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The size of virus-specific RNA synthesized in cultured mouse kidney cells infected with polyoma virus was estimated by electrophoresis and sedimentation analysis of RNA extracts from whole cells. Newly synthesized "late" polyoma-specific RNA appears as "giant" molecules of heterogeneous size, up to several times larger than a strand of polyoma DNA (1.5 X 106 daltons). Treatment with dinethylsulfoxide or urea showed that the large size of these molecules is not due to aggregation. Giant polyoma-specific RNA is strikingly similar in size distribution to "nuclear messenger-like" RNA ("heterogeneous nuclear" RNA) of the host cell. Subsequent to its synthesis, some of the giant polyoma-specific RNA appears to be cleaved to at least three smaller species.During lytic infection of mouse kidney cell cultures with polyoma virus, virus-specific RNA can be detected by hybridization with polyoma DNA (1, 2). Viral RNA transcribed after the onset of polyoma DNA replication has been designated as "late" RNA (2). The rate of synthesis of late RNA increases rapidly beyond 12 hr after infection and approaches a maximum at about 30 hr.Earlier experimental results suggested that late polyomaspecific RNA is the transcript of most or all of the genetic information contained in a strand of polyoma DNA (2). We undertook the present experiments to define more precisely the size of late virus-specific RNA found in polyoma-infected cells. Total RNA was extracted about 30 hr after infection from ['HJuridine-labeled cultures under conditions that minimize breakdown, and was analyzed by gel electrophoresis and sucrose or Me2SO-sucrose gradient centrifugation. RNA from each gel or gradient fraction was hybridized with an excess of polyoma DNA to determine the amount of virus-specific RNA present. The results show that the bulk of late polyomaspecific RNA is synthesized as "giant" molecules larger than the viral genome, and that some of this giant RNA is subsequently cleaved to smaller RNAs of specific sizes. Within 10-30 sec of lysis, total cellular RNA was extracted with redistilled phenol; this treatment was repeated twice, for a total of 8 min at 60-650C (5). Nucleic acids were precipitated with ethanol, redissolved, and incubated at 00C for 30 min with 10 jhg/ml of DNase I (ribonuclease-free, electrophoretically purified; Worthington Biochemical Corp.) inl 0.01 M sodium acetate (pH 5.1)-2 mM MnCl2. Macaloid, 100 Mg/ml, was added to adsorb DNase and was removed by centrifugation; the purified RNA was reprecipitated. One Petri dish yielded 100-150 ug RNA (taking 1 Am0 Unit to equal 42 ug RNA.) DNA-RNA hybridization Highly purified polyoma DNA I, prepared as the 53 S form by sedimentation at pH 12.5 (6), was converted into single strands by boiling for 30 min in 0.1 X SSC [SSC: 0.15 M NaCl-0.015 M Na citrate (pH 7.4)], cooled rapidly by dilution in ice-cold 6 X SSC, and fixed onto 25-mm membrane filters (Schleicher and Schuell, B6) by filtration (7). Filters 3.5 mm in diameter were cut from dried and baked (800C, 2 hr) filters. A ...

The DNA extracted from purified preparations of polyoma (Py) virus consists of three components, I, TI, and III, with sedimentation coefficients of 20, 16, and -14S. I and II are circular DNA molecules with a molecular weight of 3 X 106f and a buoyant density in CsCl of 1.709 gm cm-3.1, 2 Both I and II are infective.3Earlier observations suggested that Py DNA III might be derived from mouse cellular DNA.4, 5 In this paper we will show that most of the DNA molecules in III are linear fragments of mouse cellular DNA enclosed in Py viral capsids. Viral particles that contain mouse DNA instead of the viral genome are not infective and are referred to as Py pseudovirions.Materials and Methods.-Wild-type virus was grown on confluent MK cell cultures4 6 and was harvested at various times after infection. The crude viral lysates were extensively treated with pancreatic DNase'8 and RNase'8 and then purified by differential and CsCl density gradient equilibrium centrifugation.6 If viral preparations were subjected to a second cycle of purification, the relative amounts of Py DNA 1, II, and III remained unchanged.'8 Plaque-forming titer was generally determined on MK cultures7 and in some comparative experiments on mouse embryo cultures.8 Hemagglutination titer was determined according to Eddy et al.9' 18 DNA was extracted from purified viral preparations with 0.6% SDS-0.01 M EDTA.4For radioactive labeling of viral and cellular DNA deoxythymidine-methyl-Hi (TdR-H3, spec. act. 10-17 c/mM) and deoxythymidine-2-C14 (spec. act. 30 mc/mM) were used.'8 Radioactivity was measured on filter papers in a liquid-scintillation counter (Nuclear Chicago 6851). Unless otherwise indicated, the counting background was not subtracted.Sedimentation velocity analysis by band centrifugation and CsCl density gradient equilibrium centrifugation were performed according to methods used in earlier work.'0'" The buoyant densities for the DNA's were determined by using E. coli DNA as a standard (p = 1.710 gm cm-3)."2 The buoyant density of fractions (single drops) collected from CsCl density gradient of viral preparations was obtained from measurements of the refractive index at 250C. The value of 1.330 gm cm-' for Py virions (peak fraction) is the average from the measurement of 58 different viral preparations. DNA was examined by electron microscopy according to Kleinschmidt and Zahn."3 Spherules of polystyrene latex (Dow Chemical) were added to the preparations and their shadow was used to calibrate the magnification.'4 The preparations were photographed at a magnification of 104."Natural" mouse RNA for hybridization experiments was extracted with hot phenol"' 16 from uninfected MK cultures (2 days after plating) which had been labeled for 30 min with uridine-5-H3 (5 pc/ml; spec. act. 4.4 c/mM) prior to the extraction. "Synthetic" mouse RNA was prepared in a cell-free system with DNA-dependent E. coli RNA polymerase (sed. const. 13S; E. coli MRE 600; RNase I-) according to Pettijohn and Kamiyal7 using highly purified DNA from MK cultures'8 as t...

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