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Double-stranded RNA isolated from cells infected with the RNA bacteriophage R17 has been implicated in the replicative mechanism of viral RNA. I Two types of double-stranded RNA have been described in cells infected with RNA viruses: the intact double strand of molecular weight 2 X 106,2, 3 knowA as replicative form,4 and the population of double-stranded molecules containing nascent single strands, known as replicative intermediate.5 The description of replicative intermediate isolated from infected cells by phenol extraction and its relation to in vitro replicating RNA structures has been discussed.'-3 Besides removal of the RNA-dependent RNA polymerase from the replicating structure, phenol could possibly dissociate some of the hydrogen-bonded single strands from the double strand.3 Therefore it was necessary to investigate the localization of intracellular viral RNA-replicating structures prior to phenol extraction. These structures could be associated with cell membranes, or with ribosomes, or not associated with any recognizable cellular structures. In order to obtain meaningful data it was first necessary to obtain reproducible patterns of polyribosomes in uninfected cells. This was accomplished by adding poly-L-ornithine to the media during preparation of spheroplasts and lysate. This polyamino acid has been used to enhance titers of infectious RNA from poliovirus,6 presumably by protecting the viral RNA from degradation by nucleases. The authors are particularly grateful to Dr. S. Dales of this institute for suggesting the use of this compound. Materials and Methods.-Bacteria and virus: Escherichia coli strain 3000, a nonlysogenic Hfr strain, and RNA bacteriophage R17, free of X bacteriophage, were used in all experiments. Media and solutions: TCGl synthetic growth medium has been described.' PHO: TCGl with 0.0015% casamino acids (L/loo normal concentration); chloramphenicol (Parke, Davis and Co.), 100 ig/ml; sodium azide, 0.005 M; poly-L-ornithine (Mann Research Lab., Inc.), 10 /Ag/ml. PH 1: Tris buffer, 0.1 M, pH 8.1 (50); chloramphenicol, 100 Ag/ml; poly-L-ornithine, 10 yg/ml; 20% sucrose (Mann Research Lab., Inc., ribonuclease free). TMK: Tris buffer, 0.005 M, pH 7.4 (5°); KC1, 0.06 M; chloramphenicol, 100 jg/ml; poly-L-ornithine, 10 j~g/ml; sodium azide, 0.005 M. TMK 1: Tris buffer, 0.005 M, pH 7.4 (50); KC1, 0.06 M; MgSO4, 0.01 M. Preparation of labeled cell lysates from uninfected cells: The volumes refer to a starting volume of 1 liter of cells. E. coli was grown to a titer of 2 X 108/ml in TCG1, harvested, and resuspended in 300 ml of prewarmed TCG1 with 1/ioo casamino acids. After 5 min further incubation at 370, the cells were labeled for 3 min with C'4-amino acids at 1 /,c/ml. The amino acids were a mixture of 15 L-amino acids uniformly labeled at approximately 40 mc per milliatom of carbon (New England Nuclear Corp., NEC-445). In many experiments the cells were labeled for 15-20 sec with H3-uridine, at 1 ,uc/ml (H3-uridine, the Radiochemical Centre, Amersham; specific activity 25.4 c/mM). Other concentrations of H3-uridine and other labeling times were also used and these are specified in the tables and figure legends. The incorporation was stopped by pouring the cells over 100 ml of PHO which had been frozen at -70°. The cells were centrifuged, washed once with PH 1, and resuspended in 1.8 ml of PH 1. Lysozyme spheroplasts were then prepared at 0° by addition of lysozyme (Muramidase, 2 X crystallized, Worthington Biochemical Corp.) to a final concentration of 400 jug/ml plus 1 mM

proceedings of the national academy of sciences of the united states of america

Replication of bacteriophage ribonucleic acid: alterations in polyribosome patterns and association of double-stranded RNA with polyribosomes in Escherichia coli infected with bacteriophage R17.