Homeodomain proteins are essential to normal development and differentiation in Drosophila and humans. They act as transcriptional regulatory proteins. How they impart their specificity of action is still poorly understood. This study is designed to characterize the molecular specificity of homeodomain protein action. Phase I will characterize the DNA sequences that impart specificity of homeodomain binding. The model system used will be the Abdominal-B homeoprotein response element of the Drosophila empty spiracles gene. This newly identified element will be sequenced and then functionally important regions will be identified by an evolutionary, and mutational approach and tested for function in a transgenic fly strain. The specific Abdominal-B homeoprotein binding sites will be compared to another homeobox response element. Attempts will be made to change sequences in one response element to make it specific for another homeobox protein; thus those sequences essential for specificity of action will be determined. The principles of homeotic specificity learned will be tested for applicability to humans using homologus human homeobox genes in a transgenic fly strain. Phase II will attempt to identify and characterize novel cofactors that are likely involved in refining homeotic specificity. Once identified, these cofactors will be assayed for conservation in humans. These genes and cofactors will then be assayed for function in mammals. A comprehensive characterization of the determinants of homeotic specificity will be delineated in both Drosophila and humans.