Figure 2.

The pL335V plasmid used for transformation of the tobacco chloroplast genome. This plasmid was based on pLEV1 with the nucleotide substitutions (in bold) required to change codon 335 from Leu to Val and silent substitutions to introduce an adjacent EcoRV site for screening of transformants. The numbers refer to the sequence of the tobacco chloroplast genome (Shinozaki et al., 1986). The dashed bars joined by the dotted lines indicate the homologous flanking regions present in pL335V and available for the crossovers required to introduce the mutated region and the aadA gene into the large single-copy region of the chloroplast genome. The probe used in screening by Southern blotting and the primers for amplifying and sequencing the mutant rbcL are indicated. K, KpnI; N, NruI; E, EcoRV; T, rps16 terminator sequence.