Nucleic Acids Research, 1990, Vol. 18, No. 20 6049-6055
© 1990
CHEMISTRY |
Metal-dependent binding of a nuclear factor to the rat metallothionein-I promoter
1Department of Biological Chemistry, University of California Los Angeles, CA 90024, USA 2Laboratory of Biomedical and Environmental Sciences, University of California Los Angeles, CA 90024, USA
*To whom correspondence should be addressed
Received June 25, 1990. Revised September 12, 1990. Accepted September 12, 1990.
Genomic footprintlng studies in vivo and experiments using synthetic metal regulatory elements (MREs) in vitro suggest protein binding to the MREs of the mouse and rat metallothionein I (MT-I) genes. Using gel-retardation assays of promoter fragments, we observe a cadmium-dependent binding factor for the rat MT-I promoter in rat hepatoma cells . This factor is present in extracts from both uninduced and cadmium-induced cells, but requires the presence of cadmium to bind to the promoter. The formation of a cadmium-dependent complex is competed by an oligonucleotide containing two MREs. This competition is lost when when one of the MREs is mutated, Indicating a requirement for atleast two MREs for binding of this factor. The cadmium-dependent factor dissociates more rapidly from the MT-I promoter than does a factor that binds to a consensus Sp1 site present on the same DNA fragment. UV crosslinking analysis using nuclear extracts from cadmium induced cells, In the presence of an oligonucleotide probe containing both 5-bromodeoxyurldine and 32P-deoxycytidine, identifies a 39 kDalton protein associated with the metal inducible complex.
+Present addresses: California Institute of Technology, Division of Biology 216-76, Pasadena, CA 91125
Columbia University, Department of Microbiology, College of Physicians and Surgeons, New York, NY 10032
Universiry of California; Department of Animal Science, Davis, CA 95616, USA
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