Study of heterogeneity of chromatin endonuclease fragments, using fractionation by step-wise endonucleolysis

Isolated nuclei from rat liver were incubated at different time intervals under conditions, optimal for manifestation of the Ca, Mg-dependent endonuclease activity. After each of the 6 endonucleolyses chromatin was extracted and 6 chromatin fractions (I--VI) and "residual" chromatin were collected. A comparative analysis of the "early" (I--III) and "late" chromatin fractions revealed an increased RNA content in the "late" fractions and changes in the composition of the non-histone proteins. Electrophoresis in acrylamide gel concentration gradient demonstrated that fractions I--III predominantly contain high molecular weight fragments whereas fractions IV-VI are represented by highly fragmented chromatin. Each fraction was characterized by peculiar shapes of alkaline denaturation curves and by heterogeneity of charges of their constituent chromatin fragments.     

Studies on the stability of chromatin from hen oviduct

The stability of sheared chromatin from the magnum portion of hen oviduct has been examined during storage at 0 °C using several parameters. Chemical composition and ultraviolet spectra were unchanged after 1 month while changes in melting profiles were seen after 2 weeks. Gel electrophoretic patterns of histones indicated slight proteolysis after 4 weeks with no apparent degradation occurring at earlier times. Acidic protein gel patterns remained unchanged during the period of study. Rapid changes (within 1 to 3 days) were seen in chromatin size distributions as measured in sucrose gradients and in chromatin template activity. The mean chromatin size decreased with storage, an increasing percentage of the total population sedimenting as a 17S component. This change in chromatin size was accompanied by an increase in the amount of free protein floating on the top of gradients.Several experiments were conducted to investigate the nature of the chromatin changes occurring at early storage times. Changes in chromatin size and template activity could be attributed to neither proteolysis nor DNase activity. Diisopropylfluorophosphate, a known nuclear protease inhibitor, did not inhibit the rate of change in chromatin size with storage. Furthermore, no proteolysis was detectable by gel electrophoresis during storage times at which significant changes in chromatin size and template activity had occurred. When the size of chromatin DNA was examined, no changes were seen with storage nor were there differences in the size of DNA from fast and slow sedimenting chromatin fractions.When template activities of fast and slow sedimenting chromatin fractions were compared, the slow sedimenting fractions were found to be the most template active. The template activity of a slow sedimenting, high template activity fraction was examined with storage and was found to increase with time.     

Solubilization of Jerusalem artichoke tuber chromatin by 2,4-dichlorophenoxyacetic acid

Chromatin from the tuber of the Jerusalem artichoke (Helianthus tuberosus) was solubilized in 2,4-dichlorophenoxyacetic acid (2,4-D) solution (100 mM) at pH 7.0. This solubilization was much affected by the pH; below 6.0 less chromatin was solubilized. The elution pattern of the products on gel filtration with Sepharose 4B showed that the solubilization was caused by the dissociation of the DNA and associated proteins. The pattern of polyacrylamide gel electrophoresis of histones extracted from the chromatin solubilized by 2,4-D was quite different from those of histones extracted from the original chromatin or from NaCl (2.0M) solubilized chromatin. The F1 and F3 fractions seemed to be little affected by 2,4-D, but the F2a1, F2a2 and F2b fractions were greatly decreased. In addition, the ratios of histones and non-histone proteins to DNA changed considerably in 2,4-D solubilized chromatin in an inverse manner. None of these changes were observed with NaCl. which suggests that the behaviour of 2,4-D for the solubilization of chromatin differs substantially from that of NaCl.     

Rat liver chromatin. Distribution of histone and nonhistone proteins in eu- and heterochromatin.

Rat liver chromatin was sheared and separated into template-active (euchromatin) and template-inactive (heterochromatin) fractions by glycerol gradient centrifugation. Chromosomal proteins associated with the chromatin fractions were analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. Histone composition did not vary qualitatively, but more histone protein was consistently found associated with the euchromatin fractions. Nonhistone protein banding patterns for these chromatin fractions exhibited marked heterogeneity, with a number of bands unique to either eu- or heterochromatin.     

Distribution of estrogen receptors in subfractions of hen oviduct chromatin.

Hen oviduct chromatin was digested with DNase II and separated into two fractions. The MgCl2 insoluble chromatin fraction (43% of the total DNA) was enriched in nucleosome-like particles, which sedimented at 11 S and contained 185 base pairs of DNA. The MgCl2 soluble chromatin fraction (5% of the total DNA) was characterized by 5 S and 14 S peaks in sucrose gradients; Estrogen receptors in the chromatin fractions were labelled with [3H] estradiol using the steroid exchange assay. The concentration of receptors in the MgCl2 soluble chromatin was 4;5 times higher than that in the MgCl2 insoluble chromatinmin sucrose gradient analysis the 11 S particles displayed a negligible specific radioactivity suggesting that estrogen receptors mainly bind to extranucleosomal chromatin.     

Ultrastructural and chemical study of the chromatin during spermiogenesis of the fish Scyliorhinus caniculus (L.) (author's transl)

Electron microscopic, cytochemical and biochemical techniques were applied to study structural aspects and changes in nuclear components during the spermiogenesis of Scyliorhinus caniculus. Five major stages of nuclear differentiation were recognized and characterized by variations in the organization and chemical properties of chromatin. Stage I is analogous to a somatic nucleus with heterogeneous chromatin. At the second stage, the nuclear content is dispersed but the chromatin fibers are of the same diameter as those of the stage I. The nuclear elongation begins at stage III, the DNP fibers running preferentially parallel to the long axis of the nucleus. During these early modifications of chromatin structure appear two new basic nuclear proteins (S 1 and S 2) which migrate faster than histones but typical histones remain assosciated with these nuclei. In later elongation stage (stage IV), the chromatin fibers organize in a helical form and fuse side by side giving lamellar systems which have a reticular structure. At the end of this stage, the nuclear material has become uniformly compact. These late variations in chromatin organization are parallel to the association of chromatin with new basic nuclear proteins (S 3, S 4, Z 1, Z 2 and Z 3). The cytochemical and electrophoretical properties of one of these proteins (S 4) which appears at the end of spermiogenesis are similar to those of a protamine. In stage V, the chromatin is homogeneous and the nucleus assumes a helical configuration beginning at the posterior end. The deoxyribonucleoproteins of the mature sperm show some novel chemical characters, including the appearance of a stable nuclear acidophilia with the ALFERT and GESCHWIND method and extraction with 0.25 N HCl of one of the basic protein fractions newly appeared in late spermiogenesis (Z 3), two other fractions (Z 1 and Z 2) being extracted with a more drastic procedure. The other fractions described before are no more detectable.     

The effect of low doses of external gamma irradiation on the chromatin structure and activity of the histone-specific proteinases of rat brain cells]

Irradiation of rats with doses of 0.5 to 2 Gy was shown to cause dose-dependent changes in the sensitivity of brain cell chromatin to the effect of DNAase I that were manifested by the increased level of DNA hydrolysis and a high content of the chromatin soluble fractions. The chromatin structure was only partially restored 24 h after irradiation. Changes in the chromatin structure were accompanied by the increase in the histone-specific proteinase activity.     

Lipid peroxidation and the polymerase activities of liver chromatin fractions in rats during aging

Lipids, which enter the composition of actively transcribed and repressed chromatin fractions are found to undergo a peroxidation. This process decreased with aging and more pronounced in actively transcribed chromatin fraction. A decreased activity of DNA polymerase beta and increased activity of RNA polymerase I in this chromatin fraction with aging were observed. It is assumed that observed changes of genome function of old animals may be caused by decreased peroxidation of chromatin lipids.     

Fractionation of mechanically sheared chromatin on ECTHAM-cellulose

Chromatography of chromatin on the weak ion-exchange resin ECTHAM-cellulose was re-examined using the combined salt-pH elution conditions of Stratling, W.H., Van, N.T. and O'Malley, B.W. (1976) Eur. J. Biochem. 66, 423-433. When mechanically sheared rat liver chromatin was chromatographed on ECTHAM-cellulose the histone composition of eluted fractions was very similar, whereas early eluting fractions were enriched in non-histone proteins, including certain high mobility group proteins, and in hnRNP particles, containing newly synthesised RNA. Later eluting fractions were depleted in all of these components. The majority of hnRNP particles in early eluting chromatin were shown to be physically associated with chromatin by centrifugation in metrizamide. Hen erythrocyte chromatin contained no early eluting material. Size of DNA fragments was not a significant factor in determining the elution position of chromatin fragments. Early eluting material was not generated by endogenous nuclease and protease action. The conditions of chromatin preparation, and of elution of early chromatin fractions caused no gross disruption of chromatin structure, or dissociation of chromatin proteins, although some nucleosome sliding may have occurred. The conditions required for elution of some of the later fractions are sufficient to cause dissociation of protein, and alteration of chromatin conformation.     

Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs

A cell-free cytoplasmic preparation from activated Rana pipiens eggs could induce in demembranated Xenopus laevis sperm nuclei morphological changes similar to those seen during pronuclear formation in intact eggs. The condensed sperm chromatin underwent an initial rapid, but limited, dispersion. A nuclear envelope formed around the dispersed chromatin and the nuclei enlarged. The subcellular distribution of the components required for these changes was examined by separating the preparations into soluble (cytosol) and particulate fractions by centrifugation at 150,000 g for 2 h. Sperm chromatin was incubated with the cytosol or with the particulate material after it had been resuspended in either the cytosol, heat-treated (60 or 100 degrees C) cytosol or buffer. We found that the limited dispersion of chromatin occurred in each of these ooplasmic fractions, but not in the buffer alone. Nuclear envelope assembly required the presence of both untreated cytosol and particulate material. Ultrastructural examination of the sperm chromatin during incubation in the preparations showed that membrane vesicles of approximately 200 nm in diameter, found in the particulate fraction, flattened and fused together to contribute the membranous components of the nuclear envelope. The enlargement of the sperm nuclei occurred only after the nuclear envelope formed. The pronuclei formed in the cell-free preparations were able to incorporate [3H]dTTP into DNA. This incorporation was inhibited by aphidicolin, suggesting that the DNA synthesis by the pronuclei was dependent on DNA polymerase-alpha. When sperm chromatin was incubated greater than 3 h, the chromatin of the pronuclei often recondensed to form structures resembling mitotic chromosomes within the nuclear envelope. Therefore, it appeared that these ooplasmic preparations could induce, in vitro, nuclear changes resembling those seen during the first cell cycle in the zygote.     

Fractionation of Chromatin Depleted of Histone H1

Chromatin depleted of histone HI was prepared after extraction with 0.6 M NaCl. The salt-extracted chromatin was mechanically fragmented, then fractionated on sucrose gradients in two fractions, the fast migrating (heavy) and the slow migrating (light) fractions. The average sizes of the ONA-moieties from heavy and light fractions were found to be similar. However, heavy fractions were enriched in high temperature melting populations, whereas, light fractions exclusively included low temperature melting populations, not seen in the heavy fractions. Comparison of the derivative plots of thermally denatured whole and salt-extracted chromatin as well as heavy and light fractions of salt-extracted chromatin suggests that the DNA regions associated with histone HI in intact chromatin may be co-isolated with the light fraction.     

Participation of the genetic apparatus of the hippocampal and neocortical cells in the mechanisms of action of desglycine-arginine vasopressin on learning and memory processes in rats

The paper deals with the influence of synthetic vasopressin analogue--desglycine-arginine vasopressin (DG-AVP) on the content of RNA and fractional composition of chromatin proteins in the tissue of neocortex and hippocampus of intact white rats and after establishing of two-way avoidance reflex. Administration of the peptide alone significantly increased RNA content in hippocampal tissue, injection of the peptide 10 min before conditioning did not lead to significant changes in RNA quantity as compared to that in animals in which the conditioned reflex was established against the background of saline administration. In neocortical tissue neither learning itself nor administration of DG-AVP alone was accompanied by significant changes in RNA content, while learning against the background of peptide injection significantly increased RNA in that structure. In hippocampal and neocortical tissues quantitative changes were observed in certain fractions of chromatin proteins in all animal groups studied.     

Incorporation of 1-(14)C linoleic acid in rat liver nuclei and chromatin fractions

The incorporation of 1-(14)C linoleic acid in several chromatin fractions of rat liver nuclei was investigated using two different procedures: (1) rat liver nuclei were incubated with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid. After 40 min at 37 degrees C the chromatin obtained by sonication of nuclei suspended in 0.25 M sucrose was fractionated by differential sedimentation; (2) chromatin fractions obtained by differential sedimentation were incubated separately with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid 40 min at 37 degrees C in order to characterize the fatty acid incorporation in isolated chromatin. A comparative study of the incorporation of 1-(14)C linoleic acid in microsomes and nuclei isolated from rat liver is also presented for the purpose of comparison. Linoleic acid was incorporated into nuclear lipids as well as in chromatin fractions. The fatty acid incorporation was stimulated considerably in the acylation system when compared to control, it appears to be highly dependent on the state of condensation of chromatin, being barely detectable in the lowest density fraction. The major proportion of 1-(14)C linoleic acid was found in phospholipids and in a lesser proportion it remained esterified to triglycerides and cholesteryl esters. The distribution of radioactivity in different classes of phospholipids present in microsomes and nuclei isolated from rat liver, showed a similar profile of distribution. The major proportion of radioactivity, approximately 50% was found in phosphatidylcholine and in a lesser proportion in sphingomyelin, phosphatidylserine and phosphatidylethanolamine. When chromatin fractions were incubated separately, it was observed that the major proportion of 1-(14)C linoleic acid in phospholipids was found in heavy chromatin fractions whereas low density chromatin fraction only incorporated in a lesser proportion.     

Changes in non-histone chromatin proteins during the storage of chromatin

We have described here the changes in stored chicken reticulocyte chromatin which take place among non-histone protein fractions based on SDS-polyacylamide gel electrophoresis and hybridization of globin cDNA with RNAs transcribed on native and reconstitited chromatin templates.     

Fractionation of L-cell chromatin into DNA, RNA, and protein fractions on Cs2SO4 equilibruim density gradients

A new procedure is described for fractionation of chromatin into DNA, RNA, and total chromatin protein. By isopycnic gradient centrifugation of chromatin preparations in Cs₂SO₄ solutions containing dimethylsulfoxide and sodium sarcosyl it is possible to obtain highly purified fractions of these components. The method gives a very high yield of these chromatin fractions unlike some other methods, where irreversible binding to columns occurs. Also with this method it is possible to obtain highly concentrated fractions, which after a simple dialysis step, can be conveniently analyzed by polyacrylamide gel electrophoresis.Nuclei from L-929 cells were isolated by a method involving citric acid or by a method using a nonionic detergent. The yields of DNA obtained by both methods was compared. Chromatin was isolated from purified nuclei (prepared in either of the above ways) in two different ways also. In one method, chromatin was extracted from nuclei with 1 m NaCl. A second method involving fractionation of lysed nuclei in sucrose and metrizamide solutions was also used. The yields of DNA obtained by both methods was compared. There appears to be little nuclear membrane contamination of any of these chromatin preparations.A preliminary analysis of L-929 cell chromatin total RNA and protein fractions on polyacrylamide and agarose gels has been made. Both fractions appear to be quite complex with a wide spectrum of subcomponents of differing S values.