Targeted deletion of rpo genes from
the plastid genome. A, Deletion of the rpoA gene.
Homologous recombination events (hatched lines) between ptDNA sequences
in vector pGS95 and the tobacco plastid genome yields a genome lacking
rpoA. Probes for Southern blots in D are marked with
thick horizontal lines. Map position of the probed restriction
fragments with size in kilobases is shown below the maps.
aadA, Chimeric spectinomycin resistance gene (Svab and
Maliga, 1993); rpoA, rpoB,
rpoC1, and rpoC2, the plastid genes
encoding the α-, β-, β′-, and β"-subunits of PEP, respectively;
atpI, petD, rps2, and
rps11, plastid genes (Shinozaki et al., 1986).
Restriction endonuclease cleavage sites: H, HincII; X,
XbaI; Bg, BglII; Sc, ScaI;
P, PstI; B, BamHI; Pp,
Psp1406I; A, AccI; SI,
SacI; SII, SacII; StI,
StuI; E, EcoRV; Bs, BsrGI.
Brackets indicate restriction sites eliminated during cloning. B,
Deletion of the rpoC1 gene. Homologous recombination
events (crossed lines) between ptDNA sequences in vector pGS97 and the tobacco plastid
genome yields a genome lacking rpoC1. C, Deletion of the
rpoC2 gene. Homologous recombination events (crossed
lines) between ptDNA sequences in vector pGS99 and the tobacco plastid
genome yields a genome lacking rpoC2. D, Southern
probing demonstrates a uniform population of transformed plastid
genomes. Total cellular DNA was isolated from the leaves of plants
transformed with plasmids pGS95 (targeting rpoA), pGS97
(targeting rpoC1), and pGS99 (targeting
rpoC2), and from wild-type green leaves (WT). Data are
shown for two independently transformed lines (pGS95-2, pGS95-3), or
two plants derived from the same transformation event (pGS97-2.2,
pGS97-2.3 and pGS99-4.1, pGS99-4.4).