INFECTIOUS NUCLEIC ACIDS OF E. COLI BACTERIOPHAGES, IX. SEDIMENTATION CONSTANTS AND STRAND INTEGRITY OF INFECTIOUS M12 PHAGE REPLICATIVE-FORM RNA*

Since 1964, when several laboratories reported on phage-specific double-stranded RNA in RNA phage-infected cells (literature cited in refs. 11 and 12), different studies have been performed on the function of this RNA species. Today double-stranded RNA is widely assumed to be an intermediate in the process of replicating single-stranded phage RNA of the f2 phage class. Controversial results exist only for the replication of phage Qf3 RNA. ' 2 According to the most probable model, double-stranded molecules (replicative form, RF) replicate phagetype RNA (plus strands) semiconservatively and asymmetrically.3' 4 The intact complementary minus strand in the RF serves as a template on which plus strands are synthesized. The first plus strand synthesized is thought to be displaced by the synthesis of the following one. According to this hypothesis, a heterogeneous population of double-stranded molecules should exist composed of two classes, pure double-stranded molecules and double-stranded molecules with nascent singlestranded tails of different length (the latter class is also termed replicative intermediate4). The model described is based mainly on two lines of argument. (1) In pulse-labeling experiments, one part of the pulse passes at first through an RNase-resistant double-stranded RNA and is later found in single-stranded plus strands.4-7 Another part of the pulse stays in the double-stranded RNA and is incorporated into minusstrand material, as revealed by hybridization experiments.8 (2) The sedimentation constants of the double-stranded RNA vary widely, indicating heterogeneity of the material.4 81-0 Using the dependence of molecular weight and sedimentation constant of DNA for the calculation of the molecular weight of double-stranded RNA, the molecular weights of some of this RNA exceed twice the molecular weights of single-stranded phage RNA. Thus the RNase-resistant complexes could contain complete plus and minus strands. While these arguments are plausible, they are not necessarily convincing. It has not yet been established that the calculation of molecular weights with the formula for DNA is also applicable for double-stranded RNA. One can argue that the RNase-resistant complexes have, indeed, smaller molecular weights. They could then be only nonfunctional fragments composed of plus strands being complemented with minus counterparts of different length. But even assuming the molecular weight calculation to be correct and in good agreement with the postulated size of RF, it does not prove the continuity of the strands, i.e., single-stranded breaks in the double-stranded molecule would not be excluded. To resolve this problem, studies on size and strand integrity of M12 RF were continued. M12 RF hasalreadybeen demonstrated to be infectious.1' Itmust therefore harbor at least one intact strand, which is probably the plus strand. M12 RF is found in the infected cell prior to free single strands and does not accumulate late in the infection cycle.12 Thus, it cannot be classified as a late artifact.