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

对果蝇耐甲基丁二烯基因(MET)的产物--保幼激素受体(JH-R)以及参与JH信号转导的其他转录因子进行了分子分析。这些蛋白质被用在几种不同的组合中，用于DNA序列的缩合，作为含有MET的转录复合体的靶标。使用上述组合蛋白制剂，通过凝胶移位对具有随机区域的75bpDNA进行分级。利用哺乳动物二恶英受体成分的蚊虫同源基因AhR和ArnT对果蝇S2细胞进行细胞转染和报告基因分析：AhR的表达导致JH反应元件(XRE)控制的报告基因转录上调；AhR不结合放射性标记的JH III；AhR基因不直接结合XRE；MET的表达不影响JH诱导的报告基因系统。从蚊子中克隆了一个新的核受体家族成员(NR)，并将其用于S2细胞的细胞转染和报告基因分析。在烟曲霉JH-酯酶基因启动子下游的报告基因中，NR的表达适度提高了JH添加后的报告活性。此外，NR干扰蜕皮激素诱导的S2细胞的报告活性，提示NR通过形成不同的异源二聚体而使ECR/USP解离。

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.