Worm Breeder's Gazette 1(2): 17

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.

Levamisole-resistant Mutants and the Cut Worm Assay

J. Lewis, C.-H. Wu, J. Levine, H. Berg

We have isolated several hundred levamisole (1-tetramisole)-
resistant mutants and are characterizing them genetically and 
pharmacologically.  There are at least 8 complementation groups 
including the four most frequent discovered by Brenner (tmr-1 to 4).  
The fifth most frequent maps right next to tmr-2 and tmr-3.  Among the 
much rarer complementation groups are one corresponding to unc-50 (LG 
III) and two at least not strongly linked to dpy-5 (LG I).  Mutants in 
seven of the complementation groups all have about the same mildly 
uncoordinated phenotype, extreme tetramisole-resistance, and a 
sensitivity to osmotic shock.  The uncoordinated phenotype and 
resistance to levamisole can be phenocopied by high concentrations of 
mecamylamine, an acetylcholine blocking agent.  Rare mutants resistant 
to mecamylamine can be isolated and their properties are being 
investigated viz. levamisole resistance.  Revertants, ts mutants, etc. 
are being sought.
We have developed a cut worm assay of body muscle contraction to 
compare the pharmacology of the wild type and our mutants .  We have 
found that C.  elegans cut in half will survive happily for hours in 
Ascaris Ringer's solution.  The low sensitivity of the wild type to 
acetylcholine is greatly potentiated by esterase inhibitors (eserine, 
Aldicarb, Trichlorfon).  Tetramisole and nicotinic agonists, nicotine 
and carbachol, cause rapid contraction by themselves.  Muscarine, 
methacholine and carbamyl-B-methyl-choline, muscarinic agents, have 
little effect.  Cholinergic blocking agents, curare, mecamylamine, and 
atrophine sulfate, and local anesthetics, tricaine and procaine, block 
reversibly the effects of cholinergic agonists.  Only tricaine blocks 
contraction caused by ouabain.  The high concentrations of drugs 
required (10+E-4 to 10+E-2 M) are probably due to impermeability 
caused by intervening tissue.  Tetrodotoxin and bungarotoxin seem to 
have no effect.  Other agents are under investigation.
All uncoordinated levamisole-resistant mutants tested are very 
resistant to all acetylcholine agonists effective on the wild type.  
The mutants, however, retain sensitivity to ouabain, and are still 
paralyzed by cholinergic antagonists (we believe paralysis is an 
anesthetic side effect not directly related to blocking action).  
Strychnine, an inhibitor of inhibition, causes whole or cut wild type 
worms to convulse but has little effect (or causes rapid paralysis) on 
levamisole-resistant uncs.  Whole levamisole-resistant uncs survive 
very much better than the wild type on plates containing the esterase-
inhibitors Trichlorfon and Aldicarb but eventually die, too.
We believe that levamisole is a nicotinic depolarizing blocking 
agent and that levamisole-resistant mutants are altered 
postsynaptically, possibly in the function of acetylcholine receptors. 
We are looking into bungarotoxin binding activity and the composition 
of plasma membrane proteins by 2-D gel electrophoresis in our mutants. 
Our work implies that nematode locomotory behavior depends mostly on 
myogenic activity with inhibitory regulation, cholinergic activity 
being largely dispensable.
David Hirsh kindly sent us his Trichlorfon-resistant mutants to test 
in the cut worm assay.  Mutants resistant to esterase inhibitors might 
be altered in the esterase itself or pre- or post-synaptically and be 
distinguished by testing with inhibitor alone, inhibitor plus 
acetylcholine, and a non-degradable acetylcholine analog, e.g.  
carbachol.  Our preliminary impression is that all Trichlorfon mutants 
are altered presynaptically.  The mutants do not seem to be as tight 
pharmacologically as levamisole-resistant uncs-a difficulty possibly 
circumventable by selecting for resistance at low temperature and 
screening for lethality at high temperature.
We have not yet found any significant pharmacological abnormality 
for E933, resistant to esterase inhibitors lannate and aldicarb (Temik)