DNA-binding properties of an “ecdysteroid receptor” from epithelial tissue culture cells of Chironomus tentans

Abstract

Recently an ecdysteroid binding protein (“ecdysteroid receptor”) has been identified in extracts of epithelial tissue culture cells of the dipteran insect Chironomus tentans. We now show that this protein also displays high affinity to DNA. When using a solid phase binding assay an association rate constant of 3.3 × 10 6 M −1 s −1 and a dissociation rate constant of 2 × 10 −4 s −1 were determined for the ecdysteroid receptor-immobilized DNA complex at 0°C. An apparent equilibrium dissociation constant of 6.1 × 10 −11M could be estimated from these values indicating a high affinity binding of ecdysteroid receptors to immobilized DNA. The binding of ecdysteroid receptors to immobilized DNA was saturable. Only double stranded free DNA competed significantly for ecdysteroid receptor binding to immobilized DNA. Furthermore, DNA sequences that contain ecdysone response elements (EcREs) from hormonally regulated Drosophila genes or sequences from an ecdysteroid-inducible gene of C. tentans revealed a 10–350 times higher affinity to ecdysteroid receptors than random sequences of calf thymus DNA or from a plasmid.