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At least 523 records · Page 29Linked to original sources
PubMed1988

Persistence of a micrococcal nuclease sensitive region spanning the promoter-coding region junction of a cell cycle regulated human H4 histone gene throughout the cell cycle.

We have examined the chromatin structure of the cell cycle regulated human H4 histone gene FO108A at various times during the cell cycle, by treating nuclei isolated from synchronized HeLa S3 cells with micrococcal nuclease. Purified DNA was fractionated electrophoretically, transferred to nitrocellulose, and hybridized to small (150-250 nucleotides) radiolabeled probes from various portions of the promoter and coding regions of the gene. Our results indicate the existence of a micrococcal nuclease sensitive region located between positions -60 and +90 base pairs (bp) from the start codon of the gene, which includes the TATA box. This nuclease-sensitive region persists at all the cell cycle times analyzed. Hybridization with a 250-bp probe containing only coding region sequences reveals a disrupted nucleosomal ladder during early S phase, when this H4 histone gene replicates and exhibits an enhanced level of transcription. By mid-S phase, the regular nucleosomal structure of the coding region is restored and persists during subsequent phases of the cell cycle. The disruption of a normal nucleosomal organization in the promoter and mRNA coding regions of this H4 histone gene is also supported by the sensitivity of these sequences to S1 nuclease.

Chromatin↗
PubMed1995

The gradual reduction of viroid levels in hop mericlones following heat therapy: a possible role for a nuclease degrading dsRNA.

A high concentration of hop latent viroid (HLVd) was detected in infected mericlones of Osvald's hops grown in vitro. This concentration was about 8-fold higher than in leaves of young, field-grown plants, reaching about 30 pg/mg of fresh mass. Treatment of these in vitro-grown plants at high temperature (35 degrees C) for two weeks lead to a dramatic (about 70-90%) decrease of HLVd content. More detailed investigations performed with mericlone 6147 of Osvald 31 showed that HLVd levels decrease gradually during subsequent cycles of heat treatment. A nuclease activity capable of cleaving HLVd and fully double-stranded RNA was shown to increase significantly in hop tissues during thermotherapy cycles, or after the heat shock. The nuclease activity was found to have similar properties to those extracted earlier from tobacco anthers. This enzyme resembles a sugar-unspecific nuclease which has a maximum activity at pH 5.5. Analysis of the activity with viroid and dsRNA showed that both, endo- and exonucleolytic activities were attributable to the enzyme. A strong tissue-specific gradient of viroid (the lowest level in stem apex and the highest level in roots) was observed in young plants, showing a negative correlation with the dsRNAse activity. In senescent plants, the highest viroid concentration was observed in maturated cones and in upper stems. High nuclease activity in the upper stem tissue suggests that viroid RNA must be protected in this tissue against degradation.

Chromatography, Gel↗
PubMed1983

Extraction of androgen-receptor complexes from regions of rat ventral prostate nuclei sensitive or resistant to nucleases.

Rat ventral prostate nuclei contain androgen-binding sites which are susceptible or resistant to excision by endonucleolytic action. Those which were susceptible were associated both with oligonucleosomal and subnucleosomal particles. The sedimentation profile characteristic of a nuclear androgen-receptor complex could be obtained by exhaustive nucleolytic digestion or by treatment of fractions with KCl (0.6 mol/l). Androgen-binding sites resistant to DNAase I were also resistant to KCl, whereas those sites resistant to micrococcal nuclease were partially extractable with KCl. Nuclease-resistant sites could be extracted with heparin (10 mg/ml). Androgen-receptor complexes obtained from nuclease-sensitive or nuclease-resistant regions by extraction with KCl or heparin were indistinguishable by routine sedimentation analysis.

Animals↗
PubMed1996

cDNA cloning of rat major AP endonuclease (APEX nuclease) and analyses of its mRNA expression in rat tissues.

APEX nuclease is a mammalian DNA repair enzyme having apurinic/apyrimidinic (AP) endonuclease, 3'-5'-exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. It is also a redox factor (Ref-1), stimulating DNA binding activity of AP-1 binding proteins such as Fos and Jun. In the present paper, a cDNA for the enzyme was isolated from a rat brain cDNA library using mouse Apex cDNA as a probe and sequenced. The rat Apex cDNA was 1221 nucleotides (nt) long, with a 951-nt coding region. The amino acid sequence of rat APEX nuclease has 98.4% identity with mouse APEX nuclease. Using the rat Apex cDNA as a probe for Northern blot analysis, the size of rat Apex mRNA was shown to be approximately 1.5 kb. Its expression was compared in 9 rat organs on postnatal days 7 and 28. Although Apex mRNA was expressed ubiquitously, the levels varied significantly, suggesting organ- or tissue-specific expression of the Apex gene. The highest level was observed in the testis, relatively high levels in the thymus, spleen, kidney and brain, and the lowest level in the liver. The level of expression at postnatal day 28, with the exception of the testis, was almost the same as or lower in respective organs than that at postnatal day 7. Postnatal developmental changes of Apex mRNA expression in the testis and thymus were further studied. The expression in testis was markedly increased on postnatal days 21 and 28. The expression in thymus increased once at postnatal day 14, and then decreased. The developmental changes of Apex mRNA expression in testis and thymus suggest that APEX nuclease is involved in processes such as recombinational events.

Amino Acid Sequence↗
PubMed1993

Immobilized nucleases.

Nucleases are important analytical enzymes and are used widely for the determination of nucleic acid structure. Their application depends on the specificity and mode of action of the particular enzyme. Nucleases have also found application in the production of flavor enhancers like 5' IMP and 5' GMP, removal of nucleic acids in single cell protein preparations, and as therapeutic agents. Immobilization of nucleases and the use of immobilized nucleases for various biotechnological applications have been reviewed.

Biotechnology↗
PubMed2004

Evaluation of a 5'-nuclease (TaqMan) assay with the thin agar layer oxyrase method for the detection of Yersinia enterocolitica in ground pork samples.

A 5'-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica. Sensitivity studies of a 5'-nuclease assay for detecting Y. enterocolitica 0:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5'-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of "Oxyrase for Agar" onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (-15 degrees C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN (P < 0.05). The 5'-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.

Animals↗
PubMed2001

Excision repair of adozelesin-N3 adenine adduct by 3-methyladenine-DNA glycosylases and UvrABC nuclease.

Adozelesin is a synthetic analog of the antitumor antibiotic CC-1065, which alkylates the N3 of adenine in the minor groove in a sequence-selective manner. Since the cytotoxic potency of a DNA alkylating agent can be modulated by DNA excision repair system, we investigated whether nucleotide excision repair (NER) and base excision repair (BER) enzymes are able to excise the bulky DNA adduct induced by adozelesin. The UvrABC nuclease and 3-methyladenine-DNA glycosylase, that exhibit a broad spectrum of substrate specificity, were selected as typical NER and BER enzymes, respectively. The adozelesin-DNA adduct was first formed in the radiolabeled restriction DNA fragment and its excision by purified repair enzymes was monitored on a DNA sequencing gel. The treatment of the DNA adduct with a purified UvrABC nuclease and sequencing gel analysis of cleaved DNA showed that UvrABC nuclease was able to incise the adozelesin adduct. The incision site corresponded to the general nuclease incision site. Excision of this adduct by 3-methyladenine-DNA glycosylases was determined following the treatment of the DNA adduct with a homogeneous recombinant bacterial, rat and human 3-methyladenine-DNA glycosylases. Abasic sites generated by DNA glycosyalses were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase (Fpg). Resolution of cleaved DNA on a sequencing gel showed that the DNA glycosylase from different sources could not release the N3-adenine adducts. A cytotoxicity assay using E. coli repair mutant strains showed that E. coli mutant strains defective in the uvrA gene were more sensitive to cell killing by adozelesin than E. coli mutant strain defective in the alkA gene or the wild type. These results suggest that the NER pathway seems to be the major excision repair system in protecting cells from the cytotoxicity of adozelesin.

Alkylation↗
PubMed1991

Two isoforms of Serratia marcescens nuclease. Crystallization and preliminary X-ray investigation of the enzyme.

Two isoforms of an extracellular endonuclease, nucleases Sm1 and Sm2, were purified from culture fluid of Serratia marcescens strain BIO MI by ligand-exchange chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI-values for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The nuclease Sm1 has been crystallized from ammonium sulfate solution by the vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell constants a = 69.0, b = 106.7, c = 74.8 A, contain two molecules in an asymmetric unit, packing density Vm = 2.3 A/Da, and diffract to at least 1.5 A resolution. The Pt- and UO2-derivatives of the protein were obtained. Preliminary X-ray investigation of nuclease Sm2 crystals was carried out.

Amino Acid Sequence↗
PubMed2006

Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects.

Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic ribonuclease (RNase A), is known to display special biological activities namely cytotoxicity for human tumor cells. Because some plant ribonucleases have a similar mass weight and structure as the animal ribonuclease, effects of a commercial product of Mung bean (Phaseolus aureus) nuclease (PhA) were studied on proliferation of ML-2 human tumor cells, as well as it's aspermatogenic, embryotoxic, immunogenic, and immunosuppressive activity, and therapeutic efficiency in athymic mice bearing human melanoma tumor. Concerning the antiproliferative activity, PhA nuclease was almost non-effective in vitro on ML-2 cells and also immunosuppressive activity on human lymphocyte in mixed culture was very low compared to that of BS RNase. However, significant antitumor activity was detected on human melanoma tumor after intratumoral or intraperitoneal administration into the mice. Furthermore conjugate of PhA nuclease with polyethylene glycol (PEG) injected seven times at the dose of 10 microg intraperitoneally showed identical antitumor activity as that of bovine seminal ribonuclease (BS RNase) injected by the same way at ten times higher dose. Both PhA and BS RNases exerted strong aspermatogenic effect on the width of spermatogenic layers while RNase A administration at ten times higher concentration was ineffective. PhA nuclease when compared by means of antibody cross reaction with RNase A, BS RNase and wheat leaf neutral RNase (WLN-RNase) was found to be immunologically similar to RNase A and WLN-RNase, meanwhile BS RNase showed much higher antigenicity in comparison with them.

Animals↗
PubMedDate not supplied

[Comparison of the conformation of RNA from phage MS2 and 16S rRNA. Accessibility to nucleases S1 and SV specific to secondary structure and thermal stability].

The hydrolysis of E. coli 16S rRNA by nucleases specific to the secondary structure elements (S1 and SV), the melting of the RNA after partial hydrolysis by nuclease S1 and the electrophoretic mobility of hydrolysis products after denaturation-renaturation of RNA were studied. It was shown that the sensitivity of 16S rRNA to nuclease S1 depends on Zn or Mg ions concentration. The melting curves after partial hydrolysis by nuclease S1 were characterized by a decrease of the hyperchromic effect (by approximately 15%) and by a increase of Tm (by 3 degrees). After RNA denaturation followed by slow or fast renaturation the electrophoretic patterns of the hydrolysis products were not changed, as in the case of phage MS2 RNA. It was supposed, that the rRNA molecule has a stable "nucleus" (or "nuclei"), which is organized as an intramolecular association of parallelly oriented double-stranded fragments of this RNA. Previously, such a mode of the spatial organization was proposed by us for phage MS2 RNA.

Coliphages↗
PubMed1986

Mung bean nuclease cleaves preferentially at the boundaries of variant surface glycoprotein gene transpositions in trypanosome DNA.

Telomere-linked genes coding for the variant surface glycoproteins (VSGs) of African trypanosomes have been difficult to clone because their flanking regions frequently lack restriction sites. Therefore, we constructed a genomic DNA library of fragments generated by digestion of purified trypanosome DNA with mung bean nuclease, an enzyme that cleaves before and after genes in Plasmodium falciparum DNA (McCutchan, T. F., Hansen, J. L., Dame, J. B., and Mullins, J. A. (1984) Science 225, 625-628). Southern hybridizations with several gene probes showed that under the appropriate conditions mung bean nuclease produces discrete trypanosome DNA fragments that are as clearly resolved on an agarose gel as restriction fragments. The majority of VSG genes are on fragments of about 1.7 kilobase pairs. To examine the sites of mung bean nuclease cleavage, the insert boundary sequences of eight recombinant clones in the library containing VSG genes were determined. In general, mung bean nuclease cleaved 300-800 base pairs in front of the VSG start codon and within 50 base pairs on either side of the termination codon. These regions also form the boundaries of VSG gene conversion events indicating that the enzyme recognizes, in part, a conformational structure rather than a specific sequence. The analyzed clones included both telomere-linked and interior basic copy VSG genes indicating that the library potentially contains all of the telomere-linked VSG genes in the genome.

Animals↗
PubMed1977

Effect of neurectomy on nuclease activity in skeletal muscles of rats.

Author followed up the activity of the three enzymes involved in the catabolism of nucleic acids--acid deoxyribonulease (DNase II), alkaline ribonuclease (RNase I), and acid ribonuclease (RNase II)--in the denervated gastrocnemius and soleus muscles of rats for 28 postoperative days. The activity of both acid nucleases increased in both types of denervated muscles, compared with the respective controls. Up to the 14th postoperative day, the activity excess of both acid nucleases was more significant in the m. gastrocnemius than in the m. soleus. The RNase I ran below the control activity during the whole period in the m. soleus and up to the 14th day in the m. gastrocnemius. The role of nucleases and nuclease inhibitors in the changes of nucleic acid catabolism in neurogenic muscular atrophies is discussed.

Animals↗
PubMedDate not supplied

[Nuclease activity of blood serum from healthy persons and from patients with some hematologic impairments].

Nuclease activity was studied using as a substrate native RNA as well as synthetic polyribonucleotides polyU and polyU-polyA in blood serum of healthy persons and of patients with leukoses and myelofibrosis. In the hematologic impairments no distinct correlation was observed between the nuclease activity with either native RNA or the polyribonucleotides as substrates. The RNAase activity was markedly increased only under conditions of chronic myeloleukosis, whereas a significant increase in the nuclease activity using polyU was found in 42.8% of the patients with acute leukosis, in 61.5% of the patients with chronic myeloleukosis, in 68,7% of the patients with chronic lympholeukosis and in 77.8% of the patients with myelofibrosis; the elevation in the nuclease activity with polyU-polyA as a substrate observed in 28,5%, 15,4%, 12,5% and 33.3% of the patients, respectively.

Humans↗
PubMedDate not supplied

[Characterization of the nuclease-sensitive chromatin regions from N-nitrosodiethylamine-induced hepatoma cells].

Protein-DNA complexes released from chromatin of the intact rat liver and hepatoma induced by N-diethylnitrosamine during nuclease digestion have been subdivided into two groups of high and low molecular weight. Immunoreactive proteins are concentrated mainly in high molecular complexes. Immunoblotting of proteins from the nuclease-sensitive regions of normal and tumor chromatins allow distinguishing antigens of three main types: 1--antigens of nuclease-sensitive samples common both for normal and tumor cells; 2--antigens determined in tumor preparations only; 3--antigens usual for normal samples. Such differences in composition of immunogenic proteins in the nuclease-sensitive regions from the cells of the normal and malignant rat liver may be useful for investigating changes in active chromatin during chemical induction of tumors in animals.

Animals↗
PubMed1993

Uraemic lymphocytes: analysis of their nuclease activity.

The nuclease activity in T and B lymphocytes isolated from patients with chronic renal failure and control subjects was studied. The data obtained show a slight increase in nuclease activity in T and B cells isolated from uraemic patients as compared to the control cells. The increase in enzyme activity was limited to the group of relatively small nucleases with molecular weights ranging from 14 kDa to 18 kDa. It was documented previously that these nucleases are among the cleavage products of the largest subunit of DNA-dependent RNA polymerase I, which is responsible for ribosomal RNA synthesis. Thus, we suggest that the characteristic feature of lymphocytes isolated from uraemic patients is lowering of their metabolic activity. Amongst toxins increasing catabolism of the active enzyme, a significant correlation with the rise of lymphocyte Mg2+ and Mn2+ ions was disclosed.

Adult↗
PubMed1977

DNase-sensitive sites in nucleosomes. Their relative suspectibilities depend on nuclease used.

We have used three endonucleases having different catalytic and physicochemical properties to digest HeLa nucleosomes (chromatin core particles) which had been labeled with 32P at their 5'-DNA termini. Each endonuclease nicks nucleosome DNA at the identical sites, supporting the idea that the conformation of the DNA within a nucleosome is the major factor influencing its nuclease susceptibility and indicating that nucleases can indeed yield important information as to nucleoprotein structure. On the other hand, the relaative susceptibility of a given site can differ for each nuclease, indicating that enzyme-substrate interactions unique for each enzyme influence the course of the reaction; this limits the structural information which can be obtained by using a single nuclease to study nucleoprotein structure.

Aspergillus↗
PubMed1995

Structural specificity of nuclease from wheat chloroplasts stroma.

A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models. In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop. In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop. No primary cleavages were observed within the double-stranded stems. Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs.

Base Sequence↗
PubMed1997

Purification and some properties of a novel dsRNA degrading nuclease bound to rye germ ribosomes.

A two-step procedure including affinity chromatography for purification of rye germ ribosomal nuclease that degrades double-stranded RNA from a virus of Penicillium chrysogenum and the poly(I).poly(C) complex was developed. The specific activity towards poly(I).poly(C) of the obtained nuclease preparations was 30 times as high as that of ribosomes. The recovery of activity was 3.4% when the Octyl-Sepharose column was used, and 2.0% in the case of the Phenyl-Sepharose column. On polyacrylamide/SDS gel electrophoresis the nuclease was resolved into two proteins of molecular mass 62 kDa and 57 kDa, respectively. 2-Mercaptoehanol and Mn2+ stimulated the activity of the purified enzyme. Glycerol (20%-50% concentration) stabilized enzyme. In addition to activity towards dsRNA and ssRNA the enzyme cleaves native and denatured DNA. It is suggested that this type of a nuclease takes part in regulation of the mRNA level in cytoplasm.

Penicillium chrysogenum↗