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.

Selection for Mutants Lacking Alcohol Dehydrogenase

Valerie M. Williamson and George Theodoris

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.