Figure 4

Inversion of the Tg-110 insert. (A) Schematic diagram of inversion strategy. Two incomplete fragments of the tetracycline resistance gene (tet), te (5′ side) and et (3′ side), which contained an overlapping region e, were used for inversion. The inversion cassettes, phl-et, consisting of et and the phleomycin resistance gene (phl), and te-erm, consisting of te and the erythromycin resistance gene (erm), were sequentially inserted into the ends of the Tg-110 insert. A 0.9 kb fragment in the left end and a 0.8 kb fragment in the right end were used as homology arms. Homologous recombination between te and et in Tg-110 T/P results in the inversion of the insert to form an intact tet gene. The about positions of BamHI sites are represented by “B”. (B) Inversion was confirmed by Southern blotting using the tet gene as a probe. The genomic DNA of the represented clones was digested with BamHI. Changes in the size of tet cassette fragments represented inversion by intrachromosomal homologous recombination. The open (2.8 kb te-erm cassette and 2.3 kb phl-et plus 2.7 kb Tg-110 fragment) and closed (3.4 kb erm-e-phl cassette and 1.6 kb tet plus 2.7 kb Tg-110 fragment) arrowheads indicate signals before and after inversion, respectively.