A transcriptional role for conserved footprinting sequences within the larval promoter of a Drosophila alcohol dehydrogenase gene.

All Drosophila alcohol dehydrogenase (Adh) genes that are expressed in larvae display strong transcription in the larval fat body. To identify and characterize elements needed for Adh promoter function, footprinting analysis of the Drosophila affinidisjuncta Adh gene was performed with stage-specific nuclear proteins from embryos and larvae. Multiple sites upstream of the larval promoter were protected from deoxyribonuclease digestion by both embryonic and larval extracts. Comparison with foot-printing results for Adh genes from other Drosophila species revealed only one nuclease-protected region that is conserved in both sequence and position. Clustered point mutations in this sequence were analyzed by footprinting analysis, transient transformation and in vitro transcription. Two separate sequences in this footprinting region exerted positive effects on transcription from the Adh proximal promoter in the larval fat body. The effects of these sequences on gene expression were synergistic. One of these sequences, TGATAA, bound in vitro to Drosophila melanogaster box A binding factor protein, as shown by gel mobility shift assays. This is the first direct demonstration of specific protein-DNA interactions influencing transcription of a Drosophila Adh gene in the larval fat body.