DNA and RNA Syntheses by Intraerythrocytic Stages of Plasmodium knowlesi*

Incorporation of the nucleic acid precursors, orotic acid, adenosine, thymidine, and uridine, was studied in various stages of intraerythrocytic Plasmodium knowlesi from infected rhesus monkeys. Incubation of the parasitized erythrocytes with the precursors was for 3 hr periods using a plasma-free culture medium. The samples containing primarily rings, early trophozoites, or late trophozoites incorporated orotic acid, adenosine, and uridine into RNA; however, these stages exhibited negligible or very low levels of incorporation of any of the precursors into DNA. The sample containing late trophozoite and schizont stages incorporated orotic acid, adenosine, and uridine into RNA, and orotic acid, adenosine, and very low levels of thymidine into DNA. These results indicate that DNA synthesis (the S phase of the cell cycle) occurs very close to the time of nuclear division, and that either the G1 or G2 phase is very short in P. knowlesi. It was also observed that adenosine and orotic acid, 2 precursors which are incorporated into both DNA and RNA, are utilized differently by the intraerythrocytic parasites. Incorporation of orotic acid into RNA and DNA and adenosine incorporation into DNA were continuous for the entire incubation period, whereas incorporation of adenosine into RNA was very low during the last 2 hr of each period. It was further demonstrated that the parasites utilized exogenous uridine for synthesis of RNA, and that the older parasite stages incorporated thymidine into DNA.     

Stage-dependent effects of chloroquine on Plasmodium falciparum in vitro

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of 3H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.     

Cell cycle analysis of asexual stages of erythrocytic malaria parasites

Abstract. Intra-erythrocytic Plasmodium species can be stained with the DNA binding dye, Hoechst 33342, and the distribution of DNA content determined for parasite populations by flow cytometric measurement of fluorescence. Analysis of this distribution will determine the parasitaemia (percentage of erythrocytes infected), and the percentages of trophozoite infected red blood cells, polyparasitized (trophozoite) red blood cells, and schizont/segmenter infected red blood cells. This analysis is based on the hypothesis that the asexual parasites cycle with single G₁ period, and effectively, a single S phase with no significant G₂/M period except at schizogony when the genome DNA content is equivalent to 8 N or higher, dependent on the species. Data are presented to support this model.     

Stage-Dependent Effects of Chloroquine on Plasmodium falciparum In Vitro1

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of ³H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Abstract. Cellular uptake of [³H]thymidine ([³H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 × 10^-18 mol/cell. The pool activity of G₁ cells was unmeasurable but rose to maximum values at the border of the G₁-S phase. It decreased again during G₂. The [³H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10%× min^-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Plasmodium falciparum RuvB2 translocates in 5′–3′ direction,relocalizes during schizont stage and its enzymatic activities are up regulated by RuvB3 of the same complex

Two similar proteins RuvB like1 (Rvb1/Pontin) and RuvB like2 (Rvb2/Reptin) of AAA + family of enzymes are present in yeast to human and are well known to be involved in diverse cellular activities. The human malaria parasite Plasmodium falciparum contains three different RuvB like proteins. Thus it has been of interest to explore why P. falciparum requires three RuvB like proteins and how these enzymes are biochemically regulated. In this study, we present the detailed biochemical characterization of PfRuvB2. The complex of PfRuvB3 was immunopurified and the presence of PfRuvB2 was confirmed. The in vitro interaction study shows that PfRuvB2 interacts only with PfRuvB3 but not with PfRuvB1. The recombinant as well as endogenous PfRuvB2 contains ATPase as well as weak DNA helicase activities. The presence of PfRuvB3 in the helicase reaction of PfRuvB2 increases the helicase activity significantly. Interestingly PfRuvB2/PfRuvB3 complex preferentially translocates and unwinds DNA in the 5′–3′ direction. In vivo studies showed that PfRuvB2 is expressed in all the asexual intraerythrocytic developmental stages and localizes mainly in the nucleus during merozoite, ring and trophozoite stages while during schizont stage it relocalizes partially in the nucleus and partially towards cytoplasm. As PfRuvB3 is specific to intraerythrocytic mitosis so we interpret that PfPuvB3 interacts with PfRuvB2 during schizont/intraerythrocytic mitosis and acts as its modulator mainly for the appreciable helicase activity.     

Cytokinin-induced switch in development in excised cotyledons of radiata pine cultured in vitro

Cotyledons of Pinus radiata D. Don were cultured under shoot-forming (plus cytokinin) and elongating (minus cytokinin) conditions. Using. autoradiographic and precursor incorporation techniques, the sites and rate of macromolecular synthesis were examined during the first five days in culture. Active incorporation of ³H-thymidine, ³ H-uridine and ³H-leucine occurred. In shoot-forming cotyledons the incorporation became preferentially located in the epidermal and sub-epidermal cell layers in contact with the medium. In elongating cotyledons, in contrast, incorporation was randomly distributed, and the amount of incorporation declined with time. Biochemically, differences in DNA, RNA and total protein synthetic patterns were observed. In elongating cotyledons the rates of RNA and protein synthesis were higher during the first 48 h than in shoot-forming tissues, after which the synthetic rates were similar. Two peaks of newly formed DNA were observed in both tissues. These findings indicate that the cytokinin-induced changes in developmental pathways began within 24 h in culture.     

X-ray sensitivity and DNA synthesis in synchronous culture of Plasmodium falciparum

The relationship between parasite development and sensitivity to irradiation with X-rays was investigated during a single synchronous cycle of Plasmodium falciparum in culture. The sensitivity of the parasites to irradiation was closely correlated with the phases of DNA synthesis. Their sensitivity was greatest at the ring stage in development, but decreased at the trophozoite stage when DNA synthesis begins. Lowest sensitivity was found when DNA synthesis was most rapid as the parasites were transforming from late trophozoite to schizont forms. These findings suggest that DNA is the target of the lethal radiation damage in the parasites.     

Incorporation of Radioactive Precursors into DNA and RNA of Plasmodium knowlesi in vitro

SYNOPSIS. Cultures of the intra-erythrocytic stages of Plasmodium knowlesi incubated in vitro utilized all the pre-formed radioactive purines tested (adenine, adenosine, deoxvadenosine, guanine, guanosine and hypoxanthine) but none of the pyrimidines (thymine, thymidine, uracil, uridine, cytidine and deoxycytidine). They did, however, utilize the pyrimidine precursor orotic acid.
All precursors analysed, including deoxyadenosine, were incorporated into both DNA and RNA (in the ratio of ∼1:3) but 19% was incorporated into other unidentified compounds. ³Hadenosine was incorporated into adenine and guanine residues of both DNA and RNA.
No unambiguous evidence was obtained for any periodicity in the synthesis of DNA or RNA in our cultures, even tho cultures remained as synchronous in vitro as they are in vivo. An estimate is presented of the amount of DNA made during one cycle in vitro.     

Protein Synthesis during Photocontrolled Progression of the Cell Cycle in Single-celled Protonemata of the Fern Adiantum Capillus-veneris

Protein synthesis during photoinduced, synchronous progression of the cell cycle in single-celled protonemata of the fern Adiantum capillus-veneris was studied by tracer techniques. Nuclei of the protonemata were labelled with ³H-thymidine during spore germination so that the amount of ³H incorporated into the TCA-insoluble fraction of the cells could be used as a measure of the cell number in each sample. The rate of the incorporation of ¹⁴C-amino acids into TCA-insoluble materials was not significantly varied at different stages of the cell cycle or by treatment with blue light. Extracts of cells labelled with ³⁵S-methionine at various times after the transfer from red light condition (G₀) to darkness (G₁ to S) were analyzed by two-dimensional gel electrophoresis. At least 3 of about 200 spots showed significant changes in intensity on fluorograms. Spot A (molecular weight 20,000, isoelectric point 6.3) was detectable only in early G₁, whereas spot B (molecular weight 19,500, isoelectric point 6.3) was found only in the late G₁ and S phases. When the cells were exposed to blue light before the dark incubation, the times of disappearance of spot A and appearance of spot B were advanced depending upon the progression of the cell cycle but not upon the clock time.     

Cellular Commitment for Post-Gastrular Increase in Alkaline Phosphatase Activity in Xenopus laevis Development

Xenopus embryos were dissociated into cells and cultured in Ca²⁺-free medium to study the relationship between the cell-to-cell interaction and macromolecular synthesis. Under the conditions, cells did not aggregate at all, and remained isolated even while they were dividing actively. Synthesis of DNA and protein as studied by the incorporation of (³H)thymidine and (³H)leucine proceeded as in the aggregating cells. Also, the activity to synthesize rRNA, 5S RNA, and heterogeneous RNA as determined by the incorporation of (³H)uridine was not impaired. Such an increase in the activity of alkaline phosphatase, as occurs in embryos after the gastrula stage, was found to be inhibited greatly when early-blastula cells were cultured in the non-aggregating conditions. However, we found here that the inhibition was not observed with cells isolated from late-blastulae. Therefore, it appears that the increase in the activity of alkaline phosphatase during post-gastrular stages is dependent on some cellular commitment which may be established by cell-to-cell contact during the blastula stage.     

STUDIES ON LIVER REGENERATION: II. EFFECTS of PHENOBARBITAL ON the ONSET and PATTERN of RAT LIVER REGENERATION FOLLOWING PARTIAL HEPATECTOMY

Abstract. Partially hepatectomized rats were given a single intraperitoneal injection of a phenobarbital solution or of water immediately after surgery. At various time intervals following the operation, the animals were injected with ¹³¹ iododeoxyuridine (¹¹³IDU), sacrificed 2 hr later, and radioactivity retained in formalin-fixed liver tissue was determined as a measure of DNA synthesis at the time of administration of the labeled precursor. In control animals without phenobarbital treatment, ¹³¹IDU incorporation into liver began to increase between 14 and 16 hr after partial hepatectomy. Phenobarbital treatment (0.1 mg per g of body weight) resulted in a delay of the increase in ¹³¹IDU incorporation by several hours. This delay was observed in animals subjected to partial hepatectomy in the morning as well as in those operated on in the evening. After phenobarbital treatment, the increase of mitotic activity was either delayed or occurred more slowly. the results are compared with the reported effects of partial hepatectomy on the time course of microsomal enzyme induction by phenobarbital.     

The effects of interleukin 4 on the cell cycle of a human B-cell lymphoma line

Abstract. Exposure of Farage, a human B-cell lymphoma line, to IL-4 for 3–11 days led to inhibition of tritiated thymidine ([³H]dT) uptake by the cells. Study of the incorporation of 5-bromodeoxyuridine by Farage cells showed that IL-4 reduced significantly the number of cells in the S phase of the cell cycle and increased the proportion of cells in the G₁ phase. Limiting dilution analysis of proliferation demonstrated that IL-4 decreased the frequency of clone-forming cells by 40%. IL-4 did not reduce the viability of Farage cells. On the contrary, IL-4 diminished the spontaneous death of Farage cells in culture, as determined by pulse chase analysis of cells which were labelled with [³H]dT. Moreover, the pre-treatment of Farage cells with IL-4 prevented their death induced by exposure to a high dose of staurosporine. IL-4 abrogated the staurosporine-induced arrest of cells in the G₂+ M phase and replaced it by accumulation of cells in the G₁ phase. IL-4 protected Farage cells from the radioactive suicide caused by the uptake of [³H]dT by dividing cells. The cytokine failed to prevent the damage to Farage cells exerted by mitomycin C, which affected cellular DNA regardless of the phase of the cell cycle. The data obtained showed that IL-4 inhibited the division of B cells by arresting their progression through the early stages of the cell cycle. This inhibition of the cell efflux from G₁ phase plays an important role in the protection against cell death during further stages of the cell cycle.     

Bacterial production measured by leucine and thymidine incorporation rates in French lakes

1. Production of heterotrophic bacterioplankton was estimated monthly by the tritiated thymidine and leucine incorporation methods during the draining and filling of the mesotrophic Lake Pareloup (over a 2.5-years sampling program).
2. Rates of ³H-leucine (leu) and ³H-thymidine (thy _DNA) incorporation generally paralleled each other but the ratio of leu/thy _DNA incorporation rates was higher for the draining period (34.5 mean) than during and after filling (11.5 mean).
3. After draining, the highest ratios were observed during periods of low temperature and low bacterial specific activity, while DNA labeling by ³H-thymidine was reduced. However, bacterial production estimates obtained by ³H-leucine (BPL) and ³H-thymidine (BPT) incorporation methods were generally well correlated and the average BPL/BPT ratio was equal to 0.78.
4. In addition, both methods were applied during a diel cycle in three lakes of different trophic status. An increase of leu/thy _DNA incorporation rates was noted from the oligotrophic to the eutrophic system. In the absence of Cyanobacteria, BPL and BPT values were quite concordant on average.
5. In situations of unbalanced growth, BPL and BPT values can diverge but when considered over a sufficient period of time they were found to be in agreement.     

Proliferation of cells undergoing chondrogenesis in vitro

Abstract. Continuous exposure of chicken embryo limb bud mesenchyme cells undergoing chondrogenesis in vitro to [³H] thymidine ([³H]TdR) revealed that more than 90% of the cells synthesized DNA at least once during 120 h of culture. When cells were exposed to [³H]TdR for 24 h beginning at various times throughout the culture period, the percentage of cells which incorporated [³H]TdR during each period was approximately 92%. However, when the period for incorporation of radioisotope was limited to two hours, the number of cells which incorporated [³H]TdR was found to decline during chondrogenesis in vitro. This decline was coincident with the appearance of extracellular matrix material and occurred in those cells which had, and had not, expressed the cartilage phenotype.
We conclude from these studies that (1) practically all of the cells continue to proliferate while chondrogenesis is occurring in vitro, (2) there is an increase in the length of the cell cycle during chondrogenesis in vitro, and (3) withdrawal from the cell cycle is not required for differentiation of mesenchyme into cartilage.     

RNA Synthesis and Processing during Cytodifferentiation in Fetal Rat Pancreas

In fetal rat pancreas cytodifferentiation occurs between day 14 and day 20 of gestation and is accompanied by an exponential increase in the cellular accumulation of tissue specific proteins and an elaboration of the cellular organelles associated with their synthesis and secretion. Evaluation of RNA synthesis by [³H] uridine incorporation into trichloroacetic acid precipitable material showed that during this period the apparent rate of RNA synthesis increased 7.5 fold from 2 × 10³ dpm/μg DNA/h on day 15 to 1.5 × 10⁴ dpm/μg DNA/h on day 19; [³H] leucine uptake showed that the rate of protein synthesis increased about the same extent with the major difference being that the maximum rate of protein synthesis occurred on day 19, one day after the maximum rate of RNA synthesis. The soluble pyrimidine nucleotide pools decreased from 122 pmol/μg DNA on day 14 to 15 pmol/μg DNA on day 16 followed by an increase to 104 pmol/μg DNA on day 19; the purine nucleotide pools decreased from 367 pmol/μg DNA on day 14 to 286 pmol/μg DNA on day 16 and then increased to 635 pmol/μg DNA on day 19. These values roughly paralleled the transitions observed in the rates of RNA and protein synthesis. Agarose-acrylamide slab gel electrophoresis showed an increase in RNA synthesis and an increase in ribosomal RNA synthesis and processing with cytodifferentiation.     

Protein Synthetic Activities during Spermiogenesis in the Sea Urchin: An high Resolution Autoradiographic Study of 3H-Leucine Incorporation

Protein synthetic activity has been studied during spermiogenesis of Paracentrotus lividus by high-resolution autoradiography using ³H-leucine as a labeled precursor. Under the adopted experimental conditions ³H-leucine is incorporated during the whole spermiogenesis period. The early spermatid is the most active stage and it shows labeling over the nucleus, the cytosol and the mitochondria. Nuclear ³H-leucine incorporation progressively decreases as spermiogenesis proceeds. Cytosol labeling shows similar values at early and intermediate spermatid and it undergoes a considerable decreases at late spermatid. Mitochondrial grain density increases from early to intermediate spermatid and it remains almost constant at late spermatid.
Our results are compared with the data reported for other animal groups and possible functions of the observed protein synthesis are discussed.     

Pyrimidine deoxyribonucleotide metabolism during maturation and germination of white spruce (Picea glauca) somatic embryos: metabolic fate of C-labelled cytidine, deoxycytidine and thymidine

Pyrimidine deoxyribonucleotide metabolism was investigated during maturation and germination of white spruce somatic embryos by following the metabolic fate of [2‐¹⁴C]cytidine, [2‐¹⁴C]deoxycytidine and [2‐¹⁴C]thymidine. The de-novo pathway of deoxyribonucleotides was estimated indirectly, by the ability of the tissue to incorporate cytidine into DNA after conversion to dCTP. The salvage pathway was estimated by the utilization of labelled cytidine, deoxycytidine and thymidine for synthesis of deoxyribonucleotides and nucleic acids. Utilization of cytidine for DNA synthesis, via the de novo pathway, was always lower than that observed for RNA throughout the course of the experiment. Incorporation of cytidine into RNA was found to occur either directly, after conversion to CTP, mediated by the enzymes cytidine kinase, nucleoside monophosphate kinase and nucleoside diphosphate kinase, or indirectly, after conversion to UTP via uridine and UMP. Active incorporation of uridine into RNA of white spruce-cultured cells was demonstrated previously. Salvage of deoxycytidine and thymidine was operative in maturing and germinating white spruce somatic embryos, as label from both compounds was recovered in nucleotides and DNA. However, the utilization of these precursors by the cells was different. Salvage of deoxycytidine was always higher than that observed for thymidine, which was extensively catabolized to CO₂ at all stages of embryo development.     

AUTORADIOGRAPHIC DETECTION OF DNA POLYMERASE CONTAINING NUCLEI IN SARCOMA 180 ASCITES CELLS

A technique has been developed allowing the autoradiographic detection of incorporation of ³H-thymidine-5'-triphosphate (³H-TTP) into nuclear DNA of smears of Sarcoma-180 (S-180) mouse ascites tumors under the direction of the cell's own nuclear DNA polymerase. Dried smears are dipped into an agar solution, which strips cytoplasm from the nuclei, and are then air dried and incubated with a buffered mixture containing four nucleotide triphosphates (one labeled), Mg⁺⁺, and Ficoll, with the cell's own DNA acting as primer. The incorporation of ³H-TTP into the nuclei, like the cell free DNA polymerase assay, is largely dependent on the presence of all four nucleotide triphosphates and Mg⁺⁺ and produces a product which is DNase sensitive and RNase resistant.
DNA polymerase activity, as studied in a cell free assay, decreases with tumor age. This correlates well with a decreasing ³H-TTP labeling index in autoradiographs of aging tumors. The ³H-TTP labeling index has also been shown to exceed but parallel the in vivo ³H-thymidine (³H-TdR) pulse labeling index for all tumor ages examined.
In at least some cell systems DNA polymerase seems characteristic of cells in cycle. The autoradiographic detection of nuclei containing the enzyme offers a new tool for the study of tumor cytokinetics.     

Indole-3-acetic acid production by bacteroids from soybean root nodules

Purine nucleotide and RNA synthesis have been investigated at the different growth stages of carrot ( Daucus carota L.) cells grown in suspension cultures. At the early growth stages an increase in the content of RNA was observed, although at later stages RNA was degraded. The highest rates of incorporation of [¹⁴C]-labelled adenosine into ATP and GTP were observed at the late growth sttages. This indicated that purine slavage was more importnt at the late growth stages, while de novo synthesis was dominant during the initial growth stages. This pattern was also reflected by increased levels, in the cell dividison phase, of theenzymes glycinamide ribonucleotide synthetase (EC 6.3.1.3.) and phosphoribosylpyrophosphate amido-transferase (EC 2.4.2.14) involved in de novo purine synthesis. The activities of the purine salvage enzymes varied little during growth. Cells in the stationary phase, that were starved for sucrose and phosphate, showed a dramatic increase in cellular metabolism, as judged from a rapid uptake and incorporation of [³²P]-labelled phosphate into nucleotides and RNA, when incubated in fresh medium.