Molecular Mechanism of Dioxin Action: Molecular Cloning of the Ah Receptor Using a DNA Recognition Site Probe.

摘要

We have utilized gel retardation analysis and DNA mutagenesis to examine the specific interaction of transformed guinea pig hepatic cytosolic TCDD:AhR 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 TCDD:AhR:DRE complex formation. A putative TCDD:AhR DNA-binding consensus sequence of GCGTGNNA/TNNNC/G 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 TCDD:AhR 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 gene(s) for DRE binding proteins.