defects in adult tail morphology for treated L3 males. Comple- mentation of the uba-1(ok1374) deletion allele was determined by generating it129/ok1374 heterozygous animals and performing temperature-shift assays as described for phenotypic characteriza- tion. Transformation rescue [49] was obtained by germ line microinjection of a 6.0 kb genomic fragment of the wild-type uba-1 gene mixed with plasmid pRF4, which contains the dominant roller marker rol-6(su1006), and linearized N2 genomic DNA at concentrations of 1, 50, and 100 mg/ml, respectively. Stable roller transgenic lines were generated from uba-1(it129) hermaphrodites maintained at 15uC, then rescue of sperm-specific sterility and embryonic lethality was scored after shifting to 25uC. The molecular lesion of the uba-1(it129) allele was identified by PCR amplification of the 6.0 Kb uba-1 genomic interval from mutant worms followed by sequence determination. In situ hybridization for uba-1 germ line expression was performed on dissected gonads following fixation [78]. Digoxigenin-labeled, single-stranded sense and antisense probes were generated from a 1 kb cDNA fragment by linear amplification according to the manufacturer’s protocol (Roche, Indianapolis, IN). Following hybridization, probe detection was by colorimetric assay with alkaline phosphatase-conjugated anti-digoxigenin antibodies and NBT/BCIP substrate. Western blot analysis was performed on soluble worm lysates from age-synchronized young adult hermaphrodites shifted to 25u References 1. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67: 425–479. 2. Nakayama KI, Nakayama K (2006) Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer 6: 369–381. 3. Ardley HC, Robinson PA (2004) The role of ubiquitin-protein ligases in neurodegenerative disease. Neurodegener Dis 1: 71–87. 4. Schnell JD, Hicke L (2003) Non-traditional functions of ubiquitin and ubiquitin- binding proteins. J Biol Chem 278: 35857–35860. 5. Haglund K, Dikic I (2005) Ubiquitylation and cell signaling. EMBO J 24: 3353–3359. 6. Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315: 201–205. 7. Geetha T, Kenchappa RS, Wooten MW, Carter BD (2005) TRAF6-mediated ubiquitination regulates nuclear translocation of NRIF, the p75 receptor interactor. EMBO J 24: 3859–68. 8. Chiu RK, Brun J, Ramaekers C, Theys J, Weng L, et al. (2006) Lysine 63- polyubiquitination gurads against translesion synthesis-induced mutations. PLoS Genet 2: e116. 9. Lim KL, Chew KC, Tan JM, Wang C, Chung KK, et al. (2005) Parkin mediates nonclassical, proteasomal-independent ubiquitination of synphilin-1: implica- tions for Lewy body formation. J Neurosci 25: 2002–2009. 10. Hershko A, Heller H, Elias S, Ciechanover A (1983) Components of ubiquitin- protein ligase system Resolution, affinity purification, and role in protein breakdown. J Biol Chem 258: 8206–8214. 11. Jackson PK, Eldridge AG, Freed E, Furstenthal L, Hsu JY, et al. (2000) The lore of the RINGS: substrate recognition and catalysis by ubiquitin ligases. Trends Cell Biol 10: 429–439. 12. Starostina NG, Lim JM, Schvarzstein M, Wells L, Spence AM, et al. (2007) A CUL-2 ubiquitin ligase containing three FEM proteins degrades TRA-1 to regulate C. elegans sex determination. Dev Cell 13: 127–129. 13. Feng H, Zhong W, Punkosdy G, Gu S, Zhou L, et al. (1999) CUL-2 is required for the G1-to-S phase transition and mitotic chromosome condensation in Caenorhabditis elegans. Nat Cell Biol 1: 486–492. 14. Burbea M, Dreier L, Dittman JS, Grunwald ME, Kaplan JM (2002) Ubiquitin and AP180 regulate the abundance of GLR-1 glutamate receptors at postsynaptic elements in C. elegans. Neuron 35: 107–120. 15. Juo P, Kaplan JM (2004) The anaphase-promoting complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of C. elegans. Curr Biol 14: 2057–2062. 16. Schaefer H, Rongo C (2006) KEL-8 is a substrate receptor for CUL-3- dependent ubiquitin ligase that regulates synaptic glutamate receptor turnover. Mol Cell Biol 17: 1250–1260. 17. Frazier T, Shakes D, Hota U, Boyd L (2004) Caenorhabditis elegans UBC-2 functions with the anaphase-promoting complex but also has other activities. J Cell Sci 117: 5427–5435. 18. Bowerman B, Kurz T (2006) Degrade to create: developmental requirements for ubiquitin-mediated proteolysis during early C. elegans embryogenesis. Devel- opment 133: 773–784. UBA-1 in C. elegans Development as L2 larvae. Lysates were obtained by one freeze-thaw cycle, homogenization, and centrifugation for 10 minutes at 10,000 RCF. Protein concentration of the soluble fraction was quantified by Bradford assay. 10 mg samples were fractionated by SDS- PAGE and transferred to PVDF membranes. Ubiquitin-conjugat- ed proteins were detected by mouse anti-ubiquitin monoclonal antibody (1u) followed by HRP-conjugated goat anti-mouse IgG polyclonal antibody (2u; both Stressgen, Ann Arbor, MI). Duplicate gels were stained with Gelcode Blue (Pierce, Rockford, IL) to visualize total protein. Acknowledgments We wish to thank Diane Shakes for isolating and Sam Ward for providing the uba-1(it129) allele, Michael Stitzel of Geraldine Seydoux’s lab for providing the oma-1::GFP strain, the Caenorhabditis Genetics Center and C.elegans Gene Knockout Consortium for providing strains, Andy Golden for providing strains and suggesting the synthetic lethal experiment, Eugene Melamud for assistance with the structural prediction, Sara Hapip for assistance with the RNAi candidate screen, and members of the lab and the Baltimore/Washington worm community for fruitful discussions. Author Contributions Conceived and designed the experiments: MK HES. Performed the experiments: MK HES. Analyzed the data: MK HES. Wrote the paper: MK HES. 19. Golden A, Sadler PL, Wallenfang MR, Schumacher JM, Hamill DR, et al. (2000) Metaphase to anaphase (mat) transition-defective mutants in Caenorhabditis elegans. J Cell Biol 151: 1469–1482. 20. Davis ES, Wille L, Chestnut BA, Sadler PL, Shakes DC, et al. (2002) Multiple subunits of the Caenorhabditis elegans anaphase-promoting complex are required for chromosome segregation during meiosis I. Genetics 160: 805–813. 21. Bosu DR, Kipreos ET (2008) Cullin-RING ubiquitin ligases: global regulation and activation cycles. Cell Div 3: 7. 22. Pintard L, Willis JH, Willems A, Johnson JL, Srayko M, et al. (2003) The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature 425: 311–316. 23. Xu L, Wei Y, Reboul J, Vaglio P, Shin TH, et al. (2003) BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3. Nature 425: 316–321. 24. Kemphues KJ, Wolf N, Wood WB, Hirsh D (1986) Two loci required for cytoplasmic organization in early embryos of Caenorhabditis elegans. Dev Biol 113: 449–460. 25. Sonneville R, Gonczy P (2004) Zyg-11 and cul-2 regulate progression through meiosis II and polarity establishment in C. elegans. Development 131: 3527–3543. 26. Liu J, Vasudevan S, Kipreos ET (2004) CUL-2 and ZYG-11 promote meiotic anaphase II and the proper placement of the anterior-posterior axis in C. elegans. Development 131: 3513–3525. 27. DeRenzo C, Reese KJ, Seydoux (2003) Exclusion of germ plasm proteins from somatic lineages by cullin-dependent degradation. Nature 424: 685–689. 28. Rappleye CA, Tagawa A, Lyczak R, Bowerman B, Aroian RV (2002) The anaphase-promoting complex and separin are required for embryonic anterior- posterior axis formation. Dev Cell 2: 195–206. 29. Shakes DC, Sadler PL, Schumacher JM, Abdolrasulnia M, Golden A (2003) Developmental defects observed in hypomorphic anaphase-promoting complex mutants are linked to cell cycle abnormalities. Development 130: 1605–1620. 30. Finley D, Ciechanover A, Varshavsky A (1984) Thermolability of ubiquitin- activating enzyme from the mammalian cell cycle mutant ts85. Cell 37: 43–55. 31. Ciechanover A, Finley D, Varshavsky A (1984) Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell 37: 57–66. 32. Ghaboosi N, Deshaies RJ (2007) A conditional yeast E1 mutant blocks the ubiquitin-proteasome pathway and reveals a role for ubiquitin conjugates in targeting Rad23 to the proteasome. Mol Biol Cell 18: 1953–1963. 33. Lee TV, Ding T, Chen Z, Rajendran V, Scherr H, et al. (2008) The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autono- mously and tissue growth non-autonomously. Development 135: 43–52. 34. Jones D, Crowe E, Stevens TA, Candido EP (2002) Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin- conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins. Genome Biol 3: RESEARCH00021-15. 35. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, et al. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421: 231–237. PLoS Genetics | www.plosgenetics.org 12 July 2008 | Volume 4 | Issue 7 | e1000131