Properties of isolated frog liver and kidney nuclei

In order for a nuclear preparation to be used for analytical purposes, the method of isolation and composition of the suspension medium must be carefully examined. Accordingly, satisfactory techniques for the isolation of frog liver and kidney nuclei were developed. The medium for frog liver nuclei consisted of: 55% glycerol, 0.001 M magnesium chloride, 0.033 M sodium β-glycerophosphate and/or 0.002 M KH₂PO₄, K₂HPO₄ (pH 6.8), however, the addition of 0.15 M sucrose was essential for satisfactory isolation of kidney nuclei. Inclusion of sucrose (0.15 M ) in the isolation medium promoted nucleolar swelling and a decrease in nuclear volume in liver cell nuclei. Nucleolar migration and extrusion were noted in solutions with high cationic content. The morphological appearance of isolated nuclei was found to be extremely sensitive to the ionic strength of the isolation medium, as was the isolation procedure in toto. Effects were considered to be the result of precipitation and swelling of nucleoprotein. Dissociation of nucleoprotein was considered to be associated with temperature change. The uptake of supra-vital dyes aided in recognition of the morphological alterations and was also an indicator of nuclear viability. Trypsin readily altered the nuclear membrane and a rapid decrease in nuclear density occurred, but the nucleolus remained intact. The diverse response of liver and kidney nuclei as compared with the nucleated red blood cells (a contaminant) to treatment with trypsin was noted and its implications discussed.     

Purification of Xenopus laevis embryo and liver nuclei using metrizamide.

While the nuclei of many diverse types of tissue can be purified by centrifugation through dense sucrose solutions, Xenopus laevis embryo and liver nuclei present special purification problems due to the presence of large numbers of melanosomes in embryo and liver cells. These melanosomes were removed by isopycnic centrifugation in Metrizamide gradients. In Metrizamide, embryo nuclei banded at an average buoyant density of ρ_5°C = 1.288 ± 0.003 g/cm³. Liver nuclei separated into two peaks, one containing nonparenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.274 ± 0.003 g/cm³ and the other peak containing parenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.284 ± 0.001 g/cm³.     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

A METHOD FOR ISOLATING INTACT MITOCHONDRIA AND NUCLEI FROM THE SAME HOMOGENATE,AND THE INFLUENCE OF MITOCHONDRIAL DESTRUCTION ON THE PROPERTIES OF CELL NUCLEI

1. An improved type of ground glass homogenizer for soft tissues has been described which brings about a high degree of cell disruption and liberation of nuclei without causing appreciable damage to mitochondria. The gentleness and effectiveness of the new homogenizer in respect to isolation of mitochondria have been ascertained by comparing the ATP-ase activities of mitochondria isolated in 0.25 M sucrose solution without pH adjustment using a previous type of homogenizer with those of mitochondria isolated under the same conditions with the aid of the new homogenizer. In these experiments sucrose of 0.25 molarity without pH adjustment has been used in order to maintain the mitochondria in a rather sensitive state so as to make slightly deleterious effects of homogenization readily apparent. 2. A new method is described for the isolation of morphologically intact mitochondria and cell nuclei from the same homogenate. In this procedure the pH of the homogenate in 0.44 M sucrose is maintained at 6.0–6.2 with citric acid during the homogenization. An alternative method employing 0.44 M sucrose plus 0.005 M CaCl₂ is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The α-ketoglutarate, malate, succinate, and hexanoate oxidases of the "intact" mitochondria isolated in 0.44 M sucrose adjusted to pH 6.0–6.2 with very dilute citric acid as described in this paper have been investigated, and it has been shown that the mitochondria compare favorably to those isolated in 0.25 M sucrose by a previously described method. 4. Mitochondria have been found to contain an enzyme which causes nuclei to lose their ability to form gels in dilute alkali. This enzyme is released from the mitochondria when the latter are disrupted. 5. Some properties of nuclei isolated by the new method have been briefly discussed.     

Isolation and biochemical characterization of nuclei from chick embryo liver

A procedure is described for the isolation of enzymatically active nuclei from chick embryo liver. It consists of the homogenization of the pooled tissue in 0.32 M sucrose-3 mM MgCl₂ followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg²⁺ ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl₂. Based on DNA recovery, the nuclear fraction isolated by the procedure described contained an average of 62% of the nuclei in the original filtered homogenate. Light and electron microscope examinations showed that 90% of the isolated nuclei were derived from hepatocytes. They appeared intact with well preserved nucleoplasmic and nucleolar components, nuclear envelope, and pores. The isolated nuclei were quite pure, having a very low level of cytoplasmic contamination as indicated by cytoplasmic enzyme marker activities and electron microscope studies. The nuclear fraction consisted of 19.9% DNA, 6.2% RNA, 74% protein, the average RNA/DNA ratio being 0.32. Biosynthetic activities of the two nuclear enzymes NAD-pyrophosphorylase and DNA-dependent RNA polymerase were preserved. The specific activities of these enzymes were: NAD-pyrophosphorylase, 0.049 µmoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg²⁺ activated RNA polymerase, 4.3 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 10 min; and Mn²⁺-(NH₄)₂SO₄ activated RNA-polymerase, 136 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 45 min.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

METHODS FOR THE PURIFICATION OF THYMUS NUCLEI AND THEIR APPLICATION TO STUDIES OF NUCLEAR PROTEIN SYNTHESIS

Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C¹⁴-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.     

Isolation of a rat ventral prostate chromatin fraction exhibiting a singular melting profile

Rat ventral prostate chromatin, prepared by a gentle procedure in which marked shearing forces were avoided, was separated by sucrose gradient centrifugation into a quantitatively minor less dense (L) fraction, and 2 heavier components, (H₁) and (H₂). The (L) fraction, comprising 5–15 percent of the totäl chromatin DNA, had a unique melting profile, with a Tm_app. of 59°, compared to 82.5°, 85° and 80.5° for total, (H₁) and (H₂) chromatin. (L) chromatin has a number of other properties expected of “euchromatin”. This approach to the preparation of chromatin has been used successfully with other tissues such as liver.     

THE ISOLATION OF A CELL MEMBRANE FRACTION FROM RAT LIVER

A procedure is described for isolating cell membranes from rat liver homogenates. 20 gm. of rat liver was homogenized in a Dounce homogenizer in ice cold water buffered to pH 7.5 with NaHCO₃, rupturing all of the cells and most nuclei. The diluted homogenate was filtered through cheesecloth to remove precipitated nucleoprotein and centrifuged at 1500 g, 10 minutes, to sediment a crude membrane fraction. The membrane containing sediment was recentrifuged 3 times in conical tubes (1220 g, 10 minutes), the top layer of the 2-layered sediment being retained. Flotation in a sucrose solution d = 1.22 freed the preparation from contaminating cell fragments and nuclear membranes not previously disintegrated. The floating material ~0.4 ml. was quite homogeneous and consisted of thin amorphous membranes. Electron micrographs revealed numerous double profiles similar in shape and dimensions to apposed liver cell membranes in intact tissue.     

The Efflux of Substances from Frog Ventricles to Sucrose and to Ringer's Solutions

The frog ventricle in sucrose solution contracts for several hours at 25°C, and for as long as 24 hours at 5°G. The possibility that a fraction of the extracellular fluid remains outside of the excitable membrane was examined by measuring the efflux of tracers. The half-time for the efflux to sucrose solution at 25°C of C¹⁴ sucrose is about 1 minute, for Na²⁴ is 6.5 minutes, and for Cl⁸⁶ is 4 minutes. There is no evidence for the retention of an extracellular Na fraction. The Q₁₀ for Na and Cl efflux is about 1.3. The half-time for K⁴² efflux is about 180 minutes; the Q₁₀ is 1.7. The efflux rates of Na²⁴, Cl³⁶ and K⁴² to sucrose and to Ringer's solutions are quite similar. Ca⁴⁵ efflux is only one-fifth as fast to sucrose solution as to Ringer's; the retention of Ca⁺⁺ may be important for maintaining excitability in sucrose solution. P³² efflux is five times faster to sucrose solution than to Ringer's solution, and there is a similar increase in the rate of inosine loss to sucrose solution. The Q₁₀ for efflux to sucrose solution is 2.2 for P³²O₄ and 2.4 for inosine. We suggest that energy metabolism is abnormal in ventricles in sucrose solution and that low temperature prolongs excitability by slowing the metabolic change.     

Preparation and crystal structure of manganese(II) isothiocyanate tetrahydrate

Crystals of Mn(NCS)₂·4H₂O were isolated from an aqueous solution obtained by mixing solutions of barium thiocyanate and manganese(II) sulphate tetrahydrate. The crystals are monoclinic with a = 7.827(7), b = 9.208(1), c = 7.456(5) Å, β = 112.57(5)°, space group P2₁/c. The structure consists of discrete centrosymmetric trans-[Mn(NCS)₂(H₂O)₄] octahedra linked by hydrogen bonds.     

Two-dimensional electrophoresis of proteins of citric acid nuclei prepared with aid of a Tissumizer

Nuclei prepared from normal rat liver and Novikoff hepatoma ascites cells with the aid of a Tissumizer® in media containing 0.5 and 5 % citric acid were compared on the basis of electron microscopic appearance, DNA, RNA and protein content. Electron microscopy revealed better preservation of the nucleolar and nuclear morphology in the nuclei isolated in 0.5 % citric acid than in nuclei isolated in 5 % citric acid. Moreover, losses of protein and DNA from liver nuclei prepared by the sucrose-Ca²⁺ procedure were significantly less in nuclei treated with 0.5 % citric acid than in nuclei treated with 5 % citric acid. The preservation of nuclear morphology and the retention of the majority of types of nuclear protein were significantly better with the procedure using 0.5 % citric acid than with the procedure using 5 % citric acid. The 5 % citric acid treatment was found to alter nuclear morphology and extract specific nuclear proteins, as demonstrated by two-dimensional polyacrylamide gel electrophoresis of the proteins.     

Soluble enzymes of nuclei isolated in sucrose and nonaqueous media; a comparative study

Nuclei of calf thymus and liver and of rat liver were isolated in sucrose media and a number of their properties studied in relation to those of corresponding nuclei isolated in non-aqueous media with a view to determining their capacity to retain soluble components. The best preparations of sucrose nuclei were obtained from calf thymus. Cytochrome oxidase measurements and DNA/N ratios were far less sensitive than microscopic examination as indicators of purity when rat liver and calf thymus nuclei were compared. No satisfactory preparation of calf liver nuclei was obtained, contamination with whole cells having been appreciable; such preparations, nevertheless, could be used to advantage in the tests undertaken. DNA content of thymus nuclei isolated in sucrose was much the same as that of non-aqueous ones, pointing to a retention of soluble protein under aqueous conditions of isolation. That this net retention of protein was not due to the impermeability of the nuclear membrane was shown by the hydrolysis of the DNA upon addition of some crystalline DNAase to a sucrose suspension of nuclei. A comparative study of liver and thymus nuclei isolated in aqueous and non-aqueous media with respect to the soluble enzymes glucose-6-phosphate dehydrogenase, adenosine deaminase, and nucleoside phosphorylase yielded the following results: 1. Lyophilization of sucrose-isolated nuclei and their extraction with the organic solvents used in the non-aqueous procedure did not inactivate any of the enzymes tested. In the case of thymus the reverse was true, there being a marked increase in activity of all the enzymes studied. 2. In thymus, nucleoside phosphorylase and adenosine deaminase were active to approximately the same extent in nuclei isolated by either procedure. Glucose phosphate dehydrogenase alone was more active in sucrose-isolated nuclei, pointing to the possibility of an adsorption of this enzyme. 3. In rat liver nuclei isolated in sucrose, lyophilization and treatment with organic solvents revealed only the presence of some dehydrogenase. 4. The washing out of soluble enzymes was most markedly demonstrated in the case of calf liver. Only traces of the nucleoside enzymes were found in the sucrose-isolated nuclei, and in the case of the dehydrogenase only a half of that present in the non-aqueous nucleus remained. The main conclusions drawn were as follows:— 1. In sucrose media the nuclear membrane is ineffectual in preventing the inward or outward diffusion of protein. 2. The extent to which soluble proteins are retained by a nucleus isolated in sucrose appears to depend upon internal structural factors, such as the concentration of DNA in the nucleus. 3. With respect to determining the composition of nuclei in terms of soluble components, the sucrose isolation procedure is considered to be of indifferent merit and hence invalid for such a type of analysis.     

Effects of D2O and Osmotic Gradients on Potential and Resistance of the Isolated Frog Skin

Exposure of the outside surface of isolated frog skin (R. pipiens and R. catesbeiana) to sulfate solution made up with D₂O decreased skin potential and resistance. Exposure of the inside surface to D₂O solution decreased the potential slightly but increased the resistance. The changes were linearly related to the D₂O concentration. Since D₂O acts like a hyperosmotic solution, the skin potential and resistance were studied upon exposure to solution made hyperosmotic by addition of sucrose, mannitol, acetamide, urea, thiourea, Na₂SO₄, or K₂SO₄. Skin potential and resistance decreased when the outside solution was made hyperosmotic. The changes depended upon the concentration and the nature of the solute. Thiourea and urea solutions were the most effective. Treatment of the inside surface gave relatively small decreases in potential; the resistance either increased or remained unchanged. These effects appeared to depend upon the direction of the osmotic gradient across the skin rather than upon the value of the osmolarity compared to normal body fluids. Experiments with a series of six polyhydric alcohols from methanol to mannitol and the polysaccharides, sucrose and raffinose, showed adonitol with 5 carbons to decrease the potential the most. Smaller and larger compounds of this set gave lesser effects. As yet no consistent explanation of the effects is forthcoming, but their demonstration calls for caution in the indiscriminate use of solutes such as mannitol or sucrose "to make up the osmolality" and in the neglect of urea because "it penetrates freely."     

CYTOCHEMICAL LOCALIZATION OF 5''-NUCLEOTIDASE IN SUBCELLULAR FRACTIONS ISOLATED FROM RAT LIVER : I. The Origin of 5''-Nucleotidase Activity in Microsomes

A procedure has been developed for the cytochemical localization of 5'-nucleotidase in isolated, unfixed, rat liver microsomes. Membranes were incubated with adenosine 5'-phosphate (5'-AMP) and Pb(NO₃)₂ and then isolated on sucrose density gradients: all the phosphate released was recovered with the membranes by this procedure. Adenosine 2'-phosphate (2'-AMP) and adenosine 3', 5'-cyclic phosphate (3',5'-AMP) were shown to be competitive inhibitors, but not substrates, for purified 5'-nucleotidase and were employed to determine the specificity of the cytochemical reaction. It was found that the incubation conditions for the cytochemical assay did not affect the specificity of 5'-nucleotidase. Microsomes incubated as controls with Pb²⁺, or Pb²⁺ and 2'-AMP or 3',5'-AMP were of the same density, although slightly denser than microsomes incubated without Pb²⁺, and were unassociated with lead precipitate when examined by electron microscopy; microsomes incubated with Pb²⁺ and 5'-AMP were much denser and were stained heterogeneously with lead phosphate when examined by electron microscopy. Precipitates formed artificially from Pb²⁺ and inorganic phosphate did not resemble the reaction product. Microsomes were, therefore, separated on sucrose gradients and the subfractions were examined cytochemically. Lead precipitates were associated with the majority of rough-surfaced vesicles, and the reaction product was distributed heterogeneously in all fractions. Vesicles which stained like the membranes of the bile canaliculi in isolated plasma membranes were observed in the lightest subfraction. The reaction product was localized on the outside surface of the microsomal membranes, and was solubilized by low concentrations of ethylenediaminetetraacetic acid. It is concluded that 5'-nucleotidase is present in the endoplasmic reticulum and that the microsome fraction contains, in addition, vesicles derived from the plasma membrane.     

Studies of excitable membrane formed on the surface of protoplasmic drops isolated from Nitella III. Impedance of the surface membrane

A protoplasmic drop isolated from an internodal cell of Nitella in an initial solution composed of 70 mM KNO₃, 50 mM NaNO₃ and 5 mM CaCl₂ became electrically excitable when the drop was placed in the final solution containing 0.5 mM KNO₃, 0.5 mM NaCl, 1 mM Ca(NO₃)₂ and 2 mM Mg(NO₃)₂. The electrical impedance of the surface membrane of the drop was measured both in the initial and final solutions at frequencies between 60 Hz and 100 kHz.The impedance and admittance loci of the surface membrane fell on circular arcs. The d.c. resistance R_m°, and the d.c. capacitance C_m° were determined by extrapolating the circular arcs to the low frequency limit. R_m° thus determined was in the range of 50–200 Ω·cm² in the initial solution, and increased to a steady value of 0.4–4.0 kΩ·cm² when the external solution was replaced by the final solution. After the protoplasmic drop was isolated from the internodal cell of Nitella, C_m° decreased monotonically from about 1.5 μF/cm² within 20 min and approached $\text{1.25±0.1 μ}\text{F/cm}{}^2$ both in the initial and final solutions. No appreciable difference was observed for C_m° in these two solutions.The impedance data were discussed in relation to the process of formation of the membrane at the surface of the protoplasmic drop. After the excitable stage was reached, the drop membrane impedance was found to decrease by a factor of 10 during excitation.     

Solubilization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat and chicken liver microsomes

A simple, efficient, freeze-thaw procedure for the solubilization of liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has been developed. Microsomes of chicken or rat liver were prepared by homogenization in buffer containing 100 mm sucrose, 50 mm KCl, 40 mm KH₂PO₄, 30 mm EDTA, and 2 mm DTT, pH 7.2 (buffer A). The homogenate was centrifuged at 12,000g (15 min), and the microsomes were separated from the supernatant by centrifugation at 100,000g (60 min). The isolated microsomes were frozen, either by dry ice-acetone or by storage in a freezer at ?20°C. The frozen microsomes were permitted to thaw at room temperature, homogenized in buffer A, and centrifuged at 100,000g (60 min). The extraction was repeated and the combined supernatants contained 70 to 90% of the microsomal HMG-CoA reductase activity. The yield of enzyme activity by the freeze-thaw technique is equal to or greater than previously reported methodologies and is significantly easier to perform. This procedure is particularly suited to the preparation of large quantities of solubilized enzyme for isolation and characterization of HMG-CoA reductase. In addition, this method does not require the use of detergents, sonification, or other procedures which might partially inactivate or alter the molecular properties of the enzyme.     

DNA SYNTHESIS IN NEURONAL, GLIAL AND LIVER NUCLEI ISOLATED FROM THE ADULT GUINEA PIG

Abstract— [³H]Deoxythymidine-5′-triphosphate incorporation into P₅₁ (51% neuronal nuclei: 49% glial nuclei), P₃ (3% neuronal nuclei: 97% glial nuclei) and liver nuclear preparations, isolated from the adult guinea pig, was determined in the presence of the other three complementary deoxyribonucleo-tides. The enzymic characteristics of the DNA synthesis reaction were studied and DNA polymerase contents were estimated in neuronal, glial and liver nuclei. (1) Cerebral and liver nuclei exhibited similar enzymic properties for DNA synthesis activities with a few discrepancies. (2) P₅₁ nuclei synthesized DNA 2.4-fold more actively than P₃ nuclei. Liver nuclei carried out the most active DNA synthesis. The proportion of chromatin DNA available as template and primer was estimated by comparison with native calf thymus DNA. The available proportions found, in terms of the total chromatin DNA. were 2.39% for P₅₁ nuclei, 1.38% for P₃ nuclei and 37.6% for liver nuclei. (3) Exogenous native and heat-denatured calf thymus DNA were utilized as template and primer by DNA polymerase in nuclei in different ways depending on the nuclear species. The enzyme was saturated with native DNA by elevating the concentration and the activity reached a plateau. Denatured DNA inhibited the activity at the higher concentrations. (4) From the enzyme activities at a saturation concentration of exogenous DNA, DNA polymerase contents were estimated: P₅₁ nuclei, 39.2 ± 2.6 (s.e.m. ) units (fmol of TMP incorporated/30 min at 31°C)/μg of nuclear DNA; P₃ nuclei. 24.5 ± 1.6; and liver nuclei, 72.5 ± 8.1; the specific activity obtained on a protein basis was 1.55 times higher with P₃ nuclei than with P₅₁ nuclei. (5) Denatured DNA inhibited the nuclear DNA polymerase activity dependent on native DNA. The efficiency of inhibition was in the order: P₃ > P₅₁ > liver nuclei.     

Fractionation of L-cell chromatin into DNA, RNA, and protein fractions on Cs2SO4 equilibruim density gradients

A new procedure is described for fractionation of chromatin into DNA, RNA, and total chromatin protein. By isopycnic gradient centrifugation of chromatin preparations in Cs₂SO₄ solutions containing dimethylsulfoxide and sodium sarcosyl it is possible to obtain highly purified fractions of these components. The method gives a very high yield of these chromatin fractions unlike some other methods, where irreversible binding to columns occurs. Also with this method it is possible to obtain highly concentrated fractions, which after a simple dialysis step, can be conveniently analyzed by polyacrylamide gel electrophoresis.Nuclei from L-929 cells were isolated by a method involving citric acid or by a method using a nonionic detergent. The yields of DNA obtained by both methods was compared. Chromatin was isolated from purified nuclei (prepared in either of the above ways) in two different ways also. In one method, chromatin was extracted from nuclei with 1 m NaCl. A second method involving fractionation of lysed nuclei in sucrose and metrizamide solutions was also used. The yields of DNA obtained by both methods was compared. There appears to be little nuclear membrane contamination of any of these chromatin preparations.A preliminary analysis of L-929 cell chromatin total RNA and protein fractions on polyacrylamide and agarose gels has been made. Both fractions appear to be quite complex with a wide spectrum of subcomponents of differing S values.