PURIFICATION OF BIOLOGICALLY-ACTIVE GLOBIN MESSENGER-RNA USING CDNA-CELLULOSE AFFINITY CHROMATOGRAPHY

Abstract

A complementary DNA (cDNA) copy of mouse globin mRNA was synthesized using the RNA-dependent DNA polymerase from avian myeloblastosis virus and the oligo(dT) covalently attached to cellulose as primer. All 4 deoxyribonucleotide triphosphates, NaCl, the globin mRNA template and an oligo(dT) primer were required for optimal synthesis of cDNA. By saturating the primer sites using a 3-fold excess of mRNA, sufficient concentrations of immobilized cDNA could be synthesized to allow the hybridization reactions to be performed using an excess of globin cDNA. Conditions which permitted the annealing of globin mRNA to cDNA-cellulose were selected and the sequence specificity for hybridization to cDNA-cellulose was determined using 28 S rRNA, polyA, and mouse L-cell [transformed fibroblast] RNA. Both analytical and preparative applications of this chromatographic medium were explored. When radioactively labeled poly(A)-containing 9 S RNA isolated from nucleated erythroid cells was analyzed by affinity chromatography on globin cDNA-cellulose, 46% of the applied radioactivity hybridized to the cDNA-cellulose column. Only 1% of the labeled RNA was retained by the column during reapplication of the unbound fraction, while 96% of the bound RNA reannealed to cDNA-cellulose. Hybridizations utilizing unfractionated RNA extracts from either mouse reticulocytes or nucleated erythroid cells provided a 1 step purification method for globin mRNA sequences. The relative purity of the RNA isolated by cDNA-cellulose affinity chromatography was determined by hybridization kinetic analysis. The cDNA-bound fraction obtained from the unfractionated RNA of either cell type had a Crt1/2 [value of the product of the concentration of RNA in an annealing reaction and the time of incubation [Crt] at which 50% of the [3H] cDNA is hybridized] of 2.7 .times. 10-3. This represents a 60-fold purification of the globin sequences present in reticulocyte polysomal RNA and a 280-fold enrichment of the globin mRNA in nucleated erythroid cells. Hybridization to cDNA-cellulose did not result in any change in the sedimentation rate of globin mRNA. Experiments were performed which demonstrated that the globin mRNA isolated by hybridization to cDNA-cellulose retained its biological activity when assayed in a wheat germ cell-free lysate.