Partial Purification of the Messenger RNA for Eukaryotic Elongation Factor Tu from Artemia salina

Abstract

Polyadenylated RNA from developing Artemia salina cysts was fractionated by centrifugation through a sucrose gradient containing methylmercuric hydroxide (CH3HgOH). Aliquots of each fraction were directly added to a rabbit reticulocyte lysate to program protein synthesis in vitro. The translation products were assayed for eukaryotic elongation factor Tu (eEF-Tu) by immunoprecipitation with an antibody raised in rabbits and purified by affinity chromatography. The immunoprecipitated radioactivity was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Sequences coding for eEF-Tu sediment in the 20-S region of the gradient and form a major component of the poly(A)-containing RNA. The mRNA of the 20-S region, comprising about 10% of the poly(a)-containing RNA fractionated on the gradient, has been translated in vitro and 30% of the translation products represent immunoprecipitable eEF-Tu protein chains with an Mr of 50000.