Kreitman 9604477 Technical section The phenotypic consequences of evolutionary changes in the DNA sequence of a regulatory region controlling expression of the pair-rule gene even-skipped (eve) will be examined. Eve is expressed in early Drosophila embryos as a series of seven transverse stripes which demarcate the boundaries of future alternating segments in the fly. A 671 bp enhancer element located approximately 1.1 kb upstream of the eve transcription start site regulates strip 2 expression (s2e). Four regulatory proteins, the activators bicoid (bcd) and hunchback(hb), and repressors giant (gt) and Kruppel (Kr), each bind at multiple sites within the enhancer and interact to determine the expression of eve stripe 2 in developing embryos. Through extensive site-directed mutagenesis of binding sites and P-mediated transformation, a comprehensive understanding of s2e structure and function has recently emerged. Recently discovered extensive DNA sequence variation in the s2e within and among Drosophila species allows an exploration of the functional evolution of this regulatory sequence. The phenotypic consequences of this sequence variation on the timing, spatial localization and intensity of stripe 2 expression will be investigated. The working hypothesis to be tested is that individual evolutionary changes in binding sites (or in spacing) will have measurable effects on expression, but that combinations of such changes will have occurred to assure a near-wildtype pattern of expression in each species. Naturally occurring s2e sequences from several Drosophila species will each be linked to a reporter gene (lacZ) and inserted into the D. melanogaster genome. The expression pattern of the reporter gene in developing embryos will be precisely quantified both spatially and temporally relative to the native eve expression by doubly staining embryos for both reporter transcript and for eve protein. Chimerical s2e sequences will be constructed to evaluate phenotypic consequences of specific evolut ionary substitutions, both for the regulatory protein binding sites and for the spacing of the sites. More complex chimerical sequences will also be constructed and tested to evaluate the possibility of compensatory changes within a s2e. Non-technical section The functional consequences of regulatory sequence evolution in the even-skipped stripe 2 enhancer of Drosophila will be investigated. The work will provide a foundation for understanding microevolutionary forces governing regulatory sequence evolution and the role natural selection plays in this process. The studies should illuminate basic molecular mechanisms of gene regulation and also evolutionary mechanisms of regulatory sequence variation and change.
