ProfessorMarkSaltzman:
So,we'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]
ProfessorMarkSaltzman:
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
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