Vol. 43, No. 2JOURNAL OF VIROLOGY, Aug. 1982, p. 427-4350022-538X/82/080427-09$02.00/0

Specifically Unmethylated Cytidylic-Guanylate Sites inHerpesvirus saimiri DNA in Tumor Cells

RONALD C. DESROSIERSNew England Regional Primate Research Center, Harvard Medical School, Southboro, Massachusetts 01772

Received 4 January 1982/Accepted 8 April 1982

The restriction endonucleases MspI (CCGG), HpaII (CCGG), FnuDII (CGCG),and HaeIII (GGCC) were used to study the methylation of Herpesvirus saimiriDNA in tumor cells taken directly from tumor-bearing animals. No evidence wasfound for methylation of the 5' terminal C in the sequence CCGG or of the internalC in the sequence GGCC, but extensive methylation of CG was detected. FifteenHpaII sites and 17 FnuDII sites were detected in the unique DNA region of the H.saimiri strain used. Twenty-eight of the 32 sites were methylated in greater than90o of the viral DNA molecules in tumor cells, but the remaining 4 sites wereunmethylated in greater than 95% of the viral DNA molecules in tumor cells. Thelocations of the four specifically unmethylated sites were mapped and appeared tobe identical in the four different induced leukemias examined (one owl monkeyand three white-lipped marmosets). The nonproducer 1670 tumor cell line, incontinuous passage for over 7 years, contained four similar specifically unmethyl-ated sites. Possibilities for the physiological significance of the unmethylated sitesare discussed.

Herpesvirus saimiri naturally infects squirrelmonkeys (Saimiri sciureus), in which it causesno apparent disease. Infection of other speciesof New World primates, however, frequentlyresults in lymphoma or leukemia or both. Infec-tious virion DNA contains a high degree ofintramolecular heterogeneity in guanine plus cy-tosine (G+C) content. The centrally located,unique DNA region is 115 kilobase pairs (kb) inlength, includes the vast majority of codingsequences, and is 36% G+C (light DNA [L-DNA]). L-DNA has covalently attached at eachend repetitive DNA with 71% G+C content(heavy DNA [H-DNA]). Each repeat unit of 1.3kb is oriented in the same direction, and approx-imately 30%o of an infectious virion DNA mole-cule is H-DNA (for review, see reference 9 andB. Fleckenstein and R. Desrosiers, In B. Roiz-man (ed.), The Herpesviruses, in press). Thestructure of infectious virion DNA (M-DNA) isshown schematically in Fig. 1.5-Methylcytosine (m5C) is the only modified

residue that has been detected in mammaliancell DNA; m5C occurs by far most frequently inthe following palindromic sequence:

5' CG 3'3' GC 5'

CG is the least common of the 16 dinucleotides,and various estimates have indicated that 50 to90%6 of CG dinucleotides in mammalian cellDNA are methylated (3, 12).

Three lines of evidence indicate that H. sai-miri virion DNA produced from lytic infection ofcultured owl monkey kidney (OMK) cells isunmethylated or nearly so. (i) After hydrolysis,the nucleosides present in unlabeled, purified H.saimiri virion DNA have been analyzed by high-performance liquid chromatography (R. Desro-siers, J. Gehrke, and M. Ehrlich, unpublisheddata); m5C was below detectable levels, at least30 times less than the level in host cell (OMK)DNA. This is less than 20 methyl groups per L-DNA region. (ii) Acken et al. have labeled H.saimiri virion DNA with radioactive precursorsand were also unable to detect either m5C orm6A (1). Their reported limits of detection indi-cate less than one methyl group per virion M-DNA molecule. (iii) Virion L- and H-DNA di-gested with HpaII produced patterns that wereindistinguishable from those of MspI (6). Thesequence specificities of HpaII and MspI areshown in Table 1. Greater than 95% of theCCGG sites in virion DNA appear to be by thiscriterion unmethylated; this represents less thanone methylated CCGG per L-DNA region andless than 10 per H-DNA region.

Cell lines have been established from H. sai-miri-induced tumors, and these have been usedto study the persistence of the viral DNA. ViralDNA in cells of nonproducing lines has beenpreviously reported to be extensively methylat-ed. Since virus gene expression in these tumorcell lines is severely limited and since the level

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428 DESROSIERS

KILOBASE PAIRS

I I I I I I0 10 20 30 40 506070 80 90 100 120 130 140 150

H-DNA L-DNA H-DNA

FIG. 1. Arrangement of unique L-DNA (36%G+C) and repetitive H-DNA (71% G+C) in infectiousH. saimiri DNA. Each repeat unit in H-DNA is 1.3kbp (2).

TABLE 1. Sequence specificities of restrictionendonucleases HpaII and Mspla

Sequence HpaII MspI

CCGG + +CMGG - +MCGG + -MMGG - -

a The ability or inability to cleave the indicatedsequence is noted by a + or -, respectively; M, 5-methylcytosine.

of viral DNA methylation is greatest in cell linesnot producing virus, it was suggested that per-haps the methylation of viral DNA in tumor cellsplays some role in limiting gene expression (6).The methylation status of a number of viral andcellular genes has since been analyzed, and themethylation of at least certain CG sites generallycorrelates inversely with expression in RNA (forreview, see references 7 and 19). In this report,analyses are extended to H. saimiri DNA intumor cells taken directly from tumor-bearinganimals, and a few CG sites of many (4 of 32 sofar examined) are described that specificallyremain unmethylated in viral L-DNA in tumorcells.

MATERIALS AND METHODS

Cells and virus. The propagation of H. saimiri inOMK cells (line 637, passages 10 through 30) andpurification of virus have been described previously(2). The 1670 cell line, originally established from anH. saimiri-induced marmoset tumor, was grown inRPMI 1640 medium containing 10% fetal calf serum (5,16). Tumor cells were obtained directly from peripher-al blood of animals with leukemia induced by H.saimiri strain S295C. Blood was drawn 3 to 14 daysbefore death in the four different animals used in thisstudy. The four animals included one owl monkey(Aotus trivirgatus; death at 12 months) and threewhite-lipped marmosets (Saguinusfuscicollis-nigricol-lis; death at 2 to 3 months). Leukocyte counts at thetime DNA was prepared ranged from 20,000 to 200,000per mm3. Tumor cells were prepared by banding overFicoll-Hypaque (density = 1.076 g/cm3).

Analysis of DNA. Purified virion DNAs and tumorcell DNAs were prepared as described previously (2,5). Aliquots containing 2 ,ug of DNA were digestedwith restriction endonucleases (New England Bio-labs), as indicated in each figure, and electrophoresedthrough a 0.6% agarose gel. Control digestions ofvirion DNA contained 1 to 3 ng of the virion DNA plus2 jig of OMK cell DNA. Digestions were performedfor 4 to 20 h, with at least twice the amount of enzymethan was required to digest the DNA to detectablecompletion in 15 min. DNA fragments in agarose gelswere transferred to nitrocellulose by the procedure ofSouthern (20), with acid nicking of the DNA in the gelbefore denaturation (5). Nicking of the DNA allowsquantitative transfer even of high-molecular-weightDNA fragments. Transferred DNA was hybridized

with virion L-DNA labeled with [32P]dTI P in vitro bynick repair, rinsed extensively, and placed over KodakX-Omat film for autoradiography (5). In the experi-ments shown in Table 2, lambda DNA (0.5 ,ug) wasalso included with each DNA sample for digestion;subsequent hybridization to 32P-labeled lambda DNAdemonstrated the completeness of each digestion. Thefragment used for hybridization (see Fig. 4) was puri-fied by electroelution from an agarose gel.Tumor cells taken directly from peripheral blood of

leukemic animals contained 50 to 300 viral genomeequivalents per cell; this is similar to the numbercontained in most continuous tumor cell lines, butmore than that found in lymph nodes or spleens ofanimals with diffuse lymphoma (10, 14). Lymph nodesand spleens of tumor-bearing animals undoubtedlycontain a significant proportion of nontumor, virus-free cells. Peripheral leukocytes, after leukemia wasinduced by H. saimiri S295C, provided a rich andconvenient source of essentially pure tumor cells.

RESULTSDegree of methylation. Methylation of CCGG

sites was examined in H. saimiri DNA of sixtumor cell lines and four fresh tumor samples byusing HpaII and MspI. H. saimiri L-DNA in

TABLE 2. Degree of methylation of H. saimiriDNA in tumor cells

Degree of methyl-Origin of cellsa Producer vs ationcnonproducerb L-DNA H-DNA

1670 NP 1 170N2 NP 1 21926 P 2 477/5 P 3 4H1591 P 2 4MLC-1 P 3 4Owl monkey tumor P 1 4

a Listed here are five tumor cell lines, an in vitrotransformed cell line (H1591), and tumor cells directfrom a tumor-bearing owl monkey.

b Ability to produce virus when 106 cells werecocultivated with permissive OMK cells. NP, Nonpro-ducer; P, producer.

c 1, >85% of CG sites methylated; 2, 50 to 85% ofCG sites methylated; 3, 15 to 50% of CG sites methyl-ated; and 4, <15% of CG sites methylated.

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UNMETHYLATED SITES IN H. SAIMIRI DNA 429

1 2 3 4 5 6 7 8 9 10

'4

FIG. 2. Degree of methylation of H. saimiri L-

DNA in tumor cells. DNA from tumor cells, tumor cell

lines, and virions was digested with restriction endo-

nuclease, electrophoresed overnight through a 0.6%

agarose gel, transferred to nitrocellulose, and hybrid-ized to 32P-labeled L-DNA of strain 11. Slot 1, strain

11 virion (Hpall); slot 2, 1926 cell line (HpaII); slot 3,

1926 cell line (MspI); slot 4, 1670 cell line (Hpall); slot

5, 1670 cell line (MspI); slot 6, 70N2 cell line (Hpall);slot 7, 70N2 cell line (Mspl); slot 8, strain S295C virion

(HpaII); slot 9, strain S295C-induced tumor (HpaII);and slot 10, strain S295C-induced tumor (Mspl).

cells taken directly from a leukemic animal

yielded the same MspI fragments found in the

parental strain S295C virion DNA (compare

slots 8 and 10 in Fig. 2). The relative intensity of

fragments also did not differ detectably. Thus,little if any methylation of the 5' terminal C in

the sequence CCGG occurred in H. saimiri L-

DNA in tumor cells. Similarly, the H. saimiri L-

DNA fragments produced from HaeIlI digestionof virion DNA and tumor DNA were indistin-

guishable (data not shown); this indicates little

or no methylation of the internal C in the se-

quence GGCC (15). Very little digestion of H.

saimiri L-DNA contained within the tumor

DNA samples occurred with HpaII; most of the

hybridizing sequences were contained in materi-

al greater than 40 kb (Fig. 2, slot 9). This is

indicative of extensive methylation of the inter-nal C in the sequence CCGG. Note that aspecific HpaII L-DNA fragment of 17.5 kb waspresent in tumor DNA that did not comigratewith any parental virion DNA HpaII fragment(upper arrow in slot 9 of Fig. 2). Such a fragmentcould arise from noncontiguous CCGG sites thatwere specifically not methylated; additional ex-periments presented below confirm this inter-pretation. Analyses of H. saimiri DNA con-tained within the three other tumor samplesyielded results indistinguishable from thoseshown in Fig. 2 (slots 9 and 10), including thesize and intensity of the specific 17.5-kb HpaIIfragment.Methylation analyses of H. saimiri DNA con-

tained within tumor cell lines were slightly morecomplicated due to the deletions, rearrange-ments, and duplications that are sometimes ob-served in these continuously passaged cells (5).H. saimiri L-DNA in the producing cell line 1926(derived from an H. saimiri strain 11-inducedtumor) yielded the same MspI fragments thatwere found in the parental virion DNA (compareslots 1 and 3 in Fig. 2). The intensity of some ofthe L-DNA MspI fragments in 1926 cells differedfrom virion DNA; this more than likely was dueto duplication of some regions of the genome orpopulations of defective DNA molecules thatarose through continued passage of the cell line.HpaII digestion products ofL-DNA in 1926 cellswere very heterogeneous in size, with most L-DNA still of high molecular weight (Fig. 2, slot2). Digestion of DNA from other producing celllines with HpaII and MspI indicated similarlevels of L-DNA methylation (Table 2).H. saimiri L-DNA in the nonproducing tumor

cell lines 1670 and 70N2 yielded some of theMspI fragments that were found in the parentalvirion DNA (strain 11), but others were missing(compare slots 5 and 7 with slot 1 in Fig. 2).Previous work has demonstrated a 21-kb dele-tion in at least the majority of H. saimiri DNAmolecules in 1670 and 70N2 cells (5, 23). Sincewe do not yet have a map of virion DNA MspIfragments, we do not know whether the missingMspI fragments are in fact derived from theregion known to be deleted in 1670 and 70N2cells, but the size of missing and new fragmentsis sufficient to be accounted for by the known21-kb deletion. HpaII digestion of 1670 and70N2 cell DNA revealed the presence of high-molecular-weight H. saimiri HpaII L-DNA frag-ments and also specific H. saimiri L-DNAHpaII fragments that did not comigrate with anyvirion DNA HpaII fragments and that did notcomigrate with any L-DNA MspI fragmentsfound in 1670 or 70N2 cells (Fig. 2, slots 1, 4,and 6). Again, evidence presented below con-firms that the specific HpaII fragments arise

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430 DESROSIERS

TUiMOR DNA

0.f1 a1I1VI-,KB

17.5"16.8 .-

4.7 --

TUMOR DNAx

EcoRI +

E| Cj

0-1MI mi!.Ii l Li

KB

!(A)26 -

22 - 40

( a3) 18,7 -iAh M-M.17.5

(Ol/D) 15.2

TUMOR DNAx

KpnI +

1 ¢I=kIiKB

,/Al\7 {)

E725

V:I7 SIv --

(E)l1.6 - 46 _ 4W_

7.5 - m7,2 -- U7.0 -

V.P. 6.4 -

,"C/D)90 W 0*(F)8.6 -

(G)7.7 - :.- *

5 4 w

FIG. 3. Specifically unmethylated sites in tumor L-DNA. DNA was digested with restriction endonucle-ase(s), electrophoresed for 40 h through a 0.6% agarose gel, transferred to nitrocellulose, and hybridized to 32p_labeled L-DNA of strain S295C. A composite photo of autoradiograms from two different gels is shown. Therestriction endonuclease SmaI cleaves three times in each repeat unit of strain S295C H-DNA, but does notcleave in L-DNA (C. Mulder and B. Fleckenstein, unpublished data). Since EcoRI, KpnI, and BamHI cleaveonly in L-DNA, additional SmaI digestion was used to reveal L-DNA terminal fragments as specific bands onthese gels (EcoRI fragments A and J, Kpn fragments B and C, and BamHI fragments A and E).

from noncontiguous CCGG sites that are specifi-cally not methylated.From the degree of digestion with HpaII, the

overall degree of CG methylation among CCGGsites was estimated (Table 2).

Virus was recovered from the producing celllines 1926 and 77/5 by cocultivation with OMKcells, and purified virion DNA was analyzed byHpaII and MspI digestion. This virion DNA wasnot detectably methylated.Mapping of specifically unmethylated CG sites

in tumor cells. The specific H. saimiri L-DNAHpaII fragments observed in DNA from freshtumor cells and from nonproducing tumor celllines (Fig. 2, slots 4, 6, and 9) suggested thatspecific CG sites were not methylated apprecia-bly. It seemed necessary to confirm this inter-pretation and to map the location of specifically

unmethylated sites. To achieve these goals, dou-ble digestion with HpaII plus a restriction endo-nuclease whose cleavage sites in L-DNA areknown was used.For the analysis of methylated and unmethyl-

ated sites in L-DNA, it was desirable not to beconfined to CCGG sites so other enzymes inaddition to HpaII were used. Methylation-sensi-tive restriction endonucleases with a six-nucleo-tide recognition sequence such as SmaI, SacII,or SaiI cleave H. saimiri L-DNA too infrequent-ly (0 to 3 times) for a detailed analysis. HhaI(GCGC) does not cleave at GMGC (where M =

m5C) (15), and results with this restriction endo-nuclease are reported below. H. saimiri DNAafter stable association with a tumor cell wasfound to resist digestion by FnuDII (CGCG)(Fig. 3). The inability to cleave most FnuDII

S295CVIRION DNA

1..

SE I I I i L

5 x ..

7,2 - "aM>.6.4 -

54

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UNMETHYLATED SITES IN H. SAIMIRI DNA 431

TABLE 3. Summary of H. saimiri S295C L-DNA fragments found in tumors

Enzyme(s) Fragments' Fragments

HpalI >40, 17.5, 5.4 hetEcoRl A, B, C, D, E, F, G, H, I, J, KEcoRI-HpaII B, C, D, E, F, G, H, I, J, K 17.5, 5.4 het, 2.5KpnI A, B, C, DKpnI-HpaII A, C, D 17.5, 5.4 het, 5.0BamHI A, B, C, D, E, FBamHI-HpaII B, C, D, E, F 17.5, 11.8, 5.4 hetFnuDII >40, 3.6 hetEcoRI-FnuDII B, C, D, E, F, H, I, J, K 22, 4.2, 3.4, 3.6 hetKpnI-FnuDII C, D -40, 33, 26, 3.6 het

a Letters refer to the EcoRI, KpnI, or BamHI fragments as shown in Fig. 6. The approximate sizes offragments (in kb) are as follows: EcoRI-A, 26; EcoRI-G, 7.7; KpnI-A, 70; KpnI-B, 29; and BamHI-A, 35(reference 9).

b Numbers refer to the sizes of fragments (in kb) produced by HpaII or FnuDII digestion alone or the sizes ofthe unique fragments produced from the indicated double digestions. Het refers to the heterogeneity in thesefragments (see Fig. 3 and text).

sites in viral DNA in tumor cells is taken aspresumptive evidence for failure to cleave whenat least one of the CG dinucleotides of itsrecognition sequence is methylated. As men-tioned above, m5C is the only modified nucleo-side that has been detected in mammalian cellDNA, and no other reasonable explanation isavailable for the inability of a restriction endonu-clease with CG in its recognition sequence tocleave many sites in viral DNA after stableassociation with a cell. The sequence specific-ities of HpaII and MspI have been well charac-terized, and the inability of HpaII to cleave viralDNA in tumor cells is quite convincingly due toCG methylation. Nevertheless, the evidencewith FnuDII is presumptive until further dataare forthcoming. Also, TaqI (TCGA) was foundto produce the same viral DNA fragments intumor cells that are found in purified virions (notshown); this is consistent with other recent workwhich found that TaqI cleaves whether or notthe C of its recognition sequence is methylated(11, 21).When 32P-labeled strain S295C L-DNA was

used for hybridization after Southern transfer ofHpaII-cleaved tumor DNA, a series of L-DNAfragments starting at 5.4 kb (5.4 kb, heteroge-neous) was observed in addition to the specific17.5-kb fragment and DNA >40 kb (Fig. 3). Astrain S295C virion L-DNA fragment of 5.4 kbwas not readily apparent in the blot shown inFig. 2 (lower arrow slot 8) because of the use of32P-labeled strain 11 L-DNA for the hybridiza-tion. The left-most 7 kb of L-DNA from thesetwo H. saimiri strains show little or no cross-homology (P. Medveczky, R. Desrosiers, E.Szomolanyi, L. Falk, and C. Mulder, unpub-lished data).When tumor DNA was digested with both

EcoRI and HpaII, the left-terminal L-DNA

EcoRI fragment A could no longer be detected(Fig. 3 and Table 3); the molarity of the otherEcoRI fragments appeared to be unchanged.HpaII also cleaves approximately seven times ineach repeat unit of H-DNA. The results withHpaII and EcoRI-HpaII digestion shown in Fig.3 and Table 3 were consistent with two specifi-cally unmethylated CCGG sites within theEcoRI fragment A of L-DNA. Two lines ofevidence suggested that 5.4-kb heterogeneousfragments were derived from the left-most re-gion of the EcoRI fragment A: (i) the series ofless-intense bands above the 5.4-kb HpaII frag-ment suggested a variation in the amount of H-DNA attached to the 5.4-kb fragment due toheterogeneity of methylation in the repetitive H-DNA region immediately adjacent to L-DNAand (ii) the 5.4-kb HpaII fragment in strainS295C virion DNA and the 5.4-kb heteroge-neous HpaII fragments in tumor DNA did nothybridize significantly when 32P-labeled strain11 L-DNA was used for the hybridization probe.The left-most 7 kb of L-DNA is the only regionin which nonhomology between strains was de-tected. To further confirm the order of HpaIIsites within the viral L-DNA EcoRI fragment Ain tumor cells, a SacII fragment of 10 kb derivedfrom the left end of strain S295C L-DNA wasprepared, labeled in vitro, and used for hybrid-ization (Fig. 4). As expected, the 32P-labeled,left-terminal, 10-kb virion L-DNA fragment hy-bridized detectably only to the EcoRI fragmentA of virion and tumor DNA. The left-terminal10-kb fragment hybridized strongly to both theHpaII 17.5-kb and the 5.4-kb heterogeneousfragments of tumor cells. This demonstrates thatthere are two CCGG sites in L-DNA of tumorcells, located approximately 5.4 and 22.9 kbfrom the left H-L DNA border, that are specifi-cally not methylated. Double digestions with

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432 DESROSIERS

C,

O, 0. oc

ui :L.j LLi

X X )x X>< < <-

5.4-~~~~~~~~~~~~~~~~~~~~~~

z z z w

K B

26-

5.4-

FIG. 4. Use of purified 10-kb left-terminal L-DNAfragment for hybridization. DNA was digested withrestriction endonuclease(s), electrophoresed overnightthrough a 0.6% agarose gel, transferred to nitrocellu-lose, and hybridized to a 32P-labeled SacII fragment,which represents the left-most 10 kb of L-DNA.

KpnI-HpaII and BamHI-HpaII also confirmedthis and did not reveal any other unmethylatedCCGG sites (Fig. 3 and Table 3). (Cleavagemaps of H. saimiri S295C L-DNA are shown atthe top of Fig. 6.)

Similar experiments were performed withFnuDII (Fig. 3 and Table 3). Double digestion oftumor DNA with EcoRI-FnuDII revealed thepresence of specifically unmethylated sites with-in the L-DNA EcoRI fragments A and G. Thesuspected locations of specifically unmethylatedsites were confirmed by digestions with KpnI-FnuDII and BamHI-FnuDII (Fig. 3 and Table 3).The data for HpaII and FnuDII presented in

Fig. 3 and Table 3 represent a single tumorsample, but analyses of L-DNA contained with-

in the other three tumor DNA samples producedindistinguishable patterns (data not shown).

Results with HhaI were not as straightfor-ward. Although the EcoRI fragment A disap-peared below detection after double digestionwith EcoRI-HhaI, the intensity of some of theother fragments also appeared to decrease (Fig.3). Furthermore, new fragments (e.g., EcoRI-HhaI, 22 kb) also appeared less than molar.There may be HhaI sites with intermediatelevels of C methylation, and the use of clonedDNA fragments for hybridization and accuratequantitation will be required for a more detailedanalysis.Mapping of specifically unmethylated CG sites

in 1670 cells. Digestion of 1670 cell DNA withHpaII and FnuDII and double digestion withEcoRI-HpaII, EcoRI-FnuDII, KpnI-HpaII,KpnI-FnuDII, BamHI-HpaII, and BamHI-FnuDII were used to map the location of specifi-cally unmethylated sites in viral L-DNA in 1670cells (Fig. 5 and other data not shown). Themajority of viral DNA molecules in 1670 cellsare composed of two segments of L-DNA (Liand L2) separated by segments of H-DNA. L2represents the left-most 54 kb of virion L-DNA,and Li is approximately 92 kb, with 21 kbdeleted from the central region of virion DNA(5, 23). When 1670 DNA was double digestedwith EcoRI-HpaII, again only the left-terminalL-DNA EcoRI fragment A disappeared. Thespecific L-DNA HpaII fragment of 17.5 kb in1670 cells was not distinguishable in size byagarose gel electrophoresis from that found infresh tumor DNA. The left-most HpaII fragmentin L-DNA of 1670 cells was found to be 7.2 kb,i.e., 1.8 kb larger than that found in tumor DNA.Double digestion with EcoRI-FnuDII revealed aspecifically unmethylated site within the EcoRIfragment A and within the 7.2-kb EcoRI frag-ment in 1670 cells, which includes sequencesfrom virion EcoRI fragment G and spans the 21kb of deleted sequences.

DISCUSSIONThe analyses of fresh tumor and nonproducer

cell line DNA described in this report haverevealed the presence of a few CG sites in H.saimiri DNA that are specifically not methyl-ated. Four H. saimiri S295C-induced tumorswere analyzed, and four unmethylated CG sitesthat appeared identical in each of the four tu-mors were found. The locations of the fourunmethylated CG sites in L-DNA of tumors areshown schematically in Fig. 6. Strain S295Cvirion L-DNA contains 15 HpaII sites and 17FnuDII sites that produce fragments greaterthan 0.5 kb. In each of the tumor samples, two ofthe HpaII sites and two of the FnuDII sites were

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UNMETHYLATED SITES IN H. SAIMIRI DNA 433

STRAIN 11

VIRION DNA.-. -.

CC7

KB

1670 DNA

1-4 N-"0CLo CX c

=

2 I :: cLs-

KB

1670 DNA

EcoRI +

ElallaC

KB

1670 DNA

KpnT +

- N I C

woI2mm1 X;I Lr-KB

49- v.."29-

22.6- j 22.6-

16.8 1 7.5-16.8- W ~~16.8-

13.1 -

11.6 -

(A)26 -.

1 7.5

(E) 11.6

(F) 8.6 -

7A -

(B/C)l0.2-p(D/E) 8.8 -

7.4-

5.6- ,A

PA

_L, m

FIG. 5. Specifically unmethylated sites in L-DNA of 1670 cells. Digestion and analysis are as described in thelegend to Fig. 3.

specifically unmethylated. From the band inten-sities of the autoradiograms it was estimated thatthese four sites were unmethylated in greaterthan 95% of the molecules, and the remaining 28sites were methylated in greater than 90% of themolecules. The fact that the number and loca-tions of the unmethylated sites appeared identi-cal in the four tumor samples analyzed indicatesthat this is a reproducible, distinguishing charac-teristic. As with any restriction endonucleasemapping in the absence ofDNA sequencing, it isnot yet possible to determine whether whatappears as one site may be actually two sitesvery closely separated, for example, by 20 nu-cleotides. Such a possibility seems statisticallyunlikely in this case, in which the 115-kb stretch

of L-DNA contains only -15 HpaII sites, butDNA sequencing data will be needed to defini-tively answer this question.For a DNA with 50% G+C, the sequence

CCGG would represent approximately 0.25 x0.25 = 1/16 = 6.25% of all CG sites. For a DNAwith 36% G+C (L-DNA), the sequence CCGGwould be approximately 0.72 x 0.25 x 0.25 x0.72 = 3.24% of all CG sites. The CCGG andCGCG sites examined in this report thus repre-sent approximately 6.5% of all CG sites in L-DNA. The analysis of the remaining CG sites(other than HhaI sites) is hampered by the lackof appropriate methylation-sensitive restrictionendonucleases.The 1670 cell line has been in continuous

26-

13.1 -

11.6--

9.4- is 9.4 -

I* .

7.4 -

6A - S

(F)6.7 -

a:VW 9,..:

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434 DESROSIERS

Specifically unmethylatedsites in L-DNA of tumorsinduced with virus strainS295C

Specifically unmethylatedsites in L-DNA of the 1670cell line (strain 11 virus)

BamHI A B I D IF I C I EKpnIr A ID ICEcoRI A I FI| C/D|G|H|C/D E IB I

KILOBASEPAIRS0 10 20 30 40 50 60 70 80 90 100 110

I I I I I I I I I I I

FnuDII HpaII HpaIt FnuDII

FnuDIl H all HpaII FnuDII

LI I I I

0 10 20 30 40 50

L2L I 1 I I I

80 90 100 110

B amHI fA I C | DKpnI FB/CF AEcoRI A I F I B- I G|,IHI D

E I a-

I Dff4D/EI'E|C |

FIG. 6. Location of specifically unmethylated sites in L-DNA. The terminal cleavage site in each map is thefirst SmaI site in H-DNA. Virion L-DNA cleavage maps were taken from Fleckenstein and Mulder (9).

culture for over 7 years, yet the specificallyunmethylated HpaII and FnuDII sites in L-DNAare remarkably similar to those found in freshtumor cells (Fig. 6). The two right-most unmeth-ylated sites in 1670 cells appear equivalent tounmethylated sites in tumor cells and most likelyrepresent corresponding sites in the two strainsof viruses used. The two left-most unmethylatedCG sites in 1670 cells (H. saimiri 11) are not thesame distance from the H-L DNA border as thetwo left-most unmethylated CG sites in freshtumor cells (H. saimiri S295C). However, theseunmethylated sites are located in a region inwhich little or no homology exists between thesetwo virus strains. The fact that there are twospecifically unmethylated sites in this region ineach case underscores the similarity. The left-most 7 kb of L-DNA are particularly interestingbecause of the concentration of specifically un-methylated sites, the two groups of viruseswhich exhibit little or no homology in this re-gion, and because two independently derivednononcogenic variants have deletions in thisregion (C. Mulder, J. M. Koomey, R. C. Desro-siers, L. A. Falk, M. D. Daniel, and B. Flecken-stein, unpublished data).

Extensive analysis by electron microscopyand by indirect immunofluorescence tests oftumor cells obtained directly from tumor-bear-ing animals has not yielded evidence for anyvirus particle production or production of lateantigens (8, 13). Expression of the human her-

pesvirus, Epstein-Barr virus, is similarly limitedin tumor cells, and Epstein-Barr virus RNA intumor cells is derived principally from threeregions, representing only 5 to 10% of thegenome (4). The large number of correlations ofhypomethylated regions of DNA with geneexpression naturally prompts speculation as towhether H. saimiri RNA in tumor cells might bederived from regions containing unmethylatedCG sites. Unfortunately, nothing is known aboutthe origins of H. saimiri RNA in tumor cells;thus, future work will be directed toward deter-mining which regions of H. saimiri DNA arebeing expressed in tumor cells.

Unmethylated foreign DNA such as bacterialplasmid DNA or the thymidine kinase gene,introduced into cultured cells via transfection,usually remains unmethylatedc after stable asso-ciation with the cell (17, 18, 24). H. saimiri, aswell as adenovirus (22), contrasts with this;these viral DNAs invariably are extensivelymethylated after stable association with thegrowth-transformed cell. If cellular methylase ismethylating H. saimiri DNA, it remains to beexplained how this particular methylation pat-tern arises. The unmethylated CG sites may benaturally refractory to methylation. Alternative-ly, these methylated and unmethylated sitescould arise by chance in the millions of infectedlymphocytes in the animal; if this led to expres-sion of transformation-specific products andoutgrowth of transformed cells, this particular

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UNMETHYLATED SITES IN H. SAIMIRI DNA 435

methylation pattern could be selected for andpassed on to daughter cells. These possibilitiesof course do not exclude other explanations.

ACKNOWLEDGMENTS

I thank Sharon Tracy for expert assistance, L. Falk forproviding tumor cells, C. Mulder and B. Fleckenstein forproviding unpublished information and helpful discussion, andLinda Rennie for preparation of the manuscript.These studies were supported by Division of Research

Resources (National Institutes of Health) grant #RR00168 tothe New England Regional Primate Research Center, theMilton Fund, and by a fellowship from the Medical Founda-tion, Inc. of Boston.

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15. Mann, M., and H. Smith. 1979. Specificity of DNAmethylases from Haemophilus sp., p. 483-492. In E.Usdin, R. T. Borchardt, and C. R. Creveling (ed.),Transmethylation. Elsevier/North-Holland PublishingCo., Amsterdam.

16. Marezynska, B., L. Falk, L. Wolfe, and F. Deinhardt.1973. Transplantation and cytogenetic studies of Herpes-virus saimiri induced disease in marmoset monkeys. J.Natl. Cancer Inst. 50:331-337.

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21. Streeck, R. 1980. Single- and double-strand cleavage athalf-modified and fully modified recognition sites for therestriction nucleases Sau 3A and TaqI. Gene 12:267-275.

22. Vardimon, L., R. Newmann, I. Kuhlmann, D. Sutter, andW. Doerfler. 1980. DNA methylation and viral geneexpression in adenovirus transformed and infected cells.Nucleic Acids Res. 8:2461-2473.

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