DEV02060 Supplementary Material

Files in this Data Supplement:

• Supplemental Tables - Adobe PDF

• Supplemental Figure 1 - Fig. S1. Immunostaining of extruded germlines with anti-CAR-1 antibody. N2 and car-1(RNAi) animals were stained with affinity purified CAR-1 antibody. car-1 RNAi reduced staining to background levels. Images were acquired by fluorescent microscopy using equal exposure times.

• Supplemental Figure 2 - Fig. S2. A single CAR-1 ortholog is present in most eukaryotes. (A) Sequence structure of selected CAR-1 orthologs. Percentage identity and similarity are indicated relative to CAR-1, and were calculated using pair-wise BLAST analysis. A highly conserved domain of approximately 100 amino acids (dark shading) encompasses a Sm-like domain (approximately 70 amino acids), while the central region of the protein contains an additional region of similarity (gray shading) that includes an FDF domain, a recently described sequence motif of unknown function (Anantharaman and Aravind, 2004). Arginine-Glycine-Glycine (RGG) repeats or functional equivalents (vertical bars) are generally present near the C terminus. CAR-1, C. elegans (NP_493254); Cb, Caenorhabditis briggsae (CAE63508); Xl, Xenopus laevis (AAH42251); Hs, Homo sapiens (NP_056393); Dr, Danio rerio (AAH47185); RAP55, Pleurodeles waltl (CAA68149); At, Arabidopsis thaliana (AAG50526); Trh, Drosophila melanogaster (AAF49905); Sum2p, Schizosaccharomyces pombe (NP_595110); Scd6p, Saccharomyces cerevisiae (NP_015454). (B) Clustal W tree of CAR-1 orthologs. This dendogram was generated using the entire protein sequence from each species, but similar relationships were revealed using only the N-terminal 100 amino acids.

• Supplemental Figure 3 - Fig. S3. Alignment of the Sm-like domain from selected CAR-1 orthologs. Identical and conserved residues are shown in red and green, respectively. The characteristic five b-strands present in the Sm-like motif were predicted by PSIPRED v2.4 (http://bioinf.cs.ucl.ac.uk/psipred/psiform.html) and are indicated by the black arrows. Xl, Xenopus laevis (AAH42251); Hs, Homo sapiens (NP_056393); RAP55, Pleurodeles waltl (CAA68149); Dr, Danio rerio (AAH47185); CAR-1, C. elegans (NP_493254); Tral, Drosophila melanogaster (AAF49905); Sum2, Schizosaccharomyces pombe (NP_595110); Scd6p, Saccharomyces cerevisiae (NP_015454).

• Supplemental Figure 4 - Fig. S4. The cgh-1 deletion allele ok402 was generated by the C. elegans Gene Knockout Consortium and contains a 1043 bp deletion that removed part of the second exon, all of the third exon, and the entire coding region of the fourth exon. The predicted protein product from this allele would not be functional because it lacks a large portion of the essential DEXDc region and all of the helicase C domain.

• Supplemental Figure 5 - Fig. S5. ACS stain positively for the oocyte-specific marker RME-2. Extruded gonads from two-day-old adult N2 and car-1(RNAi) hermaphrodites were stained for DNA with DAPI, and for the yolk receptor RME-2 (Grant and Hirsh, 1999). RME-2 staining levels in N2 (A) and car-1(RNAi) (B) oocytes were similar, but ACS stained particularly strongly for RME-2 (white arrowheads).

• Supplemental Figure 6 - Fig. S6. ced-3 and ced-3;car-1(RNAi) young adult hermaphrodites initially have similar rates of gamete production. The approximate total number of gametes produced by ced-3 (n=24) and ced-3;car-1(RNAi) (n=18) hermaphrodites during the 16 hour period immediately following the L4 molt was determined by doubling the number of oocytes counted in one gonad arm, and adding the number of eggs laid per animal.