IMMUNOLOGICAL STUDIES ON THE DEVELOPMENTAL AND CHROMOSOMAL DISTRIBUTION OF ECDYSTEROID RECEPTOR PROTEIN IN CHIRONOMUS-TENTANS

Antisera were raised against different domains of a putative ecdystero id receptor (cEcRH) of Chironomus tentans. All the antisera reacted wi th a 68,000 dalton protein exhibiting DNA binding properties. Addition ally, we were able to demonstrate that the antisera immunoprecipitate protein which binds a radioactively labeled ecdysteroid (Ec), i.e., [H -3]ponasterone A, with high specificity. These properties indicate tha t the antisera recognize specifically an endogenous ecdysteroid recept or protein (cEcR) in C. tentans cells and thus are suitable for the fo llowing quantitative and qualitative immunological and immunohistochem ical investigations. The cellular level of cEcR varies during developm ent, and it is particularly low in oligopausing larvae. In polytene ch romosomes of prepupal salivary glands, cEcR is located at approximatel y 50 transcriptionally active loci. These loci include both early ecdy steroid (Ec)-inducible puff sites, such as the locus containing the ge ne coding for the homolog of the E75 protein in Drosophila melanogaste r, as well as late Ec-inducible puff-sites. The latter group comprises a locus of a gene specifying the homolog of the D. melanogaster ultra spiracle protein. However, loci of genes coding for salivary gland sec retory proteins (e.g., Balbiani ring forming chromosome regions) do no t specifically react with the antisera. Thus, the developmental regula tion of these genes is not directly controlled by Ec. Polytene chromos omes of oligopausing larvae show hardly any loci that contain cEcR. Th e few detected correspond, with few exceptions, to the most potent cEc R binding sites found in prepupae. (C) 1995 Wiley-Liss, Inc.