Gene 2 (1977) 115--132 115 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
TRANSFORMATION WITH SPECIFIC FRAGMENTS OF A D E N O ~ R U S DNAs I. ISOLATION OF SPECIFIC FRAGMENTS WITH TRANSFORMING ACTIVITY OF ADENOVIRUS 2 AND 5 DNA (In vitro transformation of rat kidney cells; T antigen; restriction endonucleases; EcoRI; BamHI; HpaI; SmaI; HsuI) ALEX J. VAN DER EB, CAREL MULDER, FRANK L. GRAHAM* and ADA HOUWELING
University of Leiden, Laboratory for Physiological Chemistry, Sylvius Laboratories, Wassenaarseweg 72, Leiden (The Netherlands) and *McMaster University, Department of Biology, 1280 Main Street, West, Hamilton, Ontario (Canada) (Received March 10th, 1977) (Revision received and accepted June 10th, 1977)
SUMMARY
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 theHsuI G fragment (molecular weight 1.7 • l 0 s for both Ad2 and Ad5 DNA). Transforming activity of both adeno DNAs was abolished by digestion with endo's R.HpaI and Sinai. This indicated that these enzymes cleave into an area essential for transformation. A number of cell lines transformed by restn'ction 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 immunofiuorescence. The T antigen pattern in the HsuI G-transformed Abbreviations: BRK, baby rat kidney; HEK, human embryonic kidney; 0-ME, O-mereaptoethanol; MEM, minimal essential medium.
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cells, however, was atypical and differed from the usual pattern, in that the fluorescence 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. INTRODUCTION
Previous studies have shown that the DNA of adenovirus type 5 (ADS) is infectious for human cells (Graham and Van der Eb, 1973a) and that it is able to transform primary rat kidney cells in vitro (Graham and Van der Eb, 1973b). Further experiments have indicated that the transforming activity o f Ad5 DNA is unaffected by shearing the DNA into half or quarter molecules, suggesting that the integrity of the viral genome is not required for transformation. More extensive studies have confirmed this assumption, and showed that the segment of the Ad5 DNA involved in transformation is not larger than about I • 10 s daltons (4--5% of the AdS genome), and is located very closely to the left, hand end of the molecule, beginning at a distance of about 1% from-the left end and extending to about 5 6% (Graham et al., 1974a). As a further extension of these studies attempts were made to isolate the transforming gene(s) of adenovirus DNA as unique fragments, ideally as small as I • 106 daltons. Specific fragments of DNA can be obtained by utilizing bacterial restriction endonucleases(endo's R. ) which cleave DNAs at unique base sequences. Recently, several endo R. cleavage maps of Ad2 and Ad5 DNA have been obtained (Mulder et al. 1974a; R.J. Robert, personal commun.; C. Mulder and Greene, unpublished rebaits). This paper describes the isolation of unique fragments of Ad2 and AdS DNA obtained by cleavage with restriction endonucleases capable of inducing transformation of primary rat kidney cells in vitro. The smallest fragments of Ad2 and Ad5 DNA with transforming activity obtained so far have a molecular weight of 1.7 • 106 (7.3% of the viral genome)*. These fragments represent the left-terminal segments of both viral DNAs, confirm. ing the results obtained in our earlier studies with AdS DNA (Graham et al., 1974a). Evidence will be presented showing that the fragment-transformed cell lines contain adenovirus subgroup-C specific T-antigen, as detected by immunofluorescence. However, the distribution of the fluore~ cence in the cell lines transformed by the 7% fragments of Ad5 DNA is atypical mid differs from the u~ual pattern observed in adenovirus transformed cells. A preliminary report of some of these results has appeared elsewhere (Graham et al., 1974b). *Preliminary results suggest that a DNA fragment smaller than 7.3% of Ad5 DNA may also contain transforming activity.
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MATERIALS AND METHODS
Cells, viruses and viral DNA KB cells, used for cultivation of adenoviruses, were grown in monolayer cultures in Eagle's MEM supplemented with 8% newborn calf serum, or in suspension cultures in Eagle's MEM for suspension supplemented with 5% horse serum. Adenovirus type 2 (strain "adenoid 6") and adenovirus type 5 (strain "adenoid 75") were obtained from the American Type Culture Collection. KB cells were infected with 20 pfu of adenovirus per cell, and were incubated in MEM plus 2% horse serum (monolayer cultures) or in MEM for suspension plus 5% horse serum (suspension cultures). The procedures for purification of the virus and isolation of viral DNA have been described (Van der Eb et al., 1969). Primary cultures of BRK cells were prepared from kidneys of 6--7
TRANSFORMATION WITH SPECIFIC FRAGMENTS OF A D E N O ~ R U S DNAs I. ISOLATION OF SPECIFIC FRAGMENTS WITH TRANSFORMING ACTIVITY OF ADENOVIRUS 2 AND 5 DNA (In vitro transformation of rat kidney cells; T antigen; restriction endonucleases; EcoRI; BamHI; HpaI; SmaI; HsuI) ALEX J. VAN DER EB, CAREL MULDER, FRANK L. GRAHAM* and ADA HOUWELING
University of Leiden, Laboratory for Physiological Chemistry, Sylvius Laboratories, Wassenaarseweg 72, Leiden (The Netherlands) and *McMaster University, Department of Biology, 1280 Main Street, West, Hamilton, Ontario (Canada) (Received March 10th, 1977) (Revision received and accepted June 10th, 1977)
SUMMARY
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 theHsuI G fragment (molecular weight 1.7 • l 0 s for both Ad2 and Ad5 DNA). Transforming activity of both adeno DNAs was abolished by digestion with endo's R.HpaI and Sinai. This indicated that these enzymes cleave into an area essential for transformation. A number of cell lines transformed by restn'ction 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 immunofiuorescence. The T antigen pattern in the HsuI G-transformed Abbreviations: BRK, baby rat kidney; HEK, human embryonic kidney; 0-ME, O-mereaptoethanol; MEM, minimal essential medium.
116
cells, however, was atypical and differed from the usual pattern, in that the fluorescence 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. INTRODUCTION
Previous studies have shown that the DNA of adenovirus type 5 (ADS) is infectious for human cells (Graham and Van der Eb, 1973a) and that it is able to transform primary rat kidney cells in vitro (Graham and Van der Eb, 1973b). Further experiments have indicated that the transforming activity o f Ad5 DNA is unaffected by shearing the DNA into half or quarter molecules, suggesting that the integrity of the viral genome is not required for transformation. More extensive studies have confirmed this assumption, and showed that the segment of the Ad5 DNA involved in transformation is not larger than about I • 10 s daltons (4--5% of the AdS genome), and is located very closely to the left, hand end of the molecule, beginning at a distance of about 1% from-the left end and extending to about 5 6% (Graham et al., 1974a). As a further extension of these studies attempts were made to isolate the transforming gene(s) of adenovirus DNA as unique fragments, ideally as small as I • 106 daltons. Specific fragments of DNA can be obtained by utilizing bacterial restriction endonucleases(endo's R. ) which cleave DNAs at unique base sequences. Recently, several endo R. cleavage maps of Ad2 and Ad5 DNA have been obtained (Mulder et al. 1974a; R.J. Robert, personal commun.; C. Mulder and Greene, unpublished rebaits). This paper describes the isolation of unique fragments of Ad2 and AdS DNA obtained by cleavage with restriction endonucleases capable of inducing transformation of primary rat kidney cells in vitro. The smallest fragments of Ad2 and Ad5 DNA with transforming activity obtained so far have a molecular weight of 1.7 • 106 (7.3% of the viral genome)*. These fragments represent the left-terminal segments of both viral DNAs, confirm. ing the results obtained in our earlier studies with AdS DNA (Graham et al., 1974a). Evidence will be presented showing that the fragment-transformed cell lines contain adenovirus subgroup-C specific T-antigen, as detected by immunofluorescence. However, the distribution of the fluore~ cence in the cell lines transformed by the 7% fragments of Ad5 DNA is atypical mid differs from the u~ual pattern observed in adenovirus transformed cells. A preliminary report of some of these results has appeared elsewhere (Graham et al., 1974b). *Preliminary results suggest that a DNA fragment smaller than 7.3% of Ad5 DNA may also contain transforming activity.
117
MATERIALS AND METHODS
Cells, viruses and viral DNA KB cells, used for cultivation of adenoviruses, were grown in monolayer cultures in Eagle's MEM supplemented with 8% newborn calf serum, or in suspension cultures in Eagle's MEM for suspension supplemented with 5% horse serum. Adenovirus type 2 (strain "adenoid 6") and adenovirus type 5 (strain "adenoid 75") were obtained from the American Type Culture Collection. KB cells were infected with 20 pfu of adenovirus per cell, and were incubated in MEM plus 2% horse serum (monolayer cultures) or in MEM for suspension plus 5% horse serum (suspension cultures). The procedures for purification of the virus and isolation of viral DNA have been described (Van der Eb et al., 1969). Primary cultures of BRK cells were prepared from kidneys of 6--7
