Worm Breeder's Gazette 14(4): 66 (October 1, 1996)
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.
Department of Biology, Faculty of Science, Okayama University, Okayama 700 Japan
To characterize excitation-contraction coupling in C. elegans, we have applied two approaches. First, we isolated a mutant having abnormal response against ketamine, an anesthetic in vertebrates. unc-68(kh30), the novel semi-dominant allele of unc-68 mutation that was initially isolated as kra-1(kh30), exhibited strict ketamine-dependent convulsions followed by paralysis (7th C. elegans meeting, 1989). Second, we cloned and analyzed the C. elegans ryanodine receptor gene, ryr-1. CeRYR had a homology to those of Drosophila and the cardiac type of rabbit at the rate of 47% and 42% respectively. Ryr-1 promoter/lacZ plasmids were expressed in body wall and pharyngeal muscles, were also expressed in neurons and intestinal cells (10th meeting, 1995). We identified the mutation sites of kra-1 and unc-68 in the ryr-1 gene (WC Meeting, 1996). The kra-1(kh30) mutation was a Ser1,444Asn substitution at a putative PKC phosphorylation site of CeRYR. The unc-68(e540) mutation was in a splice acceptor site caused a stop codon at the middle in the ryr-1 gene. The kh30 mutation locates in the most hydrophilic region of the foot structure. This mutation region may be responsible for the specific modification or interaction with other regulatory molecules. We also confirmed that unc-68 mutation was a defect of the ryr-1 gene. pCRYR1, the designed cosmid vector including the entire ryr-1 coding sequence and the essential 5'-regulatory sequences, rescued mutation phenotypes of the unc-68(e540) animal. Injected e540 animals segregated both Rol (non-Unc) and the wild type progenies. e540::ExpCRYR1 animals moved at 73% of the wild type, and recovered the sensitivity to 1 mM ryanodine. In contrast, e540 and r1158, a Tc1 induced deletion allele of ryr-1 showed only 20-23% motility of the wild type and lacked ryanodine sensitivity (Lewis et al. 1980, Maryon & Anderson, 10th meeting, 1995). The reason why it took so long to get the final result was follows; 1) poor information about the gap between the genetic and the physical map at that time, 2) the unc-68 mutation has a weak phenotype, 3) unc-68 is a huge gene that spans more than 30 kb and encodes a predicted polypeptide of 5,071 amino acid residues. Detailed analysis on the kh30 animal is useful to know the calcium signaling pathway from the voltage sensitive calcium channel to the muscle fibers.