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18 Britten, R. J., and R. B. Roberts, Science, 131, 32 (1960).'9 Crestfield, A. M., K. C. Smith, and F. WV. Allen, J. Biol. Chem., 216, 185 (1955).20 Gamow, G., Nature, 173, 318 (1954).21 Brenner, S., these PROCEEDINGS, 43, 687 (1957).22 Nirenberg, M. WV., J. H. Matthaei, and 0. WV. Jones, unpublished data.23 Crick, F. H. C., L. Barnett, S. Brenner, and R. J. Watts-Tobin, Nature, 192, 1227 (1961).24 Levene, P. A., and R. S. Tipson, J. Biol. Ch-nn., 111, 313 (1935).25 Gierer, A., and K. W. Mundry, Nature, 182, 1437 (1958).2' Tsugita, A., and H. Fraenkel-Conrat, J. Mllot. Biol., in press.27 Tsugita, A., and H. Fraenkel-Conrat, personal communication.28 Wittmann, H. G., Naturwissenschaften, 48, 729 (1961).29 Freese, E., in Structure and Function of Genetic Elements, Brookhaven Symposia in Biology,

no. 12 (1959), p. 63.

NUCLEOTIDE BASE CODING AND AM1INO ACID REPLACEMIENTSIN PROTEINS*

BY EMIL L. SMITHt

LABORATORY FOR STUDY OF HEREDITARY AND METABOLIC DISORDERS AND THE DEPARTMENTS OFBIOLOGICAL CHEMISTRY AND MEDICINE, UNIVERSITY OF UTAH COLLEGE OF MEDICINE

Communicated by Severo Ochoa, February 14, 1962

The problem of which bases of messenger or template RNA' specify the coding ofamino acids in proteins has been largely elucidated by the use of synthetic polyri-bonucleotides.2-7 For these triplet nucleotide compositions (Table 1), it is of in-terest to examine some of the presently known cases of amino acid substitutionsin polypeptides or proteins of known structure.The code appears to be universal, that is, it is the same in all species.6 It is

assumed that a mutation involving the substitution of one amino acid for anotherin a protein of the same species, e.g., human hemoglobin, represents an alterationin the triplet code in which only a single base of the three is replaced without al-teration of the sequence of the other two bases. A change of two or more bases isless likely. Moreover, in those cases for which the code for two amino acids isrepresented by the same triplet composition of bases, a substitution by an aminoacid possessing the same code composition would be unlikely, since this wouldamount to a double base substitution in order to accomplish the necessary trans-position in sequence. Various considerations suggest that the triplet code is of thenonoverlapping type; this has recently been discussed rather fully by Crick et al.,8who have also reported evidence that a triplet code is involved.At the time this present evaluation was undertaken, the codes for only 14 amino

acids were known.9 It was of interest to determine whether it was possible to pre-dict, on the basis of known amino acid substitutions, the codes for other amino acids.This proved to be valid for certain amino acids where sufficient information wasavailable. This is illustrated below for glutamic acid, aspartic acid, asparagine.and alanine. These data, as well as other, thus serve as a verification of the thesisthat simple mutations involving an amino acid substitution involve a change in asingle nucleotide base of a triplet without alteration of the sequence in the triplet.

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TABLE 1TRIPLET CODE LETTERS FOR AMINO ACIDS*

Amino acid Code Amino acid Code Amino acid CodeAlanine UCG Glycine UG2 Proline UC2Arginine UCG Histidine UAC Serine U2CAsparagine UA2 (UAC)t Isoleucine U2A Threonine UAC (UC2)Aspartic acid UAG Leucine U2C (U2A, U2G) Tryptophan UG2Cysteine U2G Lysine UA2 Tyrosine U2AGlutamic acid UAG Methionine UAG Valine U2GGlutamine UCG Phenylalanine UUU ..... ...

* This table has been adapted from Speyer, Lengyel, Basilio, and Ochoa.6 These codes are, in general, similarto the 15 recently reported by Martin, Matthaei, Jones, and Nirenberg.7 The code for glutamine is not directlyavailable. It was deduced from an amino acid replacement in HNO2 mut ant of tobacco mosaic virus.4

t Code letters in parentheses represent additional letters for the amino acid (degenerate code).6

On the basis of this concept, it is useful to examine some homologous proteins ofdifferent species in which substitution of amino acids is known; this offers the pos-sibility of determining whether the alteration has involved a change in a single baseor whether two or more base substitutions have occurred. Hence, it can be de-duced, in some instances, whether single or multiple steps in mutation have occurredduring the evolution of species-specific proteins. Thus, a definition is at hand fordirect permissible substitutions (single base change in the triplet) and for changeswhich would appear to be nonpermissible by a change in a single base. It is alsoof interest to examine briefly some amino acid substitutions as related to the knowncodes in terms of possible effects on the functions of proteins.Deduced Codes for Amino Acids.-From known amino acid substitutions, it was

possible to deduce certain codes by making use of the code information then avail-able.

Code for glutamic acid: In Table 2, there are listed the presently reported aminoTABLE 2

SOME AMINO ACID REPLACEMENTS IN HUMAN HEMOGLOBINSMutant ,_ __Mutant

HbA Amino acid Type of Hb HbA Amino acid Type of HbGlutamic acid Valine S10, 13 Lysine Aspartic acid F18Glutamic acid Lysine C", E'2 Histidine Tyrosine MBostOn MEmory'5Glutamic acid Glycine GSan Jose13 Histidine Arginine Zurich'5Glutamic acid Glutamine GHonolulu14 Glutamic acid Alanine A220Valine Glutamic acid MMilwaukeel' Serine Threonine A220Asparagine Lysine Gphiladelphia.6 Threonine Asparagine A220Glycine Aspartic acid Norfolk17

.... ...

acid substitutions in human hemoglobin (Hb). For glutamic acid, it is apparentthat this amino acid has been replaced by lysine, valine, glycine, and glutamine.By assuming that only a single base of the triplet can be replaced, the code for glu-tamic acid can be deduced. Since lysine is UA2, the code for glutamic acid mustcontain at least one A. Since the valine code is U2G, the code for glutamic acidmust also contain one U. The code for glycine is UG2; hence, the glutamic acidcode must contain, in addition, one G. Therefore, the complete code for glutamicacid is UAG. This is consistent with the code for glutamine, UGC, which can bederived from the code for glutamic acid by a single base change.

It is noteworthy that a mutant involving the change glycine to glutamic acid hasbeen found also in the A protein of the tryptophan synthetase of E. coli.21

Code for aspartic acid: Inspection of Table 2 shows that aspartic acid has re-placed lysine (UA2) and glycine (UG2). In order for the code for aspartic acid to

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have two bases in common with the codes for glycine and lysine, the code for as-partic acid must be UAG.

Code for asparagine: Asparagine has replaced threonine (UC2) in HbA2, and lysine(UA2) has replaced asparagine in HbGPhiladelphia (Table 2). This suggests that thecode for asparagine is UAC.

Code for alanine: The only known substitution for alanine in the hemoglobinseries is in HbA2 in which it has replaced glutamic acid (UAG) although this maynot be a simple mutation. Although such assumptions are somewhat risky, onemay consider the known replacements for alanine in the homologous proteins ofvarious species. The data of Pal6us and Tuppy,22 for the cytochromes c of variousspecies, show that glutamine (UCG) and leucine (U2C) replace alanine and, inanother position, that serine (U2C), glutamic acid (UAG), -and leucine (U2C) oc-cupy the same position as alanine. Also, in the insulins of various species threonine(UC2) and alanine occur in the same position.23' 24 The codes for almost all of theaforementioned amino acids, viz., threonine, serine, leucine, and glutamine, con-tain U and C. Although some of the transformations for the triplet codes of theseamino acids directly to one another and to alanine may involve more than one basechange, it appears very likely that at least some of these are permissible changes,that is, that they involve only a single base change. The foregoing informationsuggests that the code for alanine would have to contain U and C in order to havetwo bases in common with the code for most of these amino acids. Since the codesfor glutamic acid and glutamine both contain G, it is likely that the code for alanineis UCG. This has subsequently been shown to be the code for alanine.6

The Role of Uracil.-The most striking feature of the triplet base compositions forall of the amino acids thus far is that each triplet contains at least one U. Inas-much as the presence of U is the only unique feature of the base composition ofRNA, as compared to DNA, an important and specific function for U in templateor messenger RNA is implied. On the basis of a triplet code with 4 bases, there are64 possible combinations. If, however, each code for an amino acid must containone U, then all of the 27 possible codes lacking one U are excluded. This leaves forthe 20 amino acids commonly found in proteins 37 potential triplets.The presently available information on mutants suggests that there are probably

preferred positions in the triplet for the unique U although the sequences in thetriplet are unknown. It is likely, for example, that there are 16 codes for aminoacids in which U occupies the same unknown position of the triplet. This wouldrepresent all the possible codes for U in one position. This view is suggested by thefollowing considerations. Again, we must suppose that a mutant involves a replace-ment of only a single base without alteration of sequence.

Let us arbitrarily assume that U occupies the first position as a fixed one in thetriplets for the following amino acids: glutamic acid (UAG), lysine (UA2), valine(U2G), glycine (UG2), and glutamine (UCG). Since the four last named aminoacids can replace glutamic acid as a result of only a single base change, a U mustoccupy the same position in all five codes. Further, aspartic acid (UA G)and aspara-gine (UAC) can replace lysine (UA2), and asparagine can replace threonine (UC2);hence, in the codes for these three additional amino acids U appears to occupy thesame position also. The same considerations also apply to serine (U2C), which canreplace threonine. Arginine (UCG) is known to replace glycine (UG2) in trypto-

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phan synthetase (Table 3) and to replace histidine (UAC) in hemoglobin. Tyro-sine (U2A) can also replace histidine in hemoglobin. Thus, we can relate all of thesecodes with U in the same position for the aforementioned amino acids. Pheflyl-alanine (UUU) can obviously be included, making 13 codes thus far. Informationconcerning mutants in tobacco mosaic virus (see the summary by Speyer et al.6)suggests that isoleucine (U2A), leucine (U2C), and proline (UC2) complete the 16possible triplet codes with U in the same position. The above tentative group ac-counts for all but four of the commonly occurring amino acids in proteins. Amongthe remaining four amino acids are tryptophan (UG2), which must have U in adifferent position from glycine, also UG2, and methionine (UAG), which must differin its code for the position of U since the codes for glutamic acid and aspartic acidare also UAG. Similarly, since alanine, glutamine, and arginine all have the codeUCG, one of these must have U in a different position than in those mentionedabove. In view of the relationship of glutamine to glutamic acid and glycine toarginine, alanine may be the one which differs in the position of U. The code forcysteine completes the list but the relationships of its code to others are still un-known.From the above interrelationships it is apparent that when the triplet code se-

quence is established for one amino acid, where three different bases are involved,it will be possible to deduce the sequence for essentially all of the other amino acidcodes.

Note added in proof: The work of Henning and Yanofsky2l on recombinationof two mutants of E. coli to restore the normal A protein of tryptophan synthe-tase indicates that if the glutamic acid code is UAG, that for arginine is UGC.The G in each of these two amino acid codes nmust be in a different position toobtain by recombination UGG the code for glycine. This information togetherwith that for the hemoglobin mutants should -facilitate absolute assignment ofbase sequences in the triplet codes.

If in 16 amino acid codes U occupies the same position, as indicated above, onemay assume that in the four other codes U occupies the same position, this is dif-ferent from the position for the 16 related codes. This suggests that there may beone position of U in the triplet which is nonfunctional or stated in the conversemanner, each functional triplet must have U in one of two possible positions. Thus,of the 37 possible triplet codes containing U it would appear that only 28 may befunctional. This hypothesis might aid in explaining the limited number of func-tional codes.

It may be noted that Speyer et al.6 suggest that asparagine may be coded by UA2and UAC on the basis of incorporation experiments. Inasmuch as lysine (UA2)pan replace asparagine in human hemoglobin, this transformation would involvethe UAC code for asparagine. A transformation of asparagine as UA2 to lysine asUA2 would involve an unlikely double base change. Similarly, Speyer et al.6suggest additional (degenerate) codes for leucine and threonine. The reportedreplacements involving threonine (Table 2) can be explained with UC2 as the codefor this amino acid, but they can also be explained thus: serine (U2C) -- threonine(UAC) asparagine (.UA2).

Relationships among Amino Acid Code Letters.-It is of interest to compare the

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codes for certain amino acids which have functional properties in common. Wemay consider first those amino acids which possess large hydrophobic side chainsand which presumably have similar or closely related properties within a proteinmolecule, i.e., where substitution of one of these amino acids for another wouldbe expected to produce minimal disturbance in protein conformation. The code forphenylalanine is U3, for leucine U2C (U2A, U2G), for tyrosine U2A, for valine U2G,for isoleucine U2A, and for tryptophan UG2. It is noteworthy that a single basechange would permit substitution of leucine, tyrosine, valine, or isoleucine forphenylalanine, whereas it is possible to derive the code for typtophan only from thatof valine or leucine (U2G) among this group of amino acids, but not from any of theothers. The code for methionine suggests that it too may be derived from that ofvaline and possibly some of the other hydrophobic amino acids. The code for cys-teine (U2G), as well as serine (U2C), may also be derived from the code for phenyl-alanine by a single base change.A few substitutions among hydrophobic residues are presently known within the

homologous proteins of several species (Table 3). Unfortunately, such sub-stitutions are not known as yet within the same species and these are not likely tobe easily found since the techniques presently used have largely depended on dif-ferences in electrophoretic mobility and hence in the charge of the proteins or thepeptides derived from them, e.g., as in the human hemoglobins.

TABLE 3SOME AMINO ACID REPLACEMENTS IN PROTEINS*

Protein Species Amino acid Species Amino acid ReferenceCytochrome c Mammalian Valine R. rubrum Phenylalanine 22Cytochrome c Equine Threonine Bovine Serine 25Insulin Bovine sheep Valine Horse, human, Isoleucine 23, 24

pig, etc.-y-Globulins Bovine, rabbit Phenylalanine Human Tyrosine 26Tryptophan E. coli Glycine E. coli Glutamic acid 21Synthetase A ..... Glycine .... Arginine* These are in addition to those mentioned in the text.

The codes for the two dicarboxylic acids and their amides present certain interest-ing features. The codes for glutamic acid and aspartic acid prove to be both UAG.Hence, a mutation involving a single base change would not permit direct substitu-tion of one dicarboxylic acid for the other. Nevertheless, such substitution isapparent in the reported structures for ACTH of several species.27 In human hemo-globin, substitution of glutamine for glutamic acid occurs. This substitution has:also been found in the y-globulins of different species.26 Conversion of the codefor asparagine to glutamine by a single base change would appear to be possible, andthis is known to occur in the glycopeptides derived from the 'y-globulins of differentspecies.26 Thus, conversion of the code by a single base change from either glutamicacid or aspartic acid to asparagine is possible, but it cannot be possible for both ora double substitution would be involved. There is no present evidence as towhich is the likely possibility, in the absence of sequence information for the triplet.The complex relationships among the codes for the dicarboxylic acids and theiramides emphasizes the importance of determining the correct amide assignmentsin a polypeptide sequence.

Substitution involving the two hydroxy amino acids, serine and thrconine, is

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known in a number of cases (Tables 2 and 3) which are intra- as well as inter-specific.

In the insulins of various species, threonine (UC2, UAC) and alanine (UCG)occur in the same position. As noted above (see code of alanine), this could occurvia a single base change in the triplet codes for these two amino acids. There isalso known in the insulins a serine (U2C)-glycine (UG2) substitution. Clearly, onthe basis of the triplet codes for these two amino acids, such a substitution isimpossible by a mutation involving only a single base in the triplets.

There are some features of the triplet codes which are surprising in terms of pro-tein chemistry. An aspartic acid-glutamic acid direct substitution by a single basechange is, as already mentioned, not possible despite the similar properties of thetwo amino acids. The same is true for arginine and lysine, although function isnot greatly altered in known cases of such substitution-cytochromes c,22 vasopres-sins,28 and ,3-melanophore-stimulating hormones.29 One possibility is that arginine(UGC)-lysine (UAA) replacement has occurred via an intermediate, such as histi-dine, whose code (UAC) has two bases in common with the codes for both arginineand lysine. An arginine-histidine substitution has been reported for human hemo-globin (Table 2). The properties of histidine are such that it may serve functionallyin some circumstances in place of arginine or lysine, although the histidine analogof the vasopressins is only weakly active.28Amino acid substitutions in tobacco mosaic virus in relationship to the triplet

codes have already been discussed.6 The substitution of arginine for glycine in amutant of E. coli (Table 3) is of special interest in view of the profound difference inthe properties of these two amino acids.

Clearly, we are faced with several different problems in the evolution of proteinstructure. One of these is the preservation of function, in which we may envisagesubstitution in the polypeptide sequence of compatible amino acid residues whoseside chain properties are similar and critical for enzymic or other functions. Whenreplacements occurred involving amino acids of radically different properties andthese proteins survived, evidently either the substitutions occurred at loci which werenot critical for the function of the protein or new functions evolved when "activesites" were modified. Presently known cases suggest that in the hemoglobins,insulins, cytochromes c, and other proteins, many changes occurred at noncriticalsites.

In contrast to the above, we can visualize possible changes in function by aminoacid substitution, as exemplified by the polypeptide hormones of the neurohypo-physis. The primitive weakly active vasotocin of the frog30 (Table 4) could have

TABLE 4HORMONES OF THE NEUROHYPOPHYSIS

Position1 2 3 4 5 6 7 8 9

Vasotocin* CyS-Tyr-Ileu-Glu-NH2-Asp-NH2*CyS-Pro-Arg-Gly-NH2Vasopressin CyS-Tyr-Phe Glu-NH2 Asp-NH2,CyS-Pro Arg*Gly-NH2Oxytocin CyS-Tyr-Ileu-Glu-NH2-Asp-NH2-CyS-Pro-Leu-Gly-Nh2

* The half-cystine residues in positions 1 and 6 are in each case linked by a disulfide bridge.

evolved into both vasopressin and oxytocin. The change from vasotocin to ar-ginine-vasopressin involves a change in position 3 from U2A (isoleucine) to U3

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(phenylalanine), only a single compatible base change in the triplet code. Like-wise, from vasotocin to oxytocin, the change in position 8 is from UCG (arginine)to U2C or U2G (leucine) which also represents a change in only a single base of thetriplet code. It is of special interest that the chicken possesses all three compoundsin the neurohypophysis.31 For the evolution from a single hormone to the presenceof two or more hormones in the same species, we must also assume a multiplicationof the genetic material in order to have independent production of two or morehormones.The above case suggests that the investigation of the amino acid sequences of

homologous proteins of various species should contribute much to our understandingof the evolution of function in the polypeptides and proteins.Summary.-Known amino acid replacements in mutants of human hemoglobin

are consistent with a single base change in the known triplet codes. Certain aminoacid replacements in homologous proteins of various species also involve singlebase changes whereas others do not; the latter probably involve stepwise multiplemutation. The data suggest that for 16 amino acids, uracil occupies the same butunknown position in the codes; the four other codes must involve a different loca-tion of uracil in the triplet. Problems of the maintenance and evolution of peptideand protein structure and function are discussed briefly.

* Aided by grants from the National Institutes of Health, U. S. Public Health Service.t Present address: University of Utah College of Medicine, Salt Lake City, Utah.1 Abbreviations: RNA, ribonucleic acid; the capital letters U, A, C, and G are used for the

nucleotide residues present in RNA, uridylic, adenylic, cytidylic, and guanylic, respectively.2 Nirenberg, M. W., and J. H. Matthaei, these PROCEEDINGS, 47, 1558 (1961).3 Lengyel, P., J. F. Speyer, and S. Ochoa, these PROCEEDINGS, 47, 1936 (1961).4Speyer, J. F., P. Lengyel, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 63 (1962).5 Lengyel, P., J. F. Speyer, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 282 (1962).6 Speyer, J. F., P. Lengyel, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 441 (1962).7Martin, R. G., J. H. Matthaei, 0. W. Jones, and M. W. Nirenberg, Biochem. Biophys. Res.

Comm., 6, 410 (1962).8 Crick, F. H. C., L. Barnett, S. Brenner, and R. J. Watts-Tobin, Nature, 192, 1227 (1961).9 Thanks are due to S. Ochoa and J. F. Speyer for their courtesy in making available their

results to us before publication.10 Hunt, J. A., and V. M. Ingram, Nature, 184, 640 (1959).11 Ibid., 181, 1062 (1958).12 Ibid., 184, 870 (1959).13 Hill, R. L., R. T. Swenson, and H. C. Schwartz, J. Biol. Chem., 235, 3182 (1960).14 Swenson, R. T., R. L. Hill, H. Lehmann, and R. T. S. Jim, J. Biol. Chem., in press.15 Gerald, P. S., and M. L. Efron, these PROCEEDINGS, 47, 1758 (1961).16 Baglioni, C., and V. M. Ingram, Biochim. Biophys. Acta, 48, 253 (1961).17 Baglioni, C., J. Biol. Chem., 237, 69 (1962).18 Murayama, M., Federation Proc., 19, 78 (1960).19 Muller, C. J., and S. Kingma, Biochim. Biophys. Acta, 50, 595 (1961).20 Ingram, V. M., and A. 0. W. Stretton, Nature, 190, 1079 (1961).21 Helinski, D. R., and C. Yanofsky, these PROCEEDINGS, 48, 173 (1962); Henning, U., and

C. Yanofsky, ibid., 48, 183 (1962).22 Pal6us, S., and H. Tuppy, Acta Chem. Scand., 13, 641 (1959).23 Brown, H., F. Sanger, and R. Kitai, Biochem. J., 60, 556 (1955).24 Harris, J. I., F. Sanger, and M. A. Naughton, Archiv. Biochem. and Biophys., 65, 427 (1956).25 Matsubara, H., personal communication." Nolan, C., and E. L. Smith, J. Biol. Chem., 237, 446, 453 (1962).27 Lee, T. H., A. B. Lerner, and V. Buettner-Janusch, J. Biol, Chem., 236, 2970 (1961).

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28 Katsoyannis, P. G., and V. duVigneaud, Arch. Biochem. and Biophysics, 78, 555 (1958).29 1)ixon, J. S., and C. H. Li, Gen. and Comp. Endocrinol., 1, 161 (1961).30 Acher, R., J. Chauvet, M. T. Lenci, F. Morel, and J. Maetz, Biochim. Biophys. Acta, 42, 379

(1960).31 Chauvet, J., MI. T. Lenci, and R. Acher, Biochim. Biophys. Acta, 38, 671 (1960).

RIBOSOMAL LOCALIZATION OF STREPTOMYCIN SENSITIVITY*

BY JOSEPH F. SPEYER, PETER LENGYEL, AND CARLOS BASILIot

DEPARTMENT OF BIOCHEMISTRY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE

Communicated by Severo Ochoa, February 28, 1962

Erdds and Ullmann' reported that streptomycin inhibits the incorporation oflabeled amino acids into protein in cell-free preparations of sensitive strains of My-cobacterium friburgensis but not in preparations of resistant strains. More recentlySpotts and Stanier2 advanced the hypothesis that streptomycin sensitivity, resist-ance, or dependence, is the result of modifications in the structure of the ribosomesthat affect their affinity for messenger-RNA. They propose that the structure ofthe ribosomes of sensitive cells endows them with a high affinity for streptomycinand that combination with this antibiotic interferes with the attachment of mes-senger-RNA with corresponding inhibition of protein synthesis. The ribosomes ofresistant cells are supposed to have no affinity for streptomycin; consequently thedrug does not affect their function. It had been found by Hancock3 that strepto-mycin sensitive, but not resistant, bacterial cells can bind small amounts of strep-tomycin irreversibly.We wish to report on experiments showing that inhibition of protein synthesis by

streptomycin in cell-free preparations of sensitive strains of Escherichia coli is dueto interference of the drug with ribosomal function. Thus, streptomycin decreasedthe polyuridylic acid (poly U) dependent incorporation of C14-labeled phenyl-alanine into acid-insoluble products4 in a system containing (a) supernatant andribosomes of sensitive, or (b) supernatant of resistant and ribosomes of sensitiveE. coli. The drug had no effect with (a) supernatant and ribosomes of resistant,or (b) supernatant of sensitive and ribosomes of resistant cells. While these ex-periments point to the ribosomes as the site of streptomycin sensitivity, in agree-ment with the view of Spotts and Stanier, they throw no light on the actual modeof action of the drug.

Preparations and Methods.-These were as previously described4 except that the preincubationof the ribosomes with supernatant, cold amino acids, and an adenosine triphosphate generatingsystem, was left out, and for the omission of cold amino acids (only phenylalanine-C14 beingpresent) from the incubation mixture. Addition of ribosomes and streptomycin preceded thatof the remaining components of the system. Streptomycin inhibition was less pronounced ifthe drug was added last. E. coli transfer RNA was added in saturating amounts. The incuba-tion was for 30 minutes at 37°. Poly U, sample 1 (sedimentation coefficient, 10. 3 S), was used.The amount of ribosomal protein, in a final volume of 0.25 ml, was approximately 0.78 mg inthe case of streptomycin sensitive (E. coli W), and 0.61 mg in that of.streptomycin resistant cells.A culture of the latter cells (E. coli K12 F- 2341) was kindly provided by Dr. Alexander Tomasz,The Rockefeller Institute, New York. Wre are also indebted to Dr. David Perlman, The Squibb

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