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.
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.