Worm Breeder's Gazette 10(1): 50

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.

Germline Transposition of Tc2

A. Levitt and S. Emmons

As previously reported, we have looked for evidence of Tc2 
transposition in a series of Bergerac/Bristol recombinant strains 
carrying the mut-4 locus.  These strains were constructed by Ikoue 
Mori and Don Moerman in Robert Waterston's lab.  Using the Southern 
blot technique, we have detected Tc2 restriction fragments in these 
strains that are not present in either of the parental strains.
We have cloned one of these new Tc2 fragments and used a unique 
sequence from the DNA adjacent to the element to identify the 
corresponding fragments in the parental strains.  In a Southern blot 
experiment this flanking sequence probe detects identical restriction 
fragments in Bergerac and Bristol which are approximately 2.7 KB 
smaller than the fragment in the recombinant strain.  This result is 
consistent with the idea that a Tc2 element of 2.7 KB has been 
inserted at this site in the recombinant strain.
As transposition events can be detected by Southern blot, without 
any genetic assay, it appears that Tc2 movement is occurring at an 
extremely high rate.  It is possible that transposition of both Tc1 
and Tc2 is dependent on some gene product expressed by the mut-4 locus 
that is particularly potent in its effect on Tc2.  Another possibility 
is that transposition of Tc2 is mobilized by a Bristol x Bergerac 
We have cloned a second new Tc2 fragment, and are performing the 
flanking sequence experiment with this clone as well.  The next step 
will be to analyze the sequence and structure of these two elements.  
It will be interesting to see if the two active elements we have 
isolated have the same structure, especially since our previous 
results suggested that the Tc2 sequence family is quite polymorphic.  
It will also be interesting to test both of the elements for somatic 
excision activity.
Cloning an active element proved to be more difficult than we had 
anticipated.  Our first efforts yielded only a scrambled sequence (
judging by the restriction pattern) in a sick bacteria, suggesting 
that the active form of the element may contain a sequence, which 
inhibits bacterial growth.  Such 'poison' sequences have been 
encountered by other researchers working with particular plasmid 
sequences, bacterial genes, and eukaryotic genes.  Dr.  David Finnegan,
at the University of Edinburgh, has described similar difficulties in 
cloning an intact I element in Drosophila (personal communication).
We have attempted two approaches to overcome these problems.  One is 
to use a low-copy-number plasmid as our cloning vector.  The other is 
to clone a portion of the element rather than a complete Tc2 sequence, 
starting from a restriction site inside the transposon and ending at a 
restriction site within the flanking region.  The latter approach has 
worked quite well.  Both of the fragments described above were 
isolated in this way.  The flanking sequence of the cloned 'half' 
element is then used to probe for the empty site fragment in the 
parental strains, and the cloned empty site fragment is used to 
isolate the other half of the transposon.