Loss of integrated viral DNA sequences in polyoma-transformed cells is associated with an active viral A function

Cell, Vol.

17, 645-659,

July 1979,

Copyright

0 1979

by MIT

Loss of Integrated Viral DNA Sequences in PolyomaTransformed Cells Is Associated with an Active Viral A Function Claudio Basilica, Sebastian0 Gattoni, * Dimitris Zouzias and Giuliano Della Valle Department of Pathology New York University School of Medicine 550 First Avenue New York, NY 10016

Summary Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the “curing” of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for “curing” by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo “curing” with about the same frequency at 33’ or 39’C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33” than at 39°C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to l Present address: Institute of Cancer College of Physicians and Surgeons, York, New York 10032.

Research, Columbia University 701 West 168th Street, New

occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable. Introduction A variety of studies conducted on polyoma- or SV40transformed cells have led to the conclusion that such cells contain viral DNA integrated into their chromosomal DNA. Such integration is a prerequisite for the continuous expression of viral functions in the transformed cells and is generally considered very stable (reviewed by Martin and Khoury, 1976; Kelly and Nathans, 1977). Not considering chromosome segregation in hybrid cells, only a few cases of loss of integrated viral genomes have been reported (Kelly and Sambrook, 1974; Steinberg et al., 1978). and the mechanism of this loss has not been clarified. Transformed cells generally do not produce infectious virus spontaneously, but in the case of rat ceils transformed by polyoma virus we could show that such cells contained, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules (Prasad, Zouzias and Basilica, 1976). The presence of nonintegrated viral DNA was due to a spontaneous and periodic induction of viral DNA replication occurring in a minority of the cell population. We were also able to show that the free DNA originated from the integrated form, probably by a mechanism of excision and limited replication (Zouzias, Prasad and Basilica, 1977). The presence of the free viral DNA is dependent upon an active viral A function (Fried, 1965; Eckhart, 1969; DiMayorca et al., 19691, since ts-a-transformed cells lose the free viral DNA upon shifting to the nonpermissive temperature but reacquire it upon reincubation at low temperature (Zouzias et al., 1977). While the requirement for a functional A protein could have been explained merely by the fact that such a protein was necessary for replication of the excised viral DNA, the possibility existed that the A function was also necessary for “excision” itself. Our study had two main goals. First, we wanted to determine whether induction of free viral DNA synthesis in the transformed rat cells was accompanied by loss of integrated viral DNA molecules, such as occurs during the curing of lysogenic bacteria. Second, we wanted to investigate whether the polyoma A gene product was also necessary for the “excision” event necessary for the production of free viral DNA. We therefore studied the extent of “curing” taking place in polyoma ts-a-transformed cells which had been propagated at the permissive or the nonpermissive temperature. The strategy used to isolate putative “cured” cell lines was based on the selection of cells which had regained, partially or totally, a normal phenotype. We then investigated whether such cells had

Cdl 646

lost integrated viral genomes. Our results show that in polyoma-transformed rat cells, loss of the integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted to a normal phenotype. The polyoma A function is necessary for this phenomenon; in the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable. This viral function therefore seems to be necessary for excision.

growing in low serum and beyond monolayer density should be killed by FUdR, while revertants should, like normal cells, become arrested in GI and escape the effects of the drug (Pollack, Green and Todaro, 1968; Renger and Basilica, 1972). FUdR was then washed off and the cells were incubated in medium containing 10 pg/ml of thymidine for 3-4 days. At this point, if the killing by FUdR had been such that the surviving cells would have grown into distinct colonies, the medium was changed and the cultures were incubated until colonies appeared. In most cases, however, cells were replated at 200 cells per 100 mm petridish, and the resulting colonies were analyzed for the proportion of morphological “flat” revertants (Table 1). Colonies were then isolated and their properties were studied as described below. To test whether a functional A gene product was required for the appearance of morphological revertants and presumably for “curing,” rat cells transformed by the ts-a polyoma mutant were propagated at 39.5 or 33°C for 15-30 days and then subjected to FUdR selection for revertants. To avoid temperature effects on the efficiency of FUdR selection, the selection was always performed at 37°C the temperature to which the cells were brought 3-4 days before exposure to FUdR. The results of these experiments are shown in Table 1, which shows that the frequency of morphological revertants isolated from these lines after different times of growth in culture varied widely, but that in all cases the frequency of revertants was found to be much higher (50-300 fold) in ts-a-transformed cells after propagation at 33” than at 39.5%. To rule out non specific effects of growth at different temperatures other than those related to the function of the A gene product in ts-a-transformed cells, we performed a similar experiment on a line of cells transformed by wild-type polyoma Py54. Table 1 shows that in this case the frequency of revertants isolated was the same after propagation at either temperature.

Results The polyoma-transformed cell lines used were all clonal isolates derived from F2408 rat cells (Freeman et al., 1973; Prasad et al., 1976) after infection and plating in soft agar medium. Py rat 54 was transformed by wild-type polyoma at an moi of 500 pfu per cell. The ts-a 13 line was transformed by the ts-a polyoma mutant (Fried, 1965) at 1 pfu per cell and was isolated at 33’C. The ts-a H5 and H6A lines were produced by infection with ts-a polyoma at 50 pfu per cell. Cells were plated in soft agar at 33°C for 5 days and then shifted to 39%. This procedure yielded about the same number of agar colonies as did keeping the cells at 33% (Fried, 1965). The latter cell lines were always propagated at 39.5”C unless otherwise stated. The expression of the transformed phenotype in all these cells was not temperature-dependent. The cell lines were recloned in liquid medium or in agar at 39°C (H6A) before the beginning of the selection experiments. Selection of Revertants Polyoma-transformed rat cells were plated in 100 mm petri dishes so that they reached confluence 3-4 days later. 24 hr after the cultures became confluent, 5fluorodeoxyuridine (FUdR) was added at a concentration of 25 pg/ml, and the cells were incubated in its presence for 2-3 days. Transformed cells capable of

Table

1. FUdR

Selection

of Revertants

from Polyoma-Transformed Frequency” Propagated

Rat Cells

of Revertants at:

from Cultures Virus RescueNegative Clones’

Experiment

Cell Line

I

ts-a 13

15

x 10-S

100

II

ts-a H5

20

2 x 10-r

7 x 10-S

350

19/26

4/4

Ill

ts-a H5

30

6 x lo-’

2 x 10-d

333

16/16

4/4

IV

ts-a H6A

22

1 x 10-S

5 x 10-A

50

12/14

6/6

V

Py 54

15

3 x 10-S

2 x 10-S

6/i’

6/6

395°C

33°C

-5

-5

x 10-r

Ratio 33”C/ 39.5x

T AntigenNegative Clones

Days in Culture”

a The average doubling time of these cell lines was 16 hr at 39.5%. 24 hr at 33°C. b Calculated from the proportion of “flat” colonies among the cells surviving FUdR treatment per total cell number subjected to FUdR treatment). In experiment I, the cells were subjected calculated from this experiment is approximate. ’ Among T antigen-negative revertants.

0.66

6/8

5/5

(frequency flat colonies per fraction surviving FUdR to two FUdR cycles; thus the frequency of revertants

Integrated 647

Viral DNA Sequence

Loss

These results suggested that the A gene product is necessary for the generation of revertants, but did not indicate whether reversion originates from a loss of integrated viral DNA. To determine whether this was the case, we analyzed the revertants further. General Colonies

Table

Properties of the Revertants of morphological revertants

2. Properties

ts-a 13 (Parental

of “Cured”

Lines of Polyoma-Transformed Saturation

Cellsa

were

10

DensityD

isolated,

recloned in most cases and then analyzed for the expression of some in vitro transformed growth properties, the presence of polyoma T antigen(s) and the capacity to produce infectious virus upon fusion with permissive mouse cells (Table 2). The expression of the transformed phenotype in these cell lines (as judged by growth in agar, saturation density and morphology) ranged from complete

Rat Cells EOP in Agar Medium 19

(%)’

T Antigend

Virus

+

2x

-

0

Rescue 106

line)

1A

ND

1.9

38

4.4

9.2

-

0

8C

3.8

0.1

-

0

1oc

4

0.7

-

0

1OA-2

1.9

<0.005

-

0

25

+

1 x 10”

h-a H5 (Parental

10 line)

c9-1

1.7

-co.005

cs-2

1.5

<0.005

0 -

0

c3-5c

2.2

to.005

-

0

C3-6E

2.9

<0.005

-

0

c3-9

1.5

to.005

fe

0

c3-11

2.7

<0.005

-

c3-18

4.0

10.005

0

C3-20

1.8

10.005

0

Py 54 (Parental

11

0

26

+

8x

3.0

ND

+e

0

G3

2.9


H3

3.4

2.5

-

0

c9

2.8


0

D9

2.3

10.005

0

J9

2.4

<0.005

0

ts-a H6A (Parental line)

22.4

50

0

+

1.5 x 106

R9-2

5.2

0.1

0

R3-2B

8.4

1.1

0

R3-10

lo5

Ime)

83

R3-9C

by Fusion‘

9.2 11

R3-16A

8.8

Rat F2408

2.0

17

0

20

0

18 to.005

-

0 0

a Except in the case of the lines derived from ts-a 13 that were all isolated from cultures propagated at 33°C. the first numeral after the letter in the denomination of a cell line indicates whether that cell line was isolated from cultures propagated at 39.5”C t-9) or 33OC f-3). ’ Cells per 60 mm petri dish x 1 Om6. ’ Percentage of cells plated forming colonies in agar affer 14 days of incubation at 37°C. d Determined by immunofluorescence as described in Experimental Procedures. e Very weakly positive. ’ pfu/ml at 5 days afler PEG-assisted fusion with mouse 3T3 cells. Ts-a-transformed cells and their derivatives were fused at 33°C. wild-type transformed cells at 37°C. 0 = t10 pfu/ml.

Cell 646

reversion to a normal phenotype to reversion to a phenotype intermediate between that of normal cells and the parental transformed line. These “intermediate” revertants formed colonies in agar, but did so with low efficiency, and their size was small; their saturation density was also intermediate between that of the parental and normal cells. There seemed to be a tendency for a given cell line to produce certain types of revertants rather than others. For example, while ts-a H5 seemed to produce mainly complete revertants, the revertants derived from ts-a H6A were primarily of the intermediate type. The possible significance of this observation is being investigated. It is also worth noting that the expression of these properties in the revertant lines was not temperature-dependent. We examined the revertant cell lines for the presence of polyoma-specific T antigen(s) by immunofluorescence. Cells were fixed and stained after 3-4 days of growth at 33°C. We assumed that reversion due to loss of integrated viral genomes would have resulted in cells which either did not express T antigen or expressed it to a much lower degree, while a phenotypic reversion due to a host mutation might have left the expression of this viral function unaltered. As shown in Tables 1 and 2, the majority of the cell lines examined were found to be T antigen-negative or very weakly positive. In contrast, the parental cell lines used in this study were all strongly positive (Figure 1). This finding suggested that some changes leading to a lack or reduced expression of viral functions had taken place in these cells. Cells that were T antigen-negative or weakly positive were tested for their ability to yield infectious polyoma virus upon fusion with permissive mouse 3T3 cells (Prasad et al., 1976). Virus rescue was measured at 33% in the case of ts-a transformants and at 37°C in cells transformed by wild-type virus. All the cell lines examined were unable to release infectious virus after fusion (Table 2). These two findings, particularly the inability of the revertant lines to yield infectious virus by fusion, strongly suggested that these cell lines owed their properties to changes at the level of the integrated viral DNA rather than to host cell-mediated alterations in the expression of the transformed phenotype (Basilico et al., 1974). Retransformation We examined the response of the revertant cell lines derived from ts-a-transformed cells to superinfection by wild-type polyoma virus. Cells were tested for T antigen expression during acute infection and for retransformation as detected by growth in agar medium. Figure 1. Micrographs T Antigen

of TWO Polyoma-Transformed

(A) ts-a rat H5: (B) ts-a H5 derivative

C3-20:

In all the lines tested, infection with wild-type polyoma virus (20 pfu per cell) led to the appearance of T antigen in a significant proportion of the cells by 40 hr after infection (data not shown). In addition, all the cell lines tested were capable of being retransformed by polyoma infection. The efficiency of transformation varied from lower than that in normal F2408 cells to much higher (Table 3). Several retransformed clones were isolated and tested for their ability to yield virus by fusion. Invariably the virus produced was of the wild-type (data not shown). It is not clear at present whether the wide differences in susceptibility to retransformation of these cell lines are due to differences in virus uptake or whether other factors are involved. The efficiency of infection (as measured by the percentage of T antigenpositive cells at 30 hr after infection) was found to be about 3 fold higher in the cell lines C3-20 and C9-2, and 2 fold lower in C3-9, than that observed in Rat F2408 cells. Thus differences in virus uptake may be at least partly responsible for the variations in retransformation of these lines. While this specific point will have to be investigated further, our data demonstrate that these cells are not incapable of supporting the expression of T antigen or of the transformed phenotype per se. State of the Viral DNA in the “Cured” Lines To determine whether the T antigen-negative, virus rescue-negative revertants owed their loss of transformed growth properties to a loss of integrated viral genomes. we determined the amount of viral DNA associated with host DNA in these cells. To avoid the error arising from the presence of free viral DNA, all ts-a-transformed cells and their derivatives were grown at 39.5% prior to DNA extraction. Under these conditions, these cells do not produce detectable free viral DNA (Zouzias et al., 1977). The ability of cellular DNA to influence the rate of reassociation of 32Plabeled whole polyoma DNA was then tested (Sharp, Petterson and Sambrook, 1974) and the number of viral DNA equivalents associated with the cellular DNA was determined as previously described (Zouzias et al., 1977). The results obtained with a total of 17 cell lines derived from three independent ts-a-transformed rat clones are shown in Table 4. Similar to other polyomatransformed rat cells studied, all the parental cell lines contain a rather high number of integrated viral DNA copies (Prasad et al.. 1976; Zouzias et al., 1977; Birg et al., 1979). These viral DNA copies are generally arranged in tandem repeats (Birg et al., 1979; S. Gattoni and C. Basilica, manuscript in preparation). The cured lines derived from ts-a rat 13 exhibit a very

Rat Cells and T Antigen-Negative

(0 ts-a rat 13: (D) ts-a 13 derivative

Revertants

1 OA-2.

after lmmunoftuorascent

Staining

for Potyoma

Integrated 649

Viral DNA

Sequence

Loss

WI 650

Table 3. Retransformation of Cured Infection with Polyoma Virusa

Polyoma

% Cells Plated

Transformants

Forming

Colonies

upon

Table 4. Quantitation of Viral DNA in Cured Transformed Rat Cells

in Agar Phenotypea

Cells ChllS

Control

Infected

c9-2

-co.005

13.6

ts-a 13 (Parental

c3-5c

<0.005

10.7

c3-9

<0.005

0.16

c3-11

<0.005

C3-20

O.lb

1 OA-2

0.040

Rat F2408

-co.005

Lines of ts-a Polyoma-

Integrated Viral DNA Equivalent@

T

10

1A

I

6

38

I

4.5

5.6

8C

N

2.8

10.0

line)

1oc

I

5

0.21

1 OA-2

N

2.1

1.8

ts-a H5 (Parental

T

7.3

c9-1

N

0.5

c9-2

N

0.7

c3-5c

N

0.3

C3-6E

N

0.8

c3-9

N

1.1

c3-11

N

0.3

C3-18

N

C3-20

N

0.3

ts-a H6A (Parental line)

T

2.7

R9-2

N

1.6

R3-2B

I

1.4

R3-9C

I

1.4

R3-16A

I

1.4

a Cells were infected in suspension with wild-type polyoma virus at a multiplicity of 400 pfu per cell. They were then plated in soft agar at a concentration of 5 X 1 03-1 O4 cells per 60 mm plate and incubated at 37’C. Colonies were counted after 14 days. b Microcolonies.

significant decrease in the number of integrated viral DNA equivalents, ranging from about 50% for clone 1 A to 80% for clone 1 OA-2. The lines derived from tsa rat H5 seem to have lost the majority of the integrated viral DNA sequences, so that with the exception of clone C3-9 these cell lines contain less than one viral DNA equivalent per diploid cell genome. In five cases, the amount of viral DNA approached the limits of detection of DNA-DNA reassociation as determined by reconstruction experiments (- 0.5 viral DNA equivalents per cell genome). It is therefore possible that these lines have totally lost integrated viral DNA. The four cured lines derived from ts-a H6A tested have lost only about 50% of their integrated viral DNA sequences. These data show that all the T antigen-negative, virus rescue-negative clones tested have lost a significant proportion of their integrated viral DNA sequences and therefore can be considered to have undergone “curing” of integrated polyoma DNA. We also examined the production of free viral DNA by these cell lines at 33°C. In all cases tested, cells failed to produce detectable amounts of free viral DNA (data not shown). To study the arrangement of the integrated viral DNA sequences in the transformed rat cell lines and their “cured” derivatives, we used the technique of DNA blotting developed by Southern (Southern, 1975; Kettner and Kelly, 1976; Botchan, Topp and Sambrook, 1976). Cells were grown at 39.5”C; their DNA was extracted and electrophoresed on agarose gels after digestion with restriction enzymes of different specificity for polyoma DNA (Fried and Griffin, 1977) (Figure 2a). The DNA was then transferred to nitrocellulose paper and hybridized with nick-translated (Kelly et al., 1970; Rigby et al., 1977) 3’P-labeled polyoma DNA, and the bands containing viral DNA sequences were visualized by autoradiography. This paper presents only the results obtained with some

line)

<0.5

’ (T) transformed: (N) normal: (I) intermediate. b Per diploid cell genome. Determined by DNA-DNA described in Experimental Procedures.

reassociation

as

representative cell lines. Figure 2 shows the results obtained with the ts-a 13 rat line and its derivative 1 OA-2. The results obtained with the restriction enzyme Bgl II, which does not cut polyoma DNA (Birg, et al., 1979) and yields at least two bands having approximate molecular weights of 30 and 10 x lo6 daltons (Figure 2b, slot 11, suggest that ts-a 13 contains more than one insertion of polyoma viral DNA. However, digestion with Hpa I, another restriction enzyme which does not cleave polyoma DNA, yields a rather broad band whose average molecular weight is > 15 x 1 O6 (Figure 2c, slot 4). The determination of molecular weight in this region of the gels is not very accurate (Botchan et al., 1976). These figures are nevertheless consistent with the large number of integrated polyoma DNA equivalents in this cell line (Table 4). In several experiments with enzymes that do not cleave the viral genome, we always found it difficult to obtain a clear restriction pattern of the bands containing polyoma DNA in this cell line. An alternative explanation of these results is that ts-a 13 contains only one insertion of viral DNA, but that its

Integrated 651

Viral DNA Sequence

Loss

extremely large size (Table 4) makes it very difficult to avoid breakage during the DNA extraction procedure (see below). Upon digestion with enzymes that cleave polyoma DNA only once (Barn I, Eco RI), these insertions generate two types of monomeric forms of polyoma DNA molecules (Figure 2b, slot 3; Figure 2c, slot 3). One monomer is a full-length polyoma DNA molecule (3.4 x 10’ daltons) (Fried and Griffin, 1977). The second is a defective one of which about 35% is deleted (2.2 x 10’ daltons); it has lost both Hint II and one Hind Ill restriction enzyme sites. These two types of molecules also represent the main species of “free” DNA produced by this cell line (S. Gattoni and C. Basilica, manuscript in preparation). The generation of discrete viral fragments of the same size upon digestion of cellular DNA with different enzymes that cleave polyoma DNA once is indicative of a tandem “head-to-tail” arrangement of integrated viral DNA sequences. The results obtained with these enzymes also favor the hypothesis that the full-length and defective molecules are contained in a tandem arrangement in a single viral insertion. In fact, in no case could we detect in the blots more than two linker sequences of polyoma and cell DNA (see Figure 2b, slot 3; Figure 2c, slot 3; Figure 2d. slots 4 and 7) and this was also true in the case of digestion with two other restriction enzymes, Hha I and Bum I (data not shown). We therefore think it probable that ts-a 13 contains a single insertion of defective and non defective polyoma molecules. The cured derivative of ts-a 13, clone lOA, has clearly lost the insertion of non defective polyoma DNA molecules while retaining the defective ones. Thus the bands obtained with enzymes which cleave polyoma DNA-Hind Ill, Barn I and Eco RI-appear to be identical to those obtained with ts-a 13 when the insertion of full-length molecules in ts-a 13 is not taken into account. Enzymes such as Hind Ill and Eco RI give a restriction pattern apparently identical to one of those obtained from ts-a 13 (a 2.2 x IO6 linear molecule and what are presumably two linker sequences of polyoma and cell DNA); enzymes which do not cut polyoma DNA (Bgl II; Figure 2b, slots 1 and 2) yield in lOAa band of lower molecular weight than the smallest one of ts-a 13. The results obtained with Hint II (which does not cleave the defective molecule) are particularly interesting. While digestion of ts-a 13 DNA generates a linear form of polyoma DNA of about 3 x 1 O6 daltons (see Figure 2a) and a band of higher molecular weight, digestion of lOADNA not only does not yield the 3 x lo6 form, but also reveals a fragment containing viral DNA whose molecular weight is higher than that of the corresponding band of ts-a 13 (Figure 2d, slots 7 and 8). The simplest interpretation of these results seem to be the following. The higher molecular weight band in ts-a 13 DNA represents a large linker sequence of host and defective polyoma DNA (uncleaved by Hint II) in a

tandem arrangement. In lOAthis sequences is joined with cellular DNA on both ends, since the fulllength viral molecules have been excised, and thus its molecular weight is higher. This interpretation is in line with the hypothesis that ts-a 13 contains only one insertion of viral DNA. In conclusion, while the interpretation of the restriction pattern of ts-a 13 is not totally unequivocal, it is clear that the 1 OA-2 line has lost the integration of the non defective viral DNA while retaining part or all of the integrated defective molecules. Figure 3 shows the results obtained with ts-a H5 and its derivative C3-9. Ts-a H5 contains at least two polyoma DNA insertions, as shown by cleavage with enzymes that do not cut polyoma (Figure 3a, slots 2 and 5). One insertion contains tandem repeats of a full-length polyoma DNA molecule; the other contains tandem repeats of a defective molecule (molecular weight - 3 x lo6 daltons) which has lost one of the Hind Ill sites and the Barn I site (S. Gattoni and C. Basilica, manuscript in preparation). In C3-9, which has retained only a small amount of integrated viral DNA (Table 4) restriction enzyme digestion with enzymes that do not cleave polyoma DNA (Figure 3a, slots 3 and 6) reveals only one band which is of lower molecular weight than those observed in ts-a H5. Cleavage of ts-a H5 with Eco RI produces the two types of polyoma monomeric molecules mentioned above and what are presumably four linker fragments (Figure 3b, slot 4); in C3-9 the viral insertion does not appear to be cleaved by the Eco RI enzyme, suggesting that the integrated viral DNA has lost this site. Figure 4 shows the results obtained with ts-a H6A and its derivatives R9-2, R3-2B and R3-2C. H6A contains only one insertion of polyoma DNA, as shown by analysis with the Hpa I enzyme, which does not cleave polyoma DNA (Birg et al., 1979; Figure 4, slot 2). Upon digestion with Hpa I, the cured lines yield a fragment containing viral DNA which is reduced in size with respect to the parental line (Figure 4, slots 3-5). The reduction in molecular weight (from - 14.5 to 10.5 X lo6 daltons) correlates with the loss of integrated viral DNA observed in these cells (Table 4). Analysis of H6A with Eco RI shows a unit length fragment of polyoma DNA and what is presumably a linker sequence containing appreciable amounts of viral DNA, again indicating a tandem arrangement of integrated viral molecules (Figure 4, slot 7). The DNA from the cured lines cleaved with Eco RI yields a similar linker sequence, but not the unit length molecule. Hind Ill digestion also shows a large linker sequence which appears identical in the parental and revertant lines (Figure 4, slots 12-l 5). While H6A also yields two viral bands corresponding to the two polyoma DNA fragments produced by this enzyme (1.9 and 1.5 X lo6 daltons, respectively), the cured lines yield only the small fragment. Thus these lines seem to have lost the tandem repeats of integrated viral

Cell 652

12345

v a 12

345

1

23456789

Integrated 653

Viral DNA Sequence

Loss

14.5 I)

12345 dbIb“. jl c

Figure

of Integrated

3.

Analysis

The products of DNA digestion fragments containing polyoma (a) Slot (I) ts-a Py DNA marker marker and Bgl II (which does (b)Slot (I) ts-a Py DNA marker

Polyoma

DNA Sequences

6

in the DNA of Cell Lines is-a H5 and C3-9

with restriction enzymes were analyzed as in Figure 2. The molecular weights (x 1 Oe6) of some linear DNA DNA are indicated. They were calculated as described in the legend to Figure 2. digested with Hpa I (which does not cut polyoma DNA); (2) H5 DNA and Hpa I: (3) C3-9 DNA and Hpa I; (4) Py DNA not cut polyoma DNA); (5) H5 DNA and Bgl II; (6) C3-9 DNA and Bgl II. (untreated): (2) Py marker and Eco RI; (3) H5 DNA (undigested); (4) H5 and Eco RI; (5) C3-9 and Eco RI.

sequences corresponding to a complete molecule. The regions connected with the host DNA do not appear to have undergone major changes. Taken together, the results obtained with the blotting analysis of the integrated viral DNA show that the cured lines originate from the parental ones and have undergone major losses of the integrated viral DNA sequences. A common feature is their appearance of having lost the contiguous tandem arrangement of viral DNA sequences necessary to produce full-length polyoma DNA molecules. This is consistent with the inability of these cells to produce infectious virus upon fusion with permissive mouse cells. Figure

2. Analysis

of Integrated

12345

Polyoma

DNA Sequences

Rate of Curing in the Polyoma-Transformed Rat Lines To determine at what rate the loss of integrated viral genes occurred in the ts-a polyoma-transformed cell lines, we determined the rate at which morphological revertants arose in the transformed cell population and then examined the general properties of such revertants. For this experiment, cells of the ts-a H6A line were plated at 39.5” or 33°C and were kept thereafter in exponential growth at these two temperatures. At various time intervals the cells were plated for colony formation at 39X, fixed, stained and examined for the frequency of “flat” colonies. Within

in the DNA of Cell Lines ts-a 13 and 1 OA-2

-8-10 pg of cellular DNA per sample were digested with various restriction endonucleases as described in Experimental Procedures. The digestion products were fractionated on a 15 X 16 cm 1% agarose slab gel and run for 18-20 hr at 15 V. At the end of the run the DNA was denatured and transferred to nitrocellulose filters, and specific polyoma sequences were detected by DNA-DNA hybridization with denatured “Plabeled nick-translated polyoma DNA, followed by autoradiography. (a) Map of polyoma DNA (-3.4 X 10’ daltons) showing the sites of cleavage of the restriction enzymes used in these experiments (from Fried and Griffin, 1977). (b) Slot (1) m-a 13 DNA after digestion with Bgl II (which does not cut polyoma DNA); (2) 1 OA-2 and Bgl II; (3) ts-a 13 and Barn I; (4) 1 OA-2 and Barn I; (5) ts-a Py DNA marker and Barn I. (c) Slot (1) Py DNA marker and Eco RI; (2) 1 OA-2 and Eco RI: (3) ts-a 13 and Eco RI; (4) ts-a 13 and Hpa I; (5) Py marker and Hpa I. (d) Slot (1) ts-a Py DNA marker after digestion with Hpa I (which does not cut polyoma DNA); (2) lOAand Hpa I; (3) Py marker and Hind Ill (digestion was incomplete; some fOrIn Ill is present in addition to the two Hind Ill fragments); (4) ts-a 13 and Hind Ill; (5) 1 OA-2 and Hind Ill: (6) Py marker and Hint II; (7) ts-a 13 and Hint II; (8) 1 OA-2 and Hint II; (9) Py marker and Barn I. The molecular weights (X 1 Om6) of some of the linear DNA fragments containing polyoma DNA are indicated on the right side of (b) and (d) and on the left side of (c). They were calculated from the mobility of Hind Ill and Eco RI fragments of X phage DNA, which were run on the gels in parallel and detected by ethidium bromide staining. Polyoma DNA forms I and II [(d) slot 1 or (c) slot 51 migrate more quickly or more slowly, respectively, than linear polyoma DNA (form Ill).

Cell 654

12 3456789101112l3

3.4-

1.9l.S-

Figure

4. Analysis

of Integrated

Polyoma

DNA Sequences

in the DNA of Cell Lines ts-a H6A, R3-2B.

R3-9C

and Fig-2

The products of DNA dtgestion wtth restriction enzymes were analyzed as described in the legend to Figure 2. (Slots l-5) ts-a Py DNA marker, H6A. R3-2B, R3-9C, R9-2 treated with Hpa I. (Slots 6-l 0) Py marker, H6A. R3-2B. R3-9C. R9-2 treated with Eco RI. (Slots 11-l 5) Py marker, H6A. R3-2B. R3-9C. R9-2 treated with Hind III. The positions and molecular weights (X IO-? of some of the linear DNA fragments containing polyoma DNA are indicated on the left and were calculated as described in the legend to Figure 2. The high molecular weight bands ere M-tt because the transfer of high molecular weight DNA to nitrocellulose is very inefficient

the number of colonies examined, no evidence of morphological reversion was observed in the cultures propagated at 39.5”C. Even after approximately 50 division cycles, all the clones exhibited a fully “transformed” morphology and were strongly T antigenpositive. In the cultures propagated at 33”C, on the other hand, a substantial proportion of cells having a morphology varying from intermediate to normal arose with time, and their frequency increased with time of propagation (Figure 5). The proportion of such colonies became as high as 15% of the total population after approximately 40 generation times. To determine whether these morphological revertants were of the same type as the “cured” cells selected by FUdR, 58 colonies were selected and analyzed for T antigen by immunofluorescence. About one third of these colonies were either T antigen-negative or very weakly positive. We analyzed eleven of these clones for their ability to yield infectious virus by fusion with mouse 3T3 cells.

They appeared to be negative for virus rescue. Thus the general phenotype of the cells isolated without FUdR selection is similar to that of FUdR-selected cured cells in two important properties: T antigen negativity and inability to yield infectious virus upon fusion with permissive cells (Table 5). Figure 5 shows that the rate at which revertants appeared at 33°C was not constant, suggesting the possibility of a two-step phenomenon. Assuming that only a third of the cells scored as morphological revertants are cured cells (as determined from T antigen analysis), the rate of curing appears to be - 4 x 1 OV4 per cell per generation for the first part of the curve, and 3 x 1O-3 per cell per generation for the second (Hayes, 1968). Given the number of assumptions implicit in this calculation (lack of selective advantage for either cell type and so on), it may be premature to draw any specific conclusion from these numbers. It is clear, however, that revertants arise at a rather high rate in polyoma-transformed rat popu-

Integrated 655

Viral DNA Sequence

Loss

Table 5. Properties of Cured without FUdR Selection

Cells ts-a H6A (Parental line)

0~

IO

al

30

Generation

40

so

cycles

Figure 5. Rate of Appearance of Morphological Revertants in Cultures of ts-a Rat H6A Cells during Propagation at 33°C or 39.5”C (o--O) 33OC: (M) 395°C. The abscissa indicates the number of generations after which the cells were plated for colony formation, as described in the text. The number of colonies examined for each experimental point was between 4000 and 6000.

lations, and that FUdR is not responsible for their appearance. The data presented in this section suggest that, under conditions permissive for the A viral function, curing in the polyoma-transformed cell lines occurs at a high rate. Although ts-a H6A may exhibit a higher rate of curing than other lines of transformed rat cells, it is clear that this rate is higher than that which could have been inferred from the frequency of revertants isolated after FUdR selection. It thus appears that for polyoma-transformed rat F2408 cells, FUdR selection does not discriminate well between normal and transformed cells, leading only to an enrichment for the most “tight” revertants. Discussion Nature of the “Cured” Cell Lines The results described in this paper show that under certain conditions, “curing’‘-that is, partial or total loss of integrated viral DNA-can occur in the polyoma-transformed rat cells at a rather high rate. The mechanism of such curing is not totally understood, but it appears to involve excision of integrated viral DNA. The cured lines acquire a phenotype which ranges from the fully “normal” to intermediate between normal and transformed. Since the method used to isolate cured cells selected for cells which had regained a more or less normal phenotype, it is not inconceivable that a number of cells could lose integrated viral DNA without detectable phenotypic reversion. Such cells would not have been identified in our study. Our data show clearly that in some cases the parental transformed lines used and their cured derivatives are related. Thus in the case of ts-a 13 and its

Lines Isolated

from ts-a H6A Rat Cells

Saturation Density”

EOP in Agar Medium VW

T Antigen’

Virus Rescue’

22.4

50

+

1.5 x 106 0

S2-A

2.2

0.25

kd

S2-B

4.4

1.5

-

0

s2-c

3.6

2.4

*d

0

S7-A

5

1 .o

kd

0

57-B

8

co.01

-

0

SE-A

5.6


-

0

S9-B

2.5

29

kd

0

SN

1 .a

<0.005

-

0

a Cells per 60 mm petri dish X 1 Oe6. ’ Percentage of cells plated forming colonies incubation at 37°C. ’ Determined by immunofluorescence. ’ Weakly positive. e pfu/ml at 5 days after PEG-assisted fusion 33OC. 0 = <5 pfu/ml.

in agar after 14 days of

with mouse

3T3 cells at

derivative clone 1 OA-2, the data show that 1 OA-2 has retained the insertion of defective molecules of the parental line while losing the insertion of nondefective polyoma molecules. In the case of ts-a H6A and its derivatives, the cured lines appear to have lost a portion of the integrated viral DNA, including the tandem repeat of viral DNA necessary to produce one complete polyoma DNA molecule. At least one of the viral cell-DNA sequences at the junction between viral and cell DNA does not appear to have undergone major changes. Aside from the excision of integrated viral DNA, chromosome loss could be another possible mechanism of curing. This hypothesis, however, is not generally supported by our data. First, it has been shown (Prasad et al., 1976) that the karyotype of the polyoma-transformed rat cell is generally quite stable (it has a modal number of 42) and is indistinguishable from that of the untransformed rat F2408 cells. In most of the cured cells we could not detect any gross chromosomal change with respect to the parental line (our unpublished results). We have also shown that curing occurs with about the same frequency at both 33” and 39°C in wild-type transformed cells. Thus if chromosome loss is responsible for curing, it can take place at both 33” and 39°C; however, the data show that in cells transformed by the ts-a mutants, curing takes place only at 33%. Finally, some of the data obtained with the blotting analysis of the integrated DNA clearly shows that parts of the integrated viral sequences remain, while chromosome loss would be expected to cause a total loss of the viral DNA insertion.

Cdl 656

It is not improbable, however, that in cases such as those of the cured cells derived from the ts-a H5 line, a combination of chromosome loss and excision might have been responsible for the substantial loss of viral DNA in these cells. Ts-a H5 has two insertions of viral DNA. The cured line C3-9 appears to have totally lost one insertion, while the other has changed. It is possible that an excision taking place in a cell which had already lost the entire chromosome containing one viral insertion may be responsible for these properties. Consistent with this hypothesis is the observation that ts-a H5 is the only polyoma-transformed rat line studied in which we could detect a considerable degree of karyotypic variation accompanied by aneuploidy (G. Della Valle and C. Basilica, manuscript in preparation). While loss of integrated viral DNA in the polyomatransformed cells is accompanied by a variable degree of phenotypic reversion, the most distinctive phenotype of the cured lines is their lack of T antigen expression, as detected by immunofluorescence, and their inability to yield infectious virus upon fusion with permissive cells. Inability to yield virus by fusion is clearly due to the fact that these cells have lost the genetic information necessary to produce a complete copy of viral DNA. In the case of cured cells which have maintained a significant amount of viral DNA equivalents (for example, the ts-a 13 derivative clone 1 OA), this is constituted by defective molecules. The negativity for T antigen is almost certainly due in some cases to the loss or rearrangement of the regions of the viral DNA responsible for the production of these proteins (Miller and Fried, 1976; Fried and Griffin, 1977; Ito, Spurr and Dulbecco, 1977; Hutchinson, Hunter and Eckhardt, 1978; Silver, Schaffhausen and Benjamin, 1978). It should also be mentioned that T antigen negativity is not always accompanied by a full reversion of the transformed phenotype, but that in all cases tested we failed to detect any free viral DNA. This agrees with the previous conclusion that this protein was necessary for the production of free viral DNA (Zouzias, et al., 1977). A study of the regions of viral DNA maintained in the cured cells and the relevance of the presence or absence of such regions to the phenotype of the cells is now in progress. Mechanism of Curing in Polyoma-Transformed Rat Cells Our data show that loss of integrated viral DNA in polyoma-transformed rat cells requires a functional A gene product. Thus wild-type transformed cells generate cured cells at both 39.5” or 33”C, while ts-atransformed cells undergo curing at a much higher rate at 33” than at 39.5”C (the nonpermissive temperature for the ts-a mutation). The few cured cells which can be obtained after propagation at 39.5% can be explained by the leakiness of the ts-a mutant used (Fried, 1965) and by the temperature variations

experienced by the cultures, which at the time of trypsinization and replating were not rigorously maintained at the nonpermissive temperature. The hypothesis that the viral A gene function was involved not only in the replication of the induced free viral DNA, but also in the primary “excision” event, is therefore supported by our results. At the moment, however, we cannot conclusively state that induction of free viral DNA and curing take place concurrently. While the requirement for an active A function for both curing and induction of free viral DNA (Zouzias et al., 1977) is evident, it could be the case that curing never takes place in cells producing free viral DNA, such cells being killed as a result of viral DNA replication (Zouzias et al., 1977). We believe it more probable, however, that while a certain proportion of the induced cells may be killed, a fraction survive as cured cells. It is possible that the cells which survive are preferentially the ones in which the induced viral DNA has undergone only abortive replication. A model accounting for the involvement of the A viral function-that is, large T antigen (Ito et al., 1977; Hutchinson et al., 1978; Silver et al., 1978)in virus rescue by fusion, and by inference for the induction of free viral DNA synthesis in transformed cells, has been proposed by Botchan, Topp and Sambrook (1979). In this model, which is similar to that proposed for phage Mu (Bukhari et al., 1977), a viral T antigen “activated” by a permissive cell factor recognizes the origin of replication of the integrated viral DNA and initiates independent viral DNA replication. In the case of transformed cells which are not totally nonpermissive, the same activation of T antigen would occur with very low probability, resulting in the induction of a minority of the cell population at any given time. After repeated rounds of replication of the integrated viral DNA in the “onion-skin” model (Botchan et al., 1979) viral DNA would be freed by peeling of the newly replicated DNA strands. This model would explain our finding that not only replication of excised viral DNA but also excision itself seems to require an active A function (large T antigen), and also accounts for the known involvement of this protein in the initiation of viral DNA replication (Fried and Griffin, 1977). We believe, however, that at least in some cases induction does not take place by peeling off the newly replicated DNA strands (which would leave the old strands in place and thus would not result in curing), but by excision of the integrated viral DNA molecule following aberrant replication. Excision could be due to host enzymes involved in DNA repair or to T antigen itself. Alternatively, curing may take place by a mechanism of internal recombination favored by the tandem arrangement of the integrated viral DNA sequences which [as was also found by Birg et al. (1979)] seems to be a distinct feature of the polyoma-transformed rat lines. This hypothesis implies that such a recombina-

Integrated 657

Viral DNA Sequence

Loss

tion event requires the A locus product or is facilitated by replication of the integrated viral DNA. In the case of polyoma-transformed rat cells, at least for cells that were all agar-isolated (Seif and Cuzin, 1977), large T antigen not only does not appear to be necessary for the expression of the transformed phenotype, but its presence actually seems to lead to instability of viral integration, and totally stable integration may only occur in the absence of a functional T antigen. Thus our ts-a polyoma-transformed rat cells behaved as though fully transformed at high temperature, and the integration of the viral DNA into the host DNA appeared to be very stable. At low temperature, loss of the viral DNA occurred at a relatively high rate, leading to the formation of cells which had undergone major changes in the arrangement of the integrated viral DNA sequences. A similar phenomenon may be responsible for the early observation of Summers and Vogt (1971) that polyoma ts-a-transformed BHK cells lose the ability to yield infectious virus by fusion upon propagation at 32” but not at 39°C. In conclusion, our results show that polyoma-transformed F2408 cells can undergo variable losses of integrated viral DNA (curing) in the presence of a functional A gene product. Our method of study led to the selection of cells which, as a result of such loss, had reverted to a normal or intermediate phenotype, but the occurrence of losses which would leave the cell’s transformed phenotype unchanged cannot be excluded. The rate of curing appears rather high, and most cured cells have lost the ability to produce free DNA and are negative or very weakly positive in the immunofluorescent T antigen reaction. We are now studying which (if any) species of T antigen(s) (Ito et al., 1977; Hutchinson et al., 1978; Silver et al., 1978) are produced in the weakly positive cells, and we are also examining in detail the arrangement of the viral sequences which have remained part of the genome of the cured cells. We hope that this study, together with the characterization of the free viral DNA produced by the parental transformed cells, will provide information on the precise mechanism of excision of the viral DNA from its integrated state and the role of T antigen in this process. Experimental

Procedures

Transformation Assays The ability of cells to grow in agar medium was measured by plating 1O4 cells in 60 mm petri dishes in suspension in medium contaimng 0.34% agar, as previously described (Prasad et al., 1976). Infections were carried out in suspension at room temperature. Saturation density of different cell lines was measured several days after the cultures reached confluence when the cell numbers did not increase any further. Cells were maintained with regular 3 day medium changes. T Antigen Assay Cells were fixed as previously described (Basilica. Matsuya and Green, 1970). and the presence of polyoma-specific T antigen was examined by immunofluorescence using rat anti-polyoma T serum (prepared in Brown Norwegian rats) and anti-rat fluorescein-conlugated goat gammaglobulin. Cells were examined through a Zeiss ultraviolet microscope equipped with eprfluorescence illummation and an automatic camera. Virus Rescue by Fusion Transformed rat cells were mixed with 3T3 mouse cells and plated in 60 mm petri dishes at 6 X lo5 cells per plate. One day later they were treated with PEG 1000 as described by Pontecorvo. Riddle and Hales (1977). Cells were then incubated for an additional 4-6 days and harvested, and the virus was extracted and titered. Fusion was performed at 37°C for wild-type polyoma virus-transformed cells and at 33OC for cells transformed by the ts-a mutant. Determination of the Rate of Reversion The rate of appearance of revertants in the polyoma-transformed cell populations was determined by the method described by Hayes (1968). 1 O4 cells of the ts-a H6A line, which had never been propagated at low temperature, were inihally seeded in ten 100 mm dishes. Five of the dishes were placed at 39.5’C and the remaining five at 33°C. both sets being maintained in continuous exponential growth. When the cells were almost confluent, they were trypsinized and counted, the five dishes were pooled, and for each set, 5 x 1 O5 cells were plated in five 100 mm petri dishes. During the propagation period the cells were plated as described above, seeding five large plates at 5 x 1 O5 cells per plate. The medium was changed every 2 days. At the established time intervals, the pooled cells coming from both 33” and 39.5’C were seeded in 40 large petri dishes at 300 cells per plate and grown at 39°C for 10 days. The colonies were fixed in 10% formalin m saline solution (0.85% NaCI) and stained with crystal violet. To determine the frequency of the revertants, only large colonies were counted. The mutation rate was assessed from the formulam=2log,2(2-?)/g,whereM,andMz,andNland N1 are the number of “flat” and total colonies, respectively, 1 and 2. and g is the number of generation cycles.

Cells and Viruses Swiss mouse 3T3 D cells, F2408 Fischer rat fibroblasts and F2408 polyoma-transformed cells ware used in these experiments; the general properties of these lines have been described previously (Prasad et al., 1976: Zouzias et al., 1977). Py rat 54 is a polyoma wild-type transformant. Ts-a 13. ts-a H5 and ts-a H6A were transformed by the ts-a mutant of polyoma virus (Fried. 1965). Cells were grown in Dulbecco’s modified Eagle’s medium containing 10% calf serum. Polyoma virus infectivity was titered by plaque assay on 3T3 D monolayers at 37°C for wild-type virus and at 33°C for the ts-a mutant. FUdR Selection ot Revertants Cells which had grown to confluence

medium containing 1% calf serum. One day later, 25 pg/ml FUdR were added without medium change and the cells were incubated for 2-3 days. The cultures were washed extensively and incubated in the presence of 10 pg/ml thymidine until the next medium change (usually 4 days later) (Pollack et al., 1968; Renger and Basilica. 1972).

at 37°C

were

incubated

in

at times

Nick Translation “P-labeled polyoma DNA with a specific activity of 2 x 1 O* cpm/gg or greater was obtained by mck translation (Kelly et al., 1970; RIgby et al.. 1977) using the modifications indicated by Weinstock et al. (1978). The reaction mixture was incubated with DNAase I (Worthington; 2 rig/ml of reaction mixture) for 15 min at 37°C. then shifted to 15°C and supplemented with DNA polymerase I (Boehringer-Mannheim; 8 lJ/pg of DNA). The incubation was continued for 60 additional min; the reaction was stopped by adding 10 mM EDTA. The enzymes were Inactivated by incubation at 68°C for 10 min. and the nicktranslated polyoma DNA was separated from the unincorporated tnphosphates by gel filtration through a Sephadex G-50 column (Pharmacla).

Cdl 658

DNA-DNA Reassociation Kinetics DNA-DNA hybridization and separation of single- and doublestranded DNA by hydroxyapatite chromatography were carried out essentially as described by Sharp et al. (1974). 32P-labeled polyoma DNA, prepared as described above but of lower specific activity, was used as probe. This probe was treated as described by Pellicer et al. (1978) to remove the fast reassociating material. The method of calculating the equivalents of viral DNA per diploid quantity of cell DNA (3.9 x 1 O’* dattons) was described by Gelb. Kohne and Martin (1971). DNA Blotting High molecular weight chromosomal DNA was prepared as described by Gros-Bellard, Oudet and Chambon (1973) with slight modifications, and digested with the following restriction enzymes (New England Etiolabs): Hpa I and Bgl II; Eco RI and Barn I; Hind Ill and Hint II (Figure 3a). Digestion was carried out under assay conditions suggested by the manufacturer at 37°C for 5-l 0 hr. The buffer used for Hpa I was 10 mM Tris-HCI (pH 7.4), 10 mM MgCIP. 20 mM KCI. 1 mM dithiothreitol (DTT). 100 pg/ml bovine serum albumin (BSA); the buffer for Bgl II was 6 mM KCI. 10 mM Tris-HCI (pH 7.4). 10 mM MgC12, 1 mM DTT. 100 pg/ml BSA; the buffer for Eco RI was 100 mM Tns-HCI (pH 7.5). 50 mM NaCI. 5 mM MgCb, 100 pg/ml BSA; the buffer for Barn I was 50 mM NaCI, 6 mM Tris-HCI (pH 7.4). 6 mM MgC12. 6 mM 2-mercaptoethanol, 100 pg/ml BSA; the buffer for Hind Ill was 60 mM NaCI. 7 mM MgC12. 10 mM Tris-HCI (pH 7.4). 100 pg/ ml BSA; and the buffer for Hint II was 10 mM Tris-HCI (pH 7.9). 6.6 mM MgCb. 6 mM P-mercaptoethanol, 60 mM NaCI, 100 pg/ml BSA. The reaction was stopped by adding EDTA and SDS to final concentrations of 10 mM and 0.1%. respectively. The samples were loaded on a 15 X 16 x 0.4 cm 1% agarose slab gel and run for 18-20 hr at a 15 V potential. After alkali denaturation. the DNA was transferred from the gel to nitrocellulose filter paper (Southern. 1975) as described by Kettner and Kelly (1976). After the transfer, the nitrocellulose paper was rinsed in 6 x SSC and dried under vacuum at 80°C for 8-l 0 hr. Before hybridization the filter was pretreated overnight at 65°C in 6 x SSC containing 0.02% each of polyvinylpyrrolidone (PVP 360-Srgma). Ficoll 400 (Pharmacia) and BSA. as described by Denhardt (1966). The pretreatment was continued for 3 more hr after the addition of 10 pg/ml of sonicated and denatured salmon sperm DNA. The filter was then hybridized with denatured 32P-labeled nicktranslated polyoma DNA in sealed plastic bags for 24 hr at 65OC. After hybridization, the filter was rinsed at the same temperature for several hours with four changes of 2 x SSC. 1.5 mM sodium pyrophosphate, 25 mM phosphate buffer (pH 6.8). 0.1% SDS (Weinstock et al., 1978). Finally, the nitrocellulose paper was dried and exposed at -70°C against XR-2 X-ray film (Kodak) with or without an intensifying screen (Lightning-Plus. DuPont). Acknowledgments We wish to thank Drs. M. Rush and R. Carroll for helpful discussrons; Dr. T. Benjamin (Harvard Medical School) for a gift of anti-polyoma T serum; and Eva Deutsch, Pat Santanello and Lisa Dailey for their excellent assistance. This investigation was supported by grants from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

January

30. 1979;

revised

March

Botchan. M., Topp, W. and Sambrook. J. (1976). The arrangement of simian virus 40 sequences in the DNA of transformed cells. Cell 9. 269-287. Botchan, M.. Topp, W. and Sambrook. excision from cellular chromosomes. Ouant. Biol. 43. 709-719.

J. (1979). Studies on SV40 Cold Spring Harbor Symp.

Bukhari. A. I.. Ljunquist, E.. DeBruijn. F. and Khatoon. H. (1977). The mechanism of bacteriophage MU integration. In DNA Insertion Elements, Plasmids. and Episomes. A.I. Bukhari. J. A. Shapiro and S. L. Adhya. eds. (New York: Cold Spring Harbor Laboratory). pp. 249261. Denhardt. D.T. (1966). A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641646. DiMayorca, G.. Callender. J., Marin, G. and Giordano, R. (1969). Temperature-sensitive mutants of polyoma virus. Virology 38. 126133. Eckhart. W. (1969). Complementation and transformation by temperature sensitive mutants of polyoma virus. Virology 38, 120-l 25. Freeman, A. E.. Gilden. R. V.. Vernon, M. L., Wolford. R. G.. Hugunin. P. E. and Huebner, R. J. (1973). Bromo-2-deoxyuridine potentiation of transformation of rat embryo cells induced in vitro by 3-methylcholantrene; induction of rat leukemia virus gs antigen in transformed cells. Proc. Nat. Acad. Sci. USA 70, 2415-2419. Fried, M. (1965). Cell transforming ability of a temperature-sensitive mutant of polyoma virus. Proc. Nat. Acad. Sci. USA 53. 486-491. Fried, M. and Griffin, B. E. (1977). Organization polyoma and SV40. Adv. Cancer Res. 24, 67-l

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Basilrco. C.. Renger. H. C., Burstin. S. J. and Toniolo. D. (1974). Host cell control of viral transformation. In Control of Proliferation in Animal Cells, B. Clarkson and R. Baserga. eds. (New York: Cold Spring Harbor Laboratory). pp. 167-l 76.

of

Gelb, L. D.. Kohne. D. E. and Martin, M. A. (1971). Ouantitation of SV40 sequences in African green monkey and mouse virus transformed cell genomes. J. Mol. Biol. 57. 129-l 45. Gros-Bellard, M., Oudet, D. and Chambon. high molecular weight DNA from mammalian 36, 32-38.

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Ito, Y.. Spurr. N. and Dulbecco. R. (1977). Characterization of polyoma virus T-antigen. Proc. Nat. Acad. Sci. USA 74, 4666-4670. Kelly, F. and Sambrook. J. (1974). mouse cells resistant to cytochalasin Ouant. Biol. 34, 345-353.

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Kelly, R. B.. Cozzarelli. N. R.. Deutschman. N. P., Lehman. I. R. and Kornberg, A. (1970). Enzymatic synthesis of DNA. XXXII: Replication of duplex deoxyribonucleic acid by polymerase at a single strand break. J. Biol. Chem. 245, 39-45. Kelly, T. J. and Nathans. Adv. Virus Res. 2 1, 85-l

D. (1977). 73.

The genome

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virus 40.

Kettner, G. and Kelly, T. J. (1976). Integrated SV40 sequences in transformed cell DNA. Analysis using restrrction endonucleases. Proc. Nat. Acad. Sci. USA 73, 1102-l 106.

Miller, L. and Fried. M. (1976). Construction the polyoma genome. J. Virol. 18. 824-832. C., Matsuya, Y. and Green, H. (1970). virus with mouse-hamster somatic hybrid

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Pontecorvo. G.. Riddle, P. l-t. and Hales, A. (1977). Time and mode of fusion of human fibroblasts treated with polyethylene glycol. Nature 265. 257-258. Prasad, I., Zouzias. D. and Basilica. C. (1976). State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of non-integrated viral DNA molecules. J. Viral. 78. 436444. Renger, H. C. and Basilica. C. (1972). Mutation causing sensitive expression of cell transformation by a tumor Nat. Acad. Sci. USA 69, 109-l 14.

temperature virus. Proc.

Rigby. D. W. J.. Dieckmann. M.. Rhodes, C. and Berg., P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick-translation with DNA polymerase I. J. Mol. Biol. 7 13. 237-252. Seif. R. and Cuzin. F. (1977). Temperature sensitive growth regulatron m one type of transformed rat cells induced by the ts-a mutant of polyoma virus. J. Viral. 24, 721-728. Silver, J., Schaffhausen. B. and Benjamin, T. (1978). Tumor antigens induced by nontransforming mutants of polyoma virus, Cell 15, 485496. Sharp, P. A., Petterson, V. and Sambrook. J. (1974). Viral DNA in transformed cells. I. A study of the sequences of adenovirus 2 DNA in a line of transformed rat cells using specific fragments of the viral genome. J. Mol. Biol. 86. 709-726.. Southern, fragments 517.

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