California Association of Chemistry Teachers
H. Fmenkel-Conrat
University of California Berkeley, California
Ribonucleic Acid, the Simplest Inf~rmati~n-tran~mitting Molecule
Man's ascendance to a singular position on this planet can he attributed to his acquisition and perfection of various special attributes. One of these is his ability to preserve and transmit information. The complex intellectual challenge to achieve this without the need of a human carrier resulted more than 5000 years ago in the development of symbols and scripts. I n recent years attention has been focused on the fact that the problem of information transfer is not only an advanced intellectual development, but also represents one of the primordial features in the development of living organisms on the earth. Each cell transmits information to its daughter cells, and the information transmitted by sperm and ovum assures the continuity of evolution for the more complex organisms. We now regard the life process as resting on two primary pillars of function: replication and metabolism. Metabolism n+,h its associated sensory and motile functions represents and encompasses the machines and factories of the living state, while replication is assured by the blueprints or the microfilm of inheritable information. Proteins have developed as the ideal material for the metabolic processes, nucleic acids for the genetic functions. The proteins are 3dimensional bodies of varied and specific shapes, and they make up the bulk of the cellular contents. The nucleic acids, on the other hand, function as tickertape like threads, and if they occur folded it is only for the sake of convenience or safekeeping. It seems from recent studies on flatworms that nucleic acids may represent the carriers not only of genetic but also of acquired information, or memory, and it is not excluded that they may play a similar role in the more complex nervous and mental centers of learning in the higher animals and man. All cells contain the two kinds of nucleic acid, deoxyribonuckic acid (DNA), and ribonucleic acid (RNA). While the DNA may function exclusively as the storing house and primary template of genetic information, the RNA plays a variety of roles in transmitting this information to the metabolism factories in the day-to-day operation of the cell. Even the This research was aided by grant G-9745 from the National Science Foundation.
216 / lourno1 of Chemkal Education
simplest cell contains thousands of functional proteins, all described in DNA records and coordinated and controlled by RNA molecules. It is therefore evident that cellular nucleic acids must he extremely complex in nature, and uninviting to detailed chemical study. Yet, it is the chemical detail which accounts for the function of these macromolecules, just as the detail of each letter determines the meaning of words and sentences. Whenever scientists are faced with a complex natural phenomenon they attempt to simplify it, or they search for the simplest examples of the given phenomenon. I n this sense, the viruses which generally lack all metabolic activities represent ideal objects of study for those interested in nucleic acid function. Viruses may be regarded as genetic units of nucleic acid, endowed with a specific protein coat for its protection in transport from cell to cell or from host to host. They become functional only within a host cell whose machinery they can commandeer for their replication. The amount of information which a virus particle must carry is variable, as we will see, but it is always very much less than that carried by a complete cell, and the obvious consequence of this is that there is much less nucleic acid in a virus than in a cell. Thus, viruses probably represent the best material for a chemical approach to the problem of genetic informat'on transfer. Viruses may contain either DNA or RNA. All RNA containing viruses seem to contain one molecule of RNA, of a molecular weight of about 1.0-2.5 million which means that according to current concepts which we will explain later, they contain 1000-2500 genetic letters. This seems to be the minimal amount of information to describe the structure and function of a virus. The DNA viruses range from this same size up to 100 times this size, and many of the bigger ones are known to perform much more complex functions in invading thick-walled bacteria and forcing the host cell into new metabolic pathways. The Chemical Structure of Nucleic Acids
For obvious reasons, chemical research has been concentrated on the RNA and DNA of the simplest viruses. Both of these macromolecules represent unbranched sugar-phosphate-sugar-phospate. . . chains,
thc sugar being ribose for RNA aud 2' deoxyribose for DKA, and the phosphate forming diester bridges between the 3' and 5' positions of adjacent sugars (Figure 1). The feature that makes these molecules functional is the attachment of one of four possible purines or pyrimidines at the aldehydic 1' position of each sugar residue (see Figure 1). The order of these so-called bases appears to be the primary feature of genetochemistry. Three of them, adenine, guanine, and cytosine are shared by RNA and DNA. Since these do not carry an electric charge between pH 4 and 9, the term bases is really a misnomer, just as the term nucleic acids is a misnomer, since these occur and are stable only as the salts or nucleates, but not as the free acids. The fourth component, no base at all, is uracil in RWA and 5 - methyluracil (thymine) in DNA. The complete mouomer unit of base-sugar-phosphate is termed a nucleotide, and it may be looked a t in a t least two different ways. Certain enzymes degrade nucleic acid by splitting the phosphate -3' ribose bond and thus yield 5' phosphorylated nucleotides; on the other hand, acids, alkali, and other enzymes split the other way and thus produce 3' phosphorylated nncleotides. Undcr certain conditions the phosphate groups on the 3' ribonucleotides may partly rearrange to the 2' position. The availability of the 2'-OH group next to 3' phosphate ester bridge is responsible for the great alkali sensitivity of RKA (in contrast to DNA), and this may well have been the chemical cause for an evolutionary advance fromRNA to DXA as the primary genetic material of all organisms. As stated, the sequence of four symbols along the polymer chain appears to determine its function. What can we say and learn about such sequences? Unfortunately there is as yet a great lack in methods for the sequential analysis of polynucleotides. And even if good methods were available the length of the chain and the lack of characteristic features (such as rare letters in an alphabet) will make sequence analysis of a viral RNA an extremely difficult task which may Cytosine
Chemical Modification of RNA and Mutagenesis
,,
I
Adenine
n-0
...............
Guanine
uracil
ofif
...
(Ribose) H ...I.........
0
OH . .-.
% .
>
o=p
(Phosphate)
require many decades for its completion. Concerning the virus RNA which is studied most intensively in our laboratory (that of the tobacco mosaic virus) we believe we know one nucleotide a t the left end, and four a t the right end of the chain of 6400, and less is known about other viral nucleic acids (1-3). Intensive work is going on with a class of small RNA molecules (80 nucleotides long), the so-called transport-RiSA (or s-RNA). This RNA has special features facilitating sequence analysis from one end, and information about one-tenth of the chain is now being accumulated for some of these agents by P. Berg and his collaborators at Stanford University (4). Another approach is not from the ends, but through the determination of groups of "odd" sequences. Most nucleic acids are composed of similar amounts of purines and pyrimidines and one can calculate the probabilities of these occurring in groups of 2, 3 or more of the same kind, etc., for a random polymer. I n a specific and functional polymer such bunches of the same nncleotide, or of neighboring purines or pyrimidines would differ in frequency from that calculated and such differences would represent characteristic features of a given nucleic acid. Methods to determine the frequencies of neighboring purines or pyrimidines and of other specific sequences are now available, and interesting data are being accumulated, as shown by the following example. Although we know that the RNA of two different straiils of a virus must differ (for it is the ItNA that contains the information describing both the similarities and the differences between these strains), no differences were detected in the nucleotide composition of different strains of TMV. However, structural differences of the type discussed above (frequency of bunches) could be demonstrated among several of these straius (5). A completely dierent technique has enabled scientists in Kornberg's department to determine the nearest neighbor frequency of DNA, again a highly specific chemical feature of this class of macromolecules (6).
I
OH'0-
Figure 1. The structure of 2 dinucleotide., cytidylis 3'-5' adenylic acid, ond ~uonylic3'-5' vridylic acid. The three building blocks (phosphate, ribose, bore) ore delineated. The barer are shown H-bonded in pairs, or they tend to be in polynucleotides.
Whiie we are as yet unable to determine long nucleotide sequences, we do have methods to alter them by chemical means. Among these methods, which include alkylation, halogenation, etc., the most fruitful has been the deamination resulting from treatment with nitrous acid. When applied to viral nucleic acid this as all other modification reactions causes gradual loss of its biological function, an average RNA molecule being inactivated when about two of its 6400 nucleotides have become chemically altered ("hit") (7). What made this study so important was the discovery that most of the surviving molecules gave different symptoms than the original virus, which means that they were mutants, resulting from the chemical modification of the nucleic acid (8). If we now look a t the specific effect of deamination of the aminopurines, adenine and guanine, we observe that the resulting deaminated purines are unnatural nucleic acid constituents. In contrast the deamination of cytosine yields uracil, a natural component of RNA (Figure 2). Thus deamination of RNA, but not of DNA may produce chemical variants of the same species of moleVolume 40, Number
4, April 1963
/ 217
cules, and it appears probable that many of the observed mutants are progeny of such molecules in which 1, 2, or 3 cytosines have been changed to uracil. As we will see later this means of producing with high probability a specific change of the genetic script has proven of considerable importance in the elucidation of the nature of the code which relates RNA structure to protein structure. Before we can enter more deeply into the discussion of the nature of the code and its change by mutation, we must first describe the present concepts concerning the function of the genetic material, or the reading of the information. Intelligent information transmittal requires an intelligent receiver. Both writing and reading are active processes. I n contrast, chemical information script must through its chemical structure make itself read-writing must force reading. This has proven to be realized by the chemical nature of the bases. As it was first formulated by Watson and Crick on the basis of available analytical and X-ray scattering data, a remarkable fit and hydrogen bonding affinity makes the Aamino purine (adenine) bind to the 6-ketopyrimidines (uracil and thymine), and the 6-ketopurine (guanine) to the 6-amino pyrimidine, cytosine, particularly if these are in polymer linkage (Figures 1, 3). Thus, polymers of adenylic acid (poly A) form quite stable dimers, with polyuridylic or polythymidylic acid (poly U, poly T) and a polymer composed of alternating A and T residues always tends to be double-stranded, held together by H bonds between opposite A and T residues. DNA was recognized by Watson and Crick as generally occurring in the stable double-stranded form and what was first an inspired hypothesis is now a well established fact: The two strands have complementary nucleotide sequences, so that opposite each A there is a T and opposite each G there is a C, and vice versa (9). These multiple and regularly spaced interactions lead to double-stranded helm formation, and the stacking of the bases which form shelves perpendicular to the axis of the helix, further stabilizes their linkage. The double-strandedness is not a necessary structural feature for biological function, since DNA occurs in single-stranded form in certain small viruses, and RNA generally occurs in single-stranded form only. Nevertheless, this affinity for the complementary base seems
Figure 2.
21 8
/
---
CYTOSINE URACIL HYPOXANTHINE) (ADENINE XANTHINE) (GUANINE Deamination of the barer containing amino groups in RNA
Journal of Chemical Education
to be the directing force that usually ensures faultless replication of the long molecules of ilucleic acids. Thus, the presence of a polynucleotide of certain base sequence leads to the formation of a complementary strand when nucleotides are being polymerized by enzymes i n uitro. Features of the molecule other than the hydrogen bonding "surface" of the bases, such as the sugar (ribose or deoxyribose) or the methyl group in the 5 position (thymine or uracil) do not affect this principal feature of the replication process. It represented a major advance in molecular biology when Kornberg and later many other research groups began to study the reproduction of specific nucleotide sequences in cell-free systems, starting with either RNA or DNA as the template (5,10-12). The energy necessary for the linking of one nucleotide to the other in such systems is supplied by using the energy-rich 5'-triphosphates of the four nucleotides, or deoxynucleotides, pyrophosphate being released from each condensing unit (Figure 3). If we now rcturn to consider the action of nitrous acid, it is obvious that each deamination of cytosine to uracil (C U) will be replicated as such (U information being passed through A to U, instead of C G C). Deamination of A on the other hand yields a 6-oxypurine which resembles G more than A and thus might be replicated as such (A HNOl + hypoxanthine % G C G) although it might also lead to inactivation of the molecule. Finally, the deamination of G to xanthine does not appear mutagenic in terms of the H-bonding site of the purine, but may well be inactivating. Other modifications of nucleotides, such as the introduction of alkyl groups on the purines or of bromine atoms or hydroxylamine residues on the pyrimidines have been reported to cause occasional mutants. But all these reagents are of a low mutagenic efficiency, compared to nitrous acid acting on RNA. These infrequent mutations are probably the consequence of an occasional error in the replication of a base which is slightly deformed because it carries a substituent on its "back."
--
--
+
DNA Template
P-P t--RNA being formed
-
Figure 3. Schematic presentation of RNA formation on a DNA template. The vertical line represents the rugor; the letters, A,G,U,C,T, the bores; ond P the phosphate group. The polymer chain i. .how" to form from right to left. The doned lines rewesent H-bonds.
Role of RNA in Protein Structure
This was a review in a nutshell of the recent advances made in our understanding of the chemical mechauism by which nucleic acids replicate aud transmit their structure from DNA to ILNA. However, the other critical aspect of nucleic acid function lies, as we stated earlier, in their ability to transmit this structural information to the enzyme proteins which control metabolism. Concerning this mechanism we knew very little until 1961. Even without knowledge of the mechanism, however, useful information concerning the interrelationship of proteins and nucleic acids was again derived from virus research. For each virus particle consists of both the genetic material and one of its phenotypic expressions: the protein that gives it its shape and stability. It was long known that the proteins of related strains of a virus could he very similar to one another, or that they could differ in specific manner. In the case of TMV the protein coat of each virus particle consists of 2130 identical protein molecules each composed of 158 amino acid residues. Methods for the aualysis of the amino acid composition are now sufficieutly exact to permit the detection of the presence of o m amino acid more or less in such a unit. It thus has become evident that strains of TMV, be they naturally occurring or produced by one of the mutagenic reactious described above, may be identical in composition but may also differ one from another by one or several (up to 30) amino acid replacements ( I S , ISa). Replacement means that any decrease in one amino acid is always compensated by an increase in another so that the total number of 158 residues appears unchanged. The general belief that the amino acid composition is a genetically controlled feature of the virus was proven by us a few years ago when we started to disassemble and reassemble (reconstitute) virus particles. We then found that the strain origin of the nucleic acid alone determined the nature of the progeny particle, including its amino acid composition, even if the RXA was encased in vitro, through reconstitution, in protein from a differentstrain, greatly differingin compositiou (14) (Figure 4).
ORIGINAL STRAINS
DEGRADATION
RECONSTITUTION
(Wy/i!)
REPLICATION
PROTEIN
n n
Figure 4. Schematic presentation of mixed reconstitution of the RNA of rtroin 4 with the protein of rtroin B of a virus. The progeny of such mixed virus shows all the properties, including protein composition, of strain 4.
We have since gone beyond composition. For now through the work of one research team at our laboratory, and another a t Tiibingen, the complete sequence of the 1.58 amino acid residues along the peptide chain is known (15, 15a, 16) (Figure 5). We can therefore ask and answer questions, such as: which particular serine is replaced by phenylalanine in this mutant? The virus protein fulfills an important function in protecting the viability of the virus, and certain amino acid replacements may interfere with this function. Thus, those virus strains which we are able to study have passed the screening test of natural selection, and are not grossly deficient in protein quality. Yet all strains appear more or less deficieut in viability compared to the original common TMV, presumably because this is the strain best adapted to its environmental circumstances. I n one specific instance we found this fact illustrated in clear fashion. The replacement of proline #I56 (on the amino acid sequence map) by leucine in a strain resulting from nitrous acid treated RNA rendered that strain susceptible to extensive degradation by the enzyme carboxypeptidase (15). I n the common strain the presence of proline in position 156 restricts enzymatic attack to the terminal threonine (#158) which may be jettisoned without detriment to the infectivity and stability of the virus. Thus, we have here observed that a change in RNA structure, presumably a replacement of C by U, leads to a change in protein structure which affects the stability of the viral coat protein, and thus presumably the viability of the virus (IS). Mechanism of Information Transfer from RNA to Protein
Studies of the type discussed above are supplying more data concerning amino acid replacements as a consequence of mutation. However, they tell us nothing about the mechanism of information transfer from the RNA to the protein. This so-called coding problem has been discussed in theoretical manner for a number of years. Since only 4 different nucleotides can code for 20 amino acids, they must act in consort, and only triplets of nucleotides give a sufficient number of permutations to suffice for the purpose (pairs of nucleotides would give 4%= 16 symbols, while triplets give 4a = 64 possible symbols). Recently, some experimental evidence was reported by Crick and coworkers in support of the triplet theory, but other possibilities have certainly not been ruled out (17:. Regardless of this number, it appears probable that IPROGENY the base pairing principle accounts for the translation of sequence information from RNA to protein, as it does from DNA to RNA. This is possible because each amino acid is carried to the protein synthesizing components of a cell, the so-called ribosomes, by a specific transport RNA, to which it is bound by ester linkage to the ribose of a terminal adenosine. Thus a certain nucleotide sequence on the information-carrying DNA-derivedRiVA is believed to attract by base pairing a certain attaching site of the transport RKA and thereby to bring its amino acid near to that attaching at the Volume 40, Number 4, April 1963
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neighboring site (18). These neighboring amino acid residues may then exchange the ester linkage holding them to their specific transport-RNA for the amidc linkage which locks them into a peptide chain. Much of this process is still uncertain and difficult to envisage, but the principle features may be correct. Whatever the detailed mechanism, recent work by Nirenberg and Matthaei (IQ), and subsequent work by a team in Ochoa's laboratory (28) have definitely established means of studying this reaction. The protein-making factory, the ribosomes, require energy supplied by ATP, as well as the twenty different amino acids as building blocks, each with its corresponding activating enzyme and transport RNA. If one, several, or all of the amino acids are radioactive, their incorporation into peptide and protein linkage can easily be detected and measured. Besides these components, the system needs instructions as to which protein to make. Such blueprints are usually supplied by the DNA of the cell, as transmitted via messenger RNA. If no DNA is present or if it is removed by means of the enzyme DNAase, the messenger cannot be rcplaced as it becomes nsed up, and the factory grinds to a halt. It was Nirenberg's discovery that the addition of various types of RNA to the system a t that time resulted in a new spurt of production, as measured by amino acid incorporation (16). What was being produced? Obviously one would have to expect that it was the message carried by the added RNA which would be translated into protein structure. Yet, if one nsed the protein factory of a bacterium (E. colz?, and if one added an RNA as strange to this system as that of a plant virus (TMV-RNA), the idea that TMVprotein would be synthesized would seem almost too fantastic to entertain. Fortunately, Nirenberg and Matthaei refused to reject this possibility and initiated joint studies with our laboratory to characterize the nature of the radioactive protein syilthesized by their cell-free E. coli enzyme system under the direction of TMV-RNA. The result of a variety of complex tests is as follows: A good part of the protein seems to resembles TMV protein, as shown by immunological tests, and by amino acid sequence studies. Yet, a definite chemical dif-
ference was detected near one end of the peptide chain of the TMV-protein. Further, in regard to its function to form a coat for the viral RNA through aggregation of chains, the newly syilthesized protein was definitely deficient. The presence of this protein actually interfered in specific manner with the aggregation of the normal protein. It appears quite possible that a comparatively minor chemical deficiency, such as the absence of the N-terminal acetyl group, might account for this behavior. The "incomplete" protein unit might fit and attach to its neighbors in the course of the helical stacking of "good" protein subunits but, lacking the proper configuration "on its hack," the incompletely synthesized protein might thereby terminate the process of protein aggregation and resultant rod formation. Both the nature of the chemical and of the functional deficiency of the TMV protein synthesized in the cell-free E. coli system are under continuing investigation, and much remains to be learned about the mechanism of this reaction. However, the close relationship between TMV protein and the product of the enzymatic amino acid polymerization in E. coli, when directed by TMV-RKA, is suggested by serological, physicochemical, amino acid sequential, and functional tests, and thus appears to be a reality (20). The potential implicationsof this finding,if confirmed, are great and manifold. First, it would give a distinct answer to the question of the universality of the code. If the genetic language of a plant virus RNA can be understood by a bacterial system, then it would appear probable that the code is the same or nearly the same for most if not all organisms. This conclusion is borne out by several other recent findings. Further, the concept of messenger RNA, as first proposed on quite different grounds by Monad and others @I), is supported and broadened by those findings. For the nature of the added RNA appears to be the principle agent that determines the nature of the protein produced by the synthesizing factory of E. coli. A viral RXA can act as messenger, and it does so most probably by serving as the template which assures the reproduction of all that pertains to the making of a virus. It seems now certain that the viral RNA encompasses more than the temnlate for the coat vrotein. althoueh this is the only product that was looked for in the reported biosynthetic experiments. Since RKA alone is infectious, it is logically evident that it must supply the template for its own replication, and for the production of any new enzymes required for the synthesis and the spreading of the virus and the appearance of its symptoms. The 6400 nucelotides of TMV-RNA are believed, on the basis of the triplet code theory, to carry enough information for 2130 amino acid sequences while the viral coat protein consists of chains only 158 amino acids long. The incorporation of histidine and methionine by the E. coli system is stimulated to the same extent by TMV-RNA and by HR-RNA, although the common TMV lacks these amino acids in its coat protein, but the HR-strain contains them (14, 20). And finally we know that many mutants produced by chemical modi~
~
L~
-
fication of the RNA do not show any differences in their amino acid composition from common TMV from which they are clearly distinguishable by the symptoms they produce. Obviously, we must assume that chemical analysis should reveal differences in the RNA of these strains, but the methods of nucleotide analysis do not yet approach the precision necessary to find minimal changes such as the transformation of one or two out of 1200 cytosines to uracils. As a consequence of these facts and considerations, our former oversimplified concept that TMV-RKA represents nothing but the nucleic acid equivalent of the TMV-coat protein had to be abandoned for the more complex one that it carries all kind of information, much of which we are not yet able to recognize. Thus, it is not surprising that the E. coli system as directed by TMV-RNA causes the synthesis of much protein which is not related to TMV-coat protein. As usual when a new mountain of scientific knowledge has been climbed it turns into a foothill compared to the range of peaks now exposed to view, all challenging further effort. Another instance where the E. coli-protein making machinery was found to accept direction from a specific RNA, namely that of a coli-phage, has recently been reported. Be it due to the normally closer relationship of that RNA to the bacterial system, or just to luck, an appreciable fraction of the observed amino acid incorporation resulted in what seemed to he phage-coat protein. However, functional tests wcre not attempted in this instance (22). The message carried by the viral RNA has turned out to be more complex than first bargained for. It was thus fortunate for Nirenberg and Matthaei, that soon after they initiated the study of this reaction, they sought and found a simpler messenger, an isolated peak of discovery which immediately revealed a wide new area for the rapid advance of knowledge. Since the work of Grunberg, Ortiz, and Ochoa (23), it had been possible t o prepare polynucleotides by means of certain bacterial enzymes acting on nucleotide 5' diphosphates. The polymer of uridylic acid alone (poly-U), as prepared in this manner, proved very active in stimulating the E. coli system to incorporate or polymerize one and only one amino acid, phenylalanine (19). The conclusion was soon firmly established that some number of uridylic acid residues, possibly 3, in polymer linkage coded for phenylalanine. This represented the first experimental breakthrough on this crucial front of scientific progress, the correlation of nucleotide with amino acid sequences. A systematic study of the effects of polymers prepared from pure di, tri, and tetranucleotides in this system will undoubtedly be performed and will give the definitive data on the code. Unable to await the results of this laborious process, workers at Ochoa's and Nirenberg's laboratory have studied the effects of random copolymers of 2 or 3 nucleotides, and have obtained data about the nucleotide composition, though not the sequence, of possible code symbols for all amino acids (19). This work will not be discussed in detail here, for it is the talk of the scientific community and is being reviewed by others. A number of scientists not engaged in the primary discoverieshave brought
their ingenuity to bear on the problem and have built new theoretical card houses concerning coding sequences, evolutionary developments, etc. These, too, will stand or fall with the laborious accumulation of data, so distasteful to those who feel pressed to join the band-wagon. The name of that vehicle is the Proceedings of the Kational Academy of Sciences, and it travels once a month, and one month's delay in publication might mean the loss of a man's right to claim 5 years later when the data are a t hand, that he had first guessed at a particular conclusion. Mutants and Coding
We will now return to our main topic of discussion, the viruses and what we can learn from them in regard to the mechanism of replication. As discussed earlier, the effect of mutation on the amino acid composition of the protein of TMV has long been the subject of intensive study in two laboratories. Many of the mutants were the result of nitrous acid treatment of the RNA, and could thus with some probability be attributed to a change of C + U. Obviously, these data represented a good test for the validity of the coding symbols derived from the E. coli work of Kirenberg's and Ochoa's laboratory. The first observation that phenylalanine is coded by poly-U alone means that deamination (C -+ U) may lead to the replacement of other amino acids by phenylalanine, but never to the reverse process. I n accord with this, serine was found replaced by phenylalanine in several strains, while phenylalanine has not been found replaced by other amino acids (13, 1Sa). The frequent changes involving proline, the code symbol of which is rich in C, are also in accord with expectation. I n general, it can he stated that the majority, but not all, of the observed amino acid exchanges are in accord with the code symbols proposed for these amino acids, on the basis of C U transformations. A few of the others can be accounted for by the hypothesis that A, deaminated to hypoxanthin resembles and codes like G. Others that do not fit could signify erroneous coding symbols, but probably they are indicative of a strain which resulted from spontaneous mutation (or contamination) rather than being evoked by the deamination reaction. Since each strain is the progeny of a single infective molecule, these unfortunately are definite and ever-present possibilities. Seen as a whole, the agreement between the two sets of data, each weighted by its own errors and uncertainties, is as good as might be expected, and lends further support to the validity and to the universality of the code symbols as elucidated with E. coli, and tested against tobacco mosaic virus. Other systems using bacterial or bacteriphage strains promise to supply similar and possibly more incontrovertible evidence along such lines.
-
Conclusions
Let us now review the subject of this article. We have discussed the chemical nature of the nucleic acids, both RNA and DNA, which are the carriers of genetic and possibly also of other types of information in all organisms. We have considered methods by which this information can be changed through chemical modification, and how this process may result in mutation. We have described mechanisms by which genetic Volume 40, Number 4, April 1963
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information can be transferred from one type of nucleic acid to another, and from nucleic acid to protein. This mas illustrated most convincingly with the bacterial protein synthesizing system. Thus, a mixture of enzymes and ribosomes derived from E. coli makes the protein which it is directed to make by an added RNA, the so-called messenger RNA, whether this RNA is as complex as that of a virus or as simple as polyuridylic acid. We have briefly touched upon the enormous advances made in the past year in the elncidation of the code relating each amino acid to a corresponding nucleotide combination; and we have discussed the extent of agreement of these code symbols with amino acid exchange data observed in mutants of TMV and presumably due to specific nucleotide exchanges in its RNA. While this is the first mention of tobacco mosaic virus in this summary, most of the work and most of the discussion actually dealt with this virus, a fact which illustrates the versatility and general usefulness of viruses as study objects in research on the fundamental principles of molecular biology.' Literature Cited
Centre Nstionnl DP 1,s Recher~he i.. e.. n t i f-.,i~ m w. -P-. s ~." i* . . ~ ~ -S.~~ . ... ~ 1962, p. 259. RUSHIZKY, G . W., A N D KNIGHT,C. A,, Virology, 11, 236 (lofill> * - - * ,. JOSSE,J., KAISER,A. I ) . , AND KORNBERG, A,, J . nid. ~
~
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~~~
~
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GIERER,A,, A N D MUNDRY, K. W.. N a t t m , 182,1457 (1958). WATSON,J . D., AND CRICK,F. H. C., Cold Spring Havbor SymposiaQz~ant.R i d . , 18, 123 (1953).
LEHMAN, I. R., BESSMAN, M. J., SIMMS,E. S., AND KORNBERG,A,, J. B i d . Chem., 233,163(1958). WEISS, S., AND NAKAMOTO, T., PTOC.Natl. Aead. Sci., 47, 694 (1961).
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A N D DIRINGER, R., Biochem. Biophys. ResearehCommuns., 3 , 1 5 ( 1 9 6 0 ) . TSUGITA, A,, AND FRAENKEIXONRAT, H., Proc. Nat. Aead. Sci. USA 46, 636 (1960); ( a )TSUGITA, A,, J . Molec. Biol., 5,284,293 (1962). J . Mol. Riol.4,73(1962). FAENKEI~CONRAT, H., AND SINGER,B., Biochim. Biophys. Acta, 24,540(1957). ANDERER, F. A,, ~ ~ 1 . 1 0m., , WEBER,E., AND SCHRAMM, G . , Nature, 196, 922 ( 1 9 6 0 ) ; ( a ) ANUERER, F . A., A N D HAN~scanIi,H., Z. j . h~aturforsch.,17b, 536 (1962). TSUGITA,A,, GISH, D. T., YOUNG, J., FRAENKEL-CONRAT, H., KNIGHT,C. A,, AND STANLEY, W. M., PTOC. Natl. Aead.Sci. lJ.S..46.1463119601.
S., in press (1963). ( 3 ) SINGER, B., Biochemist~y,in press (1963). ( 4 ) BERG,P., AND LAGERKVIST, U.,"Acides Ribonucl6iques E t Polyphosphates Structure, SynthPse E t Fonctions,"
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' A review of this subject matter for the layman or beginning student of molecular bialoev is available in form of a. small naner back, "Design and Func&m a t the Threshold of ~ i f e : ' +he Viruses," by H. Fraenkel-Conrat, Academic Press, New York 1962.
222
JACOB, F., AND MONOD, J., J. Mol. B i d , 3,318 (1961). NATHANS, D., NOTANI,G., SCHNARTZ, J . H., A N D ZINDER, N. D., Pme. Natl.Aead. Sci. U . S . , 4 8 ,(1962). GRTJNBERG, M. M., ORTIZ,P. J., A N D OCHOA S., Biochim. Biophys. A d a , 20,269 (1956).
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