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?
