med 4耶鲁大学公开课生物医学工程Word下载.docx

med 4耶鲁大学公开课生物医学工程Word下载.docx

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regoingtocontinuetalkingtodayaboutDNA.Inparticular,we'

regoingtofocustodayonsortofhowtomanipulateanduseDNAinsomeapplications,andthisisahugeareaofscienceandtechnology.Youknowthis,youcan-it'

shardtopickupanewspaperoranewsmagazinewithouthearingsomenewapplicationofDNAtechnology.WhatI'

mgoingtodoisfocusonacouplebasicthingsthatturnouttobereallyimportantforgeneralapplications,andthenI'

lltalknotintoomuchdetailaboutafewapplicationsofDNAthatyou'

reprobablyfamiliarwithtotrytogiveyouaframeworktohangthison.

This,IthinkChapter3,describesfairlywellthethingswe'

lltalkabouttoday.You'

veprobablyalreadynoticedthatthere'

ssomematerialthat'

ssetasidefromthetextinboxes,andIencourageyoutoparticularlylookatthoseboxesforthischapter.There'

soneonDNAfingerprinting,forexample,thatgivesyoualittlebitmoredetailaboutthatspecifictechnique;

anotheroneonproductionoftherapeuticproteins.I'

lltalkaboutthesebothabittodaybutIencourageyoutoreadthoseformoreinformation.

Iwanttostartwhereweleftofflasttime.WetalkedaboutthestructureofDNA,howitworksintermsofaphysicalchemicalmodeloftheDNAmolecules.Wetalkedaboutbasepairingandhowthatleadstothisprocessofhybridizationorveryspecificmatchingbetweencomplimentarystrands.WetalkedverysuperficiallyaboutthebiologicalprocessgoingfromDNAtoprotein,sotheprocessoftranscription,RNAprocessing,andtranslationtoproduceaprotein.Iendedwiththispicturethatshowsyoualittlebitaboutcontrolofgeneexpression.Theimportantconceptisthat,whileeverycellinyourbodyhasthecapabilityofmakingalltheproteinsthatareneededthroughoutyourbody,noteverycellisdoingthatatanygiventime.Onlycertaingenesarebeingexpressedandit'

sthefamilyofgenesthatarebeingexpressedinacell,likewisethefamilythatarenotbeingexpressed,thatdetermineswhatacellislike,howitfunctions.What'

scalledthephenotypeofacellandwe'

lltalkmoreaboutthisnextweekwhenwestarttalkingaboutcellsandalittlebitaboutcellphysiology.

Therearemultiplemechanismsthatacellcanusetodecidewhichgenesitisexpressingatanyonetimeandwhichonesarenotexpressed.Ishowedyouthisinthispicturehereandthoselevelsofcontrolcanbeattheleveloftranscription.TherearemoleculesincellsthatgivetheDNAthesignalthatit'

stimetotranscribeandexpressagene,thosearecalledtranscriptionfactors,we'

lltalkaboutthemabitlater.There'

sinterferingwithRNAprocessing,andI'

mgoingtotalkaboutthatinthenextcoupleofslidesbecausethere'

sacoupleofnewmethodsfor-orpotentiallyinterferingwithgeneexpressioninlivinganimalsthathavebeendevelopedbasedonchanging-interferingwithDNAtranscriptionandtheabilityofmessengerRNAtobetranslated.Youcouldinterfereattheselaterlevelsaswell,forexample,byaugmentingRNAdegradation.IfthemessengerRNAforproteinisnotpresentinacell,thenthatcan'

tbetranslated,obviously,andtheproteincan'

tbemade.

ThesetwonewmedicaltherapiesthatImentionedarebasedoninterferingwiththebiologyofRNA,andoneisolderthantheotherandtheolderoneiscalledanti-sensetherapy.Whenyouthinkaboutageneoratranscript,themessengerRNAcopyofagene,youknowthatforeverysequenceofanucleicacidthere'

sacomplimentarysequence.Nowofthetwocomplimentarysequences,oneofthemencodesthegene.Oneofthemhastherightsequenceofcodonstospecifytheaminoacidsequenceoftheprotein,andtheotheronehasacomplimentarysequence.Youknowfromourdiscussionlasttimethatthesetwocomplimentarystrandsarenotmirrorimagesofoneanother,they'

renotidentical,they'

recomplimentary.Theyfaceintheoppositedirectionandyoucouldpredictthepropertiesofonefrombasepairingrulesoftheotherbutthey'

renotthesame.Oneofthestrandsencodestheprotein,encodesfortheprotein,theotherdoesnot.Theonethatdoesiscalledthesensestrandandtheotheroneiscalledtheanti-sensestrand,antimeaningit'

sthecomplimentanditwillhybridizetothesense.

Whatifyouknewwhatthesequenceofagenewas?

Agene,let'

ssayit'

sthegeneforinsulin.I'

llusethatoneastheexamplebecauseit'

safamiliaronetomostpeopleandyouknowthattheproteininsulinismadeonlyinyourpancreasandcertaincellsofthepancreas.SothatmeansthosecellsarecontinuallymakingmessengerRNAandthatmessengerRNAisbeingconvertedintoprotein.Well,whatifyouknewthesequenceforthemessengerRNAthatmadeinsulinandyoudesignedanothersinglestrandedDNAorRNAmoleculethatwastheexactopposite,ortheexactcompliment,Ishouldsay,ofthatstrand?

Soyoumadesomehowananti-sensepolynucleotidetotheinsulingeneorsomefractionoftheinsulingene.

Well,thatanti-sensestrandisshownhereastheredandthecellisnaturallymakingtheblueorsensestrandofmessengerRNAforaparticularprotein.Ifsomehowyoucouldtakeyouranti-sensemoleculesthatyou'

vemadeandyoucouldgetthemintocells,thenbythisprocessofhybridizationtheywouldnaturallyformapairlikethis.Theywouldnaturallyhybridizeandformaduplex,oradoublestrandednucleicacid.Whenit'

sdoublestrandedthisgenecan'

tbetranslated,becauseyouhavetohavethesinglestrandinorderforthetransferRNAtobindandforthisprocessoftranslationtotakeplace.Whatyoucoulddothen,ifyoucoulddelivertheseredcoloredmoleculeshereisyoucouldstopspecificallytheexpressionofthisparticulargeneintheseparticularcells.

Now,whatarethechallengesthere?

You'

vegottobeabletomakethisstuffandyou'

vegottobeabletomakeitinlargequantitiesandwe'

lltalkabouthowtomakenucleicacidsinlargequantitiesalittlebitlaterinthelecture.You'

vealsogottogetitintothecell.Itturnsoutthatgettinglargemoleculeslikethis,particularlylargechargedmoleculeslikenucleicacids,insideofcellsisnotsoeasy.We'

lltalkaboutthatalittlebitlateraswell.Infact,we'

lltalkaboutthatconceptthroughoutthecoursebecauseoneofthebigchallengesofmakingthesesortofnewbiologicaltherapiesworkinpeopletotreatdiseasesisgettingtherightmoleculesintotherightcells,attherightperiodoftime.

NowIgaveyoutheexampleofinsulinandyouprobablywouldn'

twanttostopinsulinproduction.Thatmightnotbeagoodthingtodo.Whatifthisisagenethat'

scausingacelltobecancerous?

Itwasagenethatwascausingacelltobemalignantandtodividewithoutcontrol,forexample.Thenyoucouldimagineblockinggeneexpressionwouldbeatherapy.

Student:

[inaudible]

Youwouldn'

twanttostopinsulin,forexample.Infactwhatyoumightprefertodoisstartinsulinproductionandwe'

lltalkaboutwaystodothatinjustaminute.Thesearewaystostopagenefrombeingexpressed,andthereturnsoutthere'

slotofapplicationsinthat,lotsofdiseasesresultfromtheunwantedexpressionofcertainkindsofgenesandcancerisprobablythebestexampleofthat,buttherearemany.

AnewerversionofthisthatworksinasimilarwaybutadifferentwayiscalledRNAinterference,anditturnsoutthatthisisanaturalmechanismthatcellshave.It'

samechanismthattheyhaveevolvedinordertopreventforeigngenesfromenteringacellandbeingexpressed.YouhavemechanismsinsideyournaturalmechanismsinsideyourcellthatallowthecellstodegradeunwantedRNAsequences.ThosemechanismsarecalledRNAinterference.Youmighthaveheardaboutthisbecauseit'

sbeenquiteanactiveareaofscience.

ItturnsouttoactivateRNAinterference,youdeliverdoublestrandedRNA.CertaindoublestrandedRNAsequenceswillcauseinthecellaprocessofdegradationofveryspecificRNAsequence.Thisinvolvesmechanismsthatarestillbeingunderstood,butifyou'

vestudiedsomebiologyorreadaboutthisyou'

veheardabouttheproteincomplexcalledDicer.DicerisaninternalcellularmechanismfordegradingRNA'

s.YoumighthavealsoheardabouttheRISCcomplex,ortheRNAsilencingcomplex,andthesearethebiologicalmechanismsthatareinvolvedhereandonlyshownbyorangearrowsonthisslide.TheendresultisyoucandesignnowveryspecificdoublestrandedRNAsequences,thatwhendeliveredintocellsagainwillactivatethisprocessofnaturaldegradationofanexistingmessengerRNA.Ofcourse,ifyoudegradethemessengerRNAatarapidratethanyou'

llstopexpressionofthecells.

Nowthenicethingaboutthisisthatthedegradationmechanismsseemtopersistforsomeperiodoftime,beyondthetimeatwhichyoudeliverthedoublestrandedRNA;

whereas,obviously,thismechanismhereisonlygoingtoexistforaslongastheanti-sensesequenceispresent.Sothismightbealongerlasting,morepermanentformofeliminationofexpressionofaparticulargene.

Ijustwantedtointroducethoseconceptsbecauseyou'

vereadaboutthem;

we'

llbetalkingmoreaboutRNAinterferenceinparticularaswegoonthroughthecourse.TherestofthetimeIwanttotalkaboutexpressionofgenes,ofnewgenes.Takingforeigngenes,genesthataren'

tnaturallyexpressedormightnotevenexistinsideacellandputtingthemthereandputtingthemthereinawaywheretheywork,andbyworkmeaningthegenegetsexpressedortranslatedintoaprotein.

I'

mgoingtostartbytalkingaboutaveryspecificandinterestingformofdoublestr