Crystalline desoxyribonuclease; isolation and general properties; spectrophotometric method for the measurement of desoxyribonuclease activity

A crystalline enzyme capable of digesting thymus nucleic acid (desoxyribonucleic acid) has been isolated from fresh beef pancreas. The enzyme called "desoxyribonuclease" is a protein of the albumin type. Its molecular weight is about 60,000 and its isoelectric point is near pH 5.0. It contains about 8 per cent tyrosine and 2 per cent tryptophane. It is readily denatured by heat. The denaturation is reversible if heated in dilute acid at pH about 3.0. The digestion of thymus nucleic acid by crystalline desoxyribonuclease is accompanied by a gradual increase in the specific absorption of ultraviolet light by the acid. The spectrophotometric measurement of the rate of increase in the light absorption can be conveniently used as a general method for estimating desoxyribonuclease activity. Details are given of the method for isolation of crystalline desoxyribonuclease and of the spectrophotometric procedure for the measurement of desoxyribonuclease activity.     

Le Macronucleus Hétéromere de Quelques Ciliés

SUMMARY. In ciliates belonging to the two gymnostome families Chlamydodontidae and Dysteriidae as well as to the order Chonotrichida the macronucleus is composed of two parts which are distinct but in very close juxtaposition. One part, here called "orthomerous," contains granules or microsomes of desoxyribonucleoprotein and several nucleoli; it thus shows the normal structure of a macronucleus of the ordinary type. The other part, "paramerous," contains desoxy-ribonucleic acid diffused, apparently, throughout a homogeneous karyolymph and possesses, in addition, several nucleoli and an endosome of desoxyribonucleoprotein.
The three groups of ciliates under discussion are closely related and constitute an assemblage of forms remarkably evolved. The "heteromerous" structure of their macronuclei must be considered as a secondary acquisition and a differentiation of the "homeomerous" type which characterizes the structure of the macronucleus in most other ciliates.     

Assembly and disassembly of RecA protein filaments occur at opposite filament ends. Relationship to DNA strand exchange

RecA protein primarily associates with and dissociates from opposite ends of nucleoprotein filaments formed on linear duplex DNA. RecA nucleoprotein filaments that are hydrolyzing ATP therefore engage in a dynamic process under some conditions that has some of the properties of treadmilling. We have also investigated whether the net polymerization of recA protein at one end of the filament and/or a net depolymerization at the other end drives unidirectional strand exchange. There is no demonstrable correlation between recA protein association/dissociation and the strand exchange reaction. RecA protein-mediated DNA strand exchange is affected minimally by changes in reaction conditions (dilution, pH shift, or addition of small amounts of adenosine-5'-O-(3-thiotriphosphate) that have large and demonstrable effects on recA protein association, dissociation, or both. Rather than driving strand exchange, these assembly and disassembly processes may simply represent the mechanism by which recA nucleoprotein filaments are recycled in the cell.     

Studies on isolated cell components: I. Nuclear isolation by differential centrifugation

A method is presented for isolating nuclei of rat and hamster liver in a high state of purity and in a condition optically similar to nuclei within living cells.The isolation procedure consists in the homogenization and differential centrifugation of fresh liver at 0–5 ° in a salt-sucrose solution buffered at pH 7.1. By layering the material to be centrifuged over a relatively large volume of a slightly denser solution the purification can be carried out in 4 or 5 centrifugations. The entire procedure can be completed in about 90 minutes. The yield as determined by measurements of desoxyribonucleic acid is about 5 per cent.The solutions contain KH₂PO₄, K₂HPO₄, NaHCO₃, and sucrose. The sucrose concentration is varied to give density differences required for layering. Salt-sucrose solutions maintain a large proportion of the isolated nuclei in a nongranular condition for 6 hours at 0 °. Pure sucrose is less satisfactory for maintenance.The protein-desoxyribonucleic acid ratio for the isolated nuclei averages 5.1 with a range of 2.7 to 8.9.     

Histone-like protein in the prokaryote Thermoplasma acidophilum.

The DNA of the prokaryote Thermoplasma acidophilum is associated with a histone-like protein that has the following properties: it has a high content (23%) of basic amino acids, is positively charged at neutral pH, is soluble in acid, and can stabilize DNA against thermal denaturation. In polyacrylamide gel electrophoresis, in the presence of either sodium dodecylsulfate or urea, it migrates at the same rate as histone IV (F2a1) of calf thymus. The amino acid composition, however, it unusually rich in the amides of acidic amino acids (16-20%), and it does not appear to be closely homologous to any of the classes of eukaryotic histones. Escherichia coli DNA, on the other hand, was associated with no detectable acid-soluble proteins, and the nucleoprotein thermally denatured at a lower temperature than pure DNA.     

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 CaCl(2) is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The alpha-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.     

Accumulation of messenger RNA of the atrial natriuretic factor in the rat left ventricle at the compensatory hypertrophy stage in pressure overload

The atria produce several peptides that have natriuretic and vasoactive properties, collectively called atrial natriuretic factor. All these peptides share a single messenger ribonucleic acid, the amount of which greatly increases in the rat left ventricle when the latter is submitted to chronic volume overload. Using the molecular hybridization technique and a desoxyribonucleic acid probe complementary to the atrial natriuretic factor messenger ribonucleic acid, we now report that a very important increase in the amount of this messenger ribonucleic acid is also observed in rat ventricle at at the compensatory stage of a pressure overload induced cardiac hypertrophy. This result suggests that the pressure overload hypertrophied rat ventricle also has the potential to itself regulate it's loading conditions via the regulation of extracellular fluid volume and vascular resistance.     

Nucleoprotein complexes of yeast and their biological effect on T-lymphocytes

Complexes of nucleic acids and acid nuclear proteins that are active toward human T-lymphocytes were isolated from cells of bakers' yeast Saccharomyces cerevisiae. The conditions of isolation of nucleoprotein complexes by acid extraction followed by microfiltration for concentration of macromolecular components were optimized. Gel filtration and electrophoresis were used to study the composition and molecular weights of components of the preparations obtained. It was shown that nucleoprotein complex had a molecular weight of 1430 kDa. However, only one zone was determined by electrophoresis of the protein component with a molecular weight of 30 kDa.     

RNA-DNA hybridization promoted by E. coli RecA protein.

RecA protein of E. coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments. The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro. These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.     

The secondary structure of bacteriophage DNA in situ. VIII. The reaction of sodium bisulphite with intraphage cytosine as a probe for studying the DNA-protein interaction

To obtain data on the viral nucleoprotein a study has been made of the reaction of sodium bisulphite with cytosine in the intraphage DNA of the phage Sd. The CHlO4 hydrolysates of the bisulphite-modified phage Sd have demonstrated a decrease of 18% in the cytosine content and the presence of the products with the properties of cytosyl-amino acids (the main amino acid responsible for the DNA-protein interaction involving cytosine is lysine). But when prior to hydrolysis the modified phage was disintegrated under mild conditions in 0.1--1 M NaCl solution or Tris-HCl buffer (pH 7), neither the decrease in the cytosine content nor cytosyl-amino acids have been found. An exception is the heating of the phage at 70 degrees C in a medium containing 0.05 M phosphate buffer (pH 7.9--8.5), when an 18% decrease in the cytosine content and subsequent appearance of cytosyl-amino acids have also been observed. The presence of cytosyl-amino acids which are the nucleotide-protein cross-links is confirmed by the results of viscometry, equilibrium centrifugation in cesium sulphate gradient and determinations of the survival percentage. It is suggested that the reaction between bisulphite and cytosine in the phage Sd stops at the stage of the intermediate product C5-C6-dihydro-C6-sulphopyrimidine whose amino group is shielded by interaction with protein (product VII). This product can exist only under in situ conditions: with disintegration of nucleoprotein (destruction of phage particles or ejection of the DNA) in phosphate-free media the product VII reverts into the initial cytosine. Under the conditions of acid hydrolysis or destruction of phage in the presence of phosphate ions product VII undergoes transamination with cleavage of SO3 and restoration of the C5-C6 double bond producing cytosyl-amino acids. The factors determining the stability of the product VII are discussed.     

Nucleoprotein Complexes of Yeast and Their Biological Effect on T-Lymphocytes

Complexes of nucleic acids and acid nuclear proteins that are active toward human T-lymphocytes were isolated from cells of baker's yeastSaccharomyces cerevisiae. The conditions of isolation of nucleoprotein complexes by acid extraction followed by microfiltration for concentration of macromolecular components were optimized. Gel filtration and electrophoresis were used to study the composition and molecular weights of components of the preparations obtained. It was shown that the nucleoprotein complex had a molecular weight of 1430 kDa. However, only one zone was determined by electrophoresis of the protein component with a molecular weight of 30 kDa.     

Characterization of alfalfa-mosaic-virus protein polymerization in the presence of nucleic acid

The polymerization of alfalfa mosaic virus (AMV) protein in the presence of homologous nucleic acids and a number of other natural and synthetic nucleic acids was studied. The conditions for optimal assembly were found to be pH 6.0 and low ionic strength (I = 0.1 M) at room temperature, irrespective of the type of nucleic acid. The resulting nucleoprotein particles exhibited the same structural characteristics as the virus. This information emerged from optical diffraction and computer filtering of electron micrographs from the reconstituted particles. Irrespective of the type of nucleic acid present the polymerization of the protein resulting in a nucleoprotein particle is a cooperative process. Evidence for this was obtained by nitrocellulose filter binding assay, sodium dodecylsulphate/polyacrylamide gel electrophoresis, sedimentation velocity and electron microscopy of the reaction mixtures. The rates and efficiencies of reconstitution were of the same order of magnitude for a number of ribonucleic acids. Sedimentation data derived from AMV protein and AMV RNA mixtures suggested the existence of a specific nucleation product in the first stage of assembly. The results are discussed in terms of a tentative model of the assembly, in which at least two different steps (nucleation and elongation) can be distinguished, each characterized by an association constant.     

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.     

Enzymatic and nonenzymatic degradation of myelin basic protein

A procedure for large scale isolation of myelin basic protein (BP) has been modified to insure BP preparations free of neutral proteinase activity. Fractions were monitored by electrophoretic analysis of BP solutions incubated under various conditions of temperature and pH. Maximum degradation of human BP prepared by the old batch procedure occurs at pH 7, 47°C. BP preparations obtained by the new procedure, as well as BP preparations purified by CM-cellulose chromatography, are stable under these conditions. The latter, however, do undergo significant breakdown at pH 9, 100°C. The results suggest that the degradation observed under these conditions is non-enzymatic in nature.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

In situ protamine release: A versatile sample preparation method for the electrophoretic analysis of nuclear proteins on acid/urea-based gels

Protamine sulfate is used to release histones and other basic proteins from the DNA of chromatin. This phenomenon becomes the basis of a versatile method for analysis of the nucleic acid and protein composition of nucleoprotein samples, which is termed here in situ protamine release. When protamine is added to a nucleoprotein sample in 5% acetic acid and 8 m urea, at a concentration of 1.0%, ≥94% of the histones are released from the DNA of chromatin, comparable to the release of histones using sodium dodecyl sulfate. This makes in situ protamine release the method of choice for the analysis of acid-soluble proteins on acid/urea-based gels, where the DNA must be removed from the protein prior to electrophoresis. Compared to DNase I release or acid extraction, protamine release is found to be the simplest, most reliable, and most effective method for removing the acid-soluble proteins from DNA. Protamine is either added to the sample (very much like the detergent, sodium dodecyl sulfate), or electrophoresed through a gel containing nucleoprotein, thus displacing proteins in its path. A serendipitous advantage of protamine is that it can also serve as a carrier for the precipitation of dilute nucleoprotein samples with ethanol, and 3 mm Mg²⁺, to concentrate the nucleoprotein in preparation for analysis. A unique feature of the in situ protamine-release method is that the DNA is not lost or destroyed and can therefore be used for subsequent analysis.     

DNA binding properties of a 110 kDa nucleolar protein.

A single strand specific DNA binding protein was purified to homogeneity from calf thymus nucleoprotein. The monomeric protein is elongated in shape and has a molecular mass of 110 kDa. Since immunocytochemistry revealed that the protein is predominantly located in the nucleolus we refer to it as the 110 kDa nucleolar protein. The protein binds not only to single stranded DNA but also to single stranded RNA, including homopolymeric synthetic RNA. We have used the single stranded DNA binding properties of the 110 kDa protein in model studies to investigate its effects on the configuration of nucleic acid. Our results are: only 50-55 protein molecules are sufficient to saturate all binding sites on the 6408 nucleotides of phage fd DNA; protein binding cause a compaction of single stranded DNA; large nucleoprotein aggregates are formed in the presence of divalent cations; this is due to protein-protein interactions which occur at moderately high concentrations of magnesium-, calcium or manganese ions; the protein induces the reassociation of complementary nucleic acid sequences. We speculate that the 110 kDa protein performs similar reactions in vivo and may have a function related to the processing and packaging of preribosomal RNA.     

Nitrite and nitric oxide treatment of Helix pomatia hemocyanin: single and double oxidation of the active site.

The reaction of nitrite and nitric oxide with Helix pomatia hemocyanin has been studied. One or both of the two copper ions in the active site can be oxidized, depending upon reaction conditions. The single oxidation of the oxygen binding site can be reversed by reduction with hydroxylamine, and the oxygen binding properties of the protein are simultaneously restored. The experiments, including electron paramagnetic resonance, indicate that nitric oxide is not a ligand of copper in the singly oxidized active site and that the oxidized copper ions is coupled to at least two nitrogen atoms of amino acid residues. The doubly oxidized protein can be reduced to a singly oxidized one with ascorbic acid or hydroxylamine; the latter reagent is again able to reduce the singly oxidized state and to restore the oxygen binding properties.     

Studies on a nucleoprotein prepared from rat liver polysomes by digestion with T1 ribonuclease

1. Treatment of rat liver polysomes in a buffer containing 2.5mm-magnesium chloride with T(1) ribonuclease at a concentration of 330units/ml. of reaction medium at 37 degrees for 2hr. leads to the production of an insoluble nucleoprotein. 2. On the bases of analysis for protein and RNA and of u.v.-absorption spectra the nucleoprotein appears to have lost approx. 60% of the structural RNA originally present in the ribosome. Degradation of (3)H-labelled polysomes (structural RNA labelled with orotic acid) with T(1) ribonuclease leads to nucleoprotein preparations retaining approx. 30% of the radioactivity originally present in the polysomes. By means of sucrose-density-gradient centrifugation it is shown that the nucleoprotein preparations are free of single 73s ribosomes and ribosomal subunits. No evidence for the presence of 28s and 18s structural RNA was obtained on examination of extracted nucleoprotein-particle RNA by means of sucrose-density-gradient centrifugation. 3. Digestion of washed polysomes carrying (14)C-labelled nascent peptide chains with T(1) ribonuclease gives a nucleoprotein particle that retains approx. 70% of the original labelled chains. Treatment of labelled nucleoprotein particles with 1mm-puromycin in the absence of transfer factors releases 20% of the labelled chains. Addition of GTP (0.48mumole) increases this release to 37%. 4. Treatment of nucleoprotein particles carrying (14)C-labelled peptide chains with either EDTA (50mm) or ammonium chloride (0.5m) brings about a small release of labelled material (approx. 15%). 5. Disruption of nucleoprotein particles carrying (14)C-labelled peptide chains with either sodium dodecyl sulphate or 2m-lithium chloride, followed by addition of transfer RNA as marker and chromatography on Sephadex G-200, show in both cases that considerable amounts of labelled peptide material move well ahead of the added transfer RNA marker. Further, if nucleoprotein particles carrying labelled peptide chains are treated with 0.3m-potassium hydroxide at 20 degrees for 24 hr., neutralized to pH7.6, and then chromatographed on Sephadex G-200, the labelled peptide material moves much closer to the added transfer RNA marker. These results suggest that a proportion of the nascent (14)C-labelled peptides on the nucleoprotein are attached to transfer RNA or large fragments of transfer RNA. 6. [(3)H]Polyuridylic acid binds to nucleoprotein particles in 1mm-magnesium chloride. The rate of binding is rapid when measured at 20 degrees .     

Feulgen nucleal reaction. I. Quantitative extraction of fuchsin from Feulgen-stained nucleoprotein

A new extraction method for the quantitative determination of the fuchsin contained in a Feulgen-stained nucleoprotein sample has been introduced. The method is based on the following facts: (1) Treatment of a Feulgen-stained nucleoprotein sample with hot acid or alkali brings about a splitting of the linkage between fuchsin-SO(2) and the hydrolyzed nucleic acid moiety of nucleoprotein through aldehyde groups. (2) It also effectuates the formation of fuchsin from the liberated fuchsin-SO(2). (3) The fuchsin is made colorless by the treatment, but is restored to its original pink colored state when the pH of the acidic or alkaline medium is adjusted to 4.6. (4) The fuchsin, either pink colored or decolorized by alkali, can be extracted from an aqueous phase by amyl alcohol. A linear relationship was found to exist between the amount of fuchsin extracted by the FEM from a Feulgen-stained nucleoprotein sample and its DNA content. This relationship holds over a wide range of DNA concentration. From experiments utilizing this method, knowledge may be gained about the mechanism of the Feulgen reaction in situ which can lead to an improvement of the reaction in the field of cytochemistry.