branes in lysates of FMDV-infected baby hamster kidney cells (3, 4). This report concerns the dissociation of the active polymerase complex from these structures, and its activity in a cellfree system. Direct analyses of the reaction mixtures which contain the soluble polymerase have shown a_ heterogeneous RNA-containing component (140 to 3005) and a 50S component containing RNA, in addition to virus-specific RNA (see 3). The 140 to 300S RNA component is found before the appearance of 37S virus RNA. The FMDV RNA polymerase was prepared according to Polatnick and Arlinghaus (3), It is known to be active in 0.5-percent deoxycholate (3). However, the virus-specific RNA synthesized in the presence of deoxycho- late was largely degraded by contami- nating nucleases, yielding 20S RNA re- sistant to ribonuclease, and 4 to 125 RNA fragments unless bentonite was present. By use of this deoxycholatebentonite polymerase mixture, antibody to an antigen associated with FMDV infection (5) was found to inhibit FMDV-RNAsynthesis by about 90 per- cent (6). We used dextran sulfate-500 (7) to inhibit rrbonuclease (8). Preliminary ex- time at which cell-free synthesis has stopped), showed that 90 percent of the radioactive RNA insoluble in trichloroacetic acid was in the pellet; no significant peak was seen in the gradient. The results were similar when the sample was centrifuged for only 2 hours. It was also determined that active polymerase forms pellets under the same conditions, These results indicate that the FMDV polymerase, as well as its attached RNA template, and its RNA products, are membrane-bound in cell lysates. Direct sucrose-gradient analysis of reaction mixtures, containing polymerase and both 0.25-percent deoxycholate and dextran sulfate (140 ,g/ml) gave the following results after 60 minutes at 37°C: The amount of *H-uridine phosphate incorporated into RNA soluble in trichloroacetic acid was to 25 percent greater than in the sence of deoxycholate and dextran sulfate. The RNA products were released from the membrane, since 60 to 70 percent of the radioactive RNA insoluble in trichloroacetic acid was found in the gradient. The optical-density profile showed peaks of 185 and 285 ribosomal RNA that originated from ribosomal subparticles present in the periments showed that dextran sulfate at 10 to 20 ug/ml caused some 30% stimulation of incorporation of ?H-uri- corporation. Addition of 0.25 percent of deoxycholate to an intermediate concentration of dextran sulfate (140 pg/ml) gave maximum incorporation. The sodium dodecylsulfate-extracted RNA products of the polymerase treated with deoxycholate-dextran sulfate in the cell-free synthesizing system contained alf three virus-specific RNA’s (3, 9): 37S virus RNA, 20S ribonu- clease-resistant RNA, geneous RNA. and a hetero- Cell-free reaction mixtures were examined directly, without prior RNA extraction, by centrifugation on linear sucrose gradients of from 5 to 25 percent in 0.01M tris(hydroxymethyl) aminomethane HCI (pH 7.5) and 0.001M MgCl., (tris-MgCl.) for 17 hours at 25,000 rev/min in the SW-25.1 rotor. The sucrose-gradient profile of the polymerase reaction mixture, containing neither deoxycholate nor dextran sulfate, after 60 minutes at 37°C (the 8 DECEMBER 1967 1600 1200 > ¥ 1200 205 = 8 800 = 400 >» 800 400 Olea. 2-0-2-9 9-9-2 £ 1600 {B 3 0 50S 375 205 800 800 400 400 10 Tube 20 Number ¥ a = om 1200 oO > => 1600 5 = 1200 30 component near the bottom of the tube, which was not found after treatment with sodium dodecylsulfate or pretreat- ment with either 0.01M_ ethylenediaminetetraacetate or 0.04M pyrophos- phate; a 37S zone containing infectious virus RNA; and 20S RNA resistant to ribonuclease. No attempt has been made to demonstrate net synthesis of 37S infectious RNA in the cell-free system. A minor peak of 26 to 305 heterogeneous RNA was always present in the reaction mixture (peak D: 3, 9), but the amount varied with the activity of the polymerase (that is, ac- tivity presumably lost by denaturation of the enzyme and not by ribonuclease). The 50S and 375 zones were made soluble in trichloroacetic acid by treatment with ribonuclease (10 »g/ml) for 30 minutes at 37°C in 0.15M KCl and 0.1M tris-HCl, pH 7.0. Also, the infectivity of the 37S zone was lost on treatment with trace levels of ribonuclease. The 20S zone was almost completely resistant to ribonuclease, and vitro Sucrose 1600 dient (for example, Fig. 1B): a 50S Fig, 1. Sucrose-gradient profiles of the iu 10% sucrose dine triphosphate in the cell-free FMDV-polymerase system (Fig. 1 legend), whereas high concentrations (1 to 2 mg/ml) strongly inhibited in- triin15 ab- polymerase preparation. This deproteinizing of ribosomes is attributed to the action of dextran sulfate (4). Three major zones were detected in the gra- chase of whole-cell, pulse-labeled, virus-specific RNA. Baby hamster kidney cells (6 x 10°) were infected as in text. The soluble polymerase complex was isolated; it contained protein at 2.8 mg/ml and 125,000 count/min mg” protein of 4C-uridine, as RNA insoluble in trichloroacetic acid, at 48-percent counting efficiency. The complete cell-free reaction mixture contained: 10 wmole of tris-HCI, pH 8.1 (23°C); 5 umole of phospho(enol)pyruvate; 20 y~g of pyruvate kinase; 25 mumole of each of adenosine triphosphate, cytidine triphosphate, uridine triphosphate, and guanosine triphosphate; 12.5 nmole of MgCl; 0.1 ml of polymerase; and water to a final volume of 0.7 ml. The mixture was incubated (see text). Casein (300 ug} and 10 ml of 5-percent trichloroacetic acid were added to each gradient fraction. After 20 minutes at O0°C, the precipitate was collected on type-B6 membrane filters (25 mm in diameter; Schleicher and Schuell), and the filter was washed five times with 5-percent trichloroacetic acid. Samples were counted in a liquid scintillation spectrometer (9). (A) The cell-free reaction mixture was held at 0°C for 60 minutes with 10 uc of *H-uridine triphosphate. The reaction mixture contained 100 wg of dextran sulfate, 0.05 percent deoxycholate, 0.1 ml of soluble polymerase complex, and all components of the cell-free system in a volume of 0.7 ml. The mixture was diluted to 2.2 mi with 0.01M tris-HC], pH 7.5, prior to layering on the gradient. Two milliliters were applied, and the tube was centrifuged for 17 hours at 20,000 rev/min on a 10- to 30-percent linear sucrose gradient in tris-MgCl. in the SW-25.1 rotor. Carbon-14 at 35,344 count/min was applied to the gradient: 21,147 count/min was in the pellet; 12,336 count/min, in the gradient. (B) The reaction mixture was the same as for (A), and the tube was incubated for 60 minutes at 37°C. chilled and treated as for (A). Carbon-14 at 32.634 count/min was applied to the gradient: 5,862 count/min was in the pellet; 24,350 count/min, in the gradient. 1321