Nucleic Acids Research, 1990, Vol. 18, No. 15 4385-4393
© 1990
MOLECULAR BIOLOGY |
Attachment of DNA to the nucleoskeleton of HeLa cells examined using physiological conditions
Sir William Dunn School of Pathology South Parks Road, Oxford 0X1 3RE, UK
*To whom correspondence should be addressed
Received May 22, 1990. Revised June 19, 1990. Accepted June 19, 1990.
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.
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