Pancreatic Deoxyribonuclease Research Articles

R-banding produced by DNase I digestion of chromomycin-stained chromosomes.

A distinct reverse (R-) banding pattern was produced on human chromosomes by digesting chromosome spreads with pancreatic deoxyribonuclease I (DNase I) in the presence of an excess of chromomycin A3 (CMA), followed by staining with Giemsa. The banding pattern corresponds with that obtained by chromomycin A3 fluorescence, and bands which fluorescence brightly with chromomycin appear darkly with Giemsa. The same relationship was observed in two plants, Scilla siberica and Ornithogalum caudatum, which have contrasting types of heterochromatin. Chromomycin bright C-bands stained darkly with the CMA/DNase I technique, whereas chromomycin negative C-bands appeared lightly stained. The digestion patterns are thought to reflect the variation in chromomycin binding capacity along the chromosome with R-bands and dark C-bands being sites which preferentially bind the antibiotic.

Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei.

Analysis of the DNA of isolated nucleosomes suggests that virtually all genomic DNA sequences are organized in this basic chromatin subunit. In this report, we demonstrate that although histones reside on the transcriptionally active ovalbumin genes in the oviduct, the organization of proteins about this gene renders it highly sensitive to deoxyribonuclease I (deoxyribonucleate 5'-oligonucleotidohydrolase, EC 3.1.4.5). Treatment of oviduct nuclei from the laying hen with pancreatic deoxyribonuclease I results in the preferential digestion of over 70% of the ovalbumin sequences when only 10% of the total nuclear DNA has been solubilized. Treatment of liver nuclei does not reveal selective sensitivity of these genes to DNase I. Furthermore, regions of DNA not actively transcribed, such as the endogenous leukosis virus genes in the oviduct, are not selectively degraded by this enzyme. Similar digestions with micrococcal nuclease, however, reveal no specific digestion of transcriptionally active chromatin. These data confirm the observations of H. Weintraub and M. Groudine [(1976) Science 193, 848-856] and suggest we are dealing with an aspect of structure that may be necessary to permit transcription of the chromatin complex.

Further characterization of a DNA polymerase activity in mouse sperm nuclei

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg 2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase α. The temperature for maximum incorporation of nucleotides was found to be 32°C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 μg/ml), ethidium bromide (25–50 μM), netropsin (5–50 μg/ml), and spermidine (0.5–2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519–525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase β) reported in several eukaryotic organisms.

Decondensation of sperm nuclei in vitro

Treatment of mouse spermatozoa with dithiothreitol and proteases, particularly trypsin, causes the nucleus to enlarge and decondense, while the acrosomal region remains relatively intact. Dithiothreitol or trypsin alone does not induce swelling, and exposure to the reducing agent is necessary before trypsin can act. Chymotrypsin, promise, and papain will substitute for trypsin, but micrococcal nuclease and pancreatic deoxyribonuclease will not. Similar results were obtained with rat, guinea pig, and rabbit sperm. These results provide the basis for a method of purifying sperm acrosomes and suggest possible mechanisms for decondensation of the sperm nucleus after penetration of the egg.

A simple and efficient method to remove ribonuclease contamination from pancreatic deoxyribonuclease preparations

An affinity chromatography method which efficiently removes the contaminating RNase activity from commercial pancreatic DNase preparations is described. The new method is compared with the iodoacetate treatment previously used for the preferential inactivation of RNase.

Chromosomal Subunits in Active Genes Have an Altered Conformation

Ten percent digestion of isolated nuclei by pancreatic deoxyribonuclease I preferentially removes globin DNA sequences from nuclei obtained from chick red blood cells but not from nuclei obtained from fibroblasts, from brain, or from a population of red blood cell precursors. Moreover, the nontranscribed ovalbumin sequences in nuclei isolated from red blood cells and fibroblasts are retained after mild deoxyribonuclease I digestion. This suggests that active genes are preferentially digested by deoxyribonuclease I. In contrast, treatment of red cell nuclei with staphylococcal nuclease results in no preferential digestion of active globin genes. When the 11S monomers obtained after staphylococcal nuclease digestion of nuclei are then digested with deoxyribonuclease I, the active globin genes are again preferentially digested. The results indicate that active genes are probably associated with histones in a subunit conformation in which the associated DNA is particularly sensitive to digestion by deoxyribonuclease I.

Attempts to Detect Agrobacterium tumefaciens DNA in Crown-Gall Tumor Tissue

Primary and secondary crown gall tissue cultures were established from sunflower plants (Helianthus annuus, variety Mammoth Russian) wound-inoculated with Agrobacterium tumefaciens (Smith and Townsend) Conn strain B(6). Growth rates of tumor tissues and habituated healthy sunflower stem section tissues on basal medium lacking auxin and cytokinin were compared to those of healthy sunflower stem section tissue grown on the same medium with added phytohormones. No difference was detected in the thermal denaturation midpoints (74.8 C) and melting profiles in 25 mm sodium phosphate (pH 6.8), or the buoyant densities in cesium chloride equilibrium centrifugation (1.687 g cm(-3)), between deoxyribonucleic acids (DNAs) isolated from crude nuclear preparations of the four tissue types. No satellite DNA was observed in equilibrium centrifugation of unsheared plant DNAs.Heterologous DNA renaturation kinetic analyses were performed in 0.14 m sodium phosphate (pH 6.8) at 70 C. Thermal stability measurements of reassociated DNA revealed less than 1% of mismatched base pairs. Reannealing of sheared, denatured, radioactive A. tumefaciens B(6) DNA (molecular weight, 325,000 daltons) in the presence of a 5400-fold excess of sheared calf thymus, healthy tissue, or secondary sunflower crown gall DNA obeyed second order kinetics, with a Cot((1/2)) of 2.8, identical to that observed when B(6) DNA was reannealed in the absence of foreign DNA.Reannealing rates of B(6) DNA in the presence of 5400-fold excesses of DNA from two lines of primary sunflower crown gall were increased 2.24- or 1.47-fold. Digestion of the tumor DNA preparations with pancreatic deoxyribonuclease I until no detectable DNA remained, followed by restoration of solution viscosity by added calf thymus DNA, failed to remove the acceleration effect of the tumor DNA preparations. Reisolation of the reannealed nucleic acid formed in this experiment, and digestion with ribonuclease A or deoxyribonuclease I revealed that the double-stranded fraction was composed entirely of DNA-DNA duplexes, with no detectable DNA-RNA hybrids.The data indicate that tumor, but not healthy tissue DNA preparations contain some factor or factors (not DNA) which accelerate the reannealing of bacterial DNA. Sunflower tumor tissue DNAs, therefore, do not contain integrated A. tumefaciens DNA sequences in amounts greater than a random (1/5) of the bacterial genome per diploid amount of plant DNA, or a complete bacterial genome per five diploid plant cell DNA equivalents. Further, the possibility of the presence of many copies of a specific portion greater than 5% of the bacterial genome is excluded.

Synthesis of subsituted glycopeptides containing at 2-acetamido-2-deoxy-beta-d-glucopyranosyl residue and the amino acid sequence 18-22 of bovine pancreatic deoxyribonuclease A.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAmino sugars. CI. Synthesis of substituted glycopeptides containing a 2-acetamido-2-deoxy-.beta.-D-glucopyranosyl residue and the amino acid sequence 18-22 of bovine pancreatic deoxyribonuclease AHari G. Garg and Roger W. JeanlozCite this: J. Org. Chem. 1976, 41, 14, 2480–2484Publication Date (Print):July 1, 1976Publication History Published online1 May 2002Published inissue 1 July 1976https://doi.org/10.1021/jo00876a030RIGHTS & PERMISSIONSArticle Views35Altmetric-Citations12LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (736 KB) Get e-Alerts Get e-Alerts

Inhibition of hemoglobin synthesis by pancreatic deoxyribonuclease in rabbit reticulocyte lysates

A pancreatic deoxyribonuclease preparation, shown to be free of significant ribonuclease activity, inhibits hemoglobin synthesis in a rabbit reticulocyte lysate. Preincubation with DNAase (15 min at 25°C) is required to obtain a marked inhibitory effect (nearly 70%). It has been shown that DNAase does not significantly interfere with the elongation or termination steps of translation, but it seems to prevent reinitiation of globin polypeptide chains allowing polysome run-off. In particular, the formation of the 40 S met-tRNA met f complex is greatly reduced in the DNAase-treated lysate. At the moment the mechanism by which DNAase inhibits initiation is not clear.

Deviations from compositional randomness in eukaryotic and prokaryotic proteins: The hypothesis of selective-stochastic stability and a principle of charge conservation

Eight proteins of diverse lengths, functions, and origin, are examined for compositional non-randomness amino acid by amino acid. The proteins investigated are human fibrinopeptide A, guinea pig Insulin, rattlesnake cytochrome c, MS2 phage coat protein, rabbit triosephosphate isomerase, bovine pancreatic deoxyribonuclease A, bovine glutamate dehydrogenase, and Bacillus thermoproteolyticus thermolysin. As a result of this study the experimentally testable hypothesis is put forth that for a large class of proteins the ratio of that fraction of the molecule which exhibits compositional non-randomness to that fraction which does not is on the average, stable about a mean value (estimated as 0.32 plus or minus 0.17) and (nearly) independent of protein length. Stochastic and selective evolutionary forces are viewed as interacting rather than independent phenomena. With respect to amino acid composition, this coupling ameliorates the current controversy over Darwinian vs. non-Darwinian evolution, selectionist vs. neutralist, in favor of neither: Within the context of the quantitative data, the evolution of real proteins is seen as a compromise between the two viewpoints, both important. The compositional fluctuations of the electrically charged amino acids glutamic and aspartic acid, lysine and arginine, are examined in depth for over eighty protein families, both prokaryotic and eukaryotic. For both taxa, each of the acidic amino acids is present in amounts roughly twice that predicted from the genetic code. The presence of an excess of glutamic acid is independent of the presence of an excess of aspartic acid and vice versa.

A study of the binding of Mn2+ to bovine pancreatic deoxyribonuclease I and to deoxyribonucleic acid by electron paramagnetic resonance.

EPR studies of Mn2+ binding to bovine pancreatic deoxyribonuclease I show that the enzyme can bind three Mn2+ ions at pH 7.5 and 2 degrees. Two sites bind Mn2+ strongly, with a Kd of 10(-4)M, and the third binds Mn2+ weakly, with a Kd of 10(-3)M. Ca2+ competes with the two strong sites, whereas Mg2+ competes only with one of them, indicating that both sites are not equivalent. Mn2+ binding to DNA has been confirmed by EPR measurements. Two types of sites, with different affinities for Mn2+ binding, were found on DNA molecules, one with a Kd of 1.2 times 10(-4)M and the other with a Kd of 10(-3)M. Mg2+ ions can displace Mn2+ from the high affinity sites, but not from the low affinity sites. These results suggest the Mn2+ binds not only to the phosphate groups, but also to the electron donor groups of the base rings.

Some effects of calcium and magnesium ions on the activity of bovine pancreatic deoxyribonuclease A

Bovine pancreatic deoxyribonuclease requires divalent metal cations for hydrolysis of DNA. The effects of calcium and magnesium, alone and combined, on the rate and kinetics of the reaction were examined. Divalent metal salts of DNA were used as substrates. The ratio of either Ca 2+ or Mg 2+ to DNA-P in these salts was 1 : 2. The Mg 2+ salt of DNA was found to have sufficient Mg 2+ for optimal DNAase activity. Addition of MgCl 2 to a large excess of Mg 2+ over DNA-P had no effect on the rate. K m for the hydrolysis of Mg 2+-DNA was 1.76 mM. K m for the hydrolysis of Ca 2+-DNA was too low to measure by our methods of assay, indicating a high affinity of enzyme for substrate. The rate of hydrolysis of Ca 2+-DNA, however, is slow compared to that of Mg 2+-DNA. By mixing the Ca 2+ and Mg 2+ salts of DNA, a synergistic effect on the activity of DNAase was observed. On the basis of kinetic studies the synergistic effect is attributed to an increased affinity of DNAase for DNA ( K m = 0.34 mM for the mixed Ca 2+, Mg 2+ salt of DNA). A 2-fold increase in DNAase activity was observed when DNAase was incubated in CaCl 2 before assay. This 2-fold stimulation was not related to the synergistic effect. DNAase I is inactive against the sodium salt of DNA. In experiments with mixed Na + and Mg 2+ salts of DNA, Na +-DNA was found to be a competitive inhibitor of the action of DNAase against Mg 2+-DNA.

Reversible inactivation of pancreatic deoxyribonuclease A by sodium dodecyl sulfate. Removal of COOH-terminal residues from the denatured protein by carboxypeptidase A.

In the course of experiments on the role of the COOH-terminal residues in pancreatic deoxyribonuclease, we undertook to ascertain whether the presence of sodium dodecyl sulfate would render the normally unavailable terminus susceptible to hydrolysis by carboxypeptidase A. When DNase A is dissolved in 0.005% sodium dodecyl sulfate the protein becomes enzymically inactive when assayed against DNA in the same sodium dodecyl sulfate concentration. The loss of activity caused by treatment with sodium dodecyl sulfate for 1 hour at 45 degrees can be fully restored if the detergent-containing solution is diluted 10-fold into 6 M guanidinium chloride and then 10-fold into a pH 7.0 buffer, 10 mM in CaCl2, prior to a 100-fold dilution for assay. The presence of Ca2+ is essential for the refolding process. If the same degree of dilution is made into sodium dodecyl sulfate-free buffer without the guanidinium chloride step, there is very little reversal of the inactivation. An almost complete loss of regenerable activity is caused by 1 hour of digestion by carboxypeptidase at 45 degrees in the presence of 0.03% sodium dodecyl sulfate. Although up to 6 amino acid residues can be removed from the COOH terminus, the loss of activity can be correlated with the removal of either 1 or 2 amino acid residues (-Leu-Thr) from the COOH-terminal sequence. Thus, DNase A is one of the several enzymes in which residues at the COOH terminus are essential to the active conformation. If the enzyme minus 2 to 6 terminal residues was mixed with a 15-residue COOH-terminal peptide (obtained by cyanogen bromide cleavage), only about 2% activity could be regenerated.

Deoxythymidine 3', 5'-di-p-nitrophenyl phosphate as a synthetic substrate for bovine pancreatic deoxyribonuclease.

Bovine pancreatic deoxyribonuclease liberates p-nitrophenol from the 3'-group of deoxythymidine 3', 5'-di-p-nitrophenyl phosphate. A similar hydrolysis occurs with deoxythymidine 3'-p-nitrophenyl phosphate 5'-phsophate, but the rate is less than 2% of that with the di-p-nitrophenyl ester. The rate of formation of the p-nitrophenol, measured spectrophotometrically at 400 nm, varies linearly with DNase concentration. The binding of the substrate is not strong (K-m(app) in the 10 mM range), but the hydrolysis is rapid; 1 mug of DNase (free from other phosphodiesterases) can be assayed in 3 min after addition to a 10 mM substrate solution at pH 7.2, 10mM in MnCl2, and 1mM in CaCl2. All four bovine pancreatic DNases (A,B,C, and D) show the same relative activities toward DNA and toward the di-p-nitrophenyl ester; both activities are lost when DNase is inactivated by iodoacetate or by trypsin. The specificity of DNase toward the di-p-nitrophenyl substrate is different from that which has been established for the enzyme's predominant action on DNA or synthetic oligonucleotides, where a monoesterified phosphate group is formed at the 5'-position.

The essential role of Ca2+ in the activity of bovine pancreatic deoxyribonuclease.

DNase requires Ca2+ for activity against DNA with Mg2+. The Ca2+ selective chelating agent, ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, (EGTA) inhibits DNase completely at pH 7 or 8, and subsequent addition of excess Ca2+ reverses inhibition in less than one second. DNase action can be stopped at any point by the addition of excess EGTA over Ca2+. Ca2+ is required for DNase to bind substrate. Gel filtration experiments fail to show DNase binding to 0.2 mg per ml of DNA at 5 mm Mg2+ and 10-4 M EGTA. The concentration of Ca2+ needed for half of maximum DNase activity decreases with increases DNA concentration, from 1.2 times 10-5 M Ca2+ at 2.3 times 10-5 M DNA-P to about 4 times 10-7 M Ca2+ at 2.3 DNA-P. Kinetic analysis by the titrametic assay of protons releases shows that V max is independent of Ca2+ concentration while Km increases from 7.7 times 10-5 M DNA-P at 5 times 10-4 M Ca2+ to 3.4 times 10-4 M DNA-P at 5 times 10-6 M Ca2+. Both of these results are predicted by a rate equation which is derived from the assumption that DNase must bind Ca2+ before it can bind DNA. The essential Ca2+ atom probably binds to the one of two high affinity Ca2+ binding sites on DNase which cannont bind Mg2+ or Mn2+. The only other divalent metal ions which can bind to this site, Sr2+ and Ba2+, are also the only metal ions which can substitute for Ca2+ in DNase action against DNA with Mg2+. Some DNase activity is obtained in the absence of added Ca2+ with Mg2+ at pH 6 or below and with Mn2+ or Co2+ at pH 8. These assay solutions are contaminated by 1 to 3 muM Ca2+, which may be sufficient to account for the observed activity.

Suggested viroid etiology for chrysanthemum chlorotic mottle disease

In the range of pH 5.5–6.5 the etiologic agent of chrysanthemum chlorotic mottle disease (ChCMV) is extremely unstable in crude leaf extracts. However, grinding of infected tissue in alkaline buffer (pH 7.5–9.5) significantly increased the stability and infectivity of the agent. Homogenization of tissue in 0.2 M borate buffer, pH 9.0, with sodium dodecylsulfate (SDS)-phenol or chloroform- n-butanol, rendered the agent as infectious and more stable than homogenization in borate buffer alone. Pancreatic ribonuclease but not pancreatic deoxyribonuclease abolished infectivity of ChCMV extracted from tissue with SDS-phenol. Following centrifugation of crude extracts, most of the infectivity was found in the 5,900 g and 120,000 g supernatant fluids, although some could be sedimented under these conditions. Rate-zonal centrifugation of leaf extracts of ChCMV in linear-log sucrose density gradients showed that the predominant infectious species sedimented between 6 and 14 S but with minor species sedimenting between 18 and 23 S and 35 and 40 S. We propose that ChCMV is a viroid.

Deoxyribonucleic acid polymerases of BHK-21/C13 cells. Partial purification and characterization of the enzymes

DNA polymerase from BHK-21/C13 cells were separated into two species, DNA polymerase I corresponding to the heterogeneous enzyme with sedimentation coefficient of 6-8S, and DNA polymerase II, corresponding to the enzyme with sedimentation coefficient of 3.3S. DNA polymerase I was purified 114-fold and DNA polymerase II 154-fold by a simple extraction procedure followed by column chromatography on phosphocellulose and gel filtration through Sephadex G-100. The purified enzymes differed markedly in respect of pH optimum, stimulation and inhibition by K+, Km for the deoxyribonucleoside 5'-triphosphates, stability to heating at 45 degrees C, and inhibition by N-ethylmaleimide. The preferred primer-template for both enzymes was "activated" DNA (DNA submitted to limited degradation by pancreatic deoxyribonuclease); native or thermally denatured DNA templates were relatively very poorly copied. When certain synthetic templates were tested, substantial differences were revealed between the two enzymes. Poly[d(A-T)] was poorly used by polymerase I but was superior to "activated" DNA for polymerase II. Poly[d(A)]-oligo[d(pT)10] was used efficiently by polymerase I but not by polymerase II. Poly(A)-oligo[d(pT)10] was not an effective primer-template although polymerase I could use it to a limited extent when Mn2+ replaced Mg2+ in the polymerase reaction and when the temperature of incubation was lowered from 37 degrees to 30 degrees C. When only one or two or three triphosphates were supplied in the reaction mixture, the activity of polymerase I was more severly diminished than that of polymerase II.

Stimulation of the uptake of soluble proteins into isolated HeLa nuclei by pancreatic deoxyribonuclease

The uptake into isolated HeLa nuclei of radioactive cytosol proteins and purified Escherichia coli ribosomal protein L7 is stimulated up to 4-fold by pancreatic deoxyribonuclease (DNase I). Similar effects are not observed with pancreatic ribonuclease A or phospholipase C. The results reported suggest that there is a general stimulatory effect of DNase on protein uptake by nuclei.

Removal of proteases from DNase I by chromatography over agarose with covalently attached lima bean protease inhibitor

Lima bean protease inhibitor (LBI) can be convalently attached to agarose using the cyanogen bromide activation procedure. The LBI-agarose complex retains full capacity to inhibit chymotrypsin and 30% capacity to inhibit trypsin; the dissociation constant for chymotrypsin bound to LBI-agarose is 3.7 × 10 −6 m. Bovine pancreatic deoxyribonuclease is contaminated by 3% by weight of chymotrypsin plus chymotrypsinogen. The contaminating proteases may be removed from DNase by sequential reaction with LBI-agarose followed by filtration. The most effective method for the removal of proteases from DNase is chromatography over LBI-agarose. DNase which has been chromatographed over LBI-agarose is at least ten times more stable than DNase prepared by earlier procedures.

Presence of Ca 2+, Mg 2+-dependent endonuclease stimulating factor in rat liver and testis nuclei

A factor was extracted from isolated rat liver and testis nuclei which stimulated rat liver and bull semen Ca2+, Mg2+-dependent endonuclease activity and Micrococcal Ca2+-dependent nuclease. The factor did not influence pancreatic deoxyribonuclease I; whereas it inhibited the Mg2+-dependent acid endonuclease of rat liver. It retained its stimulating capacity after boiling for 60 min. Its action was lost on incubation with pronase. It enhanced the ability of bull semen Ca2+, Mg2+-dependent endonuclease to activate the template of rat liver residual chromatin for DNA synthesis.