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published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The nematode <i>Caenorhabditis elegans</i> is a small invertebrate whose nervous system, general anatomy, and normal development are all (comparatively) extremely simple and reproducible, and have all been well characterised. This dissertation describes work based on two different approaches to the study of the control of neural development in <i>C. elgans</i>. In the first part the course of neural outgrowth in the region of the ventral nerve cord was followed from electron microscope reconstructions of a series of fixed embryos. Following this, neurons whose processes grew out early were removed by laser ablation of their parent cells and the effect on subsequent nerve outgrowth was assayed by electron microscope reconstruction. The first process to grow along the ventral cord is that of AVG, and its presence is required for the normal, highly asymmetrical structure of the cord. Two more examples of dependency on particular nerve processes for correct guidance can be deduced from experiments in which cells at the back of the animal were removed. The observations of normal development and the ablation experiments can in some cases be related to defects seen in <i>uncoordinated</i> mutants with defective nerve process organisation. The second approach was to establish and analyse a computer data base containing all the synaptic connectivity data obtained by White et al. (1986), who reconstructed at an electron microscope level the entire central nervous system regions of two <i>C. elegans</i> specimens. Since the circuitry is highly reproducible, comparisons of connections between equivalent pairs of cells can be used to infer properties of synapse formation. Overall, the <i>C. elegans</i> circuitry is anatomically highly directional, and what little chemical synaptic feedback that is seen is mostly part of reciprocal synaptic connections. There is also evidence for physical organisation of the nerve processes in subbundles of the main process tract in the central nervous system.

Many obese individuals and type 2 diabetes mellitus (T2DM) patients have elevated levels of insulin. Hyperinsulinemia is a major cancer risk factor in T2DM individuals and activated insulin receptor (IR) has been linked to many types of cancer and poor survival. However, the mechanisms that account for the link between the hyper-active insulin signaling and cancer risk is not well understood. PTEN plays an antagonistic role in the canonical insulin signaling pathway, and is the second most commonly mutated tumour suppressor (after p53) found in human cancers. In many cancers the PTEN gene is not deleted, but instead the protein is lost. Therefore the regulation of PTEN protein in humans is of great importance. Here we hypothesized that the activated insulin signaling down-regulates PTEN. Considering that insulin signaling is highly conserved from C. elegans to human, I used C. elegans as a model and showed that DAF-2, the worm homolog of IR, is a negative regulator of DAF-18, the worm homolog of PTEN. In addition, I showed that DAF-28, the worm homolog of insulin, also negatively regulates DAF-18/PTEN. I used western blot and immunostaining to show that the protein level of DAF-18/PTEN is increased in the daf-2/IR and daf-28/insulin mutants. I further showed that daf-18/Pten is genetically epistatic to daf-2/IR in regulating neuronal development. I then employed human cell culture experiments and reported that this negative regulation is conserved in human cancer cell lines. I showed that knocking-down IR through siRNA up-regulates PTEN, and over-expressing a gain-of-function IR down-regulates PTEN. I also showed that insulin stimulation dramatically decreased PTEN and this decrease is dependent on IR. I further confirmed a physical association between IR and PTEN in both human and C. elegans, and reported that IR could phosphorylate PTEN. To provide mechanistic insight to DAF-18/PTEN regulation, I identified another protein, which is a ubiquitin ligase, that functions in insulin signaling to down-regulate DAF-18/PTEN. Additionally, I also provided evidence that insulin signaling cross talks with Eph receptor signaling. In summary, my findings will be informative for cancer biologists to study the roles of these genes in carcinogenesis. / Thesis (Ph.D, Biology) -- Queen's University, 2013-02-04 14:37:29.376

In all organisms studied so far, the recognition of a functional poly(A) site is essential for RNA polymerase II termination at the end of nearly all genes transcribed by this enzyme (Whitelaw and Proudfoot, 1986; Guo et al., 1995; Birse et al. 1997). A number of eukaryotes have some of their genes organised in polycistronic structures which resemble bacterial operons (Davis and Hodgson, 1997; Ganot et al., 2004; Spieth et al. 1993), and in C. elegans, approximately 20% of all genes are contained within these operon-like structures (Blumenthal et al., 2002). Here, functional poly(A) sites will be synthesised and recognised by RNA polymerase II at the end of each gene within the operon, however termination of the polymerase only occurs at the final gene of the polycistronic transcription unit In these studies, we analyse the halting of RN A polymerase II transcription at the end of monocistronic genes and furthermore observe how premature RNA polymerase II termination is prevented during polycistronic transcription in the nematode C. elegans. We predominantly make use of reverse transcriptase PCR-based techniques to examine these mechanisms. We show that a large increase in pre-mRNAs stretching into the 3' flank of genes can be detected in worms depleted of the riboexonuclease XRN-2, indicating that this enzyme may have a possible role in RNA pol II termination and 3' end formation in C. elegans. Furthermore, we provide evidence that the polymerase can read into telomeric structures in the nematode. Also, we demonstrate that an RNAi-mediated knockdown of the UI-70K subunit of the UI snRNP causes a drop in polycistronic transcripts, providing a link between cis- splicing and the prevention of premature RNA polymerase II termination at operon-internal poly(A) sites. Finally, we illustrate that operon-internal poly(A) sites are capable of directing efficient 3' end formation outside of a polycistronic background. Together, these findings provide valuable insights into the mechanisms involved in directing or preventing premature RNA polymerase II transcription termination at C. elegans poly(A) sites.

The diet an organism keeps is crucial in sustaining its health and fitness. The fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans are excellent models for nutritional studies due to their small size, large progeny numbers, quick development, and modifiable laboratory diets. Here I examine these two organisms in order to better understand the complex interrelationship between an animal and its diet. Previous work has shown that in the wild numerous organisms are capable of selecting specific nutrients in a non-random manner in order to maximize fitness. However, the genetic underpinnings driving these nutrient choices remain elusive. Female fruit flies consume higher levels of protein following mating to prepare for the costs of reproduction. I examined the role of S6 Kinase (S6K), a downstream effector of the nutrient-responsive target of rapamycin pathway, in mediating this decision. I demonstrate that neuronal S6K activity and serotonin are involved in regulating protein consumption when allowed to choose nutrients freely as well as following macronutrient deprivation; suggesting that they may play a role in mediating postmating dietary switch and maintaining nutrient balance. Modulating levels of dietary components can have extensive impacts on processes such as development, fecundity, and metabolism in multiple organisms. However, the influence of dietary genetics on the consumer is virtually unknown. I performed a screen feeding single-gene mutants of E. coli to C. elegans and monitored the effects on the insulin-like signalling pathway (ILS). When mutated, genes involved in multiple processes and functions in E. coli enhanced activity of the ILS downstream transcription factor, DAF-16. One mutant strain of E. coli I pursued had a knockout of the cAMP-producing, adenylate cyclase gene. Addition of exogenous cAMP to the diet containing live, metabolically active E. coli rescued all the effects of the mutant on C. elegans; thereby suggesting that bacterial metabolism of dietary cAMP can influence the C. elegans ILS. Collectively, my work demonstrates how the nutrient-sensing pathways of the consumer can shape and be shaped by interactions with its diet. These studies contribute to a better understanding of the consumer-diet relationship, and could help guide future work to investigate the role of diet in disease, quality of life, and longevity.

Mutation is a fundamental process that drives evolutionary change; however, most new mutations are deleterious for organismal fitness and can readily propagate within populations under a broad range of conditions. Mutational processes able to counteract deleterious mutation accumulation include: 1) reversion mutation back to wildtype, 2) acquisition of generally beneficial mutations, and 3) compensatory mutations that specifically mitigate the effects of previously-acquired deleterious mutations through epistasis. The potential for any of these mutation types alters our expectations for the impact of deleterious mutation in populations, but since the fitness effects of individual mutations are rarely characterized, the relative importance of beneficial and compensatory epistatic mutations is unknown. In this thesis, I characterized the nuclear mutations that arose in a previous mutation accumulation (MA) experiment using Caenorhabditis elegans nematodes, in which mutations were allowed to accumulate under extreme drift conditions in replicate, independently evolving lines initiated from a low-fitness mitochondrial electron transport chain (ETC) mutant, gas-1. In contrast to the results of typical MA experiments, gas-1 MA lines improved fitness slightly compared to their ancestor. Here, I find that the gas-1 MA lines demonstrate little increase in among-line variance and that the gas-1 MA nuclear mutations are more narrowly functionally defined than wildtype MA nuclear mutations. When combined with evidence for zygotic or post zygotic selection these data suggest that selection--both purifying and positive--can be an extremely powerful force even in conditions of extreme genetic drift. Furthermore, functional characterization of a four-mutation set isolated from one of the gas-1 MA lines on gas-1 and wildtype backgrounds shows fitness improvements on both backgrounds. This beneficial four-mutation set is associated with a decrease in steady-state endogenous ROS on the gas-1 background while exhibiting no effect on wildtype. I also find that steady-state ATP levels associated with the beneficial four-mutation set decreased compared to wildtype suggesting that fermentation may be metabolic strategy to cope with increase oxidative stress. These findings suggest that we can detect and characterize specific genetic changes that lead to a partial recovery of fitness and phenotype in a low-fitness ETC-deficient mutant strain of C. elegans. I extended my thesis to include analyses of fitness and phenotype of 24 replicate lineages of the gas-1 ETC mutant evolved in large population (n = 1000) sizes for 60 generations--conditions optimal for selection and fitness recovery (RC). I find that two distinct gas-1 RC fitness groups emerged: one group with significantly higher average fitness than the ancestor and containing two lines that exceeded wildtype fitness levels, and another group with more modest and non-significant fitness gains. Interestingly, many lines in the first group were observed to generate appreciable numbers of males during experimental evolution--consistent with evolution of outcrossing either accompanying or driving rapid fitness recovery. Bioinformatic functional analyses of the nuclear mutations that arose in the gas-1 RC lines show the availability of potentially more paths to fitness recovery for large populations than small ones. Combined, these data allow us to identify patterns in selection and drift in gas-1 recovery under MA and RC (recovery) conditions. My research advances our understanding of the genetic bases of adaptive evolution under extremely unfavorable population genetic conditions and how mitochondrial dysfunction affects evolutionary dynamics.

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Helicases perform vital roles in the cell by unwinding D N A to make it accessible for the essential processes of replication, transcription and repair. In Caenorhabditis elegans, the DOG- 1 helicase-like protein is required for polyG/polyC-tract (G/C-tract) maintenance, as dog-l animals have a mutator phenotype characterized by deletions that initiate in G/C-tracts. DOG-1 may unwind secondary structures that form in polyguanine D N A during lagging strand replication. In order to more completely understand the role of dog-1, genetic interactors were identified, dog-1 functionally interacts with the him-6/BLM helicase. Absence of recombinational repair-implicated proteins in the dog-1 background, including HIM-6/BLM, RAD-51, BRD-1/BARD1 and HIM-9/XPF, as well as the trans-lesion synthesis polymerases polKMD po/7 increased the frequency of animals with G/C-tract deletions, indicating that these pathways are important mechanisms for repair at G/C-tracts in the absence of DOG-1. These data support the hypothesis that persisting D N A secondary structures can cause replication fork stalling, which can be resolved by deletion-free or deletion-prone mechanisms. DOG-1 has highest sequence identity to human BR1P1/FANCJ, which is mutated in patients from the Fanconi Anemia (FA) subgroup J. D N A damage sensitivity experiments indicated that, like chicken F A N C J cells, dog-1 mutants were not significantly sensitive to DNA damage from X-ray or UV-irradiation, but were extremely hypersensitive to the D N A interstrand cross-linking agent UVA-activated trimethylpsoralen. Thus, DOG-1 appears to have a conserved role in cross-link repair and is the C. elegans F A N C J homolog. Characterization of the dog-1/FANCJ-relatsd helicase, Jrh-1, revealed that mutants for this putative helicase are moderately sensitive to cross-linking agents, dog-1 jrh-1 double mutants displayed a synthetic lethal phenotype characterized by excessive recombination intermediates and mitotic catastrophe in the germline. However, absence of JRH-1 did not have any effect on G/C-tract deletions, indicating that JRH-1 does not have a redundant function with DOG-1 at G/C-tracts. Absence of JRH-1 reduced the fitness of eTl and nTl translocation hétérozygotes, but not translocation homozygotes, jrh-1 was synthetically lethal with him-6/BLM and with the endonuclease mus-81, suggesting a possible role for JRH-1 in regulating the balance between different types of repair.