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Using a differential display of mRNA technique we discovered that the juvenile hormone (JH) esterase gene (Cfjhe) from Choristoneura fumiferana is directly induced by juvenile hormone I (JH I), and the JH I induction is suppressed by 20-hydroxyecdysone (20E). To study the mechanism of action of these two hormones in the regulation of expression of this gene, we cloned the 1270-bp promoter region of the Cfjhe gene and identified a 30-bp region that is located between ؊604 and ؊574 and is sufficient to support both JH I induction and 20E suppression. This 30-bp region contains two conserved hormone response element half-sites separated by a 4-nucleotide spacer similar to the direct repeat 4 element and is designated as a putative juvenile hormone response element (JHRE). In CF-203 cells, a luciferase reporter placed under the control of JHRE and a mini- (6), juvenile hormone esterase (7,8), calmodulin (9), vitellogenin (10), and several others have been reported (11,12). Through indirect action, JH was shown to modulate 20-hydroxyecdysone (20E) action by affecting the expression of genes in a 20E-induced cascade (13,14). Totally different from the above genomic actions, in ovarian follicular epithelium, JH acts through a membrane receptor to bring about rapid enzyme activation without the need for new transcription (15).Numerous attempts have been made to identify JH receptors. Palli et al. (16) used human retinoic acid receptor cDNA as a probe and identified a steroid/thyroid superfamily member from Manduca sexta. Further characterization of this cDNA revealed that this is not a JH receptor but, rather, an ecdysoneinduced transcription factor that plays a critical role in ecdysone signal transduction and is related to Drosophila hormone receptor 3 (17), subsequently named Manduca hormone receptor 3 (18). The 29-kDa nuclear protein identified in M. sexta epidermis turned out to be a low affinity JH-binding protein (19,20).The mammalian retinoid X receptor (RXR) forms a heterodimer with several nuclear receptors including the farnesoid X-activated receptor (FXR). JH III but not JH acid or methoprene can bind/activate the FXR and RXR heterodimer (21). Methoprene and methoprene acid but not JH III can activate RXR (22). These two studies suggested that RXR or its insect homologue ultraspiracle (USP) could play an important role in signal transduction of JH or JH-related compounds. Jones and Sharp (23) showed that both JH III and JHB 3 bind to a USP homodimer from Drosophila melanogaster. Subsequent studies showed that USP from D. melanogaster can bind to the DR12 response element and a reporter gene placed under the control of the DR12 response element fused to the jhe core promoter was induced by JH III (24).A D. melanogaster mutant tolerant to methoprene (Met) was identified (25). An 85-kDa protein isolated from Met flies showed a 6-fold lower affinity than the wild-type protein for JH III (26). The Met gene was cloned and found to be a member of the basic helix-loop-helix-PER-AHR/ARNT-SIM (PAS) family of transcr...

Cloning and characterization of a Choristoneura fumiferana ultraspiracle (Cfusp) cDNA are described. First, a PCR fragment and then a cDNA clone (4.4 kb) were isolated from spruce budworm cDNA libraries. Comparison of the deduced amino acid sequence of this cDNA with the sequences in Genbank showed that this sequence had high homology with the ultraspiracle cDNAs cloned from Drosophila melanogaster (Dmusp), Bombyx mori (Bmusp), Manduca sexta (Msusp), and Aedes aegypti (Aausp). The Cfusp cDNA contained all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNA binding domain or C region was the most conserved sequence among all the usps. The A/B, D, and E regions also showed high amino acid identity with the amino acid sequences of Dmusp, Msusp, Bmusp, and Aausp. The Cfusp 4.5-kb mRNA was present in the embryos, in all larval stages, and in the pupae. The Cfusp mRNA levels in the midgut increased during the sixth-instar larval development and reached peak levels during the ecdysteroid raises for the pupal molt. However, Cfusp mRNA levels remained unchanged in the midgut of fifth-instar larvae, and in the epidermis and fat body of sixth-instar larvae indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression.

Heterodimerization of nuclear receptors is facilitated by the interaction of two dimerization interfaces: one spanning the DNA-binding (C domain) region and the adjacent hinge (D domain) region, and the other in the ligand-binding (E domain) region. Ultraspiracle (USP) heterodimerizes with ecdysone receptor (EcR) and this complex participates in ecdysone signal transduction. The natural ecdysone response elements (EcREs) discovered so far are asymmetric elements composed of either imperfect palindromes or direct repeats. However, gel mobility shift assays have shown that both symmetric (perfect palindromes) and asymmetric (imperfect palindromes and direct repeats) elements can bind to the EcR/USP complex. Therefore, we analyzed EcR/USP domains involved in heterodimerization on different types of response elements (RE). Gel shift assays using full-length and truncated EcR and USP proteins showed that heterodimerization of these two proteins in the presence of asymmetric RE (DR4 and the natural EcRE hsp27) requires both dimerization interfaces present in CD and E domains of both proteins. In contrast, the dimerization interface present in the E domain of either EcR or USP was not essential for heterodimerization on symmetric RE such as PAL1 or IR1. We conclude that the use of heterodimerization interfaces present in CD and E domains of EcR/USP depends on the nature of response elements they bind to.