Abstract
SV-40-transformed human lung fibroblasts (WI38-VA13 cells) were incubated for 4 hr with highly purified, tritium-labeled puromycin aminonucleoside (AMS), together with unlabeled AMS at a final concentration of 340 µM (100 µg/ml). Approximately 90% of AMS was found unchanged in the acid-soluble pool. Phosphorylated forms of the demethylated derivative of AMS, 3'-amino-3'-deoxyadenosine (3'-AmA) were also found; one form was shown to be the 5'-monophosphate, and the other a 5'-triphosphate. Tracer concentrations of AMS (0.066 µM) were converted to phosphorylated derivatives to a larger extent, and nonphosphorylated 3'-AmA was not found in the acid-soluble pool even at the higher AMS concentration, indicating that the demethylating step is slower than the phosphorylating reactions. Alkaline hydrolysis of the RNA from AMS-treated cells released only nonphosphorylated 3'-AmA. AMS or its derivatives were not detected in the DNA of treated cells. The results indicate that AMS is successively demethylated and phosphorylated, and that the resultant 3'-AmA triphosphate is incorporated into the terminal positions of nascent RNA chains. Further elongation of the growing RNA polynucleotide is prevented by the 3'-amino group of the analog, thus causing premature termination of RNA synthesis.
ACKNOWLEDGMENT Our thanks are due to Mr. Robert Lockwood, whose assistance was invaluable in the early stages of this work, to Mrs. Marsha Trocola for technical help, to Dr. Ethel Somberg of Rutgers University for the use of the high-voltage electrophoresis apparatus, and to Dr. Bonnie Seidel for her assistance in the operation of this apparatus.
- Copyright © 1980 by The American Society for Pharmacology and Experimental Therapeutics
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