Subcellular organelles composed of microtubules are responsible for many of the motions produced by and within cells. The function and regulation of these organelles is critical to the life and growth of both individual cells and multicellular organisms. Malfunctions in these microtubule-based organelles influence global aspects of cell behavior including chromosome segregation, cell division, cell differentiation, and tissue morphogenesis; and are reflected in numerous medical problems including cancer, congenital chromosomal syndromes, and birth defects. The long range goal of our research is to elucidate the molecular mechanisms that govern the function of microtubule-based organelles. At a basic level, the function and regulation of microtubule-based organelles depends on the motors that drive them. Microtubule motors are energy-transducing enzymes that convert the energy derived from nucleotide binding and hydrolysis into the sliding of microtubules and/or the movement of vesicles or organelles relative to the microtubule. A microtubule motor, cytoplasmic dynein, has been identified in Drosophila embryos and its motor activity characterized in vitro by microtubule gliding assays. Molecular and genetic strategies will be used to investigate the cellular and developmental functions of cytoplasmic dynein and to identify the structural and functional domains of the dynein heavy chain polypeptide that are required for its functions. DNA sequences that encode the heavy chain polypeptide of cytoplasmic dynein will be isolated by expression cloning techniques using a monospecific antibody that recognizes the dynein heavy chain. Overlapping clones containing the entire protein coding sequence will be isolated and analyzed to predict the primary sequence of the polypeptide and to identify conserved motifs shared with other mechano-chemical enzymes. Recombinant dynein polypeptides will be produced from the isolated coding sequences and will be assayed in vitro and in vivo to identify structural and functional domains of the dynein polypeptide. Antibodies against recombinant dynein fragments will be used to relate the primary amino acid sequence to the two-dimensional structure observed by electron microscopy, and to characterize the distribution of cytoplasmic dynein in Drosophila embryos. The isolated dynein clones will provide the means to begin a mutational analysis of the cellular and developmental roles of cytoplasmic dynein in vivo.
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