Worm Breeder's Gazette 14(3): 49 (June 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 Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas TX 75235-9038 (email@example.com)
We previously showed that avr-15 mutant worms lack neurotransmission by the M3 pharyngeal motor neuron. Furthermore, we were able to demonstrate that whereas depolarized pharyngeal muscles from wild type animals respond to the iontophoretic application of glutamate by hyperpolarizing, pharyngeal muscles from avr-15 mutants do not (Avery et al., WBG 13(4),72; J. Dent, 1995 International Worm Meeting, 69). These results suggested that avr-15 is necessary postsynaptically, in pharyngeal muscle, for the expression of a glutamate-gated chloride channel that mediates M3 neurotransmission. One possibility was that avr-15 codes for a component of the channel itself. Cully et al. (Nature 371,707-711) cloned two subunits of a glutamate-gated chloride channel, GluCla and GluClb, from C. elegans. GluCla encodes a subunit that, as a homomer, forms an ivermectin-gated chloride channel. Given avr-15's role in ivermectin sensitivity (see Avery et al., WBG 13(4),72) and glutamatergic neurotransmission, one possibility was that avr-15 coded for GluCla. To test this hypothesis, we probed a YAC grid with GluCla. Although the map position of GluCla was not consistent with it being encoded by avr-15, we were able to use GluCla to identify a closely related gene whose map position is consistent with that of avr-15. We isolated a cDNA encoding this related gene and found that the predicted protein was 85% identical to GluCla at the amino acid level and ~45% identical to GluClb. These homologies indicate that the new subunit and GluCla define a subclass of channel subunits and therefore we designated the new subunit GluCla2 and the original GluCla we call GluCla1. To demonstrate that GluCla2 is encoded by avr-15, we showed that a cosmid containing GluCla2 restores M3 neurotransmission when transformed into avr-15 worms. We also found that the ad1051 allele of avr-15 contains a nonsense mutation early in the open reading frame, which indi cates that ad1051 is likely to be a null allele. Finally, we were able to restore M3 neurotransmis sion to avr-15 mutants by transforming them with an avr-15 cDNA driven by the myo-2 promoter, which should drive expression specifically in the pharyngeal muscle. Thus, expression of the receptor postsynaptically in pharyngeal muscle is, as we predicted, sufficient to restore M3 neu rotransmission. In addition, we transformed the myo-2::avr-15 construct into an ivermectin resis tant avr-14;avr-15 double mutant strain and found that the transformed worms became sensitive to ivermectin. Thus, ivermectin's effect on the pharyngeal muscle, mediated by AVR-15/ GluCla2, is sufficient to kill C. elegans.