Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The endonuclease DNase II preferentially attacks a limited and tissue-specific portion of chromosomal DNA. This material may be separated from the bulk of chromatin DNA by virtue of its solubility in 2 mM MgCl2. The Mg2+ soluble fraction forms a specific subset of DNA sequences and is enriched four to sevenfold in sequences coding for cytoplasmic poly(A)-containing RNA and globin messenger RNA (in globin-producing cells). The bulk (70--90%) of rapidly labelled RNA is found associated with the Mg2+-soluble fraction. Transcriptionally active, Mc2+-soluble chromatin is organized into repeating subunits of DNA (200 +/- 5 base pairs) and histone. Mc2+-soluble active subunits differ from the subunits or nucleosomes of non-transcribed regions in many respects: namely, chemical composition (non-histone protein and RNA), sedimentation properties, differential sensitivity to DNase I and the single-strand-specific nuclease S1, and optical melting behaviour. These results suggest that chromatin subunits adopt a new configuration during the process of transcription.
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PMID:Organization of transcribed regions of chromatin. 2 80

Non-membranous HeLa cell nuclear ghosts, representing non-membranous nuclear envelope or 'skeletal' components, have been examined in whole-mount fashion by transmission electron microscopy. Major components of the ghosts include annuli with inner and outer diameters of 43 and 90 nm, respectively, which are consistent in dimensions with nuclear pore complexes. Also present are rod-like images (260 nm in length and 50 nm in width or diameter) representing either previously unobserved nuclear structures, or condensations of repeating functional units not otherwise observable. The annular and rod-like images were also observed when various steps in the ghost isolation procedure, such as the use of detergents, 0.5 M MgCl2 and polylysine attachment of the ghosts to electron-microscope grids, were circumvented. The annular and rod-like images are connected into linear and polygonal arrays by strands (15-30 nm in width) that are sensitive to DNase I and DNase II but resistant to nuclease S1. Thus, although the non-membranous ghosts from HeLa cells are composed primarily of protein, enzymic dissection indicates that their gross integrity is markedly dependent on double-stranded DNA. Nuclear ghosts prepared from a wide range of species including mammals, birds and plants, exhibited essentially the same components and organization.
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PMID:The ultrastructure of non-membranous nuclear ghosts. 70 96

A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg2+. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. The maximal difference in fluorescence between untreated and HN2-treated cells was observed after heating at 100 degrees C for 5 min in PBS containing 1.25 mM MgCl2. Higher concentrations of MgCl2 inhibited MOAB binding to HN2-treated cells and heating at lower concentrations induced binding to control cells. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl2. Thus, the presence of phosphates and MgCl2 during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S1 nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. Specific reactivity of MOAB F7-26 with deoxycytidine was established by avidin-biotin ELISA. Single-stranded conformation was necessary for the binding of MOAB to deoxycytidine on the DNA molecule. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.
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PMID:Decreased stability of DNA in cells treated with alkylating agents. 225 76

A crude in vitro transcription system which selectively transcribes DNA fragments containing the promoter region of the Tetrahymena pyriformis rRNA gene has been prepared from T. pyriformis. The system requires both an S100 fraction of lysed isolated macronuclei and an S100 extract of whole cells. When a HhaI-HindIII fragment of the promoter containing plasmid pEN 19-1 is employed as a template, transcription yields two major products of about 560 (A) and 510 (B) bases in length. The analysis of the transcription products of truncated templates showed that RNA A is a runoff transcript and RNA B is produced by nucleolytic cleavage of RNA A at a site about 50 nucleotides to the left of the HindIII cleavage site. S1 nuclease mapping was used to demonstrate that the 5' end of RNA A is identical with that predicted for a transcript which was initiated at the same site on the gene as the in vivo 35S rRNA precursor. Transcription is dependent upon the addition of promoter containing DNA, is inhibited by 1 microgram/mL actinomycin D, and is insensitive to 200 micrograms/mL alpha-amanitin. Transcription is dependent upon the salt levels in the assay exhibiting activity peaks at 58 mM KCl, 28 mM (NH4)2SO4, and 3 mM MgCl2. Several minor transcription start sites to the left of the major initiation site become active at high salt, yielding several minor longer transcripts. High salt also inhibits the RNA cleavage activity, reducing the levels of RNA B produced.
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PMID:Characterization of a crude selective PolI transcription system from Tetrahymena pyriformis. 608 6

Multivalent cations condense DNA in vitro, but it had been thought that a valence of at least + 3 was required in aqueous solution. We have found that Mn2+ can produce toroidal condensates of supercoiled plasmid DNA, but not of linearized plasmid. Mg2+ does not cause condensation, and neither MgCl2 nor NaCl can negate the effect of MnCl2, indicating that the condensation mechanism with Mn is not primarily electrostatic. Supercoiled MnDNA is more extensively digested than the linear form by S1 nuclease. Supercoiling appears to cooperate with Mn2+ in stabilizing helix distortions and also provides a "pressure" that enhances lateral association.
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PMID:Condensation of supercoiled DNA induced by MnCl2. 781 99

The mouse thymidylate synthase (TS) promoter (pTS) lacks a TATAA box and an initiator element, and has multiple transcription start points (tsp) located across a 90-bp region. We have developed an in vitro transcription system for pTS using circular templates and nuclear extracts from HeLa cells or mouse 3T6 fibroblasts. The amount of RNA synthesized and the locations of the tsp were determined by S1 nuclease protection assays. The transcription system reproduced the complex pattern of in vivo tsp, except that the downstream tsp were used preferentially. The reaction temperature, concentrations of DNA template and MgCl2, and incubation time were optimized. The pTS core region contains binding sites for the Sp1 and Ets transcription factors. Inactivation of the Sp1-binding element led to a twofold reduction in transcription and a preferential use of upstream tsp. Inactivation of the Ets-binding element, which reduced promoter activity tenfold in vivo, had only a minor effect in vitro. Addition of a strong initiator element introduced a new tsp, but did not eliminate the complex tsp pattern. To determine if pTS had bidirectional promoter activity, the promoter was inverted and analyzed for transcriptional activity. The inverted promoter was found to initiate transcription at multiple tsp and had approximately the same strength as the normal pTS.
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PMID:In vitro transcription of the TATAA-less mouse thymidylate synthase promoter: multiple transcription start points and evidence for bidirectionality. 807 17

Complex of osmium tetroxide with 1,10-phenanthroline (Os,phen) reacts with double-stranded B-DNA in contrast to osmium tetroxide, pyridine and other osmium structural probes which show a strong preference for single-stranded DNA (ssDNA) (Palecek, E. in Abelson, J.N., and Simon, M.I. (eds), Lilley, D.M.J., and Dahlberg, J.E., (volume eds.), Methods in Enzymology, Vol. 212, DNA Structures, part B., Academic Press, 139-155 (1992)). Modification of negatively supercoiled DNA (scDNA) with Os,phen changes the DNA electrophoretic mobility inducing the DNA relaxation at lower degrees of modification followed by formation of positive supercoils at higher modification extents. Electrophoretic mobility of the Os,phen-modified DNA fragments in agarose gel is almost unchanged while a strong retardation of the same fragments is observed in polyacrylamide gels. Os,phen-modified DNA is hypersensitive to nuclease S1. Cleavage of this DNA by restriction enzymes is selectively inhibited showing a preference of Os,phen for TA and AT dinucleotide steps. DNA modification by Os,phen is inhibited by low and moderate concentrations of MgCl2. The covalent binding of Os,phen to double-stranded DNA (dsDNA) is preceded by noncovalent interactions (probably intercalation) inducing DNA structural changes; the shape of the Os,phen-modified DNA molecule appears to be severely deformed.
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PMID:Complex of osmium tetroxide with 1,10-phenanthroline binds covalently to double-stranded DNA. 882 34