This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Parsons, J. T. Articles by Weissmann, C. Search for Related Content PubMed PubMed Citation Articles by Parsons, J. T. Articles by Weissmann, C.

 Previous Article  |  Next Article 

J Virol. 1973 May; 11(5): 761-774
Copyright © 1973 American Society for Microbiology. All Rights Reserved.

Quantitative Determination and Location of Newly Synthesized Virus-Specific Ribonucleic Acid in Chicken Cells Infected with Rous Sarcoma Virus

^a Institut für Molekularbiologie, Universität Zürich, Zürich, Switzerland

ABSTRACT

A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70S AMV RNA, equilibrium density-gradient centrifugation in Cs₂SO₄ gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with ³H-uridine could be quantitated by the addition of an internal standard consisting of ³²P-labeled SRV RNA prior to purification and hybridization. This quantitative assay was used to determine that, in SRV-infected chicken cells labeled for increasing lengths of time with ³H-uridine, labeled viral RNA appeared first in a nuclear fraction, then in a cytoplasmic fraction, and still later in mature virions. This observation is consistent with the hypothesis that RNA tumor virus RNA is synthesized in the nucleus of infected cells.

¹ Present address: Institut de Biologie Moléculaire, Université de Genève, Genève, Switzerland.