Haemoglobin Messenger in the Post-ribosomal Supernatant of Reticulocytes

Abstract

both give rise to two additional fragments six and seven bases long with the smaller fragment existing in twice the quantity of the larger one. The individual peaks appear on the chromatograph in the same position if re-run on DEAE-Sephadex or after a second digestion with pancreatic RNAase. This finding indicates that each fragment from the RNAase TI digestion is composed of two different oligonucleotides having five and six adenylate residues respectively, and further that there is a total of four terminal sequences in the globin mRNA. Although the pancreatic RNAase used in these experiments did not degrade poly(A) or oligo(A) under the conditions of the digestion (low salt concentration) it is known that the aggregate form of the enzyme does have activity towards poly(A) at low ionic strength. Therefore the pancreatic RNAase digestions of the fragments from the RNAase TI digestion were repeated at high salt concentrations, namely 0.1 M-NaCI, 20m~-Tris-HCl buffer, pH7.0, and ~ ~ M E D T A , at 37°C for 1 h. The results were identical, indicating that the maximum length of pure poly(A) at the 3’-hydroxyl terminus is five or six bases. The proposed sequences are summarized in Table 1. The terminally labelled RNA binds to a poly(U)-cellulose column and is eluted, along with the biological activity, between 45“ and 65”C, suggesting that there may be a longer poly(A) or A-rich region in the rabbit globin mRNA, as has been shown for mouse globin mRNA (Lim & Cannellakis, 1970) and duck globin mRNA (Pemberton & Baglioni, 1972). These results suggest that a long sequence of pure poly(A) does not occur at the 3’hydroxyl terminus of the globin mRNA but does not exclude the possibility that these adenylate residues are part of a longer adenylate-rich region.