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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.

Type

Journal article

Journal

Nucleic Acids Res

Publication Date

11/08/1990

Volume

18

Pages

4385 - 4393

Keywords

Chromatin, DNA, DNA Restriction Enzymes, Deoxyribonuclease (Pyrimidine Dimer), Detergents, Electrophoresis, Endodeoxyribonucleases, HeLa Cells, Humans, Hydrogen-Ion Concentration, Nuclear Matrix, Sodium Chloride, Temperature