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.

The ryanodine receptor gene ryr-1 rescued unc-68 mutation

Yasuji Sakube, Hiroaki Kagawa

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.