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Studies on the Mechanism of Template Stability

I. The Effect of Actinomycin on the Base Composition and Sedimentation of ³²P-Labeled RNA in Rat Liver¹

McArdle Laboratory, The Medical School, University of Wisconsin, Madison, Wisconsin

The RNA from nuclei, cytoplasm, and membranes of the endoplasmic reticulum of rat liver was extracted using phenol and ribonuclease inhibitors. The RNA was purified by passage through Sephadex G-200 columns and was characterized by sedimentation through 5–20% linear sucrose gradients. Radioactive phosphorus was administered at various times prior to the sacrifice of the animals and the effect of actinomycin D on the labeling pattern was studied. In utilizing a labeling period of less than 30 min, it was noted that the distribution of the radioactive RNA did not follow the optical density pattern. However, 3 hr after administration of the label, the patterns of optical density and radioactivity were found to be superimposable. If actinomycin D (500 µg/kg) was given prior to the ³²P, the radioactive pattern after 3 hr resembled more closely that seen in an untreated animal after a 20-min labeling period. The base ratios of the 3-hr labeled RNA of both nucleus and cytoplasm following actinomycin administration were noted to have a more DNA-like base composition than that of nontreated animals. In the liver cytoplasm of actinomycin-treated rats, a labeled RNA component with a sedimentation constant of about 18 S and a DNA-like base composition was found to be associated with the membranes of the endoplasmic reticulum and could not be removed by chelating agents. The possible relation of this membrane-bound DNA-like RNA to the stable messenger RNA templates of rat liver is discussed.

¹ This investigation was supported in part by Grant CA-07175 from the National Cancer Institute, USPHS, and P-314 from the American Cancer Society.

² Department of Medicine, University of Wisconsin Medical School, Madison, Wisconsin.

³ Predoctoral Summer Student Trainee of the USPHS. Present address: Columbia University, New York, New York.

⁴ Career Development Awardee (CA-29, 405) of the National Cancer Institute, USPHS.

Received 12/20/65. Revised 4/ 4/66.