Cloned DNA sequences complementary to mRNAs encoding precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and a chlorophyll a/b binding polypeptide

Abstract

Double-stranded c[circular]DNA was synthesized from pea poly(A)-containing mRNA and inserted into the PstI site of the bacterial plasmid pBR322 by the addition of synthetic oligonucleotide linkers. Bacterial colonies containing recombinant plasmids were detected by hybridization to partially purified mRNA and further characterized by cell-free translation of hybridization-selected mRNA. To confirm the identity of cDNA clones encoding chloroplast polypeptides, translation products derived from complementary mRNA were incubated with intact chloroplasts in vitro. After uptake, precursor polypeptides were converted to their mature size and identified by fractionation of the chloroplast stroma and thylakoid membranes. By using these procedures, cDNA clones encoding the 2 major cytoplasmically synthesized chloroplast proteins were isolated and characterized, the small subunit of ribulose-1,5-bisphosphate carboxylase and a constituent polypeptide (polypeptide 15) of the light-harvesting chlorophyll a/b-protein complex. Similarly, a 3rd cDNA clone was isolated and shown to encode a 22,000-dalton thylakoid membrane polypeptide.