PURIFICATION OF GLOBIN MESSENGER-RNA FROM DIMETHYLSULFOXIDE-INDUCED FRIEND CELLS AND DETECTION OF A PUTATIVE GLOBIN MESSENGER-RNA PRECURSOR

Procedures are described that permit the detection and isolation of a specific mRNA as well as its precursor from total cell extracts. DNA complementary to the mRNA was elongated by the addition of dCMP residues and annealed with labeled cell RNA. The elongated DNA with RNA hybridized to it was isolated by chromatography on a poly(I)-Sephadex column. The method was used to isolate 32P-labeled globin mRNA from labeled Friend cells, a mouse erythroleukemic cell line, induced with dimethylsulfoxide to synthesize Hb. 32P-labeled globin mRNA isolated by this procedure was estimated to be 80% pure by hybridization analysis and sedimented as a single peak at 10 S. Partial sequences were determined for 16 oligonucleotides derived from the purified 32P-labeled globin mRNA by RNase T1 digestion. The partial sequences for 9 oligonucleotides corresponded to those predicted from the amino acid sequences of .alpha. and .beta. globin; the other oligonucleotides were presumably derived from non-translated regions. In order to detect a possible precursor to globin mRNA, RNA from induced Friend cells pulse-labeled with [32P]phosphate for 20 min was centrifuged through a sucrose gradient and the resulting fractions were analyzed for globin-specific sequences. Two peaks of globin-specific RNA were detected, a larger one at 10 S, the position of mature globin mRNA, and a smaller one at 15 S.