GSBS Dissertations and Theses

Approval Date

January 2004

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences

Subjects

Chlamydomonas reinhardtii; Dynein ATPase; Flagella; Adenylate Kinase; Gene Expression; Academic Dissertations; Dissertations, UMMS

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 1/3rd 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 extraction, as are the ODA-DC and the outer dynein arm. However, Oda5p does not associate with this super-complex in the high salt extract as determined by sucrose gradient sedimentation. Oda5p assembles onto the axoneme independently of the outer dynein arm and the ODA-DC,demonstrating it does not require these complexes for localization. Furthermore, Oda5p assembles onto the axoneme in the oda8, but not the oda10 mutant, demonstrating a role for the Oda10 protein in localization of Oda5p. These data provide the first biochemical evidence for an interaction between Oda5p and Oda10p.

CHAPTER V reveals the discovery of a previously unrecognized phenotype exhibited in both oda5 and oda10 mutant strains: a defect in the assembly of a previously unknown flagellar adenylate kinase (AK). The protein levels of this flagellar AK are reduced in oda5 mutant axonemes, as determined by quantitative mass spectrometry. Direct enzymatic assays confirmed a reduction in AK activity in both oda5 and oda10 mutant axonemes, providing a second line of biochemical evidence supporting a complex containing Oda5p and OdalOp. The sequence of the flagellar AK gene and its cDNA were determined.

CHAPTER VI details our efforts to identify the ODA10 gene. Genomic clones were isolated, which contain sequences at, or near, the ODA10 locus. Analysis of the genomic clones yielded no insights into the identity of the ODA10 gene. The inability of these clones to rescue the Oda10-motility phenotype indicates that these clones most likely do not contain an intact ODA10 gene.

And lastly, CHAPTER VII discusses future experimentation that can be done based on the data provided by the current study.

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