Abstract
In Drosophila, the homeotic gene proboscipedia (pb) is required for the formation of the adult mouthparts. To determine the functional significance of putative pb regulatory DNA, we have performed an in vivo analysis of sequences upstream of and within pb using a series of minigenes. Additionally, we have initiated a dissection of pb's promoter and enhancer elements using lacZ reporter gene constructs. Our results establish that a conserved region located in the second intron is essential for proper formation of the adult mouthparts. A 0.5 kb fragment from this region was shown to direct lacZ expression in a pb pattern in both embryos and third instar labial discs when combined with a 600 bp pb basal promoter sequence. A 32 bp element contained within the 0.5 kb region functions as a labial disc enhancers for pb. Surprisingly, the conserved second intron pb enhancers do not function properly with a heterologous hsp70 promoter, suggesting that promoter-specific interactions occur at the pb locus. We also found redundant and cryptic enhancers in the large introns of pb that are not required for pb function. Finally, we demonstrate that the pb transcription unit does not require sequences upstream of -98 bp to provide pb function in the labial discs. Rather, pb's upstream DNA appears to contain negative regulatory DNA required for silencing PB accumulation in inappropriate domains of third instar imaginal discs. Thus, we have defined many of pb's cis-controlling sequences to an experimentally manageable size, thereby making this an attractive system for the discovery of transacting proteins and, consequently, for elucidating the mechanisms of homeotic gene regulation.
