Thesis--PlanA (M.S.)--University of Wisconsin--Stout, 2002. / Includes bibliographical references.

Mutations in the Caenorhabditis elegans maternal-effect gene clk-1 result in a highly pleiotropic phenotype, characterized by a general slow down in embryonic and larval development, as well as a slowing down of adult behaviors including defecation, pharyngeal pumping and swimming. First generation homozygous clk-1 mutants descended from a heterozygous mother are fully rescued for these mutant phenotypes. It has been shown that CLK-1 protein is a hydroxylase that acts in the conversion of demethoxyubiquinone (DMQ) to 5-hydroxyubiquinone, in the ubiquinone (Q) biosynthesis pathway. Consequently, clk-1 mutants accumulate the Q9 precursor, DMQ9 (the subscript refers to the length of the isoprenoid side chain). Here, I show that the profound maternal rescue observed in clk-1 maternally rescued animals is due to presence of the CLK-1 protein throughout larval development, in sufficient amounts to catalyze the production of Q9. clk-1 mutants have been shown to have a dietary requirement for Q8 due to their inability to synthesize Q9. I demonstrate that clk-1 maternally rescued animals have sufficient amounts of Q 9 to complete larval development and produce an almost full brood when raised on a Q8 deficient E. coli strain. I also show that prolonged arrest at the first larval stage, which is likely to result in degradation of any maternally contributed mRNA or protein, brings about a Clk mutant phenotype in maternally rescued animals. Finally, I reveal that the Clk mutant phenotype can be rescued at any larval stage by ectopic expression of CLK-1, suggesting that there is no developmental window for the rescue of clk-1 mutants by CLK-1. These results identify perdurance of maternally contributed product throughout development as the mechanism that accounts for the maternal effect observed in clk-1 mutants.

Coenzyme Q (ubiquinone or UQ) is a prenylated benzoquinone lipid that is found in membranes throughout the cell, and functions in a wide variety of enzyme-mediated redox reactions. One of the primary roles of UQ is its involvement in respiratory metabolism, where it functions in the inner mitochondrial membrane of eukaryotic cells as a transporter of electrons and protons in complexes II and III. The biosynthesis of UQ involves two separate O-methylation steps. Coq3p which is 40% identical to the Escherichia coli O-methyltransferase, UbiG, catalyzes both O-methylation steps. / In order to elucidate the function of genes involved in UQ biosynthesis, a coq-3 null mutant in the nematode C. elegans has been isolated by mean of reverse genetics. About 3,000,000 haploid genomes have been screened and a deletion in this gene isolated. Many technical difficulties have been encountered and modification concerning the details of the screening protocol was required to bypass them. The loss of coq-3 function in nematodes leads to developmental arrest and sterility, even when the worms are fed with bacteria that produce UQ. This demonstrates that UQ is required for the development and fertility of the worms.

Thesis (Ph. D.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references. Also available via the World Wide Web.

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Title from PDF of title page (University of Missouri--Columbia, viewed on Mar. 15, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisors: Dr. Ingolf U. Grün, Dr. Mengshi Lin. Vita. Includes bibliographical references.

clk-1 encodes a hydroxylase that is necessary for ubiquinone (UQ) biosynthesis. clk-1 mutants do not synthesize UQ, but instead accumulate the precursor demethoxyubiquinone (DMQ). When fed on bacteria that synthesize UQ the mutants are viable but display slow development, behaviours and aging. However, they arrest development when fed on UQ synthesis-deficient bacteria. I have taken a genetic suppressor approach to investigate the causes of the various phenotypes as well as of the dietary requirements of the clk-1 mutants. / We identified two classes of mutants that suppress the defecation phenotypes of clk-1. All of these "dsc" mutants suppress the lengthened cycle of clk-1. Class I mutants also restore the ability to react normally to changes in temperature whereas the Class II mutants do not. The characterization of the Class I mutants suggests that part of the phenotype of clk-1 is due to an alteration of lipid metabolism, likely the level of lipid or lipoprotein oxidation. dsc-4 encodes the worm homolog of the Microsomal Triglyceride Transfer Protein (MTP), a protein required for the formation of low density lipoproteins (LDL) in vertebrates, and whose absence in people leads to abetalipoproteinemia. dsc-3 appears to be allelic to tat-2, which encodes a type IV P-type ATPase that is related to a family of human aminophospholipid transporters that includes ATP8B1/FIC1, whose inactivation results in cholestatic liver disease. dsc-3 and dsc-4 appear to affect distinct aspects of lipid metabolism. A general link between the Class II mutants and clk-1 remains elusive. dsc-1, a Class II gene, encodes a paired-like homeodomain transcription factor that is necessary for the GABA sensitivity of enteric muscles. / We also identified 9 clk-1(e2519)-specific suppressors, which suppress most Clk phenotypes, including their requirement for dietary UQ. Our analysis of these suppressors reveals that it is the lack of UQ rather than the presence of DMQ that is responsible for most phenotypes. In addition, they allowed us to show that most Clk phenotypes can be uncoupled from each other. We cloned six suppressors and all encode missense tRNA(Glu) suppressor genes. To my knowledge, these represent the first missense tRNA suppressors identified in any metazoan.

Ubiquinone (UQ) is a lipid found in all cellular membranes. It is involved in multiple cellular processes, either directly or through its effect on the redox status of the cell. clk-1 encodes a highly conserved hydroxylase required for UQ biosynthesis. In C. elegans, mutations in clk-1 result in the accumulation of an UQ precursor, DMQ, and a pleiotropic phenotype in the mutants characterized by the slowing down of development, behaviors and aging. Additionally, in the absence of dietary UQ, clk-1 mutants also show a transient growth arrest and are sterile. Mutants that can suppress both sets of phenotypes in the point mutant clk-1(e2519) have been isolated. Their suppression patterns indicate that various aspects of the clk-1 phenotype can be uncoupled from each other. Furthermore, the analysis of their quinone content suggests that the phenotypes on UQ-producing bacteria are caused by the inability of dietary UQ to completely substitute for endogenous UQ. These suppressors carry mutations in tRNA genes, and thus to our knowledge, they are the first tRNA missense suppressors found in any metazoan.