This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. Genetic screens have identified about 30 genes that are involved in the process of segmentation in early Drosophila embryogenesis. These genes form a transcriptional cascade that converts broad gradients of maternal morphogens into increasingly refined patterns of gene expression. An important gene in this process is the pair-rule gene even-skipped (eve) which is expressed in a series of seven transverse stripes. Previous studies have shown that two of these stripes (#2 and #3) are regulated by discrete (about 500 bp) enhancers located in the 5' flanking region. These enhancers act as independent genetic switches that turn on transcription in response to broadly distributed activators. The borders of the stripes are set by repressors that turn off expression through a short range mechanism at the level of the enhancer. The overall goal of this application is to use the eve promoter as a model system to gain an understanding of how regulatory molecules interact at the level of the enhancer, and to learn, on a molecular level, how enhancers control pattern formation in early development. Drosophila is an excellent system for this type of analysis because changes in enhancer function can be directly visualized in the developing embryo. The proposed research will focus on five major goals. First, in vitro DNA-binding assays will be performed to test whether stripe 2 activation involves cooperative binding of activator proteins, and whether repression involves competition for DNA-binding, or protein-protein interactions between activators and repressors. Second, a combination of in vitro and in vivo approaches will be used to identify binding sites for proteins other than those identified genetically that may be important for stripe 2 enhancer function. Third, transient cotransfection assays will be used to test mechanisms of enhancer-mediated activation and repression by changing the number, affinity, and spacing of the DNA-binding sites in the enhancer. Fourth, the role of binding site number, affinity, and spacing on stripe 2 formation will be tested in vivo by P-element-mediated transformation. Finally, spacer sequences between enhancers will be tested by P-element transformation to determine whether they contain specific sequences that prevent interactions between individual enhancer. These studies should contribute significantly to our understanding of the mechanisms whereby eukaryotic enhancers mediate interactions between regulatory molecules to activate and repress transcription.