Figure 4

Rescue of PAP85 regulatory and coding regions into a plant transformation vector. (A) Schematic diagram showing the location of primers and restriction enzyme sites used for analysis of the integrity of candidate clones of pWY189 encoding PAP85 in pWY109. Boxed region represents PAP85 regulatory and genomic coding regions, and the line represents vector sequence. Arrows indicate location of diagnostic PCR primers and BamHI sites used for evaluating the correctness of the assembled construct. (B) PCR-based screening of clones potentially encoding PAP85 in pWY109. The 5'-Test (primers pCB-RB and PAP85-Prom-Rescue-3'-Test) and 3'-Test (primers tCUP2-OUT and PAP85-Term-Rescue-5'-Test) evaluates for the fusion of the vector with the 5' or 3' regulatory regions, respectively. An amplicon of 0.6 kb is expected for correct vector-gene fusion at both the 5' and 3' end. (C) Restriction enzyme analysis of candidate PAP85 clones in pWY109. Clone 1 from panel A was digested with BamHI and exhibited the predicted restriction fragments of 2.7 and 8.3 kb. M, DNA size marker (1 kb Plus ladder, Invitrogen) with representation from 0.1–1 kb (B) or 1–12 kb (C).