Copyright 0 1991 by the Genetics Society of America Perspectives

Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove

Fifty Years Ago: The Neurospora Revolution

Norman H. Horowitz

Biology Division, Calijornia Institute of Technology, Pasadena, Calgornia 91 125

HIS year marks the fiftieth anniversary of the microorganisms, but it should be pointed out that the T publication of one of the pivotal works ofmod- Neurospora discoveriesfirst described in the 1941 ern biology, the first Neurospora paper of BEADLE paper werecrucial for making bacteria genetically and TATUM(1941). This brief paper, revolutionary useful. in both its methods and its findings, changed the BEADLE hadlearned of Neurospora at a lecture by genetic landscape for all time. Where previously there B. 0. DODGEgiven at Cornel1University in 1930, existed only scattered observations (albeit with some when the former was a graduate student. DODGE,a acute insights) on the relation between genetics and mycologist at the New York Botanical Garden, was a biochemistry, this paper established biochemical ge- strong advocate of Neurospora as a genetic organism. netics as an experimental science, one in which pro- It was he who found that theascospores-the products gresswould no longer belimited by the rarity of of meiosis-requiredheat shock to induce germination. mutants with biochemically knowablephenotypes, but (He had made this discovery originally in Ascobolus, where such mutants would be generated at will and by accident, after setting down some plates of asco- where findingscould be repeated and hypotheses spores ina sterilizing oventhat he thought was turned explored, as in other experimental sciences. This pa- off.) This finding made Neurospora available for ge- per was the first in a series of fundamental advances netic studies, and DODGE worked out the basic ge- in chemical geneticsthat by 1953 had bridged the gap netics of the organism. Among other things, he inves- between geneticsand biochemistry and ushered in the tigated the inheritance of mating type, albinism, and age of molecular biology. other monogenic characters. He showed that the eight I have explained in a recent memoir of BEADLE ascospores of an ascus display a perfect Mendelian (HOROWITZ1990) how the Neurospora investigation ratio (4:4). By isolating and culturing the ascospores arose from his earlier study of the genetics of eye- in the linear order in which they occur in the ascus, color synthesisin Drosophila with BORISEPHRUSSI, he discovered the patternsof first-and second-division and I will not repeat this history here. segregations. DODGE alsounderstood the benefits that The methodological innovations of the BEADLE- haploidy offered for genetic studies. When combined TATUMpaper were twofold. First, the authors intro- with the other features of Neurospora, it convinced duced whatwas for most geneticists a new kind of him that this ascomycete was the ideal genetic orga- experimental organism-amicroorganism that was nism. He frequently pointed this out to his friend T. ideally suited for classical genetic studies but which H. MORGAN,arguing that it was actually superior to differed from the classical organisms in that it grew Drosophila (ROBBINS1962). readily ona medium ofdefined chemical composition. As its second methodological innovation, the BEA- It was actually superior in some ways to the usual DLE-TATUMpaper introduced a procedure for re- experimental species becausethe entiremeiotic tetrad covering an important classof lethal mutations, could be recovered and cultured. Thisnovel creature namely those blocking the synthesis of essential bio- was the filamentousascomycete Neurospora crassa. logical substances.These were expressed in the orga- (Neurospora sitophila was also used inthe early studies, nism as new nutritional requirements. These muta- but was abandoned before long in favor of N. crassa.) tions were crucialfor understanding the biochemistry It is well known that the investigations that led to the of gene action. They displayed in a most convincing development of molecular genetics largely employed manner the central importance of genes in biochem-

Genetics 147: 631-635 (April, 1991) 632 N. H. Horowitz Aside from its revolutionary methods, the BEADLE- TATUMpaper was remarkable for the results it re- ported. Itdescribed three X-ray-induced mutants that grew on “complete medium” (a complex, undefined mixture containing yeast extract), but that failed to grow on “minimal medium” (a mixture consisting of the minimal nutrients capable of supporting the growth of wild-type Neurospora). The presumption was that the mutations expressed in these cultures affected genes needed for the production of growth- essential compounds present in complete, but not minimal, medium. A systematic search revealed that each of the mutants required a different substance. The three substances were pyridoxine, thiamine and p-aminobenzoic acid, and the inability to synthesize them was eventually shown,in every case, to be inher- ited as a single-gene defect. (The 194 1 paper reported on the genetics of only the “pyridoxineless” mutant.) FIGURE I.-BEADLE’S lantern slide explaining the procedure for isolating biochemical mutants of Neurospora. The thiamine-requiring mutant was found to respond to the thiazole moiety of thiamine by itself, implying istry and ended forever the idea that the role of the that the mutant could synthesize the pyrimidine half genes in metabolism was somehow a subordinate one. of the molecule and showing that genes were limited Genetics,which before the Neurospora revolution in the range of their individual chemical effects. had been notably isolated from the physical sciences, The fact thatthe first three mutants found by now found itself in the mainstream of biochemistry. BEADLEand TATUMwere vitaminauxotrophs reflects, Or, more correctly, genetics and biochemistry were at least in part, the relatively high frequency of such now seen to be different aspects of the same thing. mutants recovered by their method of mutant selec- A diagram of the BEADLE-TATUMprocedure is tion. [See BEADLEand TATUM(1945), Table 5, for a shown in Figure 1. This figure is reproduced from a listing of all Neurospora mutants identified and cited lantern slide drawn and lettered by BEADLE,one of a in the literature up to that time.] In this method, set that he used in lectures in the 1940s. As the slide ascospore descendants of irradiated conidia were iso- suggests, BEADLEfavored the word “sex” rather than lated and cultured separately (see Figure l), a proce- “mating type” in his writing and speaking about Neu- dure that recovers even mutants with trace require- rospora. It should be noted that the test tube labeled ments. The mass selection procedures that came later “vitamins” alsocontained nucleic acid components. are biasedagainst such mutants becauseof cross- The essential character of the substanceswhose feeding. syntheses were affected in the Neurospora mutants- The pyridoxineless mutant, No. 299, is of special amino acids, purines, pyrimidines, vitamins-suggested interest. This was the first mutant found by BEADLE that similar mutations should occur in other microbial and TATUM,and it was one of the few that were species. This proved to be the case. In thefirst impor- recovered in N. sitophila. It was, so to speak, the tant extension of the Neurospora findings, GRAY and breakthrough mutant, the one that vindicated their TATUM(1 944) showed that “biochemical mutations” ideas about a new kind of genetics. But itsimportance could be induced in bacteria. This result solved a did not end there. Soon after the 1941 paper was fundamental difficulty that had long prevented pro- published, BEADLEreceived a letterfrom an acquaint- gress toward a genetics of bacteria-that is, the lack of ance at the Merck Research Laboratory requesting a suitable markers-and led directly to the demonstra- culture of No. 299 for the purpose of developing an tion of genetic recombination inEscherichia coli by assay method for pyridoxine. BEADLEsent a transfer, TATUM’Sstudent JOSHUA LEDERBERG.Biochemical as he invariably did once a mutant had been referred mutations were induced later in yeast and other mi- to in print. BEADLE firmly believed that this policy croorganisms. Modern microbial geneticsis to a large was in the best interest of science, a belief that was extent based on mutations of the type first described certainly confirmed in this case because,in the course by BEADLEand TATUMin their 1941 paper and on of their investigation, the Merck group discovered temperature-sensitive alleles of theseand otheressen- that No. 299 would grow without pyridoxine if the tial genes.The discovery of temperature-sensitive mu- pH of minimal mediumwas raised to 6 from its normal tants followed directly from the 1941 paper, as will value of 5 (STOKES, FOSTERand WOODWARD1943). be shown later. I recall first hearing of this unexpected result at an Perspectives 633

GEORGEBEADLE afternoon tea-break in the BEADLElab atStanford who has written a perceptive essay on the rediscovery University. In the ensuing discussion, it was decided (EDGAR1966). to learn if other environmental variables-tempera- In another, and different, early application, a tem- ture, in particular-might also affect the phenotype of perature-sensitive mutant of E. coli was used to dem- mutants in a specific way. The mutant hunt that ran onstrate that genes determine the molecular proper- more or less continuously in the lab was accordingly ties, as well as the presence or absence, of enzymes modified to includean incubation step at35” in (MAASand DAVIS1952). addition to the usual one at 25”. Soon the first tem- The Neurosporamutants, as everyone knows, perature-sensitive mutants were found.The first pub- opened a new approach to the study of biosynthetic lished description of one of these was by MITCHELL pathways, the cumulative results of which led to the and HOULAHAN (1946). These mutants turned out to one gene-one enzyme theory (BEADLE1945). This be very important-arguably moreimportant than theoryhad already been foreshadowed in the first thenonconditional auxotrophs. By modifying the paragraph of the 1941 paper, where the authors sug- gene in such a way that its activity was abolished in gest the possibility that genesmay act “by determining only part of the organism’s normal temperature the specificities of enzymes” with the further possibil- range, temperature-sensitive mutants were essentially ity of “simple one-to-one relations”between genes and unselected. That is, mutants whichin the ordinary chemical reactions. These ideas doubtless grew out of course of events would be lost because of the im- the authors’ earlierwork on Drosophila eye colors. In permeability, instability, or unavailability forany his Nobel lecture, BEADLE,in an oft-quoted passage other reason of the genetic end-product, can be re- referring to onegene-one enzyme, said, “In this long, covered as temperature-sensitive alleles. Regarding roundabout way, first in Drosophila and then in Neu- them in the light of present-day knowledge, we can rospora, we had rediscovered what GARRODhad seen see that any gene whose end-product is aprotein so clearly so many years before” (BEADLE1959; GAR- should be recoverable as a temperature-sensitive mu- ROD 1909). BEADLEwas without doubt sincere in this tant. This attribute made them useful in an early test characteristically generous remark, but was he right? of the one gene-one enzyme hypothesis (HOROWITZ And if he was right, doesthis diminish the importance 1948,1950; HOROWITZ and LEUPOLD195 1). The of the BEADLE-TATUMaccomplishment? The answer utility of temperature-sensitive mutants for problems to both questions is, I think, “No.” of this kind was rediscovered years later by EDGAR, In a penetrating discussion of the first question, 634 N. H. Horowitz

SCRIVERand CHILDS(1989) raise the question of Heredity, published in 1925, is still in print. Usually whether, at this date, we can actually know what was very clearheaded, Wilson took what can only be de- in GARROD’Smind when he wrote his great works on scribed asan exceedingly murkyview when, regarding human hereditary disease. These authors show that the role of the genes, he wrote: GARROD’Sunderstanding of genetics appears not to In what sense can the chromosomes be considered as have extended beyond 1910 (he liveduntil 1936). agents of determination? By many writers they have been They suggest that “His words could have meant one treated as the actual and even as the exclusive “bearers of thing to him when he uttered them and something heredity”; numerous citations from the literature of the else to us who are tempted to freight them with subject might be offered to show how often they have been contemporary significance.”They conclude that GAR- treated as central, governing factors of heredity and devel- ROD could hardly have had BEADLE’S“one gene-one opment, to which all else is subsidiary. . . Many writers, enzyme” ideain mind. It is hard todisagree with them while avoiding this particular usage, have referred to the when one considers the state of geneticsand biochem- chromosomes or their components[WILSON rarely used the istry at the time. The year 1909, when GARROD’S word “gene”]as “determiners”of corresponding characters; famous book was published, was the same year that but this term, too, is becoming obsolete save as a convenient descriptive device. The whole tendency of modern investi- JOHANNSEN introduced the word gene into the lan- gation has been toward a different and more rational con- guage. The chromosome theory of inheritance was ception which recognizes the fact that the egg is a reaction- still in the future. Biochemistry was also in an embry- system . . . and that (to cite an earlier statement) “the whole onic state. In a monograph published in 1914, W. M. germinal complex is directly or indirectly involved in the BAYLISSconsidered it necessary to defend the idea production of every character” (WILSON1925). that enzymes couldbe assumed to be definite chemical compounds, “at all events until stronger evidence has In an obvious and not very interesting sense, the been brought to the contrary.” The one thing that foregoing statement is correct; but in another and seemedclear was that enzymes were not proteins much more important one, it is altogether wrong. (BAYLISS1914). This question was not settled until With the Neurospora revolution, musings of this sort SUMNERcrystallized urease in 1926. on the natureof gene action faded away. The evidence The same considerations must apply with equal or for a one-to-one relation between genes and enzymes greater force to the most prescient ofall writings (actually proteins, later modified to polypeptides) now about genes and enzymes, those of the French genet- became clear, abundant andundeniable. The individ- ual gene in someway determined the specific enzyme, icist LUCIENCU~NOT. In 1903, CU~NOTdiscussed his although it was not yet seen how. The efforts of the celebrated experiments on the inheritance ofcoat pre-Neurospora workers to understand gene action color in mice in terms of mnimons (genes), enzymes, had been made with systemsoften not suited for both and achromogen (see WAGNER1989). Sadly, CU~NOT biochemical and genetic studies. BEADLEand TATUM gave up genetics and discouraged his students from changed this by founding a new science based on an entering the field; see BURIAN,GAYON and ZALLEN organism and an experimental protocol designed to (1 988). be maximally useful for the purposes of biochemical There were, of course, later antecedents of the one genetics. In doing so, they transformed biology, and gene-one enzyme principlein the writings ofWRIGHT, that is the reason we remember this fiftieth anniver- HALDANEand others, where unfamiliarity with mod- sary. ern science does not enter in. But while these works were correct in deducing that genes mustact through LITERATURECITED their effects on enzymes (and other proteins), none of them succeeded in persuading geneticists of the clas- BAYLISS,W. M., 1914 TheNature of Enzyme Action, pp. 33, 36. sical era that a direct relation betweengenes and Longmans, Green & Co., London. BEADLE,G. W., 1945 Biochemical genetics. Chem. Rev. 37: 15- proteins was real and important and was, in fact, the 96. key to understanding the organization of living mat- BEADLE,G. W., 1959 Genes and chemical reactions in Neuro- ter. According to STURTEVANT(1965), geneticists spora. Science 129 1715-1719. were disinclinedto accept simple ideas ofgene action BEADLE,G. W., and E. L. TATUM,1941 Genetic control of bio- because they were convinced that development was chemical reactions in Neurospora. Proc. Natl. Acad. Sci. USA 27: 499-506. too complex a process to be explained by any simple BEADLE,G. W., and E. L. TATUM,1945 Neurospora. 11. Methods theory. Not long before he died, STURTEVANTtold of producing and detecting mutations concerned with nutri- me that especially E. B. WILSON’Sposition on gene tional requirements. Am. J. Bot. 32: 678-686. action had carried much weight. WILSON,a cytologist, BURIAN,R. M.,J. GAYONand D. ZALLEN, 1988 The singular fate of genetics in the history of French biology, 1900-1940. J. was one of the most influential figures in American Hist. Biol. 3: 357-402. biology. Although he died in 1939, the third edition EDGAR,R. S., 1966 Conditional lethals, pp. 166-170 in Phage and of his monumental book, The Cell in Development and the Origins of Molecular Biology,edited by J. CAIRNS, G.S. STENT Perspectives 635

and J. D. WATSON.Cold Spring Harbor Laboratory, Cold mutant of Escherichia coli. Proc. Natl. Acad. Sci. USA 38: 785- Spring Harbor, N.Y. 797. GARROD,A. E., 1909 Inborn Errors OfMetabolism. Frowde, Hodder MITCHELL,H. K., and M. B. HOULAHAN,1946 Neurospora. IV. & Stoughton, London. A temperature-sensitive, riboflavinless mutant. Am. J. Bot. 33: GRAY,C. H., and E. L. TATUM,1944 X-ray induced growth 31-35. factor requirements in bacteria. Proc. Natl. Acad. Sci. USA 30 ROBBINS,d. J., 1962 Bernard Ogilvie Dodge. Biogr. Mem. Natl. 404-410. Acad. Sci. USA 36 85-124. HOROWITZ,N. H.,1948 The one gene-one enzyme hypothesis. SCRIVER,C. R., and B. CHILDS,1989 Garrod’sInborn Factors in Genetics 33: 61 2-61 3. Disease. Oxford University Press, New York. HOROWITZ,N. H., 1950 Biochemical genetics of Neurospora, STOKES,J. L., J- W. FOSTER and C. R. WOODWARD,JR., Adv. Genet. 3: 33-71. 1943 Synthesisof pyridoxin by a “pyridoxinless”x-ray mutant HOROWITZ,N. H., 1990 George Wells Beadle (1903-1989).Ge- of Neurospora sitophila. Arch. Biochem. 2: 235-245. netics 124 1-6. STURTEVANT,A. H., 1965 A History ofcenetics, p. 101. Harper & HOROWITZ,N. H., and U. LEUPOLD,1951 Some recent studies Row, New York. bearing on the one gene-one enzyme hypothesis. Cold Spring WAGNER,R. P., 1989 On the origins of the gene-enzyme hypoth- Harbor Symp. Quant. Biol. 1665-74. esis. J. Hered. 80 503-504. MAAS, W. K., and B. D. DAVIS,1952 Production of an altered WILSON, E. B., 1925 TheCell in Development andHeredity, 3rd pantothenate-synthesizing enzyme by a temperature-sensitive edition, pp.975-976.Macmillan, New York.