David S Peal et al. (2001) International C. elegans Meeting "Chemical and Genetic Inhibition of Sulfation in C. elegans"

David S Peal, Laura L Kiessling

[International C. elegans Meeting, 2001]

In order to examine the process of sulfation in C. elegans, sulfation was inhibited chemically using sodium chlorate, and genetically using the process of RNA-mediated interference (RNAi). Sodium chlorate inhibition during early larval stages resulted in a dose-dependant developmental delay. BLAST searches of characterized sulfotransferases against the worm genome resulted in the identification of 4 putative sulfotransferases: C34F6.4 and F08B4.6 (previously identified: [1] and [2]), F40H3.5, and Y34B4A.e. RNAi of the putative N-deacetylase/N-sulfotransferase F08B4.6 resulted in "stacking" of eggs in the gonad, along with eggs laid at the 2- and 4-celled stage. RNAi of the putative hexuronic 2-O sulfotransferase C34F6.4 resulted in a shortened, bulbous gonad. These initial results indicate that sulfation may be important during development of C. elegans. [1] Shworak, NW, Liu, J, Fritze, LMS, Schwartz, JJ, Zhang, L, Logeart, D, Rosenberg, RD. JBC 272: 28008-19 (1997). [2] Kobayashi, M, Sugumaran, G, Liu, J, Shworak, NW, Silbert, JE, Rosenberg, RD. JBC 274: 10474-80 (1999).

Hitoshi Suda et al. (2005) International Worm Meeting "The general relation between survival curve and energy metabolism obtained by a novel optical method"

Tetsuji Shouyama, Kayo Yasuda, Kimihiro Imamura, Hitoshi Suda, Naoaki Ishii

[International Worm Meeting, 2005]

Aging and longevity strongly correlate with energy metabolism. Thus we developed a novel tool, which is constructed from an optical detector, using an indirect method that can measure simply the energy metabolism of C. elegans. If we measure the oxygen consumption rate using this optical tool, we can easily evaluate the activity of mitochondria as an index in the aging process. However, a direct measurement of the oxygen consumption rate of C. elegans exposed in air is thought to be impossible because of the high concentration of atmospheric oxygen and the small size of the animals. We demonstrate here that we can directly detect the oxygen consumption with a small number of animals (1~40) and a short accumulation time (< 30 min) at high precision by using both an optical probe and a small chamber (~ 3 56;l). Oxygen concentration was measured using an optical oxygen sensor, a spectrometer-coupled chemical sensor for full spectral analysis of dissolved or gaseous oxygen pressure. The fluorescence method measures the partial pressure of dissolved or gaseous oxygen. An optical fiber carries excitation light (475 nm) produced by a blue LED to a thin film coated by a ruthenium complex at the probe tip. The probe collects fluorescence (600 nm) generated at the tip and carries it via another optical fiber to the high-sensitivity spectrometer. Here, we used a 300-56;m fiber-optic probe. When oxygen in the gas or liquid sample diffuses into the thin film coating, it quenches the fluorescence. The degree of quenching correlates to the level of partial pressure of oxygen. The fluorescence is related quantitatively to the partial pressure of oxygen in terms of the Stern-Volmer equation. For a given medium and at a constant total pressure and temperature, the partial pressure of oxygen is proportional to the oxygen concentration. The metabolic rate of a 4-day-old wild-type animal cultured at 25 oC, for example, was approximately 80 nW. Using mutants that have long lives and short lives, we make clear a general relation between survival curve and energy metabolism. Our result shows that the process of death coincides with the disappearance of respiratory activity. Here, we report that our novel method has the potential to become a useful technique for research on aging correlated with energy metabolism.

Jason Morton et al. (2007) International Worm Meeting "Targeted mutagenesis in C. elegans can be mediated by zinc finger nucleases."

Jason Morton, Erik Jorgensen, Dana Carroll, Wayne Davis

[International Worm Meeting, 2007]

Zinc finger nucleases (ZFNs) are chimeric proteins composed of a zinc finger DNA recognition domain fused to a nonspecific endonuclease domain. The double-strand break (DSB) produced can then be repaired either by nonhomologous end joining (NHEJ) or by homologous recombination (HR), using a marked template, to introduce targeted alterations in the DNA. Since the binding specificity of zinc fingers can be easily engineered, ZFNs can, in principle, be constructed to target any gene in a genome. We have shown that when ZFNs are expressed in nematode somatic tissues, they frequently produce mutagenic DSBs at both synthetic and genomic targets. When a ZFN target sequence was placed on an extrachromosomal array, 26% of all targets examined contained a ZFN-induced mutation - most frequently a 4-bp insertion indicative of joining of blunted DNA ends. When a unique 18-bp sequence (Nw) on the nematode X chromosome was targeted, 18% of the targets analyzed contained a mutation. Here most mutations consisted of small deletions, which can also arise by the rejoining of blunted DNA ends after resection. To investigate this repair mechanism, identical experiments were performed in lig-4- nematodes, which lack the ligase IV protein central to the NHEJ pathway. In this case, mutation frequency at both targets dropped markedly, and most mutations consisted of substitutions, consistent with an alternate synthesis-dependent DNA repair mechanism. These results indicate that DSBs are preferentially blunted and repaired by ligase IV and that, in its absence, alternate repair processes must be employed. Differences in the 4-bp insertion frequency at the synthetic and genomic targets may be due to increased HR on a repetitive array, which amplifies such insertions when they are used as templates for subsequent repair events. Experiments to examine the interplay between NHEJ-mediated repair and the HR processes required for gene targeting will be forthcoming. These results indicate that if ZFNs can be activated in the germline, targeted mutant offspring can be produced at a high frequency. Since transgene expression is often suppressed in the germline, we have developed an RFP-GFP reporter to monitor transgene activity. Expression of the yeast recombinase Flp excises an RFP coding sequence in the reporter construct and allows a myo-2 promoter to instead drive GFP. As conditions favoring germline activity are discovered, ZFNs can be placed in identical contexts in wild-type and cosuppression-deficient (hpl-2-) nematodes.