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Temperature-sensitive markers can be of value in order to elucidate the mechanism of virus-induced cell transformation, and to demonstrate the expression of virus-coded genes in transformed cells. It has been shown in previous studies with large and small plaque strains of polyoma virus (PV) that a small plaque mutant of PV is temperature-sensitive for virus replication at high temperature.' Experiments on the time of the temperature-sensitive event suggested that this corresponded to the time of virus DNA synthesis.' The present experiments were undertaken: (1) to obtain direct evidence on whether the temperature-sensitive step in the PV mutant involves a block in the synthesis of virus DNA, (2) to determine whether the temperature-sensitive mutant can induce the synthesis of cellular DNA at the high temperature, and if so (3) whether cells transformed by PV (and by simian virus 40) can be differentiated from normal cells and cells transformed by nonviral carcinogens by their ability to replicate at the high temperature. Materials and Methods.-Isotope: H3-thymidine (16,000-18,000 mc/mM) was obtained from the Radiochemical Centre, Amersham, England. Cell cultures: Primary and secondary mouse embryo cultures used for plaque assays were grown in 0.5% lactalbumin hydrolyzate in Earle's saline (LA) with 10% horse serum. Primary mouse kidney cultures and secondary mouse embryo cultures used for experiments on virus replication were grown in Eagle's medium with a fourfold concentration of amino acids and vitamins (EM) with 5% calf serum, and all other cells were grown in EM with 10% calf serum. Clones of hamster and rat cells transformed by the small plaque PV mutant SP22 and the large plaque PV strain LP11 (previously referred to as l1)3 were isolated from soft agar4 after infection of secondary embryo cells with 100 or 1000 plaque-forming units (PFU) per cell. A line of PV and a line of SV40-transformed 3T3 cells5 were kindly supplied by Drs. H. Green and G. J. Todaro, and a line of SV40-transformed hamster cells was obtained from Flow Laboratories. The two hamster cell lines transformed by nonviral carcinogens were obtained after transformation of hamster embryo cells by 3-methylcholanthrene,6 and by X irradiation with 300 R.7 Virus: Stocks of the small plaque SP22 and the large plaque LP11' strain of PV were prepared from single plaques after three successive single plaque isolations. The virus stocks were used as lysates or purified as described previously.8 Plaque and infectious DNA assay: Plaque assays3 were made on 2-day-old secondary mouse embryo monolayers in LA with 10% horse serum, and the plaques were finally counted on day 15. DNA was extracted and the infectious DNA assayed as described elsewhere.9 The cultures were incubated at 290C for 30 min after addition of the DNA, and about 17 hr later 106 secondary mouse embryo cells were added to each plate before the addition of agar as in the usual plaque assay. Hemagglutination: The hemagglutination test was carried out as previously described10 in 0.01 M Tris-saline buffer, pH 7.2. Samples were heated for 30 min at 560C before the test. Incubation at 41 and 41.50C: In order to obtain better regulation of the temperature than that obtained in an incubator, cells were incubated in closed 125-ml conical beakers (Corning Works, catalogue no. 1080) in water baths whose temperature was maintained with a Thermomix II (Braun). The tops of the conical beakers were made circular, so that they could be

Temperature sensitivity of polyoma virus, induction of cellular DNA synthesis, and multiplication of transformed cells at high temperature.