Cells producing Rous sarcoma virus contain virus-specific ribonucleic acid (RNA) which can be identified by hybridization to single-stranded deoxyribonucleic acid (DNA) synthesized with RNA-directed DNA polymerase. Hybridization was detected by either fractionation on hydroxyapatite or hydrolysis with single strand-specific nucleases. Similar results were obtained with both procedures. The hybrids formed between enzymatically synthesized DNA and viral RNA have a high order of thermal stability, with only minor evidence of mismatched nucleotide sequences. Virus-specific RNA is present in both nuclei and cytoplasm of infected cells. This RNA is remarkably heterogeneous in size, including molecules which are probably restricted to the nucleus and which sediment in their native state more rapidly than the viral genome. The nature of the RNA found in cytoplasmic fractions varies from preparation to preparation, but heterogeneous RNA (ca. 4-50S), smaller than the viral genome, is always present in substantial amounts. MATERIALS AND METHODS Materials. The sources of most reagents have been described (13, 15). Conidia of N. crassa were purchased from Miles Laboratories, Inc.; dimethylsulfoxide from Matheson, Coleman and Bell; Takadiastase (Sanzyme) was a gift from Sankyo Ltd., Tokyo, Japan. Diastase powder obtained from Sigma also contains S-1 nuclease, but the data in the present communication pertain only to the Sankyo material. All cell cultures were prepared from embryos known to be free from carrier infection with avian leukosis virus (embryonated eggs obtained from Kimber Farms, Berkeley, Calif.). Electrophoretically purified deoxyribonuclease was purchased from Worthington Biochemicals and treated with iodoacetate by the method of Zimmerman and Sandeen (34) to inactivate traces of contaminating ribonuclease. The alkylated preparations were tested for ribonuclease with 32p_ labeled 70S RSV RNA, which was denatured (11, 12) after exposure to the enzyme and was analyzed by rate-zonal centrifugation. 891

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