Molecular dissection of methoprene-tolerant gene product and survey for its target genes

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Molecular analyses were done on putative juvenile hormone (JH) receptor, namely a product of Drosophila methoprene-tolerant gene (MET) as well as on other transcription factors, involvement of which was suggested in JH signaling pathway.Proteins were synthesized in vitro from cDNA clones of MET, Gce and Tgo, all of which are the members of bHLH-PAS superfamily. The proteins were used in several different combinations for the condensation of DNA sequences as a target of MET-containing transcriptional complex. The 75 bp DNAs with a random region were fractionated by gel-shift using the combined protein preparation mentioned above. After three cycles of the condensation procedure, the DNA population enriched with the target sequences was obtained.Cell transfection and reporter gene assays were done with Drosophila S2 cells using mosquito orthologs of mammalian dioxin receptor constituents, Ahr and Arnt : expression of Ahr brought about the elevated transcription from the reporter gene under the control of xenobiotic response element (XRE) in response to JH ; Ahr dose not bind radio-labeled JH III ; Ahr dones not bind to XRE directly ; expression of MET dose not affect this JH-induceable reporter gene system. Taken together, these observations suggest the occurrence of MET-independent JH signaling pathway.A novel nuclear receptor family members (NR) was cloned from mosquitoes and used for the cell transfection and reporter gene assays in S2 cells. The expression of NR moderately elevated the reporter activity upon JH addition from a repoter placed downstream of C.fumiferana JH-esterase gene promoter. In addition, the NR interfered with the ecdysteroid-induced reporter activity in S2 cells, suggesting the NR renders EcR/USP dissociated by forming distinct heterodimers.