Isolation and characterization of ecdysone-inducible genes from Drosophila

Metamorphosis in Drosophila melanogaster is initiated by a pulse of the steroid hormone ecdysone. The genetic response to ecdysone can be visualized in the reproducible puffing patterns of the giant salivary gland polytene chromosomes of late third instar larvae. A hierarchical model to account for salivary gland puffing posits that: ecdysone exerts its effects by reversibly binding to its cognate receptor; the hormone-receptor complex functions both to activate an early set of puffs and to repress a late set of puffs; and products of the early puffs antagonize the activity of the hormone-receptor complex by activating the late puffs and repressing the early puffs. In an effort to extend this regulatory hierarchy beyond the salivary glands, a subtracted cDNA library was constructed that is enriched for genes directly induced by ecdysone in cultured larval organs. Ecdysone-inducible genes (Eigs) were identified by testing individual cDNAs from the subtracted library for their ability to detect ecdysone-inducible transcripts in cultured larval organs. Cytological mapping of the Eigs led to the identification of two previously characterized early puff genes, E75 and E63-2. The temporal patterns of Eig expression in the organ culture system and in whole animals was determined by RNA blot analysis. Tissue-specific patterns of Eig expression were determined by in situ hybridization to cryostat sections of staged larvae and prepupae. Sequence analysis of Eig cDNAs led to the identification of a novel cytochrome P450 gene and a novel C3HC4 type zinc-finger protein.