To understand the role of dynein heavy chains in the assembly of a dynein motor complex, we are analyzing a series of motility mutants in Chlamydomonas that were generated by insertional mutagenesis (Tam & Lefebure, 1993, Genetics 135375). The characterization of one of these strains, 27B3, indicates that the selectable marker inserts into the structural gene for a novel dynein heavy chain, now known as Dhc10. Northern analyses of the Dhc10 mutant strain reveal that the mutation results in a truncated Dhc10 transcript that is approximately one-third the size of the wild-type transcript. Biochemical and structural analyses from our lab and others indicate that Dhc10 mutant axonemes lack the inner dynein isoform known as the I1 complex, a multiprotein, two-headed complex that repeats every 96nm along the length of the axoneme. We have recently been able to partially rescue the motility defect in the Dhc10 mutant by transforming this strain with a wild-type fragment of the Dhc10 gene, suggesting that the Dhc10 gene product is a component of the I1 complex. We are currently using EM and image averaging techniques to determine which I1 structures have been restored to the rescued axonemes. Future experiment will address whether all subunits of the I1 complex are assembled in the rescued axonemes, and will focus on further characterizing the minimum region of the Dhc10 gene required for complex assembly. ?
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