Autoradiographic Studies of H3-Thymidine Uptake in Entamoeba histolytica in CLG Medium*

SYNOPSIS. Entamoeba histolytica grown with H³-thymidine in CLG medium took up tritium into DNase-sensitive material in the nucleus and cytoplasm. The distribution of nuclear activity indicated that the entire nucleus, including the peripheral chromatin, may possess DNA; previous investigators reported DNA only in the endosome. The penicillin-inhibited bacterial associate (Bacteroides sp.) used in the CLG medium incorporated tritium from H³-thymidine into autoradiographically detectable DNase-sensitive material. Autoradiographs of amebae fed bacteria prelabeled with H³-thymidine also revealed some nuclear and cytoplasmic label. Thus, the amount of cytoplasmic label due to ingested, prelabeled bacterial DNA and/or actual biosynthesis of cytoplasmic DNA by the amebae themselves, is not known. Also, at least some of the nuclear DNA of amebae is synthesized from ingested bacteria, or, more likely, from bacterial degradation products.     

Incorporation of Tritium from Thymidine-methyl-H3 into DNA,RNA, Lipids and Protein in Logarithmic and Stationary Phase Tetrahymena pyriformis,Strain W*

SYNOPSIS. Numerous reports may be found in the literature on cytoplasmic and non-DNA utilization of tritium from H³-thymidine. Such reports underscore the need to clarify the metabolic fate of H³-thymidine. This investigation outlines the fate of thymidinemethyl-H³ (TMH³) in logarithmic phase and stationary phase Tetrahymena pyriformis, strain W. Isotope identification by liquid scintillation spectrometry in chemically derived fractions of log phase cultures grown thruout the initial 48 hours of population growth with TMH³ revealed the majority of the radioactivity (90% of intracellular recovery) to be in the DNA fraction. The remainder of the intracellular label was recovered in the acid soluble fraction, lipid fraction, and a small amount in the RNA and cell residue. On chromatographs, tritium appeared only in the thymine moiety of the nucleic acid derivatives. Hence in dividing cells, thymidine-methyl-H³ is “essentially” specific for DNA at the dosage used although some incorporation into other compounds was detected. Fractionation of the lipid extract from the above experiment on a florisil column localized most of the label to the triglyceride and phospholipid fractions with some recovery in the cholesterol-esters. Similar scintillation counting of the various fractions of early stationary phase cells incubated for the last 48 hours of culture with TMH³ revealed limited tritium distribution in all fractions.     

Effect of Mitomycin C on Thymidine-Methyl-H3 Utilization by Entamoeba histolytica Propagated in CLG Medium*

SYNOPSIS. Autoradiographic studies were done which tested the effect of a potent DNA inhibitor, mitomycin C (MC) on the utilization of tritium from exogenous thymidine-methyl-H³ (TMH³) in Entamoeba histolytica grown with Bacteroides sp. in CLG medium. Concentrations of MC (0.0002%) which inhibited growth of amebae by ca. 50%, caused an overall depression of tritium utilization by both associate cell and amebae. However, no reduction in percent cells with nuclear activity was apparent. The effect of MC on utilization of tritium in amebae propagated with Bacteroides which were prelabeled with TMH³ was also studied. The extent of labeling and percent amebae with cytoplasmic label was not appreciably depressed by MC. MC did, however, cause a depression of the percent amebae with nuclear label. This would indicate that the utilization of bacterial DNA products for nuclear DNA (reported in a previous communication) is reduced in the presence of MC. These data on the effect of MC on use of exogenous TMH³ and prelabeled Bacteroides provide some evidence that at least some of the nuclear DNA of amebae can be synthesized from the exogenously supplied isotope. Amebae grown with exogenous TMH³ and resuspended in unlabeled medium for 24–28 hrs. with and without MC had a considerable reduction of the extent of label whether MC was present or not. This suggests that the primary effect of MC is not to degrade DNA.     

DNA SYNTHESIS IN THE OOPLASM OF DROSOPHILA MELANOGASTER

Tritiated thymidine was injected into 2-day-old Drosophila melanogaster females, and tissue sections were prepared from the ovary for radioautography with both the light and electron microscopes. Besides the expected incorporation of H³-thymidine into nuclei of nurse cells and follicle cells, there was a relatively high level of incorporation of label into ooplasmic DNA. The highest level of incorporation occurred at stage 12. At the same time, the 15 nurse cell nuclei also incorporate thymidine in spite of the fact that they are breaking down and degenerating. The label in the ooplasm is not removed by extraction with DNase (although this removes nuclear label) unless extraction is preceded by a treatment with protease. Electron microscopic radioautography revealed that 36% of the silver grains resulting from decay of H³-thymidine are found over mitochondria, with a further 28% being located within 0.25 µ of these organelles. The remaining 36% of the silver grains was not found to be associated with any organelles, and it probably represents synthesis in the cytoplasm by the "storage DNA" characteristic of many eggs. It is suggested that one mechanism acting throughout the egg chamber is responsible for the synchronous synthesis of DNA in the degenerating nurse cells, in the mitochondria of the egg, and in the "storage DNA" of the ooplasm.     

THE INCORPORATION OF TRITIUM FROM THYMIDINE INTO PROTEINS OF THE MOUSE

Tritium from methyl-H³-thymidine was found to be incorporated into proteins in mice. This incorporation in the mouse as a whole represented between 1 and 10% of the injected tritium. Tritiated water was not an intermediate. Transmethylation reactions are proposed as a means whereby certain amino acids might have acquired the tritium from thymidine at some stage of its catabolism. The initial (2 hr) ratios of DNA to protein tritium activities per milligram of wet tissue ranged from 5 in two tissues of low DNA synthetic activity (pancreas, liver) to 35 to 40 in two tissues of high DNA synthetic activity (spleen, small intestine). Labeled nuclear protein was coincident with labeled DNA in nuclei, where it constituted less than 2.5% of the total tritium. The significance of the findings is discussed.     

ACTIVITY OF MARGINAL AND PLATE MERISTEMS DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

The mitotic and biosynthetic activities of the marginal and plate meristems were studied during the entire course of leaf development of Xanthium pennsylvanicum. In contrast to statements in the literature, marginal meristem activity is long in duration, as assayed by the mitotic counts and H³-thymidine incorporation. This me istem is active 23 days. The plate meristem is active for an additional 3 days after cessation of cell division in the marginal meristem, but the total duration of its mitotic activity is also approximately 23 days. Numerous periclinal cell divisions of the plate meristem form additional cell layers and contribute to the growth of the lamina in thickness. Incorporation of H³-thymidine increased during the course of leaf development. Cells between plastochronic ages 0 and 2.0 incorporated more of the radioisotopic precursor than those of younger leaf primordia. The uptake and incorporation of H³-thymidine into nuclear DNA was more sluggish during the early stages of development than in the more expanded leaves. No DNA synthesis was demonstrated after cessation of cell division in the leaf lamina. Metabolic or endomitotic DNA synthesis after leaf plastochron index (LPI) 3.0 seems improbable. No significant differences in the incorporation of H³-thymidine could be demonstrated between the marginal and plate meristems. This would indicate no distinct biosynthetic differences between the two meristems. The definitions of the marginal and plate meristems of Xanthium leaves were formulated in view of the above findings.     

Thymidine kinase-deficient mutants of Physarum polycephalum : Biochemical characterization

Thymidine kinase (TK) and deoxycytidine kinase (dCK) activity levels, [³H]thymidine (TdR) and 5-bromo-2′-deoxyuridine (BUdR) incorporation and 5-fluoro-2′-deoxyuridine (FUdR) sensitivity have been compared in TK-deficient (TU63 and TU84) and normal (TU291 and M₃b) strains of the myxomycete, Physarum polycephalum. The mutants had about 2% of the TK and 100% of the dCK activity of wild-type (wt) strains. They incorporated some TdR into both nuclear (nDNA) and mitochondrial DNA (mtDNA) but incorporated too little BUdR to give a buoyant density shift in nuclear DNA. They grew in the presence of levels of FUdR which completely blocked DNA synthesis in TU291. The FUdR sensitivity of strain M₃b could be increased by supplementing growth medium with folic acid.     

Effect of nickel(II) on DNA-protein binding,thymidine incorporation,and sedimentation pattern of chromatin fractions from intact mammalian cells

Nuclear uptake and chromatin binding of nickel(II) was investigated in Chinese hamster ovary (CHO) cells. The cytoplasmic:nuclear ratio of nickel immediately following treatment was 5:1, but by 24 and 48 hours this ratio decreased to 4:l and 2:1, respectively, indicating that nickel is retained longer in the nucleus than cytoplasmic nickel. Chromatin was fractionated by sonication and centrifugation into fast-sedimenting, magnesium-insoluble, or magnesiumsoluble components. The magnesium-insoluble portion bound more nickel ions and retained the metal longer than either the magnesium-soluble or the fastsedimenting fractions. Treatment of cells with nickel chloride (NiCl₂) decreased the amount of DNA in the magnesium-insoluble fraction but increased the amount of DNA in the fast- sedimenting chromatin fraction. The magnesium-insoluble fraction isolated from nickel-treated cells contained approximately ten times more [35-S]-methionine–labeled protein per milligram DNA compared with untreated cells. The magnesium-soluble and the fast-sedimenting fractions isolated from the nickel-treated cells did not exhibit a similar increase in [35-S]-methionine–labeled protein per milligram of DNA. Nickel treatment suppressed [14-C]-thymidine incorporation into total DNA by 30% compared with untreated cells. However, the magnesium-insoluble chromatin fraction from nickel-treated cells had a tenfold to 20-fold increase in thymidine incorporation, while the other chromatin fractions did not exhibit an increase in thymidine incorporation. These findings indicate that nickel induced widespread alterations in chromatin conformation and preferentially interacted with an Mg-insoluble component of chromatin.     

Asynchronous Duplication of Chromosomes in Cultured Cells of Chinese Hamster

Chromosome duplication (DNA synthesis) was studied in cultured cells of Chinese hamsters by means of autoradiography following thymidine-H³ incorporation. The technique used was to expose an asynchronously dividing population of rapidly growing cells for a 10 minute interval to a medium with thymidine-H³. Cells were then transferred to a medium with excess unlabeled thymidine. The population was sampled at intervals thereafter and studies made of the frequency of labeled interphases and division figures, and the patterns of labeling of specific chromosomes. The average generation time during these experiments was about 14 hours. DNA synthesis occurred during an interval of about 6 hours and stopped 2 to 3 hours before metaphase. After metaphase the chromosomes usually begin duplication again within 5 to 6 hours. Grain counting, to estimate the amount of tritium incorporated after a short contact with thymidine-H³ and at intervals after transfer to a medium with excess unlabeled thymidine, indicated that the intracellular pool of labeled precursors was diluted within less than a minute so that further labeling would not be detected. The chromosomes labeled during the contact period retained their precise pattern of labeling through another duplication cycle and no turnover of DNA or loss of tritium was detectable. Five or 6 chromosomes of the complement have segments typically late in duplication. Two of these are the X and Y chromosomes. The long arm of the X chromosome and the whole Y chromosome are duplicated in the last half of the interval of DNA synthesis. The short arm of the X chromosome in a male strain is duplicated in the first half of the interval. In another strain (female), one X chromosome had the same timing, but the other one was all duplicated in the last half of the period of DNA synthesis. The DNA in the short arms of 2 medium sized chromosomes, as well as most of the DNA in 1 or 2 of the smallest chromosomes of the complement was replicated late. The study has led to the hypothesis that various chromosomes or parts of chromosomes have a genetically controlled sequence in duplication which may have some functional significance.     

INCORPORATION OF H3-THYMIDINE INTO SHOOT AND ROOT APICES OF CERATOPTERIS THALICTROIDES

Gifford , Ernest M., Jr . (U. California, Davis.) Incorporation of H³-thymidine into shoot and root apices of Ceratopteris thalictroides. Amer. Jour. Bot 47(10): 834–837. Illus. 1960.—The localization of tritiated thymidine in apical meristems of Ceratopteris thalictroides by the autoradiographic method is described. Intact, floating plants of the fern were placed in 1/2 strength Hoagland's inorganic nutrient solution containing H³-thymidine (10 μc/ml.) for 3 days. The material was killed, dehydrated and embedded in paraffin. Autoradiographic stripping film (AR 10 Kodak) was applied to serial sections. After an appropriate exposure period, the film was developed and the sections with the superimposed film were stained lightly with Harris' hematoxylin. The autoradiographs revealed the presence of the H³-thymidine in nuclei of the large, individualized apical cells of shoots and roots which is proof of DNA synthesis. In no instances were these nuclei unlabeled. If endomitotic reduplication is excluded the results of these studies lend support to the concept that apical cells actually do divide and perhaps at a higher rate than envisioned by other workers. Considerable cytoplasmic labeling occurred and its significance to general problems of DNA synthesis is discussed.     

Macromolecule synthesis leading to cell division in Tetrahymena pyriformis after replacement of required amino acids

Summary Tetrahymena pyriformis W were brought to a nonmultiplying state by removal of required amino acids from their growth medium. After amino-acid replacement, the incorporation rates of H³-uridine, H³-thymidine and H³-leucine were measured by the autoradiographic method. Following amino-acid replacement, the first response was detected in RNA synthesis, then protein synthesis, then DNA synthesis and, lastly, in cell division. Amino-acid deprived cells showed a 23% net increase in DNA content, a result supporting the view of others that protein synthesis is not necessary for the initiation of DNA synthesis but is necessary for the maintainance of DNA synthesis.     

DNA synthesis in the giant salivary chromosomes of Drosophila virilis prior to pupation

Both two-wavelength microspectrophotometry of Feulgen-stained whole nuclei and autoradiography of H³-thymidine incorporation by giant salivary chromosomes in Drosophila virilis demonstrate a net decrease in the relative rate of salivary DNA synthesis during the late third instar and prepupal stages of development. Amounts of DNA-Feulgen per nucleus were distributed into several classes, the means of which closely approximated values projected as geometric multiples of the basic somatic DNA level estimated from hemocyte nuclei of the same larvae. Comparison of DNA polytene class frequencies showed no statistical difference between male larvae of different development stages, although female prepupae showed a greater frequency of nuclei in higher polytene classes when compared to male prepupae of the same age. Comparison of chromosomal H³-thymidine incorporation with previously described H³-histidine incorporation suggests that the amino acid labeling, which reaches a maximum during the prepupal period, has a physiological significance distinct from chromosomal endoreplication.     

Relationship between Tracheary Element Differentiation and DNA Synthesis in Single Cells Isolated from the Mesophyll of Zinnia elegans--Analysis by Inhibitors of DNA Synthesis

Effects of inhibitors of DNA synthesis on tracheary element(TE) differentiation were investigated in a culture of singlecells isolated from the mesophyll of Zinnia elegans L. cv. Canarybird. In this system, neither mitosis nor replication of thewhole genome during the S phase in the cell cycle is a prerequisitefor TE differentiation [Fukuda and Komamine (1980) Plant Physiol.65: 61, unpublished data]. Fluorouracil (FU), fluorodeoxyuridine(FUdR), mitomycin G (MC), arabinosyl cytosine (ara-C) and aphidicolin,inhibitors of DNA synthesis, prevented the incorporation of[³H]-thymidine into nucleic acid, cell division and cytodifferentiationto TE. However, neither FUdR nor aphidicolin prevented the incorporationof [14C]-leucine into protein. Thymidine reversed the inhibitoryeffect of FUdR when given simultaneously with FUdR. These resultsshow that the inhibitors of DNA synthesis prevent TE differentiationvia blockage of the synthesis of some DNA, although replicationof the whole genome during the S phase is not a prerequisitefor cytodifferentiation. The role of DNA synthesis in TE differentiationis discussed. (Received October 13, 1980; Accepted November 17, 1980)     

Is there "Metabolic" DNA in the Mouse Seminal Vesicle?

This study was designed to answer the question: Is H³-thymidine uptake by nuclei of the mouse seminal vesicle evidence for DNA synthesis and mitosis, or does it signify some "metabolic" function of DNA unrelated to chromosome duplication? Mice were given an intraperitoneal injection of H³-thymidine. Six hours later Feulgen squashes of the seminal vesicle epithelium were made and covered with autoradiographic stripping film. The silver grains above labeled nuclei were counted, and the Feulgen dye contents of these same nuclei were determined photometrically after removal of the grains from the emulsion. Unlabeled nuclei were also measured. The dye contents of non-radioactive nuclei form a unimodal distribution, indicating that polyploidy is absent from this tissue. The radioactive nuclei fall into two groups. In the first, the average dye content is the same as that of the cold nuclei (2C). In the second, the values range from 2C to 4C. In the 2C to 4C group the grain count is proportional to the dye content, showing that incorporation is correlated with synthesis. The radioactive 2C nuclei arose by mitosis during the course of the experiment. This is shown by the following facts: (1) They frequently occur in pairs. (2) They average smaller than unlabeled 2C nuclei. (3) Their average grain count is approximately half that of the 4C nuclei. (4) Labeled division figures are found. (5) A mitotic rate estimated from the number of labeled 2C nuclei accords reasonably well with one based on the number of observed mitoses. Since the incorporation of thymidine accompanies DNA synthesis and precedes mitosis, there is no reason to postulate a special "metabolic" DNA in this tissue.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.     

THYMIDINE DEGRADATION PRODUCTS IN PLANT TISSUES LABELED WITH TRITIATED THYMIDINE

A study of the metabolic pathways of H³-thymidine utilization in buds of Lilium longiflorum and root tips of Vicia faba was undertaken in order to obtain information that might explain the binding of H³ from H³-thymidine in the cytoplasm of these plants. H³-thymidine was administered for various periods of time, the tissues were fixed and processed in the manner routinely used in preparation for sectioning and autoradiography, and the radioactivity removed in this way from the tissues was determined. It was found that the ethanol/acetic acid fixative contained the major portion of the radioactivity. Analysis of this extract by paper chromatography showed that the radioactivity was distributed among various degradation products of thymidine, principally β-ureidoisobutyric acid and β-aminoisobutyric acid. Time course experiments with Vicia showed that these degradation products rapidly appeared in the tissue during incubation with H³-thymidine, while H³-thymine appeared in the incubation medium. Preliminary studies indicated that Vicia root tips incubated with H³-dihydrothymine for 24 hours would bind a small amount of H³ non-specifically in the cells. It seems unlikely that utilization of degradation products of H³-thymidine is sufficient to explain labeling which is concentrated in the cytoplasm.     

Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

Incorporation of 5-bromodeoxyuridine into DNA of wild type Escherichia coli and its use for the enrichment of auxotrophic mutants

Summary The effect of deoxyadenosine (AdR) and 5-fluorodeoxyuridine (FUdR) on the incorporation of 5-bromodeoxyuridine (BUdR) into DNA of thymine-non requiring cells of Escherichia coli was studied. This incorporation renders the cells sensitive to irradiation at near-UV light. It was found that the combination of AdR and FUdR increases incorporation of BUdR and sensitivity to irradiation.An enrichment of auxotrophs by a factor of 10⁵ was obtained from a mixed population grown in minimal medium containing BUdR, AdR and FUdR and irradiated subsequently.Part of an M.Sc. Thesis in Microbiology submitted by A. Rosner to the Tel-Aviv University.     

INCORPORATION OF TRITIATED THYMIDINE IN THE CELLS OF CAENORHABDITIS BRIGGSAE (NEMATODA) REARED IN AXENIC CULTURE

In the rhabditid nematode Caenorhabditis briggsae the incorporation of thymidine-H³ has been studied by autoradiography after Feulgen staining, with animals maintained under axenic conditions in a medium of only partly defined composition. Labeling has been followed in adults left in the presence of thymidine-H³ for periods of from ½ to 24 hours, as well as in adults reared from larvae in the presence of the tritiated nucleoside. A massive incorporation is found in the nuclei of the gonads and intestine; also a less intense particulate cytoplasmic incorporation is clear in certain cells, especially those of the intestine. In general, all labeling has proved to be sensitive to DNase, but resistant to RNase. The label's stability has been tested by the transfer of adults into a medium containing "cold" thymidine. They remain there for up to 48 hours. A transfer for 24 hours results in a considerable decrease in the intensity of nuclear and cytoplasmic labeling; a stay of 48 hours leads to its complete disappearance from non-dividing (intestinal) as well as dividing (gonadal) nuclei. A phenomenon of DNA turnover is envisaged and discussed as a possible physiological attribute of C. briggsae.     

Isolation of a single-stranded extrachromosomal DNA from germinating wheat embryos

Newly-synthesized cytoplasmic non-mitochondrial DNA was isolated from wheat embryos which had been germinated in the presence of [¹⁴C]-thymidine for a time period not long enough to trigger the first post-dormant round of the nuclear DNA replication. This extrachromosomal DNA fraction consisted of linear single-stranded polydeoxyribonucleotide chains, corresponding in size to approximately 1.1×10⁶ daltons, and amounted to about 0.5% of the total cellular DNA content. It is suggested that the appearance of the newly-synthesized polydeoxyribonucleotide chains in the cytoplasm may be a physiological signal for the initiation of the nuclear DNA replication in germinating wheat embryo cells.