Screening of cDNA clones for plastid-targeted proteins

We present a method to isolate cDNA clones that encode plastid-targeted proteins other than those encoding proteins of photosynthetic machinery. As an example, we have isolated clones for dark-inducible chloroplast proteins. Poly(A)(+) RNA was isolated from 15-day-old radish cotyledons that had been kept in darkness for 24 h, and was used to prepare a library with the plasmid vector pBluescript. It was divided into groups of 8 or 12 clones, which were separately expressed in vitro in the presence of [S-35]methionine. The labeled polypeptides were then incubated with isolated chloroplasts to see if they are imported by the chloroplasts. The group that showed positive signals after SDS-PAGE of the lysed chloroplasts were further analyzed for individual clones in the same manner. Thus, we were successful in isolating cDNA clones encoding chloroplast-targeted proteins that were expressed in radish cotyledons specifically in the dark. The method can be used to identify nuclear genes for nonphotosynthetic plastids, if an appropriate cDNA Library is available.