Phospholipids in plant and animal chromatin

Isolated hepatic nuclei and hepatic chromatin have been analysed for their DNA, RNA, protein and phospholipid content. The protein/DNA ratio is 3 for nuclei and 1.95 for chromatin extracted from Triton X-100 treated nuclei. The phospholipids, (2.36 +/- 0.91 (S.D.) per cent of the total nuclear material), are lost during the chromatin preparation mainly during the Triton X-100 washings of the nuclei. Nevertheless, 10 per cent of the total nuclear phospholipids remain bound to the chromatin. The comparative analysis of both nuclei and chromatin shows a difference in phospholipids and fatty acid composition. Thus, the chromatin-associated phospholipid cannot be attributed simply to contaminating nuclear membrane. This is supported by the autoradiographic study of semi-thin sections of interphase nuclei from root apices of Vicia faba in which [3H] ethanolamine is clearly localized in the chromatin and nucleolar regions of the nuclei.     

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.     

Structural and functional aspects of nuclear lipids in normal and tumor cells

This review summarizes available data on the structural and functional role of neutral lipids and phospholipids in normal and tumor eukaryotic cells. The role of acidic phospholipids (cardiolipin, phosphatidylinositol, and phosphatidylglycerol) in regulation of activities of DNA- and RNA-polymerases, DNA-topoisomerases I and II, DNA-methylases, and replication initiation proteins (dnaA and T-antigen) is discussed. The role of sphingolipids is emphasized considering, on one hand, the involvement of sphingosines in signal transduction, chromatin association-dissociation, and regulation of DNA and RNA synthesis and protein kinase C and, on the other hand, participation of ceramides and dihydroceramides in apoptosis. The possible role of sphingomyelin, sphingosine, cardiolipin, and diglycerides in the contacts of DNA loops with nuclear matrix is analyzed. Lipid hormones indirectly influence supercoiled DNA conformation; the effect of hormones on metabolism of phospholipids and neutral lipids in chromatin and nuclear matrix is reviewed. Characteristics of lipid composition in chromatin and nuclear matrix of the tumor cells are discussed.     

The effect of phospholipase C on DNA synthesis, morphology and phospholipid content of isolated HeLa cell nuclei.

Isolated HeLa cell nuclei have been treated with purified phospholipase C (Bacillus cereus) and sphingomyelinase (Staphylococcus aureus). The phospholipids of untreated nuclei consisted of about 67% phosphatidylcholine, 23% phosphatidylethanolamine, 7% sphingomyelin, 2% phosphatidylserine and 1% phosphatidylinositol. Phospholipase C degraded 80-90% of the total phospholipids of the nuclei. Such nuclei seemed ultrastructurally intact, and had an average diameter and a protein loss during incubation which were not significantly different from those of controls. Their rate of DNA synthesis was only slightly reduced after treatment with phospholipase C alone and slightly more reduced when phospholipase C was used in combination with sphingomyelinase. This suggests that the polar head-groups of the nuclear phospholipids are of very limited importance in DNA synthesis. Since it has been reported that phospholipase C treatment releases nascent DNA from a membrane complex, the absence of a concommitant reduction in DNA synthesis may suggest that this complex is not necessary for the replication of DNA. Phospholipase C did not significantly influence the stability of the DNA product and gave only a slight inhibition of cytosol and nuclear DNA polymerases when tested with exogenous template.     

The loss of rapidly labelled ribonucleic acid from isolated HeLa cell nuclei

1. The loss of nucleic acids and protein from isolated HeLa-cell nuclei was studied. During 4hr. incubation at 37 degrees DNA was conserved, but appreciable amounts of RNA and protein were lost. 2. Two classes of nuclear RNA were distinguished: at least 75% of the RNA was lost from the nuclei relatively slowly through degradation to acid-soluble fragments; the rest of the RNA was lost much more rapidly, not only through degradation to acid-soluble fragments but also through diffusion of RNA out of the nuclei into the incubation medium. 3. The RNA that was preferentially lost was the fraction of nuclear RNA that was rapidly labelled when intact HeLa cells were grown in a medium containing radioactive precursors of RNA. 4. The RNA appearing in the incubation medium was apparently partially degraded and had a sedimentation coefficient of about that of transfer RNA. 5. Both the degradation of RNA and the loss of RNA from the nuclei were sensitive to bivalent cations. Low concentrations of Mg(2+) and Mn(2+) greatly increased the rate of degradation of the rapidly labelled RNA to acid-soluble fragments, and produced a corresponding decrease in the amount of RNA diffusing into the medium. At higher concentrations they suppressed both degradation and diffusion of RNA. The cations Ca(2+), Cu(2+), Zn(2+) and Ni(2+) all progressively inhibited both forms of loss of RNA. 6. Salts of univalent cations produced appreciable effects only at ionic strengths of about 0.2, when degradation to acid-soluble fragments was preferentially inhibited. 7. Both ADP and ATP inhibited loss of RNA at about 30mm. 8. It was concluded that the diffusion of rapidly labelled RNA out of the isolated nuclei was not related to the movement of RNA from nucleus to cytoplasm in vivo, but reflected the ease with which the rapidly labelled RNA detached from the chromatin and the permeability of the membranes of isolated nuclei.     

Chromatin phospholipids and DNA synthesis in hepatic cells

The synthesis of phospholipids found in microsomes, in the nuclei and in chromatin has been studied in rat liver after partial hepatectomy. [32P]O4(2-)incorporation in phospholipids has been compared with that of (3H) thymidine over a period of 48 h after operation. The presence of two peaks of DNA synthesis has been observed at 18 and 36 h; nuclear phospholipids show a continuous synthesis starting from 12 h, whereas the microsomes show two peaks at 12 and 24-30 h. The specific activity of the chromatin phospholipid fraction increases at 12h, doubles its initial value at 18 h, shows a peak at 30 h and comes back to the initial value at 48 h. It is concluded that chromatin phospholipids increase their synthesis in relation to the S phase of the cell cycle, whereas those of the nuclear membranes do not change the rate of synthesis throughout the cell cycle. The possibility is suggested that chromatin phospholipids are synthesized in the microsomes and transferred to the nucleus.     

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.     

DNA, RNA, protein and heterochromatin changes during embryo development and germination of soybean (Glycine max L.)

DNA, RNA, protein and heterochromatin were measured cytophotometrically in developing soybean (Glycine max) seeds. The average 2C DNA content for the soybean genome was 2.64 pg. The amounts of nuclear DNA in embryo axes showed no significant change during embryo development, whereas the DNA content in cotyledon nuclei increased significantly from 3.58 pg to 5.49 pg. The number of endopolyploid nuclei increased from 26% to 48% and the DNA content from 4.45 to 5.49 pg after cessation of cell division. The changes in RNA and protein content during embryo development were in general similar to those in DNA content. This can be interpreted that increased DNA levels in soybean cotyledons generated during embryogeny increase the protein synthesizing capacity. During the first 15 days of germination, the number of endopolyploid nuclei in cotyledons declined from 46% to 4%, and this decline is interpreted as DNA degradation providing a ready source of nucleosides and phosphates during early embryo growth. A later decline, however, between 15 and 20 days after germination, was age related similar to leaf senescence, because the percentage of endopolyploid nuclei remained unchanged while the number of non-viable cells increased. In senescing cotyledons, 73% and 80% of RNA and protein but only 20% of DNA were lost, as compared to dormant cotyledons. The heterochromatin (condensed chromatin) measurements indicated that nuclei of metabolically inactive dormant and senescent cotyledon nuclei contained an average of 33% more heterochromatin than nuclei from the green cotyledons of seedlings.     

Effect of the conditions of isolation and incubation of the nuclei on digestion of DNA chromatin by calcium- and magnesium-dependent endonuclease. Change in the spectrum of chromosomal proteins and possible role of DNA-protein interactions]

Digesting of chromosomal DNA of interphase rat liver nuclei by Ca, Mg-dependent endonuclease in situ in the presence of chelating agents results in the appearance of the soluble DNP--up to 30% of the total DNA. In addition, 50% of the chromatin is solubilised after mild ultrasonication. In the absence of the chelating agents the degree of fragmentation is considerably increased. The process is accompanied by a loss of some histone and nonhistone chromosomal proteins; the nonhistone proteins are lost selectively. The preliminary removal of the nuclear membrane and significant part of the proteins by tritone X-100 promotes the chromatin degradation and the appearance of low molecular weight fragments. The DNA-fragments of solubilised chromatin are similar to the DNA-fragments of residual chromatin, but in the presence of the chelating agents the latter does not contain monomeric fragments.     

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.     

An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.

Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.     

Actinomycin D binding in vitro: active chromatin preferred.

When [3H] Actinomycin D (Act. D) is used to interact with nuclei and nucleoli in vitro, it binds preferentially to nucleolar chromatin. The preferential binding is no longer detectable, when purified nuclear and nucleolar DNAs are used. In parallel, Act. D preferentially inhibits nucleolar over nuclear RNA synthesis when chromatin templates are used, and the preferential inhibition is lost when purified nuclear and nucleolar DNAs are used. It is concluded: 1) the preferential inhibition of nucleolar over nuclear RNA synthesis by Act. D is a direct reflection of the preferential binding of Act. D to the nucleolar chromatin; and 2) the nucleolar chromosomal proteins, not the nucleolar DNA, confer the preferential binding of Act. D.     

Effect of cycloheximide on lipid metabolism in cells, nuclei and subcellular fractions in the rat liver

Well coordinated stages of inhibition, restoration and stimulation of protein, DNA and RNA synthesis were observed after administration of cycloheximide (3 mg/kg). The changes in lipid synthesis and composition in the nuclei and intranuclear structures were studied at different stages of cycloheximide action. The accumulation and stimulation of lipid synthesis in the nuclei during the inhibition and restoration of protein and DNA syntheses were followed by electron microscopy and labeled precursors methods. Dramatic changes were observed in the phospholipid composition of chromatin and nuclear matrix. The accumulation of minor phospholipid fractions in intranuclear structures was observed during DNA synthesis. The sphingomyelin concentration was predominant and commensurable with those of phosphatidylcholine and phosphatidylethanolamine.