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Lecture 11

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School
University of Windsor
Department
Biological Sciences
Course
55-213
Professor
Hubberstey
Semester
Winter

Description
Lecture 11 2013-01-10 2:29 PM Important)concepts)for)week)#6)(midterm)week):) • Gene$evolution$and$alternative$splicing$ o Really$dynamic$process$ o Genes$can$be$changed$over$long$periods$of$time$under$the$right$selective$ conditions$ o Genes$can$produce$more$than$one$single$produce$ • Pseudogenes$and$genome$content$ o Pseudogene$is$essentially$a$gene$that$has$been$duplicated$a$long$time$ago,$ and$at$one$time$there$was$a$function$for$that$gene.$However,$over$the$years,$ mutations$have$built$up$in$that$gene$and$made$it$inactive$ o Sometimes,$there$are$so$many$mutations$in$one$site$over$a$long$time,$that$ you$can’t$even$tell$a$gene$used$to$be$there$ o All$of$the$genomes$that$we$have$in$our$cells$make$us$who$we$are$ o They’re$all$based$on$those$6$billion$letters$ o Slight$changes$in$those$6$billion$letters$make$us$all$unique$ o At$the$same$time,$these$sequences$tell$us$where$to$begin$in$the$past$and$a$ lot$about$ancestry$ o Thus,$genomes$tell$you$a$lot$about$ancestry$ $ Humans)and)Genes:) • Humans$have$~$25$000$genes$ o Estimated$that$there$are$>$100$000$different$proteins$expressed$in$our$bodies$ o How$is$this$possible?$ ! Can’t$assume$1$gene$product$and$1$protein$per$gene$ ! This$is$possible$due$to$alternative$splicing$ ! All$the$exons$that$are$within$a$gene$don’t$change$their$position,$ however,$the$presence$or$absence$of$a$specific$exon$in$a$mature$ mRNA$isn’t$always$predicted$ • Some$genes$can$produce$more$than$one$protein$ o This$can$be$done$through$these$2$different$processes:$ o Overlapping$reading$frames$ ! Primarily$used$in$viruses$and$mitochondrial$genes$ ! A$single$gene$can$create$multiple$overlapping$reading$frames$in$that$ gene$sequence$ ! In$that$way,$viruses$which$have$small$genes,$can$produce$a$large$ number$of$protein$products$using$these$processes$ o Alternative$splicing$ ! Splicing$out$different$exons$under$different$circumstances$ • ***Position$of$exons$is$never$changed!***$ • See$figure$4.12$"$alternative$starts$(or$stops)$generate$related$proteins$ $ $ $ $ $ $ $ $ $ $ $ $ $ o Overlapping$genes$–$same$reading$frame!$ o Gene$shown$at$the$bottom,$where$the$mRNA$is$at$the$bottom.$Green$is$start$ codons$(AUG).$in$this$case,$then$the$mRNA$is$produced,$the$ribosome$binds,$ and$as$it$starts,$it$may$skip$over$that$first$start$codon$and$keep$on$reading$ until$it$hints$the$second$start$codon.$These$2$proteins$from$this$point$on$(C$ terminus)$are$exactly$the$same.$However,$the$one$at$the$N$terminus$(full$ length)$is$different$in$sequence.$Thus,$we$produced$2$different$proteins$with$ similarities$and$differences$from$a$single$gene.$These$are$obviously$very$ related$proteins,$and$they$share$sequence,$but$they$also$show$differences.$ Here,$we$used$the$same$reading$frame$(same$codons,$it$wasn’t$changed),$ only$an$alternative$start$site$was$used$as$it$skipped$the$first$one.$$ o What$about$using$different$reading$frames$within$the$same$context$of$the$ gene$sequence?$(See$below)$ • What$happens$when$a$gene$uses$more$than$one$reading$frame?$ o Occurs$in$many$viruses$and$some$mitochondrial$genes$ o See$figure$4.13$"$overlapping$triplets$may$be$used$in$different$reading$ frames$ $ $ $ $ $ $ $ $ $ $ $ $ ! Produces$2$non[homologous$proteins$ ! Start$codon$is$in$a$different$frame$than$the$one$above$it$ ! Thus,$depending$on$which$start$codon$the$ribosome$uses,$you$can$ produce$2$completely$different$proteins$from$the$same$gene$ ! The$actual$gene$sequence$is$still$a$single$gene,$but$it$makes$2$ different$proteins$ ! As$far$as$we$know,$mammalian$systems$don’t$do$this$ ! The$virus$has$to$know$when$to$turn$on$the$upper$one$and$when$to$ turn$on$the$lower$one.$The$virus$has$ways$of$knowing$this$(seen$later$ in$the$course)$using$the$same$promoter$(utilizing$different$sequences$ within$the$promoter,$for$example)$ o Many$viruses$use$this$technique$(since$their$genomes$are$so$small,$they$have$ to$get$the$most$bang$for$their$buck$with$the$nucleotides$they$have)$as$well$as$ mitochondrial$genes.$$ o Do$viruses$mutate$faster$because$of$this$(less$chance$for$silent$mutations)?$ Yes.$A$mutation$can$affect$more$than$one$gene.$Thus,$the$virus$becomes$ more$constricted$and$a$mutation$will$probably$kill$it.$At$the$same$time,$you$ could$still$build$up$mutations$that$make$the$gene$faster,$depending$on$the$ gene.$We$know$cases,$such$as$retroviruses,$where$the$mutation$rate$is$very$ high$as$they’re$constantly$trying$to$evade$the$targeted$immune$system.$This$ has$happened$in$many$examples$where$these$kinds$of$mutations$give$viruses$ a$selective$advantage$(especially$in$cases$where$the$genes$are$ immunologically$related$"$ex:$surface$of$virus$where$the$immune$system$is$ trying$to$make$antibodies$against;$if$the$$virus$is$constantly$changing$that$ sequence,$than$it$will$always$be$one$step$ahead$of$the$target$organism)$ Alternative)Splicing:) • Troponin$T:$muscle$protein$that$produces$alpha$and$beta$forms$from$same$gene$ o Called$protein$isoforms$(see$figure$3.16)$ o Protein$isoforms$are$similar$forms$of$the$same$protein,$but$they$come$off$the$ same$gene!$There$is$only$1$Troponin$T$gene$that$produces$both$alpha$and$ beta$isoforms$through$alternative$splicing!$ • WDR1:$protein$involved$in$cell$structure$and$shape$ o Wild[type:$606$amino$acids$–$15$exons$ ! During$the$professor’s$study$cloning$this$gene,$there$was$always$a$ smaller$one$appearing$(see$the$mutation$below!)$ o WDR1Δ35:$466$amino$acids$(lacks$exons$3,$4$and$5)$ ! Delta$means$lacking$exons$3[5$(see$figure$4.14)$ ! Splices$2$and$6$together,$as$they$are$perfectly$aligned$ ! There$is$a$1$bp$at$the$end$of$exon$2$and$2$bp$at$the$start$of$exon$ 6…It’s$amazing$because$the$exons$remain$intact:$the$exon$6$in$the$ wild$type$is$the$exact$same$one$in$the$mutation!$In$addition,$ everything$after$that$deletion$remains$exactly$the$same.$ ! All$it$is,$is$a$deletion$of$140$odd$amino$acids$ ! Why$does$the$cell$have$the$ability$to$differentially$splice$these$2$ proteins$at$different$times$in$the$cell?$We$have$no$clue!$We$don’t$ know$how$it$does$it$or$whatnot.$ ! The$cell$has$some$sensing$mechanism$that$relates$to$the$splicing$ machinery$and$that$tells$it$when$to$make$the$wild$type$and$when$to$ make$the$mutation,$but$we$don’t$know$why$or$how$ ! However,$we$do$know$that$the$majority$of$human$genes$use$ differential$splicing.$This$has$made$the$interpretation$of$a$lot$of$ molecular$biology$difficult.$It$was$initially$estimated$that$differential$ splicing$was$rarely$done$(5%).$However,$as$molecular$biology$ techniques$increased$their$sensitivity,$it$was$found$that$differential$ splicing$techniques$are$used$everywhere.$It$makes$the$interpretation$ of$any$kind$of$experimental$system$difficult,$because$you$have$all$ these$different$isoforms$being$made$at$higher$or$lower$amounts$ under$different$conditions$ o Same$amino$and$carboxyl$ends$ • Estimated$that$~$60%$of$human$genes$use$alternative$splicing$ • See$figure$4.14$"$alternative$splicing$can$substitute$exons$ o Troponin$T$example$ o Promoter$at$beginning$ o Beta$is$spliced$out$to$make$the$beta$isoform$ o Alpha$is$spliced$out$to$make$the$alpha$isoform$ $ $ $ $ $ $ $ $ $ $ $ $ $ $ • See$figure$4.15$"$different$combinations$of$exons$are$used$in$alternative$splicing$$ o In$this$case,$exon$2$is$spliced$out$ o Exon$1$shown$in$blue$is$fused$to$exon$3$in$pink,$to$form$the$shorter$2$exon$ mRNA$(bottom),$whereas$the$middle$one$has$all$3$exons$intact$ o Clearly,$the$2$share$similarities$as$the$N$and$C$terminuses$are$the$same,$but$ the$middle$one$has$a$middle$part$(green)$that$is$different$than$the$bottom$ one$"$new$function,$big$differences$ • Used$extensively$in$all$organisms$ • If$you’re$differentially$splicing,$what$do$you$have$to$make$sure$you$maintain$to$get$a$ functional$protein?$ o Reading$frame!$ o When$you’re$splicing$alpha$or$beta$out,$you$still$have$to$make$sure$that$when$ the$other$ones$come$together,$they’re$still$maintaining$their$reading$frame$ $ How)did)genes)evolve?) • We$talked$about$exons$being$built$into$genes$and$how$they$can$be$used$to$form$ other$genes$through$insertion$into$other$sites$within$a$gene$in$order$to$make$a$more$ multifunctional$protein$ • In$the$late$70s,$before$a$lot$of$genomic$sequence$was$ever$done,$there$were$2$ theories:$one$that$all$genes$used$to$have$introns$and$that$some$lost$them$over$time,$ and$the$second$is$that$the$primordial$genes$didn’t$have$introns$and$they$were$ inserted$later$ • Did$genes$originate$as$interrupted$(with$introns)$or$uninterrupted?$ • Intron$“early”$IE:$introns$have$always$been$apart$of$genes$and$genes$with$no$introns$ have$lost$them$over$time$ o Look$at$yeast,$ancient$organisms$with$no$introns.$We$believe$they$lost$ introns$over$time.$In$contrast,$mammalian$systems$still$haven’t$lost$introns$as$ they$haven’t$been$around$long$enough$(in$evolutionary$timing).$ • Intron$“late”$IL:$original$genes$had$no$introns$and$they$were$added$later$during$ evolution$after$prokaryote/eukaryote$split$ o Introns$come$in$“late”$during$evolution$ o Certain$organisms$have$used$this$to$evolve$more$complicated,$larger$genes.$It$ allows$for$exons$to$be$swapped$between$different$genes$at$different$loci$of$ the$genome.$$ • Still$debated$ o Some$evidence$maybe$points$to$the$intron$“early”$theory.$The$reason$this$ one$is$slightly$more$favored$is$because:$the$earlier$you$have$introns,$the$ earlier$you$can$start$exon$swapping.$If$there$were$no$introns$early,$such$as$in$ the$intron$“late”$theory,$than$how$could$genes$have$evolved$into$more$ complex$organisms?$$ • Allows$for$exon$swapping$early$in$evolution$ • Led$to$more$combinations$of$exons$and$protein$function$ • See$figure$"$random$translocations$may$produce$functional$genes$ o Example$of$exon$swapping$ o There$are$numerous$examples$where$its$clear$that$exons$have$made$their$ way$from$their$original$genes$to$new$genes,$also$giving$that$new$gene$a$ different$function$ o On$the$top,$you$have$2$exons$with$a$large$intron$in$the$middle.$The$2$exons$ are$sliced$together.$What$happens$is$that$this$fragment$of$exon$DNA$gets$ recombined$into$the$original$bottom$figure,$to$make$a$functional$protein.$ The$new$gene$has$an$added$exon$which$probably$has$a$different$or$better$ function.$ o The$odds$of$this$happening$and$being$functional,$is$very,$very$low$ o However,$we$know$that$this$has$happened$in$the$past$(look$at$evidence$for$a$ bunch$of$different$genes)$ o There$has$to$be$some$selective$pressure$to$give$that$organism$the$advantage$ of$having$the$bottom$RNA$sequence$rather$than$the$top$ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ Some)exons)encode)similar)functional)domains)of)proteins)in)different)genes:) • Recall$the$theory$that$exons$are$building$blocks:$small,$short$functional$domains$that$ come$together$to$form$the$functional$properties$of$a$protein$ • An$example$is$secreted$proteins$ o There$are$lots$of$different$secreted$proteins$ o A$lot$of$them$are$secreted$out$of$the$cell$into$the$extracellular$space$that$ attaches$cells$to$each$other$ • Many$secreted$proteins$have$signal$sequence$within$the$N[terminus$that$is$encoded$ in$first$exon$ • Sequence$acts$independently$(from$the$rest$of$the$protein)$to$allow$for$secretion$ o The$protein$has$a$function$to$do,$and$it$has$to$do$it$in$the$right$spot,$so$it$has$ to$use$signals$to$get$there$ o Usually$what$happens$is$that$after$they$transport$the$signal$and$its$cleaved$ off$by$proteases,$then$the$protein$is$allowed$to$function$ • Since$most$exons$are$small,$proteins$are$assembled$from$smaller$modules$or$ subunits$ • An$example:$signal$peptide$is$highly,$highly$conserved$in$all$proteins$and$their$cells.$ That$sequence$is$shared$in$a$lot$of$different$proteins.$That$exon$is$believed$to$have$ come$from$these$duplication/swapping$events$that$gave$the$protein$localization,$ helping$it$do$its$job$better$ • LDL$receptor$protein$has$similar$exons$from$EGF$protein$ o Another$example$ o LDL$receptor$binds$low[density$lipoproteins.$EGF$is$a$growth$factor$hormone$ that$helps$our$cell$to$divide.$These$2$functions$are$very$different:$EGF$is$a$ secreted$protein,$while$LDL$is$a$receptor$found$in$the$cell$membrane$ • See$figure$4.17$"$exons$in$two$proteins$can$be$related$ $ $ $ $ $ $ $ $ $
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