CHARACTERIZATION OF AN ECR USP HETERODIMER TARGET SITE THAT MEDIATES ECDYSONE RESPONSIVENESS OF THE DROSOPHILA LSP-2 GENE/

The Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is uniquely expressed in the fat body tissue from the beginning of the th ird instar to the end of adult life. Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone. To study the molecular b asis for ecdysone regulated Lsp-2 activity, deletion mutants of the Ls p-2 5'-flanking region were constructed by fusion to either the Escher ichia coli chloramphenicol acetyltransferase (CAT) gene or to an hsp70 -lacZ hybrid gene encoding beta-galactosidase. Constructs transfected into Drosophila S2/M3 cells were shown to confer transient ecdysone in ducibility on the reporter genes. A single functional ecdysone respons e element (EcRE) was localized at position - 75 relative to the Lsp-2 transcription initiation site. In gel mobility shift assays using fat body nuclear extracts or nuclear receptors synthesized in vitro, a 27- bp sequence harboring the EcRE bound both the Drosophila ecdysone rece ptor and the Drosophila retinoid-X homologue, Ultraspiracle, in a coop erative manner. Competition experiments indicate that the affinity of the Lsp-2 EcRE for the ecdysone receptor complex is comparable to that of the canonical EcRE of the hsp27 gene and is at least 4-fold greate r than that of Fbpl, another fat body-specific Drosophila gene. Our re sults suggest that structural features of this EcRE determine its abil ity to induce ecdysone responsiveness at a lower ligand concentration and may form the basis for differential hormone responsiveness within the fat body.