A rationale for an analysis of RNA replication.

The intent is to trace our efforts at understanding the molecular life history of RNA genomes using the E. coli-RNA phage system.' In the process, an attempt will be made to provide a background of the thinking that went into the experimnerits to be described. Because of the usual dominance of serendipity in biological and biochemical research, it is not often. thalt it is either informative o:r useful to record the reasons for a particular set of experiments; noting the outcome is usually sufficient. However, the present instance may represent an exception. (1) Problemn of Communication between an RNA Virus and Its Host Cell.-We start with the fact that all organisms which use RNA as genetic material are mandatory intracellular parasites. They must, therefore, carry out a major part of their life cycle in cells which use DNA as genetic material and RNA as genetic messages. On entry, the viral RNA is faced with the problem of inserting itself into the cellular information flow pattern in. order to communicate its own instructions to the synthesizing machinery. A possibility one might entertain centers on whether an RNA virus employs the DNA-to-RNA pathway of information flow. This could occur either because the DNA of the host already contains a sequence homologous to the viral RNA (i.e., the "escaped genetic message" hypothesis), or because such DNA sequences are generated subsequent to infection by reversal2 of the DNAdependent RNA synthesizing reaction. It is clear that a decision on the existence or nonexistence of homology between viral RNA and the host DNA is a necessary prelude to further experiments designed to delineate the molecular life history of an RNA genome. To answer questions of this nature, Doi and Spiegelman3 employed the specific hybridization test4 combined with the subsequently developed use of RNase to eliminate "noise." The sensitivity required had already been achieved in earlier experiments which identified the DNA complements of sRNA and ribosomal RNA.'-' Under conditions where complexes between 23S rRNA and IDNA were readily observed, none were detected between the viral RNA and the infected host DNA. The negative outcoml-e of the hybridization test implies that the DNA-to-RNA pathway is not employed, from-i which it follows that these RNA viruses must have evolved a mechanism of generating RNA copies from RNA. The existence is then predicted of an enzymatic mechanism involving an RNA-dependent RNA polymerase which we have named8 "replicase" for purposes of brevity and alliterative usefulness. It seems highly unlikely that an enzyme of this sort pre-exists in the cell. All recognized cellular RNA components, including the message fraction,9 the two ribo