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No description available.

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.

Several classes of genes determine the lifespan of the nematode Caenorhabditis elegans. Our laboratory is particularly interested in the clk class of genes that is composed of clk-1, clk-2, clk-3 , and gro-1. Mutations in these genes have been shown to extend lifespan and to deregulate several developmental and behavioural processes such as pharyngeal pumping and defecation cycle length. / clk-1 encodes a 187 amino acid mitochondrial protein that is composed of two homologous TRC domains (TRC for T&barbelow;andemly R&barbelow;epeated in C&barbelow;LK-1). Interestingly, the yeast homologue of clk-1, COQ7, has been implicated in ubiquinone biosynthesis. clk-1 (e2519) lesion is a point mutation that changes a conserved amino acid (E148K) in the second TRC domain. A structural model proposed that clk-1 is a di-iron carboxylate protein. We found that mutations in the di-iron binding center abolish CLK-1 activity and modify the subcellular distribution of CLK-1 in clk-1( qm30) null mutants.

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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.

In this thesis, chromosome I rearrangements were used to study the organization of essential genes and regions important for chromosome behaviour in the nematode Caenorhabditis elegans. To facilitate the genetic mapping of mutations in essential genes, rearrangements were isolated using a procedure designed to recover derivative chromosome I duplications shortened by gamma radiation from existing duplications. Sixty-two duplications were isolated in this way. These duplications, along with three deletions isolated in this study and 9 existing deletions of the region, divided the left half of chromosome I into at least 24 regions. Protocols were developed and used to rapidly map mutations into the regions defined by the breakpoints. The techniques and results described demonstrate the feasibility of carrying out a similar analysis on the whole genome. The majority of duplications behaved as if they were free; that is they segregated independently of the euploid chromosome set. While size was an important determinant of mitotic stability, clear exceptions to a size - stability correlation were observed. For example, despite its larger size, hDp72 was lost during cell division more frequently than hDpl8, suggesting features of chromosome structure were important. Shortening of duplications in the unc-11 dpy-5 region caused greater reductions in mitotic stability than similar sized shortenings in the dpy-5 unc-13 region. Therefore, specific sequences appear to influence duplication stability. Some free duplications were also observed to break spontaneously. Breakage occurred at different frequencies for different duplications and correlated with mitotic instability. The meiotic properties of four translocations involving chromosome I were examined. No recombination was observed in any of the translocation heterozygotes along the left (let-362 - unc-13) portion of chromosome I. By isolating a half-translocation chromosome as a free duplication, I mapped the breakpoints of three of the translocations. The boundaries of cross-over suppression coincided with the physical breakpoints. These results agree with the proposal that DNA sequences at the right end of chromosome I are essential for homologue recognition followed by meiotic synapsis and recombination. The published data of other translocations and duplications indicates that each of the other five C. elegans chromosomes has DNA sequences localized to one end that are required for homologue recognition and recombination. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

This thesis describes the genetic analysis of a small region of the Caenorhabditis elegans genome. The region analyzed was defined by the 0.5 map unit interval between the breakpoints of the duplications hDp16 and hDp19, which lies within the dpy-5 unc-13 region on chromosome I. The analysis consisted of the identification and characterization of essential genes in this region. All the lethal mutations analyzed came from a set of 495 EMS-induced, sDp2-rescued lethals described in Howell (1989). The lethal mutations have been maintained as homozygotes, made viable by the presence of a wild-type allele on the sDp2 duplication. I used a combination of mapping techniques to analyze over 200 EMS-induced lethal mutations. A total of 189 new lethal mutations were positioned within the dpy-5 unc-13 interval. Three methods were used for mapping: recombination analysis, lethal rescue using duplications, and deficiency mapping. Duplication mapping was found to be the fastest and most precise method used. 178 of the new lethal mutations were mapped relative to the duplication breakpoints of hDp13, hDp16, and hDp19. These three duplications divided the dpy-5 unc-13 region into three approximately equal-sized zones. This study completes the mapping of the 495 lethals in the sDp2 set. In addition, thirty-three previously identified essential genes lying between dpy-5 unc-13 were positioned with respect to the breakpoints of the duplications hDp12, hDp13, hDp15, hDp16, hDp17, and hDp19. The dpy-5 unc-13 region carries a relatively large number of loci, therefore, I decided to concentrate on the smaller hDp16/hDp19 interval within this region. Complementation analysis was used to define the number of essential genes in the hDP16/hDp19 region. A total of eight new genes were described, six lying in the hDp16/hDP19 region, two lying just outside this region. This brings the total number of essential genes in the hDP16/hDp19 region to sixteen. In addition, as a result of my mapping data, the hDp16/hDp19 region has been subdivided into six intervals with respect to duplication and deficiency breakpoints. The stage of developmental arrest was determined for both the essential genes, and the new lethal mutations (ie. not yet defined by complementation tests), in the dpy-5 unc-13 interval. Although the number of genes studied was not great, the data suggests a relationship between map position and time of developmental arrest. The average forward mutation rate for C. elegans genes was determined to be 5.8 X 10⁻⁵mutations per gene. I have made a comparison of the forward mutation rates of the essential genes in the hDp16/hDp19 region, bli-4 was found to be the most mutable target in the region with nine mutant alleles, giving a forward mutation rate six times higher than average. In the hDp16/hDp19 region, ten of the sixteen essential genes were represented by more than one allele. The minimum estimate of the number of essential genes in the region using a truncated Poisson calculation was twenty. Therefore, the sixteen genes identified represent 80% of the essential genes in this region. This data was extrapolated to give a minimum estimate of approximately 225 essential genes in the 15 m.u. sDp2 region, and 4,500 essential genes in the C. elegans genome. This research has established the hDp16/hDp19 region as a genetically well-defined system for studying the genetic organization of essential genes, as well as the developmental regulation of gene expression, and the functional relationship between adjacent genes. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

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