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Spinach chloroplast 4S RNAs has been separated by two-dimensional polyacrylamide gel electrophoresis into about 35 species. After extraction from the gel, 27 of these RNA species were identified by aminoacylation as tRNAs specific for 16 amino acids. Individual tRNAs were labeled in vitro with 125I and hybridized to DNA fragments obtained by digestion of spinach chloroplast DNA with KpnI, PstI, SalI and XmaI restriction endonucleases. A minimum of 21 genes corresponding to tRNAs for 14 different amino acids have been localized on the restriction endonuclease cleavage site map of the DNA molecule. Of these, 15 genes corresponding to tRNAs for 12 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. Each copy of this repeat region contains a set of genes for the ribosomal RNAs and a gene for tRNA2Ile in the "spacer" sequence between the 16S and 23S ribosomal RNAs. The genes for tRNA1Ile, tRNA2Leu and tRNA3Leu also map in the repeat region, but outside the ribosomal DNA unit. At present, two more chloroplast tRNAs (for Pro and Lys) have been identified, but not mapped, while 4 unidentified 4S RNAs have been mapped in the large single-copy region of the DNA molecule. Evidence is presented that isoaccepting tRNA species can be transcripts from different loci.

The cytoplasmic prolyl-tRNA synthetase can be separated by hydroxyapatite chromatography, from the enzyme present in the chloroplasts and in the mitochondria (organellar enzyme). The cytoplasmic lysyl-tRNA synthetase can also be separated from the organellar enzyme. There are two tRNAsPro in the cytoplasm; they can be charged by the cytoplasmic enzyme, but not by the organellar enzyme or the Escherichia coli enzyme. Chloroplasts contain, in addition to the two cytoplasmic tRNAsPro, one chloroplast-specific tRNAPro, which is not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E. coli enzyme. Mitochondria contain, in addition to the two cytoplasmic tRNAsPro, two mitochondria-specific tRNAsPro, which are not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E. coli enzyme. There are two tRNAsLys in the cytoplasm. Both can be charged by the cytoplasmic enzyme, but one can be charged by the organellar or E. coli enzyme. Chloroplasts contain in addition to one cytoplasmic tRNALys, one chloroplast-specific tRNALys which can only be charged by the organellar or E. coli enzyme. Mitochondria contain, in addition to one cytoplasmic tRNALys, one mitochondria-specific tRNALys which can only be charged by the organellar or E. coli enzyme.

Leucyl-tRNA synthetase from Phaseolus vulgaris chloroplasts could be separated from its cytoplasmic counterpart upon chromatography on hydroxyapatite, but the cytoplasmic and mitochondrial leucyl-tRNA synthetases could not be distinguished. The tRNALeu species from the various plant cell compartments and from Escherichia coli were aminoacylated using either homologous or heterologous enzymes; the levels of aminoacylation and the profiles of the leucyl-tRNAs upon reverse-phase chromatography were studied. Cytoplasmic tRNALeu species could be aminoacylated by the cytoplasmic or by the mitochondrial enzymes and in both cases yielded two peaks upon reverse-phase chromatography (RPC-5). But they could not be charged by the chloroplast-specific or by the E. coli enzynes. Mitochondrial tRNALeu species could be charged by the mitochondrial or by the cytoplasmic enzymes and in both cases yielded four peaks upon reverse phase (RPC-5) chromatography. But they could not be aminoacylated using the chloroplast-specific or the E. coli leucyl-tRNA synthetases. Chloroplastic tRNALeu species can be divided into two classes: the first class contains four isoacceptor species which can be charged by the cytoplasmic or mitochondrial enzymes, but not by the chloroplast-specific or the E. coli enzymes; the second class contains three chloroplast-specific tRNALeu species which can be charged by the chloroplast-specific or the E. coli enzymes but not by the cytoplasmic or the mitochondrial enzymes. There are five isoacceptor tRNALeu species in E. coli; all are charged by the E. coli or the chloroplast-specific enzymes, while only one is aminoacylated by the plant cytoplasmic or mitochondrial enzymes.

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules. Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.