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

The objective of these experiments was to explore long-term memory in Caenorhabditis elegans. This examination of memory in a simple organism with accessible genetics and a well understood biology may permit later work to define the cellular processes that underlie long-term memory. Habituation training with a vibrational stimulus was administered on Day 1, and the retention test of a block of stimuli was given 24 h after the end of training on Day 2. Long-term retention of habituation was evident as a lower level of responding on Day 2 relative to the level of responding on Day 2 of untrained controls or the initial level of responding of worms on Day 1. In Experiments 1 and 2, a habituation training protocol that produced long-term retention of habituation was established, and the effects of stimulus number, interstimulus interval (ISI), and distribution of training on both short-term and long-term habituation were examined. In Experiment 1 (10-s ISI), there appeared to be a floor effect which resulted in a low level of responding regardless of training on Day 1; thus no evidence for long-term habituation after training at a 10-s ISI could be found. In Experiment 2 (60-s ISI), worms that received distributed and massed habituation training with 60 stimuli showed a significantly lower level of responding relative to untrained controls. The distributed habituation training appeared to be more effective at inducing long-term habituation and was used in the subsequent experiments. To characterize the effects of heat shock treatments used in the behavioral experiments that follow, the effects of heat shock on two assays, the induction of a heat shock protein gene, hsp16, and the rate of egg-laying were measured in Experiment 3. All heat shock treatments used caused the induction of hsp16. In addition, the number of eggs laid during a fixed interval after heat shock was sensitive to the heat shock treatments given in Experiments 4 through 8. In Experiments 4 through 8, the effects of heat shock on short- and long-term habituation were examined. Heat shock, which acts as a general cellular stressor, was administered at different times before, during and after training. In Experiment 4, heat shock (45 min, 32°C) was administered, ending 2 h before training on Day 1. Heat shock before training did not affect the initial level of responding on Day 1, habituation during training, short-term retention of habituation between blocks of training or long-term retention of habituation. In Experiment 5, heat shock (45 min, 32°C) was administered during the rest periods of distributed training in the 1-h interval after each training block. While heat shock during training had no significant effect on responding on Day 1, long-term habituation was blocked. In Experiment 6, the possibility that heat shock before training would prevent the disruption of long-term habituation by heat shock during training by inducing thermal tolerance was examined. This was tested by administering heat shock (45 min, 32°C) that ended 2 h before training and heat shock during training. It was found that heat shock before training did not prevent the disruption of long-term habituation by heat shock during training. In Experiment 7, the effect of heat shock that ended 2 h before the retention test on Day 2 on the retention of long-term habituation was examined. It was found that heat shock on Day 2 did not disrupt the retention of habituation. Finally, in Experiment 8, the effect of brief heat shock (15 min, 32°C) at different intervals in the rest period following the training blocks was examined in an attempt to more narrowly define a critical period for consolidation of long-term habituation. Although there was no significant effect of brief heat shock on retention of habituation, the pattern of the data suggests that there may be a period of greater vulnerability worth further investigation. In summary, heat shock given before training or before the retention test did not affect long-term habituation, while heat shock during training disrupted consolidation of long-term habituation. Taken together, these behavioral results provide the foundation for an investigation of the cellular processes underlying long-term memory in C. elegans. By exploring the dynamics of the formation of long-term habituation, intervals of time critical to the formation of long-term habituation were defined. This in turn will help to focus attention on the cellular processes whose activity during those intervals of time may be important to the consolidation of long-term memory. / Arts, Faculty of / Psychology, Department of / Graduate

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Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 123-133). Also issued on the Internet.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 59-68).

Thesis (Ph. D.)--University of Oregon, 2002. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 90-98). Also available for download via the World Wide Web; free to University of Oregon users.