Immunological studies on the developmental and chromosomal distribution of ecdysteroid receptor protein in Chironomus tentans

Abstract

Antisera were raised against different domains of a putative ecdysteroid receptor (cEcRH) of Chironomus tentans. All the antisera reacted with a 68,000 dalton protein exhibiting DNA binding properties. Additionally, we were able to demonstrate that the antisera immunoprecipitate protein which binds a radioactively labeled ecdysteroid (Ec), i.e., [3H]ponasterone A, with high specificity. These properties indicate that the antisera recognize specifically an endogenous ecdysteroid receptor protein (cEcR) in C. tentans cells and thus are suitable for the following quantitative and qualitative immunological and immunohistochemical investigations. The cellular level of cEcR varies during development, and it is particularly low in oligopausing larvae. In polytene chromosomes of prepupal salivary glands, cEcR is located at approximately 50 transcriptionally active loci. These loci include both early ecdysteroid (Ec)-inducible puff sites, such as the locus containing the gene coding for the homolog of the E75 protein in Drosophila melanogaster, as well as late Ec-inducible puff-sites. The latter group comprises a locus of a gene specifying the homolog of the D. melanogaster ultraspiracle protein. However, loci of genes coding for salivary gland secretory proteins (e.g., Balbiani ring forming chromosome regions) do not specifically react with the antisera. Thus, the developmental regulation of these genes is not directly controlled by Ec. Polytene chromosomes of oligopausing larvae show hardly any loci that contain cEcR. The few detected correspond, with few exceptions, to the most potent cEcR binding sites found in prepupae.

Indirect immune detection of ecdysone receptor (EcR) during the formation of DNA puffs in Bradysia hygida (Diptera, Sciaridae)

“…These results are in agreement with those obtained with T. pubescens (Stocker et al 1997) and with EcR localization in C. tentans (Wegmann et al 1995) in active early and late ecdysteroid-inducible puff sites. That EcR can bind to the origin of amplified genes was also shown with electromobility shift assays (EMSA) (Foulk et al 2006).…”

“…In western blots of salivary gland extracts, a polypeptide with an apparent molecular mass of 66 kDa was detected (BhEcR). In general, EcRs of insects species vary considerably in molecular mass: 105 and 80 kDa in D. melanogaster (Koelle et al 1991, Talbot et al 1993; 70, 68 and 66 kDa in Chironomus tentans (Imhof et al 1993, Wegmann et al 1995; 95 and 75 kDa in A. aegypti (Cho et al 1995); 62 kDa in Manduca sexta (Fujiwara et al 1995); 70 kDa in Bombyx mori (Swevers et al 1995); 66 kDa in Tenebrio molitor (Mouillet et al 1997); 57 kDa in Choristoneura fumiferana (Perera et al 1999); and 75 kDa in A. albopictus (Jayachandran & Fallon 2000).…”

CDK8-Cyclin C Mediates Nutritional Regulation of Developmental Transitions through the Ecdysone Receptor in Drosophila

“…Despite the tremendous progress in our understanding of the physiological and developmental effects of EcR-USP signaling, the molecular mechanism of how the EcR-USP transcription factor interacts with the general transcription machinery of RNA polymerase II (Pol II) and stimulates its target gene expression remains mysterious. EcR is colocalized with Pol II in Bradysia hygida and Chironomus tentans [ 22 , 23 ]. Although a number of proteins, such as Alien, Bonus, Diabetes and Obesity Regulated (dDOR), dDEK, Hsc70, Hsp90, Rigor mortis (Rig), Smrter (Smr), Taiman, and Trithorax-related (TRR), have been identified as regulators or cofactors of EcR-mediated gene expression [ 13 , 24 – 32 ], it is unknown how these proteins communicate with the general transcription machinery and whether additional cofactors are involved in EcR-mediated gene expression.…”

Antibodies against the D-domain of a Chironomus ecdysone receptor protein react with DNA puff sites in Trichosia pubescens

“…The anti-EcR antiserum (Wegmann et al 1995) used in this study was directed against the bacterially expressed D-domain of an EcR homolog of Chironomus tentans (Imhof et al 1993). For most of the experiments, diluted unpurified antiserum (called AScE/ D) was used since its reaction was stronger and easier to observe with low power objectives.…”

“…We have attempted to visualize the engagement of Ec with its receptor (EcR) at DNA puff sites in Trichosia by using an antiserum against the semiconserved D-domain of the Chironomus EcR (Wegmann et al 1995) in indirect immunofluorescence reactions. We wanted to examine the time course of appearance of the endogenous sciarid EcR during the processes of DNA amplification, RNA synthesis, and puffing.…”