Synthesis of tobacco mosaic virus infectivity by cell-free extracts

BIOCHIMICA ET BIOPHYSICAACTA

257

BBA 83).3

S Y N T H E S I S O F T O B A C C O MOSAIC V I R U S I N F E C T I V I T Y BY CELL-FREE EXTRACTS w. R. HUDSON*, Y. T. KIM*, R. A. SMITH" A~D S. G. WILDMAN* University o/ Cali/ornia, Los Angeles, Cali[. (U.S.A.) (Received April i6th, 1963) SUMMARY An improved and simple method is described for preparing extracts from TMVinfected leaves which engender a rise in TMV infectivity simultaneously with incorporation of [14C]ATP into a material having the properties of RNA. The rise in infectivity depends on the presence of ATP, GTP, CTP and UTP in the incubation mixture. Incorporation occurs with ATP alone, but to a greater extent when the four triphosphate nucleotides are present. 2. The incorporation and rise in TMV infectivity reactions are time dependent, inactivated by ribonuclease (EC 2.7.7.16 ), and heat.

INTRODUCTION In a previous communication 1 a method was described for obtaining cell-free extracts from TMV-infected tobacco leaves which in themselves were infectious, and which would produce a rise in TMV infectivity when incubated with ATP, GTP, CTP and UTP. The rise in infectivity was attributed to the de novo synthesis of TMV-RNA from the nucleoside triphosphates. Since then, two other investigations have reported on a similar phenomenon*, 3. The purpose of this communication is to present an improved method for isolating materials capable of TMV-RNA biosynthesis and to describe some of the properties of these extracts as measured both by infectivity and the incorporation of radioactive isotopes. The extraction method is based on the previous finding of WANG AND COMMONER4 that leaf DNA is extractable with high concentrations of NaC1. METHOD AND MATERIALS Plant material Nicotiana tabacum var. Turkish Samsun plants grown in 6-in pots are used when they are 12-18 in in height and contain about 20 well-expanded leaves. The plants are topped, and the upper surface of each expanded leaf is inoculated with a gauze pad moistened with a solution of lO-2 mg of common TMV per ml. The infection is

Abbreviations: TMV, tobacco mosaic virus; 4N, equal molar mixture of ATP, GTP, CTP and UTP. * Department of Botany and Plant Biochemistry. ** Division of Biochemistry of the Department of Chemistry. Biochim. Biophys. Acta, 76 (1963) 257-265

258

W.R.

HUDSON, Y. T. KIM, R. A. SMITH, S. C. WILDMAN

allowed to develop under glasshouse conditions for up to 3° days before the directly inoculated leaves are used for making extracts.

Method o/ extraction Infected leaves are removed from the plant and the midribs dissected and discarded. The lamina tissue is stored in a deep-freeze overnight. Freezing is not a necessary condition for obtaining active extracts, but aids in the homogenization of the leaf tissue. Working in a cold room, the frozen leaves are crushed to a coarse powder and placed in a Waring Blendor. Water containing 7 mg of bentonite per ml, and 0.2 M mercaptoethanol is added to the blendor in the ratio of two parts liquid to i part of leaves (v/w). The bentonite is prepared according to the directions of FRAENKEL-CONRAT et al. 5. The mixture is blended just long enough to produce a homogeneous slurry. The slurry is transfered to 50 ml plastic centrifuge tubes, and then spun in a Sorvall centrifuge at top speed for 20 rain. The brown supernatant solution is decanted and discarded. The green pellets are scraped out of the tubes into a mortar to which is added glycine-phosphate buffer (o.I M glycine, 0.05 M K2HP04, 0.2 M mercaptoethanol, adjusted to p H 9-5) which contains 20 % NaC1. Except for the NaC1, DIENER6 found this buffer to be an effective solvent for TMV-RNA. A volume of buffer equal to the original weight of leaves is added to the mortar, and the mixture homogenized with a pestle for about IO rain. (In our experience, blending cannot be used as a substitute for mortar and pestle homogenization at this stage in the preparation of active extracts.) The viscous, green, homogenate is transfered to plastic centrifuge tubes and spun at 7000 rev./min for IO rain in a Sorvall centrifuge. The supernatant solution (yellow and turbid with TMV-infected leaves; greenish and turbid with healthy tobacco leaves) is decanted and saved. This material is referred to as the GPS extract. The green pellets are discarded.

Chemical analyses and nucleotides RNA was estimated by the orcinol method of DISCHE7 using phenol-purified TMV-RNA as the standard. DNA analyses were performed by the diphenylamine method s, a highly purified sample of thymus Z)NA, kindly supplied by Dr. N. SIMMONS of the Laboratory of Nuclear Medicine, UCLA, serving as the standard. Protein was estimated by the Fodn-Wu method 9. [8-14C]ATP was a commercial preparation obtained from Schwarz Bio-Research Inc. ATP, GTP, CTP and UTP were obtained as sodium salts from Calbiochem.

Bioassay /or in/ectivity Nicotiana glutinosa was used for all local lesion assays for TMV infectivity. Either half-leaf comparisons were used when only two treatments were to be compared, or latin-square arrangements for more than two comparisons. With half-leaf comparisons, a solution to be tested for infectivity was applied as a drop to one half-leaf, another drop of 0.067 M phosphate buffer (pH 7) containing 500 mg of celite per inl was added to the first drop, and the combined mixture rubbed over the half-leaf surface with the aid of a glass paddle, a clean paddle being used for each inoculation. In the case of whole-leaf latin-square assays, 2 drops of the test solution together with 2 drops of phosphate buffer were rubbed over the entire leaf surface. Biochim. Biophys. Acta, 76 (I963) 257-265

S Y N T H E S I S OF T M V

259

INFECTIVITY

Latin-square assays were repeated so that each measurement of infectivity was derived from at least eight leaves randomly distributed in regard to individual plants and position of the leaves on the plants.

Radioactivity measurements Nucleic acids and proteins contained in incubation mixtures were precipitated by adding sufficient 15 % HCI04 to produce a final concentration of 6 % HCI04. 0.3 mg of carrier TMV-RNA, prepared by the phenol method 1° was then added. The mixture was allowed to stand in an ice bath for 20 rain, and then centrifuged at top speed in an International Clinical Centrifuge for 15 min. The supernatant solution was decanted and discarded. The pellets were resuspended in 2 ml of 1 % HC1Q, the centrifugation repeated, and in this manner the precipitate was washed three times with 1 % HCI04. The washed precipitate was finally dissolved in 0.5 M Nlff,OFI, an aliquot deposited on a planchet, and after drying, counted in a gas-flow counter to 5 % significance. EXPERIMENTAL RESULTS

Properties o/ GPS extracts Immediately after preparation, the GPS extract is first dialyzed for 2 h against two changes of I 1 each of 0.067 M KH2PO4-K2HPO * buffer (pFI 7) to remove excess NaC1. While some variation in composition occurs from extract to extract, an average composition of dialyzed GPS extract is around i. 3 mg per ml of protein, 0.04 mg per ml of DNA, and 0.2 mg per ml of RNA, or an equivalent yield on a gram fresh weight of leaves basis. As shown by the data in Table I, the GPS extract exhibits a rise in virus infecTABLE

I

THE EFFECT OF FOUR TRIPHOSPHATE NUCLEOTIDES ON THE INCORPORATION OF [14CIATP INTO AN ACID-PRECIPITABLE MATERIAL CORRELATED WITH A RISE IN T M V INFECTIVITY I h i n c u b a t i o n at 25 °. R e a c t i o n m i x t u r e : o.2 m l extract; o.15 m l A T P ( 2 / , m o l e s / m l ) o r 4 N (2 p m o l e s / m l each of A T P , G T P , C T P and U T P ) ; o . i ml T r i s - p h o s p h a t e buffer ( p H 7 . o ) ( i M Tris, 4 ° / z M K H z P O , , 0 . 6 / * M m e r c a p t o e t h a n o l ) ; 0.05 m l M g - M n (o.08 M MgC1 v 0.02 M MnC12). Specific a c t i v i t y A T P , 1 . 2 8 - l O s c o u n t s / m i n / p m o l e . --, n o t tested.

Nature o/ extract Expt.

No.

Spun

GPS ATP

4N

ATP

GPS

Pellet 4N

ATP

4N

16

Incorporation* Lesions'*

2.22 1574

5.80 2o97

o.41 171

1.51 296

2.47 1386

3.88 1804

0.22 I96O

0.66 2332

o. i o 262

0.59 363

o.33 I63O

1.67 I975

--

o. 15 lO 3

2.28 193

--

--

--

_

17

Incorporation Lesions 25

Incorporation

--

Lesions

--

* Incorporation: m / z m o l e s A T P fixed per 0. 5 m l reaction m i x t u r e . ** L e s i o n s : G P S and Pellet, t o t a l lesions per 16 leaves; S p u n GPS, t o t a l lesions per 8 leaves;

L a t i n - s q u a r e bioassays. Biochim. Biophys. Acta, 76 (1963) 2 5 7 - 2 6 5

260

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HUDSON, Y. T. KIM, R. A. SMITH, S. C. WILDMAN

tivity after incubation with 4N compared to incubation with ATP alone. The GPS extracts also incorporate [14C]ATP into an acid-precipitable material and to a greater degree in the presence of 4 N compared to ATP alone. GPS extracts contain a high level of infectivity without incubation, and it is probable that intact TMV nucleoprotein m a y contribute to this background infectivity, although this possibility has not been definitely established. In this connection, we have investigated the possibility that the infectivity of TMV might be "activated" when incubated with 4 N. However, when purified TMV was incubated with 4 N and also with A T P alone, neither of the treatments produced a change in the infectivity of the virus. Similarly, when phenol-prepared TMV-RNA was used instead of TMV, no change in infectivity occurred as the result of incubation with ATP or 4 N'. With the object of trying to separate the process responsible for the rise in infectivity from the process responsible for the incorporation of [14C]ATP, procedures aimed at the further fractionation of the GPS extracts were explored, none of which has been successful in separation of the two processes. It is possible to eliminate infectivity from extracts that still incorporate the isotope, but no evidence has yet been obtained to indicate that a rise in infectivity has occurred in the absence of incorporation. That infectivity could be the most labile element of the two processes is not surprising in view of the extensive research 11,~ that indicates that disruption of a single phosphodiester bond is enough to inactivate the infectivity of TMV-RNA.

Fractionation o~ GPS extracts by centri[ugation When undialyzed GPS extracts are centrifuged for I h at 4 ° ooo rev./min in a Spinco Model-L centrifuge, a pellet is deposited which can be separated from the crystal clear supernatant solution, the latter being referred to as Spun GPS extracts which have a composition of around 0.6 mg protein per g fresh weight of leaves, 0.04 mg per g I)N'A, and o.I mg per g RNA. Thus, most of the material removed as a pellet is protein and RNA. DNA has not been detected in the pellet. As shown by the data in Table I, both the pellet and the Spun GPS extracts produce a rise in infectivity as well as a greater degre~ of [14C]ATP incorporation upon incubation with 4 N as compared to ATP alone. Although the results of only three experiments are shown, m a n y other experiments have shown the Spun GPS extracts to contain infectivity which rises after incubation with 4 N. The level of infectivity contained in Spun GPS extracts is variable, and seems to depend upon the length of time the leaves were infected with TMV, and also on the seasonal behavior of the plants supporting the infectious process. With periods of infections less than 15 days during the Winter months, the Spun GPS extracts m a y be barren of infectivity, and no appearance of infectivity is induced by incubation with ATP or 4 N. When the period of infection extends from 15 to 30 days, infectivity is usually present in the extracts, and when found, a rise has invariably occurred upon incubation with 4N. Our experience so far suggests that a shorter period of infection will result in infectious GPS extracts when the plants are harvested during the period of late Spring to early Fall. The pellet obtained after high-speed centrifugation of GPS extracts contains most of the infectivity of the GPS extract. The apparent absence of I)NA in this Biochim. Biophys. Acta, 76 (1963) 257--265

S Y N T H E S I S OF

TMV

INFECTIVITY

261

material together with the evidence that it will produce a rise in infectivity as well as incorporation of ATP may indicate that TMV-RNA biosynthesis is not concerned with a DNA template.

Fractionation o/ GPS extracts by ethanol precipitation During the Summer and Fall months of 1962, most of our experimentation was performed with extracts that were obtained by precipitation of a material from GPS extracts by ethanol. When two volumes of ethanol (containing 935 ml of 95 % ethanol, 60 ml glacial acetic acid, and 5 ml of 2 N NaOH) are added to one volume of undialyzed GPS extract, a translucent, sticky material gradually forms a cohesive mass and floats towards the top of the vessel in which the solutions were mixed. After standing in the cold for 20 rain, this material can often be removed by "wrapping" it around a stirring rod. After removing excess ethanol by blotting, the product can be scraped off the rod and partially redissolved in glycine-phosphate buffer containing 20 % NaC1. This material will be referred to as E t O H - I extracts.

Properties o/EtOH-I extracts Table II contains the results of several experiments on incorporation of [x4C]ATP into an RNA-like material after incubation with E t O H - I extracts. In these experiments, the ethanol precipitate was resuspended in o.15 M NaC1 in a volume i / i o t h of that of the GPS extract. While there is considerable variability from sample TABLE

1I

INCORPORATION OF [ x 4 C ] A T P INTO AN ACID-PRECIPITABLE MATERIAL RESULTING FROM INCUBATION OF E t O H - I EXTRACTS WITH A T P AND FOUR TRIPHOSPHATE NUCLEOTIDES Incubation

c o n d i t i o n s s a m e as for T a b l e I.

mttmoles A T P fixed per o,5 ml o] reaction mixture Expt. No.

2 3 6 II 12

ATP

4N

0.35 0.38 o.Io o.17 o.62

4.1 8.4 0.27 o.75 8.o

to sample, in all cases there was more incorporation when the incubation mixtures contained 4 N compared to ATP alone. None of extracts in Table II contained infectivity either before or after incubation. However, in experiments performed in Summer and Fall before isotopes were used, infectivity was found in equivalent extracts and furthermore increased upon incubation with 4 N. Since the isotope experiments were all performed in the Winter months, it is surmised that absence of infectivity can be attributed to the season, and that there are times of the year when both kinds of activity will be found in these extracts. That RNA is the probable product into which [14C]ATP is incorporated is shown by the data in Table III. In this experiment, the E t O H - I precipitate was resuspended into o.15 M NaC1, the suspension divided into aliquots, each of which received the B i o c h i m . B i o p h y s . A c t a , 76 (1963) 2 5 7 - 2 6 5

262

W. R. H U D S O N , Y. T. KIM, R. A. S M I T H , S. C. W I L D M A N TABLE

11I

EFFECT OF VARIOUS AGENTS ON THE ACID-PRECIPITABLE PROPERTIES OF THE PRODUCT OF [14C]ATP INCORPORATION INDUCED BY INCUBATION OF E t O H - I EXTRACTS WITH A T P OR FOUR NUCLEOTIDE TRIPHOSPHATES Incubation

Conditions

ATP 4N 4N 4N 4N 4N 4N

c o n d i t i o n s s a m e a s in T a b l e I. ml~moles A T P fixed per 0.5 ml reaction mixture

Treatment

Incubate 2 h Incubate I h Incubate 2 h I n c u b a t e I h, t h e n I h with io/,g/ml lZNAase I n c u b a t e I h, t h e n I h with Io/~g/ml DNAase I n c u b a t e 2 h, t h e n o. 5 N N a O H o v e r n i g h t , 25 ° I n c u b a t e 2 h, t h e n h o t 5 ~0 H C I 0 4 f o r 20 m i n

o.62 i.o 2.2 0.7 1. 3 0. 4 0.03

treatment indicated in Table I I I . The change in the acid precipitation properties of the product of ATP incorporation by the various agents employed is consistent with the known properties of RNA. DNAase ~ does not destroy the HCI04 precipitation properties, but seems to prevent further incorporation of the isotope during incubation, an effect similar to an experiment previously reported 1 where DNAase appeared to prevent a rise in infectivity that was otherwise promoted by incubation with 4 N. The time dependency of the incorporation reaction is also shown in Table I I I , and other experiments have indicated that the reaction ends after 2 h of incubation, and the level of incorporation m a y decline with still longer periods of incubation. Other experiments indicate that optimum incorporation occurs at p H 7- The composition of E t O H - I extracts is around o.o4 mg protein, o.o2 mg I)NA and o.o4 mg RNA per gram fresh weight of leaves. Many of our experiments during the Summer and early Fall were performed with material that had been twice precipitated with ethanol. In this case, E t O H - I was resuspended in one-half the volume of glycine-phosphate buffer-2o °/o NaC1 in which it was contained as GPS. After allowing 2o-3o min for solvation, the suspension was centrifuged for IO min at 7ooo rev./min in a Sorvall centrifuge. A pellet was deposited which was discarded. One volume of the slightly turbid supernatant was mixed with two volumes of ethanol causing an almost colorless precipitate to form quickly and float towards the top of the vessel. The precipitate was removed by winding on a rod, and the material was resuspended in a volume of o.15 N NaC1 equivalent to I / I o t h to I/2oth of the volume of the GPS extract. This suspension will be referred to as EtOH-2 extract. The composition of this material is around o.o2 mg protein, o.o2 mg DNA, and o.o4 mg RNA per gram flesh weight of leaves.

Properties o/ EtOH-2 extracts All of the experiments with this material were performed b y infectivity measurements, and occurred before work with isotopes commenced. At the time of preparing " C r y s t a l l i n e d e o x y r i b o n u c l e a s e ( d e o x y r i b o n u c l e a t e o l i g o n u c l e o t i d o - h y d r o l a s e , E C 3.1.4.5) ( S i g m a Co.). B i o c h i m . B i o p h y s . A c t a , 76 (1963) 2 5 7 - 2 6 5

SYNTHESIS

OF

TMV

263

INFECTIVITY

this paper, we have not had an experiment which combines the two methods of measurement, the fact being that none of the recent EtOI-[-2 extracts has been infectious, even though they were prepared from GPS extracts that were infective. On the presumption that presence or absence of infectivity in EtOH-2 extracts is also a seasonal effect, we will present data obtained in the Summer and Fall of 1962, and which at that time were highly reproducible. Table IV contains data showing the results of four experiments utilizing EtOH-2 extracts, all of which show a rise in infectivity induced by incubation with 4 N. These four experiments are representative of at least 50 other experiments involving ethanol extracts. Also shown are similar results obtained when EtOH-2 extracts were reprecipitated to yield EtOH-3 extracts. The results with Spun GPS are inserted into this table to show that raising the concentration of ATP to the same level as the 4N does not evoke the rise in infectivity found with the 4 N, and also that the presence of ATP does not alter the level of infectivity over that found in the absence of nucleotides. TABLE RISE OF T i M

IV

INFECTIVITY I N D U C E D BY INCUBATION OF D I F F E R E N T EXTRACTS WITH FOUR TRIPHOSPHATE NUCLEOTIDES

I n c u b a t i o n c o n d i t i o n s s a m e as in Table I e x c e p t for 4 A T P w h e r e c o n c e n t r a t i o n w a s raised to 4/tmoles/ml. N u m b e r s in p a r e n t h e s e s are the t o t a l leaves used in the bioassay. --, n o t tested. Total lesions Expt. No.

66 67 68 61 8o 86 85

Nature o] extract

EtOH-2 EtOH-2 EtOH-2 EtOH-2 EtOH- 3 EtOH-3 Spun GPS

Assay procedure

H a l f leaf (16) H a l f leaf (16) H a l f leaf (12) H a l f leaf (16) H a l f leaf (8) Latin square (16) Latin square (16)

H20

ATP

4 ATP

4N

---63 --4°

41 197 58 -217 112 51

------48

69 317 91

ioo 372 203 90

The data in Table V show that the level of infectivity induced by the interaction of EtOH-2 extracts and 4N is dependent on the time of incubation. The data in Table VI provide evidence that the increase in infectivity is induced by a material in TABLE

V

EFFECT OF TIME OF INCUBATION OF E t O H - 2 EXTRACTS WITH FOUR TRIPHOSPHATE NUCLEOTIDES ON T M V INFECTIVITY

I n c u b a t i o n c o n d i t i o n s s a m e as in Table I. All ingredients m i x e d at o °, the z e r o - t i m e s a m p l e w i t h d r a w n f o l l o w e d b y i n c u b a t i o n at 25 °. R e a c t i o n s t o p p e d b y placing a l i q u o t s at o °. Latinsquare bioassay. - - , n o t tested. Time (min)

Total lesions per 8 leaves Expt. 34

Expt. 55

Expt. 58

o 15

156 206

195 --

89 --

3° 60

337 358

-342

-178

Biochim.

Biophys.

Acta,

76

(1963)

257-265

264

W. R. HUDSON, Y. T. KIM, R. A. SMITH, S. G. WILDMAN TABLE VI

EFFECT

ON

RISE

IN

TMV

AFTER

INFECTIVITY

INCUBATION

WHEN

WITH

EtOH-2

FOUR

EXTRACTS

TRIPHOSPHATE

ARE

HEATED

BEFORE

AND

NUCLEOTIDES

I n c u b a t i o n c o n d i t i o n s as in T a b l e I. L a t i n - s q u a r e b i o a s s a y . Total lesions per 8 leaves

Conditions

I h i n c u b a t i o n a t 25 ° i n a b s e n c e of n u c l e o t i d e s 5 rain h e a t i n g of e x t r a c t a t 65 °, 4 N a d d e d , t h e n i n c u b a t e I h a t 25 ° I h i n c u b a t i o n a t 25 ° w i t h 4 N I h i n c u b a t i o n a t 25 ° w i t h 4 N, t h e n h e a t a t 65 ° for 5 rain

Expt. 42

Expt. 44

428 4o2

193 191

769 784

303 351

EtOH-2 extracts which is inactivated b y heat and therefore has this attribute of an enzyme. The data in Table V I I show that both the infectivity initially present in the E t 0 H - 2 extracts, as well as the increase in infectivity induced b y incubation with 4 N is inactivated by incubation with ribonuclease*. To prevent possible confusion with the inhibitory action of ribonuclease on TMV (see ref. I3), the level of ribonuclease used was insufficient to have caused more than IO % inhibition, if all of the infectivity had been due to TMV. TABLE VII EFFECT OF R N A A s E ON TMV INFECTIVITY OF E t O H - 2 EXTRACTS I n c u b a t i o n c o n d i t i o n s as in T a b l e I. L a t i n - s q u a r e b i o a s s a y . --, n o t t e s t e d . Total lesions per 8 leaves Cond~ions Expt. 35

I I I I

h incubation h incubation h incubation h incubation bation with

w i t h no n u c l e o t i d e s with ATP w i t h 4N w i t h 4 N, followed b y I h incuI / ~ g / m l of lZNAase

Expt. 39

418 134 191 35

572 89

DISCUSSION

While there are still m a n y experiments that need to be performed with this system, the work that has been accomplished so far is consistent with the view that a de novo synthesis of TMV-RNA occurs through the agency of cell-free, enzymatic extracts interacting with ATP, GTP, CTP and UTP. Where it has been possible to measure a rise in infectivity simultaneously with incorporation of [14C]ATP into an RNAlike material, the two processes have been found to be closely correlated. However incorporation of ATP appears to occur in the absence of GTP, CTP and UTP, although to a lower level than occurs when all four nucleotide triphosphates are present together, whereas the rise in infectivity is dependent on all four ingredients. " Crystalline pancreatic ribonuclease (cyclizing), EC 2.7.7.16) (Sigma Co.).

( p o l y r i b o n u c l e o t i d e 2-ol i gonuc l e ot i do t r a n s f e r a s e

Biochim.

Biophys.

A c t a , 76 (1963) 257-265

SYNTHESIS OF T1V[V INFECTIVITY

265

It does not seem profitable to speculate on possible explanations for these differences at this time, since it is hoped that further experimentation will permit a decision to be made on whether the isotope is actually being incorporated into TMV-lZNA. In the same sense, further experiments may resolve the question of whether the incorporation and infectious processes are controlled by the presence of DNA, RNA or both. It is clear, however, that simple methods are now available for preparing reproducible GPS and Spun GPS extracts that display both incorporation and a rise in TMV infectivity, and that these are useful starting materials for the further characterization of the nature of the two phenomena. ACKNOWLEDGEMENTS

Supported by research grant E 536(CII) from the U. S. Public Health Service, and Contract AT-(II-I)-34, Project 8 from the U. S. Atomic Energy Commission. We are grateful for the advice we have received from Dr. J-I{. Wu and Mr. T. GIVEON. REFERENCES 1 y . T. HIM AND S. WILDMAN, Biochem. Biophys. Res. Commun., 8 (1962) 394. 2 G. COCHRAN, A. DHALIWAL, G'. WELKIE, J. CHIDESTER, ~¢[. LEE AND B. CHANDRASEKHAR, Science, 138 (1962) 46 . s IV[. KARASER AND G. SCHRAMM, Biochim. Biophys. Res. Commun., 9 (1962) 63. 4 T. WANG AND B. COMMONER, Proc. Natl. Aca¢~. Sci. U.S., 42 (1956) 831. 5 H . FRAENKEL-CONRAT, B. SINGER AND A. TSUGITA, Virology, 14 (1961) 54. 6 T. O. DIENER, Virology, 14 (1961) 177. Z. DlSCHE in E. CHARGAFF AND J. DAVlDSON, The Nucleic Acids, Vol. I, A c a d e m i c Press, N e w York, 1956, p. 286. 8 K. BURTON, Biochem. J., 62 (1956) 315. 9 0 . LOWRY, N. ROSEBROUGH, A. FARR AND R. RANDALL., J. Biol. Chem., 193 (1951) 265. io A. GIERER AND O. SCHRAMM, Z. Natur]orsch., I I B (1956) 138. 11 W . C*INOZA, Nature, 181 (1958) 958. 1, A. GIERER, Nature, 179 (1957) 1297. 13 H. LORING, J. Gen. Physiol., 25 (1942) 497-

Biochim. Biophys. Acta, 76 (1963) 257-265