Identification of oda 6 as a Chlamydomonas Dynein Mutant by Rescue with the Wild-type Gene

We find that two Chlamydomonas outer arm dynein assembly loci, oda6 and oda9, are located on the left arm of linkage group XII, in the vicinity of the previously mapped locus for a 70,000 Mr dynein intermediate chain protein. Restriction fragment length polymorphism mapping indicates that this dynein gene is very closely linked to the oda6 locus. A cDNA clone encoding the 70,000 Mr protein was isolated, sequenced, and used to select genomic clones spanning the corresponding locus from both wild-type and oda6 libraries. When wild-type clones were introduced into cells containing an oda6 allele, the mutant phenotype was rescued, while no rescue was observed after transformation with oda6 clones. Genetic analysis further revealed that newly introduced gene copies were responsible for the rescued phenotype and thus confirms that ODA6 encodes the 70,000 Mr dynein intermediate chain protein. The inability of oda6 mutants to assemble any major outer arm dynein subunits shows that this protein is essential for assembly of stable outer dynein arms. This is the first use of transformation with a wild-type gene to identify the product of a Chlamydomonas mutant. YNEIN ATPases are exceptionally large (1-2 x 106 Mr), multisubunit enzymes that translocate along microtubules. Flagellar dyneins generate force for flagellar beating, while the related cytoplasmic dyneins, such as MAP1C (Paschal et al., 1987), contribute to retrograde axonal transport (Paschal and Vallee, 1987) and other forms of intracellular motility (Scholey et al., 1984; Schroer et al., 1989) including, perhaps, mitotic chromosome movements (Steuer et al., 1990; Pfarr et al., 1990). One way to determine the structure-function relationships in such large multisubunit complexes is through the analysis of mutations that disrupt normal function of the complex, and much effort has been devoted to the isolation of flagellar dynein mutants, primarily in the single-celled green alga Chlamydomonas reinhardtii (Huang et al., 1979; Kamiya, 1988; Kamiya et al., 1989; Mitchell and Rosenbaum, 1985; Sakakibara et al., 1991). The recent development of a simple method for reliable high-frequency transformation of Chlamydomonas (Kindle, 1990) has now opened the way for molecular analysis of dynein mutants. The outer row of dynein arms in Chlamydomonas is composed of three •400-kD heavy chains (alpha, beta, and gamma), two intermediate chains (80 and 70 kD), and at least eight light chains (10-30 kD) (Pfister et al., 1982; Piperno and Luck, 1979). A wide variety of mutations affecting flagellar structure and function have been isolated in this organism, including mutations at 12 loci that prevent formation of functional outer row dynein arms (Mitchell and Rosenbaum, 1985; Kamiya, 1988). All but one of these loci were identified on the basis of alleles that block the transport of dynein subunits into the flageUar comparianent and/or prevent their assembly onto flagellar doublet microtubules, so that all three outer arm heavy chain subunits are missing from gels of flagellar proteins (outer arm dynein assembly, or oda mutants). The one exception, sup-pfl, was first identified as a suppressor of radial spoke and central pair defects in which the beta dynein chain has an altered electrophoretic mobility (Huang et al., 1982). Restriction fragment length polymorphism (RFLP) ~ analysis has confirmed that sup-pfl is linked to the beta dynein gene (Ranum et al., 1988) and genetic analysis shows that sup-pfl is allelic to assembly mutant oda4 (Luck and Piperno, 1989). The other eleven loci, whose gene products have not yet been identified, could potentially encode either additional key structural components of the complex or factors involved in dynein synthesis and/or assembly. Here we use genetic mapping and complementation with a genomic clone to identify one of these mutations, oda6, as a defect in the 70-kD intermediate chain gene. Materials and Methods Chlaraydomonas Strains and Genetics Chlamydomonas stocks were maintained by standard methods (Harris, 1. Abbreviation used in this paper: RFLP, restriction fragment length polymorphism. © The Rockefeller University Press, 0021-9525/91/05/835/8 $2.00 The Journal of Cell Biology, Volume 113, Number 4, May 199I 835-842 835 on A ril 3, 2017 D ow nladed fom Published May 15, 1991