Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity inseveral life stages and tissue types of D. melanogaster using P-element-mediated transformation of constructs that contain the esterase6coding region and deletions or substitutions in 5' or 3' flanking DNA.Hemolymph is a conserved ancestral site of EST6 activity in Drosophila andthe primary sequences regulating its activity lie between -171 and -25 bprelative to the translation initiation site: deletion of these sequencesdecrease activity approximately 20-fold. Hemolymph activity is alsomodulated by four other DNA regions, three of which lie 5' and one of whichlies 3' of the coding region. Of these, two have positive and two havenegative effects, each of approximately twofold. Esterase6 activity ispresent also in two male reproductive tract tissues; the ejaculatory bulb,which is another ancestral activity site, and the ejaculatory duct, whichis a recently acquired site within the melanogaster species subgroup.Activities in these tissues are at least in part independently regulated:activity in the ejaculatory bulb is conferred by sequences between -273 and-172 bp (threefold decrease when deleted), while activity in theejaculatory duct is conferred by more distal sequences between -844 and-614 bp (fourfold decrease when deleted). The reproductive tract activityis further modulated by two additional DNA regions, one in 5' DNA (-613 to-284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to+2731 bp; threefold decrease when deleted) that probably overlaps theadjacent esteraseP gene. Collating these data with previous studiessuggests that expression of EST6 in the ancestral sites is mainly regulatedby conserved proximal sequences while more variable distal sequencesregulate expression in the acquired ejaculatory duct site.