| The insect steroid moulting hormone, ecdysterone, in concert with juvenile hormone, directs the process of metamorphosis. Four of the seven heat shock protein genes in Drosophila, hsp22, hsp23, hsp26, hsp27, are selectively inducible by ecdysterone and by physical or chemical stress. To identify heat shock control elements (HSEs) and hormone responsive elements (EcREs) in the promoter, two independent approaches were used.;The first approach utilized a functional expression assay, which yields a relatively low resolution map of transcriptionally active regions in the promoter. To identify regulatory elements for the hsp22 gene, fragments spanning the promoter were cloned into a reporter vector. Each construct was transiently transfected into Drosophila S3 cells which were treated with heat shock or hormone, then assayed for induced transcription of the reporter gene. The second approach involved the use of gel shift analysis and DNA footprinting assays, with nuclear extracts, to map more finely regulatory regions.;Using the functional expression assay and heat shock conditions, an hsp22 gene promoter region containing a series of putative heat shock elements was found to be necessary and sufficient to confer heat shock inducibility to the reporter gene. This result was extended by establishing that the HSE functioned in an orientation independent manner to induce hsp22 gene expression.;Analysis of the hsp22 promoter, using the functional expression assay in the presence of hormone, did not reveal any hormone responsive element. Combinations of two hsp gene promoter regions were unable to induce reporter gene expression. Results from gel shift analysis and DNase I footprinting demonstrated that the hsp22 promoter is capable of supporting many binding activities, including a protected TATA box region, a region which coincides with three putative ecdysterone responsive elements, and a region shown in the functional hormone assays to be transcriptionally inactive. |
