Open AccessCloned DNA sequences complementary to mRNAs encoding precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and a chlorophyll a/b binding polypeptide
Author(s) -
Richard Broglie,
Guy Bellemare,
Sue G. Bartlett,
NamHai Chua,
Anthony R. Cashmore
Publication year - 1981
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.78.12.7304
Subject(s) - biology , complementary dna , microbiology and biotechnology , chloroplast , thylakoid , biochemistry , oligonucleotide , plasmid , protein subunit , chloroplast dna , dna , gene
Double-stranded cDNA was synthesized from pea poly(A)-containing mRNA and inserted into thePst I 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 mRNAs and further characterized by cell-free translation of hybridization-selected mRNAs. To confirm the identity of cDNA clones encoding chloroplast polypeptides, we incubated translation products derived from complementary mRNAs with intact chloroplastsin 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, we have isolated and characterized cDNA clones encoding the two major cytoplasmically synthesized chloroplast proteins: 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 third cDNA clone was isolated and shown to encode a 22,000-dalton thylakoid membrane polypeptide.