Worm Breeder's Gazette 8(1): 47
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 been investigating the possibility that the spontaneous unstable unc-22 alleles induced in the BO strain of C. elegans may be caused by Tc1. Our conclusion, based on two types of analysis, is that Tc1 is probably not the causative agent of unstable twitchers. To investigate the causative role of high Tc1 copy number in generating twitcher mutations we have tested several strains in addition to N2 and BO for the induction of twitcher mutations. These strains include the 'high' copy number strains, FR, BL1, and EPC-4; the 'low' copy number strains, Ga-5, PA-1, and PA-2 (Emmons et al., Cell 32:55-65, 1983; Liao et al., P.N.A.S. 80:3585-3590, 1983); and HA-8 which has not been examined for Tc1 copy number. Of these nine stains, only the BO strain exhibits the high frequency of spontaneous induction of twitcher mutations. These observations suggest that high Tc1 copy number per se is not sufficient to induce unstable unc-22 mutations. Rather there is something unique in the Bergerac background resulting in the high spontaneous twitcher frequency which, for example, could be an altered Tc1 or other factor. Our second approach to this problem consisted of examining Tc1 copy number and position in DNA from a number of different genetic strains with and without spontaneous twitcher mutations. The strains used were as follows: N2, RW7012 (contains unc-22(st136), a spontaneous twitcher isolated on a Bergerac IV chromosome in the BO strain and then outcrossed to an N2(male) strain via 8 cross intercross mating cycles), RW7018 (contains unc-22(st137), a spontaneous twitcher isolated on a Bristol IV chromosome in a BO background and then outcrossed as described for RW7012), Rw2370 (a spontaneous revertant to wild type from RW7018), RW1461 (a triple containing daf-14(m77) 136) 66),st136 derived from RW7012), RW1462 (an unc-43 ant from daf-14 136) -43(e408) 4)), RW1463 (a daf-14 recombinant from daf-14 1363 -43 )), RW1464 (a triple of daf-14 137) rived from RW7018), RW1465 (an unc-43 ant from daf-14 137) -43 4)), RW1466 (a daf-14 recombinant from daf-14 137) -43 )). In DNA from the various strains we could detect about 30 bands that hybridized to the Tc1 probe after HindIII, EcoRI, or XbaI digestion. However, we could not identify a Tc1 that was strictly associated with the mutated state of the unc-22 locus. In particular, we saw no difference between the digestion patterns of RW7018 and RW2370 for these three restriction enzymes. The strains derived from three factor crosses showed some banding heterogeneity, but again, we did not detect a band that was present in those strains containing a twitcher mutation (RW1461 and RW1464), and absent in those strains that did not (RW1462, RW1463, RW1465, and RW1466). These observations are negative in that we failed to observe a difference in Tc1 location consistent with Tc1 as the cause of the unc- 22 mutations. Of course, we cannot rule out the possibility that the critical Tc1 element is in a band comigrating with other Tc1 containing segments. Although failing to identify a Tc1 at the unc-22 site, our analysis of the recombinants has revealed two putative unc-22 linked Tc1 polymorphisms, one between daf-14 and unc-22 (a 2 m.u. interval), and one between unc-22 and dpy-4 (a 4.5 m.u. interval). The putative polymorphism to the left of unc-22 is a 2.1Kb HindIII fragment containing Tc1 which we have cloned into lambda 590. At present we are trying to isolate a unique sequence segment from this HindIII fragment so that we can use it to position the polymorphism with greater certainty. If the fragment is suitably close to unc-22 we may be able to use it to walk to the unc-22 gene. In conclusion, we feel that Tc1 is unlikely to be responsible for generating the unc-22 mutations, but linked Tc1 polymorphisms may be useful in providing us with a molecular probe for the region.