NUCLEAR MEMBRANES OF CULTURED MAMMALIAN CELLS IN THE PERIOD PRECEDING DNA SYNTHESIS

When kidney cells are cultured directly from the rabbit, the nuclear membranes undergo a change that can be measured as an increase in electrophoretic mobility. The change appears to begin immediately upon culture and is maximal 2 hours later, after which the mobility remains constant at the elevated level. Actinomycin D and p-fluorophenylalanine, but not EDTA or ionizing radiation, suppress the increase in nuclear electrophoretic mobility. With synchronously growing L cells, no change was detected in nuclei from cells taken during various parts of the division cycle.     

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.     

Increased levels of rat hepatic nuclear free and engaged RNA polymerase activities during liver regeneration.

Rat liver nuclei, seventeen hours after partial hepatectomy, showed a two to three-fold increase in total RNA synthesis $\text{in vitro}$ over the sham operated controls. When tested with exogenous synthetic template, this was found to be mainly a reflection of increased levels of both the nuclear free and engaged RNA polymerase activities $\text{per se}$. It was also observed that there was a greater stimulation of the species of RNA polymerase that are α-amanitin resistant than sensitive (3.2 μg/ml). This observation was further confirmed by DEAE-Sephadex column chromatography of the solubilized nuclear free and engaged RNA polymerases and found RNA polymerase I and IIIa were the major species greatly stimulated during this period of liver regeneration. These data suggest not only that there exists a sensitive equilibrium between the nuclear free and engaged RNA polymerases; they also suggest the possibility that RNA polymerase itself may play a positive role in the regulation of gene expression.     

REUTILIZATION OF LYMPHOCYTE DNA BY CELLS OF INTESTINAL CRYPTS AND REGENERATING LIVER

Lymphoid cells from mice injected 54 hours and 30 hours earlier with ³H-thymidine were washed and transfused into isogenic recipients at 29 to 30 hours after partial hepatectomy. The recipients were killed 28 to 30 hours later, and liver, intestine, Peyer''s patch, spleen, and the transfused cells were examined in autoradiographs exposed 6 months. Approximately 80 per cent of the labeled transfused cells were classed as lymphocytes. The labeled DNA contained in the transfused cells was partitioned to about 14 times as many recipient liver and intestinal cells, appearing in 72 to 78 per cent of hepatocyte nuclei, in 30 to 35 per cent of liver reticuloendothelial nuclei, and in 90 to 95 per cent of intestinal crypt nuclei. The label was not comparably widespread in the lymphoid organs, but was limited to a few intensely labeled lymphocytes and a somewhat larger number of very weakly labeled cells. When heat-killed cells rather than living cells were transfused, intensely labeled lymphocytes were absent from the lymphoid organs, but the labeling of cells in the recipients was otherwise identical. The results suggest that (a) reutilized DNA is derived from dead cells, (b) reutilized DNA is mainly degraded to nucleosides and nucleotides, the usual immediate de novo DNA precursors, before reincorporation into DNA, and (c) DNA reutilization may occur in the lymphoid organs, but on a less active scale than in intestine or regenerating liver.     

Protein,nucleic acids and cell structure in the regenerating mouse liver

Summary Prior to the onset of mitotic activity in the regenerating mouse liver, the concentrations of total protein and DNA, but not of RNA, decreased to 93 per cent of their levels in normal liver. During maximum mitosis (48–72 hours), the DNA and RNA concentrations were 110 per cent of their normal levels. The concentrations of liver nucleic acids 24 hours after a sham-operation were also about 110 per cent of normal values.Increased concentrations of insoluble protein were found at 12, 24 and 144 hours after partial hepatectomy. This may reflect an increase in the stability of the liver cell membranes during the regenerative period. Increased amounts of various cytoplasmic membranes were indicated by electronmicroscopy and may also have contributed to the increase in insoluble protein.A temporary increase in the measured solubility of the liver protein occurred during maximum mitosis. It is suggested that this was due to the association of protein with lipids during the depletion of accumulated fat from the regenerating liver.This investigation was facilitated by grants from the Royal. Physiographical Society.I wish to thank Professor I. Agrell and Docent B. Karlsson for helpful advice and criticism and Miss E. K. Persson for skilled technical assistance.     

Nuclear ribonucleoprotein complexes containing U1 and U2 RNA.

Nuclear ribonucleoprotein (RNP) complexes that contain the U1 and U2 RNA of chromatin of Novikoff hepatoma cells were extracted with 0.01 M Tris-HCl (pH 8.0) after the nuclei were initially washed with 0.075 M NaCl and 0.025 M EDTA (pH 8.0). These RNP complexes were purified by chromatography on Sepharose 6B columns and centrifugation on sucrose density gradients. The identity of the U1 and U2 RNA in these particles was established by their electrophoretic mobility in polyacrylamide gels and their T1 RNase fingerprints which were identical with those of authentic U1 and U2 RNA (R. Reddy et al. (1974), J. Biol. Chem.249, 6486-6494; H. Shibata et al. (1974), Mol. Cell. Biochem. 4, 3-19). The nuclear riboncleoproteins had a buoyant density of 1.47 g/ml in CsCl gradients. Two-dimensional polyacrylamide gel electrophoresis of their proteins showed these RNP complexes contain 10 polypeptide spots, of which two are phosphorylated in vivo.     

Surface changes in irradiated thymocytes studied by partition in 2-phase systems and electrophoresis

A study was made of thymocyte surface changes 1-6 hour after the irradiation in a dose of 4 Gy by means of two phase partition in dextran-polyethylenglycole systems and of electrophoretic mobility registration. A decrease in the two phase partition coefficient (by 20% per cent in one hour after irradiation) was registered. The electrophoretic mobility of irradiated cells did not change.     

MOLECULES AT THE EXTERNAL NUCLEAR SURFACE : Sialic Acid of Nuclear Membranes and Electrophoretic Mobility of Isolated Nuclei and Nucleoli

The molecules occurring as terminal residues on the external surfaces of nuclei prepared from rat liver by either sucrose-CaCl₂ or citric acid methods and nucleoli derived from the sucrose-CaCl₂ nuclei were studied chemically and electrokinetically. In 0.0145 M NaCl, 4.5% sorbitol, and 0.6 mM NaHCO₃ with pH 7.2 ± 0.1 at 25°C, the sucrose-CaCl₂ nuclei had an electrophoretic mobility of -1.92 µm/s/V/cm, the citric acid nuclei, -1.63 µm/s/V/cm, and the nucleoli, -2.53 µm/s/V/cm. The citric acid nuclei and the nucleoli contained no measurable sialic acid. The sucrose-CaCl₂ nuclei contained 0.7 nmol of sialic acid/mg nuclear protein; this was essentially located in the nuclear envelope. Treatment of these nuclei with 50 µg neuraminidase/mg protein resulted in release of 0.63 nmol of sialic acid/mg nuclear protein; treatment with 1 % trypsin caused release of 0.39 nmol of the sialic acid/mg nuclear protein. The pH-mobility curves for the particles indicated the sucrose-CaCl₂ nuclei surface had an acid-dissociable group of pK. ~2.7 while the pK for the nucleoli was considerably lower. Nucleoli treated with 50 µg neuraminidase/mg particle protein had a mobility of -2.53 µm/s/V/cm while sucrose-CaCl₂ nuclei similarly treated had a mobility of -1.41 µm/s/V/cm. Hyaluronidase at 50 µg/mg protein had no effect on nucleoli mobility but decreased the sucrose-CaCl₂ nuclei mobility to -1.79 µm/s/V/cm. Trypsin at 1 % elevated the electrophoretic mobility of the sucrose-CaCl₂ nuclei slightly but decreased the mobility of the nucleoli to -2.09 µm/s/V/cm. DNase at 50 µg/mg protein had no effect on the mobility of the isolated sucrose-CaCl₂ nuclei but decreased the electrophoretic mobility of the nucleoli to -1.21 µm/s/V/cm. RNase at 50 µg/mg protein also had no effect on the electrophoretic mobility of the sucrose-CaCl₂ nuclei but decreased the nucleoli mobility to -2.10 µm/s/V/cm. Concanavalin A at 50 µg/mg protein did not alter the nucleoli electrophoretic mobility but decreased the sucrose-CaCl₂ nuclei electrophoretic mobility to -1.64 µm/s/V/cm. The results are interpreted to mean that the sucrose-CaCl₂ nuclear external surface contains terminal sialic acid residues in trypsin-sensitive glycoproteins, contains small amounts of hyaluronic acid, is completely devoid of nucleic acids, and binds concanavalin A. The nucleolus surface is interpreted to contain a complex made up of protein, RNA, and primarily DNA, to be devoid of sialic acid and hyaluronic acid, and not to bind concanavalin A.     

Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

Semipermeability of the nuclear membrane in the intact cell

The osmotic properties of nuclei in intact cells were studied by injecting solutions into the cytoplasm of amphibian oocytes. Subsequent changes in nuclear volume were recorded photographically. The injection of solutions containing polyvinylpyrrolidone or bovine serum albumin caused changes in nuclear volume which were related to the colloid osmotic pressure of the solution injected. The concentration in which no significant nuclear volume change occurred (the isotonic range) was 1.0 to 1.5 per cent polyvinylpyrrolidone (2.0 to 3.75 x 10(-4)M). 2 per cent bovine serum albumin had no significant effect on nuclear volume, whereas 4 per cent caused a significant decrease. The significance of these findings is discussed in terms of the permeability characteristics of the nuclear membrane.     

Electrophoretic mobility of mouse cells and homologous isolated nuclei

The electrophoretic mobilities of Ehrlich ascites, sarcoma 37 ascites, mouse liver cells and their isolated nuclei were measured under similar environmental conditions. No differences in mobility were detected between cells and homologous nuclei from the same cell population and it was concluded that their surface charge densities were probably the same. The effect of neuraminidase on Ehrlich ascites and liver cells and nuclei was also determined; neuraminidase reduced the mobility of Ehrlich ascites cell nuclei as well as cells. The reduction in mobility of cells and nuclei prepared by a sucrose method was the same; however, the reduction in mobility of citric acid prepared nuclei was less than that of citric acid treated cells. The reduction in mobility of both liver cells and nuclei was small or insignificant. It is suggested that although cells and nuclei have similar electrophoretic mobilities, possibly different groups contribute to their surface charge.     

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 ± 0.1, 0.6 mM NaHCO₃, had an electrophoretic mobility of - 2.88 ± 0.01 µ/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 ± 0.02, of rat liver mitoplasts was - 1.22 ± 0.07, and of rat cerebral cortex mitoplasts - 0.91 ± 0.04 µ/sec per v per cm. Treatment of these particles with 50 µg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in µ/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 µg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK_a ∼ 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.     

RNA synthesis in maturing avian erythrocyte nuclei

The rate of RNA synthesis and its inhibition by α-amanitin in the nuclei of mature and immature avian erythrocytes are increased with the increase in ionic strength of incubation medium. Polyacrylamide gel electrophoresis indicates that heterogeneous species of RNAs are synthesised in the mature and immature erythrocyte nuclei. However, a large number of high molecular weight RNAs are synthesised in the nuclei of immature erythrocytes. Elution profiles on poly(U)-sepharose chromatography indicate that the RNAs synthesised in the nuclei of two types of cell contain poly(A) segments. Sixteen per cent of mature erythrocyte nuclear RNA syntbesised are polyadenylated, while it is 13% in immature erythrocyte nuclei. However, the total RNA synthesised is 2–3 fold higher in immature erythrocyte nuclei than that in mature erythrocyte nuclei.     

PROPERTIES OF CELL NUCLEI ISOLATED FROM VARIOUS REGIONS OF RAT BRAIN: DIVERGENT CHARACTERISTICS OF CEREBELLAR CELL NUCLEI

Abstract— Cell nuclei were isolated in yields ranging from 38 to 61 per cent from six anatomically defined brain regions of the albino rat. To provide basic information for further studies of altered genomic activity in brain cell nuclei, various properties of these isolated nuclei were measured, including counts of their number, estimates of the distribution of sizes, amounts of RNA, DNA and protein, and endogenous RNA polymerase activity. DNA content per nucleus approximated the accepted value of 6 pg per diploid set of chromosomes. Distributions of nuclear size showed a sensitivity to the concentration of divalent cation, with a shift toward larger nuclear diameters as the Mg concentration was reduced. Cell nuclei from hippocampus, hypothalamus-preoptic region, cerebral cortex, amygdala and midbrain plus brainstem were generally similar in yield, distribution of size, and RNA, DNA and protein content. Cell nuclei from cerebellum differed from those of other brain regions, in all of these parameters. The cerebellum contained a high content of DNA and had an enormous number (8 × 10⁸ per g wet wt.) of cell nuclei of predominantly very small size and characterized by lower ratios of RNA, histones and non-histone protein to DNA and lower endogenous activity of RNA polymerase than nuclei from other brain structures. These properties correlated well with properties of cerebellar tissue, namely, high content of small granule neurons and low ratio of RNA to DNA, and suggest that the small cerebellar nuclei may have relatively inactive genomes. The relationship of 'large' and 'small' cell nuclei to cell types in the brain is discussed.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

Some chemical properties of isolated pea nucleoli

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the living nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleoli; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.     

BIOCHEMICAL EFFECTS OF THYROID DEFICIENCY ON THE DEVELOPING BRAIN

Abstract— The effects of neonatal thyroidectomy on some constituents of the cerebrum, cerebellum and liver of the rat have been studied during the first 7 weeks of life. In the normal rat between the 6th and 14th post-natal days the RNA content per unit of DNA in the brain increased by 70 per cent. Although the brain continued to grow from the 14th to the 35th day, the amount of RNA relative to DNA decreased by about 20 per cent. The ratio of protein to DNA increased during the whole period studied and in the cerebral cortex it was more than trebled between the age of 6 and 35 days. The growth of the cerebellum extended over a longer period than that of the cerebrum, its weight increasing by 88 per cent between the ages of 14 and 35 days as compared with a cerebral increase of 34 per cent. The DNA content showed a 50 per cent increase during this period. Qualitatively these maturational changes were not affected by neonatal thyroidectomy. Quantitative changes, which applied equally to the cerebral cortex and brain as a whole, were observed. At the age of 35 days, the weights of the cerebral hemispheres and cerebellum were reduced by thyroidectomy by 20 per cent; the overall DNA content per organ did not change, but the amounts of protein and RNA relative to DNA decreased significantly. It is therefore inferred that thyroid deficiency affects the size of the cells in brain and cerebellum rather than their total number. Conversely, the cell population of the liver was only a quarter of that in the control. There was a small but significant decrease in the hepatic protein and RNA content in the hypothyroid animal. The activities of the following enzymes which served as markers for subcellular fractions in homogenates of cerebral cortex were determined: lactate dehydrogenase for the supernatant, glutamate dehydrogenase for the mitochondrial and glutamate decarboxylase for the synaptosomal fractions. When the activities were expressed on a fresh weight basis a significant decrease by comparison with the control values was observed only in the case of glutamate decarboxylase (—15 per cent at the age of 17–32 days); when the activities were based on DNA content all values were reduced, probably as a result of the general decrease in cell size. Pyrimidine metabolism of brain and liver, studied after the administration of [6-¹⁴C]-orotic acid, was not affected in either tissue by neonatal thyroidectomy. A small but significant reduction in the incorporation of labelled pyrimidine nucleotides in liver RNA was observed, but no significant decrease in the incorporation in cerebral RNA was found in the hypothyroid rats.     

Some Chemical Properties of Isolated Pea Nucleoli

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the living nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleoli; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.