The proteins of mutants of TMV : Composition and structure of chemically evoked mutants of TMV RNA

J. Mol. Biol. (1962) 5, 284-292

The Proteins of Mutants of TMV: Composition and Structure of Chemically Evoked Mutants of TMVRNA AxmA

TSUGITAt

Virus Laboratory, University of California, Berkeley, California, U.S.A. (Received 15 May 1962) Data concerning the amino acid composition and the location of amino acid exchanges, if any, in 55 chemically evoked mutants of TMV and in 35 progeny isolates are reported. The relation of these amino acid replacement data to the problem of the code relating nucleotide to amino acid sequences is discussed.

1. Introduction To understand the mechanism of protein synthesis, it is necessary to understand the functional relationship between the genetic material and the protein. In TMV the RNA has been shown to carry all the genetic information required to determine the symptoms in the host and the amino acid sequence of the protein coat. This was proved by the mixed reconstitution experiments (Fraenkel-Conrat, Singer & Williams, 1957), as well as by the studies of chemically evoked mutants (Tsugita & FraenkelConrat, 1962a,b). If we assume that the nucleotide sequence in the RNA is related to the amino acid sequence of the protein coat, and that the nature of the correspondence can be generalized, TMV appears ideal for the study of the "coding problem" of protein synthesis. We already possess experimental methods for the production of mutants in vitro (Gierer & Mundry, 1958; Fraenkel-Conrat, 1961) and have knowledge of the complete amino acid sequence of the protein (Tsugita et al., 1960). Unfortunately, the chemical study of the RNA is not sufficiently advanced to permit the localization of mutagenic chemical events on the polynucleotide chain. Also genetic methods are not readily applicable to TMV, and recombination has not yet been clearly demonstrated with any plant virus. The amino acid compositions of the proteins of 29 artificial mutant strains of TMV have been reported (Tsugita & Fraenkel-Conrat, 1960, 1962a). In this paper we present analyses of the amino acid composition of additional strains and the approximate or exact location of the amino acid exchanges of most of the artificial mutants studied. These experimental results will be discussed in the light of current theories and data pertaining to the problem of coding and information transfer.

t Present address, Research Institute for Protein, Osaka University, Osaka, Japan. 284

CHEMICALLY EVOKED TMV MUTANTS

285

2. Materials and Methods TMV and Tl\1V RNA were prepared b y standard m ethods. The mutagens used wer e ni trous acid (HNO z), N-bromosu ccinimide (NBSI), as we ll as alkylating reagents such as dimethylsulfate (DM S) and propyl en e oxide (PO) (Fraenkel-Conrat, 1961). The sev eral m ethods for production , dete ction and isolation of mutants were the same as previously employe d (Tsugita & Fraenkel-Conrat , 1962a). Th e virus proteins wer e isolated by the ac etic acid m ethod prior to ch emi cal study (Fraenkel-Conrat, 1957a). The amino ac id compositions of t he mutant proteins were obtained with t he Sp inc o ami n o acid analyser under t he car efu lly contr olled conditions descr ibed in a previous paper (Tsu gita & Fra enkel-Conrat, 1962a) . Additional ex pe r imen t a l data suggest that in order t o obtain va lid analytical data for se r ine and threonine on e must be particularly care ful t o main tain t he tem pe rature at 108 ± 1°C. Tryptophan was determined sp ectrophotometrica lly, and cysteine by -SH titration (Fraenk el-Conrat, 1957b). After amino acid analysis, samples of 50 to 100 m g. of t he p roteins were digested with trypsin at 40°C (ratio of en zyme t o subs tr at e was 1 to 50 or 100). The reaction was maintained at pH 8·0 with automat ic titration using 0·1 N-NaOH; it reached completion within 1 hr. After digestion, the reaction mixtures were adjusted to pH 4·5 and allowed to stand for 1 hr in an ice bath. The resultant precipitate contained mainl y the no. 1 peptidej (Ip ep t ide) with minor amounts of other p eptides, particul arly peptides 10 and 12. Longer p eriods at ODC incr eased t he minor p ep tide contaminat ion s. The precipitates were further purified eith er by column chro m atogr aphy on Duolit e A -2 (a weak b asic resin) (Tsugita, to be published) or by oxidat ion with p erformic acid (Hirs , 1956) followed by paper chromat ography. The supe rnatant fr actions fr om t he t ryptic digests wer e se p ara te d on Dowex 1 x 2 (0,9 x 150 em ) by a modified m ethod deriv ed from t hat origin all y r eported by Wit tmann & Braunitzer (1959). This modified procedure h ad t he ad vantages of automat ic control and bet t er r eproducibilit y. Figure 1 shows the p ep t id e sep aration pat t ern under t he m odified con dit ions, and t he id entifica tion of ea ch p eak in terms of t he sequential number of it s main p eptide com p onents, wh ich had no t previou sly b een r ep or ted. The m ain difficult y in this fract iona ti on was t hat several pairs of peptides were often n ot clearly sep arated an d a ll n eeded (further) purifica tion after p ooling. Anothe r difficul t y , probably due t o t he stro ng acetic ac id n eed ed for elution of some of the p ep tides from t he column, was the app earance of p ep t id e d egradat ion product s in t he course of column chrom atography. Other impuriti es were caused by some chymotryptic prot eolysis occurring in the course of tryptic digest ion, and no r eliable m ethod was found t o fr ee t he t rypsin fro m this a cti vi t y. Owing to d egr adation of some pep tides and t o irrevers ible adsorpt ion of others on the column, the yie lds of the later peaks wer e gene rally not sat isfact ory. P eptide 10 was especially difficul t to remove from the column, even with glac ial acetic acid. Further purification of peptide 10 by paper chromatography or p aper elect r oph or esis was also difficult because of its t enden cy to become irreversibly absorbed by the paper. Peptide 1 presents technical probl ems owing to its size (41 amino acids) and its abundance of labile peptide b onds (e.g. -Ser-Ser-). It al so has a t endency to adsorb other p eptide fractions as describ ed above. These facts and the lack of sp ecific degradation m ethods render the analysis an d system at ic study of p eptide 1 d ifficult. Since peptide 1 contains on e cy steine (the only cys teine in the protein) which can under go oxidation to a variable degr ee, it was found advantageous to oxidize the cysteine to cysteic acid by m eans of p erformic acid b efor e proceeding with further purifica t ion of t h is p ep tide by either p aper or column chromat ography. Samples from eac h p eak, or from bo th shoulders of eac h peak in cases where its purit y was in doubt, as we ll as a port ion of t he purified p eptide 1, were h ydrolysed for 48 hr with twice-d istilled 5·7 N-HCl in sealed evacuated t ubes at 105 t o 108°C. The p ep tide h ydroly sat es were an alysed for amino ac id com position wit h the Spinco am in o acid analyser under modified conditions. A 50 em column was used ins tead of a

t All peptides will be r eferred to b y their se quen t ia.l numbers, st a rtin g with the a.cetyl- Nt erminal I -peptide (no. 1).

AKIRA TSUGITA

286

150 cm column for acidic and neutral amino acids and a 5 em column was used instead of a 15 em one for basic amino acids. For the former column the buffer change period is 3 hr and the time for a complete analysis is 5·5 hr. A 5 cm column analysis can be completed in 1·5 hr. Advantages in this variant in the use of the amino acid analyser for analysis of peptides are that the time for analysis is but one-third that of the normal procedure and that only one-tenth as large a sample (0,01 to 0·25 fl-g) is required to obtain nearly the same accuracy (±2%) as obtained by the standard procedure.

~

" 0·90 ]8

.,

2

~

.5')

V")

.oJ

0

0·60

ci

.9 5 "0 0·30

12 6

~

'6

7 3

9

8 4

.,

<:7l

c 0

11

.s: u

v .s

"0 u..

FIG. 1. Chromatographic separation of peptides in soluble fraction of tryptic digest of TMV protein. 1. 1 % collidine-l % pyridine-fl-Sfi X 10- 3 N-acetic acid (pH 8,2) (180 ml.), 2. Gradient (byautograde) (Technicon Chromatography Co. N.Y.).

Chamber no. 1 1 % collidine 1 % pyridine 0·033 N-acetic acid (pH 7,3). no.2 same no. 3 0·1 N-acetio acid no. 4 0·5 N-acetic acid no. 5 0·5 N-acetic acid no. 6 1 N-acetic acid no. 7 2 N-acetic acid no. 8 glacial acetic acid no. 9 glacial acetic acid (each chamber contains 120 mI.) Column 0·9 x 150 cm Dowex 1 x 2 (200 to 400 mesh); flow rate 30 mI.jhr by Mini pump (Milton Roy Co.); fraction 3·3 mI.; 80 mg protein. The numbers identify the main components of each peak with the sequential peptide number on the polypeptide chain; no. 1 is not present because it is the N-terminal I-peptide removed by centrifugation prior to chromatography. The peptides which showed amino acid compositions different from those of the normal strain were subjected to sequential analysis. For structural study, further purification was generally required, since all of the peptides separated 'by the column except nos. 3 and 4 usually contained impurities. Paper chromatography, using the solvent system n-butanolpyridine-acetic acid-water (30 : 6 : 20 : 24), achieved adequate purification of almost all peptides in a one-dimensional procedure. The methods used to study these purified peptides were, as usual, dinitrophenylation, hydrazinolysis, the phenylisothiocyanate method, and enzymic digestion with leucine aminopeptidase and carboxypeptidase A and B. If necessary, partial degradation with various enzymes such as chymotrypsin, subtilisin (Crystalline bacterial A Lprotease, Nagase no. 10074) or a streptomyces protease (Pronase, Kaken) was also used. For the technical reasons discussed above localizations of changes in peptides 1 and 10 have not yet been attempted.

CHEMICALL Y EVOKED TMV MUTANTS

287

3. Results and Discussion Analytical results It was previously reported that mutants which gave systemic symptoms on N. sylvestris generally showed no amino acid replacements. On the other hand, mutants giving local lesions on N. sylvestris usually had amino acid changes (Tsugita & Fraenkel-Conrat, 1962a). This conclusion, which is supported also by the current data, enables us to select for analysis those mutants which have a high probability of showing amino acid changes. TABLE 1

Amino acid composition of chemically evoked strainst NBSI

HNO.

Asp Thr Ser GIu Pro GIy Ala Cys 1/2 Val Ileu Leu Tyr Phe Lys Arg Try Met His Total

TMV

237

18 16 16 16 8 6 14 1 14 9 12 4 8 2 11 3 0 0

18 16 16 16 8 8 14 1 14 9 12 4 8 2 9 3 0 0

158

±fromTMV

DMS

32 IB t 329

332

284

330

326

176

178

18 16

18 16 16 16 7 6 14 1 14 9

18 16 16

15 16 8 6 14 1 14 9 12 4 8 2 11 3 0 0

18 16 16 16 7 6 14 1 14 9

13 4 8 2 11 3 0 0

18 16 16 16 8 7 14 1 14 9 12 4 8 2 10 3 0 0

18 16 16 16 8 14 1 14 9 12 4 8 2 8 3 0 0

16 8 6 14 1 14 9 12 4 10 2 11 3 0 0

4 8 2 11 3 0 0

10 3 0 0

18 17 16 16 8 6 14 1 14 8 12 4 8 2 11 3 0 0

158

158

158

158

158

158

158

158

158

2

3

2

2

1

3

1

1

9

14

13

15 8 7 14

1 14 9 12 4 8

3

16

19

t Numerals in italics indicate differences from TMV. Besides these strains showing differences from common TMV, which in most instances were confirmed also by the analyses of progeny isolates, 30 other strains showed no detectable differences in composition from the parental strain. t Secondary change from 237. Most mutants showing analytical differences were subsequently tested for genetic stability by further cycles of passage through single lesions, propagation, isolation and amino acid analysis. In most cases the progeny composition agreed with that of the original strain. Occasionally, and mainly when the temperature in the greenhouse was unusually high, the progeny showed an amino acid composition different from that of the original mutant. This could be attributed to natural mutation, impurities or genetic heterogeneity in the original mutant, or some external contamination or confusion (for a more detailed discussion of this problem, see the following paper, Tsugita, 1962). To date the analysis of 90 virus isolates, consisting of 55 chemically evoked mutants and their progeny, has been completed. Of the 55 original mutant strains selected for analysis, 30 did not differ from common TMV in their amino acid composition (see Table 1, footnote, also Tsugita & Fraenkel-Conrat, 1962a, b). Two mutants of this

A K IR A TSUG I TA

288

2 Amino add changes in chemical mutant8 of common TMV T ABLE

Mutagen

Mutant n o.

Amino acid changes

HN0 2

273

Asp ] -->- Ser

P ep tide

HN0 2

282

Aspt-->- Ala Thr -+ Ser

P eptid e P eptide

H N0 2

171

Pro -->- Leu Asp] -->- Ala T h r -->- Ser

Residue 156 Peptide 10 Peptide 1

H N0 2

332

Pro -->- L eu

Peptide

Location

10

1

H N0 2

220

Ser -->- Phe

Residue 138

H N0 2

329

Ser -+ P h e Ser -+ P h e

R esidue 138 Peptide 12

HN0 2

237

Arg -+ Gly Arg -+ GIy

R esidue 61 Residue 134

H N0 2

32lB:

Arg -->- Gly Arg -+ GIy Arg-+ GIy

Residue 61 R esidue 134 Residue 122

H N0

284

GIu -->- Gly Arg -->- Lys

Residue

2

97

16 amino acids ch a n ged in many p eptides;

H N0 2 H N0 2

328} 262

NBSI

218

Pro -->- Leu

Peptide

NBSI

233§

Pro -+ L eu

Peptide

NBSI

207

Pro -->- Leu

P eptide

NBSI

235

Pro -+ Leu

Peptid e

NBSI

187§

Arg -->- GIy Asp] -->- Ser

R esidue Peptide

46 1

NBSI

326

Asp] -+ Ser Asp ] -+ Ser Aspt-+ Ser

P eptide Peptide

1 1

NBSI

330

Ileu -+ Thr

NBSI NBSI NBSI

223} 206 331

composition identical with G·TAMV (see Table 1)

1

17 changes, similar t o G·TAMV, but for one Ala-e- GIy

7 chan ges, simi lar to Y·TAMV (Table 1), b ut for on e Leu-» Ileu

D MS

214

P ro -+ Leu

Peptide

D MS

278

-P ro -+ Leu

Peptide

DMS

176

Pro -+ Leu

Peptide

DMS

215

Ser -+ Phe

Residue 138

D MS

178

PO

249

Arg-->- GIy

16 amino acid changes in many peptides; composition identical with G·TAlvIV

t Not known whether aspartic a cid or asparagine residue. t Secondary change fr om strain no. 237. § Secondary change.

CHEMICALLY EVOKED TMV MUTANTS

289

type (nos. 283 and 293) were analysed for peptide composition to check the possibility of simultaneous opposite substitutions, but none was observed. The possibility of compensating changes is thus greatly reduced but can only be completely ruled out by subjecting the mutant to complete sequence analysis, including the positions of the amide groups. However, the great number of instances in which mutants were found to have the same composition as the common TMV in both Wittmann's and our laboratory seems almost to eliminate the possibility that absence of detected differences might be due to methodological errors. CH CONH-1-2.3-. 4

5 7 1-6+ -8+ 9

10 11.12-COOH

r-~~~;~~~~~~ff~~f~f~~~i~~~f~j~~E~~~~

Deamination

1t

~~~ .::

3~~~ ====tt=t:tt:=1t==t:t=:t~~

-;::=;;:==tt=ttt:=r:=t:t=:tt== ::==:::=:tt::t=tt==!t=:t=t=tt== ;~~ _e_---1l---t+-+-++--tt--+-+--++-I~6 i67 ==:=::=tt=t:::t+=1==+=+==tt==

Bromination

.t

Methylation

t

284 218 -

233

;~:

176 215

_e_---ft---t+-+-++--tt--+-+--++-_e_---ft---t+-+-++--tt--+-+--++-~_.I-l..l.__.ll-_.l._-'--'-lt_"_-

FIG. 2. Location of amino acid replacements in chemically evoked mutants. Each horizontal line represents the peptide chain of a mutant, subdivided into its 12 tryptic peptides. Solid rectangles indicate definite location of a change, open rectangles possible alternative sites of a single change in a specific sequential peptide (nos. I to 12). Only in strain no. 326 are two of the four potential aspartic acids of peptide I transformed to serines.

In 19 mutants, one to three amino acid residues were found to be replaced. Table 1 shows the amino acid composition of those artificial mutants which showed amino acid changes, and had not previously been reported (Tsugita & Fraenkel-Conrat, 1962a). A survey of all the replacement data (Table 2) illustrates the point previously made, namely, that the same changes frequently recur, regardless of the nature of the mutagenic reaction. Thus the replacement of proline by leucine has been detected 7 times and has resulted from all three mutagenic reactions studied. The replacement of serine by phenylalanine was observed 4 times, three of which were nitrous acid mutants. Altogether the 31 changes observed to date at our laboratory have involved only eight pairs of amino acids, always proceeding in the same direction. A survey of the location of the changes on the peptide map indicates that they occur at sites scattered over the entire sequence. Yet, it would appear from the data on 19 of the strains illustrated in Fig. 2 that certain parts of the peptide sequence are more prone to change than others. Thus, the same serine (residue 138) was changed to phenylalanine in three strains and the same nearby arginine (residue 134) was replaced by glycine twice. Only one of the frequent replacements of proline by leucine has been exactly located (residue 156, Tsugita & Fraenkel-Conrat, 1960), all the others involving one or the other of the two prolines of peptide 1. That these replacements are thus also restricted to the distal segments of the chain appears significant since proline residues are known to affect the folding configuration of proteins. It must be stressed, however, that the finding of preferred sites of replacement may be due to other than genetic causes. The predominance of certain replacements might be caused by natural selection, in that changes in other parts of the chain might not give functional or stable proteins, and thus produce little or no virus progeny.

290

AKIRA TSUGITA

Apart from mutants of un chang ed or slight ly changed protein composit ion a third gr oup of mutants was found to be very markedly different from the parental strain , differing by 8 or 16 to 17 net amino acid changes and at many more sites on the peptide sequence. Members of this group closely resembled one or the other of tw o natural stra ins (Y-TAMV and G-TAMV) obtained from t omat o plants. This class is di scussed further in the following paper (Tsugita, 1962), in conjunct ion with structural st udies on natural st rains. In conclusion , no evident correlat ion was detected between the presence and the extent of amino acid exchanges and eit her the nature or the intensity of the modifying reaction. Th e only clear corre lation noted in this work was that a mino acid changes were almost always detected in mutants giving local lesions on N .sylvestris ,and detect ed only once in 60 strains in a mutant not showing this distinct biological property. Relation of results to the coding pr oblem Ever since the first realiz ation that the linear sequence of nucleotides in an RNA must carry information pertainin g to the linear sequence of amino acids in a protein, sch emes have been proposed which would account for this relat ionship . The fact that four different nucleotides can code for about 20 amino acids makes it necessary to postulate a triplet or a greater number of nucleotides as representing anyone amino acid. Various suggestions about the arrangement and mean s of functional sep aration of triplets have been proposed , some on mathemat ical grounds (Gamow, 1954 ; Crick, Griffith & Orgel, 1957), others, based on analytical data (Yeas, 1960 ; W oese, 1961). Most of these schemes have been disproven , eit her on a theoretical basis (Brenn er , 1957) or by fact s of the type reported in this paper and in our earlier publications (Tsugit a & Fraenkel-Conrat, 1960, 1962a , b). R ecently , Crick, Barnett, Brenn er & Watts-Tobin (1961) hav e supplied experimental evidence that t he code in T4 bact eriophage is most likely t riplet in nature, bu t that previously proposed coding mechani sms were no longer t enable. However, it is the first experiment al elucidation of a code sy mbol by Niren berg & Matthaei (1961) whi ch has represented the long-awaited breakthrough in this field . These investigators discovered that an amino acid in corporating system from E. coli was stimulated by the addit ion of polyuridylic acid t o syn t hesize polyphenylalanine. The work has been confirmed and extended and it is now firmly established that three or more uridylic acid residues in a polymer carry t he code for phenylalanine . The use of many mi xed random copolymers, all containin g uridylic acid, for similar experiments carried out in t wo laboratories (Lengy el, Speyer & Ochoa, 1961, Len gyel et al., 1962; Martin, Matthaei, Jones & Nirenberg, 1962), has given indications concern ing the composition of the oligonucleotides coding for most amino acids. However , the absence of sequential homogeneity in the polynucleotides, the degeneracy of the code (i.e. several nucleotide comb inations code for the same amino acid, probably with differ ent relative efficiencies ), and t echnical factors seem to lim it the number of unambiguous conclusio ns t hat can be deri ved from t he use of only stat istically homogeneous messenger RNA. That this limi tati on is not inh erent in the biosy nt het ic syste m is suggeste d by our findin g that a prot ein closely resembling TMV prot ein is synthesized by this E . coli system under the direction of added TMV RNA (Tsugita, Fraenkel-Conrat, Niren berg & Matthaei, 1962). As has been amply discussed in previous publicati ons (Tsugita, 1961; Fraenk elConrat, 1961), ni trous a cid is believed to exert its primary mutagenic action on R NA

CHEMICALLY EVOKED TMV MUTANTS

291

through its ability to change cytosine to uracil directly. Deamination of adenine to hypoxanthine, which resembles guanine in its base-pairing properties, may represent a secondary mechanism of mutation attributable to deamination. When Nirenberg & Matthaei (1961) first reported that polyuridylic acid made polyphenylalanine, and that polymers rich in cytidylic acid made polyproline, these facts were recognized as being in accord with our amino acid exchange data, and similar data obtained by C2U

CU2

C2X

CXU

XU2

CX2

X2U

Pro

Ic

2u

I~

U)

• Leu (0)

AU (0) CU2l

(N)

2

GU2 C
(N)

3(0

.. SeCU2

(0)

CGU} C
+

i

1(4)

~~~: ---- -.....-----_J!L

IIeu

Asp· (AspN)

A2U}(0)

CAuJ

(Asp) GAU (0)

Glu JCGU (N.Ol!

Arg

2'12)

ICGU (N.Oll

FIG. 3. Correlation of observed amino acid replacements with coding symbols. All amino acid replacements that have been observed in this laboratory are shown, with their frequency of occurrence. Numbers in parentheses indicate replacements resulting from reactions other than deamination, and thus unrelated to the arrangement scheme of this presentation which is based on the decreasing cytosine content from left to right. Solid lines and underlined code symbols indicate agreement of amino acid replacement data with C -+ U transformations. Nand 0 after code symbols refers to the origin of the respective identification, N standing for Nirenberg & Matthaei (1961), Nirenberg, Matthaei & Jones (1962), and Martin et al. (1962), and 0 standing for Lengyel et al. (1961, 1962) and Speyer, Lengyel, Basilio & Ochoa (1962). The symbol 2+ in the bottom line refers to the fact that a progeny isolate of this strain showed three arginines to be replaced by glycines, rather than two.

Wittmann (1960, 1961). For nitrous acid mutants frequently showed replacement of proline, and the appearance but never the loss of phenylalanine, as would be expected if the former was coded by an oligonucleotide rich in C, and the latter by poly-D. The inspection of all the replacement data obtained in our laboratory on nitrous acid mutants in conjunction with the latest coding symbols (see Fig. 3) indicates that 55

292

AKIRA TSUGITA

to 70% of the observed exchanges can be attributed to C -+ U transformation, with the rest being indicative to a similar extent of A -+ G and A-+-U transformations. Unfortunately, however, similar replacement frequencies are observed also in mutants obtained by the other two mutagens, which would not be expected to give similar alterations of the RNA. Most of the replacement data obtained by Wittmann for nitrous acid mutants (1961) also support our original hypothesis that C-+ U represents the predominant mutagenic event in the deamination of RNA, if these data are correlated with the latest coding symbols. Yet, the fact that in the interpretation of the E. coli data cognizance was taken of the TMV replacement results somewhat depreciates the significance of this agreement. The author is indebted to Dr. W. M. Stanley for his interest and encouragement; to Dr. H. Fraenkel-Conrat for the production, isolation and propagation of mutants, for the preparation of the proteins and analyses performed on these directly (tryptophan and cysteine), for the preparation of most of the hydrolysates, and for his help and counsel in the preparation of this paper. Finally, the author acknowledges with thanks the devoted technical assistance of Mr. K. Pyle and D. Barish. This work was aided by United States Public Health Service Training Grant, CRTY-5028. REFERENCES Brenner, S. (1957). Proc. Nat. Acad. Sci., Wash. 43, 416. Crick, F. H. C., Barnett, L., Brenner, S. & Watts-Tobin, R. J. (1961). Nature, 192, 1227. Crick, F. H. C., Griffith, J. S. & Orgel, L. E. (1957). Proc. Nat. Acad. Sci., Wa8h. 43, 687. Fraenkel-Conrat, H. (1957a). Virology, 4, 1. Fraenkel-Conrat, H. (1957b). In Methods of Enzymology, ed, by S. Colowick & N. O. Kaplan, vol. IV, p. 247. New York: Academic Press. Fraenkel-Conrat, H. (1961). Biochim. biophys. Acta, 49, 169. Fraenkel-Conrat, H., Singer, B. & Williams, R. C. (1957). Biochim, biophys. Acta, 25, 87. Gamow, G. (1954). Nature, 173, 318. Gierer, A. & Mundry, K. W. (1958). Nature, 182, 1457. Hirs, C. H. W. (1956). J. Biol. Chem, 219, 611. Lengyel, P., Speyer, J. F. & Ochoa, S. (1961). Proc. Nat. Acad. Sci., Wash. 47, 1936. Lengyel,P.,Speyer,J. F., Basilio, C. & Ochoa, S. (1962). Proc. Nat. Acad. Sci., Wa8h. 48, 282. Martin, R. G., Matthaei, J. H., Jones, O. W. & Nirenberg, M. W. (1962). Biochem, Biophys. Res. Comm, 6, 410. Nirenberg, M. W. & Matthaei, J. H. (1961). Proc, Nat. Acad. Sci., Wash. 47, 1588. Nirenberg, M. W., Matthaei, J. H. & Jones, O. W. (1962). Proc. Nat. Acad. Sci., Wa8h. 48, 104. Speyer, J. F., Lengyel, P., Basilio, C. & Ochoa, S. (1962). Proc, Nat. Acad. Sci., Wa8h. 48, 63. Tsugita, A. (1961). Protein, Nucleic Acid, Enzyme, 6, 385. Tokyo: Kyoritsu-Shuppan Co. Tsugita, A. (1962). J. Mol. Biol. 5, 293. Tsugita"A. & Fraenkel-Conrat, H. (1960). Proc. Nat. Acad. Sci., Wa8h. 46, 636. Tsugita, A. & Fraenkel-Conrat, H. (1962a). J. Mol. Biol. 4, 73. Tsugita, A. & Fraenkel-Conrat, H. (1962b). Progress in Molecular Genetics. New York: Academic Press. Tsugita, A., Fraenkel-Conrat, H., Nirenberg, M. W. & Matthaei, J. H. (1962). Proc. Nat. Acad. Sei., Wash. in the press. Tsugita, A., Gish, D. T., Young, J., Fraenkel-Conrat, H., Knight, C. A. & Stanley, W. M. (1960). Proc. Nat. Acad. Sci., Wash. 46, 1463. Wittmann, H. G. (1960). Virology, 12, 609. Wittmann, H. G. (1961). Nature, 24, 1. Wittmann, H. G. & Braunitzer, G. (1959). Virology, 9, 726. Woese, C. R. (1961). Nature, 190, 697. Yeas, M. (1960). Nature, 188, 209.