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.     

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.     

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.     

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.     

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.     

(ADP-ribosyl)ation pattern of chromosomal proteins during ageing.

(ADP-ribosyl)ation of chromosomal proteins was studied by incubating the nuclei of brain and liver of young and old rats with 14C-NAD+. In brain as well as in liver histone proteins show approximately 2-3 fold higher (ADP-ribosyl)ation than that of non-histone chromosomal (NHC) proteins of both the age groups. H1 seems to be the major target for (ADP-ribosyl)ation. Amongst nucleosomal histones H2B is the main acceptor of 14C-labelled ADP-ribose moieties. A sharp age related decline of (ADP-ribosyl)ation of chromosomal proteins was observed in both the tissues.     

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.     

Non-histone chromosomal proteins from virus-transformed and untransformed 3T3 mouse fibroblasts.

A peak in the non-histone chromosomal protein polyacrylamide gel electrophoresis profiles has been detected which is higher in log phase 3T3 and 3T3/SV40 cells than in density-inhibited 3T3 cells. Radioactive incorporation is substantially higher into this peak in log phase 3T3 than in 3T3/SV40 and density-inhibited 3T3 cells. Reversion of 3T3/SV40 cells with dibutyryl cyclic AMP and theophylline produces increased radioactive incorporation into the peak. Electrophoresis of non-histone chromosomal proteins extracted at different stages of the cell cycle in density inhibited 3T3 cells following serum stimulation shows a cyclic variation in the amount of this peak with maximum accumulation in late G1. In contrast the height of an equivalent peak in synchronously growing 3T3/SV40 cells remains constant throughout the cell cycle. It is postulated that the protein(s) of this peak may have a regulatory role in cell growth.     

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.     

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.     

The effect of sodium butyrate on acetylation in vitro of chromosomal proteins in three classes of liver nuclei from different ages of rats

The optimum conditions of in vitro incorporation of sodium [³H]acetate into sliced rat liver were studied. The incubations with sliced liver from three different ages of rats were performed in the presence of sodium n-butyrate. It was found that butyrate decreases the incorporation of sodium [³H]acetate into the homogenate, isolated nuclei, non-histone chromosomal proteins and histones for all age groups. The acetylations of non-histone chromosomal proteins and histones increase with age upto 2-months and decrease in 4-month-old rats both in the absence and presence of butyrate. Liver nuclei were fractionated by the simple method of zonal centrifugation into three classes, namely diploid stromal, diploid parenchymal and tetraploid parenchymal nuclei. The acetylations of non-histone chromosomal proteins and histones in three classes of nuclei of three ages of rats were studied in the presence and absence of butyrate. Butyrate can decrease the overall acetylations of non-histone chromosomal proteins and histones but increase the amount of polyacetylated histone H4 in all classes of nuclei of the three ages.     

Lipids as integral constituents of nuclear and chromatin fractions in Ehrlich ascites tumor cells

Lipid content and composition of DNA, histone and non-histone proteins of Ehrlich ascites tumor cell chromatin were investigated. All fractions contained small amounts of lipids, mostly neutral ones, in a specific distribution. According to isotopic studies with labeled lipid precursors, incorporation took place mainly in the non-histone fraction. These findings suggest that neutral lipids attached to non-histone chromosomal proteins may also contribute to the regulatory functions ascribed to phospholipids.     

A method for the fractionation of the high-mobility-group non-histone chromosomal proteins

A method is given for the preparation of four non-histone chromosomal proteins, one of which, protein 14, hitherto has not been isolated. The method also enables the preparation of histone H1 in gram quantities. The four non-histone chromosomal proteins so prepared are all polar molecules over 50% of each being composed of acidic and basic amino acids. It is also shown that protein 14 can be prepared from calf thymus without prior isolation of chromatin.     

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.     

Proteins of Friend leukemia cells. Comparison of hemoglobin-synthesizing and noninduced populations.

Proteins of Friend leukemia cells induced to form large amounts of hemoglobin by dimethylsulfoxide treatment were compared with proteins from noninduced cells by high resolution two-dimensional polyacrylamide gel electrophoresis. Approximately 98% of more than 500 proteins separated by this technique were qualitatively and quantitatively the same in both cell populations. Changes representing more than 50% of the control cell amount were detected in six non-histone chromosomal proteins, two nucleoplasmic proteins, and three cytoplasmic proteins. It is concluded that dimethylsulfoxide induces an extremely specific pattern of erythroid differentiation in these cells, which should be susceptible to detailed analysis. Comparison of protein patterns from Friend leukemia cells and HeLa cells revealed electrophoretic identity of approximately 20% of cytoplasmic proteins and 50% of non-histone chromosomal proteins.     

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.