CL`RRENT PROBLEMS IN RESEARCH

Genes and Antibodies

Do antigens bear instructions for antibody specificity
or do they select cell lines that arise by mutation?

Joshua Lederberg

An antibody is a specific globulin
which appears in the serum of an ani-
mal after the introduction of a foreign
substance, an antigen (I). Each of the
many globulins is specified by its reac-
tion with a particular antigen (2). Our
present concern is to formulate a plaus-
ible mechanism for the role of the anti-
gen in evoking large amounts of a spe-
cific complementary globulin. An impor-
tant element of any theory of antibody
formation is its interpretation of self-
recognition, the means by which an or-
ganism discriminates its own constitu-
ents from the foreign substances which
are valid stimuli of the immune re-

sponse.

Recent speculation about antibody
formation (3-8) has been dominated
by instructive theories which suppose
that the antigen conveys the instruc-
tions for the specificity of the globulin
synthesized under its governance. Elec-
tive theories date from Ehrlich (9) and
have been revived principally by Jerne
(IO), Talmage (2, II), and Burnet
(12). These postulate that the informa-
tion required to synthesize a given anti-
body is already inherent in the organism
before the antigcnic stimulus is received,
and the stimulus then functions to stim-
ulate that mechanism electively. Jerne
had proposed an elective transport of
antibody-forming templates to function-
ing sites; Talmage and Burnet have
explicitly proposed an elective function
based on cellular selection. The details
which distinguish the various proposals
are pointed out in the following dis-
cussion.

Immunology does not suffer from a
lack of experimental data, but still some
of the most elementary questions are

The author is professor of genetics at the Stan-
ford University Medical School, Stanford, Calif.
This paper was delivered as the second J. Howard
Mueller memorial lecture at Harvard Medical
School, 13 Nov. 1958.

undecided, and it is not yet possible to
choose between instructive and elective
theories. However, the latter have had
so little expression in the past few dec-
ades that a detailed exposition may serve
a useful function, if only as a target for
experimental attack. This article is an
attempt to formulate an elective theory
on the basis of genetic doctrines devel-
oped in studies of microbial populations.

Of the nine propositions given here,
only number 5 is central to the elective
theory. The first four are special postu-
lates chosen as an extreme but self-con-
sistent set; however, they might well be
subject to denial or modification with-
out impairing the validity of the elcc-
tive approach. The last four proposi-
tions are stated to account for the gen-
eral features of antibody formation in
cellular terms and may be equally ap-
plicable to instructive and elective the-
ories. If this theory can be defended,
and I know of no fatal refutation of it,
then clearly elective theories of antibody
formation perhaps less doctrinaire in de-
tail should have a place in further ex-
perimental design, each proposition be-
ing evaluated on its own merits. I am
particularly indebted to Burnet (13) for
this formulation, but But-net should not
be held responsible for some elabora-
tions on his original proposal, especially
in propositions 1 through 4. A connected
statement of the nine propositions is
given in Table 1, and each one is dis-
cussed in detail in the following sections.

Antibody Globulin

Al. The stereospecific segment of each
antibody globulin is determined by a
unique sequence of amino acids.
This assertion contradicts the more
popular notion, and the usual basis of
instructive hypotheses, of a uniform se-

quence subject to differential folding.
The chemical evidence is far from de-
cisive. For example, Karush (14) rejects
this proposition not on analytical evi-
dence but on the cogent argument that
miscellaneous antigenic compounds can
scarcely convey instructions for sequence.
But if instructive-sequence is implaus-
ible, this perhaps argues against instruc-
tion rather than differential sequence.
Karush has also demonstrated the re-
markable stability of antibody through
cycles of exposure to denaturing concen-
trations of urea. He attributes the struc-
tural continuity to stabilizing disulfide
linkages, but determinant amino acid se-
quences may also be involved.

Elective antibody formation is of
course equally compatible with sequence
or folding. In such a theory, the mecha-
nism of assembly does not have to be
specified, so long as the product (the
prospective antibody) recognizes-that
is, reacts with-the antigen. Differential
sequence is proposed (i) to stress the
ambiguity of present evidence and (ii)
as being more closely analogous to cur-
rent conceptions of genitally controlled
specificity of other proteins (15).

The direct analysis of antibody struc-
ture by physicochemical methods has
been equivocal. The fractionation of
globulins by partition chromatography
(16) might be interpreted by differen-
tial exposure of phenolic, amino, and
carboxyl groups rather than differences
in essential composition. Characteriza-
tion of amino acid composition has
given sharply different results with rab-
bit globulins, on the one hand, and
equine and human globulins, on the
other. Rabbit globulins, including vari-
ous antibodies, apparently have a uni-
form N-terminal sequence. so far identi-
fied for five residues as (17) :

Alanine-leucine-valine-aspartic-glutamyl
Various antibodies were, furthermore,
indistinguishable in over-all composition
(18). Any chemical differences would
then have to attach to a central, diffcr-
ential segment. This possibility is made
more tangible by Porter's recent finding
119) that rabbit antibody globulin could
be split by crystalline papain into three
fragments. One of these was crystalliz-
able (and presumably homogeneous),
devoid of antibody activity, but equiva-
lent as an antigen to .the intact globu-
lin. The remaining fractions were m,ore
heterogeneous and retained the antigen-
combining specificity of the intact anti-
body. AS these fractions may well corre-
spond to the differential segments, their


             Table 1. Nine propositions.
AZ. The stereospecific segment of each antibody globulin is determined by a
   unique sequence of amino acids.

A2. The cell making a given antibody has a correspondingly unique sequence of
   nucleotides in a segment of its chromosomal DNA: its "gene for globulin
   synthesis."

A3. The gcnic diversity of the precursors of antibody-forming cells arises from a
   high rate of spontaneous mutation during their lifelong proliferation.
Al. This hypermutability consists of the random assembly of the DNA of the glob-
   ulin gene during certain stages of cellular proliferation.
A5. Each cell, as it begins to mature, spontaneously produces small amounts of the
   antibody corresponding to its own genotype.
AG. The immature antibody-forming cell is hypersensitive to an antigen-antibody
   combination: it will be suppressed if it cncolmters the homologous antizen
    at this time.

A7. The mature antibody-forming cell is reactive to an antigen-antibody combi-
   nation: it ~,ill be stimulated if it first encounters the homo!ogous antigen
   at this time. The stimulation comprises the acceleration of protein synthesis
   and the cytological maturation which mark a "plasma cell."
ii??. Mature cells proliferate extensively under antizenic stimulation but are geneti-
   cally stable and therefore generate large clones genotypically preadapted
   to produce the homologous antibody.

AS. These clones tend to persist after the disappearance of the antigen, retaining
   their capacity to react promptly to its later reintroduction.

further immunological and chemical
analysis will be of extraordinary interest.

In contrast to the uniformity of rabbit
globulins, normal and antibody globulins
of horse strum proved to be grossly het-
crogencous but equally so, a wide variety
of N-terminal groups being found in all
preparations (20). This merely confirms
the concept of the plurality of antibodies
evoked by a given antigen, \vhich have
in common only the general properties
of normal gamma globulins and the
capacity of reacting with the evoking
antigen. The globulins of man, and in
particular the characteristic globulins
produced by different patients suffering
from multiple myeloma, are likewise
recognizably diflrrcnt, inter se: in amino
ac?cl composition (21).

Gene for Globulin Synthesis

AZ. The cell making a given antihod)
has a correspondingi~~ unique sequence
o/ nuclcotidcs in a segment of its chro-
mosomal 0`V.t: its "geue for globulin
synthesis."

This postulate follo\vs plausibly from
proposition Al, and would trace anti-
body-forming sprcificity to the same
source as is imputed to other specific
proteins. As the most deterministic of
genetic hypotheses, it should be the most
vulnerable to experimental test. For ex-
ample, a single diploid cell should be
capable of at most two potentialities for
antibody formation, one for each chro-
mosome.

In tests of single antibody-forming

cells from rats simultaneously immun-
ized against two Salmonella serotypes,
Xossal and I (22) could find only mono-
specific cells producing one or the other
antiflagellin. Coons (23) and White
(24) have reached a similar conclusion
in applications of fluorescent labeling
technique. However, Cohn and Lennox
(25) have convincing e\:idence for some
bispecific antibody-forming cells in rab-
bits serially immunized against two bac-
teriophages. Experiments pertinent to
the possibility of a single cell's carry-
irlg more than two antibody-forming
specificities remain to be done (26).

The chromosomal localization of anti-
body-forming specificity is uncoupled
from its elective origin in proposals
(7, 8, 27) that an antigen induces a mu-
tation in a gene for globulin synthesis,
tllough not necessarily involving a new
nucleotide sequence.

Multiple  specificity  would  stand
against a simple chromosomal basis for
antibody formation (28), leaving two
alternative possibilities:  (i) replicate
chromosomal genes or (ii) extrachro-
mosomal particles such as microsomes.
These might best be disentangled by
some technique of genetic recombination.

The differentiation of microsomes
must be implicit in a:ly current state-
ment of a theory of antibody formation
that recognizes their central role of pro-
tein synthesis. The main issue is whether
or not their specificity is dependent on
that of the chromosomal DN.4. Auton-
omy of microsomes, in contradiction to
proposition A2, is implicit in most in-
structive theories, the microsome carry-

ing either the original or a copy of the
antigenic message. On the other hand, a
powerful elective theory is generated by
substituting the term microsomal RN.1
for the terms chromosomal D:\"d and
gene in the various propositions. Since a
single cell may have millions of micl-o-
somcs, this theory \vould allow for atly
imaginable multiplicity of antibody-
forming information in a single cell. If
the potential variety of this information
approaches that of the total antibody rc-
sponsc, further instructions in an anti-
genie input Ivould become moot. In ad-
dition, the complexities of selection of
cellular populations would be com-
pounded by those of microsotnal popu-
lations lvithin each cell. These degrees
of freedom which blur the distinction
between  microsomal instruction  and
election favor the utility of the chromo-
somal hypothesis as a more accessible
target for exprrimental attack.

Genie Diversity of Precursor Cells

A3. The genie dicersity of the pre-
cursors of antibody-forming cells arises
from a high rate of spontaneous muta-
tion during their lifelong proliferation.

Three rlements of this statement
should be crnphasi7ed: (i) that anti-
body-forming cells are specialized, (ii)
that their diversity arises from some ran-
dom process, and (iii) that the diversi-
fication of these cells continues, in com-
pany \\ith their proliferation, through-
oilt the life of the animal.

Item (i) and its justification by vari-
ous experiments have already been dis-
cllsscd as an aspect of proposition AZ.
Talmage (2) also stresses the special-
ization of antibody-forming cells by re-
fcrring to their progressive diflerentia-
tion. This is entirely consistent with
propositions A3 and A4, rvhich then
postulate a specific mechanism of cellu-
lar differentiation, in this case: gene mu-
tation. If, on Talmage's model, fully
differentiated cells are ultimately left
with no more than one antibody-form-
ing specificity per chromosome? the gen-
eral consequences  will be the same
whether this final state represents the
unique activation of one among innu-
merable chromosomal loci (see 27) or
the evolution of one among innumer-
able specific alleles at a given locus.
Once again, the final resort for decision
may have to be a recombinational tech-
nique.

If the discrepancy between the experi-
ments of Nossal and Lederberg (22) and
those of Cohn and Lennox (25)) as dis-


cussed under proposition A2, is real and
depends on the timing of immunization,
it may furnish strong support for (ii),
the rxnclum origin of antibody-forming
specificity. If antibody-forming cells can
have two (or any small number of)
specificities randomly derived, only a
negligible proportion will have just the
tlvo being tested for. This would corre-
spond to tile case of simultaneous im-
munization with the two test antigens.
If. holvever, a population of cells carry-
ing one specificity is selected for, fol-
lowed by sclcction for a second speci-
ficity among all available cells, this is
the cast of serial immunization and is
prcciscly the method one would predict
to obtain a clone "heterozygous" for two
mutant alleles. Simultaneous versus se-
rial immunization \brould be analogous to
the suppression \`ersus selection of bac-
tcrial mutants resistant to two antibiotics
(29). Further experiments are needed
to escluclc more trivial reasons for the
scarcity of bispccific antiflagellin-form-
ing cells

Item (iii) diverges from Burnet's
proposal that the "randomization" of
antibody-forming cells is confined to
perinatal life, thereby generating a set
of then stable clones corresponding to
the antibody-forming potentiality of
the animal. These clones would then be
irreplaceable if lost either by random
drift or as a consequence of premature
exposure to the corresponding antigen.
The arguments against Burnet's pro-
posal are by no means decisive; how-
ever, the correspondence between cells
and antibodies is made more difficult
by having to maintain each clone at
a sufficient population size to com-
pensate for loss by random drift. Fur-
ther, the recurrence of antibody-forming
specificity is supported by experiments
showing the decay of immune tolcrancc
in the absence of the corresponding anti-
gen (30; see comment on proposition
.46). Since immune reactivity in these
experiments may return during adult
life, susceptibility to the induction and
maintenance of tolerance by the timely
introduction of the antigen may have
only a coincidental relationship to the
immunological incompetence of the new-
born animal.

Hypermutability

  A4. This hypcrmutability consists
of the random assembly of the DNA of
the "globulin gene" during certain stages
of cellular proliferation.
This ad hoc proposal is doubtless the

least defensible of the propositions, and
certainly the furthest removed from
experimental obsenration. It is stated to
illustrate that accurate rrplication rather
than mutability is the more rrmark-
able phenomenon, whatever the detailed
mechanism for the variation. If, as has
been suggested, many nucleotide trip-
lets arc nonsensical (31)) the triplets
rather than single nucleotidcs would
have to be posed as the unit of assembly
in this cast.

To carry this speculation one step fur-
ther, heterochromatin has been proposed
to be, on the one hand, a random se-
quence, and, on the other hand, a dis-
synchronously assembled segment of the
genome (32). If both views are correct,
proposition A4 might be restated: "the
globulin gent is heterochromatic during
certain stages of cellular proliferation"
(becoming by implication, euchromatic
in the mature stages of propositions A8
and A9 ) .

For the throry of microsomal election
it might be postulated that globulino-
genie microsomes are initially fabricated
as faulty replicas of the globulin gene,
but arc then capable of exact, autono-
mous replication.

Pending more exact kno\\-ledge and
agreement of opinion on the morpho-
genetic relationships of antibody-form-
ing cells, the term certain stages cannot
be improved upon. On the other hand,
as is shown under proposition A8, a
model might be constructed even on the
basis of a constant but high mutation
rate of all antibody-forming cells.

Further insight into the mechanism of
cellular diversity in antibody formation
may be won by studies on the genetic
control of reactivity to various antigens
in inbred animals (33) ; two cautions,
however, must be stated: (i) for effects
on the transport of particles of different
size, and (ii) for effects from cross-reac-
tions with gene-controlled constituents
evoking autotolerance.

Spontaneous Production of Antibody

M. Each cell, as it begins to mature,
spontaneously produces small amounts
of the antibody corresponding to its own
genotype.

Note the implication that antibody is
formed prior to the introduction of the
antigen into the antibody-forming cell.
The function of spontaneous antibody
is to mark those cells preadapted to re-
act with a given antigen, either to sup-
press these cells for the induction of im-
mune tolerance (proposition A6) or to

excite them to massive antibody forma-
tion (proposition A7). Therefore, the
antigen need participate in no type of
specific reaction with cell constituents
other than antibody itself, the one type
of reaction available to chemically di-
verse antigens that requires no further
special pleading. There is no agreement
whether the reactive globulins found in
the serum of untreated animals are pro-
duced spontaneously or by casual ex-
posure to cross-reacting antigens (see
2). Accordingly, the spontaneous anti-
body postulated in proposition A5 may
or may not be produced in the quantity
and form needed for it to be liberated
and dctcctcd in the serum. The non-
specific fragment of antibody-globulin
described by Porter raises the possibility
that the same determinant segment may
be coupled either to a diffusible or to
a cell-bound residue, the latter corre-
sponding to various aspects of cellular
immunity, including the suppression or
excitation of antibody-forming cells by
reactions lvith the corresponding antigen.

Induction of Immune Tolerance

A6. The immature antibody-forming
cell is hypersensitive to an antigen-anti-
body combination: it will be suppressed
if it encounters the homologous antigen
at this time.

This is the first of four propositions
which bear less on the source of anti-
body-forming specificity than on its sub-
sequent expression in terms of cellular
behavior. These propositions are there-
fore equally applicable to instructive
theories.

The duality of reactions of antigens
with antibody-forming cells is simply a
restatement of the experimental obser-
vations of tolerance versus immunity
(34). It seems plain that every cell of
the antibody-forming system must be
marked to inhibit its reactivity both to
the autologous antigens of the same ani-
mal and extraneous antigens introduced
and maintained from a suitably early
time of development. In the light of cur-
rent evidence for the persistence of anti-
genie molecules (5, 6) and for the loss
of tolerance t\-hen a given antigen has
dissipated (30) there are no more plaus-
ible candidates for the self-markers then
the antigens themselves. The distinction
between the function of an antigen as
inhibitor (self-marker) or as inducer of
antibody formation is then the time
when the antigen is introduced into the
potential antibody forming cell. We may
profitably define maturity in terms of


the progression of the cell from sensitiv-
ity towards reactivity.

The suppression of this process of ma-
turation is a sufficient attribute to ac-
count for tolerance, and this need not
involve so drastic an event as the de-
struction of the cell. However, the elec-
tive hypothesis proposes that only a lim-
ited number of cells will spontaneously
react with a given antigen, so that their
destruction by premature reaction can
safely be invoked as the means of their
suppression. It may be hoped that pres-
ently documented phenomena of cellu-
lar hypersensitivity may furnish a prece-
dent for cellular destruction by such
reactions. The cytotoxicity of the anti-
gen to hypersensitive cells is still contro-
versial even in the historical case of
tuberculin sensitivity (35). However, the
destruction of invading lymphocytes of
the host in the course of rejection of a
sensitizing homograft (36) supports the
speculation of some role of cellular de-
struction of immature antibody-forming
cells in the induction of tolerance.

The nature of immaturity remains
open to question. It might reflect the
morphogenetic status of the antibody-
forming cell-for example, sensitive
lymphocyte + reactive plasma cell
(37)) some particular composition of im-
mature sensitizing antibody, or merely a
very low level of antibody so that com-
plexes are formed in which antigen is
in excess.

Finally, one additional hint of an im-
plication of hypersensitivity in the early
stages of the antibody response: the
transient skin sensitivity of delayed type
(and transferable by cells) appearing in
the course of immunization, as observed
by several workers (38). If these skin
reactions reflect the destruction of some
antibody-forming cells, it would speak
for some overlapping or reversibility of
the two stages of maturation.

The implications of proposition A6 in
the elective theory may be summarized
as follows: If an antigen is introduced
prior to the maturation of any antibody-
forming cell, the hypersensitivity of such
cells, while still immature, to an antigen-
antibody reaction will eliminate specific
cell types as they arise by mutation,
thereby inducing apparent tolerance to
that antigen. After the dissipation of the
antigen, reactivity should return as soon
as one new mutant cell has arisen and
matured. As a further hopeful predic-
tion, it should be possible to induce
tolerance in clones of antibody-forming
cells from adult animals by exposing a
sufficiently small number of initials to a
given antigen.

Excitation of Massive
Antibody Formation

A7. The mature antibody-forming cell
zs reactive to an antigen-antibody com-
bination: it will be stimulated if it first
encounters the homologous antigen at
this time. The stimulation comprises an
acceleration of protein synthesis and the
cytological maturation which mark a
"plasma cell."

These principles of the cellular re-
sponse to secondary antigenic stimula-
tion are widely accepted and are readily
transposed to the primary response on
the elective hypothesis whereby some
cells have spontaneously initiated anti-
body formation according to proposi-
tion A5.

Proliferation of Mature Cells

A8. Mature cells proliferate exten-
sively under antigenic stimulation but
are genetically stable and therefore gen-
erate large clones genotypically pre-
adapted to produce the homologous
antibody.

This proposition takes explicit ac-
count of the secondary response, the
magnitude of which is a measure of the
increase in number of reactive cells
(26). However, the antigen need play
no direct part in the stabilization of anti-
body-forming genotype which might ac-
company the determinate maturation of
the cell whether or not it is stimulated.
In fact, it may be possible to dispense
with the postulate that mature cells are
less mutable by adopting a mutation rate
which is an effective compromise: to
furnish a variety of genotypes for the
primary response while selected geno-
types may still expand for the secondary
response. For example, by mutation of
one daughter chromosome per ten cell
divisions, on the average, after ten gen-
erations about 600 chromosomes of the
same type would have been produced,
together with 100 new genotypes dis-
tributed among the other 400 or so cells.
Selection must then compensate for the
mutational drift if a given clone is to
be maintained.

Persistence of Clones

A9. These clones tend to persist after
the disappearance of the antigen, retain-
ing their capacity to react promptly to
its later reintroduction.

This is a restatement of the possibly
controversial phenomenon of lifelong

immunity to viruses (4, 5). A substan-
tial reservoir of immunological memory
should be inherent from one cycle of
expansion of a given clone. Its ultimate
decay might be mitigated either by con-
tinued selection (that is, persistence of
the antigen) stabilization of genotypes.
or dormancy (to cell division or remuta-
tion, or both) on the part of a fraction
of the clone.

Discussion

Each element of the theory just pre-
sented has some precedent in biological
fact, but this is testimony of plausibil-
ity, not reality. As has already been
pointed out, the most questionable prop-
osition is A4, and it may be needlessly
fanciful to forward a too explicit hy-
pothesis of mutability for antibody for-
mation when so little is known of its
material basis anywhere.
Theories of antibody formation have.
in the past, been deeply influenced by
the physiology of inducible enzyme syn-
thesis in bacteria. In particular, instruc-
tive theories for the role of the substrate
in enzyme induction have encouraged
the same speculation about antibody for-
mation. This interpretation of enzyme
induction, however, is weakened by the
preadaptive occurrence of the enzymes.
at a lower level, in uninduced bacteria
(39).

One of the most attractive features of
the elective theory is that it proposes no
novel reactions: the only ones invoked
here are (i) mutability of DNA; (ii)
the role of DNA , presumably through
RNA, as a code for amino acid sequence
and (iii) the reaction between antibody
and antigen, already known to have
weighty consequences for cells in its
proximity. The conceptual picture of
enzyme induction would be equally sim-
plified if the enzyme itself were the
substrate-receptor. Clearly, susceptibility
to enzymic action is not a necessary con-
dition for a compound to be an inducer
-for example, neolactose and thiometh-
ylgalactoside for the fl-o-galactosidase of
Escherichia coli (39, 40), but formation
of complexes with the enzyme may be.
The picture is somewhat complicated by
the intervention of specific transport sys-
tems for bringing the substrate into the
cell (40).

Antibody formation is the one form ot
cellular differentiation which inherently
requires the utmost plasticity, a problem
for which the hypermutability of a patch
of DNA may be a specially evolved so-
lution. Other aspects of differentiation


5

may be more explicitly canalized under
genotypic control. Nucleotide substitu-
tion might still play a role here by modi-
fying the level of activity rather than
the specificity of neighboring loci, and
elective recognition of transient states
spontaneously derived then remains as a
formal, if farfetched, possibility for
other morphogenetic inductions.

3.

4.

5.

6.
7.

8.

9.

10.

Il.

12.
13.

References and Notes

This definition excludes antibody-like sub-
stances such as the hemagglutinins found in
normal human sera. These reagents do not,
however, pose the problem of the mechanism
of specific response ivhicb is the burden of this
discussion.

Talmage, in this issue of Science, discusses
various aspects of antibody specificity, includ-
ing the number of antibodies, which may be
exaggerated in current immunological thought.
For the present discussion, however, this num-
ber is left open for experimental determina-
tion, for it would embarrass a theory of cel-
lular selection only if it is large compared with
the number of potential antibody-forming cells
in the organism. To anticipate proposition Al,
ar few as five determinant amino acids would
allow for '20s = 3,200,OOO types of antibody.
L. Pauling, 1. Am. Ckem. Sot. 62, 2640
(1940).
F. M. Burnet and F. Fenner, Heredity 2, 289
(1948).
F. Haurowitz, Eiol. Revs. Cambridge Phil.
Sac: 27, 247 (1952).
D. H. Campbell, Blood 12, 589 (1957).
A. H. Coons, J. Cellular Camp. Pkyriol. 52,
Suppl. 1, 55 (1958).
R. S. Schweet and R. D. Owen, ibid. 50,
Suppl. 1, 199 (1957).
P. Ehrlich, Sfudies in Immunify (Wiley, New
York. lqlnl.

N. K. Jerne, Proc. Nafl. Acad. Sci. U.S. 41,
849 (1955).
D. W. Talmage, Ann. Rev. Med. 8, 239
(19571.
F. M Burnet, Ausfralion J. Sci. 20, 67 (1957).
I am also indebted to the Fulbright Educa-
tional Exchange Program for furnishing the

14.

15.

16.
17.

18.

19.
20.

21.

22.

23.

24.
25.

26.

27.





28.

opportunity of visiting Burnet's laboratory in
Melbourne.

F. Karush, in Serological and Biochemical
Comparisons of Profeinr, W. H. Cole, Ed.
(Rutgers Univ. Press, New Brunswick, N.J.,
1958)) chap. 3.
V. M. Ingram, Scientific American 198, No.
I,68 (1958).
R. R. Porter, Biockem. J. 59, 405 (1955).
   ibid. 46, 473 (1950) ; M. L. McFadden
and: L. Smith, J. Efiol. Ckcm. 214, 185
(1955).
E. L. Smith, M. L. McFadden, A. Stockell,
V. Buettner-Januscb, J. Biol. Ckem. 214, 197
(1955).
R. R. Porter, Nature 182, 670 (1958).
M. L. McFadden and E. L. Smith, J. Eiol.
Ckem. 216, 621 (1955).
E. L. Smith, D. hf. Brown, M. L. McFadden,
V. Buettner-Janusch, B. V. Jager, ibid. 216,
601 (1955); F. W. Putnam, Science 122, 275
(1955).
G. J. V. Nossal and J. Lederberg, Nafure 181,
1419 (1958); G. J, V. Nossal, Erif. J. Expfl.
Pafhol. 39, 54% (1958).
A. H. Coons, J. Cellular Camp. Physiol. 50,
Suppl. 1, 242 (1957).
R. G. White, Nature 182, 1383 (1958).
M. Cohn and E. S. Lennox, private corn-
munication.

An indirect measure of polyspecificity would
be the total number of antibodies multiplied
by the proportion of competent cells initially
recruited to yield a particular species. Coons
(7) has not attempted to count the antibody-
forming cells in primary response, but his
statements are compatible with an incidence
of 10-6 to lCt3 of cells forming antialbumin
in lymph nodes 4 days after inoc&ion. Nossal
(Erif. I. Exf~tl. Pathol.. in oressl found about
2 percent df yielding cells* in a primary re-
sponse after 7 days. These figurer are subject
to an unknown correction for the extent of
proliferation in the interval after inoculation.
They perhaps also raise the question whether
all tbe yielding cells are indigenous to the
lymph node, or whether circulating cells of
appropriate type can be filtered by a node
in which locally administered antigen has
accumulated.
J. Schultz, Science 129, 937 (1959). Schultz
makes an analoav between antibody formation
and serotype &termination in Pbramccium,
stressing the role of cytoplasmic feedback
mechanisms in the maintenance of specificity.
A diploid cell should be heterozygoir for .&

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.









40.

most two alleles at one locus, but strictly
speaking, this is a restriction of genotype,
not phenotype. A cell whose proximate an-
cestors had mutated through a series of dif-
ferent states might carry a phenotypic residue
of information no longer represented in its
chromosomes [see linear inheritance in trans-
duction clones: B. A. D. Stocker, J. Gsn.
Microbial. 15, 575 (1956); J. Lederberg, Gs-
netics 41, 845 (1956)]. Pending tests on clones
from single cells, bi- or polyspecificity of anti-
body-forming- phenotype remains subject to

this qualification.

V. Brvson and M. Demerec, Am. J. Med. 18,

723 (i955).

C. If. Tempelis, H. R. Wolfe, A. Mueller,
Erif. J. Expfl. Pofkol. 39, 323 (1958); R. T.
Smith and R. A. Bridges, J. Bxpfl. Med. 108,
227 (1958); P. B. Medawar and M. F. A.
Woodruff, Immunology 1, 27 (1958); G. J.
V. Nossal, Nafure 180, 1427 (1957).
F. H. C. Crick, J. S. Griffith, L. E. Orgel,
Proc. Nafl. Acod. Sci. U.S. 43, 416 (1957).
C. D. Darlington and K. Mather, Nature 149,
66 (1942); J. Schultz, Cold Spring Harbor
Symposia Quanf. Eiol. 12, 179 (1947) ; A. Ficq
and C. Pavan, Nofurc 180, 983 (1957).
J. H. Sang and W. R. Sobcy, J. Immunol. 72,
52 (1954) : M. A. Fink and V. A. Quinn, ibid.

70, 61 (1653).
hf. Cohn, Ann. N.Y. Acod. Sci. 64, 859
1,457,

,`.T"`,.

C. B. Favour, Intern. Arch. Allergy 10, 193
(1957); B. H. Waksman and M. Matoltsy, J.
Immunol. 81, 220 (1958).
J. M. Weaver, G. H. Algire, R. T. Prehn, J.
Nczfl. Cancer Inrf. 15, 1737 (1955).
J. W. Rebuck, R. W. Monte, E. A. Mona-
-ban, J. M. Riddle, Ann. N.Y. Acad. Sci. 73,
8 (1958).
L. Dienes and T. B. Mallory, Am. J. Pafkol.
8, 689 (1932) ; M. Tremaine, J. Immunol. 79,
467 (1957); J. W. Uhr, S. B. Sal&, A. M.
Pappenheimer, Jr., J. Expfl. Med. 105, 11
(1957) ; S. Raffel and J. M. Newel, ibid. 108,
823 (1958).
J. Lederberg, in Enrymer: Unifr of Biological
Sfrucfure and Function, 0. H. Gaebler, Ed.
(Academic Press, New York, l956), p, 161.
A feeble attempt in this paper to homologize
antibody formation with elective enzyme in-
duction was hindered by an uncritical rejcc-
tion of proposition Al and by the want of a
tangible cellular model such as Burnet and
Tabnage have since furnished.
J. hlonod. ibid., p. 7.


Addendum to "Genes and Anti bodies"

(SC t ENCE  129: 1649-53, 1959)

With the help of some perspective and many         A8.  That is,  if an antigen stimulates a large
discussions since this ms. was written (Nov.
1958), some obscurities in this presentation         issue from a particular cell it is not neces-
                                 sary to invoke the added element of genetic Sta-

might now be clarified.  The following comments
are also represented in discussions elsewhere
(SCIENCE, in press:  "A view of genetics"; Ciba
Foundation Symposia: Human Biochemical Genetics
(Naples, May 1959); Gel lular Aspects of lmmuni ty
(Royaumont, June 1959); Summary Comment Gatlin-
burg Symposium, J. Comp. Cell. Physiol.52, Suppl.
l:383-402, 1959).

bi I ization.  The amplification of progeny would
suffice to insure the retention and preeminence
of that clone.

Al. The dependence of the three-dimensional
shape (folding) on the amino acid sequence
should be stressed; more precisely (following
S. Brenner) the indicated sequence as the
polypeptide is formed determines the folding.
We should not overlook the possibility, how-
ever,  that DNA contains information not only
for amino acid sequence but also for fold-
ing: viz.,  through interstitial punctuation
or spacing among the (triplet?) codes for
amino acids.  The functional specificity of
the protein will, of course, finally depend
on the shape into which it has folded.

A2.  The "gene for globulin synthesis" refers
to the differential segment of the antibody
molecule, not to the common segment.  Grubb's
& gene (Ciba, Naples) is a likely candidate
for a gene for the common segment.

A2 and A3.  The discrepancy between the find-
ings of Coons, of Nossal, and of White respec-
tively,  contra those of Cohn and Lennox has not
yet been resolved.  The main difference in the
experiments seems to be the duration of immuni-
zation, which was relatively short in the former,
prolonged in the latter.  This provokes the
suggestion that bispecific cells may be sequen-
tial steps, for example, spontaneous mutations.
Prolonged immunization may also allow for ex-
change of information among cells, e.g., phag-
ocytosis with retention of the microsomes of
the eaten ccl I, not to exclude other processes
of ccl lular recombination.

A4. Undue prominence is given to this paren-
thesis on random assembly and heterochromation.
It simply illustrates one of many possible ways
to understand high mutation rates.

A5.  "Each ccl I" should be read, "Each cell of
the antibody-forming I ineage."

A6.  The importance of cytotoxicity of the
antigen in hypersensitive states is being
warmly debated, but there is no debate over
the destruction of graft cells in the homo-
graft reaction.  For this, we picture a lethal
encounter between an immune lymphocyte and an
antigenic graft ccl I.  A soluble antigen may
destroy such cells indirectly, its combination
with adherent antibody sensitizing them to
destruction by other cells.

The RNA-microsomal hypothesis should be clar-
ified.  The essence of the theory, embodied in
A5,  is the preadaptive genotypic diversity of

antibody-forming cells.   If the mutation occurs
in a typical gene,  in chromosomal DNA, this DNA
would then govern the protein via DNA-dependent
microsomal RNA.  Alternatively,  suppose autonomy
of this RNA: mutations in it would then be propa-
gated so that cells with a stable DNA gene would
include many varieties of RNA.   Rather than the
whole cell,  each of the ri bosomes would stand as
a unit of function, propagation, and selection.

  The elective role of the inducer in the syn-
thesis of D-galactosidase in E. coli has been
greatly clarified by the recent work of Pardee,
Jacob, and Monod (C. R. Acad. Sci. 246:3125-3126,
199.)   They now propose that the inducer re-
leases a preformed enzyme-forming system from
internal repression.  The de-repress ion may
depend,  in part, on competitive complex-
formation by inducers with the incipient enzyme
to faci I itate its release from its RNA template.
The same concept might apply to A7.   The intro-
duction of a homologous antigen might stimulate
an antibody-forming cell by releasing incipient
antibody from its complexes with RNA.

Neolactose is referred to in a misleading
way: this compound is a good substrate but a
poor i nducer.  The discrepancies may be even-
tually cleared up if the incipient enzyme,
bound to tt:e template, has somewhat different
specificities from the free enzyme.

Stanford University J. Lederberg June 28,195~