GLS-16His and derivatives were purified in SF+ lysis buffer [50 mM HEPES pH 7.5, 100 mM NaCl, 5% glycerol, 0.1% Triton X-100, 1 mM DTT, Protease inhibitor cocktail (Roche) and E64 (BioMol)] on Ni-NTA agarose beads (Qiagen), eluted with 250 mM Imidazole/50 mM HEPESpH 7.5/500 mM NaCl and dialysed against DB200 [20 mM Tris-Cl pH 7.5, 200 mM NaCl, 0.1% Triton X-100]. For the competition assay MBP-GLD- 36His and MBP-FBF-16His were purified on amylose beads (NEB) in SF+ lysis buffer. The N-terminal MBP fusion part of GLD- 3L6His was cleaved off by PreScission protease treatment (GE Healthcare). Western Blot bands were quantified in Adobe Photoshop CS2 by sliding an equally narrow sized box over all bands and extracting the pixel intensities. For Protein co-IPs see Text S1. GeneBank Accession Number of GLS-1: FJ610055. References 1. Thompson B, Wickens M, Kimble J (2007) Translational Control in Development. In: Mathews M, Sonenberg N, Hershey JWB, eds (2007) Translational control in biology and medicine. [3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. pp 507–544. 2. Seydoux G, Braun RE (2006) Pathway to totipotency: lessons from germ cells. Cell 127: 891–904. 3. Cinalli RM, Rangan P, Lehmann R (2008) Germ cells are forever. Cell 132: 559–562. 4. Strome S (2005) Specification of the germ line. WormBook. pp 1–10. 5. Eckmann CR, Kraemer B, Wickens M, Kimble J (2002) GLD-3, a bicaudal-C homolog that inhibits FBF to control germline sex determination in C. elegans. Dev Cell 3: 697–710. 6. Suh N, Jedamzik B, Eckmann CR, Wickens M, Kimble J (2006) The GLD-2 poly(A) polymerase activates gld-1 mRNA in the Caenorhabditis elegans germ line. Proc Natl Acad Sci U S A 103: 15108–15112. 7. Chicoine J, Benoit P, Gamberi C, Paliouras M, Simonelig M, et al. (2007) Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression. Dev Cell 13: 691–704. 8. Wang L, Eckmann CR, Kadyk LC, Wickens M, Kimble J (2002) A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312–316. 9. Mohler J, Wieschaus EF (1986) Dominant maternal-effect mutations of Drosophila melanogaster causing the production of double-abdomen embryos. Genetics 112: 803–822. 10. Mahone M, Saffman EE, Lasko PF (1995) Localized Bicaudal-C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1. EMBO J 14: 2043–2055. 11. Kimble J, Crittenden SL (2007) Control of Germline Stem Cells, Entry into Meiosis, and the Sperm/Oocyte Decision in C. elegans. Annu Rev Cell Dev Biol 23: 405–433. 12. Ellis R, Schedl T (2007) Sex determination in the germ line. WormBook. pp 1–13. 13. Kraemer B, Crittenden S, Gallegos M, Moulder G, Barstead R, et al. (1999) NANOS-3 and FBF proteins physically interact to control the sperm-oocyte switch in Caenorhabditis elegans. Curr Biol 9: 1009–1018. 14. Zhang B, Gallegos M, Puoti A, Durkin E, Fields S, et al. (1997) A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line. Nature 390: 477–484. 15. Tsuda M, Sasaoka Y, Kiso M, Abe K, Haraguchi S, et al. (2003) Conserved role of nanos proteins in germ cell development. Science 301: 1239–1241. 16. Bhat KM (1999) The posterior determinant gene nanos is required for the maintenance of the adult germline stem cells during Drosophila oogenesis. Genetics 151: 1479–1492. 17. Subramaniam K, Seydoux G (1999) nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans. Development 126: 4861–4871. 18. Kawasaki I, Shim YH, Kirchner J, Kaminker J, Wood WB, et al. (1998) PGL-1, a predicted RNA-binding component of germ granules, is essential for fertility in C. elegans. Cell 94: 635–645. GLS-1 and Germline Development Supplemental Materials and Methods. at: doi:10.1371/journal.pgen.1000494.s001 (0.07 MB Supporting Information Text S1 Found DOC) Acknowledgments We are in debt to Geraldine Seydoux for providing us with the gld-3(ax562) mutant. We are grateful to Ivonne Krienitz and the MPI-CBG antibody/ protein expression facilities for technical assistance. K. Bennett, B. Grant, T. Hyman, Y. Kohara, T. Schedl, S. Strome, and the CGC stock centre for providing reagents/strains. We also thank Dave Rudel, Sarah Crittenden and all lab members for critical reading of the manuscript. Author Contributions Conceived and designed the experiments: AR MH CRE. Performed the experiments: AR MH BJ APK MS CRE. Analyzed the data: AR MH BJ APK MS CRE. Wrote the paper: AR CRE. 19. Gruidl ME, Smith PA, Kuznicki KA, McCrone JS, Kirchner J, et al. (1996) Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans. Proc Natl Acad Sci U S A 93: 13837–13842. 20. Eckmann CR, Crittenden SL, Suh N, Kimble J (2004) GLD-3 and control of the mitosis/meiosis decision in the germline of Caenorhabditis elegans. Genetics 168: 147–160. 21. Gallegos M, Ahringer J, Crittenden S, Kimble J (1998) Repression by the 39 UTR of fem-3, a sex-determining gene, relies on a ubiquitous mog-dependent control in Caenorhabditis elegans. EMBO J 17: 6337–6347. 22. Bachorik JL, Kimble J (2005) Redundant control of the Caenorhabditis elegans sperm/oocyte switch by PUF-8 and FBF-1, two distinct PUF RNA-binding proteins. Proc Natl Acad Sci U S A 102: 10893–10897. 23. Kimble J, Hirsh D (1979) The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. Dev Biol 70: 396–417. 24. Schmid M, Kuchler B, Eckmann CR (2009) Two conserved regulatory cytoplasmic poly(A) polymerases, GLD-4 and GLD-2, regulate meiotic progression in C. elegans. Genes Dev 23: 824–836. 25. Rosenquist TA, Kimble J (1988) Molecular cloning and transcript analysis of fem-3, a sex-determination gene in Caenorhabditis elegans. Genes Dev 2: 606–616. 26. Hodgkin J (1986) Sex determination in the nematode C. elegans: analysis of tra-3 suppressors and characterization of fem genes. Genetics 114: 15–52. 27. Crittenden SL, Bernstein DS, Bachorik JL, Thompson BE, Gallegos M, et al. (2002) A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans. Nature 417: 660–663. 28. Musco G, Stier G, Joseph C, Castiglione Morelli MA, Nilges M, et al. (1996) Three-dimensional structure and stability of the KH domain: molecular insights into the fragile X syndrome. Cell 85: 237–245. 29. Lunde BM, Moore C, Varani G (2007) RNA-binding proteins: modular design for efficient function. Nat Rev Mol Cell Biol 8: 479–490. 30. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71–94. 31. Jones AR, Francis R, Schedl T (1996) GLD-1, a cytoplasmic protein essential for oocyte differentiation, shows stage- and sex-specific expression during Caenor- habditis elegans germline development. Dev Biol 180: 165–183. 32. Clifford R, Lee MH, Nayak S, Ohmachi M, Giorgini F, et al. (2000) FOG-2, a novel F-box containing protein, associates with the GLD-1 RNA binding protein and directs male sex determination in the C. elegans hermaphrodite germline. Development 127: 5265–5276. 33. Ward S, Roberts TM, Strome S, Pavalko FM, Hogan E (1986) Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins. J Cell Biol 102: 1778–1786. 34. Grant B, Hirsh D (1999) Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte. Mol Biol Cell 10: 4311–4326. 35. Kawasaki I, Amiri A, Fan Y, Meyer N, Dunkelbarger S, et al. (2004) The PGL family proteins associate with germ granules and function redundantly in Caenorhabditis elegans germline development. Genetics 167: 645–661. 36. Crittenden SL, Kimble J (1999) Confocal methods for Caenorhabditis elegans. Methods Mol Biol 122: 141–151. PLoS Genetics | www.plosgenetics.org 14 May 2009 | Volume 5 | Issue 5 | e1000494