Proc. Natl. Acad. Sci. USA, 74 (8). pp. 3409-3413.

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Abstract

Translation of injected mRNA in oocytes of Xenopus laevis has been used as a highly sensitive and quantitative assay for interferon mRNA. Injection into oocytes of polyadenylylated RNA extracted from poly(I)poly(C)induced human diploid fibroblasts (FS-4) leads to the synthesis of biologically active human fibroblast interferon over a period of 24-32 hr. There is a linear relationship between the amount of mRNA injected and the interferon yield obtained over a range of 1-20 ng of injected RNA. Injection of 40-80 ng of mRNA into each of 15 oocytes, homogenized in 0.3 ml of incubation medium, gave a titer of 128-256 interferon reference units/ml of homogenate. FS-4 cells at the peak of interferon production-i.e., approximately 2.5 hr after the beginning of induction with poly(I)poly(C-gave mRNA that yielded 24-48 interferon reference units/ml in the oocyte assay (30 ng of RNA injected per oocyte). An equivalent amount of mRNA from FS-4 cells in the shutoff phase, approximately 6 hr after induction, gave <4 interferon reference units/ml. In contrast, mRNA extracted from FS-4 cells that had been induced and maintained in the presence of 40 uM 5,6-dichloro-1-j-o-ribofuranosylbenzimidazole for 6 hr produced 64-128 interferon reference units/ml. Polyadenylylated RNA obtained from uninduced FS-4 cells did not lead to detectable interferon synthesis (<4 interferon reference units/ml). These data provide a direct verification of the hypothesis that the shutoff of interferon production in FS-4 cells involves a regulatory event leading to the posttranscriptional inactivation or degradation of interferon mRNA. Because the inactivating mechanism is sensitive to inhibition by 5,6-dichloro- 1--D-ribofuranosylbenzimidazole, a selective inhibitor of nuclear heterogeneous RNA and mRNA synthesis, it is likely that synthesis of an RNA molecule is necessary for the shutoff of interferon production.