Single-stranded, adenine-rich RNA from purified reoviruses.

Reoviruses are known to contain double-stranded (DS) RNA.' It was therefore surprising to find that RNA extracted from highly purified reovirus type 3 and analyzed without further fractionation had a purine/pyrimidine ratio of 1.5 and an adenylic acid content of more than 40 moles per cent. In an effort to understand the basis for this observation, the RNA's of all three reovirus serotypes were examined. Each type was found to contain single-stranded adenine-rich (A-rich) RNA in addition to DS RNA. The isolation and properties of this single-stranded RNA are described. AMaterials and Methods.-Reovirus types 1 (Lang), 2 (D-5 Jones), and 3 (Abney) were obtained from the American Type Culture Collection. Polyadenylic acid (poly A) and adenylate oligonucleotide of chain length seven, (Ap)7, were purchased from Miles Chemical Company. L-cell DNA was isolated by Marmur's method2 and cell RNA by phenol extraction.' E. coli sRNA was purchased from Schwarz Bioresearch, Inc. Merck & Co. kindly provided the actinomycin cl. The following enzymes were obtained from Worthington Biochemical Corporation: micrococcal nuclease, venom and spleen phosphodiesterases, and electrophoretically purified DNase I and E. coli alkaline phosphatase. Crysstalline RNase was from Sigma Company, and E. coli polymerase was purified by the procedure of Chamberlin and Berg.4 Radioactive ribonucleoside triphosphates, amino acids, and carrier-free P'2-phosphoric acid were purchased from Schwarz Bioresearch, Inc., Nuclear Chicago Corp., and International Chemical & Nuclear Corp., respectively. The procedures for growth of mouse L-929 and HeLa S3-1 cells, reovirus infection, virus purification, and extraction of viral RNA have been described.5 6 Sucrose density gradient centrifugation, isopycnic sedimentation in Cs2SO4 solutions, methylated albumin-Kieselguhr (MAK) chromatography, and base analysis of RNA were performed as described previously.1-7 Sedimentation analysis in the Spinco analytical ultracentrifuge was done at 250C and 59,780 rpm. A synthetic boundary cell was used to overlay 0.2 ml of an RNA solution (31 .ug/ml; 0.01 M phosphate buffer pH 7, 0.06 M NaCl) above 0.5 ml of the same buffer containing 0.1 Al NaCl. Ultraviolet light absorbancy patterns were photographed at 8-min intervals and S values were determined from tracings made with an Analytrol densitometer. For electrophoresis of RNA in 10% polyacrylamide gels, the general procedure of Loening8 was followed, and the gels were fixed in acetic acid and stained with 0.2% methylene blue.9 The assay conditions for amino acid binding10 and the procedure for the preparation of soluble RNA1' and an active extract from rat liver12 (kindly provided by Dr. C. Caskey) have been described previously. A mixture of 15 C"4-labeled amino acids (specific activities = 67-247 mc/mmole) less asparagine, cysteine, glutamine, methionine, and tryptophan was tested.