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It is difficult to willingly forgetabout DNA — what it looks like,how it works, what you can dowith it, and what it means as aunifying idea. It’s tricky, but it’swhat would be required to lookover Avery’s shoulder in 1943 ashe wrote up his paper on the‘transforming principle’. Forauthenticity’s sake, you couldthrow in a certain preoccupationwith proteins and theirfascinating diversity, and anunderstanding of DNA as anuncomplicated chemicalconstituent of the nucleus. At thatpoint, you’d probably besufficiently skeptical.

The work reported in Februaryof 1944 by Oswald Avery, ColinMacLeod and Maclyn McCartyhas been called the century’sgreatest biological discovery, butalso the ‘undiscovered discovery’.It broke the seal on modernbiology, but famously failed to wina Nobel prize. It showed that DNApossessed genetic informationthat could transform the heritablecharacter of cells, but the world ofbiological research in 1944 hadenough in the way of distractions,assumptions, and divisions towithhold its attention from such arevolutionary idea.

In some ways, the story wasn’tall that new, having begun with apaper sixteen years before. As anofficer of Britain’s Ministry ofHealth, the bacteriologist FredGriffith had in 1928 published theoutcome of his latest efforts atcharacterizing the puzzle ofserological types present amongpneumococci. Samples collectedfrom pneumonia patients routinelyyielded multiple pneumococcaltypes representing varioussubsets of the known serologicalcategories. The basis for thisphenomenon, and its clinicalrelevance, were unclear tomedical bacteriologists.

Pneumonia was a leading killerof the day, but bacteriologists hadfew tools with which to study thecell biology and virulence of theorganism responsible. Armed withsputum samples, chocolate bloodmedia plates and an assortment oftype-specific sera, Griffith setabout characterizing thepneumococcal types present fromsamples collected at various pointsduring illness, using sequentialantibody neutralization andinoculation into mice as an assay.Critically, though perhapssurprisingly, he did not assume asan explanation for the type resultsan etiology of multiple infection,and pursued experiments testingthe possibility of in vivo type-switching. In doing so, hesucceeded in demonstrating thattype transformation could occur inmice. Subcutaneous inoculation ofa mixture of live — but avirulent —pneumococci of a first serotype,and heat-killed, virulent cells of asecond serotype led to virulentdisease in an injected mouse;moreover, he subsequentlyisolated virulent cells of the secondserological type. As is apparent inhis discussion of the work,

Griffith’s concerns lay with theclinical treatment of pneumonia,not the chemical nature of thetransformation. Besides, asMcCarty points out in his excellentbook, The Transforming Principle,at that time ‘bacteriology haddeveloped as a science almost asif unrelated to the rest of biology. Itwas too early in its history toexpect genetic interpretations ofany phenomena encountered in thelaboratory’.

Avery was at that time involvedin characterizing the antigenicnature of the pneumococcalcapsule, working at what was thenthe Hospital of the RockefellerInstitute for Medical Research.Upon seeing Griffith’s paper hewas reportedly intrigued but notwholly convinced by the findings,at least until they were confirmedshortly afterwards by Fred Neufeldworking at the Koch Institute inBerlin. At about the time Griffith’sstudy appeared, it so happenedthat one of Avery’s associates,Martin Dawson, was writing up hisown observations regardingpneumococcal transformation.Griffith had attempted todemonstrate the transformingeffect in vitro but had failed;Dawson saw an opportunity andsucceeded with Richard Sia inrecapitulating in vitro the virulenceswitching he’d seen in mice. With a

Feature

The quiet revolutionSixty years have passed since Avery, MacLeod and McCarty publishedtheir landmark paper revealing DNA as the genetic material, writesHeather Dawes.

The transformation: This plate from Avery’s 1944 paper shows the dramatic changeassociated with transformation from ‘rough’ (and avirulent) type II cells (left), to ‘smooth’,virulent type III cells (right). (Reproduced from The Journal of Experimental Medicine,1944; 79:137-158, by copyright permission of The Rockefeller University Press.)

test-tube assay in hand, Avery’sgroup had the stage set for aconcerted biochemical analysis ofthe transformation phenomenon,but the ensuing story took nearly14 years to play out. Most yearsbrought their share of importantsteps forward, but a few sawalmost no progress. There was asignificant lull around 1934 whenAvery underwent thyroid surgeryfor the treatment of Graves’disease, and another hiatus in thelate thirties during which theproject was put aside for threeyears, possibly owing to the lack ofpublishable progress and morepressing clinical matters.

In the ’30s, progress and insightinto the nature of the transformingprinciple, as it came to be called,was accompanied by efforts tomake a complex and chronicallytroublesome assay simple andstable. A primary problem wascontamination of the system bywhat was later recognized to beDNase; once recognized for itsrelevance, DNase later proved acritical tool for proving the identityof the transforming principle. Onanother front, efforts were made

to identify the serum factornecessary to support in vitrotransformation. This was anunderstandable goal, as thestanding protocol called for adose of heated chest fluidsobtained from patient samples.Overall, according to McCarty,during those years“transformation in the test tuberemained an inconstant andmaddeningly unreliable process”.

In 1935 Avery and MacLeod hadyet to fully recognize that theywere dealing with a geneticphenomenon — thetransformation seemed to be castin one report in terms of inducedsystems of presumably extant, butlatent, synthetic enzymes. But thatyear also saw a notable advancein the finding that the transformingprinciple survived chloroformextraction, arguing against aprotein factor.

1940 marked a renewed effort,and the investigators began toappreciate an association betweenthe transforming principle andnucleic acids in their preps. Thiswasn’t an expected result. AsMcCarty has written, ‘Knowledgeof the occurrence and distributionof the nucleic acids in nature hadnot yet reached the point whereone could assume that all livingcells contained both RNA andDNA. Indeed, the notion had onlyrecently been discarded that therewere two general classes ofnucleic acid: plant nucleic acid…and animal nucleic acids…’.

Despite growing evidenceequating DNA with thetransforming principle, it wasn’tuntil the early ‘40s that the grouprecognized that the shinyprecipitate they’d been observingin their active preparations mightitself be DNA, and notpolysaccharide contaminant asthey had once assumed. Twofloors above, the proteinbiochemist Alfred Mirsky hadsucceeded in purifying anucleoprotein substance theytermed chromosin; throughinteractions with Mirsky, Avery’sgroup learned what purified DNAlooks like — fibrous, opalescent,and quite different from the brownpowdery form with which theywere acquainted. And theycontinued to gain valuable

evidence for DNA as the long-sought factor: it absorbed UV lightat the right wavelengths, actedlike a very large molecule in acentrifuge, and, most importantly,was resistant to the rightenzymes. Altogether, the evidencestrongly pointed to the conclusionthat the activity lackeddemonstrable protein, saccharide,or lipid, and was composed nearlyentirely of DNA.

They were aware they’d need tobuild a strong case. Their upstairsneighbor Mirksy was one of thefirst skeptics, saying that thetransforming principle could notpossibly be DNA because “nucleicacids are all alike”. This reflected awidely held viewpoint generatedmainly by Pheobus Levene’stetranucleotide theory, whichessentially stated that the fourbases of DNA were joined togetherin the same repeating order.Clearly this idea wasn’t compatiblewith DNA possessing much in theway of biological specificity. Inreality, there was only very limitedevidence for the tetranucleotideidea, and especially since DNAwas known to be closelyassociated with chromosomes,not everyone shared the view of its

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Oswald T. Avery: Born in Nova Scotia in1877 to a Baptist minister, Avery prac-ticed surgery in the early 1900s beforeturning to bacteriology, where he brokeground in his analysis of polysaccharidecapsules. A gifted speaker, he wasfamous for monologues he’d deliver tovisitors and new associates on a rangeof biological topics, but he was not fondof delivering talks. To close associatesand friends he was known as ‘Fess’ – forProfessor, a title he never held. (Photocourtesy of the Rockefeller UniversityArchives.)

Colin MacLeod: Also a minister’s sonborn in Nova Scotia, MacLeod skippedenough grades in school that McGillasked that he stay back a year ratherthan enter college at the age of fifteen.After his work with Avery, he headed upthe New York University Department ofBacteriology for fifteen years, later goingon to a career in science policy, workingfor the War Department, the NIH, and theNational Academies. (Photo courtesy ofthe Rockefeller University Archives.)

limited potential. But by and large,Avery’s group would be working atough room. As Hotchkiss hasstated, “to recapture the state ofbiochemical genetics around 1940,one must think separately of thebiochemists’ genetic knowledge,which was negligible, and of thegeneticists’ biochemicalknowledge, which, if sometimesmarked with great insight, wasmainly not functional”.

Avery exhibited a characteristicreserve in putting the story onrecord. The understated languageof the 1944 paper has beensuggested by some to evince alack of understanding on the partof the authors for the significanceof their findings. But according toMcCarty, Avery “held to thephilosophy that it was enough topresent the facts and leave theinterpretations to others. It wasnot that he was indifferent to theinterpretations, since he enjoyeddiscussing them …but he wasmore than reluctant to put hisspeculation in writing for publicconsumption”. This state of mindis reflected in a letter Avery writesat the time to his brother Roy, alsoa bacteriologist. He reveals hissuspicions in tell-tale statements(“Sounds like a virus — may be agene…”), but also asks Roy to bediscrete. “Talk it over with[colleague] Goodpasture but don’tshout it around — until we’re quitesure or at least as sure as presentmethod permits. It’s hazardous togo off half cocked — &embarrassing to have to retractlater.” Nonetheless, from the letter,and even his caution itself, itseems clear Avery senses he’sonto something big.

The appearance of the paper, inRockefeller’s Journal ofExperimental Medicine, wasn’tmarked by any notable events inthe way of scientific reception, butcircumstances wouldn’t havefavored it. Appearing four monthsbefore D-day, the paper greetedan audience that must have beenpreoccupied with war (in fact thewar claimed two of the story’spioneers – Griffith perished inLondon during the Blitz, andNeufeld is thought to have starvedin 1945 Berlin). There was also thedifficulty posed by the fact thatgeneticists did not generally read

JEM. This block effectivelysymbolized the larger problem thatAvery’s group was up against,namely a divide betweendisciplines that seemed to barelyallow for the possibility thatserotype transformation ofbacteria could have anything to dowith the austere concepts ofclassical genetics, and bemediated by a chemical poorlyunderstood even by biochemists.

The paper’s lack of visibility forthose who arguably needed mostto see it meant that its near-termsuccess was especially dependenton word-of-mouth and vulnerableto vocal criticism, particularly byinfluential figures. As it happened,Avery’s neighbor Mirsky becamethe work’s chief public critic,reiterating his conviction thatprotein activity had not beenconvincingly ruled out. Avery didnot feel the need to respond toMirsky, and generally eschewedinvitations to speak. Some ofthese opportunities were passedon to McCarty, but even atmeetings where sparks might haveflown – such as the 1946 ColdSpring Harbor Symposium on

heredity and variation inmicroorganisms where McCartyspoke – they didn’t, at least in anydramatic way.

But the paper was not ignored.Ultimately, it was the investigatorswho purposely set the course oftheir own work in response toAvery’s paper who brought aboutacceptance of its ideas. JoshuaLederberg, future president ofRockefeller and a young graduatestudent who had been greatlyimpressed by the paper, based hisfuture work on it, and hascontinued to be the paper’schampion ever since. Arguably themost critical positive influence wasthat of Erwin Chargaff, who turnedhis biochemical work to awholesale effort aimed atunderstanding amino acids in anenterprise that finally overturnedthe tetranucleotide hypothesis andcleared the way for acceptance ofDNA’s specificity. Forming abridge between two greatdiscoveries, Chargaff’ssubsequent work on basecomposition provided a much-needed clue to Watson and Crick.

Despite missing out on a Nobelprize, Avery did receiverecognition for his work; inparticular, the Copley Medal fromthe Royal Society, which hereceived in 1946, is seen as thefirst official recognition of hisachievement. The conservativenature of Nobel awarding did notwork in Avery’s favor, especially ashe was already in his mid-sixtieswhen the 1944 paper waspublished. The ideas needed timeto be accepted, and for many thatdidn’t occur until Hershey andChase’s work on phage DNAtransmission in 1952, and Watsonand Crick’s paper the followingyear. There was time for Avery toreceive a Nobel even after thesediscoveries, but it was not to be,and he died in 1955.

If it was indeed Watson andCrick’s ringing proclamation in1953 that made the Averydiscovery believable to stragglingdoubters, it serves to illustrate thatnot all revolutions in biology arequiet. But thanks to a missingprize, Avery’s legacy may be amore willing audience in biologyfor quiet revolutions still findingtheir voice.

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Maclyn McCarty: Born in South Bend,Indiana, ‘Mac’ was guided by well-readparents and formed an ‘AmateurResearch Chemists’ club with friends inhigh school. He studied medicine andwas a pediatrics intern before joiningAvery’s group. Following the transforma-tion work, he switched to the study ofrheumatic fever, and remained on thefaculty at Rockefeller — as did Mirsky,with whom he was admirably able tofoster a civil relationship. Now in his 90s,McCarty is Professor Emeritus at Rocke-feller. (Photo courtesy of the RockefellerUniversity Archives.)