unc-68 encodes ryr-l, a C. elegans ryanodine receptor. Ed Maryon and Phil Anderson. 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 ry r- I . First, three site-selected insenions of Tcl (rl 145, rllSI, rllS2) were isolated in a
mut-2 background using a PCR/sib selection approach (3). None of the insenion mutants had visible phenotypes, and all were paralyzed by ryanodine. Second, a deletion derivative of
rll51 (
rll58.) was isolated using a similar PCR approach (4)
rll58 homozygotes have a kinker Unc phenotype, and are weak shrinkers. The rl 158 Unc phenotype is tightly linked to the deletion polymorphism detected with PCR, and maps to a deficiency interval in which ryr-l resides (5). Southern analysis confirms that rl 158 has a 5kb deletion within ryr-l .
rll58 fails to complement alleles of
unc-68.
unc-68 resides in the same genetic interval as ryr-l, and
unc-68(
e540) has an Unc phenotype indistinguishable from
rll58. Both eS40 and rllS8 are not hypercontracted in ryanodine, even at 20 times the concentration used to paralyze wild type animals. These data suggested that ryr-l 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-l. First, EMS-treated N2 males were mated to dp y - I I u n c - 6 8 ( r I 1 5 8 ) hermaphrodites. Fl 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-l . Second, we used two ryr-l :Tcl insenion alleles described above (
rll51 and
rll52) to isolate
mut-2 induced
unc-68 mutations. rllSI and rllS2 hermaphrodites were crossed to N2 males, and the male progeny were mated to dpy-llunc-68
(rll58) hermaphrodites. Fl progeny were screened for Unc non Dpys. 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 (10-4 to 10-5(6)), but is similar to the frequency of imprecise excisions of Tc 1 in the m u t - 2 background (4). This strongly suggested that the
unc-68 phenotype of these alleles was a result of Tcl-induced mutations within ryr-l. All 5 of these new alleles had PCR or southern blot polymorphisms in ryr-l. It is likely that the Unc phenotype is the
unc-68 null phenotype. Both
e540 and rl 158 have similar Unc phenotypes when heterozygous to a deficiency (sDf20). rllS8 deletes most of the proposed membrane spanning domains of the calcium channel, and some of the new (viable) alleles also have large (2-4 kb) deletions in ry r - I . Although 2 of the 9 new alleles appear to be lethal as homozygotes, they may have large deletions or other mutations that affect linked essential genes. We are further characterizing the new
unc-68 alleles, and testing mutants for 3 H-ryanodine binding activity. l)Kim et al. Biophys. 1. 1992. 63:1379. 2)WBG 13#2 p.58, see also this gazette 3) Rushfonh et al. 1993. MCB 13:902. 4)WBG 12#5 p.20. 5)'93 Mtg Abstracts, p.303. 6) J. Collins and P. Anderson, unpublished results.