rapid removal of histone F1 and of some nonhistone proteins from the chromatin gel, but also in the slow transfer of histones F2a2 and F2b to the tRNA. A similar pattern of histone redistribution is observed when the tRNA (or DNA) is added to the soluble deoxyribonucleoprotein preparation obtained by mechanical disruption of the chromatin gel. There is no protein redistribution in the above‐mentioned soluble deoxyribonucleoprotein preparation (in the absence of exogeneous free polynucleotides). The addition of free DNA or tRNA to the soluble deoxyribonucleoprotein preparation, obtained by treatment of the chromatin gel with 2 M urea, allows one to remove quantitatively histones F1, F2a2 and F2b from the deoxyribonucleoprotein, leaving the initial distribution of the arginine‐rich histones (F2a1 and F3) on the endogeneous DNA unchanged. The redistribution of proteins (and in particular of histones) occurs in the soluble deoxyribonucleoprotein preparation obtained by urea treatment even in the absence of any exogeneous polynucleotides suggesting that conservation of the initial distribution of proteins in this soluble deoxyribonucleoprotein preparation is not complete.

An Electrophoretic Comparison of Vertebrate Histones

We have studied histones from a wide range of vertebrates with high resolution electrophoresis. There are five major electrophoretic fractions of histone in all somatic tissues in all species examined. Two fractions of histone have a constant electrophoretic mobility no matter what their source. Two fractions vary somewhat from class to class of vertebrate; and one fraction, the lysine-rich fraction, varies considerably in mobility, indicating substantial changes in its primary structure during evolution. This interpretation is confirmed by separation of the individual histone fractions and amino acid analysis. Electrophoretic heterogeneity is found within several of these fractions in good agreement with previous reports on calf histones (Panyim and Chalkley, Biochemistry 8, 3972 (1969)). The electrophoretic heterogeneity within Fractions F2al and F3 is manifested by bands which, while varying in relative intensity from species to species, nonetheless always have a constant mobility. On the other hand the extent of the electrophoretic heterogeneity of the lysine-rich histone group varies among species indicative of a much less stringent demand for a constancy of primary sequence of these molecules relative to the more arginine-rich histone molecules.

Structure primaire de l'histone riche en glycine et en arginine isolée de la tumeur de chloroleucémie du Rat

The primary structure of the glycine-rich, arginine-rich histone isolated from the chloroleukaemic tumor of the rat has been established. The amino-acid sequence deduced from the study of the tryptic peptides and of the COOH-terminal fragment resulting from cleavage with cyanogen bromide, is identical to that of homologous histones isolated from calf thymus and hog thymus. La Séquence complète des 102 résidus d'acides aminés de l'histone riche en glycine et en arginine isolée de la tumeur de chloroleucémie du Rat a été déduite de l'étude des peptides trypsiques et du fragment COOH-terminal obtenu par coupure de la protéine avec le bromure de cyanogène. La structure primaire de la GAR-histone de chloroleucémie est identique à celle des histones homologues isolées du thymus de Veau et du thymus de Porc. Die vollständige Sequenz der 102 Aminosäurereste des Glycin — und Arginin — reichen Histons, welches aus der chlorleukämischen Tumor der Ratte isoliert wurde, ist durch die Untersuchung der Trypsinpeptide und des COOH-End-Bruchstücks, welches durch Zerlegen des Proteins mit Cyanogenbromid erhalten wurde, bestimmt worden. Die primäre Struktur des « GAR-Histonsder Chlorleukämie ist mit derjenigen der homologen Histone identisch, welche aus der Kalbs — oder Schweins — Thymusdrüse isoliert werden.

Cross-linking of nucleohistone by nitrous acid

Treatment of calf-thymus nucleohistone with nitrous acid yields a product from which protein cannot be completely removed and which, on hydrolysis, shows both amino acids and nucleic acid bases. This is proposed to result from covalent cross-linking between histone and nucleic acid, arising from alkylation of DNA by the carbonium ion formed from the action of nitrous acid on amino groups of lysine. Whole nucleohistone, and partially dehistonized nucleohistone, containing argininerich histone, give cross-linking with both KNO 2 and NaNO 2. DNA-Polylysine and DNA-lysine-rich histone complexes become cross-linked with nitrous acid generated from KNO 2 but not from NaNO 2. The cross-linking may result in mutations.

Model nucleohistones: The interaction of F1 and F2al histones with native T7 DNA

Synthetic complexes of the lysine-rich histone (F1) and of the glycine- and arginine-rich histone (F2al) with native T7 DNA have been prepared by the step-gradient dialysis procedure from 5 m-urea-3 m-NaCl and from 5 m-guanidine-HCl. Data on the physical properties of these complexes were obtained relevant to their solubility, thermal stability, sedimentation coefficients, circular dichroic spectra, and ultrastructure. Complexes of F1 with T7 DNA, prepared from urea-NaCl or from guanidine, exhibited a salt-dependent structural change between 10 −3 and 10 −1 m-sodium ions. At low ionic strengths, complexes sedimented like naked DNA and appeared to be a loose network of DNA-like fibers when examined in the electron microscope. At higher ionic strengths the complexes exhibited rapid and heterogeneous sedimentation and an altered circular dichroic spectrum, and they appeared in the electron microscope to be a mixture of large (0.1 to 0.3 μ diameter) donut and stem-like structures. Complexes of F2al with T7 DNA prepared from urea-NaCl and from guanidine revealed differences with respect to melting, sedimentation, circular dichroic, and electron microscopic properties. Complexes prepared from urea-NaCl sedimented as a single rapid heterogeneous component and resembled rosettes in the electron microscope; complexes from guanidine exhibited greater thermal stability, sedimented as two components, and revealed the presence of donut structures. Neither type of complex revealed salt-dependent structural changes of the magnitude seen with F1-DNA complexes. Poly- l-lysine and T7 DNA were combined by gradient dialysis from urea-NaCl, and also appeared as donuts and short stem structures over a wide range of ionic strengths. The present studies suggest that the structure of the annealed protein-DNA complexes strongly depends on the conformation of the basic protein during the initial stages of its interaction with native DNA. A highly disordered protein conformation results in characteristic donut and stem-like structures. F2al histone, however, aggregates in high NaCl concentrations, probably due to its hydrophobic portion (residues 46 to 102), resulting in rosette structures. The instability of F1-DNA donuts and stems at decreased ionic strength may result from the high proline content in the basic portion of F1 histone, consequent disruptions of lysine-phosphate interactions, and increased susceptibility to polyelectrolyte expansion.

Nucleolar and extranucleolar chromatin of rat Novikoff hepatoma cells: A comparison of histone and non-histone protein acetylation

Histone acetylation was studied in two nuclear subfractions, nucleoli and the nuclear chromatin, which have substantially different transcriptional activity. The quantitative uptake and turnover of [1- 14C]acetate into the total histone was found not to differ significantly between the two nuclear fractions. Some variations among the relative specific activities of the individual histones were observed after analysis on acrylamide gels, but their significance is questionable due to possible changes in the F 3 dimer/monomer ratio induced by the isolation of histones. All histone fractions were labeled when rat ascites cells were exposed to 3H-labeled amino acids for 1.5 h. In a similar 1.5-h experiment, [1- 14C]acetate was incorporated primarily into the arginine-rich histones F 3 and F2a1; the lysine-rich F1 was unlabeled. Thus, the acetylation observed in these experiments occurred predominantly on pre-formed histone molecules. Nonhistone chromosomal proteins were acetylated to about 25 % of the specific activity of the histones. Only minor differences in the extent of histone acetylation in the isolated nucleoli as compared with nuclear chromatin indicate that histone acetylation is not directly related to the amount of active template in these nuclear subfractions.

Early and late histones during sea urchin development

During cleavage of sea urchin embryos, replication of the chromosomes occurs at about hourly intervals whereas at later stages of development the cells divide at a much slower rate. A single slightly lysine-rich histone predominates in the rapidly dividing chromosomes. When cell division slows, lysine-rich and argininerich histones become the major components.

Acid-extractable proteins from chick embryo muscle nuclei: Characterization of histone and studies on histone acetylation

Nuclei from developing chick muscle were isolated from 11- and 16-day-old embryos. The histones were extracted and characterized by ion-exchange chromatography, disc electrophoresis and amino acid analysis. Lysine-rich (F1), moderately lysine-rich (F2b + F2a2), and arginine-rich (F3 + F2a1) histone were present. During the development of the muscle the ratio acid-extractable protein/DNA did not change and the relative amounts of the purified fractions were very similar ε- N-Acetyllysine was present in the arginine-rich fractions. Incubation of isolated nuclei from 11- and 16-day embryos with [1- 14C]acetate showed marked differences in the uptake of acetate into the arginine-rich histone fraction.

Enzymic modification of chromosomal macromolecules: II. The formation of histone [formula omitted] by a soluble chromatin methylase

A soluble enzyme preparation devoid of histone has been isolated from Krebs 2 ascites tumour cell chromatin by extraction with 1.5 M NaCl, which catalyses the transfer of methyl groups from S- adenosyl- l-methionine to protein but only in the presence of added histone. Although the arginine-rich histone fractions are the most effective methyl group acceptors the lysine-rich fractions still suffice. As a result of this enzymic activity some lysine residues of the added histone molecules are modified to yield ε-N- trimethyl- l-lysine residues.

Proteins of chromatin in template restriction: I. RNA synthesis in vitro

1. 1. Selective extraction of the very lysine-rich histone fraction F1 does not increase the activity of chromatin as a template for RNA synthesis in vitro. Removal of either arginine-rich histones F2a and F3 or lysine-rich histones F2b (together with F1) results in an increase of the template activity of chromatin. 2. 2. (NH 4) 2SO 4 removes selectively the lysine-rich histone fractions F1 and F2b. Even in 1.0 M (NH 4) 2SO 4 the arginine-rich histones F2a and F3 remain associated with chromatin. 3. 3. (NH 4) 2SO 4 in 0.2 M concentration (as does 0.5 M NaCl) dissociates only the F1 histone fractions without simultaneous increase of the DNA-dependent RNA synthesis. Since similar concentrations of (NH 4) 2SO 4 increase the in vitro nuclear RNA synthesis several-fold, it can be concluded that the salt activation of RNA synthesis in isolated nuclei or chromatin is caused by the activation of RNA synthesis system and not by template derepression of chromatin. 4. 4. The pH of the salt solutions used to extract histones influences the solubility of nonhistone proteins. At pH 6.0 little nonhistone protein is extracted while at pH 8.0 considerable amount of nonhistone proteins is released into the solution.

Studies on the nucleic acids of human lymphocytic cells: Acetylation of histones

Histones have been isolated from human lymphocytic cells in culture and purified through column chromatography. These human lymphocytic cells deriving from various lymphoproliferative diseases incorporate 3H-acetate to differing degrees. Distribution of 3H-acetate label indicated that lysine-rich histones were the least, while arginine-rich histones were the most, extensively acetylated.

Solubilization and Partial Purification of Protein Methylase III from Calf Thymus Nuclei

Abstract An enzyme which methylates the e-amino group of lysine residues in histones was solubilized from calf thymus nuclei. The enzyme was present in nuclei and was found in all the rat organs tested. Although the enzyme was not purified to high extent, practically all of the endogenous methylation activity (enzyme activity without added exogenous histone) was eliminated. The enzyme was most effective toward various histones, and especially arginine-rich histone. Polylysine was methylated to some extent. Zinc ion at a concentration of 2 x 10-3 m abolished the enzyme activity completely, but Mg++ ions were without effect. The Km value of the enzyme for S-adenosyl-l-methionine was 3.0 x 10-6 m. A simple assay method for the enzyme activity was also determined.

In vivo incorporation of labeled amino acids into nuclear proteins of the sea urchin embryos

Analysis of acid-soluble proteins extracted from nuclei isolated from eggs and embryos at various developmental stages during early embryogenesis of the sea urchin Strongylocentrotus purpuratus indicates that arginine-rich histones are the first histone fraction to appear in the nucleus. It has been possible to time their appearance as occurring between the 4 to 8 cell stage and the early blastula. During the first few cell divisions after fertilization, fractionation of the acid-soluble nuclear proteins on Amberlite CG-50 columns using a gradient of guanidinium hydrochloride for elution revealed that no 14C-labeled amino acid entered the biosynthesis of histones. Typical lysine-rich histones appear in the nucleus only later, during hatching and gastrulation. At hatching and later stages, but not earlier, 5% perchloric acid extracted proteins containing 25% lysine could be detected. The amino acid composition of such fractions closely resembled the very lysine-rich histone fraction f 1 derived from calf thymus nuclei.

Studies on Adrenal Histones: CHARACTERIZATION, BIOSYNTHESIS, ENZYMATIC PHOSPHORYLATION, AND ACETYLATION OF HISTONES FROM A HUMAN ADRENAL CARCINOMA

Abstract Histones F1, F2a, F2b, and F3 were isolated from a human adrenal carcinoma. The fractions were purified by chromatography on carboxymethyl Sephadex and characterized by amino acid analysis and disc electrophoresis. The histones were comparable to calf thymus histones in amino acid composition and electrophoretic patterns. Adrenal carcinoma F3 showed a more complex electrophoretic pattern than calf thymus F3. To study biosynthesis, phosphorylation, and acetylation of histones, adrenal slices were incubated with lysine-3H, Na332PO4, and sodium acetate-14C at intervals between 5 and 120 min. The rate of histone biosynthesis as measured by lysine-3H incorporation was similar for the F1 and F2b fractions and did not increase further after 30-min incubation time. The arginine-rich histone F3 showed the highest rate of lysine-3H uptake, and its biosynthetic rate was about 16 times higher than the rate of F1 biosynthesis. The incorporation of Na332PO4 and sodium acetate-14C increased with time in all histone fractions. The rate of phosphorylation and acetylation as indicated by the specific activities of the histones was similar in the F1, F2a, and F2b fractions. The F3 histone showed the highest rate of 32P and 14C uptake. However, comparison of the acetylation and phosphorylation of the histones in relation to their biosynthetic rate suggests that the turnover of histone-bound phosphate-32P and acetate-14C is faster in the lysine-rich fractions F1 and F2b than in the arginine-rich histones.

A fluorometric protein assay suitable for histones based on lysine content

A new, simple, and highly sensitive method for measurement of histones is based on fluorescence after treatment with fluorodinitrobenzene. The fluorescence is related to the lysine content of the protein. The data indicate that DNP-lysyl residues in proteins are much more fluorescent than free DNP-lysine. It is posiible to detect less than 0.2 μg of lysine-rich histone or 0.4 μg arginine-rich histone by this method. Most of the commonly employed reagents for histone extraction or fractionation do not interfere. The method is valuable for monitoring chromatography of small amounts of histone, and when used in conjunction with other physical measurements permits a distinction to be made between histones and other proteins, and berween lysine-rich and arginine-rich histones.

Labelling of mouse spermatozoa with L-arginine-14C(U) monohydrochloride

Two mouse males were injected with L-arginine-14C(U) monohydrochloride, one with a dose of 100 μ Ci, the other with a dose of 500 μ Ci. Mating of both males was accomplished at regular intervals. Sperm was collected from uteri, smeared and coated for grain density autoradiography. Autoradiography revealed a sharp peak of the proportion of labelled spermatozoa in the ejaculate on the 13th day after injection followed by a rapid decrease on the 16th and 20th day. The most heavily labelled spermatozoa from the peak and ova fertilized by them were further investigated by means of track autoradiography. Spermatozoa in the ejaculate smear and spermatozoa and male pronuclei in histologic sections from the ampullar part of the oviduct were checked for the presence of tracks. The number of tracks originating from spermatozoa in the vicinity of the fertilized ova was about the same as that observed in freshly ejaculated spermatozoa. By contrast, no tracks were detected in male pronuclei. This observation is interpreted as an evidence of a possible loss of the argininerich histone of the sperm at the time of pronuclear formation.

Regional histone changes in embryos of the newt, Triturus pyrrhogaster, during early development

By means of micro-scale chromatography, histones from different regions of newt embryos in the course of development were analyzed qualitatively and quantitatively. The first appearance of lysine-rich histones occurred at the animal-ventral side of the blastula. These increased rapidly in the ectodermal regions at middle gastrula. The time of appearance of lysine-rich histones in the other regions, i. e. mesoderm and endoderm, was delayed. Arginine-rich histone, being a major component in blastula, tended to decrease in every region as development proceeded, though it was predominant in the endoderm throughout early embryogenesis. The pattern of histone subcomponents tended to converge toward a characteristic type, and the histone content in every region approached the DNA content toward the end of early embryogenesis.

Analytical study of the degradation of nucleohistone during calf thymus chromatin autolysis

The mechanism of nucleohistone degradation in calf thymus chromatin during autolysis of the latter involves a release of lysine-rich histone fractions, followed by degradation of the thus-exposed DNA portions; the latter portions are richest in adenine-thymine sequences. After this degradation process, residual nucleohistone fragments of low molecular weight (10(5)) remain. Their chemical composition is special, in that the DNA contains a larger proportion of guanine-cytosine sequences than does the initial DNA and it is combined with arginine-rich histones.