Study of calf thymus deoxyribonucleoproteins by means of gel electrophoresis,effect of ionic composition on the mode of chromatin fragmentation

Calf thymus soluble deoxyribonucleoproteins (DNP) obtained by sonication of chromatins isolated both in a “physiological” salt medium and in a buffered water were resolved into four fractions on electrophoresis. The DNA extracted from DNP obtained in a salt medium occurs in fragments of discrete sizes while the sonicated chromatin gel obtained in a buffered water gives rise to a heterogeneous population of DNA fragments upon deproteinization. It is suggested that regularly spaced “weak” points exist in native chromatin and that the regularity is destroyed during isolation procedures involving the transfer of the nuclei into water.     

Properties of chromatin subunits from developing trout testis.

When a sample of trout testis nuclei is digested with micrococcal nuclease, the DNA is cleaved almost entirely to discrete fragments approximately 200 base pairs long and multiples thereof. The same DNA fragments can be obtained when isolated chromatin, as opposed to intact nuclei, is nuclease digested. These DNA fragments can also be found in discrete chromatin "subunits" isolated from nuclease-digested nuclei. Sedimentation through sucrose gradients or velocity sedimentation in an analytical ultracentrifuge separates these chromatin subunits into 11 S (monomer), 16 S (dimer), and 22 S (trimer) etc. species. Subunits can also be fractionated on a Sepharose 2B column equilibrated and run in low salt. High salt (greater than 40 mM NaCl) or divalent cations (congruent to 5 mM) cause subunit precipitation. Chromatin subunits have a protein to DNA ratio of approximately 1.2 and contain all the histones, including the trout-specific histone T. There are, however, no detectable nonhistone chromosomal proteins. Mg-2+ precipitates of the 11 S chromatin monomers, when pelleted, are thin and clear, while oligomer Mg-2+ pellets are thick and white. This could reflect a more symmetrical or ordered packing of 11 S monomers, which are deficient in histone I. This histone may cross-link the larger oligomers, resulting in a disordered Mg-2+ complex. These results are consistent with the subunit model of chromatin structure, based on 200 base pair long regions of DNA associated with histones. These subunits would be separated by nuclease-sensitive DNA spacer regions and cross-linked by histone I.     

Subcellular distribution of glucocorticoid receptors in mouse fibroblasts.

Mouse fibroblasts contain a macromolecular binding component (receptor) which binds glucocorticoids specifically and with high affinity. This study shows that there are three different cellular forms of bound receptor and that it is experimentally possible to markedly alter the subcellular distribution of these three forms. Cells incubated with (3H)triamcinolone acetonide were broken after hypotonic shock and a 7000g hypotonic supernatant was obtained; the pellet was extracted with 0.3 M KCl, yielding a nuclear extract; the remaining pellet was resuspended in water, sonicated, and assayed for "nuclear residual" (i.e., nonextractable) radioactivity. If whole cells are incubated at 0 degrees in a growth medium, almost all of the bound steroid is located in the hypotonic supernatant fraction. Incubation at 37 degrees produces a shift of the steroid-bound macromolecule into the nuclear extractable form, while omission of glucose and addition of KCN at 37 degrees markedly increase the nuclear residual form at the expense of both the nuclear-extractable and supernatant forms. Since DNase treatment of chromatin liberates a soluble steroid-receptor complex, we believe that the nuclear residual form may be steroid-receptor complex tightly bound to chromatin. We propose a model suggesting that an energy-requiring process is required to generate free receptor from the chromatin complex to complete the normal cellular recycling system.     

Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Chromatin fine structure of the histone gene complex of Drosophila melanogaster.

We have used salt extractions of nuclei and long agarose gels to dissect the chromatin fine structure of the histone gene repeat of Drosophila melanogaster. Extraction of nuclei with 0.35 M KCl removes many non-histone chromosomal proteins but does not significantly disturb the overall nucleosome arrangement of the repeat unit. After extraction of nuclei with 0.55 M KCl, which also removes histone Hl, the basic arrangement of nucleosome core particles in the repeat unit is not greatly disturbed and the exposed DNA segments near the 5' ends of the histone genes are also retained. Extraction of nuclei with 0.75 M or higher KCl concentrations causes extensive nucleosome sliding and rearrangement with accompanying changes in the nucleoprotein organization of the histone gene complex and loss of the 5' hypersensitive sites. Our results indicate that the histone gene repeat displays a highly organized chromatin structure in vivo.     

The effect of low ionic strength on the circular dichroic spectrum of chromatin and nucleosomal subunits

Circular dichroism has been used to measure the conformation changes in the DNA of chromatin and chromatin subunits as a function of ionic strength. Transfer of chromatin from 0.15 M to 0.25 mM salt led to an enhancement of the circular dichroic bands at 275 and 285 nm. Removal of histone H1 did not appreciably affect the circular dichroic spectrum when measured in 0.15 M salt, but in 0.25 mM salt H1 depletion led to a marked increase in the ellipticity. Conformation changes due to low ionic strength were also observed with a 145- and a 172-bp chromatin subunit. A linear combination of the ellipticities of the DNA of the two domains in chromatin, namely core and linker, was successful for measurements at 0.15 M salt, but large unexplained discrepancies appeared with the data from measurements in 0.25 mM salt.     

Non-histone proteins of soluble nucleoproteins released from mouse myeloma nuclei by mild micrococcal nuclease digestion

Mono- and dinucleosomes preferentially cleaved from mouse myeloma chromatin by very mild micrococcal nuclease digestion at 0 degree C are soluble and are released from nuclei under near-physiological conditions in which normal nucleosomes containing Hl are insoluble. These nucleosomes are highly enriched in RNA, high-mobility-group proteins and a unique subset of other non-histone proteins. They are nearly devoid of histone Hl and contain DNA significantly less methylated than whole myeloma DNA, indicating that they comprise a subset of genomic sequences. Previously we have shown that this fraction is enriched in transcribed DNA sequences. Non-histone proteins that co-sedimented with readily solubilized nucleosomes included many of the most basic, low-to-moderate molecular weight chromosomal proteins. Many of these proteins were also preferentially acetylated in vivo. The residual, pelleted chromatin was highly enriched in high molecular weight proteins (greater than 60 000), and very depleted in medium molecular weight proteins. Readily solubilized nucleoproteins sedimenting like mononucleosomes were partly resolved by electrophoresis, under non-denaturing conditions, into several subfractions differing significantly in non-histone protein contents. Methods described here should be useful for identifying and isolating non-histone proteins bound to nucleosomes and other chromatin regions that are structurally and functionally unique.     

Release of nucleosomes from nuclei by bleomycin-induced DNA strand scission

Mononucleosomes were released from both isolated mammalian (hog thyroid) and protozoan (Tetrahymena) nuclei by the bleomycin-induced DNA-strand breaking reaction. Trout sperm nuclei, on the other hand, were protected from the bleomycin-mediated DNA degradation. The mononucleosomes released from the bleomycin-treated nuclei contained the core histones H2A, H2B, H3, and H4; while HMG1 and HMG2 proteins, in addition to the core histones, were detected in the mononucleosomes obtained by micrococcal nuclease digestion of nuclei. HMGs, but not H1 histone, were dissociated into the supernatant by cleavage of chromatin DNA with bleomycin, whereas both HMGs and H1 were found in that fraction by digestion of nuclei with micrococcal nuclease. HMG1 and HMG2 were exclusively dissociated from chromatin with 1 mM bleomycin under the solvent condition where the DNA strand-breaking activity of the drug is repressed. These observations suggest the possibility that bleomycin preferentially binds to linker DNA regions not occupied by H1 histone in chromatin and exclusively dissociates HMG proteins and breaks the DNA strand. The results of the effects on bleomycin-induced DNA cleavage of nuclei of various drugs including polyamines, chelating agents, intercalating antibiotics such as mitomycin C or adriamycin, and radical scavengers are also presented.     

Isolation and characterization of nuclei from Neurospora crassa.

A procedure was developed for isolating nuclei from either the conidial or germinated conidial growth phase of Neurospora crassa. A frozen conidial suspension was lysed by passage through a French pressure cell, and the nuclei were freed from the broken cells by repeated homogenization in an Omni-Mixer. Pure nuclei were obtained from the crude nuclear fraction by density banding in a Ludox gradient. The final nuclear yield was 20 to 30%. The nuclei had a deoxyribonucleic acid (DNA):ribonucleic acid (RNA):protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6-diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 micron in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70 to 75% yield by dissociation with 2 M sodium chloride and 5 M urea had a DNA:RNA:protein ratio of 1:1.05:1.7. Chromatin reconstituted from this preparation exhibited a level of RNA polymerase template activity lower than that of pure Neurospora DNA, but the maximum level of reconstitution obtained was only 10%. Fractionation of Neurospora chromatin on hydroxylapatite separated the histones from the chromatin acidic proteins. The normal complement of histone proteins was present in both the reconstituted and dissociated chromatin preparations. The acidic protein fraction exhibited a variety of bands on sodium dodecyl sulfate gel electrophoresis ranging in molecular weight from 15,000 to 70,000. The gel pattern was much more complex for total dissociated chromatin than for reconstituted chromatin.     

Study of peripheral chromatin granules--anchorosomes

The granular particles of chromatin peripheral layer, were isolated together, with the nuclear envelope by treatment of nuclei with nuclease. These particles differ from total chromatin by a decreased content of histone H1, a specific set of minor acid-soluble proteins and a low DNA methylation level. Taking account of the fact that these particles facilitate chromatin interaction with the nuclear envelope, the latter were termed as "anchorosomes". Using UV-induced cross-linking of DNA to the proteins, it was found that all anchorosome-specific acid-soluble proteins can directly interact with anchorosomal DNA. Treatment of anchorosomes with staphylococcal nuclease and electron microscopic data showed that anchorosomes have a nucleosomal organization. Five to ten per cent of anchorosomal DNA appear to be firmly bound to nuclear lamina. This DNA cannot be separated from the lamina by treatment with 2 M NaCl, 1% SDS or heparin (1 mg/ml). The bulk of DNA in the laminal fraction after treatment with the above reagents is protected from hydrolysis with DNAase I by anchorosomal proteins and thus has a high molecular weight (10,000-30,000 base pairs). After treatment of anchorosomes with 0.6 M or 2 M NaCl, DNAase I splits this DNA, predominantly to minor fragments.     

Chromatin dynamics of unfolding and refolding controlled by the nucleosome repeat length and the linker and core histones

Chromatin is composed of genomic DNA and histones, forming a hierarchical architecture in the nucleus. The chromatin hierarchy is common among eukaryotes despite different intrinsic properties of the genome. To investigate an effect of the differences in genome organization, chromatin unfolding processes were comparatively analyzed using Schizosaccaromyces pombe, Saccharomyces cerevisiae, and chicken erythrocyte. NaCl titration showed dynamic changes of the chromatin. 400-1000 mM NaCl facilitated beads with approximately 115 nm in diameter in S. pombe chromatin. A similar transition was also observed in S. cerevisiae chromatin. This process did not involve core histone dissociation from the chromatin, and the persistence length after the transition was approximately 26 nm for S. pombe and approximately 28 nm for S. cerevisiae, indicating a salt-induced unfolding to "beads-on-a-string" fibers. Reduced salt concentration recovered the original structure, suggesting that electrostatic interaction would regulate this discrete folding-unfolding process. On the other hand, the linker histone was extracted from chicken chromatin at 400 mM NaCl, and AFM observed the "beads-on-a-string" fibers around a nucleus. Unlike yeast chromatin, therefore, this unfolding was irreversible because of linker histone dissociation. These results indicate that the chromatin unfolding and refolding depend on the presence and absence of the linker histone, and the length of the linker DNA.     

Partially purified "activated" receptor-glucocorticoid complex from rat liver: regulation of nuclear, chromatin, and DNA-cellulose binding of "activated" complex by pyrophosphate and ATP

The effects of PP1 and ATP on nuclear binding of the "activated" receptor-[3H]-triamcinolone acetonide (TA) complex purified about 3,000-fold from adrenalectomized rat liver were investigated. ATP at up to 5 mM did not affect nuclear binding of the "activated" complex, but PP1 at 2-7 mM greatly enhanced it. However, ATP in the presence of PP1 decreased nuclear binding dose-dependently. Similar results were obtained in the case of chromatin binding, but PP1 alone did not alter DNA-cellulose binding of the "activated" complex, suggesting that the binding sites for chromatin and DNA on the "activated" complex are different. Furthermore, PP1 enhanced ATP-agarose binding of the "activated" complex, indicating that the PP1 binding site(s) on the receptor is different from the ATP binding site(s). The physicochemical properties of the "activated" receptor-glucocorticoid complex bound with ATP and/or PP1 were examined by sucrose density gradient centrifugation and Sephadex G-150 gel filtration. There was no detectable change in the sedimentation coefficient or molecular weight (about 4.2S; Mr approximately equal to 98,000) on binding with ATP and/or PP1. These results suggest that the binding of PP1 and PP1 plus ATP to the "activated" complex caused some allosteric change of the acceptor binding sites of the receptor, resulting in increase or decrease in its binding to nuclei, chromatin, or DNA.     

Chromatin fractionation according to the strength of its matrix bond

Extraction in low salt concentration followed by centrifugation allows rat liver nuclear chromatin to be divided into two fractions: the supernatant chromatin and matrix chromatin. The former fraction contains about 60-70% of initial DNA and about 15% of initial protein along with all five histones, and an insignificant amount of non-histone proteins. RNA synthesis in the matrix chromatin fraction is 2-3 times more intense than that in the original nuclei. The data on gradient centrifugation do not suggest the elongation of RNA molecules synthesized in the matrix fraction. The results obtained as compared with the literature data suggest that the matrix chromatin fraction is enriched with active genes.     

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.     

Structural changes in simian virus 40 chromatin as probed by restriction endonucleases.

The structure of simian virus 40 (SV40) chromatin was probed by treatment with single- and multiple-site bacterial restriction endonucleases. Approximately the same fraction of the chromatin DNA was cleaved by each of three different single-site endonucleases, indicating that the nucleosomes do not have unique positions with regard to specific nucleotide sequences within the population of chromatin molecules. However, the extent of digestion was found to be strongly influenced by salt concentration. At 100 mM NaCl-5 mM MgCl2, only about 20% of the simian virus 40 (SV40) DNA I in chromatin was converted to linear SV40 DNA III. In contrast, at lower concentrations of NaCl (0.05 or 0.01 M), an additional 20 to 30% of the DNA was cleaved. These results suggest that at 100 mM NaCl only the DNA between nucleosomes was accessible to the restriction enzymes, whereas at the lower salt concentrations, DNA within the nucleosome regions became available for cleavage. Surprisingly, when SV40 chromatin was digested with multiple-site restriction enzymes, less than 2% of the DNA was digested to limit digest fragment, whereas only a small fraction (9 to 15%) received two or more cuts. Instead, the principal digest fragment was full-length linear SV40 DNA III. The failure to generate limit digest fragments was not a consequence of reduced enzyme activity in the reaction mixtures or of histone exchange. When the position of the principal cleavage site was mapped after HpaI digestion, it was found that this site was not unique. Nevertheless, all sites wree not cleaved with equal probability. An additional finding was that SV40 chromatin containing nicked-circular DNA II produced by random nicking of DNA I was also resistant to digestion by restriction enzymes. These results suggest that the initial cut which causes relaxation of topological constraint in SV40 chromatin DNA imparts resistance to further digestion by restriction enzymes. We propose that this may be accomplished by either "winding" of the internucleosomal DNA into the body of the nucleosome, or as suggested by others, by successive right-hand rotation of nucleosomes.     

Endogenous polymers of ADP-ribose are associated with the nuclear matrix

The metabolism of nuclear polymers of ADP-ribose has been implicated in several chromatin-associated processes. However, the distribution of endogenous ADP-ribose polymers in the nucleus or within different fractions of chromatin has not been studied. Using a procedure which allowed the radiolabeling and detection of endogenous polymers of ADP-ribose, we have analyzed the nuclear distribution of these polymers in untreated cells and in cells subjected to hyperthermia, N-methyl-N'-nitro-N-nitrosoguanidine, or both. When isolated nuclei from cells subjected to any of these conditions were digested with micrococcal nuclease such that 80% of the DNA was released, 90% of the total poly(ADP-ribose) remained with the micrococcal nuclease resistant chromatin fraction. When nuclear matrix fractions were prepared by exhaustive DNase I digestion in combination with three different salt extraction procedures (2 M NaCl, 300 mM (NH4)2SO4 or 25 mM lithium diiodosalicylate), the matrices contained less than 1% of the total nuclear DNA but 50 to 70% of the total poly(ADP-ribose). These data suggest that the nuclear matrix may be a major site of poly(ADP-ribose) metabolism.