Reprinted from the PROCEEDINCB OF THE N.4~ios.1~ .\CAUEMY OP SCIEYCE~; Val. 54, So. 3, pp. 919-927. September, 1965. THE SYNTHESIS OF d SELF-PROPAGATING AND INFECl'IOU8 NUCLEIC ACID WITH d PURIFIED ENZYilIE* DEI' IRPMEIVT OF MICROBIOLOGY, I'SIVERSII'T OF ILLINOIS, URB 4% 1 Communicated July 23, 1965 The unambiguous analysis of a replicating mechanism demands evidence that thc reaction being studied is, in fact, generating replicas. If, in particular, the concern is with the synthesis of a viral nucleic acid, data on base composition and nearest neighbors are not sufficient. Ultimately, proof must be offered that the polynu- cleotide product contains the information necessary for the production of the cor- responding virus particle in a suitable test system. These conditions impose severe restraints on the type of experiments acceptable as providing information n hich is irrefutably relevant to the nature of the replicat- ing mechanism. Clearly, the enzyme system employed must be free of interfering and confounding activities so that the reaction can be studied in a siniple mixture 920 BIOCHEAIISTRI': SPIEGELJIAS ET ;1 L. Paoc. ?;. A. s. containing only the required ions, substrates, and templates. Since the biological activity of the product is likely to be completely destroyed by even one break, the elimination of nuclease activity must be rigorous indeed. The purity required im- poses the necessity that the eiizyiiiological aspects of the investigation be virtually completed before an examination of niechariisrii can be safely instituted. We have previously reported' the purification of two distinct RNA-depeiideiit- RNA-polymerases (designated "replicases" for brevity) induced in the same host by two unrelated?, RNA bacteriophages (AIS-2 and Qp). It was showii that under optimal conditions, both enzyriieq are virtually inactive with a variety of heterol- ogoui ItKA species, iiicludirig ribosomal and sRNA of tlie host. E'urther, neither replicase can fuiictioii with the other's RNA. Each erizynie recognizci the RS.\ genome of its origin aiid requires it as a template for normal synthetic activity. exhibited' the following distiiictivc features: (a) freedom from detectable levels of the DNA-depeiiderit-R~-A-l)olyiiierase, ri- bonuclease I,4 ribonuclease II,j and RXA phosphorylase; (b) coni~)lete dependence on added RX-4 for synthetic activity; (c) competence for prolonged (more than 5 hr) synthesis of RNA; (d) ability to synthesize many times the input teniplates; (e) saturation at low levels of RNA (1 y RNA/40 y protein); cf) virtually exclusive requirement for intact homologous template under optinial ionic coiiditions. The discriniiriatirig selectivity of the replicase permitted a simple test of similarity between template and product. Haruna and Spiegelmanfi showed that when reac- tioris are started at late coiiceiitratioiiq below those required to saturate the enzyme, RNA syri t h 1lo.w~ an autocatalytic curve. When tlie haturation con- centration level is re the kinetics become linear. The autocatalytic behavior below saturation of the erizynie implies that the newly synthesized product can 111 turn serve as templates for the reactioii. To test this coriclusion directly, the pro- duct was purified from a reaction allowed to proceed until a 63-fold increase of the input RIVA had The ability of the newly synthesized RNA to ini- tiate the reactioi examined in a saturation experiment arid found to be ideii- tical to RNA isolated from virus particles. It is evident that the sequences cni- ployed by the enzyme for recognition are being faithfully copied. The findingh summarized above and the state of purity of the eiizymes eiicour- aged us to enter the next phase of the investigation and examine the infectivity of the synthesized material. It is the purpose of the present paper to describe ex- periments demonstrating that the RNA produced by replicase is fully competent to program the production of complete virus particles. The data establish that the reaction being studied is indeed generating self-propagating replicas of the in- put RNA. The bacterial viruy employed is Qp, isolated by Wataiiabe.2 The host and assay organism is a mutant Hfr btrairi of E. coEz (Q13) iwlated in the laboratory of W. Gilbert by Diane Vargo. This bacterial strain ha\ the convenient property7 of lacking riboriuclease I and RNA phosphorylaae. The preparation of infected cells and the subsequent isolation and purification of the replicase follows the detailed protocol of IIaruna and Spiegelman.1 The preparation of virus stocks and the purification of RNA from them follow the methods of Uoi arid Spiegelman.* (2) The assay of enzyme activity by zncorporation of radzoactzve nucleotides: The standard rear- tion mixture is 0.25 ml and, in addition to 40 y of enzyme, contain3 the following iii pmoles: Tris ITCI, pH i.4. 21 : JIgCl?, 3.2; CTP, .4TP, UTP, arid GTP, 0.2 each. The reaction is ter- Iri summary, the purified replica mulated. Materrals and ,2lethods.-( I ) Bzological system and rnzyrne preparation: VOL. 54, 1965 HIOCHE'MISI'RY: SPIEGELMAN ET AL. Y2 1 minated in an ice bath by the addition of 0.15 in1 of iieulralized saturated pyrophosphate, 0.16 ml of neutralized saturated orthophosphate, arid 0.1 ml of 80% trichloracetic acid. The pre- cipitate is transferred to a membrane filter and washed 7 times with 5 ml of cold lOu/, TCA. The membrane is then dried aiid counted iri a liquid scintillatioil counter as described previously. UTP3* was synt,hesized as described by Haruna et aL9 It was used at :t specific: activity such t#hat, the iricorporatioii of 20,000 cpm corresponds to the synthesis of I y of RPU'A, permitting the use of 20 X samples for following the formatioii of labeled ILNA. Samples removed froni the reaclioii mixture are piaced immediately in aii ice bath arid 20 X removed for immediate assay of radioactive RNA as described in (2) above. The volume is then adjusted to 1 nil with TNI buffer (10P M Tris, 5 x 10-8 M IllgClz, pII 7.5). One mi of water-saturated phenol is then added and the mixture shaken iii heavy wall glass cetitrifuge t,ubes (Sorvall, 18 X 102 mm) at 5°C for 1 hr. After separation of t'he wat,er phase from the phenol by ceiitrifugation at 11,000 rpm for 10 min, ariot,her 1 ml of TR.1 buffer is added to t,he pheiiol which is then mixed by shaking for 15 inin at 5°C. Again, t,he pheiiol and water layers are separat>ed, aiid the two water layers combined. Phenol is eliminated by two ether ext,racLioiis, care being takeii t'o remove t'lie phenol from t'he walls of the centrifuge tsubes by conipletely filling t'heni with ether aft'er each extractiori. The ether dissolved in the water phase is theii removed with a st,reani of nitrogen. The RNA is precipitated by adding vol of potassium ace6at.e (2 M) arid 2 vol of cold absolut'e ethanol. The samples are kept for 2 hr at -20°C before being centrifuged for one hour at 14,000 rpm in a Sorvall SS 34 rotor. The pellets are drained, aiid the reniainiiig alcohol is removed by storing urider reduced pressure in a vacuuiii desiccator for 6-8 hr at 5°C. The RNA is t>lieri dissolved in 1 ml of bufler (10-2 M Tris, TCA- precipitable rtidioiiclivity is measured oii 20 A aliquot's of t.he final product from which the per cent recovery of synthesized RNA caii be determined. In the range of 1-8 y, it, was found that, in general, 65% of t8he synthesized RNA was recovered. All purified product,s were examined for the presetice of ititact, virus particles by assay 011 whole (.ells and none were found. (4) The assay for irLj'ectivify os the synthesized RNA: The procedure used is a modification of the spheroplast method of Gut hie aiid Sinsheinier.lo (a) Medium: The medium used is a modificat,ioii of the 3XU medium of Fraser arid Jerrel" and requires in grttms/liter the following: Na211P04, 2 gm; KHzPOd, 0.9 gm; NH4C1, 1 gm; glycerol (Fisher reageiit), 30 gni; Difco yeast extract, 50 mg; casamino acids (Difco vitamin- free), 15 gm; LmethioIiiiie, 10 my; U,L-leuciue, 10 mg; ILzgSO4.7 HzO, 0.3 gm. These com- ponents are mixed iri the order indimled iri 500 ml glass-dist,illed wat.er. To this is firinlly added another 500 1111 coutjairiiiig 0.3 nil ikl CaCI2. (b) Sucrose nut~ient broth (SNB) coutairis iii grarns/lil~er the following: casamino acids (Difco), 10 gm; iiutrierit brot,h (IMco), 10 gm; glucose, 1 gm; sucrose, 100 gm. After autoclaving, the followiiig are added aseptically: 10 nil 10% MgSO4, and 3.3 nil 30% bovine serum albumin (BSA) from Aruiour LaboriLtoious RNA. However, the optimal prot,aminr coticetilratmiou iti (.he present system is corisiderahly lower t,han t,hat, used by Pararichych. The RNA infection is usually carried out at rocm 1,cmperature with solutious oont,airiiug 0.5 y of RNA/Inl, :t concenl~ra.~ ion at which t8hr :tssay is iiot limit'ed by t8he number of spheroplasts per (3) Isolation 0.f syvdhesized product; lll MgCI,, pH 7.5) atid samples are renioved immediately for infectivity assay. The necessary comporierit,s are as follows: (c) Eeayenfs reyuired for fhe production os spheroplasts: !?22 HIOC'HEMISTItY: S'PIEGBLMAIV ET' .4L. PHOC. 5. A. S. infectious unit. To 0.2 ml of RNA is added 0.2 1111 of llie spheroplast st,ock coiit,aiiiitig about S X 10i spheroplasts. The samples are mixed, and iriiiiiediatJely aii aliquot, is removed arid diluted appropriately through SNB before platiiig OII ],-agar using Q1:Z as t,lie iiidic:at,or. The soft, agar (0.77,) layer eniployed (2.5 ml) coiit,aiiis 1Uy1~~ sucrose; 0.1 yo hlgSOa; and 0.01 in1 of SOY, BSA per tube plus 0.2 in1 of an overnight culture of QlS. To ol)t,aiii reproducit)ility, the spheroplast, stock is used 15-4.5 niiu after dilulioii iiito (lie SNH. 1Cfbcielicy of platiiig (e.0.p.) is usually 2-8 x Higher e&-ieiicies (>1 X IOP) can be oblaiiied if t,he sptieroplasl stock is eiiiployed imriiediately aft.er diluhii :tiid t)y iiicludiiig a stabiliaat ioti period iti the SNH dilutioti tubes, rather t,hati platiiig immediately. However, t8his higher platliiig eficieiicy decays rapidly, making it diffirult to obtain reproducible duplicat es iii repet.itive assays. Since reproducibility was of greater concerii t,hm eficieric*y, the assay riietliod delailed :hove was employed. Results.-Iii designing experiiiieiitjs which involve infectivity assays of the ctixy- rriatically synthesized RXA, it is iiiiportarit, to recogriixc tlint evcti highly purified enzymes from infected cells, although tlciiiotist rahly devoid of intjac*t (:ells, are likr:ly to include some virus particles. Chemic:ally, the coiitamiiiat ion is trivial, amourit- iiig to 0.16 y of nucleic acid :tiid 0.8 y of prot,oiii for eavh 1,000 y of eiizyiiie protein einployed in t,he present st,udies. Sinre 40 y of pivtc'iii :we used for t'wh 0.25 ml of reitctioii, t,he contributiori to the total RK;\ I)y the particles is only 0.006 y, which is to be compared with the 0.2 y of iiiput HNA :tiit1 t,lic :3-~20 y syiithesized in the usual experimetit. It wits sliowii in coiit 1.01 experinietits that, RNA frtdily extract,ed from particles in the rrac:t,ioii tiiixtrirt. is no iiiorc iirfcclive t,hati t lint ot)t:thied fro111 t,he usual piirifictl virus preparat ion. P'url her, the niatid:tt,ory i.equiretrient, for added RNA proves t hat,, withiti t tie iti(*ubat,ioti t iriies used, t.his sniall amount of RNA is either in:ttieyua(>c: or u~i~~vaiI:ihle lor thv itiitiatioti of tlitb reactioii. Thus, these particles do not, sigiiificaiit ly iiifluetic:c either the cheniiwd or (,lie eiizyiiiatic: aspects of t,he cxperiiiierit . Ho\vever, because of their higher iiifec:t,ive efficiency, even moderat,e aiiioutits of ititact, viriis cannot he tolerated iti the examinations of the synthesized RNA for infectivity. Coiisequeiitly, :t11 RXA preparations were phenol-treated [Methods, (3) ] prior to assay. Further, the phenol-purified RNA was routinely tested for wholo virris p:trtivIrs :tiid notie \vert' fourid iti the experi- ments reported. We now uridertalcc to describe experiiiieiits in wliicli the kiiietics of t,he appear- ance of new RNA and infective uiiit,s were examined in t'wo different ways. The first, shows that t8he accumulation of radioactive ltSA is accompanied by a propor- tionat,e increase in infect'ive units. The second type proves by a serial dilution ex- periment t,hat the newly synt hesizcd Rh-A is infective. To compare the appearance of new RNA and infectious uiiit,s in an extensive synthesis, 8 ml of reaction mixture was set up containing the necessary components iri t,he conceritrations specified in Methods (2). Aliquots mere taken at the times indicated for the determination of radioactive RNA and piirifieat,ion of the product for irifect'jvit,y assay. The results are summarized in Figure 1 in the form of a semilogarithmic plot against, t,ime of' t,he observed increase in both RNA and infectious units. Further details of the experiment,al protocol are given in t'he corresponding legend. The amount of RNA (0.8 y/mI) put in at zero iime is well below the saturation level of the enzyme Consequently, the RNA increases autocatalytically for about the first 90 min, followed by a synthesis which is linear with time, a feature which had been observed previously.6 It will be noted that; the increase in RNA is (I) Assay of infectivity of the putified product: VOL. 54, 1965 BIOCHEMISTRY: SPIEGELMA N ET d Id. 923 60 120 IBO 240 MINUTES FIG. 1.-Kinetics of RNA syrithe- sis and formation of infectious units. An 8-ml reaction mixturc was set up containing the components at the concentrations specified in Methods (2). Samples were taken as fol- lows: 1 ml at 0 time and 30 min, 0.5 rnl at 60 min, 0.3 ml at 90 min, and 0.2 ml at all subsequent times. 20 h were removed for assay of incor- porated radioactivity as described in Meth,ods (2). The RNA was purified from the remainder [Meth- or+ (3)], radioactivit,y being deter- mined on the final product to moni- tor recovery. Infectivity assays were carried out as in Methods (4). paralleled by a rise in the number of infectious units. During the 240 min of incu- bation, the RNA experiences a 75-fold increase, arid the infectious units experience ii 3.5-fold increase over the amount present at zero time. These numbers are in agreement. wit.hiri the awur:tcy limit,s of the infectivity test. Experimerit,s carried out wit>h other enzyme I)rcparations yielded resiilt s in caoniplet,e accord with those just described. It is clear t,hat one c:m 1)rovide cvideiice for atli iric:rease in t,he number of infectious units which parallels the appearaiic~ of newly synthesized RSR. (2) Prooj that th,? new:l?g spfhesized RNA inolecules are injective: The kind of experinient~s just descv-ihcd oficr i)lausihle rvidciiw for irifevtivit,y of the radioactive RNA. They aro not, ho~vwcr, (+oii(-lusive, siliw they do Iioi eliniinat,e the possi- bilit8y that, the agrccmerlt, observed is fort,uitoiis. One caould argue that' t'he enzyme is "artivatirig" the irifcc6vity of the input RNA while synthesizing new noninfec- tious RKA arid that the rather c.ornplex exponeritjial arid linear kinetics of the two processes happen to coinride by chance. Direct proof that, the ~iewly syrit,hesized RNA is ilifectious can in principle be obtained by experinierit,s which use S'5-H3-laheled initial tetnplat,es to geiierat,c N14-P32-labeled product. The two (:an t,hen he separated* in equilibrium density gradients of Cs2S04. Such experirnents have been carried out for ot'her purposes, arid will be described elsewhere. However, t,he steepness of t,he Cs2SOI derisi1,y gradients makes it diffic.ult to achieve :I separation clean enough to hr cwnpletely sat,isfying. There exist's, however, another approach which bypasses these technical difficul- ties atid takes advantage of the fact t,hat we are dealing wit>h a self-propagating eii- t,ity. Consider a series of tubes, eavh containing 0.25 ml of the standard reaction mixtiirc, but no added templat,e. The first tube is seeded with 0.2 y of QP-RNA and incubated for a period adeyutite for t,he synth everal y of radioactjive RNA. An aliquot, (50 A) is then transferred to nd tube which is in in turn permitted to synthesize about) the same amount of RNA, a portion of which is again transferred to a third tube, arid so on. If each successive synthesis pro- TABLE 1 SERIAL TR.4NSFER EXPERIMENT 1 Transfer no. 0 1 2 3 4 6 7 8 9 10 11 12 13 14 15 3 2 Interval (min) 0 40 4( ) 40 40 30 3 0 30 30 a0 :30 20 20 2 ) 20 20 3 Time 0 40 80 120 160 190 220 250 280 3 1 0 :340 360 380 400 420 440 -------Formation of Rh-A----? 6 7 4 5 Cpm Total x 10-3 (Y) 0 0.2 64 3 2 84 4 2 112 3 7 134 6 7 113 ,5.7 144 7.2 150 7.5 162 8.1 164 8.2 156 7.8 134 6.7 121 6.0 123 6. 1 118 5.9 13 3.6 7- A (Y) 0 3 . 0 3.6 4 . 9 .i . 6 4.4 6.1 6.1 6.6 6.6 6.2 ,5 . 1 4.7 4.9 1.7 2.4 z (7) 0 :I . 0 6.6 11.5 17.1 21.5 27.6 33.7 40.3 46.9 3.1 58.2 62.9 67.8 72.5 74.9 Formation of It? ----Concentration of Orininal Terndate----? 11 12 - 8 9 . 10 Y 2.0 x 10-1 2.0 x lo-' 4.0 x 10-2 6.i X 10-" 1.1 x 10-J 1.9 x lo-' :3. 1 x lo-" 5.1 x 10-6 8.6 x lo-' 1.4 x 10-7 2.4 x 10-8 4.0 x 10-9 6.6 x IO-lo 1.1 x 10-'0 1.8 x 10-11 :3.1 x 10-12 Strands 1.2 x 10" I .2 x 10" 2.4 x 10'" 4.0 x 109 6.6 X loH 1.1 x 108 1.8 x 107 3.0 x 106 *5,0 x 105 8.4 x 10" 1.4 x 105 2.3 x 102 :3.8 x 10' 6 I 0.16 IU 6.0 x 104 6.0 X 10' 1 2 x 10L 2 0 x 10J 3 3 x 102 55 9 15 <1 <1 <1 <1 <1 (1 <1 <1 A x 10-5 1.0 5.2 2.2 11.3 5.7 7.4 15.0 13.4 8.8 5.6 9 . 3 6 .3 6 9 3.6 10.8 2.2 2 x 10-5 1 . 0 5 , 2 6.5 17.4 21.2 27.6 36.4 48.1 54.7 58.4 66.8 73.7 84 3 89.4 102 , 0 105.0 13 Observed e.0.p x 10-7 5.5 3.2 2.0 5.3 3 . 0 3.0 3.7 5.0 5.2 2.0 4. 0 3 . 7 70 4.0 5 , 7 5.5 14 YG Recovery of P~Z-RN~ 54 2 88 3 .59 9 42 3 63 4 H2 4 52 9 51.4 92 6 34 2 74 3 46 h 49 2 79 7 65 4 ... Sixteen reaction mixtures of 0.25 in1 \yere set up, each cuntaininl: 40 5 of protein and tlie other components specified for tlie "standard" assay in Xethods. 0.2 y of template RN.i Then 50 X of tnhe 1 \yere transferred to tubr Every tube hfter tl:e transfer from a given tube. 20 X were removed Control tubes inrubated for 60 min without the Columns 1, 2, and 3 give the transfer number, the time interval permitted for synthesis. and the elapsed time from zero. re- Column 4 records the amount of radioactive RNA found in each tube at the end of tlie incubation, column 5 the total RNA in each, and 6 gives the net synthesis The decreasing concentrations of the input RNA resulting from tlie serial dilutions are recorded in The last is calculated from colnmn 9 and from an efficiency of plating (e.o.p.) of 5 X Colnmn 11 lists the increment in infections units observed during escli period of synthesis, corrected for the efficiency of recovery (col. 14). and column 12 represents the Column 13 is the plating eflicimcy (e.o.p.1 (letermined from the observed nrirnher of plaqnes (col. 11) and tlie actual amount of RNA assayed as determined were added to tubes 0 and 1: RNA was extracteil from tlie former immerliately, and the latter ,vas allon-ed to incubate for 40 min. 2, whicli \vas incribated for 40 min. and 50 h of tribe 2 then transferred to tribe 3. and so on. each step after the first involving a 1-6dilution of theinput material. was transferred from an ice bath to the 35°C water bath a few niiniites before use tir permit temperature eqiiilibration. to determine tlie amount of I'aZ-RN.\ synthesized. and tlie product nas purified from tlie remainder as described in Methods. addition of the 0.2 y nf RNA slioir-ed no detectable RN.1 synthesis, nor any formation of infectious iinits. spectively. durinr tlie time interval. terms of y (col. 8). number of strands (col. 9). and infections iinit,s (IU) per tuhe (col. 10). 10-7. corresponding sum. from rdiiinns 6 and 1-1. All recorded niimbers are normalized to 0.25 ml. Column 7 lists the cumulative synthesis of RNA. Column 14 is determined from assay8 of acid-pit.rlpitahle radionrtivity on 20 X aliqiiots of tlie final product as compared with coliimn 5. VOL. 54, 1965 HIOCHEAfIS1`RY: SI'IEGELMLIN ET .LL. 75 2 50 IT s 25 MINUTES 300 400 5c /*-- 200 l....I.... I I 5 IO 15 TRANSFERS 100 90 80 - X - 70 VJ e 50 w IT w IT 60 Z 3 40 2 30 20 IO a MO-5 FIG. 2.--RNA synthesis and forrnatiori of infectious units in a serial transfer experiment,. All details are as described in the heading to Table 1, and the data are taken from columns 7 and 11 and plotted against, elapsed time (col. 3) and corresponding transfer number (col. 1). Both ordi- nates refer to amoiints foiind in 0.25-ml aliqiiots. duces RNA which can servc t,o initiate the next one, the experiment can be con- tinued until a point is reached at which the init,ial RNA of tube 1 has been diluted to an insignificant level. In fact,, enough transfers can be made to ensure that the last tube cont,ains less t>han one st)rand of the input primer. Ij" in all the tubes, in- cludin,g the last, the number of infectious units corresponds to the amount of radioactive RNA found, convincing evidence is offered that the newly synthesized RNA is infectious. Table 1 records a complete account of such a serial transfer experiment, and the corresponding legend provides the details necessary t,o follow the assays and calcu- 11 tubes are involved, t,he first, (tube 0) heir1 gan unincubated zcro time corit,rol. It will be noted that t,he successive dilution was such (1-6) that, by t,he 8t,h tube, there was less t]han one infectious unit, ascribable t,o the initiating 0.2 y of RXA. Nevertheless, t.his same tube showed 8.8 X lo5 newly synthesized in- fectious units during the 30 niiri of its incubation. Finally, tube 15, which containcd less than one strand of the original input, produced 1.4 x 1OI2 new strands arid 3.2 X lo5 infectious units in 20 min. It should he noted that a corit,rol tube lacking added RNA was incubated for (50 niin. As compared with tube 1, which incor- porated 4800 cpm for each 20 X in 40 min, the control showed no increase above the zero time level of 80 c:prn. l+irt8her, no synt,hesis of infective unit,s was observed in such ront,rols. Figure 2 coinpares the c:umulat,ive inrreinent8s wit h hic in newly synthesized RNA (column 7) and infect,ious units (column 12). The agreement between in- crements in synt>hesized RNA and newly appearing infectious unit,s is excellent at, HIOCHEMISII`RY: SPIEGELMAN E?` -4lJ. PROC. N. .4. S. TARIdE 2 SEROLOGICAL BEHAVIOR OF VIRUS FORMED IN RESPONSE TO "SYN rHmIc'' RNA Authentic Virus from QP 1.9 X 10* 1.0 X los 0.052 1.1 X 108 1.06 X 108 08 synthetic RNA 1.5 X 10* 8.8 X lo4 0.053 1.5 X lo8 1.40 X lo8 Yd In all cases, lysate8 were made from E'. coli Q13, which was also the assay organism. .intisera were used at 1/100 dilution, and the inrnhation temperatiire was 35OC. The niimhers represent plaque formers per ml. every stage of the serial t)ransfer -atid contitiues to tjhe last, tube. Long after the initial RNA has been diluted t'o insignificant levels, t>he RNA from one t,ube serves to initiate synthesis in the next. E'urt,her, as may be seen from the comparative constancy of the infective efficiency (Fig. 2 and colurnn 13 of Table l), the new RXA is fully as competent :is the original viral RNA t,o program the snythesis of viral particles in spheroplasts. To complete t,he proof, it was necessary to show that t,heviruses produced by the synthesized RNA were indeed Qp, the original source of the RNA used as a seed in tube 1 to itlitmiate the transfer expel-imcnt,. Since Qp is a unique serological type,2, a t,his charact,eristic was chosen RS a vonvenietit, diagriost ir test.. Plaques induced by the K.NA synthesized in t,rihe 1.5 were used t,o produce lysates, and t>he resulting particles exposed t>o atnt~isera against, 14s-2 and &a. The results, briefly summarized in Table 2, show c!le:trly that, tjhe synthetJic RNA indures virus particles of the same serological type as aut#hentic Qs. Discussion.---One perhaps might have imagined that, an erizyrne carrying out a complex copying process would show x high error frequency when funct,ioning in the unfamiliar environment provided by tjhe enzymologist. Had this heeri a quantita- tively significant complication, biologically inactive st'rands should have accumu- lated as the synthesis progressed. That this is riot, the case is rather dramatically illustrated by the serial transfer experiment (Table 1 and Fig. 2). The RNA synthe- sized after the 15th transfer is as competmt biologically as the initiating "nat- ural" material derived from virus particles. The successful synt,hesis of a biologically active nucleic acid wit.h a l'urified erixyrrie is itself of obvious interest,. However, the iniplication which is niost pregnant with potential usefulness stems from tthe demonstratlion that the replicase is, in faci,, generating identical copies of the viral RNA. For the first t,ime, a system has been made available which permit>s the unambiguous analysis of the molecular basis 1111- derlying the replication of a self-propagating nucleic acid. Every step and com- ponent necessary to cornp1et)e the replication must be represent'ed in the reaction mixture described. If two enzymes are required,IY both must, be present and it should be possible either to establish their existence or t'o prove t'hat' one is sufficient. If an intermediat,e "replicating" stage int,ervcwes between the template and the ul- timate identical copy,I3 t'heri a "replicat>ive fot-ni" should he dcmoristrably present in tlhe react'ion mixture. If copying is direct, no such intermediate will be found. These and other issues of the replicat,ing mechanism will be discussed in a. subsequent publication which will detail the relevant experiments. Vor,. 54, 1965 BIOCHEMISTRY: SPIEGELMAN ET rl L. !42i Sur,Lir~a/.y.-Exi)eriiiieiit,s are described with a purified RNA-dependent,-RNA- polymerase (replicase) induced in h'. coli by the RNA bacteriophage Qp. The dah demolist rat e t,hat, the eiizynie (:ail generate ideiit)ic:al col)ies of added viral RNA. 9 serial dilutioti cxperimeiit established tbat t,he iiewly synthesized RNA is fully its coiiil)etetit as t,tie original viral RNA to program t)he synthesis of viral particles atid to serve as tetiiplat,es for the generation of` inore copies. Since the data show t>lint thc: crizynie is, in fact, geiieratitig replicas, :LII unambiguous a- nalysis of the RNA rc1)licatirig inechanisni is now possible in a siniple system con- sisting of purified rel'licase, teni\)late RNA, ribosidetriphosphales, arid Mg+ +. * This investigation was supporled by USPHS research grant CA-01094 from the National t Predoctoral trainee in Microbial and Molecular Genetics, grant USPHS 2-T1-GM-319. Cancer Inst,itute and grant GB-2169 from the Xational Science Foundation. Haruria, I., and 8. Spiegelriian, these PKO(:EEI)IN(:S, 54, 579 (196.5). Watanabe, I., Nihor~ IZirLsho, 22, 243 (1964). Spahr, P. F., and B. I<. Hollitigworth, J. Hiol. Chem., 236, 823-831 (1961). Spahr, P. F., .I. Hid. Cheni., 239, 3716-3726 (1964). 6 Haruna, I., and S. Spiegelmati, Science, in press. 7 Gesteland, I<. F., Fedetation Proc., 24, 203 (19M). 8 I>oi, Roy H., and S. Spiegelnmi, t,hese PI~OCEEDINGS, 49, 353-360 ( l!363). lo (+uttirie, C+. I)., aid K. L. Sinsheimer, J. Mol. Hid., 2, 297 (1960). l1 Fraser, I)., atid K. A. Jerrel, J. Bid. Chrni., 205, 291-295 (1053). l2 Pararic.liyc.h, W., Hiocheni. Hiophys. IZas. Coninii~n., 11, 28 (1963). l3 Ochoa, S., C. Weissmann, P. Borst,, It. H. Burdon, and R.1. A. Billeter, Federation Proc., 23, a Overby, L., G. H. Barlow, K. H. hi, M. Jaool), nnd S. Spiegelman, .I. Hacteriol., in press. (dl3 is a derivat,ive of A10, an R.Nase negative riiul ant reported in this reference. Haruna, I., K. Nozu, Y. Ohtaka, and S. Hpiegelrnan, t,hese PHOCEEDINGS, 50, 90.5-911 (1963). 1285-1296 ( 1964).