Further Verification of the Isotope Dilution Approach for Estimating the Degree of Participation of [3H]thymidine in DNA Synthesis in Studies of Aquatic Bacterial Production

The optimal concentration of [³H]thymidine (i.e., the maximal degree of participation in DNA synthesis) as determined by adding increasing amounts of labeled thymidine at the same specific activity was similar to the concentration of thymidine inhibiting the de novo pathway as determined by isotope dilution plots. These experiments provide further verification of the isotope dilution approach for determining the degree of participation of [³H]thymidine in DNA synthesis.     

Direction of DNA replication in mammalian cells

We have re-examined the direction of DNA synthesis in mammalian cells by means of pulse-labeling with [³H]thymidine and DNA autoradiography. Our results show that, whether or not the cells are treated with 5-fluoro-deoxyuridine, and whether they are labeled first with high specific activity [³H]thymidine and then with low, or vice versa, most (? 90%) of the unambiguous autoradiographic patterns can be explained by bidirectional replication but not by unidirectional replication.We also find that in autoradiographic experiments using two different specific activities of [³H]thymidine, obvious differences in grain density are obtained only when the difference in specific activity is threefold or more. Thus, the apparently contradictory findings of Lark et al. (1971) can be explained by the low difference in specific activity used by those authors.     

DNA REPLICATION: DIRECTION AND RATE OF CHAIN GROWTH IN MAMMALIAN CELLS

Using pulse labeling techniques with [³H]thymidine or [³H]cytidine, combined with DNA fiber autoradiography, we have investigated the direction and rate of DNA chain growth in mammalian cells. In general, chain elongation proceeds bidirectionally from the common origin of pairs of adjacent replication sections. This type of replication is noted whether the DNA is labeled first with [³H]thymidine of high specific activity, followed by [³H]thymidine of low specific activity or the sequence is reversed. Approximately one-fifth of the growing points have unique origins and in these replication units, chain growth proceeds in one direction only. Fluorodeoxyuridine and hydroxyurea both inhibit DNA chain propagation. Fluorodeoxyuridine exerts its effect on chain growth within 15–23 min, while the effect of hydroxyurea is evident within 15 min under conditions where the endogenous thymidine pool has been depleted by prior treatment with fluorodeoxyuridine. Puromycin has no effect on chain growth until 60 min after addition of the compound, even though thymidine incorporation is more than 50% reduced within 15 min. After 2 h of treatment with puromycin, the rate of chain growth is reduced by 50%, whereas thymidine incorporation is reduced by 75%. Cycloheximide reduces the rates of DNA chain growth and thymidine incorporation 50% within 15 min, and, on prolonged treatment, the decrease in rate of chain growth generally parallels the reduction in thymidine incorporation.     

Activity of an Attached and Free-Living Vibrio sp. as Measured by Thymidine Incorporation, p-Iodonitrotetrazolium Reduction, and ATP/DNA Ratios

Three independent techniques, [³H]thymidine incorporation, the reduction rate of p-iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA, were adapted to measure the activity of attached and unattached estuarine bacteria. In experiments employing the estuarine isolate Vibrio proteolytica, nutrient concentrations were manipulated by varying the concentration of peptone-yeast extract. In the presence of exogenous nutrients, the activity of free-living cells was greater than that of attached cells as measured by [³H]thymidine incorporation and ATP/DNA ratios. In the absence of peptone-yeast extract, however, the activity of attached cells surpassed that of free-living cells as determined by [³H]thymidine incorporation and INT formazan normalized to DNA. Of the three techniques, [³H]thymidine incorporation was deemed most sensitive for detecting changes in activity resulting from slight differences in nutrient concentration. By this technique, attached cells were much less sensitive to changing nutrient concentrations than were free-living cells. Below a threshold concentration, attached cell activity remained constant, while the activity of unattached cells decreased as a function of decreasing nutrient concentration. The results suggest that loss of cell surface area available for substrate uptake due to attachment may be an important factor in determining the relative activities of attached and free-living cells.     

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [³H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [³H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [³H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [³H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [¹⁴C]leucine or [¹⁴C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [³H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.     

Identification and characterization of a soluble suppressor factor(s) in the serum of AKR mice bearing lymphocytic leukemia

Leukemia in AKR mice was found to be associated with the presence of a serum factor(s) termed AKR leukemic suppressor factor (AKR-LSF). Suppression was quantitated by measuring the inhibition of PHA-stimulated [³H]thymidine incorporation by normal AKR spleen cells at various dilutions of leukemic mouse serum (LMS). AKR-LSF activity was expressed as units per milliliter, which is the reciprocal of the LMS dilution that inhibited [³H]thymidine uptake by 50% with respect to fetal calf serum control cultures. The amount of activity in the serum directly correlated to the rate of tumor cell growth. Mice receiving 10⁷ BW5147 transplanted leukemia cells had 130 ± 12 units of AKR-LSF activity/ml of serum compared to 40 ± 8 units/ ml for mice with spontaneous leukemia. Normal mouse serum contained 33 ± 11 units/ml. The leukemic serum exhibited no strain specificity in either phytohemagglutinin or lipopolysaccharide assays, but was found to be twofold more inhibitory against mouse spleen cells than that against rat spleen cells. Human lymphocyte blastogenesis was not inhibited by the leukemic serum. LMS did not inhibit the growth of L929 fibroblasts or murine tumor cells in vitro. Further work is necessary to determine what role the suppressor factor may play in the regulation of antitumor cell immunity.     

DNA Synthesis and Tritiated Thymidine Incorporation by Heterotrophic Freshwater Bacteria in Continuous Culture

Continuous cultivation of heterotrophic freshwater bacteria was used to assess the relationship between DNA synthesis and tritiated thymidine incorporation. The bacteria were grown on a yeast extract medium with generation times of 0.25 to 3.7 days. In six different continuous cultures, each inoculated with a grazer-free mixed bacterial sample from Lake Vechten (The Netherlands), tritiated thymidine incorporation into a cold trichloroacetic acid precipitate and bacterial cell production were measured simultaneously. Empirical conversion factors were determined by division of both parameters. They ranged from 0.25 × 10¹⁸ to 1.31 × 10¹⁸ cells mol of tritiated thymidine^-1 (mean, 0.60 × 10¹⁸ cells mol of tritiated thymidine^-1). In addition, DNA concentrations were measured by fluorometry with Hoechst 33258. The validity of this technique was confirmed. Down to a generation time of 0.67 day, bacterial DNA content showed little variation, with values of 3.8 to 4.9 fg of DNA cell^-1. Theoretical conversion factors, which can be derived from DNA content under several assumptions, were between 0.26 × 10¹⁸ and 0.34 × 10¹⁸ cells mol of thymidine^-1 (mean, 0.30 × 10¹⁸ cells mol of thymidine^-1). Isotope dilution was considered the main factor in the observed discrepancy between the conversion factors. In all experiments, a tritiated thymidine concentration of 20 nM was used. Control experiments indicated maximum incorporation at this concentration. It was therefore concluded that the observed difference resulted from intracellular isotope dilution which cannot be detected by current techniques for isotope dilution analysis.     

Studies on Deoxyribonucleotide Synthesis in Vicia faba

The incorporation of uniformly labelled [¹⁴C] cytidine into the nucleic acids was studied in root tips of Vicia faba. Cytidine was found to be incorporated into RNA and DNA and the specific activities of the individual mononucleotides were deter- mined. The pyrimidine nucleotides were degrade and the ratio between the specific activity of the pentose and the specific activity of the base was determined for each nucleotide. CMP of RNA and deoxy CMP of DNA bad almost the same pentose: base ratios as The cytidine added to the incubation medium. It was concluded that the administered cytidine or a derivative of it was reduced to the corresponding deoxycytidine compound without breakage of the bond between pentose and base. [¹⁴C)-cytidine was transformed to UMP of RNA with some loss of radioactivity from the pentose and had almost the same pentose: base ratio as deoxy TMP of DNA. This indicates that the formation of thymidine phosphates involved The reduction of a uridine compound. Furthermore the incorporation of ¹⁴C-labelled thymidine, deoxyadenosine and deoxyguanosine into DNA was studied. Deoxyguanosine was found to be incorporated only to a slight extent. This finding has been discussed in relation to previous results.     

Effect of antiserum on leukemia 15178y cells: [3H]thymidine incorporation does not reflect dna synthesis

• 1.1. The total amount of DNA present in L5178Y cells treated with antiserum in the absence of complement was decreased whereas the rate of incorporation of [³H]thymidine into DNA during a 1 hr pulse was increased as compared with those of controls.
• 2.2. This increase in specific activity of [³H]thymidine in the DNA of antiserum-treated cells appeared to be due, at least in part, to an increase in thymidine kinase activity.

     

Underestimation of DNA Synthesis by [3H]Thymidine Incorporation in Marine Bacteria

A direct comparison of [³H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [³H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [³H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [³H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [³H]thymidine incorporation and isotope dilution assays.     

Bacterial secondary production in freshwater measured by3H-thymidine incorporation method

Bacterial secondary production in lake water was measured by³H-thymidine incorporation into DNA. The application of the method to freshwater systems studied required (1) thymidine concentration > 10 nM (10–25 nM) evaluated from isotope dilution by varying the specific activity of labeled thymidine, (2) short incubation periods less than 1 hour, and (3) partial purification of the DNA fraction for measuring³H-thymidine incorporation. During 2 diel studies, bacterial productivity was compared to phytoplankton primary production and extracellular release of organic carbon. Diel changes in bacterial growth suggested substantial activity during the morning and evening. Possible mechanisms of control of bacterial growth, such as extracellular release of organic carbon, are discussed.     

Morphogenesis of albino rat lung: an autoradiographic analysis of the embryological origin of the type I and II pulmonary epithelial cells

A correlation of autoradiographic and histochemical data indicates that the type I and II pulmonary epithelial cells are endodermally-derived; and, that the interstitial pulmonary cells are mesodermally-derived. Tritiated thymidine (T-H³) was found to be an excellent cell marker for in vivo developmental studies of mammalian (rat) lung. At a dose of 3 μc per gm (specific activity, 15.6–16.9 c per mM) maternal body weight, T-H³ crosses the placenta in amounts sufficient to effect heavy labeling of dividing cells. A partial placental barrier to T-H³ was found in late stages of development. Following an injection of T-H³ on day 16 of gestation, a higher rate of endodermal cell division was reflected by higher labeling indices and a steeper slope of the endodermal dilution curve as opposed to the mesoderm. This differential in labeling was maintained through the third postnatal day. Neonatal labeling patterns of the definitive cell types (type I and II pulmonary epithelial cells, interstitial pulmonary cells) reflected those of their germ layer precursors. Histochemical analysis of the developing rat lung demonstrated large accumulations of cytoplasmic glycogen in areas of rapid cell division (endodermal cells). As the mitotic rate decreased and cellular differentiation progressed, glycogen decreased; postnatally it is not a feature of mature pulmonary cell types.     

The specific and endogenous mitotic inhibitor of lymphocytes (chalone)

Aqueous extracts of various lymphoid tissues, but not of non-lymphoid tissues, contain a species-non-specific but cell-specific inhibitor of the transformation and DNA synthesis of PHA-stimulated human lymphocytes which is apparently not cytotoxic and is reversible. This activity is found in similar molecular weight fractions from pure lymphocytes obtained in culture and hence appears to be endogenous to the lymphocyte itself. This specific and endogenous mitotic inhibitor does not appear to be a result of competitive lectin-binding, thymidine pool size dilution, phosphorylation, destruction of thymidine, or the direct immunosuppressive effects of thymidine upon the lymphocytes themselves. Rather, it appears to be a result of the effects of a protein contained in the crude ultrafiltrate from lymphoid tissues whose properties correspond to those originally described by Bullough & Laurence for a ‘chalone’. The chalone activity from thymus appears to be specific for T cells rather than B cells.     

Letter: On the source of "minor thymine" in DNA from a Novik off rat hepatoma cell line

To determine if a fraction of DNA-thymine might derive from deamination of DNA-5-methylcytosine in polymeric DNA, the occurrence of tritiated DNA-thymine residues was measured in DNA isolated from Novikoff rat hepatoma cells incubated with varying levels of unlabeled thymidine and with G-³H-labeled deoxycytidine, unlabeled deoxyguanosine, deoxyadenosine, adenosine, serine, glycine and amethopterin. At 0 and 10^?5 M-thymidine, significant incorporation of label was recovered as DNA-thymine. At higher levels of thymidine (5× 10^?5m to 10^?3m) label in DNA-thymine progressively decreased to levels that could be accounted for by deamination of DNA-thymine by the two different two-dimensional Chromatographic systems used to separate free DNA bases. “Minor thymine” in DNA of Novikoff hepatoma cells is thus generated by incomplete amethopterin blockade, insufficient dilution by exogenous thymidine and methodological deamination.     

Characteristics of spontaneous and induced thymidine and 5-iodo-2-deoxyuridine resistant clones of mouse lymphoma cells

Clones resistant to 5-iodo-2-deoxyuridine (IUdR) were isolated from P388 cells and cultured in the absence of selective medium. Thymidine kinase assays were performed on 8 clones which had arisen spontaneously and 19 isolated after exposure to X-rays or alkylating agents. All the clones tested showed significantly reduced thymidine kinase activity relative to wild-type cultures, but none showed zero levels. 14 of these clones were tested for thymidine (TdR) uptake and all showed a marked reduction in the rate of [³H]TdR incorporation into acid soluble fractions and into DNA. 7 IUdR-resistant (IUdR^r) clones were tested for revertibility as measured by growth of colonies in HAT medium. 5 of the 7 were found to revert at measurable rates either spontaneously or after a low dose of mutagen.Thymidine kinase activity was also measured in 8 thymidine resistant P388 clones (TdR^r). Initial rates of thymidine phosphorylation were not significantly altered in 5 of the 8 clones tested but significantly lower amounts of phosphorylated products were observed in 6 of the 8 clones. [³H]TdR uptake was reduced in 9 of 12 clones tested, and 2 of them showed no corresponding reduction in the thymidine kinase activity, suggesting the occurence of mutants with altered permeability for thymidine.IUdR resistant L5178Y clones could not be isolated. Thymidine resistant L5178Y clones were similar to TdR^r P388 clones, i.e. they showed changes in initial rates of thymidine kinase activity and reduced accumulation of phosphorylated products. Only one clone could be shown to be a membrane mutant. These results are discussed in relation to the genetic nature of the thymidine kinase locus in the two cell lines.     

Activity Measurements of Planktonic Microbial and Microfouling Communities in a Eutrophic Estuary

[³H]thymidine incorporation, the rate of reduction of iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA were adapted to measure the activity of attached and unattached microbial assemblages of Bayboro Harbor, Fla. Activity measurements by [³H]thymidine incorporation were made of cells attached to polystyrene culture dishes, in unfiltered water samples, and in the <1-μm-filtered fraction. In most cases, the activity of attached cells was greater than that of unattached cells either in unfiltered water samples or in the <1-μm fraction. The calculated thymidine incorporation rates for cells in the >1-μm fraction were higher than those for cells either in unfiltered water or in the <1-μm-filtered fraction. By the rate of reduction of INT to INT formazan normalized to DNA and by ATP-to-DNA ratios, attached cells were also more active than cells in unfiltered water samples. These results indicate that the microenvironment afforded by attachment is a more beneficial habitat for microbial growth. Reasons for greater activity by natural populations of attached bacteria are discussed.     

Synthesis of DNA during the cell cycle of synchronous Anacystis nidulans

Cells of Anacystis nidulans strain 1402-1 incorporate [methyl-³H]thymidine or [8-³H]adenine into DNA; in synchronous cultures (21/2 h full light, 1/2 h weak light, 5 h dark), this incorporation occurs in the dark to different extents according to the labeled precursor offered or to its specific activity. The specific activity of in vivo, uniformly labeled DNA decreases to half the initial value when the cells are grown in the absence of radioactive DNA precursors during the light phase; it does not decrease during the following dark phase. If unlabeled thymidine is given during the dark phase, the specific activity of the DNA starts to decrease at the onset of the next light phase. The time course of the decrease supports the hypothesis that all cells start their DNA replication immediately after illumination and that the first cells have completed if after 1.25 h. The slowest cells then need 3.75 h for completion of DNA replication. It is discussed whether the incorporation during the dark might be due to pool size effects.     

Inhibition by 1-beta-D-arabinofuranosylcytosine of the incorporation of N-acetylneuraminic acid into glycolipids and glycoproteins of hamster embryo cells

1-β-D-Arabinofuranosylcytosine which interferes with DNA synthesis in bacteria and mammalian cells and brings about transformation of hamster embryo fibroblasts, has been found to inhibit the incorporation of N-Acetylneuraminic acid into glycolipids and glycoproteins of both normal and transformed hamster embryo cells in tissue culture. Three hours after commencement of treatment (10^?3M ara-C), incorporation of [¹⁴C] thymidine into DNA was inhibited by 95 per cent, while incorporation of [³H] D-glycosamine (precursor of sialic acid) into glycolipids and glycoproteins was inhibited by 85 per cent. At 24 hours, the inhibition of incorporation of the two labelled components was 83 and 80 per cent respectively. In homogenates of both cell types, incorporation of [¹⁴C] N-acetylneuraminic acid was competitively inhibited by ara-CMP. Ara-C was found to have no effect on the incorporation of [¹⁴C] choline into phospholipids of cells grown in tissue culture. These results suggest that interference with DNA synthesis by ara-C may not be the only factor involved in cell transformation by this substance.     

THYMIDINE METABOLISM AND DEOXYRIBONUCLEIC ACID SYNTHESIS IN THE DEVELOPING RAT BRAIN

—In growing rat brain, the specific activity of DNA at 12 h after the subcutaneous injection of [³H]thymidine underwent a sharp rise during the first 6 days of life, dropping just as precipitously by 15 days, thereafter continuing to decrease with increasing age. When [³H]thymidine was given to 6-day-old rats, a considerable amount was taken up immediately into the brain. Thymidine taken up into the acid-soluble fraction was readily phosphorylated to its nucleotides, thymidine mono-, di-, and triphosphate (TMP, TDP and TTP) within only 30 min following injection. The highest specific activity was found in TTP. The incorporation of of [3H]thymidine into DNA took place over a longer period of time after injection.     

Polypeptide inhibitors of DNA synthesis and cellular proliferation produced by cultured BRL-3A rat liver cells

Variants of the NIL8 hamster cell line have been isolated by a procedure that selects against cells that incorporate [³H]thymidine into DNA in response to stimulation with multiplication-stimulating activity (MSA, an insulin-like growth factor produced by cultured BRL-3A rat liver cells). Some of the variant clones proved to be hypersensitive to two inhibitors of [³H]thymidine incorporation that were present in partially purified MSA preparations. The inhibitors have been found to be heat-labile polypeptides. The inhibitors copurify with MSA through ion exchange chromatography on Dowex 50, but are separable from MSA by gel filtration on Sephadex G-75. One of these factors (IN I) has been shown to inhibit DNA synthesis (as determined by autoradiography) and proliferation of the variant cells. Among several other rodent tumor cell lines tested for sensitivity to inhibition by INI, Reuber H-35 rat hepatoma cells and HSV-transformed hamster fibroblasts were not inhibited by the factor, whereas PG19 mouse melanoma cells were slightly inhibited.