43

CHAPTER III

GENERAL NATURE OF THE GENETIC CODE"

F. H. C. CRICK, University of Cambridge, Cambridge, England

  I have taken as my title "The General Nature of the Genetic Code"
because we do not yet know the genetic code in detail, although we certainly
hope to do so before very long.

  We believe that genes are made of nucleic acid. It need not concern us
at this stage whether it is DNA or RNA, double-stranded or single-stranded.
We also believe that one of the main functions of genes is to determine the
amino acid sequence of proteins. There may be other functions but this
function is certainly an important one.

In addition, proteins have to take up their correct secondary and
tertiary structure. I shall assume here without further discussion that the
folding of a protein is mainly dictated by its primary structure.

  The genetic code, then, covers the problem of how the sequence of bases
in a particular piece of nucleic acid determines the sequence of amino acids
in a particular protein. There appear to be four common bases in nucleic
acid: guanine, adenine, thymine (or uracil) and cytosine. It is true there
are a few rarer ones, most of which can be described as having additional
methyl or hydroxymethyl groups. We cannot be sure that they do not have
some important function in the genetic code, but this is not very likely.

  It is an interesting speculation as to whether we could have nucleic acid
with six bases: that is, three base-pairs instead of two base-pairs. Dr. Alex
Rich, a little time ago. did make a suggestion along these lines (Rich, 1963),
hut we can say fairly rategorirally that af the moment wc have no indication
of a third base-pair in any biological material.

  The polypeptide chains of prnleins are made from a standard set of
twenty amino acids. Again you could argue about thr: number twenty. It.
does not include hydrnxyproline, phosphnserine, and certain other amino
acids which arr certainly found in true proteins. Neverthelrss, for reasons
which I have explained elsewhere (Crick. 196.7). I think thr slanrlard set
of twenty is probably the correct one.

I shall assume, therefore, that we have four standard bases in nucleic
acids and twenty standard amino acids in proteins. Moreover the bases and

*An address presented before "The Robert A. Welch Foundation Conferences
on Chemical Research. VIII. Selected Topics in Modern Biochemistry," which
was held in Houston, Texas, November 16-18, 1964.


/1,4         GENERAL NATURE OF THh GENETIC CODE'
tlltr Illllitlt) rlc*itIx 111~ lmpi(*llIIy the same throughout Nature. It does not matter
                                                   .
wlwt orKllllitiItI OIW looks III. -- cmilnnl, plant, or micro-orgamsm - one
always linds in Lhem the same standard set of amino acids and the same
four bases.

It might be thought that solving the genetic code would be straight-
forward, but this is not so, since although it is possible, in favourable
circumstances, to determine the amino acid sequence of a protein, it is at the
moment technically almost impossible to discover the base sequence of the
corresponding piece of nucleic acid. Consequently less direct methods than a
detailed comparison of two corresponding sequences have to be used.

The first assumption I shall make is that the genetic code is fairly simple.
In other words, that the relationship between the sequence of four things in
the nucleic acid and the twenty things in the protein is not an elaborate one
but is relatively straightforward. The main justification for this is that the
code has to be translated by biochemical machinery, and although we know
that this is fairly complicated we do not think it is exceedingly ingenious
and intricate. I shall assume that for each amino acid there will be a small
group of bases on the nucleic acid which codes it, and I shall call this group
a "codon".

All codons cannot consist of just two bases, because we have only four
alternatives in each position, so we can only construct a maximum of sixteen
different pairs of dinucleotides, whereas we need twenty for the amino acids
and certainly one or two more in addition as punctuation marks. Groups of
three bases would provide sixty-four possible codons, and this is now
believed to be the correct answer.

It has been widely assumed that the genetic message and its translation
were co-linear. That is, that the order of the codnns on the nucleic acid was
the same as the order of the corresponding amino acids in the protein. Until
recently there was very little evidence for this, although it was confidently
predicted that it would sonn be found.

Strictly speaking we still do not have such evidence but it has been
shown recently by two groups of workers that the genetic map of a gene is
rn-linear with the amino acid sequence nf the protein controlled by that gene.

  A genetic map can be constructed by the standard methods of genetics.
The essence of the matter is that two different versions of the same organism,
which differ in having mutations in somewhat different places, are mated
together in snme way so that genetic recombination takes place. It is found
that when these two differences are far apart on the genetic map recombina-
lion is cniiitnon: on the other hand, when these differences are close together,

F. H. 6. CRICK                                   45

recombination is rather rare. By this method, and extensions of this method,
it is possible to put genes in order on the chromosome and in more recent
years, using micro-organisms, to put different sites within a single gene into
a linear order. The classic work on mapping within a gene is that of Benzer
on the rI1 locus of bacteriophage T4 (Benzer, 1959 and 1961). In these
genes he found several hundred different sites and by an ingenious method
(called deletion mapping) he was able to show that they could be put in a
linear order on the genetic map.

Dr. Yanofsky and his colleagues (Yanofsky et al. 1964) have worked
on the A protein of the enzyme tryptophan synthetase of Escherichiu coli.
A series of mutants was picked up, mapped genetically and thus put into a
definite order. The corresponding protein was isolated, purified and part of
its amino acid sequence determined. For each mutant the amino acid altera-
tion was pinpointed in the sequence. As was expected the two orders, that
of the mutants on the get&c map and that of the corresponding alterations
in the polypeptide chain, were the same.

Shortly after this work was published some colleagues of mine at
Cambridge, Dr. Sarabhai, Dr. Brenner and Dr. Stretton together with Dr.
Belle from Geneva (Sarabhai et al. 1964) also produced some evidence
which led to the same result.

They used a special sort of mutant called "amber" mutants. Such mutants
are found in all sorts of genes, not only in the genes of the virus, but also
in the genes of the host. They are characterized by the fact that they can
be suppressed. They were able to show that what an amber mutant does is to
terminate the polypeptide chain at a particular point. In other words, instead
of producing a complete polypeptide chain the cell makes the protein (from
the amino end), but only up to a certain point, and then releases it.

Using proteoytic enzymes they chopped the polypeptide chaiu into :I
number of peptides and they looked to see, in each case, whether certairl
peptides were made or not. By this means they were able to tell, for each
mutant, whether it made a long polypeptide chain or a short one, ad SO
they could put all the mutants in order, using only the methods of I)rotritl
chemistry. At the same time they constructed a genetic map, using IBIII!
genetic methods, and found, as expected, that the two orders were the ww.

we thus have two examples to make us feel reasuuaMy happy that tlw
gene and the protein it controls are colinear, and 1 think that in the UJIIIW
of time more evidence, from different proteins, in different organisms. will
accumulate.

  It was thought al one Lime that the genetic code might IX 4 th
overlapping type. That is, that a particular base might belong to mort~ t11u11


46            GENERAL NATURE OF THE GENETIC CODE
one codon. This now seems very unlikely, since almost all mutants which
have been studied show a change of only a single amino acid. If the code
were an overlapping one we should certainly expect to find mutants which
were changed in two or three (adjacent) places in the polypeptide chain.
The evidence comes from the work of Dr. Yanofsky already mentioned
and from studies on abnormal human haemoglobins and the protein of To-
bacco Mosaic Virus (Wittmann and Wittmann-Liebold, 1963; Tsugita, 1962.)
It is not yet certain that the bases making up a codon are adjacent on
the polypeptide chain, although we have no reason to think they are not
(Dr. Stent, in discussion, pointed out that the recent biochemical work of
Leder and Nirenberg (1964) makes it very likely that codons consist of
three adjacent bases).

If the code is of the non-overlapping type there must be something which
decides how the message is split up correctly into groups of three bases.
Otherwise the message might be read "out of phase" and thus be totally
incorrect. There are, of course, two ways of reading a triplet code out of
phase, one corresponding to beginning one base too early and the other to
beginning one too late. From evidence (Crick et al. 1961) obtained by study
ing two adjacent genes joined together by a genetic deletion we believe this
is done in the most obvious way. That is, by beginning at some fixed point
in the sequence and reading along from there in steps of three bases at a time.
This genetic work also suggested very strongly that codons consist of
three bases, rather than four or five or some other number. The evidence was
obtained using acridine mutants of the rII locus of phage T4. These mutants
behave as though they were due to the addition or subtraction of a base (or
bases) in the genetic message, rather than to one base being changed into
another, though this interpretation will be more certain when it becomes
possible to study the amino acid changes produced by such mutations.
It was found that when three mutants of like type (each the addition of
a single base, say) were combined by genetic methods into one gene, the
gene still functioned, whereas the same mutants, either singly or in pairs,
made the gene completely inactive. Such results are most easily understood
if every codon consists of three bases. The recent biochemical work of Leder
and Nirenberg (1964) appears to confirm this.
Our recent more detailed knowledge of the genetic code has come from
experiments using cell-free systems which synthesize proteins in the test-tube.
The advantages of such systems are not only that the separate steps of protein
synthesis can be more easily studied in this way, but also that one can add
molecules to them which cannot penetrate easily, if at all, into iiilact cells.
The system most used is obtained from E. coli. though other sources, includ-

F. H. C. CRICK

47

ing mammalian ones are also employed. In outline it consists of ribosomes,
S-RNA molecules, activating enzymes and various "soluble" factors, which
are probably protein, in addition to amino acids, ATP (as an energy source)
and GTP. The recent review by Watson (1964) should be consulted for
details.

In the cell the genetic information in DNA is copied onto single-
stranded RNA by the enzyme RNA polymerase, and the messenger RNA
(m-RNA) thus p ro uce IS then "read" by the ribosomes. In the cell-free
           d d '
system varitius artificial m-RNA molecules can be added and their effect on
amino acid incorporation studied. The break-through came when Nirenberg
and Matthaei (1961) showed that polyuridylic acid, used as m-RNA, pro-
moted the incorporation of phenylalanine into polypeptide chains. Thus one
codon for phenylalanine is presumably UUU. Subsequent work mainly by
Nirenberg and his colleagues and by Ochoa and his group have shown that
polyadenylic acid codes lysine, and that polycytidylic acid probably codes
proline. In addition by using mixed polynucleotides, with random or semi-
random sequences, it has been possible to allocate a fair number of the 64
possible triplets to the various amino acids, though triplets high in guanine
have proved difficult to study, since polynucleotides tend to have too much
secondary structure if their guanine content is high. Of course by these
methods alone it has not been possible to obtain the order of the three bases
in a particular codon, but only the composition of the codon. Two reviews
(Nirenberg et al. 1963; S peyer et al. 1963) provide a good summary of the
results obtained by these methods.

Among other things the evidence strongly suggested that there is, in
general, more than one codon for each amino acid; that is, that the code is
"degenerate". lt is suspected that there is one type of S-RNA for each codon.
but this has not yet been proved rigorously.
It is not yet kuowu whether the genetic code is identical in all living
things, but preliminary studies (see the article by Weinstein, 1963) show
that it is certainly rather similar in a number of rather widely separate
organisms.

In order to ohtaiu the order of bases within any codon it is necessary
Lo use polynucleotides having their bases in a known order. Khorana's group
is attempting to do this by synthesizin g
sequences.              polynucleotides with repeating
   Doty's group is also studying polyllucleotides with known or

partly known sequences. Meanwhile Nireuberg has hit upon an ingenious
method for obtaining codons usirlg Lrinucleotides. Only preliminary results
have so far been published (Letler autl Nilruberg, 1964) but it is knowu
that Nirenberg and his colleagues have already deterruined many codons ill
this way and the results will probably be irl IJrillt hefore this article appears.


&:             GENERAL NATURE OF THE GENETIC CODE
The method depends upon the binding of specific S-RNA molecules to
ribosomes in the presence of a particular trinucleotide. Thus GUU will help
to bind a species of valine RNA, whereas the trinucleotides UGU and UUG
will not. (The convention is that the 5' position is written on the left.) It is
not yet clear how clean and reliable this method will become, but it is
certainly one of great promise.

Even when the majority of codons have been obtained it will be neces-
sary to confirm them by quite different methods to make sure that no artifacts
have crept into the cell-free system. In particular the code should explain
the various amino acid changes produced by single-step mutations, of which
many different types have already been observed. Eventually this should
enable us to see how various chemical mutagens act, as direct chemical
methods may sometimes be misleading.

There is also additional evidence to be satisfied. Dr. Yanofsky, for
example, has been able to obtain genetic recombination between bases within
the same codon. In particular he has crossed a mutant having arginine at a
particular place with one that has glutamic acid there, and has obtained
a genetic recombinant having glycine at that point, which was the amino
acid there in the wild type (Yanofsky, 1963). Any allocation of codons
should be able to explain such a result.

We shall also need to know the so-called "nonsense" codons, though
my own belief is that all 64 codons will have some function or another. My
colleagues at Cambridge are studying the codon associated with the "amber"
mutants, which, you recall can in certain cells terminate the polypeptide
chain. They know that this mutant has to be read "in phase", and that it is
related, by single-step mutations, to glutamine, tryptophane and tyrosine.
When it is suppressed the amino acid inserted is serine. This and other
evidence has led Brenner and his colleagues to suspect that the anlber triplet
is UAG, and that the related nonsense triplet (now called an "ochre" mutant)
is UAA. Whether these are the usual triplets for chain termination, and
whether there are any more of them remains to be seen.

When the details of the code are fully established we shall want IO
examine its general structure. By this I mean the way in which codons are
allocated to the different amino acids, and in particular the relationship (if
any 1 between the various codous for any one amino acid (see for example,
Eck, 1963, and the review by Lanni, 196.1). Is there something in the nature
of, say; leucine which niakes it inevitable that it will have certain codousI (~1
is the present code the result of a series of historical accidents? III the formel
case it should l,e possible 10 cletluc*r the whole of the c*otlr frolll theoretical
principles. 111 the latler (`asc this would I)t* impossihlr.

r'. rk c;. CRICK                                   49

This will certainly lead us to the question of how the code originated.
It is not too difficult to see how nucleic acid synthesis could arise, but it is
almost inconceivable that the elaborate machinery for protein synthesis arose
all in one step. It must have evolved from something simpler. We do not
know whether in the primitive system there were all twenty amino acids, or
even all four bases. Perhaps only one pair of bases was present at the start.

The crucial point in all discussions about the origin of life is to see
at which point natural selection could have begun to operate. For that you
must have a replicating system of some kind, although what it was we can,
at this moment, only guess.

However, our more immediate aim should be to determine unati-
biguously the comblete code for at least one organism, say E. coli. We are
likely to have a moderately accurate, moderately complete version of the
code before long, but it is important that each triplet should be established

beyond reasonable doubt. This will open the
of which the biochemical nature of control
most important.

way to many other problems,
mechanisms is probably the

REFERENCES

Benzer, S. (1959) Proc. Nat. Acad. Sci. U.S. 45 1607.
Benzer, S. (1961) Proc. Nat. Acad. Sci. U.S. 47' 403
Crick, F. H. C. (1963) in Progress in Nucleic Aiid Rksearch Ed
son and W. E. Cohn, Vol. 1, Academic Press, Inc., New ko&. J. N. David-
Cric7g;. H.2:., Barnett, L., Brenner, S. and Watts-Tobin, R. J. (1961) Nature,
Eck, p. `V. (1663) Science, 140 477.
Lanm, F. (1963) in Advances ih Genetics, Vol. 12, Academic Press New York
Leder, P. and Nirenberg, M. W. (1964) Proc. Nat. Acad. Sci. v'.S. 52 420:
Nn-enberg, M. W., Jones, 0. W., Leder, P., Clark, B. F. C., Sly W. S'
Pestka, S. (1963) Cold Spring Harbor Symp. Q. B. Vol. 28,`~. 549: and
Nire;t;;g, M. W. and Matthael, J. H. (1961) Proc. Nat. Acad. Sci. U.S. 47,
Rich, A. (1963) in Horizons in Biochemistry, Ed. A. Kasha and B. Pullman,
Academic Press, New York.
Saraik;l,l$: S., Stretton, A. 0. W., Brenner, S. and Belle, A. (1964) Nature,
Speyer, 1. F., Lengyel, p., Basilio, C., Wahba, A. J., Gardner R S. and Ochoa,
S. (1963) Cold Sprmg Harbor Symp. Q. B. Vol. 28, p. b59'.
Tsuglta, A. (1962) J. Mol. Biol. 5, 284.
Watson, J: D. (1964) in Volume du cinquantenaire de la soci6tC de chimie
biolpglque, Maurice Declume,. Lons-le-Saunier.
Weinstein, I. B. (1963) Cold Spnng Harbor Symp. Q. B. Vol. 28 p. 579
Wlttmann, H. G. and Wittmann-Liebold, B. (1963) Cold Spring HBrbor Symp.
  Q. B. Vol. 28, p. 589.
Yanofsky, C. (1963) Cold Spring Harbor Symp. Q. B. Vol. 28, p. 581
Yanofsky, C., Carlton, B. C., Guest, J. R., Helinski, D. R. and Henning, V.
  (1964) Proc. Nat. Acad. Sci. U.S. 51. 266.


50            GENERAL NATURE OF THE GENETIC CODE
        INTRODUCTION AND DISCUSSION

Dr. Slnrzley: Ladies and gentlemen, we are ready to begin our afternoon
session with another real treat in store. Francis Crick, so far as I know, is
not particularly trigger-happy, but that word came to my mind when I was
thinking about a terminology that would describe his mind. I must say, I
have never met anybody who was so quick mentally as I've known Francis
Crick to be over the years.

I imagine this is what paid off a few years ago when Jim Watson came
to Cambridge for a little stay, and it was the interplay of these two minds
that resulted in a conception which 1 regard as being one of the truly great
ones of modern times, and that's the Watson-Crick structure, the double
helical structure, for DNA. It certainly has had a tremendous impact upon
the minds of mankind, and scientists, of course, particularly. There is an
extraordinary amount of work which has stemmed from this concept.
Dr. Crick came into this work as a physicist. Dr. Watson, who, unfor-
tunately, is not here, came in as a biologist, a microbiologist. He received
his degree with Dr. Luria. So 1 think the two were ideally equipped to deal
with the problem of the structure of DNA at that time.

Dr. Crick has ceased to become a straight x-ray man. When I was in
his laboratory in I961 for a few weeks, he was very busy pouring bacterial
plates hour after hour, sometimes until the wee hours of the morning. He
was studying acridine mutants in an attempt to establish the mode of reading
of the DNA. Nobody watching him in those days would have thought that
he had started out as a physicist, because he was an out and out biologist
at that time.

Well, I need not tell you anything more al)out Dr. Crick. You have his
I,iography here in the booklet. 1 shall uow call on I)r. Crick to give his
t~iWUsSifJl1 of lhe "(;eneral Nature of the (;enetie code":

  CThe following discussion took place after Ih-. Crick presented his
  address.]
Dr. S~nnley: Thank you very much, Dr. Crick, for this quite fascinating
exposC: on the "General Nature of the Genetic Code." I should like to make
a c~omt~~rtlt or two ahc)ut the work that 11as gcme on in the Virus 1,aboratory
with tol~acc-o mosaic virus. The active portion is, ol course, an RNA, and it
(`a11 IJc! regardrcl a~ a messrnger RNA. Tobacco iuosaic: virus, a nucleo-
protein, has hreii availal4e for years iii pure form and from this a quite
pure ac.tive RNA cau he ol~taiuetl. This viral RNA carries the message or
informatic~u for makin, o the or.otrin over'wnt c~ornpcmer~t of the virus.

  Our (,r our c)l)jrc:ti\t3. whic.11 was started some years ago. was lo obtain

F. H. C. CRICK

51

the amino acid sequence of the protein of tobacco mosaic virus. This work
was stimulated by Sanger's work on insulin. And as a result, about 1960
I think it was, we obtained the complete amino acid sequence of the 15;
amino acids of the protein component of tobacco mosaic virus.

Then Schuster in Tubingen, Germany reported that you could convert
cytosine to uracil in RNA through deamination. A great many nitrous acid
mutants of tobacco mosaic virus RNA were made in our laboratory, as well
as in the Tubingen Laboratory. This gave an opportunity, in a preliminary
way, to make some of the verifications that Dr. Crick has mentioned relating
triplets to amino acids.

We have, between the two laboratories, I think around 50 or 60 mutants,
where the exact amino acid can he pinpointed in the 158 amino acids, that
has been changed, presumably by virtue of a C to U conversion.

The difficulty with tobacco mosaic virus is that you cannot conduct
genetic experiments; you cannot do the necessary crossing experiments. This
was the reason I am particularly pleased with the recent work of Yanofsky
of Stanford, for he combined chemistry and genetics in the very beautiful
way that Dr. Crick has indicated. I had hoped that Dr. Yanofsky would be
here, but as has already been indicated, he stays in the laboratory at work
and doesn't tend to come to meetings.

Biochemists are not nearly as backward as our friend, the physicist-
turned-molecular-biologist, would indicate. I happen to know a graduate
student who, two or three years ago, had separated and purified 15 or 20
of these triplets obtained from the hydrolysis of yeast nucleic acid.

Unfortunately, things don't move quite as rapidly as sonletirucs Lltc
minds of some people move, hut I am sure that all 61. LritJlels will l)c
available eventually, and means wiII be found to test their acvtivity in amiuc,
acid incorporating systems. We already, for example, know much about the
terminal nucleotides in the tobarc-o mosaic- virus nucleic acid aud that may
provide useful iIlforlrla~icJl~.

Now, in view of the vast amount of work that Dr. Ochoa has d,,ue 0,)
the kxmetic- df=. 1 wonder if he 1V~Jdd c-are IO ruakc: :I few ~~~~~~~~~~~~~~~~ ,,,
iIS k a question.
Dr. fhero Oclmn (Speaker ), New YorX, lfrriuersily School (Jf ~lf~~/i(~i~~~~.~

lt was, as usual, a fascinating presentation by Francis Cric-k, M.hich l(,: all
have enjoyed very much. Now, as Dr. Stanley meutioned, a nurnl)er ,,r
trimdeotides are available. They have been isolated frown ribonuc,lcasr (ligc:sts
of RNA by Wendell M. Stanley, Jr..
our labtrratory.           who is a post-doctoral stucleut ill


52            GENERAL NATURE OF THE GENETIC CODE
We have not looked into the binding of aminoacyl-sRNA's by ribosomes
in the presence of nucleotide triplets very thoroughly, but as far as we went,
we had no difficulty in confirming Nirenberg's results with ApApA and
UpUpU. One does get specific binding of lysyl-sRNA and phenylalanyl-sRNA,
respectively, to the ribosomes. With ApApU we had negative results with
each of three aminoacyl-sRNA's, one of which would have been expected to
be specifically bound by ribosomes in the presence of this triplet. But, I
repeat, we have not tried very extensively.

The extent to which binding occurs is quite small. We have made a few
trials (using ApApA and UpUpU) with ribosomes from bacteria other than
E. coli but the binding was not better.

The cell-free E. coli system, referred to by Crick as purified, is really
very crude. Among other things it contains large amounts of nucleases that
cleaire the messenger RNA, whether natural or artificial. The main culprit is
an exonuclease which apparently cleaves polyribonucleotides from the 3'-end
releasing 5'-nucleoside monophosphates.

We put triplets of known sequence at the 3'-end (the right-hand side
end of the polynucleotide in the conventional way of writing these com-
pounds). The terminal base in these triplets had radioactive label. For
example, we had a poly ApApA . , . pApAC* in which C was labeled with
Cl4 or H3. When this polynucleotide was incubated with the system of
E. coli supernatant and ribosomes, the radioactivity became acid-soluble
exceedingly rapidly. This explained why we would not obtain any specific
amino acid incorporation (other than that of lysine) with such a poly-
nucleotide. We have, therefore, put some effort into getting a system
which might be reasonably free of exonuclease and of the so-called latent
ribonuclease. Albert Wahba and Wendell Stanley have to a great extent
succeeded in doing so by using a purification procedure of the E. coli
ribosomes which removes contaminating ribonuclease and exonuclease. The
purification involves treatment of the ribosomes with ammonium chloride
solutions overnight and chromatography on DEAE-cellulose columns. These
so-called "clean" ribosomes are essentially nuclease-free.
We next tried to purify the E. coli supernatant, which contains 75 per
cent of the exonuclease of the whole extract. However, to remove the nuclease
leaving behind the 20-odd amino acid activating enzymes as well as the
transfer enzymes, is not easy. We only made a few unsuccessful attempts
and rapidly dropped this approach. We had an idea that maybe organisms
other than E. coli might be free of exonuclease. The first one we tried, hcto-
bacillus w&noms, proved to tneet this specification. The II. arabinosus
supernatant proved to couple fairly well with the purified E. coli ribosomes
to give amino acid incorporation into polypeptides, at least with synthetic

F. H. C. CRICK                                  53
polynucleotides such as poly A, poly U, poly AU, and so forth. With this
system poly ApApA . . . . pApApC*, which stays intact during the incubation,
leads to the incorporation of lysine and asparagine. The ratio of asparagine
to lysine incorporated with a polymer of about 20 nucleotide residues was
in the neighborhood of 5 to 6 per cent. These results tentatively assign the
sequence AAC to one of the asparagine triplets. Experiments are in progress
to determine whether the asparagine thus incorporated into short lysine
polypeptides is C-terminal or N-terminal. This should definitely answer the
question as to the direction of translation of the message.

The combined` purified E. coli ribosomes-L.arabinosus system may be a
good one for the study of the synthesis of natural proteins with natural
messengers by cell-free systems. Since the combined system has little or
no nuclease activity the messengers should remain intact. No matter what
one does, one usually first runs into complications. Our combined "clean"
E. coli ribosomes-L.arabinosus system did not work with a natural messenger,
namely MS2 phage RNA, but neither did the usual system of (non-purified)
E. coli ribosomes + E. coli supernatant. However, as used by other people,
an extract obtained by centrifugation at 30,ooO g (the so-called E. coli S-30
extract) was fairly active with MS2 RNA. All this goes to show that a number
of difficulties will have to be overcome before one obtains systems purer
than are now available for the study of protein synthesis in vitro.

Dr. Stanley: Thank you, Dr. Ochoa. Do you want to comment now,
Dr. Crick?

Dr. Crick: I was very interested to hear about your poly A with a C
at the end, because we tried this and got very funny results. It looked as if
the message slipped.

As to your comment on UUA: let me just say there are reasons to
suspect, on genetic and mutagenic grounds, that not all the mixed U and A
triplets stand for amino acids, in spite of the result with the poly UA. We
have a strong suspicion that chain termination may occur with a triplet
containing only U's and A's. We would be very interested to know which
triplet you failed with, because that might be the one we are particularly
interested in.

Dr. Ochoa: I believe the one we tried was AAU not UUA. Your indi-
cations that one of the triplets having U and A does not stand for an amino
acid are interesting because, for a long time, we thought that in the case of
poly AIJ we had a polymer which contains hundred per cent sense, that is,
all of its triplets code for one amino acid. I was, therefore, interested to hear
what you had to say in this respect.


54             GENERAL NATURE OF THE GENETIC CODE
Dr. Crick: Bretscher, when he was in our laboratory, obtained some
(unpublished) evidence about this. He looked into the question of what
makes the polypeptide chain terminate. It is well known that if you use
poly U as a messenger, the polyphenylalanine produced is not released but
is still bound to the soluble RNA. To a large extent this is also true if you
use poly A as a messenger. Although short oligolysines are produced, they
are still bound to soluble RNA. He, therefore, used poly AU as a messenger
and obtained a release of up to 50 per cent, although it was not very
reproducible.

Dr. Stanley: Dr. Rich, I think you had a comment.
Dr. Alexander Rich (Discussion Leader), Massachusetts Institute of
Technology: At the beginning of his lecture Dr. Crick mentioned the fact
that DNA is made of four bases which form two base pairs; toward the end
of his lecture he referred to the fact that in the early history of life, it is
possible that only one base pair was found.

In fact, only one base pair is needed, because this is able to carry enough
information, to make a very complex molecule. It is like a molecular Morse
Code using, instead of dots and dashes, the two bases of the pair.
We may ask if it is possible to have a DNA-like genetic code involving
more than two base pairs. To answer that question one has to specify
several relations.

First of all, we believe that one of the reasons that these particular base
pairs are used in DNA is the fact that both pairs can fit into the double helix
in either orientation, either A and T, or vice versa, with the same geometry
and the same angles between the glycosidic bonds going to the chains.

Now, if we restrict the question to a molecule of the same geometry,
there is a possibility of using one other base pair. Only one additional base
pair would have the form which would allow it to fit into the DNA helix
and still have specificity between its hydrogen bonds. This involves a pair
of bases which are closely related to the puanine-cytosine pair. These are
the bases isoguanine and isocytosine. Whereas guanine has a carbonyl oxygen
at position 6. and an amino grnup at position 2. isoguaninc has these
reversed. Likewise, rytnsine has an amino group at position 4 and a rarbonyl
group at position 2. while these are reversed in isncytosine. These would
form a hydrogen bonded pair which could fit into the DNA helix if the
tautnmeric form of isnguanine has a proton on position N,.

So, in principle, there could be another pair that might fit into the
DNA molecule. Now, we don't know whether this can, in fact, be done.
We arc trying to tackle this problem experimentally at the present time

F. H. C. CRICK                                  55

by making essentially methylated or ethylated derivatives of isoguanine and
isocytosine. We are attempting to co-crystallize these together to see if they
will in fact form this hydrogen bonded pair. If we can do that, then we will
try to introduce them into the DNA molecule.

  Dr. Stanley: Thank you, Dr. Rich. Are there other comments? Dr.
Scheraga, we are glad to see a non-nucleic-acid chemist join us.
Dr. H. A. Scheraga (Discussion Leader), Cornell University: I would
like to ask a question about the role of binding in Nirenberg's experiments,
that is, whether binding is really the only thing that is involved?
If binding were all that is involved, this would imply that a triplet on
the S-RNA sees a triplet on the messenger, and hooks on without the amino
acid knowing anything about it. Yet you cited an experiment in which the
S-RNA took the amino acid to the site on the messenger, but it took the
wrong amino acid to the site after the amino acid was chemically modified.
This would imply that the amino acid doesn't know anything about the
binding properties.

On the other hand, mustn't the S-RNA come off the messenger; there-
fore, doesn't this imply that there may be a conformational change involved?
Dr. Crick: Of course the S-RNA must come off, but the only signal that
may be necessary for it to do so is the breaking of the link between the
amino acid and the S-RNA. Suppose we had to start from first principles.
It is obviously necessary to reject the S-RNA when you have finished with it,
but it is not at all clear how it is done. There is no reason why the triplet
has to see the amino acid to do that, though perhaps the ribosome has to
be aware of it in snme way.

  However it need not be the side chain of the amino acids which is
recognized, hut the part which is common to all amino acids. Consequently,
changing one side chain into another may make no difference. because it
has got to be a mechanism which is going to work for all amino acids.

Dr. Sfnnley: There is also an unfortunate lack of specificity in the Nirrn-
herg experiments, ton. Isn't that right ? I mean, the background is prett)
high, isn't it?

  Dr. Crick: I th' k I I1 1
               m we s mu c  re rautioue about this new technique. 141vcll
if it does work fairly well it wilt still need confirmation. I quite agree that
we need rather better discrimination. Even if you know some of the answers
in advance, and can check the method because you know the answers. you
have got to find a method of checking it without knowing the answers.

Dr. Sfanley: Which is a little difficult!


56             GENERAL NATURE OF THE GENETIC CODE
Dr. Ochoa: I think the question raised by Dr. Scheraga, on the release
of the polypeptide chain when it is finished, is a very interesting one. It
would seem that there must be an enzyme that does this job, that is, cleave
the last aminoacyl-sRNA bond when the chain is completed. Now, as Dr.
Crick mentioned, there may be at the end of the messenger, or in inter-
cistronic positions in polycistronic messengers, special release triplets. It
may be that when the polypeptidyl sRNA chain hits that spot it comes off
and now becomes accessible for the activity of the sRNA release enzyme.

In regard to the universality of the genetic code, I believe Dr. Crick
forgot one of the very dramatic experiments that bear upon this point; the
experiments of Pamela Abel in which she was able to infect B. subtilis
with vaccinia virus, so vaccinia virus multiplied inside an "unnatural" host.
This is the sort of thing one would expect from universality of the genetic
code.`If a messenger nucleic acid, viral nucleic acid let us say, can get into
a cell, any cell, it will give rise to the production of virus in a cell which
is not normally its host. The selectivity of a host is probably at the level
of the entry of the nucleic acid into the cell, and that may be why specific
viruses with specific hosts.

Dr. Stanley: I think one of the amazing things of the past few years
has been the discovery, which Dr. Ochoa just mentioned, of Pamela Abel
and Thomas Trautner on vaccinia virus growing in B. subtilis.

There are increasing evidences, as people work with viruses, that virus
nucleic acids will now grow or replicate or at least give a one-growth cycle,
in a whole variety of cells, whereas just a few years ago we hadn't the vaguest
idea that these viruses had such a potential. And now, with the isolation of
several biologically active viral nucleic acids and the preparation of proto-
plasts, you have great opportunities.

It is my own personal feeling that this whole area is just beginning to
be explored. It is really in its infancy. I think we are in for some real
surprises about nucleic acids of one kind, growing in a whole variety of
cells. And this will be very interesting, as are these experiments which have
already been referred to.

Dr. Crick: I am sorry I didn't mention Abel and Trautner, but I was
pressed for time at the end of my talk. I do agree with both of you on the
importance of it.

  I would say-going back to chain termination for the moment-that I
would be surprised if there were not an enzyme involved, although we have
discussed how it could be done without an enzyme. I think the mterestmg
question is whether there is a special soluble RNA for chain termination,
what it will be like, and how you would look for it.

F. H. C. CRICK                                 57
Dr. Gunther S. Scent (Discussion Leader), University of California:
I would just like to point out that, if Nirenberg's experiments on the ribo-
somal binding of transfer RNA by trinucleotides are correct, the interlacing
codes, of which you said that they are still possible, would be eliminated
because under such codes nucleotides no smaller than pentanucleotides would
be required for the binding reaction.

I would also like to point out that whereas the code appears to be
universal, or quasi-universal, the elements that are responsible for regulation
of enzyme synthesis are not. Experiments have been carried out in which
both structural and regulatory genes of some enzymes in one bacterial genus
were introduced into the cells of another genus, to produce a kind of partial
hybrid diploid or heterogenote. In these intergeneric heterogenotes, the regu-
latory properties of the enzymes carried by the foreign genetic element are
completely upset, showing that some specific, non-universal cytoplasmic
elements participate in these regulatory processes.

Dr. Crick: I think the question is whether any universal base sequence
is involved. We all suspect there may be proteins involved in regulation and
these might possibly have unusual features. There may be a special base
sequence which occurs at the beginning of all genes, saying where to start
reading. But in addition, there may be a special mechanism which turns on
and off that particular gene.

Dr. Stanley: Are there other comments or questions from the audience
in general that you would like to put to Dr. Crick?

Dr. F. R. Duke, Texas A & M University: Now, the only way that 1
can see that you can have these little lines drawn every three times in these
polycodons is to start at one end. And in that case it would seem that the
synthesis of these proteins would he rather slow, because you would have
to put in one amino acid, then the next and the next and the next, and I
wonder if such rate studies have been done, and whether or not they are
reasonable on the basis of this one-at-a-time mechanism?

Dr. Crick: The evidence the proteins are synthesized sequentially from
one end, one amino acid after the other is really pretty good, and shows that
it starts at the amino end.

So the next question you can ask is: Do you accumulate the amino acids
a little ahead of the growing point ? You can show quite easily this will give
you an advantage in rates. If you accumulate 10 positions ahead you can
go up to a factor of about four and a half faster. There isn't any evidence
that this happens, though I wouldn't say that the present evidence really
rules it out.


GENERAL NATURE OF THE GENETIC CODE

Given, therefore, that the protein is synthesized from the amino end one
step at a time, you can ask whether the reaction rates are reasonable. I think
that you can't make a dilemma to show that the rates are totally unreasonable,
since one doesn't know enough about the factors involved. In fact, you don't
make proteins terribly fast. What you do make terribly fast is nucleic acid.
Dr. Duke: Did you speculate that some of the triplets or codons not
necessary for fixing the amino acid sequence might be intermediate starting
points in protein synthesis?

Dr. Crick: Yes. This is one of the speculations. That is what I meant
by saying there might be a triplet for starting a polypeptide chain. That was
exactly what was implied. Whether there is such a thing, I wouldn't like to
say. I suppose I hear the thing discussed, roughly, every other day in the
laboratory over a period of six months, and I know we haven't reached a
conclusion yet. So I think everyone would agree the evidence is more sugges-
tive than conclusive, according to which way you take it, one way or the other.
Dr. Henry Eyring, University of Utah: I am afraid mine is a long
involved question with no answer. Apparently there is no direct connection
in the sense of hand-in-glove relation between the DNA carrying the genetic
code and the amino acids. Such a relation seems to be ruled out by the
things that you have told us.

The other point that seems to be true is that the genetic code is a
universal language, or at least this is probably true. This has interesting
implications. Thus, the development of the code probably came after the
development of the individual enzymes that activate the amino acids prior
to their incorporation into the proteins.

There is one other point I would like to raise. We never find anything
but the L-amino acids incorporated in the proteins of any known living
species. Now in order for the activating enzyme to distinguish between L
and D amino acids it must fit tightly against the D hydrogen of the L amino
acid. Such universality seems to bespeak an early common origin of this

selective property.

  Now. the only jvay that you car1 have the exclusive use in proteins of
/,-amino acids persisting during the entire evolutionary process is through
a dynamic stability which operates in spite of mutations. One must suppose
that the spasnmdic. mutations that occur never can produce individuals that
can successfully compete. Competition would only be possible if there were
simultaneous wholesale mutations in the organism and Its environment.

  I finally come to my point. Apparently we haven't yet reached the stage
where we can make very much out of the relative rates on the basis of how

F. H. C. CRICK                                  59

substrate and enzymes fit in selecting the amino acids. The genetic code is
apparently a completely learned language presumably developed in response
to natural selection. If so, it becomes all the more remarkable that there has
been no confusion of tongues during the whole course of evolution, and that
we should end up with a universal or nearly universal genetic code for living
things. I have said several things, I would like to be corrected at any point
where you think I'm obviously wrong.

Dr. Crick: To take the question of handedness. I think you can ask
first, why should there not be mixed L and D amino acids? The answer I
would expect is that the mechanism is such that at some stage stereochemistry
comes in, and since it's a repetitive process you want them all to be the same.
The next question you can ask is: Why are they all L amino acids?
Why couldn't they all be D? Y ou can look at protein and ask: Would it
make much difference to the way proteins fold up, if you folded up alpha
helices right or left handed? It turns out that there is not too much difference
between making a lefthanded one and a righthanded one.
However, when we look at nucleic acid the position is quite different.
YOU cannot easily make a lefthanded DNA structure using the present handed-
ness of sugar. This is because the sugar has got a number of asymmetric
carbon atoms, which are a very integral part of the backbone, so there is a
very strong handedness imposed in nucleic acid.

One has a feeling that if the choice between L and D originated by
chance, it operated on the nucleic acid rather than the protein, although I
could not defend this point if I were pressed hard.

The evolution of the code I don't understand. It would be very easy
to understand if there were a structural reason for it all. If it isn't that, if
it were a series of historical accidents, then we and the whole of nature have
probably all descended from one small population of organisms, all of which
had the same code. What isn't easy to see is the stage just hefnre that. Why
didn't the code diversify ? You can easily set up mathematical equations to
show that in a siutation like this one version of the code will get the upprr
hand. but this simplified picture is not always home nut if you look around
you. It*s not true to say that the reptiles have all disappeared hccause the
mammals are superior. Of course, if at each step there is a clear aelectivc
advantage to one choice or the other there would he no problem. Rut I find
it just a little difhcult tn believe that. that if you started with, say. half a
dozen codons, (which might he determined structurally), and expanded to
sixty, taking about fifty or so distinct steps. at each of which you added on
a new codon, that when there were two alternatives, one was selected and
the other was completely eliminated by competition.


60              GENERAL NATURE OF THE GENETIC CODE
So there are the problems, and I personally have left them a little on

one side until we know the structure of genetic code, since that may give
us a hint one way or the other.

Dr. Eyring: I have the impression that the development of secondary
structure greatly stabilized protein molecules. The irreversible destruction
of enzymes frequently involves a big activation energy. For example the
irreversible inactivation of the enzyme luciferase, important in bacterial
luminescence, occurs via a reaction having an overall activating energy. This
fantastically large inactivation energy is made up of a large heat associated
with the preliminary reversible denaturation plus an additional modest heat
of activation of the reversibly denatured molecule. The reversible denaturation
presumably involves the disappearance of some tertiary and probably some
secondary protein structure. Analogously, I wonder if the ability of nucleic
acids -to last depends on whether or not they can protect themselves by
cooperative hydrogen bonding.

yoli

Dr. Crick: Well, I think it is a protection in the present context because
tend to get enzymes developing that chop them off, but I don't know
other reason.

Dr. Eyring: But is unfolding a protection for enzymes?
Dr. Crick: Now, I wouldn't believe that chemical loss would be affected
                                                    _ __

very much by whether, let us say, DNA was single stranded or double
stranded. But you don't get a chemical breakage. All you are saying is that
there is a cooperative effect in destroying the tertiary structure.

Dr. Eyring: Excuse me. In luminescent bacteria there is an activation
energy of 91 kilocalories for destroying luciferase. Most of it is the activation
energy for destroying structure, that is, of reversible denaturation. You can
inhibit the enzyme very easily with sulfanilamide, for example, and do no
harm to it, because this inhibition is not due to reversible denaturation. But
if you inhibit luminescence with something like alcohols or ethers that make
it reversibly denatured you get an accompanying rapid destruction.

  Thus the structure that is lost in reversible denaturation is a very great
protection against subsequent reactions which irreversibly destroy the en-
zymes activity.

Dr. Crick: Well, I think I agree. The tertiary structure, of course,
stabilizes the tertiary structure, But I don't see what else you are saying.

Dr. Eyring: Oh, excuse me. Then I haven't made myself clear. The
irreversible destruction is something that is in addition to and only follows
after reversible denaturation.

F. H. C. CRICK                                   61
Dr. Crick: I agree. What's the evidence that you break the backbone?

Dr. Eyring: Whether the irreversible reactions involve something like
breaking S-S linkages or breaking the backbone I cannot say, but you can
put your light out by warming the enzyme and so reversibly denaturing it,
and during that period of reversible denaturation there is a greatly enhanced
tendency for it to break irreversibly.

So the irreversible structure has a known activation energy, a very large
one, and the overall process is something in addition to the reversible
unfolding, because the enzyme will unfold and go back again very nicely if
it is not kept too long in the reversibly inactivated state.

Dr. Crick: Let me just put you on the spot. Are you trying to say that
you can break the primary structure of a protein by unfolding it?

Dr. Eyring: No, indeed.
Dr. Crick: Then I don't see what you are saying.

Dr. Eyring: I am saying that, preliminary to what may be primary
breaking of protein structure but in any case is irreversible inactivation,
there is an unfolding which makes it vulnerable to this subsequent reaction
which destroys the enzyme.

Dr. Crick: But is there evidence that you easily break the primary
structureof course, if you digest with trypsin, it's well known.

Dr. Eyring: There is very good evidence that you can reversibly put
the enzyme out of business by something that can only be interpreted, at
least in my opinion, as a preliminary unfolding of the protein. And you can
bring about this state where it breaks very fast by warming it or with
chemical denaturants.

Dr. Crick: What breaks?

Dr. Eyring: Some chemical bond (or bonds) break essential to the
functioning of the enzyme. There is an irreversible change which follows and
is contingent upon a preliminary reversible change.

Dr. Stanley: There was a hand way back there. Would you like to ask
a question?

Mr. Ed. Peeples, l'he University of Texas: There seems to be a little
bit of a question about the physical relationships here, and relationships, say
to the ribosomes as they come off the messenger RNA, their movement, if
any movement, and also the relationship of the soluble RNA. In other words,
these things have to get to the proper place at the proper time.


GENERAL NATURE OF THE GENETIC CODE

You mentioned grouping them together in tens and so forth, but what
is the mechanism of movement here. 7 Does there need to be postulated a
movement mechanism?

Dr. Crick: The first question concerns the movement of the ribosome
along the messenger, or vice versa, since there is no important distinction.
We don't know how that happens. There are speculations that you need GTP
(which may be split in the process) and also two special enzymes to produce
the actual movement, but in fact we don't know how it happens. It is
obviously a problem we have to answer.
The second question is how the soluble RNA gets in the right place,
and this is a quite different problem. When I was a physicist I used to
worry about how the substrate got onto the enzyme, and the answer is it
just bangs around and bangs around and bangs around until it hits it in
the right way. And that is what we believe to be the answer in this case.
Whether the rates, as I was asked earlier, are reasonable, we cannot say
for sure. We know that they are not totally unreasonable.
Dr. Stanley: Well, Dr. Crick, there is evidence, is there not, on the rate
of synthesis of hemoglobin?

Dr. Crick: Yes, but you have to estimate how fast the soluble RNA will
diffuse to a ribosome and so on. Even the figures that we have on the rate
for hemoglobin synthesis are not agreed to within a factor of two. If
everything were very favorable there is no reason why synthesis shouldn't
go at the rate it does, but one can't be more precise than that.
Dr. Stanley: Are there other questions or comments?
Dr. T. R. Henderson, University of Arkansas School of Medicine: Dr.
Crick, I would like to ask you if you really think that base sequence alone
can account for many of the phenomena, such as operator regions in the
DNA and for spaces in between the gene messages? Or do you think it
would also be necessary to consider it a possibility that some of the regions
in DNA may vary from a simple double helix, that is, the type of hydrogen
bonding which I believe poly tlG and poly dC can undergo, according to
Dr. Inman? Do you think something of this type as well would have to he
considered in these mechanisms?

Dr. Crick: Let me make two points. First of all, in the last analysis.
I believe it's the base sequence that counts, in this sense: I believe the only
                                           . .
way you can copy a base sequence is by specific palrmg. Anything else.
secondary structure or methyl groups, and so on, will not affect the specificity
of that copying process.

On the other hand. WC: ran ask: Can the expression of a gene be medi-

F. H. C. CRICK

ated by something which is not strictly the base sequence, but is a result
of it? For example, by putting methyl groups on by a certain enzyme, which
puts them on only where they are certain sequences and not others, or
because of the secondary structure, in the way you have mentioned. I think
that is quite possible.

In particular there is a speculation that the base pairing might be
different if you get runs of A's and T's, and that this might be the signal
for the beginning of a gene. It is known that when you mix poly A with
poly U, you make a structure having one poly A with two poly U's, one of
which pairs by the standard base pairing and one of which pairs by a
different one.

The question you have to ask is: What do you get in a structure having
one poly A and one poly U? This is not known. It could be that in DNA
if you have a stretch of A's on one chain and U's on the other that the
structure clicks over into the other base pairing. This would expose the
normal base pairing and would be a very good way of beginning making
messenger.

Dr. Stanley: Are there other questions, or comments?

Dr. Paul D. Boyer (Discussion Leader), University of California at
Los Angeles: In connection with the last question, I wondered if the impli-
cation was that there might be linkages other than the three-five phospho-
diester that would be involved, such as the linkage in the circular phage,
+X-174. If so, might such linkages have functional genetic significance?

Dr. Crick: One always suspects there may be occasional links of that
kind which may do special things, but I think that one would expect that
these would be special. The argument in theoretical terms is this: The repli-
cation process is going to be a repetitive one, since this is very economical.
It means you can copy by the same mechanism very, very many different
sorts of genes. If you are going to do something special you have probably
got to make an enzyme to do that part of the job. Of course, there is no
reason why you shouldn't, but it is only likely to be done in special cases.
It's not likely to be general because it is acE hoc.

Dr. Boyer: I would think the implication is that the special linkage takes
a special function, like that of beginning reading or ending reading.

Dr. Crick: Yes, but there are lots of other functions. One of the things
YOU need, for example, is to keep genes quiet. The reason, I think, most things
are double-stranded is that you can't easily read messenger off them.


64               GENERAL NATURE OF THE GENETIC CODE

  Often I think these mechanisms are not so much to do something as
to prevent things happening. This falls within what you were saying, of
course, but just emphasizes a different aspect of it.

  Dr. Stanley: Most of the information in our genes presumably is being
suppressed most of the time, and we use very little of it at any one time.
Are there any more comments or questions to be put to Dr. Crick?

Dr. Alfred Mar&k, Tulane University Medical School: I wonder
whether Dr. Crick would like to comment on the possible role that DNA
might play in such structures as mitochondria and plastids.

Dr. Crick: Can I pass the topic on to Dr. Rich?

  Dr. Rich: The question is what is the DNA doing in subcellular organ-
eiles, such as chloroplasts or mitochondria?

  The facts are briefly the following: It is possible to isolate from chloro-
plasts a DNA species with a different base ratio than that found in the
nucleus of the cell. We were able to show this in our laboratory about two
or three years ago.

And more recently workers have discovered that there is also another
species of DNA in mitochondria.

Now, for many years people have believed that the chloroplast is a
self-replicating organelle, and that it had what has been called "extra
chromosomal inheritance." It is an attractive hypothesis to believe that some
of the information needed to replicate this subcellular organelle is found
in the organelle itself, in a DNA which is not integrated into the great
mass of DNA in the chromosome.

These are higher organisms, of course, and in a sense this might be
like an episome or a fragment of DNA which is not integrated into the
genome of a bacteria.

The evidence about the origin of this DNA is not very clear. I think
what has been shown clearly is that these different classes of DNA exist.
At a later stage, one would hope that people would take this DNA and try
to show that messenger RNA made from it produces some of the structural
protein of the sub-cellular organelle. At that stage one would say, that indeed,
this is the function of the extra chromosomal DNA.

  Dr. Stanley: Are there other questions?
I)r. Eyrirlg: DO you picture the appropriate folding or pairing of
chromosome-like material as a control which stops growth, and that damaging
this folding up mechanism may lead to uncontrolled growth?

F. H. C. CRICK                                 65

Dr. Stanley: The possibility of nucleic acids operating in the cytoplasm
independently of the genome is, of course, a very interesting one, having,
perhaps, implications in the cancer field. Because we now have, particularly
in work with the Rous sarcoma virus as well as with the polyoma virus,
good evidence that the viral nucleic acid replicates endlessly in cells from
which you can get no active viral nucleic acid.

  The big problem today is, what is the status of the nucleic acid in these
so-called non-producer cells? Is it integrated with the nucleic acid of the
host, or is it not? And is it there in only one copy, or is it there as several
copies? Some of the things you heard this morning, the techniques that
Dr. Ochoa has devised, I think will be useful in elucidating this particular
problem, which I think will be of extreme importance in connection with
the cancer viruses I mentioned, and quite possibly with human cancer.

If there are no further questions, then, I should like to close this session
by thanking our excellent speaker this morning, Professor Ochoa, and our
very stimulating speaker this afternoon, Dr. Crick, for their wonderful,
really brilliant presentations. I don't know when I have heard two such good
talks as I have heard today. I should like to thank the discussion leaders
for entering in, in such a very useful manner, and providing leadership in
the discussion.

Again thank you, the audience, for being with us during the day. We
hope to see you all tomorrow morning at ten o'clock.