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Dedication

ToOswaldT.Avery

PossiblyIamascientistbecauseIwascuriouswhenIwasyoung.Icanrememberbeingten,eleven, twelveyearsoldandasking,‘Nowwhyisthat?WhydoIseesuchapeculiarphenomenon?Iwouldliketounderstandthat.’

LINUSPAULING

TableofContents

Cover

TitlePage

Copyright

Dedication

Epigraph

Introduction

ChapterOne:WhoCouldHaveGuessedIt?

ChapterTwo:DnaIsConfirmedastheCode

ChapterThree:TheStoryinthePicture

ChapterFour:ACoupleofMisfits

ChapterFive:TheSecretofLife

ChapterSix:TheSisterMolecule

ChapterSeven:TheLogicalNextStep

ChapterEight:FirstDraftoftheHumanGenome

ChapterNine:HowHeredityChanges

ChapterTen:TheAdvantageofLivingTogether

ChapterEleven:TheVirusesThatArePartofUs

ChapterTwelve:GenomicLevelEvolution

ChapterThirteen:TheMasterControllers

ChapterFourteen:OurHistoryPreservedinOurDna

ChapterFifteen:OurMoreDistantAncestors

ChapterSixteen:TheGreatWildernessofPrehistory

ChapterSeventeen:OurHumanRelatives

ChapterEighteen:TheFateoftheNeanderthals

ChapterNineteen:WhatMakesYouUnique

ChapterTwenty:TheFifthElement

Bibliography

ChapterNotes

Index

Acknowledgements

BytheSameAuthor

AboutthePublisher

Introduction

Nospecialactofcreation,nosparkoflifewasneededtoturndeadmatterintolivingthings.Thesameatomscomposethemboth,arrangedonlyinadifferentarchitecture.

JACOBBRONOWSKI,THEIDENTITYOFMAN

Bronowskibeginshismorefamousbook,TheAscentofMan,withthewords,‘Manisasingularcreature.Hehasasetofgiftswhichmakehimuniqueamongtheanimals:sothat,unlikethem,heisnotafigureinthelandscape–heisashaperofthelandscape.’Butwhyshould we humans have become shapers of the landscape rather than mere figuresinhabitingit?Wedifferfrom,say,aseahorseoracheetahbecauseourgeneticinheritance,thesumoftheDNAthatcodesforus,isdifferentinhumanswhencomparedtothehorseor thecheetah.Wecall this thegenome,or, tobemore specific inourcase, thehumangenome.

Our genome defines us at themost profound level. That same genome is present inevery one of the approximately 100,000 billion cells that make us who we are asindividualmembersofthehumanspecies.Butitrunsdeeperthanthat.Inmorepersonalterms,inmyriadtinyvariationsthatweeachpossessandareindividualtousonly,itistheveryessenceofus,allthat,ingeneticandhereditaryterms,wehavetocontributetoouroffspring,and through themto thesumtotalevolutionary inheritanceofourspecies.Tounderstanditistoknow,inthemostintimatesense,whatitmeanstobehuman.Notwopeopleintheworldtodayhaveexactlythesamegenome.Evenidenticaltwins,whowillhavebeenconceivedwithexactlythesamegenome,willhavedevelopedtinydifferencesbetweentheirgenomesbythetimeoftheirbirths:differencesthatmayhaveariseninpartsoftheirgenomesthatdon’tactuallycodeforwhatwenormallymeanbygenes.

Howstrangetorealisethatthereisactuallymoretoourindividualgenomethangenesalone.Butletusputasidesuchdetailsforthemomenttofocusonthemoregeneraltheme.How could a relatively simple chemical code give rise to the complexity of a humanbeing? How could our human genome have evolved? How does it actually work?Immediatelyweareconfrontedbymysteries.

To answer these questions we need to explore the genome’s basic structure, itsoperatingsystemsanditsmechanismsofexpressionandcontrol.Somereadersmightreactwith incredulity. Surely any such exploration promises a journey into extraordinarycomplexity,onethatisfartooobscureandscientificforanon-scientistreader?Infactthisbookisaimedatexactlysuchareader.Asweshallsee,thebasicfactsareeasyenoughtograsp,andthewaytograspthemis tobreaktheexplorationintoaseriesofsimple,andeminently logical, stages. This journey will lead us through a sequence of remarkablerevelations about our human history – even into the very distant past of our ancestors’livesandtheirprehistoricexplorationofourbeautifullife-givingplanet.

Theexplorationwill raiseother, equally important,questions, too.How, forexample,does this extraordinary entity that we call the human genome enable our humanreproduction–thefertilisationofourmaternaleggwiththepaternalsperm?Howdoesitcontrolthequasi-miracleofthedevelopingembryowithinthemother’swomb?Returningtogeneralitiesforthemoment,though,wecanbesurethatanimportantingredientofthegenome,anditsessentialnature,ismemory–thememory,forexample,ofthetotalityofevery individual human’s genetic inheritance. But how exactly does it perform thisremarkable feat ofmemory?Weknow already that thiswonder chemicalwe callDNAworkslikeacode,buthowcouldanycoderecallthecomplexinstructionsthatgointothemakingofcellsandtissuesandorgans,andthenoncemade,bringthemintofunctioninthesinglecoordinatedwholethatcomprisesthehumanbeing?Eventhenwehavehardlybeguntoconfrontthemysteriesofthehumangenome.Howdoesthissameextraordinarystructureacquiretheprogrammingthatgiftsthegrowingchildwiththewonderofspeech,that bestows the related capacity for learning,writing and education, andwhichmakespossible thematuringof thenewborn to the futureadult,who then repeats thecyclealloveragainwhenheorshebecomesaparentinturn?

Thewonderisthatallofthismightbeencompassedinaminusculeclusterofchemicalsincluding, but not exclusive to, themastermoleculewe call deoxyribonucleic acid–orDNA.Thischemicalcodesomehowrecordsthegeneticinstructionsformakingus.Builtinto that codemust be the potential for individual liberty of thought and inventiveness,enablingeveryhumanartistic,mathematicalandscientificcreativity.Itgivesrisetowhateachofusthinksinnatelyasourprivateinviolate‘self’.Somehowthatsameconstructionof ‘self’made possible the genius ofMozart, Picasso, Newton and Einstein. It is littlewonderthatwelookattherepositoryofsuchpotentialwithawe.Nomoreisitsurprisingthatweshouldwanttounderstandthismysterythatliesattheverycoreofourbeing.

Onlyrecentlyhavewecometounderstandthehumangenomeinsufficientdepthandsubtlety to be able to put together itsmarvellous story – to discover, for example, thatthereisrathermoretoitthanDNAalone.ThisisthestoryIshallattempttoconveyinthisbook.

A few years ago I gave a lecture on a related theme atKing’sCollegeLondon.ThechairmanaskedmeifIplannedtowriteabookaboutit.WhenIsaidyes,heaskedmetopleasewriteitinwordsthatalayreader,likehimself,couldreadilyunderstand.

‘Justhowsimpledoyouwantmetomakeit?’

‘IwantyoutoassumethatI–yourreader–knownothingatalltobeginwith.’

This, then, I promise to do. There will be no complicated scientific language, nomathematicalorchemicalformulaeorunexplainedjargon,andIshallintroducenomorethan a handful of simple illustrations. Instead I shall begin from first principles andassume that the readers of this book know little about biology or genetics. Even non-biologistsmightrecallthemanysurpriseswhenthefirstroughdraftofthehumangenomewas announced to theworld, in 2001.The discoveries since then have confirmed that agooddealof thehumangenome– in itsevolution,structureandworkings– is far fromwhat we had earlier imagined. Those surprises do not diminish the importance of the

wealthofknowledgethathadgonebefore,butrather,likeallgreatscientificdiscoveries,theyenhanceit.Thankstothisnewunderstanding,humanityhasenteredwhatIbelievetobe a golden age of genetic and genomic enlightenment, which is already beingextrapolated tomany important fields, frommedicine to our human prehistory. I thinksocietyatlargedeservestounderstandthisandwhatitpromisesforthefuture.

one

WhoCouldHaveGuessedIt?

Thelargeandimportantandverymuchdiscussedquestionis:Howcantheeventsinspaceandtimewhichtakeplacewithinthespatialboundaryofalivingorganismbeaccountedforbyphysicsandchemistry?

ERWINSCHRÖDINGER

InApril1927ayoungFrenchman,RenéJulesDubos,arrivedattheRockefellerInstitutefor Medical Research, in New York, on what would appear to have been a hopelessmission.Tall,bespectacledandarecentgraduateofRutgersUniversity,NewJersey,withaPhDinsoilmicrobiology,Duboshadanunusuallyphilosophicalattitudetoscience.Hehadbecomeconvinced, through theworkof eminentRussian soilmicrobiologistSergeiWinogradsky, that itwas awaste of time studying bacteria in test tubes and laboratorycultures.Dubosbelievedthatifwereallywantedtounderstandbacteriaweshouldgooutandstudythemwheretheyactuallylivedandinteractedwithoneanotherandwithlifeingeneral,inthefieldsandthewoods–innature.

OngraduationfromRutgersDuboshadfoundhimselfunemployed.HehadappliedtotheNationalResearchCouncilFellowshipforaresearchgrantbuthadbeenturneddownbecausehewasn’tAmerican,butsomebodyhadscribbledahandwrittenmessageonthemarginoftherejectionletter.Duboswouldlaterreflectuponthefactthatitwaswritteninafemalehand,almostcertainlyaddedasakindlyafterthoughtbytheofficial’ssecretary.‘Why don’t you go and ask advice and help from your famous fellow countryman,DrAlexisCarrel, at theRockefeller Institute?’Dubos dulywrote toCarrel,which broughthim,inApril1927,tothebuildingonYorkAvenue,onthebankoftheEastRiver.

DubosknewnothingaboutCarrel,orindeedabouttheRockefellerInstituteforMedicalResearch,andwassurprisedonhisarrivaltodiscoverthatCarrelwasavascularsurgeon.Dubos had no academic knowledge of medicine and Carrel knew nothing about themicrobes that lived in soil. The outcome of their conversation was all too predictable;Carrelwas unable to help the youthfulmicrobiologist. Their conversation closed aboutlunchtimeandCarreldidDubosthecourtesyofinvitinghimtohavelunchwithhimintheInstitute’sdiningroom,whichhadtheattractionforahungryFrenchmanthattheyservedfreshlybakedbread.

It seemed entirely by accident that Dubos found himself sitting at a table next to asmall,slightlybuiltgentlemanwithadomedbaldheadwhoaddressedhimpolitely inaCanadian accent.Thegentleman’s namewasOswaldTheodoreAvery.AlthoughDuboslaterconfessedthatheknewaslittleaboutAveryashedidofCarrel,ProfessorAvery(hisclose associates referred tohimas ‘Fess’)was eminent inhis field,whichwasmedical

microbiology.Itwouldprovetobeameetingofhistoricimportancebothtobiologyandtomedicine.

AverysubsequentlyhiredDubosasaresearchassistant,inwhichroleDubosdiscoveredthefirstsoil-derivedantibiotics.MeanwhileAveryledhissmallteam–whowereworkingonwhathecalledhis‘littlekitchenchemistry’–onanotherquitedifferentquest,onethatwouldhelpunravelthekeytoheredity.Whythendoessocietyknowlittletonothingaboutthisvisionaryscientist?Tounderstandhowthisanomalycameaboutweneedtogobackintimetothemanhimselfandtheproblemshefacedthree-quartersofacenturyago.

*

In1927,whenDubosfirstmetAvery,theprinciplesofhereditywerepoorlyunderstood.The term ‘gene’ had been introduced into the nomenclature two decades earlier by aDanishgeneticist,WilhelmJohannsen.Curiously,Johannsenhadadoptedavagueconceptof heredity, known as ‘pangene’, that was first proposed by Charles Darwin himself.Johannsenmodified it to takeonboard thebelateddiscoveryof thepioneeringworkofGregorMendel,whichdatedbacktothenineteenthcentury.

ReadersmaybefamiliarwiththestoryofMendel–thecigar-smoking,Friar-Tuck-likeabbotofanAugustinianmonasteryinBrünn,Moravia(nowtheCzechRepublic)–whoundertook some brilliantly original studies of the peas he cross-bred in the monasteryvegetablegarden.FromthesestudiesMendeldiscoveredthebasisofwhatwenowknowasthelawsofheredity.Hefoundthatcertaincharacteristicsofthepeasweretransmittedto the offspring in a predictable manner. These characteristics included tallness ordwarfishness,presenceorabsenceofthecoloursyelloworgreenintheblossomsoraxilsoftheleaves,andthewrinkledorsmoothskininthepeas.Mendel’sbreakthroughwastorealise that heredity was stored within the germ cells of plants – and this wouldsubsequentlybeextrapolatedtoall livingorganisms–in theformofdiscretepacketsofinformation that somehow coded for specific physical characters or ‘traits’. Johannsencoinedtheterm‘gene’forMendel’spacketofhereditaryinformation.Atmuchthesametime,acombativeBritishresearcher,WilliamBateson,extrapolatedtheterm‘gene’tothediscipline he now called ‘genetics’ to cover the study of the nature and workings ofheredity.

Today,ifwevisitthefreedictionaryonline,wegetthefollowingdefinitionofagene:‘Thebasicphysicalunitofheredity;alinearsequenceofnucleotidesalongasegmentofDNA that provides the coded instructions for the synthesis of RNA, which, whentranslatedintoprotein,leadstotheexpressionofhereditarycharacter.’ButMendelhadnonotion of genes as such, and he certainly knew nothing about DNA. His discoverieslanguished in some little-read papers for forty years before theywere rediscovered andtheir importance was more fully understood. But in time his idea about the discretepacketsofhereditywenowcallgeneshelpedtoansweraveryimportantmedicalmystery:howcertaindiseasescomeaboutthroughanaberrationofheredity.

Wenowknow thatgenesare thebasicbuildingblocksofheredity inmuch the samewaythatatomsarethephysicalunitsthatmakeupthephysicalworld.Butduringtheearlydecadesof the twentiethcentury,nobodyhadany realnotionofwhatgenesweremade

from,orhowtheyworked.Buthereandthere,peoplebegantostudygenesinmoredepthby examining their physical expression during the formation of embryos or in thecausationofhereditarydiseases.Fruitfliesbecametheexperimentalmodelforpioneeringresearch in the laboratory of Chicago-based geneticist Thomas Hunt Morgan, whereresearchers locatedgenes,onebyone,onto structures calledchromosomes,whichwerethemselves locatedwithin the nuclei of the insect’s germ cells.The botanical geneticistBarbara McClintock confirmed that this was also the case for plants. McClintockdeveloped techniques that allowed biologists to visualise the actual chromosomes inmaize,leadingtothegroundbreakingdiscoverythat,duringtheformationofthemaleandfemale germ cells, thematching, or ‘homologous’, chromosomes from the two parentslinedupoppositeeachotherandthenthechromosomesswappedsimilarbitsso that theoffspring inherited a jumbled-upmixture of the inheritance from the two parents. Thiscuriousgeneticbehaviour(whichisknownas‘homologoussexualrecombination’)istheexplanationofwhysiblingsaredifferentfromoneanother.

By the early 1930s biologists and medical researchers knew that genes were actualphysical entities – chemical blocks of information that were lined up like beads in anecklacealongthelengthsofchromosomes.Inotherwords,thegenomecouldbelooselycomparedtoalibraryofchemicalinformationinwhichthebookswerethechromosomes.The discrete entities known as genes could then be compared to discrete words in thebooks.Thelibrarieswerehousedinthenucleiofthegermcells–inhumanterms,theovaandsperm.Humanshadatotalstackof46books,whichwerethesummedcomplementofovaandsperm,ineverylivingcell.Thiscameaboutbecausethegermcells–theovumandthesperm–contained23chromosomes,sothatwhenahumanbabywasconceivedthetwosetsoftheparentalchromosomesunitedwithinthefertilisedovum,passingonthefull complement of 46 chromosomes to the offspring.But this initial unravellingof the‘hereditymystery’merelyopenedupaPandora’sboxofnewmysterieswhenitcametoapplyinggeneticstothehugediversityoflifeonourfecundplanet.

Forexample,dideverylifeform,fromwormstoeagles,fromtheprotozoathatcrawledabout in the scum of ponds to humanity itself, carry the same kinds of genes in theirnuclei-boundchromosomes?

Themicroscopiccellular life forms, includingbacteriaandarchaea,donotstore theirheredityinanucleus.Thesearecalledthe‘prokaryotes’,whichmeanspre-nucleates.Allotherlifeformsstoretheirheredityinnucleiandareknowncollectivelyas‘eukaryotes’,which means true nucleates. From the growing discoveries in fruit flies, plants andmedical sciences, it was becoming rather likely – excitingly so – that some profoundcommonalities might be found in all nucleated life forms. But did the same geneticconcepts, such as genes, apply to the prokaryotes, which reproduced asexually bybudding, without the need for germ cells? At this time within the world of earlybacteriologytherewasevenadebateastowhetherbacteriashouldbeseenaslifeformsatall.Andviruses,whichwere for themostpart severalordersofmagnitudesmaller thanbacteria,werelittleunderstood.

Overtimemanyresearcherscametoseebacteriaaslivingorganisms,classifyingthemaccording to thebinomialLinnaean system; so, for example, the tuberculosisgermwaslabelled Mycobacterium tuberculosis and the boil-causing coccoid germ was labelledStaphylococcusaureus.OswaldAvery,with his extremely conservative nature, kept hisoptionsopen,eschewingthebinomialsystemandreferringtotheTBgermasthe‘tuberclebacillus’.It is instructiveforourstorythatDubos,whocametoknowAverybetterthananyothercolleague,wouldobservethat‘Fess’wassimilarlyconservativeinhisapproachtolaboratoryresearch.Sciencemustadherewithapuritanicalstringencytowhatcanbelogicallyobservedanddefinitivelyproveninthelaboratory.

In 1882German physicianRobertKoch discovered thatMycobacterium tuberculosiswas the cause of the greatest infectious killer in human history – tuberculosis. Kochconstructedacodeoflogicthatwouldbeappliedtobugswhenfirstdeterminingif theycausedspecificdiseases.Knownas‘Koch’spostulates’, thiswasuniversallyadhered to,andonceacausativebughadbeenidentifieditwasstudiedfurtherunderthemicroscope.Thusthebugwasdulyclassifiedinanumberofways.Ifitscellswereroundedinshapeitwas a ‘coccus’, if a sausage shape it was a ‘bacillus’, if a spiral shape it was a‘spirochaete’.Bacteriologistsmethodically studied the sort of culturemedia inwhich abugwouldgrowbest–whether inagar alone,or agarwithaddedoxblood, and soon.They also studied the appearance of the bacterial colonies when they were grown incultureplates–theircolours,thesizeofthecolonies,whethertheywereroughinoutlineor roundandsmooth, raisedor flat, stellate,granularordaisy-head.So the textbooksofbacteriologyextendedtheirknowledgebaseonafoundationofprecisefactualstudyandobservation.Andasunderstandinggrew,thisnewfoundknowledgewasappliedtothewaragainstinfection.

Oneoftheusefulthingstheylearntaboutdisease-causing,or‘pathogenic’,bacteriawasthatthebehaviourofthedisease,andthusofthebugitselfinrelationtoitsinfectedhost,couldbealteredbyvariousdeliberatemeans:forexample,throughrepeatedculturesinthelaboratory, or by repeatedly passing generations of the bug through a series ofexperimental animals. Through suchmanipulations itwas possible tomake the diseaseworse or less severe by making the bug either ‘more virulent’ or ‘attenuated’.Bacteriologists looked forways to extrapolate this tomedicine. InFrance, for example,theeminentLouisPasteurusedthisprincipleofattenuationtodevelopthefirstvaccinetobeusedsuccessfullyagainsttheotherwiseuniversallyfatalvirusinfectionofrabies.

Onefascinatingobservationthatcameoutofthesestudieswasthefactthat,onceabughadbeenattenuatedorbeendriventogreatervirulence,thechangeinbehaviourcouldbe‘passedon’tofuturegenerations.Coulditbethatsomefactorofthebug’sownheredityhadbeenalteredtoexplainthechangeinbehaviour?

Bacteriologists talked about ‘adaptation’, using the same term that was coming intovogue with evolutionary biologists when referring to evolutionary change in livingorganismsastheyadaptedtotheirecologyovertime.Whileitwastooearlytobesureifbacterialhereditydependedongenes,thesescientistslinkedittothephysicalappearanceof bugs and colonies, or to the bugs’ internal chemistry, and even to their behaviour in

relationtotheirhosts.Theseweremeasurableproperties,thebacterialequivalentsofwhatevolutionary biologists were calling the ‘phenotype’ – the physical make-up of anorganismasopposedtowhatwasdeterminedbythehereditarymake-up,or‘genotype’.

Bacteriologistsalsocametorecognisethatthesamebacteriumcouldexistindifferentsubtypes,which could often be distinguished from one another using antibodies. Thesesubtypeswerecalled‘serotypes’.In1921aBritishbacteriologist,J.A.Arkwright,noticedthatthecoloniesofavirulenttypeofdysenterybug,calledShigella,growingonthejelly-coated surfaces of culture plates, were dome-shaped with a smooth surface, whereascolonies of an attenuated, non-virulent, type of dysentery bug were irregular, rough-lookingandmuchflatter.Heintroducedtheterms‘Smooth’and‘Rough’(abbreviatedtoSandR)todescribethesecolonialcharacteristics.Arkwrightrecognisedthatthe‘R’formscroppedupinculturesgrownunderartificialconditions,butnot incircumstanceswherebacteria were taken from infected human tissues. He concluded that what he wasobservingwasaformofDarwinianevolutionatwork.

Inhiswords:‘Thehumanbodyinfectedwithdysenterymaybeconsideredaselectiveenvironmentwhichkeepssuchpathogenicbacteriaintheformsinwhichtheyareusuallyencountered.’

Soonresearchersindifferentcountriesconfirmedthatlossofvirulenceinanumberofpathogenic bacteria was accompanied by the same change in colony appearance fromSmoothtoRough.In1923,FrederickGriffith,anepidemiologistworkingfortheMinistryof Health in London, reported that pneumococci – the bugs that caused epidemicpneumonia and meningitis which were of particular interest to Oswald Avery at theRockefeller Laboratory – formed similar patterns of S and R forms on culture plates.GriffithwasknowntobeadiligentscientistandAverywasnaturallyintrigued.

Griffith’s experiments also produced an additional finding, one that really shook andpuzzledAvery.

WhenGriffithinjectednon-virulentR-typepneumococcifromthestrainknownastypeIintoexperimentalmice,heincludedanadditionalingredientintheinjections,aso-called‘adjuvant’, which usually pepped up the immune response to the R pneumococci. Acommonadjuvantforthesepurposeswasmucustakenfromtheliningoftheexperimentalanimalstomach.ButforsomeobscurereasonGriffithswitchedadjuvanttoasuspensionofSpneumococci,derivedfromtypeII,thathadbeendeliberatelykilledoffbyheat.Theexperimentalmice died from overwhelming infection. In the blood of these deadmiceGriffithexpectedtofindlargenumbersofmultiplyingR-typeIbacteria–thetypethathehadinjectedatthestartoftheexperiment.WhythenhadheactuallyfoundS-typeII?Howonearthcouldaddingdeadbacteriatohisinoculumhavechangedtheactualserotypeofthebacteriumfromnon-virulentR-typeItohighlyvirulentS-typeII?

Researchers, includingAveryhimself,hadpreviouslyshown thatSandR typesweredetermined by differences in the polysaccharide capsules coating the cell bodies of thebugs.Griffith’sfindingssuggestedthatthetestbacteria,initiallyR-typepneumococci,hadchanged their polysaccharide coats inside the infected bodies of themice to that of thevirulentstrain.Buttheycouldnothaveachievedthisbyjustflingingofftheoldcoatand

puttingon thenewone.Thecoatwasdeterminedby thebacteria’sheredity– itwasaninherited characteristic. Further cultures of the recovered bacteria confirmed that the Stype bred true. There appeared to be only one possible explanation: adding the dead SbacteriatothelivingRbacteriahadinducedamutationintheheredityofthelivingR-typebacteria,sotheyliterallytransformedintoS-typeII.

In the words of Dubos: ‘[At the time] Griffith took it for granted that the changesremained within the limits of the species. He probably had not envisaged that onepneumococcus type couldbe transformed into another, as thiswas then regardedas theequivalent of transforming one species into another – a phenomenon never previouslyobserved.’

*

It is little wonder that Avery was astonished by Griffith’s findings. Like Robert Kochbeforehim,Averysubscribedtotheviewthatbacterialstrainswereimmutableintermsoftheir heredity. The very concept of a mutation – that heredity was capable of anexperimentally induced change – was a highly controversial issue within biology andmedicineatthistime.Tounderstandwhy,weneedtograsptheconceptofwhatamutationmeans.

By the latenineteenthcenturyDarwinian theoryhadentereda crisis.Darwinhimselfhad been well aware that natural selection relied on some additional mechanism, ormechanisms,capableofchangingheredity,sothatnaturalselectionwouldhavearangeof‘hereditablevariation’tochoosebetween.Generationslater,intheopeningchaptersofhisinnovativebookEvolution:TheModernSynthesis,JulianHuxleyputhisfingeronthenuboftheproblem.‘ThereallyimportantcriticismshavefallenuponNaturalSelectionasanevolutionary principle and centred round the nature of inheritable variation.’ In 1900, aDutchbiologist,HugodeVries,putforwardanovelmechanismthatwouldbecapableofprovidingthenecessaryvariation:theconceptofarandomchangeinaunitofinheritance.Opportunityforchangeexistswhengenesarecopiedduringreproduction,whenarandomchange in the coding of a gene might arise from an error in copying the hereditaryinformation.DeVries called this source of hereditary change a ‘mutation’. Itwas onlywithwhatJulianHuxleytermed‘thesynthesis’ofMendeliangenetics–thepotentialforchange in the inherited genes through mutation – and Darwinian natural selectionoperating on the hereditary choices presented within a species, that Darwinian theorybecamecredibleagaintothegreatmajorityofscientists.

In timeGriffith’s findingwould be confirmed to bewhatAverywas nowwonderingabout:itwasamutation.GeneticistswouldshowthatthechangefromtheRtotheSstrainofpneumococcusinvolvedthe transferofagenefromthedeadS-typeIIbacteria to thelivingR-type I bacteria,whichwas incorporated into subsequent bacterial reproductivecycles, transforming the cells of the R-type I bacterium into the cells of the S-type IIbacterium.Itwasindeedthebacterialequivalentofachangeofspecies.AndGriffithwasprovenright in inferring thatDarwiniannaturalselectionhadoperatedeven in theshorttimeframeoftheinfectionofacohortoflaboratorymice.

Griffith’s experimental findings galvanised bacteriologists and immunologists aroundtheworld.Hisdiscoverywasconfirmedinseveraldifferentresearchcentres,includingtheRobertKochInstitute inBerlin,where thepneumococcal typeshadfirstbeenclassified.ThenewswasinevitablyahottopicofdiscussioninAvery’sdepartment,asDuboswouldrecount: ‘butwedidnoteven try to repeat themat first, as ifwehadbeenstunnedandalmostparalysedintellectuallybytheshockingnatureofthefindings’.

AtfirstAverysimplycouldn’tbelievethatbacterialtypescouldbetransformed.Indeed,he had been one of the authoritative figures who had settled the fixity of bacterialreproductionbeing true to typeyearsbefore.But from1926AveryencouragedayoungCanadianphysicianworkingintheRockefellerLaboratory,M.H.Dawson,toinvestigatethe situation.According toDubos,Dawson,unlikeAvery,wasconvinced from the startthat Griffith’s conclusion must be correct because he believed that ‘work done in theBritishMinistryofHealthhadtoberight’.

Dawson began by confirming Griffith’s findings in laboratory mice. His resultssuggested that the majority of non-virulent bacteria – the R types – had the ability incertaincircumstances to revert to thevirulentS type.By1930 theyoungCanadianwasjoined by a Chinese colleague, Richard P. Sia, and between them they took theexperimentalobservationsfurtherbyconfirmingthatthehereditarytransformationcouldbe brought about in culturemedia,without the need for passage throughmice.At thisstage,Dawsonleft thedepartmentandAveryencouragedanotheryoungphysician,J.L.Alloway,totaketheinvestigationfurther.Allowaydiscoveredthatallheneededtobringabout the transformation was a soluble fraction derived from the S pneumococci bydissolving the living cells in sodium deoxycholate, then passing the resultant solutionthroughfilterstoremovethebitsofbroken-upcells.Whenheaddedalcoholtothefilteredsolution, the activematerial precipitated out as sticky syrup.Throughout the laboratorythisstickysyrupwasreferred toas the‘transformingprinciple’.So theworkcontinued,experimentfollowingexperiment,yearbyyear.

WhenAlloway left thedepartment, in1932,Averybegan todevote someofhisowntimetothepneumococcustransformation, inparticularaimingto improvetheextractionand preparation of the transforming substance. Frustration followed frustration. Hefocused on its chemical nature. Discussion took place with other members of thedepartment,rangingfromthe‘plamagene’thatwasthoughttoinducecancerinchickens(nowknowntobearetrovirus),ortothegeneticalterationsinbacteriathatwerethoughttobecausedbyviruses.AccordingtoDubos,Allowaysuggestedthetransformingagentmight be a protein-polysaccharide complex.But by1935Averywasbeginning to thinkalong other lines. In his annual departmental report that year he indicated that he hadobtained the transformingmaterial in a form that was essentially clear of any capsularpolysaccharide.In1936,RollinHotchkiss,abiochemistwhohadnowarrivedtoworkinthedepartment,wroteahistoriccommentinhispersonalnotes:

‘Averyoutlinedtomethatthetransformingagentcouldhardlybeacarbohydrate,didnotmatchverywellwithaproteinandwistfullysuggesteditmightbeanucleicacid!’At

this stage,Dubos,whomanyyears laterwouldwriteabookaboutAveryandhiswork,dismissedthisasnomorethanasurmise.Thereweregoodreasonsforhiscaution.

Thatyearfewresearchersthroughouttheworldbelievedthattheanswertohereditylaywithnucleic acids.Thesechemical entitieshadbeendiscoveredbyaSwissbiochemist,Johann FriedrichMiescher, back in the late 1800s. Fascinated by the chemistry of thenucleus, Miescher had broken open the nuclei of white blood cells in pus, andsubsequently the heads of salmon sperm, to discover a new chemical compoundwhichwas acidic to pH testing, rich in phosphorus and comprised of enormously largemolecules.Afteralifetimeofexperimentationonthediscovery,Miescher’spupil,RichardAltmann,wouldintroducethetermnucleicacidtodescribeMiescher’sdiscovery.Bythe1920s,biochemistsandgeneticistswereawarethatthereweretwokindsofnucleicacids.One was called ribonucleic acid, or RNA, which contained four structural chemicals:guanine,adenine,cytosineanduracil,orGACU.Theotherwascalled‘desoxyribonucleicacid’,orDNA,whichwasamajorcomponentofthechromosomes.Theyhaddeciphereditsfourbases–threeidenticaltoRNA,guanine,adenineandcytosine,butwiththeuracilreplaced by thymine – making the acronym GACT. They knew that these four basesconsistedoftwodifferentpairsoforganicchemicals;adenineandguaninebeingpurines,and cytosine and thymine being pyrimidines. They also knew that they were strungtogether to form very longmolecules. At first they thought that RNAwas confined toplantswhileDNAwas confined to animals, but by the early thirties thiswasdismissedwhen itwasfound thatbothRNAandDNAwereuniversallydistributed throughout theanimalandplantkingdoms.Stilltheyhadnoknowledgeofwhatnucleicacidsactuallydidinthenucleiofcells.

A distinguished organic chemist based at the Rockefeller Institute, Phoebus AaronLevene,proposed that the structuresofDNAandRNAwere exceedinglyboring– theyformedgroupsoffourbasesthatrepeatedthemselvesintheidenticalrepetitiveformationthroughout themolecule, like a four-letterword, repeated ad nauseam. Thiswas called‘the tetranucleotide hypothesis’. Such a banal molecule couldn’t possibly underlie theexceedingly complex basis of heredity. In the words of Horace Freeland Judson, ‘thebeliefwas heldwith dogmatic tenacity thatDNAcould only be some sort of structuralstiffening,thelaundrycardboardintheshirt,thewoodenstretcherbehindtheRembrandt,sincethegeneticmaterialwouldhavetobeprotein’.

Proteins are lengthymoleculesmade up of smaller organic chemical units known asamino acids. There are 20 amino acids in the make-up of proteins, reminiscent of thenumber of letters that make up alphabets. If genes were the hereditary equivalents ofwords,onlythecomplexityofproteinscouldfashionthewordscapableofspellingoutthenarratives.Chemists,andthroughextrapolationgeneticists,notunnaturallyassumedthatonlythislevelofcomplexitycouldpossiblyaccommodatetheincrediblememorytemplatethat the complexity of heredity demanded– a lineof thought that Judson labelled ‘TheProteinVersionoftheCentralDogma’.

ThiswasthecontentiouszeitgeistthatAverynowconfronted.Asearlyas1935,inhisannualreportstotheBoardoftheInstitute,heindicatedthathehadgrowingevidencethat

the ‘transforming substance’ appeared free of capsular polysaccharide and it did notappeartobeaprotein.

Further progress on this line of research appeared to drag. In part this was becauseDubos, working in the same department, had made a breakthrough in his search forantibiotic drugs. In 1925, Alexander Fleming, at St Mary’s Hospital in London, haddiscoveredapotentialantibiotic,penicillin,buthehadbeenunabletotakehisworktothestage of useful production for medical purposes. Now, working on the philosophicalprinciple encapsulated by the biblical saying ‘dust to dust’, Dubos had pioneered thesearch formicrobes in soil thatwould potentially attack the polysaccharide coat of thepneumococcus.Bytheearly1930shewasmakingprogress.FromacranberryboginNewJerseyhefoundabacillusthatdissolvedthethickpolysaccharidecapsulethatcoatedthepneumococcuswithitsarmour-likeoutercovering.DuboswentontoextracttheenzymethattheCranberryBogbacillusproduced.HeandAveryhadreportedtheirdiscoveryinapaperinthejournal,Science,in1930.Inafurtherseriesofpapersthetwoscientistswouldreportfurtherexperiments,allaimedatextrapolatingthediscoverytohumantrialsoftheCranberryBogenzymeintreatingthepotentiallyfatalpneumoniaandmeningitiscausedbythepneumococcus.

But their researches encountered difficulty after difficulty. In part these arose from apredictable ignorance in a field of such pioneering research. A more personal, anddevastating,problemarosewhen,underthestressofitall,Averydevelopedthyrotoxicosis–adebilitatingautoimmuneillnessinwhichhisthyroidglandbecameoveractive.

Thyrotoxicosiscausesthesystemtobefloodedbythyroidhormones,whichwouldhaveinappropriatelyswitchedhismetabolismintoadangerousoverdrive.Hewouldhavefeltshaky,agitated,physicallyandmentallyrestless,sufferingdifficultieswithrelaxationandsleep–animpossiblesituationforacreativeperson.Averyhadtospendtimeawayfromthe lab undergoing surgery to remove the bulk of the ‘toxic goitre’, a procedure thatcarriedriskofside-effects,evenfatalityinaminorityofcases.Hissurgeonadvisedhimagainst any early activity, physical ormental, that provoked stress.Dubos later recalledhowAverywas away from hiswork for as long as sixmonths. AndwhileAverywasaway, the laboratory stagnated. In Dubos’ ownwords, ‘I… pursued [the research] forthree or four years. However I could not carry the work very far because there wereseriousgaps inbothmyknowledgeofgeneticsandbiochemistryand in the[prevailing]statesofthesesciencesthemselves.’

Duboswouldcontinuehisresearchesagainstsuchdifficulties,toberewarded,in1939,with the discovery of the first soil-derived antibiotic. He called it ‘gramicidin’. Butgramicidincouldnotbe takenbymouthoradministeredbyinjectionbecauseitwastootoxic.Itcouldonlybeappliedtoskinconditions.Theresearchcontinued.Butthen,allofasudden,thehopesofAveryandDuboswereovertakenbyarivalbreakthrough.WorkinginthepharmaceuticalresearchlaboratoriesoftheBayerCompanyinElberfeld,Germany,doctor Gerhard Domagk reported the discovery of a new antibacterial agent calledprontosil. The first of what would come to be known as the sulphonamide drugs, it

immediately entered the medical formulary, pioneering the treatment of a number ofhithertountreatableinfectiousdiseases.

Todaywe are apt to forget how littlewe could do to control infection in the 1930s.Epidemicssuchasscarletfever,measles,pneumonia,meningitisandpoliomyelitissweptthrough the population in regular, sometimes annual, cycles.Other notorious infectionswere everyday threats, including tuberculosis, which ravaged entire families, or boils,septic arthritis, septic osteomyelitis,which caused agonising abscesses in bone, and thecommonplace but potentially deadly streptococci capable of breaking through a septicthroat to cause abscesses in the brain. Most of the human population, whether indeveloped or developing countries, died from infections, including the insidiouspneumoniasthathitthosewhoseimmunitywasdepressed.Thetreatmentofinfectionswasthemosturgentproblemthenfacinghumanity.ForDubos,andevenmoresoAvery, thedisappointmentoffailingintheirlineofresearchwouldhavebeenshattering.

When,induecourse,Averyreturnedtowork,heswitchedtheemphasisofhisresearchtothe‘transformingsubstance’.ColinMacLeodimprovedthetechniquesofextractionsothey could now produce sizeable amounts for assay and further testing. They began tomakemorerapidprogresssothat,inareporttotheRockefellerBoardfortheyear1940–41, they were more confident in stating that even a highly purified extract of thetransformingsubstanceappearedtobeprotein-free.

That summerMacLeod left the Institute to become Professor of Bacteriology at theNewYorkUniversitySchoolofMedicine.Buthestilltookaninterestintheprojectandfrequently returned to the Institute to add his advice. A young paediatrician, MaclynMcCarty,tookMacLeod’splaceinthetransformingexperiment.McCartybroughtausefullevel of biochemical training to the laboratory. And now they had the transformingsubstanceinquantityandinstableform,heappliedhischemicalskillstofurtherprocessandidentifytheactivematerial.Hebegantoculturethepneumococciinlargebatchesof50 to 75 litres, developing a series of steps that increased the yield of transformingsubstancewhileremovingproteins,polysaccharidesandribonucleicacid.Theprevailingbeliefsabout thehereditaryprincipleclaimed thatnucleoproteinswere theanswer.Thusthetopmostpriorityinallofthiseffortwastoensurethatthefinaltestmaterialcontainednoprotein.

BynowMcCartyhadextractedconcentratedsolutionsoftheactivematerial.Hetreatedthis with a series of protein-digesting enzymes, such as the gut-derived trypsin andchymotrypsin,whichwereknowntodestroyproteins,ribonucleicacidandpneumococcalcapsularpolysaccharide.Whatremainedwasoncemoreshakenwithchloroforminafinalefforttoremoveeventhefinesttracesofprotein.

By late 1942, after repeated extraction and experiment, McCarty had come to theconclusion that the transforming activitywas confined to a highly viscous fraction thatconsistedalmostexclusivelyofpolymeriseddeoxyribonucleicacid.Whenheprecipitatedthisfractioninaflaskbyaddingabsoluteethylalcohol,dropbydrop,allthewhilestirringthesolutionwithaglassrod,theactivematerialseparatedoutofthesolutionintheformof long, white and extremely fine fibrous strands that wound themselves around the

stirring rod. Dubos would recall the excitement felt within the lab by all those whowitnessedthesightofthebeautifulfibres,whichwerethepureformofthetransformingsubstance.

Inearly1943,Avery,MacLeodandMcCartypresentedtheirfindingstodistinguishedchemistsat thePrinceton sectionof theRockefeller Institute forMedicalResearch.Thechemists must have been astonished, perhaps even nonplussed, but they offered nocontradictionoftheevidencenoraskedforfurtherproof.Theresearcherssummeduptheevidence for the Board of the Rockefeller in April of that year. Avery, MacLeod andMcCarty,all threemedicaldoctorsratherthangeneticists,werenowreadytoinformtheworld in a paper submitted to the Journal of ExperimentalMedicine in November thesameyear,whichwouldbepublishedearlythefollowingyear.Thetitleofthepaperwaslong-winded and cautious: ‘Studies on the chemical nature of the substance inducingtransformation of pneumococcal types. Induction of transformation by adesoxyribonucleicacidfractionisolatedfrompneumococcustypeIII’.

In the words of Dubos, this paper ‘had staggering implications’. The sense ofexcitement,temperedbycaution,wascapturedinaletterthatAverywrotetohisbrother,Roy,dated26May1943:

…Forthepasttwoyears,firstwithMacLeodandnowwithDrMcCarty,Ihavebeentryingtofindoutwhatisthechemicalnatureofthesubstanceinthebacterialextractswhichinducesthisspecificchange…Somejob–andfullofheartachesandheartbreaks.Butatlastperhapswehaveit…Inshort,thesubstance…conformsverycloselytothetheoreticalvaluesofpuredesoxyribosenucleicacid.Whocouldhaveguessedit?

Intheletter,‘desoxyribosenucleicacid’,inthepaper,‘desoxyribonucleicacid’:theseareolder names for what we now call deoxyribonucleic acid – commonly reduced to itsacronym,DNA.

two

DNAIsConfirmedastheCode

Lookingbackathisown failure toappreciateAvery’sdiscoveryat the time,Stentcame to theconclusion‘insomerespectsAveryetal’spaperisamoredramaticexampleofprematuritythanMendel’s’.

UTIDEICHMANN

Scientists, in the opinion of the Nobel Prize-winning Linus Pauling, were fortunatebecausetheirworldwassomuchthericherforitsmysteriesthanthosenotinterestedinsciencecouldpossiblyappreciate.Certainlyin thosedaysAvery’s labat theRockefellerMedicalInstituteforResearchwasfilledwithamoodofexpectationandexcitement. In1943OswaldAverywas65yearsold.HehadplannedtoretireandjoinhisbrotherRoy’sfamily inNashville,Tennessee, but therewas no question of his leaving the lab at thistime. He needed to continue his work on the transforming substance. In particular heneeded to convince his colleagues throughout the world of microbiology and, morewidely, the sceptical world of biochemists and geneticists, of the validity of theirdiscovery.

Averywasconservativebynature.Agenerationearlierheandacolleaguehadproposedthat complex sugar molecules, called polysaccharides, and not proteins determined theimmunological differences between different types of pneumococcal bacteria.Althoughthis theorywas eventually confirmed to be true, at the time of discovery it provoked astorm of controversy that had haunted this nervous and sensitive man. In a long andramblinglettertohisbrotherAveryhadrepeatedlyreferredtohisworryaboutthereactionto the new discovery. ‘It’s hazardous to go off half-cocked… It’s lots of fun to blowbubbles–butit’swisertoprickthemyourselfbeforesomeoneelsetriesto.’

Averyhadanadversarycloser tohome.AlfredE.Mirsky,adistinguishedbiochemistandgeneticistalsoworkingattheRockefellerInstitute,hadreactedtoAvery’sdiscoverywith incredulity. Tomakematters worse,Mirskywaswidely regarded as an expert onDNA.He had discovered that the quantity ofDNA in every cell nucleus remained thesame, establishing a principle called ‘DNAconstancy’.Henowdoubted the efficacyofMcCarty’s DNA extraction. A stickler for ‘clean’ biochemical experiment, Mirskybelieved that protein found in the nucleus, called nucleoprotein, must be the basis ofheredity.Evenaslateas1946,MirskyinsistedthatthetwoenzymesMcCartyhadusedinhis extractionswouldnot digest away all of theprotein.Mirskywasvery influential ingeneticcirclesandhisargumentimpressedtheleadinggeneticistofthetime,HermannJ.Muller,whohadbeenawardedtheNobelPrizethatsameyearforhisdiscovery,madetwodecadesearlier,thatX-rayscausedmutationsinthegenesofthefruitfly.Inalettertoa

geneticist colleague, Muller stated ‘Avery’s so-called nucleic acid is probablynucleoprotein after all, with the protein too tightly bound to be detected by ordinarymethod.’

Tosomeextentsuchdisagreementwastypicalofthesituationonemightfindanywhereinsciencewhenvariousgroupsfromdifferentscientificbackgroundsare investigatingamajor unknown. Never is the argument more acrimonious than when a new discoveryconfounds the acceptedparadigm.But thevociferousoppositionofMirsky fromwithinAvery’shomeresearchfoundationmusthavebeenparticularlydamaging.In1947Mullerpublishedhis‘Pilgrim’sLecture’asascientificpaperinwhichheconcludedthatwhethernucleicacidorproteinwastheanswer‘mustasyetberegardedasanopenquestion’.Inthe words of Robert Olby, a historian and philosopher of science, ‘Through Muller’swidelyreadPilgrimLecture,this[sceptical]influencewasspreadtoawideaudience.’

In a new series of extractions, with stringent quality checking, Avery attempted toconfoundhiscritics.McCartyleftthelaboratoryin1946,whichwasleftinthehandsof,amongstothers, themeticulousRollinHotchkiss.Hotchkissaddedseveralnewchemicalexplorationsoftheextract,allfurtherconfirmingthatitwasDNA.HedisprovedMirsky’sobjectionbypurifyingtheextracttotheextentthattheproteincontentwasbelow0.02percent and he showed that it was inactivated by a newly discovered crystalline enzymespecific toDNA:DNase.Whilemanygeneticists remainedobdurate in theiropposition,somewerebeginningtotakenotice.

InasubsequentinterviewwiththebiophysicistandfutureNobelLaureate,theGerman-bornphysicistMaxDelbrück,HoraceF. Judsonwoulddiscover that somedistinguishedresearcherswereawareofthepotentialimportanceofAvery’sdiscovery.‘Certainlytherewasscepticism,’Delbrückrecalled. ‘Everybodywholookedat itwasconfrontedby thisparadox.ItwasbelievedthatDNAwasastupidsubstance…whichcouldn’tdoanythingspecific. So one of these premises had to be wrong. Either DNA was not a stupidmolecule, or the thing that did the transformation was not DNA.’ Avery had raised amonumentally important question and the only way of resolving the dilemma was forotherresearcherstoprobeitthroughsomeformofalternativeexperimentationtofindoutifhewasrightorwrong.

In1951,twoAmericanmicrobiologists,AlfredHersheyandMarthaChase,undertooksuchanalternativeexperimentwhilestudyingthewaythatcertainvirusesusebacteriaasafactorytomakedaughterviruses.Thesevirusesarecalled‘bacteriophages’,or‘phages’for short– from theGreekphago,whichmeans to eat, because theydevour culturesofhostbacteria.Thepresence,andnumber,ofvirusescouldbemeasuredifyouseededyourhostbacteriaintoheat-softenedagarandthenaddedthevirusesinvariousdilutionstotheagarbefore spreading itovera laboratoryplate.When theagarcooled it formeda thin,even layer of jelly in the plate,which, on overnight culture,would become cloudedbygrowth of bacteria within the agar. Wherever a virus landed among the bacteria therewouldbearoundwindowoftransparencycausedbythedissolving(lysis)ofthebacteriawhichwaseasilyvisible, and thuscountable.This ‘plaque-counting technique’,which Imyselflearntfrommymicrobiologyprofessorasamedicalstudentandlatermadeuseof

inexperimentsonthenatureofautoimmunityasahospitaldoctor,iseasilylearntandthusputtousebythousandsofscientistsinagreatvarietyofexperiments.

What interested Hershey and Chase was the fact that phage viruses were known tocomposeacoreofgeneticmaterialsurroundedbyacapsuleofprotein.Infact,eachviruscloselyresembledamedicalsyringeinstructure,sothatwhenitinfectedthebacterialcellof itshost, itappearedtosqueezeout thegeneticmaterialfromthebodyof thesyringe,leaving the empty protein coat attached to the outer bacterial cellwall.Meanwhile, thegeneticmaterialwasinjectedintothebacterialcellinterior,wheretheviralgenomewouldbe replicated as part of its reproduction. Hershey and Chase invented an ingeniousexperiment that would decide whether protein or DNA was the basis of the viralreproductive system.Thiswould involveadding radioactivephosphorusand radioactivesulphur to themedia inwhichseparatebatchesof thehostbacteriaweregrowing.Afterfourhours,toallowtheradioactiveelementtobetakenupbythebacteria,theyintroducedthephageviruses.

To understand the basis of the experiment we need to grasp that DNA containsphosphorusaspartofitsmake-upbutnosulphur,meanwhiletheaminoacidsthatmakeupproteinscontainsulphurbutnophosphorus.

By inoculatingeachof these twogroupsofbacteriawithviruses,HersheyandChasederivedtwopopulationsofphageviruses–onecontainingtheradioactivephosphorusandtheothercontainingtheradioactivesulphur.Whenthevirusesinfectedthebacteria,theyleft their empty viral coats, mostly made up of protein, attached to the outside of thebacterialcellwalls,havinginjectedtheircorematerial,knowntocompriseDNA,intothebacterial bodies. Hershey and Chase used centrifugation to separate and extract emptyviral coats. Meanwhile, the infected bacteria were allowed to go through their normalreproductivecycle,whichallowedtheviralcoresinsidethemtogenerateentirenewphageviruses,rupturingthebacterialbodiesandfloodingthegrowthmediawithlargenumbersof fully formed viruses. Hershey and Chase now removed what was left of the hostbacterialbodiestogatherdenseconcentrationsoffullyformedviruses.

When they now compared the empty viral coats, made up of protein, with the fullyformedviruses,withtheircoresfullofgeneticmaterial,theyfoundthat90percentoftheradioactivesulphurwasleftbehindintheviralcoatswhenthevirusinfectedthecell,and85percentofthephosphoruswasnowpartofDNAthathadenteredthebacterialcelltocode for the future offspring of virus. This confirmedAvery’s findings: DNA, and notprotein,wasthecodeofheredity.

WemightdulynotethatthisseparationofcoatfromcoreDNAofvirusinvolvesamuchhigher degree of protein impurity than Avery’s extractions. Yet the hitherto scepticalgeneticistsappearedtobemoreconvincedbythephageexperimentthanbyAvery’swork.Perhaps the strikingly visual nature of the experimentwas a factor. Perhaps it was theadditional,quitedifferent,avenueofconfirmation.Itdidn’tharmcredibility that leadinggeneticistswerewithinthe‘phagecamp’,too.

*

Today, with the advantage of retrospect, scientists by and large see the 1944 paper byAvery,MacLeod andMcCarty as the pioneering discovery ofDNA as themolecule ofheredity.Ithasbeenportrayedasoneofthemostregrettableexamplesofadiscoverythatmerited,butwasnotawarded, theNobelPrize.There isampleevidence thatAverywasrecommendedbyseniorcolleagues,particularlywithinhisowndisciplineofmicrobiologyandimmunology–indeedhewasnominatedtwice,firstinthelate1930s,forhisworkonthepneumococcaltypinganditsrelevancetobacterialclassification,and,after the1944paper was published, he was nominated yet again for his fundamental contribution tobiology.But itwould appear that theNobelCommitteewasnot sufficiently swayed. Inretrospect, it is seen as amajor omission that causes people to scratch their heads andwonderwhy.

DubosworkedforfifteenyearsinthelabnextdoortoAvery’sand,insomuchasthereticentProfessor allowed it, hehadplentyofopportunity toget toknowAvery and tounderstandhisapproachtoscienceandhisreactiontothestressesinvolvedinpioneeringnewconcepts. InDubos’ opinion,writing in1976, the curious lackof recognitionmostlikely derived from a combination of happenstance and Avery’s own personality. Hewouldsubsequentlyremarkhow,inallthattime,Averyneverclosedthedoorofhislab,orthesmallofficethatledoffit,allowinganyofhisstafftocomeandtalktohim.ThissameeternallyopendooralsoallowedDubostowitness‘Fess’s’activitiesatthebench,tolistenin to his conversations with colleagues and to observe his interludes of introspectivebrooding.

Thisreserved,smallandslenderbachelorwouldinevitablyarriveatworkdressedinaneat and subdued style, his conservative attire somehow at one with the charm of hislively and affable behaviour. His eyes, under the domed bald head that seemed toovoluminous for the frail body, were sparkling and always questioning, and he wouldtransform the most ordinary conversation into an artistic performance with spiritedgestures, mimicry, pithy remarks and verbal pyrotechnics. Avery might have beensomewhat reticent in manner (he could be silently introspective), but in his ownquintessentialwayhewasvulnerablyhuman,andthatmadehimallthemoreinterestingandenchanting.

Iwouldsuggestthatcreativityinscienceiseverybitasintertwinedwithpersonalityasone finds in a writer, artist, ormusically gifted composer or performer. It would seemunsurprisinginanartistifheappearedunusuallyascetic,withdrawnfromthehurly-burlyworld of the surrounding New York, ensuring that he lived close enough to theRockefellerInstitutesohecouldwalktowork.Inhisways,Averycouldseemcuriouslyambivalent.He sufferedmood swings at times,when alone in the lab,when hewouldappeartobedejectedbythedifficultiesfacinghim.Afterwardshewoulddeclaim,clearlyreferring to himself, that resentment hurts the personwho resentsmuchmore than thepersonwhoisresented.Heleftmanylettersunansweredandrefusedtohaveasecretary.He refused to review, or sponsor, any scientific paper in which he had made nocontribution. In Dubos’ words, ‘Graciousness and toughness when it came to what hehimselfwasdeterminedtodo,waspartofhisnature.’Averywasaverysuccessfulteacherduringhis earlymedical career,yet inhis lateryearsheappears tohave resentedbeing

expected to lecture on his own research. In this respect, he bore some interestingsimilarities to Charles Darwin. Avery scrupulously avoided any discussion of his ownhealthandanyintrusion,howeversmall,intohisprivatelife–whichwasdevotedtohisyoungerbrother,Roy,andtoanorphanedfirstcousinwhomhesupportedallthroughhislife.Heneverexpressedresentmentaboutcriticismsofhiswork,evenwhen thesewereunjustified.Heleftnorecordofhisprivatethoughts,otherthantheletterstohisbrother.AsingleexperiencestruckDubosasbeingsignificant.

One day, in early 1934, the same year that Avery suffered the onset of histhyrotoxicosis,Dubos toldAvery thathewasabout tobemarried.The lady inquestionwasaFrenchwomanlivinginNewYork,namedMarieLouiseBonnet.Averyimmediatelyrejoiced at the news. They were conversing in the laboratory on the sixth floor of theRockefeller hospital building. During the subsequent animated conversation, Averyclimbedoutofhischair,walkedtothewindowandlookedout,asiflostforamomentindeepreflection.Returning tohischair,hementioned thathehadcontemplatedmarriageyearsbefore,butthatcircumstanceshadnotprovedfavourabletohisplans.ItseemslikelythattheladyinquestionwasanursethatAveryhadmetduringthecoursehehadtaughttostudentnursesattheHoaglandLaboratory.Averywouldhavebeenabout32yearsoldatthetime.Foramomentortwotheolderman’seyeswerefulloflonging.

‘Oneofthegreatjoysoflife,’heremarkedtoDubos,‘istogohometosomeonewhowouldratherseeyouthananybodyelse.’

Fate would prove cruel to both men. Marie Louise Bonnet subsequently died fromtuberculosis at a time when Dubos was pioneering the very antibiotics that wouldeventuallyhelptocurethesameillness.Themarriagewaschildlessandtheeffectsofhiswife’s death on Dubos were devastating. He resigned, forthwith, from his antibioticresearches,whichwerelatertakenupbyhisformerteacher,SelmanWaksmanatRutgersAgriculturalCollege,nowRutgersUniversity,andwhichledtothediscoveryofaseriesofimportantantibiotics,includingstreptomycin.ThisbreakthroughresultedinWaksmanbeingawardedtheNobelPrizeinMedicineorPhysiologyin1952.

Much of what Dubos witnessed of Avery spoke of an intense focus and purity ofpurposeinscienceandhiswork.But,increasingly,hisdevotiontohisresearchappearedtobeaccompaniedbyinsularityborderingonreclusiveness.

Scientistswhohavelabouredlongandhardatadifficultbuteventuallyrewardinglineof research are usually happy to talk about it – if not to the media or ordinary socialchannels, certainly to colleagues. They travel to scientific symposia. They take part inconferences.Theyenjoy thecamaraderie thatcomes fromsharing thesame interests. InthewordsofFrankPortugal,‘wide-rangingdiscussionswithpeersbothindividuallyandatmeetingsarepartandparcelofthescientificprocess.Itisanimportantcomponentofhowcollaborationsareformedandscientificadvancesaremadeandrespected.’Mostscientistsareonlytoogladtoacceptthe,oftenrare,honoursanddistinctiontheirworkbringstheirway.NotsoOswaldAvery.

In 1944 Avery was proposed for an honorary degree at Cambridge University, arecognitionmostscientistswouldcherish.ThefollowingyearhewasawardedtheCopley

MedalbytheRoyalSocietyofLondon.Avery’srootswereEnglish–inthelatenineteenthcenturyhisfamilyhademigratedtoCanadafromthecityofNorwich–butherefusedtovisitEnglandevenonsuchprestigiousoccasions,puttingforwardtheexcusethathisstateof health did not permit it except by travelling first class. InDubos’ opinion, this wasdisingenuous, since the respective foundations would have funded the flights. That hemighthavefeltnervous,claustrophobic,onthelengthyflightispossible.Recallingthosedarkmoods inwhichAverymightmumble tohimselfabout thedamaging inflictionsofresentment, it seemed more than likely to Dubos that he might have been unable tosuppress lingering anger at the hurtful controversy provoked years ago by hispolysaccharide typing of pneumococci. Whatever his reasons, Avery refused bothhonours.

An incident highlighted just how strong was Avery’s aversion to such formalacknowledgementofhiswork.SirHenryDale,whowasPresidentoftheRoyalSocietyinEngland,tookituponhimselftobringtheCopleyMedaltotheRockefellerInstitute,theretoconferitontheshyandretiringAveryinperson.DalewasaccompaniedbyaDrTodd,who knew Avery personally. The two highly respected English visitors arrived at theInstituteinNewYorkunannouncedandwentdirectlytoAvery’sdepartmentinthemainhospital building. Butwhen they sawAveryworking in his lab, through the ever-opendoor,theyretreatedwithoutintrudingonhispresence.

Dr Todd would later recount how Sir Henry Dale said simply: ‘Now I understandeverything.’

Bizarre as this behaviour would appear, it was in keeping with Avery’s increasinglyreclusivepersonality:amanwhoavoidedanyofthenormalpersonalcontactsoutsideofimmediate family and work colleagues. Genius can be strange. Yet such idiosyncraticbehaviour apart, itwas this sonof an evangelicalBaptist preacherwho first discoveredthat DNA was the molecule of heredity. And putting such personal matters aside, thequestionremains:whywassuchafundamentaldiscoverynotrecognisedbytheawardingoftheNobelPrize?

In his letters to his brother, Avery retained a modest outlook. Could it be that acombination of Avery’s innate conservatism, his tendency to over-caution, and hisdownplaying of the implications of his discovery in the paper of 1944 might havecontributed to his being overlooked? In Dubos’ words, the paper… ‘did not make itobviousthatthefindingsopenedthedoortoaneweraofbiology’.DuboswonderediftheNobel Committee, unaccustomed to such restraint and self-criticism ‘bordering on theneurotic’mighthavecausedthemtowaitawhileforbothconfirmationofthediscoveryand to see what the broader implications might be. To put it another way, Dubosquestioned if the papermight have been a failure not in its ownmerits, as a scientificcommunication,butfromthepublicrelationspointofview.

Thislackofrecognitionismadeallthemorepuzzlingbythefactthat,iftheimportanceofthe1944paperwasnotuniversallyrecognisedwhenitwaspublished,itbecamemoreandmoreobviouswiththepassageoftime.TheHersheyandChasepaperwaspublishedin 1952. And although he was retired by the time Crick and Watson published their

famousdiscoveryofthethree-dimensionalchemicalstructureofDNAin1953,Averywasstillalive.Hewouldn’tdieuntiltwoyearslater,in1955.

MorerecentlytheNobelauthoritieshaveallowedopenaccesstotheirearlierthinking,and this has confirmedmuch ofwhatDubos had concluded.As part of the system fordeciding who should get Nobel Prizes, the Nobel Committee receives proposals fromleadingexpertsaroundtheworld.InthewordsofPortugal,whoreviewedtheirworkingandarchives,‘ItseemsthatkeybiologicalchemistswerenotconvincedbyAvery’sclaimthatDNAwasthebasisofheredity.’NotasinglegeneticistnominatedAveryfortheNobelPrize.Inpartthismayhavereflectedadifficultyinextrapolatinghisdiscoveryinasingletypeofbacteriumtogeneticsmorewidely,buteven thosecolleagueswhodidnominatehim for the Nobel Prize tended to overlook his work on DNA in favour of hisimmunological typing of the pneumococcal capsule. Portugal alsowondered ifAvery’sownidiosyncraticbehaviour,includinghisreluctancetomeetwithandexchangefindingswith colleagues, and in particular geneticists, at scientificmeetings had unintentionallyconfoundedtheacceptanceofhisgroundbreakingdiscovery.

WeareleftwithalingeringsenseofregretthatAverywasnotaccordedtherecognitionhedeserved.Hewas67yearsoldwhenhisiconoclasticpaperwaspublished.Itwas,inthewordsoftheeminentbiochemistErwinChargaff,therareinstanceofanoldmanmakingamajor scientificdiscovery. ‘Hewasaquietman:and itwouldhavehonoured theworldmore,hadithonouredhimmore.’

But there isagreateracknowledgementofdiscoverythantheawardingofaprize,nomatterhowrespectedandprestigious.InthewordsofFreelandJudson,‘Averyopenedupa new space in biologists’minds.’By space hemeant he had unravelled amajor truth,revealing newunknowns and raising important newquestions.Avery himself had,withquintessentialmodesty, touched upon those important new questions in his letter to hisbrother:

Ifweareright,andofcoursethatisnotyetproven,thenitmeansthatnucleicacidsarenot merely structurally important but functionally active substances in determining thebiochemicalactivitiesandspecificcharacteristicsofcells–andthatbymeansofaknownchemical substance it is possible to induce predictable and hereditary changes in cells.Thisissomethingthathaslongbeenthedreamofgeneticists…Soundslikeavirus–maybeagene.ButwithmechanismsIamnotnowconcerned–onestepatatime–andthefirstis,whatisthechemicalnatureofthetransformingprinciple?Someoneelsecanworkouttherest…

three

TheStoryinthePicture

Youlookatscience(oratleasttalkofit)assomesortofdemoralisinginventionofman,somethingapartfromreal life,andwhichmustbecautiouslyguardedandkept separate fromeverydayexistence.But scienceandeverydaylifecannotandshouldnotbeseparated.

ROSALINDFRANKLIN

The discovery of the ‘transforming substance’ by Avery, MacLeod and McCarty,confirmedbyHersheyandChase’selegantexperimentwiththebacteriophage,provedthatDNAwasthemoleculeofheredity.Butbothgroupswereworkingwithmicrobes,bacteriaandviruses,whichwereknown tobemuchsimpler in theirhereditarynature than, say,animals andplants.This left hugeunknowns thatneeded tobe explored.WasDNA thekey to theheredityof all of life, orwas it just relevant tobacteria andviruses?By theearly 1950s,work inmany different laboratories had confirmed thatDNAwas amajoringredientinthenucleiofanimalsandplants.ThissupportedtheideathatDNAwasthecodingmoleculeoflife.Butifso,howdiditreallywork?How,forexample,didasinglechemicalmoleculecodeforthecomplexheredityofalivingorganism?

Biologists,doctors,molecularbiochemistsandgeneticistswerenowaskingthemselvesthe same, or similar, questions. Critical to any such understanding was the precisemolecularstructureofDNA.If,forexample,weweretoregardtheroleofDNAasakintoa stored geneticmemory, howdid thatmolecular structure enable the quality of such aphenomenallycomplexmemory?Howwasthatgeneticmemorytransferredfromparentsto offspring? How did the same stored memory explain embryological development,whereasinglecellarisingfromthegenomicunionofapaternalspermandmaternalovumgivesrisetothedevelopinghumanembryoandfutureadulthumanbeing?

Therewasanotherprofoundlyimportantquestion.

Darwinian evolution lay at the heart of biology. To put it simply, Darwin’s idea ofnaturalselectionimpliedthatnatureselectedfromarangeofvariationsintheheredityofdifferent individuals within a species. The way in which it worked was exceedinglysimple,ifbrutal.Thoseindividuals,andbyinferencetheirvariantheredities,whocarriedasmall advantage for survival and thusabetter chanceofgiving rise tooffspring,wouldthereforebemorelikelytocontributetothespeciesgenepool.Inrealitynaturalselectionworkedmorethroughaprocessofattrition.Thoselessadvantagedindividualswhodidnotcarrytheadvantageforsurvival,weremorelikelytoperishinthestruggleforexistence,andthustheywerelesslikelytocontributetothespeciesgenepool.

This is whatDarwinian evolutionary biologists refer to as ‘relative fitness’. It is themeasureoftheindividual’scontributiontothespeciesgenepool.Certainlyithasnothingtodowithracistnotionsofsuperiorityandinferiorityattachedto‘survivalofthefittest’–atermintroducednotbyDarwinbutbythesocialphilosopherHerbertSpencer.Butifwetake a pause and think about it, such variant heredity, essential for natural selection towork,mustalsocomeaboutthroughmechanismsinvolvingthiswondermolecule,DNA,whichmust lie not only at the heart of heredity but also at the absolute dead centre ofevolution.Allofthesequestionsneededtobeansweredbythescientistsnowstrugglingtounderstand the structure and, assuming structure was function, the properties of thisremarkablechemical,DNA.

Infactthefirststeptowardsansweringthesequestionshadalreadybeentakenbackin1943, in what might appear unlikely circumstances. It was taken not by a biochemist,biologistorgeneticist,butbyanAustrianphysicist.Thesparkwaslitwhen,at4.30pmonFriday 5 February, Erwin Schrödinger stepped up to the podium inDublin to deliver alecturethatisnowseenasalandmarkmomentinthehistoryofbiology.Schrödingerhadbeen awarded theNobel Prize in 1933 forwork in quantumphysics that expanded ourunderstandingofwavemechanics–butIwon’tconfusemyselformyreadersbyenteringfurtherintothephysics.ThesimplefactswerethatSchrödingerhadexiledhimselffromhis native Austria in protest at human rights abuses and had been given sanctuary inneutralIrelandbyitsPresident,EamondeValera.InDublinSchrödingerhadhelpedfoundtheInstituteforAdvancedStudies.AspartofhisdutiesinsupportoftheInstitute,hehadagreedtogiveaseriesofthreelecturesinwhichhedevelopedacentraltheme:‘WhatIsLife?’

SuchwasSchrödinger’sfamethatthelecturetheatre,whichhadaseatingcapacityfor400,couldnotaccommodateallwhowishedtoattendthelectures–thisdespitethefactthattheyhadbeenwarnedinadvancethatthesubjectmatterwasadifficultoneandthatthe lecture was not going to be pitched at an easy or popular level, even thoughSchrödingerhadpromisedtoeschewmathematics.DeValerahimselfwaspresent in theaudience, aswerehis cabinetministers anda reporter forTimemagazine.OnewonderswhatthesepoliticiansandjournalistsmadeofSchrödinger’sfocuson‘howtheeventsinspaceandtimewhichtakeplacewithinthespatialboundaryofalivingorganismcanbeaccountedforbyphysicsandchemistry’.

Schrödinger subsequently extrapolated the three lectures into a book of less than ahundredpageswiththesametitle:WhatIsLife?Thiswaspublishedthefollowingyear.Inwhat is now a very famous book, Schrödinger popularised a quantum mechanicsinterpretation of the gene that had been proposed earlier by another distinguishedphysicist,thepreviouslymentionedMaxDelbrück.

Intheopeningpagesofthefirstchapter,Schrödingerposedthequestion:‘Howcantheevents which take place within a living organism be accounted for by physics andchemistry?’ Admitting that at the time of writing the prevailing knowledge within thedisciplines of physics and chemistry was inadequate to explain this, he neverthelesshazardedtheopinionthat‘themostessentialpartofalivingcell–thechromosomefibre–

may suitably be called an aperiodic crystal’. The italicisation is Schrödinger’s toemphasise, as he further explained, that thephysics up to this timehadonly concerneditselfwithperiodiccrystals,thekindofrepetitiveatomicstructuresseen,forexample,inveryobviouscrystallinecompoundssuchasgemstones.

Whatdidhemeanbyan‘aperiodiccrystal’?

Heexplainedthiswithametaphor.Ifweexaminedtheimageswithinthepatternofawallpaper, we could see how the pattern was repeated, over and over. This was theequivalentofaperiodiccrystal.ButifweexaminedthecomplexelaborationofaRaphaeltapestry,wesawapatternof images thatdidnot repeat themselves,yet thepatternwascoherentandmeaningful.

Schrödingerintuitedfurther.

Itwasthechromosomes,ormorelikelyanaxialfibremuchfinerthanwhatwasvisibleunder the microscope, that contained what he termed ‘some kind of code-script’ thatdeterminedtheblueprintoftheindividual’sdevelopmentfromfertilisedeggtobirth–andfurtherdeterminedthefunctioningofwhatwewouldnowtermthegenomethroughoutthelifetimeoftheindividual.

That intuition would provide the drive for a naïve but highly inquisitive youngAmerican, called JamesDeweyWatson, to join forceswith a slightly older but equallyinquisitive Englishman, Francis Crick, and form what is now seen as one of the mostfamous partnerships in scientific history. Both men would take their inspiration fromSchrödingertosearchfortheaperiodiccrystalthatcodedforDNA.

*

WatsonwasanexceptionallybrightchildwholivedathomewithhisfamilyinChicagowhileattendingthelocaluniversity.Heenrolledwhenagedjust15andhegraduated,aged19,in1947withabachelor’sdegreethatincludedayearstudyingzoology.Histeacherofembryologywouldrememberhimasastudentwhoshowedlittleinterestinlecturesandmadenonoteswhatsoever,soitwasallthemorepuzzlingwhenhegraduatedtopofhisclass.Watsonwouldsubsequentlyadmittoahabituallaziness.Thoughvaguelyinterestedin birds, he had deliberately avoided any courses that involved chemistry or physics of‘evenmediumdifficulty’.Thisself-indulgentstudentleftChicagowithonlyarudimentaryknowledgeofgeneticsorbiochemistry.Aspartofhiseducationhehadattendedlecturesby the geneticist Sewall Wright, who had devised a mathematical system of studyingpopulationgenetics.Wright’scourse includedadiscussionofAvery’swork,butWatsonwould subsequently confess that he took little notice. He would also confess that theinspiration for his subsequent interest in the ‘mystery of the gene’ was Schrödinger’sbook,WhatIsLife?

Inspired by this book,Watson landed a research fellowship at IndianaUniversity, atBloomington.Hewas delighted by themove becauseNobel LaureateHermann JosephMullerwasthelocalProfessorofZoology.Asearlyas1921Mullerhadobservedthatthegenesofthefruitflyunderwentmutations–asdidthegenesofthebacteriophages–thevirusesthathadinspiredHersheyandChase.Watsonwasintriguedbythefactthatphage

virusescouldbemanipulatedintesttubes.Theirreproductivecycleswereextremelybrief– an important consideration for an impatient young scientist. There were simple testsystems that could be employed to follow their life cycles, and numbers, in away thatwouldopenupnewanglesfromwhichtoattackthegeneproblem.Allyouhadtodowascarefully design an experiment aimed at probing some particular aspect of the geneproblemandthewholeshebangcouldbecompletedinamatterofdays.Thisintimate,ifbrutal,interplaybetweenphagevirusesandtheirhostbacteriaallowedscientiststofigurethecomplexchemistryofgenes,geneticsandchromosomes.

Curiously itwould not beMuller but another phage researcher, Salvador Luria,whowouldnowgiveshapeanddirectiontotheyoungscientist’sgrowinginfatuationwiththegene.

TheItalian-bornLuriawasanotherEuropeanscientist–amicrobiologist,likeAvery–whofoundrefugeinAmericafromtheEuropeanwarzone.BynowhehadenteredintoaworkingcollaborationwithMaxDelbrück,whowasProfessorofBiologyattheCaliforniaInstitute of Technology. In 1943 Luria and Delbrück designed and conducted anexperiment that demonstrated that genetic inheritance in bacteria followed preciseevolutionaryprinciples.ThisexperimentbecameoneofthefoundationstonesofmodernDarwinism. That same year Delbrück befriended another microbiologist called AlfredHershey,whowouldsubsequentlywritethekeyDNApaperwithMarthaChase.InalettertoLuria,Delbrück summarisedHershey as follows: ‘Drinkswhiskey but not tea.Likeslivinginasailboat…Likesindependence.’Thethreescientists joinedforcestobecomethe nucleus of a cooperating and mutually supportive network of scientists that wouldbecome known as the ‘phage group’. Delbrück would subsequently explain that theywouldbeagrouponlyinthesensethattheycommunicatedfreelyonaregularbasis,andthattheytoldoneanotherwhattheywerethinkinganddoing.Inthiswayaloosecreativemovement grew around the twoEuropean expatriate scientists, allworking towards thecommonambitionoffiguringouthowgenesworked.

Luria, Delbrück and Hershey now posed some interesting questions. How does thephagevirusactuallyget into thebacterium?How,once inside,does itmultiply?Does itmultiplylikeabacterium,growingandbuddingoffdaughterviruses?Ordoesitmultiplyby an entirely different mechanism? Is this multiplication some complex physical orchemical process that could be understood in terms of known physical and chemicalprinciples?Throughmakinguseofthephagereproductivesystem,theyhopedtosolvethemysteryof thegene.Tobeginwith it all seemedsimple inprinciple,but asexperimentfollowed experiment and year followed year, they found themselves no closer to theanswer.

Upto1940orso,peoplelikeDelbrückandLuriahadassumedthatvirusesweresimple.Theyhadlittletogoonsincethemajorityofvirusesweresominusculetheycouldnotbeseenwithanyclaritythroughtheordinarylightmicroscope.Theywouldeventalkaboutthemasiftheywereakintoproteinmolecules.Luriawouldcometodefinephageviruses,in amisleading oversimplification, as extensions of the bacterial genome.Butwith theinventionoftheelectronmicroscope,bytheGermancompanySiemens,eventhesmallest

viruses,includingbacteriophages,wouldsoonbecomevisibleforthefirsttime.Andwhenthey did become visible, they proved to be more complex than the two scientists hadinitiallyconceived.

Manyphageshadaheadthatwascylindricalinshape,withanarrowsheathbelowit,astallasthehead,andabaseplatewithsixspikeswithfibresattached.Nowthattheycouldvisualisephagesintheprocessofinfectingtheirhostbacteria,somethingstruckDelbrückandLuriaasexceedinglyodd.Thevirusesdidn’tactuallypass throughthebacterialcellwall. What they appeared to do was to squat down against the wall and inject theirhereditarymaterialintothecell.In1951aphageresearchercalledRogerHerriottwouldwrite toHershey, ‘I’vebeen thinking that thevirusmaybehave likea littlehypodermicneedle full of transforming principles.’ This became the background to Hershey andChase’sexperimentinwhichtheyconfirmedthatthatwaspreciselywhathappened.Thevirusbehavedexactly likeahypodermicsyringe; the tailand itselongatedfibrilswouldattachtothebacterialwallandthephagewouldtheninjectitsviralDNAinthroughthebacterial wall to take over the bacterium’s own genetic machinery, the viral genomecompelling the bacterial genome to constructwhatwas necessary for the generation ofdaughterviruses.Ineffect,theinfectedbacteriumbecameafactoryfortheproductionofdaughterviruses.

It would be this discovery, together with many associated extrapolations tomicrobiologyandgenetics, thatwould lead to all three scientists–Delbrück,Luria andHershey–sharingtheNobelPrizein1969.

Meanwhile,backin1947,itwasthedynamicenergyandinfectiouscharmofLuria,andthe innovative genius ofDelbrück, that provedmost influential to the youthfulWatsonafterhisarrivalintoIndianaUniversity.Stillfascinatedbythemysteryofthegene,itwashis hope that the mystery might be solved without his bothering to learn any of thecomplexphysicsorchemistry.

It is instructive todiscover, fromconversationsbetweenLuriaandWatson, that therewas no ignorance at Bloomington about Avery’s discovery of DNA. Luria had visitedAveryin1943,priortothepublicationofthekeypaper,whenhehadtheopportunityofdiscussingAvery’sfindingsindetail.HewouldrecallAverytoWatsonasanutterlynon-pompousscientist,preciseinhislanguage,withatendencyashespoketoclosehiseyesand rub his bald head – ‘every bit of a chemist, even though hewas anMD’.WatsonwouldtakehiscuefromLuria,writing,inTheDoubleHelix,howAveryhadshownthathereditarytraitscouldbetransmittedfromonebacterialcelltoanotherbypurifiedDNAmolecules.Given the fact thatDNAwas known to occur in the chromosomes of everytype of living cell, ‘Avery’s experiments strongly suggested that … all genes werecomposedofDNA.’

In the autumnof 1947,Watson, still just 19, tookLuria’s course in bacteriology andMuller’sinX-ray-inducedgenemutation.FacedwiththechoiceofenteringintoresearchwithMulleronDrosophilaorwithLuriaonmicrobes,heplumpedforLuria,despitethefact that the Italian scientist had a reputation among the graduate students for having ashortfusewithdimwits.Watsonwouldsubsequentlyadopthispatron’sexample.Delbrück

was a heroic figure to Watson because he had inspired Schrödinger’s ideas in theinspirationalbook.WatsonwasdelightedwhenLuria introducedhim toDelbrückwhentheeminentGermanphysicistpaidavisittoBloomington.

LuriasetWatsonaPhDdissertationonthepathologicaleffectsonphageofexposuretoX-rays. The work proved so mundane that Watson would barely mention it in hisbiography.Buthisobsessionwiththegenewasundimmed.Bythesummerof1949,histhesis nearing completion, he had the itch to travel to Europe. Luria arranged aMerckFellowshipfromtheNationalResearchCouncil–threethousanddollarsforthefirstyear,potentiallyrenewable.InMaythefollowingyear,withhisPhDunderhisbelt,hesailedforDenmark,wherehehadbeenassignedtostudynucleotideswithabiochemistnamedHermanKalckar.Kalckarwasagiftedscientistbuthis interestwasneither thegenenorthebacteriophage.AdisenchantedWatsonswitchedhisattentionstoanotherDane,andamember of the phage group, Ole Maaløe, who was working on the transfer ofradioactively-taggedDNAfromphagestotheirviraloffspring.

Out of the blue, Kalckar accepted a short-term project in the Zoological Station inNaples.HesuggestedthatWatsonmighttagalong.Thoughhehadlittleinterestinmarinebiology,Watsonwasdelightedtoacquiesce.HehopedtowarmhimselfintheItaliansun.ButhewasdisappointedtofindNapleschilly,withnoheaterinhisroomonthesixthfloorof a nineteenth-century house. ‘Most ofmy time I spentwalking the streets or readingjournalarticles…Idaydreamedaboutdiscoveringthesecretofthegene,butnotoncedidIhavethefaintesttraceofarespectableidea.’

Here, by happenstance, he attended a lecture in the Zoological Station given by anEnglish scientist namedMauriceWilkins.The lecture couldhardlyhave excitedhim inprospect, knowing that most of it would be about the biochemistry of proteins. ‘WhyshouldIgetexcitedlearningboringchemicalfactsaslongasthechemistsneverprovidedanythingincisiveaboutthenucleicacids?’

Buthetooktheriskandattendedanyway.

Tall,bespectacled,asthenicandsomewhatdiffidentinmanner,youmighthaveexpectedWilkins’presentation tobore therestlessand impatientWatson.But itdidnot.Tobeginwith, itwas delivered in a language thatWatson readily understood.And for all of hisdiffidentmanner,Wilkinskepttothepoint.Thensuddenly,closetotheendofthelecture,a projected slide jarredWatson to full attention. On the screen was a photograph thatshowedsomethingWilkinscalledanX-raydiffractionpatternofDNAthathadbeentakenin theKing’s College laboratory in London.Watsonwould subsequently admit that heknewnothingaboutX-raycrystallography.Hehadn’tunderstoodawordofwhathehadread about it in the scientific journals and he thought that much of what the ‘wildcrystallographers’wereclaimingwasverylikelybaloney.

ButnowherewasWilkinsmentioning inpassing that thiswas theclearestpictureofDNA that he andhis colleagueshadyet obtained from theirX-ray studies. In the sameaudiencewastheLeeds-basedEnglishphysicist,WilliamAstbury,whohadpioneeredX-ray diffraction studies of biological molecules, and who had produced the first X-raypicturesofDNA.Astburywouldsubsequentlyconfirmthatnoonehadevershownsucha

sharp,discretesetof reflectionsfromtheDNAmoleculeasWilkins thenprojectedontothescreen.‘Therewasnothinglikeitintheliterature.’Inexplainingthepicture,WilkinssuggestedthatDNAmightbethoughtofasacrystallinesubstance.

WatsonwaselectrifiedtohearSchrödinger’sprophecyconfirmed.Hesat inadazeofwondermentasWilkinswentontoexplainthatifandwhenweunderstoodthestructureofDNA,thenwemightbeinabetterpositiontounderstandhowgenesworked.Watsonwasnowaskinghimselfsomepertinentquestions.WhowasthisinterestingEnglishscientist,Wilkins?AndhowcouldhegettojoinhisteamatKing’sCollegeinLondon?

*

MauriceHughFrederickWilkinswasnot, infact,English,asWatson initiallysurmised.HewasborninPongaroa,NewZealand,wherehisfather,EdgarHenry,wasapractisingdoctor. The family were Anglo-Irish in origins, hailing from Dublin, where Maurice’spaternalgrandfatherhadbeenheadmasterofthehighschoolandhismaternalgrandfatherchiefofpolice.OnleavingNewZealandthefamilyfirstreturnedtoIreland,thenheadedforLondon,whereDrWilkinswaslatertodohispioneeringworkinpublichealth.

Mauricehadhadanatural scientificcuriosityevenasaboy,and itwas thiscuriositythatledtohisstudyingphysicsaspartofhisBAatCambridgeUniversity,afterwhichheworkedforhisPhDunderJohnTurtonRandall(laterknighted),aphysicistwhoplayedaleadingroleinthedevelopmentofradarduringthewar.

As a postgraduate, Wilkins moved to the University of Birmingham, following theposting of his Cambridge tutor, Randall, where the two scientists continued theircollaborationonradar.Butthen,outoftheblue,WilkinsfoundhimselfdispatchedtotheUnitedStates towork on theManhattanProject.His purposewas to figure out how topurify suitable isotopesof uranium from impure sources, tomake them suitable for theatomicbomb.InFebruary1944WilkinscrossedthedangerouswatersoftheAtlanticontheQueenElizabeth,headingfortheUniversityofBerkeley,California.Herehemadeamodest contribution to the development of the atomic bomb. However, the subsequentdestructionofHiroshimaandNagasakiby theveryweapons thathehadworkedon leftWilkinssomewhatunsettledinconscience.

After thewarWilkinsreturnedtoEngland,whereheendedupasassistantdirectorofthe new Biophysics Unit at King’s College London, funded by the Medical ResearchCouncil, and where his former boss, Randall, was now the Wheatstone Professor ofPhysics.Thenewdepartmentalremitwastoapplytheexperimentalmethodsofphysicstoimportant biological problems. This would result in Wilkins developing a relationshipwithWatsonandCrickand joining thesearchfor themolecularcodeofDNA.Itwouldalso involve him in a somewhat infamous strainedworking relationshipwith theX-raycrystallographerRosalindFranklin.

Given thisdevelopinghistory,wemightpauseamomentor two toconsiderWilkins’personality, and its relevance to thecom-ing storm.Fromwhatonecangather fromhisbelatedlypublishedbiography,andthememoryofthosewhoknewhimandworkedwithhim,Wilkinswasaquiet,highlymoralman,somewhatQuaker-likeinsocialattitudes.As

aboyheenjoyedacloseemotionalrelationshipwithhiseldersister,Eithne,whotaughthimtodance.ButthisintimacywastornapartwhenEithnedevelopedabacterialinfectionthat turned into a septicaemia, the blood-borne infection provoking septic arthritis inmultiplejoints.Thiswouldhavebeenashockinglypainfulanddisablingcondition,which,priortoantibiotics,mighthaveprovedfatal.Shespentmonthsinahospitalbed,withherlimbsdanglingfromhoists,herjointslancedopentodrainthepus.TheunfortunateEithnesurvivedbuttheintimacywithheryoungerbrotherended.Thetraumaofthisexperiencemaywellhaveaffectedhisself-confidence,particularlyinhisrelationshipswithwomen.

While anundergraduate atCambridge, he fell in lovewith awomancalledMargaretRamsey,buthe‘wasincapableofmakingasuitableadvancetoher’.Afterhetoldherofhislove,therewasashortsilenceafterwhichshewalkedfromtheroom.DuringhisstayinBerkeley,WilkinswasattractedtoanartistnamedRuth,whohadsharedlodgingswithhim. She conceived a child and they subsequently married, but when, as the war wasending,heinformedRuththatheintendedtoreturntotheUK,sherefusedtoaccompanyhim.‘RuthtoldmeonedaythatshehadmadeanappointmentformewithalawyerandwhenIarrivedathisofficeIwasshockedtohearthatRuthwantedtoendourmarriage.’Shortlyafterthedivorce,Ruthgavebirthtoason.Wilkinswenttoseeher,andtheirbaby,inthehospitalward,beforereturningtotheUKalone.

Wilkinswouldadmittodifficultyovercominganinnateshyness,andhewouldrequireperiodicpsychotherapyinhistimeworkingatKing’s,buthesubsequentlyfoundawife,Patricia,whoappreciatedthesensitivesoulbehindthediffidentexterior,andheenjoyedahappymarriageandthejoysofrearingafamilyoffourchildren.TherewasalsoafruitfuloutcomeofhisunsettledconsciencefollowinghisworkontheManhattanProject.BeforeleavingBerkeley,oneofhisworkingcolleaguescametohisrescue…‘SeeingIwantedtofindsomenewdirection,helentmeanewbookwiththeratherambitioustitle,WhatIsLife?’

four

ACoupleofMisfits

Francislikestotalk…Hedoesn’tstopunlesshegetstiredorhethinkstheidea’snogood.Andsincewehopedtosolvethestructurebytalkingourwaythroughit,Franciswastheidealpersontodoit.

JAMESWATSON

ItissomewhatironicthatMauriceWilkinsonlyarrivedinNaplesbyhappenstance,sincehewassubstitutingforRandall,whohadagreedtopresentthetalkbuthadbeenunabletoattend. It seemsunlikely,hadRandallhimselfpresented the lecture, thathewouldhaveincludedtheDNAslide,orthathewouldhavespokenofwhatitportrayedwithsuchclearreference to Schrödinger’s book. This lecture, which so excited Watson, was on thephysico-chemical structure of big biological molecules, mostly proteins, made up ofthousands of atoms. The key photograph had been taken byWilkins, working togetherwith a graduate student called RaymondGoslingwhile using a technique calledX-raydiffraction.Oneofthethingsthistechniquewasparticularlygoodatwasfindingthesortof repetitivemolecular themesyou found in crystals, hence theother term for it:X-raycrystallography.

‘Suddenly,’asWatsonwouldlaterrecall,‘Iwasexcitedaboutchemistry.’

UptothismomentWatsonhadhadnoideathatgenescouldcrystallise.Tocrystallise,substancesmusthavearegularatomicstructure–a lattice-likestructureofatomsat theultramicroscopic level. The youthful Watson appears to have been a wonderfully freespirit journeying from one interesting encounter to another. Impulsive, impatient,egregiouslydirect,yetallthewhileonthehuntfornewadventure.

‘ImmediatelyIbegantowonderwhetheritwouldbepossibleformetojoinWilkinsinworking onDNA.’ ButWatson never got to workwithWilkins. Instead, happenstanceheadedhiminthedirectionofanotherX-raycrystallographercalledMaxPerutz,whowasworkingattheCavendishLaboratoryatCambridgeUniversity.

TheCavendishLaboratoryisaworld-famousdepartmentofphysics.Firstestablishedinthe latenineteenthcentury tocelebrate theworkofBritishchemist andphysicistHenryCavendish, one of its founders and the firstCavendishProfessor of Physicswas JamesClerk Maxwell, famous for his development of electromagnetic theory. The fifthCavendishProfessorandthedirectorofthelaboratoryatthetimeofWatson’sarrivalwasWilliamLawrenceBragg,whowasthesuccessor,asdirector,toLordErnestRutherford,another Nobel Prize-winner and the first physicist to split the atom. Bragg was anAustralian-bornphysicistwho,jointlywithhisfather,hadbeenawardedtheNobelPrizeinPhysics in1915 for establishing theuseofX-rays in analysing thephysico-chemical

structures of crystals.X-ray beams are bentwhen they pass through the orderly atomiclatticeofcrystals.Whatisprojectedontothephotographicplateisnotthepictureoftheatoms within the structure but the refracted pathways of the X-rays after they havecollidedwiththeatoms.Thisiscalled‘diffraction’andissimilartohowlightisbentwhenitpassesthroughwater.Inastructurewithhaphazardpositioningofatomsinspace,theX-rayswillbescatteredrandomlyandformnopattern.Butinastructurethatcontainsatomsinarepetitiveatomiclattice–suchasacrystal–theX-raysaredeflectedinarecognisablepatternofblobsontheX-rayplate.Fromthisdiffractionpattern,theatomicstructureofthestructurecanbededuced.

The twoBraggs–fatherandsonworkingasa teamat theUniversityofLeeds–hadconstructedthefirstX-rayspectrometer,allowingscientiststostudytheatomicstructureof crystals.At the ageof 22,Bragg Junior, nowaFellowofTrinity atCambridge, hadproduced a mathematical system, Bragg’s Law, that enabled physicists to calculate thepositionsoftheatomswithinacrystalfromtheX-raydiffractionpictures.AtthetimeofWatson’s arrival into the laboratory, Bragg’s main focus of study was the structure ofproteins.ItwasthispotentialfortheX-raydiffractionofproteinsthathadattractedMaxPerutztotheCavendishLaboratory.

BorninViennaofJewishparentage,PerutzwasanotherenforcedexilewhohadsettledinEnglandandbecomearesearchstudentattheCavendishLaboratory.HecompletedhisPhDunderBraggandsubsequentlydevotedmostofhisprofessionallifetotheanalysisofthemacromoleculeofhaemoglobin, thepigment that colours the redcells inourblood,enabling them tocarryoxygenaround thebody.Alsoworkingat theCavendishwasanunusual young scientist,FrancisCrick.TheEnglish-born scientist hadgraduatedwith aBSc inphysics fromUniversityCollegeLondonaged21,but thanks towarduty andaprofoundantipathytohisPhDproject(hewassupposedtobeworkingontheviscosityofwaterathightemperatures)he,likeWatson,foundanalternativesourceofinspirationinSchrödinger’sbook.InCrick’sownwords,‘Itsuggestedthatbiologicalproblemscouldbethoughtaboutinphysicalterms.’

Butwhatterms?

At the time Crick wasn’t as convinced by Avery’s discovery as Watson was. LikeSchrödingerhimself,Crickwasmoreinclinedtotheproteinhypothesis.Buthewaseverybit as impressed with Schrödinger’s ‘code-script’ idea asWatson.What then could hepossiblymakeofSchrödinger’sconceptionofanaperiodiccrystal?

Simplecrystalssuchassodiumchloride,thebasisofcommonsalt,wouldbeincapableofstoringthevastmemoryneededforgeneticinformationbecausetheirionsarearrangedin a repetitive or ‘periodic’ pattern. What Schrödinger was proposing was that the‘blueprint’of lifewouldbe found inacompoundwhose structurehadsomethingof theregularity of a crystal, but must also embody a long irregular sequence, a chemicalstructure thatwascapableofstoring information in theformofageneticcode.Proteinshad been the obvious candidate for the aperiodic crystal, with the varying amino acidsequence providing the code. But now that Avery’s iconoclastic discovery had beenconfirmedbyHersheyandChase,thespotlightfellonDNAasthemolecularbasisofthe

gene. Suddenly new vistas of understanding the very basics of biology, and medicine,appearedtobebeckoning.

ItwasthroughamixtureofluckandthegutreactionofPerutzthatthedilettantishCrickwas taken into thefoldof theCavendish. InPerutz’srecollection,Crickarrived in1949withnoreputationwhatsoeverinscience.‘HejustcameandwetalkedtogetherandJohnKendrewandI likedhim.’Andso the likeableCrickendedup, insuchan idiosyncraticprocess of selection, working on the physical aspects of biology – what todaywe callmolecularbiology–undertheguidanceofBragg,PerutzandKendrew,attheCambridgelaboratory.

In 1934, John Desmond Bernal, an Irish-born scientist with Jewish ancestry and astudentofBraggSenior,hadshownforthefirsttimethatevencomplexorganicchemicalmolecules,suchasproteins,couldbestudiedusingX-raydiffractionmethods.Bernalwasa Cambridge graduate in mathematics and science, who was appointed as lecturer toBragg at the Cavendish in 1927, becoming assistant director in 1934. Together withDorothy Hodgkin, Bernal pioneered the use of X-ray crystallography in the study oforganic chemicals – the chemicals involved in biological structures – including liquidwater,vitaminB1, the tobaccomosaicvirusandthedigestiveenzyme,pepsin.ThiswasthefirstproteintobeexaminedattheCavendishinthisway.When,in1936,MaxPerutzarrived as a student from Vienna, he extended Bernal’s work to the X-ray study ofhaemoglobin.

By the timeCrick joined the laboratory,SirWilliamBragghadbeen replacedbySirLawrenceBragg,andJohnKendrewandMaxPerutzhadtakenBernal’sfindingsfurthertobecomeboggeddownina ‘disastrouspaper’on thechainstructuresofproteins.AndnowwediscoversomethingdistinctlyunusualaboutFrancisCrick,somethingthatPerutzmayhaveintuitedattheirmeeting.Hehadanavidcuriosityaboutscience,readingverywidely,andhewasequippedwithamindcapableofamassinga formidableknowledgebase across different disciplines.One of the first things he did after his arrival into theCavendishwastoacquainthimselfwitheverythinghisbosseshadachieved.Juniorashewas,Cricknowtookituponhimselftoundertakealong,criticallookattheirwork.Thishe then proceeded to criticise from basic principles. At the end of his first year in thedepartment,Crickpresentedhiscriticismsintheformofanadhocseminar,borrowinghistitlefromKeatsas‘WhatMadPursuit’.Hebeganwithatwenty-minutesummaryofthedeficienciesinthedepartmentalmethodsbeforepointingoutwhathesawasthe‘hopelessinadequacy’oftheirinvestigationofthestructureofthehaemoglobinmolecule.TheX-rayanalysisofhaemoglobinwasofcoursePerutz’smainobjective.Braggwasinfuriatedbythecockybehaviourofthisupstartjuniorcolleague,butPerutzwouldsubsequentlyadmitthatCrickwasrightandproteinswerefarmorecomplicatedintheirstructuresthantheyhad initially assumed. Restless and ever-inquisitive, Crick proved to be an uneasy,sometimesdownrightembarrassingimportintothescientificpoolofthelaboratory.Andwhile Bragg and Perutz saw proteins as the great unsolved puzzle, Crick was moreinterestedinthemysteryofthegene.

As1949elidedinto1950,CrickwouldsubsequentlyconfessthathestilldidnotrealisethatthegeneticmaterialwasDNA.Butheknewthatgeneshadbeenplottedoutinlineararrays along the chromosomes by people like Barbara McClintock, and that proteins,whichhadtobetheexpressionofthegenes,werealsobeingplottedoutaslineararrays,however lengthy and complicated. There had to be some logical way in which onetranslated into the other. By 1951, two years after his arrival into the CavendishLaboratory,Crickperceivedthattheseweretwodifferent,ifnecessarilyrelated,puzzles–themysteryofhowgenesappearedabletocopythemselves,andthemysteryofhowthelinearstructuresofgenestranslatedintothelinearstructuresofproteins.

Thewide-reading,voraciouslyinquisitiveCrickneededwhatJudsontermedacatalyst.Thisarrived in the formof thegangly,equally inquisitiveWatson thatsameyear,1951.From their first meeting, it would appear that here was one of those rare workingconjunctions of two odd-ball personalities that, when they come together, make anextraordinarycreativewholethatismorethanthesumoftheindividualingenuities.Andyetitverynearlydidn’thappen.

*

WeshouldrecallthatWatsonwasextremelyjuniorwithinthedepartment.ArecentPhDgraduate,hehadarrivedintoKalckar’slaboratoryonaMerckFellowshipfundedbytheUSNationalResearchCouncil.Thetermsandconditionswerelaiddownandsignedforbackhome,butnowherehewasabandoning thosecarefully laid intentions togallivantfromtheworkinDenmarktofollowsomegiddynewinspirationinEngland,aplacehehadnevervisitedinhislifeandwhereheknewabsolutelynobody.Impulsiveandsingle-minded,Watsonwouldsubsequentlyconfessthathisheadwasfilledwithcuriosityaboutthat single DNA photograph. He had tried to engage withWilkins in Naples after thelecture,atabusstopduringanexcursiontotheGreektemplesatPaestum.Hehadeventriedtotakeadvantageofavisitfromhissister,Elizabeth,whohadarrivedtojoinhimasatouristfromtheStates.NowherewereMauriceWilkinsandWatson’ssister,Elizabeth,findingacommontabletotakelunchtogether.Watsonsensedanopportunityandbargedin,with the intentionof ingratiatinghimselfwithWilkins.But the self-effacingWilkinsexcusedhimself,toallowbrotherandsistertheprivacyofthetable.

His plans foiled,Watson refused to let go of this exciting new avenue of interest. ‘IproceededtoforgetMaurice,butnothisDNAphotograph.’

He stopped over inGeneva for a few days to talk to a Swiss phage researcher, JeanWeigle, who provoked yet more excitement by informing Watson that the eminentAmerican chemist, Linus Pauling, had partly solved the mystery of protein structure.WeiglehadattendedalecturebyPauling,wholikeBragginCambridgehadbeenworkingwithX-rayanalysisofproteinmolecules.Paulinghad justmade theannouncement thattheproteinmodelfollowedauniquelybeautifulthree-dimensionalform–hehadcalleditan‘alpha-helix’.BythetimeWatsonarrivedbackinCopenhagen,Paulinghadpublishedhisdiscoveryinascientificpaper.Watsonreadit.Thenhere-readit.Hewasconfoundedby his lack of understanding ofX-ray crystallography.The terminology, in physics andchemistry,wassofarbeyondhimthathecouldonlygraspthemostgeneralimpressionof

itscontent.Hisreactionwassochildishlynaïveastobetouching:inhisheadhedevisedtheopeninglinesofhisownimaginedpaperinwhichhewouldwriteabouthisdiscoveryofDNA,ifandwheneverhediscoveredsomethingofsimilarportent.

ButwhattodotogetonboardtheDNAgravytrain?

He needed to learnmore about X-ray diffraction studies. Ruling out Caltech, wherePaulingwould reactwithdisdain to some ‘mathematicallydeficientbiologist’, andnowrulingoutLondon,whereWilkinswouldbeequallyuninterested,WatsonwonderedaboutCambridgeUniversity,whereheknew that somebodycalledMaxPerutzwas followingthesameX-raylinesofinvestigationofthebloodproteinmolecule,haemoglobin.

‘IthuswrotetoLuriaaboutmynewlyfoundpassion…’

Theworldofsciencewassmallerin1951thanit istoday.Evenso,itwouldappearahopelesslyoptimisticambitionforthisimpulsiveyounggraduatetomerelyaskhismentortofixhisarrivalintoaleadinglaboratoryinEnglandtoengageinalineofresearchthatheknewabsolutelynothingabout.

The amazing outcome was that Luria was able to do so. By happenstance, he metPerutz’sco-worker,JohnKendrew,atasmallmeetingatAnnArbor,inMichigan,where,byasecondandequalhappenstance,therewasameetingofminds–bothscientificandsocial.Andbyathirdhappenstance,Kendrewwaslookingforajuniortohelphimstudythestructureofthemuscle-basedproteinmyoglobin,whichcontainedironatitscoreandheldontooxygen,justlikethehaemoglobinintheblood.

TwiceinhisshortcareertheyoungAmericanscientisthadleaptintotheunknownandlanded on his feet. First it had been throughLuria’s patronage inBloomington, and byextensionalsoDelbrück’s,twooftheco-foundersofthephagegroup;andnowthegiftofhappenstance extended further, again through Luria’s patronage, to Kendrew, and byproxy to theCambridge laboratoryandMaxPerutz.Watson’sarrival into the laboratorywouldbringhimunder theultimate tutelageofSirLawrenceBragg,a founderofX-raycrystallography. It would connect him directly to his future partner in DNA research,Francis Crick, and further afield – through the connection between the CambridgelaboratoryandtheX-raylaboratoryatKing’sCollegeLondon–withMauriceWilkinsanda young female scientist, Rosalind Franklin, who were working on the X-raycrystallographyofDNA.

five

TheSecretofLife

Ithinktherewasageneralimpressioninthescientificcommunityatthattimethat[CrickandWatson]werelikebutterflies flicking around with lots of brilliance but not much solidity. Obviously, in retrospect, this was aghastlymisjudgement.

MAURICEWILKINS

Intheopeningpagesofhisbrief,wittyandbrutallycandidautobiography,JamesWatsonrecountsachancemeetingin1955withascientificcolleague,WillySeeds,atthebottomofaSwissglacier.ItwastwoyearsafterthepublicationofthediscoveryofDNA.WatsonandSeedswere acquainted, Seeds havingworkedwithMauriceWilkins in probing theoptical properties ofDNA fibres.WhereWatsonhad anticipated the courtesy of a chat,Seedsmerely remarked, ‘How’sHonest Jim?’, before striding away. The sarcasmmusthavebittendeepforWatsontonotmerelyrememberitdistinctly,buteventoconsidertheterm‘HonestJim’astheinitialtitleofhislifestory,beforebeingpersuadedtoadoptthemoredescriptive alternative, ‘TheDoubleHelix’. Itwas as if the former colleaguewasquestioningWatson’srighttoberecognisedastheco-discovererofthesecretoflife.

Hehadbeentakenaback,reflectingonmeetingswiththesamecolleagueinLondonafewyears earlier, at a timewhen, inWatson’swords, ‘DNAwas still amystery, up forgrabs…Asoneofthewinners,Iknewthetalewasnotsimple,andcertainlynotasthenewspapers reported.’ It was amore curious story, one in which his fellow-discoverer,Francis Crick, would freely admit that neither he nor Watson was even supposed toworking on DNA at the time. Equally curious was the fact that up to the day of thediscovery,neitherWatsonnorCrickhadcontributedanythingmuchtothemanydifferentscientific threads and themes that, when finally put together, like the pieces of aremarkable three-dimensional jigsawpuzzle, laid themolecular natureofDNAbare forthefirsttimeinhistory.

Watson’s welcome into the Cambridge laboratory was quintessentially English in itslackofformality.HearrivedinPerutz’sofficestraightfromtherailwaystation.Perutzputhim at his ease about his prevailing ignorance of X-ray diffraction. Both Perutz andKendrewhadcometothesciencefromgraduationinchemistry.AllWatsonneededtodowastoreadatextortwotobecomeacquaintedwiththebasics.ThefollowingdayWatsonwasintroducedtothewhite-moustachedSirLawrence, tobegivenformalpermissiontoworkunderhisdirection.WatsonthenreturnedtoCopenhagentocollecthisfewclothesandtellHermanKalckarabouthisgoodluck.HealsowrotetotheFellowshipOfficeinWashington, informing them of his change of plans. Ten days after he had returned toCambridgehereceivedabombshell in thepost:hewasinstructed,byanewdirector, to

forgo his plans. The Fellowship had decided he was unqualified to do crystallographywork.HeshouldtransfertoalaboratoryworkingonphysiologyofthecellinStockholm.WatsonappealedoncemoretoLuria.

As far as Watson was concerned it was out of the question to follow these newinstructions. If theworst came to theworst, hewould survive for at least ayearon the$1,000stilllefttohimfromthepreviousyear’sstipend.Kendrewhelpedhimoutwhenhislandlady chucked him out of his digs. It was just another indignitywhen he ended upoccupyingatinyroomatKendrew’shome,whichwasunbelievablydampandheatedonlybyanagedelectricheater.Thoughitlookedlikeanopeninvitationtotuberculosis,livingwith friends was preferable to the sort of digs he might be able to afford in hisimpecuniousstate.Andtherewasacomforttobehad:

‘IhaddiscoveredthefunoftalkingtoFrancisCrick.’

Andtalktheydid.

InCrick’sownmemory:‘JimandIhititoffimmediately,partlybecauseourinterestswere astonishingly similar andpartly, I suspect, because a certainyouthful arrogance, aruthlessness, and an impatience with sloppy thinking came naturally to us both.’ Thatconversation, lasting for two or three hours just about every day for two years, wouldunravelthemostimportantmysteryeverinthehistoryofbiology–themolecularbasisofheredity.

Weneedtograspafewfundamentalstounderstandhowthishappened.Firstly,wehavetwoyoungandambitiousmen–inWatson’scaseagedjust23,inCrick’s,aged35–whowere both exceptionally intelligent and surrounded by the ambience of high scientificendeavour and achievement. We need to grasp that Watson’s interest, intense andobsessive,wasthestructureofDNAinitspotentialtoexplainthemysteryoftheworkingsof the gene, and thus the storing of heredity. We also need to grasp the slight, butimportant,differencewithCrick’sinterest,whichwasnotDNA,oreventhegeneinitself,butthepotentialofDNAtoexplainhowSchrödinger’smysteriousmolecularcodes–hisaperiodiccrystals–hadthepotentialnotonlyforcodinghereditybutfortranslatingfromonecodetoanother,fromthegenetothesecondaperiodiccrystalthatmustdeterminethestructureofproteins.

Crick would subsequently recall Watson’s arrival in early October 1951. Odile, hisFrenchsecondwife,andhewerelivinginatinyramshackleapartmentwithagreendoorthat they had inherited from the Perutzes. Conveniently situated for the centre ofCambridgeandonlyafewminutes’walkfromtheCavendishLaboratory,itwasalltheycould affordonCrick’s research stipend.The ‘GreenDoor’, as itwas thereafter called,consistedofanatticoveratobacconist’shouse,with‘twoandahalfrooms’andasmallkitchen thatwas reachedby climbing a steep staircaseoff thebackof the tobacconist’shouse.ThetworoomsservedaslivingroomandbedroomforCrickandOdile,withthehalf room providing a bedroom forCrick’s son,Michael – born to his firstwife, RuthDoreen–whenMichaelwas home fromboarding school.Thewash-roomand lavatoryopenedhalfwayupthestairsandthebath,coveredwithahingedboard,wasafeatureofthetinykitchen.

Oneday,outoftheblue,PerutzbroughtWatsontotheflat.Crickwasout.ButhewouldrecallOdile remarking thatMax had come roundwith a youngAmericanwho ‘had nohair’. The newly arrived Watson was sporting a crew-cut – a hairstyle uncommon inEnglandatthetime.Theymetwithinadayortwo…‘Irememberthechatswehadoverthosefirsttwoorthreedaysinabroadsortofway.’

Both men were impecunious, but it hardly mattered since they were uninterested inmoney.Whatmatteredwas that the deeply personal, deeply intellectual, symbiosis hadbegun.Crick brought a rowdy enjoyment of problem solving, togetherwith the hubris,bornoutofhisbackgroundinphysics,tobelievethatthebigproblemfacingthem–themysteryofthegene–wasindeedsolvable.Watson,whohadlittleknowledgeofphysicsor X-ray crystallography, brought a mine of knowledge about the way in which genesworked–thefruitsofthebacteriophageresearchesofLuriaandDelbrück.Perutzwouldsubsequently confirm that the arrivalofWatson, at thatparticularmomentof time,wasopportune for the workings of the Cavendish Lab, where his enthusiastic personalityappearedtohavegalvanisedCrick,andwherehisknowledgeofthefieldofgeneticsaddedan exotic aspect to the structural physics and chemistry that otherwise prevailed.Moreover,differentastheirbackgroundswere,CrickandWatsonsharedadeep,insatiablelevel of curiosity about the puzzle that lay at the very root of biology: they weredetermined,almostfromtheirfirstmeeting,thattheywouldsolvethemysteriousnatureofthegene.

The first creative step was to realise that the answer lay with DNA. To be moreaccurate, they realised that somehow chemical structure must parallel function: so theanswer to thegreatconundrumlay in the three-dimensionalchemicalstructureofDNA.Butnobodyreallyknewwhatshapeorformthisstructuretook.TothemindsofCrickandWatsonatthatparticularmomentintime,itwouldhaveseemednothingmorethanaghostinthemist.

Newdiscoveries insciencewillusually involvea lengthyperiodof laboratorylabour,with knowledge growing by hard-won increments, often involving contributions fromseveral,oragooddealmorethanseveral,differentsources.Inmanywaysthestruggletoget to grips with the mysteries of heredity followed exactly such a course. But themundane sweat of the laboratory aspects, the growth of knowledge by hard-wonincrements,wouldnotfalltoWatsonandCrick.Thesewouldbelefttoothers.TheCrick–Watson symbiosis would be founded on a second, equally important ingredient ofscientific advance, and one that has commonalities with the advances in the arts andhumanities:thisisthequintessentiallyhumangiftwecall‘creativity’.

Withinthehierarchyofthelab,CrickandWatsonwerethelowestcontributinglevel.InCrick’swords, ‘Iwas justa researchstudentandJimwas justavisitor.’Theyreadverywidely,imbibingthefruitsofthehardworkofothers.Theytalkedandtalked,thinkingoutloud, probing one another’s ideas and knowledge, often with Crick playing devil’sadvocate.Infacttheygossipedandarguedsomuchtheyweregivenaroomtothemselves– to avoid their interrupting the thoughts of their more senior colleagues – within thecrowdedstructureoftheoldCavendishLaboratory.TheX-raylaboratory,withitsheavy

machineryandradiationdangers,waslocatedinthebasement.JimandFranciswouldalsoshareacheapandcheerfullunch,ofshepherd’spieorsausageandbeans,atthelocalpub,the Eagle – a grubby establishment in a cobblestoned courtyard – where the creativedebatewouldsimplycontinue.

WhatlittletheyknewaboutDNAwasmadeevenmoreuncertainbythefactthatCrickbelievedthatmuchofwhatwasgenerallyassumedtobethecasewithDNAandhereditywasalmost certainlywrong. It hadbeen this attitude thathadgothim into troublewithBragg.Itmeant thathedidn’teventrust theworkofhisseniorshere in the lab.But therealreasonbehindBragg’sangerwashisresentmentofthefactthatthechemist,Pauling,haddiscoveredthealphahelixofprotein.Meanwhile,CrickwasconvincedthatthereasonwhytheCavendishhadmissedoutonthiswasbecausetheywereassumingtheaccuracyof some earlier experimentation on theX-ray interpretation of the skin protein, keratin,whichisthemainingredientofourhumannailsandaraptor’sclaws.ThewayinwhichCrick’smindworkedcanbegleanedfromarememberedconversation:

‘The point is [so-called] evidence can be unreliable, and therefore you should use aslittle of it as you can.Wehave three or four bits of data,wedon’t knowwhich one isreliable…[Whatif]wediscardthatone…thenwecanlookattherestandseeifwecanmakesenseofthat.’

*

WatsonjoinedtheCavendishinthesameyear,1951,inwhichLinusPaulingpublishedhispaperontheprotein‘alphahelix’.ThisdiscoverysorattledWatsonthatallofthetimehewasworkingwithCrick on the structure ofDNA, hewas looking over his shoulder inPauling’sdirection.

He had good reason for seeing Pauling as the supreme rival in such an exploration;awarded the Nobel Prize in Chemistry in 1954, Pauling was already being hailed byscientifichistorians asoneof themost influential chemists inhistory.Hismasterwork,thoughhecontributedagreatdealmore,wastoapplyaquantumtheoryperspectivetothechemical bonds that bind atomswithin the structure ofmolecules, extending this basicsciencetothecomplexorganicmoleculesthatarethechemicalbuildingblocksoflife.

The twentieth century has amazed us with its achievements in astronomy, in whichscientists have plotted the stars and galaxies, and the forces, such as black holes, thatgovern theUniverse.Equally important, thoughnot soeasily recognisedas suchby theordinarymanandwoman,havebeentheachievementsofthechemistsandbiochemistsinexploringthemicro-universeofatomsandmolecules.Twoforcesinparticularplayakeyroleinthewaythatatomsbindtooneanothertomakeuplife’sparticularmolecules.Oneoftheseiscalledthecovalentbond;theotheriscalledthehydrogenbond.Paulingappliedthe science of quantum mechanics to the forces involved in these two very differentchemicalbonds.

We have no need to concern ourselveswith the complexmathematics of the appliedphysics.Wejustneedtograspthebasicmechanics.Andwherebettertolookthanatthefamiliarmoleculeofwater.

Everybody knows that the chemical formula for water is H2O. This tells us that amoleculeofwatercomprisesoneatomofoxygenandtwoatomsofhydrogen.Buthowdotheylinkwithoneanothertoformthestablecompoundthatwehandleandconsumeeverydayofourlives?Themoleculeofwatermightbecomparedtoaplanet,oxygen,withtwoencirclingmoonsofhydrogen.Insuchasituation,wecanreadilyimaginehowtheforceof gravitywould hold the hydrogenmoons to their orbits around the oxygen planet. Inmolecularterms,theforcesholdingthetwohydrogenatomstotheoxygenatomarecalled‘covalent bonds’.At the ultramicroscopic level of atoms, the nucleus of each hydrogenatom contains a single positively charged protonwhile circling around the nucleus is asingle negatively charged electron. Meanwhile, the oxygen atom has eight positivelycharged protonswithin its nucleus and eight balancing, negatively charged electrons inorbitsaroundit.Theseelectronsoccupytwoorbits–twoelectronstakingupaninnerorbitandsixtakingupanouterorbit.Incomingtogethertoformamoleculeofwater,thetwoelectronsinorbitaroundeachofthetwohydrogennucleihavepairedwithtwoofthesixelectrons of the oxygen outer orbits. The paired electrons share their attraction to theprotonsofthetwoparentnuclei,sothepairedelectronsarenowequallyattractedtotheoxygen nucleus and the hydrogen nuclei. This sharing of attraction creates a stable‘covalent’bondbetweenthethreeatoms,justasgravitycreatedstableorbitsforthetwomoonsrotatingaroundourimaginaryplanetofoxygen.

Hydrogenbondsaresomethingelse.

Once again, we might take water as our example. But here we are looking at thechemical interactions between whole water molecules – the H2Os reacting with oneanother.Thereareforcesofattraction,albeit ratherweakerandlessstable thancovalentbonds,betweencertainmolecules thatcontainbothhydrogenandheavieratomssuchasnitrogen,oxygenorfluorine.Sincewatercontainshydrogenandoxygen,thesehydrogenbonds can form between molecules of water – it is this sticking together of watermolecules thatexplains thedifferencebetweenwatervapour,or steam, liquidwaterandsolidwater,orice.Inicemostofthemoleculesareattachedtooneanotherbyhydrogenbonds, toformsomethinglikeacrystal; in liquidwatervaryingamountsareattachedtoone another; and in steam, as a result of the addition of energy through heating, thehydrogen bonds linking water molecule to water molecule are broken down but thecovalentbondslinkingatomstoatomsremainintact.

We see that hydrogen bonds are weak, and thus unstable when heated, but covalentbonds are stable. These same two bonds, covalent and hydrogen bonds, are importantingredients in the structure of organic chemicals such as proteins. And they are alsoimportantinthestructureofDNA.

Between 1927 and 1932, Pauling published some fifty scientific papers in which heconducted X-ray diffraction studies, coupled with quantum mechanical theoreticalcalculations,leadinghimtopostulatefiverules,knownasPauling’srules,thatwouldhelpscience topredict thenatureof thebonds thatheld togetheratomswithinmolecules.Atleast three of these rules were based on Bragg’s own work, the purloining of whichprovoked Bragg to fury. It was now inevitable that there would be ongoing scientific

rivalrybetweenthetwoscientists.Pauling’sworkintothenatureofchemicalbondingwassooriginal, andpioneering, thathewasawarded theNobelPrize inChemistry in1954.Meanwhile,thisnewlevelofunderstandingenabledPaulingtovisualisethepreciseshapeand dimensions of molecules in three-dimensional space. Working at Caltech, Paulingapplied this to thehugemoleculesofproteins,using the techniquesofX-raydiffractionanalysispioneeredbytheBraggs.Heshowed,forexample,thatthehaemoglobinmolecule–thefocusofPerutz’sresearch–changeditsphysicalstructurewhenitgainedorlostanoxygen atom. And Pauling continued to apply his rules to researching the molecularstructureofproteins.

PioneeringX-raypicturesof fibrousproteinshadbeenobtained someyearsbeforeatthe University of Leeds by William Thomas Astbury, the physicist who had attendedWilkins’talkinNaples,butitwasassumptionsbasedontheseX-raydiffractionpicturesthatCrickwasnowquestioningattheCavendishLaboratory.FormanyyearsPaulinghadtriedtoapplyquantummechanicscalculationstoAstbury’sX-raypictures,buthefoundthatthingsjustdidn’taddup.Itwouldtakehimandtwocollaborators,RobertCoreyandHermanBranson,fourteenyearsbeforetheymadethenecessarybreakthrough.

Allproteinshaveaprimarystructurethat ismadeupofanaminoacidcode,withtheletters made up of twenty different amino acids. The chemical bonds that join up theamino acids into the primary chain are called ‘peptide bonds’. Pauling and hiscollaboratorsnowrealisedthatpeptidesbondedtogetherinaflattwo-dimensionalplane–theycalledthis‘aplanarbond’.AproblemwithoutdatedequipmenthadcausedAstburytomakeacriticalerror in takinghisX-raypictures: theproteinmoleculesbecametiltedawayfromtheirnaturalplanes,skewingthemathematicalextrapolationsoftheirstructure.Once theyhadcorrectedAstbury’serror,Paulingandcodiscovered thatas thechainofaminoacidsgrew,toformtheprimarystructureofproteins,itnaturallyfollowedtheshapeof a coiled spring, twisting to the right – the so-called ‘alpha helix’. This was thediscoverythathadexcitedWatsononhisreturntripfromNaples.

Back in Cambridge, Sir Lawrence Bragg was bitterly disappointed when Pauling’sgroupbeathistothediscoveryoftheprimarystructureofproteins.Buttherewasasilverliningtothecloud:PerutznowusedPauling’sbreakthroughtoreappraisehisownworkonthe haemoglobin molecule, a reappraisal that would solve the structural puzzle ofhaemoglobinandgarnerhisNobelPrize inChemistry in1963.Pauling’sdiscoveryalsoalarmedWatsonwho, fromhis arrival atCambridge, had assumed that theyhad a veryknowledgeable and powerful rival in what was now a race to discover the three-dimensionalstructureofDNA.

*

But the problem, as Crickwould point out in their day-to-day sharing of thoughts andincessant debate, was that they couldn’t even assume that Pauling’s data was right. InCrick’s words, ‘Data can be wrong. Data can be misleading.’ So Crick and Watsonattempted to construct their physical model with a sceptical eye on prevailingexperimentaldata.Toput itanotherway, theyrelied justasheavilyoncreative leapsoftheirownimaginationasonexistingexperimentaldata.

Crick andWatson were now asking themselves if DNA, like proteins, had a helicalstructure,andWatsoninparticularwasconvincedthattheyshouldalsotaketheircuefromPauling, who liked to construct three-dimensional models of the molecules he wasattempting to envisage. To do so they would have to think, as Pauling did, about theatomic structures thatmadeup the chemistry ofDNA– to fit themolecules,with theircomponentatoms,andthebondsbetweenthem,intoacomplexthree-dimensionaljigsaw.Theyknewthattheyweredealingwiththefournucleotides–guanine,adenine,cytosineand thymine– togetherwith themolecule of the sugar, called ribose, and the inorganicchemical,phosphate,allofwhich,whencorrectlyfittedtogether,mustsomehowmakeupthemysteriousthree-dimensionaljigsawpuzzle.

Tworelevantquestionsnowloomed.Firstly, if thestructurewashelical,whatkindofhelix was involved? And secondly, where did the phosphate molecule fit into thestructure?Calciumphosphateisthemineralofbones,ofshells,ofrocksformedfromtheremains of livingmarine organisms – limestone. The presence of phosphate suggestedsomekindofstrengtheningoftheDNAchain–achemicalscaffold–maybeaspine?Butwhere did this spine lie in relation to the presumptive and as yet unknown spiral?Andwhere,orhow,did thesugarfit in?Thecodeitselfmustsurely liewith thenucleotides,actingperhapsassomethinglikeletters.Eachwasakeyingredient,buthowonearthdidthewholethingassembleinawaythatmadesense?

An important clue must come from the X-ray diffraction patterns. That meant theyneededthehelpofMauriceWilkinsandRosalindFranklin–‘Rosy’,asWatsonreferredtoher in his autobiography – who were conducting X-ray analyses of DNA fibres at theKing’sCollegeLondonlaboratory.

*

RosalindElsieFranklinwasborninLondontoaprosperousJewishfamilyin1920.Fromanearlyagesheshowedbothabrilliantlyincisivemindandthestubbornnessnecessarytomakeadistinguishedmarkforherself.Shealsoshowedanaggressivelycombativesidetoherpersonalitythatmightproveamixedblessinginovercomingtheprevailingprejudicesagainst Jews in society, as well as against women being in higher education and thescientific workplace. It didn’t help that her father, who appeared to be a similarlycombative character to his daughter, opposed her notion of a career in science. In hersecondyear atNewnhamCollege,Cambridge, he threatened to cut off her fees, urgingthatsheswitch tosomepracticalapplication insupportof thewareffort.Onlywhenhewasdissuadedbyhermotherandauntdidherelentandallowhertocontinuehercourse.

Franklin studied physical chemistry, which involved lectures, extensive reading andlaboratory experience in physics, chemistry and the mathematics that applied to thesedisciplines.Oneof themandatory texts she readwasLinusPauling’sTheNatureof theChemicalBond.

The youthful Rosalind Franklin was disappointed when she ended up with a goodsecond, and not a first, ‘bachelor’s’ degree in 1941. Even then, suchwas the lingeringprejudiceagainstfemalegraduatesinsciencethatshewasforcedtowaitinanunseemly

uncertainty, one shared with all previous female graduates of Newnham, until her duequalificationwasformallygranted,retrospectively,in1947.

Like Francis Crick, Franklin was seconded to National Service during the SecondWorldWar,studying thedensityandporosityofcoal foraPhD, inwhichshehelped toclassifydifferenttypesofcoalintermsoffuelefficiency.Post-war,shefollowedthisupwith a research stintworking under the direction of JacquesMering at the LaboratoireCentral des Services Chimique de l’Etat, in Paris. Here, Mering introduced her to theworld of X-ray crystallographywhich he used to study the structure of fibres, such asrayon.‘Withhishightartarcheekbones,greeneyesandhaircombedrakishlyoverhisbaldspot’,Franklinwas surprised todiscover thatMeringwas Jewish, aswell asbeing ‘thearchetypalseductiveFrenchman’.Thestillyouthful,andperhapsnaïve,RosalindFranklinappearstohavefalleninlovewithMering,whowasalreadymarried,butwhosewifewas‘nowhereinevidence’.

BrendaMaddox, one ofFranklin’s biographers,would draw attention to the fact thatFranklin’smostimaginativeandproductiveresearchwasconductedwhenshewasteamedupwithmalescientistsofJewishbackground.Meringalsoappearedtobeattractedtothetrim, slender youngwoman,with the lustrous dark hair and glowing eyes. Theywouldspendentiredaysandon into theeveningsdeep indiscussionandargumentover likelymeaningsofX-rayplatesandatomicstructures.

However, Franklin’s infatuation with Mering would be painfully halted when, inJanuary1951, she tookupapostas researchassociateatKing’sCollegeLondon in theMedical Research Council Biophysics Unit, directed by John Turton Randall. Herappointment happened to coincide with a major post-war rebuilding within thedepartment, designed to accommodate new ambitions within the nascent field ofbiophysics. The precise nature and purpose of her appointment has since become thesubjectofdebate.InpartsomeconfusionhasarisenbecauseRandallchangedthescopeofherappointment inbetweenfirstconfirming itandFranklin takingup thepost.ShehadinitiallyagreedtocarryoutX-raydiffractionstudiesofproteins,butRandallwrotetoherbeforeshetookupherappointment,suggestingthatshechangedirectiontothestudyofDNA. According toMauriceWilkins, this was at his suggestion.Whether atWilkins’suggestion orRandall’s own idea, Franklin agreed. Shewas offered the assistance of apromisinggraduate student,RaymondGosling, toworkwith.But therewasan inherentproblemwiththisnewdirection.

Wilkins,whowasDeputyDirectoroftheMRCUnitbasedatKing’sCollege,wasthesame scientist who had first lit the fuse of inspiration forWatson in the 1950 Napleslecture.WilkinshadinitiatedtheresearchintoDNAinthedepartment,buthappenedtobedeputisingonceagainforRandallinAmericaatthetimeofFranklin’sappointment.UptonowGoslinghadbeenworkingwithWilkinsonDNA;evenafterhisreturnfromAmerica,Randall failed to informWilkins about the terms he now proposed for Franklin’s jobdescription. This led to what Franklin’s later research colleague, Aaron Klug, woulddescribe as ‘an unfortunate ambiguity about the respective positions of Wilkins and

Franklin,which later led to dissension between them and about the demarcation of theDNAresearchatKing’s’.

This is a short quote from the typed letter from Randall to Franklin, specifying herworkingconditions:

…asfarastheexperimentalX-rayeffortisconcernedtherewillbeatthemomentonlyyourselfandGosling,togetherwiththetemporaryassistanceofagraduatefromSyracuse,Mrs.Heller…

While this clearly suggests that Franklinwas expected to take on theX-ray diffractionwork, thequalification‘at themoment’ is toovague to interpret.But there isnothing inthis letter tosuggest thatFranklinshouldignoretheworkperformedbyWilkins,or thatshe should refuse to collaboratewith the rest of the department in her approach to theDNAproblem.

Wilkins,workingwithGosling,had initiated theX-raydiffractionstudiesonDNAinthedepartment,andinparticularobtainingthebestresolutiondiffractionphotographsthatexisted up to this date. They had demonstrated a key property ofDNA – that it had aregular, crystal-like molecular structure. In Paris Franklin had learned, and improvedupon,X-raydiffractiontechniquesfordealingwithsubstancesoflimitedorder.ButevenKlug,anardentsupporterofFranklin,admittedthatinrelationtotheworkconductedbyFranklin in Paris, ‘It is important to realise… Franklin gained no experience of suchformalX-raycrystallography.’

Back inearly1950Wilkinshadcomplainedofpoor-qualityX-rayapparatus thatwasnotdesignedforthescrutinyofexquisitelyfinefibres.Athissuggestion,thedepartmenthadpurchasedanewandbetter-qualityX-raytubetobesetupinthebasement,butithadlain there forayearormoreunusedwhileWilkinswasdistractedby themultiple tasksthat fell to a busy deputy director of the unit.Onher arrival, Franklin, not unnaturally,believedthatshewastheretotakeovertheDNAworkasherpersonalproject.However,the returningWilkins expected thatFranklinhadbeenbrought in ashis collaborator, totake up the research from where he had already developed it. He would subsequentlyadmit that he was unqualified to take the X-ray diffraction work further and neededexactly such a dedicated and qualified collaborator. ‘That’s why we hired RosalindFranklin.’

Unfortunately,FranklinandWilkinsnowdisagreedastoherrole.Evenso,rancourwasneither necessary nor inevitable between the two scientists, personally or scientifically.Thesedifficulties,provokedbyRandall’svagueness,mighthavebeen readilyovercomewithgoodwillonbothsides,butFranklin,intheopinionofbothherbiographers,wasnotinclinedtocooperate.

Muchhasbeenwrittenaboutprejudicialattitudestowomeninscienceatthistime.Inparticular anAmerican journalist, andpersonal friendofFranklin’s,AnneSayre,wouldwrite a biography of her in which she suggested that King’s College was particularlyunfriendlytofemalescientists,withFranklinstrugglingtoassertherpresenceinadomainthatwasalmostexclusivelymale.ButwhenanotherAmericanjournalist,HoraceFreelandJudson,lookedintothisclaim,hediscoveredthatofthe31staffworkingatKing’satthistime,eightwerefemale,includingsomeworkinginaseniorpositioninFranklin’sunit.A

second biography of Franklin, byBrendaMaddox, confirmed thatwomenwere, on thewhole,welltreatedatKing’sCollege.Crickmadethesamepointinhisbiography–andCrickhadcometoknowFranklinwellintheyearsfollowingtheDNAdiscovery.EveninSayle’smoretrivialcomplaint–thatthemaindiningroomwasexclusivelyforbiddentowomen, who were thus precluded from lunchtime conversation – is misleading. Therewere two dining rooms. One was limited to men, but this, in the main, was used byAnglican trainees. The main dining room, used by the departmental staff, includingRandallhimself,wasopentoall.

ThefrostyrelationshipbetweenWilkinsandFranklinwasnottheresultofanti-femaleprejudice – it even seemsunlikely to be the result ofRandall’s peculiarwording in theletter–but itappears tobemoredirectlyrelated toapersonalityclashbetween the twoscientists. Of the two, only Wilkins ever seems to have made any attempt atcompromising. He asked other colleagues what he should do, but Alexander (Alec)Stokes,hisclosestcolleague,wasevenmeekerthanhewas.InBrendaMaddox’sopinion,the twoshouldhavegotonwell;Wilkinswasgentle inmannerand,despitehis lackofself-confidence,wasattractivetowomen.HewasmathematicallyfluentandimmersedintheveryproblemsthatconcernedFranklin.But‘confrontation’,inMaddox’swords,‘wasFranklin’stactic,whenevercornered’.Inanearlierconfrontationwithherprofessor,R.G.W.Norrish,whenworkingon a postgraduate researchproject atCambridge, shewouldconfide,‘WhenIstooduptohim…wehadafirst-classrow…hehasmademedespisehimsocompletely I shallbequite impervious toanythinghemaysay in the future.Hegavemeanimmensefeelingofsuperiorityinhispresence.’

Sayre,whochampionedherfriend,wouldadmitthatFranklin’sogrishdepictionofherprofessorwasunkindand inaccurate.ProfessorNorrishwasawarded theNobelPrize inChemistryin1967.

Sayre had a correspondencewithNorrish inwhich she describedFranklin as ‘highlyintelligent … and eager to make her way in scientific research’, but also ‘stubborn,difficult to supervise’ and, perhaps most tellingly, ‘not easy to collaborate with’. InMaddox’sopinion, ‘IfRosalindhadwished, she couldhave twistedWilkins aroundherlittlefinger.’Thefactisshehadnowishtocollaboratewithhim.ThisleftWilkinsisolatedlocallysoinsteadheturnedtoCrickandWatsonatCambridge.ItalsomeantthatFranklinwasequally isolated.To thecommonsensicalCrick, thismayhavebeenacrucial factorwhenitcametoworkingoutthemolecularstructureofDNA.‘Ouradvantagewasthatwehadevolved…fruitfulmethodsofcollaboration,somethingthatwasquitemissingintheLondongroup.’

InthatsameyearofFranklin’sappointment,justbeforeWilkinsheadedforAmerica,heasked his colleague,AlecStokes – anotherCambridge-educated physicist – if he couldworkoutwhatkindofdiffractionpatternahelicalmoleculeofDNAwouldprojectontoanX-rayplate.IttookStokesjusttwenty-fourhourstodothemathematics,largelyfiguringitoutwhiletravellinghomeonthecommutertraintoWelwynGardenCity.AhelicalmodelfittedverycloselywiththepictureGoslingandWilkinshadobtainedintheirdiffractionpicturesofDNA.ItwouldappearthatifanybodyfirstconfirmedthatDNAhadahelical

structure, thecreditsmustsurely includeWilkins,GoslingandStokes– thelatterwouldsubsequentlylamentthat,inretrospect,hemighthavemerited1/5000thofaNobelPrize.

In November 1951, Wilkins told Watson and Crick that he now had convincingevidence thatDNAhadahelicalstructure.WatsonhadonlyrecentlyheardFranklinsaysomethingsimilarinatalkaboutherresearchduringaKing’sCollegeresearchmeeting.ThisinspiredWatsonandCricktoattempttheirfirsttentativethree-dimensionalmodelforDNA.

Butwheretobegin?

TakingtheircuefromLinusPauling,WatsonandCrickdecidedthattheywouldattempttoconstructathree-dimensionalphysicalmodeloftheatomsandmoleculesthatmadeupDNAwiththeircovalentandhydrogenbondlinkagestooneanother.Onthefaceofit,thestructurewasmadeupof avery limitednumberofdifferentmolecules.Therewere thefournucleotides–guanine,adenine,cytosineandthymine–but theyalsoknewthat thestructure contained a sugar molecule, deoxyribose, and a phosphate molecule. Thephosphatewaslikelytobeplayingastructuralrole,perhapsholdingthethreadtogether,much as phosphate is a key structural component of our bony human spine. In thecolloquium atKing’s, attended byWatson, suchwas his lackadaisical absence of focusthathecompletelymissedtheimportanceofFranklin’sstatementthatthephosphate-sugar‘spines’were on the outside,with the coding nucleotides, theGACT, on the inside.Asusual,hehadeschewedmakingnotes.AllthatseemedtointrigueWatsonwasthefactthattheKing’speoplewereuninterestedinthemodel-buildingapproachdeveloped,withsuchaplomb,byPauling.

In 1952 Franklin appears to have undergone a drastic change of heart in her ownthoughts on the structure of DNA. She had in her possession a brilliantly clear X-raypictureofDNA,takenbyGosling,thatclearlyshowedahelicalstructuretothemolecule.Shecalledthisher‘wetform’,andalsoher‘Bform’.Butshehadevenclearerpicturesofa different structure of the same molecule in its ‘dry form’, or ‘A form’, that did notappeartosuggestahelix.ThecontrastbetweenthetwoformscausedFranklintoditherastowhethertheDNAmoleculewashelical.Thereisasuggestionthatshemayhaveaskedthe opinion of an experienced French colleague, who advised her to place her bets onwhichever formgave the clearest pictures. Shemust have been altogether aware of theadvice her ignored colleague,Wilkins, would have given. Unfortunately, she ended upputtingtheBformintoadrawer,meanwhilefocusingmostofherresearchoverthatyearintotheAform.

EarlythatsameyearWatsonandCrickmadeafirstattemptatbuildingatriple-strandedhelical model of DNA, with a central phosphate-sugar spine. When Wilkins broughtFranklinandGoslingup toCambridge toview themodel, theybrokeout into laughter.Themodelwasabsoluterubbish.ItdidnotfitatallwiththeX-raydiffractionpredictions.Thanks to Watson’s lackadaisical focus, and his failure to take notes at Franklin’scolloquium, he hadmade the cardinal error of putting the phosphate-sugar spine at thedead centre of their helix and not on the outside, as Franklin and Gosling had clearlydeduced.

Sayre, who rightly defended Franklin from the egregious caricature depicted byWatson’s book, loses track of the contribution of Wilkins and Gosling. It is true thatFranklin andGosling had produced some of the clearest pictures yet of the B form ofDNA, pictures of such clarity that they did come astonishingly close to the truth of itsmolecularstructure.Butthen,confusedforayearbythetwoseeminglydifferentpatternsof theAandBforms,Franklinveeredawayfromherownearlierconclusionsandforayear she took the view thatDNAwasn’t helical at all. Sayre appears to refute this, butGoslingwould subsequently confirmWilkins’ account of how,onFriday18 July1952,FranklingoadedWilkinswithaninvitationtoawake.Theinvitationcardannounced,withregret, the death of the DNA helix (crystalline) following a protracted illness. ‘It washoped thatDrM.H. F.Wilkinswould speak inmemory of the deceased.’At the timeWilkins assumed itwas typical ofGosling’s sense of humour.Butmany years later hewould discover that it was Franklin who had written the card, and it confirmed herrefutationofanyhelicalstructureofDNAinthatconfusedyear.

*

Inthemiddleofthesameyear,1952,CrickstruckupaconversationwithalocalyoungWelshmathematiciancalled JohnGriffith,whomhemetoneeveningafter a talk at theCavendish by theoretical astronomer Thomas Gold. Gold had captured Crick’simaginationwith the notion of ‘the perfect cosmological principle’.Wondering if theremightbesomeequivalent‘perfectbiologicalprinciple’,CrickpressedGriffith,whowasinterested in how genes replicated, about the work of an American chemist, ErwinChargaff,whohaddiscoveredthatthenucleotidesinDNAformedflatlinkageswithoneanother.ItwascuriouslyreminiscentofPauling’sdiscoveryofhowtheaminoacidsthatmadeuptheprimarychainsofproteins,knownas‘peptidebonds’,alsojoineduptoformflattwo-dimensionalplanes.InCrick’sminditinvokedavaguenotionthatthismightbesomethingtodowithDNAreplicatingitself.HeaskedGriffithifhecouldworkoutwhichof the four nucleotides would pair off with which. Griffith confirmed that the likelypairingwasCwithGandAwithT…

Buteventhenthepennydidnotdrop.

ErwinChargaffwasyetanotherAustrianscientistwhofledEuropeintheyearsleadinguptoWorldWarTwoandheadedtotheUS,wherehebecameProfessorofBiochemistryatColumbiaUniversity.Hisinterestwasthestudyofnucleicacids.Wemightrecallthatmuch of the disbelief aroundAvery’s discovery centred on the fact that geneticists hadbeen misled by the erroneous notion of Levene’s ‘tetranucleotide hypothesis’, whichproposed that DNA comprised repeats of the same cluster of four nucleotides. Such asimpleformulawouldbeincapableofstoringthevastmemorydemandedforthemoleculeofheredity,soitwaswronglyassumedthatDNAcouldnotbetheanswertothegene.

Chargaff didn’t give a damn what the geneticists thought of Avery: he was deeplyimpressedbyhisfindings.AndifAverywasright,andDNAwasthemoleculeofheredity,theDNAsequencesofahorse,say,wouldbedifferentfromthatofacat,oramouse,orahuman being. In Chargaff’s words, ‘There should exist chemically demonstrabledifferences between [their] deoxyribonucleic acids.’ These differences should be

demonstrable in theproportionsof the fourdifferent nucleotides. Itmight appear that afour-lettercodewouldbelimitedindelimitingthewidevarietyofgenesthatoccurredinnature,butifweweretoregardthenucleotidesaslettersinafour-letteralphabet,andthegenes aswords, thepotential fordifferent arrangementsof just four ‘letters’ inwords athousandormoreletterslongcouldeasilyexplainthecomplexityneededforthemake-upofgenes.

Technology was limited in the late 1940s and early 50s, but Chargaff modified atechnique, known as paper chromatography, to read off the different proportions of thefournucleotidesinanygivensampleofDNA.

After fouryearsof laboriousexperiment,analysing theDNAofyeast,bacteria,oxen,sheep,pigsandhumans,Chargaffhadhisanswer: thefournucleotidesthatmadeuptheword of the gene were not present in the equal proportions one might expect fromLevene’shypothesis.Forexample,humanDNA,extractedfromaglandinthechestcalledthethymus,yielded28percentadenine,19percentguanine,28percentthymineand16percentcytosine.Thetetranucleotidehypothesiscouldnowbeditched.ButChargafftookitfurther.Hedemonstratedthattheproportionsofthenucleotidesvariedbetweenspecies–meanwhile,theproportionofnucleotideswasalwaysthesameformembersofthesamespecies,andindeedwasthesamefromorgantoorganandtissuetotissuewithinthesamespecies.Healsonoticedsomethingelse:byinference,thesumofthemoleculesofadenineandthyminewasverysimilartothesumofthemoleculesofcytosineandguanine.

Thiswasagroundbreakingdiscovery.

InMay 1952, by remarkable happenstance, Chargaff arrived in person at CambridgeUniversity where Kendrew introduced him, over lunch, toWatson and Crick. Chargaffwas offended by how little they knew about hiswork. In his opinion they appeared toknownexttonothingabouttheactualchemistryofnucleotides.InChargaff’ssubsequentdescriptiontoJudson:‘Iexplainedourobservations…[that]adenineiscomplementarytothymine, guanine to cytosine.’ But as far as Chargaff could see, all that preoccupiedWatsonandCrickwastheracetoconstructaDNAhelixtorivalPauling’salphahelixforproteins.WatsonwouldsubsequentlyrecallChargaff’sopenscornforthe‘twomenwhoknewsolittle–andaspiredtosomuch’.

Chargaffwas largely right in his assessment ofWatson andCrick’s ignorance of thebiochemistryatthetime.CrickknewnothingaboutChargaff.Nomoredidheunderstandthat thepairingof thenucleotides involvednot the covalent chemicalbonding found instablemoleculesbuttheweakerhydrogenbonding.WhatthenwashetomakeofChargaffexplaining the importanceof theone-to-oneratiosofcytosineandguanine,andadenineandthymine,inthemoleculeofDNA?

Crickthenhadabrainwave:whatifthissignifiedanaturalchemicalattractionbetweenthesenucleotides?

Might this not play a vital role when an existing strand of DNA copied itself to adaughterstrand?EveryCwouldattractaG,andeveryAwouldattractaTinthedaughtersequence–andthewholethingwouldreverttothematernalsequencewhenthedaughter

strand replicated in its turn. He took it a stage further. What if DNA comprised twothreads, complementing one another in exactly this way? If and when the two threadsbrokeapartandcopiedthemselves,theywouldcreateanidenticalpairofthreads,anewidenticalchain.

It seemed too incredible to be true that the great and profound mystery of hereditymight be explained on the basis of these simple chemical couplets, with their specificattractionstooneanother.

ThenCrick andWatsonmade amistake, not in scientific terms, but in human terms.Theysatbackandthoughtaboutallthattheyknewbutdidnothingaboutconstructinganew model. It almost cost them everything. In December 1952, Peter Pauling, son ofLinus,thenworkingasagraduatestudentattheCavendish,informedWatsonthathehadjustreceivedaletterfromhisfathertosaythathehadworkedoutthestructureofDNA.Thefollowingmonth,Petershowedeverybodyanadvancecopyofthepaper,whichwasscheduledtobepublishedinFebruary1953intheProceedingsoftheNationalAcademyofSciences.WatsonandCrickwouldlaterconfesswithwhatsinkingheartstheyreadthepaper,whichproposeda triplehelixwith thephosphate-sugar spineat thecentre.Forabrief interval they were dumbfounded, wondering if their own model, dismissed byWilkinsandFranklin,mighthavebeencorrectafterall.ButthentheyrealisedthatallofthescornheapedonthembytheX-raycrystallographersalsoappliedtoPauling’smodel.Thistimeitwasthegreatchemist’sturntoblunder.

Theracewasnowonagaintogetitright.WheretheCambridgeduohadagreedtostayawayfromDNA,Watsonwasnowconvincedthatiftheydidso,Paulingwouldbeatthemalltotheprize.

A few days after reading Pauling’s paper, Watson took it down to King’s CollegeLondon,where,accordingtohisbiography,hetalkedaboutitfirstwithFranklin–who,accordingtoWatson,flewintoarage.InWatson’sopinionherragewasprovokedbyhiscriticism of her rejection of helical structures. But it would also appear that Watsondeliberately provoked Franklin into that response. Discovering that … ‘Rosy was notabouttoplaygameswithme,Idecidedtoriskafullexplosion.WithoutfurtherhesitationIimpliedthatshewasincompetentininterpretingX-raypictures.’

ItwashardlysurprisingthatFranklinflewintoarage.

Muchhasalsobeenmadeofthefactthat,withoutconsultingFranklin,WilkinsshowedWatson thephotographiccopyof theparticularlyclearX-raypictureof thewet formofDNAobtainedthepreviousMay–apicturethatconfirmedwithoutashadowofadoubtthatthemoleculeofDNAwasahelicalstructure.Infact,Watson,CrickandWilkinswerealreadylongconvincedofthehelicalstructureofDNA.Wilkinsmakesclearinhisbelatedbiography, published in 2003, just a year before his death, the X-ray photograph thatWatson crowed aboutwas not stolen fromFranklin butwas passed to him byGosling,whohadtakenthephotographinthefirstplace,andwhowouldhaveassumed,nowthatFranklin was leaving, that she could have no objection to his doing so. Gosling stillneeded to complete hisPhD thesiswithout the departingFranklin’s supervision, and sohadeveryreasontoshowhisownworktothedeputydirectoroftheunitwhowouldnow

betutoringhim.Goslinghimselfwouldconfirmthat,‘Mauricehadaperfectrighttothatinformation.’GoslingwasclearlyfedupwiththerancourprovokedbyFranklin’srefusalto collaboratewithWilkins, bemoaning a timewhen, ‘Therewas somuch going on atKing’sbeforeRosalindcame.’

Atthetime,FranklinwaspreparingtoleaveKing’stojointhestaffattheBiomolecularResearch Laboratory at Birkbeck College, London, where she would work under thedirectorshipofJ.D.Bernal.Tohercredit,inhertwoyearsatKing’sFranklinhadmadeaseries of original discoveries about DNA. Her research included revealing that DNAexisted in two different forms, which she had labelled A and B; that one form couldreadilyturnintotheother;andthatshehadhardproofthatthephosphatespinewasontheoutside. This latter revelation established that it was readily exposed to take up watermolecules which wrapped the molecule in a protective sheath within the nuclearenvironment, keeping it relatively free from the interaction of neighbouring moleculeswhilemakingstretchingofthemoleculeeasier.

Once ensconced at Birkbeck, Franklin appears to have settled into a fruitful andamicableworkingroutinewithherboss,Bernal,andgraduatestudentAaronKlug.Hereshe ceased to work on DNA fibres and instead focused on the molecular probing ofviruses,producingsomeofherfinestwork.Onherlatertragicanduntimelydeath,whenshe left her worldly possessions to Klug and his family, her scientific obituary wasadmiringly and respectfully written by Bernal in The Times and the scientific journal,Nature:

Her life is an example of single-minded devotion to scientific research…As a scientistMiss Franklinwasdistinguishedbyextremeclarityandperfection ineverythingsheundertook.Herphotographsareamong themostbeautifulX-rayphotographsofanysubstanceevertaken.

NeitherFranklinnorWilkinswasawareat thetimewhenWatsonstormedinwiththePauling paper, that he andCrickwere nowdetermined to construct a newmodel of itsthree-dimensionalstructure.Followingthetriplehelixdebacle,BragghadforbiddenthemdoinganymoreworkonDNA.Throughall theemotionalballet, recalled sovividlybyWatson,weshouldacknowledge thatWatsondidkeep theKing’sgroup informedabouthis and Crick’s thinking, and he had attempted to acquaint Franklin with a relevantpublication,comingfromamajorpotentialrival.Wemightalsonotethatuptothefinaldecipherment exercise,Watson, Crick andWilkins had communicated openly with oneanother.IfFranklinwasnotprivytothesediscussions,itwasatherchoosing.InneitherofthebiographiesofFranklinistherementionofherbeinginspiredbySchrödinger’sbookorhis theoryofanaperiodiccrystal.ShehadnotchosenDNAasherresearch theme, ithad been suggested to her by Randall – though she evidently saw it as a challengebefittinghergrowingexpertiseandfascinationwithX-raycrystallography.

Intheirenthusiasmfor themodel-buildingapproach,WatsonandCrickhadexplainedallabout it toWilkins. Inpassingover thereinsof theDNAresearch tohim, theyevenlenthimtheirjigsformakingthenecessarypartsofthemodel.ButnotonlyhadFranklinrebuffed cooperation withWilkins, the King’s group had eschewed the opportunity oftakingupthePauling-inspiredmodellingapproach.Andnow,atwhatmusthaveappearedacriticalmomentintime,whatwereCrickandWatsontomakeofthefactthatFranklin

wasleavingKing’s,abandoningherworkontheDNAfibre,andatthesametimeWilkinshad also stoppedworkingonDNA,waiting, as he confessed, for thedust ofFranklin’sdeparturetosettlebeforevaguelystartinganew.

WatsonhadeveryreasontoassumethatPauling,bruisedbyhisownpublishederrorofatriplehelixstructureforDNA,mustnowbemoreintensivelyengagedthaneverwiththeproblem – he must surely be formulating a newmolecular approach. After the heatedencounteratKing’s,WatsonandWilkinssharedamealandabottleofChablis.Buttheirconversation over dinner produced no new inspiration. ForWatson the key theoreticaldifficultywasnotwhether theDNAmoleculewas a helix, butwhether thehelixwas atriple or a double chain.Wilkins still favoured three chains over two, but in so far asWatsoncouldtell,Wilkins’reasoningwasnotfoolproof.Bythetimehehadcycledbackfrom the station inCambridge and climbedover the backgate tomake a late return tocollege,‘Ihaddecidedtobuildtwo-chainmodels.’HemusthavechuckledatahumorousinspirationhewouldsubsequentlypassontoCrickthenextmorning.Franciswouldjusthavetoagree.‘Heknewthatimportantbiologicalobjectscomeinpairs.’

This inspiration – and there appears to be no otherword forwhyWatson decided tofocusonadoublehelix–wouldprovetobeexactlywhatwasrequiredtofittheChargaffdataandCrick’sideasonhowDNAmightreplicateitself.GiventhenewstateofaffairsatKing’s,evenBraggsawthesenseofallowinghisunrulyyoungscientiststoreturntothemysteryofthegene,mostparticularlysosinceitmightgivehisgrouptheadvantageofatriumphoverhisacademicrival,Pauling.

Themodellingwent intooverdrive,withWatsonputting together scalemodelsof thedifferentchemicalsinvolvedinthestructureofDNA,thefournucleotides–G,A,CandT– the phosphatemolecule and the sugarmolecule, deoxyribose.Obstinate in his notionthat the spine, which probably involved the phosphate and sugar, had to be internal,Watson attempted to construct a newmodelwith the phosphate-sugar spine still on theinside.ButCrick,playingsymbioticdevil’sadvocate, insistedthisjustdidnotfit theX-raydata.Indeed,FranklinandGoslinghadbothinsistedthatthephosphatespinemustbeontheoutside.Watsonnowconfessedthathehadsimplyrefusedtotakethisintoaccountbecause it made the modelling too easy and introduced an enormous variety ofpossibilities. But now, persuaded by Crick, he switched to putting the phosphate-sugarspine on the outside – as an exoskeleton, like one sees in the insectworld – and thenattachingthenucleotidessotheyprojectedintothemiddleofthedoublehelixmadebythespirallingphosphate-sugar spines. In spite ofChargaff’swork, and in spite ofGriffith’sadvicetoCrick,Watsonpersistedinattemptingtoattachlikewithlike,forexampleAtoA,GtoG.Itjustdidn’twork.

In the middle of all this, happenstance again contributed to the story. An Americanscientist,JerryDonohue,aformerprotégéofPauling’s,paidavisittotheCambridgelab.Anexpertonhydrogenbonding,Donohuenowcorrectedtheirmodelstosuitthequantumimplications.

Watson andCrick now feltmore confident that it had to be a double-stranded helix,withthetwostrandsreadingincounterdirections–thesenseandanti-sensewetakefor

grantedtoday.Thetwostrandshadtolineup,withthecomplementarynucleotideslinkingtooneanotherthroughhydrogenbonding.Watsonsatdownathisdeskandcutoutpiecesof stiff cardboard in the shape of the nucleotide molecules, looking at how the actualshapesfittedwithoneanother,hopingtoseesomepairingpossibilities.

SuddenlyIbecameawarethatanadenine-thyminepairheldtogetherbytwohydrogenbondswasidenticalinshapetoaguanine-cytosinepairheldtogetherbyatleasttwohydrogenbonds.

Todaywerecognisethatthelatterisheldtogetherbythreehydrogenbonds.IfwepeeratthebasicshapesinthefigurebelowwecanseewhatbecameobvioustoWatson.

The sceptical Crick, on arrival at the lab to examine Watson’s matching shapes ofcardboard, almost immediately agreed with Watson’s brainwave. The complete modelcouldnowbeassembledinthree-dimensionalspace–aconflagrationofbitsofwire,cuttotherightlengthstorepresentcovalentandhydrogenbonds,themoleculesconstructedofthecompositeatoms,andthewholeassemblagesuspendedbyclampsfromtallverticalsteelrods.Theresultingdoublehelixcoiledaroundthecentralrods,risinginaspectacularconflationofwiresandhand-cut,molecular-shapedplatesfromthelabbenchupwardstotheceiling.

Everybodywhosawthesubsequentmodelreactedwithawe.Itwasasifinthebriefestlook at it they saw immediately that it had to be right. Itwasn’tmerely right: itwas aspectacularly gorgeous creation – a beauty to behold. All the more so since it wasimmediatelyobvioustoeverybodywhosawitthatitexplainedallthatwasdemandedofthemysteryofthegene,intermsofchemicalmemoryandthecopyingnecessaryforthegene to reproduce itself, from cell to cell, from parent to offspring. It was capable ofprovidingthecodingthatwasnecessarytopassthesecreton,generationaftergeneration,for the immense complexity of biodiversity, and for the complex evolving lineages ofevolution.Ittrulywasthesecretoflife.

*

Watson and Crick’s first paper on the structure and function of DNA appeared in thejournalNatureon25April1953,andwasaccompaniedbytwopapersinthesameissuefrom the crystallographers atKing’sCollegeLondon– the first byWilkins,Stokes andWilson; the secondbyFranklinandGosling.Nobody’scontributionwasexcluded.Fiveweeks laterWatsonandCrickpublishedasecondpaper,again inNature,on thegeneticimplications of the structure of DNA. A short sentence in the 25 April edition wouldcapturetheattentionofscientiststhroughouttheworld:‘Ithasnotescapedournoticethatthe specific pairing we have postulated immediately suggests a possible copyingmechanismforthegeneticmaterial.’

Thosepaperswouldinevitablychangetheworldofbiology,evolutionarybiologyandmedicine. Indeed, the ramifications are still echoing throughourworld, andpenetratingmuchwideranddeeperintosocietythanWatsonandCrickcouldhavepossiblyimagined.

It is extraordinary to realise that, less than two years after the start of their ad hocpartnership,Watson andCrickhad correctly figuredout the three-dimensional chemicalstructureofDNA.Crickwas37andstillhadnotcompletedhisPhD,andWatsonwasstillapostgraduatestudent,agedjust25.Atasuperficialglance,therewouldappeartobenological reason why these two seeming misfits should be the discoverers of the three-dimensional chemical structure of DNA. They had done little or no lab work in theprelude to the discovery. They were lowly in their positions within the lab – Crick aresearch assistant and Watson a graduate student. They were impecunious, living inimpoverishedsurroundings,yetuncaringaboutall that.Theyhadonlybelatedlyrealisedtherelevanceofdiscoveriesmadebyotherscientificcontributors.TheirofficialdutieshadnothingtodowithDNA.Crickwasstill tryingtocompletehisPhDthesisontheX-raydiffraction of polypeptides and proteins while Watson was supposed to be helpingKendrew to crystallise the molecule of myoglobin. The head of their department, SirLawrenceBragg,was,throughmostoftheirefforts,opposedtotheirworkonDNA.Inthemannerinwhichsciencenormallyworks,thepairshouldneverhavemadetheirdiscovery.Therewere colleagues, likeWilly Seedswho insultedWatson at the foot of the Swissglacier,whothoughtthatWatsoninparticulardidn’tdeservetheacclaim.Butthefactis,theybothdid.

Thedetractors aremissing the point:whatWatson andCrick achievedwas an act ofsublimecreativity, like theplaysofShakespeare,DaVinci’sMonaLisa,orBeethoven’sNinth Symphony. Admittedly this was not artistic creativity. Rather, like Newton’sdiscovery of gravity, Darwin’s discovery of natural selection and Einstein’s Theory ofRelativity, this was an act of scientific creativity that opened a new window ofunderstandingontoourunderstandingofLifeitself–and,atthemostprofoundoflevels,whatitistobehuman.

In1962,Crick,WatsonandWilkinssharedtheNobelPrizeinPhysiologyorMedicineforthediscoveryofthestructureofDNA.TheonlyoneofthethreetomentionRosalindFranklinwasWilkins,whoalsoacknowledgedAlexanderStokes’1/5000thcontribution.Tragically,Franklinhaddiedofovariancancersomefouryearsearlier,atatimewhenher

workonviruseswasbecominggloballyrecognisedasamongthefinestachievements inX-raycrystallography.Some,includingSayle,havequeriedifFranklinmighthavetakenWilkins’placeontherostrumhadshelived.It’samootquestion,butIpersonallythinkitunlikely.WilkinsinitiatedtheDNAresearchatKing’s,inspired,likeWatsonandCrick,bySchrödinger’sbook.HisX-raydiffractionpicture–actually takenbyGosling–inspiredWatson’sarrival intotheCambridgelab.HiscooperationwithWatsonandCrickwassoclose and formative in the discovery that Watson wanted to include his name in thefamous first paper. ItwasWilkins’modesty and integrity that caused him to refuse thehonour.ThisiswhyIdoubtthatFranklinwouldhavereplacedWilkinsontherostrumin1962.ButIdobelievethattheremighthavebeenasecond,rathermorelikely,opportunityforrecognitionofthecontributionofRosalindFranklintoX-raycrystallography,onethatissuggestedinthegreatadmirationfeltforherworkbysuchaneminentfigureasBernal.

When she moved to Birkbeck College Franklin found a happy working relationshipwith a Lithuanian Jewish chemist and biophysicist, Aaron Klug, who, followinggraduationinSouthAfrica,hadarrivedintheUKonaresearchfellowshiptocompletehisdoctorate inX-raycrystallographyatTrinityCollege,Cambridge, in1953.Thiswas,ofcourse, the year of publication of theDNA discovery.AtBirkbeck Franklin tookKlugunderherwing,formingacloseworkingrelationshipandfriendshipthatwouldcontinuefor the rest of her life. We know that after Franklin’s untimely death Klug took hertechniquesfurthertoberewardedwiththeNobelPrizeinChemistryin1982.TheofficialdeclarationofhismeritingthePrizewas:‘forhisdevelopmentofcrystallographicelectronmicroscopy and his structural elucidation of biologically important acid-proteincomplexes’.Howlikelyisitthat,hadRosalindFranklinlived,shewouldhavesharedthepodiumwithAaronKlugfortheircooperativeeffort?

*

Somenineyearsearlier,on12August1953,fivemonthsafterCrickandWatsonhadfirstmodelledthedoublehelix,FrancisCrickwrotealettertoErwinSchrödingerinwhichhethankedhimfortheinspirationofhisbook.Intheletterhedescribedhow,inthestructureofDNA,theyhadindeeddiscoveredthe‘aperiodiccrystal’thathehadpredictedwouldbethemolecularcodeforlife.

six

TheSisterMolecule

Ihavethefeelingthatifyourstructureistrue,andifitssuggestionsconcerningthenatureofreplicationhaveanyvalidityatall,thenallhellwillbreakloose,andtheoreticalbiologywillenterintoamosttumultuousphase.

MAXDELBRÜCK,WRITINGTOWATSON

Judson,widelyrecognisedasthehistorianoftheDNAstory,describedtheelucidationofits structure as ‘a siege, a conquest’. Given the discovery of the three-dimensionalstructure of DNA, with its four-letter code for the storing of heredity, onemight haveanticipated enlightenment, but instead the prevailing atmospherewas one of confusion.WatsonandCrick’sdiscoveryhadprovokedastormofnewquestions.Tobeginwith,wasDNAtheanswertothecodingofhereditytoalloflife?Atleastthisquestionwasalreadyanswered;Averyhadset theball rollingbydiscovering it inbacteria.Thephageschoolworked on it in viruses. Chargaff had confirmed the same in a range of different lifeforms. DNA was universal. The next major question was this: how did its incrediblysimplefour-lettercode–G,A,CandT– translate into thecomplexityof theestimated80–100,000 proteins thatwere essential for the structure and functioning of our humanbodies,andthebodiesofeveryotherlivingcreature?

Crickwould later recall that theyalreadyhadanoutlineof the answer to theproteinenigma.Sincethespineofthehelixwasmadeupofsugarandphosphaterepeats,theonlychemicals capable of coding for heredity, and translating to proteins, were the fournucleotides–otherwiseknownasbases,orbasesequences–GACT.Someadvanceshadalready been made into this mystery. Thanks to the pioneering evolutionary biologistThomas Hunt Morgan, working in his fruit fly laboratory at Columbia University, weknew that the genome was divided into chromosomes. Thanks toMorgan,Muller andothers,weknewthat thechromosomeswere themselvesparcelled intodiscretesections,calledgenes.Afurtherstep,thatagenecodedforaspecificprotein,wasfirstpostulatedbyaBritishdoctor,ArchibaldE.Garrod,asearlyas1908,whenhefiguredout that theinheritedillnessknownasalkaptonuriawasprobablytheresultofadefectiveenzyme.Anenzymeisaproteinthatspeedsuptherateofachemicalreactioninlivingsystems.ButGarrodcouldn’ttakeitthatvitalstepfurtherandprovethatthedefectiveenzymewastheexpression of a defective gene. The essential link between genes and proteins wasconfirmedbytwoAmericans–ageneticist,GeorgeW.Beadle,andabiochemist,EdwardL. Tatum – who were working on the heredity of eye colour in fruit flies. By 1941,shiftingtheirfocustoafungusthatinfectedmouldybread,theyhadshowedthatasinglegene coded for a specific enzyme involved in themould’s living chemistry. Itwas this

discoverythatresultedinthemaxim:‘onegeneoneprotein’.Buthowdidthefour-letterDNAcodeofthegenetranslatetothe20-letteraminoacidcodeoftheprotein?

ForFrancisCrickthiswastheveryenigmathathadinspiredhisown‘madpursuit’afterreadingSchrödinger’sbook.Followingthediscoveryof thedoublehelix,WatsonwouldsoonbeforcedtoreturntotheUnitedStates,havingrunoutoffunding.ButCrickwouldcontinuetoinvestigatetheextrapolationtoproteins.

SincetheDNAwascontainedinthenucleusofthecellandproteinmanufacturealwaystookplaceintheregionoutsidethenucleus,knownasthecytoplasm,thecodeofthegenehad tobecopied in someway thatallowed it tobesentoutof thenucleusand into thecytoplasm.ThismadeCrickconsiderasistermoleculeofDNAknownasribonucleicacid,orRNA.

Thereareobvioussimilaritiesbetweenthetwomolecules.Botharenucleicacids,madeupofvaryingsequencesoffournucleotides.WhereDNAismadeupofguanine,adenine,cytosineandthymine,orGACT,RNAismadeupofguanine,adenine,cytosineanduracil–GACU.RNAalsodiffersfromDNAinthefactthatitisnotadouble-strandedhelixbut,atleastinmostofitsroles,issingle-stranded.ItalsodiffersfromDNAinitssugar,whichisribosewhereDNA’ssugarisdeoxyribose.AtthetimeofWatsonandCrick’sdiscoveryof the 3-D structure of DNA,molecular biologists and geneticists were also becomingincreasingly interested in this sister molecule, RNA. Immediately prior toWatson andCrick’smonumentaldiscovery,manyscientistswerebeginningtothinkthatRNAmustbeplayinganimportantroleinthewaylivingcellsworked.

AtthesametimetherewassomethingelusiveaboutRNA.WheretheamountofDNAin different cells of the body, say a brain cell or a liver cell,was always the same, theamount ofRNA seemed to vary. To add to the confusion,DNAwas only found in thenucleus, meanwhile RNAwas found in both the nucleus and the non-nuclear territory,known as the cytoplasm – the part of the cell where most of the active biologicalchemistry takesplace.Toconfuse thingsevenmore, theamountofRNAinagivencellalso varied, depending on how active the cell was. A growing cell, or a cell that wasproducinglotsofnewprotein,hadmoreRNAthanacellthatwasmatureandchemicallyquiet.Livercells,forexample,whichwerethoughtofasthefactoryformakingproteins,were packed with RNA. Moreover, RNA was also found in the same parts of thecytoplasm – the small round bodies known as ribosomes – where protein wasmanufactured.

ItwasnowbecomingincreasinglylikelythatifDNAwasthestoredcodeforheredity,andsomehowwastranslatedintotheaminoacidsequencesthatmadeupproteins,RNAhadsomethingtodowiththeactualmanufactureofthosetranslatedproteins.Itwaseasyto see how a stretch of DNA couldmake an RNA copy – all it would take is for thethymine, orT, ofDNA to be replaced by uracil, orU, during the copying.As early as1947, two Strasbourg-based scientists, André Boivin and Roger Vendrely, had alreadyproposedthattheGACT-basedsequencesofaDNAgenewouldbecopiedinthiswaytotheGACU-basedRNAmessenger,whichwouldferry thecodingout into thecytoplasmwhere thecorrespondingproteinswouldbemanufacturedat theribosomes.All thatwas

leftwastofigureouthowthefourlettersofGACUcouldtranslatetothe20-letteraminoacidcodeoftheproteins.

Crickwas intrigued by a letter from aRussian theoretical physicist,GeorgeGamow,whicharrivedoutof theblue in the summerof1953, soonafterCrickandWatsonhadpublishedtheirfirsticonoclasticpaperonthestructureofDNA.Gamow,whowaspartofthegroupwhohadcomeupwiththe‘BigBang’theoryfortheoriginoftheUniverse,hadbeenintriguedbythedoublehelix.Inhisletterheproposedawayinwhichthefour-lettercodeofDNAmighttranslatetotheaminoacidprimarysequencesofproteins:tripletsofthefournucleotides–G,A,CandT–mightcodeforsingleaminoacids.ButtoCrickthisdidn’t add up. Sixty-four different triplet assortments would result from the randommixingderivedfromfourcodingletters,whereastherewereonly20aminoacidsfoundinnaturalproteins.Gamowhadthoughtthisthrough,proposinganingeniousoverlapofthetriplets,sothatwhatcodedforoneaminoacidmightinpartcodeforanother.Crickdidn’tbuyit,butheandWatsontookGamow’sletterwiththemwhentheyretired,asusual,totheEagle for lunch. If nothing else,Gamow’s interventionprovoked theDNApioneersintoareneweddebateonhowtheDNAtoproteinmysterymightbecracked.

TherewouldbelittlefurtheropportunityforthemtoswapideasthatyearonceWatsonleftCambridge forAmerica. In fact, therewould be little progress in themystery for anumberofyears.

In thesummerof1954,CrickandWatsonteamedupagainfor threeweeksatWoodsHole,Massachusetts.Gamowandhiswifewerethere.MostafternoonsCrickandWatsonwouldjointheGamowsdownbythewater’sedge,watchingtheRussianphysicistdocardtricksandchattingaboutthesamemystery.Intheinterim,sincewritingtheletter,Gamowhadcollectedtogetherthenamesofanumberofpeopleinterestedinsolvingtheproblem.Somehoworother,anditwaslikelythatbothWatsonandDelbrückwereattheheartofthe joke, a ‘whisky, twisty RNA party’ was called for, with invitations presumablyaddressedtopartiesinterestedintheenigma.ThisbecametheinspirationforwhatcametobecalledtheRNATieClub,limitedto20members,toparallelthenumberofaminoacids.In addition to Crick,Watson and Gamow, the club members includedMartynas Ycas,AlexRichandanOxford-educatedSouthAfrican,SydneyBrenner.Inthespringof1953,BrennerhadbeenoneofapartyofyoungscientistswhohaddrivenoverfromOxfordtoCambridge to see Watson and Crick’s 3-D model of DNA. At the time, Brenner wasconducting a bacteriophageproject for hisPhD inmolecular biology. In a garden strollwithWatson,BrennerhadlearntabouttheHershey–Chaseexperiment.Thesedayshewasworkingasapost-docattheMedicalResearchCouncil’sMolecularBiologyLaboratoryinCambridge,buthehadmaintainedthatearlyinterestinDNAandgenetics.Eachmemberoftheclubreceivedatie,madetoGamow’sdesignbyahaberdasherinLosAngeles.Thetiesinturnwerepinnedbyindividuallydesignedshortformsofeachspecificaminoacid–Crick’spin read ‘tyr’ for tyrosine. Itwasall a fantasy; theclubnevermet,but, like thephagegroup,itactedasarallyingfocusforthegroup,whowouldcirculateanypapersornewsofcommoninteresttomembers.InthewordsofBritishjournalistandauthor,MattRidley,whowroteabiographyofFrancisCrick,theEnglishscientistwas‘thedominanttheoreticalthinker…theconductorofthescientificorchestra’.

Brenner showed, mathematically, that the overlapping triplet idea was a non-starter.CrickandLeslieOrgelwere joinedbyCrick’s friendandcollaborator, theyoungWelshmathematician,JohnGriffith,whotriedhishandatrulingoutspecifictripletsofthefourletters that simplywouldnotwork.For example, they ruledoutAAAbecause itwouldcauseconfusionifpositionednexttoanotheridenticalletter,A.Byexcludingtripletsthatwouldcauseconfusion,theycalculatedthatitonlyleft20‘sense’permutations.Thiswasduly published as a paper in theProceedings of the National Academy of Sciences, in1957.Unfortunately,itwasutterlywrong.

Nevertheless,bynowsomeusefulideaswerebeginningtoemerge.

Agene,withitslongthread-likemolecule,madeupofaspecificsequenceofG,A,CandT,oftenathousandormoreletterslong,codedforaspecificproteinwhoseprimarystructure was again a long thread, made up of the 20 amino acids in a very specificsequence. They even knew by now that sickle-cell disease, in which there was anabnormaloxygen-carryinghaemoglobinintheredcells,wascausedbyamutationinthegenethatcodedforthebetaglobin.Themutationinthegenehadtranslatedintoafaultycoding for the haemoglobin protein. Crick now focused on ideas that were coalescingfromseveraldifferentquarters,whichboileddowntothefactthattherewerelikelytobetwo quite different forms of RNA involved in the translation from nuclear-basedDNAgenes to the coding for protein assembly in the ribosomes. One form – now calledmessengerRNA,ormRNA–copied thecode fromtheentiregene in thechromosomeswithinthenucleusandcarriedthecodeoutofthenucleustotheribosomes.Interestingly,messengerRNAwas found by a group of researchers atHarvard,working inWatson’snewlaboratorythere.Meanwhile,asecondformofRNA,calledtransportRNA,ortRNA,pickedupsingleaminoacidsand,guidedbythecodingcarriedonthemessengerRNA,addedtherightaminoacids,onebyone,totheassemblingproteinchain.Inthiswaythenucleic acid coding of the gene translated to, and was ferried into, the ribosomes toconstructthecorrespondingproteinchain.

The coding triplets were eventually discovered through trial and error by scientistsincludingMarshallNirenberg,GobindKhoranaandSeveroOchoa.Today,weknowthattripletsofDNA,nowcalled‘codons’,codeforspecificaminoacids,butasingleaminoacidcanhavemorethanonecorrespondingcodon.Forexample, theaminoacidleucinehassixdifferenttripletcodons:CTT,CTC,CTA,CTG,TTAandTTG;phenylalaninehastwo, TTT and TTC;whilemethionine has just one, ATG.Moreover, there are specifictriplepermutations–TAA,TAGandTGA–thatcodenotforaminoacidsatallbutforthegeneticequivalentofafullstop.Thesehalttheproductionofaproteinatthefullstop,andsoareknownas‘stopcodons’.

Thiswasanothermajorstepinunderstanding,but,onceagain,itbeggednewquestions.Thefactory-likemechanismsofproteinproductionhad tobecontrolled.Howdidacelldecidewhichproteintomake?Howdiditdecidewhentomakethespecificproteininthelifeofthecell?Howdiditturnproteinproductiononandoff?

*

WemightrecallthegroupwhohadearliercontributedtotheWatson–Crickdiscovery(aworld-based cooperative of scientistsworkingwith the viruses that infect bacteria), thephagegroup.A trioofParis-based scientists,AndréMichelLwoff, JacquesMonodandFrançoisJacob,wereconductingresearchonphagesandtheirhostbacteriaatthePasteurInstitute.Theyfocusedonthebacteriumthatwasthehostforallofthephageexperiments,abugcalledEschurichiacoli–E.coliforshort–whichisthebacteriummostcommonlyfoundinthehumanintestine.Whatinterestedthem,tobeginwith,wasadiscoverymadebytheirAmericancolleaguesJoshuaLederbergandEdwardTatum,whichsuggestedthat,contrary to the prevailing ideas, bacteria had a kind of sex life. Normally bacteriareproduceasexuallythroughadaughterbacteriumsimplybuddingoffthematernalstrain–ratherlikeonesausagebeingsqueezedoffinthemiddletoformtwo–butnowandthenabacteriumwouldfashionapenis-likeextrusionthroughwhichitwouldinjectitsgeneticmaterialintoanotherbacterium.Thescientistsjokilyreferredtoitas‘coitus’.

In 1955, Jacob, workingwith a colleague called ÉlieWollman, explored theway inwhichgenetic informationwaspassedon fromonebacterium to another.Realising thatbacteriahadgenesmadeupofDNA,justlikeallotherlifeforms,theyalsoknewthatthebacterialgeneswerethreadedalongasinglelengthychromosomethattooktheformofaring, which had a point of attachment to the inside of the bacterial wall. Jacob andWollman nowdiscovered that during coitus the chromosomewas very slowly extrudedfromthe‘male’andthroughthecellwallintothebodyofthe‘female’bacterium.Whilebacterial reproduction by budding took only twenty minutes, bacterial sex lasted forroughly two hours. This allowed Jacob and Wollman to conduct a series of ‘coitusinterruptus’experimentsinwhichtheyhaltedtheprocessattimedintervalsalongthetwo-hourprocess.Sincethebacterialchromosomealwayscamethroughinthesameorderofgenes, theycould, through looking for theeffectsof specificmutatedgenes,plotwherealong the course of the bacterial chromosome the genes for various different propertieswerelocated.

But now the French scientists took the experiment a step further; they set out todiscoverhowthosegeneswerecontrolledwithinthebacterium.

Theyfocusedonthreegenesthatallowedthebacteriumtotransportthesugar,lactose,intothebodyofthebacteriumandtheredigestitintoitstwosmallercomponentsugars,glucoseandgalactose.Itwouldbewastefulfor thebacteriumtoactivate thesegenesallthe time,evenwhen therewasno lactose in itsenvironment.What theydiscoveredwasthatthegeneticchemistryoperatedasystemofcontrol.Whenthereisnolactosearound,this triggered the activation of a ‘repressor’, which halted the production of the threerelevantgenes.Whenlactosewaspresent, therepressorwasremovedandageneticareaalongsidethegenes,knownasthe‘promoter’,activatedtheexpressionofthegenes.

Wedon’tneedtoworryabouttheprecisegeneticdetails.Allweneedtograspis thatthere are regulatory systems that switchgeneson andoff in every life form.Moreover,thesesystemshavewaysofdetectingkeysignalscomingfromoutsidethegenome–intheabovecasetheyarecapableofdetectingthepresenceofthesugar,lactose,inthebacterialenvironment. This was the first scientific demonstration of what we now call genetic

‘regulatory’control.ItwouldresultinLwoff,MonodandJacobsharingtheNobelPrizeinPhysiologyorMedicinein1965.

*

Thetimehascometointroducealittlemagic.WhatIhaveinmindisamaidenvoyageinamysterytrain.Imaginethatwehaveshrunktoultramicroscopicsize–athousandtimessmallerthanaretrovirus,sothatahumancellwouldappearthesizeofamajorcityandwheretheindividualnucleotidesthatmakeupDNAareeasilydiscernible.Wecan,intheblinkofaneye,climbaboardthemostexcitingpartofit,thechuggingengine.

Withatootonthewhistle,weareoff.Upaheadweseeaglowingspirallingshape,aspectacularlybeautifuldoublehelix,spinningawaythroughtheetherfromlefttoright.Asweapproachthedoublehelixflattensout,stillglowing,stillrunningacrossthedream-likelandscapeinhorizontalfashion,fromlefttoright.Wenowseethatittakestheformofarailwayline,withtwinrailsspacedbycloselysethorizontallyplacedsleepers.Foradizzymomentor two,wegazeontheextraordinarystructureofDNAfromthisclose..ThenIslow the engine down to a halt. We are now hovering in a steam-filled stillnessimmediatelyabovetherailwayline.Wehopoutsowecantakeagoodlookatwhereweare.

WetakeashortstrollalongtheglowingDNAmolecule,inthedirectionthatthenow-stationarytrainispointing.

Whatwe took tobe rails areactuallybanded structures,madeupof alternating four-pointedstarsandpentagonsatrightanglestothesleepers.Thesheergorgeousnessofitisoverwhelming. The stars and pentagons aremade up of glowing spheres connected bylinesofforce.

‘So,’yougazealittlecloser,inwhatIimaginetobethesamewonderthatIamfeeling,‘thespheresaretheatomsthatmakeupthecomponentmolecules?’

‘Yes.’

‘Thecrossesandpentagons…?’

‘The pentagons are the deoxyribose sugars. The stars are the supporting phosphatemolecules.’

‘Betweenthemtheymaketherails?’

‘Thephosphatestarsmakeup theexternalspine thatWatsonandCrickarguedabout.Eachsugarconnectsthephosphatespinetoasleeper.’

‘Thelinesofforcebetweentheatomsarethestablecovalentbonds?’

‘Yes. The phosphates hold the whole thing together. The sugars are the connectionbetweenthespineandthesleepers.Time,perhaps,totakeacloserlookatthesleepers.’

Iallowyoutheleisureofastrollalongthetrack,examiningsleeperaftersleeper.

‘Thesleepersareattachedtotheinneranglesofeverysugarpentagon?’

‘Takeacloserlookatthem…’

‘Therearetwoshapes,joinedtogetherinthemiddle.’

‘Twocomplementarynucleotides,yes–butthejoinisnotexactlyinthemiddle.’

‘It has to be a trifle eccentric since the complementarynucleotides are unequal.Thisjoinhereisclosesttotheupperrail.Inthefollowingsleeperitisclosertothelowerrail.’

‘The purines, guanine and adenine –G andA – arewider because they contain twocontiguousatomicrings.Thepyrimidines,thymineandcytosineareshorterbecausetheyonlycontainasinglering.’

‘So,onewayoranother,thesleeperisalwaysmadeupofapurineandapyrimidine?’

‘Yes.Ithaseverythingtodowithshapes.Takeagoodlookatthejunctioninthemiddleof the sleepers.Lookathow the shapesof thenucleotidesmeet.Does it remindyouofanything?’

‘It’slikethemeetingoftwopiecesofajigsawpuzzle.’

‘Exactly.’

‘Sothat’swhytheyarecomplementary?’

‘Absolutely.Andnowyouknowwhythemoleculehastobeconstructedexactlyasitis.’

‘So the real DNA – the nucleotides – is like beads on the string of phosphates andsugars?’

‘No.Anotherscientist,Ithinkamathematician,saidexactlythattoCrick.Buthewaswrong.CricktoldhimthatDNAwasitselfthestring.’

‘TheDNAhastoincludethephosphatesandsugars,aswellasthenucleotides?’

‘Theconstructionhastobethewholething,exactlyasitis.Canyouseewhy?’

You take another short stroll, getting the hang of this idea. ‘So, the nucleotides, thebases,don’tmakecontactalongthethread?’

‘Their only meeting point is one-to-one within the sleepers. And always with theircomplementarypartner,AtoT,andGtoC,orviceversa.’

Yougazedownatthiswonder,blinkingforamomentortwo.‘Sothecodeliesinthesleepers?’

‘That’s right. And the sleepers also explain how the code replicates to form a newdaughterstrandofDNA.Theyalsoexplainhowcodeofprotein-codinggenestranslatestoproteins.Whatyouneedtograspisthecodeiscontainedinjustasinglerail.Inthiscaseifwe take the uppermost of the two rails, the code is in the sequence of the uppermostportions of the sleepers.You can read it off if you stroll along the rail and name eachnucleotideasyoucometothem,likeaseriesofletters.’

‘I’mreadingthem:A,A,C,T,G,C…IthinkI’mgettingthepicture.Butwhythenisthereasecondrail?’

‘Thecodehasalreadycopieditselftoadaughterthread.Whatyouseeontheoppositerailisthiscopy.’

‘Ah!So–thedoublehelixisactuallytwocopiesofthecodingDNA?’

‘Yes,twocomplementarysequences.Wouldyouliketoseeitcopyitself?’

‘I’dlovetoseethat.’

Westandbackandourengineevaporates.Thelinebeginstovibrate.

‘What’shappening?’

‘Tocopyitselfthedoublehelixmustpartintoitstwocomponenthalves.Thisnormallyhappensthroughtheactionofanenzyme,butitcanbedonejustbyheatingthesystemup.Heataddsenoughrandomenergytobreakopenthebondswithinthesleepers.’

‘Sothosebondsholdingthesleeperstogetherarenotstable?’

‘No.ThesearetherelativelyweakhydrogenbondswecameacrosswhentalkingaboutLinusPaulingandhisstudyofchemicalbonds.’

Aswewatch,thesleeperscomeapart,likepiecesofajigsawseparating.Acloud-likemassappearsoutofthedistanceanditbeginstomoveoverthenow-separatedupperrail,withitsexposedhalfsleepers.

‘What’sthat?’

‘Thecloudisanenzyme–aproteincalledasynthetasethathelpsDNAtoreplicate.’

Wewatchasthecloudmovesalongthedetachedrail,fromeasttowest.Itappearstodiscover thenucleotides itneedsfromtheteemingbackground,andas itpassesalongitattachesthecomplementarynucleotides,AtoT,CtoG,TtoA,GtoC,thensomeotherelement in the cloud, perhaps another enzyme, or enzymes, grabs the necessaryphosphatesandsugarstomakeupthespine.

You’retoodazzledbythespeedatwhichthecloudisshuttlingalongtosayaword.Inwhatseemsnomorethanafewmoments,thehiveofactivityhaslongpassedusbyandthenewtwintrackistherebeforeusandgleamingintothedistance.

‘That’sit?’

‘Almost.Ihaveonemorepointtomakebeforeweheadforhome.Firstweneedtotakeajourneyalongthisnewstretchoftrack.’

In the twinklingofaneyeourpuffingenginehas reappearedbeforeus, ready to roll,andwehoponboard,tootlethewhistleandheadeastwardsatarattlingrate.

‘Keepyoureyespeeledforaredlightupahead.’

Afterwhatcouldhavebeenquiteafewmiles,youspotit.Apinpoint,redglowinthedistance.‘It’sinthetrack,toourright.’

‘Yes.Itwouldhavetobeinthenewdaughtercopy.’Iexplainthatthetrackclosesttousiscalledthe‘sense’track,becauseitistheoriginal.Thedaughtercopyontheotherrailisthe ‘anti-sense’ track.The geneticmachinery reads thiswhile travelling in the oppositedirection.Islowtheenginetoahaltsowecanseewhattheredlightindicates.‘Lookatthesleepers.’

Yougetdownontoyourhaunches tohavea closer look.At first youcan’tmakeoutanythingwrong.Thesplitinthesleeperappearstobeasbefore,withtheshortsectiontotheleft,thentheshortsectiontotheright.Thenyougasp.‘TheoneontheleftisaC,sotheotherhalfofthesleepershouldhavebeenaG.Butitisn’t.It’sanA.’

‘So?’

‘Thecopyingmechanismhasmadeamistake.’

‘Yes.’

‘Sothisis…amutation?’

‘Yes, it is. To be precise, it’swhat is called a pointmutation,whichmeans a singlenucleotide has beenmis-copied.But if andwhen the anti-sense strand copies itself, themutatednucleotidewillattractathyminetoattachtoit–inotherwordsthemutationwillnowbefixedintothedoublehelix.Themutationwill,inthisway,perpetuateitself.Ifthisweretohappenduringtheformationofthegermcells,thespermortheovum,thatgermcellwouldcarrythemutationintothegenomeofthenewgeneration.’

‘Arethesemutationscommon?’

‘Much more common than one might think. But thankfully there are compensatingmechanisms in that moving cloud that will usually recognise and correct them. Butmutationsdogetthroughfromtimetotime.’

‘Andthiswillcausedisease?’

‘Mostmutationsdon’tcausedisease.TheyonlydosoiftheyaffectapartoftheDNAthatfulfilsanimportantroleintheoffspring’sinternalgenetics,ortheyseriouslyaffectthecodingofaprotein-codinggene.’

*

In the early years of the twentieth century a Dutch botanist, Hugo de Vries, made theconceptualbreakthroughthatMendel’sdiscretepackagesofhereditaryinformationcouldbechangedbymutations.AmazinglyhedidsobeforeweknewanythingabouttheactualstructureofDNAorwhatconstitutedagene.Aswehavejustwitnessed,amutationisanerror in thenucleotidesequencemadeduring thecopyingofDNA.Mutationscanarise,albeitrarely,duringthenormalprocessofDNAcopying.TheycanbeinducedatamuchhigherrateiftheDNAreplicationisdamagedbyexternalinfluences,suchasexposuretotoxicchemicalsorexcessivedosesofradiation.

There are many different types of mutation.What we have witnessed is one of thesimplest,inwhichasinglenucleotidehasbeensubstituted–apointmutation.Aso-called‘frame-shiftmutation’wouldresultfromthesimpledeletionofanucleotide.Ifweimagine

whatthiswoulddotothesucceedingtriplecodonswerealisethatitwouldinterruptallthetripletsequencesfollowingit,andthuswouldplayhavocwiththetranslatedprotein.Evena pointmutation in a protein-coding genemight result in a changed amino acid in thecoded-for protein. This is what causes sickle cell anaemia. In this case the mutationreplaces what should be an adenine in the beta-globin gene for a thymine.When thistranslates to thebeta-globinprotein, theaminoacidglutamicacid is replacedbyvaline.This makes the abnormal haemoglobin in the red cells that causes the disease. If theoffspringgetsjustonecopyofthemutatedgene,theysufferamilderformofthediseasethatincidentallyprotectsthemfrommalaria.Iftheygetadoubledoseofthemutatedgene,inotherwordsiftheygetamutatedgenefromboththeirparents,theygetasevereformofthediseasethatcanbefatalearlyinlife.Mutationsaffectingthecellsoftissuesandorgansinthebody,asopposedtothegermcell,areanintegralpartoftheunderlyingcausesofmanydifferentformsofcancer.

There are a few additional terms I need to explain to present an outline of basicgenetics. Other than the sex chromosomes, X and Y, we inherit 22 non-sex-connectedchromosomesfromeachofourparents.Thesearecalled‘autosomes’.Thismeansthatweall, both males and females, inherit two copies of every gene that is found on theautosomal chromosomes, which amounts to the bulk of our genes. When a mutationaffectsanautosomalgeneduring theformationof theovumorspermitwillonlyaffectoneofthetwocopiesintheoffspring.Iftheremainingnormalcopyofthegeneisenoughtosupplythebody’sbiochemicalneeds,therewillbenoupsetintheinternalchemistry–noclinicaldisease.Thistypeiscalleda‘recessive’genemutation.Butsometimesjustonebadgeneisenoughtogiverisetoseriousdisturbanceintheinternalchemistry,despitethefacttheothergeneisnormal.Thisiscalleda‘dominant’genemutation.Whenamutation,whetherdominantorrecessive,givesrisetoadiseasethisisreferredtobydoctorsas‘aninheriteddisorderofmetabolism’or‘aninbornerrorofmetabolism’.

Many medical conditions arise from dominant genes, for example, Huntington’sdisease, a condition in which the affected person may develop a progressive cerebraldeteriorationlaterinlife.Theinheritanceofonerecessivegeneisn’tenoughtocauseaninherited disease ofmetabolism, but if both parents are carrying one copy of the samerecessivemutatedgene,thenthereisaoneinfourchanceoftheoffspringbeingunluckyenoughtoinheritmutatedversionsofthegenefrombothparents.Sincethereisnonormalcopy,thiswillthengiverisetodisease.

One in every 2,500 babies born to Caucasian parents suffers from cystic fibrosis,making it one of the commonest of hereditary diseases. It is caused by a variety ofmutationsaffectingaregulatorgene,whichisknownasthecysticfibrosistransmembraneregulator gene, or CFTR, located in the region q31–32 of human chromosome 7, andwhichcodesforanionchannelinvolvedintransportacrossmembranes.Cysticfibrosisisperhaps themostfamiliarexampleofanautosomalrecessivecondition.Therearemanyotherrecessivegeneticdisordersthatmightpotentiallybecuredbytheadditionofasingle‘normal’gene,andtheseconditions,includingcysticfibrosis,arethesubjectofintensivecurrentinvestigationaimedat‘genetherapy’.

Anotherpatternofmutationgivesrisetoasex-associatedrecessivecondition.Femaleshavetwoof thesex-associatedchromosomescalled‘X’chromosomes,whilemalesonlyhave one X, always inherited from the mother. This means that a recessive gene thathappenstobecarriedontheXchromosomewillusuallyhavenoseriouseffectsinfemalesbut it will behave like a dominant gene if inherited by amale. A sex-linked recessivemutant gene is the cause of haemophilia, a condition that ravaged some of the royalhouses of Europe. It is also the cause of the red-green colour blindness that affectsbetween7percentand10percentofmen,aswellasseveraltypesofmusculardystrophy.

Suchsingle-genemutationswillusuallybeinheritedalongMendelianlines,suchasthedominantly inherited achondroplasia andHuntington’s disease, the recessively inheritedcystic fibrosis, and the sex-chromosome-linked disorders. To date, geneticists haveidentifiedmore than5,000 single-genedisorders inhumans causedbymutations.Somemutations can change the number of chromosomes, as inDown’s syndrome, or delete,duplicate, fragment,orotherwisedamagethestructureofchromosomes,givingrise toavarietyofsyndromes.Asmentionedabove,mutationisalsoacommonfeatureofcancers,which usually arise in fully developed tissues long after embryogenesis. Otherchromosomalabnormalitiesaffectthegermcells,wheretheygiverisetoawiderangeofdisorders including aberrant embryological development, with resultant congenitalabnormality,aswellasagreatmanyinbornerrorsofmetabolism.Inallsuchcases,aclearunderstanding of the genetic cause, or causes, is the basis for medical prevention andtherapy.

Themedicalapproach tomutation includesgeneticcounselling, forexampleenablingcouplesatriskofparticulardisorderstohaveessentialinformationsotheycanmaketheirown decisions on matters of reproduction, and public education about the risks ofincreasingmaternalage,avoidanceofriskfactorssuchasirradiationofthegermcellsandfoetus, caution with respect to drug and chemical exposure, such as thalidomide, andvaccinationagainst rubella.Newermeasures, suchaspreimplantationgeneticdiagnosis,involve thegenetic screeningof the foetus at the16-or32-cell stages, followedby theselection and implantation of healthy embryos. This is only suitable for a geneticabnormalitythatispredictable,andtheavailabilityofasuitablescreeningtestinisolatedembryologicalcells.Itnotonlyreducestheriskofsevereabnormalityinchildreninveryhigh-risk circumstances but also removes the mutation, and thus the risk pedigree, infuturegenerations.Thereare,ofcourse,importantethicalandmoralprinciplesinvolvedinsuchtherapyforbothdoctorandpatientsinwhatessentiallyamountstoapositiveformofeugenics.

Cancerisanotherarenainwhichintensivestudyofthemutatedgenesoffersthehopeofdevelopingmoreefficienttherapies.Herethegeneticabnormalitiesaremorecomplexthanin the inheriteddiseasesandveryoften involvemultiplemutationsaswell as importantlinks to environmental factors. At genetic level, cancer involves a series of steps thatinvolve multiple mutations that deregulate regulatory pathways. New lines of researchsuggestthatthesemutationsmustcooperatewitheachotherforthecancertodevelop,sothatresearchaimedatdeterminingtheprecisenatureofthecooperatingmutationsandtheregulatorypathwaystheyaffectisamajorchallenge.Thedecodingofthehumangenome

hashighlightedthegeneticalterationsthatunderliecancers insuchunprecedenteddetailthatithasledtwoAmericanoncologists,VogelsteinandKinzler,todeclarethat‘canceris,inessence,ageneticdisease’.

Some 15 to 20 per cent of women with breast cancer have a family history of theconditionand5percentofallbreastcancershavebeenlinkedtomutationsinthegenesBRCA1 and BRCA2. Geneticists can further predict that women who carry thesemutationshavean80per cent riskofdevelopingbreast cancerduring their lifetime, sothat there are various options that help to reduce the risk, including prophylacticovariectomy,regularbreastscreeningandthepotentialofpre-emptivesurgery.

In2006,asystematicmulti-centreAmericanstudypioneeredthescreeningofmorethan13,000genestakenfromhumanbreastandcoloncancercells.Giventhe‘normal’humangenome,theywereinapositiontocomparethegenestheyfoundinthetwocancerswiththenormal,revealingthatindividualtumoursaccumulateanaverageof90mutantgenes.Itseemsthatamuchsmallernumberoftheseactuallyplayapartinthecancerprocess,intheirestimationperhaps11mutationsforeachofbreastandcoloncancer.Encouragedbythese findings, the US National Institutes of Health is drawing up an atlas of cancergenomes – The Cancer Genome Atlas Project, or TCGA. The aim is to decode thegenomesofeveryhumancancerand,bycomparing these to thenormal,extrapolate thegeneticabnormalitiesthatunderlieallcancers.Apilotstudyhasbegunwithcancersofthelung,brainandovaries.This isfarfrompie in theskyresearch;alreadycancer isbeingforced back on many different fronts and today some forms of cancers are eminentlytreatableby surgery, focused radiotherapyandchemotherapyor immunotherapy, so thatwhat might formerly have been a death sentence has become more a chronic butcontrollableailment.

seven

TheLogicalNextStep

Ofthethreemainactivitiesinvolvedinscientificresearch–thinking,talking,anddoing–Imuchpreferthelastandamprobablybestatit.Iamallrightatthethinking,butnotmuchgoodatthetalking.

FREDERICKSANGER

Inthelate1960sIwasprivilegedtobeamedicalstudentat theUniversityofSheffield.WatsonandCrickwerestillrelativelyyoungmen,theirdiscoveryhavingbeenmadejustfifteenorsixteenyearsearlier.IcanremembermyownsenseofwonderasourteachersexplainedthestructureofDNA,andtheelegancewithwhichitsfour-lettercodetranslatedtoproteins.Wehadlecturesongeneticsinwhichwelearnthowmutationwasamajorstepin our understanding of many different hereditary diseases, including the so-called‘inherited errors of metabolism’. We also had lectures on the importance of the samediscoveriestothesisterdisciplineofevolutionarybiology.Icanrecalltheprevailingsenseof excitement that camewith the feeling that the biological andmedical scienceswereenteringanewparadigm,basedonthegrowingunderstandingofDNAanditsmolecularextrapolations, an understanding that clearly had implications notmerely for biologicalscientistsanddoctors,butforallofhumanity.Butatthatstagemanyimportantquestionsremainedtobeanswered.

Oneveryobviousquestionwashowdidthefertilisedegg,or‘zygote’,developintothecomplexwonderofahumanbaby?Howcould thisextraordinarychemical,DNA,storenot only the heredity of the individual but also the instructional blueprint that wasnecessaryforthesinglecellofthezygotetogiverisetothedevelopingembryo,withitswiderangeofdifferentcellsandtissuesandorgansthatwentintomakingthefuturebaby?

Whilemuchwasknownaboutthetissuechangeswithintheembryo,littlewasactuallyknown of the relevant genetics at this time. The work of the scientists at the PasteurInstituteinFranceofferedusthefirstglimpseintothisquandary:theyhadpioneeredourunderstanding of how a gene is activated by switching on its ‘promoter’ sequence, andhowitisinactivatedbyswitchingoffthepromoter.Thiswasthefirststeptowardswhatwenowcallgenetic‘regulation’.

Backthenwealsoknewthatthecellsthatmakeupthedifferenttissuesandorgansinthehumanbody,suchas thebraincells,or thewhitecells that fightoff infection in thecirculatingblood,orthecellsthatmakeupthekidney,orliver,heartorlung,allcontainedexactlythesameDNAintheirnuclei.Thedifferencesinstructureandfunctionbetweenthesecells,andthusthemake-upofthevarioustissuesandorgans,mustsomehowinvolvedifferences in the expression of genes. This provoked two new questions: was the

differencebroughtaboutbyspecificgenesthatwereonlyswitchedoninspecificorgans,or was it brought about by different profiles and timing of the expression of the samegenes?

Thequestionsdidnotstopthere.

Whatever the explanation, whether special genes for particular cells, or differentprofilesofexpressionofthesamegenes,therehadtobeasystemthatdecidedwhatgene,orwhatprofile,wouldbeexpressedinthedifferentcells,tissuesandorgans.Thismustbeakeyelementintheplanningandregulationofthedevelopinghumanembryo–andverylikelytherewouldbeverysimilarpatternsofregulationofembryogenesisinallanimals–andmaybeplantsaswell.

WemightrecallhereSydneyBrenner,whocametoworkwithCrickattheCavendishLaboratoryon the translationofgenes toproteins. In1973,when still employedby theMRC Laboratory in Cambridge, Brenner published a paper that addressed this verysubject.Itopenedwiththelines,‘Howgenesmightspecifythecomplexstructuresfoundinhigherorganisms isamajorunsolvedprobleminbiology.’Heexplained thatbynowmanyofthemolecularmechanismspreviouslyshowninmicrobeswerefoundtoexistinmuchthesameformandfunctionineukaryoticcells–thenucleatedcellsofanimalsandplants.Thegeneticcodewasuniversalandthetranslationofthatcodetothemechanismsof protein synthesis appeared to be equally universal. Meanwhile, ‘Although there aremanytheoriessuggestinghowthe[DNAofhigherorganismsmightcontrolsuch]complexgenetic regulation, the problem is still opaque.’Brenner chose a newmodel system forresearchintohowanimalgeneswerecontrolledandorganised.Inhispaperheintroducedhis new model, a minuscule round-worm, Caenorhabditis elegans, which was just amillimetrelongandacommoninhabitantoftemperatesoilenvironments.C.eleganshadanumber of attractive properties for this type of research. It was non-parasitic, so itwouldn’tinfectlaboratoryworkers;itwasverysimpleinstructure,withtheentirewormcomprisingjust959cells;itcouldeasilybebred;itwasconvenientlytransparentsoonecouldpeerinsideitthroughamicroscope;ithadatinygenomecomprisingjustfivepairsof autosomes and one pair of sex chromosomes; and it comprised two sexes –hermaphrodite and male. In a nutshell, it presented geneticists with an ideal modelexperimentalanimal,beingeasytobreed,safetostoreinlargenumbers,withasexualityandageneticsthatcouldeasilybemanipulated.

In his paper, Brenner showed how he had used experimentally inducedmutations insome 300 of the worm’s genes to show how these genes contributed to the worm’sbiological make-up and behaviour. But even in a creature as simple as the worm, thegeneticsprovedtobemorecomplexthanBrennerhadimagined.Astaggering77differentgeneswere involved in its simplewrigglymovements.Nevertheless, studyof thewormsoonconfirmedhis choiceof experimentalmodel.Herewas anewexperimentalmodelcapable of figuring out what genes did, and in particular how they regulated themysterious and profound changes that took place during embryological development,when those extraordinary pluripotent cells of the early embryo began the processes of

changethatwouldultimatelygiverisetothecellsofthemanydifferentbodytissuesandorgans.

Brenner’smodel proved to be an inspired choice. It was taken up inmany differentscientificcentres,andasknowledgegrewtheC.elegans,which itselfcomplemented theearlierfruitflyresearch,wascomplementedbypioneeringexplorationsofgenefunctionandgeneregulationinfish,frogs,lanceletsandmammalsintheformofmice–aswellasagrowingvarietyofplants.

Thehumanbodycontainsabout200differenttypesofcell,formedintolimbs,tissuesandorgans,eachspecialisedtoperformdistinctfunctions.Forthezygotetodevelopintoallof these, itmustbegin its lifeasa ‘totipotent’cell–acell thatcandifferentiate intoeverypossiblehumantissue,includingtheplacentaaswellasthedevelopingfoetus.Thefirst differentiation is from this stage of totipotent to ‘pluripotent’ cells –whichmeanscellswithmultiplebutnottotalpotency.Thepluripotentcellsarethecellsthatnowgiverisetothemorecomplexshapesandcellulardifferentiationthatwillbegintofashionthedistinct tissuesandorgans.Thesesamepluripotentcells,alsoreferredtoas‘stemcells’,remain with us for the rest of our lives, replacing damaged tissues in the constantrecycling that is necessary for normal physiological functioning and health. For such aremarkable transformation tooccur in the embryowith suchpredictableprecision, eachcellmustknowitsownfate.Thisisdeterminedbyacarefullycontrolledbureaucracyofgeneticelementsincludingepigeneticregulation,whichweshallcometoinasubsequentchapter,aswellasentitiesknownas‘regulatorygenes’.

By the late1980sgeneticistsworkingwith the fruit flydiscoveredabatteryofgenesthat determined the identity of the different segments of the insect body in strict orderfrom the head to the ‘tail’ end during the embryological formation of the insect bodywithin the egg. They called these ‘homeobox’ or ‘Hox’ genes (the name of a gene isconventionallyitalicisedwhereasthenameofitstranscribedproteiniswritteninordinaryscript). Subsequent researchwoulddiscover that these sameHox genes in similar orderalongaspecificchromosomewerecriticallyimportanttothedevelopmentoftheanimalbody plan during embryological development.We humans, like all vertebrate animals,followaHox-determineddevelopmentalplanthatcreatesarightandaleftside.Thisgivesus our ‘bilateral’ symmetry. We might compare this, for example, to the exoticallybeautifulseacreatures,calledechinoderms,suchasstarfishandseaurchins,whichhavearadialpatternofsymmetrysimilartothesegmentsofanorangeortheflowersofadaisy.

Asthehumanembryogrowsfromtheinitialballofcells,ourHoxgeneswilldetermineatwhich point to place the head, andwithin the head the eyes, the nose, the jaws; andthen, vertebra by vertebra, the seven bones that constitute the neck. Then, vertebra byvertebra again, the twelve bones that designate the thorax, with the offshoots of upperlimbsandribs;insimilarfashion,thefivelumbarvertebraethatwillsupporttheabdomen;andfinallythefusedvertebraeofsacrum,whichsupportsthepelvisandlowerlimbs–allinappropriatepositionalongthecentralaxisofourbilateralbodyplan.TheevolutionofHoxgeneswasclearlyavitalevolutionarystepintheevolutionoftheanimalkingdom.Sovitalistheirfunctionthattheyhavebeensteadfastlyconservedbynaturalselectionover

vasttimeperiodsofevolution.Forexample,eventhoughthecommonancestorofhumansandinsectsinhabitedtheoceansroughly600millionyearsago,ifweweretoreplacetheHox gene for the specificationof the insect eyewith its humanequivalent in the insectzygote,thehumangenewouldstilllocatethedevelopmentoftheinsecteye.

Hoxgenescodeforproteins,butHoxproteinsarenotenzymes,nordotheyfunctioninastructuralcapacity–suchasmakingupskin,orthestructuresofkidneys,heartorbone.Instead, they regulate the expression, or ‘transcription’, of other genes.Hence they aredubbed‘transcriptionfactors’.TheHox-codedproteinsbindthemselvestokeynucleotidesequenceswithin thechromosomes,knownas ‘enhancers’,where theyactby switchingthetargetgenesofforon.Intimescientistscametodiscovermanysuch‘regulatorygenes’thatplayimportantrolesduringembryologicaldevelopment,aswellasinnormalhumanphysiologythroughoutlife.Keygenes,suchastheHoxcluster,initiateaprocessthatleadsto a series of developmental steps, involving signalling hormones and transcriptionfactors. Key to understanding such systems is the fact that one key genemay activatemany secondary genes, each further activatingmultiplemore, so that it ends upwith acascade of hundreds of genes that constitutewhat is called a ‘developmental pathway’.This ensures that a region in the embryo becomes the brain, or a limb, a kidney or atoenail.Infact,ifweconsidertheconstructionofacomplextissue,suchasakidneyoralimb, it is evident that this will contain many different kinds of cells and tissues. Forexample,adevelopinglegwillincludetheconstructionofskin,muscle,bone,nervesandbloodvessels–soitsembryologicaldevelopmentmustinvolvethecoordinationofmanydifferentregulatorypathways,perhapswithlocalsignallingbetweentissues.Afailureofany individual component is likely to lead to catastrophe. Thalidomide was a popularover-the-counter drug used to treat nausea in pregnancy in the 1950s and early 1960s.Within a few years of widespread use approximately 10,000 children were born withseverely malformed limbs – so-called ‘phocomelia’. The failure of the normal bloodvesseldevelopmentwithinthedevelopinglimbbudswastherootcauseofthethalidomidetragedy.

AtthetimeofpublicationofBrenner’spaper,intheearly1970s,weunderstoodlittleofthisgeneticregulationofhumandevelopment.Weknew,ofcourse,thatthehumanbrainwasstillrelativelyundevelopedatbirth,continuingtogrowanddevelopforperhapstwoor three years into infant life.Andwhilewewere aware of the glandular changes thatbroughtaboutandresultedfrompuberty,wehadlittleornounderstandingofitsgeneticregulation.Todayweknowthatpubertyinvolvesveryprofoundchanges,bothatgeneticandepigeneticlevel–ineffect,wereturntothecontrolledturmoilofembryogenesis,inwhat geneticists now recognise to be a major and dramatic phase of ‘postembryonicdevelopment’.Therearesomanysimilarities in thegenetic regulationand theprofoundbodilychangesbetweenhumanpubertyandtheastonishingmetamorphosesweseeinthelives of moths and butterflies, some scientists regard puberty as a variety ofmetamorphosis.

Prepubertal boys and girls have much the same proportion of muscle mass, skeletalmassandbodyfat.Butnow,with thereawakeningof thesamepowerfuldevelopmentalepigeneticandgeneticpathwaysthatcontrolledembryologicaldevelopment,thebodiesof

boys and girls undergo dramatic physical change, including rapid growth and majorchangesinmuscleandfatdistributionthatdifferbetweenthesexes.Bytheendofpuberty,menhave1.5timestheskeletalandmusclemassofwomen,whereaswomennowpossesstwice as much body fat as men. These obvious physical changes are accompanied bycellularandtissuechangesinvolvingsexualorgansandrelatedorgans,suchasthebreastsinwomenandtheprostateglandsinmen.Pubertyisbroughtaboutbysignallingpulsesofgonadotrophin-releasinghormone(GnRH),whichisreleasedbythehypothalamicportionofthebrain.Thisstimulatesthemastergland,thepituitary,toincreasethesecretionandreleaseof the sexgland stimulatinghormones, or gonadotrophins,which travel throughthebloodstreamtotheovariesandtestes,wheretheyprovokeincreasingbloodlevelsofoestrogens and androgens. If sometimes the pubertal adolescent appears moody andconfused,itishardlysurprisinggiventhemassivephysicalandhormonalchangesthatarecoming about. Only recently have we recognised that puberty is also associated withhormone-drivenmajor rewiringof the neural circuits of the adolescent brain, triggeringchangestowardsadultbehaviour.

Some psychologists have proposed that individual differences in adult behaviour andthe risk of sex-related psychopathies may derive from variations in timing and in theinteractionsbetweenthehormonesofpubertyandthebrainrewiringatthiscriticalperiodofadolescence.

*

Bytheearly1990sbiologistshadabasicunderstandingofhowgenesworked.Theyknewthat genes coded for several different types of protein. Some played key roles in ourinternal chemistry, as enzymes, while others were structural components in cellmembranes, the tissues of skin, eyes, hair and nails. Geneticists had plotted wherehundreds of genes were to be found on the 46 human chromosomes. They wereaccumulatingsubstantialknowledgeaboutgeneticregulation.Theywerealsobeginningtorealise that therewere additional systems of regulation thatwere notDNA-determined.There was growing evidence of non-DNA-based systems that could influence theexpressionofDNAfromoutsidethegenome–systemsthatmightbecapableofchangingduring the life and experience of the individual. These would in time come to beunderstoodaspartoftheepigeneticregulatorysystem,whichwillbedescribedinalaterchapter.

Therevolutionthathadbegunin1953withthediscoveryofthestructureofDNA,hadgivenrisetothenoveldisciplineofmolecularbiology,withitsmanifoldextrapolationstomedicineandbiology.Inafewdecades,wehaddiscoveredmoreofthelabyrinthineinnerworkings of human heredity, embryological development and the workings of cells,tissues and organs at biochemical level, than in all of previous history. Therewas alsogrowingevidencethatviruseshadinvadedourhumangenome,fromthepresenceofviralgeneticsequencesandevenwholeviralgenomes.Whilesomegeneticistsregardedtheseasjunk,thefossilsofinfectionfromlongago,othersbelievedtheymightbecontributinginsomefunctionalways.

Thousandsofgeneshadbeendiscoveredusingthelaborioustechniquesofmutationinexperimental animalmodels.But, given that thehumanbody incorporatedanestimated80,000to120,000proteins,onthebasisofone-gene-one-proteinthereshouldbeasmanygenes encoding all those proteins. This suggested that there must be vast numbers ofunknown genes yet to be discovered. What geneticists now needed to know extendedbeyondthesequencingofindividualgenes.Thenextlogicalstepmustbetheexplorationof the structure of every chromosome, and beyond that the exploration of the entirenuclear genome. Without such exploration we could not determine how the systemworked as a whole. Only with the sequencing of the entire human genome would weunderstandwhatlayatthecoreofourbeing–toparaphraseBronowski,whatgeneticgiftsmadeussouniqueamongtheanimals.Allthatweneededtotakethisfinalalmightystepwas the political will to fund the exercise, together with more efficient techniques ofreadingDNAsequences.

Back in themid-1970saCambridge-basedBritishbiochemist,FredSanger,alreadyaNobelLaureate inChemistry forworkon thestructureofproteins,hadpioneerednoveltechniques of automated DNA sequencing that were subsequently named after him:‘Sangersequencing’.Sangerusedthesetechniquestodeterminethefirstcompletegenomeofanorganism–thesameorganismIstudiedasastudent,anddoctor,thebacteriophagevirusknownasΦX174.ThiswonhimasecondNobelPrizeinChemistry,theonlyNobelLaureateever towin twoprizes inChemistry.WhileSanger’smethodologybecame thestandardtechniqueforDNAsequencinginlaboratoriesthroughouttheworld,discoveringthestructureoftensofthousandsofgenes,itwas,inSanger’sownadmission,slowandlaborious to conduct, requiring the scientists to read off the results on printouts, anddemanding wasteful quantities of radioactive phosphorus which was used to label thenucleotides. In the mid-1980s Leroy Hood and colleagues at Caltech, in America,introduced a faster and easier methodology which labelled the nucleotides with fourdifferent-colouredfluorescentdyesthatcouldbereadbyalaserwithinamachine.OthersdevelopedtechniquesofreplicatinggeneticsequencesusingculturesofE.colibacteria,sothat smallamountsofDNAcouldbeamplified tomakesequencingeasier.Thegenomecould now be broken down into smaller sequences which could be amplified to manycopiesinbacteria,andthensequencedinautomatedmachines.

In 1984, the political component achieved critical mass when the United StatesDepartmentofEnergyproposedthattheywouldfundthesequencingoftheentirehumangenome,withits6.6billionnucleotidesequences.Thenameoftheprojectwasdecidedbyacommittee:itwouldbe‘TheHumanGenomeProject’.

Thescopeoftheprojectwasdaunting,butitwasalsofantasticallyambitious,inspiringand exciting.By1987 the proposal had beendebated and clarified,with a crystal clearstatement of purpose: ‘The ultimate goal of this initiative is to understand the humangenome,knowledgeofwhichisasnecessarytothecontinuingprogressofmedicineandotherhealth sciences asknowledgeofhumananatomyhasbeen for thepresent stateofmedicine.’

ExclusivelyAmerican to startwith, the project later expanded to includemanyothercountries,ultimatelygrowingintothelargestcollaborativebiologicalprojectinscientifichistory.Withsomanydifferentscientistsandscientificgroupsinvolved,itwasinevitablethattherewouldbeconflictingopinionsonhowtogoaboutit.Somethoughtweshouldundertake a chromosome at a time, but thiswould stretch tomaybe ten or even fifteenyears to complete. Some politicians failed to grasp the importance of the project andbridledat the likely financialcost,whichwould rise tobillionsofdollars.Somepeoplemay have been somewhat daunted by the prospect of such amonumental step into theunknown.

Butbytheearly1990sthediewascast.In1990,twomajorfundingagencies,theDOEand theNational Institutes ofHealth (orNIH), coordinated their plans.That same yearJamesDeweyWatson,jointdiscovererofthestructureofDNA,wasappointedheadoftheNIHprogramme.Watson’sprestige,thebackingoftheUSNationalAcademyofSciences,thesupportofmanyacademicallyinfluentialmolecularbiologists,andthegovernmentalandotherofficialsponsorshipofsomethinglike$2.6billion,nowunderpinnedtheproject.Watsonimmediatelyencouragedtheinternationalisationof theirplans,enlistingthehelpoftheUK,GermanyandFrance,withcontributionsfrommanyotherEuropeancentres,aswellasJapan,ChinaandAustralia.IntheUK,theWellcomeTrustbecameamajorcharitypartnerwiththeUSpublicbodies.

Withthemajoropusnoworganised,coordinated,fundedandreadytoroll,thebanksofcomputers and the automated sequencing machines kicked into action. It was still thegeneralassumption that theprojectwould takefifteenyears tocomplete,but thatwouldchange with the intervention of an unexpected rival from the business sector – anAmerican commercially sponsored organisation named ‘Celera Genomics’. Thecompetition from a rival, privately sponsored organisation would throw an unwelcomespannerintosomeofthemostconservativelyorganisedplans.

eight

FirstDraftoftheHumanGenome

Ifeelthisisanhistoricmoment.Thisisthemostimportantscientificeffortthathumankindhasevermounted…Itwillchangebiologyforalltime.

FRANCISCOLLINS

On Sunday 12 February 2001, the two rival organisations, Celera Genomics and thepublicly fundedHumanGenomeProject–embodyingmanydifferentgovernmentalandmajor charitable bodies in the US, UK, Germany, Japan and France – announcedsimultaneously that they had completed the first comprehensive analysis of the humangenome. It created a wave of excitement throughout the world’s media. In the UnitedStates,PresidentClintonledthechorusthatwouldbejoinedbyPrimeMinisterTonyBlairintheUK,andbysimilarnationalleadersandleadingscientificfiguresineverycountry,proclaimingthatanewepochofknowledgeandscientificexplorationhadarrived.IntheUK, Roger Highfield, science editor of The Daily Telegraph, put it bluntly: ‘ScienceRivals Open the Book of Life’. In the words of Andy Coghlan andMichael Le Page,writingforNewScientist,thegenomewouldsoonbeasfamiliartoschoolchildrenastheperiodic table of the elements. There could be no doubting that it signalled a newmilestone in genetic science, themost powerful and logical development to follow thebreakthroughs onDNA.And like theDNA story, therewere new issues of rivalry andconflictbetweenthediscoveringgroups.

Asdirectorof theHumanGenomeProject,Watsonhad internationalised thescopeoftheundertaking,meanwhileengenderingthegratitudeanddedicatedsupportofacademicscientiststhroughouttheworld.Hehadalsodevotedasmallbutsignificantallocationoffunds to include sociological, religious and ethical concerns in the minds of layintellectuals and politicians. Celera Genomics was seen by some within scientificacademiaasabrashinterloper,ledbyanentrepreneurialscientist,J.CraigVenter–buttohiscreditVenterhad,throughinsightandsheerforceofpersonality,alreadysucceededina litany of impressive breakthroughs involving new fields of genetic discovery. LikeWatson, Crick and Wilkins, Venter would also admit that he had been inspired bySchrödinger’sbook.

Growingupaspartofacademia,Venterhadworkedfor theUSNational InstitutesofHealthinalabimmediatelybelowthatofMarshallNirenberg,whohadcontributedtothediscovery of the histone code. In 1992, impatient with the slow progress of academia,Venter set up his own commercial laboratory, The Institute for Genomic Research, orTIGR. He was now free to combine automated sequencing with his group’s newly

invented ‘shotgun’ approach, in which incredibly lengthy genetic sequences found inlivinggenomescouldbebrokendownintosmallerfragments.Bybreakingdownthesamegenomeagainandagain,andbreakingthesequencesindifferentregions,histeamcoulduse the inevitableoverlappingsections topiece together thefragmentsso that theentiresequenceof, say, amicrobe, or a human chromosome and soon, to the entire genome,couldeventuallybestitchedtogether.

This ‘shotgun sequencing’ technique had the potential to speed things up but it wasderided as potentially inaccurate by Venter’s academic rivals. Nevertheless, by 1995,Venterwasreadytopublishhisfirstsuccess–thefirstevercompletelysequencedgenomeof a bacterium,Haemophilus influenzae, which causes respiratory and other infections.Thiswas followedby thegenomeof theulcer-causingbug,Helicobacterpylori, and inMarch2000, thefirst insectgenomicsequence–thatofThomasHuntMorgan’sfamousexperimentalsubject,thefruitfly.Thescepticalworldofacademiawasrockedbackontoitsmetaphoricalheels.

InMay1998VenterhadteamedupwithPerkinElmertoamalgamatehisInstituteforGenomic Research with Elmer’s PE Corporation, and formed a new company, CeleraGenomics.Celera, as itsLatinderivative implies, conveyed theobjectiveof ‘speed’. Itsremit, as Venter made clear, was not biotechnology per se but the provision ofinformation.In thewordsofJamesShreeve,whowrote thehistoryof thisextraordinaryperiod,Celera’smarket productwould be amassive database ofDNA,with the humangenomesequenceasitsheart.ThusVenter,andthenewcompany,hadastheirveryraisond’êtreavestedinterestinrivallingthepubliclysponsoredHumanGenomeProject.

In1992,JamesWatsonhadamajordisagreementwithBernadineHealy,thendirectorof theNational InstitutesofHealth,whohadbeenput inchargeof theHumanGenomeProject. Healy supported the dictates of Congress that NIH discoveries should, wherepossible,besupportedbypatents.Watsonheatedlydisagreed.WatsonderidedHealyuntil,‘tired of his insulting remarks’, she fired him. That same yearWatsonwas replaced asdirectoroftheHumanGenomeProjectbythemorediplomaticallysavvyFrancisCollins.In theUK, theWellcomeTrust had set the ball rolling by funding amajor sequencinglaboratory near Cambridge, the Sanger Centre, which would work in a coordinatedsynchronicitywithNIHontheHumanGenomeProject.

TheambitiousCeleracommissioned200ofthefastestautomatedsequencingmachines,which would combine the speed of mass production with Venter’s shotgun method,blastingthe46chromosomes(containingsome6.4billionnucleotides)intomuchsmallerfragmentswhichwouldbecapableofdeciphermentbythebanksofthesequencersbeforebeing reassembled to complete thewholegenome.TheCelera approach, asVenternowplanned it, would reduce the time needed to complete the project from the ten yearsproposed by his rivals to seven. Meanwhile Collins, on behalf of the many scientistsinvolvedinthepubliclyfundedHumanGenomeProject,arguedthatthistechniquewouldlead to unacceptable inaccuracy.The academics now raised newworries – that, despiteVenter’sreassurances, thecommercialmindsetwouldleadtounacceptablelimitationsin

thefreedomofaccesstothegenomicdata,hamperingfutureresearch.Takentoextremes,somescientistsfearedthatCeleramightattempttocopyrightourhumangenes.

Thisacrimonyanddebatestillrankledbetweentherivalsandinfusedthemedia,atthetimeofthetwindeclarationsofdiscovery,in2001,withtheCeleraresultspublishedintheAmericanflagshipmagazine,Science,andtheGenomeProject’sresultspublishedin theBritish equivalent, Nature. In effect we now had two readout versions of the samegenome.WhileCeleramadeclearthattheywouldpermitfreeaccesstotheirfindingstoacademicscientists, thiswouldnotextrapolate tocommercialapplicationsandpotential.After all, they had spent hundreds of millions of dollars in the exploration and, as acommercialcompany,theyneededtorecouptheircostsandmakesomeprofitoutoftheenterprise.Meanwhile, thepublicly fundedgroupmadeexplicit thatallof their findingswerenowuniversallyavailable.

Some readers might feel indignant that commercial interests should intrude intosomethingassacredasourgeneticmake-up,butinfactthisparryingbetweencommercialandpublic interest is commonplace inbiological andmedical research.While itmayattimesbetrickytodrawanyhardandfastlinebetweenthetwoverydifferentapproaches,inpracticeresearchintothemostimportantofarenas,suchasvaccination,antibioticsandthe treatment of cancer, has always involved an uneasy balance between opposinginterests.

Thebreakthroughactuallycameaboutthroughbothavenuesofresearch,andwithequalplauditstothetwoopposingsides.ThroughthetwinpublicationsofNatureandScience,theworldofscience,andhumanityingeneral,wasnowprivilegedtolearn,on15and16Februaryrespectively,about theenormouslycomplexmolecularstructures that lieat thegeneticheartofus.Thedeciphermentwasepochalinwhatitpromisedfuturegenerationsofbiologicalandmedicalscientists–indeed,inwhatitpromisedhumansociety–butitalso proved to be mind-blowing in its unexpected revelations. If, as newspapers andmagazines proclaimed, here was the basic genetic landscape at the core of life, thatlandscapewasnowrevealedtobeavastterraincognita.

Theword‘breakthrough’ isoftenmisused in relation toscientificdiscovery,buthere,indeed,wasrealbreakthroughafterbreakthrough.Andthebreakthroughspresentedaverymuch unprepared world of science with not one but three major surprises, each achallengingnewmystery.Thesewillbecomeevident ifweexamine thepiechartof the2001humangenomebelow.

I should make clear that this pie chart is a metaphor of sorts, summing up thepercentage contributions of different distinct genetic elements to the genome withoutreference towhere things are actually situated throughout the 46 chromosomes.At thisstage in our knowledgemost geneticistsweremainly interested in genes that coded forproteins,soit is inthisaspect, thepartof thegenomethatcodesforproteins,wherewediscoverthefirstofthethreemysteries.

Biochemistshadarrivedataroughassessmentthatthereweresomethinglike100,000proteinsinvolvedinthestructureandfunctioningofthehumanbody.Thusweanticipatedthat there would be the same number of protein-coding genes.What many geneticistswantedtoknowwasexactlyhowmanyprotein-codinggenestherereallywere,andwheretheyweresituatedonthechromosomes.Itwasanalmightyshocktodiscover that theseprotein-codinggenesamounted to less than2per centof theentiregenome,perhapsaslittle as 1.5 per cent. It hardly seemed possible that this minuscule inheritance couldpossibly translate to the 100,000 different proteins that went into the make-up of thehumanbody.

Howhadwegotthingssoverywrong?

*

Thismodest 1.5 per cent of the genome coding for protein-coding geneswas found tocompriseroughly20,500genes.Forgeneticists,andbiologistsingeneral,thisobservationwas astonishing. According to Beadle and Tatum’s maxim of one gene one protein –universally believed up to this moment – there should have been 80,000 to 100,000protein-codinggenes.Itappearedtomakenosense.Toconfusemattersfurther,anotherofthe dawning implications of the first draft of the genome was the fact that, in CraigVenter’sestimation,at least40percentof theprotein-codinggenestheyhaddiscoveredhad no known function. ‘We have no idea what they do. They have not been seen inbiologybefore.’Hewentontoadmit:‘Itisincrediblyhumbling.’

Thepaltry20,500genesseemeddownrighthumiliating.Toputit intoperspective,wehadroughly ten timesasmanygenesas theaveragebacterium,four timesasmanyasafruitfly,andjusttwiceasmanyasanematodeworm.Intermsofgenes,weseemedhardlymorecomplexthanthesehumblelifeforms.Arelatedrevelationwasthenumberofgeneswe have in common with these simpler organisms. We now discovered that we share

2,758ofourgeneswiththefruitflyand2,031withthenematodeworm;andthethreeofus—human,flyandworm—have1,523genesincommon.

Darwin had been the first to dare to imagine that all of life onEarthwas intimatelyrelated,throughtheprocessofevolutionthathehadhimselfpioneered.Hereatoncewasboththeconfirmationofhisbrillianceinthelettersofthecodeoflife,ourhumanDNA,butalsoanewandastonishingincongruity.

How could science possibly explain how roughly 20,500 genes could code for theestimated100,000proteins?

Uptothispointwebelievedthattheprotein-codinggenes,madeupoflongstrandsofDNA,werecopied to theirexactmatches in termsofcomplementarymessengerRNA–with the exception that the fourth nucleic acid, thymine in the DNA, was replaced byuracilintheRNA–andthismatchinglongstrandofmessengerRNAwasthenferriedoutofthenucleusandtakentotheprotein-manufacturingribosomesinthecytoplasm,whereit was translated, using the triplet codes, to proteins whose amino acids correspondedfaithfullytotheoriginalDNAcodeofthegeneinthenucleus.Thusthenumberofgenesshouldcorrespondtothenumberofproteins.

Thekeytothisenigmaprovedtobeastartlingdiscovery,madebytwoscientistsbackin1977.

Richard J. Roberts graduated frommy own almamater, the University of Sheffield,with a Bachelor of Science degree in chemistry, completing his PhD in 1965. Hesubsequentlywent towork atColdSpringHarborLaboratory,NewYork.PhillipAllenSharp graduated at theUniversity of Illinoiswith aPhD in chemistry in 1969.He alsoended up working at Cold Spring Harbor. Roberts and Sharp were exploring how thegenesofavirus,calledadenovirus2,wereexpressedasproteinwithinthecellsoftissuecultures.WhattheydiscoveredwasthattheactualmessengerRNAstrandthatarrivedatthe ribosomes ready to code for the protein was significantly shorter in terms of itsnucleotidesequencethantheDNA-basedgeneintheviralcore.Thistoldthemthatonlyaportionoftheso-calledprotein-codinggeneactuallycodedfortheaminoacidsequencesofthetranslatedprotein.Somethingverystrangemusthavetakenplaceduringthechainof communication from the viral gene,within the viral core, and the expression of thatgenewithinthehostcellinthetissueculture.

Aswiththephageresearchagenerationearlier,thetiniestofmicrobes,theviruses,hadopened up a window onto a more general biological truth. Roberts and Sharp haddiscoveredwhatwenowcall ‘introns’and ‘exons’and the importanceof their role inageneticmechanismknownas ‘splicing’–discoveries that led to their sharing theNobelPrizeinPhysiologyorMedicinein1993.

Whatthenareintronsandexons?Andhowdotheysolvethepuzzleofthediscordancebetweenthenumberofprotein-codinggenesandtheanticipatednumberofproteinscodedbythehumangenome?

Perhaps it is timeweclimbedbackaboardour imaginary train to takeanew journeyinto that ultramicroscopic landscape, with its astonishing twin track of alternating

phosphatesanddeoxyribosesugars,andthoseall-importantsleepers.

*

Wearriveatourdestinationintheblinkofaneyetofindourselveschuggingalonglargestretches of a chromosome. We know that within this chromosome there are distinctstretchesofDNAcalledgenes.Sincethisisawonderland,withmagicalpotential,wecanwishthatsomeforthcominggeneshouldshowitselfbyglowingwithagreenlight.Withthis inmindwe slowdown sufficiently to see that exactly such a stretch is looming infrontus, pulsating abeautiful emeraldgreen,which tells us thatwehavearrived at thebeginningofagene.We throw theengine into lowgearand travelalong the twin-trackrails,observingthatthegreenglowisactuallycomingfromthesleepers.Afterawhileweseethatthetrackhasrevertedtothenormalbrownofsleepersagain.Imustnowsuggestthatwehaven’tactuallycome to theendof thegene.Thegreen-glowing trackwehavejusttraversedismerelythefirstexon.

Youareinclinedtoask:‘Sowhereexactlyarewenow?’

‘Thenormalstretch,withthebrownsleepers,isthefirstintron.’

Aswechugslowlyalongthissection,wefinditis,ifanything,longerthanthegreen-glowingprevioussection.Thenittooendsabruptly,aswearriveatanothergreen-glowingsection–a secondexon.Aswecontinueour journey,wecount some three stretchesofexons with two intervening stretches of introns. There are no further green-glowingsections. So what we have been looking at is a protein-coding portion of a genecomprising three separate exons with two introns, somewhat like spacers, in betweenthem.Itreallyisthatsimple.WhatRobertsandSharpediscoveredisthatthewholeDNAof a single ‘gene’ does not necessarily code for a single protein. The gene is actuallybrokendownintosmallerchunks,theexons,separatedbyinterveningintrons.Tocodeforaspecificproteinonlyaparticularclusterofthegene’sexonswillbeexpressed–theywillbecopiedtomessengerRNA,completewiththeinterveningintrons,buttheinterveningintrons will be removed from the coding sequence before the exons are then ‘spliced’togethertofashionthefinalmessengerRNAthatwillcodeforaprotein.

Itmight help us remember ifwe think of the exons as ‘exiting’ the nucleus tomakeproteins,while the introns stay ‘in’ the nucleus. The total number of exons in any onehumangeneisveryvariable,withanaverageof8.4.Soinordertomakeaspecificproteinthegenomemustknowhowtopickouttherightgene,andthen,withinthegene,mustbecapableofchoosingwhichexonstosplicetogethertocodefortherelevantprotein.

Take,forexample,ourhumanbeta-globin,whichispartofthemoleculehaemoglobin.Wenowknowthathaemoglobincontainsasingleironatomatitscore,surroundedbytwoalphaproteinsubunitsandtwobetaproteinsubunits.Sotheproteinasawholeismadeupoffourdifferentparts–it’saso-calledquaternaryprotein.Nowifwelookatoneofthetwoidenticalbeta-haemoglobinsubunits,thesameproteinsubunitthatismutatedinsicklecelldisease,we find that theDNA that codes for thesecomprises threeexonswith twointerveningintrons.

Itmighthelpatthisstagetoknowhowageneisactivated.

Ifwewere to alight fromour train and take a lookat the actual stretchofDNA thatcodesforbeta-haemoglobin,wewouldfindthat,somewhereclosetothestartofthefirstexon(remember that thedecodingmechanismmovesfromtheleftandmovesalongtheDNA molecule to the right) we find a section of DNA known as the ‘promoter’.Somewheremoredistant–maybeatsomeconsiderabledistance–thereareotherstretchesof DNA that act as ‘up-stream regulatory elements’ – another office or maybe severaloffices full of administrative bureaucrats. The bureaucrats send a signallingwire to thepromotertosay,‘Timetoexpressthegene.’Whetherornotaspecificgeneisexpressedwillvaryfromcelltocell,tissuetotissue,organtoorgan,withinthehumanbody,andsowill the timing of gene expression and the amount of gene expressed. That’s what thebureaucratscontrol.ThepromotertheninstructsthegenetoexpressitsDNA.Inthecaseofthebeta-globinprotein,thethreeexons,togetherwiththetwointerveningintrons,areconvertedtothematchingmessengerRNA,afterwhich,andstillwithinthenucleus,thetwointronsareexcisedand theremaining threeexonsare joinedup together.Onlynowdoes the messenger RNA leave the nucleus and travel to the protein-manufacturingribosomesinthecytoplasm.

The largestknownhumangenecodes foraproteincalled ‘dystrophin’,whichhas79exons separated from one another by 78 introns. Dystrophin is important for normalmusclefunction.Aswithsicklecelldisease,mutationsaffectingthisverylongproteincangiverisetoaninheritedformofdisease.Forexample,inBeckerandDuchennemusculardystrophies, awhole exon is usuallymissing.This damages themembrane surroundingthemusclefibre,resultinginimpairedmusclefunction.

Understandingofthegeneticsofdiseaseslikethesecanhelpmedicalscientiststoworkon a treatment and, perhaps in the not too distant future,work towards a genetic cure.Moreover,understandingofhowexonsand intronsworknowaffordsanexplanationofhowjust20,500genescouldpossiblycodefor80,000to100,000proteins.

Agenewhich,forexample,had14exonsseparatedby13introns,islikelytocodeformore than one protein. All that is necessary is that the regulatory mechanisms, whichdecideonwhichexons tosplice together tomake themessengerRNA,choosedifferentcombinationsofexons.Wenowknowthatthisisexactlywhathappens.Theabilityofasinglegenetocodeformorethanoneproteinisknownas‘alternativesplicing’.Wealsoknowthatthisisubiquitousineukaryoticlife–includingalltheanimals,plants,fungiandsimplerformswhosegenomeiscontainedwithinanucleus.

NowweunderstandwhytheNobelauthoritiesdecidedtoawardRobertsandSharpwiththe Nobel Prize in Physiology or Medicine in 1993. In 2005 a multi-million-poundexpansion to the chemistry department of the University of Sheffield, where I oncestudied,wasnamedafterRichardJ.Roberts.

*

Aswehaveseen, thefirstof themajorenigmasthrownupbythe2001blueprintof thehumangenomehada readysolution.But theother two, thevastvirus-relatedsegments,andtheempty50percent,willtakeagooddealmoreexplaining.Beforewejourneyintothesemoredifficult territories,werequireabasicunderstandingof themechanisms that

arecapableofchangingthegenomesofexistingspecies,andindoingso,ofcreatingnewlifeforms.Thiswillrequireabasicunderstandingofevolutionarybiologytogetherwithsomeveryrecentdiscoverieswithinthisbroad,excitingdiscipline.

nine

HowHeredityChanges

…inadozenyears,TheOriginofSpecieshasworkedascompletea revolution inbiological scienceas thePrincipiadidinastronomy–andithasdoneso,because,inthewordsofHelmholtz,itcontains‘anessentiallynewcreativethought’.

THOMASHENRYHUXLEY

When,in1859,Darwinfirstpublishedhistheoryofevolutioninhisbook,TheOriginofSpecies byMeans of Natural Selection, it provoked a tsunami of shock throughout thecivilisedworld.Althoughhemade little orno reference tohumanevolution in this, hisfirstbook,theimplicationsforhumanevolutionwereimplicit ineverythoughtandline.Giventhattherewasnorealunderstandingofhowheredityworked,histhinkingremainsremarkablyprescienttoday.Inessenceheproposedthatnatureselectsforkeycharacters,or ‘traits’, that enhance the potential for survival in the same way that breeders ofdomestic animals and crops had long selected for traits such as size of kernel, coat ofwool,meatinessofmuscle, resistance todisease,ordrought,andsoon.Thewaynaturedidsowasbrutal,though:itwasthroughattrition.Mostparents,forexampleinanimalsorplants, produced far more than two offspring. Yet by and large the numbers within aspecies stayed roughlyconstant.Darwin realised that theoffspringhad tocompetewithoneanotherforscarceresourcesortoavoidpredators.Thiscreatedfiercecompetitionforsurvival;thosewhohadaslightedgeinthetoothandclawofnatureweremorelikelytosurvive. If thisedgewasdeterminedbyheredity, thesurvivorswouldpass iton to theiroffspring. In time – andDarwinwaswell aware that this wouldmost likely involve agradual and incremental sum of small advantages over very long periods of time – theadvantagedwouldbemorelikelybothtomultiplyandeventuallytogeneratedescendantssufficiently different from the original parental strain as to give rise to a new species.Dilution of the hereditary advantage would be reduced if the emerging species wasgeographicallyisolatedfromtheparentalstrain–forexamplethroughisolationonislands,or through separation bymountains ormajor rivers. In time the new specieswould besufficiently different physically, and reproductively, to breed true within its ownpopulation.

Naturalselectionwasaverysimpleandconvincinghypothesis.Darwinhadobserveddifferences in the beaks of the birds on the different Galapagos islands. Soon othernaturalists–what todaywecallbiologists–wouldobserveandconfirmhis findings inanimals and plants, fungi, protists (what we once called protozoa) and much simplerorganisms,suchasbacteriaandviruses.

While many scientists were intrigued by and largely supportive of Darwin’s theory,some,suchasthedistinguishedSwiss-AmericanJeanLouisRodolpheAgassiz,whohadperformedlandmarkworkonglaciersandextinctfishes,wereadamantlyopposedtoanytheoryofevolutionon religiousgrounds.Darwin’s former friendSirRichardOwen, therenowned naturalist and founder of the Natural History Museum in London, is alsopresentedasopposingevolutionarytheoryonreligiousgrounds,butitwouldappearthathehadhisown theoriesabout itandsimplydisagreedwithDarwin’sconceptofnaturalselection combinedwith gradual change. Darwinwaswell aware that natural selectioncouldonlyworkifthereweremechanismscapableofcreatingchangesintheheredityoflivingorganisms.Toputitanotherway,naturalselectionrequireshereditaryvariationforit towork.Someoftheresistancefromwithinscienceitselfderivedfromtheprevailinglackofunderstandingaboutthenatureofheredity.InthesubsequentopinionofSirJulianHuxley,grandsonofThomasHenryHuxley,whochampionedDarwin inhis lifetime, itwasthislackofunderstandinginparticularthatdoggedconfidenceinDarwiniantheoryassciencemovedtowards theendof thenineteenthcentury. In theopeningchaptersofhisbook,Evolution:TheModernSynthesis,JulianHuxleyputhis fingeron theheartof theproblem: ‘The really important criticisms have fallen upon Natural Selection as anevolutionaryprincipleandcentredroundthenatureofinheritablevariation.’

Itwas hardly a criticismofDarwin that he could not explain howhereditary changemight come about – next to nothingwas known about it in his day.He speculated thathereditaryvariationarose fromakindof ‘blending’of thepedigreesof the twoparents.ThefirsttwochaptersofTheOriginaredevotedtoexplaininghowblendingworked,bothinanimalsanddomesticatedcrops.Butover time,Darwinhimselfbecame lessand lessconvinced that blending was a sufficient explanation. In the words of the leadingAmericanDarwinian,thelateErnstMayr:‘Theoriginofthisvariationpuzzledhimallofhis life.’ Today we know that what Darwin implied by ‘variation’ suggests somemechanismormechanisms thatgiverise tohereditarygenetic,orgenomic,change.TherediscoveryofMendel’slawsofheredityproducedabreakthroughintheunderstandingofhow heredity actually operated: specific characters, or traits, were inherited as discretegeneticunits–whatwenowcall‘genes’.In1900aDutchbotanist,HugodeVries,tookthisanimportantstepfurtherwhenhehadtheinspirationthathereditycouldbealteredifmistakes were made during the copying of these genes. The obvious opportunity wasduringreproduction–hereamistakeincopyingagenewouldgiverisetowhatdeVriescalleda‘mutation’.

In the 1920s and 1930s the reality of mutations was confirmed by the laboratoryexperiments of evolutionary biologists such as Thomas Hunt Morgan, BarbaraMcClintockandHermannJ.Muller.Mutationwasnolongeratheoreticalpossibilitybutafactand,verylikely,acommonenoughfacttobemathematicallypredictable.Anumberofinvestigatingscientiststhroughouttheworldbegantopiecetogetheramathematicallybased synthesis of how natural selection would be enabled through such ‘germ-linemutations’ofgenes.TheseincludedpioneeringgeneticistssuchasRonaldAylmerFisherand John Burdon Sanderson Haldane in Britain, Sewall Wright and TheodosiusDobzhanskyintheUnitedStatesandSergeiSergeevichChetverikovintheSovietUnion.

In time geneticists found that most of the mutations in DNA sequences during theformationofthehumanovaandspermhadlittleornoeffectonthefunctionofproteins,andthusseemedunlikelytocontributetoevolutionortodisease.Thosethatdidgiverisetochangeinproteins,orregulatoryfunction,mostlydidsofortheworse.Theywerethecausesofinheriteddiseases.Butasmallminorityofmutationsalteredtheheredityoftheoffspring inways thatmight potentially improve the chances of survival. For example,thereisgrowingevidencethatasmallnumberofmutationsinageneknownasPrx1mayhavecontributedtotheelongationoftheforelimbskeletonthatenabledtheevolutionofthemembranouswingsofbats.

Fromamedicalperspective,mutationsofDNAcanalsoariseduring thecelldivisionthatisanormalpartofthereplenishmentprocessesinthemanytissuesandorgansduringlife.Theseso-called‘somaticmutations’areimportantinthecausationofvarioustypesofcancers,fromtheleukaemiasandlymphomasofbloodandlymphatictissuestocancersofbreast, skin, kidney and bowel, and so on. The reality is a little more complex. Thegenomesoftheeukaryoticlifeforms–thosewithnucleatedcells,includinganimalsandplants – have mechanisms for correcting these copying errors as they arise, but thesemechanismscansometimesfailorbeoverwhelmed.

Medical geneticists can now list thousands of germ-linemutations that give rise to arange of inherited problems affecting the internal chemistry of the affected offspring.Manyof these ‘errorsofmetabolism’arise fromamutationaffectinga singlegene,butsome can result from mutations affecting clusters of genes, aberrant sections ofchromosomes or the loss or gain of a whole chromosome. In an earlier chapter wewitnessed therecessivemutationaffectingbeta-globin thatcausessicklecelldisease.Atthisstagewemighthopaboardourmagicaltraintovisitthegenomeofanindividualwhohas had themisfortune to inherit a dominantmutation, and take a look in a littlemoredetailathowthismutationhascomeabout.

Eachofour46humanchromosomesis,inourmodel,aseparaterailwayline.Trainscanonly run from start to finish – they cannot switch lines, since each chromosome is aseparate linear structure. On this occasion we choose to travel on Line 4 – humanchromosome 4 .We chug along until we come to a stretch of track that is signposted‘Huntingtin’. Ifwealighthereandexamine theadjacent trackcarefully,weobserve thetypical gene structure we saw in an earlier trip. Here is the section of DNA, with itsnucleotide sleepers, that announces itself as the Huntingtin gene ‘promoter’. Thissequence,whichisusuallyadjacenttothestartofthegene,isthegeneticswitchthatturnsthegeneoffandon.Fromherewestrollfurthereastwards,moving‘sense-wise’alongthetrack,tillwearriveatthefirstexonofthegene.Aswestrollalittlefurtheralongtheexontrack,wecomeacrosssomethingveryodd;weseethatatripletofnucleotidesequences,cytosine-adenine-guanine – CAG – appears to be repeating itself over and over insuccessivesleepers.

‘Goahead,’Isuggest.‘Countthenumberofrepeats.’

Youaresurprised todiscover that thereare45CAGrepeats,oneafteranother, in thefirstexonofthegene,Huntingtin.

‘This mutation is the cause of the illness called Huntington’s disease, which causescerebraldeteriorationduringadultlife.’

‘Youmeanthereshouldbenorepeats?’

‘It’salittlemorecomplex.Curiously,weallhavemanyrepeatsoftheCAGsequenceinthefirstexonof thegene,Huntingtin. It’s theactualnumber thatdetermineswhetherornotweinherit thecondition.Ifwehavebetween6and34repeats,wedonotinherit thedisease. The more repeats above this number the more likely we are to inherit thecondition. Above 40 repeats means disease in nearly every case. And the higher thenumbertheyoungertheonsetofsymptoms.’

‘Sowhatwefindhereisbadnewsforthisunfortunateindividual?’

‘I’mafraid it is.Allhumanshave twoversionsofchromosome4,one inherited fromourmotherandone fromour father. Ifwewere togoandvisit theotherversionof thegeneonthematchingchromosome,we’ddiscoverthatitwasnormal.’

‘Inotherwords,Huntington’sdiseaseis,what…adominantlyinheritedmutation?’

‘That’sright.Italsomeansthatifmedicalsciencecouldfindawayofswitchingoffthisdamaged gene, the remaining normal gene would take over and the condition would,hopefully,becured.’

At first people only thought of mutations as affecting these types of protein-codinggenes. But as geneticists came to understand the importance of genes that coded forregulatory genetic sequences, including genes that coded for proteins that wereintrinsically involved in gene regulation, they realised that a mutation that affected aregulatory sequence, for example a sequence that affected embryological development,couldalsoaffectthephysicalandmentaldevelopmentoftheoffspring.Weshall lookatthisinmoredetail inlaterchapters.AtthispointImerelywishtoexplainthatthesamepatterns ofmutationwill sometimes change the hereditary nature of an individual in abeneficialway–awaythatenhancestheindividual’schancesofsurvival.Andsincethisishereditary,thatbeneficialmutationwillbepasseddowntotheindividual’soffspringandfuture generations. This doesn’t just apply to humans; it applies to all animals, plants,fungi – indeed to every livingorganism.This is integral to theway inwhich evolutionoperates.

For almost a century, evolutionary geneticists have been recording howmutations inprotein-codingand regulatory regionshave contributed to thediversityof lifeonEarth,from the evolution of whales and dolphins from original land-living creatures to theorigins of flight in insects and birds. They have also found some evidence for theevolutionofgenesthatmayhavecontributedtotheexpansioninsize,andcomplexity,ofthehumanbrain.Butmutationsdidn’thavetobeasdramaticasthis.Asmallchangethataffected, say, the duration of effectiveness of a digestive enzyme such as lactase inhumans,iscapableoftellingusagreatdealaboutourownmigratoryhistory.Asweshalldiscover in later chapters, evolutionary genetics appears to be entering a golden age,where the genomes of long-dead ancestors, including supposedly extinct humans, arebeing resurrected and subjected to intensive study. Soon we shall be in a position to

determinewithclinicalaccuracywhypeopleofEuropeanorigins foundaway todigestcow’s and goat’smilk throughout lifewhile those fromAsian ancestry did not.We arealready capable of determining through resurrected genomeswhen and howEuropeansdevelopedblueeyesandfair,orred,hair,inthesamewaythatwecandetermine,throughthe genomic examination of fossil bones, how dark-skinned our ancestors were – orthrough analysis of teeth, how fast they matured during childhood and what diet theyconsumed.

The inspiration, and subsequent study,ofmutationhasprovidedevolutionarybiologywithatreasuretroveofinformationonhowlifeevolvedanddiversifiedonEarth.Butthefact that themutationsoccur randomly– and this randomaccumulationofmutations iseasilymeasured–isonlypartofit.Randommutationonitsownwouldnotbeenoughtocreatebiodiversity.Thekeytounderstandingis thatnaturalselectionisoperatingonthevariationbeingpresentedbytherandommutations.Andtheoperationofnaturalselectionis not random; it chooses those mutations that favour survival, and through survival,reproduction.

Mutation plus selection was soon recognised to be a very important mechanism inevolution.Ithasplayedamajorroleintheevolutionofthehumangenome.Italsohasaseductivemathematicalattraction:sincemutationsarethoughttoariseatafairlyregularrate–givingrisetowhatweshallsubsequentlyencounterastheso-calledmolecularclock– mutation plus selection lent itself to calculus-based mathematical extrapolations thatwere increasingly seen as the major, if not the exclusive, mechanism of evolutionarychange. This came to be viewed as the centralmechanism ofmodernDarwinism, alsocalledneo-Darwinism.Todaymanyschoolandcollegeteachersstillteachthatthisisthemain,ifnottheonly,sourceofthehereditarychange,butwenowknowthatmutationisnottheonlymechanismofcreatinghereditarychange.Onthecontrary,mutationisoneofa number of different naturally occurringmechanisms that are capable of changing theheredityoflivingorganisms.

For close to a century biologists and molecular geneticists have been gatheringinformationonthreeothermechanismsthatalsogeneratethehereditarychangenecessaryfor evolution to take place. These include epigenetic inheritance systems, geneticsymbiosis and hybridisation, which, together with mutation, I have gathered under theconvenientumbrelladesignationof‘genomiccreativity’.IchosemywordscarefullywhenIcoinedthephraseinapaperpublishedintheBiologicalJournaloftheLinneanSocietybecauseIwantedtoemphasisethatthesefourmechanismsarecreativeinthemselves.AndIusedtheword‘genomic’ratherthan‘genetic’becausetheverydefinitionofepigeneticsystemsdefinesthemasnon-genetic.Eachofthethreeothermechanismsisverydifferentfrom mutation, and their genetic and genomic implications are also quite different.Following publication of the same ideas in my book, Virolution, Gordon N. Dutton,Emeritus Professor at Glasgow Caledonian University, suggested I use the easilyremembered acronymMESH for these four distinctmechanisms:mutation, epigenetics,symbiosis and hybridisation. Thank you Professor Dutton, henceforth I shall. As weoriginally saw with mutation, all four mechanisms work hand in glove with Darwin’sconceptofnaturalselection.

ten

TheAdvantageofLivingTogether

Had there not been a lack of communication between my teachers and colleagues at Berkeley … and myquantitative friends at theBacteria andVirus Laboratories, Imight not have foundmyself gropingwith theproblemswhosepossiblesolutionispresentedinthisbook.

LYNNMARGULIS

ThestudyofnaturehasamplyconfirmedDarwin’sinsight–theland,airandoceansarerepletewithexamplesofthestruggleforsurvival.Competitionforresources,theneedforcamouflage,thearmourofprotection,themassingofnumbers,suchasweseeinthegreatherds of herbivores, shoals of fish and themagnificent flocks of birds, are all evolvedstrategies for survival in apredatoryworld.From theseveryobvious adaptations to themicroscopic mutations affecting genes, the evolutionary processes are now seen to beuniversal. In 1976, Richard Dawkins, while based at the University of Oxford,consolidatedtwodecadesofevolutionarystudyinhisiconoclasticbook,TheSelfishGene,which was seen by many scientists as the perfect modern encapsulation of Darwin’soriginalvision.However,while the conceptof competition–which is themaindrivingforceinthevisionofbothDawkinsandDarwin–iscommonplaceinnature,itisnottheonlydrivingfactorinthestruggleforsurvival.

In 1878, at a timewhenDarwinwas still alive, aGerman professor,Anton deBary,drewattentiontothefactthatdifferentlifeformssometimesgainedanadvantagethroughliving together. He called such living interactions ‘symbioses’. It was hardly a newobservation.Herodotus described how the ploverwas known to take leeches out of themouthsofcrocodiles,Aristotleobservedasimilarrelationshipbetweenabivalvemolluscandacrustacean,andCicerowassoimpressedbymanysuchexamplestodrawthemoralthathumansmight learn fromsuchfriendships innature.Honeybeesappear tohaveanintimaterelationshipwithfloweringplants,withtheplantsupplyingthebeeswithnectar,meanwhile the bees assist in the transfer of pollen to other plants, thus enhancing theirsuccessinreproduction.Inthecleanerstationsoftheoceans,predatorssuchassharksandgroupers lineup as if arriving at a taxi rank, to haveparasites anddebris cleaned fromtheirskinsandmouthsbytinyfishorshrimps.Anywhereoutsidethecleanerstationsandthesmallfishandshrimpswouldbeseenasfood.

Inthelate1800sdeBaryandanotherGermannaturalist,AlbertBernhardFrank,putthestudy of symbiosis onto a more firm scientific footing, defining the concept andpioneering the study of the biological and evolutionary implications. It is a commonmistake to think of symbiosis exclusively in terms of mutualism. Let us immediatelyclarify the fact that symbiosis is not aboutMrNiceGuy,who comes along and shakes

handswithMsNiceGirl and everything is hunky-dory from then on.Only one of thepartners needs to benefit for the association to be regarded as a symbiosis. In fact,symbiosis often begins with outright parasitism, which may progress to mutualism.Biologists who study symbiosis today see many examples that would be placedsomewhereinbetweenthetwoextremes.Eveninitsmutualisticform,symbiosisisabouttoughbargainingandhardcompromising,with survivalof thepartnership, and thus thepartners,dependingontheoutcome.

Oneofthefirstsuchlivingassociationstobestudiedbynaturalistswerethelichensthatcoat rocks and stones, like themonumentsofStonehenge.Lichenshadpreviouslybeencategorisedasaformalbranchofthebiologicaltree,withavarietyofdifferentgeneraandspecies.But now theywere shown to be not species at all but intimate partnerships ofalgaeandfungi.

Frankdiscoveredsomethingveryimportantabouttheassociationoffungiandplantsingeneral.Whenfolksgotoagardencentretobuysomeseedlingplantsintheirpots,theyhavelittle ideathatwhat theyassumearerootswhentheyshaketherootballoutof theplasticpotareforthemostpartaballoffungus.Allofthelandplantshavefungalpartnersthatgrow into their roots to fashionan intimate symbiosis,with theplant supplying thefunguswithcarbohydratesforenergyandthefungussupplyingtheplantwithwaterandminerals.Thisarrangementiscalleda‘mycorrhiza’,whichliterallymeansafungalroot.Some woods are underpinned by a vast mass of fungi underground that extends as acontiguouslivingsystemtofeedtheentirewood.

There are a few simple terms we need to grasp. The study of symbiosis is called‘symbiology’andthebiologistswhoworkinthisdisciplinearecalled‘symbiologists’;theinteractingpartnersinasymbiosisarecalled‘symbionts’;andthepartnershipasawholeis called a ‘holobiont’. As we have seen, symbiosis includes the smash-and-grab ofparasitism,whereonlyoneofthepartnersbenefitsattheexpenseofitspartner,aswellasmutualism,inwhichtwoormorepartnerssharethespoils.Todayweknowthatsymbiosesareomnipresentinnature,fromthecoralreefstotheprairiesandfromtherainforeststothe wind-blasted valleys of Antarctica. From its inception, the definition of symbiosisimplied that it was a force in evolution, referred to as ‘symbiogenesis’. Symbioticpartnerships also include different types of partnerships, depending on what is beingshared. The root symbioses of plants involve the sharing of the products of livingchemistry, or ‘metabolism’, of plant and fungus, so these are called ‘metabolicsymbioses’. Other metabolic symbioses include the partnership of alga and fungus inlichens and the gut bacteria that play an important role in human nutrition andimmunology.Meanwhile, thecleanerstationsymbioses involveasharingofbehaviours,sothesearecalled‘behaviouralsymbioses’.

Symbioses,astheybecomeestablishedoverlongperiodsoftime,willinevitablybringabout genetic changes in the partners. Take, for example, some 319 species ofhummingbirds,whicharewidelydistributedthroughoutthewarmerpartsoftheAmericas–theselivealmostentirelyonnectar,whichisprovidedbyflowers.Specialisedjointsinthewingsofhummingbirdsenablethemtobeatsofasttheyarepracticallyinvisible;this

‘adaptation’ enables them to hover with pinpoint accuracy in front of the appropriateflower. In this symbiotic partnership, the Columnia plant has changed the shape of itsflower to suit the elongated and curved bill of the violet sabrewing hummingbird thatpollinatesit;meanwhilethehummingbirdhaschangedthelengthandshapeofitsbilltoexactlyfittheflower.Ifonesitsbackforamomenttothinkaboutthis,birdandplantarenow influencing one another’s evolution to accommodate the symbiosis. To put this inevolutionaryterms,naturalselectionisnowoperating,toasignificantdegree,atthelevelofthepartnership–theholobiont.

The benefit of such a mutualism is clear. Only the violet sabrewing bill fits theColumniaflower:onlytheColumniaflowerislikelytobefertilisedbythedabofpollentransferredfromflowertofloweronthebrowofthesabrewinghummingbird.

A third typeof symbiosis,knownas ‘genetic symbiosis’, ismorepowerful still asanevolutionaryforce.

The most abundant element in the atmosphere is gaseous nitrogen, which must beboundupintomorecomplexchemicalcompoundstobeusefulfortheinternalchemicalprocessesof life.Thechemical fixationofatmosphericnitrogen isanessential step thatmakes thefreeatomicelementavailable toeveryanimalandplant,yet theability tofixnitrogenisimpossibleforallanimalsandplantsworkingbythemselves.Itisonlyfoundinbacteria.Legumes,suchaspeasandclover,formsymbioticunionswithnitrogen-fixingbacteria,knownas rhizobia, that live innoduleswithin their roots.The rhizobiaget thehigh energy they need from their plant host while the host gets nitrogen in a suitableorganicformforitsinternalchemistryfromthebacteria.

But there isanadditionalwrinkle to thenitrogencycle.Most speciesof the rhizobialbacteria that live in soil are not capable of fixing nitrogen. They only become capablewhen a ‘symbiotic island’ comprising a package of six genes is transferred into theirgenomes from a nitrogen-fixing species. This transfer of pre-evolved and ready-to-gogenesfromonespeciestoanotherisaverydifferentmechanismofhereditarychangefromwhatwesawwithmutation.It’sanexampleofwhatiscalleda‘geneticsymbiosis’.

Unlike the accidental nature of mutations, genetic symbiosis adds genes with pre-evolvedpotentialtoadifferentevolutionarylineage.Somebiologistswilldescribethisas‘horizontal gene transfer’, which indeed it is. But this is a generically collective termrather than a scientifically definitive concept.The concept of genetic symbiosis definesand explains exactly how the transferred gene came about, and how themechanism oftransfer operates. Likemutation, this genetic change is hereditary: the offspring of thechanged rhizobial species will inherit the symbiosis island. And again, just as withmutation,thegeneticsymbiosiswillonlybecomeevolutionarilysignificantifandwhenitbecomes incorporated into the evolving species gene pool by natural selection.Geneticsymbiosis, working hand in glove with natural selection, has obvious potential forevolutionarychange.Atitsmostpowerfullevel,whereitinvolvestheunionofentirepre-evolved genomes, genetic symbiosis will create a novel ‘holobiontic genome’, whichbrings together the pre-evolved interactive genetic potential from two, or more, quitedifferentevolutionarylineages.

*

BetweenthreeandtwobillionyearsagotheEarthhadnosignofthegreenlifeofplantswe are familiar with today. It was populated by the first cellular life forms, whichcomprised bacteria and bacteria-like organisms, called archaea. The atmosphere at thistime contained no oxygen. But many of the genetic and biochemical pathways nowcommontolifeevolvedduringthismicrobialstage,soitisn’taltogethersurprisingthatallof life today hasmanygenes, and biochemical pathways, in common.Then, about twobillion years ago, life underwent two enormous changes that were described by theeminent evolutionary biologist JohnMaynard Smith as major transitions. A variety ofocean-bound bacterium, known as the cyanobacteria, evolved the ability to capture theenergyofsunlight–theprocesswecallphotosynthesis.Intimethosecyanobacteria,andavarietyofotherphotosyntheticmicrobes,becamepartoftheevolutionofthekingdomofplants,wherethemicrobeshaveevolvedtotheorganellesinthecellsoftheleavesthatwecallchloroplasts.Asaby-productofphotosynthesisthebacteriaexcretedgaseousoxygeninto the oceanicwater andultimately the atmosphere.Todaymost of the oxygen in theEarth’s atmosphere finds its way through the photosynthesis of plants, algae and thecyanobacteria that still grow with great abundance in just about every terrestrial andaqueous environment. But this proved to be a catastrophe for the sulphur-breathingbacteriaandarchaeathatoriginallyinhabitedthesurfacewatersoftheoceans,forwhomoxygenprovedtobealethalpoison.Todaythedescendantsofthesesulphur-breathersareforced to eke out an existence in placeswhere oxygen cannot get to them, such as theinsidesofanimalintestines,ordeepinanaerobicmudorbetweenthelayersofrockmilesundertheground.

Perhaps two billion years ago another species of bacteriamade the leap to breathingoxygen.Andnowasecondmajorgeneticsymbiosiscameabout,leadingtoallofthelifeformsthatbreatheoxygentoday,includingplants,animals,fungiandavarietyofsingle-celledorganisms.

Howdoweknowabouttheseextraordinarysymbioticeventsfromtheveryfardistantpast?Weknowbecausethechloroplastsinthegreenleavesinplantsstillretainenoughoftheiroriginalmicrobialstructuresandgenomestotellus–andbecausethemitochondriainthecytoplasmofourhumantissuecellsalsoretaintheirbacterialshapesandstructures,and the residuum of their original bacterial genomes. We also know that whereas theevolution of chloroplasts happened again and again, involving different photosyntheticmicrobes, the symbiotic union that led tomitochondria only ever happenedonce.Or atleast only one such union gave rise to the mitochondria that populate the cells of allanimals, plants, fungi and the oxygen-breathing protists that are found throughoutbiodiversity today. My late friend, LynnMargulis, pioneered our understanding of thesymbiotic origins of chloroplasts and mitochondria, through the serial endosymbiosistheory, or ‘SET’,which shepublished in a pioneeringbookon theorigins of nucleatedcells.

Thissymbioticoriginofourhumanmitochondriaisimportanttoourunderstandingofhow the twogenomes,mitochondrial andnuclear, still functionasa ‘holobiontic’union

eventoday.

*

At the time of first symbiotic union, the ancestral bacterium would have probablypossessedroughly1,500to2,000genes.Today,asaresultofnaturalselectionworkingatthelevelofholobionticunion,thegenomeofthemitochondrionhasbeenwhittleddowntoaresiduumof37genes.Atsomestage in thepast,approximately300of theoriginalbacterialgenesweretransferredtothenucleus,wheremanycontinuetoplayapartinthenucleus-mitochondrialgenetic linkage that isnecessary fornormal function.Ourhumanmitochondriapopulatethecytoplasm,thepartofthecelloutsidethenucleus,wheretheyhaveevolvedtosausage-shapedorganellesthatlookexactlyliketheoriginalbacteria.Themitochondria also reproduce themselvesbybacterial-stylebudding independentlyof thereproductionofthenucleus.

This changes the inheritanceof diseases that comeabout throughmutations affectingthemitochondrial genes.Where the nuclear genome is inherited fromboth our parents,andfollowsthetypicalMendelianlawsofinheritance(includingthepatternsofrecessive,dominant and sex-linked inheritance we saw in an earlier chapter), the mitochondrialgenomeisinheritedexclusivelyfromourmothersanditfollowsnon-Mendelianpatternsofinheritance.

Mitochondria fulfil an enormously important cellular function – enabling our livingcells to breathe oxygen. This is further linked to multiple cellular functions, includingenergyproduction,thegenerationoftoxicfreeradicalsthatareby-productsofrespiration,andtheregulationofprogrammedcelldeath,orapoptosis,whichisanecessarypartofthecyclingof cells in tissues andorgans.Since themitochondrial genome ismuch smallerthan the nuclear genome – some 16,500 nucleotide pairs compared to 6.4 billionnucleotide pairs – we might anticipate fewer mutations and thus a low prevalence ofgeneticallyinduceddisease.However,wheremostofournuclearDNAdoesnotcodeforfunctional proteins, so thatmutations are less likely to cause disease, nearly all of ourmitochondrialDNAiscodingandthusmutationsaremuchmorelikelytocausedisease.Moreover, because it comprises bacterial genes, which are more error-prone thanvertebrate genes,mutations inmitochondrial genes are about ten to twenty timesmorecommon than would be expected. This is further complicated by the fact thatmitochondrial disease can also result from mutations affecting those 300 genes thatcrossed over into the nucleus. All of this means that we are particularly intolerant ofmitochondrialmutations,whichareapttocauseseriousdifficultieswiththeoxygenationofourlivingcells.

Mitochondrialdiseasesarecomplexandtendtobehighlyspecifictotheindividual,orfamily,ranginginseverityfrommildtofatal.Itishardlysurprisingthatthecomplexityoftheunderlyinggenetics,coupledwiththevariationindiseasepresentation,canmakethegeneticbasisofsuchdiseaseshard todiagnoseand trace.Roughly1 in7,600birthsareaffected by genetic abnormalities affecting the mitochondria, contributing a significantproportion of inborn errors of metabolism in newborn children. Mutations, leading tosignificant disease, havebeen identified inmore than30of the 37mitochondrial genes

and in more than 30 of the related nuclear genes. The illnesses include ‘Complex Ideficiency’, which accounts for roughly a third of all ‘respiratory chain deficiencies’.Oftenpresentingatbirthor in early childhood, affected individuals suffer aprogressivedegenerative disorder of the brain and nervous system, accompanied by a variety ofsymptomsinorgansandtissuesthatrequirehighenergylevels,suchasbrain,heart,liverandskeletalmuscle.Anothermitochondrialsyndrome,presentinginadultlife,isLeber’shereditary optic neuropathy, which is one of the commonest inherited forms of eyedisease.MostcasesofLeber’ssyndromearecausedbymutationsinmitochondrialgenes.

Thereisgrowingevidencethatmitochondrialdysfunctionplaysasignificantroleinamuch broader spectrum of diseases, and perhaps even the ageing process. Given theadvances ingenetics,wemay in timedevelopeffectivegene-based therapy for someofthese conditions, but any such therapeutic approachwill need to consider the symbioticevolutionary origins ofmitochondria and the complex genetic andmolecular dynamicsthatarisefromsuchaninheritance.

Thereisanothermicrobethatisquintessentiallyadapted,throughthenatureofitslifecycle,toenteringintoholobionticgeneticunionswiththegenomesofitshosts:thisistheratherstrange,andinmyview,ratherextraordinary,microbeweknowasaretrovirus.

eleven

TheVirusesThatArePartofUs

WhenIamaskedwhetherpoliovirusisanon-livingoralivingentity,myanswerisyes.

ECKARDWIMMER

EckardWimmerisadistinguishedGerman-bornvirologistwhohasspenthisprofessionallife working in America. In 2002 he astonished the world when he and his group ofcolleaguesreconstructedthepoliovirusfrommail-ordercomponentstheyreconstructedinthelaboratory.Twentyyearsearlier,Wimmerhadbeenthefirsttosequencethepoliovirusgenome.Eventoday,ashisdefinitionsuggests, it ishead-scratchinglydifficult todefinewhatwemeanbyavirus.Thisdefinitionhasnotbecomeanyeasierwiththepassingoftheyears,adifficultycompoundedbytherecentdiscoveryofgiantviruseswith1,000ormoregenes,makingthemgenomicallymorecomplexthansmallbacteria.Perhapsratherthan attempting to defineviruses amore sensible approach is to examine someof theirbasicproperties.

All viruses are codedbygenomes– just as in all of life, frombacteria tomammals.Most viral genomes are DNA based, but some have genomes based on RNA. In fact,virusesaretheonlyorganismsthatuseanRNAcode.Thismakessomebiologistswonderif RNA viruses might date back to a purported stage in evolution known as the RNAworld,which,ifthistheoryiscorrect,wouldhaveprecededthepresentDNA-basedworld.RNA,unlikeDNA,iscapableofreplicatingwithoutthehelpofproteinenzymes.Thusitwouldhaveentailedasmallerstepintheoriginsoflifefromthepurportedambientsoupof chemicals forRNA-based self-replicators to set the ball rolling.Viruses are obligateparasites; they are invariably born within the cells of their hosts. They can die – likebacteria they canbekilled throughheating and anumberofother toxic agencies.Theyalso go through ‘life cycles’ that involve a stage of reproduction, another basiccharacteristic of living organisms. The next, and perhaps most important, question ispredictable:dovirusesevolvethroughtheestablishedevolutionarymechanisms?

Theanswerisyes–theymostcertainlydo.

Viralgenomesmutatefasterthanthoseofanyotherknownorganism.Thisispartoftheexplanationwhyourimmunesystemfindsitsodifficult tocounteractHIV-1onceithasgot inside our bodies. Within a year or two of infection there are literally billions ofdifferentevolvingstrainsoftheviruswithinasingleinfectedperson.Whilevirusesdonotcontaintheirownepigenetic inheritancesystems, theywillsometimestakeadvantageofhostepigeneticsystemswhentheyinvadethenucleus.Aretheycapableofhybridisation?Again, they are the prime examples – it is theway inwhichnewpandemic flu virusesemergetoprovokemayhemaroundtheworld.Arevirusescapableofsymbioticevolution– in the jargon, genetic symbiogenesis? As I shall soon explain, they are the ultimateexampleofthis.

Whythendosomescientistsinsistthatvirusesdonotbelonginthetreeoflife?AsfarasIcanseethisappearstoderivefromhistoricalreasonsdatingbacktomistakennotionsofhowvirusescameintobeing.

Whenlifewasdefinedinaboutthemiddleofthetwentiethcentury,atatimewhenweknewalot lessaboutviruses,aconsensusofbiologists tooktheviewthat theminimumrequirementwasanenclosingcellmembranecontaining theenzymatic andbiochemicalmeansofconductingitsowninternalchemistry.Tomymindthissuggeststhatthedefinerstook pains to invent a definition that specifically excluded viruses. Why should lifedemandacellmembraneasadefiningboundarywhileexcludingaviralenvelope,whichis theviralequivalentofacellmembrane?Andas to the requirement fora life formtocarry out its own internal chemistry, only a limited number of so-called ‘autotrophicbacteria’ are capable fully of carrying out their own chemistry. All other life forms,includingwehumans,aredependentforsurvivalonahostofotherlivingentitiesforouressentialaminoacids,fattyacidsandvitamins.Othersappeartohaveruledoutvirusesaslifeformsbecausetheyareinevitablyparasites–thisdespitethefactthatsotooaremanydifferenttypesofbacteria.

Another mistaken idea adopted at the time of the cellular definition of life was thenotion that certain viruses, such as bacteriophages and retroviruses, evolved fromwanderingpiecesofthehostgenomethatacquiretransmissiblecharacteristics.Ithinkthatgiventhepresentunderstandingofvirallineages,thisisnolongercredible.Theevidencepoints to bacteriophages and retroviruses evolving out of exceedingly ancient virallineages – albeit these viral lineages, like many others, have evolved in an intimatesymbioticinteractionwiththeirhosts–whatvirologiststerm‘co-evolution’–throughouttheaeons.Atthetimeoftheoriginaldefinitionbiologistshadnoknowledgeofthemake-upofgenomes.Nowthatwedohavethisinformationthereisaverysimplewayinwhichwecanputthisideatobedonceandforall.Ifphagevirusesandretrovirusesweretrulyoffshootsofthehostgenome,theviralgenomewouldlargelyconsistofsimilargenestothe host genome. Instead we find the very opposite – the majority of viral genes areexclusively found inviral lineages.Viruses are incredibly creative evolutionary entities,capable of manufacturing new genes all by themselves. And where there are genuinegeneticcommonalitiesbetweenvirusandhost,theexchangeofgenesisfarheavierinthedirectionfromvirusestotheirhosts.

AIDS is the pandemic of our age. The causative virus, HIV-1, is a retrovirus. Evenamong theviruses,whichhavemany strangeandcuriousmembers, the retroviruses areremarkable. As the ‘retro’ of their name suggests, they contradict the now-outmodeddogmaoftheinexorableprogressionfromgenetoprotein,viamessengerRNA.NotonlydoretroviruseshaveagenomethatisbasedonRNAratherthanDNA,theyalsohavetheirownenzymescapableofconvertingtheviralRNAtoitscomplementarysequenceofDNAbeforetheyinjecttheirconvertedgenomeintothehostcell’snucleus.Thisisalsothekeytounderstandinghowretrovirusesarecapableofchangingtheevolutionaryhistoryofthehosts that they infect.Toput it inevolutionary terms, retrovirusescan invade theirhostgermlinesandtherebyenterintogeneticsymbioseswiththeirhoststhroughthecreation

ofanewholobionticgenome–onethat,inourcase,ismadeupofasymbioticunionofretrovirusandthehumangenome.

HIV-1, themain cause of AIDS, spreads by unprotected sexual intercourse, whetheranal,vaginalororal,when thevirus findsaway through thesurface tissues. Itcanalsoenterthebloodstreamdirectlywhenpeoplesharecontaminatedinjectionequipment,andalsofromamothertoherbabyduringpregnancy,birthorthroughbreastfeeding.Evenatthisepidemicstage,whenthevirusisbehavingasaselfishgeneticparasite,asymbioticpatternofevolutionhasalreadybegun.Animportantinternationalresearchinvestigationhasshownthatthatrateofdiseaseprogressionininfectedpeopleislinkedtosubtypesofahuman gene, known as HLA-B. This is one of the genes that determines immuneresponsesandtissueduringorgantransplants.ThedistributionofHLA-Bsubtypesinthehuman population changes the evolution of the virus: meanwhile the virus, throughlethalityforspecificsubsetsofthesamegenesubtypes,changesthehumangenepool.

Justaswesawwithhummingbirdsandflowers,virusesandhumansarechangingoneanother’s evolution. This is the pattern one would expect in a symbiotic evolutionarysituation.

Itdoesn’timplythatthevirusisnotalsoevolvingselfishly,anymorethanitimpliesthesameforthehumanpopulation.Atoneandthesametime,naturalselectionisoperatingselfishlyinvirusandinhumanbutithasalsobeguntoactatthelevelofthepartnership.Virologists term this pattern of parasitic interaction a ‘co-evolution’. From asymbiologicalperspective,wearewitnessinghowsymbiosesoftenbeginwithparasitismbuttheevolutionarysituationcanprogress,insomecases,tooneofmutualism.

The HIV-1 virus selectively hunts down an immune cell known as a CD+T helperlymphocyte. This cell has a key immunoglobulin type of chemical on its surfacemembrane,knownasCD4,whichallowstheviralsurfaceenvelopetofusewiththecellmembrane.Theviralgenomenowentersthecellnucleuswherethevirus’sownchemicalenzyme, known as ‘reverse transcriptase’, copies the viral RNA genome to its DNAequivalent, and this, with the help of another viral enzyme, known as ‘integrase’,integrates the viral genome into the cell’s nuclear genome. This remarkable virus–hostgenomic fusion is an essential step before the virus can instruct the host genome tomanufacture daughter viruses that will spread to other cells, and repeat the process;meanwhilethevirusspreadswidelythroughthebloodstreamandtissuesoftheinfectedindividual.

Wenote in passing the importance of the retroviral capsular envelope in evading thehostimmunity,thenfindingthetargetcellandfusingwiththecellmembranetoallowthevirustoinvadethehostcell.Aspartofthisprocessofspread,theviruswillagainmakeuseoftheenvelopetoevadethehumanimmunitythatistryingtofightit,atthesametimeinfecting and killing more and more CD4 cells. As the disease progresses, the virusreaches the stagewhere billions of newviruses are created every day,meanwhile thesedaughterprogenyarealsomutating,throughcopyingerrors,atanextraordinaryrate.Itisthisvastproductionandsimultaneousmutationalevolutionoftheviruswithinitsinfectedhost thatmakes it sodifficult for thehuman immunesystemtodefeat theviruswithout

medical treatment.Andduring thisproliferationphase, theviruswill alsopreferentiallyfinditswaytothegonads,theovariesandtesticles,anditwillfinditswaytotheglandsthatmakeseminalfluid,vaginalsecretionsandsaliva,tomaximiseitspotentialforspreadtootherhosts.

InthesamewaythataretrovirusiscapableofinsertingitsgenomeintotheCD4cells,manyretroviruseshavetheastonishingpotentialofinsertingtheirgenomesintothegermlineoftheirinfectedhosts,theovumandthesperm.Weareobservingthishappeningrightnow in a retroviral epidemic that first infected koalas in the eastern side of Australiaroughlyacenturyago.Wewitness the terrifyingeffectivenessof sexual transmissionofthisso-called‘emerginginfection’atfirsthand,withvirologistsconfirmingthatalloftheanimalsinthenorthareinfected,andthewaveoftransmissionpassingsouthwards,where,other thanisolatedislandpopulations,allof thekoalasarelikelytobeinfectedwiththevirus in time. It is causing a horrific wave of mortality, from leukaemia andlymphosarcoma.ButthoughbiologistswereinitiallyworriedthattheretroviralepidemicmightcausetheextinctionoftheAustraliankoala,itisnowunlikelythatthiswillhappen.Already the retrovirus is inserting into thegermcellsof thekoala, so that livingkoalashaveanythingupto40or50virallociintheirchromosomes,whichwillnowbepasseddown as part of the inheritance of future generations. Since this holobiontic genomicunion is taking place within the nuclear genome, unlike that of the mitochondrial, thekoalaretrovirusinsertswillbeinheritedinclassicalMendelianmanner.

Todate,HIV-1hasnotbeenseentoinvadethehumangermline.Somevirologistshadbelieved that this would prove impossible because HIV belongs to a subgroup of theretroviruses, called lentiviruses, which were not known to ‘endogenise’. But recentlylentiviruseswerefoundinthegermlinesofEuropeanrabbitsandMadagascarlemurs,thelatteraprimate.WhetherHIVwilleventuallybecomepartofusremainstobedetermined.Amultitude of other retroviruses have entered the human andpre-humanprimate germlines,tocontributetotheevolutionofthehumangenomeinthisway,sothatroughly9percent of our human genome is nowmade up of retroviral DNA.Retroviruses that haveinvadedthegenomesoftheirmammalianhostsareknownas‘endogenousretroviruses’orERVs, as opposed to free-ranging infectious viruses, which are known as ‘exogenous’retroviruses. Our human endogenous retroviruses are known as human endogenousretroviruses, or ‘HERVs’. HERVs comprise between 30 and 50 families, depending ondefinition, and these families are further subdivided intomore than 200 distinct groupsandsubgroups.Themostrecentoftheselineagestoinvadethepre-humangenome,knownasHERV-Ks,includetensubtypesthatareexclusivetohumans.

Each of these HERV families and sub-families appears to represent an independentgenomiccolonisationevent–andthereforeagenomicinvasionduringahistoricretroviralplague that infected our ancestors. Given what we have seen of AIDS and the koalaretrovirusepidemic, itsuggestsagrimstoryofancestralsurvival throughepidemicafterepidemic.Whentwodifferentsetsofscientistsrecreatedthelikelyoriginalgenomeofourmost recent human retrovirus invader, the human endogenous retrovirusHERV-K, theydiscovered a highly infectious exogenous retrovirus with pathogenic potential in tissue

cultures.It’ssalutarytoreflectthatwearethedescendantsofthesurvivors.Butnowweneedtoconsidertheconsequencesofretrovirusesenteringtheevolvinghumangenome.

Whenaretrovirusinvadesagermlinecellitdoessoasaselfishlydrivenparasite.Thehostgenomewillfightbackagainstthealieninvader.Thisbattlewillcontinue,evenifthedefensiveweaponrymustchange,whentheviralgenomehascolonisedthegermline tocreate ‘viral loci’ scattered throughout the chromosomes. Antibodies are no longereffectiveherewithinthegenomebutothermeasures,aimedatshuttingdowntheviralloci,willcomeintoplay.Onesuchmeasureis‘epigeneticsilencing’(Ishallexplainmoreaboutthis inasubsequentchapter).Butsuchepigeneticmeasuresas‘methylation’of thevirallocus are not a permanent solution to suppressing an infectious pathogenic virus.Permanent silencingwill requiremutations,whether through damage to the viral genesandregulatoryregions,orthroughtheinsertionofanunwantedgeneticsequenceintotheviralgenome.Meanwhile,thecontinuingpresenceofviralgenomewithinthehostgermline, often in many copies distributed throughout the chromosomes, introduces a newpossibilityforsymbioticgeneticinteractionbetweenthetwoverydifferentgenomes.Overthefullnessofevolutionarytime,manysuchopportunitieswillarise.

Weshouldrecallthatwhilevirusandhostareseparateevolutionaryentities,withverydifferentevolutionarypathways,theyarenotunknowntooneanother.Infacttheysharean intensely interactiveparasitichistory.During thishistory thevirushasevolvedmanydifferentstrategies formanipulatinghost immunityandcellularphysiology,strategies inwhich the viral envelope, coded by the viral env gene, has played an important role.Meanwhile the human genome, and in particular its protective immune systems, someinnate and some highly changeable and adaptive, has also evolvedmany strategies forhuntingdownanddisablingthevirus,itsalienproteinsanditsaliengenes.

WithintheHERVloci–whichcomprisewholeviralgenomesembeddedinthehumanchromosome–manyviralgeneshavebeensilencedovermillionsofyearsbymutations.Thisledanearliergenerationofgeneticiststodismissallviralcomponentsas‘junkDNA’,butnowweknow thatmanyviral locihave remained ‘active’, inanumberofdifferentways. Retroviruses have their own regulatory sequences, known as ‘long terminalrepeats’,or ‘LTRs’. In theviral lociembedded in thechromosomes, theseareborderingstretches of DNA enclosing the viral genes. Retroviral LTRs are regulatory dynamoscapable of taking over the bureaucratic control of nearby human genes. They are alsocapableof interactingwithothergenetic sequences, includingepigenetic and regulatorysequences.WealsoknowthatthehugechunksofthegenomeknownasLINEsandSINEsare structurally related toHERVs and, as shownbyProfessorVillarreal, they appear toworkinacomplexcoordinationwiththeHERVcomponent.Betweenthem,theHERVs,LINEs and SINEs account for some 45 per cent of our human DNA. This begs someimportant questions.What role has this vast retroviral legacy played in the holobionticevolutionof thehumangenome?What role is itplaying inourhumanembryology,ourday-to-dayphysiology,includingoursusceptibilitytodiseases?

*

In2000,ayearbeforethedrafthumangenomewasreleased,DrJohnM.McCoyandhiscolleagues in the United States and Dr François Mallet and his colleagues in Francediscoveredthatahumanproteintheycalled‘syncytin’iscodedbytheenvelopegeneofaretroviruslocus,calledERVWE1,whichisembeddedinhumanchromosome7.Wemightrecallthatthisgenecodesfortheenvelopeproteinthatnotonlycoatstheviruswithakindof protectivemembranebut also plays a key role in the ability of the virus to find andpenetrate thehost targetcellmembrane,meanwhileevadingandoutwitting thecomplexwilesofthehumancellularbarriersandourwhitecellandantibodyimmunedefences.Notonlyissyncytincodedbytheviralenvelopegene,orenv, itsexpressioniscontrolledbythevirus’sownpromoterwithintheviralregulatoryLTR.Inotherwords,thevirallocusisfunctioningasaviralgenomicunitwithin theoverallhumangenome.Theviralprotein,syncytin, does not code for an enzyme or structural protein, as many of our humanproteinsfunction.Syncytinchangesthefateofthecellsintheplacentalinterfacebetweenthematernal and foetal circulations, so that a cell called a ‘trophoblast’ is turned into a‘syncytiotrophoblast’. This enables the human placenta to create a fused multi-cellularmembrane,calleda‘syncytium’,thatactsasanextremelyfinefilterbetweenthematernaland foetal circulation, so that nutrients frommother to foetus andwaste products fromfoetus tomotherareobligedtopass throughcellularcytoplasm.Thishelps tocreate themostdeeplyinvadingofallknownmammalianplacentasaswellasbeingthefinestbarrier–microscopicallythinasonlyasinglecelllayercanbe.

Further research confirmed that the original retrovirus, which endogenised into ourgenomeasthelocusERVWE1,invadedtheprimatecelllineroughly30millionyearsago.Since itarrivedbefore thedivergenceof theevolutionary linesof thegreatapes fromacommon primate ancestor, we humans share the ERVWE1 locus, and its placentalfunction, with chimpanzees, gorillas and orangutans. This is why there is no ‘H’ for‘human’inthelocusname.Wereitexclusivetohumans,wewouldaddtheH,sotheviralnamewould now readHERV-WE1.And that tells us that the virus is amember of theHERV-Wgroup.Todaywealsoknowthat thevirusinserteditselfsome650orsotimesinto the genome. But the remaining 649 viral loci, spread over many differentchromosomes,haveallhadtheirenvelopegenesswitchedoffthroughmutationsundertheinfluence of natural selection. This is not merely necessary as a precaution againstunwanted invasive viral emergence, it is also vitally important to avoid a conflict ofenvelope gene expression in such a pivotal role as reproduction. I would go further toinsist that it must be this way because the ERVWE1 locus, and its expressed syncytingene,mustberecognisedas‘self’byourhumanimmunitythroughoutourlifetime.

Withinafewyearsofthediscoveryofsyncytin,twomorevirallociwereidentifiedascontributingtheirexpressedproteinstoplacentalstructureandfunction.ThevirusHERV-FRD was found to code for a protein designated ‘syncytin-2’, which appears to helpprotectthefoetusfrommaternalimmuneattackthroughtheplacentalbarrier.ERV3wasfound tocomplement thecell fusion roleof syncytin,nowredesignated ‘syncytin-1’. Intimeafourthvirus,amemberofthehuman-associatedHERV-Ks,wasfoundtocontributetoplacentalfunction.ItseemslikelythatthereisaprotectiveoverlapinfunctionbetweenthefourretrovirusessinceasmallnumberofthepopulationhaveamutationintheERV3

envgene yet they appear to be protected from sterility by the overlapping cover of theotherviralgenes.

Today we can list at least twelve different viral loci that contribute in one way oranother tohuman reproduction.Westilldon’tknowwhat someof thesevirusesdo,butone of these expresses its gene if a mother has a Caesarean delivery, while anotherexpressesitsgeneduringanaturalbirth.FurtherresearchonthevirusesHERV-FRDandERV3alsoconfirmsthesamepatternofmutationalsuppressionofallpotentiallyrivallociofthesevirusesscatteredthroughoutthehumanchromosomes.Onlytheselectedlocicanberegardedasself.

InMay2012 itwasmypleasure tomakea trip to thehistoricUppsalaUniversity, inSweden, famous for its associationwithCarl Linnaeus,who devised the system of theclassificationoflifestillusedbybiologists.Icamehereattheinvitationofmyfriendandcolleague, Erik Larsson, Professor of Pathology at the University Hospital, who is aninternationalexpertonhumanendogenousretroviruses.IwasalreadyawarethatProfessorLarsson and his colleagues had conducted important pioneering research on the role ofHERVs inhumanembryology, andon their contribution to important aspectsofnormalphysiology,particularlyinrelationtoplacentationandhumanreproduction.Iwroteaboutthisextensivelyinmybook,Virolution.

ProfessorLarsson’sresearchpointedtoamajorroleofHERVsinhumanevolutionaswellasinhumandiseases,suchascancersandtheautoimmunediseases.Togetabetterunderstanding of both these roles, we needed to know what viral genes might becontributingtonormalhumanembryologyandphysiology.Inparticularweneededbettertechniques of searching for viral gene expression in different human cells, tissues andorgans.Until recently scientists had been limited to looking for the expression of viralgenesasmessengerRNA.Thishadgivenusimportantcluesastowhatwashappeningattissue level, butwe needed to develop accurate deep sequencing techniques thatwouldallowus to showviral proteins atwork in the human cells.This has been the focus ofresearchinUppsalaformanyyears.

Duringmyvisit, itwas alsomypleasure tomeetProfessorLarsson’s colleagues, themolecularbiologists,ProfessorFredrikPonténandDrPer-HenrikEdqvistoftheRudbeckLaboratory.Oneofthegreatmysteriesofhumanembryologicaldevelopmentishow,fromtheoriginalpluripotentcellsofthefertilisedovum,allthedifferentcellsthatmakeupthevarioustissuesandorgansarise.Formanyyears,PonténandEdqvisthavebeenworkingwithotherSwedishmolecularbiologistsinexploringthismystery.Inparticulartheyhavescreened the cells of different human tissues and organs to see how the expression ofproteinsmightdifferbetween,say,anervecellandabloodcell,oracellfromtheliverorkidney.Thisenormousundertaking,whichisanaturalfollow-upto theHumanGenomeProject,isknownasthe‘HumanProteomeProject’.Atthetimeofmyvisit,itwasnearingcompletionandithadprovidedfascinatinginsightsintohowtheday-to-daymachineryofthedifferenttissuesandorganswork.Inessencetheyarrivedattworelatedconclusions:each specific tissue cell had a small number of proteins thatwere exclusive to the celltype,onaverageperhapssixorseven,but themajordifferencebetween thecellsof the

varioustissuesandorganswasinthevariationsoftheoverallexpressionprofileofaverywiderangeofproteinsthatwerecommontomost.

Pontén and Edqvist also cooperated with Professor Larsson and his Department ofPathology,inlookingfortheexpressionofHERV-derivedproteins.Todosotheydevisedanewsysteminvolvingextensivetissuescreeningwithapairofantibodiesraisedagainstprotein sequences derived from two different sections of HERV envelope gene. TheUppsala-basedscientistsnowextrapolatedthissystemtostudythreeretrovirallociinthehumangenome,ERVWE1,ERV3andHERV-FRD, lookingforsignificantexpressionoftheir envelope genes in a wide range of human cells, tissues and organs. What theydiscovered was original and astonishing. These virus-derived envelope proteins, pre-evolvedtointeractatadeeplevelwithourhumanphysiology,werebeingexpressedatasignificant level–one that suggestedphysiological function– inmanydifferenthumancells,tissuesandorgansotherthantheplacenta,includingthebrain,theliver,thebowel,skeletalmuscle, the heart, the skin, the adrenal glands, the salivary glands, the insulin-secretingisletsofLangerhansinthepancreas,andthetestis,aswellasinmultinucleatedinflammatorycellsfoundinthebloodaspartofthereactiontoforeigninvaders,suchasbacteriaandinfectiousviruses.

Thiswaspowerfulsupportiveevidenceforvirus–humansymbiosisatgenomiclevel.Ineachcasetheviralenvelopegenewasunderthecontroloftheviralpromotersequences,which were conserved by natural selection within the virus’s own regulatory LTRs. ItseemslikelythatthesepioneeringSwedishcolleagues,andothers,willextendthistypeofstudytoothervirallociwithinthehumangenome.Westillhaveagreatdealtolearnaboutwhatsuchviralproteinsmightbedoinginthesemanydifferenttissuesandorgans,bothintermsofnormalphysiologicalfunctionandintermsofdisease.

So-calledbecause it showsupasastarryshapewithin thesubstanceof thebrain, theastrocyteisasupportcellinvolvedinlocalimmuneresponseswithinthebrainandcentralnervoussystem.TheSwedishscientistsconfirmedthatsyncytin-1,theenvelopeproteinofERVWE1, is normally expressed in modest amounts in these cells. Other scientists inFrance,Italy,GermanyandAmericahavediscoveredthatthesameviralproteinappearstobeoverexpressedinthelocalastrocyteswithinthedisease-affectedpartsofthebrainandcentral nervous system in patients with multiple sclerosis. Italian scientists have alsoshown that during the first acute attack of MS, a virus closely resembling ERVWE1appearsinhighlevelsofintensityintheblood.Inotherlaboratorytests,ithasbeenshownthattheaffectedastrocytessecreteachemicalthatislethaltoatypeofbraincellcalledtheoligodendrocyte,whichmanufacturesmyelin,thesubstancethatcoatsnervecellsliketheinsulationonanelectricalcable.DamagetomyelinisthecentralpathologyofMS.Thissame virus falls to unmeasurable levels in patientswho respondwell to beta-interferontherapy. Could it be that a defective regulatory control of the ERVWE1 viral envelopegeneexpressionintheastrocytesisplayingsomeroleinthepathologyofMS?

While the evidence for a causative role is not yet strong enough tobedefinitive, thepossible association between this virus – now labelled MSRV/HERV-W – and MS isundergoing extensive further testing, so that in time we shall have an answer to this

important question – and perhaps to more of the questions that are beginning toaccumulate about the potential viral contribution to various cancers as well as a widerange of illnesses that are included under the umbrella description of the autoimmunediseases.

*

In2001,whenthefirstdraftofthecompletehumangenomeshowedthatroughly45percentofthehumangenomeappearedtobemadeupofretroviruses,orvirus-likeentities,such as LINEs and SINEs, some biologists dismissed this huge genetic inheritance asjunk,thegraveyardofpastviralinfections.Buttodaywehavebecomeagooddealmorecautious in our interpretations. The 2001 papers inScience andNature had shown thatsome50percentofthegenomewasaccountedforbetweentheprotein-codinggenesandthevariousvirus-relatedsections,butthepapershadalsorevealedthatapproximately50per cent of ourDNA appeared to code for nothing thatwe recognised at this time.Ofcourse, somebiologistsoncemore labelled it junk,butotherswerenowmorecautious.Thisnewmysterywouldbecometheraisond’êtreofadeliberateinvestigativeenterprise,inspiredbytheveryshockofourexposedignorancebeyondthetiny1.5percentfractionof protein-coding genes. This investigation, involving a consortium of research groupsworldwide,wouldbeencouragedandfundedbyapublicresearchprojectlaunchedbytheNationalHumanGenomeResearchInstitute in theUnitedStates inSeptember2003. Itsacronymwasderived from theverynatureof themystery: the ‘EncyclopaediaofDNAcodingelements’–ENCODE.

twelve

GenomicLevelEvolution

Thefactthatselectioncanworksimultaneouslyonbothgeneticandepigeneticvariationcomplicatesmattersevenfurther…modelsthatincorporatetheeffectsofepigeneticvariations…showhow[this]leadstodifferentevolutionarydynamics.

EVAJABLONKAANDMARIONJ.LAMB

Ifwelookatwhathappenswhenafertilisedeggdevelopsintothecomplexwonderofthehumanbaby, logicwould tellus that theprocessofembryologymustbedirectedbyanintegrated and coordinated system of control. The fertilised ovum, or ‘zygote’, is apluripotentcell–acellthatcandevelopintoanyoftheorgansandtissuesthatmakeupthe individual human being.When the zygote first begins to divide, the daughter cellsretain this pluripotency throughout the very early divisions. If cells separate from theembryo at this stage, each cell is still capable of giving rise to a complete healthyindividual.Thisishowidenticaltwins,tripletsorquadrupletsusuallyarise.Butsoonthedevelopingmassofcellsdevelopsintotwodifferententities,anencirclinghollowballofcellsthatwillbecometheplacentaandaninnercellmassthatwillbecomethefoetus.Atthis stage the symbiotic endogenous retroviruses kick in and express their envelopeproteins, which help to create the deeply invasive placenta that, in a quasi-parasiticpattern, burrows into the maternal uterine wall and constructs the fused cell interfacebetweenthematernalandthefoetalcirculations.Whilethisishappening,acomplexarrayofsignalstakesoverthecontroloftheinnercellmass,instructingthecellstodivideandmultiply, but also recognising very early the need for the cell types to change, so thatselectedembryoniccellsbegintochangeintotheforerunnercellsofthedifferenttissuesandorgans.

Herewefacetheenigmathatall thecells inanorganismhavethesameDNA,whichincorporatesthesamesumofgenes.Thereforedifferentorganandtissuecelltypesmustbedeterminedbymechanismsotherthanthesumofallthegenestheycontain.FromthestudiesoftheSwedishscientistsdescribedinthepreviouschapter,wenowknowthatthedifferencebetweenabraincelland,say,akidneycelloracirculatingbloodcell,comesmainly from theprofileof expressionofgeneswithin the cell.Wealso recall that eachtissue-type cell also expresses a limitednumberof genes that appear tobeparticular tothat cell – perhaps about half a dozen for each cell type. This fate of cells, and theorganisation of these cells into the growing complexity of form and function that willmakeupthedifferenttissuesasorgansoftheheadandbodyparts,iscontrolledbywhatgeneticistscalltheepigeneticsystem.

Some readersmightbecomea trifleworriedat thispoint, since there appears tobeageneral notion that epigenetics is immensely complicated. It is even fair to say that theworldofepigeneticsseemedconfusingtoscientistsuntilrecently–butthereasonforthiswas that the definition and scope of epigenetics were undergoing a rapid evolution inthemselves. As our understanding has grown, the basic principles have, thankfully,becomemuch easier to grasp. In particular, our growing understanding of the so-callednon-coding RNAs has clarified things so that not only can we redefine epigenetics insimplertermsbutwecanalsoseehowitprovidesafascinatinganswertotheremainingmysteryofthathugeunknownsectionofourhumanDNA.

Theepigeneticsystemisessentiallyasystemofregulatorycontrolofthefunctioningofthegenome.Itcomprisesanumberofdifferentmechanismsthatactinanintegratedandcoordinatedmannertocontroltheactivationandclosingdownofgenes.Butitsrolealsoextends beyond genes to work in what might be compared to a housekeeping andregulatorycoordinationaffectingtheentiregenome.Theeasiestwaytounderstandthisistoexaminehowthevariousmechanismsoperate.

Wehaveseenhowageneis thegeneticsequencethatcodesforaprotein,orperhapsmorecorrectly,aparcelofdifferentproteins.Ourgenomecontains roughly20,500suchgenes.Wehavealsoseenhowaspecificcelltype,andthusthemake-upofthedifferenttissuesandorgansofthebody,isdeterminedmostlybytheprofileofexpressionofalargenumberofgenes.Theepigeneticsystemdecides thatprofileofgenes,controllingwhichgenesswitchon,whentheydoso,whatquantityofproteintheyexpress,andsoon.Beforewe examine how it does so, I would like to explain something interesting about thisepigeneticsystemofcontrol.

TheDNAthatcodesforgenesandtheotherfunctionalgeneticsequencesofthegenomeis fixed when the parental germ cells unite to form the fertilised cell, or zygote. Thisaspectofyourgenomeremainsexactlythesamethroughoutyourlifeunlesschangedbyamutationoraninvadingvirus.Butyourepigeneticsystem,anditsregulatoryeffects,isnotfixedthroughoutyourlife.Itiscapableofchangingitsregulatorycommands,forexamplebyrespondingtosignalscomingfromyourinternalphysiology,andeventhroughsignalscomingfromyourenvironment.Inplants,forexample,itistheepigeneticsystemthattellsthemthatspringhasarrived.Andinanimals,includinghumans,theepigeneticsystemissimilarlysensitivetoimportantchangesinyourlivingcircumstances,suchastheimpactofdisease,protractedstress,severepainorstarvation.Inotherwords,althoughyourgenesstaythesamethroughoutyourlife, theexpressionofthosegenesinyourvarioustissuesandorganscan,andwill,changebecauseofsignalsarrivingintothesystemsofepigeneticcontrol. The implications go even deeper: your epigenome is capable of learning fromexperienceandchangingtoaccommodatethatexperience.Moreextraordinarystill,thosechanges will sometimes be inherited by future generations of offspring throughmechanismsknownasepigeneticinheritancesystems.

The inheritance of epigenetic changes through epigenetic inheritance systems meansthat epigenetics has evolutionary potential: this is why I included it as one of themechanisms of ‘genomic creativity’. Moreover, the potential for outside influences to

bringaboutepigeneticchangeoffersexcitingpotentialformedicine.Forexample,itmightlead to future medical therapies aimed at changing the expression of disease-causinggenes.

Todaywe recognise fourmain epigenetic control systems, which can be seen in thefigurebelow:

Would you like to join me in a new journey on our metaphorical train? We findourselvesreversingalongthetwintrackoftheDNAofagene,makingourwaybackpastthefirstexon, toarriveat thenearbystretchofDNAthat is the‘promoter’– theregionthat switches the gene on andoff.As before,we hopdown sowe can observewhat ishappeningasoneoftheepigeneticmechanismsswingsintooperation.

Wehearadeepbuzzingsoundnearbyandarethenstartledasasmallcloudloomsintoview,buzzinglikeabee.ThecloudisaproteincalledaDNA-methyltransferase.Weseethat it isbearing tinyclustersofatoms, resemblingchemicalbeads– theseare ‘methyl’chemicalgroups,madeupofacarbonatomattachedbycovalentbondstothreehydrogenatoms. As we watch, the cloud attaches a methyl bead to a nucleotide in one of thesleepers.

‘Goahead–checkwhichnucleotide.’

‘It’sacytosine–aC.’

‘Okay–sowiththemethylchemicaltaggedontothisitisnowamethylatedcytosine.Believeitornot,thissimplechemicalchangeisallthereistooneofthemostpowerfulepigeneticregulatorymechanisms.’

Wefollowtheprogressoftheproteincloudasitmovesalongthepromoter,attachingmoreandmoremethylbeads,alwaystocytosines,untilmostofthesewithinthepromotersequencehavebeenmethylated.

‘That’sit–thepromoterhasbeencloseddown.Sonowthegenecan’tbeswitchedon.’

‘Youmeanit’scloseddownforgood?’

‘Nothingintheepigeneticsystemisquiteasfixedasthat.Toclosethepromoterdownreally hard will require some additional silencing – using a second mechanism ofepigeneticshutdown.ButnowIwantyoutotakealookathowmethylationcanbecomeanepigeneticinheritancesystem.Tounderstandthisyouneedtotakeacloserlookatthenucleotidesadjacenttothecytosines.’

‘Youmeantheotherhalvesofthesleepers?’

‘No–wealreadyknow that theywillbeguanines.Becausecytosinealwaysbinds toguanine.Lookattheadjacentsleepers.’

Ittakesyouaminuteortwo,becauseyoudon’tseethepatternuntilyouhaveexaminedhalfadozenorso.

‘Therealwaysseemtobeguaninesadjacenttothemethylatedcytosines.’

‘That’s right. These cytosine-guanine “couplets” are the key to how themethylationstatusofgenesisinheritedtonewgenerations.Ifwenowclimbbackaboardourtrainwecanactuallywatchithappen.’

Intheblinkofaneyewefindourselvesenteringthegenomeofagerm-formingcellthatisintheprocessofcopyingitsgenomeintoaspermoranovum.Wehopdownagainsowecanwatchwhatishappeningtoanotherpromoterregionasitisintheprocessofbeingcopied.Andherewenoticesomethingveryinterestingtakeplace.

Tobeginwith,IdrawyourattentiontooneoftheC-Gcouplets.Wecanhardlymissthefact that theC ismethylated– it has its smokybead attached.Wewatch as the doublehelix cleaves apart, and then the process of copying begins, with nucleotides beingmatchedwithoneanotherandthenewrailforming.

Wefollowthecopyingfromthe‘sense’strandtothenew‘antisense’strandand,withachuckle of recognition, we observe that wherever there is a C-G couplet on the sensestrand,itcreatesamirrorimagecopyofG-Cinthedaughterstrand.Ibidyouwaitsowecanwitnessanothersurprise.

Withamazingspeed,weobservethearrivalofanotherbuzzingcloud,whichappearstonoticetheunmethylatedcoupletsonthedaughterstrandthatstandoutincontrastwiththemethylatedcoupletsoppositeonthematernalstrand.Withthatsameefficiencyasbefore,it moves along the daughter strand, methylating every complementary couplet on thedaughterline.

‘Themethylationstatushasbeentransferred?’

‘Wehavewitnessedtheoperationofan“epigeneticinheritancesystem”whichisawayinwhichachangeinthemethylationcodecanbeinheritedbyfuturegenerations.What’smore, our train analogy has allowed us to get close enough to witness somethingdefinitive.We have seen how methylation can switch off the expression of a gene. Ifinherited by future generations, this would have evolutionary potential since it wouldchangetheheredityofthosefutureindividualsthroughalteringgene-profileexpression.Inotherwords,methylationstatuscanbringabouthereditarychange.It’saforcewithinmyumbrellaofgenomiccreativity.Yet,aswesee,itcomprisesaddingasimplechemicaltoa

pre-existingnucleotide,cytosine.ThereisnoactualchangeinanyDNAsequence.Everyotherforceofgenomiccreativitythatwehaveseentodate–mutation,geneticsymbiosis,hybridisation – acts through changing DNA sequences, yet this epigenetic mechanismchangeshereditywithoutchanginggenes.Anepigeneticinheritancesystemisagenomicforcebutitisnotageneticmechanism.ThisiswhyIcoinedtheterm“genomic”creativityratherthan“genetic”creativity.’

Methylation is a very important epigenetic mechanism during the formation of theembryo in the mother’s womb. The vitamin folic acid plays an important role in thisprocess ofmethylation during early embryological development. This iswhy a lack offolicacidinthemother’sdietinthoseearlymonthsofpregnancycandamagethefoetusand increase the propensity for developing spina bifida. Extensive defects in themethylationpatternsthroughoutthegenomeisalsoafeatureofmanyformsofcancer,afindingthatisbeingextensivelyinvestigatedinthehopethatitwillprovideenlightenmentandpotentialavenuesoftreatmentinthefuture.

Anothersituationwheremethylationstatusmaybeimportantismorbidobesity,withitstendency to maturity onset diabetes. Some studies have shown that epigenetic factors,particularly changes inmethylation status in key areas of the genome,may be playingsomepartinthis.

‘Canwedonothingtohelpourselves?’

‘Yes,wecan.Unlikethefixityofgenes,epigeneticregulatorysystemsareamenabletochange.Andsomethingassimpleasregularexercisecanchangethingsbacktoahealthierepigeneticcode.’

‘But,holdon!What’shappening?Weappeartobemovingagain.’

‘Time for another trip. I want you to observe another epigenetic mechanism as itactually happens. But this time we are going to restore the double helix in all of itssplendour.’

Wewatchasourtrainmovesawayfromthegenomeintheultramicroscopiclandscape,sufficienttoobservethespectacularbeautyofDNA’snaturaltwist.

‘This timewe need to observe the actual structure of the chromosome– in this casehuman chromosome 6, the chromosome that contains that supremely important MajorHistocompatibilityComplex.And our first surprisewill be to discover, in passing, thatthose early geneticistswhomade life difficult forOswaldAverymay have had a pointwhentheyinsistedthatproteinshadsomethingtodowiththemysteryofthegene.’

*

Wediscovertoourdelightthatourmagicalsteamengineiscapableofhoveringwithintheultramicroscopiclandscapeatsufficientdistanceforustoobservethedoublehelixgrowsmallenoughtoappearasafinethreadinthedistance–farenoughawayforustonoticethingsthatwerenotapparentbefore.

Not only does the incredibly long molecule of DNA spiral within its molecularstructure,thetwintrackthencoilsforasecondtimeinabroadspiralaroundsomestrange

globular structures, that from this distance resemble tennis balls. The tennis balls areproteins,calledhistones,andtheyarepackedtogetherasstructuralunitsofeightballs,inracksoffouronfour.Theseeight-packsarethemselveswoundaroundacentralspineofanotherlinearprotein,acentralspineunlikethephosphatespinesoftheDNAmolecule.This cruder secondary spiral, of the DNA thread winding around the eight-packs ofhistones,extendstheentirelengthofthechromosome,windingawayintothefathomlessdistance.

Youappeartobenonplussed.

‘It is extraordinary toglimpse thegargantuanwonderof this secondarychromosomalstructure–ifmymentalarithmeticisanywherenearaccurate,chromosome6issomethinglike150millionnucleotideslong.’

‘Whereareweheaded?’

‘TothestretchthatcodesfortheMajorHistocompatibilityComplex.’

Nowyouturntomewithanewquestiononyourlips:‘What’sthisnewtipabout?’

‘We’regoingtotakealookatasecondepigeneticsystem,calledthehistonecode.Andlikethemethylationstatus,it’sreallyverysimple.’

Youlookatrifledubious.

‘It’sallaboutthingstheepigeneticistscallhistonetails.’

Theenginechugsclosertoasinglesectionofthechromosome,ataplacewherewecanexamine the structurewhere the eight-packs of tennis balls are inclined to us, side on.These appear to be packaged together like newly harvested onions, bound tight to thecentralstringofthespine.

Thetightpackagingof thehistoneeight-packssuddenlyopensup.WewatchhowthethreadofDNAloosensupastheindividualeight-packsarenowteasedoutintoalooserarrangement.

Iedgetheenginecloser.‘Lookmorecloselyattheeight-packs.’

‘There’ssomethingpokingout…They’resportingtails.’

‘Chemicaltails–yes.’

Sincethehistonesoftheeight-packsareproteins, theyaremadeupoflongstringsofaminoacids.These tails, trailing from thehistones,are sidechainsofaminoacids.Thekey to understanding is that these amino acid tails poke out beyond the broad spiral ofDNA thread that is wrapped around the histones. As we watch, one of those buzzingproteincloudshovesintoview,haulingoneof thosechemicalbeadswesawbefore.Wewatchinsilenceasitattachesthebeadtooneofthedanglingtails.Immediatelythewholearrangementbeginstochangeagain.Theloosenedstructureofthehistonepacksbeginstotightenintotheonionpackagain.

‘It’scomingtogether.’

‘The histone proteins are exceedingly sensitive to the attachment of certain chemicalmoleculestotheirtails.’

‘Likemothstopheromones?’

Nowyouhavemechucklingwithyou.‘Sometimesit’souroldfriend,themethylgroup.But it can be an acetate group, or a phosphate – there’s a range of different simplechemicalgroupsthatcantriggerthechange.Andthechangecanonlybeoneortheother,a local loosening up of the histone packs, or a tightening up. The chemicals attach tospecificaminoacidswithinthehistone.Forexample,itmightbetheaddingofanacetylgrouptotheaminoacidlysine,ortheaddingofaphosphategrouptoserine,ortheaddingofamethylgroupthistimenottocytosineoftheDNAbutlysineonthehistonetail.’

TogetherwewatchhowthetightlywoundspiralofDNAthreadthatiswrappedaroundthetightlypackedhistonesloosensupagain.

‘Soyouknowwhathasjusthappened?’

‘Thegene–orwhateversequenceiscodedbythisstretchoftheDNA–iscloseddownwhenthethreadispackeduptight.’

‘Andreadyfortranslationwhenituncoils–exactly!’

‘Sothehistonecodeswitchesageneonoroff,justlikethemethylationstatus?’

‘It may look very simple, but there is nothing remotely accidental in the acetyl,phosphateormethylgroupscosyingup to the tails. It isunderaverycarefulcontrolbyother elements within the epigenetic control system that would make the secret policeforceofadictatorshiplooklikeamateurs.Andjustlikethemethylationstatus,thehistonecodeisalsoamenable tochangewithin the lifetimeof the individual. It isresponsivetostimulienteringthegenome,throughenvironmentalinfluences.And,likemethylation,ithas the potential to change heredity, and thus bring about evolutionary change,withoutchangingtheDNAofthegeneticcode.’

‘Justhowpowerful,’youask,‘isthishistonecode?’

‘Letmegiveyouasingleexample.Thatproteincloudwesawisactuallyanenzymecalleda“deacetylase”.Whatitdidwasremovetheacetylchemicalgroupfromthetailsofasinglehistonepack.Itsname,inthejargon,isdeacetylaseHDAC11,andwewatcheditswitch off a gene that codes for a protein involved in the body’s immune system.Thatproteindecideswhetheryouor Iwill respond to a certain antigen as self or foreign. Inmedicalterms,thissingle“epigeneticmark”willinfluenceinanimportantwayourfutureimmune tolerance – in other words, how we might respond to a dangerous invadingmicrobeorhow,ifwesufferedanorganfailure,wemightreacttoanorgantransplant.’

I can explain through another example. Identical twins – in the jargon ‘monozygotictwins’–areconceivedasclonesofoneanother.Thustheyareconceived,andliveouttheirlives, with identical genomes. The sum of all of the epigenetic systems in the body isknownasthe‘epigenome’.Identicaltwinsareconceivedfromthesamepluripotentcells,so they begin as embryos with identical epigenomes. We formerly believed that thisimpliedthatidenticaltwinswerealsobornwithidenticalepigenomes,butnowweknow

thatthisisnottrue.Theepigenomeofeveryfoetus,includingthoseofidenticaltwins,hasalready begun to change by the time of birth in response to environmental influencesarrivingintothephysiologyofthefoetusduringdevelopmentinthewomb.Ofcourseitdoesn’tstopthere.AstudyinSpainshowedthat,dependingonthecircumstancesinwhicheachoftwoidenticaltwinsgrowsupandlivesouttheirlives,theycontinuetoaccumulatetheseepigeneticdifferences.

In practice, the epigenetic silencing of genes through methylation will often bereinforcedbyasilencinghistonecodebeingappliedtothesamegene’spromoter–abeltandbracesguaranteethatthegeneremainssilent.

Perhapswe should takeabreather. Iwantyou suitably restedbeforeweconfront thenewlydiscoveredandevenmoreextraordinaryepigeneticstoryofwhatsomegeneticistsformally, and somewhat disparagingly, called DNA’s Cinderella sister – that secondnucleotidemoleculecalledribonucleicacid.Inshort,RNA.

Ofcourse,wehavealreadycomeacrossRNA.Outshonebyitsstellarsistermolecule,DNA,we ratherassumed thatRNAhadhad itsday in someghastlyearlymodelof theEarth, a time before the greening of the planet, when life was still bogged down in achemicalstageof itsevolution,withself-replicatorscompetingwithoneanother for thegrubbychemicalstheyneededinthedirtoftheprimevalplanet.Itwasunderstandable,inretrospect,thatamidthefabulouscatalogueofdiscoveriesderivingfromthediscoveryofDNAandhowitcodedforproteins,scientiststhoughtofgenes,andthehumangenome,moreorlessexclusivelyintermsofDNAasthemastermolecule.

Today,however,werecognise that thiswasablinkeredvision–and itwas thissameblinkered vision that led to half the human genome remaining blank in that pie chartdating back to 2001. The solution to that enigma lay in themore recent discoveries ofextraordinarynewrolesforRNAintheburgeoningnewdisciplineofepigenetics.Thisischangingmuchofwhatweformerlyassumedaboutgenetics,biology,molecularbiologyandmedicine.Thesenewdiscoveriesaresobracingandchallengingthatweareobligedtorethink our views on how the genomeworks. This dilemma is throwing up some veryfundamentalquestions.What,forexample,dowereallymeanbyagene?Ifweadheretotheconceptofthegeneastheunitofhereditythenweshallhavesomeredefiningtodo.Forexample,ThomasGingeras,oneoftheinvestigatingexpertsinvolvedintheENCODEProject,goessofarastoarguethatthefundamentalunitofthegenome–thebasicunitofheredity–shouldnolongerbethegeneatallbuttheRNAtranscriptdecodedfromDNA.

thirteen

TheMasterControllers

WehadmanydiscussionsonDNA,forIhadcometoOxfordwithtwohalfideas,bothofwhichweremorethanhalfwrong.

SYDNEYBRENNER

Thisnewchapterofdiscoverybeganaslongagoas1991withadiscoverybyAmericanbiologistsVictorAmbros,RosalindLeeandRhondaFeinbaumwhentheywerestudyingasingle gene, called lin-14, which regulates development in the worm, C. elegans. WemightrecallthatthistinywormwastheexperimentalsubjectchosenbyCrick’sfriendandcolleague,SydneyBrenner, forhispioneeringexperiments into thegenesandmolecularbiologyofdevelopment.Wemightalsorecall that this tinywormprovedsohelpfulandamenabletoBrennerandhiscolleaguesthatitsubsequentlybecamethetestorganismforthousands of laboratory experiments all over the world. Brenner’s own studies wereextended by the biologists Robert Horvitz, in the United States, and John Sulston, inEngland,wheretheireffortswerecrownedbytheNobelPrizeinPhysiologyorMedicinein 2002. In the press release from the Nobel Institute, the award was made for theirdiscoveriesinthe‘geneticregulationoforgandevelopmentandprogrammedcelldeath’.

Theitalicsaremine,becauseIwanttodrawattentiontowhatthosewordsmightimply.

In an adult human being more than a thousand billion cells are created every daythroughcelldivision,or‘mitosis’.Ineverysuchcelldivisiontheentiregenomeiscopied.Atthesametimeanequalnumberofcellsdiethroughaformofcontrolledsuicide.Thisiswhat is referred to as ‘programmed cell death’, or ‘apoptosis’. It is amazing,whenwethinkabout it, thatdeathaswell as life isprogrammed intoourgenome;andBrenner’swork led to our first understanding of the genetics involved in bringing about death.Specific regulatory genes, and genetic pathways, are involved in this darker side ofprogramming. And yes, RNA – that strange, almost quixotic, sister molecule – theCinderellaofthenucleotidesisters–isinvolvedinthiscuriousregulation.

AvarietyoftinyRNAmolecules,between20and30nucleotideslong,wasdiscoveredbackin1991,butscientistswereunsurewhattheyrepresented.Afewyearslater,theUK-basedbotanistDavidBaulcombe, togetherwithhis colleague,AndrewHamilton, foundthat some small interactive RNA molecules, or siRNAs, were somehow capable ofsilencingmessengerRNAmolecules.IntheUnitedStatestwogeneticists,CraigC.MelloandAndrewZ.Fire,adoptedtheC.elegansmodeltostudythisinfinerdetail.Focusingonthegeneticcontrolofamuscleproteinthatwasimportantintheworm’snormalsinewymovement, theyinjectedthegonadswithsiRNAmoleculesandwatchedhowitaffected

theworm’smovement.Tostartwith,theybrokedownthedouble-strandedsiRNAintoitstwo strands,knownas ‘sense’ and ‘anti-sense’, first testing the sense strand– theRNAthat exactlymatched the original geneticDNAcoding – and then the anti-sense strand.Neitherhadanyeffectontheworm’smovements.Onlywhentheyinjectedboththesenseand anti-sense RNA at the same time did something happen. The worm developed anabnormaltwitch–thesamedysfunctionalmovementthattheysawwhentherelevantgenewasdamagedbyamutation.

This led to the startling realisation that these small RNAmoleculeswere capable oftakingoutspecificmessengerRNAs.Inotherwords,evenafterthetranslationhadalreadytakenplace–withthemessengerRNAcopiedfromthegene,theintronsremovedandtheexons spliced together for the final messenger RNA molecule to move out into thecytoplasmreadytocodefortheprotein–thesetinyRNAmoleculeswouldterminatetheprocess.

InpassingwerealisewhytheSwedishscientiststhoughtitsoimportanttolookforviralproteins notmerely asmessenger RNA transcripts but as the actual expressed proteinswithinthecells.

Thisepigeneticmechanismcametobecalled‘RNAinterference’,or‘RNAi’.Itwasyetanothermechanismofepigenetic control.The implicationswere startling.TheseRNAisrecognisedkeysequencesinthespecificmessengerRNAmolecule,sotheycouldhomeinto inactivate or even to destroy it entirely. In 2006, Fire andMello were awarded theNobelPrizeinPhysiologyorMedicinefortheirdiscovery.

Fromtheearliestdaysofgenetics,scientistshadfocusedonwhatwasperceivedtobedogma – that genes invariably coded for proteins. Then scientists discovered that thisrequiredRNAasthemessengermolecule,mRNA.ItalsorequiredadifferenttypeofRNAforthetransportofaminoacidstotheribosomes,theso-calledtransferRNA,ortRNA,aswellasathirdvarietyofRNAthatwaspartofthebasicribosomestructure,theso-calledribosomal RNA which somehow read off mRNA while translating its coding to theprotein.However,inthoseearlyyearstheseroleswereperceivedassecondary,oratbestintermediary,tothenobleGene-to-Proteincentralaxis–thecentralparadigm.ButnowwehadafourthtypeofRNA,theseterminatorRNAimolecules!Ofcourse,thethreedifferentvarieties of RNA, other thanmessenger RNA,must be codedwithin the nuclear-basedchromosomes by sequences of matching DNA. But these coding zones, the DNAsequences that coded for these non-messenger forms of RNA, could hardly be calledgenes.Theydidnotcodeforproteins,onthecontrary,theRNAmoleculestheycodedforwereendpointsinthemselves.

At this point geneticists were faced with a dilemma. Howwere they to classify thegenetic sequences that coded for these RNA upstarts? And now that RNAi had beendiscovered, a number of other small ‘non-coding’ RNAs were further challenging theparadigm.

Somegeneticiststoyedwiththeideaofan‘RNAgene’,agenethatcodedforanend-pointRNA.ButothershadreservationsabouttheveryideaofRNAgenes.Whatevertheterminologicalinconsistencies,therecouldbenodoubtingthefactthatthehumangenome

coded for a surprising variety of RNA molecules that did not code for proteins, butneverthelesshadimportantrolestoplayinthecontrolandexpressionofgenes.

RNAinhibitionbysmall,non-coding,double-strandedRNAmoleculeshasprovednotonly important in theory but also of practical usefulness to biologists and geneticists,allowingthemtoexaminetheroleofspecificgenesbyobservingwhathappenstocellsorlife formswhen thegene is ‘knockedout’.Thepotential formedical therapy is equallyimportant. Some women bear the frightening burden of inheriting one or other of thebreast- andovarian-cancer-associatedgenemutations,BRCA1andBRCA2,whileotherpatients presentwith the early symptoms ofHuntington’s disease. All it would take toalleviate the suffering and distress of such patientswould be to switch off the relevantmutatedgenes.Sometimeinthefuture,perhapssoonerthanwemightimagine,moleculargeneticistswill findaway todo this.Moreover,RNAisarenot the solecontributionofRNAtotheregulationofgenes.Adifferentgroupofsmallnon-codingRNAs,knownasPiwi-interacting RNAs, or piRNAs, appear to be playing an important role in theepigeneticsilencingofdangerousviralsequencesinthehumangenome.Moreover,thereis another, perhaps evenmore astonishing class of non-codingRNAs that regulates thehumangenome,arelativelynewdiscoverythatexplainsthatmysteriousblackholeinthe2001draftgenome–the50percentofourhumanDNAthatwasleftabafflingblank.

*

Wemammalshaveevolvedsexuallydifferentiatingchromosomes,theXandtheY,sothatfemalesinherittwocopiesoftheX,onefromeachparent,andmalesinheritanXfromthemother and a Y from the father. In addition we inherit 22 non-sex-differentiatingchromosomes,called‘autosomes’fromeachparent,makingupatotalnucleargenomeof46 chromosomes. While the Y chromosome contains an estimated 78 protein-codinggenes, largely concerned with testicular development as well as the male physique,fertilityandspermproduction, theXchromosomecontains roughly2,000genes, fewofwhich have anything to do with sexuality. This chromosomal discordance between thesexesledtoapotentialimbalanceinregulationduringembryologicaldevelopment.Ifthesex-linkedchromosomeswere tobe fully expressedduringembryologicaldevelopment,femaleembryos–andfemalesthroughoutlife–wouldbesubjecttodoublethedoseoftheX-linkedgenes,whilemaleembryos–andmalesthroughoutlife–wouldbesubjecttoasingle dose of those same X-linked genes. This could lead to unwelcome regulatoryclashes.

In1961MaryF.Lyon,aformerpupiloftheepigeneticpioneerConradH.Waddington,realisedthatasolutiontothisdevelopmentalriddlemightbetoswitchoffoneofthetwoX chromosomes in females. Lyon was duly vindicated when geneticists subsequentlyconfirmed‘X-inactivation’infemaleembryosonaboutthesixteenthdayofembryologicaldevelopment.Curiously,theinactivationdoesnotselectfortheXchromosomefromanyparticular parent; it appears to choose randomly between the maternally or paternallyinheritedXchromosomesanditdoesnotswitchoffalloftheinactivatedchromosome,butsomethingmorelike60percentofitsgenes.Theremaining40percentareimportantinprotecting females fromdiseases caused by recessivemutations on theX chromosome.

Thisiswhyfemalesarerarelyaffectedbycolourblindnessorhaemophilia–theywouldneedadoubledoseofthemutatedrecessivegenes–butmalesneedonlyasinglecopyontheirsolitaryXchromosome.

In 1991, some thirty years after Lyon came up with the idea, scientists working atStanford University discovered that a single gene on the inactivated X chromosomeplayedakeypartintheprocessofX-inactivation.TheycalledthegeneXistaftertheroleitencoded,asthe‘Xinactivespecifictranscript’.Theyalsoassumeditmustworkthroughtranslating to a correspondingXist protein. But when they looked for the protein theycouldn’t find it. This was baffling, since they could trace the gene’s expression to therelevantmessengerRNA,whichwassplicedtoremovetheintronsandtheexons,whichwerecobbledtogetherasusual.ButthemRNAfailedtomoveouttotheribosomes,whereonewouldexpecttheproteintobemanufactured.Ithinkitmightbetimelyforustomakeanother exploration on the train, to observe one of the most mind-blowing of recentdiscoveriesaboutourhumangenome.AsweenterthemagicallandscapeIdirectyoutooneoftwosimilartracks,runningparalleltooneanother–thetwoXchromosomes.Wehaveenteredthegenomeofafemalefoetusonthecriticalsixteenthdayofembryologicaldevelopment.

Wewatch cell division taking placewithin the early embryo,with replication of thegenome,thedoubletracksofthetwoXchromosomesunzippingalongtheweakhydrogenbondsoftheinterlockingjigsawsofthesleepers,toliberatethesensefromtheanti-sensestrandsofDNA.Thespeedofcopying is impressive.Ablizzard isapproaching,but thecomposite flakes are not snowbut theRNA-boundnucleotides,G,A,C andU.Aswecontinuetowatch,sectionsofthesensestrandbegintoglowindifferentcolours.Itisallpartofthemagicthatenablesustomakeoutsequencesthatmarkoutgenes,orpromoters,orviralsections,orsectionswecurrentlyknowdiddleysquatabout.Theprocessisverysimilar to what we saw with the coding for a protein, with the stretch of DNA beingcopiedtoitsmatchingstretchofmessengerRNA,butherethecopyingappearstogoonand on, extending far more extensively than the thousand or so nucleotides we wouldexpectforasinglegene.AhugemoleculeofRNAisbeingfashioned,comprisingsome17,000 nucleotides. It appears to be peculiar also in its intrinsic structure, with theequivalent of genetic full stops, or ‘stop codons’, at intervals throughout its length.Wehaveneverseenanythingremotelylikethisstructurebefore.

‘Whatisit?’

‘It’salongnon-codingRNA–theproductofwhatsomegeneticistscallanRNAgene.ThescientificnameforitisXistRNA.’

We watch as the RNA molecule flows like a leaking pipe over the targeted Xchromosome,changing thegene-activatinghistoneepigeneticmarks inaway thathaulstogether the histone packs into a tight non-coding formation and calling inmethylationproteincloudstoswitchoffthecytosine-guaninecouplets.

‘What’sitdoing?’

‘It’sswitchingeverythingoffbutnotthewholechromosome,justtheunwanted60percent.’

Xistwasdulyrecognisedasthefirstofaremarkablenewclassofepigeneticcontrollers– what we now call the ‘long non-coding RNAs’, or lncRNAs. But soon afterwards asecond, very powerful, lncRNA was discovered and it explained another epigeneticmystery.

Geneticists had already observed that the genome can recognise the specific parentaloriginsof thematchingpairsof chromosomes.For example, it could select the specificpaternal, or the maternal, chromosome when allowing certain genes, or even wholeclusters of genes, to be expressed. This epigenetic mechanism, which is known as‘imprinting’,isakeyfactorinthegeneticcausationofdiseasessuchasPrader–WilliandAngelmansyndromes,becauseitselectsadamagedchromosomeaccordingtoaspecificparentoforigineventhoughthechromosomeinheritedfromtheotherparentoforiginisperfectlynormal.Geneticistsdiscoveredthatakeymechanismofimprintingwascausedbyepigeneticsilencingofawholeregionofthenon-chosenchromosomebyanotherlongnon-codingRNA,knownasAir.

Inspired by these discoveries, scientists began to search formore of these long non-coding RNA molecules to discover that they are pervasively transcribed throughoutmammalian genomes. In time, lncRNAs were duly recognised as part of a newlyrecognisedandverypowerfulepigeneticregulatorysystem,givingrisetoanexplosionofnew research.This exciting newventure is still taking place as Iwrite, but alreadyweknowthatourhumangenome,likethatofallplantsandanimals,containsvastnumbersoflong and small non-codingRNAswithinwhich the lncRNAs comprise a class of theirown, ranging in size from 200 to more than 100,000 nucleotides long. And what it isrevealing,intermsofthecodingfortheselncRNAs,isatfirstglancebizarre,butyetalsowonderfullylogical.

There is a second, utterly different, reading from the entirety of the genome. Thisreadingisnotconcernedwithnormalboundariesofgenes,orregulatorysequences.Itcancodefromanystretch,whetherremainingconfinedto,say,anexon,agroupofexons,apromoter region, or a combination of promoter and exons, or the regulatory LTR of avirus, or all of these in a single sequence.And the transcripts are all non-codingRNAmolecules.

Thisistheexplanationfortheunknown50percentofthegenome.

Igazeatyourpuzzledface,awarethatwearestillaboardourmagicalmysterytrain,onourwaybackintothenormalworldofthesepages.

‘The puzzle was an artefact caused by how they actually derived their genomicsequences. The 2001 reading of the draft full genome was based on compiling all themessengerRNAsequencesfoundinthehumancell,throughatechniquecalled“expressedsequence tags”, or ESTs. Themessenger RNA is reversed to its complementary DNA,knownascDNA.Sothe2001piechartwasbasednotontheDNAofthehumangenomebutthesumcodingofallthemessengerRNAthatisexpressedfromthatDNA.’

Youarestillshakingyourhead.

‘Thewholegenome,ormostof, is actually translated twice– in twoutterlydifferentwayswhenitcomestogeneticsequences…’

‘Ah,thewholegenome–it’scopiedtwice.’

‘Exactly.Thatwaswhytheblackholewasroughly50percentofthegenome.Itwasthemissingsecondtranslationintonon-codingRNAs.’

*

Weseenowhowthatolderattitudetothegenome,withitsexcessiveemphasisonprotein-codingDNAgenes,blinkeredourvisiontothebiggerpicture.Thismorecomprehensiveunderstandingisstillbeingfurtherevaluated.

Thenomenclatureofnon-codingRNAsissimpleandpredictable;theyarenamedafterthesequencewithinthegenomethatencodesthem.Soasequencebasedonasingleexon–orintron–iscalledan‘exonic’or‘intronic’lncRNA.Asequencebasedonageneisa‘genic’ lncRNA,andsoon.The lncRNAcanbederivedfromthesensestrandofDNA,from a regulatory region, a promoter sequence, from an entire gene, including all theexons and introns, or even from the intervening sequencesbetweendifferent genes thatincludesupstreamregulatory regions. Itcanalsobecoded inmuch thesamewayalongthe anti-sense DNA strand. Some are coded in both directions, being known as‘bidirectionaltranscripts’.ThereareevenmitochondriallncRNAs,andvirusorLINE-orSINE-associatedRNAs,knowncollectivelyasrepeat-associatedlncRNAs.Theirpurposeisepigeneticcontrolofthegenome,sothat,asthisextraordinaryrepertoirewouldsuggest,lncRNAscontrolagreatvarietyofdifferentgenomicfunctions.

Onesuchfunctionistofocusonwhatareknownasregulatoryproteins–proteinsthatswitchgenesonoroff.The lncRNAwill fix to theDNAthreadatanappropriatepoint,grabholdof the regulatoryproteinanddirect it towhere itneeds tobe to influence theappropriategene.Althoughtheresearchisstillongoing,wealreadyknowthat lncRNAsare involved in the epigenetic, genetic andwholegenomic regulationofmanydifferentandsometimesverycomplicatedbiologicalprocesses.Theyappeartobeimportantduringthe embryonic stage of development,where they play a pivotal role in embryonic stemcells– thepluripotent cells thatmakeup theveryearly embryo.Here the lncRNAsareinvolvedinthedifferentiationofthesestemcellsintothosethatwilldifferentiateintothedifferent tissues and organs. As we have seen with the Prader–Willi and Angelmansyndromes, theyappear toplay important roles in thehereditaryaspectsof someof theinherited disorders of metabolism. They are also thought to play some role in manydifferent cancers affecting breast, bladder, colon, prostate, lung, bone, brain, aswell asmelanomas and leukaemias. In addition it is thought they may contribute to theautoimmune disorders, coronary artery disease, neurological disorders such aspinocerebellarataxia,fragileXsyndrome,Alzheimer’sdisease,and,possibly,theageingprocess.

Wecannowfillintheblankspacetocreateanewpiechartofthegenome:

BreakdownoftheHumanGenome2012

Howtrulyextraordinaryisthecomplexityofthisstructurethatliesattheverycoreofourbeing!Thesedifferentgeneticentitiesarenotparcelledupneatlyindifferentpartsofthegenomeasweseeinthepiechart,theyarealljumbledup–virusandvertebrategene,theDNA that translates to long non-coding RNA, ignoring the supposed function of othercoding stretches and cutting into it or straddling several in one stretch. The motleyassemblagesitscheekbyjowlorpiledontopofoneanotherthroughoutthechromosomes.Andhiddenwithinthisremarkableandmessyreservoiroftheheredityofeachandeveryoneofusisasecretnarrativeofourhumanhistory,fromthemostdistantancestorsfromlongbeforethehumanstageofourexistencerightuptotheimmediatepresent.

ItistothisnewmysteryIwouldnowliketodirectourjourney.

fourteen

OurHistoryPreservedinourDNA

Thisscienceappealstousverydifferentlyfromphysics.Itdirectlyinformsourunderstandingofourselves.Itsmysteriesoncedeemeddangerousandforbidden:itsconsequencespromisetobepractical,personal,urgent.

HORACEFREELANDJUDSON,THEEIGHTHDAYOFCREATION

On13February2014thejournalNaturepublishedanarticlewiththetitle,‘ThegenomeofaLatePleistocenehuman fromaClovisburial site inwesternMontana’.TheClovisculture is a prehistoricAmerican culture named after distinct stone tools found at sitesnearClovis,NewMexico,inthe1920sand1930s.DatingbacktotheendofthelastIceAge, roughly 13,000 to 12,600 years ago, the Clovis people are believed by manyAmericanpalaeontologiststobetheancestorsoftheNativeAmericansofNorthandSouthAmerica. At the time of publication the origins of the Clovis people were still beingdebated, with most anthropologists believing they came from Asia although someproposedanalternativeroutefromsouthwesternEurope,followingthemarginsoftheicesheets across the Atlantic Ocean. The Montana burial site was already of historicimportance.Firstdiscoveredin1968onlandownedbytheAnzickfamilyatthefoothillsoftheRockyMountainsnearWilsall,itcontainedtheskullandotherskeletalremainsofamaleinfant,roughly12–18monthsold,nowknownasAnzick-1.ItwasprizedastheonlyknownClovisburialthatalsoincludedaconsiderableassemblageofstonetoolsandbonetoolfragments.

Thechild’s remainshadalreadybeencarbondated to roughly12,700yearsold– theoldestknownburialtodateinNorthAmerica.This,togetherwiththecharacteristictools,suggestedthathebelongedtotheearliestphaseofClovisimmigration.Sothesequencingofhisgenomemightprovideinvaluableinformationontheethnicandgeographicoriginsof the earliest Native Americans. The sequencing work was undertaken by a team ofDanish evolutionary biologists together with experts based at the National HistoryMuseumandUniversityofCopenhagen.

What thenwere thescientists really lookingfor in thegenomeof thischild,whohaddiedduringthelastgreatIceAge?

The answer is that they hoped to learn something about our human origins andmigrations back in a time when all of humanity was still dependent on hunting andgathering for survival,when theonly toolsandweaponsweremadeofwood,boneandstone – a time when there were no national boundaries, no empires, no cities, noagriculture.

To get a clearer grasp of what these palaeogeneticists were looking for we need tounderstandwhattheymeanbySNPs–anacronymfor‘singlenucleotidepolymorphisms’.Itsoundscomplicatedbut,asweshallsee, it issimplicity itself– ifwehopaboard thatnow-familiarmagical steam train tomake a newexploration along the railway trackofDNA.InthiscasewechoosetosteamalongastretchofDNAinagermcellduringtheformation of the sperm or the ovum, when I suggest we take a closer look at whatsometimeshappenswhenDNAreplicates. Ihardlyneed to remindyou that thesleepersaremadeupofcomplementarynucleotides,Calwaysjoininginthemiddle,throughthatweakcementofthehydrogenbond,withG,AwithT,andviceversa.Now,aswewatchthecycleofreplicationtakeplace,thetwosidesoftherailseparateatthehydrogenbondlinkagesinthesleepersandthelongstrandsdisentangletobegintheprocessofcopying.Atmysuggestion,wefollowthishappeningalongthelowermostrail–theso-called‘anti-sense’strand.Wenowheadeastwards,overthousandsofsleepersbeforeIstoptheengine.Wegetdownoffthetraintotakeacloserlookatasinglesleeper.

‘I should explain that this is a section ofDNA in a so-called non-coding part of thegenome.Soitisnotpartofaprotein-codinggene.’

‘Whatarewelookingfor?’

‘Amistakeinthecopying.’

Justaswhenwelookedformutationsinprotein-codinggenesearlier,youspottheerror.WhereaGintheincompletesleepershouldhaveattracteditscomplementaryCbeforethesleepersrejoinedtoformthecomplete track, therehasbeenamistake.Athymine,orT,has taken the place that should have a cytosine, or C. This is another point mutation.Clearly this one nucleotide will not quite fit the G, so the sleeper is buckled. But insubsequentreplicationcycles,theTmistakewillnowattractamatchingadenine,orA,incopyingtoanewsensestrand.ThischangeintheDNAsequencewillbepassedontothegerm cells, to be inherited by the offspring of the individual, and so on to futuregenerations.Thisiswhat isreferredtoasasinglenucleotidepolymorphism–or‘SNP’,nicknameda‘Snip’.

Since the mutation is in a non-coding sequence, it won’t affect the health of theoffspring.Snipslikethisareignoredbynaturalselection–ortousethejargon,theyare‘selectively’neutral.Thefactthattheyareselectivelyneutralmeansthattheyareinheritedwithout bias or favour throughout all future generations.Over time,more andmore ofthese Snips accumulate within an interbreeding species population, creating genetic‘markers’ in specific places within chromosomes that identify that particular geneticlineagefromthattimeonwards.

There are millions of Snips in every human genome. And they show significantvariationfromoneindividualtoanother,andagreatdealmorevariationbetweendifferenthumanpopulations.ParticularSnipsgatherasidentifiableclustersinaspecificregionofachromosome,where they tend to be inherited together as a group, or ‘haplotype’, evenremainingundisturbedduringtheswappingofbitsofmatchingchromosomesduringthesexualrecombinationthattakesplaceduringtheformationofthegermcells.Ishouldalsoexplain, inpassing, that theoriginaldefinitionofhaplotypereferred toclustersofgenes

that tended to be inherited as a closely linked collection, but this definition had to bemodifiedwhenwediscoveredthatmostofthehumangenomeisnotactuallymadeupofgenes.Ifyouaremale,yourY-chromosomehaplotypeshouldbethesameasyourfather’s,and to the generations ofmales going back through time in your paternal lineage. Thesameruleswouldapply tomitochondrialhaplotypesandmaternal lineages, thoughbothmales and females inherit theirmitochondrial lineages exclusively through thematernalline.

Geneticists also employ another grouping, called a ‘haplogroup’, which some use togroup haplotypes into more distant common groups that share an overall commonancestor. However, I should voice caution here, because some geneticists ignore thisdistinction,using‘haplotype’or‘haplogroup’tomeanmuchthesamething.Forexample,Celtic males, such as the original Irish, Welsh and Basques, share a Y-chromosomehaplogroup, as do males of Germano-Nordic origins. But if we go further back, mostEuropeanmales, or females, coalesce into a common haplogroup of still earlier originswhencomparedwith,say,malesorfemalesofeastAsianorigins.Thushaplotypestendtobeusedformorecloselyrelatedfamilytreesandhaplogroupsformoredistanthistoricandarchaeologicalpopulationgeneticstudies.

Ahaplogroup(orhaplotype)beginswitha‘root’or‘founder’mutation,whichhasbeenlocatedbyacombinationofarchaeologicalandpalaeogeneticstudiestoaspecifichistorichumanpopulation.Thisisthenaddedtobysubsidiaryselectivelyneutralmutationswithinthesame regionof thechromosome,creating recognisablegenetic subgroupsover time.The root or founder mutations are usually given a capital letter and the subsequentmutations,whichcomeabout throughadditionalSnips, aregivennumbersor lowercaseletters.Thusthelineagesappearlikethebranchesofatree–itbeginswithatrunk,thenmajor branches, and then finer and finer branches, representing different subgroupsradiatingfromthefoundergroupoverthousandsortensofthousands,orevenhundredsofthousandsofyears.

One such ancient haplogroup, found exclusively inmitochondrialDNA, is called the‘D’rootor ‘clade’.Thisoriginatedasa founderSnip inapopulation living innortheastAsia,includingpresent-daySiberia,roughly48,000yearsago.OvertimeadditionalSnipsarose in themitochondrialDNAof thedescendantsof theDpopulation, leading to fourdivergent clades or branches, calledD1 toD4, and additionalmutationswithin the stillmigratingcladesgaverisetofurthersub-branchingovertime.Eachnewbranch,orsub-branch,wouldcorrespondtoageographiclocationandtimingsofpopulationmovement,which can be cross-referenced to archaeological findings, such as carbon 14 dating, sopopulationgeneticistscanplot thehistoricmovementsand interactionsofpeoplewithinthesecladesalloverAsia,EuropeandinduecoursetoNorthandSouthAmerica.

Returningtothechild,Anzick-1,werecallthattheskeletonwascarbondatedto13,000to 12,600 years ago, which places him to very early in the human colonisation of theAmericas.HismitochondrialhaplogroupwasfoundtobeD4h3a,whichisoneoftherarelineagesspecifictoNativeAmericans.GiventhedateandthehaplogrouptheresearchersconcludedthatAnzick-1belongedtoanethnicgroupthatmustbeclosetothefounderof

theD4h3asub-lineageandthushispeoplewerethoughttobedirectlyancestralto80percentofallNativeAmericanpeoples,andclosecousinstotheremaining20percent.Thestudy ofAnzick-1’s genome also turned up some distant commonalitieswith Europeanhaplotypes.

In the same journal, a paper by the same group of genetic and archaeologicalinvestigatorsdescribedayoungboy’s remains,datingback24,000years, fromanupperPalaeolithicburial site inSiberia.Theoldestburialofanymodernhumandiscovered todate, study of his haplotype revealed that he belonged to an even older mitochondrialhaplogroupthanAnzick-1–abasallineageofhaplogroupR.Todaythislineageisfoundinpeople living inwesternEurasia,southAsiaand theAltai regionofsouthernSiberia.SisterlineagesofhaplogroupRformahaplogroupQ,whichisthecommonesthaplogroupinNativeAmericans, and in Eurasia theQ haplogroup lineages closest to those of theNativeAmericansarealsofoundintheAltairegionofsouthernSiberia.IntheopinionofDanishpalaeontologistEskeWillerslev,wholedthesequencingofbothsetsofremains,‘AtsomepointinthepastabranchofeastAsiansandabranchofwesternEurasiansmeteach other and they widely interbred.’ Their descendants headed east, across the landbridgebetweenAsiaandNorthAmerica,discoveringtwohugeandbounteouscontinentsthathadneverbeenpopulatedbyhumansbefore.TheygaverisetothemajorityofNativeAmericansweseetoday,includingAnzick-1.WhilenoteverybodyagreeswithWillerslev,thecombinationofthetwoinfantboydiscoverieswouldexplainhowNativeAmericansshare14to38percentoftheirgenomeswithwesternEurasians.

*

Snips,haplotypesandhaplogroupsarenotexclusivetoournucleargenome.IreferredtomitochondrialDNAinboththesegeneticexplorationsandnowwecanhopbackaboardourtrainforanothertripintothatmysteriousultramicroscopicworldtoprobeyetanothermystery.Butourdestinationonthistripisnolongerthelandscapeofthenucleargenome;thistimeweareheadingintotheterritorythatliesoutsidethenuclearmembrane,intotheequally intriguing landscape of the cytoplasm, moving carefully through an incrediblycongested and frenetically busy space which might be compared to an industriallandscape,inwhichfreshproteinsarebeingmanufactured,ageingproteinsbeingbrokendown for recycling, andwhere hugemachines, resembling free-floating sausage-shapedjuggernauts,areextractingenergyfromgaseousoxygenandpackagingitinstorableform,soitcanbeusedineverylivingcell.Theseareourvitallyimportanthumanmitochondria.

Aswewatch,amitochondriondevelopsaconstrictionaboutitscentre,andbeforeourstartled gazes it buds, the parentmitochondrion cleaving itself into two clones.As oursteam engine carries us deeper into the ultramicroscopic world of the genes, we findourselvespassingthroughtheouterwallofoneofthesausage-shapedjuggernauts,which,aswemagicallycontractinsize,hasnowgrowncomparativelygigantic,fullytothesizeof a city. Within its cavernous spaces we come upon another railway track, with itsgleamingrailsofdeoxyribosesugarandstiffeningphosphates,andthefamiliarsleepers,withtheir interlockingnucleotidebases,headingawayintotheblurrydistance.Wehave

enteredtheworldofthemitochondrialgenome,withitsverydifferentevolutionaryoriginfromthatofthenuclearchromosomes,anenigmawithinthegreatermystery.

‘Iknowyoutalkedaboutmitochondria–aboutwheretheycamefrom…’

‘A genetic union between what was once a free-living parasitic bacterium and thesingle-celledforerunnerofallcomplexlifeonEarth.Themitochondriastillretainquitealot of their bacterial origins. They retain enough of their genome to reproduce bythemselves,whichiswhat thatmitochondriwasdoing.Whileeachofusinheritshalfofour nuclear genome from our father, we inherit more than that from our mother. Inadditiontohalfhernucleargenome,wealsoinheritthephysicalstructureofacell–theovum–whichincludesthemitochondria.’

‘Whichiswhyweall,malesandfemales,getourmitochondrialgeneticsonlyfromourmothers?’

‘Yes. And that explains why our mitochondrial inheritance doesn’t follow theMendelian laws of nuclear inheritance, such as recessive and dominant behaviour ofgenes.Themitochondriaalsoreproducemuchmorefrequentlythanthenucleargenome,andbecausetheyarebacterialgenes,andthuslessabletocorrectmistakes,theyareevenmoreliabletomutation.’

‘So–that’showyougetmitochondrialSnips?Haplotypes–andhaplogroups?’

‘You’vegotit!Andthesewillbeexclusivelyinheritedthroughourmotherssotheywillruntruetothematernalline,rightbacktotheyeardot.’

‘So there was a woman, back in Elizabethan times, who would have had the samemitochondrialDNAasmymother?’

‘Thesameasyou–otherthanwhateverSnipshaveaccumulatedinthemeantime.Andyoucangomuchfurtherback than that.Whynot toRomantimes,or thebeginningsofagriculture in theFertileCrescent? In fact, youcouldgomuch furtherback still, to theovariesofthematernallinetotheveryoriginsofHomosapiens.’

Aswe tootle ourwhistle and steamalong this new railwayofmitochondrialDNA, Iwould like to explain a littlemore about thewayourhumanhistory iswritten intoourgenome–or,ifyoulike,ourtwosymbioticallylinkedgenomes.Oneofthekeythingstograspisthatwehave,inessence,threedifferentpartsofourholobionticgenomethatarelibrariesofthreedifferentgenetichistories.Oneisthemitochondrialgenome,whichtellsusthestoryofthematernalgeneticline–thematrilineage.TheYchromosome,whichispartofthenucleargenome,tellsusthestoryofthepaternalgeneticline–thepatrilineage.And the remainingnuclear lineage,which is by far the bulk of our genetic inheritance,tellsusfarmoreaboutourgenetichistoryasaspecies.

All the while we’ve been rattling along this mitochondrial railway, we have beenglimpsing subtle differences in how this bacterial genome works. For example, themitochondrial genome is much smaller than that of a single chromosome within thenucleus.Thetotalmitochondrialgenomeconsistsof16,600nucleotidepairs–sleepers–where thenucleargenome,evenin thehalvedgenomeof thegermcells,consistsof3.2

billion.Thereisnoshake-upofthemitochondrialchromosomes,aswefindduringsexualreproduction involving the nuclear genome. In fact, the mitochondrial DNA isn’tcomposed of linear chromosomes at all. Themitochondrial railway consists of a singlecirculartrack,aswewouldfindinabacterialgenome,sothat,ifwetakealengthyjourneyalongthemitochondrialrailwaytrack,itwilleventuallybringusrightbacktowherewebegan.

‘Thissymbiosis–theevent,asyoucallit,thatgaverisetomitochondria–yousaiditonlyeverhappenedonce?’

‘We know this for certain because all the mitochondria, from every animal, plant,fungus and the oxygen-breathing less complex organisms, are clearly derived from asingleancestor.’

‘Youcandeterminethisfromthemitochondrialgenes?’

‘Yes.’

‘Whythen,ifthishappenedsoverylongago,havethetwogenomes,themitochondrialandnuclear,notjoinedup?Wouldn’tthathavemadesense?’

‘You’reright.Itwouldhavemadesense.Infact,mostofthestructuralproteinsinwhatwenowcall themitochondrialorganellesarecodedbynuclear-basedgenes.Webelievethatatleast300ofthenucleargeneswereformerlymitochondrialgenesthattransferred.’

‘Butsomedidn’t?’

‘Thosegenesthatstayedwithinthemitochondriaareall,ornearlyall,involvedintherespirationofoxygen.Oxygenisanextremelytoxicelement.It’spossiblethatithadtobehandled within separately walled-off organelles to prevent its toxicity affecting theremainderofthelivingcell.’

‘Butwasn’toxygenalwaysaround,aspartoftheatmosphere?’

‘No.Atmosphericoxygenisproducedbyplantsandcyanobacteria.Itisaby-productoftheir internal chemistry. We might recall that the early arrival of oxygen into Earth’satmosphere proved calamitous tomany of the oceanic and shore-dwelling life forms atthat time. Only thosewho could breathe oxygen could survive its toxic presence. Andevenwithinthecellsofthosewhoinheritedthemitochondrialabilitytobreatheoxygen,ithadtobecontainedawayfromthedelicategenomicmachinerywithinthenucleus.Ithadto be locked away inside the original invading cells, the former bacteria that had nowevolvedintothemitochondrialorganellesthatwerealreadyresistanttothetoxiceffectsoftheoxygen.’

‘Didn’tweseesomethingverysimilarwiththeviruseswithinthechromosomes?’

‘Wedid!Thosevirusesthatwereexpressingtheirgenesasproteinsappearedtoretaintheiroriginalgenomicstructure,includingthecontrollingpromoters.’

But to return to the use of mitochondrial genetics in palaeontology, the uniquelymaternallineageofmitochondriahasinevitablyprovidedapowerfultoolforevolutionarygeneticistsinexploringpopulationgeneticsandthecomplexweaveofhumanmovement

throughout history. Thus we are not surprised to discover that the D4h3a haplotypecomponent that linkedtheClovischild tohisSiberianancestorscamefromthestudyofAnzick-1’smitochondrialDNA.

Asmentioned above, another equally powerful tool for population geneticists comesfromtheYchromosomewithinthenucleargenomeofmales.LikethemitochondrialDNAanditsexclusivelinktothematernalgeneticline,theYchromosomeisexclusivelypasseddown from fathers to sons. Since it has no matching chromosome to recombine withduring sexual recombination, it doesn’t undergo the jumbling of bits from onechromosometoanother.ThisprovidesaveryusefulmeansoffollowingthepatternofkeymutationalhaplotypesindifferentY-chromosomepopulationsovertime.Forexample,inthecaseoftheClovisinfant,Anzick-1,the‘foundingQhaplotype’onhisYchromosomewasfoundtobelongtosubgroupL54*,whichthegeneticistspredictedhadseparatedoutfrom another founding haplotype subgroup, M3, dating to approximately 16,900 yearsago. This confirmed thatAnzick-1 belonged to an ethnic population grouping thatwascloselyrelatedtothefirsthumanstoarriveintotheAmericas.

*

Justhowaccurateisthisexplorationofhaplotypesandhaplogroups?KingRichardIIIofEnglandwasmadeinfamousbytheShakespeareplaythatbearshisname.ThelastofthePlantagenet dynasty, Richard was killed in the Battle of Bosworth Field on 22August1485, during the bloody War of the Roses. Shakespeare’s play portrays Richard as ahunchbackedvillain,whomurderedhisbrotherandtwoyoungnephewsbeforeattemptingto marry his niece. But his supporters, including Philippa Langley of the Richard IIISociety, defended his reputation, claiming that Shakespeare had defamed Richard insupportoftheTudormonarchswhohadsupplantedhimandwhoreignedatthetimewhenShakespearewrotehisplay.

Inanexquisitestoryofdetectivesleuthing,LangleytracedthehistoricalrecordstofindthatRichard’sremainshadbeenburied,withoutcoffinordignity,inanAugustinianFriaryin the city of Leicester.Having obtained a small amount of funding she persuaded thearchaeologistsof theUniversityofLeicester toconductadig inwhatwasapresent-daycarparkwhichwasthoughttooverlietheformerFriaryhighaltar.TheretheydiscoveredahumanskeletonwhichfittedthehistoricaldescriptionsofRichard.Itwasthatofamaninhis thirtieswith a severe curvature in his thoracic spine, known as a scoliosis – fittingShakespeare’s description of Richard’s hunched back. It also showed signs ofmultiplewounds, indicating that theman had died in battle. Radiocarbon from two independentsourcesdatedthebonestobetween1430–1460and1412–1449.ThisseemedtooearlyforRichard,butmassspectrometrycarriedouton thebonessuggested that theirownerhadeaten a good deal of seafood, which can skew radiocarbon dating. A corrected dateworkedout at between1475and1530,whichwould fit nicelywith thehistoricdateofRichard’sdeath.However,uncertaintiesremainedand,evenwiththelocation,thephysicalanatomy and the radiocarbon dating, controversy still reigned as to whether Richard’sremainshadbeenfound.

A new line of genealogical research managed to identify a woman, Joy Ibsen (néeBrown),whowasadirectmatrilinealdescendantfromRichard’smother.Ingeneticterms,Mrs Ibsenshouldbecarrying thesamemitochondrialhaplotypeasRichardhimself,butshe had emigrated to Canada after the Second World War and died there in 2008.Fortunatelyshehadgivenbirthtoason,Michael,whowaspreparedtogiveasampleofhis DNA for testing. The controversy was resolved whenMichael Ibsen was found tosharetheraremitochondrialhaplotypeJ1c2cwiththeexhumedskeleton.TherecouldnolongerbeanyreasonabledoubtthatthesewerethebonesofthelastPlantagenetking.

As this genetic exploration of our human inheritance continues, we have becomeincreasingly awareof howaccuratelyour genome reflects hugely importantmovementsandevolutionaryeventsinourhumanancestryandhistory.Ifsomeresearchersaretobebelieved,wemayevenhavediscoveredthegeneticAdamandEve.

fifteen

OurMoreDistantAncestors

Ourobsessionwith fossilshasdistractedus fromamuch richer sourceof evolutionary information:geneticdata…

LUIGILUCACAVALLI-SFORZA

Weareallmorecloselyrelatedtooneanotherthanwemightimagine,asalittlethoughtexperimentwillquicklydemonstrate.Ifweweretoconsider,say,fournewgenerationsofourancestorspercentury,wecanreadilyconstructabranchingtreewiththenumbersofancestors doubling with each generation as we travel backwards. Four grandparentsdescended from eight great-grandparents, who in turn descended from 16 great-greatgrandparents, and so on. Two centuries ago, or eight generations, we discover that wedescended from some256multi-great ancestors living in that single generation. In fourcenturiesthenumberrisesto65,536.Ineightcenturiesthenumberrisesto4,294,967.296,–whichisvastlymorethantheentirepopulationoftheworldatthattime–probablymorepeoplethanhadeverlivedonEarthuptothen.Weneedhardlygofurtherbacktorealisethat there is something seriously amiss with this line of thinking. Since we could notpossiblyhavesomanyancestorstherehastobeanotherexplanation.

The answer is simple: we all have a great many common ancestors. This can beexplored at genetic level by constructing haplotype trees, and it can be extended evenwider geographically, and deeper into the past, if we construct haplogroup trees. Thecloserpeoplearetooneanother, themorehaplotypesandhaplogroupstheywillhaveincommon. And each distinguishing haplogroup is a marker – a genetic signpost – to asingle ancestor in a specific place and a specific time who was the first to inherit therelevantSnip.Fromthishaplogroupmarkerspecificpopulationgroupscanbeidentifiedandtheirsubsequentmovementsandmigrationsplotted.

TheItaliangeneticistLuigiCavalli-Sforza,whowasaprofessoratStanfordUniversity,devoted his life to gathering this type of genetic information on different humanpopulations. In his bookGenes, Peoples and Languages, Cavalli-Sforza dismantled theidea of different races, arguing that the differences we saw between Africans, Asians,EuropeansandAustralasians,weresuperficialevolutionaryadaptationstolocalconditionssuchasclimate.Geneticstudiesofdifferentpeoplesthroughouttheworldhaveconfirmedthat we are all part of a single species, in which our commonalities overwhelm ourdifferences.Archaeological studies of fossilised bones, commonalities of tools, and thepatterns of habitation and culture all point to the likelihood that our human ancestorsoriginatedinAfrica,mostlikelysub-SaharanEastAfrica.Thesetraditionalarchaeological

disciplinesarenowreaffirmedandaugmentedbygeneticstudies thatallowus togathermuchdeeperperspectivesonourhumanhistorythanwaspossiblebefore.

Wehavealreadyglimpsedhowspecificclustersofmutations,knownasSnips, in themitochondrial genome and on the Y chromosome, enable geneticists to trace genetichaplotypeandhaplogrouplineages,andthuscharthumanpopulationmigrations,backintoprehistorictimes.Similarhaplotypesandhaplogrouplineagescanbefoundinthe22pairsofhumanchromosomesunalliedtosexualdifferences,theso-called‘autosomes’,enablinga third line of genetic tracing of human lineages and population movements. ThedistributionofspecificendogenousretrovirusescanbetreatedinthesamewaytolocatetheAfricanoriginsofHomosapiensandthesubsequentglobalmigrationsofourspecies,whichatthisearlystagearecalled‘earlymodernhumans’.Thegenomicvirusesalsohavearoletoplayinsuchgeneticdeterminations.Forexample,thedistributionoftwohuman-specificendogenousretroviruses,HERV-K113andHERV-K115,areaddingtothehistory.

UnlikemostoftheotherHERVs,thesetwoappeartohaveenteredthegenomeaftertheprimarymigrationofearlymodernhumansoutofAfrica.Thus,whilethegreatmajorityofHERVsarecommontoallofus,thesetwoarefoundinahighpercentageofpresent-day people who hail from East Africa, Arabia and further east into Asia, but in lowpercentages,ornotatall,inpeoplehailingfromEurope.Tosomegeneticiststhissuggeststhat there may have been more than one migration of modern humans out of Africa,perhaps with expansions and retreats that may have been precipitated by significantenvironmentalorclimatechange.

Aswehaveseen,mitochondrialgeneticsoffersaseriesofmutationaltagsthatmakeitpossibletofollowsomeofthesecomplexpopulationmovements.Withouttheinfluenceofevolutionarychange,everydaughterwill inheritexactlythesamemitochondrialgenomefrom her mother, again and again, throughout all of history. If this were the case, mymitochondrialgenome,inheritedfrommymother,wouldbeidenticaltothatofacommonancestor in Africa, say, 200,000 years ago. But we have seen how the mitochondrialgenomehasbeenalteredbycopyingmistakes,or‘mutations’,duringthebuddingstyleofreproduction that mitochondria undergo. Sometimes these copying mistakes causeimpairmentoffunctionofthemitochondrialgenome,whichwouldhaveverylikelygivenrisetodisease.Butsuchpathologicalmutationswouldnotbecomeestablishedaslineagemarkersbecausetheresultingdiseasewouldresult inreducedreproductivefitness.Onlymutations that had no significant effect on reproductive fitness would have becomeincorporatedaslineagemarkers.Thesehavebecomepartofhaplotypesandhaplogroups,which, having no effect on survival, are ignored by natural selection, so they surviveunchangedovervasttimeperiods.

Snips like this crop up at reasonably predictable intervals – a feature that enablesgeneticists to compare the numbers ofmutations in a given stretch of the genome to a‘molecularclock’.Wehaveseenhowkeyclustersofmutations incertainregionsof themitochondrial genome – haplotypes and haplogroups – can be linked to founderindividuals, in place and time, then spread into thedescendant population, allowing themigrationsandmovementsofthispopulationtobeplottedingeographyandtime.Looked

atfromadifferentangle,differencesbetweenhaplogroupsaremarkersofdifferenthistoricpopulations.Andwherewefind,albeitrarely,asinglehaplogroupthatiscommontoverymanywidelydispersedpopulations, this isseenasanimportantmarker linkingall thesepopulationstoacommonfounderancestor.

WhatthenifweweretodiscoveramitochondrialhaplogroupthatiscommontoeverymanandwomanonEarth today?Would thisnotpoint toawomanwhowasacommonancestorofallofus–ageneticEve?

Inthe1980sthiswaswhatagroupofgeneticists,ledbyAllanWilson,oftheUniversityofCalifornia,Berkeley, had inmindwhen,with thehelpof his doctoral studentsMarkStoneking and Rebecca L. Cann, he conducted an examination of mitochondrial DNAfrom 147Americans coming from awide variety of racial and ethnic groupings. Theywerelookingforevidenceofsharedanddivergenthaplogroupsthatwouldenablethemtoconstructahereditarytreeforallofhumanity.Adecadeearlier,Wilsonhadbeenjoinedbyanotherpioneeringgeneticist,WesleyM.Brown,whohaddevelopednewtechniquesforscreeningmitochondrialDNA.BetweenthemthesescientistsdiscoveredthatmutationofmitochondrialDNAwas 5–10per cent faster than in nuclearDNA. ItwasWilsonwhofirstthoughtoftheideaofthemolecularclock,basedonthefairlypredictableoccurrenceofmutationsinthehumangenomewithtime.Nowtheywereconvincedthattheyhadthetoolstoinvestigatethepotentialofmitochondrialmutationsasameasureofevolutionaryrelationshipsovertime.

Inthepaper,Wilsonandhiscolleaguesfiguredthattheglobalhumanpopulationbrokedownintotwobroadmitochondrialhaplogroups.OneofthesewasconfinedtoAfrica;theother included someAfricangroups aswell as the rest of humanity.Theydrew severalconclusions. For a start, it appeared to confirm the ‘out ofAfrica’ theory, proposed bysome palaeoanthropologists for the origins of Homo sapiens, while contradicting thealternative ‘multi-regional theory’ which proposed that modern humans had not comedirectlyoutofAfricabuthadevolvedovervast timeperiods in themajorcontinents. Italso supportedwhat is now known as the ‘recent common origins’ ofmodern humans,whichproposes thatallof thepeopleonEarth todayarepartofasingle,closelyrelatedpopulation that emerged from Africa some time in the last 200,000 years. Theirextrapolationswent further; theAfricanhaplogrouphad thegreatest genetic diversity, afindingthathassincebeenamplyconfirmedbyotherstudies.Therecanbemoregeneticdiversity,asdefinedbySnips,betweenneighbouringAfricanpeoples,forexample,acrossamajorriver, thanwefindacrosstheentireEurasianlandmass.IfwethenconsiderthatSnipsarisethroughmutationsatafairlypredictablerateovertime,thisimpliesthatHomosapienshaslivedinAfricaforfarlongerthananywhereelseonEarth.

However, there was an additional, altogether surprising extrapolation. Wilson andcolleaguesalsoclaimedthattheyhadfoundgeneticevidenceforafemale‘lastcommonancestor’ofmodernhumans–awomaninAfricawhowasthefirsttoacquirethefoundermitochondrial haplogroupmutation that is common to all of the people onEarth.Theyfigured that this woman, dubbed by themedia as ‘mitochondrial Eve’, contributed thecommon founder haplogroup to the human pedigree some time between 140,000 and

200,000years ago.The ideaof amitochondrialEve created front-pagenewsheadlines,proving both exciting and controversial at the time. It was, perhaps inevitably,misunderstood by many lay people, including religious groups, to imply thatmitochondrialEvewasthesinglefemaleancestorofallofus.

Weshouldmaintainaprudentcautionhere–forreasonsthatwillbecomeclearalittlelater. For themoment, let us celebrate our commonmatrilineal ancestorwith a visit tomeether inherhunter-gatherercommunity, and lookathowshehasbeen linked to theentireworld.

We can reasonably assume that shewould have appeared little different to the otherwomeninhersmall,close-knitcommunity.Somebelievethatshewouldhaveresembledthe San people, who, until very recently, still followed a hunter-gatherer existence insouthernAfrica,with themenspearing fish in the shallowsorhunting landanimals formeat,thewomendiggingforrootsorforagingalongtheshorelineforshellfish.Weknowthat she had the gift of language,with all of the social potential that conveys.We alsoknow that shemight have painted her skin or clothing with patterns in ochre.We canhazardaguessatwhatmighthavebeenherclothing–perhapsacoveringskirtfromthewaistdownmadefromplantfibres,oranimalhides.AstudybytheUniversityofFloridafoundevidence that humansmayhavebegun towear clothes as early as170,000yearsago.Sheisalsolikelytohavedecoratedherneck,wrists,orclothingwithbeadsofsmall,similarly sized sea shells,whichwere brightly colouredwith natural pigments.We canalso prettymuch assume that the older females taught the children, and younger adultfemales,whattodo,inforagingthroughforestandseashore,andinthebearingandcaringforchildren.

MitochondrialEvewasnottheultimategreat-grandmotherofallhumanity.Shewouldhavebeenoneofmanyfemalesaliveandreproductivelyactiveat the timesheacquiredthefounderhaplogroup.Allof theseotherreproductivewomenwouldhavebeenjustaslikely to contribute to the species gene pool, but she was the only one whosemitochondrialgenomefounditswayintoallmodernhumans.Letmeexplainhowthisislikelytohavearisen.

Let us say that there are ten reproductively activewomen in a single hunter-gatherergroup.Onlyoneofthese,Eve,hasthefoundermutation,orsmallclusterofmutations,inher mitochondrial genome. Perhaps eight of the women are reproductively successful,givingrisetotwoorthreesurvivingoffspringperwoman.Eve,forexample,mighthavegivenbirthtotwosurvivingdaughtersandason.Allthreewillinherithermitochondrialgenome, but her sonwill not pass it on –males do not contribute to themitochondriallineage.Otherwomeninthesamegroupmay,throughchance,havehadnodaughters,sotheywillnothavecontributedtothemitochondriallineage.Insubsequentgenerationsthesame actions of chancewill continue to operate. Generation after generation, for some140,000to200,000years,Eve’sdescendantsmusthavegivenbirthtoadaughterineverygenerationforthistoresultinanunbrokenmatrilineallineageoverthevasttimeperiodtothepresentday.

So now we understand how really it was a game of chance that resulted inmitochondrial Eve drawing thewinning ticket. But this does notmean that those othermothers–andfathersforthatmatter–havenotcontributedtousallgenetically.Wehavealreadyseenhow,usingsimplemathematics,weshareverymanycommonancestors,whowillhavecontributedtootheraspectsofourgenome.

Mitochondrial Eve would have almost certainly lived in Africa, although where inAfrica is uncertain; perhaps somewhere in the region of modern-day Tanzania. Eve’sfounder mitochondrial haplogroup is the ‘macrohaplogroup’ L and it probably didoriginate somewhere between 120,000 to 200,000 years ago. Current thinking suggeststhat her matrilineal line first spread out over the remainder of Africa, the original LhaplogroupevolvingtoregionalsubgroupsL0andL1toL6.AlthoughwetendtoimaginetheseancestralpopulationsradiatingoutfromEastAfricaintotheMiddleEast,andfromthere radiatingwest toEurope and northeast and southeast toAsia,Australasia and theAmericas,infactthegeneticevidencesuggestsacomplexadmixtureandmovementwithwavesofadvanceandreturnbefore,perhaps,anewmigration,startingabout60,000yearsago, saw themitochondrial haplogroupL3 first diversify into haplogroupsM andN inEast Africa before crossing into the Arabian Peninsula, from where it spread anddiversified,perhapsthroughacoastalmigration,intoAsia,Eurasia,EuropeandtheNewWorld.ThiswouldimplythatallthemitochondriallineagesoutsideofAfricadescendedfromtheMandNlineages.

ThesebasalMandNlineageshavenowbeentracedalongthesouthernAsianshoreline.A combination of archaeological and genetic evidence has also revealed that as theexpansionandmigrationprogressedover thousandsofyears, theseMandNsubgroupsacquiredfurtherdefiningsub-subgroups,asthepopulationsmoved,andthelineagessplitintosmallerandsmallerbranches.Forexample,ifwescreenmodernpopulationswecaninfer that those with mitochondrial haplogroups H, I, J, N1b, T, U, V and W are ofEuropeanoriginandthosewithA,B,CandDareofAsianandtheNewWorldorigins,withG,YandZpredominantlyassociatedwithwesternAsia.

*

Itisinthenatureofscientiststobesceptical,andthescientistinmeasksthequestion:Arewebeingoverly simplistic inassuming that this sequenceofmitochondrialhaplogroupsextrapolatestoactualpopulationmovementsandpresenthumandiversity?

Twogeneticists,BrigittePakendorfandMarkStoneking(thelatteroneoftheoriginalWilson group at Berkeley), have warned us that the studying of mitochondrialhaplogroups has limitations when it is extrapolated to explain major populationmovements. They don’t deny that it is a useful tool, but they advocate expanding thesearchestotheanalysisoftheentiremitochondrialgenomeandfurther,toamuchwidergeneticanalysis.Averyobviousnextstepwouldbetoextendthegeneticanalysistothepatrilinealancestry.SowhereinallthishistoricexplorationisthegeneticevidencefortheancestralAdam?

Just as the mitochondrial inheritance passes entirely through the maternal geneticlineage,weassumethattheY-chromosomenuclearinheritancepassesentirelythroughthe

paternal genetic lineage. This is based on theY chromosome having no correspondingpartnertorecombinewithduringtheformationofthespermcells,soitissubjectedtonomixing of chromosomal elements from both parents during the formation of the germcells.Infact,thisisnotcompletelytrue.Some5percentoftheYcananddoesrecombinewith a corresponding part of the X chromosome during germ cell formation. Butgeneticistsgetaround thisbyfocusingon the95percent that is invariablypassedfromfathertoson.Thisiscalledthe‘malespecificregion’oftheYchromosome,or‘MSRY’.

Acronyms–brrrh!

Perhaps, likeme,youhaveaninstinctiveaversiontoacronyms?Alas,weneedtogetthe hang of yet another two that geneticists are fond of flinging into the heated air ofdebatewhen itcomes tomatrilinealandpatrilineal lineages–namelyLCAandMRCA,which in fact denote exactly the same thing: the ‘last common ancestor’ and the ‘mostrecentcommonancestor’.

Brrh–andbrrhagain!

Incontrast to themitochondrialgenome,whichcomprises roughly16,000basepairs,theY chromosome comprises 60million. Thismeans that the study of Y-chromosomemutations, those Snips and haplotypes and haplogroups, is more complex than that ofmitochondrial mutations. But the consolation is that since they focus on two differentgenomes, with different evolutionary and genetic origins and thus different mutationalrates and properties, the sum effect of combining the two adds significantly to theaccuracyofallthosearchaeogeneticcalculations.

For a start, studies of Y-chromosome haplogroups also point to Africa as the placewhere modern humans evolved. But in this case it suggests either eastern or southernAfricaas theplacewhere‘Adam’, theearliestdetectablecommonmaleancestor,or ‘Y-MRCA’,wasborn.

A basal haplogroup lineage, conveniently labelledHaplogroupA, ismore frequentlyfoundinmalesfromAfricathananywhereelseintheworld.Adam’sarrivalonthescenewas variously estimated as 188,000 years ago – or 270,000, or 306,000, or 142,000 or338,000yearsago.Someofthisdisagreementmayhavecomeaboutfromdifferentwaysofcalculatingtheso-called‘molecularclock’,butitmayalsohaveresultedfromproblemswiththegeneticanalysisofverylongDNAsequences.MorerecentlyastudybyG.DavidPoznikandcolleaguesin69malesfromnineverydifferentpopulations,andonebyPaoloFrancalacci and colleagues from 1,204 Sardinian males, arrived at estimates of the Y-MRCA, the mean of which tallied a little more closely with the purported age of ourcommonmaternalancestor.Theseofferedarangeoffrom120,000to300,000yearsago.

Y-chromosome Adam clearly did not inhabit an African Eden at the same time asmitochondrialEve.Nevertheless, throughsuchgeneticstudies theevidence thatmodernhumansoriginated inAfricagatheredmomentum.Butwherewemighthave looked forhelpful confirmation from the fossil record over the same key period of 100,000 to300,000 years, this proved somewhat elusive. Chris Stringer, at the Natural HistoryMuseum in London, drew attention to this prevailing lack of palaeoanthropological

information in an article in the journal Nature in 2003. But he was pleased to drawattentiontotworeportsinthesameissueofthemagazine,whichdescribedthreefossilisedskullsfromclosetothevillageofHertoinEthiopiathatwere,inhisopinion,someofthemostsignificantpalaeontologicaldiscoveriesofearlyHomosapienstodate.

The skulls, which included adults and one juvenile, were almost complete and theirantiquity,atabout160,000yearsold,talliedremarkablywellwiththegeneticdating.Theywereassociatedwithstonetoolsoftheso-calledAcheulean,orMiddleStoneAge,types.The palaeontologists also found evidence that the heads of the adults and juvenile hadbeenremovedfromthebodiesafterdeath,withthelowerjawsdeliberatelydisarticulatedand the skulls systematically defleshed. While this could have been associated withcannibalism, which is sometimes found in human fossils, there were additional ‘moredecorative’cutmarks,incisedbyaverysharpandfineblade,thatsuggestedanalternativeexplanation.These,combinedwith thepolishingofsomeof theroundedsurfacesof theskull, suggested formalmortuary practices followed by cultural or ritualistic treatment.ThepatternsofthemarksweresimilartowhatisseenintheskullsofsomemuchmorerecentNewGuineacrania,whichwereknowntoplayaroleinritualmortuarypractices.

*

This palaeoanthropological evidence, combinedwith the historic evidence storedwithinourhumanDNA,isprovidingpowerfulcorroborativeevidencefortheoriginsofmodernhumansinAfrica.Yetfurthermysteriesneedtoberesolved.WhendidourdistantAfricanancestorsmoveouttopopulatetherestoftheworld?Didtheydosoinasinglehistoricmigration?Or,givenhumanmigratorybehaviourinmorehistorictimes,togetherwiththemajor climatic variability produced by the comings and going of IceAges and naturalcalamitiessuchasvolcaniceruptions,isitmorelikelythattherewasaseriesofebbsandflows, like smaller wavelets overlapping two or more major waves of migrations overtime?

TheWilson studymentioned above, andmany subsequent studies of human geneticdiversity, highlight what may be a related puzzle. If we compare our human geneticdiversity to, say, that of our evolutionary cousin, the chimpanzee, we discoversignificantlylessgeneticdiversityinthehumangenome.Thisisparticularlynoticeableifwecomparetheimportantgeneticregionknownasthemajorhistocompatibilitycomplex,or MHC, which, as we saw earlier, determines immunological and biological self andplaysavitalroleinourimmunologicalreactiontoinvadinginfectiousorganisms,suchasvirusesandbacteria.Thislossofdiversityisasignificantfinding.Itsuggeststhatatsometime in our evolutionary history – and some geneticists believe that itwas a time veryclose to theoriginsofearlymodernsasa species,orperhapsclose to the timingof thespreadofthespeciesoutofAfrica–thattheancestralhumanpopulationwassubjectedtoa near-extinction event. This created a genetic bottleneck that reduced the founderpopulationtolessthan10,000individuals,andsomeeventhinkitmayhavebeenasfewas1,000.Whatpossibledisastercouldhavebroughtaboutsuchashockingcull?

One suggestion was the explosion of a volcano calledMount Toba on the island ofSumatra, which is believed to have erupted 70,000 years ago. But if this distant

catastrophewascapableofslate-wipingpopulationsextendingfromeastAsiatoAfrica,itwould have wiped out the human population closer to the epicentre. The survival ofpopulations in India, as shown by primitive stone tool assemblages in layers above theash, brings this into question. Moreover, the distinguished palaeontologist Sir PaulMellars,at theUniversityofCambridge,hasproducedconvincingevidencethatmodernhumansaremorelikelytohavereachedAsia,throughacoastalmigration,atleast10,000years after the volcanic explosion, whichwould cast additional doubts onMount Tobabeingthecausativecatastrophe.Thereis,however,anothercontender,onethatbringsusback to the endogenous retroviruses that account for roughly 9 per cent of our humanDNA.

We recall that these endogenousviruses entered thepre-humanandhumangerm lineduringretroviralepidemics.ThemostrecentofthegenomicviralinvadersarecalledtheHERV-Ks,agroupthatfirstinvadedtheancestralprimategenomesomewherearound30millionyearsago.TheevolutionaryvirologistLuisP.Villarreal,basedattheUniversityofCalifornia,at Irvine,believes that thearrival,andexplosivecolonisation,of theprimategenomebyHERV-Kswasawatershedeventinprimateandsubsequenthumanevolution.Itcoincidedwiththeswitchingoffofanearlierviralcolonisationofthehumangenome,byDNA-basedvirusesor so-called ‘transposons’.Mostof theHERV-Ksarepresent, inthe same chromosomal distributions, in all of us,many nowhaving entered into usefulholobionticfunctionwiththerestofthegenome.AtleasttensubgroupsoftheHERV-Ksinvadedthehumangermlineafterourseparationfromchimpanzees,sotheseHERV-Ksareexclusivetothehumangenome.Fourofthesearebelievedtohaveenteredthehumangenomeduring the lastmillionyears, includingHERV-K106,HERV-K113,HERV-K115andHERV-K116.Basedonthemolecularclock–inthiscaseweareapplyingittoDNAmutations in the regulatory regions of the viruses, known as long terminal repeats, orLTRs – HERV-K115 inserted into human chromosome 8 roughly a million years ago,whileHERV-K113insertedintochromosome19roughly800,000yearsago.HERV-K116,whichinsertedintochromosome1,andHERV-K106,whichinsertedintochromosome3,have no mutations in their LTR regions. This suggests that their insertions, and therelevant exogenous retroviral epidemics,weremuchmore recent thanHERV-K115 andHERV-K113.

In 2011, Jha and colleagues reported the research outcome of six different groups ofAmerican genetic and evolutionary scientists who combined forces to examine thedistributionofHERV-K106in51Americansofdiverseethnicorigins.Thisallowedthemtoseparatethetestpopulationsintofourdifferenthaplogroups.TheyconcludedfromthehaplogroupevidencethatHERV-K106insertedintothehumangenomeroughly91,000to154,000yearsago.Thismusthaveresultedfromaretroviralepidemicaffectingthehumanpopulationat thesametime.YetunlikeHERV-K115andHERV-K113,HERV-K106wasfound to be universally present in the genomes of their test subjects, suggesting itwasferociously infectious. Judging from the patterns of the two prevailing retroviralpandemics,HIV-1inhumansandthekoalapandemicinAustralia,exogenousretrovirusesare extremely efficient in their spread as emerging infections within a geographicallycontiguousspecies.Andweknowhowtheybehaveinrelationtotheirnewhosts.Thinkof

AIDSinavirginhumanpopulationwithnoknowledgeofepidemiologyandnotherapy;for its endogenousversion tobeuniversal, the exogenousK106 is likely tohave sweptthrough all of the human population that contributed to the descendant people whopopulate theEarth today.And thedateofvirus insertionappears tocoincide fairlywellwith the approximate dates drawn from both themitochondrial and themore recentY-chromosomecalibrations,aswellasthefossildiscoveriesfromMiddleAwashinEthiopia.In time, study of ancient human genomes, including a careful examination of theendogenous viral components, may help to answer whether or not HERV-106 was theculpritforthehumancullingthatproducedtheapparentgeneticbottleneck.

*

Putting aside such speculations, the weight of evidence is pointing strongly tomodernhumanshavingoriginated inAfricaatadate roughlyabout150,000yearsago.But thisalsoleavesuswithanewsetofquestions.WhendidmodernhumansmigratefromAfricatopopulate the restof theworld?Was there just a singlemajormigration,orwas theremorethanone?Weknowthatourancestorsencounteredother,so-called‘archaic’humanspecieswhen they enteredEurope andAsia, sowhat happenedwhen theymet upwiththem?

sixteen

TheGreatWildernessofPrehistory

…inthesecondstagewehadtogointothegreatwildernessofprehistorytoseewhethertherewereelementsofinternalconsistencywhichwouldleadonetobelievethatthemethodwassoundornot.

WILLIAMF.LIBBY

Historyfascinatesusbecauseittellsushowwe,andthesocietyinwhichwelive,cametobe.Formostofusthistendstobeadistinctlyparochialfascination,thetownorcountyinwhichweliveor,inthewidestsense,thecountryorcontinentwefeelthatwebelongto.Thisisaltogethernaturalsinceitistheworldwearefamiliarwith.Butthereisadeeperhistory,olderbyfarthannationalorevencontinentalboundaries–onethattakesusbackto a timewhen life was simpler and yetmore challenging. There were no schools, noemployers and employees, no farms, noherded animalswith their suppliesofmilk andreadymeat,noshopswiththeirimplicitexchangeintheformofmoney,andaboveallnometal tools – no machines. This lost world is what Libby, the pioneer of radiocarbondating, refers toas the ‘greatwildernessofprehistory’. It isadeeply fascinatingworld,andone thatweknowvery little about. It is also aworld that constituted an extremelyimportantphaseinourhumanhistory,onethatgoesbeyondnational,ethnicandindeedalldivisionalboundaries,becauseitisaworldthateverysinglehumanbeingonEarthlivingtodayhasincommon.WearealldescendantsoftheseearlymodernhumansandthuswehaveacommoninterestinhowtheseancestorscametoevolveinAfrica,andhow,outofAfrica,theycametocolonisetherestoftheplanet.

Crucialtounderstandingthisstageinourhistoryisthearchaeologicalexplorationofthetimingof the firstevolutionaryemergenceofearlymodernhumans,a topicwe touchedupon in thepreviouschapter.And from there it is equally crucial to establisha reliableframework,indateandgeography,ofthemovementsofthesepioneeringancestorsduringtheirmigration,ormigrations,outofAfrica.Thishasprovedextremelychallenging forarchaeologists, in part because of the paucity of archaeological evidence discovered todate,andbecausemostsuchsitesaresituatedinwarmerareasofAfrica,EuropeandAsia,wherepreservationoforganicremainstendstobepoorandthereforenoteasilydated.Butthis is now changing; windows of opportunity are opening up through new scientifictechniquesofdatingandthegeneticextractionfromanimalbonesandhumanfossils,aswell as through a broadening of geographic horizons. On 1 May 2014, I travelled toOxfordtointerviewKaterinaDouka,oftheOxfordRadiocarbonAcceleratorUnit,inthehopeofgettingtoknowmoreabouttheseinterestingdevelopments.

DrDoukawasborn inGreece;aftergraduating from theUniversity inAthenswithabasicdegreeinarchaeologyandarchaeologicalsciencesshewenttoOxfordtostudyforaMastersdegreeand,ultimately,aPhD.Hertopicwasthedispersalofmodernhumansoutof Africa and into Europe, and within this broad theme her primary interest wasradiocarbon dating. It was one of her published papers that introduced me to Libby’sconceptof thegreatwildernessofprehistory,andher interest in radiocarbondatingwasone of the keys that might open the lock onto this wilderness. I asked her if she hadbecome interested inarchaeologyat school–or ifperhapsamemberofher familyhadstirredhercuriosity.

‘No. If you come from Greece you are a little bit overwhelmed with archaeologybecauseit iseverywhere.I’mjust interestedinthepast,whetherit is twohundredyearsagoortwohundredthousandyearsago.’

Perhaps,Iventured,itwaspeoplewhoreallyinterestedher?

‘Yes–thepastanditsrelationtopeople.’

The theme of DrDouka’s PhD thesis was: ‘The dispersal ofmodern humans out ofAfricaandintoEurope–fromaradiocarbon-datingperspective.’

In2006SirPaulMellarsproducedwhatisnowafamouspaperinwhichhedescribedhowmodernhumanswere likely tohaveenteredEurope. IfMellars is right, a seriesofbreakthroughs in technology took place in Africa roughly 60,000 to 80,000 years ago.Powerful evidence for this was found in layers of the Blombos Cave in South Africa,whichdatedtoroughly75,000to55,000yearsago.Theadvancesincludednewpatternsoffinestonebladetechnology,scrapersforworkingskinsandhides,toolsfortheshapingofboneandwood,bonepointsforthetipsofthrowingspearsandsharplypointedawls,andcarefullyshapedartefactsforthrowingspearsandeven,conceivably,thefirstarrows.Thesewerefoundinthesamesedimentsasperforatedshellsusedforpersonalornament,the earliest such items ever found, as well as large quantities of imported red ochre,includingpieces incisedwithgeometricornamentation.Allof thiswas accompaniedbyevidenceoflarge-scaleexchangeanddistributionoverdistances.Equallysignificantwastheevidence,gatheredbyotherscientists,ofanaccompanyingrapidpopulationgrowthintheancestralAfricanpopulation,between60,000to80,000yearsago.

ThesespecificAfricantoolsandculturalornamentationshowastrikingresemblancetowhatwasappearinginarchaeologicalsitesthroughouttheMiddleEast,EuropeandAsia,taken as evidence for the migration of modern humans out of Africa and into theselandscapes,roughly45,000to50,000yearsago.Itwas,ofcourse,thisveryexpansionoftheancestorsofmodernhumansintoEurasiathathadbeenthefocusofKaterinaDouka’sPhDandithasfurtherextendedtoherpresentfieldofscientificinterest.

Oneofherrecentscientificpapers,whichborethetitle‘Exploring“thegreatwildernessof prehistory”’, described how the transition from a Neanderthal-dominated westernEurasiatoacontinentinwhichmodernhumanswerenowthesoleinhabitantsmarkedoneof thebiggest transformationseverwitnessed in thisvast region. Iwasaware that somescientists believed that there had been at least two major migrations of early modern

humans out of Africa: one about 120,000 years ago, related to some hominin fossildiscoveriesintheSkhulandQafzehcavesites, theninPalestine,andanothermigration,dated to roughly 45,000 to 39,000 years ago, linked to a famous rock shelter site inLebanon, known as theKsarAkilwhichwas also associatedwith hominin fossils.Thelatter site was widely regarded as a key staging post in the most important migration,startinginorpassingthroughtheNearEast.Tothisend,KaterinaDoukaledateamthatsetouttoapplymodernradiocarbondatingtothehumansettlementsatKsarAkil.

IaskedheraboutLibby’sintriguingphrase:thewildernessofprehistory.Whathadhemeantbyit?Didhemeanthatprehistoryinitselfwasawildernessinthesensethattherewasverylittleknownaboutit?

‘Libby is one of the greatest, if not the greatest, figure in radiocarbon dating. Hedeveloped the ideas that led to carbon dating while working as a physicist on theManhattanProject,andfortheseideashelaterreceivedtheNobelPrizeinChemistry.’

RadiocarbondatingisbasedontheknownrateofdecayofthecarbonisotopeCarbon14.Basically, the atmosphere contains different isotopes of carbon in the form of carbondioxide,whichincludethemainstableisotope,C12,aswellastheunstableisotope,C14,whicharepresentinroughlyconstantproportions.Theseisotopesaretakenupbyplantsandmicro-organisms,fromwheretheyenterthelivingbodiesofotherlifeforms.Onceanorganismdies, there isnopossibilityof replenishing the carbon isotopes, and from thatmoment the ratioofC14 toC12willprogressively fall over timeas theunstable isotopedecays to the stable one. Measurement of the ratio of these isotopes is the basis ofradiocarbondating,whichhasrevolutionisedthedatingofPalaeolithicarchaeologysites.This,Ipresumed,wasLibby’scontributiontoexploringthewilderness.

‘That’sright.Ifwelookatscientificpaperspre1960,thereisnoabsolutedating.Peoplearetalkingaboutafewthousandyears,ortensofthousands,orhundredsofthousands,buttherewasnowayofquantifyingthisasfarastimewasconcerned.’

‘Howfarbackcanyouaccuratelygowithradiocarbondating?’

‘Fiftythousandyears.’

‘Butthere’sabigchunkoftimebeforethat?’

‘That’sour actual limitation.But in factweare luckybecause theupper limitof thismethodallowsustocalibratethelastyearsoftheNeanderthalsandtheirinteractionwithmodernhumansinEuropeandmorebroadlyEurasia.Forpreviousepochsweneedtouseothermethods,suchasthermoluminescence,whichcantakeusback200,000or300,000,maybeevenhalfamillionyears.’

IwasinterestedtoknowmoreoftheworkshedidforherPhD.

Theidea,asDoukaexplained,wastofocusontheMediterraneanrimandtrytotracemigrationsdeepintoEurope.Thereweretwobroadpossibilities:oneroutewaslikelytohave followed the Mediterranean coastline; the other would most likely have headednorthwest,alongtheDanubeRivercorridor.FormanyyearspeoplehadassumedthatthelikelycrossroadsfromAfricatoEurasiahadbeenthroughtheNearEastbuttheevidence,

and dating, for such a crossroads was scanty. Perhaps it was time that Palaeolithicarchaeologistsexaminedthisagain?

‘Forme,’Douka laughed, ‘itwas great timing. I had just startedmyPhD and IwasinterestedinfollowingupMellars’ideas.Sotheideawastodateshellbeadsfromaroundthe Mediterranean to see if we could date modern human expansion. TraditionallyNeanderthalsarenotthoughttohaveusedornamentsinthesameway–oressentiallynotmanufacturedshellbeadsanyway.KsarAkilwasoneofthefirstsitesIlookedat.’

Iwaskeentoknowmoreaboutthepotentialofsuchasimpleandcommonresourceasseashells. ‘Doyoufind theseshellbeadsvery frequently inmodernhumansitesdatingfromthisperiod?’

‘Yesyoudo–especiallyaroundtheMediterranean.Theyarenotrarefindings,buttheyhave exciting potential. Andwhen you do find them, they are often in great numbers.We’retalkingabouthundreds.’

‘WhenyousayaroundtheMediterranean–inwhatsortofplaces?’

‘Lebanon,southernTurkey,southernGreece,ItalyandallalongtheFrenchandSpanishcoastsextendingalsointothenorthofSpain.’

‘Whatsortsofshellsarewetalkingabout?’

‘Essentially they are small, about 1 to 2 centimetres diameter and perforated with astonetool.’

‘Toosmalltobeconsideredfood?’

‘These are not food residue. These people were very selective in what species theygathered.Mostlikelytheycollectedtheshellsdirectlyfromthebeachasemptyshells,butonlyfreshlydead.Theythenpiercedtheshells–thisishowweknowthattheywereusedasbeads,whetherasjewelleryordecorationforclothing…Sotheideaformyresearchwas to go back to the sites, go back to the original collections, and date thismaterial,whichamountedtoseveralhundredshellbeads.’

Carbondatingcanmakeuseofmanydifferentmaterials,includingsofttissues,suchasskinandderivedleather,bone,charcoal,seeds–anythingthatexchangescarbonwithitsenvironment.Doukahasmadeaspecialstudyofseashellsasareliablesource,whichsheappliedtoanewappraisaloftheKsarAkilsiteinLebanon,workinginassociationwithcolleagues,includingProfessorsRobertE.M.HedgesandThomasF.G.HighamfromtheOxfordRadiocarbonAcceleratorUnit,DrChristopherH.BergmanfromCincinnati,andDrFrankP.Wesselingh,fromLeidenintheNetherlands.

There were considerable problems that needed to be overcome, though. A majorhandicapwasthefactthathumanfossilremainsfromtheregionhadbeenscantyeveninthe original excavations, and much of this had been lost over the decades since.ExcavationofKsarAkilhadbegunaslongagoas1937byagroupofAmericanJesuits,andithadextended,inphases,andwithdifferentexpertsconductingtheexcavations,until1975.Theworkover thisperiodof timehad revealedmore than30 stratigraphic layerssome23metresdeep.Theearliestexcavationhaddiscoveredtheskeletonfossilplayfully

named‘Egbert’.Thefossilisnowlostandsoit isonlyknownthroughphotographsandcasts made at the time. When these casts were examined at the British Museum, byBergmanandStringer,theysuggestedthattheskull,whichissmallanddelicatelybuilt,islikelytobethatofagirlagedbetweensevenandnineyears.Asecondhumanfossil,thatof amore primitive-lookingupper jawwith a single canine tooth,was playfully namedEthelruda–perhapsafteranAnglo-Saxon femalesaint.Thiswas initially thought tobethejawofaNeanderthalbutisnowconsideredverylikelytobethatofamodernhuman.WhileEgbertremainslost,EthelrudawasrediscoveredintheArchaeologicalMuseumofLebanonbutappearedtolackcollagen, theusualsourceofcarboninsuchfossilswhichenables radiocarbondating, so analternative indicatorwasneeded.Douka switchedherattentiontoseashells.Asithappened,themolluscancollectionfromKsarAkilwasoneofthe largest ever discovered at a Palaeolithic site, containing approximately 2,000specimens, the majority of which showed evidence of human modification such asperforationforornament,orthepresenceofochrepigmentsappliedtothem.

Theseshellshadalsobeenlostbackinthe1960s,butin2006Doukaandhercolleaguestraced them to theNetherlands.Theywere thenable to locate the levels layerby layer,from the levels they knewwere associated with Neanderthals to those associated withmodern humans. The shells from these layers could now be transported to the Oxfordlaboratory for preparation and testing. Recent research into ways of excludingcontamination when dating shell carbonates now proved useful in the preparation andgrinding down of a small piece of each shell into a fine dust that she could use toradiocarbondatethesiteand,byinference,itsassociatedhumanfossils.

‘Whatdatesdidyoufind?’

‘Fromtheshellbeadswegotaradiocarbondatingfortheearliestmodernhumanlayersataround37,000radiocarbonyears,whichconvertstoabout42,000calendaryears.’Thisallowed Douka to use a probability mathematical calculation, known as Bayesianmodelling,todatetheskullofEgberttobetween40,800and39,200yearsoldandthejawofEthelrudatobetween42,400and41,700yearsold.

This established time frame had additional implications; it put the arrival ofmodernhumans into the Levant somewhat later than had been previously assumed. There wasevidenceelsewhere that thebigmigrationoutofAfricadated to several thousandyearsearlier than this. So Douka’s findings suggested new possibilities. Themainmigrationroutemighthavebeenelsewhere,perhapsdirecteastfromnortheastAfricathroughArabiaandCentralAsia, later curling towards theNearEast. ‘Weneeded to cast the netmorewidelytoincludeotherplacesandmigrationroutesinthehistorichumandispersaloutofAfrica.’

WhetherKsarAkilhadbeenonthecrossroads,orwhetherthemigrationhadarrivedatKsar Akil a few thousand years later, the site remains a very important source ofinformation,bringing togetherhominin fossilswithevidenceof theprevailingculture. Ihadmorequestionstoaskbeforemovingfurtherafield.

‘Whatwere thesepeople like?Whatwas their society like?Whatdid theyeat?Whatsortofclothesdidtheywear?’

‘FromKsarAkil,andothersitesalongtheMediterranean,weknowthat thesepeoplewerehunter-gathererswithaveryvarieddiet.Throughouttheyeartheywerehuntinglargeanimals,suchasdeer,whateverwasavailable.Buttheyalsoateawidevarietyofdifferentfoods,includingawidearrayofplants,nutsandfruits.Wearestillworkingonthis,butwe think they may have been collecting shellfish when other dietary resources weresparse,probablyinlatewinterorearlyspring.’Intermsofpopulationsize,Doukathoughtwe were probably looking at groups of about 80 people living in relatively closeproximity.

But there was something else that puzzled me.While accepting that Ksar Akil waslinkedtothemainmovementofmodernhumansoutofAfricaandintoEurasia,Irecalledthattherewassupposedtohavebeenanearliermigration,judgingfromotherevidenceintheNearEast.What,Iwondered,hadhappenedtothatearliermigration?Whydidn’ttheypopulateEurasia?Iknew,ofcourse,thatinEurasiatherehadbeenamajorproblemwithrecurrent Ice Age pulsations, starting about 2,580,000 years ago and extending to thecurrent interglacial period. These big freezes must have played havoc with humanhabitation andmovements inEurasia, inparticularwith a freeze calledRiss, that lastedfrom180,000to130,000yearsago,andonecalledtheWürm,thatlastedfrom70,000toroughly10,000years ago.Might this suggest that climate amelioration, beginning soonafter theendof theRissglaciation,hadencouraged that earlierhumanmigrationoutofAfrica?Butwhatthenhappenedtothatpurportedearliermigration?

*

Someof the best evidence for an earliermodern humanmigration out ofAfrica comesfromthetwofossilsitesEsSkhulandQafzeh.EsSkhulisacaveontheslopesofMountCarmelandQafzehisarockshelterinLowerGalilee.Thesetwositeshaveprovedrichinearlyhomininfossils,whichhavebeententativelydatedatbetween80,000and120,000years old using techniques suitable for this level of antiquity, known as electron spinresonance and thermoluminescence. In particular the fossils include a number of well-preserved skulls ofmale and female individuals, and of a variety of ages. These skullsshow a mix of archaic and modern features, with heavy brow ridges and a projectinglower face, similar to theNeanderthals, but the brain case and upper skull ismore theshapeassociatedwithmodern-dayhumans.

Themixtureoffeaturesissostrikingthatatfirstthesewereinterpretedasevidenceforapartial evolution fromNeanderthals tomodernhumans, but thenobviousNeanderthalremainswerediscoveredinthenearbyKebaracavethatweredatedtoamuchlaterperiod,roughly61,000to48,000yearsago,disprovinganysuchidea.Someanthropologistsnowproposed that the Es Skhul andQafzeh hominins represent an early exodus ofmodernhumansfromAfrica,around125,000yearsago,sothattherobustfeaturesrepresentedamorearchaicH.sapiensthatwouldintimeevolvetowhatweseetoday.Ifso,perhapstheSkhul/Qafzeh people may have represented a true earlier out-of-Africa migration thatsimplystoppedintheNearEast.Inotherwords,amigrationthatcametoadeadendhere,withnocontributiontothepeopleofEurasia.

‘Yes,’Doukamused,‘weknowtherewasanIceAgecoming.PerhapsinEurasiaatthatearliertime,theNeanderthalpopulationwasatitspeak.Perhapstheywereholdingstrong.But then again it is very difficult to knowwhy – it might just have been an effect ofrelativepopulationsizes.Again,theearliermigrationmightnothavebecomecompletelyextinct…’

A set of teeth discovered in the nearby Tabun Cave, in 2005, was thought to showNeanderthalfeatures.Theseteethweretentativelydatedtoaround90,000yearsago.This,together with the discovery of more Neanderthal remains, again tentatively dated to120,000 years ago, suggests that Neanderthals and modern humans might have madecontact in the Near East at this time. This introduced the possibility that what we areseeing at Skhul/Qafzeh is a sexual crossing between the two species, giving rise to ahybridpeoplewhowerepartmodernhumanandpartNeanderthal.

DrDoukashrugged: ‘This is somethingweare trying to lookat.Weknowvery littleabout any earliermigration from fossil evidence. I think a lot ofweight is put into theNearEastwhenitmightnotbethemostlikelyplaceweshouldbelookingat.Butwenowhave a major five-year project under way that is looking at all the possibilities fromEasternEuropeallthewaytoCentralAsia,includingSiberia.’

As we shall see in a subsequent chapter, some remarkable new genetic evidence isbecoming available from the fossil remains of early humans, including Neanderthals,whichislikelytorevolutionisepalaeoanthropology.Itwillclearlyhelpwhenmorearchaicgenomesfromthe fossil recordcanbeadded to thegeneticpicture, since thesewillnotonly allow genomic study for haplogroups but will also allow calibration of genomicdating with fossil-dating methods such as Carbon14, electron spin resonance andthermoluminescence.Intime,thiswillshedmorelightonthetimingandmigrationsofourancestors,whethertheyactuallywentnorththroughtheLevantoreastthroughArabia–oreven directly across the Mediterranean Sea from North Africa. Some scientists arepersuasivelyarguingthatfuturestudyshouldincludemoreoftheextraordinaryvariationfoundinthehumangenome.Thespreadofendogenousretroviruses,suchasHERV-106,HERV-113 and HERV-115, and the genetic and epigenetic regulatory regions of thegenome, including those all-important non-coding RNAs, will also help to pin downmovementsanddatesmoreaccurately.Ithinkweshouldbepreparedforthetruerecordtoprove complex, with allowance for population advances and retreats, with mixing ofdifferentpopulations,andinparticularwiththeproblemsofaharrowingIceAgecausingmajorupheavalsinlargeswathesoftheEurasianlandmass.

IaskedDoukaifwehadanyideaofthelikelysizeofthemigratingpopulations.

‘Ireallydon’t thinkwehaveanytrueidea,becausewedon’treallyhavemuchinthewayof hard data that could lead us to calculate population size.But if you look at thegeneticdatacomingouton theNeanderthalgenome, itproposesaneffectivepopulationsize of between 1,500 and 3,000 reproductive Neanderthal women. Based on that youcouldbetalkingaboutatotalNeanderthalpopulation,intheEuropeancontext,ofroughly10,000 individualsat the time the fossilswerecollected. Ifyouaskme, Iwouldhazardevenlessthanthat.’

‘Andcontemporaneousmodernhumans–Ipresumetheywouldalsohaveruntosmalltotalpopulations?’

‘Modernhumansareconsideredatropicalspeciesofhumans,wherepopulationsizeisthoughttohavebeenmuchgreater.Plus, theideais thatastheycameoutofAfricaanddispersed across Asia, they maintained a wide networking of groups, with associatedgeneticexchange.Soitisnotunlikelythatthepopulationofmodernhumanshadahigherturnover,andtheirnumberswerelarger.’

Ina recentpaper in the journal,Science,MellarsandhisCambridge-basedcolleague,JenniferC.French,assessedrelativepopulationsofNeanderthalstomodernhumansusinga combination of genetic and traditional archaeological techniques. They comparedmitochondrialDNAdiversityamongpresent-dayEuropeanpopulations tomitochondrialDNA diversity derived from Neanderthal remains. They also analysed various‘archaeological proxy evidence’ for intensity of occupation over the Neanderthal tomodernhuman transition in thewell-studied south-westofFrance.Theyconcluded thatthe numbers of modern humans settling the area showed a nine-fold increase on thenumbers of Neanderthals that had previously occupied the same area. There are someobvious assumptions in such a technique, but their conclusions do pose an interestingquestion: what if the Neanderthal disappearance from Eurasia was brought about bysimplenumericalsupremacyofarrivingmodernhumans?

Asweshalldiscoverinthesucceedingchapter,this,perhapscombinedwithsomeotheremerging discoveries, might answer the question that has intrigued scientists and laypublicalikeformorethanacentury.Butforthemoment,weshallmaintainourfocusonthecolonisationofEuropebyarrivingmodernhumans.

IpressedDrDouka,‘SoitseemsthatyougetmodernhumansarrivingintoEurope–orEurasia – and from that point on there appears to be a fairly rapid cultural evolution,perhapsstartingaround20,000yearsago?’

‘Iwouldn’thaveputitat20,000.Formeit’sprobablyearlier,morelikelyabout40,000to 45,000 years ago. Some of these early modern humans would have occupied smallpocketsofEurope,southernItalyandwesternFrance,butafterthat,between33,000and30,000yearsago,wereallyseesomethingcompletelydifferent.ThismarksthearrivaloftheGravettians.’

‘AnothermigrationoutofAfrica?’

‘Wedon’tknowwhere theycome fromorwhether their culture firstdevelopswithinEurope and then expands all the way to Russia, or vice versa. These humans appeararound33,000yearsago.Andthereisacompletelynewwayofdoingthings.Theyburytheirdead–andtheyburythemwiththousandsofbeads.Someof theseareshellbeadsbutothersaresmallivorybeads.Theyalsousethecaninesofreddeer,whichtheymakeintobeads.Theyproducedwonderful sculptural figurines.Sowe’re talkingaboutmajorculturalchange.’

‘Wedon’tknowwhetherit’sanideaspreading,orwhetherit’sanewpeoplebringinginthenewideas?’

‘That’sright.’

‘Perhapsintimethegeneticswillanswerthis?’

‘Perhaps,yes.AtpresentwehaveverylittleinthewayofDNAfromthesepeople,butthisisabouttochange.Whatweknow,however,isthatfrom33,000onwardstherewasanewbloomingofhumanculture.’

Theseculturalandpopulationchangestookplaceatatimeofmonumentalclimateandecological change in Eurasia. These severe climatic variations were accompanied byperiodsofmajorpopulationmovementandgrowthintheareasaffectedbytheIceAge.Aprolonged cold period, known in the scientific jargon as theLastGlacialMaximum,or‘LGM’,occurredbetween26,000and19,000yearsago.ThiscoolingperiodisthoughttohavecausedamassivepopulationdeclineinEurope,with thesurvivors takingrefugein‘climatesanctuaries’,or‘refugia’,thefourmajorexamplesofwhichwerenorthernIberiaandsouthwestFrance, theBalkans, theUkraineand thenortherncoastof theBlackSeaandItaly.

The Last GlacialMaximummaywell have reduced the genetic diversity of Europe,which in turn would complicate any assessment of arrival and dispersal based on thehaplogroups of current Eurasian inhabitants. As the glaciers began to pull back, about16,000to13,000yearsago,peoplebegantorepopulatethedevastatedlandscapefromthefour geographic refuges, so that anthropological geneticists can expect to see not onlygeneticsignatures thatdatefrombefore theLGMbutalso thegeneticsignaturesarisingfromtheisolatedpopulationsofthefourrefuges,whichwouldhaveexpandedtofill thelandscapeastheglaciersmelted.

For example, 80–90 per cent of males in Ireland, Wales, Scotland, the Basques innorthern Spain and western French share the male-specific-region Y-chromosomehaplogroup,or‘MSRY’,knownas‘R1b’,asdo40–60percentofthemalepopulationofEngland,France,Germanyandmostof therestofWesternEurope.Thismakes it likelythat their patrilineal ancestors took refuge in the northern Iberian climate sanctuary. InsoutheasternEuropeR1bdropsbehindarelatedhaplotype,R1a,intheareainandaroundHungary and Serbia. Another MSRY haplogroup, labelled ‘I’, is found in its highestfrequenciesinBosniaandHerzegovina,Serbia,Croatia,aswellasNordiccountries,suchas Sweden and Norway, and parts of Germany, Romania and Moldova. The samehaplogroupcladeishighlyEuropean-based,makingsomegeneticiststhinkitcoulddatetobefore the last IceAge. These, andmany other different haplogroup clades, do help totracepopulations and theirmovements, but I should add that, aswemight expect fromhistoricsources,theyalsoshowhugepopulationmixing.

Curiously,whenwelookatthematrilineal-basedmitochondrialhaplogroupsinEuropewe see what appears to be a very different pattern from what we saw in the male-associated Y chromosome. When compared to the males, the female-associatedhaplogroups show much less geographic patterning, which seems to indicate thatEuropeanwomen share amore common ancestry.How fascinating if thismight reflectdifferentsocio-culturaltraditionsaffectingthemobilityofmenandwomen.

Some99percentofallEuropeanmitochondrialhaplotypesfallwithinthecategoriesH,I,J,K,M,T,U,V,andWorX.HaplotypeHisthecommonest,beingfoundinnolessthan50percentofallEuropeans,andsixoftheabovehaplogroups,H,I,J,K,TandWareonlyfoundinEuropeanpopulations.Thelattersuggeststhatthesehaplogroupsarosein the ancestral Caucasoid populations after they had separated from the ancestors ofmodern Africans and Asians. As historical records show, these ancestral genomes willhavebeenblendedwithmanydifferentgeneflowsfromeasternAsia,southernSiberiaandAfrica, but the ancestral patterns are still readily detectable even in modern genomes.Geneticists are currently assessing whether Europeans are mostly descended fromPalaeolithicorNeolithicancestorsbylookingatmoreandmoregenomesthatdatefrom15,000yearsagoorolder.

These early ancestorswerevery similar tous, but theyweren’t the same.Theuseofgeneticdatatodiscernaspectsofhumanprehistoryisknownas‘archaeogenetics’.Oneoftheintriguinginsightsthatiscomingoutofsucharchaeogeneticstudiesinrecentyearsisthefactthatsignificantevolutionarychangehastakenplaceinourhumangenomeinthelast50,000years–akeytimeinthehumanmigrationsoutofAfrica,andthecolonisationbymodernhumansofEurope,Asia,AustralasiaandtheAmericas.Palaeoanthropologistshaveraisedthepossibilitythatthishasbeendrivenbytheevolutionofculture.

Perhapsweshouldn’tbetoosurprisedatthis.Cultureisaquintessentialfacetofhumanlifeandexperience.Onceagainthegeneticistshavelookedtomutationalchange,andinparticular to the acquisition of new Snips that seem to mark out specific culturalpopulations. The theory is that some of these Snips in the autosomal chromosomes areconservedbecausetheyhappentobeclosetoaparticularvariantofagene(inthejargon,a specific ‘allele’) that is already present, even in just a small number of individuals,perhaps dating back to a single ancestor, that has been favoured by natural selectionbecauseitgivesasurvivaladvantage.

RobertMoyzisandhiscolleaguesattheUniversityofCalifornia,atIrvine,searchedforsuchevolutionarychangeamongsome1.6millionSnips scattered throughout theentirehumangenome.Theyconcludedthatroughly1,800geneshadbeeninfluencedinthiswayover the last10,000 to40,000years–or, toput it anotherway, some7per centof thegenomehad been specifically targeted by evolutionary forces during the expansion andsettlement of humans in this phase of human history. The populations studied includedAmericans ofEuropeanorigins,Americans ofAfricanorigins, andAmericans ofAsian(Han Chinese) origins. Key areas of the genome that appeared to be under intenseselective pressure included some of the most important aspects of human internalchemistry.Theseincludedourabilitytofightoffinfectiousdiseases,sexualreproduction,DNAchemistryandcopyingaspartofthecellcycle,proteinmetabolismandthefunctionofthenervecellsthatformourbrainandcentralnervoussystem.

The authors also concluded that we have undergone evolutionary change affectingmanydifferentphysicalattributes,verylikelyextendingbeyondthosethattheytestedfor.This study suggested that human evolution can involve widespread physiological andphysical change inwhat, from an evolutionary perspective,would be a relatively short

space of time. How likely is it that the same rapidly moving evolutionary selectionpressuresarestilloperatingonustoday?

*

Asourjourneyprogresseswebecomeincreasinglyamazedathowtheexplorationofourhumangenomerevealssomuchthatwasformerlymysteriousinourpersonalandculturalhistory. The potential for such exploration has undergone a sea change in recent years,presentinguswithpossibilitiesthatwouldhaveseemedimpossibleagenerationago.Aswe shall nowdiscover, it has becomepossible to learn from the study of genomes thatdivergedfromourhumanancestrallinemorethanhalfamillionyearsago.

seventeen

OurHumanRelatives

It’s thequestionsandnot theanswersthatareinteresting,becausethesearequestionsthathavenoanswers.Buttheyareinterestingquestionstothinkaboutbecausetheysomehowreflecthowwethinkaboutdifferencesbetweenusandourancientancestors.

SVANTEPÄÄBO

SvantePääboisaSwedishevolutionarygeneticistwhoworksattheMaxPlanckInstitutefor EvolutionaryAnthropology in Leipzig,Germany. The son of biochemistKarl SuneDetlof Bergström, who shared the Nobel Prize in Physiology orMedicine in 1982 fordiscoveries related to theprostaglandins,Pääbo founded thenew investigative scientificdiscipline known as palaeogenetics.Until recently geneticists had assumed that readingthegenomesofextinctspeciesofanimalsandplants,aspopularisedbyMichaelCrichtoninhisnovelJurassicPark,wouldbepractically impossiblebecauseDNAdegradesovertime.Theolder the fossilisedbones, themoredegraded theDNA.Thus ancient fossils,datingbacktensorevenhundredsofthousandsofyears,wereassumedtocontainlittleorno residual DNA and were thus thought to be an unlikely source of useful geneticinformation.Butover thedecadessincethe1980s,Pääboandhiscolleaguesat theMaxPlanckInstitutebegantomakeinroadsintowhathadhithertoseemedimpossible.

Through their work we have discovered that ancient DNA, though degraded, willsometimes survive the ravages of time. In pioneering this revolutionary new science ofpalaeogenetics, Pääbo has perfectedmethods of amplifying and then extracting geneticinformationfromfossilbonesandotherancientremains,makingitpossibletoexplorethegenomesoflong-deadanimalsandplantsand,mostintriguingofall,ofancientspeciesofhumans.Inaninterviewwiththeonlinewebsite,Edge,Pääboadmittedthathestartedoutnaïvelythinkingitwouldbeeasytostudythegenomesoflong-deadindividuals.Tobeginwith his interest was the more recent past, and in particular the mummified bodies ofancientEgyptians,assumingthesewouldbemoreamenablethanthevastlyolderextincthominins.Hewouldsubsequentlyconfess:‘Iwasdrivenbydelusionsofgrandeur.’Buthepersistedinhisdreamand,thoughitprovedtobefarfromeasy,heeventuallysucceeded.

Perhapsthesimplestwaytoexplainhowhedidsowouldbetoclimbbackonboardourtrainandpayabriefvisittothegenomiclandscapeofsuchancientfossils.Boneandtoothhave turned out to be two of the best sources of ancientDNA in humans, so thiswilldetermineourchoiceofdestination–thefossilisedboneortoothfromalong-deadhuman.Hereweenteralandscapeverydifferentfromanythingwevisitedbefore.Wearriveatnoneat railway line running away into the distance, east and west, but a confusion offragments that, fromadistance, resembleanexplosion ina spaghetti factory.Pulling in

closer, it still takes us a littlewhile to recognise thatwhatwe are looking at ismyriadhaphazard shards of decayed genome. We are dismayed at the sight of these brokensectionsandpieces,whichappeartobemeaninglessintermsofreadingtheoriginalcodeinthepatternofsleepers.Itseemstoconfirmwhatgeneticistshadlongthoughtaboutthestateofaffairsinsuchancientfossils…

Butallisnotquiteasitseems.EachindividualfragmentofspaghetticontainsasmallbutgenuinefragmentofDNAsequence–inouranalogyabrokenpieceofrailwaytrack.And now, as we look at the vast scatter of fragments more closely, we see that eachfragment contains anything from a few sleepers to perhaps a few hundred. Hardlyreassuring, one might still be inclined to think, especially when the assembled humangenomecomprises6.4billionsleepers.Indeed,ifalltherewereinthisfossilbonewasthefragmented remains of a single copy of the genome, the task of reading that genomewouldbehopeless.Thekeytounderstandingis that thesemyriadfragmentsarenotbitsand pieces of a single copy of the genome, these are the fragmented remains of vastnumbersofthesamegenome,theresidueofthebillionsofindividualcellsthatmadeuptheboneduringlife.

All of those different copies of the genome will have broken up into differentfragments. And just as in the original Human Genome Project, when the genome wasdeliberatelyfragmentedtoreduceittomoremanageablegeneticbites,thishugevarietyoffragments,withbreaksindifferentplaces,willcontainDNAsequencesthatoverlap.Thequestionnowisjusthowmuchofanoverlapdoweneedtobesurethattheoverlaphasn’tjustresultedfromchance?Infact,wecaneasilyworkoutthemathematicswithasimplecalculation. What’s the likelihood of, say, three, or four, or six, or eight nucleotidesfollowinganidenticalsequenceinarow?Sincetherearejustfournucleotides,thechanceofthenucleotidematchinggivesustherandompossibilityofoneinfour–G,A,CorT.Forthefirsttwoinsequencetobethesamegivesusarandompossibilityof4x4–aonein 16 chance.With each subsequent nucleotidewemultiply by an additional 4.By thetimewehavecalculatedthemathematicsforeightsleepersfollowingidenticalsequences,the chance of this happening by accident is one in 65,536 – in otherwords, extremelyunlikely.Wenowhaveasystemthatworks.

The first step is obvious. We need to sequence every fragment and bank thisinformation in a computerwith a very largememory. The second step is to search thebanked sequences formatching sections thatwill pickout areasofoverlapbetween thedifferentfragments.Fromtherewecanbeginthelaboriousprocessofstitchingsequencestogetherusingtheoverlapstoidentifycontiguousareas.Infact,wearereproducingprettymuchthewaythefirstdraftofthehumangenomicsequencewascalibrated,exceptwearedealingwithavastcollectionofmuchsmallersequencesthatmustnowbepainstakinglyknittedtogether.

To this breakthroughPääbo enlisted twoothers.The first of thesewasKaryMullis’sNobelPrize-winningdiscoveryofthepolymerasechainreaction.Thisensuredthateveniftheindividualfragmentswerepresentinveryfewcopies,tootinybyfartobedetectablebytheautomatedsequencingmachines,theywouldbeduplicatedoverandoveruntilthey

becameidentifiable.NextcamethebrainwaveofteamingupwithaninnovativeAmericanbiotechnologycompany,454LifeSciences,whichhadbeensetupbyJonathanRothbergtodevelopmachines thatwere capable of automatedhigh-throughputDNAsequencing.ThecompanyhadbeentakenoverbythepharmaceuticalgiantRochein1997.Theentireprocesscouldnowbeautomated.ThemachinescouldautomatetheextractionofancientDNA,amplifyitusingPCR,andthusgenerateasoupofsmallbitsandpiecesoftheentiregenome,fromwhichtheycouldbankandthenknititalltogetherbyaligningtheoverlaps.

Ofcourse,therewereotherproblemstobeovercome,suchascontaminationofthetestsamples by unwanted sources – human, animal and bacterial. They found ways ofremovingcontaminatingDNAwithenzymes.Theyalsoavoidedcontaminatingspecimenswith their own DNA by working in a designated ‘clean room’ and by taking strictbiosafetyprecautions.DuringtheactualsequencingtheydiscoveredthatoneoftheDNAnucleotides,cytosine,sometimesdegradedtouracil,thenucleotidethatreplacesthyminein RNA. Then it became even more complicated; sometimes cytosine that had beenepigenetically tagged with a methyl group degraded not to uracil but to thymine. Thiscreated confusion until they realised that, by accident, they had made a significantdiscovery.In2009oneoftheteam,AdrianBriggs,figuredoutamethodfordistinguishingthyminesderivedfrommethylatedcytosinesfromoriginalthymines.Nowtheycouldreadoffsomeoftheoriginalepigeneticprogrammingofthegenome.Soithappenedthat,littleby little, the scientists advanced and perfected their DNA extraction techniques andimprovedthequalityofthereadouts.

The applications were taken up by other laboratories and extended to the woollymammoth, cavebear andcoelacanth, aswell as fossilplants.MeanwhilePääboandhisteam addressed the singlemost exciting challenge that had faced his lab from the verybeginningofthispioneeringresearch:theywouldattempttheextractionofthegenomeofourlong-losthumancousin,NeanderthalMan.Time,perhaps,forustotakeabrieflookatwhatisknownofourhumanevolutionaryhistory.

*

Inhisnotebook,duringthedecadesinwhichhegraduallyassembledhisthoughtsonhowa new species might arise from an existing one, Charles Darwin sketched somethingresembling the branching pattern of a tree. This branching pattern has since then beenamplyconfirmedbybiological studyand thesamepatternneatly fitswith theLinnaeanclassificationof life intospecies,genera, families,andsoon– thedisciplinesbiologistscall ‘phylogenetics’ and ‘taxonomy’. Humans are no exception to this; as recently as70,000yearsagosomefivedifferentspeciesofhumanscohabitedtheEarth.Allfivehaddirectlyorindirectlydescendedfromasingleancestralspecies,knownasHomoerectus.Oneof thefivewasourdirectancestors, ‘earlymodernhumans’–or togive themtheirformalLinnaeanclassification,Homosapiens.BothH.sapiensand thestill-evolvingH.erectus shared the planet with Neanderthal Man, or Homo neanderthalensis, whoinhabited largeareasofEurasia; theso-called‘Hobbit’,orHomofloresiensis,who livedon the Asian island of Flores; and a mysterious species only recently mooted, calledDenisovanMan,orDenisovahominins,whoappearstohaveinhabitedpartsofAsia.All

members of the human evolutionary tree that evolved after our divergence from thecommon human-chimpanzee ancestor are referred to as ‘hominins’. We need todistinguish this term from a second umbrella term, ‘hominids’, which includes allhomininsaswellasallmodernandextinctGreatApes, includingchimpanzees,gorillasandorangutans.

A good place to begin our exploration of this human evolutionary tree is with thecommon ancestorHomo erectus, who first evolved in Africa approximately 2 millionyearsago.AnalmostcompleteskullofanearlyindividualwasunearthedinKenyaandanalmostcompleteskeletonofa juvenilewasdiscoveredneartoLakeTurkana,Kenya,byRichardLeakey’steam.Thisjuvenileskeletonwasattributedtoaboywho,judgingbyhisfive-foot-threestatureandskeletaldevelopment,wasinitiallythoughttobe12or13yearsofage.Lateronhisagehadtobeadjustedtoeightyearswhenscientistsdiscoveredthemeticulousaccuracyofcountingthegrowthlinesinteeth.ThisintriguedscientistssinceitsuggestedthatchildhoodinHomoerectuswasmuchshorterthanthatofhumanchildrentoday.

Homo erectus individuals were as tall as modern humans and they were even morerobustly built. They controlled fire in hearths and manufactured stone tools of the‘Acheulean’set,includingbeautifullysculpturedhandaxesandcleavers,whichdemandedthe ability for mental imagery and planning and would have enabled them to kill andbutcheranimals.Thereisalsosomeevidencethattheymayhavecaredfortheirweakandelderly.TheskullofH.erectuswasprimitive in its features,withaheavybrowridge,alow,flatvaultandprotrudingjaws,includingapoorlydevelopedchin.Thebrainvolumewasabouttwicethesizeofachimpanzee’s,atapproximately850ml,whichcomparesto1,300ml inanaveragemale today.Homoerectuscontinued toevolvewithinAfricabut,rather surprisingly, alsomigrated out ofAfrica at a very early stage in its evolution topopulate the Eurasian landmass in the earliest-known hominin migration. Famous H.erectus fossils include skulls and jaws fromDmanisi inGeorgia, dating to roughly 1.8millionyearsago.OtherfossilevidencefindsH.erectuscolonisingAsiaclosetoamillionyearsagoandtheMiddleEastandSouthernEuropeabout730,000yearsago,where,asinAfrica, the speciescontinued toevolvebiggerbrains longafter ithadspawned the fourdescendantspecies,includingourown.Fromthisoriginaldispersaltherearetwoschoolsofthoughtastohowmodernhumansmighthavecomeonthescene.

The‘multi-regionaltheory’pioneeredbyMilfordH.WolpoffsuggeststhatsubsequenthumanevolutionofHomoerectus in regionssuchasAfrica,Europe,AsiaandAustraliagave rise to distinct local lineages, albeit with some mixing, through mating, betweenregional groups and perhapswith peoplesmigrating out ofAfrica. The ‘out ofAfrica’theoryproposesthatallcurrentlylivinghumansaredescendedfromearlymodernhumanancestorsthatevolvedinAfricaroughly180,000yearsago,andwhomigrated,perhapsina series of waves, out into the rest of the world. Until recently these competingevolutionary theories were largely based on the fossil record and their associatedpalaeoarchaeology.Aswehaveseen,thehaplogroupgenetictracingtendsbyandlargetofavour the out-of-Africa scenario, but this leaves open the possibility of subsequentadmixtureofmodernhumanswithotherregionallydistributedspeciesofhumansthathad

evolvedfromearliermigrationsoutofAfrica,thusperhapsaddinginaregionalaspecttoourevolution.

Palaeontologists had assumed that it was exceedingly unlikely that they would everhavethechancetostudythegenomesoftheseancientcousinspecies,butnowthepicturewasradicallychangedbyPääbo’sspectacularbreakthroughingenetictechnology,withitspioneering of awhole new armof genetic investigation – including a newname for it:palaeogenetics.

Ininterviews,Pääboadmitsthathisworkinrelationtopalaeontologywasinspiredbythedesire toanswermanyquestions.Tobeginwith,wasitpossibletoextractanyDNAfrom such ancient fossils? Even if he managed to extract useful sequences, perhapsconfinedtomitochondrialDNA,whatwouldthistellusaboutourhumanevolution?Whatifhecouldextractsignificantnucleargenomicsequences?Wouldthishelptoclarifythedebate as to why the Neanderthals became extinct? Would it also answer importantunknownsaboutourownspecies’evolution?Forexample,wouldithelptoclarifywhichofthecompetinghypothesesforourownhumanoriginswascorrect?

Many of the most interesting and provocative questions were directed at theNeanderthals.What do we really know about them?Were they the stupid cavemen ofpopularprejudice?Inwhatlandscapeandecologydidtheylive?Howdidtheysurviveintermsoffoodandshelter?Whatwastheirsocietylike?Wouldtheyhaveunderstoodlove,family, friendship? How were they like us – and how did they differ? What possiblecatastrophecouldhavesobefallenapeoplewhohadsurvivedforaquarterofamillionyearsintheEurasianlandscapeonlytobecomeextinctwithintentofifteenthousandyearsofthearrivalintoEurasiaofmodernhumans?

*

Neanderthals are so named because one of the early fossilswas found in a cave in thevalley of the Neander River, in Germany. The ‘th’ diphthong is pronounced ‘t’, in theGermanmanner,soNeanderthalispronounced‘Neandertal’.TheearliestfeaturestypicalofNeanderthalsfirstappearintheEuropeanfossilrecordabout400,000yearsago,whichprobablymarkssomethingclosetotheirevolutionaryorigins.Theyaregenerallythoughtto have descended from an intermediary species,Homo heidelbergensis, which in turndescendedfromHomoerectus.NeanderthalfossilsandtoolsarefoundwidelythroughoutEuropeandwesternAsia,as fareastas southernSiberiaandas far southas theMiddleEast, before disappearing from the fossil record about 28,000 years ago,with themostrecent remains found in a cave inGibraltar.Andalthough theyareusually classed as aseparatespeciesfrommodernhumans,evolutionarybiologistsregardthemasourclosestandmostrecenthumancousins.

Whatdidtheylooklikeandhowdotheycomparetous?

Onaveragetheyappeartohavebeenshorterandstockierthanus,inparticularhavingshorterlegsandforearms.Theirstatureisthoughttobeanadaptationtolivinginacoldclimate.Theirskullswerelong,moreflattenedontopthanourown.Theyhadmuchmoreprominent eyebrow ridges and their noses were also much larger than we find in the

averagemodernhuman–Stringerimaginedthattheirnosesmusthavebeen‘remarkablyprominent’.Insomecasesthenasalbonejuttedoutnearlyhorizontallyunderthebrows.Theirfrontteethwereverylargeandoftenheavilywornevenwhencomparedwiththoseof their own ancestors,H. heidelbergensis. Stringer and Gamble have queried if thesefacialappearancesareanadaptationthatcameaboutbecausetheNeanderthalsmayhaveusedtheir incisor teethasanextraappendage.ManyadultNeanderthalsdisplayincisorsworndowntomerestubs,suggestingtheymighthaveusedthemlikeaportablevicewhileworkingsomematerial,perhaps,intheopinionofSharaBaileyatNewYorkUniversity,the processing of skins tomake leather goods.Their cheekswere swept back on eithersideofthenose,givingthecentralpartofthefaceamarkedprotuberance.Thelowerjawfollowedthisforwardprojectionoftheupperfaceand,likeH.erectus,thisresultedintheloss of a pointed chin.When compared to modern humans, Neanderthals also showeddifferencesintheirchestshape,thepelvicbonesandthelimbbones,whichtendedtobethicker, with wider, stronger joints, all of which suggest that the Neanderthals wereadapted tomore powerful physical activities and stresses thanwas the case with earlymodernhumans.

Contrary to theunenlightenedearlier interpretations,Neanderthalswereneitherstupidnor brutal. TheNeanderthal brain is slightly bigger in volume than ourmodern humanbrain, even today.Casts of their brains taken from fossil skulls show that they had thesametendencytoberight-handed.Intriguingly,theireyesocketswerelargerthanoursandtheoccipitallobesoftheirbrainswerealsolarger,suggestingthat,perhaps,theyhadbetternightvisionthanus,whichmighthavegiventhemasurvivaladvantageintheirhuntinglifestyle in the murky twilight of the cold northern climate, with its dark and drearywinters. Another intriguing difference was the time taken for childhood growth andmaturation.AswesawwithH.erectus,studiesbasedondentalgrowthlinessuggestthatNeanderthalchildrenmayalsohavematuredmorequicklythanmodernhumanchildren.

Weneedtoavoiddrawingderogatoryinferencesfromsuchobservations.Forexample,weshouldcompareNeanderthalchildren’sgrowthnottothatofmodernhumanchildrenbut to the fossils of early modern children, dating to the same time period as theNeanderthalsunderstudy.Butifthisprovestocarrytrue,thisshortenedchildhoodmighthave important cultural implications since so much learning takes place during theextendedperiodofdevelopmentofourchildren.

OneofthemorequestionabletheoriestoemergeinthepasthasbeenthesuggestionthatNeanderthalsdidnotpossesslanguage.Theymusthavehadlanguage,albeititwouldverylikely have been simpler thanwe humans possess today. They lived in hunter-gathererbands of perhaps a dozen or two, very likely extended families. Although fewNeanderthals appear to have lived beyond 40 years, there is some evidence that theypossessedknowledgeofherbalmedicine,cared for theirelderlyand lookedafter infirmindividuals.

WearecurrentlyinthemiddleofanextensivereappraisalofNeanderthalevolutionandculture inwhich some palaeontologists draw attention to the fact that theNeanderthalssurvived in Europe for roughly 250,000 years, despite very testing climatic conditions,

colonising a vast geographic territory. And now, as the first breakthroughs in geneticsbecameavailable,wefindthat theywerenotswarthyandcoveredwithblackhair,as inthe early drawings and models, but more likely fair-skinned, as we might have betterimaginedaspecieslongadaptedforsurvivalinthecoldnorthernclimatesofEurope.Still,thedie-hardsinsistedthatNeanderthalslackedtheonekeyfeaturethatlayattheverycoreof our advanced civilisation: they lacked the higher cognitive function that enabledcomplex language and symbolic thinking, the quintessentially human cognitivebreakthroughs that enabled symbolic art and subtleties of reasoning that came withlanguage.

In2001,ahumangenewas identifiedbyOxfordUniversitygeneticistsSimonFisherand Anthony Monaco, now known as FOXP2, which is important in our ability toarticulatelanguage.Mutationsofthegenegaverisetodifficultywithmusclecontrolinthevocal cords, tongue, and lips thatwereneeded to speak.Thismade scientistswonder ifthisparticulargenewasakeyacquisitionexclusivetomodernhumansandresponsibleforourevolutionoflanguage.

However, a singlegene is unlikely to code for our ability to speak.The evolutionofspeechandlanguageinvolvedcomplexchangesinthestructuresofthehumanvoicebox,or larynx, the throatand themobilityof thehuman tongueand lips,allofwhichwouldalso have required long-term and complex modification of the areas of the brain thatcontrol thought, sensation,movement and the coordination of these various body parts.Weknow that speech is dependenton thedevelopmentof specific regionsof thebrain,such as Broca’s area,whichwould hardly have evolved over the 200,000 years of ourseparationfromNeanderthals.Fromstudyofferalchildren,weknowthatsocloselyisthis‘speechmodule’ linked to culture that if a child is not exposed to picking up languagefrom parents and those teaching him or her up to around the age of seven, they neverdevelop proper speech. Remarkably, the late neurobiologist and avian scientist, PeterMarler,discoveredthatsomethingsimilarappliestothesongofbirds.

SociallythereissomeevidencethattheNeanderthalhunter-gathererbandsweresmallerinnumberthanthoseofearlymodernhumans,andtheyappeartohavebeenlessmobileandpossiblylessconnectedwithotherpopulationgroups.Theirtoolswereadaptedfromlocal resourceswhere the earlymoderns appeared to trade, andmove,morewidely.Atfirstanthropologistsbelievedtheydidnotproduceoriginalart,intermsofcavepaintingorpersonal ornaments, such as necklaces and sculptured items. But a modern reappraisalsuggeststhattheNeanderthalsweremoreinventivethanpreviouslyassumed.Theymadeuseofstringsome90,000yearsago.ThepresenceofNeanderthaltoolsonMediterraneanislandsconfirmedthat theyknewhowtocross thesea insomekindofcraft.Where theappearanceofcomplextoolsandshellorbeadbodyadornmentinNeanderthalsettlementswere thought to have been copied from arriving early moderns, a collection ofsophisticated bone tools, known as lissoirs, were discovered at two sites in southwestFrancethatweredatedtobetween45,000and51,000yearsago,which,iftrue,isseveralthousand years before earlymoderns arrived there. Some anthropologists still proposedthat,ontheweightofevidence,Neanderthalslackedthehighermentalevolutionimplicitinsymbolism,pointing to thebeautifully illustratedcavepaintingsofFrance,Spainand

much further afield in Australia; but others have cautioned that the wonderful cavepaintingswerenotproducedbyearlymodernhumansatthetimeoftheNeanderthals,butpaintedsome20–30,000yearsaftertheNeanderthalshaddisappeared–plentyoftimeforasubsequentculturalevolution.

In2010,agroupofscientistsfrommanydifferentuniversitycentrespublishedapaperonthesymbolicuseofmarineshellsandmineralpigmentsbyIberianNeanderthals.Theirpaper, headed by João Zilhão, reported the discovery of two Neanderthal-associatedMiddlePalaeolithicsites,onea largecavenear to theMediterraneanandanotherarockshelterby theMulaRiver,both inpresent-daySpainanddated to roughly50,000yearsago. Here the investigators discovered pigment-stained marine shells that had beendeliberatelyperforated to allow them tobe strung together into somekindof decoratedadornment. They also discovered lumps of yellow and red colorants and residuespreserved inside a Spondylus shell, together with many other tools and artefacts. Thesymbolic use of perforated and pigmented shells, used for necklaces, contradictedprevious cultural assumptions. It suggested that Neanderthals may have had a similarabilityforsymbolic thought toearlymodernhumans– theymightevenhavetaughtourarrivingearlymodernsathingortwoaboutthesophisticatedmanufactureanduseofbonetools.

As part of this reappraisal some palaeoanthropologists are coming round to a newperspective on theNeanderthals, arguing that theywere little different in their culturalevolution fromearlymoderns at the time they first emerged fromAfrica.For example,two American experts, P. Villa and W. Roebroeks, suggest that much of the earlierprejudicehadresultedfromafalsecomparisonbetweenNeanderthals,basedondigsitesdatingfromtheearlytomiddleStoneAge,andmodernhumansfrommuchmorerecenttimes.OthersarguethatevenifmodernhumansweremoreadvancedintheirculturethanNeanderthals, this does not necessarily reflect superiority in genetic or intellectualpotential,butthesortofdifferencesincultureseeninourmodernhumanhistory,broughtaboutbythedevelopmentandtransmissionofideas.

However, none of this debate reveals why the Neanderthals vanished off the globalmap, facing uswith themystery of their disappearance.Were they exterminated in thesamewaythatsomenativepopulationswereexterminatedbyarrivingEuropeancolonists?WeretheywipedoutbydiseasescarriedintoEurasiabymodernsarrivingoutoftropicalAfrica?Were they simply out-competed through the cultural superiority of the arrivingmodernhumans?Thesequestionshaddoggedpalaeoanthropologists foracenturyandahalf.SvantePääbo’snewdisciplineofpalaeogeneticswasabouttosuggestacompletelydifferentexplanationfortheextinctionoftheNeanderthals.

eighteen

TheFateoftheNeanderthals

EarlyinouranalysisofthehumanremainsfromLagarVelho,weproposedthatthechild’sskeletonpresentedevidence of prior blending of local Neanderthal and arriving early modern human populations in westernIberia.Ourinterpretationhasbeenwidelyacceptedasbothinterestingandreasonable,beingrejectedapriorionly by those who are intellectually immune to the idea of Neanderthal–modern human productiveinterbreeding.

JOÃOZILHÃOANDERIKTRINKAUS

There are, as we have seen, two quite different genomes within each human cell, themitochondrial and the nuclear. Since there are hundreds ofmitochondria in every cell,eachwith its own bacterium-derived genome, by far themost copiousDNA in a fossilboneisgoingtobemitochondrial.Themitochondrialgenomeisalsomuchsmallerthanthe nuclear genome. So the logical place to begin the exploration of the NeanderthalgenomewaswiththeextractionanddeciphermentofthemitochondrialDNA.

After some preliminary sequence discoveries over a number of years, in 2008Pääboand his team at theMaxPlanck Institute,working in cooperationwith colleagues fromAmerica, Croatia and Finland, published the first complete sequencing of NeanderthalmitochondrialDNA,which they had extracted from a 38,000-year-old fossil bone. Thediscoverywasgroundbreaking,notmerelyinthehistoricsensebutalsothesignificanceoftheir findings: ‘It establishes that the Neanderthalmitochondrial DNA falls outside thevariationofextanthumanmitochondrialDNAs.’

Thisconfirmedthelong-heldviewthatNeanderthalswereadifferentevolutionarylinefrommodern humans. In applying the customary search for nucleotide polymorphisms,Green and colleagues discovered far more Snip-type differences between Neanderthalsand humans thanwe see across the globalmodern human divide.Given thatmutationsoccuratroughlypredictableintervals,asinthemutationalclock,‘[It]allowsanestimateofthedivergencedatebetweenthetwolineagesof660,000plusorminus140,000years.’This came close to the divergence date previously proposed by palaeontologists on thebasisoffossilsandarchaeologicalfindings.

ThepaperalsopredictedsomethingthatmightprovehelpfulinworkingoutthefateoftheNeanderthalsafter thearrivalofearlymodernhumans intoEurope, theMiddleEastandAsia: ‘There is evidence that… the effective population size ofNeanderthalswassmall.’ By effective population size, the scientists were referring to the Neanderthalspecies gene pool – or, to put it another way, the genetic diversity of the Neanderthalspecies.Weneedtobecautiousininterpretingsomethingassweepingasthisfromafirstdraftoftherelativelysmall,andentirelyfemale-associated,mitochondrialgenome.Butif

this were to be confirmed from study of the nuclear genome, it would have importantimplications for the impactof interbreedingbetweenNeanderthals andmodernhumans.For themoment letusflag thisupassomethingtokeepinmind.MeanwhileweshouldconsiderthesuggestedtimelinefortheseparationofthemodernhumanandNeanderthallineages.

Both the palaeontological and genetic evidence is pointing to a divergence of themodernhumanandNeanderthal lineagesroughlyhalfamillionyearsago.Basedon thestudyofothermammals, this is a relatively short time forcomplete separation into tworeproductively distinct and separate species, which is generally thought to requiresomethinglikeamillionyears.Soletusflagthatupasasecondconsiderationtokeepinmind.Somereadersmighthaverealisedtheoddityposedbythistimingofdivergence;ifthe Neanderthals and modern humans began their separate evolutionary trajectoriesroughly 500,000 years ago, how do we explain the purported African origins of themodernhumanevolutionarylineat180,000,perhapsatmost200,000yearsago?

In fact there is no contradiction. There is a common consensus that both speciesevolved, albeit throughdifferent lineages, froma common ancestor,H.erectus. For thefirst 300,000 years after separation, both lineages would have continued to evolve,presumablywithoutcontactwithoneanother,modernhumansinAfricaandNeanderthalsinEurasia.And thus,by180,000yearsago theyhadbecomesufficientlydifferent tobeconsidereddistinctevolutionarylineages,headingtowardsbecomingseparatespecies.InNeanderthal terms, there isageneralconsensus that the intervening intervalbetweenH.erectus and the Neanderthals was filled by the intermediate ancestral lineage of H.heidelbergensis.ThisalsoleavesopenthequestionastowhetherH.sapiensevolvedfromH.heidelbergensis–albeitgivenadifferentname–inAfrica.

Puzzledaboutthis,IwrotetoProfessorStringer,thehumanoriginsexpertattheNaturalHistory Museum in London, and essentially he confirmed that there were indeed twoalternativetheories.OnetheoryproposedthatboththeNeanderthalandthemodernhumanlineageswentthroughtheintermediatestepofH.heidelbergensis,probablyseparatelyinAfricaandinEurope.TheothertheoryproposedthatonlytheNeanderthal lineagewentthrough theH.heidelbergensis stage inEurope:meanwhileH.sapiens evolved directlyfromH.erectusinAfrica.Herethenisanothersituationthatwemightkeepinmindwithrespecttoanyfuturegenomicrevelations.

All along Pääbo had made clear that it was his ambition to reconstruct the entireNeanderthalnucleargenome.In2007,ayearbeforethemitochondrialannouncement,andthesameyearhewasnamedoneofTimeMagazine’s100most influentialpeopleof theyear, European researchers reported that Neanderthals had a gene mutation that wasassociatedwith the fair skin seen inmodernEuropeans. This samemutationmay havegivensomeofthemredhair.Thefollowingyear,anotherpaperreportedthatNeanderthalshadthesameObloodgroupasmodernhumansandtheysharedthelanguage-associatedgene,FOXP2,withus.In2009,attheannualmeetingoftheAmericanAssociationfortheAdvancementofScience,itwasannouncedthattheMaxPlanckInstituteforEvolutionaryAnthropology had completed the first rough draft of the Neanderthal nuclear genome,

whichincludedsomefourbillionoftheestimatedtotalof6.4billionnucleotide‘sleepers’ofDNAextractedfromthefossilbonesof threeindividuals.Thiswasdulypublishedin2010.

Thefirstandthepredictableoutcomewasthatmorethan99percentoftheNeanderthalgenomewasidenticaltoourown.Thiswashardlysurprisingsincewehadevolvedfromacommonancestoronlyhalfamillionyearsearlier.Muchofourgenome,andinparticulartheprotein-codingportion, has todowith everyday aspects of our inner chemistry, cellstructures, cell repair, cell death and removal, and cell regeneration, as well asimmunologicaldeterminationofselfandtheconstantbattleagainstmicrobialdisease.Wewouldexpecttheseportionsofthetwogenomestobelargelyidentical.Amoresurprisingdiscovery was that the Neanderthal genome showed more commonality with modernhumanswhohadevolvedinEuropeandAsiathanwithmodernhumanswhohadevolvedinAfrica.Inscientificparlance,‘aparsimoniousexplanationfortheseobservationsisthatNeanderthalsexchangedgeneswith theancestorsofnon-Africans’. InordinaryEnglish,ourmodernhumanancestorsinterbredtoasignificantdegreewithNeanderthals.Toputitmorebluntlystill,someofourdistantgrandparentswereNeanderthals.

ThefindingssuggestedthatpeopleofEuropeanoriginsinheritedbetween1and4percentoftheirnucleargenomicDNAfromNeanderthalancestors.ThepreliminaryevidencesuggestedthatpeopleofAsianoriginsmayhaveinheritedevenhigherpercentagesoftheirDNA from their distantNeanderthal ancestors.As the paper expressed it,Neanderthalsappeared to be as closely related toChinese andPapuaNewGuineans as theywere toEuropeans,despitethefact thatNeanderthalfossilshaveonlybeenfoundinEuropeandwestern Asia. Perhaps early moderns had already mixed with Neanderthals part waythrough theirmigration, taking theirNeanderthal ancestrywith them in their expansionintoEurasia.

Justhowcommonplacewastheinterbreedingbetweenthetwopopulations?

Previous studies of hybridisation in evolutionary biology had suggested that when acolonising new population encountered a resident population, even a small number ofbreedingeventsalongthewaveofinteractioncanresultinsubstantialintroductionofnewgenes into the colonising population. It’s all a question of population numbers andsubsequentpopulationexpansion.Itseemsthattheincominggenes–Neanderthalinthissituation–can‘surf’tohighfrequencyasthecolonisingpopulationexpands.Thereis,ofcourse, an alternative, simpler explanation. Perhaps interbreeding between modernhumansandNeanderthalswastrulycommonplace?

ThenewsthatEuropeansandAsianswerepartNeanderthalintheirgeneticoriginshitthe popular media like a bombshell. What should have been anticipated, really, givenhumanbehaviour,nowprovokedastonishment.

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Fromthefirstdiscoveryoftheirfossils,Neanderthalshadbeenthesubjectoffascinationasarivalspeciestoourown–buttheyhadalsobeenthesubjectofenormousprejudice,and this, alas,was true of professionals and laymedia alike.MarcellinBoule, the first

experttoreviewtheirfossilremains,paintedapictureofabrutallyapishcreatureinwhichthere could only be ‘rudimentary intellectual faculties’ and in which ‘all traces of anypreoccupations of an aesthetic or of a moral kind’ were unimaginable. This pejorativepatterndominatedtheopinionofNeanderthalsforthefirsthalfofthetwentiethcentury.Itwas an unfortunate accident of fate that Boule’s post-mortem examination featured thepartialskeletonofanoldermalewhohadbeenbadlymaimedinsomeaccident,provokingsevere secondaryosteoarthritis.Thisprejudicial interpretation fromsuchadistinguishedsource influenced opinion far beyond the objective world of archaeology andpalaeontology.

H.G.Wellswroteastoryfeaturingthedeservedexterminationofwhathelabelledthe‘grisly folk’.Whilemore recentwriters, such asWilliamGolding and JaredDiamond,presentedamoresympatheticviewoftheNeanderthals,theyalsoassumedtheirextinctionat the hands of our more advanced ancestors, even though there is no hardpalaeontologicalevidencetosupportit.

This bigotry prevailed until relatively recently, with anthropologists arguing thatNeanderthalscouldn’tspeak,oriftheydidsoitwasinapeculiarvoice,thisinspiteofthefacttheyhadbrainsslightlylargerthanourownandthespeechareaoftheirbrains,knownasBroca’sarea,waslittledifferenttoo.Thelargebrainwasdownplayedaspoorinquality.Theirtools,typicalofwhatiscalledtheMousterianculture,werelesssophisticatedthanthoseofmodernhumans.Inparticulartheyappearedtolackthrowingspears,compellingthem to face up directly to the huge prey they hunted, such aswoollymammoths andrhinoceroses.

There were, of course, very real differences in skeletal morphology betweenNeanderthalsandearlymodernhumans,butresearcherschosetofocusonthedifferenceswhile ignoring the similarities. In the last two decades a growing number ofpalaeontologists have re-evaluated the evidence for Neanderthal society and culture,concludingthatpreconceivednotionsmayhaveledearlierresearcherstoignoretheactualevidenceforNeanderthalinventivenessandculture.Theirbrainshadlargefrontallobes–the regions associated with intelligence and culture. Ralph Holloway at ColumbiaUniversity inNewYorkhas studiedhundredsof brain casts fromNeanderthal skulls toconfirmthattheyhadthesamelevelofdevelopmentofthespeechareaaswedo.SitesinFrancehaveconfirmedthatNeanderthalsdidn’tjustoccupycavesandrockoverhangsbuttheyerectedshelters, leavingtracesofthesupportivewoodenposts.Theirtoolsarenowseen as difficult to manufacture, involving planning, vision and great skill. There isgrowingevidencethattheyworeclothesandthattheyadornedthemselveswithsymbolicartefacts, including perforated and painted shells. They buried their dead, perhapswithceremony,andmayhaveplayedmusic.Andalthoughtheyhuntedformidableprey,suchas rhinos, mammoths and bison, they also adapted their strategies to suit differentenvironments,trappingandhuntingbirdsandrabbitsandgatheringseafood.

Penny Spikins, and her colleagues at York University, identified at least three sites:WansuntRoadinKent,FoxhallRoadinIpswich,andRhenenintheNetherlands,whereminiatureNeanderthalhandaxes,whichwere likely tobechildren’s toys,havecometo

light.InArcy-sur-CureinFrance,andasiteinBelgium,otherpalaeoarchaeologistshavediscoveredcollectionsofexpertlychippedstonetoolsnexttosomeveryinexpertattempts,which suggestNeanderthal adults teaching children how tomake them in a StoneAgeequivalent of schooling. The role of emotions, such as compassion, is central to thebehavioursandintimaterelationshipsthatdefinehumansociety,butpalaeontologistshavebeen cautious about extrapolating emotional significance to archaeological findings.Spikinsandhercolleagueshaveattemptedtoexplorescientificconstructsthatmightoffera basis for such studies, looking for the evidence of empathy and compassion inarchaeologicalcontexts fromtheearliestarchaichumans tomodernsociety.Theyfoundsuchcompassion,incaringforsickandinjuredindividualsinNeanderthalsitesfrom‘theOldManofShanidar’,whosufferedfromterribleinjuries,totheSimadelosHuesos–thepit ofbones–where a child suffering fromahereditarydisease affecting the skullwascaredforuptoitsdeathattheageofeight.

Given this accumulation of evidence, Erik Trinkaus at Washington University in StLouis, Missouri, has concluded: ‘If you look at the archaeological evidence ofNeanderthalsandmodernhumans…theyareverysimilar.Neanderthalswerepeople,andtheyprobablyhadthesamerangeofmentalabilitieswedo.’

Ofcourse,noteverybodyagrees.Somedistinguishedresearchers,suchasMellars,thinkthatNeanderthals,while skilled enough to survive in theirEurasian landscape formorethan 200,000 years, were less cognitively equipped than contemporaneous modernhumans.HearguesthatbythetimemodernhumansarrivedintoEurope,theyhadbettertechnology,better socialorganisationand,by inference,betterbrains.StevenMithen,atthe University of Reading, agrees with him. Theymay be right. However, we need todistinguish social evolution from the hereditary ability for intellectual thought.We alsoneed to be careful to compare likewith like – in otherwords, to compareNeanderthalculturewithmodernhumancultureduringthesameera.Oneonlyneedstoreflectonthemassive cultural differences between different modern human populations a century ortwoago–whichweremoreassociatedwitheducationand the inheritanceof ideas thandifferencesinheredityorinherentintellectualabilitybetweenpopulations.

The encouraging momentum that is now sweeping like a tsunami of potentialadjudicationthroughsuchpassionatedebateis that, thankstoPääboandhisdiscoveries,the hard facts of genetics can now be combined with the equally hard facts ofarchaeologicaldatingtechniques,anditwillbefactsandnotprejudicialsuppositionthatwillultimatelydefinefuturebelief.

Meanwhile,therearesomerelevanthardfactsthatshouldbetakenfullyintoaccountinthefast-developingscenario.

*

Hybridisation,or sexualcrossingbetweendissimilar species–or subspecies– isoneofthe four major mechanisms that give rise to the hereditary genetic change that makesevolutionpossible. It can,where therearemajorgeneticdifferencesbetween thehybridpartners,giverisetoseriousgeneticdysfunction,includinginfertility.Scientistswhostudyhybridisation in animals andplantshave found that the closer the evolutionary lineages

involved, the shorter the time of separation from a common ancestor, and thus the lessdifferentthetwogenomesandthereforethemorestablethegeneticoutcomes.ThegeneticinterbreedingbetweenNeanderthalsandourmodernhumanancestorshardlygaverisetoinfertility sincewe are the hybrid descendants.And theNeanderthal component of ourEurasiangenomestodayisnotsmall.Atitsupperrangeof4percent,thisisthegeneticlegacy you or I would have inherited from a great-great-great grandparent from just acenturyago.AtDNAlevel,thisishuge.Oneneedonlydivide6.4billionsleepersby25toseehowmanysleepersourtrainwouldhavetotraverseintheexplorationofthisgenomiccontribution – 260million – all making up distinctive genes, viral sections, regulatorysequencesandnon-codingRNAs.

Someauthorsappeartobemissingthepointininterpretingsuchamajor‘hybridisationevent’.Thepoolingoftwodifferentevolutionarylineageswillgiverisetoanimmediatedramatic increase in genetic diversity in the hybrid offspring, an increase that will beinheritedbythedescendantsinallfuturegenerations.

Wemight considerwhat this implies: that the two genomes contain differing genetichistories, includinggenetic adaptations,withpotential advantages for survival that havebeen hard won in different ecologies and environments. Studies of the effects of such‘hybrid creativity’ in nature suggest that the hybrid offspring may be better able towithstandtoughenvironmentalconditions, includingharshclimates, thanthenon-hybridparental lineages.Naturalselectionwillnotchooseone lineageoveranother.Sincebothlineagesarenowcommontoasinglegenome,selectionwillnowworkatthelevelofthehybridgenome,justasitworkedattheleveloftheholobionticgenomeincasesofgeneticsymbiosis.Geneticsequencesthatimpairsurvival,regardlessoftheirspeciesorigins,willbe selected against; meanwhile, genetic sequences that enhance survival, regardless oforigin,willbefavoured.ThefactthatwestillretainahugeNeanderthalcomponenttoourgenomic make-up speaks for itself. It strongly suggests that our ancestors did gain anadvantageforsurvivalinthesexualcrossingwithNeanderthals.Moreover,itisn’thardtoseewhythismightbeso.

Ourmodernhumanancestorshadevolved in the tropicalheat andbright sunshineofAfrica. They encountered Neanderthals when moving into a much colder, less sunnyenvironment inwhich thewinters inparticularwouldbe long, cloudyandgloomy.Onekey effect of thiswould have been a failure tomake enough vitaminD in their darkerskins.GiventheeffectsofvitaminDdeficiencyinsofteningbones,orinweakeningtheimmune system, thisNeanderthal contributionwould have given the hybrid offspring abetterchanceofsurvival.

But thebenefitsofcooperationbetween the twopopulationsmaywellhaveextendedfarbeyondincreasedgeneticdiversity.Howlikelywasitthattherewouldhavebeensocialandculturaladvantagesofexchangebetweenthetwodifferentpopulations?ThiswasnomeetingofEuropeansbearing the fruits ofRenaissance enlightenment, or the IndustrialRevolution, into aStoneAgehunter-gatherer society.Both populationswere still at theStoneAgehunter-gathererstage.Theexchangeofknowledgeof the localgeography,offlora and fauna, of seasonal availability of foods, sources of shelter, perhaps evolved

techniques ofworking skins or other items of clothing, herb lore – and, perhaps,moresymbolicaspectsofculture,suchasdecoration,music–wouldhaveworkedbothways.

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During the search for Neanderthal fossils that grew up around Pääbo’s genomicexploration,RussianarchaeologistshadbeendigginginthefloorofagiganticcavernhighupintheAltaiMountainsofsouthernSiberia.Theclimateonthenorthernslopeswherethe cave is situated is arid andcold,with ameanyear-round temperatureof about zerodegreesCentigrade.In2010thearchaeologistsdiscoveredatoebonefromaNeanderthalfemale from a layer dated to approximately 50,000 years ago. This bone proved to beincredibly rich in archaic DNA, enabling Pääbo’s group to extract the best-qualityNeanderthal nuclear genomic sequences to date. In January 2014 scientists frommanydifferent centres and laboratories combined forces with Pääbo’s group to analyse andreport the complete Neanderthal nuclear genome of this individual, comparing andcontrasting it with the human genome and with what was already known from theprevious examination of Neanderthals from widely different geographic localities,including three Neanderthal individuals from the Vindija Cave in Croatia and aNeanderthalinfantfromtheMezmaiskayaCaveintheCaucasus.

Werewetoreturntoourmagicaltrain,wouldwerecognisethesectionsoftrackthatweowetotheNeanderthalpartofourheritage?

In March 2014 three different Harvard-based groups of evolutionary geneticists,includingDavidReichandcolleaguesbasedattheDepartmentofGeneticsattheHarvardMedicalSchool,andgroupsattheBroadInstituteandHowardHughesMedicalInstitute,combinedforceswithPääbo’sgroupattheMaxPlanckInstituteinGermanytopublishanoverview on the genomic contribution of Neanderthals to present-day humans. In theopinionof theseauthors, theanswer tomyquestion is adefiniteyes.Althougha lotoftimehasgonebysincethehybridisationevent,theNeanderthalchunksoftrackarereadilydistinguishablebecauseoftheirhaplotypes.Whereatthetimeofhybridisationfullyhalfthegenomeof the firstoffspringwouldhavebeenNeanderthal, thedilutionaleffectsoftimeandasuccessionofwithin-speciesmatingshasreducedtheNeanderthalsectionstosequencesofDNAlessthan100,000sleeperslong–smallperhapsagainsttheoverallsizeofthegenomeat6.4billionsleepers,butenoughtoofferaseriesoflengthyjourneystoour magical steam train. In the words of the authors: ‘Neanderthal haplotypes aredistinctive enough that several studies have been able to detectNeanderthal ancestry atspecificloci.’Whatthendidtheyactuallydiscoverwhentheyvisitedthegenomesofsome1,004present-dayhumans, examining the track inminute detail and taking a longhardlookattheseregionsthatareidentifiablyNeanderthal?

TheReichgroupconcludedthatregionsofourmoderngenomethatareparticularlyrichinNeanderthalgenes includedthosecodingfor theproteins thatmakekeratinfilaments.Keratinisthestructuralmaterialthatmakesuptheouterlayerofourskin,andinmodifiedformisthemainstructuralcomponentofourhairandnails.Oneofthespecificgenestheymayhavebequeathedus isBNC2,which is involved in skin pigmentation.Researchersbased in theUniversity ofArizona also discovered that a proportion of Eurasians, and

especially Melanesians, had inherited the genetic region known as STAT2 fromNeanderthalancestors.STAT2ispartofthesystemthatdeterminestheidentityofselfandourabilitytofightinfections.

This samegenetic analysis confirmed that theNeanderthal skin colourwas light, butprobablyasvariableasweseeinmodernEuropeans.Theireyecolourrangedfrombrowntoblue,orblue-greenorpossiblyhazel.Thoseofuswhohaveinheritedthelighterskinsof Western Europe may have inherited part of our appearance from our Neanderthaldistantgrandparents.Wemayalsohaveinheritedourinclinationtoredhairandfreckledskin,withthetendencytosunburnthatgoeswithit.Moreover,asareportintheSundayTimes newspaper explained, part of our Neanderthal legacy may include a particularvariant of theMajorHistocompatibilityComplex– the part of our genome that definesselfanddealswithforeign invaders– that increases thegeneticriskofspecificdiseasessuchastype2diabetes,lupusandCrohn’sdisease.

Onalighternote,whenProfessorStringertestedBritishcomedianBillBaileyandBBCsciencepresenterAliceRobertsforthelevelsoftheirNeanderthalancestry,hefoundthatBaileyhadinheritedabout1.5percentofhisgenomefromtheNeanderthals,andRobertshad inherited2.7per cent.Stringer’sown legacy lay inbetween, at 1.8per cent. Inhisbook,Pääbowouldrelatesomecomicalextrapolationsofthehybridisationgoingpublic,withmen andwomenwondering if theirNeanderthal inheritance explained the odditiestheyhadobservedintheirownappearance,orbehaviour,ortheodditiestheyattributedtotheirspouses.

Thedeeper explorationofourhybridorigins andevolution isonly just beginning. InparticularscientistshaveonlybeguntoexaminethepotentialNeanderthalcontributiontomore subtle aspects of our physiology, immunological identity of self, our ability tocounteract disease, including infections, and the differences, if any, between modernhumansandNeanderthalsintermsofintrinsicbraindevelopment,withitsapplicationstocognition,creativityandthemanysocialandculturalaspectsofsocietalmake-up.

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Asurprisingdiscovery in thegenomeof theAltai femalewas apeculiarly low levelofgeneticdiversity.Sheappearedtobetheoffspringofinbreedingparentswhowererelatedatthelevelofhalf-siblings.Occasionalinbreedingisafeatureofhunter-gatherersocieties,includingearlymodernhumans.ButthisfindingintheAltaigenomeraisedanimportantquestion:weretheNeanderthals,whoappearedfromthearchaeologicalrecordtoliveinrelatively small groups, with little evidence of wider mobility when compared tocontemporaneousmodernhumans,moreatriskofinbreedingthanearlymodernhumans?Thiswill need to be explored furtherwith high-quality sequencing of awider range ofNeanderthal genomes, but if inbreeding was significantly commoner in NeanderthalgroupsitmightexplaintheimpoverishmentingeneticdiversityseenintheAltaigenome.Thisinturnwouldhaveincreasedtheriskofinbornerrorsofmetabolism.Anindividualinheriting a single recessive gene fromone of her parentswould be protected from thediseaseifsheinheritedanormalvariantofthegenefromtheotherparent.Ifbothparents

werecloselyrelated,as intheAltaifemale’scase, therewouldbeasignificantlygreaterriskthatshewouldinheritthedefectivegenefrombothparents.

Another importantrevelationof theAltaigenomewasitssuggestionofasurprisinglylow population of Neanderthals at this late stage in their occupation of the Eurasianlandmass.Itraisesavitallyimportantquestion:whatweretherelativepopulationsizesofNeanderthalsandmodernhumansatthetimewhenthetwopopulationsmetinEurasia?

OfpotentialrelevanceistheclimaticcatastropheknownastheLastGlacialMaximum,which afflicted the ecology of the Neanderthals roughly 48,000 years ago, a freeze soseverethatvastareasofthelandwereburiedunderglaciersmilesdeepandvasttractsofthe northern Atlantic Ocean were similarly frozen over. There is evidence that thisdecimatedthepopulationofanimalsandpeoplesurvivingthroughoutEurasia.Couldthisexplainthereducednumbers,andtendencytoinbreeding,ofthesurvivingNeanderthalsatthe time, a catastrophe that still afflicted their numbers some five thousand years laterwhenourmodernhumanancestorsarrived?

We are now in a position to take a more measured look at what might really havehappenedtotheNeanderthals,whoweremoresimilartoourselvesthanwehadpreviouslythought. Their populations may have been reduced to relatively small and infrequenthunter-gatherer bands by the time our ancestors met them. This makes all the morerelevantMellars’andFrench’ssuggestionthatatthetimeofearlyco-habitationofEuropebythetwopeoples,ourancestorsmayhaveoutnumberedtheNeanderthalspopulationbyroughly ten to one.Whilewe cannot rule out skirmishes, or even the extermination ofsomepopulationgroups,wemightquestionhowpurposefulhostilitiesormassacresmighthave been in such circumstances.We know that the two populations interbred, perhapswith less inhibition and on a larger scale than some would like to imagine. Suchinterbreedingbetweenasmallandamuchlargerpopulationislikelytoresultinthebulkofthesmallerpopulationbeingassimilatedintothelarger.

CouldthisthenbetheexplanationforthemysteriousfateoftheNeanderthals?

ItwouldcertainlyexplainwhywefindnoneofthepathognomonicskeletalfeaturesintheEurasianpopulationwithin10,000to15,000yearsofmodernhumanarrival–thisisplenty of time for the Neanderthal cranial and skeletal features to be melded into andswallowedupwithintherapidlyexpanding,andstillevolving,largerpopulationofearlyHomosapiens.Ifthisiswhathappened,theNeanderthalsdidnotbecomeextinct–oratleast not in the way we previously imagined. They disappeared as a separate,distinguishablepopulationbutliveonasanintegralpartofourownhereditarypedigree.

InApril2014,PaoloVilla,fromtheUniversityofColorado,andWilRoebroeksfromLeidenUniversityintheNetherlands,teameduptowriteanoverviewofwhattheytermed‘the Modern Human Superiority Complex’, by which they meant the overweeningassumptionbymanyscientistsandpublicalikeastooursuperiorityovertheNeanderthalcave men and women. They concluded: ‘This systematic review of the archaeologicalrecordsofNeanderthalsandtheirmodernhumancontemporariesfindsnosupportforsuchinterpretations,astheNeanderthalarchaeologicalrecordisnotdifferentenoughtoexplainthe demise in terms of inferiority in archaeologically visible domains.’ Instead they

proposed thatcomplexprocessesof interbreedingandassimilationwas indeed themorelikelyexplanationforthedisappearanceoftheNeanderthalskeletalandphysicalfeaturesfromthefossilrecord.

*

It is an extraordinary story, brought about by extraordinary scientific breakthroughs ingeneticresearch,butstillthereweremoresurprisestocomeduringthefruitfulperiodinwhichPääbo’sbreakthroughwasbeingappliedtohithertorefractoryfossilbones.

InJuly2008,aRussianarchaeologist,AlexanderTsybankov,wasdigginginthefloorofthe same cathedral-like cavern high up in the Altai Mountains of southern Siberia.Working through deposits dating back 30,000 to 50,000 years, Tsybankov discovered afragmentofasinglefingerbone,anunprepossessingchipfromthetipofthelittlefinger.Whenheshowedhisfindtohisboss,AnatolyDerevianko,thelatterthoughtthatthebonewasprobablymodernhuman.Thiswouldexplainhowitcametobeinthesamedepositassomesophisticatedartefacts,includingabraceletofpolishedgreenstone.ButNeanderthalremainshadpreviouslybeenfoundinthesamecavesoDereviankochoppedthefragmentofboneintotwoparts,puttingthesmallerofthetwointoanenvelopeandarrangingforittobehanddelivered toPääbo inGermany for genetic analysis.The tiny sliver of bonearrived just as Pääbo’s team were about to complete the first draft sequence of theNeanderthal nuclear genome, and the team were already very busy, with a backlog offossilswaitingtobeexamined.TheRussianbonewenttothebackofthequeue.

Itwasn’tuntil late2009thatPääbo’scolleague,JohannesKrause,assistedbyChinesegraduatestudentQiaomeiFu,foundthetimetoperformapreliminaryscreenoftheAltaibone’smitochondrialDNA.Minuscule as the fossilwas, it proved to be incongruouslyrich in DNA and it was also relatively uncontaminated. And what they found was sostartling that they were obliged to repeat the analysis. The strange findings wereunchanged.AnexcitedKrausepickedupthephoneandcalledPääbo,whowasattendingameeting atColdSpringHarborLaboratory inNewYork.Krause began by asking him:‘Areyousittingdown?’

Hewasn’tsittingdown.

‘Maybeyouhadbetterfindachair?’

Pääbowouldsubsequentlyconfessthathehadindeedfoundhimselfachairbecausehefearedthatsomethingterriblehadhappened.ThemitochondrialgenomeextractedbyFuwas not that of aNeanderthal. It was so surprising thatKrause had counter-checked itbecausehecouldn’tbelieveFu’sfindings.Hehadtheninsistedoncomparingittoallsixversions of theNeanderthalmitochondrial genomes now filed in their records.Withoutquestion,itwasnotNeanderthal.Nomorewasitthemitochondrialgenomeofanyofthemodern humans that had been sequenced from all around the world. Whereas theNeanderthal mitochondrial genome had differed from that of modern humans in 202nucleotides,orSnips,thisdifferedin385.Thestaggeringtruthwasthatitwasn’tlikeanymitochondrialgenomethey,oranybodyelse,hadeversequencedbefore.

TheimplicationsflashedthroughPääbo’smind.Coulditbe that theyhadturnedupahitherto unknown species of human? Working on the basis of the Snips, if theNeanderthalshadsplitfrommodernhumansaround500,000yearsago,thenthisspeciesmusthavesplitfromacommonancestormaybe800,000yearsago.YetamemberofthissamespeciesmusthavebeenaliveinSiberiasome50,000to30,000yearsago?InPääbo’srecollection:‘Myheadwasspinning.’

WhenhegotbacktotheMaxPlanckInstitute,somethreedayslater,hetalkedoverthefindingswithKrause.Thesliveroffingerbonewas,itseemed,almostmiraculouslyrichinDNA.Forexample,theverybestsourceofNeanderthalDNAinthefossilbonestheyhadtestedhadyielded4percent,butthenewboneyielded70percent.Notonlyhaditcome from an amazing human source, it had experienced an equally amazing level ofpreservation.Nevertheless,PääboinsistedthatKrauseandFurepeattheanalysisonwhatlittlewas left of the tiny bone.The resultswere exactly the same.He emailedAnatolyDereviankoandtheyarrangedtomeetattheInstituteofArchaeologyandEthnographyinAkademgorodok, a Russian city that had been purpose-built for science by the Sovietregime in the 1950s. Pääbo arrived in deepwinter, with an ambient temperature of 35degrees below zero.He knew that the sliver of bone had been part of a slightly largerwholeandheaskedfortheremaindertoworkonthenucleargenome.ButtheytoldhimtheyhadsentittoacolleagueinAmerica,whoappearedtohavelostit.

Chagrined, the German team returned to Leipzig, though they had succeeded inbringingbackamostunusualtooththattheRussianarchaeologistshadrecoveredfromthesame cavern in the Altai. The tooth was unusually large in size and primitive inappearancewhencomparedtothatofamodernhumanoraNeanderthal.

On10April2010,Pääbo’steamandtheirRussiancolleaguespublishedtheirfindingsoftheextraordinarymitochondrialgenomeintheLetterssectionofthejournal,Nature.Itwas a unique paper, the first time an extinct human species had been discovered bygenomicanalysisofafossilbone.Inthescientists’opinion,themostlikelyexplanationoftheAltaifingerboneisthatitrepresents‘ahithertounknowntypeofhominin’–onethat,averylongtimeago,sharedacommonancestorwithmodernhumansandNeanderthals.Therewere some ‘exceptionally archaic features’ to themitochondrialDNA that talliedwiththearchaicnatureofthetwoteeth.Thedifferencesbetweenthisnewhomininandtheothertwoweresomarkedthattheyconsideredformallyannouncingitasthediscoveryofanewspecies,butthen–prudently,asitturnedout–theychangedtheirminds.

The cave where the sliver of bone was discovered had been inhabited by a hermitnamedDenis back in the eighteenth century. So they decided that theywould label thenewlydiscoveredpeoplethe‘Denisovans’.

Andstillthelitanyofsurpriseswasgrowing…

ThetinychipofbonewassorichinDNAthattheymanagedtoextractahigh-qualitynuclear genome from it. InDecember of the same year, Pääbo’s team combined forceswithDavidReich, ofHarvardMedical School, and colleagues from a large number ofother institutes inAmerica, Germany, Spain, China, Canada and Russia, to publish theDenisovannucleargenome.Theycouldnowconfirm that at the timewhenourmodern

human ancestors emerged in Africa, around 180,000 years ago, a number of cousinspecies shared the world with them. First they dealt with the modern human andNeanderthalsplit.Theygauged,fromthetwodifferentgenomes, that thehumanlineageand Neanderthal lineages had separated into separate lines a little later than they hadearlier surmised, between270,000 and440,000years ago.This confirmed that the splitwas probably too recent for complete species separation, thus allowing interbreedingbetween the two emerging species with fertile hybrid offspring. Examination of theDenisovan genome and comparison with that of Neanderthals suggested that theDenisovans had also shared a common ancestor with the Neanderthals, but the splitbetween these lineages had been much earlier than that of Neanderthals and modernhumans, at roughly 640,000 years ago. The last shared ancestor of the Denisovan andhumanancestrallineageswasputevenfurtherback,atroughly800,000yearsago.

ThecomparativegeneticsshowedthattheDenisovansweremorecloselyrelatedtotheNeanderthalsthantheyweretous,butnotsoclosethattheyextensivelymatedwiththem.It also confirmed that theywere notmerely a subgroup ofNeanderthals but a separatespeciesvery likely inhabitingawidegeographicareaofAsia, andwithanevolutionaryhistory distinct from that of modern humans and Neanderthals. Some of the geneticsequencesintheDenisovangenomelookedsoprimitivethatitmadethemwonderiftheDenisovans had acquired these sequences through hybrid crossingwith amore archaicspeciesstill.Thelatterwasdescribedasanas-yet-unidentifiedspecies,butthemostlikelycandidateisfascinating:itcouldsignifyourfirstglimpseintothegenomeofthecommonancestor of all three species – modern humans, Neanderthals and Denisovans – theamazingglobaltravellerandpioneerofearlyhumanity,Homoerectus.

Like theNeanderthals, theDenisovans had interbredwith earlymodern humans, butwhere theNeanderthalshadcontributed tomostEuropean lineages, theDenisovanshadonly contributed to Asian lineages, and in particular to native peoples of Polynesia,MelanesiaandAustralia.ThegeneticistsfoundthattheDenisovanshadcontributedsome4 to 6 per cent of the genome toMelanesianswho currently inhabit areas of southeastAsia,suggestingthattheDenisovansinhabitedalargegeographicareainAsialongago.Thepaperendedwithanemergingpictureofadistantperiodofhumanevolution,knownas the Upper Pleistocene, in which ‘gene flow among different hominin groups wascommon’. Not species genocide then, but mutual sharing of culture and geneticinheritance.

WithinafewyearsoftheextractionoftheDenisovannucleargenome,aninternationalgroup of geneticists confirmed the advantage of hybrid genomic novelty in terms ofsurvival when the going got tough. One of the most celebrated examples of such anadaptation in humans is the ability ofTibetans to survive extremehigh altitude in theirHimalayanhomeland.GeneticistshadalreadydiscoveredthatTibetanspossessedaunique‘hypoxia pathway gene’, known as EPAS1, that lowered their haemoglobin levels inconditionsoflowoxygenation–theveryoppositeofwhathappenswhenanon-Tibetanisexposed to such a low-oxygen atmosphere.The normal thickening of bloodunder suchconditionswouldputpeopleat riskof life-threateningbloodclots.Tibetansonlysharedthe geneEPAS1with one other group of people – the Denisovans. In the researchers’

opinion: ‘Ourfindings illustrate thatadmixturewithotherhomininspecieshasprovidedgeneticinformationthathelpedhumanstoadapttonewenvironments.’

Thesurprisesjustkeptoncoming.

*

TheAtapuercaMountains,inthenortheastSpanishprovinceofBurgos,arehoneycombedwith caves containing hominin fossils and artefacts. One of these caves, known as theSimade losHuesos– the‘pitofbones’–hasyieldedoneof thegreatestassemblageofhomininboneseverfound,includingtheremainsofatleast28individualsthathavebeendated tomore than 300,000 years old. The skeletons have some features that resemblethoseoftheNeanderthals,butmanyoftheirfeaturesaremoretypicalofthemorearchaicHomoheidelbergensis,thoughtbysometobetheancestorofNeanderthals,andbyothersto be the ancestor of both modern humans and Neanderthals. The promise of thisarchaeologicaltreasuretrovewasheightenedbythefactthattheseboneswereremarkablywell preserved, suggesting they might be a good source of archaic DNA. SpanishpalaeontologistssuppliedPääbowithacompletefemurinremarkablygoodcondition,andthiswasdulydrilled tosupply1.95gramsofpowderedbone.Asearlier, thegeneticistsbegan by sequencing the mitochondrial DNA, to produce yet another round ofastonishment when they revealed the results in a Nature article, published online inDecember2013andinprintedformthefollowingJanuary.

Theexpertshadanticipatedamitochondrialgenomecloselyrelatedto,andverylikelyancestral to, the Neanderthal sequences. But what they found was a genome that wascloser to that of theDenisovans than to either theNeanderthals or tomodern humans.Theyhadprovokedyetanothermystery thatappeared toshakeupourprior ideasaboutourhumanorigins.

In a covering article inNature, the authors confessed that theywere now scratchingtheir heads to explain the surprising discovery. Everyone seemed to have their owndifferentideasastotheexplanation.AsCliveFinlayson,anarchaeologistattheGibraltarMuseum,commented,thefindingswereactuallysomewhat‘soberingandrefreshing’.Toomanyideasabouthumanevolutionhadbeenderivedfromlimitedsamplesandoverblownnotions. From now on the real truth would be revealed by the genetics, which, inFinlayson’swords,‘doesn’tlie’.InPääbo’sadmission,hewasasbemusedaseverybodyelse.‘Myhopeisthateventuallywewillbringnotturmoilbutclaritytothesituation.’

What a refreshing light palaeogenetics is shining on the real history of our distantancestors!

While we cannot rule out skirmishes, even limited violence, between the differentspecies and populations, it would appear that the different evolving species of humanswerenot intentonexterminating their evolutionary rivals.They surelycameacrossoneanotherfromtimetotime–theymayevenhavelivedcheekbyjowlinsomegeographicareas –when, to judge from recognisable human behaviour, theywould probably havebeen intensely curious about one another. They must have recognised their commonhumanity, held conversations with one another, looked at each other’s traditions. They

may have learnt from one another, too, picking up ideas about hunting or foraging, orinformationonthemanufactureoftools,howtheyoperatedasgroups,orthetraditionsoffamilylife,therulesofsexualpartnership,howtheytookcareofandeducatedchildren,how they decorated their bodies, manufactured their clothing and homes, how theyworshipped,orgrievedforanddealtwiththeirdead.

ThisiswhatPääboandhiscolleagueswanttoknowmoreabout.ItisalsowhatyouandIwant to knowmore about – the real human history, the story that is forever retainedwithin and being constantly added to anew in that mysterious world of our commonhumangenome.

nineteen

WhatMakesYouUnique

The preservation of favourable variations and the rejection of injurious variations, I callNatural Selection.Variations neither useful nor injurious would not be affected by natural selection, and would be left afluctuatingelement…

CHARLESDARWIN

InthecelebrationaryissueofTheDailyTelegraph,onMonday12February2001,RogerHighfield, the newspaper’s science editor, forecast that cracking themysterious codeofthehumangenomemadeeveryhumanbeingspecial.Hewasabsolutely right. Justhowspecialis,ofcourse,stillintheprocessofbeingunravelled.

Itwillhavelongbeenapparentonthisjourneythatweallsharethegenomiclegacyofafascinating and extraordinary evolutionary history. It is a history that straddles the verybeginnings of life onEarth andwhat is turning out to be an epochal age, inwhich ourspeciesisbeginningtoexploretheUniversebeyondourocean-girdledplanet.Wecaughtasnapshotofthishistorywhen,in2001,thefirstdraftofourhumangenomerevealedthatwesharethousandsofgeneswithmanyotherformsoflife,andnotjustthegreatapesoreven themammals, butwith reptiles, fish, the fruit fly and the nematodeworm.And itgoes even deeper than this. My late friend and distinguished scientist Lynn Margulisshowed us howwe have inherited somuch of our distant prehistory, andmuch of ourfundamental internal chemistry, from the bacterial stage of life – in the jargon, theProterozoic stage – that pioneeredmany of the genes andmetabolic pathways that lifedepends on today. And, chapter by chapter, we have discovered how all four of themechanisms that give rise to hereditary change, those same mechanisms I groupedtogetherundertheumbrellaconceptof‘genomiccreativity’,haveprovidedthe‘variation’necessary for Darwin’s pioneering idea of evolution to mould our wonderful humangenome.

Wehave seen how the symbiotic union of the genomes of former parasiticmicrobeswiththegenomesofourancestorshascontributedtothisinexorableevolution,fromthecapture of the energy of sunlight by cyanobacteria, and the production of high-energyoxygenasaby-product,totherespirationoftheoxygenbythebacterialforerunnersofthemitochondriathatnowaddasecondgenometoourlivingcells,aswellastheinvasionoftheendogenousretroviruses thatarestillchangingthewayourgenomeworks.Wehaveseenhow,asthegenomebecamemoreandmorecomplex,powerfulsystemsofepigeneticregulationhavebecomeintricatelyinvolvedwiththebureaucraticsystemsofgovernanceofgenes andmanyother aspectsof thegenome.Some scientistswouldnow regard thegenes as hardware and the regulatory systems as software, with the implications that

wherethehardwareisforordinarypurposesfixed,thesoftwareiscapableofchanginginrelation tosignalsfromtheenvironment inevery individualhumanbeing.Andwehaveseenhowsexualcrossesbetweencousin specieshaveaddedmajor injectionsofgeneticdiversitytoourevolvingancestralgenome.

Allofthismayappearaconfusionofcompetingforces–andsoitwouldbeifevolutionwasrandom,butthankstoDarwinwerealisethatitisnotrandom.Thereisanadditionalpowerfuleditorial force–Darwin’sbrilliantconceptofnaturalselection–whichselectsforthosechangesinhereditythatenhancesurvivalandselectsagainstthosethatthreatensurvival.Survival,andthroughitreproduction,governseverymechanismthatcontributestowhatmightappearaconfusionofcompetingforces.Andyes,builtintothishistoryofour constantly evolving genome is the inevitability that every one of us is genomicallyunique.

Weareunique, tobeginwith,becauseeachofus,other thangenetic twins, inheritsarandomisedmixtureofthegenomesoftwodifferentindividuals–ourparents.Themixingis inherent in the way the germ cells are created within the ovaries and testes of ourmothersandfathers.Itisbroughtaboutduringtheprocessknownas‘meiosis’,whenthechromosomeslineupinparalleltooneanotherandthenthesimilarchromosomesbreakupintofragmentsandswapthesematchingfragmentswithoneanother.Thisprocessofsexual homologous recombination explains why brother is not identical to brother andsister to sister, even though they share the same parents. Only identical, or so-called‘monozygotic’,twinsshareidenticalgenes,becausetheydevelopfromthesamefertilisedegg.Butnow,followingourexplorationofepigenetics,weknowthatevenidenticaltwinsarealreadydevelopingdifferencesintheirepigeneticsystemsofregulatorycontrolbythetimetheyareborn.Andifwelookedreallycloselyattheirgenomesthroughoutlife,wewoulddiscoverthattheybecomeincreasinglydifferent,becausetheirepigeneticsystemshavebeenrespondingtodifferentenvironmentalstimuli.

A key region inmaking every human being unique is a portion we have repeatedlyvisited on our journey, the major histocompatibility complex, or MHC. Located onchromosome6,thiscontainsmorethanahundredprotein-codinggenes,anditdeterminesour immune defences as well as our antigenic identity, for example when it comes tobloodtransfusionsandorgantransplants.Nopartofourgenomesotellinglydefinesusas‘self’.Thispersonalgeneticidentitybeginswithinthedevelopingembryointhemother’swomb and it continues to update itself, through interaction with invading microbes,throughout all of our lifetime. It is through somedamageor aberrationof this complexrecognition of self that autoimmune diseases such as rheumatoid arthritis, lupus andjuvenileonsetdiabetesarise.

WehaveseenhowtinyerrorsaremadeeverytimetheDNAofourgenomeiscopiedtogive rise to the ovum and sperm– thesemutations in parts of ourDNA that are of noconsequence tonatural selectiongive rise to theSnips,haplotypesandhaplogroups thatenablegenetichistorianstotraceoriginsandmovementsofhistoricpopulations.

Thusatconceptionweshare roughlyhalfourgenes, including thosevastnumbersofsinglenucleotidepolymorphisms,orSnips,witheachparent.Wealsoshareroughlyhalf

of our genes and Snips with our siblings. If we have identical twins, we begin ourembryological development by sharing all of our genes and Snips with our twins. Insimilarfashionweshareaquarterofthesamewithourgrandparents,aneighthwithourgreat-grandparents,andsoonbackintime.Butthereisalreadypotentialforchangeevenin this seeminglywell-orderedsystem.Sovast is thegenome that there isameasurablepotential for smallmistakes during its copying.And thosemistakes guarantee that tinypartsofuswillbedifferentevenfromthegeneticsequenceswewouldhaveexpectedtoinheritfromthegenomesofourparents.

Whole genome sequencing has now established the mutation frequency for wholegenomes.Fromonegenerationtothenext–inotherwordsfromparentstochild–thereare,onaverage,about70newmutations.Thevastmajorityofthesearenotlocatedinthe1.5percentprotein-codingfractionofthegenome,wheretheaverageisasinglemutationfor every three parent-to-child generations. Instead, themajority are to be found in theviralandepigeneticregulatoryregions.Weshallreturntothisshortly,butIwouldliketocontinuetofocusonmutationalchange.Asanintegralpartofthispredictablemutationalchange, you and I can expect to haveSnips unique to our genomes. Something closelyrelatedtothishascontributedtotheuniquenessofindividualgenomesthatisessentialtowhatweknowasDNAfingerprinting.

*

WearefamiliarwithDNAfingerprintingasameansofdeterminingfamilyrelationships,for example in paternity testing, or identifying the perpetrator of a crime.Up until the1980s, accurate forensic identification had largely relied on fingerprinting, but inmanycrimes there was no fingerprint evidence. DNA profiling offered the same accuracy ofindividualidentification,whetherfromsaliva,oraspotofbloodorsemen,orasampleoftissueofanykind,includingbone.Butfirsttherewasaworkadaymethodologicalproblemthatneeded tobeovercome;abusy forensic servicecouldnotbeexpected to screenanentirehumangenome,withallofits6.4billionnucleotides,inthesearchfortheelusiveevidenceofindividualpeculiarities.Whatwasneededwasasimpleandreliablesystemofautomatedscreeningcapableofspottingdifferencesbetweenindividualstothesamehighfidelityasfingerprinting.In1985,aLeicester-basedBritishgeneticistcalledAlecJeffreysprovidedthis.

JeffreysmadehisdiscoverybyaccidentwhenexploringdifferencesinDNAsequencesbetween individual family members of one of his laboratory technicians. He had beenexaminingodd-lookingDNAsequencesfromthe‘repeat’sectionsofthegenome–thosehuge chunks of virus-related sequences that were scattered widely throughout thechromosomes.HereandthereheobservedregionsofDNAcontainingrepeatsofthesamehandfulofnucleotideletters.Theseso-calledrepeatswerehardlyuncommoninthehumangenome, but in certain locations within the chromosomes the actual number of themseemedtovaryfromoneindividualtoanother.

Ifwewere topayone such regionavisitonourmagical steam train,wewould findourselves hopping down to walk along a highlighted section of track, noting that thesequencebeganwith,say,foursleepers,thatreadperhapsT,C,AandG.Aswecontinue

to perambulate the track, we find the same sequence, TCAG, repeating itself, possiblythreetimes.Sincetheserepeatsoccurintandemalongthetrack,theyarecalled‘tandemrepeats’.As far as Jeffreys could see, they served no purpose in the sense of theDNAtranslatingtoprotein–or,inthesemoreenlightenedtimes,togenomicregulation,geneticorepigenetic.Theseweretypicalofthesortofsequencethatwouldbeignoredbynaturalselection because theywouldmake no difference to individual survival or reproductivecapability. So if we were to examine many different individuals within a population,throughchancealonethenumbersoftandemrepeatsatthesesiteswouldbeveryvariable.Theymightshowcloserthanaveragesimilarityamongsiblings,otherthanidenticaltwins,but even siblings would show some differences as a result of sexual homologousrecombination. In the jargon, these siteswere locations of ‘variable number of tandemrepeats’,orVNTR.

WhatJeffreysdidnextwastodevelopasimplemethodologybasedonthenumbersofrepeatsattendifferentVNTRlocationsscatteredthroughoutthechromosomes.Whyten?Wemightindulgeinthesamesimplemathematicsweusedearliertodeterminehowmanynucleotides we needed in an overlap between fragments of chromosomes for it to besignificant beyond reasonable doubt. Ten loci, say with variation from 1 to 4 repeats,proved to bemore than enough to identify an individual beyond phenomenal levels ofreasonabledoubt.Jeffreysthenaddedasimplegenetictestthatwoulddeterminewhetherthe forensic evidence came from amale or a female. Aswemight have expected, theeffectiveness of the actual genetic screening is greatly improved by using PCR, whichneedsonlytraceamountsofanindividual’sDNAtofindamatch.Thusforensicscientistsweregivenanincrediblyaccuratenewtoolofindividualidentification,basedontheveryfact that every human being really is genomically unique; and that evidence could begleanedfroma traceofblood,orbodyfluids, fromasinglehair,or thecellsshedfromskin,indeedfromaverywidevarietyofpersonalidentificationleftbehindatagreatmanydifferent crime scenes. It just remained to be demonstrated that the new geneticmethodologywouldprovetobeeverybitashelpfulastraditionalfingerprinting.

OneoftheearliestapplicationsofgeneticfingerprintingwasinthesearchinthecountyofLeicestershireforarapistkilleroftwoteenagegirls.Notonlydidthisprocessdiscovertherealmurderer,italsoexoneratedaninnocentmanwhountilthenhadbeenconsideredthe prime suspect. Since then Jeffreys’ methodology has been taken up by forensiclaboratories around theworld, helping to solve a vast array of family pedigree geneticenquiriesaswellascriminalcases.ButweshouldnotconfuseDNAfingerprintingwiththecompleteDNAsequencingofanindividualhumangenome.Thisremainsaformidableundertaking, although it is much easier to conduct these days, with high-throughputcomputer-assisted sequencing machines. Whole genome sequencing is becomingincreasinglycommon,forvariouspurposes,andthishashighlightedhowuniqueeachandevery individual human being really is at levels that go far beyond variable tandemrepeats.

*

Asingleendogenousretrovirusinsert,orlocus,isroughly10,000nucleotideslong.PeoplehailingfromAfricaortheNearEastaremuchmorelikelytocontainthelociofHERV-113andHERV-115intheirgenomesthanpeopleoriginatinginWesternEurope,orAsia.Meanwhile,thosesameWesternEuropeansarefarmorelikelytocontaintractsofDNAofNeanderthaloriginsthanpeoplehailingfromsub-SaharanAfrica.AsiansandPolynesiansarealso likely tocontain someNeanderthalDNA,and tocontainevenmoreDenisovanDNA,amountingtoasmuchasone-sixteenthofalltheirgenome.Howmuchinformationwill themore detailed explorationof these differences provide of our humanhistory oforigins and migrations that was previously thought lost to the dust and fossils ofprehistory? We are only beginning to explore the implications of these two majorhybridisation events. Yet such differences do not feed into the slanted viewpoints ofracists.Rather,theyconfirmandextendwhattheearliestgeneticists,suchasLuigiLucaCavalli-Sforza,wereatpainstoemphasiseandcelebrate:ouronenessnotonlyasaspeciesbutasahumanfamily.

Wholegenomicsequencingmust,bydefinition,includeboththemitochondrialandthenucleargenome.Thishas led to surprisingdifferenceswhenwescreenpopulations.Wehave already seen significant differences betweenmales and femaleswhenwe conducthaplogroup screening of European populations, with females showing a much greaterhomogeneity throughout allpopulationswhencompared tomales.Sexualdifferences inhaplogroup movements are also seen in much more recent population screenings, forexample in thepeoplingof thevariousnationsamong theBritish Isles.Wholegenomicsequencingmayhelptoclarifywhatthismightmean.AswesawwiththeNeanderthals,screeningof themitochondrialgenome,orperhaps thesimilarly limitedscreeningofY-chromosomesequences,mayactuallybegivingussubtlydifferent informationfromthescreening of the entire nuclear genome. How interesting if this translates to realdifferencesintheprehistoricmovementsofthetwosexeswithinancientsocieties!

SomeofthefirstwholepersonalgenomestobesequencedincludedJ.CraigVenter,theentrepreneurialscientistwholedthecommercialgrouptothefirstdraftsequenceof2001,andJamesWatson,theco-discovererofDNA.WhenKoreanresearcherscomparedtheirgenomestothoseofaHanChinese,aYorubanNigerian,afemaleleukaemiapatientandaKorean-originated scientist, the researchers were astonished to discover that the twoAmericans had more sequences in common with the Korean than with one another. Ishouldadd thatwhat theKoreanresearchersactuallycomparedwerenotwholegenomeDNAsequencesbutwholegenomepatternsofSnips,whentheydiscoveredsome420,083novel,single-nucleotidepolymorphismspreviouslyunknowntotheSnipdatabaseaswellasthestartlingsimilaritiesanddifferencesmentionedabove.

Another interesting personal example was the 2008 sequencing of themitochondrialgenomeofÖtzi,theTyroleanIceman,whose5,300-year-oldmummifiedremainshadbeendiscovered with the thawing of an Alpine glacier. This revealed that he belonged to abranch of the mitochondrial haplotype K1 that had not been identified in Europeanpopulationsuptothispoint.Butwhen,in2011,thiswasfollowedbySnipanalysisofhisnuclear genome, it showed a recent common ancestry with the inhabitants of theTyrrhenianSea,which ispartof theMediterranean immediatelywestof Italy, including

thecoastofTuscanyand the islandsofCorsicaandSardinia.The report,byKellerandcolleagues, suggested that he had brown eyes, belonged to blood groupO,was lactoseintolerantandhadageneticpredispositiontocoronaryheartdisease.

When Pääbo and his colleagues were screening archaic genomes they made somestartlingadditionaldiscoveries.Thedifferentsectionsofourchromosomeshavedifferentpalaeontologicaloriginsintermsofpeopleandtime.Itwouldappearthatwhenwevisitthese different regions of the genomic landscape in our magical train, we really areglimpsing something of those different ancient peoples in their prehistoric worlds. Arelated discovery was that while some portions of our genome have a very modernevolution, other portions appear to have stayed the same for millions of years. WhenPääboandhiscolleaguescalculatedbacktothelikelydateofthecommonancestorofthe‘reference modern genome’ and the Neanderthals, the results suggested an LCA some830,000yearsago.ButwhentheydatedthelastcommonancestorofthesamereferencemoderngenomeandtheSanpeopleinAfrica,theycameupwithadateof700,000years.Even stranger, when they dated last common ancestors for specific present-day DNAregionsofchromosomesfrompeoplefromdifferentgeographiclocationsovertheEarth,they found some regionswhere they shareda commonancestor just30,000or soyearsago,butotherregionsthatsuggestedalastcommonancestor1.5millionyearsago–thetimeofearlyHomoerectus.InPääbo’sownwords:‘IfsomebodycouldtakeawalkdownoneofmychromosomesandcompareittobothaNeanderthalandthereaderofthisbook,that chromosomalpedestrianwould find that sometimes Iwouldbemore similar to theNeanderthal than to the reader, sometimes the reader would be more similar to theNeanderthal,andsometimesthereaderandIwouldbemoresimilar.’

ForPääbo’swalk,myreaderandImightsubstitutea trainride,andI thinkwemightshareasmileforamoment.

Itisstrangetoconsiderthatdifferentregionsofourgenomehavedifferentevolutionaryorigins.But perhapswe shouldn’t be sooverly surprisedby itwhenwe realise thatweshareathousandormoregeneswithwormsandfruitfliesand,intheopinionofmyfriendandcolleague,theeminentevolutionaryvirologist,LuisVillarreal,wehaveinheritedkeyDNAandRNAmanagementgenesfromviral lineagesthatdatetobillionsofyearsago.Sometimesanevolvedgeneticorgenomicsystemsimplyworkssowellthatthepassageoftimecannotimproveonit,overmillions,orevenbillions,ofyears.YetthiswasexactlythepointmadebyDarwin, in his ruminationsbefore thepublicationof his iconoclasticbook.Ourcommonancestors,generationbygeneration,speciestofamilies,andbackevenbeyondPhylaandKingdoms,gobacktotheveryoriginsoflifeonEarth.

twenty

TheFifthElement

IwantedtotakeusintoaneweraofbiologybygeneratinganewlifeformthatwasdescribedanddrivenonlybyDNAinformationthathadbeencreatedinthelaboratory.

J.CRAIGVENTER,LIFEATTHESPEEDOFLIGHT

Thephilosophersamongtheancientsbelievedthatmatter,andthustheEarth,wasmadeupoffourelements:Earth,Air,FireandWater.Theyfurtherbelievedthatthestarsinthefieryheavensweremadeofafifthandmorewondrouselementwhichwasinfusedwiththe celestial power over life. These metaphysical elements are not the same as thechemicalconceptsofelementstoday,whicharethebuildingblocksofmolecules,buttheydo bear an ideational comparison since, in a more holistic sense, the elements of theancients were, in their imaginations, the building blocks of worlds. And we mightcontinuethecomparisoninextrapolatingthefifthmetaphysicalelementtotheremarkablesibling molecules, DNA and RNA, which make possible the evolution, heredity anddevelopment of life. How daunting then to open our own imaginations wider still, toacknowledgethat,forgoodorforbad,thatquasi-miraculousfifthelementhasnowfallenintotheambitiousgraspofhumankind.

Tinkeringwiththeprocessesoflifeisnotnewtohumanity.AslongagoastheStoneAge farmers learnthow to select the seedgrainsofwheat andother cereal crops togetfatter, more nutritious kernels. Today virtually all of the seed grains ‘husbanded’ byfarmers are the results of hybridisation – that same evolutionary mechanism of sexualcrossingbetweendifferentspecies.Humanshavebeenlearningfrom,andalso intrudinginto,thesecretsofnatureforaverylongtime,butonlyveryrecentlycouldwebesaidtohaveaddedafifthtothehithertofournaturalmechanismsofgenomiccreativity–thosesamemechanismsthatnaturalselectionreliesontoenabletheevolutionoflifeonEarth.Thatfifthmechanismisthecalculatedgeneticengineeringoflivinggenomes.

Whereinthepastanyhuman-inducedgeneticmodificationswerebroughtaboutastheaccidentaleffectsofbreedingfarmedanimals,petsandcrops;now,thankstothegoldenage of genetics, we are poised to take the reins of deliberate genetic and epigeneticcontrol.Thisisnotsomefearful,orwonderful,thingthatwilleventuallycometobe;ithasalreadybeenhappening for a generation in termsof the genetic engineeringof animalsandplants.Iftodateithasnotintrudedintothehumangenome,I’mafraidthatlogicallyitseemsmerelyamatteroftimebeforeitbeginstobeappliedtohumans.

Intheinitialmediaresponsetothe2001publicationsofthedraftgenomewewitnessedthatitheraldedanewwayoflookingatourselves.Whatelsehavewebeendoinginthis

book other than looking at ourselves anew? It is difficult to consider such possibilitiesdispassionately. Yet it would appear timely to do so. Scientists, including moleculargeneticists, are neither amoral nor unethical. The obvious applications of the dawninggolden age of ‘creative genetics’ – or, taking on board the dawning importance ofepigenetic regulation, shouldwe call it ‘creative genomics’? –will be for the potentialgood of humanity, in terms of medicine, for improved provision of food and, lessexplicitly,aspartoftheongoingexplorationofthewondersofnature.

Whatcouldbemorejustifiedthanunderstandingthegeneticbasisofdisease,sowecanmakeuseofsuchunderstandingtotreataffectedindividualsaswellaspreventingdiseaseinfuturegenerations?Thesetwinaimshavealreadybegunandarerapidlyexpandinginterms of preimplantation genetic diagnosis and the selection of healthy embryos. Somemembersofsocietywillhaveethicalorreligiousobjectionstothis.Pioneersofmoleculargeneticsand‘recombinantDNA’,suchasJamesWatson,SydneyBrennerandPaulBerg,pointed out that the prudent way to encompass such concerns is to ensure that non-scientists understand and are thus ‘intelligently aware’ of such scientific enterprise andambition so that the safety, moral and ethical implications are routinely taken intoconsideration.

A good deal of themodern extrapolation of genetics and genomics does not involveworrisome genetic engineering. Much of the pharmaceutical research into epigenetics,including non-coding RNAs, is aimed at medical therapies that change the epigeneticcontrolfor thebetter.This isalreadythebasisof linesofresearchintocancer therapy.Icanpredictthatitwillalsobecomethebasisofmanylinesofresearchintoautoimmunediseases.

Aswehaveseen,ourphysicalandmentalhealthiscloselylinkedtogeneticaswellasenvironmental factors. Genetic and epigenetic differences between individuals maydeterminethepotentialforaddictiontodrugsandalcohol.Asimilarindividualvariationmaybeimportantinourpredispositiontomanydifferentdiseases.Thisisusheringinnewfields of investigation and prediction of disease, such as the closely related ‘personalgenomics’and‘predictivemedicine’.Personalgenomics–alsoreferredtoas‘integratedpersonalomicsprofiling’– isanambitiousprogrammeofresearchaimedatprovidingadynamic assessment of the physiology and health of an individual over time.One suchinvestigation is the brain-child of Michael Snyder, Professor of Genetics at StanfordUniversity, inwhich volunteers are subjected to genomic, transcriptomic and proteomichigh-throughput readouts, combined with screening of the individual’s metabolic state,andchangesinauto-antibodyprofiles.Theideaistospotkeychangesinandinteractionsbetween genome, epigenome and internal physiology during normal health and in thelead-uptodisease.

Somethingsimilarishappeninginothercountries.IntheUK,between2006and2010aregisteredcharitycalledUKBiobankrecruited500,000peopleagedbetween40and69yearstoundergomedicalexaminationsandtodonatebloodforDNA,aswellasurineandsalivasamplesforfutureanalysis.Theaimistocreateadatabankthatwillimproveourabilitytoprevent,diagnoseandtreatawiderangeofseriousandlife-threateningillnesses

– including cancer, heart diseases, stroke, diabetes, arthritis, osteoporosis, eyedisorders,depressionandformsofdementia.In2005,intheUnitedStates,DrGeorgeM.Church announced the creation of the Personal Genome Project, aimed at recruiting100,000volunteersfromtheUS,CanadaandtheUKwhowouldbeagreeabletohavingtheir entire genomes sequenced and stored. This large collection of ‘genotypes’, or fullDNA sequence of all 46 chromosomes, would be published, along with extensiveinformationaboutthemedicalrecords,variousphysicalmeasurements,MRIimages,andso on, so that researchers will be able to study the links between genotype, theenvironment and so-called phenotype – the physical make-up and progress of thevolunteers.Notonlywillthishelptoplotgeneticlinkstodisease,researchersareplanningto examine the reaction of society, including insurers and employers, to suchextrapolations from the genotype to future health predictions. Despite the potential fordiscrimination, it seems that recruitment has been very successful. It seems likely thatsimilarwide-ranginggeneticandepigeneticscreeningprogrammeswillbeconducted inmanyothercountries.

Intimesuchpersonalgenomeprojectsmayenablepredictivemedicine,whichisbasedon the notion that, by predicting the probability of serious disease from an individual’sgenome,thiswillallowactivemeasurestoreducetheriskofdiseaseinthefuture.Anotherway in which this might prove useful would be to predict the likelihood of iatrogenicdisease–diseasecausedbyside-effectsofmedical therapy.Inarecentstudyofadversedrugreactionsinvolving5,118childrenadmittedforbothmedicalandsurgicaltherapytoaUK-based hospital, 17.7 per cent experienced at least one adverse drug reaction.Theauthors thought it likely that the actual incidence of side-effectsmay have been higherbecause they excluded ‘possible’ but unproven cases. Opiates and anaesthetic drugsaccountedformore than50percentof thereactionsand0.9percentcausedpermanentharmor requiredadmission toahigher levelofcare. It is important tograsp thatmanysuch side-effects were not life-threatening, for example vomiting after a generalanaesthetic,buttheseexperienceswillhavebeenfrighteningandmemorableforchildrenandwould be better avoided, if possible.Another obvious potential for troublesome oreven life-threatening side-effects is the long-term treatment of a very wide variety ofdiseases, both under hospital and general practitioner management. Some of the mostserious side-effects may become predictable, and thus preventable by deciding from achoiceofmedicationattheoutsetoftherapythroughmodern‘omics’investigation.

Membersofthepublicarealsodoingthingsforthemselves.Moreandmorepeoplearepaying tohave theirpersonalgenomes sequenced, somemerely throughcuriosity abouttheir genetic background, others because they want to know about their own geneticpredispositiontodisease.Forexample,awomanwhofears,fromherfamilyhistory,thatshemightbemoresusceptible tobreastorovariancancermightwant toknow if she iscarryingspecificgenesthatwouldincreaseherriskofthesediseases,suchasBRCA1andBRCA2.Thismightallowher,inconsultationwithherdoctors,toplanacourseofactionthatwouldmitigateherrisk.

Allsuchinvestigations,aswellasthetherapeuticoptionsthatcomefromthem,mightinvokeethical,moralorreligiousdilemmas.Weliveinarapidlychangingworldinwhich

complexpersonal and social issues arebeingbrought intoquestion inwaysourparentsandgrandparentswouldneverhave imaginedpossible,with increasingneed forgeneticcounselling,genomicpredictionand,perhapssoon,thepotentialforgeneticengineering.

Eventodaysomefolkareworriedthatthisgrowingunderstanding,andmanipulation,ofgeneticswillleadtoanewpotentialforeugenics.Alreadysomepeoplemightarguethatpreimplantationgeneticdiagnosis,withrejectionofgeneticallycompromisedembryos,isanunacceptableformofeugenics,eventhoughmostaffectedfamilieswouldprobablyseeitasthepreferredoptioninverypainfulanddifficultcircumstances.Acommercially-runclinicinCaliforniaisalreadyprovidingtowould-beparentsapre-determinedsexofchild.Whatelsedoesthefuturehold?Isitgoingtobecomepossibletogeneticallymanipulateembryos to change their physical appearance, their stature, their athleticism, theirintelligence?Willfuturegenerationsofparentsorgovernmentsinstructscientiststomakeuse of DNA technology to breed what they regard as desirable genetic and epigeneticbreedsofchildren?

*

I set out to write this book from the premise that it would attempt to provide a non-scientificreaderwithabasicunderstandingofhowhisorherowngenomeworks.IcanonlyhopethatIhavesucceededinthataim.Theverynotionthatwemightunderstandtheevolution, structural make-up and detailed function of the genomes that code for life,includingourownhumangenome,isofepochalimportancenotonlyforsciencebutalsoforallofus.Ihopethatithasbecomeclearthatsuchunderstandingisimportant,sinceitmustbeforsocietyingeneral,andnotscientistsalone,todecidewherewegofromhere.Natural selection, nature’s powerful force that decides what genetic novelty will movethrough a population to change the species gene pool, does not aim for any kind ofperfection.AsDarwinhimselfpatientlyexplained,itisdeterminedbyonething:survival,or failureofsurvival,of individuals,and implicit in this is thefateas towhetherornottheyreproduceoffspringandthuscontributetothespeciesgenepool.Thereisnohigherobjective involved in themoral, philosophical or religious sense – no forward planningwhatsoever in thewayhuman reasonandambitionmight conceive.Butour capacity toaltergenomesatourownwhimchangesthat.Geneticengineering,ifitisintroducedintothehumangenome,willinjectexactlysuchreasonedforwardplanning.Thishasimportantimplications; the potential for the treatment and prevention of serious disease willobviously benefit society, but there will be other potentials that some might view asdangers.Themoral andethical implications are thus important. It is no exaggeration tosay thatwhatwas previously the domain of science fiction is now increasingly sciencefact.

Geneticengineeringofcropsandfarmanimalsbeganinthe1970s.Fromthebeginningthis process encountered societal resistance, some protests being more emotive thanrational.Butscientists,andgoverningbodies,werepersuadedbythepotentialforbenefit–purportedlythefeedingofthehungryinpartsoftheworldstrickenbyadverseclimatesandecologies.CriticssawthepotentialforharmthroughGM-modifiedgenes‘escaping’fromthegeneticallymodifiedfieldstoenterthesurroundingecologyinunwantedways.

Thecrossingofgenesfromonespeciestoanotheriscalled‘horizontalgenetransfer’.Aswehaveseen,geneticsymbiosis,involvingbacteriaandviruses,andhybridisationeventsarepotentexamplesofthiscrossingofevolutionaryboundariesinnature.

In1976theUSNationalInstitutesofHealthsetupanadvisorycommitteetoadviseonthe putative dangers of ‘recombinant-DNA’, and this was followed by ‘complex butrelaxed’ regulation from offices including the United States Department of Agriculture(USDA), the Environmental Protection Agency (EPA), and the Food and DrugAdministration(FDA).ThisledtotheestablishmentofacommitteeundertheaegisoftheOffice of Science and Technology, which approved GM plants under the continuingregulationandcontrolofthevariousregulatorybodies.In2000theCartagenaProtocolonBiosafetycame intobeingasan international treaty togovern the transfer,handlinganduse of genetically modified organisms. One hundred and fifty-seven countries aremembers of this protocol,which is seen as a de facto trade agreement.GMcropswill,usually, have built-in modifications aimed at preventing sexual crosses with non-GMcrops. They also have ‘traceability’ built into their genomes, which would enablegeneticists to discover the source of origin if GM-modified genes escape into theenvironment. In 2010, a study byUS scientists showed that about 83 per cent ofwildcanola plants in the hinterland of GM crops contained genetically modified resistancegenes. While the scientists involved in GM research and agriculture now saw nosignificantrisktotheenvironmentorhumansfromthisestablishedescape,thoseopposedtogeneticengineeringremainunconvinced.

A European deal in June 2014 opened up the possibility of GM crops being growncommerciallyinanycomponentstatewhoseauthoritiesdecidedtosanctionthis.ThedealwassupportedbyallofthememberstatesotherthanBelgiumandLuxembourg.CountrieslikeFrance,whichissaidtoopposeGMcrops,willbefreetoeschewthem,meanwhileEngland will be free to introduce them, even though other parts of the UK, includingScotlandandWales,maydecidetoopposethem.Itistooearlytospeculatewhoisprudentandwhoisnot.

Thepotentialforfuturegeneticmodificationofthehumangenomeisevenmorelikelytoexcitecontroversyanddebate.

Mostdoctorswouldprobablyfavourmodificationofthegenomeofpeoplewhoareathighriskofgeneticallyprovokedserious,orpotentiallyfatal,diseases–ifandwhensuchmodificationbecomesavailable,andsafe.Whowouldn’twishtosaveyoungwomentheriskofdevelopingbreastorovariancancer,orachildfromcysticfibrosis,haemophilia,orHuntington’s disease? But once the technology tomodify the human genome becomesmorereadilyavailable,howfarwilltheapplicationsextend?Webeganthisjourneywiththeaimofconfrontingthemysteriesofthehumangenome,butnowthisverytrail,andthesolutions it has provided, may have opened a Pandora’s box for future scientists, andsocietymoregenerally.

Meanwhile,whatofnature?Naturehasnocompunctionsaboutchanginggenomes.Sothequestionthatisincreasinglyaskedofscientistsisasfollows:isthehumangenomestillnaturallyevolvingtoday?

*

Ourmodern humanhistory has been accompanied by dramatic changes in environmentand lifestyle.Wehavebeenbombardedby lethal infectiousdiseases, includingmalaria,tuberculosis, yellow fever, pneumococcal pneumonia, meningococcal meningitis,whooping cough, measles, poliomyelitis, diphtheria. Many of these swept throughpopulations on a regular basis, as well as notorious everyday bugs such as thestaphylococcusandstreptococcusthatcauseboils,cellulitis,rheumaticandscarletfever,andboneanddentalabscesses.InmylifetimeIhavetreatedhumanbeingssufferingfrommanyoftheseillnesses.Susceptibilitytodiseasesisoneofthemostpowerfulofexternalpressures for adaptive genomic change,most particularly affecting the evolution of theMajor Histocompatibility Complex as well as the epigenetic portions of the genome.Meanwhile the lingeringpresenceof resident retroviruses, and thehybridisation-derivedNeanderthalandDenisovangenomicintrogressionswillinevitablybestillworkingatthelevelofthespeciesgenepool.

In 2006, Voight and colleagues from the Department of Human Genetics at theUniversityofChicagodevelopedanewanalyticalmethodforscanningforSnipsinwholegenomesurveysthatwascapableofsearchingforrecentevolutionarypressures.Inthreebroadgeographicpopulations–eastAsians,northernandwesternEuropeansandAfricansbased onYorubans from Ibadan,Nigeria –Voight and his team discoveredwidespreadsignalsthatdenotedrecentevolutionarychange.Theseincludedgenesrelatedtomalaria,lactose sensitivity, salt sensitivity in relation to climate, and genes involved in braindevelopment. They also discovered signals of selective sweeps – so-called ‘geneticbottlenecks’–thatappearedtobestillinprogressandwerepresumablyrelatedtodiseaseliability.

So, dear me – no! I do not imagine for a moment that we humans have stoppedevolving.

Evolutionisintrinsictolife.NewviralplaguessuchasAIDS,hepatitisA,BandC,aresweeping through us. Natural calamities, including some that are man-made, arethreateningus.Weshouldrecallthatresponsivenesstotheenvironmentispartofthewayin which the epigenetic system evolves. And that epigenetic system is akin to anexquisitely sensitive and constantly changing software that governs how the genetichardwareworks.Amoresubtlepressuremaybe themassive increase inknowledgeandlengtheningperiodofeducationofouryoung,this,coupledwiththedramaticchangeswehavewitnessedinjustthelasttwodecadesintermsofhowmodernsocietyworks:thinkoftheintrusionofcomputerisedmachines,socialmedia,theglobalvillage,allmaximallyimpactingonouryoung–astageinwhichthehumanphysiology,andepigenome,arestilldeveloping. Canwe doubt that such overwhelming change is not already affecting ourhuman evolution?How likely is it that suchhuge changes in behaviour and systemsoflearning,broughtaboutbytheITrevolution,willaffectfuturebraindevelopment?

Meanwhile there is another related development, a potential change thatmay be themostastonishingofall:thisisthecapabilityoffuturegeneticengineerstocreateartificiallifeforms.

*

CraigVenter,thescientistwhofoundedCeleraGenomics,producedthefirstcommerciallyfunded draft of the human genome in 2001, his team inventing several importantinnovationsanddevelopingtheconceptofESTsandshotgunsequencingalongtheway.Inabluntandhighly-entertainingautobiography,Venterdeclaresthatsciencehasalwayssetouttomasterlife:‘Forcenturiesaprincipalgoalofsciencehasbeen,first,tounderstandlifeatitsmostbasicleveland,second,tolearntocontrolit.’Venteranticipatesafutureinwhichscientistswillengineernewlifeforms,aswellasmodifythehumangenome,tosuithumanandsocietalneeds.Hehasalreadytakenwhatheseesasthepreliminarystepstodoexactlythat.

Byanystretchoftheimagination,CraigVenterisaninterestingindividual.Amanwhotook little interest in his early schooling inSaltLakeCity, preferring to spendhis timesurfingandboating,hewouldsubsequentlyputthisdowntohispersonalattentiondeficitdisorder,whichhehadtostruggletoovercome.ThoughopposedtotheVietnamWar,hewasdrafted,enlistingintotheUSNavy,whereheworkedasanintensivecareassistantinafieldhospital.WhileinVietnam,heattemptedsuicidebyswimmingoutintotheocean,then changed his mind whenmore than a mile out. His experiences persuaded him toconsideracareerinmedicine,buthesubsequentlychangedcoursetobiomedicalresearch.Aggressivelyambitiousbynature,Venterprovedtohaveaprofoundlyinnovativegiftasascientist,combinedwithanirrepressibleentrepreneurialspirit.In2007and2008hewaslistedinTimemagazine’s100mostinfluentialpeopleintheworld.Twoyearslaterhewaslisted fourteenth in theNew Statesman’s roll call of ‘the World’s 50 Most InfluentialFigures’.

VenterwassackedbyCelerain2002,ayearafterthehumangenomewasmadepublic,reputedly because of differences in opinion with the main investor. He is currentlyPresidentoftheJ.CraigVenterInstitute,whichhastwomainfieldsofenterprise.Thefirstof these is to pioneer a discipline he labels ‘synthetic biology’, in which he and hiscolleaguesaimtoproduceartificiallyengineeredorganismsdesignedtoservehumanandsocietal needs. He first began to work towards this goal with a company, SyntheticGenomics, that he founded in the early 2000s. He moved on to explore the minimalgenomenecessityforcellularlifebeforesynthesisingthebareminimumofthegenomeofone of the smallest living bacteria, Mycoplasma genitalium, which causes urethralinfections inhumans.Inessencehereconstructedtheminimalgenomeinstages,first inhiscomputerandtheninalaboratorysynthesiser.Priortothis,thelargestgenomeseverartificiallyassembledinthiswayhadbeenthemuchsmallergenomesofviruses,thefirstbeingthepoliovirusassembledbyEckardWimmerandhiscolleagues.TheMycoplasmagenomewastwentytimeslarger.Overcomingmanyobstacles,Venter’sgroupsucceededinreplacingthenaturalgenomeofalivingbacteriumwithhisownsynthesisedequivalenttocreatealivingbacterialcell–abreakthroughheandhiscolleaguespublishedin2010.Thispavedthewaytothedeliberatecreationofcellularlifeformstoorder.

The mystery has not ended, though. The extraordinary story of exploration of ourmysterioushumangenomehas,asever,thrownupanewraftofveryimportantquestions.

IsVenter right in suggesting that Science has always been determined notmerely tounderstand lifeat itsmostbasic levelbutalso to learnhowtocontrol it? It’ssomethingoneisobligedtothinkdeeplyabout.Ican’tsaythatIamsureastotheanswer,butIrathersuspectthatheis.Whythenisthisso?Isitbecausewehumansarearrogantenoughtojustthink that we can? Or is it because we think that there are important reasons why weshould?IfVenterisright,wehavegonebeyondthestageofmusingaboutthisquestion.Itismucheasier togeneticallyengineeragermcell,oranewlyfertilisedembryo, than tomodifythegenomeofadevelopedhumanbeing.Wehavealreadygeneticallyengineeredanimals and plants in this way. In April 2015 the human embryo was deliberatelyengineeredinascientificexperimentforthefirsttime.Ibelievethatthisisasgreataleapas the discovery of gravity by Newton, relativity by Einstein, and the extrapolation ofEinstein’s discovery to the atomic bomb.And just aswith those epochal discoveries, itcarrieswithitthepotentialforgreatgoodandgreatharm.

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chapternotes

Frontispiecequote:Pauling,L.:25.

Introduction

Bronowski,J.:4.

Chapter1

Chapterheadquote:Schrödinger,E.:3.

ThestoryofDubosandhiscontributiontotheantibioticstory:Ryan,F.,1992.

Formore detail about Griffith, Arkwright, Dawson, Alloway, and so on: Dubos, R. J.,1976.AlsoOlby,R.,1994.

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TheletterfromAverytohisbrotherisreproducedinDubos,R.J.,1976.

Chapter2

Chapter head quote:Deichmann,U.Early responses toAvery et al’s paper onDNAashereditarymaterial.Historical Studies in the Physical and Biological Sciences 2004;34(2):207–232.

OnAverynotgetting theNobelPrize:Portugal,F.OswaldT.Avery:NobelLaureateornobleluminary?PerspectivesinBiologyandMedicine,2010;53(4):558–70.

TheHersheyandChasepaper:Hershey,A.D.&Chase,M.Independentfunctionsofviralproteinsandnucleicacidingrowthofbacteriophage.JGenPhysiol1952;36:39–56.

ThewarmerreceptionofHershey/Chaseexperiment,Olby,R.:318.

ThebombshellofGriffith’s1928paper:Dubos,R.J.:132–133.

For Alfred E Mirksky: Olby, R., 1994. Also Cohen, S. S. Alfred Ezra Mirsky: Abiographicalmemoir.NationalAcademyofSciences.

Avery’sresponsetoDubos,andDubos’responsetothedeathofMarieLouise:Ryan,F.,1992.

Chapter3

Chapterheadquote:Maddox,B.:60–61.

Watson’searlylifeandconfessionsofinnatelaziness:Watson,J.D.:21.

Watson’searlyinterestinphages:Judson,H.F.:49.

Hershey‘Drinkswhiskeybutnottea’:Judson,H.F.:53.

WatsononAvery:Watson,J.D.:13–14.

Chapter4

Chapterheadquote:BBC4documentary,TheCodeofLife.

FormoreaboutWilkinsandGosling:Wilkins,M.,2003.

Watsonquotes:Watson,J.D.,1968.

‘WhatMadPursuit’:thisbecamethetitleofCrick’sautobiography,1988.

Braggwasinfuriatedbytheupstartjunior:Crick,F.,1988.

Chapter5

Chapterheadquote:BBC4documentary,TheCodeofLife.

Crick–‘JimandIhititoff’:Crick,F.:64.

Meringas‘thearchetypalseductiveFrenchman’:Maddox,B.:96.

FranklinworkingbetterwithJewishmalecolleagues:ibid:96.

Chargaff’sscorn:Judson,H.F.:142.SeealsoWatson,J.D.,1968andCrick,F.,1988.

Aaron Klug’s comments about the terms of Franklin’s appointment: Klug, A. ThediscoveryoftheDNAdoublehelix.JMolBiol2004;335:3–26.

StokesfiguringoutthelikelyX-raydiffractionofahelicalstructure:Maddox,B.:152.

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Chargaff’s paper: Chargaff, E. Chemical specificity of nucleic acids andmechanism oftheirenzymicdegradation.Experientia1950:6:201–09.

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Wilkins,M.H.,Stokes,A.R.,Wilson,H.R.Molecularstructureofdeoxypentosenucleicacids.Nature1953;171:738–40.

Franklin,R.E.,Gosling,R.G.Molecularconfigurationinsodiumthymonucleate.Nature1953;171:740–41.

Chapter6

Chapterheadquote:Judson,H.F.:230.

ForthechemicalstructureofDNA:Hartl&Jones,2000.

BackgroundofDNAtoprotein,seeJudson,H.F.,1995;Olby,R.,1974.

ForbiographicalinformationonSydneyBrenner:Brenner,S.,2001;Friedberg,E.,2010.

Fordifferenttypesofmutations:Hartl&Jones,2000.

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Puberty and brain rewiring: Sisk, C. L. & Zehr, J. L. Pubertal hormones organise theadolescentbrainandbehaviour.FrontiersinNeuroendocrinology2005;26:163–74.

Chapter8

Chapterheadquote:Shreeve,J.:236.

MoreaboutJ.CraigVenter:Venter,J.C.,2013.

RogerHighfieldquote:TheDailyTelegraph:4.

TheNatureandSciencepapers:

InternationalHumanGenomeSequencingConsortium.Initialsequencingandanalysisofthehumangenome.Nature2001;409:860–921.

Venter,J.C.,Adams,M.D.etal.Thesequenceofthehumangenome.Science2001;291:1304–51.

Chapter9

Chapterheadquote:Huxley,T.H.,1893.ChapterV,MrDarwin’sCritics:120.

Mutations and bat wings: Cooper, K. L. & Tabin, C. J. Understanding of bat wingevolutiontakesflight.GenesandDevelopment2008;22:121–24.

Definitionofgenomiccreativity:Ryan,F.P.Genomiccreativityandnaturalselection:amodernsynthesis.BiolJLinneanSoc2006;88:655–72.

Chapter10

Chapterheadquote:Margulis,L.,1970:Preface.

TheSelfishGene:Dawkins,R.,1976.

Ihavewrittenalotmoreaboutthehistoryofsymbiosisanditsdevelopmentinmybook,Darwin’sBlindSpot,2002.

ForMaynardSmithandsymbiosis:Smith,J.M.&Szathmáry,E.,1999.

ForLynnMargulis,chloroplastsandmitochondria:Margulis,L.,1970.

Chapter11

Chapterheadquote:Wimmer,E.Thetest-tubesynthesisofachemicalcalledpoliovirus.EMBOReports2006;7:special issue:S3–S9.ThefirstsequencingofpolioviruswaspublishedinKitamura,N.,Semler,B.L.etal.Primarystructure,geneorganisationandpolypeptideexpressionofpoliovirusRNA.Nature1981;291:547–53.

Theviralcomponentsofthehumangenome:Ryan,F.,2009.

HIV-1andhumanHLA-B types:Kiepiela,P.,Leslie,A. J. et al.Dominant influenceofHLA-B inmediating the potential co-evolution ofHIV andHLA.Nature 2004;432:769–74.

Screening forviralproteins invarious tissues:Chen,F.,Atterby,C.etal.ExpressionofHERV-R ERV3 encoded Env-protein in human tissues: introducing a novel protein-antibody-basedproteomics.JRoySocMed2013;107(1):22–29.

Chapter12

Chapterheadquote:Jablonka,E.&Lamb,J.M.:vi.

C.elegans:Brenner,S.,2001.

Chapter13

Chapterheadquote:BrennerNobelspeech,2002.

Chapter14

Chapterheadquote:Judson,H.F.:10.

Clovisdiscovery:Rasmussen,M.,Anzick,S.L.etal.ThegenomeofaLatePleistocenehumanfromaClovisburialsiteinwesternMontana.Nature2014;506:225–29.

Siberian child discovery:Raghaven,M., Skoglund, P. et al.UpperPalaeolithicSiberiangenomerevealsdualancestryofNativeAmericans.Nature2014;505:87–91.

Richard III story: Ehrenberg, R. A king’s final hours, told by his mortal remains.Sciencenews.org,9March2013.

Chapter15

Chapterheadquote:Cavalli-Sforza,L.:33.

FirstWilsonpaper:Cann,R.L.,Stoneking,M.,Wilson,A.C.MitochondrialDNAandhumanevolution.Nature1987;325:31–36.

For a more recent overview: Pakendorf, B. & Stoneking, M. Mitochondrial DNA andhumanevolution.AnnRevGenomicsHumGenet2005;6:165–83.

Stringer’sdrawingattentiontolackofagreementondateofAdamandEve:Stringer,C.OutofEthiopia.Nature2003;423:692–95.

Y–chromosomesequencing:Poznik,G.D.,Henn,B.M.etal.SequencingYchromosomeresolves discrepancy in time to common ancestor of males versus females. Science2013;341:562–65.AlsoCruciani,F.,Trombetta,B.etal.Arevisedrootforthehuman

Y chromosomal phylogenetic tree: the origin of patrilineal diversity in Africa.Am JHumGenetics2011;88:814–18.

MountTobaandAsiandispersal:Mellars,P.,Gori,K.C.etal.Geneticandarchaeologicalperspectiveson the initialmodernhumancolonizationof southernAsia.PNAS 2013;110(3):10699–704.

HERV-K106:Jha,A.R.,Nixon,D.F.etal.HumanendogenousretrovirusK106(HERV-K106)wasinfectiousaftertheemergenceofanatomicallymodernhumans.PLoSOne2011;6:e20234.

EarlymodernhumanmigrationintoAsia:Brown,P.RecenthumanevolutioninEastAsiaandAustralasia.PhilTransRoySocLonBioSci1992;337:235–42.

Aggressive symbiont: I have explained and defined what a viral aggressive symbiontcomprises in threebooks,VirusX,Darwin’sBlindSpot, andVirolution, aswell as inmanyscientificpapers.

Chapter16

Chapterheadquote:takenfromLibby’sNobelLecture.

The timingof earlymodernhumansenteringAsia:Mellars,P.Whydidmodernhumanpopulations disperse from Africa ca. 60,000 years ago? A new model. PNAS 2006;103(25):9381–86.

Douka,K.Exploring‘thegreatwildernessofprehistory’:thechronologyoftheMiddletotheUpperPaleolithic transition in theNorthernLevant.MitteilungenderGesellschaftfürUrgeschichte2013;22:11–40.

Douka,K.,Bergman,C.A.etal.ChronologyofKsarAkil(Lebanon)andimplicationsforthe colonization of Europe by anatomically modern humans. PLoSOne 2013; 8(9):e72931:1–10.

Mellars, P. & French, J. C. Tenfold population increase in Western Europe at theNeanderthal-to-modernhumantransition.Science2011;333:623–27.

Evidenceforwidespreadrecentevolutionarychangeinthehumangenome:Wang,E.T.,Kidama,G. et al.Global landscape of recent inferredDarwinian selection forHomosapiens.PNAS2006;103:135–40.

Chapter17

Chapterheadquote:from‘AconversationwithSvantePääbo’.Edge,7April2009.

Sequencinggenomesofanimalsandplants:Miller,W.,Drautz,D.I.etal.Sequencingthenucleargenomeoftheextinctwoollymammoth.Nature2008;456:387–90.Dabney,J.,Knapp,M. et al. Completemitochondrial genome sequence of aMiddle Pleistocenecave bear reconstructed from ultrashort DNA fragments. PNAS 2013;doi/10.1073/pnas.1314445110. Amemiya, C. T., Alföldi, J. et al. The Africancoelacanthgenomeprovides insights into tetrapodevolution.Nature2013;496: 311–16.

ForNeanderthalfossilsandappearances:Stringer,C.&Gamble,C.,1994.

Neanderthalsandart:Abadía,O.M.,&GonzálezMorales,M.RedefiningNeanderthalsand art: an alternative interpretation of the multiple species model for the origin ofbehaviouralmodernity.OxfordJArchaeology2010;29(3):229–43.Seealso,Zilhão,J.Symbolic use ofmarine shells andmineral pigments by IberianNeanderthals.PNAS2010;107:1023–28.

SuperioritycomplexwithregardtoNeanderthals:Villa,P.&Roebroeks,W.Neanderthaldemise: an archaeological analysis of the modern human superiority complex.PLoSOne2014;9(4):e96424.

Chapter18

Chapterheadquote:fromZilhão,J.&Trinkaus,E.Eds.PortraitoftheArtistasaChild:TheGravettianHumanSkeletonfromtheAbrigodoLagarVelhoanditsArcheologicalContext.TrabalhosdeArquelogia2002;22:9.ISBN972-8662-07-6.

Neanderthal mitochondrial draft genome: Green, R. E., Malaspinas, A. S. et al. AcompleteNeanderthalmitochondrialgenomesequencedeterminedbyhigh-throughputsequencing.Cell2008;134:416–26.

FirstdraftNeanderthalnucleargenome:Green,R.E.,Krause,J.etal.AdraftsequenceoftheNeanderthalGenome.Science2010;328:710–22.

ABObloodgroup:Lalueza-Fox,C.Geneticcharacterisationof theABObloodgroupinNeandertals.BMCEvolutionaryBiology2008;8:342.

Neanderthalsandmodernhumanssharethesamevariantofthelanguagegene:Krause,J.etal.ThederivedFOXP2variantofmodernhumanswassharedwithNeandertals.CurrBiol2007;17:1908–12.

Neanderthalsfoundtohaveredhairandfairskin:Lalueza-Fox,C.,Rompler,H.etal.Amalocortin 1 receptor allele suggests varying pigmentation among Neanderthals.Science2007;318:1453–55.

Neanderthal ancestry in Asians: Wall, J. D., Yang, M. A. et al. Higher levels ofNeanderthalancestryinEastAsiansthaninEuropeans.Genetics2013;194:199–209.

The reappraisal of Neanderthal culture and society: Abadía, O. M. & Morales, R. G.RedefiningNeanderthals and art: an alternative interpretation of themultiple speciesmodelfortheoriginofbehaviouralmodernity.OxfordJArchaeol2010;29(3):229–43.

Teaching Neanderthal children: Spikins, P., Hitchens, G. et al. The cradle of thought:growth,learning,playandattachmentinNeanderthalchildren.OxfordJArchaeol2014;33(2):111–34.

Compassion,cultureofNeanderthals:Spikins,P.A.,Rutherford,H.E.&Needham,A.P.From homininity to humanity: compassion from the earliest archaics to modernhumans.TimeandMind:TheJofArchaeology,ConsciousnessandCulture2010;3(3):303–26.

ThecompleteNeanderthalgenomefromtheAltaiCave:Prufer,K.,Racimo,F.etal.ThecompletegenomesequenceofaNeanderthal from theAltaiMountains.Nature2014;505:43–49.

GenomiccontributionofNeanderthalstopresent-dayhumans:Sankararaman,S.,Mallick,S.etal.ThegenomiclandscapeofNeanderthalancestryinpresent-dayhumans.Nature2014:507:354–57.

Neanderthallegacyintermsofdisease:Leake,J.Neanderthals’revenge:thegiftofdeadlygenes.TheSundayTimes,26.01.14:19.

DisparityinhumantoNeanderthalpopulationswhentheymet:Mellars,P.&French,J.C.Tenfold population increase inWestern Europe at the Neanderthal-to-modern humantransition.Science2011;333:623–27.

Discovery of theDenisovan genome.Marshall,M.MysteryRelations.New Scientist: 5April2014:34–38.

Denisovanmitochondrial genome:Krause, J., Fu,Q. et al. The completemitochondrialDNAgenomeofanunknownhomininfromsouthernSiberia.Nature2010;464:894–97.

The Denisovan nuclear genome: Reich, D., Green, R. E. et al. Genetic history of anarchaichominingroupfromtheDenisovacaveinSiberia.Nature2010;468:1053–60.

Tibetan inheritance from the Denisovans: Huerta-Sánchez, E., Jin, X. et al. Altitudeadaptations inTibetanscausedby introgressionofDenisoval-likeDNA.Nature2014;doi:10.1038/nature13408.

DNA fromSima de losHuesos: The genome of a fossil from the Sima de losHuesos:Meyer, M., Fu, Q. et al. A mitochondrial sequence of a hominin from Sima de losHuesos.Nature2014;505:403–36.

Chapter19

Chapterheadquote:Darwin,C.,1859.ChapterIV:NaturalSelection:131.

Jeffreys’discoveryofDNAfingerprinting:Jeffreys,A.J.,Wilson,V.etal.Hypervariable‘minisatellite’regionsinhumanDNA.Nature1984;314:67–73.

Ouronenessasaspecies:Cavalli-Sforza,L.L.,2001.

References to haplogroup differences between the sexes, in Europe and in WesternEurope, includingtheBritishIsles:Wilson,J.F.,Weiss,D.A.etal.GeneticevidencefordifferentmaleandfemalerolesduringculturaltransitionsintheBritishIsles.PNAS2001;98:5078–83.Seealso,Capelli,C.,Redhead,N.etal.AYchromosomecensusoftheBritishIsles.CurrBiol2003;13:979–84.

Venter andWatsonmore similar toKorean genome: Barbujani,G. et al. Human races.CurrBiol2013;23(5):R185–R187.See also,Ahn,S.-M.,Kim,T.-H. et al.The firstKorean genome sequence and analysis: full genome sequencing for a socio-ethnicgroup.GenomeResearch2009;19:1622–29.

ÖetzitheIceman:Ermini,L.,Olivieri,C.etal.CompletemitochondrialgenomesequenceoftheTyroleanIceman.CurrBiol2008;18:1687–93.Seealso,Keller,A.,Graefens,A.et al. New insights into the Tyrolean iceman’s origin and phenotype as inferred bywhole-genome sequencing.NatureCommunications 2012: doi: 2-.1038/ncomms1701|www.nature.com/naturecommunications.

Theoriginsofdifferentsectionsofthehumangenome:Pääbo,S.:186–187.

Chapter20

Chapterheadquote:Venter,J.C.:110.

For more lay-directed discussion of scientists and ethics involved see Duncan, D. E.,2006.

Adversedrugreactions:Thiesen,S.,Conroy,E.J.etal.Incidence,characteristicsandriskfactorsofadversedrugreactionsinhospitalizedchildren–aprospectiveobservationalcohort study of 6,601 admissions. BMC Medicine 2013; 11: 237.http://www.biomedcentral.com/1741-7015/11/237.

Chen, R., Mias, G. I. et al. Personal Omics profiling reveals dynamic molecular andmedicalphenotypes.Cell2012;148:1293–307.

Regulation of GM crops: see Wikipedia article, ‘Genetically modified organismcontainmentandescape’.

Wildcanolafoundtocontaingeneticallymodifiedgenes:Ibid.

We are still evolving: Voight, B. F., Kudaravalli, S. et al. A map of recent positiveselectioninthehumangenome.PLoSBiology2006;4(3):e72,0446–58.

Constructing the first artificial genome:Gibson,D.G., Glass, J. I. et al. Creation of abacterialcellcontrolledbyachemicallysynthesizedgenome.Science2010;329: 52–56.

Humanembryodeliberatelyengineeredinascientificexperiment:LiangP,XuY,ZhangX,etal.CRISPR/Cas9-mediatedgeneediting inhumantripronuclearzygotes.ProteinCell2015doi10.1007/s13238-015-0153-5.

index

Thepagenumbersinthisindexrelatetotheprintedversionofthisbook;theydonotmatchthepagesofyoureBook.YoucanuseyoureBookreader’ssearchtooltofindaspecificwordorpassage.

ΦX174(bacteriophagevirus)121

Acheulean(MiddleStoneAge)stonetools220,249

achondroplasia109

adenine17–18,69,77,80,81,87,94,102,107,142,199

adeno-virus2,131

‘adjuvant’13

Africa:humanancestry/migrationpatterns211–12,214–24,226–42,249–50,256,259,260,261,267,276,288,290,301

Agassiz,JeanLouisRodolphe138

AIDS159,162,223,301

Air(longnon-codingRNA)192

alkaptonuria93

Alloway,J.L.16–17

alpha-helix,protein56–7,64,65,68–9,81

AltaiMountains,Siberia202,267–8,270–1,273–6

Altmann,Richard17

Ambros,Victor185

AmericanAssociationfortheAdvancementofScience260

aminoacids18,28,53,68,79,93,95–6,97,98,107,131,158,181,187

Angelmansyndrome192,194

antibiotics6,19,20,32,48,128,316

anti-senseDNA105–6,177,194,199

Anzick-1198,202–3,207

apoptosis(programmedcelldeath)153,185–9

ArabianPeninsula212,217,233,236

archaea151–2

archaeogenetics219,240

ArchaeologicalMuseumofLebanon232

Aristotle147

Arkwright,J.A.12

art,Neanderthalproductionof254–6

Asia:humanancestry/migrationpatterns143,197–8,201,202,203,211,212,214,217,218,222,224,226,228,229,230,233,234,235,236,237,238,239,240,241,248,249,261,277,288,301

Astbury,William46,68

astrocyte169–70

AtapuercaMountains,Burgos,Spain278–9

AtlanticOcean198,271

Australia:

humanancestryin249,255,277

HumanGenomeProjectand123

koalaretrovirusepidemicin161–2,223

autosomes107,108,114,189,212,241

Avery,ProfessorOswaldTheodore‘Fess’10,41

Allowayand16

character10,25,30–5

Chargaffand79,92

classificationofTBgermand10

CopleyMedal,RoyalSociety,awarded32

CranberryBogBacillusand19

Crickand53

Dawsonand15–16

DNAdiscovery13,14,15–17,19,21,22–3,24,25–7,29–35,36,40,43–4,53,79,92

DNAdiscoverydisputed25–7,29,34–5,79,179

Dubosand6–7,10,17,19,20,29–30,31,32,33,34

Griffith’sexperimentsand13,14

health19–20,21

honorarydegree,CambridgeUniversity32–3

Kochand10,14

Luriaand43–4

recognitionofachievement,lackof29–30,31,33,34–5

‘Studies on the chemical nature of the substance-inducing transformation of pneumococcal types: induction oftransformationbyadesoxyribonucleicacidfractionisolatedfrompneumococcustypeIII’22–3,24,29,34–5

Watsonand40,43–4

bacteria5

adaptation11–12

archaeaand151–2

autotrophic158

Avery’sdiscoveryofDNAand13–17,23,36,44,92

bacteriophages/phagesandseebacteriophages/phages

classificationof9–10,25,29

‘coitus’andchromosome99–100

cyanobacteria151–2,206,282

definitionofvirusand156–7

DNAsequencingand121

heredity10–17,41,42

Dubosand5,10,15,16

firstcompletelysequencedgenomeofabacterium126

killingof157

Koch’spostulates10–11

aslivingorganisms10

mitochondrialgenomeand154,204,205–6

‘morevirulent’or‘attenuated’11,12

nitrogencycleand150

oxygenbreathing,first152

pathogenic(disease-causing)10–11,12–13

polysaccharideand13–14,17,19,21,22,25,33

prokaryotes9–10

rhizobia150

serotypes12,13

SmoothandRough(SandR)12–14,15,16

symbioticunionand149,150,151–2,153

VentersynthesisestheMycoplasmagenitaliumgenome303–4

Venter’sgroupcreatealivingbacterialcell304

bacteriophages/phages56,96

discoveryofDNAstructureand41–4

evolutionoutofexceedinglyancientvirallineages158–9

firstcompletegenomedetermined(ΦX174)121,132,158–9

geneticinformationpassedfromonetoanother,processof99–100

HersheyandChaseconfirmdiscoveryofDNAwithexperimentwith27–9,36

LuriaandDelbruckinvestigateinterplaywithhostbacteria41–3,63,92

phagegroup41–5,57,97,99

Watsonand41,44,45

Bailey,Bill269–70

Bailey,Shara252

Bary,ProfessorAntonde147

Bateson,William8

Baulcombe,David186

BayerCompany,Elberfield,Germany20

Bayesianmodeling232–3

Beadle,GeorgeW.93,130

BeckerandDuchennemusculardystrophies134–5

Berg,Paul294

Bergman,DrChristopherH.231–2

Bergström,KarlSuneDetlof243

Bernal,JohnDesmond53–4,83,84,90

beta-globin97–8,107,133–4,141

bidi-rectionaltranscripts194

BiologicalJournaloftheLinneanSociety145

Birkbeckcollege,BiomolecularResearchLaboratory83–4,90–1

Blair,Tony124

BlombosCave,SouthAfrica227–8

BNC2(gene)269

Boivin,André95

Bonnet,MarieLouise31–2

Boule,Marcellin262

Bragg,SirWilliamLawrence51–2,53,54–5,56,64,67,68–9,84,86,89

Bragg,SirWilliamHenry51,52,53–4,68

Bragg’sLaw52,89

Branson,Herman68

BRCA1andBRCA110,188,297

Brenner,Sydney96–7,114,115,118,185,186,294

Briggs,Adrian247

BritishMuseum231

BroadInstitute268

Broca’sarea254,263

Bronowski,Jacob121

TheAscentofMan1

TheIdentityofMan1

Brown,WesleyM.214

Caenorhabditiselegans(roundworm)114–15,185,186–7

CaliforniaInstituteofTechnology(Caltech)41,57,67–8,121

CambridgeUniversity32–3,48,51–5,56,57,58,60,62,63,64,65,68,70,75,76,78–9,82,86,90,91,97,114,121,222,237seealsounderindividuallaboratoryorcollegename

cancer16

bladdercancer195

bonecancer195

bowelcancer141

braincancer195

breastcancer110,111,141,188,195,297,300

coloncancer110,111,195

geneticengineeringand295,297,300

HERVsand167,170

IncRNAsand194–5,294

kidneycancer141

lungcancer111,195

methylationpatternsand178–9

mutationsand107,109,110–11,141,188

ovariancancer90,188,297,300

prostatecancer195

research128

skincancer141

somaticmutationsand141

Cann,RebeccaL.213–14

Carbon-14dating236

Carrel,DrAlexis6

CartagenaProtocolonBiosafety,2000299

Cavalli,Sforza,ProfessorLuigiLuca210,211,288

Genes,PeoplesandLanguages211

cavepaintings254–5

CavendishLaboratory,CambridgeUniversity51–5,57,58,60,62,63,64,65,68,78–9,82,86,90,114

Cavendish,Henry51

CD+Thelperlymphocyte160

CD4160,161

CeleraGenomics123,124,125,126,127–8,302,303

cell:

division(mitosis)140–1,186,190–1

DNAamountwithin94–5,113,173

eukaryotic114

germcell7,8,9,10,106,107,109,162,174,198–9,200,205,218,283,304

nucleargenomewithinseenucleargenome

pluripotent(‘stem’)cells115–16,168,172,183,194

programmedcelldeath(apoptosis)153,185–9,260

regenerationandrepair260

RNA,variationofamountwithin94–5

symbiosesand151,152–4,282

totipotent115

virusesand156–69

zygoteandseezygote

CFTR(cysticfibrosistransmembraneregulatorgene)108

Chargaff,Erwin35,79–81,86,92

Chase,Martha27–8,34,36,53

chemicalbonds65,66–8,71,79,81,104

Chetverikov,SergeiSergeevich140

chloroplasts151,152

chromosomes:

autosomes107,108,114,189,212,241

Avery’sdiscoveryofDNAand44

bacterialtransferofDNAand99–100

chromosome3223

chromosome4141,142

chromosome6179–82,283–4

chromosome7108,165

chromosome8223

chromosome19223

commonmaleancestorand218–21

CrickandWatson’sdiscoveryofstructureofDNAand55,93,98

discoveryof8–9

genotypesand295

haplotypes/haplogroupsand200–1,204,205,206–7,211–12,218–19,220,222,223,224,229,239–40,289–90

homologoussexualrecombination9

Hoxgenesand116,117

lncRNAs(longnon-codingRNAs)and191–3

MajorHistocompatabilityComplex(MHC)and179,180–4,269283–4,301

meiosis283

MSRY(malespecificregion)ofYchromosome218,239–40

mutationand107,108–9,141–2,200,211,241seealsomutationsandSnips

prokaryotesand9–10

retrovirusesand162,163,164,165,166,167,206–7,222–3

Schrödinger’sWhatIsLife?and39–40

sequencingofentirehumangenomeand120,122,126,127,129,132

Snipsand200,211,241seealsoSNPs(‘singlenucleotidepolymorphisms’)(‘Snips’)

VNTRand286–7

X-inactiviationinfemaleembryos189–91

Church,DrGeorgeM.295

chymotrypsin22

Cicero147

clades201,240

Clinton,Bill124

Clovisculture197–8,207

co-evolution158,160

Coghlan,Andy125

ColdSpringHarborLaboratory,NewYork131,274

Collins,Francis124,127,128

colourblindness109,190

ColumbiaUniversity79,93,263

Columniaplant149–50

‘ComplexIdeficiency’154–5

Corey,Robert68

Corsica289

covalentbond65–6,67,102,176

CranberryBogBacillus19

creativegenomics293–4

creativity:

creativegenomics293–4

Crick-WatsondiscoveryofstructureofDNAand63–4,69,88,90

DNAand3

genomic145,175,178,282,293,304

hybrid266

Neanderthal270

personalityandscientific20,30–1

Crichton,Michael:JurassicPark243

Crick,Francis34,71,74

Avery’sdiscoveryofDNAand53,55

background52

BrennerandseeBrenner,Sydney

CavendishLaboratoryandseeCavendishLaboratory,Cambridge

character54–5,64–5

Chargaffand80–1,86

DNAextrapolationtoproteins,investigates93–4,95–8,114

Franklinand74,76–8,84,85

Gamowand95–6

Griffithand78–9,86,97

Pauling’spaperonstructureofDNAand82–3,85

‘perfectbiologicalprinciple’and79

RNATieCluband96,97

Schrödinger’sinspires40,52,52,91,93,125

secondpaperongeneticimplicationsofthestructureofDNA,195388

structureofDNA,investigationintoanddiscoveryof34,40,53,55,59,60,54–6,58,61–5,68,69–70,74,76–91,92,93,94,99,102,103,112

Watsonand40,55–6,58,60,61–5,69

WhatIsLife?and52–3

‘WhatMadPursuit’(ad-hocseminar)54–5

Wilkinsand47,75–6,83

Crick,Michael62

Crick,Odile62

cyanobacteria151–2,206,282

cysticfibrosis108,109,300

cytoplasm94,95,131,134,152,153,165,187,203–4

cytosine17–18,69,77,80,81,87,94,102,142,176,177,178,181,191,199,247

‘D’root(haplogroup)201

D4h3a(haplogroup)202,207

DailyTelegraph124,281

Dale,SirHenry33

DanubeRiver230

Darwin,Charles7,12,14,15,31,37,41,90,130,137–9,144,145,146–7,247–8,281,282,283,291,298

Darwinism41–2,144

Dawkins,Richard:TheSelfishGene146–7

Dawson,M.H.15–16

DeValera,Eamon38–9

deacetylaseHDAC11182

Deichmann,Uti24

Delbruck,Max26,39,41–2,43,44,57–8,63,92,96

Denisovanman(Denisovahominins)248,276–8,279,288,301

DepartmentofAgriculture,US.(USDA)299

DepartmentofEnergy,US.122

Derevianko,Anatoly273,275

developmentalpathway117–18

Diamond,Jared262

disease8,10–11,12–13,20,97–8,106–11,112,120,134–55,160,161,164,167,169,170,175,188,190,192,195,212–15,242,256,261,264,269,270,271,284,289,294–7,298,300–2seealsounderindividualdiseasename

Dmanisi,Georgia249

DNA(deoxyribonucleicacid)3

Anti-sense105–6,177,194,199

cDNA(complementaryDNA)193

codingtriplets,searchfor94–9

codons98–9,106–7,191

constancy25

correspondenceofnumberofgenestonumberofproteins93,130,131–5

discoveredbyAvery,194414–35,36

discoveryconfirmedbyHersheyandChase’sexperimentwithbacteriophage,195127–8,34,36,53

epigeneticsandseeepigenetics

exonsand132,133,134,135,142,187,190,193

extractiontechniques21–2,25,26,28,29,226,244,246,247,250,257,258,260,268,274,276,277

extrapolationtoproteinsfirstexplored92–111,112–23

fingerprinting285–7

GACT(guanine,adenine,cytosine,thymine)bases17–18,77,93,94,95

geneticsengineering/creativegenomicsand292–304

genomiclevelevolutionand172–85

humanancestryand197–291

intronsand132,133,134,135,187,190

junk164,170

mutationsofseemutations

naturalselectionandseenaturalselection

numberofprotein-codinggenesinhumangenomeand130–1,135

profiling285

promotersequences100,113,134,141–2,165,169,176–7,183,191,193,194,207

questionsoverthediscoveryof24–9

recombinant294,299

RNAandseeRNA

sensestrand177,186,191,194,199

sequencingof120–3,124–36,167,198,202,246–7,258,270–1,278,

284–5,287,288–9,302

splicing130–5,87

structurediscoveredbyCrickandWatson,195338–49,50–8,59–91,92,93,95,112,113

sugar–deoxyribose94,102

symbiosisandseesymbiosis

tetranucleotidehypothesisand18,79,80

up-streamregulatoryelements134

virusesandseeviruses

DNA-methyltransferase176

Dobzhansky,Theodosius140

Domagk,Dr.Gerhard20

Donohue,Jerry86–7

Douka,DrKaterina226–38

Down’ssyndrome109

Dubos,RenéJules5–7,10,14,15,16–17,19–21,22,23,29–30,31,32,33,34

Dutton,ProfessorGordonN.145

dystrophin134–5

EastAfrica211,212,222

Edge(website)244

Edqvist,DrPer-Henrik167,168

Egbert(skeletonfossil)231–3

Egypt244

electronmicroscope,inventionof42

electronspinresonance234,236

Elmer,Perkin126

embryo/embryogenesis2,8,37,40,109–10,113–19,120,143,164,167–8,172–3,174,178,183,189,190–1,194,284,294,297,304

ENCODE‘EncyclopaediaofDNAcodingelements’171

EnvironmentalProtectionAgency(EPA)299

enzymes19,22,25,26,54,93,104,117,119,143,157,158,159,160,165,182,247

EPAS1(gene)277–8

epigenetics116,118–19,120,145,157,163,164,172,173–84,187,189,191,192,194,236,247,282,283,285,286,293–4,295,296,297,301

discoveryof96–106,113–16

epigeneticmark182–3

epigeneticsilencing163,176–7,183,186,189,192

epigeneticsystem173–84

epigenomeand175,183,295,302

evolutionarypotential175,178,182–3

fourepigeneticcontrolsystems175–84

geneticengineeringand293,294,295,296,297

genomiccreativityand175,178

histonemodificationand175,180–3,191

HoxgenesandseeHoxgenes

imprinting192

IncRNAsand192,194

LTRsand164

meiosisand283

methylationand163,175,176–9,180,181,182,183,191,247

mutationlocationand285

non-codingRNAsand173–4,175,183–4,185–96,236,266

pubertyand118–19

RNAiand187–9

tandemrepeatsand286

EsSkhulcavesite,MountCarmel,Palestine228,234–5

Eschurichiacoli–E.coli99,121

ESTs302

Ethelruda(fossil)232,233

Ethiopia220–1,224

eugenics110,297

eukaryotes9–10,114,135,141

Eurasia,humanancestry/migrationpatternsand202–3,214,217,228,229,230,234,235,236,237,238,239,248,249,251,256,259,261,264–6,269,271,272

Europe:humanancestry/migrationpatternsand143,144,197–8,200–1,202,211,212,217–18,224,226,227,228,229,230,236,237,238,239–41,249,251,253,256,258,260,261,262,264–5,267,269,271–2,277,288,289,299–300,301

evolution:

continuing301–2

DarwinandtheoryofseeDarwin,Charles

genomiclevel172–84

seealsounderindividualareaofevolution

evolutionarybiologists11–12,37,140,198,251

exons132,133,134,135,142,176,187,190,193,194

Feinbaum,Rhonda185

fingerprinting,DNA285–7

Finlayson,Clive279

Fire,AndrewZ.186,187

Fisher,RonaldAylmer140

Fisher,Simon254

Fleming,Alexander19

Flores,islandof248

FoodandDrugAdministration(FDA),US.299

454LifeSciences246

FoxhallRoad,Ipswich,Neanderthalhandaxesfoundat264

FOXP2(gene)254,260

Francalacci,Paolo219–20

France11,165,169

GMcropsand300

Homosapienancestryand230,237,238,239,255,255,263,264

HumanGenomeProjectand123,124

Frank,AlbertBernhard147,148

Franklin,Rosalind36,47,58

background70–2

BiomolecularResearchLaboratory,Birkbeckcollege,moveto83,84,90–1

BiophysicsUnit,King’sCollege,roleat72–6

character71–2

Crickand74,76

death90,91

discoveryofstructureofDNA,rolein47,58,70,72–8,82–5,86,88,90

Meringand71–2

Sayreand74–6,78

scienceandeverydaylife,onseparationof36

Watsonand43–4,57,60–1,63,70,82–3,84–5

Wilkinsand47,72–8,82–5,90

French,JenniferC.237,271

fruitflies8,10,25,41,93,116,126,130,290

Galapagosislands138

Gamble,Clive252

Gamow,George95–6,97

Garrod,ArchibaldE.93

gene:

definitionof7–10

expression2,8,55,93,100,113–14,117,120,131,134,165,166,167,168,170,173,174,175,178,188,190seealsoepigenetics

therapy108,109,110,155,175,188,294,297

geneticbottlenecks221–2,224,301

geneticengineering293–300,302

genetics,birthofdiscipline8

genomiccreativity(mutation,epigenetics,symbiosisandhybridisation)145,175,178,282,293,304

genotype12,295–6

GibraltarMuseum279

Gibraltar,Neanderthalremainsin251

GlasgowCaledonianUniversity145

GM(geneticallymodified)298–300

Gold,Thomas78–9

Golding,William262

gonadotrophin-releasinghormone(GnRH)119

Gosling,Raymond50,72,73,76,77,78,83,86,88,90

Gravettians238

GreatApes165–6,248,281

Griffith,Frederick12–14,15,16

Griffith,John78–9,86,97

guanine17–18,69,80,81,87,94,102,142,177,191

haemoglobin52,54–5,57,68,69,97–8,107,133–4,278

haemophilia108,190,300

Haemophilusinfluenza(bacterium)126

Haldane,JohnBurdonSanderson140

Hamilton,Andrew186

haplotypes/haplogroups200–9,210–21,223,236,239–40,250,268–9,284,288,289

HarvardUniversity98,268

HarvardMedicalSchool268,276

Healy,Bernadine126–7

Hedges,ProfessorRobertE.M.231

heredity,principlesof7–15,17,18,24,25,29,33,34,36,37–8,61–2,63,64,79,81,92,93,95,106,113,120,137–45,153,162,178,182,184,196,204,265,283,292

Herriott,Roger43

Hershey,Alfred27–8,34,36,41–2,43–4,53,96

Herto,Ethiopia220–1

Higham,ThomasF.G.231

Highfield,Roger124,281

histonemodification125,175,180–3,191

HIV-1157,159–60,162,223

HLA-B(gene)160

Hodgkin,Dorothy54

Holloway,Ralph263

holobiont148–9

holobionticgenome151,153,155,159,162,164,205,222,266

hominids244,248

hominins228,233,234,235,244,248,249,275–6,277,278

Homoerectus248–50,251,252,259,260,277,290

Homofloresiensis248

Homoheidelbergensis251,252,259–60,278

Hood,Leroy121

horizontalgenetransfer150–1,299

Horvitz,Robert185

Hotchkiss,Rollin17,26

HowardHughesMedicalInstitute268

Hoxgenes116–18

humanendogenousretroviruses(HERVs)162–70,173,212,222–4,236,282,288

ERV3166–7,168

HERV–FRD166–7,168–9

HERV–Ks162,163,166,212,222–4,236,288

HERV–K106223–4,236,288

HERV–K113212,223,236

HERV–K115212,223,236,288

HERV–K116223

HERV–Wgroup166,170

MSRV–HERV–W170

seealsoretroviruses

humangenome:

amountofprotein-codinggenes(20,500)discoveredinhumangenomesequencing129–6,170–1,174

DNAdiscovered14–35,36,53

DNAstructurediscovered38–49,50–8,59–91,92,93,95,112,113

DNAextrapolationtoproteinsfirstexplored92–111,131–5

DNAbreakdownof129–30,195–6

epigeneticsand172–96seealsoepigenetics

geneticengineering/genomiccreativityand292–304

genomiclevelevolutionof172–84

HERVsandseeHERVs

humanoriginsandmigrationsand197–291

mappingof120–3,124–36,170,193,281,293

mutationsandseemutations

naturalselectionand137–45seealsonaturalselection

numberofgenesincommonwithotherorganisms130,281–2

percentageofretroviralDNAin162

symbiosisand146–55seealsosymbiosis

uniquenessofeach283–91

unknown50percentof,explanationfor135–6,170–1,189,193–6

virussymbiosiswith159–71seealsoretrovirusandvirus

wholegenomesequencing,increaseinnumberof287–8

seealsounderindividualcomponentsofhumangenome

HumanGenomeProject,The122–3,124–30,168,245

humanoriginsandmigrations197–291

AfricanoriginsofHomosapiens211–24,226–42,248,249–50,256,259–61,267,276,288,290,301

autosomesand212,241

commonancestors200,210–24,248,259,260,265–6,274,275,276,277,290,291

extractionandassessmentofancientDNA,advancesin226,229,234,236,243–7,250,257,258,260,268,274,276,277

firstevolutionaryemergenceofmodernhumans226,243–80,289–91

haplotypes/haplogroupsand200–3,204,205,206–9,211–12,213–14,218–19,220,222,223,224,229,239–40,289–90

HERVdistributionand212–13,222–4,236,288

hominidsand228,235,244,248

homininsand233,244,248,249,275–6,277,278

Homoerectusand248–50,251,252,259,260,277,290

Homofloresiensisand248

Homoheidelbergensisand251,252,259–60,278

humanevolutionarytree/speciesevolutionand248–56,257–80

hunter-gatherers215,216,233,267,270,271

IceAges/climateand197,198–9,221,233–4,235,236,238–40,251,252,253,266,267–8,271

LastGlacialMaximum(‘LGM’)and239–40,271

lossofdiversityandnear-extinctionevent221–4

MajorHistocompatabilityComplex(MHC)and221,283–4,301

mitochondrialDNAand203–9,212–24,237,240,250,257,258,260,273–4,275–6,278,279,282,288–9

MSRYand239–40

‘multi-regionaltheory’and249–50

NeanderthalevolutionandseeNeanderthals

radiocarbondatingand208,226–7,228,229,231,232,236

RichardIIIand207–9

sitesofexcavationandfossilnamesseeunderindividualsitelocation/name

SNPs(‘singlenucleotidepolymorphisms’)(‘Snip’)and198–201,203–7,211–15,219,241,258,274,284,285,289,301

wholegenomesequencingand288–91

HumanProteomeProject168

hummingbird149–50,160

Huntington’sdisease108,109,141–2,188,300

Huxley,Julian:Evolution:TheModernSynthesis14,15,139

Huxley,ThomasHenry137,139

hybridisation:

genomiccreativityand145,178

humanancestryand235–6,261–2,265–80,288,293,299,301

virusesand157

hydrogenbond65–7,76,81,86,87,88,104,191,199

Iberia239,255,257

Ibsen,Joy(néeBrown)209

Ibsen,Michael209

IceAges197,198–9,221,234,235,236,239,240

identicaltwins(‘monozygotictwins’)2,172–3,182–3,283,284,286

India222

IndianaUniversity,Bloomington40–1,43–4,57

InstituteforAdvancedStudies,Dublin38

InstituteofArchaeologyandEthnography,Akademgorodok,Russia275

integrase160

introns132,133,134,135,187,190,194

J1c2c(mitochondrialhaplotype)209

Jablonka,Eva172

Jacob,François99–100,101

Jeffreys,Alec285–7

Johannsen,Wilhelm7–8

JournalofExperimentalMedicine22–3

Judson,HoraceFreeland18,26,35,55,74,81,92

TheEighthDayofCreation197

Kalckar,Herman45,55–6,60

Kebaracave,Palestine235

Kendrew,John53,54,57,58,60,61,80,89

Kenya248

keratin64,269

Khorana,Gobind98

King’sCollege,London:MedicalResearchCouncilBiophysicsUnit45,46,47,48–9,58,70,72–6,77,82,83,84,85,86,88,90

Klug,Aaron72–3,84,91

Koch,Robert10–11,14,15

‘Koch’spostulates’10–11

Krause,Johannes273–4,275

KsarAkilrock-shelter,Lebanon228,230–3

LaboratoireCentraldesServicesChimiquedel’Etat,Paris71

LagarVelho,Portugal257

LakeTurkana,Kenya248

Lamb,MarionJ.172

Langley,Philippa208

language:

humanevolutionand215,253–5,260

Neanderthal254–5

Larsson,Erik167,168

LastGlacialMaximum(‘LGM’)239,271

LCA(lastcommonancestor)219,290

LePage,Michael125

Leakey,Richard248

Leber’shereditaryopticneuropathy155

Lederberg,Joshua99

Lee,Rosalind185

LeidenUniversity,Netherlands231,272

Levant233,236

Levene,PhoebusAaron18,79,80

Libby,WilliamF.225–6,227,228–9

lin-14(gene)185

LINEs(virus-likeentity)164,170,195

Linnaeansystem10,247–8

Linnaeus,Carl167

Luria,Salvador41–4,57–8,60,63

Lwoff,AndréMichel99,101

Lyon,MaryF.189–90

MacLeod,Colin21,22–3,29,36

Maddox,Brenda71,74,75

MajorHistocompatabilityComplex(MHC)and179,180–4,221–2,269,283–4,301

Mallet,DrFrancois165

ManhattanProject47,49,229

Margulis,Lynn146,152,282

Marler,Peter254

MaxPlanckInstituteforEvolutionaryAnthropology,Leipzig,Germany243,244,258,260,268,273–5

Maxwell,JamesClerk51

Mayr,Ernst139

McCarty,Maclyn21–3,25,26,29,36

McClintock,Barbara8–9,55,140

McCoy,DrJohnM.164–5

MedicalResearchCouncil47,72,97

Mediterranean230,233,236,255,289

meiosis283

Melanesians269,277

Mellars,SirPaul222,227,230,237,264–5,271

Mello,CraigC.186,187

Mendel,Gregor7,8,15,24,106,109,139,153,162,204

meningitis12–13,19,20,300

Mering,Jacques71–2

MESH(mutation,epigenetics,symbiosisandhybridisation)145

methylchemicalgroups176,181,247

methylation163,175,176–9,180,182,183,191,247

methyltransferase,DNA–176

MezmaiskayaCave,Caucasus268

MiddleAwash,Ethiopia224

MiddleEast217,228,249,251,258

Miescher,JohannFriedrich17

MinistryofHealth,London12–13,16

Mirsky,AlfredE.25,26

Mithen,Steven265

mitochondria152–5,162,194,200–7,209,211,212–19,224,237,240,250,257–9,260,273–6,278,279,282,288–9

haplotypes/haplogroupsand200–7,209,211–15,219,237–8,240,250,258,274,289

mitochondrialdisease154–5

‘mitochondrialEve’215–17,220

mitochondrialgenome153–4,203–4,205,211–13,216,218,219,257–9,274,275,279,288–9

mitochondrialIncRNAs194

mitochondrialsnipsseeSNPs(‘singlenucleotidepolymorphisms’)(‘Snip’)

nucleargenomeandseenucleargenome

palaeontology,useofmitochondrialgeneticsin200–7,209,211–15,219,237–8,240,250,258,274,289

symbioticoriginof(SET)152–5,205–6

mitosis(celldivision)140–1,186,190–1

‘ModernHumanSuperiorityComplex,the’272–3

molecularbiology37,53,94,96,97,120,122,167,168,184,185

molecularclock144,213,214,219,223

Monaco,Anthony254

Monod,Jacques99,101

Morgan,ThomasHunt8,93,126,140

MountToba,Sumatra222

Mousterianculture263

Moyzis,Robert241

MRCMolecularBiologyLaboratory,Cambridge97,114

MRCA(mostrecentcommonancestor)219–20

MS(multiplesclerosis)169–70

MSRY(malespecificregion)ofYchromosome218,239–40

Muller,HermannJ.25,26,41,44,93,140

Mullis,Kary246

‘multi-regionaltheory’214,249–50

musculardystrophy109,135

mutation:

autosomesand107,108

concept/discoveryof13–15,25,41,44,105–7,137–40

diseaseand107–11,112,115,120,134–55,190,212–15seealsounderindividualdiseasename

dominantgenemutation107,108,141–2

errorinthenucleotidesequence104–6

evolutionofHomosapiensand198–201,203–7,211–15,219,241,258,274,284,285,289,301

FOXP2and254

frame-shiftmutation106–7

frequencyforwholegenomes284–5

genetherapyandseegenetherapy

geneticsymbiosisand150–1

genomiccreativityand145

haplotypes/haplogroupsand198–201,203–7,211–15,219,241,258,274,284,285,289,301

LCA/MRCAand200–1,215,216,219

mitochondrialdiseaseand153–5,212–15seealsomitochondria

mutationalclock258

naturalselectionand137–45

Neanderthal258,260

pointmutation105–7,199

recessivegenemutation107–9,141,190

retrovirusesand157,161,163,164,166–7,223seealsoretroviruses

sex-linkedrecessivemutantgene108–9

single-genedisordersand109

SNPs198–201,203–7,211–15,219,241,258,274,284,285,289,301

somaticmutations140–1

structureofDNAdiscoveryand97–9,100,105–6

X-rayinducedgene25,44

Mycobacteriumtuberculosis(tuberculosisgerm)10

Mycoplasmagenitaliumgenome303–4

myelin169–70

NationalAcademyofSciences,US.122

NationalHumanGenomeResearchInstitute,U.S.171

NationalInstitutesofHealth(NIH),US.111,122,125,126,299

NationalResearchCouncilFellowship,US.6,44,56,60

NativeAmericans197,198,202–3,207

NaturalHistoryMuseum,London138,198,220,259

naturalselection14–15,37–8,90,117,137–45,149,151,153,160,166,169,199–200,213,241,266,281,283,284,286,293,298,304

Nature84,88–9,127,128,170,197–8,220,275,278–9

Neanderthal(Homoneanderthalensis)228,229,230–8,247,248,250–6,257–80,288,290,301

NearEast228,230,233,234,235–6,288

near-extinctionevent,lossofhumandiversityand221–4

neo-Darwinismand144

Neolithicera240

NewGuinea221,261

NewScientist125

NewYorkUniversity252

SchoolofMedicine21

Nirenberg,Marshall98,125

nitrogencycle150

NobelPrize24,25,26,29,32,33,34–5,38,41,43,51,65,67,69,75,76,90,91,101,121,132,135,185,187,229,243,249

Norrish,ProfessorR.G.W.75

NorthAfrica236

nucleargenome121,153–4,160,162,188,189,203,204,205,206,207,214,218,250,257,258–9,260,261,268,273,275,276,277,288,289

nucleicacid17–18,79

obesity,methylationand179

Ochoa,Severo98

Ötzi,theTyroleanIceman289

Olby,Robert26

OldManofShanidar264

oligodendrocyte(braincell)169

organdevelopment,geneticregulationof185

Orgel,Leslie97

ovum9,37,106,107,168,172,177,198–9,204,284

Owen,SirRichard138–9

OxfordRadiocarbonAcceleratorUnit226,231,232

OxfordUniversity96,146,185,226–7,231,232,254

oxygen52,57,66,67,68,97–8,151–2,153,154,203,206,278,282

Pääbo,Svante243,244,246,247,250,256,258,260,265,267,268,270,273,274,275,276,278,279,280,289–90

Pakendorf,Brigitte218

Palaeolithicera202,229,230,232,240,255

‘pangene’7

paperchromatography80

PasteurInstitute99,113

Pasteur,Louis11

Pauling,Linusix,24,56–7,64,65,67–9,71,76,77,79,81,82,84,85,86,104

Pauling,Peter82

PCR(polymerasechainreaction)246,287

PECorporation126

penicillin19

‘perfectcosmologicalprinciple’79

PersonalGenomeProject295–6

personalgenomics294–5

Perutz,Max51,52,53,54,55,57,58,60,62,63,68,69

phagegroup41–5,57–8,92,97,98

phenotype12,295

phocomelia118

photosynthesis151–2

phylogenetics248

placentalstructureandfunction115,165–6,167,168–9,172–3

pluripotentcells115–16,168,172,183,194

pneumococci12–17,19,21–2,23,25,29,33,35,300

pneumonia12–13,19,20,21,300

poliovirus156,304

Polynesia277,288

polysaccharide13–14,17,19,21,22,25,33

Pontén,ProfessorFredrik167,168

Portugal,F.32,34,35

Poznik,G.David219–20

Prader–Willisyndrome192,194

predictivemedicine294,296

prehistory,thegreatwildernessof225,226,227,228

preimplantationgeneticdiagnosis109–10,294,297

ProceedingsoftheNationalAcademyofSciences82,97

profiling,DNA285,294–5

prokaryotes9–10

promotersequences100,113,134,141–2,165,169,176–7,183,191,193,194,207

proteins8

DNAdiscoveryand18–19,21–2,25–9,53,54,55

DNAstructurediscoveryand42,45,50,52,53,54,55,56–7,62,64,65,67–9,72,79,81,89,91

epigeneticsand173,174,176,179,180,181,182,183,186,187–8,189,190,191,193,194

extrapolationofDNAtoproteinsfirstexplored92–111,114,116,117

genomesequencingand119–21,129–35,170–1,174,284–5

humanancestryand199,203,206,207,242,260–1,269

mutationsinDNAand140,142–3,154,157,159,164

numberofprotein-codinggenesinhumangenomeand129–35,170–1,174,284–5

onegeneoneproteinmaxim,BeadleandTatum’s93,130,131–5

structureof50,52,53–7,64,65,68–9,79,81

virusesand157,159,164,165,166,167,168,169

X-rayanalysisofproteinmolecules50,52,53–5,56–7,64

prontosil20

‘ProteinVersionoftheCentralDogma,The’18

Prx1(gene)140

puberty118–19

purines18,102

pyrimidines18,102

Qafzehcavesite,LowerGalilee,Palestine228,234–5

QiaomeiFu273–4

quantumtheory/mechanics38,39,65,67–8,86

radiocarbondating208,226–7,228,229,231,232

Ramsey,Margaret48

Randall,JohnTurton47,50,72–3,74,75,84–5

recombinantDNA294,299

Reich,David268,269,276

retrovirus16,101,155

co-evolutionand158–9

constantchangewithinhumangenomeand282,301

endogenous(ERVs)162–71,173,212,222,236,282

epidemic,easternAustraliakoala161–2,223

epigeneticsilencingand163

exogenous162,163,223–4

humanendogenous(HERVs)seehumanendogenousretroviruses(HERVs)

longterminalrepeats(LTRs)and164,165,169,193,195,223

originsofHomosapiens,distributionofusedtolocate212,222–4,236,288,301

retrovirallegacyroleinholobionticevolutionofhumangenome164–71,173,212,222

RNAbasedgenome159

symbioseswithhost159–62,222,223

viralenvgene164,165,166

viralloci162,163,164,165–7,168,169

Rhenen,Netherlands264

Rich,Alex96

RichardIII,King207–8

Ridley,Matt97

Riss(IceAge)234

RNA(ribonucleicacid)8

discoveryof17–18

epigeneticsand184,185–96,236,247,266,291,292,294

GACUstructuralchemicals(guanine,adenine,cytosine,uracil)17,94,95

lncRNAs(longnon-codingRNAs)191–3

mRNA(messengerRNA)98,131,133,134,135,159,167,186,187,188,190,191,193

non-coding173–4,187–9,191–6,199–200,236,266

piRNAs(Piwi-interactingRNAs)189

programmedcelldeathand186–9

riboseand94

ribosomal187

‘RNAgene’,ideaof188

RNAi(RNAinterference)187–9

roleingeneextrapolationtoproteinsfirstexplored94–5,96,98,131–5

single-strandedhelix94

siRNAs(smallinteractiveRNAmolecules)186–7

tRNA(transport/transferRNA)98,187

varianceinamountofindifferentcells94

viralgenomesand157,159,160,173–4,183–4seealsoretrovirus

RNATieClub96

RobertKochInstitute,Berlin15

Roberts,Alice269–70

Roberts,RichardJ.131,132,133,135

Roche246

RockefellerInstituteforMedicalResearch,NewYork5,6,13,15–16,18,19,21,22–3,24,25,30,31,33

Roebroeks,Wil256,272

Rothberg,Jonathan246

rubella,vaccinationsagainst109

RudbeckLaboratory167,168

Russia238

RutgersUniversity,NewJersey5,6,32

Rutherford,LordErnest51

Sanpeople215,290

SangerCentre,UK127

Sangersequencing121

Sanger,Frederick112,121,127

Sardinia219–20,289

Sayre,Anne74–6,78,90

Schrödinger,Erwin5,38–9

WhatIsLife?39–41,44,46,49,50,52–3,61–2,84,90,91,93,125

‘WhatisLife?’lectureseries38–9

Science19,127,128,170,237

seashells,humanancestryandcollectionof216,230–1,232,255–6

Seeds,Willy59,89

sensestrandofDNA177,186,191,194,199

sequencing,genome/DNA120–3,124–36,167,198,202,246–7,258,270–1,278,284–5,287,288–9,302

automated121,123,125,127,246,285

firstcompletegenomeofanorganism(bacteriophagevirus–ΦX174)121

firstinsectgenomicsequence(fruitfly)126

firstcompletelysequencedgenomeofabacterium(Haemophilusinfluenzae)126

humanancestorsand198,202,246–7,258,270–1,278,284–5,287,288–9,302

humangenomefirstsequenced122–3,124–36,170,193,281,293

mutationfrequencyforwholegenomes284–5

Neanderthalgenome258,270–1,278,288–9

Sangersequencing121

shotgunsequencing125–6,127,302

wholegenome284–5,287,288–90seealsoHumanGenomeProject

serialendosymbiosistheory(SET)152–3

Shakespeare,William90,207–8

Sharp,PhillipAllen131,132,133,135

Shigella(dysenterybug)12

shotgunsequencing125–6,127,302

Shreeve,James126

Sia,RichardP.16

Siberia,humanancestryin201,202,207,235,240,251,267–8,270–1,273–4

sickle-celldisease97–8,107,141

Siemens42

SimadelosHuesos(‘pitofbones’),Burgos,Spain264,278

SINEs(virus-likeentity)164,170,195

Smith,JohnMaynard151

SNPs(‘singlenucleotidepolymorphisms’)(‘Snips’)198–201,203–7,211–15,219,241,258,274,284,285,289,301

Snyder,Michael295

Spain,humanancestryin230,239,255–6,276,278–9

Spikins,Penny264

splicing132,133–5,187,190

Spondylusshell255–6

StMary’sHospital,London19

StanfordUniversity190,211,295

Staphylococcusaureus(coccoidgerm)10

STAT2(geneticregion)269

stemcells116,194

Stokes,Alexander(Alec)75,76,88,90

StoneAge220,256,264,267,293

Stoneking,Mark213–14,218

streptomycin32

Stringer,Chris220,231–2,251,252,259–60,269–70

sulphonamidedrugs20

Sulston,John185

SundayTimes269

symbiosis:

behaviouralsymbioses149

co-evolutionand158–64,169,282

conceptof147–55

cyanobacteriaevolutionand151–2

discoveryof147–8

firstsymbioticunion153

geneticengineeringand299

geneticsymbiogenesis149,157

geneticsymbiosis150–1

genomiccreativityand145,178

holobiont/holobionticunionand148–50,151,153,155,159,162,164,205,222,266

horizontalgenetransferand150,299

humanancestryand282

metabolicsymbioses149

mitochondria,symbioticoriginof(SET)152–5,205–6

mutualism,benefitsof148–50,160

mycorrhiza148

parasitismand148,160

rhizobiabacteria150–1

serialendosymbiosistheory(SET)152–3

symbiology148

symbionts148

symbioticisland150

virus–human157–64,169,173,282

syncytin165–6,169

syncytiotrophoblast165

syncytium165

syntheticbiology303

SyntheticsGenomics303

TabunCave,Palestine235

tandemrepeats285–7

Tanzania217

Tatum,EdwardL93,99,130

taxonomy248

tetranucleotidehypothesis18,79,80

thalidomide109,118

TheCancerGenomeAtlasProject(TCGA)111

TheInstituteforGenomicResearch(TIGR)125–6

thermoluminescence229,234,236

thymine17–18,69,77,80,81,87,91,94,95,102,105–6,107,131,199,247

thyrotoxicosis19–20,31

Tibetans:‘hypoxiapathwaygene’EPAS1277–8

Time38

Todd,Doctor33

tools,stone197,198,211,220,222,227,228,230,249,251,254,255–6,263,264,279

transposons195,222

Trinkaus,Erik257,264

trophoblast165

trypsin22

Tsybankov,Alexander273

tuberculosis10–11,21,32,61,300

TyrrhenianSea289

UKBiobank295

UniversityofArizona269

UniversityofAthens226–7

UniversityofBerkeley,California47,48,49,146,213,218

UniversityofBirmingham47

UniversityofCalifornia,atIrvine222,241

UniversityofChicago40,301

UniversityofColorado272

UniversityofCopenhagen198

UniversityofFlorida215

UniversityofIllinois131

UniversityofLeeds46,52,68

UniversityofLeicester208

UniversityofReading265

UniversityofSheffield112–13,131,135

UpperPleistocene277

UppsalaUniversity,Sweden167–9

uracil17–18,94,95,131,247

Vendrely,Roger95

Venter,J.Craig:

CeleraGenomicsand125–6,127,130,303

geneticengineeringand292,302–4

humangenomesequencingand125–6,127,130,289,302

J.CraigVenterInstituteand303–4

LifeattheSpeedofLight292,302

personalgenomesequencingand289

Villa,Paolo256,272

Villarreal,LuisB.164,222,291

VindijaCave,Croatia268

Virolution(Ryan)145,167

viruses10,16

bacteriophages/phagesseebacteriophages/phages

co-evolutionof158–9,160

deathof157

definitionof156–7

discoveryofDNAand27–9,36,41,42,43,92

DNAandRNAbasedgenomes157

firstcompletegenomeofanorganismsequenced(bacteriophagevirus,ΦX174)121

geneticsymbiogenesisand157–60,162,222seealsoretrovirusesandsymbiosis,genetic

genomecodingof156–7

intronsandexonsdiscoveryand131–2

invasionofhumangenome120

lifecycles157

mutations157

Pasteurand11

retrovirusseeretrovirus

viralgeneexpressioninhumancells,tissuesandorgans,searchingfor167–70

seealsounderindividualvirusname

vitaminDdeficiency267

VNTR(variablenumberoftandemrepeats)286–7

Vries,HugoDe14–15,106,139–40

Waddington,ConradH.189

Waksman,Selman32

Wansuntroad,Kent,Neanderthalhandaxesfoundat264

WashingtonUniversity,StLouis,Missouri264

Watson,Elizabeth56

Watson,JamesDewey:

autobiography44,59–60,70,82

background40–1

CavendishLaboratoryand51,52,55–8,60,90

Chargaffand80–1

Crickand50,52,53,55–6,58,61–5,76,96

Delbruckand43–4,57–8

DNAextrapolationtoproteins,investigates96,98

Europe,firsttravelsto44–5

Franklinand70,76,77,78,82–3,84–5

Gamowand95,96

Harvardand98

Kalckarand45,55–6

Luriaand43–4,57,58,60–1,63

Maaløeand45

MerckFellowship,NationalResearchCouncil44,56,60–1

NIHHumangenomeProjectand122–3,125,126–7,289

Pauling’sdiscoveryofthe‘alpha-helix’and56–7,69–70

Pauling’striplehelixconceptand82–3,84–6

Researchfellowship,IndianaUniversity,Bloomington40–1

returnstoU.S.afterrunningoutoffunding93–4

RNATieCluband96

Schrödinger’sInspires40–1,46,50,52,125

structureofDNA,roleindiscoveryof34,40–1,43–6,50,51,52,53,55,56–8,59,60–5,68,69,76–7,78,80–2,84–90,91,92,94,95,99,102,112,122,294

Wilkinsand45–6,47,51,56,57,58,59,72,76,77,78,82–3,84–5

WillySeedsand59,60

X-raycrystallography,firstcomesacross45–6,51,56,72

ZoologicalStation,Naples,attendsWilkinslectureat45–6,56,72

Weigle,Jean56

WellcomeTrust123,127

Wells,H.G.262

Wesselingh,DrFrankP.231

WesternEurope197–8,239,269,288,301

Wilkins,Eithne48

Wilkins,Maurice45–9,50,51,56,57,58,59,68,70,72–6,77–8,82,83,84,85,88,90,125

Wilkins,Patricia48–9

Willerslev,Eske202–3

Wilson,Allan213–15,218,221

Wimmer,Eckard156,304

Winogradsky,Sergei5

WoodsHole,Massachusetts96

Wollman,Élie99–100

Wright,Sewall40,140

Würm(IceAge)234

X-raycrystallography45–6,47,50–1,54,56–7,58,60,62–3,71,73–4,82,85,88,90,91

X-raydiffraction45,46,50,51–2,54,57,60,67–8,70,72,73,74,78,89,90

X-rayspectrometer52

Xistgene190,191–2

Y-MRCA(earliestdetectablecommonmaleancestry)219–20

Ycas,Martynas96

YorkUniversity264

Zilhão,João255–6,257

ZoologicalStation,Naples45–6,56,68,72

zygote(fertilisedovum)113,115,117,172,174

acknowledgements

Manyscientificcolleagueshavecontributed tomy thoughtson thehumangenomeoverthe last two decades, andmanymoremembers ofmy audiences in the lectures I havegiven on various aspects of this fascinating theme. In particular I am indebted to thekindness and generosity of Erik Larsson and his colleagues at Uppsala, and KaterinaDoukaattheOxfordRadiocarbonAcceleratorUnitforreasonsthatwillbecomeobviousinthetext.Iwould,ofcourse,thankmypublisher,MylesArchibaldatHarperCollins,forthemanycommunicationsandconversationsthatledtothescopeandformatofthebook.It’sagreatpleasurealsotoacknowledgethehelp,andpracticalsuggestions,ofmyagent,Jonathan Pegg, aswell as the ever-supportive editor, JuliaKoppitz, atHarperCollins. Ithankyouallwithfondnessandgratitudeforyourinterestandstimulation.

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