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.
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 cross. 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 Methods 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.