INHIBITION OF INTRACTABLE NUCLEASES WITH RIBONUCLEOSIDE-VANADYL COMPLEXES - ISOLATION OF MESSENGER RIBONUCLEIC-ACID FROM RESTING LYMPHOCYTES

Human lymphocyte lysates prepared by detergent treatment of intact, normal resting cells contain ribonucleases that are insensitive to many inhibitors commonly used with eucaryotic cells. Phenol-extracted ribonucleic acid (RNA) obtained directly from unfractionated cytoplasm is sometimes degraded, but after fractionation of the cytoplasmic material by sucrose density gradient centrifugation, the polyadenylated RNA, in particular, is inevitably destroyed. An extensive survey of ribonuclease inhibitors, undertaken as a consequence, indicated that the complexes formed between the oxovanadium ion and the four ribonucleosides were unique in their ability to suppress lymphocyte nuclease activity. It proved possible to isolate intact ribosomal RNA and polyadenylated messenger RNA from lymphocyte cytoplasm fractionated on sucrose gradients when 2.5 mM each of the four ribonucleoside-vanadyl complexes was used throughout the procedure. The data showed that the size distribution of poly(A)-bearing RNA remained unchanged, with a peak at~16 S under denaturing conditions, regardless of whether the mRNA was originally associated with polysomes or was nonpolysome bound. The cytoplasmic RNAs were completely free of contamination by either intact nuclear RNA or nuclear fragments. Furthermore, exogenous globin mRNA mixed with lymphocytes and reisolated together with endogenous cytoplasmic polyadenylated RNA was fully translatable only when ribonucleoside-vanadyl complexes were employed during the preparation. The use of this inhibitor should therefore be considered for all tissues in which ribonucleases impede isolation of intact RNA. © 1979, American Chemical Society. All rights reserved.