Functional Analysis of Even-Skipped Regulatory Evolution

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Research into the structure/function relationship and the evolution of the even-skipped stripe two (eve stripe 2) element of Drosophila melanogaster, arguably the best characterized of all eukaryotic regulatory elements will be continued. Considerable evolutionary change has occurred in the eve stripe 2 element including changes in functionally important binding sites for trans-acting transcription factors. Novel binding sites can be seen to emerge de novo and pre-existing sites can be seen to disappear. The length of stripe 2 element also evolves, thus changing the spacing between transcription factor binding sites. Yet despite these structural changes the stripe 2 elements from several different species, when placed in D. melanogaster, express a reporter gene at identical and indistinguishable times and locations as the native eve stripe 2. Thus functional conservation is maintained despite structural change in the architecture of the element, a strong indication that stabilizing selection is the dominant mode of selection governing this element's evolution. However a chimeric eve stripe 2 element composed of the left and right halves of two functionally conserved elements does not express correctly and raises the possibility that epistatic changes are involved in maintaining functional stasis in the native element. The specific aims of this work are:1. To further investigate functional differences in native and chimeric eve stripe 2 elements by phenotypic analysis in late embryos, larvae and adults using two different "rescue" techniques2. To initiate functional studies of evolved differences in bicoid and kruppel binding sites in the melanogaster subgroup species to determine if compensatory evolution has also occurred in these regulatory loci3. To quantitate expression levels of a lacZ reporter gene when driven by native and chimeric eve stripe 2 elements from different species of Drosophila and to determine the molecular basis of evolved differences in those levels 4. To evaluate the generality of results by conducting comparative sequence analysis in other Drosophila species as well as in the mosquito and housefly.The long-term objective of the proposed research is to understand cis-regulatory sequence evolution in eukaryotes, including the mutational and functional mechanisms by which regulatory evolution occurs and the selective and non-selective forces at work. Deciphering the rules governing the evolution of regulatory sequence evolution is likely to benefit efforts to decode whole genome sequences, including humans and therefore is expected to have a major impact on advancing knowledge in experimental and computational genomics. It will also serve as fertile training ground in these important new areas of biological science.