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Nucleic Acids Research, 1990, Vol. 18, No. 7 1797-1803
© 1990

MOLECULAR BIOLOGY

In situ nucleoprotein structure involving origin-proximal SV40 DNA control elements

Li Zhang and Jay D. Gralla*

Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California 405 Hilgard Avenue, Los Angeles, CA 90024-1 569, USA

* To whom correspondence should be addressed

Received December 4, 1989. Revised February 25, 1990. Accepted February 25, 1990.

Nucleoprotein structures at the SV40 GGbox and adjacent AT-rich region have been probed by nucleases in permeabiilzed 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 pattem 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.


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