Worm Breeder's Gazette 13(4): 74 (October 1, 1994)
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.
Laboratory of Genetics, 445 Henry Mall, Univ. of Wisconsin, Madison WI 53706
Ryanodine receptors (RYRs) are a family of intracellular calcium channels found in a number of cell types, including muscle fibers, neurons, and oocytes. RYRs open after depolarization of the plasma membrane, and flood the cytoplasm with calcium ions. The name ryanodine receptor is based on the high-affinity binding of the drug ryanodine to the channels. Ryanodine locks open the channels, causing contractile paralysis in muscle due to elevated calcium levels in the myoplasm. Treatment of C. elegans with ryanodine causes contractile paralysis, and a ryanodine binding activity from C. elegans copurifies with ion channels similar to mammalian RYRs (1). C. elegans has a RYR homolog on LGV called ryr-1 (2). We used reverse genetic methods to isolate a deletion within ryr-1 .First, three site-selected insertions of Tc1 ( r1145 , r1151 , r1152 )were isolated in a mut-2 background using a PCR/sib selection approach (3). None of the insertion mutants had visible phenotypes, and all were paralyzed by ryanodine. Second, a deletion derivative of r1151 ( r1158 .)was isolated using a similar PCR approach (4) r1158 homozygotes have a kinker Unc phenotype, and are weak shrinkers. The r1158 Unc phenotype is tightly linked to the deletion polymorphism detected with PCR, and maps to a deficiency interval in which ryr-1 resides (5). Southern analysis confirms that r1158 has a 5kb deletion within ryr-1 . r1158 fails to complement alleles of unc-68 . unc-68 resides in the same genetic interval as ryr-1 ,and unc-68 ( e540 )has an Unc phenotype indistinguishable from r1158 .Both e540 and r1158 are not hypercontracted in ryanodine, even at 20 times the concentration used to paralyze wild type animals. These data suggested that ryr-1 and unc-68 are the same gene. We did two noncomplementation screens for new alleles of unc-68 ,and examined the new alleles for PCR and/or Southern blot polymorphisms in ryr-1 . First, EMS-treated N2 males were mated to dpy-11 unc-68 ( r1158 )hermaphrodites. F1 progeny were screened for Unc non-Dpy animals. At least 4 new unc-68 alleles were isolated, and one of the new alleles had a deletion within ryr-1 .Second, we used two ryr-1 : Tc1 insertion alleles described above ( r1151 and r1152 )to isolate mut-2 induced unc-68 mutations. r1151 and r1152 hermaphrodites were crossed to N2 males, and the male progeny were mated to dpy-11 unc-68 ( r1158 )hermaphrodites. F1 progeny were screened for Unc nonDpys. At least 5 new unc-68 alleles were isolated in this screen at a frequency of about 1 in 500. This frequency is dramatically higher than random spontaneous mutations in the same mut-2 background (10+E-4 to 10+E-5 (6)), but is similar to the frequency of imprecise excisions of Tc1 in the mut-2 background (4). This strongly suggested that the unc-68 phenotype of these alleles was a result of Tc1 -inducedmutations within ryr-1 .All 5 of these new alleles had PCR or southern blot polymorphisms in ryr-1 .