The Unwinding of the DNA Molecule

The Unwinding of the DNA MoleculeAuthor(s): Peter FongSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 52, No. 2 (Aug. 15, 1964), pp. 239-246Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/72429 .

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VOL. 52, 1964 MICROBIOLOGY: P. FONG 239

518-mu compound as a catalyst of photosynthesis, but they do question its locali- zation.

Summary.-The properties of a newly observed light-induced absorbance change in the 600-mu region in green plants are described. The spectrum of this absorbance change fits very well with the oxidized-ninus-reduced spectrum of purified plastocyanin in vitro. It is also demonstrated that the light-induced increase of absorbance corresponds to an oxidation and that far-red light, 713 mu, is more effective than red, 651 mni, in causing it. An antagonistic effect of red and far-red light on this absorbance change is also reported. It is suggested that plastocyanin might be a link between the two photochemical systems of photosynthesis.

It is a pleasure to thank Dr. A. Muller who gave one of us (D. F.) much valuable advice which greatly facilitated the initial assembly of the absorbance-change apparatus.

I Katoh, S., Nature, 186, 533 (1960). 2Katoh, S., I. Suga, I. Shiratori, and A. Takamiya, Arch. Biochem. Biophys., 94, 136 (1961). 3 Katoh, S., and A. Takamiya, Plant Cell Physiol., 4, 335 (1963). Kok, B., and G. Hochl, in La Photosynthese (Paris: Centre National de la Recherche Scien-

tifique, 1963), p. 93. ' Kok, B., in Photosynthetic Mechanisms of Green Plants, NAS-NRC Pub. No. 1.145 (1963),

p. 35. 6 Norrish, R. G. W., and G. Porter, Nature, 164, 658 (1.949). 7 Witt, H. T., R. Moraw, and A. Muller, Z. Physik. Chem. Neue Folge, 20, 193 (1959). 8 Spoehr, H. A., and H. W. Milner, Plant Physiol., 24, 120 (1949). 9 Fork, D. C., Plant Physiol., 38, 323 (1963).

11 Witt, H. T., A. Miiller, and B. Rumberg, in La Photosynthese (Paris: Centre National de la Recherche Scientifique, 1963), p. 43.

1 Muller, A., D. C. Fork, and H. T. Witt, Z. Naturforsch., 18b, 142 (1963). 2 Green, L. F., J. F. McCarthy, and C. G. King, J. Biol. Chem., 128, 447 (1939).

13 de Kouchkovsky, Y., Physiol. Veg., 1, 15 (1963). 14 Duysens, L. N. M., in La Photosynthese (Paris: Centre National de la Recherche Scienti-

fique, 1963), p. 75. L" Lichtenthaler, H. K., and R. B. Park, Nature, 198, 1070 (1963). 16 Fork, D. C., and Y. de Kouchkovsky, submitted to the 4th Intern. Congr. Photobiology, Oxford,

England, July 26-30, 1964.

THE UNWINDING OF THE DNA MOLECULE

BY PETER FONG

PHYSICS .DEPARTMENT AND LABORATORY OF NUCLEAR STUDIES, CORNELL UNIVERSITY

Communicated by Robert R. Wilson, June 5, 196.4

Introduction. The unwinding of the DNA molecule has been discussed by Delbriick, Gamow,2 Platt, Bloch,4 Levinthal and Crane, Kuhn,6 Longuet-Higgins and Zimm,7 and Fixman,s mostly in connection with the replication process. It is true that based on the principle of complementary replication the unwinding of the parent DNA is unavoidable in the replication process, yet it is not absolutely necessary that the parent be completely unwound first. It may well be that the

unwinding of the parent, the duplication of the strands, and the rewinding of the two daughter DNA's proceed simultaneously as first suggested by Watson and


518-mu compound as a catalyst of photosynthesis, but they do question its locali- zation.

Summary.-The properties of a newly observed light-induced absorbance change in the 600-mu region in green plants are described. The spectrum of this absorbance change fits very well with the oxidized-ninus-reduced spectrum of purified plastocyanin in vitro. It is also demonstrated that the light-induced increase of absorbance corresponds to an oxidation and that far-red light, 713 mu, is more effective than red, 651 mni, in causing it. An antagonistic effect of red and far-red light on this absorbance change is also reported. It is suggested that plastocyanin might be a link between the two photochemical systems of photosynthesis.

It is a pleasure to thank Dr. A. Muller who gave one of us (D. F.) much valuable advice which greatly facilitated the initial assembly of the absorbance-change apparatus.

I Katoh, S., Nature, 186, 533 (1960). 2Katoh, S., I. Suga, I. Shiratori, and A. Takamiya, Arch. Biochem. Biophys., 94, 136 (1961). 3 Katoh, S., and A. Takamiya, Plant Cell Physiol., 4, 335 (1963). Kok, B., and G. Hochl, in La Photosynthese (Paris: Centre National de la Recherche Scien-

tifique, 1963), p. 93. ' Kok, B., in Photosynthetic Mechanisms of Green Plants, NAS-NRC Pub. No. 1.145 (1963),

p. 35. 6 Norrish, R. G. W., and G. Porter, Nature, 164, 658 (1.949). 7 Witt, H. T., R. Moraw, and A. Muller, Z. Physik. Chem. Neue Folge, 20, 193 (1959). 8 Spoehr, H. A., and H. W. Milner, Plant Physiol., 24, 120 (1949). 9 Fork, D. C., Plant Physiol., 38, 323 (1963).

11 Witt, H. T., A. Miiller, and B. Rumberg, in La Photosynthese (Paris: Centre National de la Recherche Scientifique, 1963), p. 43.

1 Muller, A., D. C. Fork, and H. T. Witt, Z. Naturforsch., 18b, 142 (1963). 2 Green, L. F., J. F. McCarthy, and C. G. King, J. Biol. Chem., 128, 447 (1939).

13 de Kouchkovsky, Y., Physiol. Veg., 1, 15 (1963). 14 Duysens, L. N. M., in La Photosynthese (Paris: Centre National de la Recherche Scienti-

fique, 1963), p. 75. L" Lichtenthaler, H. K., and R. B. Park, Nature, 198, 1070 (1963). 16 Fork, D. C., and Y. de Kouchkovsky, submitted to the 4th Intern. Congr. Photobiology, Oxford,

England, July 26-30, 1964.

THE UNWINDING OF THE DNA MOLECULE

BY PETER FONG

PHYSICS .DEPARTMENT AND LABORATORY OF NUCLEAR STUDIES, CORNELL UNIVERSITY

Communicated by Robert R. Wilson, June 5, 196.4

Introduction. The unwinding of the DNA molecule has been discussed by Delbriick, Gamow,2 Platt, Bloch,4 Levinthal and Crane, Kuhn,6 Longuet-Higgins and Zimm,7 and Fixman,s mostly in connection with the replication process. It is true that based on the principle of complementary replication the unwinding of the parent DNA is unavoidable in the replication process, yet it is not absolutely necessary that the parent be completely unwound first. It may well be that the

unwinding of the parent, the duplication of the strands, and the rewinding of the two daughter DNA's proceed simultaneously as first suggested by Watson and



240 MICROBIOLOGY: P. FONG PltOC. N. A. S.

Crick' and later expounded by Levinthal and Crane.5 In this nmechanism only infinitesimal unwinding (about 360) is involved in the replication of one base pair. On the other hand, complete unwinding takes place in the melting process, i.e., the process of thermal denaturation. Partial unwinding may be involved in the

transcription process. In this paper a general theory of unwinding is developed which may be applied to complete, partial, and infinitesimal unwinding as

special cases. In particular, we determine the time of complete unwinding of a DNA of 2 X 104 turns, which is the number of turns of T2 bacteriophage. Experi- mentally, the replication time for T2 phage is 2-3 mnin and the nmelting time for coli DNA at 100?C is about I min.

The qualitative discussions'-5 will not be reviewed here; none of them is without difficulties. Kuhn's mechanism of translational Brownian nmotion is not efficient enough: it would take 10 days to unwind 2 X 104 turns. Incidentally, Kuhn has also shown that it takes 150 days to unwrap turn by turn from the end a DNA molecule of 900 turns. Fixman's work is a generalization of Longuet- Higgins and Zimn's; the mechanism of the latter will be briefly discussed. They proposed that there exists a torque tending to unwind from the ends, originating froum the increase of entropy when the bonded base pair is broken and the two bases and the sections of chains thus made loose can freely rotate about a total of ten valence bonds, each having a few positions of minimum potential energy. Once an unwinding torque exists, the process of unwinding becomes directional and proceeds much more rapidly than a random process. However, a question may be raised on their calculation from thernmodynamic considerations. The work done in a thermodynamic process may be calculated from the increase of entropy only when the process is reversible. In irreversible and spontaneous processes the work done is smaller than that in reversible processes and may even be zero, such as in the free expansion of a gas against vacuum. The torque they calculated applies only to a reversible process in which the unwinding torque would be balanced by an external torque and the DNA would unwind quasi-statically and reversibly. However, the actual unwinding process is obviously not reversible and therefore less work is done which we do not know how to calculate. Their result of the

unwinding time is thus the lower limit. Another difficulty which they noted is that the unwinding torque is likely to change the tightly wound double helix to a

configuration of two loosely intertwined strands, and additional mechanism is re-

quired to unravel the remaining windings. According to this theory the time to unwind 2 X 104 turns would be at least 12 min, which is too long.

An important point in considering the unwinding of DNA is that the winding number of a double helix is a topological invariant as long as the ends are fixed. Therefore any mechanism of unwinding, barring the cutting of links, must involve the rotating of one end some 2 X 104 turns relative to the other. Without intelli-

gent supervision how can a molecule keep on turning always in the same direction for so many turns? Thus, besides the physical and chemical problems, we have an additional information problem. It is true that unwinding is a process in the direction of increasing entropy and therefore may be accomplished eventually by any random processes; but the time required by a random process is usually very lolg. Our problem is thus to find a mechanism efficient enough so as to lead to the correct time of replication and melting, both of the order of a minute. In the following we




propose a mechanism of unwinding. The application to infinitesimal unwinding will be discussed in another paper. ?

Mechanism of Unwinding.-The unwinding of the DNA molecule is preceded by the breaking of the hydrogen bonds between the base pairs. In the melting process the bonds are broken by increasing temperature. Changing the pH value may produce the same effect. Before the bonds are broken, the molecule may be regarded as a rigid rod and may rotate with respect to its own axis with an energy determined by the principle of equipartition of energy. This energy fluctuates in the course of time according to statistical mechanics. Once the bonds are broken, the rod may no longer be considered as rigid and rotation of one end may not be transmitted instantaneously to the other. For the sake of argument, let us say that a few bonds are broken at the midpoint; then each of the two sections of the rod may pursue an independent rotation with an independent fluctuation. Each angular velocity may be positive or negative. One half of the time the two angular velocities of the two sections have the same sign, resulting in no change of the winding number. When the signs are opposite, one half of the time they result in unwinding while another half of the time they result in overwinding. Now, the DNA molecule is a closely packed one and thus cannot overwind. When there is kinetic energy tending to overwind, the energy will be converted into

potential energy of squeezing the atoms closer to one another, and then the potential energy will tend to unwind, just as a ball bounces back from a wall. The magnitude of the angular velocity remains the same, but the direction is changed from overwind to unwind. Thus, at the beginning of the unwinding process, one half of the time the molecule tends to unwind.

Once a sufficiently large number of turns has been unwound, the situation is different. Now the two sections can afford to rewind as far as the unwound turns are used up in rewinding. Thus the two sections of the DNA will unwind and rewind at random, and the situation may be compared with the random walk problem. Even by random walk alone the DNA may completely unwind itself. The actual situation is better than that because rewind cannot proceed indefinitely; it is sub-

ject to the condition of no overwind. Furthermore, at the point of overwind the

angular velocity is reversed, which tends to unwind. The random walk is thus a biased one, the bias favoring unwinding.

The physical random walk takes place in the momentum space but the bias is set up in the configuration space. The involvement of the two spaces complicates the solution. Our interest lies in the configuration space-we want to find the time when the molecule is completely unwound. The mathematical problem is discussed below.

Mathematical Theory.-Definition: The winding number N is defined as the number of revolutions rotated through by a vector formed by the two points of in- tersection made with the two strands of the double helix by a plane perpendicular to the helical axis, when the plane moves from one end of the helix to the other.

The winding nunmber at any timle t is designated by N(t); the initial winding nunber, i.e., the number of turns of the DNA molecule is designated by No. The relative angular velocity of the two sections of DNA at time t is designated by - @(t).



242 MICROBIOLOGY: P. FONG PROC. N. A. S.

THEOREM (topology). The winding number is a topological invariant as long as the ends are fixed.

ASSUMPTION (physics). The DNA molecvle cannot overwind. Any attempt to overwind results in reversing of angular velocity o (t). Thus,

N(t) <No, for t > 0, (1)

and

o(t_) = -c(t+), when N(t) No. (2)

PROBLEM. Find the unwinding time T such that

foT c(t)dt = -2wNo; (3)

the fluctuation of co(t) is governed by statistical mechanics subject to the bias of equation (2) (which implies equation 1).

In this paper we do not attelnmpt to solve comlipletely the biased raandomlm walk problem. Instead we shall calculate the upper and lower limits of T; this is sufficient for the biological aspect of the problem as far as the informatioln difficulty is concerned. We introduce a quantity, the unwinding angular velocity W(t) which is the average of w(t) over a short time interval containing lmany fluctuations. It is a monotonically decreasing function of tinme (this corresponds to the fact that it takes more than twice the time to unwind a double'helix of twice the number of turns).

The lower limit of the unwinding time T may be obtained by replacing o((t) with its maximum value co(0), its initial value. Let the rotational angular velocity of one section of the DNA about its axis due to thermal nmotion be coo. When the two sections tend to unwind, the value of co(O) equals 2wo on the average. Since the DNA unwinds one half of the time at t = 0, we conclude co(0) equal to ? w(O) or coo. We calculate oo by the principle of equipartition of energy,

I o2 = - kT, (4)

where I is the moment of inertia about the helical axis of one section of the DNA, k is the Boltzmann constant, and T is the absolute temperature of the solution in which the DNA molecule lies. We consider a DNA molecule of 2 X 104 turns, corresponding to a molecular weight of 1.2 X 108, consisting of 2 X 105 base pairs. The radius of gyration about the helical axis is calculated from the latest model of DNA-Model 3 of Langridge et al.t' The value is 6.68 A. The mass of one section of the DNA is 6 X 107 molecular weight; the mom-ent of inertia may be calculated. At 20 ?C the value of coo is found to be

co = 3.0 X 10s rad/sec. (5)

Using this value for C(0), we calculate the lower limit TI of the unwinding time,

TI = 27rNo0/(0) = 4.2 X 10- sec. (6)

The upper limit of the unwinding tinme 7' may be obtained by considering the problem as a pure random walk probleim without the benefit of the bias. We simplify further by considering the unwinding time Tu of one section of the DNA molecule (1 X 104 turns) with respect to the midpoint of the molecule which is



VoL. 52, 1964 MICROBIOLOGY: P. FONG 243

assumed to be fixed; the other half of the molecule will unwind in the same time by the same mechanism. In the calculation we meet the difficulty that the kinetic theory of gases is not applicable and the kinetic theory of liquid is little known. Fortunately, the difficulty may be circumvented by using the empirical pressure of the solution without a detailed knowledge of the structure of the liquid state of water. First we consider the random walk in the momentuln space. We assume the DNA molecule to be immersed in water at 20?C exposed to one atmosphere pressure. By the principle of equipartition of energy we calculate and obtain the following: L = average angular momentum of rotation of a DNA section = 1.34 X 10-22 cgsu. p = average linear momentum of the water molecule = 1.90 X 10--1 cgsu. Multiplying p by the radius of the DNA molecule, we get the order of magnitude of the average angular momentum transfer per collision from water to DNA, I = average angular momentum transfer per collision = 1.71 X 10-25 cgsu. Define m as the ratio of L to 1,

L m -- 7.84 X 102. (7)

Since m is a large number, it takes many collisions to change the angular momentum of the DNA appreciably; in particular, to change it from L to zero. Let us calculate the time necessary for the fluctuation to do so. The number of collisions necessary is just m2. The number of collisions per unit time by water molecules on the DNA may be calculated from the pressure of water (one atmosphere), the dimension of the DNA molecule (radius = 9.05 A, pitch = 33.6 A),1 and the average linear momentum of water molecule p; the result is 2.04 X 10k5 collisions per sec. The time required for m2 collisions is thus calculated to be 3.01 X 10-1? sec, which is the time required to change the angular momentum from L to 0. The mean time X between two consecutive zeros of the fluctuating angular momentum is just twice this amount because of symmetry with respect to time in fluctuation,

X = 6.02 X 10-1? sec. (8)

We now consider the problem in the configuration space. Within the time period X the angular velocity of rotation of the DNA does not change sign, and therefore the molecule keeps on winding or unwinding in the same direction. Consequently, we may consider the time period X and the number of turns n wound or unwound during this period as a basic step, and the problem in the configuration space may be regarded as a random walk problem in which the basic steps so defined are compounded randomly. n may be calculated as follows:

n = woX = 2.88 X 10-2 turns. (9)

The time required to unwind 104 turns may thus be calculated as follows:

T = (104/n)X = 73 sec. (10)

The unwinding time T is thus between the upper and lower limits, 73 and 4 X 10-4 sec. For winding number No >> 1 it can be shown that T is very close to the

upper limit. Thus T ~ 73 sec for 2 X 104 turns. The unwinding time is thus comparable to the melting time of 1 min and reasonably small compared to the

replication time of 2-3 min.



244 3MICROBIOLOGY: P. FONG PROC. N. A. S.

Discussion.--The topological theorelim minakes it clear that, barring the cutting of the links, the unwinding must be associated with a rotation of one end relative to the other by No turns. This still leaves the freedom of choosing the point where the unwinding is to be started. To start unwinding from the ends involves the following difficulties which were pointed out by Levinthal and Crane.5 The unwound strands whip through the fluid, causing large viscous drag. Furthermlore, the loose unwound strands may entangle each other. This leaves the unwinding to start from the middle, as proposed here, the most likely mechanism for which the above difficulties do not appear. There are no loose ends to entangle. The unwound parts do not whip around in the solution; it is the wound parts that rotate in the solution. The viscous drag on the unwound parts thus does not appear and that on the wound parts is small, as Levinthal and Crane have shown.

It is not likely that the ends should remain intact until the very end of the process. The Longuet-Higgins and Ziumm mechanism or others may cause a small number of turns to be unwound at the ends. Mlost likely the loose ends, once sufficiently long, do not rotate in the fluid and therefore do not contribute to the unwinding any further.

The present nlechanism makes use of a random process in the miomentum space, which is more efficient than a random process in the configuration space, such as proposed by Kuhn, because of the fact that within the time period X the angular velocity fluctuates in magnitude but does not change direction so that the angular displacements in the period X are added. in the same direction instead of being compounded at random, which is less efficient.

The present mechanism is efficient because it makes use of the rotational motion of the wound part of the DNA molecule, which is a collective motion. The wound turns are coordinated with one another in rotation, and this reduces the information requirement in directing the unwinding. The information requirement is finally reduced to zero by making use of the random walk mnechanism.

One question may be raised at this point. After the hydrogen bonds are broken, the DNA molecule becomes flexible. There exists the possibility that each turn or each small section of the DNA may execute independent rotation thus destroying the advantage of the collective motion employed in the present mechanism. The justification of treating the unwinding molecule as two connected rigid rods lies in the following fact: a single turn or a section of turns in the middle of the flexible molecule cannot execute independent rotation. If it does, it decreases the winding number on one side of it but increases the winding number on the other side. According to the physical assumption of no overwinding, the increase of winding number cannot take place. Thus it cannot rotate independently. Even after the bonds are broken, the two sections behave as if they were rigid. For the whole molecule there can be only two possible independent rotations, each corresponding to one end of the molecule; this is just what we assumed.

There still remains the possibility that after a large number of turns has been unwound in the middle, the two rigid sections will now have a total of four ends and four independent rotations may develop, resulting in four rigid sections connected by unwound strands. Further fragmentation may develop as unwinding proceeds. When many sections are present, the rotation of those in the middle does not contribute to the unwinding according to the topological theorem, each one un-




winding at one end while rewinding at the other with no net change of the total

winding number. The unwinding is achieved by the rotation of the two sections at the two ends, for which the theory has been developed. Once an end section is

unwound, we have two loose strands. They may not rotate in the fluid as pre- viously discussed. Furthermore, once fragmentation takes place, it will not stop, the length of each fragment section is likely to be short, and the loose strands at the ends will be short accordingly. They will remain short as the sections in the middle diffuse outward and rewind them. The entanglement difficulty thus may not appear. The important point that concerns us here is whether the fragmentation process speeds up or slows down the unwinding process. The answer is that it

speeds up. What makes unwinding a slow process is that there is a large number of unwound turns between two rigid sections which makes rewind and unwind equally possible. The fragmentation process decreases the number of unwound turns between any two rigid sections; this prevents indefinite rewinding and increases the chance of angular velocity reflection (eq. 2) which helps unwinding. Thus

fragmentation tends to speed up the unwinding process. Furthermore, the un-

winding time of an end section is proportional to the cube of the section length; fragmentation shortens the length of the section to be unwound at a time and thus also shortens the total unwinding time for the whole molecule. As long as we are interested in an upper limit, we need not consider fragmentation. In a more detailed study the statistics of fragmentation should be worked out.

One effect which was neglected and should be considered in a more detailed study is the change of the moment of inertia of a section of DNA as unwinding proceeds and the resulting change of the other dynamical quantities involved. However, this effect should not change the order of magnitude of the result.

For DNA molecules of a different molecular weight M, the dependence of Tu and T, on M may be determined by dimensional analysis: Tu is proportional to MI whereas Ti is proportional to M312.

Finally a comment on the transcription process. The manufacture of the

messenger RNA is likely to be preceded by the opening up of a section of the DNA double helix. The mechanism of unwinding discussed here is suitable for this

purpose; it allows a section in the middle to be opened without disturbing the other parts of the DNA, and the time required to do so is extrermely short. If the unwinding is to proceed from the ends, it would be quite inefficient to unwind the whole molecule to use just a small section of it. Furthermore, such a process would take a long time.

This work was undertaken after discussions of preliminary ideas with Professor John R. Platt of the University of Chicago, whose initiation of the author into molecular biology and whose sug- gestions are gratefully acknowledged. The author also wishes to thank Professor Philip Morrison for many discussions and criticisms on the manuscript, and Professor H. C. Longuet-Higgins for valuable discussions.

Delbruck, M., these PROCEEDINGS, 40, 783 (1954). 2 Gamow, G., these PROCEEDINGS, 41, 7 (1955). 3 Platt, J. R., these PROCEEDINGS, 41, 181 (1955). 4 Bloch, D. P., these PROCEEDINGS, 41, 1058 (1955). ' Levinthal, C., and H. R. Crane, these PROCEEDINGS, 42, 436 (1956). 6 Kuhn, W., Experientia, 13, 301 (1957). 7 Longuet-Higgins, H. C., and B. H. Zimm, J. Mol. Biol. 2, 1 (1960).



246 MICROBIOLOGY: WECKER AND LEDERHILGER PROC . N. A. S.

8 Fixman, M., J. Mol. Biol., 6, 39 (1963). 9 Watson, J. D., and F. H. C. Crick, Nature, 171, 737, 964 (1953); Crick, F. H. C., and J. D.

Watson, Proc. Roy. Soc. (London), A223, 80 (1954). 10 Fong, P., "The replication of the DNA molecule" these PROCEEDINGS, in press (Sept. issue). 1 Langridge, R., et al., J. Mol. Biol., 2, 38 (1960).

CURTAILMENT OF THE LATENT PERIOD BY DOUBLE-INFECTION WITH POLIOVIRUSES*

BY E. WECKER AND G. LEDERHILGERt

THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY, PHILADELPHIA

Communicated by Warren H. Lewis, May 19, 1964

The events taking place during the latent period of small RNA-containing animal viruses have been the subject of several recent investigations. It has been shown that infection with Mengovirus and poliovirus causes the inhibition of cellular RNA as well as protein synthesis within the first 2 hr.1-9 The appearance of a new viral RNA polymerase and its increasing activity with time has also been demonstrated. 0-x2 These mechanisms inply the involvement of newly formed proteins since they can be prevented by inhibition of protein synthesis.9 13-15

Although the virus undoubtedly initiates the synthesis of these proteins, nothing is known about their virus specificity.

The present study deals with the question 9o- of whether the prior infection of a cell with

one virus can shorten the latent period of a Control subsequently infecting second virus (timed

8-0- ............ ...... double-infection). The experiments were conducted with

...........T35 .two closely related virus strains: a type 3

/ poliovirus strain, Habel 24, which is highly / sensitive to guanidine (Gs3), and a mutant

population obtained from Habel 24 which re- '' ........................ T3 quires guanidine for optimal growth (Gr3).

60- . In further experiments, two different types of poliovirus were used, a guanidine-sensi-

'.t.... tive type I strain M\ahoney and a guanidine- oI-_ ,T, _ , __"""-T2 . requiring type 3 strain Habel 24 (Gsl and

2 3 TI 6 Hours

Gr), and the results indicated that the latent

I 1 . period of the superinfecting viruses could be FIG. 1.--The interruption of Gs virus

formation by guanidine. Control samples significantly shortened both in homotypic were harvested at the times (T) indicated and heterotypic double-infection. It was and assayed for the presence of Gs virus. At the same times guanidine.HCl (100 thereby observed that the superinfecting ,ug/ml as in all other experiments where virus seems to replicate by using the mecha- guanidine was used) was added to aliquots of the cells. These aliquots were then fur- nlsm initiated by the first infecting virus. ther incubated until time 6 hr. Time 0 Cords and Holland, independently, have. ob- represents the time when the cells were first exposed to the virus. tained similar results.6

246 MICROBIOLOGY: WECKER AND LEDERHILGER PROC . N. A. S.

8 Fixman, M., J. Mol. Biol., 6, 39 (1963). 9 Watson, J. D., and F. H. C. Crick, Nature, 171, 737, 964 (1953); Crick, F. H. C., and J. D.

Watson, Proc. Roy. Soc. (London), A223, 80 (1954). 10 Fong, P., "The replication of the DNA molecule" these PROCEEDINGS, in press (Sept. issue). 1 Langridge, R., et al., J. Mol. Biol., 2, 38 (1960).

CURTAILMENT OF THE LATENT PERIOD BY DOUBLE-INFECTION WITH POLIOVIRUSES*

BY E. WECKER AND G. LEDERHILGERt

THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY, PHILADELPHIA

Communicated by Warren H. Lewis, May 19, 1964

The events taking place during the latent period of small RNA-containing animal viruses have been the subject of several recent investigations. It has been shown that infection with Mengovirus and poliovirus causes the inhibition of cellular RNA as well as protein synthesis within the first 2 hr.1-9 The appearance of a new viral RNA polymerase and its increasing activity with time has also been demonstrated. 0-x2 These mechanisms inply the involvement of newly formed proteins since they can be prevented by inhibition of protein synthesis.9 13-15

Although the virus undoubtedly initiates the synthesis of these proteins, nothing is known about their virus specificity.

The present study deals with the question 9o- of whether the prior infection of a cell with

one virus can shorten the latent period of a Control subsequently infecting second virus (timed

8-0- ............ ...... double-infection). The experiments were conducted with

...........T35 .two closely related virus strains: a type 3

/ poliovirus strain, Habel 24, which is highly / sensitive to guanidine (Gs3), and a mutant

population obtained from Habel 24 which re- '' ........................ T3 quires guanidine for optimal growth (Gr3).

60- . In further experiments, two different types of poliovirus were used, a guanidine-sensi-

'.t.... tive type I strain M\ahoney and a guanidine- oI-_ ,T, _ , __"""-T2 . requiring type 3 strain Habel 24 (Gsl and

2 3 TI 6 Hours

Gr), and the results indicated that the latent

I 1 . period of the superinfecting viruses could be FIG. 1.--The interruption of Gs virus

formation by guanidine. Control samples significantly shortened both in homotypic were harvested at the times (T) indicated and heterotypic double-infection. It was and assayed for the presence of Gs virus. At the same times guanidine.HCl (100 thereby observed that the superinfecting ,ug/ml as in all other experiments where virus seems to replicate by using the mecha- guanidine was used) was added to aliquots of the cells. These aliquots were then fur- nlsm initiated by the first infecting virus. ther incubated until time 6 hr. Time 0 Cords and Holland, independently, have. ob- represents the time when the cells were first exposed to the virus. tained similar results.6



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