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

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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.