ISOLATION AND DEVELOPMENTAL EXPRESSION OF THE ECDYSTEROID-INDUCED GHR3 GENE OF THE WAX MOTH GALLERIA-MELLONELLA

Three degenerate primers were designed to match the most conserved regions within the DNA-binding domains of several selected members of the steroid hormone receptor family. Use of these primers in the polymerase chain reaction with cDNA from Galleria mellonella prepupae detected a 177 bp fragment that had 87% identity to the Manduca sexta gene MHR3 and 75% to the Drosophila melanogaster DHR3 gene, and therefore was named ''GHR3''. Screening of a Galleria penultimate instar cDNA library with this fragment yielded a cDNA clone that contained a 557 codon open reading frame, predicting a 62.3 kDa protein. The deduced amino acid sequence of GHR3 showed 92% overall identity with the MHR3 protein and 97 and 70% identity with DHR3 in the putative DNA- and ligand-binding domains, respectively. Hybridization of whole body RNA revealed high GHR3 mRNA levels during both the larval and pupal molts, coincident with the molt-inducing ecdysteroid pulses, and low or undetectable levels during the first half of the last instar, During the larval-pupal transformation, no GHR3 mRNA was found at the beginning of the stemmatal pigment retraction at the onset of the ecdysteroid rise; maximal levels were observed 4 h later, coincident with the peak ecdysteroid titer (over 2.3 mu g 20E equivalents/ml hemolymph). Two mRNAs (4.6 and 3.6 kb) were detected when the ecdysteroid titer was high. Injection of 2 mu g/gm 20E into isolated final instar larval abdomens induced the appearance of the 4.6 kb mRNA within 1.5 h; the mRNA level then reached maximum by 3 h and declined by 6 h. No 3.6 kb mRNA was detectable during that time. A 10-fold lower 20E dose caused only trace induction by 3 h.