May 14, 1970 - does not depend upon the persistence of SV40 DNA in transformed cells. The existence of an apparently virus-induced antigen associated with ...
Vol. 6, No. 2 Printed in U.S.A.
JOURNAL OF VIROLOGY, Aug. 1970, p. 199-207 Copyright @ 1970 American Society for Microbiology
Virus-Specific Deoxyribonucleic Acid in Simian Virus 40-Exposed Hamster Cells: Correlation with S and T Antigens1 ARTHUR S. LEVINE, MICHAEL N. OXMAN, PATRICK H. HENRY, MYRON J. LEVIN, GEORGE T. DIAMANDOPOULOS, AND JOHN F. ENDERS Medicine Branch, National Cancer Institute, Bethesda, Maryland 20014; Research Divisioni of Infectious Diseases and Clhildren's Cancer Research Foundation, Children's Hospital Medical Center, Boston, Massachusetts 02115; and the Departments ofBacteriology and Immunology, and Pathology, Harvard Medical School, Boston, Massachusetts 02115 Received for publication 14 May 1970
Several homologous hamster embryonic cell lines, transformed in association with simian virus (SV) 40 infection, were examined for the presence of deoxyribonucleic acid (DNA) complementary to SV40 ribonucleic acid (RNA) made in vitro. The methods employed permitted the detection of 10-5 ,ug of viral DNA in 100 ,ug of cellular DNA, corresponding to one-fifth of an SV40 DNA molecule per cell. Those lines which contained both the SV40 surface (S) and tumor (T) antigens also contained DNA complementary to SV40 RNA synthesized in vitro. In contrast, neither of two lines which contained S, but not T, antigen contained detectable DNA complementary to SV40 RNA. These findings suggest that the production of S antigen does not depend upon the persistence of SV40 DNA in transformed cells. The existence of an apparently virus-induced antigen associated with the surface of simian virus (SV)40-transformed hamster cells (26) and of cells acutely infected by SV40 virus (13) has been well documented. This surface antigen (S) differs from the SV40 tumor antigen (T) and the SV40 transplantation antigen (25). S antigen appears only in embryonic hamster cells transformed by SV40 virus, but not in homologous cells which have undergone transformation spontaneously or have been transformed by other viruses (9). These observations suggest that the synthesis of S antigen depends upon exposure to the virus. We have previously reported that in transformed cell lines containing the S, but not the T, antigen, no virus-specific ribonucleic acid (RNA) can be detected (15). However, the viral genome might persist in S +T - cells and be responsible for the production of S antigen by some mechanism not mediated by virus-specific RNA. The present studies were undertaken to examine this hypothesis. The embryonic hamster cell lines employed in the present experiments are those utilized in our earlier study of virus-specific RNA (15). They consist of genetically homologous cells which
underwent transformation spontaneously or after SV40 infection. SV40-exposed lines contain either the S antigen alone or both the S and T antigens. The spontaneously transformed cells contain neither S nor T antigen. To detect and quantitate SV40 deoxyribonucleic acid (DNA) sequences in these cell lines we have utilized a DNA-RNA hybridization technique. Molecular hybrids were formed in solution (19) between tritium-labeled in vitrosynthesized SV40-complementary RNA (3HSV40 cRNA) and the DNA extracted from transformed cells. Existing techniques were modified to permit the detection of an amount of SV40 DNA equivalent to less than one viral genome per cell.
MATERIALS AND METHODS
Cell lines. The tumor cell lines used in these studies were derived from the first hamster passage of SV40exposed or unexposed hamster embryonic cell lines (E, M, P, and 2Ps) (9). Methods of propagation and harvesting have been described previously (15). Included in the study were two SV40-exposed lines which contained both the S and T antigens, two SV40-exposed lines which contained only the S antigen, and two spontaneously oncogenic control lines (Table 1). All of these lines reached cell densities in 1 Preliminary report of this work was presented at the annual meeting of the American Association for Cancer Research, excess of 3 X 105 cells per cm2 in roller bottle cultures. The lines were virus-free, and no virus was induced Philadelphia, Pa., 9-11 April 1970. 199
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LEVINE ET AL.
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TABLE 1. Cell lin2es studied for the presence of SV40-specific DNA Cell line
Description
Antigensa
SV40-specific RNA
producedb 1809 2671 1807
2672 1808
2673 2675 SV3T3479 H-50 Py3T3
Embryonic hamster, spontaneously transformed Same as for 1809 Embryonic hamster, transformed after exposure to SV40 virus (nonvirus yielding)c Same as for 1807 Embryonic hamster, transformed after exposure to SV40 virus (nonvirus yielding)c Same as for 1808 Embryonic hamster, transformed after exposure to SV40 virus (virus yielding) SV40 virus-transformed mouse cells (virus yielding) Derivative of an SV40-induced hamster tumor Polyoma virus-transformed mouse cells
S-T-
No
S-T-
S+T-
No No
S+TS+T+
No Yes
S+T+ S+T+
Yes Yes
S-T+d
Yes
S+T+
Not tested
No SV40 antigens
Not tested
a Presence of S and T antigens was tested at the beginning and during the experiments reported herein by techniques described in the text. I Correlation between S and T antigens and virus-specific RNA has been reported previously (15); this RNA was detected by hybridization of cellular RNA with SV40 DNA. Lines failing to yield virus were negative on co-cultivation and Sendai virus fusion with AGMK cells. d Although SV40 can be recovered from this cell line by co-cultivation with AGMK cells, the mouse cells did not have detectable S antigen. -
when transformed cells were co-cultivated or fused with African green monkey kidney cells (AGMK). Also examined were a virus-free line of SV40transformed hamster embryo cells (2675), containing both the S and T a.itigens but yielding infectious virus on co-cultivation with AGMK cells; a virus-free line of SV40-transformed mouse cells (SV3T3479) yielding virus on co-cultivation with AGMK cells; a virus-free line of polyoma-transformed mouse cells, received from Thomas Benjamin; and a derivative from an SV40-induced tumor, H-50, received from Fred Rapp (12). All lines were periodically tested for the presence of T antigen by indirect fluorescent-antibody staining (20) by using sera from hamsters bearing virus-free SV40 tumors. Of cells which were T antigen-positive, 100%'o exhibited characteristic bright, finely granular fluorescence, filling the nucleus but sparing the nucleolus. Cells which were T antigen-negative exhibited no nuclear fluorescence. S. S. Tevethia and F. Rapp kindly assayed coded samples of all cell lines for the SV40 S antigen during the course of these studies. This antigen was also detected by an indirect immunofluorescent test. Cells in suspension were reacted with sera from SV40-vaccinated hamsters which had resisted a transplant of cells transformed by the homologous virus. Only cells showing a bright green fluorescence in the form of a ring around the cell were counted as positive. [Sera are called positive only
when more than 50%0 of H-50 cells show specific immunofluorescence (24).] SV40 viral DNA. SV40 strain 777 (3) was maintained by serial passage in BSC-1 cells. Cells and fluids were harvested when the cell monolayers were completely destroyed; the virus pools were centrifuged at 60,000 X g to sediment cells and virions, and the pellets were frozen and thawed three times. The pellets were resuspended in Eagle's minimal essential medium (with 2% agamma-globulinemic calf serum, penicillin, streptomycin, and glutamine), treated with 1% sodium deoxycholate-0.1% trypsin to disperse aggregates, and cushioned on saturated KBr for 2.5 hr at 23,000 rev/min. The cushioned virus was collected, dialyzed overnight at 4 C, and banded in CsCl for 16 to 20 hr at 35,000 rev/min. Virus bands were dialyzed again, and viral DNA was extracted from the purified virus by papain digestion, followed by sodium dodecyl sulfate (SDS)-phenol extraction. The methods of purification of the virus and of viral DNA extraction will be detailed extensively elsewhere (M. Levin et al., suibmitted for publication). Viral DNA was stored in 0.1 X SSC (SSC: 0.15 M sodium chloride and 0.015 M sodium citrate, pH 6.9) at -20 C. This DNA was employed both in the reconstruction experiments and as the template for the synthesis of complementary RNA. At least 50% of the SV40 DNA extracted under these conditions was determined to be form 1
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by sedimentation in an alkaline sucrose gradient. The buoyant density of the DNA was 1.700 + 0.002 g/ml, the per cent guanine plus cytosine (GC) was 40, and the Tm value was 67.9 C, in 0.1X SSC (C. Crumpacker et al., submitted for publication). Cellular DNA. DNA from whole cells propagated in tissue culture or from animal tissues was extracted by the method of Marmur (17) with omission of Pronase and with the following additions: DNA dissolved in 0.1 X SSC was treated with 50,4g of pancreatic ribonuclease per ml and 25 units of ribonuclease T, per ml for 30 min at 37 C; this treatment was followed by incubation of the DNA with a protease, subtilisin, 10 ug/mi, for 15 min at 37C. Cnicken liver DNA was the gift of Bert O'Malley and was reextracted prior to use. Each sample of DNA was extracted repeatedly with chloroformisoamyl alcohol until no protein was visible at the interface; after this, two further deproteinizations were performed. The ratio of optical density at 260 to 280 nm was 1.90 or greater for all samples of DNA used in these experiments, and protein determinations performed by Lowry's method (16) demonstrated that 1 mg of DNA contained less than 5 ,ug of protein. Cellular DNA samples were stored in 0.1X SSC at -20 C with the addition of one or two drops of chloroform. Cellular and viral DNA samples were quantitated by the diphenylamine reaction, as modified by Burton (7), with a calf thymus DNA standard (Calbiochem, Los Angeles, Calif.). Enzymes. DNA-dependent RNA polymerase was the gift of Richard R. Burgess. The highly purified enzyme preparation, from Escherichia coli K-12, consisted of fraction 4, obtained after centrifugation in a high salt glycerol gradient ("complex" polymerase, containing a factor). The enzyme was stored for as long as 1 month at 4 C, with no loss of activity, in 1.0 M KCI, 0.01 M tris(hydroxymethyl)aminomethane buffer (Tris) (pH 7.9), 0.01 M magnesium acetate, 10-4 M ethylenediaminetetraacetate, 10-4 M dithiothreitol, and 15% glycerol. The protein concentration was 1 mg/ml (6). Pancreatic ribonuclease (Sigma Chemical Co., St. Louis, Mo.; type XII-A) and ribonuclease T1 (Calbiochem, Los Angeles, Calif.; B grade) were both heated at 80 C for 10 min to inactivate traces of deoxyribonuclease. Electrophoretically purified deoxyribonuclease (ribonuclease-free) was obtained from the Worthington Corp., Freehold, N.J., and subtilisin (Nagarse) from the Enzyme Development Corp., New York, N.Y. Synthesis of viral complementary RNA. Radioactive RNA complementary to SV40 DNA or to adenovirus 12 DNA (used as a control) was synthesized in vitro by a modification of the method of Chamberlin and Berg (8). The reaction mixture contained, in 125 uliters: 10 Ag of DNA-dependent RNA polymerase; 10 ,g of native SV40 or adenovirus 12 DNA; 10 nmoles each of tritiated cytidine 5'-triphosphate, 12.6 Ci/mmole; uridine 5'-triphosphate, 14.8 Ci/mmole; adenosine 5'-triphosphate, 18.5 Ci/mmole (all purchased from Schwarz BioResearch Inc., Orangeburg, N.J., in a concentration of 0.5 nmoles/uliter, in 2% sterile ethanol); and unlabeled guanosine 5'-triphos-
201
phate (obtained from Mann Research Laboratories, New York, N.Y.). Also in the reaction mixture were: KCl, 5 ,umoles; MgCI2, 1 MAmole; Tris buffer (pH 7.9), 3 /umoles; 2-mercaptoethanol, 0.7,umoles; and dialyzed bovine serum albumin, 10 ug. After an incubation time of 1 hr at 37 C, the reaction was stopped by adding 40 ,g of deoxyribonuclease (15 min, 37 C), followed by the addition of SDS at a final concentration of 0.5%. One milligram of yeast RNA (Worthington Corp.) was added as a carrier, and the mixture was dissolved in 2 ml of 0.05 M Tris buffer (pH 7.9). The RNA was then extracted with 1 ml of phenolm-cresol (7.9:1.0, v/v) containing 1 mg of 8-hydroxyquinoline per ml. Extraction was carried out at 60 C for 3 min, with vigorous shaking, and the mixture was then cooled rapidly in a dry ice-alcohol bath. This step was followed by centrifugation for 10 min at 2,000 rev/min in an Intemational Centrifuge (PR-2) at 4 C. To the aqueous phase were added 0.10 volume of 20% potassium acetate (pH 5.4) and 2 volumes of ethanol. RNA was then precipitated by storage at -30 C for 60 min, and the RNA was collected by centrifugation. The pellet was dissolved in 2X SSC containing 0.05% SDS and stored at -30 C. Calculation of the amount of radioactive complementary RNA formed was based on the assumption that the four nucleoside triphosphates were incorporated equally. On this basis, the yield of a reaction mixture was estimated to be 17 Ag of radioactive complementary RNA ('H-cRNA), with a specific activity of 1.94 X 107 disintegrations per min per ug. This represented 32% of the entire input of nucleoside
triphosphates.
Formation and detection of DNA-RNA hybrids. A number of modifications were made in the method of Nygaard and Hall (19). A trace amount of thymidine-2-'4C-labeled control hamster cell DNA (line 1809) was added to each cellular DNA to be tested for the presence of SV40 genetic material. The assumption was made that the trace amount of radioactive DNA (specific activity, 2.4 X 104 disintegrations per min per ug) would be denatured and retained to the same extent by nitrocellulose membrane filters as would the large amounts of unlabeled cellular DNA being tested. Thus, the specific activity of each mixture of tested DNA-tracer DNA could be determined, and this information could be used to calculate the amount of tested DNA present on each filter after the hybrid material was applied, ribonuclease-treated, and washed. The ratio of tested DNA to tracer DNA was 4 X 103. DNA mixes were denatured in 0.01 X SSC at a concentration of 300 Ag/ml in a boiling water bath for 15 min. The denatured DNA was quickly quenched in a dry ice-alcohol bath and added in 100-,4g amounts to glass ampoules containing the 3H-cRNA. The DNA-RNA mixture was brought to a final concentration of 2X SSC and 0.05% SDS, in a volume of 0.5 ml per ampoule. Ampoules were sealed and incubated at 65 C for 20 hr, after which the reaction mixture in each ampoule was pipetted into 15 ml of 3X SSC at 4 C. The hybrid material was filtered through nitrocellulose filters (HAWP, 47 mm, 0.45 ,m pore size; Millipore Corp., Bedford, Mass.) which had been presoaked overnight in cold 3X SSC
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LEVINE ET AL.
and prewashed with 10 ml of 60 C 3X SSC. The ampoule contents were applied by gravity, and the filters were then washed with 150 ml of 3X SSC at 60 C under suction filtration. The filters were treated with 10 ml of ribonuclease solution (20 pg of pancreatic ribonuclease per ml and 10 units of ribonuclease T1 per ml) for 1 hr at room temperature. Finally, the filters were agitated sequentially in three beakers of 3X SSC at 60 C and again washed by suction filtration with 150 ml of 3 X SSC at 60 C. The filters were then dried and counted directly in a toluene phosphor (Liquifluor; Nuclear-Chicago Corp., Des Plaines, Ill.), by using a Packard Tri-Carb scintillation spectrometer. Tritium was counted with an efficiency of 13.6% in the presence of I4C; 14C was counted with an efficiency of 41.8% in the presence of tritium. The percentage of 14C disintegrations/min counted in the tritium channel was 10.8. Samples were counted for a sufficient period of time to achieve a statistical accuracy of at least i- 1% in all channels. More than 95% of the cellular DNA in a hybridization ampoule was present on the membrane filters after the final washing, and the variability between replicate filters made from a given DNA mixture was less than 5%. The exact amount of DNA present was determined from the disintegrations/min of 14C retained and the known specific activity of the DNA mixture. The amount of 3H-cRNA retained by the filters was normalized to 100 jg of DNA.
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TABLE 2. Hybridization between IH-S V40 cRNA and DNA from various vertebrate and inzvertebrate cells Source of cellular DNA
Disintegrations (3H)perm 100 g of DNAa
Hamster liver ....................... 721 i Mouse L1210 leukemia cells ......... 777 i Monkey (Vero line) ................. 498 + Chicken liver ....................... 608 + Mycoplasma pneumoniae (Friendship 3 strain) .................... 436 4± Escherichia coli ..................... 455 4t
171 165 14 32 46 82
a Hybridization was performed under standard conditions, and tritium counts/min retained by membrane filters were normalized to 100 pg of cellular DNA and expressed as disintegrations per minute. Results are given as arithmetic means i standard deviations. Each type of DNA was tested with a minimum of three replicate filters. Input of cRNA: 22 X 106 disintegrations/min. Of this input, approximately 100 disintegrations/min were retained by membrane filters when no DNA was present in a hybridization reaction.
This assumption is supported by the observation that there is no homology between the SV40 DNA used in these experiments and tritiumlabeled monkey cell RNA (M. Levin et al., submitted for publication). Thus, there is no evidence confirming the reported presence in SV40 DNA of sequences homologous with monkey cell DNA
RESULTS Background homology between SV40 cRNA and hamster DNA. Radioactive RNA complementary to SV40 DNA was initially incubated with DNA from normal hamster liver cells and (2). Calibration of the hybridization system. The with DNA from other vertebrate and invertebrate species so that the degree of homology with these amount of 3H-SV40 cRNA needed to saturate nucleic acids could be quantitated. In the reaction all available SV40 DNA sites was determined by between 3H-SV40 cRNA and normal hamster adding increasing amounts of RNA to a mixture DNA, 0.03 % of the total input RNA was retained of 100 ,ug of control hamster cell DNA (sponby the membrane filters (Table 2). Although taneously oncogenic line 1809) and 10-3 ,ug of some homology between hamster cell DNA and SV40 DNA (corresponding to the equivalent of SV40 DNA has been previously noted (21), the 20 viral genomes per cell). Approximately 2 X 106 fact that the amount of hybridization occurring disintegrations/min of viral cRNA were needed between 3H-SV40 cRNA and E. coli or myco- to saturate this amount of viral DNA (Fig. 1). plasma DNA (both phylogenetically distant About 2 X 104 disintegrations/min were bound from vertebrates) was more than 85 % of that by the DNA at saturation, corresponding to 10-s occurring between this RNA and monkey cell Ag of 3H-SV40 cRNA. In repeated experiments, DNA casts some doubt upon the specificity of an RNA:DNA binding ratio of approximately the background hybridization. The data in Table 1:1 was observed, suggesting that the comple2 do not reveal significant differences between mentary RNA made in vitro contained informareactions of 3H-SV40 cRNA with the various tion copied from both strands of the template. vertebrate and invertebrate DNA samples studied. However, it is not possible to make a precise In addition, the absence of significant excess quantitative interpretation of the observed bindhybridization between the SV40 cRNA and DNA ing ratio because the true specific activity of the extracted from monkey cells (the species in which RNA is unknown. Moreover, since 50% of the the SV40 virus was propagated) demonstrates SV40 DNA employed in these experiments conthat the template SV40 DNA was free of cellular sisted of the rapidly sedimenting form 1, this DNA contamination, assuming that any con- portion might reanneal rapidly and be unavailtaminating DNA would be efficiently transcribed. able for hybridization with SV40 cRNA. If so,
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the labeled products were made under identical conditions. Approximately 3 ,ug of the unlabeled cRNA inhibited more than 95% of the binding of SV40 late RNA (labeled from 24 to 40 hr 15,000 o postinfection) to 0.03 ,ug of SV40 DNA. Input of the late RNA was 150 ,ug, which was a satum< 10,000 rating amount, and, in the absence of unlabeled competitor RNA, 4,845 counts/min were bound oT per Ag of DNA (M. Levin et al., submitted for 5000 publication). The hybridization-competition study indicates that the 3H-SV40 cRNA contains information transcribed from all viral DNA sequences that are transcribed in SV40-infected 0 055 22 2.75 1.65 3.3 3.85 INPUT OF 'H -cSV40 RNA (DPM xIO") cells, assuming that all SV40 genes are transcribed FIG. 1. Hybridization of SV40 DNA with homolo- with equal frequency in a productive infection. gous 3H-cRNA. 10-3 ,g of SV40 DNA was mixed When reconstruction experiments were perwith 100 MAg of DNA extracted from spontaneously formed (Table 3) with saturating amounts of 20,000
G
z 6
Z0
> > U)
L.
c
4.4
transformed embryonic hamster cells (line 1809). This ratio ofSV40 to control DNA is equivalent to 20 viral genomes per cell (see calculations, legend to Table 3). Hybridization with the stated amounts of cRNA was performed under standard conditions. The disintegrations per minute specifically hybridized were obtained by subtracting the background disintegrations per minute bound to equal amounts of spontaneously transformed hamster cell DNA alone. Each point represents the arithemitic mean of three replicate filters; standard deviation was less than 5%.
the real RNA :DNA binding ratio would be 1 :2 (the observed 1 :1 ratio could be an error resulting from our inability to determine directly the specific activity of the cRNA, although a twofold error is highly unlikely). To determine whether a portion of the SV40 DNA was in fact unavailable for hybridization with cRNA, a sample of the DNA was treated with deoxyribonuclease (8 x 10-4 Ag per 100 MAg of DNA; 60 min at 30 C) as described by Westphal and Dulbecco (27). Under these conditions, more than 90% of SV40 form 1 is converted to form 2, which would not reanneal rapidly after heat denaturation. When SV40 DNA so treated was employed in saturation experiments identical to those previously described, the counts/min of SV40 cRNA bound at saturation were no higher than when untreated viral DNA was used, indicating that the conditions employed in these experiments appear to permit the availability of all SV40 DNA for hybridization with cRNA, even when a significant portion is form 1. Previously, we have reported that unlabeled SV40 cRNA competes completely with the hybridization reaction between SV40 DNA and radioactive RNA obtained from cells productively infected by SV40 virus ("late RNA"). The amount of unlabeled cRNA used in this hybridization-competition study was estimated from the known incorporation of labeled triphosphates into radioactive SV40 cRNA; the unlabeled and
TABLE 3. Reconstruction experiments: hybridization between 3H-SV40 cRNA and increasing amounts of SV40 DNA Viral Expected DNA Amt (pg) of disintegra- Observed (net) disintegia- equivaSV40 DNAa tionsb tions/min (3H) c lents/ min (H) bcelidi
lo-5 5X 10- 5 104 5,X 10-4 10-
194 967
245 1,153 1,934 2,293 9,670 11,392 19,338 21,090
4f ± i i i
33 (2; 5) 0.2 93 (2; 6) 1 113 (4; 11) 2 536 (1; 3) 10 1212 (5; 12) 20
a Each hybridization ampoule contained the viral DNA plus 100 gg of spontaneously transformed hamster cell DNA (line 1809) and 2.2 X 106 disintegrations/min of SV40 cRNA. b Expected hybridization values were predicted on the basis of the estimated specific activity of the SV40 cRNA and the apparent RNA to DNA binding ratio of 1:1 observed in saturation experiments (Fig. 1). c Arithmetic means 4 standard deviations are given; in parenthesis are the number of experiments performed and the total number of filters. Observed (net) disintegrations per minute were calculated by subtracting the disintegrations per minute bound to control (spontaneously transformed hamster cell) DNA in a given experiment from the disintegrations per minute bound to the virus DNA-cell DNA mixtures in that experiment. Mean of control values for all experiments: 610 ±t 111 disintegrations per min/100 ,g of DNA. d If we assume that the DNA of one hamster cell nucleus weighs 1I-- Ag and that of one SV40 molecule weighs 5 X 10-2, Mg, the proportion of SV40 DNA to cell DNA in a cell which contains one molecule of viral DNA would be 5 X 107. On this basis, 5 X 10-' ,ug of SV40 DNA in 100
,ug of cell DNA corresponds to one viral genome per cell.
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LEVINE ET AL.
3H-SV40 cRNA and various quantities of SV40 DNA, as little as 10-5 Mg of viral DNA could be reliably detected, corresponding to one-fifth of one viral DNA molecule per cell. With 5 x 10-5 Mug of viral DNA (corresponding to one viral genome per cell), the disintegrations/min (3H) retained by the filter was approximately triple that retained by 100 Mg of control hamster cell DNA alone. Using an expected RNA to DNA binding ratio of 1:1, and the estimated specific activity of the 3H-SV40 cRNA, data were tabulated for ideal hybridization between the saturating amounts of cRNA and varying amounts of SV40 DNA (Table 3). The data for expected and observed hybridization were plotted on a log-log graph (Fig. 2). The observed hybridization is a linear function of the quantity of SV40 DNA present on the filter and closely approximates expected hybridization. SV40-specific DNA in SV40-exposed and unexposed cell lines. DNA (100 ;g) from each of the various cell lines (Table 1) was hybridized with that amount of 3H-SV40 cRNA which had saturated 10- Mug of SV40 DNA (mixed with 100 Mig of control cellular DNA). Cell lines 1807 and 2672, as well as the control line 1809, were each studied at two different passage levels. The results are presented in Table 4. In each experiment, the control value observed for hybridization with the spontaneously transformed cell line has been subtracted from the values observed with the
J. VIROL.
virus-transformed lines. Table 4 also presents the number of viral genome-equivalents detected per cell. These data are derived from a comparison of the net 3H disintegrations/min bound to each cellular DNA with the calibration data (Table 3, Fig. 2). The results of hybridization of DNA samples from the homologous embryonic hamster lines demonstrate that cells containing T antigen also contain SV40-specific DNA. Line 1808 had the equivalent of two viral genomes per cell. Line 2673, an analogue of 1808 which also yields no
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zz
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