Cellular distribution of the hamster liver specific nucleolar antigens

The immunochemical localization of hamster liver nucleolar antigens in subcellular fractions (nuclei, 10,000 x g pellet, 100,000 x g pellet and supernatant), nuclear substructures (chromatin, nuclear matrix, nuclear envelope, nucleoli, RNP particles and nucleosomes), and three classes of nonhistone chromosomal proteins with different affinities to DNA (NHCP1, NHCP2 and NHCP3) from nuclease-sensitive and nuclease-resistant chromatin fractions of hamster liver were studied. Six main nucleolar antigens with mol. wts 27,000; 29,000; 30,000; 36,000; 45,000; and 46,000 were found in subcellular fractions, nuclear substructures and classes of non-histone proteins of hamster liver. The antigens with mol.wts of approx. 27,000; 29,000; and 36,000 which were absent in hamster pancreas, spleen and Kirkman--Robbins hepatoma nuclei, seem specific for liver tissue.     

Diversity of non-histone protein fraction NHCP2 from hamster Kirkman-Robbins hepatoma and liver

Non-histone protein fraction NHCP2 eluted from hydroxyapatite with 100mM phosphate buffer (pH6.8) of undigested, nuclease-sensitive and nuclease-resistant nuclei of hamster Kirkman-Robbins hepatoma and liver was studied by two-dimensional gel electrophoresis and microcomplement fixation test in the presence of antibodies elicited against NHCP2 of examined tissues. The NHCP2 of undigested nuclei as well as from two chromatin fractions with different susceptibility to nuclease of both tissues, besides many common components, showed some differences in their non-histone patterns especially within molecular weights of 17 000–24 000, 36 000–44 000 and 60 000–90 000. Immunological analysis confirmed the high specificity of hepatoma non-histone components of the NHCP2 fraction. However, these components appeared not to be exclusively localized either in nuclease-sensitive or nuclease-resistant part of chromatin of neoplastic tissue.     

Specificity of Kirkman-Robbins hepatoma non-histone chromatin proteins: electrophoretic and immunological analyses

The specificity of Kirkman-Robbins hepatoma and hamster liver non-histone chromatin proteins has been studied by comparing polypeptide patterns in polyacrylamide gel electrophoresis and by their immunological activity in the complement fixation test. Non-histone proteins were separated from DNA with a polyethylene glycol-dextran mixture and fractionated by hydroxylapatite chromatography into three classes named NHCP1, NHCP2, and NHCP3. Electrophoretic analysis indicated that among the non-histone proteins of Kirkman-Robbins hepatoma and hamster liver differences mainly of a quantitative nature can be observed. However, the polypeptides with molecular weight 25 000, 31 000, 36 000, 73 000 in NHCP1; 20 000, 40 000 in NHCP2 and 20 000, 23 000, 32 000, 38 000, 44 000, 75 000, 80 000 in NHCP3 were found to be specific for hepatoma chromatin. Application of antibodies against NHCP1, NHCP2 and dehistonized chromatin of Kirkman-Robbins hepatoma revealed that the highest specificity of NHCP2 eluted from hydroxylapatite with 100 mM phosphate buffer at pH 6.8. The NHCP1 of hepatoma shares some common antigenic determinants with analogous proteins of liver. On the other hand non-histone proteins specific for hepatoma dehistonized chromatin can be localized in the NHCP3 and partially in the NHCP1 fractions.     

Molecular characterization of non-histone chromatin proteins from experimental tumours

Using two-dimensional (2-D) electrophoresis, two non-histone chromatin protein fractions (NHCP1 and NHCP2) from three animal tumours (Kirkman-Robbins hepatoma, Morris hepatoma 7777 and Ehrlich ascites cells) and normal hamster liver were analyzed. Apart from many common components several tissue specific polypeptides of the NHCP1 and NHCP2 fractions were detected. It was found that some spots present in electropherograms of non-histone proteins of tumour cells (M X 10(-3)/pI): 17-24/4.9-6.5 (NHCP1 and NHCP2); 34-41/4.9-6.0 (HCP1 and NHCP2); 44-46/5.3-7.5 (HCP2); 46-49/5.0-7.5 (NHCP1); 49/5.9-7.5 (NHCP2) and 102-134/5.6-7.0 (NHCP1) were absent from normal liver.     

Molecular and functional diversity of non-histone protein fraction NHCP1 from hamster Kirkman-Robbins hepatoma and liver

Non-histone protein fraction NHCP1 of micrococcal nuclease-sensitive and nuclease-resistant chromatin from Kirkman-Robbins hepatoma and hamster liver was studied by two-dimensional electrophoresis followed by Coomassie and silver staining and by microcomplement fixation technique in the presence of antibodies elicited against NHCP1 of both tissues. Apart from many common spots several tissue specific components associated with either nuclease-sensitive or nuclease-resistant chromatin were found. The presence of tissue specific components among NHCP1 from hepatoma and liver was confirmed by immunological analysis. It was stated that these components are exclusively localized in nuclease-resistant part of chromatin from neoplastic and normal tissues thus suggesting their structural function.     

Integrity of proteins in reconstituted chromatin.

Rat liver chromatin reconstituted from fractionated histones, chromosomal non-histone proteins, and DNA is extensively degraded by chromatin-bound protease.     

Growth-related changes of non-histone chromatin proteins from Kirkman-Robbins hepatoma

1. Non-histone chromatin protein fractions NHCP1 and NHCP2 eluted from hydroxyapatite with 50 and 100 mM phosphate buffer (pH 6.8) from nuclei of Kirkman-Robbins hepatoma from 4th, 7th and 9th day of growth were analysed by one- and two-dimensional gel electrophoresis as well as Western blot technique in the presence of antibodies elicited against NHCP1, NHCP2 and dehistonized chromatin of hamster hepatoma and liver. 2. The presence of electrophoretically and immunologically specific components among NHCP1 and NHCP2 fractions during Kirkman-Robbins hepatoma growth was stated.     

Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1.

Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.     

Fractionation of micrococcal nuclease-digested chromatin solubilized at physiologic ionic strength

When mouse brain nuclei are optimally digested with micrococcal nuclease, most of the chromatin is soluble in a 180 mM salt/1 mM EDTA buffer [1]. At this ionic concentration, chromatin maintains its native structure [2]. In an attempt to selectively extract different fractions of chromatin from digested nuclei, we have examined the differential solubility of chromatin in the 180 mM salt buffer containing concentrations of MgCl2 ranging from 2 to 0 mM. The results suggest that digested chromatin may be fractionated into specific soluble chromatin fractions which correspond to nuclease-sensitive chromatin, bulk chromatin, and heterochromatin. These soluble fractions have a high molecular weight (up to 20 kbp), and contain a full complement of histones as well as a complex assortment of non-histone proteins. The residual insoluble fraction may be equivalent to a native, nuclear matrix-bound chromatin fraction.     

An electrophoretic comparison of non-histone proteins from rat liver total chromatin and chromatin depleted of 0.35 M NaCl soluble proteins

• 1.1. A procedure of isolation of non-histone proteins from rat liver chromatin in mild conditions provided 3 groups of these proteins, i.e. NHCP1, NHCP2 and NHCP3.
• 2.2. The investigated proteins are devoid of DNA and revealed various influences on RNA synthesis in vitro.
• 3.3. The extraction of rat liver chromatin with 0.35 M NaCl (pH 7.5) removed about 30% of examined proteins. Electrophoretic patterns of 3 groups of non-histone proteins from total chromatin and chromatin depleted of 0.35 M NaCl soluble proteins are compared.

     

Chromosomal proteins from the spruce budworm Choristoneura fumiferana

The electrophoretic properties of histones and non-histone chromosomal proteins (NHCP) were examined in second instar larvae of the spruce budworm, Choristoneura fumiferana. The five main histone fractions, typical of most organisms, were present. Subfractions were not observed for the very-lysine-rich F1 histone. By contrast to the histones, the NHCP displayed considerable heterogeneity with no fewer than 38 bands. The molecular weights of the NHCP ranged from 9000 to over 110,000 daltons.     

Studies on erythrocruorin. V. Nuclear magnetic resonance quadrupole relaxation study of sodium, calcium and chloride binding.

Metabolically labeled non-histone chromosomal proteins of high specific activity were fractionated on the basis of their sequential extractability from Krebs II chromatin with urea/salt solutions according to Bekhor et al. (1974a). The binding of each of these NHCP² classes to protein-free DNA and histone-DNA complexes (nucleohistone) was measured and compared to the binding to DNA substituted with 5-bromo-2′-deoxyuridine. After reconstitution of the interacting components, the binding of NHCP and histones was measured according to Scatchard formalism by titration of fixed amounts of DNA with increasing inputs of protein ligands under stringent conditions of 0.25 ionic strength, pH 8.0. Histone binding to either native DNA or BrUrd-substituted DNA was found to be essentially the same. In the presence of histones, the binding for all NHCP classes, except for medium 3 NHCP, was enhanced by an order of magnitude over the binding values for NHCP to DNA in the absence of histones. The binding of NHCP to DNA was thus strongly influenced by histones bound to DNA. A general and significant decrease in histone content in the complexes relative to increased NHCP binding was also apparent, with medium 3 NHCP having the greatest activity to weaken histone interaction with DNA and medium 0 the least. Enhancement in NHCP binding to BrUd-substituted DNA in the presence of histones was decreased to about 50% of the binding to control DNA. The distribution and quantity of DNA binding and non-DNA binding NHCP was also estimated by photochemical attachment to 33% BrUrd-substituted DNA in tryptophan-labeled chromatin and by direct binding assays. We have obtained 30% crosslinking for either histones or NHCP to DNA in stringently formed complexes. In histone-NHCP-DNA complexes, histone crosslinking remained unchanged, while that of NHCP increased to 70%. This is further evidence for a modification in the binding of NHCP to DNA in the presence of histones. The percentage of NHCP crosslinked to DNA in native chromatin ranged from 24% for medium 0 NHCP to 50% for medium 1 and 3 NHCP with an average of 35% for total NHCP. These results plus the direct binding assays indicate that NHCP, in addition to high affinity DNA binding, also interacts non-specifically to DNA and to proteins in chromatin. A mechanism is also being proposed to account for the observed BrUrd effects in chromatin.     

Effect of non-histone proteins on thermal transition of chromatin and of DNA.

The effect of chromatin non-histone protein on DNA and chromatin stability is investigated by differential thermal denaturation method. 1) Chromatin (rat liver) yields a multiphasic melting profile. The major part of the melting curve of this chromatin is situated at temperatures higher than pure DNA, with a distinct contribution due to nucleosomes melting. A minor part melts at temperatures lower than DNA which may be assigned to chromatin non-histone protein-DNA complex which destabilized DNA structure. 2) Heparin which extracts histones lowers the melting profile of chromatin and one observes also a contribution with a Tm lower that of pure DNA. In contrast, extraction on non-histone proteins by urea supresses the low Tm peak. 3) Reconstitution of chromatin non-histone protein-DNA complexes confirms the existence of a fraction of chromatin non-histone protein which lowers the melting temperature when compared to pure DNA. It is concluded that chromatin non-histone proteins contain different fractions of proteins which are causing stabilizing and destabilizing effect on DNA 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.     

Proteinase activity of nuclei from various tissues towards endogenous histones]

The proteinase activities of nuclei isolated from tissues differing in their mitotic activities (brain, thymus, liver, ascite lymphoma) towards the histones and non-histone acid -- extractable proteins were studied. The sensitivity of different histone fractions to nuclear proteinase depends on temperature and time of nuclei incubation under conditions providing for complete dissociation of chromatin proteins from DNA (2 M NaCl--5 M urea). The proteinase activity in the brain and thymus nuclei is revealed only under prolonged (43 hrs) incubation of the nuclei at 25 degrees C, which is accompanied by partial proteolysis of histone H1. Histone H4 from brain nuclei and histone H2a from thymus nuclei are preferably degraded. In the nuclei isolated from the mice ascite cell lymphoma NK/ly and from rat liver the enzyme activity is revealed mainly towards the arginine-enriched histones H3 and H4. The proteolysis of the arginine-enriched histones in tumour cell nuclei is more complete. A high sensitivity to proteolysis was observed for non-histone acid-extractable proteins with low electrophoretic mobility, which were found in brain and tumour cell nuclei.     

Chromosomal proteins of conifers

During imbibition and germination of jack pine seeds, the composition of the total extractable chromatin varied. Relative to DNA, the histone levels decreased as the nonhistone chromosomal proteins (NHCP) increased. New chromosomal proteins were synthesized after 2 days of imbibition as judged by recovery of ¹⁴C-amino acids from the major protein fractions. Phosphorylation of histones from ³²P-phosphoric acid was detected before the incorporation of ¹⁴C-amino acids. In the seed the synthesis and relative changes of chromatin coincided with a fall in total soluble protein and free arginine N. By contrast, adenylate energy charge, free glutamine N and in vitro template activity of chromatin increased during chromatin protein synthesis. When seeds had germinated for 4 days after the start of imbibition more radioactivity, derived from free ¹⁴C-amino acids, was recovered from the NHCP than from the histones. The percentage amino acid composition of most histone fractions remained stable, whereas the composition of NHCP changed more with time. The phosphorylation of NHCP was 8- to 41-fold greater than that of the histones. Phosphorylation of histone H4 was not detected at any stage of germination. Correlations between recovery of radioactivity (³²P and ¹⁴C) from chromosomal proteins and higher adenylate energy charge were positive.     

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.     

The synthesis and decay of histone fractions and of deoxyribonucleic acid in the developing avian brain

1. The turnover of cerebral histones and DNA after injection of [4,5-(3)H]leucine or [methyl-3-(3)H]thymidine, respectively, was studied in the developing chick. 2. Chromatin was prepared from chick nuclei that had been purified by centrifugation through 1.9m-sucrose. 3. Nuclear proteins were fractionated into three major histone classes, F1 (lysine-rich), F2(b) (slightly lysine-rich) and [F3+F2(a)] (arginine-rich), and a non-histone protein residue. 4. The proportions of the histone classes remained constant throughout the period of development studied. 5. All histone fractions decayed at a similar rate, initially with a half-life of around 5 days, later with a half-life of 19 days. 6. Non-histone proteins from chromatin decayed in a heterogeneous manner with a wide range of half-lives. 7. Short-term labelling studies showed that all histone fractions were synthesized at the same rate. 8. Some non-histone proteins were very rapidly synthesized relative to histones. 9. DNA had a longer half-life than any histone fraction studied. A biphasic exponential decay curve with half-lives of 23 and 50 days was found. 10. It was concluded that the turnover of histones can occur independently of that of DNA and that different histone classes have similar rates of synthesis and decay.