COOPERATIVITY OF THE MORPHOGENETIC PROTEIN BICOID

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The long-term goal of this grant proposal is to understand the molecular mechanisms of embryonic pattern formation and gene expression in Drosophila, focusing on the morphogenetic protein Bicoid, which controls the development of anterior structures. The Bicoid protein, distributed as a gradient in the embryo with the highest concentration at the anterior, is a transcriptional activator. One target gene of Bicoid protein is hunchback, which, in response to the Bicoid protein gradient, is expressed uniformly in the anterior half of the embryo with a sharp posterior border. This grant application proposes to study how the Bicoid protein gradient results in such an all-or-none response of the target gene hunchback, focusing on the aspects of cooperative DNA binding and protein-protein interaction. A lack of any biochemical studies which are essential to the understanding of this fundamental biological problem has been due to a lack of large quantities of the protein: Various groups have attempted unsuccessfully to isolate an intact Bicoid protein in large quantities. This laboratory has recently successfully generated large amounts of Bicoid protein in the SF-9 insect cells using the baculovirus expression system. This laboratory has demonstrated that the recombinant Bicoid protein binds cooperatively to multiple sites in the hunchback enhancer element. This grant application proposes to further study the molecular mechanisms of cooperativity of Bicoid protein both biochemically and in Drosophila embryos. There are four specific aims in this grant proposal. Specific Aim 1 proposes to further study cooperative DNA binding of Bicoid protein. The recombinant Bicoid protein will be purified further and other derivatives will be generated to define the domains important for cooperative DNA binding. Kinetic studies will be performed to determine the molecular mechanisms of cooperative DNA binding and the formation of stable protein/DNA complexes. Specific Aim 2 will further study the mechanisms of the interaction between Bicoid molecules, an important property of Bicoid that has recently been demonstrated in this laboratory. More deletion derivatives will be generated to narrow down the regions important for protein-protein interaction. These derivatives will be generated to narrow down the regions important for protein-protein interaction. These derivatives will also be used to determine how these domains interact with each other. Specific Aim 3 proposes to isolate specific mutants of Bicoid protein defective in protein-protein interaction and cooperative DNA binding. These mutants will be tested in Drosophila embryos to determine their effects on gene expression and pattern formation. Specific Aim 4 will determine consensus Bicoid binding sites. These synthetic Bicoid binding sites will be used to determine the correlation between in vitro affinity and in vivo activity. Experiments are also proposed to address the question of how Bicoid protein can interact with apparently different types of binding sites. Because many developmentally important mammalian proteins, like the Drosophila morphogenetic protein Bicoid, contain homeodomains, the studies proposed in this grant application will have great impact on the understanding of mammalian development and gene expression.