A fraction of mouse RNA containing messenger RNA coding for mouse interferon was translated with high efficiency in a wheat germ system into a fully active product. This product fulfills the criteria for mouse interferon, namely: (1) it was active against vesicular stomatitis virus and herpes simplex virus in mouse cells; (2) its antiviral activity was species specific; (3) its activity was completely neutralized by mouse anti-interferon serum. The synthesis of interferon in this cell-free system requires the presence of spermine.Interferon is a species-specific protein with high antiviral activity that is induced in most vertebrate cells by either virus or double-stranded RNAs. Using metabolic inhibitors, it was shown that interferon mRNA appears very soon after induction (1). We have previously described a biological assay of interferon mRNA using heterologous intact cells (2).We report herewith that the mouse interferon message can be efficiently translated in vitro by wheat germ extracts. The product thus synthesized possesses both antigenic and antiviral properties of mouse interferon as well as its species specificity. MATERIALS AND METHODSInduction of Cells for mRNA Extraction. Mouse C-243 cells, a line transformed by murine sarcoma virus (3), were induced with poly(I)-poly(C) in large petri dishes (150 mm diameter); they received 10 ml of MEM (Eagle's minimum essential medium) without serum containing 30 ,ug/ml poly(I)-poly(C) and 100 ,g/ml of DEAE-dextran. The inducer was left for 2 hr on the cells and then removed; the cells were washed once with warm MEM and 30 ml of MEM containing 3% calf serum were then added. Nine hours after the onset of induction, the cells were harvested and frozen at -70°until the time of RNA extraction.RNA Extraction was performed according to the two-step technique previously described (2). A typical extraction, in which cells harvested from 15 confluent dish cultures were utilized, yielded 3550 .g of RNA in the A phase (RNA solubilized in aqueous layer after sodium dodecyl sulfate and phenol extraction), and 2360,ug in the I phase (RNA solubilized only after Pronase treatment). The I phase was further fractionated by sedimentation over a 10-20% sucrose gradient for 30 min at 30,000 rpm in a SW 41 rotor. The bottom half of the gradient contains rapidly labeled nuclear RNA in an aggregated form, whereas the upper half (called 13) contains nuclear RNA as well as cytoplasmic poly(A)-containing mRNA (L. Montagnier and H. Collandre, in preparation). Since this latter fraction was found to be the most active, using the mRNA assay in intact Vero (African green monkey) cells, it was exclusively utilized for the in vitro translation experiments.Preparation of Wheat Germ Extractsk. Commercial wheat germ, mechanically extracted and not toasted, was supplied by Grands Moulins de Paris, Paris (France). Before use, the wheat germ was pre-washed with the grinding buffer: 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) (pH 7.6); 100 mM potassium chloride; 1 mM magn...

Native Escherichia coli polynucleotide phosphorylase can be retained on blue-dextran -Sepharose. The bound enzyme cannot be displaced by its mononucleotide substrates such as ADP, UDP, CDP, G D P and IDP, but it is easily eluted by its polymeric substrates. Under identical conditions, lactate dehydrogenase, bound on blue-dextran -Sepharose, is not eluted by poly(1) but can be specifically displaced by NADH. On the other hand, the trypsinized polynucleotide phosphorylase, known to be an active enzyme which has lost its polynucleotide site, does not bind to the affinity column. The native polynucleotide phosphorylase can also be tightly bound to poly(1) -agarose and displaced from it only by high salt concentration. The trypsinized enzyme is not bound at all on poly(1) -agarose. Moreover, the native enzyme linked on blue-dextran -Sepharose, remains active indicating a free access of nucleoside diphosphates to the active center. These results taken together show that the dye ligand is not inserted onto the mononucleotide binding site and suggest rather that it binds to the polynucleotide binding region. The implications of this study and the application of blue-dextran -Sepharose affinity chromatography to other proteins having affinity for nucleic acids are discussed.Blue-dextran -Sepharose has been widely used as an affinity chromatography tool for studies and purification of enzymes which have a nucleotide as substrate or co-factor, such as dehydrogenases, kinases and nucleotidyltransferases (cf. [l I). Among the first two classes, many enzymes have the 'dinucleotide fold' structure [2,3]. It becomes tempting to associate the binding property of a protein to blue-dextran -Sepharose with the characteristic of having such a dinucleotide fold. We have extended the blue-dextran -Sepharose chromatography to studies of another class of enzymes that includes polynucleotide phosphorylase, RNA polymerase and aminoacyl-tRNA synthetases. Such enzymes have, in addition to the mononucleotide binding site, other site(s) for binding of oligonucleotide or polynucleotide (RNA). Results of the present work show that polynucleotide phosphorylase binds to blue-dextranSepharose through a site other than the nucleoside diphosphate binding site and suggest that the bluedextran -Sepharose . enzyme binary complex results from the binding of the dye to the polynucleotide binding site of the enzyme. (Due to lack of information on the precise structure of the polynucleotide binding site, this term refers only to the area of the enzyme -~

SUMMARYMouse interferon (IFN) was produced to high titres after induction of Ehrlich ascites tumour cells with Sendai virus by using an improved procedure. The 1FN molecules were labelled during their synthesis by the incorporation of [3H]leucine and [3H]lysine. Electrophoretically homogeneous labelled IFN with a molecular weight of 34000 was obtained after a two-step purification procedure using poly(I)-agarose and octylSepharose column chromatography followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The specific radioactivity of this IFN was about l0 ct/min/IFN unit.

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