Inhibition by a-Amanitin of Simian Virus 40-Specific

Simian virus 40 (SV40) grows lytically in monkey cells. Throughout the entire infection, ribonucleic acid (RNA) can be detected which is complementary to about 30% of the sequences of one of the strands of viral deoxyribonucleic acid (DNA): transcripts corresponding to about 70% of the sequences of the other strand appear only at late times after infection (5; G. Khoury and M. Martin, Nature, in press; J. Sambrook, P. A. Sharp, and W. Keller, J. Mol. Biol., in press). Whether this early-late shift in SV40specific RNA species reflects a change in the DNA template, in RNA processing, or in the enzymes transcribing the DNA is not known. The experiments described in this paper are designed to determine which of the two major DNA-dependent RNA polymerases found in mammalian cells (1, 8) is responsible for transcribing SV40 DNA. The principal forms of RNA polymerase, I and II, differ in their chromatographic properties, cation requirements, salt optima, intranuclear location, and sensitivity to a-amanitin (9, 11). a-Amanitin is a toxin extracted from the poisonous mushroom Amanita phalloides which inhibits the activity of enzyme II, whereas that of polymerase I is unaffected. This differential sensitivity provides a simple way to distinguish the two polymerase activities both in vivo and in vitro. The work of Ledinko (4) suggests that some types of primary cells in culture are permeable to a-amanitin. However, several experiments in this laboratory indicated that different batches of primary monkey kidney cells showed variable sensitivity to ac-amanitin, and we therefore chose to use isolated nuclei from a continuous line of green monkey kidney cells in which a-amanitin consistently shows an inhibitory effect. Isolated nuclei have been employed successfully by Price and Penman (7) and Wallace and Kates (13) to investigate transcription of adenovirus 2 in HeLa cells. MA 134 cells (obtained from J. Pagano) were grown at 37 C in 5% CO2 on 100-mm plastic tissue culture dishes in Dulbecco modified Eagle medium (2) (Gibco) supplemented with 10% fetal bovine serum (Gibco). Semi-confluent monolayers of the cells were infected at a multiplicity of 20 plaque-forming units per cell with a stock of SV40 grown at low multiplicity. Thirty hours later, nuclei were isolated from infected and mock-infected cells as follows. The cells were removed from the dishes with trypsin, washed in ice-cold phosphate-buffered saline (3), and collected by centrifugation for 7 min at 2,000 rev/ min in a Sorvall SS34 rotor at 0 C. The cells were washed once with ice-cold swelling buffer (SB0.01 M potassium phosphate, pH 7.9, 0.002 M MgCl2) and were resuspended at a concentration of 1.5 X 106 cells/ml. After about 1 min at 0 C, Triton-X was added to a final concentration of 0.5% and dithiothreitol to 0.001 M, and the cells were lysed by gentle pipetting for 3 min at 0 C. The nuclei were sedimented for 3 min at 800 X g and resuspended in SB at 107 to 4 x 107 nuclei/ml; 0.05-ml samples of nuclear suspension were used per assay. RNA polymerase activity was measured by following incorporation of 3H-uridine triphosphate (UTP) into trichloroacetic acid-precipitable material. Standard assays consisted of 4% glycerol, 0.001 M dithiothreitol, 0.01 M MgCl2, 0.01 M -tris(hydroxymethyl)aminomethane (Tris)-hydro-