Proteins from different classes of liver nuclei in normal and thioacetamide-treated rats

1. In normal rats the amounts of each of the main types of nuclear protein, i.e. soluble proteins, histones, non-histone chromosomal proteins and residual proteins, vary within the different classes of rat liver nuclei fractionated by zonal centrifugation. 2. Heterogeneity is observed in the non-histone chromosomal proteins prepared from different classes of liver nuclei. These differences were observed by analysis of the proteins both by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and electrofocusing electrophoresis. They are most evident between the non-histone chromosomal proteins obtained from stromal and parenchymal nuclei. However, some differences are also found for the parenchymal nuclei, between the diploid parenchymal and the tetraploid parenchymal, and between them and the nuclei involved in the synthesis of DNA respectively. 3. Drastic alterations in the nuclear proteins are found after the administration of thioacetamide. The changes observed are complex and not uniform. They vary with the age of the animal and the type of nucleus. In general an increase in the soluble proteins and non-histone chromosomal proteins and a decrease in the residual proteins is observed. There is a decrease in the specific radioactivity of soluble and residual proteins. 4. Electrophoretic analysis of the non-histone chromosomal proteins showed that specific changes occurred after administration of thioacetamide, which are different in adolescent and young adult rats.     

Immunospecificity of non-histone chromosomal proteins in 89Sr-induced osteogenic sarcoma (mouse).

Antibodies against non-histone chromosomal proteins for 89Sr-induced osteogenic sarcoma (mouse) were prepared by immunization of rabbits. The immunoreactivity of this antigen was then compared with those of non-histone chromosomal proteins from Ehrlich ascites tumor, normal mouse liver, and calf thymus by the method of quantitative microcomplement fixation. The non-histone chromosomal proteins of 98Sr-induced osteogenic sarcoma, fractionated by hydroxylapatite chromatography, exhibited significant affinity for the antibodies. Similar proteins from Ehrlich ascites tumor, normal mouse liver, or calf thymus were virtually inactive, indicating the tissue-specificity of 89Sr-induced osteogenic sarcoma proteins.     

Stimulation of non-histone chromosomal protein synthesis in simian virus 40-infected simian cells.

The pattern of synthesis of non-histone chromosomal proteins in simian virus (SV) 40-infected African green monkey kidney cells was analyzed by polyacryl-amide gel electrophoresis to see whether the changes in chromosomal protein metabolism are involved in the viral-induced synthesis of cellular DNA and mRNA. During the prereplicative phase of infection, the rate of histone synthesis was decreased until 15 h postinfection, whereas that of non-histone protein synthesis was increased after 5 h postinfection and reached a maximum at 10 to 15 h postinfection when viral-induced synthesis of cellular DNA and mRNA began to be observed. Stimulation of non-histone protein synthesis was also observed in the infected cells treated with cytosine arabinoside and was dependent on the multiplicity of infection. Stimulation occurred in almost all species of non-histone proteins. These results suggest that the stimulation of non-histone protein synthesis is caused by an early SV40 function and occurs prior to the viral-induced synthesis of cellular DNA and mRNA. During the replicative phase of infection, a marked increase in the rate of synthesis was observed in the non-histone proteins with molecular weights of about 48,000, 35,000, and 23,000, which were subsequently found to be SV40 capsid proteins.     

Studies on histones and non-histone proteins from rats treated with dimethylnitrosamine.

A study has been made of the histone and non-histone chromosomal proteins of rat liver after treatment in vivo with dimethylnitrosamine (DMN) (2 mg/kg). DMN was found not to affect histone turnover, as measured by 3H-labelled amino-acids incorporation. A decrease was observed in specific activity of the histones with time after injection of [14C]DMN or [14C]-formate and this was attributable to demethylation of both abnormal and normal methylation sites in these proteins. In the case of the non-histone proteins, DMN was found to increase greatly the turnover of those non-histone proteins loosely associated with chromatin DNA and RNA; turnover of those non-histone proteins tightly bound to chromatin DNA and RNA was unaffected. Demethylation of both normal and abnormal methylation sites was found to take place from both non-histone protein fractions. In the case of the loosely bound non-histone proteins a lower rate of demethylation was observed after DMN treatment.     

The characterization of the non-histone chromosomal proteins of the main classes of nuclei from rat brain fractionated by zonal centrifugation

1. Non-histone chromosomal proteins were isolated from the cell nuclei of whole rat brain and nuclei from different types of brain cells. 2. Brain nuclei were fractionated by zonal centrifugation into five zones deriving from five main categories of brain cells. These are the neuronals, astrocytes I, astrocytes II, oligodendrocytes I and oligodendrocytes II. 3. The non-histone chromosomal proteins were analysed by (a) sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, (b) electrofocusing electrophoresis and (c) two-dimensional electrophoresis. The results of this analysis showed a limited specific pattern of non-histone chromosomal proteins from the different classes of nuclei. Differences were found to exist between the proteins from neuronal and glial nuclei. In particular one polypeptide band with mol.wt. 10000 and pI8.5 was found to be present in the non-histone protein fractions of neuronal nuclei, and absent from the corresponding fractions of nearly all the other classes of nuclei. 4. Two other classes of nuclear proteins, buffered-saline-soluble and 0.35m-NaCl-soluble, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis along with the non-histone chromosomal. The similarities and differences among these groups of proteins are discussed. 5. The patterns of non-histone chromosomal proteins during development were investigated by studying them in two age groups of animals: in infant rats (10 days old) and adult rats. The polypeptide that was found to be specific for the proteins of neuronal nuclei of adult rats is present in all the classes of nuclei of infant rats.     

Fractionation of chromosomal proteins of pea seedlings using procedures which avoid denaturation

Chromatin was prepared from the buds and cotyledons of Alaskapea seedlings. The dissociated chromosomal components in thepresence of 2 M NaCl and 5 M urea were completely fractionatedinto DNA and proteins with a Bio-Gel A50 column. The proteinswere recovered by (NH₄)₂SO₄ and further fractionated into histonesand non-histone proteins using a Bio-Rex 70(Na⁺) column. Thedifference in the ratios of histones to non-histone proteinsbefore and after chromatography with the Bio-Rex 70 was lessthan 10%. The histones and non-histone proteins thus preparedshowed typical protein absorption spectra. Polyacrylamide gelelectrophoresis of histones showed that the histone compositionsin buds and cotyledon were similar, but the amount of HI histoneswas a little less in cotyledons than in buds. Unlike histones,non-histone proteins fractionated by SDS-polyacrylamide gelelectrophoresis indicated distinct differences between the twotissues. Buds had more heterogeneous non-histone proteins, atleast 13 polypeptides, than cotyledons did. On the other hand,non-histone proteins of cotyledons showed less heterogeneityand lacked proteins of high molecular weight which were foundin buds. (Received May 6, 1976; )     

Rapid turnover of non-histone chromosomal proteins in skeletal muscle

Incorporation of ³H-leucine into histones and non-histone chromosomal proteins was investigated in liver, a tissue in which proteins generally turn over rapidly, and in muscle, a tissue in which proteins turn over slowly. Incorporation into histones was low in both tissues. Incorporation into non-histone chromosomal proteins which, in liver, proceeded at about the same rate as into soluble cytoplasmic proteins was, in muscle, considerably more rapid than into any other cytoplasmic or nuclear protein fraction investigated. The significance of the relatively high incorporation rate into the non-histone chromosomal proteins in muscle is not known. However, autoradiographic experiments suggest that in muscle all nuclei display a high rate of incorporation into these proteins, and gel electrophoretic experiments indicate that a high rate of turnover is characteristic of many of the proteins comprising this fraction.     

Distinct effects of histone H1 and non-histone chromosomal proteins on the structure of nucleosomes and the chromatin fibre in solution

In this study we attempt to differentiate between the effects of the non-histone chromosomal proteins and histone H1 on the structure of the nucleosomes and the chromatin fibre in solution. The properties of chromatin preparations with different histone H1 and non-histone protein compositions were compared using circular dichroism and flow linear dichroism and the following conclusions were drawn. When histone H1 is absent the non-histone proteins partially prevent the unfolding of the nucleosomes at low ionic strength. The complete blocking of this unfolding, however, is accomplished only in the presence of histone H1. The non-histone proteins do not affect the orientation of the nucleosomes along the fibre axis. Only histone H1 can maintain the positive anisotropy of the chromatin fibre.     

Interphase chromosomal deoxyribonucleoprotein isolated as a discrete structure from cultured cells

A new procedure is described for the preparation of interphase chromatin from cultured mouse cells (line P815). The primary objective of this procedure was to eliminate exchanges of histones between deoxynucleoprotein molecules; this objective is shown experimentally to have been attained. The chromatin is released from cells by the non-ionic detergent Nonidet P40 in medium of low ionic strength (0.1 mM-KNa₂PO₄), and may then be sedimented as a structure which conserves the general form and ultrastructural characteristics of chromatin within the cell. The nuclear envelope cannot be detected in these structures by electron microscopy, and their content of choline-containing phospholipids is less than 10% of that of nuclei. The maintenance of form in this structure must thus depend on properties of the chromatin itself, and possibly on the more compact peripheral chromatin.Soluble DNP² prepared by shearing these structures has the same relative contents of DNA, histones, non-histone proteins and RNA as DNP prepared by standard methods. Analyses by electrophoresis on polyacrylamide gels of the non-histone proteins reveals certain differences from the pattern of these proteins in DNP prepared by a salt precipitation method. The template activity for RNA synthesis, in the presence of Escherichia coli RNA polymerase of sheared, soluble DNP prepared by this procedure, is comparable to that of DNP prepared by other methods. However, in the absence of exogenous RNA polymerase the rate of RNA synthesis by structured (unsheared) chromatin is about ten times higher than the rate using sheared DNP.The rapid removal of the nuclear envelope in this lysis procedure allowed experimental examination of the origin of the histones and non-histone proteins of DNP. When DNP was prepared from a mixture of two populations of cells, one containing DNA distinguishable by a density label and the other containing radioactively labelled proteins, radioactive proteins were found exclusively in DNP of normal density, and not in dense DNP and vice versa. It is concluded that the proteins of DNP prepared in this way are not acquired during the preparation procedure but were already associated with DNA in vivo, and that other proteins are not bound non-specifically to DNA during the preparation of DNP. When a mixture of DNP molecules prepared, in this way is precipitated in 150 mm-NaCl and redissolved, some radioactively labelled histones migrate onto dense DNA molecules.This procedure is suitable for routine, quantitative isolation of chromosomal DNP from small numbers of cells; it is also applicable to cells of other cultured lines.     

Hepatocyte chromosomal non-histone proteins in developing rats.

Rat hepatocytes taken a different stages of the perinatal period were partially purified. On sodium dodecylsulphate acrylamide electrophoresis chromosomal non-histone proteins showed important variations in complexity during development. Chromosomal phosvitin kinase strongly increased during the last days of fetal life; it strongly decreased just after birth and increased again for a short time, while the cytosol phosvitin kinase increased more significantly after birth. Chromosomal non-histone proteins prepared at varoius stages were incubated with [gamma-32P]ATP and resolved on polyacrylamide gel. The incorporation was very low in sample taken at the 15th say of the fetal life. A dramatic increase was observed at the 17th day. This incorporation strongly decreased in the samples taken thereafter and it was negligible in proteins from adult rats. The variations in protein kinase and in 32P incorporation into non-histone proteins were correlated with the pattern of appearance of enzymes in this period of life, with cell growth and with the hormone-induced maturation.     

Protein kinases and their protein substrates associated with chromatin and ribosomes in SV40-transformed rat cells.

The level of endogenous protein phosphorylation in non-histone chromosomal and ribosomal wash proteins is 7--10 times greater in SV40-transformed rat cells than in untransformed parental cells. Protein kinase activity in these proteins was fractionated by either phosphocellulose or DEAE-cellulose chromatography. One major and one minor component were detected in non-histone proteins and only one component in ribosomal wash proteins when the activity in each fraction was measured with an exogenous substrate, casein. These enzymes prefer casein to whole histone as substrate and are cyclic AMP-independent. The enzyme activity in a major peak of non-histone proteins and in ribosomal wash proteins measured with casein as substrate is 3 times greater in transformed cells than in untransformed cells, whereas pH optimum, cation requirements and apparent Km values for casein and ATP are identical or very similar in the two cell types. No significant phosphatase was detected in non-histone and ribosomal wash proteins from the two types of cell. The patterns of endogenous protein phosphorylation in these protein fractions analysed by gel electrophoresis are significantly different between these cells. These results suggest that the high level of endogenous protein phosphorylation in non-histone and ribosomal wash proteins from SV40-transformed cells is caused mainly by the increased activity of protein kinase and the nature of protein substrates.     

Changes in chromatin of Daucus carota cells during embryogenesis

Cells of carrot (Daucus carota var. Rote Riesen) were cultured on media inductive and non-inductive for embryogenesis and analyzed for differences in their chromosomal proteins and chromatin template activity. Non-histone proteins were prepared from dehistonized chromatin and their properties were investigated. Non-histone proteins proved to be acidic and associated easily with calf thymus histone. Non-histone proteins were able to counteract the inhibitory effect of histone on DNA-directed RNA synthesis in vitro. Almost the same rate of restoration occurred regardless of the interaction between DNA and protein, when sufficient amounts of non-histone proteins were added. However, once the histone-DNA complex was established, the restoration by non-histone proteins at comparably lower concentration was poor. Another acidic protein, bovine serum albumin, had no effect on histone inhibited RNA synthesis. Also non-histone proteins enhanced the chromatin directed RNA synthesis more than 100%. The template activity of chromatin changed after the inductive treatment of embryo formation and induced cells showed higher template activity than non-indiiced controls after embryo cells were formed. Histone components were the same in inductive and non-inductive cells. On the other hand, there was a correlation between template activity and the stimulation by non-histone proteins of histone-inhibited RNA synthesis.     

Native state of high mobility group chromosomal proteins 1 and 2 is rapidly lost by oxidation of sulfhydryl groups during storage

Oxidized forms of non-histone chromosomal proteins high mobility group 1 (HMG1) and HMG2 were detected by high-pressure liquid chromatography of preparations stored at 4 degrees C for 1 day. The oxidized form of each was found to have two free sulfhydryl groups, while the freshly prepared native form of each contained four. The native, reduced state could be maintained during storage by the addition of ethylenediaminetetraacetic acid or reducing agents.     

Phosphorylation of nuclear proteins

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.     

Differential effects of polyamines on the phosphorylation of chromatin-associated proteins

Studies are presented on the nature of chromatin-associated phosphoproteins whose phosphorylation is influenced by polyamines. After labelling with 32P, chromatin-associated proteins were separated into four fractions. Fraction I comprised neutral and basic non-histone phosphoproteins, including high-mobility-group non-histones; fraction II consisted mostly of histones; fraction III consisted of a class of (salt-soluble) acidic non-histone phosphoproteins; and fraction IV consisted of residual (salt-insoluble) acidic non-histone phosphoproteins. The average relative distribution of protein in the four fractions (I-IV) was about 1:4:2:1 for both liver and prostate. However, tissue-dependent differences were observed in the incorporation of 32P in various protein fractions. In the presence of polyamines (e.g. 1 mM-spermine or 2 mM-spermidine) maximal stimulation of phosphorylation was observed in non-histone proteins of fraction I (160-180%), followed by that in non-histone proteins of fraction III (80-110%). The phosphorylation of residual non-histone proteins in fraction IV, and the small extent of phosphorylation of histones in fraction II, remained unaltered in the presence of polyamines. Thus polyamines do not stimulate the phosphorylation of all non-histone proteins; their stimulative effect is most prominent in the phosphorylation of neutral and basic non-histone proteins and a class of salt-soluble acidic non-histone proteins. In accord with our hypothesis, these differential effects of polyamines on phosphorylation of endogenous non-histone proteins may relate to the conformation of these substrates rather than to endogenous kinases.     

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.