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1、FigureAppendix2FIGUREAPPENDIXFigure2-9ConstructionandanalysisofSAGElibraries.Instep1,acDNAlibraryisconstructedfromthecellsortissueofinterest,andthecDNAsareimmobilizedonmagneticbeadsattheir3'ends.Instep2,thecDNAsaresubjectedtorestrictionenzymedigestionw
2、ithaso-calledanchoringenzyme.Thisanchoringenzymeisa“frequentcutter”restrictionendonuclease(usuallyNlaIII)thatensuresthatallthecDNAsarecutatleastonce.Instep3,cleavedcDNAsaredividedintotwopools.Shortoligonucleotidelinkersareligatedtothenewlycut5'endsofth
3、etags.Adifferentlinkerisusedforeachpool(linkersAandBasshown).Theseoligonucleotidelinkerscontainarecognitionsitefora“taggingenzyme.”ThistaggingenzymeisatypeIIrestrictionendonuclease(usuallyBsmfI)thatcutsatsomedistancetothe3'sideoftheactualrecognitionsit
4、e.Instep4,“SAGEtags”arereleasedbycleav-agewiththetaggingenzymeandfurtherprocessedtoyieldbluntends.Instep5,thefree,blunt-endedSAGEtagsaredimerizedtoyieldditags.TheseditagsareamplifiedbyPCRusinglinkerAandBprimers.Notethateachditagisflankedbytherecognitio
5、nsiteforthefrequentcutteranchoringenzymeusedinstep2.Theseflankingrecogni-tionsitesserveas“punctuationmarks”forsequenceanalysisoftheconcatenatedSAGEtags.Oncesufficientamountsofditagsaregenerated,theyareligatedtogetherinlineararrayscontainingseveralditag
6、s(ie,theyarecon-catemerized)andsubclonedintoaplasmidthatcanbeusedastemplateforautomatedsequencing.Eachsequencedplasmidcanyielddataon30to35SAGEtags.DataareanalyzedbyusingtheSAGE-softwarethatreadsthesequenceobtained,derivestheSAGEtags,matchesthemtotheirc
7、ognatecDNA,andgivesthegeneexpressionprofileinanumericformat.FIGUREAPPENDIX3Figure4-1PathwaysforCaspaseactivation.ThemajorpathwaysforCaspaseactivationinmammaliancellsarepresented.Theextrinsic(left)isinducedbymembersoftheTNF-familyofcytokinereceptorssuch
8、asTNFR1,Fas,andtheTRAILReceptors.TheseproteinsrecruitadapterproteinstotheircytosolicDDs,includingFADD,whichthenbindDED-containingpro-Caspases,particularlypro–Caspase-8,induc-ingtheiractivation.CTLsandNKcellsintroducetheproteasegranzymeB
