Synthesis of chromatin phospholipids

The presence of a phospholipid fraction associated with chromatin has been demonstrated by biochemical technique in rat hepatocytes. The composition of this fraction determined by chromatography with respect to that of the nuclei is characterized by low content of phosphatidylserine and high content in phosphatidylethanolamine. Also the synthesis and turnover studied after injection of [32P]O4(2-) show a different behaviour: the peak of activity is after 6 hrs in nuclei and microsomes, whereas in chromatin it occurs after 9 hrs. A second peak is evident after 24 hrs in chromatin and microsome phospholipids. Differences have been also shown by analyzing the single phospholipid radioactivity in time. The behaviour of chromatin phospholipids has also been studied during DNA premitotic synthesis in regenerating liver. It has been shown that there is no difference in synthesis in relation to that of DNA in nuclear phospholipids, whereas the specific activity of chromatin phospholipids begins to increase twelve hours after hepatectomy and continues throughout the period of the first mitotic wave, thus bringing to a summation with the beginning of the second wave. The role of this phospholipid fraction in relation to DNA synthesis and gene expression is discussed.     

Phospholipids in chromatin: incorporation of [32P]O4(2-) in different subcellular fractions of hepatocytes

Nuclear, chromatin and microsomal fractions were isolated from hepatocytes prepared from rats injected with [32P]O4(2-) and killed subsequently at times between 1 and 48 h. Specific activities of the total phospholipids (PL) were determined for each subcellular fraction. The major points noted were the initial specific activity of the chromatin PL was half that of both nuclear and microsomal PL at 1 h; the first peak of labelling occurred at 6 h in both nuclear and microsomal PL, but was 3 h later (9h) in the chromatin PL; and a second peak of labelling occurred in the chromatin and microsomal PL, but not in those of the nuclei. On fractionation of the PL, the major and most metabolically active components were phosphatidylcholine + phosphatidylethanolamine, whilst sphingomyelin accounted for only about 8 per cent of the total PL. The chromatin and microsomal fractions were somewhat similar in their labelling patterns though with a delayed peaking of activity in the chromatin. This is indicative of a synthesis and transport of PL from the microsomes to the chromatin.     

Comparative sensitivity of the nuclear and mitochondrial DNA syntheses to X-irradiation and to the administration of a sulfhydryl radioprotector (AET) in normal and regenerating rat liver

Summary Incorporation of thymidine into nuclear and mitochondrial DNA has been measured in the livers of normal rats and of rats killed 17 h or 24 h after partial hepatectomy. When total-body X-irradiation (500 or 1,500 R) is given at increasing intervals (up to 30 h) before sacrifice, a progressive decrease followed by a plateau of low level of incorporation is observed in the nuclear DNA (N-DNA) whereas the synthesis of mitochondrial DNA (M-DNA) first decreases until a minimal value is reached after 4 h in normal liver and 12 h in regenerating liver, then recovers at an exponential rate, quite independently of the nuclear DNA synthesis.The results suggest that irradiation can interfere with the enzymatic processes of both M-DNA and N-DNA syntheses, but that the persistence of the inhibition of N-DNA synthesis must be ascribed to a temporary block in the cell cycle.The SH-protector AET injected to non-irradiated rats exerts a very small inhibitory effect on normal liver N-DNA and M-DNA synthesis. In regenerating liver, both syntheses are affected rather similarly suggesting a common mechanism of inhibition through impairment of some step in the synthetic process.     

CHOLINE BASE EXCHANGE ACTIVITY IN RAT HEPATOCYTE NUCLEI AND NUCLEAR MEMBRANES

Previous investigations have demonstrated the presence of phospholipids as a component of chromatin; however the mechanism of their synthesis, namely if they are synthesized in the nuclei or in the cytoplasm (microsomal fraction), from where they may eventually be transported to the nucleus, has not yet been clarified. The phosphatidylcholine, for example, can be formed, albeit in a limited amount, by an interconversion reaction between bases. The aim of the present research was to ascertain the presence of the enzyme complex responsible for this reaction in hepatocyte nuclei and in isolated nuclear membrane. The incorporation of [¹⁴C]-choline in phosphatidylcholine was assayed in microsomes, hepatocyte nuclei, liver nuclei and nuclear membranes of rat liver. The reaction was Ca²⁺-dependent and the specific activity was higher in microsomes but was present, albeit at a low level, also in nuclei and in nuclear membranes. Possible contaminations were excluded by specific microsomal markers and by the reaction time course. In fact, the nuclear reaction reached the maximum level slowly with respect to microsomes. Since the phosphatidylcholine extracted from the nuclei show an enrichment in unsaturated fatty acids of monoenoic fraction, such as oleic acid, the difference in reaction kinetics has been tentatively explained as due to the phosphatidylcholine fatty acid content. The presence of this base exchange enzyme complex may allow a fast change in chromatin phospholipid composition.     

A temperature-sensitive mutant of cultured mouse cells defective in chromosome condensation

A temperature-sensitive mutant, designated ts85, was isolated from a mouse mammary carcinoma cell line, FM3A. The ts85 cells grew at 33 °C (permissive temperature) with a doubling time of 18 h, which was almost the same as with wild-type cells, whereas the cell number scarcely increased at all at 39 °C (non-permissive temperature). When the ts85 cells were shifted from 33 to 39 °C, their DNA synthesis fell to below 1% of the initial value in 14 h. RNA or protein synthesis, however, was maintained at the initial levels for at least 14 h at 39 °C. Cytofluorometric analysis of asynchronous cultures and studies with synchronous cultures suggested that the bulk of the cells cultured at 39 °C for 12–18 h were arrested in late S and G2 phases. Electron microscopic observations revealed that chromatin was abnormally condensed into fragmented and compact forms, particularly around nucleoli, in about 80% of cells of an asynchronous culture incubated at 39 °C for 16 h. Cells in mitosis were not detected in such cultures and nuclear membrane and nucleoli were still intact. Such abnormal chromosome condensation was not observed in the ts85 cells at 33 °C or in wild-type cells at either temperature. Since these findings suggest that a ts gene product of ts85 cells is necessary for chromosome condensation, ts85 cells may represent a useful tool for establishing the mechanisms of chromosome condensation. The interrelationship between abnormal chromosome condensation and reduction in DNA synthesis of the ts85 cells is discussed.     

Effect of diet on the lipid composition of cell nuclei and liver mitochondria during ontogenesis of the rat

The possible influence of the type of dietary fat on quantitative changes of different lipids in microsomes, nuclei and chromatin in hepatic cells of albino rats in ontogenesis was studied. While the type of diet had no significant influence on the levels of different phospholipids and fatty acids of nuclei, the type of dietary fat exerted definite effect on the levels of whole phospholipids, cholesterol of microsomes, nuclei and fatty acids of chromatin. The age specificity was observed in nuclear structures.     

Sphingomyelin synthase in rat liver nuclear membrane and chromatin.

The presence of phospholipids in chromatin has been demonstrated, as well as the difference in composition and turnover compared to those present in the nuclear membrane. Recently, some enzymes were also evidenced in chromatin: the base exchange protein complex and neutral sphingomyelinase. The latter has a particular relevance, since sphingomyelin is one of the phospholipids more represented in chromatin. We therefore decided to study the synthesis of sphingomyelin in chromatin and in nuclear membrane isolated from liver nuclei. The evaluation of the enzyme was made (i) using [(3)H]phosphatidylcholine as donor of radioactive phosphorylcholine and (ii) by identifying the product isolated by thin layer chromatography. In both fractions the enzyme phosphatidylcholine:ceramide phosphocholine transferase or sphingomyelin synthase was present, although with higher activity in nuclear membrane. The enzyme present in the chromatin differs in pH optimum and K(m), showing a higher affinity for the substrates than that of nuclear membrane. The results presented show that sphingomyelin synthase is present not only in the cytoplasm at the level of the Golgi apparatus, but also in the nuclei, at the level of either the nuclear membrane or the chromatin.     

The effect of X-rays and artificial hypotermia on lipids in rat neocortex

Lipid content of tissue and of fraction of microsomes in neocortex of Wistar rats was studies under artificial hypothermia, after X-ray irradiation in dose 8 Gy under conditions of normothermia and artificial hypothermia in 48 h. The condition of artificial hypothermia get by cooling of rats to 15-18 degrees C. It was shown, that in fraction of microsomes of hypothermia rats the content of phosphatidylinositol was decreased, and in 48 h after cooling of rats the amount of protein, total and individual phospholipids was increased. The lipid content in tissue and in fraction of microsomes of rats, which were irradiated in normotermia, had no changes after 48 h. In fraction of microsomes of rats, which were irradiated after hypothermia, the amount of protein, total phospholipids, sphingomyelin, phosphatidylcholine and phosphatidylserine is increased trustworthy. Thus, we think, that radioprotective effect of hypotermia may be connected with the accumulation of proteins and of phospholipids in the endoplasmic reticulum membranes of neocortex.     

Cytochemical and autoradiographic studies of the localization and turnover of chromatin and nucleolar associated phospholipids in plant and animal tissues

The localization of chromatin-associated phospholipids has been demonstrated on chromosomes and on chromatin of interphase nuclei by cytochemical methods either in plant and in animal tissues. Three methods of fixation are suggested which can be combined which two cytochemical methods for phospholipids detection. An additional method is represented by autoradiographic technique after incorporation of a radioactive precursor such as [3H] ethanolamine. This method has been used with good results in nuclei of lateral root apices of Vicia faba on 1 micron thick section, thus avoiding any possible contamination by nuclear membrane. In these nuclei the phospholipids appear associated with the chromatin and more intensely with the nucleolar regions. The positivity of the cytochemical reaction disappears after extraction of fixed tissue with acidified methanol chloroform and after treatment of unfixed sections with phospholipase D. The use of phospholipid precursor has allowed the study of chromatin-phospholipids synthesis in root apices of Vicia faba with respect to timings of the cell cycle. The results show that there is a strong case for a pattern of chromatin phospholipid synthesis which operates during S phase. Concerning the role of phospholipids it is suggested that they may be linked to acidic protein and may have a structural function, particularly on the nucleoli.     

Incorporation of 1-(14)C linoleic acid in rat liver nuclei and chromatin fractions

The incorporation of 1-(14)C linoleic acid in several chromatin fractions of rat liver nuclei was investigated using two different procedures: (1) rat liver nuclei were incubated with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid. After 40 min at 37 degrees C the chromatin obtained by sonication of nuclei suspended in 0.25 M sucrose was fractionated by differential sedimentation; (2) chromatin fractions obtained by differential sedimentation were incubated separately with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid 40 min at 37 degrees C in order to characterize the fatty acid incorporation in isolated chromatin. A comparative study of the incorporation of 1-(14)C linoleic acid in microsomes and nuclei isolated from rat liver is also presented for the purpose of comparison. Linoleic acid was incorporated into nuclear lipids as well as in chromatin fractions. The fatty acid incorporation was stimulated considerably in the acylation system when compared to control, it appears to be highly dependent on the state of condensation of chromatin, being barely detectable in the lowest density fraction. The major proportion of 1-(14)C linoleic acid was found in phospholipids and in a lesser proportion it remained esterified to triglycerides and cholesteryl esters. The distribution of radioactivity in different classes of phospholipids present in microsomes and nuclei isolated from rat liver, showed a similar profile of distribution. The major proportion of radioactivity, approximately 50% was found in phosphatidylcholine and in a lesser proportion in sphingomyelin, phosphatidylserine and phosphatidylethanolamine. When chromatin fractions were incubated separately, it was observed that the major proportion of 1-(14)C linoleic acid in phospholipids was found in heavy chromatin fractions whereas low density chromatin fraction only incorporated in a lesser proportion.     

Synthesis and phosphorylation of histones and nonhistone proteins as affected by irradiation and serotonin in cycloheximide-synchronized hepatocytes

Phosphorylation and synthesis of histones and nonhistone proteins were studied after the inhibition of translation by sublethal cycloheximide doses. Activation of the chromatin protein phosphorylation was noted: (1) at the stage of recovery and stimulation of the protein synthesis (18-24 h), and (2) at the stage of activation of the replicative DNA synthesis (30-60 h). Phosphorylation and synthesis of the chromatin proteins depended upon the individual or combined effect of X-radiation and serotonin. The authors discuss the possible role of the chromatin protein phosphorylation in the response of the nuclear apparatus to the effect of radiation and serotonin the latter being used as a radioprotective agent.     

Intranuclear localization of phospholipids by ultrastructural cytochemistry.

The presence of phospholipids within the interphase nucleus and in isolated chromatin, previously demonstrated by analytical biochemical methods, has been only rarely documented by cytochemical procedures, especially at the ultrastructural level. By means of a gold-conjugated phospholipase technique, we investigated the fine localization of endogenous phospholipids in the different nuclear domains in rat pancreas and in cell cultures. To reduce possible removal or displacement of phospholipids, different specimen preparation procedures such as cryofixation, cryosectioning, and freeze-fracturing were utilized. Apart from slight differences in efficiency among these methods, phospholipids have been cytochemically identified in the same nuclear domains: the interchromatin granules and fibers and the dense fibrillar component of the nucleolus. These results suggest that the phospholipids are an actual nuclear component, not randomly distributed in the nucleoplasm but mainly localized in the nuclear domains involved in the synthesis, maturation, and transport of ribonucleoproteins.     

Diurnal changes in the fraction of 3-hydroxy-3-methylglutaryl-CoA reductase in the active form in rat liver microsomal fractions.

'Initial' and 'total' activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in cold-clamped samples of liver from rats at 2h intervals throughout the 24h light/dark cycle. Initial activities were obtained in microsomes (microsomal fractions) isolated and assayed in the presence of 100mM-KF, whereas 'total' activities were measured in microsomes prepared from the same homogenates but washed free of KF and incubated with exogenous partially purified rat liver protein phosphatase. The initial/total-activity ratio for HMG-CoA reductase underwent a diurnal cycle, which had a nadir 4h into the light phase (when initial activity was 28% of total activity) and a peak 12h later, i.e. 4h into the dark phase (when initial activity was 80% of total activity). These low and high points of the cycle were separated by gradual steady changes in the ratio. The characteristics of this diurnal cycle were different from those of the cycle observed for total activity, which had a plateau of high activity between 2 and 10h into the dark cycle preceded and succeeded by a very rapid increase and decrease, respectively, in the total activity of HMG-CoA reductase. The combination of the two cycles resulted in the dampening of the resultant cycle for the initial or effective activity of HMG-CoA reductase, such that the changes in initial activity around the beginning and and end of the dark phase were more gradual than would otherwise have been the case if the initial/total-activity ratio for HMG-CoA reductase were constant throughout the diurnal cycle. The physiological implications of the observed diurnal variation in the fraction of hepatic HMG-CoA reductase in the active form are discussed.     

DNA synthesis and endomitoses in the giant nuclei of the silkgland of Bombyx mori.

At the first symptoms of organisation of the silkgland in the embryo, mitoses stop and nuclei start to grow. Through autoradiographic studies, performed after sequenced labeling with [3H] and [14C] thymidine, the durations of the different phases of DNA synthesis cycles (T = 42 to 48 h, S = 22 to 25 h, G = 20 to 23 h) are established. These durations are in fact identical during the second and the third instar, and the same, whatever region is concerned : posterior, middle or anterior parts. A model has been established to account for the distribution of the S phases during the second and third instars. The DNA synthesis in all nuclei of a given region has been followed during the first four instars by labelling with [3H]thymidine. The activity goes through maxima and minima, depending on the percent of nuclei synthesizing DNA and their synchronism, both being characteristic of each region; long resting periods are observed during the molting stages of the first three instars in the middle and the anterior parts. The coincidence is obvious between the maxima and minima and respectively the S and G phases of the model. DNA assays agree with the distribution of cycles established by autoradiography if one admits that each cycle does lead to a doubling of the amount of DNA. The overall DNA synthesis from the diploid value is estimated to correspond to 18-19 endomitoic cycles in the nucleic of the posterior part, 19-20 cycles of those of the middle part and 13 in those of the anterior part. The analysis of chromosome structures, by squashing the nuclear content, shows that existence of a complete endomitotic cycle: the doubling of chromosomes is associated with condensed structures, alternating with a decondensed state of chromatin, responsible for the DNA synthesis. The female heterochromatin undergoes a restricted morphological cycle delayed with respect to bulk chromatin. It is characterized by a late DNA duplication and by non dispersed daughter chromosomes. Some of these aspects are, to a lesser extent, reproduced in groups of autosomic chromosomes.     

Changes of chromatin organization induced by phospholipids

Isolated nuclei represent a suitable model for studying the influence of exogenous phospholipids, normally found as minor chromatin components, on the nuclear structure, which, in turn, could be related to the observed modifications of DNA and RNA synthesis. The morphological modifications induced on chromatin RNP granules and nuclear matrix have been analyzed both with conventional thin sectioning and with an original method based on image analysis of freeze-fractured and replicated nuclear samples. The results obtained support the hypothesis that anionic phospholipids, by removing histone H1, induce a transition of the chromatin from solenoid to nucleosome conformation and favour the RNA polymerizing activity which results in an increased release of RNP particles, while neutral phospholipids, probably affecting the matrix structure, partly impare the RNP maturation and transport, with consequent increase of chromatin condensation.     

Phosphatidylcholine-dependent phospholipase C in rat liver chromatin

Phosphatidylcholine-dependent phospholipase C is an enzyme which hydrolyses phosphatidylcholine giving origin to diacylglicerol and phosphorylcholine. Diacylglicerol has many effect and activates also protein kinase C. Since the presence of protein kinase C in the hepatocyte nuclei and the existence of a phospholipidic fraction in the chromatin have been demonstrated, we investigated if phosphatidylcholine-dependent phospholipase C could be present in the nuclei. The results obtained have shown the presence of this enzyme in the chromatin fraction which differs with respect to that of nuclear membrane in pH and Km. The activity has been also evaluated during liver regeneration. In the chromatin an increase of activity has been shown 12 h and 30 h after hepatectomy, i.e. at the beginning of hepatocyte S-phase. No similar behaviour has been observed in the nuclear membrane. It has been suggested that diacylglicerol, produced by the hydrolysis of chromatin phosphatidylcholine, may have a role in initiating DNA synthesis through the prolonged activation of the nuclear form of protein kinase C.     

Daily rhythms of nuclear protein fractions in rat liver

Abstract

Daily changes of a number of nuclear functions of rat liver were analysed in rats kept in a light‐dark (LD 12 : 12) cycle and constant temperature. Measurements of the DNA content of rat liver with diphenylamin revealed a mean value of about 3 mg/g liver freshweight without showing significant daily rhy thmicity. When related to mg DNA, no significant rhythmicity could be observed in the total protein content and only a slight rhythmicity in the nuclear protein content. Injection of cycloheximide(2mg/100gbody weight) 10 h before killing the animals resulted in an about 10–20% decrease of the protein content of the tissue as well as of the nucleus and probably in a loss of cell water. Nuclear proteins were separated into nuclear sap proteins, chromatin proteins and restproteins, the first 2 fractions of which were further fractionated by means of polyacrylamide SDS electrophoresis. Considerable differences in the protein content of some of the bands were observed: some bands appeared only at a certain time of day (at 6 h), other bands showed a high amplitude rhythmicity with a maximum at 18 h, whereas other bands— as for example the histone containing bands— varied only slightly during 24 h.     

Kinetics and localization of wound-induced DNA biosynthesis in potato tuber

Tuber wounding induces a cascade of biological responses that are involved in processes required to heal and protect surviving plant tissues. Little is known about the coordination of these processes, including essential wound-induced DNA synthesis, yet they play critical roles in maintaining marketability of the harvested crop and tubers cut for seed. A sensitive “Click-iT EdU Assay” employing incorporation of the thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU), in conjunction with 4′,6-diamindino-2-phenylindole (DAPI) counter labeling, was employed to objectively identify and determine the time course and spatial distribution of tuber nuclei that were wound-induced to enter S-phase of the cell cycle. Both labeling procedures are rapid and sensitive in situ. Following wounding, EdU incorporation (indicating DNA synthesis) was not detectable until after 12 h, rapidly reached a maximum at about 18 h and then declined to near zero at 48 h. About 28% of the nuclei were EdU labeled at 18 h reflecting the proportion of cells in S-phase of the cell cycle. During the ∼30 h in which induced cells were progressing through S-phase, de novo DNA synthesis extended 7–8 cell layers below the wound surface. Cessation of nuclear DNA synthesis occurred about 4 d prior to completion of wound closing layer formation. Initiation of wound periderm development followed at 7 d, i.e. about 5 d after cessation of nuclear DNA biosynthesis; at this time the phellogen developed and meristematic activity was detected via the production of new phellem cells. Collectively, these results provide new insight into the coordination of wound-induced nucleic acid synthesis with associated tuber wound-healing processes.