CLAVATA3, a multimeric ligand for the CLAVATA1 receptor-kinase

Science

Volumn 289, Issue 5479, 2000, Pages 613-617

  Trotochaud, Amy E ^a   Jeong, Sangho ^a   Clark, Steven E ^a  

^a UNIVERSITY OF MICHIGAN   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

LIGAND; POLYPEPTIDE; PROTEIN; PROTEIN KINASE; RECEPTOR;

ARABIDOPSIS; ARTICLE; CELL DIFFERENTIATION; CELL PROLIFERATION; COMPLEX FORMATION; ENZYME ACTIVITY; LIGAND BINDING; NONHUMAN; PRIORITY JOURNAL; PROTEIN EXPRESSION; RECEPTOR BINDING;

ALLELES; ARABIDOPSIS; ARABIDOPSIS PROTEINS; BIOPOLYMERS; BRASSICA; CELL MEMBRANE; GENES, PLANT; IMMUNE SERA; LIGANDS; MEMBRANE PROTEINS; MERISTEM; MOLECULAR WEIGHT; PLANT EXTRACTS; PLANT PROTEINS; PROTEIN BINDING; RECEPTOR PROTEIN-TYROSINE KINASES; RECOMBINANT FUSION PROTEINS; SIGNAL TRANSDUCTION;

ARABIDOPSIS; BRASSICA OLERACEA VAR. BOTRYTIS;

EID: 0034725902     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.289.5479.613     Document Type: Article

References (23)

1. H. J. Franssen, Curr. Opin. Plant Biol. 1, 384 (1998).
2. S. E. Clark, M. P. Running, E. M. Meyerowitz, Development 119, 397 (1993); Development 121, 2057 (1995).
3. S. E. Clark, M. P. Running, E. M. Meyerowitz, Development 119, 397 (1993); Development 121, 2057 (1995).
4. S. E. Clark, R. W. Williams, E. M. Meyerowitz, Cell 89, 575 (1997).
5. R. W. Williams, J. M. Wilson, E. M. Meyerowitz, Proc. Natl. Acad. Sci. U.S.A. 94, 10467 (1997).
6. J. M. Stone, A. E. Trotochaud, J. C. Walker, S. E. Clark, Plant Physiol. 117, 1217 (1998).
7. J. C. Fletcher, U. Brand, M. P. Running, R. Simon, E. M. Meyerowitz, Science 283, 1911 (1999).
8. A. E. Trotochaud, T. Hao, W. Guang, Z. Yang, S. E. Clark, Plant Cell 11, 393 (1999).
9. S. Jeong, A. E. Trotochaud, S. E. Clark, Plant Cell 11, 1925 (1999).
10. J. M. Stone, M. A. Collinge, R. D. Smith, M. A. Horn, J. C. Walker, Science 266, 793 (1994)
11. K. Hofmann and P. Bucher, Trends Biochem. Sci. 20, 347 (1995).
12. J. Li, G. P. Smith, J. C. Walker, Proc. Natl. Acad. Sci. U.S.A. 96, 7821 (1999).
13. CLV3 mature protein coding sequence was generated by polymer chain reaction (PCR) amplification from Ler Arabidopsis genomic DNA using primers 3B3 (5′-TTCCCGGGAGATCTCACTCAAGCTCATGCTCACGTTCAAG) and 3B2 (5′-TCATCATCATCTTGCGGTTGGAAAGTCCTTGAACGTGAGCAT) for exon 2, and 3C1 (5′-CAACCGCAAGATGATGATGATGAAAATGGAA) and 3C2 (5′-CTCTAGATCAAGGGAGCTGAAAGTTGTTT) for exon 3 using Vent polymerase (New England Biolabs, Beverly, MA). The two products were then used to prime each other in a fusion PCR reaction. The resulting fragment was cloned into the pMalK vector (New England Biolabs), verified by sequencing, and expressed in E. coli. The expressed protein was purified using amylose resin (New England Biolabs) and separated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The purified protein was then band-isolated and used for anti-sera production in rabbits (Cocalico Biologicals, Reamstown, PA). All proteins for Western analysis were separated on 4 to 20% gradient polyacrylamide gels (Bio-Rad, Hercules, CA). All protein analysis was carried out as described (7).
14. Supplemental material is available at www.sciencemag. org/feature/data/1048126.shl
15. J. A. Pogany et al., J. Plant Res. 111, 307 (1998).
16. A. E. Trotochaud and S. E. Clark, data not shown.
17. M. Grossman, B. D. Weintraub, M. W. Szkudlinski, Endocr. Rev. 18, 476 (1997).
18. 4 (pH 9.4), 1.2 M sorbitol, 1× protease inhibitor cocktail, 50 mg/ml zymolase] and incubated for 30 min at 30°C. The cells were then collected by centrifugation for 5 min at 3000g. The cells were gently resuspended in 50 ml of cleared cauliflower extract and rocked gently for 2 hours at room temperature. The cells were centrifuged at 5000g for 10 min. The pellet was resuspended in a minimal volume of 50 mM Hepes (pH 7.4), 1 mM EDTA, 0.1% Triton, and 1× protease inhibitor cocktail; ground using a mortar and pestle on ice; and centrifuged at 5000g for 10 min. The supernatant was removed. Finally, 30 μl was loaded on a 4 to 20% gradient SDS-PAGE (Bio-Rad Ready Gels) and transferred to 0.2 μm of nitrocellulose (Bio-Rad) for a Western blot.
19. J. Casanova and G. Struhl, Nature 362, 152 (1993).
20. Y. Chen and G. Struhl, Cell 87, 553 (1995).
21. A. Hajnal, C. W. Whitfield, S. K. Kim, Genes Dev. 11, 2715 (1997).
22. D. Mumberg, R. Müller, M. Fink, Gene 156, 119 (1995).
23. A. Bevan, C. Brenner, R. S. Fuller, Proc. Natl. Acad. Sci. U.S.A. 95, 10384 (1998).