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Nucleoprotein structures at the SV40 GC-box and adjacent AT-rich region have been probed by nucleases in permeabilized cells at nucleotide level resolution. The patterns of nuclease protection and hypersensitivity in these permeabilized cells that allow initiation of RNA and DNA synthesis are quite different from those observed in isolated nuclei that are inactive. Whereas simple DNA protection by factors is found in nuclei, the pattern in permeabilized cells includes very strong nuclease hypersensitive sites. Their arrangement suggests that the region exists as a higher order nucleoprotein complex in vivo, which is disturbed during the preparation of nuclei. The pattern is also found to be disturbed in permeabilized cells when T-antigen is inactivated by temperature-sensitive mutation. Since T-antigen origin binding sites and the GC-box region have been shown previously to interact functionally, the existence of a higher order structure involving both components provides a likely physical basis for the functional interaction of separate control elements.

In situnucleoprotein structure involving origin-proximal SV40 DNA control elements