Our research program is focused upon the function of the genes and proteins of the notch family. These proteins are transmembrane receptors that mediate cell fate decisions in Drosophila and in vertebrates. Human notch-1 (TAN-1) is truncated in some T-cell leukemias, and the intact gene is expressed in adult lymphoid organs and in bone marrow """"""""stem"""""""" cells. 1) We have obtained recombinant expression of the putative ligand binding region of human notch-1 (rh1112). To our knowledge, this represents the first example of a protein with six disulfide bonds expressed in E. coli in soluble, oxidized form. 2) Polyclonal antibodies generated against the recombinant rh1112 recognize intact notch-1 in human lymphoid cell lines. These antibodies detect both the intact and truncated form of TAN-1 in human lymphoblastoid cell lines. In addition, notch is detectable in human peripheral blood mononuclear cells. 3) We have shown that a recombinant protein corresponding to the intracellular region of human notch-1 binds transcription factors of the NF-kB family and functions in an IkB-like fashion. The interaction of TAN-1 with NF- B demonstrates specificity for the p50 subunit. We have shown by co-immunoprecipitation with antibodies to p50 and p65 that the intact notch receptor is associated with NF-kB in Jurkat cells. Notch-NF We have shown by co-immunoprecipitation with antibodies to p50 and p65 that the intact notch receptor is associated with NF-kB in Jurkat cells. Notch-NFkB interaction is functional in transfected cell models in which TAN-1 protein regulates HIV-LTR-kB-dependent transactivation in vitro.4) We have obtained clones encompassing the entire intracellular region of mouse notch-1, and created constructs which mimic the truncation of notch observed in T-cell leukemias. These constructs will be used for transfection studies of the mechanisms of notch-mediated signal transduction and inhibition of differentiation, as well as for the creation of transgenic mice. 5) We have obtained antisense mouse notch-1 constructs, which will be used in transfection studies and to generate an antisense transgenic mouse.
