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PMID: 2167466 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Attachment of DNA to the nucleoskeleton of HeLa cells examined using physiological conditions.

Nucleic acids research ·Vol. 18 ·No. 15 ·1990-08-11 ·Pages 4385-93

Jackson DA, Dickinson P, Cook PR

Abstract

Although it is widely believed that eukaryotic DNA is looped by attachment to a nucleoskeleton, there is controversy about its composition and which sequences are attached to it. As most nuclear derivatives are isolated using unphysiological conditions, the criticism that attachments seen in vitro are generated artifactually has been difficult to rebut. Therefore we have re-investigated attachments of chromatin to the skeleton using physiological conditions. HeLa cells are encapsulated in agarose microbeads and lysed using Triton in a 'physiological' buffer. Then, most chromatin can be electroeluted after treatment with a restriction enzyme to leave some at the base of the loops still attached. Analysis of the size and amounts of these residual fragments indicates that the loops are 80-90kbp long. The residual fragments are stably attached, with about 1kbp of each fragment protected from nuclease attack. This is very much longer than a typical protein-binding site of 10-20bp.

MeSH Terms
Chromatin/metabolism DNA/metabolism DNA Restriction Enzymes/metabolism Deoxyribonuclease (Pyrimidine Dimer) Detergents Electrophoresis Endodeoxyribonucleases/metabolism HeLa Cells Humans Hydrogen-Ion Concentration Nuclear Matrix/metabolism Sodium Chloride/pharmacology Temperature
Chemicals
Chromatin Detergents Sodium Chloride DNA Endodeoxyribonucleases DNA Restriction Enzymes Deoxyribonuclease (Pyrimidine Dimer)
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Jackson D A
Sir William Dunn School of Pathology, Oxford, UK.
Dickinson P
Cook P R
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/18.15.4385
Published
1990-08-11
Pages
4385-93
Language
English
Region
England
NLM ID
0411011
PMCID
PMC331255
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/18.15.4385 PMC Full Text View journal details
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