Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans - PubMed (original) (raw)

Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans

M E Gruidl et al. Proc Natl Acad Sci U S A. 1996.

Abstract

Two components of the germ-line-specific P granules of the nematode Caenorhabditis elgans 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 stage 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.

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Figures

Figure 1

Figure 1

Alignment of GLH-1 and GLH-2. N-terminal glycine-rich imperfect repeats are shaded in grey. CCHC zinc fingers are indicated with black backing. The zinc finger consensus in the two GLH proteins is PxxCFNCxxxGHRSxxCPEP; these amino acids are found in at least 7/10 fingers. Comparisons of specific zinc fingers are shown at the bottom with differences in black. The conserved motifs found in all DEAD-box RNA helicases (26) are boxed. The 22-amino acid peptides used to produce GLH-1 and GLH-2-specific peptides are underlined (N termini). Sites of introns in the corresponding glh-1 and glh-2 genomic sequences are indicated with arrowheads. The 58 amino acids encoded by a 168-nt internal_Bam_HI–_Bam_HI fragment were absent in the original report of the glh-1 cDNA (18); the corrected sequence is GenBank accession no. L19948L19948.

Figure 2

Figure 2

In situ hybridization to C. elegans adults and embryos. glh-1 hybridization to a splayed hermaphrodite (A) and male (B) using a 253-nt antisense probe from the glh-1_-specific 5′_Eco_RI–_Bam_HI fragment (18). (C) Sense strand of the same glh-1 probe as a negative control. The gonads of the splayed worms are indicated with arrowheads. The 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei are blue. All glh-1 slides were hybridized with 5 × 105 dpm and exposed for 7 days. (D and_E) Antisense glh-2 RNA hybridization to a hermaphrodite (D) and male (E). The probe used was 340 bp long, including 130 bp of the 3′-most coding region and the entire 210-bp glh-2 3′ UTR, minus the poly(A) tail. This probe was determined to be specific for glh-2 by both Northern and Southern blot analyses (data not shown). Exposures for_glh-2_ were 14 days, using 106 dpm. This_glh-2_ signal results from use of 2-fold higher probe concentration and exposure relative to the glh-1 conditions. (F) Antisense glh-1 hybridization to whole-mount embryos. From top to bottom, the embryo stages are: 8-cell stage, >60-cell stage, and 1-cell stage. (G) Antisense glh-2 hybridization to a whole mount 1-cell embryo (Left) and a 12- to 14-cell embryo (Right). Embryos were exposed for 7 days with 106 dpms. (Bars: A–E, 50 μm;F and G, 20 μm.)

Figure 3

Figure 3

Western blot analysis using chicken yolk and mouse serum antibodies. Total C. elegans protein homogenate was resolved for 1100 V·h on an SDS/8% polyacrylamide gel and transferred to nylon-supported nitrocellulose membrane (Optibind-NC, Schleicher & Schuell). Strips were cut in half and incubated with anti-GLH antibodies as follows. Lanes: 1, preimmune yolk from one of the chickens immunized with GLH-1 peptide; 2, anti-GLH-1 chicken yolk; 3, preimmune yolk from one of the chickens immunized with GLH-2 peptide; 4, anti-GLH-2 yolk; 5, preimmune serum from one of the mice immunized with GLH-1 fusion protein; 6, anti-GLH-1 mouse serum. Prestained high molecular weight markers (GIBCO/BRL) of 202, 103, and 68 kDa are indicated.

Figure 4

Figure 4

Immunofluorescence staining of embryos with OIC1D4 and anti-GLH-1 and anti-GLH-2 antibodies. Embryos are oriented with anterior left and ventral down. (A). Embryo stained with affinity-purified chicken anti-GLH-1 antibodies (Left), mouse monoclonal antibody OIC1D4 (Center), and DAPI (Right). (B–D). Embryos stained with affinity-purified mouse anti-GLH-1 (Left), chicken anti-GLH-2 (Center), and DAPI (Right). (A and B) Two-cell embryos. P1 is in mitosis, and P granules are segregated to the posterior cortex destined for P2. (C) Seven-cell embryo. P2 is in mitosis, and P granules are segregated to the ventral region destined for P3. (D) Late-stage embryo showing P granules in Z2 and Z3. (Bar = 10 μm.)

Figure 5

Figure 5

Immunofluorescence staining of adult gonads using anti-GLH-1 and anti-GLH-2 antibodies. Each row shows a sample stained with affinity-purified mouse anti-GLH-1 antibodies (Left), chicken anti-GLH-2 (Center), and DAPI (Right). Gonad arms are oriented with distal left. (A) Distal gonad arm from a wild-type hermaphrodite. (B) Gonad arm from a wild-type male. Sperm present at the far right of each panel fail to stain with anti-GLH-1 and anti-GLH-2. (C) Gonad arm from a sterile hermaphrodite worm produced by a mother injected with antisense RNA to glh-1. (Bar = 10 μm.)

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