The Chlamydomonas reinhardtii ODA3 Gene Encodes a Protein of the Outer Dynein Arm Docking Complex

Abstract

We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the outer dynein arm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737– 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83.4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme.

Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase

“…Flagellar beat frequency was determined as described previously (Kamiya, 2000). Kamiya, 1988;Takada et al, 2002ODA2 ␥DHC Kamiya, 1988Mitchell and Rosenbaum, 1985;Wilkerson et al, 1994ODA3 DC1 Kamiya, 1988Koutoulis et al, 1997ODA4 ␤DHC Kamiya, 1988Mitchell and Brown, 1994;Sakakibara et al, 1993 ODA5 This study Kamiya, 1988ODA6 IC2 Kamiya, 1988Mitchell and Kang, 1991ODA7 ? Kamiya, 1988ODA8 ?…”

“…Chlamydomonas reinhardtii strains used in this study include the following: CC-2454 (cw15, nit1-305, mtϪ), mtϩ), mtϪ), CC-2229 (oda1, mtϩ), CC2233 (oda3-1, nit1, nit2, AC17, mtϪ), and137C (nit1-137, nit2-137, mtϩ), all from the Chlamydomonas Genetics Center (Department of Biology (Duke University, Durham, NC); oda5ϩ (oda5-1, mtϩ), oda3ϩ (oda3-1, mtϩ), oda8ϩ (oda8, mtϩ), oda9ϩ (oda9, mtϩ), oda10ϩ (oda10-1, mtϩ) (Kamiya, 1988), V87.2 (oda10-2, nit1::NIT1, NIT2, agg1, mtϪ) (Koutoulis et al, 1997), oda9-V5 (oda9, mtϩ) (Wilkerson et al, 1995), and 45BO3 (oda5-2, cw15, nit1-305::NIT1, mtϪ), an insertional allele of oda5.…”

“…RNA was cross-linked to the membranes by using a Stratalinker (Stratagene). Chlamydomonas genomic DNA isolations were performed as described previously (Koutoulis et al, 1997). Genomic DNA was separated on 0.8% agarose gels.…”

“…All transformations were done using the glass bead method as described previously (Kindle et al, 1989;Koutoulis et al, 1997). The insertional mutant 45BO3 was generated by transforming CC-2454 cells with plasmid pMN24 containing the Chlamydomonas NIT1 gene.…”

“…The correct positioning of the outer arm is due at least in part to its association with the outer dynein arm-docking complex (ODA-DC), which is required for attachment of the outer arm onto the A-tubule. The ODA-DC, a heterotrimeric complex comprised of subunits DC1 (Koutoulis et al, 1997), DC2 (Takada et al, 2002), and DC3 (Casey et al, 2003), assembles onto the site normally occupied by the outer arm even in the absence of the latter structure.…”