A method for mapping intranuclear protein-DNA interactions and its application to a nuclease hypersensitive site.
We have devised a method 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. However, we detect within the domain two well-defined regions that are protected against attack, indicating the presence of one 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.
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002642:Chick Embryo; D002645:Chickens; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D005796:Genes; D005914:Globins / Q000235:genetics; D009698:Nucleoproteins / Q000378:metabolism*
D009698:Nucleoproteins; D005914:Globins; D004247:DNA; D003850:Deoxyribonuclease I