The ultimate objective of this research project is the elucidation of the molecular mechanism of leukemogenesis by avian myeloblastosis virus (AMV). We propose to characterize the structure and subcellular location of the AMV oncogene product, a nuclear protein of 48,000 Mr, p48myb, and of its normal homolog encoded by the cellular protooncogene (proto-myb), and to investigate the respective functions of these two molecules in leukemogenesis and normal hematopoiesis.
Specific aims i nclude: 1) Characterization of p48myb. Purification and molecular analysis (e.g., post-translational processing, kinetics of synthesis, possible role in DNA replication or transcription); subnuclear localization (e.g., nuclear subfractionation, intermolecular associations, immuno-electron microscopy); identification of biologically active site(s) by site-directed mutagenesis (e.g., construction of AMV recombinant clones with altered oncogene, transfection, virus recovery, and analysis of the mutants for their role in the induction and maintenance of transformation). 2) Molecular basis of hematopoietic specificity of AMV transformation. Characterization of p48myb expressed in cells that are not target cells for transformation; exchange of the LTR and C-regions of AMV for RAV-2 LTR and C-regions, and effect of these alterations upon transformation in vivo and in vitro; comparative studies of AMV and the related E26 virus which contains a subset of the proto-myb sequences transduced by AMV, but which transforms fibroblasts while AMV does not; construction of a murine myb virus by inserting the AMV oncogene into a Moloney murine leukemia virus and determining its transforming capabilities. 3) Relation of AMV oncogene to the proto-oncogene. Unequivocal identification of p75 as the cellular homolog of p48myb (e.g., peptide mapping, use of antisera against purified p48myb and against the product of a myb bacterial expression vector); intracellular location and function of the cellular homolog; determination of alterations in the proto-myb gene which are required for leukemogenesis (e.g., exchange of homologous regions of myb with proto-myb cDNA in recombinant clones of AMV, and insertion of longer segments of proto-myb cDNA, including 5' and 3' coding sequences which are not present in the AMV oncogene).
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