Worm Breeder's Gazette 15(3): 36 (June 1, 1998)
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 Molecular Biology and Oncology, UT Southwestern Medical Center, Dallas, Tx. 75235
In the presence of an abundant supply of bacteria, worms eat as much as they can by initiating rapid pharyngeal pumping. Previously we showed that the motorneuron MC is necessary for this rapid pharyngeal pumping (1,2). Genetic and pharmacological evidence suggests that the MC neurotransmitter is acetylcholine (ACh) and that it acts directly on pharyngeal muscle (3).
eat-2 is a gene identified in a screen designed to find mutations that eliminate neurotransmission from MC. eat-2 worms are unable to pump rapidly in the presence of food and they are starved. Two results implicate eat-2 in MC neurotransmission. First, electrical recordings from the pharynxes of eat-2 mutants were similar to recordings from MC- worms. Also, when MC was ablated in two possible loss of function alleles, pharyngeal pumping rate did not decrease when compared to the same mutants in which MC was intact (3). Fourteen independent recessive mutations were identified in eat-2 that all caused a starved appearance and reduced pumping rate. There is complex intragenic complementation among the 14 eat-2 alleles as well as allele specific interactions between eat-2 and a semidominant allele of another slow pumping mutant, eat-18 (3). eat-18 is MC- by the same criteria as eat-2. A ! model consistent with these data is that eat-2 and eat-18 interact in a protein complex that is involved in ACh neurotransmission.
Mapping experiments placed eat-2 at the end of the right arm of LGII near unc-52, a region of the genome that is currently being sequenced. Eric Jorgenson pointed out to us that a recent sequence update contained a nicotinic ACh receptor subunit in an area consistent with the map position of eat-2. Transformation of eat-2 mutants with a YAC containing the nicotinic receptor subunit rescued the slow pumping and starved phenotypes. To determine if the nicotinic receptor was responsible for rescuing eat-2 we used PCR to amplify 10kb of genomic DNA that included the coding region of the receptor and 4kb of upstream sequence. This 10kb PCR product was sufficient to rescue eat-2, showing that the nicotinic receptor is encoded by eat-2. We also found that the eat-2 allele ad451 contains a missense mutation that changes a glutamate to a tyrosine in the amino-terminal extracellular region of the protein. No specific func! tion is known for this amino acid but it is highly conserved among nicotinic receptors. We were able to rescue MC nuerotransmission in an eat-2 mutant by expressing an eat-2 genomic clone in pharyngeal muscle using the myo-2 promoter. Rescue by this construct shows activity of eat-2 in pharyngeal muscle is sufficient to restore MC neurotransmission. Comparison of eat-2 with other receptor subunits shows it is most similar to vertebrate alpha7 subunits of neuronal nicotinic ACh receptors. However it does not have the vicinal cysteines present near the ACh binding site characteristic of all alpha subunits and is therefore a non-alpha subunit.