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.     

Chromosomes and DNA-replication of rat kangaroo cells (PtK2)

The karyotype, chromosomal measurements, and the time course of DNA replication during the S-phase were determined in metaphase chromosomes of non-synchronized monolayer cultures of PtK₂ cells (CCL 56) derived from Potorous tridactylis. The karotype was the same as originally determined for this cell line. Chromosomal measurements differed from data for primary bone marrow cells of this species published by Shaw and Krooth. PtK₂ cells and chromosomes showed maximal incorporation of tritiated thymidine (³H-TdR) halfway through the S-phase. Chromosome Y₁ showed a second peak of ³H-TdR-incorporation at the end of the S-phase in addition to the peak halfway through S. Comparison of grain densities for chromosomal arms showed late replication of the short arms of chromosomes 1, 3, and X. The time course of incorporation of ³H-TdR was changed when cells were treated for 1 h with fluorodeoxyuridine (FUdR) prior to the ³H-TdR-pulse. FUdR-treated cells showed maximum incorporation of ³H-TdR immediately after the beginning of the S-phase, which was followed by a second peak halfway through the S-phase. This indicated that ³H-TdR-incorporation was partially synchronized by treatment of cells with FUdR. Total radioactivity of FUdR-treated cells had increased by 77% in comparison to cells not treated with FUdR, which indicates that approximately 44% of the TdR-precursors of the latter cells may have originated from cellular precursor pools.     

Tritiated thymidine effects on DNA, RNA, and protein synthetic rates in synchronized L-cells

Exponentially growing L -cells were synchronized by the double thymidine-block method and exposed to high specific activities of tritiated thymidine. DNA, RNA, and protein synthetic rates were measured through one cell cycle with 1-hour pulses of the appropriate C¹⁴-labelled precursors. Equivalent doses of tritiated water were substituted for tritiated thymidine in some experiments. Total amounts of DNA and histones per nucleus were determined photometrically in Feulgen and fast-green stained cells. It was observed that incorporated tritiated thymidine has an effect distinct from that of tritiated water and that it enhances the incorporation of the precursors at specific stages of the cell cycle, to a degree roughly proportional to the dose. Photometric data indicated an increase in DNA net synthesis and a metabolic instability of histones in the H³-thymidine-treated cells, resulting in higher DNA:histone ratios.     

Transfection of mouse cells with thymidine kinase gene of herpes simplex virus

A mouse cell line (LP1-1) was established from the murine L cells deficient in thymidine kinase (L-M(TK ^–)) by prolonged selective culture on the hypoxanthine-aminopterine-thymidine (HAT) medium following transfection with the thymidine kinase gene of herpes simplex virus type-I (HSVTK). Southern blot analysis has shown that the viral TK gene was integrated into one of the chromosomal loci by a single copy. From this established cell line, the 5-bromo-2-deoxyuridine (BrdU) resistant revertant was brought out at a frequency of 1×10^–6 and from these BrdU resistant revertants (LP1BU), one out of 1×10⁵ cells could return to the HAT-resistant phenotype. The established LP1-1 cell line showed a typical biphasic nature of DNA synthesis as determined by the ³H-thymidine incorporation test. The activity of thymidine kinase was shown to be equivalent to that of the DNA polymerase- when the whole nuclear fraction or the nuclear matrix were used for examination. These results indicate that the transfected viral TK gene can be expressed under the normal cell-cycle regulation and its gene product can act as a component of the multienzyme complex which is responsible for DNA replication.     

Cell cycle and DNA synthesis in a marine dinoflagellateAmphidinium carterae

Summary The incorporation of tritiated thymidine into the DNA ofAmphidinium carterae in synchronized cultures has been analyzed by nucleic acid separation and CsCl centrifugation. The phases of the cell cycle are well defined In spite of an unusual characteristic: superimposed on the S phase, synchronous for nuclear and plastidal DNA's, a residual incorporation is observed whenever a labelling pulse is given. The corresponding continuous DNA replication is insensitive to ethidium bromide.Abbreviations BET ethidium bromide - CPM radioactive counts per minute - HAP hydroxylapatite - J₁, J₂, ..., J₉: 1st 2d, ... 9th day of a culture - L, D Light, Dark periods - mM milli-moles - pM picomoles - MF macromolecular fractionation - PB phosphate buffer - SSC standard saline citrate (0.15 M NaCl, 0.015 M Na citrate, pH 7).     

Nucleolar DNA in oocytes of Salmo irideus (Gibbons)

The amplification of ribosomal genes has been studied in oocytes from Salmo irideus. In situ nucleic acid hybridization showed that the synthesis of nucleolar DNA begins in oogonium and proceeds slowly through leptotene and zygotene when a small amount of extrachromosomal nucleolar DNA is produced. In early pachytene there is a rapid build-up of nucleolar DNA demonstrable by rapid incorporation of tritiated thymidine. Synthesis stops completely in early diplotene when nucleolar DNA becomes dispersed over the inner surface of the nuclear envelope in the form of small Feulgen-positive granules. Photometric measurements of Feulgen stained nuclei showed that the final amount of amplified nucleolar DNA synthesized in each oocyte is approximately 20 g. The amplified DNA does not form a heterochromatic mass. The buoyant density of the amplified nucleolar DNA calculated from analytical centrifuge tracings in relation to DNA from Micrococcus luteus ( = 1.731 g cm^–3) is 1.715 g cm^–3 and corresponds to a G + C content of 57%. There are indications that the buoyant density of the somatic nucleolar DNA is lower than that of amplified nucleolar DNA.Similarities and differences between ribosomal gene amplifications in oocytes of Salmo irideus and the corresponding process in Xenopus are discussed.     

Effects of beta-lactam antibotics on thymidine incorporation of bacteria

Five strains each ofEscherichia coli, Proteus mirabilis, andStaphylococcus aureus were grown in Trypticase soy broth (TSB) with or without penicillin, oxacillin, ampicillin, amoxicillin, or cefamandole. [³H]Thymidine incorporation and the optical density of the cultures were determined at an hourly interval for the duration of incubation. All strains ofP. mirabilis showed after 2 and 3 h of incubation of 5-to 16-fold increase in the specific activity of [³H]thymidine incorporated in the presence of MIC of the antibiotics as compared to controls grown without the drugs.E. coli andS. aureus showed smaller increases in thymidine incorporation than didP. mirabilis. After 4–5 h the antibiotics produced an inhibition of [³H]thymidine incorporation. At the MAC, the responses were of a smaller magnitude. Regardless of whether these changes are the result of specific interference or just lack of [³H]thymidine incorporation, they are directly related to the antibiotic activity of agents known not to affect DNA synthesis. The monitoring of [³H]thymidine incorporation detects early antibiotic activity probably earlier than other current systems which are used for this purpose.     

DNA Synthesis Cycle in Blepharisma*

SYNOPSIS. Observations made with tritiated thymidine uptake indicate that Blepharisma cannot utilize this precursor directly from the organic medium. An intermediary, such as a food organism (bacteria or Tetrahymena, is needed. These food organisms are not only capable of incorporating thymidine from the medium, but must also break it down or prepare it in a form that is utilizable for Blepharisma. DNA synthesis phase in Blepharisma occurs 17 hours after division. Maximum incorporation is obtained 18 and 19 hours postdivision and lasts thru 24 hours. This is followed by a 1–2 hour postsynthesis phase and division.     

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.     

LIMITATIONS IN THE USE OF [3H]THYMIDINE INCORPORATION INTO DNA AS AN INDICATOR OF EPIDERMAL KERATINOCYTE PROLIFERATION IN VITRO

The validity of using the incorporation of [³H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [³H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [³H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [¹⁴C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [³H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Effect of aging on EGF-stimulated replication of specific genes in rat hepatocytes

EGF-stimulated replication of specific genes was examined in primary hepatocyte cultures from mature (6 months) and senescent (24 months) rats. Basal and EGF-stimulated [³H]thymidine incorporation and DNA polymerase α activities, as well as total cellular DNA, were also assessed. The genes examined were dihydrofolate reductase (DHFR) and c-myc, as well as total mitochondrial DNA (mt DNA). Although [³H]thymidine incorporation, DNA polymerase α activity, total cellular DNA, DHFR, and c-myc gene specific DNA replication stimulated by EGF are reduced with age, mt DNA replication is not affected by either EGF or age. Chromosomal DNA replication is mediated mainly by DNA polymerase α while mt DNA replication is mediated by its own DNA polymerase γ. Thus, the age-related decline in stimulated DNA replication appears to be associated mainly with the DNA polymerase α activation pathway. J. Cell. Physiol. 176:32–39, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Uptake of 125I-iododeoxyuridine in mouse organs during deuteration of body water: Evidence for a thymus-specific effect

The effects of body water deuteration on mammalian DNA synthesis $\text{in vivo}$ during the deuterium equilibration period in the body were studied. Young adult mice were given 15% or 30% D₂O in the drinking water for 4, 10 or 21 days. Control mice were given distilled water. Eighteen hours prior to sacrifice, ¹²⁵IUdR, a conveniently monitored synthetic analogue of the DNA precursor thymidine, was injected intravenously. Although neither radioiodine activity of the total body nor body weight varied significantly among the three groups, thymic radioactivity per g tissue was significantly lower in mice given 30% D₂O and, to a lesser extent, in mice given 15% D₂O than in the control group. In contrast, intestine and hemopoietic bone marrow displayed minor changes in ¹²⁵IUdR incorporation. This reduction of ¹²⁵IUdR incorporation is discussed in relation to the particular importance of thymidine reutilization in the thymus.     

SOME MORPHOLOGIC AND KINETIC STUDIES OF THE DEVELOPING ERYTHROID CELLS OF THE COMMON GOLDFISH, CARASSIUS AURATUS

Developing erythroid cells of the goldfish Carassius auratus were obtained from kidney prints and from smears of the peripheral blood. All preparations were stained with the May-Grunwald Giemsa technique. Developing cells were divided into six different stages. The criteria used to stage the cells were degree of chromatin condensation, degree of basophilia, nuclear:cytoplasmic ratio, and cell shape. The morphology of the maturation sequence for erythroid cells in this organism was similar to that found by other workers in other non-mammalian vertebrates. Fish received intraperitoneal injections of tritiated thymidine, tritiated uridine or tritiated leucine so that the stages involved in DNA synthesis, RNA synthesis and protein synthesis could be determined by means of autoradiography. For the tritiated thymidine studies the per cent labeled cells per stage from four different series receiving 0.5, 1.0, 3.0 or 6.0 μCi/g body weight were pooled, since subjecting the average per cent labeled cells per stage at the lowest and at the highest dosages to Student's t-test showed no significant differences. In all four series the fish were killed 2 hr, 12 hr and daily, 1–8 days post-injection. The ³H-TdR studies showed that stages I-IV were engaged in DNA synthesis; they also showed that about 5 days were required for the stage V cell to become a mature erythrocyte (stage VI cell). Tritiated uridine was injected at a dosage of 5.0 μCi/g body weight and animals were killed 1/2, 1, 3 and 6 hr post-injection. Grain counts showed that stages I-IV are engaged in RNA synthesis and that the rate of this synthesis decreased as maturation proceeded. Tritiated leucine was administered at a dosage of 5.0 μCi/g body weight, and fish were killed 45 min and 3 hr post-injection. Grain counts indicate that stages I-V are engaged in the synthesis of protein (assumed to be globin). The fact that DNA and RNA synthesis ceased with stage IV cells while protein synthesis continued into stage V cells indicated that the mechanism responsible for protein synthesis in stage V cells was produced at an earlier stage and was self-sustained for about 5 days.     

TheB locus (MHC) in the chicken: Association with the fate of RSV-induced tumors

The fate of tumors induced by Rous sarcoma virus (RSV) was determined in anF ₂ population segregating at three alloantigen loci. TheF ₁ resulted from crossing tumor-resistant RPRL line 6₁ (B ² B ² D ³ D ³ I ² I ²) with tumor-susceptible RPRL line 15₁ (B ⁵ B ⁵ D ⁴ D ⁴ I ⁸ I ⁸). Among theF ₂ segregantsB ² B ²,B ² B ⁵, andB ⁵ B ⁵, the percentage of chicks dying of terminal tumors (by 70 days post-inoculation) was 5, 26, and 93, respectively (P0.01). NeitherD orI genotypes nor sex significantly affected tumor growth. In chickens with terminal tumors, the incidence of metastatic lesions was also significantly associated withB genotypes. Thus, the MHC chromosomal region in the chicken appears to exert a crucial role in determining the outcome of RSV-induced tumors.     

SYNTHESIS OF CHLOROPLAST DNA IN ISOLATED CHLOROPLASTS

Chloroplasts isolated from Euglena gracilis incorporated both tritiated thymidine 5'-triphosphate and tritiated deoxyadenosine 5'-triphosphate into an acid-stable fraction. The incorporation was dependent on the presence of all four deoxynucleoside triphosphates and was sensitive to treatment with deoxyribonuclease and actinomycin D. It was demonstrated that bacterial contamination could not account for the incorporation of label. Extraction of DNA from the chloroplasts and subsequent density gradient centrifugation of the DNA in CsCl₂ showed that the incorporation was into chloroplast DNA (ρ = 1.686) of high molecular weight.     

Modulation of DNA synthesis in aortic smooth muscle cells by dinitrotoluenes

Studies were conducted to determine if in vivo exposure to dinitrotoluenes (DNT), which is associated with circulatory disorders of atherosclerotic etiology in humans, is associated with alterations of vascular smooth muscle cells (SMC) consistent with the atherogenic process. Sprague-Dawley rats (150-180 g) were injected IP for 5 days/week for 8 weeks with 2,4- or 2,6-DNT (0.5, 5, or 10 mg/kg) or medium chain triglyceride (MCT) oil. Histopathologic evaluation of aortae from animals exposed to either isomer showed dysplasia and rearrangement of SMC at all doses tested. Reduced ³H-thymidine incorporation was observed in primary cultures of aortic SMC from DNT-exposed animals relative to vehicle controls. This inhibitory response was maintained for up to two passages in culture after which a significant increase in thymidine incorporation was observed. Exposure of SMC from naive animals to DNT in vitro (1–100 µM) did not alter the extent of thymidine incorporation in cycling or growth-arrested cultures. In contrast, exposure to 2,4- or 2,6-diaminotoluene (DAT) (1–100 µM), carcinogens which share toxic metabolic intermediates in common with DNT, inhibited replicative DNA synthesis and stimulated unscheduled DNA synthesis in cycling and growth-arrested cultures of SMC, respectively. Our results suggest that modulation of DNA synthesis in aortic SMC by DNT metabolites generated in vivo contribute to the development of vascular lesions.Abbreviation DAT diaminotuluene - tDNT technical grade dinitrotoluene - DNT dinitrotoluenes - HU hydroxyurea - IP intraperitoneal - LDH lactate dehydrogenase - MCT oil medium chain triglyceride - NPTC non-protein thiol content - RDS replicative DNA synthesis - SEM standard error of the mean - SMC smooth muscle cells - UDS unscheduled DNA synthesis     

Genome differentiation in Aegilops. 3. Evolution of the D-genome cluster

 Six polyploid Aegilops species containing the D genome were studied by C-banding and fluorescence in situ hybridization (FISH) using clones pTa71 (18S-5.8S-26S rDNA), pTa794 (5S rDNA), and pAs1 (non-coding repetitive DNA sequence) as probes. The C-banding and pAs1-FISH patterns of Ae. cylindrica chromosomes were identical to those of the parental species. However, inactivation of the NOR on chromosome 5D with a simultaneous decrease in the size of the pTa71-FISH site was observed. The N^v and D^v genomes of Ae. ventricosa were somewhat modified as compared with the N genome of Ae. uniaristata and the D genome of Ae. tauschii. Modifications included minor changes in the C-banding and pAs1-FISH patterns, complete deletion of the NOR on chromosome 5D^v, and the loss of several minor 18S-5.8S-26S rDNA loci on N^v genome chromosomes. According to C-banding and FISH analyses, the D^cr1 genome of Ae. crassa is more similar to the D^v genome of Ae. ventricosa than to the D genome of Ae. tauschii. Mapping of the 18S-5.8S-26S rDNA and 5S rDNA loci by multicolor FISH suggests that the second (X^cr) genome of tetraploid Ae. crassa is a derivative of the S genome (section Emarginata of the Sitopsis group). Both genomes of Ae. crassa were significantly modified as the result of chromosomal rearrangements and redistribution of highly repetitive DNA sequences. Hexaploid Ae. crassa and Ae. vavilovii arose from the hybridization of chromosomal type N of tetraploid Ae. crassa with Ae. tauschii and Ae. searsii, respectively. Chromosomal type T1 of tetraploid Ae. crassa and Ae. umbellulata were the ancestral forms of Ae. juvenalis. The high level of genome modification in Ae. juvenalis indicates that it is the oldest hexaploid species in this group. The occurrence of hexaploid Ae. crassa was accompanied by a species-specific translocation between chromosomes 4D^cr1 and 7X^cr. No chromosome changes relative to the parental species were detected in Ae. vavilovii, however, its intraspecific diversity was accompanied by a translocation between chromosomes 3X^cr and 3D^cr1. Received July 24, 2001 Accepted October 1, 2001     

Delays in prophase induced by adenosine 2-deoxyriboside and their relation with DNA synthesis

The rate of DNA synthesis in the course of the division cycle in root meristem ofAllium cepa growing under constant temperature and aeration conditions has been studied by means of treatment with AdR, as a specific inhibitor of the synthesis, as well as by the incorporation of tritiated thymidine. The one-hour treatment with AdR or tritiated thymidine was given at various hours in the course of the interphase of a synchronous population of binucleate cells induced by caffeine. In the case of AdR, sensitivity to the inhibition of DNA synthesis was studied by recording the delays produced by the treatment in the appearance of biprophases and bitelophases. The selection by the use of caffeine, of spontaneously synchronous populations of cells going through the telophase and becoming binucleate and the detection of the first biprophases in the subsequent mitosis provide a highly synchronized system with which to study the incorporation of tritiated thymidine during the interphase. The curves representing sensitivity to the inhibition of DNA synthesis by AdR and the rate of tritiated thymidine incorporation coincide, so that we can regard the delays, under our conditions, as proportional to the rate of DNA synthesis at the moment of the AdR treatment. This rate, in the S period, was found to be variable by both methods, being higher in the first and the last thirds of the S period (S₁ and S₃) and lower in the middle third (S₂).     

Fixation of CO2 and incorporation of thymidine under heavy metal stress inClosterium moniliferum

The unicellular green algaClosterium moniliferum was sensitive to the action of divalent Ni, Cu, Hg and Cd chlorides both in CO₂ fixation and in thymidine incorporation into DNA. At 0.08 ppm, Cd²⁺ was the most potent inhibitor (86 % inhibition of both processes), followed by Hg²⁺ and Cu²⁺, and finally by Ni²⁺, thymidine incorporation being generally more affected than CO₂ fixation.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.