Analysis of in Vivo Nucleosome Positions by Determination of Nucleosome-Linker Boundaries in Crosslinked Chromatin
Identifieur interne : 003D92 ( Main/Exploration ); précédent : 003D91; suivant : 003D93Analysis of in Vivo Nucleosome Positions by Determination of Nucleosome-Linker Boundaries in Crosslinked Chromatin
Auteurs : Gilberto Fragoso ; Gordon L. HagerSource :
- Methods [ 1046-2023 ] ; 1997.
English descriptors
- Teeft :
- Acid extraction, Assay, Core histones, Core particle, Crosslinked, Crosslinking, Crosslinks, Digestion, Edta, Formaldehyde, Formaldehyde treatment, Histone, Histone octamer, Loading buffer, Metha, Methodology, Methods fragoso, Methods nucleosome boundaries, Mnase, Mnase digestion, Mononucleosomal, Mononucleosomal material, Nucleic acids, Nucleosomal, Nucleosomal material, Nucleosome, Nucleosome boundaries, Octamer, Primer, Primer extension, Primer extension assays, Primer extension reaction, Primer extensions, Promoter, Thermal reversal, Various members.
Abstract
Abstract: We describe a procedure for the determination of nucleosome boundaries that utilizes single-stranded mononucleosomal DNA obtained from fixed cells as the template in a primer extension assay. The procedure entails treatment of cells with formaldehyde, a reversible protein–DNA crosslinking agent used with the object of fixing the histone octamers to DNAin vivo,followed by preparation of nucleosomal DNA with micrococcal nuclease, reversal of the crosslinks, and isolation of the mononucleosomal material. Full-length single-stranded mononucleosomal DNA is then prepared and used as a template in a linear amplification primer extension assay. The use of single-stranded DNA templates eliminates interference from nicked DNA present in double-stranded preparations. Because of its reversibility, the use of formaldehyde permits the preparation of DNA suitable as a template in DNA synthesis. We present evidence demonstrating the efficiency of histone–DNA crosslinking and the reversibility of the crosslinking reaction as applied to the regeneration of native DNA, active in DNA synthesis. Use of this methodology removes the impact that mobility of the histone octamer and the presence of nicks on nucleosomal DNA have on the determination of nucleosome boundaries.
Url:
DOI: 10.1006/meth.1996.0411
Affiliations:
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Hager</name><affiliation><wicri:noCountry code="subField">20892-5055</wicri:noCountry></affiliation></author></analytic><monogr></monogr><series><title level="j">Methods</title><title level="j" type="abbrev">YMETH</title><idno type="ISSN">1046-2023</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1997">1997</date><biblScope unit="volume">11</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="246">246</biblScope><biblScope unit="page" to="252">252</biblScope></imprint><idno type="ISSN">1046-2023</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1046-2023</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Acid extraction</term><term>Assay</term><term>Core histones</term><term>Core particle</term><term>Crosslinked</term><term>Crosslinking</term><term>Crosslinks</term><term>Digestion</term><term>Edta</term><term>Formaldehyde</term><term>Formaldehyde treatment</term><term>Histone</term><term>Histone octamer</term><term>Loading buffer</term><term>Metha</term><term>Methodology</term><term>Methods fragoso</term><term>Methods nucleosome boundaries</term><term>Mnase</term><term>Mnase digestion</term><term>Mononucleosomal</term><term>Mononucleosomal material</term><term>Nucleic acids</term><term>Nucleosomal</term><term>Nucleosomal material</term><term>Nucleosome</term><term>Nucleosome boundaries</term><term>Octamer</term><term>Primer</term><term>Primer extension</term><term>Primer extension assays</term><term>Primer extension reaction</term><term>Primer extensions</term><term>Promoter</term><term>Thermal reversal</term><term>Various members</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: We describe a procedure for the determination of nucleosome boundaries that utilizes single-stranded mononucleosomal DNA obtained from fixed cells as the template in a primer extension assay. The procedure entails treatment of cells with formaldehyde, a reversible protein–DNA crosslinking agent used with the object of fixing the histone octamers to DNAin vivo,followed by preparation of nucleosomal DNA with micrococcal nuclease, reversal of the crosslinks, and isolation of the mononucleosomal material. Full-length single-stranded mononucleosomal DNA is then prepared and used as a template in a linear amplification primer extension assay. The use of single-stranded DNA templates eliminates interference from nicked DNA present in double-stranded preparations. Because of its reversibility, the use of formaldehyde permits the preparation of DNA suitable as a template in DNA synthesis. We present evidence demonstrating the efficiency of histone–DNA crosslinking and the reversibility of the crosslinking reaction as applied to the regeneration of native DNA, active in DNA synthesis. Use of this methodology removes the impact that mobility of the histone octamer and the presence of nicks on nucleosomal DNA have on the determination of nucleosome boundaries.</div></front></TEI><affiliations><list></list><tree><noCountry><name sortKey="Fragoso, Gilberto" sort="Fragoso, Gilberto" uniqKey="Fragoso G" first="Gilberto" last="Fragoso">Gilberto Fragoso</name><name sortKey="Hager, Gordon L" sort="Hager, Gordon L" uniqKey="Hager G" first="Gordon L." last="Hager">Gordon L. Hager</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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