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An RNA sedimenting at 10 S with a molecular weight of 2.3 X 106, was isolated from rabbit reticylocyte polyribosomes. When this RNA is added to a cell-free extract from Krebs II ascites cells, both a and ,B chains of rabbit hemoglobin are synthesized; g chains are made in about 50% excess over a chains.Isolation and characterization of the messenger RNA for a and # chains of rabbit hemoglobin has been reported by several groups (1-7). The informational content of this RNA has been assayed by adding it to a eukaryotic cell-free extract that was already synthesizing a homologous hemoglobin (3, 7) or myosin (5), or to a bacterial cell-free extract (4). The protein made in these extracts was analyzed by separation of the polypeptide chains into those whose synthesis was directed by endogenous messenger RNA and those whose synthesis was the result of added RNA. We report here the synthesis of both a and 13 chains of rabbit hemoglobin when 10S messenger RNA from rabbit reticulocytes is added to a cell-free extract from Krebs II ascites cells. This cell-free extract can be incubated to eliminate the synthesis of endogenous protein, while it retains the ability to respond to added viral RNA (8,9). Our results extend the use of previously incubated extracts of Krebs II ascites cells to characterization of proteins encoded by isolated mammalian messenger RNAs. METHODSThe 10S messenger RNA was prepared from rabbit reticulocytes by the method of Labrie (2). Polyribosomes were dissociated in EDTA, and the ribonucleoprotein particle that contained 10S messenger RNA was separated from ribosomal subunits on a sucrose density gradient (2). The RNA was then precipitated by adjusting the appropriate fractions to 0.1 M NaCl and 0.5% sodium dodecyl sulfate (SDS), followed by the addition of 3 volumes of ethanol. After 2 hr at -20'C, the precipitate was collected by centrifugation, dissolved in 0.1 M NaCl-0.5% SDS-1 mM EDTA-0.01 M Tris [pH 7.4 (SDS buffer)], layered over a 15-30% sucrose density gradient made in the same buffer, and centrifuged for 21 hr at 22°C and 27,000 rpm in the Beckman SW 27 rotor. The gradient was monitored for Aseonm; fractions containing RNA sedimenting at 10 S were pooled and precipitated with ethanol.The precipitated RNA was dissolved in SDS buffer and extracted with phenol-chloroform, as described by Penman (10). After precipitation with ethanol, the RNA was washed at least three times with ethanol at -20°C to remove SDS, dissolved in water at a concentration of 1 mg/ml, and stored 2716 at -70°C. 40 ug of purified 10S RNA was prepared from 10 mg of polyribosomes. Maintenance of Krebs II ascites cells in mice has been described (8). A cell-free extract was prepared from these cells according to a method communicated to us by M. B. Matthews, and used by him and others (8, 9). Mice bearing tumors were killed 7-10 days after inoculation, and the ascites cells were removed from the peritoneal cavity into 0.146 M NaCl-35 mM Tris (pH 7.5). They were collected by centrifugation at 800 rpm (120 X g) for 2 ...

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