Figure 3.

TnpI-mediated relaxed and constrained recombination between directly and inversely repeated core sites in vivo. Reporter plasmids containing different arrangements of the IR1–IR2 core sites joined to directly repeated DR1–DR2 sequences or not were transformed into E. coli TOP10 cells harbouring the TnpI expression vector pGIV004 (ev) or the control plasmid pCB104 (c). Plasmid DNA was isolated from a pool of transformants and run uncut on a 0.8% agarose gel. Intramolecular recombination of the substrate monomer (S) yielded monomeric resolution (mR) products in which the DNA fragment between both recombination sites was deleted. Unconstrained recombination generated multimers of the substrate and/or resolution product, together with a mixture of high molecular weight products arising from both inter and intramolecular recombination reactions. Reporter plasmids are diagrammed on the top of the figure, with the IR1 and IR2 core motifs represented by filled and open triangles, respectively; and the DR1 and DR2 accessory motifs by shaded triangles. Arrows show the orientation of the core sites as determined by the central spacer sequence.