We plan to use monoclonal antibodies to identify and characterize the molecular species on the skeletal myoblast membrane that are germane to muscle development. Using one such monoclonal antibody we have identified, H36, a muscle-specific, developmentally regulated, integral membrane protein. We propose to use H36 as a model system to understand the nature, functions and interactions of surface membrane molecules in myogenesis. The structure of H36 protein and its possible species- and stage-specific isoforms will be determined using peptide analysis and immunochemical techniques. The complete primary sequence of H36 will be deduced from nucleotide sequence analysis; secondary structure and functional domains will be determined from the protein sequence, from immunological peptide mapping and from inhibition studies. Recombinant DNA technology will be used to study the structure and the mechanism of regulation of expression of the H36 gene. The physiologic role of H36 and other cell surface antigens will be determined by using antibodies and flow cytometry to select mutants lacking specific membrane molecules. These mutants, and antisense vectors, will be used to study the effects of loss of individual molecular species on myoblast development. Flow cytometry utilizing antibodies to H36 protein will be used to define and isolate cells of the myogenic lineage. These cells will be used to examine the biochemistry of earlier stages of myogenesis that have heretofore been difficult to study. This combined genetic and immunochemical approach will be extended to other antigens of interest, leading to identification of membrane components with their respective functions and to an understanding of how membrane structure and function relate to myoblast differentiation.
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