The F2 progeny were scored to identify animals that lacked GFP ex

The F2 progeny were scored to identify animals that lacked GFP expression under normoxic conditions. To map n5500, the polymorphic Hawaiian CB4856 strain was crossed with n5500; nIs470 animals to obtain F2 progeny for SNP mapping ( Davis et al., 2005). To map the n5500 suppressor n5515 using genetic markers, n5500 II; nIs470 IV; n5515 males were crossed with n5500 II; nIs470 IV; dpy-6(e14) egl-15(n484) X hermaphrodites. Seven out of fifteen Compound Library ic50 Egl non-Dpy F2 progeny segregated GFP-negative n5500-suppressed animals. Refined mapping using SNP analysis further positioned n5515 between dpy-6 and egl-15 in an interval between the SNPs pk6127 and pk6138. To map the dominant suppressor

n5535, n5500; nIs470; n5535 hermaphrodites were crossed with n5500; nIs470 males, and GFP-positive F2 animals were isolated for SNP mapping. Locomotive responses to step changes of O2 were measured using a custom-built multiworm tracker and a gas-flow system controlled in real-time by MATLAB (see Figure S1). The gas flow consisted of pre-mixed 20%, 10%, selleck compound 5%, or 0% O2 balanced by N2. Well-fed young adult hermaphrodites (50–100 per assay) were transferred to a Petri plate freshly seeded with the bacterium OP50 and allowed to stabilize for 1 hr before the assay at 20°C. Worm-tracking

videos were analyzed later using MATLAB to calculate instantaneous locomotion speeds and other behavioral parameters. A hypoxia chamber (Coy Laboratory) that contained 0.5% O2 balanced by N2 was used for experiments involving hypoxia experience. After 24 hr of hypoxia exposure, animals

were allowed to recover in room air at 20°C for 2 hr preceding the acute behavioral assay. For experiments involving H2S exposure, 1 μl of 0.1M NaHS, an established H2S donor that releases 3-mercaptopyruvate sulfurtransferase H2S from solution, was dropped on the edge of agar-containing Petri plates and immediately sealed with tape to ensure airtight conditions. To obtain optimal effects, 24 hr duration of H2S exposure was used for behavioral experiments; 12 hr duration was used for GFP induction; and 1 hr duration was used for biochemical interaction experiments and quantitative real-time PCR. We thank Jo Anne Powell-Coffman, Yuichi Iino, Rene Garcia, Michael Hengartner, Mark Roth, Andy Fire, and Erik Jorgensen for reagents; the Caenorhabditis Genetics Center for strains; Na An, Rita Droste, and Tove Ljungars for technical assistance; Ales Hnizda, Jakub Krijt, and Milan Kodicek for help with characterization of purified CYSL-1; Viktor Kozich for helpful discussion; and Shunji Nakano, Takashi Hirose, Nick Paquin, Howard Chang, and Shuo Luo for comments. This work was supported by grant GM24663 to H.R.H from the NIH. R.V. was supported by grant No. 21709 from Grant Agency of Charles University, Prague, Czech Republic and by the Research Project of Charles University No. MSM0021620806. H.R.H.

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