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At least 613 records · Page 34Linked to original sources
PubMed1984

In vitro rabbit mammary gland chromatin replication studied by micrococcal nuclease digestion.

Isolated nuclei from pregnant rabbit mammary glands were labelled with [3H]dTTP under conditions for DNA synthesis and subsequently digested with micrococcal nuclease. Replicating chromatin was found to exhibit increased susceptibility towards the nuclease. Analysis of chromatin digestion products by sucrose density gradient centrifugation demonstrated the association of in vitro replicated DNA with nucleosomes. Furthermore, the distribution of DNA polymerizing activity was studied in isolated nuclease-digested mammary gland chromatin. About 90% of all recovered nuclear DNA polymerizing activity cosedimented with nucleosomal particles, mainly with mononucleosomes. The distribution of DNA polymerases alpha and beta in chromatin isolated from the mammary glands of pregnant and lactating rabbits was compared. In these physiological states the mononucleosome-associated DNA polymerase alpha activity varied in accordance with the rate of DNA synthesis.

Animals↗
PubMed1993

[Liberation of nuclease by cells of Staph aureus].

In supernatants of washed, resting cells of investigated strain, suspended in buffered solutions of adenine, adenosine, xanthosine and uridine, a nucleolytic activity was observed which was increasing during 3 hours of incubation at 37 degrees C. This activity was observed only in presence of determined concentration of these substances. Optimal concentration for liberation of nuclease activity in cell supernatants were: for adenine 0.2-0.4, adenosine 0.2, xanthosine-0.5 and uridine 0.3 mg/ml. Supernatants of cell suspensions were tested for presence of urease, an intracellular enzyme, which appears in the cellular environment only after disintegration of cells. Ureolytic activity in supernatants of cells suspended in solution of the above listed substances was not detected. These data exclude possibility of appearance of nucleolytic activity (nuclease) in supernatants as a result of cell disintegration and they indicate participation of adenine and some nucleosides in liberation of nuclease from cells.

Micrococcal Nuclease↗
PubMed1998

DNA protection from extracapsular nucleases, within chitosan- or poly-L-lysine-coated alginate beads.

DNA was immobilized within alginate matrix using an external or an internal calcium source, and then membrane coated with chitosan or poly-L-lysine. Membrane thickness increased with decreasing polymer molecular weight and increasing degree of deacetylation (chitosan). Beads were exposed to a 31,000 molecular weight nuclease to determine the levels of DNA protection offered by different membrane and matrix combinations. Almost total hydrolysis of DNA was observed in alginate beads following nuclease exposure. Less than 1% of total double-stranded DNA remained unhydrolyzed within chitosan- or poly-L-lysine-coated beads, corresponding with an increase in DNA residuals (i.e. double- and single-stranded DNA, polynucleotides, bases). Chitosan membranes did not offer sufficient DNA protection from DNase diffusion since all of the double-stranded DNA was hydrolyzed after 40 min of exposure. Both chitosan and poly-L-lysine membranes reduced the permeability of alginate beads, shown by enhanced retention of DNA residuals after DNase exposure. The highest level of DNA protection within freshly prepared beads was obtained with high molecular weight (197,100) poly-L-lysine membranes coated on beads formed using an external calcium source, where over 80% of the double-stranded DNA remained after 40 min of DNase exposure. Lyophilization and rehydration of DNA beads also reduced permeability to nucleases, resulted in DS-DNA recoveries of 60% for chitosan-coated, 90% for poly-L-lysine-coated, and 95% for uncoated alginate beads.

Alginates↗
PubMed1987

Structural transition in inactive Balbiani ring chromatin of Chironomus during micrococcus nuclease digestion.

We have analysed by micrococcus nuclease digestion the chromatin structure of genes in the Balbiani ring (BR) regions of a Chironomus cell line. Gel electrophoresis of the DNA fragments reveals a repeating structure which consists of two repeat sizes, a long repeat seen in the large fragments and a small repeat seen in the small fragments. The two repeats hardly overlap, except in a narrow transition zone which is at a different fragment size in the BR 2.2 and the BR 2.1 gene. The sizes of the large repeats fit the repeat of the underlying DNA sequence. The short repeats are between 170 and 180 bp, and after H1 depletion the short repeat in the BR 2.2 gene is 160 bp. Our most favoured interpretation of these data is that in intact chromatin the nucleosomes in the BR genes are phased with respect to the repeating DNA sequence, whereas micrococcus nuclease digestion leads to loss of a nucleosome-positioning constraint and hence to rearrangement of the nucleosomes. Our results imply a possible artefact of nuclease digestion of chromatin, which has to be taken into account in mapping nucleosome positions.

Journal Article↗
PubMed1990

The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease.

The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins.

Amino Acid Sequence↗
PubMed1994

Sequences required for enhancer blocking activity of scs are located within two nuclease-hypersensitive regions.

The Drosophila 87A7 heat shock locus is bordered, on the proximal and distal sides, by two special chromatin structures, scs and scs'. Each structure is characterized by two sets of nuclease-hypersensitive sites, located within moderately G/C-rich DNA, flanking an A/T-rich nuclease-resistant region. scs and scs' have been shown to insulate a white reporter gene from position effects and to prevent enhancer-promoter interactions. These and other properties suggest scs and scs' might function as chromatin domain boundaries. To identify the DNA sequences which are essential for the insulating activity of scs we used an enhancer blocking assay based on the white gene. Sequences capable of suppressing activation of white by its upstream enhancer elements reside within a 900 bp DNA fragment corresponding to the scs chromatin structure. Within this region, DNA fragments associated with the two nuclease-hypersensitive regions are essential for full enhancer blocking activity, while the central A/T-rich region is dispensable. Deletions which remove part of the hypersensitive regions result in intermediate levels of white activity. Insulating activity can, however, be reconstituted by multimerizing DNA fragments from either hypersensitive region. Our results suggest that the scs boundary is assembled from a discrete number of functionally redundant DNA sequences located within both hypersensitive regions and that boundaries act by decreasing the frequency of enhancer-promoter interactions. We also show that certain types of position effects, like those involved in dosage compensation, are not efficiently blocked by scs.

ATP-Binding Cassette Transporters↗
PubMed1993

Spermidine-induced alteration in the gene-spacer discrimination of nucleases in protonated DNA.

The sequence preference of a Drosophila lysosomal DNase was studied on the Drosophila hsp 70 heat-shock and histone recombinants, which carry six different genes, and the surrounding spacer sequences. The distribution of cleavage sites was random in respect of the locations of gene and spacer sequences. However, in the presence of 10 mM spermidine, a major transition was observed: the coding sequences became more susceptible than the spacer regions to nuclease attack. A similar transition was induced in the sequence preference of DNase I if the digestion was performed in the presence of spermidine at pH 5.2. At pH 7.5, spermidine does not influence the sequence preference of DNase I, which indicates the involvement of DNA protonation in this transition. In the presence of spermidine, the distributions of preferred and protected sequences were almost indistinguishable for these nucleases, suggesting that the protonated DNA, and not the enzymes, is the target of spermidine. A Drosophila embryonal protein was detected and partially purified which induced the same transition as observed in the presence of spermidine. The purified protein preferentially protected the spacer DNA sequences against acid DNase or DNase I cleavage in the hsp 70 heat-shock and histone gene recombinants. The protection was concentration dependent and occurred only at pH 5.2. The transition of nuclease specificity is probably due to a conformational change in the protonated DNA, induced by the binding of either the embryonal protein or spermidine.

Animals↗
PubMed1995

Molecular states of fungal nuclease composed of heterogeneous subunits as estimated from the effects of urea and chelating agents.

Fungal nuclease composed of subunits with molecular weights of 8.0 x 10(4) (80K subunit), 5.0 x 10(4) (50K subunit), and 2.5 x 10(4) (25K subunit) (K. Ito, Y. Matsuura, and N. Minamiura (1994) Arch. Biochem. Biophys. 309, 160-167) was inactivated by urea and dissociated into its subunits. The urea inactivation depended on the concentration of urea, the incubation period and the temperature. The urea-inactivated enzyme had about 25% activity restored by removal of urea, and the native form of the enzyme was also reconstituted. The urea inactivation and the dissociation of subunits were almost completely prevented by Ca2+ but not by glycerol. The enzyme was also inactivated by ethylenediaminetetraacetic acid (EDTA). From this method of inactivation, the 50K and 25K subunits were still associated, but the complex showed no nuclease activity. About 80% of the activity of the EDTA-inactivated enzyme was restored by the addition of Ca2+ or Sr2+ and 20-40% by Mn2+, Ba2+, Mg2+, or Co2+. The reactivation of the enzyme by these metal ions was accompanied by the reconstitution of the native form of the enzyme. The enzyme was inactivated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) at much higher concentrations compared with the inactivation by EDTA. On the other hand, dissociation of the subunits by EGTA proceeded in a manner similar to that of the inactivation of the enzyme by EDTA. The 50K and 25K subunits were still associated, and the complex showed nuclease activity. These results indicated that the enzyme contains two kinds of metal ions. One metal ion, represented by Ca2+, is thought to stabilize the quanternary structure of the enzyme, especially to connect the 80K subunit and the complex of the 50K and 25K subunits. Another metal ion, represented by Mg2+, is thought to be bound to the complex of the 50K and 25K subunits and to be required for activity appearance of the complex. Along with these results, possible molecular states of the enzyme under various conditions are proposed.

Aspergillus↗
PubMed2002

Self-quenched covalent fluorescent dye-nucleic acid conjugates as polymeric substrates for enzymatic nuclease assays.

A fluorescent method is described for assessing nuclease activity. The technique is based on the preparation of quenched fluorophore-nucleic acid covalent conjugates and their subsequent dequenching due to degradation by nucleases. The resulting fluorescence increase can be measured by a spectrofluorometer and exhibits subpicogram per milliliter sensitivity level for RNase A and low picogram per milliliter level for DNase I. The method is adaptable for quantitative nuclease inhibitor testing.

Binding, Competitive↗
PubMed1997

Purification and characterization of the precursor tRNA 3'-end processing nuclease from Aspergillus nidulans.

The precursor-tRNA 3'-end processing nuclease activity was purified homogeneously about 15,300 fold from the heat-treated fraction. The precursor-tRNA 3'-end processing nuclease was a single polypeptide of 160,000 Da. This nuclease generates a mature 3'-end of nuclear tRNA(Asp) of Aspergillus nidulans by the endonuclease activity and prefers the 5'-end processed tRNA(Asp) rather than primary precursor-tRNA(Asp) as a substrate. However, this enzyme did not process both primary mitochondrial precursor-tRNA(His) and 5'-end processed mitochondrial precursor-tRNA(His) of A. nidulans.

Aspergillus nidulans↗
PubMed1998

Identification of the nuclear factor HMG2 as an activator for DFF nuclease activity.

DNA fragmentation during apoptosis is mediated by a heterodimeric protein complex, DFF45/ICAD and DFF40/CAD/CPAN. Purified DFF40 alone possesses intrinsic nuclease activity that is inhibited by its association with DFF45. The proteolytic cleavage of DFF45 by caspase-3 frees the DFF40 subunit to function as a nuclease. In the course of identifying factors that stimulate DFF activity, we have isolated a nuclear factor and identified it as the high mobility group protein 2 (HMG2). We found that bacterially expressed HMG2 is able to enhance the nuclease activity of DFF. As HMG2 has DNA bending activity (6), our data suggest that HMG proteins may augment DNA fragmentation during apoptosis through changes in chromosome structure.

Amino Acid Sequence↗
PubMed1993

Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks chi-specific, but not non-specific, nuclease activity.

RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme, designated recB2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain. The recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced approximately 2 to 3-fold relative to the wild-type enzyme. The ATP-dependent dsDNA exonuclease activity of recB2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme. Despite its ability to degrade linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking activity at chi sites, which are hotspots for genetic recombination. Since this interaction with chi significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.

Adenosine Triphosphate↗
PubMed1998

A novel class of supercoil-independent nuclease hypersensitive site is comprised of alternative DNA structures that flank eukaryotic genes.

The cell makes a fundamental distinction between genes and non-gene sequences, which mechanistically underlies the process of gene regulation. Here, we describe the properties of a novel class of genetic sites that reproducibly flank and delineate the coding regions of the eukaryotic genes tested. Defined in vitro reaction conditions that include altered solvation and elevated temperature rendered the sites hypersensitive to nuclease cleavage. Consequently, the complete coding regions of the Drosophila genes tested were quantitatively excised from genomic DNA or genomic clones by this treatment. Identical reaction products were generated from linear or supercoiled DNA substrates. Chemical modification and fine-structure analysis of several cleavage sites flanking Drosophila genes showed that the cleavage sites were stable nucleic acid structures that contained specific arrangements of paired and unpaired nucleotides. The locations and properties of the cleavage sites did not correspond to previously known nuclease hypersensitive sites nor to known alternative DNA structures. Thus, they appear to represent a new class of genetic site. In a deletion analysis, the minimal sequence information necessary to direct in vitro nuclease cleavage 3' to the Drosophila GART gene co-localized with the signal required for termination of transcription in vivo. The data suggest that a novel class of DNA site with distinct structural properties encodes biological information by marking the boundaries of at least some gene expression units in organisms as diverse as Plasmodium and Drosophila.

Animals↗
PubMed2000

The pressure-temperature free energy-landscape of staphylococcal nuclease monitored by (1)H NMR.

The thermodynamic stability of staphylococcal nuclease was studied against the variation of both temperature and pressure by utilizing (1)H NMR spectroscopy at 750 MHz in 20 mM Mes buffer containing 99.9 % (2)H(2)O, pH 5.3. Equilibrium fractions of folded and unfolded protein species were evaluated with the proton signals of two histidine residues as monitor in the pressure range of 30-3300 bar and in the temperature range of 1.5 degrees C-35 degrees C. From the multi-parameter fit of the experimental data to the Gibbs energy equation expressed as a simultaneous function of pressure and temperature, we determined the compressibility change (Deltabeta), the volume change at 1 bar (DeltaV degrees ) and the expansivity change (Deltaalpha) upon unfolding among other thermodynamic parameters: Deltabeta=0.02(+/-0.003) ml mol(-1) bar(-1); Deltaalpha=1.33(+/-0.2) ml mol(-1) K(-1); DeltaV degrees =-41.9(+/-6. 3) ml mol(-1) (at 24 degrees C); DeltaG degrees =13.18(+/-2) kJ mol(-1) (at 24 degrees C); DeltaC(p)=13.12(+/-2) kJ mol(-1) K(-1); DeltaS degrees =0.32(+/-0.05) kJ mol(-1) K(-1 )(at 24 degrees C). The result yields a three-dimensional free energy surface, i.e. the free energy-landscape of staphylococcal nuclease on the P-T plane. The significantly positive Deltabeta and Deltaalpha values suggest that, in the pressure-denatured state, staphylococcal nuclease forms a loosely packed and fluctuating structure. The slight but statistically significant difference between the unfolding transitions of the His8 and His124 environments is considered to reflect local fluctuations in the native state, leading to pre-melting of the His124 environment prior to the cooperative unfolding of the major part of the protein.

Calorimetry↗
PubMed1997

The potential of 5' nuclease PCR for detecting a single-base polymorphism in Orthopoxvirus.

A fluorogenic 5' nuclease PCR assay was evaluated for its ability to specifically detect and differentiate DNA of two Orthopoxvirus species. A pair of consensus primers that target a DNA segment of the Orthopoxvirus haemagglutinin gene, and two oligonucleotide probes; each labelled with a different fluorescent reporter dye and the same quencher dye, were used in a single-tube assay. The assay is based on the 5'-->3' nuclease activity of AmpliTaq DNA polymerase that cleaves a fluorescein-labelled hybridized probe. Probe cleavage generates specific fluorescent signals whose intensity can be quantified by fluorometry. After evaluating the effects of various annealing temperatures and probe concentrations and normalizing the emission intensities of the reporter dyes, it was possible to detect and differentiate monkeypox and vaccinia virus DNAs on the basis of a single-base polymorphism. The sensitivity of the 5' nuclease PCR assay is comparable to the sensitivity of ethidium bromide-stained gels, but the assay provides higher specificity and virtually eliminates the need for laborious post-PCR processing.

DNA Primers↗
PubMed2001

Automated 5' nuclease assay for detection of virulence factors in porcine Escherichia coli.

This paper describes the development of a 5' nuclease assay for detection of virulence factor genes responsible for colonization factors and toxins in Escherichia coli isolated from pigs. Colonization factors were F4, F5, F6, F18, F41 and the outer membrane protein intimin. Toxins were heat stable (STa, STb, EAST1) and heat labile (LT) enterotoxins and the verocytotoxin variant 2e (VT2e). To correctly identify false negative results, an endogenous internal control targeting the E. coli 16 S rRNA gene was incorporated in each test tube. The assay was evaluated using a collection of E. coli reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coli field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains. When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory.

5'-Nucleotidase↗
PubMed1978

Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases.

The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and non-denaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosomic DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50-75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multi-nucleosomic chromatin segments containing a full complement of histones.

Animals↗
PubMed1979

Studies in heterochromatin DNA: accessibility of late replicating heterochromatin DNA in chromatin to micrococcal nuclease digestion.

Heterochromatin DNA in cactus mouse (Peromyscus eremicus) replicates in the late S phase of cell cycle. A method of obtaining cells which contain DNA preferentially labeled at heterochromatic areas by a pulse-labeling of late replicating DNA is described. When the nuclei of P. eremicus cells containing radioactively labeled DNA in heterochromatin were digested with micrococcal nuclease and the resultant nucleosomal DNA was separated by gel electrophoresis, it was found that the repeat length of nucleosomal DNA in the heterochromatin DNA is not different from that of the bulk of the genomic DNA. Furthermore, there was no significant difference in the accessibility to digestion by micrococcal nuclease between the late replicating heterochromatin DNA and the total DNA under our digestion conditions. Two dimensional gel electrophoresis patterns of nucleosomal DNAs isolated from micrococcal nuclease digested nuclei from P. eremicus, P. collatus, and P. crinitus cells in culture were very similar. Cytogenetic data showed that these three species are different in heterochromatin but similar in euchromatin.

Animals↗