Presence of non-histone proteins in nucleosomes

It has been established that nucleosomes are made of histones and DNA fragments. The purpose of this work to establish whether some non-histone proteins are also present in these chromatin subunits. We have found that nucleosome preparations contain phosphorylated non-histone proteins and protein kinases by sucrose gradient analysis. In order to establish whether these proteins are actually bound to nucleosomes or if they represent unbound or aggregated proteins, the following experiments were performed. (a) Free non-histone proteins and proteins released from chromatin by DNase overdigestion were analyzed by sucrose gradient centrifugation. No phosphoproteins but some phosvitin kinase activity was found in the part of the gradient which contained the nucleosomes. It could be assumed that part of the phosphoproteins are bound to nucleosomes. (b) A digestion of nucleosomes with DNase I suppressed the phosvitin kinase activity in the 11-S region of the gradient. (c) High ionic strength, which extracted non-histone proteins, suppressed the phosvitin kinase activity in the nucleosome region. Part of phosvitin kinase and of nuclear phosphoproteins are therefore bound to nucleosomes and are released by nuclease digestion and by high ionic strength.     

Nematode chromosomal proteins--I. Isolation of chromatin and preliminary characterization of the chromosomal proteins of Caenorhabditis elegans.

1. A procedure is described which gives clean chromatin preparations from the free-living nematode Caenorhabditis elegans. It involves homogenization using glass beads, collection of the precipitate from a low speed centrifugation, removal of cell membranes with Triton X-100, several washes with 0.14 M NaCl, sucrose density gradient centrifugation, a cycle of extraction and reprecipitation using dilute Tris buffer and 0.14 M NaCl respectively, and final extraction of the purified deoxyribonucleoprotein in 10 mM Tris-HCl (pH 8). 2. Acidic urea gel electrophoresis of the histones from C. elegans yielded 4 main groups which were preliminary identified as H1, H2a (+ H3?), H2b, H4 and moved on the gels in that order of increasing mobility. the coincidence of histone H3 with H2a was putative, but its presence was firmly suggested by the generation of a dimeric form in oxidizing conditions. 3. By SDS-Tris-glycine gel electrophoresis of the non-histone chromosomal proteins of C. elegans, about 18 proteins were distinguished with molecular weights ranging from 15,000 to 100,000 daltons.     

Nuclear proteins : IV. Deficiency of non-histone proteins in condensed chromatin of Drosophila virilis and mouse

Drosophila virilis egg nuclei were fractionated by a technique of multiple sonication and centrifugation in an isotonic buffer containing 0.15 M NaCl, Mg²⁺ and Ca²⁺. This allowed the condensed chromatin to remain tightly condensed. By Hoechst 33258 staining this procedure resulted in brightly fluorescing and poorly fluorescing fractions. The brightly fluorescing fraction was enriched in satellite DNA. Examination of the non-histone proteins by SDS slab gel electrophoresis showed that this fraction was markedly deficient in non-histone proteins and contained no unique major non-histone proteins. The poorly fluorescing fractions were enriched in non-histone proteins. Similar results were obtained with mouse liver nuclei. Comparison of the non-histone proteins of D. virilis (40% satellite DNA), D. americana (35% satellite DNA), and D. ezoana (no satellites) confirmed the absence of major, satellite specific, non-histone proteins. These results, suggesting condensed chromatin is primarily a DNA-histone complex, agree with published cytochemical studies.     

Porcine follitropin. The amino-acid sequence of the beta subunit

By treatment with tRNA in the presence of 1 mM MgCl2, a chromatin preparation was obtained containing all five major histone fractions but lacking a considerable portion of non-histone proteins. This chromatin preparation as well as chromatin extracted with 0.6 M NaCl (depleted of H1 histone and some non-histone proteins) were characterized in respect of solubility and chromatin DNA accessibility. Both samples possessed practically the same solubility in the presence of 0.15 M NaCl and 1 mM MgCl2. The solubility of tRNA-treated chromatin in 5 and 10 mM MgCl2 was higher than that of salt-extracted chromation. The accessibility of the DNA of these chromatin preparations was tested with DNA-dependent RNA polymerase of Escherichia coli as a probe, using procedure that permits measurement of binding site frequency. Both tRNA-treated and salt-extracted chromatin contained as many as 33% and untreated chromatin as few as 4% of the number of binding sites found on protein-free DNA. These results demonstrate that at least in part the non-histone proteins are responsible for salt-induced insolubility and low DNA accessibility of chromatin, thus revealing the importance of non-histone proteins in the maintenance of an overall chromatin structure.     

Biochemical characterization of chromatin fractions isolated from induced and uninduced friend erythroleukemia cells

Summary Chromatin fractions from Friend erythroleukemia cells after induction of differentiation by dimethylsulfoxide (DMSO) were compared in their biochemical characteristics to fractions from uninduced cells. Fractions were prepared by extracting chromatin from nuclei after mild micrococcal nuclease treatment with increasing concentrations of NaCl according to Sanders [1]. This procedure has been found to release chromatin containing hyperacetylated histones preferentially [2]. The fractions obtained by this procedure were analysed in respect to the amount of chromatin released, the amount of histone H1, the degree of acetylation of histone H4, the presence of non-histone proteins and the concentration of transcribed and non-transcribed sequences. It was found that the fractions differ in the amount of histone H1 present, in several non-histone proteins and in the acetylation of histonie H4, regardless whether induced or uninduced cells were analysed. The distribution of transcribed sequences versus non-transcribed sequences among the fractions was the same, demonstrating that this fractionation procedure, although leading to fractions with biochemical differences, is not able to discriminate functional states of chromatin and that the biochemical characteristics of the fractions may be common to both, active as well as inactive states of chromatin.     

The association of a magnesium-dependent endonuclease activity with a nucleosome fraction from rat-liver nuclei

The nucleosomes released by the incubation (autodigestion) of rat-liver nuclei were fractionated by sucrose-density gradient centrifugation, and subjected to nuclease assay with heat-denatured 3H-DNA from Escherichia coli as an exogenous substrate. With increasing incubation time, the nuclease activity was enhanced and localized in the mono/tetra-, hexa/hepta-, and long-chain oligonucleosome fractions. In contrast, independent of the nucleosome size, the activities of 0.35 M NaCl-soluble fractions from them were found to be almost equal in terms of specific activity (dpm/nucleosomal DNA). Such nuclease activity was not detected in the sucrose gradient (top region) lacking nucleosomes and/or chromatin. When the chromatin was dialyzed against a 0.35 M NaCl buffer and then fractionated in a sucrose gradient containing 0.35 M NaCl, most of the nuclease activity was solubilized into the above top region. On gel filtration of the mononucleosome fraction in the 0.35 M NaCl buffer, the nuclease activity was eluted at the position of 36,000 daltons. This nuclease cleaved heat-denatured DNA more rapidly than the native DNA in the presence of Mg2+, and had the ability to make both single-strand nicks and double-strand cuts in pBR322 DNA; in other words, it had an endonucleolytic activity. Moreover, four different classes of mononucleosomes were fractionated by electrophoresis of the nucleosomes released by autodigestion of the nuclei. These mononucleosomes also showed nuclease activity with the heat-denatured DNA. Thus, the present studies suggest that an Mg2+-dependent endonuclease of about 36,000 daltons is associated with the nucleosome particle(s) in rat-liver nuclei.     

Decrease in ADP-ribosylation of HeLa non-histone proteins from interphase to metaphase

Variations for non-histones in the ADP-ribosylating activities of interphase and metaphase cells were investigated. 32P-Labeled nicotinamide adenine dinucleotide ([32P]NAD), the specific precursor for the modification, was used to radioactively label proteins. Permeabilized interphase and mitotic cells, as well as isolated nuclei and chromosomes, were incubated with the label. One-dimensional and two-dimensional gels of the proteins of total nuclei and chromatin labeled with [32P]NAD showed more than 100 modified species. Changing the labeling conditions resulted in generally similar patterns of modified proteins, though the overall levels of incorporation and the distributions of label among species were significantly affected. A less complex pattern was found for nuclear scaffolds. The major ADP-ribosylated proteins included the lamins and poly(ADP-ribose) polymerase. Inhibitors of ADP-ribosylation were effective in preventing the incorporation of label by most non-histones. Snake venom phosphodiesterase readily removed protein-bound 32P radioactivity. A fundamentally different distribution of label from that of interphase nuclei and chromatin was found for metaphase chromosome non-histones. Instead of 100 or more species, the only major acceptor of label was poly(ADP-ribose) polymerase. This profound change during mitosis may indicate a structural role for ADP-ribosylation of non-histone proteins.     

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis.

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine.     

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.     

Non-histone chromosomal proteins easily extractible from chick erythrocytes.

Those non-histone chromosomal proteins which are easily extractible from chick erythrocytes differ substantially from proteins similarly extracted from other tissues of various species. Although a protein P1 was isolated along with histone H1 by extraction of calf thymus chromatin with HC1O4, the same procedure did not extract this protein from chick erythrocyte chromatin of either normal or regenerating blood. Likewise , non-histone proteins extracted with 0.35 M NaCl from calf thymus differed from those of normal chick erythrocytes, which were qualitatively identical but quantitatively inferior to those of regenerating blood. The major protein of about 25 000 molecular weight, totally extracted with 0.35 M NaCl from calf thymus, was not found in chick erythrocyte chromatin, but rather another major protein of about 35 000 molecular weight was partially extracted from erythrocytes.     

Role of non-histone proteins in chromatin solubility

Treatment of chromatin gel with low ionic strength solution of tRNA has produced the dioxyribonucleoprotein (dnptRNA) in which only part of non-histone proteins was removed without loss of any major histone fraction. The solubility of DNP in the presence of 0.15 M NaCl and 1 to 5 mM MgCl2 was considerably higher than that of initial untreated chromatin. It has been assumed that the solubility of chromatin depended primarily on some non-histone proteins and not on H1 histone.     

Separation of nucleic acids and chromatin proteins by hydrophobic interaction chromatography

A procedure by which chromatin proteins (histones and non-histones) can be rapidly separated from nucleic acids by hydrophobic interaction chromatography is described. The procedure is carried out under non-rigorous conditions that must be assumed to induce little irreversible change in the biological properties of most proteins. More than 90% (w/w) of the chromatin proteins can be retained by hydrophobic interaction while nucleic acids pass quantitatively through the columns. By gradient elution of the columns the histones can be divided into fractions containing H1, H2A/H2B and H3/H4, and at the same time a subfractionation of the non-histone proteins is obtained. Protein recovery depends on the type of column used, but exceeds 80% (w/w) with even the most strongly binding hydrophobic matrix investigated.     

A new cAMP independent protein kinase tightly bound to DNA, in rat liver nuclei

A protein kinase has been characterized among the proteins tightly bound to DNA. It is not extracted with 1 M NaCl and is released by extensive DNase I digestion. This enzyme is able to phosphorylate nucleosomal histones, essentially H2B and H3, and several non-histone proteins associated with DNA, on serine residue(s). It does not phosphorylate protamine, casein, phosvitin and the chromosomal non-histone proteins extracted with 1 M NaCl and is cAMP independent. This protein kinase can be distinguished from the previously described enzymes.     

Nucleoprotein chromatin subunit from Physarum polycephalum

The nucleoproteins resulting from digestion of the nuclei of the true slime mold Pysarum polycephalum with micrococcal nuclease have been resolved according to the size classes in linear sucrose gradients containg 0.5 M NaCl, and analysed for DNA, RNA and protein content. The basic nucleoprotein subunit has been found to contain a DNA fragment of about 150--170 base pairs complexed with an approximately equal amount, on a weight basis, of basic proteins and a relatively small amount of non-histone proteins (about 35% of the amount of DNA). Higher nucleoprotein oligomers were shown to contain spacer DNA fragments between adjacent subunits and a considerably higher ratio of non-histone proteins to DNA than the basic subunit. Both the basic subunit and higher nucleoprotein oligomers of Physarum chromatin contain some amount of tightly bound RNA. However, in contrast to the distribution of the non-histone proteins, the ratio of RNA to RNA is similar in both fractions.     

Removal of degradation products from calf thymus high mobility group non-histone chromatin proteins by chromatography on immobilized double-stranded DNA

The substantial protease activity in calf thymus chromatin inevitably produces some degradation of high mobility group (HMG) non-histone proteins in NaCl extracts of calf thymus chromatin. We have found that proteins considered to be degradation products can be conveniently and cleanly separated from intact high mobility group proteins 1 and 2 by chromatography on double-stranded DNA-cellulose in 0.2 M NaCl/1 mM Tris-HCl (pH 7.5). Under those conditions, only the presumptive degradation products are retained by the column.     

Comparative study of lymphocyte non-histone proteins

The non-histone chromosomal proteins of bovine lymphocytes were investigated by the two dimensional gel electrophoresis of O'Farrell. The 0.35 M NaCl extractable proteins from lymphocyte nuclei, the high mobility group proteins (HMG) and some proteins released from nuclei by DNase I were compared on the basis of their electrophoretic patterns.     

A computer program for the calculation of sedimentation coefficients and molecular weights of nucleic acids

A procedure is described for the purification of nuclei and identification of chromatin proteins in transformed epithelial cell lines from mammalian bladder and salivary gland. Nuclear purification was performed by homogenization, in hypotonic buffer containing polyamines to stabilize the nuclear structure, followed by 0.1–0.2% Triton X-100 washing and centrifugation through 2.2 m sucrose. Chromatin was liberated from nuclei by freeze-thawing in hypotonic buffer and the chromatin proteins were extracted with 7 m urea/3 m NaCl. The chromatin proteins were identified using NEPHGE two-dimensional electrophoresis and fluorographic autoradiography. This procedure enabled detection of histones and a range of basic nonhistone chromatin proteins, following cell culture in the presence of low levels of l-(4,5-³H)leucine.     

The presence of glycosyltransferases on chromatin acceptors in monkey liver nuclear membranes (author's transl)

Nuclei were prepared from monkey hepatocytes by centrifugation of the homogenate on a cushion of 2.3 M sucrose, during 45 min at 100000 X g. The yield was 2.2 x 10(7) nuclei per g of liver, and 70% of te homogenate DNA was recovered in these nuclei. An electron microscopic study as well as a biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. A mannosyltransferase and an N-acetylglucosaminyltransferase, working on endogenous glycoproteic acceptors, are present in the nuclei for 1.4 and 6.5% of the homogenate activities, respectively. The nuclei are hydrolysed by DNAse I. The suspension, adjusted in 1.9 M sucrose, was centrifuged for 2 h at 100000 X g, under buffer layer. Purified nuclear membranes were collected at the interface. These membranes did not contain any more endoplasmic reticulum enzyme activities, but the mannosyl and N-acetylglucosaminyltransferase activities were still present. They essentially work on an exogenous chromatin acceptor, prepared by lysis of the nuclei. The eventual role of these glycosyltransferases in the glycosylation of non-histone proteins is discussed.