Figure 1. Molecular characterization of
klp-18, which encodes a C. elegans kinesin-like protein. (A) Schematic illustration of the
klp-18 transcript (3059 nt; GenBank accession no. AY211948). The
klp-18 gene contains seven exons and is trans-spliced to SL1 at the 5' end. AUG and UGA mark the translation initiation and termination codons, respectively. The 3'-UTR (273 nt) contains two putative polyadenylation signals (AGUAAA and AACAAA) upstream (19 and 15 nt, respectively) of the poly-A tail. (B)
klp-18 encodes a protein of 932 aa. The N terminus contains the predicted motor domain (aa 2-338). The C terminus is predicted to form an extensive alpha-helical coiled-coil (Lupas plot; Lupas et al., 1991). P, a putative
cdc2 kinase phosphorylation site (SPAR). The bar marks the protein fragment used for generation of anti-KLP-18 antibodies. (C) RT-PCR amplified a 1.1-kb fragment of
klp-18 but not a 0.8-kb fragment of a related predicted gene, C33H5.4/klp-10. The darker shaded regions of
klp-18 are absent from C33H5.4. The region with an asterisk was used to produce
klp-18-specific dsRNA for RNAi. (D) Northern blot and Western blot analyses both detected only a single band, corresponding to the
klp-18 transcript (3.2 kb) and protein (110 kDa), respectively. (E)
klp-18 transcript levels in wild-type adult hermaphrodites and temperature-sensitive mutants raised at restrictive temperature:
glp-4(
bn2ts) hermaphrodites contain a severely underproliferated germ line;
fem-2(
b245ts) hermaphrodites contain only oocytes; and
fem-3(
q20ts) hermaphrodites contain only sperm. For comparison of relative transcript levels, the ribosomal protein mRNA (
rpp-1) served as a loading control.