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

本文对果蝇耐甲氧苯二烯基因（MET）的产物JH受体进行了分子分析，并对JH信号通路中可能涉及的转录因子进行了分子分析。利用bHLH-PAS超家族成员MET、Gce和Tgo的cDNA克隆体外合成蛋白。这些蛋白质以几种不同的组合用于DNA序列的凝聚，作为含met转录复合物的靶标。采用上述组合蛋白制备，用凝胶移位法分离具有随机区域的75bp dna。经过三个循环的浓缩过程，得到了富集目标序列的DNA群体。利用哺乳动物二恶英受体成分Ahr和Arnt的蚊子同源物对果蝇S2细胞进行细胞转染和报告基因检测：在JH作用下，Ahr的表达引起报告基因在异种反应元件（XRE）控制下的转录升高；Ahr不与放射性标记的jhiii结合；Ahr不直接绑定到XRE；MET的表达不影响jh诱导报告基因系统。综上所述，这些观察结果表明存在met独立的JH信号通路。从蚊子中克隆了一个新的核受体家族成员（NR），用于S2细胞转染和报告基因检测。NR的表达适度地提高了JH的报告活性，而JH是由放置在fumiferana JH-酯酶基因启动子下游的报告基因添加的。此外，NR干扰了甾体激素诱导的S2细胞报告活性，表明NR通过形成不同的异源二聚体使EcR/USP分离。

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.