Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated the association of the triiodothyronine (T3) nuclear receptor with rat liver chromatin by the use of selective endonuclease digestion and differential solubilization. The T3 receptor was found in a fraction of chromatin having some of the characteristics of active chromatin: (a) It is highly sensitive to DNase I and micrococcal nuclease digestion; (b) it is enriched in nonhistone proteins and depleted of histone (H-1). Micrococcal nuclease and pancreatic DNase I excised two receptor-containing fragments from chromatin, a minor (12--14 S) form and a major (5.5--6.0 S) form. The latter structure has a Stokes radius of 42 +/- 2 A and an estimated molecular weight of 95400 when a partial specific volume of 0.725 cm3/g for protein is used. It contains DNA but no histones. Similar receptor-containing fragments were excised from chromatin of other rat tissues, including brain, kidney, and heart. Both the 5.5--6 S and the 12--14 S receptor-containing chromatin structures are converted by 0.5 M KCl to the 3.5 S form (R0 35 A molecular weight 50500). Titration with micrococcal nuclease and pancreatic DNase I revealed that the 5.5--6 S form is preferentially excised by endonuclease. Neither receptor occupancy nor thyroidal status had an apparent effect on the susceptibility of chromatin to endonucleolytic digestion nor did they influence the distribution of T3 receptors in chromatin. Our results suggest that T3 receptors are not tightly associated with nucleosomes, the basic subunit of chromatin, but are associated with the DNA-linking nucleosomes in structurally modified regions of chromatin in rat liver nuclei. The T3 receptor-containing fragment may well represent a higher order of structural complexity necessary for T3 action at the cellular level.
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PMID:Association of the thyroid hormone receptor with rat liver chromatin. 627 79

Simian virus 40 nucleoprotein isolated from the nuclei of infected cells contains a nuclease-sensitive site adjacent to the viral origin of replication (between 0.66 and 0.73 map unit). Nuclear extracts were subfractionated by sucrose gradient centrifugation to yield provirions (200S) and simian virus 40 chromatin (80S). The 80S fraction was cleaved either by DNase I or by an endonuclease endogenous to BSC-1 cells with high preference for the 0.66 to 0.73 region. The 200S fraction was treated to release core particles that were sensitive to nuclease cleavage; however, DNase I showed little or no preference for the 0.66 to 0.73 region of the provirion core nucleoprotein.
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PMID:Nuclease-sensitive sites in the two major intracellular simian virus 40 nucleoproteins. 630 35

We have determined the nucleotide sequence of a 4.0-kilobase DNA fragment containing the genes of the PstI restriction-modification system. Two large open reading frames were identified within the sequence and were ascribed to the restriction enzyme and methylase by the analysis of a series of deletion mutants. The two genes are encoded on opposite DNA strands, and hence must be transcribed from separate promoters rather than as a polycistronic message. The sequence of the first 10 amino acids of the restriction endonuclease was determined by sequential Edman degradation of the purified protein, permitting the alignment of the polypeptide with the DNA sequence. The NH2 terminus of the modification enzyme was established by sequential Edman degradation of the protein synthesized in bacterial minicells with different radiolabeled amino acids. The initiation codons of the two genes are separated by 130 base pairs. The deduced amino acid sequences indicate that the restriction endonuclease contains 326 amino acids with a calculated Mr = 37,370; the modification enzyme is composed of 507 amino acids with a calculated Mr = 56,830. There is no significant homology between the two proteins at the level of the primary structure. Antibody raised against the purified restriction endonuclease did not immunoprecipitate the modification enzyme. The transcription initiation sites were mapped using mung bean nuclease. Both of the transcripts begin with adenosine. The initiation sites are separated by only 70 base pairs. This close proximity suggests that the promoters for the two divergent genes overlap. DNase I protection experiments show that Escherichia coli RNA polymerase has a higher affinity for the methylase promoter than for the restriction enzyme promoter.
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PMID:The organization and complete nucleotide sequence of the PstI restriction-modification system. 633 92

Some characteristics of the postirradiation degradation of chromatin in the thymuses of mice were studied. The results proved that the main wave of chromatin degradation becomes evident between 2 and 4 h postirradiation, when considerable amounts of degradation products leach from nuclei during their isolation and are solubilized by lysis of nuclei. Similarly the degradation is manifested in the increase of salt-soluble chromatin fraction as well as of the fractions released from chromatin by various solutions (EDTA, heparin, deoxycholate, alkaline buffer). Later on, within 24 h after irradiation, only little changes in the relative amounts of the degradation products take place. Evidently only a certain thymocyte population is involved. Electrophoretic analyses of DNA fragments from various fractions in native and denatured state demonstrated that chromatin was degraded into nucleosomes and their oligomers by an endonuclease activity. The DNA bears, however, no signs of intranucleosomal regular single-strand fragmentation. This fact makes improbable the participation in this process of DNase I, DNase II and Ca,Mg-dependent endonuclease. No appreciable amount of smaller DNA fragments (products of further degradation of nucleosomes) were found even at 24 h postirradiation interval. Thus the nucleosomes and their oligomers must be considered as the only "long-lived" chromatin fragments in damaged lymphoid cells.
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PMID:On the degradation of chromatin to nucleosomes in the thymocytes of X-irradiated mice. 637 91

Treatment of rats with the hepatocarcinogen, N-2-acetylaminofluorene (AAF) results in development of malignant tumors derived primarily from hepatic parenchymal cells. Following administration of AAF, or its N-hydroxy derivative, in vivo nuclei from parenchymal cell and non-parenchymal cell populations (NI and NII nuclei populations, respectively) were isolated and treated with the endonuclease DNase I. The binding of carcinogen residues to the DNA of nuclease-accessible vs. nuclease-resistant regions of chromatin was evaluated on the basis of the selectivity of DNase I for transcriptionally active DNA. Under the experimental conditions employed DNase I digested approx. 50-60% of the genome of NI nuclei while only 10-20% of the DNA from non-parenchymal cell nuclei (NII) was susceptible to this enzyme. When the DNA of NI and NII nuclei were nick translated following limited digestion with DNase I, a greater degree of transcriptional activity (nuclease accessibility) was found in parenchymal cell nuclei (NI). Following a single injection of rats with [ring-3H]AAF or its N-hydroxy metabolite (N-[ring-3H]-OH-AAF) (1.8 mumol carcinogen/100 g), adducts were preferentially associated with DNA of DNase I resistant regions of target cell nuclei (NI), while preferentially associated with nuclease-accessible regions of non-target cell nuclei (NII). Damage following a single injection persisted for up to 3 days in DNase I-resistant DNA of NI nuclei, carcinogen adducts were rapidly lost from DNase I-accessible DNA of NII nuclei. These studies stress the importance of investigating specificity of carcinogens for particular regions of the genome of cell subpopulations within the target organ.
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PMID:Non-random interaction of N-2-acetylaminofluorene and its N-hydroxy metabolite with DNA of rat hepatic parenchymal and non-parenchymal cell nuclei following in vivo administration of carcinogen. 669 93

In order to investigate the regulation of actin gene transcription during early sea urchin development, a specific hybridization probe for actin sequences is required. Such a probe was produced by cloning cDNA transcribed from a sea urchin poly(A)-containing mRNA preparation enriched for actin message. Double-stranded DNA was ligated into the BamHI restriction site of plasmid pBR322, and the resulting hybrid molecules were used to transform the Escherichia coli strain ML100. After preliminary screening of bacterial colonies by antibiotic sensitivity and hybridization back to the original cDNA, clones containing sea urchin DNA were further characterized by a positive translation assay in which total sea urchin mRNA was hybridized to plasmid, and the hybridized message then was eluted and translated in a reticulocyte cell-free protein-synthesizing system. In this way, one clone (pSA38) was found to hybridize selectively to sea urchin mRNA coding for a protein of 43,000 daltons. This protein was identified as actin by three criteria: electrophoretic migration in two-dimensional polyacrylamide gels, affinity for DNase I, and peptide mapping. Restriction endonuclease and heteroduplex mapping of pSA38 indicate that it contains a 1.5-kilobase-pair insert and is therefore likely to contain a large portion of the actin coding sequence. By using pSA38 as a hybridization probe, it has been found that the level of actin-specific RNA sequences increases dramatically during early sea urchin development.
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PMID:Cloning of sea urchin actin gene sequences for use in studying the regulation of actin gene transcription. 692 77

An endodeoxyribonuclease has been purified to near homogeneity from rat small intestinal mucosa by a procedure involving Con A-Sepharose affinity chromatography. During the initial steps of purification, the presence of 5 mM CaCl2 was essential for stability of the enzyme activity. The enzyme has a molecular weight of 32 000 and an isoelectric point of 4.7. NaCl, sulfhydryl reagents, and iodoacetate strongly inhibited the reaction, but tRNA did not. The enzyme required divalent cations for activity and had a pH optimum of pH 6.2 with Co2+ and pH 7.7 with Mn2+. In both optimum conditions, the enzyme hydrolyzed native DNA more rapidly than denatured DNA, and the average chain lengths of limit digestion products of native and denatured DNA were 8 and 10, respectively, at pH 6.2 and 9 and 11, respectively, at pH 7.7. The enzyme activity to produce acid-soluble fractions from linear DNA substrate was similar in the two optimum conditions, but the activity to nick double-stranded, superhelical circular DNA substrate was significantly higher at pH 6.2 than at pH 7.7. The endonuclease formed single-strand breaks making 5'-phosphoryl and 3'-hydroxyl termini, and deoxythymidine was present at the 5' termini with a frequency of about 50% in both optimum conditions. Bovine pancreatic DNase I antibody and G-action inhibited the enzyme activity. Thus this endonuclease is classified as a DNase I.
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PMID:Purification and properties of a neutral endodeoxyribonuclease from rat small intestinal mucosa. 707 91

All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L1210 leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonuclease micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM [14C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest. 64, 1103 (1979)]. In contrast, the ratio of alkylation (L1210:bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210:bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.
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PMID:Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites. 710 31

Few techniques exist for quantitating DNA fragmentation during apoptosis. Our aim was to develop a quantitative assay for DNA fragmentation in apoptosis by enzymatically labeling DNA with a fluorescent dideoxynucleotide. Terminal deoxynucleotidyl transferase was used to enzymatically label 3'-OH DNA ends with fluorescein-12-dideoxyuridine triphosphate in an assay referred to as fluorophore end-labeling. Because only one labeled dideoxynucleotide can be added per 3'-OH end of DNA, the fluorescence intensity is directly proportional to the number of DNA strand breaks. The sensitivity and validation of this approach were first established in isolated calf thymus DNA treated with the endonuclease, DNase I; and excellent correlation was observed between fluorophore end-labeling and an isotopic approach to quantitate 3'-OH ends of DNA. Quantitation of DNA strand breaks was then obtained in nuclei isolated from hepatocytes undergoing apoptosis using fluorescent digitized microscopy, flow cytometry, and fluorometry. In addition to its quantitative aspects, fluorophore end-labeling proved to be quite sensitive as it detected DNA strand breaks prior to the morphologic changes of apoptosis or the development of the hypodiploid state as assessed by fluorescence microscopy and flow cytometry, respectively. This assay should prove useful for studying the molecular mechanisms leading to DNA cleavage during apoptosis.
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PMID:A fluorometric assay for quantitating DNA strand breaks during apoptosis. 748 77

By using a band mobility shift assay, deoxyinosine 3'-endonuclease, an Escherichia coli enzyme which recognizes deoxyinosine, AP site, urea residue, and base mismatches in DNA, was shown to bind tightly to deoxyinosine-containing oligonucleotide duplexes. Two distinct protein-DNA complexes were observed, the faster migrating complex (complex I, Kd = 4 x 10(-9) M) contained one molecule of deoxyinosine 3'-endonuclease, while the slower migrating complex (complex II, Kd = 4 x 10(-7) M) contained two molecules of the protein bound to every molecule of duplex DNA. The endonucleolytic activity of deoxyinosine 3'-endonuclease paralleled the formation of the complex I. Interestingly, deoxyinosine 3'-endonuclease exhibited similar affinities for both the substrate and the nicked duplex product and thus remained bound to the DNA after the cleavage reaction. The formation of a stable complex required the presence of a duplex structure 5' to the deoxyinosine residue. DNase I footprinting revealed that deoxyinosine 3'-endonuclease protected 4-5 nucleotides 5' to the deoxyinosine, and when complex II was formed, at least 13 nucleotides 3' to deoxyinosine were protected. Based on these results, a model is proposed for the interaction of deoxyinosine 3'-endonuclease with DNA containing deoxyinosine.
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PMID:Interaction of deoxyinosine 3'-endonuclease from Escherichia coli with DNA containing deoxyinosine. 749 77


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