Isolation of a biologically active messenger RNA: preparation from fish pancreatic islets by oligo(2'-deoxythymidylic...

RNA was extracted from the pancreatic islets of channel catfish in the presence of the ribonuclease inhibitor, diethyl pyrocarbonate (oxydiformate). High molecular weight RNA was observed on sucrose gradient analysis. enrichment of mRNA was achieved by oligo(2'-deoxythymidylic acid)-cellulose affinity chromatography. The mRNA fraction stimulated incorporation of [35S]methionine into protein up to 30-times the background in the wheat-germ cell-free system. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed two major proteins corresponding to molecular weights of 27 000 and 11 000. These proteins were not observed in the absence of mRNA or in the presence of mRNAs from other tissues. They were also synthesized in the ascites tumour cell-free system. No protein co-migrating with proinsulin or insulin was detected in either the ascites or wheat-germ cell-free systems. Pancreatic islet slices also synthesized the proteins of 27 000 and 11 000 molecular weight and smaller ones as well.