d. Mol. Biol. (1986) 188, 517-528 An Ecdysterone-responsive Puff Site in Drosophila Contains a Cluster of Seven Differentially Regulated Genes Linda L. Restifo and Gregory M. Guild¢ Department of Biology~G5 University of Pennsylvania Philadelphia, PA 19104, U.S.A. (Received 2 July 1985, and in revised form 18 November 1985) We have determined the molecular organization of an ecdysterone-responsive puff site in Drosophila melanogaster. The 71E puff site contains a tightly linked cluster of at least seven genes within a neighborhood of 10 x 103 base-pairs. All the genes are expressed in a tissue- specific manner in either the larval or the prepupal salivary gland. However, these genes can be divided into two groups on the basis of their temporal pattern of transcription. Six of the genes are expressed only in prepupal salivary glands and are arranged as three divergently transcribed pairs. Nestled within this region is one gene expressed primarily in late third-instar salivary glands. We conclude that this developmentally complex puff site contains six members of the ecdysterone-induced "late"-gene set and one member of the ecdysterone-regulated "intermolt"-gene set. Additional complexity is found at the transcript level: a heterogeneously sized population of RNA molecules arises fi'om each of the seven genes. 1. Introduction The larval and prepupal salivary glands of Drosophila melanogaster offer a model system for studying co-ordinated gene expression during development (for a review, see Ashburner & Berendes, 1978). Transcriptional activity in the salivary gland genome can be monitored by observing changes in puffing patterns of the giant polytene chromosomes. Cytological observations have shown that the expression of three dispersed sets of puffs is altered in response to a developmentally specific concentration increase of the steroid molting hormone ecdysterone~: that occurs shortly before the end of the third, and final, larval instar. The three sets of ecdysterone-sensitive loci in salivary glands differ in the time-course of their response to the hormone and their sensitivity to hormone concentration. Following the increase in ecdysterone titer, a small number of previously active larval chromosomal puffs regress (Ashburner, 1972a, 1973). t Author to whom all correspondence should be addressed. ~:Abbreviations used: eedysterone, 20-hydroxy- ecdysone; kb, 103 bases or base-pairs; SGS, salivary gland secretion. 0022-2836/86/080517-12 $03.00/0 These puffs represent the "intermolt" genes, some of which encode protein components of the salivary gland "glue" (Muskaviteh & Hogness, 1980; Crowley et al., 1984; Guild & Shore, 1984). Within minutes of ecdysterone stimulation, a small number of puff sites (representing the "early" genes) are induced and persist for several hours. A large number of sites (representing the "late" genes) become puffed several hours following exposure to the hormone; that is, during the prepupal period, whose onset is marked by characteristic behavioral and morphological changes (Bainbridge & Bownes, 1981). By this time, ecdysterone titers are declining (Handler, 1982). Experiments using salivary gland organ culture techniques, in which the ecdysterone-responsive puffing sequence can be mimicked and manipulated, have clarified the differences between the responses of the early and late puff sites to the hormone. In general, the puffing of early sites requires the continuous presence of ecdysterone, is independent of protein synthesis, and varies in amplitude in direct proportion to hormone concentration. In contrast, the response of late-gene sites requires protein synthesis, is essentially all-or-none with respect to hormone concentration, and can occur prematurely (for some late sites) if ecdysterone is removed from the culture medium (Ashburner, 517 © 1986AcademicPress Inc. (London)Ltd.