Functionally distinct, sequence-specific replicator and origin elements are required for Drosophila chorion gene amplification

To meet the demand for the rapid synthesis of chorion (eggshell) proteins, Drosophila ovarian follicle cells amplify the chromosomal loci containing the chorion gene clusters up to 60-fold. Amplification occurs by repeated firing of one or more origins located within each gene cluster. Deletion analyses of transgenic constructs derived from the third chromosome cluster have identified a 320-bp amplification control element (ACE3) required for amplification, as well as several stimulatory amplification enhancing regions (AERs). Two-dimensional (2D) gel analyses have identified multiple DNA replication initiation sites (origins) that partially overlap in location with ACE3 and the AERs. To further study sequence requirements for amplification, a vector was used in which transgenic constructs are protected from chromosomal position effects by flanking insulator elements, the suppressor Hairy-wing protein binding site (SHWBS). Using the buffered vector, the 320-bp ACE3 and an 884-bp element designated ori-beta were found to be necessary and sufficient for amplification. Two-dimensional gels revealed that ori-beta was acting as the origin. In contrast, origin activity could not be detected for AGES. An insulator placed between ACE3 and ori-beta inhibited amplification, indicating that ACE3 activates ori-beta in cis. The results suggest that ACE3 acts as a replicator and support and extend the replicator model for the organization of metazoan chromosomal replicons.