Identification of Juvenile hormone-dependent transcription factors by using yeast system

作为鉴定幼龄激素（JH）依赖性转录因子的第一步，我们对蚊子ecdysone （20E）信号传导的潜在共激活因子和基础转录因子进行了cDNA克隆、序列分析和功能解剖，得到以下结果：对Bx42、GCN5、TBP、CBP和MBF1的蚊子同源物进行了cDNA克隆。对Bx42、GCN5和TBP进行了全长cDNA克隆，并进行了序列分析和系统发育分析。通过将由20E响应元件驱动的荧光素酶报告基因构建体导入哺乳动物和果蝇细胞系，建立了瞬时转染系统，在培养基中添加配体或类似物后，报告基因构建体的转录能力大大增强。利用上述瞬时细胞转染系统，Bx42和GCN5均被证明在20e依赖性基因激活中起共激活因子的作用。哺乳动物双杂交实验显示GCN5的Bx42蛋白与蜕皮激素受体蛋白相互作用。用表达Bx42- egfp融合蛋白的构建体进行细胞转染实验，证实了Bx42的核定位。

As an initial step toward the identification of Juvenile hormone(JH)-dependent transcription factors, cDNA cloning, sequence analysis and functional dissection of potential coactivators and basel transcription factors involved in ecdysone (20E) signaling in mosquitoes were done, giving the following results :1. cDNA cloning mosquito homologs of Bx42, GCN5, TBP, CBP and MBF1 were taken place. As for Bx42, GCN5 and TBP, full length cDNA clones were isolated, and their sequence and phylogenetic analyses were performed.2. By introducing a luciferase reporter construct driven by 20E response elements into mammalian and Drosophia cell lines, transient transfection systems, where the transcription from the reporter construct was enhanced greatly upon the addition of the ligand or its analog in culture media, were established.3. By utilizing the transient cell transfection systems mentioned above, both Bx42 and GCN5 were proved to function as coactivators in 20E-dependent gene activation.4. Mammalian two-hybrid assay revealed the protein-protein interaction of Bx42 of GCN5 against ecdysone receptor protein.5. Nuclear localization of Bx42 was confirmed by cell transfection experiments with a construct which expresses Bx42-EGFP fusion protein.