Notch is the best characterized of several Drosophila 'neurogenic' genes that control a wide range of cell fate determinations that start in the early embryo. Notch serves as a cell membrane-bound receptor. It is cleaved following cell-surface association with ligand, to produce a soluble, intracellular protein fragment that functions in nuclei. Our studies will be extended as follows: (a) Properties of constitutively active Notch receptors allow us to map metalloprotease Kuzbanian function during Notch proteolysis and signaling. We will characterize the step in Notch cleavage regulated by Kuzbanian, the subcellular location of Notch/Kuzbanian interaction, and the timing of Kuzbanian function in relation to other factors influencing Notch activity, especially Presenilin. (b) Two novel genetic screens will be employed to search for factors regulating Notch cleavage and signaling. Certain Notch transgenes produce dominant male lethality, and a selection for mutations that rescue this phenotype will be the basis for one strategy. A second genetic screen will involve the simultaneous over-expression of Delta and each of about2,000 randomly chosen target genes in a Notch-dependent developing tissue, the eye. (c) A cultured cell assay has been developed that allows signaling by various activated Notch proteins to be measured quantitatively and qualitatively. Proteins implicated by genetic screening will be directly tested in cultured cells for their correlated effects on a Notch-dependent transcriptional response, and Notch proteolysis. (d) We will determine the function of 4 newly identified Drosophila relatives of kuzbanian. One approach will involve a recently devised method for stably repressing each gene's function by tissue-specific expression of inverted-repeat RNAs. (e) Factors regulating Notch transport to and from the cell surface, and controlling nuclear translocation of Notch will be identified and characterized using certain transport deficient Notch proteins.
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