A VIEW OF GENETICS.
By
JOSHUA LEDERBERG.
Nobel Lecture, May 29, 1959.
The Nobel Statutes of 1900 charge each prize-winner to give a public
lecture in Stockholm within six months of Commemoration Day. That I
have fully used this margin is not altogether ingenious, since it furnishes
a pleasant occasion to revisit my many friends and colleagues in yourbeautiful city during its best season.
The charge might call for a historical account of past "studies on
genetic recombination and organization of the genetic material inbacteria", studies in which I have enjoyed the companionship of manycolleagues, above all my wife. However, this subject has been reviewed
regularly (36, 37, 38, 41, 42, 45, 49, 54, 55, 58) and I hope you will sharemy own inclination to assume a more speculative task, to look at thecontext of contemporary science in which bacterial genetics can bebetter understood, and to scrutinize the future prospects ofexperimental genetics.
The dispersion of a Nobel award in the field of genetics symbolizes theconvergent efforts of a world-wide community of investigators. That
genetics should now be recognized is also timely — for its axial role inthe conceptual structure of biology, and for its ripening yield for thetheory and practice of medicine. However, experimental genetics isreaching its full powers in coalescence with biochemistry: in principle,
each phenotype should eventually be denoted as an exact sequence ofamino acids in protein (79) and the genotype as a corresponding
sequence of nucleotides in DNA (a, 63). The precise demarcation ofgenetics from biochemistry is already futile: but when genetics has been
fully reduced to its molecular foundations, it may continue to serve inthe same relation as thermodynamics to mechanics (69). The coordination of so many adjacent sciences will be a cogent challenge to theintellectual powers of our successors.

a. No reader who recognizes deoxyribonucleic acid will need to be reminded what
DNA stands for.

Fig. I. Primary structure of DNA — a segment of a polynuclotide sequence CGGT.
From (13).

That bacteria and their genetics should now be so relevant to general
biology is already a fresh cycle in our scientific outlook. When thoughtof at all, they have often been relegated to some obscure byway of
evolution, their complexity and their homology with other organisms
grossly underrated. "Since Pasteur's startling discoveries of theimportant role played by microbes in human affairs, microbiology as ascience has always suffered from its eminent practical applications. By
far the majority of the microbiological studies were undertaken to
answer questions connected with the well-being of mankind" (30). The
pedagogic cleavage of academic biology from medical education has
helped sustain this distortion. Happily, the repatriation of bacteria andviruses is only the first measure of the repayment of medicine's debt tobiology (6, 7, 8).
Comparative biochemistry has consummated the unification of biology
revitalized by Darwin one hundred years ago. Throughout the living
world we see a common set of structural units — amino acids,
coenzymes, nucleins, carbohydrates and so forth — from which everyorganism builds itself. The same holds for the fundamental process ofbiosynthesis and of energy metabolism. The exceptions to this rule thus
commandspecialinterestasmeaningful

tokensofbiologicalindividuality, e. g., the replacement of cytosine by hydroxymethyl
cytosine in the DNA of T2 phage (12).
Nutrition has been a special triumph. Bacteria which required novitamins had seemed simpler than man. But deeper insights (32, 61)interpret nutritional simplicity as a greater power of synthesis. The
requirements of more exacting organisms comprise just thosemetabolites they can not synthesize with their own enzymatic
machinery.
Species differ in their nutrition: i f species are delimited by theirgenes, then genes must control the biosynthetic steps which arereflected in nutritional patterns. This syllogism, so evident once told,
has been amplified by BEADLE and TATUM from this podium. Its
implications for experimental biology and medicine are well-known:among these, the methodology of bacterial genetics. TATUM has related
how his early experience with bacterial nutrition reinforced thefoundations of the biochemical genetics of Neurospora. Then, disregarding the common knowledge that bacteria were too simple to havegenes. TATUM took courage to look for the genes that would indeed
control bacterial nutrition. This conjunction marked the start of my own
happy association with him, and with the fascinating challenges ofbacterial genetics.
Contemporary genetic research is predicated on the role of DNA as thegenetic material, of enzym3 proteins as the cell's working tools, and of
RNA as the communication channel between them (63). Three lines ofevidence substantiate the genetic function of DNA. Two are related tobacterial genetics; the third and most general is the cytochemical
observation of DNA in the chromosomes, which are undeniably stringsof genes. But chromosomes also contain other constituents besides
DNA: we want a technique to isolate a chromosome or a fragment ofone, to analyse it and to retransplant it to verify its functional capacity.
The impressive achievements of nuclear transplantation (29) should
encourage the audacity needed to try such experiments. The
constructive equivalent to chromosome transplantation was discoveredby a bacteriologist thirty years ago (20) but the genetic implications ofthe "pneu-mococcus transformation" in the minds of some of GRIFFITH'S
successors were clouded by its involvement with the gummy outercapsule of the bacteria. However, by 1943, AVERY and his colleagues had
shown that this inherited trait was transmitted from one pneumococcal
strain to another by DNA. The general transmission of other traits by
the same mechanism (25) can only mean that DNA comprises the genes(b).

b. One might be tempted to write: "One DNA molecule = one gene." However, thequanta of factorial genetics, based on mutation, recombination and enzymaticfunction are all smaller than the DNA unit of molecular weight ~ 6 x 106 (4).
There is increasing evidence that such a molecule is a natural unit rather than an
artefact of fragmentation (64).

To reinforce this conclusion, HERSHEY and CHASE (23) proved that thegenetic element of a bacterial virus is also DNA. Infection of a host cellrequires the injection of just the DNA content of the adsorbed particle.
This DNA controls not only its own replication in the production of newphage but also the specificity of the protein coat, which governs theserological and host range specificity of the intact phage.
At least in some small viruses, RNA also displays genetic functions.
However, the hereditary autonomy of gene-initiated RNA of thecytoplasm is now very doubtful — at least some of the plasmagenes thathave been proposed as fulfilling this function are now better understoodas feedback-regulated systems of substrate-transport (65, 72, 81).
The work of the past decade thus strongly supports the simple
doctrine that genetic information is nucleic, i. e., is coded in a linear
sequence of nucleotides. This simplification of life may appear too facile,and has furnished a tempting target for agnostic criticism (37, 41, 44,74). But, while no scientific theory would decry continual refinement
and amplification, such criticism has little value if it detracts from theevident fruitfulness of the doctrine in experimental design.
The cell may, of course, carry information other than nucleic either inthe cytoplasm or, accessory to the polynucleotide sequence, in thechromosomes. Epinucleic information has been invoked, without being
more

precisely

defined,in

many

recentspeculationsoncytodifferentiation and on such models of this as antigenic phasevariation in Salmonella (71, 52, 56, 47). Alternative schemes have somuch less information capacity than the nucleic cycle that they are more
likely to concern the regulation of genie functions than to mimic their
specificities.
D N A as a SubstanceThe chemistry of DNA deserves to be exposed by apter craftsmen (86,31, 13) and I shall merely recapitulate before addressing its biologicalimplications. A segment of DNA is illustrated in Fig. 1. This shows alinear polymer whose backbone contains the repeating unit:││[— O — P (OO-) — O — CH2 — CH — CH —] O — P(OO-) — O — CH2 —[diester phosphate

C´5

C´4

C´3]

diester phosphate . . . .

The carbon atoms are conventionally numbered according to theirposition in the furanose ring of deoxyribose, which is coupled as an
N-glycoside to one of the nucleins: adenine, guanine, cytosine orthymine, symbolized A, G, C or T, the now well-known alphabet inwhich genetic instructions are composed. With a chain length of about
10 000 residues, one molecule of DNA contains 20 000 "bits of
information", comparable to the text of this article, or in a page ofnewsprint.
Pyrophosphate-activated monomer units (e. g. thymidine
triphosphate) have been identified as the metabolic precursors
of DNA (31). For genetic replication, the monomer units must be
assembled in a sequence that reflects that of the parent
molecule. A plausible mechanism has been forwarded by W ATSON

Fig. 2. The scheme of Watson and Crick for DNA replication. "Unwinding andreplication proceed pari passu. All three arms of the Y rotate as indicated." (14).

and CRICK (87) as a corollary to their structural model whereby DNA
occurs as a two-stranded helix with the bases being centrally oriented.
When their relative positions are fixed by the deoxyribose-phosphatebackbones, just two pairs of bases are able to form hydrogen bonds
between their respective NH and CO groups; these are A: T and G: C.
This pairing of bases would tie the two strands together for the length of
the helix. In conformity with this model, extensive analytical evidenceshows a remarkable equality of A with T and of G with C in DNA fromvarious sources. The two strands of any DNA are then mutuallycomplementary, the A, T, G and C of one strand being represented by T,
A, C and G, respectively, of the other. The information of one strand istherefore equivalent to, because fully determined by, the other. The
determination occurs at the replication of one parent strand by thecontrolled stepwise accretion of monomers to form a complementarystrand. At each step only the monomer which is complementary to thetemplate would fit for a chain-lengthening esterification with theadjacent nucleotide. The model requires the unravelling of theintertwined helices to allow each of them to serve as a template. Thismight, however, occur gradually, with the growth of the daughter chain— a concept embedded in Fig. 2 which symbolizes the new Cabala. The
discovery of a single-stranded configuration of DNA (85) makes
complete unravelling more tenable as an alternative model.
For the vehicle of life's continuity, DNA may seem a remarkably undistinguished molecule. Its overall shape is controlled by the uniform
deoxyribose phosphate backbone whose monotony then gives X-ray
diffraction patterns of high crystallinity. The nucleins themselves are
relatively unreactive, hardly different from one to the other, and in DNA
introverted and mutually saturated. Nor are any of the hydroxyls of
deoxyribose left unsubstituted in the polymer. The structure of DNA
befits the solipsism of its function.
The most plausible function of DNA is ultimately to specify the aminoacid sequence in proteins. However, as there are twenty amino acids tochoose among, there cannot be a one: one correspondence of nucleotide
to

amino

acid.

Takingaccountofthecode-duplication

incomplementary structures and the need to indicate spacing of the words
in the code sequence, from three to four nucleins may be needed to spell
one amino acid (19).
While a protein is also defined by the sequence of its monomeric units,
the amino acids, the protein molecule lacks the "aperiodic crystallinity"(80) of DNA. The differentiæ of the amino acids vary widely in size,
shape and ionic charge (e. g., H •; CH3 •; H2N • CH2 • CH2 • CH2 • CH2 •;COOH • CH2 • CH2 • ; HO • C6H4 • CH2 • ) and in the case of proline,
bond angles. The biological action of a protein is, therefore, attributable
to the shape of the critical surface into which the polypeptide chain folds

(73). The one-dimensional specificity of the DNA must therefore betranslated into the three-dimensional specificity of an enzyme or
antibody surface. The simplest assumption would be that the aminoacid sequence of the extended polypeptide, as it is released from theprotein-building template in the cytoplasm, fully determines the folding
pattern of the complete protein, which may, of course, be stabilized by
non-peptide linkages. If not we should have to interpose some accessory
mechanism to govern the folding of the protein. This issue has reached a
climax in speculations about the mechanism of antibody formation. If
antibody globulins have a common sequence on which specificity issuperimposed by directed folding, an antigen could directly mold thecorresponding antibody. However, if sequence determines folding, itshould in turn obey nucleic information. As this should be independentof antigenic instruction, we may look instead to a purely selective role ofantigens to choose among nucleic alternatives which arise by
spontaneous mutation (8, 50).
The correspondence between amino acids and clusters of nucleotides
has no evident basis in their inherent chemical makeup and it now
appears more probable that this code has evolved secondarily andarbitrarily to be translated by some biological intermediary. The coding
relationship would then be analogous to, say, Morse-English (binary
linear) to Chinese (pictographic). Encouragingly, several workers havereported the enzymatic reaction of amino acids with RNA fragments(22, 75). Apparently each amino acid has a different RNA receptor andan enzyme whose twofold specificity thus obviates any direct
recognition of amino acid by polynucleotides. The alignment ofamino-acyl residues for protein synthesis could then follow controlledassembly of their nucleotidates on an RNA template, by analogy withthe model for DNA replication. We then visualize the following modes of
information transfer:(1) DNA replication — assembly of complementary deoxyribonucleotides on a DNA template.
(2) Transfer to RNA by some comparable mechanism of assembling
ribonucleotides.

Our

understandingof

uncertainties of the structure of RNA (16).

this

is

limitedby(3) Protein synthesis:(a) Aminoacylation of polynucleotide fragments;(b) Assembly of the nucleotidates on an RNA template by analogy
with step (1);(c) Peptide condensation of the amino acid residues.
Some workers have suggested that RNA is replicated in step (3)concurrently with protein synthesis, in addition to its initiation fromDNA.
The chief difference in primary structure between DNA and RNA is thehydroxylation of C´2 in the ribose, so that a reactive sugar hydroxyl isavailable in RNA. This may prove to be important in the less orderedsecondary structure of RNA, and in its function as an intermediary toprotein. It remains to be determined whether the aminoacyl
nucleotidates are esterified at C´2 or at C´3 which is also available in theterminal residue. From this resume we may observe that the DNA
backbone constitutes an inert but rigid framework on which thedifferential nucleins are strung. Their spatial constraint lends specificity
to the pattern of hydrogen bonding exposed at each level. This extended
pattern is a plausible basis for replication; it is difficult to visualize any
reagents besides other nucleotides to which this pattern would be
relevant. These conditions are quite apt for a memory device — rubber
and guncotton are poor choices for a computing tape.
D N A and Bacterial Mutation
The ignis fatuus of genetics has been the specific mutagen, the reagent
that would penetrate to a given gene, recognize and modify it in a
specific way. Directed mutation has long been discredited for higherorganisms and the "molar indeterminacy" of mutation established bothfor its spontaneous occurrence and its enhancement by X-rays (68).
However, the development of resistance apparently induced by drugs
revived illusions that bacterial genes might be alterable, an inference
that would inevitably undermine the conception of "gene" for theseorganisms. No wonder that the mechanism of drug resistance has
excited so much controversy (89)!

What sort of molecule could function as a specific mutagen, a reagent
for a particular one of the bacterium's complement of genes, which canhardly number less than a thousand targets? On the nucleic hypothesis,
the smallest segment capable of this variety would be a hexanucleotide,
all possible configurations of which must be discriminated by the
specific mutagen. How could this be generally accomplished except byanother molecule of conforming length and periodicity, that is ananalogous polynucleotide? Certainly there is nothing in the chemistry of
penicillin or streptomycin to support their direct intervention in nucleic
instructions.
In addition, we recognize no chemical reagent capable of substituting
one nuclein for another in the structure of existent DNA. However, as
the modification of a nuclein, even to give an unnatural base, could havemutagenic effect, the chief limitation for specific mutagenesis is therecognition of the appropriate target.
Of course the origin of drug resistance, for all its theoretical
implications, poses an experimental challenge of its own. Concededly,
experiments cannot decide untried situations. Nevertheless, themechanism whereby resistant mutants arise spontaneously and are thenselected by the drug can account for every well-studied case of inherited
resistance (5, 10). Furthermore, in favorable instances the spontaneous
origin of drug-resistant mutants can be verified unambiguously bycontriving to isolate them without their ever being exposed to the drug.
One method entails indirect selection. To illustrate its application,
consider a culture of Escherichia coli containing 109 bacteria per ml. By
plating samples on agar containing streptomycin, we infer that one per
million bacteria or 103 per ml produce resistant clones. But to countthese clones they were selected in the presence of streptomycin which
hypothetically might have induced the resistance. We may however
dilute the original bacteria in plain broth to give samples containing 105
per ml. Since 10-6 of the bacteria are resistant, each sample has amathematical expectation of 0,1 of including a resistant bacterium. The
individual bacteria being indivisible by-dilution, nine samples in ten will
include no resistants; the tenth will have one, but now augmented to 1 :105. Which one this is can be readily determined by retrospective assay
on the incubated samples. The procedure can be reiterated to enrich forthe resistant organisms until they are obtained in pure culture (11). The
same result is reached more conveniently if we spread the original
culture out on a nutrient agar plate rather than distribute samples into
separate test tubes. Replica plating, transposing a pattern of surface
growth from plate to plate with a sheet of velvet, takes the place ofassaying inocula distributed in tubes (53). Dilution sampling and replica
plating are then alternative methods of indirect selection whereby thetest line is spared direct contact with the drug. Selection isaccomplished by saving sublines whose sibling clones show the resistantreaction. This proof merely reinforces the incisive arguments that had
already been forwarded by many other authors.
If mutations are not specific responses to the cellular environment,
how do they arise? We still have very little information on the proximate
causes of spontaneous, even of radiation and chemically induced
mutation. Most mutagenic chemicals are potent alkylating agents e. g.
formaldehyde or nitrogen mustard, which attack a variety of reactive
groups in the cell. Similar compounds may occur in normal metabolism
and account for part of the spontaneous mutation rate; they may also
play a role as chemical intermediates in radiation effects. For the mostpart, then, studies on mutagenesis, especially by the more vigorous
reagents, have told us little about the chemistry of the gene. Probably
any agent which can penetrate to the chromosomes and have a localizedchemical effect is capable of introducing random errors into the genetic
information. If the cell were not first killed by other mechanisms most
toxic agents would then probably be mutagenic.
Another class of mutagenic chemicals promises more information:
analogues of the natural nucleins which are incorporated into DNA. For
example, bromouracil specifically replaces thymine in phage DNA whenfurnished as bromodeoxyuridine to infected bacteria. FREESE has shown,by genetic analyses of the utmost refinement, that the loci of resulting
mutations in T4 phage are distributed differently from the mutants of
spontaneous origin or those induced by other chemicals (18). Thismethod presumably maps the locations of thymine in the original DNA.
In order to account for wide variations in mutation rate for different
loci, further interactions among the nucleotides must be supposed. So
far, these studies represent the closest approach to a rational basis forchemical mutagenesis. However, every gene must present many targets
to any nuclein analogue and the specificity of their mutagenesis can bedetected only in systems where the resolution of genetic lociapproximates the spacing of single nucleotides (4). At present this isfeasible only in microorganisms; similar studies with bacteria and fungi
would be of the greatest interest.
More specific effects might result from the insertion of oligo- andpolynucleotides, a program which, however, faces a number of technicaldifficulties: even if the requisite polymers were to be synthesized, there
are obstacles to their penetration into cells. The use of DNA extracted
from mutant bacteria to transfer the corresponding genetic qualities isdiscussed as "genetic transduction".
RNA is the one other reagent that may be expected to recognize
particular genes. As yet we have no direct evidence that the transfer of
information

from

DNA

to

RNA

is

reversible.

However,theanti-mutagenic effect of nuclein ribosides (21, 71) may implicate RNA in
mutation. The reversibility of DNA ' RNA information is also implicit
in STENT'S closely reasoned scheme for DNA replication (82). The
needed experiment is the transfer of DNA information by some isolatedRNA. Although not reported, this has probably not been fairly tried.
One motivation for this approach is the difficult problem of finding
sources of homogeneous nucleic acids. DNA occurs biologically as setsof different molecules presumably in equimolar proportions. (A useful
exception may be a remarkably small phage which seems to be
unimolecular (85).) The species of RNA, however, may vary with thepredominant metabolic activity of the cells. If so, some molecularspecies may be sufficiently exaggerated in specialized cells to facilitate
their isolation. A purified RNA would have many potential applications,among others as a vehicle for the recognition of the corresponding DNA
implied by our theory of information transfer. Pending such advances,
specific mutagenesis is an implausible expectation.
Adaptive mutations, of which drug resistance is a familiar example,
are crucial to the methodology of microbial genetics. Once having
connected adaptive variation with gene mutation (78), we could proceed
to exploit these systems for the detection of specific genotypes in verylarge test populations. The genotypes of interest may arise, as in theprevious examples, by mutation: the most extensive studies of thephysiology of mutation now use these methods for precise assay. For, inorder to count the number of mutants of a given kind, it suffices toplate large numbers of bacteria into selective media and count thesurviving colonies which appear after incubation. In this way, mutation
rates as low as one per 109 divisions can be treated in routine fashion.
Genetic Recombination in Bacteria
The selective isolation of designed genotypes is also the most efficient
way to detect genetic recombination. For example, the sexualmechanism of Escherichia coli was first exposed when prototrophic(nutritionally self-sufficient) recombinants arose in mixed cultures of
two auxotrophic (nutritionally dependent) mutants (35, 84, 57). At first
only one recombinant appeared per million parental bacteria and theselective procedure was quite obligatory. Later, more fertile strains were
descovered which have been most helpful to further analysis (45, 51).
This has shown that typical multinucleate vegetative bacteria unite by aconjugation bridge through which part or all of a male genome migrates
into the female cell (43). The gametic cells then separate. The
exconjugant male forms an unaltered clone, surviving by virtue of its
remaining nuclei. The exconjugant female generates a mixed clone
including recombinants (1, 46). WOLLMAN, JACOB and HAYES (88) havesince demonstrated that the paternal chromosome migrates during
fertilization in an orderly, progressive way. When fertilization isprematurely interrupted, the chromosome may be broken so that only
anterior markers appear among the recombinants. All of the genetic
markers are arranged in a single linkage group and their order can beestablished either by timing their passage during fertilization or by theirstatistical association with one another among the recombinants.
Finally, the transfer of genetic markers can be correlated with thetransfer of DNA as inferred from the lethal effect of the radioactive
decay of incorporated p32 (27).
Sexual recombination is one of the methods for analysing thegene-enzyme relationship. The studies so far are fragmentary but they
support the conception that the gene is a string of nucleotides whichmust function as a coherent unit in order to produce an active enzyme

(4, 15, 33, 67, 90). However, metabolic blocks may originate throughinterference with accessory regulatory mechanisms instead of thefundamental capacity to produce the enzyme. For example, many"lactase-negative" mutants have an altered pattern of enzyme induction
or a defective permease system for substrate-transport (55, 65). Several
laboratories are now working to correlate the relative sequence ofgenetic defects with the sequence of corresponding alterations inenzyme proteins; this may be the next best approach to the coding
problem short of a system where a pure DNA can be matched with its
protein phenotype.
At first these recombination experiments were confined to a single
strain of E. coli, K-12. For many purposes this is a favorable choice ofmaterial—perhaps the main advantage is the accumulation of a libraryof many thousands of substrains carrying the various markers called forby the design of genetic tests. However, strain K-12 is rather unsuitable
for serological studies, having lost the characteristic surface antigens
which are the basis of serological typing. In any event it would be
important to know the breeding structure of the group of entericbacteria. Systematic studies have therefore been made of theinter-fertility of different strains of bacteria, principally with aconvenient tester of the K-12 strain (39, 93). About one fourth of theserotype strains of E. coli are fertile with strain K-12, and in at least
some instances with one another. Whether the remaining three fourthsof strains are completely sterile, or whether they include different,
closed, breeding groups (i. e., different genetic' species) has not beensystematically tested, partly because of the preliminary work needed toestablish suitable strains.
E. coli K-12 is also interfertile with a number of strains of Shigella spp.
(59). Finally, although attempted crosses of E. coli with many
Salmonella types and of Salmonellas with one another have usually
failed, BARON has demonstrated crosses of E . coli with a unique strainof Salmonella typhimurium (3). This may be especially useful as ameans of developing hybrids which can be used to bridge the studies ofsexuality in E . coli and transduction in Salmonella.

Genes and Viruses
Bacteria furnish a unique opportunity to study the genetic
relationships with their host cells. Another treasure of strain K-12 wasfor a time hidden, it carries a temperate phage, λ, which is technicallyquite favorable for genetic work. In accord with BURNET'S early
predictions we had anticipated that the provirus for λ would behave as a
genetic unit but Dr. ESTHER LEDERBERG'S first crosses were quite
startling in their implication that the prophage segregated as a typicalchromosomal marker (34). This was shown quite unambiguously by the
segregationoflysogenicity

versussensitivity

frompersistentheterozygous cells, a test which bypassed the then controversial detailsof fertilization. The viability of such heterozygous cells supports the
hypothesis that lysogenicity depends in part on the development of acytoplasmic immunity to the cytopathic effects of infecting phage as aresult of the establishment of the prophage in a bacterial chromosome.
This picture is also brought out by "zygotic induction" (26), whereby thefertilization of a sensitive cell by a prophage-bearing chromosome may
provoke the maturation and aggressive growth of the phage and lysis of
the complex. On the other hand, the introduction of a sensitive
chromosome into a lysogenic bacterium does not result in thisinduction. The mode of attachment of prophage to its chromosomal site
is as unsettled as the general picture of the higher organization of DNA,but most students favor a lateral rather than an axial relationship forthe prophage. The isolation of intact chromosomes of bacteria would
give a new approach to this question but has so far been inconclusive.
Another infectious particle that has jumped out of our Pandora's box
determines the very capacity of E. coli to function as a male partner in
fertilization (51). For lack of a better inspiration, we call this particle"F". Two kinds of male strains are now recognized according to whether
the F particle has a chromosomal or a cytoplasmic location. F + strains,
like the original K-12, are highly contagious for F and will rapidly
convert populations of female, F— strains in which they are introduced.
Hfr males, on the other hand, have a chromosomal localization of the F
factor resulting from occasional transpositions in F+ strains. The
different localization of the F particle in the two cases is diagnosed
primarily by the behavior of the particle in crosses. In addition, HIROTA

and IIJIMA (24) found that the F particle could be eliminated from F+
strains by treatment with acridine dyes. Hfr clones are unaffected byacridine orange, but when they revert to the F+ state, as occasionallyhappens, the F particle again becomes vulnerable to the dye. The
accessibility of extrachromosomal F is paralleled by several otherexamples of plasmid disinfection (reviewed in 40); perhaps the mostnotable is the bleaching of green plant cells by streptomycin (17, 76). No
reagent is known to inactivate F or prophage while bound to thechromosome.
The virus λ and the plasmagene F are analogous in many features (28,48). Their main differences are:(1) Cytopathogenicity. A bacterium cannot long tolerate λ in its
cytoplasmic state and remain viable. The vegetative λ must
promptly reduce itself to a chromosomal state or multiply
aggressively and lyse the host bacterium. F has no known
cytopathic effect.
(2) Maturation. Vegetative λ organizes a protein coat and matures
into an infective phage particle. F is known only as an intracellularvegetative element; however, the coat of the F+ cell may beanalogous to that of the phage.
(3) Transmission. λ is infective, i. e., forms a free particle which canpenetrate susceptible cells. F is transmitted only by cell-to-cellconjugation.
(4) Fixation. λ has a fore-ordained site of fixation on the bacterial
chromosome; F has been identified at a variety of sites. However, thisdifference may be illusory. In special situations, F does havepreferential sites of fixation (77), and generally, translocations of F todifferent sites are more readily discovered than those of λ would be.
(5) Induction. Exposure of lysogenic bacteria to small doses ofultra-violet light causes the prophage to initiate a lytic cycle with theappearance first of vegetative, then of mature phage (62). Hfrbacteria make no analogous response. However, the kinetics of thereversion, Hfr  F+, has not been carefully studied.
The genetic function of bacteriophages is further exemplified bytransduction whereby genes are transferred from cell to cell by theintervention of phage particles (42, 91). In our first studies weconcluded that the bacterial genes were adventitiously carried in normal
phage particles (66, 83, 92). Further studies favor the view that thetransducing particle has a normal phage coat but a defective phage
nucleus. This correlation has suggested that a gene becomestransducible when a prophage segment is translocated to its vicinity (2,9, 60).
Transduction focuses special attention on the phenomenon of specificpairing of homologous chromosome segments. Howsoever a transduced
gene is finally integrated into the bacterial genome, at some stage it
must locate the homologous gene in the recipient chromosome. For intransduction, as in sexual recombination, new information is not merely
added to the complement; it must also replace the old. This must
involve the confrontation of the two homologues prior to the decision
which one is to be retained. Synapsis is even more, puzzling as between
chromosomes whose DNA is in the stabilized double helix and thenfurther contracted by supercoiling. Conceivably gene products rather
than DNA are the agency of synaptic pairing.
The integration of a transduced fragment raises further issues (41).
The competing hypotheses are the physical incorporation of thefragment in the recipient chromosome, or the use of its information
when new DNA is replicated. The same issues still confound models ofcrossing over at meiosis in higher forms; once again the fundamentals
of chromosome structure are needed for a resolution.

Virus Versus Gene
The homology of gene and virus in their fundamental aspects makes'
their overt differences even more puzzling. According to the simplest
nucleic doctrine, DNA plays no active role in its own replication otherthan furnishing a useful pattern. Various nucleotide sequences should
then be equally replicable. What then distinguishes virus DNA, which
replicates itself at the expense of the other pathways of cellularanabolism? For the T even phages, the presence of the uniqueglucosylated hydroxymethylcytosine furnishes a partial answer (12).
However, other viruses such as λ display no unique constituents;
furthermore, as prophage they replicate coordinately with bacterialDNA. Does the virus have a unique element of structure, either chemicalor physical, so far undetected? Or does it instruct its own preferential
synthesis by a code for supporting enzymes?

The Creation o f L i f e
The mutualism of DNA, RNA and proteins as just reviewed isfundamental to all contemporary life. Viruses are simpler as infective
particles but must, of course, parasitize the metabolic machinery of thehost cell. What would be the least requirements of a primeval organism,
the simplest starting point for progressive replication of DNA in terms
of presently known or conjectured mechanisms? They include at least:(1) DNA(2) The four deoxyribotide pyprophosphates in abundance.
(3) One molecule of the protein, DNA polymerase.
(4) Ribotide phosphates as precursors for RNA.
(5) One molecule of the protein RNA polymerase.
(6) A supply of the twenty amino acyl nucleotidates.
(a) Failing these, each of the twenty enzymes which catalyse thecondensation of an amino acid and corresponding RNA
fragments together with sources of these components.
(7) One molecule of the protein aminoacyl-RNA polymerase.
In principle, this formidable list might be reduced to a single
polynucleotide polymerized by a single enzyme. However, any scheme
for the enzymatic synthesis of nucleic acid calls for the coincidence of aparticular nucleic acid and of a particular protein. This is a far more
stringent improbability than the sudden emergence of an isolated DNA
such as many authors have suggested, so much more so that we must
look for alternative solutions to the problem of the origin of life. These
are of two kinds. The primeval organism could still be a nucleic cycle if
nucleic

replication

occurs,howeverimperfectly,

without

theintervention of protein. The polymerase enzyme, and the transfer of
information from nucleic acid to protein, would then be evolved
refinements.

Alternatively,

DNA

has

evolvedfromasimpler,

spontaneously condensing polymer. The exquisite perfection of DNA
makes the second suggestion all the more plausible.

The nucleoprotein cycle is the climax of biochemical evolution. Its
antiquity is shown by its adoption by all phyla. Having persisted for ~109 years, nucleoprotein may be the most durable feature of thegeochemistry of this planet.
At the present time, no other self-replicating polymers are known or
understood. Nevertheless, the nucleic system illustrates the basic
requirements for such a polymer. It must have a rigid periodic structurein which two or more alternative units can be readily substituted. It
must allow for the reversible sorption of specific monomers to the units
in its own sequence. Adjacent, sorbed monomers must then condense toform the replica polymer, which must be able to desorb from thetemplate. Primitively, the condensation must be spontaneous but
reliable. In DNA, the sorption depends on the hydrogen bonding of
nuclein molecules constrained on a rigid helical backbone. This highly
specific but subtle design would be difficult to imitate. For the moreprimitive stages, both of biological evolution and of our own
experimental insight, we may prefer to invoke somewhat cruder
techniques of complementary attachment. The simplest of these isperhaps the attraction between ionic groups of opposite charge, forexample, NH3+ and COO- which are so prevalent in simple, organic
compounds. If the ingenuity and craftmanship so successfully directed
at the fabrication of organic polymers for the practical needs of mankind
were to be concentrated on the problem of constructing aself-replicating assembly along these lines I predict that theconstruction of an artificial molecule having the essential function ofprimitive life would fall within the grasp of our current knowledge oforganic chemistry.
Conclusions
The experimental control of cellular genotype is one of the measures ofthe scope of genetic science. However, nucleic genes will not be readily
approached for experimental manipulation except by reagents thatmimic them in periodic structure. Specifically induced mutation, if everaccomplished, will then consist of an act of genetic recombination
between the target DNA and the controlled information specified by thereagent. Methods for the step-wise analysis and re-assembly of nucleic

acids are likely to be perfected in the near future in pace with theaccessibility of nucleic acid preparations which are homogeneousenough to make their use worth-while. For the immediate future, it islikely that the greatest success will attend the use of biological reagents
to furnish the selectivity needed to discriminate one among
innumerable classes of polynucleotides. Synthetic chemistry is,however, challenged to produce model polymers that can emulate theessential features of genetic systems.

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The experimental work from my laboratory summarized in this paper has been
generously supported by research grants from the National Institutes of Health,
U. S. Public Health Service, the National Science Foundation, the Rockefeller
Foundation, the Wisconsin Alumni Research Foundation, the University of
Wisconsin and, most recently, Stanford University. It is also a pleasure to record
my thanks to the Jane Coffin Childs Fund for Medical Research for a research
fellowship which supported my first association with Professor E. L. TATUM.