STUDIES ON INVITRO TRANSCRIPTION AND TRANSLATION OF VESICULAR STOMATITIS-VIRUS MESSENGER-RNA

Optimal conditions are described for the preparation of transcribing vesicular stomatitis virus [VSV] ribonucleoprotein [RNP] cores, and for the coupled in vitro transcription and translation of VSV mRNA using RNP cores as a generating source of mRNA. The system was used to study some aspects of the mechanism and regulation of VSV mRNA synthesis. The effect of varying ionic strength and of extracts from eukaryotic cells [rabbit reticulocytes, mouse Krebs 2 ascites cells, wheat embryos] on size and composition of VSV in vitro RNA were examined. Coelectrophoresis in formamide-polyacrylamide gels of VSV in vitro and in vivo RNA showed that RNA synthesized in vitro under standard conditions (100 mM salt) was much more heterogeneous and most of it considerably smaller than in vivo RNA. Either high ionic strength (250-500 mM salt) or the presence of a wheat embryo extract were required for the synthesis of RNA with molecular weights and molar ratios comparable to that of in vivo RNA. In the presence of the wheat embryo extract RNA species corresponding in size to complete copies of the entire VSV genome were reproducibly synthesized in vitro. Some of the findings are consistent with a model of the transcription mechanism involving initiation of the various VSV mRNA at 1 common promotor site and generation of messenger size RNA by a cleavage process.