Figure 1.

Structures of the wild-type genomic PPO locus, the T-DNA repair gene-targeting (GT) construct and the targeted PPO locus. (a) The sequence of the zinc finger nucleases (ZFN) target site is shown, the target half sites for ALP and ARP are boxed and the spacer is in small letters. The triplet coding for tyrosine in the endogenous PPO gene (and changed in the repair construct) is shown in a black box. (b) The coding region of the PPO gene is shown as a grey bar; the ZFN target site in the PPO gene as a triangle. The GT repair construct, missing the C-terminal region of the PPO gene including the first 364 bps of the coding sequence, contains base pair substitutions leading to two amino acids changes (S305L and Y426M; indicated by asterisks) causing insensitivity for the herbicide butafenacil and a KpnI site for detection of GT events. The BAR gene on the GT repair construct is used to determine the transformation frequency. Primers used for PCR detection of GT events are shown. Sizes of DNA fragments expected after digestion with selected restriction enzymes are indicated. Probes used for Southern blotting are shown as black bars.