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 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.

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.

Comparison of the cyanelle DNA from two different strains of Cyanophora paradoxa.

The cyanelle DNA from two different strains of Cyanophora paradoxa (strain LB555UTEX and strain 1555) was investigated.The cyanelle DNA from both strains showed a buoyant density in neutral CsCI gradients of 1.692 g/cm(3). The total molecular weight, as judged by restriction endonuclease analysis, of the two cyanelle DNAs differed. In strain LB555UTEX the size of the cyanelle DNA was equivalent to 127 ± 1 kb whereas in strain 1555 a size of 138 ± 1 kb was consistently found. The sizes of individual DNA fragments and the number of recognition sites for a particular restriction endonuclease appeared largely unrelated.A high amount of cross hybridization, as judged by reciprocal heterologous DNA hybridizations, however indicated a high degree of sequence homology between the two cyanelle DNAs. Under comparable conditions, cyanelle DNA hybridized nearly exclusively with the dG+dC-rich rRNA transcription units from plastid DNAs. Up to now conserved restriction endonuclease recognition sites between the two cyanelle DNAs were only observed within the cyanelle rRNA genes which are present twice on both cyanelle DNAs.

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.

The subcellular localization of DNA components from Cyanophora paradoxa, a flagellate containing endosymbiotic cyanelles.

Cyanophora paradoxa, a unicellular flagellate, contains cyanelles which are supposed to be cyanobacterial origin. DNA was isolated from subcellular fractions and separated according to density components in CsC1 density gradients. The main DNA component, comprising more than 90% of the total DNA, has a buoyant density of 1.724 g X cm-3. Several subsfractions in the range from 1.718 g X cm-3 to 1.735 g X cm-3 are contained in this component. This DNA of high complexity was considered to be host nuclear DNA. The DNA from the endosymbiotic cyanelles, which were isolated, treated with DNase, and purified by sucrose density gradient centrifugation exhibited a buoyant density of 1.692 g X cm-3 in one strain and 1.695 g X cm-3 in a second strain. Both cyanelle DNAs (cyDNA) have a complexity of approximately 126 X 10(3) base pairs and comprise about 5% of the total cellular DNA content. Two additional DNA components of low complexity were isolated from crude cyanelle pellets obtained without DNase treatment. The larger of these, approximately 48 X 10(3) base pairs in size, had a density of approximately 1.688 g X cm-3. The second component, about 15 X 10(3) base pairs in size, banded in the density range between 1.710 g X cm-3 and 1.720 g X cm-3. The latter is associated with nuclear DNA. The 48 X 10(3)-base-pair component was located in the cytosol and could be obtained after CsC1/ethidium bromide density gradient centrifugation at the position of covalently closed circular DNA. Both these components amounted to approximately 0.5-1% of total DNA. A further DNA component with a complexity of more than 150 X 10(3) base pairs, enriched in fractions where mitochondria are expected, was not characterized further. The density was intermediate between cyDNA and nuclear DNA (1.710-1.720 g X cm-3) and it amounted to 1-2% of the total DNA. Our results indicate that the DNA from cyanelles, believed to be endosymbiotic cyanobacteria, is not more complex than higher plant chloroplast DNAs.

Fractionation and identification of Euglena gracilis cytoplasmic and chloroplastic tRNAs and mapping of tRNA genes on chloroplast DNA.

The cytoplasmic and chloroplast tRNAs of Euglena gracilis Z strain were fractionated by two-dimensional gel electrophoresis and identified by aminoacylation. Purified chloroplast tRNAs, labeled in vitro with |(32)P|, were hybridized to endonuclease restriction fragments of chloroplast DNA, allowing the corresponding tRNA genes to be localized on the physical map of Euglena chloroplast DNA.

Physical mapping of differences in chloroplast DNA of the five wild-type plastomes in Oenothera subsection Euoenothera.

1) DNA has been isolated from the five genetically distinguishable plastid types of Oenothera, subsection Euoenothera. DNA of plastomes I to IV was obtained from plants with identical nuclear backgrounds containing the genotype AA of Oenothera hookeri whereas the DNA of plastome V came from Oenothera argillicola (genotype CC). 2) The DNAs of the five basic Euoenothera wild-type plastomes can be distinguished by restriction endonuclease analysis with Sal I, Pst I, Kpn I, Eco RI and Bam HI. The fragment patterns exhibit distinct common features as well as some degree of variability. 3) Physical maps for the circular DNAs of plastome I, II, III and V could be constructed using the previously detailed map of plastome IV DNA (Gordon et al. 1981). This has been achieved by comparing the cleavage products generated by restriction endonucleases Sal I, Pst I and Kpn I which collectively result in 36 sites in each of the five plastome DNAs, and by hybridization of radioactively labelled chloroplast rRNA or chloroplast cRNA probes of spinach to Southern blots of appropriate restriction digests. The data show that the overall fragment order is the same for all five plastome DNAs. Each DNA molecule is segmentally organized into four regions represented by a large duplicated sequence in inverted orientation whose copies are separated by two single-copy segments. 4) The alterations in position of restriction sites among the Euoenothera plastome DNAs result primarily from insertions/deletions. Eleven size differences of individual fragments in the Sal I, Pst I and Kpn I patterns measuring 0.1-0.8 Md (150-1,200 bp) relative to plastome IV DNA have been located. Most changes were found in the larger of the two single-copy regions of the five plastomes. Changes in the duplication are always found in both copies. This suggests the existence of an editing mechanism that, in natural populations, equalizes or transposes any change in one copy of the repeat to the equivalent site of the other copy. 5) Detailed mapping of the two rDNA regions of the five plastomes, using the restriction endonucleases Eco RI and Bam HI which each recognize more than 60 cleavage sites per DNA molecule, disclosed a 0.3 Md deletion in plastome III DNA and a 0.1 Md insertion in plastome V DNA relative to DNA of plastome IV, I and II. These changes are most probably located in the spacer between the genes for 16S and 23S rRNA and are found in both rDNA units.

Compositional heterogeneity of the chloroplast DNAs from Euglena gracilis and Spinacia oleracea.

The chloroplast genomes of Euglena gracilis and Spinacia oleracea were investigated in their compositional heterogeneity, by using different experimental approaches. Euglena chloroplast DNA has a dG + dC content of 28%. Preparations averaging 20 x 10(6) in molecular weight exhibit a gross heterogeneity in their elution profiles from hydroxyapatite and in their buoyant densities because the rRNA genes have a high rG + rC content. Finer analysis by melting, buoyant density of restriction fragments and micrococcal nuclease degradation have revealed an extended compositional heterogeneity. From micrococcal nuclease digestion data, approximately 30% of the chloroplast genome is as low as 12% in its dG + dC content, whereas 10% is higher than 60% dG + dC. Since the average dG + dC content of large restriction endonuclease fragments varied to a lesser extent, most of dA + dT-rich sequences must occur in short stretches interspersed with dG + dC-rich stretches. Spinach chloroplast DNA (dG + dC = 36.5%) did not exhibit any gross compositional heterogeneity in its hydroxyapatite elution or in its buoyant density profile. But the higher resolution methods of melting, bouyant densities of restriction fragments and micrococcal nuclease degradation revealed a high degree of heterogeneity which appears to be due to interspersion of short DNA stretches of different base composition. About 30% of genome is as low as 22% in dG + dC, while 10% is higher than 60% in dG + dC.

The mitochondrial genomes of Ustilago cynodontis and Acanthamoeba castellanii.

Mitochondrial DNA from Ustilago cynodontis has been investigated in several of its properties. Its dG + dC content is equal to 33.5%; its buoyant density (1.698 g/cm3) is higher, by 5 mg/cm3, and its melting temperature (82.5 degrees C) is lower than expected for a bacterial DNA having the same base composition; the first derivative of its melting curve indicates a large compositional heterogeneity, its molarity of elution from hydroxyapatite is high, 0.28 M phosphate, and allows its partial separation from nuclear DNA. Degradation by micrococcal nuclease indicates that about 25% of the DNA is formed by stretches having no more than 15% dG + dC. Finally, the unit size of mitochondrial genome is about 50 X 10(6). In most of its properties, the mitochondrial genome of U. cynodontis presents strong analogies with that of Saccharomyces cerevisiae. A parallel investigation on mitochondrial DNA from Acanthamoeba castellanii which has as genome unit size of only 27 X 10(6), has shown that this shares with the former the dG + dC content (32.9%), the melting temperature (82.5 degrees C), a large compositional heterogeneity and a very similar pattern of micrococcal nuclease degradation; its buoyant density (1.692 g/cm3) and its molarity of elution from hydroxyapatite (0.25 M phosphate) are, however, normal, probably because of a different short-sequence pattern and the fact that its dA + dT-rich stretches are shorter, on the average.

Restriction endonuclease cleavage site map of chloroplast DNA from Oenothera parviflora (Euoenothera plastome IV).

1) More than 50 cleavage sites produced by the restriction endonucleases Sal I, Pst I, Kpn I, Sma I and Eco RI have been physically mapped on the 47 µm circular DNA molecule of the Euoenothera plastome IV. This plastome (= plastid genome) is considered to be the phylogenetically oldest of the subsection. 2) The DNA molecule is segmentally organized into four regions represented by a large duplicated sequence in inverted orientation whose copies are separated by two single-copy segments. The single-copy regions comprise about 14 and 57 Md in size, respectively. 3) The size of the inverted repeat, about 15 Md, was determined by restriction site mapping, by mapping of genes for ribosomal RNAs and by hybridization of a cRNA transcribed from a homologous part of Spinacia oleracea chloroplast DNA which appears to be phylogenetically conserved. 4) Hybridization of radio-iodinated spinach 16S, 23S and 5S chloroplast rRNA species to Southern blots of restricted plastome IV DNA has localized the rDNA to the inverted repeat regions, in the order given. The genes for 16S and 23S rRNA are separated by a 2.4 kbp spacer. 5) The physical map of the plastome IV DNA serves as basis for comparison with the DNA from the four other, closely related Euoenothera plastomes.

Homologies among ribosomal RNA and messenger RNA genes in chloroplasts, mitochondria and E. coli.

Labelled chloroplast rRNAs from Spinacia oleracea were hybridized to restriction endonuclease digests of chloroplast DNA from Oenothera hookeri and Euglena gracilis, to mitochondrial DNA of Acanthamoeba castellanii, and to DNA of the E. coli rrn B operon in the transducing phage lambda rifd 18. The degree of homology is greatest for the 16S rRNA gene. Greater than 90% occurs between the two higher plant genes, 80% homology to the lower plant gene, 60%-70% homology to the bacterial gene, and 20% homology to the mitochondrial gene. The degree of hybridization varied considerably for the 23S and the 5S rRNA genes. Very high homology exists between the two higher plant genes, only about 50% homology for both the Euglena and bacterial genes, and no significant homology for the mitochondrial genes. These results show that any chloroplast (or E. coli) rRNA may be used as a probe to identify rRNA genes in other ctDNAs. Two RNA populations, each enriched for a different ctDNA-encoded mRNA, proved useful in the location of these genes on both higher plant ctDNAs. No significant hybridization was obtained using these probes to the Euglena ctDNA which seems to be too distantly related.

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.

The Euglena gracilis chloroplast genome: analysis by restriction enzymes.

1. Chloroplast DNA was isolated from autotrophically and mixotrophically grown Euglena gracilis cells. 2. Aliquots of chloroplast DNA were mechanically degraded to an average molecular weight of 4-7 X 10(6) and G+C-rich DNA fragments (density 1.701 g/cm3) were separated from the bulk DNA (density 1.685 g/cm3) using preparative CsCl density gradients. 3. Total chloroplast DNA and its DNA subfractions, which first were characterized with respect to average G+C content and hybridization capacity for chloroplast rRNA, were hydrolysed with restriction endonucleases (endo R-EcoRI, end R-HindII, endoR-HindIII, endo R-HindII+III, endoR-Hpal, endo R-HpaII and endoR-HaeIII). The fragments were separated on gels under a variety of electrophoretic conditions. 4. With each enzyme tested, a rather large number of bands was obtained. In all cases, different banding patterns were obtained for total DNA, and the DNA subfractions. 5. Chloroplast DNA from autotrophically and mixotrophically grown cells gave identical banding patterns. 6. Digestion of total DNA with the endoR-HaeIII yielded 51-52 fragments separated in the gels in a total of 36 bands of which 11-12 bands were composed of 2-3 fragments as estimated by densitometry. The molecular weights of all fragments combined was 87 X 10(6) or 95% of the genome (92 X 10(6)). 7. Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome (Mr92 X 10(6)). These cistrons are located on seven different types of endo R-HaeIII fragments. The hybridising fragments are preferentially found in the G+C-rich subfraction and in bands which are composed of 2-3 fragments.

The mitochondrial genome of Euglena gracilis.

Mitochondrial DNA from Euglena gracilis has been investigated in its chemical and physical properties. Its G + C content is equal to 25%; its buoyant density in a CsCl density gradient (1.690 g/cm3) is higher, by 5 mg/cm3, than expected for a bacterial DNA having the same base composition. The buoyant densities of denatured and renatured DNA are higher than that of native DNA by 10-12 mg/cm3 and 6 mg/cm3, respectively. The melting temperature, Tm, is 77 degrees C in standard saline citrate; the first derivative of the melting curve shows a striking multimodality. Degradation of the DNA by micrococcal nuclease indicates that about 40% of the DNA is formed by stretches lower than 10% in G + C. In all its properties the mitochondrial DNA from Euglena gracilis is strikingly similar to that of Saccharomyces cerevisiae.