Removal Of Nucleic Acids Research Articles

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.

A procedure for the extraction and high resolution two‐dimensional gel electrophoresis of total nuclear phosphoproteins from isotonically purified nuclei

Methods are described for the extraction and preparation of total nuclear proteins for high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The conditions for protein extraction and preparation limit both protease and phosphatase activity, allowing application of this technique to the reliable analysis of changes in nuclear protein composition and nuclear protein phosphorylation as well as other forms of post-translational modifications. Unlike other procedures for 2-D PAGE analysis of nuclear proteins the technique allows solubilization and extraction of all nuclear proteins along with removal of nucleic acids which interfere with isoelectric focusing and autoradiography of 32Pi-labeled proteins. It avoids lengthy dialysis in which precipitation of nuclear proteins often occurs and does not require precipitation and resolubilization of nuclear proteins to obtain sufficient protein concentrations for 2-D PAGE analysis; often impractical steps in which complete resolubilization of all proteins is not possible. It produces high resolution 2-D PAGE analysis in which identification of even low abundance proteins can be made, based on isoelectric point and molecular weight, allowing comparison with other studies.

DNase activity ofMicrococcal endonuclease insolubilized on corn cob

The endonuclease from S. aureus has been immobilized on ground maize cob, previously activated with tosyl chloride. Pretreatment of the support on acid before tosylation yielded the best insoluble enzyme derivatives. The catalytic activity has been evaluated as percent of total hydrolysis attained in a batch reactor using DNA as a model substrate. The derivatives prepared are very resistant to high temperatures under conditions of catalysis (24 h at 45 degrees C). For these long reaction times, the extent of hydrolysis in the presence of small amounts of organic solvent (dimethyl sulfoxide at 2 percent) is larger than in plain buffer (Tris). This type of derivative could be very useful for the removal of nucleic acids from single-cell protein concentrates.

Removal of nucleic acids from yeast nucleoprotein complexes by sulfitolysis

Removal of nucleic acids from yeast nucleoprotein complexes by sulfitolysis

Nematode chromosomal proteins—IV. The nonhistones of Caenorhabditis elegans

1. 1. A method has been developed which yields reasonably clean nuclei from the free-living nematode Caenorhabditis elegans. Nonhistones were prepared from these nuclei by gentle extraction with 2M NaCl and analyzed by one- and two-dimensional electrophoresis using a nonequilibrium pH gradient electrophoresis in the first dimension after removal of nucleic acids by phenol extraction. 2. 2. The extraction procedure yielded nonhistone chromatin proteins and proteins associated with nuclear RNA and a background of polypeptides derived from the nuclear matrix and pore-lamina complex. 3. 3. Over 40 polypeptides were distinguished on silver stained one-dimensional slab gels. 4. 4. The presence of actin, tropomyosin and tubulin was demonstrated by immunoprobing after transfer of the polypeptides to nitrocellulose. 5. 5. Bands co-migrating with myosin and α-actinin gave no specific when probed with antibodies raised against the respective proteins from vertebrate tissues. 6. 6. Over 200 spots were visible on two-dimensional gels stained with silver nitrate.

Purification of a DNA polymerase-DNA primase complex from calf thymus glands.

An immunoabsorbent column, prepared by covalently linking mouse monoclonal anti-calf thymus DNA polymerase-alpha to Protein A-Sepharose, was used as the primary purification step for rapid isolation of DNA polymerase-alpha from calf thymus-gland extracts. In a 4-step procedure consisting of the removal of nucleic acids by protamine sulfate precipitation, chromatography on the immunoabsorbent column, desalting on Sephadex G-50, and removal of bovine immunoglobulins on Protein A-Sepharose, DNA polymerase-alpha activity was purified about 5000-fold from the crude extract with greater than 40% recovery of total enzyme activity. The antibody column-purified DNA polymerase-alpha fraction contains a DNA primase activity that is efficient in replication of single-stranded DNA and poly(dT) when rNTPs are included in the replication reactions. Synthesis by calf thymus DNA polymerase-primase is totally dependent on added template. Complete replication of circular single-stranded phage DNA is achieved with polymerase-primase producing a nicked circular DNA containing oligoribonucleotide primer in the final product. Primers synthesized with single-stranded phage DNA as template were up to 10 nucleotides long when dNTPs were omitted from the reaction and 8 or less nucleotides long when dNTPs were present. Primers synthesized using poly(dT) consisted of three populations when dATP was absent from the reaction, averaging 20 nucleotides, 10 nucleotides, and 3-4 nucleotides. The 20-nucleotide population was not found when dATP was included in the reaction.

Nucleolar hypertrophy as an indicator of transcription in cells infected with second generation meronts of Eimeria tenella.

Changes in nuclei and nucleoli of cells of chicken cecum infected with Eimeria tenella were studied in living cells by interference microscopy and in fixed and stained tissues using light level microscopy. As soon as merozoites began to transform into second generation meronts, there was an increase in the size of both the nucleus and the nucleolus of the host cell. The dry weight of the nucleus increased somewhat, but there was a greater increase and a correlation of the dry mass of the nucleolus with the size of the parasite as measured by interference microscopy. In fixed and stained tissues, there was a correlation between the area of the nucleolus and the area of the parasite. Removal of nucleic acids with DNase and/or RNase showed high concentrations of both in the nucleoli and a residue of protein. The increased nucleolar size indicates a high level of transcription in infected cells and allows the conclusion that the parasite somehow induces transcription to occur. Since transcription is a highly specific process, the high degree of host and site specificity shown by nearly all coccidia is consistent with a hypothesis that the coccidia share a portion of the host genome.

Purification and properties of gentamicin nucleotidyltransferase from Escherichia coli: Nucleotide specificity, pH optimum, and the separation of two electrophoretic variants

Gentamicin nucleotidyltransferase, AAD 2″, catalyzes the transfer of a nucleotide to many aminoglycoside antibiotics, which are the drugs of choice in the treatment of gram-negative bacterial infections. The transfer is accompanied by the production of pyrophosphate, which is coupled to three other enzymes so that an increase in absorbance at 340 nm of NADPH can be monitored continuously as a quantitative assay of activity. A purification method was developed for this enzyme using all common principles of protein purification. These include selection of a desirable source of enzyme (choice of plasmid pMY 10), maximizing cellular yield of enzyme (controlled and monitored growth of Escherichia coli pMY 10/W677), selective extraction of protein (modified osmotic shock), removal of nucleic acids (precipitation with streptomycin sulfate), concentration of protein (precipitation with ammonium sulfate), removal of low-molecular-weight impurities (chromatography on Bio-Gel P-2), separation of proteins on the basis of charge (ion-exchange chromatography on DEAE-Bio-Gel A), separation of proteins according to a biospecific property (affinity chromatography on gentamicin-Affi-Gel), and separation of proteins according to size (gel filtration on Ultrogel AcA 54). Purification to near-homogeneity revealed the presence of two related forms of enzyme. The first had a specific activity of 0.134 units/mg, bound rapidly and tightly to gentamicin-Affi-Gel, eluted as a function of ionic strength from Ultrogel, and migrated faster during electrophoresis in both the presence and absence of sodium dodecyl sulfate. It has an isoelectric point of 5.7 ± 0.2 and consists of a single polypeptide of 32,500 Da. Kinetic characterization showed a pH optimum of 9.5 and Michaelis constants of 2.76 ± 0.35 μ m for tobramycin, 404 ± 28 μ m for Mg-ATP, 2008 ± 260 μ m for Mg-CTP, 30 ± 3 μ m for Mg-dATP and Mg-dGTP, and 90 ± 7 μ m for Mg-dCTP and Mg-dTTP. The second form had a specific activity of 0.274 unit/mg. It also bound tightly to gentamicin-Affi-gel but the onset of binding was time dependent. This form migrated slower during polyacrylamide gel electrophoresis in both the presence and absence of sodium dodecyl sulfate. It has an isoelectric point of 6.0 ± 0.2 and consists of a single polypeptide of 31,500 Da. The exact relationship between the two forms has not been elucidated. It is probable that they have a recent common ancestor or are the same polypeptide because the amino acid compositions and polypeptide chain lengths are essentially identical. An intriguing possibility of the relationship of the two forms is that they are the same polypeptide but that they have been differentially conjugated to either sugars or lipids, which in turn changes their chromatographic, electrophoretic, and kinetic properties.

Studies on Basidiomycetes. I. Antitumor polysaccharide from bagasse medium on which mycelia of Lentinus edodes (Berk.) Sing. had been grown.

From bagasse medium on which mycelia of Lentinus edodes (BERK.) SING. had been grown, an acidic polysaccharide (fraction C-1-2) was isolated by deproteinization, removal of nucleic acid, fractionation with barium hydroxide and DEAE-cellulose column chromatography. The purified polysaccharide consisted mainly of xylose, arabinose, mannose, galactose, glucose and uronic acid in a molar ratio of 1.4 : 0.7 : 2.0 : 0.9 : 1.0 : 1.5. The tumor inhibition ratio of the polysaccharide against solid sarcoma 180 was 87.9% in mice given doses of 100 mg/kg×10 d by intraperitoneal administration and the tumors in 5 of 10 mice completely regressed.

Changes in the composition of digesta during passage through the small intestines of steers.

1. Dry matter (DM), organic matter (OM), nitrogenous constituents, carbohydrate constituents and pH were estimated in digesta taken from eight different sites in the small intestines of slaughtered steers given different diets. Amounts of constituents passing different sites were compared using cellulose as a non-digestible marker. 2. Amounts of the different constituents entering the small intestines varied with the type of diet given but the patterns of removal of each constituent were similar regardless of diet. 3. DM and OM were uniformly down the length of the small intestines. Net digestibilities of 0.62 and 0.63 for DM and OM respectively of the amounts measured in segment 1 digesta (first 3 m post-pylorus) were found at the terminal ileum. DM and OM contents of segment 1 digesta were 5 and 8% higher respectively than in abomasal digesta. Significantly smaller amounts of digesta were found in the proximal quarter of the small intestines than in more distal sections. There was a 10% addition of water to digesta in segment 1 compared with abomasal digesta. Of the water entering the small intestines a net 65% was absorbed therein, most absorption occurring in the first half. 4. Ammonia nitrogen was removed uniformly down the length of the small intestines to a net extent of 75% of that in segment 1 digesta. There was an apparent increase of approximately 18% in the total N (TN) content of segment 1 digesta compared with abomasal digesta. There was an approximate net removal of 80% of the TN measured in segment 1 digesta during passage down the small intestines. Over 95% of the digestible TN was removed in the section of tract between 3 and 15 m from the pylorus. Net removal of nucleic acid N (NA-N) up to the distal ileum was 78% of that in segment 1 digesta, most removal occurred in the proximal quarter of the small intestine. Little or no net loss of diaminopimelic acid-N (DAP-N) occurred in the small intestine. 5. Of the carbohydrates entering the small intestines, there was little or no removal of rhamnose, arabinose or xylose. Net removal of ribose, mannose, galactose and 'starch' glucose up to the distal ileum was 82, 76, 54 and 70% respectively of the amounts found in segment 1 digesta. All disappeared uniformly down the length of the small intestine.

13] Purification and properties of HindII and HindIII endonucleases from Haemophilus influenzae Rd

Publisher Summary This chapter discusses the purification and properties of HindII and HindIII endonucleases from Haemophilus influenzae Rd. Haemophilus influenzae Rd is the source for two restriction endonucleases,HindII and HindlII, that may be separated and purified using conventional ion exchange chromatography media. The basic steps in the procedure are removal of nucleic acids from the cell extract, separation of the two activities by DEAE-cellulose chromatography, and chromatography of each activity on phosphocellulose as a combined purification and concentration step. The chapter also presents separation of HindlI and HindllI activities by DEAE-cellulose chromatography. In addition, properties are also overviewed, such as enzyme stability. HindlII is stable for at least 2 years at -20 °. Under reaction conditions it retains activity for hours at 37 ° and for several minutes at 65 °.

Investigation of the unit operations involved in the continuous flow isolation of β‐galactosidase from Escherichia coli

AbstractA 1000 liter fermentor has been used to produce a continuous feed of Escherichia coli containing a high level of β‐galactosidase. We have investigated the individual unit operations for the isolation of the enzyme: cell disruption, nucleic acid removal, protein precipitation, and solid–liquid separation after each stage. Using the information obtained we have been able to operate a semicontinuous process which when fully continuous would yield 100 g protein/hr, comprising 23% β‐galactosidase.

Biospecific fractionation matrices for sequence specific endonucleases.

Fractionation of several type II specific restriction endonucleases was achieved by separation on two novel biospecific matrices. The matrices are pyran, a copolymer of divinyl ether of maleic anhydride, and Cibacron Blue F3GA, a blue dye commonly used for the calibration of molecular sieves. Both compounds are insolubilized by coupling to sepharose through a cyanogen bromide linkage and in their soluble form inhibit the restriction endonucleases which we have tested. These affinity matrices can be used to obtain restriction endonucleases from crude extracts after removal of nucleic acids. They have also proven to have a high capacity when used as subsequent steps in enzyme purification. Their additional advantage is the rapid development time and reusability of columns packed with the two matrices.Images

A simplified SDS-gel electrophoresis procedure with improved resolution of chromosomal proteins

1. 1. The simplified SDS-electrophoresis procedure reported here allows application of total chromatin on the gel. 2. 2. The chromosomal proteins are extracted directly on the gel, without prior removal of nucleic acids. 3. 3. Either histones or nonhistones can be resolved completely on gels with this procedure.

Cytochemical evaluation of the Guard procedure a regressive staining method for demonstrating chromosomal basic proteins. I. Effects of fixation, blocking reactions, selective extractions, and polyacid "differentiation".

Appropriately fixed preparations stained by a modification of the Guard (1959) reaction for "sex chromatin" display selective staining of interphase chromatin and mitotic or meiotic chromosomes. This is a regressive staining method which seems to depend on the selective displacement of an acidic dye from less basic structures, and retention of the dye at more basic sites. The results obtained with the reaction can be controlled by the length of time that the preparations are "differentiated" in solutions containing phosphomolybdic and phosphotungstic acids (polyacids). After three- or four-hour exposures to polyacid solutions, all chromatin is stained. However, with longer differentiation, "condensed" chromatin can be stained preferentially. Of a number of fixatives investigated, only 10% formalin, ethanol-acetic acid (3:1), and Bouin's solution proved useful. Others resulted in diminished specificity or a total loss of selectivity. The most intense staining was obtained after formalin fixation. Less intense dyebinding was observed after fixation in 3:1 - probably due to extraction of some histone fractions-and the least amount of dye was bound in Bouin's-fixed chromatin - probably due to blockage of arginine residues by picric acid. The reaction was not affected by enzymatic removal of nucleic acids or the extraction of lipids. It was diminished by treatment with trypsin or weak acetylation, and it was completely prevented by strong acetylation, deamination, or extraction of basic proteins with HCl. The results presented suggest that the modified Guard (1959) procedure selectively demonstrates basic nucleoproteins. Further, by the use of regressive differentiation in polyacid solutions, the retention of dye in more condensed chromatin can be favored.

The Isolation of Two Different Lipopolysaccharide Fractions from Various Proteus mirabilis Strains

Four distinct Proteus mirabilis strains were extracted by the phenol/water procedure. After ultracentrifugation of the dialyzed water phase, the pelleted lipopolysaccharide was purified and analyzed. The sugar composition of this lipopolysaccharide fraction I was similar for all four strains, containing only small amounts of strain-specific constituents. A second lipopolysaccharide fraction was isolated from the supernatant above (termed L1 fraction) after removal of nucleic acids. DEAE-cellulose chromatography indicated that this material is not a polysaccharide but rather a water-soluble lipopolysaccharide containing strain-specific constituents such as uronic acids, amino acids, amino sugars, neutral sugars, ethanolamine and phosphate, depending on the strain from which lipopolysaccharide II was isolated.

Binding of [ 14C]aflatoxin B 1 to cellular macromolecules in the rat and hamster

The uptake and binding of ring-labelled [-14C]aflatoxin B1 (AFB1) by rat and hamster liver and kidney has been studied, the former species being extremely sensitive to the carcinogenic action of AFB, whereas the latter is resistant. In contrast to an earlier report (Lijinsky et al, Cancer Res., 30 (1970) 2280-2283, binding of the carcinogen to nucleic acids was far greater than that to protein. Rat liver DNA bound ten times and rRNA twenty times more carcinogen than protein. There were also differences in the amount of carcinogen bound to rat liver nucleic acids compared to those of the hamster, the latter species binding lower amounts of the carcinogen. Rat liver DNA bound four times and rRNA ten times as much AFB1 6 h after carcinogen administration whereas liver protein bound AFB1 was similar for the two species. Not only was there a difference in the amount of AFB1 bound but whereas in the rat, liver nucleic acid bound carcinogen decayed with time, no such fall was seen in the hamster, this remaining at a low level throughout the 48-h time period studied. In contrast, reaction of the carcinogen with kidney macromolecules was similar for the two species. The much higher binding of AFB1 to nucleic acids than to protein might account for the potent carcinogenicity of this compound in the rat, particularly since liver protein binding does not differ between a susceptible and a resistant species. A further important factor in determining carcinogenic sensitivity may be the removal of nucleic acid bound radioactivity with time, a possible repair process.

The removal of nucleic acids from microbial extracts by precipitation with lysozyme

It is usually necessary to remove nucleic acids from microbial extracts in order to avoid their interference with the isolation of enzymes from the extract. This may be particularly important where the purification procedure includes chromatography on an anion-exchange column such as DEAE-Sephadex (1). Methods that have been used have included precipitation with Mn, destruction of the nucleic acids with nucleases, and precipitation with basic substances, usually protamine sulfate or streptomycin sulfate. It is likely that there may be advantages, in some cases at least, in using other basic proteins for this purpose, since the results obtained with the previous methods have not always been satisfactory. The procedure described in this paper utilizes high concentrations of lysozyme to precipitate nucleic acids from a bacterial extract. The separation obtained with lysozyme was efficient, reproducible, and superior to the separation obtained with protamine sulfate.

Initial steps in the large-scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase

A procedure for the partial removal of nucleic acids from extracts of E. coli is described. It consists of adding DEAE-cellulose directly to an E. coli extract. Separation of the DEAE-cellulose removes 25% of the nucleic acid in the extract with no loss in RNA polymerase activity and facilitates subsequent fractionation of the enzyme. RNA polymerase purified to homogeneity by the procedure of Berg, Barrett, and Chamberlin (Berg, D., Barrett, K., and Chamberlin, M. (1971) Methods Enzymol. 21, 506), but utilizing DEAE-cellulose treatment instead of DNase to remove nucleic acids, contains much less contaminating DNase activity. This procedure can be used as a general step in the purification of other E. coli proteins.

Enzyme fractionation and simultaneous nucleic acid removal from crude cellular extracts by preformed gradient ion exchange gel filtration

A crude cellular lysate (S30) can be fractionated on DEAE Sephadex A-25 gel into major classes including an excluded protein class. The protein class itself can be fractionated simultaneously by applying a salt gradient to the A-25 gel before applying sample. The procedure is rapid, that is, complete in one column liquid volume (or less) of column development. In addition, the fractionating conditions are very mild permitting very high recovery of input enzyme activity in a volume representing minimal dilution of the original sample.