Chlamydomonas Reinhardtii ODA5 Encodes an Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Flagellar Adenylate Kinase: A Dissertation

Abstract

The first type of dynein identified, axonemel dynein (Gibbons and Rowe, 1965), slides adjacent microtubules within the axoneme, generating the force necessary for ciliary and flagellar beating. The outer dynein arm is an important component of the flagellar axoneme, providing up to 60% of the force for flagellar motility. In the absence of the outer arm, cells swim with a slow-jerky motion at about 1/3 rd the speed of wild-type cells, and the flagellar beat frequency is markedly reduced. Sixteen genes (ODA1-ODA16) have been identified that are required for outer arm assembly in Chlamydomonas reinhardtii . In addition, PF13, PF22, and FLA14 are required for outer dynein arm assembly, but their phenotypes are pleiotropic, suggesting that they affect additional flagellar components. Of the uncloned genes, ODA5, ODA8, and ODA10 are of particular interest because they do not encode subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC). Mutant alleles of these genes are unable to complement in temporary dikaryons, suggesting that the gene products interact with each other (Kamiya, 1988). Since the genes encoding all of the known components of the outer dynein arm and the ODA-DC have been characterized, it is of great interest to identify the gene products of these additional, uncloned ODA alleles. The first chapter provides an introduction to the Chlamydomonas flagellum, the dyneins in general, the outer dynein arm in particular, and mutations that impinge on the assembly and regulation of this important axonemal structure. The second chapter addresses the identification and isolation of genomic DNA containing the ODA5 gene. Utilizing a NIT1-tagged oda5-insertional mutant, I identified sequences flanking the site of the inserted NIT1 gene. These sequences were used to isolate wild-type genomic clones spanning the ODA5 gene. When transformed into the oda5 mutant, the wild-type clones rescued the mutant phenotype. These results demonstrated the successful isolation of the ODA5 gene. The third chapter describes the identification of the ODA5 gene and its corresponding cDNA. The rescuing genomic fragments were sequenced. Gene modeling was used to predict intron-exon splice sites. Primers to predicted exons were designed and used to obtain the ODA5 cDNA. The gene structure of Oda5 was analyzed and its predicted amino acid sequence deduced. Secondary structure predictions indicate that Oda5p is likely to contain a series of coiled-coil domains, followed by a poly-glycine sequence and a short, highly charged region. Northern analysis demonstrated that ODA5 gene expression is upregulated by deflagellation, a hallmark of many flagellar mRNAs. Data in CHAPTER IV further characterize the Oda5 protein and its association with the axoneme. Oda5p localizes to the flagellum, consistent with the enhancement in mRNA levels in response to deflagellation. Within the flagellum, Oda5p is an axonemal component that is released from the axoneme upon high salt…