The action of mutagens on MS2 phage and on its infective RNA. V. kinetics of the chemical and functional changes of the genome under hydroxylamine treatment

Abstract The kinetics of chemical and functional changes induced in the genome of bacteriophage MS2 by hydroxylamine under the conditions of predominant modification of either cytidine (pH 5.0) or uridine (pH 8.0) have been studied. Comparison of the kinetics of chemical modifications of monomeric nucleotides with those of bacteriophage inactivation at pH 8.0 and 5.0 made it possible to estimate the effective number of exposed cytidine and uridine residues in the intra-phage RNA ( B eff c and B eff u ). The B eff u was close to that expected and increased from 70 to 130 as the temperature rose from 0 to 30°. The B eff c was much greater than that expected on the basis of the results with the monomer, suggesting that side reactions are involved in the inactivation of the phage at pH 5.0. A significant increase of the frequency of mutation occurs only under the conditions of predominant modification of cytidine (pH 5.0) at 0°. No such effect was observed at 30°. This was probably due to the increased contribution of inactivating side reactions. The effect of hydroxylamine on the phage under the conditions of predominant uridine modification (pH 8.0) did not lead to an increase in frequency of mutants. Incubation of the intact phage in acetate buffer resulted in considerable inactivation and mutations. Inactivation was inhibited by magnesium ions. Incubation at pH 5.5, of the phage inactivated by hydroxylamine treatment at pH 8.0, resulted in a considerable increase of the inefectivity with no effect on the frequency of mutants. The infectivity and the mutation frequency of the phage treated with hydroxylamine at pH 5.0 did not change as a result of incubation at pH 4.0 after the removal of the reagent.