High molecular weight fragments of R17 bacteriophage RNA produced by pancreatic ribonuclease

Abstract

Limited digestion of R17 RNA with pancreatic ribonuclease A (0.4mμg RNase/100 μg RNA for 30 min at 0 °C) results in the production of at least ten fragments seen as discrete bands when analyzed by polyacrylamide gel electrophoresis. Molecular weights of the fragments were calculated from their mobilities using the mobilities and molecular weights of 16 and 23 s Escherichia coli ribosomal RNA for calibration. Fragments derived from the 3′-end of the parent molecule were identified, (1) by reacting sodium periodate oxidized R17 RNA with [ 14C]isonicotinic hydrazide, digesting with RNase, and analyzing the distribution of radioactivity after sucrose gradient centrifugation; or (2) by reducing periodated R17 RNA with [ 3H]NaBH 4, digesting, and then analyzing the distribution of radioactivity in nucleosides after polyacrylamide gel electrophoresis. Four fragments, all larger than half molecules are derived largely from the 3′-end of the parent molecule. Of the six fragments which are smaller than half molecules, only one (mol. wt = 410,000) appeared to have originated primarily from the 3′-end of the original genome. The molecular weights and the distribution of 3′-termini suggest that six of the fragments are the results of three scissions in the 5′-half of the molecule.