This project uses both molecular biology and genetics to study the structure and function of flagellar dynein ATPases, which are large, multi- subunit complexes that support motility along both cytoplasmic and flagellar microtubule frameworks. The long-term objective of this work are to determine the mechanism of dynein force generation and its regulation. To determine primary structure and predict higher-order structure in the catalytic heavy chain subunits, genomic clones for the Chlamydomonas reinhardtii outer arm dynein alpha and beta heavy chains will be sequenced. Sequence analysis will also be used to look for nucleotide and microtubule binding domains and homology with other mechanochemical enzymes. Transformation of Chlamydomonas dynein assembly mutants will be developed as a method to express cloned dynein genes. Expression of hybrids between cloned wild-type and sup-1 mutant beta heavy chain genes will be used to pinpoint the sup-1 mutation, which defines a region of this heavy chain involved in motility regulation by radial spoke-central pair microtubule interactions. Sequences analysis will reveal the molecular defect responsible for the suppressor phenotype. Genomic alpha heavy chain and 70 k intermediate chain clones will be used to map the genetic loci encoding each protein by restriction fragment length polymorphism (RFLP) analysis. Previously-identified dynein assembly mutants will then be tested for allelism to these structural gene loci by a combination of RFLP mapping and transformation with cloned genes.
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