clk-1 encodes an enzyme involved in the biosynthesis of ubiquinone (UQ), a redox active lipid that is found in all cellular membranes, including in the mitochondrial respiratory chain. In the nematode Caenorhabditis elegans clk-1 mutants display slow and deregulated physiological rates, including slow embryonic and post-embryonic development, retarded germline development, slow behaviours, and an increased lifespan. clk-1 slow germline development phenotype was previously linked to clk-1 altered ROS metabolism and its effect on lipid oxidization and the let-60/ras pathway. I have taken a genetic suppressor approach to further investigate the causes of the slow germline development phenotype of the clk-1 mutants. / Through this approach one mutant that suppresses the germline phenotype of clk-1 was identified. This suppressor, gsc-1(qm216), restored clk-1 germline development to slightly faster rates than wild type worms. This effect was specific to clk-1 and gsc-1 did not speed germline development rates in wild type worms. Furthermore, this effect appeared to be additive to lowering cholesterol levels but not to increasing cytoplasmic ROS levels. gsc-1 by itself appeared to have a deleterious effect on brood size and to increase lifespan. Neither of these effects were additive to the clk-1 phenotype and were therefore believed to affect similar mechanisms. The genetic mapping of gsc-1 precisely located the mutation to the center of chromosome II and linked it tightly to the lin-5 mutation. However, none of the transgenic lines managed to complement the gsc-1 mutation and its identity was not discovered. / In addition, to determine the role of reactive oxygen species (ROS) in the Clk phenotype, I have been analyzing all clk mutants (clk-1 to -10), by increasing ROS levels through the disruption of sod-1 and sod-2 genes, and scoring Clk phenotypes. I found that, although several clk mutants appear to have altered ROS levels, the phenomenon does not apply to all clk worms and does not correlate with lifespan. The disruption of either sod-1 or -2 affects growth and embryonic viability: sod-2 tends to exacerbate the mutant phenotypes, while sod-1 shows both weakly enhancing or weakly suppressing effects. Interestingly, only one mutant, clk-4, and only one phenotype of this mutant, slow post-embryonic development, is suppressed by sod-2 (but not sod-1). Furthermore, disrupting the expression of the sod-1 gene has only moderate or no effect on the lifespan of wild type worms, while sod-2 was shown to extend lifespan. On the whole, our results suggest that low superoxide levels do not participate in extending lifespan and are not the common process inducing the Clk phenotype in these mutants. Yet, several of the mutants analyzed have a dramatically increased lifespan and specifically behave like mutants which affect mitochondrial electron transport such as isp-1. Thus, our findings suggest that electron transport has a crucial role in longevity and developmental rates that is independent of superoxide generation.

Caenorhabditis elegans clk-1(qm30) and isp-1(qm150) mutants exhibit highly pleiotropic phenotypes that include slow development and long lifespan. clk-1(qm30) and isp-1(qm150) correspond to loss of function mutations in genes necessary for ubiquinone biosynthesis and complex III electron transport, respectively. Previous research has lead to the hypothesis that altered levels of cellular reactive oxygen species (ROS) may underlie clk-1(qm30) and isp-1(qm150) mutant phenotypes. To test this hypothesis RNA interference (RNAi) by feeding was used to indirectly alter cellular ROS levels by knocking down genes that encode ROS detoxification enzymes. Specifically, genes that detoxify ROS using glutathione or thioredoxin, both of which are important cellular thiol-redox molecules, were knocked down to examine the role of ROS in determining clk-1(qm30) and isp-1(qm150) lifespan, post-embryonic development, and germline development. In summary, knocking down ROS detoxification genes does not severely appear to affect the phenotypes that were studied. ROS detoxification gene knockdowns consistently induced mild decreases in wild type, clk-1(qm30), and isp-1(qm150) lifespan. However, knocking down NAD+-dependent isocitrate dehydrogenases, which are not closely involved in ROS detoxification, similarly affected lifespan, indicating that decreases are not specific to ROS detoxification. Of note, knocking down gcs-1, which is required for glutathione biosynthesis, induced lethal intestinal abnormalities in wild type, c1k-1(qm30), and isp-1(qm150) worms. Overall, findings do not support that low ROS underlies the clk-1(qm30) and isp-1(qm150) mutant phenotypes.

Five allelic, maternal-effect mutations which affect developmental and behavioral timing in Caenorhabditis elegans have been identified. They result in a mean lengthening of embryonic and post-embryonic development, the cell cycle period, and life span, as well as the periods of the defecation, swimming, and pumping cycles. These mutants also display a number of additional phenotypes related to timing. For example, the variability in the length of embryonic development is several times larger in the mutants than in the wild-type, resulting in the occasional production of mutant embryos developing more rapidly than the most rapidly-developing wild-type embryos. In addition, the duration of embryonic development and the length of the defecation cycle of the mutants, but not of the wild-type, depends on the temperature at which their parents were raised. Finally, individual variations in the severity of distinct mutant phenotypes are correlated in a counter-intuitive way. For example, the animals with the shortest embryonic development have the longest defecation cycle and those with the longest embryonic development have the shortest defecation cycle. Most of the features affected by these mutations are believed to be controlled by biological clocks, and we therefore call the gene defined by these mutations clk-1, for "abnormal function of biological clocks".

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'The rate of living' hypothesis of aging speculates that the metabolic rate of a species ultimately determines its life expectancy. Using the nematode worm Caenorhabditis elegans as model system, mutation in twp-1 (t&barbelow;ime w&barbelow;arp) gene was found to significantly delay biological timing and remarkably increase mean and maximum life span. The rate of living in twp-1 is dramatically delayed in all the biological processes we tested, including rates of rhythmic adult behaviors, development, and reproduction. Oxygen consumption, which indicates metabolic rate of an organism, is reduced to approximately two-fold in twp-1 mutant. According to my study, twp-1 and dauer genes, daf-2 and daf-16, interact to determine biological timing and adult life span. twp-1 mutation prolongs life span in a way that is at least partially different from dauer formation mutants, whose longevity might due to their high resistance to stresses, especially oxidative stress. twp-1 gene is cloned and found to encode iron-sulfur protein (ISP) in complex III, which is the major site of mitochondrial superoxide radical production, of the mitochondrial respiratory chain. This suggests that twp-1 may live long because they produce less reactive oxygen species (ROS), and thus, result in less oxidative damage. mts-1 (mitochondrial twp-1 suppressor) mutation can fully or partially rescue most of the biological timing in twp-1 mutant, including both developmental and behavioral rates, but except life span. mts-1 encodes another subunit of complex III, cytochrome b, which normally interact with ISP during function. mts-1 might somehow restore the activity of complex III, and consequently, accelerate the rate of living. Paraquat, a herbicide that induces the formation of superoxide, was used to provide an acute oxidative stress to animals. twp-1; mts-1 was found to be highly resistant to paraquat, indicating that twp-1 animals are well capable of coping with oxidative stress. According to o

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