Worm Breeder's Gazette 12(4): 34 (October 1, 1992)

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.

unc-50 ,A Gene Required for Acetylcholine Receptor Function, Encodes an Unfamiliar Protein

Michael Hengartner[1], Nancy Tsung[1], Jim Lewis[2], Bob Horvitz[1]

[1]HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA
[2]Division of Life Sciences, U. of Texas at San Antonio, San Antonio, TX 78249

Animals homozygous for recessive mutations in the unc-50 gene are resistant to the potent anthelminthic drug levamisole, which acts as an acetylcholine (ACh) agonist in nematodes. The levamisole-sensitive acetylcholine receptor (AChR) is critical to early juvenile motor behavior, and unc-50 mutants are highly uncoordinated as young larvae. Lewis et al. (J. Neurosci. 7, 3059-3071, 1987) have shown that unc-50 mutants lack levamisole-binding activity, indicating that the unc-50 gene is necessary for the function of the AChR. In principle, the unc-50 protein might be a subunit of the AChR or might be required for proper expression, assembly, or functioning of the multi-subunit receptor.

To determine the nature of the unc-50 protein, we have cloned unc-50 .Genetic mapping places unc-50 very close and to the left of unc-69 and ced-9 .Cosmid C15B3 recognizes allele-specific polymorphisms in three unc-50 mutant strains (1/7 EMS-, 1/2 y-ray-, and 1/1 mutator-induced mutations). By microinjection of wild-type DNA into unc-50 mutants, we localized a rescuing activity to a 5 kb fragment of genomic DNA, which also recognized the previously identified RFLPs. A 1.2 kb subclone from this region hybridizes to a 1.5 kb transcript on a Northern blot. We used this 1.2 kb fragment to isolate a 1.3 kb cDNA that could encode a 301 amino acid protein. This putative unc-50 protein shows no significant similarity to any proteins currently in the databases. Secondary structure analysis suggests that unc-50 might contain several membrane-spanning domains. The unc-50 gene contains four exons and three introns. The first intron is composed almost entirely of 28 direct repeats of a short 40 bp unit of sequence CCTCCT(c/t)AAAAAGTAGGTCA(g/t)GACCTAGTTTTTAACGTGA, that itself is an imperfect inverted repeat of a 20 bp core element. A second type of repetitive element, also observed in ced-9 , ced-3 , lin-12 , glp-1 ,and several of the cosmids from the worm sequencing project (Shaham, Hengartner, and Horvitz, WBG, 12 (3), 26, 1992), is found in the third intron and in the region downstream of the gene.

Since unc-50 apparently does not encode a subunit of the AChR, the question arises as to why there is no receptor activity in these mutants. We are currently trying to determine the fate of known AChR subunits (encoded by unc-29 , unc-38 ,and lev-1 )in unc-50 mutants using both immunocytochemistry and Northern and Western blot analyses. One possibility is that unc-50 might be required for proper assembly of the AChR.

Literature Cited:

Lewis et al., J. Neurosci. 7, 3059-3071, 1987.

Shaham, Hengartner, and Horvitz, WBG, 12 (3), 26, 1992.