Worm Breeder's Gazette 9(1): 27
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 sequenced the insertion sites of nine independent insertions at unc-54. Our purpose is to determine the exact structure of Tc1- induced mutations and to assess the target site specificity of Tc1 transposition. The results of this analysis are summarized below. Each insertion occurs within a TA dinucleotide (underlined). The numbering system is that of Karn et al. (PNAS 80:4253-4257 (1983)). [See Figure 1] All of the insertions are located in exons. The data indicate that Tc1 insertion is highly site-specific. Five of the nine insertions are in the exact same TA dinucleotide. Furthermore, two of the remaining four insertions are located in another identical TA dinucleotide. Combined with the sequences of Rosenzweig et al. for two natural Tc1 insertions in Bergerac (N.A.R. 11:7137-7140), the following consensus sequence emerges: [See Figure 2] It is difficult to judge the significance of parts of this consensus. Only the TA dinucleotide is absolutely conserved. Clearly, the sequences of additional insertion sites are needed, especially those that occur at high frequency. We have determined the sequence of a germ-line, wild-type revertant of unc-54(r323::Tc1). We were surprised to discover that this revertant (TR462) contains a four base pair insertion remaining at the site of excision: This structure (...TATGTA...) is the same as that reported at the worm meeting by K. Ruan and S. Emmons for a somatic excision product of a natural Bergerac Tc1 element. The TG dinucleotide might be derived from the two nucleotides at one end of Tc1 (as indicated by the asterisks). An exon insertion of four base pairs would disrupt the unc-54 reading frame. Therefore, the wild-type phenotype of this revertant is unexpected. One possible explanation is as follows: The target TA dinucleotide in r323 is one base pair removed from the 5' splice site of IVS#3. We suggest that the inserted bases in TR462 generate a new splice site four base pairs 5' of the normal site. In this way, the reading frame is restored. The sequence of the proposed new splice site fits fairly well with the C. elegans splice site consensus sequence compiled by Tom Blumenthal: The suggested new 5' splice is indicated by the vertical line. The wild type splice site is indicated by the apostrophe ('). The similarity of this new site to the consensus may allow sufficient splicing at this location to generate a wild type phenotype. We do not know whether TR462 constitutes a 'typical' excision event. If excisions of this type are the most frequent, mutants having Tc1 inserted within exons may not generally revert at high frequency. It may require 'atypical' excision events for such mutants to visibly revert. We have detected partial revertants for r323::Tc1 (indicating excisions of other types), but their sequences have not been determined. An intriguing feature of this revertant sequence is that the TA target site, coupled with the terminal TG of Tc1 left behind, always yields upon excision four of the highly conserved nucleotides that constitute a donor splice site. Perhaps many Tc1 excisions generate new donor splice sites?