Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans (DEAD-box proteinsyCCHC zinc fingersyglycine-rich repeatsygene duplicationsychicken antibodies)

Two components of the germ-line-specific P granules of the nematode Caenorhabditis elegans have been identified using polyclonal antibodies specific for each. Both components are putative germ-line RNA helicases (GLHs) that contain CCHC zinc fingers of the type found in the RNA-binding nucleocapsid proteins of retroviruses. The predicted GLH-1 protein has four CCHC fingers; GLH-2 has six. Both GLH proteins localize in the P granules at all stages of germ-line development. However, the two glh genes display different patterns of RNA and protein accumulation in the germ lines of hermaphrodites and males. Injection of antisense glh-1 or glh-2 RNA into wild-type worms causes some offspring to develop into sterile adults, suggesting that either or both genes are required for normal germ-line development. As these very similar glh genes physically map within several hundred kilobases of one another, it seems likely that they represent a fairly recent gene duplication event. Embryos of the free-living soil nematode Caenorhabditis elegans generate distinct founder cells via a series of asymmetric cell divisions. At each division, the germ-line daughter cell inherits distinctive non-membrane-bound particles, called P granules (1–3). P granules are partitioned to the primordial germ cell P4 of the 16to 24-cell embryo and become perinuclear. P granules persist around the nuclei of all germ cells, until gametogenesis, at which point they are excluded from mature sperm and become dispersed within the cytoplasm of mature oocytes in preparation for cytoplasmic localization in the embryo. Although the distribution pattern of nematode P granules has been well-studied, the identity and function of P-granule components have yet to be determined. Germ granules are found in many species (4, 5). The germ-line-specific polar granules of Drosophila melanogaster have been well-studied, with a number of different genes identified that are required for polar granule assembly and germ-cell formation, including vasa, staufen, valois, oskar, tudor, mago nashi, and germ-cell-less (6–15). With the exception of vasa, these genes encode novel proteins. Vasa, however, is a member of a family of proteins with recognizable motifs and predictable function. Vasa is an RNA helicase of the DEAD-box family (8, 9) whose ATP-dependent RNA helicase activity has been demonstrated in vitro (16). As polar granules contain RNA as well as protein (11, 12, 15, 17), a germ-linespecific RNA helicase may function to bind and unwind RNAs necessary for germ-line development. Several potential vasa homologues have been cloned, including glh-1 (germ-line helicase 1) from Caenorhabditis, Xvh (Xenopus vasa homologue), mvh (mouse vasa homologue), and rvh (rat vasa homologue) (18–21). glh-1 in C. elegans is unique among RNA helicase genes reported, including vasa, in that its predicted product contains four retroviral-like zinc fingers (18). We have identified a second C. elegans germ-line RNA helicase gene, glh-2, that also encodes zinc fingers. Immunolocalization with GLH-1 and GLH-2-specific antibodies demonstrates that both GLH-1 and GLH-2 localize to P granules. Thus, to our knowledge, GLH-1 and GLH-2 are the first components of C. elegans germ granules to be identified. glh-1 and glh-2 differ in their patterns of RNA and protein accumulation in the germ line, suggesting that these genes may have distinct functions. Injection of either glh-1 or glh-2 antisense RNA into the germ line of wild-type hermaphrodites results in sterile progeny, leading us to predict that mutations in glh-1 and glh-2will result in a germ-line-defective mutant phenotype. MATERIALS AND METHODS Strains. Wild-type worms were C. elegans strain N2 variety Bristol. Worms were grown using standard methods (22). Sequence Analysis. Both strands of genomic glh-1 and glh-2 clones and the glh-2 cDNA were sequenced using the chaintermination method with either Sequenase II (United States Biochemical) or with SequiTherm polymerase (Epicentre Technologies, Madison,WI). In addition, preliminary genomic sequence was obtained courtesy of the C. elegans sequencing consortium at Washington University (23). The glh-2 cDNA sequence is derived from a partial 2.3-kb cDNA isolated from a mixed-stage cDNA library made by S. Kim (Stanford University) and from a 927-bp PCR product generated with an upstream primer corresponding to the putative translation start site in the glh-2 genomic sequence (59-CGAAGATGTCTGACGATTGG-39) and a downstream primer corresponding to the 59 end of the 2.3-kb cDNA (59-CGCGGGATCCTTTCGGCCTTCACCCGGT-39). Several different cDNA libraries yielded the same-sized PCR product. In Situ Hybridization. Whole-mount embryos permeabilized by freeze cracking were fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS) as described (24). Splayed adult worms were fixed in 4% paraformaldehyde in PBS. Fixed specimens were acetylated with acetic anhydride, dehydrated through an alcohol series, and stored at 2808C until needed. All probes were prepared by in vitro transcription of linearized templates of 59and 39gene-specific cloned fragments in the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: GLH, germ-line helicase; UTR, untranslated region; DAPI, 49,6-diamidino-2-phenylindole. Data deposition: The sequences reported in this paper have been deposited in the GenBank data base (accession nos. glh-2 cDNA, U60194; glh-2 genomic, U60449; glh-1 cDNA, L19948; glh-1 genomic, U62772). †Present address: Department of Pathology, Moffitt Cancer Center, University of South Florida, Tampa, FL 33612. ¶Present address: Bristol–Myers Squibb, Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543. §To whom reprint requests should be addressed. e-mail: bennettkl@ medsci.mbp.missouri.edu.