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 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.     

Incorporation of tritiated thymidine into mitochondrial DNA of the liver and kidney cells of chickens and mice in tissue culture

Summary ³H-thymidine incorporation into mitochondrial DNA of the liver and the kidney cells of chick embryos and newborn mice in tissue culture was shown by means of electron microscope radioautography with accurate localization. In these cells, about 20% of all the mitochondria were labeled at their matrices between the cristae within 4 hours in contact with the radioisotope, which were removed by DN'ase.From the results, it is clear that the mitochondria of avian and mammalian cells in tissue culture synthesize DNA.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

Incorporation of tritiated thymidine by marine bacterial isolates when undergoing a starvation survival response

Bacterial DNA synthesis, as measured by the incorporation of [methyl-³H] thymidine, was examined during conditions of decreasing biomass and non-growth of three heterotrophic marine bacteria. High rates of [³H] thymidine incorporation were recorded during the initial phase of starvation and two strains exhibited a net increase in DNA during the first few hours of starvation. The decreased rate of [³H] thymidine incorporation with the time of starvation, was in agreement with the decrease in the percentage of the total population that showed uptake of labelled thymidine, as seen by a combined autoradiography-epifluorescence technique. It is suggested that new rounds of replications were initiated after cells had been starved for times that well exceeded the time for replication of genomes during growing The initial increase in cell numbers upon transfer of growing cells to a starvation regime was inhibited by nalidixic acid, suggesting that DNA synthesis, rather than an excess of nuclear bodies, allow for the fragmentation process in these strains.     

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 tritiated adenosine into mouse ovum RNA

The total RNA of ovulated mouse ova has been examined by polyacrylamide gel electrophoresis. The amount of RNA present in the two main peaks observed, 28 S and 18 S ribosomal RNA, has been estimated as 0.20 ng.The RNA of ovulated mouse ova was labeled by exposure of growing mouse oocytes to adenosine-8-³H in vivo. For this purpose a small volume of a concentrated solution of the precursor was injected into the ovarian bursa, and ova were collected by superovulation at various subsequent times. The major growth phase of the oocyte is known to lie between 20 and 6 days before ovulation. Significant incorporation into egg RNA was observed when bursal injection was performed between 19 and 7 days, but not between 5 days and 1 day before ovulation.The types of labeled RNA in ova ovulated at five intervals between 19 and 7 days after bursal injection of adenosine-8-³H or uridine-5,6-³H were analyzed by polyacrylamide gel electrophoresis. The distribution of label on the gels demonstrated that the bulk of the label appeared in ribosomal RNA and transfer RNA. In addition labeled heterogeneous RNA was estimated to represent 10–15% of the total incorporation.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Incorporation of 18O2 into thymidine 5'-aldehyde in neocarzinostatin chromophore-damaged DNA

Strand scission of DNA by the chromophore of neocarzinostatin converts the 5'-hydroxyl of deoxyribose to a 5'-aldehyde. The origin of the aldehydic oxygen has now been elucidated by mass spectrometry. DNA-associated thymidine 5'-aldehyde produced by treatment of DNA with neocarzinostatin chromophore in 2H218O/16O2 or in 2H216O/18O2 was reduced, liberated by nuclease treatment, permethylated, and analyzed by gas chromatography-mass spectrometry. The data clearly show that molecular oxygen is the only source of the 5'-aldehydic oxygen. The addition of molecular oxygen at C-5', possibly via a reactive form of neocarzinostatin chromophore, must be involved; a carbonium ion intermediate at C-5' is ruled out.     

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: Effect of malnutrition

Incorporation of [³H]thymidine into DNA and of [³⁵S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [³H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18–21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.