On the production of highly labeled tumor virus deoxyribonucleic acid.

This work had as its objective the production of polyoma virus (PV) DNA of high specific radioactivity. Such a highly labeled tumorogenic virus DNA would have many applications in physical, chemical, and biological studies where high sensitivity of detection is essential-for example, for studies on the properties and fate of the viral gene after infection of a cell. This material has been producedl-4 by adding tritium-labeled thymidine (TdR-H3) to the growth media of PV-infected cell cultures, since TdR-H3 label appears only in DNA.5 6 We investigated the effect of 5-fluoro-2'-deoxyuridine (FUdR), an inhibitor of TdR synthesis,', 8 in promoting cellular utilization of exogenous high specific activity TdR-H3. FUdR inhibits DNA synthesis in bacteria7' 8 and in animal cells.'-" Sheinin" showed that FUdR inhibited DNA synthesis and PV production in dividing mouse embryo cells. Subsequent substitution of equimolar TdR for the inhibitor reversed this inhibition. No data was presented to show the effect of different levels of FUdR on DNA synthesis in infected cells. Weil"2 observed that addition of FUdR to confluent, PV-infected monkey kidney cell monolayers increased uptake of 5-bromo-2'-deoxyuridine-C'4 (BUdR-C14), a TdR analogue. Hirt'3 likewise noted that FUdR increased substitution of TdR by BUdR in DNA in PV-infected mouse kidney cultures. We therefore investigated the effect of different FUdR levels on dividing, uninfected and PV-infected, mouse embryo cells. DNA synthesis was then restored by adding TdR-H3 to the inhibited system. Virus was purified from the cell cultures, its DNA extracted, and its specific activity determined. We found that labeling of PV DNA was increased by more than threefold in the presence of the drug, to a level nearly sixfold greater than the previously reported. '