Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Dec;78(12):7304–7308. doi: 10.1073/pnas.78.12.7304

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

Richard Broglie 1, Guy Bellemare 1,*, Sue G Bartlett 1,†, Nam-Hai Chua 1, Anthony R Cashmore 1
PMCID: PMC349254  PMID: 16593132

Abstract

Double-stranded cDNA was synthesized from pea poly(A)-containing mRNA and inserted into the Pst 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 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, 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.

Keywords: chloroplast protein, leaf cytoplasmic mRNA, cDNA synthesis, hybridization selection, chloroplast uptake and processing

Full text

PDF
7304

Images in this article

7306Image
on p.7306
7306Image
on p.7306
7307Image
on p.7307
7307Image
on p.7307
7307Image
on p.7307
7307Image
on p.7307
7307Image
on p.7307

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Apel K., Kloppstech K. The plastid membranes of barley (Hordeum vulgare). Light-induced appearance of mRNA coding for the apoprotein of the light-harvesting chlorophyll a/b protein. Eur J Biochem. 1978 Apr 17;85(2):581–588. doi: 10.1111/j.1432-1033.1978.tb12273.x. [DOI] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blair G. E., Ellis R. J. Protein synthesis in chloroplasts. I. Light-driven synthesis of the large subunit of fraction I protein by isolated pea chloroplasts. Biochim Biophys Acta. 1973 Aug 24;319(2):223–234. doi: 10.1016/0005-2787(73)90013-0. [DOI] [PubMed] [Google Scholar]
  4. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. Cashmore A. R., Broadhurst M. K., Gray R. E. Cell-free synthesis of leaf protein: Identification of an apparent precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase. Proc Natl Acad Sci U S A. 1978 Feb;75(2):655–659. doi: 10.1073/pnas.75.2.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cashmore A. R. Reiteration frequency of the gene coding for the small subunit of ribulose--1,5--bisphosphate carboxylase. Cell. 1979 Jun;17(2):383–388. doi: 10.1016/0092-8674(79)90164-8. [DOI] [PubMed] [Google Scholar]
  7. Chua N. H., Bennoun P. Thylakoid membrane polypeptides of Chlamydomonas reinhardtii: wild-type and mutant strains deficient in photosystem II reaction center. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2175–2179. doi: 10.1073/pnas.72.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chua N. H., Schmidt G. W. Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6110–6114. doi: 10.1073/pnas.75.12.6110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Coen D. M., Bedbrook J. R., Bogorad L., Rich A. Maize chloroplast DNA fragment encoding the large subunit of ribulosebisphosphate carboxylase. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5487–5491. doi: 10.1073/pnas.74.12.5487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  11. Derman E., Krauter K., Walling L., Weinberger C., Ray M., Darnell J. E., Jr Transcriptional control in the production of liver-specific mRNAs. Cell. 1981 Mar;23(3):731–739. doi: 10.1016/0092-8674(81)90436-0. [DOI] [PubMed] [Google Scholar]
  12. Genge S., Pilger D., Hiller R. G. The relationship between chlorophyll b and pigment-protein complex II. Biochim Biophys Acta. 1974 Apr 23;347(1):22–30. doi: 10.1016/0005-2728(74)90196-0. [DOI] [PubMed] [Google Scholar]
  13. Goodman H. M., MacDonald R. J. Cloning of hormone genes from a mixture of cDNA molecules. Methods Enzymol. 1979;68:75–90. doi: 10.1016/0076-6879(79)68007-2. [DOI] [PubMed] [Google Scholar]
  14. Huber S. C., Hall T. C., Edwards G. E. Differential Localization of Fraction I Protein between Chloroplast Types. Plant Physiol. 1976 May;57(5):730–733. doi: 10.1104/pp.57.5.730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kawashima N., Wildman S. G. Studies on fraction I protein. IV. Mode of inheritance of primary structure in relation to whether chloroplast or nuclear DNA contains the code for a chloroplast protein. Biochim Biophys Acta. 1972 Feb 23;262(1):42–49. doi: 10.1016/0005-2787(72)90217-1. [DOI] [PubMed] [Google Scholar]
  16. Kirchanski S. J., Park R. B. Comparative Studies of the Thylakoid Proteins of Mesophyll and Bundle Sheath Plastids of Zea mays. Plant Physiol. 1976 Sep;58(3):345–349. doi: 10.1104/pp.58.3.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kung S. D., Thornber J. P., Wildman S. G. Nuclear DNA codes for the photosystem II chlorophyll-protein of chloroplast membranes. FEBS Lett. 1972 Aug 1;24(2):185–188. doi: 10.1016/0014-5793(72)80763-4. [DOI] [PubMed] [Google Scholar]
  18. Smith D. F., Searle P. F., Williams J. G. Characterisation of bacterial clones containing DNA sequences derived from Xenopus laevis vitellogenin mRNA. Nucleic Acids Res. 1979 Feb;6(2):487–506. doi: 10.1093/nar/6.2.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ullrich A., Shine J., Chirgwin J., Pictet R., Tischer E., Rutter W. J., Goodman H. M. Rat insulin genes: construction of plasmids containing the coding sequences. Science. 1977 Jun 17;196(4296):1313–1319. doi: 10.1126/science.325648. [DOI] [PubMed] [Google Scholar]
  20. Woolford J. L., Jr, Rosbash M. The use of R-looping for structural gene identification and mRNA purification. Nucleic Acids Res. 1979 Jun 11;6(7):2483–2497. doi: 10.1093/nar/6.7.2483. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES