Cloning, mapping, and in vitro transcription-translation of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase from spinach chloroplasts

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RESUMO

An 11.2-kilobase pair (kbp) BamHI restriction nuclease fragment from spinach chloroplast DNA has been found to contain the gene for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [RuP2 carboxylase; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39]. The gene was located by hybridization of cloned chloroplast DNA fragments containing the maize LS gene (Bedbrook, J. R., Coen, D. M., Beaton, A. R., Bogorad, L. & Rich, A. (1979) J. Biol. Chem. 254, 905-910) to spinach chloroplast DNA cleaved with restriction nucleases. The 11.2-kbp BamHI fragment has been inserted into the BamHI site of the plasmid pBR322. The resulting recombinant plasmid, pSoe3101, was used to direct the synthesis of a protein, which was immunoprecipitable with antibody to RuP2 carboxylase, in a partially defined in vitro transcription-translation system derived from Escherichia coli. The product synthesized in vitro has a molecular weight identical to that of authentic spinach LS. By using pSoe3101 DNA cleaved at various positions with restriction nucleases, and the in vitro transcription-translation system, the LS gene has been mapped to a 1.5-kbp region located at one end of the 11.2-kbp BamHI fragment. The direction of transcription of the LS gene on the plasmid as well as on the chloroplast chromosome has also been determined. The position of the LS gene on circular spinach chloroplast DNA is approximately 27 kbp from the start of one of the inverted repeat regions and 180° from one of the rRNA-coding regions.

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