Specificity in transcription of the reovirus genome.

Reovirus contains double-stranded ribonucleic acid with a molecular weight of approximately 107 daltons. 1 2 This RNA exists in the virion as a continuous linear structure, but it is extraordinarily susceptible to breakage.3-5 When isolated by any of the commonly used extraction techniques, the major portion of viral RNA appears as fragments with lengths of 0.35 ,u, 0.6 gA, and 1.1 jA, corresponding to molecular weights of 0.8 X 106, 1.6 X 106, and 2.4 X 106 daltons respectively.3 In previous work we have shown that the three fragments do not arise through random breaks; rather, there are specific weak spots in the RNA chain where the breaks occur during isolation.6 Further, the virus-specific single-stranded ribonucleic acid (ssRNA) formed in infected cells, and shown previously to be messenger RNA (mRINTA) ,7 8 was separated by sucrose gradient sedimentation into three clearly defined fractions whose sedimentation rates coincided with those found for the three double-stranded (ds) RNA fragments after denaturation.6 For each of the three classes of dsRNA a similar length of mRNA could thus be isolated, suggesting that each mRNA might be copied from the corresponding segment of dsRNA in the infected cell. This proposition is shown to be correct in the present paper. Each class of ssRNA hybridizes exclusively with its corresponding length of denatured dsRNA and is, therefore, transcribed uniquely from it in the infected cell. Methods.-Reovirus was propagated in suspension cultures of L, cells.9 In such cultures two species of virus-induced RNA commence to form at approximately five hours after infection and continue to be synthesized for a further ten hours or so.810 One RNA is double-stranded and is viral progeny RNA. Its extraction and separation into three fractions by chromatography on columns of methylated bovine albumin-kieselguhr (M\IAK) have been described.6 These fra(tions are designated dsRNA-1, dsRNA-2, and dsRNA-3, in order of elution from the column, and they have molecular weights of 0.8 X 106, 1.4 X 106, and 2.4 X 106 daltons, respectively. The other RNA formed in infected cells is single-stranded.9 Its separation into three fractions by sucrose gradient sedimentation has been described.6 Molecular weights of the three classes are 0.4 X 106, 0.7 X 106, and 1.2 X 106 daltoIls, and they are designated ssRNA-1, ssRNA-2, and ssRNA-3.6 Preparation of virus-specific RNA: Cellular RNA synthesis in infected cultures was suppressed by addition of 0.5 u~g per ml of actinomycin D at the time of infection.9 Double-stranded RNA was labeled with uridine-H3 (0.2 ,uc per ml) or uridine-C14 (0.02 jc per ml) added four hours after infection. After a further 16 hours, the dsRNA was extracted with phenol and purified on a I\IAK column using a steep NaCl gradient that eluted all three dsRNA fragments in a single peak.9 The eluate was concentrated by dialysis against Carbowax and was then dialyzed against 0.01 M STE buffer (0.01 1f NaCl; 0.01 Mll Tris-HCl, pH 7.4; 0.001 M ethylenediaminetetracetate (EDTA))." This preparation contained dsRNA-1, dsRNA-2, and dsRNA-3, and will be referred to as dsRNA.