Worm Breeder's Gazette 8(3): 85

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An Update on Tc1 Induced unc-54 Mutations

D. Eide, P. Anderson

We have continued our analysis of spontaneous insertion mutations 
affecting unc-54.  We reported previously that 11 of 19 spontaneous 
unc-54 mutations isolated in the Bergerac genetic background are 
insertions, and that most of these insertions are probably copies of 
Tc1.  Ten alleles have now been cloned into lambda vectors.  DNA 
sequence and Southern blot analysis proves that all ten inserts are 
copies of Tc1.  Among the ten cloned Tc1 elements, we have observed 
only one case of restriction site heterogeneity.  One element contains 
a HindIII site, and may be a copy of Tc1(Hin), a Tc1 variant described 
by Harris et al.  (Newsletter 8,1:48).  Several different target sites 
have been observed within unc-54, but there is considerable clustering 
of independent insertions.  Seven of our inserts map to a 600bp region 
within unc-54.  Sequence data for three of these alleles indicate that 
the insertions occur at precisely the same site.  As has been observed 
at other Tc1 insertion sites (Rosenzweig et al., N.A.R.  20:7137), 
these inserts are flanked by TA dinucleotides.  The question of target 
site duplication, therefore, remains unanswered.
We are examining the frequencies of germline and somatic reversion 
for these unc-54 ::Tc1 insertions.  Germline reversion results in wild-
type animals that contain precise or nearly-precise excisions of Tc1.  
Somatic reversion results in mosaic animals in which the sex muscles 
and certain of the body-wall muscles are revertant.  Such animals are 
egg-laying proficient, and this provides a convenient genetic screen 
for somatic reversion.  Although we have no direct evidence that these 
somatic reversion events are due to Tc1 excision, we feel that the 
high-frequency somatic excisions described by Emmons & Yesner (Cell 
36:599) are the most likely explanation.  Germline reversion 
frequencies range from 2x10+E-5 to less than 1x10+E-6.  Somatic 
reversion frequencies also show a range of values, often as high as 
6x10+E-3.  We hope to determine whether the differences in reversion 
frequencies relates to features of the target site or the individual 
Tc1 elements themselves.
Germline reversion, but not somatic reversion, is sensitive to 
genetic background.  Substitution of an unc-54 ::Tc1 mutant into the 
DH424 genetic background (a high-copy strain in which Tc1 does not 
transpose) results in germline stabilization of the mutation, but the 
frequency of somatic reversion is unaffected.  This observation is 
similiar to that of D.  Moerman et al.  (Newsletter 8,2:47) and 
suggests that germline and somatic excision of Tc1 are regulated 
differently or are mechanistically distinct.