A VIEW OF GENETICS I`hr~ Kctl~l Statutes of Iqou charge each prize-winner to give a public lccturr7 in Stockholm within six months of Commemoration IIal.. That I have full! used this margin is not altogether ingenious, since it furnishes a pleasant occasion to revisit my manv friends and colleagues in !*oru- lxzautiful tit\, during its best season. The charge might call for a historical account of past "studies on genetic rt>combination and organization of the genetic material in bacteria", studies in which I have enjoyed tht> companionshil) of man\- collcagucs, al~\~ all In\- wifr. However, this subject has been reviewed regularly (30, 37, $4, 41. 4% 45, 44. 54, S.5, 58) and 1 1 lope \;ou will share rn\p own inclination to assume a more speculati\~e task, too look at the context of contrmporar!. science in which bacterial genetics can bc better understood, and to scrutinize the future prospects of experimental genetics. `1`11~ dispersion of a Nobel award in the field of gcnctics symholizcs the convergent efforts of :I world-wide communit!. of investigators. That genetics should now he recognized is also timel), ~ for its axial role in the conceptual structure of hiolog~-, and for its ripening yield for the theor>. and practice of mcdicinr. Howe\-er, experimental genetics is reaching its full powers in coalesce~~c~~ with hiochemistr).: in principle, each phenotype should c\.entuall\. 1~ denoted as an exact stxluencc of amino acids in protein (;;(I) and the genot!y as a corresl)onding sequence of nucleotides in DNA (a, 03). `The precise dc- marcation of genetics from biochemistr\. is already futile: hut when genetics has 11rrn fully reduced to its molecular foundations, it may continue to ser1.r in the same relation as thermodvnamics to mechanics (OCJ,. The coordination of so many adjacent sciences will he a cogent challenge to the intellectual powers of our successors. 171 4- o- b- Fig. I. l'rimary 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 thought of at all, the>. have often been relegated to some obscure byway of evolution, their complexit> and their homology with other organisms grossly underrated. "Since Pasteur's startling discoveries of the important role played bv microbes in human affairs, microbiology as a science has always suffered from its eminent practical appli- cations. 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 and viruses is only the first measure of the repayment of medicine's debt to biology (6, 7, 8). Comparative biochemistry has consummated the unification of biolog!- revitalized by Darwin one hundred years ago. Throughout the living world we see a common set of structural units - amino acids, coenzvmes, nucleins, carbohydrates and so forth - from which every organism builds itself. The same holds for the fundamental process of biosynthesis and of energy metab- olism. The exceptions to this rule thus command special interest as meaningful tokens of biological individuality, e. g., the replacement of cytosine by hydr- oxymethyl cytosine in the DNA of Tz phage (12). Nutrition has been a special triumph. Bacteria which required no vitamins 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 those metabolites they can not synthesize with their own enzymatic machinery. Species differ in their nutrition: if species are delimited by their genes, then gelzes must control the biosynthetic steps which aye reflected in nutritio~zal patterns. This syllogism, so evident once told, has been amplified by BEADLE and 172 TATUM from this podium. Its implications for experimental biology and medi- cine are well-known: among these, the methodology of bacterial genetics. TATCM has related how his early experience with bacterial nutrition rein- forced the foundations of the biochemical genetics of Neurospora. Then, dis- regarding the common knowledge that bacteria were too simple to have genes. TXTUM 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 of bacterial genetics. Contemporary genetic research is predicated on the role of DNA as the genetic material, of enzy-me proteins as the cell's working tools, and of RSA as the communication channel between them (63). Three lines of evidence substantiate the genetic function of DNA. Two are related to bacterial genetics; the third and most general is the cytochemical observation of DNA in the chromosomes, which are undeniably strings of genes. But chromosomes also contain other constituents besides DNA: we want a technique to isolate a chromosome or a fragment of one, to analyse it and to retransplant it to verify its functional capacity. The impressive achievements of nuclear transplanta- tion (29) should encourage the audacity needed to try such experiments. The constructive equivalent to chromosome transplantation was disco\.ered by a bacteriologist thirty years ago (20) but the genetic implications of the "pneu- mococcus transformation" in the minds of some of GRIFFITH'S successors were clouded by its involvement with the gummy outer capsule of the bacteria. However, by 1943, AVERT and his colleagues had shown that this inherited trait was transmitted from one pneumococcal strain to another by DI\;A. The general transmission of other traits by the same mechanism (25) can only mean that DNA comprises the genes (b). To reinforce this conclusion, HERSHI+- and CHASE (23) proved that the genetic element of a bacterial virus is also DNA. Infection of a host cell requires the injection of just the DNA content of the adsorbed particle. This DNA controls not only its own replication in the production of new phage but also the specificity of the protein coat, which governs the serological 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 the cytoplasm is now very b. One might be tempted to write: "One DNA molecule = one gene." However, the quanta of factorial genetics, based on mutation, recombination and enzymatic function are all smaller than the DNA unit of molecular weight - 6 x ro6 (4). There is increasing evidence that such a molecule is a natural unit rather than an artefact of fragmentation (54). I73 doubtful - at least some of the plasmagenes that have been proposed as ful- filling this function are now better understood as 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 nu- cleotides. 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 the evident fruitfulness of the doctrine in ex- perimental design. The cell may, of course, carry information other than nucleic either in the cytoplasm or, accessory to the polynucleotide sequence, in the chromosomes. Epinucleic information has been invoked, without being more precisely defined, in many recent speculations on cytodifferentiation and on such models of this as antigenic phase variation in Salmolzella (71, 52, 56, 47). Alternative schemes "have so much less information capacity than the nucleic cycle that they are more likely to concern the regulation of genie functions than to mimic their specificities. IlN.4 as a Substance The chemistry of DNA deserves to be exposed by apter craftsmen (SO, 31, 13) and I shall merely recapitulate before addressing its biological implications. rl segment of DNA is illustrated in Fig. I. This shows a linear polymer whose backbone contains the repeating unit: [m-O--P (OO-)-0-CH,-CH-CH-] 0-P(OO-)-0-CH2- Ldiester phosphate C', C', C',] diester phosphate . . . The carbon atoms are conventionally numbered according to their position in the furanose ring of deoxyribose, which is coupled as an N-glycoside to one of the nucleins: adenine, guanine, cytosine or thymine, symbolized A, G, C or T, the now well-known alphabet in which genetic instructions are com- posed. With a chain length of about IO ooo residues, one molecule of DNA contains 20 ooo "bits of information", comparable to the text of this article, or in a page of newsprint. Pyrophosphate-activated monomer units (e. g. thymidine triphosphate) have been identified as the metabolic precursors of DSA (31). For genetic replica- tion, the monomer units must be assembled in a sequence that reflects that of the parent molecule. -4 plausible mechanism has been forwarded by \~ATSON I74 Fig. 2. The scheme of \vATSOx and CRICK for DNA replication. "Unwinding and replication proceed pari pnssu. 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 rel- ative positions are fixed by the deoxyribose-phosphate backbones, 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 evidence shows a remarkable equality of A with T and of G with C in DNA from various sources. The two strands of any DN,4 are then mutually complementary, the A,T, G and C of one strand being represent- ed by T, ,4, C and G, respectively, of the other. The information of one strand is therefore equivalent to, because fully determined by, the other. The deter- mination occurs at the replication of one parent strand by the controlled stepwise accretion of monomers to form a complementary strand. At each step only the monomer which is complementary to the template would fit for a chain-lengthening esterification with the adjacent nucleotide. The model requires the unravelling of the intertwined helices to allow each of them to serve as a template. This might, however, occur gradually, with the growth of the daughter chain - a concept embedded in Fig. 2 which symbolizes the new 17.5 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 un- distinguished molecule. Its overall shape is controlled by the uniform deoxy- ribose 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 amino acid sequence in proteins. However, as there are twenty amino acids to choose among, there cannot be a one : one correspondence of nucleotide to amino acid. Taking account of the code-duplication in complementary 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" (So) of DNA. The differem% of the amino acids vary widely in size, shape and ionic charge (e. g., H. ; CH, * ; H,N'CH;CH;CH;CH2.; COOH*CH2*CH,*; HO * C,H, . CH, *) 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 specificit> of the DKA must therefore be translated into the three-dimensional specificity of an enzyme or antibody surface. The simplest assumption would be that the amino acid sequence of the extended polypeptide, as it is released from the protein-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 specula- tions about the mechanism of antibody formation. If antibody globulins have a common sequence on which specificity is superimposed by directed folding, an antigen could directly mold the corresponding antibody. However, if sequence determines folding, it should in turn obey nucleic information. As this should be independent of antigenic instruction, we may look instead to a purely selective role of antigens 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 176 probable that this code has evolved secondarily and arbitrarily 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 have reported the enzymatic reaction of amino acids with RNA fragments (22, 75). Apparently each amino acid has a different RNA receptor and an enzyme whose twofold specificity thus obviates any direct recognition of amino acid by polynucleotides. The alignment of amino- acyl residues for protein synthesis could then follow controlled assembly of their nucleotidates on an RNA template, by analogy with the model for DNA replication. We then visualize the following modes of information transfer: (I) DNA replication - assembly of complementary deoxyribonucleotides on a DNA template. (2) Transfer to RNA by some comparable mechanism of assembling ribo- nucleotides. Our understanding of this is limited by uncertainties of the structure of RNA (10). (3) Protein synthesis: (a) Aminoacylation of polynucleotide fragments; (b) Assembly of the nucleotidates on an RNA template by analogy with step (I); (c) Peptide condensation of the amino acid residues. Some workers have suggested that RN,4 is replicated in step (3) concurrently with protein synthesis, in addition to its initiation from DNA. The chief difference in primary structure between DKA and RNA is the hydroxylation of C', in the ribose, so that a reactive sugar hydroxyl is available in RX4. This may prove to be important in the less ordered secondary struc- ture of RNA, and in its function as an intermediary to protein. It remains to be determined whether the aminoacyl nucleotidates are esterified at C'2 or at C', which is also available in the terminal residue. From this resume we may observe that the DNA backbone constitutes an inert but rigid framework on which the differential nucleins are strung. Their spatial constraint lends specificity to the pattern of hydrogen bonding exposed at each level. This cstended 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. =77 OX;1 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 higher organisms and the "molar indeterminacy" of mutation established both for 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 these organisms. No wonder that the mechanism of drug resistance has excited so much controversy (89)! \Vhat sort of molecule could function as a specific mutagen, a reagent for a particular one of the bacterium's complement of genes, which can hardl! number less than a thousand targets? On the nucleic hypothesis, the smallest segment capable of this variety would be a hexanucleotide, all possible con- figurations of which must be discriminated by the specific mutagen. How could this be generally accomplished except by another molecule of conforming length and periodicity, that is an analogous polynucleotide? Certainly there is nothing in the chemistry of penicillin or streptomycin to support their direct inter- vention 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 modi- fication of a nuclein, even to give an unnatural base, could have mutagenic effect, the chief limitation for specific mutagenesis is the recognition 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, the mechanism whereby resistant mutants arise spontaneously and are then selected by the drug can account for every well-studied case of inherited resistance (5, IO). Furthermore, in favorable instances the spontaneous origin of drug-resistant mutants can be verified unambiguously by contriving 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 10~ bacteria per ml. By plating samples on agar containing streptomycin, we infer that one per million bacteria or ro3 per ml produce resistant clones. But to count these clones they were selected in the presence of streptomycin which hypothetically might have induced the resistance. We may however dilute the original bac- 12-593407. Les prix Nobel en 1958. 178 teria in plain broth to give samples containing 10~ per ml. Since IO-~ of the bacteria are resistant, each sample has a mathematical expectation of O,I of including a resistant bacterium. The individual bacteria being indivisible b> dilution, nine samples in ten will include no resistants; the tenth will have one, but now augmented to I : 10~. Which one this is can be readily determined by retrospective assay on the incubated samples. The procedure can be reiter- ated to enrich for the resistant organisms until they are obtained in pure culture (II). 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 of assaying inocula distributed in tubes (53). Dilution sampling and replica plating are then alternative methods of indirect selection whereby the test line is spared direct contact with the drug. Selection is accomplished by saving sublines whose sibling clones show the resistant reaction. 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, hoa do they arise? We still have very little information on the proximate causes of spontaneous, even of radiation and chemically induced mutation. Most muta- genic chemicals are potent alkylating agents e. g. formaldehyde or nitrogen mustard, which attack a variety of reactive groups in the cell. Similar com- pounds may occur in normal metabolism and account for part of the spon- taneous mutation rate; they may also play a role as chemical intermediates in radiation effects. For the most part, then, studies on mutagenesis, especiall!- 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 ha\-e a localized chemical 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 DSA. For example, bromo- uracil specifically replaces thymine in phage DNA when furnished 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 in- duced by other chemicals (IS). This method 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 be detected only in systems where the resolution of genetic loci approximates the spacing of single nucleotides (4). At present this is feasible only in micro- organisms; similar studies with bacteria and fungi would be of the greatest interest. More specific effects might result from the insertion of oligo- and poly- nucleotides, a program which, however, faces a number of technical difficulties: 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 is discussed as "genetic trans- duction". 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, the anti-mutagenic effect of nuclein ribosides (21, 71) may implicate RNA in mutation. The reversibility of DNA 2 RNA information is also implicit in %XT'S closely reasoned scheme for Dh'A replication (Sz). The needed experiment is the transfer of DNA informa- tion by some isolated RNA. 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 sets of different molecules presumably in equimolar proportions. (A useful exception mav be a remarkably small phage which seems to be unimolecular (Sj).) The species of RNA, however, may vary with the predominant metabolic activity of the cells. If so, some molecular species 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 very large test populations. The genotypes of interest may arise, as in the previous examples, by mutation: the most extensive studies of the physiology of mutation now use these methods for precise assay. For, in order to count the number of mutants of a given kind, 180 it suffices to plate large numbers of bacteria into selective media and count the surviving colonies which appear after incubation. In this way, mutation rates as low as one per IO' 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 sexual mechanism of Escherichia coli was first exposed when prototrophic (nutritionally self-sufficient) recombinants arose in mixed cultures of two auxotrophic (nutritionally de- pendent) mutants (35, 84, 57). At first only one recombinant appeared per million parental bacteria and the selective 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 vege- tativc bacteria unite by a conjugation 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 (I, 46). WOLLMAN, JACOB and HAYES (88) have since demon- strated that the paternal chromosome migrates during fertilization in an orderly, progressive way. When fertilization is prematurely 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 be established either by timing their passage during fertilization or by their statistical association with one another among the recombinants. Finally, the transfer of genetic markers can be correlated with the transfer 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 the gene-enzyme relationship. The studies so far are fragmentary but they support the conception that the gene is a string of nucleotides which must function as a coherent unit in order to produce an active enzyme (4, 15, 33, 67, go). However, metabolic blocks may originate through interference with accessory regulatory me- chanisms instead of the fundamental 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 of genetic defects with the sequence of corresponding alterations in enzyme proteins; this may be the next best approach to the coding problem short of 181 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 of material-perhaps the main advantage is the accumulation of a library of many thousands of substrains carrying the various markers called for by the design of genetic tests. However, strain K-12 is rather unusitable 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 enteric bacteria. Systematic studies have therefore been made of the inter- fertility of different strains of bacteria, principally with a convenient tester of the K-12 strain (39, 93). About one fourth of the serotype strains of E. coli are fertile with strain K-12, and in at least some instances with one another. Whether the remaining three fourths of strains are completely sterile, or whether they include different, closed, breeding groups (i. e., different genetic species) has not been systematically tested, partly because of the preliminary work needed to establish 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 SaJmonellas with one another have usually failed, BARON has demon- strated crosses of E. coli with a unique strain of Salmonella t@himtirizsm (3). This may be especially useful as a means of developing hybrids which can be used to bridge the studies of sexuality 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-IZ was for a time hidden, it carries a temperate phage, I,, which is technically quite favorable for genetic work. In accord with BURICET'S early predictions we had antici- pated that the provirus for 1 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 typical chromosomal marker (34). This was shown quite unambiguously by the segregation of lysogenicity versus sensitivity from persistent heterozygous cells, a test which bypassed the then controversial details of fertilization. The viability of such heterozygous cells supports the hypothesis that lysogenicity depends in part on the development of a cyto- plasmic immunity to the cytopathic effects of infecting phage as a result of the establishment of the prophage in a bacterial chromosome. This picture is also brought out by "zygotic induction" (26), whereby the fertilization of a sen- IQ2 sitive 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 3, lysogenic bacterium does not result in this induction.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 for the 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 fer- tilization (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. Ff strains, like the original K-m, are highly contagious for F and will rapidly convert populations of female, I;-- strains in which they are introduced. Hfr males, on the other hand, have a chromosomal localization of the F factor resulting from occasional trans- positions 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 elimi- nated from F+- strains by treatment with acridine dyes. Hfr clones are un- affected by acridine orange, but when they revert to the F+ state, as oc- casionally happens, the F particle again becomes vulnerable to the dye. The accessibility of extrachromosomal F is paralleled by several other examples of plasmid disinfection (reviewed in 40); perhaps the most notable is the bleaching of green plant cells by streptomycin (17, 76). Ko reagent is known to inactivate F or prophage while bound to the chromosome. The virus 3. and the plasmagene F are analogous in many features (28, 4s). Their main differences are: (I) Cytopathogenicity. A bacterium cannot long tolerate 2, in its cytoplasmic state and remain viable. The vegetative 3, must promptly reduce itself to a chromosomal state or multiply aggressively and lyse the host bac- terium. F has no known cytopathic effect. (2) Maturation. Vegetative i organizes a protein coat and matures into an infective phage particle. F is known only as an intracellular vegetative elrment; however, the coat of the F+ cell may be analogous to that of the phage. (3) Transmission. ,I is infective, i. e., forms a free particle which can penetrate susceptible cells. F is transmitted only by cell-to-cell conjugation. 183 (4) Fixation. I has a fore-ordained site of fixation on the bacterial chromo- some; F has been identified at a variety of sites. However, this difference may be illusory. In special situations, F does have preferential sites of fixation (77), and generally, translocations of F to different sites are more readily discovered than those of 1 would be. (5) Induction. Exposure of lysogenic bacteria to small doses of ultra-violet light causes the prophage to initiate a lytic cycle with the appearance first of vegetative, then of mature phage (62). Hfr bacteria make no analogous response. However, the kinetics of the reversion, Hfr + F+, has not been carefully studied. The genetic function of bacteriophages is further exemplified by iransductiorz n-hereby genes are transferred from cell to cell by the intervention of phage particles (42, 91). In our first studies we concluded that the bacterial genes were adventitiously carried in normal phage particles (66, 83, gz). Further studies favor the view that the transducing particle has a normal phage coat but a defect& phage nucleus. This correlation has suggested that a gene becomes transducible when a prophage segment is translocated to its vicinit? iz, 9, 64. Transduction focuses special attention on the phenomenon of specific pairing of homologous chromosome segments. Howsoever a transduced gene is finally integrated into the bacterial genome, at some stage it must locate the homol- ogous gene in the recipient chromosome. For in transduction, 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 DSA is in the stabilized double helix and then further contracted by supercoiling. Conceivably gene products rather than DN,4 are the agency of synaptic pairing. The integration of a transduced fragment raises further issues (41). The competing hypotheses are the physical incorporation of the fragment in the recipient chromosome, or the use of its information when new DNA is replicated. The same issues still confound models of crossing over at meiosis in higher forms; once again the fundamentals of chromosome structure are needed for a resolu- tion. T ~ivus Versus Gene The homology of gene and virus in their fundamental aspects makes their overt differences even more puzzling. According to the simplest nucleic doc- 184 trine, DNA plays no active role in its own replication other than 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 cellular anabolism? For the T even phages, the presence of the unique glucosylated hydroxymethylcytosine furnishes a partial answer (12). However, other viruses such as 3L display no unique constituents; further- more, as prophage they replicate coordinately with bacterial DNA. Does the virus have a unique element of structure, either chemical or physical, so far undetected? Or does it instruct its own preferential synthesis by a code for supporting enzymes? The Creation of Life The mutualism of DNA, RNA and proteins as just reviewed is fundamental to all contemporary life. Viruses are simpler as infective particles but must, of course, parasitize the metabolic machinery of the host cell. What would be the least requirements of a primeval organism, the simplest starting point for progressive replication of DKA in terms of presently known or conjectured mechanisms? They include at least: (I) 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 the con- densation of an amino acid and corresponding RNA fragments together with sources of these components. (7) One molecule of the protein aminoacyLRN4 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 a particular 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 sug- gested, 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, however imperfectly, without the intervention of protein. The polymerase enzyme, and the transfer of information from nucleic acid to protein, would then be evolved refinements. Alternatively, DNA has evolved from a simpler, spontaneously condensing polymer. The exquisite perfection of DN,4 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 * 10~ years, nucleoprotein may be the most durable feature of the geochemistry of this planet. ilt the present time, no other self-replicating polymers are known or under- stood. Nevertheless, the nucleic system illustrates the basic requirements for such a polymer. It must have a rigid periodic structure in 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 to form the replica polymer, which must be able to desorb from the template. 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 more primitive stages, both of biological evolution and of our own experimental insight, we may prefer to invoke somewhat cruder techniques of complemen- tary attachment. The simplest of these is perhaps the attraction between ionic groups of opposite charge, for example, ?jH,+ 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 man- kind were to be concentrated on the problem of constructing a self-replicating assembly along these lines I predict that the construction of an artificial molecule having the essential function of primitive life would fall within the grasp of our current knowledge of organic chemistry. Conclusions The experimental control of cellular genotype is one of the measures of the scope of genetic science. However, nucleic genes will not be readily approached for experimental manipulation except by reagents that mimic them in periodic structure. Specifically induced mutation, if ever accomplished, will then consist of an act of genetic recombination between the target DNA and the controlled information specified by the reagent. Methods for the step-wise analysis and re-assembly of nucleic acids are likely to be perfected in the near future in pace with the accessibility of nucleic acid preparations which are 186 homogeneous enough to make their use worth-while. For the immediate future, it is likely 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 the essential features of genetic systems. REFERENCES AND NOTES I. A4sDERsoN, `I-. F., 1958. Recombination and segregation in Escherichia coli. Cold Sping Harbor Symposia Quant. Biol. 23, 47-58. 2. =\RBER, W., 1955. Transduction des caract&res Gal par le bactPriophage Lambda. Archives des Sciences, Sot. Phys. Hist. Sat. Gen+ve II, 259-338. 3. BARON, L. S., W. F. CAREY, and W. M. SPILMAN, 1958. Hybridization of Salmonella species by mating with Escherichia coli. 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PREISS, J., 1'. BERG, E. J. OFESGAND, F. H. BERGMASN, and M. DI~cI~M~s~-, 1959. 1sq LES PRIX NOBEL EN 1958 Your Majesty, with Your gracious permission, the toast. 4+$l The toast is to the Nobel Laureates - to genius, devotion, courage. Long live the Nobel Laureates! Leve de! Le premier & repondre & ce discours fut M. LEDERBERG qui s'exprima en ces termes: Your Majesties, Your Royal Highnesses, Your Excellencies, Ladies and Gen- tlemen. Pride is-humbled as humility is exalted in the dignity and splendor of this occasion. Who would deny his pride in the appreciation of his fellows and to join therein with GEORGE W. BEADLE and EDWARD L. TATUM whose exploration and teaching have inspired a generation of discovery. Here pride must merge with humility in the same contemplation of the webs of interdependence of each investigator in the global community of scientific research, of each elusive fact in the continuum of human knowledge. But formal eclat and public attention are so unaccustomed a distinction that a scholar may ask by what lasting motive he is elevated from the simpler satisfactions of academic life. We must concede that' some aspects of Nobel's dedication have been deflected by the force of history. His contrition for chemical inhumanity is shaded in the gloom of cosmic insecurity. ManJ sciences, and genetics in particular, have germinated and flowered only since his time. The growing complexity of science and the reticulation of its ad- vances must make the. task of singular choice ever more difficult and arbitrary. But if Nobel's honors are celebrated in Stockholm his passion is enacted in Oslo. His zeal for peace and international understanding is further es- pressed in his testamentary "wish that in awarding the prizes no consideration whatever shall be given to the nationality of the candidates". The merit the prizes have won is in the faith and courage of this trust. The illumination of human aspirations in intellect and in charity which transcend nationality is then the enduring warrant of Nobel's legacy. Our presence honors his hopes for the fraternity of mankind. My greatest pride and humblest gratitude is to join in this dedication.