Molecular Mechanism of Dioxin Action: Molecular Cloning of the Ah Receptor Using a DNA Recognition Site Probe
Final rept. 1 Sep 1990-31 Aug 1991
MICHIGAN STATE UNIV EAST LANSING DEPT OF BIOCHEMISTRY
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We have utilized gel retardation analysis and DNA mutagenesis to examine the specific interaction of transformed guinea pig hepatic cytosolic TCDDAhR complex with a dioxin responsive element DRE. Sequence alignment of the mouse CYPIA1 upstream DREs has identified a common invariant core consensus sequence of TNGCGTG flanked by several variable nucleotides. Competitive gel retardation analysis using a series of DRE oligonucleotides containing single or multiple base substitutions has allowed identification of those nucleotides important for TCDDAhRDRE complex formation. A putative TCDDAhR DNA-binding consensus sequence of GCGTGNNATNNNCG has been derived. The four core nucleotides, CGTG, appear to be critical for TCDD-inducible protein-DNA complex formation since their substitution decreased AhR binding affinity by 200- to 2000-fold the remaining conserved bases are also important, albeit to a lesser degree 3- to 5-fold. Our results indicate that the primary interaction of the TCDDAhR complex with the DRE occurs with the conserved core sequence, although nucleotides flanking the core also contribute to the specificity of DRE binding. The optimal DRE consensus sequence is being utilized for screening of cDNA libraries in an attempt to directly clone the genes for DRE binding proteins.
- Anatomy and Physiology