INFECTIOUS NUCLEIC ACIDS OF ESCHERICHIA COLI BACTERIOPHAGES .11. DIFFERENTIATION BETWEEN CONSERVATIVELY AND SEMICONSERVATIVELY REPLICATING RNA INTERMEDIATES OF BACTERIOPHAGE M12 BY FORMALDEHYDE INACTIVATION

Replicative intermediate RNA was purified from cells infected with M12. The infectivity of this replicative intermediate was inactivated with formaldehyde using conditions in which the infectivity of double-stranded RNA is not affected. Two types of replicative intermediate RNA could be distinguished by their different sensitivities to formaldehyde. 80% (or more) of the total infectivity was inactivated at a rate similar to that of single-stranded RNA. This indicates that the single-stranded portion of the replicative intermediate is part of the complete (infectious) viral strand (semiconservative replication mechanism). The infectivity of the residual 20% (or less) was more resistant to formaldehyde, indicating that the viral strand remains in the duplex (conservative replication mechanism). The ratio of five to one in favour of the semiconservative type was found to be constant throughout the replication cycle. Dimethyl sulphoxide was used to reduce the degree of secondary structure of single-stranded RNA. In 45% dimethyl sulphoxide, different rates for the formaldehyde inactivation of replicative intermediate RNA could be demonstrated: the rate increased with the length of the single-stranded portion of the infectious strand. Treatment of the replicative intermediate with 60% dimethyl sulphoxide converted the conservative type into a completely formaldehyde-resistant form with the sedimentation properties of pure double-stranded replicative form RNA. The semiconservative type molecules, not denatured by 60% dimethyl sulphoxide, were shown to contain not more than 0.5% formaldehyde-resistant viral strands. This excludes the possibility of major amounts of replicative intermediate RNA replicating complementary (minus) strands semiconservatively. © 1969.