Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026937-08
Application #
3274393
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1979-09-28
Project End
1987-08-31
Budget Start
1986-09-01
Budget End
1987-08-31
Support Year
8
Fiscal Year
1986
Total Cost
Indirect Cost

  Institution

Name
University of Arizona
Department
Type
Earth Sciences/Resources
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85722

  Publications

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
Bonham-Smith, P C; Bourque, D P (1989) Translation of chloroplast-encoded mRNA: potential initiation and termination signals. Nucleic Acids Res 17:2057-80
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