Worm Breeder's Gazette 10(3): 75

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.

Analysis of Tc1 Excision in vitro

Henri G.A.M. van Luenen and Ronald H.A. Plasterk

Figure 1

We previously reported that we found Tc1 excision in vitro.  
Extracts were made from a worm strain that had nuc-1 crossed into 
Bergerac;  this strain is transpositional proficient and DNase minus.  
The extract is incubated with a plasmid that carries Tc1 engineered 
into the bacterial lacA gene.  Plasmid DNA was recovered after the 
reaction and used to transform E.  coli.  With a frequency of 1 in 10,
000 Lac+ colonies were found.  Without incubation no revertants were 
found.  Twenty independent revertants were analyzed.  As expected, 
they had all undergone a deletion that removed Tc1.  The junctions 
were sequenced and this gives the following 
[See Figure 1]
All deletion endpoints fall within the window that is expected for 
lacZ reversion (no essential lacZ sequences can be removed and so much 
of Tc1 must be deleted that no stop triplets remain).  The surprising 
observation is that in all cases, the deletion is between very short 
direct repeats that are inevitably found in any stretch of DNA.  This 
raises the issue whether we are really looking at an event that is 
related to Tc1 transposition.  We doubt that.  The deletions could 
also be stimulated by single-strand nicking during the incubation (we 
see a strong relaxation activity in all extracts)  which could 
stimulate recombination after transformation in E. coli.  Since there 
is reason to doubt even if somatic excision of Tc1 in vitro is related 
to real transposition (see Beckers and Plasterk, this issue) we will 
for now not continue on this research line.

Figure 1