Detection and identification of a tumor-associated heteroorganic antigen of Zajdela hepatoma in non-histone proteins of chromatin

By polyacrylamide gel electrophoresis, a phosphoprotein with mol. weight of 42 kDa was detected in non-histone proteins (NHP) of chromatin of Zajdela ascitic hepatoma cells eluted from phosphocellulose with 0.4-0.5 M NaCl. A protein of the same mol. weight is present in narrow fractions of rat kidney chromatin, but is absent in rat liver. It is suggested that the revealed protein corresponds to the tumor-associated heteroorganic NHP antigen detected earlier in NHP chromatin of rat tumor cells. By MALDI mass spectrometry, this phosphoprotein was identified as ERK2/mitogen-activated protein kinase.     

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.     

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide. II. Phosphorylation.

Stimulation of mouse lymphocytes with the B lymphocyte specific mitogen lipopolysaccharide results in an increased rate of phosphorylation of non-histone chromatin proteins. An initial small increase in phosphorylation occurs during the first 2 h and a much larger increase after 24 h of culture with mitogen. The phosphorylated nuclear and cytoplasmic proteins were analysed by polyacrylamide gel electrophoresis and the stimulation index of each prominent peak measured. It was inferred that selective stimulation of the phosphorylation of individual proteins had occurred from: (1) the range of stimulation indices for different proteins, and (2) the appearance, after 8 h stimulation of an apparently newly phosphorylated non-histone chromatin protein of molecular weight 115 000. The pool size of ATP was monitored and showed only small changes during the first 24 h of exposure to lipopolysaccharide. Phosphatase activity was found to be associated with lymphocyte chromatin and nucleoplasm and may help to regulate the level of phosphorylation of non-histone chromatin proteins in vivo. To preserve phosphorylated proteins during their isolation phosphatase activity was inhibited by Na2MoO4. The selective changes in phosphorylation of nuclear proteins precede, and continue during, the stimulation of immunoglobulin and DNA synthesis. Our results are thus consistent with the hypothesis that phosphorylation of non-histone chromatin proteins plays a role in the regulation of gene expression in B lymphocytes.     

Monoclonal antibodies to a phosphoprotein from chromatin of rat liver.

Three monoclonal antibody subclasses (IgG1, IgG2a, and IgM) were raised to the phosphoprotein B2 (Mr 68000, pI6.5-8.2) which has been shown previously to be associated with the nucleosomes of rat liver nuclei. These antibodies do not show any significant cross reactivity with CM-cellulose 'unbound' non-histone chromosomal proteins, bovine serum albumin or histones. Further verification of the specificity of these antibodies to this phosphoprotein was carried out using both 'dot' blot and immunological transfer analysis ('Western blot'). The monoclonal antibodies (IgG1 and IgG2a) could also be used to semi-quantify the phosphoprotein B2 in rat liver nuclei. The high specificity and unlimited availability of this type of probe provides a means to study the role(s) of this phosphoprotein in the overall scheme of actively transcribed chromatin.     

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide.

The synthesis of non-histone chromatin proteins and nucleoplasmic proteins has been followed during lipopolysaccharide-induced division and differentiation of murine B lymphocytes. Synthesis was measured by pulse labelling with [3H]leucine, extraction of proteins was under conditions designed to prevent proteolysis and analysis of labelled proteins was by polyacrylamide gel electrophoresis. The average specific activity of non-histone chromatin proteins increased 3-fold, to a maximum, after culture for 24 h with lipopolysaccharide. Comparison of the relative synthesis of individual proteins (stimulation index) reveals three distinct responses: (1) those in the largest group show low stimulation indices, generally less than two; (2) a group of four proteins have indices between 4 and 5; (3) two proteins (molecular weights 21 000 and 22 000) both show an index of 5 at 24 h rising to between 7 and 8 by 48 h when the average specific activity is falling, coinciding with the period of rapid differentiation to high rate IgM secretion. Additionaly at this time, a newly labelled protein (Mr = 36 500) appears in the nucleoplasm followed by a second protein (Mr = 63 000) appearing between 48 and 72 h. The patterns of change are consistent with an overall increase in non-histone chromatin proteins synthesis, necessary for cell division, with superimposed specific changes in synthesis of non-histone chromatin proteins which could be related to regulation of cell differentiation.     

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.     

Isolation of chromatin fragments rich in non-histone protein after endonuclease digestion of rat liver nuclei

Detergent-washed rat liver nuclei, prepared in the presence of a protease inhibitor, were incubated for up to 60 min at 37 °C. The action of endonucleases produced chromatin fragments which could be removed from the nuclei by extraction with 8 M urea 50 mM phosphate, pH 7.6, 15% of the total nuclear DNA being extracted. No DNA could be detected in this extract after incubation of nuclei at 4 °C. The chromatin fragments were sedimented by centrifugation at 90000 g or by chromatography on Sepharose 4B. The DNA fragment sizes were similar to those found in nucleosome particles but the protein/DNA ratio was approx. 5.3:1. The nuclei were prelabelled with [³H]tryptophan and 60% of the label present in the 8 M urea extract was found to sediment with the chromatin fraction. SDS polyacrylamide gel electrophoresis of the latter showed the presence, in addition to histones, of at least 25 polypeptide species of tightly bound non-histone proteins with molecular weights in excess of 30000.     

Interaction of non-histone proteins with DNA and chromatin from Drosophila and mouse cells. Specificity, isolation and analysis of complexes.

The specificity of the binding of purified non-histone proteins to DNA has been investigated through two types of experiments. Using a nitrocellulose filter assay at a low protein/DNA ratio, the binding of mouse non-histone proteins to mouse DNA was twice as great as the binding of mouse non histone protein to Drosophila DNA. The reverse experiment using Drosophila non-histone protein confirmed the interpretation that some protein . DNA complexes were specific. Protein . DNA complexes isolated by gel filtration chromatography indicated that 20% or 10% of the non-histone protein was bound to homologous or heterologous DNA respectively. Purified non-histone proteins bound with lower efficiency (15%) than unpurified but with higher specificity to soluble chromatin than to naked DNA. This binding did not result from an exchange between chromatin non-histone proteins and purified non-histone proteins added in excess. DNA-bound and chromatin-bound proteins were analysed on polyacrylamide gels. Whereas no major qualitative differences were observed with DNA-bound proteins, some proteins bound to homologous mouse chromatin were different from those bound to heterologous Drosophila chromatin. These results suggest a possible role of DNA-bound non-histone proteins in the regulation of gene expression.     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.     

Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone

A phosphorylated glycoprotein was purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 M EDTA in 4 M guanidinium chloride. A high level of purity for the preparation was indicated by a single band on sodium dodecyl sulfate (SDS)-gradient gel electrophoresis, sedimentation equilibrium ultracentrifugal data, and by automated Edman degradation results. The molecular weight of the phosphoprotein was shown to be about 44,000 by sedimentation equilibrium analyses in 4 M guanidinium chloride, even though an Mr of 75,000 was obtained by 5-15% SDS-polyacrylamide gel electrophoresis. Subsequent analysis by 15% SDS-polyacrylamide gel electrophoresis gave an Mr of 45,000. Analytical data showed that the protein contained 16.6% carbohydrate, possibly including 1 N-linked oligosaccharide and 5-6 O-linked oligosaccharides. The aspartic acid- and glutamic acid-rich protein contained about 300 amino acid residues including 1 phosphothreonine and 12 phosphoserine residues. Alkaline beta-elimination/NaBH4 reduction data showed that the phosphate obtained by complete acid hydrolysis prior to amino acid analysis was equivalent to the phosphate subject to alkaline beta-elimination. In this experiment, the losses of serine plus threonine exceeded the amount of phosphate liberated by 5-6 residues/protein. These serine and threonine residues probably represent O-linked oligosaccharides, since the protein contained about this number of N-acetyl-galactosamine residues. That the phosphoprotein is synthesized and secreted by osteoblast-like cells was shown with cultures of clonal rat osteosarcoma cells. After pulsing with 32PO4 the proteins secreted into the medium were precipitated with trichloroacetic acid and the radiolabeled proteins were immunoadsorbed. A protein migrating in the same position, on 5-15% SDS-polyacrylamide gel electrophoresis (i.e. with an Mr = 75,000) and on 15% gels (Mr = 45,000), as the phosphoprotein obtained from bone could be specifically immunoprecipitated.     

NUCLEAR PROTEIN PATTERNS IN DEVELOPING AND ADULT BRAIN AND IN ETHYLNITROSOUREA-INDUCED NEUROECTODERMAL TUMOURS OF THE RAT1,2

In a comparative study, the patterns of histones and non-histone proteins were analysed in the chromatin of foetal (18th day of gestation), 10-day-old, and adult BD IX-rat brain, as well as in the chromatin of two ethylnitrosourea-induced neuroectodermal tumours (TV1A1 and GV1A1) and the corresponding malignant cell culture lines TV1C1 and GV1C1. Separation of nuclear proteins at high resolution was obtained by electrophoresis in 15% and 10% polyacrylamide gels containing urea (2·5 m or 6·25 m ). In spite of an overall similarity, significant quantitative and qualitative differences were observed between the respective non-histone proteins banding patterns of normal brain and the neoplastic cells analysed. The non-histone protein banding patterns of brain (∼40 different bands) at different stages of development revealed both quantitative differences and the presence of particular bands characteristic of foetal or adult brain, respectively. Both the‘foetal’and‘adult’non-histone protein bands also appeared in the electrophoretograms of the neoplastic neuroectodermal cells.     

Non-histone chromatin protein fractions associated with ‘active’ chromatin in embryonic chicken liver

Non-histone chromatin proteins synthesized during chicken embryonic liver development were labeled with [3H]tryptophan and [3H]methionine and characterized by electrophoresis. During embryonic development protein/DNA ratio in chromatin was low (1.30-1.62) but synthesis of non-histone protein was high. Especially one characteristic fraction K (MW 18 000), tightly bound with DNA was preferentially associated with DNAase II sensitive, active transcribed sequences. In 7-day old and adult chicken synthesis of all non-histone proteins was low, fraction K was absent or synthesized only in small amounts in association with non-active sequences, however protein/DNA ratio in chromatin was high (2.30-2.33).     

The heterogeneity of rat high density lipoproteins.

Ubiquitin was first found in nuclei in protein A24 where its carboxyl terminal is covalently bound to histone 2A by an isopeptide linkage (Goldknopf, I. L. and Busch, H. (1977) Proc. Natl. Acad. Sci. USA $\text{74}$, 864–868). Two-dimensional polyacrylamide gel electrophoresis of the 0.4 N H₂SO₄ soluble proteins from fractionated rat liver chromatin showed that protein A24 and histones H1, H2A, H2B, H3 and H4 were present in fractions P1 and P2 and markedly diminished in relative amounts in fraction S2. Conversely, a spot designated Ub was found in fraction S2 along with an increased amount and number of non-histone proteins. The Ub spot was not found in chromatin fractions P1 and P2. Ub was identified as ubiquitin by migration on two-dimensional gels and after purification by preparative polyacrylamide gel electrophoresis by its methionine NH₂-terminal amino acid and its amino acid composition.     

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.     

Stability of liver nuclear proteins in inbred strains of mice

1. A remarkable similarity in the gel patterns of liver nuclear proteins between four inbred strains of mice (A.CA, B10.A, CBA and DBA/2) was observed. 2. Only a very few quantitative differences were detected in the protein spot patterns of nucleoplasmic (spot of about 41 kDa) and chromatin (spot of about 37 kDa) non-histone proteins between those strains of mice. 3. Comparison of two-dimensional gel patterns of non-histone proteins from males and females revealed a few sex-linked spots. Nucleoplasmic protein with molecular weight of about 59 kDa and chromatin proteins with molecular weights of approximately 47 and 57 kDa were more abundant in liver nuclei of male mouse.