#3_manipulation of genes

8/20/2019 #3_Manipulation of Genes

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TH MAIPULATIO OF GENES

Techniques for leavng DNA. and splicg it in a ier m

make i possble to transfer geetic noratin from one i

a unrele n. ere e D relicates an exresses t

M

hology s fll of hybrd crea-urs such s h Sphnx, hMnoaur nd he Chmera, bu

h real world

no; s populaed byrganisms ha hav been shapd no byhe non of charactersics drvd fomvr dssimlr organisms b b volu-n whn spcs ha ran hr bascn nraon fr gneraon. Thss cas r ar naral barrers hanrmll rvn h xchang of gne- frmo en nrlad rgass rrers r sl oorl n-rs, h ar f fndamenallgcl poranc

h asc n of bological relatd-

ss s h speces, nd n organsms harc sexally speces are dned bly of heir members o breedh n anoher Speces e deernd nd dned by he genes heyarry, so ha n organisms ha repo-dce asexually h concp of specis de-ns on ntr's abliy o preven hbologically sgnican echange of genetc maeralthe nuclec acd DNAbetee nelated oups

he pesistenc o genetc uiuenessis pehaps mos remakable n simple

oraisms such as bacter. Een heny occupy he same habta mos baceial speies do n ehane geeic inomation Een rathe simila species obactera do not odialy ehane thgenes on heir chromosmes, the stucures ha r os of heir genetcnormaon Ther r exeptions, hower. There re bts o DNA, called plas-mds, ha xs apar om h chomo-somes n som baceri Sometimes alasmd can pc p a short sgme oDN from h chromosom of ts on

cel n ansf o cll of rela-d ctral ss, and someimes hls n sgn f chromosomal n com aed no hcomsom f h cpen cell. hs

y Stanley Cohen

ransfer of genes bewen spces byrachromosomal lmens has srelyplayed some role n baceral evolion,bu apparenly has no been widespead n nature. Otheris h charcterscs of h common baceral spe-ces wold no have remaned so largelyn oer h hg nmber of bceralgneraions ha hav xised drng hra of modern bacerology.

n 197 nn Y Chang nd he Sanor Unversy School of Mdcne nd rber W Boyr and obrB ellng h nersy of CalfornSchool f Medcn San rancisco

repoed he cosction n a tes ub

of bologcally funconal N molecules ha combned genetc normaionfom to dieent souces We made hemolecles by splcng ogether segmentsof wo dieen plasmids found n hecolon bacls Escheicia cli and ennserng h compose DNA no E clicells, where t rplicatd sel and e-pessed he geeic nomaion of bohpaen plasmds. Son aeard e inodced plasmd genes rom a e-laed baceral species, Staphylcccuaureus nto E cli where hey oo ex-

pessd he bological popetis heyhad dsplayed n hei oiinal hos; hen,applyng th same pocedues ith JohnF. Moo of Staord and Hoard Goodma n San aisco, e eeale t nse nto . col some geesm n aimal: the tad Xenopu laevs

e calle our cmposite moleculesDNA chimeas because hey ere conceptually similar to h mytholoialChmea (a ceatue ith he head o lo, he body o a goa ad th tail o aserpent and ee e mleular coute

pats o hybrd plan chimeas poduedby aricultual grati he poedue descibed has since been used adetended by wokers n seeral laboa-ores. has been called plasmid en

gineeg, because tilzes plasmids noduce he foregn genes, and moleclar cloning, becas provides a way popagat a clone, or lne of geneticallalke organisms, all cotaining dencalcompos DN moleculs. Becaus fh mho's poenal for creatng wd vre f novel geneic combnaons n mcroorgansms s also knows genc ngneerng and gnec mnplaon Th procdr call consss of svral dsnc bochemcal nbologicl manplaons ha wer madpossbl by sres of ndependen dscovers mad n rpd sccesson n la 1960's and early 970's. Ther ar

for essenal elemens method beakig and jonig DNA moleules d ried fom diee soces; a suitablgee caier ha can repliate both itselfand a eig DNA see liked o ;a means of troducig h composiDNA molecule, or chimera, into a funcional bacterial cell, and a method ofselecting om a lage populatio o cellsa clo o repien cells th has ac-uied he mleclar himea

n 967 DA liasesenzymes ha can

repair beaks n DNA and under ceran cditions can ji ogether theloose eds o DNA standsee dis-coered almost simultaneusly in elabatries A DNA stad s a chin ofnuleotides, each consising o a deoxy-bse sugar ri, a phosphat goup andone o ur oanic bases adnne, hy-mie, uaie ad cytosine. The sugarsad phosphates m he backbo ofthe stand, rom which h bases proje he nddal nucleod bldnblocks ae coected by phsphodester

bds betee te cabon aom a pos-ti 3 on oe sugar n e carbnatm at position No5 on djacensua Doublesand , h forud mos organsms, consists f w

© 1975 SCIENTIFIC AMERICAN, INC


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hans of nuotes nke by hyogeons between the pojeting baseshe bases ae opeentay: aenne is aways osite hyme aguanne (G) is aways opposte ytosne The funton of the gase s to epanks" o beaks n snge D stansby synthesng a phosphoeste bonbetween ajonng nuetes l-lrn v

In 97 a goup wokng in the aboatoy of H Gobn hoana who was

hen at he nivesty of Wsonsfoun hat the gase poue by thebateia vus T4 ou soetes atalye the entoen nkage of opetey sepaate oubestan D egments The eatin equie ha hees of two segents be abe o neah othe suh pstonng of two Dmoeues was a matte of hane an the eato was neen I wasea tha eient joing of D moeues eque a mehansm fo hoinghe w D ens togethe so that he

igase ou at genous way of apshghs wa evee a ese iepeenty w aboatoie a tafo:by ete Lobba a A ae Kaise a

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by Davi Jakso obe yons anPau Beg aie wok by othes hashow ha he e of the D oeue eta batea vuses an beoie by baepaiing between ompemeay sequenes of nueotes haae auay esen o sngesaegme jeig fo he ens ohose meue: pa with 's 'sa wih 's an he moeues ae heogethe by hyoge bons tha fombeween the pai The ppe of ink

ing D moeue by means of thegestan ojetons ha bee eoie Khoana's aoatoy fo joiig sh ythe equenes of ueoes into onge segmets of D

The tanfo grups kew too tha enyme emia ansfease wou ataye he stepwise aito speaya wha e ad he 3 es f ingera o NA, eie of ieiaueie I he eyme woke asowith oubetra DN he a bok ofiea ueie u be ae o

oe ouatio f DN moeues an abk he ompementay nueoeou be e anothe uaofom anohe oue Moeues hewo ouain he b eae

by hyoge bonig a eae oehe by D igase The metho was oentay apabe oig any w speies of D Whie obba an Kaseeste the teinatansfease poeuewth the D of he batea vius P22,Jakson yos an Beg appe heoeue to k he DN he analvus V 4 to baeaviu DN

he V40 an baeavius Doeues Beg' gou woke wth aeose ops a o a be eave ovie ea moeuewith fee e fuhe poessnga ikage llutrtn n pptepJ. s i happene the patiuaenye hose o eave the oops wasthe RI eueae whih waae o be ue i ieen peueo akng r biogiay funiona gene ombiai he tme,oweve he eyme eia oey

pouig memeay ingean en a by ie ha o e beesovee

The eave nea meue weeeate with an enyme poue by thebaeia vus amba ae an eonuease beause i peae by utting oueotie at he e o a D moeue The amba eouease hewebak the ens of DN moeues athus ef pojeng singestan ensha ha 3 emii to wh the bokf opementay nueoes ou be

ae The ne tep was o a wthhe he of temina tanfease a bokof ' a the 3 en of one of the twoD spees to be nke an bok of' at the ens of the othe speesThe spees wee e ogethe agens havng opemetay boks atthe ens ou n eah othe ne upan beoe anneae by hyogen boning thus fong ombie oeuesTo the gaps at the es of the ogina segents he investgaos suppeueoties a wo moe enyes eo

uease III an D poyease ay the niks in the oeues weeseae wth D gase

The etho of akng ohesve ten fo jong D moeues nthe st suessfu genetanipuatoepeents was onepuay an opeatonay een om he tenatansfease poeue I was aso muhspe I epee o e abity oone of a goup o eymes ae estition enonueases make ompemen

tayene fagmen ug he eavage N a i i e moeue insea of equirig h aition ofew boks of memeay uie DN emii



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Vue own on cetan an of E.coli wee known to be rected n ablt to ow ubeuent on otetan Inetaton ad own tatt etcton wa due to bactea nzm at rconz ecc te ona foren ra DNA and clae ta

DNA (To otect t own DNA e bactea cel ake odcaon enzmta add met rou to nucleotdconttutn t rconton te fo te etcton endonuceae, makn tem etant to cleaae) ecton endonuceae (and odcaton metae

ar wderad mcooganm; enefor makn wee found on achomoo nd acomooa lamd DNA w a on an bactera chomoome Dun e ea970 te nucotd euence at ceaae te econzed b ea r

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triction ndoncass wr dntd.n vr instanc, dvlod, tlavag wa a r nar an axs f rota-tional symmtr a alindrome where thenucleotd bas squences read th samen both strands in e 5to3 dirction[see illutration below]

n som nstances th breaks in thN strands mad b rsiction nms wr posit ach ther. On

artclar ndonclas, howver, th nm isola b ober NYsori in Bors laborator in Sanrancisc, had port that was fcia intrs. Unlik h other nu-las nown a h tim, this nymnodcd bra h two DNAands a wr sarad svralclotids. Bcas f th smmtical,alindromic arrangmnt of th nclo- in rgon f lavag s a-aon f av n t wands N in wit r-ng mlmnar cloid c i" ortisandtno r

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ge (b) yels DNA faments wth comple. entay, erlapng sgltran en Aa reut th d f y DN fragent rodce by o cleavage can anneal withy ther fragent prouce by the enzye

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mini. Te Eco RI nzyme thus poduedin one step DNA molecules that wrfunctionally equialent to the coesiend molecules roduced by th oplicated teminaltanserase pocedure.

Th xperimnts that led to the discoer of the capabilities of Eco RI wer reported independentl and siulta-neousl in November, 1972, by JanetMet and onald W. Dais o Stanord

and b anothr Stanfod inestigatoVittorio Sgaramella Sgaraela foundta molecules of th bacteial irus P22could be cleaved with Eco RI and wouldthen link up nd to nd to fom DNAsegments equal in length to two o moreviralDNA mlecules. Mert and Daisobsrved that closedloop SV40DNmolcles cleaved b Eo I wold rform thmselvs into circlar molecules hdrogn bonding and could bald with N ligas; th reconsti-ttd molculs wr infetious in ani-mal lls growing in issu cultur. Borand his collags analyd th nuleoid sqnc a th N trmini ro- I, an thr vidnconrmd th omplmntar natr ofh rmini, whic accontd for thircohesi activit

n lat 192, tn, svra thowr availal which n olin doblsan mlcl of NTa was a major n dvlon f sstm for manilating gnsMor was ncssar, owvr. Most sg-mns f N do no av an inhrntcapacity for slfrplication; ordr to reproduc thmsls in a biological ss-em the nd b integratd intDNA molcules that can replicate in thparticula system ven a DNA segmentthat can replicate in its original host wasnot likely to hav th specic geneticsignals required for repliation in a different nironent If foreign DNA wasto be propagated in bacteria as had long

been poposed in peulatie senaiosof genetic engineeing a suitable eileor arrier, was required A compositeDNA molecul consisting of the veicleand th desied foreign DNA would haeto b introducd into a population offnctional host bacteia Finall itwould be necessa to selet, or identiy,tose cells in th bacteial populationthat took up the DNA cieas. In 1972it still seeed posible that the geneticinformation on totall foreign DA molcules might produc an aberrant situa

tion that would preent the popagationf hybrid molecules in a new host.Moleclar biologists had focused or

man ars on virses and their relations with bacteria, and so it was nat

ral that bacterial virses wer thoughtf as the most likely vehicles for geneticmanipulation. For soe tie there hadbeen speculation and disusion aboutusing iruses, suh as labda, that occasionall acquie bits of the E. coichoosome by natual recobinationehaniss for loning DNA fo oreign soures It as not a ius oever,but a plasmid tat rst seed as a ve

hile for intoducing foreign genes intoa bacterium and that poided a ehanism for the replication and selection ofte oeign DNA.

A ubiquitous group of plasmids thatconfer n heir host acteria h abilitto resist a nuber of antibiotics had beenstudied intensiely for moe than a decade Antibioticreistant E. coli isolatedin many parts of th world, for xample,wer found to contain plasmids, designated factors (fr resistanc") carring h genetic information for productsthat in one wa or another could intefre with the action of specic antibiotics[s Infctios rg sistanc," bTsutomu Watanab; NTF -N Dcembr, 196] Doublstandirclar molculs f factor DNA hadbn searatd from bacterial chromo-omal DN b cnifugation in densitgradients and had bn characterid bbiocemical and hysial tchniques[s Th Molecul f Inftios Drugsistance" b oston Cloes;NTF N ril, 193].

In 1970 Morton Mandel and A. Higaf th Uniersity of Haaii School ofMdicin had discoered that treatmentf E. coli ith calcium salts enabled thebacteia to take up vial DNA At Stanford Chang and I, with Leslie Hsufound tht i we made te cell membanes o E. coli pereable by teatingte ith caliu chloide puied Rfactor DNA could be intodued intothe [e ilutrtin on oppite pag.The Rfator DNA is taken up in tis

tansforation process b only about onebacteial cell in a million, but thos fecells can be seleted because the lieand multiply in the presence o the antibiotis to hich th R factor cones resistance, hereas other cells die Eachtransoed cell gie rie to a lone thatcontains exat replias of te paent pasmid DNA moleules, and so e eaonedtat plasids igt sere as eiles opopagatig new geneti inoation ina line of E. coi ells

In an eort to explore the genetic

and moleular poperties o aious regions of the Rfactor DNA we had begn to tak lasmids aar b shearing their DNA mechanicall and thentransforming . with th rslting



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fragets. S fterward we bega cleave the plas ds wth the co RI ezye, whch had bee show t prdceultiple sitespecc breaks severalviruses t ght therefore be coued t leave all lecules f a baceralplasmi i the sae way, so that ayparticular species f D oul yield aspecic set f cleavage fragments, a s reproucibly. The fragments coul

the be separate an entied according to the diere rates at whch theywld igrate thrugh a gel uner theinuence of an electric current.

When the D termini pruce byco RI edoclease ere foud tobe chesive, Chag ad I, cllabrat with Byer and Helling SaFracisco, prceeded to search fr aplasd hat he ezye would clevewithout aecg he plasmis abil

eplcate r fer atibiotic ress

nce. We hped ha f sch a plsduld be fd, we l ser ege f freg at he co RIcleavage ste, d ha igh be ssible to prpagate the foreig l.

I our collection at Stanford there wasa small plasmid, O, that hd beeisolated fllwg he echaical shearng f a large plasd bearng gees frultiple atibitc resstace. It wa essthan a telfth as long as the pare plasi, bu t di reta he genet fr-

aio fr its replcat coli dfor coferring ressace oe atbc,tetracycle. Whe we subected OD cleavage b co RI d lyze he prdcs by gel electrphress,we fund tha he ezye had heplasmid olecule ly oe place, pr-dcig a sgle iear frage. Wewere able o in the ends f hat fragent agai by hyroge boing and reseal the with D ligase, and whewe itrouced the reconstitute circularD olecules nt l by tras

fratio, they were bilogically fctona pasmis they replicate an conferre tetracyclie resistance.

The net step was to see if a fragentf freig D uld be iserted a hecleavage site without nterferig wthreplication r epressio f etracycieresstance and thus destroyig the plsms ability serve as a cnig e-hcle. We ied he D of another coli lasd, whch carred resistace he atib kanayc, wth O D We sbeted the xed to cleavage by co RI ad the lgtio, trasfred l wth he re-sltig ad fd ha se erasfred bacer were deed resis

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he wo DNA species akes he iula mi DNA enser han he homsNA he lamis o iin ban enifugaio i eium hore gean can be seaae bttm t) Te plami DNA is mixe wih baceia ells h e o reian o eaycine h have been mae pemeable by eamen wih aual Te D ene e cell, eplicae ee an mae e cell eian o ecye

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FOEIGN N is spliced into e pSCIO plasmid and itroduced

ith the pasi nto the be heh l The asdis claved y he endonucleae Ec RI at sgle site hat des nttrfere ith e plasmid's enes for replicatin o fo retace traccline (top f). e nceotde sequence recgzed c R is esent also in othe NA so tat a fregn N ex·ped to te endnucease is cleaed au ce evr 4, to

6, uceotide pars o a ram ass (tp igh) Fragmets ofcleaved foreig N are aealed to the asmid y hdro.

gn din of the complementary base airs and the new com· oite molecules are seled b N gase Te chras,

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a t bth eracyclie and kaamyciThe plasmids slaed from such transfrmats ctaied the entire pSCI0DNA segmet and also a second DNAfragmet that carried the iformatiofor kaamycin resistance, although itlacked eplicatio functios of its onThe results meat that the pSCI0 coulserve as a cloning vehicle for introucing a least a onreplicating segment of

a related DNA ito col An the rocedure as eraordinarily simple.

Could gees from ther species be i-troduced it c plasmis, hoever?There migh be geetic signals o foreig DNA ha uld prevent its propa-ga r expressi co e decded ry cmbine DNA from aplasid f ather bacterium, he plasmid f tahycocus ureus, ithr riga E lasmid. The saphylccca lasid had already beesdied n several laboratories e had

fud ha as cleavd ito four DNAfragmes by Ec RI. Sice as aive E r t related bacera, cld its o propagatei hs. Ad t as kno tcarry a gee fr resistace to still atheraibic, peicill, hat ud serve as arer fr elecg ay trasformedc. c ressace, lke cmbid rea eracycle ad ka-ayc, a arady desread agE rai are. That as ipr-a e r bacterial peces ca ray exchage geecifrma he cl bacillus ere be dced , as essetialtha hey carry ly abiticresistacera a ere aready prevalet erse e ld be etedge species aibiticresistace capabil-e.)

ha ad repeaed the eerimetha ad bee sccessfl ith kids E ad, b his time e didi ith a ixture f he col' SC-101 d h

aylcccal 258: e

cleaved he ixed lasmis ith c Reduclease, reaed hem ith ligasead he asfred co Net eislaed rasfrmed bacteria that epressed he peicilli resistance codefr by he S areus plasmid as ell ashe etracyclie resistance f the oliplasi. These dubly resistat cellsere foud to contai a ne DNA secies that had he molecular characteristics f the staphylococcal plasmid DNAas ell as the characteristics of pSCI01

The replicatio ad epressio i co f gees derved from an organismrdinarily uite unable to echangegenes ith co represente a breachi he barriers tha rmally searate

biological speces The bulk of the genetic informatio epressed i the transformed bacteria deed it as ol, butthe transformed cells also carrie relicaing DNA molecules that had molecular and biological charateristics derivedfrom an unrelate scies, S ureusThe fact that the foreign genes ere oa plasmid meant that they oul be easyto isolate and purify in large quantities

for further sudy Morever, here asa pssiblity that ne might itroducegenes int the easytogo col thatspecify a ide variety of metabolic orsynthesizing functions (such as photo-sythesis or antibiotic productio) adhat are digeous to other biologicalclasses Ptetially the pSC101 plasmidad the molecularcloning prcedurecoul serve to introuce DNA moleculesfrom comple higher organisms it bac-terial hosts, making it possible o applyrelatively simle bacterial geetic and

biochemical techiques to the study faimalcell gees.

could animalcell genes in fact be inroduced t bacteria, ad uld they

eplicate there? Byer, Chag, Hellingad , together ih rro and Goma, immediately undertok t oute picked certai gees ha had beenell studied ad characterized ad ereavailable, puried, i uatiy: he geeshat code for a precursr f the ribosomes(the structure n hic proteis are syn-hesized the ad Xenp laevs Thegenes had prpertie at d eables t idetify the f e succeeded ingettig hem rpagate i bacteriaThe tad DNA as able fr aothereason: althugh e ld be costructing a vel blgical cmbiatio contaiig gees fr bth aimal cells adbacteria, e ad hers epected that nohazard uld resl fro trasplatigthe highly puried ribosomal genes f atoad

Ulike the foreig DNAs of our ear-lier eperiments, the toad genes did notepress traits (such as antibiotic resistance that could help us to select bacteria carrying plasmid chimeras Thetetracycline resistance conferred bypSC101 ould make it possible to selectrasformed cloes, hoever, ad ecould then proceed to eamine the DNAisolated from such clones o see if ayclones contained a oreign DNA havinghe molecular proerties of toad ribo-somal DNA The edonucleasegener

aed fragments of toad ribosomal DNAhave characteristic sizes ad base com-osiions DNA from the transformedcells coul be tested for those characteristics The genes proagated i bac

teria could also be tested for nucleotideseuence homology ith DNA isolateddrectly from the toad

hen e id the eperiment and analyzed the resulting transforme cells, efound that the animalcell genes ere indeed reproducing themselves i gen-eration after generatio of bacteria bymeas of the plasmids replicatio fuc-tios n aitio, the ucleotide se

uences of the toad DNA ere beingtrascribed ito a NA produc i thebacterial cells.

ithi a very fe mths after therst DNAcloig eperiments the pr-cedure as beig used in a umber oflaboratories t clone bacterial and ai-malcell DNA from a variety of sources.Soo to plasmids other than pSCI0ere discovered that have a single cI cleavage site at a locatio that oesnt interfere ith essetial gees. neof these plasmids is present in may

copies in the bacterial cell, making iossible to amplify," or multily maytimes, ay DNA fragments linked to it.vestigators at the Uiversity f di-burgh an at Stanfor ent develop mutats f he viru labd(hich rdinarily ifecs c) hmade the virus to a eecive clivehicle. ther resricti edceaeere discovered ha als ake chesitermini b that cleave DNA a dersites from the c R ezymes, chromosomes ca be ake aarand put together i varius ay.

The ivestigative possibilities f clonig are already bei expred tesively Some orkers have islaefrom comple chromosomes certai re-gions that are iplicated in particularfunctios such as replication thers areaking plasmids order ith speciprperties hat shuld larfy aspect etrachromosomalDNA biolgy thahave bee hard t stuy. The orgaiza-ti of complex chromosomes, such athose of the fruit y Dsopla, is beistuie by cloning he animal genes ibacteria. ithi the past fe monthmethos have bee develoe for selec-tively cloning secic gees of higherorganisms through he use of raioac-tively labeled NA probes instead purifyig the gees to be studied befreintroucing them into bacteria, oe catransform baceria ith a heterogeeopulation of animlcell DNA ad heisolate those gees tha produce a particular species of NA. t is als pssible

to isolate groups f gees that are ex-pressed concurretly a a particular staei the aimals develome

The potential eems t be eve broad-er. ene maipulatio opes he pro

31



8/9

pec of consutng bactea ceswhich can e gown easy an inepenivel, tha w synthesie a vaiey f

ilgay pouce substnces suh asaniiots an homones, o enymesha can cnve sungh iecty nooo sustances o usabe enegy Pe

haps t even poves an epeimenabasis fo inoucing new genetc infomaion into pant o anima ces

I as een cea fom he begnnng ofexementation in moecua coningha he constuction of some kins f

novel gene combinatons ma have a poenial fo bolgca aa, an he sc

ec community has move quicky mae cetain ha reseac n genecmanipuation wou not enange thepublic Fo a me afer u nial expei-mens the 101 plasmid wa he nlyveice known o e sabe fr lningregn DNA in co and ur l-

eages aske for spples with which purse stues we kn wee f marcentc an mecal importance I

vesigatos nomay aciiae the eeexcange of bacteia an oher expermental stans they ave saed r devee bu Chan ad I ere n-cee tha manipulain ceragenes cou gve rise novel ansmswse infecious poperes an lical eects cl n e prdced agreing o pvie e asmd herefoe aske fo asuance hat rclleagues woul neir inouce -mor viuses into baceria n cea anbiiesistance cmbnans a ern aeay pesen in nare; w alsased he eciiens n sen h las-md n ter labrars, a cl keep ack of is sribion

hen st he cning vehices wee

PASD DA

T OAD DAFRAG MET O.1

TAD DARAGMN O.3

CLEAVED T OADRIBOSOMA DNA

scvee, became apaen ha ame geneal mecanm f ensur ex-perimenal safe enemanilainesearc wa aviabl he rndwrkfo sh cnl a een esabliseeaer: h aina Aaemy f Sci-ence ad bee re consier hepssily tha tealy hazadoconeqences mig resl fom wespead r ndici se" f hese tech

niqes and ha ased Pau Ber t frman avisy cmmiee ha l consier isse Ber had bee cn-cened ab he enal aards fceta n expermena rsome as an ad himsel ecded abad lan r nrdue enesfom mr vr S0 n baceriabeca f sible aner f e ex-erimen were ccesful

r r er a nmbr nvesar d m e drc lvd mlecar cln-

, r 197 a rrtreeaed l ad a ler eadnrfena ral mmbs mmte xred er cncernab l ra ns-e ndrmna aa" q ad rall aed allvar em vlarrrn xerme ad a mar a a a ) Exr vlvd ram a ma xrd aabe r a-ra d ar 2 xrm vvd d A rm mr vr r a ara mm e a c rcmbinanml m mr a m

CEAVED PASMID HMERAS

nae bacteria ulains in humansa her ie, ad mg h ncease he incece f cancer or theiseases"

he Acaemy cmmitee was cn·cerned large because f nailiyo assess he azars erain expeiments accuaely before the epementswre neaken Gene for safetyad ln been avaiable n the aeas of

poeniay hazadus research, such assie invving knwn seasecausngbactea an viuse, raacive isooesr xic chemicas ecase f he new-ness of he mcobal enemanipatinmethds, n sc delne had yebee eveope fr w h aeawever hee was h ssiby thatpenialy aao experimes mghtreed befo aprpra delinescld b cnsidere an mpemenee recgnzed tha ms w w thenew meh id and wld i

vve epemen a azars naue we reommended e deeal fe an Type II exerimens nil heazar wee moe caefy assesse,nl t was etemned wheer r note wk cul be neraen sae anunl aequae sae precain eeavalable The cmmie as rsea an intenaonal meeing b arl in 195 o conser the maer moelly

S a meeting was d in Ferara Aslmar erence Cr nearac Grve, Ca brgh ehe86 Ameran bi a 53 nvesigar rm 16 er cne, senree ad a alf da revein rgess h eld f mlelar lnn and foma de a wld alwms e e erediar aracter-s b nrod n baceia an

CD 2 CD 0 4 D 18

EAVEDpS0

PS MID

58 :z

�w

. � 9 �C

uz

3 0

Q0

L LH monae he preence of toadA n m pla amn of DNA mgrate though a

l at n a uner he nuence of an electc curet, depnn n r Lnea molule of plm DNA (ig)

an lava pou of oa rbooma DNA (et) therefore

have characec e an mgae harac an n agven me he ban of DNA, vuale by a uorent ye a

photographed uraoet A ve chmerc pam (ent)cota pamd NA moece; n adton each chmera n

cude oe or moe fragmet chaacerc of ogna toad DNA

2



9/9

ly. v ce m ld l d � cd dcs decs Am, l repeseves f ecs d Fdel s cec esech; h mees ee pe ss d ee ull red. hssues ee cmlex d hee e ddece my em,

bu e csesu e mjrs. Fs, ly deld cl md r sc del de vey m scc d medcl blem s ell he blems ble scey, sucs evmel llu d d dey ses. Secd, he ccdeldsem ce vel blclcmbs my e vy deees f el sk. csuc c cmbs hld cdly de rdd c

s, clly blcl d ysclb, deqe eve escf y zdu sm; h xe e cl sk hld b exld byxeme cdced d c cm cd. d, sme exe elly zd b cd ese, ve crl cme. F c d xec my my l zd cd l d lbbl sc.

l, d drd rc ld b b d y c b mdd l me rk c.

Pycl cm ber vl be d U.S. cxlr rm mmz e sbly cm by eesl mcbes. Cme cd l mlyd rly ec lby ke d blc frm hzd sscd rdcv se d xc chmcl d

k h dseec bced vuss. h Aslm mee muled e ddl cce blgcl be, hc lv fdcl ecle h ble e ly eclze d qully d bcel bl l xce dr elby cs.

I s he cec cmmys cmmly lced s cs mlly, d ehcl cce elfmpse es. Usully, b

lys, scey lge ls cderd blc llb dm klede bd bybc ec eec hld b ld. Exesve ublc scry d

REGION OFTOAD DNA

�

PASID DNA KINONA

PASMID HIMERA DNA

HODPLX ANALY enties regions of a toa DNA (bk) tha t have be en ncoporate chimeic asmi NA moecue DNA soate fom toa eggs a heDA of the chimea ae enature that s, each nata oubestan oecue s spt nto

to singe strans of DNA, by ai reatment The toa an the cher N's re e together, an any compementary sequences are aoe to n each other he toa DNA

incopoate in the chimeas has nuceoti sequences that ae compementay to sequces in the DNA taen iecty fom the anima souce hose homoogous sequences anea

to fom heteoupex oubeta DNA that can be ientie n eecton miogaphs

e dscs by c d c sbl rk d b

rcl l bc rc b r, , c) zd r lyl d, r r, ycl. rcl r ll l lr cm b

l dscus d by cesrk c ml ll b.

c rrg c rr dds h b dd r rec cl rcmb A mleculesl m a r c rl blc d vrmel y

P O OAD DA in to sepaa hiei pami moecues s emontae by an eectron icogaph mae by Joh F orro a he Stanfor ers Sc ofMeicine As s nicate n the aing (bttm) there ae DNA trans from o s mis an a stan of toa DNA The microgaph shos hickene egons f N he

nueotie sequecs ae homoogous an o singe stans he ee annee

toa DNA n the himeras coes fo ribosomes, an the space eee the heteruex egions i compatibe ith the spacing of mutipe rboso ees DN

33

#3_manipulation of genes

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