Fate of donor DNA in pneumococcal transformation

Abstract

The molecular fate of homologous (pneumococcal) and heterologous ( Escherichia coli) [ 32P]DNA was examined after introduction into pneumococcal cells. In both cases the composition of 32P-containing products at the earliest time of observation was the same. About 50% of the incorporated donor material was in the form of single strands, identified by their density in alkaline CsCl gradients. Of the incorporated 32P, 20 to 30% was in the form of native DNA of pneumococcal composition. Since this was true for donor DNA from E. coli as well as for donor DNA from pneumococcus, it appears that this label entered native DNA by way of de novo synthesis of DNA, presumably from nucleotides. The remaining 32P in the cell was in the form of dialyzable fragments composed of large amounts of inorganic phosphate and l-α-glycerophosphate and smaller amounts of the four 5′-deoxynucleotides. A plausible origin of labeled inorganic phosphate is the dephosphorylation of nucleotides. The labeled glycerophosphate, in turn, may be derived from inorganic phosphate, since artificially introduced [ 32P]phosphate rapidly equilibrated with a large pool of intracellular glycerophosphate. Thus, the initial products of entry are envisioned to be equal amounts of single strands and 5′-deoxynucleotides. This would be compatible with their origin in the degradation by a pneumococcal exonuclease of one strand of an incoming DNA duplex. On subsequent incubation, single strands of heterologous origin were slowly degraded; their breakdown products were used for de novo DNA synthesis. The disappearance of homologous single strands was much faster and corresponded to an increase of 32P in native DNA as well as to the recovery from eclipse of donor marker-transforming activity. It is concluded that single strands are precursors of genetically integrated DNA and that recombination involves the interaction of homologous single strands with host DNA. The ratio of genetically integrated DNA to total DNA incorporated suggests that only half, or somewhat more, of the single-stranded DNA was inserted as intact sequences. Inhibition of DNA synthesis by aminopterin gave results which were compatible with these conclusions.