Isolation of globin pre-messenger RNA on thiol-agarose by terminally mercurated complementary DNA

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Abstract
A mercurated DNA complementary to globin mRNA was prepared by the addition of mercurated poly(dC) tails to the 3′-end of the molecule using the enzyme terminal deoxynucleotidyl transferase. The mercurated complementary DNA was retained efficiently on thiol-agarose from which it was eluted by 2-mercaptoethanol. Hybridization of the mercurated probe to globin mRNA led to a specific selection of the latter from a mixed population of RNA through hybrid retention on thiol-agarose. In some pilot experiments this technique was applied for the isolation of globin gene-specific pre-mRNA. Pulse-labeled RNA up to 3 x 106 MW was thus isolated with prominent peaks of 1.5 kb (‘15 S’), 4.0 kb (‘28 S’) and up to about 10 kb. Electron microscopical analysis revealed pre-mRNA molecules of up to 1.2 μ (about 4.5 kb) in length isolated by hybridization to (Hg)cDNA; in control experiments, hybridization of this high MW RNA was competed out by highly purified globin mRNA. These data provide another indication for the existence of globin gene transcripts in the 10 kb range, i.e. transcripts larger than the about 1 500 nucleotides long ‘15 S’ pre-mRNA, the substrate to final splicing. Such ‘giant’ transcripts can be interpreted as either the obligatory primary pre-mRNA or only facultative transcripts of the globin genes.