Composition and synthesis of DNA in synchronously growing cells of Chlorella pyrenoidosa

Summary The composition and synthesis of DNA in synchronous cultures of Chlorella pyrenoidosa strain 211/8b has been investigated. Analytical CsCl density gradient centrifugation gave a homogenous major DNA component with a (G+C) content of 51% and a minor component containing 28% (G+C). The (G+C) contents derived from melting profiles were 2–3% lower. A second minor component with approximately 41% (G+C) content was inferred from banding patterns of labelled DNA in preparative CsCl density gradients. ¹⁴C-uracil was readily incorporated into the pyrimidine moieties of the major (nuclear) DNA between the 10th and 18th hour after beginning of the light period, but not at any other time. ¹⁴C-uracil incorporation into the minor (satellite) component was low but continuous throughout the whole cell cycle. The incorporation is correlated with an increase in the proportion of satellite DNA from 6% up to 20% during the time when no nuclear DNA replication takes place. The results suggest that different regulatory mechanisms exist for the nuclear and for satellite DNA synthesis.     

Malic Dehydrogenase Heterogeneity in Malaria (Plasmodium lophurae and P. berghei)*

SYNOPSIS. Molecular heterogeneity of malic dehydrogenase (MDH) in malaria was shown by zone electrophoresis in potato starch, starch gel, and by enzymatic activity with analogs of the coenzyme diphosphopyridine nucletide. A single anodal peak of MDH characterized the normal duck red blood cell whereas P. lophurae free of the host cell had a cathodal form of the enzyme. Infected duck erythrocytes had a combination of these electrophoretic forms. The isolated enzymes had different pH optima with oxaloacetate as substrate: pH 7.4 for the duck red cell and 6.4 for the plasmodial enzyme. The Km of each enzyme for oxaloacetate varied with the pH. The Km at pH 7.4 was 4.1 and 4.4 × 10⁵ M for parasite and host, respectively, whereas at 6.4 it was 2.0 × 10⁵ M for P. lophurae and 6.3 × 10⁵M for the duck erythrocyte. At pH 7.4 both enzymes were inhibited by oxaloacetate concentrations greater than 10^?4 M. P. berghei MDH also had a different electrophoretic character from that of the mouse red blood cell. Quantitatively, MDH activity increased with parasitization, and erythrocyte-free P. lophurae contained approximately twice the activity found in the uninfected duck erythrocyte. The quantity of MDH activity of the infected cell was ca. 50% less than the sum of the activities of the parasite and the uninfected cell. It is suggested that these properties of the parasite MDH may give it a physiologic advantage over the red cell under the conditions which prevail intraerythrocytically.     

Cultivation of avian malaria parasites in mammalian liver cells

The exoerythrocytic stages of Plasmodium lophurae and P. fallax were grown in cell cultures derived from embryonic mouse livers. Liver cell monolayers were maintained in continuous culture with frequent subculturing for extended periods of time. Morphologically, the main cell type was a large, flat cell which closely resembled in shape the mouse parenchymal cell, although small colonies of spindle-shaped cells could also be found. Mammalian cells were labelled with thymidine-³H prior to infection with avian merozoites so they could be positively distinguished from any avian cells which might be present in the merozoite inoculum as contaminants. Merozoites were observed inside the labelled mammalian cells within three hours and large forms were seen at 48 hr. The mammalian cells supported parasite growth comparable to that observed in avian cell cultures.     

Effects of Ethidium Bromide and Several Acridine Dyes on the Kinetoplast DNA of Leishmania tropica*†

Whole cell DNA from Leishmania tropica has 2 peaks when banded by CsCl equilibrium density centrifugation. The main band has a buoyant density of 1.721 and the satellite band a buoyant density of 1.705, with Clostridium perfringens DNA (ρ= 1.6915) used as a reference. The satellite band has been identified as the kinetoplast DNA by purifying DNA from isolated kinetoplasts. L. tropica has the highest G + C content of both nuclear and kinetoplastic DNA thus far reported for trypanosomatids. The effects of ethidium bromide, acriflavin, proflavin, and 5-aminoacridine on the kinetoplast of L. tropica have been compared. Ethidium bromide and acriflavin, but not proflavin or 5-aminoacridine, induce dyskinetoplasty. L. tropica is one of the most sensitive trypanosomatids to ethidium bromide and acriflavin. Examination of the DNA from drug-treated cells in CsCl gradients revealed a loss of the satellite band after ethidium bromide or acriflavin treatment, but not after proflavin or 5-aminoacridine treatment. Cell division was required to produce these effects on the kinetoplast.     

Non-random incorporation of 5-bromodeoxyuridine in rat cell DNA

Secondary cultures of rat embryo cells were exposed for 24 hrs. to 10-⁷M [³H] thymidine (TdR) or 10^?7M [³H]5-bromodeoxyuridine (BrdU) in order to localize and compare the distribution of the isotopes in DNA. DNA was extracted, sheared, and centrifuged to equilibrium through neutral and alkaline CsCl density gradients. The DNA band from each gradient type was separated into a “heavy” and “light” fraction, and DNA-DNA reassociation hybridizations were performed on each sample. Renaturation profiles revealed that each fractionated DNA sample was representative of the complete rat cell genome, except for the “light” [³H]BrdU-DNA prepared by centrifugation through alkaline CsCl gradients. This fraction was predominantly depleted of labeled late repetitive and intermediate sequences. Uncentrifuged rat DNA was sequentially fractionated during reassociation into rapidly, intermediate, and slowly reassociating sequences by hydroxyapatite chromatography. Relative specific activities of each component revealed a non-uniform distribution of [³H]BrdU moieties as compared to [³H]TdR. These results suggest a nonrandom incorporation of 10^?7M BrdU into rat cell DNA sequences.     

Purine and Pyrimidine Synthesis by the Avian Malaria Parasite,Plasmodium lophurae*

SYNOPSIS. Purine and pyrimidine biosynthesis in the avian malaria parasite Plasmodium lophurae and its host cell, the duck erythrocyte, were investigated in vitro. Pyrimidine synthesis, as measured by the incorporation of C¹⁴-NaHCO₃ into cytosine, uracil and thymine was slight in uninfected duck erythrocytes, whereas infected erythrocytes and erythrocyte-free parasites had high rates of incorporation of NaHCO₃ into these bases. In addition, orotidine-5′-monophosphate pyrophosphorylase and thymidylate synthetase, 2 enzymes of the pyrimidine biosynthetic pathway, were found in cell-free extracts of the plasmodia. Purine synthesis was measured by determining the extent of incorporation of C¹⁴-Na-formate into adenine and guanine. Uninfected and infected erythrocytes had similar rates of Na-formate incroporation into adenine. whereas free parasites incorporated little of this compound into adenine, or guanine. On the other hand, the incorporation of Na-formate into guanine was 54% higher in infected erythrocytes than in uninfected erythrocytes. It is suggested that P. lophurae synthesizes purines to a limited extent, and derives most of its purines from the host erythrocyte. The greater incorporation of Na-formate into guanine by infected cells, and its low incorporation into free parasites may be accounted for by parasite conversion of host cell adenine (in the form of ATP) into guanine. Pyrimidine biosynthesis in infected cells can be accounted for by de novo synthesis by the parasite itself.     

DNA of Akodon (Rodentia,Cricetidae). I. Biophysical characterization

Buoyant density in CsCl, melting temperature, and G + C base content of the DNA from four species of Akodon (Rodentia, Cricetidae) were determined. The buoyant density values of 1.699–1.701 g/cm³ were in accordance with the data reported for other cricetids. No satellite bands were seen in neutral CsCl. The T _m values determined in 1 × SSC ranged from 86.2 to 87.0 C, which corresponds to G + C contents of 41.2–43.2%. There was good agreement in DNA base composition of the four species, although values were slightly higher in A. obscurus, suggesting a certain degree of interspecies variability.This study was supported by grants from Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, and Organización de Estados Americanos.     

Pantothenic Acid Metabolism During Avian Malaria Infection: Pantothenate Kinase Activity in Duck Erythrocytes and in Plasmodium lophurae*

SYNOPSIS. The initial enzymatic reaction in the pathway of coenzyme A genesis from pantothenic acid, i.e., the conversion of pantothenic acid to phosphopantothenic acid catalyzed by pantothenic acid kinase, was found in extracts of avian erythrocytes but not in extracts of Plasmodium lophurae or intact parasites. The activity of the kinase was significantly higher in extracts prepared from normal duck erythrocytes than those infected with P. lophurae. The apparent absence of pantothenic acid kinase in P. lophurae is corroborative support for nutritional studies(7,9) which suggest that the avian malarial parasite is dependent for its supply of coenzyme A on the host cell.     

The DNAs of the A and B chromosomes of the mealy bug,Pseudococcus obscurus

When the DNA of mealy bugs carrying B chromosomes (+ B:DNA) was compared to the DNA of individuals not possessing Bs (-B:DNA), no significant differences were found using isopycnic centrifugations in CsCl or thermal denaturation analyses. Both DNAs had buoyant densities of 1.693 g/cm³ in neutral CsCl gradients and 1.748 g/cm³ in alkaline CsCl gradients. Satellite DNAs were not detected. The average T_m of +B:DNA was 67.9° C in 0.1 SSC while -B:DNA had an average T_m of 67.4° C in the same solution. However, in situ molecular hybridizations with complementary RNAs (cRNAs) transcribed in vitro from each type of DNA showed considerable differences with regard to the amount of labeling of B chromosomes. Using cRNA to +B:DNA, the average number of silver grains over a B chromosome was 2.1 × the average number of silver grains over individual non-B chromosomes (A chromosomes). In contrast, the ratio (B/A) using cRNA to -B:DNA was less than 0.14. The results are interpreted as meaning that very little DNA is shared in common by both A and B chromosomes.     

The Purification of DNA from the Genomes of Paramecium aurelia and Tetrahymena pyriformis1

SYNOPSIS. Methods are described for the recovery of highly purified DNA from Paramecium aurelia and Tetrahymena pyriformis in high yields. Our DNA is only slightly contaminated with RNA and carbohydrate, and little or no protein can be detected. We could not reduce the RNA (orcinol-positive material) by further treatment (sephadex or hydroxyapatite chromatography and preparative CsCl gradients). At the extreme our DNA is contaminated with 15–20% RNA but the real value is most likely considerably lower than this. The DNA we have prepared from Paramecium and Tetrahymena shows all the properties of double-stranded, high molecular weight DNA when characterized by temperature melting, CsCl density gradient centrifugation and hydroxyapatite and sephadex chromatography. When denatured, it absorbs to nitrocellulose filters. The 2 major results of importance from our work reported here are: (1) There is similarity in base composition of DNA from different syngens of Paramecium (28% G+C for syngens 1, 2, 4, 5, and 9 and 29–30% G+C for syngen 8) while there is variation between the syngens of Tetrahymena (24–31% G+C for syngens 1, 4, 7, 10, 11, and 12); (2) the density of any Paramecium DNA varies depending upon whether the cells are grown in the presence of bacteria or in axenic medium. Our results are compatible with observations previously reported for Tetrahymena but contradict those made for Paramecium. The earlier reports of differences in base composition between syngens of Paramecium are probably due in large part to the use of stocks grown on bacteria.     

Mitochondrial DNA synthesis during the cell cycle of Saccharomyces cerevisiae

Logarithmically growing cultures of Saccharomyces cerevisiae were separated into fractions of increasing average age by zonal centrifugation. Nuclear and mitochondrial DNA labeled for both long and short periods were examined at different times in the cell cycle following isolation of the DNA on CsCl gradients. The data thus obtained were used to determine the relative times of replication. In S. cerevisiae the synthesis of nuclear and mitochondrial DNA was found to occur at the same time in the cell cycle. Some data indicate that mitochondrial DNA synthesis was completed before nuclear DNA synthesis.     

Visual selection and maintenance of the cell lines with high plant regeneration ability and low ploidy level in Dianthus acicularis by monitoring with flow cytometry analysis

Efficient plant regeneration system from cell suspension cultures was established in D. acicularis (2n=90) by monitoring ploidy level and visual selection of the cultures. The ploidy level of the cell cultures closely related to the shoot regeneration ability. The cell lines comprising original ploidy levels (2C+4C cells corresponding to DNA contents of G1 and G2 cells of diploid plant, respectively) showed high regeneration ability, whereas those containing the cells with 8C or higher DNA C-values showed low or no regeneration ability. The highly regenerable cell lines thus selected consisted of compact cell clumps with yellowish color and relatively moderate growth, suggesting that it is possible to select visually the highly regenerable cell lines with the original ploidy level. All the regenerated plantlets from the highly regenerable cell cultures exhibited normal phenotypes and no variations in ploidy level were observed by flow cytometry (FCM) analysis.     

Effect of heat on the viability of Schizosaccharomyces pombe 972h growing in synchronous cultures

The viability of synchronous cultures of the fission yeast Schizosaccharomyces pombe 972h⁻ has been examined after exposure to temperatures of 49 °C. Synchronous cultures were established by continuous flow size selection. Samples were taken at 20 min intervals over two cell cycles and heat shocked for 15 min. The cells showed different sensitivities to heat treatment during the cell cycle. The sensitive stage lasted from nuclear division to a point in early G2. The position in the cell cycle and duration of the heat sensitive stage of S. pombe are similar to those reported for the response of this organism to ultraviolet light, γ radiation, and to suicide labelling with ³²P.     

Fractionation and characterization of satellite DNAs of the kangaroo rat (Dipodomys ordii)

Nuclear DNA from liver cells of the kangaroo rat species Dipodomysordii was fractionated and characterized with the aid of buoyant density gradients in neutral and alkaline CsCl and in Ag⁺-Cs₂SO₄. More than one-half of the DNA was present in three density satellites, a greater proportion than in any other species yet reported; the purified satellite DNAs were denser than principal DNA. All satellite fractions revealed sharp isopycnic bands and narrow denaturation profiles. Two had identical buoyant densities but differed substantially in T_m, base composition, and reassociation kinetics. In alkaline CsCl all three satellites, as well as a shoulder of intermediate repetitive DNA on the heavy side of the principal band, revealed unique strand densities. The most highly repetitive satellite was unusually rich in (G + C) and contained 6.7% of 5-methylcytosine. A survey of internal organs and spermatozoa of an adult male revealed no significant differences in distribution of the satellites among tissues.     

Distribution of P1 Type Nuclease in the Genus Penicillium

P₁ type nuclease, which hydrolyzes RNA and heat-denatured DNA completely into 5’-mononucleotides and also shows 3’-nucleotidase activity, was widely distributed among various species belonging to the genus Penicillium such as P. expansum, P. notatum, P. steckii and P. meleagrinum. P₁ type nucleases isolated from these strains were produced in a form of complex with malonogalactan when molds were grown on wheat bran. These enzymes showed similar characters in heat-stability (stable at 60°C), temperature optimum (60 to 70°C for RNA and heat denatured DNA, and 70°C for 3’-AMP) and sensitivity to EDTA. The enzymes from P. steckii and P. expansum were immunologically co-related to nuclease P₁.

In addition, many strains of Penicillium produced base-nonspecific RNases forming 3’-mononucleotides via 2’: 3 ’-cyclic nucleotides. These RNases showed similarity in heat-lability (completely inactivated at 60°C), temperature optimum (45 to 50°C), sensitivity to Zn²⁺ and Cu²⁺, and relative hydrolysis rate toward 2’: 3’-cyclic nucleotides (A?C>U?G).     

Timing of mitochondrial DNA synthesis during meiosis in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) synthesis was examined during meiosis in Saccharomyces cerevisiae using an aneuploid strain disomic (n + 1) for chromosome III. The aneuploid has the advantage over true diploid strains in that it completes early meiotic events, including premeiotic chromosome replication, but does not form mature ascospores. Thus, differential extraction problems, resulting from the simultaneous presence of both unsporulated cells and spores in the population, are eliminated. The kinetic of mtDNA synthesis was monitored by determining the actual mtDNA content of cells following analytical CsCl centrifugation of cell extracts. MtDNA synthesis started soon after the cells were placed in sporulation medium and continued at an approximately constant rate until 24 h, resulting in slightly more than a doubling of the mitochondrial DNA content per cell. [¹⁴C]uracil was incorporated into mtDNA during the entire developmental period. Extensive preferential labeling of mtDNA occurred between 24 and 50 h, when no net DNA synthesis was observed.     

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd²⁺ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd²⁺-induced impairment of cell division is connected with DNA damage. The [³H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd²⁺ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd²⁺ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd²⁺ on DNA of soybean cells. We suggest that Cd²⁺ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA     

Purine Uptake and Utilization by the Avian Malaria Parasite Plasmodium lophurae*

SYNOPSIS. Plasmodium lophurae cannot carry out extensive de novo purine biosynthesis, and depends upon the host erythrocyte for a supply of preformed purines. Exogenous purines taken up by the parasitized erythrocyte may constitute a major source of preformed purines for parasite nucleotide biosynthesis. The uptake of exogenous radioactive purine compounds and their incorporation into nucleic acids by duck erythrocytes parasitized with P. lophurae, uninfected erythrocytes, and erythrocyte-free parasites were studied. P. lophurae was found to have a remarkable ability, both intracellularly and extracellularly, to take up and utilize certain exogenous purines such as adenosine, inosine, and hypoxanthine. Incorporation studies indicated that this species has a functional purine salvage pathway by which inosine, hypoxanthine, and adenosine can be converted to both adenine and guanine nucleotides.     

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.