Worm Breeder's Gazette 15(2): 19 (February 1, 1998)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Antibodies Based Functional Analysis of The Ryanodine Receptor in Caenorhabditis elegans

Tomoyo Hamada, Yasuji Sakube, Hiroaki Kagawa

Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, JAPAN

The ryanodine receptor in C. elegans (CeRYR) is encoded by unc-68 gene consisting of 46 exons. CeRYR having 5071 amino acid residues has 40-45% homology to that of Drosophila, cardiac- and skeletal-type of mammals. Unc-68(x14), unc-68(e540) and unc-68(r1161) have a stop codon in the internal or Tc1 induced deletion in the ryr-1 gene. Although the ryanodine receptor gene in C. elegans is only one, these mutants aren't lethal. Unc-68(kh30) isolated as a ketamine response abnormal(Kra) phenotype has a mutation site at Ser1444Asn, a putative phosphorilation site of PKC. Unc phenotype of unc-68(e540) is rescued with complete ryr-1 gene by microinjection experiment. We have reported that the promoter/lacZ fusion plasmids expressed in muscles and non-muscles under the control of 5' upstream sequences.

To know the real localization of CeRYR, we raised six antisera against region- specific fusion proteins and did Western blot and immunohistochemical analysis. Affinity-purified antibodies were prepared by adsorption with a corresponding band of the fusion proteins. Three of six purified antibodies reacted to a band of 360 kDa from N2, unc-68(kh30) and unc-68(e540):;pCRYR1 animals. But the band corresponding to 360 kDa was not detectable in unc-68 deletion mutant animals. Interestingly these antibodies crossreacted to a band of 270 kDa only in unc-68(r1161). One of three antibodies was raised against the region corresponding to kh30 mutation site. This antibody mainly stained the body-wall, pharyngeal and vulval muscles of N2 but not that of unc-68 deletion mutant animals. Intestine staining was also observed in N2 and unc-68 deletion mutant animals. This result come from the existence of nonspecific antibody or a 150 kDa protein having a common antigenicity to CeRYR in the worm. We continue to isolate a specific antibody of CeRYR for knowing the real location and the function of the protein. Fusion protein encompassing two EF-hand motif was shifted its mobility in the presence of 5mM CaCl2.

These region specific antibody approaches are useful to analyze the functional domains of the ryanodine receptor molecules of C. elegans and other animals.


Sakube et al., (1997) J. Mol. Biol. 267, 849-864.