Applied Evolution PDF

Applied Evolution
Author(s): J. J. Bull and H. A. Wichman

Source: Annual Review of Ecology and Systematics, Vol. 32 (2001), pp. 183-217
Published by: Annual Reviews
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Annu.Rev.Ecol. Syst.2001. 32:183-217

? 2001 byAnnualReviews.All rightsreserved
Copyright
APPLIED EVOLUTION
J.J.Bull1andH. A. Wichman2
'SectionofIntegrative
Biology,Institute
ofCellularandMolecularBiology,University
Texas78712-1023;e-mail:bull@bull.biosci.utexas.edu
ofTexas,Austin,
2Department
ofBiologicalSciences,University
ofIdaho,Moscow,Idaho 83844-3051;
e-mail.hwichman@uidaho.edu
Key Words artificial
selection,directedevolution,phylogenetics,
resistance,
evolutionary
computation
* Abstract Evolutionary
biologyis widelyperceivedas a disciplinewithrelevance
thatlies purelyin academia. Untilrecently,
thatperceptionwas largelytrue,except
forthe oftenneglectedrole of evolutionary
of agriculbiologyin the improvement
turalcrops and animals.In thepast two decades, however,evolutionary
biologyhas
assumeda broadrelevanceextendingfaroutsideits originalbounds.Phylogenetics,
thestudyof Darwin'stheoryof "descentwithmodification,"
is now thefoundation
of
of species in medical,pharmacological,or
disease trackingand of theidentification
conservation
underliesbioinformatics
settings.It further
approachesto theanalysisof
genomes.Darwin's "evolutionby naturalselection"is beingused in manycontexts,
fromthedesignofbiotechnology
protocolstocreatenewdrugsandindustrial
enzymes,
to theavoidanceofresistant
of new computer
pestsand microbes,to thedevelopment
foreducationofthepublicandfor
technologies.Theseexamplespresentopportunities
nontraditional
careerpathsin evolutionary
biology.They also providenew research
materialforpeople trainedin classical approaches.
OVERVIEW
in thepast
Evolutionarybiology has undergonean expansion and transformation
few decades. Despite occasional claims to the contrary,
thebig changes in evolutionarybiologyhave come fromimprovementsin understandingmechanismsthat
are fullycompatiblewithDarwinism;descentwithmodificationand naturalselectionare stilltheconceptualfoundationsof thediscipline.For example, a veritable
explosion of studiesestimatingthe relationshipsamong different
species has reof evolutionaryhistory,butthemodem versionof thetree
finedourunderstanding
of lifehas manysimilaritiesto old ones and certainlysupportsa Darwinianmodel.
The sequencing of genes and genomes has yielded insightsinto genetic mechanisms underlyingevolution,and we are even progressingtowarda genetic understandingof themajor developmentaland morphologicaltransitions-advances
thataugmentearlierideas about thesetransitions.
Theoriesbased on naturalselectionhave led to revolutionsin understanding
behavior,parasitism,and a wealthof
0066-4162/01/1215-01
83$14.00
183
184
BULL * WICHMAN
northe
neither
theindividual
thatbenefit
mechanisms
geneticandphysiological
population.
acain evolutionhas occurredoutsidethetraditional
Some of therevolution
bioloas evolutionary
biology.Thosewhostarted
ofevolutionary
demicboundaries
ofthefieldintooneof
a transformation
giststwoormoredecadesago arebeholding
withcreatisconcerned
industry
Muchofthebiotechnology
broadsocialrelevance.
enterprise
Thisgoal-oriented
functions.
ingbiologicalmoleculesthathavespecific
ofmolecules
todirecttheevolution
principles
has quicklyembracedevolutionary
expandedthehorizonandrelevance
intesttubesand,in so doing,hasprofoundly
ofmulthematerial
principles
aresuddenly
biology.Evolutionary
ofevolutionary
to new drugsand other
biochemists
timilliondollarpatents,leadingindustrial
afterlong
front,
themedicalestablishment,
commercial
molecules.On a different
microbes,
hasseen
ofdrug-resistant
is facedwithan onslaught
evolution,
ignoring
monkeyvirusesjumpintohumansand accelerateintoepidemics,andmustnow
ofpathogens.
theworldwide
dynamics
tounderstand
principles
use evolutionary
The themethatunitestheexamplesin thispaperis thatevolutionandevolusuch
Therearetwomainreasonsforwriting
biologyaresociallyrelevant.
tionary
ofevolutionary
biologyis notup todatewiththe
a paper.First,publicperception
discipline."Evolution"is stilla bad wordto manypeople,notonlybecauseitis
withsomereligiousviews,butalso becauseitis widely
perceivedas conflicting
sciencewithno social value.Acceptanceof evolutionviewedas an irrelevant
arybiologyis farmorelikelywhenthepublicrealizesthatit holdsthekeyto
G.C. Williamsin hisworkon
[a viewthatmotivated
manysocialimprovements
biologyto medicine(Nesse & Williams,1994;G.C.
ofevolutionary
applications
we needtouse
As evolutionary
biologists,
Williams,personalcommunication)].
and the
theseexamplesof relevancewhenexplainingevolutionto our students
inertiainthetrainthispaperis thathistorical
public.A secondreasonforwriting
ina lackofexposuretosociallyrelevant
hasresulted
biologists
ingofevolutionary
willhavemuchtooffer
as evolutionary
biologists
trained
Individuals
applications.
Career
buttheyneedtobe awareoftheseapplications.
in solvingtheseproblems,
outside
biologistsmayalreadybe moreplentiful
forevolutionary
opportunities
academiathaninsideit.
to someexamplesofsociallyrelevant
evolutionThispaperis an introduction
arybiology.We have chosentopicswithwhichwe are familiarand in which
biologyhas alreadybeenusedto producean outcomeorto affecta
evolutionary
manageselection(in biotechnology),
resistance
artificial
policy:phylogenetics,
to socially
of evolutionary
Otherapplications
principles
ment,and computation.
relevant
problemsincludeDarwinianmedicine(Lappe 1994,Nesse & Williams
et al. 1999),infectious
diseases(Ewald 1994,Morse1994),and
1994,Trevathan
humanimpacton evolution(Palumbi2001). An excellentoverviewof thesocial relevanceof evolutionhas been assembledand endorsedby eightscientific
societies(Futuyma1999).
ofevoluThe topicsin thisreviewfitlogicallyintotheconceptualframework
tionary
biology.The firsttwosectionsare based on Darwin'stheoryof descent
APPLIEDEVOLUTION
185
history
forbiomedical
is aboutestimating
evolutionary
withmodification:
Thefirst
modelsin
andotherapplications,
andtheseconddiscussestheroleofevolutionary
variation
seenintheburgeoning
genomedatabases.The
interpreting
themolecular
fornatural
selection.
framework
lastthreesectionsarebasedontheneo-Darwinian
theelementsofnatThe first
describesexamplesinwhichhumanshavemodified
The nextis somewhat
uralselectionto producevariousbiologicalcommodities.
to
ofnaturalselectionis recruited
thereverseof that,in whichan understanding
touses ofmodelsof
The lastsectionis a briefintroduction
trytoblockevolution.
computer
programs.
naturalselectionin designing
PHYLOGENETICS:USING THE TREE OF LIFE

has been a centraltenetof
The premisethatall life sharescommonancestry
evolutionary
biologysinceDarwin.Ifwegobackfarenoughintime,thegenealogy
of everyorganismalive todaycan be tracedto a pointthatunitesit withthe
closelyrelatedspecies
alivetoday.By definition,
genealogyofanyotherorganism
whereasthecommonancestors
ofdistantly
related
haverecentcommonancestors,
theprocessis continual.
Today'sspecies
speciesgo farbackintime.Furthermore,
arethemselves
oflineagesthatcontinuetodiverge.
comprised
is thestudyoftheseevolutionary
genealogies.DespitetheantiqPhylogenetics
hasmatured
imthefieldofphylogenetics
principle,
uityofthecommon-ancestry
and
technology
menselyinthepast10 to 15 years,owingtoadvancesincomputer
ofexplicittheoriesandmethodDNA sequencingas well as to thedevelopment
Most phylogenetic
methodsuse DNA
reconstruction.
ologiesforphylogenetic
canuse morsequences,protein
sequences,orRNA sequences,butsomemethods
differ
phologicaldata,whicharevitaltotheanalysisoffossils.Notonlydomethods
inthetypesofdatathatcanbe analyzed,methods
usingthesametypesofdatamay
in theassumptions
usedtoconvert
thedataintoevolutionary
history.
also differ
the
The outputof a phylogenetic
treethatrepresents
analysisis a branching
of thelineagesbeingstudied.The treeprovidesnotonlya
evolutionary
history
nestedhierarchy
of commonancestorsgoingback in time,butalso quantitative
on theamountofchangebetweenthedifferent
pointsinthetree.The
information
reachbackovera billionyears,
treemayincorporate
taxawhosecommonancestors
or thetreemaybe limitedto a groupofviruseswhosecommonancestorexisted
methodsto socially
ofphylogenetic
onlyweeksormonthsago. The applications
relevant
likewiseoccurat severaltimescales.
problems
DiseaseTracking:
MolecularEpidemiology
and in
in identifying
diseasereservoirs
has becomeindispensable
Phylogenetics
thestep-by-step
transmission
of someviruses.The conceptualbasis of
tracking
thisworkis as follows.We wanttoknowthesourceofa virusinfecting
personX.
A, B, C, andD.
possiblesourceshavebeenidentified:
Supposethatfourdifferent
186
BULL * WICHMAN
infected
individuals
whohadcontactwithX,
Thesefoursourcescouldbe different
theycouldbe different
geographic
locationsthatpersonX visited,or theycould
be fourspeciesofmammalslivingin thevillageofpersonX thataresometimes
withthetypeofvirusinX. Theprobleminfiguring
infected
outthesourceis that
noneofthevirusesinA, B, C, andD willnecessarily
havethesamesequenceas
thevirusinX. Phylogenetic
the
analysisgetsaroundthatproblembyestablishing
ofvirusesfromeach ofthepossiblesources(Figure1).
relatedness
evolutionary
The analysisnotonlyindicateswhichofthevirusesin A-D are mostsimilarto
thatin X (sourceD in Figure1), it also indicateshowcloselyrelatedtheviruses
areand,hence,whether
thesourcemightbe otherthanA-D.
Below
Phylogenetic
analysisisnowa standard
partofanydiseaseepidemiology.
we offer
a fewofthemanyapplications.
methods
X is compared
Figure1 Thevirusacquiredbyindividual
byphylogenetic
The
for
could
be
withviruses
from
context this
fourpossiblesources(A-D).
anyofthe
orshared
with
who
had
sex
individuals
infected HIV
following.
(a) A-D aredifferent
thevirus
with
and
we
to
find
who
transmitted
needles
aretrying
out
individual
X,
in their
A-D
with
rabies
viruses
to X. (b)
aredifferent
mammalspecies
circulating
mammal
transmitted
andX is a human
whodiedofrabies.Which
populations,
species
from
theinfection?
NewMexico,Texas,California,
(c) A-D aremicewithhantavirus
andNevada.X is a Texasresident
whorecently
traveled
acrosstheUnitedStatesand
withhantavirus.
thevirus?In thishypothetical
becameinfected
WheredidX contract
the
virus
in
most
to
the
virus
from
X is
sourceD, whichgives
example,
closelyrelated
of
information
aboutthelikelysource(andplace) transmission.
APPLIED EVOLUTION
187
ERADICATION OF WILD-TYPE POLIOVIRUS FROM THE AMERICAS Poliomyelitis

is a
paralyzing,
occasionally
fatal,diseasecausedbyanRNA virus.On recovery
from
a personcarrieslifetime
infection,
immunity,
buttherearethreeformsofthevirus,
andimmunity
to one formdoesnotconferimmunity
to theothers.Less than1%
of thoseinfected
by thevirusactuallydevelopthedisease,buttheincidenceof
diseaseperinfection
is thought
to increasewithage. It has thusbeenspeculated
thatthewidespread
outbreaks
ofpoliodiseasethatoccurred
inthefirst
partofthe
twentieth
century
werea consequenceofsocialhygiene,
becauseimprovements
in
socialhygienedelayedtheaverageage ofinfection
etal. 1995,Garrett
(Nathanson
2000).
Over38,000cases ofpoliomyelitis
werereported
intheUnitedStatesin 1954.
The first
vaccinewas approveda yearlater,andwithin2 decades,thenativepolio
viruswas thought
to havebeeneradicatedfromtheAmericas.The onlyknown
hostforpolioviruswas humanbeings,makingeradication
seema feasiblegoal.
However,isolatedcases of polio continueto occurin theAmericas.Withthe
exceptionof an outbreakin a religiouscommunity
thatavoidedvaccinesand a
recentoutbreakin theDominicanRepublic(Greensfelder
2000), theseisolated
cases failedtomaterialize
intoepidemicsbecausethepopulation
maintained
high
levelsofvaccination
(whichcontinues
today),buttheyraisedthepossibility
that
nativepolio strainsmightstillbe present.The alternative
forthese
explanation
introduced
frompartsoftheworld
sporadiccases was eithernonnative
poliovirus
whereitwas stillendemicora vaccinestrain.
The vaccineinuse after1961was a
live,attenuated
virusthatwascapablenotonlyoftransmission,
butalsoofevolving
intoa morevirulent
form.Phylogenetic
analysisshowedthatthevirusesthatgave
rise to isolatedcases and epidemicswereinvariably
eithervaccinederivedor
wildstrains
fromoutsidetheAmericas;no native,American
originating
virushas
beenfound,anditis presumedtohavebeeneradicated(Rico-Hesseet al. 1987).
An assaultto eradicatepoliovirusworldwidecontinuesto thisday,aidedby the
thatthevirusdoesnotlayhiddenin nonhuman
knowledge
reservoirs.
ORIGIN OF HIV The mostnotorious
humaninfectious
diseaseto havearisenin
the1900sis AIDS (acquiredimmunedeficiency
untilabout
Unknown
syndrome).
1981,20yearslateritwasestimated
thatmorethan30 millionpeoplewereinfected
worldwide
andmorethan16millionhadalreadydied.Thisdiseaseis causedbythe
retrovirus
HIV (humanimmunodeficiency
virus),whichexistsintwobasicforms,
HIV-1 and HIV-2.AlthoughtheHIV epidemicis now drivenby human-human
evidenceindicatesthatHIV-1 originally
came from
transmission,
phylogenetic
andHIV-2(whichis less abundant
andless oftenfatalthanHIV-1)
chimpanzees
camefroma typeofmonkey
knownas thesootymangabey
(Gao etal. 1999,Hahn
et al. 2000). Phylogenetic
evidencesupports
introductions
ofHIV-1and
multiple
HIV-2intohumansthiscentury
(Korberet al. 2000).
HIV TRANSMISSION BETWEEN PEOPLE One ofHIV's unusualproperties
is thatit
evolvesrapidly,evenfora virus.Thatproperty
one fromthe
is an unfortunate
188
BULL * WICHMAN
perspective
ofcuringan infection
orcreating
a vaccine,becausethevirusevolves
quicklyinresponsetodrugtreatment
andimmune
attack(Coffin1996,Colgrove&
Japour1999,Mosier2000).However,
itsrapidevolution
enablesfine-scale
analysis
withmolecularepidemiology
thatis notpossiblewithmostotherviruses.Starting
froma singlevirusin a person,theinfection
willblossomintoa miniature
treeof
life,thevirallineseverexpandingas theinfection
continues.
Consequently,
two
virusesfrom
onepersonusuallyhavedifferent
genomesequences,butsequencesof
virusfromonepersonwillbe moresimilartoeachotherthantheyaretosequences
ofvirusesfromotherpeople.Thisproperty
allowsdetermination
potentially
ofthe
individual
whotransmitted
thevirus.
OneoftheearlyanalysesofHIV transmission
revolvedarounda Floridadentist
withAIDS whosepatients
exhibited
anunusually
highincidenceofHIV infection
(10 patientswereeventually
discoveredto be HIV+). By itself,theclustering
of
HIV+ patients
associatedwiththisdentist
might
havebeenregarded
as evidenceof
butthisclusterwas discovered
dentist-to-patient
intheearliestdays
transmission,
ofunderstanding
ofunknown
HIV,andthepossibility
routesofinfection
hadtobe
considered.
If thedentist
was notthesourceoftheinfection,
itwas important
to
discoverthesourcetohaltfurther
transmissions.
On theotherhand,ifthedentist
was thesource,theimplications
forhealthcare practiceswereenormous.The
stakeswereveryhigheitherway.Phylogenetic
thatthepatient
analysissuggested
viruseswereclose relativesof thedentist'svirusin all buttwocases, and those
twopatients
had otherknownriskfactors(Ou et al. 1992,Hillis& Huelsenbeck
was likelythesourceofinfection
1994,Hillisetal. 1994).Thusthedentist
forthe
withno knownriskfactors.
eightpatients
case ofHIV molecularepidemiology
Perhapsthemostsensational
was a criminal case in Lafayette,
LA. A physicianwas accused of injectinghis former
mistresswithblood containing
HIV. He had been givinghervitaminB injections,and it was supposedlythefinalinjectionin August1994 thatcontained
theblood withHIV. Whenthewomanwas diagnosedwithHIV and hepatitis
C in December1994, she suspectedthephysician'sinjectionas thesource;at
the timeof a blood donationin April 1994, she had been negativeforboth
viruses.Thiscase was unusualin thatthepersoninfecting
thewoman(thephysician) was nothimselfinfected,
so it was necessarybothto locatethepatient
whoseHIV infectedthewomanand to demonstrate
thatthephysicianhad access to thispatient'sblood. Recordswerediscoveredin thephysician'soffice
indicating
thatbloodhadbeendrawnfromtwopatients
duringtheweekin quesknownto be HIV+ and theotherpositivefor
tion;one patientwas previously
hepatitis-C.
Phylogenetic
analysesshowedthatthe HIV sequencesof the exmistress
clustered
thoseofthepatient
herstory(State
within
withHIV,supporting
ofLouisianaCriminal
Dockett#96CR73313;D. Hillis,personalcommunication).
The physicianwas convictedof attempted
murder
and sentenced
second-degree
to a 50-yeartermin prisonin thisfirstuse of phylogenetics
in a U.S. criminal
court.
APPLIED EVOLUTION
189
Predicting
Evolution
evolution-wasintroduced
in
analysis-predicting
A novelstepin phylogenetic
A, therespiratory
virusthatcauses fluepidemicseveryyear.
workon influenza
in thehumanpopulation.
typesarecirculating
At anyone time,severalinfluenza
A viraltypeis definedbyhowthevirusreactswitha setof antibodies(Webster
determine
theviral
hemagglutinin
andneuraminidase,
1993).Twoviralproteins,
type.
bytheCenters
forDisease
Forsomedecades,influenza
isolateshavebeenstored
inhumanpopulations.
Control;theseisolatesincludelineagesthatarenowextinct
weredevelopedto obtainviralgenesequences,itwas possible
Whentechniques
circulating
lineages,
history
fromcurrently
notonlyto estimatetheevolutionary
typestoobtaindetailedinsight
butalsotousethestoredisolatestosequenceextinct
discovered
fromthisworkis
ofinfluenza
intoancestral
states.Anunusualproperty
hasnotprogressively
expandedovertime(Fitch
thatthelevelofsequencediversity
existed
etal. 1997,Bushetal. 1999).Atanypointoverthepast15 years,variation
thatwas nevermorethana fewyearsold.Thus,
arounda recentcommonancestor
viralextinctions
occurredas fastas new typesevolved,withonlyone ultimate
winner.Each winnerwouldcontinueto generatenewvariantsthatcompetedin
ofthis
thepopulation,
andonlyoneofthemwouldwin,andso on.Thephylogeny
andlotsofshort(dead-end)sidebranches.
processwas a treewitha longtrunk
The studythatpredicted
influenza
used thehemagglutinin
gene fora single
meansthatitmay
viraltype,HA3. The factthatonlyonelineageofHA3 survives
in a populationwill give
circulating
be possibleto predictwhichofthevariants
ata few
risetothevirusesofthefuture.
Fitchetal. (1997) observedrapidevolution
was facilitated
bytheanalysisof
residuesinthehemagglutinin
gene.Thisfinding
atcommonsiteshadotherwise
historical
samples,becausetherepeatedevolution
erasedearlierevolution
atthosesites.Thoserapidlyevolvingresiduesprovidedan
at
obviousyardstick
viruses:Ofthevirusespresent
bywhichtocomparedifferent
at thosesiteswerethecandidates
anyone time,theoneswiththemostevolution
as progenitors
of futurelineages.Bush et al. (1999) developedthispredictive
worked
data.The prediction
andapplieditretrospectively
totheexisting
statistic
be
it awaitsa trulya prioritest(whichwillcertainly
exceedingly
well,although
carriedoutinthecomingyears).
Will thismethodlead to betterpredictions
forfluvaccines?Not necessarily.
indecidingeachyearwhichfluvaccinetoproduceliesinpredicting
Thedifficulty
whichofthecirculating
viraltypeswillinfectthemostpeople.Thephylogenetic
prediction
is, instead,ofwhichviraltypewillprevailin thelongtermand,thus,
thanis necesofviralvariation
coversa longertimescaleanda smallermagnitude
theworldwide
pandemicsofinfluenza
(that
saryforvaccinedesign.Furthermore,
different
infectandkillmorepeoplethanaverage)arecausedbya fundamentally
(Webster1993).
typeofsequencechangethanwas studiedintheHA3 predictions
methodpointstowarda newlevelofutility
thephylogenetic
prediction
However,
forthiskindofevolutionary
biology.
190
BULL * WICHMAN
on theTreeofLife:
SpeciesIdentification
and OtherMethods
Ribotyping
frombacteriato humans(butnotviruses)carrygenes
The cells of all organisms
Partsoftheribosome
to assembleproteins.
thescaffolding
thatbuildribosomes,
maybe
areproteins,
andotherparts(rRNA)areRNA moleculeswhoseancestry
"RNA world,"beforeDNA was the
tracedall thewayback to thehypothetical
PortionsoftheserRNAgenesevolveslowly,so theirsequences
geneticmaterial.
aremore
relatedorganisms;
otherportions
areverysimilarevenbetweendistantly
variable.The essentialnatureof rRNAgenescombinedwiththewiderangeof
stretches
of themoleculesmakes
in ratesof evolutionacrossdifferent
variation
thetreeoflife.Thisapproachto assessingthe
themideallysuitedforestimating
of andrelationships
amongall lifeon thesamescale was pioneeredby
diversity
Woese(2000). Thistreeoflifeshowsthreemaindomains:Bacteria,Archaea,and
to a modest
ofplantsand animalsis confined
diversity
Eukarya.The impressive
branchin one ofthesethreedomains,theEukarya(Figure2).
thoucovering
Overtheyears,therRNAtreeoflifehas beenfilledin densely,
ofmanyofthetaxain thistreeoflife
sandsoftaxa.Of course,therelationships
butmanytaxahavebeenaddedwhoserelawithearliertheories,
are consistent
is thefactthatthistreeprowerepreviously
obscure.Equallyimportant
tionships
forcomparing
anylifeformto anyother(whichwas
videsa universalstandard
oftherRNAtree
forexample).The utility
notpossiblewhenusingmorphology,
of different
setsof polymerase
of lifehas been facilitated
by thedevelopment
partsof rRNAgenes
thatcan be used to amplify
chainreaction(PCR) primers
or amplify
themfromonlyspecifictaxa.It is nowroutineto
fromanyorganism
taxonandto use itsrRNAsequencesto
acquireDNA orRNA froman unknown
Thistechnique(known
itsclosestknownrelatives.
identify
it,orat leastidentify
ofthedifusefulbecauseitallowsidentification
can be extremely
as ribotyping)
eventhoughit maybe impresentin a community,
ferent
speciesof organisms
or recognizethemby anyothermethod(Pace
possibleto culturetheorganisms
1997).
Permethod
usedforspeciesidentification.
is nottheonlymolecular
Ribotyping
is thatitcanbe usedacrossthefullspectrum
ofribotyping
hapsthemainadvantage
beingtyped.But
of life(excepting
knowledgeoftheorganisms
viruses)without
whenworking
withinnarrowtaxonomicgroups
othermethodsmaybe preferred
(e.g., mammals,or at a finerlevel,whales).Thus,noncodingregionsof mitothanrRNAsequencesin distinguishing
chondrialDNA are oftenmoresensitive
sequences
closelyrelatedspeciesand subspecies,because thesemitochondrial
a species,
methods
arenotonlyusefulinidentifying
Thesesensitive
evolvefaster.
locationofthetaxonas
aboutthegeographic
theymayalso provideinformation
well(theDNA "zip codes"ofBaker1994).
Sevother
causes.Ribotyping
and
genotyping
eraldiseaseshaveno knowninfectious
USING THE TREE OF LIFE: PATHOGENIDENTIFICATION AND DISEASE ETIOLOGY
APPLIED EVOLUTION
EUKARYA
191
STAMENOPILES
RE
&
PROTISTA
spo
BACTERIA
Ores
ngPe
Dinoflagellates
discoidewn
ANINIALIA
;
Dictyostelamn
Xll
PCifPqAcomplexans
PlantChloroplasts
Mycoplasma
FUNGI
Entanoebae
istvadesti
/
Amoebamastigote
Cot
capricoiw
anobactera
/ grubei,i t
N^
Entramoebae
Naegleria histolyuica
Bodonids
Kinetoplaatids
Agrobacteriurn
tutewefaciens
Euglenoids
Physanum
polycepha\in
PlantMiltochondria
necatrix
testosteroni
Pseudomnonas
|aitintorpha
lTichotrtontasfoetuis
Escherichiacoli
Sulfalobussolfataricus
Thernoplasmaacidophillim
AMfethanococczus
vannielii
Tricizomonas
vaginiallis
Giar-dialamvblia
Halobacteria
ARCHAEA
Figure 2 A treeof life,based on 18S rRNA sequences.The treeshowsthethreedomains
in each domain.Notewhat
(Bacteria,Archaea,andEukarya)as well as manyrepresentatives
littledivergenceis represented
by theanimalsand plantsrelativeto theentiretree.The 18S
sequencesfromhundredsof lifeforms(butnotviruses)have been obtainedand can be resolved on thistree,withclosest relativessharingthe mostrecentcommonancestors(the
is simplytoocrowdedto portray
themanyspeciesthathavebeenanalyzedin this
figure
The 18S sequencesfromsamplesofunknown
canthusbe mappedonto
fashion).
identity
inthesample.(Figurecourtesy
thistreetodiscover
whattypesoflifeforms
werepresent
of
MitchSogin.)
methodsoffera wayof identifying

diseasedtissuesare associatedwith
whether
anymicrobe(Relman& Falkow1992,Relman1998). If thecausativeagentis
a previously
unknownspecies,evolutionary
biologyplaysa role in identifying
itsclosestknownrelatives.
Once a
itis, i.e.,in identifying
whattypeoforganism
suspectmicrobehas beenidentified,
stepscan be takento treatwithappropriate
havegreatly
facilitated
drugs.Hadthisapproachbeenavailableatthetime,itmight
Relman(1999) listed
thediscovery
thatulcerswereassociatedwitha bacterium.
fiveinfectious
diseaseswithcausativeagentsrecently
discoveredbygenotyping
Anextension
ofthisapproachis touseribotyping
toassessthemicrobial
methods.
oforganisms,
as in animalgutsandotherpassages.
ofa community
composition
a predisposition
to
ofa community
Changesin thecomposition
mayforeshadow
diseaseortheonsetofdisease(Kroesetal. 1999).
192
BULL * WICHMAN
Concernforendangeredspecieshas led to widespreadadoptionof laws limitingtheharvesting,

andevenpossessionoftissuesandotherproducts
ofthoseorganisms.
marketing,
Althoughsomederivatives
of a speciesareunmistakable
(e.g.,a sea turtleshell,
an elephant
tusk),othersarenotso obvious.In manycases,an endangered
species
has close relatives
thatarenotendangered
and are legitimately
marketed.
If the
harvesting
occursata remotesite,farfromtheeyesofconcerned
observers,
bythe
timethemeatofan endangered
it maybe indistinguishspeciesreachesmarket,
ablefromthemeatofa legalspecies.Usinggenotypic
methods,
Baker& Palumbi
(1996) andBakeretal. (2000)reported
thesaleofmeatfromendangered
speciesof
whalesinAsianmarkets.
Ribotyping
andsimilarmolecular
methods
allowsimple
andportablemeansofidentifying
endangered
speciesin themarketplace.
USING THE TREE OF LIFE: CONSERVATIONAND FORENSICS
GENOMICS AND BIOINFORMATICS

Fromtheperspective
ofhumanhealth,a majorgoalofgenomicsworkis tounderstandnotonlythefunction
ofhumangenes,butalso theimpactof mutations
in
thosegenes,andhowdrugscan be designedto modifyorrepairthosefunctions.
variablenoramenableto exYet we humansareneither
sufficiently
genetically
fromjustour
thatthefunction
ofmostgenescouldbe ascertained
perimentation
benefit
to understanding
humangeneticscomesfrom
species.An immeasurable
workon otherspecies-modelorganisms.
As we nowknow,workon thegenetics
of othereukaryotes
can oftenbe extrapolated
to humans.One recentexampleis
theidentification
of a humangeneresponsible
fora sleepdisorderbasedon the
Drosophilacircadianclockgeneper (Tohetal. 2001).
The extrapolation
of information
betweenspecies will acceleratenow that
completegenomesequencesare availableformanyspecies.Rapid advancesin
havetakenus fromthefirstcompletesequenceof a smallDNA
biotechnology
viralgenome(Sangeretal. 1977)tothecompletesequenceofthehumangenome
in less than25 years(Venteret al. 2001). Duringthesameperiod,thenumber
of transistors
percomputer
processorhas increased6000-fold.The marriageof
increasedcomputing
powerand largebiologicaldatasetshas spawnedthefield
ofbioinformatics,
whichis dominated
bytheanalysisofnucleicacid andprotein
data.Bioinformatics
is firmly
rootedinevolutionary
biology.Fromtheinitialstep
of carrying
outa BLAST search(Altschulet al. 1990) of a databaseto identify
relatedsequences,to multiplesequencealignment
(Higgins& Sharp1988),to
theidentification
of orthologous
genesin otherspecies(DeBry& Seldin 1996,
is a comparative
exTatusovet al. 1997,Chamberset al. 2000), bioinformatics
and mostessential
is one of its inherent
ercise,and descentwithmodification
assumptions.
is botha plus and a
Molecularevolution,specifically
sequencedivergence,
minusin bioinformatics.
in yeastwillnot
A genein humansand itscounterpart
havethesameDNA sequenceevenifthefunction
thesame
ofbothhas remained
APPLIED EVOLUTION
193
to
sincetheircommonancestor.Divergencecan be so greatthatit is difficult
ofhomolgenes.However,oncealignments
alignorevenrecognizehomologous
The
divergence
can be veryinformative.
ogousgenesareachieved,evolutionary
equivalent
ofanexis theevolutionary
divergence
ofgeneswiththesamefunction
important
toidentify
thefunctionally
inwhichgenepositions
aremutated
periment
time,but
ones-neutralor nearlyneutralchangesaccumulateoverevolutionary
ofnaturalselection.
genefunction
areweededoutbythefilter
changesthatdisrupt
for
aminoacidresiduesareimportant
The conceptthatthemosthighlyconserved
inmolecular
biology(Benner1995,Golding&
is firmly
entrenched
genefunction
forfunctionare highly
Dean 1998). But not all residuesthatare important
signatures
conserved-adaptive
evolution
is achievedbygeneticchange.Statistical
to
ofpastadaptiveevolutioncan be recognizedas highratesofnonsynonymous
substitutions
amonghomologoussequences(McDonald& Kreitman
synonymous
changesbetween
1991,Boyd & Hartl1998,Crillet al. 2000) or as correlated
different
residues(Gutellet al. 2000).
ARTIFICIALSELECTION
Artificial
Selectionin thePast
thouNearlyall thecommonanimalsandplantswe use todayweredomesticated
sandsofyearsago, some(sheep,goats,dogs,wheat,andrice)atleast9000 years
as a processoftaming,
thenofcaptivebreedprobablystarted
ago.Domestication
ofselecting
forspecific
traits.
Theseearlydomestications
maywell
ing,andfinally
inappliedevolution.
Theirimpactwas so profound
havebeenthefirst
experiments
and
possiblebyenablingsocietiesto switchfromhunting
as to makecivilization
toagriculture
1999).Although
(Ucko& Dimbleby1969,Clutton-Brock
gathering
in thepastmillennium,
we havecontifewnewspecieshavebeendomesticated
nuedto refinetheold ones. The successof artificial
selectionis evidentin its
oftheoriginal
ofselectedphenotypes
welloutsidetheextremes
ultimate
creation
SaintBemards,pitbulls,andgoldenretrievers
species.Forexample,Chihuahuas,
differ
inbothappearance
andnothing
likeanyofthesebreedswould
andbehavior,
ofdomestic
ofwilddogs.Fruitsofmodemstrains
havebeenfoundina population
plantsaremuchlargerthanthoseof theirancestors;corn(maize) is profoundly
different
fromitsancestor,
teosinte.
A modelof evolutionby naturalor artificial
selectionhas threecomponents:
success(Lewontin
and (c) differential
reproductive
(b) inheritance,
(a) variation,
ofvariation
largelyon an understanding
1970).Darwin'stheorywas formulated
ofinheranddifferential
successbuta relatively
poorunderstanding
reproductive
with
voidbegantobe filledearlyin thetwentieth
century
itance.The inheritance
therediscovery
ofMendel'swork,enhanced(in theWest)by a close association
selecto improvemethodsof artificial
of evolutionary
biologywithagriculture
traitlocus
tion.Thisproductive
continues
todaywiththeuse quantitative
marriage
(QTL) mappinginbothfields.
194
BULL * WICHMAN
Another
culturalrelationship
betweenevolutionary
theory,
geneticsand agriculture,
was notso productive,
however.In the1930s,Trofim
Lysenkorejected
Darwin'stheory
ofnaturalselectionin favorLamarck'stheory
oftheinheritance
of acquiredtraits.Lysenko'ssubsequent
risein poweras thehead of theSoviet
ofAgriculture
anddecimated
Ministry
directly
destroyed
geneticresearch
agriculturein theSovietUnion(Soyfer1994,Garrett
2000). The history
ofLysenkoism
thedangerof letting
highlights
politicalandothernonscientific
ideologydictate
scientific
practice.
artificial
of differential
Historically,
selectionwas a manipulation
reproductivesuccess.Parentsclosestto thedesiredphenotype
werechosentoproducethe
nextgeneration,
thatfellshortoftheidealwereomitted
andindividuals
fromthe
of variationand inheritance
werepresent
breedingpopulation.The components
butwereoftennotmanipulated
fromtheirnaturalstates,exceptfortheoccasional
introduction
of novelstrainsor wildrelativesintothebreedingstock.Mutation
rateswereseldom,ifever,purposely
manipulated,
butitis possiblethatsomeof
the"widecrosses"usedto introduce
variation
intothebreeding
population
could
haveincreasedmutation
ratebyinducing
transposition
orothermutagenic
mechanisms.Althoughthemechanisms
of inheritance
in artificial
selectionwerenot
fundamentally
different
frominheritance
duringnaturalselection,
themanipulationofcrossesfrequently
increasedthelevelofinbreeding
andthustheabilityto
selectfortheexpression
ofrecessivetraits.
Artificial
selectionmovedintoa new realmwithbiotechnology
(Kauffman
1993). In contrastto agriculture,
biotechnology
specializesin evolvingsmall
inbiotechnology
methods
have
things,
i.e.,moleculesandmicrobes.
Evolutionary
muchin commonwithclassical artificial
selection.The same threefactorsBut
variation,
inheritance,
anddifferential
reproductive
success-are manipulated.
itis no longertheartificial
selectionof old. The methodsusedin biotechnology,
ofmolecincluding
DNA sequencing
oftheentireevolvedgenome,determination
ularstructures,
andmonitoring
geneexpression
levels,provideunparalleled
levels
of analysisof evolution.The combination
of experiments,
replication,
productoriented
and analysisof resultshas allowedrapidattainment
of a new
research,
levelofscientific
in evolutionary
accomplishment
biology.
DirectedEvolution
used to fashionnucleicacids
Variousprotocolsbased on evolutionarecurrently
The directed
evolu(ribozymes
andaptamers)
orproteins
withspecificfunctions.
ofwholegenomes
tionofnovelbiologicalpathways
andevolutionary
engineering
weremade possibleby changing
may notbe faroff.These accomplishments
of variation,
and differential
success.
thecomponents
inheritance,
reproductive
Naturecomes close to some of thesemethodologies,
and indeed,severalare
fromnature,
borrowed
usingmicrobesandvarioustypesofparasticgeneticelements.Biotechnology
createdunnaturalmeans of evolving
has, nonetheless,
molecules.
APPLIEDEVOLUTION
195
Perhapsthemostextremeformof directedevolutionis thein vitroreplicaThe earliestsystemof thissortwas

tionof nucleicacids outsideof life-forms.
of theQ/3genomewiththeQ/3RNA-dependent
RNA
Spiegelman'sreplication
polymerase
(Spiegelmanetal. 1968). Q/3is a phagewhosegenomeencodesfour
genes,one beingthepolymerase
enzymethatmakesRNA copiesofthephage's
enzymeandthenplacingthe
RNA genome.By isolatingthephage'sreplication
RNA genomein a cocktailofnucleotides
plustheenzyme,thegenomewas replinoneofitsgeneswerebeingexpressed-the
catedonitsown.In thatenvironment
genomewas just a moleculethatwas evolvingto copyitselfin an environment
Itrapidlyevolvedto a smallsize.
whereitsgeneswereirrelevant.
any
tobe directed
toward
TheSpiegelman
invitrosystem
didnotallowevolution
Perhapsthefirst
purelyinvitrosystem
thatdid
goalotherthanfastself-replication.
allowdirected
evolution
wastheSystematic
EvolutionofLigandsbyEXponential
enrichment
developedbytwodifferent
(SELEX) systemthatwas simultaneously
labs(Ellington
& Szostak1990,Tuerk& Gold 1990).Thiselegantsystem
allowed
oneto startwitha synthesized
pool ofnucleicacids(Figure3). The endsofthose
thesequencesofwhichmatchedsequences
nucleicacidswere"constant"
regions,
Thispool
ofprimers
Themiddleregionswererandomized.
forPCR amplification.
molecule
ofmoleculeswas thenwashedacrossmanycopiesofa particular
target
(e.g.,a protein).The moleculesthatboundthetargetstayedbehind,andtherest
werewashedoff.The boundmoleculeswerethenelutedintoa separatetube,
thecycleagain.In thisway,a nucleicacidamplified
byPCR, andpassedthrough
molecule
target
bindingspecies("aptamer")couldbe obtainedforanyparticular
(Tuerk& Gold 1990,Gold et al. 1995,Osborne& Ellington1997,Famulok&
Mayer1999).
variationson thisthemehas sincebeen developed.
A varietyof interesting
schemeto
Beaudry& Joyce(1992) used an in vitroselectionand amplification
of a ribozyme,
an RNA moleculewithenzymatic
activity.
modifythefunction
The starting
produced
ribozymecleavedRNA molecules,butdirectedevolution
a ribozymethatcleavedDNA molecules.Breaker& Joyce(1994) thenevolved
DNA moleculesthatcouldcleaveRNA.Thesemethods
weresimilartotheSELEX
methodin usingPCR to amplifymoleculesthatsurvivedtheselection,butthe
methodsdiffered
fromSELEX in theselectionitself.Table 1 liststheimpressive
varietyof unnatural
ribozymes(notknownfromnature)thathavebeenevolved
nucleicacids,directed
thesefewexampleshighlight
bythesemethods.
Although
evolutionalso manipulates
proteinsand entiremicrobes.In manyways,these
a special
fromnaturalevolutionas to warrant
newtechnologies
are so different
or technovolution?).
term(e.g., techno-evolution
However,theterm" directed
the
whichis nowin wideuse,does havetheadvantageof conveying
evolution,"
of
arebasedonthefoundations
applications
messagethatthesesociallybeneficial
standard
evolutionary
biology.
of the
DIFFERENTIAL REPRODUCTIVE SUCCESS: REPRODUCTION Reproduction
progress.In thepast,one had
thingsbeingselectedis essentialto evolutionary
196
BULL * WICHMAN
~~~~~5
V
selecinvitro,
schemeofevolution
Figure3 SELEX,a purely
bynatural
nonliving
is
of
at
the
tion.(1) A heterogeneous
(thesquiggles
top) passed
pool oligonucleotides
ofanchored
molecules
a columnm
(darkovals).Alloligoshaveidentical
target
through
sequenceson theirendsbutvaryin thesequencesoftheirmiddleregion.(2) Oligo
whosesequencesfacilitate
whereas
variants
remainbehindin thecolunmn,
binding
variants
thatdo notbindpass through
andarewashedaway.(3) Bindingoligosare
orpH ofthesolution.
thesaltconcentration
eluted,bychanging
(4) Theoligosfrom
3
are
The
is
the
PCR.
(5)
poolof
step
amplified
by
cycle repeated
bypassing amplified
are
limited
the
a
few
the
over
After
colunmn.
remaining
oligos
cycles, oligosequences
tothosecapableofbinding
molecules.
thetarget
create
butnowwe can literally
to workwithan organism
capableofreproducing,
in therightcocktailofenthemselves
andthenevolvemoleculesthatreproduce
nucleicacid
Themostcommonmethodofreproducing
zymesandothernutrients.
molecules is PCR. A similar but less popular method is self-sustained sequence
are
andtheirprogeny
(3SR). In PCR, DNA moleculesaretheparents,
replication
complementaryDNA molecules. In 3SR, RNA molecules are theparents,cDNA
of theseDNA moleculescreates
moleculesare theprogeny,
and transcription
cyclesofreproduction,
RNA copiesas thegrandchildren.
PCR uses synchronized
inwhichall moleculesinthetubereproduce
onceandthenstopuntilthenextcycle
APPLIED EVOLUTION
197
TABLE 1 Unnatural
ribozymes
evolvedbydirected
evolutiona
Ribosyl2'-0-mediatedcleavage
Mg2+-dependentcleavage
Pb2+-dependentcleavage
Ribosyl2',3'-cyclicP hydrolysis
Pb2+ dependent
RNA ligation
3',5' Ligation(class 1)
2',5' Ligation
5',5' Ligation
RNA phosphorylation
Class 1 (ATP-S)
Class 1 (ATP)
Self-aminoacylation
Acyltransfer
reaction
Acyltransfer
transfer
Aminoacyl
Self-nitrogen
alkylation
Sufluralkylation
Biphenylisomerization
Prophyrin
metalation
aRNA moleculeswiththeseactivitiesare
unknown
fromnature.(FromJaeger1997.)
is initiated.
3SR is a methodinwhicheverything
happenscontinuously
(Fahyetal.
1991).
Formanypurposes,
thedirected
evolution
ofproteins
andmanyothertypesof
moleculesstillrequiresanorganism.
A geneis expressed
inanorganism
(typically
a bacteriumor yeast),butthe gene maybe removedfromthatorganismand
beforebeingreturned
tothesameora different
subjectedtovariousmanipulations
but
thusallowsa genetohitchhike
as partofanorganism
organism.
Biotechnology
thenbe divorcedfromthatorganism
at will.However,twonewmethodsallowin
vitrotranslation
tocouplea protein
tobe evolved
to itsmRNA,enablingproteins
in a SELEX-like fashion(Wilsonet al. 2001). Limitedformsof peptideselfhavealsobeenproduced,
ofthepeptidecopying
thechemistry
replication
although
mechanisms
is fundamentally
fromthatofnucleicacids(Lee etal. 1997).
different
DIFFERENTIAL
REPRODUCTIVE
SUCCESS:
DIFFERENTIAL
SUCCESS
A challenge in
out directedevolutionis to come up witha powerfulmethodto macarrying

successof themoleculesunderselection.
nipulatethedifferential
reproductive
The goal is to choosegeneswhosephenotypes
as
offerthegreatest
improvement
198
BULL * WICHMAN
forthenextroundofselection.Thiscanbe donedirectly
parents
through
selection
or indirectly
through
screening.
Trueselectionoccurswhenmoleculeswiththe
desiredphenotypes
reproduce
thanmoleculeswithlessdesiredphenotypes;
faster
in theextreme
case, moleculeswiththedesiredphenotypes
aretheonlyonesto
survive.In screening,
thegood andbad survive;thephenotype
ofeach molecule
is individually
assessed-forexample,through
a colorimetric
enzymeassay-and
genesforthemostdesiredmoleculesarechosenas parents
forthenextgeneration.
Thedistinction
betweenselectionandscreening
indirected
evolution
thusappears
to be functionally
thesame as betweennaturalselectionand artificial
selection
(D. Futuyma,
personalcommunication).
Trueselectionis generallymuchmore
powerful
thanscreening
becausethenumber
ofvariant
moleculesthatcanbe subjectedto selectionis generally
muchlargerthanforscreening
(Arnold& Volkov
1999).However,schemesfortrueselectioncan be difficult
to design.
The formof selectionusedin muchofbiotechnology
is whatquantitative
gereferto as truncation
neticists
selection-reproducing
onlythosemoleculesthat
meeta certainstandard,
in thesamewaythata livestockbreedermatesonlyanimalsthatachievea certainweightorfatcontent.
In biotechnology,
theselection
be determined
might
celltogrowona toxicorantibiotic
bytheabilityofa bacterial
In othercases,as in aptamer
substrate.
selection,
reproductive
successis basedon
adherenceto a targetmolecule.In someprotocolsforDNA enzymeevolution,
inthereverse
reproductive
successis determined
fashion-escapingfroma bound
stateis thegatewayto future
reproduction.
Oneoftheadvantages
inbiotechnology
ofevolution
is thattruncation
selection
can achieveastronomical
levels.The limiting
factorsin anytruncation
selection
are(a) therangeofvariation,
whichis limited
ofdifferent
bythenumber
genotypes
thatexist,and(b) thefecundity
ofthoseorganisms
betweenboutsofselection.If
an asexualorganism
producesonlytwooffspring
betweenboutsof selection,
for
example,thena 50% cullingis thelong-term
upperlimit,whereasifthefecundity
is 1000,thena 99.9% cullingis possible.The latterallowsmuchmorerapidevolution,
geneticvariation
permitting.
Methodssuchas cloninggenesintoselectable
bacterialplasmidsandPCR allowamplifications
ofas highas 109
(reproduction)
betweenboutsofselection.Suchlevelscouldbe achievedonlyin specialcases of
naturalselection(e.g.,withmicrobesevolvingin a newenvironment
orinvading
a newhostspecies).
Perhaps
thegreatest
deviations
fromnaturalevolutionary
processesusedinbiotechnology
havecomefrommanipulating
Theseadvancesincludeelevating
geneticvariation.
andrecombining
molecules.
geneticvariation
VARIATIONAND INHERITANCE:ELEVATINGLEVELS OF GENETIC VARIATION
Therateofevolution
inmanyexperiments
is limitedbygeneticvariselectionthatmaybe applied
ation,largelybecausethehighlevelsoftruncation
cantakeadvantage
oflevelsofvariation
thatlie ordersofmagnitude
beyondanyofapproaches
To raisemutation
havebeendeveloped.
thingnatural.
rates,a variety
MUTATION
APPLIEDEVOLUTION
199
The mostextreme
is simplyto incorporate
highlevelsofvariation
in synthesized
molecules.Poolsofoligonucleotides
can be synthesized
witharbitrary
base compositionsat specificsites.These moleculescan be used as thedirecttargetof
selection(as in aptamer
evolution
studies)or as materialclonedintovectorsthat
thenbecomestranslated
intoprotein.At theextreme,
thesequenceof theinitial
selectivepopulation
is completely
randomized
andall bases haveequal frequencies.Current
technology
limitsthepool sizetoabout1016,so an exhaustive
search
of"deeprandom"is limitedto a sequenceof27 bases (Landweber1999).When
largerstretches
ofrandomized
sequencesareused,thislimitation
meansthatindependent
experiments
usingthesameprotocolmayarriveat different
solutions
becauseonlya smallfraction
ofthepossiblesequencesarepresentin theinitial
inthesecases is dominated
pool ofmolecules.Evolution
bythe"tyranny
ofsmall
motifs"
pattern
(Ellington
1994),suchthatanysolutions
totheselectivechallenge
thatare specifiedby a smallnumberof residueswill certainly
be presentin the
pool of randomized
molecules,whereassolutionsspecifiedby a largenumber
of residuesmaynot.So theevolvedsolutionstendto be thesimplerones,not
thebestones.
necessarily
is nottheonlymeansofelevating
Oligo synthesis
mutation
rates.Ifmolecules
arenotsynthesized,
thentheyarecopiedfrompreexisting
Naturalentemplates.
zymesandprocessesareinvariably
andtheirinherent
usedforthisreplication,
error
ratesareusuallymuchlowerthanwanted.Othermethodsofgenerating
variation
orintroducing
relyoninflating
theerror
rateduring
errors
intothetemreplication
plateitself.The standard
methodofelevating
mutation
rateshas
whole-organism
beentoexposetheorganism
toa chemicalorphysicalmutagen.
Thatmethoddoes
notallowprecisecontroloverthegenomiclocationsofthemutations,
it
although
allowssomecontroloverthetypesof geneticchanges,becausedifferent
agents
tendto causedifferent
Morerecentmethodsinvolveinflating
typesofmutations.
theerrorratesduringreplication:
(a) PCR withunnatural
bases,withasymmetric
base compositions,
withmanganese;(b) amplification
and/or
thatinvolvesalternatelycopyingbetweenDNA andRNA becausethetranscription
step(DNA into
et al. 1995).
RNA) is highlyerrorprone(e.g.,Fromant
RECOMBINATION One of themostpowerful
used in directedevolutechniques
tionis recombination
amongvariantsof thesameor similarmolecules.Several
methodsforperforming
in vitrorecombination
are now routine(knownin the
fieldas geneshuffling
or molecularbreeding).PimStemmer
of Maxygendevelmethod(Stemmer1994),whichinvolves
oped thefirstpurelyin vitroshuffling
poolingDNA templates,
nickingthemto cut strands,
denaturing
them,and letbeforepatchingthemup. Moleculesrequireonly
tingthemcomebacktogether
shortregionsofidentity
torealign,so recombination
(strandexchange)can occur
betweenmoleculesthatdiffersubstantially.
The variantsto be shuffled
can be
thosealreadyimprovedduringearlierroundsof selection,or theycan be natuof species-familyshuffling
rallyoccurring
homologousgenesfroma diversity
etal. 1998).Familyshuffling
hasthesameadvantage
as increasing
(Crameri
genetic
200
BULL * WICHMAN
variation
byintroducing
novelstrains
orwildrelatives
intothegeneticstock:Naturalselectionhasalreadyweededoutthedeleterious
mutations
fromthesevariants.
In at leastsomecases familyshuffling
radicallyacceleratestheprocessofdirectedevolution.
Forexample,whencephalosporinase
genesfromfourmicrobial
specieswereindependently
evolvedthrough
one roundof DNA shuffling,
they
showedup to an eightfold
improvement
in theirresistance
to theantibiotic
moxalactam.A singlecycleof familyshuffling
thatcombinedall fourgenesyielded
a 540-foldimprovement
in resistance.
The evolvedgeneshowingthegreatest
increaseinresistance
was madeofeightfragments
fromthreeofthefourgenesand
hadan additional33 aminoacid substitutions
(Crameriet al. 1998).
Reproductivesuccessis an ultimatenecessityin anyformof evolutionby naturalor
artificial
selection.Yet in a narrowsense,reproduction
mayhavenothing
to do
withthephenotype
soughtby artificial
selection.The reproductive
requirement
oftenlimitstheprogress
thatcan be achievedin artificial
selection.Forexample,
mostpeopleprefer
toeat seedlesswatermelons,
butseedsareneededforthenext
generation.
are createdas triploidhybridsfromcrosses
(Seedless watermelons
betweendifferent
One ofthebigsuccessesinbiotechnology
hasbeenthe
strains.)
reduction
of suchconstraints,
so thatmoleculeswithdesiredphenotypes
can be
propagated
without
concernfortheircorrelated
negativeeffects
on reproduction.
Severaltricks
facilitate
thedivorcebetweenreproductive
constraints
andselection
on thedesiredphenotype.
VARIATIONAND INHERITANCE: VARIANTSWITH REDUCED CONSTRAINTS
Whena nucleicacid (RNA or DNA) moleculeitselfis

selectedforthephenotype,
as in aptameror ribozymeevolution,
amplification
by PCR is notonlytheeasiestmethodof reproducing
themolecule,it also entailsminimalconstraints.
Theendsofthemoleculemustmatchprimer
sequences,
butintervening
sequencesarelargelyirrelevant.
Earliermethodsofamplification
theDNA moleculein a plasmid,and a plasmid'srepro(cloning)incorporated
ductionis tiedto thatof itshost.Not onlywas cloninga cumbersome
method
ofamplification,
iftheclonedsequenceswereincompatible
withcell growth,
the
wouldnotwork.PCR is a nonselective
amplification
such
amplification
method,
thattheentirepool ofnucleicacids(withsuitableends)experiences
minimalevolutionduringtheamplification
(Bull & Pease 1995).ThuswithPCR, differential
successcanbe virtually
reproductive
removed
from
theamplification/reproduction
stage.
REPRODUCTION BY PCR
LIMITING THE DELETERIOUS CONSEQUENCES OF PHENOTYPE EXPRESSION Inmany

cases, thedesiredphenotype
requiresproteinexpression.The standardway to
createproteins
is toputthegeneinsidecells andletthecellularmachinery
build
theprotein.This approachcan be a problemiftheproteinis incompatible
with
cellularfunction.
A compatibility
canbe overcome
problem
byplacingthegenein
APPLIED EVOLUTION
201
vectorsthatoffer
control
overgeneexpression.
Geneexpression
canbe suppressed
untilthehosthas reproduced,
so thatevenifthecell is killedbyexpression,
the
DNA vectorfromselectedcoloniescan be recovered
andputintonewcells.The
new methodsforin vitrotranslation
and couplingof thepeptideto its mRNA
avoid theproblementirely,
however(Wilsonet al. 2001). Anothermechanism
forlimiting
deleterious
consequencesappliesat theproteinlevelinsteadof the
genomelevel.Whenevolvingpeptidesequences,it is oftendesirableto couple
theevolvablepeptidewitha reporter
orperhapsas partof
peptide(forscreening
of a reporter
theselection).Activity
withsequence
peptidewillbe incompatible
insertions
at manypositions,butthereare oftenregionsthatcan toleratesmall
or evenlargepeptideadditionswithout
destroying
activity,
suchas proteinends
orloops.Thusa fusionprotein
is createdthathas boththereporter
activity
anda
variableregionthatcanbe selectedfora different
function.
Thisrationale
underlies
thehighlysuccessfulmethodsoftheyeasttwo-hybrid
assay(Chienet al. 1991),
phagedisplay(Smith1985),andsomeothersystems.
The synthesis
ofunnaturalmolecules,whichis nowroutine
has
affected
artificial
selectionin
chemistry,
twoways:New typesof moleculescan be evolved,and old typesof molecules
can be evolvedtowardnew targets.Replicationof new kindsof nucleicacids
is possiblewithmodified
knownas base analogs.The ribonubases,sometimes
cleotidesinnaturalRNA containthebasesadenine,uracil,guanine,andcytosine.
Severaltypesofmodified
baseshavebeenincorporated
intoribonucleotides,
and
a fewtypeshave been incorporated
intodeoxyribonucleotides.
These modified
bases have novelgroupsattachedthatdo notinterfere
withhydrogen
bonding
butthatcan alterthecharacteristics
of thenucleicacid in otherways.Provided
will acceptthebase analog,it is straightforward
thepolymerase
to createa mix
of bases thatwillresultin a nucleicacid withone or moreof thenaturalbases
replacedby theanalog.These nucleicacids maythenbe subjectedto theusual
inthehopethattheproduct
willbe superior
tothenatural
nucleicacids
selections,
(Tarasowetal. 1997,Wiegandetal. 1997,Sakthivel
& Barbas1998,Battersby
etal.
1999).
Novelmoleculesmayalso serveas selectiveagents.Thisphenomenon
hasbeen
visitedmanytimesinhumanhistory,
as pesticideswereappliedtocontrol
pestsor
were
used
treat
and
the
offended
synthetic
drugs
to
microbes,
organisms
responded
withtheirownevolution
uses of
(see sectionbelow).One ofthemoreinteresting
novelmoleculesas a selectiveagentis based on thesymmetry
of mirror-image
of aminoacids and D-forms
of nucleicacids,
molecules.Life uses theL-forms
The novel
of
can
be
butthemirror
forms
both
image
synthesized
chemically.
a
of
D-nucleic
acidsas
method
usesa synthesized
as
a
D-peptide target
against pool
whereas
D-nucleic
acids
the
is
the
unnatural
the
potential
aptamers; D-peptide
form,
directed
When
areofthenatural
formso thattheycanbe evolvedthrough
evolution.
a successful
is evolved,itsL-form
is synthesized
D-aptamer
(whichis unnatural).
VARIATIONAND INHERITANCE:UNNATURALMOLECULES
202
BULL * WICHMAN
thesynthetic
L-aptamer
recognizesthe
Because of themirror-image
symmetry,
techniquewas used to createan
L-peptide
thatis foundin nature.Thistwo-step
was
L-formDNA aptamerthatinhibitsvasopressinand,unliketheD-aptamer,
resistant
tonucleasedegradation
(Klussmannetal. 1996,Williamset al. 1997).
ExamplesofDirectedEvolution
Santoro& Joyce
RNA
(1997) evolveda shortDNA moleculethatcleaveda specificsitein a target
betweenthisDNA enzymeanditstarget,
molecule.Froma sequencecomparison
theDNA enzymeappearedto containa catalyticcoreflankedon bothsidesby
RNA.Thisdiscovery
regionsthatformed
Watson-Crick
base pairswiththetarget
againstalmostanyRNA
suggestedthata DNA enzymecould be synthesized
theRNA sequenceat
merelyby changingitsflanking
sequencesto complement
thedesiredtarget.
In oneapplication,
theDNA enzymewas designedtocleavethe
mRNAoftheEgr-1 gene,whoseexpression
causesunwanted
proliferation
ofthe
Arterial
(ballooninflation).
arterialwall in responseto damagefromangioplasty
is toexpandthe
is counterproductive
proliferation
becausethegoalofangioplasty
arterial
narrows
it.Testsina ratmodelshowedthatthe
canal,andtheproliferation
(Santiago
DNA enzymehad thedesiredeffectof reducingarterialproliferation
etal. 1999).
A DNA ENZYME TO LIMIT ARTERIALDAMAGEFROM ANGIOPLASTY
ENZYMES FOR HOUSEHOLD AND AGRICULTURALUSE The year2000 markedthe

5thAnnualWorldCongresson EnzymeTechnologies,
organizedbytheIBC (International
BusinessCommunications).
The sponsorswerethebiotechcompanies
This meetingwas dominated
by
Maxygen,Genencor,
Diversa,and Thermogen.
in specificsettalkson usingdirected
evolution
toimproveenzymeperformance
wastein the
cellulaseactivity.
Agricultural
tings.One industrial
goal is improved
offers
a potential
windfall
ofethanol
formofcornstalksandotherplantmaterial
if cellulosecan be digestedwithcheap enzymes.Anothergoal is
production,
theimprovement
of enzymes(proteases,lipases)to use in laundrydetergents
to
removestainsand otherdirt.Cellulases,proteases,and lipaseshave been isolevelsaretoolowunderapplied
latedfromnumerous
organisms,
buttheiractivity
conditions
tojustifytheiruse. Forexample,naturally
occurring
proteasesdo not
in warm,soapywater.Directedevolutionis beingused to improvethat
function
to thislargearea of research,see Marrset al.
performance
[foran introduction
& Arnold(1999),Voigtet al. (2000)].
(1999),Schmidt-Dannert
of
EVOLVING PEPTIDES THAT LIMIT CRYSTALSIZE Phagedisplayis theinsertion
ofa bacteriophage
(bacterial
virus)so thatthe
peptidesequencesintocoatproteins
inthephage'senvironment.
The size and
peptideinsertis abletocontactsurfaces
With
locationofthepeptideinsertis chosenso thatitpermits
phagereproduction.
insert
randomized
a phagedisplaylibrary
maycontainbillionsofdifferent
inserts,
Usinga
sequencesthatcan be selectedforbindingto manytypesof substrates.
APPLIEDEVOLUTION
203
peptideepitopes
commercial
phagedisplaylibrary,
Whaleyetal. (2000) recovered
different
forms
thatboundgalliumarsenate
crystals;
someepitopesdiscriminated
of thecrystal.
The peptideinsertwas a mere12 aminoacidslong,yetit seemed
couldbe achievedwithevenfewerthan12 aminoacids.
thatcrystalrecognition
themanufacture
of
This discoverymaybe an important
stepin miniaturizing
semiconductors
(nanotechnology)
because by bindingthecrystallattice,these
a simplewayofreproducibly
controlling
crystalgrowth.
peptidesmayoffer
BLOCKING HIV EXPRESSION Manydrugshavebeendevelopedto suppressHIV,
demandfornew
butevolutionof HIV resistance
to thesedrugsfuelsa continual
involving
RNA
drugs.Some recentapproachesto inhibitHIV use technologies
RNA(RNAsthatarecomplemenmolecules.Thesenewmethods
includeantisense
(RNA enzymesthatcleave
taryto thesingle-stranded
viralmRNAs),ribozymes
theviralmRNAs),andaptamers
The antisense
RNAsand
thatbindHIV proteins.
of HIV genomeseribozymescan be developedmerelyfroman understanding
needtobe evolvedina SELEX-likemanner.
When
quences,whereastheaptamers
different
agentscreatedbythesethreemethodswerecomparedfortheirefficacy
was obtainedonlywithaptamers
successful
inhibition
againstHIV incellculture,
(Good et al. 1997). Assumingthatan anti-HIVaptamercouldbecomean effeccould
tivedruginvivo,theencouraging
aspectofthisresultis thatnewaptamers
totheold aptamers.
possiblybe evolvedeachtimethevirusevolvedresistance
OtherApproaches
Evolution
isjustoneofseveraltechnology-driven
methods
formodifying
molecules
dividedintoaparegenerally
forspecificuses.Methodsforprotein
improvement
design(Arnold1997,Arnold&
proachesknownas rationaldesignandirrational
of
engineering
bymodification
Volkov1999).Rationaldesignconsistsofprotein
andmechspecificaminoacid residuesbasedon knowledgeofproteinstructure
we are currently
anisticdetails.Although
thisapproachis potentially
powerful,
farfromhavingenoughinformation
aboutmostproteins
to applyrationaldesign,
andfunction
ofproteinstructure
and in generalwe lack thedeepunderstanding
thatwouldallowus to predicttheoutcomeof specificaminoacid substitutions
(Tobinet al. 2000). The "irrational"
approachesof directedevolutiondo notrebecausetheyrelyon selectiontoreachthedesired
quiresuchdetailedknowledge
outcome.
notonlyoffera newrelevanceforevoluin biotechnology
Theseapplications
forbiologistswithclassical
tionary
biology,theyalso providenewopportunities
witha
in evolution.
The industrial
training
approachof large-scaleexperiments
focuson productsand detailedmolecularanalysisis oftendonein ignoranceof
lies aheadforthe
classicalframework.
A trulygoldenopportunity
theunderlying
withindustrial
interests.
Whatlevelofrecombination
ofclassicaltheory
marriage
andmutagenesis
is optimal?Howruggedis thefitness
landscape?Itshouldbe posreachitsgoals
andhelpindustry
sibleto builda newlevelofevolutionary
theory
204
BULL * WICHMAN
of
inthisnewareaofdirected
evolution,
allowingforappropriate
accommodation
academia.
between
industry
and
conflicting
goals
thesometimes
RESISTANCEMANAGEMENT
indeveloping
chemistry
to
(including
humans)havebeencreative
Manyorganisms
Forhumans,
thepestsmaybe cancercells,incompetitors.
deterorkillunwanted
all our
As is knownall toowell,virtually
sects,mites,worms,weeds,ormicrobes.
tothoseagents
pestswithchemicalagentshaveledtoresistance
attempts
tocontrol
is so routinethatit seemsinevitable,
(Garrett
1994).The evolutionofresistance
in
examples,suchas theAmericanchestnut,
a fewnoteworthy
notwithstanding
failedtoevolvereapparently
whichan entirespecieswithbillionsofindividuals
sistancetoaninvading
pathogen
(Newhouse1990).Thequestionis howanunderorevenprevent
ofresistance.
canhelpus retard
theevolution
ofevolution
standing
The evolutionof resistanceis nota newproblem.It accompaniedtheintroto thisproblemfrom
andpesticides50 yearsago. Attention
ductionofantibiotics
biologistshas lagged,however.Partof thereasonforthisslowreevolutionary
limitless
supplyofnewchemicals.Whenthe
sponsemayhavebeentheseemingly
windfall
ofdifferent
a virtual
drugswas
first
wereisolatedfromnature,
antibiotics
whennewdrugsbecame
was inconsequential
ofresistance
found.The evolution
availablefasterthanold onesfailed.Likewise,newpesticidesmayhaveseemed
in thedaysbeforegovernment
Now,however,
many
regulation.
easyto engineer
oftheold technologies
havereachedlimits,andthecostofobtaining
government
approvalis enormous(e.g.,on theorderofhalfa billiondollarsfora newdrug).
Therearethusampleincentives
to use chemicalswiselyandprolongthelivesof
whatworksnow.
One role of evolutionary
biologistsis to educatethepublic.A widespread
is thatan individual
whoabusesthemwill
antibiotics
concerning
misconception
That
so thedrugswillno longerworkforthempersonally.
developa tolerance,
assumethattheperson'sbodychangesinresponseto
is, somepeoplemistakenly
encouragesevoantibiotic
misuse.The trueproblemis thatmisuseofantibiotics
lutioninthebacterium
so thatitis no longeraffected
bythedrug.Ifthebacterium
no matitareatriskofan untreatable
infection,
spreads,thenpeoplecontracting
Resistance
terhowconscientious
theyhavebeenin theirpastuse of antibiotics.
quicklybecomesa globalproblemcausedbyevolution.
is affected
The evolutionof resistance
by themannerin whichwe applythe
toxins.These factorsare underour controland allow us at leastto impedethe
ofresistance.
evolution
The RightDose
theevolutionof reNo doubtthemostwidelyacknowledged
factorinfluencing
to
at least,are awareof theadmonition
sistanceis thedose. ManyAmericans,
APPLIEDEVOLUTION
205
takethefullcourseofprescribed
antibiotics,
lesttheinfection
in a more
return
resistant,
less easilytreated
form(thisis especiallya problemwithtuberculosis
infections,
inwhicheradication
ofthebacterium
froma personrequiresmonths
of
treatment).
Evolutionary
sinceDarwinhavebeenawarethatweakselecbiologists
tioncanultimately
yieldphenotypes
thatlie welloutsidetherangeofphenotypes
inthepopulation,
currently
whereasimmediate
selectionforthoseextremes
would
failandthereby
extinguish
thepopulation.
A low dose ofa drug-low enoughto
allowsurvival
ofsomesensitive
individuals-isa formofweakselection.
Itsmain
detriment
is thatitfavorsindividuals
withpartialresistance;
as partialresistance
is moreeasilyattained.
evolves,fullresistance
A secondcomplication
witha low
dose is thatitleavessensitive
survivors
thatmaymutateto resistance
beforefurtherdosesareapplied.The evolution
ofbacterialresistance
is nowa factorwhen
recommending
levelsofsomeantibiotics
(Blondeauetal. 2001),andvariations
in
dose,bothtemporally
andwithin
thepatient,
areusedtohelpunderstand
theevolutionofresistance
(Baquero& Negri1997,Baqueroet al. 1997,Blondeauet al.
2001). "Dose" is likewisea consideration
whenplanting
insect-toxic,
transgenic
plantsto avoidinsectresistance
(see below).
SelectiveApplication
aremorelikelythansmallonestocontainresistant
Largerpopulations
genotypes,
so limiting
thesize of thepopulationtreatedreducesthechancethatresistance
will evolve.A simpleway to limitthetreatedpopulationyetstillbe effective
is to treatonlythosepestscausingdamage.Withantimicrobial
agents,selective
wouldconsistoftreating
application
an infection;
onlythosepatients
manifesting
withpesticides,selectiveapplication
wouldconsistof spraying
onlythosecrops
experiencing
economicinjurylevelsof pests.Althoughthismodelis simplein
forsocialandtechnical
reasonsitis oftendifficult
principle,
toinstitute.
Withantiofbacterialinfections,
biotictreatment
a strict
adherence
to selectiveapplication
wouldmeanthatpatientsare notgivena druguntiltheirinfecting
microbehas
beendiagnosedas a strainsensitive
to thedrug.Thispracticeis neither
patientfriendly
norsafeinallcases,as aninfection
canworsenduring
thetimerequired
for
diagnosis.Evenwhenitis clearthatantibiotic
treatment
is unwarranted,
a patient's
demandsfora drugmayoverride
concernfortheeventual
anyphysician
evolution
of resistance.
The problemis a classicexampleof a phenomenon
describedby
in thatthecost/benefit
Hardin(1968), "TragedyoftheCommons,"
ratiofromthe
individual'sperspective
favorsantibiotic
overuse,in oppositionto thecommon
concernsfurther
contribute
to antibiotic
overuse
good (Palumbi2001). Industrial
in our environment
because antibioticfood supplements
yieldfasterlivestock
The agricultural
of greatermeatproduction
has so farbeenthe
growth.
interest
inlivestock
victoroverproposalstorestrict
antibiotics
food,despiteclearevidence
thatantibiotic
foodsupplements
resistance
encouragetheevolutionof antibiotic
inhumanpathogens
(Garrett
1994).
206
BULL * WICHMAN
Does limited
use (selectiveapplication)
reduceresistance
levels?Itis ofcourse
clearthatantibiotic
to thewidespread
use led directly
evolutionof antibiotic
resistancein manybacteria,as indicatedby geographic
and temporal
correlations
betweendrugresistanceand antibiotic
use (Garrett1994,2000; Granizoet al.
2000). Limitingantibiotic
use duringthepastfourdecadeswouldat leasthave
slowedtherateat whichresistance
evolved.Butselectiveapplication
also causes
current
resistance
levelstodrop(Cristino1999).
Combination
Therapy
Simultaneous
a smallpopulation,
applicationof multipleagentsmayextinguish
evenwhenhighdoses of singleagentswouldnotwork,forthesamereasonthat
highdoses of a singleagentarebetterthanlow doses:The chancethatresistant
individualsoccurin theinitialpopulationdecreaseswiththemagnitude
of the
imposedmortality.
If resistanceto a singleagentcan be conferred
by a single
mutation,
thenmultipleagentsmayoffertheadvantagethatno singlemutation
confers
completeresistance.
Perhapstheoriginaluse ofcombination
therapy
was
in thetreatment
oftuberculosis
withthefirst
antibiotics
(Ryan1993). Treatment
witha singleantibiotic
resultedin theevolutionof drugresistancewithinthe
patient.Simultaneous
treatment
witha combination
of threedrugsallowedthe
infection
to be curedand thewithin-patient
evolutionof resistanceprevented.
More recently,
thesimultaneous
use of multipledrugsto treatHIV infections
has resultedin prolongedavoidanceof AIDS becausethevirusis so effectively
curtailed
withtheharshtreatment
(Coffin1995,Matsushita
2000). The long-term
successofmanyantiviral
vaccinesmaylikewisestemfrom
theimmunity
generated
againstmultiple
targets
through
boththehumoralandcell-mediated
components.
Withothergoals,however,
treatment
simultaneous,
multidrug
maynotbe thebest
practice(Bonhoeffer
etal. 1997).
theCourseofResistance
Charting
Whenmoredrugsareavailablethancanbe giventoa singlepatient,
thechoiceof
whichdrugstotakeshouldbe basedon theevolution
ofresistance.
for
Screening
resistance
preexisting
tothedrugscanensurethatthecombination
ofdrugsadminis maximally
istered
Forexample,anti-HIVdrugs(ofwhichtherearenow
effective.
ofdrugsareoften
basedon
many)areso harshonthepatient
thatdosesandnumbers
tolerance
levels.Therearethreemajorclassesofanti-HIVdrugs(protease
patient
inhibitors
andtwokindsofreverse-transcriptase
butmanydrugoptions
inhibitors),
willtake
existwithin
eachclass.A patient
onhighlyactiveantiretroviral
treatment
onedrugfromeachclasstomaximizethenumber
ofviraltargets,
butwithineach
class,thedrugthatis takenis somewhat
optional.Protocolsarenowbeingtested
in whicha patient'sviralpopulation
is assessedforresistance
withthe
mutations,
drugchoicebasedon thatgeneticinformation
(O'Meara et al. 2001,MacArthur
themolecular
2000).Itis obviousthatthiskindofapproachrequires
understanding
basis of resistance.
It also requiresa technicalmeansof assessinglow levelsof
APPLIED EVOLUTION
207
resistance
inthetarget
population
(e.g.,on theorderof 1% oreven0.1%), as such
levelsarenoteasilydetectable
yetcan changerapidlyinresponseto selection.
Refugia
in agriculture
One of themoreexcitingnew developments
is the engineering
of transgenic
cropsexpressing
a genefromthebacterium
Bacillusthuringiensis
(Bt),theproteinofwhichis highlytoxicto butterflies
andmoths.Thistoxin(Bt)
has longbeen a favorite
of organicfarmers,
used as a spraypriorto transgenic
killsonlya smallsubsetofinsectsandtypically
technology,
becauseitselectively
doesnotharmtheparasiticandpredatory
controlagents.Cornandcottonaretwo
majorBttransgenic
cropsinwideusenow,andthepotential
forreducing
pesticide
applications
to theenvironment
fromthemaloneis impressive.
Despitea public
backlashagainstgenetically
modified
foodsandtheconsequent
withdrawal
ofone
Bt cornstrainfromthemarket,
approximately
onequarter
oftheU. S. corncropis
in 2001 (F. Gould,personalcommunication).
expectedto be Bt transgenic
There
is thusa hugemarket
forBt strains.
in several
AllelesforBt resistance
arealreadyfoundat moderatefrequencies
in some cases at frequencies
pestpopulations,
greaterthan10% (Gould et al.
1997,Tabashniket al. 2000). These pestsare thuspoisedfora rapidresponse
levelsof resistant
insectsare muchlower
to Bt transgenic
plants.Fortunately,
thanlevelsof resistant
allelesbecausetheresistance
allelesarerecessive,hence
Thesesurprisingly
resistant
insectsoccuratthesquareoftheallelefrequency.
high
frequencies
ofresistance
allelesareunexplained
in atleastsomespecies,because
thereis no knownhistory
ofexposureto agricultural
Bt toxin(F. Gould,personal
communication).
The commercial
of Bt resistanceare obviousand haveinspired
implications
theU. S. Department
ofAgriculture
andslowits
andseedcompaniestoanticipate
evolution(Gould 1998). An antiresistance
evolutionstrategy
is beingemployed
thatdependson recessiveresistance.
FarmersgrowingBt cropsaremandatedto
planta certain
amountofnon-Btcropsas well-untilrecently
4%; nowitis higher.
Thesenon-Btcropsareplantedin separatefields(refugia)close to theBt crops,
toprovidea safehavenforpeststhatarenotresistant
tothetoxin.
ofresistance
toBt as follows.Pestsintherefugia
Refugiaimpacttheevolution
survive
oftheirresistance
tothetoxin.PestsintheBtfieldssurviveonly
regardless
iftheyareresistant,
fortheresistance
allele.As longas resistance
i.e.,homozygous
allelesareuncommon,
mostinsectswillbe sensitive.
Thus,refugiawillproduce
manymoreinsectsthantheBt fields.Withrandommatingbetweenpestsbornin
to the
willbe sensitive
therefugiaandpestsbornin theBt fields,mostoffspring
toxin(becausetheyare eitherhomozygoussensitiveor heterozygous).
Refugia
resistance
allelesatlowfrequencies
forlongperiodsoftimebecause
willmaintain
dilutetheresistance
theycontinually
genesand keep themfrombeingselected.
Resistancewilleventually
ascend,buttheascentis greatlydelayedwithrefugia
(Figure4). This resultis a specialcase of thepopulationgeneticsprinciplethat
208
BULL * WICHMAN
No refugia
4% refugia
1% refugia
0)
0)
2 0.5*
cn
LL
10
20
30
40
50
60
Generation
Figure 4
Effectof refugiaon thedelayofBacillus thuringiensis

(Bt) resistanceevolution.
(b) 1% refugia,
and(c)
evolution
areshown:(a) norefugia,
Threecurvesofgenefrequency
becausetheonlysurvivors
response
tonorefugia
is immediate,
4% refugia.
Theevolutionary
are recessivehomozygotes.The ascentof theresistanceallele is progressively
delayedwith
for
becauserefugia
arenotselective
forgenotype
andthusweakenselection
larger
refugia
therecessive,resistanceallele. These simulationsassumedcompletemixingof individuals
betweenrefugiaandBt cropswhenmatingandwhenovipositing.
Theyalso assumedthatthe
of all heterozygotes
and of sensitivehomozygotesin
onlysourceof selectionwas mortality
Bt fields,thatgenerations
werediscrete,and thattheinitialfrequency
of theresistanceallele
donot
was0.001.Theeffect
ofrefugia
wouldbemodified
totheextent
thattheseassumptions
oftheresistance
andthemixing.
Also,thecurves
phenotype
hold,especially
therecessivity
theactualnumbers
of surviving
pestswouldbe an important
giveonlyallelefrequencies;
factor
toconsider
intheeconomic
toa crop,butthatfactor
is notincluded
here.These
injury
curveswerecalculatedbyiteration
Waa+ 2pqWAa
oftheequationp' = (p2Waa+ pqWAa)/(P2
wherep is thefrequency
oftherecessive
resistance
alleleingeneration
t,andp'
+ q2WAA),
is itsfrequency
ingeneration
t+ 1.Thefitness
valuesweredetermined
as Waa= 1,WAa=
R is theproportion
ofcropsintherefugia.
WAA= R,where
weakselectionon a recessivetraitis ineffective

whentherecessivealleleis rare
size.
of selectiondecreaseswithrefugium
(Crow& Kimura1970); thestrength
insects
Onereasonnon-BtcropsarenotmixedinfieldswithBtcropsis toprevent
intermediate
frommovingbetweenthetwotypesofplantsandthusexperiencing
dosesofthetoxin(whichmightreducetherecessivity
oftheresistance
alleles).
EvolvableDrugs
offers
newdrugsfromnewclassesofmoleculesbutit also offers
Biotechnology
new drugsfromsomeold molecules-proteinsand nucleicacids. (Some types
APPLIEDEVOLUTION
209
of RNA-baseddrugswerediscussedabovein a paragraph
aboutblockingHIV.)
Proteinsandnucleicacidsin essencehavetheirowngenome,so theoretically
it
shouldbe possibleto evolvethosedrugs.Evolvinga drugmightprovidea way
ofproducing
newformsofthedrugthatovercomepestsresistant
to theoriginal
formof thatdrug.At themoment,
thisapproachis futuristic.
It is analogousto
theuse of combinatorial
chemistry
to producenewgenerations
of antibiotics
by
modifying
subgroups
ofold drugs.Drugevolution
was attempted
in a modelsystemofantisense
RNA usedto inhibit
a virus(Bull et al. 1998).Antisense
nucleic
acids workon thesimpleprinciple
ofbeingcomplementary
to a targetsequence
(inthiscase a regulatory
sequenceofthevirus)andthusblockinggeneexpression.
Ifthetargetsequenceinthevirusevolvesto a resistant
form,
antisense
molecules
to thenewtargetare theneasilycreated.This "armsrace" can
complementary
thuspotentially
be continued
indefinitely
becausea new antisenseRNA can be
createdforeverystepthevirustakesin evolvingresistance.
The empirical
testof
thistheorysupported
someoftheassumptions
butnotall: New antisenseRNAs
couldbe createdthatinhibited
individual
virusesresistant
totheoriginalantisense
RNA. However,theviralpopulationexhibiteda varietyof escape mutants,
and
no singlenewantisense
RNA couldcontroltheresistant
viralpopulation.
Resistancepolymorphism
in theviralpopulationthusthwarted
controlbythesecond
generation
ofdrugs.
Othervariationsof thisapproachhave also been developed,again without
success.Djordjevic& Klaenhammer
(1997) andBulletal. (2001) eachdeveloped
a suicideplasmidcassetteagainsta bacterial
a toxic
virus.Eachplasmidcontained
of a promoter
genedownstream
expressedby thevirus.Infection
expressedthe
toxicgene,which,in turn,killedtheinfection.
A priori,it seemedlikelythatan
armsracecouldbe wagedagainstthevirusbyinserting
a newtoxicgeneintothe
cassetteeachtimethevirusevolvedresistance
to theold toxicgene.However,in
bothsystems
thevirusevolvedresistance
bya changeinitstranscriptional
activity,
fromtheplasmidregardless
of whichtoxicgene
selectively
reducing
expression
was carriedby theplasmid.In a further
to keeppace withtheresistant
attempt
virus,Djordjevic& Klaenhammer
(1997) createda newplasmidthatcontained
multiplecopies of theviralpromoter;
thisplasmidpartially
restoredinhibition
cell (perhapsthrough
againstthevirus,althoughat somecostto theuninfected
lowlevelsofconstitutive
expression).
It is notclearhowwidelytheprincipleof drug"evolution"
can be appliedto
blockresistance.
Drugsthatsharea commonmolecularancestorwill likelyuse
inthepestcanprotect
thesamemolecular
thetarget
andrender
Ifevolution
target.
reverseit inaccessible,
thendrugevolutionwillfail.The case of nonnucleoside
inhibitors
transcriptase
(NNRTI) in HIV providesan analogyto drugevolution.
VariousNNRTIdrugshavebeendeveloped,
buttheyall function
bysimilarmechanisms,bindingtothesamepocketintheviralreversetranscriptase
(Emini1996).
If
toseveraloftheothers.
Resistancetooneofthesedrugsoftenconfers
resistance
oneregards
thedifferent
ofevolveddrugs,thenan
NNRTIdrugsas theequivalents
virusthan
evolveddruginthiscase affords
muchlessprotection
againsta resistant
210
BULL * WICHMAN
All thedifferent
evolved
mechanism
of inhibition.
does a drugusinga different
ofinhibition,
justas different
formsofa drugmayuse thesamebasicmechanism
in whichcase viralresistance
to one drug
NNRTIdrugsuse similarmechanisms,
willatleastpartlyoverlapwithresistance
totheothers.
EVOLUTIONARYCOMPUTATION
Introduction
ofusingevolunorhasthebenefit
is notrestricted
tobiologicalsystems,
Evolution
ofevolutionapplication
tionto solveproblems
beenlimitedtobiology.Although
tocomputer
programming
precededtheworkofFogeletal. (1966),
aryprinciples
Thegoalofthisworkwas lofty-"that
theideatotheforefront.
theirbookbrought
meanswill be foundfor
a replication
of specificaspectsof evolution,
through
of an artificially
automata... capableof solvingprobthegeneration
intelligent
almostas
ways."The fieldgainedsophistication
undiscovered
lemsinpreviously
andbythemid-1970s,Holland(1975) was
quicklyas itsbiologicalcounterpart,
inthecontext
ofcompuinteractions
discussing
coadaptedgenesetsandepistatic
computation
(EC)
tation.Manyconceptsandmuchofthelanguageofevolutionary
fromthebiologicalworld.Bitsare "loci,"potentialsolutionsto a
are borrowed
orgenomes,andtheyarechanged
chromosomes,
individuals,
aretermed
problem,
A collectionof variantsolutions,
termeda
and "recombination."
by "mutation"
The
somemeasureof"fitness."
is subjected
to"selection"
byimposing
population,
they"reproduce"
forthenextgeneration,
bestsolutionsareselectedas "parents"
toproducea newpopulation
thatis subjectedto
withmutation
andrecombination
andso on.
selection,
inthe
ofartificial
existsin severalformsthatdiffer
intelligence,
EC, a subfield
emofevolution
oftheproblem
thatis evolvedandthemechanisms
representation
executable
programs
ployed(Foster2001). Forexample,ingeneticprogramming,
theparameters
thatdescribe
themselves
areevolved,whereasingeneticalgorithms,
recombinaa potential
solutionto a problemareevolved(Koza 1992).Although
itis
andgeneticprogramming,
tionis commonly
employedin geneticalgorithms
evolutionary
programming
notemployedin theothertwoEC varieties,
generally
whichinsteadrelyon large-scale
mutations.
andevolutionary
strategies,
ofbiologicalsystems,
researchintheareaofEC
Like studiesoftheevolution
In thiscase themodelsareproblemsto be solved.
makesuse of modelsystems.
salesmanproblem,"an NP-complete
problem(i.e.,
Theseincludethe"traveling
the
difficult
a class ofcomputationally
problems)in whichonetriesto determine
routefora salesmantraveling
betweena largenumberofcities.Another
shortest
modelproblemis the"iteratedPrisoner'sDilemma,"in whichone
interesting
decideswhenit is bestto cooperateand whenit is bestto defectin a seriesof
oftheevoluThisproblem
hasbeenexploredfromtheperspective
confrontations.
tionofcooperation
(Axelrod& Hamilton1981) andis nowa classroomstandard
in EC (Vogel 1995). Anothercommonmodelproblemin geneticprogramming
APPLIEDEVOLUTION
211
whereone mustevolvea program

toreproduce
theinputis symbolicregression,
function
selectedinputsand
givenonlyrandomly
outputbehaviorofanunknown
function
values.
Evolvedprograms
haveinteresting
properties
thatinsomecasesaresharedwith
viarecombination
ofa geneticprogram
Forexample,evolution
biologicalsystems.
by"codebloat"(Langdonet al. 1999).Likebiologicalgenomes,
is accompanied
suchcomputer
genomesalmostalwaysaccumulate"junk,"and it is noteasy to
ones.In
ofthegenomefromthenonfunctional
components
separatethefunctional
ofcode
thegenomewillleadtotheevolution
junkfrom
fact,a defensethatremoves
thatevadestheeditingalgorithm
(Soule & Foster1998). However,genomesize
forlargergenomes.Thisis
a fitness
penalty
directly
bycharging
canbe constrained
in whichmicrobial
morestreamsimilarto biologicalsystems
genomesmaintain
presumably
becauseof
organisms,
linedgenomesthanthoseofmostmulticellular
selectionforrapidreplication.
WhatKindsofProblemsareBestExploredwithEC?
inprinciple
canbe appliedtoanycomputational
evolutionary
algorithms
Although
towhichthere
problems-problems
theyarebestsuitedtoverydifficult
problem,
As withbiologicalevolution,
EC maynot
areno otherknownefficient
solutions.
For
findtheoptimalsolutionbecauseitis a stochastic
algorithm.
approximation
salesman
witha verylargepotential
solutionspace,suchas thetraveling
problems
N citiesis N!, EC will
problemin whichthenumberofpossibleroutesthrough
landscapeis
notexploreall possiblesolutions.Butifthetopologyof thefitness
is an efficient
peaks.
wayto findandexplorefitness
rough,evolution
BiologicalproblemsarenaturalsforEC. On one hand,thisapproachcan be
suchas predicting
orRNA
usedto buildtoolsto solvedifficult
protein
problems,
DNA orprotein
sequences
inferring
phylogenies
(Lewis1998),oraligning
folding,
& Higgins1996).On theotherhand,itcanbe usedtomodelcomplex
(Notredame
orcells(Adami1998).
suchas theimmune
system,
ecosystems,
biologicalsystems,
havefoundapplications
forsomeverypracticalproblems,
Geneticalgorithms
suchas scheduling
andconstructing
In suchcases,theremay
complextimetables.
butperhapsnot
be othermethodsthatfindequallygood orevenbettersolutions,
have also been applied
and speed.Geneticalgorithms
withthesameflexibility
suchas processingsignalsfromradar,sonar,and
to nonlinear
filtering
problems,
GPS satellites
(Whitelyet al. 2000).
(written)
programs.
Evolvedprograms
tendtobe morerobustthanuser-created
thatis analogous
a property
totalfailure,
moredamagewithout
Theycanwithstand
is notalwaysclear,butatleastin
Thebasisforrobustness
tobiologicalhomeostasis.
inevolvedprograms
somecases,itis notmerely
(Masneretal. 2000).
redundancy
Different
user-created
maytendtouse thesameapproachtoa particular
programs
potential
problem,butevolvedsolutionswill tendto be unique.An important
is fault
andtheuniquenatureof evolvedprograms
applicationoftherobustness
tolerance.
Wherecomputer
failurecan havecatastrophic
consequences(i.e., on a
212
BULL * WICHMAN
ofanairplane),
agreement
betweenmultiple
system
spaceshuttle
orthenavigation
offailureofthesystem.
is used to reducetheprobability
independent
programs
"fault"becausetheywerewritten
havethesameunderlying
Butifall theprograms
agreement
maynotassureagainst
principles,
according
tothesameprogramming
failure.Evolvedprogramsmayprovidegreaterfaulttolerancethando written
And it is notout of thequestionto imagineprograms
thatevolveto
programs.
personalcommunication).
whentheydetecta fault(J.A.Foster,
repairthemselves
CONCLUSION
herearebuta smallfraction
of theapplications
of evoThe examplespresented
lutionary
biologyto sociallyrelevantissues.We havelimitedthisreviewto exandmethodshaveactuallybeenusedto
principles
amplesin whichevolutionary
is beingapproached
withevosolveproblems.
An evenrichervariety
ofproblems
to
thenear
and
some
of
these
efforts
are
likely
yield
fruit
in
lutionary
perspectives,
future.
willperhapsprovetobe an ephemeral
A research
focuson "appliedevolution"
fora disciplineas broadas evoluone.It doesnotoffer
a conceptualorganization
toariseandprosper,
and
will,ofcourse,continue
tionary
biology.Newapplications
andinfluencing
students'
career
funding
applications
maybe usefulin garnering
thedefining
basisforappliedevolution
is ignorance.
choices.Butatthemoment,
unaware
that
both
scientists
and
the
are
biology
evolutionary
public,
Manypeople,
theteaching
receive
ofevolution
yetattackstosuppress
hasbecomeveryrelevant,
will
atthelocal level.We hopethatignorance
ofapplications
widespread
support
be short-lived,
theexamples,and
as textsandthenewsmediabegintodisseminate
withwidespread
publicaccepbiologyshouldemergein thefuture
evolutionary
ofevolution,
butitis also
tance.Thisreviewhas focusedon positiveapplications
thesepotentialapplications
to guardagainstotheruses
to understand
important
ofthistechnology,
suchas production
ofbiologicalweaponsofmassdestruction
viruses.The timehas arrivedforwide
or theevolutionof damagingcomputer
of
the
and
relevanceofevolutionary
biologyin
understanding
importance
public
lives.
everyday
ACKNOWLEDGMENTS
discussionswithcolthrough
Manyoftheexamplesin thispaperwereidentified
M. Robertson,
B. Levin,L.
leagues.We thankD. Hillis,F. Gould,A. Ellington,
R. Bush,
S. Palumbi,D. Futuyma,
W. Maddison,I. Matsumura,
Ancel,L. Vawter,
andJ.Fosterfordiscussions,
M. Courtney,
I. Eckstranol,
references,
or,in some
of
cases,personalaccounts.MitchSoginprovidedFigure2. Editorialcomments
theprose.Thetopicsinthispaperalso
D. Futuyma
andD. Fautinhelpedimprove
we organizedon appliedevolutionat the2000 meetoverlapwitha symposium
We also arevery
ingsof theSocietyfortheStudyof Evolution(Bloomington).
APPLIEDEVOLUTION
213
grateful
forthesupport
oftheNIH (GM38737andGM 57756) andtheNSF (DEB
9726902) duringthetimewe prepared
thisreview.
VisittheAnnualReviewshomepage at www.AnnualReviews.org
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Applied Evolution

Author(s): J. J. Bull and H. A. Wichman

Annu.Rev.Ecol. Syst.2001. 32:183-217

PHYLOGENETICS:USING THE TREE OF LIFE

ERADICATION OF WILD-TYPE POLIOVIRUS FROM THE AMERICAS Poliomyelitis

USING THE TREE OF LIFE: PATHOGENIDENTIFICATION AND DISEASE ETIOLOGY

methodsoffera wayof identifying

Concernforendangeredspecieshas led to widespreadadoptionof laws limitingtheharvesting,

GENOMICS AND BIOINFORMATICS

Perhapsthemostextremeformof directedevolutionis thein vitroreplicaThe earliestsystemof thissortwas

out directedevolutionis to come up witha powerfulmethodto macarrying

VARIATIONAND INHERITANCE:ELEVATINGLEVELS OF GENETIC VARIATION

Whena nucleicacid (RNA or DNA) moleculeitselfis

LIMITING THE DELETERIOUS CONSEQUENCES OF PHENOTYPE EXPRESSION Inmany

ENZYMES FOR HOUSEHOLD AND AGRICULTURALUSE The year2000 markedthe

Effectof refugiaon thedelayofBacillus thuringiensis

weakselectionon a recessivetraitis ineffective

whereone mustevolvea program

FamulokM, Mayer G. 1999. Aptamersas

Kew OM. 1987. Geographicdistribution

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