VOL. 48, 1962      BIOCHEMISTRY: J0NE.S' .LVD YIRENBERG

2115

QCALITATIVE St-RVEY OF RiVA CODEWORDS*

BY OLIVER W. JONES, JR.? urn M~RSITL~LL W. SIRESBERG

XYATIONAL HEART INSTITUTE, ?I.ITION.%L INSTITUTES OF HEALTH, BETHESDA, YARYIA.YD

Camwunicated by Richard B. Roberts, O&her 29, 1962

h degenerate genetic code was suggested a number of years ago by Cramowt and
by Crick.?  In such a code, an amino acid may be directed into protein by two or
more codewords.  Previous work, demonstrated that C%mino acids were directed
into protein by synthetic polynucleotides in cell-free E. cnti extracts." and that
leucine incorporation was stimulated by either poly c'G,`r I-C'. or L-?l.++ Thus
the code was shown to be degenerate with respect to leucine.4a

Initially. all of the codewords found contained V.  However. assuming a triplet
code, the proportion of L compared with other nucleotides in rodewords seemed
unusually high, for natural template F&.-L such as viral RX-\, did not contain such
a preponderance of I-.  To resolve this paradox. a more degenerate code was pro-
posed with both non-I: and U cont.aining codewords.5  An alternative hypothesis
was advanced by Roberts, who suggested a doublet code, for in such a code the
proportions of nucleotides would be within the range found in viral RSA.`. 8

The existence of non-r codewords was suggested when poly AC preparations
were found to direct small amounts of proline and threonine into protek9* ?`)
Recently, in a careful study, Bretscher and Grunberg-Manago clearly demonstrated
coding by non-U words. lo  Several poly -K! preparations were reported to code well
for proline, threonine, histidine, and glutamine.  This work indicated that other


2116           BIOCHEMISTRY: JOh`ES Ah-D NIREh-BERG     PROC. x. A. s.

non-l' polynucleotidee might have template activities. In this communication,
further qualitative analysis of coding by such polynucleotides will be reported.

Mate&z& and M-.-The preparation of E. coli extracts (preincubated, DSAase-treawd
L&?O fractions). and the techniques used in aashing, plating, and counting protein precipitatee
have been rep~ti.~. 6  Polyribonucleotider were synthesized according to the method of Singer
and GIIBB."

The base-ratio of each polynucleotide preparation was determined by incubation in 0.4 .T KOH
at 25" for 18 hours.  Under these conditions. little deamination occurred.lz  Such mild conditions
were not sufficient to hydrolyze certain polymers: however. in such cases inrubation in 0.3 .Y K?)H
at 37" for 18 hours resulled in complete bydrolysis.`J  Mononucleotidr products u-ere separated
either by paper elertrophorek IFVhatman 30. 3 MM paper. O.OS+ ammonium formate. pH 3.7 '
or bv dtscending paper chromatigrapby (Whatman ."io. 3 M%f pawr and s sokent s,wten!
conkming 0.1 M sodium phosphate, pH 7.0. and 3 M ammonium sulfate!.  Two per rent or
greater contamination of pol,vnurleotides bx IY would have been detH%pd.  .Vo contamination t)!.
U was found.

Reartinn mixtures used t.o determine Cl'-amino acid incorporation int,(> protein contained thr
following components: 0.1 M Tris (hpdrox!meth~lamincRthane`~ pH i.8: 0.01 M magnesiurr
state: 0.05 M h-Cl: 6 X lO-s M mercaptoethanol: 1 X IO-" M ATP; 5 X 10-s M potassium
phosphoenolpyruvate; 20 ~g /ml of crystalbne phosphoenolpyruvat kinasr ( California Bicrhem.
Corporation); 0.8 X IO-' M C'*(-amino acid (approximately 60.000 rounts /minute ireaction mix-
turr); 3.2 X IO-' M each of I!, CL%-amino acids minus the C"-amino acid: 10 M of polvnurlr~+
tide/reartion mixture where swi6ed: and E. &i preincubated p30 extrart,s I 1-2 mg protein
reaction mixture). Total volume of each IWLCtitJn mixture was 0.3 ml. &action mixtures were
incubated at. 37' for 30 minutes; thus total incorporation rather than rate xvw8p measured.

Stimuti oj amino acid inwrp~ration Iry polynueieotides containing jaw Gasc~s:
The data of Table 1 demonstrate that synthetic polynucleotides containing four
bases stimulate the incorporation of a large number of amino acids into protein.  In

TABLE 1

       .!h-IN~LATION OF kXN0 -4CID k!ORPORATlON BY POLI' UGa4C
Pdynucleotidc     UGAC    1CGAC    UGAC    UGAC    UGAC    IiGAC
            I-  56    IT.56    P 3    I?  23    r  27    I-  27
      2  32      26
      2 1:      45
        2 4:    G  21    G  22
*ratio
m&d pi3 can     c  2    c       c    i? 524    A  22  2 `2:
                                        cm c 9

Dtai&Bti0Il
C'cmnino acid
Alanine
Arginiqe
$J$c=Y;s
Glvcine
Hikidine
laoleucine
Leuhne
k$%&ine
Ph&ahnine
Serine
Threonine
Typtophw
Tvrosine
&line
Total

-4p231

110
  69
  10
ii:
  20
  68
168
  10
1s;
  SO
1 T!l
  15
  23
1:
I .Oi5

-4~232    A~233    Ap234    JU2&8    JuZ510
   Incorporation above control (4,u~molesl*
127
270  Fii     152     31      5
              212     99      5;
2  12 9   IO 9  25 14      11
                            ,.
663     25            12
30:    90 ii  13 0  i 0

  54     l!l      78     44      0
  F1     16
6E  67 11  70 1:   A  `0 13
5.@36    867    2.651  5z     10;


VOL. 18, 1962      BIOCHEMISTRY: JONES .-LVD .VIRlLVBERG            2117

the last column is given the basal level of C1%mino acid incorporation obtained in
the absence of polynucleotide: other figures refer to the net increase above basal
incorporation due to addition of polynucleotide.  The base-ratios of the poIynucleo-
tides vary widely.  The hfth polynucleotide (Ju-258) contains approximately equal
proportions of U, G, A, and C, whereas the other polynucleotides contain predomi-
nant amounts of two or three nucleotides.  -411 of the polynucleotides were active in
directing amino acid incorporation except polynucleotide Ju-2510. Although 10 erg
of polynucleotide were added to each reaction mixture, the total amount of CW
amino acid directed into protein by each polynucleotide varied more than N-fold.
As we have shown previously, the template activity of polynucieotides is dependent
upon factors other than nucleotide sequence.  For example, large polymersof-chain  
length greater than 100 units are considerably more active than shorter ones."
Single-stranded polynucleotides are active, whereas double or triple stranded poly-
mers are not.J  In addition, randomly mixed copolymers which have a high degre
of secondary structure are inactive in coding.=  In particular, polymers containing
much G have little activity, possibly because of G-G interactions. Thus, the
relative inactivity of the last poly UGAC preparation (Ju-2510) should not be
ascribed necessarily to the presence of a high proportion of nonsense nucleotide
sequences. Such considerations make it diflicult to compare with validity the
relative abilities of different polynucleotides to code for the same amino acid; thus,
`such comparisons shovld be made ,urith caution.  The fact that polynucleotides con-
taining four bases coded so well for so many amino acids strongly suggests that most
nucleotide sequences can be read.  In addition, a high proportion of U clearly was
not required for messenger Rs-1 activity.

St-imdat&m of am&o acid i~wn-poration by pdy ACG:  The coding activities of
polymers which did not contain I' are given in Table 2.  Base-ratio analysis of each
poly ACG preparation failed to detect contamination by U. Poly ACG preparations

                       TABLE 2

     %&WLATION OF h.VINO kID hCOFIPfXWl'ION BY POLY ACG
Polvnucleotide          MY3       ACC      ACC      ACG

     Designation           J251       M  76     M  75     x 74
    C~Qmino acid               Incorporation shove control (4.rmoIes)*
   Alanine             123       45       56       85         8
  Arginine             1'28       30       40       7.4
  Aspartic acids          .-
         .f&    0"        0       34    1:
  Glutamic acid"           i:       .x3        21
  Glycine               5
  Histidine             71   sit
               `LI 0        9   9:         1:;
                                                       5
   Lsoleucine              0                0    ii        20
   Leucine                     10                         40
  Lysine      84        5    x       23
   Metbionine             1  320 :        0
P$;;iaIanine    14;      1% 1  42 9    1:
                       :
  .Serine             is?
            256       24       30       .5.j        45
  Tbreonine                     11       1R       11
;ww&m
V"Sne       0 1    0 4    1:    x    4:
                                                     18
                       1         -         -   45.i          1
    Total           2.;m   xii   33;       22

o ~\ur.Molea reprenata the difference between C"-emino acid incorporation intn protein in the presence
and sbaence of polynucleotides.  &say procedures we described tinder .Katsn& and Methods.


2118          BIOCHEMISTRY: JOSES -4?i-D NREKBERG     hoc. K. A S.

stimulakd the incorporation of many amino acids t&ed, including alanine. argininc,.
glutamic acid, lysine? proline, and threonine.  Such high incorporations of glutamic
acid, lysine, and ohreonine were not observed previously. Jh  -4 number of amino acids
did not appear to be coded by any -4CG preparations, wbicb suggested that I- ma!
be an absolute requirement in coding for some amino acids.

Stim.ulation 0-f amino acid incurporatitm by polynwleotides cont.aininij two bases:
The polynucleotides in Table 3 are listed in order of decreasing C content.  In
accord with the findings of Bretscher and Grunberg-Manago.`" poly -4C s:imulaird
incorporatiou of proline. threonine. and histidine. In a&$ion. poly -4C n-as found
to direct aspartic arid. glutamir acid, and l+ne int,o pro:ein.3E  Poly _4C (J-104 :
contained 9; per rent C. yet actively directed proline into protein.  Thus. it appear'
that one codeword for proline may wntain only C.  Relatively large amounts of
lysine were directed int,o protein by ,4C J-109 and J-108 whic*h contained 67 and 80
per cent ,4 respectively.  Tbus. the lysine codcu-ord may contain only 9.
The data of Table 4 demonstrate the e5ects of poly CG and AG preparat,iona in
directing amino acids int.o protein.  The first three CG poleymers comained high
proportions of C and directed alanine, arginine, and proline into prot>ein.  The lasr
poly CG preparation (F-135) contained 91 per cent G, and xas inactive s template
RX;A.  Poly ,4G directed incorporation of glutamic acid and Iysine into protein.
Quantitahe aspeck oj data:  A comparative study of polynucleotides of I-arying
base-ratios is helpful in evaluating amino acid incorporation data. for relative amino
acid incorporations can be easily correlated with changes in base-ratio.  Occasional
inexmsistencies and the significance of minor incorporations become apparent.
Isotope dilution experiments were performed routinely to detect, the possible
presence of radioactive impurities in C1kmino acids.  The presence of Cl*-impuri-
ties seemed unlikely, for incorporation of a Cl'-amino acid was reduced sharply if
the reaction mixture contained both a Cl*-amino acid (0.05 pmole) and the same

TABLE 3

~TIMTTLATIOX OF AMINO ACIII INCORPOFLATIO!~ BT POLT At?

Polynuclechide
Base-ratio
moleA per cent
Designation
C~+muno acid
Alanine
Arginine
hpartic acid*
Glutamic acid-

Glpcine
Hihiine
em;>e
Lysine
Methiontie
Ph'h;;~alanine
Swine
Threonine
p.w.w~~"
V'aline
Total

AC

5104

8
cl

4
5

0
i
x
62:
ii!
23
14
0
723

AC
L!4

6103

1
4

0
  19
  16
"0
1:
  10
  0
1,132
i!'
x
1.27:

AC      AC      AC      AC

A 12    A  30    A  Si    A  80
cf43    c  70    c  33    c  20

5102     JlOl     JIOR     JlO8
Incorporatmr. above rontroi i ~Irpmolrs~*
  0       0      0      0

75     170     176    105
1
f   (:   :,   i
              0     0
793  1.64:   1.616    70;

  Mmw
polynuclrot!df
  eonfroi

11
12
24

15
6
g

3
5

10
I1
9
46


VOL. 48, 1962      BZOCHEMZSTRY: JOSES A-h-D YZREMBERG             a119

    .
Designation     kiMI41    41  71
C'camino acld
Alanine        30     20
Arginine       39     16
Aspartic acid S           0
Glutamic acid=  8      0
Glvcine          -
Hiitidine    ii   1:

Isoleucine
Leuciue
Lvaine
kiethionine
Phenylnlanine
Proline
Seriue
Threonine
pY!Jw!J;feh~"
V&e
Total

Fl20     F 135     J 106     .I 107
Incorpwation above control i &mmle3~+
t;      0   10 0   s 0
0"  0 ;   44 12    10 -

        0       2   ;
        0       0      0

39
7
13
1-I
8
42
4?l
14
4
332

* ~&f&s represents the difference berween C'+mino acid incorporation into prutein in the presence and
absence of polynuciwtides.  Detads of tbe assay procedures are described under Matmds and Methoda.

Cl?-amino acid (1.0 pmole).  The purity of each Cl*-amino acid also was deter-
mined by paper electrophoresis followed byradioautographyas described previouslyy5
Limiting amounts of polynucleotides were added to reaction mixtures and total
ammo acid incorporations were measured rather than rates of ammo acid incorpora-
tions. E. cola' extracts contain nucleases which rapidly degrade synthetic poly-
nucleotides and the nuclease content may vary from one preincubated S-30 prepa-
ration to another.  Since many difjFerent enzyme extracts were used in this study,
the data are not useful for quantitatioe analyses.  Comparisons between theoretical
fretiuencies of triplets, etc., in polynucleotides and relative ammo acid incorpora-
tions have not been presented because the data do not permit such calculations to be
made with ~curncy.  The data demonstrate only qualitative aspects of the code:
that is, nucleotide caompositions of words and the degree of code degeneracy.

SlLmmary oj Incorpcrration D&z.-Table .5 summarizes all of the coding data
previously pubiishedR-6, S- bn and rhose obtained in this study.  Only polynucleo-

C'%mllno WI11
Phenvldxune
ProI&
Lysine
Threonine
se l-he
Vdine
Leucine
Uycine
CpthW
Glutamic acid

31

C"-ammo uxd drimulsced by
      pdy-*
Isoleucine   Clc 8 )
Trvptoph;m CG(ti!
TqToslne    Cli 9 j
Irginine   CG( 15)
Msthionine  CAC( I )
Hi&dine   hC( 101
Alanine    CG(l1;
Aspartic acids AC!8)

* Polymen used for these caieuistions soutain the mitimd number of bases ~leceeawy to direct the Cl'-amino
acid8 mm pl0tein.

t Xumberg ;a parentheses refer to: ,+mules of amino acid incorporsted X lOO/Sum of ineurporstion of all amino
ecd~ af the optimal bree ratio assayed.


2120

BIOCHEMISTRI': JO.?`ES A.VD h-IRE-+-BERG     PROC. K. -4. \

tides containing t,he minimum number of bases capable of stimulating an amint,
acid into protein are given in Table 5.  The roding of proline by poly C and I>-&
by poly -4 was suggested by the poly A4C experiment.> presented in Table 2.  l'h,.
fact that poly C and poly A code so weakly may be due to inhibitory effect> 0;
secondary structure.  In solut,ion at avid pH. poly A is double-stranded.!Y 3 ant!
at neutral pH. both poly A and pofy C have parGaIly ordered strurl ure.?;

-4 surprising wnclusion drawn from this summary is that almost every amino a~+i
tested could be coded by a polymer cxont,aining only two bases.  Mrthioninr c*ouitf
be coded only by poly I-G.4 as previously reportrd,$. lc but sinw the amoun: O:
methionine dirwwd into protein was small. this c*od&wd remain: qurstionat)lr~.
Assuming a triplet (*ode. a summary of codewords estimat,ed t,hus far is prr+nl~~ll
in Table 6.  Prel-iously. poly I-CG was found t,o direct alanine and arginint in: I;

AGU_

cc.4

protein and codewords containing U, C, and G were proposed for these amino
aci&.43 5, 1:. 18 The observed frequencies of incorporations5 suggest coding of
alanine and arginine by either I-CG or CCG. but not b\- both codeword-, ln
addition, the data of Table 4 show that poly CG c&es for alanine and argininc,.
thus, codewords corresponding to t,hese amino acids do not appear to contain V.

S~IIW it is not possible at this time t,~ d&@&h between triplet and doublet
codes. etc., the assignments in Table 6 represent current approximations of code-
WONTS.  It seenw probable that additional codewords w-ill be found.
Di~sitm.-Codeword specijkity in protein synthesis: The term degencrar!!

refers to the phenomenon whereby one amino acid is coded by two or man' ~d(`-
words. This term is inadequate when applied to the mechanism of coding. for it
does not indicate codeword speci$city -4 degenerate code may halve high or 1~
specificity depending upon the fidelity of protein synthesis.  In most cases lb('
fidelity of protein s.ynthesie in viva appears to be high, and amino acid rrplacvmc'n:`
other than those due to muiat.ion, have not been found.  H~ww~. ahho& th('
amino acid sequenrk analyses would reveal mist.aS;es at one sit.t occurring v%tI i1


VOL. 18, 1962      BIOCHEMISTRY: JONES .4AVD YIRE.VBERG            2131

frequency higher than I or 2 per cent, they would not reveal occasional mistakes
occurring at different sites.  Thus, occasional coding errors of 1 or 2 per cent, dii
tributed at random over entire protein molecules, might not be detected.

In the in L&O system, coding during protein synthesis displays striking specihc-
ity.22 In Table 3, for example, poly AC preparations do not direct the incorporation
into protein of alanine, arginine, glycine, isoleucine, leucine, methionine, phenylala-
nine, tryptophan, tyrosine, or valine. The specificity of coding by poly CG and AG
preparations in Table 4 is equally apparent.  Such negative data clearly demon-
strate the very high fidelity of codeword recognition during protein synthesis.

The codewords corresponding to both leucine and valine contain U and G.&-"
-4lthough the nucleot.ide content of these codewords is identical, each word was
shown to code only for the appropriate amino arid.??  Thus. nucleotide sequence a~
well as chemical structure confers specificity upon codewords.

However, one unclarified example of ambiguity has been found.  Poly U directs
about 3-S per cent as much leucine into protein as phenylalanine.S  Bretscher and
Grunberg-Manago also have reported this phenomenoni In the absence of
phenylalanine poly G coded for leucine about 50 per cent as well as it would code for
phenylalanine.`23

E$iciency of synthetic RLVA4 in coding:  In spite of the previously mentioned
di&ulties in comparing template activities of RNA preparations with Merent
chain lengths and degrees of secondary structure, it seems clear that synthetic
poiynucleotides containing 4, 3, or 2 bases code as well in this system as natural
template RXA obtained from viruses.3, 24-s The efficiency in coding displayed
by synthetic polynucleotides suggests that most nucleotide sequences direct amino
acids into protein, and that few nonsense nucleotide sequences are present.

Although aiternative explanations of coding efficiency, such as nonrandom
polynucleotides or nonsequential reading of template RSA, may be considered,
such efficiency cannot be ascribed simply to random error in directing amino acids
into protein, for amino acids are coded with marked specificity.

Considerations such as these may be used to approximate the coding ratio.
In a doublet code only 16 base permutations are possible.  Thus, the information
content would be insufficient to code specifically for all amino acids.  Triplet and
quadruplet codes would contain 64 and 2.56 codewords respectively.   Since
almost every amino acid ixsted was found to be coded by polynucleotides con-
taming only 2 bases, specific and efficient coding by quadruplet words would
not seem likely. Tue data sugge
        0% either coding of all amino acids by tripiers
or coding of some by triplets and  ers by doublets (mixed doublet-triplet code).
Recently Weisblum, Benzer, and Hohey" have established a molecular basis of

degeneracy by demonstrating that multiple species of transfer RX-1 recognize
different codewords with specificity.  Multiple peaks of transfer RX-1 correspond-
ing to at least four amino acids have been found independently by Holley et al.,%
Sueoka el a1.,?g and Doctor et aLa

If a triplet code is assumed, each cell would require almost 6-l transfer RX-\
species. Alternatives which do not require so many transfer RNA species deserve
consideration.  For example, Donohue and others have described many models
other than Watson-Crick pairing. 71  The demonstrated interaction between poly
-1 and poly I,"? and the type of base-pairing suggested by Hoogsteen33 also might be


2122           BIOCHEMISTHY: JO.?-ES 9A-D a~?REA-BERG     PROC. ?;. -4. 4

cited. Theories which require recognition of either the 2- or &substituents of
basesS4 are not supported by the demonstration that hypoxant,hine functions in
codeword< like G.15. 35
       L       The Z&amino group of G does not appear t,o be required.
A triplet code ma\- be construct,ed xherein correct hydrogen bonding brtnwn
two out of three nucleotide pairs may, in some cases. suffiw for coding.  0xrw.t
pairing of a base at one position in the triplet sometimes may be optional. II
should be noted that a triplet code of t,hie type in some respects would bear a super-
ficial resemblance t'c! a doublet code and would he in aword n-it h all of t hr data
-4ny theory concerning t#he physical hasi? of the vode$nust a; t,empt IO explair~
the following esperiment,ally obtained da.ta : ; 1) high coding, efficiency by sync hp; 11'
polvnucleotides; (~2) marked codeword specificity : (3, degvnerat,e codewords.
(4)`the P-amino group of G is not essential for proper coding: (5) RX-$ with a high
degree of secondary struct#ure has little ability t.o code: and ;6,1 almost al! amincb
acids t,ested can be poded bg polynucleotides containing only two bases.
Summary.-Synthetic polynucleot,ides containing 4. 3, or 2 bases have twn
found t,o direct amino acids into prot,ein with high efIiriency and specificity.  Milan>.
additional RX.4 codewords which do not contain uridylic acid haw been found
Almost all amino acids could be coded by polynucleotidee containing on!y 2 haw-
These results have been discussed in terms of the general nature of the code.

It is a pleasure to thank Lynn Hurwitz, Linda Greenhowe. and Glenn5 Baile? for vahwblr
technical assistsme.

* Presented at the Symposium on l~form&onul Mac~omol~rules. !Cew Brunanick. Xl`ew J~;w
Sept. 1962 (proceedin@ in press:.

t The following abbreviations are used: A, adenylir acid; G: guanylir acid; C. cytidylic arid
V, uridylic acid.

I Gamow, G., Xature, 173, 318 (19541.
* Crick, F. H. C., in Stwcture and Function o-f Genetic Elements. Brookhaven S;!-rnwi:i iri
Biology, No. 12.35 (1959).

* Nirenberg, M. R.. and J. H. Matthaei, these PROCEEDINGS. 47, 1588 t 1961:.
' Martin. R. G., J. H. Matthaei, 0. W. Jones, and M. W. ?&&erg. Bznchem Rzo,vh?/,+ Kr,
(lomm., 6, 410 (19621.

6 Matthaei, J. H., 0. a. Jones, R. G. Martin and M. IV. ?iirenbwg. these PROCEE~~~~(:~~ 46.
666 (1962).

6 Speyer. J. F., P. L+ngyel. C. Basilio, and S. Ochna. thee PROCEEDINGS, 48, 63 ( IW
`Roberts, R. B.. thew PROCEEDINGS. 48, 8!X (1962:.
8&d., 1245 (1962,.
SJones, 0. W., and R. G. Martin, Pad. Proc., 21, 414 (1962).
~Bretscher, M. S., and M. Grunberg-Manago. .\-ature, 195, 283 (1962).
I1 Singer, M. F., and J. K. Guss, J. Biol. Chem., 237, 182 ! 19621.
I* We thank Dr. M. Grunberg-Manago for this protocol.
I* Davidson, J. X., and R. M. S. Sm&rj Rio&m. J.. 52, 594 :`I952 I.
I4 Unpublished observations.
U Singer, M. F.. 0. W. Jones, and M. VI-. Kirenberg. unpublished nbsen-ationf.
I6 Lengvel. P., J. F. Spever, and S. Ochoa. these PROCEEDIKGS, 47, 1936 I 19611.
Ii Leng&l, P.. J. F. Sp&er, C. Baeilio. and S. Ochoa, these PROCEEDINGS. 48, 2% ( INil
18 Speyer, J. F., P. Lengyel. C. Basilio. and S. Ochoa, these PROCEEDINGS. 48, 44 1 ( lW;L'
I9 Fresro, J. R., and P. Dot,?, J. -4m. rhem. Sot., 79, 3928 [1957:.
9 Rich, -4., D. R. Davies. F. H. C. Crick, and J. D. Watson. J. Mol. Bzoi.. 3, 71  1~'
81 Fresco, J. R.. Trans. .V. J-. dead. SC%., Se&~ II, 21, 653 (1959:.
** ?;irenbwg. M. H.. ,I. H. Matthaei, 0. W. Jones, S. H. Barondek: and R. G %faVt,i'r "`!
Proc., in press.


VOL. 48  196"
     , -       BfOf`HEMfSTRY: K.lPL-4-V .i.VD Cd H.V

21`23

?a Nireoberg, M. W , J. H. Mstthaei, and 0. W. Jones, unpublished observations.
?+ Ofengand, J., aand R. Haselkorn, Biocha. Biophys. Res. Corm., 6, 469 ( 19tZ ).
a Tsugita, A., H. Fraenkel-Conrat, M. W. Sirenberg, and J. H. .Matthaei, these PROCEEDINGS,
48, 34-16 ( 1962).

1 Nathans. D., G. Yotsni. J. H. Schwartz, and N D. Zinder, these PROCEEDIXGS, 48, l-U4
19623.
n Weisblum, B., S. Benaer, and R. W. Halley, these PROCEEDINOS, 48, 1449 (196&Z).
a Halley. W. H.. B. P. Doctor, S. H. Merrill, and F. M. Saad, Biochim. Biophys. dctu, 35,
252 (1959).

`!g Sueoka, X., and T. Yamane, these PROCEEDINGS, 48, 1154 (196Z2!.
m Doctor, B. P.. J. hpgar. and R. W. Halley, b. Bid. C&m., 236, 1117 ( 1961).
31 Donohue, J., these PROCEEDINGS, 42, 60 (1956).
J* Rich, A., Suture, 181, $21 I' 1958).
23 Hwgsteen. K.. .1&z C'rysf.. 12, 82`2 i 1959).
YJ' Wtwse. C. R.. .Vntrrre. 194, 111-L I 196Y2!.
s Basilio. C.. A. Wahha. P. Lengyel, J. F. Bpeyer, and 8. Ochoa, these PROCEEDINGS, 48, 613
1962).
a Poly .IC is reported to code for glutamine.*'  We have been unable to obtain Cl'-glutamine
:hnd C'Qqaragine. Therefore, we cannot ditrerentiate between codewords corresponding to
,+ach frc?t? acid and its respective amide.