Partial transformation of primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA

Abstract

This paper reports the identification and partial characterization of a transforming activity associated with the 1 × 10 6-dalton HpaI E fragment of the DNA of human adenovirus type 5. This fragment represents the left-terminal 4.5% of the viral genome, which essentially corresponds to early region EIa. The transforming activity of this fragment is approximately fivefold lower than that of larger DNA fragments, as measured by focus formation in primary cultures of baby rat kidney (BRK)cells. The HpaI E-transformed cell lines were found to contain between 0.8 and 16.4 copies of fragment HpaI E per diploid amount of cell DNA and only very low concentrations of virus-specific T antigen, as detected by immunofluorescence. An analysis of some of the clones for the presence of viral RNA transcripts showed that each line contained cytoplasmic RNA hybridizing with fragment HpaI E. The size of the RNAs, however, differed from that of RN As normally transcribed from this segment of the genome. A study of the phenotypical properties of HpaI E-transformed cells showed that they can be distinguished from BRK cells transformed by larger DNA fragments by (1) a more or less fibroblastic appearance, (2) a tendency to grow in parallel orientation (a property characteristic of untransformed fibroblasts), (3) a low growth rate, (4) the inability to reach high cell densities in monolayer culture. Some of these properties changed gradually upon prolonged passaging in vitro, in particular the growth rate and, to a lesser extent, the saturation density gradually approached those characteristic for transformed cells. Apart from these differences in properties, all HpaI E-transformed cells have at least two characteristics in common with other transformed cells: they all grow as immortal cell lines and they are all aneuploid. The results indicate that the leftmost 4.5% of Ads DNA is able to convert diploid cells in a primary culture into established aneuploid cell lines which still lack several of the properties characteristic for adenovirus 5-transformed cells.