JOURNAL OF VIROLOGY, March 1977, p. 986-995Copyright © 1977 American Society for Microbiology

Vol. 21, No. 3Printed in U.S.A.

Isolation ofthe Mouse Mammary Tumor Virus Sequences NotTransmitted as Germinal Provirus in the C3H and RIII

Mouse StrainsW. DROHAN, R. KETTMANN, D. COLCHER, AND J. SCHLOM*

Meloy Laboratories, Springfield, Virginia 22151, and National Cancer Institute, Bethesda,Maryland 20014*

Received for publication 13 October 1976

Radioactive 60-70S RNA from the mouse mammary tumor virus (MMTV)produced by the C3H mouse mammary tumor cell line (Mm5mt) hybridized to agreater extent, and at a lower Cotj/2 value, to the DNA ofC3H mammary tumorcells than to the DNA of C3H liver cells. The 125I-labeled MMTV(C3H) 60-70SRNA was annealed to a vast excess ofDNA from C3H livers, and single-strandedRNA was eluted from hydroxylapatite and recovered. This "recycled RNA" didnot hybridize to the DNA ofthe apparently normal organs tested from normal orfrom mammary tumor-bearing C3H mice, but hybridized extensively to both theDNA from the C3H mammary tumor cell line and the DNA from spontaneousC3H mammary tumors. This hybridization could be competed out by the addi-tion of unlabeled MMTV 60-70S RNA but was unaffected by the addition ofunlabeled 60-708 RNA ofC3H type C virus. Similar experiments were conductedwith the RIII mouse strain. We therefore report on the isolation ofthe sequencesof the RNA genomes of the MMTVs from C3H and RIII mice that are transmit-ted by some mechanism other than via the germ line. These studies furtherdefine the differences, via molecular hybridization, between the MMTV-S andthe MMTV-L in both C3H and RIII mice.

The C3H and RIII mouse strains are amongthe most widely used in the study of virus-mediated mammary oncogenesis. Both strainscontain high mouse mammary tumor virus(MMTV) titers in their milk, termed "MMTV-S," and mammary tumors arise at a high fre-quency "early" (at-approximately 9 months) inlife (2, 18). Foster nursing of offspring on moth-ers free of overt MMTV in their milk (e.g.,mothers of the C57BL strain) results in a lowincidence of mammary tumors that arise "late"(at approximately 15 months) in the life oftheseC3Hf and RIIIf mice. The MMTV found in theearly mammary tumors of C3H and RIII micehas also been termed "MMTV-S," whereas theMMTV found in the late-occurring mammarytumors of C3Hf and RIIIf mice has been termed"MMTV-L" or NIV (10, 18, 21). A central ques-tion in this virus-mediated mammary oncogen-esis is: are the "MMTV-S" and "MMTV-L" thesame virus transmitted via different mecha-nisms, or do substantial differences exist be-tween them?

Reports have claimed (23, 30) that there areno detectable differences by nucleic acid hybrid-ization between MMTV-S and MMTV-L, andthat no detectable qualitative differences inMMTV proviral sequences exist in numerousmouse strains examined, including those of

both high and low mammary tumor incidence.These studies, however, were conducted withradioactive complementary DNA probes thatconstituted a disproportionate representation ofthe MMTV genomes used.Using the technique of competitive molecular

hybridization, which used the entire 60-70SMMTV RNA genome in question (radioactivelylabeled), we were previously able to demon-strate distinct differences between MMTV-Sand MMTV-L in C3H mice (16). The techniqueof saturation hybridization, using tritiated 60-70S RNA of MMTVs from early mammary tu-mors of both C3H and RIII mice, has also indi-cated that the DNA of early mammary tumorsof both C3H and RI mice contains someMMTV-S proviral sequences that are not foundin the DNA ofan apparently normal organ (theliver) of those same tumor-bearing mice (16,17). Liver cells were chosen because of theirlack of detectable MMTV antigens (11, 19).We report here the isolation of the portions of

the MMTV-S genomes that are present in theDNA of early mammary tumors of C3H andRIII mice and are absent in the DNA of severalapparently normal organs of those same tumor-bearing animals. These results are consistentwith the concept that early mammary tumori-genesis in these two mouse strains results from

986

ISOLATION OF NONGERMINAL MMTV SEQUENCES 987

the introduction of a portion of the MMTV ge-nome that is not present in the germ line ofthose mice.

MATERIALS AND METHODSViruses and tissues. C3H MMTV-S was obtained

from supernatant fluids of the C3H mouse mam-mary tumor cell line Mm5mt/c, (9, 20) and desig-nated MMTV(C3H). The cell line was kindly sup-plied by D. Fine (Frederick Cancer Research Cen-ter) at passage 23. Cells were seeded at 3.3 x 107 per490-cm2 plastic roller bottle (Corning) in Dulbeccomodified Eagle medium with 10% fetal calf serum(GIBCO, Grand Island, N.Y.), 2 ,ug of dexametha-sone per ml, and 10 gg of insulin per ml. When cellsreached 90 to 100% confluency, 24-h virus fluid har-vests were collected. Collections were obtained onlyfrom passages 29 to 31 of the cell line. The mediumwas clarified by low-speed continuous-flow centrifu-gation to exclude material larger than 10,000S. Vi-rus in the supernatant fluid was purified by sedi-mentation into a 20 to 60% (wt/wt) linear sucrosegradient buffered with 0.05 M Tris, pH 7.8, by usinga Beckman CF32 rotor (5 liters/h) at 32,000 rpm.Fractions were eluted from the rotor after 0.5 h ofequilibration. Buoyant densities and optical densi-ties (at 254 nm) were determined for each fraction.Density regions between 1.14 and 1.19 g/ml werediluted and pelleted at 143,000 x g. Pellets wereresuspended in 0.01 M Tris, pH 8.3, at a 1,000:1concentration of starting supernatant fluid volume.No type C virus was detected by electron microscopyor by divalent cation preference ofDNA polymerase(Mg2+/Mn2+ ratio of 23:1) using oligo(dG):poly(rC) astemplate (9, 12).Primary cultures of spontaneous mammary tu-

mors of 7- to 12-month-old RIII mice were preparedas previously described (12, 13). The RIII mice wereobtained through the Office of Resources and Logis-tics of the Virus Cancer Program of the NationalCancer Institute. Tumor cells were plated at a den-sity of 106 cells/cm2 in 75-cm2 plastic flasks (Falcon)and incubated for 5 to 7 days. To obtain radioac-tively labeled MMTV-S of the RIII strain[MMTV(RIII)], the standard tissue culture mediumwas removed and replaced with 10 ml of tissue cul-ture medium containing 0.12 mCi of [3H]uridine and[3H]cytosine per flask for approximately 14 h. Three6-h harvests were collected the following day andreplaced with cold medium. This cycle was repeatedfor approximately 8 days. All isotopes were obtainedfrom New England Nuclear Corp. Supernatantfluids were stored at -20°C. After thawing, theywere clarified at 6,000 rpm for 10 min in a J10Beckman rotor. The virus containing supernatantfluid was layered over, and pelleted through, a layerof 20% sucrose and collected on top of a 70% sucrosepad (25,000 rpm at 4°C for 90 min in a SW27 rotor).The virus band was collected and diluted 1:1 withbuffer (0.02 M Tris, pH 8.3; 0.3 M NaCl; 0.02 MEDTA) and immediately used for RNA extraction.An established cell line of mouse embryo fibro-

blasts, C3H/10T1/2, kindly supplied by U. Rapp andG. Todaro (22), was used as a source of C3H type Cvirus. Virus was purified from a 3-day harvest of 3.3

liters of supernatant fluid. Virus banding on a su-crose gradient between a density of 1.12 to 1.17 g/mlwas collected and resuspended in 3.3 ml of buffer(0.01 M Tris, pH 8.2).C3H/HeN mice were obtained from the Small An-

imal Resources section of the National Institutes ofHealth. C3H mammary tumor-bearing animalswere kindly supplied by George Vlahakis of theNational Cancer Institute. Sheep lung and bovineovary tissue were purchased from Pel Freeze, Inc.(Rogers, Ark.). Calf thymus DNA, type 1, was pur-chased from the Sigma Chemical Co., St. Louis, Mo.

Purification of viral RNAs. The procedure forisolating viral RNAs was as described previously (6,25) and was the same for all viruses. After sedimen-tation, the viral pellets were resuspended in a solu-tion of 0.01 M Tris-hydrochloride (pH 8.3), 0.1 MNaCl, and 0.01 M EDTA and lysed by the addition ofsodium dodecyl sulfate (SDS) to a 1% final concen-tration. After the suspension cleared, 0.75 mg ofself-digested Pronase (2 h, 37°C) per ml and 1%mercaptoethanol were added and incubated at 37°Cfor 30 min. This mixture was then extracted twicewith 2 volumes of PCC (phenol-cresol-chloroform[7:1:8, vol/vol/vol] containing 8-hydroxyquinoline[0.185 g/100 ml], pH 8.3). The aqueous phase wasremoved and adjusted to 0.4 M LiCl, and the RNAwas precipitated by the addition of 2 volumes of coldethanol. After 16 h at -20°C, the RNA was pelletedat 17,000 x g for 30 min at -20°C and dissolved in100 ,ul ofTNE buffer (0.01 M Tris-hydrochloride [pH8.3], 0.15 M NaCl, 0.002 M EDTA). The 60-70S com-ponent was isolated by glycerol gradient sedimenta-tion in a 10 to 30% linear glycerol gradient in TNE(centrifuged at 200,000 x g for 3 h at 4°C [SpincoSW41 rotor]). Fractions were collected dropwise, andthe 60-70S RNA peak ofthe gradient, determined byabsorbance at 260 nm, was precipitated with ethanolas described above. The 60-70S [3H]RNA peak ofMMTV(RIII) was determined by counting aliquotsof each fraction in a Beckman liquid scintillationcounter.

Isolation of poly(A)-enriched RNA. Thirty gramsof frozen C3H kidneys was placed into a mortarcontaining liquid nitrogen and then ground to a finepowder. This mixture was added to 25 ml of extrac-tion buffer (0.1 M NaCl; 0.01 M Tris-hydrochloride,pH 8.5; 0.001 M EDTA; 1% SDS) per g of tissue. Thesolution was then put into a Waring blender andunderwent three pulses of 20 s each at the highestspeed. After stirring for 30 min, the aqueous phasewas extracted two or three more times as statedabove. The aqueous phase was removed; 1/9 volumeof 2 M sodium acetate was mixed in well, and 2volumes of absolute ethanol was added. DNA wasspooled out on a glass rod and treated as othercellular DNAs (see below), whereas the alcohol su-pernate (containing the RNA) was stored overnightat -20°C.The RNA was pelleted and dissolved in 0.01 M

Tris-hydrochloride, pH 7.4, and 0.02 M MgCl2.DNase I, RNase-free (Worthington BiochemicalsCorp., Freehold, N.J.), was further purified by glyc-erol gradient sedimentation, and then added to theRNA solution at a final concentration of 15 ,g/mland incubated at 37°C for 20 to 30 min. The solution

VOL. 21, 1977

988 DROHAN ET AL.

was then extracted one or two times with an equalvolume of PCC, ethanol precipitated, and storedovernight at -20°C.The RNA was pelleted, dissolved in sterile water,

and further purified by adding LiCl to a final con-centration of 2 M. The solution was stirred at 4°C for24 h. The RNA precipitate was collected by centrifu-gation at 10,000 rpm for 15 min at 4°C in a BeckmanJS13 rotor. The RNA was then dissolved in buffer A(0.01 M Tris-hydrochloride, pH 7.4; 0.3 M NaCl;0.001 M EDTA) and added to poly(U) Sepharose 4B,equilibrated in the same buffer. This poly(U) Sepha-rose was previously shown to remove 98% of the[3H]poly(A) added to 20 mg of cold chicken RNA.After stirring at room temperature for 1 h, the solu-tion was put over a water-jacketed column at 25°C,and the unbound RNA was removed with buffer A.The column temperature was then raised to 45°C,and the bound RNA was eluted with a buffer con-sisting of 0.01 M Tris-hydrochloride, pH 7.4, 0.001 MEDTA, and 0.2% SDS. The poly(A)-enriched RNAwas precipitated with alcohol and stored at -20°C.Five micrograms of this RNA was pelleted and iodi-nated to a specific radioactivity of approximately 2x 107 cpm/,g. The RNA was then used in liquidhybridization experiments with normal mouseDNA.

Iodination of viral RNA. The iodination proce-

dure is a modification of that of Commerford (7) as

described by Colcher et al. (6). Carrier-free 125I wasobtained from Amersham/Searle (pH 8 to 11, 100mCi/ml). Thallium perchlorate, obtained from AlfaProducts, was dissolved at 4 x 10-3 M in 0.05 Msodium acetate buffer (pH 4.2).

Reactions were performed in siliconized 50-,1 cap-illary pipettes. Six hundred microcuries of 125I wasmixed with 1 t, of sodium sulfite and incubated at25°C for 15 min. To this solution, 2 ,ul of thalliumperchlorate and 1 ul ofMMTV 60-70S RNA (5 ,ug/,ul)were added and mixed well. The capillary was thensealed, and the reaction was allowed to proceed at68°C for 15 min, diluted with 200 ul of 0.5 M sodiumphosphate buffer (NaPB), pH 6.8, containing 0.01 M,/-mercaptoethanol, and then incubated for another45 min. The RNA was separated from free iodine bycolumn chromatography using Sephadex G-50 (10-ml column) in 0.05 M NaPB, pH 6.8. The RNAsample was then adjusted to 30% ethanol in 0.01 MTris-hydrochloride, pH 8.3, 0.15 M NaCl, and 0.002M EDTA, and layered on a 1-ml column of CF11(Whatman) equilibrated in this buffer. The columnwas washed with 20 ml of the same buffer, and theRNA was eluted with TNE. The specific activity ofthe RNA was approximately 2 x 107 cpm/,ug.

Purification of cellular DNA. DNA was extractedfrom pools of tissues by a modification of the methodof Baluda et al. (1). Approximately 1 g of tissue wasadded to 9 ml of extraction buffer (0.1 M NaCl; 0.01M Tris, pH 8.5; 0.001 M EDTA), and this mixturewas disrupted by a 5-min pulse on a Waring blender.SDS and mercaptoethanol were then added to a 1%final concentration, and the mixture was incubatedfor approximately 30 min at 37°C. Predigested Pro-nase was added to a final concentration of 0.5 mg/ml. The solution was incubated for 2 to 12 h. Then

0.5 volume of chloroform and 0.5 volume of water-saturated phenol were added to the DNA solution,and the solution was shaken for 15 to 30 min. Theaqueous phase was separated by centrifuging thesolution at 4VC for 15 min at 6,000 rpm on a BeckmanJS7.5 rotor. One-ninth volume of 2 M sodium ace-tate (pH 5.5) solution and 2 volumes of ethanol wereadded to the aqueous phase to precipitate the DNA.The DNA was then spooled out on a glass rod, dis-solved in 0.3 N KOH, and incubated at 370C for 16 hto hydrolyze any contaminating RNA. The solutionwas neutralized with a solution of 2 N HCl in 0.4 MTris-hydrochloride and then sonically treated (Soni-fer Cell disruptor, model W185, Heat System Ultra-sonics, Inc.; highest setting) for 3 min at room tem-perature. The DNA solution was precipitated withalcohol as described above, pelleted, and dissolvedin extraction buffer. At this time the solution wasextensively reextracted with a phenol-chloroformmixture until no interphase was observed betweenthe aqueous and organic phases. The aqueous phasewas separated from the phenol phase, and the DNAwas precipitated with alcohol as described above andthen dissolved in 0.001 M NaPB (pH 6.8) at a concen-tration of 6 mg/ml; DNA prepared in this way had a260/280-nm ratio of at least 1.70, and usually above1.80. The sedimentation coefficient, as determinedby alkaline sucrose sedimentation, was between 6and 9S.

Molecular hybridization. Cellular DNA was hy-bridized to iodinated or tritiated 60-70S MMTV RNAunder the following conditions: cellular DNA, 3 mg/ml in 0.4 M NaPB (pH 6.8), 0.05% SDS, and variableamounts of labeled MMTV RNA. The DNA (at 6 mg/ml) was first boiled for 1 min in a bath of ethyleneglycol and then put in a water bath at 680C. The saltand labeled RNA were mixed with the DNA, and theentire solution was incubated at 680C until the de-sired Cot had been attained. (Cot values are cor-rected to 0.12 M NaPB [5].) At appropriate times,aliquots of 0.17 ml (500 lug) were removed and di-luted into 8.5 ml of 2x SSC (lx SSC is 0.15 M NaCl,0.015 M sodium citrate). The sample was divided inhalf, and 4 ml was added to each of two tubes con-taining an additional 1 ml of 2 x SSC (DNA concen-tration is now 50 ,ug/ml). RNases A and T1 wereadded to one-half of the sample at final concentra-tions of 15 ,ug/ml and 3 U/ml, respectively. Bothtubes were then incubated at 37°C for 30 min. Thesamples were chilled for 10 min and adjusted to 10%trichloroacetic acid. After being kept on ice for 30min, the acid-insoluble material was collected on0.45-,.m nitrocellulose filters (Gelman, Inc.). A zero-time control, which was boiled for 1 min in a bath ofethylene glycol before the addition of RNase, wasincluded in all experiments. The counts per minuteof radioactivity present in the RNase-treated por-tion of this sample was deducted from the counts perminute obtained on all other samples. The percent-age of hybridization was determined by dividing thecounts per minute present in the RNase-treatedsample by the counts per minute present in theuntreated sample, after deducting the zero-timebackground.Thermal stability of hybrids. The technique for

J. VIROL.

ISOLATION OF NONGERMINAL MMTV SEQUENCES 989

analysis ofthermal stability ofDNA-RNA hybrids isessentially that of Kohne and Byers (15). The hy-bridization solution contained 1 mg of cellular DNAand approximately 6,000 cpm of 1251-labeled MMTVRNA. The conditions of hybridization were the sameas delineated above. After hybridizing to a Cot of15,000, the DNA was diluted to a concentration of 50,ug/ml and applied to a 4-ml column of packed hy-droxylapatite maintained at 60'C in 0.006 M NaPB,pH 6.8. Under these conditions approximately 75 to95% of the cellular DNA remains bound to the col-umn. While at 600C, the column was washed twicewith 9-ml samples of 0.12 M NaPB, pH 6.8, contain-ing 0.01% sodium N-lauryl sarcosinate (SLS). Thisprocedure was repeated at 50C increments until atemperature of 100'C was attained. The thermaldissociation ofthe DNA-DNA hybrids was measuredby monitoring the absorbance at 260 nmn in the ef-fluent fractions, and that of the 1251-labeled RNA-DNA hybrids was measured by determining theirtrichloroacetic acid precipitability and radioactivityin effluent fractions.

Recycling of viral RNAs. The technique is a mod-ification of that of Shoyab et al. (27, 28). DNA wastreated with 0.01% diethyl pyrocarbonate (Calbi-ochem, San Diego, Calif.) and mixed for 5 min at4VC. The diethyl pyrocarbonate was removed byheating the DNA at 680C for 1 h. A total of 300,000cpm of 1251-labeled MMTV(C3H) 60-70S RNA washybridized to 30 mg ofC3H liver DNA (unless other-wise stated) to a Cot of 15,000 to 20,000 under theconditions described above. At the completion ofhybridization, the sample was diluted to 15 j.g ofDNA per ml with sterile water and then heated to60°C. The solution was applied to a column of 150 mlof packed hydroxylapatite at 600C (DNA grade, Bio-Gel, HTP, lot 14557, Bio-Rad Laboratories, Rich-mond, Calif.). The sample that came through wasreloaded, and the column was rinsed with a solutionof 0.01 M NaPB, pH 6.8, in 0.01% SLS until nomaterial eluted which absorbed at 260 nm. Single-stranded DNA and RNA were then eluted with asolution of 0.14 M NaPB, pH 6.8, in 0.01% SLS. Thedouble-stranded DNA and hybridized RNA wereeluted with a solution of 0.4 M NaPB, pH 6.8, in0.01% SLS. The single-stranded fraction was di-alyzed (Spectrapor no. 1 or no. 3) against threechanges of 6 liters of water containing 0.1% SLS.After dialysis, 1 to 2 mg of yeast carrier RNA wasadded, and the RNA was precipitated with 2 vol-umes of alcohol. The RNA was pelleted and dis-solved in 1 ml of 0.001 M NaPB and stored at -20°C.[3H]MMTV RNA from RIII mice was recycled ex-actly as above except that 100,000 cpm of viral RNAwas recycled against 30 mg of cellular DNA.

RESULTSKinetics of hybridization of 125I-labeled

MMTV(C3H) 60-70S RNA to cellular DNAs.MMTV(C3H) was isolated from supernatantfluids of the Mm5mt C3H mammary tumor cellline. The 60-70S RNA from these virions waspurified as described in Materials and Methods

and was iodinated to a specific activity of ap-proximately 2 x 107 cpm/,ug; this RNA was 99%acid precipitable and 98% RNase sensitive. ThisRNA was then hybridized at various Ct valuesto DNA from Mm5mt C3H mammary tumorcells and DNA from an apparently normal C3Hliver; hybridization to sheep DNA was used as acontrol. As assayed by resistance to RNase Aand T. digestion, hybridization to sheep DNAremained consistently at 5 to 6% up to a Cot of35,000 (Fig. 1) and was thus scored as nonspe-cific background. Hybridization between the io-dinated MMTV(C3H) 60-70S RNA and DNAextracted from the Mm5mt/c, C3H mammarytumor cell line reached a maximum of 60%(Fig. 1). This value was about 10% more thanthe maximum extent of hybridization betweenthis MMTV(C3H) RNA and DNA from livers ofC3H mice (Fig. 1).The Cot,,2 value of the hybridization between

'25I-labeled MMTV(C3H) 60-70S RNA and theC3H mammary tumor cell line DNA was ap-proximately 380, and the C-t112 value of thehybridization to DNA from C3H liver was ap-proximately 440. For comparison, poly(A)-en-riched C3H cellular RNA, selected by poly(U)Sepharose chromatography, was also iodinatedand hybridized to C3H liver DNA (see Materi-als and Methods). The Cot1/2 value obtainedusing this RNA was approximately 3,100 (Fig.1). As an additional control, the poly(A)-en-riched [1251]RNA was also hybridized to calf

A

0~~~~~~~~

20 A

FIG. 1. Hybridization of '251-labeled MMTV-(C3H) 60-70S RNA to various cellular DNAs. Hy-bridization conditions were as described in Materialsand Methods. The hybridization mixtures contained125j-labeled MMTV(C3H) 60-70S RNA and DNAfrom the C3H mammary tumor cell line Mm5mt (A);normal C3H liver (0); and sheep lung (0). For com-parison, hybridizations were performed between 125Jlabeled poly(A)-enriched mouse RNA and C3H liverDNA (A) and calf thymus DNA (U).

VOL. 21, 1977

990 DROHAN ET AL.

thymus DNA; no significant hybridization wasobserved up to a Cot of 35,000.The results depicted in Fig. 1 demonstrate

that both the C3H mammary tumor cell lineand C3H liver contain MMTV proviral se-quences in the low repetitive range (4). Thelower CAt1M2 value obtained with the C3H mam-mary tumor cell line DNA suggests that thereare more MMTV proviral sequences in thisDNA than in the DNA of the C3H liver. Thedifference in final percentage of hybridization(i.e., approximately 60% for the C3H tumor cellline DNA and approximately 50% for the C3Hliver DNA), however, may indicate two phe-nomena: (i) there are quantitatively moreMMTV proviral sequences in the mammarytumor DNA than the liver DNA; and (ii) theDNA of the C3H mammary tumor cells con-tains a portion of the MMTV genome that is notfound in the DNA ofC3H liver cells. To answerthis question, recycling experiments were per-formed.Recycling of 125I-labeled MMTV(C3H) 60-

70S RNA. To determine whether there are anyMMTV sequences that are not present in theDNA of an apparently normal organ, i.e., theliver of a C3H mouse, iodinated MMTV(C3H)60-70S RNA was first hybridized to a vast ex-cess ofC3H liver DNA. A total of 300,000 cpm ofMMTV(C3H) 60-70S RNA was first annealed to30 mg of normal C3H liver DNA at 680C to a Cotof 20,000. The unhybridized single-stranded[125I]RNA eluting from the hydroxylapatite col-umn at 0.14 M sodium phosphate was termed"recycled RNA." The recovery of this recycledRNA for five different experiments is outlinedin Table 1. This RNA was then concentratedand reannealed to C3H mammary tumor DNAand to C3H liver DNA to a Cot of 20,000 asdescribed above. The recycled MMTV(C3H)RNA failed to hybridize above background lev-els to the DNA of normal C3H livers (Fig. 2).This demonstrates that the recycling experi-ment effectively removed all portions of the 125I_labeled MMTV(C3H) RNA that were comple-mentary to the DNA of normal C3H liver. Thesame low level of hybridization could be seenbetween the recycled MMTV RNA and sheeplung DNA. The recycled [125I]RNA hybridizedmore than 50%k, however, with DNA frommammary tumor cells (Fig. 2).The above results were obtained with DNA

from a C3H mammary tumor cell line and DNAfrom the liver of an apparently normal animal.To determine whether similar results could beobtained with naturally occurring "early"mammary tumors, DNA was extracted fromspontaneous mammary tumors of C3H miceand from livers of those same tumor-bearing

animals. Livers were examined grossly anddemonstrated no evidence of metastatic lesions.No significant difference was observed in per-

TABLE 1. Recycling of 1251-labeled MMTV(C3H)RNA against C3H liver DNA

Acid-precipitable cpm(X 10-3) % Acid-pre-

Expt cipitableno. RNA 0.14 M RNA in 0.14RNA NaPB elu- M elutionainput" tione

ld 320 40 132 401 57 143 349 46 134 313 61 205 344 60 17

a This was calculated by dividing the acid-precip-itable RNA in the 0.14 M NaPB elution from hy-droxylapatite by the acid-precipitable RNA added tothe initial hybridization reaction to C3H liver DNA.Only 75% of the total input counts per minute in theinitial hybridization reaction, however, was re-covered after incubation at 68°C to a Cot of 20,000.

b Counts per minute of trichloroacetic acid-pre-cipitable MMTV(C3H) [125I]RNA hybridized to 30mg of C3H liver DNA.

e 0.14 M NaPB elution from hydroxylapatite col-umn (see Materials and Methods).

d The total 125I-labeled MMTV(C3H) RNA countsper minute x 10-3 (uncorrected for acid precipitabil-ity) at different steps of the recycling procedure areas follows: input to initial hybridization to C3Hliver, 395; hydroxylapatite load, 302; 0.14 M NaPBelution, 71; 0.4 M NaPB elution, 230.

60

A

A A

!20 A

20

log CotFIG. 2. Hybridization of recycled 1251-labeled

MMTV(C3H) RNA to murine cellular DNAs. Iodi-nated MMTV(C3H) 60-70S RNA was extensively hy-bridized to normal C3H liver, and the unhybridizedfraction was recovered by hydroxylapatite columnchromatography as described in Materials and Meth-ods. The recycled RNA was then hybridized to thefollowing cellular DNAs: C3H mammary tumor cellline Mm5mt (A); C3H liver (@); and sheep lung(0). Hybridization conditions are as described inMaterials and Methods.

J. VIROL.

ISOLATION OF NONGERMINAL MMTV SEQUENCES

centage of hybridization to a Cot value of 15,000when 125I-labeled MMTV(C3H) 60-70S RNAwas hybridized to the DNA of spontaneous C3Hmammary tumors before (55%) or after (53%)recycling against C3H liver DNA (Table 2).This same [1251]RNA, however, hybridized 49%to the DNA of livers from the tumor-bearinganimals before recycling, but hybridized to onlybackground levels (5 to 6%) to this same DNAafter recycling. These data again demonstratethat there are proviral sequences in "early"C3H mammary tumors that are not transmit-ted via the germ line.When 125I-labeled MMTV(C3H) RNA was re-

cycled against C3H mammary tumor DNA (us-ing the exact protocol used for C3H liver), only3% of the input RNA was recovered in the 0.14M NaPB elution from the hydroxylapatite col-umn. This RNA, however, did not hybridizeabove background levels (6%) to either C3Hmammary tumor DNA or C3H liver DNA.Thermal analyses of hybrids. It is important

in the interpretation of the recycling experi-ments to determine whether the hybridsformed between recycled MMTV(C3H) and cel-lular DNAs are composed of well-matched com-plementary nucleic acid sequences. Therefore,the DNA-RNA hybrids formed were analyzedfor thermal stability by hydroxylapatite col-umn chromatography. Figure 3 depicts thethermal dissociation of renatured DNA fromthe spontaneous C3H mammary tumors andthe Mm5mt/c, mammary tumor cell line andthe dissociation of hybrids formed betweenthese same DNAs and recycled 1251-labeledMMTV(C3H) RNA. A T. of 830C was measuredfor the renaturation of both the DNA-DNA du-plexes. The T. values of both the 125I-labeledRNA-DNA hybrids were 75.50C. This difference

TABLE 2. Hybridization of 1251-labeled MMTV(C3H)60-70S RNA, before and after recycling against

C3H liver DNA, to murine DNAs

Percent hybridizedSource of DNA Before re- After recy-

cycling cling

C3H spontaneous mammary 55 53tumor

C3H mammary tumor cell 61 58line (Mm5mt)

C3H liver from mammary tu- 49 6mor-bearing animal

C3H liver from a non-tumor- 49 5bearing animal

Bovine ovary 6 6a All values refer to an average percentage of

hybridization of duplicate points assayed at a Cot of15,000.

in Tm between DNA-RNA and DNA-DNA hy-brids is in accordance with that reported byothers (14).

Competition of hybridizations involving recy-cled MMTV(C3H) RNA. To determine whetherthe single-stranded [125I]RNA recovered by re-cycling is indeed part of the MMTV(C3H) ge-nome, purified unlabeled 60-70S RNAs ofMMTV(C3H) and MuLV(C3H) were added tothe hybridization between recycled ['251]MMTV-(C3H) RNA and mammary tumor DNA. Addi-tion of 0.2 or 1.5 ,g of MuLV(C3H) RNA did notinhibit the hybridization (Table 3). However,0.2 ,ug of MMTV(C3H) 60-705 RNA inhibitedthis hybridization by 87%, and addition of 1.5,g of this RNA resulted in a greater than 98%inhibition of the hybridization. These resultsfurther demonstrated that the radioactive se-quences obtained by recycling against C3Hliver DNA are not germ-line-transmittedMMTV(C3H) sequences.

Recycling of 125I-labeled MMTV(C3H)against a pool of C3H organs. 1251-labeledMMTV(C3H) was recycled against DNA fromC3H livers because liver cells do not appear toexpress detectable amounts of known MMTVantigens (11, 19). However, to determine whetherother organs of C3H would be suitable for recy-cling experiments, a DNA preparation was

,000

2 0

& 40-

20

.0i 70 80 90 100Temperature (IC)

FIG. 3. Thermal stability of hybrids formed be-tween recycled '251-labeled MMTV(C3H) RNA andmurine DNAs. Hybridization conditions and ther-mal elution from hydroxylapatite were performed asdescribed in Materials and Methods. Dissociation ofhybrids formed between recycled 1251-labeledMMTV(C3H) RNA and DNA from C3H sponta-neous mammary tumors (0), and DNA from theMm5mt mammary tumor cell line (0). Dissociationofreannealed DNA-DNA duplexes from spontaneousC3H mammary tumors (U) and the Mm5mt cell line(0).

991VOL. 21, 1977

992 DROHAN ET AL.

made from a pool of five tissues of apparentlynormal C3H mice: brain, spleen, lung, kidney,and heart. A total of 300,000 cpm of 125I-labeledMMTV(C3H) was hybridized to 30 mg of DNAfrom these tissues, and the recycled single-stranded RNA was purified and concentratedas described in Materials and Methods. Thisrecycled RNA hybridized substantially to DNAfrom the mammary tumor cell line both beforeand after recycling (Table 4). However, afterrecycling, the ['25I]RNA failed to hybridizeabove background levels to DNA from livers orkidneys from normal C3H mice. This indicatesthat pools of other organs from normal C3Hmice can also be used for recycling to obtain thenon-germ-line-transmitted MMTV sequencesfound in the DNA of C3H mammary tumors.

TABLE 3. Competition of the hybridization ofrecycled '25-Ilabeled MMTV(C3H) RNA and C3H

mammary tumor DNA

Ag of corn- PecnCompetitor RNA petitor PercentRNA tionaadded to

None 0 53MuLV(C3H), 60-7&Sb 0.2 54

1.5 51MMTV(C3H), 60-70Sc 0.2 7

1.5 1a The hybridization between recycled 125I-labeled

MMTV(C3H) RNA (recycled before use by hybridi-zation to C3H liver DNA) and C3H mammary tumorcell line DNA (Mm5mt) was as described in Materi-als and Methods. The 60-70S competitor RNAs wereadded after boiling of the DNA. The 6% backgroundhybridizaton to bovine DNA has been subtractedfrom all values.

b MuLV purified from the C3H/1OT1/2 cell line.e MMTV purified from the Mm5mt cell line.

TABLE 4. Hybridization of'251-labeled MMTV(C3H)60-70S RNA before and after recycling againstDNA

from a pool ofC3H organsa

Percent hybridizationSource of DNA Before re- After recy-

cycling clingC3H mammary tumor 60 54

cell line (Mm5mt)C3H liver 47 7C3H kidney NTb 7Bovine ovary 6 6

a '25I-labeled MMTV(C3H) 60-70S RNA was recy-cled against a pool of C3H organs (lung, kidney,heart, brain, and spleen). The conditions of hybridi-zation and processing of hybrids are as described inMaterials and Methods.bNT, Not tested.

Hybridization of recycled MMTV(RIII) RNAto cellular DNAs. Not only the C3H strain ofmice but also the RIII strain carries an MMTV-S that is involved in the induction ofmammarytumors which occur at a high frequency early inthe life span of the mouse (at approximately 9months) (2, 18). Therefore, experiments wereundertaken to determine whether the DNA ofspontaneous RII mammary tumors containsMMTV(RIII) proviral sequences that are notfound in the DNA of all cells of RII mice. Thispostulate was again investigated by recycling3H-labeled MMTV(RIII) 60-70S RNA, whichwas purified from virions produced by primaryexplants of RIII mammary tumors (13), againstRIII liver DNA (as described in Materials andMethods and the legend to Table 5). These cul-tures have been shown to contain less than 1%type C viruses as compared with MMTV by avariety of criteria (12, 13). Before recycling, the[3H]MMTV(RIII) 60-70S RNA hybridized 56%to DNA from spontaneous RIII mammary tu-mors and 10% lower to the DNA of livers fromthese tumor-bearing mice or DNA from normalRUTI livers. However, after recycling againstnormal RIII liver DNA, the resultant [3H]RNAfailed to hybridize to DNA from normal RIIIlivers or livers from tumor-bearing RIII mice.The recycled [3H]RNA, however, still hybrid-ized well to DNA from RIII mammary tumorcells (Table 5), demonstrating that there arealso MMTV(RUI) proviral sequences in theDNA of RUI mammary tumors that are nottransmitted via the germ line of that mouse.

DISCUSSIONThe results presented here demonstrate the

isolation of the sequences of the RNA genomesof the MMTV-S of both the C3H and the RIIImouse strain that are transmitted by some

TABLE 5. Hybridization of 3H-labeled MMTV(RIII)60-70S RNA to RIIIDNAs before and after recycling

against RIII liver DNA

Source of DNA

Percent hybridiza-tiona

Before re- After recy-cycling cling

RIII mammary tumor 56 46RIII liver (from a non-mam- 46 5mary tumor-bearing ani-mal)

RIII liver (from a mammary 46 5tumor-bearing animal)

Bovine ovary 5 5a All values refer to an average percentage of

hybridization of duplicate points assayed at a Cotof 15,000.

J. VIROL.

ISOLATION OF NONGERMINAL MMTV SEQUENCES

mechanism other than as a germinal provirus.The "recycling" procedure permits experimentsthat eliminate possible misconceptions thatmay be generated by relying solely on quantita-tive analysis ofresults. As can be seen in Fig. 1,the lower Cot,,2 value obtained in the hybridiza-tion of 125I-labeled MMTV(C3H) 60-70S RNA tothe DNA of C3H mammary tumor cells than tothe DNA of C3H liver cells indicates that thereare more MMTV proviral sequences in theDNA of mammary tumor cells; it is difficult todetermine, however, whether the lower per-centage of hybridization observed to the DNAof the C3H liver cells is due solely to the factthat liver cells contain fewer MMTV-S proviralsequences or due to a lack of part ofthe MMTV-S RNA genome. After recycling the '25I-labeledMMTV(C3H) 60-70S RNA to the DNA of livercells, no hybridization above background is ob-served when the recycled RNA is hybridized tothe DNA of C3H liver, even to Cot values ofgreater than 20,000 (Fig. 2); substantial hybrid-ization is observed, however, when this RNA ishybridized to the DNA ofC3H mammary tumorcells (Fig. 2). The fidelity of these hybrids isconfirmed by the assays used to score them,i.e., resistance to RNase, and by their Tm val-ues (Fig. 3). This technique of recycling hasbeen used successfully to demonstrate thatavian myeloblastosis virus is not transmittedas a germinal provirus in chickens (27, 28) andto demonstrate that Rauscher MuLV is nottransmitted as a germinal provirus in BALB/cmice (29).To further demonstrate that the recycled

RNA sequences that hybridize to the DNA ofC3H mammary tumors are indeed MMTV se-quences and not MuLV sequences, unlabeled60-70S RNAs of MMTV(C3H) and a type CMuLV from C3H mice were added as competi-tors to these reactions. Since a substantial ho-mology was reported between all type C viruses(3, 8) and no competition was observed, usingthe 60-70S RNA from the type C virus from thesame strain as competitor (Table 3), there isgood evidence that the recycled RNA is not typeC in origin; furthermore, unlabeled MMTV 60-70S RNA competed completely in this reaction(Table 3).These same experiments were also conducted

with the RIII mouse strain. TritiatedMMTV(RIII) 60-70S RNA was obtained fromvirions from supernatant fluids of primary cul-tures of spontaneous "early" RIII mammary tu-mors. 3H-labeled MMTV(RIII) RNA, recycledagainst a vast excess of DNA from RHI liver,was not able to hybridize to RIII liver DNA butdid hybridize substantially to the DNA of a

spontaneous "early" RIII mammary tumor.These results indicate that in the RIII mousestrain, as in the C3H strain, there are MMTVsequences found in the DNA of "early" mam-mary tumors that are transmitted by somemechanism other than via the germ line.To rule out the possibilities that some C3H or

RIII mice carry their respective MMTV-S ge-nomes as a germinal provirus and that thoseparticular mice are the ones that develop mam-mary tumors, we hybridized the recycledMMTV RNA in both systems specifically to theDNA of apparently normal livers from mam-mary tumor-bearing mice (Tables 2 and 5);since these results were negative, this possibil-ity has been ruled out.The exact distribution of these recycled

MMTV sequences within a given mouse is ob-viously difficult to determine. Therefore, oneshould be cautious at this time about callingthese "mammary tumor-specific" sequences.Recycling using DNA from pooled organs of aC3H mouse worked as effectively as did recy-cling using DNA from livers (Table 4). No hy-bridization above background was observed tothe DNA of liver or kidney using this recycledRNA. Since these organs are heterogeneous innature with respect to cell type, we cannoteliminate the possibility that some cells insome apparently normal C3H or RHI mice con-tain these recycled sequences. It is probable,furthermore, that a population of normal or"preneoplastic" mammary cells in the appar-ently normal mammary gland contains thesesequences. These experiments can best be ac-complished when the technology for cloningand propagating normal murine mammarycells is established. Further studies concerningthe distribution and expression of these recy-cled sequences are now in progress.When C3H newborn mice are foster-nursed

on mothers free of overt MMTV in their milk(i.e., mothers of the C57BL strain), the fre-quency of the mammary tumor decreases andthe latent period increases in these so-calledC3Hf mice. These mammary tumors do containMTV particles (10, 18, 21) that have been re-ferred to as NIV or MMTV-L. We have shown(R. Kettmann, W. Drohan, D. Colcher, and J.Schlom, manuscript submitted for publication)that the DNAs of these tumors contain MMTVproviral sequences when MMTV-S tritiatedcomplementary DNA or iodinated 60-70S RNAis used as a probe. When recycled ['25I]RNA isused as a probe, however, no hybridizationabove background to the DNA of these C3Hftumors is detected. This agrees with the resultsreported here and previously (16, 17, 24) that a

VOL. 21, 1977 993

994 DROHAN ET AL.

difference can be readily detected by molecularhybridization between MMTV-S and MMTV-L.We have also shown in this same study (Kett-mann et al., submitted for publication) thatmammary tumors and livers of BALB/c mice,and livers of C57BL/6N and C57BL/1OSCNmice, contain MMTV proviral sequences but donot contain the recycled sequences of theMMTV-S of the C3H mouse. The DNA frommammary tumor cells and from normal livercells of GR mice, however, both contain se-quences related to the recycled MMTV-S se-quences. These data support the genetic studiesof Bentvelzen (2) that the MMTV of the GRmouse strain is being transmitted as a ger-minal provirus.The results of the studies reported here

should be a caveat to those who study the con-trol ofMMTV on the transcriptional and trans-lational level in mouse strains such as C3H:there is information for at least two MMTVs inthe DNAs of the cells from "early" mammarytumors.The origin of the MMTV sequences isolated

as a result of recycling remains to be deter-mined. It is difficult to conceive that they arethe result of a germinal or somatic recombina-tion event, for they would also be present inanother location in the DNA of normal cellssuch as the liver if this were the case. Theycould have been acquired, however, as the re-sult of the recombination of the information ofthe endogenous MMTV of C3H, i.e., theMMTV-L, with the DNA from a progenitor ofMus musculus, or the DNA of another species,similar to the model proposed for the origin ofthe rat sequences in the Kirsten murine sar-coma virus (26). The origin and expression ofthe recycled sequences found in the DNA ofearly mammary tumors of C3H and RIII miceare currently being investigated.

ACKNOWLEDGMENTSWe thank G. Gibson, D. Joiner, and S. Jones for their

technical assistance. We thank J. Young for her help insome experiments, and G. Vlahakis and S. Spiegelman forhelpful suggestions.

R. K. is "Aspirant du Fonds National belge de la Re-cherche Scientifique" and received a fellowship from theRotary Foundation. This study was supported in part byPublic Health Service contract NO1-CP-43223, Virus Can-cer Program, National Cancer Institute.

LITERATURE CITED1. Baluda, M. A., and W. Drohan. 1972. Distribution of

deoxyribonucleic acid complementary to the ribonu-cleic acid of avian myeloblastosis virus in tissues ofnormal and tumor-bearing chickens. J. Virol.10:1002-1010.

2. Bentvelzen, P. 1972. The biology of the mouse mam-mary tumor virus. Int. Rev. Exp. Pathol. 11:259-297.

J. VIROL.

3. Benveniste, R. E., and G. J. Todaro. 1973. Homologybetween type-C viruses of various species as deter-mined by molecular hybridization. Proc. Natl. Acad.Sci. U.S.A. 70:3316-3320.

4. Bishop, J. 0. 1972. DNA-RNA hybridization, p. 247-276. In E. Diczfalusy (ed.), V Karolinska symposia onresearch methods in reproductive endocrinology. Ka-rolinska Institutet, Stockholm.

5. Britten, R. J., and J. Smith. 1970. A bovine genome.Carnegie Inst. Washington Yearb. 68:378-386.

6. Colcher, D., W. Drohan, and J. Schlom. 1976. Mason-Pfizer RNA genome: relationship to the RNA of mor-phologically similar isolates and other oncornavi-ruses. J. Virol. 17:705-712.

7. Commerford, S. L. 1971. Iodination of nucleic acids invitro. Biochemistry 10:1993-1999.

8. East, J. L., J. E. Knesek, J. C. Chan, and L. Dmo-chowski. 1975. Quantitative nucleotide sequence re-lationships of mammalian RNA tumor viruses. J.Virol. 15:1396-1408.

9. Fine, D. L., L. 0. Arthur, J. K. Plowman, E. A. Hill-man, and F. Klein. 1974. In vitro system for produc-tion of mouse mammary tumor virus. Apple. Micro-biol. 28:1040-1046.

10. Heston, W. E., and G. Vlahakis. 1968. C3H-A'Y -a highhepatoma and high mammary tumor strain of mice.J. Natl. Cancer Inst. 40:1161-1166.

11. Hilgers, J. H. M., G. J. Theuns, and R. Van Nie. 1973.Mammary tumor virus (MTV) antigens in normaland mammary tumor-bearing mice. Int. J. Cancer12:568-576.

12. Kimball, P. C., R. Michalides, D. Colcher, and J.Schlom. 1976. Characterization of mouse mammarytumor viruses from primary tumor cell cultures. II.Biochemical and biophysical studies. J. Natl. CancerInst. 56:119-124.

13. Kimball, P., M. B. Truitt, G. Schochetman, and J.Schlom. 1976. Characterization of mouse mammarytumor viruses from primary cell cultures. I. Immuno-logical and structural studies. J. Natl. Cancer Inst.56:111-117.

14. Kohne, D. E. 1968. Isolation and characterization ofbacterial ribosomal RNA cistrons. Carnegie Inst.Washington Yearb. 67:316-320.

15. Kohne, D. E., and M. J. Byers. 1973. Amplification andevolution of deoxyribonucleic acid sequences ex-pressed as ribonucleic acid. Biochemistry 12:2373-2378.

16. Michalides, R., and J. Schlom. 1975. Relationship innucleic acid sequences between mouse mammary tu-mor virus variants. Proc. Natl. Acad. Sci. U.S.A.72:4635-4639.

17. Michalides, R., G. Vlahakis, and J. Schlom. 1976. Abiochemical approach to the transmission of mousemammary tumor viruses in the mouse strains RIIIand C3H. Int. J. Cancer 18:105-116.

18. Nandi, S., and C. M. McGrath. 1973. Mammary neopla-sia in mice. Adv. Cancer Res. 17:353-414.

19. Nowinski, R. C., N. Sarkar, L. J. Old, D. H. Moore, D.I. Scheer, and J. Hilgers. 1971. Characteristics of thestructural components ofthe mouse mammary tumorvirus. Virology 46:21-38.

20. Owens, R. B., and A. J. Hackett. 1972. Tissue culturestudies of mouse mammary tumor cells and associ-ated viruses. J. Natl. Cancer Inst. 49:1321-1332.

21. Pitelka, D. R., H. A. Bern, S. Nandi, and K. Deome.1964. On the significance of virus-like particles inmammary tissues of C3Hf mice. J. Natl. Cancer Inst.33:867-885.

22. Rapp, U. R., R. C. Nowinski, C. A. Reznikoff, and C.Heidelberger. 1975. Endogenous oncornaviruses inchemically induced transformation. Virology 65:392-409.

ISOLATION OF NONGERMINAL MMTV SEQUENCES 995

23. Ringold, G. M., P. B. Blair, J. M. Bishop, and H. E.Varmus. 1976. Nucleotide sequence homologiesamong mouse mammary tumor viruses. Virology70:550-553.

24. Schlom, J., D. Colcher, W. Drohan, P. Kimball, R.Michalides, and G. Schochetman. 1976. Biochemicalcharacterization of mouse mammary tumor virusesand related isolates: Mason-Pfizer virus and theBUdR-induced guinea pig virus, p. 11-51. In J. C.Henson, W. H. Mattheiem, and M. Rozeneweig (ed.),Breast cancer: trends in research and treatment.Raven Press, New York.

25. Schlom, J., D. Colcher, S. Spiegelman, S. Gillespie,and D. Gillespie. 1973. Quantitation of RNA tumorviruses and virus-like particles in human milk byhybridization to polyadenylic acid sequences. Science179:696-698.

26. Scolnick, E. M., R. J. Goldberg, and D. Williams. 1976.Characterization of rat genetic sequences of Kirsten

sarcoma virus: distinct class of endogenous rat type Cviral sequences. J. Virol. 18:559-566.

27. Shoyab, M., and M. A. Baluda. 1975. Acquisition ofviral DNA sequences in target organs of chickensinfected with avian myeloblastosis virus. J. Virol.16:783-789.

28. Shoyab, M., R. M. Evans, and M. A. Baluda. 1974.Presence in leukemic cells of avian myeloblastosisvirus-specific DNA sequences absent in normalchicken cells. J. Virol. 14:47-49.

29. Sweet, R. W., N. C. Goodman, J.-R. Cho, R. M. Ru-precht, R. R. Redfield, and S. Spiegelman. 1974. Thepresence of unique DNA sequences after viral induc-tion of leukemia in mice. Proc. Natl. Acad. Sci.U.S.A. 71:1705-1709.

30. Varmus, H. E., J. M. Bishop, R. C. Nowinski, and N.Sarkar. 1972. Mammary tumor virus specific nucleo-tide sequences in mouse DNA. Nature (London) NewBiol. 238:189-191.

VOL. 21, 1977