Precipitation Of Nucleic Acids Research Articles

A single temperature amplification technique applied to the detection of citrus tristeza viral RNA in plant nucleic acid extracts

A procedure for the successful detection of citrus tristeza virus (CTV) RNA in total crude nucleic acid extracts of infected citrus whole leaves and bark is described. The method requires the isolation and precipitation of total nucleic acids from either infected whole leaf or bark tissue. The CTV viral RNA is then specifically amplified using a single temperature RNA Self-Sustained Sequence Replication technique (3SR TM) performed at 42°C for 60 minutes. The amplified negative-sense viral RNA product can subsequently be detected by fixing a portion of the reaction mixture onto a nylon membrane and hybridizing with positive sense tristeza specific DNA oligonucleotide probes. Central California isolates of CTV were readily detected by this method. Denatured viral specific dsRNA was also a suitable template for the specific detection of CTV.

Rapid preparation of DNA from phytopathogenic mycoplasma-like organisms for polymerase chain reaction analysis

A simple procedure for the processing of MLO (mycoplasma-like organisms)-infected plant samples for PCR analysis is described. It is based on differential centrifugation and removal of enzyme inhibiting agents with the aid of a resin. Results of the amplifications are comparable with those achieved with more complex DNA extraction procedures. The method does not require any extraction with organic solvents or alcoholic precipitation of nucleic acids.

High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity.

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment. Upon heat induction, the 832-amino-acid construct produced 1-2% of total protein as Taq Pol I. The induced 544-amino-acid construct produced 3% of total protein as Stoffel fragment. Enzyme purification included cell lysis, heat treatment followed by Polymin P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography. For full-length 94-kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste. For the 61-kD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight cell paste. The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCl2 and 10-55 mM KCl. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgCl2 is the preferred cofactor compared to MnCl2, CoCl2, and NiCl2. The full-length Taq Pol I has an activity half-life of 9 min at 97.5 degrees C. The Stoffel fragment has a half-life of 21 min at 97.5 degrees C. Taq Pol I contains a polymerization-dependent 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity. A comparison is made among thermostable DNA polymerases that have been characterized; specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest reported.

Production of multiple forms during purification of Streptomyces aureofaciens DNA polymerase: a study using nondenaturing polyacrylamide gradient gel electrophoresis

A modified method for the detection of DNA polymerases in cell extracts and purified enzyme preparations after electrophoresis in polyacrylamide gradient cylindrical gels is described. The technique, which is based on direct assay of activity in a reaction mixture during elution of DNA polymerases from gel slices, was applied to the pursuit of enzyme forms of Streptomyces aurofaciens DNA polymerase during purification procedure. In a crude extract of S. aureofaciens mycelium many catalytically active forms of DNA polymerase ranging from 35 to 860 kDa were detected: After purification, that included mycelium homogenization, precipitation of nucleic acids by polyethylene glycol, DEAE-Sephadex, QAE-Sephadex and DNA-Sepharose chromatography, higher molecular mass forms of more than 172 kDa have not been found. The lower molecular mass forms were separated into two groups by DNA-Sepharose chromatography. On the basis of their characterization, it is assumed that the lower molecular mass forms are produced by proteolysis and the major form found after purification of S. aureofaciens DNA polymerase in the presence of suitable proteinase inhibitors should be a protein of 172 kDa.

Precipitation of nucleic acids with poly(ethyleneimine).

Removal of nucleic acids from cell extracts is a common early step in downstream processing for protein recovery. We report on the precipitation of nucleic acids from a homogenate of Saccharomyces cerevisiae by addition of the cationic polyelectrolyte poly(ethyleneimine) (PEI), focusing on the effect of PEI dosage on particle size, protein loss, and extent of nucleic acid removal in both batch and continuous mode. Better than 95% removal of nucleic acids from yeast homogenates was achieved by means of precipitation with PEI with protein losses of approximately 15% with or without previous removal of cell debris. The coprecipitated protein is predominately large molecular weight material and exhibits both low and high isoelectric points. Such treatment does not aggregate the cell debris; size distribution of the precipitated particles from a continuous precipitator is very similar to that for protein precipitation.

Purification of the cysB protein from Salmonella typhimurium.

Using an assay based on polypeptide mobility in one- and two-dimensional polyacrylamide electropherograms, we purified the Salmonella typhimurium cysB protein from an Escherichia coli strain carrying plasmid pGBK14, in which the S. typhimurium cysB gene is under transcriptional control of the strong promoter PL from phage lambda. cysB protein constitutes approximately 4% of total soluble protein in such cells and was obtained with good yield at a purity of 85% after precipitation of nucleic acids with streptomycin sulfate, ammonium sulfate fractionation, and hydrophobic chromatography on methyl agarose. Material with 95% purity was obtained with poor yield by ion-exchange chromatography of native protein and with good yield by size exclusion chromatography of denatured protein in sodium dodecyl sulfate. Physicochemical studies of the purified cysB protein show that it is a tetramer of identical 36-kDa subunits with a pI, amino acid composition, amino-terminal sequence, and carboxyl-terminal amino acid content identical to those known from previous studies or deduced from the cysB DNA sequence. Although O-acetyl-L-serine is believed to bind to cysB protein to form a complex that stimulates transcription of genes of the cysteine regulon, we were unable to demonstrate such an interaction using methods that would have detected binding with a Kd of less than 0.1 mM.

Purification and some properties of the D-lactate-2-sulphatase of Pseudomonas syringae GG.

A soil bacterium grown on propan-2-yl sulphate as sole source of carbon and sulphur yielded extracts containing an enzyme capable of liberating sulphate from racemic lactate-2-sulphate. The enzyme was purified to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, batch treatment with DEAE-cellulose, and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was monomeric with an Mr of 55 000-60 000. The enzyme activity was specific for D-lactate-2-sulphate (Km 6.6 nM; maximal specific activity 14.3 mumol/min per mg of protein) and showed no activity towards the L-isomer. The products of the enzyme's action were inorganic sulphate and D-lactate which were released in equimolar amounts and stoicheiometrically with the amount of ester hydrolysed. No L-lactate was formed. Retention of configuration implied cleavage of the O-S bond of the C-O-S ester link and this was confirmed by 18O-incorporation experiments in which 18O from 18O-enriched water in the incubation medium was incorporated exclusively and quantitatively into inorganic sulphate. Only two other esters (serine-O-sulphate and p-nitrophenyl sulphate) of a total of 29 compounds tested were substrates for the enzyme. D-Lactate, L-lactate-2-sulphate and the substrate analogues glycollate-2-sulphate and butyrate-2-sulphate were significantly inhibitory.

A method for the purification of E. coli plasmid DNA by homogeneous lysis and polyethylene glycol precipitation.

A procedure is described for the isolation and purification of E. coli plasmid DNA by polyethylene glycol precipitation. The method is rapid, simple, inexpensive and amenable to both small and large scale manipulation. This procedure involves lysis of bacterial cells by treatment with pronase in sodium dodecyl sulfate, removal of chromosomal DNA by centrifugation, precipitation of residual nucleic acids with polyethylene glycol and removal of RNA by precipitation with LiCl. Plasmid DNA purified as described is pure enough for restriction endonuclease analysis, for use as a vector for the cloning of cDNA or synthetic DNA, or for use as a template in an E. coli transcription-translation cell-free system.

Nucleic acid-binding glycoproteins which solubilize nucleic acids in dilute acid. Re-examination of the Ustilago maydis glycoproteins.

Holloman ((1975) J. Biol. Chem. 250, 2993-3000) reported the isolation from Ustilago maydis of a glycoprotein which prevented the precipitation of nucleic acids in cold 5% trichloroacetic acid. Two glycoprotein fractions from U. maydis with this nucleic acid-solubilizing activity were isolated in our laboratory using improved purification procedures. The activity was not due to nuclease contamination. The glycoproteins are distinguished by: their ability to bind to concanavalin A-Sepharose; their differential binding to double- and single-stranded deoxyribonucleic acid, and to ribonucleic acid; their molecular weights (46,000 and 69,000); and the relative amounts present in growing versus nongrowing cells. Both fractions required sulfhydryl-reducing conditions for optimal yields, specific activity, and stability. Nucleic acid binding was cooperative, the minimum number of glycoproteins required to make a native T7 DNA molecule soluble in dilute acid being estimated at 2 and 15, respectively.

Purification of Bacillus subtilis RNA polymerase with heparin-agarose. In vitro transcription of phi 29 DNA.

We have devised a new procedure for the purification of highly active preparations of Bacillus subtilis RNA polymerase holoenzyme. A column of heparin-agarose A-15m is used to rapidly and quantitatively adsorb RNA polymerase from the initial crude extract fraction. This affinity procedure obviates the necessity of including nucleic acid precipitation or partitioning steps and allows for rapid separation of RNA polymerase from proteolytic activity. The enzyme is further purified by preparative glycerol gradient centrifugation resulting in an overall purification in 200-fold in 24 h with near quantitative recovery of polymerase protein and activity. RNA polymerase holoenzyme is obtained by chromatography on single-stranded DNA-agarose. The in vitro transcription products made by purified preparations of B. subtilis and Escherichia coli RNA polymerase holoenzymes in response to B. subtilis phage phi 29 DNA have been analyzed, and an in vitro transcription map is presented. The E. coli RNA polymerase holoenzyme initiates transcription from three promoter sites not efficiently utilized by the B. subtilis holoenzyme under optimal conditions for RNA synthesis.

A simple, general procedure for purifying restriction endonucleases.

A simple, general method for purifying restriction endonucleases is described. The method employs precipitation of nucleic acids from crude extracts with polyethyleneimine followed by affinity chromatography on columns of heparin covalently linked to agarose. Most of the sixteen enzymes tested could be purified to a degree sufficient for DNA sequencing work by this method sometimes supplemented by at most one step of ion exchange chromatography.

The differential precipitation of nucleic acids and proteins from aqueous solutions by ethanol

The addition of an appropriate mixture of ethanol, water and sodium perchlorate to crude extracts of some biological material results in the selective precipitation of nucleic acids. Detergent extracts of tissue-cultured plant cells treated with this reagent yielded as much as 95–100% of the nucleic acid while a similar percentage of the total protein remained in solution.

Isolation of nucleolar proteins by ethanol precipitation of nucleic acids

Almost all of the DNA, RNA, and protein of Novikoff hepatoma ascites cell nucleoli can be solubilized in 6M guanidine hydrochloride. Under proper conditions, the nucleic acids may then be precipitated (more than 99%) with ethanol, while 85% of the protein remains in solution. Electrophoretic analysis of the proteins from the ethanol-soluble and -insoluble fractions shows that no major species of protein appear to be lost during this precipitation.

The preparation of an immobilised glucose isomerase. I. The production and selected properties of the partially‐purified enzyme from Lactobacillus brevis

AbstractA strain of Lactobacillus brevis producing glucose isomerase was grown in a xylose‐containing medium. Production of intracellular glucose isomerase was four times that previously reported. Methods for the release of enzyme were compared, and a process for the production of an enzyme concentrate proposed involving (i) heat autolysis of the cells at 40 °C for 8 h; (ii) precipitation of nucleic acids with manganese chloride; (iii) precipitation of enzyme with ammonium sulphate, collecting the fraction precipitated between 60 and 85% saturation, and (iv) heat treatment of the dissolved enzyme at 55 °C in the presence of Co2+ for 10 min.Enzyme concentrates prepared using steps (i) to (iii) only showed considerable competing activity, and produced sigmoid kinetics. The use of enzymes prepared using the full method showed that substrate inhibition was apparent at glucose concentrations above approximately 30% w/v. The apparent KM value was calculated as 10 to 12% w/v (glucose), based on the definition of KM as KM= So at Vo ‐ Vmax(observed)/2. The equilibrium concentrations of fructose and glucose were 62 and 38% respectively, and were independent of temperature between 35 and 60 °C.

Precipitation of nucleic acids with polyethyleneimine and the chromatography of nucleic acids and proteins on immobilised polyethyleneimine

Nucleic acids in microbial extracts can be efficiently precipitated with a cationic polymer, polyethyleneimine under conditions where there is little protein precipitation or enzyme denaturation. The binding of nucleic acids and proteins to immobilised polyethyleneimine at low ionic strength and their subsequent chromatography is also described.

Precipitation of nucleic acids with cetyltrimethylammonium bromide: A method for preparing viral and cellular DNA polymerase products for cesium sulfate density gradient analysis

Precipitation with cetyltrimethylammonium bromide followed by precipitation with ethanol:salt provides a quick, simple, and effective method for preparing labelled nucleic acid products of DNA polymerase reactions for analysis by Cs 2SO 4 equilibrium density gradient centrifugation. Results obtained using this treatment show that this approach is superior to other methods of preparing products of endogenous DNA polymerase reactions from relatively crude particulate fractions such as disrupted virions.

D-mannonate: NAD oxidoreductase from Escherichia coli K12. Purification, properties and specificity

D-Mannonate : NAD oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: precipitation of nucleic acids with protamine sulfate; ammonium sulfate precipitation; ion-exchange chromatography on DEAE-cellulose and Sephadex G-200 gel filtration. The overall procedure results in a 53-fold purification with a yield of 70%. Mannonate oxidoreductase is an inducible enzyme: glucuronate and fructuronate are good inducers, galacturonate is not. Optimal activity is obtained at pH 6.3 for fructuronate reduction, and at pH 8.4 for mannonate oxidation. The apparent Michaelis constants for the purified enzyme have been determined: for fructuronate, the Km is 0.5 mM; for NADH 0.03 mM; for mannonate, 1.8 mM and for NAD+, 0.2 mM. Mannonate oxidoreductase and altronate oxidoreductase have been separated on DEAE-Sephadex in order to study mannonate-oxidoreductase specificity. After separation, mannonate oxidoreductase is found to be active with both fructuronate and tagaturonate as substrates. Kinetic studies, differential inhibition of activities by p-chloromercuribenzoate and thermal inactivation studies confirm this double specificity. Other carbohydrates tested are unsuitable as substrates. Mannonate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 0.7 mM. Kinetic inhibition studies of the enzyme by direct and reverse reactional products suggest a random mechanism for mannonate oxidoreductase. Enzyme inhibition specificity appears to be restricted. The value of the energy of activation is 34 kJ/mol (8 kcal/mol); the Q10 is 1.5. The results of thermal inactivation of mannonate oxidoreductase (53°C) indicate homogeneousenzyme activity with fructuronate in the crude extract. In the absence of inducer, basal activity is dependent on mannonate oxidoreductase only. Thermal inactivations also indicate that the same enzyme, mannonate oxidoreductase, catalyzes the direct reactions with NADH or NADPH, and the reverse reaction. Enzyme substrates, such as NADH, NADPH, fructuronate, tagaturonate, NAD+, mannonate and altronate protect enzymatic activity from degradation by heat. NADH in particular, is effective as a stabilizer; this ability is proportional to its concentration. p-Chloromercuribenzoate is an active inhibitor: at 5 μM, activity is 100% inhibited. Inhibition is non-competitive, with an apparent Ki of 0.1 μM. The end product of the reaction of mannonate oxidoreductase with fructuronate has been identified by means of paper chromatography as D-mannonate.

Factors affecting the extractability and MAK chromatography of nucleic acids from woody plant tissue

Factors affecting the yield of the different classes of nucleic acids extracted from woody plant tissues were investigated using MAK chromatography. Sodium lauryl sulfate (2·0%) improved the yields of nucleic acids by 187 per cent. Shaking of the homogenate for 30 min increased yields an additional 40 per cent, probably by allowing sodium lauryl sulfate to more effectively dissociate nucleic acids from protein and membranes. In Korean boxwood leaves, the addition of phenol before the liberation of nucleic acids by sodium lauryl sulfate reduced yields of DNA and r-RNA. Lyophilization of tissues permitted the use of dry grindign techniques which improved cell breakage and increased yields of nucleic acids. Nucleic acid degradation during lyophilization, due to the thick tissue cuticle which prevented evaporative cooling, was minimized by maintaining the sample at −20° during drying. Precipitation of nucleic acids from dogwood bark with 2-ethoxyethanol at 0–4° improved MAK separation by eliminating interfering substances which competed with nucleic acids for binding sites on the MAK column. Degradation of precipitated nucleic acids by sodium lauryl sulfate was prevented by removing it with ether prior to precipitation.

Studies on a glycoprotein of fetal calf serum that protects nucleic acids from acid precipitation.

SummaryFetal calf serum contains large amounts of a glycoprotein that inhibits the acid precipitation of nucleic acids. The glycoprotein has been purified by gel filtration, acetone precipitation, and polyacrylamide gel electrophoresis. Its protective activity is insensitive to extremes in pH and temperature, but sensitive to proteolytic digestion and low concentration of bivalent metals.

Reduction of aromatic acids to aldehydes and alcohols in the cell-free system. 1. Purification and properties of aryl-aldehyde: NADP-oxidoreductase from Neurospora crassa

An enzyme catalyzing the reduction of the carboxyl group of aromatic acids to the respective aldehydes has been isolated from mycelia of the fungus Neurospora crassa grown in the presence of salicylic acid as an inducer. This protein has been purified about 300-fold by precipitation of nucleic acids with protamine sulfate, ammonium sulphate fractionation, adsorption on calcium-phosphate gel and chromatography on TEAE-cellulose (at pH 7.6 and 8.4) and on hydroxylapatite. The enzyme, the molecular weight of which was estimated to be about 120,000 by chromatography on a calibrated Sephadex G-200 column requires NADPH and ATP as cofactors. The Km-values for salicylic acid as a substrate are 1.67 × 10−4 M and for ATP 1.52 × 10−4 M. The presence of Mg-ions and of a reduced thiol in the incubation mixture is necessary to achieve full activity. The purified enzyme is most active around pH 8.0 and 35°. It shows a broad range of substrate specificity towards aromatic acids, especially to phenyl carboxylic and phenyl acrylic acids, but does not catalyze the reduction of aliphatic carboxyl groups. During the reaction ATP is split into ADP and inorganic phosphate, while stoichiometric amounts of aldehyde are produced from the corresponding acid. NADPH used as a reductant, the formation of the carbonyl group involves the oxidation of the hydrogen donor in a molar ratio of 1:1. Since no intermediate could be detected and there was no evidence for a reverse reaction we formulate the reaction as R–COOH + ATP + NADPH + H+→ R-CHO + ADP + NADP++ PO43-+ H2O; the systematic name aryl aldehyde: NADP oxidoreductase (ADP) is proposed; the enzyme belongs to EC group 1.2.1. By supplying α-, or β-[3H]NADP the enzyme was shown to be of the “B-type”.