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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.

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.

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.

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.

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.