Molecular characterization of tagged line ET2-17. (A) Schematic representation of probe (thick lines) and primer (short arrows) positions for the different tagged sequences in line 17 transformed with promoter tagging vector pETKUL2. The position of the codon-optimized luciferase (luc+) and neo gene cassettes are shown with respect to the right (closed triangle) and left (open triangle) T-DNA border. Long arrows mark the direction of transcription. Dotted lines represent plant genomic DNA flanking the right (RB) and left (LB) T-DNA border, denominated 5' and 3' region, respectively. The drawing is not precisely according to scale. (B) Southern hybridization analysis of the luc+ gene and the cloned 5' regions. Ten micrograms of total DNA were digested with HindIII, separated fragments were hybridized with a DIG-labeled luc+ probe (862 bp) and rehybridized with a 5'-tagged sequence-specific probe (seq. 17-1: 422 bp, seq. 17-2: 425 bp, seq. 17-3: 435 bp and seq. 17-4: 165 bp). C: untransformed control plant. 17: tagged line ET2-17. MW: DIG-labeled DNA molecular marker III (Roche). The tagged luc+ inserts are marked by arrows. (C) PCR confirmation in line ET2-17 of continuous tagged sequences with primers specific for 5'-tagged sequences (17-LinkRB-1F, 17-LinkRB-2F, 17-LinkRB-3F and 17-RT-4 for seq. 1–4, respectively) in combination with reverse primers specific for 3'-tagged sequences (17-LinkLB-1R, 17-LinkLB-2R, 17-LinkLB-3R and 17-LinkLB-4R). MW: SmartLadder (Eurogentec, Seraing, Belgium).