Photosensitized reaction of polyribonucleotides

J. Mol . Biol. (1965) 12.50-59

Photosensitized Reaction of Polyribonucleotides I. Effects on Their Susceptibility to Enzyme Digestion and Their Ability to act as Synthetic Messengers MELVIN

I.

SIMON. LAWRENCE GROSSMAN .AND HELEN VAN VUNAKIS

Group in Biochemical Sdencee, Princeton University, Princeton, N.J. , U.S.A. and Graduate Department of Biochemistry, Brandeis University WaUham, Mass., U.S.A. (Received 18 December 1964) It had previously been shown that the primary effect of the photosensitized

oxidat ion of DNA and RNA was the preferential destruction of the guanine residue. With synthetic polyribonucleotides, absorbance chan ges occu r only in t hose polymer s which contain guanine. The capacity of guani ne-co nt ain in g polyribonucleotides to direct either the in corporation of amino ac ids int o pol y peptides or t he specific binding of a mino acyl S-RNA's to ribosom es is al tered during irradiat ion . After irradiation of pol y UG, the a ctivities med iat ed in t he amino ac idincorporating system by eit her a combination of guanine and uracil residues or b y uracil residues al one are depressed, indicating t hat the destruction of a guanine residue affects the continuity of coding to the same extent . Irradiation of poly UG does not affect the binding of phenylalanyl S-R NA, but markedly inhibits t he binding of valyl-S-RNA. The enzyme RNase T 1 wh ich is specific for guanine residues can not attack bonds in poly UG adjacent to the product(s) of photosen sitization. Nor can the amino acid-incorporating system in troduce any new amino acid after irradiation. Thus the products of photo-oxidation cannot be recognized by specific com plementing sites. either on the enzyme or in the amino acid-in corporating system.

1. Introduction Methylene blue has been shown to sensitize the specific photo-oxidation of guanine residues in nucleic acids (Simon & Van Vunakis 1962,1964; Simon, 1963). The reaction appears to be mediated by the nature of the purine base , since ribose and deoxyribose derivatives were found to be equally reactive and the guanine residues in both DNA and RNA were rapidly attacked. Synthetic polyribonucleotides should therefore also serve as substrates for the reaction. The effect of irradiation on the chemical and biochemical properties of guanine-containing polymers has been studied. This paper describes some of the results obtained when irradiated polyribonueleotides are used as : (1) substrates for pancreatic RNase and for RNase T 1 ; (2) synthetic "messenger" RNA's directing either the incorporation of amino acids into polypeptides (Nirenberg & Matthaei, 1961; Lengyel, Speyer, Ba silio & Ochoa , 1962); or (3) the specific binding of amino acyl S-RNA to ribosomes (Leder & Nirenberg. 1964). The results to be presented confirm the marked specificity of the photodynamic reaction for guanine and suggest that the reaction may be useful in exploring the events involved in polypeptide synthesis. 50

PHOTO· OXIDATION OF l'OL YIUBONUCL:EOTIDEt;

51

2. Materials and Methods Radioactive amino acids were obtained from Schwartz Bioresearch Inc. or from the New England Nuclear Corp. Nucleoside diphosphates were obtained from Schwartz Bioresearch Inc. Methylene blue was a zinc-free preparation from Coleman-Matheson & Bell. RNase 1'1 was generously provided by Dr David Strauss of Princeton University. Pancreatic RNase was purchased from Worthington Biochemicals.

Polymers Synthetic polyribonucleotides were prepared with polynucleotide phosphorylase isolated from Micrococcus lysodeikticus according to the procedure of Steiner & Beers (1961). Large-scale preparations of polymers were carried out in Ostwald viscometers as described by Grossman (1962) and purified by the method of Steiner & Beers (1961). Solutions containing the partially purified polymers were passed through columns of Sephadex G100 and the fractions containing the first peak eluted by 0·05 M-NaCl (measured by the absorb. ance at 260 rnu] were pooled. Since some of the experiments involving poly UG required relatively large amounts of polymer, a number of different preparations was used in the course ofthis work. They contained from 20 to 25% guanine and their chain length ranged from 50 to 100 residues per chain. Poly UC contained 25% cytosine. Base compositions were determined after alkaline hydrolysis and electrophoresis (0,05 M·ammonium formate, pH 3,5, 4 hr at 800 v), or descending paper chromatography using isopropanol-HCl-water (170:41:39) as the solvent system (Wyatt & Cohen, 1953).

Amino acid incorporation The "s-30 fraction" of Nirenberg & Matthaei (1961) was prepared from Escherichia coli by the procedures described. The s-30 fraction was dialyzed overnight at 5°C against 100 vol. of standard buffer (0,01 M-tris (pH 7'8), 0·1 M-magnesium acetate, 0·06 M-potassium chloride and 0·006 M-mercaptoethanol). Unless otherwise stated, the s-30 fraction was used without pre-incubation, The assay mixtures were the same as described by Nirenberg & Matthaei (1961) except that the final volume was 0·3 ml. The reaction mixtures were incubated at 37°C for 45 min and the reaction was stopped by adding 0·5 ml. of 10% TCA. t The precipitates were washed according to the methods of Siekevitz (1952) and counted with a Tracerlab windowless flow counter. In all the experiments described, control assays were performed in the absence of polynucleotide, and the results of the assays are expressed as ctsjrnin ineorporatedjmg of protein minus the control value. The binding of amino acyl S-RNA to ribosomes was measured by the method described by Leder & Nirenberg (1964). Determinaion of chain length Total phosphorus was measured by the method of Chen, Toribara & Warner (1956). For determination of monoesterified phosphorus, about 250 jLg of polymer were incubated at 37°C with 5 jLg of E. coli alkaline phosphatase (obtained from Worthington Biochemical Corp.) in 0·1 M-tris buffer (pH 8,3) for 3 hr. An equal volume of 2% PCA or IN-sulfuric acid was added at O°C and the mixtures were centrifuged at 10,000 g for 15 min. Inorganic phosphate in the resulting supernatant fluid was measured by the method of Chen et al. (1956). Irradiation The apparatus and procedures used for irradiation in the presence of methylene blue were the same as previously described (Simon & Van Vunakis, 1962).

3. Results (a) Abeorbomce properties of irradiated polyribonucleotides

Synthetic polynucleotides of different base composition were irradiated in the presence of methylene blue and their characteristic ultraviolet absorbance spectra were measured during the course of the reaction (Table I). No changes were observed

t

Abbreviations used: TCA, trichloroacetic acid; PCA, perchloric acid.

52

M. I. SIMON, L. GROSSMAN AND H. VAN VVNAKIS TABLE

1

Effect of methylene blue on the absorbance of synthetic polynucleotides

Polymer

Poly tro

Time of irradiation (hr)

o 1-5

3·5 7·0

Poly VC

o 32

Poly VA

o 32

Poly VAC

o 12

260m!, O.D.--

230m!,

2·85 2·62 2·16 1·50 2·25 2·21 3·33 3·33 1·81 1·83

The reaction mixtures contained 700 !,g/ml. of each of the polynucleotides and 18 !,g/ml. of methylene blue (0'1 M-tris buffer, pH 8'35). They were irradiated at 4000 ft-candles and oxygen was bubbled through the mixtures during the course of the reaction. Samples were removed, and diluted with distilled water. Their ultraviolet absorbance spectra were measured with the Cary automatic recording spectrophotometer.

in the absorbance spectrum of poly UC, poly UA or poly UAC. There were, however, marked changes in the absorbance spectrum of poly UG. The absorbance decreased between 300 miL and 240 miL and increased at wavelengths below 240 miL (Fig. l(a)). These changes are consistent with spectral differences observed as a result of the photosensitized oxidation of guanine derivatives (Simon & Van Vunakis, 1962). The decrease in absorbance at high wavelengths corresponds to the destruction of the purine, and the increased end absorbance is due to the products of the reaction. If the spectrum of poly UG after irradiation is subtracted from that of the starting material (corrected for the presence of methylene blue), the resulting difference spectrum closely resembles that of guanosine from 300 to 240 miL (Fig. l(b)). The spectral changes observed can be completely accounted for by the specific destruction of guanine residues. (b) Susceptibility to RNase digestion

In order to test further the effects of photosensitization on the properties of the polynucleotides, the enzymes pancreatic RNase and RNase T 1 were used (Fig. 2). Irradiation of poly UG in the presence of methylene blue had no effect on the susceptibility of poly UG to hydrolysis by pancreatic RNase, which acts specifically at phosphodiester bonds adjacent pyrimidine residues in RNA (Schmidt et al., 1951). On the other hand, the capacity of irradiated poly UG to be hydrolyzed with RNase T 1 decreased rapidly. Sato & Egami (1957) had shown that this enzyme specifically cleaves phosphodiester bonds adjacent to guanine residues. After 120 minutes of irradiation, the polymer was almost completely resistant to hydrolysis by RNase T 1 • These observations suggest that the destruction of guanine residues in the polymer results in products that can no longer be recognized as guanine residues by this specific enzyme.

PH OTO -OXIDATI O N OF POLYRIBONUCLEOTIDES

53

0'0

\

(0)

+ 0 ·200

I I 0·6

~,

\

g0' 4

(b)

I~

+ 0·100

\ \ \

.... .... o ~

0

oI

l

•

~

\,J

./1 \ ---J-------

.., 0

0

o -100 0-2

_o~

)

» • -200

0 240

220 240 260 2BO 300

320 " (mp,)

FIG . 1. Effect of irradiation on the absorbance of po ly

U~.

The rea ction m ixture containe d 600 iLg/ml. of poly UO and 13 iLg/ml. of m ethylene blue in 0 ·1 M t ris buffer (pH 8-3). It was irradiated at 4000 ft-cand les . Oxygen was bub bl ed through the solution during t he course of the reac t ion . Samples were removed and d ilu t ed with d istilled water. The absorption spectra w er e measured on t he Car y automati c recording spectrop h ot ometer . (a) Ab sorbance b efore irradiation, - - ; after 22 hr of irradiat ion , - - --. (b) Differ en ce in absorbance between poly UO b efore and after 22 hr of irradiati on. - -, Differ en ce spectrum; - - --. absorbance of guanosine . The guanosin e absorbance was cal culated b y normalizing the spectral data of Beaven, Holiday & J ohnson (1955 ) t o t he difference spectrum at 260 rnu.

(c) Ami1W acid incorporation

Irradiated polynucleotides were tested for their capacit y to direct amino acidincorporation in vitro. There was little or no change in the activity by the polynucleotides lacking guanine, Le. poly U, poly UC or poly UAC (Table 2). On the other hand, poly UG, which is sensitive to photo.oxidation, rapidly lost its capacity to direct amino acid-incorporation. Pretreatment of the polymer with methylene blue in the dark did not affect its subsequent "messenger" activity. There was no further increase in this activity when concent rations of the irradiated polymer were either increased to 200 JLg or decreased to 3 JLg per reaction vessel. Grossman (1962) and Wacker, Jacherts & Jacherts (1962) found that ultraviolet irradiation of poly U resulted in inhibition of its capacity to stimulate incorporation of phenylalanine. Accompanying this loss of t he capa city to incorporate phenylalanine, there was a concurrent stimulation of the incorporation of serine. In order to determine if the photosensitized reaction resulted in an alteration of the coding specificity of the polymer, irr adiate d poly UG was tested for its capacity to stimulate the incorporation of a variety of amino acid s. There was, however, no increase in the incorporation of the 15 amino acids tested (Table 2).

64

M. I. SIMON, L. GROSSMAN AND H. VAN VUNAKIS

., ::0 :::J 0 100

x--x

x

'"

~

'u ...

., ...E

~

80

d

6

.! ., u

...c

.0 L-

0

-c'"

..,

.0

c

., a. U

L-

0

FIG. 2. Susceptibility of irradiated poly UG to RNIIBe digestion. The reaction mixtures containing 330 p,g/mI. of poly UG and 9 p,g/ml. of methylene blue (O·ll1:. tria, pH 8'5) were irradiated at 7000 ft-eandles, while air WIIB bubbled through the mixture. Samples were removed during the course of the reaction and their ultraviolet absorbance was meaaured. Fifty p,g of polymer were incubated with 20 p,g of pancreatic RNase or RNase T 1 for 15 and 60 min at 37°0. One mg of bovine serum albumin and O·ll1:.perchloric acid were added to a final volume of l·OmI. The tubes were incubated at 0°0 for 30 min and centrifuged to remove the precipitate. The absorbance at 260 mp, of the supernatant fluids was measured and corrected for the loss in absorbance that results from irradiation (Fig. 1). Each value was multiplied by 0.D'260 0 min ------,::=-.:-. The results of enzymic digestion for 15 min and for 60 min were similar and the O.D'260 Pmm average values are presented. - X - - X - , Pancreatic RNIIBe; -e--e-, RNase T 1 •

The molecular size of the irradiated polymer was tested to determine if loss of "coding" ability was correlated with destruction of the polymer. There was, however, little if any change in the sedimentation coefficient or in the ratio of terminal to total phosphate (Table 3). Furthermore, there was no significant change in the pattern of elution of the polymer from Sephadex GlOO by 0·05 M·NaC!. Irradiation, therefore, has little or no detectable effect on the chain length of the polyribonucleotide. The results plotted in Fig. 3(a) show that the rates of inactivation of poly UG with respect to incorporation of valine and phenylalanine are similar. In other experiments, phenylalanine, valine and leucine activity were all found to decrease at about the same rate (Simon, 1963). The kinetics of phenylalanine incorporation directed by poly UG after various times of irradiation are shown in Fig. 4. The decrease in. total incorporation reflects both a decrease in the rate of incorporation and in the extent of the incorporation reaction. The incorporation reaction proceeds linearly for about 21 minutes when unirradiated polymer is used. Mer the polymer has been irradiated for 125 minutes, it stimulates phenylalanine incorporation at half the control rate and incorporation continues for only six minutes. The inactivation of the capacity of the polymer to induce polypeptide synthesis proceeds five times faster than the attack on guanine residues (Fig. 3(b)). The destruction of 8% of the guanine residues in the polymer leads to 50% loss of amino acidincorporation. On the basis of the average chain length of the polymer and its base

PHOTO-OXIDATION OF POLYRIBONUCLEOTIDES TABLE

55

2

Effed of irradiation in the presence of methylene blue on the capacity of polynucleotides to stimulate amino acid-incorporation

Polymer

Time of irradiation (hr)

Amino acids (cts/min/mg) A

Phe

Leu

810 0

Poly UG

0 12

2398t 22

Poly UC

0 12

5950t 5900

PolyU

0 10·5

4600t 3800

Poly UAC

0 12

Amino acid mixturea A

y

Ser

Lys

III

II

I

242 0

29 0

18 0

670 819

98 80

The conditions of irradiation were the same as those described in Table 1. Samples were removed at various times during the reaction. Dowex 50, Na + form, was added to remove the methylene blue. The samples were tested for the capacity to stimulate amino acid-incorporation. The total volume of the assay mixture was 0·3 ml., containing 70/Lg of polynucleotide. The assays were performed as described in Materials and Methods. The specific activities (me/mole) of the amino acids used were as follows: phenylalanine,'] 46; phenylalanine.f 125; leucine, 247; serine, 17; lysine, 5·8. The amino acid mixtures were the same as those described by Grossman (1962). Mixture I contained alanine, arginine, aspartic acid, glutamic acid and glycine. Mixture II contained histidine, isoleucine, lysine, methionine and proline. Mixture III contained serine, threonine, tryptophan, tyrosine and valine.

TABLE

3

Effect of irradiation on the integrity of poly UG Time of irradiation (hr)

[liC]Phe incorporated (cts/min/mg)

0 1 3·6

1740 520 57 0 0

9

22

Total PI Terminal

50

Sedimentation coefficient (a)

2·1

46 40

2·2

The poly VG and the conditions of irradiation were the same as described in Fig. 1. The assay mixtures contained 15 /Lg of polymer. The sedimentation coefficients were measured in solutions containing 40 J.LgJml. of polymer in 0·1 M-NaCI and 0·01 M.phosphate buffer (pH 7,0). The ratio of total to terminal phosphate was determined by the methods described above.

56

M. 1. S IMON, L . GROSSMAN AN D H. VAN VUNAK IS

10,0 00 , - - - , - - y - - - , - - . . , - - r - - - - , - - r - - ,

5000

en c:

1000

'c '0 E

c

~

e a.

'';:;

'2

c:

0

500

~

0

en

a.

E

(;

2-

v .s /0

~

...'" U

10

~

~

v

0

0

\3

0

c:

~

'E

-c

100

~

.se

~

;;-.e

.s .s'0" e

5

5 x

50

(0)

30

60

90

120

180

210

Time of irradiation (min)

FIG. 3(a) . The loss of amino acid-incorporating act iv it y of poly UG after irradiation under the conditions desc ri bed in Table 1. The s -30 fract ion was pre-incubated as described b y Nirenberg & Matthaei (1961) and 8 p,g of poly UG were added to each rea ct ion mixture. Phenylalanin e incorporat ion ; - X - - X- , valine in corp or at ion . (b) The percen t age amino acid -incorporating cap acity and percentage guanine remaining in poly UG after irradiat ion. The base compositions were determined by alkaline hydrolysis and ch ro matography. -0--0-, Percentage guanine; - 0 - - 0 -, percentage phenylalanine incorporation; - X- - X - , percentage valine incorporation remaining.

- e--e-,

composition, it can be calculated that after the destruction of an average of one guanine resid ue per chain, only 55% of the capacity for amino acid-incorporation remains. Leder & Nirenberg (1964) showed that synthetic oligonucleotides could stimulate the specific binding of amino acyl S-RNA to ribosomes . They have further defined the unique sequence of bases in the triplets which specify the binding of specific ami no acids. Figu re 5 shows the effect of irradiation in the presence of methylene blue on t he

PHOTO-OXIDATION OF POLYRIBONUOLEOTIDES

57

9000 c:

.~

a.. OJ

E

~ 6000 +'

l:

o

E-

8 .s

c

~

60min


~

3000

o'C 1'J ~

0

___0 - - - - -o0 - - - - - 125 min

12

8

16 20 Time (min)

24

28

32

FIG. 4. Kinetics of amino acid incorporation. The reaction mixtures containing 500 fIog/ml. of poly UG and 12 fIog/ml. of methylene blue (0,1 M·tris, pH 8,3) were irradiated at 3500 It-candles for the times shown on the graph. Oxygen was bubbled through the mixture and samples were removed during the course of the reaction and measured as described. The incorporation mixtures were incubated at 34DC and 10 flog of polymer were added to each.

100 -u

c:

:J

0

.D

80

«

z a::

60 >.. u 0

\

\ \

\ \

0

c:

'E

« 40

~

\ \ \

20

\ \

b

0

FIG. 5. The binding of amino acid RNA to poly UG after irradiation in the presence of methyl. ene blue. The conditions of irradiation were the same as for Fig. 2. Amino acyl RNA binding was measured by the method of Leder & Nirenberg (1964). Reaction mixtures contained 5 flog of polymer, 2·5 O.D. '60 units of ribosomes and 0·8 D.D'260 unit of amino acyl S-RNA. They were incubated at 24°0 for 20 min. Amino acid-incorporation into polypeptide was measured as described above. - X - - X - , Phe S·RNA binding; - 0 - - 0 - , Val S-RNA binding; - 0 - - 0 - , incorporation into polypeptide.

58

M.1. SIMON. L. GROSSMAN AND H. VAN VUNAKIS

capacity of poly UG to induce the binding of phenylalanyl and valyl S-RNA's. There is little if any effect of irradiation on the binding of phenylalanyl S-RNA. On the other hand, the capacity of the polymer to induce the binding of valyl S-RNA decreases with time of irradiation. The rate of loss of valyl S-RNA binding is four times slower than the loss of incorporation of amino acids into polypeptides.

4. Discussion The primary effect of irradiation of polyribonucleotides in the presence of methylene blue is the destruction of guanine residues in the polymer. This conclusion is based on the following evidence. (1) Irradiation of synthetic polyribonucleotides causes absorbance changes only in those polymers which contain guanine. These changes can be completely accounted for in terms of the specific destruction of guanine residues. (2) Irradiation does not significantly affect the chain length of the polymers. Even after extensive destruction of guanine, no marked changes are observed in the sedimentation coefficient, the ratio of total to terminal phosphate, the pattern of chromatography on Sephadex or the ultraviolet absorbance of acid-soluble material. The amount of degradation that could result from irradiation is clearly very small compared to the damage to guanine residues. (3) The capacity of guanine-containing polynucleotides to direct the incorporation of amino acids into polypeptides decreases progressively as irradiation proceeds, whereas the activity of polymers which do not contain guanine is unimpaired. Studies of the kinetics of inactivation of poly UG show that the activity of the polymer with respect to phenylalanine and valine incorporation is depressed at the same rate. The incorporation of phenylalanine is directed by uracil residues, while valine incorporation requires sequences which include guanine residues (Lengyel et al., 1962). Thus while irradiation directly affects only the guanine residues, the activity mediated by the uracil residues in the amino acid-incorporating system is also depressed. The similarity in the rates of inactivation for different amino acids indicates that the destruction of a guanine residue affects the continuity of coding to the same extent. The products of the photosensitized reaction of guanosine have not been completely characterized. Their chemical, chromatographic and electrophoretic properties, however, suggest that the purine structure is not retained (Simon & Van Vunakis, 1964; Sussenbach & Berends, 1964; Simon & Van Vunakis, 1962; and Simon, 1963). The enzyme RNase T 1 cannot attack bonds in the polymer adjacent to the product of photosensitization, and the amino acid-incorporating system does not introduce any new amino acid after irradiation. This suggests that the products of photo-oxidation cannot be recognized by specific complementing sites either on the enzyme or in the amino acid-incorporating system. If the coding units in poly VG are translated in a sequential manner, then the presence of the product of the photosensitized reaction in the polymer chain may serve to terminate polypeptide synthesis and thus interfere equally with the incorporation of valine and of phenylalanine. Support for this interpretation is derived from experiments measuring the binding of amino acyl S-RNA to ribosomes induced by poly VG. While the amino acidincorporation assay measures the total activity of the polymer, the binding assay requires only the information contained in a single coding unit. Irradiation of poly va

PHOTO·OXIDATION OF POLYRIBONUCLEOTIDES

ri9

did not affect the binding of phenylalanyl S-RNA, but markedly inhibited binding by valyl S-RNA. These results are consistent with the idea that destruction of a guanine residue converts a readable code unit into one which cannot be read. The presence of a "nonsense" word could terminate polypeptide synthesis by inhibiting the binding of the proper amino acyl S·RNA or by releasing the synthetic polymer from its binding site on the ribosome. This interpretation has been suggested by a number of workers in order to explain the effects of group-specific reagents and purine and pyrimidine analogues on the amino acid-incorporating activity of synthetic polynucleotides (Wahba et al., 1963; Ludlum, Warner & Wahba, 1964; Michelson & Grunberg-Manago, 1964). Photosensitization is a simple and specific method for affecting the properties of polyribonucleotides. The observations presented suggest a number of applications of this technique. For example, RNase T 1 cannot cleave phosphodiester bonds adjacent to guanine residues that have been altered by irradiation in the presence of methylene blue; thus partial photo-oxidation of RNA followed by treatment with RNase T 1 and chromatography should provide a series of oligonucleotides that would increase the precision of determination of base sequence. This and other questions of the effect of the reaction on the template activity of polynucleotides are currently being investigated. During the course of this research, Melvin 1. Simon held a predoctoral (GPM.13, 346·C2) and postdoctoral (5·72.GM.13.346.02) fellowship from the National Institutes of Health. His new address is Department of Biology, University of California, San Diego, California. Helen Van Vunakis is a recipient of a Public Health Service research career award (5.K6. AI.2372) from the National Institute of Allergy and Infectious Disease. This work W88 supported in part by grants from the National Institutes of Health (E.2792) and the National Science Foundation (B-1671 and G-883). This paper is Publication No. 347 of the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts. REFERENCES Beaven, G. H., Holiday, E. R. & Johnson, E. A. (1955). In The Nucleic Acid8, ed, by E. Chargaff & J. N. Davidson, vol. 1. New York: Academic Press Inc. Chen, P. S., Toribara, T. Y. & Warner, H. (1956). Analyt. Ohern. 28,1756. Grossman, L. (1962). Proc, Nat. Acad. Sei., Wa8h.48, 1609. Leder, P. & Nirenberg, M. W. (1964). Science, 145, 1399. Lengyel, P., Speyer, J. F., Basilio, C. & Ochoa, S. (1962). Proc. Nat. Acad. s«, Wash. 48, 282. Ludlum, P., Warner, R. C. & Wahba, A. J. (1964). Science, 145, 397. Michelson, A. M. & Grunberg-Manage, M. (1964). Biochim. biophy8. Acta, 91, 92. Nirenberg, M. W. & Matthaei, J. H. (1961). Proc, Nat. Acad. s«, Wa8h. 47, 1588. Sato, K. & Egami, F. (1957). J. Biochem., Tokyo, 44, 753. Schmidt, G., Cubiles, R., Zollner, N., Hecht, L., Strickler, N., Seraidarian, K., Seraidarian, M. & Thannhauser, S. J. (1951). J. Biol. Ohem, 192, 719. Siekevitz, P. (1952). J. Biol. Ohem. 195, 549. Simon, M. 1. (1963). Doctorate thesis, Grad. Dept. of'Bioehem., Brandeis University, Waltham, Mass. Simon, M. 1. & Van Vunakis, H. (1962). J. Mol. Biol. 4, 488. Simon, M. 1. & Van Vunakis, H. (1964). Arch. Biochem. Biophy8. 105, 197. Steiner, R. & Beers, R. (1961). In Polynucleotidea. Amsterdam: Elsevier Pub. Co. Sussenbaoh, J. S. & Berends, W. (1964). Biochem. Biophy8. Res. Oomm. 16, 263. Wacker, A., Jacherts, D. & Jacherts, B. (1962). Angew. Ohem. International Edition, 1, 509. Wahba, A. J., Gardner, R. S., Basilio, C., Miller, R. S., Speyer, J. F. & Lengyel, P. (1963). Proc, Nat. Acad. Sci., Wash. 49, 116. Wyatt, G. R. & Cohen, S. S. (1958). J. Biochem. 55, 774.