Worm Breeder's Gazette 8(3): 85
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 continued our analysis of spontaneous insertion mutations affecting unc-54. We reported previously that 11 of 19 spontaneous unc-54 mutations isolated in the Bergerac genetic background are insertions, and that most of these insertions are probably copies of Tc1. Ten alleles have now been cloned into lambda vectors. DNA sequence and Southern blot analysis proves that all ten inserts are copies of Tc1. Among the ten cloned Tc1 elements, we have observed only one case of restriction site heterogeneity. One element contains a HindIII site, and may be a copy of Tc1(Hin), a Tc1 variant described by Harris et al. (Newsletter 8,1:48). Several different target sites have been observed within unc-54, but there is considerable clustering of independent insertions. Seven of our inserts map to a 600bp region within unc-54. Sequence data for three of these alleles indicate that the insertions occur at precisely the same site. As has been observed at other Tc1 insertion sites (Rosenzweig et al., N.A.R. 20:7137), these inserts are flanked by TA dinucleotides. The question of target site duplication, therefore, remains unanswered. We are examining the frequencies of germline and somatic reversion for these unc-54 ::Tc1 insertions. Germline reversion results in wild- type animals that contain precise or nearly-precise excisions of Tc1. Somatic reversion results in mosaic animals in which the sex muscles and certain of the body-wall muscles are revertant. Such animals are egg-laying proficient, and this provides a convenient genetic screen for somatic reversion. Although we have no direct evidence that these somatic reversion events are due to Tc1 excision, we feel that the high-frequency somatic excisions described by Emmons & Yesner (Cell 36:599) are the most likely explanation. Germline reversion frequencies range from 2x10+E-5 to less than 1x10+E-6. Somatic reversion frequencies also show a range of values, often as high as 6x10+E-3. We hope to determine whether the differences in reversion frequencies relates to features of the target site or the individual Tc1 elements themselves. Germline reversion, but not somatic reversion, is sensitive to genetic background. Substitution of an unc-54 ::Tc1 mutant into the DH424 genetic background (a high-copy strain in which Tc1 does not transpose) results in germline stabilization of the mutation, but the frequency of somatic reversion is unaffected. This observation is similiar to that of D. Moerman et al. (Newsletter 8,2:47) and suggests that germline and somatic excision of Tc1 are regulated differently or are mechanistically distinct.