Targeted replacement of rbcL-T in
the tobacco plastid genome with the rbcL-S gene. A,
Plastid-targeting region in plasmid pIK83 (plastid DNA is underlined)
and the cognate region of the plastid genome (Nt-ptDNA) and of the
Nt-pIK83-1 line. atpB (Shinozaki et al., 1986) and
accD (Sasaki et al., 1993) are plastid genes.
aadA is the plastid-selectable spectinomycin-resistance
gene. Recombination endpoints (1–4) discussed in the text are marked
by vertical arrows. Horizontal arrows represent mRNAs detected by the
rbcL (P1) and aadA (P2) probes.
Restriction enzyme recognition sites: P, PvuII; R,
EcoRV; Xh, XhoI. B, Wild-type plastid
genome copies are absent in four plants regenerated independently
(lanes 1–4) from the Nt-pIK83-1 line. Data for wild-type tobacco (T)
are also shown. DNA blots of EcoRV-digested total
cellular DNA (1 μg per lane) were hybridized with the
rbcL (P1) and aadA (P2) probes. The
rbcL probe hybridized to a 7.1-kb DNA fragment of the
wild-type Nt-ptDNA and a 4.2-kb fragment of the transplastome (Fig.
1A). The 4.2-kb transgenic fragment also hybridized with the
aadA probe. Note the lack of wild-type 7.1-kb DNA
fragments in the plastid transformants.