Structural and functional conservation of the Caenorhabditis elegans timing gene clk-1

Science

Volumn 275, Issue 5302, 1997, Pages 980-983

  Ewbank, Jonathan J ^a   Barnes, Thomas M ^a   Lakowski, Bernard ^a   Lussier, Marc ^a   Bussey, Howard ^a   Hekimi, Siegfried ^a  

^a MCGILL UNIVERSITY   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN;

AMINO ACID SEQUENCE; ARTICLE; CAENORHABDITIS ELEGANS; GENE DUPLICATION; GENE FUNCTION; GENE MUTATION; GENE STRUCTURE; GENETIC COMPLEMENTATION; GENETIC CONSERVATION; LONGEVITY; MOLECULAR CLONING; NONHUMAN; PHENOTYPE; PRIORITY JOURNAL; SEQUENCE HOMOLOGY;

AMINO ACID SEQUENCE; ANIMALS; CAENORHABDITIS ELEGANS; CAENORHABDITIS ELEGANS PROTEINS; CELL AGING; CHROMOSOME MAPPING; CONSERVED SEQUENCE; EXONS; FUNGAL PROTEINS; GENES, HELMINTH; GENETIC COMPLEMENTATION TEST; GLYCEROL; HELMINTH PROTEINS; HUMANS; LONGEVITY; MICE; MOLECULAR SEQUENCE DATA; PHENOTYPE; PROTEIN STRUCTURE, SECONDARY; PROTEIN STRUCTURE, TERTIARY; RNA SPLICING; SACCHAROMYCES CEREVISIAE PROTEINS;

CAENORHABDITIS ELEGANS; EUKARYOTA;

EID: 0031030678     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5302.980     Document Type: Article

References (42)

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2. A. Wong, P. Boutis, S. Hekimi, Genetics 139, 1247 (1995); A. Wong, thesis, McGill University, Montréal, Canada (1994).
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7. Defecation was scored as previously described (1). Five worms of each genotype were scored for five cycles at 20°C. The means of individual animals' mean defecation period, ± standard deviation, in seconds, were as follows: N2, 44.1 ± 2.1; e2519, 55.4 ± 5.4; e2519; qmEx81[ZC400, pRF4], 46.2 ± 1.6; and e2519; qmEx96[pRA40, pRF4], 43.6 ± 1.5. The defecation period of clk-1(e2519) worms is both longer and more irregular than that of wild-type worms (1). Both aspects of the phenotype are rescued in the transgenic animals.
8. Genetic and physical map data for C. elegans were obtained by anonymous FTP from the public repository at ftp://nlm.nih.gov/repository/acedb/celegans and viewed by using the program ACeDB [F. H. Eeckman and R. Durbin, Methods Cell Biol. 48, 583 (1995)], obtained from the same source. The sequence of cosmid ZC395, with predicted genes, made available by the C. elegans genome sequencing consortium [J. Sulston et al., Nature 356, 37 (1992); R. Wilson et al., ibid. 368, 32 (1994)] can also be obtained from GenBank (accession number U13642).
9. Genetic and physical map data for C. elegans were obtained by anonymous FTP from the public repository at ftp://nlm.nih.gov/repository/acedb/celegans and viewed by using the program ACeDB [F. H. Eeckman and R. Durbin, Methods Cell Biol. 48, 583 (1995)], obtained from the same source. The sequence of cosmid ZC395, with predicted genes, made available by the C. elegans genome sequencing consortium [J. Sulston et al., Nature 356, 37 (1992); R. Wilson et al., ibid. 368, 32 (1994)] can also be obtained from GenBank (accession number U13642).
10. Genetic and physical map data for C. elegans were obtained by anonymous FTP from the public repository at ftp://nlm.nih.gov/repository/acedb/celegans and viewed by using the program ACeDB [F. H. Eeckman and R. Durbin, Methods Cell Biol. 48, 583 (1995)], obtained from the same source. The sequence of cosmid ZC395, with predicted genes, made available by the C. elegans genome sequencing consortium [J. Sulston et al., Nature 356, 37 (1992); R. Wilson et al., ibid. 368, 32 (1994)] can also be obtained from GenBank (accession number U13642).
11. Genetic and physical map data for C. elegans were obtained by anonymous FTP from the public repository at ftp://nlm.nih.gov/repository/acedb/celegans and viewed by using the program ACeDB [F. H. Eeckman and R. Durbin, Methods Cell Biol. 48, 583 (1995)], obtained from the same source. The sequence of cosmid ZC395, with predicted genes, made available by the C. elegans genome sequencing consortium [J. Sulston et al., Nature 356, 37 (1992); R. Wilson et al., ibid. 368, 32 (1994)] can also be obtained from GenBank (accession number U13642).
12. i. The genes produce mRNAs with 3′ untranslated regions of 367 and 176 nucleotides for toc-1 and clk-1, respectively. For amplification of the 5′ end of the clk-1 message we used the nested primer pairs SL and SHP12 and then SL and SHP10 [where both trans-spliced leaders SL1 and SL2 were tested [D. A. Zorio, N. N. Cheng, T. Blumenthal, J. Spieth, Nature 372, 270 (1994); J. Spieth, G. Brooke, S. Kuersten, K. Lea, T. Blumenthal, Cell 73, 521 (1993)]} on cDNA that had been synthesized by priming with SHP12. For amplification of the 5′ end of toc-1, SHP15 and SHP14 replaced SHP12 and SHP10, respectively. Methods were essentially those given in (23). The sequences of primers are available on request.
13. i. The genes produce mRNAs with 3′ untranslated regions of 367 and 176 nucleotides for toc-1 and clk-1, respectively. For amplification of the 5′ end of the clk-1 message we used the nested primer pairs SL and SHP12 and then SL and SHP10 [where both trans-spliced leaders SL1 and SL2 were tested [D. A. Zorio, N. N. Cheng, T. Blumenthal, J. Spieth, Nature 372, 270 (1994); J. Spieth, G. Brooke, S. Kuersten, K. Lea, T. Blumenthal, Cell 73, 521 (1993)]} on cDNA that had been synthesized by priming with SHP12. For amplification of the 5′ end of toc-1, SHP15 and SHP14 replaced SHP12 and SHP10, respectively. Methods were essentially those given in (23). The sequences of primers are available on request.
14. i. The genes produce mRNAs with 3′ untranslated regions of 367 and 176 nucleotides for toc-1 and clk-1, respectively. For amplification of the 5′ end of the clk-1 message we used the nested primer pairs SL and SHP12 and then SL and SHP10 [where both trans-spliced leaders SL1 and SL2 were tested [D. A. Zorio, N. N. Cheng, T. Blumenthal, J. Spieth, Nature 372, 270 (1994); J. Spieth, G. Brooke, S. Kuersten, K. Lea, T. Blumenthal, Cell 73, 521 (1993)]} on cDNA that had been synthesized by priming with SHP12. For amplification of the 5′ end of toc-1, SHP15 and SHP14 replaced SHP12 and SHP10, respectively. Methods were essentially those given in (23). The sequences of primers are available on request.
15. i. The genes produce mRNAs with 3′ untranslated regions of 367 and 176 nucleotides for toc-1 and clk-1, respectively. For amplification of the 5′ end of the clk-1 message we used the nested primer pairs SL and SHP12 and then SL and SHP10 [where both trans-spliced leaders SL1 and SL2 were tested [D. A. Zorio, N. N. Cheng, T. Blumenthal, J. Spieth, Nature 372, 270 (1994); J. Spieth, G. Brooke, S. Kuersten, K. Lea, T. Blumenthal, Cell 73, 521 (1993)]} on cDNA that had been synthesized by priming with SHP12. For amplification of the 5′ end of toc-1, SHP15 and SHP14 replaced SHP12 and SHP10, respectively. Methods were essentially those given in (23). The sequences of primers are available on request.
16. T. Blumenthal, Trends Genet. 11, 132 (1995).
17. Such differences have been shown to be a confounding factor in the reported extended longevity of spe-26 mutant worms [D. Gems and D. L. Riddle, Nature 379, 723(1996)].
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21. i. Both mouse and human internal products were entirely sequenced, and the longer mouse 3′ amplification product was partially sequenced. The human and mouse sequences have been deposited into GenBank with accession numbers U81276 and U81277, respectively. A mouse expressed sequence tag (GenBank accession number AA030846) corresponds to the gene amplified here, and in the region of sequence overlap there were no discrepancies. The mouse sequence in Fig. 3 is a fusion of our sequence and the expressed sequence tag. The sequences of primers are available on request.
22. The CLK-1 sequence contains one potential protein kinase C phosphorylation site, one casein kinase II phosphorylation site, and three N-myristoylation sites [A. Bairoch, P. Bucher, K. Hofmann, Nucleic Acids Res. 24, 189 (1995); http://expasy. hcuge.ch/sprot/prosite.html ]. All of these are common among proteins and none of them is also present in both the yeast and rat homologs, suggesting that they may not be functional. Two cysteine residues are conserved between the three species, suggesting that they could have a structural or functional role (17).
23. The CLK-1 sequence contains one potential protein kinase C phosphorylation site, one casein kinase II phosphorylation site, and three N-myristoylation sites [A. Bairoch, P. Bucher, K. Hofmann, Nucleic Acids Res. 24, 189 (1995); http://expasy. hcuge.ch/sprot/prosite.html ]. All of these are common among proteins and none of them is also present in both the yeast and rat homologs, suggesting that they may not be functional. Two cysteine residues are conserved between the three species, suggesting that they could have a structural or functional role (17).
24. The protein is predicted to be largely helical [B. Rost and C. Sander, Proteins 19, 55 (1994)], but prediction-based threading [B. Rost, in Protein Folds, A Distance-Based Approach, H. Bohr and S. Brunak, Eds. (CRC Press, Boca Raton, FL, 1995), pp. 132-151; B. Rost, in The Third International Conference on Intelligent Systems for Molecular Biology (ISMB), C. Rawlings et al., Eds. (AAAI Press, Menlo Park, CA, 1995), pp. 314-321] fails to identify any other proteins of similar structure; http://www.embl-heidelberg.de/predictprotein/predictprotein.html.
25. The protein is predicted to be largely helical [B. Rost and C. Sander, Proteins 19, 55 (1994)], but prediction-based threading [B. Rost, in Protein Folds, A Distance-Based Approach, H. Bohr and S. Brunak, Eds. (CRC Press, Boca Raton, FL, 1995), pp. 132-151; B. Rost, in The Third International Conference on Intelligent Systems for Molecular Biology (ISMB), C. Rawlings et al., Eds. (AAAI Press, Menlo Park, CA, 1995), pp. 314-321] fails to identify any other proteins of similar structure; http://www.embl-heidelberg.de/predictprotein/predictprotein.html.
26. The protein is predicted to be largely helical [B. Rost and C. Sander, Proteins 19, 55 (1994)], but prediction-based threading [B. Rost, in Protein Folds, A Distance-Based Approach, H. Bohr and S. Brunak, Eds. (CRC Press, Boca Raton, FL, 1995), pp. 132-151; B. Rost, in The Third International Conference on Intelligent Systems for Molecular Biology (ISMB), C. Rawlings et al., Eds. (AAAI Press, Menlo Park, CA, 1995), pp. 314-321] fails to identify any other proteins of similar structure; http://www.embl-heidelberg.de/predictprotein/predictprotein.html.
27. The protein is predicted to be largely helical [B. Rost and C. Sander, Proteins 19, 55 (1994)], but prediction-based threading [B. Rost, in Protein Folds, A Distance-Based Approach, H. Bohr and S. Brunak, Eds. (CRC Press, Boca Raton, FL, 1995), pp. 132-151; B. Rost, in The Third International Conference on Intelligent Systems for Molecular Biology (ISMB), C. Rawlings et al., Eds. (AAAI Press, Menlo Park, CA, 1995), pp. 314-321] fails to identify any other proteins of similar structure; http://www.embl-heidelberg.de/predictprotein/predictprotein.html.
28. T. E. Creighton, Proteins: Structures and Molecular Properties (Freeman, New York, ed. 2, 1993).
29. A. Schöler and H. J. Schüller, Mol. Cell. Biol. 14, 3613 (1994); M. Proft, D. Grzesitza, K. D. Entian, Mol. Gen. Genet. 246, 367 (1995).
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31. 3, which contains ethanol (11). The sequences of the primers are available on request.
32. 3, which contains ethanol (11). The sequences of the primers are available on request.
33. 3, which contains ethanol (11). The sequences of the primers are available on request.
34. 3 medium (24)]. The Δcat5/coq7 strain transformed with the CAT5/COQ7-containing plasmid did not grow as well as the wild-type yeast strain on nonfermentable carbon sources. When the yeast gene was reintroduced in the context of its own promoter on a centromeric vector, full restoration of wild-type growth was obtained (24). CAT5/COQ7 is known to be involved in the regulation of its own expression (11); presumably, the presence of excess Cat5p/Coq7p perturbs the normal metabolic balance of yeast. The sequences of the primers are available on request.
35. E. W. Jones, J. R. Pringle, J. R. Broach, Eds., The Molecular and Cellular Biology of the Yeast Saccharomyces (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1992).
36. J. Campisi, Cell 84, 497 (1996); L. Guarente, ibid. 86, 9 (1996).
37. J. Campisi, Cell 84, 497 (1996); L. Guarente, ibid. 86, 9 (1996).
38. T. M. Barnes, Y. Jin, H. R. Horvitz, G. Ruvkun, S. Hekimi, J. Neurochem. 67, 46 (1996).
39. B. Lakowski and J. Ewbank, unpublished data.
40. By picking Sma non-Dpy recombinant progeny of dpy-17(e164) sma-4(e729)/unc-79(e1030) clk-1(e2519) Ion-1(e185) hermaphrodites, we were able to position clk-1 more precisely: dpy-17 25/79 clk-1 27/79 Ion-1 27/79 sma-4. To interpolate the physical position of clk-1, we estimated that the separation between ced-4 and dpy-17 is ∼0.2 centimorgans on the basis of data in the database ACeDB (7). By using linked double mutants, we also directly determined (24) the two point distances between dpy-17 and sma-4 (0.85 cM), dpy-17 and Ion-1 (0.5 cM), and Ion-1 and sma-4 (0.35 cM). Details of the mapping data can be found in ACeDB (7).
41. The sequencing of allele qm11, which has a phenotype essentially identical to e2519 (1), revealed an identical lesion. The low probability of independently obtaining the same mutation twice suggests that the original allele was lost. Sequencing of qm47 failed to reveal a mutation. Subsequent reexamination of the phenotype of qm47 homozygotes and new complementation tests suggest that qm47 is not a clk-1 allele.
42. We thank A, Coulson for cosmids; K. Kemphues for strains; J.-C. Labbé, A Kothari, and J. Mes-Mason for nematode, mouse, and human RNA, respectively; and A. Wong, A.-M. Sdicu, and R. Durbin. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. Supported by a Royal Society-National Science and Engineering Research Council of Canada exchange fellowship and a Medical Research Council of Canada fellowship to J.J.E., a Medical Research Council of Canada grant to S.H., a Canadian Genome Analysis and Technology grant to H.B., and by fellowships to B.L. from the J. W. McConnell Foundation and Fonds pour la Formation de Chercheurs et l'Aide à la Recherche Québec.