gamma-Irradiation in vitro apparently blocked a plasma-membrane associated, superoxide-producing, NADPH oxidase in rat thymocytes. Differential centrifugation of the mixed thymocytes indicated the smaller lymphocytes (approx. 6 microns diameter) to be the radiosensitive population. The oxidase system co-isolated in part with thymus nuclei and could be solubilized by detergent treatment [Bellavite, Jones, Cross, Papini & Rossi (1984) Biochem. J. 223, 639-648]. Endogenous NADPH was the rate-limiting component for superoxide formation in vitro. The level of NADPH was lowered by gamma-irradiation, an effect mimicked by GSSG in the presence of 50 microM-ZnCl2 to inhibit GSSG reductase. These findings are suggested as the metabolic basis for interphase death of small lymphocytes exposed to ionizing radiation.

1. Cyclic AMP-independent casein kinase 1 in liver cytoplasm and nuclei was inhibited by Be2+ in vitro (Ki 2.5 microM and 29 microM respectively). Casein kinase 2 (phosvitin kinase) and cyclic AMP-dependent protein kinase were unaffected. 2. The inhibition of casein kinase 1 by Be2+ was competitive with respect to the protein substrate; at non-saturating concentrations of casein, inhibition was non-competitive with respect to ATP. 3. In rats given LD50 doses of Be2+ 24 h before death, the activities of cytoplasmic and nuclear casein kinase 1 in livers from partially hepatectomized animals were diminished approx. 50%; with intact rats, nuclear casein kinase 1 was inhibited at concentrations of casein less than the Km.

Initiation sites were enumerated in rat liver nucleosomes with Echerichia coli RNA polymerase. One site was present in approx. 3.5 mononucleosomes and in a 1200-base-pair length of polynucleosomes. S-phase nuclei or normal nuclei phosphorylated in vitro showed increased numbers of sites; the elongation rates were unchanged.

Liver nuclei were prepared through the first cell cycle in partially hepatectomized young rats showing 30% parenchymal cell synchrony. To determine if nucleosome structure altered during this period, liver nuclei from sham-operated rats were compared with nuclei isolated at various times after partial hepatectomy. These nuclei were exposed to deoxyribonuclease I (EC 3.1.4.5), deoxyribonuclease II (EC 3.1.4.6) or micrococcal nuclease (EC 3.1.4.7) and the nucleosome-associated DNA length was ascertained. In no case was a difference in the DNA lengths associated with nucleosome structure observed. Differences were observed with regard to the histones and their relative association with nuclear material. When nuclei from normal rat livers were incubated in hypo-osmolar medium 9% of histone 1 and 4% of the other histones were released. These released histones, unlike those remaining bound to the nuclei, showed high [3H]adenosine and [3H]acetate uptakes in vivo. [32P]P1 uptake was also much greater into released than bound histones 1 and 3, but was not different for histone2A. At 3.5-4.5 h after partial hepatectomy, the release of histone 1 was trebled and that of histone 4 doubled. By 13.5 h, when phosphorylation of the bound forms of histones 2A and especially 1 was increased, no further changes in histone release in hypo-osmolar medium were found. The released histones from partially hepatectomized livers had indistinguishable [3H]adenosine uptakes from controls. The roles are discussed of phosphorylation and ADP-ribosylation in labilizing histone binding.

When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].

The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32P uptakes than did histones from resting liver nuclei; the other histones all showed 32P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [gamma-32P]ATP was incorporated into non-histone proteins, including protein P1, and into the ADP-ribosylated form of histone 1; negligible 32P was incprporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein P1 was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. gamma-Irradiation decreased 32P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed.

1. ADP-ribose is found in rat liver nuclei covalently bound to histone F1, to a non-histone protein, and to a small peptide. 2. A single unit of ADP-ribose, covalently bound to phosphoserine, was isolated from an enzymic hydrolysate of histone F1. ADP-ribose-bearing peptides were isolated from a tryptic digest of the histone. 3. It is proposed that the 1′-hydroxyl group of ADP-ribose is linked to the phosphate group of phosphoserine in histone F1. 4. The incorporation of 32P into ADP-ribose on histone F1 a parallels the DNA content through the cell cycle. An increased incorporation of the nucleotide into the other derivatives is observed during S phase. 5. It is suggested that the ADP-ribose derivative of histone F1 has a role in maintaining the G0 state and that one or both of the other derivatives is concerned with control of DNA synthesis.

1. At 1h after operation livers from partially hepatectomized rats showed a 60–100% increase in the capacity to concentrate 3 H radioactivity from orotate, thymidine or uridine with respect to the radioactivity in plasma. Uptake of [ 3 H]cytidine into liver was unaffected, as was entry of any precursor studied into any tissue other than liver. 2. This increase in intracellular radioactivity was detectable 10min after operation with both orotate and thymidine. With orotate the augmentation had disappeared by 3 days, but with thymidine it was still evident 8 days after partial hepatectomy, when [ 3 H]thymidine incorporation into DNA was no longer increased. Competition studies established that orotate was not entering the liver by the same mechanism as thymidine. 3. In the soluble fraction of the liver all the 3 H radioactivity from orotate was present as uridine nucleotides. Thymidine was not phosphorylated, and was believed to be catabolized.

1. At 0.5–1.0h after partial hepatectomy the intracellular acid-soluble fraction of rat liver took up twice as much radioactivity from [ 3 H]orotic acid, [ 3 H]uridine and [ 3 H]thymidine as did similar fractions from sham-operated animals. This increase in penetration was not prevented by adrenalectomy or actinomycin, both of which decreased precursor uptake into nuclear RNA at this time. The increase in entry was still shown by thymidine at 22h after operation, at the height of the S period. Adenine penetration was not increased 1h after partial hepatectomy. 2. Plasma concentrations of ornithine and possibly methionine, tyrosine and lysine were raised 1.5h after partial hepatectomy. [ 3 H]Lysine entry into regenerating liver at this time was increased by 60% [ 3 H]valine uptake was unaffected. Intracellular amounts of tyrosine, phenylalanine and ornithine in the liver were also increased. 3. The relation of these events to the start of liver regeneration is discussed.

1. The activity of the soluble phosphokinase for histone F1 increases in regenerating rat liver during the first period of DNA synthesis after partial hepatectomy. The increase probably represents new enzyme synthesis. 2. A dose of 500rd of γ-irradiation given early in G1 decreases the amount of histone F1 phosphokinase found 22h after partial hepatectomy by 60–70%. 3. The enzyme preparations also contained a histone F1 phosphatase; the presence together of the kinase and phosphatase caused a disproportion between net 31 P uptake and 32 P incorporation into histone F1. 4. All four subclasses of histone F1 could accept phosphate from ATP. 5. Crude enzyme preparations transferred more 31 P into histone F1 with an initially low phosphate content than into one with a high phosphate content; conversely, more 32 P was transferred into the latter.

1. Liver cell fractions were prepared by non-aqueous procedures and nuclei were also obtained in a hyperosmotic sucrose medium. Histone phosphokinase activity, assayed with histone F1 as substrate, was present in the soluble fraction of the cytoplasm and also bound on to the chromatin fraction of the nucleus. 2. The activity of the enzyme increased sixfold in nuclei from regenerating livers 22h after partial hepatectomy. 3. The enzyme bound in the nucleus was only marginally activated by 1μ m -3′:5′-cyclic AMP which stimulated the cytoplasmic soluble enzyme fourfold. 4. Nuclei prepared by the non-aqueous technique were also able to phosphorylate histones F2a and F3 and showed histone phosphatase activity with histone F1 phosphate as substrate.

1. Changes in the microstructure of histones from the pancreas were followed in rats given ethionine while on a protein-free diet. The lysine-rich histone F1 and the other histones extracted subsequently with 250m m -hydrochloric acid were isolated. When the ethionine-treated rats returned to the stock diet, regeneration of the pancreas was accompanied by an immediate increase in phosphate content of the total histone extract from which histone F1 had been removed. After 3–4 days there was a further increase in the phosphate content of this extract, and also in that of histone F1. 2. Similar changes in the phosphate and thiol+disulphide content of pancreas histones other than F1 were produced by starvation and re-feeding.

Four subfractions of histone F1 from rat thymus were obtained by preparative polyacrylamide-gel electrophoresis. These subfractions are closely related in primary structure but show marked differences in radioactivity when derived from rat thymus nuclei labelled in vitro with [ 32 P]phosphate.

1. Two minor protein fractions (B and C) were separated from histone F1 by chromatography on DEAE-cellulose. Fraction B was acidic. 2. Uptake of 32 P in vivo into histone F1 but not into fractions B and C was stimulated by partial hepatectomy. 3. It is suggested that partial hepatectomy causes an increase in the number of histone F1 molecules phosphorylated.

1. DNA synthesis in Echinus esculentus eggs kept at 10°C takes place just after fusion, 0.75–1.5h after fertilization, and at telophase at about 2.67–3.33h after fertilization. 2. An increase in the thiol/thiol+disulphide ratio in acid extracts from washed nuclear fractions of the eggs is found at fusion, at early stages of mitosis and at telophase. When DNA is being synthesized, the relative amount of thiol in the extracts increases. 3. There are at least five thiol-containing histones in the acid extract together with a diffusible thiol peptide containing methyl-lysine and 3-methylhistidine and a thiol-containing acidic protein.

1. The proportion of thiol groups in the total thiol+disulphide of histone extracts from fertilized eggs from Echinus and Psammechinus was increased during periods of structural alterations in the nucleus. 2. The probable start of DNA synthesis in the fertilized eggs coincided with periods of maximum thiol content. 3. Histone extracts from rat liver and regenerating liver were predominantly in the thiol form and no significant variations could be detected during the first 30hr. after partial hepatectomy. 4. An assay system was developed to follow the phosphorylation believed to be associated with the arginine-rich histone F3. Phosphorylation increased by 50% at 1 and 2hr. after partial hepatectomy. The phosphate content also increased during the period of DNA synthesis. 5. The increased phosphorylation found 1hr. after partial hepatectomy was not prevented by actinomycin or prior irradiation. 6. The phosphate content of histone F1 was very high in livers from foetal rats and declined in neonatal rats similarly to the decline in DNA synthesis.

1. The capacity of the histone–DNA complex to act as a template for RNA synthesis is increased by phosphorylation of histone F1. 2. In regenerating liver, DNA synthesis is preceded by phosphorylation of histone F1. 3. Exposure to ionizing radiation in vivo decreases and delays the phosphorylation of histone F1 in regenerating liver, and decreases it in the hyperplastic kidney. 4. Histone F1 is phosphorylated to a greater extent in dividing than in resting tissues.

1. In regenerating rat liver the phosphate content of the lysine-rich histone F1, but not that of the more arginine-rich histone F3-1, increases during the period of DNA synthesis. 2. The phosphorylation of histone F1 in this ‘S period’ is decreased by γ-irradiation, but, like phosphate uptake into DNA, is affected to an even greater extent if the irradiation is given in the presynthetic period. 3. Histones from three species of sea-urchin eggs show similarities to the F2 and F3 groups of histones from mammalian thymus gland. 4. The proportion of thiol to total thiol plus disulphide in acid extracts from sea-urchin eggs varies from less than 20% in mature unfertilized eggs to 59% just before cleavage. 5. The phosphorylated forms of histones F1 and F3 are less effective in decreasing DNA synthesis by DNA polymerase than the non-phosphorylated forms. 6. Oxidation of thiol groups on histone F3-1 does not affect its capacity to decrease DNA synthesis in vitro .