Worm Breeder's Gazette 8(1): 46
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.
The excision events that Tc1 elements in Bergerac undergo at high frequency occur in somatic cells and not in germ cells. We drew this conclusion earlier from experiments showing that Tc1 elements were stably inherited during long-term propagation. We have now been able to demonstrate directly that excision is confined to somatic cells by studying the amount of excision that has occurred as a function of age in staged populations of worms. We synchronized Bergerac cultures by isolating embryos from young, gravid hermaphrodites with hypochlorite. These embryos were allowed to develop and at various stages samples were taken for DNA isolation. In each DNA preparation the amount of excision that had taken place at three Tc1 sites was assessed using flanking-sequence probes. (We used pCe1001, isolated in our laboratory, pCe(Br)T1, isolated in Hirsh's laboratory, and pCe1003, a subclone from phage lambda 3-8 isolated by Bill Sharrock.) For all three probes the fraction of empty sites was found to steadily increase during larval development from as little as 0.6% in embryos to as much as 10% in late larvae. The fraction in adults was lower, 2-3%, as expected from growth of germ cells and internal embryos. Most particularly, however, the level in embryos of the next generation fell once again to the lowest level, indicating that empty Tc1 sites were not inherited. Preliminary results with worms arrested by starvation suggest that excision occurs at a steady rate independent of developmental stage, since the amount of excision was found to correlate with time since fertilization rather than with stage. We have been able to detect extrachromosomal copies of Tc1 that may be the excised elements. When Bergerac DNA is fractionated on an agarose gel without treatment with a restriction endonuclease, and hybridized on a Southern to a Tc1-specific probe, three low molecular weight species can be seen with mobilities that are consistent with a 1.6 kb linear, relaxed circle, and supercoil. The level of hybridization to these species in DNA from L4 larvae corresponds to about one copy per cell, and appears to increase during development, suggesting a relationship to the excision process. We have isolated these molecules on sucrose gradients and begun to analyze their structure with restriction endonucleases. Results so far confirm that the relaxed circle and linear are free copies of Tc1. The linear species has unique (not permuted) ends that correspond to the ends of an inserted Tc1 element. 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.