The replication of bacteriophage MS2: 1. Transfer of parental nucleic acid to progeny phage

Abstract

The genetic information of the small bacteriophage MS2 is stored in a single-stranded RNA molecule. This thesis is an analysis of the mechanism whereby the MS2 genetic material is replicated. In Part I, the presence of phage-specific double-stranded RNA in MS2-infected cells is demonstrated. The kinetics of conversion of the input parental strand to a duplex form, and of the formation of progeny duplexes, is also described. In Part II, the replication of phage-specific RNA duplexes is studied by means of density, labeling and CsC1 equilibrium density sedimentation. Neither progeny nor parental duplexes are conserved, which is strong evidence that the duplex plays some role in replication. The above experiments were performed on material isolated after ribonuclease digestion of the nucleic acids from infected cells. In Part III, two other procedures are described which permit identification of phage-specific replicative intermediates (RI) without use of ribonuclease: the host cell RNA synthesis can be preferentially blocked by use of the antibiotic, Actinomycin D; or RI can be separated from the other infected cell components by a centrifugal method based on studies of RNA denaturation with the organic solvent DMSO. These procedures lead to the conclusion that the true RI is a heterogeneous, approximately 15S component which is only partially double-stranded. In Part IV, use is made of this partial double-stranded nature to purify and fractionate the RI on columns of benzoylated, naphthoylated DEAE cellulose, which seem to fractionate nucleic acids on the basis of secondary structure. From measurements of the ribonuclease-resistance and the buoyant densities of purified RI fractions it was concluded that the RI consists of a duplex of constant length to which is attached one single-stranded of length less than the viral RNA. The RI is only infective in the spheroplast assay when denatured. This structure for the RI is interpreted to mean that the nascent strand is still attached after purification of the RI. It was therefore possible to determine the mode of replication by determining whether the consisted of the nascent strand (conservative replication) or the displaced portion of the viral strand (semi-conservative replication). Appropriate labeling experiments indicated that the tail could arise from either origin, equally often. It was concluded that double-stranded RNA is involved in phage RNA replication and that replication is both conservative and semi-conservative, equally often