Vol. 17, No. :3 Printed in U.S.A.

JOURNAL OF VIROLOGY. Mar. 1976. p. 1009-1026 Copyright © 1976 American Societ% for Microbiology

Proposed Structure of Two Defective Viral DNA Oligomers Produced in 3T3 Cells Transformed by the ts-a Mutant of Polyoma Virus MARGUERITE VOGT,* LEE T. BACHELER, AND LuBELLE BOICE The Salk Institute, San Diego, California 92112 Received for publication 2 September 1975

The various oligomeric viral DNA species produced at 32 C by two related sublines of ts-a-transformed mouse 3T3 cells were characterized. Results from the analysis of the cleavage products observed after digestion with restriction endonucleases from Haemophilus parainfluenzae, Escherichia coli RI, and Haemophilus suis are consistent with the assumption that in both sublines, the major oligomeric component is a dimer from which a segment of different length is deleted. The major oligomeric (27S) component in subline 1 was estimated to be 1.77 times the size of the viral monomer, and the major (25.5S) component in subline 15 was estimated to be 1.54 times the size of the viral monomer. These size estimates were confirmed by electron micrograph measurements. The larger oligomers produced by both sublines were found to be multiples of the major oligomeric component of each subline. Mouse 3T3 cells transformed by the thermosensitive mutant ts-a of polyoma (Py) virus (5, 6) only rarely synthesize viral DNA or release ts-a virus at 39 C. Upon shift to 31 C, the permissive temperature for the growth of the ts-a mutant, a large proportion of the ts-a 3T3 cells is induced to liberate virus. The intracellular viral DNA pools of such induced ts-a 3T3 cells are unusual in that they contain a significant amount of viral DNA larger than the size of a Py DNA monomer (2). In two clonal sublines, 1 and 15, originally derived from the same ts-a 3T3 transformant, TRF2, the oligomeric viral DNA forms the major portion of the intracellular viral DNA pool (17). An analysis of the structure of the oligomeric DNA in these two sublines is the subject of this study. Thi' work forms part of a broader study aimed at comparing the oligomeric DNA produced by different ts-a 3T3 cells with the organization of the viral sequences integrated in the corresponding host DNAs.

mately 12 h after seeding. The cultures were grown at 32 C for 48 h and labeled for 24 h before harvesting with [3H]thymidine (6.7 Ci/mmol) at a final concentration of 10 uCi/ml. (Generally only 100 out of 400 plates were labeled.) Viral DNA. Viral DNA from ts-a 3T3 cells was extracted selectively by the method of Hirt (10) from total cells or from nuclei obtained after lysis of the cells with 0.5%o Nonidet P-40-0.01 M Tris-hydrochloride (pH 7.4)-0.00,3 M MgCl2. The Hirt extracts were phenol extracted, ethanol precipitated, and purified by two successive equilibrium centrifugations in CsCl gradients containing ethidium bromide. Fractions containing the heavier band of DNA were monitored by 3H counts, pooled, extracted four times with isopropanol to remove the ethidium bromide dye, dialyzed extensively against 0.01 M Tris-hydrochloride (pH 8.0)-0.001 M EDTA at 4 C, and stored over chloroform at 4 C or as ethanol precipitates at - 20 C. 32P-labeled Py DNA was prepared by infecting primary baby mouse kidney cell cultures with plaquepurified wild-type stock in low-phosphate medium in the presence of 32P; (10 to 100 ,Ci/ml). The cultures were extracted 40 to 60 h after infection, and the DNA was isolated as described above and further purified on neutral sucrose gradients. The specific activity of the 3H-labeled viral DNA varied from 105 to 1.2 x 105 counts/min per gg, and that of the 32P-labeled viral DNA ranged from 1.2 x 105 to 1.8 x 106 counts/min per gg. Unlabeled viral DNA was isolated from Py-infected 3T6 cells and purified as described for 32P-labeled polyoma DNA. Mitochondrial DNA. 32P-labeled mitochondrial DNA was isolated from a non-virus-producing deriva-

MATERIALS AND METHODS Cells. The derivation of sublines 1 and 15 from the ts-a-transformed 3T3 clone TRF2 was described previously ( 17). Cells of' sublines 1 and 15 were grown at 38.5 C in reinforced Eagle medium (22) plus 10% calf serum and receptor-destroying enzyme (Microbiological Associates, Inc.) at a concentration of 1/200 (vol/vol). For the isolation of viral DNA, cultures showing 50% confluency were shifted to 32 C approxi1009

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VOGT, BACHELER, AND BOICE

tive of subline 15. Cultures showing 50% confluency were shifted to 32 C. The cultures were grown for 48 h in reinforced Eagle medium containing 2 x 10-5 M phosphate, 10% dialyzed calf serum, and 8 uCi of H 32PO4 per ml. Mitochondrial DNA was extracted selectively by the method of Hirt (10) and purified by the procedure described for viral DNA. The mitochondrial peak sedimented in a neutral sucrose gradient at 35S. The specific activity was 3.2 x 10' counts/min per Ag. Cell DNA. Mouse DNA was purified from the livers, kidneys, and spleens of Swiss mice by the method of Schmeckpeper and Smith (19). Calf thymus DNA (Sigma) was further purified by phenol and chloroform extraction and ethanol precipitations. After sonic treatment, the cell DNAs were alkaline digested by boiling in 0.3 N NaOH for 10 min, neutralized and ethanol precipitated, and exhaustively dialyzed against 0.01 M Tris-hydrochloride (pH 8.0)-0.001 M EDTA. DNA hybridizations. DNAs for hybridization were dissolved in 0.01 M Tris-hydrochloride (pH 8.0)-0.001 M EDTA and reduced to a uniform piece size by sonic treatment for 2 min in 30-s intervals in a Branson sonifier (model S 125) equipped with a microtip at a power setting of 5. All hybridizations were carried out in a buffer containing 0.5 M NaCl, 0.01 M TES buffer (pH 7.0), 0.001 M EDTA, and 0.1% sodium dodecyl sulfate (SDS). Samples containing only viral DNAs contained in addition 100 Atg of yeast tRNA per ml. DNAs were denatured by boiling and incubated at 68 C. At various times, aliquots were withdrawn and diluted into a buffer the final composition of which was 0.05 M sodium acetate, pH 4.5, 0.005 M ZnSO4, 0.25 M NaCl, 25 Mg of denatured calf thymus DNA per ml, and 2.5 Mg of native calf thymus DNA per ml. Samples were digested with the single-strand-specific nuclease S1 (21) for 1 to 3 h at 45 C, precipitated by cold 5% trichloroacetic acid in the presence of 40Mg of tRNA carrier per ml, collected on membrane filters (Millipore Corp., HAWP), and counted in Liquifluor scintillation fluid (New England Nuclear). Enzyme and enzyme reactions. Haemophilus parainfluenzae II (Hpa II) restriction endonuclease was kindly provided by John Morrow and Joe Sambrook. The reactions were performed in 0.01 M Trishydrochloride (pH 7.4)-0.01 M MgCl2-0.5 mM dithiothreitol-100 Mg of gelatin (Sigma) per ml. Aliquots of enzyme stocks (in 50% glycerol [vol/vol]-0.005 M KPO4 [pH 7.41-0.125 M KCl-0.5 mM dithiothreitol) were usually added to 10 to 20 volumes of DNA in reaction buffer. A three- to six-fold excess of enzyme over the amount required for complete digestion was used in the reactions. After incubation for 4 h at 37 C, the reactions were terminated by the addition of EDTA (to 0.04 M) and SDS (to 0.1%), and the samples were subsequently phenol extracted and ethanol precipitated. Small reactions were terminated by adding one-half volume of a mixture containing 0.12 M EDTA, 45% glycerol, and 0.03% bromophenol blue and were layered immediately onto the gels. E. coli RI (Eco RI) restriction endonuclease was a gift from R. Helling. The enzyme stock (in 0.01 M KPO4 [pH 7.01-0.2 M NaCl-0.001 M EDTA-0.007 M

J. VIROL.

2-mercaptoethanol-0.2%c Nonidet P-40 was added to 30 volumes of DNA in enzyme buffer (0.01 M Trishydrochloride-0.01 M MgCl2), and the reaction mixture was incubated for 1 h at 37 C. The reaction was terminated by the addition of EDTA (to 0.04 M). H. suis I (Hsu I) restriction endonuclease was a gift of Carel Mulder. The same reaction buffer used for Hpa II enzyme was used for Hsu I enzyme. The reaction mixtures were incubated for 6 h at 37 C. The reactions were terminated by adding SDS (to 1%) and the EDTA-glycerol-bromophenol blue mixture as described above. Gel electrophoresis. Electrophoresis of the viral DNA digests was carried out through 3.5 and 5% polyacrylamide gels or through composite 2.2% polyacrylamide-0.7% agarose gels (18). Stock solutions and gels were prepared as described by Edgell et al. (4). Buffer E (0.04 M Tris-0.02 M sodium acetate-0.001 M EDTA, adjusted to pH 7.2) (1) was used both in the gels and as electrode buffer. The DNA digests were applied to tube gels (29.5 by 0.6 cm) and electrophoresed at constant voltage (100 to 125 V) for 14 to 18 h. The gels were subsequently fractionated in water with an Autogeldivider (Savant) by the method of Maizel (16). The fractions were dried in an oven, and the radioactivity was counted in a scintillation mixture of NCS Solubilizer (Amersham/Searle), NH4OH, Liquifluor, and toluene. Recovery of DNA fragments for hybridization. Viral DNA fragments were isolated by the continuous electroelution method as described by Lee and Sinsheimer (15). Tube gels (13 by 1 cm) were prepared with wells (0.5 cm deep) in the upper end to minimize streaking. To optimize the recovery of the fragments, SDS (to 0.2%) was added to the electrophoresis buffer. To shorten the overall time of the electroelution, a higher voltage was applied in the first hours of electrophoresis (migration rate of bromophenol blue, 2 to 3 cm/h); subsequently the voltage was reduced to allow a better separation of the fragments. The extent of the reduction in the migration rate was measured by applying a second (DNA free) bromophenol blue sample to the gel and following its migration. The pumping speed used for the elution varied from 5.0 to 5.5 ml/h. Lower speeds led to appreciable losses into the anode buffer chamber. The fractions containing the fragments were extracted once with phenol, once with chloroform-isoamylalcohol (24/1), and (after the addition of tRNA [to 40 Mg/ml ] and potassium acetate [to 0.2 M]) precipitated with 2 volumes of ethanol. Neutral sucrose gradients. Samples (0.2 ml) of the purified DNA solution were layered on top of a 5 to 20% sucrose gradient in 1 M NaCl-0.01 M Tris-hydrochloride (pH 8.0)-0.005 M EDTA (total volume 10.2 ml). The gradients were centrifuged in an SW41 rotor of a Beckman L2-65B ultracentrifuge at 20 C for 260 min at 36,000 rpm. Ten-drop fractions were collected from the bottom of the tubes. Alkaline sucrose gradients. Alkaline 5 to 20% sucrose gradients were formed from a 5% sucrose solution containing 0.2 N NaOH and a 20% sucrose solution containing 0.3 N NaOH in 0.9 M NaCl-0.01 M Tris-hydrochloride (pH 8.0)-0.005 M EDTA. Samples (0.1 ml) were denatured with 0.2 N NaOH at

DEFECTIVE OLIGOMERS

VOL. 17, 1976 room temperature and layered on top of 3.4-ml gradients. The tubes were centrifuged in an SW56 rotor at 20 C for 100 or 180 min at 50,000 rpm. Fractions of 6 drops were collected and neutralized, and the radioactivity was counted in scintillation fluid containing Triton X-100, 2,5-diphenyloxazole, and toluene. Electron microscopy of viral DNA. Open circular oligomeric and monomeric viral DNA obtained by limited DNase digestion was mounted for electron microscopy by the formamide method of Davis et al. (3). Micrographs were taken with a Hitachi HU-11B electron microscope. Contour length measurements were made using a Hewlett-Packard 9864A digitizer and 9820A calculator with a calculation program which gave an accuracy of +0.5%. The use of this equipment was kindly provided by N. Davidson.

RESULTS Sedimentation pattern of superhelical DNA in sublines 1 and 15. The intracellular pools of superhelical closed-circular DNA produced by cells from sublines 1 and 15 grown at 32 C for 48 h were fractionated by sedimentation in a neutral sucrose gradient (Fig. 1). As will be shown below, these DNA pools are mainly viral in nature and only slightly contaminated with mitochondrial DNA. Both DNA pools contain oligomeric DNA in addition to monomeric (21S) viral DNA (Fig. 1). The major

1011

oligomeric component in subline 1 sediments at 27S, whereas the major component of subline 15 is slightly smaller, sedimenting at 25.5S. Both lines also produce smaller amounts of larger oligomers. To analyze in more detail the structure of the various oligomeric species in both DNA pools, fractions corresponding to regions I to IV (Fig. 1) were separately pooled, purified by a second sedimentation in a neutral sucrose gradient, and subsequently digested with H. parainfluenzae II (Hpa II) restriction endonuclease. Hpa II cleavage products of the 27S and 25.5S oligomer. Hpa II restriction endonuclease has been shown to cleave Py DNA into eight fragments (9). The cleavage products obtained after digestion with Hpa II enzyme of the two major DNA components with sedimentation values of 27S and 25.5S, respectively (Fig. 1, pools II), were analyzed by electrophoresis in polyacrylamide gels. To all reaction mixtures, wild-type monomeric (32P-labeled) Py DNA was added as a reference marker. The Hpa II digest of both the 27S and 25.5S oligomer forms nine major bands (Fig. 2). The slow-migrating minor components present in less than molar amounts near the top of the gels are assumed to be of mitochondrial origin (see below). Of the nine

0

I

FIG. 1. Fractionation of superhelical closed circular DNA of subline 1 (A) and subline 15 (B) in neutral sucrose gradients. The superhelical DNA was labeled in vivo with ['H]thymidine and extracted as described. A total of 40 sg of subline 1 DNA or of 10 jyg of subline 15 DNA was sedimented through a 5 to 20% neutral sucrose gradient in an SW41 Beckman rotor for 260 min at 36,000 rpm. Fractions of 10 drops (200 gIl) were collected, and 1-jsl aliquots of each fraction were counted on glass-fiber filters in a liquid scintillation counter. Fractions corresponding to pools I to IV were collected as indicated. The pools were sedimented a second time through a neutral sucrose gradient in an SW56 rotor for 100 to 120 min at 50,000 rpm to achieve a better separation of the different size classes (not shown).

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FIG. 2. Gel electrophoresis pattern of the 27S (A) and 25.5S (B) oligomer after cleavage with Hpa HI restriction endonuclease. A mixture of sH-labeled (0) 27S or 25.5S DNA and 32P-labeled ('0) Py DNA was digested with Hpa II enzyme and electrophoresed for 13.75 h (A) or 14.9 h (B) in 3.5% polyacrylamide gels at 125 V. Each gel was fractionated with a Maizel Autogeldivider into 123 fractions, and the radioactivity in each fraction was counted as described. The numbers above the peaks indicate the Hpa II fragments 1 to 8 described by Griffin et al. (9). 1012

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bands, eight correspond in their migration to the eight fragments of Py DNA. (Slight differences in the migration of bands 3 and 5 as compared to bands 3' and 5' of the marker DNA are also found in certain wild-type plaque stocks and are therefore not specific to ts-a DNA [M. Fried, personal communication; our own unpublished data].) The 9th band, band 0 in the 27S digest from subline 1, migrates slower than band 1, and band A in the 25.5S digest from subline 15 migrates slower than band 5. Using the reported relative sizes for the various Hpa II fragments of Py DNA (9) as references, the average size estimates for fragments 0 and A equal 0.32 and 0.09 genome length, respectively. A comparison of the height of the various peaks in Fig. 2 with those of the marker DNA suggests that fragments 3, 4, 5, 7, and 8 are represented twice in the oligomers. Graphs from various gels in which the counts (relative masses) in each band were plotted semilogarithmically against fragment mobility confirmed this conclusion. Figure 3 shows an example for a composite acrylamide agarose gel which allows a better separation of the larger fragments. Under these conditions, fragment 8 runs out of the gel and is therefore not included in the graphs. Using the line (n = 1) through the points for bands 1, 2, and 6 as reference, it can IC

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FIG. 3. Curves relating fragment mobility to mass (measured by integrated radioactivity counts in each band). (A) Data for the Hpa II fragments of a 27S digest. The fragments were eluted from the gel by a continuous electroelution device (see Materials and Methods). (B) Data for the Hpa II fragments of a 25.5S digest (0); data from an Hpa II digest ofpool IV of larger oligomers shown in Fig. 13D (x). The lines 1 are drawn through bands containing one fragn ment per band. The lines n 2 are the mass-versusmobility lines calculated for bands containing two fragments. =

=

1013

be seen that the values for bands 3, 4, 5, and 7 fall close to the parallel line (n = 2) predicted for bands containing two fragments. Similar plots (not shown) for 3.5% acrylamide gels indicate that fragment 8 also is present in two doses in both oligomers. In contrast, band 0 and band A fall on the line (n = 1) and are therefore only represented once in each oligomer. Size of the 27S and 25.5S oligomers. Knowing the number and size of each fragment in the Hpa II digests of both oligomers, it is possible to obtain an estimate of the size of these oligomers. Adding to one genome the length estimate of 0.45 genome, which represents the combined length of the duplicated fragments 3, 4, 5, 7, and 8, and the length estimate of 0.32 genome for fragment 0 or 0.09 genome for fragment A, we obtained a total length of 1.77 times the viral genome for the 27S oligomer and 1.54 for the 25.5S oligomer, respectively. These values agree well with the relative molecular weights 1.77 and 1.55 calculated by Hobom and Hogness (11) from the sedimentation values obtained in neutral sucrose gradients using a value of 21S for monomeric DNA. The values are smaller than the values 2.0 and 1.6 calculated previously (17) from the sedimentation rates in alkaline CsCl gradients by applying the formula of Studier (20) for linear DNA to cyclic coils. The correctness of the newer estimates was confirmed by electron micrograph measurements: the average contour length of 54 27S molecules equaled 1.77 i 0.01 genome lengths and that of 58 25.5S molecules equaled 1.54 ± 0.01 genome lengths. Cleavage of the 27S and 25.5S oligomers by Eco RI endonuclease. The 27S and 25.5S oligomers each contain two copies. of Hpa II fragments 3, 4, 5, 7, and 8 (Fig. 2 and 3). The duplicated fragments form a contiguous sequence on the physical map of Py DNA (Fig. 4). Therefore, it appears likely that the two oligomers can be considered as dimers from which a segment of different length has been deleted in the region of Hpa II fragments 1, 6, and 2 (Fig. 5). Under this assumption, the deleted fragments are replaced by the new Hpa II fragments 0 and A, which consist of parts of Hpa II fragments 1 and 2 fused on each side of the deletion. To test this assumption, the facts that Hpa II fragment 2 carries the Eco RI cleavage site and that Hpa II fragments 1 and 2 carry both one Hsu I cleavage site (9) were utilized. Both oligomers were digested with Eco RI or Hsu I endonuclease, and the cleavage products were analyzed. Figure 6 shows the cleavage products of the

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VOGT, BACHELER, AND BOICE

FIG. 4. Map of the polyoma genome with cleavage sites of the restriction endonucleases Hpa II (solid lines), Eco RI (arrow) and Hsu I (broken lines) from Griffin et al. (9). Also shown are the sites of initiation (OR) and termination (T) of replication. The dark areas indicate the fragments represented twice in the 27S and 25.5S oligomers. The lightly-shaded areas are regions of fragments 1 and 2 in fragment 0, as deduced from cleavage with Hsu I enzyme.

27S and 25.5S oligomer after Eco RI digestion and sedimentation in alkaline sucrose gradients. It can be seen that both the 27S and the 25.5S oligomer are cleaved into a larger 16S component and a smaller component with a sedimentation value of 14.5S and 12.5S, respectively. Using the formula of Studier (20) for alkali-denatured DNA, the relative sizes of the smaller components calculated from their sedimentation values agree closely with the values of 0.77 and 0.54 viral lengths expected after excision of a complete viral genome from the oligomers. The generation by RI cleavage of two molecular species, one of which is of unit viral length, indicates a tandem head-to-tail orientation of the duplicated region in each oligomer (Fig. 5). The formation of two RI cleavage products requires the presence of two RI sites in both oligomers. To determine the location of the two RI sites, oligomers cleaved by RI enzyme were further digested with Hpa II enzyme, and the resulting fragments were analyzed by gel electrophoresis. The Hpa II fragments (2, 0, and A) migrate faster than their counterparts in digests not previously cleaved by RI enzyme (Fig. 7), indicating the presence of an RI site in each of these fragments. From the difference in migra-

J. VIROL.

tion, it can be estimated that all three fragments, (2, 0, and A) are shorter by the same amount (about 0.02 genome length). This places the RI site at 0.02 genome length from one end of each fragment, a position which agrees well with the published value of 0.015 for the RI site in fragment 2 (9). We can therefore conclude that both fragments 0 and A contain at one of their ends the section of fragment 2 which carries the RI site and is adjacent to the Hpa II-2.7 junction (Fig. 5). It should be mentioned that Fig. 7C shows in addition a new band in front of fragment 8. It contains the expected cleavage products of 0.015 genome size generated by RI from fragments 2 and A. (The corresponding band migrated off the gel in Fig. 7A but was present in gels of 27S digests run for shorter times.) Cleavage of the 27S and 25.5S oligomers by Hsu I endonuclease. Endonuclease R Hsu I, an isoschizomer of endonuclease R Hind III (8), cleaves Py DNA into two fragments of about 0.44 and 0.56 genome length (9). One cleavage site is located in Hpa II fragment 1 at about 0.09 genome length from the Hpa II-1.3 junction; the other cleavage site is located in Hpa I fragment 2 at about 0.03 genome length from the Hpa II-2.7 junction (9). The cleavage products obtained after Hsu I digestion of the 27S and 25.5S oligomers are shown in Fig. 8. As can be seen, Hsu I cuts the 27S oligomer into four fragments: two fragments of 0.56, one fragment of 0.44, and one fragment of 0.20 genome length. The generation of four fragments requires the presence of four Hsu I sites. Since Hpa II fragments 1 and 2 are only represented once in the oligomer, it appears likely that Hpa II fragment 0 contains the regions of fragments 1 and 2 which carry the Hsu I sites. Assuming that fragment 0 arose by fusion of parts of Hpa II fragments 1 and 2, this would place the two Hsu I sites at opposite ends of fragment 0, approximately at 0.09 and 0.03 genome length from each end, respectively. Cleavage of fragment 0 by Hsu I enzyme would therefore generate a new fragment of 0.32 - 0.09 - 0.03 = 0.20 genome length, a size which agrees well with the estimated length of 0.20 genome for the new fragment (Fig. 8A, inset). The same length estimate was obtained with an Hsu I digest of the 27S oligomer electrophoresed in a 3.5% polyacrylamide gel (not shown). No new fragments of smaller size were observed in this gel. Figure 8B shows the cleavage pattern of the 25.5S oligomer after Hsu I digestion. Two bands can be seen. The width of the first band

DEFECTIVE OLIGOMERS

VOL. 17, 1976

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HsuI-

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FIG. 5. Proposed scheme of derivation of the 27S and 25.5S oligomers from a polyoma dimer. Hpa II fragments 1, 2, and 6 are replaced by fragment 0 in the 27S oligomer and by fragment A in the 25.5S oligomer. For further details see text. Symbols are as in the legend to Fig. 4.

indicates the presence of two fragments. The length of both fragments is close to the predicted genome lengths of 0.56 and 0.54, respectively. The second band represents a fragment of about 0.44 genome length. Since Hpa II fragments 1 and 2 are also only represented once in the 25.5S oligomer, it is likely that the third Hsu I site is located in fragment A. The small size of fragment A suggests that it is the Hsu I site normally present in Hpa II fragment 2. To determine whether the additional Hsu I sites are located as postulated in fragments. 0 and A, the 27S and 25.5S oligomers were digested simultaneously with Hpa II and Hsu I enzyme, and the resulting fragments were analyzed by gel electrophoresis. In the 27S digest

fragment 0 was absent and three new fragments of about 0.20, 0.09, and 0.03 genome in size were observed (gels not shown). The two smaller fragments co-migrated with the corresponding smaller cleavage products of Hpa II fragments 1 and 2. In the 25.5S digest, fragment A was absent and two new fragments of about 0.06 and 0.03 genome length were present. The smaller fragment co-migrated with the smaller cleavage product of Hpa II fragment 2. These findings support, therefore, the assumption that fragment 0 contains at its opposite ends the Hsu I sites of Hpa II fragments 1 and 2 and that fragment A contains the Hsu I site of Hpa II fragment 2. The cleavage maps of the 27S and 25.5S

VOGT, BACHELER, AND BOICE

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oligomers as derived from the foregoing data are illustrated in Fig. 5. The position and orientation of fragments 0 and A, as suggested in Fig. 5, satisfy both the Eco RI cleavage pattern (Fig. 6) and the Hsu I pattern (Fig. 8). All cleavage results are consistent with a head-to-tail orientation of the duplication in both oligomers. The proposed structure of the oligomers does not exclude the possibility that parts of Hpa II fragment 6 are present in fragments 0 and A. This seems, however, unlikely, since both Hpa II sites defining fragment 6 are lost in both oligomers. The presence of a single deletion loop of approximately 0.23 genome length in hetero-

duplex molecules between the two oligomers (Fig. 9) lends further support to the deduced structures for the two oligomers. Test for cellular DNA sequences in Hpa II fragment 0. As described above, the cleavage by Eco RI and Hsu I enzyme of the 27S oligomer leaves a region of 0.20 genome length in fragment 0 uncharacterized. The possibility of the presence of cellular sequences within this region was therefore tested in hybridization experiments. The rate of reassociation of purified fragment 0 was measured alone or in the presence of additional unlabeled viral or cellular DNAs. The reassociation of Hpa II fragment

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FIG. 6. Sedimentation through alkaline sucrose gradients of the 27S (A) and 25.5S (B) oligomer after cleavage with Eco RI restriction endonuclease. A mixture of 3H-labeled (0) 27S or 25.5S DNA and 32P-labeled (') Py DNA was cleaved by Eco RI restriction endonuclease as described. The cleavage products were centrifuged in an SW56 rotor at 20 C for 100 min (A) or 180 min (B) at 50,000 rpm. Fractions of 6 drops were collected and assayed for radioactivity.

FIG. 7. Gel electrophoresis pattern of Hpa II digests after cleavage by Eco RI restriction endonuclease. 3H-labeled (0) oligomer DNA was cleaved by Eco RI enzyme. The cleavage products were phenol extracted, ethanol precipitated, and resuspended in reaction buffer. "P-labeled (Z) Py DNA was added to the DNA suspensions. The mixtures were further digested with Hpa II enzyme, and the digests were subsequently electrophoresed in a 2.2% polyacrylamide -0.7% agarose gel for 15.25 hat 120 V (A) or in a 3.5% polyacrylamide gel for 14 h at 125 V (C). The gels were fractionated as described in the legend to Fig. 2. (A) Unfractionated oligomer DNA of subline 1 after Eco RI and Hpa II digestion; (B) after Hpa II digestion without prior cleavage by Eco RI enzyme; (C) 25.5S DNA of subline 15 after Eco RI and Hpa II digestion. The arrows indicate changes in the migration of fragments 0, 2, and A after cleavage with Eco RI enzyme. Asterisk indicates new fragments generated by cleavage of fragments 2 and A.

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80 60 100 FRACTION NUMBER FIG. 8. Electrophoretic analysis in 2.2% polyacrylamide-0.7% agarose gels of the cleavage products generated from the 27S and 25.5S oligomer by Hsu I restriction endonuclease. 3H-labeled (0) 27S and 25.5S oligomer DNA were cleaved by Hsu I enzyme as described. 32P-labeled ('c) Py DNA, cleaved by Hpa II enzyme, was then added to both DNAs. The mixtures were applied to the gels and electrophoresed for 18 h and 10 min at 100 V. The gels were fractionated as described in the legend to Fig. 2. (A) 27S oligomer DNA. (Inset) Plot of fragment mobility versus fragment size. Arrow indicates fragment of 0.20 genome size. (B) 25.5S oligomer DNA. Arrow indicates fragment of approximately 0.54 genome size. 1018

DEFECTIVE OLIGOMERS

VOL. 17, 1976 9

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FIG. 9. Representative heteroduplex molecule between the 27S and 25.5S oligomer. A 40% concentration of formamide was used in the spreading solution, and a 10% concentration was used in the hypophase. Double-stranded monomeric Py DNA (Py) and single-stranded OX DNA (OX) were used as standards. The deletion loop is indicated by arrow. The average contour lengths for 10 heteroduplex molecules equalled 1.54 ± 0.02 genome lengths for the double-stranded region and 0.23 + 0.02 genome length for the single-stranded region.

1 was also studied as an example of a pure viral DNA sequence of nearly equal size. The addition of purified polyoma DNA accelerated the reassociation of fragments 0 and 1 to similar extents (Fig. 10). Since the reassociation of at least 69% of fragment 0 (the highest point measured) is accelerated by the addition of polyoma DNA, at least this fraction of fragment 0, corresponding to 0.22 genome length, is viral in nature. When the reassociation of fragment 0 was measured in the presence of large amounts of cellular DNA, no significant differences could be detected between the initial rates of reassociation in the presence of calf DNA and mouse DNA (Fig. 11). If the uncharacterized remaining 31% of fragment 0 (corresponding to 0.1 genome length) had been complementary to a unique cellular sequence of the mouse genome, the cellular DNA would have been in sufficient weight excess to cause a 0.3-fold increase in the initial rate of reassociation. The absence of any

detectable increase suggests that fragment 0 contains few if any cellular sequences. Hpa II cleavage products of larger oligomers. The intracellular pools of closed circular DNA of sublines 1 and 15 contain, in addition to the major 27S and 25.5S oligomers, smaller amounts of larger oligomers. To compare the structure of the larger oligomeric species with those of the 27S and 25.5S components, the unfractionated intracellular DNA pools comprising all sizes of supercoiled molecules and the purified pools corresponding to regions I to IV in Fig. 1 were digested by Hpa II enzyme, and the cleavage products were analyzed by gel electrophoresis. The results for the different DNA pools of subline 1 are illustrated in Fig. 12. A comparison of the fragmentation patterns shows the following. (i) Pool I (Fig. 12B), which represents the region of monomeric D NA in the intracellular pool of subline 1, shows a pattern essentially identical to that of

VOGT, BACHELER, AND BOICE

1020

the monomeric control DNA. A slight contamination with the 27S pool is indicated by the presence in submolar concentrations of fragment 0 and by a slightly elevated number of counts in peaks 3 and 4. (ii) The unfractionated DNA pool (Fig. 12A) and the pool of larger oligomers (Fig. 12C) show nine bands with the same electrophoretic mobilities as the bands 0 to 8 of the 27S oligomer. (iii) In both oligomer pools, bands 3, 4, and 5 show approximately twice the number of counts and therefore twice the number of fragments as compared to bands FRAGMENT 4.0

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3, 4, and 5 of the monomeric control DNA. (The same was found for bands 7 and 8 in gels run for shorter times.) From the identity of the cleavage pattern of the larger oligomers and the 27S component, it can be concluded that the larger oligomers are multiples of the 27S oligomer. The cleavage patterns of the various DNA pools of subline 15 are shown in Fig. 13. A conclusion similar to that drawn for the DNA pools of subline 1 can be drawn for the DNA pools of subline 15 with the restriction that the basic unit for the larger oligomers is the 25.5S and not the 27S oligomer (see also Fig. 3B). Nature of slowly migrating bands in intracellular DNA pools. In discussing the Hpa II cleavage pattern of the intracellular DNA pools of sublines 1 and 15, the slowly migrating bands near the top of the gels were purposely disregarded. This appeared justified for DNA pools of subline 1 in which the contribution of the slowly migrating bands to the total DNA was small. In contrast, the proportion of slowly migrating components was larger in subline 15 and became significant in pool IV (Fig. 13D), which comprises the larger oligomers. To verify that the slowly migrating components were mitochondrial in nature, mitochondrial DNA, purified under the same conditions used for the purification of oligomer DNA, was digested by Hpa II endonuclease, and the digest was coelectrophoresed with the unfractionated (Hpa II FRAGMENT 0

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FIG. 10. Reassociation kinetics of purified Hpa II fragments 0 and 1. A 0.019-,gg amount of fragment 0 or 0.017 7ig of fragment I was reassociated alone (0) or in the presence of purified unlabeled Py viral DNA (A). Data are plotted as Co/CO - Ca, the initial concentration divided by the fraction remaining single stranded versus time, according to Wetmur and Davidson (23).

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FIG. 11. Reassociation kinetics of fragment 0 in the presence of cell DNA. A 0.0086-,gg amount of fragment 0 was reassociated in the presence of 4.16 mg of calf thymus DNA (0) or mouse DNA (U). Data are plotted as described in the legend to Fig. 10.

FIG. 12. Gel electrophoresis profiles of superhelical DNA pools of subline 1 after cleavage by Hpa II restriction endonuclease. 32P-labeled (Z) Py DNA was added to all 3H-labeled (0) DNA pools, and the mixtures were digested with Hpa II enzyme. The electrophoresis was performed at 120 V for 15 h in 2.2% polyacrylamide-0.7% agarose gels (A and C), or at 125 V for 13.75 h in a 3.5% polyacrylamide gel (B). (A) Unfractionated pool of oligomer DNA; (B) pool I = monomer DNA; (C) pool III = larger oligomers.

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VOL. 17, 1976

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cleaved) oligomer pool of subline 15. The major slow component of the oligomer DNA of subline 15 co-migrates with the main band formed by the Hpa II digest of the mitochondrial DNA (Fig. 14). Its mitochondrial origin therefore appears certain. The presence of submolar bands in the mitochondrial DNA indicates a heterogeneity of the mitochondrial DNA. This may account for some of the differences between the minor slowly migrating components of the two DNAs compared in Fig. 14.

DISCUSSION The subject of this paper is a characterization of the various oligomeric species of superhelical viral DNA produced at 32 C by cells of two Py ts-a-transformed clonal sublines. Estimates of the size of the two major oligomeric components of both sublines were obtained from their sedi-

mentation values, from the number and size of fragments generated by cleavage with Hpa II restriction endonuclease, and from electron micrograph measurements. The major oligomeric (27S) component in subline 1 was found to be 1.77 times the size of the viral monomer, and the major (25.5S) component in subline 15 was found to be 1.54 times the size of the viral monomer. Results from the analysis of the cleavage products observed after digestion with Hpa II, Eco RI, and Hsu I endonucleases are consistent with the assumption that both oligomers are head-to-tail dimers from which a segment of different length has been deleted (Fig. 5). Since sublines 1 and 15 were derived from the same ts-a 3T3 transformant, TRF 2 (17), the finding that both oligomers show a duplication of the same fragments points to the possibility that the smaller 25.5S oligomer originated from the larger 27S oligomer.

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VOL. 17, 1976

The intracellular pools of superhelical DNA in sublines 1 and 15 contain, in addition to the 27S or 25.5S oligomer, larger oligomeric species (Fig. 1). The Hpa II cleavage patterns of all pools of larger oligomers were found to be identical, with respect to number and size of fragments, to those of the 27S (or 25.5S) pools. It can therefore be concluded that the larger oligomers are multiples of the 27S oligomer in subline 1 and multiples of the 25.5S oligomer in subline 15. Although the broad distributions shown by the larger oligomeric species in neutral sucrose gradients do not allow any accurate estimate of sedimentation rates, the distributions indicate that the sedimentation rate of most (and possibly all) larger oligomers does not exceed that expected for molecules twice the size of the 27S and 25.5S oligomer (36.6S and 34.5S, respectively). Most larger oligomers, therefore, appear to be "dimers" of the 27S and 25.5S oligomers. These arise most likely from replication errors (7, 14). The distributions of the larger oligomeric species in neutral sucrose gradients show a peak around 32S (subline 1) and 28S (subline 15), which persists upon recentrifugation. There are several possibilities to account for these peaks. (i) A certain proportion of the "dimers" are catenated dimers (13) that have been nicked once, yielding molecules which consist of a closed and a relaxed circular submolecule. Such molecules show sedimentation values intermediate between those of doubly closed and doubly open circular dimers (12). (ii) The peaks represent nicked higher multiples of the 27S or 25.5S oligomer. The relatively high proportion of these molecules (as indicated by the height of the peaks) seems unusual for higher oligomers (11, 13) and seems therefore to argue against this possibility. (iii) The peaks correspond to "dimers" from which a Py monomer has been excised. This third possibility has to be considered for the following reasons: both the 27S and the 25.5S components are infectious (17), and therefore can generate monomeric DNA; and the intracellular pools of both sublines contain monomer (21S) DNA (Fig. 1). Molecules generate by excision of monomeric DNA from molecules twice the size of the 27S or 25.5S oligomer would have a length of 2.54 and 2.08 viral genomes and have sedimentation values equal to 32S and 28S, respectively, agreeing well with the observed S values of the two peaks. However, Hpa II digestion of such molecules should generate three copies of the fragments represented twice in the 27S and 25.5S oligomers. However, no indication of a larger representation of the duplicated fragments was detected in

DEFECTIVE OLIGOMERS

1025

any of the pools of larger oligomers. (iv) The peak of 28S in subline 15 is formed by molecules twice the size of the viral monomer. If such molecules contributed significantly to the 28S peak, all fragments in an Hpa II digest of the 28S region should be represented in an equimolar concentration. However, all fragments represented twice in the 25.5S oligomer were also represented twice in the digest of the 28S region (Fig. 13C). Possibility (i) seems therefore to explain best the 32S and 28S peaks observed in the sedimentation profiles. However, it should be noted that the fragment patterns of the Hpa II digests do not exclude the presence, in small amounts, of such molecules as suggested by possibilities (iii) and (iv). The pools of oligomeric DNA species produced at 32 C by sublines 1 and 15 revealed a striking uniformity in the basic structure of their components. This uniformity has been maintained through many passages and subclonings at 39 C. Independent of the physical state in which we assume that the viral genomes are maintained at 39 C-as autonomous plasmids or as structures integrated into the host DNA (2)-the constancy over many cell passages and subclonings of the basic structure of the 27S and 25.5S oligomer suggests that in the cells of sublines 1 and 15 recombination events within the viral genomes occur rarely at 39 C. This may be in contrast to the situation which prevailed in the cells of the early passages of the transformed clone, TRF 2, the parent to sublines 1 and 15. As previously described (17), cultures of clone TRF 2 showed a high incidence of cell death at 39 C for a 2-month period after the isolation of the clone. This could indicate that abortive viral replication occurred during this culturing period, which led first to dimerization and subsequently to the formation of the defective 27S and 25.5S dimers. The processes leading to the defective dimers may have been the same that generate defective viral monomer molecules during lytic infection. ACKNOWLEDGMENTS We would like to thank Jeff Brown and Candy Haggblom for expert technical assistance. This research was supported by Public Health Service grants no. CA-13068 and CA-14195 from the National Cancer Institute, American Cancer Society Fellowship no. PF 928 to L. B. Bacheler, and Public Health Service Special Fellowship no. CA-53485 from the National Cancer Institute to L. B. Boice. LITERATURE CITED 1. Bishop, D. H. L., J. R. Claybrook, and S. Spiegelman. 1967. Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Mol. Biol. 26:373-387. 2. Cuzin, F., M. Vogt, M. Dieckmann, and P. Berg. 1970.

1026

:3.

4.

5. 6.

7. 8.

9.

1(). 11. 12.

VOGT, BACHELER, AND HOICE

Induction of virus multiplication in 3T3 cells tralsformed by a thermosensitive mutant of polyoma virus. II. Formation of'oligomeric polvoma DNA molecules. J. Mol. Biol. 47:317-:3383. Davis, R. W., M. Simon, and N. Davidson. 197 1. Electron microscope heteroduplex methods for mapping regions of base sequence homology in tlucleic acids. p. 41:3-428. In I. Grossman and K. Molda\e (ed.), Methods in enzymolog. viol. XXID. Academic Press Inc., New York. Edgell, M. H., C. A. Hutchison, III, and M. Sclair. 1972. Specific endionuclease R fragments if bacteriophage OX1T4 deoxyribonucleic acid. J. Virol. 9:574-582. Fried, M. 1965. Isolation of' temperature-sensitise mutants of polyoma virus. Virology 25:669- 671. Fried, M. 197(0. Characterization of a temperature-sensitive mutant of polyoma virus. Virology 40:605 617. Goebel, W., and D. R. Helinski. 1968. Generation of higher multiple circular DNA forms in bacteria. Proc. Natl. Acad. Sci. U.S.A. 61:14(16-141:3. Graham, F. L., P. J. Abrahams, C. Mulder, H. L. Heijneker, S. 0. Warnaar, F. A. J. deVries, W. Fiers, and A. J. van der Eb. 1975. Studies on in L'itro transformation by DNA and DNA fragments of human adenoviruses and simian virus 40. Cold Spring Harbor Symp. Quant. Biol. 39:6:37-65). Griffin, B. E., M. Fried, and A. Cowie. 1974. Polyoma DNA: a physical map. Proc. Natl. Acad. Sci. t S.A. 71:2077 -2081. Hirt, B. 1967. Selective extractiom if polyomia DNA from infected mouse cell cultures. .J. Mol. Biol. 26::365:-369. Hobom, G., and D. S. Hogness. 1974. The role of' recombination in the formation of circular iiligomers of' the \ dv1 plasmid. J. Mol. Biol. 88:65-87. Hudson, B., and J. Vinograd. 1969. Sedimentation

,J. V IROIL-

velocitY

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15. 16.

17.

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20. 21.

22.

2:3.

properties of complex mitochondrial DNA. Nature (London) 221::332-:3:37. Jaenisch, R., and A. Levine. 1971. DNA replication in S\'40-infected cells. V. Circular and catenated ligoimers of S\V40 DN A. \Virology 44:48(0-49:1. Jaenisch, R., and A. Levine. 1973. DNA replication of S\'40-infected cells. \'II. Formation of S\'40 catenated and circular dimers. .J. Mol. Biol. 73:199- 212. Lee, A. S., and R. L. Sinsheimer. 1974. A continuous electroelution method for the recovery of DNA restriction enzyme fragments. Anal. Biochem. 60:64(0-644. Maizel, J. V., Jr. 1971. Polyacrylamide gel electrophoresis of viral proteins, p) 179- 246. In K. Maramorosch and H. Koprowski led.). Methods in virologv. \ol. 5. Academic Press Inc., New Yiork. Mulder, C., and M. Vogt. 197:3. Production of non-defective and defective oligomers of' iral 1)NA in mouse :3T:I cells transformed b! a thermosensitise mutant ofi polvoma virus. ,J. Mol. Biol. 75:6(31-608. Peacock, A. C., and C. W. Dingman. 1968. Molecular w eight estimation and separation of ribonucleic acid h! electrophoresis in agarose-acrs larnide com posite gels. Biochemistrv 7:668-674. Schmeckpeper, B. J., and K. D. Smith. 1972. U'se of formamnide in nucleic acid reassociation. Biochemistr\ 11:1319-1:326. Studier, F. W. 1965. Sedimentation studies of the size and shape of DN\A. ,J. Mol. Biol. 11::373-:390. Sutton, W. D. 1971. A crude nuclease preparation suitable for use in DNA reassociation experiments. Biochim. Biophys. Acta 240:522-5:11. Vogt, M., and R. Dulbecco. 196:3. Steps in the neoplastiC transformation of hamster embryo cells by polvnoma virus. Proc. \atl. Acad. Sci. U.S.A. 49:171-179. Wetmur, J. G., and N. Davidson. 1968. Kinetics i)f renaturation of I)NA. J. Miol. Biol. 31:349 :37(0.