Enhancers with this kind of structure have been described for many genes (14). Healy et al. (12) have identified putative 5' regulatory elements for Est-6 and related loci in several species that appear to have some of these characteristics. In addition to the antennae and maxillary palp sites identified in this work, Healy et al. (12) have located putative ejaculatory bulb and hemolymph sites in D. pseudoobscura. Our procedures did not allow us to detect expression in the hemolymph, but occasional lines did show expression in the ejaculatory bulb (Fig. 1), suggesting that the element identified by Healy and colleagues may function when other regulatory regions are not present.
The elements essential for the expression of Est-6 in D. melanogaster in both antennae and maxillae lie within 400 bp immediately 5' of the Est-6 coding sequence (Fig. 4). This region of the genome has remarkably low levels of polymorphism and divergence within several species of the melano gaster subgroup (15). Brady et al. (3) found that the 174 bp immediately 5' of the start site in Est-5B was the most highly conserved 5' region between D. melanogaster and D. pseudoobscura. Karotam et al. (15) argue that strong directional selection or functional constraint would be necessary to retain such a low level of sequence variation.
The conservation of esterase expression in the antennae and the maxillary palps suggests that esterase 6 may have a role in olfaction. Vogt and Riddiford (16) identified esterases in the antennae of silk moths, Antheraea polyphemus, that were capable of degrading the female sex pheromone of this species. Carlson and his colleagues (8, 17) have demonstrated that the antennae and maxillary palps of D. melanogaster both are olfactory organs and that a single gene affects the response of these organs to a variety of odorants. The effect of the Est-6 null allele on several mating behaviors (2) and our previous demonstration (18) that esterase 6 is capable of hydrolyzing a Drosophila pheromone suggest that an investigation of the effect of Est-6 alleles on the olfactory responsiveness of antennae and maxillary palps would be fruitful.
Developmental geneticists have proposed that major evolutionary changes in morphology can be the result of a few changes in homeotic genes that can affect the development of body segments (19). Population genetic analysis of the eve gene-controlling segmentation in arthropods and vertebrates (13) supports a neo-Darwinian model that postulates that major changes in morphology are the result of numerous genetic changes both in structural and regulatory genes. Our analyses of the regulation of the Est-6 locus in Drosophila support the neo-Darwinian model for the evolution of gene regulation. While the expression of the Est-6 homologue in D. pseudoobscura is markedly different from that seen in the D. melanogaster group, a number of small changes in elements regulating the expression of these loci can readily account for these differences.
We thank Susan Brandon for technical assistance, T.Kozlova and Bruce Cochrane for many useful discussions, and Marion Healy for sharing unpublished data. This work was supported by a National Science Foundation grant to R.C.R.
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