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

---

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

Conditions were established where the thallium-catalyzed iodination of random coil DNA proceeded 100-200 times faster than for native DNA. This reaction was explored as a probe for localized regions of disrupted base pairs in duplex DNA. A heteroduplex was constructed between DNA fragments produced by Hind II + III cleavage of phi80 plac DNA and phi80 plac DNA containing the Ll deletion (73 nucleotides in length). This heteroduplex incorporated twelve times as much iodine as the parent homoduplex fragments. Hence the technique could reveal the presence of a few (two or more) nonpaired cytosines, if they existed within an otherwise helical DNA fragment 789 base pairs long. Iodination studies were performed on superhelical SV40 DNA and on linear lambdaplac DNA. Analysis of the relative amount of iodine in restriction endonuclease fragments of these DNA's revealed the absence of localized single-stranded regions.
...
PMID:Iodination as a probe for small regions of disrupted secondary structure in double-stranded DNAs. 108 8

The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Features of apoptotic cells measured by flow cytometry. 133 43

Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.
...
PMID:Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei. 137 3

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-diisopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.
...
PMID:Sequence- and strand-specific cleavage in oligodeoxyribonucleotides and DNA containing 3'-thiothymidine. 155 Aug 25

The ability of glucocorticoids to induce apoptosis or programmed cell death in mouse thymocytes is well-established. Measurement of apoptosis-associated internucleosomal DNA fragmentation through determination of the percentage of fragmented DNA by electrophoresis or centrifugation of whole cell lysates is by far the most common means of quantifying apoptosis. Since these methods measure DNA fragmentation in whole cell lysates rather than intact cells, they have severe limitations, particularly with heterogeneous cell populations. When mouse thymocytes were incubated with glucocorticoids, fixed, stained with propidium iodide and analysed flow cytometrically for cell cycle distribution, a distinct subpopulation of cells was observed to form below the G0/G1 region, denoted as the A0 region. The presence of cells in this region was consistent with the presence of internucleosomal DNA fragments as determined by gel electrophoresis. Inhibitors of transcription, translation and endonuclease activity, and a glucocorticoid receptor antagonist prevented accumulation of cells in this region. Irradiation of mouse thymocytes also produced a population in the A0 region. Cells in this region are believed to have undergone glucocorticoid-induced DNA fragmentation. This method represents a useful alternative to whole cell lysate assays, since apoptosis can be evaluated on an individual cell basis.
...
PMID:Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry. 165 18

We describe a subtractive hybridization protocol which is designed to permit subtractions between cDNA libraries. The method uses single-stranded phagemids with directional inserts as both the driver and the target. We modified the M13 phagemid vector pBluescript for the directional cDNA cloning and subtractive hybridization. Two simplified methods for efficient construction of directional cDNA libraries are also described. Using a model system, we found that one round of subtractive hybridization results in a 5,000-fold specific subtraction of abundant molecules. We used two methods to quantify the efficiency and verify the specificity of the subtraction. In order to obtain these subtraction efficiencies, it was necessary to develop a method to purify the single-stranded DNA to homogeneity. The single-stranded purification involved using potassium iodide (KI) density centrifugation, restriction endonuclease digestion and phenol extraction in the presence of magnesium. We describe the several advantages of using directional inserts for the subtraction procedure.
...
PMID:Subtractive hybridization system using single-stranded phagemids with directional inserts. 216 39

DNA fragments of defined sequence have been used to determine the sites of cleavage by gamma-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus gamma endonuclease and analyzed on high resolution, denaturing, polyacrylamide gels. Gamma endonuclease was found to cleave irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to gamma radiation.
...
PMID:Sequence specificity of DNA cleavage by Micrococcus luteus gamma endonuclease. 398 67

The synthesis and characterization of an octanucleotide, d(GGsAATTCC), containing the recognition sequence of the EcoRI restriction endonuclease with a phosphorothioate internucleotidic linkage at the cleavage site are described. Two approaches for the synthesis of the RP and SP diastereomers of this octamer by the phosphite method are presented. The first consists of the addition of sulfur instead of H2O to the phosphite at the appropriate position during chain elongation. This method results in a mixture of diastereomers that can be separated by high-performance liquid chromatography after 5'-terminal phosphorylation. The second uses the presynthesized and diastereomerically pure dinucleoside phosphorothioate d[Gp(S)A] for the addition to the growing oligonucleotide chain as a block. The products are characterized by digestion with nuclease P1, fast atom bombardment mass spectrometry, 31P NMR spectroscopy, and conversion to d(GGAATTCC) by desulfurization with iodine. Only the RP diastereomers of d(GGsAATTCC) and its 5'-phosphorylated derivative are cleaved by EcoRI endonuclease. The rate of hydrolysis is slower than that of the unmodified octamer. The phosphorothioate octamer will be useful for the determination of the stereochemical course of the EcoRI-catalyzed reaction.
...
PMID:Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site. 608 94

The state of rabbit (Shope) papilloma virus DNA in virus-induced nonproducing tumors on domestic rabbits was investigated. Virus-specific sequences were resolved into many distinct bands by one-dimensional agarose gel electrophoresis. Two-dimensional gel electrophoresis, CsCl/propidium iodide density equilibrium centrifugation, partial digestion with a restriction endonuclease, and S1 nuclease digestion permitted us to identify the bands as free viral episomes representing circular molecules of increasing size. In some tumors (both papillomas and carcinomas), up to 25% of the virus-specific DNA was linear and comigrated with cellular DNA. Integration of at least some of these sequences was suggested by the detection of viral-cellular junction bands in one tumor after digestion of DNA with EcoRI and Sal I, enzymes that cut Shope DNA once. Finally, the physical states of viral DNA in papillomas and carcinomas were found to be similar, although free episomes were generally larger in carcinomas.
...
PMID:Variable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some. 627 94

The preferred dye binding sites and the microenvironment of known nucleotide sequences within mitochondrial and plasmid pBR322 DNA was probed in a gross fashion with restriction endonucleases. The intercalating dyes, ethidium bromide and propidium iodide, do not inhibit a given restriction endonuclease equally at all of the restriction sites within a DNA molecule. The selective inhibition may be explained, in part, by the potential B to Z conformation transition of DNA flanking the restriction site and by preferred dye binding sites. Propidium iodide was found to be a more potent inhibitor than ethidium bromide and the inhibition is independent of the type of cut made by the enzyme.
...
PMID:Selective inhibition of restriction endonuclease cleavage by DNA intercalators. 631 80


1 2 3 4 Next >>

---