NATURE VOL. 226 JUNE 27 1970 in, the presence of the four common ribonucleoside ‘triphosphates, the enzyiue incorporated *H-GMP exten sively". At thia pH, however, in the prosoneo of the four deoxyribonueleoside triphosphates, no HLM incorpora- tion was domonstrable (Fable 4). Furthermore, replace. mont of even a singlo ibonnclestide by its homologous deoxyribonneleotide led to no detectable synth ‘unpublished observation), At pH_8-3, the opti the MLV DNA polymerase, the VSV polymerase cata lysed much loss ribonucleotide incorporation and no significant dooxyribonucleotide incorporation could be detected. ‘le 8. aren oF augue ow. nuk DNA rokennasss screens moles tre onions coor {Xe pineabation 200 ‘Preated wth mo adttion 230 FPretecabatod with 30 tin, Ibonusease oe ‘retnabato ih 9 hg, bemtlease oat Proivcibated with 200 le rbopscense OOS Prenat with eto 300 ‘Preiscbated with 0 ol Hbonuseane os Drcnebated with 0 yam soryne 307 Premeabated with 0 pal estocrumee 37 nnn eo rh wi id te ented amu Be ae ale Thexeporation tn 4 lg nce) Procanae a ete tititine hae STEER at ure SS ke TER CKER wan BEB 8 ene hoge de SPREE? aura sane casi teat ala Sitar, Saal Bao itt ‘Sitar eg a emotes Vstindar inndna Ot pr ede neon Siok oe roles Abe MI aatays coutainnt 12 pit of Vial tots ‘he RAMLY per oan only aos slice Rares Pt es ere dn A opin of SEE he ERE Genel gaat aPOe titteBentn ie pte Fe me eid Pa Euan fet eb Snyder ai DNA Polymerase in Rous Sarcoma Virus A preparation of the Pragae strain of Rous sarcoma viras was inssayed for DNA polymerase activity (Luble 3). Incorporation of radiowetivity from *HLTTP was demon: strable and the activity was severely reduced by omission. of either My?* or ATP from the roaetion mixture. RNA dependent DNA polymerase is therefure probably @ eon: stituent of all RNA tumour viruses, "Those experiments indicate that the virions of Rauscher mouse leukaomia virus and Rous sarcoma virus contain & DNA polymerase. The inhibition of its activity by ribo- nuclease suggoats that the enzymo ia an RNA-dopendont DNA polymerase. It seems probable that all RNA tumour viruses havo such an activity. Tho existenes of this enzyme strongly supports the earlier suggestions!” thet taut genetically specific DNA synthesis is an early event in tho replication eycle of the RNA tumour viruses and that DNA is the template far viral RNA synthesis, Whether tho viral DNA (“provirus”)! is integrated into the host genome or remains as @ free template for RNA synthesis will require further study. Tt will also be nocessary to, determine whether the host DNA-dependent RNA poly- ‘erase or @ virus-specific enzyme catalyses the synthesis of viral RNA from the DNA. ‘Tals 5. pxoreemts oF PE nos s4ncoua vines DNA posuEMASE drole TI Reaction aston, Snore fa 18) la omplste 205 Wittont magostum acctste on Without dar oy rity nn eho on om sharma basis ero Ot be aes ne ive ch" prepatsiln was coast andthe pallet daly Sieh ity Guar eare sen eee ce i Se ieee re, 2 Seat hae cia atasrcecine etree SEcuaseh Ghia uses So areas EEA Roane I thank Drs G. Todaro, I. Rauscher and R, Holdenreid for their assistance in providing the mouse leukuemia virus. ‘This work was supported by grants from the US Public Health Service and the Amerioan Caneer Society and was fearried out during the tenure of an American Society Paoulty Research Award, Davin Baxmntore Department of Biology’, Massashusetts Institute of Technology Cambridge, assachusotts 02139, eceid Fane 9, 1970, * Geen, M.A. Ree. Bish. 39 (1970, the pe) 2 Benin Hae Peta 88 4800) * baer 8, Virloy 3 4421988 ‘isle, and Gol, A, Plan 8, B11 (106) " Duosbire iésand Vari, Pe Ke Bow, US Nat ead Si, 8 999 (1960. ‘Remi I ti olny of Lange RSA Virus (a, hy Nar Tey a8 “Says in) (Ate Ma Lay 20 al Acd. Sei, 62,1488 (1068). "Tochom. Boe Be. Coma, 38, 5 aMirianl,S., Yatwe, 26,1211 (1970) followin atl). bo temin “Oto By Racor, dy and ele Ty See 44 C4 afb, TV. and Crawford B-, Virlyy, 18, 227 eH). ‘at Hobinsony We 8 Proc. U6 Net dead. Sel, 8, 310 2 Dui ead Vor, BK, Veto, 88,18 (196), "Damen “does RNA-dependent DNA Polymerase in Virions of Rous Sarcoma Virus InrzoTio of sensitive eolls by RNA. sarcoma viewses requires the synthesis of new DNA different from that synthesized in the S-phase of the eoll eyelo (refs. 1, 2 and unpublished results “of D, Boettiger and HT. M. T.); production of ENA tumour viruses is sensitive to actino mycin D®'; and cells transformed by RNA tumour viruses have new DNA which hybridizos with viral RNAS. ‘These are the basie observations essential to tho DNA provirus hypothesis— replication of RNA. tumour ‘viruses takes place through a DNA intermediate, not122 through an RNA intermediate a4 docs tho replication of ‘other RNA virnsos?, Formation of the provirus is normal in stationary chicken colls exposed to Rous snreoma virus (RSV), eve! in the presence of 0-5 ygjiml. cycloheximide (our un \blished results). ‘This finding, together with Giscovery of polymerases in virions of vaceinia virus and of renvirus*, suggested that an enzyme that would, gynthosize DNA from an RNA template might be prosent. in virions of RSV.” We now report data supporting the existones of such ‘an enzyme, and wo learn that David Baltimore has independently discovered a similar enzyme in vitiona of Bauschor leukwoonin virus, 4 o° wa 3 wo ins tn) om gt, Kluge of ingcrparaton. ‘lhiginrttel ato" Cand tneubated us Seid taal as ap pole feet it a is, Pi sit "Tho soures of virus and methods of eoncontration have toon desorbed! All poparations were eeried out i orte conditions. ‘Concentrated. vis was placed fayer of 18 per cent eucrose and centrifugal ae 25,000 pun, for 1 f'n the ‘SW 20,1 rotor of tho Spinco slr Conitige on to a cushion of OD per cent avers. The ‘ius band was clleted from the interphase und further Durifled By equilibrium sucrose denny gradiont conte Eaton.” Vitus furiher puriied "by ‘tuerose velocity naity gradient centifagtion gave the sane resus ‘Table 1. acrivaniox oF usa SLEEP sr system poated (aan) ‘Visions erpted with Nook” AtoreDIT 00 Ato Der S20 Ateeprr 6.000 Aco oDrr Peart staat atten Sais ‘Fatal (DI) Sion) wore aye tn the waar polymerase ‘The polymerase amay consined of 0125 ymoles euch of dab, dCTP, and GIP (Cabbie) “in 002 3t ‘Taig buler at pH 890, containing 0:39 M EDTA and Tr mM 2mercaptocthanl)y 1-25 gioeles of MgCl, ane NATURE VOL. 226 JUNE 27 1970 2-5 moles of KCl; 2-5 ug phosphoenolpyravate (Calbio. chem); 10 pg pyruvate Innase (Calbiochem); 2-5 Ci of SH-TTP (Schwarz) (12 Ci/minole); and 0025. mul. of fenzyine (10* focus forming units of disrupted Schmidt. ‘Ruppin virus, Asie 9m = 0-30) in a total volume of 0-125 a1 Tneubation was at 40° C for Lh. 0-025 ml. of the reaction mixture swat withdrawn and assayed for acid-insalable ‘cunts hy the method of Furlong "To observe full activity of the enzyme, it was necessary to treat the virions with a nonionic detergent (Tables 1 and 4), Tf the irealment was at 40° C the presence of dithiothreitel (DTT) was necessary to recover activity ‘Tn moat preparations of virions, howovor, there was some activity: 5-20 per gent of the disrupted virions, in the “absence of detergent treatment, which probably ropresents disrupted virions in the preparation, Tt is known that virions of RNA tumour viruses arc easily dlistupted!*", $e that the activity i probably preseat i the nuclei of ‘eble 2. wagumnNsre FoR EsaOHR ACHE LTP noo system porated apn) Complete sens ‘without Mac 180 Withont MC eh Ma 0 Without Mac th Ca 18 Without aT? s ‘wineat aor 2100 “Virus treated with ‘Nona a alata at O° was iocatate in tog sitar palfmerane asey with the Stet se ‘Tho kinotics of incorporation with disrupted virions ars shown in Fig. 1. Incorporation is rapid for 1h. Other experiments show that incorporation continues at about tho same rate for tho second hour. Preheating disrupted virus at 80" C prevents any incorporation, and 50 «loos pretreatment of disrupted virns with erystalline trypsin. ym (109 . ow! gC coeanraton (m1) ig, 2, Matta eauinment, Views teat witn “ould” eu de ifaliy AUSe Sait Ine staan paso say hy tel ae OT Cw nt eoncenesone af Ma Fig, 2 demonstrates that thore is an absolute require: ‘ment for MCly, 10 mM being tho optimum eoncontration. ‘The data in Table 2 show that MnCl, can substitute for ‘MgCl, in the polymerase assay, but CaCl, cannot. Other experiments ‘show that a monovalent. cation’ is not required for activity, although 20 mM KCl eauses 2 15 perNATURE VOL. 226 JUNE 27 1970 ‘cont. stimulation. Higher concentrations of KCI are inhibitory: 60 per cent inhibition was observed at 80 mM. ‘When the amount of disrupted virions prosent in the polymerase assay was varied, the amount of ineoeporation Naried with second-order kinetics. When incubation was, carried! ont at different temperatures, @ broad optimum, between 40° C and 50° C was found. (The high tempera- turo of this optimum may relate to the fact that the normal host of the virus isthe chicken.) When incubation ‘was carried out at different pHs, a broad optimum at pH 8— 8:5 was found. ‘Table 2 demonstrates that all four dooxyribonucleotide triphosphates are required for full activity, but some tetivity was present when only three deosyribonucleatide triphosphates wore added and 10-20 per cent of fall activity was still present with only two deoxyribonucleo: tide triphosphates. The activity in tho presence of three dooxyribonucleotide triphosphates is probably the result bf the presence of deoxyrihonucleotide triphosphates in the vition. Other host components are known to. be incorporated in the virion of RNA tumour viruses!®™, ‘able 5. TENA pevespasce op rourumnase scmvine LTTE cor porated as) oun0 2850 ‘rratmnt {Nov-aeated daunted wien Dhanupted vino pevdnonbatsd with boneless A (50 fraind) at 50°C tor th ala ite lacie wth nam 60 aim at Gt ee gested with tonite A Bell ‘ 1 9800 en, Sue ik ceca A, ea {lstbtation i fe peciod onions, a wear polymerase toy = rhe data in Table 8 demonstrato that incorporation of thymidine triphosphate was more then 99 per cont abolished if tho virions were pretreated at 0° with 1 mg Tibonsiclease per ml. Treatment with 50 ug/ml. ribo- nuclease at 20° C did not prevent all incorporation of thymidine triphosphate, which suggosts chat tho RNA of tho virion may be masked by protein. (Lysozyme was added as « control for non-specife binding of ribonuclease to DNA.) Because the ribonueleaso was heated for 10 min at 80° C or 100° C before use to destroy: deoxyribe rmuolease it seoms that intact RNA is necessary for incor poration of thymidine triphosphate ALTP neon Ssouce porated ain) Visions of SRY 110 Disrupted visors of SRV 573 irons of MY 2819 Disrpted iio of ADV 1 's0 Disrpted pellet om supernatant iad ale ‘irony of Seamt-upyin vu (SHY) were prepare as before (exer sucat SE Eatie" Wins of aan ried Str ci) a ei fiom atoted ele were. topes renal sna Letptt prperatlom wee utted with Moridet a dition Mt PENS ed samantha nes he ail ae et {ie wh realy fom oP for SRY SOI Tor AM aod Bor nieces el ‘Vo detoriaine whether the onzyane is present in super- natants of normal cells or in RNA leukaemia virases, the experiment of ‘Table 4 was performed. Normal cell Supematant did not contain activity even after treatment With "Nonidet’. " Virions of avian myeloblastosis virus (AMY) contained activity that was increased ten-fold by treatment with ‘Nonidet. ‘The nature of the product of the polymerase assay was investigated hy treating portions with deoxyribomelease, i2ia ribonuclease or KOH. About 80 per cent of the product, ‘was made acid soluble by treatment with deoxyribo, nuclease, and the product was resistant to ribonuclease, and KOH (Table 3). ‘Table 6 NATURE oF PRODUCE eid aitinateie TEE apm.) ‘Treatment Eegetineat k Exponent ar 000 8800 Deosstbonuclease on 1.500 boneless 103900 Zao uname ected Se wa tin a Sra mance ath ate aati Rls geo eaamarca tenuate ‘emanated for ae sole soma . ‘To detormine if the polymerase might also make RNA, Aisrupted virions wore sneubated with the four ribo nucleotide triphosphates, including *H-UTP (Schwarz, 32 Ci/mmole). With either MgCl, or MaCl, in the incu: Dation mixture, no incorporation was detected. In a parallel incubation with deoxyribonucleotide triphos- phates, 12,200 d.p.m. of *H-TTP was incorporated. "Those results demonstrate that there ita new poly- ‘merase inside the virions of RNA tumour viruses. Ttis not present in supernatants of normal eolls but is present. in, vinous of avian sarcoma and leukaemia RNA tumour viruses. The polymerase seems to catalyse the ineorpora- tion of deoxyribonucteotide triphosphates into DNA from. an RNA template. Work is being performed to charae- terize further the reaction and the product. Tf the present. rowults and Baltimore's results! with Rauscher leukemia, virus are upheld, they will constitute strong evidence that the DNA provirus hypothesis is correct, and that RNA ‘tumour viruses heve a DNA’ genome whon they are in cells and an RNA genome wher they are in virions. This result would have strong implications for theories of viral carcinogenesis and, possibly, for theories of information transfer in other biological systems ‘This work was supported by e US Public Health Servico research grant’ from the National Cancer Tnstitute, HM. T. holds @ research enresr development award frot ‘tho National Cancor Institute. Howarn M, Taxus Sarosii. Mizczant McArdle Laboratory for Cancer Research, University’ of Wisconsin, Madison, Wisconsin 58706. esived Sune 15,1070, "Tomi HM, Caner Be 8, 1855 (4086. 4 Meta Hy a em, 3, acer J Cee (the pes) ‘umn He Polo 20,677 lb) ‘alud, At aod Nayak, DPJ. Voy 4554 1000), ‘Tani HL Me” Proc. 0S Nat dd. Se 88, 989 (1068) "ud td agate ily of Dae J 4 Vir et ‘Bate .2and Suhy, hj Academie Pas Londen Ato) “ {tei Ht, Kats Cancer rt ona 2,65 60 s7.s14080, eat St 38 Seno, Jai Gata, A. 8, Blom, Bopha, Rt, Comm 9 86 ‘sunt, A J and Sine, Da, Poe. CS Not, dead Se, 482 1088. (nein sti). * balino‘e, iy are 228, 1200 C07 Aland To ak Vit 4008. “Ronan, Ws. lta, A and Rainy BC, Proc. 8 Net. Acad Set, SAE bad : oslo, Nea Meth, Cane Be 82 ‘No Po de Prune, 2081965) Bates He, a Sar, War Vita 2, 494 (180). ‘Bauer, HE) Z, Datereh Bt, (30 G98) ‘gun, I, italy 9,124 4900). ‘Pela, Hey Porap.Bil. Md. in E pres).