Reprinted from the PROCELDlVGb OY llIL NATIONAL *\CADhMY OF SrlLhCtR Vol. 58, No. 1, pp. 217-224. July, 1967. AN EXTRACELLULAR DARWINIAN EXPERIMENT WITH A SELF-DUPLICA TING NUCLEIC ACID MOLECULE* BY D. R. i\lILLs,t R. 1,. PETERSON, AND S. SPIEGELMAN DEPARThtENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS, URBANA Communicated Afay 18, 1967 The replicases (RNA-dependent RNA polymerases) induccd by two unrelated'. bacteriophages (AIS-2 arid Qp) have been isolated arid shown to require their intact3 homologous RNA as te~iiplate.~. It was further demonstrated that the RNA molecules synthesized could serve as templates for further synthesis6 and were fully competent to program the synthesis of complete virus particles in pro- toplasts.' Finally, when Qp-replicase is presented with either of two genetically distinct Qp-RNA molecules, the RNA synthesized is identical to the initiating template.* This specific response of the same enzyme preparation to the particular template added proved that the RNA is the instructive agent in the replicative process and hence satisfies the operational definition of a self-duplicating entity. An opportunity is thus provided for studying the evolution of a self-replicating nucleic acid molecule outside of a living cell. It should be noted that this situation mimics a11 early precellular evolutionary event, when environmental selection presumably operated directly on the genetic material. The comparative simplicity of the system arid the accessibility of its kriowri chemical components to manipula- tion permits the imposition of a variety of selection pressures during growth of the replicating molecules. We wish to report here one example of the experiments thus made possible. In the universe provided to them in the test tube, the RNA molecules are liberated from many of the restrictions derived from the requirements of a complete viral life cycle. A restraint iniposcd is that they retain whatever sequences are involved in the recognition mechanism employed by the replicase. Thus, sequences which code for the coat proteins arid replicase components may now be dispensable. Under these circumstances, it is of great interest to design an experiment which attempts an answcr to the following question: "What will happen to the RNA molecules if the only demand made on then1 is the Biblical injunction, multiply, with the biological proviso that they do so as rapidly as possible?" The conditions required are readily attained by a serial transfer experiment7 in which the intervals of synthesis between transfers are adjusted to select the first molecules completed. It is the purpose of the present paper to detail the results of this type of selection. Thc outcome is what might have been expected on a priori grounds. The smaller the polynucleotide chain, the shorter the time required for its completion. Con- sequently, if the initial Qp-RNA molecules possess sequcrices which are dispensable under the conditions of the experiment, their elimination could confer a selective advantage. In accordance with this expectation, it was found that as the experi- ment progressed, the multiplication rate increased and the product became smaller. By the 74th transfer, 53 per cent of the original genome had been eliminated. Aside from their intrinsic interest, it is evident that such experiments generate molecules potentially useful for the resolution of a variety of problems. These in- clude elucidation of the mechanism of replication and the identification of the recog- 217 218 I,'lOCHEAIISl'RY: MILLS ET AL. Puoc. N. A. 8. riitiori device which allows the replicase to select the molecule to be replicated. Finally, thcsc abbreviated niolccules opcri up a novel pathway for a highly selectivc iriterfererice with the replication of the complet'e viral gcrioinc. Synthesis of radioactive ribo- nucleotide triphosphates and liquid scintillatioil coiiiitiiig of labeled RNA on membrane filters have been detailed previon~ly.~ RNA from a temperature-sensitive mutant of Qp (ts-1) was ex- tracted from the virus as tlescribed previoiisly.a Thc first reactioii in the series was iiiitiated at a conceiitrat,ioii of 0.2 pg/O.25 ml of a standard5 reactiori. The same replicase preparation purified through the CsCl arid sucrose stepsg was iised iii all the steps of tlie traiisfer experiments to be described. Aliquols (0.01-0.10 ml) were withdraw1 from variolis reactions arid xdjrist,ed to 0.276 (by weight) with respect to sodium tlotlecyl sulfate (SIIS). Each sample was diliited to a filial volume of 0.20 ml in TE buffer (0.01 ill Tris, pII 7.4, and 0.003 A?' EDTA), then layered on a 5-nil hear gradieiit, of sucrose (2-20Oj, in 0.10 M Tris, pII 7.4, aiid 0.003 nl EDTA). These gradieiits were centrifuged in a Spirico SW-39 rotor at 39,000 rpm at 4°C for 5 hr. k'ractions of 0.25 ml were co1lect)ed dropwise, precipitated with IOS;, trichloro- acetic acid (TCA), washed oiito cellulose nitrate membrane filters, and coiinted in a Packard liquid scintillation counter. (c) Gel electrophoresis: Unswollen ethyleiie diacrylate cross-linked polyacrylamide gels (3.G%) and preswolleii N,N'-methylerie-bis-acrylamide cros-liuked polyacrylamide gels (2.4%,) were prepared as described previoiisly.1° Electrophoresis runs were made at room temperittiire for 90 miii, at 5 ma/gel and 50 volts for gels 0.7 cm in diameter and 10 ma/gel for gels 0.9 cm in diameter and 9 cm in length. Optical density measiirements of gels were performed by scanning each gel (transferred to a quartz cell 0.5 cm in depth) with traiismitted ultraviolet light iii a Joyce high-resolution "chromo- scan" eqiiipped with a 266-mp iiiterference filter. Frozen gels were sectioned in 0.5-mm slices with the use of a carbon dioxide-cooled microtome.'" Successive pairs of 0.5-mm sections were placed in vials aiid eluted in TE or SSC (0.015 111 sodirirn chloride and 0.015 ill sodium citrate) buffers with gentle agitation for 12 hr at 5°C. Aliquots were removed from each elution, pre- cipitated with cold l0yo TCA, washed onto celliilose nitrate membrane filters, and coiinted in a Packard liqiiid scintillation counter. Samples from each gel were adjusted to 0.15 ill sodiiim chloride aiid 0.015 M sodium citrate, 20 pg/ml pancreatic ribonuclease, arid 20 pg/ml TI ribo- nuclease. After a 2-hr incubation at 35"C, each sample was washed onto a cellulose nitrate mem- brane filter with cold 105; TCA, and counted in the Packartl liqiiid scintillation counter. Heated (100°C for I min) aiid qiiick-cooled (in ice) samples were cont,aiiied in TE biiffer which was theii adjusted to 0.15 M sodirim chloride :%rid 0.015 A1 sodium citrate for ribonuclease assay. Samples were withdrawn and set aside for sedi- mentation analysis or gel electrophoresis from 0.125-ml reaction volume (or half standard repli- case reaction). Samples for infectjivitjy assays were diluted into 0.003 A1 EDTA and t,reat,ed as described by Pace and Spiegel~nan.~ (f) Base cornposition anal:ysis: In addition to the standard co~npoiieiits,~ reaction solution for base composition analysis contained the four rihonncleotide triphosphates (labeled in the a- phosphorus with Paz) at a specific activity of 7.53 X lo7 cpm/0.2 p32 for each triphosphate. The volume was 1.0 ml aiid coiitained 160 pg of replicase. The reaction was initiated with 0.3 pg of gel purified single-stranded variant RNA obtaiiied from the 74th transfer. After incubation at, 35°C for 40 miu, the replicase reaction was terminated by rapid chilling to 0" and addition of SDS to a final concentration of 0.2(x,. The terminated reaction was dialyzed 12 hr at 5°C against 500 ml TE biiffer. This dialyzed soliition was theii reduced in volume to about 0.1 to 0.2 ml with fine grade (2-25 Sephadex and subjected to gel electrophoresis. RNA in the peak single-strand region was pooled and repiirified by gel electrophore The peak single-strand regions were again pooled. To remove any residue of labeled riboside triphosphate, bidk E. coli RNA was added to the major portion of the pool, precipitat,ed with a solution of saturated sodium pyro- phosphate, saturated sodium biphosphate, and saturated TCA (1 : 1 : 1 by volume), and washed onto a cellulose nitrate membrane filter with cold 10% TCA. The membrane was then cut into Afaterials and ilfethods.-(a) Enzyme, substratcs, and asstcys: (b) Sedimentation anul,ysi.s of produck: (d) Ribonuclease resistance assays: (e) Synthesis of XLVL4 and infectio?rs units: VOL. 58. 1967 BIOCHEMISTRY: MILLS ET AL. 219 small pieces and eluted with 0.3 M aqueous potassium hydroxide. Three 1-ml washes with 0.3 M KOH were used. These were pooled and incubated 12 hr at 35". Chromatographic analysis of the resultiiig 2'-3'-nucleotides was performed on a Tlowex-formate column as detailed by Hayashi arid Spiegelman. l1 An account of a transfer experi- ment involving 75 serial reactions is illustrated in Figure 1. The first reaction (0th) was allowed to proceed 20 minutes at 35"C, whereupon a 20 X aliquot was used to seed the second, arid so on for the first 13 reactions. The incubation periods were then reduced as detailed in the legend of Figure 1. These periodic reductions in the incubation intervals between transfers were instituted in an attempt to maintain the selection pressure for the most rapidly multiplying molccules. As may be seen from the inset, the synthesis of biologically competent RNA ceased betwcen the fourth arid fifth transfers. Second, a dramatic increase in the rate of incorporation of PJz- UTP into RNA occurred between transfers 8 arid 9. I,ast, an apparent decrease in the rate of RKA synthcsis, coinciding with the reduction in the incubation time from 15 minutes to 10 minutes, occurred after trarisfer 29. The RNA products from the reactions indicated by arrows in Figure 1 were expanded by using them to initiate ncw replicase reactions which were coritinued for 40 minutes at 3.5"C. The resulting products were then examined in sucrose gradients. The product obtained from thc reaction initiated by the 0th transfer Results.-(a) Selection during serial transfer: Three outstanding features of Figure 1 may be noted. 6- 5- n- I a: -4- - c3- %2 - x E- "- I- a 0- N- n- w- 0 5 IO 15 20 25 30 40 55 74 TRANSFERS Fig. 1.Serial transfer experiment. Each 0.25-ml standard reaction mixt11re5 contained 40pg of Qp replicase purified through CsCl and silcrose cent,rifiigation, and (P32) UTP (ilritiiiie triphos- phate) at, a specific act,ivit,y silch that, 4,000 cpm corresponds to pg of synthesized RNA. The first, react,inn (0 transfer) was initiated by t,he addition of 0.2 pg ts-1 (temperatiire-serrsitive RNA) and incubated at 35°C for 20 min, wherelipon 0.02 ml was drawn for connting and 0.02 ml was used to prime t,he second reaction (1st transfer) and so on. Aft,er the first 13 reactions, the in- citbation periods were reduced to 15 miri (transfers 14-29). Transfers 30-38 were incubated for 10 min. Transfers 39-52 were inciibnted for 7 min, and transfers 53-74 were incubated for 5 min. The arrows above transfers (0, 8, 14, 29, 37, 53, and 73) ind e where 0.01-0.1 of product' was removed arid used to prime reactions for sedimentat ion anal on siicrose (see Figs. 2-5). The inset examines both irifect,ious and t,otal RNA. The results show that biologically competent RNA ceases to appear after tjhe 4t)h transfer. 220 BIOCHEMISTRY. MILLS ET AL. PROC. N. A. S. let TRANSFER 20 X . IO .a 0 N' n - IO 20 FRACTION (Above) FIG. 2.-Sedimentation analysis of 1st transfer reaction. As described in Afethods, 0 02 ml of the 0 reaction was used to initiate a reaction for a 1st transfer reaction product. After completion, this reaction was adjusted to 0.ZC% SDS, an aliquot was withdrawn, diluted to 0.2 ml in TE buffer (0.01 M Tris pII 7.4, 0.003 M EDTA), and then layered onto a 5-ml linear sucrobe (2-20yo in 0.1 M Tris, pH 7.4, and 0.003 M EIITA) arid run as desrribed in Methods (section b). H%belecl bulk RNA of E. coli was inchitled as iiiteriial size markers. (Right) FIG. 3.-Sedimentation analysis of 9th transfer (A) and 15tJh transfer (B) reac- tion products. Details are as in Fig. 2. IO 20 FRACTION FRACTION shows (Fig. 2) thc 2SX pcak charactcristic of QP-RNA as well as thc pcalis cor- responding to thc usual complexes observed during the in vitro synthesis.I2 Com- parison with subsequent transfers reveals, however, dramatic charigcs in the nature of the replicating cntity. Thus, by the ninth transfer (Fig. 3A) there is no material synthesized corresponding to the original 2SS viral RNA. In its place we see a major component at about 20s and a minor one at about 15s. This pattcrn is es- sentially maintained through the 15th trmsfer (Fig. 3B). By the 30th transfer (Fig. 4A) thc major component has decreascd to 15s arid the mitior one to about 14s. The product of thc SSth transfcr shows variant RNA which 110 longer splits into two peaks, a feature rctairicd through subscquent trans- fers. It will be noted (Fig. 5A arid B), howcver, that the sirigle pcali niovcs more slowly so that by the 74th transfer it is at about 12s. Gel electrophoresis oj variant RNA: At this point it was decided to ex- amine the nature of the variant in greater detail. Transfcr 75 was expanded with replicase to a total of 120 pg of RKA arid subjected to analysis by polyacrylamide gel electrophoresis (Fig. 6). Clearly, the apparcntly homogeneous peak of Figure 5B is composed of at least two distinct RNA species. As may be seen froni Figure 6, the major component is sensitive to ribonuclease whereas the minor om is resis- tant. It would appear that the faster componcnt is thc single-strmded variant and that the slower minor peak contains a mixture of the Hofs~hricidcr~~ arid Frank- (b) VOL. 58, 1967 BIOCHEMISTRY: MILLS ET AL. 221 IO 20 FRACTION 54 th TRANSFER 10 20 FRACTION FRACTION FRACTION FIG. 4.--Sedimentation analysis of the 30th transfer (A) and the 38th transfer (B) reaction products. Details are as in Fig. 2. products. Details are as in Fig. 2. FIG. 5.--Sedimentation analysis of the 54th transfer (A) and the 75th transfer (13) reaction lin14 structures observed first in vivo and seen in in vitro synthesis of QP-RNA with purified rcplicase.'2s l5 Wc have previously shown'" that thc relative electrophoretic mobility (IZERI) is linearly relatcd inversely to the molecu- lar weight of single-stranded RNA. Consequently, to determine the molecular weight, the single-stranded variant RNA was subjected to gel electrophoresis with seven internal marker RNA's of known size. The rcsults are shown in Figure 7 arid indicate that the variant RNA has a molecular weight of about 1.7 X lo5 daltons. Base composition of variant RNA: To determine its basc composition, a standard rcaction mixturc was initiated with the variant isolatcd by gel elec- trophoresis. In this reaction, all four riboriuclcotide triphosphatcs were labeled with P3? at the a-position (Methods, section f). The RNA product of this rcaction was purified twice by gel electrophoresis, hydrolyzed, and analyzed as described in Methods (section f). Comparison with the base composition of the original QP-RSA (Table 1) indicates that there has been a corisiderablc (5 mole yo) increase in the G content in the variant RNA. On the othcr hand, A and C have decreased by 2.4 mole per cent, thc uridine content remaining constant. (c) Molecular weight of variant RNA: (d) 222 BIOCHEMISTRY: MILLS ET AL. PROC. N. A. 8. c H3 VARIANT IO 20 30 40 DISTANCE MOVED, MM FIG. 6.-Gel electrophoresis of H3 CTP-labeled 75th transfer reaction product. A preswollen N,N'-methylene-brs-acrylamide cross-linked gel (2.47,) was prepared and run as described in Methods (section c). Samples for electrophoresis were about 0.1 ml in E buffer and sucrose. One-mm sections were eliited in 0.5 ml TE buffer (0.01 M Tris, pTI 7.4, and 0.003 Al EDTA) for 12 hr at 4°C with gentle shaking. Aliqnots for ribonuclease were withdrawn and treated as in Methods (section d). All data are represented as cpm/0.05 ml. 23 s RNA BMVL RNA 75 th TRANSFER P R 0 DUCT I.III.1.I I I 2 3 4 5 6 104 RELATIVE ELECTROPHORETIC MOBILITY (REM). CM FIG. 7.--RIdecular weight of the variant RNA determiried by electrophoretic mobility. The relative electrophoretic mobility (REM j is plotted against molecular weight. TJnswollen ethylene diacrylate cross-linked polyacrylamide gels (3.6cj, j as described in Methods (section c) were used in these determinations. E. colz (H3j bulk RNA (238, 16S, 5S, and 4s) and home grass mosaic virus RNA (BhIVj, which gives three distinct size components (BMV-I, BMV-2, BMV-3), kindly donated by Ilr. Paul Kaesberg. Nucleic acid markers include: VOL. 58, 1967 BIOCHEMISTRY: MILLS ET AL. N 9- ?- z v- 223 TABLE 1 RASE COMPOSITION OF VAIUANT RNA RNA C A u G Variant Q@-lINA-l 84-RNA-2 22.3 25.0 24.7 19.7 22.5 22.1 29.3 29.5 29.1 28.7 23.0 23.7 Variant RNA uniformly labeled with P32 was prepared, purified, and analyzed as described in Methods (section j). TO monitor the quantitatiye adequacy of the analysis a parallel experiment was carried out with a slmilarly prepared and unl- formly labeled 28s Qp-RNA (second line, Qp-!AN'\-1). The last line (Qg-RN.4-2) gives for com- parison the base composition of RN.4 isolated from virus particles.'. 2 Numbers represent mole per cent of the corresponding bases. The resultinx data are given in the first line. (e) Kinetics of QP and variant RNA: A comparison of the kinetics of synthesis at saturation of the 75th variant arid the original ts-Qp-RKA reveals (Fig. 8) some interesting differences. It will be rioted that the Qp-RNA shows the usual six minutes of riorilinear synthesis which precedes the linear phase. The variant has decreased this apparent lag to 1.5 minutes. Further, the slope of the linear portion of the variant synthesis is 2.6 times that of the original Qp-RNA. Since the variant is only 17 per cent of the original size, its growth rate in terms of the production of new individuals is 15 times that of the coniplete viral RNA molecules. Discussion.--The primary purpose of the present paper was to demonstrate the potentialities of the replicase system for examining the extracellular evolution of a self-replicating nucleic acid molecule. Further, the experimental situation provides its own paleology; every sample is kept frozen arid can be expanded at will to yield the comporieiits occurring at that particular evolutionary stage. While only seven such saniples are detailed here, they iri- dicate that progress to a small size occurs in a series of steps. It should be rioted that we have learned how to modify the enzyme reaction so that this process is greatly ac- celerated. This involves changing the pro- portions of the two coniponentsl6 of the Qp replicase and will be reporLed subsequently. The last product examined in the present study is a molecule which has cliniinated 53 per cent of its original length and has ex- perienced a sigriificarit change in base com- position. The fact that it replicates 15 times faster than the complete viral RNA sug- gests that in addition to becoming smaller, the variant has increased the efficiency with which it interacts with the replicase. In any event, the findings reported establish that neither the specific recognitiori nor the replicating nicchariisni requires the coni- plete original sequence. In this cotinec- tion, it should be rioted that although ab- breviated, these variants are not equivalent to randoni fragments. The latter are un- able to complete the replicative / 1 7 OB RNA p' PRIMED / I> 2 4 6 8 IO 12 14 16 MINUTES FIG. 8.-A comparison of the kinetics of synthesis of the 74th variant aiid the original ts-QP-RNA. Two 0.25-ml stan- dard reactions (as detailed in Methods, section a), me primed with gel purified single-stranded variant RNA (74th traiis- fer) arid the other primed with ts-QP-RNA (both above saturations), were initiated at 35°C. Aliqiiots of 0.02 ml were drawn at times indicated aiid assayed for iii- corporation of Pae-UTP. Data are re- presented as cpm/0.02 ml. 224 BIOCHEMISTRY: AlILLS ET AL. PROC. N. A. S. The availability of a molecule which has discarded large and unnecessary seg- ments provides an object with obvious experimental advantages for the analysis of many aspects of the replicative process. Finally, these abbreviated RNA molecules have a very high affinity for the replicase but are no longer able to direct the synthesis of virus particles. This feature opens up a novel pathway toward a highly specific device for interfering with viral replication. It should not escape the attention of the reader that the situation described places at our disposal a completely novel method for the resolution of a variety of interest- ing problems. Potentially, other selective stresses can be imposed on the system to generate RNA entities which exaggerate other molecular features. SU~LVLU~J- Experiments were performed to explore the evolutionary coiise- qucrices for a sclf-duplicating nucleic acid molecule put under selection pressure for fast growth. As the cxperinient progresscd, the rate of RKA synthesis in- creased and the product became smaller. By the 74th transfer the replicating molecule had elirninatcd S3 per cent of its original genome, becoming the smallest known self-duplicating entity. Aside from their intrinsic interest, such studics can provide insight into a number of central issues. Thus, they can tell US the smallest self-duplicating entity which can be constructed by such devices and provide much simpler objects for analyzing the replication process. Further, the sequences involved in the recognition mech- anism between template arid enzyme are enriched in the sniallcr molecules which evolve. Finally, these abbreviated molecules have a very high affinity for the replicase but are no longer able to direct the synthesis of virus particles. This feature opens up a novel pathway toward a highly specific device for interfering with viral RNA replication. * This iiivestigation was supported by USPTTS reiearch grant CA-01094 from thc National t Predoctoral trainee, USPI-IS graiit F-TOl-GM-319, in Microbial and Molecrdar Genetics. zZbid., 92, 739 (1966). J ITaruiia, I., and S. Spiegelman, these PROCEEDINGS, 54, 1189 (1965). Ilarruia, I., K. Nozu, Y. Ohtaka, and S. Spiegelman, these PROCEEDINGS, 50, 905 (1963). Hariuia, I., and S. Spiegelman, these PROCEEDINGS, 54, 579 (1865). IIarnria, I., and S. Spiegelman, Science, 150, 884 (1965). Caiicer Iiistitute and grant GB-4876 from the Natioiial Science Foundation. Overhj , L., G. Rarlow, R. hi, RI Jarob, and S. Spiegelmari, J. Bacterial, 91, 442 (1966). ' Spiegelniaii, H., I. I-Iaruna, I. B. Holland, C. Bcaudreau, and I). Rl~lls, these PROCEEDINGS, 8 Pace, N. 11., arid S. Spiegelmari, Science, 153, 64 (1966). 9 Pace, N. It., and S. Spiegelman, these PROCEEDINGS, 55, 1608 (196G). l1 IIayashi, ll., and 8. Spiegelmarr, these PROCEEDINGS, 47, 1564 (1961). 12 Rlills, D., N. R Pace, arid S. Spiegelman, these PI~OCEEDINGS, 56, 1778 (1966). 1J Fiancke, B., and P. H. Hofschrieider, J. Mol. Zlzol., 16, 544 (1966). l4 Fraiiklin, It. AI., these PROCEEDINGS, 55, 1504 (1966). l5 Bishop, D. IT. L., J. R. Claybrook, N. R. Pace, and S. Spiegelman, these PROCEEDINGS, 57, 16 Eikhom, T. S., and S. Spiegclman, these PILOCEEDINGS, 57, 1833 (1967). 54, 919 (1965). Bishop, D. TI. L., J. 11. Claybrook, and S. Spiegelman, J. Mol. Bzol., in press (1967). 1474 (1967).