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In helminths, including the free-living nematode, Caenorhabditis elegans, serotonin (5-HT) acts as an important neuroactive agent and is associated with carbohydrate metabolism, glucouse utilization, motility, feeding and reproductive behaviour. In mammals and other organisms, 5-HT is synthesized through the action of tryptophan hydroxylase (TPH). TPH is the rate limiting enzyme in the biosynthesis of 5-HT, and as such sets the pace for the formation of 5-HT. TPH is a member of a family of enzymes that hydroxylate aromatic amino acids and have an absolute requirement for the pterin cofactor, tetrahydrobiopterin (BH4). It is unknown if this same enzyme catalyzes the synthesis of 5-HT in C. elegans and other helminths. / Based on sequence information from the C. elegans Genome Data Base and RT-PCR, we have cloned a full-length C. elegans TPH cDNA (CeTPH) that shows high homology to mammalian TPH. The predicted coding sequence of CeTPH was subcloned into the prokaryotic expression vector, pET-15b, and the resulting construct was introduced into E. coli (BL21 DE3 pLys strain) for IPTG-inducible expression of CeTPH protein. Results show that CeTPH expressed in E. coli has TPH activity and also shows an absolute requirement for the cofactor, BH4, just as shown previously for the mammalian enzyme. It has been well established that 5-HT is present and is biologically active in the tissues of C. elegans. By way of characterizing furthur CeTPH, we examined the localization of TPH in whole mounts of C. elegans by immunofluoresence using a polyclonal antibody against TPH. / Taken together, the results of this thesis characterize at the structural, functional and in situ levels one of the most primitive forms of TPH enzyme ever cloned.

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Proper cell fate specification is crucial for development, and dysregulation in cellular signaling pathways can lead to deleterious effects like cancer. The conserved LIN- 12/Notch signaling pathway mediates fate specification in many cellular contexts, and multiple regulatory mechanisms ensures appropriate LIN-12/Notch activity in each context. Here, I have identified several cis-regulatory domains and trans-acting factors that contribute to the negative regulation of LIN-12/Notch in Caenorhabditis elegans. In this thesis, I find that LIN-12/Notch requires binding to LAG-1/CSL and association with the nuclear complex for protein turnover in the C. elegans vulval precursor cells (VPCs). I also identify two layers of negative regulation in the VPCs and their descendants. The E3 ubiquitin ligase SEL-10/Fbw7 mediates degradation of LIN-12/Notch via the PEST domain in the VPCs, while a novel structural conformation in the C-terminal end of the LIN-12/Notch intracellular domain is required for downregulation in the descendants. Through an RNAi screen for negative regulators, I isolated 13 conserved kinases that downregulate LIN-12/Notch activity. Of these 13 kinases, CDK-8 had been previously implicated in Notch turnover, while the other 12 are novel negative regulators. I provide evidence that 5 of the kinases regulate LIN-12/Notch through modulation of the intracellular domain. Furthermore, I conduct a deeper investigation into CDK-8, which is the kinase component of the Mediator complex. I determine that CDK-8 acts with the rest of the Cdk8 Kinase Module and independent of the Mediator core to negatively regulate LIN-12/Notch, and that CDK-8 kinase activity is required for this process. Lastly, I find that sur-2/MED23 and lin-25/MED24 are required for LIN-12/Notch ligand transcription, independent of the Cdk8 Kinase Module and Mediator Head and Tail components.

clk-1 mutants of the nematode C. elegans have been phenotypically characterized in previous work and found to have decreased rates of development and of periodic adult behaviours, such as defecation, pumping and swimming (Wong et al., 1995). In this study, the defecation periods in older wild type and clk-1 mutant worms were found to be similar despite their differences in young adults. Transgenic strains which express high levels of clk-1 were characterized phenotypically. Over-expression of the clk-1 gene has no observed effect on physiological rates as measured conventionally in young adults but rather has an effect on defecation rates in an age-dependent manner. A proportion of ageing transgenic animals over-expressing clk-1 did not have decreased defecation period, as seen in ageing wild type and clk-1 mutants. This suggests that the action of CLK-1, which functions in controlling physiological rates, is reduced in older wild type worms. As previously characterized, clk-1 mutants have lengthened life spans compared to wild type (Wong et al., 1995). Consistent with this effect, we found transgenic strains with high clk-1 expression to have a shortened average life span. Furthermore, no effect on average life span was found in transgenic control strains in which clk-1 expression was not altered, confirming that the presence of the transgene and the phenotype marking its presence do not influence the observed effects in strains with high clk-1 expression. Together, the findings from both the studies on physiological rates and life span suggest that clk-1 plays a role in determining the rate at which worms age.

The Caenorhabditis elegans gene gro-1 belongs to the Clock group of genes. The four known genes making up this class are believed to be involved in a general mechanism acting to coordinate the time-dependent processes in the organism. Mutation of these genes alter the timing of many disparate processes. This results in the mean lengthening of embryonic and post-embryonic development, as well as in a lengthening of the periods of a number of adult behaviours including pharyngeal pumping, swimming and the defecation cycle. These mutants also exhibit a significantly increased life span. The gene gro-1 has been cloned and encodes a metazoan N6-(Delta2) isopentenyl PPi: tRNA isopentenyl transferase. In S. cerevisiae and bacteria this enzyme has been shown to modify tRNAs that code for codons begining with U. This modification consists of the isopentenylation of the adenine residue at position 37, adjacent to the anti-codon. Interestingly, gro-1 is the fifth member of an operon. Preliminary expression studies with GFP reporter constructs suggest that as in yeast, GRO-1 is expressed in the cytoplasm and mitochondria, as well as in the nucleus.

The gene clk-2 of C. elegans is required in both the germline and the soma, for subsequent embryonic viability, and for developmental and behavioural rates, respectively, clk-2 encodes a protein that is homologous to Tel2p in yeast, which is required for the telomere length regulation. It has been demonstrated that clk-2 affects telomere length also in worms and human cells. By now the exact biochemical function of CLK-2 is unknown. In order to shed light onto the function of the gene clk-2, a two-hybrid screen was carried out to identify the interactors of the protein CLK-2. A potential interactor of CLK-2, Y105C5B.19, was identified in the screen. Y105C5B.19 is a novel gene and does not have homologues in other species. Y105C5B.19 contains an MSP (major sperm protein) domain, therefore it is possible that it could be involved in the processes that regulate oocyte maturation, gonadal sheath contractions, or sperm mobility. Interestingly, given that clk-2 is required for subsequent embryogenesis at some point during a narrow time between the end of oocyte maturation and the 2-cell stage, it is tempting to speculate that the interaction between CLK-2 and Y105C5B.19 might be functionally relevant. The lethality of clk-2(qm37) mutants might result from delayed consequences of defects in ovulation and/or fertilization, and perhaps such defects could result from the disruption of the interaction between CLK-2 and Y105C5B.19. The amino acid substitution C772Y resulting from the clk-2(qm37) mutation was found to disrupt the interaction between CLK-2 and Y105C5B.19 in a two-hybrid assay, lending support to the idea that the interaction between CLK-2 and Y105C5B.19 takes place in vivo.

In Caenorhabditis elegans, mutations in the clk-1 gene result in delayed embryonic and post-embryonic development, a slowing down of rhythmic behaviors and an extended life span. CLK-1 encodes the demethoxyubiquinone (DMQ or DMQn) hydroxylase in the ubiquinone (CoQ or Qn) biosynthesis pathway. Thus, clk-1 mutants produce DMQ instead of CoQ. In order to understand the relationship between the CoQ biosynthesis defect and the pleiotropic phenotype of clk-1 mutants, I isolated a deletion mutant, coq-4(qm143), in C. elegans. In Saccharomyces cerevisiae, mutants in COQ4, the coq-4 homologue, do not produce ubiquinone, like those in COQ7, the clk-1 homologue. coq-4(qm143) is a non-strict maternal-effect lethal mutation. Most of the progeny from a homozygous coq-4(qm143) hermaphrodite die during embryogenesis. However, homozygous coq-4(qm143) hermaphrodites from a heterozygous mother can develop and behave normally until adulthood. As adults, they become uncoordinated and paralytic, and are defective in egg-laying. Unlike hermaphrodites, homozygous coq-4(qm143) males are fully maternally rescued. The qm143 is a 1469 base pair deletion, which completely removes the coq-4 gene and does not affect the coding sequence of any other gene. By performing germline transformation, I also showed that the non-viable phenotype of coq-4 (qm143) is indeed due to the removal of the coq-4 gene itself. The preliminary study of COQ-4 expression pattern by using a COQ-4::GFP fusion protein indicates that COQ-4 is expressed in mitochondria of the worm.

In an effort to find new genes involved in development and the biological timing, I carried out a new screen for viable maternal-effect mutations similar in protocol to the previous screen in which 24 such genes have been identified. I screened 10,600 genomes and isolated 6 slow-growing mutations and 5 behavioral and morphological mutations. Another maternal-effect slow-growing mutation is isolated from a screen for both maternal-effect and non maternal-effect slow development mutations. Genetic mapping suggests that none of the seven slow growing mutations corresponds to previously identified genes, so seven new clk genes (clk-4, clk-5, clk-6, clk-7 clk-8, clk-9, clk-10) have been identified. Because most identified clk genes (clk-2, clk-4 to clk-10 and gro-1) are defined by single allele, we believe that these genes have not yet been saturated. Mutants of seven new clk genes have typical Clk phenotypes: a mean lengthening of embryonic development, postembryonic development, defecation cycle and life span. As the screening procedure did not involve any measure of life span, it is suggested that slow life is sufficient for long life. As expected, all seven mutations can be maternally rescued.