Concentrations Of DNase Research Articles

Light-regulated changes in DNase I hypersensitive sites in the rRNA genes of Pisum sativum

We have examined the rDNA chromatin of Pisum sativum plants grown with or without exposure to light for the presence of DNase I hypersensitive sites and possible developmental changes in their distribution. Isolated nuclei from pea seedlings were incubated with various concentrations of DNase I. To visualize the hypersensitive sites, DNA purified from these nuclei was restricted and analyzed by gel blot hybridization. We find that several sites exist in both the coding and noncoding regions of rDNA repeating units. Several of the sites in the nontranscribed spacer region are present in the light but are absent in the dark. Conversely, the hypersensitive sites within the mature rRNA coding regions are present in the dark but absent in the light. There are two major length variants of the rRNA genes in P. sativum var. Alaska. The sites in the nontranscribed spacer region that appear during the light treatment occur only in the shorter of these two length variants in this cultivar.

Evidence for the transcription os physiologically inactive rat-liver nucleolar chromatin by Escherichia coli RNA polymerase.

Rat-liver nucleoli (10-15 micrograms DNA) were digested with either 0.6 or 3 units of DNase I for various times (up to 1 h). RNA synthesis was then measured in the absence or presence of 3 units of Escherichia coli RNA polymerase. It was found that the nucleolar chromatin supporting the endogenous engaged RNA polymerase I transcription was completely destroyed in 3 min with either concentration of DNase I. The nucleolar chromatin template transcribed by E. coli RNA polymerase retained 50% of its original capacity even 60 min after 3 units of DNase I digestion. When hybridization experiments were conducted, it was found that the DNAs derived from both levels of DNase-I-digested nucleoli were incapable of forming hybrids with the labelled nucleolar RNA synthesized by the engaged RNA polymerase I from the untreated nucleoli. Since the engaged RNA polymerase I transcribes only the physiologically active genes of the nucleolar chromatin, and the RNA transcripts represent active gene product, these data suggest that DNase I digestion has completely destroyed the active genes of the nucleolar chromatin, and E. coli RNA polymerase is able to transcribe the inactive nucleolar chromatin template.

Murine erythroleukemia cell differentiation: DNase I hypersensitivity and DNA methylation near the globin genes.

The sensitivity to digestion by DNase I of chromatin containing the alpha- and beta(major)-globin genes and the pattern of DNA methylation near these genes was examined during hexamethylenebisacetamide (HMBA)-mediated erythroid differentiation of murine erythroleukemia cells (MELC). In uninduced and induced cells, the chromatin regions containing the alpha- and beta-(major)-globin genes are more sensitive to digestion by DNase I than is the region containing an immunoglobulin gene (Igalpha) not expressed during erythroid differentiation. However, at low concentrations of DNase I, a 6- to 10-fold increase in site-specific cleavages was generated in chromatin regions near both the alpha- and beta(major)-globin genes in cells induced to differentiate by HMBA. The DNase I hypersensitive site near the beta(major)-globin gene maps to a small region near the 5' terminus of the gene. No detectable change in the pattern of DNA methylation around either the alpha- or beta-globin genes was observed during HMBA-mediated erythroid differentiation. Of the potentially methylated sites assayed and mapped near the beta(major)-globin gene, one site is fully methylated, one is partially methylated, and one is unmethylated both in uninduced and induced cells. Many (but not all) sites assayed near the alpha-globin genes are unmethylated in both uninduced and induced cells. These results show that specific alterations of chromatin structure occur during MELC differentiation and suggest that these changes may not involve alterations in the pattern of DNA methylation.

DNAse I digestion of fixed chromosomes specifically reveals NORs in man

Chromosomal preparations were digested with different concentrations of DNAse I for various periods of time and then stained with Giemsa. It was found that this endonuclease rapidly modifies the structure of the chromosomes which then lose most of their stainability. However, small chromosomal segments corresponding to the nucleolar organizers (NORs) in man have been consistently observed. Although DNAse I treatment has permitted us to observe that some NORs are stained less intensely than others, comparisons with silver nitrate-stained NORs have shown a strict correspondence, in metaphase as well as in interphase. Chromosomal proteins seem responsible for this staining.

Sequence analysis of nuclear matrix associated DNA from rat liver

The DNA associated with the nuclear matrix of rat liver was studied. The amount of DNA associated with the matrix varied with the concentration of DNase used in its preparation, but approached a plateau value of 1% of total nuclear DNA. We next studied the sequence complexity of this residual DNA. Using C 0 t analyses, it was found that the complexity of matrix DNA was the same as that of total nuclear DNA, indicating that matrix DNA does not represent any specific sequence class. However, using 125I-labeled ribosomal RNA (rRNA) as a probe, we found that the genes for rRNA are significantly enriched in the residual matrix DNA. The implications of these results are discussed.

Nick translation of active genes in intact nuclei

Transcriptionally active genes in chromatin are maintained in a conformation such that they are exceedingly sensitive to attack by the enzyme DNase I. We have exploited this observation to introduce radioactive nucleotides specifically into active regions of the chromosome by performing nick translation reactions on intact nuclei. Nuclei are treated with low concentrations of DNase I such that active genes are nicked rather than solubilized. The subsequent addition of DNA polymerase I results in the incorporation of nucleotides at these nicked sites. This reaction results in the introduction of label into nucleosomal structures. Annealing reactions with the products of nuclear nick translation reactions and total cellular RNA or DNase I-treated DNA indicate that over 85% of the label is incorporated into sequences transcribed in the oviduct. Three enzymatic processes participate in this reaction. We have demonstrated that the discrimination we observe for active regions of chromatin is imparted by the nicking action of DNase I. These studies may now provide the technology necessary to effect the successful isolation of active genes from chromatin.

Synthesis of poly(adenosine diphosphate ribose) in synchronized Chinese hamster cells

Abstract Chinese hamster ovary cells were synchronized by mitotic selection and used to study the relation of poly(adenosine diphosphate ribose) synthesis to DNA synthesis and the different phases of the cell cycle. DNA synthesis was measured in cells rendered permeable to exogenously supplied nucleotides. Poly(ADPR) synthesis was also measured in permeable cells in the presence of both minimum and maximum DNA damage. The maximum DNA damage was produced by treating the cells with saturating concentrations of DNase. As anticipated, the DNA synthesis complex showed its maximum activity during S phase and showed 4–5-fold less activity during the other phases of the cell cycle. The basal level of poly(ADPR) synthesis was elevated during G1, fell to its lowest level during S phase, then increased during G2 and rose to its highest level during G1. The DNase responsive activity of poly(ADPR) synthesis was relatively constant thru the cell cycle but showed a peak at the end of S phase; then the activity decreased during the subsequent G2-M period.

An improved filter-disc assay for 3H-DNA and serum deoxyribonucleases

1. The filter-paper disc assay for tritium-labeled nucleic acids was modified to reduce washing and manipulation. Evaluation of the procedure indicated greatly enhanced counting efficiency for tritium, complete removal of nuclease digestion products and retention of even nanogram levels of radioactive DNA on the discs in the presence of protein. The modified assay was quite sensitive and had a coefficient of variation of 5.2%. Quite low concentrations of DNase I and endogenous human serum nuclease activity were readily measured by the improved technique, 22 sera yielding a mean value equivalent to about 0.5 ng DNase I/ml serum. We are now using the disc assay to follow serum DNA and DNase levels in patients with autoimmune disorders and various types of cancers.

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.

Histochemical detection of in vitro DNA polymerase activity.

A significant level of nuclear DNA polymerase activity (deoxyribonucleoside-triphosphate: DNA deoxyribonucleotidyltransferase EC 2.7.7.7) can be detected in (a) cryostat sections and (b) toluene treated tissues of onion seed radicles by autoradiography with 3H-TTP. Incorporation was optimal in the presence of all four deoxyribonucleoside triphosphates and dithiothreitol but sensitive to (a) DNase (b) high concentrations of KCl and (c) the sulfhydryl reagents p-chloromercuribenzoate and N-ethyl maleimide. Attempts to activate incorporation with the inclusion of (a) small concentrations of DNase and (b) ATP in the incubating medium failed. The percentage of cells displaying enzyme activity in vitro was similar to the number observed in vivo suggesting that incorporation was associated with S-phase cells and is genuine DNA replication as opposed to repair synthesis.

Bacterial Deoxyribonuclease Production and Its Possible Influence on Mastitis Detection

Various hemolytic streptococci, hemolytic staphylococci, and micrococci were tested for production of deoxyribonuclease (DNase). In general, beta-hemolytic pathogenic streptococci of human origin produced highest concentrations of DNase. However, some beta-hemolytic bovine strains were also highly productive. Alpha-hemolytic strains produced a maximum of 7.6 units/milliliter. None of the staphylococci or micrococci produced detectable DNase. The possibility of correlating DNase production with pathogenicity of the organism is suggested.Addition of DNase to milk abolished the California Mastitis Test (CMT) reaction only after leucocytes had been disrupted by the surface active agent.Possible effects of bacterial DNase production in milk on mastitis screening tests such as the CMT are discussed.

Infection of Bacillus subtilis with phenol-extracted bacteriophages

A bacteriophage capable of infecting transformable strains of Bacillus subtilis was isolated from soil and designated as SP3. Concentrated, partially purified suspensions of this virus were prepared and extracted with water-saturated phenol. The resulting aqueous solutions of a material that had the properties of free deoxyribonucleic acid were shown to induce the production of plaque-forming units when incubated with competent B. subtilis cultures. Phage production was not detected when the bacteriophage extracts were incubated with identical noncompetent bacteria. The infectivity of the phage extracts for B. subtilis was resistant to RNase, trypsin, and to the antiserum prepared against homologous intact SP3 bacteriophages, but was completely destroyed by incubation with low concentrations of DNase.

Studies of extracellular and intracellular bacterial deoxyribonucleic acids.

Summary: Highly polymerized bacterial deoxyribonucleic acids (DNAs) obtained from extracellular and from intracellular locations were analysed. DNAs from two strains (Staphylococcus aureus and S. epidermidis) were of the type having high adenine and thymine contents; two others (Alcaligenes faecalis and Pseudomonas Jluorescens) had a high content of guanine and cytosine. For each micro-organism the composition of extracellular DNA was essentially the same as that of the intracellular DNA. Conditions associated with the extracellular accumulation of DNA in cultures of 3 strains were investigated. S. aureus elaborated a deoxyribonuclease (DNase) which required calcium ion and high pH for activity. In a culture medium of c. pH 6 and insufficient calcium, the DNase was inactive and DNA slime accumulated. In cultures of P. fluorescens a specific ribonuclease-sensitive DNase inhibitor protected slime DNA from depolymerization by culture DNase. In cultures of A. faecalis, also, both a DNA slime and a low concentration of DNase may occur. This enzyme, detected by studying its action against solutions of calf thymus DNA prepared from dried fibres, was activated by various cations within the pH range of cultures. Unlike other DNases, however, it has little capacity to attack the slime-layer DNA, which presumably is more nearly native.

The Effect of a Single Systemic X-Irradiation of the C57BL Mouse on the Nucleodepolymerases of the Thymus

In a study of thymic nucleodepolymerases in radiation-induced thymoma in the C57BL mouse, we noted that early postirradiation changes in activity appeared to be independent of the carcinogenic process. A detailed study of these early changes with low doses of X-irradiation was carried out as described in this paper. Nucleodepolymerase changes in mammalian tissues after whole-body X-irradiation have been reported. After 600 r to rats, there were variable changes in acid and alkaline RNase2 concentrations in whole-liver homogenates and liver mitochondria and depression of a soluble inhibitor tested with pancreatic RNase. These changes were noted 1 to 5 days after treatment (1). A twoto threefold increase in acid DNase concentration of spleen was found 24 hours after doses of 50 to 500 r to rats. The increase was not observed at earlier periods tested, and there was no change in spleen content (2, 3). Finally, increases in urinary (4) and serum (5) acid and alkaline DNases have been observed after 700 r to rats. The response of other enzymes of spleen and thymus to whole-body irradiation has been reviewed by DuBois and Petersen (6). Thymic concentration of various respiratory enzymes showed little or no change. The most marked effect observed was an increase in concentration of adenosinetriphosphatase after relatively small doses of X-irradiation to the rat. The increase was maximal in both spleen and thymus 3 to 5 days postirradiation with doses as low as 50 r in spleen and 100 r in thymus. It was two to three times normal in both tissues after 200 r (6). After 650 r to the mouse, however, splenic content of adenosinetriphosphatase was below normal from about 4 hours to 18 days (7).

Inactivation of deoxyribonuclease by x-rays. II. The kinetics of inactivation in aqueous solution

1. 1. The following three phenomena were observed in relation to the inactivation of concentrations of DNase by X-rays: 1.1. (a) The D 37 dose decreases as the enzyme concentration diminishes and becomes constant in the lower concentration range where 20% of the enzyme (0.5 μg./ml.) was inactivated by 100 r. of x-ray. 1.2. (b) The ionic yield decreases with decreasing enzyme concentration. The “dilution effect,” i.e., a constant ionic yield with varying concentrations of solute, is found in the intermediate concentrations of the enzyme (8.14 × 10 −5 M and 8.14 × 10 −6 M). 1.3. (c) The degree of enzyme inactivation relative to the x-ray dose is an exponential function at concentrations ranging from 8.14 × 10 −9 to 8.14 × 10 −5 M. 2. 2. The three afore-mentioned phenomena can be interpreted satisfactorily on the basis of the kinetics developed on the assumption of a prevailing steady state. 3. 3. The ionic yield at concentrations at which the “dilution effect” occurs is about 0.14 and the G value at the same range of concentrations is approximately 0.42. The kinetics suggest that only one in 9.3 free radicals which react with one enzyme molecule is effective in causing loss of enzymatic activity. 4. 4. The deactivation reaction of free radicals, which is the reaction competing with the enzyme for the destruction of the free radicals, was of the first order rather than the second order. 5. 5. The per cent of the effective free radicals, i.e., 100 times the ratio of free radicals which reacted with the enzyme to total number of free radicals produced, increased with the concentration.