The Incorporation of Radioactive Uridine into the Hepatitis B Antigen of a Chimpanzee 1

Abstract

Summary

Radioactive (³H) uridine was incorporated into RNA isolated from the HB_sAg of a chimpanzee carrier. HB_sAg was purified by precipitation as an immune complex with the IgG fraction of chimpanzee anti-HB_s. RNA was extracted from the washed complex with buffered phenol precipitated with alcohol and four nucleotides were identified by thin layer chromatography after alkali degradation.

We acknowledge with appreciation the time and assistance of Dr. Manfred E. Bayer for the electron microscopic examination of the purified HB_sAg—anti HB_s complexes.

Addendum. After this work was prepared for publication we became aware of the possibility that radioactive uridine could have been incorporated into uridine diphosphoglucose and not RNA. We have since repeated the procedure for the isolation of nucleic acid described in methods but included two additional steps. (1) The chimpanzee plasma was treated with 25 μg/ml of RNAse (4000 units/mg, Worthington, Freehold, NJ) and incubated for 15 min at 37°. A control containing ¹⁴C labeled RNA was shown to be completely degraded under the same conditions of treatment. The treated plasma was then extracted with buffered phenol as described in the Materials and methods section. (2) The alcohol precipitated and redissolved RNA was then precipitated with perchloric acid (final concentration 0.2 N). The perchloric acid precipitate, adhering to a fiberglass filter, contained the same radioactive count as the KOH hydrolyzed material originally described under Materials and methods. Uridine diphosphoglucose is not precipitable under these conditions.

To further exclude the possibility that the material we were dealing with was not RNA an aliquot of ethanol precipitated material (approx one-fifth of the total yield described in methods) was dissolved in distilled water and treated with 80 μg of RNAse for 15 min at 37°. Perchloric acid was added to a final concentration of 0.2 N.