It was of interest to examine the RNA associated with the polymerase preparation; one could then discriminate between free. unbound, virus-specific RNA and active protein-RNA complexes (polymerase-template). The latter is the structural complex that replicates virus RNA. The polymerase was harvested, 3.5 hours after infection, from FMDV-infected baby ham- ster kidney cells pulse-labeled with 14C-uridine under such conditions that only virus-specific RNA was labeled. Actinomycin D (5 g/ml) was added 30 minutes before harvest, and 20 yc polymerase complex was nent is not found after extraction with sodium dodecylsulfate; after treatment The soluble polymerase complex la- minutes at 37°C; or in reaction mixture incubated for 60 minutes. In all the soluble stable for long periods at —60°C, even after repeated freezing and thawing. beled with '4C-uridine was incubated 50% sucrose 1405 + 805 tube was kept at O°C; another was and FMDV polymerase) appears to be incubated for 1 hour at 37°C. The reaction mixture was analyzed directly + 700 > 6000 3 & = 5 soo 400 Us > «> 2 2 5 oo 35 E 2 = 200 °o = a a foo o 5 Fig. 2. Sucrose-gradient profile of the 140 to 3005S RNA containing component of the soluble polymerase complex. The soluble polymerase complex (0.9 mg) was incubated in 2.1 mi of the complete cell- free reaction mixture (Fig. 1) for 5 minutes at 37°C, with 30 uc of *H-uridine triphos- phate. The solution was rapidly chilled and adjusted to 0.1M tris-HCl, pH 8.1 (0°C). After addition of ammonium sul- fate to 50 percent saturation and standing for 20 minutes at 0°C, the suspension was centrifuged for 15 minutes at 10,000. The precipitate was suspended in 2.2 ml of tris-MgCl,, and 2 ml was layered on a 10- to 50-percent linear sucrose gradient in tris-MgCl». The gradient was centrifuged for 17 hours at 15,000 rev/min in the SW-25.1 rotor. Tritium at 18,375 count/ min was applied to the gradient; 4032 count/min was in the pellet; 12,662 count/ min. in the gradient. Samples were pre- cipitated Fig. 1, with trichloroacetic acid as in identical with the heterogeneous RNA (peak D; 3, 9). Difficulties in demon- stration of the 140 to 3005S component have been encountered because of ag- min, gregation in solutions containing magnesium. It should be noted that intact ribosomesare not detected in the soluble The whole-cell labeled with material, pulse- 1!4C-uridine, in soluble polymerase complex, contained both 375 and 20S virus-specific RNA, and a small amount of the 50S component in the pellet (S- Tate exceeding 100). After incubation in the cell-free system at 37°C, nearly all thé whole-cell RNA labeled with ™“C-uridine was found in the gradient in the three ma- jor zones (Fig. 1B). This test consti- tuted a true chase experiment, since total counts per minute of “C in both the zero-time and the incubated sample were identical; only the distribution of radioactivity varied. This fact suggests that the pellet material (exceeding 1005) is a precursor to the 50S, 37S, Tube Number 300S complex (presumably of RNA (no RNA extraction) on a 10- to 30- percent linear gradient at 20,000 rev/ activity was found 800 three instances all the RNA product occurred only at the top of the gradient. The RNA contained in the 140 to %H-uridine triphosphate—specific activity, 400 »c/ymole (Fig. 1 legend). One ever, 65 percent of the 44C_ radio- 10% sucrose with ribonuclease at 1 pg/ml for 5 in the complete cell-free system with appeared to be present (Fig. 1A). How- 0.4 3005, and another is at the top of the tube. The 140 to 3005 RNA compo- and 20S components, Moreover, nearly all the polymerase complexes active in the whole cell (labeled with MCuridine) are active im the cell-free system. The product of the cell-free synthesis of RNA @H-RNA) gave an identical profile, and 85 percent of the *7H-RNA was found in the gradient (Fig. 1B). Since the polymerase complex incorporates *H—uridine triphosphate into RNA for only 60 minutes at 37°C in the cell-free system, analysis of products formed early (after 5 to 10 minutes) should identify precursors to 375 virus RNA. Therefore a soluble polymerase complex was incubated for 5 minutes in the cell-free system containing dextran sulfate at 140 pg/ml. The reaction mixture was rapidly chilled to polymerase complex (Fig. 2). The methods of both isolation and storage of the soluble polymerase complex in dextran sulfate at 1 mg/ml and 0.5percent deoxycholate destroy ribosomes and polyribosomes. These results suggest that the 140 to 3005S RNA containing component is a precursor to FMDV 375 RNA.Studies with poliovirus in intact HeLa cells indicated that synthesis of poliovirus RNA takes place in complex structures possessing an average S-rate value of 250 (2). RALPH B. ARLINGHAUS JEROME POLATNICK Plum Island Animal Disease Laboratory, U.S. Department of Agriculture, Greenport, New York 11944 References and Notes 1. L. Daigarno, E. M. Martin, 5S. L. Liv, T. S. Work, J. Mol, Biol. 15, 77 (1966); D. Baltimore and R. M. Franklin, J. Biol. Chem, 238, 3395 (1963). - M, Gerard, D. Baltimore, J. F. Darnell, J. PN (4). Analysis of the precipitate material ‘by zonal centrifugation gave the profile shown in Fig. 2. A peak of radioactivity is found ranging from 140 to Mol. Biol. 24, 59 (1967). . J, Polatnick and R. B. Arlinghaus, Virology 31, 601 (1967). R. B. Arlinghaus and J. Polatnick, in preparation. K. M. Cowan and J. H. Graves, Virology 30, 528 (1966). . J. Polatnick, R. B. Arlinghaus, J. H. Graves, K. M. Cowan, ibid. 31, 609 (1967). From Pharmacia Fine Chemicals, Inc, L. Katz and S. Penman, Biochem. Biophys. Res, Commun, 23, 557 (1966). . R. B. Arlinghaus, J. Polatnick, G. F. Vande Woude, Virelogy 30, 541 (1966). 10, J. Polatnick and R. B. Arlinghaus, J, Virol., in press. 11, We thank Barbara Montgomery for technical assistance. wR we overlaps the 20S double-stranded RNA PH 8.1. A partially sensitive to ribonuclease. and reduction in production of polymerase (10). The enzyme was prepared as usual (3) and made 1 mg/mlin dextran sulfate and 0.5 percent in deoxycholate before storage at —60°C; this preparation will be referred to as the “soluble polymerase complex.” Activity of 0°C and adjusted to 50 percent of saturation with ammonium sulfate at oa tained larger amounts of the above- mentioned heterogeneous RNA. This heterogeneous RNA (peak D; 3, 9) is of 4C-uridine was added 15 minutes before harvest (9). This schedule of addition of actinomycin D caused no @ most likely is double-stranded RNA. It has been reported that the 205 zone is only partially resistant to ribonuclease (3, 9); however. such results were obtained from RNA, extracted with sodium dodecylsulfate and phenol, that con- 20 October 1967