Conformational changes in rat liver chromatin after liver regeneration.

N-Pyrenemaleimide, a fluorescent probe that specifically labels histone H3 of rat liver chromatin in situ, was used to monitor the accessibility of histone H3 in chromatin isolated from rat liver at different times during degeneration. At times of maximum DNA synthesis (18--24 h after hepatectomy), the accessibility of the probe was found to be markedly (40--50%) increased. This increase is abolished, however, by treatment of the chromatin fibres with high salt (2 M-NaCl) or detergent. Tryptophan fluorescence was also enhanced at points of maximum DNA synthesis, suggesting that some non-histone tryptophan-containing protein was being synthesized. The polarization of the labelled histone H3 is not markedly altered, suggesting that fibre aggregation or dissociation does not occur. Mononucleosomes extracted from sham-operated and hepatectomized animals did not exhibit any difference in binding to the probe. Also, analysis of the chromatin protein by electrophoresis on detergent- and acid/urea/ Triton-X-100-containing polyacrylamide gels showed no detectable difference in histone H3 : 1, H3 : 2 or H3 : 3 subclasses.     

Nonhistone chromatin proteins of B-lymphocytes stimulated by lipopolysaccharide Two-dimensional gel electrophoresis analysis of proteins synthesized and phoshorylated during the initiation of lymphocyte differentiation

Synthesis and phosphorylation of nonhistone chromatin and nucleoplasmic proteins during the first 24 h of activation of mouse B-lymphocytes by the B-cell mitogen lipopolysaccharide have been studied by two-dimensional gel electrophoretic analysis. Although little change occurs in the nucleoplasmic proteins, it has been shown that the incorporation of [³⁵S]methionine into nonhistone chromatin proteins is selectively stimulated. The degree of stimulation and the kinetics of synthesis are characteristic for each individual protein; some proteins exhibit increased incorporation only 4 h after addition of mitogen, while others are synthesized de novo between 8 and 24 h. After 72 h stimulation, the majority of nonhistone chromatin protein synthesis occurs in the highly differentiated lymphoblasts and plasma cells actively secreting IgM, very little synthesis taking place in the small lymphocytes. Analysis of nuclear proteins from lymphocytes stimulated for 2 h showed no selective stimulation of phosphorylation. These observations suggest that nonhistone chromatin proteins play an important role in the regulation of gene expression in B-lymphocytes.     

Chromosomal protein synthesis during erythropoiesis in the mouse spleen

Induced erythropoiesis in the mouse spleen was employed to study chromosomal protein synthesis during erythroid cell development. Splenic erythropoiesis occurring after phenylhydrazine induced hemolysis can be divided into an early phase during which nuclear RNA polymerase activity and RNA production are maximal and a late phase in which hemoglobin synthesis and DNA accumulation are maximal. Chromatin was isolated from splenic tissue during both the early and late phases of erythropoiesis as well as from non-anemic animals. The total protein content of chromatin from the early erythroid phase was greater than that of chromatin from the late erythroid phase or from non-anemic controls. The increase was due to a coordinate increase in the concentration of both histone and nonhistone proteins. During late erythropoiesis, the concentration of each returned to pre-anemic levels. Total histone synthesis increased 2.6-fold during early erythropoiesis as compared with the pre-anemic state and remained elevated in late erythropoiesis. The increase in histone synthesis was due to an increase in the synthesis of all five major histone proteins. Nonhistone protein synthesis was more active than that of histones in the pre-anemic spleen and rose only slightly during early erythropoiesis, returning to preanemic levels during late erythropoiesis. Fractionation of nonhistone proteins on SDS-urea polyacrylamide gels revealed complex patterns with significant differences between the pattern of erythroid spleen non-histone proteins and that of the pre-anemic spleen. Analysis of the incorporation of ³H-valine into the non-histone proteins indicated that during early erythropoiesis there was a generalized increase in nonhistone protein synthesis. During the late erythroid phase, the decline in non-histone protein synthesis was most marked for the higher molecular weight proteins.     

Expression of nuclear matrix proteins in rat liver tissue

We have studied the synthesis of nuclear matrix proteins as it occurs in the rat liver. To investigate their kinetics in tissue, nuclear matrix proteins were prepared from liver of rats injected with radioactive methionine. Synthesis of lamins was not observed in quiescent hepatocytes although they were the principal proteins of this subcellular fraction, suggesting that lamins are very stable in the liver. When hepatocytes were stimulated to divide by partial hepatectomy, only synthesis of lamin B was initiated. Many proteins not visible on Coomassie blue-stained gels were detectable by autoradiography. In the nuclear matrix extracts of quiescent hepatocytes, one of the most prominently labeled ones was a protein of 70 kDa. After hepatectomy, an additional protein of 62 kDa was detectable. These proteins were visible 1 h after the injection of radioactivity, but were no longer observed in nuclear matrices prepared 24 h after injection. These experiments indicate that in addition to lamins, two nuclear matrix proteins are present in the rat liver that were not detected previously, perhaps because of their rapid turnover.     

Early Events in Lymphocyte Transformation by Phytohaemagglutinin

Synthesis and phosphorylation of three main nuclear protein fractions were studied in human lymphocytes stimulated by phytohaemagglutinin (PHA). The first fraction to be synthesized and phosphorylated after induction was that of the acidic proteins, followed by that containing the soluble proteins. Synthesis of histories commenced 24 h after exposure to PHA. Distinctive patterns of both synthesis and phosphorylation of the acidic proteins were recorded at different times in the cell cycle, which may reflect activation or suppression of specific cellular functions. Phosphorylation of the histones also occurred, as an early event during lymphocyte transformation and also much later, at the time of DNA synthesis.     

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.     

Metabolic activities of histones in rat liver and spleen.

Synthesis and turnover of histone I and II in normal rat liver and spleen were studied by Amberlite CG 50 column chromatography. Histone I was separated into three or four subfractions, each of which showed a different rate of incorporation of [3H]lysine. This was verified by a more shallow gradient chromatography developed by Kinkade and Cole [3] for very lysine-rich histone (F1), which showed tissue specific differences between liver and spleen in both the elution pattern and synthetic rates. These subfractions were distinguished from each other by dodecylsulphate electrophoresis. The turnover, or disassociation of histone I and II in chromatin was measured by double-labelling of normal rat liver with [3H] and [14C]lysine. A good correspondence was found between the synthesis and turnover patterns of individual histone I fractions, while the histone II synthesized was conserved for over a month. From consideration of the turnover in relation to the cell population of normal liver tissue, which consists of a very small fraction of growing cells and a very large fraction of resting ones, it was concluded that turnover of histone I must occur even in resting cells. When DNA synthesis in the spleen was completely inhibited by hydroxyurea, the synthesis of histone II was inhibited but that of histone I was only partially inhibited. The remaining synthesis seemed to occur in cells in the resting state. It was concluded tentatively, the continuous replacement of very lysine-rich histones of chromatin must occur even in resting cells in which DNA synthesis has ceased. The biological significance of disassociation of histones from chromatin was discussed.     

Histone gene expression in early development of Xenopus laevis

Abstract. This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis , and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3.
Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.     

Electrophoretic analysis of the stored histone pool in unfertilized sea urchin eggs: quantification and identification by antibody binding

A maternal store of histones in unfertilized sea urchin eggs is demonstrated by two independent criteria. Stored histones are identified by their ability to assemble into chromatin of male pronuclei of fertilized sea urchin eggs in the absence of protein synthesis, suggesting a minimum of at least 25 haploid equivalents for each histone present and functional in the unfertilized egg. In addition, electrophoretic analysis of proteins from acid extracts of unfertilized whole eggs and enucleated merogons reveals protein spots comigrating with cleavage stage histone standards, though not with other histone variants found in later sea urchin development or in sperm. Quantification of the amount of protein per histone spot yields an estimate of several hundred haploid DNA equivalents per egg of stored histone. The identity of some of the putative histones was verified by a highly sensitive immunological technique, involving electrophoretic transfer of proteins from the two-dimensional polyacrylamide gels to nitrocellulose filters. Proteins in amounts less than 2 x 10(-4) micrograms can be detected by this method.     

Individual histone messenger RNAs: Identification by template activity

Newly synthesized polysomal messenger RNAs from cleavage stage embryos of the sea urchin Arbacia punctulata and Lytechinus pictus that contain putative histone mRNAs have been fractionated on 6% polyacrylamide slab gels. At least 8 RNA species with unique electrophoretic mobilities have been recognized. The complex of RNAs has been eluted from the gels in three groups, A, B, and C, in increasing order of mobility. The template activity of the three fractions and the unfractionated starting material was examined in the mouse Krebs II ascites tumor cell-free protein synthesizing system. The unfractionated messenger complex programs the synthesis of proteins that coelectrophorese exclusively with sea urchin histones in both sodium dodecyl sulfate and acid urea gel systems. The products of in vitro protein synthesis stimulated by the individual polyacrylamide gel RNA fractions were similarly examined. Each stimulated protein synthesis and was enriched for specific histone templates. We conclude that RNA fraction A is template for histone f1, C is template for histone f2a1, and B serves as template for f2b, f2a2, and f3 histones. A minor degree of contamination of the A and B RNA fractions was obvious from the production of other histones by each template. The co-electrophoresis of specific template activity with specific radiolabeled RNAs supports the concept that most or all of the labeled RNAs are indeed themselves the histone mRNAs.     

NUCLEAR CHROMATIN PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER: SYNTHESIS AND PHOSPHORYLATION

Abstract— In order to investigate synthesis and phosphorylation of the various fractions of nuclear proteins. [³H]leucine and [³²P] phosphate incorporation were studied with tissue slices in vitro. Cerebral cortex and cerebellum were used to delineate the similarity and dissimilarity within CNS, and liver was taken to compare the extraneural organ. There were significant differences in [³H]leucine incorporation into nuclear proteins among those tissue sources examined, while [³²P]phosphate incorporation showed very similar results among them. Although the acidic chromatin protein demonstrated high activity in each tissue source for both synthesis and phosphorylation, 0.14M-NaCl soluble protein showed the activity as high as or even higher than the acidic chromatin protein. Both [³H]leucine incorporation and [³²P]phosphate incorporation were relatively low in histone. When the acidic chromatin protein was further fractionated with SDS-acrylamide gel electrophoresis, significant difference was found between CNS tissue and liver for synthesis and phosphorylation. However, considerable difference was also observed even between cerebral cortex and cerebellum. The present investigation demonstrated complicity and diversity of nuclear chromatin proteins in different organs, not only for their protein constituents but also for their synthesis and phosphorylation.     

Variations in some molecular events during the early phases of the Reuber H 35 cell cycle. II.-Chromatin protein phosphorylation and protein kinases

Reuber H 35 hepatoma cells were synchronized by transfer in a serum free medium. Growth was re-initiated by addition of serum. Under these conditions DNA synthesis exhibited a maximum after 24 hours. Chromatin non-histone proteins prepared from cells at various phases of the cell cycle were incubated with [gamma-32P] ATP and the radioactive pattern of protein bound 32P was analysed by electrophoresis on polyacrylamide gels. No radioactive peak was observed in G0. Several peaks appeared 3 hours after the addition of serum. The radioactivity progressively increased until the cells reached the S phase. When most of the cells were in the S phase the radioactivity strongly decreased. Chromatin protein kinase activities were found to increase in late G1 and continued to increase in the S phase. The increase was 65% when phosvitin was the substrate, 100% with casein and histone H1. It is suggested that chromatin phosphorylated proteins could be involved in the mechanism which initiates DNA synthesis in G1 phase cells.     

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.     

Kinetics of accumulation and depletion of soluble newly synthesized histone in the reciprocal regulation of histone and DNA synthesis

Procedures are presented which permit the identification and analysis of cellular histone that is not bound to chromatin. This histone, called soluble histone, could be distinguished from that bound to chromatin by the state of H4 modification and the lack of H2A ubiquitination. Changes in the levels of newly synthesized soluble histone were analyzed with respect to the balance between histone and DNA synthesis in hamster ovary cells. Pulse-chase protocols suggested that the chase of newly synthesized histone from the soluble fraction into chromatin may have two kinetic components with half-depletion times of about 1 and 40 min. When protein synthesis was inhibited, the pulse-chase kinetics of newly synthesized histone from the solubl fraction into chromatin were not significantly altered from those of the control. However, in contrast to the control, when protein synthesis was inhibited, DNA synthesis was also inhibited with kinetics similar to those of the chase of newly synthesized histone from the soluble fraction. There was a rapid decrease in the rate of DNA synthesis with a half-deceleration time of 1 min down to about 30% of the control rate, followed by a slower decrease with an approximate half-deceleration time of 40 min. When DNA synthesis was inhibited, newly synthesized histone accumulated in the soluble fraction, but H2A and H2B continued to complex with chromatin at a significant rate. Soluble histone in G1 cells showed the same differential partitioning of H4/H3 and H2A/H2B between the soluble and chromatin-bound fractions as was found in cycling cells with inhibited DNA synthesis. These results support a unified model of reciprocal regulatory mechanisms between histone and DNA synthesis in the assembly of chromatin.     

Synthesis of cytoskeletal and contractile proteins by cultured IMR-90 fibroblasts

Models of the assembly of cytoskeletal and contractile proteins of eukaryotic cells require quantitative information about the rates of synthesis of individual component proteins. We applied the dual isotope technique of Clark and Zak (1981, J. Biol. Chem., 256:4863-4870) to measure the synthesis rates of cytoskeletal and contractile proteins in stationary and growing cultures of IMR-90 fibroblasts. Fibroblast proteins were labeled to equilibrium with [14C]leucine over several days, at the end of which there was a 4-h pulse with [3H]leucine. Fractional synthesis rates (percent per hour) were calculated from the 3H/14C ratio of cell protein extracts or protein purified by one- or two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of medium-free leucine. The average fractional synthesis rate for total, SDS- or urea-soluble; Triton-soluble; and cytoskeletal protein extracts in stationary cells each was approximately 4.0%/h. The range of values for the synthesis of individual proteins from total cell extracts or cytoskeletal extracts sliced from one-dimensional gels was similar, though this range was greater than that for major proteins of Triton-soluble protein extracts. Three specific cytoskeletal proteins--actin, vimentin, and tubulin--were synthesized at similar rates that were significantly slower than the average fractional synthesis rate for total protein. Myosin, on the other hand, was synthesized faster than average. Synthesis rates were the same for beta-and gamma-actin and polymerized (cytoskeletal extract) vs. Triton-soluble actin. The same was true for alpha- and beta-tubulin and two different forms of vimentin. Synthesis rates were uniformly higher in growing cells, though the same pattern of differential rates was observed as for stationary cells. Synthesis rates in growing cells were higher than the rate necessary to maintain the growth rate, even for those cytoskeletal proteins being synthesized slowly. Therefore, there appears to be some turnover of these cytoskeletal elements even during growth. We conclude that proteins in cytoskeletal extracts may have nonuniform rates of synthesis, but at least one important subclass of cytoskeletal proteins that comprise filament subunits have the same synthesis rates.     

DNA replication, chromatin structure, and histone phosphorylation altered by theophylline in synchronized HeLa S3 cells

The onset of DNA replication normally is coincident with an increase in histone 1 phosphorylation and a relaxation in chromatin structure. In this paper we show that 5 mM theophylline, added 2 h after selective detachment to synchronized HeLa-S-3 cells, delays the onset and reduces the rate of DNA synthesis while theophylline treatment beginning at 8 h has no effect on subsequent DNA synthesis. These actions of theophylline are accompanied by an inhibition of histone 1 phosphorylation and a prevention of the normal relaxation in chromatin structure between G1 and S phases as revealed by image analysis of Feulgen-stained nuclei. The time courses of intracellular cyclic AMP levels, nonhistone protein phosphorylation, and [3H]lysine incorporation are also compared in the same treated and untreated synchronized HeLa cells. Comparison with experiments using 1-beta-D- arabinofuranosylcytosine (Ara-C) shows that the above phenomena are not a direct result of inhibition of DNA synthesis. We interpret our results as evidence that the associations between histone 1 phosphorylation, chromatin relaxation, and the onset of DNA synthesis are temporally and causally related.     

The Ag-NOR proteins and transcription and duplication of ribosomal genes in mammalian cell nucleoli

The relationship between the Ag-NOR (silver-stained Nucleolar Organizer Region) proteins and the functional-structural organization of the nucleolar ribosomal chromatin was studied in regenerating and cortisol-stimulated rat hepatocytes. Statistical analysis of Ag-NOR proteins, carried out with an automated image analyzer, indicated that in regenerating rat hepatocytes the quantity of Ag-NOR proteins mainly increased between the 4th and 12th h of regeneration, reaching a level twice that of resting hepatocytes. Also the synthesis of pre-ribosomal RNA (pre-rRNA) was stimulated after the 4th h of regeneration. Cycloheximide administered to rats at a dose of 0.025 mg/100 g body weight (bw) prevented any increase in Ag-NOR proteins but did not hinder the stimulation of pre-rRNA synthesis. In 8 h cortisol-stimulated hepatocytes no significant change in amount of Ag-NOR protein was observed whereas pre-rRNA synthesis was highly increased as in 12 h regenerating hepatocytes. These results indicated that in rat hepatocytes Ag-NOR proteins and stimulation of pre-rRNA synthesis are not related. The relationship between the Ag-NOR proteins and the distribution of the completely extended intranucleolar ribosomal chromatin was also studied in regenerating rat hepatocytes. At 12 h after partial hepatectomy an increased amount of completely extended ribosomal chromatin was observed, contemporaneously with an increased quantity of Ag-NOR proteins. These ribosomal chromatin changes preceded the beginning of DNA synthesis and were prevented by cycloheximide-induced inhibition of protein synthesis.     

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.