Estimation by reassociation assay of viral DNA copies in three polyoma virus transformed cell lines.

We have determined the number of polyoma virus (P V ) genome equivalents in PV transformed cell lines. The method used for estimation was the re­ association assay1 of denatured 3H-labelled DNA from transformed cell lines. 3H-labelled PV DNA was obtained by labelling of PV infected (polyoma wild type virus, a gift from Dr. J. Zemla, Bratislava) secondary mouse embryo cells with [6-3H] thymidine [50 /<Ci ml-1 in the presence of fluorodesoxyuridine (1 0 jug ml-1) ] and DNA isolation according to H irt2. Purification of PV DNA was achieved by ribonuclease treatment, a two-fold ethanol precipitation, and three centrifuga­ tion steps (CsCl sedimentation, alkaline sucrose sedimentation, and CsCl ethidium bromide equili­ brium centrifugation)3. We used form I of PVD NA. The DNA was sheared by sonication (4 to 6S) and denatured by heating for 10 min at 100 °C and rapid cooling. Cell DNA was prepared according to Colter et al. 4 from three PV transformed cell lines. The lines PyBHK and Pyts3-BHK were obtained from Dr. H. Werchau (University Bochum), and line PyBHK R.C. from Dr. H. Tiirler (Geneva). After shearing and denaturation, the DNA was reassociated with 3H-labelled PV DNA at 66 °C in one-ml probes containing sodium phosphate (pH 6.9) and 0.4% SDS. After different incubation periods, aliquots were drawn from the incubation mixtures and diluted with H20 to obtain a phosphate concentration of 0.14 m . DNA reassociation was monitored by DNA fractionation on hydroxyapatite columns (2 ml volume; DNA grade Biogel HTP from BioRad Laboratories). Single-stranded DNA was obtained in the 0.14 M phosphate (0.4% SDS) column eluates and double-stranded D NA in the 0.4 M phosphate eluates. After DNA precipitation with TCA on glass