Worm Breeder's Gazette 9(1): 35

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.

Spontaneous Levamisole-Resistant Mutants Of The TR679 Strain Of C. elegans

J.A. Lewis

Figure 1

We have isolated 29 spontaneous levamisole-resistant mutants arising 
from the TR679 strain of C. ly provided by 
Phil Anderson.  We find spontaneous levamisole-resistant mutants arise 
roughly five to ten times more frequently in the TR679 strain than 
they do in the Bergerac strain of C.  elegans.  Mutants were isolated 
by our variation of Brenner's tetramisole selection scheme (Lewis, et. 
al., Genetics 95: 905-928 (1980)), putting 40 adult TR679 
hermaphrodites out per large plate.  A mutant can be found on almost 
every selection plate.  The relative infertility of TR679 allows the 
parent strain to struggle along for a number of generations on a 1 mM 
levamisole plate, so a resistant mutant may appear on a selection 
plate after several generations of growth even if one is not found 
initially.  The table below compares the number of mutants isolated by 
EMS mutagenesis of the Bristol wild-type strain (~several hundred 
selection plates) with the number of spontaneous mutants found for the 
Bergerac strain (163 selection plates) and for the TR679 strain (36 
selection plates).  Spontaneous mutants of the Bergerac strain are 
found predominantly in the unc-38 and unc-63 genes.  With the TR679 
strain, unc-63 is still a frequent target but unc-38 is decidedly less 
of a target and the overall mutational spectrum is a closer 
approximation of that found using ethylmethane sulfonate.
[See Figure 1]
The ability to easily isolate multiple spontaneous mutants of a gene 
by levamisole resistance selection is a powerful asset.  Since 
nematode genes contain few introns and are therefore relatively small, 
the number of different possible new Tc1-containing restriction 
fragments that might arise by mutational insertion of Tc1 into a gene 
should be small.  For example, we have found after backcrossing and 
recombination with flanking genetic markers in the Bristol strain that 
four of four Bergerac unc-38 mutants have a 4.6 Kb Tc1-containing 
HindIII restriction fragment not found in five of five control 
constructs of the same region made from the Bergerac parent strain.  
Reference to wild-type control constructs of the same region provides 
a way to readily differentiate flanking elements from possible novel 
inserts into a gene.  Repeated observation of the same novel Tc1 
insert in several mutants provides assurance that the insert is not an 
irrelevant insertion into a nearby flanking region.  The flanking 
elements found in the mutant constructs must be a subset of those seen 
in the control constructs, a goal which can be achieved by putting the 
mutant constructs through additional cycles of recombination, if 
necessary, to remove flanking elements.
Phil Anderson has impressed upon us the utility of obtaining a 
revertant of any possible Tc1-caused mutation to substantiate a cause-
and-effect relationship between the appearance of a novel Tc1 element 
and mutation of a gene.  None of our Bergerac spontaneous mutants, 
however, has shown great willingness to revert.  Several of our TR679 
spontaneous mutants have reverted in just a few generations on stock 
plates.  We have found it possible to readily revert a Bergerac unc-38 
mutation by crossing the mutation into a TR679 background and then 
picking a mutant homozygote that segregates twitchers at high 
frequency as a starting strain from which to select revertants.  After 
cloning mutant genomic DNA from the region flanking the novel Tc1 
insert seen in the Bergerac unc-38 mutants, we plan to examine the 
genomic DNA of the revertant directly without further manipulation for 
loss of 1.6 Kb from the restriction fragment hybridizing with the 
mutant genomic DNA.

Figure 1