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.     

Immunochemical study of chromatin non-histone proteins

Summary Rabbit antibodies against rat thymus and liver chromatin are obtained. Antithymus IgG are found to interact only with homologous chromatin, while antiliver IgG interact with both liver and thymus chromatin. After preincubation of antiliver IgG with thymus chromatin the antibodies interact with homologous chromatin only. Thus chromatin of both organs contains tissue-specific proteins. Antiliver and antithymus IgG are used to investigate a distribution of tissue-specific and tissue-non-specific immunogenic proteins in thymocyte nuclei. It is shown that these proteins are practically lacking in nuclear membranes, matrix, nuclear sap, nucleous chromatin and do not participate in attaching DNA to the nuclear matrix.Abbreviations NHP non-histone chromatin proteins - IgG immunoglobulins G - PMSF PhMeSO₃     

Conformation of DNA in chromatin protein-DNA complexes studied by infrared spectroscopy.

The following observations concerning the DNA secondary structures in various nucleohistone complexes were made by infrared spectroscopy: 1/ in chromatin, chromatin extracted by 0.6 M NaCl, nucleosomes, and histone-DNA reconstituted complexes, the DNA remains in a B type conformation at low relative hygrometry; 2/ in chromatin extracted by tRNA and in non histone protein-DNA reconstituted complexes, the DNA can adopt an A type conformation. Infrared linear dichroism data show that in NHP-DNA complexes the low relative hygrometry conformation of DNA may be modified and that the infrared parameter -1090 is close to that measured for RNA's or DNA-RNA hybrids. It is concluded that the histones block the DNA in a B form and that some of the NHP could be involved in the control of the secondary structure of DNA in chromatin.     

Mechanics of chromatin template activation. Physical evidence for destabilization of nucleoproteins by polyanions.

Thermal denaturation profiles were used to quantitate the destabilizing activity of five classes of polyanions in an effort to explain previously reported differences in activity among apparently similar polymers. This physical assay for polyanion action on chromatin is free of the biochemical complications associated with template assay systems. Most polyanions tested caused a thermal destabilization of the normal nucleohistone complexes of rat thymus chromatin and exposed free or weakly complexed DNA. Polyphosphates dissociated chromatin even when their net charge was as low as 6, and activity per unit of weight increased with chain length only for polymers containing less than about 20 phosphate units; pyrophosphate was inactive. Two polymers which had no influence on chromatin had a low negative charge density. Some, such as polycytidlate and denatured DNA, possessed a high charge but caused only minor changes in the shape of denaturation profiles and exposed little or no free DNA. The highly active nucleic acids, ribosomal RNA and polyguanylate, were distinguishable from inactive nucleic acids by having more secondary structure and potentially less steric hindrance for anionic interaction with chromatin. There was evidence that some polyanions dissociate certain histones before others. Although data obtained show that some histones may be redistributed from chromatin onto added double-stranded DNA in a medium containing urea, this does not appear to interfere significantly with the interpretation of typical thermal denaturation profiles of chromatin.     

PHYSICAL STUDIES OF ISOLATED EUCARYOTIC NUCLEI

The degree of chromatin condensation in isolated rat liver nuclei and chicken erythrocyte nuclei was studied by phase-contrast microscopy as a function of solvent pH, K⁺ and Mg⁺⁺ concentrations Data were represented as "phase" maps, and standard solvent conditions selected that reproducibly yield granular, slightly granular, and homogeneous nuclei Nuclei in these various states were examined by ultraviolet absorption and circular dichroism (CD) spectroscopy, low-angle X-ray diffraction, electron microscopy, and binding capacity for ethidium bromide Homogeneous nuclei exhibited absorption and CD spectra resembling those of isolated nucleohistone. Suspensions of granular nuclei showed marked turbidity and absorption flattening, and a characteristic blue-shift of a crossover wavelength in the CD spectra. In all solvent conditions studied, except pH < 2 3, low-angle X-ray reflections characteristic of the native, presumably superhelical, nucleohistone were observed from pellets of intact nuclei. Threads (100–200 A diameter) were present in the condensed and dispersed phases of nuclei fixed under the standard solvent conditions, and examined in the electron microscope after thin sectioning and staining Nuclei at neutral pH, with different degrees of chromatin condensation, exhibited similar binding capacities for ethidium bromide. These data suggest a model that views chromatin condensation as a close packing of superhelical nucleohistone threads but still permits condensed chromatin to respond rapidly to alterations in solvent environment.     

The non-histone proteins of the rat liver nucleus and their distribution amongst chromatin fractions as produced by nuclease digestion.

The search for proteins involved in maintaining higher order chromatin structures has led to a systematic examination of the non-histone proteins (NHP) of rat liver nuclei in the context of nuclease digestion studies. 40-45% of the 3H-tryptophan labelled NHP originally present could be removed by extensive washing in a "physiological" buffer, incubation at 37 degrees C with or without nuclease and a further wash step. Nuclei at this stage had a remarkably constant NHP content (ca. 0.73 micrograms/micrograms DNA), independent of the degree of digestion with micrococcal nuclease or HaeIII. The solubilized chromatin produced by limited digestion with either nuclease contained 0.3-0.5 microgram NHP/microgram DNA, this value falling to ca. 0.16 after more extensive cleavage. Insoluble chromatin fractions were between 2-fold (very limited digestion) and 16-fold (extensive digestion) richer in NHP than the corresponding soluble fractions. Gel electrophoresis revealed about 12 NHP bands in soluble fractions, the most prominent of M.Wt. 41.400, while the insoluble material had at least 50 components. These properties were independent of whether lysis of nuclei occurred in 0.2 or 50 mM ionic strength. The large disparity in NHP content between complementary soluble and insoluble chromatin fractions is considered in terms of chromatin organization in vivo and the possible role of NHP migration.     

Differences among subfractions of H1 histone in retention of linear and superhelical DNA on filters

Four kinds of rabbit thymus H1 histone differ among themselves in their ability to retain DNA on nitrocellulose filters. This is true for linear, or superhelical DNA, but the order of effectiveness of the different H1 histones depends on the physical conformation of the DNA. For linear DNA the binding efficiencies of the H1 histones are: RTL2 = RTL3 greater than RTL4 greater than RTL1. This order of effectiveness parallels the effectiveness of the H1 histones previously found for the condensation of linear DNA as observed by circular dichroism and viscosity. The binding efficiencies of the various histones toward superhelical DNA were: RTL4 greater than RTL3 greater than RTL1 greater than RTL2. The variation in amino acid sequence between different rabbit thymus H1 histones might thus introduce structural variations in nucleohistone fibers and perhaps in chromatin.     

Two-dimensinal gel electrophoresis of rat liver nuclear washes,nuclear matrix,and hnRNA proteins

The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by two-dimensional gel electrophoresis. The inclusion in the gels of six common protein standards of carefully selected molecular weight and isoelectric point allowed us to clearly follow the distribution of specific proteins during nuclear extraction. In the nuclear washes and chromatin, we observed five classes of proteins: (a) Exclusively cytoplasmic proteins, present in the first saline-EDTA wash but rapidly disappearing from subsequent washes; (b) ubiquitous proteins of 75,000, 68,000, 57,000, and 43,000 mol wt, the latter being actin, found in the cytoplasm, all nuclear washes and the final chromatin pellet; (c) proteins of 94,000, 25,000, and 20,500 mol wt specific to the nuclear washes; (d) proteins present in the nuclear washes and final chromatin, represented by species at 62,000, 55,000, 54,000, and 48,000 mol wt, primarily derived from the nuclear matrix; and (e) two proteins of 68,000 mol wt present only in the final chromatin. The major 65,000- 75,000-mol wt proteins seen by one-dimensional gel electrophoresis of nuclear matrix were very heterogeneous and contained a major acidic, an intermediate, and a basic group. A single 68,000-mol wt polypeptide constituted the majority of the membrane-lamina fraction, consistent with immunological studies indicating that a distinct subset of matrix proteins occurs, associated with heterochromatin, at the periphery of the nucleus. Actin was the second major nuclear membrane-lamina protein. Two polypeptides at 36,000 and 34,000 mol wt constituted 60% of the hnRNP. Approximately 80% of the mass of the nonhistone chromosomal proteins (NHP) from unwashed nuclei is contributed by nuclear matrix and hnRNPs, and essentially the same patterns were seen with chromatin NHP. The concept of NHP being a distinct set of DNA- bound proteins is unnecessarily limiting. Many are derived from the nuclear matrix or hnRNp particles and vary in the degree to which they share different intracellular compartments.     

Conformation of control and acetylated HeLa stripped chromatin after reassociation with H1

The effect of histone H1 on the conformation and stability of control and acetylated HeLa high-molecular-weight chromatin that had been stripped of H1 and nonhistone proteins was compared by circular dichroism (CD) and thermal denaturation measurements. Two different preparations of H1, originating from rat thymus and chicken erythrocyte, were used in the reconstitution studies. The control and acetylated stripped chromatin had identical CD and thermal denaturation properties, as did their reconstitutes with rat thymus H1. Reconstitutes of the two chromatins with chicken erythrocyte H1 had similar CD properties, but thermal denaturation studies showed that the acetylated reconstitute was destabilized compared to the control reconstitute. Reconstitutes of both chromatins with chicken erythrocyte H1 had a more condensed and stabilized structure than the reconstitutes with rat thymus H1. Thus, acetylation caused a decrease in the stability of chromatin in the presence of erythrocyte H1, but more marked differences were detected in the structure of stripped chromatin after reassociation with different H1 preparations.     

Effect of narrow fractions of chromatin non-histone proteins on the expression of membrane tumor-associated antigen MA-50 and phosphorylation of proteins of cultured rat hepatocytes

As earlier reported, the main component of narrow fractions of chromosomal non-histone proteins (NHP) of kidney and of Zaidel hepatoma cells has its own protein kinase activity, and is identified as a heteroorgan NHP-antigen, which is intrinsic to the definite renal tissue and absent in the liver. Effects of narrow fractions of kidney and Zaidel hepatoma NHP on biosynthetic processes and sizes of hepatocytes were studied in vitro. It has been shown that as a result of a 5 h incubation of rat hepatocytes with a narrow fraction of renal NHP the proportion of small hepatocytes increases approximately by 12% as compared with that of cells cultivated without NHP. Besides, binding of organ-specific anti-kidney immune serum with a small hepatocyte population rises by more than 20%, which results from the expression of tumor-associated heteroorgan kidney-specific antigen on the hepatocyte surface. According to immunoprecipitation and subsequent electrophoresis, the molecular mass of a membrane heteroorgan antigen on the surface of hepatocytes amounts approximately to 65 kDa, and an active phosphorylation of cellular proteins takes place. The same effect on hepatocytes is produced by a narrow NHP fraction of chromatin of Zaidel hepatoma cells, whereas no phosphorylation is observed in the presence of liver NHP as well as in the absence of NHP. It is suggested that the heteroorgan NHP-antigen induces biosynthetic processes including synthesis of membrane tumorassociated antigen on the surface of hepatocytes cultivated in vitro by activation of cellular protein phosphorylation, which can lead to changes in size of cultivated cells.     

Hydroxylapatite thermal fractionation of chromatin and DNA

Chromatin and DNA from developing muscle cultures were fractionated by hydroxylapatite thermal chromatography on the basis of differential thermal stability. A thermal chromatography system was developed in which protein mediated thermal stability of chromatin DNA was maximally expressed. The resulting chromatin and DNA elution profiles were similar to thermal denaturation profiles in low ionic strength solution. Additional studies showed this system was able to detect protein stabilization of DNA in in vitro nucleohistone preparations. Although some protein remained bound to hydroxylapatite during chromatin thermal elution, it did not affect the denaturation or elution behavior of free DNA on the same column. These studies show that fragments of chromatin or DNA can be segregated on the basis of differential thermal stability by hydroxylapatite chromatography.     

Marked differences in the ability of distinct protamines to disassemble nucleosomal core particles in vitro

In accordance with the results of classical experiments performed in vitro with calf thymus chromatin and the fish protamine salmine, we have observed that this highly basic, small molecular weight protamine cannot cause major displacement of histones from nucleosomal core particles at concentrations several times higher than physiological (arginine/nucleotide ratios 1-8) and that hyperacetylation of histones facilitates nucleosome disassembly. However, the avian protamine galline, with molecular weight and number of arginine residues almost twice those of common fish protamines, is able to displace the nucleosomal core histones from DNA in vitro at concentrations (arginine/nucleotide ratios 0.6-1.2) within the physiological range (0.8). Our results suggest that the binding of the avian protamine galline to chromatin could be directly involved in the rapid disassembly of nucleosomes that takes place during the nucleohistone nucleoprotamine transition in chicken spermiogenesis.     

Rat liver chromatin non-histone proteins and glucocorticoid binding

We have previously characterized a specific corticosterone binding protein in chromosomal non histone proteins (NHP) from rat liver. In this paper, we present evidence that a relationship exists between this protein and the cytoplasmic glucocorticoid receptor. The binding capacity of NHP is reduced by 40 p. cent when this fraction is isolated from adrenalectomized animals. Incubation of isolated nuclei with the glucocorticoid hormone receptor complex results in a decrease in the specific radioactivity of the cytoplasmic proteins and simultaneously in a rapid uptake of the isotope by the nucleus; radioactive hormone was extracted along with the NHP. Evidence is presented that the NHP component binding the hormone is closely related or identical to the cytoplasmic receptor-proteins. Progesterone and corticosterone compete similarly for the binding of dexamethasone to nuclear and cytoplasmic forms of the receptor. However the nuclear form of the receptor has a higher affinity for corticosterone (K_a : 6 × 10⁹ M⁻¹) than for dexamethasone (K_A : 10⁸ M⁻¹) in vitro.A mixture of rat liver NHP and cytosol was shown to bind specifically more corticosterone than when the two proteins were incubated separately with the hormone. The Scatchard analysis shows that the enhancement of binding is due to an interaction of nuclear and cytoplasmic proteins leading to the appearance of a stable protein-protein complex which has a high affinity for the hormone (K_a : 2 × 10⁸ M⁻¹). KCl prevented this interaction. Complex formation does not require the presence of the hormone. The experiments presented here favor the hypothesis of the existence of a regulatory protein in the nucleus. This protein associated with the binding protein to reveal or enhance the active form of the receptor.     

Chromatin changes related to dedifferentiation and differentiation in tobacco tissue culture (Nicotiana tabacum L.)

Non-histone chromosomal proteins (NHP) were isolated from different stages of Nicotiana tabacum L. pith dedifferentiation to callus and callus redifferentiation. The NHP were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis on slab gels and analyzed by densitometry. Simultaneous histological changes are reported. In both processes, some high molecular weight protein (HMWP) bands increase drastically in an induction period, previous to cell proliferation, and decrease when cell division declines. Some low molecular weight protein bands, intense in pith tissue, decrease early when callus is forming and increase when cells differentiate. chromatin template activity is high when cells proliferate, coinciding with maximum HMWP-bands intensity.Abbreviations HMWP high molecular weight proteins - IAA indole-3-acetic acid - LMWP low molecular weight proteins - NHP non-histone proteins - TA template activity     

Nucleosomal structure of sea urchin and starfish sperm chromatin. Histone H2B is possibly involved in determining the length of linker DNA.

Comparison has been made between sea urchin and starfish sperm chromatin. The only protein by which chromatins from these sources differ significantly is histone H2B. Sea urchin sperm H2B is known to contain an elongated N-terminal region enriched in Arg. Analysis of the micrococcal nuclease digests of sea urchin and starfish nuclei in one- and two-dimensional electrophoresis has shown that sperm chromatin of both animals consists of repeated units similar in general features to those of rat thymus or liver. However, DNA repeat length in chromatin of sea urchin sperm (237 bp) is higher than that of starfish sperm (224 bp), while the core DNA length does not differ and is the same as in the chromatin of rat liver or thymus. A suggestion has been made that the N-terminal region of histone H2B is associated with the linker DNA and is responsible for the increased length of sea urchin linker DNA.     

Acidic pentapeptide phosphorylated in vitro by calf thymus protein kinase NII binds to DNA in the presence of Mg2+ cations

The pentapeptide pyroGlu-Ala-Glu-Ser-Asn has been synthetized and phosphorylated in vitro at level of serine by protein kinase NII isolated from calf thymus chromatin. It is noteworthy that the calf thymus kinase NII shows a remarkable affinity for this peptide. The [32P]peptide is able to bind to several DNAs in the presence of Mg2+ (lambda phage, calf thymus, pBR540 plasmid). This binding appears not specific with regard to the type of DNA and its base sequence. These data support the hypothesis that phosphorylated acidic domains of nuclear nonhistone proteins could bind directly to DNA in the presence of Mg2+ cations.     

Effect of norepinephrine on chromatin protein phosphorylation in C-6 glioma cells

Rat C₆ glioma cultures were exposed to labelled sodium phosphate after treatment with NE with or without propanolol. Histones and non-histone proteins (NHP) were extracted from chromatin and there was no significant change in the specific activity of the total pool of histones and NHP between control and other two groups. However, after electrophoretic separation F₂a₂ histone showed a 60% increase while F₂b and F₃ histones exhibited a 40% decrease in phosphorylation in response to NE. There was no significant change in the gel pattern of NHP from different groups on SDS-PAGE. However, the 30k dalton NHP showed an increase in phosphorylation in response to NE and this increment was blocked by propanolol. The possible role of β-receptors on nuclear protein phosphorylation and genomic expression is discussed.     

Non-histone chromatin proteins from the sea urchin Strongylocentrotus droebachiensis sperm and embryo

Non-histone chromatin proteins (NHP) from sperm and gastrula of the sea urchin Strongylocentrotus droebachiensis have been studied. The results obtained show that the total amount of the NHP in the sperm chromatin is one-sixth of that in the gastrula chromatin. Certain notable differences in the electrophoretic banding patterns of the NHP have also been observed. SDS polyacrylamide gel electrophoresis of NHP revealed one major specific component of molecular weight 17000 in the sperm chromatin and three major specific fractions with molecular weights 14000; 15000 and 35000 in gastrula chromatin. Furthermore, the gastrula chromatin NHP contains about ten minor specific fractions in the molecular weight range 25 000 to 65 000. The relevance of these results to the control of gene activity is discussed.