MOLECULAR BIOLOGY OF CHLOROPLAST RIBOSOMES

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We propose to study 1) the site of translation of the genes which code for the proteins of chloroplast ribosomes, 2) the intracellular location of these genes, and 3) the physical location of these genes on chloroplast DNA. The site of synthesis of chloroplast ribosomal proteins will be examined by an in vivo mistranslation assay and by translation inhibitor studies. Isolated chloroplast and cytoplasmic fractions will be tested for endogenous synthesis of chloroplast ribosomal proteins in vitro. Messenger RNA fractions from cell cytoplasm and chloroplasts will be examined for ability to program in vitro synthesis of chloroplast ribosomal proteins by translation with 1) E. Coli ribosomes, 2) a reconstituted Nicotiana chloroplast system, 3) wheat germ ribosomes and 4) a reticulocyte lysate system. Isolated fractions with mRNA activity for specific chloroplast ribosomal proteins will allow physical mapping of genes for chloroplast ribosomal proteins. Chloroplast DNA genes will then be physically located by a combination of DNA-RNA hybridization, restriction enzyme mapping procedures, ribosome binding to chloroplast DNA, and coupled transcription-translation studies using individual restriction fragments of chloroplast DNA.