Tc3 and TcS elements transpose and excise in the germ line of mutator strain TR679 [
mut-2(
r459)]. Germ-line activity of Tcl is elevated in this background as well, suggesting that these different element families might move by a common mechanism in response to the same factor. If so, we would expect the structure of sites of insertion and excision of each element to be similar. To address this question we have determined the nucleotide sequence at the junctions of several insertions of Tc3 and TcS in the
unc-22 gene, and the empty sites resulting from excision of each element. Using PCR, we amplified from genomic DNA the regions containing these sites, then directly sequenced the PCR products. Three independent
unc-22::Tc3 alleles were examined: in each case the element inserted at the sequence TA, and the insertion is flanked by this TA dinucleotide. These results support the above model, since all Tcl insertions examined to date exhibit the same features. Also consistent with this idea is the fact that the termini of Tcl and Tc3 are nearly identical. Analysis of TcS complicates things: two independent TcS insertions in
unc22 inserted at the sequence TAA, and the insertion is flanked by this three nucleotide sequence. While different from Tcl and Tc3, this is the same sequence reported for a Tc4 insertion site (Yuan and Horvitz). Further complicating things, a TcS insertion in
mec-7 [
mec-7(
u388::Tc5) - Savage and Chalfie] has a TGA target site. Interestingly, the termini of TcS and Tc4 are nearly identical, and differ from Tcl and Tc3. Collectively, these results suggest that the transposon families in C. elegans form two subgroups, Tcl/Tc3 and Tc4/TcS. To examine empty sites resulting from excision of Tc3 and TcS, we amplified and sequenced appropriate genomic regions from wild-type revertants of most insertion-containing mutants described above. For both elements, some excision events were precise (wild-type
unc-22 sequence restored), others created small, in-frame insertions at the site of excision. None of the sites analyzed resembled the four- nucleotide 'footprint' characteristic of most Tcl excision sites. However, many more excision events must be examined before the significance of this observation can be assessed. We are also using PCR to address the question of somatic activity of Tc3 and TcS. Using templates prepared from single L2
unc-22::Tc3 or
unc-22::TcS mutants, and primers flanking the insertion site, we have observed amplification products of sizes consistent with element excision. Work at present is directed at determining the sequence of these PCR products and analyzing the frequency of these events.