An in vitro analysis of a replicating molecule.

RNA viruses must carry out a major part of their life cycle in cells which use DNA as their genetic material and RNA as ge etic 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 to protein 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 that DNA sequences are generated subsequent to infection by reversal ?f the DNA dependent RNA synthesizing reaction. Either mechanism would predict homology between viral RNA and some segment of DNA derived from infected cells. 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 Spiegelman ('62) employed the specific hybridization test (Hall and Spiegelman, '61) com bined with the subsequently developed use of RNAase to eliminate "noise." The sensitivity required had already been achieved in earlier experiments which identified the DNA complements of ribosomal RNA (Yankofsky and Spiegelman, '62a, b; '63) and s-RNA (Giacomoni and Spiegelman, '62; Goodman and Rich, '62). Under conditions where com plexes between 23S r-RNA and DNA were readily observed, nono were detected between the viral RNA and the infected host DNA. The negative outcome of the hybridization test implies that the DNA to RNA pathway is not employed. We must conclude that these RNA viruses have evolved a mechanism of generating RNA copies from RNA. We predict then the existence of an enzymatic mechanism involving an RNA dependent RNA Polymerase which we have named "replicase" for purposes of brevity and alliterative usefulness.

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