A method for mapping intranuclear protein-DNA interactions and its application to a nuclease hypersensitive site.

Abstract

A method was devised for mapping sites on DNA within the nucleus that are protected against nuclease attack by interaction with bound protein or other factors. This footprinting method uses an end-labeled sequence-specific DNA probe, which is annealed to the DNA from nuclear digests under carefully controlled conditions. The annealed complexes are treated with single-strand-specific nuclease, the resulting duplex molecules are electrophoresed on gels, and the gels are autoradiographed. The high sensitivity and resolution of the method have made it possible to obtain a detailed map of DNase I cutting patterns in the 5'' flanking sequence of the chicken adult .beta. (.beta.A)-globin gene within nuclei from various tissues. In nuclei from adult erythrocytes, this domain is hypersensitive to nucleases. Within the domain 2 well-defined regions were detected that are protected against attack, indicating the presence of 1 or more bound factors. Nuclei from oviduct or 5-day-old embryonic erythrocytes, in which the domain is not hypersensitive, show limited and different patterns of protection.