Transformation with specific fragments of adenovirus DNAs I. Isolation of specific fragments with transforming activity of adenovirus 2 and 5 DNA

Abstract

DNA of human adenoviruses 2 and 5 was cleaved by the restriction endonucleases HsuI, BamHI, HpaI, and SmaI. The resulting fragments were separated and tested for their ability to transform primary baby rat kidney (BRK) cells, using the calcium technique. Fragments with transforming activity were obtained with endo's R·EcoRI (fragments A), BamHI (fragments B of Ad2 and A of Ad5 DNA), and HsuI (fragments G). The transforming fragments all represented the left terminal fragments of the respective restriction endonuclease cleavage products. The smallest fragment found to contain transforming activity was the HsuI G fragment (molecular weight 1.7 · 10 6 for both Ad2 and Ad5 DNA). Transforming activity of both adeno DNAs was abolished by digestion with endo's R·HpaI and SmaI. This indicated that these enzymes cleave into an area essential for transformation. A number of cell lines transformed by restriction endonuclease fragments were established and some of their properties were studied. All adeno DNA fragment-transformed lines were found to grow to only a very low level in 0.33% agarose medium (cloning efficiency < 1%), irrespective of the size of the fragment used to transform the cells. All transformed cell lines were found to react with antiserum against adenovirus subgroup-C specific T antigen, as detected by immunofluorescence. The T antigen pattern in the HsuI G-transformed cells, however, was atypical and differed from the usual pattern, in that the fluorencence was largely localized in the cytoplasm. Selection of HsuI G-transformed cells in 0.33% agarose medium resulted in cell populations containing the typical adenovirus T antigen pattern.