Persistence of freely replicating SV40 recombinant molecules carrying a selectable marker in permissive simian cells

Cell, Vol. 30, 499-508,

September

1982,

Copyright

0 1982

by MIT

Persistence of Freely Replicating SV40 Recombinant Molecules Carrying a Selectable Marker in Permissive Simian Cells Lap-Chee Tsui, Martin L. Breitman, Louis Siminovitch*+ and Manuel Buchwald*+ Research Institute The Hospital for Sick Children *and Department of Medical Genetics +and Department of Medical Biophysics University of Toronto 555 University Avenue Toronto, Ontario, Canada M5G 1X8

ase, allows mammalian cells to utilize xanthine when de novo synthesis of GMP is inhibited (Mulligan and Berg, 1981 a). Since pSV2 lacks the gene encoding the large T antigen of SV40, it cannot replicate in normal simian cells. To circumvent this, a SV40-transformed simian cell line, COSl (Gluzman, 19811, which endogenously expresses T antigen, has to be used to allow replication of the vector. Results

Summary We have demonstrated that a SV40-pBR322 recombinant vector (pSVP-gpt) carrying a bacterial gene of selectable phenotype (Eco-gpt) may persist extrachromosomally in COSl cells, a simian cell line that endogenously produces SV40 large T antigen. The amount of circular (supercoiled) recombinant DNA was estimated to be between 5 and 2DDO copies per cell among several pSVP-transformed COSI clonal lines examined. Complete pSV2 molecules were found in the majority of the transformants, although some of the pSV2 DNAs recovered were shown to have deletions in the pBR322 region. Our results indicate that removal of the pBR322 “inhibitory sequence” in pSV2 is not necessary for stable maintenance of these recombinant molecules in COSl cells. In addition, large amounts of pSVP-related high molecular weight DNAs, probably concatemers of pSV2, were detected in the transformed lines. Introduction SV40 has been widely used as a eucaryotic cloning vector for the study of gene expression in mammalian cells. The various aspects of the use of SV40 recombinant vectors have been reviewed recently (Hamer, 1980; Elder et al., 1981). Most genes carried on SV40 replicating vectors are able to be expressed properly after introduction into mammalian cells. However, previous studies have only demonstrated transient gene expression shortly after transduction or transfection. It would be of great practical importance to show that SV40 recombinant molecules can be maintained indefinitely as extrachromosomal structures inside a mammalian cell. Such a system would allow one to clone genes with available biochemical selection procedures, and to obtain large quantities of gene products for various purposes. We demonstrate that high copy numbers of pSVPgpt (Mulligan and Berg, 19801, a SV40 recombinant vector that carries the Escherichia coli gpt (Eco-gpt) gene (Figure 11, can persist episomally inside a permissive cell. This bacterial gene provides an excellent biochemical selection system because the enzyme it encodes, xanthine-guanine phosphoribosyl transfer-

Replication of pSV2-gpt in COSl Cells Various investigators (Myers and Tjian, 1980; Gething and Sambrook, 1981; Gluzman, 1981) have shown that DNA molecules containing the SV40 origin of replication can multiply freely inside COSl cells, in which the SV40 T antigen, which is required for viral DNA replication (Tegtmeyer, 19721, is provided by an integrated defective SV40 (Gluzman, 1981). The recombinant plasmid pSV2-gpt (Mulligan and Berg, 1980) contains a functional SV40 origin of replication and should therefore be able to replicate under these conditions. To examine this question, we introduced circular pSVBgpt molecules into COSl cells by the calcium phosphate coprecipitation method (Graham and Van der Eb, 1973). Low molecular weight DNAs were isolated from the transfected cells by the procedure of Hirt (19671, subjected to electrophoresis on agarose gels, blot-transferred to nitrocellulose filters (Southern, 1975) and visualized by hybridization to a 32P-nick-translated pSV2 probe with subsequent autoradiography. Figure 2A shows the fate of the input pSV2 DNA at various times after transfection. The amount of circular forms I and II of pSV2 DNA observed at day 2 was substantially greater than that at day 1, and since no such increase was observed when control pBR322 DNA was used (Figures 28 and 2C), it is clear that pSV2 is able to replicate in COSl cells. Assuming that 5%-l 0% of the cell population had been successfully transfected by pSV2 (see below) and that the difference in the amount of pSV2 between day 1 and day 2 represents replicated pSV2 DNA, the average number of pSV2 molecules at day 2 was estimated to be about lo3 per cell. In comparison, transfection by wild-type SV40 DNA into permissive simian cells under similar conditions generally gives rise to approximately lo6 copies of viral DNA per cell (Lusky and Botchan, 1981). The relatively poor efficiency of replication of pSV2 in COSI cells could be due in part to the presence of the pBR322 “poison sequence” in the molecule (see below). It could also be the result of low T-antigen content inside the cell (Myers and Tjian, 1980; Gething and Sambrook, 1981). The replication of pSV2 in COSl cells seemed to be a transient event, because the amount of pSV2 in the Hirt supernatant prepared from the transfected cells decreased gradually with respect to time after 2

Cell 500

A.

B.

psv2

pBR322

C.

psv2 + I 1

09’ I

Figure

1. Physical

I

\

pBR322

1-w 2

3

7

12 lng

1

2

3

1wJ

123

7

Hmdm

Map of pSVS-gpt

The restriction enzyme recognition sites indicated on this 5.2 kb circular molecule are based on published information (Sutcliffe. 1978; Mulligan and Berg, 1980, 1981 b) and our unpublished data. Open box: the pBR322 ampicillin resistance (B-lactamase) gene. Solid box: the DNA sequence containing the E. coli gpt gene. The SV40 early promoter is located between the origin of replication (on) of SV40 and the Hind Ill site. The SV40 splicing and poly(A) addition signals for small t antigen mRNA are located between the end of the E. coli sequence and the Barn HI site. The Hinf I recognition sites are indicated on the inserted inner circle: numbers: distances (in base pairs) between adjacent sites. Asterisks: the two fragments containing the “poison sequence” (see text).

days, so that very little DNA could be detected on day 7 (Figure 2A). The sensitivity of our assay did not allow us to determine unequivocally if a small number of pSV2 molecules had been maintained inside some of the transfected cells. Transient Survival of pSV2-gpt-Transfected COSl Cells under Drug Selection The pSV2-gpt molecule Used in this study carries the xanthine-utilizing gene of E. coli (Eco-gpt) linked to the SV40 early promoter that permits the mammalian cell that ha8 acquired this gene to grow in a selective medium containing hypoxanthine, aminopterin, thymidine, mycophenolic acid and xanthine (Mulligan and Berg, 1981 a). This property of pSVP-gpt allowed us to investigate whether a small number of pSV2-gpttransfected COSl cells in which the Eco-gpt gene was maintained for an extended period of time could be selected. The transfected cells were therefore transferred to selective medium 2 days after transfection. It was clear that COS-1 cells that were not transfected with pSV2-gpt were unable to grow in selective medium (Figure 3A), whereas cells transfected with pSV2-gpt were able to survive (Figure 38). An appreciable percentage (5%-10%) of this latter cell population was able to survive for up to 7 days in selective medium, suggesting that the Eco-gpt gene

Figure

2. Replication

of pSVP-gpt

in COSl

Cells

Approximately 1 X 10’ COSl cells in each 100 mm dish were transfected with 20 ng pSVP-gpt (A). 20 ng pBR322 and 20 ng pSVPgpt (B) or 20 ng pBR322 with carrier DNA as described in the Experimental Procedures. Low molecular weight DNAs were isolated from each dish at day 1, 2. 3, 7 and 12, as indicated at the top of each lane. Half of the extracted material from each dish was subjected to electrophoresis on a 0.7% agarose gel and analyzed by filter blotting hybridization. (Lanes 1 ng) Circular forms (I and II) of pSVPgpt (1 ng) and pBR322 (1 ng) were included in the gel as markers.

was being expressed inside the cells. This finding is consistent with our observation of transient replication of pSV2 in COSl cells. Although most of the DNAtransfected cell8 eventually died, a number of colonies in could be detected after 3-4 Week8 of maintenance selective medium (Figure 3C). These colonies, corresponding to approximately O.Ol%-0.1% of the initial transfected cell population, were isolated and grown into mass culture for further study. The resulting transformed line8 were designated C/4, C/5, etc. Free pSVP-gpt Molecules in Stably Transformed COSl Cells It was first of interest to determine the state of the Eco-gpt-containing DNA that was responsible for the “stable” transformation of COSl cells. In previous studies, in which mammalian cells have been stably transformed by exogenous DNA carrying selectable markers, the DNA sequences that confer the selected phenotype are found exclusively associated with high molecular weight cellular DNA (Pellicer et al., 1978; Mulligan and Berg, 1981 a; Robins et al., 1981; Scangos and Ruddle, 1981; Breitman et al., 1982). However, since circular pSV2 molecules can multiply freely inside COSl cells, an alternative possibility was that transformation of the COSl cells was due to the persistence of freely replicating forms of pSV2-gpt. To examine these alternatives, we prepared low molecular weight DNAs from several pSV2-gpt-trans-

Persistence 501

Figure

of an Extrachromosomal

3. Transient

Survival

SV40

Recombinant

of pSVP-gpt-Transfected

COSl

Cells under

Drug Selection

Approximately 5 x 1 O5 COSl cells in each 100 mm dish were transfected with IO pg pSVP-gpt DNA. Transfected cells were replated of 1 to 3 x 1 O5 cells per 100 mm dish in drug-containing medium 3 days later. (A) Mock-transfected culture at 1 week after exposure medium: (6) oSV2-gpt-transfected culture at 1 week after exposure to selective medium; (G) a colony of “pSV2-gpt-transformed” observed at 3’h weeks after exposure of the culture to selective medium.

formed COSl lines (C/4, C/5, C/6, C/7 and C/9) and analyzed them by agarose gel-blotting hybridization. As shown in Figure 4, circular pSV2 molecules (forms I and II) were found in the Hirt supernatants in all of the five lines examined, indicating that persistence of the transformed phenotype might be associated with the existence of free replicating forms of pSV2. Although the pSV2 molecules detected were somewhat smaller than the parental molecule in some of the transformed COSl lines, further evidence (see below) confirmed their pSV2 identity. In addition, no such molecules could be detected in the low molecular weight DNA prepared from nontransfected COSl cells. Figure 4 also shows that the number of pSV2 molecules in each transformed line varied significantly; these differences were consistently observed when the lines were retested. The average number of supercoiled pSV2 molecules in each transformed line was estimated by comparison of the intensity of the band hybridizing to the 32P-labeled probe with those of various reconstructed amounts of pSV2 DNA (data not shown). The values ranged from approximately 5 (C/4) to as many as 2000 (C/7) copies per cell. Since the efficiency of DNA extraction was not measured, these amounts probably represent the lower limits of the copy number per cell. The sizes of the pSV2 molecules isolated from the COSl transformants were then examined by digestion of the Hirt supernatant DNA with Pvu II, a restriction enzyme that cuts pSVP-gpt at a single site (see Figure l), followed by analysis of the DNA as described above. As shown in Figure 5A, the majority of the pSV2 molecules from C/6 and C/9 were apparently identical in size to wild-type pSV2 (5.2 kb). The predominant pSV2 molecules in C/4 had suffered a dele-

at a density to selective COSl cells

tion of approximately 0.2 kb, and those in C/5 had lost about 1.5 kb. Two major forms of pSV2 molecules were detected in C/7; the slightly more abundant species was wild-type in size, and the other one was 0.55 kb smaller. The location of the deletions in the smaller-sized pSV2 molecules found in C/4, C/5 and C/7 were mapped by additional restriction enzyme analysis. When native circular pSV2-gpt DNA was digested with Pvu II and Barn HI (see Figure 1). two DNA fragments of 3.1 and 2.2 kb in size were obtained (Figure 5B), and after additional digestion with Eco RI, the 3.1 kb fragment was cleaved into two fragments of 2.3 and 0.75 kb (Figure 5C). The result of double enzyme digestion with Pvu II and Barn HI on low molecular weight DNAs recovered from C/4, C/5 and C/7 is shown in Figure 56. The 2.2 kb pSV2 fragment was intact in all these lines, but the 3.1 kb fragment was replaced by smaller-sized fragments, indicating that the deletions all had occurred in the 3.1 kb fragment of pSV2. The result of additional digestion with Eco RI (Figure 5C) revealed that all deletions mapped in the 2.3 kb fragment of pSV2-that is, in the sequence derived from pBR322 (see Figure 1). This more detailed restriction enzyme analysis also confirmed that the pSV2 molecules of wild-type size in C/6, C/7 and C/9 had not suffered detectable deletions. Additional species of pSVP-related DNAs were also observed in the Hirl supernatant of transformed COSI cells. These molecules were generally smaller than monomeric pSVP-gpt and were present in relatively low quantities. For example, C/5 and C/6 contained a low molecular weight species that was resistant to digestion with Barn HI and Eco RI (see Figures 5A and 5B). These species might have arisen by deletion or rearrangement of the parental pSV2-gpt molecule. In

Cell 502

psv2 cos.1

c/a

c/5

Cl6

c/7

c/9

generated by double restriction enzyme digestion of the pSV2 molecules contained in COSl cells with Pvu II and Barn HI. The result of such an analysis (Figure 5B) shows that the pSV2 molecules in all the transformed lines contained this 2.2 kb fragment. This result, of course, would be expected in those lines that contained a total pSV2 molecule. However, the fact that the DNA segment containing the Eco-gpt gene and its flanking sequences was intact even in the molecules that had suffered extensive deletions argues strongly that the expression of this gene was responsible for survival of the cells in the presence of the drug.

2QP9

23.5II

9.56.54.4-

0

5

200

Copies Figure 4. pSVP-gpt-Related COSI Cells

20 2000

20

per cell DNA Detected

in Transformed

Lines

of

Low molecular weight DNA isolated from several representative pSV2transfected COSl cells were subjected to electrophoresis on a 0.7% agarose gel and analyzed by filter-blotting hybridization. DNA was extracted from 2 x 10’ cells for COSl and C/4; from 5 X 1 O5 cells for C/5, C/6 and C/9; and from 5 X 10’ cells for C/7. Numbers at bottom: amount of pSV2 DNA in each transformed cell, estimated by use of various amounts of pSV2 form I and II DNAs as references (data not shown). Size markers (in kilobase pairs) were Hind Ill fragments of phage h DNA (data not shown).

any event, since these molecules are too small to accommodate the entire Eco-gpt gene (see below), their existence is probably not required for survival of the cells in selective medium. Intact Eco-gpt gene in Replicating pSV2-gpt Molecules There is strong evidence that the expression of the bacterial Eco-gpt gene in mammalian cells requires the presence of the flanking SV40 sequences as constructed in pSV2 (Mulligan and Berg, 1981 a; Breitman et al., 1982). This supposition could be tested in our system by analysis of the structure of the replicating incomplete pSV2 molecules. The integrity of the Eco-gpt gene and its flanking sequences would be indicated by the presence of a 2.2 kb DNA fragment

Retention of pBR322 Poison Sequence in PSV29Pt Previous investigators (Myers and Tjian, 1980; Lusky and Botchan, 1981) have indicated that SV40pBR322 recombinants that have been propagated in simian cells and then reestablished as plasmids in E. coli frequently suffer deletions within a segment of pBR322 DNA. This segment of DNA maps between the pBR322 origin of replication and the Pvu II site, and has been referred to as the “poison sequence” (Lusky and Botchan, 1981). SV40-pBR322 recombinant molecules lacking this sequence replicate more efficiently in COSl cells (Lusky and Botchan, 1981). In addition, molecules that contain the inhibitory sequence show a reduction by a factor of 100 in their ability to transform E. coli after replication in simian cells; removal of this sequence restores the transformation ability. It was therefore interesting to examine whether the pSV2 retained in the COSl transformants had undergone such a deletion. Low molecular weight DNAs prepared from Hirt supernatants of COSl transformants were used to transform E. coli, and ampicillin-resistant colonies were isolated. Some of the pSV2 molecules from C/7 and C/9 were able to reestablish as plasmids in E. coli cells. The efficiency of transformation was between 2 and 20 colonies per nanogram of pSV2, as estimated by use of the total number of ampicillinresistant colonies obtained and the amount of the predominant pSV2 species present in the Hit? supernatants (see Figure 4). In comparison with the transformation efficiency of pSV2 molecules isolated from E. coli (approximately 1 O3 colonies per nanogram of DNA), this poor transformation efficiency might be an indication that the pSV2 molecules isolated from the COSl transformants were not “detoxified.” Restriction enzyme Hinf I, an enzyme that cuts pSV2-gpt at multiple sites (see Figure 1), was then used to analyze the plasmid DNA recovered in E. coli. According to published data on the location of the poison sequence (Sutcliffe, 1978; Lusky and Botchan, 1981) detoxified pSV2 molecules would have lost one or both of the Hinf I fragments of 75 bp and 742 bp in size. Analysis of the plasmid DNAs reestablished from C/7

Persistence 503

of an Extrachromosomal

SV40 Recombinant

A.

8.

PWII

hull

r

C.

+ s.alnnr

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+

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l

Figure 5. Restriction Enzyme Analysis of Low Molecular Weight DNAs isolated from pSVPgpt-Transformed COSl Cells

ECOII

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C/S

C/b

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Cl9

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C/4

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-

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-

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-

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*

and C/9 showed diagnostic DNA fragments indistinguishable from those of wild-type pSV2 (Figure 61, confirming that the poison sequence had not been removed from those molecules. When the Hirt supernatant of C/4, containing about 150 pg pSV2 DNA, was used to transform E. coli, a single colony was isolated. Analysis of the plasmid DNA by digestion with the Hinf I restriction enzyme revealed that it had suffered a deletion of 1.7 kb in total, spanning the S/40-splicing and poly(A) addition signals and part of the Eco-got sequence (see Figure 6). This molecule is obviously different from the major pSV2 species, which has a 0.2 kb deletion in the pBR322 sequence; it probably represents one of the minor species of low molecular weight molecules found in C/4. The fact that the more abundant pSV2 species in this line could not be established as a plasmid in E. coli suggests that the 0.2 kb deletion in that molecule might have affected the integrity of some essential region of pBR322. We were also unable to establish the pSV2 molecules of reduced size found in C/5 and C/7 as plasmids in E. coli. Since the deletions in those molecules in C/5 and C/7 were 1.5 and 0.55 kb in size, respectively, and since they were totally within the pBR322 sequence, it is conceivable that the origin of replication or the ampicillin resistance gene, or both, of pBR322 had been destroyed. However, whether the poison sequence was present in these pSV2 DNAs could not be readily determined. Concatenated pSV2-gpt Molecules in COS Transformants Materials strongly hybridizing to the 3”P-labeled pSV2 probe that had high molecular weights were detected

The low molecular weight DNAs isolated from C/4. C/5, C/6, C/7 and C/9. and pSV2 DNA. were digested with Pvu II (New England BioLabs) (A), Pvu II and Barn HI (New England BioLabs) (B), or Pvu II, Barn HI and Eco RI (Boehringer Mannheim) 0. then fractionated on a 0.7% agarose gel and analyzed by filterblotting hybridization. Numbers on right and left: expected fragments of pSV2 (in kilobase pairs) upon sequential digestion with the enzymes. The presence of the 3.7 kb band of C/5 in (B) and the presence of the 3.0 kb band in (C) were the result of an incomplete digestion with Barn HI.

consistently in the Hirt supernatants of the COSl transformants (see Figure 4). When total DNA was prepared from either C/5 or C/7, digested with Xba I (a restriction enzyme that does not cut pSV2) and analyzed by gel-blotting hybridization (Figure 7A), the amount of pSV2-related high molecular weight DNA was highly enriched. The hybridizing band intensities also indicated that the amount of pSV2-related high molecular weight DNAs far exceeded the amount of low molecular weight, circular pSV2 DNAs. However, the high intensities of hybridization were clearly not due to pSV2 molecules that had been trapped in the high molecular weight region of the gel, because reelectrophoresis of the material extracted from the gel gave an identical band pattern (Figures 78 and 7C). Since concatemeric SV40 DNAs have been observed at the late stage of the viral lytic growth cycle (Rigby and Berg, 19781, we suspected that these pSV2-related high molecular weight DNAs represented equivalent structures for pSV2. To analyze the arrangement of pSV2 sequences within the high molecular weight DNAs, we subjected the material extracted from the preparative gel to limited digestion with Hind Ill (a restriction enzyme that cuts pSV2-gpt at a single site). As shown in Figures 78 and 7C. discrete pSV2-related bands were obtained upon enzyme digestion. For C/5 a band of 3.8 kb, corresponding to the size of free pSV2 in this line, could be observed. Bands corresponding to dimers and trimers of this 3.8 kb molecule were also detected after limited Hind Ill digestion, and more importantly, they could all be converted to the 3.8 kb monomeric form upon complete digestion. Similar results were obtained when high molecular weight DNA from C/7 was analyzed (Figure 70. Monomers, dimers, trimers and

Cell 504

DNAs found in C/5 and C/7 monomeric pSV2 molecules.

are concatemers

of

Role of SV40 T Antigen in the Persistence of Supercoiled pSV2 Molecules in COSl Cells It has been well established that DNA replication of SV40 is controlled by large T antigen, encoded by the early region of the viral genome (Tegtmeyer, 1972). In our study, the T antigen is provided by an integrated defective SV40 in the COSl cells. Since the amount of supercoiled pSV2 molecules varied significantly among different transformed COSl cell lines, we examined the possibility that T antigen might play some role in regulating the copy number of monomeric pSV2. T antigen was therefore assayed in three of the pSV2-gpt-transformed COSl cell lines (C/4, C/5 and C/7) by immunoprecipitation of 35S-methionine-labeled cell extracts. As may be seen in Figure 8, although no significant difference in the amounts of T antigen could be ascertained in C/4 and C/5, considerably higher levels of T antigen (molecular weight approximately 90,000) were detected in C/7. Hence there seems to be a relationship between the level of T antigen and the amount of monomeric pSV2 DNA in these cell lines (see Figure 4). Discussion

Figure 6. Hinf I Digest of pSV2 DNAs Recovered from COSl formants and Reestablished As Plasmids in E. coli

Trans-

Plasmid DNAs were prepared as described in the Experimental Procedures, digested with Hinf I (New England 6ioLabs.J and analyzed on a 5% polyacrylamide gel. (Lanes pSV2) Parental pSVP-gpt DNA: (lane pC4-1) DNA from plasmid reestablished from C/4; (lanes pC71 through pC7-5) DNA from plasmids reestablished from C/7; (lanes pC9-1 and pC9-2) DNA from plasmids reestablished from C/9. Arrows: the two fragments containing the pBR322 poison sequence. The total size of the deletion (1.7 kb) in pC4-1 was estimated from the difference between the sum of the Hinf I fragments of this plasmid and the sum of the Hinf I fragments of the parental pSVP-gpt; its location was determined by use of the map shown in Figure 1.

tetramers of the more abundant pSV2 species (wildtype in size) in this line were readily detected upon limited Hind Ill digestion, and only monomers were obtained after complete digestion. To demonstrate further the relationship between the free monomeric pSV2 DNAs and the concatemers, we digested the total DNA from C/5, which was highly enriched in concatemeric structures, doubly with Pvu II and Barn HI. As shown in Figure 7A, double digestion of concatemers from C/5 generated two bands of 2.2 and 1.6 kb in size that were identical to those obtained from double digestion of the free circular DNAs in this line (see Figure 5B). Taken together, these data indicate that the pSV2-related high molecular weight

Our results have shown that circular pSV2-gpt can persist as free molecules inside COSl cells. The average number of supercoiled pSV2 molecules per cell ranged from 5 to 2000 copies in different transformed lines. The amount of T antigen inside one pSV2-transformed line (C/7) was much higher than that in the other two lines examined (C/4 and C/5) and in the parental COSl cells. This observation probably reflects the inherent heterogeneity of expression of endogenous T antigen in COSl cells (Gething and Sambrook, 1981; Gluzman, 1981). The fact that the line containing the largest number of copies of supercoiled pSV2 contained much higher levels of T antigen suggests that T antigen may play a role in regulating the extent of pSV2 replication. The pSV2-gpt-transformed COSl lines seem to grow best at high cell densities in selective medium, and attempts to subclone these lines have been unsuccessful so far. We therefore cannot comment on whether there is heterogeneity in the number of pSV2 molecules among individual cells within the population. For the same reason, we cannot determine whether the two major species of pSV2 molecules in C/7 coexist within the same cell. The difficulty in subcloning these transformed COSl lines is probably a special characteristic of the pSV2-COSl combination, since this problem was not encountered for pSV2-gpt-transformed CVl cells. It has been shown previously that SV40-pBR322 recombinants carrying a deletion of the pBR322 inhibitory sequence replicate 1 OO-fold more efficiently than

Persistence 505

of an Extrachromosomal

SV40

Recombinant

c/5 PVUII +

BamHI C/5 c/7 XboI I XbaI ‘T

0’

1’

‘T

5’15’60’

0’

1’ 5’ 15’ 60”

23

- (41 Z(3)

d3l

9

(21

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6

II’-

4

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-Ill II’-

-2.3 -2.0

Figure

7. Restriction

Enzyme

2 2

Analysis

of High Molecular

Weight

pSV2-gpt-Related

DNAs from C/5

and C/7

(A) Five micrograms of total DNA isolated from C/5 or C/7 was digested with Xba I (New England BioLabs) or with Pvu II and Barn HI, subjected to electrophoresis on a 0.6% agarose gel and analyzed by filter-blotting hybridization. (6 and C) Total DNA (2Opg) isolated from C/5(B) or C/7(C) was first digested with Xba I and fractionated on a 0.6% agarose gel. High molecular weight pSV2-related DNAs were electroeluted from the appropriate region of the gel with Hind Ill fragments of phage h DNA as markers. The extracted DNAs were then digested with Hind III for 0, 1, 5. 15 or 60 min (lanes 0’. l’, 5’. 15’ and 60’, respectively) and analyzed on a 0.7% agarose gel. (Lanes T) Total DNAs (4 pg each of C/5 and C/7). included in the gel for comparison. I and II (or I’ and II’): circular pSV2 molecules in the two transformed COSl lines. Numbers in parentheses: monomers, dimers. trimers or tetramers of the respective pSV2 molecules in each line.

the parental molecule (Lusky and Botchan, 1981). If similar deletions of pSV2 were occurring in our transformed COSl lines, such “detoxified” molecules would have predominated during propagation of the culture. Since molecules indistinguishable from parental pSV2-gpt DNA were observed in the majority of the pSVP-gpt transformants, loss of the pBR322 poison sequence did not appear to be a frequent event. However, it is also possible that detoxified molecules do arise but that cells replicating these DNAs have a growth disadvantage. The pSV2 DNAs found in several “stably” transformed COSl lines (C/4, C/5 and C/7) have suffered deletions in the pBR322 region. Since these molecules are not maintained at a higher copy number in the transformed cells than wild-type pSV2, we suspect that they still contain the pBR322 inhibitory sequence. Further mapping data should resolve this point. The fact that the parental vector persists in most of the transformants argues against the possibility that there is a constant progression of pSV2 towards smaller

sizes. Therefore, these viable deletions probably occur early after transfection. Stable transformants were also found to contain a large amount of high molecular weight pSV2 structures. Restriction enzyme analysis on these structures suggest that they are concatemers of the major species of pSV2 monomer found in each transformed COSl line. These concatemers are not detected early after transfection (see Figure 2A); the establishment of these structures is perhaps related to the persistence of pSV2 in the stable transformants. Similar concatemers of SV40 have been observed in permissive cells late after infection (Rigby and Berg, 1978). It has been suggested that these structures arise by rolling-circle DNA replication, and in at least some instances, concatemers have been demonstrated to result from homologous recombination (Goff and Berg, 1977; Chia and Rigby, 1981). Our observations suggest that concatemers of pSV2 may form by similar mechanisms. Furthermore, it is not clear whether the presence of monomeric forms of pSV2-gpt in the

Cell

506

CVIY

c/4

c/5

NT

NTNT

Cl7

cos-1

cules in these transformants (data not shown). The decrease in the amount of concatemers was even more drastic, since such structures could not be detected at this time. We take this rapid disappearance of pSV2 concatemers as indirect evidence of the lack of stable association of these structures with cellular DNA% although the alternative possibility, that cells containing these structures are lost, cannot be ruled

---II-

NT

92.5

-

68

-

43

-

Figure

NT

8. Levels

of T Antigen

in pSVP-gpt-Transformed

COSl

Cells

Cells were labeled with ?S-methionine and extracts were prepared and immunoprecipitated. as described in the Experimental Procedures. (Lanes CV/y) Extracts from a pSV2-transformed CVl cell line. lmmunoprecipitates obtained with nonimmune rabbit antiserum (lanes N) or with rabbit anti-T antiserum (lanes T) were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and fluorographed. Molecular weight markers used (data not shown) were: myosin heavy chain (200 kd). phosphorylase 6 (92.5 kd), bovine serum albumin (68 kd) and ovalbumin (43 kd).

COSl transformants results from replication or DNA excision from concatenated high molecular weight structures. Excision of pSV2 from tandemly integrated DNA has previously been documented (Breitman et al., 1982). The fact that the pSV2 circles and concatemers found in each transformed COSl line are structurally related argues that one must be the precursor of the other. Whether there is a constant conversion from one form to the other remains to be determined. When high molecular weight DNAs from transformed cells were digested with Hind Ill, an enzyme that cuts pSV2 at a single site, several pSV2-related bands in addition to the linear form of the major species were also detected (see Figures 78 and 70. Because of the strong intensities of these bands, it is unlikely that they are derived from single pSV2 molecules that have integrated into cellular DNAs. It is therefore probable that these bands came from their corresponding concatenated forms. In fact, the integrated copies of the defective SV40 genome in the parental COSl cells were not detected under the experimental conditions used here. It would be interesting to know whether the pSV2 concatemers are integrated into cellular DNAs. We have noticed a drastic reduction of the amount of pSVP-related DNAs shortly after the transformed cell lines (C/4, C/5 and C/7) were removed from drug selection. After 6 weeks of growth in nonselective medium, there was a significant decrease in the amount of low molecular weight, circular pSV2 mole-

The major discovery in this study, that pSV2 can persist as extrachromosomal structures inside COSl cells, bears comparison with earlier observations by Mulligan and Berg (1981 a). These authors investigated the physical state of vector DNA following transfection of permissive TC7 simian cells with pSVSgpt, a derivative of pSV2-gpt carrying the early region of SV40. Although pSV3 was found to replicate transiently in TC7 cells, selection and subsequent analysis of stable transformants showed that the pSV3 sequences were associated exclusively with high molecular weight cellular DNA. Moreover, the pSV&gpttransformed cell lines were unable to produce SV40 T antigen, suggesting that alteration had occurred within the corresponding region of the vector. In our system, T antigen is provided endogenously by COSl cells, and inactivation of its expression is probably a rare event. The transfected COSl cells are therefore able to replicate free pSV2 molecules under drug selection. Experimental

Procedures

Materials The sources of pSV2, E. coli C600 r- m+ and COSl cells have been described previously (Breitman et al., 1982). Plasmid pBR322 was originally obtained from K.-L. Lee. CVl cells were obtained through the courtesy of J. Hassel. Rabbit antiserum raised against gel-purified SV40 T antigen was provided by R. Hand, and has been described in detail (Baumann et al., 1981). DNA Transfection and Cell Culture Plasmid DNAs used for DNA transfection were prepared by standard procedures described previously (Breitman et al., 1982). COSl and CVl cells were maintained in (I minimal essential medium (Stanners et al., 1971) supplemented with 10% fetal calf serum (Gibco). For the study of replication of pSV2 in COSI cells, 20 ng supercoiled pSV2 DNA was mixed with 20 pg carrier DNA prepared from Chinese hamster ovary (DR31) cells and added to each 100 mm dish containing 1 X 10’ COSl cells by calcium phosphate coprecipitation as described previously (Graham and Van der Eb, 1973; Breitman et al., 1982). As controls, cells were exposed to 20 ng pBR322 DNA or 20 ng pSV2 DNA and 20 ng pBR322 DNA with 20 pg carrier DNA. The next day, DNA-containing media were removed and replaced with fresh O! minimal essential medium supplemented with 10% fetal calf serum. Cells were subcultured 1:3 on the third and seventh day after transfection. For the selection of stable transformants. 100 mm culture dishes containing 5 x 1 O5 COSl cells were transfected with 10 gg supercoiled pSV2 DNA. Medium was changed the next day as described above. The cultures were trypsinized 48 hr later and replated at a density of 1 to 3 x 1 O5 cells per 100 mm dish in u minimal essential medium containing 10% dialyzed fetal calf serum, 15 pg/ml hypoxanthine, 2 pg/ml aminopterin, 10 lg/ml thymidine. 250 pg/ml

Persistence 507

of an Extrachromosomal

SV40 Recombinant

xanthine and 25 pg/ml mycophenolic acid (a gift from Eli Lilly). Medium was changed every 3-4, days and surviving colonies were isolated after 25-35 days. Cloned lines of stably transformed cells were propagated to high cell densities in the drug-containing medium described above and subcultured I:3 with frequent medium changes at 3-4 day intervals. Clonal lines of pSV2-transformed CVl cells were derived similarly. DNA Preparation Low molecular weight DNAs were isolated from dishes of cultured cells by the Hirt (1967) extraction procedure modified by Yang et al. (1960). After treatment with self-digested pronase (100 Kg/ml) for 2 hr at 37’C, the solutions containing low molecular weight DNAs were extracted three times with equal volumes of phenol-chloroform-isoamyl alcohol (25:24:1). Nucleic acids were then precipitated out from solution by addition of 2.5 volumes of cold ethanol and storage at -20°C overnight. Precipitates were collected by centrifugation. dissolved in 400 pl of 0.3 N sodium acetate and transferred to 1.5 ml Eppendorf-type microtest tubes. RNAase A (10 as/ml) was usually added to remove RNAs from the DNA preparations, which was followed by repeated phenol-chloroform-isoamyl alcohol extraction. The low molecular weight DNAs were precipitated again in 70% ethanol, pelleted by centrifugation and dissolved in 20-50 pl TE buffer (10 mM Tris-HCI [pH 7.51, 1 mM EDTA). Total DNAs from cultured cells were prepared essentially as described by Breitman et al. (1982), except that DNAs were collected by centrifugation following precipitation with ethanol at -20°C overnight. Restriction endonuclease digestions of DNA samples were performed under conditions recommended by the suppliers of the enzymes. Agarose Gel Electrophoresis and Filter-Blotting Hybridization DNA samples were subjected to electrophoresis on 0.7% agarose (Bethesda Research Laboratories) gels in TBE buffer (90 mM Trisborate [pH 8.31, 3 mM EDTA) containing 1 Kg/ml ethidium bromide, transferred to nitrocellulose filters (Schleicher and Schuell BA85) and hybridized to 35P-labeled nick-translated pSV2 probes (5 X 10’ cpm/ pg DNA) as described previously (Breitman et al., 1982). Extraction of DNA from Agarose Gels and Limited Restriction Enzyme Digestion DNA bands of interest were located on agarose gels by use of Hind Ill-digested phage X DNA fragments as markers. DNAs were electroeluted from agarose gels with dialysis membranes as described by Yang et al. (1979). The DNA solutions were recovered from the wells, adjusted to 400 pl each with 0.3 N sodium acetate, chilled at 0°C for 20 min. clarified by centrifugation (12.000 X g for 5 min) and extracted several times with phenol-chloroform-isoamyl alcohol. DNAs were then precipitated by ethanol with 25 pg yeast tRNA as carrier, rinsed with cold ethanol, vacuum-dried and redissolved in 50 pl TE buffer. Limited restriction enzyme digestion was carried out by incubation of the extracted DNA with 1 U Hind III (Boehringer Mannheim) at 37°C. Aliquots of the reaction mixture were taken at 1, 5. 15 and 60 min after addition of enzyme, and the reactions were terminated immediately by addition of 1 /I 0 volume of 100 mM EDTA (pH 8) followed by heating at 65°C for 10 min. Bacterial Transformation and Rapid Plasmid DNA Isolation Low molecular weight DNAs isolated from pSV2-transformed COSl cells were used to transform E. coli C600 r- mc cells by the method described previously (Breitman et al., 1982). Ampicillin-resistant colonies were picked, grown into 50 ml overnight cultures in L broth containing 100 pg/ml ampicillin and amplified with 300 gg/ml spectinomycin (Upjohn). Plasmid-containing bacteria were harvested by centrifugation, resuspended in 200 gl of 25% sucrose, 50 mM TrisHCI (pH 7.5). 50 mM EDTA. and 40 cl of 10 mg/ml lysozyme (Calbiochem) in 0.25 M Tris-HCI (pH 8) was added. After incubation for 10 min at room temperature, cells were lysed by addition of 400

pl of 0.5% Triton X-100, 50 mM Tris-HCI (pH 8). 50 mM EDTA and brief mixing at room temperature for 5 min. Bacterial debris and cellular DNAs were removed by high-speed centrifugation (12,000 x g for 40 min) at 4°C. Supernatants were collected. adjusted to 1% SDS and 1.4 M NaCl and clarified again by high-speed centrifugation. The supernatants containing plasmid DNAs were then extracted several times with phenol-chloroform-isoamyl alcohol until free of proteins. Nucleic acids were collected by ethanol precipitation and redissolved in 50 f.rI TE buffer. Aliquots of these plasmid DNA preparations were subjected to restriction enzyme digestion, and contaminating RNAs were removed by brief treatment with RNAase A prior to gel electrophoresis. Polyacrylamide Gel Electrophoresis Restriction-enzyme-digested plasmid DNA fragments were separated on a 0.5 mm thick, 5% polyacrylamide gel (29:l acrylamide to bisacrylamide: Biorad) with TBE buffer in avertical gel electrophoresis apparatus (model V16; Bethesda Research Laboratories) at 100 V until the tracking dye (bromophenol blue) migrated to approximately 1 cm from the bottom of the gel. DNA bands were stained with ethidium bromide (10 Kg/ml in TBE buffer) and visualized on an ultraviolet transilluminator. Assay for T Antigen Levels of SV40 T antigen in pSV2-transformed COSl cells were assayed essentially as described by Carroll and Smith (1976). Semiconfluent cultures were labeled for 24 hr in 5 ml drug-containing medium containing one tenth of the normal concentration of methionine and 20 @/ml ?S-methionine (1000 Ci/mmole; New England Nuclear). Parental COSl cells were labeled as described above in drug-free medium. After labeling, cells were washed twice with phosphate-buffered saline lacking Mg2+ and Ca*+, detached with 0.02% EDTA (pH 8). collected by centrifugation and resuspended in 200 pl lysis buffer (20 mM Tris-HCI [pH 81, 80 mM NaCI, 20 mM EDTA. 1 mM dithiothreitol. 1% Nonidet-P40). Extracts were freeze-thawed twice and cleared by centrifugation at 12.000 x g for 30 min at 4’C. Supernatants were collected, and approximately 1 X 1 O6 cpm acidinsoluble material was incubated with 10 Kl normal rabbit antiserum for 2 hr at 4’C. Immune complexes were removed by absorption with 100 pl of a 10% solution of formaldehyde-fixed Staphylococcus aureus (Calbiochem) (Kessler. 1975). This preabsorption procedure was repeated twice. The cleared supernatants were divided into two equal portions and treated with 10 KI normal rabbit antiserum or rabbit anti-T antiserum (provided by R. Hand) overnight at 4’C. Immune complexes were precipitated by absorption with S. aureus and washed sequentially with 0.5% Triton. 0.5% sodium deoxycholate and 0.1% SDS in 10 mM Tris-HCI (pH 7.5), 100 mM NaCI. 10 mM EDTA. lmmunoprecipitates were boiled in SDS electrophoresis buffer for 5 min. clarified by centrifugation and analyzed on a 10% SDS, polyacrylamide gel (Laemmli, 1970). Radioactive protein bands were visualized by fluorography (Banner and Laskey. 1974) with Kodak XARS film. Acknowledgments This work was supported by grants from the Medical Research Council and National Cancer Institute of Canada. L.-C. T. is a recipient of a Fellowship from the Sellers foundation. M. L. B. holds an MRC Postdoctoral Fellowship. 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

March

31, 1982;

revised

June 3. 1982

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