Copyright 0 1991 by the Genetic? Society of America Perspectives Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove The Gene (H. J. MULLER 1947) Joshua Lederberg Rockefeller University, New York, Nm York 10021 66 T HE Gene" was H. J. MULLER'S Pilgrim Trust lecture, delivered before the Royal Society of London on November 1, 1945. World War II was barely over, but sea travel was still hazardous. A storm had dislodged a number of floating mines, and the transit to port of SS Queen Mary was something of an adventure (CARLSON 1981). Published in 1947, "The Gene" is the finest exposition of the state of develop- ment of genetics at the very dawn of its turning molecular in the decade spanned by AVERY, MACLEOD and MCCARTY (1944)and WATSON and CRICK(~~~~). The explosive transition was of course closely linked to contemporaneous history: the burgeoning commit- ment to scientific research, now strongly supported by government in a style inherited from wartime experience. I will be reviewing this paper in a retro- spective mood: "now" (as opposed to "today") will mean 1945. The contrast of 1945 with 1991 gives us a chance to reflect how much we have learned in 46 years, and how much was anticipated. It is especially instructive to reread this work in company with DU- BOS's The BacteriaE Cell (1945), a noted microbiolo- gist's synthesis that almost converges with "The Gene." MULLER'S leading argument is whether there is "even such a thing as genetic material at all, as distinct from other constituents of living matter." He responds that the simplest observation of the developmental life cycle points to some conserved invariant that persists from fertilization, through embryonic development and the formation of gametes, returning to the fertil- ized egg. This is then complicated by the requirement for accurate duplication of that invariant, whatever it may be, under its own influence. Discrete mutations are then further evidence of correspondingly discrete particles as the material basis of inheritance. However, the knowledge of chromosomes now enables an appeal to much more direct pragmatic evidence, if not yet of the material composition of the gene, at least of its Cknetm 129: 313-316 (October, 1991) cytological location. Most of the genetic research dur- ing 1900-I 945 was indeed devoted to chromosome mechanics; today we view Mendelian ratios less as a fundamental law of biology than of the idiosyncrasies of chromosome partition in material carefully chosen for the avoidance of particularities like meiotic drive, nondisjunction, gene conversion or paternal imprint- ing. MULLER turns to the chemical composition of chro- mosomes as predominantly "nucleoprotein, a com- pound of protein with nucleic acid, [as] was shown in analyses of sperm chromosomes by MIESCHER, 1897." The reference is to a compilation of MIESCHER'S work for the previous 30 years. MULLER remarks that "only recently has it become reasonably certain-through the analogous finding in viruses-that it is really this major component rather than some elusive accompa- niment of it which constitutes the genetic material itself." Protein, rather than monotonous nucleic acid, is presumably the information-bearer; however, "nu- cleic acid also exists in highly polymerized form . . . as may be very significant." Much of MULLER'S own research had concerned mutagenesis, including that induced by X-rays. Again, the gene is a particle with highly circumscribed local- ity. Mutation can alter one allele and leave its homol- ogous partner "lying but a fraction of a micron away. . . undisturbed. " "Blindness and molar indeter- minacy" characterize mutation. How this can lead to constructive evolution is usually through the action of natural selection on ensembles of mutations each with small effect, and therefore unlikely to be disastrous. MULLER reaches hard to extract useful hints on the chemistry of the gene from X-ray mutagenesis. At least it is internally nonrepetitive or "aperiodic" (per SCHR~DINGER; but most molecules are), on the feeble argument that mutation is a discrete event, not a protracted instability that might speak for continued internal reshuffling. 3 1 4 ,J. Lederberg News of chemical mutagenesis was just trickling in, especially of the war gas, mustard (AUERBACH, ROB- SON and CARR 1947). These studies were inspired by similarities between mustard gas burns and radiation damage. MULLER was aware of the mustard gas work at the time of his lecture, but military restrictions prevented his mentioning it (CROW 1990). MULLER also recited more dubious claims from Caltech of antibody-induced mutations in Neurospora (EMERSON 1944) which, together with PAULINC and CAMPBELL'S (1942) claims for antibody synthesis by protein folding in vitro, have been consigned to oblivion. Alkylation mutagenesis remains a lively research topic today and, indeed, "These . . . experiments constitute the first decided break in the impasse that had developed in studies directed toward the chemistry of the mutation process." Nevertheless, genetic chemistry has contrib- uted more to the rather intricate and still problemat- ical mechanism of mutagenesis than the converse (DRAKE 1989). Historically, the validation of chemical mutagenesis seems long overdue, considering that almost every molecule is suspect today. This can be attributed in part to the tediousness of methods, requiring elabo- rate statistical validation, before bacterial systems were developed. Furthermore, few of MCTLLER'S con- temporaries were intellectually positioned to be able to marry concepts from genetics and chemistry; MULLER was by no means a sophisticated chemist, but used an aggressive and insightful imagination in bor- rowing from the insights of other disciplines. The new horizon of chemical mutagenesis offered no obstacle as yet to the concept of evolutionary indeterminacy. Despite effects on "the frequency of gene mutation in general, . . . each individual muta- tion remains a chance and uncontrollable event, from the macroscopic standpoint." This has remained ge- netic orthodoxy to the present day, bolstered by re- vulsion about the criminal excesses of the Lysenkoist counter-doctrine, It deserves reexamination in the light of the intricacies of DNA conformation and its secondary structure, which are indisputably coupled to regulated gene expression (DAVIS 1989; LEDER- BERG 1989). MULLER'S consideration of heterochro- matin position effect as a &-acting influence of chro- matin coiling on gene expression is a harbinger of today's second look. Despite the molar indeterminacy of evolution, and the disruptive "bad" consequence of most mutations, "the Maxwell demon of natural selection. . brings order out of mutation's chaos despite itself." MULLER could not yet know of the plethora of phenotypically silent mutations in DNA which today support a much greater role of mutation pressure and genetic drift in evolution (KIMURA 1991). MULLER then turns to nonchromosomal genes. Chloroplasts are the best worked out; but animal cells can do without them. Uniparental inheritance con- strains the diversification of chloroplast genomes, and their limited content suggests they have a correspond- ingly small role in evolution. The chloroplast probably "had a common ancestry with the chromosomal genes, dating back to the period before the latter had become organized into typical nuclear chromosomes." This conjecture was voiced on the brink of a new and successful cycle of evolutionary attribution of chloro- plasts to endosymbiotic cyaiobacteria (LEDERBERG 1952; MARGUIJS 1981). LINDEGREN and SPIEGEL- MAN'S yeast "cytogenes" are also mentioned, but with cautious reservations about the "links of the evidence" for the regular production of self-reproducing replicas from a chromosomal gene. This caution was amply vindicated. However, proviruses, from lambda to HI\ today, securely occupy that niche. Some exceptional instances of gene amplification may follow a similar pattern (STARK et al. 1989). For nonchromosomal genes in animal cells, MULLER attends to DARLINGTON and ALTENBURG'S specula- tions about plasmagenes and viroids. These had some support from SONNEBORN'S work 011 kappa in Para- mecium, and this is extensively discussed. But while "The Gene" was in press, SONNEBORN revised his prior formulation of a chromosomal origin of kappa, and MULLER footnotes this. The polemics about such par- ticles being viruses, symbionts or genes were the im- mediate stimulant for the overarching concept of plas- mids (LEDERBERG 1952), which has largely dissolved the controversy. Gene duplication within the chromosome is uncon- troversial. After subsequent divergent mutation, "the germ plasm becomes not merely more compound but more complex and . . . the possibilities of organiza- tional complexity for the body in general should rise also." MULLER was the first high peer in genetics to enun- ciate that "virus particles . . . which fulfil the deflni- tion of genes in being self-determining in their repro- duction and capable of transmitting their mutations, are composed of. . . nothing but nucleoprotein." He finds appealing DELBRCCK'S early ideas (1941) about polypeptide template-directed assembly "by means of a resonance . . . at peptide links followed up by a finishing up of the peptide connections, and associated undoing of the" bonding of the old and new chains. To be sure, there is no evidence that gene synthesis involves peptide links: and protamines are too simple. However, some kind of steric complementarity might pertain between basic proteins and acid DNA. By 1953, WATSON and CRICK would model DNA-DNA complementarity as the core of modern molecular genetics. In 1947, chromosome synapsis was a possible clue to recognitional mechanisms in gene duplication, Perspectives though MULLER is quick to emphasize that karyologi- cal synapsis involves forces over microscopically visible distances. Especially perplexing (then and today) is meiosis in Neurospora, where "the chromosomes come into contact while still in a condensed, closely coiled condition . . ." MULLER'S response here is an invocation of temporal vibrations, a premonition of holography, better left without further comment, though similar ideas still recur in neurobiological spec- ulation. Many aspects of synapsis, and the example of Neurospora's ability to scan for duplicated segments (SELKER 1990), remain a challenging mystery today. 47 P ROY SOC LOND 6 610 W 1 ABOLINS L MP CELL RE 3 1 52 ANDERSON TF BEADLE GW 15 17 z 10 BOTAN REV ANN R BIOCH ANN R PHYSL FORTSCH CH P NAS US ANN A BICCH BLOOD NATURE T FARAD SOC ANN R EIOCH NATURE 464 49 727 48 17 48 Akin to virus replication is pneumococcal transfor- mation. MULLER'S endorsement was an important tes- timonial for geneticists of that decade: In my opinion, the most probable interpretation of these virus and Pneumococcus results then becomes that of actual entrance of the foreign genetic material already there, by a process essentially of the type of crossing over, though on a more minute scale . that is, there were, in effect, still viable bacterial "chromosomes" or parts of chromosomes floating free in the medium used. These might, in my opinion, have penetrated the capsuleless bacteria and in part at least taken root there, perhaps after having undergone a kind of crossing over with the chromosomes of the host. In view of the transfer of only a part of the genetic material at a time, at least in the viruses, a method appears to be provided whereby the gene constitution of these forms can be analyzed, much as in the cross-breeding test on higher organisms. However, unlike what has so far been possible in higher organisms, viable chromosome threads could also be obtained from these lower forms for in vitro observation, chenlical analysis, and determination of the genetic effects of treatment. This emboldened me to posit a close analogy to the newly discovered phenomenon of genetic exchange in bacteria (LEDERBERG 1947). But he could not yet accept that the transforming activity had been proven to be pure DNA (contra "nucleoprotein"`). PHOEBES LEVENE had laid the groundwork of DNA chemical structure with the elucidation of the constituent de- oxyribonucleotides and their linkage through phos- photriester bonds. But the model closest to hand was that of a monotonous tetranucleotide, which left little room for genetic informational variety. MULLER left several hints that larger polymers might alter our perceptions, but he had no platform for more detailed chemical modeling or experiment. How do genes work? MULLER cautions against too facile a depiction of the gene or its primary product as an enzyme. I translate his two arguments: that the known primary gene product is another gene, and this has properties not shared by known enzymes; and that developmental pathways will almost always show pleiotropic complications, viz. several genes affecting one enzyme even if this is not seen in initial surveys ("new methods will be needed before the primary gene products can be identified"). I shared this skep- ticism about the ultimate rigor of the "one-gene:one- BLUMEL J BROWN GB BURT NS CHAYEN J COULSON CA DAVIDSON JN DEVI P ESSRIG IM FRANKEL OH GULLANO JM HARAlSdN JA HERSHEY AD H0FFMANN.H HhOWlTZ NH HUSKINS CL JEHLE H .I KNIGHT CA LANHAM UN LAVELLE A LE0ERBER.J II LEWIS EB LILLIE RS LWOFF A MAZIA D MEDAWAR PB MULLER HJ PONTEC0R.G I. ROPER JA SCHMITT FO SPARROW AH SPIEGELM S ,, STONE WS VOGT M WATSON JD WITKIN EM ZINDER ND N ENG J ME0 HEREDITY COLD S HARB ANN R MICRO ADV GENETIC 2 NATURFO B ADV GENETIC AM NATURAL NATURE P NAS US SCIENCE ADV VIRUS R AM NATURAL ANAT REC COLD S HARB GENETICS PHYSIOL REV ADV GENETIC AM NATURAL ANN IN PAST P NAS US BIOL REV AM J HU GEN ADV ENZYMOL ADV GENETIC SYM SOC EXP NATURE ANN R PHYSL AM J BOTANY COLD S HARB SYM SOC EXP P NAS US Z INDUKT AB COLD S HARB II J BACT 5 A zz 6 171 48 R 240 4 12 i 6 R 3 81 161 M 1:: R 8; 119 16 82 78 R zi 2 13 5 6 166 k 10 300 48 561 48 141 53 906 51 472 53 777 52 155 49 503 47 15 49 103 50 95 47 19 47 a9 53 63 51 33 50 401 47 80 48 238 50 454 50 153 54 213 52 305 54 413 51 505 47 403 52 73 50 5 48 711 50 521 54 360 47 111 50 121 52 141 53 218 52 956 50 i 48 439 47 2ii 47 286 48 59 47 324 50 18 123 53 12 256 47 64 679 52 enzyme" theory, to the irritation of BEADLE and HOROWITZ (LEDERBERG 1956). In retrospect, it ws an indispensable heuristic, and complications like the intervention of mRNA, RN.4 splicing and editing, and post-translational modifications could be left for later historical superimposition on the initial skeleton of colinearity of DNA with protein. MULLER was among the first to extrapolate from basic scientific knowledge of genetic mutation and evolution to their human implications. Mutational disorder will eventually afflict the human genome as a result of the blunting of natural selection by culture; but this process will take centuries and \ve have time to educate ourselves in countermeasures of genetic hygiene. His estimate of one lethal equivalent as the 3 1 6 J. Lederberg genetic load of recessive mutation in the contempo- rary human still stands. Meanwhile, we should be cautious about exposure to X-rays and to "special chemicals." It is curious that he makes no reference here to nuclear explosions. Fallout was yet to enter the lexicon (after the H-bomb tests), and even then MULLER was concerned that its effects might be exaggerated in contrast to other radiation hazards, and in ways that might erode nu- clear deterrence of Soviet aggression (CARLSON 198 1). Horrified by the "terrible Nazi perversion of ge- netics," he believes that "any conscious guidance over our own genetic processes" be deferred for voluntary concern, understanding, and better developed social consciousness. Many psychological traits, in particular, are attributed to "training or by largely unwitting conditioning." But eventually social wisdom should allow "the self-reproduction of the gene and the self- reproduction of intelligence [to] reinforce one an- other in an ascending curve." It will illustrate the impact of this article to list the citations that appeared in 1947-l 954 (Figure 1). LITERATURE CITED AUERRACH, C., J. M. RORSON and J. G. CARR, 1947 The chemical production of mutations. Science 105: 243-247. AVERY, 0. T., C. M. MACLEOD and M. MCCARTY, 1944 Studies on the chemical nature of the substance inducing transforma- tion of pneumococcal types. J. Exp. Med. 79: 137-158. CARLSON, E. A., 1981 Genes, Radiation, and Society. The Lzj2 and Work of H. J. Muller. Cornell University Press, Ithaca, N.Y. CROW, J. F., 1990 H. J, Muller, scientist and humanist. Wis. Acad. Rev. 36(3): 19-22. DAVIS, B. D., 1989 Transcriptional bias: a non-Lamarckian mech- anism for substrate-induced mutations. Proc. Natl. Acad. Sci. USA 86: 5005-5009. DELBRUCK, M., 1941 A theory of autocatalytic synthesis of poly- peptides and its application to the problem of chromosome reproduction. Cold Spring Harbor Symp. Quant. Biol. 9: 122- 126. DRAKE, J. W., 1989 Mechanisms of mutagenesis. Environ. Mol. Mutagen. 14(16): 11-15. DUBOS, R. J., 1945 The Bacterial Cell. Harvard University Press, Cambridge, Mass. EMERSON, S., 1944 The induction of mutation by antibodies. Proc. Natl. Acad. Sci. USA 30: 179-183. HAYNES, R. H., 1989 Genetics and the unity of biology. Genome 31: l-7. KIMURA, M., 1991 Recent development of the neutral theory viewed from the Wrightian tradition of theoretical population genetics. Proc. Nat]. Acad. Sci. USA 88: 5969-5973. LEDERBERC, J., 1947 Gene recombination and linked segregations in Escherichia coli. Genetics 32: 505-525. LEDERBERG, J., 1952 Cell genetics and hereditary symbiosis. Phys- iol. Rev. 32: 403-430. LEDERBERG, J., 1956 Comments on gene-enzyme relationship, in Enzymes: Units of Biological Structure and Function (Ford Hos- pital Interational Symposia), edited by 0. H. GAEBLER. Aca- demic Press, New York. LEDERBERG, J., 1989 Replica plating and indirect selection of bacterial mutants: isolation of preadaptive mutants in in bac- teria by sib selection. Genetics 121: 395-399. MARGULIS, L., 1981 Symbiosis in Cell Evolution. Freeman Press, San Francisco. MULLER, H. J., 1947 The gene. Proc. R. Sot. Land. B 134: l-37. PAULING, L., and D. H. CAMPBELL,, 1942 The manufacture of antibodies in vitro. J. Exp. Med. 76: 2 1 l-220. SELKER, E. U., 1990 Premeiotic instability of repeated sequences in Neurospora crassa. Annu. Rev. Genet. 24: 579-6 13. STARK, G. R., M. DEBATISSE, E. GIULOTTO and G. M. WAHL, 1989 Recent progress in understanding mechanisms of mam- malian DNA amplification. Cell 57: 901-908. WATSON, J. D., and F. H. C. CRICK, 1953 Molecular structure of nucleic acids. A structure for deoxyribose nucleic acid. Nature 171: 737-738.