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Nucleic Acids Research, 1980, Vol. 8, No. 13 2895-2906
© 1980

MOLECULAR BIOLOGY

Mapping sequences in loops of nuclear DNA by their progressive detachment from the nuclear cage

P.R. Cook and I.A. Brazell

Sir William Dunn School of Pathology, University of Oxford South Parks Road, Oxford OX1 3RE, UK

Received May 29, 1980. Nuclear DNA is organised into loops, probably by attachment to a supramolecular structure. We describe a method which enables us to map the position of sequences within a loop relative to the point of attachment. Nuclear DNA is isolated unbroken by lysing HeLa cells in 2M NaCl to release structures which retain many of the morphological features of nuclei. Their DNA is supercoiled and so must remain unbroken and looped during lysis. Nucleoids are digested to various degrees with a restriction endonuclease and the cages- and any associated DNA - sedimented free from unattached DNA. The cage-associated DNA is purified and completely fragmented using the same restriction endonuclease. Equal weights of fragmented DNA are separated by gel electrophoresis, transferred to a filter and the relative amounts of the {alpha},ß and {gamma} globin genes on the filter determined by hybridisation to the appropriate probes. The {alpha} genes, unlike the ß and {gamma} genes, resist detachment from the cage and so must lie close to the point of attachment to the cage. Our ability to map these genes implies that sequences cannot be attached at random to the cage; rather, specific sequences must be attached, so looping the DNA.


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