Digestion Of Nuclei Research Articles

Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo.

Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.

Analysis of chromatin of skeletal muscle of developing rats using micrococcal nuclease and DNase I.

Conformational changes in the chromatin of skeletal muscle of 3-, 14- and 30 day-old developing rats have been studied using DNase I and micrococcal nuclease (MCN). Purified nuclei were digested separately by MCN and DNase I. The rate and extent of digestion by MCN decreases gradually as development proceeds. The electrophoretic pattern of MCN digested DNA, however, shows no change. The kinetics of digestion of nuclei by DNase I show no change with development. However, the electrophoretic pattern of DNase I digested DNA shows a gradual decrease in the amount of 10-30 bp fragments with progressive development. These studies show that the chromatin of the skeletal muscle undergoes certain conformational changes during postnatal development, and such changes in chromatin may be necessary for terminal differentiation of this tissue.

Increased thermal stability of solubilized chromatin after poly(ADP-ribose) synthesis.

A hyperthermic shift in the hyperchromicity curve of thermally denatured swine aortic-smooth-muscle-cell chromatin solubilized by digestion of nuclei with micrococcal nuclease was observed after the chromatin was incubated under conditions to allow poly-(ADP-ribose) synthesis by the endogenous poly(ADP-ribose) polymerase. When the order of solubilization and poly(ADP-ribosyl)ation was reversed, a smaller proportion of the solubilized chromatin exhibited greater thermal stability. Nuclease digestion of nuclei preincubated for poly(ADP-ribose) synthesis revealed no difference in kinetics of digestion or fragment size distribution compared to that of control nuclei. Poly(ADP-ribose) synthesis in these nuclei was proportionately greater in the chromatin fraction most resistant to solubilization by micrococcal nuclease treatment.

Histone Acetylation in Chromatin Containing Mouse Satellite DNA

Histone acetylation in transcriptionally inactive chromatin has been studied with chromatin containing mouse satellite DNA. The latter was obtained by digestion of nuclei from Ehrlich ascites tumor cells with the restriction nuclease Bsp, which degrades main-band DNA but leaves satellite DNA intact. The enzyme-resistant material was separated by gel filtration. Satellite DNA amounted to 65% of the total DNA in this fraction. When the cells were grown in the presence of sodium n-butyrate to inhibit histone deacetylation, a few, if any, hyperacetylated forms of core histones were found in satellite chromatin. Conversely, the highest quantity of tetraacetylated H4 molecules was found in the fractions containing the most extensively degraded chromatin.

Phosphorylation of nonhistone proteins during the HeLa cell cycle. Relationship to DNA synthesis and mitotic chromosome condensation.

Cell cycle variations in the phosphorylation of chromatin-associated nonhistones were determined. Cells were radiolabeled with [32P]orthophosphate and chromatin was obtained by mild digestion of nuclei with micrococcal nuclease. The experiments were performed in the presence of a substrate inhibitor of alkaline phosphatase, beta-glycerophosphate. The results show that, while similar molecular weight species of phosphorylated nonhistones are associated with interphase chromatin through the HeLa cell cycle, the incorporation (32P cpm/micrograms of protein) profiles of selected major phosphononhistones show substantial changes. The most prominent peaks of specific radioactivity occur in the DNA synthesis phase (S phase). The phosphorylation states of the proteins of isolated metaphase chromosomes were also determined. Nonhistone proteins of isolated metaphase chromosomes are strikingly dephosphorylated, especially in comparison to histone H1. The phosphorylation of the major phosphononhistone of chromatin, which has a molecular weight of 55,000, was further characterized by techniques that included one-dimensional peptide mapping in sodium dodecyl sulfate-polyacrylamide gels and nonequilibrium pH gradient slab gel electrophoresis. Phosphoproteins are also components of the nuclear scaffold, and cell cycle variations in these proteins were investigated. The primary phosphorylated species has a molecular weight of 119,000. As with chromatin-associated nonhistones, this nuclear scaffold protein shows substantial incorporation of 32P in S phase, and a high level of incorporation also occurs close to mitosis.

Acetylation and calcium-dependent phosphorylation of histone H3 in nuclei from butyrate-treated HeLa cells.

In HeLa nuclei, 1 microM Ca2+ stimulates 3-fold the phosphorylation of histone H3. Prior treatment of cells with Na butyrate increases the degree of H3 phosphorylation and reveals a correlation between the extents of H3 acetylation and phosphorylation. Acetylation of H3 increases its accessibility to the calcium-dependent kinase. Phosphorylation of H3 occurs at a serine residue located in the trypsin-sensitive region of the protein. Brief digestion of nuclei with DNase I preferentially releases the phosphorylated form of H3 from chromatin.

Antigens in chromatin associated with proliferating and nonproliferating cells

Xenoantisera were raised to total chromatin from the leukemia cell line K562, or materials released through limited deoxyribonuclease I digestion of nuclei or during the control incubation of nuclei without enzyme. The peroxidase-antiperoxidase method of antibody-antigen detection was employed to visualize individual antigens resolved on one-dimensional polyacrylamide gels following transfer to sheets of nitrocellulose (immunotransfers). Each antiserum contained multiple antigen specificities as evidenced by the diverse patterns of reactive bands displayed on the immunotransfers. The most striking difference in antigens recognized between the antisera was observed in the molecular weight region below 50,000, where two highly reactive bands were seen mainly with antiserum to nuclear materials released by deoxyribonuclease I digestion. The antigens detected with all of the antisera were present in chromatins prepared from proliferating cells, while the levels of antigens present in chromatin from non-proliferating peripheral blood lymphocytes were greatly reduced or not detected. Antigens in chromatin from proliferating cells that migrated with apparent molecular weights of 37,000 and 100,000 were not lost once the activities to antigens in lymphocyte chromatin were absorbed out. These two activities were absorbed from antisera with the same amount of chromatins from proliferating cells. Two antigens migrating at molecular weight 52,000 and 76,000 appeared more active in the chromatin from unstimulated lymphocytes than in chromatin from proliferating cells.

Nucleosomes containing histones H1 or H5 are closely interspersed in chromatin.

The distribution of histones H1 and H5 along chromatin fibers has been examined in the nucleated hen erythrocyte. Nucleosome oligomers, produced by micrococcal nuclease digestion of nuclei, were sequentially reacted with affinity-chromatography purified rabbit anti-H5 and sheep anti-rabbit antibodies. Quantitation of the relative amounts of H1 and H5 in the precipitated and supernatant fractions as a function of the oligomer number was consistent with a close interspersion of both types of histones, probably a random one. This conclusion was supported by the immunoprecipitation of longer chromatin fibers. This pattern of distribution appears to apply both to bulk chromatin and to chromatin inactivated during the maturation of the erythrocyte.

Chromatin organization in the rat hypothalamus during early development.

The organization of chromatin in neuronal and glial nuclei isolated from different brain regions of rats during development was studied by digestion of nuclei with micrococcal nuclease. A short chromatin repeat length (approx. 176 base-pairs compared with that of glial nuclei from foetal cerebral cortex (approx. 200 base-pairs) was present in hypothalamic neurons throughout the ages studied, which was similar to the repeat length of cortical neurons from 7- and 25-day-old animals (approx. 174 base-pairs). Whereas in cortical neurons the chromatin repeat length shortened from approx. 200 base-pairs in the foetus to approx. 174 base-pairs in the first postnatal week, the short chromatin repeat length of hypothalamic neurons was already present 2 days before birth, indicating that hypothalamic neurons differentiate earlier than cortical neurons during brain development.

The variation with age of nuclear phosphoproteins released during micrococcal-nuclease digestion and nucleosomal phosphoproteins in three cell types from rat liver.

The nucleosomal non-histone phosphoproteins, and phosphoproteins released during the digestion of nuclei by micrococcal nuclease, were studied in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2- and 4-month-old rats with increasing proportions of parenchymal tetraploid nuclei. Qualitative and quantitative differences in nucleosomal phosphoprotein band patterns were found among different types of nuclei and ages. More phosphoprotein bands were found in nucleosomes derived from parenchymal than stromal nuclei. The number of phosphoproteins released during micrococcal-nuclease digestion increased with age for parenchymal nuclei. The significance of these results, considered in conjunction with the increase of DNA repeat length and decrease of nuclease accessibility with age, is discussed.

Protein distribution amongst chromatin particles following DNAase II digestion

When active chromatin is released as a Mg-soluble fraction following digestion of nuclei with DNAase II, a concomitant release of HMG proteins, and hnRNP particles occurs. Release of HMG 14 and 17 is dependent on active chromatin release, whereas HMG 1 and 2, and hnRNP particles are released in an independent process. The Mg-soluble fraction comprises a heterogeneous mixture of particles of less compact conformation than normal nucleosomes, and prone to protein-induced aggregation. Histone H1, and HMG 14 and 17 appear to be associated with these particles in a reversible manner, whereas HMG 1 and 2 are unbound.

Hb switching in chickens

We have taken advantage of the preferential digestion of active genes by DNAase I to investigate the chromosomal structure of embryonic and adult β-globin genes during erythropoiesis in chick embryos, and in particular to examine the question of hemoglobin switching during development. DNA in isolated red cell nuclei was mildly digested with DNAase I to about 10–15 kb, purified and restricted with a variety of restriction enzymes. The DNA was then separated on agarose gels, transferred to nitrocellulose filters and hybridized with an adult-specific β-globin cDNA clone or a genomic clone containing the genes coding for both an embryonic and an adult β-globin chain. Preferential sensitivity of the respective globin genes was monitored by the disappearance of specific restriction bands after DNAase I digestion of nuclei. In embryonic red cells, both adult and embryonic β-globin genes are very sensitive to DNAase I; however, in adult erythroid lines, the embryonic β-globin gene becomes relatively more resistant but the adult gene remains highly sensitive. Controls showed that all globin genes were resistant to DNAase I in brain nuclei and nuclei from lymphoid cells. Thus the switch from embryonic to adult globin expression is associated with an apparent change in the chromosome structure of the embryonic globin gene as reflected in the gene becoming less accessible to DNAase I in adult red cell nuclei. Our results also show that the chromosomal structure of both adult and embryonic genes is altered in embryonic red cell nuclei; thus the nonexpressed globin gene (that is, the adult gene in embryonic red cells) has already been “recognized” to some degree and marked by the erythroid compartment. The sensitivity of the adult globin gene in embryonic cells may represent a “pre-activation” state of the chromosome.

Accessibility of expressed and non-expressed genes to a restriction nuclease.

Nuclei of mouse liver and an immunoglobulin producing myeloma were digested with HaeIII or its isoschizomer BspRI. The DNA fragment patterns after electrophoresis, blotting, and hybridization were very similar for the two types of nuclei when a probe for the non-expressed beta-globin gene was used. The constant (C) region of the kappa light chain gene, on the other hand, was more accessible to the nuclease in myeloma than on liver nuclei. In myeloma nuclei the BspRI sites were about equally sensitive, in fact the pattern resembled a partial digestion pattern of free DNA. In contrast, in liver nuclei some sites were much more protected than others. This is interpreted by assuming that this single copy non-expressed gene region is covered by nucleosomes which are preferentially located on certain DNA sequences. Restriction nuclease digestion of nuclei seems to be a promising method for the analysis of genes in their expressed and non-expressed states.

Hemoglobin switching in sheep: Only the γ gene is in the active conformation in fetal liver but all the β and γ genes are in the active conformation in bone marrow

Differential expression of the closely linked gamma, beta(A) (or beta(B)), and beta(C) globin genes in sheep results in the production of fetal hemoglobin (Hb F, alpha(2)gamma(2)) during gestation and the adult hemoglobins (Hb A, alpha(2)beta(2) (A), and Hb B, alpha(2)beta(2) (B)) after birth. Erythropoietic stress in certain animals leads to production of Hb C (alpha(2)beta(2) (C)). The molecular mechanism of differential expression of these genes in nuclei of fetal and adult erythroid cells has been investigated by analysis of their susceptibility to digestion by DNase I (genes that are in the conformation associated with active transcription are sensitive to this nuclease). The concentration of globin gene sequences in DNA from control and DNase I-digested nuclei was determined by annealing to synthetic DNAs and analogous cDNA probes derived from recombinant plasmids containing one of the sheep globin genes. In nuclei from sheep fetal liver erythroid cells, the gamma genes but not the beta genes were digested by DNase I; the gamma locus was open but the beta(A) or beta(C) loci was closed, consistent with synthesis of only Hb F by these cells. DNase I digestion of nuclei from bone marrow of anemic sheep making only Hb C or Hb B resulted in equivalent digestion of the beta and gamma gene sequences, although gamma mRNA was not detected in these cells. Digestion by DNase I did not decrease the globin gene sequence concentration in residual DNA of spleen nuclei. As a further control, DNA from digested bone marrow and spleen nuclei were shown to anneal equally well to a cDNA prepared from liver polysomal mRNA. Differential expression of the gamma and beta globin genes in sheep fetal erythroid cell appears to be based on differences in chromatin structure. The gamma globin gene remains in the active conformation in adult erythroid cells; failure of gamma mRNA to accumulate in these cells probably reflects transcriptional or post-transcriptional regulation.

Nucleosome-associated proteins and phosphoproteins of differentiating Friend erythroleukemia cells.

Mononucleosomes derived from brief digestion of uninduced Friend cell nuclei with micrococcal nuclease contain a set of non-histone chromosomal proteins which are partly or altogether missing in the oligomeric nucleosomes. On the other hand, the latter contain a protein of Mr 190,000 not seen in the mononucleosomes. Longer digestion removes most of these non-histone proteins, excepting the Mr 190,000 protein. Brief digestion of nuclei from Friend cells induced by DMSO or by n-butyrate removes most of the non-histone proteins from the nucleosomes, as did the prolonged digestion of uninduced nuclei. The Mr 190,000 protein remains, while a protein of Mr 27,000 is increased. The rate of phosphorylation of histone H1 associated with mononucleosomes was 3 to 4-fold greater in cells induced with DMSO. The major phosphoprotein and most of the other phosphorylated non-histones were modified at the same rate in control and induced cells. However, a Mr 95,000 protein was less phosphorylated in the induced cells.

Cross-linking of DNA with trimethylpsoralen is a probe for chromatin structure

Me 3-psoralen (4,5′,8-trimethylpsoralen) undergoes a photochemical reaction with DNA, resulting in the formation of covalent monoadducts and interstrand cross-links. DNA was photoreacted with 3H-me 3-psoralen inside mouse liver nuclei to the extent of one covalently bound me 3-psoralen molecule per 114 or per 246 base pairs (bp) on the average. The photoreacted nuclei were digested with micrococcal nuclease, which is known to degrade the DNA preferentially between nucleosomes (subunits of chromatin containing approximately 200 bp of DNA). The digestion of nuclei with micrococcal nuclease produced DNA fragments of the same molecular weights whether or not the nuclei had been reacted with me 3-psoralen. The rate of acid solubilization of covalently bound 3H-me 3-psoralen was much greater than that of the bulk DNA when the digestion was performed in nuclei, even though me 3-psoralen-containing regions of DNA were digested more slowly in control experiments with deproteinized DNA. At the digestion limit, when 45% of the DNA had been degraded to acid solubility, 92% of the 3H-me 3-psoralen had been released from chromatin by micrococcal nuclease. Throughout the nuclease digestion, the me 3-psoralen was found to be covalently bound to DNA fragments with the molecular weights previously characterized for micrococcal nuclease digestion products. We conclude that the major site of me 3-psoralen reaction in nuclei is with the DNA most susceptible to micrococcal nuclease—that is, with the DNA between nucleosomes. Because me 3-psoralen cross-links can be located in DNA by electron microscopy, this probe provides a possible method for determining the in vivo location of chromosomal proteins along high molecular weight DNA, such as a transcription unit.

Solenoidal model for superstructure in chromatin.

Chromatin prepared by brief digestion of nuclei with micrococcal nuclease, and extracted in 0.2 mM EDTA, appears in the electron microscope as filaments of about 100 A diameter which coil loosely. In 0.2 mM Mg++ these "nucleofilaments" condense into a supercoil or solenoidal structure of pitch about 110 A corresponding to the diameter of a nucleofilament. It is proposed that the x-ray reflections at orders of 110 A observed in chromatin originate in the spacing between turns of the solenoid rather than that between nucleosomes along the nucleofilament. The solenoidal structure appears to need histone H1 for its stabilization. Under certain conditions, isolated nucleosomes can also aggregate into a similar structure. The solenoidal structure can be correlated with the "thread" of diameter about 300 A observed by other workers in nuclei.

The subunit structure of sea urchin sperm chromatin: A kinetic approach

Many reports have appeared showing that digestion of chromatin DNA by endonucleases results in formation of fragments, apparently multiples of a unit length Hewish and Burgoyne [l] initially reported the formation of these fragments in experiments of autodigsstion of rat liver nuclei. The evidence for an oligomeric structure of the histones [2] led to the proposal of a repeating unit for the chromatin structure consisting of eight histones and 200 base pairs of DNA [3]. DNA fragments of about 200 base pairs in length have been obtained by adding micrococcal nuclease to rat liver nuclei [4], to mouse liver nuclei [5] and to developing trout testis nuclei [6] . In experiments with chromatin from duck reticulocyte nuclei [7] , with chromatin from pea seedlings [8] and with chromatin from yeast [9], the monomer DNA has been found to be about 130 base pairs in length. Recently it has been shown that digestion of nuclei or of native chromatin from rat liver gives similar results [lo] At variance with these results are those on trout sperm chromatin that show no fragmentation pattern upon digestion [6] . We wish to compare here the DNA fragmentation pattern, obtained by digestion of the chromatin of nuclei of sea urchin sperms with different DNases, and the time course of the digestions. The electrophoretic size distributions of the DNA fragments observed at different digestion times with micrococcal nuclease suggest that sperm chromatin is homogeneously structured as a highly regular series of histone oligomers along the DNA molecule. The DNA length, protected by each oligomer, depends on the percent DNA digested and ranges from about 160 base pairs, when about 3% DNA is

The association of [6,7-3H]oestradiol with a nuclear protein.

SUMMARY The binding of [6,7-3H]oestradiol to nuclei from rat dimethylbenzanthracene-induced (DMBA) mammary tumour, uterine epithelium, anterior pituitary and kidney has been studied. Digestion of nuclei labelled in vivo with [6,7-3H]oestradiol, with RNase, DNase and trypsin indicated that only the last enzyme released tritium from the nucleus, indicating that the receptor is a protein. The optimal concentration of sodium chloride for extracting the oestradiol-receptor complex from tumour nuclei was 1-molar. This concentration was required even after enzymic removal of most of the DNA and RNA. Sodium deoxycholate also extracted oestradiol from nuclei but at the same time dissociated the oestradiol-receptor complex. An attempt was made to fractionate the oestradiol-receptor complex on Sephadex and Biogel columns.