Reprinted from the PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES
Vol. 54, No. 3, pp. 919-927. September, 1965.

THE SYNTHESIS OF <A SELF-PROPAGATING AND INFECTIOUS
NUCLEIC ACID WITH A PURIFIED ENZYME*

By 8. SPIEGELMAN, I. Haruna, I. B. Hotianp, G. BEAUDREAU, AND D. Mist
DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS, URBANA
Communicated July 23, 1965

The unambiguous analysis of a replicating mechanism demands evidence that the
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 which 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 simple mixture
920 BIOCHEMISTRY: SPIEGELMAN ET AL. Proc. N. ALS.

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 enzymological aspects of the investigation be virtually
completed before an examination of mechanism can be safely instituted.

We have previously reported! the purification of two distinct RNA-—dependent-
RNA-polymerases (designated “replicases” for brevity) induced in the same host
by two unrelated? ? RNA bacteriophages (MS-2 and Q@). It was shown that under
optimal conditions, both enzymes are virtually inactive with a variety of heterol-
ogous RNA species, including ribosomal and sRNA of the host. Further, neither
replicase can function with the other’s RNA. Each enzyme recognizes the RNA
genome of its origin and requires it as a template for normal synthetic activity.

In summary, the purified replicases exhibited the following distinctive features:
(a) freedom from detectable levels of the DNA-dependent-RNA-polymerase, ri-
bonuclease I,? ribonuclease II, and RNA phosphorylase; (6) complete dependence
on added RNA for synthetic activity; (c) competence for prolonged Gnore than 5
hr) synthesis of RNA; (d) ability to synthesize many times the input templates;
(e) saturation at low levels of RNA (1 y RNA/40 y protein); (f) virtually exclusive
requirement for intact homologous template under optimal ionic conditions.

The discriminating selectivity of the replicase permitted a simple test of similarity
between template and product. Haruna and Spiegelman® showed that when reac-
tions are started at. template concentrations below those required to saturate the
enzyme, RNA synthesis follows an autocatalytic curve. When the saturation con-
centration level is reached, the kinetics become linear. The autocatalytic behavior
below saturation of the enzyme implies that the newly synthesized product can in
turn serve as templates for the reaction. To test this conclusion directly, the pro-
duct was purified from a reaction allowed to proceed until a 65-fold increase of the
input RNA had accumulated. The ability of the newly synthesized RNA to ini-
tiate the reaction was examined in a saturation experiment and found to be iden-
tical to RNA isolated from virus particles. It is evident that the sequences em-
ployed by the enzyme for recognition are being faithfully copied.

The findings summarized above and the state of purity of the enzymes encour-
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.

Materials and Methods——(1) Biological system and enzyme preparation: The bacterial virus
employed is Q8, isolated by Watanabe.? The host and assay organism is a mutant Hfr strain of
E. coli (Q13) isolated in the laboratory of W. Gilbert by Diane Vargo. This bacterial strain has
the convenient property’ of Jacking ribonuclease I and RNA phosphorylase. The preparation
of infected cells and the subsequent isolation and purification of the replicase follows the detailed
protocol of Haruna and Spiegelman.!. The preparation of virus stocks and the purification of
RNA from them follow the methods of Doi and Spiegelman.*

(2) The assay of enzyme activity by incorporation of radioactive nucleotides: The standard reac-
tion mixture is 0.25 ml and, in addition to 40 y of enzyme, contains the following in ymoles:
Tris HCl, pH 7.4, 21: MgCh, 3.2; CTP, ATP, UTP, and GTP, 0.2 each. The reaction is ter-
Vou. 54, 1965 BIOCHEMISTRY: SPIEGELMAN ET AL. 921

minated in an ice bath by the addition of 0.15 ml of neutralized saturated pyrophosphate, 0.15
ml of neutralized saturated orthophosphate, and 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 10% TCA. The
membrane is then dried and counted in a liquid scintillation counter as described previously.
UTP* was synthesized as described by Haruna ef al. It was used at a specific activity such
that the incorporation of 20,000 cpm corresponds to the synthesis of | y of RNA, permitting the
use of 20 A samples for following the formation of labeled RNA.

(3) Isolation of synthesized product: Samples removed from the reaction mixture are piaced
immediately in an ice bath and 20 A removed for immediate assay of radioactive RNA as described
in (2) above. The volume is then adjusted to 1 ml with TM buffer (107? M Tris, 5 X 107-3 M
MegCh, pH 7.5). One ml of water-saturated phenol is then added and the mixture shaken in
heavy wall glass centrifuge tubes (Sorvall, 18 X 102 mm) at 5°C for 1 hr. After separation of
the water phase from the phenol by centrifugation at 11,000 rpm for 10 min, another | ml of TM
buffer is added to the phenol which is then mixed by shaking for 15 min at 5°C. Again, the
phenol and water layers are separated, and the two water layers combined. Phenol is eliminated
by two ether extractions, care being taken to remove the phenol from the walls of the centrifuge
tubes by completely filling them with ether after each extraction. The ether dissolved in the
water phase is then removed with a stream of nitrogen. The RNA is precipitated by adding 3/15
vol of potassium acetate (2 M) and 2 vol of cold absolute 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, and the remaining alcohol is removed by storing under reduced pressure in a
vacuum desiccator for 6-8 hr at 5°C. The RNA is then dissolved in 1 ml of buffer (10-2 M Tris,
10? M MgCh, pH 7.5) and samples ure removed immediately for infectivity assay. TCA-
precipitable radioactivity is measured on 20 d aliquots of the final product from which the per
cent recovery of synthesized RNA can be determined. In the range of 1-8 y, it was found that,
in general, 65% of the synthesized RNA was recovered. All purified products were examined for
the presence of intact virus particles by assay on whole cells and none were found.

(4) The assay for infectivity of the synthesized RNA: The procedure used is a modification of the
spheroplast method of Guthrie and Sinsheimer.!° The necessary components are as follows:

(a) Medium: The medium used is a modification of the 3XD medium of Fraser and Jerrel!!
and requires in grams/liter the following: Na,HPO,, 2 gm; KH»2PO,, 0.9 gm; NH,Cl, 1 gm;
glycerol (Fisher reagent), 30 gm; Difco yeast extract, 50 mg; casamino acids (Difeo vitamin-
free), 15 gm; L-methionine, 10 mg; D,L-leucine, 10 mg; MgSOv7 H.0, 0.3 gm. These com-
ponents are mixed in the order indicated in 500 ml glass-distilled water. To this is finally added
another 500 ml containing 0.38 m! M CaCh.

(b) Sucrose nutrient broth (SNB) contains in grams/liter the following: casamino acids (Difco),
10 gm; nutrient broth (Difco), LO gm; glucose, 1 gm; sucrose, 100 gm. After autoclaving, the
following are added aseptically: 10 ml 10% MgSO,, and 3.3 ml 30% bovine serum albumin
(BSA) from Armour Laboratucies.

(c) Reagents required for the production of spheroplasis: The followings olutions are required for
the production of spheroplasts: lysozyme (Sigma) at 2 mg/ml in 0.25 M Tris, pH 8.0; protamine
sulfate from Eli Lilly and Co., 0.1%; and sterile solutions of 30% BSA; 0.25 M Tris (Trizma),
pH 8.0; 0.01 M@ Tris, pH 7.5 and pH 8.0; 0.5 M sucrose; 0.4% EDTA in 0.01 M Tris, pH 7.5.

For the preparation of spheroplasts, an overnight culture of Q13 in 3XD medium is first diluted
into a fresh medium to an ODevo of 0.06. The culture is allowed to grow to an ODee between 0.2
and 0.22 at 30°C, and the cells are spun down at room temperature. The pellet from 25 ml of
cells is first suspended in 0.35 ml of 0.5 M sucrose plus 0.1 ml of 0.25 M Tris, pH 8.0. Then 0.01
ml of lysozyme is added followed by 0.03 ml EDTA. After 10 min at room temperature, when
conversion to spheroplasts is 99.9%, 0.2 ml of this stock is diluted into 3.8 ml SNB, and 0.025 ml
of protamine sulfate is added. The spheroplast stock must be examined microscopically before
proceeding. The presence of even 5% breakage of spheroplasts indicates a preparation which
will give » low efficiency of plating. In agreement. with Paranchych,!? we have found that. pro-
famine increases the efficiency of detection of infectious RNA. However, the optimal protamine
concentration in the present system is considerably lower than that used by Paranchych.

The RNA infection is usually carried out at room temperature with solutions containing 0.5 y
of RNA/ml, a concentration at which the assay is not limited by the number of spheroplasts per
922 BIOCHEMISTRY: SPIEGELMAN ET AL. Proc. N. ALS.

infectious unit. To 0.2 ml of RNA is added 0.2 ml of the spheroplast stock containing about 3 X
107 spheroplasts. The samples are mixed, and immediately an aliquot is removed and diluted
appropriately through SNB before plating on L-agar using Q13 as the indicator. The soft agar
(0.7%) layer employed (2.5 ml) contains 10% sucrose; 0.1% MgSO,; and 0.01 ml of 30% BSA
per tube plus 0.2 ml of an overnight culture of Q1I3. To obtain reproducibility, the spheroplast
stock is used 15-45 min after dilution into the SNB. Efficiency of plating (e.o.p.) is usually
2-8 X 10-7. Higher efficiencies (>1 & 107) can be obtained if the spheroplast. stock is employed
immediately after dilution and by including a stabilization period in the SNB dilution tubes,
rather than plating immediately. However, this higher plating efficiency decays rapidly, making
it difficult to obtain reproducible duplicates in repetitive assays. Since reproducibility was of
greater concern than efficiency, the assay method detailed above was employed.

Results —In designing experiments which involve infectivity assays of the enzy-
matically synthesized RNA, it is important to recognize that even highly purified
enzymes from infected cells, although demonstrably devoid of intact. cells, are likely
to include some virus particles. Chemically, the contamination is trivial, amount-
ing to 0.16 y of nucleic acid and 0.8 y of protein for each 1,000 y of enzyme protein
employed in the present studies. Since 40 y of protein are used for each 0.25 ml of
reaction, the contribution to the total RNA by the particles is only 0.006 +, which
is to be compared with the 0.2 y of input RNA and the 3-20 y synthesized in the
usual experiment. It was shown in control experiments that RNA freshly extracted
from particles in the reaction mixture ix no more infective than that obtained from
the usual purified virus preparation. Further, the mandatory requirement for
added RNA proves that, within the incubation times used, this small amount of
RNA is either inadequate or unavailable for the initiation of the reaction. Thus,
these particles do not significantly influence either the chemical or the enzymatic
aspects of the experiment. However, because of their higher infective efficiency,
even moderate amounts of intact virus cannot be tolerated in the examinations of
the synthesized RNA for infectivity. Consequently, all RNA preparations were
phenol-treated [Methods, (3)] prior to assay. Further, the phenol-purified RNA
was routinely tested for whole virus particles and none were found in the experi-
ments reported.

We now undertake to describe experiments in which the kinetics of the appear-
ance of new RNA and infective units were examined in two different ways. The
first shows that the accumulation of radioactive RNA is accompanied by a propor-
tionate increase in infective units. The second type proves by a serial dilution ex-
periment that the newly synthesized RNA is infective.

(1) Assay of infectivity of the purified product: To compare the appearance of new
RNA and infectious units in an extensive synthesis, 8 ml of reaction mixture was
set up containing the necessary components in the concentrations specified in
Methods (2). Aliquots were taken at the times indicated for the determination of
radioactive RNA and purification of the product for infectivity assay. The results
are summarized in Figure 1 in the form of a semilogarithmic plot against time of
the observed increase in both RNA and infectious units. Further details of the
experimental protocol are given in the corresponding legend.

The amount of RNA (0.8 y/ml) put in at zero time is well below the saturation
level of the enzyme present.* 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.’ It will be noted that the increase in RNA is
Vou, 54, 1965 BIOCHEMISTRY: SPIEGELMAN ET AL. 923

110 f coe Jioo

ae . RNA

r - | Fie. 1.—Kineties of RNA synthe-
Ce -neeCTWE Units sis and formation of infectious units.
r 1 An 8-ml reaction mixture was set up

A containing the components at the

10? FF 4 “J 10 concentrations specified in Methods

/ 4 (2). Samples were taken as fol-

I lows: 1 ml at 0 time and 30 min, 0.5

ml at 60 min, 0.3 ml at 90 min, and

0.2 ml at all subsequent times. 20

4 » were removed for assay of incor-

vy porated radioactivity as described

wot / a in Methods (2). The RNA was

J purified from the remainder [Meth-

ods (3)], radioactivity being deter-

mined on the final product to moni-

tor recovery. Infectivity assays
were carried out as in Methods (4).

PLAQUE FORMERS / CSmt
ne rrr
SS

——.
#9 RNAi

 

 

60 120 leo 240
MINUTES.

paralleled by a rise in the number of infectious units. During the 240 min of incu-
bation, the RNA experiences a 75-fold increase, and the infectious units experience
a 35-fold increase over the amount present at zero time. These numbers are in
agreement within the accuracy limits of the infectivity test. Experiments carried
out with other enzyme preparations yielded results in complete accord with those
just described.

It is clear that one can provide evidence for an increase in the number of infectious
units which parallels the appearance of newly synthesized RNA.

(2) Proof that the newly synthesized RNA molecules are infective: The kind of
experiments just. described offer plausible evidence for infectivity of the radioactive
RNA. They are not, however, conclusive, since they do not eliminate the possi-
bility that the agreement observed is fortuitous. One could argue that the enzyme
is “activating” the infectivity of the input RNA while synthesizing new noninfec-
tious RNA and that the rather complex exponential and linear kinetics of the two
processes happen to coincide by chance.

Direct. proof that the newly synthesized RNA is infectious can in principle be
obtained by experiments which use N'-H*1labeled initial templates to generate
N*4-P*labeled product. The two can then be separated® in equilibrium density
gradients of CspS8O.. Such experiments have been carried out for other purposes,
and will be described elsewhere. However, the steepness of the Cs:SO, density
gradients makes it difficult. to achieve a separation clean enough to be completely
satisfying.

There exists, however, another approach which bypasses these technical difficul-
ties and takes advantage of the fact that we are dealing with a self-propagating en-
tity. Consider a series of tubes, each containing 0.25 ml of the standard reaction
mixture, but no added template. The first tube is seeded with 0.2 y of QB-RNA
and incubated for a period adequate for the synthesis of several y of radioactive
RNA. An aliquot (50 \) is then transferred to the second 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, and so on. If each successive synthesis pro-
TABLE 1

SERIAL TRANSFER EXPERIMENT

 

 

14
—--——— Formation of RNA Formation of IU 13 %
2 3 4 5 6 7 ————-- Concentration of Original Template 11 12 Observed Recovery
Transfer Interval Cpm Total A = 8 9 10 A = e€.0.p oO
no. (min) Time x 1073 (y) (y) (y) y Strands IU x 1075 x 10-5 x 10-7 P2RNA

0 0 0 0 0.2 0 0 2.0 X 107! 1.2 * 10" 6.0 & 10 1.0 1.0 5.5 pie
1 40 40 64 3.2 3.0 3.0 2.0 X 107} 1.2 * 10) 6.0 & 10! 5.2 5.2 3.2 54.2
2 40 80 84 4.2 3.6 6.6 4.0 X 107? 2.4 X 10 1.2 & 10! 2.2 6.5 2.0 88.3
3 40 120 112 5.7 4.9 11.5 6.7 X 1073 4.0 X 10° 2.0 & 10° 11.3 17.4 5.3 59.9
4 40 160 134 6.7 5.6 17.1 1.1 XK 10 6.6 X 108 3.3 X 10 5.7 21.2 3.0 42.3
5 30 190 113 5.7 4.4 21.5 1.9 X 107+ 1.1 X 108 55 7.4 27.6 3.0 63.4
6 30 220 144 7.2 6.1 27.6 3.1 xX 107% 1.8 X 10° 9 15.0 36.4 3.7 82.4
7 30 250 150 7.5 6.1 33.7 5.1 X 1078 3.0 X 108 1.5 13.4 48.1 5.0 52.9
8 30 280 162 8.1 6.6 40.3 8.6 X 1077 5.0 X 105 <1 8.8 54.7 5.2 51.4
9 30 310 164 8.2 6.6 46.9 1.4 k 107 8.4 X 10° <l 5.6 58.4 2.0 92.6
10 30 340 156 7.8 6.2 53.1 2.4 * 107% 1.4 X 105 <t 9.3 66.8 4.0 54,2
11 20 360 134 6.7 5.1 58.2 4.0 X 10 2.3 X 10? <1 6... 73.7 3.7 74.3
12 20 380 121 6.0 4.7 62.9 6.6 X 107 3.8 X 10} <1 6.9 84.3 7.0 46.8
13 20 400 123 6.1 4.9 67.8 1.1 X& 107" 6 <i 3.6 89.4 4.0 49.2
14 20 420 118 5.9 4.7 72.5 1.8 X 107! I <1 10.8 102.0 5.7 79.7
15 20 440 75 3.6 2.4 74.9 3.1 &* 107? 0.16 <1 3.2 105.0 5.5 65.4

Sixteen reaction mixtures of 0.25 ml were set up, each containing 40 y of protein and the other components specified for the “standard” assay in Methods. 0.2 7 of template RNS
were added to tubes 0 and 1; RNA was extracted from the former immediately, and the latter was allowed to incubate for 40 min. Then 50 of tube 1 were transferred to tube
2, which was incubated for 40 min, and 50 A of tube 2 then transferred to tube 3, and so on, each step after the first involving a 1-6 dilution of theinput material. Every tube
was transferred from an ice bath to the 35°C water bath a few minutes before use to permit temperature equilibration. After the transfer from a given tube, 20 \ were removed
to determine the amount of P32-RNA synthesized, and the product was purified from the remainder as described in Metheds. Control tubes incubated for 60 min without the
addition of the 0.2 y of RNA showed no detectable RNA synthesis, nor any formation of infectious units.

‘All recorded numbers are normalized to 0.25 ml. Columns 1, 2, and 3 give the transfer number, the time interval permitted for synthesis, and the elapsed time from zero, re-
spectively. Column 4 records the amount of radioactive RNA found in each tube at the end of the incubation, column 5 the total RNA in each, and 6 gives the net synthesis
during the time interval. Column 7 lists the cumulative synthesis of RNA. The decreasing concentrations of the input RNA resulting from the serial dilutions are recorded in
terms of (col. 8), number of strands (col. 9), and infectious units (IU) per tube (col. 10). The last is calculated from column 9 and from an efficiency of plating (e.0.p.) of 5 X
10-7. Column 11 lists the increment in infectious units observed during each period of synthesis, corrected for the efficiency of recovery (col. 14), and column 12 represents the
corresponding sum. Column 13 is the plating efficiency (e.0.p.) determined from the observed number of plaques (col. 11) and the actual amount of RNA assayed as determined
from coluinns 6 and 14. Column 14 is determined from assays of acid-precipitable radioactivity on 20 \ aliquots of the final product as compared with colimn 5.

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Vou. 54, 1965 BIOCHEMISTRY: SPIEGELMAN ET AL. 925

 

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TRANSFERS

Fig. 2.—RNA synthesis and formation 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 amounts found in 0.25-ml aliquots.

duces RNA which can serve to initiate the next one, the experiment can be con-
tinued until a point is reached at which the initial RNA of tube 1 has been diluted
to an insignificant level. In fact, enough transfers can be made to ensure that the
last tube contains less than one strand of the input primer. Jf in all the tubes, in-
cluding 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 to follow the assays and calcu-
lations. Sixteen tubes are involved, the first (tube 0) bein gan unincubated zero
time control. It will be noted that the successive dilution was such (1-6) that, by
the 8th tube, there was less than one infectious unit ascribable to the initiating 0.2
y of RNA. Nevertheless, this same tube showed 8.8 X 105 newly synthesized in-
fectious units during the 80 min of its incubation. Finally, tube 15, which contained
less than one strand of the original input, produced 1.4 X 10)? new strands and 3.2
X 10° infectious units in 20 min. It should be noted that a control tube lacking
added RNA was incubated for 60 min. As compared with tube 1, which incor-
porated 4800 cpm for each 20 d in 40 min, the control showed no increase above the
zero time level of 80 cpm. Further, no synthesis of infective units was observed in
such controls.

Figure 2 compares the cumulative increments with time in newly synthesized
RNA (column 7) and infectious units (column 12). The agreement between in-
crements in synthesized RNA and newly appearing infectious units is excellent at
926 BIOCHEMISTRY: SPLIEGELMAN ET AL. Proc. N. AL&,

 

 

 

 

TABLE 2
SEROLOGICAL BEHAVIOR oF Virus FormMED In REsPoNsE 'to “SyntTHETIC’ RNA
a ae + + — Antisera —
TA QB ——— Anti-MS-2
Virus 0 Time 10 Min Survivors 0 Time 10 Min Survivors
Authentic
QB 1.9 X 108 1.0 X 105 0.052 1.1 X& 108 1.06 X 108 96
Virus from
synthetic
RNA 1.5 X 108 8.8 X 104 0.053 1.5 X 108 1.40 X 108 93

In all cases, lysates were made from B. coli Q13, which was also the assay organism. Antisera were used at
1/100 dilution, and the incubation temperature was 35°C. The numbers represent plaque formers per ml.

every stage of the serial transfer--and continues to the last tube. Long after the
initial RNA has been diluted to insignificant levels, the RNA from one tube serves
to initiate synthesis in the next. Further, as may be seen from the comparative
constancy of the infective efficiency (Fig. 2 and column 13 of Table 1), the new RNA
is fully as competent as the original viral RNA to program the snythesis of viral
particles in spheroplasts.

To complete the proof, it was necessary to show that. the viruses produced by the
synthesized RNA were indeed Q§, the original source of the RNA used as a seed in
tube 1 to initiate the transfer experiment. Since Q8 is a unique serological
type,” * this characteristic was chosen as a convenient diagnostie test. Plaques
induced by the RNA synthesized in tube 15 were used to produce lysates, and the
resulting particles exposed to antisera against MS-2 and Qs. The results, briefly
summarized in Tabie 2, show clearly that the synthetic RNA induces virus particles
of the same serological type as authentic Qs.

Discussion.—One perhaps might have imagined that an enzyme carrying out a
complex copying process would show a high error frequency when functioning in the
unfamiliar environment provided by the enzymologist. Had this been a quantita-
tively significant complication, biologically inactive strands should have accumu-
lated as the synthesis progressed. That this is not 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 competent biologically as the initiating “nat-
ural” material derived from virus particles.

The successful synthesis of a biologically active nucleic acid with a purified enzyme
is itself of obvious interest. However, the implication which is most pregnant with
potential usefulness stems from the demonstration that the replicase is, in fact,
generating identical copies of the viral RNA. For the first time, a system has been
made available which permits the unambiguous analysis of the molecular basis un-
derlying the replication of a self-propagating nucleic acid. Every step and com-
ponent necessary to complete the replication must be represented in the reaction
mixture described. If two enzymes are required,!* both must. be present and it
should be possible either to establish their existence or to prove that one is sufficient.
If an intermediate “‘replicating” stage intervenes between the template and the ul-
timate identical copy,!’ then a “replicative form” should be demonstrably present
in the reaction mixture. If copying is direct, no such intermediate will be found.
These and other issues of the replicating mechanism will be discussed in a subsequent
publication which will detail the relevant experiments.
Vow. 54, 1965 BIOCHEMISTRY: SPIEGELMAN ET AL. 927

Sunmary.—Experimenis are described with a purified RNA-dependent-RNA-
polymerase (replicase) induced in /. cold by the RNA bacteriophage Qs.

The data demonstrate that the enzyme can generate identical copies of added
viral RNA. A serial dilution experiment established that the newly synthesized
RNA is fully as competent as the original viral RNA to program the synthesis of
viral particles and to serve as templates for the generation of more copies. Since
the data show that the enzyme is, in fact, geuerating replicas, an unambiguous a-
nalysis of the RNA replicating mechanism is now possible in a simple system con-
sisting of purified replicase, template RNA, ribosidetriphosphates, and Mg++.

* This investigation was supported by USPHS research grant CA-01094 from the National
Cancer Institute and grant GB-2169 from the National Science Foundation.

+ Predoctoral trainee in Microbial and Molecular Genetics, grant USPHS 2-T1-GM-319.

1 Haruna, I., and 8. Spiegelman, these Procerpinas, 54, 579 (1965).

2 Watanabe, I., Nihon Rinsho, 22, 243 (1964).

3 Overby, L., G. H. Barlow, R. H. Doi, M. Jacob, and 8. Spiegelman, J. Bacteriol., in press.

‘Spahr, P. F., and B. R. Hollingworth, J. Biol. Chem., 236, 823-831 (1961).

5 Spahr, P. F., J. Biol. Chem., 239, 3716-3726 (1964).

6 Haruna, L., and 8. Spiegelman, Sczence, in press.

7 Gesteland, R. F., Federation Proc., 24, 293 (1965). Q13 is a derivative of A19, an RNase
negative mutant reported in this reference.

8 Doi, Roy H., and S. Spiegelman, these Procexpines, 49, 353-360 (1963).

9 Haruna, L., K. Nozu, Y. Ohtaka, and 8. Spiegelman, these PRocEEpINGs, 50, 905-911 (1963).

” Guthrie, G. D., aud R. L. Sinsheimer, J. Mol. Biol., 2, 297 (1960).

1 Fraser, 1)., and K. A. Jerrel, J. Biol. Chem., 205, 291-295 (1953).

12 Paranchych, W., Biochem. Biophys. Res. Commun., 11, 28 (1963).

18 Ochoa, 8., C. Weissmann, P. Borst, R. H. Burdon, and M. A. Billeter, Federation Proc., 23,
1285-1296 (1964).