The structure of Sipunculus nudus erythrocyte chromatin

The structure of the Sipunculus erythrocyte chromatin has been characterized by electron microscopy and nuclease digestion (staphylococcal nuclease and pancreatic nuclease). Contrary to previous results [1], we were able to isolate and characterize a histone H_2B in sipunculid nuclei. Though the histones H_2A and H_2B were markedly different from their vertebrate homologues, the subunit structure of the chromatin is the same. But the length of the repeat unit of DNA in the chromatin, is 177 ± 5 bp for the sipunculid erythrocyte nuclei, close to that reported for the chromatin of some lower eukaryotes.     

Tissue-specific histones in the erythrocytes of chicken and turtle

Mature erythrocytes from Leghorn chickens contain lysine-rich histone F1 and a tissue-specific histone F2c. The composition of the F1 fraction was found to be similar to the F1 histones in higher vertebrates. In the erythrocytes of a sea turtle (Chelonia mydas), only lysine-rich histones F1 could be detected. One of these fractions (F1b) differed in amino acid composition from the typical F1 histones described in the literature. The F1b histone fraction was not found in turtle liver. Chromatographic analysis of tryptic peptides of the chicken erythrocyte F1 and F2c histones and of the turtle erythrocyte F1a and F1b histones revealed considerable similarities between these four fractions, thus indicating their possible phylogenetic relationships.     

H1 histone variants in Xenopus laevis

The H1 histones from erythrocytes, livers, intestines, testes, and embryos of Xenopus laevis have been examined electrophoretically. This species has been found to contain at least five electrophoretically resolvable lysine-rich histones in addition to the presumptive H5 histone of erythrocytes. Quantitative and qualitative distinctions between the H1 histones from each source were readily observed. Three H1 histones (H1A, H1B, and H1C) were found in both embryos and adult tissues, although in varying amounts. Two other H1 histones (H1D and H1E) were found only in adult tissues. Comparative SDS gel V8 protease cleavage maps of the lysine-rich histones from testes and erythrocytes have demonstrated that the “adult-specific” H1D and H1E are not artifacts of proteolysis and may be closely related to the presumptive H5 histone. Spermatogenic cells were found to be similar to embryonic cells in being deficient in H1D and H1E. These observations suggest that H1D and H1E are enriched in cell types with low rates of cell division similar to the mammalian H1° histone. The results presented here demonstrate a previously unrecognized degree of developmental and cell-specific variance in the H1 histones of Xenopus laevis.     

Occurrence of histone H10-related protein fraction in trout liver

The lysine-rich histones of trout liver were studied in order to see whether a protein similar to mammalian histone H1₀ exists in this lower vertebrate. The biochemical methods used involved SDS and acid-urea polyacrylamide gel electrophoresis, gel exclusion and ion-exchange chromatography. Specific rabbit antibodies were elicited against chromatographically-purified mouse liver H1₀ and applied for further characterization of the presumptive trout-liver H1₀ fraction. The results from the indirect immunofluorescence on nitrocellulose-immobilized crude H1 subfractions and from the complement fixation test supplied additional evidence for the existence of an H1₀-like protein in trout liver.     

Induction of DNA synthesis by 2,4-dichlorophenoxyacetic acid during callus induction in Jerusalem artichoke tuber tissues

During the induction of DNA synthesis in Jerusalem artichoke (Helianthus tuberosus L.) tuber by 2,4-D, the 2-¹⁴C-2, 4-D from the agar medium rapidly incorporated into the ethanol soluble and insoluble fractions. Although the 2,4-D level in the ethanol soluble fraction decreased on transplantation of the tissue from the 2-¹⁴C-2,4-D medium to medium without the auxin, its level in the buffer-soluble and -insoluble macromolecular fractions increased. The purified, buffer-insoluble macromolecules were chromatin. The 2,4-D binding to chromatin particularly increased during DNA synthesis. The histone contents of chromatin decreased as DNA synthesis progressed. The polyacrylamide gel electrophoretic patterns of the histones showed a decrease in the moderately lysine-rich histone fraction as compared to other fractions. Thus, the decrease in the histone level caused by 2,4-D and the presence of the 2,4-D moderately lysine-rich histone complex may be closely related to the induction of DNA synthesis by 2,4-D in cells.     

Nature of the phosphorylated 26 000 molecular weight protein extracted from dog thyroid with contractile proteins

Dog thyroid contractile proteins are characterized by their ATPase activity at high KCl concentration. In the presence fo Ca²⁺, 80 nmol ATP are hydrolyzed per min per mg protein. This Ca²⁺-ATPase activity is inhibited by Mg²⁺ but not influenced by sodium azide.The 26 000 molecular weight protein which is present in thyroid contractile protein preparations and the phosphorylation of which is stimulated by thyroid stimulating hormone (TSH) is suggested to be identical to the lysine-rich histones (H₁). Indeed, radioactive thyroid H₁ histones added to unlabelled thyroid slices copurify with the contractile proteins and migrate at the same level as the 26 000 molecular weight protein when submitted to electrophoresis in polyacrylamide sodium dodecyl sulfate gels of different acrylamide concentrations.     

H5 Histone and DNA-relaxing enzyme of chicken erythrocytes. Interaction with superhelical DNA.

The interaction of closed circular duplex DNA with the lysine-rich H5 histone fraction of avian erythrocytes has been studied. H5, like H1 histone, interacts preferentially with superhelical DNA. The extent of interaction increases with increasing negative or positive superhelicity. Salt-extracted lysine-rich histones show the same specificity for interaction with superhelices as do acid-extracted preparations. Chicken erythrocyte nuclei contain DNA-relaxing enzyme. This enzyme is extracted from the nuclei at lower salt concentrations than those required to extract H1 and H5 histones and is, therefore, probably a function of a protein distinct from H1 and H5 histones.     

Unambiguous identification of histone H1 in Trypanosoma cruzi

The existence of histone H₁ has been questioned in Trypanosomatids. We report here the presence of a histone H₁ in the chromatin of Trypanosoma cruzi. This protein was purified by narrow-bore reversed phase HPLC and its amino acid composition analyzed and compared with histones H₁ from other species. Furthermore, the purified chromosomal protein was digested with proteases and the amino acid sequences of the resulting peptides were analyzed by the automated Edman degradation. The sequences obtained were found to present a high degree of homology when compared to the carboxy terminal domain of other known histones H₁.     

Electron microscopic and biochemical evidence that chromatin structure is a repeating unit

Electron microscopic and biochemical studies demonstrate that the fundamental structure of chromatin depleted of lysine-rich histones is composed of a flexible chain of spherical particles (nucleosomes), about 125 Å in diameter, connected by DNA filaments. Such a chromatin preparation can be separated by centrifugation into two fractions which differ in the spacing of the nucleosomes. In one fraction almost all of the DNA is condensed in nucleosomes, while the other fraction contains long stretches of free DNA connecting regions where the nucleosomes are closely packed. The isolated nucleosomes contain about 200 base pairs of DNA and the four histones F2a₁, F2a₂, and F2b, and F3 in an overall histone/DNA ratio of 0.97. In such a structure the DNA is compacted slightly more than five times from its extended length. The same basic structure can be visualized in chromatin spilling out of lysed nuclei. However, in this latter case the nucleosomes are very closely packed, suggesting that histone F1 is involved in the superpacking of DNA in chromosomes and nuclei. The chromatin fiber appears to be a self-assembling structure, since the nucleosomal arrangement can be reconstituted in vitro from DNA and the four histones F2a₁, F2a₂, F2b and F3 only, irrespective of their cellular origin.     

The histones of rat testis

Polyacrylamide gel electrophoresis of the histones of the nuclei of seminiferous epithelial cells of rat testis revealed the five principal histone fractions which are found in liver and other somatic tissues, but, in addition, three unusual bands (desginated X₁, X₂, and X₃) were observed. Fraction X₁ had a mobility slightly less than that of F1 and was isolated with F1 in the fractionation procedure of Johns. F1 and X₁ were separated by chromatography on carboxymethylcellulose, and they were shown by amino acid analyses to be closely related lysine-rich histones. However, X₁ had lower content of lysine and alanine and higher content of arginine, aspartic acid, serine, proline, valine and leucine than F1. Both of these fractions had blocked amino-terminal residues, and both had a lysine residue at the carboxyl terminus. These fractions had similar molecular weights by electrophoresis on sodium dodecyl sulfate gels.Fraction X₂ migrated between histone fractions F1 and F3 on electrophoresis while X₃ migrated between fractions F2b and F3. Fraction X₃ was isolated with F2b during fractionation by the Johns procedure. Fraction X₂ has received minimal study, and this fraction may not be unique to the testis inasmuch as a faint band in approximately the position of X₂ can be seen in electrophoretic patterns of rat liver histones.The results of the treatment of the histone fractions with alkaline phosphatase indicated that the electrophoretic differences between X₁ and F1, or X₃ and F2b are not attributable to phosphorylation.     

Basic nuclear proteins in the Oömycete fungus Achlya bisexualis

Summary A comparison was made of the basic proteins extracted from the chromatin and nuclei of Achlya bisexualis, Blastocladiella emersonii, and Pisum sativum. Extraction of purified chromatin and nuclei of the Chytridiomycete, B. emersonii followed by gel electrophoresis produced no detectable protein bands. Extractions of purified nuclei and chromatin by either mineral acid or CaCl₂ from the Oömycete A. bisexualis resulted in several protein bands following separation by disc gel electrophoresis. Monitoring the nucleic acids during the nuclear isolation procedure as well as comparing electropherograms of basic nuclear proteins with basic ribosomal proteins suggests no significant contamination of the nuclear preparations with ribosomes. Carboxymethyl cellulose chromatography of the A. bisexualis nuclear basic proteins resolved three distinct fractions. Gel electrophoresis of the CM cellulose fractions indicated heterogeneity of each fraction. Amino acid analysis of the CM fractions showed that they were all lysine-rich and meet the basisity requirements of histones.     

Affinity labeling of rabbit skeletal muscle phosphorylase kinase by 5′-(p-fluorosulfonylbenzoyl)adenosine

Simple mixing of acid purified histones H₃ and H₄ in equimolar quantities at low ionic strength near pH 7 does not yield the tetramer but rather a high M_r aggregate. Dialysis of acid extracted total or core histones into 2 M NaCl 150 mM phosphate (pH 7.4) followed by fractionation of the histone complexes at lower ionic strength (150 mM NaCl) results in an H₃H₄ tetramer of a structure identical to that derived from salt-extracted histones. Dialysis of acid extracted total or core histones directly into the lower ionic strength buffer with subsequent fractionation, results in H₃H₄ tetramer of closely similar structure.     

Amphibian Lens Histones and Their Relation to the Cell Cycle

Histones have been electrophoretically separated from acid extracts of the frog lens for the first time. The five conventional histone fractions, representing four electrophoretic bands (f1; f2b, f3; f2a2; and f2a1), are present in both the epithelial and fiber cells. In addition, a fifth fraction was isolated from both sources and the evidence suggests that it may be a tissue-specific histone, possibly related to the lysine-rich f2c fraction found previously only in nucleated erythrocytes. The epithelial cells contain a substantially greater amount of histone than the fiber cells. Moreover, the fibers, unlike the epithelium, manifest no net histone synthesis or turnover following lenticular explantation. Microspectrophotometric, radioautographic, and gel electrophoretic studies indicate that the histones are synthesized in frog lenses concurrently with DNA. Inhibition of DNA synthesis does not completely abolish that of histones but reduces it by about one-half. In the early stages of culture (prior to their synthesis and that of DNA) the histones appear to undergo alterations which are prevented by treatment with cycloheximide.     

METHODS FOR THE PURIFICATION OF THYMUS NUCLEI AND THEIR APPLICATION TO STUDIES OF NUCLEAR PROTEIN SYNTHESIS

Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C¹⁴-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.     

Lysine-rich histone H1 kinase from soybean hypocotyl.

A protein kinase (ATP: histone phosphotransferase) with high specificity for the phosphorylation of the very lysine-rich histone H1 has been partially purified and characterized from soybean hypocotyl. The enzyme has a molecular weight of about 48,500. Its activity and sedimentation behavior are refractory to cyclic nucleoside monophosphates. No significant amount of cyclic AMP or cyclic GMP binding activity could be detected in the crude or partially purified enzyme preparations. Km for ATP and histone H1 are 0.4 μM and 0.7 μM, respectively. The enzyme requires Mg²⁺ or Mn²⁺ for activity, while addition of 0.5 mM Ca²⁺, Zn²⁺ or Hg²⁺ results in 50% inhibition. Arginine-rich histones H3 and H4 are inhibitory to histone H1 phosphorylation; these histones affect the Vmax of the enzyme, but not the Km for histone H1.     

Studies on poly (adenosine diphosphate-ribose). X. Properties of a partially purified poly (adenosine diphosphate-ribose) polymerase

The enzyme catalyzing the synthesis of poly (adenosine diphosphate-ribose) with an average of eight repetitions of ADP-ribose was purified 10-fold from rat liver nuclei in 15% yield. The enzyme required DNA, histone, MgCl₂, and dithiothreitol for activity. DNA could not be replaced by polyanions such as poly (U), poly (A), poly (C), RNA, polyvinyl sulfate, methyl dextran sulfate, or heparin. The enzyme was as active on native DNA as on heat-denatured DNA and on poly [d (A-T)], but less active on poly(dG)·poly(dC) and on acid-soluble oligodeoxyribonucleotide. Whole histones of calf thymus or of rat liver, lysine-rich histone of calf thymus, and arginine-rich histone were similarly effective in stimulating the reaction. Casein, bovine serum albumin, cytochrome c, and spermidine did not replace lysine-rich histone. CaCl₂ or MnCl₂ was as effective for the reaction as MgCl₂. Dithiothreitol could be replaced by 2-mercaptoethanol and by glutathione. Polyanions, such as RNA, poly(U), poly(C), poly(A), and polyvinyl sulfate inhibited the enzyme activity. The molecular weight of the enzyme was determined to be 78,000 by sucrose density gradient centrifugation.     

Duplicated nucleosome repeat generated in the erythrocyte chromatin by DNAse I. The role of lysine-rich histones

Dinucleosome periodicity of DNA fragmentation produced by DNAse I in nuclei of pigeon and trout erythrocytes differing in the content of histones H1 and H5 has been investigated. In spite of differences in the content of histone H5 (H1 to H5 ratio is approximately equal to 0.5 and 2 in pigeon and trout erythrocytes respectively) the double-nucleosome repeat was revealed clearly in pigeon and trout erythrocyte nuclei. To elucidate the role of lysine-rich histones we carried out the selective extraction of histone H1 from erythrocyte nuclei by a solution containing 0.3-0.35 M NaCl (pH 3.0) or cleavage of histones H1 and H5 by mild trypsinization in the presence of Mg2+ ions. It was shown that lysine-rich histones play a principal role in formation and maintenance of the so-called dinucleosomal chromatin structure.     

Mode of action of antitumour antibiotics-III: Modulation of permeability of nuclear membrane in the presence of the antibiotics

The binding characteristics of the antibiotics to nuclei and their effect on the permeability of nuclear membrane with respect to histones and ribonucleic acids have been investigated. The binding constant for chromomycin A₃ was found to be 1.4 × 10⁴M^?1 and number of binding sites was equal to 3.48 ± 1.08 × 10¹² molecules/nuclei. The antibiotic chromomycin A₃ enhanced the uptake of lysine-rich histone, actinomycin D decreased the uptake and ethidium bromide had no effect. Chromomycin A₃ also enhanced the release of acid insoluble fraction containing RNA from the nuclei, actinomycin D and ethidium bromide inhibited the release of acid insoluble fraction containing RNA. The relevance of this finding to the role of nuclear envelope in understanding the mechanism of action of the antibiotic has been discussed.     

Aggregation of mono- and dinucleosomes into chromatin-like fibers

Three classes of chicken erythrocyte chromatin particles differing in their content of lysine-rich histones and/or spacer DNA have been studied in order to determine their ability to aggregate into complexes resembling those observed in native chromatin. The complexes have been obtained in the presence of MgCl₂ and NaCl and studied by electron microscopy. Mononucleosomes, containing spacer DNA and histones H1 and H5, give rise to thick (about 70 nm) ellipsoidal particles in the presence of 0.5 mM MgCl₂. These particles are disrupted by the addition of small amounts of NaCl (5–20 mM). On the other hand in 0.5 mM MgCl₂ dinucleosomes give rise to regular fibrous complexes of about 40 nm in diameter which are very similar to native chromatin fibers. These complexes are much more stable when NaCl is added. We conclude that for the stability of nucleosomal aggregates, similar to native chromatin fibers, a continuity of DNA structure is not required, but the presence of divalent cations, spacer DNA and lysine-rich histones is essential.     

Histone-acetylating enzyme of brain

1. Acetylation of histones by an enzyme system derived from rat brain and liver (histone acetylase) was studied by using [1-(14)C]acetyl-CoA as the acetyl group donor. 2. The activity of this enzyme was largely confined to the nucleus. 3. Histone-acetylating activity of cerebral nuclei purified by centrifugation through 1.9m-sucrose was not altered by the presence of the cytoplasmic fraction. 4. Cerebral nuclei from adult rats exhibited greater histone-acetylating activity than did the corresponding preparation from newborn animals. 5. Nuclear acetylating activity was higher in brain than in liver of adult rats but not in newborn animals. 6. The partially purified enzyme from cerebral nuclei, prepared by ammonium sulphate fractionation of an acetone-dried powder, specifically catalysed histone acetylation. 7. Polylysine, protamine, serum albumin and gamma-globulin were not enzymically acetylated by this preparation. 8. Soluble acetylating preparations from both brain and liver nuclei were more active towards arginine-rich F3 and slightly lysine-rich F2a and F2b histone fractions than towards the lysine-rich F1 fraction. 9. Enzymic acetylation of chromatin-bound proteins was much less extensive than that of free histones. 10. The high histone acetylase activity in mature brain may reflect the importance of this process in the genetic control of cerebral function.