Worm Breeder's Gazette 11(4): 77
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.
Studies of the enzyme alcohol dehydrogenase (ADH) and the genes that encode it have provided a wealth of information about gene regulation in a spectrum of organisms, most notably, yeast, maize and Drosophila. The utility of ADH for these studies has been in large part due to the availability of strong, and relatively easy, selections for and against ADH expression. These selection systems have allowed isolation of mutants with altered regulation or structure of this gene. In addition, the availability of these selections has provided a valuable assay for the insertion and excision of transposable elements in vivo as well as a selectable marker for transformation. A survey of the literature showed that little work had been done on ADH in C. elegans. We found that ADH activity could reproducibly be extracted from worms grown on NG plates with OP50. Briefly, worms are separated from bacteria by floating on sucrose, washed in 0.1 M Tris- HCl, pH 7.6, then stored frozen at -70 C. The rest of the procedure is essentially the same as that used to extract ADH from yeast (Denis and Young, MCB 3:360). Worms are broken by vortexing with glass beads, then assayed spectrophotometrically for ADH activity. The specific activity of ADH in N2 is 200-300 mU/mg total protein. Allyl alcohol selection has been used successfully to obtain ADH- null mutants in a number of organisms including yeast and maize. The basis of the selection is that allyl alcohol is converted by ADH into the toxic compound acrolein. In the presence of allyl alcohol only mutants in ADH expression survive. We found that wild type worms are sensitive to allyl alcohol and die when put on NG plates containing 0. 3% allyl alcohol. We then selected EMS mutagenized worms for ability to survive in the presence of allyl alcohol. Thirteen independent mutants which survived repeated screening on allyl alcohol were identified. We have assayed ADH activity in seven of these. Six have no detectable ADH activity (<10 mU/mg) and one has reduced activity ( 15 mU/mg). Thus, it appears that allyl alcohol is an effective and highly specific agent for identifying mutants with reduced ADH activity in worms. The ADH-null worms seem normal with no obvious differences from N2 in appearance or in growth on NG plates. However, in the presence of 2% ethanol, the mutant worms appear to have a greater reduction in motility than does wild type. We are pursuing this further as it will be interesting to see if ADH is a limiting factor in ethanol tolerance in worms. Our next goal is to determine the number of complementation groups represented by these mutations and to map ADH.