Hybridization of bean, spinach, maize and Euglena chloroplast transfer RNAs with homologous and heterologous chloroplast DNAs. An approach to the study of homology between chloroplast tRNAs from various species.

Chloroplast tRNAs from two dicotyledons (spinach and bean), a monocotyledon (maize) and a green alga (Euglena) have been fractionated by two-dimensional gel electrophoresis. The individual tRNAs have been identified, albeled with 125I or 32P, and used in tRNA-DNA hybridization experiments. Spinach chloroplast tRNAs hybridize as well, and maize chloroplast tRNAs almost as well as bean chloroplast tRNAs to bean chloroplast DNA, thus suggesting a high degree of homology between the chloroplast tRNAs from the two dicotyledons and between the tRNAs from the two dicotyledons and those of the monocotyledon. But Euglena total chloroplast tRNA hybridizes very poorly to bean chloroplast DNA, and among the 14 individual tRNAs tested, only one, Euglena chloroplast tRNAPhe, hybridizes to both maize and bean chloroplast DNAs, which is in good agreement with the fact that Euglena and bean chloroplast tRNAsPhe have almost identical primary structures.

Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes.

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.

Fractionation and identification of spinach chloroplast transfer RNAs and mapping of their genes on the restriction map of chloroplast DNA.

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.

Transfer RNAs and tRNA genes of Vicia faba chloroplasts.

Isolated chloroplasts from broad bean and common bean were found to contain a minimum of 31 and 32 tRNA species, respectively. These individual chloroplast tRNAs were (32)P-labeled in vitro and hybridized to DNA fragments obtained upon digestion of broad bean and common bean chloroplast DNAs with various restriction endonucleases. At least 30 tRNA genes were localized on the physical maps of the two chloroplast genomes. Comparison of the broad bean tRNA gene map to that of common bean revealed DNA sequence rearrangements, such as inversions, insertions/ deletions and duplications, within these two members of the Legu minosae family.

Chloroplast transfer RNAs and tRNA genes of wheat.

Fractionation (by two-dimensional polyacrylamide gel electrophoresis) of total tRNA from wheat chloroplasts yields about 33 RNA spots. Of these, 30 have been identified by aminoacylation as containing tRNAs specific for 17 amino acids.Hybridization of labeled individual tRNAs to cloned chloroplast DNA fragments has revealed the location of at least nine pairs of tRNA genes in the segments of the inverted repeat, at least twelve tRNA genes in the large single copy region and one tRNA gene in the small single copy region.A comparison of this wheat chloroplast tRNA gene map to that of maize and of other higher plants suggests that gene rearrangements have occurred during evolution, even within cereal chloroplast DNA. These rearrangements have taken place within the inverted repeat, within the large single copy region and between the inverted repeat and the large single copy region.

Divergence of chloroplast gene organization in three legumes: Pisum sativum, Vicia faba and Phaseolus vulgaris.

Isolated chloroplasts from Pisum sativum were found to contain at least 32 tRNA species. Hybridization of in vitro labeled, identified, chloroplast tRNAs to Pisum chloroplast DNA fragments revealed the locations of the tRNA genes on the circular chloroplast genome. Comparison of this gene map to the maps of Vicia faba and Phaseolus vulgaris showed that the chloroplast genomes of Pisum and Phaseolus are otherwise more closely related than either genome is to the chloroplast genome of Vicia. Furthermore, the results suggest how possible recombination events could be involved in the evolution of these three closely related, but divergent, chloroplast genomes.

Transfer RNA genes ofZea mays chloroplast DNA.

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA2 (Ile) and tRNA(Ala) map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs(Leu) and the three for tRNAs(Ser), as well as the two isoaccepting species for tRNA(Asn), tRNA(Gly), tRNAs(Ile), tRNAs(Met), tRNAs(Thr), are shown to be encoded at different loci.Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were(32)P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.