Isolation and Characterization of IFN-Gamma mRNA Derived from Mitogen-Induced Human Splenocytes

Abstract

Human splenocytes from large cultures (5 1) were stimulated with Staphylococcus enterotoxin A (SEA). After 2.5 d of conditioning, poly A⁺ RNA was isolated from these cultures and its in-vitro translational capacity was evaluated in a wheat germ extract. Injection of the RNA into Xenopus laevis oocytes yielded a low but reproducible amount of IFN activity, which was neutralizable only by an antiserum to gamma interferon (IFN-γ) and not by antisera to alpha interferon (IFN-α) and/or beta interferon (IFN-β). Formamide sucrose gradient centrifugation analysis indicated that the mRNA which codes for IFN-γ sediments at around 15 S. The profile, however, suggested a size heterogeneity of IFN-specific mRNA. Electrophoresis of cDNA synthesized on these mRNA fractions indicated that the mRNA coding for gamma IFN has a length of between 900 and 1400 nucleotides.