Drosophila development is controlled by a hierarchical network of regulatory genes. The correct spatial and temporal expression of these genes is required to specify the basic body plan of the fruit fly. Our long term goal is to provide a molecular description of how the expression of these regulatory genes is controlled. Thus far, our studies have focused on a biochemical analysis of proteins regulating the transcription of one of the best characterized developmental control genes, Ultrabithorax (Ubx). We have demonstrated that the homeodomain containing protein even-skipped (eve) represses Ubx transcription in vitro. Genetic analysis has previously identified eve as a repressor of Ubx and other homeodomain proteins as either activators or repressors of Ubx. The major portion of this proposal aims to study the molecular mechanisms by which eve and other homeodomain proteins regulate the transcription of Ubx and other developmental control genes in the embryo. We will overproduce and purify eve and other homeodomain proteins in bacteria and insect cells. An endogenous homeodomain- like DNA binding activity we have discovered in Drosophila embryo nuclear extracts will also be purified and characterized. These various purified reagents will then be used to compare and contrast the relative DNA binding and in vitro transcriptional activities of homeodomain proteins. This should reveal the basic biochemical properties of these key regulatory molecules. We also propose to map the functional domains of eve protein required for transcriptional repression and to identify which factors eve interacts with, to elucidate mechanisms of transcriptional repression in eucaryotes. Finally, we will employ cotransfection of these proteins to their regulation of gene expression in the embryo. Our studies should provide a molecular description of how homeodomain proteins act as transcription factors to execute their developmental regulatory function. We have also identified three proteins that activate Ubx transcription in vitro (the zeste, GAGA and ddcITF proteins). These three proteins bind to a series of closely spaced and overlapping DNA elements upstream of the Ubx RNA cap site. We propose to study the protein/DNA interactions between these three factors and the Ubx promoter and to determine whether or not they act independently of each other as they activate transcription. Zeste is also a genetically defined of Ubx, and this data combined with our biochemical experiments using zeste protein, has led ut to propose that it acts to mediate the control of Ubx expression by regulatory elements located 10 to 60 kb from the RNA cap site. We will use cotransfection and P-element transformation assays to test this idea and also to study the role of GAGA and ddcITF in controlling Ubx expression in the embryo. By studying these three proteins, in addition to homeodomain proteins, a more complete description of how Ubx expression is regulated should be derived.