This project will test the hypothesis that regulation of expression of chloroplast ribosomal protein genes which are coded by Nicotiana tabacum chloroplast DNA occurs at the level of transcription and translation. We analyze rps12-rps7 transcript processing and fully characterize the trans-splicing event which takes place in the maturation of rps12 mRNA. We will also establish gene-gene product relationships for chloroplast DNA encoded ribosomal proteins by identifying the polypeptides which are synthesized in vitro from cloned genes and their functional mRNAs. We will assess the importance of transcriptional regulation of these genes by measuring mRNA levels in vivo and in vitro experiments, examine the stability of the mRNA for several chloroplast ribosomal proteins, assess the contribution of promoter sequences to transcriptional regulation, and probe for evidence of protein factors which might regulate transcription of these genes. Translational control mechanisms will be assessed by examining differences in mRNA levels and their rates of translation for intron-containing and intron lacking ribosomal protein genes. Whether translation of chloroplast ribosomal genes is regulated by a feedback inhibition by the ribosomal proteins themselves will be tested. Ribosome binding site efficiencies of several of these genes will be compared and the significance of the binding site sequences examined by site specific mutagenesis experiments followed by measurements of translational efficiencies in vitro. The function of the rps12 gene product will be examined and we will attempt to utilize this gene to develop a selectable marker for chloroplast transformation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Cytology Study Section (CTY)
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University of Arizona
Schools of Earth Sciences/Natur
United States
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Elhag, G A; Thomas, F J; McCreery, T P et al. (1992) Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor. Nucleic Acids Res 20:689-97
Elhag, G A; Bourque, D P (1992) Nuclear-encoded tobacco chloroplast ribosomal protein L24. Protein identification, sequence analysis of cDNAs encoding its cytoplasmic precursor, and mRNA and genomic DNA analysis. J Biol Chem 267:21705-11
Elhag, G A; Bourque, D P (1992) Nuclear-encoded chloroplast ribosomal protein L27 of Nicotiana tabacum: cDNA sequence and analysis of mRNA and genes. Biochemistry 31:6856-64
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Hildebrand, M; Hallick, R B; Passavant, C W et al. (1988) Trans-splicing in chloroplasts: the rps 12 loci of Nicotiana tabacum. Proc Natl Acad Sci U S A 85:372-6
Hewlett, N G; Bourque, D P (1986) Improved preparative methods for isolation and purification of tobacco chloroplast ribosomes, ribosomal proteins, and rRNA. Methods Enzymol 118:201-12
Bard, J; Bourque, D P; Hildebrand, M et al. (1985) In vitro expression of chloroplast genes in lysates of higher plant chloroplasts. Proc Natl Acad Sci U S A 82:3983-7
Hildebrand, M; Jurgenson, J E; Ramage, R T et al. (1985) Derivation of a physical map of chloroplast DNA from Nicotiana tabacum by two-dimensional gel and computer-aided restriction analysis. Plasmid 14:64-79