Vol. 22, No. 3 Printed in U.S.A.

JOURNAL OF VIROLOGY, June 1977, p. 794-803

Copyright C 1977 American Society for Microbiology

Episomal Viral DNA in a Herpesvirus saimiri-Transformed Lymphoid Cell Line FRED-JOCHEN WERNER, GEORG W. BORNKAMM, AND BERNHARD FLECKENSTEIN'* Institut fur Klinische Virologie der Universitat, Erlangen-Nurnberg, 8520 Erlangen, Germany Received for publication 7 December 1976

The lymphoid cell line #1670 has been derived from the infiltrated spleen of a tumor-bearing marmoset monkey infected with Herpesvirus saimiri. The cells contain both types of H. saimiri DNA, unique light (L-) DNA (36% cytosine plus guanine) and repetitive heavy (H-) DNA (71% cytosine plus guanine), without producing infectious virus. Viral DNA was found to persist in these cells as nonintegrated circular DNA molecules. Closed circular superhelical viral DNA molecules were isolated by three subsequent centrifugation steps: (i) isopycnic centrifugation in CsCl, (ii) sedimentation through glycerol gradients, and (iii) equilibrium centrifugation in CsCl-ethidium bromide. The isolated circles had a molecular weight of 131.5 + 3.6 x 106. This is significantly higher than the molecular weight of linear DNA molecules isolated from purified H. saimiri virions (about 100 x 106). Partial denaturation mapping of circular molecules from #1670 lymphoid cells showed uniform arrangement of H- and L-DNA sequences in all circles. All denatured molecules contained two L-DNA regions (molecular weights of 54.0 ± 1.8 x 106 and 31.5 ± 1.3 x 106) and two H-DNA regions (molecular weight of 25.6 ± 1.9 x 106 and 20.0 ± 0.8 x 106) of constant length. Maps of both L-regions suggested that the sequences of the shorter LDNA region were a subset of those of the longer region. The sequences of both L-regions had the same orientation. Circular molecules from H. saimiri-transformed lymphoid cell line #1670 appeared to represent defective genomes, containing only 75% of the genetic information present in L-DNA of H. saimiri virions.

Herpesvirus saimiri, a natural inhabitant of squirrel monkeys (Saimiri sciureus), is highly oncogenic in various New World primate species (8, 11). The DNA of the virus is characterized (i) by an unusual heterogeneity in base composition and (ii) by containing highly repetitive sequences. The predominant type of H. saimiri DNA molecules (M-genome, M-DNA) isolated from purified virions consists of about 70% unique DNA and 30% repetitive DNA (4, 5). Unique DNA (light [b-] DNA) with a guanine plus cytosine (G+C) content of 36% has a constant length of 71.6 x 106. The L-DNA region is inserted between two stretches of repetitive DNA (heavy [H-] DNA) which are variable in length. H-DNAs (71% G+C) consist of identical repeat units (molecular weight 830,000) in tandem orientation (1). Isolated M-DNA is infectious in cell culture (5) and oncogenic upon transfection in marmoset monkeys (6). A number of H. saimiri-transformed lymph' Present address: New England Regional Primate Research Center, Harvard Medical School, Southborough, MA 01772.

oid cell lines had been derived from neoplastic cells in infiltrated organs and blood of tumorbearing marmoset monkeys (3, 15). H. saimiritransformed cells revealed T-cell markers (18). All lymphoid cell lines produced infectious virus after explantation into cell cultures. Three cell lines (70N2, 70BM2, and #1670) which have been cultured over a period of several years subsequently stopped virus synthesis, converting to "nonproducer" cell lines. No infectious virus could be rescued from nonproducer cells by co-cultivation with permissive monolayer cells; attempts to extract infectious DNA were unsuccessful (unpublished data). Nonproducer cells could not be induced by halogenated nucleotide analogues to form infectious virus (R. Neubauer, personal communication), and no viral antigens were demonstrated in these cells by different methods of immunofluorescence (L. Falk, personal communication). Though virus-specific gene functions have not been detected in H. saimiri-transformed nonproducer cells, these cells contain a rela794

V OL. 22. 1977

EPISOMAL HERPESVIR US SAIMIRI DNA

795

tively high concentration of H. saimiri-specific many for 2 h at 37'C. The proteinase K had been DNA sequences. Nucleic acid hybridizations preincubated for 1 h at 370C. The lysate was diluted with 50 mM Tris-hydrohave shown that nonproducer cell lines conbuffer ipH 9.0 to a final volume of 60 ml. tained 0.07 to 0.23%c repetitive H-DNA of H. chloride '77.5 CsCl g was dissolved in the lysate at 20°C to n[, Lviral saimiri and more than 0.07%c unique = 1.4005. After centrifugation to equilibrium in a DNA sequences (B. Fleckenstein. I. M.1iller. Spinco type 60 Ti rotor for 60 h at 33,000 rpm and and J. Werner. Int. J. Cancer. in press. 207C. fractions of 0.5 ml were collected from the In situ hybridizations of virus-specific com- bottom through an 18-gauge 1' 2 syringe needle at a plementary RNA cRNA) with DNA from non- restricted flow rate. The DNA banding in a density range from 1.700 producer lymphoid cell lines indicated an association of persisting viral DNA sequences with to 1.730 was pooled and dialyzed against a solution 1 M NaCl. 20 mM Tris-hydrochloride metaphase chromosomes of host cells 6). It containing 8.0). and mM EDTA. The DNA concentration ipH suggested that there exists a certain similarity was measured1 by taking the UV spectrum between to the mode of virus genome persistence in 220 and 320 nm. Epstein-Barr virus (EBV-transformed lymphA maximal amount of 15 Ag of DNA was loaded oid B-cell lines of human origin 19). More re- on 10 to 30%'vol vol' glycerol gradients in 1 M cent studies have shown that EBV-transformed NaCI-20 mM Tris-hydrochloride (pH 8.0)-i mM lymphoid cells contain nonintegrated viral EDTA and was centrifuged in a Spinco SW 27 rotor DNA 12. 17. A cell line derived from a Burkitt for 10 h at 15.000 rpm. 20CC. DNA of about 110S was lymphoma and fresh biopsy materials from collected, dialyzed against 1 M NaCl-20 mM Trishydrochloride 'pH 8.0)-1 mM EDTA. and then di.uch tumors and nasopharyngeal carcinomas alyzed twice against 20 mM Tris-hydrochloride ' pH contained nonintegrated covalently closed cir- 8.0)-i mM EDTA for 90 min. The DNA of four to six cular EBV DNA molecules 9. 10. glycerol gradients was concentrated to a final volThese results stimulated us to search for ume of a ml by dialysis against dry polyethylene viral plasmids in H. saimiri-transformed cells. glycol 6000. followed by dialysis against two changes The present report describes the isolation of of TNE '50 mM 'aCl. 20 mM Tris-hvdrochloride nonintegrated circular H. saimiri DNA mole- [pH 8.0]. 1 mM EDTA'. Solid CsCl .5.2 g'. 300 ng of-H-labeled BK virus cules from a lymphoid cell line #1670i and their structural analysis by partial denatura- DNA form I 'specific activity. 40,000 dpm Ag), and TNE buffer were added to obtain a solution with a tion mapping. of 1.60 g'ml ' nD = 1.39015a in a final volume (This material was presented in part at the density of 9.4 ml. In subdued light. 0.6 ml of ethidium bro10th 'Meeting of the European Tumor Virus mide '6 mg ml, was added. DNA was spun to equiGroup. Grindelwald, Switzerland. 1976. librium for 60 h at 40.000 rpm. 20-C. in a Spinco type -MATERIALS A-ND METHODS

Virus and cell culture. H. saimiri-transformed #1670 cells were grown as suspension cultures in RPMI 1640 medium with 10% heat-inactivated fetal calf serum and 80 jA.g of gentamicin ml. The cells were kept in closed Erlenmeyer flasks and subcultured at 4-day intervals. The cell line was checked repeatedly for mycoplasma contamination by isolation procedures on agar plates under anaerobic conditions as described elsewhere 'Fleckenstein et al.. in press'. The cells were consistently found to be free from mycoplasma. Isolation of circular viral DNA from transformed cells. The procedure for isolation of circular H. saimiri DNA followed the method described for episomal EBV DNA 10). About 6 x 107 actively growing #1670 cells were harvested by low-speed centrifugation 2,000 rpm for a min at 20'C. The cells were washed twice with isotonic PBS iphosphate-buffered saline. pH 7.01. resuspended in 28.5 ml of 75 mM Tris-hydrochloride ipH 9.0)-25 mM EDTA, and lysed by addition of 1.5 ml of 20% wt/volb sodium lauryl sarkosinate (Sarkosyl NL). The lysate was gently rolled for 10 min at 20'C and then incubated with 3.5 ml of a 0.1% proteinase K solution (Merck, Darmstadt. Ger-

.50 Ti rotor. Fractions of 0.5 ml were collected from the bottom. and the position of the superhelical DNA was determined by counting samples of each fraction. The fractions containing H. saimiri DNA were pooled. extracted twice with isoamyl alcohol and diethyl ether. and then dialyzed against 20 mM Tris-hydrochloride 'pH 8.5a-0.2 M sodium acetate-1 mNI EDTA. The DNA was precipitated with 2.5 volumes of ethanol. collected in a Spinco SW 56 rotor tube by centrifuging at 35.000 rpm for 45 min at OC. and redissolved in 30 A1 of 20 mM Tris-hydrochloride 'pH 8.5)-i mM EDTA. DNA'A.cR.NA hybridization. Fractions from glycerol gradients were monitored for the presence of H. saimiri-specific DNA sequences by DNA-cR.NA hvbridization. Fractions were diluted with 0.1 x SSC 'SSC = 0.15 !M N'aCl. 15 mMN sodium citrate' to a volume of 3 ml. and DNA was denatured by adding 1 ml of 1 M NaOH and heating to 80-C for 10 min. The solution was neutralized with 1 ml of 1 M HCI-0.5 M Tris-hydrochloride-10x SSC. and the DNA was immobilized on nitrocellulose filters iSartorius. Gottingen. Germany: SM 11306' by drying the filters at 80C for 4 h. L-DNA from H. saimiri was purified and transcribed in vitro into 3H-labeled cRNA ' specific activity. about 4 x 107 dpm. ,ug as reported previously

796

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WERNER, BORNKAMM, AND FLECKENSTEIN

About 120,000 dpm of heat-denatured cRNA in 1.0 ml of 2.5x SSC-50% formamide-G.05% sodium dodecyl sulfate was added to each filter. After incubation at 450C for 7 days under gentle shaking, filters were washed with 2 x SSC. Treatment of filters with 20 Ag of RNase/ml (Merck, Darmstadt) for 30 min at 370C was followed by additional washing with 2 x SSC. Wet filters were dissolved by vigorous shaking in 10 ml of toluene-Triton X-100 scintillator (2:1) for 20 min at 40C before counting. Partial denaturation and electron microscopy of DNA. Viral DNA molecules were partially denatured essentially as described in a previous report (1). DNA was incubated for 30 min at 300C in 7 M sodium perchlorate, 1.04 M formaldehyde, 10 mM sodium phosphate (pH 7.5), and 1 mM EDTA. Subsequently, the DNA molecules were passed through a Sephadex G-100 column (5 by 0.5 cm), eluted with 1 mM EDTA (pH 7.0), and spread by the formamide technique (2). Bacteriophage PM2 DNA (6.4 x 106 molecular weight [14]) was added as marker immediately before spreading. The spreading solution contained 0.1 mg of cytochrome c per ml, 40% (vol/ vol) formamide, 0.1 M Tris-hydrochloride (pH 8.5), and 10 mM EDTA; the hypophase was a 10% (vol/ vol) formamide solution with 10 mM Tris-hydrochloride (pH 8.5) and 1 mM EDTA. The grids were stained with uranyl acetate (2) and rotary-shadowed with Pt/Pd, and electron micrographs were taken in a Zeiss EM 10 electron microscope. Length measurement and computer analysis of partially denatured molecules. The lengths of duplex and denatured parts of partially denatured molecules were determined with a mechano-electronic X-Y measuring stage (LM-1, Bruhl, Nurnberg, Germany), which allows reproducible length measurements with a standard deviation of less than 0.4%. For analysis in a CD 3300 computer tUniversity Erlangen-Nurnberg), each molecule was divided into equal segments of 20 nm (about 60 base pairs). Each segment received a code for single or double strandedness, and the denaturation pattern of each molecule was recorded on punch cards. Molecules were standardized to the length of the longest molecule out of a homogeneous class. The computer compared two molecules by moving one relative to the other, segment by segment, until optimal correspondence in the right polarity was achieved. The molecules were optimally aligned if the number of corresponding segments with the same code native versus denatured) reached a maximum. All molecules were aligned successively. This program can be used for constructing partial denaturation histograms of linear or circular molecules.

miri has a density of 1.7045 g/ml (5), the density difference between host DNA and presumed H. saimiri episomes with a mean base composition similar to that of M-DNA is smaller. Hybridization of each fraction of a CsCl gradient containing DNA from H. saimiri-transformed lymphoid cells with cRNA complementary to H. saimiri H- or L-DNA revealed most viral sequences in the bulk of cellular DNA (data not shown). Therefore, most cellular DNA was included in the next preparation step. Density centrifugation in CsCl was regarded as a safe and efficient deproteinization step rather than a procedure for enriching viral sequences. Attempts to isolate viral genomes by sedimentation in glycerol gradients without an initial CsCl centrifugation were unsuccessful. The CsCl-purified DNA was sedimented through glycerol gradients to separate circular DNA from the bulk of cellular DNA, analogous to the purification of EBV episomal DNA (10). The position of H. saimiri-specific sequences in these gradients was determined by hybridizing DNA of each fraction with L-DNA-specific cRNA (Fig. 1). A peak of fast-sedimenting DNA similar to that observed for superhelical EBV DNA circles contained H. saimiri-specific sequences. A second peak containing H. saimiri-specific DNA migrated at a slower rate. The exact sedimentation constants of both types of viral DNA have not been determined with internal size markers. Fractions with fast-sedimenting DNA were pooled, and the DNA was subjected to a CsClethidium bromide centrifugation. DNA band-

RESULTS Presence of closed circular DNA in #1670 cells. The first step in the preparative isolation of episomal EBV DNA was an isopycnic centrifugation in a cesium chloride density gradient (10), taking advantage of the density difference between EBV DNA (1.718 g/ml; 16) and hostcell DNA (1.700 g/ml). Since M-DNA of H. sai-

FIG. 1. Velocity sedimentation of DNA from lymphoid cell line #1670 in a 10 to 30% (vollvol) glycerol gradient. Virus-specific sequences in each fraction are detected by filter hybridization with cRNA. The arrow indicates the position of fast-sedimenting viral DNA with a sedimentation coefficient of about 110S, estimated from parallel sedimenta-

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VOL. 22, 1977

EPISOMAL HERPESVIRUS SAIMIRI DNA

797

ing in the density range of covalently closed a molecular weight significantly higher than circular DNA was spread by the formamide that of linear H. saimiri M-genomes isolated technique (2). from virus particles (Fig. 4). The standard deUpon electron microscopy, large circular viation of contour length measurements obDNA molecules were found, partly as superhel- tained from 59 molecules was ±2.7%. This variices (Fig. 2) and partly as relaxed circles (Fig. ability is compatible with the assumption that 3).

Molecular weight of episomal DNA. The length of circular molecules was measured by use of bacteriophage PM2 DNA as an internal size marker. The molecular weight of PM2 DNA is 6.4 x 10 (14). Circular molecules were found to have an average molecular weight of 131.5 x 10k. This is

all circles isolated from #1670 cells have the length. Arrangement of heavy and light sequences in circular molecules. Circular molecules isolated by the three centrifugation steps were partially denatured at low temperature and neutral pH under conditions which allow the denaturation of 90% of LDNA sequences in

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VOL. 22, 197 7

EPISOMAL HERPESVIR US SAIMIRI DNA

20 -01

molecular W*ight

FIG. 4. Histograms shooting the contour lengths of *59 episomal molecules 'CC, from #1670 cells. compared with the length distribution of .57 linear .MD.VA molecules from H. saimiri Lirions.

linear H. saimiri M-genomes. Spreading of this D.NA revealed molecules differing remarkably in the degree of denaturation. Of a total of 65 molecules observed. 46 did not exhibit any single-stranded loop: 19 molecules showed denatured regions comprising 22.4 to 53.8% of the total length. Under the conditions used for partial denaturation by sodium perchlorate and formaldehyde. the mass to length unit ratio of single-stranded segments appeared not to be significantly altered in comparison with duplex segments.

In all partially denatured molecules, singlestranded segments were clustered in two distinct regions. resulting in the characteristic picture shown in Fig. 5. This meant that these circles consisted of two regions with a high and two regions with a low G-C content. In analogy to the base sequence heterogeneity in linear H. saimiri DNA. the denatured regions in the circles were designated L-DNA regions and the duplex parts were designated H-DNA regions. Size of H-DNA and L-DSNA regions in DNA circles. The lengths of individual H- and LDNA regions in circular molecules were determined with duplex PMN2 DNA as a size standard. The two H-regions and the two L-regions were distinguishable in size. Figure 6 shows histograms displaying the size range of each region.

Short L-regions had contour lengths corresponding to molecular weights of 31.5 1.3 x 10"; long L-regions corresponded to 54.0 1.8 x 10". Thus, both L-regions are shorter than the L-region in linear M-DNA 'molecular weight. 71.6 x 10"; 1'. The lengths of the short and long H-regions

799

in the circles corresponded to molecular weights of 20.0 - 0.8 x 10" and 25.6 - 1.9 x 10". respectively. The length of H-stretches in the circular molecules is thus more constant than that of the terminal H-regions in linear Mgenomes 1'. The minor variations in length may be explained by differences in the extent of denaturation in different circular molecules if it is assumed that the length of each of the four distinct regions in circular molecules is constant. A histogram was constructed from 19 circular molecules by computer analysis: the corresponding maps are shown in Fig. 7. In this histogram the polarity of both L-regions is determined by the positions of the long and the short H-regions. Relation between short and long L-DN- A regions in circular molecules. The validity of the partial denaturation patterns for the identification of distinct sequences in the L-regions was tested by constructing denaturation histograms of L-regions without predetermining their orientation. All long L-regions except one were aligned in the same polarity Fig. 8 '. revealing two distinct areas containing preferentially native DNA positions 0.12 and 0.51 fractional length) and three areas containing preferentially single-stranded D.NA positions 0.06. 0.45. and 0.56 fractional length). To find a correlation between long and short L-regions, the denaturation maps of short Lregions of 18 circles were aligned independently to the denaturation histogram obtained from the long L-regions of the same 18 molecules. All of the short L-regions were placed by the computer into the same position with identical orientation. The denaturation maps of the small L-regions were found to fit into the left part of the long L-region. corresponding to 0 to 60% of its fractional length ' Fig. 8 '. This suggests that the short L-region and the left part of the long L-region represent corresponding L-D-NA sequences. This implies that the genetic information of total L-D.NA in the circles does not exceed the complexity of 54 x 106 molecular weight. Two possibilities exist for the arrangement of two regions of identical sequences in a circle. They can be arranged in identical or inverted orientation. Aligning of the short L-DNA maps to the long L-region indicated that the two Lregions of #1670 cells have the same polarity ' Fig. 7 and 8'.

DISCUSSION This studv has shown that covalentlv closed circular DNA molecules of high molecular weight i131 x 10") are present in H. saimiri-

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EPISOMAL HERPESVIRUS SAIMIRI DNA

VOL. 22, 1977

801

transformed #1670 cells. There is a series of specific DNA sequences in glycerol gradients is arguments suggesting that the circles are com- that expected for superhelices and relaxed circles and is similar to that of episomal EBV posed of viral sequences: DNA. (i) The sedimentation profile of H. saimiri(ii) It is unlikely that the circles represent plasmids of mycoplasma. The cell lines were consistently found to be free from mycoplasma. L-DNA All Mycoplasma strains isolated from infected HDNA tissue culure cells contain DNA with a low G+C content, and the G+C content never exceeds 41% (13). (iii) Each episomal molecule from H. saimiriE transformed cells analyzed by partial denaturaE tion consisted of four clearly distinct duplex DNA regions, two of them with a low G+C content and two of them with very high G+C content. To our knowledge, such a heterogeneity N 4N 25 3D 40 10 20 30 1s 50 in base composition has never been observed in length percent FIG. 6. Histograms showing the lengths of H- any microorganism besides oncogenic primate DNA and L-DNA regions in 19 episomal molecules herpesviruses. (iv) The relative proportion of heavy sefrom #1670 cells. Standard deviations are given in quences in circular molecules is higher than in the text. 14-

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J. VIROL.

Hence it is assumed that the large-molecular-weight circles isolated from #1670 cells represent H. saimiri DNA. The circular molecules as well as the individual H- and L-regions showed a remarkable homogeneity in size, compared with linear viral M-genomes. The partially denatured molecules were fitting into one denaturation pattern, indicating again that the circles belong to a homogeneous class of molecules. The 46 molecules which remained completely duplex under the denaturing conditions probably represented molecules in which denaturation was not possible because of intramolecular tension in superhelices. The equal length of native and denatured circles and the variation in the degree of denaturation favor this interpretation. Alternatively, the duplex circles might consist of Hsequences exclusively. A definite answer to this question is expected from partial denaturation with agents preferentially denaturing Gi+Crich sequences. Both L-regions in circular molecules were smaller (molecular weights, 31 x 10' and 54 x 10") than the L-DNA region of linear M-genomes derived from infectious virions (molecular weight, 71.6 x 10"). Partial denaturation maps of both L-regions in the circles suggested that the sequences of the shorter L-region were a subset of those of the longer region. This implies that only 75C/ of the genetic information of H. saimiri is represented in the circles of #1670 cells. It confirmed the earlier data obtained by reassociation kinetics, which indicated that in some H. saimiri-transformed cell lines part of the L-sequences was missing (Fleckenstein et al., in press). The presence of defective genomes and the apparent absence of intact genomes explains why infectious virus could never be rescued from #1670 cells. Attempts are in progress to correlate the L-sequences of episomal viral genomes with the known physical gene maps of linear viral molecules (Mulder, Fleckenstein, and Delius, manuscript in preparation). Episomal viral molecules derived from different H. saimiri-transformed cell lines have to be characterized in order to understand whether the circular structure found in #1670 cells was generated rather accidently and subsequently selected by growth or whether it is typical for all other H. saimiri-transformed cells. Episomal EBV DNA in transformed human cells has a molecular weight virtually identical to or slightly smaller than that of the linear genome from virus particles. These EBV circles may have originated from circularization of one complete linear viral genome. The circles from #1670 cells, however, are significantly longer

VOL. 22, 1977

EPISOMAL HERPESVIR US SAIMIRI DNA

and must have been formed by intermolecular recombination. It is not known whether the episomal state of viral DNA in EBV- or H. saimiri-transformed cells is substantially correlated to the transformation event or whether integrated viral sequences are necessary for the oncogenic transformation by herpesviruses. Nonintegrated episomal viral DNA molecules may possibly play an important role in herpesvirus persistence within transformed and nontransformed cells. The possibility of isolating viral genomes from transformed cells for a structural analysis may provide further prospects for the study of the mechanism of transformation by herpesviruses. ACKNOWLEDGMENTS We thank Harald zur Hausen, Hajo Delius, and Carel Mulder for their helpful discussions and advice. The excellent technical assistance of Ingrid Muller is gratefully ac-

knowledged. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 118). LITERATURE CITED 1. Bornkamm, G. W., H. Delius, B. Fleckenstein, F.-J. Werner, and C. Mulder. 1976. Structure of Herpesvirus saimiri genomes: arrangement of heavy and light sequences in the M genome. J. Virol. 19:154-161. 2. Davis, R. W., M. Simon, and N. Davidson. 1971 Electron microscopy heteroduplex methods for mapping regions of base sequence homology in nucleic acids, p. 413-482. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 21. Academic Press Inc., New York. 3. Falk, L. A., L. G. Wolfe, and F. Deinhardt. 1972. Demonstration of Herpesvirus saimiri-associated antigens in peripheral lymphocytes from infected marmosets during in vitro cultivation. J. Natl. Cancer Inst. 48:523-530. 4. Fleckenstein, B., and G. W. Bornkamm. 1975. Structure and function of Herpesvirus saimiri DNA, p. 145-149. In G. de The, H. zur Hausen, and E. M. Epstein (ed.), Oncogenesis and herpesviruses II. IARC Press, Lyon. 5. Fleckenstein, B., G. W. Bornkamm, and H. Ludwig. 1975. Repetitive sequences in complete and defective genomes of Herpesvirus saimiri. J. Virol. 15:398-406. 6. Fleckenstein, B., G. W. Bornkamm, and F.-J. Werner. 1976. The role of Herpesvirus saimiri in oncogenic transformation of primate cells. Bibl. Haematol. (Basel) 43:308-312.

803

7. Fleckenstein, B., and H. Wolf. 1974. Purification and properties of Herpesvirus saimiri DNA. Virology 58:55-64. 8. Hunt, R. D., L. V. Melendez, N. W. King, C. E. Gilmore, M. D. Daniel, M. E. Williamson, and T. C. Jones. 1970. Morphology of a disease with features of malignant lymphoma in marmosets and owl monkeys inoculated with Herpesvirus saimiri. J. Natl. Cancer Inst. 44:447-465. 9. Kaschka-Dierich, C., A. Adams, T. Lindahl, G. W. Bornkamm, G. Bjursell, G. Klein, B. C. Giovanella, and S. Singh. 1976. Intracellular forms of EpsteinBarr virus DNA in human tumor cells in vivo. Nature (London) 260:302-306. 10. Lindahl, T., A. Adams, G. Bjursell, G. W. Bornkamm, C. Kaschka-Dierich, and U. Jehn. 1976. Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line. J. Mol. Biol. 102:511-530. 11. Melendez, L. V., R. D. Hunt, M. D. Daniel, F. Garcia, and C. E. 0. Fraser. 1969. Herpesvirus saimiri. II. Experimentally induced malignant lymphoma in primates. Lab. Anim. Care 19:378-386. 12. Nonoyama, M., and J. S. Pagano. 1972. Separation of Epstein-Barr virus DNA from large chromosomal DNA in non-virus-producing cells. Nature (London) New Biol. 238:169-171. 13. Normore, W. N., and J. R. Brown. 1970. Guanine plus cytosine (G+C) composition of bacteria, p. H-24-H-74. In H. A. Sober, R. A. Harte, and E. K. Sober (ed.), Handbook of biochemistry, selected data for molecular biology. The Chemical Rubber Co., Cleveland. 14. Pettersson, U., C. Mulder, H. Delius, and P. A. Sharp. 1973. Cleavage of adenovirus type 2 DNA into six unique fragments by endonuclease R.R,. Proc. Natl. Acad. Sci. U.S.A. 70:200-204. 15. Rabson, A. S., G. T. O'Conor, D. E. Lorenz, R. L. Kirschstein, F. Y. Legallais, and T. S. Tralka. 1971. Lymphoid cell culture line derived from lymph node of marmoset infected with Herpesvirus saimiri. Preliminary report. J. Natl. Cancer Inst. 46:10991109. 16. Schulte-Holthausen, H., and H. zur Hausen. 1970. Parital purification of the Epstein-Barr virus and some properties of its DNA. Virology 40:776-779. 17. Tanaka, A., and M. Nonoyama. 1974. Latent DNA of Epstein-Barr virus DNA: separation from high-molecular-weight cell DNA in a neutral glycerol gradient. Proc. Natl. Acad. Sci. U.S.A. 71:4658-4661. 18. Wallen, W. C., R. H. Neubauer, H. Rabin, and J. L. Cicmanec. 1973. Nonimmune rosette formation by lymphoma and leukemia cells from Herpesvirus saimiri-infected owl monkeys. J. Natl. Cancer Inst. 51:967-975. 19. zur Hausen, H., and H. Schulte-Holthausen. 1972. Detection of EB viral genomes in human tumor cells by nucleic acid hybridization, p. 321-325. In P. M. Biggs, G. de The, and L. N. Payne (ed.), Oncogenesis and herpesviruses. IARC Press, Lyon.