Viral DNA in Polyoma- and SV40-Transformed Cell Lines
It is clear that cells transformed by the small DNA-containing viruses, polyoma virus (Py) and SV40, harbor the viral DNA since they contain viral mRNA,1 and some lines even yield infectious virus after fusion with cells able to support the multiplication of the virus.2, 3 Very little is known, however, about the state of the viral DNA in these cells. In the past, several attempts were directed at determining the average number of copies of the viral DNA per cell by molecular hybridization of the DNA extracted from transformed cells with either viral DNA, or RNA made in vitro by copying the viral DNA. These efforts, however, were handicapped by two main technical difficulties. One is to prepare viral DNA uncontaminated by cellular DNA; the other difficulty is to have available a hybridization procedure capable of approaching the detection of one viral DNA molecule per cell, that is, one part of viral DNA among 20106 parts of cellular DNA. In the experiments to be described below, both requirements have been met. Pure viral DNA was obtained by isolating the supercoiled component I4–6 which is uncontaminated by cellular DNA. The hybridization procedure between cellular DNA and RNA made in vitro on purified component I DNA was that of Gillespie and Spiegelman,7 with a number of small improvements which have increased its sensitivity and reproducibility. Fewer than five viral DNA molecules per cell can now be unambiguously identified.
KeywordsSodium Dodecyl Sulfate Infectious Virus Polyoma Virus Reconstruction Experiment Uridine Triphosphate
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