Studies on tritiated thymidine incorporation into DNA molecules by electron microscopic autoradiography.

The incorporation of tritiated thymidine into DNA molecules was studied by electr.:m microscopic autoradiography. To make autoradiogram in electron microscopic level, DNA was extracted from rat ascites hepatoma (AH 130) cells after in vitro incubation with tritiated thymidine. Extracted DNA samples were rotary shadowed with platinum palladium and covered with emulsion. Silver grains demonstrated on autoradiogram indicated tritiated thymidine to have incorporated into DNA molecules themselves. The incorporation was further confirmed by liquid scintillation counting of TeA soluble and insoluble fractions after DNase or RNase treatment of the DNA preparations. ∗PMID: 4334468 [PubMed indexed for MEDLINE] Copyright c ©OKAYAMA UNIVERSITY MEDICAL SCHOOL Acta Med. Okayama 25, 37-41 (1971) STUDIES ON TRITIATED THYMIDINE INCORPORATION INTO DNA MOLECULES BY ELECTRON MICROSCOPIC AUTORADIOGRAPHY Hajime OGURA and Takuzo ODA Department of Biochemistry, Cancer Institute, Okayama University Medical School, Okayama, Japan (Director: Prof. T. Oda) Received for publication, November 12, 1970 CAIRNS studied the mechanism of DNA replication in E. coli chromosomes by light microscopic autoradiography using tritiated thymidine (1, 2) and proposed a theory on the replication of circular DNA molecules. Parallel relationship between autoradiographic grain numbers and radio· activity counts was shown by HUNT et al. (3), but there are some reports about the contamination of tritiated thymidine (4, 5) or incorporation of tritium from thymidine (6) into substances other than DNA molecules themselves. If the radioactive count of DNA preparations is due to tritiated thymidine incorporated into DNA molecules themselves and not due to contamination, autoradiography of extracted DNA in the electron microscopic level will be useful for the study on the mechanism of DNA replication. With this point in mind we have carried out the present study and demonstrated the incorporation of radioactive thymidine into DNA molecules by the electron microscopic autoradiography of DNA extracted from rat ascites hepatoma (AH 130) cells after in vitro incubation with tritiated thymidine. MATERIALS AND METHODS The following materials served for the present study: rat ascites hepatoma (AH 130) cells; tritiated thymidine obtained from the Radiochemical Center, England (specific activity 5CjmM) ; autoradiographic emulsion (SAKURA NR. M2 and NR-H2) of Konishiroku, Japan; DNase and RNase (Worthington); and cytochrome c (Boehringer). Labeling of DNA: Ten ml of ascites containing logarithmically growing AH 130 cells were taken out from the peritoneal cavity of a Donryu rat 6 days after intraperitoneal transplantation and transferred in a shaking flask without delay. One mC of tritiated thymidine was added immediately and incubated for 2 hr at 37°C. Control AH 130 cells were also incubated without labeling. Isolation of DNA: DNA was extracted according to MARMUR'S method (7) and purified by RNase treatment.