Fig. 2.

Variegated plants contain rearranged amplified ptDNA regions. (A) DNA gel blot (10 μg per lane) of total leaf DNA digested with HindIII and hybridized with the probes indicated below the gels. The probe numbers refer to the nucleotides of the published Arabidopsis chloroplast genome (42). Expected fragments from restriction analysis of Col-0 ptDNA and the size of new fragments observed in variegated lines are presented below the probes. Restriction and gene maps of the reorganized regions in variegated lines are presented in Figs. S4 and S9. A lower exposition of the second panel allowing better visualization of the amplified bands in VarA is presented in Fig. S5. (B) Schematic of the possible arrangements of the reorganized ptDNA in variegated lines. A head–tail dimer and a monomeric circular molecule are represented for Var A (red) and Var B (green). Oligonucleotides used for the PCR amplification of the junctions of reorganized ptDNA are represented by small black arrows. (C) (Lower) PCR amplification of fragments containing the junctions of reorganized ptDNA in Var A and B plants. (Upper) DNA from leaves 4 (variegated) and 8 (nonvariegated) was isolated. The plastidial ycf2 gene was used as a loading control. (D) DNA gel blot analysis showing the arrangement of amplified ptDNA in the Var A line. DNA from the indicated genotypes was digested with the indicated restriction enzymes and separated on an agarose gel. The DNA was hybridized with the probe depicted in E. A 9.2-kb band corresponding to the WT DNA fragment appears in all samples digested with XhoI and PstI restriction enzymes. A band of 9.4 kb expected from digestion of the WT plastid genome with KpnI was found in all genotypes. The asterisks indicate putative circular molecules. (E) Restriction map of the reorganized regions in the Var A line. The red arrow represents the amplified region in Var A. The probe used is represented as a blue line. A portion of ptDNA is represented as a black horizontal line. The restriction sites are indicated by vertical black lines. K = KpnI; P = PstI; X = XhoI. A circular monomer is represented on the right with the expected linear digestion product of this molecule.