Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma

DNA isolated from purified nuclei of Polytoma obtusum has a buoyant density of 1.711 g/ml in CsCl, a Tm of 91.3° C in SSC, and a G + C content of 52.5% as determined by base composition analysis. Thermal dissociation and reassociation studies indicated that this nuclear DNA contains a considerable amount of heterogeneity. Under appropriate reannealing conditions for denatured DNA, about 15% of the DNA reannealed to form a satellite peak at a density of 1.711 g/ml within one hour. Native DNA fractions of different average buoyant densities, ranging from 1.723 to 1.708 g/ml were also obtained in a preparative CsCl gradient, indicating the presence of intermolecular heterogeneity at a molecular size of 8.5×10⁶ daltons. The nuclear DNA reassociated as three distinct classes. The very fast species constituted about 20 % of the total hyperchromicity, the class of intermediate rate comprised roughly 10% of the nuclear DNA, while the remaining 70% consisted of unique sequences. The haploid genome set was estimated by renaturation kinetics studies to contain 5.0×10¹⁰ daltons of DNA or 7.5×10⁷ nucleotide pairs. The analytical complexity of the total nuclear genome was found to be 9.35×10¹⁰ daltons, thus indicating that vegetative cells of P. obtusum are diploid.     

The DNA of Acanthamoeba spp.; A Method for Extraction and Its Characterization

SYNOPSIS. A method is described which permits the recovery of a high yield of undegraded DNA from Acanthamoeba castellanii. The DNA of A. castellanii is comprised of 2 species: the buoyant density of the major component is 1.720 and of the minor component, 1.693. The renaturation characteristics of the minor component suggest that it is of mitochondrial origin. Two components are also present in the DNA of A. palestinensis, A. polyphaga and A. astronyxis. The buoyant densities of major and minor components differ between species.     

The ribosomal cistrons of the water mold Achlya bisexualis

Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm^?3 and a minor component with a density of 1.685 g cm^?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm^?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA.     

Divergence of the mitochondrial electron transport chains from the green alga Chlamydomonas reinhardtii and its colorless close relative Polytomella sp

Compelling evidence exists that the colorless algae of the genus Polytomella arose from a green Chlamydomonas-like ancestor by losing its functional photosynthetic apparatus. Due to the close relationship between the colorless and the green chlorophyte, Polytomella sp. appeared as a useful indicative framework for structural studies of Chlamydomonas reinhardtii mitochondria. However, comparative studies reported here unexpectedly revealed significant differences between the mitochondrial respiratory systems of the two algae. Two-dimensional blue native/SDS-PAGE of isolated mitochondria indicated that cytochrome-containing respiratory complexes III and IV in the two chlorophytes contrast in size, subunit composition and relative abundance. Complex IV in Polytomella is smaller than its counterpart in C. reinhardtii and occurs in two forms that differ presumably in the presence of subunit COXIII. The cytochrome c and the iron-sulfur Rieske protein of both chlorophytes revealed structural differences on the amino acid sequence level. Under comparable culture conditions, the colorless alga exhibits lower levels of cytochrome c and complex IV but a higher respiratory activity than the green alga. Cytochrome c levels were also found to be differently regulated by the growth conditions in both algae. The divergence between the respiratory systems in the two related chlorophytes can be viewed as a consequence of the loss of photosynthetic activity and/or of the adaptation to the environment via the acquisition of a more flexible, heterotrophic metabolism. Our understanding of mitochondrial function and evolution is expected to be greatly enhanced via further parallel studies of photosynthetic/non-photosynthetic algae, for which this study forms an incentive.     

Variation in satellite DNA from some higher plants

Pure satellite DNAs were prepared as minor components after centrifugation to equilibrium on CsCl gradients. A single satellite DNA band was isolated from flax (Linum usitatissimum) DNA and two bands were resolved in cucumber (Cucumis sativus) DNA. These apparently homogeneous components of the plant genomes were further analyzed by thermal denaturation and renaturation. The flax satellite DNA appeared homogeneous on thermal denaturation but was shown to contain several components of renaturation. The two cucumber satellite DNAs were different from each other, but both showed at least two components in denaturation and renaturation analyses. Renaturation in the three satellites, particularly in flax, was inaccurate, indicating a considerable degree of sequence divergence. Although each satellite contained quite large amounts of simple repetitious sequences, a residual heterogeneous DNA fraction was always present. It is considered that this was too large a portion of the satellite DNA to be due to organelle or ribosomal DNA in cucumber. The latter possibility is precluded in flax, where the satellite is completely resolved in buoyant density from both organelle and ribosomal DNA.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. III. Ribosomal RNA cistrons of the nucleus and leucoplast

The colorless alga Polytoma obtusum has been found to possess leucoplasts, and two kinds of ribosomes with sedimentation values of 73S and 79S. The ribosomal RNA (rRNA) of the 73S but not the 79S ribosomes was shown to hybridize with the leucoplast DNA (rho - 1.682 g/ml). Nuclear DNA of Polytoma (rho = 1.711) showed specific hybridization with rRNA from the 79S ribosomes. Saturation hybridization indicated that only one copy of the rRNA cistrons was present per leucoplast genome, with an average buoyant density of rho = 1.700. On the other hand, about 750 copies of the cytoplasmic rRNA cistrons were present per nuclear genome with a density of rho = 1.709. Heterologous hybridization studies with Chlamydomonas reinhardtii rRNAs showed an estimated 80% homology between the two cytoplasmic rRNAs, but only a 50% homology between chloroplast and leucoplast rRNAs of the two species. We conclude that the leucoplasts of Polytoma derive from chloroplasts of a Chlamydomonas-like ancestor, but that the leucoplast rRNA cistrons have diverged in evolution more extensively than the cistrons for cytoplasmic rRNA.     

Studies with deoxyribonucleic Acid from blue-green algae

DNA from two blue-green algae was isolated and characterized. The buoyant densities, thermal denaturation and renaturation, thermal melting values, base compositions, sedimentation coefficients, and molecular weights were determined. Blue-green algal DNA renatured extensively and at a comparable rate to that of bacterial DNA. The similarities among the kinds of DNA from bacteria and blue-green algae were interpreted to reflect a close relationship.     

Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis

Euglena chloroplast DNA has a buoyant density in CsCI of 1.686. Shearing this DNA produces a satellite band at density 1.700. The satellite, easily lost during preparative CsCI gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 × 10⁶ daltons of DNA.     

Bovine galactosyl transferase. Substrate.managanese complexes and the role of manganese ions in the mechanism.

The dG+dC-rich fractions obtained by density gradient centrifugation of bovine DNA in Cs2SO4/BAMD [J. Cortadas, G. Macaya & G. Bernardi (1977) Eur. J. Biochem. 76, 13--19] were centrifuged in Cs2SO4/Ag+ density gradients. These experiments led to the preparation of the DNA components which had been detected (by analytical centrifugation in CsCl) in the Cs2SO4/BAMD fractions, and also of DNA components which had identical behaviors in Cs2SO4/BAMD gradients and identical buoyant densities in CsCl. A total of eight satellite components and 11 minor components, accounting for 23% and 4% of the bovine genome, respectively, were thus isolated and charcterized in their relative amounts and buoyant densities. The implications of these results on the interpretation of renaturation kinetic data on the bovine genome are discussed.     

Phosphorylase isoenzymes in Polytoma uvella

Starch phosphorylase isoenzymes in growing cultures of Polytoma uvella were isolated by gel electrophoresis and DEAE-cellulose chromatography. Two forms (A and B) were found. These were not detected in the lag phase of the cultures but both forms were present in the log and early stationary phase. Initially form A was more prominent than form B. In the early stationary phase, form A decreased and only form B could be found in the older cultures.     

The distribution of CR1, an Alu-like family of interspersed repeats,in the chicken genome

We have studied the distribution of CR1, a family of short interspersed repeats, in the chicken genome; this family is homologous to the AluI family of man and to the B1-B2 families of mouse. Hybridization with a suitable probe showed that the vast majority of CR1 are located on the heaviest major component (1.708) of the genome which only represents 9% of chicken DNA. Some repeats were also found on the 1.702 and 1.704 components, but none on the 1.699 component (components are denoted by their buoyant densities in CsCl). The GC content of the repeats, 48%, matches that, 47%, of the major component mainly harboring it.     

Sterols of Goniotrichum elegans

The sterol fraction was examined from cultures of the filamentous red alga Goniotrichum elegans. Brassicasterol accounted for nearly half of the total sterol and was accompanied by cholesterol (24%) as well as a number of minor components. This is the first record of brassicasterol as a major sterol in the Rhodophycophyta. The occurrence of this C₂₈ major sterol may be of taxonomic importance in determining the relationship of the Goniotrichales to the other red algae of which have C₂₇ major sterols.     

Role of Aldolase in Photosynthesis. II Demonstration of Aldolase Types in Photosynthetic Organisms

Spinach leaves and photoautotrophically grown Euglena and Chlorella possess fructose 1,6-diphosphate aldolases inhibited by p-chloromercuribenzoate but insensitive to K⁺ or ethylenediamine tetraacetate (Type I). Dark grown Euglena and Chlorella have aldolases inhibited by p-chloromercuribenzoate and ethylenediamine tetraacetate but stimulated by K⁺ (Type II). The red alga, Chondrus, and the golden-brown alga, Ochromonas, appear to possess both types. Bean, pea, and spinach seeds and the leaves and cotyledons of etiolated bean seedlings contain a p-chloromercuribenzoate insensitive, apparently non-sulfhydryl variant of Type I. Sensitivity of leaf aldolase to p-chloromercuribenzoate occurs in etiolated bean seedlings only after an extended period of illumination. Type II aldolase activity in cell-free extracts of 4 blue-green algae has been demonstrated.     

Occurrence of steryl glycosides and acylated steryl glycosides in some marine algae

There is some controversy concerning the presence of steryl glycosides and acylated steryl glycosides in eucaryotic algae. These two classes of sterol compounds were investigated in species belonging to the three major groups of eucaryotic algae: green algae (Ulva gigantea, Cladophora rupestris), brown algae (Fucus vesiculosus, Ascophyllum nodosum), and red algae (Rhodymenia palmata, Porphyridium sp.). All these algae contain both steryl glycosides and acylated steryl glycosides. The sterol components of these compounds vary according to the alga but they are always the same as the free sterols of the alga in question. The most common sugar moiety is glucose. In the acylated steryl glycosides, the fatty acid is mainly palmitic acid. The percentage of these compounds (as a percentage of the total sterol content) is often low.     

Divergence of the mitochondrial electron transport chains from the green alga Chlamydomonas reinhardtii and its colorless close relative Polytomella sp.

Compelling evidence exists that the colorless algae of the genus Polytomella arose from a green Chlamydomonas-like ancestor by losing its functional photosynthetic apparatus. Due to the close relationship between the colorless and the green chlorophyte, Polytomella sp. appeared as a useful indicative framework for structural studies of Chlamydomonas reinhardtii mitochondria. However, comparative studies reported here unexpectedly revealed significant differences between the mitochondrial respiratory systems of the two algae. Two-dimensional blue native/SDS-PAGE of isolated mitochondria indicated that cytochrome-containing respiratory complexes III and IV in the two chlorophytes contrast in size, subunit composition and relative abundance. Complex IV in Polytomella is smaller than its counterpart in C. reinhardtii and occurs in two forms that differ presumably in the presence of subunit COXIII. The cytochrome c and the iron-sulfur Rieske protein of both chlorophytes revealed structural differences on the amino acid sequence level. Under comparable culture conditions, the colorless alga exhibits lower levels of cytochrome c and complex IV but a higher respiratory activity than the green alga. Cytochrome c levels were also found to be differently regulated by the growth conditions in both algae. The divergence between the respiratory systems in the two related chlorophytes can be viewed as a consequence of the loss of photosynthetic activity and/or of the adaptation to the environment via the acquisition of a more flexible, heterotrophic metabolism. Our understanding of mitochondrial function and evolution is expected to be greatly enhanced via further parallel studies of photosynthetic/non-photosynthetic algae, for which this study forms an incentive.     

An analysis of the bovine genome by Cs2SO4-Ag density gradient centrifugation

Calf DNA preparations having molecular weights of 5 to 7 × 10⁶ have been fractionated by preparative Cs₂SO₄—Ag⁺ density gradient centrifugation into a number of components. These may be divided into three groups: (1) the main DNA component (1.697 g/cm³; all densities quoted are those determined in CsCl density gradients), the 1.704 and 1.709 g/cm³ components form about 50, 25 and 10% of the genome, respectively; they are characterized by having symmetrical CsCl bands and melting curves, both of which have standard deviations close to those of bacterial DNAs of comparable molecular weight, and by their G + C contents being equal to 39, 48 and 54%, respectively; after heat-denaturation and reannealing, their buoyant densities in CsCl are greater than native DNA by 12, 10 and 3 mg/cm³, respectively. (2) The 1.705, 1.710, 1.714 and 1.723 g/cm³ components represent 4, 1.5, 7 and 1.5% of the DNA, respectively, and exhibit the properties of “satellite” DNAs; their CsCl bands and melting curves have standard deviations lower than those of bacterial DNAs; after heat-denaturation and reannealing, their buoyant densities are identical to native DNA, except for the 1.705 g/cm³ component, which remains heavier by 5 mg/cm³; in alkaline CsCl, only the 1.714 g/cm³ component shows a strand separation. (3) A number of minor components, forming 1% of the DNA, have been recognized, but they have not been investigated in detail; two of them (1.719 and 1.699 g/cm³) might correspond to ribosomal cistrons and mitochondrial DNA, respectively.     

New insights into the unique structure of the F0F1-ATP synthase from the chlamydomonad algae Polytomella sp. and Chlamydomonas reinhardtii

In this study, we investigate the structure of the mitochondrial F(0)F(1)-ATP synthase of the colorless alga Polytomella sp. with respect to the enzyme of its green close relative Chlamydomonas reinhardtii. It is demonstrated that several unique features of the ATP synthase in C. reinhardtii are also present in Polytomella sp. The alpha- and beta-subunits of the ATP synthase from both algae are highly unusual in that they exhibit extensions at their N- and C-terminal ends, respectively. Several subunits of the Polytomella ATP synthase in the range of 9 to 66 kD have homologs in the green alga but do not have known equivalents as yet in mitochondrial ATP synthases of mammals, plants, or fungi. The largest of these so-called ASA (ATP Synthase-Associated) subunits, ASA1, is shown to be an extrinsic protein. Short heat treatment of isolated Polytomella mitochondria unexpectedly dissociated the otherwise highly stable ATP synthase dimer of 1,600 kD into subcomplexes of 800 and 400 kD, assigned as the ATP synthase monomer and F(1)-ATPase, respectively. Whereas no ASA subunits were found in the F(1)-ATPase, all but two were present in the monomer. ASA6 (12 kD) and ASA9 (9 kD), predicted to be membrane bound, were not detected in the monomer and are thus proposed to be involved in the formation or stabilization of the enzyme. A hypothetical configuration of the Chlamydomonad dimeric ATP synthase portraying its unique features is provided to spur further research on this topic.     

Identification of three distinct Polytomella lineages based on mitochondrial DNA features

Polytomella is composed of colorless green algae closely related to Chlamydomonas reinhardtii. Species in the genus have been used in diverse fields of biological research, most recently to study mitochondrial function and mitochondrial genome evolution in the Chlorophyceae, but the phylogenetic relationship between the various available taxa has not yet been clarified and it is not known whether they also possess fragmented mitochondrial genomes, as reported for Polytomella parva. We therefore examined cox1 sequence from seven Polytomella taxa with the goal of establishing their phylogenetic relationships and relating this information to their mitochondrial DNA (mtDNA) fragmentation pattern. We found that the Polytomella isolates examined fall into three distinct lineages, two of which possess fragmented mitochondrial genomes. The third and earliest branching lineage, represented by Polytomella capuana, appears to possess an intact mtDNA. In addition, there is evidence for variation in both size and number of mtDNA fragments between various Polytomella isolates, even within the same lineage. The considerable amount of sequence divergence between lineages seems to correlate with the geographic origin of the strains, leading us to believe that greater amounts of sequence divergence could be uncovered by a broader sampling of Polytomella.     

Reciprocal induction of cell division by cells of complementary mating types in Tetrahymena

Chick embryo fibroblasts in monolayer culture were synchronized by contact inhibition and serum starvation. Nuclear DNA isolated from the [³H] thymidine pulse-labelled cells throughout the period of DNA synthesis (S phase) was analysed by hydroxylapatite chromatography after renaturation at different C₀t values. It is shown that repeated sequences having different frequencies of reassociation, replicate differently throughout the S period. In order to study the distribution of the repeated sequences, DNA isolated during the S period was fractionated according to its buoyant density. It is shown that only some of the highly reiterated sequences which are included in the high buoyant density DNA fractions, replicate equally well during the early and the late S periods. By contrast, reiterated sequences of the low buoyant density DNA fractions replicate mainly during the late S period.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. II. General characterization of organelle nucleic acids.

Polytoma obtusum has a main band DNA (alpha) with a buoyant density in CsC1 of rho = 1.711 g/ml and a light DNA satellite (beta) with rho = 1.682 g/ml. beta-DNA was substantially enriched in a fraction containing small leucoplast fragments and some mitochondria, which was obtained in a pellet sedimenting between 3,000 g and 5,000 g. A crude mitochondrial pellet was also obtained by sedimenting at 12,000 g to recover particulates remaining in the supernate after 10 min at 5,000 g. This fraction contained a third DNA component (gamma) with rho = 1.714 g/ml. We have concluded that the leucoplasts of P. obtusum contain the beta-DNA (1.6882) and the mitochondria possess the gamma-component (1.714). Two distinct classess of ribosomes were isolated and separated by sucrose density gradients, a major 79S species and a minor species at 75S. The major species possessed the 25S and 18S ribosomal RNA (rRNA), characteristic of cytoplasmic ribosomes, and these particles co-sedimented in sucrose gradients with the 79S cytoplasmic ribosomes of Chlamydomonas reinhardtii. The minor species was present in about 2% of the total ribosomal population but showed an eight-to-ninefold enrichment in the leucoplast pellet, suggesting that it was of organelle origin. These 73S particles had RNA components migrating very closely with the 18S and 25S species of the 79S ribosomes, but the base composition of the rRNA from these two classes of ribosomes was significantly different; the rRNA from the 79S ribosomes had a G+C mole ratio of 50.0%, while the rRNA from the 73S class had a ratio of 47.5%. By comparison, chloroplast ribosomes of C. reinhardtii were found to sediment at 70S and contain rRNA molecules of 23S and 16S, with a G + C content of 51.0%. These findings support the concept that the Polytoma leucoplast possesses characteristic genetic and protein-forming systems.