Genetic Transduction

*

By JOSHUA LEDERBERG
Lhiversity of Wisconsin, Madison, Wisconsin


Rep&ted from AMERICAN SCIENTIST, Vol. 14, No. 3, July 1056

GENETIC TRANSDUCTION'

  By JOSHUA LEDERBERG
University of Wisconsin, Madison, Wisconsin

T HE recombination of genes stands on a par with mutation and
selection as a cardinal element of biological variation. In the labora-

tory, recombination furnishes the experimental test of mutational
change in genetic units. In nature, it leads to their fulfillment by gen-
erating a multitude of different combinations which are then sifted by
natural selection. Until recently, genetic recombination has been closely
identified wit,h sexual reproduction: indeed geneticists consider it to be
the principal biological function of sexuality, but other processes are
now recognized as ahernatiye means to the same end. In sexual repro-
duction, the fertilization of one intact cell or gamete by another pre-
cedes t,he formation of the new zygote and assures the union of a full
complement of genes from each of two parents. In genetic transduction,
by contrast, one cell receives only a fragment of the genetic content of
another. As we shall see, the fragment call be defined not> only in genetic
but also in physical, chemical, or viroloyical terms. That is, the fragment
may be associated with subcellulnr constituents which are equally
notable as virus particles or as macromolecules of DNA (desoxyri-
bonucleic acid). So far genetic transduction is only known, with cer-
tainty, in bacteria but whether this limitation is one of fart or of tech-
nology must still be found out.

Historical Background

Scattered observations that might be attributed to transduction have
been recorded since the infancy of modern microbiology in the last
century but could not be understood or coordinated in the incomplete
genetic theory of the time. During the past twenty years, however, an
infusion of quantitative method has nourished bacterial genetics, and
this discipline has enlarged that basic concordance of all living forms
which comparative biochemistry had firmly substantiated.

In higher plants and animals, the concept of the gene originated from
recombination studies, but in bacteria it arose and for some time de-
pended exclusively on mutation studies. This concept simply states that
the hereditary quality, the intrinsic differences of organisms, can be
analyzed in terms of unit factors, markers or genes. Detailed factorial
analysis of bacterial heredity has, of course, had to wait upon the recog-

* Paper No. 606 from the Department, of Genetica, University of Wisconsin.  The
research in bacterial genetics in this department has been supported by grants
(C-2157) from the National Cancer Institute, Public Health Service, and from the
Research Committee, Graduate School, University of Wisconsin with funds allotted
by the Wisconsin Alumni Research Foundation.

264

Copyrighted 1956, by The Society of the Sigma Xi
and reprinted by permission of t,he copyright owner


                 QENETIC! TFLANBDTJCTION             265
nition of recombinational techniques, be they sexual or transductional.
Unit factors that are subject to mutation, and can be used for genetic
studies of bacteria, are related to such diverse traits as pigmentation,
resistance to antibiotics and to bacterial viruses, biochemical aspects
of nutrition and fermentation enzymes, cellular morphology, and
antigenic specificity. The most distinctive and advantageous develop-
ments in microbiological technique are selective methods for the easy
discovery and isolation of rare individuals of a specified genetic con-
stitution. For an obvious example, a streptomycin-resistant cell, or at
least a clone of its descendants, can be isolated from any number of
sensitive cells among which it may be submerged, merely by plating
the entire population into a culture medium containing the drug. Such
efficient detective techniques were first applied to the study of mutation,
but are readily adaptable to a search for recombinational processes.
The idea of transduction of hereditary fragments is still difficult to
reconcile with our well established knowledge of highly organized
chromosomal systems of heredity, but the problem can now be stated
in terms clear enough that the synthesis, if not yet certain, can at least be
experimentally sought. Earlier generations of microbiologists were,
however, untroubled by subtleties of a genetic theory that had not
yet been elaborated. For example, in 1893, San Felice 111 reported that
nonpathogenic, "pseudo-tetanus" bacilli might acquire the ability to
produce tetanus toxin by being grown in a culture medium that had been
impregnated with metabolic products of the true tetanus bacillus.
That bacteria might absorb toxins from their medium is at least plausi-
ble, but San Felice had taken pains to show that the treated bacteria
would continue to produce the toxin after many generations of growth.
A new principle of genetics would be implied by that experimental
result,, but at that time even the name "genetics" had not been coined
for a science that was still dormant. Remarkably enough, although
San Felice played an important part in the scientific classification of
the tetanus bacilli, and these toxin-forming bacteria have been in-
tensively studied for many years, there is no record of later attempts
to confirm his "transduction" experiments. It would be easy to dismiss
them as technical fallacies in the sterilization and control of a notori-
ously difllcult group of bacteria, but these BO-year-old. experiments
warrant re-examination especially in the light of recent findings on toxin
formation in diphtheria 121.
Similar notions of transduction by the passive absorption of "re-
ceptors" by different bacteria in mixed cultures or from filtrates doubt-
less underlay an extensive literature under the heading of "parag-
glutination" which dates from about 1910-1935 (reviewed, 13, 41). The
discovery that bacteria, when injected into rabbits, would evoke specific
antibodies that would agglutinate the bacteria, led to the use of this


  266                 AMERlCAN SCIENTlST
technique as a means of classifying bacterial strains by what we now
call their "serotype." At first, the reactions were believed to be absolutely
specific, but exceptions were soon found whereby antiserums against
one strain might sometimes agglutinate quite different bacteria. Such
cross-reactions are even found between certain types of human red
blood cells and certain strains of pneumococcus, and are now ascribed
to fortuitous similarities in chemical make-up; in the early 1900's, how-
ever, many workers sought to find a bioIogica1 basis for serological cross-
reactions. Perhaps the most striking cross-reaction is that between the
bacterium, Proteus 0X-19, and the rickettsia which are responsible for
typhus fever. Most authorities today believe that this reaction, which
is the basis of the famous Weil-Felix diagnostic test, is a coincidence of
chemistry of no biological significance and that the Proteus ia a harmless
saprophyte; a few still speculate on the possibility of a closer relation-
ship. One recurrent proposal has been that an indifferent Proteus ac-
quired the rickettsial antigens by residing in infected animals where
it might be exposed to the antigens or other products, i.e., by transduc-
tion, but the genetic implications of such proposals were rarely thought
through. Similar proposals were backed by rather indecisive experi-
ments to account for the cross-reactions of various enteric bacteria
(SaZnzoneZla; Shigella; Escherichia colzJ and the scores of papers that
were published on paragglutination have had very little direct influence
on the further development of bacterial genetics.
        The ~(Pneumococcus Transformafion"
  A new turn of events was marked by Grifhth's announcement 151 in
1928 (the same year as Muller's proof that X-rays caused mutations)
of the transformation of serological types in the pneumococcus. Al-
though he nowhere alluded to paragglutination or other conceptual
precedents, it is hard to doubt that Griffith was influenced by the earlier
reports. He had been working for some years on the serotypic classihca-
tion of pneumococci, which was important for therapy as well as for
diagnosis, since antiserum was almost the only specific treatment known
at that time. The most prominent antigen of virulent pneumococci
consists of a polysaccharide capsule that envelopes each cell, and is
readily detected by an apparent "Quellung" (swelling) reaction in the
presence of specific antiserum. Many serotypes can be differentiated
by the specific chemical and serological reactions of characteristic
polysaccharides. The presence of the capsules conditions not only the
high virulence of these organisms, but also the appearance of their
colonies on agar media, whence these are called "smooth" types. "Rough"
variants are also known: these are relatively avirulent and lack the     .
specific capsular substances. Some rough variants were more or less
unstable and would occasionally revert to the parental smooth form;


                 GENETIC TRANSDUCTION            267
others appeared to be absolutely stable. Griflith was impressed by the
occasional occurrence of more than one serotype in a single sputum,
and speculated on the possible interconvertibility of the types. His
experimental design was fundamentally similar to that of earlier at-
tempts at transduction: he prepared heat-killed suspensions of smooth
bacteria, and inoculated mice with a mixture of this vaccine and some
living, rough cells. Neither the killed vaccine nor the rough bacteria,
separately, would be expected to generate an active infection in the mice,
and none did in control experiments, but in a few inoculations, the
mixture of the two gave a virulent infection from which living smooth
pneumococci could be isolated. In further experiments, he also showed
that the serotype of the recovered bacteria depended on the type of
bacteria used to prepare the vaccine, rather than on the parentage of
the rough cells. He had therefore justified the claim that rough bacteria,
originally of one type, had been transformed to a different smooth type.
The mouse played the part of a selective agent in obtaining a specific
genotype, namely a new virulent form. To explain this transformation,
Grifllth did not use genetic language, but adopted what amounts to a
transduction hypothesis, namely that the substances responsible for the
formation of the specific capsules had been transferred via the vaccines
to the deficient, rough cells. Some later workers have erroneously at-
tributed the transformation to the polysaccharides themselves, but Grif-
fith had already noted theoretical and experimental arguments against
such views.
Grifiith also studied another quite different " transformation," namely
the production of rough variants by the action of antiserum on smooth
types. The serum effect has not been reinvestigated in detail, but can
hardly be a transduction, and is most likely a selective action of the
serum that permits spontaneous rough variants to manifest them-
selves.
Fortunately, Griffith's observations were saved from limbo by their
prompt trial in several other laboratories where they could be confirmed
and extended. The problem was taken up in the laboratories of the
Rockefeller Institute by a group of workers under the leadership of the
late 0. T. Avery, who had already contributed much of our knowledge
of the immunochemistry of the pneumococci. Their extensive researches
culminated in the report MI in 1943, that the active material in the
"pneumococcus transformation" was a macromolecular form of desoxy-
ribonucleic acid, DNA, a substance that had already been found to
occur characteristically in the nuclei and chromosomes of the cells of
higher organisms, and was therefore believed to be intimately con-
nected with the stuff of heredity. These workers had also developed
more precise quantitative techniques for assaying the frequency of trans-
formations, and methods by which this could be accomplished in vitro.


268                  AMERICAN SCIENTIST

Griflith had been rather lucky in his earlier experimental design. The
living mouse proved to have two functions : a selective action, as already
mentioned, and to furnish protein adjuvants from its serum that play
an obscure part in conditioning the bacteria to enable them to take up
the DNA particles.
Considerable progress has been made recently in the chemical analy-
sis of DNA, and it is known to consist of extended chains of nucleotide
units (probably arranged in a double spiral 171). Each unit contains
phosphoric acid; a five-carbon sugar (desoxyribose) ; and a purine or
pyrimidine base (adenine, guanine, thymine, or uracil). The specificity
of DNA is believed to depend on the sequence of these four alternatives;
it is not surprising that very large molecules are required to store bio-
logical information of ultimate complexity in a language with such a
simple alphabet. Actually, the true molecular size of pneumococcal
DNA is not accurately known, for such long linear macromolecules are
the most difficult to study by the physicochemical methods that have
been developed for the proteins. It is generally accepted, however, that
the material extracted from the pneumococci is a mixture of active and
inactive material, and that the active constituents would consist of
chains with hundreds or thousands of nucleotide units. The same
difficulties have stood in the way of rigorous proof that only DNA
is involved in the transforming substances, though this has been es-
tablished to the limits of current analytical methods. One of the most
challenging and exciting problems of research in molecular biology will
be the resolution of these DNA preparations into homogeneous frac-
tions with specific biological activity, for, as we shall see, this represents
the most direct approach to the chemistry of the genes ~8, 91.
These spectacular discoveries in biochemistry ran far ahead of the
genetic study of the pneumococcus transformation, which relied on the
capsule as a sole genetic marker. Until this study was broadened about
1951 with experiments on drug resistance and other markers Is' 9r, a
variety of opinions were forwarded (mostly on a purely speculative level)
on the biological interpretation of Griffith's finding. They included the
following versions of the transforming substance:
1.  It was a specific mutagen with a special ability to direct a par-
ticular gene to mutate in a definite direction.
2.  It was a polysaccharide autocatalyst (perhaps as a complex with
DNA) that primed an enzymatic reaction for polysaccharide synthesis.
3.  It was a bacterial virus, which on infecting the bacteria provoked
capsular synthesis as a host reaction.
4.  It was an autonomous cytoplasmic gene or a morphogenetic in-
ducer.
5.  It might be acting at a distance without penetrating the bac-
terium.


                GENETIC TRANSDUCTION            269

6. It was a fragment of the genetic make-up of the bacterium, the
only one to have been tested to that time.
7. It was an element sui gene& for which no general conception
should be adduced.
In a more sophisticated sense, these hypotheses are not absolutely
incompatible with one another, but they had to be sorted out from
one another as the basis for genetic experimentation. The main dis-
tinction of the sixth hypothesis, later named transduction, is that it
led to the study of additional markers. The subsequent success of this
approach is enough justification for classifying these hypotheses. The
transduction hypothesis had been ably stated by H. J. Muller in 1947
in the speculation that the DNA might correspond to "still viable parts
of chromosomes (which might) undergo a kind of crossing over with the
chromosomes of the host" ~101. But this conception was not unanimously
adopted, and other workers have been less willing to correlate the
pneumococcus transformations with other genetic systems.
During the past five years, transductional systems have been a topic
of renewed interest, engaging many workers in the comparative study
of several organisms. The first common link is perhaps the well-known
chemical fact of the distribution of DNA as a major constituent of
chromosomes in all organisms. However, so long as a single trait of a
single organism was all that was being studied there could be no apprecia-
tion of the general biological significance of the pneumococcus transfor-
mation. This impasse unfortunately obtained for twenty years after
Griflith's announcement. The much more detailed analysis that has
been developed more recently owes its vigor in part to the tacit ac-
ceptance of transduction as a working hypothesis for similar phenomena
in the pneumococcus and several other organisms.
The essential purpose of multi-factorial experiments is to measure
the bounds of the genetic unit that is being transferred. In Grifllth's
own experiments, he was, for example, obliged to admit the (far-fetched)
possibility that the apparently sterile suspensions of heat-killed bac-
teria still contained damaged but viable cells that were not detected in
control experiments, and could begin to grow only in the presence of
actively growing cells. However, if the donor' and recipient differ in
two or more markers, the isolation of recombinant clones which differ
in part from both "parents" easily rules out this kind of experimental
fallacy. (Unfortunately, this possibility is not so far-fetched in some
recent claims of transduction which have not passed this genetic test.)
Further studies with multiple marker differences, for example, of resist-
ance to penicillin, resistance to streptomycin, and capsular antigens
have shown that each marker is transmitted independently of the
others, a finding which is the principal basis for classifying the "pneumo-
coccus transformation" as an example of transduction. The idea of trans-


270                  AMERICAN SCIENTIST

duction also had to bc, and has been, supported by a demonstration
that the potency of a DNA extract depends on the genetic quality of its
source. That is to say, streptomycin-resistance can be transferred only
from a resistant donor; one can only recover in any recombination sys-
tem the elements that have been put into it. This criterion implies that
a reported effect [III of nucleic acids (among other substances) from
penicillin-resistant staphylococci in transforming the .same bacteria to
penicillin-sensitivity must be explained by some other mechanism, still
unknown, and not by transduction. This order of specificity of DNA
goes even further than the reassuring observation that DNA from unre-
lated sources, e.g., beef spleen, has no transforming activity on bacteria.
The success of Griffith and Avery has, of course, inspired many inves-
tigators to look for genetic effects of DNA with other bacteria. The
greatest success to date has been met with in the influenza bacillus
(ffemophilus in&znzae) where again a capsular antigen and streptomy-
cin resistance proved to be useful markers [IZI. This system is closely
parallel to the pneumococcus transformation, a result which is important,
in itself, in confirming the generality of the potency of DNA in transduc-
ing activity. On the other hand, many trials with other bacteria have
been definitely unsuccessful, though negative results do not, of course,
receive equal attention. However, it is never possible to be sure that
some technical detail (as the use of serum with pneumococci) will not
turn the trick. This experience suggests that the critical problem may be
to assure the penetration of undenatured DNA into the recipient
cells, the mechanism of which is still quite unknown even in the pneu-
mococcus. Unfortunately, the largest number of reports ~4, 51 of possible
transduction are still indecisive, the effects being either poorly repro-
ducible or unsupported by the solid experimental evidence t,hat has
been accumulated for the pneumococcus and Hemophilus.

           Transduction by Phage
A second mode of transduction, mediated by bacterial viruses rather
than chemically extracted DNA, was discovered independently of the
pneumococcus researches in the course of studies on Salmonella ~31.
This group of bacteria is of medical interest because of their connection
with diseases such as food-poisoning and enteric and typhoid fever.
However, they are closely related to Escherichia coli, a species which
has been shown to undergo recombination by a sexual mechanism, that
is one that involves intact cells and the exchange of large blocks of genes
114, 151. Originally, sexuality could be demonstrated in only one bacterial
strain (K-12) but after suitable methods were developed, the same proc-
ess could be found in many different strains of E. coli. It was therefore
natural to extend these researches to other related organisms, of which
Salmonella was a logical choice.


                  QENETIC TRANSDUCTION             271
The principal criterion for sexual rather than transductive recombina-
tion is the concurrent exchange of several factors, a result that was also
considered technically more reliable than the change of a single marker,
for which simple mutation must always be considered. This caution led
to many indecisive experiments: indications of exchange with single
markers could not be confirmed in multi-factorial experiments. We
eventually realized that genetic exchange was taking place in certain
combinations of Salmonella strains, but in a different pattern from cross-
ing in E. coli, as only single markers were being exchanged at each event.
This realization was promptly followed by the corollary discovery that
filtrates of certain mixed cultures could be freed of intact cells, and still
transform individual traits of a recipient strain. The term "transduction"
was introduced at this point for the hypothesis that genetic fragments
were being transmitted from one strain to another, via cell-free filtrates,
as seemed to occur in the pneumococcus too. However, Salmonella
proved to be far more amenable to genetic study than the pneumococcus,
and generalizations on this transduction could be based on studies with
thirty or forty different markers in a relatively short time. The advan-
tages of Salmonella in genetics were, however, compensated for in bio-
chemistry. At first, the most important information about the trans-
forming substance of Salmonella was that its activity was not destroyed
by desoxyribonuclease, in distinction to the pneumococcus experiments.
Filtration and sedimentation experiments then connected the activity
with particles about 0.1 micron in diameter and hence just beneath
microscopic visibility. (For comparison, an E. coli cell is about 1 micron
wide and 3 to 5 microns long.) These particles were later identified as
bacteriophage, or bacterial virus. The lack of effect of desoxyribonuclease
was then easily explained, if the true agent of transduction were pro-
tected inside the skin of the virus particle. The inner agent might very
well be DNA-but chemical studies so far cannot distinguish the
bacterial genes from the DNA nucleus of the virus itself, and we must
rely for this guess on the analogy with the pneumococcal transduction.
But where did the virus come from in these bacterial filtrates? One
of the Salmonella strains proved to be "lysogenic," that is to say is
infected by a latent virus. The latent virus, or "prophage" [MI is
ordinarily transmitted as a hereditary quality, during the multiplication
of the lysogenic cells. It would remain undetected indefinitely except
for an occasional accident through which the prophage develops into
active virus, kills its host, and is released into the medium. Once it is
freed, the virus can infect other cells. Here it may behave alternatively
as a typical lethal parasite, and grow rapidly at the expense of the host,
or re-enter its latent form, and render the bacterium lysogenic. While
empirical means have been discovered by which to influence these
decisions, the actual steps that determine (a) whether a lysogenic bac-


272                AMERICAN SCIENTIST
terium will continue to reproduce as such, or break down and release
active virus, and (b) whether a newly infecting virus will kill its host, or
combine with it to form a lysogenic symbiosis are more speculated about
than understood. We shall have to give further consideration to the
genetics of the latent virus, but we may take this occasion to recapitu-
late the results of transduction genetics so far, by analysing the stages as
follows (see Fig. 1).

DONOR    FRAGMENTS  RECIPIENT "HETEROGENOT~' SYNAPSIS

A. PHYSICAL BREAKAGE

B. COPY CHOICE, 4
BELLING,`33
(DIFFERENTIAL
  JOINING1

C. COPY CHOICE
(DIFFERENTIAL
   REPLICATION 1

:.-.
; j)
.' . . . . :: . .
;.P*)
. . . . ..*

Fig. 1. Hypothetical schemes for the incorporation of transduced fragmente. The
top line shows the preliminary stages of fragmentation and reintroduction of frag-
menta into recipient cells, common to all three
              *if ropoeals. The fate of the residual
fragments, enclosed in dotted circles, is unspea ed. From II-W

The first step is the disruption of the donor bacterium, and the forma-
tion of genetic fragments. This can be accomplished either by the
chemist, as when he dissolves the pneumococcal cells in bile salts, or by
a bacteriophage as it feeds on its host. In experimental practice, the
next step is to purify these suspensions, either by filtration or by chemi-
cal precipitation; the main purpose of this step is to remove possible
inhibitory substances and any remaining viable cells of the donor strain.
Since the transducing agent, be it DNA itself, or encased in phage par-
ticles, is far smaller than the bacterium, this is not difficult.

The second major step is the introduction of the agent inside the
bacterial cells. With DNA, it is not at all clear how this comes about
but occurs when serum-treated cells are exposed to DNA solutions. On
the other hand, viruses could not infect if there were not some special
mechanism for their penetrbw. and recent studies suggest the follow-


GENETIC TRANSDUCTION            273

ing stages: the virus attaches to the bacterial surface, usually by a pro-
tein appendage; there is then some enzymatic softening of the cell sur-
face; then the DNA nucleus of the phage (and any passengers) enters
the bacterium, leaving the phage skin outside 1171. These tricks of infec-
tion simulate the fertilization of an egg by a sperm, but whether the
viruses or the cells discovered them first is an unanswered, if not a mean-
ingless question of viral evolution.

After the agent has penetrated the bacterium, it may in due course
exchange with the homologous genes of the recipient. How this is accom-
plished touches intimately on the very mechanism of genetic reduplica-
tion. Some possible formulations are presented in Figure 1, but it must
be admitted that feasible experiments to decide among these alternatives
have not yet been carried out. It is, however, virtually certain that this
exchange step takes place promptly after penetration. In Salmonella,
for example, this can be shown by means of transductions which involve
flagellar antigens: when a new antigen has been effectively transduced,
the previous homologue never reappears in the same clone WI. If ho-
mologous replacement of genes thus takes place, we have to acknowledge
some mechanism by which the homologues associate to precede the
decision of which one will be kept. Cytologists had long since deduced a
high order of genie specificity from the way in which whole chromosomes
synapse with one another during the reduction-divisions, but it is none
the less remarkable that in the large reaches of the cell a bit of DNA can
find and recognize its opposite number as it must do to displace it.
The datum' of homologous replacement is also a strong indication that
the genetic system of the recipient bacterium is organized (viz. into one
or more chromosomes). If each one of the very many kinds of trans-
ducible fragment corresponded to an independent chromosomelet, it
would be hard to see how an orderly process of homologous replacement
could be managed, not to mention the problem of keeping each p and q
in order during ordinary cell division. This intuition has, however, been
substantiated by more explicit evidence, as we shall see. The dilemma of
whether to postulate a disintegrated genotype in the whole life cycle of
the bacterium, or an unknown mechanism by which a fragment could
exchange with an integrated chromosome, was possibly the chief obstacle
to bridging transductional genetics to the general theory of the science
(for example, cf. 1191). The generality of homologous replacement in
transduction experiments has given a definite direction to further
research in this field in terms of integrated genie systems, i.e., chromo-
somes.

In the Salwwnella system, the genetic fragment has accompanied a
bacteriophage, but in the transformed cell they appear to act independ-
ently 1201. A fraction of the bacteria are killed-an obvious condition of
a workable sy$em is the survival of a reasonable proportion of the in-


274                  AMERICAN SCIENTIST

fected cells, either by the formation of lysogenic complexes, or by the
suppression of the phage. One means of separating transduction from
active virus infection is to treat the phage suspensions with ultraviolet
light. This inactivates viral infectivity much more rapidly than the ge-
netic action, so that many particles are formed that will transduce their
contained fragments without transmitting viable virus. The same result
is achieved by the use of bacterial strains to which the virus is poorly
adapted. The ultimate fate of the viral residues is unknown, as is that of
the leftovers shown in Figure 1.

The conclusion may be simply stated that the pneumococcus and
Salmonella both manifest types of genetic transduction. In the latter,
bacteriophage acts as passive carrier of the genetic (DNA?) fragments,
but the viral nucleus has no other demonstrable relationship with its
host companions. In both cases, any genetic marker that could be tested
for was amenable to transduction.

Genetics of Prophage

The role of prior work on E. coli, for the development of information
on Salmonella has already been outlined. While a sexual system (viz.
recombinat,ion between intact cells of whole blocks of genes) has not yet
been found in Salmonella, it was an obvious and early question whether
transduction could be demonstrated in E. coli. This would be of particu-
lar use and interest just because of the genetic analysis that had been
and could be done by sexual crosses, which had, for example, given some
insight into the genetic nature of latent viruses. The latency of the virus
in a lysogenic bacterium is very well exemplified by strain K-12 of E.
coli, which had hcen used as a classroom type strain for twenty years,
and an object of intensive genetic research for another five before this
was appreciated. The virus, which was named lambda, is latent both in
the sense of concealment, and potentiality for development. It was
actually detected only when a mutant strain was discovered that was
sensitive to lambda, and therefore could serve as an indicator for the
free virus. The free virus, as already intimated, is released by about one
cell per million in an ordinary culture; as Lwoff has shown, this release
can be enormously accelerated by treating the bacteria with ultraviolet
light. To account for the genetic quality of lysogenicity, many authors
have long since tacitly assumed that the phage had somehow combined
with the genetic make-up of the cell. This assumption was made even
more necessary when it was learned that lysogenic bacteria, even when
artifically opened, did not contain typical, infective virus particles.
The term "prophage" has thus been given to the latent developmental
phase of the bacterial virus, to the element of genetic continuity which
distinguishes a lysogenic bacterium WI.

Before the development of crossing methods, the genetic localization


                  QENETIC TRANSDUCTION            275
of the prophage could only be speculated about. On the analogy of the
hereditary "kappa" particles in Paramecium, it was thought possible
that the symbiotic virus might also live in the cytoplasm (whence the
analogous symbol "lambda"). However, when lysogenic (lambda-
carrying) bacteria were crossed with nonlysogenic strains, a regular
segregation for this trait was found, which ruled out the cytoplasmic
hypothesis. It was then found that the lambda-prophage was closely
linked to one of the genetic markers of the bacterium, concerned with
galactose-fermentation. Finally, hybrids were produced which segre-
gated lysogenicity and galactose-fermentation as a pair of linked mark-
ers. One can even refer to the prophage as a dominant gene, since the
hybrid is lysogenic, and like typical lysogenic bacteria is immune to
the attack of the free virus, although it segregates both lysogenic and
virus-sensitive, nonlysogenic offspring. It was concluded that the ge-
netics of the prophage could not be distinguished from that of other ele-
ments of the bacterial genotype; the main distinction is that a bacterium
which carries the prophage is liable to a sporadic transformation which
results in the death of the bacterium and the appearance of infective
virus. This result could be most readily understood on a dualistic version
of the prophage: in the lysogenic bacterium, it is a part of the bacterial
chromosomes; in the infective phage particle, it is the nucleus of an au-
tonomous parasite, the virus WI.
In the absence of similar crossing studies there is no certainty that
other phages in E. coli or other organisms have a similar duality-
whether they have a predetermined site in the bacterial chromosome or
any at all-it would be premature to generalize from a single instance,
but this kind of question is part of the catechism of any virus. As soon
as the genetic functions of Salmonella bacteriophage were appreciated,
the possible transducing ability of lambda in E. coli was examined, b\lt
at first with purely negative results on a number of tested markers.
Later, M. L. Morse looked more closely at the matter, and it was dis-
covered that lambda could indeed transduce, but only a single grol~p
of genetic markers, namely the galactose-factors to which the prophage
itself was linked 12.21. This specific relationship is quite different from
the transductions previously analyzed, in which any marker was trans-
ducible and it speaks again for the proximity of the prophage to the
galactose-genes. Again, only about one phage particle per million is
effeclive, that is, will transform a galactose-negative recipient bacterium
to the gnlactose-positive form, when the phage has been generated from
galactose-positive bacterial hosts.
It ws mentioned earlier that in the other examples of transduction,
the newly transduced fragment promptly underwent exchange with the
homologous elements of the recipient. The lambda system is different in
a useful and interesting way, for it can be shown that here the fragment


276                  AMERICAN SCIENTIST
does not quickly exchange, but may maintain its integrity for a long time
and reproduce in parallel with the reproduction of the cell (see the
" heterogenote" stage, Fig. 1).

           Linkage in transduction
The further study of the fragment depends on linkage in transduction,
which has not been mentioned so far. While the chief distinction of
transduction is the transfer of a single marker, our discussion has repre-
sented this as a "fragment" rather than a "single gene." This caution
follows from the fact that a bacterium must have thousands of genetic
factors, only a fraction of which can have been detected by their mutant
forms. It is therefore premature to label the unit of transduction of
known mutants as an absolute "single gene," particularly in view of
theoretical controversy as to the operational meaning of this concept
~9, 231. It would be more correct to state that transduction involves a
unit small enough that in most cases it has been experimentally marked
by only one factor. This reservation has been borne out by a few
instances, both in Salmonella and in the pneumococcus, of linked trans-
duction where the investigators have evidently been lucky enough to
find markers so closely associated that they can be transmitted on the
same fragment. More recently, still another transduction has been
described in E. coli, by another phage, in which it has been possible to
correlate linkage in transduction with linkage in sexual crossing far].
Evidently, problems such as are raised by the exchange of a fragment
with the chromosome are better studied with groups of linked markers.
For this purpose, we have unfortunately been unable to find any genes
other than the galactose-markers which are closely linked to lambda-
prophage. However, these have proved to be genetically distinguishable
but linked to one another and therefore serve the purpose. The diversity
of the galactose genes was first revealed by crossing experiments. When
galactose-negative mutants that had been obtained in different strains
were crossed with one another, about one galactose-positive recombinant
per thousand was found. It was therefore necessary to distinguish the
mutants by different numbers: Gab; Gal*; Gal*; and so forth, to show how
the same character may be affected by different genes. The result of the
test cross would then be symbolized as: Gall-Galz+ X Gall+ Gal*-,
which can give some galactose-positive, Gall+ Galz+, recombinants as
an application of a standard procedure for determining the identity
or difference of mutant genes.
The same distinctions were revealed by transduction experiments.
That is, phage grown on the Gall- mutant would produce galactose-
positives from GaIz- cells. This may be symbolized Gall- Gal*+ -X
Gall+ GaL- to give Gall+ Gab+, and amounts to the transduction of the
Gal,+ gene. However, the galactose-positive clones so obtained were


                 GENETIC TRANSDUCTION             277
unstable for this character and would throw off occasional galactose-
negative cells in the course of further growth. When these were typed,
either by transduction or by crossing tests, they fell into three classes:
the parentals, Gall+ GaL- and Gall- Galz+, and a new type Gall-
Galz-. The formation of galactose-negatives is therefore a segregation
process, and by adding up the markers we infer that, the transduced frag-
ment, Gall- Galz+ has remained intact in the same cell as the
recipient chromosome 1251. When the fragment is lost or exchanges
with the chromosome, the various negative types are generated. The
Gall- GaL- combination is of particular importance in relating the
exchange of fragment and chromosome to crossing-over as illustrated
in Figure 1. The same sequence may be postulated for other transduc-
tion systems in which the pace of events is too fast for their separation.
        Virus as gene; Lysogenic conversion
As in the Salmonella system, only a small proportion (about one per
million) of the lambda particles carry Gal genes with them. However,
when the lambda is obtained from bacteria which are already carrying
supernumerary fragments, from previous transduction, these fragments
are incorporated into virtually all of the phage particles. Since the frag-
ments all trace their ancestry directly to the contents of an earlier infect-
ing phage particle, this is tantamount to equating the fragment itself
with the prophage. The analogy between the two elements is at least
striking enough to justify classifying the virus infection as a special
case of transduction, i.e., of that genetic element of the donor bacterium
which we have called the prophage. In fact, experiments have recently
been reported on transduction, by other viruses, of lambda prophage as
a marker linked to Gal 124, 261.
The equivalence of virus and gene is here a formality of the means of
hereditary t,ransmission. The prophage-gene may be related more or
less closely to the rest of the genetic system, but in the cases so far can
be readily separated, e.g., by crossing-over or by a cycle of parasitic
growth of the virus. In other situations, however, a prophage may act
as a gene of the host bacterium not only in a formal but in a functional
sense. For example, certain Salmonella strains may be classified into the
serotypes El and Ez. Phage grown on Ez cells will convert El into Ez.
This conversion is, however, inseparable from infection by the phage,
which is both a necessary and sufficient condition for the conversion
~71. The same phage may casually and sporadically transduce other
markers, but the Ee serotype is invariably associated with the phage
and vice versa ~281. The prophage may therefore be considered as the
gene for Ez. This lysogenic conversion is an example of transduction in
the sense that it is t,ransduction of the prophage, rather than of other
genes by the phage. An equally striking example of lysogenic conversion


278                  AMERICAN SCIENTIST

has been described in the diphtheria ba,cterium, where phage in filtrates
of toxin-forming strains will convert nontoxin-forming cells so that
they will also form the toxin 121. If San Felice's observations UI are
vindicated and the same pattern holds for tetanus toxin, our excursion
among viruses and genes will have turned full circle both in concept and
in experiment.

          Applications and Prospects
The study of transduction in bacteria has been the labor of many
scientists of whom only a few are listed in the references here. But the
story has only well begun. Of the thousands of bacteria species, only a
few have been examined at all, and each study has revealed a new facet.
So far, only a minority of attempts to demonstrate recombination
mechanisms (sexual or transductive) in bacteria have been successful
It is a fair caution that the first essential is a selective technique, a
workable means of detecting new types even when they are extremely
rare. Partly for this reason, and partly because the experimental organ-
isms have usually been economically unimportant, recombinational
techniques have had more analytic than pract,ical utilit#y. (Artificial
serotypes in Salmonella have been of some use in the preparation of
diagnostic serums.) The vast applications of genetics in practical agri-
culture are, however, a sufficient portent of what may be accomplished
in due course with the microbes that are important in medicine and
industry.
Some of the hopes that have been expressed, however, are too extrav-
agant in the light of present knowledge, for example for the massive
transformation of virulent or drug-resistant bacteria in an infected host
to more innocuous forms. To be therapeutically effective, such trans-
formations would have to involve virtually every bacterium, which is
too much to ask of a recombination process (barring the exceptional
examples of lysogenic conversions). A similar misconception has pro-
voked the suggestion [291 that transduction could account for the spread
of drug-resistance from one mutant cell to a large population (a result
that needs no elaborate explanation other than selection). In every case
so far, genetic transduction is achieved at the expense of the life of the
donor cell: in the most favorable cases, the DNA or the lambda from
one or a few bacteria has been enough to transform a single recipient,
which speaks for the recovery of much of the original genetic material.
So far, no definite case of transduction has been reported for higher
organisms. Claims that DNA from tumor cells would induce tumors in
normal mouse tissues are controversial 130, 311 but they do illustrate the
impact of transduction on experimental cancer research. The concept
may also have some bearing on mysterious changes in tumor cells that
are transplanted to new hosts. And speculations correlating the pneu-


                GENETIC TRANSDUCTION            279
mococcus transformation to embryonic inductions perhaps have to be
inverted and reviewed with the genetic understanding of the former.
However, before a convincing search for transduction in higher organisms
can be executed, efficient selective methods will have to be developed
as they have been for bacteria.

The question of whether transduction is unique to the bacteria, or
occurs more generally, is important for its bearing on general genetic
theory. It has been suggested that the postulated "chromosomes" of
bacteria and viruses are chemically and structurally less elaborate than
the cytogenetically verified chromosomes of higher plants and animals.
However, the generalization of concepts and techniques learned from
these organisms has been the most productive approach to the analysis
of transduction in bacteria. Conversely, transduction has pointed up
the weaknesses of some traditional formulations of chromosome behav-
ior. In crossing-over, for example, can we believe that two chromosomes
will regularly break at precisely corresponding points? The impending
translation of genie differences as chemical (or grosser structural)
differences in DNA has also provoked a reexamination of the concept of
the "single gene" WI. We are reminded again of the first principle of
genetics, that we cannot recognize genes directly but only their differ-
ences. In turn, we should not insist on genes as self-reproducing units,
but as units or markers of a more complex self-reproducing system.
Nevertheless, the representation of genie differences in chemically puri-
fiable DNA is the closest approach to the reduction of genetics to bio-
chemistry, an enterprise which can challenge the skills and imaginations
of specialists in a dozen sciences.

1.

2.
::
::

7.

8.

9.

10,
11.

12.

13.

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