FUNCTIONAL-ANALYSIS OF EVE STRIPE 2 ENHANCER EVOLUTION IN DROSOPHILA - RULES GOVERNING CONSERVATION AND CHANGE

Experimental investigations of eukaryotic enhancers suggest taht multi ple binding sites and trans-acting regulatory factors are often requir ed for wild-type enhancer function. Genetic analysis of the stripe 2 e nhancer of even-skipped (eve), an important developmental gene in Dros ophila, provides support for this view. Given the importance of even-s kipped expression in early Drosophila development, it might be predict ed that many structural features of the stripe 2 enhancer will be evol utionarily conserved, including the DNA sequences of protein binding s ites and the spacing between them. To test this hypothesis, we compare d sequences of the stripe 2 enhancer between four species of Drosophil a: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our ana lysis revealed a large number of nucleotide substitutions in regulator y protein binding sites for bicoid, hunchback, Kruppel and giant, as w ell as a systematic change in the size of the enhancer. Some of the bi nding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanoga ster appears to be recently evolved. We, therefore, investigated possi ble functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revea led that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. mel anogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native e ve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. W e argue that stripe 2 eve expression in Drosophila evolution can be vi ewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We furt her hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly de leterious and adaptive mutations in regulatory protein binding sites a s well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.