Incorporation of tritiated thymidine into DNA of rat liver: a critique of various evaluation methods]

We have measured the incorporation of 3H-(methyl)-thymidine by cell cultures of rat foetal liver and in vivo by the livers of young rats stimulated by casein, in order to compare three methods for the extraction of DNA. The DNA was extracted by three different techniques: perchloric acid precipitation, trichloroacetic acid precipitation and phenol extraction, and its specific activity was determined. The radioactive labelling was also determined for the lipid, ribonucleic acid and protein fractions for the two first methods, in both of which 70 p. cent of the incorporated tritium is found in the DNA fraction and about 10 p. cent in each of the other fractions. The determination of the specific radioactivity of DNA gives similar results for the three extraction methods. However, since larger yields were obtained by both acid precipitation techniques than by phenol extraction, we believe them to be more suitable for studies on cell cultures.     

Incorporation of thymidine into the DNA of actinomycetes. I. Incorporation of exogenous thymidine into the DNA of Thermoactinomyces vulgaris]

The incorporation of exogenous thymidine and thymine into acid-insoluble material of Thermoactinomyces vulgaris has been studied during germination and subsequent growth. Thymine is not incorporated. The incorporation of thymidine stops after a short time due to the rapid breakdown of thymidine to thymine and deoxyribose-1-phosphate by the inducible thymidine phosphorylase. Deoxyadenosine enhances the incorporation of thymidine as well as of thymine and prolongs the tine of uptake. Uridine stimulates only the incorporation of thymidine but not of thymine. These effects can be explained by the function of these substances within the salvage pathway. Deoxyadenosine acts as donor of deoxyribosyl groups being necessary for the conversion of thymine to thymidine by thymidine phosphorylase and uridine inhibits thymidine phosphorylase, and thereby it prevents the degradation of thymidine to thymine. Thymidine is incorporated into alkali-, RNase-and protease-stable, hot TCA-soluble and DNase-sensitive material. That means that the cellular DNA of T. vulgaris can be specifically labelled by radioactive thymidine in the presence of deoxyadenosine and uridine, respectively.     

Low level incorporation of tritiated thymidine into the nuclear DNA of Purkinje neurons of adult mice.

Adult mice were pulse labeled with tritiated thymidine [3H]TdR and killed 9 hr later. A low level incorporation of [3H]TdT into the nuclear DNA of Purkinje neurons was found in autoradiographs. Enzymatic digestions with DNase and with RNase in combination with autoradiographic grain counts indicate that a portion of nuclear DNA is not stable in the Purkinje nucleus. These results are discussed in light of reports of the stable nature of DNA in Purkinje neurons of adult mice.     

Incorporation of tritiated thymidine into testicular deoxyribonucleic acid of the rat. Effect of inhibin]

tritiated thymidine is incorporated into DNA of spermatogonia type B as proved by autohistoradiography when injected in vivo three hours before the sacrifice. Maximum binding and specific activity (labelled thymidine expressed in DPM per mg DNA) are obtained in pubertal rats aged 42 days and weighting 150 g. Inhibin preparation extracted from rete testis fluid (RTF3) specifically inhibits tritiated thymidine into testicular DNA. Thus, no modification of incorporation into hepatic DNA is observed and the preparation loses its inhibitory effect when denatured by heating and trypsin digestion. Tritiated thymidine incorporation into testicular DNA is poor in normal adult rats and in pubertal hypophysectomized animals, RTF3 does not modify the thymidine incorporation in both conditions. The reasons for this lack of effect are discussed. An experimental condition of spermatogonial regeneration is obtained by testicular irradiation. Inhibin preparation inhibits the regenerative DNA synthesis.     

Incorporation of thymidine and iododeoxyuridine into the DNA of mouse tissues.

Mice were injected intravenously with a solution containing tritiated thymidine (TdR) and iodine-labelled iododeoxyuridine (IUdR). The ratio of 3H/125I activities was measured in the acid-soluble fraction and in the DNA of several tissues at various times from 0.08 to 24 h after injection. There did not appear to be any discrimination in favor of TdR in the acid-soluble fraction of the tissues. The amount of TdR incorporated into the DNA was four to five times greater than the amount of IUdR incorporated; moreover, this value remained relatively constant throughout the period of DNA synthesis under the conditions used. Although IUdR was destroyed more rapidly than TdR in the body, particularly at high concentrations of both precursors, this factor did not account for the major portion of the discrimination observed with tracer amounts of the two DNA precursors. Discrimination in favor of TdR as a precursor for DNA must, therefore, occur at some stage in the utilization of intracellular precursor.     

Incorporation of thymidine into DNA of the gonads in the normal and chemosterilized yellow-fever mosquito

Effect of the chemosterilants, apholate and hempa, on the incorporation of tritiated thymidine in DNA of the gonads of Aedes aegypti was studied. Larval treatment with sterilizing doses of the chemosterilants caused inhibition of DNA synthesis in the gonads. The inhibition was found to be more in testes than in ovaries. The significance of these findings in relation to the mode of action of these chemosterilants is discussed.

---

Zusammenfassung Die Wirkung der Chemosterilantien Apholate und Hempa auf den Einbau von tritiummarkiertem Thymidin in die DNS der Gonaden von Gelbfiebermücken wurde untersucht. Behandlung der Larven mit sterilisierenden Dosen der Chemosterilantien verursachte Hemmung der DNS-Synthese in den Gonaden. Die Hemmung erwies sich in den Hoden als stärker als in den Ovarien. Die Bedeutung dieser Befunde in Beziehung zur Wirkungsweise dieser Chemosterilantien wird diskutiert.

     

Stimulation of uptake of tritiated thymidine into mouse marrow cells in culture by a factor from L-cell conditioned medium

Uptake of ³H-thymidine into suspension cultures of mouse marrow cells was stimulated by the addition of serum-free conditioned medium harvested from cultures of mouse L-cells. Characterization of the “conditioning factor activity” (CFA) by gel filtration, ion exchange chromatography, velocity sedimentation, polyacrylamide gel electrophoresis and susceptibility to trypsin digestion indicated that the CFA detected by stimulation of ³H-thymidine uptake is the same as the CFA detected previously by its ability to stimulate colony formation by marrow cells in vitro. The ³H-thymidine uptake assay was used to investigate the kinetics of disappearance of CFA as a function of time in the presence of mouse marrow cells. The CFA recoverable from the cultures decreased rapidly during the first day, and approached background levels by the fifth day. There was no evidence of inhibitory substances in the depleted media. Even if the cultures continued to receive fresh conditioned medium at daily intervals, ³H-thymidine uptake decreased sharply after the fifth day, indicating that the marrow cells had lost their capacity to respond to CFA.