Nucleic Acids Research, Vol 25, Issue 20 4041-4047, Copyright © 1997 by Oxford University Press
RF Ketting, SEJ Fischer and RH Plasterk
The Tc1 transposon of Caenorhabditis elegans always integrates into the
sequence TA, but some TA sites are preferred to others. We investigated a
TA target site from the gpa-2 gene of C.elegans that was previously found
to be preferred (hot) for Tc1 integration in vivo . This site with its
immediate flanks was cloned into a plasmid, and remained hot in vitro ,
showing that sequences immediately adjacent to the TA dinucleotide
determine this target choice. Further deletion mapping and mutagenesis
showed that a 4 bp sequence on one side of the TA is sufficient to make a
site hot; this sequence nicely fits the previously identified Tc1 consensus
sequence for integration. In addition, we found a second type of hot site:
this site is only preferred for integration when the target DNA is
supercoiled, not when it is relaxed. Excision frequencies were relatively
independent of the flanking sequences. The distribution of Tc1 insertions
into a plasmid was similar when we used nuclear extracts or purified Tc1
transposase in vitro , showing that the Tc1 transposase is the protein
responsible for the target choice.
ARTICLES
Target choice determinants of the Tc1 transposon of Caenorhabditis elegans
Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
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