Analysis of ecdysone-pulse responsive region of BMWCP2 in wing disc of Bombyx mori

βFTZ-F1 and Broad-Complex positively regulate the transcription of the wing cuticle protein gene, BMWCP5, in wing discs of Bombyx mori

“…AB262389), we found five cuticle protein genes located tandemly ( Fig. 1; Nita et al, 2009). The transcription start site was estimated from the sequences of several identical cDNA clones of BMWCP5 and the promoter predictor of NNPP version 2.2 (http://www.fruitfly.org/seq_tools/promoter.…”

“…Studies with mutants have shown that bFTZ-F1 was required for normal larval cuticle production (Yamada et al, 2000). BmbFTZ-F1, an ortholog of bFTZ-F1 of the silkworm Bombyx mori, was expressed during larval, pupal molts and adult development, in coincidence with an ecdysone pulse (Sun et al, 1994;Nita et al, 2009). Although an earlier study proposed that BmbFTZ-F1 was a possible factor directing the stage-specific expression of the peptide gene BmACP-6.7 (Shiomi et al, 2000), the detailed role of BmbFTZ-F1 in cuticle formation has not been elucidated.…”

“…According to Nita et al (2009) we used W2 wing discs for in vivo reporter assay system. After the constructs were introduced into wing discs at W2, wing discs were cultured for 48 h with 20E (Non-pulse treatment) or without 20E (Pulse treatment).…”

“…Transient expression of the reporter constructs Transient expression of the reporter constructs in wing discs was performed according to Nita et al (2009). All reporter constructs were prepared using the Qiagen Miniprep Kit (Qiagen).…”

“…Nuclear exacts were prepared as previously described (Ueda and Hirose, 1990;Nita et al, 2009). Wing tissues were extracted from B. mori at P0, washed twice with PBS, and then minced using scissors in five volumes of a low-salt buffer containing protease inhibitors (50 mM KCl, 20 mM Tris, 0.1 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 2 mM DTT, 0.1 mM PMSF, 1 mg/ml aprotinin, and 1 mg/ ml pepstatin A).…”