Target choice determinants of the Tc1 transposon of Caenorhabditis elegans.
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.
Ketting
R F
RF
Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
Fischer
S E
SE
Plasterk
R H
RH
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004251:DNA Transposable Elements; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D017353:Gene Deletion; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D019895:Transposases
D004251:DNA Transposable Elements; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D004247:DNA; D009713:Nucleotidyltransferases; C081454:Tc1 transposase; D019895:Transposases