Worm Breeder's Gazette 12(1): 44 (September 1, 1991)
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.
Comparison of the reversion frequencies and of the sequences of reversion sites in homozygous versus heterozygous Tc1 mutants suggested a model for Tc1 transposition (Plasterk, EMBO J. 10, 1919-1925): Tc1 excises by two double strand DNA breaks at the ends of the element, and the cells deals with the break in the chromosome by template directed double strand break repair. This model is similar to that proposed for Drosophila P elements (Engels et al., Cell 62, 515-525).
We recently did the following experiment: a homozygous Tc1 mutant of unc-22 was made transgenic for a plasmid that contains a 3 kb region of the unc-22 gene corresponding to the region flanking the Tc1 insertion. The transgenic region of the gene has been marked by 5 silent polymorphisms. The point of this experiment is twofold: 1. If we can show that the polymorphisms are copied into the endogenous unc-22 gene after Tc1 excision then this is proof for the proposed model for Tc1 jumping. 2. This may provide a strategy for the introduction of defined alterations into the genome of Caenorhabditis elegans.
The marked template plasmid was mixed with pRF4 ( rol-6 (D))and injected into the gonads of mut-6 ( st702 ) unc-22 (st192:: Tc1 )IV animals. unc-22 Rol-6 lines were obtained, containing transgenic DNA in extrachromosomal arrays. These animals were found to revert for unc-22 at frequencies that were comparable to those found for the non transgenic strain (1O+E-4 -1O+E-5: usually at least one per 10 cm plate). From non transgenic segregants of revertants the DNA was analyzed. The numbers are still a bit low, but we thought you might want to know anyway: 14 were analyzed: 6 had the traditional Tc1 footprint, and 8 had apparently used the transgene as template for repair. The repair track was short (less than 25 nucleotides) for 3; the other 5 had picked up all the 5 polymorphisms form the transgene (implying the repair track is longer than 200 nucleotides; this is looking at only one side of the break).
This shows that breaks in chromosomes resulting from Tc1 excision are indeed repaired in a template instructed fashion. DNA in transgenic extrachromosomal arrays can be used as template, though not as frequently as the homologous chromosome (don't be fooled by the numbers here: 6 out of 14 showed a Tc1 footprint, but these are only the interrupted cases of allelic repair. An estimate of the frequency of repair that uses the homologous chromosome as template comes from previously described experiments, it is at least 100x higher than this). This method can be used for the directed alteration of the worm genome: many genes have been cloned by Tc1 tagging, and therefore for many genes Tc1 alleles are available. In other cases the method developed by Alice Rushforth and Phil Anderson for PCR detection of Tc1 insertions can be used (WBG 11/5, 65 and seen elsewhere in this issue). Starting with a nearby Tc1 insertion a desired alteration can now be introduced through transgene instructed gap repair. The frequency we found is high enough to allow a PCR sib selection procedure (and this is not even in a TR679 high hopper strain). The distance between Tc1 insertion site and mutation site can be more than 200 bp, probably quite a bit more with some loss in frequency. It may also be possible to set up a selection for the targeting event (e.g. by co-insertion of a selectable gene into an intron of the target gene together with introduction of the desired mutation). The repair probably copies anything that is flanked by homology to the break site.