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A substance produced by various types of cells can induce from single hematopoietic cells the formation of colonies of normal macrophages and granulocytes. This inducer has been purified 600-fold from serum-free conditioned medium from a tissue culture line of mouse cells. It is shown that the inducer is a protein, with a molecular weight of 65-70,000, whose inducing activity was about 1 ng per colony. Acrylamide gel electrophoresis of this material gave four bands, only one of which contained inducing activity. The purified protein was inactive. Activity was regained by the addition of a low molecular weight cofactor that is present in conditioned medium. Under certain conditions, activity was also regained by the addition of adenosine 3′:5′-cyclic monophosphate (cyclic AMP).

Mixing chromatographic fractions containing simian virus 40 (SV40) T In this report, we will describe a method for identifying a DNA binding activity in T-antigen-containing fractions which depends upon an interaction of this protein(s) with radioactive DNA. The assay depends upon the observation that unliganded, double-stranded DNA will not be efficiently trapped by a nitrocellulose filter except when bound to a protein that, itself, has affinity for the filter. This method was first employed in the assay of certain bacterial DNA binding proteins such as DNA-dependent RNA polymerase and the lac and X repressors (9-11).The present DNA binding assay has the advantage of being more rapid than the standard complement fixation test. Moreover, with the appropriate application, it may help shed some light on the biological function of T in vivo.

Messenger RNA was purified from human foreskin fibroblasts FS11, a high interferon-producer line, after induction with synthetic double-stranded RNA. The mRNA was translated in a cell-free protein-synthesis system from rabbit reticulocytes. The translation products, containing biologically active human interferon, were immunoprecipitated by a serum from rabbits immunized against partially purified interferon. Analysis of the immunoprecipitate by polyacrylamide gel electrophoresis in dodecylsulfate shows that the product of human fibroblast interferon mRNA is a 23000-M, polypeptide. Methods are described for the synthesis and rapid identification of this polypeptide, which should be useful for structural analysis of interferon and isolation of its mRNA.Interferon mRNA from human cells has been translated in heterologous intact cells [l,21, in Xenopus luevis oocytes [3, and in a variety of cell-free systems [3-51. In all these cases, as found also for mouse interferon mRNA [9-141, biologically active interferons, showing the correct species specificity and immunological properties, were obtained. The translation of human fibroblast interferon mRNA was reported to be low in cell-free protein-synthesis systems as compared to oocytes [5]. Use of cell-free extracts, such as nuclease-treated reticulocyte lysates [15], would however be advantageous to study the proteins synthesized, because of the high efficiency and low background of this translation system. This work describes the identification by immunoprecipitation and polyacrylamide gel electrophoresis, of a highly labeled 23 000-M, polypeptide which appears as the specific product of human fibroblast interferon mRNA translation in reticulocyte lysates. This method should allow the structural analysis of this polypeptide and facilitate the purification of its mRNA. MATERIALS A N D METHODS Growth oj'FS1 I Cells and Induction of' InterferonHuman foreskin fibroblast F S l l cell cultures were established in our laboratory by Dr D. GurariRotman. These diploid cells, grown from foreskin Abbreviations. Poly(r1) . poly(rC), poly(inosate) . poly(cytidy1-ate) double-stranded RNA; NaCI/Pi, phosphate-buffered saline; Hepes, 4-(2-hydroxyethyl)-l-piperdzineethdne sulfonic acid ; tricine, A'-tris(hydroxymethy1)-methyl-glycine; p.f.u., plaque-forming units; vsv, vesicular stomatis virus; CM, carboxymethyl. ~~ explants taken 8 days after birth, were selected among 15 individual isolates for their capacity to produce high titers of interferon. The cells were grown in Eagle's minimum essential medium with 10 o/, fetal calf serum in 5 % CO;?, 95 % air at 37 "C and maintained by subculture at 1 : 5, in 90-mm plastic dishes. For interferon production [16], five such plates of cells were seeded in 4-pint (2.27-1) roller bottles with 80 ml minimum essential medium containing 10% fetal calf serum. Three days after confluency (8th day) the cultures were exposed to 100 pg/ml poly(r1) . poly(rC) and 50 pg/ml cycloheximide in 25 ml medium without serum. After 3.5-4 h actinomycin D was added...