CLONING AND DEVELOPMENTAL EXPRESSION OF THE ECDYSONE RECEPTOR GENE FROM THE SPRUCE BUDWORM, CHORISTONEURA-FUMIFERANA

Degenerate oligonucleotides were designed on the basis of conserved am ino acid sequences in the DNA and ligand-binding regions of the member s of the steroid hormone receptor superfamily. Using these oligonucleo tides in RNA-PCR, a cDNA fragment was isolated from the spruce budworm , Choristoneura fumiferana. Comparison of the deduced amino acid seque nce of this cDNA fragment with the members of the steroid hormone rece ptor superfamily suggested that this PCR fragment is a region of the e cdysone receptor from C. fumiferana. Using this cDNA fragment as a pro be, 10 clones were isolated from a cDNA library that was constructed u sing the RNA from 4- and 5-day old embryos of C. fumiferana. Two cDNA clones (1.3 and 3 kb) that overlap and show amino acid identity with D rosophila melanogaster ecdysone receptor B-l isoform (DmEcR) were char acterized and sequenced. The longest open reading frame had 539 codons and covered the complete EcR coding region. The deduced amino acid se quence of this open reading frame had all five of the regions typical for a steroid hormone nuclear receptor. The C domain or DNA binding re gion showed the highest identity with EcR proteins from D. melanogaste r, Chironomus tentans, Aedes aegypti, Manduca sexta, and Bombyx mori. The A/B region, D domain or hinge region, E domain, or ligand binding region also showed significant amino acid similarity with the EcR prot eins from the five insects mentioned above. The C. fumiferana ecdyster oid receptor (CfEcR) cDNA probe detected a 6.0-kb mRNA that was presen t throughout the development of C. fumiferana. The CfEcR mRNA increase s in abundance at the time of the ecdysteroid peak during the molting phase in the embryonic, larval and pupal stages but remains low during the intermolt period. In the 6th instar larvae, the 6-kb CfEcR mRNA w as detected in the epidermis, fat body, and midgut and maximum express ion was observed during the prepupal peak of ecdysteroids in the hemol ymph. CfEcR mRNA was induced in ecdysone treated CF-203 cells as well as in the epidermis and midgut of larvae that were fed the nonsteroida l ecdysteroid agonist, RH-5992. The induction occurred within an hour and reached maximum levels around 3 hr, after which it decreased to th e basal level by 6 hr. In vitro transcription and translation of the C fEcR cDNA yielded a 67-Kda protein that bound to the ecdysone response element (EcRE) as a heterodimer, along with the ultraspiracle protein . (C) 1995 Wiiey-liss, Inc.