Chapter5OrganizationandExpressionofIgGenesOct26&31,2006你需要學習的課題:抗體基因是如何組成的？抗體基因重組(rearrangement)的機制抗體的多樣性(diversity)是如何產生的？細胞膜上的抗體如何轉變為分泌性抗體？抗體的類別(class)如何變換？-classswitchingCentralFeatureofAbMolecules:VastdiversityofAbspecificitiesAvariable(V)regionattheN-terminalendandaconstant(C)regionattheC-terminalendofAbmoleculesDifferentclasses(orisotypes)ofAb(e.g.,IgGandIgM)withidenticalV-regionsequences(antigenicspecificity)TheTwo-genemodelofDryerandBennett(1965)TwoseparategenesencodeasingleIg

HorLchain,onegenefortheVregionandtheotherfortheCregion.

Thesuggestionthattwogenesencodedasinglepolypeptidecontradictedtheexisting

onegene-onepolypeptide

principleandwaswithoutprecedent(先例)inanyknownbiologicalsystem.VerificationoftheDryerandBennetHypothesis

(byTonegawaandHozumi,1976)FirstdirectevidencethatseparategenesencodetheVandCregionsofIgandthatthegenesarerearrangedinthecourseofB-celldifferentiation.-TonegawawasawardedtheNobelPrizeforthisworkin1987.DemonstrationofDNADeletionatanIgLocusNon-Bcells:spermorlivercellsB-cellsDemonstrationofDNADeletionatanIgLocusdeletedsequence比大，因此在電泳時跑得比較慢MultigeneorganizationofIggenesl-ChainMultigeneFamilyMouse:Vregion:2Vlgenesegments4Jl

genesegments(3arefunctional)Cregion:4Cl

genesegmentsHuman:30Vl,4Jland4Clsegmentsκ-ChainMultigeneFamilyMouse:Vregion:~85Vkgenesegments5Jk

genesegments(4arefunctional)Cregion:1Ck

genesegmentHuman:40Vk,5Jkand1CksegmentsH-ChainMultigeneFamilyMouse:Vregion:~134VHgenesegments13DHgenesegments4JH

genesegmentsCregion:8CH

genesegmentsHuman:51VH,27DH,6JHand9CHsegments

V-RegionGeneRearrangements-TheH-chainV-regiongenesrearrangefirst,thentheL-chainV-regiongenes.Therearrangementsoccurinanorderedsequence,buttheyarerandomevents.ThearrangementsofIgandTCRgenesarethe

onlyknownsite-specificDNArearrangementsinvertebrates.H-ChainDNAUndergoesV-D-JRearrangements(1strearrangement)(2ndrearrangement)Amature,immunocompetentBcellexpressesbothIgM&IgDwith

identicalantigenicspecificity

onitssurface.L-ChainDNAUndergoesV-JRearrangementsintronsareremovedMechanismofV-regionDNARearrangementsTwouniquerecombinationsignalsequences(RSSs)

flankingeachgerm-lineV,D,andJgenesegmentOne-turnRSS:locatedat3’toeachVk,5’toeachJl,and

bothsidesofeachDH

genesegmentTwo-turnRSS:

locatedat3’toeachVl&VHand

5’toeachJk&JH

genesegment

RecombinationSignalSequences(RSS)CACAGTG23ntACAAAAACCGTGTCAC12ntTGTTTTTGG

Vλ

Jλ

//Oneturn/two-turnjoiningruleTheruleensuresthatVH,DH,andJHsegmentsjoininproperorderandthatsegmentsofthesametypedonotjoineachother.GeneSegmentsAreJoinedbyRecombinases-Recombination-ActivatingGenes:RAG-1,RAG-2-TheproteinsencodedbyRAG-1andRAG-2actsynergisticallyandarerequiredtomediateV-(D)-Jjoining.

Terminaldeoxynucleotidyl

transferase(TdT),anotherlymphoid-specificgeneproduct,isalsoinvolvedinV-(D)-Jrearrangement.ProcessofRecombinationofIgGeneSegmentsDoubleStrandBreakRepairTerminaldeoxy-nucleotidyl

Transferase

DefectsinIg-GeneRearrangementsRAG-1-/-orRAG-2-/-mice:-lackRAG-1orRAG-2-cannotstarttherecombinationprocessSCID(severecombinedimmunodeficiency)mice:-lackdoublestrandbreakrepair(DSBR)enzymes-cancarryoutsynapsis,introduced.s.breaks-cannotproperlyjointhecodingsequencesImpreciseJoining

-productiveandnonproductiverearrangementsproductiverearrangementin

onealleleisenoughIfrearrangementisnotproduced,theBcelldiesbyapoptosis.Ig-geneRearrangementsMayBeNonproductive!!!!

Only1/3attemptsatVL–JLjoining,and1/3subsequentattemptsatVH–DHJHjoining,areproductive.Asaresult,<1/9(11%)oftheearly-stagepre-BcellsinthebonemarrowprogresstomaturityandleavethebonemarrowasmatureimmunocompetentBcells.AllelicExclusionEnsuresaSingleAntigenicSpecificityAsingleBcellisonlyspecificforasingleepitope!!!

Onceaproductiverearrangementisattained,itsencodedproteinisexpressedandthepresenceofthisproteinactsasasignaltopreventfurthergenerearrangement.(1)(2)*activeallelesGenerationofAbDiversityAntibodyDiversity

SevenmeansofgenerationofAbdiversity:1.Multiplegerm-lineV,D,andJgenesegments2.CombinatorialV-(D)-Jjoining3.Junctionalflexibility4.P-regionnucleotideaddition(P-addition)5.N-regionnucleotideaddition(N-addition)6.Somatichypermutation7.CombinatorialassociationoflightandheavychainsJunctionalFlexibilityAddsDiversity-4differentjoiningsofVk21-Jk1inpre-Bcelllines

(Flexible)(Precise)Since

CDR3

makesamajorcontributiontoAgbindingbytheAb

molecule,aminoacidchangesgeneratedbyjunctionalflexibilitycanmakeamajorcontributiontoAbdiversity.P-AdditionAddsDiversityatPalindromicSequences{Palindromicsequences}N-AdditionAddsConsiderableDiversitybyAdditionofNucleotidesaddnew(N)-nucleotides-Upto15N-nucleotidescanbeaddedtoboththeDH-JHandVH-DHJHjoints.Thus,acompleteH-chainVregionisencodedbyaVHNDHNJHunit.NregionsappearstoconsistofwhollyrandomsequencesP-nucleotide及

N-nucleotideaddition有些什麼優缺點？SomaticHypermutationAddsDiversityinAlready-rearrangedGeneSegment

-Somatichypermutationoccursonlywithingerminalcenters,structuresthatforminsecondarylymphoidorganswithinaweekorsoofimmunizationwithanAgthatactivatesaT-cell-dependentB-cellresponse.-Somatichypermutationoccursatafrequencyapproaching

10-3/bp/generation.Thisrateisatleast100,000-foldhigherthanthespontaneousmutationrate,about10-8/bp/generation,inothergenes.-Bcellswithhigher-affinityIgreceptorswillbepreferentiallyselectedforsurvivalbecauseoftheirgreaterabilitytobindtotheAg.-----AffinityMaturation

ExperimentalEvidenceforSomaticMutationinVregionofIgGenesMostofthemutationsareclusteredinthe

CDR1

and

CDR2

hypervariableregion.

AntibodyDiversity

SevenmeansofgenerationofAbdiversity:1.Multiplegerm-lineV,D,andJgenesegments2.CombinatorialV-(D)-Jjoining3.Junctionalflexibility4.P-regionnucleotideaddition(P-addition)5.N-regionnucleotideaddition(N-addition)6.Somatichypermutation–afterAgstimulation7.CombinatorialassociationoflightandheavychainsClassSwitchingAmongC-RegionGenesOrganizationofHchain

VregionCregionAfterantigenicstimulationofaBcell,theH-chainDNAcanundergoafurtherrearrangementinwhichtheVHDHJHunitcancombinewithanyCHgenesegment.Thisprocessiscalledclassswitching.

Class(isotype)switchingClass-specificswitchrecombinasesmaybindtoswitchregionsandfacilitateDNArecombination.-CytokinessecretedbyactivatedTHcellshavebeenshowntoinduceBcellstoclassswitchtoaparticularisotype.-IL-4,forexample,inducesclasss