There are three main projects. 1) The construction and study of several recombinant retroviruses that should permit us to learn more about promoter interference and trans-acting promoter repression in embryonic cells. 2) The study of protein/viral DNA interactions in embryonic cells. The emphasis is to determine what proteins are involved in viral promoter """"""""repression"""""""" in embryonic cells and to determine their mechanism of action. These experiments will utilize DNase hypersensitivity, footprinting, and Exo III mapping techniques. 3) The microinjection of in vitro synthesized viral RNA in cultured cells and embryos. The thrust is to learn more about the viral replication cycle and to develop a gene delivery system that will permit efficient insertion of genes, that lack viral promoter, enhancer, and repressor binding sequences into transgenic mice in single copy. An effort will be made to refine the system to eliminate the need for viral packaging and reinfection. Finally, in vitro synthesized RNA microinjection will be used in an attempt to localize the viral sequences responsible for splicing regulation in retroviruses. The experiments are significant because they are likely to uncover a potent trans-acting repressor in mammalian cells. This repressor may have a more fundamental role than the inhibition of retroviruses, which will enlarge our understanding of transcriptional control. The experiments plan to localize the viral sequences responsible for promoter inactivation. This information should be relevant to those using retroviruses for gene transfer into stem cells for gene therapy. Lastly, the understanding of a mechanism responsible for retroviral inactivation may prove useful in the designing of future therapies for HTLV mediated diseases.
