Fate of Transforming Deoxyribonucleate following Fixation by Transformable Bacteria: II

In the genetic transformation of pneumococcus, it has been demonstrated that newly introduced transforming deoxyribonucleate (DNA) is fully integrated in the genetic sense with the host genetic material very soon after its irreversible fixation by the transformable bacteria. This process has been shown to require little, if any, net DNA synthesis for its completion.' In addition, it has been demonstrated that from the time integration is completed newly introduced DNA produces specific replicates at a rate which parallels the further growth of the transformed population.2 Observation of the loss of biological activity of DNA as a consequence of the disintegration of incorporated radioactive phosphorus atoms provides a tool for further examination of these processes.3 Transforming DNA carrying a high specific activity of radioactive phosphorus (P32) has been used to transform populations of pneumococcus. Following fixation of DNA by recipient cells, the total DNA was reisolated from the transformed bacterial population. The biological activity of a genetic marker present in the original radioactive transforming DNA and representing the newly introduced DNA in the reisolated material from the transformed bacteria can be precisely determined. The rate at which this activity is lost as a consequence of p32 decay has been measured. A comparison between the rates of inactivation of a marker when present in the original DNA and that same marker after it has been introduced into a bacterial host and reisolated has permitted an assessment of the minimum size of the fragment of DNA integrated into a bacterial genome in the course of the transformation process. The biological activity of radioactive DNA that has been fixed by transformable bacteria and allowed sufficient time prior to reisolation so that integration is practically complete is not less sensitive to inactivation by p32 than was biological activity of the original DNA. The retention of the same target size in the reisolation DNA excludes the possibility of any specific DNA synthesis in the physical region of the newly introduced marker during the integration process. It also excludes the possibility that any fragmentation occurs during this process, within the size limits of the observed target, of the DNA entity responsible for transformation. A streptomycin-resistant strain of pneumococcus was grown on a medium containing p32 as orthophosphate at a specific activity of 710 millicuries per milligram phosphorus. The measurement of the specific activity of the phosphorus in DNA yields a number in close agreement with that obtained from the ratio of carrier free p32 to the quantity of inorganic phosphate in the growth medium. After allowing a 20-25 fold increase in bacterial density, the culture was chilled, and both a large excess of inorganic phosphate and carrier "wild type" bacteria were added. DNA was isolated after lysis with deoxycholate, by the chloroform gel method and alcohol precipitation. The DNA preparation contains a mixture of P32-con-