JOURNAL OF VIROLOGY, Mar. 1974, p. 712-720Copyright ( 1974 American Society for Microbiology

Vol. 13, No. 3Printed in U.S.A.

Characterization of an Endogenous RNA-Dependent DNAPolymerase Associated with Murine Intracisternal A Particles

STRINGNER SUE YANG AND NELSON A. WIVELLaboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014

Received for publication 2 November 1973

An RNA-dependent DNA polymerase associated with intracisternal A parti-cles has been characterized. The enzyme required Mg2+ or Mn 2+, dithiothreitoland the presence of all four deoxyribonucleoside triphosphates for the expression

of maximal activity. Sensitivity of the endogenous RNA-dependent DNApolymerase activity to a low concentration of pancreatic ribonuclease in thepresence of a high concentration of NaCl suggested that the enzyme might beutilizing the A particle endogenous RNA as template. Evidence in support of thiswas provided by analyses of early and late DNA products of the endogenousreaction by Cs2SOI isopycnic gradient centrifugation and hybridization of purified60 to 70S and 35S RNAs of A particles with the purified DNA product.

Recent reports strongly suggest that intracis-ternal A particles represent some expression of aviral genome, since they contain a high molecu-lar weight, 60 to 70S RNA as the endogenousnucleic acid, and a group-specific structuralprotein of 70,000 molecular weight (14, 35).

Their occurrence has been frequently ob-served in tumors of the gerbil (29), rat (19),guinea pig (18), man (25), and in both normaland neoplastic mouse tissues (6-8, 33). Suchparticles are most probably vertically transmit-ted from oocyte to the fertilized embryo, aphenomenon analogous to murine C-type vi-ruses, and their transient bursts of proliferationduring early embryonic development have beendescribed (5).

In mice they are most abundant in certaintumors and can be obtained in significantquantities from such neoplasms as the plasmacell tumor, squamous carcinoma, and neuro-blastoma. Advances in the biochemical studiesof intracisternal A particles have been difficultbecause of problems involving cellular contami-nation inherent in their isolation from cytoplas-mic cisternae. The cryptic expression or the lossof biochemical function, as a result of strenuouspurification procedures, must be considered. Anearlier report described the existence of a novelDNA polymerase activity associated with intra-cisternal A particles that lacked endogenousactivity, that did not require detergent activa-tion, and that was solely dependent on thesynthetic template oligo dT12 -8 -poly rA. It hasbeen referred to as a poly (dT) polymerase (32).In this communication definitive evidence is

presented to demonstrate the existence of anendogenous RNA-dependent DNA polymeraseassociated with intracisternal A particles. Thispolymerase utilizes its own endogenous RNA astemplate; it synthesizes a DNA product that isin the form of an RNA-DNA hybrid at the endof a short-term reaction, and primnarily DNA atthe end of a long-term reaction. The DNAproduct hybridizes back to A particle RNA.

MATERIALS AND METHODS

Tumor and cells. A mouse plasma cell tumor(MOPC-104E) was kindly supplied by M. Potter ofthe National Cancer Institute, and was maintained bysubcutaneous transplantation in BALB/c mice in thislaboratory. Neuroblastoma (N-18), a continuoustumor cell line (35), was cultured in Dulbecco'smodified Eagle medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (50 units/ml),and streptomycin (25 vg/ml) in a 5% CO2 atmosphereat 37 C. Cells were fed on alternate days. All tissueculture materials were from GIBCO.

Isolation and purification of intracisternal Aparticles. Preparation of purified intracistemal Aparticles has been previously described (15, 35).Briefly, cytoplasmic extracts from tumor homoge-nates were centrifuged at 13,000 x g for 20 min toobtain a pellet of mitochondria and microsomes. Aparticles were freed from microsomnal vesicles throughthe use of Triton X-100 and mechanical shearing. Thefreed particles were further purified through threecentrifugation cycles in discontinuous sucrose (25 to48%) gradients. Finally, A particles were subjected toisopycnic centrifugation in linear gradients utilizing33 to 68% (wt/vol) sucrose or 35 to 85% glycerol, andbanded at 1.21 to 1.23 g/cm3. The appearance of arepresentative preparation is depicted in Fig. 1. It

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*, sQii *

C- * B. -;k-A .

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FIG. 1. Thin section preparation of gradient purified intracisternal A particles. In addition to completesingle particles there are numerous elongated forms made up of multiple buds (arrows). x70,000. The barrepresents 0.1 Aim.

should be emphasized that N-18 neuroblastoma cellsshowed no C-type viruses as examined ultrastructur-ally, nor did the A particle isolates contain C-typeviral antigens (35). Although the whole cells fromMOPC-104E plasma cell tumor contain C-type viralantigens, the purified A particles do not (35). Further-more, the purification scheme rules out the chancesfor contamination by C-type viruses as demonstratedby a reconstitution experiment in which Rauscherleukemia virus (RLV) labeled with 3H-uridine wasadded to the initial tumor cell suspension prior to theisolation and purification of intracisternal A particles.All RLV-associated tritium counts were removed atthe initial discontinuous sucrose gradient centrifuga-tion (35). A particles purified by a final linear gradi-ent centrifugation step therefore exhibit biochemicalproperties that can be appropriately considered asintrinsic to A particles alone.DNA polymerase assays. Details of DNA polym-

erase assay have been described earlier (2, 28, 34)except for some minor variations used to optimize theassay conditions. Briefly, in a final volume of 50gliters, the assay mixture contained Tris-hydrochlo-ride, pH 8.1, 50 mM; Mg acetate, 10 mM, or MnCl2,0.8 mM; dithiothreitol (DTT), 8 mM; KCl, 75 mM;and approximately 5 ug of purified intracisternal Aparticles treated with 0.018% Triton X-100 or X-102for 10 min at 0 C. For assaying the endogenousreaction, cold dCTP, dGTP, and dATP at a finalconcentration of 8 x 10' M were added along with 5ACi of 3H-TTP, and the reaction was carried out at37 C. For assays with synthetic templates, a template

concentration of 50 ug/ml was used along with theappropriate substrates at 30 C. The reaction wasterminated by additions of 2 ml of ice-cold 0.08 Msodium pyrophosphate in 12.5% trichloroacetic acidand 20 ug of yeast nucleic acid as carrier. Theradioactive precipitates were then collected on Mil-lipore filters, washed extensively with 5% trichloroa-cetic acid, washed with 80% ethanol, dried, and thencounted in Liquifluor by a liquid scintillation method.

All chemicals, unless specified, were obtained fromCalbiochem, Sigma, and Fisher Scientific. TritonX-102 and X-100 were obtained from Sigma and fromPackard Instrument Co., respectively. All isotopeswere purchased from New England Nuclear Corp.Bovine pancreatic RNase and DNase were obtainedfrom Worthington Biochemicals. RNase at 0.25mg/ml was boiled for 10 min to inactivate thecontaminant DNase prior to use.

Virus purification. Avian myeloblastosis virus(AMV) was kindly supplied by J. W. Beard and D. P.Bolognesi through the Special Virus Cancer Programof the National Cancer Institute. Further purificationof AMV was carried out by the methods describedearlier (4). AMV materials banding at a density of1.16 g/cm3 were used for RNA extraction. Highlypurified RLV (10" particles/ml) was obtained fromElectronucleonics Inc. and subjected to isopycniccentrifugation in a 35 to 85% glycerol gradient asdescribed. Materials banding at the 1.16 g/cm3 areaand coincident with reverse transcriptase activitywere used for RNA extraction.RNA extraction and purification. High molecular

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weight RNA was extracted from purified AMV andfrom RLV, and purified by velocity gradient centrifu-gation prior to being used as templates in DNApolymerase assays. Details of the extraction andpurification procedures have been documented else-where (35). The majority of the viral RNA from eitherAMV or RLV, when purified to homogeneity, sedi-mented at the 60 to 70S area. This fraction wasconcentrated by precipitation in ethanol-NaCl andthe RNA was resuspended in 0.1 M NaCl and 5 mMMgCl2 prior to use as templates.

Templates. Natural templates, unless specified,were purchased from Miles laboratory. Salmon spermDNA was activated with a limited DNAase digestionat 37 C for 15 min as described elsewhere (21),followed by extensive purification as described belowfor DNA products. Copolymers and homopolymers ofvarious synthetic oligonucleotides with specifiedchain lengths were obtained from either Miles Labo-ratory or Collaborative Research Corp.

Purification and analysis of DNA products ofthe endogenous RNA-dependent DNA polymerasereaction. The radioactive products from the endoge-nous RNA-dependent DNA polymerase reaction wereadjusted to 0.3 M Na acetate, pH 5.0, purified byphenol-SDS extraction twice, ether extraction fourtimes, and then ethanol-NaCl precipitation overnightat -20 C. The DNA precipitates were then collectedby centrifugation, redissolved in 0.3 M Na acetate,and applied to a sulfopropyl Sephadex C-50 column.Fractions eluted at the void volume were pooled andreprecipitated in ethanol-NaCl prior to Cs2SO4 iso-pycnic centrifugation analysis at 40,000 rpm for 60 hin the SW 50 rotor. Gradient samples were weighed fordensity determination prior to precipitation in 12%trichloroacetic acid and processed for radioactivedetermination.

Hybridization reactions and analysis of theRNA-DNA hybrids with S-l exonuclease. 3H-DNAproduct of the endogenous RNA-dependent DNApolymerase of intracisternal A particles was preparedin the presence of 5 Mg of actinomycin D per ml asdescribed in the previous sections. The 3H-DNA waspassed through a Sephadex G50 column (180 by 0.8cm) instead of an SP Sephadex C50 column. The3H-DNA recovered at the void volume was adjusted to0.3 N KOH and was hydrolyzed at 100 C for 10 min toremove any RNA. The sample was then neutralizedwith HCl and adjusted to 3 x SSC (1 x SSC-0.15 MNaCl-0.015 M trisodium citrate) and 50% formamide(Eastman-Kodak) for hybridization. Purified intra-cisternal A particle 60 to 70S RNA and 35S RNA atabout 2.0 Mg was then added to 200 Aliters of thehybridization mixture, and the reaction was carriedout at 37 C for 18 h. Control samples were prepared inthe same manner without or with heterologous RNA,such as QI3 RNA, at the same concentration. At theend of the hybridization reaction, the samples wereadjusted to a final concentration of 25 mM Naacetate (pH 4.5), 0.5 mM ZnSO4, 0.15 M NaCl, and20 Aliters of purified S-1 exonuclease. The nucleaseassay was then continued at 45 C for 90 min. At theend of the assay, the reaction was terminated withprecipitation in 12% trichloroacetic acid with 20 Mg

of yeast transfer RNA (Miles Laboratory) as carrier.The radioactivity was determined as describedearlier.

Preparation of S-1 exonuclease. S-1 exonucleasewas purified from crude a-amylase powder (SigmaChemical Co.) from Aspergillus oryzae according tothe procedures described by Sutton (26). It has aspecific activity of hydrolyzing about 1,000 counts/min of 3H-DNA (single stranded) per 60 min per 2Aliters of the enzyme preparation at 45 C.

Protein determination. Protein was determinedby the Lowry method (16). Since glycerol, sucrose,and Triton all interfere with the colorimetric determi-nation, samples were first precipitated with 10%perchloric acid (PCA) and washed three times withPCA prior to alkali digestion; and then the proteindetermination was carried out.

RESULTSAssociation ofan endogenous RNA-depend-

ent DNA polymerase with intracisternal Aparticles. Figure 2 demonstrates the associa-tion of an endogenous RNA-dependent DNApolymerase activity with purified intracisternalA particles which sedimented at the density of1.21 to 1.23 g/cm3 across a linear sucrose gradi-ent. When each fraction of the gradient wasassayed under optimal conditions, as describedin the next section, the endogenous RNA-dependent DNA polymerase activity peakedsharply and uniformly at 1.21 to 1.23 g/cm3. Itshould be noted that there were no other con-taminating materials such as C-type particlessince no polymerase activity was observed at

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1.21

1.193

1.17

1.15

1.13

FIG. 2. Linear sucrose gradient analysis of an en-dogenous RNA-dependent DNA polymerase activityassociated with murine intracisternal A particles. Allexperimental procedures were described in Materialsand Methods.

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1.16 g/cm3. This was confirmed by the electron-microscopy presented in Fig. 1. The possibilityof contaminating C-type particles that mightconfuse the biochemical studies of intracister-nal A particles was ruled out by the immunolog-ical studies and the reconstitution experimentreported earlier (35). The endogenous RNA-dependent DNA polymerase activity that sedi-mented at the 1.21 to 1.23 g/cm3 area can

therefore be regarded as intrinsic to intracister-nal A particles especially when the followingevidence is considered.

Characteristics of an endogenous RNA-de-pendent DNA polymerase associated withintracisternal A particles. Table 1 summa-

rizes the conditions essential for optimal activ-ity of the endogenous DNA polymerase associ-ated with intracisternal A particles. Similar tothe characteristics of other DNA polymerases,the requirements for a complete endogenousreaction include DTT, divalent cation, Mg2t or

Mn2+ (see Fig. 4), monovalent cation, Na+ or

Kt, and the presence of all four deoxyribonu-cleoside triphosphates. The recovery of theendogenous DNA polymerase reaction in theabsence of Na+ or K+ was 62 to 64% probablydue to the significant amount of Na+ ionpresent in all four deoxyribonucleoside triphos-phates. Total depression of activity was

achieved by omitting three deoxyribonucleosidetriphosphates from the reaction. Among thefour DNA precursors, dATP seems to be themost essential, since its deletion led to a reduc-tion of 80% of the activity. This characteristic ofthe endogenous reaction of intracisternal Aparticles DNA polymerase resembles that re-

ported for the reverse transcriptase of Roussarcoma virus (28) and Mason-Pfizer monkeyvirus (1).Detergent activation. This endogenous

DNA polymerase activity is dependent on de-tergent activation to some degree. Withoutpreincubation of the particles with Triton X-102, only 26% of the control activity was ob-served (Table 1), a phenomenon reminiscent ofreverse transcriptase of murine sarcoma

viruses (13). Clearly, this suggests that deter-gent activation is required either for solubiliz-ing the DNA polymerase for reactivity or toprovide accessibility of the substrates, or

both. At this point, it should be clarified thatthe Triton X-100 used in the isolation proce-dure is essential to free the A particles frommicrosomal vesicles. At the chosen concentra-tion (0.2%), the function of the detergent is notto lyse the microsomal vesicles, but to sensitizethe membranes to disruption by mechanicalshear. This allows for the release of completely

TABLE 1. Characteristics of endogenousRNA-dependent DNA polymerase activity associated

with intracisternal A particlesa

Acid-insolu-Conditios.ble 9H-TMP Control

incorpora- (%)

tionb

Complete .................. 13.34 100Minus Mg2" .............. 0.38 3Minus DTT ................ 0.27 2Minus Nat ................. 8.61 64Minus Kt. ............ 8.20 62Minus dATPC ............. 2.65 20Minus dGTPC .......... 6.20 47Minus dCTPc .............. 8.30 63Minus dA, G, C, TP ........ 0.64 5Minus Triton X-102 treat-ment .................... 3.50 26

Preincubation withRNase .................. 0.35 3

aAll assay conditions are described in the text.Complete condition refers to inclusion of all criticalrequirements such as K+ or Na+, Mg2+, DTT, and allfour deoxyribonucleoside triphosphates, and 0.018%Triton X-102 treatment. Each assay contained 24 tsgof A particle protein (MOPC-104E plasma cell tu-mor). Preincubation with RNase (25 gg/ml) wascarried out in 0.15 M NaCl at 37 C for 2 h; 12,970counts/min = 1.0 pmol.

b Measured in picomoles per hour per milligram ofprotein.

c Acquired from Sigma Chemical Co. in the form ofdisodium salt.

formed A particles which are morphologicallyintact and still exhibit significant intrinsic bio-chemical properties such as 60 to 70S RNA(35). However, there are many budding forms,and shearing them free of the membranes leavesopen-ended particles (Fig. 1); these incompleteparticles are obviously susceptible to leakageof intrinsic RNA and DNA polymerase as aresult of the isolation procedure. It has to beconsidered that the DNA polymerase activitybeing assayed is that primarily derived from thepopulation of completely formed intracisternalA particles.RNase sensitivity. RNase digestion experi-

ments indicated that indeed the endogenousDNA polymerase of A particles most probablywas dependent on the intrinsic RNA as tem-plate (Table 1 and Fig. 3). RNase at lowconcentration, 25 ,ug/ml, but in the presence ofhigh salt, 0.15 M NaCl, for the most part digestsonly single-stranded RNA (20). Under theseconditions, intracisternal A particles previouslytreated with Triton X-102 and incubated at 37 Cfor 2 h lost all endogenous RNA-dependentDNA polymerase activity (Fig. 3D). Under thesame conditions but using an even lower RNase

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FIG. 3. Sensitivity of the endogenous RNA-depend-ent DNA polymerase activity of intracisternal Aparticles to pancreatic ribonuclease digestion at highNaCl concentration. Approximately 125 ug of intra-cisternal A particles were preincubated with TritonX-102 at a final concentration of 0.018% in thepresence of 0.15 M NaCl. A, At 0 C for 2 h, 0; B, at37 C for 2 h, 0; C, at 37 C with 16.6 ,gg of pancreaticRNase per ml for 1 h, A; and D, at 37 C with 25.0 ,g ofpancreatic RNase per ml for 2 h prior to polymeraseassays, A. Each sample was adjusted to completeassay conditions to a final volume of 250 gliters with 5MCi each of all four 'H-deoxyribonucleotide triphos-phates (10 Ci/mmol). Samples (50,Mliters each) werewithdrawn at specified times, precipitated in 12%trichloroacetic acid-pyrophosphate and processed asdescribed in the text.

concentration, 16.6 ug/ml, and a shorter periodof incubation, 60 min, A particles still lost about87% of their endogenous activity at the end of a30-min reaction (Fig. 3C). Further proof of theinvolvement of intrinsic RNA in the endogenousDNA polymerase reaction of intracisternal Aparticles is evident when the nature of the DNAproduct is considered below.

Kinetics of the RNA-dependent DNA po-lymerase reaction. Under optimal conditions,the rate of incorporation of acid-insoluble 3H-TMP in an endogenous reaction by detergenttreated intracisternal A particles varied amongpreparations. Figure 4A-1 depicts the kinetics ofa more active preparation (number 1) in whichthe endogenous reaction is linear up to 30 to 40min at 37 C before it slowed. However, A

particle preparations varied in the extent oftheir endogenous reaction even at the sameprotein concentration (Fig. 4A-1 versus 4A-2).

A This was probably due to the availability of theRNA template; leakage of RNA from A parti-cles due to isolation procedure could account forthe paucity of an endogenous reaction in one

preparation of A particles as compared withanother. A particles isolated and purified fromfrozen tumor tissues frequently exhibited much

B lower endogenous activity (Fig. 4B-3), but giventhe presence of the synthetic hybrid template,dT,2 18poly rA, the initial rate of the RNA-dependent DNA polymerase activity was signif-icantly increased (Fig. 4B-1 and 2). The kineticsof the polymerase reaction in the presence of thedT121 8 poly dA, the DNA-DNA template, weremuch slower (Fig. 4B-4). This seems reasonablesince dT12 U8.poly dA did not stimulate theintracisternal A particle DNA polymerase.Template preference. A variety of natural

c RNAs and DNAs, including activated salmon:L.D sperm DNA, T2 phage DNA, AMV 70S RNA,30 RLV 70S RNA, MS-2 RNA, vesicular stomatitis

0

z

U

z

0 10 200 30 0 10 20 30MIN. AT 37 MIN. AT 30°

FIG. 4. Kinetics of the RNA-dependent DNA po-lymerase of intracisternal A particles with and with-out exogenous synthetic templates. All assay condi-tions were described in the text. In A, the amount ofpurified A particles from MOPC-104E plasma celltumor used in the assay depicted by curve number 1(0) was 15 Mg, whereas 12.5 Mg of purified A parti-cles from N-18 neuroblastoma cells was used in theassay depicted by curve number 2 (0). In B, 12.5sg of another purified preparation of A particlesisolated from previously frozen MOPC-104E plasmacell tumor tissues was used in each kinetic study. Ki-netics of A particle DNA polymerase in the presenceof dT,2-,8.poly rA was depicted by curve number 1(A), at 50 /ug/ml, and by curve number 2 (A), at 25jgg/ml. Curve number 3 (0) depicted kinetics of theendogenous activity of the same A particles. Curvenumber 4 (U) depicted kinetics of A particle DNApolymerase with dT,2- I.poly dA, 50 ,g/ml as tem-plate.

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MURINE INTRACISTERNAL A PARTICLES

virus RNA, influenza virus RNA, and Q# RNA,were used to assess template preference. In allcases the copying of these nucleic acids wasweak or nonexistent.Table 2 summarizes the effects of various

synthetic templates on the RNA-dependentDNA polymerase activity of intracisternal Aparticles. At high concentration, 100 jig/ml,dT,2,8. poly rA is the most effective template,among all the synthetic oligonucleotides used inthis study, stimulating the A particle DNApolymerase to 9.4-fold greater activity. At thesame concentration, dT,2,8.poly dA did notstimulate the DNA polymerase activity. Whenthese activities under the stimulation by thetwo templates are compared, a ratio(dTI2 8 -poly rA/dTI2- 8 poly dA) of 9.9 wasobserved, which is characteristic of viral reversetranscriptase (20). The same preferential ratio,although somewhat lower at 8.3, was observedwhen the template concentration was 50 jg/ml.Poly dT -poly rA, a template that stimulatedboth cellular and viral DNA polymerases (10,20, 21), also stimulated the activity of A particleDNA polymerase. Both the DNA copolymer,poly d(A-T), and the RNA-RNA duplex, polyrA -poly rU, failed to stimulate A particle DNApolymerase to a higher activity. In all theserespects, the DNA polymerase of intracisternalA particles and the poly dT polymerase (32)are similar.

Interestingly, the RNA-DNA hybrid tem-plate, dG12-18 -poly rC, that was reported asspecific for viral reverse transcriptase (1, 20, 22)also stimulated the DNA polymerase of intra-cisternal A particles to about 300% of thecontrol activity. Much greater stimulation wasobserved with other A particle preparations bythis template. Poly dT polymerase was alsoreported to be stimulated by dG12 I8 poly rC(32). Similar to other C-type viral reverse tran-scriptases (1), intracisternal A particle DNApolymerase is not stimulated by dC12-18 polyrG.

Divalent cation requirement. The endoge-nous RNA-dependent DNA polymerase activitywas preferentially activated by Mn2+ ratherthan by Mg2+. Figure 5 shows a divalent cationconcentration dependence of the endogenousDNA polymerase activity of A particles. Opti-mal Mg2` concentration varied from about 5.5to 10 mM; however, the extent of the DNApolymerase activity at optimal Mg2` concentra-tion was much less than that observed atoptimal concentration of Mn2 . The optimalconcentration for Mn2+ was rather sharp andwas found to be at 0.8 mM for the activation ofthe endogenous RNA-dependent DNA polymer-

TABLE 2. Comparison of stimulatory effects ofvarious synthetic templates on DNA polymerase

activity of intracisternal A particles

Incorporation of

Templates acid-insoluble Controldeoxyribonu- (%)

cleotidea

None-endogenous(jig/ml) 0.17b 100dT,2,8- polyrA(100) 1.59b 935dT2-,8-polydA(100) 0.16b 94dT,2-,8 poly rA (50) 1.20b 706dT,2-,-8poly dA (50) 0.14" 83poly dT-poly rA (50) 0.69" 406poly d(A-T) (50) 0.15" 90polyrA-polyrU(50) 0.11b 65

None-endogenous 0. 14c 100dC,2 ,8 -poly rG (50) 0.17c 125

None-endogenous 0.16d 100dG,2,8-poly rC (50) 0.48d 300

a Measured in picomoles per 50 glliters per 30 min.All assay conditions are described in Materials andMethods. Approximately 12 jig of intracisternal Aparticle protein (MOPC-104E) and Mn2+ instead ofMg2+ were used in each assay at 30 C. A particle DNApolymerase reaction was linear up to 40 min in theseexperiments. Specific activity for each isotope was:3H-TTP, 12,900 counts per min per pmol; 3H-dCMP,4,080 counts per min per pmol; 3H-dGMP, 2,560counts per min per pmole. For each assay 5 gCi ofisotope was used.

I Incorporation of 3H-TMP.c Incorporation of 3H-dCMP.dIncorporation of 'H-dGMP.

3000-2+Mn

'-3

C=

0.-2000 /

100

0 10 20mm

FIG. 5. Optimal concentration of divalent cationrequired for activation of A particle RNA-dependentDNA polymerase. All assay conditions were describedin Methods and Materials.

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ase of intracisternal A particles. Both the opti-mal concentrations for Mg2" and for Mn2+ werefound similar to those reported for murine andavian C-type viral reverse transcriptases (9, 13,23). The Mn2" preference of intracisternal Aparticle endogenous RNA-dependent DNA po-lymerase suggests that it resembles the viralreverse transcriptase, since the stimulation byMn2" is a characteristic of most viral reversetranscriptases (9, 10, 13, 23). It is noteworthythat Mn2" actually inhibits cellular DNA po-lymerase (10). In this respect the endogenousRNA-dependent DNA polymerase of purifiedintracisternal A particles differs significantlyfrom the poly dT polymerase (32) which resem-bled the cellular enzyme associated with murinetissue culture cells described by Weissbach etal. (10, 31) by virtue of Mg2" preference.

Analysis of the products of the endogenousRNA-dependent DNA polymerase reaction.The nature of the DNA products differed de-pending on the duration of the endogenousDNA polymerase reaction as shown in thecomposite Cs2SO4 analyses (Fig. 6). The endog-enous reaction of the purified intracisternal Aparticles used in these experiments was linearup to about 30 to 40 min. The 3H-DNA synthe-sized at the end of the first 5 min was clearlylinked to the endogenous RNA; depending onthe proportion of the newly synthesized DNA toendogenous RNA, the product sedimented atthe RNA density of 1.65 g/cm3 and at the

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FIG. 6. Analysis in Cs2SO4 gradients of the prod-ucts of the endogenous reaction of intracisternal Aparticle DNA polymerase. About 2.0 mg of purifiedintracisternal A particles were assayed for endogenousactivity at 37 C with 100 JACi of 3H-TTP (12.960counts per min per pmol) and 8 x 10- I M of all threeother deoxyribonucleoside triphosphates in a finalvolume of 1.0 ml. Samples of 200 Aliters each werewithdrawn at 5 and 10 min, and the remaining 600;liters were withdrawn at 30 min. The reaction wasterminated by phenol-SDS extraction of the radioac-tive DNA product. Purification of the product andthe Cs2SO4 analysis were described in Materials andMethods.

DNA-RNA hybrid region from 1.50 to 1.55g/cm3 at a ratio of 42:58 with respect to thedistributions of acid-insoluble 'H-DNA counts.At the end of 10 min of endogenous reaction, the3H-DNA counts shifted more towards the hy-brid region (1.55 g/cm3) and the single-strandedDNA region of 1.44 g/cm3 (17, 30), althoughsome counts were still associated with theendogenous RNA (1.65 g/cm3). The ratio ofdistribution of the 3H-DNA counts in the RNAregion versus the hybrid and DNA regions wasnow about 25:75. At the end of 30 min of theendogenous reaction, the 3H-DNA product wasfound to be primarily free DNA. The productsedimented sharply at 1.44 g/cm3 with onlysome overlapping at the 1.42 g/cm3 area.

This product was found totally sensitive toDNAase digestion, and hybridized primarilywith the high molecular weight RNA fromintracisternal A particles. The pattern of ap-pearance of the newly synthesized DNA prod-ucts of the endogenous RNA-dependent DNApolymerase of intracisternal A particles alsoresembles those reported for avian and murineC-type viruses (11, 17, 24, 27, 30).Hybridization analysis with S-1

exonuclease. S-1 exonuclease degrades the un-hybridized region of single-stranded DNA toacid-soluble counts. Its action seems to bespecific for the single-stranded DNA and theenzyme does not digest the RNA-DNA hybridregion (3). Table 3 summarizes the result in ahybridization analysis of the 3H-DNA productof the endogenous RNA-dependent DNA po-lymerase reaction of intracisternal A particleswith the purified RNAs from the same source.Approximately 69 and 88% of the 3H-DNAsynthesized in the presence of actinomycin Dwere found to hybridize back to the 60 to 70SRNA and 35S RNA of intracisternal A particles,respectively. The 3H-DNA product in its singlestranded condition after alkali treatment at 100C was totally degraded by S-1 exonuclease.Neither did it hybridize with heterologous RNAsuch as Q,B RNA. The DNA product of theendogenous RNA-dependent DNA polymeraseof intracisternal A particles therefore showed acomplementarity towards the high molecularweight RNA of the A particles. Detailed analy-sis of the nature of this DNA product synthe-sized by the A particle RNA-dependent DNApolymerase will be reported elsewhere.

DISCUSSIONThe success of demonstrating the association

of an endogenous RNA-dependent DNA polym-erase activity with intracisternal A particles

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TABLE 3. Hybridization analysis of 3H-DNA productof endogenous RNA-dependent DNA polymerase with

RNAs of intracistemal A particles

3H-DNA bound to RNA

Conditions Counts permin per hy- Hybridizedbridization (%)reactiona

3H-DNA input ............. 3,340 1009H-DNA + S-1 exonu-

clease ................... 24 0(3H-DNA-70S RNA) + S-1exonuclease ............. 2,300 69

(3H-DNA-35S RNA) + S-1exonuclease ............. 2,900 88

(3H-DNA + Q,B RNA) +S-1 exonuclease ........ .. 25 0

a All experimental conditions were described inMaterials and Methods. Background count was 22counts/min.

clearly depends on the nature of the particles atthe end of the exhaustive isolation and purifica-tion procedures which free them from cytoplas-mic cisternae. The technical pitfalls enumer-ated above all contributed to the observation ofwhether intracisternal A particles possess com-plete DNA polymerase activities with bothendogenous RNA-directed reaction and adT,2-18 -poly rA-directed reaction or only a polydT polymerase activity under the direction ofdT,218 poly rA. To further complicate the tech-nical problems, the replication of intracisternalA particles cannot be synchronized in either theMOPC-104E tumor tissues or the neuroblas-toma N-18 tissue culture line. These A particlesare continuously budding into the cisternae.The probability of obtaining open or buddingforms of intracisternal A particles potentiallydeficient in high molecular weight RNA, andtherefore endogenous RNA-dependent DNA po-lymerase activity, was great; such open formshave been described in a previous electronmicroscope study (15). Albeit, evidence thus farsupports the fact that intracisternal A particlesdo possess an endogenous RNA-dependentDNA polymerase similar to other viral reversetranscriptases. It utilizes the A particle endoge-nous RNA and synthetic RNA-DNA hybrids astemplates. It possesses characteristics typical ofreverse transcriptase from oncornaviruses suchas Mn2+ preference, a template ratio ofdT 12-18 poly rA/dT,2-15 poly dA greater than9.9, and a moderate stimulation by dG,2 18 polyrC. The purified DNA products of the endoge-nous reaction synthesized in the presence ofactinomycin D showed 70 to 90% base com-

plementarity with the A particle 35 and 70SRNA. Currently, we are examining the homol-ogy of the DNA products of intracisternal Aparticle RNA-dependent DNA polymerase withhigh molecular weight RNAs of several C-typeoncornaviruses. It is possible that the informa-tion derived will demonstrate more definitivelythe genetic relationship of intracisternal A par-ticles with other RNA tumor viruses.

ACKNOWLEDGMENTS

We thank Marian Smith and Harold Peart for theirexcellent technical assistance. We especially appreciate thehelpful suggestions from David Baltimore in the preparationof this manuscript.

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