ANTIBODIES AGAINST THE D-DOMAIN OF A CHIRONOMUS ECDYSONE RECEPTOR PROTEIN REACT WITH DNA PUFF SITES IN TRICHOSIA-PUBESCENS

An antiserum (called AScE/D) against the semiconserved D-domain of a C hironomus tentans ecdysone receptor protein (cEcR) gave indirect immun ofluorescence signals at DNA puff sites in Trichosia pubescens. The si gnals varied in maximum intensity at different DNA puff sites. Control experiments using the secondary rhodamine-labeled anti-rabbit IgG alo ne, preimmune serum, affinity purified AScE/D (called pABcE/D) and ASc E/D preabsorbed with expressing bacterial extract or highly purified b acterially expressed cEcR indicated that the signals obtained at these chromosomal sites were likely to be due to specific interaction betwe en an endogenous sciarid EcR and antibodies against cEcR. This conclus ion was supported by observation of signals at certain Ec-inducible pr imary RNA puff sites. AScE/D signals began to appear at DNA puff sites during L3, the stage when amplification initiates, but at most sites their mean intensity was low and not statistically significant. Sites with AScE/D signals of significant mean intensity at this stage alread y showed evidence of transcription. The number and strength of transcr iption signals increased during L4. Comparison of the developmental co urse of signals for AScE/D, DNA synthesis, RNA presence/synthesis, and puff size for several DNA puffs during late larval-prepupal developme nt showed a closer relationship of AScE/D signals with the initiation of RNA synthesis than with the initiation of DNA synthesis. Therefore, although we cannot absolutely eliminate a direct involvement of EcR i n the amplification process at some sites, this investigation gives st ronger support for its direct involvement in transcription. Since AScE /D signals are observed at DNA puff sites from the time the latter beg in amplification/transcription through their regression, it appears th at Ec and EcR are necessary as a sustained stimulus at these regions.