- Email: [email protected]
Biologic activity of oligomeric forms of SV40 DNA
VIROLOGY
87, 239-246
(1978)
Biologic Activity of Oligomeric MARK Laboratory
A. ISRAEL,
of Biology
of Viruses,
JANET
C. BYRNE,
Forms of SV40 DNA MALCOLM
AND
National Institute of Allergy and Infectious of Health, Bethesda, Maryland 20014 Accepted
January
A. MARTIN Diseases,
National
Institutes
27, 1978
We have analyzed the biologic activity of supercoiled oligomeric forms of SV40 DNA during productive and transforming infection. These DNA molecules had equal infectivity as assayed by plaque formation; however, the ability of such DNA molecules to transform nonpermissive cells increased linearly with their size. Oligomeric linear and circular forms of SV40 DNA produced by in vitro ligation of linear SV40 monomeric DNA had similarly enhanced transforming activity. Nucleic acid hybridization studies suggest that the amount of viral DNA in rat cells transformed by the variously sized oligomeric DNAs is similar. INTRODUCTION
In a recent report we characterized the covalently closed oligomeric SV40 DNA molecules (Martin et al, 1976), which are regularly synthesized during productive infection of African green monkey kidney (AGMK) cells (Rush et al., 1971; Jaenisch and Levine, 1971). Similar polyoma DNA molecules were previously identified by Cuzin et al. (1970), who described the appearance of supercoiled dimers and trimers following the shift of mouse cells transformed by a polyoma tsa mutant from 38.5 to 3 lo. Oligomers of pol yoma DNA (Mulder and Vogt, 1973) in sublines of such transformed cells were extensively characterized in a subsequent report by Vogt et al. (1976). Jaenisch and Levine (1972) studied the circular oligomeric and catenated forms of SV40 DNA produced during productive infection and suggested that they were not formed by recombination but were probably synthesized during viral DNA replication. Our previous experiments, on the other hand, indicated that such molecules arose by recombination rather than by an aberration in viral DNA replication (Martin et al., 1976). Although the precise biological role of such DNA molecules during lytic infection is presently unclear, their association with SV40 transcriptional complexes (Shani et al., 1977) suggests that they can function as templates for viral mRNA.
In this study we have analyzed the biologic activity of supercoiled oligomeric forms of SV40 DNA during productive and transforming infection. As expected from previously reported results (Cuzin et aZ., 1970; Jaenisch and Levine, 1971) we found oligomeric supercoiled SV40 DNA (monomers through tetramers) to have equal infectivity as assayed by plaque formation in AGMK cells. In sharp contrast, we observed that the ability of such oligomeric DNA molecules to transform nonpermissive cells increased linearly with their size. This was true of oligomeric DNA purified from lytically infected cells as well as molecules generated by in vitro ligation of linear SV40 monomeric DNA. Nucleic acid hybridization studies suggest that the amount of viral DNA in rat cells transformed by the various sized forms of oligomerit DNA is similar. MATERIALS
AND
METHODS
Cells and virus. Primary AGMK cells were propagated in Earle’s basal salt solution medium supplemented with 0.5% lactalbumin hydrolysate and 10% fetal calf serum. The plaque-purified small plaque morphology variant of SV40 (strain 776) was used in all studies. Primary’ cultures of baby rat kidney (BRK) cells were prepared by trypsinization of minced kidneys from 5- to 7-day-old 239 0042-6822/78/0872-0239$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
240
ISRAEL,
BYRNE,
Fischer rats and propagated in Dulbecco’s modified Eagle’s minimal essential medium containing 10% fetal calf serum. Assays for biological activity of viral DNA. The infectivity of various viral DNA preparations was measured by plaque assay on primary AGMK cells in the presence of 1 mg/ml of diethylaminoethyldextran (DEAE-dextran) (MW 2 x 106, Pharmacia) as previously described (Aaronson and Martin, 1970). Transformation assays were carried out in primary BRK cells as described by Graham and Van der Eb (1973). Approximately 3 weeks folowing infection of the monolayers with 0.2 pg of viral DNA in 6-cm petri dishes, the cells were fixed and stained with Giemsa stain. Transformed foci were scored as dark bluestained areas of densely packed cellular overgrowth. Preparation of SV40 DNA. Unlabeled and 32P-labeled virus were prepared by infection of primary AGMK cells at a multiplicity of 1 to 3 PFU/cell as previously described (Gelb et al., 1971). For the preparation of 32P-labeled virions, infected AGMK cells were maintained in phosphate-free Eagle’s minimal essential medium (MEM) containing carrier-free E3”P]orthophosphate (100 or 10 &i/ml). Unlabeled and 32P-radiolabeled SV40 DNAs were prepared from purified virions by incubation with 1% Sarkosyl for 1 hr at 50” and isopycnic centrifugation in CsCl containing ethidium bromide as previously described (Gelb et al., 1971). Supercoiled forms of oligomeric SV40 DNA were prepared from primary AGMK cells infected at a multiplicity of 100 to 200 PFU/cell as previously described (Martin et .aZ., 1976). Closed circular DNA purified by isopycnic centrifugation in CsCl containing ethidium bromide was sedimented through a 5 to 30% (w/v) neutral sucrose gradient. Peak fractions sedimenting at 21, 27, 32, and 36 S (corresponding to monomeric through tetrameric supercoiled SV40 DNA) (Martin et al., 1976) were pooled and used to infect primary AGMK or BRK cells. In vitro ligation of R.Eco.Rl-digested viral DNA. 32P-SV40 DNA I (sp act., 2: 4 x lo” cpm/pg), 50 pg, was digested with
AND MARTIN
R.Eco.Rl in a l.O-ml reaction mixture containing 0.1 M Tris (pH 7.9), 0.05 M NaCl, 0.1 mM EDTA, 0.012 M MgC12, and 5 U of R.Eco.RI (Miles Laboratories) for 45 min at 37”. The digestion was stopped by incubating the mixture at 68” for 8 min, at which time it was adjusted to 10 n-&f dithiothreitol, 1 r&f EDTA, 0.1 n&f ATP, and 1 pg/ml of bovine serum albumin. T4 ligase (Miles Laboratories), 2.5 U, was then added and the reaction mixture was incubated at 10’ for 1 hr, deproteinized with an equal volume of phenol containing 0.1% 8-OH quinolone, extensively dialyzed against 0.01 M Tris (pH 8.0), and sedimented through a 5 to 30% (w/v) sucrose gradient at 23,000 rpm for 16 hr at 10’ in a Beckman SW 41 rotor. Preparation of transformed cell DNA. Transformed cell lines were derived from foci resulting from the infection of primary BRK cells with supercoiled forms of monomeric, dimeric, trimeric, or tetrameric SV40 DNA. Cellular DNA was prepared from such preparations following 6 to 10 passages in culture as previously described (Gelb et al., 1971) and mechanically fragmented at 50,000 lb/in’ in a Ribi cell fractionator. Agarose gel electrophoresis. Electrophoresis through 0.7% (w/v) agarose slab gels (17 x 12 x 0.3 cm) was carried out at 150 V for 3.5 hr at 10’ as previously described (Hayward and Smith, 1972). Following visualization of ethidium bromide-stained DNA by long-wave uv illumination (Sharp et al., 1973), specific DNA bands were eluted from the agarose by homogenization of gel fractions in a solution containing 0.1x SSC, 0.1% SDS, and 1 mM EDTA (Howley et al., 1975). The eluted DNA was further purified on Sephadex G-160 containing Dowex 5OW-X8 (Trilling and Axelrod, 1970). Autoradiograms were made from dried gels by direct contact with Kodak RP/RB X-ray film. DNA-DNA reassociation. 32P-labeled (sp act., 1.2 x lo6 cpm/pg) fragmented denatured SV40 DNA was allowed to reassociate in the presence of a 1 M excess of unlabeled, fragmented, denatured, transformed cell DNA or an equal amount of salmon sperm DNA (Calbiochem) in 1.0 M
OLIGOMERIC
FORMS
sodium phosphate buffer at 68”. Samples were removed at various times and analyzed for single- and double-stranded DNA by hydroxyapatite chromatography as previously described (Gelb et al., 1971). RESULTS
Biological merit
Activity of Supercoiled OligoSV40 DNA in AGMK Cells
In the first group of experiments to be described, the biological activity of oligomerit forms of supercoiled viral DNA was assessed in primary AGMK cells. Confluent monolayers were infected with several dilutions of purified preparations of monomeric, dimeric, trimeric, or tetrameric forms of supercoiled SV40 DNA and assayed for plaque formation as outlined under Materials and Methods. The results of such an experiment are shown in Table 1. One microgram of dimeric, trimeric, or tetrameric viral DNA produces approximately one half, one third, and one quarter the number of plaques, respectively, compared to 1 pg of inoculum of monomeric DNA. Since such an oligomeric series contains one half, one third, and one quarter the number of molecules present in an equal mass of monomeric DNA, the data indicate that supercoiled oligomeric DNA molecules are equally effective in establishing productive infection. This result is in agreement with those of Cuzin et al. (1970) and Jaenisch and Levine (1972), who found that the infectivity of small oligomeric supercolied forms of either polyoma or SV40 DNAs in permissive cells was directly related to the number of DNA molecules used in the infection. Indeed, this is not an unexpected result if one assumes that the infecting oligomers are not converted to multiple smaller infectious molecules before entering the cell. Transformation of BRK Cells with Supercoiled Oligomers of SV40 DNA
To evaluate the ability of supercoiled oligomers of SV40 DNA to transform animal cells, we exposed BRK cells to purified preparations of viral DNA as described under Materials and Methods. Visible foci of cellular overgrowth were observed 14 to 18 days following DNA infection. Microscopi-
OF
SV40
241
DNA TABLE
INFECTIVITY Oligomeric
OF SV40 form
Monomer Dimer Trimer Tetramer
1 OLIGOMERIC
DNA
Plaque-forming per microgram 3.8 2.0 1.25 0.75
x x x x
unite of DNA lo5 lo5 lo5 lo5
tally, these foci consisted of densely overgrown epithelioid cells which stained deep blue with Giemsa stain. In plates which had been exposed to salmon sperm DNA, areas of overgrowth were not observed. As shown in Table 2, the number of transformed colonies per microgram of DNA is very similar for each of the DNA species examined. The variation in the number of foci for a given inoculum between the two experiments may be related to the condition and degree of confluency of the cells at the time of infection. These results strongly suggest that the ability of this SV40 oligomeric DNA to effect transformation is related to the number of molar equivalents of viral genome rather than to the actual number of SV40 DNA molecules in the inoculum. This is in contrast to the results obtained following the infection of permissive cells with such DNA preparations (i.e., that biological activity correlates with the number of DNA molecules rather than the amount of DNA in the inoculum). Transformation merit DNA Vitro
of BRK Cells with OligoProduced by Ligation in
The finding that tetrameric supercoiled viral DNA was four times more efficient in producing transformed foci than monomeric SV40 DNA suggested that large viral DNA molecules may transform BRK cells more efficiently than smaller DNA molecules. On the other hand, since the monomeric components present in these oligomerit viral DNA molecules are known to be arranged in a head-to-tail fashion (Martin et al., 1976), our results could be interpreted as suggesting that the arrangement of SV40 monomers as a repeating unit in a circular molecule endowed the multimeric supercoiled DNA with enhanced transforming activity. It is known that full-
242
ISRAEL, TABLE
form
88 106 120 94
1
Expt
pool AE pool BE
-
25s
4 I-B---rkA+l
i$
21s
*
400
2
197 149 250 149
33s
4T
of
II
350 :
II
6
Ii
II-PI
300 r
In Vitro Oligomer Oligomer
MARTIN
OF SV40
Foci per r$cqram Expt
In Vivo Monomer Dimer Trimer Tetramer
AND
2
TRANSFORMATION EFFICIENCY OLIGOMERIC DNA Oligomeric
BYRNE,
H
116 179
length linear forms of SV40 DNA transform cells with the same efficiency as either the supercoiled or relaxed circular form (Abrahams et al, 1975). We therefore decided to evaluate whether the arrangement of the monomeric constituents within oligomeric viral DNA plays a role in the enhanced transforming activity observed. Accordingly, we examined the transformation efficiency of linear and circular oligomeric forms of SV40 DNA prepared in vitro by the ligation of restriction enzyme-cleaved SV40 monomeric DNA (DeVries et al., 1976; Dugaiczyk et al, 1975). The monomeric constituents of such oligomeric DNA molecules would be arranged in both headto-tail and head-to-head orientations (Ferguson and Davis, 1975). SV40 DNA was cleaved by R.Eco.Rl and ligated with T4 ligase, described under Materials and Methods. This DNA preparation consisted of oligomeric linear and circular molecules. The ligated DNA was sedimented through a 5 to 30% (w/v) sucrose gradient and pooled into six fractions (I-VI) as indicated in Fig. 1. Aliquots of these fractions were analyzed in a 0.7% agarose slab gel, the autoradiogram of which is shown in Fig. 2. Lane A in this autoradiogram contains the reaction products of R.Eco.Rl digestion of SV40 DNA I. The prominent band is fulllength linear SV40 DNA and the fainter faster moving band is residual uncleaved supercoiled SV40 DNA. Lane B contains an aliquot of the reaction products generated by ligation of the R.Eco.Rl-digested SV40 DNA prior to centrifugation. Lanes
rat.
‘k
I OOW
50 FRACTION
FIG. 1. Sedimentation in neutral sucrose of oligomerit forms of SV40 DNA prepared by ligation in vitro. R.Eco.Rl-cleaved SV40 DNA was ligated with T4 ligase, phenol extracted, extensively dialyzed, and sedimented through a 5 to 3W (w/v) neutral sucrose gradient as described under Materials and Methods. Fractions were collected from the bottom of the tube and radioactivity was determined by Cerenkoff counting of the individual fractions. The position of closed circular SV40 DNA is indicated by the arrow labeled 21 s.
C, D, E, F, G, and I-I correspond to aliquots of the pooled fractions I-VI from the sucrose gradient shown in Fig. 1. We have characterized these fractions by alkaline sucrose density gradient centrifugation and electron microscopy and found each of them to contain linear and circular oligomers of progressively increasing size up to at least 10 times the size of monomeric SV40 DNA (data not shown). For instance, such an analysis of the DNA bands depicted in Lane D indicates that the most rapidly moving band is supercoiled SV40 DNA I which had not been cleaved by
OLIGOMERIC
FORMS
OF
SV40
DNA
243
ABCDEFGH
FIG. 2. Electrophoresis of oligomeric forms of SV40 DNA SV40 DNA I (lane A) was ligated with T4 Iigase. An aliquot of extracted, extensively dialyzed against 0.01 M Tris (pH 8.0), fractions (I-VI) of the sucrose gradient depicted in Fig. 1 were The electrophoresis was in 0.7% agarose for 3.5 hr at 150 V migration is from top to bottom; the autoradiogram was made X-ray film.
R.Eco.Rl. The slowest moving band corresponds to closed circular dimers of viral DNA. The series of faint bands moving ahead of this slowest moving band represent dimeric forms of closed circular DNA of increasing superhelicity. The darker
prepared by ligation in u&o. R.Eco.Rl-digested the reaction products of this ligation were phenol and analyzed in lane B. Aliquots of the pooled dialyzed and analyzed in lanes C-H, respectively. as described under Materials and Methods. The from the dried gel by direct contact with Kodak
band of intermediate mobility running in the midst of the supercoiled dimeric circular DNA corresponds to trimeric linear viral DNA. This agrees closely with the findings of DeVries et al. (1976), who have published a similar analysis of the products generated
244
ISRAEL,
BYRNE,
by this reaction. Following this analysis, fractions from the sucrose gradient were divided into two pools: Pool A contained all DNA species sedimenting from 20 to 24 S; pool B contained all DNA species sedimenting between 25 and 38 S (Fig. 1). Each of these pools was then separately electrophoresed in a 0.7% agarose slab gel. Bands corresponding to linear and circular dimeric and linear trimeric viral DNA were eluted from pool A and designated pool AE. Bands corresponding to linear molecules 4 to 10 times the size of monomers and circular molecules at least tetrameric in size were identified following the preparative electrophoresis of pool B, eluted, and designated pool BE. The transforming efficiencies of these two pools of SV40 DNA oligomers LEGEND
2.8 r 2.6
2.4
AND
MARTIN
prepared by ligation in vitro are shown in Table 2. One microgram of DNA from either pool Az (small oligomers) or pool BE (large oligomers) yields a number of transformed foci similar to that observed with 1 pg of a purified preparation of supercoiled oligomeric viral DNA prepared from productively infected monkey cells. This enhanced transforming efficiency of oligomerit viral DNA cannot be related to the sequential arrangement of the SV40 monomeric components since linear or circular oligomers of SV40 DNA generated in vitro contain relatively few molecules whose monomeric constituents are oriented solely head-to-tail. Quantitation of Viral DNA Sequences in Cells Transformed by Oligomeric Viral DNAs Our finding that the transformation effrciency of oligomeric SV40 DNA molecules varied directly with the size of the molecules throughout the range examined suggested that there might be a difference in the number of viral DNA equivalents present in the cell lines resulting from these transformation experiments. To examine this possibility, we prepared DNA from BRK cells transformed by preparations of supercoiled oligomers of viral DNA purified from lytically infected AGMK cells. Figure 3 shows that the DNA from cells transformed by each of the oligomeric DNA forms examined accelerated the reannealing of fragmented 32P-labeled SV40 DNA. With one exception, the preparations of transformed cellular DNA examined conTABLE
0
.2
I .4
I .6
I .8
I 1.0
I 1.2
%2
FIG. 3. Kinetics of reassociation of 32P-labeled SV40 DNA in the presence of unlabeled DNAs from salmon sperm (0) and BRK cells transformed by SV40 monomeric (O), dimeric (A), trimeric (X), and tetramerit (A) supercoiled DNA. “P-labeled fragmented denatured SV40 DNA (sp act., 1.2 X 10” cpm/ag) was allowed to reassociate in the presence of a 1.0 M excess of fragmented denatured unlabeled salmon sperm or transformed cell DNA as described under Materials and Methods. The fraction of radiolabeled DNA remaining single stranded (fss) was assayed by hydroxyapatite column chromatography (Gelb et aZ., 1971).
3
QUANTITATION OF SV40 DNA SEQUENCES IN OLICOMERIC DNA-TRANSFORMED BABY RAT KIDNEY LINES Cellular DNA AccelerEquivat1/2 ation lents of (l-4 factor viral DNA per diploid genome Monomer transformed Dimer transformed Trimer transformed Tetramer transformed Salmon sperm
5.5 4.4 5.5 5.7 16.2
2.0 2.7 2.0 1.9 -
2.0 2.7 2.0 1.9 -
OLIGOMERIC
FORMS
tamed 2.0 viral DNA equivalents per diploid genome (Table 3). DNA from cells transformed by dimers contained 2.7 viral equivalents per diploid genome. We do not consider this variation to be significant and conclude that the amount of viral DNA present in cells transformed by the various oligomeric SV40 DNA species is similar and unrelated to the particular oligomeric species used for transformation. DISCUSSION
While the biologic activity of SV40 oligomeric DNAs in permissive cells has been previously described (Jaenisch and Levine, 1971), the capacity of this DNA to transform animal cells in culture has not been studied. Jaenisch and Levine (1971) found that SV40 dimeric molecules were about as infectious as monomeric DNA molecules. That is, a given mass of dimers yields one half the number of plaques produced by the same mass of monomers. Using highly purified preparations of SV40 monomeric, dimerit, trimeric, and tetrameric DNA molecules, we confirmed this result, Crmly establishing that infection of monkey cells with oligomeric SV40 DNA molecules is a function of the number of molecules in the inoculum rather than the amount of inoculated DNA. The pattern of interaction between SV40 oligomeric DNA molecules and nonpermissive cells was quite different from the one observed with permissive cells. We found the ability of SV40 oligomeric DNA to transform BRK cells to be a function of the amount of DNA rather than the number of DNA molecules in the inoculum. That is, the number of tetramers necessary to achieve a specific frequency of transformation is one quarter the number of monomers required to produce a similar effect. Our results shed no light on the mechanism by which oligomers larger than monomeric size effect enhanced transformation efficiency. However, a number of possibilities can be entertained. Monomerization of the trimer or tetramer forms of viral DNA would yield three or four times the number of transforming units per infecting molecule. Our finding similar amounts of SV40 DNA in cells
OF
SV40
DNA
245
transformed by either monomeric, dimeric, trimeric, or tetrameric DNA molecules further suggests the possibility of a “common pathway” by which transformation could be effected by each of these molecular forms. However, since the increased transformation efficiency we observed is essentially linear over the range examined, monomerization of larger viral DNA molecules, if it occurred, would have to involve a precise and symmetrical cleavage yielding monomeric units of equal transforming efficiency, an efficiency identical to that of prototype SV40 monomeric DNA. Such a monomerization could be envisioned as occurring by efficient intramolecular recombination, although such a process has yet to be demonstrated in animal cells. It seems unlikely to us that rat cells would possess other mechanisms for such specific cleavage. The possibilities remain that there is some unique advantage conferred on the oligomeric forms of viral DNA by virtue of either its size or the physical orientation of the monomer subunits in a circular molecule. Our finding that the multimeric products of the in vitro ligation of linear SV40 DNA also had increased transforming capacity would seem to rule out the possibility that the head-to-tail arrangement of SV40 genomes in the oligomers isolated from lytically infected AGMK cells conferred any advantage on these DNA forms. On the other hand, the increased size per se of oligomeric viral DNA could lead to an enhanced transformation efficiency either by providing the cell with molecules containing multiple copies of specific viral DNA sequences or simply by providing a larger molecule to participate in the transforming event. In the former case, additional copies of certain SV40 DNA sequences could provide higher concentrations of specific sites involved during integration into the host cell chromosome or perhaps higher concentrations of viral gene products necessary for transformation. This is an interesting possibility in that it suggests that a threshold for viral sequences or virally coded products is required for transformation. Such a concept would be compatible with the finding by Graessman et al. (1976) that the inten-
246
ISRAEL,
BYRNE,
sity of T-antigen fluorescence following injection of SV40 DNA into 3T3 cells was dose dependent. The increased size of the oligomeric viral DNA molecule may itself be responsible for the increased efficiency of transformation we observed. This could also explain the findings of Mulder and Vogt (1973) who noted that while the infectivity of a defective polyoma genome about 1.6~ the size of a monomer was only one tenth that of monomers, the efficiency in transforming hamster cells was about twice that of monomers. Such a “size effect” would also be compatible with our findings that the organization of the genomic units within the oligomer is unrelated to this enhanced effect and that the amount of viral DNA present in transformed cells was independent of the particular oligomeric species used to transform those cells. So little is known of the molecular events involved in transformation that to speculate whether such an advantage is due to increased configurational flexibility, enhanced availability of sites critical for integration into cellular DNA, or any other property dictated by molecular size would be pure conjecture. Experiments currently in progress will hopefully determine the molecular basis of this enhancement and will also provide insight concerning the process of viral-induced transformation. REFERENCES AARONSON, S. A., and MARTIN, M. A. (1970). Transformation of human cells with different forms of SV40 DNA. Virology 42,848-854. ABRAHAMS, P. J., MULDER, C., VAN DE VOODE, A., WARNAAR, S. O., and VAN DER EB, A. J. (1975). Transformation of primary rat kidney cells by fragments of simian virus 40 DNA. J. Viral. 16,818823. CUZIN, F., VOGT, M., DIECKMANN, M., and BERG, P. (1970). Induction of virus multiplication in 3T3 cells transformed by a thermosensitive mutant of polyoma virus. J. Mol. Biol. 47,317-333. DEVRIES, F. A. J., COLLINS, C. J., and JACKSON, D.A. (1976). Joining of simian virus 40 DNA molecules at endonuclease RI sites by polynucleotide ligase and analysis of the products by agarose gel electrophoresis. Biochim. Biophys. Acta 435.213-227. DUGAICZYK, A., BOYER, H. W., and GOODMAN, H. M. (1975). Ligation of Eco.Rl endonuclease-generated DNA fragments into linear and circular structures. J. Mol. Biol. 96, 171-184.
AND
MARTIN
FERGUSON, J., and DAVIS, R. W. (1975). An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs. J. Mol. Biol. 94, 135-149. G~LB, L. D., KOHNE, D. E., and MARTIN, M. A. (1971). Quantitation of simian virus 40 sequences in African green monkey, mouse and virus-transformed cell genomes. J. Mol. Biol. 57,129-145. GRA~SSMAN, A., GRAESSMAN, M., and MUEI,LER, C. (1976). Regulatory mechanism of simian virus 40 gene expression in permissive and in nonpermissive cells. J. Viral. 17.854-858. GRAHAM, F. L., and VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52.456-467. HAYWARD, G. S., and SMITH, M. G. (1972). The chromosome of bacteriophage T5: I, Analysis of single stranded DNA fragments by agarose gel electrophoresis. J. Mol. Biol. 63, 383-395. HOWLEY, P. M., MULI~ARKEY, M. F., TAKEMOTO, K. K., and MARTIN, M. A. (1975). Characterization of human papovavirus BK DNA. J. Viral. 15,173-181. JAENISCH, R., and LEVINE, A. J. (1971). DNA replication in SV40-infected cells: V, Circular and catenated oligomers of SV40 DNA. Virology 44, 480-493. JAENISCH, R., and LEVINE, A. J. (1972). DNA replication in SV40 infected cells: VI, The effect of cyclohexamide on the formation of SV40 oligomeric DNA. Virology 48,373-379. MARTIN, M. A., HOWLEY, P. M., BYRNE, J. C., and GARON, C. F. (1976). Characterization of supercoiled oligomeric SV40 DNA molecules in productively infected cells. Virology 71, 28-40. MULDER, C., and VOGT, M. (1973). Production of nondefective and defective oligomers of viral DNA in mouse 3T3 cells transformed by a thermosensitive mutant of polyoma virus. J. Mol. Biol. 75,601~608. RUSH, M., EASON, R., and VINOGRAD, J. (1971). Identification and properties of complex forms of SV40 DNA isolated from SV40 infected cells. Biochim. Biophys. Acta 228,585-594. SHANI, M., SEIDMAN, M., and SALZMAN, N. P. (1977). In uiuo transcription of oligomeric simian virus 40 (SV40) DNA. Virology 83, 110-119. SHARP, P. A., SUGDEN, B., and SAMBROOK, J. (1973). Detection of two restriction endonucleases in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12,3055-3063. TRILLING, D. M., and AXELROD, D. (1970). Encapsidation of free host DNA by simian virus 40: A simian virus 40 pseudovirus. Science 168,268-271. VOGT, M., BACHELER, L., and BOICE, L. (1976). Proposed structure of two defective viral DNA oligomers produced in 3T3 cells by the m-2 mutant of polyoma virus. J. Viral. 17, 1009-1026.
87, 239-246
(1978)
Biologic Activity of Oligomeric MARK Laboratory
A. ISRAEL,
of Biology
of Viruses,
JANET
C. BYRNE,
Forms of SV40 DNA MALCOLM
AND
National Institute of Allergy and Infectious of Health, Bethesda, Maryland 20014 Accepted
January
A. MARTIN Diseases,
National
Institutes
27, 1978
We have analyzed the biologic activity of supercoiled oligomeric forms of SV40 DNA during productive and transforming infection. These DNA molecules had equal infectivity as assayed by plaque formation; however, the ability of such DNA molecules to transform nonpermissive cells increased linearly with their size. Oligomeric linear and circular forms of SV40 DNA produced by in vitro ligation of linear SV40 monomeric DNA had similarly enhanced transforming activity. Nucleic acid hybridization studies suggest that the amount of viral DNA in rat cells transformed by the variously sized oligomeric DNAs is similar. INTRODUCTION
In a recent report we characterized the covalently closed oligomeric SV40 DNA molecules (Martin et al, 1976), which are regularly synthesized during productive infection of African green monkey kidney (AGMK) cells (Rush et al., 1971; Jaenisch and Levine, 1971). Similar polyoma DNA molecules were previously identified by Cuzin et al. (1970), who described the appearance of supercoiled dimers and trimers following the shift of mouse cells transformed by a polyoma tsa mutant from 38.5 to 3 lo. Oligomers of pol yoma DNA (Mulder and Vogt, 1973) in sublines of such transformed cells were extensively characterized in a subsequent report by Vogt et al. (1976). Jaenisch and Levine (1972) studied the circular oligomeric and catenated forms of SV40 DNA produced during productive infection and suggested that they were not formed by recombination but were probably synthesized during viral DNA replication. Our previous experiments, on the other hand, indicated that such molecules arose by recombination rather than by an aberration in viral DNA replication (Martin et al., 1976). Although the precise biological role of such DNA molecules during lytic infection is presently unclear, their association with SV40 transcriptional complexes (Shani et al., 1977) suggests that they can function as templates for viral mRNA.
In this study we have analyzed the biologic activity of supercoiled oligomeric forms of SV40 DNA during productive and transforming infection. As expected from previously reported results (Cuzin et aZ., 1970; Jaenisch and Levine, 1971) we found oligomeric supercoiled SV40 DNA (monomers through tetramers) to have equal infectivity as assayed by plaque formation in AGMK cells. In sharp contrast, we observed that the ability of such oligomeric DNA molecules to transform nonpermissive cells increased linearly with their size. This was true of oligomeric DNA purified from lytically infected cells as well as molecules generated by in vitro ligation of linear SV40 monomeric DNA. Nucleic acid hybridization studies suggest that the amount of viral DNA in rat cells transformed by the various sized forms of oligomerit DNA is similar. MATERIALS
AND
METHODS
Cells and virus. Primary AGMK cells were propagated in Earle’s basal salt solution medium supplemented with 0.5% lactalbumin hydrolysate and 10% fetal calf serum. The plaque-purified small plaque morphology variant of SV40 (strain 776) was used in all studies. Primary’ cultures of baby rat kidney (BRK) cells were prepared by trypsinization of minced kidneys from 5- to 7-day-old 239 0042-6822/78/0872-0239$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
240
ISRAEL,
BYRNE,
Fischer rats and propagated in Dulbecco’s modified Eagle’s minimal essential medium containing 10% fetal calf serum. Assays for biological activity of viral DNA. The infectivity of various viral DNA preparations was measured by plaque assay on primary AGMK cells in the presence of 1 mg/ml of diethylaminoethyldextran (DEAE-dextran) (MW 2 x 106, Pharmacia) as previously described (Aaronson and Martin, 1970). Transformation assays were carried out in primary BRK cells as described by Graham and Van der Eb (1973). Approximately 3 weeks folowing infection of the monolayers with 0.2 pg of viral DNA in 6-cm petri dishes, the cells were fixed and stained with Giemsa stain. Transformed foci were scored as dark bluestained areas of densely packed cellular overgrowth. Preparation of SV40 DNA. Unlabeled and 32P-labeled virus were prepared by infection of primary AGMK cells at a multiplicity of 1 to 3 PFU/cell as previously described (Gelb et al., 1971). For the preparation of 32P-labeled virions, infected AGMK cells were maintained in phosphate-free Eagle’s minimal essential medium (MEM) containing carrier-free E3”P]orthophosphate (100 or 10 &i/ml). Unlabeled and 32P-radiolabeled SV40 DNAs were prepared from purified virions by incubation with 1% Sarkosyl for 1 hr at 50” and isopycnic centrifugation in CsCl containing ethidium bromide as previously described (Gelb et al., 1971). Supercoiled forms of oligomeric SV40 DNA were prepared from primary AGMK cells infected at a multiplicity of 100 to 200 PFU/cell as previously described (Martin et .aZ., 1976). Closed circular DNA purified by isopycnic centrifugation in CsCl containing ethidium bromide was sedimented through a 5 to 30% (w/v) neutral sucrose gradient. Peak fractions sedimenting at 21, 27, 32, and 36 S (corresponding to monomeric through tetrameric supercoiled SV40 DNA) (Martin et al., 1976) were pooled and used to infect primary AGMK or BRK cells. In vitro ligation of R.Eco.Rl-digested viral DNA. 32P-SV40 DNA I (sp act., 2: 4 x lo” cpm/pg), 50 pg, was digested with
AND MARTIN
R.Eco.Rl in a l.O-ml reaction mixture containing 0.1 M Tris (pH 7.9), 0.05 M NaCl, 0.1 mM EDTA, 0.012 M MgC12, and 5 U of R.Eco.RI (Miles Laboratories) for 45 min at 37”. The digestion was stopped by incubating the mixture at 68” for 8 min, at which time it was adjusted to 10 n-&f dithiothreitol, 1 r&f EDTA, 0.1 n&f ATP, and 1 pg/ml of bovine serum albumin. T4 ligase (Miles Laboratories), 2.5 U, was then added and the reaction mixture was incubated at 10’ for 1 hr, deproteinized with an equal volume of phenol containing 0.1% 8-OH quinolone, extensively dialyzed against 0.01 M Tris (pH 8.0), and sedimented through a 5 to 30% (w/v) sucrose gradient at 23,000 rpm for 16 hr at 10’ in a Beckman SW 41 rotor. Preparation of transformed cell DNA. Transformed cell lines were derived from foci resulting from the infection of primary BRK cells with supercoiled forms of monomeric, dimeric, trimeric, or tetrameric SV40 DNA. Cellular DNA was prepared from such preparations following 6 to 10 passages in culture as previously described (Gelb et al., 1971) and mechanically fragmented at 50,000 lb/in’ in a Ribi cell fractionator. Agarose gel electrophoresis. Electrophoresis through 0.7% (w/v) agarose slab gels (17 x 12 x 0.3 cm) was carried out at 150 V for 3.5 hr at 10’ as previously described (Hayward and Smith, 1972). Following visualization of ethidium bromide-stained DNA by long-wave uv illumination (Sharp et al., 1973), specific DNA bands were eluted from the agarose by homogenization of gel fractions in a solution containing 0.1x SSC, 0.1% SDS, and 1 mM EDTA (Howley et al., 1975). The eluted DNA was further purified on Sephadex G-160 containing Dowex 5OW-X8 (Trilling and Axelrod, 1970). Autoradiograms were made from dried gels by direct contact with Kodak RP/RB X-ray film. DNA-DNA reassociation. 32P-labeled (sp act., 1.2 x lo6 cpm/pg) fragmented denatured SV40 DNA was allowed to reassociate in the presence of a 1 M excess of unlabeled, fragmented, denatured, transformed cell DNA or an equal amount of salmon sperm DNA (Calbiochem) in 1.0 M
OLIGOMERIC
FORMS
sodium phosphate buffer at 68”. Samples were removed at various times and analyzed for single- and double-stranded DNA by hydroxyapatite chromatography as previously described (Gelb et al., 1971). RESULTS
Biological merit
Activity of Supercoiled OligoSV40 DNA in AGMK Cells
In the first group of experiments to be described, the biological activity of oligomerit forms of supercoiled viral DNA was assessed in primary AGMK cells. Confluent monolayers were infected with several dilutions of purified preparations of monomeric, dimeric, trimeric, or tetrameric forms of supercoiled SV40 DNA and assayed for plaque formation as outlined under Materials and Methods. The results of such an experiment are shown in Table 1. One microgram of dimeric, trimeric, or tetrameric viral DNA produces approximately one half, one third, and one quarter the number of plaques, respectively, compared to 1 pg of inoculum of monomeric DNA. Since such an oligomeric series contains one half, one third, and one quarter the number of molecules present in an equal mass of monomeric DNA, the data indicate that supercoiled oligomeric DNA molecules are equally effective in establishing productive infection. This result is in agreement with those of Cuzin et al. (1970) and Jaenisch and Levine (1972), who found that the infectivity of small oligomeric supercolied forms of either polyoma or SV40 DNAs in permissive cells was directly related to the number of DNA molecules used in the infection. Indeed, this is not an unexpected result if one assumes that the infecting oligomers are not converted to multiple smaller infectious molecules before entering the cell. Transformation of BRK Cells with Supercoiled Oligomers of SV40 DNA
To evaluate the ability of supercoiled oligomers of SV40 DNA to transform animal cells, we exposed BRK cells to purified preparations of viral DNA as described under Materials and Methods. Visible foci of cellular overgrowth were observed 14 to 18 days following DNA infection. Microscopi-
OF
SV40
241
DNA TABLE
INFECTIVITY Oligomeric
OF SV40 form
Monomer Dimer Trimer Tetramer
1 OLIGOMERIC
DNA
Plaque-forming per microgram 3.8 2.0 1.25 0.75
x x x x
unite of DNA lo5 lo5 lo5 lo5
tally, these foci consisted of densely overgrown epithelioid cells which stained deep blue with Giemsa stain. In plates which had been exposed to salmon sperm DNA, areas of overgrowth were not observed. As shown in Table 2, the number of transformed colonies per microgram of DNA is very similar for each of the DNA species examined. The variation in the number of foci for a given inoculum between the two experiments may be related to the condition and degree of confluency of the cells at the time of infection. These results strongly suggest that the ability of this SV40 oligomeric DNA to effect transformation is related to the number of molar equivalents of viral genome rather than to the actual number of SV40 DNA molecules in the inoculum. This is in contrast to the results obtained following the infection of permissive cells with such DNA preparations (i.e., that biological activity correlates with the number of DNA molecules rather than the amount of DNA in the inoculum). Transformation merit DNA Vitro
of BRK Cells with OligoProduced by Ligation in
The finding that tetrameric supercoiled viral DNA was four times more efficient in producing transformed foci than monomeric SV40 DNA suggested that large viral DNA molecules may transform BRK cells more efficiently than smaller DNA molecules. On the other hand, since the monomeric components present in these oligomerit viral DNA molecules are known to be arranged in a head-to-tail fashion (Martin et al., 1976), our results could be interpreted as suggesting that the arrangement of SV40 monomers as a repeating unit in a circular molecule endowed the multimeric supercoiled DNA with enhanced transforming activity. It is known that full-
242
ISRAEL, TABLE
form
88 106 120 94
1
Expt
pool AE pool BE
-
25s
4 I-B---rkA+l
i$
21s
*
400
2
197 149 250 149
33s
4T
of
II
350 :
II
6
Ii
II-PI
300 r
In Vitro Oligomer Oligomer
MARTIN
OF SV40
Foci per r$cqram Expt
In Vivo Monomer Dimer Trimer Tetramer
AND
2
TRANSFORMATION EFFICIENCY OLIGOMERIC DNA Oligomeric
BYRNE,
H
116 179
length linear forms of SV40 DNA transform cells with the same efficiency as either the supercoiled or relaxed circular form (Abrahams et al, 1975). We therefore decided to evaluate whether the arrangement of the monomeric constituents within oligomeric viral DNA plays a role in the enhanced transforming activity observed. Accordingly, we examined the transformation efficiency of linear and circular oligomeric forms of SV40 DNA prepared in vitro by the ligation of restriction enzyme-cleaved SV40 monomeric DNA (DeVries et al., 1976; Dugaiczyk et al, 1975). The monomeric constituents of such oligomeric DNA molecules would be arranged in both headto-tail and head-to-head orientations (Ferguson and Davis, 1975). SV40 DNA was cleaved by R.Eco.Rl and ligated with T4 ligase, described under Materials and Methods. This DNA preparation consisted of oligomeric linear and circular molecules. The ligated DNA was sedimented through a 5 to 30% (w/v) sucrose gradient and pooled into six fractions (I-VI) as indicated in Fig. 1. Aliquots of these fractions were analyzed in a 0.7% agarose slab gel, the autoradiogram of which is shown in Fig. 2. Lane A in this autoradiogram contains the reaction products of R.Eco.Rl digestion of SV40 DNA I. The prominent band is fulllength linear SV40 DNA and the fainter faster moving band is residual uncleaved supercoiled SV40 DNA. Lane B contains an aliquot of the reaction products generated by ligation of the R.Eco.Rl-digested SV40 DNA prior to centrifugation. Lanes
rat.
‘k
I OOW
50 FRACTION
FIG. 1. Sedimentation in neutral sucrose of oligomerit forms of SV40 DNA prepared by ligation in vitro. R.Eco.Rl-cleaved SV40 DNA was ligated with T4 ligase, phenol extracted, extensively dialyzed, and sedimented through a 5 to 3W (w/v) neutral sucrose gradient as described under Materials and Methods. Fractions were collected from the bottom of the tube and radioactivity was determined by Cerenkoff counting of the individual fractions. The position of closed circular SV40 DNA is indicated by the arrow labeled 21 s.
C, D, E, F, G, and I-I correspond to aliquots of the pooled fractions I-VI from the sucrose gradient shown in Fig. 1. We have characterized these fractions by alkaline sucrose density gradient centrifugation and electron microscopy and found each of them to contain linear and circular oligomers of progressively increasing size up to at least 10 times the size of monomeric SV40 DNA (data not shown). For instance, such an analysis of the DNA bands depicted in Lane D indicates that the most rapidly moving band is supercoiled SV40 DNA I which had not been cleaved by
OLIGOMERIC
FORMS
OF
SV40
DNA
243
ABCDEFGH
FIG. 2. Electrophoresis of oligomeric forms of SV40 DNA SV40 DNA I (lane A) was ligated with T4 Iigase. An aliquot of extracted, extensively dialyzed against 0.01 M Tris (pH 8.0), fractions (I-VI) of the sucrose gradient depicted in Fig. 1 were The electrophoresis was in 0.7% agarose for 3.5 hr at 150 V migration is from top to bottom; the autoradiogram was made X-ray film.
R.Eco.Rl. The slowest moving band corresponds to closed circular dimers of viral DNA. The series of faint bands moving ahead of this slowest moving band represent dimeric forms of closed circular DNA of increasing superhelicity. The darker
prepared by ligation in u&o. R.Eco.Rl-digested the reaction products of this ligation were phenol and analyzed in lane B. Aliquots of the pooled dialyzed and analyzed in lanes C-H, respectively. as described under Materials and Methods. The from the dried gel by direct contact with Kodak
band of intermediate mobility running in the midst of the supercoiled dimeric circular DNA corresponds to trimeric linear viral DNA. This agrees closely with the findings of DeVries et al. (1976), who have published a similar analysis of the products generated
244
ISRAEL,
BYRNE,
by this reaction. Following this analysis, fractions from the sucrose gradient were divided into two pools: Pool A contained all DNA species sedimenting from 20 to 24 S; pool B contained all DNA species sedimenting between 25 and 38 S (Fig. 1). Each of these pools was then separately electrophoresed in a 0.7% agarose slab gel. Bands corresponding to linear and circular dimeric and linear trimeric viral DNA were eluted from pool A and designated pool AE. Bands corresponding to linear molecules 4 to 10 times the size of monomers and circular molecules at least tetrameric in size were identified following the preparative electrophoresis of pool B, eluted, and designated pool BE. The transforming efficiencies of these two pools of SV40 DNA oligomers LEGEND
2.8 r 2.6
2.4
AND
MARTIN
prepared by ligation in vitro are shown in Table 2. One microgram of DNA from either pool Az (small oligomers) or pool BE (large oligomers) yields a number of transformed foci similar to that observed with 1 pg of a purified preparation of supercoiled oligomeric viral DNA prepared from productively infected monkey cells. This enhanced transforming efficiency of oligomerit viral DNA cannot be related to the sequential arrangement of the SV40 monomeric components since linear or circular oligomers of SV40 DNA generated in vitro contain relatively few molecules whose monomeric constituents are oriented solely head-to-tail. Quantitation of Viral DNA Sequences in Cells Transformed by Oligomeric Viral DNAs Our finding that the transformation effrciency of oligomeric SV40 DNA molecules varied directly with the size of the molecules throughout the range examined suggested that there might be a difference in the number of viral DNA equivalents present in the cell lines resulting from these transformation experiments. To examine this possibility, we prepared DNA from BRK cells transformed by preparations of supercoiled oligomers of viral DNA purified from lytically infected AGMK cells. Figure 3 shows that the DNA from cells transformed by each of the oligomeric DNA forms examined accelerated the reannealing of fragmented 32P-labeled SV40 DNA. With one exception, the preparations of transformed cellular DNA examined conTABLE
0
.2
I .4
I .6
I .8
I 1.0
I 1.2
%2
FIG. 3. Kinetics of reassociation of 32P-labeled SV40 DNA in the presence of unlabeled DNAs from salmon sperm (0) and BRK cells transformed by SV40 monomeric (O), dimeric (A), trimeric (X), and tetramerit (A) supercoiled DNA. “P-labeled fragmented denatured SV40 DNA (sp act., 1.2 X 10” cpm/ag) was allowed to reassociate in the presence of a 1.0 M excess of fragmented denatured unlabeled salmon sperm or transformed cell DNA as described under Materials and Methods. The fraction of radiolabeled DNA remaining single stranded (fss) was assayed by hydroxyapatite column chromatography (Gelb et aZ., 1971).
3
QUANTITATION OF SV40 DNA SEQUENCES IN OLICOMERIC DNA-TRANSFORMED BABY RAT KIDNEY LINES Cellular DNA AccelerEquivat1/2 ation lents of (l-4 factor viral DNA per diploid genome Monomer transformed Dimer transformed Trimer transformed Tetramer transformed Salmon sperm
5.5 4.4 5.5 5.7 16.2
2.0 2.7 2.0 1.9 -
2.0 2.7 2.0 1.9 -
OLIGOMERIC
FORMS
tamed 2.0 viral DNA equivalents per diploid genome (Table 3). DNA from cells transformed by dimers contained 2.7 viral equivalents per diploid genome. We do not consider this variation to be significant and conclude that the amount of viral DNA present in cells transformed by the various oligomeric SV40 DNA species is similar and unrelated to the particular oligomeric species used for transformation. DISCUSSION
While the biologic activity of SV40 oligomeric DNAs in permissive cells has been previously described (Jaenisch and Levine, 1971), the capacity of this DNA to transform animal cells in culture has not been studied. Jaenisch and Levine (1971) found that SV40 dimeric molecules were about as infectious as monomeric DNA molecules. That is, a given mass of dimers yields one half the number of plaques produced by the same mass of monomers. Using highly purified preparations of SV40 monomeric, dimerit, trimeric, and tetrameric DNA molecules, we confirmed this result, Crmly establishing that infection of monkey cells with oligomeric SV40 DNA molecules is a function of the number of molecules in the inoculum rather than the amount of inoculated DNA. The pattern of interaction between SV40 oligomeric DNA molecules and nonpermissive cells was quite different from the one observed with permissive cells. We found the ability of SV40 oligomeric DNA to transform BRK cells to be a function of the amount of DNA rather than the number of DNA molecules in the inoculum. That is, the number of tetramers necessary to achieve a specific frequency of transformation is one quarter the number of monomers required to produce a similar effect. Our results shed no light on the mechanism by which oligomers larger than monomeric size effect enhanced transformation efficiency. However, a number of possibilities can be entertained. Monomerization of the trimer or tetramer forms of viral DNA would yield three or four times the number of transforming units per infecting molecule. Our finding similar amounts of SV40 DNA in cells
OF
SV40
DNA
245
transformed by either monomeric, dimeric, trimeric, or tetrameric DNA molecules further suggests the possibility of a “common pathway” by which transformation could be effected by each of these molecular forms. However, since the increased transformation efficiency we observed is essentially linear over the range examined, monomerization of larger viral DNA molecules, if it occurred, would have to involve a precise and symmetrical cleavage yielding monomeric units of equal transforming efficiency, an efficiency identical to that of prototype SV40 monomeric DNA. Such a monomerization could be envisioned as occurring by efficient intramolecular recombination, although such a process has yet to be demonstrated in animal cells. It seems unlikely to us that rat cells would possess other mechanisms for such specific cleavage. The possibilities remain that there is some unique advantage conferred on the oligomeric forms of viral DNA by virtue of either its size or the physical orientation of the monomer subunits in a circular molecule. Our finding that the multimeric products of the in vitro ligation of linear SV40 DNA also had increased transforming capacity would seem to rule out the possibility that the head-to-tail arrangement of SV40 genomes in the oligomers isolated from lytically infected AGMK cells conferred any advantage on these DNA forms. On the other hand, the increased size per se of oligomeric viral DNA could lead to an enhanced transformation efficiency either by providing the cell with molecules containing multiple copies of specific viral DNA sequences or simply by providing a larger molecule to participate in the transforming event. In the former case, additional copies of certain SV40 DNA sequences could provide higher concentrations of specific sites involved during integration into the host cell chromosome or perhaps higher concentrations of viral gene products necessary for transformation. This is an interesting possibility in that it suggests that a threshold for viral sequences or virally coded products is required for transformation. Such a concept would be compatible with the finding by Graessman et al. (1976) that the inten-
246
ISRAEL,
BYRNE,
sity of T-antigen fluorescence following injection of SV40 DNA into 3T3 cells was dose dependent. The increased size of the oligomeric viral DNA molecule may itself be responsible for the increased efficiency of transformation we observed. This could also explain the findings of Mulder and Vogt (1973) who noted that while the infectivity of a defective polyoma genome about 1.6~ the size of a monomer was only one tenth that of monomers, the efficiency in transforming hamster cells was about twice that of monomers. Such a “size effect” would also be compatible with our findings that the organization of the genomic units within the oligomer is unrelated to this enhanced effect and that the amount of viral DNA present in transformed cells was independent of the particular oligomeric species used to transform those cells. So little is known of the molecular events involved in transformation that to speculate whether such an advantage is due to increased configurational flexibility, enhanced availability of sites critical for integration into cellular DNA, or any other property dictated by molecular size would be pure conjecture. Experiments currently in progress will hopefully determine the molecular basis of this enhancement and will also provide insight concerning the process of viral-induced transformation. REFERENCES AARONSON, S. A., and MARTIN, M. A. (1970). Transformation of human cells with different forms of SV40 DNA. Virology 42,848-854. ABRAHAMS, P. J., MULDER, C., VAN DE VOODE, A., WARNAAR, S. O., and VAN DER EB, A. J. (1975). Transformation of primary rat kidney cells by fragments of simian virus 40 DNA. J. Viral. 16,818823. CUZIN, F., VOGT, M., DIECKMANN, M., and BERG, P. (1970). Induction of virus multiplication in 3T3 cells transformed by a thermosensitive mutant of polyoma virus. J. Mol. Biol. 47,317-333. DEVRIES, F. A. J., COLLINS, C. J., and JACKSON, D.A. (1976). Joining of simian virus 40 DNA molecules at endonuclease RI sites by polynucleotide ligase and analysis of the products by agarose gel electrophoresis. Biochim. Biophys. Acta 435.213-227. DUGAICZYK, A., BOYER, H. W., and GOODMAN, H. M. (1975). Ligation of Eco.Rl endonuclease-generated DNA fragments into linear and circular structures. J. Mol. Biol. 96, 171-184.
AND
MARTIN
FERGUSON, J., and DAVIS, R. W. (1975). An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs. J. Mol. Biol. 94, 135-149. G~LB, L. D., KOHNE, D. E., and MARTIN, M. A. (1971). Quantitation of simian virus 40 sequences in African green monkey, mouse and virus-transformed cell genomes. J. Mol. Biol. 57,129-145. GRA~SSMAN, A., GRAESSMAN, M., and MUEI,LER, C. (1976). Regulatory mechanism of simian virus 40 gene expression in permissive and in nonpermissive cells. J. Viral. 17.854-858. GRAHAM, F. L., and VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52.456-467. HAYWARD, G. S., and SMITH, M. G. (1972). The chromosome of bacteriophage T5: I, Analysis of single stranded DNA fragments by agarose gel electrophoresis. J. Mol. Biol. 63, 383-395. HOWLEY, P. M., MULI~ARKEY, M. F., TAKEMOTO, K. K., and MARTIN, M. A. (1975). Characterization of human papovavirus BK DNA. J. Viral. 15,173-181. JAENISCH, R., and LEVINE, A. J. (1971). DNA replication in SV40-infected cells: V, Circular and catenated oligomers of SV40 DNA. Virology 44, 480-493. JAENISCH, R., and LEVINE, A. J. (1972). DNA replication in SV40 infected cells: VI, The effect of cyclohexamide on the formation of SV40 oligomeric DNA. Virology 48,373-379. MARTIN, M. A., HOWLEY, P. M., BYRNE, J. C., and GARON, C. F. (1976). Characterization of supercoiled oligomeric SV40 DNA molecules in productively infected cells. Virology 71, 28-40. MULDER, C., and VOGT, M. (1973). Production of nondefective and defective oligomers of viral DNA in mouse 3T3 cells transformed by a thermosensitive mutant of polyoma virus. J. Mol. Biol. 75,601~608. RUSH, M., EASON, R., and VINOGRAD, J. (1971). Identification and properties of complex forms of SV40 DNA isolated from SV40 infected cells. Biochim. Biophys. Acta 228,585-594. SHANI, M., SEIDMAN, M., and SALZMAN, N. P. (1977). In uiuo transcription of oligomeric simian virus 40 (SV40) DNA. Virology 83, 110-119. SHARP, P. A., SUGDEN, B., and SAMBROOK, J. (1973). Detection of two restriction endonucleases in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12,3055-3063. TRILLING, D. M., and AXELROD, D. (1970). Encapsidation of free host DNA by simian virus 40: A simian virus 40 pseudovirus. Science 168,268-271. VOGT, M., BACHELER, L., and BOICE, L. (1976). Proposed structure of two defective viral DNA oligomers produced in 3T3 cells by the m-2 mutant of polyoma virus. J. Viral. 17, 1009-1026.