Isolation of episomal bovine papillomavirus chromatin and

JOURNAL OF VIROLOGY, May 1983, p. 567-574 Vol. 46, No. 20022-538X/83/050567-08$02.00/0Copyright C) 1983, American Society for Microbiology

Isolation of Episomal Bovine Papillomavirus Chromatin andIdentification of a DNase I-Hypersensitive Region

FRANK ROSL, WALDEMAR WALDECK, AND GERHARD SAUER*Institute for Virus Research, German Cancer Research Center, 69 Heidelberg, West Germany

Received 15 November 1982/Accepted 9 February 1983

The investigation of papillomavirus chromatin has been hampered by theunavailability of a tissue culture system for vegetative growth of these viruses. Wehave used, therefore, bovine papillomavirus type 1-transformed hamster embryofibroblasts containing 200 to 250 episomal genome equivalents per cell as a sourceof viral chromatin. The selectively isolated chromatin was shown to be slightlylarger (80S) than the mature simian virus 40 chromatin, which was cosedimentedin a sucrose density gradient. Both Fo I and Fo II were present in the bovinepapillomavirus type 1 chromatin. A fast-sedimenting fraction, whose structure isstill unknown, also contained oligomeric bovine papillomavirus type 1 DNA. Byin situ DNase digestion of isolated nuclei and subsequent cleavage of the bovinepapillomavirus type 1 DNA with various restriction endonucleases, a majorDNase-hypersensitive region was detected in the chromatin. This region, com-prising approximately 320 base pairs, is located between the relative physical mappositions 0.88 and 0.92.

During recent years, the structure of chroma-tin has been investigated in great detail to detectproperties which distinguish active from inactiveregions. By using various nucleases, in particu-lar the DNase I with little sequence specificity, itwas found that vulnerable regions referred to ashypersensitive appear to be located close to or atthe 5' end of actively transcribed genes (forreviews, see references 10 and 33). The chroma-tin of simian virus 40 (SV40) and polyoma, forexample, display hypersensitive regions adja-cent to the origin of DNA replication (24, 28,29), which were shown to be devoid of nucleo-somes (13, 23). These regions include DNAsequences which are required for the transcrip-tion of viral genes (3), and they have beenanalyzed now in more detail to elucidate theirpossible role in the regulation of transcription(12). Thus, the identification of hypersensitiveregions may shed some light on possible regula-tory functions of such areas. This may be partic-ularly useful in the case of viral genomes whichare not very well studied, such as the group ofpapillomaviruses. The difficulty hampering theirinvestigation arises from the lack of a tissueculture system for their vegetative growth. Onlyvery recently have some data become availableconcerning their transcription patterns (2, 11),and sequence data are now either available (5, 7)or in the process of being completed. We havestarted to investigate the chromatin structure ofbovine papillomavirus type I (BPV-1) which is,for a number of reasons, an excellent model

system. As it is not feasible to isolate viralchromatin selectively from solid wart tissue (theonly permissive environment available), wehave resorted to BPV-1-transformed hamsterembryo fibroblasts (HEF) as a source of BPV-1chromatin. It has been established that BPV-1persists both in transformed cells and in equinesarcoid tumors as a free episome (1, 15, 16, 20,21). We had hoped, therefore, to obtain byselective isolation (31) BPV-1 chromatin in suffi-cient amounts for an analysis of its biophysicalproperties. As shown in this report, we haveisolated for the first time episomal viral chro-matin. Furthermore, by in situ application ofDNase I in isolated nuclei of BPV-1-transformedcells, we have identified one major hypersensi-tive region on the viral chromatin. In agreementwith the results concerning other papovaviruses,such as SV40 and polyoma, the hypersensitiveregion was confined to a position of the physicalmap where regulatory elements are located (5).

MATERIALS AND METHODS

Transformed cells. BPV-1-transformed HEF cells,the properties of which have been described recently(2), were used in passage 40 for the isolation of viralchromatin. The labeling of SV40 chromatin was per-formed as described (35).

Isolation and characterization of BPV-1 chromatin bysucrose density gradient centrifugation. Three daysafter seeding 2.5 x 106 cells per 14.5-cm petri dish, thenuclei were isolated as initially described by Su andDePamphilis (26) with some modifications (31). Five

567

568 ROSL, WALDECK, AND SAUER

petri dishes were harvested, and the chromatin was

purified by sedimentation through a sucrose densitygradient (10 to 30%, with a 2-ml 70% sucrose cushionfor 60 min at 40,000 rpm in an SW41 rotor at 0°C in a

Beckman centrifuge). Fifteen drops per fraction were

collected. To reveal the distribution of BPV-1 DNAsequences, the individual fractions were subjected toRNase treatment (pancreatic RNase, 50 p.g/ml; 15 minat 37°C) followed by proteinase K treatment (50 pg/ml;15 min at 37°C after adjustment of the reaction mixtureto 1% sodium dodecyl sulfate). Then, after adjustmentof the mixture to a final concentration of 20 ,ug ofsalmon sperm carrier DNA per ml and 0.3 M NaCl, theDNA was precipitated by addition of 2.5 volumes ofethanol overnight at -20°C.The precipitated DNA was suspended in 20 mM

Tris-hydrochloride (pH 7.5)-i mM EDTA for furthercharacterization after gel electrophoresis as describedbelow.

Agarose gel electrophoresis, blotting, and hybridiza-tion. After post-DNase I restriction analysis, the DNAwas analyzed by electrophoresis in horizontal 1 to1.4% agarose gels. Electrophoresis buffer was used as

described (27). The DNA was transferred from the gels(after treatment with 0.25 N HCI for 30 min beforedenaturation) to nitrocellulose filters (Schleicher &Schull) as described (25). The filters were preincuba-ted at 67°C in 5 times the concentration of Denhardtsolution (8). Hybridization with 32P-labeled nick-trans-lated (22) cloned BPV-1 DNA or BPV-1 Clal fragmentC DNA (cloned in pBR322) was carried out as de-scribed (14). After hybridization, the washing condi-tions for the filters were as follows: six times for 10min in 0.1% SDS in threefold-concentrated 0.15 MNaCl plus 0.015 M sodium citrate (SSC) supplementedwith Denhardt solution (8); 20 min in 0.1% SDS and0.1-fold-concentrated SSC; and three times for 10 minin threefold-concentrated SSC. All steps were carriedout at 60°C. The filters were dried and covered withKodak XAR-5 X-ray films with intensifying screens.

The recombinant DNA fragment Clal-C of the BPV-1genome was cloned in pBR322 (4) according to stan-dard procedures (3a).Mapping DNase I-hypersensitive regions. Nuclei

were isolated from BPV-1-transformed HEF cells (fivedishes with 2 x 107 cells each, 3 days after seeding)according to the method of Worcel et al. (34). The cellswere lysed by addition of the nonionic detergentNonidet P-40. The nuclei were suspended in 3 ml ofreticulocyte standard buffer as described by Wein-traub and Groudine (32). A 450-pI portion, whichserved as an untreated control, was withdrawn beforeincubation with DNase I and immediately lysed in 20mM EDTA-0.5% SDS. The remaining suspension (2.5ml) was incubated at 37°C and digested for increasingperiods of time with 2.5 pug of DNase I (WorthingtonDiagnostics) per ml. After 3, 5, 10, 15, and 30 min, 450-Rl portions were withdrawn and the DNase I digestionwas terminated by addition of 20 mM EDTA-0.5%SDS (final concentration). The lysate was then digest-ed for 2 h at 37°C with 100 p.g of proteinase K per ml.The total DNA was extracted with phenol-chloroformand precipitated with ethanol overnight at -20°C.After centrifugation, the DNA was dissolved in 200of 20 mM Tris-hydrochloride (pH 7.4)-l mM EDTAand digested for 1 h at 37°C with 50 p.g of pancreaticRNase per ml. After adjustment of the mixture to 0.5%

SDS and subsequent digestion with proteinase K (50p.g/ml, 2 h at 37°C), the total DNA was again extractedwith phenol-chloroform and precipitated with ethanol.The pellet was dissolved in 50 p.l of 20 mM Tris-hydrochloride (pH 7.4)-l mM EDTA and digestedwith various restriction enzymes.

RESULTS

Isolation and sucrose density gradient centrifu-gation of BPV-1 chromatin. An established lineof BPV-1-transformed HEF cells, which hasbeen described recently (2), was found to be asuitable source for viral chromatin, as an assess-ment of their BPV-1 DNA content had revealedthe presence of 200 to 250 viral genome equiva-lents per cell (data not shown). The BPV-1chromatin was selectively eluted from purifiednuclei according to the modified procedure of Suand DePamphilis (26) as has been detailed else-where (29, 31). As a marker, in vivo-labeledSV40 chromatin was also selectively isolated,mixed with the nuclear eluate from BPV-1-transformed HEF cells, and sedimented into asucrose density gradient. After fractionation,portions were electrophoresed into an agarosegel and blotted to nitrocellulose, and BPV-1DNA sequences were revealed by autoradiogra-phy after hybridization with nick-translatedcloned BPV-1 DNA (Fig. 1, lower part). Theautoradiograph shows in fraction 6 a strongpreponderance of fast-sedimenting BPV-1 nu-cleoprotein structures which contain mainly Fo Iand Fo II. This may perhaps be due to aggrega-tion of chromatin structures. In addition, somelarger forms of BPV-1 DNA were found. Thelatter can be converted to unit-length Fo III bycleavage with single-cut restriction endonucle-ases (data not shown) and, hence, representoligomers which are known to be present intransformed cells (16). The actual structure andcomposition of the fast-sedimenting material,however, is not known and awaits further eluci-dation. The bulk of the mature BPV-1 chromatinis contained in fraction 18 of the gradient, with ashoulder toward the faster-sedimenting fraction14. The size of the BPV-1 chromatin can bedirectly compared with the cosedimenting SV40chromatin in the upper part of Fig. 1. The curvedisplaying the typical sedimentation pattern ofSV40 chromatin (30) is superimposed upon thecurve representing an estimation of the relativeamounts of BPV-1 DNA sequences that werepresent in each fraction of the same gradient.The estimation of the minute amounts of BPV-1genome equivalents was made by densitometryof replicas from the autoradiographs (lower partof Fig. 1) and by their comparison with densi-tometries of autoradiographs made from appro-priate reconstruction experiments in which vari-ous amounts of authentic BPV-1 DNA were

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PAPILLOMAVIRUS CHROMATIN 569

9cq

0

o

UCL 6C)

z

I 3

I0

1 5 9 13 17 21 25

FRACTION NUMBER

0.6 p

I(5D

0.4

z

Q2C

B -OLIGO.

_-- - -FO E

a* E s,z,_-FOI

1 5 9 13 17 21 25

FRACTION NUMBER

FIG. 1. Sedimentation profile of BPV-1 chromatin. BPV-1 chromatin was eluted from isolated BPV-1-transformed HEF nuclei and sedimented together with 3H-labeled SV40 chromatin into a sucrose densitygradient. The direction of sedimentation was from right to left. Alternate fractions were used either for (A)determination of the radioactivity by liquid scintillation counting (solid line) or for (B) agarose gel electrophoresisand autoradiography after blotting and hybridization with 32P-labeled BPV-1 DNA. The relative amount of BPV-1 DNA (Fo I plus Fo II) sequences was estimated by densitometry of the autoradiograph (A, dotted line).

employed. In evaluating the accuracy of suchestimations, it should be borne in mind that weare dealing here only with traces of papilloma-virus DNA. Given a certain inaccuracy, thecurve which emerged from the above-describedestimation does, nevertheless, comply with theexpected sedimentation behavior of BPV-1chromatin, which, owing to the larger size of itsDNA, is supposed to sediment slightly fasterthan the smaller SV40 chromatin. This is, in-deed, the case. The faster sedimenting shoulderof the BPV-1 chromatin probably represents thereplicating structures. Previrions (30), as in thecase of SV40 (fraction 11), are, of course, miss-ing, since capsid proteins fail to be expressed inBPV-1-transformed cells. A first attempt to sub-ject episomal BPV-1 chromatin to electron mi-croscopic analysis is shown in Fig. 2. For thispurpose, from a separate gradient in which SV40marker chromatin was omitted, fractions 14 to18, comprising the bulk of the chromatin, were

pooled and analyzed. Figure 2 shows two chro-matin structures in which the DNA strands areabutted with nucleosomes. We believe that ei-ther two chromatin structures happen to besuperimposed on each other, or, what appears tobe even more likely, that replicating BPV-1chromatin is depicted here. Further efforts willhave to be made to resolve individual BPV-1chromatin structures.

Identification of a DNase-hypersensitive regionin BPV-1 chromatin. The nuclei from BPV-1-transformed HEF cells were isolated and sub-jected to DNase I treatment in situ as described(6) to reveal putative hypersensitive regions orsites on the BPV-1 chromatin. For the establish-ment of the kinetics of the reaction, a givenamount of DNase was reacted for various peri-ods of time with the nuclei. The effect of thenuclease treatment on the viral chromatin wasthen monitored by digestion of the isolated DNAwith single-cut restriction endonuclease fol-

VOL. 46, 1983


qL f7-9i-*^^ ,t.w\>s +t0 t .

chromtin)werepooldandexamned.Magnficaton, 80,00. Te bar rerset I. ,um

Southernblotsahor p.(Fig. 3e).. Hwe with*~~J4. **'~~,4 . * 6

Vq. 6 I 4-0~~~~~.

. .. ~~. G.6

p t:~~

FIG.s2.teleto micrographe oBPV-1 chromatin. BPnchromasigtinmwa purfieicbytonsucos denitogadientve

EcoRI was used as a single-cut endonuclease (a bands were accumulated which were either 4.8physical map is drawn on top of Fig. 3), the bulk (60%o) or 3.1 (38.8%) kilobases (kb) in size. Asof the viral DNA, which consisted mainly of Fo I the sum equals the size of BPV-1 Fo III, thisand Fo II (not shown), was converted to linear shows that part of the BPV-1 chromatin hadFo III, regardless of whether the incubation with been cleaved open by the DNase treatment,

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Hpa I EcoR I

0.00 0.27

a b c d e

Hpa

1.0(

f g h

FIG. 3. Detection of a DNase-hypergion in BPV-1 chromatin by post-DNawanalysis with single-cut EcoRI endonuclelated BPV-1-transformed HEF nucleiwith DNase I for (min): a, 3; b, 5; c, 10; d0; g, 30 (incubation with 20 mM EDTA). LDNA; i, virion DNA digested with EccDNA digested with TaqI. A 5-,g sampleloaded in each track before electrophotrack g, 7 ,ug); the arrows point out theThe sizes of the fragments are indicatedwere determined with calibration curvconstructed with the TaqI fragments (j).

ment of the BPV-1 genome as a nick-translatedprobe. The ClaI-C fragment was chosen, whichcovers the hypersensitive region extendingsomewhat beyond either end (see scheme in Fig.

° 5), because it might, in further experiments, beuseful for the assessment of the biological activi-ties of this area of the genome. The two single-cut endonucleases EcoRI and BamHI were usedtogether. In agreement with the previous results,the appearance of two novel bands which werealmost equal in size (2.9 and 2.75 kb) can be seen

*4,8 Kb in Fig. 5a through d. The sum of their molecular--3.8 Kb weights, when added to the weight of EcoRI-

3.1 Kb BamHI fragment B, approximates the molecularweight of the BPV-1 genome, and, hence, con-firms the localization of the DNase hypersensi-tive region. The incubation time of 30 min withDNase (Fig. Se) turned out in this experiment tocause a generalized hydrolysis of the DNA. Also

-1.4 Kb contained in Fig. 5 is the evidence for the purityof the cloned ClaI-C fragment, which failed, asexpected, to hybridize with the EcoRI-BamHI

rsensitive re- fragment B (Fig. 5a through e). This fragmentse restriction was revealed when the complete BPV-1 DNA-ase. The iso- was used as a labeled probe (Fig. 5h, lowerwere treated band).i, 15; e, 30; f,_ane h, virion DISCUSSION9RI; j, virionof DNA was The regulation of transcription of eucaryoticoresis (except genes has been studied extensively in recentnovel bands.in kb. They

es that were Hpa I

0.00

owing to the presence of a hypersensitive re-gion. The controls show that Fo III is a prioriabsent in the nuclei both before DNase treat-ment (Fig. 3f) and after 30 min of incubation inthe presence of EDTA (Fig. 3g). To determinethe polarity of the hypersensitive region on thephysical map, another single-cut endonuclease,BamHI, which cleaves BPV-1 DNA at the rela-tive position 0.56, was used for the post-DNaserestriction analysis (Fig. 4). Again, as wasshown already in Fig. 3, the kinetics of accumu-lation of novel bands can be observed (Fig. 4athrough e). Their sizes, which were evaluatedwith the help of the internal markers (lanes h andi), were, on the average, 5.1 (63.8%) and 2.8(35%) kb, respectively, revealing, when takentogether, the size of Fo III. This allows us tolocalize the DNase-hypersensitive region ap-proximately between the relative physical mappositions 0.88 and 0.92. The extension of theregion was estimated from the width of the novelbands. As an additional control experiment apost-DNase double-digestion analysis was per-formed with a small molecularly cloned frag-

BamH I

0.56

Hpa I-11.00

a b c d e f g h

*& -E1

-*--mi--IIQ4 5,1 Kb

_ -3,8 Kb

4 42,8 Kb

_ -1,4 Kb

FIG. 4. Detection of a DNase-hypersensitive re-gion in BPV-1 chromatin by post-DNase restrictionanalysis with single-cut BamHI endonuclease. Lanes athrough e, treatment of the isolated nuclei withDNAase for 3, 5, 10, 15, and 30 min, respectively.Lane f, 0 min; g, virion DNA; h, virion DNA cleavedwith BamHI; i, virion DNA cleaved with TaqI. Forfurther details, see the legend to Fig. 3.

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:' A \-

/

a~~~~~~~~~~~~~~~~~~~~bcd e

a a a _n -mII

am - -EcoR I + BamH I A5.7Kbr 2.9 Kb~- 2.75 Kb

- - EcoR I + BamH I B 2.3 Kb

FIG. 5. Mapping a DNase-hypersensitive region by post-DNase I double-digestion with EcoRl-BamHI andprobing with the cloned BPV-1 DNA fragment ClaI-C. The isolated nuclei, lanes a through e, were digested withDNase for 3, 5, 10, 15, and 30 min, respectively. Lane f, 0 min; g, 0 min (digested only with EcoRI); h, double di-gest for lanes a through f, but probed with BPV-1 DNA sequences. For further details see the legend to Fig. 3.The scheme depicts the cleavage pattern, the position of the nick-translated ClaI-C fragment (bold line), and theapproximate position of the DNase-hypersensitive region (empty boxed region of the physical map).

years. Control elements residing in particularand often strongly conserved DNA sequenceshave been detected, which determine either thecorrect initiation of transcription or the efficien-cy with which RNA sequences are copied (18).In particular, it was found that cis-acting se-quences, which are crucial for the efficiency oftranscription, are located upstream from the-position where transcription initiation takesplace (3, 9, 17, 19). This area, 50 to 400 nucleo-tides upstream from the TATA sequence, coin-cides with a region in which the chromatin ofactively transcribing genes is particularly vul-nerable to both endogeneous and other DNases(24, 28, 29, 32). It appears that this area is devoidof nucleosomes (13, 23), and hence may repre-sent a regulatory element which could be used asan entry point by the RNA polymerase (17). Inview of the evidence which has been accumulat-ed regarding the correlation between DNase I-hypersensitive regions and transcriptional con-trol signals (10), the hypersensitive region in theBPV-1 chromatin described in this area of thegenome might reflect the presence of such ele-ments in its vicinity. The availability of completesequence data of the BPV-1 DNA permits theestablishment of a correlation between theDNase-hypersensitive region and the presence

of two TATAAA promoter elements which arelocated at bases 7,108 through 7,113 and 58through 63 (5), corresponding to the relativephysical map units 0.89 to 0.90 and 0.007 to0.008 in Fig. 6. Whether only one of bothtranscription initiation signals is being used orwhether both can be utilized is presently notknown. Furthermore, more accurate data con-

0.92

FIG. 6. Map of the DNase I-hypersensitive regionin the BPV-1 genome.

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cerning, for example, the precise positions of the5' ends of the early RNA species are needed todecide between these different possibilities.The detection of DNase-I hypersensitive re-

gions is subject to problems arising from overex-

posure of the chromatin to DNase I, wherebyafter prolonged digestion, less sensitive regionsmay be cleaved, and, ultimately, nucleosomespacer regions will be subjected in increasingnumbers to hydrolysis. Under the reaction con-

ditions chosen in our experiments, i.e., up to 30min, a moiety comprising 30% of the chromatinwas cleaved open. Although further linearizedchromatin structures were noted upon pro-

longed digestion, the background was increasedat the same time to the extent that a preciseevaluation of the data was no longer possible.Some minor bands such as the 4.0- and 6.2-kbbands in Fig. 3 and 4 may well be derived from a

less sensitive region, which can be tentativelyconfined to the relative physical map positions0.37 to 0.39. Transcriptional control elementsare missing in this region of the genome (5). Weare currently attempting to characterize thestructure of purified BPV-1 chromatin in greaterdetail with the help of restriction endonucleases.

ACKNOWLEDGMENTS

We thank H. Zentgraf for providing the electron micrographand E. Amtmann for the labeled BPV-1 DNA probe. Thetechnical assistance of M. Theobald is appreciated.

LITERATURE CITED

1. Amtmann, E., H. Muller, and G. Sauer. 1980. Equineconnective tissue tumors contain unintegrated bovinepapilloma virus DNA. J. Virol. 35:962-964.

2. Amtmann, E., and G. Sauer. 1982. Bovine papilloma virustranscription: polyadenylated RNA species and assess-

ment of the direction of transcription. J. Virol. 43:59-66.3. Benoist, C., and P. Chambon. 1981. In vivo sequence

requirements of the SV 40 early promotor region. Nature(London) 290:304-310.

3a.Bolivar, F., and K. Backman. 1979. Plasmids of Esciherich-ia coli as cloning vectors. Methods Enzymol. 68:245-267.

4. Bolivar, F., R. L. Rodriquez, P. J. Greene, M. C. Betlach,H. L. Heynecker, H. W. Boyer, I. H. Crosa, and S. Falkow.1977. Construction and characterization of a new cloningvehicle II. A multipurpose cloning system. Gene 2:95-113.

5. Chen, E. Y., P. M. Howley, A. D. Levinson, and P. H.Seeburg. 1982. The primary structure and genetic organi-zation of the bovine papillomavirus type 1 genome. Na-ture (London) 299:529-534.

6. Cremisi., C. 1981. The appearance of DNAase I hypersen-sitive sites at the 5' end of the late SV 40 genes iscorrelated with the transcriptional switch. Nucleic AcidsRes. 9:5949-5964.

7. Danos, O., M. Katinka, and M. Yaniv. 1982. Humanpapillomavirus la complete DNA sequence: a novel typeof genome organization among Papovaviridae. EMBO(Eur. Mol. Biol. Organ.) J. 1:231-236.

8. Denhardt, D. 1966. A membrane-filter technique for thedetection of complementary DNA. Biochem. Biophys.Res. Commun. 23:641-646.

9. Dierks, P., A. van Oyen, N. Mantei, and C. Weissman.1981. DNA sequences preceding the rabbit ,3-globin gene

are required for formation in mouse L-cells of ,B-globin

RNA with the correct 5'-terminus. Proc. Natl. Acad. Sci.U.S.A. 78:1411-1415.

10. Elgin, S. C. R. 1981. DNAase I-hypersensitive sites ofchromatin. Cell 27:413-415.

11. Heilman, C. A., L. Engel, D. R. Lowy, and P. M. Howley.1982. Virus-specific transcription in bovine papilloma-virus-transformed mouse cells. Virology 119:22-34.

12. Herbomel, P., S. Saragosti, D. Blangy, and M. Yaniv.1981. Fine structure of the origin-proximal DNAase I-

hypersensitive region in wild-type and EC mutant poly-oma. Cell 25:651-658.

13. Jakobovits, E. B., S. Bratosin, and Y. Aloni. 1980. Anucleosome-free region in SV 40 minichromosomes. Na-ture (London) 285:263-265.

14. Krieg, P., E. Amtmann, D. Jonas, H. Fischer, K. Zang,and G. Sauer. 1981. Episomal simian virus 40 genomes inhuman brain tumors. Proc. Natl. Acad. Sci. U.S.A.78:6446-6450.

15. Lancaster, W. D. 1981. Apparent lack of integration ofbovine papillomavirus DNA in virus-induced equine andbovine tumors and virus-transformed mouse cells. Virolo-gy 108:251-255.

16. Law, M. F., D. R. Lowy, I. Dworetzky, and P. M. Howley.1981. Mouse cells transformed by bovine papillomaviruscontain only extrachromosomal viral DNA sequences.Proc. Natl. Acad. Sci. U.S.A. 78:2727-2731.

17. McKnight, S. L., E. R. Gravis, R. Axel, and R. Kingsbury.1981. Analysis of transcriptional regulatory signals of theHSV thymidine kinase gene: identification of an upstreamcontrol region. Cell 25:385-398.

18. McKnight, S. L., and R. Kingsbury. 1982. Transcriptionalcontrol signals of a eucaryotic protein-coding gene. Sci-ence 217:316-324.

19. Mellon, P., V. Parker, Y. Gluzman, and T. Maniatis. 1981.Identification of DNA sequences required for transcrip-tion of the human et 1-globin gene in a new SV 40 host-vector system. Cell 27:279-288.

20. Moar, M. H., M. S. Campo, H. M. Laird, and W. F. H.Jarrett. 1981. Unintegrated viral DNA sequences in a

hamster tumor induced by bovine papilloma virus. J.Virol. 39:945-949.

21. Pfister, H., B. Fink, and C. Thomas. 1981. Extrachromo-somal bovine papillomavirus type 1 DNA in hamsterfibromas and fibrosarcomas. Virology 115:414-418.

22. Rigby, P., D. Rhodes, M. Dieckmann, and P. Berg. 1977.Labeling deoxyribonucleic acid to high specific activity invitro by nick-translation with DNA polymerase 1. J. Mol.Biol. 113:237-251.

23. Saragosti, S., G. Moyne, and M. Yaniv. 1980. Absence ofnucleosomes in a fraction of SV 40 chromatin between theorigin of replication and the region coding for the lateleader RNA. Cell 20:65-73.

24. Scott, W. A., and D. J. Wigmore. 1978. Sites in simianvirus 40 chromatin are preferentially cleaved by endonu-clease. Cell 15:1511-1518.

25. Southern, E. M. 1975. Detection of specific sequencesamong DNA fragments separated by gel electrophoresis.J. Mol. Biol. 98:503-517.

26. Su, R. T., and M. L. DePamphilis. 1976. In vitro replica-tion of simian virus 40 DNA in a nucleoprotein complex.Proc. Natl. Acad. Sci. U.S.A. 73:3466-3470.

27. Tegtmeyer, P., and F. Macasaet. 1972. Simian virus 40deoxyribonucleic acid synthesis: analysis by gel electro-phoresis. J. Virol. 10:599-604.

28. Varshavsky, A. J., 0. Sundin, and M. Bohn. 1979. Astretch of late SV 40 viral DNA about 400 bp long whichincludes the origin of replication is specifically exposed inSV 40 minichromosomes. Cell 16:453-466.

29. Waldeck, W., B. Fohring, K. Chowdhury, P. Gruss, andG. Sauer. 1978. Origin of DNA replication in papovaviruschromatin is recognized by endogenous endonuclease.Proc. Natl. Acad. Sci. U.S.A. 75:5964-5968.

30. Waldeck, W., and G. Sauer. 1981. Papovavirus chromatinassociated cellular endonuclease which introduces onedouble-strand cut in superhelical deoxyribonucleic acid.

VOL. 46, 1983


Biochemistry 20:4203-4209.31. Waldeck, W., U. Spaeren, G. Mastromei, R. Eliasson, and

P. Reichard. 1979. Replication of polyoma DNA in nucle-ar extracts and nucleoprotein complexes. J. Mol. Biol.135:675-689.

32. Weintraub, H., and M. Groudine. 1976. Chromosomalsubunits in active genes have an altered conformation.Science 193:848-856.

33. Weisbrod, S. 1982. Active chromatin. Nature (London)297:289-295.

34. Worcel, A., S. Han, and H. L. Wong. 1978. Assembly ofnewly replicated chromatin. Cell 15:969-977.

35. Zenke, M., and G. Sauer. 1982. Spliced and unsplicedvirus specific RNA sequences are associated with purifiedsimian virus 40 chromatin. Nucleic Acids Res. 10:4543-4550.

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Documents

Isolation of episomal bovine papillomavirus chromatin and