cDNA cloning, gene structure, and expression of Broad-Complex (BR-C) genes in the silkworm, Bombyx mori.

To clarify the molecular mechanisms of metamorphosis, we analyzed the Broad-Complex (BR-C) gene in the silkworm, Bombyx mori. We cloned cDNAs for the full coding regions of the Z1, Z2, and Z4 isoforms of BR-C. The Z3 zinc finger sequence was found in the 3'UTR of the Z2 isoform. The predicted amino acid sequence showed high homology with Drosophila and Manduca BR-C proteins. Five bacterial artificial chromosome (BAC) clones were screened from a Bombyx BAC library. Restriction enzyme cleavage maps of 170 kb regions were constructed, and a total of 25.8 kb were sequenced. The BAC analysis showed that the 5'UTR of the BR-C gene consists of the first two exons, while the coding region contains a core region domain with five exons and four zinc finger exons in the order Z1, Z4, Z2, and Z3. Expression analysis revealed 9.5, 6.5, and 5.5 kb BR-C transcripts. These increased during the spinning ecdysone peak on day 6 of the fifth instar when pupal commitment occurs in the Bombyx epidermis. In addition, a small amount of BR-C mRNA was detected in the epidermis before this peak. BR-C mRNA was also expressed in the fat body from day 1 in the fourth instar to day 7 in the fifth instar.