Worm Breeder's Gazette 17(1): 58 (October 1, 2001)

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.

Isolation and characterization of suppressors of the ryanodine receptor gene unc-68 mutants

Ryota Adachi, Hiroaki Kagawa

Graduate school of Natural Science and Technology, Okayama University, Okayama 700-8530 JAPAN

unc-68 encodes the Caenorhabditis elegans ryanodine receptor consisting 5,071 amino acid residues. Most of deletion mutants move slower than the wild-type N2. Only one missense unc-68(kh30) mutant is isolated as a ketamine-response abnormal (Kra) showing convulsions with intermittent paralysis in 30 mM ketamine. The unc-68(kh30) worm produced a full-sized protein having an amino acid substitution at Ser1,444 to Asn which is a putative phosphorylation site of a protein kinase C (Sakube et al., J. Mol. Biol., 267, 849-864, 1997).

To investigate molecules interacting with the ryanodine receptor, we isolated revertants from unc-68(e540) and unc-68(kh30) animals. Seven revertants were isolated from unc-68(e540), a nonsense mutant having splicing defect, by choosing faster moving animals. Although the motility of these animals was recovered, the brood size was not. Wild-type N2 bred 330 +/- 49 eggs and 99 % was hatched while unc-68(e540) bred 102 +/- 12 eggs and 87.6 % was hatched. The brood size of revertants was similar to that of unc-68(e540) and hatching rate was about 70 % of the wild-type. We also determined the egg laying rate and defecation cycle. Egg laying rate of the wild-type and unc-68(e540) animals were 7.3 and 1.4 per hour respectively. Egg laying rate in each revertant was two to three times higher than that of unc-68(e540). Although we have not analyzed more details about these unc-68(e540) revertants, these results suggest that in C. elegans, only one ryanodine receptor has different function in body-wall and vulval muscles. On the other hand, five revertants were isolated from unc-68(kh30) by choosing a paralyzed phenotype in 30 mM ketamine like as the wild-type. We tested responses of revertants against some drugs. From the sensitivities to 50 mM caffeine which induces ryanodine receptors to the open-state, five revertants assigned to three groups; the first had high sensitivity, the second had lower sensitivity than the unc-68(kh30), and the last had medium sensitivity between wild-type and unc-68(kh30). We also observed sensitivity to ryanodine; open the channel of ryanodine receptors, levamisole; agonist of acetylcholine receptor, and ouabain; N+, K+-ATPase blocker. We assume that the revertants showing lower sensitivity to drugs have abnormal Ca2+ control, and one of them showing slow movement and high sensitivity to drugs may be very low Ca2+ concentration in body-wall muscle. The suppressor gene of the latter was mapped to X chromosome. Using three-factor analysis and cosmid rescuing, this gene was mapped to the region of 0.5 map unit neighbor of lin-15.