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The C. elegans maternal-effect gene clk-2 is essential for embryonic development, encodes a protein homologous to yeast Tel2p and affects telomere length

Claire Bénard, Brent McCright^*,, Yue Zhang, Stephanie Felkai, Bernard Lakowski and Siegfried Hekimi^¶

Department of Biology, McGill University, 1205 Avenue Dr Penfield, H3A 1B1, Montréal, Québec, Canada
^* Present address: The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA
Present address: Genzentrum, Ludwig-Maximilians Universitaet, Feodor-Lynen-Str. 25, D-81377, Munich, Germany
These authors contributed equally to this work

View larger version (82K): [in a new window]   Fig. 1. The lethal embryonic phenotype of clk-2(qm37) at 25°C. (A-D) Wild-type embryos; (E-I) qm37 mutant embryos. The very early embryonic development of the mutant (E,F) is indistinguishable from wild-type development (A,B); however, clk-2 embryos invariably arrest at different stages of embryogenesis and die subsequently with obvious morphological abnormalities (G-I), compared with the wild type (C,D).

View larger version (18K): [in a new window]   Fig. 2. Positional cloning of the gene clk-2. (A) clk-2 is inseparable from lin-39 by genetic mapping. (B) Cosmids in bold rescue clk-2(qm37). (C) Fragments and subclones of C07H6; rescuing clones are in bold. Coordinates refer to bases on C07H6. (D) Genomic structure of clk-2 and cux-7, which is the upstream gene in the operon of clk-2. cDNA clone yk215f6 does not contain the entire 3' end of cux-7, stopping at 35040; we use the in-frame stop codon, at 34999, as the 3' end in the drawing. (Accession Numbers: clk-2, AF400665; cux-7, AF400666.) The qm37 mutation is indicated.

View larger version (108K): [in a new window]   Fig. 3. An alignment of the predicted amino acid sequence of CLK-2 with its homologues from S. cerevisiae (Tel2p), A. thaliana (AtCLK-2) and humans (hCLK-2). Sequences were aligned using DNAMAN 4.1 and adjusted by hand. Similarities are shown in red when they include a residue in Tel2p, and in blue when the similarity is among sequences from multicellular organisms only. When CLK-2 is used to initiate a search by Psi-blast (http://www.ncbi.nlm.nih.gov/blast/psiblast.cgi), the algorithm finds five predicted proteins to be similar to CLK-2 and to each other (E-values are given in brackets): the Drosophila protein CG13854 (e-177); the human protein KIAA0683 (e-169); the Arabidopsis protein CAB88328 (>e-177); the S. pombe protein CAB93845 (e-106); and the S. cerevisiae Tel2p (e-105).

View larger version (35K): [in a new window]   Fig. 4. The expression pattern of clk-2. (A) Northern and western analyses of clk-2 at all developmental stages (E, embryos; L1-L4, larval stages; A, young adults; glp-4(bn2ts) adults). (B) clk-2 mRNA and CLK-2 protein levels in mutant backgrounds glp-4(bn2ts), fem-2(b245ts) and fem-3(q20ts) at 25°C. (C) CLK-2 protein levels in the wild type and clk-2(qm37) mutants at three temperatures. For northerns, actin is used as a loading control; for westerns, equal loading was confirmed by Coomassie Blue staining of identical gels (not shown). As expected, the clk-2 band detected in the northerns is at 2.8 kb, and the CLK-2 band detected in westerns is at 100 kDa.

View larger version (133K): [in a new window]   Fig. 5. Subcellular localization of a functional CLK-2::GFP fusion. Bright fluorescence is found in the cytoplasm of a variety of cells, and appears excluded from their nuclei (arrows). (A) Neurons in ganglia of the head. (B) Three neurons in the pre-anal ganglion. (C) A distal tip cell and its projections (arrowheads). (D) A two-cell embryo. Only one nucleus is in the plane of focus, but fluorescence was observed to be extranuclear in the second cell as well. (E) A region of the hypodermis. Scale bars: 10 µm.

View larger version (69K): [in a new window]   Fig. 6. The telomere-lengthening phenotype of clk-2(qm37) mutants. (A-C) In C. elegans, genomic DNA hybridization to telomeric probes after restriction digestion with HinfI reveals smears that correspond to the terminal fragments of the chromosomes containing the telomeres, and discrete bands that correspond to fragments containing internal tracts of telomeric repeats (see text). (D,E) Hybridization with telomere-specific probes reveals a smear. For XL, discrete bands at 1.6, 1.9, and 2.4 kb are also detected by the subtelomeric probe and correspond to internal genomic fragments containing non-telomeric repetitive DNA located on other chromosomes that cross-hybridize with the probe. Worms were grown at 20°C (A,B) and at 25°C (C-E). Each panel represents an independent experiment (distinct worm cultures, DNA extractions and enzymatic digestions). MQ691 is a strain carrying a clk-2(+) transgene in a clk-2(qm37) background. MQ931 was derived from MQ691 by loss of the extrachromosomal array, and thus lacks clk-2(+). The sizes indicated are in kb.