|Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1997: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
Ecdysone, the insect molting hormone, penetrates into the target cells and binds to the ecdysone receptor (EcR) in the nuclei. this complex (ecdysone-EcR/USP) regulates a special set of gene expression and finally causes molts and metamorphosis of insects. I studied the molecular mechanisms of tissue differentiation during metamorphosis, mainly focused on relationship between EcR expression and the network of gene expressions induced by ecdysone. For this purpose, we first identified two isoforms of the silkworm EcR,BmEcR-A and BmEcR-B1, using by 5^1-RACE.Expression patterns of two EcR isoforms in different tissues during metamorphosis were analyzed by northern hybridization. Further, we determined the transcriptional initiation site for each EcR isoform and their flanking sequences. Interaction between these regions and putative transcription factors were studied by gel shift assay.
Expression of EcR mRNA was induced by a very low dose of ecdysone (20 hydroxyecdysone). In the silkworm, Bombyx mori, BmEcR-B1 was expressed mainly during metamorphosis in most tissues only except anterior silk glands. This result is not consistent with the observation in Drosophila ; EcRB1 is predominant in larval tissues while EcRA is major in adult tissues. Thus, the relationship between developmental fate of tissues and patterns of EcR isoform expression is different in a lepidopteran insect. The start sites of transcription for BmEcR A and B1 are located more than 20kb from each other and we could not find homologous structure between them. In upstream regions of both start sites, there are initiator sequences instead of TATA box. Gel shift analysis implied that many factors are involved in transcriptional regulation and alternative promoting of BmEcR isoforms.