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Nonintegrated viral DNA in rat cells doubly transformed by SV40 and polyoma virus
VIROLOGY
85,328-331
Nonintegrated
ISHWARI Department
(19’78)
Viral DNA in Rat Cells Doubly Transformed and Polyoma Virus PRASAD,’ of Pathology,
DIMITRIS
ZOUZIAS,
New York University New York, New Accepted
November
AND
CLAUDIO
School of Medicine, York 10016
by SV40
BASILIC02
550 First
Avenue,
17,1977
Rat F2408 cells transformed by polyoma virus contain, in addition to integrated viral genomes, a small number (an average of 20-50 copies per cell) of nonintegrated viral DNA molecules. On the other hand, SV40-transformed rat cells contain only integrated viral genomes. SV40-transformed rat cells also differ from the polyoma transformants, in that they grow in soft agar medium at a much slower rate. Cells doubly transformed by polyoma and SV40 can be easily isolated following polyoma superinfection of SV40transformed rat cells, as their rate of growth in agar is enhanced. These doubly transformed cells yield polyoma or SV40, respectively, after fusion with cells permissive for each virus. However, only polyoma-specific DNA sequences can be detected in these cells in a “free” state.
We have reported (I) that F2408 rat cells transformed by polyoma virus contain an average of 20 to 50 copies per cell of nonintegrated (free) viral DNA molecules in addition to viral DNA integrated into the host DNA. The free viral DNA molecules were not due to a virus-carrier state and were observed in all clones examined. The presence of the free DNA molecules appeared to be due to a spontaneous induction and limited replication of integrated viral genomes and was confined to a minority of the transformed cell population at any given time (1,2). Since rat cells are not only transformable by polyoma virus but also by SV40, it was considered interesting to determine whether the SV40-transformed rat cells also contained integrated and free viral DNA and to determine the state of heterologous viral genomes in rat cells doubly transformed by polyoma and SV40. Thus, the aims of the present studies were to determine (i) whether F2408 rat cells
transformed by SV40 contain nonintegrated viral DNA, and (ii) whether the situation found in the cells singly transformed by polyoma or SV40 was in any way altered in rat cells containing both viral genomes. Fischer rat libroblasts of the line F2408, Swiss mouse 3T3 cells (clone D), and African green monkey kidney BSC-1 cells, which have all been described previously, were used (I, 3). The cells were grown in Dulbecco-modified Eagle’s medium containing 10% calf serum. Wild-type SV40 and polyoma viruses were grown at 37” in BSC-1 and 3T3 D cells, respectively. The transformed cells were isolated on the basis of their ability to grow in suspension in soft agar (4). The cells were infected with plaque-purified SV40 or with polyoma viruses at a multiplicity of infection of 500 PFU/cell in TD buffer (I ). Adsorption was carried out in suspension for 1 hr at room temperature. The final agar concentration in the medium was 0.34%. The infected cells were incubated for from 2 to 4 weeks at 37”. The frequency of SV40 transformation was much lower (about 20-fold) than that obtained with polyoma virus (1). In addi-
1 Present address: Department of Pathology, S.U.N.Y. Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203. * Author to whom requests for reprints should be addressed. 326 0042-6822/78/0851-0328$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
tion, while polyoma-transformed cells formed 3- to 4-mm size colonies after 3 weeks of incubation, SV40-transformed cells formed similar size colonies after only 5 to 6 weeks. When polyoma- and SV40transformed colonies were isolated and cultured, the growth rate and morphology of both types of,transformants under normal conditions appeared similar. Like the polyoma transformants (I ), SV40-transformed cells grew to high saturation densities and exhibited a “transformed” morphology. These cells contained SV40-specific T antigen as determined by immunofluorescence and yielded SV40 after fusion with permissive BSC-1 cells (Table 1). When the transformed cells from the independent clones were again inoculated into agar medium, the plating efficiency and the rate of growth varied among clones, although the growth rate of the transformed cells was always much slower than that of the polyoma transformants. The presence of nonintegrated viral genomes in the SV40-transformed cells was tested as previously described (1). Low molecular weight viral DNA was extracted by the method of Hirt (5) and TABLE PRODUCTION Clone
no. (1
OF INFECTIOUS
sv40 Rat No. 3s
(Y 0 0 0 0 -
purified. The number of free viral DNA equivalents was estimated by measuring the ability of the DNA preparations to increase the rate of reassociation of 32Plabeled SV40 DNA (1, 6). None of the five SV40 transformants tested contained detectable free viral genomes. In addition, the possible presence of nonintegrated viral DNA molecules was tested by determining the infectivity of the low molecular weight DNA extract, which was plated on BSC-1 cells in the presence of DEAE-dextran (7). No plaques or cytopathic effects were observed. It would therefore seem that SV40-transformed rat cells do not contain significant amounts of nonintegrated viral DNA. We took advantage of the slow growth of SV40-transformed rat cells in the agar medium to isolate cells doubly transformed by SV40 and polyoma. SV40-transformed cells (clone 3) were superinfected with polyoma at a multiplicity of 100 PFU/ cell in suspension. Then the cells were inoculated into agar. After two weeks of incubation, the colonies which grew faster and were therefore larger and more compact were isolated and cultured. All of the 1
IN RAT CELLS DOUBLY
Spontaneousb Py ”
1 2 3 4 5
VIRUS
329
COMMUNICATIONS
TRANSFORMED
BY POLYOMA
Fusion’ sv40
Py
0 0 0 0 0 0
100’
15 2.5 x 103 1 x 103 75 -
AND SV40
T antigen” sv40 8 3 2.5 4 2 1
x x x x x x
PY lo2 102 104 lo’ 102 103
+ + + + + -
sv40 + + + + + +
a Independently isolated transformants. b The cells were grown at 37”. The virus was extracted from 2 x 10” cells and titered on BSC-1 cells to determine SV40 infectivity and on 3T3 cells to determine polyoma infectivity, respectively. c Transformed cells (IO61 were fused with BSC-1 (lo6 cells) or 3T3 (lo6 cells) separately with the help of inactivated Sendai virus. The fused cells were incubated for 4 days at 37”. The virus extracted from the BSC-1 fusion was titered on BSC-1 cells to determine SV40 infectivity and the virus extracted from 3T3 fusion was titered on 3T3 to determine polyoma infectivity (5,6). rl The cells were grown on coverslips. SV40 and polyoma T antigen were determined on separate coverslips by immunofluorescence as described (101 using antiSV40 T hamster serum and anti-hamster fluorescent globulin, or anti-polyoma T rat serum and anti-rat fluorescent globulin. 1’ Py, polyoma. ‘The data for the spontaneous and fusion cultures are expressed as plaque forming units per culture. A value of zero indicates fewer than 10 PFU/culture. B Parental SV40-transformed rat line.
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putative doubly transformed cells appeared to be morphologically similar. They contained SV40- and polyoma-specific T antigens as determined by immunofluorescence, and their properties were not modified by extended cultivation or growth in the presence of antipolyoma serum. Infectious polyoma or SV40 virus was rescued from these cells following fusion with mouse 3T3 or BSC-1 monkey cells, respectively (Table 1). To test the efficiency of our method of selection of doubly transformed cells, in one experiment SV40-transformed clone 3 cells were superinfected with polyoma (200 PFU/cell), plated in agar at 10,000 cells/ petri dish, and 10 days later one large colony and six small colonies were isolated. After propagation they were tested for the presence of SV40 or polyoma T antigen by using immunofluorescence. Only the cells from the large colony contained both polyoma and SV40 T antigens, while all of the cells were positive for SV40 T antigen. To determine whether any interaction between the polyoma and SV40 genes or gene products in the doubly transformed cells affected the state of the viral DNA, low molecular weight DNA was extracted from these cells as described (I), purified, and tested by DNA-DNA reassociation kinetics for the presence of polyoma and SV40 DNA sequences. The results are shown in Table 2. The clones examined contained from 4 to 33 genome equivalents of nonintegrated polyoma DNA per cell. The variation among clones in the number of free polyoma genomes per cell was not very different from that obtained earlier in different clones of polyoma-transformed rat cells (1). We could not, however, detect nonintegrated SV40 DNA molecules in these cells, a result consistant with the observation that SV40-transformed cells did not contain free viral genomes. The nonintegrated polyoma DNA molecules were infectious when tested by plaque formation on monolayers of mouse 3T3 D cells, although their specific infectivity was low (1). The above results show that F2408 cells can be singly transformed by SV40 and by
TABLE
2
FREE VIRAL DNA MOLECULES IN RAT CELW DOUBLY TRANSIWRMED BY POLYOMA AND SV40” Transformed clone Polyoma ge- SV40 genome equivalents nome equivalents per per cell cell SV40 SV40 SV40 SV40 SV40
Py Py Py Py Rat
Rat Rat Rat Rat No.
No. No. No. No. 3*
1 3 4 5
33 13 24 4 -
co.5 co.5 co.5 co.5 co.5
” Nonintegrated viral DNA equivalents were estimated by measuring the effect of low molecular weight DNA preparations from transformed rat cells on the rate of reassociation of 32P-labeled polyoma or SV40 DNA as described in (11. Low molecular weight DNA was extracted according to Hirt (5). Purified DNA from the Hirt supernatant was dissolved in a small volume of 0.01 M phosphate and 0.001 M EDTA (pH 6.81 and dialyzed extensively against the same buffer. Before hybridization, the DNA was fragmented by boiling it with the polyoma 132PlDNA or SV40 lz2PlDNA probe for 10 min in 0.3 M NaOH. DNA-DNA reassociation kinetics and hydroxyapatite chromatography were done according to Sharp et al. (61. Nonintegrated polyoma DNA equivalents per cell were calculated from the formula: Number of polyoma DNA equivalents/cell = a x 2 x lO”/A, where a = micrograms of unlabeled viral DNA in the preparations, A = the number of cells, and 2 x 10” = the number of polyoma DNA molecules per microgram. * Parental SV40-transformed rat line.
polyoma, but the transformed cells, although they both presumably carry viral genomes in a stable association with the host DNA, differ in two properties: (i) SV40-transformed cells grow in soft agar much more slowly than do the polyoma transformants. However, when SV40transformed F2408 cells are “supertransformed” by polyoma, they grow in soft agar at the same rate as do the polyomatransformed cells. Todaro et al. (8) have suggested that in mouse 3T3 cells doubly transformed by SV40 and polyoma, each of these viruses acts independently and has a distinct effect on the host cell. However, Takemoto and Habel (9) showed that when SV40 tumor cells were superinfected with polyoma, superinfection caused increased oncogenicity. Thus our results could be explained as a summation of the
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331
COMMUNICATIONS
effect of the two viruses, a property which is also evident in the very fast growth rate of these cells in liquid medium. (ii) The other main difference appears to be that the polyoma-transformed cells contain integrated and nonintegrated viral genomes, while the SV40-transformed cells contain only integrated ones. It was therefore of interest to examine the state of the viral genomes in doubly transformed cells. The results showed that in these cells, the only “free” genomes found are those of polyoma virus. It is therefore suggested that either the association of SV40 DNA with the host DNA in the rat cells is much more stable than that of polyoma DNA, or that the rat cells are more nonpermissive for SV40 DNA replication. Excision of the two viral genomes could then occur with the same frequency, but in the case of SV40 DNA, the lack of any independent viral DNA replication would not allow the accumulation of a detectable number of free viral DNA molecules. Alternatively, as previously suggested (2), it is possible that viral DNA excision and replication may be coupled. According to this hypothesis, the same host cell factors which promote replication would be responsible for induction, and our results could be interpreted as being due to the complete lack of the factors necessary for SV40 replication in rat cells. These doubly transformed cells could be
useful for studying the characteristics of integration of the polyoma or SV40 viral genomes into transformed cells and for investigating the possibility that they may share integration sites. The ease with which these cells can be isolated should facilitate studies of the effect of viral mutations upon the transformed cell phenotype. ACKNOWLEDGMENTS This investigation was supported by PHS Grants CA 11893 and CA 11642 from the National Cancer Institute. We thank Dr. T. Benjamin for the gift of anti-polyoma T serum. REFERENCES 1. PRASAD, I., ZOIJZIAS, D., and BMILICO, C. J. Viral. 18, 436-444 (1976). 2. ZOUZIAS, D., PRASAD, I., and BASILICO, C., J. Virol. 24, 142-150 (1977). 3. PRASAD, I., ZOUZIAS, D., and BASILICO, C., J. Viral. 16, 897-904, (1975). 4. MACPHERSON, I., and MONTAGNIER, L., Virology 23, 291-294 (1964). 5. HIRT, B. J., Mol. Biol. 26, 365-369 (1967). 6. SHARP, P. A., PETTERSON, U., and SAMBROOK., J., J. Mol. Biol. 86, 709-726 (1974). 7. PAGANO, J., MCCUTCHAN, J. H., and VAHERI, A., J. Viral. 1, 891-897, (1967). 8. TODARO, G., HABEL, K., and GREEN, H., Virology 27, 179-185 (1965). K. K., and HABEL, K., Virology 30, 9. TAKEMOTO, 20-28 (1966). 10. BASILICO, C., and ZOUZIAS, D., Proc. Nat. Acad. Sci. USA 73, 1931-1935 (1976).
85,328-331
Nonintegrated
ISHWARI Department
(19’78)
Viral DNA in Rat Cells Doubly Transformed and Polyoma Virus PRASAD,’ of Pathology,
DIMITRIS
ZOUZIAS,
New York University New York, New Accepted
November
AND
CLAUDIO
School of Medicine, York 10016
by SV40
BASILIC02
550 First
Avenue,
17,1977
Rat F2408 cells transformed by polyoma virus contain, in addition to integrated viral genomes, a small number (an average of 20-50 copies per cell) of nonintegrated viral DNA molecules. On the other hand, SV40-transformed rat cells contain only integrated viral genomes. SV40-transformed rat cells also differ from the polyoma transformants, in that they grow in soft agar medium at a much slower rate. Cells doubly transformed by polyoma and SV40 can be easily isolated following polyoma superinfection of SV40transformed rat cells, as their rate of growth in agar is enhanced. These doubly transformed cells yield polyoma or SV40, respectively, after fusion with cells permissive for each virus. However, only polyoma-specific DNA sequences can be detected in these cells in a “free” state.
We have reported (I) that F2408 rat cells transformed by polyoma virus contain an average of 20 to 50 copies per cell of nonintegrated (free) viral DNA molecules in addition to viral DNA integrated into the host DNA. The free viral DNA molecules were not due to a virus-carrier state and were observed in all clones examined. The presence of the free DNA molecules appeared to be due to a spontaneous induction and limited replication of integrated viral genomes and was confined to a minority of the transformed cell population at any given time (1,2). Since rat cells are not only transformable by polyoma virus but also by SV40, it was considered interesting to determine whether the SV40-transformed rat cells also contained integrated and free viral DNA and to determine the state of heterologous viral genomes in rat cells doubly transformed by polyoma and SV40. Thus, the aims of the present studies were to determine (i) whether F2408 rat cells
transformed by SV40 contain nonintegrated viral DNA, and (ii) whether the situation found in the cells singly transformed by polyoma or SV40 was in any way altered in rat cells containing both viral genomes. Fischer rat libroblasts of the line F2408, Swiss mouse 3T3 cells (clone D), and African green monkey kidney BSC-1 cells, which have all been described previously, were used (I, 3). The cells were grown in Dulbecco-modified Eagle’s medium containing 10% calf serum. Wild-type SV40 and polyoma viruses were grown at 37” in BSC-1 and 3T3 D cells, respectively. The transformed cells were isolated on the basis of their ability to grow in suspension in soft agar (4). The cells were infected with plaque-purified SV40 or with polyoma viruses at a multiplicity of infection of 500 PFU/cell in TD buffer (I ). Adsorption was carried out in suspension for 1 hr at room temperature. The final agar concentration in the medium was 0.34%. The infected cells were incubated for from 2 to 4 weeks at 37”. The frequency of SV40 transformation was much lower (about 20-fold) than that obtained with polyoma virus (1). In addi-
1 Present address: Department of Pathology, S.U.N.Y. Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203. * Author to whom requests for reprints should be addressed. 326 0042-6822/78/0851-0328$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
tion, while polyoma-transformed cells formed 3- to 4-mm size colonies after 3 weeks of incubation, SV40-transformed cells formed similar size colonies after only 5 to 6 weeks. When polyoma- and SV40transformed colonies were isolated and cultured, the growth rate and morphology of both types of,transformants under normal conditions appeared similar. Like the polyoma transformants (I ), SV40-transformed cells grew to high saturation densities and exhibited a “transformed” morphology. These cells contained SV40-specific T antigen as determined by immunofluorescence and yielded SV40 after fusion with permissive BSC-1 cells (Table 1). When the transformed cells from the independent clones were again inoculated into agar medium, the plating efficiency and the rate of growth varied among clones, although the growth rate of the transformed cells was always much slower than that of the polyoma transformants. The presence of nonintegrated viral genomes in the SV40-transformed cells was tested as previously described (1). Low molecular weight viral DNA was extracted by the method of Hirt (5) and TABLE PRODUCTION Clone
no. (1
OF INFECTIOUS
sv40 Rat No. 3s
(Y 0 0 0 0 -
purified. The number of free viral DNA equivalents was estimated by measuring the ability of the DNA preparations to increase the rate of reassociation of 32Plabeled SV40 DNA (1, 6). None of the five SV40 transformants tested contained detectable free viral genomes. In addition, the possible presence of nonintegrated viral DNA molecules was tested by determining the infectivity of the low molecular weight DNA extract, which was plated on BSC-1 cells in the presence of DEAE-dextran (7). No plaques or cytopathic effects were observed. It would therefore seem that SV40-transformed rat cells do not contain significant amounts of nonintegrated viral DNA. We took advantage of the slow growth of SV40-transformed rat cells in the agar medium to isolate cells doubly transformed by SV40 and polyoma. SV40-transformed cells (clone 3) were superinfected with polyoma at a multiplicity of 100 PFU/ cell in suspension. Then the cells were inoculated into agar. After two weeks of incubation, the colonies which grew faster and were therefore larger and more compact were isolated and cultured. All of the 1
IN RAT CELLS DOUBLY
Spontaneousb Py ”
1 2 3 4 5
VIRUS
329
COMMUNICATIONS
TRANSFORMED
BY POLYOMA
Fusion’ sv40
Py
0 0 0 0 0 0
100’
15 2.5 x 103 1 x 103 75 -
AND SV40
T antigen” sv40 8 3 2.5 4 2 1
x x x x x x
PY lo2 102 104 lo’ 102 103
+ + + + + -
sv40 + + + + + +
a Independently isolated transformants. b The cells were grown at 37”. The virus was extracted from 2 x 10” cells and titered on BSC-1 cells to determine SV40 infectivity and on 3T3 cells to determine polyoma infectivity, respectively. c Transformed cells (IO61 were fused with BSC-1 (lo6 cells) or 3T3 (lo6 cells) separately with the help of inactivated Sendai virus. The fused cells were incubated for 4 days at 37”. The virus extracted from the BSC-1 fusion was titered on BSC-1 cells to determine SV40 infectivity and the virus extracted from 3T3 fusion was titered on 3T3 to determine polyoma infectivity (5,6). rl The cells were grown on coverslips. SV40 and polyoma T antigen were determined on separate coverslips by immunofluorescence as described (101 using antiSV40 T hamster serum and anti-hamster fluorescent globulin, or anti-polyoma T rat serum and anti-rat fluorescent globulin. 1’ Py, polyoma. ‘The data for the spontaneous and fusion cultures are expressed as plaque forming units per culture. A value of zero indicates fewer than 10 PFU/culture. B Parental SV40-transformed rat line.
330
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COMMUNICATIONS
putative doubly transformed cells appeared to be morphologically similar. They contained SV40- and polyoma-specific T antigens as determined by immunofluorescence, and their properties were not modified by extended cultivation or growth in the presence of antipolyoma serum. Infectious polyoma or SV40 virus was rescued from these cells following fusion with mouse 3T3 or BSC-1 monkey cells, respectively (Table 1). To test the efficiency of our method of selection of doubly transformed cells, in one experiment SV40-transformed clone 3 cells were superinfected with polyoma (200 PFU/cell), plated in agar at 10,000 cells/ petri dish, and 10 days later one large colony and six small colonies were isolated. After propagation they were tested for the presence of SV40 or polyoma T antigen by using immunofluorescence. Only the cells from the large colony contained both polyoma and SV40 T antigens, while all of the cells were positive for SV40 T antigen. To determine whether any interaction between the polyoma and SV40 genes or gene products in the doubly transformed cells affected the state of the viral DNA, low molecular weight DNA was extracted from these cells as described (I), purified, and tested by DNA-DNA reassociation kinetics for the presence of polyoma and SV40 DNA sequences. The results are shown in Table 2. The clones examined contained from 4 to 33 genome equivalents of nonintegrated polyoma DNA per cell. The variation among clones in the number of free polyoma genomes per cell was not very different from that obtained earlier in different clones of polyoma-transformed rat cells (1). We could not, however, detect nonintegrated SV40 DNA molecules in these cells, a result consistant with the observation that SV40-transformed cells did not contain free viral genomes. The nonintegrated polyoma DNA molecules were infectious when tested by plaque formation on monolayers of mouse 3T3 D cells, although their specific infectivity was low (1). The above results show that F2408 cells can be singly transformed by SV40 and by
TABLE
2
FREE VIRAL DNA MOLECULES IN RAT CELW DOUBLY TRANSIWRMED BY POLYOMA AND SV40” Transformed clone Polyoma ge- SV40 genome equivalents nome equivalents per per cell cell SV40 SV40 SV40 SV40 SV40
Py Py Py Py Rat
Rat Rat Rat Rat No.
No. No. No. No. 3*
1 3 4 5
33 13 24 4 -
co.5 co.5 co.5 co.5 co.5
” Nonintegrated viral DNA equivalents were estimated by measuring the effect of low molecular weight DNA preparations from transformed rat cells on the rate of reassociation of 32P-labeled polyoma or SV40 DNA as described in (11. Low molecular weight DNA was extracted according to Hirt (5). Purified DNA from the Hirt supernatant was dissolved in a small volume of 0.01 M phosphate and 0.001 M EDTA (pH 6.81 and dialyzed extensively against the same buffer. Before hybridization, the DNA was fragmented by boiling it with the polyoma 132PlDNA or SV40 lz2PlDNA probe for 10 min in 0.3 M NaOH. DNA-DNA reassociation kinetics and hydroxyapatite chromatography were done according to Sharp et al. (61. Nonintegrated polyoma DNA equivalents per cell were calculated from the formula: Number of polyoma DNA equivalents/cell = a x 2 x lO”/A, where a = micrograms of unlabeled viral DNA in the preparations, A = the number of cells, and 2 x 10” = the number of polyoma DNA molecules per microgram. * Parental SV40-transformed rat line.
polyoma, but the transformed cells, although they both presumably carry viral genomes in a stable association with the host DNA, differ in two properties: (i) SV40-transformed cells grow in soft agar much more slowly than do the polyoma transformants. However, when SV40transformed F2408 cells are “supertransformed” by polyoma, they grow in soft agar at the same rate as do the polyomatransformed cells. Todaro et al. (8) have suggested that in mouse 3T3 cells doubly transformed by SV40 and polyoma, each of these viruses acts independently and has a distinct effect on the host cell. However, Takemoto and Habel (9) showed that when SV40 tumor cells were superinfected with polyoma, superinfection caused increased oncogenicity. Thus our results could be explained as a summation of the
SHORT
331
COMMUNICATIONS
effect of the two viruses, a property which is also evident in the very fast growth rate of these cells in liquid medium. (ii) The other main difference appears to be that the polyoma-transformed cells contain integrated and nonintegrated viral genomes, while the SV40-transformed cells contain only integrated ones. It was therefore of interest to examine the state of the viral genomes in doubly transformed cells. The results showed that in these cells, the only “free” genomes found are those of polyoma virus. It is therefore suggested that either the association of SV40 DNA with the host DNA in the rat cells is much more stable than that of polyoma DNA, or that the rat cells are more nonpermissive for SV40 DNA replication. Excision of the two viral genomes could then occur with the same frequency, but in the case of SV40 DNA, the lack of any independent viral DNA replication would not allow the accumulation of a detectable number of free viral DNA molecules. Alternatively, as previously suggested (2), it is possible that viral DNA excision and replication may be coupled. According to this hypothesis, the same host cell factors which promote replication would be responsible for induction, and our results could be interpreted as being due to the complete lack of the factors necessary for SV40 replication in rat cells. These doubly transformed cells could be
useful for studying the characteristics of integration of the polyoma or SV40 viral genomes into transformed cells and for investigating the possibility that they may share integration sites. The ease with which these cells can be isolated should facilitate studies of the effect of viral mutations upon the transformed cell phenotype. ACKNOWLEDGMENTS This investigation was supported by PHS Grants CA 11893 and CA 11642 from the National Cancer Institute. We thank Dr. T. Benjamin for the gift of anti-polyoma T serum. REFERENCES 1. PRASAD, I., ZOIJZIAS, D., and BMILICO, C. J. Viral. 18, 436-444 (1976). 2. ZOUZIAS, D., PRASAD, I., and BASILICO, C., J. Virol. 24, 142-150 (1977). 3. PRASAD, I., ZOUZIAS, D., and BASILICO, C., J. Viral. 16, 897-904, (1975). 4. MACPHERSON, I., and MONTAGNIER, L., Virology 23, 291-294 (1964). 5. HIRT, B. J., Mol. Biol. 26, 365-369 (1967). 6. SHARP, P. A., PETTERSON, U., and SAMBROOK., J., J. Mol. Biol. 86, 709-726 (1974). 7. PAGANO, J., MCCUTCHAN, J. H., and VAHERI, A., J. Viral. 1, 891-897, (1967). 8. TODARO, G., HABEL, K., and GREEN, H., Virology 27, 179-185 (1965). K. K., and HABEL, K., Virology 30, 9. TAKEMOTO, 20-28 (1966). 10. BASILICO, C., and ZOUZIAS, D., Proc. Nat. Acad. Sci. USA 73, 1931-1935 (1976).