DNA Phosphorus Research Articles

The Effect of Cupric Ions on the Indole Reaction for the Determination of Deoxyribonucleic Acid

Abstract The presence of cupric ions or ferric ions was found to enhance the development of color in the indole reaction for the colorimetric determination of deoxyribonucleic acid. The addition of calcium, cobaltous, magnesium, manganous, nickel, or zinc ions did not produce this effect. In the absence of added cupric ions, the indole reaction gave variable results, and the color developed to less than half of the optimum level. It is recommended that the reaction mixture for this assay include cupric ions at a concentration of 15 µm. Under these conditions, the molar extinction coefficient (based on the molarity of DNA phosphorus) of the colored product at 490 mµ is equal to 15,800 m-1 cm-1. The assay conforms to Beer's law until the concentration of DNA exceeds 25 µg per ml (A1 cm490 mµ = 1.25) in the reaction vessel.

Effect of disease on chemical constituents of the human thyroid gland.

ABSTRACT Estimations of RNA, DNA, protein and lipid phosphorus were made in a series of diseased thyroid glands removed surgically and normal glands obtained within 8 hr post mortem. Colorimetric methods for nucleic acid estimation were found to be unsuitable because of interference from other chromogens. The extent of this interference was variable and was related to the colloid content of the tissue. RNA was best measured by a UV method accompanied by correction for peptide contamination, and DNA by estimation of phosphorus after removal of all other phosphorus-containing compounds. The chemical composition of thyroid adenomas did not differ from that of the normal post-mortem samples, but in thyrotoxicosis and even more so in Hashimoto's thyroiditis the RNA, DNA and lipid phosphorus content of the tissue was raised per unit weight. Relative to DNA, there was a decrease in the protein content and an increase in the RNA content of the tissue in thyrotoxicosis and thyroiditis. These changes are consistent...

Measurement of DNA and RNA in Human Peripheral Blood Lymphocytes

Abstract A method is described, modified from a published technic, for obtaining lymphocytes from human peripheral blood. The cell preparations containing greater than 90% lymphocytes were analyzed for their DNA and RNA content, using a modified Schmidt-Thannhauser procedure. The peripheral blood lymphocytes of 40 normal adults were examined, and normal values established for the 2 compounds. The mean lymphocyte DNA was 0.73 pg DNA phosphorus per million cells, and the mean lymphocyte RNA was 0.19 pg RNA phosphorus per million cells. These values are consistent with results published for human leukocytes. The ratio RNA phosphorus:DNA phosphorus is proposed as a reliable index of changes in the RNA content of the blood lymphocytes.

Inflorescence Initiation in Lolium Temulentum L. IX. Some Chemical Changes in the Shoot Apex at Induction

Plants of L. temulentum were induced to flower by exposure to 1 long day. Shoot apices harvested from vegetative plants prior to the long�day exposure (day I) and others harvested on days IV and VI were analysed for length, fresh weight, total and residual (after extraction for solubles and lipids) dry weight, total and residual nitrogen content, residual phosphorus, and RNA and DNA phosphorus content. Cell number per apex was also established for two harvests.

RNA, DNA and other phosphorus fractions in soybeans as affected by high phosphorus nutrition

RNA, DNA and other phosphorus fractions in soybeans as affected by high phosphorus nutrition

Interaction of aminoacridines with deoxyribonucleic acid: viscosity of the complexes.

AbstractThe intrinsic, viscosities at zero shear rate of defined complexes of proflavine, 9‐aminoacridine, and 9‐amino‐l,2,3,4‐tetrahydroaeridine with calf thymus DNA have been determined at, various ionic strengths by means of rotating cylinder viscometers. By controlled adjustment, of the composition of the mixtures, the amount of bound acridine (r moles/g.‐atom DNA phosphorus) was maintained constant at different dilutions. The intrinsic viscosities of the complexes increased with r up to r values (ca. 0.16–0.20) corresponding to the end of the process of strong binding of the acridinium cations. However, complex formation between the acridines and thermally denatured DNA caused either a marked decrease in viscosity (at the low ionic strengths of 0.0015 and 0.005) or no change at all (ionic strength 0.1). These results are discussed in the light of presently available hydrodynamic theories relating the intrinsic, viscosity of DNA to its molecular extension.

Some physical properties of RNA polymerase.

purchased from Worthington Biochemical Corp., were dialyzed overnight against a solution containinlg 0.2 M KCI and 0.01 M potassium phosphate buffer, pH 7.0, before use. Solutions: 0.1 M KCl buffer: 0.1 M KCI, 0.01 M potassium phosphate buffer, pH 7.0, 1 mM 2-mercaptoethanol and 0.5 mM EDTA; 0.5 M KCl buffer: 0.5 M KCI, 0.01 M potassium phosphate buffer, pH 7.0, 1 mM 2-mercaptoethanol, and 0.5 mM EDTA. Miscellaneous: Hydroxylapatite prepared by the method of Tiselius, Hljerten, and Levin5 was a generous gift from Dr. Bruce Alberts. Dialysis tubing was boiled twice for 15 mi in 5%/ Na2CO3, rinsed thoroughly with water, then boiled for 15 min in 0.05 M EDTA, pH 7. Boiled dialysis tubing was stored at 2?C in 0.05 M EDTA, pH 7. Calf thymus DNA assay: The calf thymus DNA assay reactioln mixture, in 0.25 ml, con-tained 0.04 M tris-HCl buffer, pH 7.9, 0.05 KCI, 5 mM MgCl2, 2 mMI MnCl2, 0.16 mM each of UTP, CTP, and GTP, 0.12 mM ATP-8-C14, 120 jig/ml calf thymus DNA (350 mgtmoles/ml in DNA phosphorus), 4 mM 2-mercaptoethanol, 0.1 mM EDTA, 0.5 mg/ml bovine serum albumin (BSA), and enzyme. After incubation for 10 or 20 min at 37?C, the reaction was stopped by adding 3 ml of cold 5% trichloroacetic acid. After 10 min at 0?C, the precipitate was collected and washed two times with 3-ml portions of cold 2% trichloroacetic acid on a Millipore HA filter. The filter was affixed to an aluminium planchet with a drop of 1% BSA, dried and counited in an end-window, low background, gas-flow counter. T7 DNA assay: The T7 DNA assay was the same as the calf thymus DNA assay except that 60 gig/ml of T7 DNA was used in place of calf thymus DNA, and MnCl2 was omitted. With purified enzyme, the T7 DNA assay gave from two- to threefold higher rates of incolporation than the calf thymus DNA assay. A unit of enzyme activity is defined as the inlcorporatiorl of 1 /hmole of total nucleotide into an acid-insoluble product in 1 min at 37?C. Sedimentation velocity measurements of RNA polymerase activity on sucrose gradients: Sedimen

Studies on synchronously dividing cultures of Euglena gracilis Klebs (strain Z). II. Patterns of biosynthesis during the cell cycle.

AbstractDry weight, total protein, chlorophyll a, carotenoids, soluble protein, RNA, DNA, and total phosphorus were determined at intervals of two hours during synchronous growth of Euglena at a cell concentration of 5–10 × 104 cells/ml maintained by periodic dilution. Detailed analyses of the soluble proteins (DEAE‐cellulose fractionation) and of the intracellular distribution of phosphorus were made also. In general, a linear doubling of each of these major components occurred during the light period; there was no net synthesis during the dark. Since cell number doubled during the dark period, a halving of the amounts of the parameters in each cell occurred. The most notable exception was the stepwise synthesis of DNA during the light period: DNA replicated during the last 6 hr only of the light period, commencing at 8 hr after the onset of light. Although DNA replication was a necessary condition for cell division to occur, it was not necessarily a sufficient one. It was found that different absolute rates of synthesis exist for the different compounds, with a doubling of a given parameter sometimes being completed before the end of the light period.

Electrophoresis of DNA. III. The effect of several univalent electrolytes on the mobility of DNA

AbstractThe electrophoretic mobility of DNA in the presence of tetramethylammonium and alkali metal ion chlorides has been studied as a function of ionic strength. Each cation exhibits a characteristic behavior in accord with the idea that the order of inter‐action with DNA is Li+ > Na+ > K+ > TMA+. The application of theories of the electrophoresis of polyelectrolytes is discussed, leading to an attempt to calculate the fractional charge per DNA phosphorus from the mobility data. Over the range 0.05–0.4M a constant and unique value of the DNA charge is obtained in the presence of each cation. Values of the zeta potential and of the friction factor per monomer unit are also calculated.

Microelectrophoretic determination of deoxyribonucleic acid content and base composition in microscopic tissue samples

A procedure for the determination of the content and base composition of DNA in amounts of 0.2 mμg by electrophoresis in a microscopic cellulose fiber is described. Individual microscopic tissue units, usually nuclei, are isolated by microdissection under microscopic control. Subsequent to the removal of non-DNA nucleotides the DNA purines are extracted with hydrochloric acid. The purines are separated in the extract by microelectrophoresis and the adenine/guanine ratio determined by a photographic-photometric procedure in monochromatic ultraviolet light. This ratio also defines other base quotients in a complementary DNA. The absolute amount of DNA is determined with the aid of a measured amount of a reference compound, cytidine, which is included in the extract and subjected to electrophoretic-spectrophotometric analysis. Control experiments and comparisons with results obtained by independent techniques indicate that the method gives reproducible and quantitative results although it cannot be stated with full certainty that data on DNA (or DNA phosphorus) content represent accurately the quantities in absolute terms, mainly because there is no biological object available for investigation for which the DNA content is precisely known and confirmed. The value for the coefficient of variation is seven on the average for adenine/guanine ratios as well as DNA contents.

Etude sur la Toxicité de l'Ethanol. Modifications des Concentrations Tissulaires de Certaines Fractions Phosphorées dans le Foie et le Pancréas du Rat

Wistar rats, with an average body weight of 150 g, were given an intraperitoneal injection of ethanol (either 2 or 3 g/kg) 1 hr before killing. The principal phosphorus-containing fractions of the liver and pancreas (inorganic phosphorus, ATP, AMP, sugar phosphate esters, phospholipids, RNA and DNA phosphorus) were analyzed against controls injected with 0·85% NaCl. It was observed that (1) with an injection of 2 g/kg there was an increase in the adenine nucleotides and RNA content of the liver (relative to the DNA phosphorus) while there was no effect at all in the pancreas; (2) with an injection of 3 g/kg (toxic dose) there was a fall-off of all fractions in the pancreas, while in the liver the amounts of phospholipids and RNA (relative to the DNA) fell by 30–50%. These facts are assessed in connexion with the theory of the oxidation of ethanol in vivo by the xanthine-oxydase-catalase system when the ethanol is injected in doses of 2 g/kg or above.

Density gradient isolation of rat liver nuclei with high DNA content.

Rat liver nuclei, after preliminary isolation in 2.2 molar sucrose solution, were separated into density classes by centrifugation at 95,000 g for 45 to 85 minutes in a sucrose density gradient (density range, 1.28 to 1.33). Nuclei from normal liver separated into three bands with average DNA phosphorus content per nucleus of 0.67, 0.84, and 0.93 picogram for top, middle, and bottom bands, respectively. Nuclei from regenerating liver (26 hours after one-third hepatectomy) yielded three bands and a pellet fraction with average DNA phosphorus content per nucleus of 0.76, 1.02, 1.38, and 1.51 picograms (top to bottom of tube). This method appears capable of yielding nuclei which have increased their DNA content prior to mitosis, and this procedure should be valuable in studies of biochemical changes which occur in nuclei preparing for mitosis.

Studies on the nucleic acid content and the heterogeneity of ribonucleic acid in the rabbit lens

The contents of ribonucleic acid phosphorus, deoxyribonucleic acid phosphorus, protein, lipid phosphorus and acid-soluble ribose have been studied in the rabbit lens. The RNA * fractions released into the aqueous phase by treatment with phenol have been separated by chromatography on ecteola adsorbent. In addition, the heterogeneity of ribonucleic acid treated with ribonuclease has been studied. * The following abbreviations are used: RNA, ribonucleic acid; RNA-P, ribonucleic acid phosphorus; DNA, deoxyribonucleic acid; DNA-P, deoxyribonucleic acid phosphorus; PCA, perchloric acid.

Liver nucleotides in experimental hepatic porphyria.

The importance of purine metabolism in experimental hepatic porphyria has been reinvestigated. RNA and DNA phosphorus and acid-soluble ribonucleotides have been estimated in the liver of rats after treatment with 2-allyl-2-isopropylacetamide, 2-propyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethyl-pyridine and hexachlorobenzene for different lengths of time. These values have been related to the porphobilinogen level in the liver and to the urinary excretion of porphyrin and porphyrin precursors. There was no evidence of impaired purine metabolism in rats with experimental hepatic porphyria: no significant change in the level of RNA and DNA and in the total adenine purine nucleotide concentration was observed in porphyric animals. A fall in liver ATP with an increase in liver AMP occurred not only after the administration of 2-allyl-2-isopropylacetamide but also after 2-propyl-2-isopropylacetamide, a compound which has been reported not to produce porphyria. The significance of these results is discussed.

Liver nucleotides in experimental hepatic porphyria

The importance of purine metabolism in experimental hepatic porphyria has been reinvestigated. RNA and DNA phosphorus and acid-soluble ribonucleotides have been estimated in the liver of rats after treatment with 2-allyl-2-isopropylacetamide, 2-propyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethyl-pyridine and hexachlorobenzene for different lengths of time. These values have been related to the porphobilinogen level in the liver and to the urinary excretion of porphyrin and porphyrin precursors. There was no evidence of impaired purine metabolism in rats with experimental hepatic porphyria: no significant change in the level of RNA and DNA and in the total adenine purine nucleotide concentration was observed in porphyric animals. A fall in liver ATP with an increase in liver AMP occurred not only after the administration of 2-allyl-2-isopropylacetamide but also after 2-propyl-2-isopropylacetamide, a compound which has been reported not to produce porphyria. The significance of these results is discussed.

Studies on the nucleotide arrangement in deoxyribonucleic acids. V. Pyrimidine nucleotide clusters: isolation and characterization.

Abstract The isolation and characterization of the pyrimidine oligonucleotide clusters occuring in DNA are described. The method is based on the fractionation of acidic hydrolysates of DNA on DEAE-cellulose and on the subsequent two-dimensional paper chromatography of these fractions. Pyrimidine oligonucleotide clusters of up to nine pyrimidine units in length, and individual pyrimidine oligonucleotides up to the pentanucleotide level, have been isolated and characterized with the use of their distinctive spectral ratios and, in many instances, their composition. The fractionation method accounts for nearly all of the original DNA phosphorus.

Studies on the nucleotide arrangement in deoxyribonucleic acids V. Pyrimidine nucleotide clusters: Isolation and characterization

The isolation and characterization of the pyrimidine oligonucleotide clusters occuring in DNA are described. The method is based on the fractionation of acidic hydrolysates of DNA on DEAE-cellulose and on the subsequent two-dimensional paper chromatography of these fractions. Pyrimidine oligonucleotide clusters of up to nine pyrimidine units in length, and individual pyrimidine oligonucleotides up to the pentanucleotide level, have been isolated and characterized with the use of their distinctive spectral ratios and, in many instances, their composition. The fractionation method accounts for nearly all of the original DNA phosphorus.

Influence of age and N, N'-diethylene-N"-phenethyl-phosphoramide (PEDP) upon nucleic acid and ribonuclease activity in tumor tissue.

SummaryAs the Walker 256 tumor increased in size and approached maximum development, the ribonuclease activity per gram of tumor or per mg of deoxyribonucleic acid phosphorus (DNA-P) also increased. The slower growing, larger tumor had a markedly decreased ribonucleic acid phosphorus (RNA-P) and protein concentration. Treating the tumor-bearing rat with N, N'-diethylene-N”-phenethylphosphoramide (PEDP) resulted in reduced rate of growth of the tumor, increased ribonuclease activity, and decreased RNA-P.

The Metabolic Stability of Deoxyribonucleic Acid Phosphorus in Regenerating Rat Liver

The Metabolic Stability of Deoxyribonucleic Acid Phosphorus in Regenerating Rat Liver

Leukocyte Kinetics Studied by Nitrogen Mustard Induced Alterations in DNA Phosphorus Labeling

AbstractThe effect of a large dose of nitrogen mustard on total and differential leukocyte counts in rabbits has been studied in conjunction with the effect on DNA labeling with inorganic radiophosphorus. Considered together, the findings indicate that this dose of nitrogen mustard has a direct lethal effect on lymphocytes and effectively blocks P32 incorporation into leukocyte DNA for several days by killing the elements capable of division. The post-mitotic granulocyte reservoir seems to remain intact and is of sufficient size to replace those lost from the circulation for about 56 hours. Granulocyte circulating half-time under the conditions of these experiments falls between two and seven hours. The post-mitotic granulocyte reservoir is calculated to be 40 times the size of the circulating pool. During the period of rapid granulocyte recovery, leukocytes have been shown to exhibit a decreasing concentration of DNA label per element and maintainence of the same level of label per element depending on the availability of precursor P32. At present no conclusion about DNA labeling characteristics with inorganic P32 under steady state conditions is warranted.