Isolation of nuclear encoded plastid ribosomal protein cDNAs

J. Stephen Gantt, Joe L. Key

Research output: Contribution to journalArticlepeer-review

39 Scopus citations

Abstract

A pea leaf cDNA library was constructed in the expression vector λgt11 and screened with antisera raised against proteins extracted from 30S and 50S ribosomal subunits and 70S ribosomes prepared from isolated pea chloroplasts. Six recombinant phage were identified that encoded fusion proteins containing plastid ribosomal protein antigenic determinants. Phage-induced cell lysate proteins, containing the fusion proteins, were bound to nitrocellulose membranes and used as affinity matrices to prepare monospecific antibodies. These antibodies were then used to identify by Western blotting which plastid ribosomal protein shared antigenic determinants with the fusion proteins. cDNA inserts from the antigen-producing phage were used to hybrid-select complementary mRNAs. The cell-free translation products of these mRNAs were added to a pea chloroplast in vitro transport system and imported proteins analyzed by two-dimensional gel electrophoresis. The imported proteins comigrated with the plastid ribosomal proteins that were identified as being antigenically related to the fusion proteins produced by the corresponding recombinant phage. The imported proteins were 3,500-5,500 daltons smaller than their precursors.

Original languageEnglish (US)
Pages (from-to)186-193
Number of pages8
JournalMGG Molecular & General Genetics
Volume202
Issue number2
DOIs
StatePublished - Feb 1986

Keywords

  • Chloroplast
  • Protein transport
  • Ribosomal proteins

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